• OVULATION

    •

  • by

  • Lawrence L. Espey & H. Lipner

    •

  • in

  • The Physiology of Reproduction

  • edited by

  • E. Knobil and J.D. Neill

  • Raven Press, New York, pp. 725-780, 1994

    •
  •  
  • OUTLINE OF THIS REVIEW ON OVULATION

  • INTRODUCTION

  • HISTORICAL BACKGROUND

  • The Early Years

  • The Smooth Muscle Theory

    •

  • The First Quarter of This Century

  • The Middle of This Century

  • The 1970's Surge in Interest

  • After the Surge

  • Summary

  • The Pressure Theory

    •

  • Superficial Observations

  • The Osmotic Pressure Hypothesis

  • Hydrostatic Pressure Measurements

  • Summary

  • The Proteolytic Enzyme Theory

    •

  • The Earlier Studies

  • The Initial Enzyme Assays

  • Intrafollicular Injections of Proteases

  • Change in Follicular Distensibility

  • Efforts to Measure Collagenase

  • Summary

  • FUNCTIONAL ANATOMY OF THE OVARIAN FOLLICLE

  • General Morphology

  • Surface Epithelium

    •

  • General Features

  • Cytoplasmic Granules

  • Polymorphous Nuclei

  • Scanning View of follicular Apex

  • Tunica Albuginea

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  • General Features

  • The Collagen Matrix

  • Multivesicular Structures of Follicular Fibroblasts

  • Theca Externa

    •

  • General Features

  • Thecal Fibroblasts

  • Ovulatory Changes in the Thecal Connective Tissue

  • Theca Interna

    •

  • General Features

  • Steroid Synthesis

  • Vascular System

  • Granulosa Layer

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  • General Features

  • Granulosa Gap Junctions

  • Other Anatomical Considerations

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  • Follicular Fluid

  • Innervation of Ovarian Follicles

  • BIOCHEMICAL EVENTS IN OVULATION

  • Membrane Phenomena and Related Events

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  • The Gonadotropin Surge

  • Gonadotropin Receptors

  • Cyclic AMP and Other Second Messengers

  • Protein Kinases

  • Mobilization of Membrane Phospholipids

  • RNA and Protein Synthesis

  • Vasoactive Agents and Related Substances

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  • Histamine

  • Bradykinin

  • Angiotensin II

  • Prostaglandins

  • Lipoxygenase Products

  • Ovarian Steroids

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  • Earlier Studies

  • Response to the Gonadotropin Surge

  • Sites of Steroid Synthesis

  • Importance of Steroids in Ovulation

  • Inhibitors of Steroidogenesis and Ovulation

  • Luteinized Unruptured Follicles

  • Other Considerations

  • Proteolytic Enzymes

    •

  • Plasminogen Activator

  • Kallikrein and Kinin-Generating Activity

  • Collagenolytic Activity

  • Other Considerations

    •

  • Follicular Inflammation

  • Fibroblast Proliferation

  • Growth Factors and Ovulation

  • Leukocytes and Ovulation

  •  

  • AN OVERVIEW OF THE OVULATORY PROCESS

  • INTRODUCTION
    •
  •     Mammalian ovulation is a distinct biological phenomenon that requires the rupture of healthy tissue at the surface of the ovary. In most mammals, the whole follicle protrudes markedly from the ovarian surface at the time of ovulation, and in many instances a thin translucent stigma, the macula pellucida, forms at the apex of the follicle as the final sign of impending rupture. This unique morphological change has left a striking impression on those who have actually observed it. Kelly (1) was so fascinated while observing a rabbit follicle near rupture he stated "as tension within the follicle increases, the transparent portion around the pole begins to bulge...It now stands out like the nipple on a breast." The moment of rupture sometimes appears as an explosive event, leading observers to compare it to a "volcano erupting" (2), or a "blister that bursts" (3).
  •     Some years ago, Walton and Hammond (4) provided a detailed account of the macroscopic changes that can be observed in a laboratory animal like the rabbit. The changes that take place in the tissue at the site of rupture are pathophysiological in that they involve a fracture in the dense layers of collagenous tissue which encapsulates the follicle, and there is invariable hemorrhage in the vicinity of this ovarian lesion. However, the structural modifications are not limited only to the apically protruding stigma of the follicle. There is ample evidence that the ovulatory surge in gonadotropic hormones transforms the entire ovarian follicle into a highly secretory corpus luteum. In fact, there is reason to believe the primary action of the gonadotropins is luteinization, and that rupture of the follicular surface is somewhat of a fortuitous event that is contingent on the local softening of tissues and proliferation of fibroblasts that occur during the early stages of remodeling of the ovarian follicle into a corpus luteum (5).
  •     This chapter provides a chronological account of the basic research on ovulation up to the 1970's, and then it concentrates on the information that has accumulated during the past several decades on the biochemical events of ovulation. The contents are not intended to be all-inclusive. For example, this chapter will not cover as many details about ovarian innervation and vascularity as the chapter on ovulation in the previous edition of these volumes (6).
  •     Innumerable reviews on various aspects of ovulation have been written during the past 60 years. Some of these reviews are listed in the following sentences of this paragraph, while others are identified at more appropriate sites in this chapter. In 1932, Hartman (7) wrote the first comprehensive review of the work on ovulation, and his account provides many interesting insights into the early history of this subject. In addition, there are a number of other reviews of the earlier work on ovulation (3, 8-18). In more recent times, there has been an exponential growth in the number of studies on ovulation. Much of this more recent work has been described in detail in innumerable reviews that have appeared in the past 15 years. These include reviews of a comprehensive nature (19-26), on ovarian follicular development and ovulation (27, 28), on the structure and morphology of follicles (29-32), on the biochemistry of ovulation (33-36), on ovarian smooth muscle (37), on applications of the ovarian perfusion model (38), on ovulation and the immune system (39, 40), on the molecular aspects of ovulation (41-43), on signal transduction processes in ovulation (44), on ovulation as an inflammatory process (45, 46), and on ovulation in relation to the menstrual cycle (47, 48).
  •     An initial note on terminology is also in order. It is common to use the expressions "preovulatory," "ovulatory," and "ovulation" all in reference to the entire gonadotropin-induced process. However, in this chapter, the adjective "ovulatory" will be applied to the entire process, while the term "preovulatory" will be used in reference to mature follicles that have not yet been stimulated to enter the ovulatory process. The term "ovulation" may indicate either the process, or the moment of egg release, but in instances where clear delineation of the latter phenomenon is of primary importance, then the term "follicular rupture" will be used for clarity.
  •  
  • HISTORICAL BACKGROUND
    •
  •     In the beginning, ovulation was studied mainly in vivo, simply with the naked eye. Observations of the follicular contents "oozing" or "bursting" from the ovary made a strong impression on the early investigators who managed to catch a glimpse of the phenomenon. The initial theories about the mechanism of ovulation keyed on the possible role of smooth muscle and the potential for such tissue to create intrafollicular pressures of sufficient magnitude to cause healthy follicular tissue to rupture. When light microscopy was first applied to studies of the microanatomy of visceral organs, the pioneering anatomists labeled much of the cellular component of the follicle as smooth muscle tissue. However, it is now clear that most of this tissue in and around mature ovarian follicles is collagenous connective tissue, which has arisen from resident fibroblasts. Nevertheless, the first 100 years of investigation into the mechanism of ovulation centered on the smooth muscle and the pressure theories. Therefore, the segments of this chapter that deal with the history of ovulation key on these two early theories and how they eventually gave way to the so-called "enzyme theory" of ovulation.
  •  
  • The Early Years
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  •     Ovarian follicles were first identified in 1672, when de Graaf examined human ovaries and observed vesicles on their surface which he mistakenly referred to as ova (49). Over a century later, Cruikshank (50) discovered ova in the fallopian tubes of rabbits, and eventually von Baer (28) realized that the mammalian oocyte was quite unlike the large avian and amphibian eggs, but instead was a relatively small cell within the follicular mass.
  •     In the middle of the 19th century, von Kolliker (51) was the first to mention that smooth muscle was a structural constituent of the ovary. In 1858, Rouget (52) suggested that ovarian smooth muscle activity impaired venous return and caused congestion and "erection" of ovulatory follicles in a manner that increased intrafollicular pressure and caused rupture. That same year, von Luschka (53) concluded that the follicular fluid was derived primarily from secretions of the granulosa cells and secondarily from fluids transported to them from the blood vessels of the theca interna. A year later, Pfluger (54) observed the motion of frog ovaries and concluded that the "peristalsis" which they exhibited was responsible for ovulation, but he was apparently unaware of the fact that the amphibian ovary is in a state of constant movement even out of the sexual season (11). Two years later, Aeby (55) used acetic and nitric acid staining techniques to identify what he considered to be smooth muscle cells in the theca externa of avian and mammalian follicles, and he suggested that similar cells were responsible for the "peristaltic" contractions of the frog ovarian stroma. Several years later, Grohe (56) described the course of the muscle fibers in the ovary of the pig, and his observations led him to support the idea that congestion from impaired venous return caused rupture of mature follicles. In 1870, Waldeyer (57) agreed with Luschka's view of fluid formation, and he went on to hypothesize that a rapid hypertrophy of the theca interna increased intrafollicular pressure and caused rupture. There was little additional work on ovulation for the remainder of the 19th century, but the "smooth muscle" and "pressure" theories of ovulation were already firmly established by that time.
  •  
  • The Smooth Muscle Theory
    •
  • The First Quarter of This Century
    •
  •     The smooth muscle theory of ovulation has been one of the most controversial issues in the field of reproductive physiology. The controversy has been perpetuated mainly by persistant reports that the dominant cell-type in the theca externa layer of the follicle wall is a typical smooth muscle cell. At the beginning of the present century, von Winiwarter and Sainmont (58) demonstrated what they called smooth muscle in the wall of the cat and human follicle, even though they were unable to demonstrate that either electrical or chemical stimuli could cause the follicle to rupture. In 1919, Thomson (59) carried out extensive histological studies and concluded that muscular contractions on an engorged follicle naturally increases the intrafollicular pressure and causes the follicle to rupture. In contrast, Corner (60) reported that same year his findings from an extensive study of the histology of the granulosa and thecal layers, and he concluded that the theca externa consisted of collagenous fibrils and long spindle-shaped "fibroblasts" that became mitotic just before rupture. Two years later, Guttmacher and Guttmacher (61) again reported histological evidence of thecal smooth muscle in the sow follicle, and they claimed that strips of these follicles contracted in response to solutions of HCl, barium chloride, and physostigmine sulfate. However, they also noted after 72 injections of ovarian and uterine arteries with saline, that "even though one braced himself against a wall and pushed the piston of the injection syringe with all the physical strength available," rupture did not occur in a single instance, although one could see the follicular vessels wash out clearly. In the end, they admitted that their experiments were so inconsistent that they were unable to conclude that muscle tissue had a role in the rupture of a follicle.
  • The Middle of This Century
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  •     Over a quarter of a century later, in 1947, Kraus (9) conducted an extensive study of ovulation in frogs, hens, and rabbits and found that attempts to induce contractility and ovulation with smooth muscle stimulants or electrical impulses invariably failed. That same year Claesson (62) attempted to resolve the smooth muscle issue by examining cow, swine, rabbit, guinea pig, and rat ovaries under a polarization microscope. By this method, he failed to find the high intrinsic birefringence that is characteristic of muscle, but instead found birefringence that is typical of connective tissue. The reliability of the method negated virtually all previous histological evidence, including Claesson's own, of smooth muscle in the follicle wall and threw doubt on the reliability of any physiological data. However, some years later, in 1960, Lipner and Maxwell (63) supported the smooth muscle theory by demonstrating visible muscle-like contractions in the follicles of ovarian tissue autotransplanted into the anterior chamber of the rabbit's eye. On the other hand, several years later, Espey (12) attempted to duplicate the earlier experiments by the Guttmachers. He found (i) that strips of sow follicles would contract only in response to HCl, (ii) that such contractions were tetanic in nature, (iii) that the tissue would relax only if it was exposed to alkaline solutions, and (iv) that such contractility could be attributed to collagen fibrils in the thecal layers of the follicle. Three years later, Espey (64) used transmission electron microscopy to confirm the distribution of collagenous connective tissue and fibroblasts in the theca externa of the follicle, and he conducted further studies to verify that follicular collagen contracts when it is exposed to acidic solutions (65).
  • The 1970's Surge in Interest
    •
  •     The smooth muscle controversy became more intense during the 1970's, when an avalanche of new reports claimed, on the basis of various kinds of evidence, that smooth muscle activity is an integral part of the ovulatory process. The reader is referred to an earlier review for enumeration of these reports (37). In brief, this review concluded that typical smooth muscle tissue is confined mainly to the hilar and medullary regions of the mammalian ovary. The numerous reports of myoid tissue in peri- and parafollicular areas are probably due to the overenthusiastic identification of "smooth muscle" tissue primarily on the observation of cytoplasmic filaments in the cells of the theca interna, without realizing that such structures are also a normal component of thecal fibroblasts (66-68). At the time of ovulation and especially during luteinization, there may be some differentiation of these thecal fibroblasts into myofibroblasts to facilitate wound healing and the removal of granulation tissue during luteolysis, but there is no convincing evidence that such tissue has an essential role in the mechanism of ovulation (37).
  • After the Surge
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  •     In spite of substantial evidence that ovarian contractions are not necessary for ovulation, there have been a number of more recent reports which continue to support the idea that smooth muscle is an important component in the mechanism of ovulation. Most of the effort to preserve the theory have come from Talbot, Martin, Schroeder, and collaborators (69-75), who continue to claim that there is a discrete layer of smooth muscle cells at least in the basal hemisphere of the follicle, and that such muscular tissue is necessary for extrusion of the oocyte. This lingering view is supported by further reports (i) that myocytes are present in the follicles of many mammals (76), (ii) that such cells are syncytially linked by several types of surface junctions (77), (iii) that these cells contain actin fibrils characteristic of smooth muscle cells (78), and (iv) that ovarian contractility and ovulation can be influenced by a wide range of agents that agonize and antagonize smooth muscle contractions (71, 79-82).
  •     Also, Schroeder and Talbot (83) have reported that intrafollicular pressure changes during preovulatory contractility in the follicle. However, two decades earlier, Espey and Lipner (84) pointed out that such rhythmic fluctuations in intrafollicular pressure are rare and are not essential for ovulation. More recently, Kobayashi et al. (85) have also shown that perfused rabbit ovaries can ovulate in the absence of ovarian smooth muscle contractions, and Lofman et al. (86) have found that perfused rat ovaries can ovulate with no visible circumfollicular muscular activity.
  •     Thus, there is still no convincing evidence that ovarian contractile activity is necessary for ovulation. Nevertheless, the existing data suggest that some kind of rhythmic motion is occasionally expressed by ovarian tissue near the time of ovulation, and the nature of this mechanical activity has not been adequately explained. Negligible attention has been given to the possibility that the rhythmic pulsations might be the consequence of vascular spasms in the blood vessels that enter the hilar region of the ovary (37). Also, it is possible that intraovarian pressure could fluctuate as a result of spasms in the tubo-uterine vasculature, because vessels of this origin anastomose with ovarian arteries (87). In considering this alternative explanation, it may be relevant to note that Markee (88) has observed that the uterine vasculature sometimes undergoes rhythmic spasms at 15-20 sec intervals, depending on the "hormonal" conditions of the tissue. Therefore, it is possible that an agent like prostaglandin F2a , which is known to stimulate contraction of ovarian arterial smooth muscle (89), and which is known to increase markedly in the ovary during ovulation (33), may be promoting spasms in the ovarian vasculature near the time of ovulation.
  •     Finally, it is worth mentioning that lutein follicles have been likened to granulation tissue (44, 45). A granuloma is a firm nodular mass which forms in conjunction with angiogenesis and fibroplasia that develops during the wound healing process in injured and inflamed tissues (90-93). Such granulation tissue usually contains myofibroblasts that contract and relax in a similar way to smooth muscle cells (94). If this information is considered in conjunction with earlier ovarian studies which suggest that corpora lutea in particular may contain well-defined coats of smooth muscle (95), then it is also possible that some of the reported ovarian contractility may be associated with myo-fibroblasts within luteinized, unruptured follicles. Thus, there may be certain aspects of the smooth muscle theory that deserve further investigation.
  • Summary
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  •     In summary, there are still differing opinions about the extent to which smooth muscle cells are distributed in and around mammalian follicles. Some investigators continue to believe that the theca externa consists of typical smooth muscle cells, while others report that the theca externa is predominantly a matrix of collagenous connective tissue with typical fibroblasts making up the cellular component. In any event, it is now rather clear that ovarian contractility is not a necessary feature of the ovulatory process. Still, there remains the possibility that some of the fibroblasts begin to differentiate into myofibroblasts as a ruptured follicle transforms into a corpus luteum, and the potential role of such myoid tissue in luteinization, or luteolysis, may deserve further investigation.
  •  
  • The Pressure Theory
    •
  • Superficial Observations
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  •     At the beginning of this century, additional attention was given to the hypothesis that an increase in intrafollicular pressure might be responsible for rupture of a mature ovarian follicle. Heape (96) stressed the role of increasing pressure as the cause of ovulation, and he believed that vasodilation and bursting blood vessels contributed to the ovulatory pressure. However, Schochet (97) did not think the follicular circulation could be responsible for intrafollicular pressure because he noted that follicle pressure probably was greater than the blood pressure within the capillaries since the capillaries appeared to be compressed by the antral pressure. On the other hand, Wester (98) suggested that some kind of pressure "necrosis" of the follicular wall was the final cause of rupture. However, the Guttmacher twins (61) subjected sow follicles to unusually high pressures of 300-350 mm Hg for hours without inducing ovulation, and the negative results of this test do not support a role for pressure in the mechanism of ovulation.
  •     The impressive studies of rabbit ovulation in 1928 by Walton and Hammond (4) demphasized that the first sign of approaching ovulation is the gradual formation of the stigma (i.e., the macula pellucida) at the apex of a follicle. They reported that the whole follicle protrudes more markedly from the ovarian surface, and then the stigma suddenly "blows out as a pimple" in a manner that suggested an increase in internal pressure. The small blood vessels at the base of the stigma were seen to rupture before the stigma broke open, and they noted some extravasation of blood into the follicular antrum near the time of follicular rupture. They concluded that the process was comparable to "the formation and rupture of a boil", and that an increase in internal pressure was probably of primary importance in causing rupture. These observations were supported 3 years later by Kelly (1). Several years after that report, Hill et al. (2) made the first movies of ovulation, using the rabbit as their subject. These investigators emphasized that follicular rupture "is truly an explosive phenomenon." Likewise, in sheep, McKenzie and Terrill (99) reported that in some cases rupture occurred with "a decided spurt." They also noticed that the apical most area of a follicle usually became very clear and transparent about an hour before rupture, and that one or more small cones about 1-3 mm high ballooned out from this clear area a few minutes before rupture. In comparison, 10 years later, Kraus (9) reported that injections of pressurized saline into rabbit follicles could promote rupture, but without stigma formation. Furthermore, she noticed that in such cases of artificial induction of ovulation the edge of the rupture site was fine and smooth, whereas in normal ovulation the rupture point was circular and the edge of the follicle opening was blunt and rough. Also, unlike previous observers, Kraus (9) only saw the follicular fluid "ooze" from the rupture site instead of spurt out. In view of these characteristics, she concluded that the pressure theory was unable to explain the observable morphological changes. Similarly, Hartman (7) pointed out that follicles of pigs, cattle, and sheep become flabby a few hours before ovulation, and this fact does not favor the idea of an increase in intrafollicular pressure.
  • The Osmotic Pressure Hypothesis
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  •     Another version of the pressure theory was based on the idea that osmotic pressure might increase in ovarian follicles near the time of ovulation. While performing the Friedman pregnancy test on rabbits, Smith (100) became interested in what caused the tiny ovarian "blebs to swell up and burst within the course of a few hours". Upon measuring the total osmotic pressure of rabbit follicular fluid, he found a moderately higher osmolarity in ovulatory follicles when compared to unstimulated follicles. In a subsequent study, Smith (101) speculated that so-called "Call and Exner" bodies of the granulosa layer of the follicle contained a glycogen-like substance that depolymerized near the time of ovulation and "contributed something to the follicle fluid which increases its osmotic tension so that it takes up fluid from the surrounding tissue and so increases the content of the follicle until it reaches the bursting point." Twenty years later, Zachariae and Jensen (102, 103) advanced the osmosis hypothesis by reporting that pre-ovulatory hyaluronidase activity depolymerized the acid mucopolysaccharides of the follicular fluid, "resulting in an increase in the intrafollicular colloid-osmotic pressure, increased volume, and follicular rupture." However, the presence of a follicular mucopolysaccharidase was never confirmed, and this theory of ovulation has faded.
  • Hydrostatic Pressure Measurements
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  •     Eventually, as technologies improved, Espey and Lipner (84) utilized an ultrasensitive Statham pressure transducer to measure the hydrostatic pressure within the follicular antrum during ovulation. Continuous monitoring clearly demonstrated that there is no increase in intrafollicular pressure during the hours preceding rupture, and that the existing steady pressure of 15-20 mm Hg is directly dependent on the mean hydrostatic pressure of the ovarian arterial supply. Concomitant with this study, Blandau and Rumery (104) adapted a water manometer to make static pressure measurements in the antra of rat follicles at different stages of development, and they found that the intrafollicular pressure remained within the range of capillary blood pressure during the ovulatory process. The results of these two experiments, which demonstrated a constant intrafollicular pressure, were confirmed a year later by a similar study conducted by Rondell (105). Some 15 years later, Bronson et al (106), also could not detect any increase in pressure in pig follicles as the time of ovulation approached. Thus, it has been firmly established that follicular rupture is not caused by an increase in intrafollicular pressure.
  • Summary
    •
  •     In summary, intrafollicular pressure does not increase during ovulation, but remains at a relatively constant 15-20 mm Hg that is dependent on the hydrostatic pressure in the extensive capillary network of the theca interna.
  •     This moderate pressure probably exerts a constant force on the follicle wall and may be an important factor in the ballooning of the stigma in a morphologically weaker area of the follicular apex. Also, the question of whether the moment of rupture is explosive or passive may have arisen from the fact that the superficial appearance of the phenomenon probably depends on the extent to which the arterial supply has been impaired during surgical exposure of the ovaries for visual observation during ovulation. If blood flow is reduced, then intrafollicular pressure would be low and the contents of the follicle would ooze from the stigma area; whereas, if the vascular supply is of normal patency, then the moderate intrafollicular pressure may be of sufficient force to cause a weakening follicle wall to rupture under what appears to be a relatively strong head of pressure.
  •  
  • The Proteolytic Enzyme Theory
    •
  • The Earlier Studies
    •
  •     As it gradually became apparent that neither the smooth muscle theory, nor the pressure theory, adequately explained the mechanical events leading to ovulation, more and more attention was given to the possibility that the morphological changes that occur at the apex of an ovulatory follicle might be the result of enzymic degradation of the thecal connective tissue. Descriptions of the disappearance of thecal tissue at the stigma and of the erosion of the rupture point by early investigators such as Long and Mark (107) made it clear that ovulation was not the consequence of any violent tearing of the follicular wall. In view of this kind of information, a professor Irving Hardesty suggested to Schochet (97) that the liquor folliculi might "exert some special digestive action on the resisting tissues" of the follicle wall. With this idea in mind, Schochet collected follicular fluid randomly from large sow follicles and conducted several crude chemical tests that led him to conclude that the fluid was indeed the source of "a proteolytic ferment or enzyme" which contributes "to the digestion of the theca folliculi." Although his experiment has never been confirmed, and although it is doubtful that the follicular fluid is the source of the proteases that degrade the follicle wall, Schochet is usually given credit for the proteolytic enzyme theory of ovulation.
  • In testing the enzyme hypothesis, Rugh (108) found that the external application of solutions of pepsin and hydrochloric acid initiated follicular rupture in frogs. A year later Markee and Hinsey (109) noticed that ovulatory rabbit follicles develop an avascular area surrounded by dilated vessels, suggesting that local morphological changes were a prerequisite for stigma formation. At about the same time, McKenzie and Terrill (99) reported similar morphological changes in the sheep, noting a thinner more transparent area at the apex of ovulatory follicles. A decade later Moricard and Gothie (110) revived Schochet's hypothesis with tenuous evidence that gonadotropins cause the secretion of a "diastase" (a term that means any kind of enzyme in French) having proteolytic activity that digests the various layers of the follicle wall and results in rupture. A year later, Kraus (9) confirmed the earlier report of Rugh by showing that immersion of frog ovaries in an acidic solution of pepsin caused erosion of the follicle wall, but the amphibian ova were not fully extruded. Furthermore, all of her attempts to identify a proteolytic enzyme in frog follicles failed, and her applications of proteolytic enzymes to the follicles of hens and rabbits were without effect. As a result of these negative findings, Kraus concluded that neither the pressure nor the enzyme theory fit the facts and that the immediate cause of ovulation remained a mystery.
  • The Initial Enzyme Assays
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  •     As various assays for proteolytic enzymes became available around the middle of this century, some investigators began attempting to identify specific enzymes in follicular tissue. One of the first major efforts was by Jung and Held (111), who evaluated proteolytic enzymes and alkaline and acid phosphatases in small, intermediate, and large follicles of the pig. All three enzymes were found in significantly greater concentrations in the small follicles, compared to the larger ones, and most of the activity was at pH 2.5-3.5. That same year, Jung and Kides (112), reported that human follicular fluid contained a significant amount of cathepsin-like activity at acid pH, but no trypsin activity at neutral pH. In a subsequent report (113), these same investigators assayed for endopeptidase, leucine-aminopeptidase, and glycyl-glycyl-dipeptidase and found that all of these ovarian proteases were more abundant in small follicles compared to large ripe follicles. They concluded that such activity must be dependent on FSH, rather than LH, and that the role of these enzymes in ovulation was uncertain. At about this same time, Reichert (114) roughly estimated ovarian proteinase activity in rats by homogenizing and extracting the ovaries and then measuring the optical absorbance of the extracts at 280 nm. He found that ovaries at diestrus and estrus contained significantly more "protease" activity than ovaries of pregnant and pseudopregnant animals. However, this crude method did not demonstrate any increase in acid or alkaline proteolytic activity on the morning of estrus, when the assays were conducted on tissue that was taken closest to the time of ovulation. Lee and Malvin (115) detected similar acid protease activity in follicular homogenates of sows. However, Espey (116) reported that the pH of sow follicular fluid remains in the alkaline range during ovulation, and therefore it is doubtful that the acid proteases reported in the above studies have any significant role in ovulation.
  • Intrafollicular Injections of Proteases
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  •     Further support for the enzyme theory came from experiments in which Espey and Lipner (13) injected small quantities of concentrated enzyme preparations directly into the antrum of rabbit follicles and showed that such injections rapidly induced morphological changes similar to normal swelling, stigma formation and rupture of the follicles. In the initial studies, clostridiopeptidase-A (a bacterial collagenase), nagarse and pronase (also microbial enzymes) were the most effective in inducing rupture. Later experiments revealed that injections of mammalian collagenase or trypsin could also induce rupture of rabbit follicles (unpublished observations).
  • Change in Follicular Distensibility
    •
  •     In 1964, Rondell (105) studied the elasticity of rabbit follicles by simultaneously inserting two micropipettes into follicles. One pipette was for injecting set quantities of saline, while the other pipette was for pressure measurements. By measuring the pressure increase after injection of a unit quantity of saline, he was able to demonstrate a substantial increase in the distensibility of rabbit follicles that were near rupture. He concluded that such a change in the physical characteristics of the follicle wall were an essential part of the mechanism of ovulation. Several years later, Espey (65) also measured the tensile strength of the wall of sow follicles and found that follicles close to rupture are more labile to dissociate when subjected to physical stress. He also showed that treatment of preovulatory follicles with a solution of collagenase caused the follicle walls to dissociate when they were subjected to only slight tension. Several years later, Espey (116) further demonstrated that elastase, trypsin, a general protease extract, a -chymotrypsin, and to a lesser extent ß-chymotrypsin could also significantly reduce the tensile strength of the sow follicle. Thus, it became evident that the follicle wall becomes progressively weaker as the time of rupture approaches and that treatment of strips of the follicle wall with various preparations of proteolytic enzymes can mimic this preovulatory degradation of the follicular connective tissue.
  • Efforts to Measure Collagenase
    •
  •     In view of much of the above information, Espey and Rondell (117, 118) attempted to measure collagenolytic activity in sow and rabbit follicles by using a synthetic peptide substrate. However, the results revealed a decrease in enzyme activity, rather than an increase as the time of rupture approached. In a further effort to assay collagenolytic activity, Espey and Coons (18) cultured sections of rabbit follicles on gels composed of reconstituted collagen. Although the follicular tissue digested the collagen gels when incubated in either acid or alkaline media, the results did not clearly demonstrate any increase in this activity during the ovulatory process. The following year, Morales et al. (119) used a more specific synthetic substrate to identify collagenase activity in rat follicles. Several years later, Morales et al. (120) applied an assay based on the digestion of endogenous collagen to measure "true" collagenase in rat ovaries, but they were unable to detect any significant change in such activity during ovulation. Thus, the measurement of an increase in ovarian collagenolytic activity has been a rather elusive goal. This difficulty is not particularly surprising since it is well known that mammalian collagenases are quite difficult to assay (16, 18). Several more recent attempts to detect ovarian collagenolytic activity will be discussed later in this chapter in the section on chemical events of the ovulatory process.
  • Summary
    •
  •     In summary, it is firmly established that the collagenous layers of the theca externa and tunica albuginea at the apex of a follicle must be degraded before a follicle can rupture. Furthermore, it is apparent that this follicular connective tissue becomes flaccid and distensible near the time of ovulation. Several in vivo and in vitro studies have shown that a number of metalloproteinases and serine proteases can decompose the follicle wall, but it has been difficult to measure collagenolytic activity in the ovary during normal ovulation. Part of the reason for the ellusive nature of this activity may be due to the fact that mammalian collagenases are highly destructive enzymes that must be inactivated rapidly in order to confine the damage to a localized area.
  •  
  • FUNCTIONAL ANATOMY OF THE OVARIAN FOLLICLE
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  • General Morphology
    •
  •     Earlier reviews containing information on the anatomy of ovarian follicles include those by Hartman (7) and Harrison (121). The first report that dealt specifically with the ultrastructure of ovulatory follicles was in 1967, by Espey (64). Several years later, Bjersing and Cajander (122-127) published a series of papers that contain a much more complete analysis of the ultrastructure of the ovarian follicle of the rabbit, and Parr (128) published a vivid description of the ovarian follicle of the rat. Additional reviews on the morphology of ovarian follicles, have been published by Lipner (15), Mossman and Duke (129), Bjersing (29), and Espey (19), and more recently by Balboni (30), Guraya (31), Lipner (6), and Espey (32).
  •     Ovarian follicles begin as primordial spheres consisting of a single layer of flattened epithelial cells which surround individual oocytes (121). These primordial follicles persist for years in the outer cortical layers of the ovary until they eventually are selected to develop under the influence of gonadotropin stimulation. In the early stages of development, the granulosa cells increase in number and begin to form layers (130). Eventually, the granulosa cells promote the organization of a sheath of stromal fibroblasts that encapsulate the developing follicle and form the thecal layers around the follicle. As the follicle continues to grow, antral spaces begin to form within the sphere of granulosa cells, and these fluid filled cavities eventually coalesce and expand into a single large cavity that makes the mature follicle appear like a blister on the surface of the ovary. In a given species of mammal, the size of a mature follicle is usually proportional to the body weight of the adult female.
  •     The anatomy of rabbit follicles has been studied quite extensively, and most of the following description is based on the morphology of ovarian tissue in this species (Fig. 1). At the apex of a mature follicle, the outer portion of the follicle wall consists of a surface epithelium, which is a single layer of cuboidal or low columnar epithelial cells that adhere to the surface of a thin basal lamina. This epithelial covering around the ovary is probably a modified coelomic epithelium, i.e., a continuation of the peritoneum (121). The tunica albuginea which lies just inside the surface epithelium is a layer of dense collagenous connective tissue that also surrounds the entire ovary. The theca externa is the distinct layer of collagenous connective tissue that delineates the follicle from the surrounding ovarian stroma. At the follicular apex where rupture normally occurs, these two connective tissue layers, i.e., the tunica albuginea and the theca externa, blend together in a fashion that makes it difficult to distinguish one from the other, except that the tunica layer has more extracellular collagen (64). The tensile strength of these two layers of collagenous connective tissue must be degraded in order for a follicle to rupture.
  •     The theca interna is a thin layer of large, oval cells that differentiate from thecal fibroblasts during follicular maturation (19). This layer is the principal site of estrogen synthesis during the final maturation of a follicle (131, 132). One of the most prominent features of the theca interna is the extensive network of large capillaries that line the inner border of these steroid secreting cells. These capillaries do not normally penetrate the basal lamina (i.e., the membrana propria) that separates the theca interna from the granulosa layer which lines the inside of the follicle wall (19, 29). The granulosa cells are relatively small polyhedral cells that are linked together by an elaborate system of gap junctions (133). The granulosa cells that lie closest to the theca interna are columnar in shape and appear to be firmly attached to the basal lamina which separates these two layers of the follicle wall. The innermost granulosa cells are more cuboidal. At some point along the inner circumference of the follicle, clusters of granulosa cells project toward the center of the follicular antrum and form the cumulus oopherous, which contains the oocyte. This egg-bearing pedestal is surrounded by follicular fluid. The follicular fluid is a mixture of exudate from the granulosa tissue and transudate from the thecal blood supply (19, 134). The same general morphology is characteristic of mature follicles in ovaries that have been perfused in an in vitro system (135).
  •  
  • Surface Epithelium
    •
  • General Features
    •
  •     All mammalian ovaries are covered by a single layer of cuboidal cells that are loosely attached to a thin basal lamina at the surface of the connective tissue sheath (i.e., the tunica albuginea) that surrounds the ovary (19, 32, 136). In a cross-sectional view of the follicular apex, these cells contain large, indented nuclei (64). Their cytoplasm has relatively small, yet conspicuous, mitochondria. However, the most striking feature of these cells is the dense cytoplasmic spheres of mucin-like material that oftentimes dominate the basal side of the cells. A massive network of microvilli project from the peritoneal surface of this epithelial layer. The cells are held together at their lateral surfaces by zona occludens (64).
  • Cytoplasmic Granules
    •
  •     In her studies on mice, Byskov (137) reported that the ovulatory degradation of the follicular apex starts at the outside and successively progresses to the interior of the follicle wall. Five years later, Bjersing and Cajander (122-124, 138) reported a similar pattern of degradation in the ovulatory rabbit follicle and concluded that the surface epithelium is the source of hydrolytic enzymes that cause ovulation. In their series of papers on the surface epithelium they described a rapid increase in the number and size of granules in the cells of the surface epithelium of rabbit follicles that had been induced to rupture. Furthermore, they concluded that many of these granules were lysosomes that released their enzymes toward the underlying tunica albuginea a few hours before follicular rupture. This hypothesis received indirect support from an earlier study by Rondell (14), who reported that collagenase-like activity exudes from the surface of follicles that are about to rupture.
  •     This idea that cells of the surface epithelium contain massive lysosomes which extrude their proteolytic contents into the thecal layers of the follicle is intriguing, but the hypothesis has not been supported by other studies. Although the surface epithelium is intact in some follicles at the time of ovulation, more often this layer is no longer a constituent of the follicle wall at the site where rupture occurs (17, 64, 123, 139, 140). Also, if the surface epithelium is scraped from mature follicles, some of the follicles will still ovulate after stimulation by gonadotropin (141). Furthermore, contrary to the impression of Bjersing and Cajander (124), Rawson and Espey (141) carried out a quantitative study of the concentration of dense granules in the epithelial cells and found a two-fold increase in these granules during the ovulatory process, rather than a decrease. In addition, Narimoto et al. (142) reported that the granules do not react like lysosomes when the follicular tissue is treated with Gomori's lead medium for acid phosphatase. Therefore, it is difficult to conclude that the surface epithelium has a significant role in the mechanism of ovulation. Nevertheless, future studies that might identify the chemical composition of the cytoplasmic granules in this layer could contribute significantly to the existing knowledge about the function of the surface epithelium.
  • Polymorphous Nuclei
    •
  •     As a matter of routine, almost every study of the ultrastructure of ovarian follicles has been conducted on tissues that have been oriented in a manner that shows a cross-sectional view of the follicle wall. Such orientation allows the investigator to see each of the follicle layers from the outer surface epithelium to the inner stratum granulosum. From such a "side" angle, the cells of the surface epithelium usually contain what appear to be large, somewhat notched, nuclei (19, 64, 124). However, if one orients the tissue so that tangential sections can be cut through the apex of ovulatory follicles, then the nuclei of the epithelial cells appear to be multinucleate (32). In view of this characteristic, Espey (32) has compared these cells to polymorphonuclear leukocytes and has suggested that they might function as an outer defense mechanism to protect the vital procreative elements of the ovary from peritoneal microbes. Still, the precise function of the surface epithelium remains to be determined.
  • Scanning View of Follicular Apex
    •
  •     Transmission electron microscopy has revealed that the surface epithelial cells become grossly necrotic and tend to "slough off" when follicles are about to rupture (64). Later studies using scanning electron microscopy made it clear that the surface cells become detached from the stigma region of ovulatory follicles in the rabbit (123, 140, 143), rat (140, 144), mouse (140, 145), and hamster (145, 146). Tsujimoto, et al (144) have stated that the apical regions without surface epithelial cells display flattened and densely arranged fibroblasts of the tunica albuginea.
  •  
  • Tunica Albuginea
    •
  • General Features
    •
  •     The tunica albuginea is a layer of dense collagenous connective tissue that surrounds the entire ovary (64, 126). The outer border of this layer is delineated by the basal lamina that separates it from the cells of the surface epithelium. This layer consists almost exclusively of fibroblasts and an extracellular matrix of collagen fibers that are embedded in an amorphous matrix of ground substance. In most mammals, the fibroblasts are about 5-7 cells deep. These cells contain prominent oblong nuclei that have relatively smooth surfaces. Their cytoplasm is dominated by rough endoplasmic reticulum that is probably involved in the synthesis of tropocollagen. It appears that cytoplasmic processes are a common feature of the distal ends of these fibroblasts.
  • The Collagen Matrix
    •
  •     At the site where rupture normally occurs at the apex of a follicle, the collagenous tissue of the tunica albuginea (and to a lesser extent the theca externa) provide most of the strength of the follicle wall. Prior to hormonal stimulation of the ovulatory process, this dense connective tissue is quite tenacious. Such strength is to be expected, since individual collagen fibrils have a tensile strength estimated to be greater than that of cast iron (147). However, as Gross (148) pointed out three decades ago, the tenacity of a matrix of thecal connective tissue depends not only on the strength of the collagen fibrils, but also on the mucopolysaccharide matrix that "cements" these fibrils into collagenous bundles. Therefore, it may not be necessary for the proteolytic enzymes that degrade the follicle wall to meet all the criteria of a "true collagenase" (i.e., they may not necessarily be enzymes that specifically hydrolyze the collagen fibrils). Instead, it is possible that less specific serine proteases like plasminogen activator, kallikrein, or trypsin-like enzymes may digest the extracellular ground substance and allow the collagen fibrils to dissociate from one another under the force of a low, but constant, intrafollicular pressure (19, 44).
  •     In 1974, Parr (128) reported that rat follicles are exceptional in that they have no collagen at their apex where rupture normally occurs. This report implied that the term "thecal tissue" might be a misnomer in the rat follicle because, by definition, thecal tissue should contain fibrous collagen. Nevertheless, on the basis of his observations, Parr (17) concluded that collagenolytic activity may not be important in the mechanism of ovulation since collagen was not detectable in his electron micrographs. However, also in 1974, Rhodin (149) described collagen in the rat follicle. The discrepancy in these publications was clarified later by evidence that glutaraldehyde fixatives can cause collagen to loose its usual resistance to penetration by an electron beam (150). By staining thin sections of the follicle wall of the rat with a 1% solution of phosphotungstic acid, the collagen fibrils of the tunica albuginea and theca externa become much more visible in electron micrographs. Such staining reveals that the apex of a rat follicle has a substantial amount of extracellular collagen distributed evenly among the fibroblasts of the tunica albuginea and the theca externa.
  • Multivesicular Structures of Follicular Fibroblasts
    •
  •     One of the more interesting features of ovulatory follicles is the multivesicular structures that protrude from the surface of the plasma membranes of the fibroblasts in the collagen layers (19, 32, 151, 152). These unusual structures are especially common at the leading edges of the pseudopod-like processes that extend from the cytoplasm of the proliferating fibroblasts in follicles that are about to rupture. Espey (153) has noted that the concentration of these structures increases approximately nine-fold during the ovulatory process. In some instances, it is evident that the extracellular matrix of collagen is decomposed in the vicinity of these multivesicular bodies (151, 154). The general features of these structures strongly suggest that they may contribute in some important way to the opening of interstitial pathways for the movement of proliferating fibroblasts in ovulatory follicles. However, these unique entities are difficult to preserve for cytochemical analysis, and there is negligible information about their chemical composition (32). Therefore, their potential role in the mechanism of ovulation remains uncertain.
  •  
  • Theca Externa
    •
  • General Features
    •
  •     The theca externa is an outer sphere of collagenous connective tissue that delineates a mature follicle from the surrounding ovarian stroma (19, 32, 64, 152). Like the tunica albuginea, this layer of connective tissue appears to contain only one type of cell, namely fibroblasts. At the apex of a follicle, where the elements of the tunica albuginea and the theca externa are adjacent to one another, it is difficult to distinguish between these two layers in rabbit ovaries except for the fact that the theca externa contains fewer collagen fibers (32, 64). In contrast, in the rat, the density of collagen fibers appears to be evenly distributed between the theca externa and the tunica albuginea (150).
  • Thecal Fibroblasts
    •
  •     As mentioned earlier, there continues to be some controversy over whether the cells of the theca interna are fibroblasts, or smooth muscle cells. The controversy has arisen in part from the fact that a significant portion of the cells appear to be spindle-shaped, if the thin sections for transmission electron microscopy are cut perpendicular to the ovarian surface (64). However, if the thin sections are cut on a plane that is tangential to the ovarian surface, then it becomes apparent that the cells of both the theca externa and the tunica albuginea are platter-shaped fibroblasts rather than spindle-shaped muscle cells (32). In addition to the oval appearance of the fibroblasts, the tangential sections render the extracellular collagen more conspicuous, and it is more obvious that the collagenous fibrils are secretory products of these cells. Thus, fibroblasts are the dominant cell type in both the theca externa and the tunica albuginea.
  • Ovulatory Changes in the Thecal Connective Tissue
    •
  •     During the first several hours after initiation of the ovulatory process, there is very little change in the appearance of the theca externa and tunica albuginea (64, 126, 152). The first signs of change occur at about 4 hours after stimulation of the process in rabbit follicles, at which time edema begins to develop in the theca interna and the fibroblasts in this area begin to dissociate (126). As the ovulatory process progesses, there is surprisingly little change in the appearance the follicle wall until the final hour before rupture. During this last hour, the fibroblasts and collagenous elements undergo marked dissociation in the apical region of the follicle wall (64, 126, 152). The concentration of collagen fibrils per square micron of cross-sectional area decreases to only about 28% of the value that was present before induction of the ovulatory process (64). Also, in the final minutes before rupture, the average depth of the collagenous tissue at the apex of a rabbit follicle decreases to only 27% of its original value (64). Thus, at the apex where rupture normally occurs, the follicle wall becomes thinner and the connective tissue elements within the follicle wall become sparser. By this stage, extravasated blood cells are common within the extracellular spaces around the fibroblasts (152). This internal hemorrhaging is an indication that rupture is imminent. Espey (19, 32, 152) has reported that the tenacious collagenous layers of the theca externa and tunica albuginea are usually the final tissue to break.
  •  
  • Theca Interna
    •
  • General Features
    •
  •     The theca interna, which is approximately two cells thick, is bounded on the outer side by the fibroblasts of the theca externa and on the inner side by the basal lamina (membrana propria) that separates it from the granulosa layer (64, 127). The embryological origin of the cells of the theca interna has been debated for many years (121). Erickson et al. (155) have reviewed most of the literature on the development and function of these cells and have concluded that they are derived from ovarian stromal cells. These "interstitial" cells of the theca interna have large oval nuclei with very prominent nucleoli. Their cytoplasm is dominated by lipid droplets, numerous mitochondria, some Golgi networks, and a few small lysosomal-like bodies. Such cytological features are highly characteristic of active steroidogenic cells (156, 157). The lipid droplets, which are a conspicuous feature of these cells, are filled with cholesterol to provide substrate for steroid biosynthesis.
  • Steroid Synthesis
    •
  •     It has been well established that a developing ovarian follicle produces more and more estrogen as it approaches maturity, i.e., as it acquires the capacity to respond to an ovulatory surge in LH (or exogenous chorionic gonadotropin (CG)) (158). Earlier studies on the histochemistry and cytochemistry of ovarian steroid synthesis have been reviewed by Guraya (159). Since that time, a number of investigators have reported that the estrogens, principally 17ß-estradiol, are actively secreted by the cells of the theca interna, while the granulosa cells appear to be relatively inactive (132, 136, 160-163). However, in 1959, Falck (164) transplanted various combinations of granulosa and theca interna tissue into the anterior chamber of the rat eye and assessed subsequent estrogen secretion. He found that estrogen secretion occurred only when thecal interstitial cells and granulosa cells were transplanted together. Thus, on the basis of this synergistic-like action of the two follicular tissues, he concluded that two types of cells were necessary for estrogen synthesis in the ovarian follicle--with the theca interna carrying out the initial biosynthetic steps of androgen production and the granulosa cells converting the androgens to estrogens. Several years later, Bjersing (165) modified this "two-cell-type" theory by proposing that side chain cleavage of cholesterol and 17a -hydroxylation are probably limited to the thecal interstitial cells, whereas aromatization to 17ß-estradiol may take place in both theca and granulosa cells. On the other hand, the ultrastructure of the different layers of the follicle wall certainly make it appear as though the interstitial cells of the theca interna are the most active steroidogenic cells in preovulatory follicles (32, 64, 127). Therefore, further assessment of the steroid composition and relevant mRNA transcripts in the theca and granulosa cells may be required to confirm the two-cell theory of ovarian steroid secretion.
  •     When a mature follicle has been stimulated by an ovulatory surge in gonadotropin, the local elevation in cyclic AMP mediates several changes in follicular steroidogenic activity. Within 1 hour, estrogen and androgen secretion increases even further, but several hours later the ovarian level of these two types of steroids declines sharply, while ovarian progesterone synthesis begins to rise significantly (161, 163, 166-177). By the time of ovulation, there are changes in the ultrastructure of the granulosa cells that reflect this increase in progesterone synthesis (32, 159). The potential role of progesterone in the mechanism of ovulation will be discussed later in this chapter in the section on ovarian steroids.
  • Vascular System
    •
  •     Most of the ovarian blood supply circulates to the mature ovarian follicles and to the wreath of large capillaries that lie along the inner border of the theca interna (19). This dense network of capillaries delivers nutrients to the steroidogenic cells of the theca interna, and it indirectly supplies the metabolic needs of the avascular granulosa layer, the cumulus oophorus, and the oocyte. The first detailed account of this elaborate blood supply was described at the turn of this century by Clark (178). The next half century of work on the changes in this vascular pattern during the sexual cycles of mammals has been nicely summarized by Bassett (179) and by Burr and Davis (180). More recently, Ellinwood et al. (181) made a thorough review of the ovarian vasculature, and Lipner (6) has updated this information.
  •     The ovarian blood supply has been studied by a variety of experimental methods. One approach has been to prepare corrosion casts for scanning electron microscopy of the follicle vasculature (182-185). Resin leakage during preparation of such casts has provided indirect evidence to support reports that the permeability of the thecal capillary network increases significantly during the ovulatory process (102, 186, 187).
  •     Macroscopic observations of ovulatory follicles are all that are needed to detect the vascular blushing and hyperemia that are characteristic of ovulatory follicles. In their studies of rabbit ovulation in 1935, Hill et al. (2) noted that the capillary blood supply increases in ovulatory follicles. Also, in 1951, Burr and Davies (180) referred to the "hyperemia" that occurs in the rabbit ovary prior to ovulation. However, a number of attempts to measure this increase in ovarian blood flow have not provided a very consistent picture of the vascular changes that occur. By following the movement of radioactive microspheres into the ovarian vascular compartment, Murdoch et al. (188, 189) have concluded that the supply of ovarian blood to the follicle wall of the ewe initially increases after the LH surge, but then it declines during the 10-12 hours preceding follicular rupture. However, Brown et al. (190) used the microsphere technique and found that capillary blood flow in the ewe ovary is significantly greater near ovulation. In the rabbit, studies with radioactive microspheres (191-193) have shown that ovarian blood flow increases significantly as early as 10 minutes after LH/hCG administration, reaches a maximum approximately 4 hours later, and remains elevated throughout the ovulatory process. In contrast, in the gonadotropin-primed immature rat, Damber et al. (194) reported that the relative follicular blood flow did not increase during the ovulatory process. However, by using radioactive isotopes to estimate ovarian fractional blood flow, Wurtman (195) claimed there was a rapid increase in ovarian hyperemia following the injection of LH into the rat. Similarly, Abisogun et al. (196) measured an increase in ovarian and follicular blood flow with the most pronounced change occurring at 1.5 hours after administering hCG into rats. A number of other studies have used either drop-flow counters (197), thermocouples (198), perfusion systems (199), ultrasonography with color-flow mapping (200), or transmission electron microscopy (201, 202) to estimate ovarian hyperemia, but these methods have not clarified the changes in ovarian blood flow and blood volume during the ovulatory process.
  •     Studies on the relationship between ovarian hyperemia and the well known increase in prostaglandins during ovulation have been no more enlightening. Some reports have suggested that prostaglandins increase ovarian blood flow and/or blood volume (193, 203), others have indicated that prostaglandins decrease ovarian blood flow (188, 204), and yet another has concluded that eicosanoids have no effect on blood flow, but may decrease permeability of the ovarian vasculature (196). In a recent study on the gonadotropin-primed immature rat, Tanaka et al. (205) used a spectrophotometric method to systematically measure hemoglobin in ovarian extracts during ovulation. This method showed that ovarian blood volume increases significantly beginning 4 hours after hCG administration, and it reaches a peak that is approximately 7-fold greater than the control level at 10 hours after hCG, i.e., at the time when follicles begin to rupture in the rat. In this model, indomethacin partially inhibited the increase in ovarian blood volume, suggesting that eicosanoids might function as mediators of ovarian hyperemia during the ovulatory process.
  •     One of the more distinct changes in the vascular pattern of ovulatory follicles is the angiogenic activity that develops near the time of ovulation. One-half century ago, Bassett (179) reported that "shortly after rupture of a follicle, the granulosa is invaded at many points by vascular sprouts from the inner capillary wreath." This early observation has been confirmed by electron microscopy (185, 202, 206). Kranzfelder et al. (207) have suggested that products from the granulosa cells regulate this angiogenic response, while Rose and Koos (208) have suggested that a combination of factors in serum and in follicular fluid can govern the mitogenesis of new blood vessels in ovulatory follicles.
  •  
  • Granulosa Layer
    •
  • General Features
    •
  •     The stratum granulosum is a multilayer of epithelial cells which extend inward from the membrana propria (basal lamina) that delineates the granulosa from the theca interna (32, 64, 125, 137, 209). There are several thorough reviews on the morphology of this innermost layer of the follicle (29, 210). The granulosa is usually about 5-10 cells deep, except for the cumulus mass which protrudes randomly from any part of this stratum and suspends the oocyte more toward the center of the follicular antrum. Over a century ago, Sobotta (211) realized that the position of the cumulus in the follicle is entirely one of chance. The granulosa cells that are adjacent to the membrana propria are usually more columnar in shape, while the remainder of the cells in this layer are polyhedral or cuboidal (19, 64). Granulosa cells of mature follicles contain some smooth endoplasmic reticulum, Golgi networks, granular mitochondria, and occasional lipid granules. Bjersing (29) has reported that these cells undergo hypertrophy near the time of ovulation, and Espey (19) has noted that they contain a greater number of lipid granules, which may reflect an increase in their steroid metabolism in response to gonadotropic stimulation. However, the most conspicuous morphological changes in this layer occur shortly after ovulation, when large lipid droplets begin to dominate the cytoplasm of the cells of the lutein granulosa, when thecal fibroblasts begin to proliferate into the area to lay down new luteal connective tissue, and when the thecal blood vessels begin to penetrate this stratum for the first time.
  •     In species like the rabbit and sheep, which form ovulatory stigma, the granulosa layer is oftentimes absent from the apical most area of follicles that are about to rupture (7, 64, 125, 128). Apparently, as the follicle wall begins to balloon out during the final stages of the ovulatory process, the thin membrana propria between the granulosa layer and the theca interna dissociates and retracts in a circular pattern at the base of the developing stigma. In the process of this retraction, the apical granulosa cells and the thecal vascular tissue also disassociate and leave a thinner stigma that is void of blood vessels. As the remaining collagenous tissue undergoes enzymatic decomposition during the final minutes before rupture, the stigma becomes translucent. At the base of the stigma, the broken blood vessels and extravasated blood may form a "rosette" around the pending site of rupture (4, 7, 99).
  • Granulosa Gap Junctions
    •
  •     One of the more interesting features of the stratum granulosum is the elaborate network of gap junctions that link this layer of epithelial cells into a continuum. These membrane junctions were originally described by Merk et al. (212, 213) and by Espey and Stutts (133). As these investigators pointed out, besides providing intercellular cohesion, the tight junctions might (i) be necessary for the transport of ions and nutrients across the granulosa layer, (ii) be involved in routing the primary follicular fluid from the granulosa cells to the antrum, or (iii) be important in conducting fluctuations in membrane potentials throughout this stratum and perhaps even to the oocyte. In addition, Espey and Stutts (133) noted that cytoplasmic invaginations commonly occur along the site where two cell membranes fuse into a gap junction. Such invaginations can be pinched off, or "phagocytized" in a manner that can result in complete incorporation of a section of a gap junction into one of the two adjacent cells. This phagocytic activity results in the formation of a so-called "annular gap junction" completely within the cytoplasm of the recipient cell. This unusual phenomenon results in an exchange of cytoplasm between the two cells that shared a common gap junction. The physiological value of this transfer of cytoplasm remains uncertain. It was initially thought that internalization of the gap junctions might increase during ovulation and that such a process might loosen the granulosa cells and the cumulus mass by the time of follicular rupture (133). In examining this hypothesis, Bjersing and Cajander (125) presented data from rabbits showing a decrease in the intercellular sharing of these gap junctions and a simultaneous increase in the internalized spherical gap junctions in the cytoplasm of individual granulosa cells as follicles progressed toward ovulation. Coons and Espey (214) conducted a more quantitative analysis of the distribution of these gap junctions during ovulation, and although they measured a significant decrease in the surface area of gap junctions situated between adjacent granulosa cells of follicles about to rupture, they also observed a decrease in the number of internalized gap junctions. These observations suggest that ovulation may be preceded by a separation of the gap junctions between cells, rather than by internalization of these junctions. Subsequent studies have further characterized the morphology of the granulosa gap junctions (162, 215-221), but the specific function of these structures remains uncertain. They probably unify the granulosa cells into a syncytial layer during follicular growth and development, but it is not clear whether they have any important role in the chemical and/or electrical coupling of these cells during ovulation. Forty years ago, Zuckerman (222) stated that the oocyte is the dynamic center of follicular activity, and that it maintains a constant influence on the membrana granulosa. Thus, it is possible that gap junctions couple the granulosa cells electrically into a syncytial network that influences the resumption of meiosis of the oocyte during ovulation, or, conversely, it is possible that a meiotically rejuvenated oocyte might exert some ovulatory influence on the cumulus cells around it and this signal might be transmitted to the remainder of the granulosa layer via the gap junctions.
  •  
  • Other Anatomical Considerations
    •
  • Follicular Fluid
    •
  •     Mature ovarian follicles in the vast majority of mammalian species contain an antral cavity filled with follicular fluid. As early as 1918, Robinson (223) recognized different kinds of follicular fluid within developing follicles. He said that a "primary" viscous fluid is secreted from the granulosa cells during follicular growth and this liquid eventually coalesces to form the central antral cavity. A mature follicle contains a more serous "secondary" liquor that rapidly increases in volume shortly before ovulation. Some years later, Burr and Davies (180) recognized that the secondary liquor is probably a transudate from blood. The actual composition of the follicular fluid has been thoroughly reviewed by McNatty (134) and by Lipner (6). When a primordial follicle begins to grow, the hyperplastic granulosa cells secrete proteoglycans and related gelatinous materials which form the primary follicular fluid. The secondary fluid that transudes into the follicular antrum during ovulation is similar to blood plasma except that it contains much lower levels of fibrinogen, a slightly lower pH, and the protein, carbohydrate, and steroid profile of the fluid can fluctuate with the metabolic stage of the follicle (6, 134).
  •     The specific function of follicular fluid is unknown. As McNatty (134) has pointed out "the formation of a fluid-filled cavity within enlarging follicles provides a means by which intrafollicular cells of one follicle may be exposed to an environment that is different from that in adjacent follicles and from that in peripheral blood," and this may "provide an intraovarian mechanism to limit the number of follicles that can go on to ovulate." It is also feasible that the follicular fluid could serve to isolate the cumulus oophorus from blood borne growth factors that might otherwise disturb the dormant meiotic state of the oocytes in preovulatory follicles. Yet, the more relevant question that remains unanswered is whether the follicular fluid contributes in some important way to the mechanism of ovulation. One possible function that has received negligible attention is that the follicular fluid, along with the zona pellucida and the corona radiata cells which surround the oocyte, may serve as defense mechanisms to protect the delicate egg from the acute onslaught of proteolytic activity that disrupts the collagenous layers of the follicle wall during ovulation. A second possibility is that the lubricative texture of the fluid may facilitate the smooth extrusion of the cumulus mass and oocyte out the rupture point. A more remote possibility is that the follicular fluid might contribute in some positive way to the postovulatory healing and luteinization processes that occur after ovulation. However, the precise functions of the fluid remain to be determined.
  • Innervation of Ovarian Follicles
    •
  •     Innervation of the mammalian ovary by sympathetic and parasympathetic fibers has been reviewed by several investigators (6, 37, 224, 225). The sympathetic nerves originate from the lower thoracic region of the spinal cord and pass through the celiac plexus and ovarian ganglia before entering the hilar region of the ovary. The parasympathetic fibers, which are probably of vagal origin, also enter the hilus along with the ovarian blood vessels. These autonomic nerves converge, for the most part, on the ovarian vasculature (224), especially around vessels in the hilum and medulla (95, 226, 227). However, a portion of the nerves pass to the cortex of the ovary and form a plexus around the ovarian follicles. Walles (228-230) has reported that this follicular innervation is extensive, but McReynolds et al. (231) has reported that it is essentially nonexistent. Within the follicle, nerves can be found in both the theca externa and the theca interna, but not inside the granulosa (224). In addition to making contact with the thecal vasculature, some of the nerves may terminate on myofibroblasts in this area of the follicle (232-234). Many investigators believe that the nerves regulate ovarian "contractility" and thereby contribute to the mechanism of ovulation. Espey (37) has reviewed these various reports and has noted that a -adrenergic agonists, ß-adrenergic antagonists, and cholinergic antagonists all appear to promote some kind of contractile response in ovarian tissues. Since that review, there have been several additional reports that catecholamines and/or adrenergic receptors may participate in the process of ovulation (235-240). However, there is no convincing evidence that nerve excitation is a crucial event in the ovulatory process (37). In a series of studies almost two decades ago, Weiner et al. (87, 241, 242) showed that denervation of the rabbit ovary did not reduce the number of ovulations, pregnancies, or corpora lutea. Likewise, Wylie (243) froze the sympathetic nerve supply to the rat ovary and found that follicular rupture was not affected by the absence of this innervation. Furthermore, it has been demonstrated repeatedly that ovulation can occur in the in vitro perfusion system (38, 244), and ovulatory like changes can be induced within cultured ovaries (245), even though such experimental models totally lack innervation. Therefore, although one cannot completely rule out the possibility that adrenergic agents might have some modest influence on ovulation, it is, nevertheless, quite apparent that nervous activity is not an essential component of the ovulatory mechanism.
  • BIOCHEMICAL EVENTS IN OVULATION
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  • Membrane Phenomena and Related Events
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  • The Gonadotropin Surge
    •
  •     The ovulatory process is initiated at the moment when follicular tissue is stimulated by a surge of pituitary gonadotropins. The pituitary surge in LH secretion can result in as much as a hundred-fold increase in the circulating level of the hormone (246). In the earlier years, it was generally presumed that LH was solely responsible for initiating the ovulatory process (8), but it is now becoming clear that other hormones can serve as substitutes for this LH action. Besides the well known capacity of highly homologous chorionic gonadotropin to induce ovulation (247), it is now evident that FSH can also cause follicles to rupture. FSH is best known for its role in follicular development and maturation (248,249), but it now appears that FSH also contributes to induction of the ovulatory process. As early as 1973, Nalbandov et al. (250) concluded that "the ovulation-inducing hormones consist of a mixture of FSH and LH and that it is not LH alone." Subsequently, Greenwald and Papkoff (251) found that FSH preparations were more potent than LH in inducing ovulation in the hamster. More recently, Galway et al.(252) have treated hypophysectomized rats with recombinant FSH and have demonstrated that this pure hormone preparation can, by itself, induce both follicle development and ovulation. Equally interesting is the growing evidence that gonadotropin-releasing hormone (GnRH) can induce ovulation in hypophysectomized animals (253-258). Yet, there is at least one report (259) that GnRH inhibits LH-induced ovulation in hypophysectomized rats. In any event, in normal conditions both LH and FSH are probably the principal hormones that are responsible for initiating the ovulatory process.
  •     The complementary action of LH and FSH in ovulation is not so difficult to understand, now that it is clear that (i) they are usually released in unison in response to GnRH, (ii) they have relatively homologous peptide sequences, and (iii) their respective receptors display considerable sequence similarity (247, 260). These two hormones, along with CG and TSH, are members of a family of heterodimer glycoprotein hormones composed of a common a -subunit that is connected to a distinct ß-subunit that confers receptor binding specificity (247).
  • Gonadotropin Receptors
    •
  •     Ovulation occurs only in mature ovarian follicles that have acquired an adequate concentration of LH receptors (42, 247, 260). Two decades ago, Midgley and coworkers (261, 262) used 125I-hCG to localize the site of LH binding in mature follicles. Their autoradiographs revealed that the majority of the binding was on the plasma membranes of the thecal interna cells of the rat ovary, while the granulosa cells were generally free of the isotope-labeled gonadotropin. A subsequent study that utilized fluorescence localization of hCG seemed to support this view (263). However, further investigations have shown that, while the highest density of labeled hormone may be in the theca interna, the layers of granulosa cells closest to the membrana propria also contain LH receptors (28, 264-269). On the other hand, the cumulus cells and oocyte appear to be devoid of LH receptors (264, 267). Acquisition of a sufficient concentration of LH receptors is dependent on the actions of estradiol, FSH, and probably some LH itself (28, 266, 268).
  •     A number of investigators have shown that the natural LH receptor has a molecular size of approximately 93 kD (270-273). McFarland et al. (247) have recently isolated and sequenced cDNA for the rat LH receptor. Decoding this cDNA has yielded a 674 residue polypeptide with a molecular size of 75 kD and six unoccupied, N-linked glycosylation sites within its domain that account for the difference in its size when compared to the natural receptor. The receptor has a relatively large 341-residue extracellular domain, and a 333-residue membrane-spanning region that is characteristic of the G-protein receptor family with seven transmembrane segments. Leung and Steele (260) have recently reviewed the literature on the nature of this receptor, its relationship to the FSH receptor, and the intracellular signaling pathways by which it stimulates phospholipase activity in the gonads. The initial response to hormone/receptor coupling appears to be an elevation in intracellular cAMP (42, 260).
  •     The possible involvement of other membrane receptors in the ovulatory stimulus is less clear. With regard to prolactin (PRL), Piquette et al. (274) have reported that this gonadotropin induces an increase in LH receptors in granulosa cells, and such an effect should increase the responsiveness of follicles to the ovulatory surge in gonadotropins. However, several other reports suggest that PRL interferes with ovulation (275-277). With regard to serotonin, this neurohumoral agent binds, like the gonadotropins, to a G protein-coupled receptor (247). Although this agent has been implicated in the mechanism of ovulation (278, 279), there is at least one report that it does not increase significantly in human follicles at the time of ovulation (239), and its role remains uncertain. Other such agents that might influence ovulation include vasoactive intestinal peptide (VIP) (260, 280, 281), neuropeptide Y (NPY) (240), oxytocin (282, 283), and growth hormone-releasing factor (GRH) (260).
  • Cyclic AMP and Other Second Messengers
    •
  •     Two decades ago, Marsh et al. (284-287) demonstrated that rabbit follicles produce cyclic AMP and suggested that this second messenger might be involved in the steroidogenic action of LH. Shortly thereafter, a number of other studies confirmed that LH action on ovarian steroidogenesis and luteinization is mediated by cyclic AMP (199, 250, 288-299). This second messenger increases within 10 min after exposure of a follicle to LH, and it increases to a peak within several hours (199, 292, 293, 295). In contrast, there is a reciprocal decline in cyclic GMP (174, 300). Cells of the theca interna are the principal source of cyclic AMP, but granulosa cells also respond in a similar manner to LH stimulation (293, 294, 296, 301, 302). Cyclic AMP acts through protein kinases to induce cholesterol side-chain cleavage cytochrome P450 (P450scc) mRNA and promote enzyme activity that catalyzes the first rate-limiting step in follicular progesterone synthesis (42, 301, 303-311). In addition to its stimulatory effect on follicular progesterone synthesis, some reports suggest that cyclic AMP increases follicular prostaglandin synthesis (38, 286, 312, 313), but at least one study has come to the opposite conclusion (314). Equally confusing are the reports that prostaglandins (particularly prostaglandin E2) may (293,295, 314), or may not (312, 315) promote the formation of cyclic AMP in ovulatory follicles.
  •     The role of other second messengers in the signal transduction processes that occur in ovarian follicular cells following an ovulatory surge in LH is not yet clear. LH probably provokes a sustained increase in inositol triphosphate (IP3) and diacylglycerol (DAG) (316, 317), and Veldhuis (318, 319) has reported that the formation of these second messengers may be influenced by prostaglandin synthesis in granulosa cells. Also, the stimulation of progesterone synthesis by LH and cAMP is probably affected by intracellular Ca2+ (307, 320-322), but the precise role of this cation in ovarian steroidogenesis has not been established. Intracellular Ca2+ is considered to be a second messenger because it increases in cells shortly after ligand-receptor coupling, and because it is required for activation of protein kinase C and for the subsequent phosphorylation of a number of metabolically active proteins (44).
  • Protein Kinases
    •
  •     When a hormone like LH binds to membrane receptors on follicular cells, the events of signal transduction activate phosphorylation processes in which the energy within phosphate bonds flows in an organized pattern through a number of effector enzymes and regulatory proteins that are sequentially energized to undergo conformational changes that bring about characteristic cellular responses (44, 323). The kinds of protein kinases that are generated by gonadotropic hormone stimulation of ovarian follicles are only beginning to be deciphered. Hunzicker-Dunn and Jungmann (324, 325) conducted several of the earliest studies on protein kinases in ovarian follicles. They found that mature follicles contained only a single species of protein kinase, and that the induction of ovulation and luteinization promoted the appearance of two additional species of protein kinases. Richards and coworkers (298, 326-330) have studied in detail a regulatory subunit (RII51) of cAMP-dependent protein kinase type II and have found that this inhibitor of the catalytic subunit of cAMP-dependent protein kinase increases in granulosa and theca cells in response to FSH and estradiol as a follicle reaches maturity. The LH/hCG surge causes a dramatic decrease in this regulatory subunit (298), but the significance of this change to the events of ovulation is uncertain except that the decline coincides with the decrease in ovarian estrogen and androgen synthesis during ovulation.
  •     Other studies have shown that protein kinase C activity, which usually increases following activation of phospholipase C and formation of DAG, has an inhibitory effect on ovarian progesterone synthesis (307, 331), and this action might be related to the stimulation of prostanoid synthesis by this protein kinase (332-334). In contrast, other reports indicate that protein kinase C stimulates progesterone synthesis in differentiated granulosa cells, and this action might be mediated by tumor necrosis factor-a (335-338). Thus, the actions of protein kinases in ovulation are uncertain. Nevertheless, it seems quite likely that protein phosphorylation plays an important intermediary role in the ovulatory process (339).
  • Mobilization of Membrane Phospholipids
    •
  •     In the course of hormone binding to G protein-coupled receptors like the LH receptor, there is usually stimulation of adenylyl cyclase and a local increase in intracellular cyclic AMP (247). The phosphorylation of such receptors and their associated G proteins also leads to the activation of phospholipases, which serve as signal amplifying enzymes by hydrolyzing common membrane phospholipids that serve as the substrates for second messenger formation (44, 340, 341). Phospholipase C and phospholipase A2 are two such amplifying enzymes that have been studied extensively in a variety of experimental models. There is now indirect evidence that these enzymes also contribute to the biochemical events of ovulation, and their potential roles have been reviewed recently by Espey (44) and by Leung and Steele (260).
  •     Phospholipase C hydrolyzes phosphatidyl inositol-4,5-biphosphate, a complex lipid that comprises as much as 5-10% of the total membrane phospholipids in animal cells (Fig. 2) (44, 342). The products of this enzyme action are IP3 and DAG, two second messengers that release calcium ions from the endoplasmic reticulum and activate protein kinase C, respectively (44, 260). DAG can be further hydrolyzed by DAG lipase to yield arachidonic acid, the principal substrate for eicosanoid metabolism in ovarian follicles (44). In comparison, phospholipase A2 can act directly on phosphatidylethanolamine and phosphatidylcholine, which together comprise about 75% of the phospholipids in the membranes of most mammalian cells, to generate arachidonic acid (44). Since this type of enzymatic activation of the arachidonate "cascade" occurs following almost any condition that stimulates the plasma membrane (343-346), similar de-esterification of membrane phospholipids is almost certain to occur in ovarian follicles that have been stimulated to ovulate. The liberated arachidonic acid is then available for the cyclooxygenase and lipoxygenase pathways of eicosanoid metabolism in ovulatory follicles, as discussed later in this chapter.
  •     In closely related metabolic events, after phospholipase A2 has hydrolyzed phosphatidylcholine into arachidonic acid and an alkyllysoglycerophosphocholine, this modified phosphocholine can then be acetylated at the sn-2 position to form platelet-activating factor (PAF) (44, 347, 348). PAF is actually a family of acetylated glycerophospholipids that usually form in acute inflammatory reactions and tissue injury (349-353). This family of phospholipids is formed by a wide variety of cells, including epithelial and endothelial cells, fibroblasts, mast cells, and leukocytes (352, 354-357). In inflammatory processes, they are potent inducers of vascular permeability, and they elicit acute extravasation of plasma proteins and the local accumulation of platelets and neutrophils (350, 358, 359). In rat ovaries, PAF is released (or labelized) during the first several hours after gonadotropic hormone stimulation of the ovulatory process, and the changes in this bioactive phospholipid are characteristic of what happens to PAF in inflamed tissues (360). Also, there are reports that antagonists of PAF activity can suppress ovulation in a dose dependent manner (361, 362). Therefore, it is possible that PAF and related membrane phospholipids mediate some of the earlier events of the ovulatory process.
  • RNA and Protein Synthesis
    •
  •     It is natural to expect the signal transduction processes initiated by the gonadotropin surge to lead to transcription and translation activity in the theca and granulosa cells. The first indirect evidence of such activity was provided by Pool and Lipner (363, 364), who found that actinomycin D (an inhibitor of mRNA synthesis) and cycloheximide (an inhibitor of protein synthesis) both blocked ovulation. These early observations were confirmed by a number of subsequent studies which demonstrated that protein synthesis is an essential part of the ovulatory process (365-370). The increase in protein synthesis is associated with an increase in mRNA formation that begins as early as 1 hour after LH stimulation of mature follicles (365, 366) and persists until a few hours before the follicles begin to rupture (371, 372). The sites of synthesis of mRNA and protein appear to be mainly in the theca interna and the granulosa layer. The types of mRNA and protein are only beginning to be deciphered. Richards and coworkers have conducted pioneering studies on the nature of the nucleic acids and enzymes responsible for ovarian steroid (42, 302, 310, 311, 373, 374) and prostanoid (42, 375-380). Also, there is preliminary information about ovarian mRNA transcripts for kallikrein (381), plasminogen activator (252, 382-384), collagenase (385), and metalloproteinase inhibitors (385-388), and these factors might be involved in the regulation of follicular proteolytic activity. In addition, mRNA for a variety of growth factors have been identified in developing follicles, but it is not yet clear whether these cytokines contribute to the mechanism of ovulation (389-393).
  •  
  • Vasoactive Agents and Related Substances
    •
  • Histamine
    •
  •     The first physical event that is known to occur in ovulatory follicles is the hyperemia that was described earlier in this chapter. Such an increase in blood flow and/or blood volume to a local area of irritation is usually associated with a release of histamine stores from mast cells and basophils (45). A major source of the ovarian histamine may be the mast cells in the walls of the large blood vessels in the ovarian hilum (394, 395), and there is evidence that histamine H1 and H2 receptors exist in these principal ovarian vessels (396). Szego and Gitin (397, 398) originally suggested that the release of histamine might be a physiologically significant event in ovulation. Their original observations were supported by subsequent reports that anti-histamines such as chlorpheniramine and pyrilamine (H1 receptor antagonists) and cimetidine (a H2 receptor antagonist), either separately or in combination, can inhibit ovulation in vivo and in vitro (395, 399-402). Further support came from evidence that histamine, by itself, could induce ovulation in vitro in perfusion systems (395, 401, 403, 404). This information, along with measurements suggesting the degranulation of mast cell histamine during ovulation led to conclusions that this vasoactive agent might act as a paracrine mediator of the ovulatory process (405). However, the importance of histamine in ovulation has not been firmly established. Over 20 years ago, Lipner (406) used histamine releasing drugs and antihistamines to show that this agent may not have a major role in ovulation. Furthermore, a number of other studies could not confirm the reported anti-ovulatory action of various H1 and H2 receptor antagonists (403, 407-409). Also, several studies have revealed that histamine cannot induce the characteristic increase in prostanoid synthesis in follicular tissue (408, 410), and this information casts further doubt on the idea that histamine by itself can mediate the ovulatory process. The question that remains unanswered is whether histamine is a causative agent in the mechanism of ovulation, or whether this agent, and the associated hyperemia it generates, is a local response to the degradative events of the ovulatory process.
  • Bradykinin
    •
  •     The nonapeptide bradykinin and other mammalian kinins are commonly generated at the site of tissue injury or inflammation (44-46). These kinins are produced by proteolytic cleavage of plasma kininogens by kallikrein (411, 412). These vasoactive agents mediate a broad spectrum of biological responses including vasodilation, increased vascular permeability, prostaglandin synthesis, cell proliferation, and inflammation (413-416). The first suggestions that bradykinin might be involved in ovulation came from a report by Smith and Perks (417) that plasma kininogen decreases in several mammals around the time of ovulation, and the review by Espey (45) which hypothesized that kinins might mediate the inflammatory-like events of the ovulatory process. Now, there is a variety of evidence that kinin-forming enzymes increase in the ovary at the time of ovulation (81, 381, 413, 418-422). Also, there are reports that bradykinin can induce ovulation in vitro in perfused ovaries (423-425). Of particular interest are the reports that bradykinin can stimulate prostaglandin synthesis in mature ovarian follicles (423, 425), although one report (410) raises some question about this action of kinins on follicular prostanoid formation. Furthermore, there is evidence that bradykinin can promote the synthesis and release of eicosanoids in other experimental models (426-428), and this correlation suggests that kinins might be responsible, at least in part, for the well known increase in eicosanoid metabolism in ovulatory follicles.
  • Angiotensin II
    •
  •     Angiotensin II is well known as a regulator of systemic blood pressure and fluid homeostasis. This vasoactive octapeptide is indirectly related to bradykinin in that the enzyme which converts angiotensin I into angiotensin II also acts as a kininase to degrade bradykinin (429). Angiotensin II has recently been implicated as a mediator of the ovulatory process because prorenin which forms the enzyme to convert angiotensinogen into angiotensin I increases in follicles in response to gonadotropic stimulation (430), angiotensin I-converting enzyme is detectable on the surface of granulosa cells (431), receptors for angiotensin II are present in ovarian follicles (432-435), and especially because of reports that the angiotensin antagonist saralasin blocks ovulation (436-438). However, the importance of angiotensin II in ovulation remains uncertain because the converting enzyme responsible for this agents formation does not increase during the periovulatory period (431), the converting enzyme inhibitor captopril does not affect ovulation (431), and several extensive efforts to confirm the reported anti-ovulatory action of saralasin were unsuccessful (439, 440).
  • Prostaglandins
    •
  •     With the possible exception of ovarian steroids, no group of agents has been studied as extensively as the prostaglandins during the past two decades of work on ovulation. Prostaglandins are produced from arachidonic acid that is formed from membrane phospholipids in response to virtually any type of environmental condition that disturbs the plasma membrane (44, 344, 345). The prostanoids are commonly associated with inflammatory reactions, but they also form during immune reactions and hormonal responses. While this group of eicosanoids are generally thought to mediate pro-inflammatory processes, there is also evidence that they might exert some anti-inflammatory action (441-447). Prostaglandin E1, in particular, may be effective as an anti-inflammatory agent, and this prostanoid may act by inhibiting collagenase gene expression (446, 448). These paradoxical pro- and anti-inflammatory theories of prostaglandin action may not be as contradictory as they appear; instead, the differences may reflect the complex nature of inflammatory reactions. Such reactions simultaneously involve degradative and reparative metabolic processes, and the prostanoids may be contributing to both of these processes in a temporal of biphasic pattern that has not yet been clearly deciphered (45). It does appear that the E-type prostaglandins have an effect on the vascular supply to irritated and inflamed tissues. This subclass of prostanoids causes a variety of vascular responses, including the mediation of vasodilation and local hyperemia (45, 46, 442, 445).
  •     The first association of prostanoids to the mechanism of ovulation was two decades ago when Labhsetwar (449, 450) reported that prostaglandin F2a could induce ovulation in several different laboratory animals. However, his studies did not make it clear whether this agent was acting directly on the ovary or indirectly via stimulation of LH secretion. Within a year of these first reports, a number of investigators demonstrated that indomethacin, a well known inhibitor of prostaglandin synthesis, could prevent ovulation in several different laboratory animals (451-455), and this anti-ovulatory action of indomethacin has been confirmed many times (176, 205, 407, 413, 456-473). Shortly thereafter, LeMaire and coworkers (474-477) reported a marked increase in prostaglandins E and F in rabbit follicles during the ovulatory process, and this association of prostanoid synthesis with the ovulatory process has also been confirmed many times (176, 458, 459, 468, 478-495). The early work on prostaglandins and ovulation has been reviewed by LeMaire and Marsh (496) and by Armstrong (497).
  •     Even though prostaglandins have been studied extensively, their importance and functions in ovulation have not been fully established. Most investigators have keyed on prostaglandin F2a as the more prominent prostanoid in the mechanism of rupture, while several studies (455, 469, 488, 498, 499) have suggested that prostaglandin E2 may be more important. After Marsh and coworkers (284-286) first demonstrated that LH induces cyclic AMP synthesis in mature ovarian follicles, several studies suggested that prostaglandins (particularly prostaglandin E2) might function to augment this action of LH on follicular cyclic AMP formation (295, 500). However, other reports indicated that prostaglandins do not mediate the action of LH on cyclic AMP synthesis (314, 315, 460, 462, 501, 502), and it now seems rather clear that the reverse is the case, i.e., that cyclic AMP mediates LH-induced prostaglandin synthesis in ovulatory follicles (286, 292, 312, 313, 469, 503). There is also indirect evidence to suggest that protein kinase C might augment the production of various prostaglandins in granulosa cells (332-334). Initial reports indicated that the LH-induced signal transduction process in ovulatory follicles results in an increase in prostaglandin synthesis mainly in granulosa cells (480, 482, 504), but other studies have suggested that cells of the theca interna are equally important as a source of prostaglandins during ovulation (505-507). More recent work based on the techniques of molecular biology has shown that the granulosa, rather than the theca, is the follicle layer that responds to LH by forming mRNA for prostaglandin synthetase (375, 377-379).
  •     Considerable attention has been given to the relationship between follicular prostaglandin synthesis and steroid synthesis. The earliest studies indicated that prostaglandins do not mediate gonadotropin-induced steroido-genesis and luteinization of mature ovarian follicles (453, 508), and this observation has been confirmed many times (193, 292, 404, 458, 460, 463, 472, 473, 492, 505, 509-519). However, in contradiction, some investigators have reported that prostaglandins, especially of the E-type, have at least a minor (520, 521) and possibly a major (504, 522-525) effect on ovarian progesterone synthesis during ovulation. Also, there are reports that prostaglandin E2 stimulates progesterone synthesis in corpora lutea, while prostaglandin F2a inhibits such steroid synthesis (526, 527).
  •     There have been a number of attempts to determine whether ovarian prostaglandins are related either to the vasoactive agents that cause follicular hyperemia, or to the proteolytic agents that have been implicated in the ovulatory process. However, most of these efforts have led to inconsistent or contradictory conclusions. Ovarian perfusion studies have shown that prostaglandin F2a or histamine individually can induce ovulation (401, 408), and these observations suggest a metabolic relationship between these two agents. Yet, the perfusion studies also showed that prostaglandin-induced ovulation was not blocked by antihistamines, and histamine-induced ovulation was not blocked by inhibitors of prostaglandin synthesis. Therefore, prostaglandins and histamine do not appear to have an interdependent effect on ovulation (408). This conclusion is further supported by a report that histamine has no stimulatory effect on the synthesis of prostanoids (410). With regard to bradykinin, one report (423) states that this vasoactive agent can stimulate the production of F-type prostaglandins in perfused ovaries, but such bradykinin-induced prostanoid synthesis is not essential for ovulation. Several other studies have found that bradykinin has little, or no, stimulatory effect on prostanoid synthesis (410, 425). With regard to ovarian proteolytic activity, several experiments suggest that prostaglandins may be involved in the regulation of plasminogen activator production in the ovary (528, 529), but other studies indicate that eicosanoids have a relatively insignificant effect on such proteolytic activity in the ovary (421, 530-532), and this latter observation is consistent with the results from other experimental models (533). Similarly, some reports conclude that prostaglandins mediate follicular collagenolysis during ovulation (473, 492, 534), while other studies have arrived at the opposite conclusion (535, 536). In addition, there are studies which suggest that prostaglandins selectively inhibit collagen synthesis at the apex of developing follicles to facilitate rupture at this site (537), or that prostaglandins promote collagen synthesis and the healing process that sets in after ovulation (538, 539).
  •     Indomethacin has been very widely used in studies on ovulation because of its consistent inhibition of ovarian prostaglandin synthesis and follicular rupture (451-473). Some studies have examined the effect of indomethacin on changes in the ultrastructure of ovulatory follicles (152, 465, 529, 540-542). Initial reports on the action of indomethacin ignored the fact that this compound is a potent anti-inflammatory agent, and it is now clear that any strong non-steroidal anti-inflammatory drug can inhibit ovulation (407, 470). Such drugs are known to suppress acute inflammatory reactions, while steroidal anti-inflammatory drugs like dexamethasone that have little effect on acute reactions do not inhibit ovulation (407, 543). Indomethacin is effective when administered before or after initiation of the ovulatory process by gonadotropins, and it can be given almost up to the time of anticipated rupture (421, 466, 468). This wide-ranging effect may be due at least in part to the fact that the drug has a half-life of at least five hours. Also, indomethacin has a very rapid effect on ovarian prostaglandin metabolism. When the drug is administered intravenously during the ovulatory process, the normally elevated levels of prostaglandins E2 and F2a decline significantly within 1-2 minutes (468). The inhibitory action of indomethacin on ovulation is dose-dependent, rather than an all-or-none effect. One of the more peculiar features of indomethacin-induced blockage of ovulation is that the highest possible doses of the drug still permit a limited number of follicles to rupture (468, 543, 544). Even more puzzling is the observation by Espey and coworkers (468, 470, 543, 544) that marginal doses of indomethacin which inhibit the normal preovulatory rise in ovarian prostaglandins have no significant effect on the ovulation rate in rabbits and rats. Conversely, cycloheximide can strongly inhibit ovulation while having minimal effect on follicular prostaglandin levels (370). This poor correlation between ovarian prostaglandin levels and ovulation rate has raised questions about the specificity of indomethacin as an inhibitor of prostanoid synthesis and about the importance of prostaglandins in the mechanism of ovulation.
  •     As mentioned above, the implication of prostaglandins in the mechanism of ovulation has been based, in part, on the measurable increase in this group of compounds in follicular tissue during ovulation, and on the capacity of indomethacin-like drugs to inhibit prostaglandin synthesis and ovulation. In addition, there have been numerous efforts to confirm the importance of prostaglandins by administering various prostanoids to induce ovulation or to overcome inhibition of ovulation by indomethacin and other agents. However, collectively, the experiments that have utilized exogenous prostaglandins have led to confusion, rather than clarification of the role of this group of compounds in the mechanism of ovulation. Several earlier reports state that mixtures of prostaglandins E2 and F2a can overcome the anti-ovulatory action of indomethacin (451, 524), but a more recent study contradicts this observation (545). Other reports conclude that prostaglandin E2 alone can promote follicular rupture (498, 499) and reverse the inhibitory effect of indomethacin (455, 469, 525, 546, 547), but these observations are neutralized by studies which show that prostaglandin E2 cannot induce or restore ovulation that has been inhibited by indomethacin (404, 517, 541, 548, 549). Furthermore, several tests suggest that prostaglandin E2 might actually inhibit ovulation (545, 550). This same kind of confusion arises from the studies with prostaglandin F2a , alone. There are reports that this prostanoid can induce ovulation in perfused ovaries (408, 515, 529) and can overcome the anti-ovulatory action of indomethacin (515, 517, 525, 541, 546, 548, 549, 551-553) or ergocornine (554), yet several studies conclude that prostaglandin F2a is relatively ineffective in restoring ovulation (498, 547), and that ovulation can proceed unabated when production of this prostanoid is significantly inhibited by indomethacin (468, 470, 472, 543, 544) or prednisolone (555). Still, in support of a role for prostaglandin F2a , there are several claims that anti-sera to this prostanoid can inhibit ovulation (457, 556). Other prostanoids, such as thromboxane (492) and prostacyclin (557), have also been implicated in ovulation, but their potential role(s) in ovulation remain even more uncertain than the E- and F-type of prostaglandins. In view of all the conflicting information, it may be important to keep in mind that the ovulatory process involves degradative events that lead to ovulation, yet a healing phase sets in shortly after the follicle ruptures (45, 46). Therefore, it is possible that the prostanoids contribute to either the damage, or the repair, or to both of these processes. In future studies, it might be worthwhile to examine the effects of prostaglandin E1 on ovulation, because this agent is known to suppress collagenolytic activity and inflammatory-like processes (446, 448).
  • Lipoxygenase Products
    •
  •     Arachidonic acid serves as a precursor not only for the prostaglandins but also for the biologically important leukotrienes and lipoxins that arise from various lipoxygenase-catalyzed reactions (346, 558-560). The prostanoid products of arachidonic acid have been given the most attention in ovulation studies probably because of early accessibility of radioimmunoassay materials for their measurement and because of the initial interest in the inhibition of prostaglandin synthesis and ovulation by indomethacin (545). However, in recent years, some attention has been given to the lipoxygenase products of arachidonate metabolism. Reich et al. (561, 562), initially reported that nordihydroguaiaretic acid (NDGA) and 3 amino-1-(3 trifluro-methyphenyl)-2-pyrazoline hydrochloride (BW755c), two presumedly specific inhibitors of lipoxygenase activity, can both block ovulation. The same laboratory has recently reported that esculetin and caffeic acid also inhibit the lipoxygenase pathway and ovulation (563). In support of these observations, Yoshimura et al. (564) have found that NDGA blocks hCG-induced ovulation in a perfusion system. In contrast, Hellberg et al. (565) have reported that NDGA and caffeic acid both actually increase, rather than decrease, the ovulation rate in the perfusion system. In addition, several in vivo tests have failed to detect any anti-ovulatory action by NDGA (472, 566). On the other hand, there is confirmation that BW755c can reduce the ovulation rate, but the specificity of this inhibitor may be questionable (472). Likewise, there is now evidence that indomethacin, which has been commonly presumed to be a specific inhibitor of prostanoid synthesis, may also reduce the action of 12- and 15-lipoxygenase (472, 544, 567, 568). Therefore, the specificity of eicosanoid-inhibiting agents may not be as reliable as previously thought.
  •     The family of enzymes that make up the lipoxygenases include 5-lipoxygenase for leukotriene formation and 15-lipoxygenase for lipoxin production (346, 472, 494, 543). These eicosanoids have been implicated in inflammatory reactions, where they may have diverse effects on blood vessels, leukocytes, glandular tissues, smooth muscles, and sensory neurons (346, 560, 569-571). Therefore, since the process of ovulation has been likened to an inflammatory reaction (45), and especially since it is not yet clear whether the prostaglandins contribute more the degradative or the reparative events in ovulatory follicles (545), it is important to explore the potential role(s) of various lipoxygenase products in the ovulatory process. The significance of ovarian leukotrienes is presently the least understood. Several reports suggest that 5-lipoxygenase activity and the leukotrienes increase only slightly during the first half of the ovulatory process and decline before follicles actually begin to rupture (494, 543). Other reports claim a 2-5-fold increase in 5-lipoxygenase activity beginning midway through the process, with enzyme activity continuing to rise up to the time of ovulation (561, 562). Still, another study could not detect a significant rise in leukotrienes until after follicular rupture (566). More recent measurements of relatively stable metabolites of lipoxygenase enzymes have revealed a rather modest and transient increase in 5-hydroxyeicosatetraenoic acid (5-HETE) during the early stages of the ovulatory process, while 12- and 15-HETE increase over 12-fold by the time follicles begin to rupture (494, 544). The ovulatory increase in 15-HETE can be inhibited in a dose-dependent manner by indomethacin (472, 544) and by epostane (472). The optimum time to administer the latter agent to block ovulation is during the first one-third of the ovulatory process (494, 572). Collectively, the present information indicates that the lipoxygenase products of arachidonate metabolism may contribute in some way to the ovulatory process. While their precise role(s) have not yet been established, there is preliminary evidence to suggest that they might stimulate progesterone and prostaglandin formation (260, 573), or promote collagenolysis in the follicle wall (534, 574, 575).
  •  
  • Ovarian Steroids
    •
  • Early Studies
    •
  •     One of the first reviews of the role of steroids in mammalian ovulation was at a 1960 conference on the "Control of Ovulation" which keyed on the ability of steroids to inhibit the gonadotropin surge that initiates ovulation (576). In the printed discussions following that report, Frederick Hisaw pointed out that a better understanding of steroid actions might provide answers to "the physiology of the follicle itself." The following year, at another conference, Hisaw (577) concluded his discussion by again stressing that the accumulation of progesterone over estrogen "might take a responsible part in the process of ovulation". He also noted that his thoughts were "supported by the observations of several workers who report the induction of experimental ovulation, in vivo and in vitro, in several species by the addition of progesterone--but not estrogen." A decade later, Lipner and coworkers (406, 578), along with Ying and Greep (579), began confirming this hypothesis by demonstrating that agents like aminoglutethimide and cyanoketone that interfere with steroid synthesis also inhibit ovulation. This earlier work has been reviewed by Lipner (15), Rondell (580), and LeMaire (496). More recent reviews on the status of steroids in ovulation have been presented by Tsafriri et al. (35), Richards and Hedin (42), and Espey (36).
  • Response to the Gonadotropin Surge
    •
  •     It is common knowledge that mature ovarian follicles secrete a relatively large amount of estradiol, along with some androgens. It is also generally understood that the ovarian synthesis of these steroids briefly increases and then declines while ovarian progesterone synthesis increases significantly within a few hours after the ovulatory process has been initiated by appropriate gonadotropic stimulation. As described earlier, this steroidogenic activity is probably mediated by cyclic AMP (284-299), and may be influenced by the calcium/calmodulin system (320-322). Although this is the usual "textbook" account of ovarian steroid output during ovulation, the data in the literature reveal many variations in this basic pattern. The reported differences may be due, at least in part, to the fact that various studies have been conducted on different species of both immature and adult animals in vivo and in vitro, and the steroid assays have been performed either on whole ovaries, follicle walls, follicular fluids, ovarian perfusates, ovarian venous blood, or peripheral blood (36). Also, it is possible that some of the variability is related to the type and amount of gonadotropin(s) that were used to initiate the ovulatory process. For example, when relatively pure FSH has been given to induce ovulation in hypophysectomized hamsters (251), or in perfused rat ovaries (581), this hormone stimulated 17ß-estradiol levels without initiating the usual ovulatory increase in progesterone. However, when LH or hCG is used in in vivo studies, the synthesis of 17ß-estradiol, testosterone, androstenedione, and 17a -hydroxyprogesterone declines to negligible levels by the time of ovulation, while progesterone synthesis increases substantially within a few hours into the ovulatory process (169, 171-176, 494, 572, 582, 583), although there may be some moderation in the output of this progestin near the time of follicular rupture (167, 170, 192, 492, 494, 523, 573). Saidapur and Greenwald (173) have reported that progesterone causes the decline in estrogen synthesis in vivo, whereas this progestin enhances 17ß-estradiol production in vitro (173). This correlation seems to also hold true for in vitro perfusion studies, where progesterone and 17ß-estradiol are usually secreted in parallel (256, 469, 485, 489, 515, 584-588). On the other hand, in vitro studies of "cultured" follicular tissue show a reciprocal relationship in the secretion of progesterone and 17ß-estradiol (174, 177, 589-591). Higuchi and Espey (244) have presented evidence that the differences in steroid secretion patterns of perfused ovaries versus non-perfused ovarian tissues may be related to adsorption properties of the perfusion system, or to the large volume of the perfusion reservoir.
  • Sites of Steroid Synthesis
    •
  •     The sex steroids are synthesized by common pathways that originate with cholesterol (157). Cholesterol is converted into pregnenolone by cholesterol side chain cleavage enzyme (P450scc) and then into progesterone by 3ß-hydroxysteroid dehydrogenase (3ß-HSD); progesterone is converted into androgens, including testosterone, mainly by the actions of 17a -hydroxylase (P45017a ) and C17-20-lyase; and, testosterone is converted into 17ß-estradiol by the action of aromatase (P450arom). Evidence of the cytochrome enzymes P450scc, P45017a , and P450arom within experimental cells and tissues is commonly used to identify the sites of progesterone, androgen (especially androstenedione and testosterone), and estrogen (especially 17ß-estradiol) synthesis. Such enzymes are frequently identified simply by the type(s) of steroids a given cell produces, or, in recent years, by the detection of specific mRNA transcripts.
  • Over three decades ago, Falck (164) conducted histological studies which led to the conclusion that both granulosa and theca interna cells might be required for estrogen formation. This hypothesis quickly lead to a number of modified versions of the so-called "two-cell theory" of granulosa-thecal cell steroidogenesis. Earlier accounts of the various forms of this theory have been summarized by Moor (592), Bjersing (29), and Ryan (593). Although it is difficult to establish a composite theory from these accounts, the preponderance of the earlier evidence seems to suggest that in mature ovarian follicles only thecal cells have the requisite enzymes for androgen synthesis, and therefore any estrogen synthesis by the granulosa is dependent on diffusion of precursor steroids from the theca interna. (However, it should be noted that this might be a "species variable" process (593, 594).) The theory is supported by more recent evidence that the thecal cells of mature follicles produce substantially more androstenedione than granulosa cells (591, 595-597), that granulosa cells lack P45017a required for androgen formation (303, 598), and that granulosa cells nevertheless generate 17ß-estradiol (597). These observations do not rule out simultaneous estrogen synthesis by thecal cells, and, in fact, when aromatizable androgens are absent from the culture media, theca interna cells can produce substantially more estrogen than granulosa cells (591). Also, it should be noted that the "two-cell theory" is not supported by a reports that granulosa cells have a potent P45017a system (599). Furthermore, there are morphological studies which describe the stratum granulosum as a steroidogenically inactive layer in mature follicles (32, 132, 600). In summary, perhaps the most relevant insight for a discourse on the mechanism of ovulation is that a mature ovarian follicle is basically an androgen and estrogen secreting gland prior to stimulation by the ovulatory surge in gonadotropin(s).
  •     The pattern and sites of steroid synthesis during the ovulatory process have been established with greater certainty. There is a variety of physical (32, 131, 601, 602) and chemical evidence (301-303, 305, 374, 591, 603, 604) that ovulatory follicles lose their capacity to produce 17ß-estradiol, and that both the theca interna and the granulosa layers begin producing substantial amounts of progesterone. There is limited evidence that the granulosa might be the initial site of this progesterone synthesis, while the thecal tissue becomes more active as the follicle progresses toward luteinization (605). Also, there is evidence that interleukin-Ia might mediate this steroidogenic activity in the theca interna (606). In any event, once this cyclic AMP-dependent progesterone synthesis has been initiated, the P450scc gene no longer requires LH/hCG or cyclic AMP and continues to transcribe the enzyme for progesterone production by independent mechanisms (310, 311, 607). Also, as luteinization progresses, the granulosa cells develop the capacity to form P450arom mRNA and synthesize 17ß-estradiol in a similar independent manner (608).
  • Importance of Steroids in Ovulation
    •
  •     The modest increase in synthesis of 17ß-estradiol, androstenedione, and testosterone in follicles during the first hours of the ovulatory process raises the question of whether these steroids might contribute in some way to the ovulatory process. Ying and Greep (579) reported that when ovulation was blocked by aminoglutethimide, this inhibitory action could be overcome by the administration of exogenous 17ß-estradiol or testosterone. However, several more recent studies have found that 17ß-estradiol is not essential for ovulation (588, 609, 610), and current opinion is that at least this steroid is not involved in the mechanism of ovulation. Presumably the androgens are not important either, but there is negligible evidence to support this common assumption. It has also been suggested that the well known decline in 17ß-estradiol synthesis during the first one-half of the ovulatory process might be a prerequisite for follicular rupture (611). However, this idea is not supported by tests which show that the maintenance of artificially high 17ß-estradiol levels throughout the ovulatory process does not impair normal ovulation rates (612).
  •     Considerably more attention has been given to the role of progesterone in ovulation, since follicles produce substantial amounts of this steroid during the last one-half of the ovulatory process. A wide range of experimental approaches have provided evidence that supports a significant role for progesterone (35, 170, 275, 370, 406, 472, 492, 523, 524, 572, 578, 580, 613-617), but other studies appear to contradict this conclusion (586, 588, 618-623). Still, there is now little doubt that progesterone makes some important contribution to the events that lead to degradation of the follicle wall and rupture. One of the first experiments to firmly establish a role for this steroid was conducted by Snyder et al. (615), who clearly demonstrated that the inhibition of follicular steroidogenesis and ovulation by epostane could be overcome upon treatment of the experimental animals with exogenous progesterone. This observation was highly complementary to the less convincing evidence of Ying and Greep (579) that progesterone could overcome the blockage of ovulation by aminoglutethimide. More recently, it has been established that the optimum time to administer epostane is during the hour preceding the ovulatory increase in progesterone synthesis, that this inhibitory agent severely impairs all steroidogenic activity in the ovary within a few minutes after its injection, that it causes only minor inhibition of follicular prostaglandin synthesis, that its anti-steroidogenic effect is transient, and that ovarian progesterone synthesis approaches normal levels by the time that inhibited follicles would have normally ruptured (472, 494, 572). Thus, these studies provide several potentially valuable bits of information about ovarian steroid metabolism during ovulation. First, they show that when ovarian steroid synthesis is blocked by a strong inhibitor like epostane (and probably by aminoglutethimide), the residual steroids rapidly dissipate from the ovarian tissue. This is a significant observation because it indicates that exogenous steroids probably need to be administered in extraordinarily large quantities in order to mimic the locally high levels within the follicular cells where the specific hormones are normally produced. Secondly, the existing data reveals that there is a relatively narrow "temporal window" in the early stages of the ovulatory process (i.e., at approximately the time when the progesterone levels begin to rise) during which it is vital for follicular steroidogenesis to proceed unabated. In contrast, it should be mentioned that when an animal like the ewe is treated for an extended period of time with moderate amounts of epostane, this synthetic steroid increases the number of developing follicles and actually increases the ovulation rate (621, 623, 624).
  • Inhibitors of Steroidogenesis and Ovulation
    •
  •     As just indicated, it is now clear that epostane is an effective inhibitor of ovarian steroid synthesis and ovulation (35, 472, 494, 572, 615). Epostane is reportedly a competitive inhibitor of 3ß-hydroxysteroid dehydrogenase (615, 621, 625), and therefore it probably exerts its anti-ovulatory action by blocking the conversion of pregnenolone into progesterone. In view of the effectiveness of this inhibitor, it is unfortunate that it is not readily available on the commercial market. A related synthetic steroid, trilostane, also inhibits 3ß-hydroxysteroid dehydrogenase (626), and this compound can also inhibit ovulation (unpublished observation). However, trilostane is much weaker than epostane, and the quantities that are required to block ovulation make it less suitable as an experimental agent. Aminoglutethimide is probably a more suitable anti-steroidogenic and anti-ovulatory agent for experimental purposes. Since its original use by Lipner and Greep (578) and by Ying and Greep (579), at least one other laboratory has found it to be an effective agent for inhibiting ovulation in vivo (176), but it did not affect ovulation in an in vitro perfusion system (620). It should also be noted that the inhibition of ovarian steroids by aminoglutethimide causes only a minor reduction in follicular prostaglandin synthesis (176, 627).
  •     Along with epostane and aminoglutethimide, the progesterone antagonist RU486 is in interesting anti-ovulatory agent. This agent has been used successfully a number of times to delay or block ovulation (35, 628-634), and in only one known instance did it fail to effect ovulation (622). While RU486 has been assumed to act by blocking progesterone receptors in ovulatory follicles, one report has suggested that this agent may act at least in part by reducing the amount of LH release, but such an explanation cannot account for its anti-ovulatory effect in experimental animals in which the ovulatory process has been initiated by exogenous hCG (630, 632). Another possibility is that RU486 may inhibit 3ß-hydroxysteroid dehydrogenase, based on recent evidence that isolated follicles that have been treated with this agent loose some of their capacity to convert pregnenolone into progesterone (634). Finally, in addition to these inhibitory agents, there are a variety of other substances that reportedly interrupt progesterone action and block ovulation (370, 613, 614, 616, 635-637), and in at least two of these cases the inhibitory effect was overcome by exogenous progesterone (616, 636).
  • Luteinized Unruptured Follicles
    •
  •     There is ample evidence that gonadotropin-stimulated follicles that have been exposed to ovulation-inhibiting doses of indomethacin continue to produce normal amounts of progesterone (176, 193, 292, 312, 460, 472, 473, 492, 516, 519), and the unruptured follicles undergo luteinization (453, 458, 463, 508, 510, 511, 514, 517, 518, 542). There is also evidence that indomethacin treatment fails to affect established luteal function (513), or to shorten the normal length of pseudopregnancy (509), although progesterone secretion by the luteal tissue might be somewhat less than normal (521). Occasionally, unruptured follicles spontaneously luteinize in the absence of indomethacin treatment (638), and such anovulatory activity could be associated with an inadequate gonadotropin surge (639).
  •     This phenomenon of luteinized unruptured follicles, which has been studied most extensively by Armstrong and coworkers (458, 460, 463, 508, 510), provides several valuable pieces of information about the mechanism of ovulation. First, the phenomenon makes it perfectly clear that the luteinization process that is normally initiated by a gonadotropin surge can proceed virtually unabated even when a stimulated follicle does not physically rupture. Secondly, the phenomenon makes it apparent that, while ovulation and luteinization are both normally initiated by the same gonadotropic stimulus, the two processes can be at least partially separated from one another. However, when this information is coupled with the fact that the ovulatory process is blocked by agents that inhibit follicular progesterone synthesis (i.e., that inhibit the central event of the luteinization process), the existing data strongly suggest that follicular rupture is fundamentally dependent on steroidogenic activity that occurs during the early stages of luteinization.
  • Other Considerations
    •
  •     There are several other features of steroid metabolism that have received only limited attention, but may be worthy of further examination in future investigations. First, there are several reports that suggest some kind of relationship between prolactin (PRL) and progesterone. It has been noted that progesterone stimulates PRL release (640). On the other hand, it has been reported that PRL inhibits ovarian progesterone synthesis and ovulation (275, 641), yet some studies conclude that the anti-ovulatory action of PRL does not influence ovarian progesterone secretion (276, 277). Secondly, although it is quite clear that prostanoids have negligible influence on follicular steroid metabolism, there are several reports that arachidonic acid (642), or lipoxygenase products of arachidonic acid (472, 494, 572, 573), might influence ovarian steroid metabolism during ovulation. Now that more convenient methods for assaying these eicosanoids, the elucidation of their possible contribution(s) to the mechanism of ovulation should be forthcoming. Thirdly, many years ago, Pincus (576) pointed out that "one of the possibilities very much overlooked is that some of these [sex] steroids are mitosis-stimulating and others mitosis-inhibiting, and, more recently, Clarke and Sutherland (643) have reviewed the mechanism by which progestins affect cell proliferation. Therefore, it might be worthwhile to examine in more detail the potential relationships between the ovulatory increase in progesterone and common growth factors and cytokines. In this regard, progesterone might influence, not only cellular proliferation within the luteinizing theca interna and granulosa, but it is also possible that this steroid might regulate the activation and proliferation of fibroblasts that occurs within the connective tissue layers of ovulatory follicles (32). Lastly, it should be noted that progesterone may play an anti-inflammatory role in arthritis (45, 644) and in other models of inflammation (645, 646). Therefore, although it is now quite apparent that progesterone contributes during the early stages of the ovulatory process to the degradative events within the follicle, it is still possible that this steroid might have some role in regulation of the healing process that sets in shortly after ovulation (36, 44).
  •  
  • Proteolytic Enzymes
    •
  • Plasminogen Activator
    •
  •     As described earlier in this chapter, a mature ovarian follicle contains a tenacious collagen matrix at the apex where rupture occurs, and it is generally thought that proteolytic enzymes weaken this connective tissue at the time of ovulation. Plasminogen activator (PA) has been given considerable attention as a contributing agent in this process because this serine protease has been implicated in many types of tissue degradation and cellular movement (647) and it reportedly is secreted from fibroblasts in association with collagenase (648, 649). In 1975, Beers and Strickland (650-652) conducted the first studies on the role of PA in ovulation. In initial experiments, it appeared as though FSH might be the principal gonadotropin that regulated ovarian PA activity, but now there is evidence that FSH (252, 384, 528, 653-658), LH/hCG (276, 421, 531, 532, 655, 659-663), or GnRH (383, 384, 663) can promote follicular PA activity, while prolactin reportedly inhibits the expression of this enzyme (276, 277, 664). The active gonadotropins mainly promote tissue-type PA activity, rather than urokinase-type PA. The stimulatory effect of gonadotropins is probably mediated by cAMP (651, 652, 655, 657), although there is one study which suggests that the use of phosphodiesterase inhibitors to increase cAMP levels may actually depress PA activity, rather than increase it (665).
  •     Most of the studies on ovarian PA activity have been conducted on granulosa cells (382-384, 528, 650-652, 655, 657, 658), or the closely related cumulus mass (656, 659). It has been reported that this enzyme is mainly in the granulosa layer (666), although molecular techniques have failed to find tPA mRNA in gonadotropin-stimulated granulosa (386). Other work indicates that the theca interna may also contain a significant amount of PA (660, 663, 667, 668). In fact, some studies conclude that PA activity is distributed throughout most of the cells in the follicle wall and that it may be especially abundant in fibroblasts in the apical area where rupture normally occurs (661, 667, 669). In general, it appears that PA activity increases just before or very close to the expected time of ovulation (421, 531, 660, 663, 667, 669-674), although there is one report that maximum activity is expressed during the first one-third of the ovulatory process and then declines (662).
  •     Unfortunately, the highly relevant experiments to assess the effects of eicosanoids and steroids on ovarian PA activity arrive at conflicting conclusions. In their original studies, Strickland and Beers (652) found that prostaglandins of the E-type effectively stimulated granulosa cells to produce PA, while prostaglandins of the F-type were without effect, yet Miyazaki et al. (529) have recently reported that prostaglandin F2a activates PA activity in the follicle wall during ovulation. Similar contradiction is found in comparing a report that indomethacin suppresses PA secretion (528) with several other studies which claim that doses of indomethacin that significantly inhibit ovulation have no apparent effect on ovarian PA activity (421, 530-532). The results of studies with steroids are equally confusing. Earlier studies concluded that none of the principal ovarian steroids effected PA activity (652). More recent studies suggest that 17ß-estradiol may be especially important in inducing granulosa cell PA activity, while progesterone and testosterone appear to have only a minor role (35, 532, 658, 666). Other reports suggest that testosterone (653) progesterone (421, 636) may be at least partially responsible for the expression of ovarian PA activity, while still another study suggests that ovarian prostaglandin synthesis may not be essential to the expression of this activity by granulosa cells (654). Experiments with a variety of agents that reportedly are specific inhibitors of PA activity have not clarified the above confusion about the role of this enzyme in ovulation (531, 675-677). Thus, in summary, the nature of ovarian PA activity and its relationship to ovarian eicosanoids and steroids in the ovulatory process are not yet fully understood.
  • Kallikrein and Kinin-Generating Activity
    •
  •     Kallikreins are a family of serine proteases that are usually activated by tissues that have become irritated or inflamed (678-681). In addition to their capacity to generate kinins, as discussed earlier in this chapter, kallikreins also convert procollagenase into its active form (682, 683). In view of this action of kallikreins, 15 years ago Espey (684) suggested that such enzymes might be involved in the activation of procollagenase in ovulatory follicles. However, only a limited number of experiments have been carried out on this potentially important protease. Espey et al. (420) have used a relatively non-specific chromogenic peptide substrate in studies which indicate that follicular kallikrein activity may increase about 6-fold in gonadotropin-primed immature rats (420). This ovulatory increase in activity is partially (but, significantly) reduced by indomethacin treatment (413, 420, 421), which suggests that it might be related to eicosanoid metabolism in the follicle. Ovarian kallikrein activity is also suppressed by epostane treatment, and this inhibitory action can be reversed by treatment of the experimental animals with exogenous progesterone (421).
  •     More precise information about the contribution of ovarian kallikrein activity to the ovulatory process may be forthcoming from new studies based on the methods of molecular biology. For example, Clements et al. (381) have recently found that the granulosa cells of gonadotropin-treated rats express mRNA for several kallikrein gene family members, and further work should reveal which of these kallikrein-like enzymes are responsible for the ovarian kallikrein activity that peaks at about the same time as the onset of follicular rupture. Also, there is evidence that the subunits of nerve growth factor and epidermal growth factor have peptide sequences that are similar to tissue kallikrein (428, 685), and therefore it might be worthwhile in future studies to assess whether the internalization of proteolytic fragmentation of growth factor/receptor complexes might generate kallikrein activity during ovulation (44). In addition, future studies might examine whether several other novel proteases that have been implicated in the ovulatory process (686, 687) happen to belong to the kallikrein family of enzymes.
  • Collagenolytic Activity
    •
  •     The thecal layers of the follicle wall contain a substantial amount of collagenous connective tissue, and there can be little doubt that such tissue must be significantly weakened in order for a follicle to rupture under the force of a relatively modest intrafollicular pressure of approximately 20 mm Hg. The first evidence that collagenolytic enzymes might be involved in this process came about when Espey and Lipner (13) injected minute amounts of bacterial collagenase into rabbit follicles in situ and observed rupture of the follicles only a few minutes after the injections. At about this same time, there were several reports with indirect (105) and direct (65) evidence that the tenacity the follicle wall declines during the ovulatory process. Shortly thereafter, it became apparent that mammalian collagenase, elastase, trypsin, and chymotrypsin also can significantly reduce the tensile strength of the follicle wall (116). Morphological studies also showed that the collagen and cellular elements of the follicle wall dissociate and the wall becomes thinner at the apex as the time of ovulation approaches (64, 126, 688). However, initial attempts by Espey and Rondell (117, 118) to measure an increase in ovarian collagenolytic activity were unsuccessful. Similarly, Parr (689) was unable to detect any neutral protease activity in ovulatory rat follicles, nor could he find any collagen fibers in the follicle wall (128). On the basis of this information, he concluded that rupture of the ovarian follicle is not mediated by collagenolytic enzymes. To the contrary, it is now clear that the thecal tissue in rat follicles does contain significant amounts of collagen (150), and numerous additional efforts have been made to identify the nature of enzymes that might degrade this connective tissue during ovulation. The earlier work on ovarian proteolytic and collagenolytic activity has been summarized in several reviews (16, 18).
  •     More recent efforts to detect follicular collagenolytic activity have used assays based on the digestion of reconstituted collagen gels (18), of 3H-labeled collagen (120, 534, 676, 690, 691), of synthetic peptide substrates (119, 692, 693), of substrates for gelatinase and proteoglycanase (694). Collectively, the results from these various studies indicate there is a substantial increase in ovarian collagenolytic enzymes during the ovulatory process. Complementary to these findings, Reich et al. (385) have recently reported the detection of an ovarian mRNA for collagenase that increases 25-fold during ovulation. Curry and coworkers ( 387, 388, 695) have reported that such activity might be locally regulated at least in part by tissue-derived inhibitors of metalloproteinases (TIMPs) which are produced by follicular cells in increasing amounts during the ovulatory process, and their observation has been supported by several other studies (385, 696). Also, there is evidence that the serum anti-protease a 2-macroglobulin might function in ovulation to restrict collagenolytic activity to the vicinity of the mature follicles that are destined to rupture (386, 387, 697). Further support for the hypothesis that collagenolytic enzymes are important in ovulation come from additional studies which demonstrate that other agents known to inhibit collagenase also inhibit ovulation (698-701). There is no data on whether these various inhibitory substances can effect the relaxin-induced collagenolytic activity that has been associated with ovulation (536, 702, 703).
  •     The cytological origin of ovarian collagenolytic activity has not been firmly established. Several studies indicate that such enzyme activity arises in the granulosa (385, 692), while other work provides evidence that thecal fibroblasts may be the principal source (5, 151, 153, 154). It is possible that the enzymes which degrade the follicle wall arise from more than one cell type, but the fibroblast in particular must be considered as a probable source since it has been recognized for some time now that such connective tissue cells are not only the origin of collagen, but also of the proteolytic enzymes that degrade this tenacious extracellular material (648, 649, 704-706). The cytological structures that have been most frequently associated with ovarian collagenase activity are the multivesicular structures that protrude from the surface of follicular fibroblasts (5, 151, 153, 154), and the limited number of lysosomes that are present within different cells in the follicle (29, 64, 142, 707, 708). The relationship between collagenolytic activity and ovarian eicosanoid production is not clearly defined. A variety of experiments which have indicated an inhibitory effect of indomethacin on such proteolytic activity in gonadotropin-stimulated ovarian tissues suggests that eicosanoids may mediate the degradation of ovulatory tissue (18, 152, 385, 473, 492, 534, 574), yet several other reports conclude that prostaglandins have no effect on ovarian collagenolytic activity (535, 536). This contradiction is not resolved by results obtained from other experimental models because, for example, one report claims that prostaglandin synthesis by bone cells may mediate collagenase activity (709), another concludes that prostaglandins of the E-type enhance (but do not induce) macrophage collagenase production (710), and a more recent report shows that prostaglandin E1 (but not E2 or F2a ) can selectively reduce collagenase mRNA levels in a dose-dependent fashion in rabbit synoviocytes and human fibroblasts (448).
  •     With regard to the relationship between ovarian collagenolytic activity and steroid hormones, there is only limited information. Several studies have suggested that progesterone may have a negligible effect on such activity (18, 536). Other reports have pointed to a positive correlation between the ovulatory increase in progesterone levels and the local production of TIMP by follicles (386, 695). In addition to this information, it is worth noting that progesterone is a potent inhibitor of collagenase expression in the uterus (711, 712) in the pubic symphysis ligament (713), and by macrophages (714). In this same line, Espey (44) has recently suggested that progesterone may have functions in the ovary that are comparable to the actions of this steroid in the uterus. Specifically, progesterone acts by inducing the formation of uteroglobin which inhibits phospholipase A2 activity and the metabolism of arachidonic acid in a manner that suppresses the proteolytic activity characteristic of inflammatory reactions (715, 716). Thus, it is possible that, after exerting its acute pro-ovulatory effect during the first half of the ovulatory process, progesterone may impose a secondary effect that causes the decline in ovarian eicosanoid metabolism and proteolytic (including collagenolytic) activity within a few hours after a follicle ruptures. That is to say, the chronic effect of progesterone on reproductive tissues might be comparable to the anti-inflammatory actions of glucocorticoids that are mediated by lipocortins in other tissues (717-719).
  •  
  • Other Considerations
    •
  • Follicular Inflammation
    •
  •     During the past 12 years, much of the work on ovulation has keyed on assessment of the hypothesis that ovulation is an inflammatory process. This idea that the ovulatory surge in gonadotropin induces an inflammatory reaction in mature follicles arose from a long pedigree of studies that reached maturity by the end of the 1970's. For more than a century, hyperemia and related vascular changes have been recognized as the cardinal signs of inflammation (45). In comparison, follicular hyperemia at the time of ovulation has been noted for at least one-half a century (8), and some of the more recent studies on this topic have been summarized in an earlier section of this chapter which keys on the vascular system of the theca interna. Also, as early as 1951, Burr and Davies (180) noted the swelling and edema that occurs in association to the ovulatory hyperemia in rabbit follicles. Likewise, Bjersing and Cajander (122) and Motta et al. (140) have mentioned edema as a relevant factor in the final decomposition of ovulatory follicles. Historically, it is interesting to note that 32 years ago John Hammond, Jr., while comparing a follicle to the formation of a blister during discussions at a symposium on ovulation (720) stated, "You burn yourself, and a fluid is liberated from the blood vessels of the dermis, yet in like manner, the fluid accumulates in the epithelium of the epidermis." Thus, at least three decades ago, the ovulatory process was passingly compared to an inflammatory reaction.
  •     Two of the more significant contributions to the "inflammation hypothesis" arose in the early 1970's when simultaneous experiments by innumerable investigators showed that prostaglandins increase markedly in ovulatory follicles and that indomethacin can block prostaglandin synthesis and ovulation. That work set the stage for some of the conclusions drawn from a series of studies by Parr (128, 689, 721). In his morphological work, Parr (128) noticed that the walls of follicles about to rupture contained fibrin, which is commonly present in inflamed tissues. He coupled this observation with the previously overlooked fact that prostaglandins are an integral part of inflammatory reactions and that indomethacin is a potent anti-inflammatory agent. However, he did not consider the ovulatory process to be a complete inflammatory reaction. Instead, he concluded that ovarian prostaglandins might contribute to "the early vascular phase of an inflammatory response", but that the "inflammation never progresses to the cellular stage" (17). Consequently, he was unable to explain "the weakening of follicular tissue which precedes rupture" (17), especially since he concluded in several reports that "ovulation is not mediated by a proteolytic enzyme" (689), or by "a collagenase" (17). The latter deduction arose from his erroneous report that the thecal layers of the rat follicle do not contain any collagen for an enzyme to digest (128, 150). Perhaps because of these significant erroneous deductions, Parr's highly relevant observations of inflammatory-like changes in ovulatory follicles received little attention, except in several review papers (18, 19). Even Parr himself may not have realized the significance of his observations about inflammatory-like changes in ovulatory follicles, because in the concluding remarks of his review on ovulation he did not mention this topic, but instead, stressed that future studies should key on "the possible role of ascorbic acid in follicle rupture" (17)--a point which had been made repeatedly throughout the 1970's (16, 18, 19, 116). Several years later, Espey (45) reviewed the existing literature on ovulation and inflammation and concluded that both the vascular and the cellular (i.e., biochemical) changes in ovulatory follicles were comparable to an inflammatory reaction. A recent reassessment of this hypothesis summarizes a significant portion of the work that has been conducted on the mechanism of ovulation during the past 12 years (46).
  • Fibroblast Proliferation
    •
  •     As early as 1919, Corner (60) reported that the theca externa "is composed chiefly of collagenous fibrils and their associated fibroblasts". The existence of such connective tissue cells in the follicle wall has been confirmed by many other studies (32, 64, 95, 128, 144, 152, 222). However, the follicular fibroblasts have been given little attention in biochemical studies of the ovulatory process, probably because they are imbedded in the collagen layers of the follicle and are relatively inaccessible to isolation compared to cells of the granulosa and theca interna. Nevertheless, the potentially prominent role of fibroblasts in the mechanism of ovulation should not be overlooked. It has been well established that fibroblasts produce collagenase and regulate collagen metabolism (704-706, 709). Therefore, it is quite possible that the final stages of the ovulatory process involve activation and proliferation of the thecal fibroblasts. In this regard, it is interesting that, in his early observations of the morphological changes during ovulation, Corner (60) also noted "just before rupture there are many mitotic figures in the cells of the theca externa, but only occasional signs of cell division in the theca interna and the granulosa." Espey (32) has also reported such mitotic activity and has suggested that "the fibroblasts transform into actively proliferating cells and become quite long." O'Shea (722) has said that after ovulation the proliferating fibroblasts migrate "from their original sites into the deeper, granulosa-derived areas of the luteal tissue." Therefore, since the thecal fibroblasts are embedded in a framework of collagen, it seems almost inevitable that these cells must produce enzymes to weaken the extracellular matrix before they can migrate toward the luteinizing granulosa. The cytological scenario may not be too unlike what happens in bone matrix where, in response to low calcium levels and parathyroid hormone secretion, osteoblastic cells (which are closely related to fibroblasts) become motile and secrete collagenase to dissolve the connective tissue matrix in which bone salts are precipitated (157, 723, 724).
  •     The important point is that within the mechanism of ovulation it appears quite likely that the thecal fibroblasts undergo transformation from a quiescent resting state to a motile proliferating state, yet there is virtually no information about the biochemical events which initiate this highly significant activity within the densest connective tissue in the follicle wall. In other experimental models, there are earlier reports that prostaglandins influence the proliferation of fibroblasts (725-728), with some evidence that prostaglandin F2a in particular might initiate this activity (729). This information suggests that the fibroblasts in ovarian follicles might also be activated by the eicosanoids that increase so markedly during the ovulatory process. The prostaglandins could be generated by cells of the granulosa and/or theca interna, or they could arise from within the fibroblasts themselves (730). It is also possible that indomethacin and other non-steroidal anti-inflammatory agents might inhibit ovulation by blocking such prostaglandin-induced activation of follicular fibroblasts. Espey (46) has recently suggested that indomethacin may act by inhibiting "the transformation of the thecal fibroblasts from a dormant, resting state to a motile, proliferating stage," and that "without sufficient movement of the fibroblasts, ultimate dissociation of the thecal collagen may not occur." There is morphological evidence to support this hypothesis (152).
  •     Other ovarian factors that could influence proliferation of the thecal fibroblasts are steroid hormones (since fibroblasts contain sex steroid receptors (731-734)), cytokines such as interleukin 1 and lymphokines (735-737), proteolytic enzymes such as plasmin, trypsin, chymotrypsin, and elastase (649), and growth factors such as EGF and PDGF which are associated with the expression of proto-oncogenes to exert local mitogenic effects and promote collagenolytic activity (723, 738-740). In addition, blood serum contains factors that promote the proliferation of fibroblasts, and this relevant fact has been recognized for some time (741). More recent work has concentrated on identification of the genes that are expressed by serum stimulated fibroblasts and some of these include the fos, jun, and myc proto-oncogenes (742-744). These actions of serum are particularly interesting since they raise the question of whether the gonadotropin-induced increase in vascular permeability and hyperemia (that were described earlier in this chapter) might permit serum factors to diffuse into the connective tissue layers of the follicle and transform the fibroblasts into proliferating cells.
  •     Lastly, as mentioned earlier, fibroblasts transform into myofibroblasts under certain circumstances such as the development of granulation tissue in chronically inflamed areas (66, 94, 745). Although ovarian luteal tissue has been compared to granulation tissue (45), no efforts have been made to determine whether some of the fibroblasts in the follicle do indeed develop into myofibroblasts in the corpus luteum. The identification of actin and myosin contractile proteins alone will not confirm such a transition because contractile proteins like actin and myosin are natural components of motile cells like fibroblasts (68), and the genes for these proteins are expressed when quiescent fibroblasts are stimulated with serum (743). On the other hand, there is a recent report that the expression of a single gene may be all that is necessary to convert fibroblasts into myofibroblasts (746), and evidence of such expression in connective tissues in ovulatory or post-ovulatory follicles might help to clarify this issue.
  • Growth Factors and Ovulation
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  •     Growth factors are hormone-like peptides that have predominantly paracrine and autocrine functions in promoting mitogenic activity in local tissue proliferation and remodelling (323, 747), like that which occurs in the transformation of an ovarian follicle into a corpus luteum (44). In recent years, there has been considerable interest by Hsueh, Adashi and others (393, 657, 748-751) in the action of growth factors on ovarian folliculogenesis. It has been suggested that ovarian thecal cells produce epidermal growth factor (EGF) and transforming growth factor-a (TGF-a ) which exert a paracrine effect to promote the proliferation of granulosa cells in growing follicles (389, 752). In comparison, it has been suggested that granulosa cells are the site of production, reception, and action of insulin-like growth factors (IGFs) (750), with activity being regulated by gonadotropins and local steroids (392). On the other hand, the follicular source of fibroblast growth factor (FGF) is uncertain, but it may not originate from granulosa cells (390, 391, 753). The potential role of growth factors in ovulation is especially intriguing since there is evidence these mitogenic agents may be involved in inflammatory processes (754-761), progesterone formation and other steroidogenic activity (338, 393, 749, 750, 762-764), protease (including collagenase) synthesis (384, 723, 738, 739), angiogenesis (391, 753, 765, 766), and possibly prostanoid synthesis (767, 768), all of which are characteristic of ovulatory follicles.
  • Leukocytes and Ovulation
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  •     In inflamed tissues, the insulted cells produce chemotactic agents which attract a variety of leukocytes and migratory cells into the area as a line of defense against possible infection (45, 46). There is also a local release of histamine, eicosanoids, platelet-activating factor and other vasoactive agents to increase the blood flow to the affected area. In some instances, the polymorphonuclear leukocytes that infiltrate the area may release stores of proteolytic enzymes including plasminogen activator, elastase, collagenase and other neutral proteases that can soften the connective tissue elements and enhance proteolytic and phagocytic action against any invading microorganisms. Since these are physical and chemical changes that also occur during ovulation, some consideration has been to the possible contributions of leukocytes and related cells to the ovulatory process. As early as 1958, Zachariae et al. (769) noted that basophils migrate into the ovary at the time of ovulation. Since that time, there has been additional direct and indirect evidence that various leukocytes, along with thrombocytes, might increase in ovulatory follicles (32, 202, 473, 770-774). In support of this data, there are several recent reports that ovulatory follicles produce a chemotactic factor that attracts leukocytes into the area (473, 775), and Murdoch and McCormick (772) may have isolated some such chemoattractant peptides. Also, Hellberg et al. (776) have reported that the addition of leukocytes to the perfusion medium supplying rat ovaries slightly, but significantly, increased the number of LH-induced ovulations in vitro. However, Murdoch and Steadman (777) have recently found that prednisolone-induced eosinopenia failed to impair ovulation in ewes, and they concluded that such leukocytes may not contribute to the mechanism of ovulation. Quantitative studies on the temporal and spacial distribution of these types of cells in mature follicles during ovulation would help elucidate the extent to which they might be involved in the degradation of the follicle wall. While leukocytes might make some contribution to the process, it still appears more likely that the thecal fibroblasts are the principal source of proteolytic enzyme activity during ovulation.
  • AN OVERVIEW OF THE OVULATORY PROCESS
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  •     In a final analysis of the above information about ovulation, it is important, first, to key on luteinization as an ovarian process. When an ovulatory surge in gonadotropin(s) stimulate a mature ovarian follicle, the principal cellular response is luteinization of the theca interna and granulosa layers of the follicle wall. That is to say, the basic function of luteinizing hormone is, as the name implies, to initiate the luteinization process. The early events of membrane signal transduction generate cyclic AMP in the target cells, and this second messenger is an important mediator of the events that follow. Less is known about other second messengers and the protein kinases that may be generated during the early stages of luteinization, but it is becoming more and more evident that the signal transduction processes lead to a significant increase in cytochrome P450scc that promotes a substantial elevation in progesterone synthesis. Simultaneously, due to the rising progesterone levels or other unknown factors, there is a reciprocal decline in cytochromes P45017a and P450arom, and ovarian androgen and estrogen secretion drops quite sharply. Once P450scc mRNA has been expressed, cyclic AMP-independent mechanisms are established and progesterone synthesis and luteinization proceed as a single, self-perpetuating process. The luteinization process is virtually unaffected by anti-ovulatory agents like indomethacin, and is only transiently attenuated by anti-ovulatory agents like epostane. Thus, it is difficult to interrupt the intrinsic, constitutive changes that are established during the earliest stages of luteinization. Furthermore, it is now rather clear that rupture of a follicle is not a prerequisite for luteal formation.
  •     It is also now evident that a number of inflammatory-like changes occur in stimulated follicles during the early stages of the luteinization process. In particular, it is apparent that follicles become hyperemic within a few hours after the gonadotropin surge. This hyperemia is probably mediated by vasoactive agents like histamine, kinins, prostaglandins, and possibly lipoxygenase products of arachidonate metabolism. The specific relationship between ovarian eicosanoid formation and progesterone synthesis has not been deciphered, but on the basis of the simultaneous onset of eicosanogenic and steroidogenic activities it would appear that there is some interdependence in these two major metabolic events in the follicle. In addition, there are numerous reports that serine proteases and metalloproteinases like plasminogen activator, kallikrein, and collagenase activity increase in follicles as the luteinization process progresses. However, the interrelationships between these proteolytic enzymes and the eicosanoids and steroids mentioned above have not been worked out either. Nevertheless, more and more knowledge about the timing of these events is being gained in certain experimental models like the gonadotropin-primed immature rat, and it should be only a matter of time before molecular techniques and other methods are successful in deciphering the relationships among ovarian progesterone, prostanoid, and protease metabolism.
  •     In essence, the ovulatory process that has been referred to throughout this chapter is actually the first 10-40 hours (depending on the species of mammal) of the luteinization process. Recognition of this duality and separation of the two processes may be important in further efforts to unravel the remaining questions about the mechanisms of ovulation. Realization of this duality raises the possibility that all of the events of the luteinization process may not be essential to the ovulatory process, and vise versa. The existing data make it apparent that the early stages of progesterone synthesis (i.e., the principal aspect of the luteinization process) are necessary for ovulation, but that progesterone synthesis can be inhibited during the second half of the ovulatory process without blocking ovulation. On the other hand, it is also now apparent that eicosanoid metabolism is required almost up to the time that a follicle ruptures, yet luteinization can occur in the absence of any elevation in ovarian eicosanoids.
  •     In the past several decades, considerable knowledge has been gained about the timing of the biochemical events that occur during the ovulatory phase of the luteinization process. While this "temporal" analysis of the ovulatory process has been very productive, it now may be appropriate to concentrate more on "spacial" analyses of the events of ovulation. That is to say, it may be important to key more on identifying the cellular locations of the various metabolic changes that arise during the ovulatory phase. It seems obvious enough that the principal events of luteinization are centered in the stratum granulosum and the theca interna (along with the vascular endothelial cells of the latter layer). On the other hand, it may be that the principal events that lead to degradation of the collagenous connective tissue and ultimate rupture of a follicle may take place in the connective tissue cells themselves, namely in the fibroblasts. If this is true, if the secret to understanding the mechanism of ovulation lies in decoding the events that convert quiescent fibroblasts into proliferating fibroblasts, then it becomes equally imperative to distinguish the specific events within luteinization of the granulosa and theca interna that activate the fibroblasts. While it is obvious that progesterone is directly or indirectly involved, fibroblast activation might also be caused by serum factors within exudate from the thecal capillary network, or by action of eicosanoids of granulosa or theca interna origin. This latter idea that eicosanoids might contribute to the activation of thecal fibroblast, if true, would help explain how anti-inflammatory agents like indomethacin can block eicosanoid synthesis and ovulation without interfering in the luteinization process.
  •     In summary, mammalian ovulation is considered to be an inflammatory-like process that occurs during the early stages of the luteinization process which is initiated by a surge in gonadotropic hormones from the pituitary gland. Although the luteinization process does not require ovulation, it appears that the ovulatory process is an integral part of the early stages of luteinization. While both ovulation and luteinization are dependent on events in the theca interna and granulosa layers, ultimate rupture of a follicle may also depend on activation of the fibroblasts in the thecal connective tissue of a mature follicle. Therefore, any comprehensive analysis of the physiology of ovulation requires not only on information about the timing of the principal events, but also an assessment of the spacial distribution of these events within the various cellular components of a follicle.
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