Draft Copy

 

CHAPTER 12:  Ovulation

Lawrence L. Espey1 and JoAnne S. Richards2

in

The Physiology of Reproduction, 3rd Edition

edited by

J.D. Neill, Elsevier, St. Louis, pp. 447-496, 2006

 

1Department of Biology, Trinity University, San Antonio, Texas 78212

2Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030

 


I.  INTRODUCTION

Mammalian ovaries have two principal functions.  They produce sex steroids to prepare the adult female for reproduction, and they release eggs at appropriate intervals during the fertile years of the organism [1].  The latter function is commonly referred to as ovulation, which is the topic of this chapter.  The ovulatory process in mammals is a distinct biological phenomenon that begins when gonadotropic hormones stimulate mature ovarian follicles and it ends when the follicles rupture and release eggs into the oviduct (Figure 1).  The main focus of this chapter is on the molecular events that lead to the release of eggs, i.e., on the biochemical changes that occur in the ovary as the result of gene expression in response to gonadotropic hormones that excite specific ovarian cells and cause ovulation.

It is generally thought that the underlying mechanisms of hormone action that cause ovulation are homologous in all mammals.  Initially, it was assumed that the process was a relatively simple phenomenon, involving a small number of regulatory factors that were activated by a surge in the secretion of pituitary gonadotropins.  For more than one hundred years, reproductive biologists “stressed the role of increasing intrafollicular pressure as the cause of rupture,…and the change in pressure was ascribed to the action of smooth muscle in the ovarian stroma” [2].  However, in recent decades, it has become clear that intrafollicular pressure does not increase significantly during the hours preceding rupture [3], and the ovary is not endowed with any functional smooth muscle tissue [4].  Instead, the current hypothesis is that the ovulatory surge in gonadotropins induces an acute inflammatory reaction that involves the stratum granulosum and the thecal layers of mature follicles [5-7].  The inflammatory process generates protease activity in the granulosa and/or thecal layers of the follicles and this proteolytic activity degrades extracellular matrices within the connective tissue in the ovary.  The degraded elements of the follicle wall dissociate and rupture under the force of a steady intrafollicular pressure, resulting in release of the cumulus cell-enclosed oocyte complex (COC).  The current chapter is primarily a review of the cascade of ovarian gene expression that induces local inflammation that results in follicle rupture via proteolytic disintegration of the connective tissue elements of the follicle wall and the expansion of the COC associated with production of a specialized extracellular matrix.  Each of these processes (rupture and COC expansion) is critical for successful release of fertilizable oocytes, hence successful ovulation.  However, the chapter will initially summarize the basic morphology of an ovarian follicle and it will update contemporary views on the roles of certain biochemical agents such as progesterone and the prostaglandins in ovulation.

Reproductive biologists who have an enduring interest in ovulation research are encouraged to examine the exceptional review by Carl Hartman in 1932 [8].  His account provides many interesting insights into the lineage of earlier research on this subject.  Details about ovarian innervation and vascularity can be found in the chapter on ovulation in the initial edition of The Physiology of Reproduction [9].  A more comprehensive review of the literature on ovulation is available in the second edition of these volumes [10].  The present review will concentrate on information that has become available during the decade since publication of that second edition.

Innumerable reviews on topics related to ovulation have been written during the past 10-12 years.  These include reviews of a generally allied nature [11-13], on prostaglandins [14-16], on progesterone [17-19], on proteases [20-29], on angiogenesis [30] and especially on cytokines [31-37] and leukocytes [38-40].  In addition, there are a number of reviews that itemize the various genes that are uniquely expressed in the ovary in association with the ovulatory process [7, 41-47].  Some of the reviews emphasize the inflammatory nature of the ovulatory process [6, 7, 46].  The contents of this chapter are based in part on information from the above assortment of reviews and in part from a number of different primary research papers.  Because of the sheer volume of information it has been impossible to include all relevant primary literature.

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 preovulatory will be used in reference to mature follicles that have not yet been stimulated by the ovulatory surge of gonadotropins, whereas the term ovulatory will be applied to the entire 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 important, the term follicular rupture will be used for clarity.

II.  MORPHOLOGICAL FEATURES OF OVULATION

            Mammalian ovulation is an exceptional biological phenomenon in that it requires the physical disintegration of healthy tissue at the surface of the ovary.  The disturbance begins when a mature ovarian follicle is stimulated by an ovulatory surge in pituitary gonadotropins that react with G-protein-coupled receptors on the plasma membranes of the two innermost layers of cells in the follicle, namely the granulosa cells and the theca interna cells [1, 10, 11, 41-43, 47] (Figure 2).  There is a variety of evidence to indicate that the molecular response to gonadotropic stimulation begins rapidly, and these biochemical changes occur rather evenly throughout the follicle wall, i.e., rather uniformly around the entire circumference of the follicle [7, 41-47].  It is generally assumed that the site of rupture is in the apical most region of the follicle because this area happens to be, morphologically, the weakest portion of the follicle wall [1, 10].  The present section of this chapter summarizes the ultrastructure of the apical region of a rabbit follicle before it has been stimulated by an ovulatory dose of gonadotropin and at several stages near the time of follicular rupture. 

The ovulatory process in rabbits requires approximately 10 hours.  During the first several hours after a mature rabbit follicle has been stimulated by an ovulatory surge in pituitary gonadotropins, there is no conspicuous change in the macroscopic appearance of the follicle.  However, by 4 hours into the process, a follicle will begin to blush [1].  There is clear evidence that the capillaries in the follicle wall have dilated, and the tissue becomes hyperemic [48].  There is no other morphological indication of pending rupture until 1-2 hours before the follicle wall will actually burst.  As the time of rupture nears, the apex of a mature follicle protrudes more and more above the surface of the ovary and the follicle wall itself gradually becomes thinner.  Eventually, in the last minutes before rupture, the apical most portion of the follicle becomes translucent and rapidly balloons above the normal curvature of the ovary to form a stigma.  A follicle usually ruptures within several minutes after the stigma forms.  Ovulation is complete when the expanded cumulus-oocyte complex or (COC) is discharged, usually within 1-2 minutes after the follicle wall bursts.  Although the release of the oocyte is assumed to be passive, the formation of the hyaluronan COC-derived matrix is critical for egg release. Therefore, it is perhaps worth considering the notion that the COC and its surrounding matrix contribute along with the mural granulosa to follicular rupture via delivery of proteases attached to the matrix or via the matrix molecules themselves [49].  This matrix also contributes to the viability of the COC in the oviduct (Figure 3).

The anatomical description that follows will highlight the apical most area where rupture normally occurs in a follicle.  Specifically, this analysis will examine morphological aspects of the five main layers of the follicle wall before initiation of the ovulatory process (i.e., at 10 hours before rupture of a rabbit follicle), at approximately 1 hour before rupture, and at less than 5 minutes before rupture.

A.  Follicular Apex at the Beginning of the Ovulatory Process

1.  Surface Epithelium

            The surface epithelium is a single layer of cuboidal epithelial cells that cover the entire surface of the ovary (Figure 4).  They are loosely attached to a thin basal lamina at the surface of the collagenous connective tissue that comprises the tunica albuginea, a layer that also surrounds the ovary (Figure 5).  A dominant feature of the cells of the surface epithelium is the large granules that are normally present in their cytoplasm.  The composition of these dense granules is unknown.  Another distinct characteristic of the cells of the surface epithelium is the polymorphous nature of their nuclei.  The multiple lobes of the nuclei are more conspicuous when the tissue sections for transmission electron microscopy are cut on a plane that is tangential to the surface of the ovary (Figure 6).

            Three decades ago, Bjersing and Cajander [50, 51] concluded that the surface epithelium is the source of hydrolytic enzymes that cause ovulation.  More recently, Murdoch and others [23, 25, 28, 52, 53] have reported that the surface epithelium produces plasminogen activator (PA) to catalyze the activation of proteolysis of follicular connective tissue and cause ovulation.  However, there is no ultrastructural evidence that the surface epithelium has any role in ovulation.  In fact, this outer layer of cells is usually sloughed from the stigma region of an ovulatory follicle before it ruptures [54-56].  Furthermore, the surface epithelium can be scraped from the apical area of mature follicles, yet the follicles can still ovulate following stimulation by gonadotropin [57].  Therefore, considering the evidence that ovulation can occur in follicles that lack the surface epithelium, it is doubtful this outer layer has an essential role in the process.

2.  Tunica Albuginea

The whitish tunic of collagenous connective tissue known as the tunica albuginea forms an outer layer of protection for the ovary (Figure 4).  This tenacious tissue is the component in the apical area of the follicle wall that represents the stratum of greatest resistance to rupture.  It is composed almost entirely of fibroblasts and associated collagen fibrils that are imbedded in the extracellular matrix; the thickness of which is species dependent.  For many years, the fibroblasts were considered to be spindle-shaped smooth muscle cells that might play an active role in the mechanism of ovulation [4], but the era of electron microscopy has made it evident that the cells are fibroblasts.  If the follicular tissue is cut on a plane that is tangential to the surface of the ovary (instead of the usual cross-sectional cut), the cells in the tunica albuginea appear oval or round (Figure 7).  Upon considering this third dimension of the cells, it becomes clear that they are platter-shaped, rather than spindle-shaped.  Such a flat structure is characteristic of fibroblasts in layers of thecal connective tissue [1, 4, 58].  Also, it is easy to locate follicular fibroblasts that appear to be actively secreting tropocollagen, a molecule that rapidly polymerizes into distinct collagen fibrils when it is exposed to the ionic components of the extracellular environment (Figure 8).  Thus, the predominant cells in the tunic albuginea are fibroblasts and the depth of this layer varies among species, e.g., there is more in the human and rabbit ovary than in the rodent.

3.  Theca Externa

The theca externa is another layer of collagenous connective tissue that is somewhat similar in composition to the tunica albuginea (Figure 4).  However, this tissue is limited to the outer layer of a mature follicle and is contiguous with the tunica albuginea only at the apical most segment of the follicle wall.  One distinction between these two layers of connective tissue is that the amount of collagen in the theca externa is usually less than in the tunica albuginea [54].  This difference might be due, at least in part, to the fact the collagenous tissue of a follicle that has just reached maturity is less established than the connective tissue that surrounds the ovary.

4.  Theca Interna

            The theca interna is a thin layer of steroid-secreting cells that are supplied nutrients and oxygen by numerous large capillaries that collectively receive most of the blood from the ovarian arterial supply (Figure 4).  Fibroblasts and collagen are sparse in this thin layer just inside the theca externa [10].  The cytoplasm of these cells is dominated by lipid droplets, numerous mitochondria and Golgi networks that are distributed throughout their smooth endoplasmic reticulum.  In regard to the theca interna, it is noteworthy that it has become common practice to refer to “theca interstitial cells” [59-62], but it is not always clear whether this terminology is being applied specifically to the secretory cells of the theca interna, to cells of both the theca interna and theca externa, to interstitial cells in the ovarian stroma, or to indefinite cells in all of these areas.  In any case, since ultrastructural studies have established that the theca interna consists primarily of distinct, steroid-secreting cells (along with capillary endothelial cells), this chapter will use the terminology theca interna cells in reference to the steroid-secreting cells that are characteristic of this layer.

5.  Stratum Granulosum

            The innermost layer of follicular cells is the stratum granulosum, which extends inward from a basement membrane (i.e., the membrana propria) at the inner margin of the theca interna (Figure 4).  The first layer of granulosa cells adjacent to the membrana propria are more columnar in shape, while the remaining cells that extend toward the follicular antrum are cuboidal.  Collectively, the cells of the stratum granulosum are metabolically integrated by an extensive labyrinth of gap junctions that couple this layer into a syncytium (Figure 9).  It has been suggested that the ovulatory surge in gonadotropins that react with the abundance of G-protein-coupled receptors on the granulosa cells might initiate action potentials that are propagated via the gap junctions throughout this layer [63].  In any case, the granulosa cells appear to be the primary site of onset of the ovulatory process.

6.  Cumulus granulosa cells and oocyte microenvironment

            The cumulus granulosa cells (i.e. those surrounding and directly in contact with the oocyte) comprise the innermost microenvironment of the follicle.  Cumulus cells have specific functions and exhibit specific patterns of gene expression in the ovulating follicle [46,47].  In particular, cumulus cell expression of the hyalruonon rich matrix and hyaluronan binding molecules and inflammation-related products during COC expansion is essential for release of the oocyte (Figure 3) and will be discussed in more detail below.

B.  Follicular Apex at less than One Hour before Rupture

            Within one hour from rupture, there are conspicuous changes in the ultrastructure of an ovulatory follicle (Figure 10).  The cells of the surface epithelium develop necrotic-like vacuoles in their cytoplasm, and they appear to be less firmly attached to the tunica albuginea.  The fibroblasts in the tunica albuginea and theca externa are more elongate and appear to have transformed from quiescent to motile cells.  In the theca interna, the secretory cells look basically the same as earlier, but the adjacent capillaries contain more leukocytes and the lumenal surface of the capillary endothelial cells have blood platelets adhering to them (Figure 11).  As the apical region of the follicle wall balloons and the tissue begins to dissociate, the wall becomes thinner.  This stretching of the apex of the follicle results in sloughing of some of the granulosa cells into the follicular antrum.  A striking new development in the cytoplasm of the granulosa cells is the formation of lipid droplets, which suggests that the cells have become more active steroidogenically during the hours preceding follicular rupture.

C.  Follicular Apex at less than Five Minutes before Rupture

            Shortly before a follicle ruptures, only traces of the surface epithelium remain clinging to the disintegrated tunica albuginea at the apex of the follicle (Figure 11).  The extracellular matrix of the tunica albuginea and theca externa is sparse, and the fibroblasts in these layers are quite dissociated from one another.  The fibroblasts appear to be more motile and occasionally they exhibit amoeboid-like movement around extravascular red blood cells (Figure 12). The capillaries in the theca interna usually have either ruptured or contain thrombi that impair blood flow in the local area.  By this stage, essentially all of the granulosa cells have sloughed into the follicular fluid, or have retracted toward the base of the stigma that oftentimes forms during this final phase of the ovulatory process.  Rupture ultimately occurs at the apical most area of the follicle simply because this is, morphologically, the thinnest point along the segment where a mature follicle is contiguous with the tunica albuginea at the ovarian surface.

III.  BIOCHEMICAL REACTIONS IN OVULATION

A wide variety of biochemical studies related to ovulation have been conducted during the past several decades.  Many of these investigations have explored the possibility that mediators of inflammatory reactions are components of the ovulatory process [5, 6, 10].  While this approach has provided useful information, it has not been as unbiased or as definitive as the recent molecular methods that have detected precise changes in gene expression and identified protein products of ovulation-related genes.  However, the current status of the knowledge that has been gained from modern molecular biology will be discussed in detail later in this chapter.  This section of the chapter on ovulation will summarize information about the three groups of biochemicals that have received the most attention during the past one-half century, namely progesterone, prostaglandins, and proteases (and related hydrolytic enzymes) (Figure 13).

A.  PROGESTERONE AND OVULATION

            Progesterone was initially associated with the corpus luteum because of the enormous production of this steroid in lutein tissue after ovulation.  However, it is now clear that the ovary begins to produce a significant amount of progesterone within only a few hours after the ovulatory process has been initiated by gonadotropin (Figure 14) [64-68].  In essence, what this means is that the luteinization process also begins from the time a mature follicle is stimulated by luteinizing hormone (or its equivalent).  Thus, in a certain sense, the ovulatory process can be considered as an initial phase of the protracted luteinization process [1, 7].  However, if the luteinization events precede those of ovulation or if the timing is altered, luteinization can proceed without ovulation and result in corpora lutea with entrapped oocytes.  Several mutant mouse models have this type of anovulatory phenotype; including mice null for the progesterone receptor (PR) [69], cyclooxygenase-2 (COX-2) [70], the prostaglandin E2 receptor subtype EP2 [71], phosphodiesterase 4 (PDE4) [72], CAAT enhancer binding protein beta (C/EBPbeta) [73] as well as cyclin D2 [74]  (Figure 15).  That is to say, follicular rupture is a biologically programmed morphological phenomenon that must occur at a precise interval during the transformation of an ovarian follicle into a progesterone-producing corpus luteum.

            The sites of ovulation-related progesterone synthesis are the theca interna and the granulosa layer [75-77] where most of the LH/hCG receptors have been identified [78-83].  Additionally, at the time of ovulation, cumulus cells also make progesterone, in response to either external or COC-derived factors [84, 85]. Predictably, in the granulosa and theca cells, the activated G-protein-coupled LH receptor initiates cyclic AMP signal transduction pathways that mediate progesterone synthesis and ovulation [86-91].  This increase in progesterone synthesis requires the transport of free cholesterol to mitochondria where P450scc and 3b-HSD sequentially convert the cholesterol to pregnenolone and progesterone, respectively [92-94].  The shuttling of cholesterol from the cytoplasm into mitochondria for steroidogenesis is highly dependent on steroidogenic acute regulatory protein (StAR) [93, 95-98].  In addition, the transfer of electrons to the mitochondrial forms of cytochrome P450 during steroid hormone synthesis is dependent on the iron-sulfur protein adrenodoxin, which is normally expressed in conjunction with P450scc and StAR [76, 99, 100].  By a mechanism that is unclear, once progesterone synthesis has been established, persistent steroidogenesis during the early stages of luteinization does not require sustained LH/hCG stimulation of the follicle or constant cyclic AMP generation in the follicle [77, 101, 102].  Hence, spontaneously luteinized granulosa cells in culture can be maintained for weeks without adding LH or cAMP derivatives.  However, the lengthy luteal phase of ovarian function in vivo is maintained by factors and hormones that are species specific.  Although LH is the luteotropin in many mammals, rodents such as the mouse and rat utilize prolactin or prolactin-like molecules and steroid receptors to maintain the corpus luteum and pregnancy (see Chapter XX).

            Besides initiating ovarian progesterone synthesis, the LH surge concomitantly induces the expression of mRNA for progesterone receptor (PR), a nuclear receptor transcription factor [7, 103-106].  PR mRNA and protein expression appears to be upregulated specifically in the granulosa cells of mature follicles in response to LH or hCG [103-107] (Figure 16).  The gonadotropin activates Sp1/Sp3 binding sites within the mouse PR proximal promoter, but the molecular mechanisms by which this activation occurs is unknown [108].  Nonetheless, it is clear that the ovulatory effects of the gonadotropin surge are mediated at least in part by induction of the PR, because mice lacking PR fail to ovulate [105] (Figure 16).

            During the first several decades after its discovery, progesterone was not considered as a component of the ovulatory process.  Then, in 1961, Frederick Hisaw [109] stressed during the proceedings of a conference that the accumulation of progesterone over estrogen “might take a responsible part in the process of ovulation.”  Eventually, the experiment that firmly established a role for this steroid was conducted by Snyder et al. [110] who clearly demonstrated that the inhibition of follicular steroidogenesis and ovulation by epostane could be overcome by treatment of the experimental animals with exogenous progesterone.  Subsequently, it was reported 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 its anti-steroidogenic effect is transient, and that ovarian progesterone synthesis rises back almost to normal levels by the time follicular rupture would have normally occurred in the animals that received an ovulation-inhibiting dose of epostane [68,].  Thus, the evidence 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.  However, in spite of this obvious participation of progesterone in the mechanism of ovulation, treatment of animals or follicles with progesterone, alone, does not induce follicular rupture [111, 112].  That is to say, the ovulatory surge in LH (or the injection of an analog of LH) induces other metabolic changes (e.g., the induction of PR) that are just as obligatory for ultimate rupture of the follicle [69, 103-105]

The specific action of progesterone in ovulation had been rather uncertain until recently.  It is now clear that the LH surge induces transcription and translation of the ovarian gene for a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-1), the expression of which is dependent not only on LH but also on increases in both progesterone [113] and PR [105, 106] (Figure 17). For example, the inhibition of ovarian progesterone synthesis and ovulation by epostane also involves inhibition of ovarian ADAMTS-1 expression, and this interference can be overcome by treating the animals with exogenous progesterone [113].  Furthermore, genetically deficient mice that lack PR fail to ovulate [105] (Figure 13).  ADAMTS-1 was first discovered in association with inflamed intestinal tissue [114], but a non-steroidal anti-inflammatory drug such as indomethacin (which can inhibit ovulation) does not block expression of the ADAMTS-1 gene during ovulation [113].  Nevertheless, ADAMTS-1 appears to be an important enzyme for ovulation, and it is currently being studied more extensively [115-118].  One known substrate of ADAMTS-1 is versican, a hyaluronan (HA) binding proteoglycan, that like HA is induced in granulosa cells of preovulatory follicles by LH [116].  The exact function of these inflammation-related matrix components in the ovulation process and the obligatory requirement for ADAMTS-1 remain to be determined.  However, it is clear that the ovulation cone (stigma) contains vascular elements (Figure 18) and exhibits intense localization of ADAMTS-1 and ADAMTS-4 (Figure 19).   These results indicate that these two ADAMTSs might be involved in regulating angiogenic activity (49).  In addition, since ADAMTS-1 localizes to the expanded COC matrix, it may contribute to a protective shield around the oocyte (Figure 20).  In addition, recent observations in the porcine ovary indicate that PR antagonists can disrupt expression of ADAMTS-1 and cumulus expansion in cultured COCs [119].  As noted below, several structurally-related ADAMTS proteases are expressed in the ovary and may have either specific or redundant functions [49,115].  In addition to progesterone action on ADAMTS-1 expression, this important steroid is involved in regulating ovarian expression of cathepsin L (cat L), which is an elastinolytic cysteine protease [43, 105, 120], and the action of this enzyme presumably contributes in some way to proteolytic degradation of the follicle wall.  Also, ovarian expression of rat pituitary adenylate cyclase activating polypeptide (PACAP), which is structurally and functionally related to vasoactive intestinal peptide (VIP), is dependent on the ovulatory increase in progesterone [44, 121, 122].  However, the function of PACAP in ovulation has not been well defined.  Most likely, other genes regulated by PR will be identified in future studies.

B.  PROSTAGLANDINS AND OVULATION

            Prostaglandins are produced from arachidonic acid, which is formed from membrane phospholipids in response to virtually any type of environmental condition that disturbs the plasma membrane [123].  These molecules are classically associated with sites of inflammation and thus their presence in ovulating follicles lends credence to the idea that ovulation is an inflammatory-like process.  The first indication that prostaglandins might be generated in the ovary in response to an ovulatory surge in gonadotropin arose circuitously from five simultaneous reports that indomethacin, a nonsteroidal anti-inflammatory agent that is commonly used to inhibit prostaglandin synthesis, could prevent ovulation when administered to various laboratory animals [124-128].  Shortly thereafter, LeMaire, Marsh and coworkers [129, 130] measured a marked increase in prostaglandins E2 and F2a in rabbit and rat follicles during the ovulatory process, and their observations have been confirmed many times.  In the immature rat model, ovarian prostaglandin levels begin to increase sharply at 3-4 h after the administration of hCG to induce ovulation, and then they start to decline even before the follicles actually rupture [66, 131].  Characteristically, prostaglandin of the E-type is about twice as concentrated as the F-type in the ovary during ovulation.

            The prostanoid pathway by which arachidonic acid is metabolized to bioactive prostaglandins is regulated primarily by cyclooxygenase (COX) enzymes (also known as prostaglandin H2 synthases) [132].  COX-1 is constitutively present in many tissues, including the ovary, but it is not upregulated in response to the LH/hCG surge [133-136].  In contrast, COX-2 is an inducible form of the enzyme, and in the ovary it is expressed in ovarian granulosa cells and cumulus cells after the LH/hCG surge in both rat and mouse models [133-139] (Figure 13, 20).  The enzyme is translated from COX-2 mRNA, which is undetectable in mature follicles that have not yet been stimulated to ovulate.  However, by 4 hours after the administration of an ovulatory dose of hCG to immature rats and mice, the expression of mRNA for COX-2 reaches a peak and then decreases dramatically in granulosa cells by 6 hours [137, 139]. However, the peak in ovarian COX-2 protein that enzymatically converts arachidonic acid to the various prostaglandins lags about 1-3 h behind COX-2 mRNA expression, and the enzyme is still present at elevated levels at 12-14 h after hCG at the time when follicles begin to rupture, but also at a time when COX-2 mRNA in the granulosa cells is low [67, 131, 139]. However, COX-2 is highly expressed in the COCs immediately prior to ovulation (12 hours post-hCG) as well as in ovulated COC within the oviduct (16 hours post-hCG) as well as in COC exposed to hormone in culture (Figure 21) [137, 138, 140]. Collectively, these observations indicate that newly translated COX-2 enzyme persists in an active form in the ovary for several hours after the COX-2 mRNA has been down regulated in granulosa cells and that other mechanisms in conjunction with the LH surge control the expression of COX-2 mRNA and protein in the cumulus cells prior to and after ovulation.  Most recently, the EGF-related factors amphiregulin, epiregulin and betacellulin have been shown to regulate COX-2 message in cumulus cells [72].   Of relevance, the expression of COX-2 in the COCs closely mirrors that of HAS-2 in the mouse [140] and likely in other species [141-143].

            On a related note, it has been hypothesized that there is a possible correlation between the time of peak expression of COX-2 and the species-specific duration of the ovulatory process [143-145].  Limited data from the rat, cow, mare and monkey indicate that in all of these species there is approximately a 10-hour interval between the onset of ovulation-related ovarian prostaglandin synthesis and the time of follicular rupture, even though the length of the ovulatory process varies from a low of 12-14 hour in the rat to a high of 32-40 hour in the monkey.  That is to say, the duration of the ovulatory process in a given species might be a function of the interval between the LH/hCG surge and the time of onset of ovarian COX-2 mRNA expression.

            Some of the components of the complex signaling pathway that begins at the LH/hCG receptor on the surface of granulosa cells and ends with the expression of COX-2 to generate bioactive prostaglandins have been determined.  The LH/hCG receptor is coupled to a Gs-protein that naturally activates adenylyl cyclase, which catalyzes the formation of cyclic AMP and leads to transient activation of protein kinase A [134, 144, 146].  Other elements of the signaling pathway have not been deciphered, but it is known that the promoter of the rat COX-2 gene contains a CAAT/enhancer binding protein (C/EBP) consensus site, and CEBPb mRNA and protein are induced rapidly in granulosa cells in vivo following an ovulatory dose of hCG [144, 146, 147].  Moreover C/EBPb KO mice are infertile and exhibit impaired ovulation [73].

            It is generally accepted that prostaglandins have a fundamental role in the mechanism of ovulation [10, 15, 43, 47, 146].  However, even though ovarian prostaglandins have been studied almost as extensively as progesterone, their functions in the rupture of a follicle are not as well established as is their critical role in COC expansion [46, 47, 138, 140, 150].  The expansion process is essential for release of the oocyte from an ovulatory follicle as confirmed by results in four mutant mouse models, namely COX-2 [70]; EP2 [71]; IaI [148, 149] and TSG-6 [150].  Suggested roles for the prostaglandins have included wide-ranging ideas such as activation of follicular proteolytic enzymes [151], stimulation of follicular angiogenesis [152] and promotion of nitric oxide synthase activity [153].  A promising new experimental approach that might help elucidate the role of prostanoids in ovulation is the assessment of specific prostaglandin receptors.  It has been reported recently that, 3 hours after the injection of hCG into mice, ovarian expression of mRNAs for the two Gs-coupled prostaglandin E2 receptors (i.e., EP2 and EP4) increased significantly in both granulosa cells and cumulus cells [154].  Furthermore, mice that are null for either COX-2 or EP2 exhibit impaired ovulation [70, 71] and have reduced expression of mRNA for tumor necrosis factor-stimulated gene-6 (TSG-6) [138].  TSG-6 is a hyaluronan-binding protein that is expressed temporally and spatially in granulosa cells and cumulus cells with hyaluronan synthase-2 (HAS-2) [140] (Figure 22).  In the follicle, the long hyaluronan polymers provide the structural backbone of the extracellular matrix surrounding the oocyte and appear to be stabilized by various hyaluronan binding proteins (Figure 20).  TSG-6 is one of these factors.  TSG-6 is also essential for delivery and covalent linkage of the heavy chains of the serum-derived protein inter-alpha trypsin-inhibitor (IaI) to hyaluronan.  Therefore, it has been proposed that one possible function of prostaglandin E2 is the induction of ovarian TSG-6 expression during the ovulatory process and that TSG-6 along with IaI stabilizes the COC matrix.   Proof of this hypothesis has been obtained by several approaches. Mice null for TSG-6 or IaI exhibit impaired ovulation that can be reversed by providing exogenous TSG-6 or IaI, respectively [148-150].  To support these in vivo data, FSH-induced expansion of COCs in culture can be disrupted by peptide specific antibodies to TSG-6 [138]. Collectively, these data indicate that the formation and stabilization of the expanded matrix is critical for oocyte release.  It is possible that the formation of expanded matrix with all of the attached HA binding proteins is essential to provide a protective shield around the oocyte. Stabilization of this hyaluronan COC-derived matrix is also essential following ovulation and is dependent on the oocyte-derivied factors GDF-9/BMP15 and their induction of pentraxin-3, an inflammation-related factor that binds specifically to TSG-6 [155. 156].  The effects that the matrix exerts on the surface epithelium or other components of the rupture process remain to be determined. However, an inviting hypothesis is that the matrix around the cumulus-enclosed oocyte plays a significant role in the final steps of ovulation, perhaps by delivering proteases such as ADAMTS-1 and ADAMTS-4 to the surface of the ovary or by providing a protective shield for the oocyte. In any event, further information about other downstream responses to activation of the prostaglandin receptor will improve the chances of deciphering other basic role(s) of prostaglandins in the mechanism of ovulation.

            Experiments that assess the metabolic effects of inhibitors of prostaglandin synthesis also have the potential of providing information about the function(s) of prostaglandins in ovulation.  For example, it has been reported that indomethacin significantly decreases the ability of interleukin-1b (IL-1b) to upregulate ovarian IL-6 transcripts in dispersed cells taken from whole ovaries of rats, and this inhibitory action could be overcome by addition of prostaglandin E2 to the cultured cells [157].  Furthermore, recent experiments with indomethacin continue to support the earlier evidence [10] that indomethacin can inhibit ovarian prostaglandin synthesis and impede ovulation without obstructing ovarian progesterone synthesis and luteal development [158-160].  Thus, it is clear that the syntheses of prostaglandin and progesterone in ovulatory follicles are two distinctly independent phenomena that activate different genetic programs that converge on the COC matrix formation and function (Figures 12, 15, 20).

            Like the inhibitory action of epostane on ovarian progesterone synthesis, the inhibitory effect of indomethacin on ovarian prostaglandin synthesis has served as an interesting subject for ovulation-related research.  The drug has a very rapid effect on ovarian prostaglandin metabolism.  When indomethacin is administered intravenously during the ovulatory process, the normally elevated levels of prostaglandins E2 and F2a decline to almost naught within only a few minutes [161].  However, in spite of the powerful inhibitory action of indomethacin on prostaglandin synthesis, it is intriguing that excessively high doses of this non-steroidal anti-inflammatory drug still permits the rupture of a limited number of follicles [161-163].  Even more puzzling is the observation that relatively low doses of indomethacin that significantly inhibit the normal increase in ovarian prostaglandins during ovulation have no significant effect on the ovulation rate in rabbits and rats [161-163].  This anomoly might be explained by the fact that induction of COX-2 mRNA and protein in the COCs is sustained at 12-16 hours post hCG and may not be inhibited easily by entrance of the drug into the follicle [137]. However, it is relevant to note that indomethacin is not as specific of an inhibitor of COX-2 as originally thought.  There is evidence that this drug also affects the activity of lipoxygenase enzymes that convert arachidonic acid into biologically important leukotrienes and lipoxins [163-165].  In view of this information, a more thorough analysis of the potential roles of ovarian lipoxygenase products in the ovulatory process is warranted.

 

C.  PROTEASES AND OVULATION

            In 1916, Irving Hardesty first suggested to Schochet [166] that the ovarian follicular fluid might “exert some special digestive action on the resisting tissues” of the follicle wall.  However, after a number of unsuccessful attempts during the next three decades to demonstrate a role for proteases in ovulation, it was concluded that the ‘enzyme theory’ did not fit the facts and that the immediate cause of ovulation was a mystery [167].  During the next two decades, several additional attempts to detect ovarian proteolytic activity did not yield convincing results [10].  Then, in the mid 1960s, it was demonstrated that small quantities of collagenolytic enzymes could be injected into rabbit follicles and cause rupture (but not necessarily oocyte release) [168], and that the collagenous tissue in the follicle wall of sows became weaker at the time of ovulation [169].  Since that time, most of the work on the so-called ‘enzyme theory’ of ovulation has focused on assessments of the potential roles of PA, matrix metalloproteinases (MMPs), and ADAMTS enzymes.

1.  Plasminogen Activators

PA is a serine protease that has been implicated in many types of tissue degradation [170], and it is secreted from fibroblasts in association with collagenase activity [171].  PA of the tissue type (tPA) was first studied in relation to ovulation in 1975 by Beers, et al. [172].  However, in the first two decades of research on the potential role of this protease in ovulation, there was considerable incongruity among the various studies [10].  It has been concluded that this enzyme is mainly in the granulosa, in significant amounts in the theca interna, or especially abundant in the fibroblasts in the apical follicle wall.  There were contradictory reports that granulosa cell production of tPA is stimulated chiefly by prostaglandins of the E type or chiefly by prostaglandins of the F type.  Also, there were conflicting reports that indomethacin treatment of experimental animals suppresses the secretion of tPA, or that indomethacin has no apparent effect.  There were differing reports on whether progesterone has a minor role or a significant role in the expression of ovarian tPA activity.  Thus, there has been considerable confusion about ovarian tPA in the past, and the most recent decade of research on this enzyme has not established the significance of this enzyme in ovulation.  The data on ovarian expression of tPA inhibitor type-1 (PAI-1) is equally confounding.  It has been reported both that PAI-1 is expressed in the rat ovary during the ovulatory process [173] and that it declines dramatically in the mouse and monkey ovary as the time of ovulation approaches [62, 174].

            It has been reported that the urokinase type of PA (uPA) from ovarian surface epithelial cells is important for ovulation [28], and TNF-a and IL-1b have been found to drastically increase the secretion of this enzyme from ovarian surface epithelial cells [175].  However, as stated earlier in this chapter, during the ovulatory process the surface epithelium sloughs from the apex of the follicle and is not attached to the ovary at the site of rupture.  Therefore, this thin layer of cells does not appear to be essential for ovulation.  Nevertheless, there continue to be reports that the tissue type variety of PA is generated in the granulosa cells (and surrounding connective tissues) of rats [173] and monkeys [61], and that such activity is important for follicular rupture.  Yet, mice lacking tPA and/or uPA gene function exhibit little or no impairment of ovulation efficiency [176, 177], and plasmin does not appear to be required for follicular rupture in mice [178].  Thus, the issue of whether PAs are important for ovulation would seem to be immaterial except, perhaps, for the evidence in other experimental models that such enzymes have a central role in activation of the four classes of inflammatory MMPs discussed in the following paragraph [179].

2.  Matrix Metalloproteinases

            Most of the original work on ovarian MMPs and tissue inhibitors of MMPs (TIMPs) has been carried out by Curry et al. [26, 29, 61, 180-182].  The four categories of MMPs that have been studied in ovarian tissues are the collagenases, gelatinases, membrane-type MMPs, and stromelysins.  The granulosa cells of ovulatory follicles appear mainly to transcribe and translate MMP-2 [182, 183] and MMP-19 [184], whereas thecal and stromal tissues are associated with gonadotropin-induced elevations in collagenase-3 [185], MMP-2 [182, 183], MMP-9 [26, 182, 183], and MMP-19 [184].  However, a recent study of MMPs in equine follicles failed to detect any increase in MMP-2 or MMP-9 in follicular fluid or in explant cultures of stromal tissue at the time of ovulation [186].  The conclusion from this latter report was that MMP-2 and MMP-9 are not key regulators for the changes in follicular shape immediately prior to ovulation.  Another recent study of MMP-2 and MMP-9 in human follicular fluid yielded too much variability among the samples to clarify whether these proteases change significantly in the ovary during the ovulatory process [187].  A novel approach to the assessment of MMPs in ovulation is the recent study of membrane type-1-MMP (MT1-MMP; MMP14) in ovulatory follicles [61].  MT1-MMP hydrolyzes type I collagen, and it cleaves pro-MMP-2 in order to render it active [188, 189].  In view of this action, it is relevant that high expression of MT1-MMP mRNA occurs in the “theca-interstitial layer” of rat follicles near the time of ovulation [61].  However, on a cautious note, there is a recent report that the phenotype of MT1-MMP deficient mice does not entirely mimic that of mice deficient in MMP-2 [190], suggesting that MT1-MMP might have functions other than the activation of pro-MMP-2.  Nevertheless, it appears likely that MMPs have a significant function in the degradation of the collagenous connective tissue in the follicle wall.  That MMP2 and MMP9 null mice are fertile and ovulate may indicate that these two proteases have some overlapping and redundant functions in the ovulating follicle [191]. It is also worth noting that the expression patterns and activation of MMP2 and MMP9 are normal in PRKO null mice that fail to ovulate [105].

3.  ADAMTS Enzymes

            ADAMTS-1 and other members of this novel family of metalloproteinases differ from the usual MMPs by the fact that they are readily secreted and thereafter either bind extracellular matrix components, or attach to the cell surface via specific regulatory mechanisms [114, 192].  By means of the differential display method for gene discovery, ADAMTS-1 mRNA was first detected in rat ovarian tissue by Espey, et al. [113].  This initial report revealed that ADAMTS-1 mRNA is expressed primarily in the granulosa cells of ovulatory follicles, that transcription of the mRNA is significantly impaired by epostane treatment of the animals, and that injection of progesterone to the experimental animals overcomes the inhibitory action of epostane.  Shortly after this demonstration that ADAMTS-1 gene expression is dependent on progesterone, it was shown that ovulatory expression of ADAMTS-1 mRNA is also dependent on PR, and that expression of ADAMTS-1 mRNA and ovulation is diminished significantly in PR knockout mice [44, 105, 106, 116] (Figures 15 and 16).  It should be noted that, in addition to the substantial expression of ADAMTS-1 mRNA in the stratum granulosum of ovulatory follicles, ADAMTS-1 is expressed in varying degrees in cells in the thecal connective tissue and ovarian stroma [115, 117 253]. In granulosa cells, the ADAMTS-1 protein normally increases greater than 10-fold during the ovulatory process, but less in PR knockout mice [116]. It appears that one function of this enzyme in ovulation might be to cleave versican in the expanded cumulus-oocyte-complex. ADAMTS-1 may also provide antiangiogenic effects to prevent vascularization of the expanded COC matrix that would impede release of this complex. A more comprehensive study of the ADAMTS family of metalloproteinases in the bovine ovary has revealed that more than one ADAMTS subtype might be involved in the ovulatory process [49, 115].  In the dominant follicle of the cow ovary, the ovulatory surge in gonadotropins induces an increase in ADAMTS-1, -2, and -5 mRNA levels in the granulosa cells and ADAMTS-1, -3, and -9 mRNA levels in thecal cells.  In the mouse and rat ADAMTS-4 and ADAMTS-9 also increase in granulosa cells and theca cells of preovulatory follicles following administration of hCG, whereas ADAMTS-5 does not [49].  ADAMTS-19 is highly expressed in the embyonic gonad [193]. Thus, the regulation of ADAMTS expression in the ovary is complex, and this family of novel enzymes will undoubtedly be investigated in greater detail in future studies on ovulation.

IV.  OVARIAN GENE EXPRESSION DURING OVULATION

            Over the years, there have been innumerable attempts to identify the basic biochemical components of the ovulatory process.  Most of the efforts to differentiate the principal mediators of this process have been conducted under the assumption that ovulation might be a relatively straightforward series of chemical reactions—involving a reasonably small number of regulatory factors that are activated by an ovulatory surge in gonadotropin.  However, this simplified notion was dispelled by the hypothesis that the biochemical events of ovulation are comparable to an inflammatory process [5].  Since the introduction of that provocative idea 25 years ago, there have been a number of discoveries of ovarian agents (e.g., various cytokines that are well known components of acute inflammatory reactions), which are now considered to be important factors in ovulation [7, 36, 46].  Still, the supposition that has driven much of the research since 1980 has been that appropriate experiments would elucidate uncomplicated signaling pathways starting with G-protein-coupled receptors for LH/hCG and leading to activation of kinases that could promote transcription and translation of one or more collagenolytic enzymes to degrade the follicle wall and cause rupture.  The greatest experimental challenges appeared to be merely the identification of one or more ovulation-specific collagenases and the delineation of the roles that ovarian prostaglandins and progesterone play in the expression of such enzymes.  Although a number of studies have provided useful information about the signaling pathways and the collagenolytic enzymes that likely play a critical role in ovulation, progress toward a comprehensive picture of the ovulatory process has been slow.

            The recent development of novel molecular techniques to detect tissue-specific gene expression has promoted greater expectations of gaining a more precise understanding of the biochemical changes that occur in the ovary at the time of ovulation.  However, in spite of an exponential growth in the number of ovulation-related genes that have been discovered during the past decade, the contemporary image of the ovulatory process seems to have become more convoluted rather than clearer.  Therefore, the aims of the present section of this chapter are to identify some of the reasons for this unexpected complexity, to catalog most of the ovulation-related genes that have been reported to date, and to briefly summarize the state of our current knowledge about gene expression pathways (i.e., gene cascades) in the ovary at the time of ovulation.  The genes listed in Table 1 include those that are induced by the ovulatory surge of LH/hCG.  It excludes the genes that are expressed in the proliferative phase of follicular growth or are turned off as a consequence of the ovulatory surge in gonadotropin.  Although the down-regulation of certain genes may be important for the transition of an ovulatory follicle into a viable corpus luteum, for reasons of simplicity these genes have not been included herein. 

A.  COMPLEXITY OF INTEGRATING GENE EXPRESSION DATA

            The scope and diversity of genes that have been described as participants in the ovulatory process were quite unpredictable.  To date, there are at least 85 ovulation-related genes that have been reported (Table 1), and this compilation probably excludes some genes that were inadvertently overlooked within the voluminous literature.  (The term ovulation-related is meant to indicate there is direct or indirect evidence to suggest that the gonadotropin surge induces expression of a given gene that is considered to be a contributing factor to the interwoven processes of ovulation and luteinization.)  Ideally, it would be nice if each of the newly discovered ovulation-related genes could be assigned to a known position in one of the established cascades of gene expression in some complex process such as an acute inflammatory reaction.  In some cases, as with the ILs and TNF-a, this has been feasible.  But, for most of the genes that have been discovered, it has not been possible to relate them to a common pathway.  Thus, the mounting challenge in the field of ovulation research is the task of delineating meaningful relationships among the numerous genes and their protein products.

            The magnitude of the mission of integrating all of the data is not merely a matter of managing the ever-expanding number of genes that have been linked to ovulation.  There are certain problems inherent in the multiplicity of experimental designs that are currently being used to identify ovulation-related genes.  First, it is difficult to place the individual genes into a chronological order of expression because the diverse experimental organisms have included rats, mice, horses, cows, monkeys and humans, which have as much as a 3-fold variation in the lengths of their ovulatory processes—ranging from 12 hours (in the rodent; i.e rat and mouse) to 36 hours (in the human).  Second, a number of the studies have been based on a very limited number of time-points during the ovulatory process (e.g., a 0-hour control group and only a 12-hour experimental group in rat experiments) that detected gene expression, but did not reveal the time of onset of expression of the given gene.  While the detection of any previously unidentified gene is certainly important, a single time-point does not allow assignment of a precise time of onset of expression of the gene in any kind of meaningful chronological order in relation to other ovulation-expressed genes.  Third, some of the work has been conducted in vitro on cultured granulosa cells, or on cells of the theca interna, or on other cell types in the ovary.  While this sort of single-cell study can provide certain useful information, the utility of the information is limited by the fact that the data does not reveal whether the expression of a given gene extends beyond the individual cell type.  Furthermore, the spatial demarcation of ovarian gene expression is difficult to decipher from some reports because of inconsistent use of terminology, e.g., the use of “ovarian interstitial tissue” versus “ovarian stroma”, or merely referring to “thecal cells” rather than “theca interna cells” or “theca externa cells.”  Thus, the typical diversity of experimental designs in different research laboratories is a certain impediment to integrating the information from different reports on assorted genes.

            One of the original objectives of this chapter was to integrate as much of the data as possible in Table 1 into one grand schematic that would cover all of the principal pathway(s) of ovarian gene expression during the ovulatory process.  However, that endeavor was reduced to the schematic (Figure XX) presented above on the progesterone and prostaglandin related cascades in part because the problems identified in the previous paragraph quickly became apparent, and in part because too many of the genes simply do not fit into any of the established cascades of gene expression.  Before other investigators attempt to integrate the information in Table 1 into a comprehensive flow chart, they are encouraged to examine (as starting points) several preliminary attempts by Espey et al. [7, 45] to categorize at least some of the genes in Table 1, and a number of efforts by Richards et al. [41-44, 46, 47, 74, 300] to schematically depict the sequential and spatial relationships among small clusters of these genes.  In such efforts to assemble the growing information into more meaningful graphics, a paradoxical question that arises is whether sufficient numbers of the full compliment of genes have been discovered to complete the picture.  The current data from random methods of gene discovery, such as the differential display procedure [45], tends to suggest there might be hundreds of additional ovulation-related genes that remain to be identified.  This deduction is based on the awareness that the last 20 genes that have been detected by the differential display procedure have less than a 25% chance of having been discovered previously (unpublished observation).  That is to say, as many as 75% of the genes that are actually involved in ovulation might still remain to be discovered.  Therefore, in metaphorical terms, the question is whether enough of the pieces of the jigsaw puzzle have been placed on the assembly table to begin a serious effort to unite those pieces.  The optimum time to assemble a comprehensive flowchart might not have arrived, yet.  Such an endeavor would be facilitated by a more complete list of the relevant genes, by a greater understanding of the functional relationships among those genes, and by more experience with the new computer software that is just becoming available to assist in literature searches that can better establishment connections among the diverse genes that have been detected so far.  In the meantime, it is the aim of this chapter to summarize pertinent information about most of the genes that have been discovered to date.

B.  CATALOG OF CURRENT GENES

To conserve space, some of the closely related genes that are listed in Table 1, are considered together in this section.

 

1.  ADAMTS Enzymes

            The ADAMTS family of enzymes consists of at least 20 secreted proteins that have multiple domains with disintegrin and metalloproteinase activities that are involved in inflammation, angiogenesis, development and coagulation [115, 301- 303].  The diverse members of this family have variable substrates, including aggrecan, brevican, versican, procollagen I and procollagen II [115, 116, 118, 302].  During the ovulatory process, a number of the ADAMTS genes are expressed in different areas of the ovary and the ovarian follicle, but the most abundant transcription appears to be in the stratum granulosum (see Table 1) [105, 106, 113, 117].  It is clear that at least ADAMTS-1 is dependent on the ovulatory increase in LH, ovarian progesterone and PR [105, 106, 113].  Moreover, mice null for ADAMTS-1 exhibit impared ovulation [118]

2.  Adrenodoxin

            ADX is a soluble, cAMP-regulated ferredoxin that transports electrons from NADPH-dependent adrenodoxin reductase to P450scc as well as to several other mitochondrial cytochromes that are involved in the steroid biosynthetic pathway [99, 304-306].  The expression of this electron carrier is upregulated mainly in the granulosa layer of rat follicles that have been stimulated with hCG [45].  Ovarian ADX follows a temporal and spatial pattern of expression that is parallel to the expression of StAR (another steroidogenic regulatory molecule) during the hours of the ovulatory process; however, after a follicle ruptures, ADX transcription down-regulates back to its 0-hour control level while StAR continues to increase during the luteinization process [45].

3.  5-Aminolevulinate Synthase

            ALAS catalyzes the production of 5-aminolevulinate, which also functions in the process of transporting electrons to cytochrome P450 enzymes that are involved in steroidogenesis [307, 308].  Transcription of the ALAS gene in granulosa cells is one of the earliest responses to hCG stimulation of rat ovaries [45].  ALAS expression has been associated with the acute phase response, which is a cascade of gene expression that is initiated in parallel with an acute inflammatory reaction and might function as a delay mechanism to moderate the degradative events of inflammation [309].

4.  Amphiregulin

            AR is a member of the EGF family of growth factors that are initially expressed as transmembrane precursor molecules that are cleaved by proteases to form active growth factors in the extracellular domain [310].  This particular EGF family member has been associated with the inflammatory-like reaction that occurs in the endometrial stroma at the time of embryonic attachment [311, 312].  Thus, it is interesting to note that AR is transiently expressed in granulosa cells of the mouse ovary during follicular remodeling in response to an ovulatory dose of LH [72].  Also, of note, AR null mice are fertile [194], perhaps because of redundant functions with epiregulin and betacellulin.

5.  Apolipoprotein-E

            ApoE is a 34 kDa segment of various lipoproteins that serves as the ligand for a cellular lipoprotein receptor engaged in the regulation of cholesterol transport and steroid metabolism [313-316].  The promotion of steroidogenesis by apoE appears to diminish the intensity and duration of the acute inflammatory response [314, 316].  However, the significance of apoE in ovulation is not clear, because one report claims that apoE mRNA increases in granulosa cells of rats during ovulation [195], while another study concludes that apoE mRNA is not detectable in the granulosa during ovulation in the same animal [313].

6.  Aryl Hydrocarbon Receptor

            AHR has been described as a relatively promiscuous receptor because of a structural design that allows it to be bound and activated by many divergent chemicals ranging from numerous naturally occurring endogenous ligands to a variety of manmade environmental toxins such as xenobiotics [317-319].  Once AHR is activated by a ligand, it can operate in conjunction with NF-kB as a transcription factor with the capacity to induce an array of genes associated with inflammation and/or an oxidative stress response that can either intensify or diminish the local disturbance [320, 321].  AHR is expressed in granulosa cells and the oocyte of growing follicles in the mouse [196]. In regards to ovulation, there is a significant increase in AHR mRNA in primate granulosa cells by 12 hours after the administration of hCG [197] and AHR might also have a role in rodent ovulation, although this possibility has not been explored extensively [198].

7.  Betacellulin

            BTC was initially analyzed in association with its unusually high expression by the beta cells of the pancreas [322].  Similar to other members of the EGF family of growth factors, it is proteolytically processed from a larger membrane-anchored precursor and functions as a potent mitogen when it binds to ErbB receptors that are present on a wide variety of cell types.  Like AR and EPI, BTC is expressed in ovarian follicles that have been stimulated to ovulate, and it may play a key role in cumulus expansion [72].

8.  cAMP-Response Element Binding Protein

            CREB is one of three ubiquitous genes (including CREM and ATF-1) that yield binding proteins that bond with cAMP response elements (CREs) in the promoters of various genes to stimulate transcription [323, 324].  As a component of the protein kinase A pathway, and in conjunction with glucocorticoid-induced kinase (Sgk), it appears that a transient increase in a phospho-CREB can be induced in granulosa cells under in vitro conditions that mimic the ovulatory transition to lutein tissue [199].  It is possible that the ovarian expression of CREB is involved in the regulation of Egr-1 expression in granulosa cells during the ovulatory process [325].   This of course is just one of many sites at which CREB is likely to act.

9.  cAMP-Response Element Modulator

            As mentioned in the previous paragraph, CREM is a gene that is commonly expressed concurrently with CREB in response to cAMP elevation in a cell [326].  And, like CREB, CREM is activated by PKA and binds to CRE in the promoter region of target genes.  However, in contrast to CREB, CREM is an autoregulatory gene that encodes the inducible cAMP early repressor (ICER), and this product of the CREM gene governs the down-regulation of its own expression, along with down-regulation of the expression of CREB and other early response genes [323, 327].  Thus, the gonadotropin-induced expression of ovarian CREM that encodes ICER in the granulosa cells of ovulatory follicles of rats probably represents a negative feedback mechanism to modulate the intense metabolic changes that cause the follicle to rupture [200].  The repressor action of ICER presumably serves to reduce CREB to basal levels and terminate the signaling cascade initiated by the LH/hCG-receptor-mediated elevation in local cAMP. However, despite the expression of CREM and ICER in the ovary, female CREM knockout mice are fertile whereas males are infertile [201]

10.  Carbonyl Reductase

            CBR is an aldo-keto reductase with broad specificity for converting carbonyl compounds into alcohols [328].  In the immature rat ovary, CBR mRNA is significantly elevated in ovarian thecal and stromal tissue (but not in the granulosa cells) beginning 4 hours after initiating the ovulatory process with hCG, and this increase persists through the time that follicles begin to rupture at 12 hours after hCG [202].  Another aldo-keto reductase, mouse vas deferens protein (MVDP) is also induced in granulosa cells of preovulatory follicles by LH/hCG [203].   It has been suggested that ovarian CBR and MVDP could function to reduce the local toxicity that might develop from the aldo-keto functional groups on the progesterone and prostaglandin molecules that are copiously generated in the ovary at the time of ovulation [202-203].

11.  Cathepsin L

            CatL is important for degradation of elastic tissue in the extracellular matrix during an inflammatory response [329, 330].  It is also involved in alteration of the extracellular matrix at the invasive margins of tumors, and its expression in tumors is in conjunction with a number of protease-related genes that have been associated with ovulation, including MMP-2, MMP-9, MT1-MMP, PAI-1, and several members of the ADAMTS family [331].  As mentioned earlier, ovarian expression of catL during ovulation is dependent in part on progesterone and PR as well as Sp1/Sp3 and CREB [44, 105, 120].  However, unlike ADAMTS-1 null mice, catL null mice are fertile [204].

12.  CCAAT/Enhancer-Binding Protein-b

            C/EBP-b is a bZIP transcription factor that seems to be involved in inflammatory responses to injury, as well as to target tissue responses to certain hormonal stimulation [332,333].  Interestingly, many of the AP-1 genes that require the inflammatory cytokines IL-1 and IL-6 for their induction have adjacent C/EBP and NF-kB motifs in their promoter regions, suggesting cooperation between these two families of transcription factors [332].  C/EBP-b is rapidly induced in the granulosa cells of rats after they have been injected with an ovulatory dose of hCG [147, 205, 206], but there are conflicting reports as to whether expression of this transcription factor does [147], or does not [206], have an important role in regulating the induction of proinflammatory COX-2 expression in granulosa cells during the ovulatory process.  Importantly, mice null for C/EBPb are infertile and exhibit corpora lutea with entrapped oocytes [73].

13.  CD63 (cell surface antigen)

            CD63 is a member of the tetraspanin superfamily of activation-linked cell surface antigens that is known for its abnormally high levels on the surface of activated basophils [334], on proliferating mast cells [335, 336], and on the surface of endothelial cells in inflamed tissue [336].  This antigen has also been identified as one of the two principal proteins in the membranes of so-called Weibel-Palade bodies, which are lysosome-related secretory organelles associated with inflamed endothelial cells [337, 338].  Before the induction of ovulation in immature rats, CD63 is expressed constitutively in follicular thecal cells and stromal interstitial cells, whereas expression in the granulosa layer must be induced by an ovulatory dose of hCG [45].  This unusual spatial pattern of expression of an ovulation-related gene might be associated in some way to the fact that the stratum granulosum is avascular prior to ovulation and then experiences endothelial cell infiltration during luteal angiogenesis.

14.  Corticotropin-Releasing Hormone Receptor

            CRHR was first characterized as the target for CRH, which serves as the principal regulator of the hypothalamic-pituitary-adrenal stress response axis.  However, CRHR is now recognized as a member of a growing family of CRH ligands, CRH receptors, and CRH-binding proteins that are expressed in peripheral inflammatory sites, especially in female reproductive tissues [207,339-343].  It has been more than a decade since CRHR1 was detected in ovarian stromal cells and in the thecal cells surrounding ovulatory follicles in the rat [207, 208].  The potential role of this proinflammatory factor in ovulation is worthy of further investigation, but future efforts will likely require conditional knockout approaches since mice null for CRHR1 die after birth due to lung dysplasia and adrenal abnormalities [209].

15.  Cutaneous Fatty Acid Binding Protein

            C-FABP was named because of its induction in epidermal tissue following skin irritation and cutaneous inflammation, and because of its roles in the synthesis and transport of fatty acids [344, 345].  C-FABP mRNA is expressed in the thecal layer (i.e., in the ‘skin’) of follicles in the ovaries of gonadotropin-primed rats that have been injected with hCG [210], and the significance of expression of this binding protein during the ovulatory process also deserves further evaluation.

16.  Cyclooxygenase-2

            COX-2 is the readily-inducible, rate-limiting enzyme for the conversion of arachidonic acid to prostanoids in most cells and tissues that have been activated by inflammatory cytokines, trophic hormones, or tumor promoters [346-349].  Over the past three decades, ovarian COX-2, along with PGE2 and PGF2a, has received enormous attention in a wide variety of ovulation studies [10, 41-43, 47, 67, 68, 70, 131, 136-140, 147].  In many different species of mammals, it has been demonstrated consistently that an ovulatory dose of LH/hCG stimulates a very marked but very transient increase in COX-2 mRNA expression in granulosa cells [45], whereas the synthesis of ovarian PGE2 and PGF2a persists through the time of follicular rupture [68, 131].  The specific inflammation-mediating action(s) of the ovarian prostanoid products of this enzyme are beginning to emerge. Based on recent evidence it is now clear that COX-2 and EP2 are essential for expression of the hyaluronan binding protein TSG-6 that is known to be essential for expansion of the COC and ovulation [138, 140, 150].  Ovarian prostaglandins may also be involved in the ovarian hyperemia, although this has not been definitively shown [48].  Mice null for COX-2 exhibit other abnormal reproductive functions [70, 211].

17.  Cytochrome P450 Side Chain Cleavage

            Mitochondrial P450scc enzyme carries out the first step of steroidogenesis by converting cholesterol to pregnenolone, which is then metabolized to progesterone by the action of 3b-HSD [93, 95, 350].  During the ovulatory process there is an enormous increase in ovarian progesterone synthesis that is associated with expression of P450scc in the granulosa cells in most mammals that have been studied including rat, horse, and monkey [84, 91, 101, 212, 213, 350]. Importantly, there is also an increase in P450scc expression in cumulus cells of ovulating COCs [84, 85], thus providing a ligand for PR expressed in these same cells and appears in the pig to regulate ADAMTS-1 [49, 119].

18.  Cytokine-Induced Neutrophil Chemoattractant

            CINC is a member of the CXC chemokine family that is induced by the inflammatory cytokines IL-1b, IL-6, and TNF-a to attract neutrophils into sites of inflammation [35-354].  Although the data on ovarian expression of CINC during ovulation is not very clear, based on preliminary studies with rat and human tissues it appears that CINC is expressed primarily in granulosa cells, and possibly in thecal tissue as early as 6 hours after the ovulatory process has been induced by hCG [216-218,355].  As a side note, it is somewhat interesting that a Japanese herbal medicine Unkei-to, which promotes ovulation, can stimulate ovarian steroidogenesis and other ovulation-like events by inducing the secretion of CINC with IL-1b and TNF-a in vitro [356].  The role of the immune cells in ovulation is just beginning to unravel and no doubt other factors will soon be identified that impact ovulation.  For example, T- cells have been found in cumulus cells and produce IL-4 [219].

19.  Early Growth Response Protein-1

            Egr-1 is an inducible zinc-finger transcription factor that binds to specific GC-rich enhancer elements on an estimated 80-100 other genes that comprise the Egr-1-induced cascade that promotes acute inflammation, vascular hyper-permeability, and angiogenesis [220, 357-360].  Two of the principal targets for transcription are interleukin-1b (IL-1b) [359] and tumor necrosis factor-a (TNF-a) [360], which are central components of the inflammatory cascade [291, 361].  Of all the ovulation-specific genes that have been discovered to date, mRNAs for Egr-1 and ALAS are the two earliest transcripts to be upregulated in the ovary following onset of the ovulatory process [45, 360].  Like ALAS, Egr-1 is expressed in significantly greater amounts in the granulosa layer within 30 min after the ovulatory process has been initiated by hCG [221].  Egr-1 null mice are infertile most likely due to the key role of this transcription factor in the pituitary gonadotropes where it is essential for transcription of the LH beta subunit gene [220-223]. Altered ovarian function may be a consequence of chronically low levels of LH that would alter the growth of preouvlatory follicles by reducing/preventing theca cell differentiation and the production of thecal cell androgen precursors for aromatase.  Alternatively, loss of Egr-1 may also alter the regulation of genes critical in the ovulation process. 

20.  Epiregulin

            EPI is another member of the EGF family of growth factors that are characterized by a six-cysteine consensus motif that forms three intra-molecular disulfide bonds crucial for binding of the growth factor to an ErbB receptor [322, 362].  While it has cytokinetic-promoting properties like the other growth factors, it is interesting to note that EPI has been associated with other genes such as Egr-1 [363] and NF-kB [364] that are common components of acute inflammatory reactions.  An increase in EPI gene expression in ovulatory follicles has been reported in both the rat [45] and the mouse [72], and there is speculation that there might be some relationship between EPI and COX-2 during COC expansion and ovulation [45, 72].

21.  FOS-like Antigen-2

            Fra-2 is related to the family of FOS genes that encode proteins that dimerize with proteins of the JUN family to form a group of transcription factors known as activator proteins (e.g., AP-1) [365, 366].  Such FOS-JUN dimers function as local regulators of gene expression for cell proliferation, differentiation, and transformation [224].  AP-1 dimers that include Fra-2 as one of the proteins are abundant in transformed fibroblasts of chickens and rodents [365].  However, in ovaries that have been stimulated by LH, Fra-2 and JunD are rapidly induced in granulosa cells that are differentiating into luteal cells, rather than in the thecal fibroblasts in the outer layers of the follicle [224].

22.  Frizzled G-Protein-Coupled Receptors

            Fzs are true members of the G-protein coupled receptor family that have 7-transmembrane segments and associate with heterotrimeric G-proteins to propagate intracellular signaling [367-368].  Upon stimulation by the Wnt family of ligands, Fz receptors signal well-known effector responses that include the canonical pathway in which beta-catenin is activated as a transcription factor as well as a pathway in which intracellular Ca++ is mobilized.  In the ovaries of PMSG/hCG-treated mice, Fz-1 mRNA increases first in the cells of the theca interna and then in the granulosa cells of ovulating follicles [225].  However, during transformation of a ruptured follicle into a corpus luteum, Fz-1 declines and is replaced by Fz-4 [225] (Figure 23).  The downstream signaling targets of Fz-1 and Fz-4 remain to be clearly defined but it is most likely that Fz-1 activates the beta-catenin pathway whereas Fz-4 may activate other pathways.  Interestingly, female Fz-4 mice are infertile but the underlying causes are not yet entirely clear (Richards, unpublished observations).

23.  Frizzled-Related Protein-4 (secreted)

            sFRPs are a group of antagonists (or in some instances agonists) of the Wnt signaling pathways that act extracellularly by binding both the Wnt ligands and the Fz membrane receptors of those ligands [369-371].  This binding action might promote apoptosis by modulating the cell survival signals that are usually transduced by Wnt-stimulated Fzs.  The expression of ovarian sFRP-4 mRNA and protein during ovulation reportedly increases in the granulosa cells of mouse follicles [226], but it increases in the thecal layers of ovulatory follicles of the rat [227].  The ovarian increase in this Wnt signaling regulator is presumably important for modulating Wnt-frizzled signaling that occurs during and after ovulation, especially in the process of luteinization [226, 227] (Figure 23).

24.  g-Glutamylcysteine Synthetase

            g-GCS is a zinc metalloprotein enzyme that synthesizes glutathione from its three constituent amino acids glycine, glutamate, and arginine [372-374].  A principal redox function of glutathione is to remove the excessive amounts of toxic peroxides and reduce the oxidative stress that can be harmful to cells during times of tissue inflammation or other forms of metabolic stress [375-377].  During the inflammatory conditions of the ovulatory process, g-GCS mRNA is expressed in large follicles from 2-8 hours after treating the animals with hCG [45].  It is expressed in an irregular pattern, mostly in the theca externa layer, but also in the stratum granulosum.

25.  Glutathione S-Transferase

            GST is a member of a family of enzymes that function to detoxify hydrophobic electrophiles, i.e., compounds that contribute to oxidative stress because they contain electron deficient atoms such as are found in the unsaturated carbonyl groups in some steroids and eicosanoids [378-381].  Specifically, GST functions by catalyzing the conjugation of glutathione (generated by g-GCS action) to any of a wide variety of endogeneous electrophilic compounds [382].  In view of the functional relationships among GST, glutathione and g-GCS, one would predict that the pattern of expression of GST would parallel that of g-GCS.  However, as indicated in the previous paragraph, g-GCS is expressed transiently during the middle of the ovulatory process, whereas GST begins to be expressed only after g-GCS mRNA levels have returned to 0-hour control values, i.e., after the follicles have ruptured [45].  Nevertheless, GST is included in this catalog of ovulation-specific genes because it is induced as a consequence of the original ovulatory surge in gonadotropin.

26.  G-protein-Coupled Receptor-54

            The GPR54 gene encodes a protein that is a galanin-like G-protein-coupled receptor that binds metastin, a 54-amino-acid peptide encoded by the metastasis suppressor gene KISS1 [382, 383].  Activation of the GPR54 receptor stimulates signaling events that result in Ca++ mobilization, phosphatidyl inositol diphosphate hydrolysis, arachidonic acid release, and ERK and p38 MAPK phosphorylation [383].  GPR54 mRNA has been detected in placental, hypophyseal, pancreatic, and spinal tissue  [383], and it is also highly expressed in the theca externa and stromal tissue of the rat ovary at 4-12 hours after initiation of the ovulatory process with hCG [45].  It might be noted that the gene has been proposed as a regulator of puberty because GPR54-deficient mice exhibit hypogonadotropic hypogonadism, with delayed vaginal opening and an absence of folliculogenesis—abnormalities that can be overcome by exogenous gonadotropins [384, 385].  In view of its expression as an ovulation-related gene, it would be interesting to know whether gonadotropin-treated GPR54-deficient animals exhibit a normal ovulation rate.

27.  3a-Hydroxysteroid Dehydrogenase

            3a-HSD belongs to the aldo-keto reductase superfamily that has, among its functions, the responsibility of inactivating metabolically disturbing aldehyde and ketone functional groups that are components of most bioactive steroids and eicosanoids [386,387].  3a-HSD mRNA increases in the granulosa layer of the gonadotropin-primed immature rat ovary as early as 2 hours after hCG administration, and it remains elevated for the duration of the ovulatory process [228].  Since non-steroidal anti-inflammatory drugs like indomethacin have been shown to be potent inhibitors of mammalian 3a-HSD [386-389], it would be useful to know whether part of the anti-ovulatory action of indomethacin is due to the inhibition of ovulation-specific 3a-HSD activity. 

28.  3b-Hydroxysteroid Dehydrogenase

            It is well established that 3b-HSD catalyzes the formation of progesterone from pregnenolone.  Thus, it is one of the two principal gateway enzymes that regulate the shuttle of cholesterol into the wide array of bioactive mineralocorticoids, glucocorticoids, and sex steroids.    3b-HSD mRNA reportedly increases in the granulosa cells of the rat [91], monkey [215] and  equine ovary [212] during ovulation.  It would be useful to have a more comprehensive description of the temporal and spatial patterns of expression of this important gene in one of the common rodent models for ovulation studies.

29.  11b-Hydroxysteroid Dehydrogenase

            11b-HSD is a reductase that is thought to suppress inflammatory reactions as a result of its ability to elevate local levels of the anti-inflammatory glucocorticoid cortisol by regenerating this bioactive steroid from cortisone [229,390, 391].  Since the ovulatory process has been likened to an acute inflammatory reaction, it is interesting that elevated 11b-HSD mRNA has been detected in rat granulosa cells [230] and in human surface epithelial cells [229] that have been stimulated with either LH or with the inflammation-promoting ILs.  In any case, it is noteworthy that 11b-HSD, 3a-HSD, and CBR proteins are all anti-inflammatory reductases that are upregulated in different parts of the ovary at the time of ovulation [45, 229].

30.  17b-Hydroxysteroid Dehydrogenase, type 4

            17b-HSDs exist as a number of different isoforms in a wide variety of tissues [392].  Each isoform has selective substrate affinity that can lead to either reductive catalysis of some steroids, or oxidative catalysis (requiring NAD+ as a cofactor) of other steroid substrates [392, 393].  Ovarian 17b-HSD4 is an oxidase that is induced by LH/hCG action on cultured theca interna cells and especially on granulosa cells [231].  The expression of this particular HSD in the equine ovary appears to function in reducing the circulating levels of 17b-estradiol during follicular luteinization.

31.  IGF-Binding Proteins

            IGFBPs function as modulators of IGF-mediated cellular growth, survival and differentiation by binding to IGF-I and -II and thereby antagonizing the coupling of these growth factors to their respective receptors [348, 394, 395].  It has been suggested that elevated levels of ovarian IGFBP-4 might inhibit ovulation by interfering with the actions of IGF-I and –II [232].  Although the precise role(s) of these binding agents in ovulation remain to be determined, they are readily detected in virtually all regions of the ovary, with IGFBP-4 [232] and IGFBP-5 [59] being especially abundant in ovulatory follicles of the primate.

32.  Interleukins

            IL-1b and IL-6 are primarily proinflammatory cytokines, with their most salient and relevant properties being their ability to initiate expression of COX-2, phospholipase A2, eicosanoids, iNOS, tPA, MMPs, NF-kB–inducing kinase, receptor activator of NF-kB (RANKL), and a myriad of other mediators of inflammatory reactions [37,396-400].  IL-1b and IL-6 gene expression in the ovary has been studied extensively, and transcripts of these genes increase in the granulosa and surrounding theca interna within 4 h after stimulation of the ovulatory process in rats [36, 157, 233-236, 397].  Il-4 has recently been shown to be expressed by T-cells localized in cumulus cells and thus may play a role in COC expansion [219].

33.  Interleukin-4 Receptor-a

            IL-4Ra has been detected in the plasma membrane of a wide range of cells, and it is especially common in fibroblasts [401].  IL-4Ra is the receptor for IL-4 which is distinctly different from the other IL cytokines because when it couples with IL-4Ra it initiates a signaling process that suppresses inflammation [402-404].  IL-4Ra mRNA is expressed in the thecal fibroblasts (or T cells) of follicles in the ovaries of gonadotropin-primed rats that have been injected with hCG [210].  Although its specific function in ovulation has not been established, IL-4Ra might be involved in terminating the ovarian inflammatory response and setting the stage for T-cell-mediated wound healing after rupture of the ovarian surface.

34.  JunD Proto-oncogene

            As mentioned above, JunD dimerizes with proteins of the FOS family (e.g., Fra-2) to form the group of transcription factors known as activator proteins (e.g., AP-1) [365, 366].  Such FOS-JUN dimers function as local regulators of gene expression for cell proliferation, differentiation, and transformation [224].  Like Fra-2, ovarian JunD is induced by LH and remains elevated through ovulation and luteinization [224].  It is not clear whether this concurrent expression of ovarian Fra-2 and JunD might be induced by transforming growth factor-b—as occurs in epithelial and carcinoma cells [405].

35.  Leptin and Leptin Receptors

            Ob (i.e., leptin), a common product of the so-called obesity gene in adipocytes, is now considered to be a marker for the inflammatory response, and it is regulated by inflammatory cytokines such as IL-1 and TNF-a [406-410].  Ob mRNA increases markedly in whole ovaries of immature rats within two hours after the animals have been injected with an ovulatory dose of hCG [237].  Several hours later, when Ob mRNA expression is declining, there are substantial increases in mRNA for both the short form (Ob-Ra) and the long form (Ob-Rb) of the leptin receptor.  The OB-Rb protein is especially high in oocytes, endothelial cells and thecal cells at the time of ovulation [238].  These observations suggest that Ob could have a significant role in the ovarian inflammatory reaction during the ovulatory process. Of note, Ob null mice are subfertile [239 ] .

36.  Liver Receptor Homolog-1

            LRH-1 is a member of a nuclear receptor superfamily that participates in the regulation of steroid metabolism.  It reportedly might function as the principal transcriptional regulator of ovarian P450arom [240], or of ovarian StAR gene expression [241].  In any case, LRH-1 mRNA and protein are significantly upregulated in rat and human granulosa cells after gonadotropic stimulation [240-242].   Although the specific ovarian role of LRH-1 versus  SF-1 remains to be clearly established, a conditional knockout of SF-1 in granulosa cells renders  females infertile. [243]. These results indicate that LRH-1 is not redundant to SF-1 in the female gonad, an observation that lends support to a report that  LRH-1 protein is much lower in granulosa cells than is SF-1 [242]. 37.  a2-Macroglobulin

            a2-M is a nonspecific proteinase inhibitor that is abundant in the plasma and body fluids of vertebrates [411, 412].  It functions as a broad-spectrum protease-binding protein that inactivates MMPs, growth factors, cytokines, and other mediators of inflammation and tissue remodeling [244-246, 413].  a2-M mRNA and protein are detectable in granulosa cells of rat follicles at the time of ovulation, and this proteinase inhibitor probably functions to control damage in the ovary during the time when an ovulatory follicle is remodeled into a corpus luteum [244-247].  Mice null for a2-M appear to be fertile [248]

38.  Macrophage Migration Inhibitory Factor

            MIF was named after its initial identification as a T-cell cytokine, but then it was rediscovered as a protein that is released by pituitary cells when they are exposed to endotoxins (414).  Now, MIF is further established as a powerful proinflammatory cytokine that acts in concert with glucocorticoids to control both the set point and the magnitude of the inflammatory response [414-417].  MIF might be a component of the proinflammatory cascade of ovarian gene expression during ovulation, because hCG can induce MIF expression in cultured granulosa cells [249].

39.  Matrix Metalloproteinases

            MMPs comprise a large family of zinc-dependent proteinases that degrade numerous proteins of the extracellular matrix during the tissue remodeling that occurs in a wide variety of physiological and pathological processes including angiogenesis, tumorogenesis, inflammation, and wound healing [26,418, 419].  Various reports in the literature use confusing and inconsistent nomenclature for the MMPs, but a convenient index of the most common terminology is available in a recent review by Curry and Osteen [26].  Since the ovulatory process has characteristics similar to an inflammatory reaction, and since luteinization requires substantial vascular proliferation into the lutein granulosa, it is not surprising that there is considerable expression of MMPs in ovulatory follicles.  Ovarian MMP-2, along with MMP-9 and -19, appear to be the most elevated in granulosa cells as well as in thecal and stromal tissues in response to LH/hCG [26, 182-185].  The specific functions of each protease that might be critical to ovulation remain to be delineated, because mice that are null to individual MMPs are fertile [191].

40.  Membrane Type-1 Matrix Metalloproteinase

            MT-1-MMP is an integral membrane proteinase that functions to regulate degradation of the local extracellular matrix and promote cell migration and invasion [420].  One of its specific functions is to activate other cell surface proteinases such as proMMP-2, the active form of which can degrade type IV collagen [421].  Thus, it is relevant that MT1-MMP RNA increases in the thecal connective tissue of rat ovarian follicles beginning 4 hours after hCG, and that pro-MMP-2 increases by 12 hours after hCG, when the follicles are just beginning to rupture [251].  MT1-MMP null mice have severe developmental defects [252].

41.  Metallothionein-1

            Met-1 is a small, cysteine-rich protein that is a member of the single most abundant group of intracellular zinc-binding proteins that are conserved evolutionarily in virtually all animals, eukaryotic plants, bacteria and fungi [422-424].  The hallmark of Met genes is the fact they are expressed in an unusually broad assortment of cells and tissues in response to an extraordinarily wide variety of chemical and physical stimuli, including agents that are known to mediate inflammation [422-426].  The metabolic significance of the Mets has not been firmly established, but the three most common hypotheses are that they control zinc availability to proteins that require zinc to function, they protect cells from oxygen-free radicals, and/or they absorb accumulating heavy metals that might otherwise cause local toxicity [422-426].  Ovarian Met-1 gene expression is a latent response to LH/hCG stimulation, because mRNA transcripts are not detectable in any significant amount in the stratum granulosum until after the follicles have ruptured and the luteal tissue is flourishing [45, 253].  Nevertheless, even though Met-1 is expressed only after rupture, it appears to be a unique marker of progesterone-secreting corpora lutea, and clarification of the role of this zinc-binding protein in luteal tissue might help to elucidate the full significance of progesterone as both a mediator of the ovulatory process and a persistence attribute of the corpus luteum.

42.  Mitogen Activated Protein Kinase

            MAPK consists of three major pathways of kinases, namely extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK), and p38 MAP kinase, that function in regulating gene expression related to cell growth, proliferation, and differentiation [426].  Activation of these MAPK pathways by cytokines IL-1 and TNF-a induces the expression of transcription factors that promote the transcription of genes in the inflammatory cascade [427].  In the ovary, gonadotropins operate through cAMP/PKA signaling mechanisms to activate MAPK pathways [254-257, 4278].  One function of ovarian MAPKs appears to be the regulation of StAR expression and progesterone synthesis [255, 428, 429].

43.  Myeloid Cell Leukemia-1

            Mcl-1 is an anti-apoptotic member of the Bcl-2 family of about 20 homologues of pro- or anti-apoptotic agents that regulate programmed cell death by either disrupting or by preserving, respectively, the permeability of mitochondrial membranes [430, 431].  Mcl-1 expression can be upregulated through activation of the transcription factor NF-kB, a central component of inflammatory cascades [432].  Once the Mcl-1 protein is translated, it can exert its anti-apoptotic effect by complexing with pro-apoptotic members of the Bcl-2 family (e.g., Bak) and rendering them inoperative [431, 433].  During the ovulatory process in PMSG/hCG-primed rats, Mcl-1 expression increases in granulosa and thecal tissue [257]—presumably for the purpose of moderating the pro-apoptotic momentum of the acute inflammatory reaction that traumatizes the follicle and causes it to rupture.

44.  Nerve Growth Factor

            NGF, the founding member of the neurotrophin family of growth factors, was initially a focus of intense investigation because of its ability to prevent apoptosis in peripheral neurons [434-436].  Now, NGF is also recognized for its associations with cytokines [437-440], its involvement in inflammation and tissue repair [441-443], its expression by and activation of fibroblasts [437, 438, 444], and its contributions to endothelial cell survival and angiogenesis [439, 441, 445]  Taking into account the reports that NGF is expressed in fibroblasts and is involved in inflammatory reactions, it is not totally surprising that the site of NGF mRNA elevation in ovulatory rat follicles is in the fibroblast-containing tissues of the thecal and stromal areas of the ovary and that this expression is intensified by the inflammatory cytokine IL-1b [259].

45.  Nerve Growth Factor Tyrosine Kinase Receptor

            NGF induces cellular responses by binding two principal receptors, namely high affinity TrkA and low affinity p75 neurotrophin receptor (p75NTR), which is a receptor that resembles members of the tumor necrosis factor receptor family [434, 439, 446, 447].  During ovulation, TrkA expression is detectable not only in the thecal and stromal areas of rat ovaries, but also in the stratum granulosum [260].

46.  Nitric Oxide Synthases

            NOSs consist mainly of endothelial eNOS and inducible iNOS, which both function to oxidize L-arginine and produce nitric oxide [448], a free radical that causes vasodilitation and edema and promotes other characteristics of inflammation [449-451].  Based mainly on immunohistochemical studies that have detected eNOS and iNOS in rat and mouse ovaries, eNOS expression appears to increase mainly in the theca externa, ovarian stroma, and luteal tissue, as well as in the granulosa and the oocyte [261-265], whereas iNOS is predominantly in the granulosa and the oocyte [262, 264, 265].

47.  p53 Tumor Suppressor

            p53 has emerged as a sequence-specific DNA-binding protein that induces the expression of target genes that functions not only to mediate tumor suppression but also to facilitate the repair and survival of damaged cells [452-455].  The expression of this transcription factor has been associated with the moderation of oxidative stress arising from inflammatory conditions [453, 456-458], as well as with the promotion of angiogenesis [459].  p53 mRNA expression reportedly increases significantly in granulosa cells of the mouse ovary near the time of ovulation [266], and its possible function is to promote gene expression that will facilitate the survival of follicular tissue that undergoes severe cellular stress during the inflammatory events of the ovulatory process.

48.  Pancreatitis Associated Protein-III

            PAP-III, a so-called secretory stress protein, was first detected because of its over-expression in inflamed pancreatic tissue—with highest expression localized in pancreatic acini [460, 461].  Although the function of PAP proteins has not been firmly established, the current hypothesis is that they perform in some way as endogenous protective agents in inflamed tissues [461, 462].  During the inflammatory-like conditions of ovulation, the ovarian increase in PAP-III mRNA is confined spatially to the endothelial lining of blood vessels and to small secondary follicles that are distributed randomly throughout the ovarian stroma, rather than to the ovulatory follicles [45, 267].  In view of the hypothetical role of PAP proteins as constituents of a defense mechanism in inflamed tissues, it is possible that the intense expression of PAP-III in small ovarian follicles is for the purpose of protecting future crops of ovulatory follicles from excessive oxidative stress at the time of ovulation.

49.  (cAMP-Specific) Phosphodiesterase

            PDE4 belongs to one of 11 major families of phosphodiesterases that hydrolyze intracellular cAMP and cGMP to their inactive 5’ metabolites.  PDE4 is specific for cAMP [463],  has been related to inflammatory conditions [463-465] and appears to be directly or indirectly involved in the activation of NF-kB, a key transcription factor in inflammatory cascades [464, 466, 467].  During the past decade, PDE4 has been linked to ovarian thecal cells [268] and to granulosa cells [269].  mRNA transcripts of this phosphodiesterase increase in the ovary within 2 hours after the administration of hCG to rats, and they remain elevated for the duration of the ovulatory process [45].  Therefore, since there is evidence that disruption of expression of the PDE4 gene significantly impairs ovulation [269, 468], it would be interesting to know whether a PDE4 inhibitor, such as rolipram [466, 467]. would suppress ovulation.  It is also worth noting a recent finding that disruption of PDE4 gene transcription markedly reduces the expression of ovarian COX-2, PR, and other ovulation-related genes, and this impairment of PDE4 gene expression results in the inhibition of follicular rupture without blocking ovarian progesterone synthesis and luteinization, i.e., it leads to the formation of so-called luteinized unruptured follicles in which luteinization occurs in the absence of release of the oocyte [72, 269, 468].

50.  Pituitary Adenylate Cyclase Activating Polypeptide

            PACAP is actually a neuropeptide that is classified in the VIP/secretin/glucagon family of peptides [469].  However, over the past decade this polypeptide has been firmly established as a potent anti-inflammatory factor that exerts its attenuating effect on inflammation in a variety of tissues by stimulating the production of anti-inflammatory mediators while inhibiting proinflammatory mediators [469-471].  During the inflammatory events of the ovulatory process, PACAP mRNA is expressed quite transiently as a progesterone-dependent message in the stratum granulosum from approximately 4 to 8 hours after administration of hCG to rats [45, 121, 122]. Somewhat unexpectedly, ovarian PACAP mRNA returns to the 0-hour control level by 12 hours after hCG, when rat follicles first begin to rupture and when the ovary would presumably be experiencing the most intense inflammation. Of interest, PACAP has also been shown to induce tPA [270].

51.  (tissue) Plasminogen Activator

            tPA, along with urokinase-PA (uPA), enzymatically converts plasminogen into the protease plasmin, which then contributes to inflammatory reactions by activating MMPs [179].  Only tPA is included in this catalog of ovulation-related genes because it appears to be more significant in the mechanism of ovulation.  The roles of PAs in ovulation have already been described in this chapter and will not be detailed again, here.  In brief, tPA activity increases throughout most of the follicular tissue in rats [ 172, 173, 472] and monkeys [62], reaching a peak near the time of follicular rupture.

52.  Plasminogen Activator Inhibitor-1

            PAI-1, which is the main inhibitor of fibrinolytic enzymes such as tPA and uPA [472], plays an essential role in tissue remodeling by suppressing inflammation and blood clotting in irritated and damaged tissues [473-477].  Information on the ovarian expression of this potentially important regulator of inflammatory reactions is not very consistent, but it appears that PAI-1 declines to minimal levels in the rat [472] and monkey [62] at the time when follicles are actually rupturing.

53.  Prepronociceptin

            The ppN gene (also known as orphanin FQ), which encodes the precursor form of an endogenous agonist that ligates to opioid receptor-like-1 [478, 479], contains a promoter region that appears to bind Sp1 transcription factor [480] and has two cAMP response elements (CRE) near the start site of transcription [481].  ppN expression is induced in acutely inflamed tissues and probably mediates pro-inflammatory features of inflammatory reactions [480, 482-484].  ppN mRNA is expressed in the thecal layer of follicles in the ovaries of gonadotropin-primed rats that have been injected with hCG [210].

54.  Progesterone Receptor(s)

            There are actually two isoforms of PR, i.e., PR-A and PR-B, that vary mainly in the length of the processed protein and appear to have differing functional activities in a wide variety of biological processes [103, 105, 270, 271, 485].  Based on studies of the mouse ovary, both isoforms of PR are highly expressed in granulosa cells of preovulatory follicles in response to an ovulatory surge in gonadotropins, and they may also be present in thecal cells [107. 271].  The importance of the PR receptor in ovulation has been demonstrated by the anovulatory state of mice that lack a functional PR gene [105].  An extensive review of the PRs has been published recently [485].

55.  Prolactin Receptor

            PRL-R is expressed in a variety of cells in and out of the reproductive system, and it might be an important link in the defense reaction known as the acute phase response that helps re-establish homeostatic conditions in insulted tissues that become acutely inflamed [486-488].  The coupling of PRL to this receptor initiates various signaling cascades involved in regulating cell differentiation, proliferation, and survival [489].  In inflamed ovarian follicles, PRL-R mRNA increases significantly in the granulosa cells as they differentiate into luteal cells [273-275].  PRL receptor null mice exhibit severe defects in luteinization [275].

56.  Receptor Interacting Protein-140

            RIP-140 is reportedly a nuclear receptor corepressor that suppresses the expression of several genes, but its regulation of nuclear receptor action is currently under debate [490, 491].  Among its array of functions, RIP-140 reportedly controls steroidogenic factor-1-dependent transcription [491], AP-1-mediated transcription [492], and vitamin A-regulated transcription [493], as well as appearing to have an indispensable role in interacting with the TNF-R1 receptor to induce NF-kB activation during inflammatory reactions [494-496].  Ovarian expression of RIP-140 is reportedly essential for ovulation, even though mice that are null for this protein can nevertheless develop follicles that undergo luteinization without ever having ruptured [276-278].  This phenotype is similar to the characteristics of mice null for PR and COC-2.

57.  Regulator of G-protein Signaling Protein-2

            RGS proteins are GTPase activating proteins (GAPs) that enhance the intrinsic rate at which the a-subunits of certain heterotrimeric G-proteins hydrolyze GTP to GDP and thereby limit the time-span that a-subunits incite the activity of downstream effectors [497-499].  In simpler terms, RGS2 regulates the duration of signaling by G-protein-coupled receptors.  During ovulation in the immature rat model, RGS2 mRNA begins to increase significantly in the granulosa layer within 2-4 hours after the animals have received hCG, but it is already declining by the time the follicles actually begin to rupture [279].  RGS-2 null mice appear to be fertile [280].

58.  Steroidogenic Acute Regulatory Protein-1

            StAR has a critical role at the onset of steroid hormone synthesis because it is responsible for the transfer of cholesterol from the outer mitochondrial membrane to the inner membrane where this primary substrate can be converted to pregnenolone by P450scc [93, 95, 96, 281, 282, 350, 500].  Therefore, predictably, ovarian StAR expression is upregulated during the peri-ovulatory period in a temporal and spatial pattern that correlates with the known pattern of steroid synthesis.  It is mainly present in the steroid-secreting cells of the theca interna at the beginning of the ovulatory process, it increases significantly in the granulosa layer when ovulatory follicles begin producing substantial amounts of progesterone, and it dominates the steroidogenically intense lutein granulosa after ovulation [41, 45, 96, 281].It was noted in an earlier section of this chapter that LRH-1, which is also upregulated in the ovary at the time of ovulation, can significantly induce StAR promoter activity in a dose-dependent manner [242].  Mice null for StAR exhibit severe adrenal abnormalites and eventual premature ovarian failure [282].

59.  (manganese) Superoxide Dismutase

            Mn-SOD exists as a tetramer that is initially synthesized with a leader peptide that restricts this enzyme exclusively to the mitochondrial matrix where it functions to efficiently catalyze the dismutation of superoxide anions [501, 502].  Expression of this superoxide scavenger can be induced by the cytokines IL-6 and TNF-aľpresumably in anticipation of the oxidative stress that is generated by these two mediators of acute inflammatory reactions [502-506].  During the ovulatory process in the rat, Mn-SOD reportedly is upregulated in the granulosa layer [283-285, 507] and in the theca interna [284, 295].  SOD knockout mice die perinatally due to mitochodrial injury and several cardiac and neurological abnormalities that preclude any chance of performing analyses on the mature ovary [286].

60.  Tissue Inhibitors of Metalloproteinases

            TIMPs are a group of four related endogenous inhibitors that form high affinity complexes with the active sites of MMPs in a 1:1 stoichiometry, but their bonding to different MMPs is not particularly selective [508].  The integrity of the extracellular matrix at any given time in any given tissue is determined by the local ratio between TIMPs and MMPs [508-510].  During ovulatory degradation of the extracellular matrix of the follicle wall, it is mainly TIMP-1 and TIMP-3 mRNAs that increase in the thecal connective tissue, but TIMP-1 also increases somewhat in the granulosa layer [45,182, 287,-289].  TIMP null mice have a relatively mild ovarian phenotype, suggesting possible redundant functions among some of the inhibitors [290, 291].

61.  Tumor Necrosis Factor-a

            TNF-a is a homotrimer consisting of 157 amino acid subunits that collectively activate two distinct cell surface receptors, namely TNF-R1 and TNF-R2, that trigger signaling pathways leading to activation of major transcription factors such as NF-kB, AP-1, and c-Jun, all of which regulate the inflammatory cascade [395, 511, 512].  Considering the frequency with which cytokines such as TNF-a are mentioned in relation to the inflammatory events of the ovulatory process, and considering the common use of TNF-a, IL-1b and IL-6 as experimental probes in studies on ovulation, it is surprising how difficult it is to find specific information in the literature to clarify the temporal and spatial patterns of expression of ovarian TNF-a in any of the common experimental models in ovulation studies.  The literature research for this chapter located one review that contained limited information [292] and one primary research report that focused on the effect of TNF-a on expression of uPA and MMP-9 in the ovarian surface epithelium [175].  TNFa null animals exhibit premature ovarian failure [293].

62.  Tumor Necrosis Factor-Stimulated Gene-6

            TSG-6 was originally cloned from diploid human fibroblasts that were stimulated with TNF-a [513].  TSG-6 expression is upregulated in many cell types in response to a variety of proinflammatory stimuli, but its actual function might be anti-inflammatory since it appears to be a component of a negative feedback loop that down-regulates the inflammatory response [513, 514].  Specifically, TSG-6 binds to hyaluronan in inflamed tissues via its link module.  TSG-6 also forms a complex with the heavy chains of inter-a-trypsin inhibitor (IaI), and in this manner “delivers” IaI heavy chains to hyaluronan. IaI then forms a covalent bond with hyaluronan to help stabilize it.  Although the interaction of TSG-6 and IaI results in the release bikunin, a protease inhibitor, evidence for bikunin in the ovary has not been reported  [138, 140, 510, 515, 516].  During ovulation in the rat, TSG-6 mRNA is upregulated throughout the granulosa layer, and, especially in the cumulus mass around the oocyte, and the translated protein appears to play a critical role in stabilizing the complex hyaluronan matrix that surrounds the oocyte [138, 140 295, 517].  As indicated above, mice null for either TSG-6 or IaI are infertile, a phenotype that can be reversed by restoring TSG-6 or IaI, respectively [126-128].  Furthermore, TSG-6-blocking antibodies disrupt the normal pattern of COC expansion in culture [138].

63.  Vascular Endothelial Growth Factor

            VEGF was originally detected as a result of its ability to induce vascular permeability and stimulate mitosis of vascular endothelial cells  However, it is now recognized as a ubiquitous growth factor that promotes neovascularization during any morphological remodeling of tissues, including the changes that occur during the growth of tumors and the trauma of acute inflammation [518-522].  In the context of this review, it is relevant that the inflammatory cytokines TNF-a and IL-1b can induce VEGF expression [236, 523].  During the transformation of an ovulatory follicle into a corpus luteum, VEGF transcripts are expressed primarily in ovarian granulosa cells, and to a lesser extent in thecal and stromal cells [296, 297].

64.  Versican (Proteoglycan M)

            Versican is a large extracellular matrix proteoglycan that readily binds with hyaluronan to provide strength and elasticity to tissues and to influence the adhesion and migration of cells within tissues [288, 524].  In rat and mouse ovaries, versican mRNA increases as much as 10-fold in granulosa cells and cumulus cells after hCG treatment of the animals [49, 116, 298].  It is relevant to ovulation that versican appears to be a substrate for several members of the ADAMTS family [116].  Versican null mice are embryonic lethal [116]. 65.  Wingless-Type MMTV Integration Site Family Member-4

            Wnts consist of a structurally-related family of secreted glycoprotein ligands that have essential roles in cell adhesion, migration, proliferation, and differentiation during morphological movement and development of tissues [367, 368, 525].  Members of this family of signaling proteins bind to the frizzled family of serpentine receptors (e.g., Fz-1 and Fz-4) that initiate a number of intracellular signaling pathways. Wnt-4 is best known for its critical role in specifying embryonic development of the ovary [299]. Wnt-4 null mice exhibit sex reversal of the gonad at birth; i.e. genotypic XX null mice have gonads that are like a testis, only lacking germ cells.  Wnt-4 mRNA is also detectable in ovulatory follicles and increases significantly during the morphological transformation of a ruptured follicle into luteal tissue [226].  The role of Wnt-4 in luteal tissue has not yet been clearly defined.

C.  ADDITIONAL COMMENTS ABOUT OVARIAN GENE EXPRESSION

            The above list of ovulation-related genes represents a wide range of agents that have been associated with a number of different gene expression cascades.  Most of the genes are components of pathways that have already been identified as parts of the ovulatory process.  These include genes that are required for steroidogenesis and for inflammation, as well as genes that are known to minimize the side effects of acute inflammatory reactions, such as oxidative stress [7].  On the other hand, some of the genes such as ALAS do not seem to fit into any of the established cascades of gene expression, while other factors such as NF-kB that appear to be predictable components of the ovulatory process have not yet been studied.  Therefore, this closing section provides several additional comments that might be useful during the two principal challenges of the next decade of ovulation research—namely the discovery of other essential genes in the ovulatory process and the integration of as much of the information as possible into a comprehensive cascade of ovulation-specific gene expression and action.

1.  The Potential Role of Nuclear Factor-kB

            Based on the literature search for this review, many of the genes listed in Table 1 are related in some way to NF-kB.  Therefore, this ubiquitous transcription factor merits special attention.  NF-kB is responsible for the expression of a wide variety of genes that control the inflammatory response [512, 526, 527].  This transcription factor normally exists in essentially all cells as a dimer that is restrained in an inactive state by bondage to an inhibitory subunit known as I-kB  [528-530].  The bioactive form of NF-kB can be rapidly released from I-kB when cell signaling from any of a number of proinflammatory stimuli produce I-kB kinase (IKK), which phosphorylates the inhibitory subunit I-kB and releases NF-kB [527, 529-532].  Thus, an elevation in active NF-kB is not brought about by de novo transcription and translation, but by release of the preexisting NF-kB dimer from its bound state with I-kB.  Although the term NF-kB is generally used in reference to any of the possible combinations of the dimer subunits, the p50-p65 dimer is by far the most common in most cell types [529].

            Inflammatory cytokines such as IL-1b and TNF-a, along with a number of other inflammatory agents such as NOS, activate MAPK and lead to the activation of NF-kB, AP-1, and other transcription factors [511, 512, 526, 527, 533, 534].  It is now recognized that “NF-kB is at the heart of the acute inflammatory response” [528].  Once the NF-kB dimer is released from I-kB, it operates through a positive feedback mechanism to promote further expression of proinflammatory cytokines, including IL-1b, IL-6, and TNF-a [529, 531, 535].  Furthermore, the NF-kB pathway leads to production of COX-2, MMPs, cell adhesion molecules, acute phase proteins, and other inflammation-related agents [527, 529, 535-537].  Useful diagrams of the signaling pathways related to NF-kB are available in the literature [529, 531, 535].

            It is also well known that anti-inflammatory steroids act by inhibiting the transactivation of NF-kB-dependent genes.  Therefore, it is relevant to note that 13 years ago, it was suggested that the ovulatory increase in ovarian progesterone might be a part of an anti-inflammatory response [63].  Now, progesterone has been firmly established as an inhibitor of NF-kB, and the action of this sex steroid appears to be either by stimulating synthesis of I-kB, or by competing with NF-kB for recognition sites on the target genes for this transcription factor [536].  It is also worthy of mention that ovulation-related PACAP (along with vasoactive intestinal peptide) might exert its anti-inflammatory action by interfering with NF-kB action [538, 539].

            In summary, it appears that NF-kB has a central role in the cascade of ovarian gene expression that is induced by an ovulatory surge in gonadotropin, and this transcription factor (along with AP-1 and other inflammation-related transcription factors) merit more attention in future studies on ovulation.  The potential role of this agent in ovulation was suggested recently [7], and one interesting report has appeared in the literature [540].  It was shown that the gene for serum amyloid A3 (SAA3), a principal protein in the acute phase response to inflammatory stimuli, can be induced in mouse granulosa cells by TNFa.  It was further demonstrated that two SAA3 promoters that are responsive to TNFa are also responsive to p65, i.e., to one of the common subunits of the NF-kB dimer.  Thus, based on the literature search for the present review, this is the first evidence (albeit indirect) that NF-kB might be activated in ovarian granulosa cells during ovulation.  In conclusion, it would appear to be worthwhile in the future to use techniques based on immunohistochemistry with NF-kB-specific antibodies, or based on incubation of tissue sections with labeled probes containing kB-specific binding sites [539], to further analyze the temporal and spatial patterns of ovarian NF-kB activation during the ovulatory process.

2.  Thoughts about Organizing Ovarian Gene Expression into Pathways

            As mentioned earlier, it will be a challenging task to integrate all of the ovulation-related genes into meaningful pathways.  Nevertheless, a significant number of genes have already been discovered, and several preliminary attempts to commensurate the relationships among those genes have already begun.  In any such efforts, it is necessary to initially organize the different genes into practical groupings that can facilitate the construction of pathways of gene interaction.  Therefore, four possible ways to group the genes are identified, below.

a.  Categorizing by temporal pattern of expression.

            Ovulation is a process that begins at the moment a surge in gonadotropins(s) couples with the LH/hCG receptors on the plasma membranes of cells in the granulosa layer and the theca interna.  This process has an equally distinct ending when the stimulated follicles rupture and release ova into the oviduct.  Based on the current information, some genes such as ALAS and Egr-1 begin to be expressed as early as 30 minutes after the start of the ovulatory process, while other genes such as ADAMTS-1 and TIMP-1 begin to be expressed several hours later.  Yet, still other genes such as Mt-1 and g-GCS, do not begin to be expressed until after the follicles rupture [45] and probably represent genes that are only indirectly related to the gonadotropin surge that initiates the ovulatory process.  Therefore, any effort to establish pathways of ovulation-related gene expression would be less problematical if all of the genes listed in Table 1 had been studied in the same experimental animal and if all of the time points of ovarian extraction were at a number of consistent intervals.  Obviously, most of the work to date has been carried out using the gonadotropin-primed immature rat model, and data from such studies will likely be the most useful in establishing pathways of gene expression.  However, even with this common model, there is only limited value to studies that measure gene expression at one control time and only one experimental time somewhere within the 12-hour ovulatory process of the rat.  The most useful data comes from studies that have examined ovarian gene expression at either 0, 3, 6, 9, 12, and 24 hours after the animals received hCG to initiate ovulation, or at 0, 2, 4, 8, 12, and 24 hours after hCG.  The 0/3/6/9/12/24-hour protocol has the advantage of even distribution throughout the principal 12 hours of the ovulatory process, but it has the disadvantage of possibly missing the earliest gene expression (e.g., it would have missed ovarian ALAS expression that peaks at one hour after hCG and is declining by two hours [45]).  The 0/2/4/8/12/24-hour protocol allows a little more scrutiny of the first four hours of the ovulatory process, i.e., during the interval when progesterone and the prostaglandins are first beginning to increase in the ovary.

b.  Categorizing by spatial distribution of expression.

            In situ hybridization and immunocytochemistry have provided useful information about the intraovarian location of each gene that is expressed during the ovulatory process.  At least 72% of the 86 genes listed in Table 1 are expressed in the granulosa layer of the follicle, whereas approximately 20% have also been reported in cumulus cells.  Obviously,  these are the sites of the greatest response to the ovulatory surge in gonadotropin.  The magnitude of the response at this site in the follicle is to be expected since it is the location of most of the LH/hCG receptors.  Granulosa cells are also the location of the two gene transcripts (namely, ALAS and Egr-1) that are first detectable during the ovulatory process.  Thus, until genes that are expressed earlier are identified it would seem acceptable to predict that the ovulatory process begins in the granulosa layer of the follicle.  One can also predict that the ovulatory process continues in the COC where specific functions related to production of the ECM are critical.  Therefore, any graphical illustration of the ovulatory cascade of gene expression must begin with the granulosa cells and continue to cumulus cells , and only those genes that are detected in these areas of the follicle should be considered for integration into granulosa/cumulus-specific pathways.  In some cases, it is difficult to decipher from the published data whether a given gene is expressed only in the granulosa, or also in the adjacent, thin layer of cells belonging to the theca interna.  Also, for genes that were detected in vitro (usually in cultured granulosa cells), it would be useful to know whether such expression occurs exclusively in the granulosa cells, or also in cumulus cells, in theca interna cells, and/or in other ovarian cells.  In any case, it is reasonable to conclude that the inflammatory-like events of the ovulatory process begin primarily in the granulosa layer.

The response of the fibroblasts of the theca externa and the surrounding ovarian stromal tissue appears to be secondary to the gene expression in the granulosa cells.  Therefore, another challenge for the next decade is to determine whether certain protein products of the ovulation-specific genes in the granulosa are having a direct effect on the thecal fibroblasts, or whether arousal of these quiescent fibroblasts is a result of action by serum proteins that have exuded from hyperpermeable vessels in the vicinity of the theca interna of ovulatory follicles.

c.  Categorizing by nature/function of the protein product.

            It is also possible to categorize most of the genes based on the characteristics of the protein products that are generated by transcription and translation.  From the ovulation/luteinization-related genes listed in Table 1, there is generated a diverse array of ligands (e.g., ApoE, ppN, Wnts, and PACAP, and membrane receptors (e.g., CRHR1, Fz-1, Fz-4, GPR54, IL-4Ra, LH, PRL-R and TrkA ) that are not distinctly related to one another.  There are a number of G-protein-coupled receptor (GPCR)-signaling regulators (e.g., CREM (i.e., ICER), PDE4, PACAP, RGS2, and sFRP4) that might regulate the duration and intensity of the signaling cascade that originates from the activation of the LH/hCG receptor; but, it is also possible that these diverse signaling regulators might be affecting signaling pathways associated with some of the other GPCRs (mentioned above) that are upregulated during the ovulatory process.  Some of the genes produce growth factors (e.g., AR, BTC, EPI, NGF, and VEGF), and various types of binding proteins (e.g., C-FABP, CREB, IGFBPs, a2-M, versican, and TSG-6) that interact with many different target molecules in order to carry out their metabolic functions.  Another important class of ovulation-related agents is a group of transcription factors (e.g., AHR, CREB, LRH-1, C/EBP-B, Egr-1, Fra-2, JunD, p53, PR), along with several factors that significantly influence transcription (e.g., CBP, MAPK, p53, and RIP140).  Central to the ovulation cascade of gene expression are several common cytokines (e.g., IL-1b, IL-6, TNF-a, and MIF), along with at least one chemokine (e.g., CINC).  A number of the downstream products of the expression cascades include a variety of proteolytic enzymes (e.g., ADAMTSs, , MMPs, tPA, catL and MT-1-MMP) and protease inhibitors (e.g., the versatile a2-M, along with PAI-1, and the TIMPs).  Ovarian steroid metabolism involves several steroidogenic enzymes (e.g., P450scc, 3b-HSD, 17b-HSD, and StAR) and several electron transport proteins (e.g., ADX and the ALAS product 5-aminolevulinate) that function in steroidogenic activity.  Also, several genes for aldo-keto reductases (e.g., 3a-HSD, 11b-HSD, and 17-b-HSD), along with several genes for other enzymes (e.g., COX-2, g-GCS, GST, MAPK, and NOS) with highly diverse functions are expressed in the ovary during ovulation.  Finally, there are still other proteins (e.g., Met-1, PAP-III, Mn-SOD,) that do not fit readily into any of the above categories of gene products.

d.  Categorizing by relevance to functional processes/pathways.

            A fourth possibility is to classify the genes in Table 1 into categories of biological processes with which they have been commonly associated.  In attempting to sort in this manner, one is immediately faced with the fact that many of the ovulation-related genes (e.g., the cytokines) have been associated with multiple gene expression cascades and/or signaling pathways.  On the other hand, if one groups the genes according to their associations with several established biological processes that are known to occur in the ovary during ovulation, it turns out that the vast majority have been linked to the inflammatory response (e.g., ADAMTSs, AR, ApoE, AHR, C/EBP-B, CD63, CRHR, C-FABP, COX-2, Egr-1, EPI, Fra-2, Fz-1, IGFBPs, IL-1b, IL-6, IL-4Ra, Ob, a2-M, MIF, MMPs, MT-1-MMP, Met-1, MAPK, Mcl-1, NGF, NOS, p53, PAP-III, PDE4, PACAP, tPA, PAI-1, ppN, PRL-R, PR, RIP140, TIMPs, TNF-a, TSG-6 and VEGF) with some of the proteins exerting pro-inflammatory actions, and others having anti-inflammatory effects [7].  Some of the other genes in Table 1 fall into less congested functional groups such as oxidative stress (e.g., AHR, CBR, g-GCS, GST, 3a-HSD, 11b-HSD, and Mn-SOD),  steroidogenesis (e.g., ADX, ALAS, P450scc, 3b-HSD, LRH-1, Met-1, p53, PR, and StAR), and angiogenesis (e.g., ADAMTS-1, ADAMTS-4, Egr-1, MMPs, NGF, and VEGF).  Thus, the challenge of the future is to integrate this omnium gatherum of genes (as well as other novel genes that remain to be discovered) into a meaningful conglomeration of gene expression that might be entitled, simply, the “ovulation cascade.”

V.  CONCLUDING SUMMARY

            Ovulation is a complicated cascade of molecular events that is initiated when LH (or an analogous gonadotropin) couples with LH/hCG receptors located in the membranes of granulosa cells as well as theca cells of mature ovarian follicles.  These G-protein-coupled receptors operate through cAMP/PKA signaling pathways to induce the expression of a repertoire of genes in granulosa cells that are associated with the up-regulation of inflammatory cytokines such as IL-1b, IL-6, and TNF-a. Within this inflammatory blitz of up-regulated gene expression is the inducible form of cyclooxygenase, namely COX-2, which catalyzes the conversion of arachidonic acid into several prostanoids including the vasodilatory prostaglandin E2.  One known, critical site of PGE2 action (based on the phenotypes of COX-2 and COX-1 null mice) occurs during COC expansion where this prostaglandin induces TSG-6, a factor that is obligatory for stabilizing the extracellular hyaluronan-rich COC matrix (Figure 20). A possible additional consequence of the biological action of prostaglandins (combined possibly with the cooperation of other vasoactive agents) is the hyperemic response that occurs during the first hours of the ovulatory process.  As a result either of the exudation of serum proteins into the thecal connective tissue surrounding ovulatory follicles, or of the diffusion of unknown signaling agents from the inflamed granulosa layer of the follicles, the fibroblasts in the ovary undergo a transformation from a quiescent state to a more active, proliferating state.  This arousal of the fibroblasts induces gene expression for tPA, catL, MT-1-MMP, and a number of MMPs that weaken the extracellular matrix in the connective tissue surrounding the follicle.  This proteolytic degradation of the collagenous tissue in the follicle wall may be augmented by the action of the progesterone-regulated ADAMTS-1 enzyme.  In addition, ADAMTS-1 that is secreted by the granulosa cells and cumulus cells and localizes to extracellular hyaluronan-rich matrices of the expanded COCs may act to help provide a protective shield around the oocyte during its release and transport within the oviduct.  Eventually, the integrity of the follicle is destabilized by proteolytic events and, under the force of a steady intrafollicular pressure, the apex of the follicle wall begins to thin out and protrude to the point where it ruptures.  Thus, the ultimate cause of ovulation is a diverse array of proteolytic activity that originates from several different ovarian tissues in and around a mature follicle.

 

ACKNOWLEDGMENTS

            This effort was supported in part by NSF Grant #0234358 (L.L.E.), in part by an endowment to Trinity University from Mrs. Ruth C. and Dr. Andrew G. Cowles, and in part by NIH Grants HD-16229, HD-07495 (Project III) (J.S.R.).  The assistance of Karla Moncada and Rebecca Garcia with certain organizational aspects of this manuscript is greatly appreciated. We also thank Dr. Derek Boerboom for reading and commenting on the text and figures.

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90.  Hedin, L., McKnight, G. S., Lifka, J., Durica, J. M., and Richards, J. S.  (1987).  Tissue distribution and hormonal regulation of mRNA for regulatory and catalytic subunits of adenosine 3’, 5’-monophosphate-dependent protein kinases during ovarian follicular development and luteinization in the rat.  Endocrinology 120, 1928-1935.

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106.    Doyle, K.M., Russell, D.L., Sriraman, V., and Richards, J.S. (2004). Coordinate transcription of the ADAMTS-1 gene by LH and PR. Mol Endocrinol. (in press).

107.  Cassar, C. A., Dow, M. P., Pursley, J. R., and Smith, G. W.  (2002).  Effect of the preovulatory LH surge on bovine follicular progesterone receptor mRNA expression.  Domest. Anim. Endocrinol. 22, 179-187.

108.  Sriraman, V., Sharma, S. C., and Richards, J. S.  (2003).  Transactivation of the progesterone receptor gene in granulosa cells:  Evidence that Sp1/Sp3 binding sites in the proximal promoter play a key role in luteinizing hormone inducibility.  Mol. Endocrinol. 17, 436-449.

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112.  Stouffer, R. L.  (2002).  Pre-ovulatory events in the rhesus monkey follicle during ovulation induction.  Reprod. Biomed. 3 (Suppl), 1-4.

113.  Espey, L. L., Yoshioka, S., Russell, D. L., Robker, R. L., Fujii, S., and Richards, J. S.  (2000).  Ovarian expression of a disintegrin and metalloproteinase with thrombospondin motifs during ovulation in the gonadotropin-primed immature rat.  Biol. Reprod. 62, 1090-1095.

114.  Kuno, K., Kanada, N., Nakashima, E., Jujiki, F., Ichimura, F., and Matsushima, K.  (1997).  Molecular cloning of a gene encoding a new type of metalloproteinase-disintegrin family protein with thrombospondin motifs as an inflammation associated gene.  J. Biol. Chem. 272, 556-562.

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116.  Russell, D. L., Doyle, K. M., Ochsner, S. A., Sandy, J. D., and Richards, J. S.  (2003).  Processing and localization of ADAMTS-1 and proteolytic cleavage of versican during cumulus matrix expansion and ovulation.  J. Biol. Chem. 278, 42330-42339.

117.  Boerboom, D., Russell, D. L., Richards, J. S., and Sirois, J.  (2003).  Regulation of transcripts encoding ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin-like motifs-1) and progesterone receptor by human chorionic gonadotropin in equine preovulatory follicles.  J. Mol. Endocrinol. 31, 473-485.

117.      Mittaz, L., Russell, D. L., Wilson, T., Brasted, M., Tkalcevic, J., Salamonsen, L. A., Hertzog, P. J., and Pritchard, M. A.  (2004).  Adamts-1 is essential for the development and function of the urogenital system.  Biol. Reprod. 70, 1096-1105.

119.  Shimada, M., Nishibori, M., Yamashita, Y., Ito, J., Mori, T., and Richards, J.S. (2004). Down-regulated expression of ADAMTS-1 by progesterone receptor antagonists is associated with impaired expansion of porcine cumulus-oocyte complexes.  Endocrinology (in press).

120.  Sriraman, V., and Richards, J. S.  (2004).  Cathepsin L gene expression and promoter activation in rodent granulosa cells.  Endocrinology 145, 582-591.

121.  Ko, C., In, Y. H., and Park-Sarge, O. K. (1999).  Role of progesterone receptor activation in pituitary adenylate cyclase-activating polypeptide gene expression in rat ovary.  Endocrinology 140, 5185-5194.

122.  Park, J. I., Kim, W. J., Wang, L., Park, H. J., Lee, J., Park, J. H., Kwon, H. B., Tsafriri, A., and Chun, S. Y.  (2000).  Involvement of progesterone in gonadotrophin-induced pituitary adenylate cyclase-activating polypeptide gene expression in pre-ovulatory follicles of rat ovary.  Mol. Hum. Reprod. 6, 238-245.

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125.  Armstrong, D. T., and Grinwich, D. L.  (1972).  Blockade of spontaneous and LH-induced ovulation in rats by indomethacin, an inhibitor of prostaglandin biosynthesis.  Prostaglandins 1, 21-28.

126.  O’Grady, J. P., Caldwell, B. V., Auletta, F. J., and Speroff, L.  (1972).  The effects of an inhibitor of prostaglandin synthesis (indomethacin) on ovulation, pregnancy, and pseudopregnancy in the rabbit.  Prostaglandins 1, 97-106.

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130.  LeMaire, W. J., Leidner, R., and Marsh, J. M.  (1975).  Pre and post ovulatory changes in the concentration of prostaglandins in rat graafian follicles.  Prostaglandins 9, 221-229.

131.  Espey, L. L., Tanaka, N., Adams, R. F., and Okamura, H.  (1991).  Ovarian hydroxyeicosatetraenoic acids compared with prostanoids and steroids during ovulation in rats.  Am. J. Physiol. 260, E163-E169.

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133.  Hedin, L., Gaddy-Kurten, D., Kurten, R., DeWitt, D. L., Smith, W. L., and Richards, J. S.  (1987).  Prostaglandin endoperoxide synthase in rat ovarian follicles:  Content, cellular distribution, and evidence for hormonal induction preceding ovulation.  Endocrinology 121, 722-731.

134.    Wong, W. Y., DeWitt, D. L., Smith, W. L., and Richards, J. S.  (1989).  Rapid induction of prostaglandin endoperoxide synthase in rat preovulatory follicles by luteinizing hormone and cAMP is blocked by inhibitors of transcription and translation.  Mol. Endocrinol. 3, 1714-1723.

135.    Wong, W. Y., and Richards, J. S.  (1991).  Evidence for two antigenically distinct molecular weight variants of prostaglandin H synthase in the rat ovary.  Mol. Endocrinol. 5, 1269-1279.

136.  Sirois, J., and Richards, J. S.  (1992).  Purification and characterization of a novel, distinct isoform of prostaglandin endoperoxide synthase induced by human chorionic gonadotropin in granulosa cells of rat preovulatory follicles.  J. Biol. Chem. 267, 6382-6388.

137.    Joyce, I.M., Pendola, F.L., O’Brien, M., and Eppig, J.J. (2001). Regulation of prostaglandin-endoperoxide synthase 2 messenger ribonucleic acid in mouse granulosa cells during ovulation.  Endocrinology 142, 3187-3197.

138.    Ochsner, S.A., Day, A.J., Rugg, M.S., Breyer, R.M., Gomer, R.H., and Richards, J.S. (2003).  Disrupted function of tumor necrosis factor –a-stimulated gene 6 blockes cumulus cell-oocyte complex expansion.  Endocrinology 144, 4376-4384.

139.  Sirois, J., Simmons, D. L., and Richards, J. S.  (1992).  Hormonal regulation of messenger ribonucleic acid encoding a novel isoform of prostaglandin endoperoxide H synthase in rat preovulatory follicles.  Induction in vivo and in vitro.  J. Biol. Chem. 267, 11586-11592.

140.    Ochsner, S. A., Russell, D. L., Day, A. J., Breyer, R. M., and Richards, J. S.  (2003).  Decreased expression of tumor necrosis factor-a-stimulated gene 6 in cumulus cells of the cyclooxygenase-2 and EP2 null mice.  Endocrinology 144, 1008-1019.

141.    Schoenfelder, M., and Einspanier, R. (2003). Expression of hyaluronan synthases and corresponding hyaluronan receptors is differentially regulated during oocyte maturation in cattle.  Biol. Reprod. 69, 269-277.

142.    Kimura, N., Konno, Y., Miyoshi K., Matsumoto, H., and Sato, E. (2002). Expression of hyaluronan synthases and CD44 messenger RNAs in porcine cumulus-oocyte complexes during in vitro maturation.  Biol. Reprod. 66, 707-717

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