ABSTRACT

In recent years, there have been a number of efforts to identify genes that are expressed in mature ovarian follicles in response to an ovulatory dose of LH, or its homologue hCG. This review keys on 20 ovulation-related genes that we have identified by the molecular procedure known as differential display. The objective is to use this sampling of genes to illustrate the diversity in the temporal and spatial patterns of expression of genes in the ovary following the stimulus of this gonadal target tissue by a single glycoprotein hormone. The specific genes that are surveyed include 5-aminolevulinate synthase, early growth response protein-1, g-glutamylcysteine synthetase, cyclo-oxygenase-2, epiregulin, pituitary adenylate cyclase-activating polypeptide, tumor necrosis factor-stimulated gene-6, regulator of G-protein signaling protein-2, adrenodoxin, steroidogenic acute regulatory protein, 3a-hydroxysteroid dehydrogenase, CD63, a disintegrin and metalloproteinase with thrombospondin motifs, tissue inhibitor of metalloproteinase-1, carbonyl reductase, a G-protein-coupled receptor, pancreatitis-associated protein-III, glutathione S-transferase, and metallothionein-1. The ovulatory expression of these different genes is predominantly within the granulosa layer of mature follicles. However, there were also instances of expression in the thecal and stromal tissue of the ovary, as well as in vascular endothelial cells and in luteal tissue. The overwhelming impression is that the molecular events of ovulation are far more complex, and therefore more highly ordered, than originally imagined.

INTRODUCTION

During the past five decades there has been an increasing effort to decipher the principal factors that cause mature ovarian follicles to rupture in response to an ovulation-stimulating surge in gonadotropin. Initially, it was assumed that the mechanism of ovulation was a relatively simple phenomenon—involving a small number of regulatory factors activated by a surge in the secretion of gonadotropin. Approximately 40 years ago, when it became clear that follicular rupture was not caused by any significant increase in intrafollicular pressure [1], it was proposed that a collagenolytic enzyme might degrade the follicle wall and cause ovulation [2]. In subsequent years, much of the work on ovulation focused on the search for such a proteolytic enzyme, under the assumption that there might be a relatively simple chain of events between the initial action of gonadotropin on the membrane receptors in mature follicles and the ultimate rupture of the follicle wall. However, several decades passed without the identification of a specific collagenolytic enzyme capable of causing ovulation.

In the meantime, radioimmunoassays became available for the detection of gonadotropin-induced increases in ovarian steroids and prostanoids. These new analytical approaches led to considerable efforts to link progesterone and several prostaglandins to the mechanism of ovulation. To date, Boolean searches of MEDLINE identify more than 7,000 publications that associate “progesterone” with “ovulation” and more than 1600 publications that associate “prostaglandin” with “ovulation.” Yet, all of this effort has yielded very little information about the specific roles of progesterone and prostaglandins in the biochemical mechanisms controlling ovulation. Nevertheless, enumerable reports of inhibition of ovarian prostaglandin synthesis and ovulation by non-steroidal anti-inflammatory agents has led to the hypothesis that the ovulatory process involves ovarian changes that are comparable to an acute inflammatory reaction.

More recently, molecular biology and recombinant DNA technologies have led investigators to a number of novel agents that contribute to the ovulatory process. These new discoveries include the detection of gene expression for TIMP-1 [3], VEGF [4], IL-1 [5], and other agents. Also, 7 years ago, we began identifying additional ovulation-related genes by applying the molecular procedure known as differential display [6]. Subsequently, other investigators have reported similar success by using the microarray procedure [7]. The net result of these studies, combined with information from recent knockout-mouse models (and especially knockout models for progesterone receptor) [8-12] has brought about a dramatic change in our conception of the ovulatory process. Ovulation is now considered to be the consequence of a highly complex assortment of gene expressions [13-17].

In the present report, we review the temporal and spatial patterns of expression of 20 ovarian genes during the ovulatory and post-ovulatory period. This minireview is not intended to be comprehensive. It does not cover all of the genes that we have identified, nor does it assess the patterns of expression of numerous genes that other workers have recently identified as components of the ovulatory process. Instead, the focus is on those specific genes that we reported at the SSR Meeting in Ottawa in 2001. The genes are presented in an order starting with those that were transcribed at the earliest phase of the ovulatory process and ending with those that were not expressed until after ovulation, when the follicles were transforming into corpora lutea. They are categorized under subheadings based on their principal site of transcription in the ovaries. In instances when a gene, such as g-glutamylcysteine synthetase (GCS) was expressed in more than one cell type, the secondary site of transcription is so indicated in the text. Also, in view of the inflammatory-like nature of the ovulatory process, the text stresses those genes that have been associated with inflammatory reactions.

The methodology for inducing ovulation in immature rats has been described elsewhere [18]. In brief, animals weighing 40-50 g (i.e., approx 24-25 days old) were administered 10 IU of eCG sc to induce folliculogenesis. Subsequently, 48 hours later, the ovulatory process was initiated by injecting 10 IU of hCG sc. This treatment usually results in a superovulation rate of 60-70 ruptured follicles, with most of the ovulations occurring at 12-14 h after administration of the hCG. In the present report, experimental tissue included ovaries taken at specific intervals between 0-72 h after hCG (Table 1).

Details about the differential display procedure that was used to discover the genes included in this review have also been described elsewhere [18]. In brief, differential display is an rtPCR-based method that utilizes special primers that amplify radioactively labeled cDNAs representing subpopulations of mRNAs from control and experimental tissues. The PCR primers and other differential display components were obtained from GenHunter Corporation (Nashville, TN). The amplified cDNAs were displayed by PAGE, and any differentially amplified cDNA bands were extracted from the acrylamide gels and used as probes in Northern analyses to confirm ovulation-specific gene expression. The intensity of the signals from the different lanes of the Northern blots provided reliable information about the temporal pattern of expression of each of the genes. The pattern of the signal from in situ hybridization of radioactively labeled probes to histological sections of rat ovaries provided information about the spatial pattern of expression of each of the genes.

Collectively, the information gained from Northern analyses and from in situ hybridization has led to a number of useful deductions about the nature of the ovarian target tissue response to a single dose of gonadotropin. Ten of these insights are enumerated at the conclusion of this review.

GENE EXPRESSION IN THE GRANULOSA LAYER

5-Aminolevulinate synthase

Among the 20 genes covered in this review, 5-aminolevulinate synthase (ALAS, i.e., aminolevulinic acid synthase) was one of the first whose transcription was up-regulated in follicular tissue during the early stages of the ovulatory process. In control ovaries (i.e., at 0 h after hCG) there was a limited amount of ALAS expressed randomly in the granulosa layer, as well as in the theca interna of large follicles. ALAS began to increase in the granulosa layer as early as 30 minutes after hCG, and it reached a peak at 1 h after the administration of hCG (Table 1). Subsequently, it declined sharply, returning to a level below the 0-h control value by 12 h after hCG, at the time when the mature ovarian follicles are beginning to rupture. However, preliminary data from our in situ hybridization studies suggest there is a secondary phase of moderate, but significant, expression of the ALAS gene in the corpora lutea that develop from ruptured follicles.

ALAS is perhaps best known for its role in catalyzing the condensation of glycine and succinyl-CoA to yield 5-aminolevulinate, a universal precursor of tetrapyrrole compounds that function in a variety of reactions including the biosynthesis of heme, the transport of single electrons, and the catalysis of redox reactions [19-23]. It is particularly relevant to note that ALAS has been associated with mitochondrial P450 cytochromes and with steroid metabolism, [19, 23, 24], i.e., with factors that are known to be involved in ovulation. Also, there is limited information to suggest that ALAS expression is associated with acute inflammatory reactions [25, 26]. It is possible that ALAS expression is an early event in the so-called “acute phase response,” which is a cascade of overlapping inflammatory pathways that are activated by tissue injury and/or infection [27, 28]. (This acute phase response includes gene expression of both pro-inflammatory and anti-inflammatory mediators.)

Early growth response protein-1

Similar to ALAS, early growth response protein-1 (Egr-1) mRNA begins to increase in the granulosa layer within 30 min after injection of hCG into the experimental animals [29]. However, Egr-1 mRNA reached a peak several h after ALAS, i.e., at 2-4 h after hCG (Table 1). Subsequently, Egr-1 gene expression remained moderately elevated through 12 h after hCG (when the follicles begin to rupture), but it declined to the 0-h control level by 24 h after hCG (when the ruptured follicles were transforming into fresh corpora lutea).

Egr-1 is a zinc-finger transcription factor that serves as a master switch to promote the expression of numerous genes important in inflammation, vascular hyperpermeability, coagulation, and other events associated with tissue damage [29-32]. After it is translated, the Egr-1 protein translocates into the nucleus and regulates the expression of an estimated 80-100 other genes, increasing their transcription rates as much as 100-fold [33, 34]. Downstream target genes include proinflammatory factors such as interleukin-1b [32] and tumor necrosis factor-a (TNF-a) [31], two agents that have been implicated as mediators of the ovulatory process [35, 36].

g-Glutamylcysteine synthetase

A moderate amount of g-glutamylcysteine synthetase (GCS) mRNA was distributed randomly (i.e., non-uniformly) in the granulosa layer of mature follicles even before the ovulatory process was induced by hCG (Table 1). In addition, there was limited, but distinct, expression of GCS mRNA in the theca interna surrounding the granulosa cells. After stimulation of the ovaries with hCG, the onset of ovarian GCS mRNA expression in mural granulosa cells was slightly later than that of ALAS and Egr-1. There was a transient increase during the first several hours of the ovulatory process in the granulosa of most, but not all, of the follicles that appeared to be large enough to progress to rupture. However, expression was minimal in most follicles at 12 h after hCG, when the follicles began to rupture. GCS expression was substantially elevated between 4-8 h after hCG specifically in the thecal tissue as well as in some localized areas of the ovarian stroma.

GCS is a zinc metalloprotein that serves as the rate-limiting enzyme for synthesis of glutathione [37, 38]. Glutathione is a ubiquitous tripeptide that protects cells against oxidative stress during the acute-phase response of inflammatory reactions and other conditions that cause cellular injury [39-42]. There is limited evidence that glutathione is synthesized in the ovary near the time of ovulation [43, 44].

Cyclo-oxygenase-2

Ovarian cyclo-oxygenase-2 (COX-2) mRNA increased sharply at 4 h after hCG, and it declined just as rapidly—approaching the 0-h control level by 8 h after hCG (Table 1). COX-2 mRNA expression was confined to the granulosa layer of the mature follicles, but its distribution within the individual follicles was not as uniform as the pattern of transcription of some of the other genes.

COX-2 (but not the isoform COX-1) has been firmly established as a component of the ovulatory process [45]. Beyond its expression and action in the mural granulosa, COX-2 reportedly has a functional role in cumulus cells [12, 46, 47]. It is well known that COX-2, which is induced predominantly during inflammation, is the key enzyme for prostaglandin synthesis [48]. Current thinking is that acute inflammatory reactions are self-limiting reactions that resolve themselves [49, 50]. That is to say, although COX-2 and prostaglandins appear to mediate pro-inflammatory effects as the reaction develops, they are thought to exert anti-inflammatory properties as the reaction resolves.

Epiregulin

The temporal pattern of expression of epiregulin was identical to that of COX-2 (Table 1), and its distribution within the granulosa layer was also somewhat irregular. Epiregulin is a relatively new member of the epidermal growth factor (EGF) family [51, 52]. Although its functions are not yet clear, it has been associated with Egr-1 mRNA expression [53], and it reportedly increases the expression of COX-2 mRNA and protein [54].

Pituitary adenylate cyclase-activating polypeptide

Pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA increased sharply between 2 and 4 h after treatment of the animals with hCG (Table 1). It declined just as rapidly between 8 and 12 h after hCG. The PACAP mRNA appeared as two distinct isoforms on the Northern blots. In situ hybridization showed that PACAP was distributed unevenly in the granulosa cells of the large ovarian follicles.

PACAP is a 38-amino acid peptide that belongs to the VIP, GHRH, glucagon, secretin superfamily [55-57]. Although it was initially isolated from hypothalamic extracts, it is widely distributed in body tissues. The present data on the pattern of expression of PACAP mRNA in ovulatory follicles confirms other recent studies on ovarian PACAP [58-61]. These reports, along with our own observation that a progesterone-inhibiting dose of epostane impairs expression of the ovarian PACAP gene (unpublished observation), indicate that PACAP mRNA expression is dependent on the hCG-induced expression of progesterone during the ovulatory process. It is also worth noting that PACAP reportedly inhibits the production of interleukins, nitric oxide, and TNF-a, and thereby attenuates the inflammatory response in experimental tissues [62].

Tumor necrosis factor-stimulated gene-6

Transcription of tumor necrosis factor-stimulated gene-6 (TSG-6) was up-regulated substantially at 4-8 h after the ovaries had been stimulated by hCG [63, 64] (Table 1). However, by 12 h after hCG, when the follicles were just beginning to rupture, the TSG-6 mRNA expression had down-regulated to only 20% of its maximum level. The newly expressed TSG-6 mRNA was distributed rather evenly throughout the granulosa layer and the cumulus cells.

The cytokines interleukin-1 and tumor necrosis factor-a activate a number of inflammation-related genes, including TSG-6 [63, 65]. Even though TSG-6 is a common component of inflammatory reactions, this agent might actually exert an anti-inflammatory action by linking to inter-a-inhibitor (IaI), which is known to covalently bind to and stabilize hyaluronan in the cumulus matrix [66, 67]. Therefore, since hyaluronan promotes inflammation, it is possible that TSG-6 exerts a negative feedback action by neutralizing hyaluronan-induced inflammatory stress on the extracellular matrix of the cumulus cell-oocyte complex during the ovulatory process [63].

Regulator of G-protein signaling protein-2

Regulator of G-protein signaling protein-2 (RGS2) gene expression in the rat ovary followed a temporal and spatial pattern that was similar to TSG-6, except the RGS2 mRNA appeared to be especially dense along the antral border of the mural granulosa as well as within the cumulus mass surrounding the oocyte[68] (Table 1). RGS2 has been characterized as a GTPase-activating protein that is thought to attenuate cell signaling by hydrolyzing GTP to GDP on the a-subunit of an activated G-protein [68]. However, it is possible that RGS proteins might also influence signaling pathways in ways beyond the mere re-coupling of G-protein subunits [69, 70]. In any event, it is evident that hormonal stimulation of the ovulatory process induces a significant increase in ovarian RGS2, and it is likely this signaling modulator has important function(s) in the mechanism of ovulation.

Adrenodoxin

Adrenodoxin mRNA was expressed moderately throughout the ovaries even before the experimental animals were treated with hCG (Table 1). This transcript began to increase significantly in the granulosa layer within 2 h after the administration of hCG. Adrenodoxin mRNA expression continued in post-ovulatory lutein tissue after the follicles had ruptured.

Adrenodoxin is an iron-sulfur protein that transfers electrons to the mitochondrial forms of cytochrome P450 during cAMP-mediated steroid hormone biosynthesis [71-74]. This steroidogenic function includes chemical cross-linkage with cholesterol side-chain cleavage cytochrome P450 (P450scc) [75]. Adrenodoxin and cytochrome P450scc are co-expressed with steroidogenic-acute regulatory protein (StAR) [76, 77].

Steroidogenic acute regulatory protein

StAR mRNA was expressed in a pattern comparable to adrenodoxin, except there was negligible transcription in the 0-h control ovaries (Table 1). The expression of this steroidogenesis-related gene reached an initial peak at 4 h after the administration of hCG. Subsequently, it declined at 12-24 h after hCG, (i.e., during the first hours of luteinization). However, StAR mRNA eventually reached a second, higher peak at 144 h (data not presented in Table 1) after the administration of hCG, when the corpora lutea are synthesizing substantial amounts of progesterone.

StAR is an indispensable protein that regulates the rate-limiting step in steroid synthesis, namely the conversion of cholesterol into pregnenolone. It shuttles cholesterol into mitochondria where cytochrome P450scc activity converts this steroid substrate into pregnenolone [78, 79]. The involvement of StAR in the ovulatory increase in ovarian progesterone synthesis has been firmly established [80, 81].

3a-Hydroxysteroid dehydrogenase

A slight, but discernible, amount of 3a-hydroxysteroid dehydrogenase (3a-HSD) mRNA was visible in the thin layer of theca interna cells of mature follicles even before the ovaries were stimulated with hCG [82]. Expression increased significantly in the granulosa layer beginning 2 h after treatment of the animals with hCG (Table 1). 3a-HSD mRNA reached a peak at 8 h and declined thereafter, although a limited amount was still visible in luteal tissue.

Mammalian 3a-HSD is quite different from 3b-HSD, which is well known for its anabolic role in the synthesis of ovarian progesterone at the beginning of the ovulatory process [82]. 3a-HSD acts with NADPH to catalyze the oxidoreduction of steroids and other compounds that possess aldehyde and ketone functional groups [83, 84]. Therefore, a possible function of the oxidoreductase enzyme that is translated from the 3a-HSD mRNA might be to reduce the toxic aldehyde and ketone components of the steroids and eicosanoids that accumulate in the mammalian ovary at the time of ovulation.

CD63

Cell surface antigen CD63 mRNA was expressed constitutively in the thecal and stromal tissue of the ovary, but not in the granulosa layer. However, this mRNA increased significantly in the granulosa cells in a transient pattern between 4-8 h after hCG treatment of the animals (Table 1). CD63 is a member of the tetraspanin superfamily that has been associated with activation of membrane proteins [85]. Although little is known about the specific mechanisms of action of such cell surface antigens, these proteins have been associated with diverse processes such as cell differentiation and proliferation [86]. It might be relevant that CD63 reportedly is rapidly expressed on the surface of endothelial cells in areas of vascular injury and inflammation [87], because it is well known that the ovulatory transformation of a mature ovarian follicle into a mass of luteal tissue is an inflammatory process that involves substantial modification of the ovarian vasculature [88].

Cyclic AMP-specific phosphodiesterase

Cyclic AMP-specific phosphodiesterease (cAMP-specific PDE) mRNA increased in the ovary beginning approximately 2 h after the administration of hCG (Table 1). cAMP-specific PDE expression reached a peak at 8 h after hCG, and then abated to the 0-h control level by 24 h after hCG. Our preliminary data from in situ hybridization tests have not made it clear whether cAMP-specific PDE mRNA is expressed primarily in the granulosa or in the thecal layer of the follicles. Other reports indicate that such a PDE may be associated with granulosa cells [89] and/or be induced by a tyrosine kinase in LH-stimulated thecal interstitial cells from rats [90]. In view of this uncertainty, based on Northern blotting, the temporal pattern of cAMP-specific PDE expression is tentatively grouped in the text and table of this manuscript with genes that are known to be expressed in the granulosa layer. In any case, the evidence that this gene is important for fertility in mice [89] and is involved in inflammatory reactions [91, 92] supports the idea of a significant role for such a PDE in the ovulatory process.

A disintegrin and metalloproteinase with thrombospondin motifs

The temporal pattern of expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-1) mRNA was different from the previously mentioned genes. Although it began to increase significantly in the granulosa layer by 4 h after hCG, ADAMTS-1 expression did not reach a peak until 12 h after hCG, at the time when rat follicles begin to rupture [93] (Table 1). Also, unlike all of the other genes in this survey except PACAP, ADAMTS-1 transcription was dependent on the usual increase in ovarian progesterone synthesis and on the up-regulation of ovarian progesterone receptor during ovulation [9, 93].

ADAMTS-1 is a recently discovered member of the ADAM family of metalloproteinases [94]. However, ADAMTS-1 differs from the regular members of the ADAM family, not only by the multiple copies of thrombospondin-type-1 repeats it carries, but also by the fact that it is readily secreted into the extracellular matrix instead of remaining anchored to the cell surface [94, 95]. Experiments that show ovarian progesterone synthesis is essential for the expression of this metalloproteinase gene (along with the cathepsin L gene [9]) provide circumstantial evidence that such proteolytic enzymes are important in ovulation [9, 93]. Furthermore, the reported association of ADAMTS-1 with various inflammatory processes [94, 95], is additional support for the idea that this metalloproteinase has a critical role in the digestion of follicular connective tissue at the time of ovulation.

GENE EXPRESSION IN THE THECAL LAYERS AND STROMA

Tissue inhibitor of metalloproteinase-1

Tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA is expressed in a temporal pattern that is similar to ADAMTS-1 mRNA, with a peak in expression at 12 h after hCG administration (Table 1). However, TIMP-1 is quite different from ADAMTS-1 in that most of the transcription of this inhibitor takes place in the thecal and stromal tissue of the ovary rather than in the granulosa layer. The increase in ovarian TIMP-1 at the time of ovulation has been recognized for quite some time [96-98], and its probable role in ovulation is to moderate the degradative action of ADAMTS-1 and other metalloproteinases in the ovary.

Carbonyl reductase

Carbonyl reductase (CBR) mRNA was hardly detectable at 0 h after hCG (Table 1). However, transcription began to increase by 2 h after hCG administration, reached a peak at 8 h, and declined to 0-h control level by 24 h after hCG [18]. Its spatial distribution was limited to the ovarian thecal and stromal areas, with negligible expression in the granulosa layer.

Similar to 3a-HSD, CBR is an NADPH-dependent aldo-keto reductase with broad specificity for converting carbonyl compounds into alcohols [99]. Therefore, analogous to the proposed function of 3a-HSD in the granulosa layer, the ovulatory increase in CBR might serve as a local protective response to the substantial increases in steroids and prostanoids in the interstitial tissue of the ovary at the time of ovulation [18].

G-protein-coupled receptor

In a temporal and spatial pattern quite similar to CBR, the differential display procedure revealed ovarian transcription of an mRNA that codes for a G-protein-coupled receptor (GPCR) (Table 1). The ligand for this receptor has not been identified, and there is almost no information about the properties and functions of this membrane receptor. A recent study of this GPCR in rat brain indicates that the receptor is a 422-amino acid protein that is induced by glucocorticoids [100]. However, the regulation of this receptor in the ovary and its potential role in the ovulatory process remain to be determined.

GENE EXPRESSION IN VASCULAR ENDOTHELIUM

Pancreatitis-associated protein-III

The temporal pattern of expression of mRNA for pancreatitis-associated protein-III (PAP-III) was comparable to the other genes that exhibited maximum expression at 8 h after the administration of hCG (Table 1). However, the spatial pattern of expression of PAP-III was completely different from any of the other genes that have been considered in this review. PAP-III mRNA expression was limited mainly to the hilar region of the ovarian stroma, with most of the signal emanating from endothelial cells that lined the inner walls of blood vessels, and from some of the small secondary follicles in the ovarian stroma.

PAP-III, a member of the C-type lectin supergene family [101], and related genes are reportedly expressed in a variety of tissues other than the ovary [102]. However, the physiological role of PAP-III remains to be established. There is some evidence to suggest that PAP-type proteins might act as endogenous protective agents during infection and inflammation [103, 104]. Therefore, it is plausible that ovarian PAP-III mRNA might be expressed in conjunction with a protective response to the hyperemia, exudation, proteolysis, and inflammation that characterize the pathophysiological events of ovulation.

GENE EXPRESSION IN THE CORPUS LUTEUM

Glutathione S-transferase

As described above, there was evidence from this study that several of the genes (namely, ALAS and StAR), which were expressed during the ovulatory process, were also transcribed in substantial amounts in post-ovulatory lutein tissue. In contrast, glutathione S-transferase, (GST) mRNA was hardly detectable during the peri-ovulatory period (Table 1). However, transcription of this glutathione-related gene increased progressively in the maturing lutein tissue.

GST is a member of a large family of soluble enzymes that catalyze the conjugation of glutathione to electrophilic compounds such as unsaturated carbonyls that periodically cause local toxicity in all forms of organisms [105, 106]. The particular isoform of GST reported here is of the T-1 variety. The conjugation of GSTs to electrophiles is critical for cellular protection against oxidative stress that arises during naturally occurring protective responses such as inflammatory reactions [107-109]. However, it is worth noting that conjugation of substrates with glutathione can also result in bio-activation of compounds [110].

Metallothionein-1

Similar to GST, metallothionein-1 (MT-1) mRNA was not expressed in any significant amount between the time when hCG was administered to the animals and the time the follicles began rupturing (i.e., about 12 h after hCG) (Table 1). Nevertheless, this gene was transcribed at a high rate after ovulation—reaching a peak at 6 days after hCG and remaining elevated in the luteal tissue in the pseudopregnant rats until at least 12 days after hCG.

After 45 years of investigation of MTs, and after more than 5,000 publications that discuss this thiol agent, the precise biological role of MT-1 remains uncertain. MT-1 is a rather ubiquitous zinc-binding protein that can chelate and release heavy metals during the enzymatic regulation of cellular homeostasis [111]. Therefore, it is tempting to speculate that MT-1 functions to regulate the activity of zinc-requiring metalloproteinases such as ADAMTS-1 during the ovulatory process. On the other hand, it is also possible that ovarian MT-1 is involved in some way with steroid synthesis and/or with inflammatory processes [111-113]. In any case, its latent expression is clearly initiated by an ovulatory surge in gonadotropins that act on the ovary.

CONCLUSIONS

The above information about gene expression in the rat ovary provides the basis for a number of generalizations about the nature of the ovulatory process in mammals. Ten of these conclusions are itemized below.

(1) Ovulation is a more complicated process than originally thought. The gonadotropin surge (or hCG) induces the sequential transcription of a large number of genes. Eventually, we may find there to be more than 100 genes differentially expressed (either as up-regulated or down-regulated) in the ovary during the ovulatory process. In any case, the process involves a cascade of highly orchestrated gene expression in response to stimulation of the ovary by a single hormone.

(2) From this survey, some patterns of ovarian gene expression are beginning to emerge. A number of the genes (e.g., ALAS, PACAP, adrenodoxin, StAR and MT-1) have been linked to steroid metabolism, while others (e.g., COX-2 and epiregulin) are involved with prostanoid synthesis. Several genes (e.g., ADAMTS-1 and TIMP-1) are commonly associated with metalloproteinase activity. Others (such as GST, GCS, 3a-HSD and CBR) are concerned with glutathione metabolism and/or protection against oxidative stress. Therefore, as more and more ovulation-specific genes are identified, it might be practical to establish a number of functional categories to help decipher and organize the diverse, but interconnected events within the overall process.

(3) Based on the 20 genes covered in this review, the data suggest that the majority of the genes that respond to the LH/hCG surge are in the granulosa layer, beginning with the transcription of genes such as ALAS and Egr-1 within 1 h. This is not surprising since a substantial proportion of the ovarian LH/hCG receptors are located in the plasma membranes of the granulosa cells that comprise this inner area of the follicle. However, this does not preclude or exclude any significant role for LH action via its receptors on the thecal cells—action that might impact the function of granulosa cells and vice versa during ovulation.

(4) The spatial pattern of expression of genes in the stratum granulosum indicates that most of the response to stimulation by gonadotropin occurs rather uniformly throughout this inner layer of the mature follicles. This homogeneous pattern suggests that the biochemical events of ovulation are not limited to the apical-most region of the follicle, where rupture occurs. That is to say, there is no apparent metabolic polarity between the apex and the base of ovulatory follicles.

(5) The data show that mRNA expression is also uniform in the mural granulosa versus the cumulus mass. This implies certain functional similarities between these two spatially distinct follicular cells. However, it should be noted that in one case, namely TSG-6, the signal for gene expression appeared to be significantly more intense in the cumulus cells than in the mural granulosa.

(6) A number of the genes (e.g., TSG-6, RGS2, epiregulin, COX-2, PACAP, 3a-HSD, CD63, CBR, PAP-III, adrenodoxin, cAMP-specific PDE and GPCR) are transcribed in a transient pattern, with maximum expression occurring at approximately 4-8 h after the ovulatory process is initiated by hCG. This pattern is comparable to the temporal pattern of prostaglandin synthesis in ovulatory follicles, suggesting that some of the genes might have a correlation with (but not necessarily a dependency on) ovarian prostanoid production at the time of ovulation.

(7) It is well known that indomethacin (an inhibitor of prostanoid synthesis) and epostane (a blocker of steroid synthesis) are effective inhibitors of ovulation. In parallel studies to determine whether either of these two anti-ovulatory agents could impair transcription of any of the ovulation-related genes in this study, only expression of PACAP and ADAMTS-1 genes have been identified as dependent on an elevation in ovarian progesterone levels.

(8) Some of the genes (e.g., ALAS and StAR) exhibited transcription with preliminary peaks before the follicles ruptured, followed by secondary peaks throughout the post-ovulatory luteal phase. In contrast, the ‘luteal gene’ MT-1 was not activated until after the follicles had ruptured. In either case, the latent expression of genes in luteal tissue must be directly, or indirectly, dependent on the original stimulus that initiated the ovulatory process.

(9) It is generally believed that acute inflammatory reactions are complicated by paradoxical metabolic processes that simultaneously cause damage and repair of the affected tissue [15]. At the time of follicular rupture, the more destructive events are thought to be dominant; but, subsequently, wound-healing processes presumably intensify in order to repair the ovarian damage that occurred during the ovulatory process. The present data do not make it clear whether early gene expression might contribute primarily to the destructive events, while post-ovulatory gene expression might be more important for tissue repair. Another consideration that requires further assessment is whether the protein products of genes that are expressed in the granulosa layer might be responsible primarily for destruction of the follicle wall, while the products of genes transcribed in the thecal tissue and ovarian stroma might be more relevant to the healing process.

(10) The scientific literature reveals that most of the genes that have been included in this assessment have been implicated as components of inflammatory reactions. Genes such as Egr-1, ALAS, GCS, COX-2, cAMP-SP, PAP-III, ADAMTS-1 and MT-1 reportedly contribute to inflammation, while GST, TSG-6, TIMP-1 and possibly PACAP and CD63 appear to moderate and/or protect against the stress of acute inflammatory reactions. Collectively, the evidence for expression of these genes provides further support for the hypothesis that an acute inflammatory reaction occurs in the ovary at the time of ovulation.

ACKNOWLEDGMENTS

The authors are indebted to Bogdan Vladu, Molly Skelsey, Takeshi Ujioka, Shinya Yoshioka, Janie Healy, Darrell Russell, and Claire Lo for their reliable assistance in obtaining the data presented in this review.

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TABLE 1. Temporal and Spatial Expression of mRNA during the Peri-Ovulatory Period