cAMP response element-binding (CREB) signalling and ovarian surface epithelial cell survival.

cAMP response-element binding (CREB) transcription factors transduce cell survival responses to peptide hormones and growth factors in normal tissues and mutant CREB proteins are implicated in tumorigenesis. Ovarian cancer most frequently arises from the ovarian surface epithelium (OSE), possibly due to repeat inflammation-associated injury-repair episodes that promote neoplasia. We asked if post-receptor signalling involving the CREB family of proteins plays a role in OSE cell survival. In an ovine ovulation model, abundant expression of phospho-CREB/activating transcription factor (ATF) protein was detected immunohistochemically, strongly localised to OSE cells in the proximity of pre-ovulatory follicles. Treatment of primary sheep OSE cell cultures with LH stimulated cAMP accumulation and reduced apoptosis (caspase 3/7 activity) in response to serum withdrawal. When OSE cells were infected with an adenovirus containing a CRE-luciferase construct, exposure to LH and FSH induced CRE-directed transcription. Finally, when a non-phosphorylatable mutant of CREB (Ad CREB(S133A)) was adenovirally expressed, apoptosis measured by activation of caspases was increased several fold relative to that caused by transfection with wild-type CREB (Ad CREB(WT)) or lacZ (Ad lacZ). To test the potential clinical relevance of these findings, we expressed mutant CREB protein in normal human OSE cells from four women and a series of cell lines derived from human ovarian cancers. Infection with Ad CREB(S133A) markedly increased apoptosis in normal human OSE but had no detectable effect on apoptosis in any of the cancer cell lines. We conclude that CREB/ATF signalling is important for the maintenance of OSE cell survival in vitro and is altered in human cell lines derived from ovarian cancers.

Synergism between FSH and activin in the regulation of proliferating cell nuclear antigen (PCNA) and cyclin D2 expression in rat granulosa cells.

Follicular development is associated with both proliferation and differentiation of granulosa cells under the control of FSH. We show that regulation of genes involved in cellular proliferation by FSH can be functionally separated from the regulation of genes involved in granulosa cell differentiation by synergistic actions of activin and T. Incubation of undifferentiated rat granulosa cells with FSH, forskolin, activin-A, or T alone did not influence either the expression of the proliferation-associated genes cyclin D2 and proliferating cell nuclear antigen or the differentiation-associated genes P450 aromatase, LH receptor, P450 cholesterol side-chain cleavage enzyme, and 3 beta-hydroxysteroid dehydrogenase. However, when granulosa cells were stimulated with either FSH or forskolin in the presence of activin-A, significant increases (P < 0.05) were observed for cyclin D2 and proliferating cell nuclear antigen at both the mRNA and protein levels as well as mRNAs for P450 aromatase, LH receptor, P450 cholesterol side-chain cleavage enzyme and 3 beta-hydroxysteroid dehydrogenase. Although T synergized with FSH to increase the expression of mRNAs for P450 aromatase, LH receptor, P450 cholesterol side-chain cleavage enzyme, and 3 beta-hydroxysteroid dehydrogenase, it did not interact with FSH to increase the expression of mRNAs for cyclin D2 and proliferating cell nuclear antigen. The differences in the actions of activin and T could provide a cellular mechanism by which FSH-regulated granulosa cell proliferation could be functionally separated from FSH-regulated granulosa cell differentiation.

Follicle selection in primates: "many are called but few are chosen".

During the follicular phase of humans and most nonhuman primates, a single preovulatory follicle usually matures each menstrual cycle. The observation that numerous preovulatory follicles may be stimulated to mature when exogenous gonadotropins are administered indicates that there must be a precise and highly reproducible mechanism by which only one of the many follicles capable of ovulating actually does so. The goal of this review is to summarize past and current research which indicates that follicle selection in primates is the result of an exquisitely sensitive interplay between gonadotropin secretion by the pituitary gland, steroid production by the ovary, and maturation-dependent alterations of the ovary's responsiveness to gonadotropins.

Elements involved in the regulation of the StAR gene.

The steroidogenic acute regulatory protein (StAR) mediates the transfer of cholesterol from the outer to the inner mitochondrial membrane, the regulated step in steroidogenesis. A most interesting facet of this protein is the manner in which its expression is acutely regulated. In this regard, a number of studies have concentrated on the search for consensus cis regulatory elements within its promoter, and, more importantly, on whether these elements are involved in its expression. This short review will summarize some of the findings that have been reported concerning the nature of how the expression of this gene is regulated.

Adenovirus-directed expression of functional luteinizing hormone (LH) receptors in undifferentiated rat granulosa cells: evidence for differential signaling through follicle-stimulating hormone and LH receptors.

This study was conducted to determine the feasibility of using replication-defective adenovirus vectors to express receptors for LH. Two vectors were constructed, one that directs the expression of wild-type human LH receptor (LHr; AdRSVLHrwt) and another that directs the expression of the constitutively activated D578H mutant human LH receptor (AdRSVD578HLHr). When infected with AdRSVwtLHr and AdRSVD578HLHr, COS-1 cells expressed LH/hCG-binding sites as reflected by specific binding of [(125)I]hCG. To determine the ability of the vectors to confer LH responsiveness, undifferentiated rat granulosa cells, which possess only FSH receptors, were infected with AdRSVwtLHr and AdRSVD578HLHR: Expression of the constitutively activated D578H LHr increased basal (gonadotropin-independent) estrogen and progesterone production. Expression of the wild-type LHr in granulosa cells did not stimulate basal steroid production, but conferred responsiveness to exogenous LH. For both wild-type LHr and D578HLHr, the absolute levels of steroid production were dependent upon the input of viral titers. Using these vectors, we compared effects of FSH and LH receptor activation in undifferentiated granulosa cells. Stimulation of undifferentiated granulosa cells by FSH and D578HLHr, as well as activation of wild-type LHr with LH resulted in comparable production of progesterone. In contrast, estradiol production in cells stimulated with FSH was greater than that in cells that expressed either D578H receptors or wild-type LHr in the presence of LH. Analysis of messenger RNAs (mRNAs) revealed that activations of FSH and the LH receptors were comparable in the induction of alpha-inhibin and 3betahydroxysteroid dehydrogenase mRNAS: However, activation of FSH receptor led to significantly greater expression of P450 aromatase and LHr mRNAs than did activation of LHR: These results suggest that activation of FSH and LH receptors in granulosa cells may differ with respect to activating intracellular signaling pathways and stimulating gene expression.

Modifications in gonadotropin signaling: a key to understanding cyclic ovarian function.

The enigma in understanding the regulation of ovarian function throughout the menstrual cycle is that both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) act on the ovary, at least in part through the cyclic adenosine monophosphate (cAMP) intracellular signaling pathway. If cAMP is a common intracellular messenger of gonadotropic stimulation during both the follicular and the luteal phases, how can the responses of the follicle and the corpus luteum to their respective trophic hormones differ so dramatically? The actions of FSH and LH on the ovary are summarized and a mechanistic hypothesis is provided as to how ovarian cyclicity (initiation of follicular development, selection of a single preovulatory follicle, corpus luteum function, corpus luteum regression, and corpus luteum rescue during early pregnancy) could be controlled by a single intracellular messenger: cAMP.

Adenovirus-directed expression of a nonphosphorylatable mutant of CREB (cAMP response element-binding protein) adversely affects the survival, but not the differentiation, of rat granulosa cells.

Although usually considered to be a constitutively expressed protein, in the primate ovary the expression of CREB (cAMP response element-binding protein) is extinguished after ovulation, and its loss is temporally associated with the cessation of proliferation of luteal cells and the ultimate commitment of the corpus luteum to undergo regression. To determine the cellular consequences of the loss of CREB expression, we expressed a nonphosphorylatable mutant of CREB (CREB M1) in primary cultures of rat granulosa cells using a replication-defective adenovirus vector. Expression of CREB M1 did not block granulosa cell differentiation as assessed by acquisition of the ability to produce estrogen and progesterone in response to FSH or forskolin. However, granulosa cells expressing CREB M1, but not adenovirus-directed beta-galactosidase or enhanced green fluorescent protein, exhibited a 35% reduction in viability that was further reduced to 65% after stimulation with 10 microM forskolin. These results demonstrate that the trophic effects of cAMP (proliferation and survival) on ovarian granulosa cells are functionally separate from the effects of cAMP on differentiation and provide novel evidence that CREB may function as a cell survival factor in the ovary. The separation of signaling pathways that govern differentiation and survival in the ovary thereby provides a mechanism by which progesterone production, which is absolutely essential for the maintenance of pregnancy, can continue despite the cessation of proliferation of luteal cells and their commitment to cell death (luteolysis).

Ovarian responses in women to recombinant follicle-stimulating hormone and luteinizing hormone (LH): a role for LH in the final stages of follicular maturation.

During the follicular phase of the menstrual cycle, FSH stimulates follicular growth, granulosa cell aromatase activity, induction of LH receptors on the granulosa cell membrane, and estradiol secretion. As a result of negative feedback of estradiol on the pituitary, serum FSH concentrations decline. Despite the fall in FSH concentrations, the maturing follicle continues to develop to the preovulatory stage. In a prospective randomized trial, we tested the hypothesis that a key mechanism by which the dominant follicle continues to develop in the face of decreasing concentration of FSH is by acquiring LH responsiveness. In 24 women, pituitary gonadotropin secretion was down-regulated with a GnRH agonist. Follicular growth was then stimulated with recombinant human FSH (r-hFSH) until a 14-mm follicle was identified by ultrasound. The women were then randomized to 1 of 4 groups for a 2-day period: continued r-hFSH treatment, substitution of r-hFSH with saline, low dose r-hLH (150 IU, twice daily), or high dose r-hLH (375 IU, twice daily). Serum estradiol concentrations in the women receiving saline declined by the end of the 2-day randomization period. In contrast, serum estradiol concentrations continued to rise in women receiving either r-hFSH or r-hLH compared with those in the saline-treated group (P < 0.05). Pregnancies occurred in each of the gonadotropin treatment groups. These findings indicate that once FSH initiates follicular growth, either FSH or LH is capable of sustaining follicular estradiol production. Extrapolating these findings to the normal menstrual cycle suggests that the maturing follicle may continue to develop in the presence of diminishing FSH concentrations by acquiring the capacity to respond to LH.

In vivo responses of the primate corpus luteum to luteinizing hormone and chorionic gonadotropin.

Although it is well established that the secretory activity of the corpus luteum absolutely depends on the presence of pituitary-derived luteinizing hormone (LH), it is unknown why the life span of the corpus luteum is extended during early pregnancy by the placental production of chorionic gonadotropin (CG) but regresses in the presence of LH despite the fact that CG and LH have similar actions on the corpus luteum. To compare the responses of the corpus luteum to LH and human CG (hCG), cynomolgus monkeys whose endogenous gonadotropin secretion was blocked during the luteal phase of the menstrual cycle with a gonadotropin-releasing hormone antagonist were i.v. infused with either LH or CG. Infusion of LH at a constant rate overcame the gonadotropin-releasing hormone antagonist-mediated premature luteal regression but failed to prolong the functional life span of the corpus luteum. Continuous infusions of hCG did not effect a pregnancy-like pattern of gonadotropin secretion, but the functional life span of the corpus luteun was extended in two of three animals. Infusion of either LH or hCG in an exponentially increasing manner prolonged the functional life span of the corpus luteum beyond its normal duration. These results indicate that luteal regression at the termination of nonfertile menstrual cycles is caused by a large reduction in the responsiveness of the aging corpus luteum to LH, which can be overcome by elevated concentrations of either LH or CG.

Cyclic adenosine monophosphate signaling in the primate corpus luteum: maintenance of protein kinase A activity throughout the luteal phase of the menstrual cycle.

Recent studies from our laboratory (Endocrinology 136:4762-4768, 1995) demonstrating that the expression of cAMP-dependent nuclear transcription factor CREB (cAMP response element binding protein) is lost following ovulation in macaques has revealed a novel mechanism by which the cytoplasmic and nuclear actions the cAMP-protein kinase A (PKA) intracellular signaling system may be regulated independently. Implicit in this hypothesis is the assumption that PKA activity is maintained throughout the luteal phase of the menstrual cycle, yet to date there have been no published reports regarding PKA activity in the primate corpus luteum. PKA activity was assessed by the incorporation of 32P from radiolabeled ATP into a PKA-specific peptide substrate (kemptide) in the presence or absence of cAMP. Luteal cytosolic fractions were obtained from corpora lutea collected during the spontaneous luteal phase (days 3-5, 7-8, 10-11, 13-15, and postmenses) or obtained from animals on days 11 or 16 of the luteal phase after the animals received seven days of exogenous human CG (hCG) treatment. Examination of PKA activity in luteal slices from various aged CL maintained in short-term organ culture in the presence or absence of recombinant cynomolgus monkey LH was also performed. There were no significant differences in basal or cAMP-stimulated PKA activities in corpora lutea collected throughout the spontaneous luteal phase. Further, Western immunoblot analyses of the catalytic subunit of PKA (PKA C alpha) in corpora lutea collected throughout the luteal phase revealed immunoreactive protein bands with similar intensities. In vitro addition of recombinant cynomolgus LH and dibutyryl cAMP stimulated PKA activity in corpora lutea collected during the early, mid, and late luteal phases. In corpora lutea obtained from animals treated with hCG during the midluteal phase, basal PKA activity was decreased 65% as compared with untreated day 11 controls and in late luteal phase, hCG-exposed CL basal PKA activity was decreased 30% as compared with untreated day 16 controls. However, there were no measurable differences in cAMP-stimulated PKA activity in CL exposed to prior hCG treatment in vivo and Western immunoblot analyses for PKA C alpha in these tissues revealed immunoreactive protein bands that were comparable with corpora lutea collected from untreated animals. Further, immunoblot analyses for CREB in corpora lutea collected from hCG-treated animals revealed that CREB immunoreactivity remained undetectable following a treatment regimen with hCG that mimics early pregnancy. These results demonstrate that, although CREB expression ceases following ovulation, PKA activity is maintained throughout the luteal phase, which provides a mechanism by which the acute steroidogenic actions of LH may be separated from longer term trophic actions that may rely the transcriptional activity of CREB.

Vascular permeability factor and vascular endothelial growth factor in ovarian hyperstimulation syndrome: a preliminary report.

OBJECTIVE: To determine whether serum levels of vascular permeability factor (VPF) are elevated in patients with ovarian hyperstimulation syndrome (OHSS) and to determine if luteinizing granulosa cells may be a source of VPF. DESIGN: Prospective observational study. SETTING: University IVF and GIFT program. PATIENTS: Eight consecutive IVF and GIFT patients at high risk for OHSS. MAIN OUTCOME MEASURES: Vascular permeability factor concentration in serum and follicular fluid. RESULTS: Serum VPF was significantly higher (15.2 +/- 4.0 pM; mean +/- SEM) on day +14 in the group who developed severe OHSS compared with those who did not. Follicular fluid VPF (171.5 +/- 18.5 pM) was approximately 100-fold greater than serum (1.7 +/- 1.3 pM) or peritoneal fluid (2.5 +/- 1.3 pM) 36 hours after hCG administration. CONCLUSION: Vascular permeability factor is elevated in patients with severe OHSS and the ovary may be a source of VPF secretion.

Luteinization in primates is accompanied by loss of a 43-kilodalton adenosine 3',5'-monophosphate response element-binding protein isoform.

Although granulosa cell differentiation and corpus luteum function are both regulated by cAMP, there are development-dependent differences, particularly at the level of gene expression and cell proliferation, between the responses of follicular granulosa cells and luteal cells to trophic hormone stimulation. In this study, we sought to determine whether these differences could be due to changes in the cellular expression of cAMP response element (CRE)-binding protein (CREB). Immunocytochemical analysis of macaque ovaries revealed a development-related alteration in the subcellular distribution of CREB-immunoreactive material. Immunoreactive CREB was present in nuclei of follicular granulosa cells from maturing follicles, whereas after ovulation and luteinization, no CREB-immunoreactive proteins were visualized in luteal cell nuclei. Anti-CREB immunoblotting of granulosa cell extracts from macaque preovulatory follicles as well as extracts of granulosa cells from luteinizing human follicles revealed a 43-kilodalton (kDa) protein, a size typical of native CREB. In contrast, whole cell extracts of monkey corpora lutea collected during the early, mid-, and late luteal phases completely lacked a 43-kDa CREB signal. The absence of 43-kDa CREB isoforms in corpora lutea was confirmed using three different antisera directed against different regions of CREB. Using a human collagenase gene CRE to probe Southwestern blots, a 43-kDa CREB was observed in follicular cell extracts, whereas no CRE-binding activity was found in corpora lutea extracts using this probe. We also sought to determine whether the loss of expression of the 43-kDa CREB isoform may be functionally correlated with the cessation of cellular proliferation that accompanies luteinization. Expression of proliferating cell nuclear antigen (PCNA), an obligatory component of DNA polymerase delta, is essential for proliferation and has been shown by others to be CRE dependent. Immunoblotting of follicle cell and luteal cell extracts with an anti-PCNA monoclonal antibody revealed PCNA expression in granulosa cells and no detectable PCNA expression in corpora lutea. These findings indicate that as follicular granulosa cells progress from the proliferative state to terminally differentiated luteal cells, there is a cessation of expression of a 43-kDa member of the CREB family of transcription factors, and there may be an association between the loss of CREB isoforms and cessation of PCNA expression.

Endometriosis impairs the efficacy of gamete intrafallopian transfer: results of a case-control study.

OBJECTIVE: To determine whether pelvic endometriosis impairs the efficacy of GIFT. DESIGN: Matched follow-up study. SETTING: University-based assisted reproduction program. PARTICIPANTS: Patients undergoing GIFT between 1987 and 1991. Cases had a primary diagnosis of endometriosis. Controls had no endometriosis and were matched with cases according to age, number of mature eggs transferred, and sperm grade. INTERVENTION: Gamete intrafallopian transfer was performed in all patients in an identical manner independent of their underlying diagnosis. MAIN OUTCOME MEASURES: Pregnancy and delivery rates. RESULTS: Of 114 laparoscopic egg retrievals performed in the endometriosis group, there were 37 pregnancies (32.5%) and 27 deliveries (23.7%). Of the 214 retrievals in the control group, there were 101 pregnancies (47.2%) and 76 deliveries (35.5%). Mantel-Haenszel estimates of relative risk indicated that endometriosis significantly impaired pregnancy and delivery rates. There was no statistically significant difference in pregnancy rates according to severity of disease among endometriosis cases. There was no statistically significant difference in pregnancy rates according to severity of disease among endometriosis cases. CONCLUSIONS: Our finding that GIFT pregnancy rates were lower in women with a primary diagnosis of endometriosis than in matched controls suggests that endometriosis is associated with reduced efficacy of GIFT.

Sperm recovery and survival: two tests that predict in vitro fertilization outcome.

OBJECTIVES: To determine if human sperm recovery during swim-up and sperm survival after 24 hours, as obtained from a screening semen specimen, are predictive of subsequent IVF and clinical pregnancy rates (PRs) and to determine if these techniques can identify men with normal semen analysis parameters and poor IVF success. DESIGN: Historical prospective study. SETTING: All semen evaluations and IVF cycles were performed at the University of Pittsburgh, Magee-Womens Hospital, Pittsburgh, Pennsylvania. PATIENTS, PARTICIPANTS: Couples undergoing IVF at Magee-Womens Hospital from August 1988 through June 1993. INTERVENTIONS: A screening semen analysis and swim-up procedure were performed on all couples undergoing IVF. The number of spermatozoa recovered after swim-up and the percentage of motile spermatozoa present after a 24-hour incubation were recorded. MAIN OUTCOME MEASURES: Fertilization and PRs were compared according to the parameters obtained from routine semen analysis, the number of spermatozoa obtained with swim-up, and the percentage of motile spermatozoa at 24 hours. RESULTS: Using chi2 or Fisher's exact test, fertilization rates were significantly different according to the number of spermatozoa recovered after swim-up (< or = 2.0 and > 2.0 x 10(6) spermatozoa recovered, 48.3% versus 71.4%) as were PRs (16.9% versus 29.8%). Similarly, the percentage of motile spermatozoa present at 24 hours (< or = 20% and > 20%) discriminated between fertilization rates (45.9% versus 65.8%) and PRs (16.4% versus 36.5%). Among a subset of men with normal semen analyses and total motile sperm counts > or = 40 x 10(6), the results from swim-up and survival discriminated between men with high and low fertilization and PRs. Receiver operating characteristic analysis revealed that swim-up results better discriminated between pregnant and nonpregnant IVF patients than sperm motility, but that the percentage of motile spermatozoa present at 24 hours was no better in this regard than sperm motility. CONCLUSIONS: The number of spermatozoa recovered after swim-up and the percentage of spermatozoa that maintain their motility after 24 hours were both helpful in assessing IVF and PRs and may be helpful in altering physicians to a subset of men having normal semen analysis parameters yet poor IVF success.

Cellular aspects of corpus luteum function in the primate.

The primate corpus luteum produces progesterone for a period of 14-16 days, at which time, in non-fertile cycles, steroidogenesis ceases and the tissue regresses. Studies completed in this laboratory have established that while luteinizing hormone (LH) is necessary to maintain luteal steroidogenesis, changes in LH secretion are not causal to luteolysis. The studies presented here demonstrate that luteal cell synthetic capacity, as reflected in steady-state levels of messenger ribonucleic acid (mRNA) encoding steroidogenic enzymes, is maximal shortly after ovulation and steadily declines thereafter, independently of progesterone and LH secretion. In addition, the loss of luteal mRNA expression for steroidogenic enzymes following LH withdrawal occurs 24 h after the decline in progesterone levels. Finally, the detection of mRNA encoding vascular endothelial growth factor within the corpus luteum throughout the luteal phase in the subhuman primate may provide the first identification of a potential secreted, non-steroidal factor responsible for the vast degree of angiogenesis that occurs within the corpus luteum.

Noncoordinated expression of luteal cell messenger ribonucleic acids during human chorionic gonadotropin stimulation of the primate corpus luteum.

In nonfertile cycles, the absolute steroidogenic capacity of the primate corpus luteum, as reflected in the expression of messenger RNA (mRNA) for the progesterone biosynthetic enzymes cytochrome P450 cholesterol side-chain cleavage (P450SCC) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), progressively declines until luteal regression. Despite this progressive loss in luteal cell function, the elaboration of CG by the implanted blastocyst is able to prolong the functional lifespan of the corpus luteum. It was the purpose of this study to investigate the relationship between aging of the primate corpus luteum and the cellular mechanisms by which the decline in luteal cell function is arrested by CG. Corpora lutea were obtained from cynomolgus monkeys on days 11 or 16 of the luteal phase after a 7-day treatment period with increasing doses of human CG (hCG) given intramuscularly beginning on days 5 or 10. Corpora lutea were also obtained from control animals on days 5, 10, 11, and 16 of the luteal phase. Human CG treatment significantly (P < 0.05) elevated both serum progesterone and estradiol levels throughout the treatment period; however, progesterone production in animals treated with hCG late in the luteal phase (days 10-16) steadily declined after the third treatment day. Expression of mRNA for P450SCC and 3 beta-HSD was markedly stimulated (P < 0.05) by hCG treatment early in the luteal phase. However, 3 beta-HSD message levels in corpora lutea from animals treated with hCG on days 10-16 were not different from those of day-16 control corpora lutea, whereas P450SCC mRNA was only minimally stimulated. There was a dramatic (P < 0.05) increase in mRNA levels for the aromatase enzyme and low density lipoprotein receptor in animals given hCG in both the early and the late luteal phase. In conclusion, there appears to be a differential responsiveness of the primate corpus luteum to hCG stimulation dependent upon luteal age. The loss in responsiveness to hCG in terms of maintenance of mRNA levels is reflective of the inability of the late luteal phase corpus luteum for continued progesterone biosynthesis in the face of heightened luteotropic stimulation.

Role of luteinizing hormone in the expression of cholesterol side-chain cleavage cytochrome P450 and 3 beta-hydroxysteroid dehydrogenase, delta 5-4 isomerase messenger ribonucleic acids in the primate corpus luteum.

It is well established that LH has an obligatory role in the acute production of progesterone by the primate corpus luteum in vivo because interruption of LH support to the corpus luteum at any time during the luteal phase is accompanied by an immediate and sustained fall in serum progesterone concentrations. However, recent studies have demonstrated that maximal steroidogenic capacity of cultured human luteal cells and maximal levels of messenger RNAs (mRNAs) for cholesterol side chain cleavage cytochrome P450 (P450scc) and 3 beta-hydroxysteroid dehydrogenase, delta 5-4 isomerase (3 beta-HSD) in luteal tissue are observed shortly after luteinization and decline thereafter throughout the remainder of the luteal phase. These findings would suggest that the role of LH in the acute regulation of progesterone production may differ from its role in the expression of mRNAs for steroidogenic enzymes. We initiated the current studies to define the role of LH upon the expression of mRNAs for P450scc and 3 beta-HSD by the primate corpus luteum. For this purpose, we treated cynomolgus monkeys with a potent GnRH antagonist for 1, 2, and 3 days during the luteal phase of the menstrual cycle and measured levels of mRNAs for P450scc and 3 beta-HSD in corpora lutea. Treatment of monkeys with the GnRH antagonist reduced bioactive LH concentrations to less than 5 ng/ml by 48 h of treatment, and LH concentrations remained less than 5 ng/ml thereafter. Serum progesterone concentrations were reduced by 74% after 1 day of antagonist treatment, 88% after 2 days of antagonist treatment, and by more than 95% after 3 days of GnRH antagonist treatment. Although progesterone secretion was markedly diminished after 24 h of antagonist treatment, there were no differences in mRNAs for P450scc and 3 beta-HSD between antagonist-treated and control animals. However, mRNAs for P450scc and 3 beta-HSD were significantly (P < 0.05) reduced after 2 days of antagonist treatment and were nearly nondetectable after 3 days of antagonist treatment. These results demonstrate a temporal dissociation of the effects of LH on the acute regulation of progesterone secretion and the maintenance of specific mRNAs involved in progesterone production. Nonetheless, the results clearly show that LH is required for the continued expression of mRNAs for P450scc and 3 beta-HSD by the primate corpus luteum.

Vascular endothelial growth factor messenger ribonucleic acid expression in the primate ovary.

We studied the distribution of messenger RNA (mRNA) that encodes for vascular endothelial growth factor (VEGF) within the primate ovary by in situ hybridization and Northern analysis to determine if the presence of mRNA for this angiogenic factor is associated with structures within the ovary in which angiogenesis is thought to play a role in development and/or function. In situ hybridization to sections of cynomolgus ovaries with a 35S-labeled antisense RNA probe revealed specific tissue localization within the follicle as well as the corpus luteum, but not stromal tissue. Intense expression of mRNA for VEGF during the late follicular phase was confined to the maturing follicle which, we presume, was destined for ovulation. Hybridization within the corpus luteum exhibited a punctate pattern suggesting that there may be specific cells within the corpus luteum that express mRNA for VEGF. The expression of mRNA for VEGF during the early and late luteal phase of the menstrual cycle was studied by Northern analysis. Messenger RNAs were detectable at approximately 3.7 and 5.0 kb positions in corpora lutea collected during the early luteal phase of the menstrual cycle (days 3-5 postovulation). No hybridization signals were observed with RNA prepared from regressing corpora lutea (1-2 days following the onset of menses). The gonadotropic regulation of the expression of mRNA for VEGF in the corpus luteum was studied by treating monkeys with a potent GnRH antagonist during the midluteal phase of the menstrual cycle. Administration of the antagonist for 1 or 2 days did not alter the expression of mRNA for VEGF in comparison to corresponding controls. However, a 3-day treatment regimen brought about a significant reduction in the levels of mRNA for VEGF (P less than 0.01). These studies demonstrate a development-related expression of mRNA for VEGF in the ovary during the menstrual cycle and are consistent with the hypothesis that VEGF may play important roles in follicle selection and corpus luteum function in primates.

Characterization of the levels of messenger ribonucleic acid that encode for luteinizing hormone receptor during the luteal phase of the primate menstrual cycle.

The present study was designed to characterize the expression of LH receptor messenger RNA (mRNA) in the primate corpus luteum throughout the luteal phase of the menstrual cycle. We obtained corpora lutea from cynomolgus monkeys at defined stages of the luteal phase. LH receptor mRNA was demonstrated in monkey ovarian sections by in situ hybridization with a 35S-labeled antisense RNA probe derived from rat LH receptor complimentary DNA. The hybridization signals were confined to thecal layers of antral follicles and corpus luteum. Using the same LH receptor cDNA, the pattern of expression of mRNA encoding for LH receptor during the luteal phase was determined by Northern blot analysis. Four species of mRNA migrating at 1.0, 4.0, 7.5, and 8.0 kilobase (kb) were identified; the 4.0 kb size mRNA species was more abundant than the other three species. Quantitative analysis of the 4.0 kb band of mRNA throughout the luteal phase by densitometry revealed that the levels of LH receptor mRNA were low during the early luteal phase (days 3-5 of the luteal phase). A progressive increase in the message levels was observed from the early luteal phase to the end of the luteal phase. By days 11-12, there was a significant increase in the message levels (less than 0.05) which further increased during the late luteal phase (days 13-15). After menstruation, the levels became undetectable. In contrast, mRNA levels for 3 beta-hydroxysteroid dehydrogenase, a key enzyme involved in luteal steroidogenesis, were high shortly after ovulation and declined throughout the remainder of the luteal phase. These results indicate that after ovulation and luteinization, the expression of mRNAs that encode for specialized luteal cell proteins is differentially regulated.

Expression of messenger ribonucleic acids that encode for 3 beta-hydroxysteroid dehydrogenase and cholesterol side-chain cleavage enzyme throughout the luteal phase of the macaque menstrual cycle.

To study further the control of the primate corpus luteum, we obtained corpora lutea from cynomolgus macaques at defined stages of the luteal phase and examined steady state mRNA levels in these corpora lutea by Northern analysis for the two major enzymes involved in progesterone biosynthesis, cytochrome P450 cholesterol side-chain cleavage (P450SCC) and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD). mRNAs for both P450SCC and 3 beta HSD were maximal or near maximal shortly after ovulation and luteinization (days 3-5 of the luteal phase). mRNA for P450SCC exhibited a slight, but nonsignificant (P greater than 0.05) decline throughout the remainder of the luteal phase and was undetectable upon luteal regression. Steady state levels of 3 beta HSD mRNA were significantly lower (P less than 0.05) from corpora lutea removed during the midluteal phase (days 7-8 of the luteal phase) than those in newly formed corpora lutea and declined to 10% of early luteal phase values by days 13-15 of the luteal phase. 3 beta HSD mRNA levels fell to nondetectable values upon luteal regression. These results reveal a paradoxical relationship between the steroidogenic activity of the primate corpus luteum in vivo and the steady state levels of the mRNAs that encode for the major enzymes involved in progesterone biosynthesis. Unlike serum progesterone concentrations, which are very low immediately after ovulation and then rise during the midluteal phase, the steady stale levels of P450SCC mRNA and 3 beta HSD appeared to be maximal or near maximal shortly after ovulation and declined throughout the remainder of the luteal phase. These findings are consistent with the notion that luteal lifespan is set at the time of ovulation and luteinization, and the decline in luteal function may be due in part to decay of specialized luteal cell mRNAs with finite half-lives.