Demonstration of the insulin receptor in vivo in rabbits and its possible role as a reservoir for the plasma hormone.

Based on studies of the interaction of insulin with its receptors in vitro, we calculated that a receptor compartment should be measurable directly in vivo. For this purpose, rabbits were injected intravenously with a labeled insulin that has low affinity for receptors in combination with a radioiodinated insulin that has high affinity for receptors. Plasma concentrations of labeled insulins were measured at selected intervals after injection. Apparent volumes of distribution were calculated by extrapolation of plasma distribution were calculated by extrapolation of plasma disappearance curves; high affinity insulins consistently distributed into spaces that were two-three times greater than those of the low affinity insulins. Injections of unlabeled pork insulin before tracer insulins decreased the distribution space of the high affinity insulin in a dose-dependent manner while having little or no effect on the distribution space of the low affinity labeled insulin. When unlabeled insulin was injected after the tracer insulins, there was an immediate rise in the plasma concentration of the high affinity insulin with only a slight change in the plasma concentration of the low affinity insulin. These results demonstrate that high affinity insulins distribute into a body compartment which has many properties of the insulin receptor previously studied in vitro. This receptor compartment: (a) recognizes insulins based on their biological potencies; (b) is saturated by elevated concentrations of insulin; and (c) insulin bound to receptors is in equilibrium with free hormone in plasma. Further, the bound to free ratios for hormone, calculated from these data, suggest that in vivo greater than 50% of the extrapancreatic insulin is bound to receptors during normal physiological states.

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.

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).

Localization of inhibin/activin subunit mRNAs within the primate ovary.

In order to gain further understanding of the physiology of inhibin and activin in the primate, the expression of inhibin/activin subunit mRNAs in the monkey ovary was examined by in situ hybridization. Granulosa cells of small antral follicles were found to express mRNA for the beta B subunit, which decreased to undetectable levels in dominant follicles. In contrast, expression of alpha and beta A subunit mRNAs was detected in granulosa cells of dominant follicles and in corpora lutea, but not in small antral follicles. These results indicate that the expression of the beta A and beta B subunits is differentially regulated during the growth and development of ovarian follicles in the monkey.

Premature elevation of systemic estradiol reduces serum levels of follicle-stimulating hormone and lengthens the follicular phase of the menstrual cycle in rhesus monkeys.

The concentration of estradiol in peripheral serum was prematurely elevated on days 3-6 of the rhesus monkey menstrual cycle to approximate the estradiol rise typically seen during the midfollicular phase. Associated with an estradiol increase (from 60 to approximately 90 pg/ml) was a significant (P less than 0.01) reduction in FSH levels and a lengthening of the follicular phase of the menstrual cycle. These results reveal the existence of an exquisitely sensitive negative feedback relationship between estradiol and FSH during the follicular phase of the menstrual cycle, in doing so, these findings suggest that the selected follicle, by way of its secretion of estradiol, indirectly inhibits the maturation of other follicles by reducing gonadotropin levels.

Ovarian responses in macaques to pulsatile infusion of follicle-stimulating hormone (FSH) and luteinizing hormone: increased sensitivity of the maturing follicle to FSH.

This study investigated the relationship between plasma gonadotropin concentrations and the initiation and maintenance of preovulatory follicular growth in macaques. Eight adult cynomolgus monkeys were treated with a GnRH antagonist [AcD2Nal1-4ClDPhe2, DTrp3, DArg6, DAla10]GnRH X HOAc to block endogenous gonadotropin secretion. In four animals, a pulsatile infusion of human FSH and human LH (hLH) (one 3-min pulse/h) was initiated, and the amount of hFSH delivered per pulse was increased every 3-4 days until serum estradiol concentrations rose. Thereafter, the amount of FSH delivered per pulse was reduced by 12.5%/day for 5 days, whereas the amount of LH delivered per pulse was not altered. Results indicated that plasma FSH concentrations in the range of 15-20 mIU/ml were associated with the initiation of estrogen production; in addition, a progressive reduction in plasma FSH concentration to 8-10 mI/ml over the subsequent 5 days was accompanied by continued rises in estradiol concentrations and preovulatory follicular growth. In contrast, in four control animals, maintenance of plasma FSH concentrations at 8-10 mIU/ml for 13 days did not result in elevation in serum estradiol concentrations or antral follicular growth. These observations demonstrate that after stimulation by elevated FSH concentration, follicles can continue to mature in the presence of FSH concentrations which are unable to support the growth of less mature follicles. Thus, this may account for the mechanisms by which the maturing follicle continues to develop during the mid-through late follicular phase of the menstrual cycle, whereas other less mature follicles undergo atresia.

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.

Progesterone does not inhibit gonadotropin-induced follicular maturation in the female rhesus monkey (Macaca mulatta).

Female rhesus monkeys were treated with exogenous human menopausal gonadotropin (Pergonal) in the presence of exogenous systemic progesterone provided by Silastic implants and during the luteal phase of the menstrual cycle to determine if progesterone influences gonadotropin-induced follicular maturation. Peripheral serum and ovarian vein plasma 17 beta-estradiol concentrations were used to assess the response to gonadotropin treatment. When systemic levels of progesterone were elevated to 6--10 ng/ml in eight animals during the midfollicular phase of the menstrual cycle, each animal responded to exogenous gonadotropin with a sustained increase in serum 17 beta-estradiol concentration, which reached levels of approximately 800 pg/ml during treatment. To determine if the corpus luteum itself influences the ovarian response to exogenous gonadotropin, animals were treated with exogenous gonadotropin for 7 days, commencing during either the follicular phase of the cycle or the midluteal phase of the cycle. Regardless of the time of initiation of gonadotropin treatment, peripheral serum 17 beta-estradiol levels rose to approximately 1 ng/ml in all animals studied. 17 beta-Estradiol levels were elevated in ovarian venous blood from the ovary, which contains the active corpus luteum and the contralateral ovary. These observations demonstrate that exogenous gonadotropins can stimulate follicular maturation in the presence of progesterone.

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.

The corpus luteum of the primate menstrual cycle is capable of recovering from a transient withdrawal of pituitary gonadotropin support.

To further our understanding of the role of pituitary gonadotropin secretion in the control of corpus luteum function during the primate menstrual cycle, we have used an experimental model which enables us to directly control pituitary gonadotropin secretion throughout the luteal phase. Specifically, we have asked whether cessation of progesterone secretion, or functional luteolysis, resulting from a 3-day withdrawal of gonadotropin support, culminates in an irreversible loss of luteal responsiveness to further gonadotropic stimulation; and do the effects of gonadotropin deprivation vary with the age of the corpus luteum? Endogenous gonadotropin secretion was abolished in seven adult rhesus monkeys by placing radiofrequency lesions in the arcuate nucleus region of the medial basal hypothalamus. Endogenous gonadotropin secretion and ovulatory menstrual cycles were then restored by chronic pulsatile infusion of GnRH (1 pulse/h). Control luteal phases supported by this GnRH regimen exhibited typical plasma progesterone patterns and ranged from 14-17 days in length. In experimental cycles, endogenous gonadotropin secretion was interrupted for a 3-day period during the early (days 2-5), mid (days 8-11), or late (days 13-16) stages of the luteal phase. During the GnRH deprivation period, bioassayable and immunoreactive serum LH was undetectable. The disappearance of circulating LH was followed by a rapid fall in plasma progesterone levels regardless of the stage of the luteal phase. The restoration of gonadotropin secretion resulted in a resumption of progesterone secretion when the gonadotropin deprivation period was imposed during the early or midluteal phase. In each instance, the resumption of progesterone secretion continued for a period of time which effectively completed the typical 14- to 17-day functional lifespan of the corpus luteum of the menstrual cycle. Thus, the luteal phase was neither shortened nor lengthened by a 3-day interruption of luteal function resulting from withdrawal of gonadotropic support. When gonadotropin secretion was interrupted during the late luteal phase (days 13-16), restoration of gonadotropin secretion on day 16 did not result in resumption of progesterone secretion. Our findings confirm our earlier demonstration that progesterone secretion during the luteal phase of the non-fertile menstrual cycle is dependent on pituitary gonadotropic support.(ABSTRACT TRUNCATED AT 400 WORDS)

The rhesus monkey corpus luteum is dependent on pituitary gonadotropin secretion throughout the luteal phase of the menstrual cycle.

Experiments were conducted in rhesus monkeys to determine whether the corpus luteum of the menstrual cycle requires pituitary gonadotropin for progesterone production and normal functional lifespan. Eight adult females were rendered anovulatory by placement of radiofrequency lesions in the arcuate region of the medial basal hypothalamus. Endogenous gonadotropin secretion and ovulatory cycles were reestablished by chronic pulsatile infusion of GnRH. Control luteal phases exhibited typical plasma progesterone patterns and ranged from 14-17 days in length. In experimental cycles, endogenous gonadotropin secretion was interrupted during the luteal phase by stopping the infusion of GnRH. When the GnRH infusion was stopped in the early luteal phase (3 days after the preovulatory estradiol peak; day 3), plasma LH fell to undetectable levels within 90 min. Plasma progesterone concentrations (1.5 +/- 0.4 ng/ml) declined to undetectable levels (less than 0.2 ng/ml) by the afternoon of day 5 (P less than 0.05). Premature menses occurred 2-5 days later. When the GnRH infusion was stopped in the midluteal phase (day 8), plasma LH fell below the limits of detectability within 150 min. Circulating progesterone (4.5 +/- 1.0 ng/ml) declined to undetectable levels by the afternoon of the following day. Premature menses occurred 3 days after the fall in plasma LH, 11 days after the preovulatory estradiol peak. Plasma LH and progesterone remained undetectable as long as exogenous GnRH was withheld (18 days), and progesterone did not reappear until the next GnRH-induced ovulatory cycle. These results demonstrate that the normal functional lifespan of the primate corpus luteum requires the presence of circulating pituitary gonadotropin during both the early (developmental) and middle (fully functional) stages of the nonfertile luteal phase.

Characterization of ovarian folliculogenesis during the luteal phase of the menstrual cycle in rhesus monkeys using [3H]thymidine autoradiography.

The characteristics of the early stages of ovarian follicular maturation were investigated in rhesus monkeys during the luteal phase of the menstrual cycle using a novel procedure to supply low levels (10 microCi) of [3H]thymidine directly to ovaries in vivo. With this labeling procedure, plasma levels of [3H]thymidine did not exceed 8 cpm/microliters and labeled ovarian cells were readily identifiable by autoradiography. Results from four animals studied during the luteal phase of the cycle indicated that all sizes of preantral follicles contained granulosa cells that incorporated [3H]thymidine. There was a progressive increase in the percentage of labeled follicles in direct relationship to follicular size; 10% of the single granulosa cell layer follicles were labeled and the labeling frequency increased to greater than 60% in the large (> 6 granulosa cell layer) preantral follicles. The labeling frequency of all size classifications of preantral follicles during the luteal phase of the menstrual cycle was similar to that of an animal which was studied during the follicular phase of the menstrual cycle. The results suggest that growth of ovarian follicles to the preantral stage occurs during the luteal phase of the menstrual cycle in rhesus monkeys, and thus the cessation of preovulatory folliculogenesis during the luteal phase of the cycle is not due to a defect of growing preantral follicles.

Effect of reduced luteinizing hormone concentrations on corpus luteum function during the menstrual cycle of rhesus monkeys.

To further define the relationship between plasma LH concentrations and progesterone secretion by the primate corpus luteum, we examined luteal function in rhesus monkeys in response to reduced LH concentrations during the luteal phase of the menstrual cycle. Five anovulatory rhesus monkeys received a pulsatile infusion of synthetic GnRH (6 micrograms/pulse; one pulse per h, iv) to restore menstrual cyclicity. During the early luteal phase (4-5 days after ovulation), the amount of GnRH administered per pulse was reduced to 1/250th or 1/750th of the standard GnRH infusion regimen. Plasma LH concentrations, determined by bioassay, were reduced by approximately 50% during cycles maintained by reduced GnRH concentrations compared with the standard GnRH dosage. Serum progesterone concentrations were maintained for 5-6 days after GnRH reduction and declined thereafter, and premature menstruations were observed in four of seven cycles maintained by the 1/250th GnRH reduction and four of six cycles maintained with the 1/750th GnRH reduction. These results are consistent with the hypothesis that luteal regression during the nonfertile menstrual cycles of primates is due primarily to an alteration in luteal cell responsiveness to LH, rather than a reduction in the gonadotropic drive to the corpus luteum per se. When plasma LH concentrations were reduced during the early luteal phase to values below those found during the onset of luteal regression in control cycles, luteal function was maintained for 5-6 days. However, as the luteal phase progressed, the reduced LH concentrations were unable to sustain progesterone secretion, and premature menses occurred in some, but not all, animals.

Effects of different gonadotropin pulse frequencies on corpus luteum function during the menstrual cycle of rhesus monkeys.

In the nonfertile menstrual cycle, the frequency of episodic LH secretion declines from approximately 1 pulse/h in the early luteal phase to 1 pulse/4-8 h in the mid- to late luteal phase, but the relevance of this phenomenon to the initiation of functional luteal regression is not completely understood. We investigated whether a reduction in LH pulse frequency causes a decline in luteal progesterone production by experimentally reducing LH pulse frequency during the early luteal phase, and measured the effects on the subsequent plasma progesterone pattern and the onset of luteal regression. Rhesus monkeys were rendered anovulatory by placing radiofrequency lesions in the arcuate region of the medial basal hypothalamus or surgically transecting the hypothalamic-pituitary stalk. Endogenous gonadotropin secretion and ovulatory menstrual cycles were restored by pulsatile infusion of synthetic GnRH at a frequency of 1 pulse/h. Commencing on days 3-6 of the luteal phase, GnRH frequency was changed to either 1 pulse/8 h (four animals) or 1 pulse/24 h (four animals), or maintained at the standard 1 pulse/h frequency (four animals). Luteal phases of 13- to 17-day duration were observed in all animals kept on the 1 pulse/h frequency and in three of four animals in which the frequency was changed to 1 pulse/8 h on day 3 of the luteal phase. Daily midluteal phase (days 5-10) plasma progesterone levels observed in response to the 1 pulse/h and 1 pulse/8 h infusion regimens were similar (mean +/- SE, 4.1 +/- 0.4 vs. 3.2 +/- 0.4 ng/ml; P greater than 0.1). In contrast, short luteal phases were observed in all animals after the LH pulse frequency was reduced to 1 pulse/24 h. Comparison of plasma LH responses to a representative GnRH pulse of each GnRH infusion regimen revealed that the maximal LH levels attained in response to 1 pulse/8 h (47.5 +/- 11.5 ng/ml) were significantly greater (P less than 0.05) than the maximal LH levels attained in response to 1 pulse/h (30.5 +/- 3.2 ng/ml) or 1 pulse/24 h (27.2 +/- 5.0 ng/ml). Progesterone levels remained elevated for 140-200 min after the LH pulse resulting from the 1 pulse/8 h infusion regimen. In response to the 1 pulse/24 h infusion regimen, plasma progesterone levels remained elevated for 60 min after the LH pulse.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

In the rhesus monkey (Macaca mulatta), the negative feedback regulation of follicle-stimulating hormone secretion by an action of testicular hormone directly at the level of the anterior pituitary gland cannot be accounted for by either testosterone or estradiol.

In the male rhesus monkey, the negative feedback regulation of gonadotropin secretion by the gonad appears to involve a specific inhibitory action of testicular hormone on FSH release at the level of the anterior pituitary gland. The purpose of the present study, which used the hypophysiotropic clamp preparation, was to determine whether circulating testosterone (T) or estradiol (E) comprises a major component of the testicular FSH-inhibiting factor in this species. Endogenous hypothalamic GnRH secretion was abolished or severely compromised in five adult male rhesus monkeys by placement of radiofrequency lesions in the region of the arcuate nucleus. Subsequently, an episodic pattern of activity in the pituitary-Leydig cell axis of these animals was restored by a chronic and unchanging intermittent iv infusion of GnRH (0.1 microgram/min for 3 min ever 3 h), which appears to provide the gonadotropes of lesioned animals with a hypophysiotropic drive comparable to that produced by the hypothalamus of animals with an intact central nervous system. Treatment of three animals with a specific anti-E-gamma-globulin fraction resulted in a marked and sustained rise in E-binding activity in serum, but this neutralization of circulating E did not elicit hypersecretion of FSH. In all five animals, initiation of T replacement on the day of orchidectomy, which maintained circulating T concentrations in the high testis-intact control range, failed to prevent the postcastration hypersecretion of FSH that is evoked after removal of testis in the hypophysiotropic clamp preparation. As expected, the changes in circulating LH levels during immunoneutralization and after orchidectomy and T replacement were unremarkable. These findings indicate that neither circulating T nor E can account for the testicular inhibition of FSH secretion in the rhesus monkey and thus lead, by a process of exclusion, to the view that in this species the negative feedback regulation of FSH release by the testis is mediated directly at the level of the gonadotrope by an inhibitory action of a nonsteroidal hormone, most probably the recently identified gonadal peptide inhibin.

Gonadotropin-binding sites in the rhesus monkey ovary: role of the vasculature in the selective distribution of human chorionic gonadotropin to the preovulatory follicle.

These experiments were initiated to determine if differences exist in the vasculature of individual follicles in the rhesus monkey ovary during the late follicular phase of the menstrual cycle and to determine whether differences in vascularity result in differential exposure of certain follicles to gonadotropic hormones. The density of blood vessels within the thecal layer of the dominant follicle and other antral follicles was determined in ovaries from four animals removed on day 9 or 10 of the menstrual cycle. Blood vessels were identified using a histochemical stain for hemoglobin. Morphometric analysis indicated that the percentage of the thecal layer occupied by blood vessels in the dominant follicles (48%) was significantly greater (P less than 0.005) than that of other smaller antral follicles either within the same ovary as the dominant follicle (24%) or in the contralateral ovary (26%). To determine if differences in vascularity result in a differential supply of gonadotropins to the dominant follicle, we studied, by autoradiography, the in vivo and in vitro binding of [125I]hCG in four rhesus monkeys on day 9 of the menstrual cycle. Results of in vitro binding studies indicated that the thecal layer of virtually every antral follicle possessed hCG-binding sites. However, when [125I]hcg was injected iv into animals and allowed to distribute via the vasculature, the dominant follicle was heavily labeled while other smaller antral follicles accumulated little, if any, radioiodinated hCG. These observations indicate that increased vascularization of individual follicles results in preferential delivery of gonadotropins, and suggest that blood flow to individual follicles may play an instrumental role in the selective maturation of the preovulatory follicle in the rhesus monkey.

Independence of steroidogenic capacity and luteinizing hormone receptor induction in developing granulosa cells.

The relationship between FSH-induced acquisition of LH/hCG receptors and the steroidogenic capacity of granulosa cells from estrogen-primed hypophysectomized rat ovaries has been examined. Granulosa cells harvested from the immature preantral follicles of animals not treated with FSH (controls) displayed negligible specific human [125I]iodo-hCG binding and produced only minimal amounts of progesterone during 48 h of culture in vitro. Addition of highly purified hFSH or prostaglandin-E2 (PGE2) to the culture medium elicited substantial increases in progesterone production which were not accompanied by measurable increases in [125I]iodo-hCG binding. Treatment with oFSH in vivo for 24 h led to the initiation of antrum formation in many follicles and was accompanied by an 8-10-fold increase in hCG binding by freshly isolated granulosa cells. Basal, hFSH-, and PGE2-stimulated progesterone production during culture was also greater than controls. In contrast, cells from animals receiving oFSH in vivo for only 12 h showed no increase in hCG binding either before or after culture, yet basal and stimulated progesterone production in vitro was significantly greater than controls, indicating that the initiation of steroidogenesis was antecedent to LH/hCG receptor induction. Only those cells obtained after the 24-h in vivo treatment with oFSH produced elevated amounts of progesterone when incubated in the presence of hCG, thereby showing that the observed increases in [125I]iodo-hCG binding reflected the induction of functionally active LH/hCG receptors. Pharmacological stimulation of steroidogenesis by cell suspensions with N,O'-dibutyryl cAMP resulted in consistently high levels of progesterone production irrespective of previous treatment with FSH in vivo. This uniform expression of in vitro steroidogenic capacity occurred in the complete absence of measurable increases in LH/hCG receptors, suggesting that these two fundamental developmental processes are independent phenomena which may be under separate regulation in vivo.

Developmental expression of Ca++/Mg++-dependent endonuclease activity in rat granulosa and luteal cells.

A Ca++/Mg++-sensitive endonuclease has been associated with programmed cell death in a variety of endocrine and non-endocrine dependent tissues. In view of the remarkable similarity of the process of the regression of the corpus luteum and other models of programmed cell death, we studied the occurrance of this endonuclease activity in granulosa cells at different developmental states and the corpus luteum. Our results show that while undifferentiated granulosa cells do not express this endonuclease activity, treatment with pregnant mares serum gonadotropin results in a rapid increase in endonuclease activity that is maintained following ovulation and luteinization of granulosa cells.