Expression of estrogen receptor alpha and beta in the rhesus monkey corpus luteum during the menstrual cycle: regulation by luteinizing hormone and progesterone

There are conflicting reports on the presence or absence of estrogen receptor (ER) in the primate corpus luteum, and the discovery of a second type of estrogen receptor, ERbeta, adds an additional level of complexity. To reevaluate ER expression in the primate luteal tissue, we used semiquantitative RT-PCR based assays and Western blotting to assess ERalpha and beta messenger RNA (mRNA) and protein levels in corpora lutea (n = 3/stage) obtained from adult female rhesus monkeys at early (days 3-5), mid (days 6-8), mid-late (days 10-12), and late (days 14-16) luteal phase of the natural menstrual cycle. ERalpha mRNA levels did not vary across the stages of the luteal phase, and ERalpha protein was not consistently detected in luteal tissues. However, ERbeta mRNA and protein levels were detectable in early and mid luteal phases and increased (P < 0.05) to peak levels at mid-late luteal phase before declining by late luteal phase. To determine if ERbeta mRNA expression in the corpus luteum is regulated by LH, monkeys received the GnRH antagonist antide either alone or with 3 daily injections of LH to simulate pulsatile LH release. Treatment with antide alone or concomitant LH administration did not alter luteal ERbeta mRNA levels. When monkeys also received the 3beta-hydroxysteroid dehydrogenase inhibitor trilostane to reduce luteal progesterone production, luteal ERbeta mRNA levels were 3-fold higher (P < 0.05) than in monkeys receiving antide + LH only. Replacement of progestin activity with R5020 reduced luteal ERbeta mRNA levels to those seen in animals receiving antide + LH. Thus, there is dynamic ERbeta expression in the primate corpus luteum during the menstrual cycle, consistent with a role for estrogen in the regulation of primate luteal function and life span via a receptor (ERbeta)-mediated pathway. Increased ERbeta expression in the progestin-depleted corpus luteum during LH exposure suggests that the relative progestin deprivation experienced by the corpus luteum between LH pulses may enhance luteal sensitivity to estrogens during the late luteal phase of the menstrual cycle.     

Induction of luteal phase defects in rhesus monkeys by follicular fluid administration at the onset of the menstrual cycle

Administration of charcoal-extracted porcine follicular fluid (pFF) to rhesus monkeys on days 1-3 of the menstrual cycle suppressed serum FSH, but not LH, during the early follicular phase. Although timely midcycle gonadotropin surges occurred in 5 of 6 pFF-treated monkeys, the preovulatory rise in serum estradiol was markedly diminished and serum progesterone (P) levels were subnormal at midluteal phase. The wet weight of the corpus luteum excised from pFF-treated monkeys at midluteal phase was less than that from controls. Moreover, both basal and gonadotropin (hCG)-sensitive P production by short-term suspensions of luteal cells from pFF-treated monkeys was significantly less than that by control cells. These findings provide direct support for the concept that FSH-dependent events during the early follicular phase are important determinants of the subsequent function of the corpus luteum of the menstrual cycle. Since pFF-induced luteal dysfunction was strikingly similar to spontaneous luteal phase defects found in monkeys and women, this primate model permits study of the mechanism(s) whereby FSH deficiency during recruitment and selection of the dominant follicle portends defective luteal function and infertility in women.     

The effect of intraluteal infusion of deglycosylated human chorionic gonadotropin on the corpus luteum in rhesus monkeys

Removal of the carbohydrates from hCG results in an antagonist (degly-hCG) that competitively inhibits hCG/LH-stimulated adenylate cyclase in macaque luteal tissue in vitro, but its effect in vivo is controversial. To examine the effect of degly-hCG on the lifespan and steroidogenic activity of the primate corpus luteum, the antagonist was administered to female rhesus monkeys (n = 7) beginning at the midluteal phase of the menstrual cycle. In a control cycle the saline vehicle was infused via an osmotic minipump directly into the corpus luteum. In a subsequent cycle, one of three dose rates of degly-hCG (0.001, 0.009, and 0.09 nmol/h) was infused into the corpus luteum. Pump implantation and infusion began 5-9 days after the midcycle LH surge and continued for 7 days. Peripheral venous blood was collected daily from day 8 of the cycle until menses, and serum progesterone levels were determined by RIA. Progesterone levels and patterns were similar in animals that received either the saline vehicle or degly-hCG, and the length of the luteal phase in monkeys receiving any dose of degly-hCG (16.4 +/- 0.5 days) was not different from that in animals receiving a control infusion (16.1 +/- 0.9 days). In a corollary study, an intraluteal infusion of degly-hCG (0.009 nmol/h) in the midluteal phase did not prevent stimulation of progesterone levels after im injection of hCG (15 IU/day for 5 days). We conclude that whereas degly-hCG is a useful tool to examine gonadotropin action in vitro, it is not a potent gonadotropin antagonist in vivo.     

Follicular fluid treatment during the follicular versus luteal phase of the menstrual cycle: effects on corpus luteum function

Administration of charcoal-extracted porcine follicular fluid (pFF) to rhesus monkeys at the time of menses impairs the subsequent function of the corpus luteum of the menstrual cycle. The following studies were performed: 1) to characterize the luteal phase defect induced by pFF treatment at menses, and 2) to determine whether pFF treatment in the luteal phase alters corpus luteum function. Adult, female rhesus monkeys were injected sc for 3 days with pFF (10, 5, and 5 ml) beginning on day 1 (n = 5) or day 18 (n = 4) of the menstrual cycle. Femoral venous blood was collected daily throughout the treatment cycle and during the posttreatment cycle of day 18 to 20-treated monkeys. Serum LH, FSH, 17 beta-estradiol (E2), and progesterone (P) were measured by RIA. After pFF treatment on days 1-3, FSH and E2 levels in the early follicular phase were less (P less than 0.05) than those of control cycles (n = 7). Serum LH was not suppressed by pFF treatment. Moreover, the preovulatory rise in circulating E2 and the amplitude of the LH/FSH surge were similar in control and pFF-treated monkeys. Although timely midcycle gonadotropin surges occurred in four of five pFF-treated monkeys, serum P was markedly reduced (P less than 0.05) during the first half of the luteal phase. Circulating P increased to control levels during the late luteal phase before normal onset of menses 16.3 +/- 1.0 (SE) days after the LH surge. Treatment with pFF on days 18-20 of the cycle reduced the levels of circulating FSH, but serum LH, E2, P, and the length of the luteal phase remained comparable to control cycles. Moreover, the hormonal patterns and the length of the follicular and luteal phases in the posttreatment cycle indicated normal ovarian function. Thus, pFF treatment at menses results in an aberrant ovarian cycle characterized by an insufficient, rather than short, luteal phase. Whereas pFF treatment in the early follicular phase vitiates development of the dominant follicle and the related corpus luteum, similar treatment at midluteal phase does not suppress concurrent luteal function or subsequent folliculogenesis.     

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)     

Intraluteal infusion of a prostaglandin synthesis inhibitor, sodium meclofenamate, causes premature luteolysis in rhesus monkeys

The physiological significance of locally produced prostaglandins (PGs) in the regulation of the functional lifespan of the primate corpus luteum is unknown. In the current study, the PG synthesis inhibitor sodium meclofenamate was administered to adult female rhesus monkeys beginning in the midluteal phase of the menstrual cycle. Meclofenamate was infused continuously for 7 days into the corpus luteum (100 micrograms/h, n = 6) or the jugular vein (100 micrograms/h, n = 3; 1000 micrograms/h, n = 3) via osmotic minipump. As controls, PBS was infused into the corpus luteum (n = 7) or jugular vein (n = 5). In some of the monkeys receiving intraluteal infusions, chronic aortic and utero-ovarian venous catheters were implanted, and blood samples were collected on alternate days for the measurement of PGE and PGF2 alpha by RIA. Saphenous venous blood was collected daily, and progesterone and cortisol levels were determined by RIA. LH levels were determined by the mouse Leydig cell bioassay. Progesterone levels over 5 days preceding treatment were not different among groups. A decline in progesterone levels on day 1 after surgery was observed in all treatment groups and was accompanied by a 1-day elevation in cortisol levels. Thereafter, five of seven monkeys who received intraluteal infusions of PBS displayed normal progesterone patterns during treatment and normal luteal phase lengths of 15.4 +/- 1.2 days (mean +/- SEM). In six monkeys that received intraluteal infusions of meclofenamate, progesterone levels typically fell to less than 1 ng/ml within 72 h after initiation of infusion; progesterone levels during 7 days of intraluteal infusion were significantly lower (P less than 0.01) in meclofenamate- vs. PBS-treated monkeys. Meclofenamate infusion into the corpus luteum significantly shortened (P less than 0.01) the luteal phase to 10.5 +/- 1.0 days. In contrast, progesterone levels during 7 days of meclofenamate infusion into the jugular vein did not differ from those in PBS-treated monkeys, and the length of the luteal phase was unaltered. LH levels, measured daily, did not differ among groups either before or during treatment. Although an venous/arterial gradient in PGE was detected at the time of surgery, we were unable to detect a significant gradient across the ovary in PGE or PGF2 alpha at any time after surgery in monkeys treated with either PBS or meclofenamate. The present data suggest an obligatory luteotropic role for locally produced metabolites of arachidonic acid, but a physiological role for either PGE or PGF2 alpha in regulating the primate corpus luteum remains equivocal.     

Oxytocin in the corpus luteum of the cynomolgus monkey (Macaca fascicularis)

To determine if oxytocin (OT) is present in cynomolgus monkey corpus luteum, OT was measured by a specific and sensitive RIA in 13 corpora lutea, ovarian venous plasma on the ipsilateral side and peripheral venous plasma at different stages of the luteal phase. Serial dilution of acetic acid extract of the corpus luteum showed parallelism with standard OT in the RIA. Total content of OT in corpus luteum was 1.9 +/- 0.5 ng (mean +/- SEM) with a content of 0.4-0.8 ng in early luteal phase, 1.0-6.2 ng in midluteal phase, and 0.4-0.7 ng in late luteal phase. OT concentrations in corpus luteum were 21.0-75.2 ng/g wet wt in early luteal phase, increasing to 34.4-602.5 ng/g in midluteal phase; and declining to 3.4-117.4 ng/g in late luteal phase. OT concentrations per mg protein in the corpus luteum were 0.05-19.6 ng with peak concentrations of 14.7-19.6 ng/mg protein on day 22. Sephadex G-25 column chromatography of the corpus luteum extract revealed a single peak for binding activity similar to that of synthetic OT on the RIA. Ovarian vein blood from the same side as the corpus luteum had a significantly higher OT concentrations of 161.2 +/- 29.7 pg/ml on days 15-24 than 16.8 +/- 3.6 pg/ml on days 25-28 (P less than 0.01) and peripheral plasma OT levels of 23.2 +/- 3.4 pg/ml (P less than 0.025). Our findings indicate that OT is present and probably produced by monkey corpus luteum with peak OT concentrations found in midluteal phase. Thus OT may play a role in primate corpus luteum function.     

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.     

Inhibition of folliculogenesis in the monkey following early follicular phase administration of an LRH agonist

This study was undertaken to determine if early follicular phase administration of a synthetic luteinizing hormone releasing hormone (LRH) agonist would produce luteal phase defects in the monkey. [D-His(im-Bzl)6,Pro9]LRH N-ethylamide was administered to groups of rhesus monkeys on days 1-3 of the menstrual cycle. Two responses were observed: a) anovulatory menstrual cycles of less than 14 days duration, and b) ovulatory menstrual cycles characterized by unusually long follicular phases. All 4 monkeys with shortened menstrual cycles had prominent increases in serum gonadotrophin and oestradiol concentrations during treatment with the LRH agonist; early menses in these animals was attributed to uterine bleeding upon oestrogen withdrawal. Serum FSH concentrations declined, serum LH concentrations were unaltered, and only 2 of 8 monkeys had elevations in serum oestradiol during ovulatory menstrual cycles. The mean interval from cessation of treatment with the LRH agonist to the next preovulatory gonadotrophin surge was 21.5 +/- 3.2 days in ovulatory menstrual cycles. Corpus luteum function was normal following treatment with the LRH agonist in ovulatory cycles. The results indicate that both the long and short menstrual cycles observed following early follicular phase administration of the LRH agonist to monkeys can be attributed to a profound inhibition in follicle recruitment. [D-His(im-Bzl)6,Pro9]LRH N-ethylamide did not alter corpus luteum function in the monkeys.     

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.     

Asymmetrical ovarian function during recruitment and selection of the dominant follicle in the menstrual cycle of the rhesus monkey

Despite similar exposure to pituitary gonadotropins by perfusion of both ovaries with the same peripheral blood, only 1 of the 2 ovaries sponsors the single dominant follicle in the typical menstrual cycle. In the present study was examined the initiation of asymmetrical ovarian function during recruitment and selection of the dominant follicle in the primate ovarian cycle by comparison of steroid hormones in the ovarian venous effluent. Thirty-four adult female rhesus monkeys were selected because of high estimated fertility based on their reproductive performance. These monkeys underwent laparotomy for ovarian inspection and collection of ovarian venous blood on 1 of days 1, 3, 5, 7, 9, and 11 after the onset of menses. In addition, femoral blood was collected daily. Repeat laparotomies were performed in the midluteal phase to assess the location of the functional corpus luteum. Concentrations of 17 beta-estradiol, androstenedione, and progesterone were determined in all sera, as well as LH and FSH in peripheral sera, by RIA. In all, 17 of 19 ovulatory monkeys manifested clear asymmetry of 17 beta-estradiol 5 days before the LH/FSH midcycle surges. Often, asymmetry of androstenedione levels was not apparent until 3 days before the midcycle gonadotropin surge. Uniformly, in ovulatory monkeys, the ovary associated with significantly greater concentrations of 17 beta-estradiol and androstenedione in ovarian venous serum ultimately bore the functional corpus luteum observed in the midluteal phase and confirmed by elevated progesterone in peripheral serum. We interpret these findings to indicate that asymmetrical ovarian steroid secretion, especially of 17 beta-estradiol, may be among the earliest indicators that the dominant follicle, or at least the ovary destined to bear it, is already selected by 5 days before the preovulatory FSH/LH surge in the typical menstrual cycle.     

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.     

Is the decrease in the hypophysiotropic signal frequency normally observed during the luteal phase important for menstrual cyclicity in the primate?

The two phases of the ovulatory menstrual cycle of the primate are characterized by divergent activities of the GnRH pulse generator. During the luteal phase, LH pulse frequency is significantly reduced below that observed during the follicular phase. In this report we investigate whether the decrease in pulse frequency during the luteal phase is of physiological relevance to normal menstrual cyclicity. We have tested the effect of a pulsatile GnRH infusion given iv at hourly intervals for a period of 8-10 days during the luteal phase on the subsequent three to five cycles in eight female rhesus monkeys. Three of the eight animals received two treatment courses. Amounts of GnRH infused were 1.5 micrograms/pulse (n = 2 trials); 3.0 micrograms/pulse (n = 7); and 4.0 micrograms/pulse (n = 2). LH response to GnRH pulses of 1.5 and 3.0 micrograms resembled spontaneous LH pulses observed during the luteal phase. During the GnRH infusion period, the monkeys were fitted with a primate vest and tethered. Eleven control experiments were performed in these monkeys under similar conditions. GnRH therapy during the luteal phase affected subsequent cycles significantly, while no differences were observed in the control experiments. Overall mean follicular phase length in the control cycle was 13.4 days; it was significantly increased (P less than 0.005) in all post-GnRH treatment cycles to reach 34.4 (+/- 10.9), 43.9 (+/- 12.7), 40.4 (+/- 13.0), and 23.1 (+/- 4.8) days (+/- SE) in the first to fourth post-GnRH cycles, respectively. Progesterone secretion was significantly lower (P less than 0.05) in the first two post-GnRH cycles than in the control cycles: progesterone, 46.4 (+/- 2.1) in all control cycles, decreased to 27.7 (+/- 3.7), 24.8 (+/- 4.3), 34.0 (+/- 5.4), and 32.0 (+/- 6.5) surface units (+/- SE) from the first to fourth post-GnRH cycles, respectively, while luteal phase length remained relatively unchanged. The data indicate that significant disturbances in the menstrual cycle of the rhesus monkey follow imposed changes in the normal frequency pattern of the GnRH hypophysiotropic signal during the luteal phase and suggest that the naturally occurring slowing of GnRH-LH pulse frequency during the luteal phase is a relevant phenomenon in the sequence of events which control menstrual cyclicity.     

Adenylate cyclase in the corpus luteum of the rhesus monkey. II. Sensitivity to nucleotides, gonadotropins, catecholamines, and nonhormonal activators

The sensitivity of the adenylate cyclase of the primate corpus luteum to various nucleotides, gonadotropins, catecholamines, and nonhormonal activators was assessed in homogenates of luteal tissue obtained from rhesus monkeys at the midluteal phase of the menstrual cycle. The conversion of [alpha-32P]ATP to [32P]cAMP was used to monitor adenylate cyclase activity. GTP, the GTP analog 5'-guanylyl-imidodiphosphate, and ITP stimulated adenylate cyclase activity in the presence or absence of exogenous hormone; however CTP, UTP, GMP, and guanosine did not. The gonadotropins, human (h) LH and hCG, stimulated cAMP production in a dose-dependent manner. Maximal stimulation of adenylate cyclase was achieved at 100 nM hLH and hCG, and the activation constant was 20 nM for both hormones. The addition of GTP increased maximal activation of adenylate cyclase by hLH or hCG, but did not alter sensitivity to the hormones. Neither hFSH nor the isolated subunits of hCG stimulated cAMP production. Deglycosylated hCG (native hCG with 70% of the carbohydrate moieties removed) did not stimulate adenylate cyclase activity. However, hLH and intact hCG failed to enhance cAMP production in the presence of an equimolar amount of deglycosylated hCG. The adenylate cyclase of macaque luteal tissue did not respond to the addition of isoproterenol, epinephrine, or phenylephrine. Furthermore, these catecholamines did not affect hCG stimulation of adenylate cyclase. The nonhormonal activators of adenylate cyclase, forskolin and fluoride, stimulated cAMP production in a dose-dependent manner, with maximal stimulation at 100 microM and 10 mM, respectively. Thus, the macaque corpus luteum at the midluteal phase of the menstrual cycle contains a guanine nucleotide-regulated adenylate cyclase which is equally sensitive to the pituitary and placental gonadotropins, hLH and hCG. However, removal of carbohydrate moieties from hCG endows the molecule with gonadotropin-antagonistic properties in the primate. The adenylate cyclase system of the macaque corpus luteum was not responsive to catecholamines; thus, the primate may lack a potential mechanism for control of luteal function that is available to many nonprimate species.     

Changes in adenylyl cyclase activity of the human and nonhuman primate corpus luteum during the menstrual cycle and pregnancy

Adenylyl cyclase (AC) activity in membrane particles of corpora lutea (CL) from humans and cynomolgus monkeys was examined at various stages of the menstrual cycle and pregnancy. AC activity was monitored by the conversion of [alpha-32P]ATP into [32P]cAMP under basal conditions and in the presence of several activators: NaF (10 mmol/L) plus forskolin (100 mumol/L); hCG (10 micrograms/mL); guanyl 5'-yl-imidodiphosphate [GMP-P(NH)P; 100 mumol/L]; and hCG (10 micrograms/ml) plus GMP-P(NH)P (100 mumol/L). The groups of human CL were midluteal (n = 10), late luteal (n = 4), following cycle (old CL; n = 5), and early pregnancy (6-11 weeks; n = 10). The groups of monkey CL were early luteal (n = 4), midluteal (n = 5), and pregnancy at term (n = 3). Luteal AC activity changed significantly during the menstrual cycle. In newly (less than 48 h after ovulation) formed CL, the enzyme was unresponsive to hCG, and total AC activity, as determined by NaF plus forskolin, averaged 86.5 +/- 28.9 (+/- SE) pmol cAMP/min.mg protein. As the CL developed, AC activity increased. Thus, in the midluteal phase, maximal hCG responsiveness in the presence of guanine nucleotide was 125 +/- 27 and 232 +/- 15 pmol/min.mg in human and monkey CL, respectively. No hCG responsiveness was detected in the late luteal phase or in the old CL. Maximal AC activity was also high in the midluteal phase (382 +/- 56 and 256 +/- 28 pmol/min.mg in human and monkey CL, respectively); the activity remained fairly high during the late luteal phase and then declined to less than 100 pmol/min.mg in the follicular phase of the next cycle. During early pregnancy, luteal AC was unresponsive to hCG stimulation, yet basal levels, maximal activity, and the characteristics of stimulation by nonhormonal activators were similar, if not identical, to those at the midluteal phase of the menstrual cycle. At term pregnancy, the enzyme remained unresponsive to hCG. However, basal activity and stimulation by NaF and forskolin were remarkably elevated, being between 2- and 7-fold higher than corresponding stimulations in the midluteal phase. We conclude that 1) AC activity in human luteal membranes is highly dependent on hormonal changes and functional state of the ovary, 2) the activity of luteal AC is similar in the CL of humans and cynomolgus monkeys, and 3) the AC system in the primate CL is functionally active during and at the end of pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)     

Suppression of luteal function by a luteinizing hormone-releasing hormone antagonist during the early luteal phase in the stumptailed macaque monkey and the effects of subsequent administration of human chorionic gonadotropin

In previous studies a single sc injection of the LHRH antagonist [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Trp3,D-hArg(Et2)6,D-Ala10 ]LHRH during the luteal phase of the stumptailed macaque menstrual cycle caused a transient suppression of serum LH and progesterone concentrations. To investigate whether a more prolonged suppression of LH release during the early luteal phase could result in a sustained suppression of progesterone, 10 monkeys were treated with 3 consecutive daily injections of 300 micrograms LHRH antagonist/kg beginning on days 0 (n = 2), 1 (n = 1), 2 (n = 1), 3 (n = 2), 4 (n = 2), and 5 (n = 2) after the LH surge. When the antagonist was administered on the day of the LH surge, serum concentrations of bioactive LH were still elevated on the following day, but then fell to low levels. Serum progesterone concentrations were subnormal in these monkeys for the next 10 days, but recovered toward the late luteal phase. In the 8 monkeys receiving antagonist starting between days 1-5 after the LH surge, serum concentrations of bioactive LH were suppressed to near the detection limit of the assay for 4 days after the first injection. Seven of the 8 monkeys demonstrated a progressive decline in serum progesterone concentrations to undetectable values which remained for the duration of the luteal phase. In the remaining monkey the decline in progesterone was less marked; this animal presented a normal progesterone profile 3 days after the last antagonist injection. Premature menses occurred in all 8 monkeys; the next ovulation occurred 18.9 +/- 0.3 days after the last antagonist injection. To test luteal function after antagonist treatment during the early luteal phase and to mimic the rescue of the corpus luteum during a fertile cycle and assess the contraceptive effects of antagonist, hCG in daily doses of 30, 60, 90, 180, and 360 IU was administered starting on day 7 of the luteal phase to monkeys previously treated with three daily injections of 300 micrograms antagonist/kg during the early luteal phase. Control monkeys received hCG injections alone. In the controls, hCG administration elevated serum progesterone concentrations to 15-20 ng/ml. In three monkeys in which antagonist administration did not commence until day 5 or 6, hCG overcame the suppressive effect of the antagonist. However, in seven monkeys in which antagonist administration began on days 1-4, hCG caused only a small progesterone rise (maximal range, 1.8-4.9 ng/ml), about 20% of that observed in control monkeys receiving hCG.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Menstrual cycle characteristics in chronically hemiovariectomized cynomolgus monkeys (Macaca fascicularis).

Circulating levels of LH, FSH, estradiol, and progesterone were measured by RIA in daily serum samples throughout the menstrual cycle in five regularly cycling, chronically hemiovariectomized, adult cynomolgus monkeys. The hormonal patterns, the lengths of the follicular and luteal phases, and the overall cycle length were nearly indistinguishable from those observed in intact cycling monkeys. The findings accord with the notions 1) that in intact monkeys, the contralateral ovary contributes little, if at all, to the regulation of the function or lifespan of the corpus luteum, and 2) that the corpus luteum after spontaneous luteolysis has no local residual effect inimical to new follicle growth.     

Bioassay of circulating luteinizing hormone in the rhesus monkey: comparison with radioimmunoassay during physiological changes.

The concentration of biologically active LH in rhesus monkey (Macaca mulatta) serum was measured by a highly sensitive bioassay based upon testosterone production by dispersed rat interstitial cells. The sensitivity of the in vitro bioassay was equal to or higher than that of radioimmunoassay, with detection limits of 0.1 mIU of human menopausal gonadotropin (hMG) or 10 ng of a rhesus pituitary gonadotropin preparation (LER-1909-2). Parallel dose-response curves were obtained for hMG and rhesus monkey pituitary gonadotropin. The method permits bioassy of LH in 20-100 micronl of serum from adult male monkeys, and from female monkeys during the follicular and luteal phases of the menstrual cycle. Bioactive LH concentrations could be assayed in 0.25 to 5 micronl of serum from mid-cycle, postmenopausal, and castrated female monkeys. Serum LH was undetectable in two hypophysectomized adult female monkeys and six intact immature animals, and was 13+/-6 (SD) mIU/ml in adult male monkeys. In adult females, follicular phase LH levels ranged from 17 to 169 mIU/ml, with a mean of 76+/-52 mIU/ml. The midcycle LH peak was 1738+/-742 mIU/ml and the luteal phase values ranged from 6-47 mIU/ml, with a mean of 35+/-5 mIU/ml. Serum LH concentrations ranged from 100 to 900 mIU/ml in two menopausal females, and from 590-1480 mIU/ml in castrated females. Treatment of castrated female monkeys with estrogen plus progesterone produced an initial two-fold rise in serum LH within 3 days, followed by a gradual decline to one-fourth to one-tenth of the initial levels after 10 days of treatment. Serum LH was suppressed to undetectable levels during the third week, and remained so for the duration of the 60-day treatment period. Bioactive serum LH levels were comparable to levels determined by radioimmunoassay during the follicular and luteal phases of the menstrual cycle, with increased bio-immunoratio at the midcycle peak. The concentrations of biologically active serum LH in rhesus monkeys were similar to those in the human female during the follicular and luteal phases of the menstrual cycle, and were higher at midcycle and after castration. Serum LH levels measured by the interstitial cell bioassay in the rhesus monkey showed appropriate physiological changes and responses to gonadal steroid administration. Furthermore, the bioassay did not detect the LH-like material measured by heterologous radioimmunoassay in the serum of hypophysectomized, immature and steroid-suppressed monkeys. Thus, the rat interstitial cell assay provides a sensitive and valid procedure for measurement of biologically active LH in the serum of these non-human primates.     

Adenylate cyclase in the corpus luteum of the rhesus monkey. III. Changes in basal and gonadotropin-sensitive activities during the luteal phase of the menstrual cycle

The activity of adenylate cyclase was examined in corpora lutea (CL) obtained from rhesus monkeys at specific stages in the luteal phase of the menstrual cycle [3-5, 6-8, 9-12, 13-15, and 16 days (menses) after the midcycle LH surge]. The conversion of [alpha-32P]ATP to [32P]cAMP was used to monitor adenylate cyclase activity. cAMP production in luteal homogenates was assessed in the absence (basal activity) and presence of maximum stimulatory doses of forskolin (100 microM), 5'-guanylylimidodiphosphate [GMP-P(NH)P; 50 microM], GTP (50 microM), and GTP plus increasing doses of hLH and hCG. Basal activity was low in the early luteal phase (days 3-5; mean +/- SE, 1.2 +/- 0.2 pmol cAMP/mg protein X min), increased (P less than 0.05) by the midluteal phase (days 6-8 and 9-12, 2.1 +/- 0.4 and 2.0 +/- 0.3 pmol/mg X min, respectively), and then declined (P less than 0.05) during the late luteal phase (days 13-15 and 16-menses, 1.6 +/- 0.3 and 1.2 +/- 0.5 pmol/mg X min, respectively). Activity stimulated by GTP and GMP-P(NH)P [e.g. GMP-P(NH)P approximately 12 times basal level] followed the same pattern as basal activity during the luteal phase. In contrast, cAMP production in the presence of forskolin did not change significantly throughout the luteal phase. In the midluteal phase (days 6-8 and 9-12; n = 12), hCG and human LH (hLH) stimulated adenylate cyclase in a similar dose-dependent manner. Maximal stimulation of cAMP production by hCG was about 10% greater (P less than 0.05) than that by hLH; the activation constant was 12.3 nM for hCG and 28.3 nM for hLH. The maximal response to hLH and hCG as well as the sensitivity of adenylate cyclase to activation by hLH were greater (P less than 0.05) in the midluteal phase than in the early or late luteal phase. Decreased basal, gonadotropin-stimulated, and guanine nucleotide-stimulated cAMP production and diminished sensitivity of adenylate cyclase to hLH correlated with a decline (P less than 0.05) in circulating progesterone and luteal weight during the late luteal phase. Thus, the adenylate cyclase system of the rhesus monkey CL undergoes significant changes during the luteal phase which are associated with the development and regression of the CL of the menstrual cycle. Mechanisms that modulate gonadotropin and nucleotide activation of adenylate cyclase without interfering directly with the catalytic unit are implicated in the changes that accompany luteolysis.