Dynamics of circulating concentrations of gonadotropins and ovarian hormones throughout the menstrual cycle in the bonnet monkey role of inhibin A in the regulation of follicle-stimulating hormone secretion.код для вставкиСкачать
American Journal of Primatology 71:817–824 (2009) RESEARCH ARTICLE Dynamics of Circulating Concentrations of Gonadotropins and Ovarian Hormones Throughout the Menstrual Cycle in the Bonnet Monkey: Role of Inhibin A in the Regulation of Follicle-Stimulating Hormone Secretion P.S. SURESH1 AND R. MEDHAMURTHY1,2 1 Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India 2 Primate Research Laboratory, Indian Institute of Science, Bangalore, India In higher primates, increased circulating follicle-stimulating hormone (FSH) levels seen during late menstrual cycle and during menstruation has been suggested to be necessary for initiation of follicular growth, recruitment of follicles and eventually culminating in ovulation of a single follicle. With a view to establish the dynamics of circulating FSH secretion with that of inhibin A (INH A) and progesterone (P4) secretions during the menstrual cycle, blood was collected daily from bonnet monkeys beginning day 1 of the menstrual cycle up to 35 days. Serum INH A levels were low during early follicular phase, increased significantly coinciding with the mid cycle luteinizing hormone (LH) surge to reach maximal levels during the mid luteal phase before declining at the late luteal phase, essentially paralleling the pattern of P4 secretion seen throughout the luteal phase. Circulating FSH levels were low during early and mid luteal phases, but progressively increased during the late luteal phase and remained high for few days after the onset of menses. In another experiment, lutectomy performed during the mid luteal phase resulted in significant decrease in INH A concentration within 2 hr (58.372 vs. 27.373 pg/mL), and a 2- to 3-fold rise in circulating FSH levels by 24 hr (0.2070.02 vs. 0.5370.14 ng/mL) that remained high until 48 hr postlutectomy. Systemic administration of Cetrorelix (150 mg/kg body weight), a gonadotropin releasing hormone receptor antagonist, at mid luteal phase in monkeys led to suppression of serum INH A and P4 concentrations 24 hr post treatment, but circulating FSH levels did not change. Administration of exogenous LH, but not FSH, significantly increased INH A concentration. The results taken together suggest a tight coupling between LH and INH A secretion and that INH A is largely responsible for maintenance of low FSH concentration seen during the luteal phase. Am. J. Primatol. 71:817–824, 2009. r 2009 Wiley-Liss, Inc. Key words: Bonnet monkey; corpus luteum; LH; FSH; INH A; P4; GnRH-R antagonist INTRODUCTION The main functions of the ovary, viz, steroidogenesis and gametogenesis, are regulated principally by pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). However, the female gonad in addition to secretion of steroids is also a site of synthesis and secretion of proteinaceous hormones such as inhibin, relaxin, oxytocin etc. Inhibin (INH) is a heterodimeric glycoprotein that comprises of a-subunit and one of the two different b subunits [bA and bB subunits to form inhibin A (INH A) (a and bA) and INH B (a and bB) isoforms], produced primarily by granulosa and granulosa-lutein cells (luteal cells) in the ovary [De Jong, 1988; Mather et al., 1992, 1997; Ying, 1988]. By definition, both INH isoforms suppress basal as well as gonadotropin releasing hormone (GnRH)stimulated secretion of pituitary FSH with effects on LH secretion has been suggested to be minimal r 2009 Wiley-Liss, Inc. [Burger, 1993; Plant et al., 1991]. Several studies have established that INHs have potentially complex interdependent endocrine, autocrine and paracrine functions essential for the regulation of growth and development of the ovulating follicle [de Kretser et al., 2002; Laven & Fauser, 2004]. During the macaque menstrual cycle, it was observed that immunoreactive INH levels were highest at the mid luteal phase and the levels could be suppressed by Correspondence to: Dr. R. Medhamurthy, Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore-560012, India. E-mail: firstname.lastname@example.org Received 11 December 2008; revised 15 April 2009; revision accepted 20 April 2009 DOI 10.1002/ajp.20709 Published online 18 May 2009 in Wiley InterScience (www. interscience.wiley.com). 818 / Suresh and Medhamurthy treatment with GnRH receptor antagonist, suggesting that INH is synthesized in the CL and that its secretion is integrated to the gonadotropin control of luteal function [Fraser et al., 1989]. Furthermore, the lutectomy experiment performed during mid luteal phase in monkeys clearly established the link between the presence of CL and circulating immunoreactive INH [Bassetti et al., 1990]. However, following development of specific assays for INH A and INH B, it was established that INH A secretion is highest during the luteal phase, while the high INH B circulating concentrations are observed only during the follicular phase, which originated from antral follicles and the dominant follicle destined for ovulation in women [Groome et al., 1996; Welt et al., 1999]. In higher primates, it is well established that circulating LH is essential for the maintenance of structure and function of CL [Zeleznik & Benyo, 1994]; however, whether LH controls INH A secretion during the luteal phase remains to be determined. Although FSH is regarded as the principal stimulus for INH secretion [Brannian et al., 1992], its role in the regulation of INH A secretion during the luteal phase is not known. In monkeys and women, administration of GnRH receptor antagonist has been shown to disrupt pituitary gonadotropin secretion and termination of the luteal phase [Duffy et al., 1999; Fraser et al., 1999; Yadav & Medhamurthy, 2006]. Previous work in the laboratory has confirmed that molecular markers of luteolysis including down regulation of expression of genes associated with steroidogenesis were demonstrable in CL collected 24–48 hr post GnRH receptor antagonist treatment [Yadav & Medhamurthy, 2006]. Recently, employing the GnRH receptor antagonist-induced model, we have demonstrated rescue of CL function with respect to progesterone (P4) secretion by exogenous LH replacement [Priyanka et al., 2009]. In light of the above information, the present experiments were carried out with the following objectives: (i) to establish various endocrine hormone profiles throughout the menstrual cycle in the bonnet monkey with emphasis on characterization of circulating concentrations of INH A and FSH prior to ovulation and during the period of late menstrual period, (ii) to study the effects of lutectomy on dynamics of ovarian hormones and FSH secretion and, (iii) to examine the effects of replacement of exogenous LH or FSH on INH A secretion during the GnRH antagonist-induced luteolysis. METHODS Animals Experiments involving monkeys in the study were cleared by the Institutional Animal Ethics Committee of the Indian Institute of Science, Bangalore. The experimental procedures followed are based on guidelines formulated by Indian National Science Academy and Indian Institute of Am. J. Primatol. Science, India. Adult female bonnet monkeys (Macaca radiata) weighing 3.8–4.5 kg and with a history of regular menstrual cyclicity (27–29 days length) were housed in individual stainless steel cages (eight monkeys of both sexes per room). The animals were maintained under a controlled photoperiod (12L:12D, lights on 0600 hr and lights off 1800 hr). During the study period performed during August to January months of the year the temperature in the animal rooms with provision for continuous change of fresh air, ranged from 22–281C (dry-bulb temperature) to 17–221C (wet-bulb temperature) maximum and minimum, respectively. Monkeys in the colony were fed a single daily meal between 1100 and 1200 hr that consisted of pellet food and supplemented with carrot, banana and sweet lime as been described previously [Medhamurthy et al., 2007]. Monkeys were also provided nutritional supplements such as Iron syrup preparation and high protein biscuits at monthly intervals. Experiment 1: Hormone Concentrations During the Normal Menstrual Cycle Four adult female bonnet monkeys were monitored daily for the onset of menses. Blood samples (1.5 mL) through femoral venipuncture were collected between 0900 and 1000 hr daily beginning day 1 of the menses for up to 35 days. Blood samples were stored overnight at 41C and the serum was separated by centrifugation and stored at 201C until assayed. Experiment 2: Effects of Lutectomy on Dynamics of Ovarian Hormone and FSH Secretions Adult female bonnet monkeys were observed daily for the onset of menses and blood samples were collected daily from day 8 to 12 of the menstrual cycle for determining the onset of estradiol (E2) and LH surges. The day of peak LH surge was designated as day 0, and one day after day 0 of peak LH surge was designated as day 1 of the luteal phase. Additional blood samples were collected on day 3 and 5 of the luteal phase to confirm for the rise in circulating P4 for the presence of functional CL. Lutectomy (n 5 4) was performed on day 8 of the luteal phase by accessing the ovaries through mid ventral laparotomy performed on ketamine hydrochloride (15 mg/kg body weight (BW) and pentobarbital sodium (8–12 mg/kg BW) anesthetized monkeys under aseptic conditions. Blood samples were collected at different time intervals before and after lutectomy for monitoring circulating hormones. In another group of monkeys (n 5 4) not subjected to surgical procedures, blood samples were collected at similar intervals to that of lutectomy group. Dynamics of Inhibin A and FSH Secretion / 819 Experiment 3: Concentrations of Circulating Ovarian Hormones and FSH After Treatment with GnRH Receptor Antagonist: Effects of LH and FSH Replacements on INH A Secretion Employing the induced luteolysis model system reported recently from the laboratory [Priyanka et al., 2009], circulating concentrations of E2, FSH and INH A were determined. Also, effects of replacement with LH or FSH during induced luteolysis on secretion of INH A were examined. For this purpose, monkeys were treated with 5.25% glucose solution (VEH, n 5 4) or CET (n 5 4) 150 mg/kg BW s.c., on day 7 of the luteal phase, and blood samples were collected immediately before and at different time intervals after VEH/CET treatment for five days. Another group of monkeys (n 5 6) were treated with CET on day 7 of the luteal phase for 24 hr, three of the monkeys were treated with a single injection of recombinant human (rh) LH (20 IU/kg BW i.v.). The dose was previously shown to stimulate the secretion of circulating P4 in the CET-treated monkeys suggestive of rescue of CL function [Priyanka et al., 2009]. Another three CET-treated monkeys received Gonal-Fs (recombinant hFSH preparation; 50 IU s.c.) as exogenous FSH supplementation sufficient for the stimulation of follicular growth in monkeys. Blood samples were collected at 8 hr postexogenous gonadotropin treatments. Hormone Assays Steroids (E2 and P4) in serum were determined by specific RIA as reported previously [Selvaraj et al., 1996]. The E2 (GDN ]244) and P4 (GDN ]337) antisera were kindly provided by Professor G. D. Niswender, Colorado State University, Fort Collins, CO. The sensitivities of the assays for E2 and P4 were 39 pg/mL and 0.1 ng/mL, respectively. Interand intra-assay coefficients of variation for both the hormones were o10%. Gonadotropins All samples from a series for a given animal were analyzed in one assay. Serum LH was quantified using a RIA kit supplied by the National Hormone and Pituitary Program, USA. It consists of antirecombinant cynomolgus LH (AFP342994) raised in rabbit and recombinant cynomolgus LH preparation, NICHD-Rec-Mo-LH-RP-1 (AFP 6936A), which was used both for iodination and preparation of standards. The antibody was used at a final assay tube dilution of 1:750,000 in a total assay set-up volume of 300 mL. For construction of the standard curve, the hormone standards employed ranged from 0.2 ng/mL (minimum) to 10 ng/mL (maximum). To rule out the possible serum effects (i.e. binding of tracer to antibody is influenced by the presence of nonspecific serum components), the reference standards were prepared initially in the monkey gonadotropin-free serum, but later replaced with female ovine serum (heterologous sera as a substitute for monkey gonadotropin free-serum). The average sensitivity of the assay was 0.2 ng NICHDRec-Mo-LH-RP-1/mL and the inter- and intra-assay coefficients of variation were o10%. Serum FSH was estimated using a monkey RIA kit supplied by the National Hormone Pituitary Program, USA. It consists of antirecombinant cynomolgus FSH (AFP782594) raised in rabbit and rec-moFSH–RP-1 (AFP-6940 A), which was used both for the preparation of standards and iodination. The antibody was used at a final tube assay dilution of 1:750,000 in a total set-up volume of 300 mL. The standard curve was generated employing hormone standards that ranged from 0.125 ng/mL (minimum) to 8 ng/mL (maximum). It was observed that serum influenced the binding by way of suppression of binding. To rule out the serum effects, the hormone standards were prepared in the bovine serum after confirming that bovine gonadotropins did not cross react with the antibody. The average sensitivity of the assay was 0.125 ng NICHD-Rec-Mo-LH-RP-FSHRP-1/mL, and inter- and intra-assay coefficients of variation were o10%. Inhibin A Concentrations of INH A in serum were measured using a commercially available Human ELISA kit (cat] DSL-10-28100, Diagnostic System Laboratories, Webster, TX). The sensitivity of the assay was 1 pg/mL, and inter- and intra-assay coefficients of variation were 7.6 and 6.2%, respectively. Statistical Analysis Wherever applicable, data were expressed as mean7SEM. The data of multiple groups were analyzed by one-way ANOVA, followed by the Newman-Keuls multiple comparison test (PRISM Graph pad, version 4; Graph Pad Software, Inc., San Diego, CA). A P value of o0.05 was considered statistically significant. Comparison between two groups for each time point was done with Student’s t-test. RESULTS Endocrine Hormone Concentrations Throughout the Menstrual Cycle in Female Monkeys Circulating mean (7SEM) serum E2 and LH concentrations throughout the menstrual cycle and few days after the onset of second menses are represented in Figure 1A. For calculation and representation of hormone concentrations, the day of peak LH surge was identified in each monkey and Am. J. Primatol. 820 / Suresh and Medhamurthy 25 P4 INH A 2.0 1.0 1.5 0.75 250 150 10 5 50 -10 -8 -6 -4 -2 0 2 4 6 100 1.0 0.5 50 0.5 FSH (ng/ml) 15 P4(ng/ml) 200 E2, INH A (pg/ml) 20 100 0.25 8 10 12 14 16 3.5 100 3.0 80 -6 60 2.0 40 1.5 1.0 20 0.5 0 -5 -4 -3 -2 -1 0 1 2 Days before and after menses 2.5 FSH (ng/ml) P4(ng/ml) IINH A (pg/ml) E2 FSH 150 300 LH (ng/ml) E2(pg/ml) 350 Fig. 2. The hormone data 5 days prior to the onset of menses (see Fig. 1) and hormone data from blood samples collected after the onset of menses are plotted to indicate patterns of FSH and ovarian hormone secretion during start of a new menstrual cycle. 0.0 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 Days before and after peak LH surge Fig. 1. Top and bottom panels, circulating mean (7SEM) concentrations of LH (solid square symbol), E2 (solid triangle), FSH (inverted solid triangle), P4 (solid diamond) and INH A (open circle) throughout the menstrual cycle of bonnet macaques. The day of peak LH surge concentration in each monkey was identified and designated as day 0 (a vertical dotted line is drawn between peak LH concentration and day 0) and circulating LH concentrations on different days before and after peak LH surge were represented in relation to the peak concentration. The days before peak LH surge were represented as decreasing days (with negative notation). was designated as day 0 of peak LH surge and all data points before and after day 0 (of peak LH surge) within each monkey was determined. The peak LH surge occurred 12.370.33 days after the onset of menses. The mean (7SEM) peak LH surge was 16.273.2 ng/mL (Fig. 1A), and the mean peak E2 concentration on the day of peak LH surge was 282.5733.5 pg/mL. Of the four monkeys, three had their peak E2 levels on the day of LH peak surge, but one monkey had high peak E2 surge level (320 pg/mL) a day preceding the peak LH surge. Mean (7SEM) hormone concentrations for each day was calculated after aligning the day of peak LH surge from all four monkeys. Circulating FSH concentrations fluctuated during the follicular phase (i.e. days preceding the onset of LH surge), but increased coincident with the LH surge and peak surge concentrations were 1.770.38 ng/mL (Fig. 1B). After the surge period, mean FSH concentrations remained low for most part of the luteal phase of menstrual cycle, but increased few days immediately before and after the onset of menses (Fig. 1B). Circulating mean (7SEM) of P4 and INH A concentrations throughout the menstrual cycle are shown in Figure 1B. As can be seen from the figure, serum P4 concentration was low on days preceding the onset of LH surge, and progressively increased to reach maximum concentrations 2.9370.20 ng/mL 7 days after peak LH surge, but the concentrations began to decline thereafter and were lowest on the Am. J. Primatol. day of menses. Circulating INH A concentrations were undetectable or low before the onset of gonadotropin surge. Circulating INH A concentrations determined immediately prior to LH surge and throughout the luteal phase revealed a pattern of secretion similar to P4 with highest concentrations seen 10–11 days postpeak LH surge, but thereafter began to decline to reach lowest concentration on the day of menses (Fig. 1B). Dynamics of P4, INH A, E2 and FSH Secretion During Late Menstrual Cycle and Menses To establish interrelationship between FSH and various ovarian hormones, circulating FSH concentrations along with P4, INH A and E2 concentrations were assayed and plotted 5 days before and 2 days after the onset of menses (Fig. 2). Circulating FSH concentrations progressively increased before occurrence of menses, but declined 1 day prior to the onset of menses. Circulating P4 concentrations 5 days prior to the onset of menses was 1.6270.39 ng/mL and declined to 0.3670.03 ng/mL on the day of menses. Circulating concentrations of INH A 5 days prior to the onset of menses were 81.9712.6 pg/mL, fell to 3.872.3 pg/mL on the day of menses and became undetectable after the onset of menses (Fig. 2). Circulating concentrations of E2 showed a progressive rise during the late luteal phase of the menstrual cycle and after the onset of menses. Effects of Lutectomy on Dynamics of FSH and Ovarian Hormone Secretions Circulating P4, INH A, E2 and FSH concentrations immediately prior to and at different time points after lutectomy are presented in Figure 3. Circulating concentrations of P4 declined immediately after performing lutectomy on day 8 of the luteal phase and the concentrations were significantly lower (Po0.05) throughout the 96 hr period monitored compared with concentrations in monkeys not subjected to lutectomy Dynamics of Inhibin A and FSH Secretion / 821 5 3 * * 2 * b 1 b b 0.8 FSH (ng/ml) * * 0.6 b 40 20 a * b * a 72 L11 * b c * b 160 b 48 L10 60 c a 0 12 24 L8 L9 80 bb 0.4 0.2 INH A (pg/ml) ** a E4(pg/ml) P4(ng/ml) 4 100 96 L12 Time (h) post lutectomy 120 80 40 a a a 0 12 24 L8 L9 48 L10 72 L11 96 L12 Time (h) post lutectomy Fig. 3. Circulating mean (7SEM) concentrations of FSH, INH A, P4 and E2 in monkeys subjected to lutectomy (solid line) on day 8 of the luteal phase of the menstrual cycle. The concentrations of various hormones in monkeys not subjected to lutectomy (dotted line) are also shown. Bars with different letters above them are significant (Po0.05) from each other. Po0.05 when analyzed with student’s t-test in comparison to control and lutectomy animals, df 6 for each time points. (Fig. 3). Circulating INH A concentrations, similar to P4, also decreased (Po0.05) 2 hr after lutectomy and remained low thereafter (Fig. 3). During the first 24 hr after lutectomy, circulating E2 concentrations were not significantly different from monkeys not subjected to lutectomy, but the concentrations were significantly (Po0.05) higher at 48 hr following lutectomy, and remained high at 72 and 96 hr postlutectomy (Fig. 3). Lutectomy on day 8 of the luteal phase of the menstrual cycle resulted in rise in FSH concentration 24 hr postlutectomy and remained high (Po0.05) after 48 hr compared with concentrations in monkeys not subjected to lutectomy (Fig. 3). Patterns of FSH Secretion and Ovarian Hormones Following Induction of Luteolysis Administration of CET, a GnRH receptor antagonist, led to significant decrease in P4 concentration within 12 hr after treatment (3.45 vs. 1.5 ng/mL at 0 and 12 hr, respectively; Fig. 4) and the levels continued to decline throughout the observation period (Fig. 4). Circulating concentrations of INH A also decreased within 24 hr after CET treatment and was 3.6471.07 pg/mL at 72 hr (Fig. 4). All monkeys exhibited menses 96–120 hr post CET treatment. Circulating E2 concentrations were lower (Po0.05) after CET treatment, but tended to increase after 48 hr (Fig. 4). In contrast to the ovarian hormone secretion patterns, concentrations of FSH although tended to be lower at some time points, but did not change significantly (Po0.05) throughout the observation period (Fig. 4). In CET-treated monkeys, circulating INH A concentrations were 82.970.46, 73.276.39 and 36.573.87 pg/mL at 0, 12 and 24 hr post treatment, respectively. In 5.25% glucose-treated monkeys (VEH for CET treatment), INH A concentrations were 65.9714.8, 7174.0 and 6872.0 pg/mL at 0, 12 and 24 hr, respectively. The INH A concentrations were significantly (Po0.05) lower 24 hr after CET treatment compared with VEH treatment. Administration of exogenous rhLH 24 hr after CET treatment led to significant (Po0.05) increase in INH A concentrations i.e. 62.075.2 pg/mL at 8 hr after rhLH injection. However, administration of rhFSH 24 hr after CET treatment did not result in significant (P40.05) change in INH A concentration at 8 hr after injection. INH A concentrations were 32.476.8 and 30.874.8 pg/mL before and 8 hr after rhFSH administration, respectively. DISCUSSION Expectedly, the patterns of gonadotropins and ovarian hormones, excepting INH A throughout the menstrual cycle in adult female bonnet monkeys, appear strikingly similar to women [Vande Weile et al., 1970] and the rhesus macaque [Knobil, 1974]. The INH A secretion patterns observed throughout the luteal phase in this study is also similar to that reported in women [Groome et al., 1996]. The findings of simultaneous occurrence of mid cycle peak E2 and LH surge concentrations are essentially similar to that previously reported for the rhesus macaque [Hotchkiss et al., 1971], but different from the observation of occurrence of E2 peak surge one day preceding the LH peak surge in women [Vande Weile et al., 1970]. Although bonnet monkeys Am. J. Primatol. 822 / Suresh and Medhamurthy 100 5 a 3 * *** 2 b 1 c c c c c * c * a 75 * * b 25 c c 0.6 100 0.5 80 0.4 0.3 0.2 a ** 60 40 b b 20 0.1 * 50 c E2 (pg/ml) FSH (ng/ml) a ** INH A (pg/ml) P2 (ng/ml) 4 b b b b b b 0 24 48 72 96 120 0 24 48 72 96 120 L7 L8 L9 L10 L11 L12 L7 L8 L9 L10 L11 L12 Time (h) post CET treatment Time (h) post CET treatment Fig. 4. Circulating mean (7SEM) concentrations of P4 (top, right), INH A (top, left), FSH (bottom, right) and E2 (bottom, left) in monkeys subjected to VEH (dotted line) or CET (150 mg/kg BW) treatment (solid line). Bars with different letters above them are significant (Po0.05) from each other. Po0.05 when analyzed with student’s t-test in comparison to VEH- and CET-treated animals, df 6 for each time points. have been extensively used as surrogates for various aspects of research related to humans, this is a first study that provides comprehensive information on patterns of secretions of gonadotropins and ovarian hormones during the menstrual cycle in the bonnet macaque. The FSH secretion data, especially the rise in FSH secretion that occurs few days prior to the onset of menses and on the day of menses, are very similar to women [Lahlou et al., 1999]. Circulating INH A concentrations in bonnet monkeys have not been reported earlier and this is the first study that provides information on INH A secretion throughout the menstrual cycle, especially during the luteal phase. In addition to the wellcharacterized endocrine and paracrine actions of ovarian inhibins, clinically they have also been recognized as important biomarkers of conception and as markers for early detection of reproductive tissue tumors [Groome and Evans, 2000]. It is interesting to note that secretion of P4 peak 2–3 days preceding the peak INH A secretion period, which is also observed in Japanese macaques [Shimizu et al., 2002]. The significance of occurrence of different time peak secretions between P4 and INH A during the luteal phase is not clear, but it may be that the transcription and translation processes of INH A require time for the ultimate expression of the protein and/or its clearance as compared with simpler twostep enzymatic synthesis of P4 together with its very short half-life. The physiological significance of increased secretion of INH A by the CL and subsequently by the placenta in the event of pregnancy is not clear. Understanding factors that regulate INH A secretion during the luteal phase is essential for examining its role in the development Am. J. Primatol. and function of CL during nonpregnant and pregnant cycles. Systemic and intra-luteal administration of INH A during mid luteal phase in rhesus monkeys readily caused suppression of FSH without altering the CL function [Stouffer et al., 1994]. It is of interest to note here that mouse with INH-a gene knockout has abnormal growth of CL and increased occurrence of ovarian tumors, suggesting perhaps INH expression is essential for CL regression [Burns et al., 2003]. It is unique that CL appears to be the primary site of synthesis of INH in primates [Bassetti et al., 1990]. Expressions of INH a and INH bA subunits have been reported in the CL [Eramaa et al., 1993; Smith et al., 1991]. Several experiments were carried out in this study to examine whether CL is the primary source of INH A expression. First, lutectomy resulted in precipitous fall in INH A levels. Second, administration of GnRH receptor antagonist to mid luteal phase monkeys caused fall in INH A concentration and third, administration of exogenous LH but not exogenous FSH to monkeys subjected to induced luteolysis resulted in restoration of INH A concentration that also resulted in rescue of CL function. Although it is well established that FSH regulates secretion of both forms of INH, but in this study, exogenous FSH treatment did not result in elevated INH A concentrations. This finding is not surprising, since it is well known that FSH receptor expression becomes down regulated or undergoes extinction after ovulation and luteinization [Minegishi et al., 1997]. We have observed low mRNA expression of FSH receptor in the CL throughout the luteal phase, and this may have contributed to lack of response to exogenous FSH treatment and thus absence of increase in INH A concentration. Dynamics of Inhibin A and FSH Secretion / 823 The results from this study suggest an inverse relationship between circulating INH A and FSH concentrations during the luteal phase in the bonnet monkey and inter-cycle rise in FSH during the lutealfollicular transition was observed after the significant decline in circulating P4 and INH A concentrations. These findings are similar to the previously reported findings in women in which a gradual but significant decline in circulating P4 and INH A concentrations were observed prior to the FSH rise [Groome et al., 1996; Roseff et al., 1989]. Before the onset of FSH rise around the menstrual period, a gradual but significant decline in circulating P4 and INH A levels was observed. It is clear therefore that the inter-cycle rise in FSH starts during the late luteal phase as a result of reduced negative feedback mechanisms responsible for the suppression of pituitary FSH secretion. Although it can be argued that the role played by P4 in the regulation of inter-cycle rise in FSH cannot be ignored, however, P4 appears to mediate its action mainly via its effect on the hypothalamic GnRH secretion, where it is thought to reduce the frequency, but increase the amplitude of LH pulse significantly during the luteal phase of the cycle [Nippoldt et al., 1989; Soules et al., 1984]. The decreased P4 level during the late luteal phase of the bonnet monkey as observed in this study, and as has been reported previously [McCartney et al., 2002] increase frequency of GnRH pulses leading to perhaps increased FSH secretion. However, it has been observed that the increased frequency of GnRH pulses is not solely responsible for inter-cycle rise in FSH in women [Welt et al., 1997]. The rise in circulating activin A concentration, a member of TGF-b super-family, appears to be another molecule responsible for the inter-cycle FSH rise [Muttukrishna et al., 2000]. To further gain insights into the mechanisms of feedback regulation of pituitary FSH secretion by CL during the luteal phase in the bonnet monkey, lutectomy performed during mid luteal phase caused a significant drop in the levels of circulating INH A and P4 with a concomitant rise in FSH within 24–48 hr. It appears then that during the luteal phase, the increased concentrations of INH A and P4 play an important role in the maintenance of low FSH secretion in the bonnet monkey. In women during the mid luteal phase, following ovariectomy, a significant drop in the levels of E2 and P4 was observed within the first 24 hr but the concentrations of FSH and LH increased gradually [Alexandris et al., 1997]. On the contrary, our data did not show any significant drop in the levels of E2 after lutectomy, rather E2 levels appeared to rise after 24 hr postlutectomy. The findings that GnRH secretion appears to be essential for maintaining basal FSH secretion was further confirmed by the observation that administration of GnRH receptor antagonist, CET, did not result in alterations in FSH concentrations, suggesting importance of GnRH secretion and INH A in the control of pituitary FSH secretion during the mid luteal phase of the bonnet monkey. In summary, we have characterized endocrine hormone profiles including INH A secretion throughout the menstrual cycle of the bonnet monkey. The results from the experiments described in this study suggest that INH A and P4 secreted during the luteal phase regulate FSH secretion. The results also suggest that increased GnRH secretion is essential for the rise in FSH following withdrawal of inhibition of regulation by P4 and INH A. ACKNOWLEDGMENTS We thank Dr. AF Parlow, NHPP for distribution of monkey LH and FSH RIA kits. The staff of PRL is gratefully acknowledged for their help with blood sampling. 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