Cisplatin- and cyclophosphamide-induced primordial follicle depletion is caused by direct damage to oocytes.

It is well established that DNA-damaging chemotherapies can cause infertility and ovarian endocrine failure by depleting the ovarian reserve of primordial follicles. Currently, no effective pharmacological therapies exist for the preservation of long-term fertility and ovarian function in female cancer patients, due to a limited understanding of the mechanisms of chemotherapy-induced follicle depletion. This study investigated the cellular targets, molecular mechanisms, and temporal course of ovarian reserve depletion following treatment with commonly used chemotherapeutic drugs. Adult female C57BL/6 mice were injected i.p. with saline, cisplatin (5mg/kg), or cyclophosphamide (300mg/kg); ovaries were harvested after 8 or 24 hours. Follicle counts showed depletion of all follicular stages 24 hours after administration of cisplatin or cyclophosphamide. Eight hours post-treatment, H2A histone family member X (γH2AX) immunofluorescence showed DNA double-stranded breaks at all follicular stages, including within primordial follicle oocytes. This staining was resolving by 24 hours, indicating that primordial follicle oocytes begin to undergo either apoptosis or repair in this timeframe. γH2AX-positive follicles were further examined to identify the specific cell types damaged. In primordial, transitional, and primary follicles, only oocytes sustained DNA damage, whereas in secondary and antral follicles, only somatic cells were affected. TUNEL staining confirmed that apoptosis occurs in these targeted cell types. Whilst multi-drug and multi-dose regimens were not examined, this study conclusively shows that cyclophosphamide and cisplatin cause direct damage to primordial follicle oocytes, which then undergo apoptosis. Therefore, future pharmacological strategies to prevent chemotherapy-induced infertility in females must specifically prevent primordial follicle oocyte death.

The primordial follicle reserve is not renewed after chemical or γ-irradiation mediated depletion.

Reports indicate that germ-line stem cells present in adult mice can rapidly generate new oocytes and contribute to the primordial follicle reserve following conditions of ovotoxic stress. We further investigated the hypothesis that adult mice have the capacity to generate new oocytes by monitoring primordial follicle numbers throughout postnatal life and following depletion of the primordial follicle reserve by exposure to doxorubicin (DXR), trichostatin A (TSA), or whole-body γ-irradiation. We show that primordial follicle number remains stable in adult C57BL/6 mice between the ages of 25 and 100 days. However, within 2 days of treatment with DXR or TSA, primordial follicle numbers had declined to 65 and 51% respectively (P<0.05-0.01 when compared to untreated controls), with no restoration of follicle numbers evident after 7 days for either treatment. Furthermore, ovaries from mice subjected to sterilizing doses of γ-irradiation (0.45 or 4.5 Gy) revealed complete ablation of all primordial follicles 5 days after treatment, with no indication of follicular renewal. We conclude that neo-folliculogenesis does not occur following chemical or γ-irradiation mediated depletion of the primordial follicle reserve.

Estrogen signaling in the regulation of female reproductive functions.

Estrogens influence fertility and infertility in animals. This chapter reviews the use of estrogen as a contraceptive through the regulation of its production and action. It is concluded that the use of specific agonists and antagonists of estrogen action that avoid the global and unwanted side effects of estrogen offers new potential methods of contraception.

A randomized controlled trial of treatment options for troublesome uterine bleeding in Implanon users.

BACKGROUND: Pilot data have indicated that both doxycycline alone and mifepristone combined with ethinyl estradiol (EE) are effective in stopping episodes of bleeding in Implanon users with troublesome bleeding. We compared four treatments against a placebo in Implanon users and tested whether repeated treatment improved subsequent bleeding patterns. METHOD: Implanon users aged 18-45 years were randomized to treatment with (i) mifepristone 25 mg given twice on day 1 followed by 4 days of EE 20 microg; (ii) doxycycline 100 mg twice daily for 5 days; (iii) mifepristone 25 mg given twice on day 1 plus doxycycline 100 mg twice daily for 5 days; (iv) doxycycline 100 mg twice daily with EE 20 microg daily; and (v) placebo twice daily for 5 days. The primary end-point was the number of days of bleeding/spotting immediately following initiation of the first 5-day course of each therapy, compared with placebo. RESULTS: There were 204 women assigned to treatment. Mifepristone in combination with either EE or doxycycline was significantly more effective in stopping an episode of bleeding (mean 4.0 days (CI 3.5-4.6) and 4.4 days (CI 3.8-5.2), respectively) than doxycycline alone or in combination with EE, or placebo (6.4 days (CI 4.4-9.2), 6.4 days (CI 4.8-8.6) and 6.4 days (CL 5.1-8.0), respectively). CONCLUSION: Mifepristone combined with either EE or doxycycline was significantly more effective than placebo in terminating an episode of bleeding in Implanon users. However there was no improvement in subsequent bleeding patterns. TRIAL REGISTRATION NUMBER: ACTR # 012605000206628.

HtrA3, a serine protease possessing an IGF-binding domain, is selectively expressed at the maternal-fetal interface during placentation in the mouse.

Hemochorial placentation involves highly regulated interactions between fetal- and maternal-derived cells. HtrA3, a novel serine protease containing an insulin-like growth factor (IGF) binding domain, was previously shown to increase during early pregnancy in the mouse uterus, being dramatically upregulated post-implantation. The present study examined the regulation of HtrA3 gene in the mouse uterus from post-implantation to late gestation. Both mRNA and protein of HtrA3 were localized specifically in the maternal decidua. In contrast, HtrA3 expression was below detection in trophoblasts, including the giant cells that are in direct contact with the decidua. This pattern persisted from the early stages of placentation to near term. The level of decidual HtrA3 mRNA and its protein gradually decreased as the placenta matured. In the decidua, only the maternal decidual cells, but not blood vessels or uterine NK cells that are present in large numbers, were positive for HtrA3. The specific localization of a protease possessing an IGF-binding domain at the maternal-fetal interface suggests that HtrA3 plays a critical role in mediating maternal decidual remodelling and maintenance, likely in association with the IGF system, in placental development and function.

The N-terminal peptide of the inhibin alpha subunit What are its endocrine and paracrine roles?

alphaN inhibin (molecular mass 23 24 kD) is present in the pro-alphaN-alphaC subunit of inhibin and can be released by cleavage at the flanking arginine residues during posttranslational processing. Although the alphaN protein isolated from bovine follicular fluid has no inhibinlike (FSH suppressing) activity, alphaN is present in high molecular weight forms of biologically active inhibin found in follicular fluid and plasma. alphaN may modify the biological activity of inhibin by influencing its half-life or access to its receptor. alphaN may also play a role in regulating fertility through a local action on ovulation by the ovary that is independent of the actions of inhibin. The evidence suggests a unique physiological significance for the precursor peptides of the inhibin-alpha subunit in both the endocrine and paracrine control of fertility.

An inhibitory effect of transferrin on differentiation of rat granulosa cells in vitro.

The effects of human transferrin (TRF) on granulosa cell function were examined using serum-free cultures of rat granulosa cells obtained from immature, diethylstilbestrol-treated rats. The results show that TRF had dose- and time-dependent inhibitory effects on FSH-induced inhibin and progesterone production with the half-maximal inhibitory dose of 6.1-6.3 micrograms/ml. The inhibitory effect of TRF on FSH-induced inhibin and progesterone production was not reversed by removing TRF and changing medium after 48 h of treatment. TRF also inhibited insulin- and insulin-like growth factor-I (IGF-I)-induced inhibin production in a dose-dependent manner. TRF did not inhibit forskolin- and 8-bromo-cAMP-induced progesterone production but did inhibit inhibin production induced by these agents. TRF had no effect on basal production of inhibin and progesterone. On the other hand, high concentrations of insulin and cortisol completely counteracted the inhibitory effect of TRF on FSH-induced progesterone production but only partially counteracted the inhibitory effect of TRF on FSH-induced inhibin production. Our data suggest that: 1) TRF may be an important negative modulator of the stimulatory actions of FSH or IGF-I and other factors acting on granulosa cells; 2) the inhibitory effects of TRF require the presence of FSH or other factors such as IGF-I or insulin, which facilitate granulosa cell differentiation; and 3) different mechanisms are involved in the modulating effects of TRF on inhibin and progesterone production.

Activin-A inhibits oxytocin and progesterone production by preovulatory bovine granulosa cells in vitro.

The aim was to examine the effect of activin on luteinization of preovulatory bovine granulosa cells in vitro. Bovine activin-A was found to inhibit the production of oxytocin (OT) and progesterone by bovine granulosa cells from individual preovulatory follicles cultured in serum-free medium. The minimal response on OT production (25% inhibition) occurred with 0.1-1 ng/ml activin-A, and the maximal inhibition (83%) occurred with 10 ng/ml activin-A after 2-3 days in culture. Progesterone showed a similar response (30% inhibition for 0.1-1 ng/ml and 74% for 10 ng/ml). Inhibin production was not consistently effected by activin-A. Inhibin (75 U/ml) had no detectable effect upon OT or progesterone production. When activin-A was withdrawn from the cell culture after 72 h and the incubation continued for a further 72 h, a recovery in OT was seen on day 4 and 5 after activin-A doses of 0.1-1 ng/ml, but not after higher doses (3 and 10 ng/ml). Progesterone did not show a recovery, but the levels remained constant for 3 days (0.1 and 0.3 ng/ml activin-A) or for 1 day (1-10 ng/ml activin-A) and then fell to control levels by day 6 of culture. We conclude that bovine activin-A has an autocrine action on bovine granulosa cells in vitro, to inhibit basal production of OT and progesterone, consistent with the role of activin-A in delaying the process of luteinization.

The effect of follicle-stimulating hormone-suppressing protein or follistatin on luteinizing bovine granulosa cells in vitro and its antagonistic effect on the action of activin.

The time- and dose-dependent effects of bovine FSH-suppressing protein (FSP)/follistatin and human recombinant activin A (hr-Act) on oxytocin (OT) and progesterone (P) production, markers of luteinization, were studied in mature and immature bovine granulosa cells (GC), using three forms of FSP (31, 35, and 39 kDa) and a FSP pool consisting of 35, 39, and 45 kDa forms. FSP alone had no detectable effect on OT and P production when added to cultures of fully differentiated bovine GC. On the other hand, all FSP forms (10-100 ng/ml) enhanced and prolonged OT and P production of immature GC induced by bovine LH (10 ng/ml). Overall, 35 kDa FSP was more effective than the other forms tested. Hr-Act alone had a dose-dependent inhibitory effect on OT and P production on LH-stimulated immature GC. All four forms of FSP (30 or 100 ng/ml) added to cultures treated with hr-Act, reversed the inhibitory effect of hr-Act, with a significant increase (25%) above control levels using the 35 and 39 kDa FSP forms. In conclusion, FSP enhanced and prolonged the luteinization process, as indicated by OT and P production induced in immature GC by bovine LH, and was able to antagonize the inhibitory effect of hr-Act in this system. These studies suggest a physiological role for activin and FSP, as modulators of folliculogenesis and luteinization in the ovary. We propose that activin and FSP act in an autocrine fashion on GC in the ovarian follicle to regulate folliculogenesis and luteinization.

Effects of activin and follicle-stimulating hormone (FSH)-suppressing protein/follistatin on FSH receptors and differentiation of cultured rat granulosa cells.

The aim of this study was to investigate the actions of both activin and FSH-suppressing protein (FSP)/follistatin either alone or in combination on FSH receptor number and on the responsiveness of granulosa cells to FSH and LH. Granulosa cells were harvested from diethylstilbestrol-treated immature Sprague-Dawley rats and cultured 48 h in serum-free medium with or without treatment. Activin treatment alone (3-100 ng/ml) resulted in a 4-fold increase in FSH receptor number with no change in binding affinity. This effect of activin was inhibited 31% by FSP (100 ng/ml) treatment which alone had no effect on FSH receptor number. Treatment with activin (100 ng/ml) prevented FSH-induced down-regulation of FSH receptor number, whereas at lower concentrations (3-30 ng/ml) activin enhanced down-regulation of FSH receptor number by 20% (P less than 0.05). In contrast, FSP alone prevented FSH-induced down-regulation by increasing FSH receptor number up to 40-50%. Pretreatment of granulosa cells with activin, but not FSP, for 24 h increased the responsiveness of cells to FSH (20 ng/ml) and LH (40 ng/ml) shown by increases in aromatase activity, progesterone, and immunoreactive inhibin production over and above control in a manner which depended upon activin doses. We conclude that 1) activin enhancement of FSH action on rat granulosa cells may be mediated in part via regulation of FSH receptor number, and 2) the effects of FSP on granulosa cells are likely to be due to its activin binding properties.

Interaction between activin and follicle-stimulating hormone-suppressing protein/follistatin in the regulation of basal inhibin production by cultured rat granulosa cells.

There is evidence that FSH-suppressing protein (FSP) antagonizes the action of activin on the differentiation of rat granulosa cells by binding activin in vitro. We tested the interaction of activin and FSP in this in vitro system by examining the effects of FSP on activin dose-related stimulation of immunoreactive inhibin release by rat granulosa cells. Granulosa cells (2 x 10(5) viable cells/well) from diethylstilbestrol-treated immature rats were cultured for 48 h in McCoy's 5a serum-free medium with additives and increasing doses of bovine FSP (0-30 nM) and human recombinant activin (0-20 nM). Inhibin was measured in the medium by RIA. Activin caused a dose-related increase in basal inhibin production, which was maximal between 4-10 nM activin (ED50, 0.6 nM). With the addition of FSP, an apparent increase in the ED50 of the activin dose-response curves was observed, but there were no changes in the maximum response. This pattern closely resembled that of chemical antagonism of an agonist by an agent that binds with relatively high affinity to form a biologically inactive complex. Based on this premise, apparent high affinity activin binding to FSP was determined by Scatchard analysis to have a Kd of 0.13 +/- 0.07 nM (mean +/- SD) and to occur in a 2:1 or greater FSP/activin molar ratio. These data support the proposition that the antagonistic effect of FSP on activin is due to the formation of an inactive complex.

The effects of the preimplantation blastocyst in vivo and in vitro on protein synthesis and secretion by cultured epithelial cells from sheep endometrium.

Epithelial endometrial cells were isolated on day 13 from nonpregnant (NPr) and pregnant (Pr) ewes and cultured in the presence or absence of conditioned media from 15-day blastocysts (BM) under optimized conditions in the presence of [35S]methionine (S-met). The incorporation of S-met into secreted protein was analyzed, and individual proteins were identified by autoradiography of two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Incorporation of S-met into secreted protein was higher (P less than 0.05) for cells from Pr than NPr animals. Secretion by cells from NPr ewes was increased in three of four cases by addition of BM to the culture medium. Autoradiography of two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis showed five secreted proteins [mol wt range 74,000-120,000; isoelectric point (pI) less than 6.5] which were either absent from or present in only small amounts in secretions from cells from NPr animals. The proportion of these proteins was greatly increased in secretions from cells from Pr animals. The presence of BM in cultures from NPr ewes enhanced the secretion of these same proteins, this effect being maintained even after heat treatment of the BM. One of these proteins had previously been shown to be maximally induced by the presence of estrogen and progesterone. Three other similarly controlled proteins were also enhanced, though to a lesser extent, by the presence of a blastocyst in vivo but were not stimulated by BM in vitro. It is concluded that endometrial epithelial cells from Pr ewes are metabolically more active than those from ewes on day 13, and the blastocyst and its secretions induce the secretion of several specific proteins by epithelial cells, some of these proteins being the same as those controlled by the combination of estrogen and progesterone.

Immunization against the N-terminal peptide of the inhibin alpha 43-subunit (alpha N) disrupts tissue remodeling and the increase in matrix metalloproteinase-2 during ovulation.

Immunization of ewes against the N-terminal peptide of inhibin alpha 43 (alpha N) reduces fertility; this is thought to be due to impaired oocyte release at ovulation. This study further investigates the effect of alpha N immunoneutralization on the ovulatory process. Light microscopy was used to examine the effects of alpha N immunization of the tissue-remodeling process during ovulation and formation of the corpus luteum (CL) structure. Changes in follicular levels of matrix metalloproteinase-2 (MMP-2) with approaching ovulation were also investigated in normal and alpha N-immunized ewes. Differences in structure of 2-day-old CL were observed between control and alpha N-immunized ewes. Control CL had confluent luteal tissue throughout the internal structure and invaginations of theca and vasculature were common and penetrated deep into the luteal tissue. Immunized ewe CL had large fluid-filled antra, giving them a cystic appearance; luteal tissue remained a thin 10- to 15-cell layer lining the wall surrounding the antrum. Infolding of the surrounding tissue was incomplete, and thecal/vascular invaginations were rare and failed to penetrate into the luteal tissue. Morphologically normal rupture stigma were seen at the apex of both control and alpha N-immunized CL. Gelatin-digesting activity in follicular fluid collected 0, 12, and 24 h after hCG administration in control ewes increased significantly as the time of ovulation approached (827 +/- 182, 842 +/- 159, and 1230 +/- 89 mU/ml, respectively, in Exp 1; 743 +/- 32, 1182 +/- 98, and 1306 +/- 91 mU/ml at the same times in Exp 2). alpha N immunization reduced follicular gelatinase activity at each time in Exp 1 (533 +/- 132, 740 +/- 67, and 809 +/- 147 mU/ml) and Exp 2 (587 +/- 21, 768 +/- 27, and 891 +/- 53 mU/ml); the reduction was significant at 24 h in Exp 1 and at all times in Exp 2. Gelatin zymography of follicular fluid revealed bands of gelatinase of 72/67 kilodaltons, consistent with latent and active MMP-2. The area digested by both latent and active MMP-2 increased with approaching time of ovulation and was reduced by alpha N immunization. These data suggest that MMP-2 has a role in the tissue-remodeling processes of ovulation and CL formation in the ewe and that immunization against alpha N, which impairs fertility, effects the preovulatory cascade of intrafollicular proteolytic activity, reducing MMP-2 levels and disrupting normal CL formation.

Regulation of follistatin production by rat granulosa cells in vitro.

The aims of this study were to apply enzyme-linked immunosorbent assays (ELISA) for human follistatins (FS) to measure total immunoreactive (ir-) rat FS and free rat FS, and investigate the regulation of production of total ir-FS and free FS by rat granulosa cells (GC) in vitro. Production of ir-inhibin was monitored as an index of GC function. The ELISAs for total ir-FS, based on an immunoradiometric assay developed recently for human FS, and free FS, based on capture of FS by a monoclonal antibody and detection by activin A binding, had sensitivities of 0.4 and 0.8 ng recombinant human (rh-) FS 288/ml, respectively, and did not cross-react with inhibin A, rLH, or FSH. rh-Activin did not cross react in the total ir-FS ELISA, but interfered with the measurement of free FS. Dilutions of GC-conditioned medium were parallel to the standard curve of rh-FS 288 for each assay. The values obtained in the free FS assay were 10- to 20-fold higher than those in the total ir-FS ELISA, suggesting that rat FS may be recognized by the antibodies differently than the human standard. Both total ir-FS and free FS production by undifferentiated GC from diethylstilbestrol (DES)-treated, immature rats increased with cell number and time in culture and were stimulated dose dependently by FSH, rh-activin A (except free FS, which was not measured because of interference), forskolin, and phorbol 12-myristrate. The effects of FSH and activin on FS production by undifferentiated GC were additive. There were significant effects of degree of differentiation of GC on basal FS production and responsiveness to FSH, LH, and rh-activin A. Both total ir-FS and free basal FS production increased up to 4-fold with the degree of differentiation of GC, produced by treating rats in vivo with DES (undifferentiated), DES plus FSH (partially differentiated), or DES plus FSH plus hCG (fully differentiated). The addition of FSH in vitro increased FS production by undifferentiated and partially differentiated GC, but not by fully differentiated GC. The only detectable effect of LH on FS production was on partially differentiated GC. Activin A stimulated total ir-FS production by undifferentiated and partially differentiated GC, but inhibited total ir-FS production by fully differentiated GC. Ir-inhibin production in these experiments was similar to that of FS with the following exceptions; phorbol 12-myristrate inhibited ir-inhibin production by undifferentiated GC, basal ir-inhibin decreased in fully differentiated GC, FSH stimulated ir-inhibin only in undifferentiated GC, and rh-activin A stimulated ir-inhibin at all stages. It is concluded that 1) FS protein production by cultured undifferentiated rat GC is up-regulated by FSH and activin, possibly via both protein kinase A and C pathways; 2) increasing GC differentiation is associated with a significant increase in basal FS production by rat GC and a change in the hormonal regulation of FS production; and 3) FS and ir-inhibin production by cultured rat GC can be differentially regulated. The results are consistent with the hypothesis that activin tone decreases within follicles as they develop due to increased production of the activin-binding protein FS.

Inhibin concentrations in ovarian and jugular venous plasma and the relationship of inhibin with follicle-stimulating hormone and luteinizing hormone during the ovine estrous cycle.

A heterologous RIA for ovine inhibin was developed which was sufficiently sensitive and specific to describe the peripheral concentrations of immunoreactive inhibin (iINH) during the estrous cycle of the ewe and to examine the effects of cautery of ovarian follicles on concentrations of iINH in ovarian and jugular venous plasma. Parallel logit-log dose-response lines were observed among ovine follicular fluid, ewe plasma, and pure native ovine (31 kDa) and bovine (31 kDa) inhibin. iINH could not be detected in ovariectomized ewe plasma, and there was no apparent cross-reactivity with a variety of structurally related and unrelated hormones and peptides, except a monomeric form of the alpha-subunit of INH, iINH in follicular fluid was 10(4)-fold higher than that in ovarian venous plasma, which was 3-fold higher than that in peripheral plasma. Cautery of the follicles resulted in a 35% reduction in iINH and an 81% reduction in estrogen concentrations in the ovarian vein within 10 min. During the estrous cycle, iINH and FSH were inversely related in samples taken over 30 h in the luteal phase (r = -0.69; P less than 0.001) and in the pre- and postovulatory phases (r = -0.45; P less than 0.001). iINH and LH were not related in the luteal phase, but were weakly positively correlated in the follicular phase (r = 0.31; P less than 0.01). iINH and estrogen concentrations in the follicular phase were also weakly correlated (r = 0.30; P less than 0.001). Furthermore, iINH concentrations rose in the follicular phase and decreased within 3-6 h of the preovulatory surges of LH and FSH, reaching a nadir around the time of the second rise in FSH 24-48 h later. It is concluded that 1) large antral follicles are a major source of peripheral iINH during the ovine estrous cycle; 2) iINH levels increase in the follicular phase with the growth of the dominant follicle and may be inhibited by the preovulatory surge of gonadotropin; 3) the fall in inhibin after the LH surge may be responsible for the second rise in FSH; and 4) the inverse relationship between FSH and iINH is consistent with the hypothesis that inhibin is involved in the feedback regulation of FSH.

The effects of estrogen and progesterone in vivo on protein synthesis and secretion by cultured epithelial cells from sheep endometrium.

Ovariectomized ewes were treated with either nothing or implants of estrogen (E), progesterone (P), or E + P. Epithelial and stromal cells from caruncular and intercaruncular regions of sheep endometrium were dispersed by collagenase digestion and enriched by Ficoll gradient separation. Verification of cell types was by electron microscopy, keratin staining (epithelial cells), cell size, and appearance in culture. Epithelial cells were cultured under optimized conditions with [35S]methionine (S-met) and uptake of label by cells and its incorporation into cellular and secreted protein determined. Protein in the medium and lysed cells was analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cells from E-treated animals had higher S-met uptake and incorporation into proteins (cellular and secreted) than cells from ewes treated with nothing and P-treated animals. E effects were not significantly reduced in the presence of P. When secreted protein was expressed as a percent of total incorporated S-met, P treatment either alone or with E increased the proportion of labeled protein secreted by cells. There were no significant differences between caruncular and intercaruncular. Two-dimensional polyacrylamide-gel electrophoresis of secreted proteins showed one major glycoprotein (mol wt, 46,000, isoelectric point, 5.8-6.5) and four minor proteins induced by E + P greater than E, and five minor proteins inhibited by the steroids. Both induction and inhibition of cellular proteins were also apparent, though of lesser magnitude. Overall, whereas E treatment in vivo influenced the rate of incorporation of S-met into proteins by epithelial cells in vitro, P treatment increased the proportion of newly synthesized protein which was secreted. Steroids caused significant alterations in the individual proteins secreted by ovine endometrium.

The radioimmunoassay of follicle-stimulating hormone (FSH)-suppressing protein (FSP): stimulation of bovine granulosa cell FSP secretion by FSH.

A RIA for bovine (b) FSH-suppressing protein (FSP) was developed using an antiserum raised in a rabbit to purified 39-kDa bFSP, iodinated 35-kDa FSP as tracer, and purified 35-kDa bFSP as standard. Purified 35-kDa FSP was iodinated using the iodogen procedure, and the iodinated FSP was purified by dye affinity chromatography. After a logit log-dose transformation of the dose-response curves, parallel displacement lines were observed between 31-, 35-, and 39-kDa FSP, bovine follicular fluid, bovine granulosa cell culture medium, and medium from bovine granulosa cells stimulated with bFSH. The specificity of the assay was investigated by comparing the immunoassay levels of FSP with in vitro bioassay levels based on the ability of FSP/inhibin to suppress FSH in rat anterior pituitary cell cultures in fractions obtained throughout the purification procedure of FSP from bovine follicular fluid. This demonstrated that 1) the FSP immunoactivity was associated with in vitro bioactivity in all fractions of the purification procedure; 2) a number of inhibin-related and other proteins showed low (less than 0.5%) or nondetectable cross-reactivity in the RIA; and 3) the in vitro biological to immunological ratios for 31-, 35-, and 39-kDa FSP were similar, indicating that the RIA detects all forms of purified bFSP. The secretion of FSP by bovine granulosa cells in culture was investigated in the presence and absence of bFSH and bLH, respectively. FSP production was proportional to granulosa cell number and decreased from highest levels at 24 h to lowest levels at 96 h of culture. The addition of either bFSH or 8-bromo-cAMP to the culture medium stimulated FSP production by a factor of 2-3 at 48 and 72 h of culture, while the addition of bLH had no effect on FSP production. Theca interna tissue cultured under the same conditions did not produce FSP. In contrast to FSP, stimulation of bovine granulosa cells with bFSH or bLH had no effect on inhibin production during the 96 h of culture, while the addition of bFSH and bLH caused a stimulation of progesterone production at 48 and 72 h of culture. It is concluded that 1) the RIA described here is specific for all mol wt forms of bFSP; 2) FSP was secreted by bovine granulosa cells and not thecal cells in vitro; and 3) FSP secretion by bovine granulosa cells in vitro is regulated by bFSH and not bLH.

Effect of purified 31K bovine inhibin on the specific binding of gonadotropin-releasing hormone to rat anterior pituitary cells in culture.

The mechanism by which inhibin decreases the responsiveness of the pituitary gonadotroph to GnRH in terms of secretion of gonadotropins is largely unknown. We studied the effect of pure 31K bovine inhibin on the specific binding of GnRH to rat anterior pituitary cells in culture using iodinated Buserelin as tracer. Results showed that treatment of cultured anterior pituitary cells from adult male rats with inhibin (0-30 U/ml) for 72 h decreased Buserelin binding in a dose-dependent manner. In the presence of a maximally inhibiting dose of manner. In the presence of a maximally inhibiting dose of inhibin, Buserelin binding decreased progressively with time, reaching a minimum of 42% of the control value after 3 days. Exposure of pituitary cells for 3 days to the inhibin-related peptides transforming growth factor-beta (up to 400 pM) and Müllerian inhibitory substance (up to 100 nM) did not decrease binding of Buserelin, suggesting that the effect was specific to inhibin. Inhibin did not compete with iodinated Buserelin for GnRH-binding sites when they were added to the assay tube simultaneously. In addition, treatment with inhibin halved the number, but did not change the affinity, of GnRH-binding sites and had no effect on either cell number of cell viability. It is concluded that the reduction by inhibin of rat gonadotroph responsiveness to GnRH may be partly related to a decrease in the number of GnRH receptors on the cell surface.

Inhibitory effect of pure 31-kilodalton bovine inhibin on gonadotropin-releasing hormone (GnRH)-induced up-regulation of GnRH binding sites in cultured rat anterior pituitary cells.

Primary cultures of enzymatically dispersed rat anterior pituitary cells were used to examine the effect of pure 31 kilodalton bovine inhibin on GnRH-induced up-regulation of GnRH binding sites. After 2 days in culture, the cells were exposed to stimuli with or without test substances for 10 h, followed by evaluation of GnRH binding sites using iodinated GnRH-A (Buserelin) as tracer. Inhibin suppressed GnRH-induced up-regulation of GnRH binding sites in a dose-dependent manner with an IC50 of 0.13 U/ml (5.5 pM). The inhibin-related peptides transforming growth factor-beta, and Müllerian inhibitory substance had no detectable effect (stimulatory or inhibitory), suggesting that the action is specific to inhibin. In addition, inhibin inhibited the calcium ionophore A23187-induced up-regulation of GnRH binding sites, indicating that this effect of inhibin can occur, at least in part, at a stage subsequent to Ca2+ mobilization. Inhibin did not compete with iodinated GnRH-A for GnRH binding sites. In conclusion, pure 31 kilodalton bovine inhibin suppressed GnRH-induced up-regulation of GnRH binding sites in cultured rat anterior pituitary cells, providing direct evidence that inhibin modulates delayed actions of GnRH.

Large luteal cells the source of luteal oxytocin in the sheep.

To determine the cellular origin of oxytocin produced by the cyclical corpus luteum (CL) of the sheep, enriched fractions of enzymatically dispersed small and large luteal cells from 12 CL were prepared on a Ficoll 400 gradient. Oxytocin was measured by RIA. Large luteal cells contained 1.08 +/- (SD) 0.70 fg/cell oxytocin, which was congruent to 30 X the content of small luteal cells. Endothelial cells contained little if any oxytocin. During a 12-h incubation, large luteal cells produced 0.28 fg/cell.h oxytocin: small luteal cells did not produce measurable amounts of oxytocin. It is concluded that the large luteal cells are the source of the oxytocin produced by the CL of the sheep.