Rapid and profound suppression of messenger ribonucleic acid encoding follicle-stimulating hormone beta by inhibin from primate Sertoli cells

We showed previously that inhibin, partially purified from cynomolgus monkey Sertoli cell culture medium (primate Sertoli cell inhibin referred to as pSCI), selectively suppressed basal FSH secretion from dispersed rat pituitary cells and decreased total cellular FSH, but not LH content, suggesting a decrease in FSH biosynthesis. In order to investigate the mechanism of action of inhibin at the molecular level, we have now examined the effects of pSCI on steady state levels of the subunit mRNAs encoding LH and FSH and correlated these with release and intracellular content of LH, FSH, and glycoprotein alpha-subunit. Dispersed pituitary cells from 7- to 8-week-old adult male rats were cultured in the presence of pSCI or control medium for 2-72 h. FSH secretion was reduced significantly by 6 h (P less than 0.05) and reached a nadir (38% of control) by 48 h. LH secretion was unchanged, while release of the alpha-subunit was decreased to 89% of control at 72 h (P less than 0.05). Also by 72 h, cell content of both FSH (73% of control) and alpha-subunit (81% of control) were significantly suppressed (P less than 0.001, P less than 0.01), while LH was slightly affected. Total RNA was extracted from the pituitary cell cultures, electrophoresed in 1.2% agarose-formaldehyde gels, transferred to nylon membranes, and hybridized with 32P-labeled cDNA probes for the rat alpha-, LH beta-, and FSH beta-subunits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancement of in vitro release of FSH from rat pituitaries by a synthetic nonapeptide fragment of human seminal plasma inhibin

A synthetic C-terminal nonapeptide fragment of human seminal plasma inhibin preferentially enhances the basal release of FSH from rat pituitaries incubated in vitro, which indicates a direct action of the peptide on the pituitary. However, in the presence of LHRH, both FSH and LH release was increased particularly at higher doses of the nonapeptide. There was no change in prolactin release at 5 and 50 ng/ml but prolactin release was suppressed significantly at 500 ng/ml.     

Similar effects of inhibin and cycloheximide on gonadotropin release in superfused pituitary cell cultures

The actions of two inhibin preparations and cycloheximide on gonadotropin release were investigated in superfused pituitary cell cultures. Pituitary cells isolated from 18-day-old male rats were grown in Matrigel-coated superfusion chambers in chemically defined medium. After stationary culture for 4 days, the cell monolayers were superfused at a constant speed (0.25 ml/min) and were intermittently stimulated (6 min/h) with 10 nM gonadotropin-releasing hormone (GnRH). Groups of cultures were exposed to the test substances for varying time periods during stationary culture and/or during superfusion. Inhibitory effects of both inhibin preparations on the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in response to GnRH pulses were observed after 2 h of exposure and became maximal after about 6 h. Basal secretion of FSH between GnRH pulses was also suppressed, whereas the basal interpulse secretion of LH was not changed. When exposure to inhibin was discontinued, the secretion of both FSH and LH progressively increased and returned to control values by approximately 6 h. Cycloheximide (500 ng/ml) affected gonadotropin release with dynamics similar to those observed for the inhibin preparation. These data support the hypothesis that inhibition of gonadotropin synthesis may be an important step in the molecular mechanism of action by which inhibin regulates gonadotropin release.     

Complete amino acid sequence of human seminal plasma beta-inhibin. Prediction of post Gln-Arg cleavage as a maturation site

The complete sequence of a 94 amino acid human seminal plasma polypeptide exhibiting inhibin-like activity is presented. This molecule, called beta-inhibin, selectively and specifically suppresses the release of pituitary FSH in vivo as well as in vitro. It does not affect the secretion of LH. Such a novel acidic protein contains a very basic C-terminal segment which is easily cleaved by mild tryptic digestion. It is predicted that the FSH inhibiting activity may reside within this region of the molecule. This would imply a post Gln-Arg cleavage to release the basic C-terminal active moiety.     

Regulation of pulsatile gonadotropin secretion by estrogen, inhibin, and follistatin (activin-binding protein) in ovariectomized rats.

The following study was conducted to examine the effects of estrogen and polypeptides, given either alone or in combination, on pulsatile gonadotropin secretion. One week after ovariectomy, rats received s.c. injections of oil or various doses (0.5, 5, 20 micrograms) of estradiol benzoate (EB) followed 1 day later by i.v. administration of 60 micrograms purified porcine follistatin, 10 micrograms recombinant inhibin, or the appropriate vehicle. Four hours after injection of the nonsteroids, blood was collected at 10-min intervals for 2 h, and the effects on pulsatile hormone release were assessed. Administration of EB alone dose-dependently suppressed mean and trough (lowest point between two pulses) FSH levels and all parameters of pulsatile LH release. Both follistatin and inhibin at the doses employed suppressed mean FSH levels to an equivalent extent (40%). Follistatin, but not inhibin, suppressed FSH pulse amplitude, while neither polypeptide alone influenced FSH pulse frequency or any parameter of pulsatile LH release. The effects of follistatin and EB on mean FSH levels were additive at all EB doses, whereas the effects of inhibin and EB were additive only at the middle EB dose. Follistatin in combination with the lowest EB dose significantly suppressed mean LH levels. These studies are the first to demonstrate that combined treatment with estrogen and the nonsteroids follistatin and inhibin is more efficacious in suppressing FSH release than treatment with either agent alone, thereby indicating that both steroids and nonsteroids are probably important in the physiological regulation of FSH secretion in rats. The additive effects of these compounds on FSH secretion could form the basis for exploring novel contraceptive interventions.     

Isolation of porcine follicular fluid inhibin of 32K daltons

Purification of ovarian inhibin from porcine follicular fluid was performed by using an bioassay based upon the suppression of spontaneous FSH release from cultured cells of rat anterior pituitary. The presence in the follicular fluid of four molecular forms of inhibin activity corresponding to Mr 100K, 80K, 55K and 32K was revealed by SDS-gel electrophoresis under non-reducing conditions. The smallest inhibin amongst them, named 32K inhibin, eliciting about 70% of the total activity in the follicular fluid, was separated by gel filtration in the presence of 8 M urea. By subsequent ion-exchange chromatography, followed by RP-HPLC, 32K inhibin was purified to homogeneity with a 8,000 fold purification factor in a yield of 12%. The purified 32K inhibin was found to comprise two polypeptide subunits (Mr 20K and 13K), linked by disulfide bridges and to specifically suppress the secretion of FSH, but not of LH from the pituitary cells.     

In vivo suppression of pituitary and circulatory follicle stimulating hormone by human seminal plasma inhibin

Human seminal plasma inhibin (HSPI), of prostatic origin, has been shown to bring about a dose dependent suppression in pituitary and circulatory FSH concentrations in intact rats. No significant changes in LH levels either in pituitary or in circulation were observed at the doses used. This has further been substantiated by an immunocytochemical staining. A marked reduction in staining intensity for FSH was observed in the pituitary of inhibin treated rats as compared to the controls. None of the purified inhibin peptides from other sources have so far been reported to act on pituitary FSH in vivo. This study thus, for the first time demonstrates an in vivo effect of inhibin (HSPI) on pituitary FSH concentration and secretion.     

Effect of uni- and bilateral cryptorchidism on testicular inhibin and testosterone secretion in rats

The effect of uni- and bilateral cryptorchidism on testicular inhibin and testosterone secretion and their relationships to gonadotropins were studied in rats. Mature Wistar male rats weighing approximately 300 g were made either uni- or bilaterally cryptorchid. Testicular inhibin and testosterone content and plasma levels of LH and FSH were examined 2 weeks later. A similar remarkable decrease in testicular inhibin content was found in uni- and bilaterally cryptorchid testes. On the other hand, the testicular testosterone content was significantly decreased only in unilaterally cryptorchid testis with an inverse increase in the contralateral testis. Plasma testosterone levels were normal and plasma LH and FSH increased significantly in both of the cryptorchid groups. These results showed that cryptorchidism impairs both Sertoli and Leydig cell functions. While testosterone production was compensated by increased LH for 2 weeks, neither inhibin secretion nor storage changed in cryptorchid or contralateral testes during the same period.     

Regulation of gonadotropin secretion and number of gonadotropin-releasing hormone receptors by inhibin, activin-A, and estradiol.

Primary cultures of ovine pituitary cells were used to characterize the effects of inhibin and activin on the secretion of gonadotropins and on the regulation of number of GnRH receptors in the presence or absence of estradiol. Number of GnRH receptors was determined by the specific binding of a saturating dose of [125I]des-Gly10-D-Trp6-GnRH-ethylamide (GnRH-A). Recombinant human inhibin-A (rh-inhibin-A) or inhibin in porcine and bovine follicular fluid (pFF and bFF, respectively) decreased secretion of FSH in a dose-dependent manner, with maximum inhibition at an inhibin concentration of approximately 0.1 nM. Neither pFF or bFF affected secretion of LH, although rh-inhibin-A caused a modest decrease (p less than 0.05) in secretion of LH. Treatment of cells with rh-inhibin-A, bFF, or pFF approximately doubled the number of GnRH receptors. Scatchard analysis indicated that increases in GnRH-A binding were due to an increase in receptor number rather than a change in affinity. Additionally, rh-inhibin-A, at a dose that doubled numbers of GnRH receptors, increased GnRH-induced LH release above that caused by GnRH alone, indicating that the increase in receptor number leads to increased responsiveness to GnRH. Recombinant human activin-A (rh-activin-A) increased secretion of FSH but did not affect secretion of LH. Number of GnRH receptors was not affected by lower concentrations of rh-activin-A but was decreased (p less than 0.05) by 3.0 nM activin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions between inhibin, oestradiol and progesterone in the control of gonadotrophin secretion in the ewe

Experiments were carried out to test the hypothesis that inhibin and oestradiol act synergistically to inhibit the secretion of FSH, to test for effects of progesterone, and to compare the FSH and LH responses to ovarian feedback. In Exp. 1, with 11 ovariectomized and 12 intact Romanov ewes during the anoestrous season, doses of oestradiol (administered by means of subcutaneous implants) that restored normal LH pulse frequencies were insufficient to restore normal concentrations of FSH. In Exp. 2, with 48 ovariectomized Welsh Mountain ewes during the breeding season, a factorial design with 4 ewes per cell was used to assess the responses in LH and FSH to 3 doses of oestradiol (s.c. implants) and 4 doses of bovine follicular fluid ('inhibin', 0.2-1.6 ml s.c. every 8 h). This was done initially in the absence of progesterone and then after 7 days of treatment with progesterone (s.c. implants). Analysis of variance revealed a significant synergistic interaction between oestradiol and inhibin on the plasma concentrations of FSH. Progesterone had little effect. In contrast, there was a significant synergistic interaction between oestradiol and progesterone on the concentrations of LH. 'Inhibin' also inhibited LH secretion but this effect was independent of the two steroids. We conclude that there are basic differences in the way that ovarian feedback acts to control the secretion of LH and FSH in the ewe. FSH secretion appears to be primarily controlled by the synergistic action of oestradiol and inhibin on the anterior pituitary gland, while the secretion of LH is inhibited during the follicular phase by an effect of oestrogen at pituitary level and during the luteal phase by the synergistic action of oestradiol and progesterone at the hypothalamic level. Inhibin, or another non-steroidal factor in follicular fluid, may also play a minor role in the control of LH secretion.     

Measurement of inhibin and an index of inhibin production by rat testes during postnatal development

Our previous studies have shown that inhibin activity in rat testes can be measured using an in vitro inhibin bioassay. In animals that have undergone unilateral efferent duct ligation (EDL), the difference in inhibin content between the ligated and nonligated testis can be used as an index of the rate of inhibin production in vivo. In the present study we examined postnatal changes in inhibin content in the testes, and the production rates of inhibin and seminiferous tubule fluid in groups of neonatal, immature, and adult rats from 1 to 80 days old. We detected inhibin activity in testes of 1-day-old rats; the activity level rose linearly to Day 8, subsequently increasing markedly from Day 30 until it reached a plateau at Day 45. Increments in the content of inhibin and weight of the testis after unilateral EDL, interpreted to represent production of inhibin and seminiferous tubule fluid, commenced at Day 20 and increased rapidly between Days 30 and 50, decreasing thereafter to Day 80. The increase in content and production of inhibin was directly correlated to the rise in serum follicle-stimulating hormone (FSH) and testosterone, suggesting that these two hormones are important in controlling inhibin secretion. In addition, the changes in serum FSH from the high, postnatal levels to those typical of adults were inversely correlated to the content and production rate inhibin in the testes and concentrations of testosterone in the serum. These data support the hypothesis that inhibin is specifically involved in the feedback control of pituitary FSH secretion during postnatal development, although a role for testosterone or a synergistic interaction between the two hormones cannot be excluded.     

Follicle-stimulating hormone secretion in monosodium glutamate-lesioned rats: response to unilateral gonadectomy or porcine follicular fluid (inhibin)

The purpose of this study is to determine the acute response of pituitary FSH and LH release to unilateral gonadectomy in the MSG-treated rat, and to determine whether pFF (inhibin) can act effectively on pituitary FSH secretion in the MSG-lesioned rat. MSG (4 mg/kg B.W.) or saline was injected subcutaneously on postnatal days 2, 4, 6, 8, and 10 to male and female littermates which were used in the experiments after postnatal day 60. In the first experiment male and female littermates were bilaterally gonadectomized and bled serially for the next 72 h. At 0 h plasma FSH concentrations in MSG-treated rats were lower (p less than 0.05) than those in saline-treated controls, and for the 72 h immediately following bilateral gonadectomy FSH levels increased parallel to those of the controls, but after a significant delay. In the second experiment, MSG-treated male and female littermates were injected with 0.5 ml of pFF at several intervals following bilateral gonadectomy and decapitated 6 hours later. Injection of pFF significantly suppressed circulating FSH titers in all groups without affecting LH levels. In a third experiment, rats were unilaterally gonadectomized and blood samples were obtained at various intervals for 48 h. Following unilateral gonadectomy there was a significant transient increase in FSH levels in male or female MSG-treated rats as compared to their 0 h values; however, the absolute levels attained were barely equal to the basal concentrations observed in the saline-treated control rats. The conclusions from these data are: insufficient FSH secretion in response to unilateral gonadectomy may be responsible for the lack of compensatory gonadal hypertrophy in MSG-lesioned rats, pituitary response to inhibin is apparently unaltered by MSG toxicity, and the MSG-lesioned rat is a useful model to study the differential control mechanisms of FSH and LH secretion.     

Changes in plasma inhibin levels following pulsatile gonadotrophin-releasing hormone therapy in a man with idiopathic hypogonadotrophic hypogonadism

Changes in circulating inhibin levels were related to changes in testosterone (T) and the gonadotrophins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in a hypogonadotrophic hypogonadal man before and during pulsatile gonadotrophin-releasing hormone therapy which resulted in normal spermatogenesis. Before treatment, the plasma inhibin levels in the patient (210 +/- 50 U/l; mean +/- SD of four samples) were lower than in normal controls (552 +/- 150 U/l; p less than 0.01), as were T (1.1 nmol/l) and gonadotrophin (less than 1.0 IU/l) levels. Within 1 week of gonadotrophin-releasing hormone treatment, plasma LH (14.1 +/- 0.7 IU/l) and FSH (14.4 +/- 0.6 IU/l) reached supraphysiological levels. In response, T and inhibin concentrations increased progressively to reach high normal levels (27.7 +/- 1.6 nmol/l and 609 +/- 140 U/l) at 4 weeks, by which time the gonadotrophin levels stared to decline and gradually returned to the normal range between 12 and 24 weeks of treatment. There was a concomitant decrease in T and inhibin levels which remained within the normal range. The decline in the FSH level following the rise in testicular hormones was earlier and steeper than that of LH (37.5% decrease at 4 weeks vs. 30.4% at 12 weeks), suggesting that T and inhibin may act together to inhibit pituitary FSH secretion as opposed to LH secretion which is primarily controlled by T. It is concluded that, in man, during maturation of the pituitary-testicular axis, changes in circulating inhibin parallel those of T, and quantitatively normal inhibin secretion is dependent on gonadotrophin stimulation. FSH secretion may be regulated through negative feedback control, by both T and inhibin.     

Influence of the degree of stimulation of the pituitary by gonadotropin-releasing hormone on the action of inhibin and testosterone to suppress the secretion of the gonadotropins in rams

This experiment determined if the degree of stimulation of the pituitary gland by GnRH affects the suppressive actions of inhibin and testosterone on gonadotropin secretion in rams. Two groups (n = 5) of castrated adult rams underwent hypothalamopituitary disconnection and were given two i.v. injections of vehicle or 0.64 microg/kg of recombinant human inhibin A (rh-inhibin) 6 h apart when treated with i.m. injections of oil and testosterone propionate every 12 h for at least 7 days. Each treatment was administered when the rams were infused i.v. with 125 ng of GnRH every 4 h (i.e., slow-pulse frequency) and 125 ng of GnRH every hour (i.e., fast-pulse frequency). The FSH concentrations and LH pulse amplitude were lower and the LH concentrations higher during the fast GnRH pulse frequency. The GnRH pulse frequency did not influence the ability of rh-inhibin and testosterone to suppress FSH secretion. Testosterone did not affect LH secretion. Following rh-inhibin treatment, LH pulse amplitude decreased at the slow, but not at the fast, GnRH pulse frequency, and LH concentrations decreased at both GnRH pulse frequencies. We conclude that the degree of stimulation of the pituitary by GnRH does not influence the ability of inhibin or testosterone to suppress FSH secretion in rams. Inhibin may be capable of suppressing LH secretion under conditions of low GnRH.     

The effect of stage of estrous cycle and follicular maturation on ovarian inhibin production in sheep

Twenty-four Scottish Blackface ewes (mean weight 50.0 +/- 0.1 kg with ovulation rate 1.3 +/- 0.1) were randomly divided into 4 groups of 6 animals. Under general anesthesia, following the collection of a timed sample of ovarian venous blood, the ovaries of these animals were collected either on Day 10 of the luteal phase or 12, 24, and 48 h after a luteolytic dose of a prostaglandin (PG) F2 alpha analogue (cloprostenol 100 micrograms i.m.) administered on Day 10. All follicles greater than 3 mm were dissected from the ovaries and incubated in Medium 199 (M199) at 37 degrees C for 2 h, following which the granulosa cells were harvested and incubated in triplicate for 24 h in M199 with or without ovine FSH or ovine LH. Plasma and culture media samples were assayed for inhibin, estradiol (E2), androstenedione (A4), and testosterone (T) by specific RIA. After correcting for hematocrit, ovarian secretion rates were calculated from the product of the plasma concentration and flow rate. The rate of ovarian inhibin secretion during the luteal phase was similar from ovaries categorized on the basis of presence of luteal tissue (1.0 +/- 0.3 and 0.9 +/- 0.5 ng/min for CL present and absent, respectively), confirming that the ovine CL does not secrete appreciable amounts of inhibin. Inhibin secretion was higher (p less than 0.05) at 12 h after PG-induced luteolysis but not at 24 or 48 h compared to values for luteal phase control ewes. Although ovaries containing large estrogenic follicles (greater than or equal to 4 mm in diameter and classified as estrogenic from in vitro criteria) secreted the most inhibin (55%; p less than 0.05), both ovaries containing large nonestrogenic follicles (33%) and small (11%; less than 4 mm in diameter) follicles secreted appreciable amounts of inhibin. This contrasted strongly with E2 where greater than 80% of the steroid was secreted by large estrogenic follicles. The rate of ovarian inhibin secretion was positively correlated (p less than 0.05) with the rate of E2, A4, and T secretion. Overall, there was no significant effect of stage of cycle on follicular inhibin content after 2 h incubation in vitro, release of inhibin by follicles incubated in vitro, or synthesis of inhibin by granulosa cells cultured in vitro. FSH and LH had no effect on the production of either inhibin or estradiol by cultured granulosa cells. Follicular diameter was positively correlated (p less than 0.001) with follicular inhibin and steroid release. Follicular inhibin content after 2 h incubation in vitro was more highly correlated with inhibin release by incubated follicles (r = 0.7; p less than 0.001) than with inhibin synthesis by granulosa cells in vitro (0.4; p less than 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibin production by Sertoli cells in culture.

Studies on the secretion of inhibin and its mode of action were carried out in vitro, utilizing cell cultures. Isolated rat Sertoli cells secreted an inhibin-like heat-labile, non-dialysable substance, Sertoli Cell Factor (SCF), which could selectively suppress FSH secretion by rat anterior pituitary cells. SCF selectively suppressed the basal and GnRH-stimulated FSH release as well as the de-novo synthesis of FSH by acting directly on the pituitary cells. In 1 out of 5 experiments, SCF also suppressed the synthesis of LH, possibly by affecting the overall protein synthesis. Under similar culture conditions, Sertoli cells isolated from animals between 18 and 90 days of age secreted comparable amounts of SCF. In contrast, anterior pituitary cells from adult rats (60-90 days old) were considerably more sensitive to SCF than pituitary cells obtained from younger (18-33 days old) animals, suggesting that decline in circulating FSH level, occurring at approximately 35 days of age, may result from increased pituitary sensitivity to inhibin. Besides identifying the Sertoli cells as the site of inhibin production in the testis, these studies demonstrated direct action of inhibin at the pituitary cell level, resulting in suppression of FSH synthesis and release.     

Inhibition of LHRH-induced LH and FSH release by gonadotrophin surge-attenuating factor (GnSAF) from human follicular fluid

It has been suggested that in superovulated women the endogenous LH surge is attenuated by a non-steroidal factor, called gonadotrophin surge-attenuating factor (GnSAF), which reduces gonadotrophin secretion in response to LHRH. To determine whether human follicular fluid (hFF) from superovulated women contains GnSAF activity, the secretion of LH and FSH by cultured sheep pituitaries was studied. After charcoal extraction of steroids, hFF was treated by heparin/Sepharose chromatography, which reversibly binds inhibin. The effects of whole hFF and the bound and unbound fractions on basal and LHRH-induced gonadotrophin secretion were then assessed. Steroid-free hFF significantly reduced basal FSH, but not basal LH, secretion, and significantly attenuated the LH and FSH responses to LHRH. The bound (inhibin) fraction significantly decreased both basal and LHRH-induced FSH secretion but did not affect LH release. The unbound fraction had no effect on basal LH or FSH secretion, but significantly attenuated LHRH-induced secretion of both LH and FSH. We conclude that the unbound fraction of hFF from superovulated women contains GnSAF. It has been demonstrated that GnSAF is a non-steroidal factor and its activity is distinct from that of inhibin.     

Effect of inhibin from primate Sertoli cells and GnRH on gonadotropin subunit mRNA in rat pituitary cell cultures

Partially purified inhibin from primate Sertoli cell culture medium (pSCl) suppresses both LH and FSH secretion from cultured rat pituitary cells stimulated with GnRH. To examine the mechanism of action of pSCl, we have measured steady state levels of mRNAs for the gonadotropin subunits in pituitary cell cultures exposed to 10 nM GnRH for 6 h in control or pSCl-containing medium (short term) and after 72-h pretreatment with pSCl or control medium (long term). Messenger RNA levels were determined by Northern analysis using specific cDNA probes for rat FSH beta, LH beta, and the common alpha-subunit. In the long term experiments, pSCl inhibited GnRH-stimulated release of FSH (47.4 +/- 3.3% of control), LH (69.2 +/- 2.3%), and free glycoprotein alpha-subunit (74.2 +/- 4.5%), and intracellular FSH declined to 88.4 +/- 3.5% of control. Concentrations of the subunit mRNAs were all decreased: FSH beta to 54.4 +/- 5.0%, LH beta to 79.6 +/- 9.4%, and alpha to 70.8 +/- 8.7% of control. In the short-term experiments, pSCl also suppressed FSH, LH, and alpha-subunit secretion to 75.9 +/- 3.6%, 79.5 +/- 2.1%, and 90.9 +/- 1.8% of control, respectively. Intracellular LH and alpha-subunit levels were significantly increased in cells treated for 6 h with GnRH and pSCl (155 +/- 18%, 145 +/- 14% of control), while FSH was comparable to control. After 6 h, pSCl selectively reduced the level of mRNA for FSH beta (56.5 +/- 5.8% of control).(ABSTRACT TRUNCATED AT 250 WORDS)     

Elevated inhibin concentration in the follicular fluid of dairy cows with chronic cystic ovarian disease

This study compared serum and follicular fluid inhibin and gonadotropin profiles between chronic cystic ovarian diseased (CCOD) and normal cyclic dairy cows. Blood samples and follicular fluid were collected from CCOD cows (n=15) and cyclic cows in the follicular phase of the estrous cycle (control, n=6) and analyzed for inhibin, follicle stimulating hormone (FSH) and luteinizing hormone (LH) concentrations. There was a significant increase in inhibin and a decrease in FSH and LH concentrations in the follicular fluid of CCOD cows compared with those of cyclic cows (P < 0.05). Mean serum inhibin, FSH and LH concentrations between CCOD and cyclic cows were not differnt (P > 0.05), however, there was a tendency for serum inhibin to be higher and FSH to be lower in CCOD cows compared to cyclic animals (P < 0.1). The FSH pulse frequency also was lower in CCOD cows than in cyclic cows (P < 0.05). These data suggest that increased production of inhibin from cystic follicles of CCOD cows alters pituitary FSH secretion and subsequently reduces the concentration of FSH in follicular fluid. As a result, decreased FSH stimulation at the ovarian level could ultimately lead to the reduction in follicular LH and FSH receptor concentrations, resulting in abnormal follicular steroidogenesis in CCOD dairy cows.