Immunisation against the amino-terminal peptide (alpha N) of the alpha 43 subunit of inhibin impairs fertility in sheep.

Processing of the 58 kDa to the 31 kDa form of inhibin (Inh) involves cleavage of the amino-terminal peptide (alpha N) from the alpha 43-subunit. We show that active immunisation of female sheep against a recombinant bovine alpha N impairs their fertility. In Exp 1, 5 treated (Group 1; 300 micrograms alpha N) and 6 control ewes (Group 2; adjuvant only) were immunized (Day 1) and given boosters on Days 22 and 56. In Group 1, mean +/- SEM binding of 125I-31 kDa Inh was less than 0.5% on Days 33 and 44, whereas binding of 125I-58 kDa Inh was 4.9 +/- 0.7 and 6.2 +/- 0.6%, respectively. In Group 2 binding of both tracers was less than 0.5%. The corpora lutea (CL)/ewe in Group 1 on Days 44 and 82 were 1.8 +/- 0.2 and 2.8 +/- 0.9, respectively, and were not different from those in Group 2 (1.7 +/- 0.3 and 1.5 +/- 0.2, respectively). One ewe in Group 1 versus 5/6 ewes in Group 2 were diagnosed pregnant. In Exp 2, 18 treated and 16 controls were immunized as in Exp 1. The binding of 125I-58 kDa Inh in treated ewes (2.4 +/- 0.3%) was greater than in controls (less than 0.5%) on Day 56. The CL/ewe in treated ewes (1.8 +/- 0.2) was similar to that in controls (2.0 +/- 0.1) on Day 76. All 16 control ewes but only 7/17 treated ewes were subsequently diagnosed pregnant. The plasma progesterone concentrations were similar in treated ewes which did (7.6 +/- 1.2 nmol/L) and did not (7.0 +/- 0.7) become pregnant. Neither basal nor GnRH-stimulated concentrations of LH, nor basal concentrations of Inh differed between treated and controls in Exp 2. Similarly, there were no differences in FSH, except that basal concentrations were higher in the luteal phase of treated ewes. We conclude that immunisation of ewes against alpha N results in a significant reduction in fertility.

Gonadotropic stimulation of inhibin secretion by the human ovary during the follicular and early luteal phase of the cycle.

We studied the pattern of secretion of inhibin bioactivity from the ovary into peripheral blood during the follicular and early luteal phase of the menstrual cycle in women receiving gonadotropin therapy. Multiple follicular development was stimulated in 5 women undergoing in vitro fertilization and embryo transfer for tubal infertility using three different treatments designed to vary the concentration of FSH and LH (14 cycles). The women received clomiphene citrate (150 mg/day) from days 2-6 alone or supplemented with either exogenous human menopausal gonadotropin (28 IU/3 h) or pure FSH (28 IU/3 h) from day 6 until the day of follicle aspiration. Inhibin concentrations increased 10-fold in parallel with those of estradiol, from 0.2-0.3 U/mL on day 2 (before the onset of treatment) to 4-5 U/mL on day 14 of the cycle (time of the peak LH level). Coincidental to the LH surge, the inhibin concentration declined 2- to 3-fold before increasing again early in the luteal phase. The concentration of inhibin was higher in the gonadotropin-treated group (clomiphene plus human menopausal gonadotropin/FSH) than in the group treated with only clomiphene during the follicular phase. The number of follicles stimulated was significantly higher (P less than 0.001) in the group given exogenous gonadotropins [4.8 +/- 0.4 (SE)] than in the clomiphene alone group (2.2 +/- 0.4). These data strongly suggest that both the Graafian follicles and the corpus luteum secrete inhibin, which together with estradiol and progesterone may play a role in the regulation of FSH secretion during the luteal phase.

Comparison of inhibin immunological and in vitro biological activities in human serum.

A comparison of serum inhibin levels in men and women was undertaken using a sensitive sheep pituitary cell in vitro bioassay and a newly developed heterologous RIA. The RIA was based on an antiserum raised to bovine 31K inhibin using [125I]31K inhibin as tracer. Bovine inhibin alpha- and beta-subunits, bovine activin-A, transforming growth factor-beta, and Mullerian inhibitory substance did not cross-react in the RIA. In both assays, dilutions of serum gave response lines parallel to that of the partially purified human follicular fluid inhibin preparation used as standard. Negligible levels of both bio (B)- and immuno (I) activities were found in serum from women with premature ovarian failure or castrated men. In ovulation-induced cycles, serum B inhibin levels increased progressively from the early to the late follicular phase and remained at the late follicular phase level during the early and midluteal phases. Serum I inhibin levels also rose during the follicular phase, but declined during the early luteal phase before increasing again in the midluteal phase. As a consequence, inhibin B:I ratios varied during the treatment cycle, with high ratios in early follicular (2.86) and early luteal (2.25) phases and a low ratio in the midluteal phase (1.09). Similar changes in serum B:I ratios also occurred during the midcycle and midluteal phases of normal cycles. The B:I ratio was lower (0.35) in normal men. We conclude that the largely similar pattern of inhibin biological and immunological activities in serum obtained during a variety of physiological conditions support the validity of the RIA procedure, and the B:I ratio of serum inhibin varies during the follicular and luteal phases of the cycle and is low in men. Potential reasons for these changes in B:I ratio include the presence of interfering substances in either the bioassay or the RIA, the presence of inhibin isoforms, and/or modulation of secreted forms by sex steroids.

Manipulation of inhibin during the luteal-follicular phase transition of the primate menstrual cycle fails to affect FSH secretion.

The pattern of inhibin concentrations in blood during the menstrual cycle in primates has suggested an endocrine role of inhibin in the negative feedback control of FSH secretion during the luteal phase. Conversely, the fall in inhibin during the late luteal phase may play a role in the rise in serum FSH during the luteal-follicular phase transition. This hypothesis was examined by determining the effects of manipulation of inhibin on FSH secretion in stumptailed macaques. During the mid-luteal phase the putative inhibin feedback was inhibited by i.v. administration of 20 ml of ovine antiserum to human recombinant inhibin in 4 macaques. FSH secretion was unaffected during the initial 24 h period post-treatment and the timing of the rise in FSH which occurred during the subsequent luteal-follicular phase transition was normal. To determine whether the elevated serum concentrations of FSH observed during the early follicular phase could be reduced by administration of inhibin, 5 cyclic macaques were treated with 200 micrograms of recombinant human inhibin i.v. Serum FSH concentrations were unaltered. These results suggest that inhibin does not play a major role in modulating FSH secretion during the luteal-follicular phase transition.

Production of inhibin bioactivity by human granulosa-lutein cells: stimulation by LH and testosterone in vitro.

Granulosa-lutein cells from human preovulatory ovarian follicles were cultured for up to 12 days to determine their capacity for production of inhibin in vitro. Using a highly sensitive sheep pituitary cell bioassay we observed time-related changes in basal inhibin production, maximal during the first 4 days of culture (48 +/- 15 units/million cells every 2 days, means +/- S.E.M.; n = 5 patients) falling to values five times lower by day 12. After 4-6 days of culture in the presence of human LH (hLH) inhibin production was enhanced in proportion to the hLH dose (maximum five fold at 10 ng/ml); hFSH over the same dose-range had no effect. Progesterone production in response to hLH followed a similar pattern to that of inhibin and was also unresponsive to hFSH. In the absence of exogenous aromatase substrate, basal and gonadotrophin-stimulated oestradiol production was negligible after the first 4 days. Addition of testosterone (1 mumol/l) to the culture medium increased oestrogen formation several hundred-fold with no effect on progesterone production. Inhibin production was also increased by 50-100% in the presence of testosterone. These results demonstrate that LH and testosterone stimulate the production of inhibin by granulosa-lutein cells in vitro. It is suggested that inhibin production occurs under hormonal control in the corpus luteum as well as in the preovulatory follicle in the human ovary.

Inhibin bioactivity and pituitary cell mitogenic activity from cultured chicken ovarian granulosa and thecal/stromal cells.

A sensitive bioassay for inhibin based on the suppression of FSH release from cultured sheep anterior pituitary cells was used to determine whether inhibin is present in the preovulatory follicle in the domestic hen. Granulosa and thecal/stromal layers were separated from the five largest (F1-F5) yellow yolky follicles in the ovary and incubated in culture medium for 18 h. Inhibin was found predominantly in the media in which granulosa layers had been incubated. There was a progressive increase in the amount of inhibin produced per mg granulosa layer protein during the 5-6 days before ovulation. The ovary was observed to contain a growth factor which stimulated the proliferation of ovine pituitary cells. Thecal/stromal layer-conditioned medium (ThCM) but not granulosa layer-conditioned medium had a dose- and time-dependent mitogenic effect on cultured sheep pituitary cells. The maximal mitogenic effect achieved for ThCM was four to fivefold greater than control media and was significantly higher than the maximal mitogenic effects of epidermal growth factor (250 ng/ml; 1.5 x control) and transforming growth factor-beta (500 ng/ml; 1.2 x control). It is concluded that inhibin is produced by the granulosa layers in the large yellow yolky preovulatory ovarian follicles of the domestic hen. The thecal/stromal layers in these follicles produce a potent mitogenic factor, not produced by the granulosa layers, which stimulates the division of ovine anterior pituitary cells in vitro.

Inhibin secretion by the sheep ovary during the luteal and follicular phases of the oestrous cycle and following stimulation with FSH.

The secretion of oestradiol and inhibin were measured during the follicular and luteal phase of the cycle by a sensitive bioassay using sheep pituitary cells in culture in four ewes in which the left ovary had been autotransplanted to the neck. On day 12 of the cycle, premature luteal regression was induced with an injection of 100 micrograms cloprostenol (prostaglandin F2 alpha analogue; PG) and ovarian venous blood was collected every 4 h for 72 h. These same four ewes were infused in the ensuing cycle with NIH-oFSH-S14 at 10 micrograms/h for 48 h immediately after an injection of PG and sampled as above. During the luteal phase (-2 h before PG) both in the control and FSH-infused cycles the inhibin secretion rate (SR) was 27-45 units/min. After PG injection, the inhibin SR declined with time to reach 3.6-5 units/min at the onset of the LH surge (60 h after PG) in the control cycle. In contrast, in the following cycle infusion of FSH after PG injection caused a slight increase in the inhibin SR which then remained raised at 42-50 units/min for up to 60 h after PG. In the late follicular phase the oestradiol SR was greater in the FSH-infused than in the control cycles, indicating multiple follicular development. In the FSH-infused cycle the preovulatory surges of LH and FSH were markedly attenuated. These data demonstrate that (1) inhibin SR is high during the luteal phase suggesting that the sheep corpus luteum secretes inhibin, (2) in the control cycle inhibin SR declines during follicular maturation at a time when oestradiol SR is increasing but FSH levels are decreasing, and (3) exogenously administered FSH stimulates the secretion of inhibin from the ovary during the follicular phase.

Measurement of exogenous and endogenous inhibin in sheep serum using a new and extremely sensitive bioassay for inhibin based on inhibition of ovine pituitary FSH secretion in vitro.

An extremely sensitive and reliable bioassay for inhibin based on inhibition of ovine pituitary FSH secretion in vitro was developed and used to measure exogenous and endogenous inhibin activity in the ewe. The sheep inhibin bioassay is 30- to 40-fold more sensitive than conventional rat inhibin bioassays. The minimum sensitivity of each bioassay in the measurement of inhibin activity in 1 ml of sheep serum is 220 mu. and 4080 mu. in the sheep and rat bioassays respectively. This sensitive inhibin bioassay has permitted, for the first time, the measurement of endogenous inhibin in the peripheral and ovarian vein blood of the sheep, as well as exogenously administered inhibin. The half-life of exogenously administered ovine inhibin (in follicular fluid) in the sheep was calculated as two components (18-24 and 50-60 min) from the inhibin profiles of six ewes. Inhibin contained in the ovine follicular fluid, given as a bolus i.v. injection, increased to maximum levels after 5 min and then remained increased for 10-32 min depending upon the dose administered, before exponentially decaying. The time for inhibin to exert its effect ranged from 3 to 6 h after injection and appeared to be dose-related. The bolus injection of inhibin, apart from causing suppression of FSH, evoked a large rebound increase of FSH up to 400% of preinjection levels. The development of the sheep bioassay will allow the measurement of biologically active inhibin in the peripheral circulation and ovarian vein blood of sheep with the possibility of extending this to man.

Inhibin bioactivity in human testicular extracts.

Inhibin bioactivity was measured in human testicular extracts by a sensitive sheep pituitary cell bioassay. The relationship between testicular inhibin bioactivity, daily sperm production (DSP) and plasma concentrations of FSH, LH, testosterone and oestradiol were examined. The mean level of testicular inhibin bioactivity was 4.4 +/- 1.3 U/g (mean +/- SD) with a significantly lower value in those who received radiotherapy (3.2 +/- 1.4 U/g) than in the untreated group (4.8 +/- 1.1 U/g). In contrast to the rat, human testicular inhibin bioactivity was not significantly correlated to FSH or DSP. These findings suggest that inhibin may have a complex role in normal and/or pathological testicular function.

FSH causes a time-dependent stimulation of preovulatory follicle growth in the absence of pulsatile LH secretion in ewes chronically treated with gonadotrophin-releasing hormone agonist.

The study investigated the relationship between the plasma concentration of FSH and the stimulation of preovulatory follicle growth in vivo in ewes chronically treated with the gonadotrophin-releasing hormone (GnRH) agonist buserelin (HOE 766). Welsh Mountain ewes with regular oestrous cycles were treated for 6 weeks with two discs implants placed s.c., each containing 5 mg of the agonist in a matrix of polyhydroxybutyric acid. Treatment with the agonist for 35 days produced a sustained suppression of the plasma concentration of FSH, stopped the pulsatile release of LH and prevented follicular development beyond 2.5 mm diameter. There was no difference between the total number of follicles greater than 1.0 mm diameter present in the ovaries of GnRH agonist-treated ewes and day 8 luteal phase control ewes. During the sixth week of agonist treatment ewes were infused with ovine FSH (6 micrograms NIADDK-oFSH-16/h) in the presence of only basal concentrations of LH. After 24, 48, 72 or 120 h of FSH infusion, the mean number of follicles greater than 1.0 mm diameters per ewe was not significantly different between treated and control animals. Infusion of FSH caused a time-dependent increase in (1) the number of follicles per ovary greater than 2.5 mm, (2) the mean diameter of these follicles and (3) the proportion of the large follicles which could be classified as oestrogenic (greater than 3.7 nmol oestradiol/follicle per h in vitro). Injection of human chorionic gonadotrophin (750 IU i.m.) after 120 h of FSH infusion caused the majority of these large follicles to ovulate and form apparently normal corpora lutea. These results indicate that, in the absence of pulsatile LH, FSH stimulates the growth of normal large oestrogenic follicles which, when stimulated, ovulate to produce viable corpora lutea.

The antagonistic effect of exogenous LH pulses on FSH-stimulated preovulatory follicle growth in ewes chronically treated with a gonadotrophin-releasing hormone agonist.

The hypogonadotrophism model induced by the chronic administration of gonadotrophin-releasing hormone (GnRH) agonist was used to investigate the effects of different concentrations of FSH with or without LH pulses on the stimulation of follicular development in the ewe. Continuous administration of an agonist (buserelin) by osmotic minipump to thirty-six Welsh Mountain ewes from the early luteal phase for 5 weeks resulted in a sustained suppression of the plasma concentration of FSH and inhibited the pulsatile release of LH. The inhibition of gonadotrophin secretion was due to the desensitization and/or down-regulation of pituitary gonadotroph function, since the agonist-treated animals showed no response to a challenge of 1 microgram GnRH. During week 6 of agonist treatment, ewes were infused with either 4-hourly pulses of ovine LH (9 micrograms/pulse), low concentrations of ovine FSH (3 micrograms/h) or high concentrations of FSH (9 micrograms/h) alone or with 4-hourly pulses of LH. After 5 days of gonadotrophin infusion, there was no difference between the mean number of follicles per ewe from the animals treated with LH alone, low concentrations of FSH with or without LH pulses or the high concentration of FSH alone compared with the mean number of follicles from control ewes on day 8 of the luteal phase. Infusion of the high concentration of FSH alone stimulated the development of an increased number of large oestrogenic follicles (follicles greater than 2.5 mm in diameter and secreting greater than 3.7 nmol oestradiol/h in vitro) compared with control ewes. The addition of high-amplitude LH pulses to the infusion of the high concentration of FSH prevented follicles developing beyond 2.5 mm in diameter, but doubled the number of small follicles (less than or equal to 2.5 mm) present in the ovaries. These results show that normal follicular development can be induced by physiological concentrations of FSH alone in the absence of pulsatile LH release. The addition of high-amplitude LH pulses antagonized this stimulatory effect of FSH on follicle growth in the ewe.

The sheep corpus luteum secretes inhibin.

An experiment was performed in 20 Merino ewes in which ovarian venous blood was collected by venepuncture at surgery and at two stages of the oestrous cycle. The ovarian venous concentrations of inhibin, oestradiol-17 beta and progesterone were determined. The results demonstrate that during the luteal phase of the oestrous cycle the ovarian venous blood draining an ovary containing luteal tissue contains significantly more inhibin bioactivity than ovarian venous blood from an ovary not containing luteal tissue. During the follicular phase the concentration of inhibin bioactivity in ovarian venous blood was reduced compared with the luteal phase. From this data we conclude that the sheep corpus luteum secretes inhibin bioactivity into the ovarian venous blood.

Effect of level of food intake of ewes on the secretion of LH and FSH and on the pituitary response to gonadotrophin-releasing hormone in ovariectomized ewes.

The effect of level of food intake on LH and FSH profiles and pituitary sensitivity to gonadotrophin-releasing hormone (GnRH) was investigated in two groups of 12 ovariectomized ewes. Ewes with a high intake (group H) had a mean daily intake (+/- S.E.M.) of 1.99 +/- 0.075 kg dry matter (DM)/head per day while ewes with a moderate intake (group M) consumed a mean of 1.02 +/- 0.021 kg DM/head per day. Ovaries were surgically removed from six ewes of each group on day 11 of the luteal phase and from the remainder 30 h after an injection of 100 micrograms prostaglandin analogue given on day 11 to induce luteolysis. During both the luteal phase and the follicular phase, mean LH and FSH concentrations and LH pulse frequencies and amplitudes were unaffected by the level of intake but mean plasma prolactin concentrations were higher (P less than 0.05) in group H than in group M ewes in the follicular phase. Mean LH and FSH concentrations at day 2 after ovariectomy were unaffected by treatment while mean prolactin concentrations were higher (P less than 0.05) in group H than in group M ewes. At day 7 after ovariectomy, mean LH and FSH concentrations were lower (P less than 0.05) in group H than in group M ewes although mean LH pulse frequencies and pulse amplitudes were not significantly affected by the level of intake at either time.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in the plasma concentrations of inhibin throughout the normal sheep oestrous cycle and after the infusion of FSH.

A radioimmunoassay for inhibin was developed using a peptide containing the 1-26 amino acid sequence of the N-terminus of the alpha-chain of 32 kDa porcine inhibin as immunogen, and 125I-labelled tracer. Evaluation of this assay using Sephadex column chromatography, chromatoelectrophoresis and immunoblotting confirmed that it measured all forms of inhibin present in sheep follicular fluid and was suitable for measurement of inhibin in sheep plasma. There was no evidence of the presence of free alpha-subunit in either sheep follicular fluid or ovarian vein plasma. The concentration of inhibin in jugular plasma throughout the follicular and luteal phases of four ewes with ovarian autotransplants was measured. The ovarian secretion of inhibin and oestradiol were also measured simultaneously throughout the follicular phase in a spontaneous cycle and after infusion of NIH-oFSH-S14 at 10 micrograms/h for 48 h following premature luteal regression induced by prostaglandin. The results showed: (1) no change in the peripheral concentration of inhibin throughout the cycle except an increase related to the periovulatory increase in FSH and LH. (2) Following luteal regression, the concentration of FSH fell as the secretion rate of oestradiol increased. During this time there was no significant change in the peripheral concentration of inhibin or ovarian inhibin secretion rate. (3) Following the infusion of FSH there was a marked increase in the concentration of inhibin in both ovarian and peripheral plasma and an increase in ovarian inhibin secretion rate. (4) The calculated metabolic clearance rate of inhibin, 20.3 ml/min, is similar to that of FSH. We conclude that in the ewe the ovarian inhibin secretion rate is stimulated by FSH and, although inhibin may modulate the basal secretion of FSH, a change in its secretion does not account for the fall in FSH which occurs during the follicular phase of the sheep oestrous cycle.

Inhibition of FSH-stimulated granulosa cell function by a synthetic fragment of the porcine inhibin alpha-subunit: evidence for involvement of GnRH receptors.

The bioactivity of a synthetic peptide fragment which mimics the N-terminal sequence of the 134-amino-acid porcine inhibin alpha-subunit (pl-alpha 1-26-Gly27Tyr28-OH) was tested and compared with the bioactivity of GnRH in rat granulosa cell cultures. Granulosa cells from immature female rat ovaries were cultured with hFSH and testosterone to stimulate the production of cyclic AMP, progesterone and oestradiol. Addition of pl-alpha 1-26-Gly27Tyr28-OH to the culture medium caused a dose-dependent suppression of all three parameters (ID50 700-1,000 nmol/l). GnRH caused similar but higher-potency inhibition (ID50 2-4 nmol/l). Suppression of granulosa cell function by both peptides was fully reversible by a synthetic GnRH antagonist. Moreover, specific binding of the porcine inhibin fragment to ovarian GnRH receptors was demonstrated by radioreceptor assay. This is evidence that the porcine inhibin alpha-subunit fragment suppresses FSH-induced rat granulosa cell function via a mechanism of action similar to that of GnRH.

The effect of acute immuno-neutralisation of inhibin in ewes during the early luteal phase of the oestrous cycle on ovarian hormone secretion and follicular development.

Ewes with ovarian autotransplants received either inhibin antiserum (10 ml i.v. raised in sheep against recombinant 32 kDa human inhibin; n = 6) or sheep serum (10 ml i.v.; n = 5) on day 3 of the luteal phase with additional daily injections (1 ml i.v.) from 48 h after the initial bolus until day 13. Jugular and ovarian venous blood samples were taken 4-hourly over days 2-13 of the luteal phase. Blood samples were also taken at more frequent intervals (every 10-15 min for 2-3 h) to examine pulsatile secretory responses from the ovary to endogenous and gonadotrophin-releasing hormone-induced (150 ng i.m.) LH pulses on days 4, 6, 8, 10 and 12 of the luteal phase. Plasma FSH levels, ovarian steroid secretion and ovarian follicular development were measured. The ovarian follicle population was estimated daily by real time ultrasound scanning. Immunisation against inhibin resulted in a 3- to 4-fold increase (P < 0.001) in plasma FSH levels within 8 h with levels remaining elevated over controls for 6-7 days. Within 24 h of immunisation there was an increase in the number of small ovarian follicles (P < 0.05) and by 3 days after treatment immunised ewes had 4-6 large ovarian follicles/ewe with this increase in the total number of large follicles being maintained for the rest of the experimental period (P < 0.05). Mean ovarian oestradiol secretion during intensive bleeds was not different from controls 24 h after immunisation, but by 3 days after immunisation it was elevated 4- to 5-fold (P < 0.001) over controls with this increase being maintained throughout the experiment. Similar responses to immunisation against inhibin in androstenedione secretion were observed although mean androstenedione secretion was not elevated until 7 days after treatment. In vitro antibody titres in immunised ewes remained elevated but declined steadily (P < 0.001) over the experimental period. We conclude that the initial stimulation of follicle development and ovarian steroid secretion following passive immunisation against inhibin can be attributed to increased blood FSH. However, the fact that with time FSH declined but increased follicle development was sustained, despite maintenance of high circulating antibody titres, suggests that on a longer term basis inhibin immunisation may stimulate ovarian function by interfering with the modulation of follicle development by inhibin at an ovarian level.

Factors affecting the secretion of immunoactive inhibin into testicular interstitial fluid in rats.

Immunoreactive inhibin was measured in testicular interstitial fluid (IF) from rats during sexual maturation or after impairment of spermatogenesis induced by ethane dimethanesulphonate (EDS), unilateral cryptorchidism or local heating (43 degrees C, 30 min) of the testes, to ascertain its usefulness as a marker of changing Sertoli cell function. Cultures of isolated seminiferous tubules were also studied. Inhibin was measured by a radioimmunoassay directed towards the first 26 amino acids of the N-terminus of the alpha-subunit, and the results confirmed for selected pools of IF by in-vitro bioassay using dispersed ovine pituitary cells. During puberty, IF levels of immunoactive inhibin fell by more than 90% (P less than 0.001) between 30 and 60 days of age, a decrease paralleled by the levels of androgen-binding protein (ABP), another Sertoli cell product secreted into IF. These changes also paralleled, but preceded, the fall (60%; P less than 0.001) in serum levels of FSH between 40 and 70 days, while the serum and IF levels of testosterone increased more than two-fold over this period. When adult rats were injected with EDS to destroy the Leydig cells, testosterone levels in IF and serum were undetectable at 3 and 7 days after treatment, were just detectable at 14 days and thereafter returned slowly towards normal by 42 days. The initial androgen withdrawal following EDS treatment caused a progressive reduction in testicular weight up to 21 days and this was accompanied by a significant increase in the serum levels of FSH and a two- to threefold increase in the IF levels of immunoactive inhibin (and also of ABP). Serum FSH and IF levels of immunoactive inhibin returned to within the normal range by 42 days when testosterone levels had normalized. In contrast, in two other experimental situations in which a marked decrease in testicular weight coupled with an increase in IF levels of ABP occurs, different results for the IF levels of immunoactive inhibin were obtained. Thus, in rats exposed to local heating of the testes, IF levels of immunoactive inhibin remained unchanged from control values at 21-40 days after treatment, a finding confirmed by bioassay results. In rats made unilaterally cryptorchid for 10 months, levels of immunoactive inhibin in IF were reduced by 60% (P less than 0.01) in the abdominal compared with the contralateral scrotal testis.(ABSTRACT TRUNCATED AT 400 WORDS)

Secretion of bioactive inhibin by the ovary of the Booroola Merino ewe with or without a copy of the fecundity (F) gene.

The secretion rates of bioactive inhibin, oestradiol and progesterone were measured during the mid-luteal phase and at various times during the follicular phase of the cycle by a sensitive bioassay using sheep pituitary cells in culture in 12 Booroola ewes with and without copies of the Fecundity (F) gene in which the left ovary had been auto-transplanted to the neck. Inhibin secretion was high during the luteal phase and fell in the early follicular phase in all genotypes (P less than 0.01). In Booroola ewes with a F/- genotype, inhibin secretion then increased again, towards luteal rates, in the mid and late follicular phases. In Booroola ewes without a copy of the F gene (+/+) inhibin secretion remained low at all three sampling times in the follicular phase. The secretion rate of inhibin at 36 h (P less than 0.1) and 48 h (P less than 0.01) were significantly lower in ewes from the +/+ (no copy of the gene) ewes than in F/- (one copy of the gene) ewes. Oestradiol secretion was low during the luteal phase and increased steadily during the early (24 h) to a plateau in the mid (36 h; P less than 0.01) and late (48 h; P less than 0.05) follicular phase. Progesterone secretion was high during the luteal phase, and decreased to a very low rate by 24 h after prostaglandin (PG) treatment (P less than 0.001) and remained low. At 24 h after PG the concentration of FSH was significantly lower (P less than 0.01) than that during the luteal phase and remained suppressed until the onset of the LH surge.(ABSTRACT TRUNCATED AT 250 WORDS)

The roles of inhibin and gonadotrophin-releasing hormone in the control of gonadotrophin secretion in the ewe.

The respective roles and relative importance of ovarian inhibition and hypothalamic stimulation in the differential control of the secretion of FSH and LH were studied in the ewe. In the first experiment two groups of ten intact ewes were injected i.v. twice daily with 9 ml charcoal-extracted bovine follicular fluid (bFF), a preparation rich in inhibin (3.65 ku./ml), throughout the luteal phase of the oestrous cycle. Compared with the control ewes, this treatment significantly reduced pituitary and plasma FSH concentrations and increased the frequency and amplitude of the LH pulses, but did not affect pituitary LH concentrations. In a second experiment, five control and five bFF-treated ewes from experiment 1 were ovariectomized and the injection regime was altered to 2.5 ml s.c. every 8 h. This treatment was maintained for 21 days. In control ewes, plasma FSH concentrations rose significantly within 12 h and continued to rise for 3-4 days. Treatment with bFF abolished this increase and maintained plasma FSH concentrations below those observed in intact ewes. The rise in mean plasma LH concentrations evoked by ovariectomy was also partially inhibited in the bFF-treated ewes. The response to the gonadotrophin-releasing hormone (GnRH) agonist buserelin (5 micrograms i.v.) was measured 6, 12 and 18 days after ovariectomy. In control ewes the agonist consistently evoked large surges of both hormones but in bFF-treated ewes the FSH response was completely blocked and the initial phase of the LH response (the first 'pool') was greatly reduced. In experiment 3, six ewes were ovariectomized and passively immunized against GnRH 3 days after oestrus. The increase in plasma LH which normally follows ovariectomy was completely abolished and mean concentrations remained very low and did not change over the following 14 days. In contrast, mean FSH concentrations rose significantly within 12 h of ovariectomy and continued to rise until the third day, after which they fell gradually. Treating three of the ewes with bFF (2.5 ml s.c. every 8 h) 8 days after ovariectomy and immunization further reduced the FSH concentrations. When the ewes were injected repeatedly (200 ng i.v., hourly for 5 h) with [D-penicillamine-(But)6]-GnRH(1-9)nonapeptide-ethylamide, a synthetic GnRH analogue which does not bind to the antiserum, there was a rapid rise in the secretion of LH in both control and bFF-treated animals but, as with the responses to buserelin, the initial response was significantly lower in bFF-treated than in control ewes.(ABSTRACT TRUNCATED AT 400 WORDS)

Follicle selection in cyclic guinea pigs with active immunization against inhibin alpha-subunit.

Experiments were conducted to elucidate the mechanisms of active immunization against inhibin on ovarian follicular development and selection in guinea pigs. Estrous cycle was synchronized in experimental guinea pigs by implanting progesterone containing tubes. Antibodies that bound 125I-labeled bovine inhibin were produced by all guinea pigs receiving the inhibin vaccine (recombinant ovine alpha-subunit in oil emulsion) without any effects on duration of the estrous cycle. Active immunization against inhibin increased the plasma concentrations of progesterone during the luteal phase and the plasma concentrations of estradiol but failed to increase the plasma concentration of follicle-stimulating hormone (FSH) during preovulatory period. The treatment also increased the number of corpora lutea (from 1.3+/-0.3 to 7.0+/-1.6 per each ovary), and preovulatory sized follicles (from 1.8+/-0.6 to 7.0+/-1.6 per each ovary), and follicles stained positively for inhibin alpha-subunit (from 2.3+/-0.5 to 6.3+/-1.3 per each ovary) significantly. The results indicate that active immunization against inhibin enhances ovulation rate by affecting the follicle selection and only dominant follicle can be stained for inhibin alpha-subunit in guinea pigs. This study is firstly to provide direct evidence that inhibins play important role in follicle selections in guinea pigs.