ABSTRACT

To stimulate a follicular-phase pattern of pulsatile LH release, gonadotrophin-releasing hormone (GnRH; 5 μg) was infused (i.v.) hourly into heifers for periods of 5–11 days during the luteal phase of the oestrous cycle, and also when plasma progesterone levels were increased artificially by means of a progesterone-releasing intravaginal device.

Plasma oestradiol-17β concentrations increased from basal (EEE 2·5 pmol/l) to preovulatory peak levels (20–30 pmol/l) during the first 3 days of GnRH treatment. They were maintained at these values before returning to basal levels within 24 h of cessation of infusion. This response occurred regardless of the source of progesterone (endogenous or administered). Follicular development was observed by ovarian palpation (per rectum) in some heifers at the time of maximum secretion of oestradiol-17β. There was no detectable cervical mucus secretion or oestrous behaviour during these periods of high oestradiol-17β levels and ovulation did not occur. Treatment with GnRH did not affect plasma progesterone concentrations or oestrous cycle length.

The study shows that oestradiol-17β secretion and follicular development (and the accompanying oestrus and ovulation) are suppressed during the luteal phase of the cycle by high concentrations of plasma progesterone, and provides strong indirect evidence that such inhibition is associated with a reduction in the pulse frequency of LH release.

J. Endocr. (1987) 112, 77–85

ABSTRACT

The enhancing effect of bovine FSH monoclonal antibody (bFSH-MAb) on the gonadotrophic activity of FSH was investigated in dwarf mice using a uterine weight bioassay.

Increasing doses of bovine FSH (NIH-FSH-B1; 3.3, 10 or 30 μg/day) were administered for 5 days to dwarf mice (groups of five) with or without administration of a bFSH-MAb preparation (USDA-bFSH-MC28; 100 μg protein/day) which at a dilution of 1:15 000 bound 50% of ¹²⁵I-labelled bFSH (USDA-bFSH-BP3). The bFSH, at the doses given, gave no increases in uterine weight; when, however, these doses were given with bFSH-MAb, significant (three- to four fold) increases in uterine weight resulted.

ABSTRACT

Chronically ovariectomized prepubertal heifers were used for a comparison of the effects of highly purified bovine inhibin (M _r 32 000) and steroid-free bovine follicular fluid (bFF) on the secretion of FSH and LH. In view of the limited availability of highly purified inhibin, an initial study was undertaken to establish the optimal method for administration of bFF inhibin activity. In comparison with the FSH response to a single large i.v. bolus injection of bFF (50 ml; 3250 mg protein), a far more effective suppression of plasma FSH concentrations was achieved when considerably less bFF (6·3 ml; 410 mg protein) was administered gradually over an extended time-period (2 days) either as a continuous i.v. infusion or as a series of 2-hourly i.v. injections. Following a single i.v. bolus injection of bFF, immunoreactive inhibin was cleared rapidly from the circulation (half-life 51 ± 8 (s.e.m.) min, n = 5), presumably accounting for its limited ability to suppress FSH secretion when administered in this manner.

In a second experiment, treatment of ovariectomized heifers (three per group) with highly purified M _r 32 000 bovine inhibin at a dose rate of 15 μg/2 h for 2 days significantly (P < 0·05) suppressed plasma FSH concentrations, which reached their minimum values (40% suppression) during day 2 of treatment. At a lower dose rate (5 μg/2 h), inhibin did not significantly affect plasma FSH levels. Administration of bFF was also associated with a dose-dependent suppression of FSH secretion. For each of three dose rates tested (three heifers per group), plasma FSH concentrations were maximally suppressed during day 2 of treatment (65 mg/2 h, 86% suppression, P < 0·001; 21·7 mg/2 h, 66% suppression, P < 0·001; 7·2 mg/2 h, 15% suppression, P > 0·05). Neither highly purified inhibin nor bFF significantly affected mean plasma LH concentrations, LH pulse frequency or LH pulse amplitude. Thus we have shown for the first time that highly purified M _r 32 000 bovine inhibin does possess in-vivo biological activity in cattle, promoting a selective suppression of plasma FSH concentrations qualitatively similar to that evoked by steroid-free bFF. Quantitatively, the inhibin preparation had an in-vivo biopotency about 1000 times greater than that of bFF, a value which accords well with its biopotency (1176 × bFF) in the in-vitro rat pituitary cell bioassay.

Journal of Endocrinology (1990) 125, 21–30

ABSTRACT

It has been shown previously that treatment of seasonally anoestrous ewes with steroid-free bovine follicular fluid (FF), a crude inhibin-containing preparation, leads to a decrease in plasma FSH level which is accompanied by a marked increase in pulsatile LH secretion. Since FF contains several factors (e.g. activin, follistatin, unidentified components) other than inhibin, which might act to modify gonadotrophin secretion, it was of interest to establish whether these concurrent effects of FF on FSH and LH secretion persisted in ewes which had been actively immunized against a synthetic peptide replica of the α subunit of bovine inhibin. In June 1989 (anoestrous period) groups of inhibin-immune and control ewes (n = 5 per group) received 6-hourly s.c. injections of either bovine serum (2 ml) or one of two doses of FF (0·5 ml or 2 ml) for 3 days. Blood was withdrawn at 6-h intervals for 6 days beginning 24 h before the first injection. On the final day of treatment, additional blood samples were withdrawn at 15-min intervals for 8 h to monitor pulsatile LH secretion. Ewes were then challenged with exogenous gonadotrophin-releasing hormone (GnRH; 2 μg i.v. bolus) to assess pituitary responsiveness. In control ewes, FF promoted a dose-dependent suppression of basal (maximum suppression 65%; P < 0·01) and post-GnRH (maximum suppression 72%; P < 0·01) levels of FSH in plasma. This was accompanied by an increase (P < 0·01) in LH pulse frequency from 1·40±0·24 (s.e.m.) to 3·20±0·37 pulses/8 h. In contrast, FF did not affect secretion of either FSH or LH in inhibin-immunized ewes. However, mean plasma LH levels in immunized ewes were significantly lower (43%; P < 0·02) than in control ewes, irrespective of treatment. These findings indicate that in the anoestrous ewe the ability of FF to suppress plasma FSH is due entirely to its content of inhibin, that FF-induced enhancement of pulsatile LH secretion is mediated by inhibin, rather than some additional component of FF, and that immunoneutralization of endogenous inhibin can reduce LH secretion.

Journal of Endocrinology (1991) 128, 403–410

ABSTRACT

Previous reports indicate that administration of steroid-free bovine follicular fluid (bFF) to intact heifers suppresses plasma FSH levels and delays the timing of ovulation. In addition, cessation of bFF treatment is associated with a rebound hypersecretion of FSH. To test the hypothesis that these effects of bFF are mediated by inhibin, we have compared the responses to bFF treatment in heifers actively immunized against the N-terminal sequence of inhibin α subunit bIα(1–29)Tyr³⁰-ovalbumin) with those in ovalbumin-immunized controls.

Oestrous cycles were synchronized, and inhibin-immune (n = 10) and control (n = 10) heifers were subdivided into two groups which received either 5 ml bFF (n = 5) or 5 ml bovine serum (n = 5) every 4 h for a 60 h period starting 8 h before prostaglandin (PG)-induced luteolysis. Blood was withdrawn every 4 h for 10 days starting 30 h before luteolysis and ovaries were examined daily by ultrasonography. Overall, mean ovulation rate in bIα(1–29)Tyr³⁰-immunized heifers was 44% higher (P < 0·02) than in controls. Inhibin antibody titres tested in bIα(1–29)Tyr³⁰-immunized heifers before (19 ± 3%), during (19 ± 3%) and after (20 ± 3%) bFF treatment did not differ. In the pretreatment period (i.e. mid-luteal phase), plasma FSH levels were 32% (P < 0·05) higher in inhibin-immunized than in control heifers. During administration of bFF to control heifers, plasma FSH was suppressed to a level 40% lower than in serum-treated heifers (P < 0·02). Unexpectedly, bFF suppressed FSH to a similar extent in inhibin-immunized heifers (37% lower than in the serum-treated group; P < 0·025). Similarly, a post-bFF rebound hypersecretion of FSH was observed in both control (P < 0·05) and inhibin-immunized (P < 0·05) heifers, although the FSH rebound lasted about 24 h longer in the latter group (P < 0·001). The timing of the preovulatory LH surge in control (86 ± 7 h post-PG) and immunized (81 ± 6 h post-PG) groups treated with serum was similar as was the timing of the preovulatory rise in plasma oestradiol and the subsequent rise in plasma progesterone. However, bFF treatment delayed (P < 0·001) the preovulatory surges of LH and oestradiol and the subsequent rise in plasma progesterone to a similar extent (> 4 days) in both control and inhibin-immunized groups. Ultrasonography confirmed that bFF delayed the emergence of the wave of dominant ovulatory follicles by 5 days and also showed that inhibin immunization and bFF treatment were both effective in promoting the development of more follicles during the preovulatory period.

These results lead us to conclude that a non-steroidal factor(s) in bFF other than inhibin is responsible for the observed delay in ovulation. Like inhibin, this factor acts to suppress pituitary FSH secretion and this could fully account for its ability to delay the onset of preovulatory follicular development and thus delay ovulation. However, the further possibility of a direct inhibitory action of certain bFF components on the ovary cannot be ruled out at this stage.

Journal of Endocrinology (1993) 136, 137–148

ABSTRACT

Two experiments were conducted to explore the effectiveness of synthetic peptide-based vaccines for active and passive autoimmunization of sheep against inhibin. In the first experiment, adult Romney ewes (n = 20) were actively immunized against a synthetically produced peptide that corresponded to the N-terminus of the α-subunit of bovine inhibin (bIα(1–29)-Tyr³⁰). This peptide was conjugated to tuberculin purified protein derivative (PPD) to increase its antigenic properties. Control groups comprised non-immunized (n = 10) and PPD-immunized (n = 10) ewes. Primary immunization (400 μg conjugate/ewe) was followed by two booster immunizations (200 μg conjugate/ewe), given 5 and 8 weeks later. Following synchronization of oestrus using progestagen sponges, ovulation rates were assessed by laparoscopy. Weekly blood samples were taken throughout the experiment. All inhibin-immunized ewes produced antibodies which bound ¹²⁵I-labelled bovine inhibin (M _r 32 000), and ovulation rate in inhibin-immunized ewes (2·15 ± 0·22; mean ± s.e.m.) was significantly (P<0·01) greater than in both non-immunized (0·90 ± 0·23) and PPD-immunized (1·20 ± 0·13) control groups. Immunization against the peptide, but not against PPD alone, resulted in a modest rise in plasma FSH, with mean levels after the second boost being significantly (P<0·025) higher (22%) than those before immunization. Moreover, when blood samples were taken (2-h intervals) from randomly selected groups of control (n = 7) and inhibin-immunized (n = 7) ewes for an 84-h period following withdrawal of progestagen sponges, the mean plasma concentration of FSH during the 48 h immediately before the preovulatory LH surge was 37% greater (P< 0·025) in immunized than in control animals. However, more frequent blood sampling (every 15 min for 12 h) during follicular and mid-luteal phases of the oestrous cycle revealed no significant differences between treatment groups in mean plasma concentrations of FSH. In addition, neither mean concentrations of LH nor the frequency and amplitude of LH episodes differed between immunized and control ewes. However, the mean response of LH to a 2 μg bolus of gonadotrophin-releasing hormone, given during the luteal phase, was significantly (P<0·05) less in immunized than in control ewes. These findings indicate that active immunization of Romney ewes against a synthetic fragment of inhibin can promote a controlled increase in ovulation rate, but this response cannot be unequivocally related to an increase in plasma levels of FSH.

In the second experiment, passive immunization of seasonally anoestrous ewes (mule × Suffolk crossbred; n = 6 per group) against inhibin, using an antiserum raised in sheep against a synthetic peptide corresponding to the N-terminus of the α-subunit of human inhibin promoted a rapid (<3 h), dose-dependent rise in plasma levels of FSH which remained increased (2·5-fold; P<0·001) for up to 30 h. Plasma concentrations of LH, however, were unaffected by treatment with the antiserum. It is deduced from this observation that, even in the seasonally anoestrous ewe, the ovary secretes physiologically active levels of inhibin, which exert an inhibitory action on the synthesis and secretion of FSH.

Journal of Endocrinology (1990) 124, 167–176

ABSTRACT

Immunization against inhibin consistently results in an increase in ovulation rate in sheep, but the effects that this treatment has on follicle development are unknown. In order to determine the influence of inhibin, parameters of follicle development were assessed in ewes that had been actively immunized against a synthetic peptide homologous to the N-terminal sequence (α^1–29, Tyr³⁰) of the a subunit of bovine inhibin, a treatment that neutralizes the biological activity of endogenous inhibin. The final stages of preovulatory follicle development that culminate in ovulation were induced in seasonally anoestrous ewes, and follicles were recovered prior to the predicted time of ovulation. After priming with progestagen, inhibin-immunized and control ewes were treated with gonadotrophin-releasing hormone (GnRH) by continuous infusion (200 ng/h). The ovaries were recovered at slaughter 24 h after the start of GnRH treatment and all follicles ≥ 2·0 mm diameter were dissected out and their capacity to produce oestradiol in vitro was assessed. Further groups of similarly treated animals were blood-sampled daily to determine luteal function following GnRH-induced ovulation. The ovaries were recovered from these ewes at slaughter 10 days after the start of GnRH treatment, the corpora lutea were dissected out and their progesterone content was assessed.

There were more (P < 0·01) follicles of 5–6 mm diameter (3·2 ± 0·45 (s.e.m.) compared with 1·1 ± 0·25 follicles/ewe) and more (P < 0·001) follicles of > 6 mm diameter (2·8 ± 0·56 compared with 0·9 ± 0·17 follicles/ewe) in inhibin-immunized than in control ewes. In addition, the mean number of the antral follicles that were oestrogenic was greater (P < 0·05) in immunized than in control ewes (2·8 ± 0·66 compared with 1·3 ± 0·25 follicles/ewe).

In animals slaughtered 10 days after the start of GnRH treatment, mean ovulation rate was greater (3·17 ± 0·65 and 1·14 ± 0·14, P < 0·01) in inhibin-immunized ewes. Although there was more (P < 0·01) total luteal tissue/ewe in the immunized group, both the mean weight and progesterone content (ng/mg tissue) of individual corpora lutea were similar between treatment groups. Mean plasma progesterone levels increased earlier and reached higher (P < 0·01) mean concentrations in immunized than in control ewes.

These results demonstrate that immunization against inhibin increases the number of preovulatory follicles during the follicular phase, and that steroidogenesis within these follicles and the resultant corpora lutea appears to be normal.

Journal of Endocrinology (1992) 133, 413–419

ABSTRACT

To study the effects of immunoneutralization of endogenous inhibin on gonadotrophin secretion and ovarian function, prepubertal heifers (n = 6) were actively immunized against a synthetic peptide replica of the N-terminal sequence of bovine inhibin α subunit bIα(1–29)Tyr³⁰) coupled to ovalbumin. In contrast to ovalbumin-immunized controls (n=6), bIα(1–29)Tyr³⁰-immunized heifers had detectable inhibin antibody titres (% binding to ¹²⁵I-labelled bovine inhibin at 1:2000 dilution of plasma) of 17 ± 3% (s.e.m.) at puberty, rising to 31 ± 5% by the end of the study period 7 months later. Neither age (immunized: 295 ± 8 days; controls: 300 ± 5 days) nor body weight (immunized: 254 ± 13 kg; controls 251 ± 9 kg) at onset of puberty differed between groups. Although the difference did not reach statistical significance, mean plasma FSH concentrations recorded in inhibin-immunized heifers remained 35–40% higher than in controls throughout the 12-week period leading up to puberty (P = 0·14) and during nine successive oestrous cycles studied after puberty (P=0·10). Plasma LH concentrations did not differ between groups at any time during the study. Inhibin immunization had no effect on oestrous cycle length (immunized: 19·8±0·5 days; controls: 19·9±0·5 days). However, in comparison with controls, inhibinimmunized heifers had more medium sized (≥0·5 to <1 cm diameter) follicles during both the preovulatory (95%, P<0·001) and post-ovulatory (110%, P < 0·05 waves of follicular growth and more large (>1 cm diameter) follicles during the preovulatory wave (49%, P<0·05). In addition, the number of corpora lutea observed during the post-ovulatory phase of each cycle was significantly greater in the inhibin-immunized group (43%, P<0·01), as was the recorded incidence of cycles with multiple ovulations (19/56 in the inhibin-immunized group compared with 0/54 in controls; P<0·001). All six inhibinimmunized heifers had at least one cycle with multiple ovulation whereas none of the control heifers did so.

These results support the conclusion that immunoneutralization of endogenous inhibin using a synthetic peptide-based vaccine can enhance ovarian follicular development and ovulation rate in heifers. Whether this ovarian response is dependent upon the expected increase in secretion of FSH remains to be established.

Journal of Endocrinology (1992) 134, 11–18