ABSTRACT

Oestrus was synchronized in 15 naturally cyclic ewes by the administration of a prostaglandin F_2α analogue. Groups of five ewes were then treated i.v. with either small doses of gonadotrophin releasing hormone (GnRH; 125 or 250 ng/injection) or saline, at 2-h intervals from day 14 of the subsequent cycle until 24 h after the onset of oestrus.

Treatment with GnRH induced episodic LH release which continued until the onset of a preovulatory LH surge. Mean plasma LH concentrations over this period were significantly (P< 0·001) higher in animals receiving 250 ng GnRH (2·44±0·11 μg/l) than in those receiving either 125 ng GnRH (1·17±0·06 μg/l) or saline (1·14±0·05 μg/l). However, GnRH treatment did not influence the timing of oestrus or mean ovulation rates.

J. Endocr. (1984) 101, 365–370

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

The role of FSH in gonadotrophin-releasing hormone (GnRH)-induced follicular development in anoestrous ewes was investigated using injections of bovine follicular fluid (bFF) to reduce plasma FSH levels. Groups of five animals were treated for 12 h with GnRH (250 ng at 2-h intervals) alone, GnRH plus bFF or saline alone, or for 36 h with GnRH alone, GnRH plus bFF or bFF alone. The administration of bFF (1·5 ml s.c. at 8-h intervals) significantly (P<0·05) reduced mean plasma FSH levels, but with the exception of animals treated with bFF alone, had no effect on LH levels. Treatment with bFF alone for 36 h resulted in a significant (P<0·05) increase in LH concentrations. There was considerable variation in the number of follicles ≥2 mm in diameter in the treatment groups. The mean diameter, oestradiol secretion and number of 'oestrogenic' follicles were significantly (P<0·01) reduced in ewes treated with GnRH plus bFF or bFF alone for 36 h compared with those treated with GnRH alone. Testosterone secretion by the follicles was not affected by treatment.

These results confirm previous findings that treatment with bFF decreases circulating FSH levels in anoestrous ewes and, moreover, that concurrent administration of bFF and GnRH inhibits the follicular maturation that is induced by treatment with GnRH alone, suggesting that FSH as well as LH is required for follicular maturation in the ewe.

J. Endocr. (1988) 119, 95–100

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