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 Blood was withdrawn every 4 h for 10 days starting 30 h before luteolysis and ovaries were examined daily by ultrasonography.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

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