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Summary. Aromatase activity was measured in granulosa cells using a 1-h in-vitro assay. This activity correlated with the concentration of oestradiol-17β and the ratio of oestradiol-17β to testosterone in follicular fluid of individual follicles ranging from 1·5 to 7·0 mm diameter. These data show an 8–10-fold difference in aromatase activity between small and large follicles and that aromatase activity per cell increased in small non-atretic follicles (<3·5 mm) whereas it remained relatively constant in large nonatretic follicles (≥3·5 mm). Aromatase activity was much lower in follicles at more advanced stages of atresia. Atresia was assessed using the morphological and the morphometric methods (% of maximum number of granulosa cells/follicle). Although the morphological method of assessment was preferable to the morphometric method, it did not differentiate a decrease in aromatase activity as a very early event in the atretic process. We believe this is due to the inability of these methods to detect follicles in the initial stages of atresia.
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Immunization of ewes against the amino-terminal peptide (αN) of the pro-α-subunit of inhibin has been shown to reduce fertility, thought to be due to disruption of ovulation. The aims of this study were to examine the effects of active immunization of ewes against αN on circulating concentrations of FSH, LH and on ovarian inhibin and progesterone, and to relate these observations to number of corpora lutea and oocyte recovery rates. Ewes were immunized against one or both of two recombinant full length bovine-αN immunogens (FP1 and FP2). Three experiments were performed in which jugular venous plasma was sampled from control and immunized ewes: (1) hourly across the oestrous surge of gonadotrophins (Expt 1); (2) daily for one entire oestrous cycle, and in the subsequent cycle, oviducts were flushed to recover ovulated eggs (Expt 2); and (3) samples were taken at 10 min intervals during the follicular and luteal phases (Expt 3). Binding of 125I-labelled αN1–26 to serum was greater (P < 0.05) in immunized groups than in controls for all experiments. The number of eggs per corpus luteum recovered from the oviducts was lower (P < 0.05) in the αN-immunized groups (39%) than in controls (88%). There were more (P < 0.05) corpora lutea per ewe in FP2 immunized groups 4 (1.8 ± 0.45) and 5 (1.75 ± 0.5) than in the control group (1.13 ± 0.13), but no increase in group 3 (FP1; 1.4 ± 0.24). The oestrous surge of LH, basal values across cycle and the frequency of LH pulses were similar in treated and control groups. Circulating FSH concentrations were higher (P < 0.05) than those of controls in all immunized groups that had binding greater than 10% at all stages of the cycle, except during the oestrous surge. A corresponding decrease (P < 0.05) in circulating inhibin concentrations was observed in most immunized groups, with a significant negative correlation between inhibin values and αN-binding in the follicular phase of the cycle. The pattern of progesterone production during the cycle was similar, with a slight non-significant increase in immunized compared with control ewes. These data confirm the previous observation that ovulation is impaired in ewes immunized against the amino-terminal peptide of inhibin α43 and also suggest that the mechanism of this effect does not involve disruption of pituitary function, implying a role for αN in the intraovarian events of ovulation.
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Summary. Follicles of various sizes at the surface of the ovary were ablated by electrocautery at the time of cloprostenol-induced luteolysis in ewes and the interval from cloprostenol treatment to the onset of the LH surge determined as an index of the time from luteolysis to ovulation. When follicles 2–4 mm or > 4 mm diameter remained in the ovaries, the interval from cloprostenol treatment to the onset of the LH surge was similar to that in sham-operated (control) ewes (55–60 h), whereas when the only follicles remaining were < 2 mm, the interval was extended by 24 h (P < 0·05). This study demonstrates that follicles capable of ovulating can be selected from those ≥ 2 mm diameter at luteolysis, emphasizing the flexibility of the sheep ovary in its final selection of the ovulatory follicle.
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Summary. An in-vitro bioassay for inhibin based on FSH content or release by rat pituitary cells was validated for measuring inhibin activity in ovine plasma and lymph. Dose-dependent increases in inhibin activity were detected in peripheral plasma of 4 ovariectomized ewes 1 min after i.v. injections of ovine follicular fluid, and the half-life of inhibin in plasma for 2 ewes was 45 and 50 min, respectively.
Inhibin was detected in ovarian lymph but not in ovarian or jugular venous plasma, even after treatment of ewes with PMSG to induce folliculogenesis. Destruction of visible follicles (> 0·5 mm diameter) on the ovaries of 4 PMSG-treated ewes by electrocautery was followed by a rapid and sustained decline in secretion of inhibin in ovarian lymph for up to 4 h. Ovarian lymph flow rates were either unchanged or slightly increased after cautery. Oestrogen concentrations in peripheral venous plasma declined within 15–30 min of cautery, but concentrations remained well above baseline. There was a significant decrease in peripheral progesterone concentrations in these same samples, but not until 2–3 h after cautery. FSH in peripheral plasma was depressed or non-detectable in PMSG-treated ewes and neither FSH nor LH concentrations in peripheral plasma were significantly altered up to 4 h after cautery of ovarian follicles.
It is concluded that (a) antral follicles (>0·5 mm) are the source of inhibin present in ovarian lymph, and (b) the ovarian lymphatic system is a route by which inhibin could reach the peripheral circulation, particularly in the luteal phase when ovarian lymph flow rates are high.
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Summary. Adult rats (16–18/group) received a single intratesticular injection of 25, 100 or 400 μl glycerol solution (7:3 in distilled water, v/v). Half of the rats in each group were given implants of testosterone, a testosterone-filled Silastic capsule (1·5 cm length) to provide serum values of testosterone within the normal range. After 1 week all animals were killed by decapitation. Serum concentrations of gonadotrophins, testosterone and immunoactive inhibin as well as testicular concentrations of testosterone and bioactive inhibin were determined. Testicular histology was studed in Paraplastembedded tissue stained with PAS and haematoxylin–eosin. Glycerol treatment caused a dose-dependent ablation of spermatogenesis in a distinct area around the site of injection. Serum concentrations of FSH increased proportionally with increasing spermatogenic damage while serum LH and testosterone remained unaltered except with the highest glycerol dose. The rise in serum FSH was significantly correlated with serum (r = −0·70, P < 0·001) and testicular (r = −0·66, P < 0·001) concentrations of inhibin. A less pronounced correlation was found between LH and serum inhibin (r = 0·48). No correlation was found between the concentrations of LH and testicular inhibin or between serum concentrations of FSH and serum testosterone in the 25 and 100 μl groups. Maintenance of low to normal serum testosterone concentrations by means of Silastic implants blocked the elevation of FSH in glycerol-treated animals but failed to affect significantly serum FSH in untreated rats. In all testosterone treated rats testicular inhibin concentrations were markedly reduced in the presence of lowered concentrations (7–14%) of testicular testosterone and unaltered serum FSH concentrations.
These findings indicate that (i) inhibin is involved in the in-vivo regulation of FSH secretion in the adult male rat, (ii) testicular inhibin content, at least in part, may be regulated at the testicular level, and (iii) serum inhibin concentrations to some extent reflect the extent of damage to spermatogenesis and Sertoli cells.
Keywords: inhibin; testosterone; FSH; feedback; testis; rat
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Summary. Immunization of gilts in a commercial piggery against a fusion protein of the α subunit of bovine inhibin, produced by recombinant DNA methods, resulted in mean ovulation rate increases of 35% at the oestrus at which, under the piggery's management practices, they would have been mated. Sera from two immunized groups showed mean binding of 6·6% and 4·9% when assayed, at 1:800 final dilution, against iodinated bovine inhibin (M r 31 000). Ovulation rates of immunized gilts were highly correlated with the ability of serum to bind iodinated native inhibin (r = 0·62; P < 0·001), particularly when weight and age were included in the correlation (r = 0·72; P = 0·001), and inhibin binding accounted for 38% of the total variation in ovulation rate. Immunization caused no deleterious effects on growth rate or onset of oestrus. These results demonstrate the potential for use of such immunization to increase prolificacy in gilts and young sows.
Keywords: inhibin; fusion protein; immunization; ovulation rate; gilts
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To study the effect of re-immunization against inhibin on ovarian response and hormonal profiles, Japanese beef heifers (n = 5) were re-immunized three times with inhibin vaccine (recombinant ovine inhibin α-subunit in oil emulsion, 125 μg ml−1) one year after the primary immunization. Control heifers (n = 5) were injected with placebo (Montanide: Marcol adjuvant alone). Oestrous cycles were synchronized by using prostaglandin F2α (PGF2α) and ovarian response was monitored daily by ultrasonography. Blood samples were collected by jugular venipuncture for assessment of hormonal levels and inhibin antibody titres. In contrast to controls, inhibin re-immunized heifers generated antibodies against inhibin rapidly reaching a peak level 9 days after the first booster injection. The mean concentrations of FSH in re-immunized cows increased significantly in comparison with controls. In addition, there was a significant increase in oestradiol-17β and progesterone levels in re-immunized cows compared with controls. Inhibin re-immunized heifers had a significant increase in small (≥4 < 7 mm), medium (≥7 < 10 mm) and large (≥10 mm in diameter) sized follicles. Moreover, the mean ovulation rate was 5.0 ± 1.1 after the third booster injection in re-immunized heifers compared with control heifers (single ovulation). These results clearly demonstrate that re-immunization of inhibin can be used to enhance ovarian follicular development and ovulation rate. Furthermore, the great number of follicles is a potential source of oocytes that could be harvested for in vitro fertilization and embryo transfer programmes.
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Summary. Inhibin activity was measured by bioassay in follicular fluid of 99 individual ovine follicles ranging from 1·4 to 6·8 mm diameter (used to calculate volume) and in various stages of atresia. Treatment of samples before assay with charcoal concentrations of > 1 mg/ml resulted in significant loss of inhibin activity.
The inhibin content of follicular fluid from individual follicles varied with follicular fluid volume but not with the degree of atresia, as assessed by morphological criteria. Inhibin concentration was not related to atresia, but was correlated with follicular fluid volume. However, aromatase activity in granulosa cells and oestradiol-17β concentration of follicular fluid, considered to be good indices of atresia, were highly correlated with both inhibin content and concentration in follicles ≥ 3·5 mm diameter.
Inhibin in ovine follicular fluid shows marked variation between follicles and it is suggested that this reflects a combination of the number and activity of granulosa cells within the follicle and the exit rate of inhibin from the follicle.
