ABSTRACT

The relative importance of inhibin and oestradiol in the control of FSH and LH secretion in the ewe was investigated by passive immunization in intact animals and by hormone replacement therapy following acute ovariectomy, in the same experiment. Mature Scottish Blackface ewes on day 10 of the luteal phase were allocated to nine groups of four to five animals. Four groups were ovariectomized and immediately treated with either progesterone alone or in combination with steroid-stripped ovine follicular fluid ('inhibin') and/or oestradiol. Three further groups of ewes were left intact and injected with antibodies to the 1–26α peptide fragment of porcine inhibin and/or oestradiol-17β. Two groups of animals were either ovariectomized alone with no further treatment, or were left intact and treated with normal sheep plasma to act as controls. Blood samples were collected at 2 h intervals from 12 h before until 48 h after ovariectomy/immunization, and from 12 to 24 h after treatment, blood samples were collected at 10-min intervals.

After ovariectomy there was a large rise in the peripheral concentration of LH (P < 0·001) which was not affected by treatment with progesterone alone but was completely prevented by treatment with progesterone and oestradiol. Treatment with inhibin had no effect on this post-castrational rise in LH. In intact ewes, immunization against oestradiol, alone or in combination with inhibin, resulted in a rise in the concentration of LH, while immunization against inhibin had no effect on LH concentration. The peripheral concentration of FSH showed a significant (P < 0·001) increase after ovariectomy which was not affected by treatment with progesterone alone. Treatment with inhibin or oestradiol alone caused a significant (P < 0·01) reduction in this rise, while treatment with inhibin and oestradiol together completely prevented this post-castrational rise in FSH concentration. Passive immunization against inhibin or oestradiol alone resulted in a transitory (P< 0·01) rise in the peripheral concentration of FSH, while immunization against the two hormones in combination resulted in a significantly (P < 0·01) larger rise. During the 14-h period after treatment, the rise in the concentration of FSH in this combined immunization group was not significantly different from that seen in the control ovariectomized group.

These results provide evidence that FSH secretion is under the control of both oestradiol and inhibin, while reinforcing the hypothesis that inhibin is not involved in the regulation of LH production, which is under the dual control of oestradiol and progesterone.

Journal of Endocrinology (1992) 133, 381–391

ABSTRACT

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 < 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 < 0.1) and 48 h (P < 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 < 0.01) and late (48 h; P < 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 < 0.001) and remained low. At 24 h after PG the concentration of FSH was significantly lower (P < 0.01) than that during the luteal phase and remained suppressed until the onset of the LH surge. There were no significant differences in LH concentrations. We conclude that (1) the secretion of inhibin by the ovary is highest in the luteal phase and (2) inhibin secretion is significantly raised during the mid to late follicular phase in Booroola ewes with a copy of the Fecundity gene compared with those without.

ABSTRACT

Passive immunization was used to investigate the importance of inhibin in the negative feedback loop regulating the production of FSH in sheep. An antiserum raised to the 1–26 peptide fragment of the N-terminus of the α-chain of porcine inhibin was first shown to neutralize the suppressive effects of inhibin on the production of FSH by dispersed ovine pituitary cells in vitro. Groups of five mature Scottish Blackface ewes on day 8 of the luteal phase of the oestrous cycle were then injected with either 10 ml plasma from normal ewes (control) or 10 ml ovine inhibin antiserum. On day 10, luteal regression was induced by an i.m. injection of cloprostenol (100 μg), and ovulation rate determined 6 days later by laparoscopy. Peripheral plasma samples were collected throughout the experimental period.

Following treatment, there was no change in the peripheral plasma concentration of LH in either group. Following injection of the inhibin antiserum, the concentration of FSH rose significantly (P<0·001) compared with the control group. The concentration of FSH rose from 1·42 ± 0·06 to a maximum of 2·58 ± 0·23 (s.e.m.) μg/l by 5·6 ±0·9 h, this maximum lasting 9·0±1·1 h. By 32·8 ±6·9 h, the concentration of FSH had returned to pretreatment levels, while the titre of free antibody in the plasma of treated ewes was still high. In the treated ewes, there were one single and four double ovulations compared with three single and two double ovulations in the control group, indicating that the inhibin immunization may have resulted in an increase in ovulation rate.

We conclude that the marked rise in the plasma concentration of FSH following injection of inhibin antiserum provides strong evidence that inhibin is an important factor in the regulation of FSH production by the pituitary gland at this time.

Journal of Endocrinology (1989) 123, 383–391

Abstract

Insulin-like growth factor-binding protein (IGFBP) extraction protocols were tested for their efficacy in removing IGFBPs from bovine plasma and bovine granulosa cell culture medium compared with standard acid exclusion chromatography. Traditional extraction methods, acidification, Sep–Pak, ethanol:acetone:acetic acid (EAA) and EAA-cryoprecipitation (EAA-C), failed to remove all the IGFBPs from both granulosa cell culture medium and plasma. However, EAA and EAA-C treatment of plasma samples did give values similar to those obtained by acid exclusion HPLC, when corrected for extraction efficiency. There was an inverse relationship between insulin-like growth factor-I (IGF-I) concentration in plasma samples, as measured using HPLC chromatography, and IGF-I concentration after EAA extraction. Furthermore, the interference caused by residual IGFBPs differed between samples taken from animals given various treatments that altered peripheral IGF-I concentrations.

As for plasma samples, EAA was the most effective extraction method for culture media, but residual IGFBPs caused an overestimation of IGF-I concentrations. In culture media, but not plasma, it was possible to block the interference of IGFBPs in the IGF-I assay, in both extracted and non-extracted culture samples, by the addition of excess IGF-II. Using this assay procedure, no IGF-I production by bovine granulosa cells was detected. This was confirmed by HPLC acid chromatography.

It is concluded that HPLC extraction is needed for the accurate measurement of peripheral IGF-I concentrations. For granulosa cell culture media it is possible to measure IGF-I concentrations in non-extracted samples if the IGFBPs are blocked by adding IGF-II. Using either this assay, or after HPLC acid chromatography, no IGF-I was detected in culture media, suggesting that IGF-I is not produced by non-luteinised bovine granulosa cells.

Journal of Endocrinology (1997) 153, 231–240

ABSTRACT

Immunization against inhibin or oestradiol causes an increase in the peripheral plasma concentration of FSH. In this study we have investigated the effect of this post-immunization rise in FSH concentration on follicular development by means of real-time ultrasonography, in ewes in which an ovary and its vascular pedicle had been autotransplanted to a site in the neck. Groups of ewes on day 10 of the luteal phase of the oestrous cycle were injected with a single 10 ml i.v. bolus of plasma from normal ewes (control; n = 4), antiserum to the 1–26 peptide fragment of the N-terminus of the α chain of porcine inhibin (n = 5) or antiserum to oestradiol-17β (n = 4). The plasma concentration of FSH was unaffected by treatment in the control group but showed a significant (P < 0·001) rise following treatment in both immunized groups (inhibin-immunized 175% over 19 h; oestradiol-immunized 138% over 22 h as a per cent of the original value). This rise in FSH concentration was accompanied by a significant (P < 0·001) rise in the total number of follicles > 2·0 mm per ovary in both immunized groups (inhibin-immunized 5·5±1·0 to 13·6± 1·4; oestradiol-immunized 4·6±0·5 to 11·5±1·0). In the oestradiol-immunized group, this rise was due to an increase (P < 0·001) in the number of small follicles (<3·5 mm) alone while, in the inhibin-immunized group, there was a rise in both the number of small follicles (P < 0·001) and the number of medium-sized follicles of 3·5–4·5 mm (1·3 ±0·5 to 3·2± 1·0, P <0·05). During the period from 12 to 20 h after immunization, when FSH concentrations were falling following the post-immunization rise, there was a significant (P <0·05) rise in the ovarian secretion rate of inhibin in the oestradiol-immunized group, and in the inhibin-immunized group the mean secretion rate of oestradiol was double that in the control group (2·7±0·5 vs 1·3 ±0·3 pmol/min, P <0·06). The temporal relationships suggest that the rise in FSH induced by passive immunization against oestradiol or inhibin stimulates the development of many antral follicles which contribute to the increased ovarian secretion of inhibin and oestradiol.

Journal of Endocrinology (1993) 136, 225–233

Subfertility that will respond to appropriate copper supplementation is a widespread problem in the national dairy herd. The aims of this study were to determine the effect of copper and/or copper chelating thiomolybdates on LH-induced differentiation by looking at the effects on androgen production, steroidogenic enzymes (cytochrome P450 17α-hydroxylase and cytochrome P450 side-chain cleavage) and lysyl oxidase mRNA expression in cultured theca cells maintained under serum-free conditions.

The effect of thiomolybdates and copper on LH differentiation was investigated by supplementing (ammonium) tetrathiomolybdate to optimum theca cell culture media at 0–100 μg/ml, copper (chloride) at equimolar concentrations (0–51.6 μg/ml) or equimolar combinations of both media. Lysyl oxidase mRNA expression was investigated with semi-quantitative RT-PCR, whilst expression of cytochrome P450 17α-hydroxylase and cytochrome P450 side-chain cleavage mRNA was examined using real time PCR. Both PCRs used bovine specific primers and cell lysates obtained from bovine theca cells cultured for 6 days and in the presence or absence of the 100 μg/ml dose of thiomolybdate and equimolar copper thiomolybdate.

Thiomolybdate depressed androstenedione production in a dose-dependent manner at doses greater than 1 μg/ml at 96 h (P<0.05); doses above 20 μg/ml were all greatly reduced at all time points and at 192 h when related to numbers of cells (P<0.001). Copper alone had no effect at physiological doses, but the use of the equimolar copper thiomolybdate reversed the effect of tetrathiomolybdates on androstenedione production at the 20 μg/ml dose. Thiomolybdate supplementation, with and without copper, had no significant effect on the level of lysyl oxidase or cytochrome P450 side-chain cleavage expression. However, cytochrome P450 17α-hydroxylase expression was significantly increased (P<0.05) by tetrathiomolybdate, possibly due to a local regulatory system.

In conclusion, these results demonstrate that thiomolybdates can prevent LH-induced differentiation of bovine theca cells in vitro and that these effects can be ameliorated by copper supplementation. Our results also indicate that it is unlikely that the effects of thiomolybdate are mediated at the transcriptional level and further work is required to determine if thiomolybdate exerts its effects through post-translation processing or some other unrelated mechanism. Overall, these data support the hypothesis that copper responsive subfertility results from perturbation of the normal pattern of ovarian steroidogenesis.