Abstract

Recombinant human inhibin A (rhInh) or steroid-free bovine follicular fluid (bFF) were infused into the ovarian artery of anoestrous ewes with ovarian autotransplants induced to ovulate with a pulsatile regimen of GnRH applied after a 10-day pretreatment with progestagen sponges. In the period 12–24 h after sponge withdrawal ewes received ovarian arterial infusions of saline (n=6), 0·3 μg rhInh/h (n=5), 1·6 μg rhInh/h (n=5) or 25 μl bFF/h (n=4).

Controls had a normal follicular phase with an LH surge 43 ± 3 h after sponge withdrawal which resulted in ovulation (six out of six). Both doses of rhlnh increased ovarian venous inhibin concentrations in a dose-related fashion (P<0·05) but resulted in depressions (P<0·05) in FSH concentrations of similar magnitude. Both doses of rhInh acutely inhibited ovarian oestradiol and androstenedione secretion (P<0·01) but at the end of rhInh infusion oestradiol secretion was quickly re-established without a corresponding increase in FSH. LH surges were detected in five out of five and three out of five ewes infused with low and high doses of rhInh respectively, and progesterone concentrations during the subsequent luteal phase were depressed (P<0·05). Infusion of bFF had no effect on inhibin or FSH concentrations but resulted in acute inhibition (P<0·01) of ovarian oestradiol, androstenedione and inhibin secretion, a delay (P<0·05) in the time to the LH surge and a depression (P<0·05) in luteal-phase progesterone concentrations.

In conclusion, while the depression in FSH induced by rhlnh cannot be excluded as a cause for the inhibitory effects of rhInh treatment on ovarian function, such a mechanism cannot fully explain the ovarian responses obtained to rhInh infusion. These results therefore support a direct ovarian role for inhibin in the modulation of ovarian function in addition to its indirect role in controlling FSH. This conclusion is supported by the demonstration that bFF can induce similar inhibitory effects on ovarian function without changing FSH.

Journal of Endocrinology (1996) 149, 531–540

Abstract

Transforming growth factor α (TGFα) inhibits hormone production by cultured follicular cells but evidence of an effect of TGFα on ovarian hormone secretion in vivo is still required. Eleven ewes with an autotransplanted ovary received, by ovarian arterial infusion, either 5 μg/h recombinant rat TGFα (n=6) or placebo (n=5) for 12 h on day 10 of the luteal phase. Two hours before the start and 1 hour before the end of the infusion each ewe received a single injection of gonadotrophin-releasing hormone (GnRH; 150 ng i.v.). Two hours after the end of the infusion luteal regression was induced with prostaglandin F_2α (PGF_2α; 125 μg i.m.). Ovarian and jugular venous blood samples were taken at 10-min, 15-min or 4-h intervals from 2 h before the start of the infusion until 96 h after PGF_2α and the rates of secretion of ovarian oestradiol, inhibin, progesterone and androstenedione were determined. Jugular venous concentrations of LH and FSH were also measured and follicle populations monitored by real-time ultrasound scanning.

Infusion of TGFα resulted in a significant (P<0.05) depression in the amplitude of the pulsatile response of oestradiol and androstenedione secretion to the GnRH-induced LH pulse at the end of the infusion. Ovarian inhibin secretion was acutely suppressed by TGFα infusion (P<0·001) and remained lower than controls for the period of the experiment. Luteal phase progesterone secretion was also acutely inhibited (P<0·001) by infusion of TGFα and in one treated ewe progesterone secretion was elevated 48–84 h after PGF_2α. Jugular venous concentrations of FSH in TGFα-treated ewes were significantly (P<0·001) elevated over controls during the first 48 h of the follicular phase and the LH surge was delayed for about 10 h (P<0·05). Infusion of TGFα caused a marked decline (P<0·05) in the number of large follicles within 12 h of the end of the infusion. Two of the six treated ewes, including the one with high follicular phase progesterone, had unusually large (8·7 and 10 mm) follicles present from 48–96 h after PGF_2α.

We conclude that direct arterial infusion of TGFα results in acute inhibition of ovarian steroid and inhibin secretion that is associated with induction of atresia in the population of large follicles. The lack of feedback of ovarian hormones results in a rebound increase of FSH which stimulates the growth of more ovarian follicles and the eventual re-establishment of ovarian hormone secretion and normal cyclicity.

Journal of Endocrinology (1994) 143, 13–24

ABSTRACT

An experiment was conducted to examine the effect of steroid-free ovine follicular fluid (oFF) on ovarian hormone secretion. Eight Merino × Finnish Landrace ewes in which the left ovary and vascular pedicle had been autotransplanted to a site in the neck were studied during the breeding season. Luteal regression was induced in all animals by injection of cloprostenol (100 μg, i.m.) on day 10 of the luteal phase. Four of the eight animals were treated with steroid-free oFF (3 ml, s.c.) in the early follicular phase, 24 and 36 h after injection of cloprostenol. Samples of both ovarian and jugular venous blood were collected at 4-h intervals from 20 h before until 96 h after injection of cloprostenol. Ovarian and jugular venous blood samples were also collected at 10-min intervals from 48 to 52 h after injection of cloprostenol to investigate the pattern of pulsatile secretion of ovarian hormones. Samples were assayed for oestradiol, androstenedione, testosterone and inhibin and the ovarian secretion rates calculated.

Both injections of oFF resulted in a fourfold increase in the concentration of inhibin in jugular venous plasma within 4–8 h of administration (P < 0·01) with concentrations remaining increased (P < 0·05) until 56 h after cloprostenol (32 h after the first oFF injection). Following oFF injection there was a profound (100%; P < 0·001) and prolonged decrease in the peripheral concentration of FSH until 60 h after cloprostenol at which time the concentration of FSH increased five- to sixfold (P < 0·001) to a peak lasting 24 h. In contrast to FSH, the concentration of LH in jugular venous plasma rose immediately following oFF treatment and continued to increase, exhibiting a profile similar to that described for FSH. No preovulatory LH surge was detected in any of the oFF-treated ewes while untreated ewes had an LH surge within 58·0±1·2 (s.e.m.) h. Within 8 h of the first injection of oFF the ovarian secretion rate of oestradiol, androstenedione and inhibin began to decline to reach a nadir of less than 1 ng/min within 32–36 h (56–60 h after cloprostenol; P < 0·01). Testosterone secretion, already barely detectable, did not change significantly following injection of oFF but remained low for 36 h following oFF and did not exhibit the increase observed over this period in controls. After injection of oFF the episodic secretion of oestradiol, androstenedione, testosterone and inhibin was markedly suppressed in spite of numerous pulses of LH. Re-establishment of inhibin, androstenedione and testosterone secretion began from around 36 h after injection of oFF and continued to increase for the remainder of the experimental period (P < 0·001). The re-establishment of oestradiol secretion, however, took until 60 h after oFF treatment (84 h after cloprostenol). This increase in ovarian hormone secretion was temporally related to the decrease in the concentration of FSH and LH in jugular venous plasma that was observed at the end of the experimental period.

We conclude that treatment of ewes with steroid-free oFF during the follicular phase of the oestrous cycle results in the immediate inhibition of the ovarian secretion of oestradiol, inhibin, androstenedione and testosterone. This effect can most probably be attributed to the depression in FSH that occurs following oFF injection, although the possibility exists that other factors present in oFF are acting directly on the ovary to inhibit follicular growth.

Journal of Endocrinology (1990) 127, 23–32

ABSTRACT

In sheep, secretion of oestradiol by the ovary is stimulated by pulses of LH but the factors controlling ovarian inhibin secretion are not well understood. We have investigated the effect of a single injection of LH on the ovarian secretion of inhibin. Six anoestrous Finn–Merino ewes which had one ovary autotransplanted to a site in the neck had jugular and timed ovarian venous blood samples collected at 10-min intervals for a total of 5 h.

The secretion rates of both inhibin (1–3 ng/min) and oestradiol (0·5–8 ng/min) were similar to those observed during the breeding season indicating significant follicular development in these animals.

After injection of 2·5 μg NIH-LH-S25 intravenously the concentration of LH in plasma rose from a baseline of 1·8 ±0·1 (s.e.m.) μg/l to a peak of 3·9 ±0·3 μg/l (P<0·01). This LH pulse stimulated a corresponding increase (P<0·01) in oestradiol secretion from a basal level of 0·9±0·2 ng/min to a peak of 4·6±0·6 ng/min that occurred within 30 min of injection. Although inhibin secretion was episodic in nature, increases were not related to either exogenous or endogenous LH pulses.

We conclude that, in contrast to oestradiol, the secretion of inhibin by the ovary is not controlled acutely by changes in plasma levels of LH during anoestrus.

Journal of Endocrinology (1989) 123, 173–179

ABSTRACT

The potential direct ovarian effects of immunoneutralization of inhibin, which increases, and follicular fluid treatment, which inhibits, follicle development in normal ewes was investigated in a sheep model in which endogenous FSH and LH secretion was suppressed thus removing any potential effects of treatment-induced alterations in endogenous FSH or LH secretion. Eighteen Welsh Mountain ewes were treated with two agonist implants containing 3·3 mg buserelin giving a total of 6·6 mg buserelin per animal. During week 5 of treatment all ewes were given a 72-h continuous infusion of ovine FSH (5 μg/h) starting at 09.00 h. Six ewes were treated with antiserum to the 1–26α peptide fragment of porcine inhibin 0 h and 24 h after the start of the FSH infusion, and a further six ewes were treated with charcoal-stripped ovine follicular fluid (oFF) as a source of inhibin, at 09.00 and 17.00 h throughout the 72 h of FSH infusion.

The plasma concentrations of both FSH and LH were significantly reduced in all ewes after 5 weeks of treatment with buserelin, and no large follicles > 2·5 mm in diameter were present. Treatment with inhibin antiserum or oFF had no effect, compared with control ewes, on the plasma concentrations of either FSH or LH during the FSH infusion period. After 72 h of FSH infusion there was no difference in the number of small follicles (<2·5 mm in diameter) or large follicles (> 2·5 mm in diameter) or the size of the largest follicles between control ewes and ewes treated with either inhibin antiserum or oFF. However, large oestrogenic follicles from ewes treated with oFF secreted significantly (P<0·01) more testosterone in vitro and had a significantly (P<0·01) higher follicular fluid concentration of testosterone than both control ewes and ewes treated with inhibin antiserum.

These results show that the effects of inhibin immunization in increasing follicle growth and oFF in inhibiting follicle growth in normal sheep are not related to a direct effect of these treatments on the ovary, but are mediated by the effects of treatment on the plasma concentrations of FSH. The effect of oFF in increasing thecal androgen production suggests an increase in thecal sensitivity to LH. In the sheep model used in the present study, pulsatile LH secretion was abolished and large follicles were induced to develop under the influence of FSH in the presence of only basal concentrations of LH. The results suggest a potential interaction between oFF components and pulsatile LH secretion in modulating FSH-induced preovulatory follicle growth through an alteration in thecal sensitivity to LH.

Journal of Endocrinology (1991) 131, 401–409

Abstract

Ewes with ovarian autotransplants received either inhibin antiserum (10 ml i.v. raised in sheep against recombinant 32 kDa human inhibin; n=6) or sheep serum (10 ml i.v.; n=5) on day 3 of the luteal phase with additional daily injections (1 ml i.v.) from 48 h after the initial bolus until day 13. Jugular and ovarian venous blood samples were taken 4-hourly over days 2–13 of the luteal phase. Blood samples were also taken at more frequent intervals (every 10–15 min for 2–3 h) to examine pulsatile secretory responses from the ovary to endogenous and gonadotrophin-releasing hormone-induced (150 ng i.m.) LH pulses on days 4, 6, 8, 10 and 12 of the luteal phase. Plasma FSH levels, ovarian steroid secretion and ovarian follicular development were measured. The ovarian follicle population was estimated daily by real time ultrasound scanning.

Immunisation against inhibin resulted in a 3- to 4-fold increase (P<0·001) in plasma FSH levels within 8 h with levels remaining elevated over controls for 6–7 days. Within 24 h of immunisation there was an increase in the number of small ovarian follicles (P<0·05) and by 3 days after treatment immunised ewes had 4–6 large ovarian follicles/ewe with this increase in the total number of large follicles being maintained for the rest of the experimental period (P<0·05). Mean ovarian oestradiol secretion during intensive bleeds was not different from controls 24 h after immunisation, but by 3 days after immunisation it was elevated 4- to 5-fold (P<0·001) over controls with this increase being maintained throughout the experiment. Similar responses to immunisation against inhibin in androstenedione secretion were observed although mean androstenedione secretion was not elevated until 7 days after treatment. In vitro antibody titres in immunised ewes remained elevated but declined steadily (P<0·001) over the experimental period.

We conclude that the initial stimulation of follicle development and ovarian steroid secretion following passive immunisation against inhibin can be attributed to increased blood FSH. However, the fact that with time FSH declined but increased follicle development was sustained, despite maintenance of high circulating antibody titres, suggests that on a longer term basis inhibin immunisation may stimulate ovarian function by interfering with the modulation of follicle development by inhibin at an ovarian level.

Journal of Endocrinology (1995) 145, 479–490

ABSTRACT

Active immunization of sheep against androstenedione results in an increase in ovulation rate that is associated with increased plasma levels of LH and progesterone, but not FSH. Although immunized ewes have more activated follicles the secretion rate of oestradiol is not increased. An experiment was conducted to examine the effect of androstenedione immunity on the ovarian secretion and peripheral plasma concentrations of inhibin.

Merino ewes in which the left ovary had been autotransplanted to a site in the neck were divided into control (n = 5) and androstenedione-immune (n = 6) groups. Ovarian and jugular venous blood was collected every 10 min at two stages of the follicular phase, 21–27 h and 38–42 h after a luteolytic dose of an analogue of prostaglandin F_2α (PG), and every 15 min for 6 h on day 10 of the subsequent luteal phase. The ewes were monitored regularly for luteal function by measurement of the concentration of progesterone and preovulatory LH surges. The concentration of inhibin in jugular and ovarian venous plasma was determined by radioimmunoassay and ovarian secretion rates and peripheral concentrations are expressed as pg of 1–26 peptide fragment of the α chain.

The ovarian secretion rate of inhibin tended to be greater in androstenedione-immune ewes at all stages of the oestrous cycle measured, with this difference being statistically significant (P <0·05) during the luteal phase (100±40 and 260±80 (s.e.m.) pg/min for control and immune groups respectively). The pattern of ovarian inhibin secretion exhibited pulsatile-like fluctuations which were not associated with LH pulses. Peripheral concentrations of inhibin were generally higher in immunized than in control ewes with this difference being significant (P < 0·01) from day 4 to 14 of the luteal phase (59±5 and 110±7 ng/1 for control and immune respectively). The ovarian secretion rate of immunoactive inhibin was greater (P <0·01) during the follicular phase than during the luteal phase in both groups of ewes, and peripheral concentrations of inhibin increased (P < 0·001) following injection of PG in ewes from both treatment groups.

We concluded that androstenedione immunity results in an increase in ovarian inhibin secretion, an effect that can probably be attributed to the greater number of large oestrogenic follicles present in the ovaries of these ewes. Furthermore, this increase in the concentration of inhibin may override any decrease in the negative feedback effects of ovarian steroid produced by immunization and, hence, explain the paradoxical findings of normal concentrations of FSH and raised concentrations of LH in ewes which are immunized against androstenedione.

Journal of Endocrinology (1990) 127, 285–296

ABSTRACT

Passive immunization was used to investigate the importance of inhibin and oestradiol in the control of FSH production during the follicular phase of the oestrous cycle in the sheep. Four groups of five mature Scottish Blackface ewes were injected with normal sheep plasma (control), antiserum to the 1–26α peptide fragment of porcine inhibin, antiserum to oestradiol-17β, or a combination of the two antisera, 24 h following cloprostenol-induced luteal regression. There was no difference in the concentration of LH in jugular venous plasma between the control and inhibin-immunized groups following the injection of normal sheep plasma or inhibin antiserum, with both groups exhibiting normal LH surges. In both the groups immunized against oestradiol, the basal concentration of LH rose by 25–30% (P<0.05) during the 96-h period following injection, while the LH surge and consequent formation of a corpus luteum was inhibited. In all three immunized groups there was a significant (P<0.001) rise in the concentration of FSH starting 3.8–4.8 h after the injection of antiserum. The duration of the rise was similar in the groups injected with oestradiol antiserum alone (43.6±12.8 h) or in combination with inhibin antiserum (40.6 ± 11.7 h), but was significantly (P<0.05) shorter in the group immunized against inhibin alone (17.0 ± 0.5 h). The rise in FSH was similar in the groups immunized against inhibin (142 ± 6%) or oestradiol (143±4%) alone, and was significantly (P<0.01) greater in the group injected with both antisera (195± 17%). These results provide evidence that both oestradiol and inhibin play a role in regulating the concentration of FSH during the follicular phase of the oestrous cycle, while reinforcing the hypothesis that inhibin is not involved in the regulation of LH production.

Journal of Endocrinology (1990) 125, 417–424

ABSTRACT

By selective removal and replacement of LH stimulation we sought to examine the relative importance of inhibin and oestradiol in controlling FSH secretion, and the role of LH in the control of ovarian hormone secretion, during the follicular phase of the oestrous cycle. Eight Finn–Merino ewes which had one ovary removed and the other autotransplanted to a site in the neck were given two injections of a gonadotrophin-releasing hormone (GnRH) antagonist (50 μg/kg s.c.) in the follicular phase of the cycle 27 h and 51 h after luteal regression had been induced by cloprostenol (100 μg i.m.). Four of the ewes received, in addition, i.v. injections of 2·5 μg LH at hourly intervals for 23 h from 42 to 65 h after GnRH antagonist treatment. Ovarian jugular venous blood samples were taken at 10-min intervals for 3 h before and 5 h after the injection of antagonist (24–32 h after cloprostenol) and from 49 to 53 h after antagonist (74–78 h after cloprostenol). Additional blood samples were taken at 4-h intervals between the periods of intensive blood sampling.

The GnRH antagonist completely inhibited endogenous pulsatile LH secretion within 1 h of injection. This resulted in a marked decrease in the ovarian secretion of oestradiol and androstenedione (P<0·001), an effect that was reversible by injection of exogenous pulses of LH (P<0·001). The pattern of ovarian inhibin secretion was episodic, but removal or replacement of stimulation by LH had no effect on the pattern or level of inhibin secretion. Peripheral concentrations of FSH rose (P<0·01) within 20 h of administration of the antagonist and these increased levels were maintained in ewes given no exogenous LH. In ewes given LH, however, FSH levels declined within 4 h of the first LH injection and by the end of the experimental period the levels of FSH were similar to those before administration of antagonist (P<0·01).

These results confirm that ovarian oestradiol and androstenedione secretion, but not inhibin secretion, is under the acute control of LH. We conclude that oestradiol, and not inhibin, is the major component of the inhibitory feedback loop controlling the pattern of FSH secretion during the follicular phase of the oestrous cycle in ewes.

Journal of Endocrinology (1990) 126, 377–384

ABSTRACT

The pattern of pulsatile secretion of inhibin, oestradiol and androstenedione by the ovary at different stages of the oestrous cycle in sheep was studied in five Finn–Merino ewes in which the left ovary had been autotransplanted to the neck. The ewes had jugular venous blood samples collected at 4-hourly intervals from 42 h before the induction of luteolysis by i.m. injection of cloprostenol (100 μg) on day 10 of the oestrous cycle, until day 3 of the following cycle. There were five periods of intensive blood sampling, when both ovarian and jugular venous blood samples were collected, as follows: (a) mid-luteal phase, before the second injection of cloprostenol on day 10 (15-min intervals for 6 h); (b) early follicular phase, 24 h after the second injection of cloprostenol (10-min intervals for 4 h); (c) late follicular phase, 48 h after the second injection of cloprostenol (10-min intervals for 4 h); (d) after the LH surge on day 1 of the cycle, 76 h after the second injection of cloprostenol (10-min intervals for 4 h); (e) early luteal phase on day 3 of the cycle, 120 h after the second injection of cloprostenol (10-min intervals for 3 h). Plasma was collected and the samples assayed for LH, FSH, progesterone, oestradiol, androstenedione and inhibin. The ovarian secretion rates for oestradiol, androstenedione and inhibin were calculated.

All ewes responded normally to the luteolytic dose of cloprostenol with the preovulatory surge of LH occurring within 56·4±1·6 h (mean ± s.e.m.) followed by the establishment of a normal luteal phase. The pulse frequency of LH, oestradiol and androstenedione increased in the transition from the luteal to the follicular phase (P<0·01). On day 1 of the cycle LH secretion consisted of low-amplitude high-frequency pulses (1·0±0·1 pulse/h) to which androstenedione, but not oestradiol, responded. On day 3 of the cycle LH secretion was similar to that on day 1 but both androstenedione and oestradiol secretion were pulsatile in response to LH, indicating the presence of oestrogenic follicles. The stage of the cycle had no significant effects on LH pulse amplitude and nadir but the ovarian secretory response to LH stimulation did vary with the stage of the cycle. Prolactin pulse frequency, amplitude and nadir were higher (P<0·05) during the follicular phase than the luteal phase. Prolactin pulse frequency was depressed (P<0·05) on day 1 of the cycle but increased to follicular phase levels on day 3. Prolactin pulse frequency was significantly correlated to oestradiol pulse frequency (r = 0·54; P<0·01).

During the luteal phase there were insufficient oestradiol pulses to obtain an estimate of pulse amplitude and nadir but both these parameters reached their highest level during the late follicular phase, fell to negligible levels on day 1 and increased to early follicular phase levels on day 3. Androstenedione pulse amplitude and nadir exhibited similar but less marked variation. Inhibin secretion was episodic at all stages of the cycle examined but did not exhibit significant variation with stage of cycle in any of the parameters of episodic secretion measured. Inhibin pulses were not related to either LH or prolactin at any stage of the cycle. FSH secretion was not detectably pulsatile but jugular venous concentrations of FSH at each stage of the oestrous cycle were negatively correlated with mean oestradiol (r= −0·52; P<0·01 but not inhibin secretion (r = 0·19).

We conclude that (i) LH secretion is pulsatile at all stages of the oestrous cycle but the steroidogenic responses of the ovary varies with the stage of the cycle, reflecting changes in characteristics of the follicle population, (ii) ovarian inhibin secretion is episodic and displays little change with the stage of the oestrous cycle and (iii) episodic inhibin secretion is not related to either pulses of LH or prolactin. The aetiology of these inhibin pulses therefore remains unknown.

Journal of Endocrinology (1990) 126, 385–393