Abstract

We have investigated the effects of oestradiol-17β on the development of the luteolytic signal (prostaglandin F_2α (PGF_2α) release to an oxytocin challenge) using a steroid-treated ovariectomised cow model. Long-term ovariectomised cows were pre-treated with progesterone for 14 days followed by oestradiol-17β for 2 days to induce oestrus (day 0), and were then treated for a further 16 days with physiological doses of progesterone and oestradiol-17β to simulate a luteal phase. On day 11 oestradiol was either withdrawn (low group; n=4), maintained (normal group; n=4) or increased (high group; n=4), generating mean (± s.e.m.) plasma concentrations of oestradiol from days 12–16 of 0·9±0·1, 2·3±0·4 and 3·7±0·3 pg/ml respectively. Basal and oxytocin-stimulated PGF_2α secretion was monitored by measuring concentrations of the principal metabolite of PGF_2α, 13,14-dihydro-15-keto PGF_2α (PGFM), in plasma samples collected before and after daily i.v. injection of 50 IU oxytocin from days 12–16. Basal PGFM was unaffected by the level of oestradiol treatment. In the normal group significant rises in plasma PGFM were seen following oxytocin on days 12, 13 and 14 (P<0·05) as well as days 15 and 16 (P<0·01). In the low group significant elevations in PGFM were only seen on days 15 and 16 (P<0·05), and were smaller (P<0·05) than those seen in the normal group. In the high group two cows showed no elevation in PGFM on any day while in two cows a rise was seen on all days which by day 16 was 84% greater than that seen in the normal group. The results demonstrate that a low level of oestradiol over the second half of the luteal phase leads to a weaker luteolytic signal, while a high level can be stimulatory or inhibitory.

Journal of Endocrinology (1995) 145, 175–180

Abstract

In intact cyclic ewes intrauterine infusion of conceptus secretory proteins results in the suppression of both endometrial oxytocin receptor concentrations and oxytocin-induced prostaglandin F_2α release. However, similar infusion in progesterone-treated ovariectomized ewes, while suppressing endometrial oxytocin receptors, does not fully inhibit oxytocin-induced prostaglandin F_2α release. To examine whether this anomaly resulted from an inadequate simulation of the luteal phase in the ovariectomized ewe treated with progesterone alone, the effects of additional treatment with two other ovarian hormones, oestradiol-17β and oxytocin, was investigated. Rather than permitting conceptus secretory protein to successfully inhibit oxytocin-induced prostaglandin F_2α release, treatment with oestradiol-17β in addition to progesterone actually resulted in an advancement in the timing of release. However, treatment with oxytocin, alone or in combination with oestradiol, permitted the full inhibition of oxytocin-induced prostaglandin F_2α release. To confirm that this effect did not result from the action of oxytocin alone, independently of the action of conceptus secretory protein, a second experiment was undertaken using a similar protocol but without the infusion of conceptus secretory protein. In this situation, oxytocin-induced prostaglandin F_2α release was only partially inhibited suggesting that both luteal oxytocin and conceptus secretory proteins are necessary to facilitate the full inhibition of luteolysis during early pregnancy in the ewe.

Journal of Endocrinology (1996) 150, 473–478

ABSTRACT

The physiological importance of circulating as opposed to locally produced insulin-like growth factor-I (IGF-I) has not been determined. By using a passive immunoneutralization technique, our objectives were to evaluate the role of circulating IGF-I in the regulation of animal growth and pituitary GH content.

A monoclonal antibody (MAb) to IGF-I, generated in our laboratory, has an affinity (K _a) of 0·13 litres/pmol for recombinant human IGF-I (rhIGF-I). Cross-reactivities of recombinant des-tripeptide IGF-I and recombinant bovine IGF-II were approximately 40 and 8% respectively. This MAb inhibited binding of purified hIGF-I to human placental membranes. In a radioimmunoassay based on displacement of ¹²⁵I-labelled rhIGF-I from the MAb, displacement curves generated with dilutions of acid–gel chromatography extracts of guinea-pig serum and rhIGF-I standards were parallel.

Twenty-four, 3-week-old male guinea-pigs were treated with the IGF-I MAb, a bovine herpes virus-I (BHV-I) MAb (control MAb) or vehicle (phosphate-buffered saline) (n = 8 per group). Treatments were administered i.p. every 3 days for 24 days at a dose of 20 mg/kg body weight. Blood was obtained on day 23 (48 h after treatment) and on day 25 (24 h after treatment). In a liquid-phase assay, serum from the IGF-I MAb-treated group bound 38 ± 8% (mean ± s.e.m.) (day 23) and 56 ± 7% (day 25) of an ¹²⁵I-labelled rhIGF-I trace at a final dilution of 1:10 000. Because of the development of an anti-mouse immune response in the guinea-pigs, these parameters would probably have been much greater during the first 2 weeks of the trial. Of the total IGF-I in serum, 50 ± 5% and 61±4% could be immunoprecipitated with an excess of rabbit anti-mouse immunoglobulin in samples from days 23 and 25 respectively. Comparisons between the groups treated with IGF-I MAb and BHV-I MAb revealed no significant differences in whole animal growth rate, growth of individual tissues, or pituitary GH content. Mean serum concentrations of IGF-I were 69 and 99% greater in IGF-I MAb-treated group than in the BHV-I MAb-treated group on days 23 and 25 respectively. These differences probably resulted from an extension of the half-life of IGF-I in serum of animals treated with the IGF-I MAb.

The lack of effect of treatment with the IGF-I MAb suggests that local production of IGF-I is generally sufficient to maintain normal growth or that local production or activity of IGF-I is increased in a compensatory fashion.

Journal of Endocrinology (1990) 124, 403–415

ABSTRACT

The source of inhibin secretion by the ovary in the sheep at different stages of the oestrous cycle was investigated by in-vivo cannulation of the ovarian veins. Twenty-four Scottish Blackface ewes were allocated to four groups of six ewes, i.e. those operated on during the luteal phase (day 10), and those operated on during the follicular phase 24–30, 36 and 60 h following an injection of 125 μg cloprostenol on day 10 of the luteal phase. Samples of jugular and timed ovarian venous blood were collected under anaesthesia before and after enucleation of the corpus luteum. Ovaries were then removed and follicles dissected out.

Following injection of cloprostenol, luteal regression occurred as indicated by a fall in the secretion of progesterone. The concentration of inhibin in jugular venous plasma and its ovarian secretion rate were similar at all stages of the follicular phase and during the luteal phase. In contrast, the secretion rate of oestradiol rose from 2·68 ±0·73 pmol/min during the luteal phase to 8·70± 2·24 pmol/min 24 h after injection of cloprostenol (P<0·05). Following enucleation of the corpus luteum the secretion rate of progesterone fell from 809 ± 270 pmol/min to 86 ± 30 pmol/min (P<0·001). There was also a smaller, artifactual fall in the secretion rate of oestradiol following enucleation of the corpus luteum, which was of similar size to a fall seen in the secretion rate of inhibin. This resulted in a significant (P<0·001) fall in the ratio of progesterone to inhibin, while the oestradiol to inhibin ratio remained unchanged. The secretion rate of inhibin from ovaries containing luteal tissue was similar to that from the contralateral side without luteal tissue (1·41±0·30 compared with 1·32±0·30 ng/min), while ovaries with large antral follicles secreted significantly (P< 0·001) more inhibin than those with no follicles ≥3 mm (2·28 ± 0·36 compared with 0·25 ±0·06 ng/min).

From these results we conclude that, in the sheep, large antral follicles are responsible for most, if not all, the secretion of inhibin by the ovary at all stages of the oestrous cycle, and that the corpus luteum secretes little or no immunoactive or bioactive inhibin. Due to the fact that, unlike inhibin, the secretion rate of oestradiol rises during the follicular phase of the cycle, when the concentration of FSH is suppressed, it seems likely that oestradiol rather than inhibin is the major ovarian factor modulating the change in FSH secretion seen at this stage of the oestrous cycle.

Journal of Endocrinology (1989) 123, 181–188

ABSTRACT

This experiment was undertaken in order to investigate the production of inhibin, oestradiol and androstenedione by ovarian follicles at different stages of the oestrous cycle in sheep. Twenty-four Scottish Blackface ewes were allocated to four groups of six ewes, i.e. those operated on during the luteal phase (day 10), and those operated on during the follicular phase 24–30, 36 and 60 h after the induction of luteal regression by an injection of 125 μg cloprostenol on day 10 of the luteal phase. Samples of jugular and ovarian venous blood were collected under anaesthesia and ovaries were then removed and all follicles larger than 3 mm diameter dissected out and incubated in medium for 2 h.

After injection of cloprostenol, luteal regression occurred as indicated by a fall in the secretion rate of progesterone. The ovarian secretion rate of inhibin was similar at all stages of the follicular phase and during the luteal phase while, in contrast, the secretion rate of oestradiol was significantly (P < 0·05) elevated in the group 24 h after injection of cloprostenol. There was good correlation between the in-vivo ovarian secretion rate and production rate during incubation in vitro for both inhibin (r = 0·57) and oestradiol (r = 0·60). When follicle diameter was compared with in-vitro hormone production there was good correlation for inhibin (r = 0·72) with larger follicles producing more inhibin, while the value for oestradiol was somewhat lower (r = 0·57) owing to the presence of large atretic follicles with low oestradiol production. Androstenedione production showed a lower correlation with follicle diameter (r = 0·39). When the four time periods were compared separately, there were significantly (P < 0·05) more follicles with high in-vitro oestradiol production (> 90 fmol/min) in the group at 36 h than in the other three groups, while inhibin release in relation to follicle size was similar in the four groups. Large oestrogenic follicles were responsible for 90% of the total oestradiol production during culture while only providing 55% of the total inhibin production, with large non-oestrogenic and small follicles contributing 33% and 12% of inhibin production respectively.

From the results of this study we conclude that while oestradiol is mainly produced by the large oestrogenic follicles, a considerable amount of inhibin is also produced by large non-oestrogenic and small follicles. We also found that a lack of variation in inhibin secretion rate in the intact animal was paralleled by a lack of variation in the pattern of inhibin produced from individual follicles.

Journal of Endocrinology (1992) 132, 225–234

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

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

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

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

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