Effect of epidermal growth factor on reproductive function of ewes

Adult Merino ewes were infused via the jugular vein with either saline (n = 5) or epidermal growth factor (EGF) (4.2 micrograms/kg per h, n = 6) for 24 h in either the luteal phase or the follicular phase of the oestrous cycle and reproductive function was examined. Infusion of EGF during the luteal phase caused no detectable change in plasma progesterone or prolactin concentrations over a 7-day period compared with the controls. Infusion of EGF during the follicular phase suppressed the oestrous rise in plasma oestradiol. Luteinizing hormone pulse amplitude was increased and pulse frequency was decreased by the end of the infusion. All control ewes had a pro-oestrous LH surge and mated, but the LH surge and oestrus were prevented by EGF infusion. Nevertheless, plasma progesterone levels rose subsequently in the EGF-infused ewes in parallel with the control ewes, suggesting that the preovulatory follicle had luteinized. Both LH and FSH rose over the 7 days after EGF infusion to levels similar to those in ovariectomized ewes. Thus EGF appears to inhibit follicular oestradiol production, although it does not affect luteal progesterone production or follicular luteinization. We suggest that the alteration in gonadotrophin secretion patterns results from a disturbance of feedback mechanisms between the ovary and the hypothalamopituitary axis, although a direct effect in the brain or the pituitary gland cannot yet be excluded.     

The antagonistic effect of exogenous LH pulses on FSH-stimulated preovulatory follicle growth in ewes chronically treated with a gonadotrophin-releasing hormone agonist

The hypogonadotrophism model induced by the chronic administration of gonadotrophin-releasing hormone (GnRH) agonist was used to investigate the effects of different concentrations of FSH with or without LH pulses on the stimulation of follicular development in the ewe. Continuous administration of an agonist (buserelin) by osmotic minipump to thirty-six Welsh Mountain ewes from the early luteal phase for 5 weeks resulted in a sustained suppression of the plasma concentration of FSH and inhibited the pulsatile release of LH. The inhibition of gonadotrophin secretion was due to the desensitization and/or down-regulation of pituitary gonadotroph function, since the agonist-treated animals showed no response to a challenge of 1 microgram GnRH. During week 6 of agonist treatment, ewes were infused with either 4-hourly pulses of ovine LH (9 micrograms/pulse), low concentrations of ovine FSH (3 micrograms/h) or high concentrations of FSH (9 micrograms/h) alone or with 4-hourly pulses of LH. After 5 days of gonadotrophin infusion, there was no difference between the mean number of follicles per ewe from the animals treated with LH alone, low concentrations of FSH with or without LH pulses or the high concentration of FSH alone compared with the mean number of follicles from control ewes on day 8 of the luteal phase. Infusion of the high concentration of FSH alone stimulated the development of an increased number of large oestrogenic follicles (follicles greater than 2.5 mm in diameter and secreting greater than 3.7 nmol oestradiol/h in vitro) compared with control ewes. The addition of high-amplitude LH pulses to the infusion of the high concentration of FSH prevented follicles developing beyond 2.5 mm in diameter, but doubled the number of small follicles (less than or equal to 2.5 mm) present in the ovaries. These results show that normal follicular development can be induced by physiological concentrations of FSH alone in the absence of pulsatile LH release. The addition of high-amplitude LH pulses antagonized this stimulatory effect of FSH on follicle growth in the ewe.     

Inadequate function of corpora lutea following the induction of ovulation with monensin and FSH in seasonally anoestrous ewes

Sixteen ewes in mid-seasonal anoestrus were stimulated to ovulate using sequential injections of FSH (total dose 10 mg) over a 4-day period. Half of the ewes received a dietary growth promotant (monensin) known to enhance the ovarian response to exogenous gonadotrophins. The ewes were ovariectomized on day 5 or 11 (day 0 = the initiation of FSH treatment). Serial blood samples were taken in half of the ewes to determine peripheral concentrations of LH and a single sample of ovarian venous blood was collected before ovariectomy. All luteal structures were dissected from the ovaries, counted and incubated in vitro to determine progesterone production. The luteal structures were then examined histologically for the abundance of luteal cells. The physical appearance of the ovary, along with plasma concentrations of LH and ovarian venous oestradiol indicated that the monensin-treated ewes ovulated before control ewes. The corpora lutea from control ewes produced significantly (P less than 0.05) more progesterone than did the corpora lutea from the monensin-treated group. Furthermore, only 7% of the remaining luteal structures in the monensin-treated group produced significant amounts of progesterone on day 11, whereas 61% of the luteal structures in the control group were actively secreting progesterone. The mean number of granulosa cells in the follicles was similar at ovulation in the two groups, but the mean numbers of large and small luteal cells were significantly (P less than 0.05) lower in luteal structures from the monensin-treated ewes than in those from the control ewes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in FSH and the pulsatile secretion of LH during treatment of ewes with bovine follicular fluid throughout the luteal phase of the oestrous cycle

Treatment of Damline ewes with twice-daily i.v. injections of bovine follicular fluid during the luteal phase for 10 or 2 days before prostaglandin-induced luteolysis resulted in a delay in the onset of oestrous behaviour and a marginal increase in ovulation rate. During the treatment cycle, blood samples were withdrawn at 15-min intervals for 25 h from 08.00 h on days 1, 6 and 10 (day 0 = oestrus). At all three stages of the luteal phase, plasma FSH concentrations were suppressed relative to controls 3 h after the 09.00 h injection of follicular fluid and remained low until 06.00 h on the following day. In the 10-day treatment group LH pulse amplitude was significantly greater than that of controls on days 6 and 10. Pulse frequency remained high throughout treatment and was significantly higher relative to controls on day 10 despite normal progesterone levels. The results suggest that the higher pulsatile LH secretion during the luteal phase is due to reduced negative feedback effects of oestradiol occurring as a result of the follicular fluid-induced reduction in FSH.     

Changes in the secretion of LH pulses, FSH and prolactin during the preovulatory phase of the oestrous cycle of the ewe and the influence of treatment with bovine follicular fluid during the luteal phase

It has previously been shown that treatment of ewes with bovine follicular fluid (bFF) throughout the luteal phase of the oestrous cycle lowers plasma levels of FSH but increases the frequency and amplitude of the pulses of LH. Under these conditions, ovarian follicles grow to a maximum diameter of 2.7 mm and have a reduced capacity to release oestradiol. We have examined the nature of the gonadotrophin signals controlling follicular development in the normally cycling ewe and have investigated the effects of previous exposure to bFF on these signals and the follicular responses to them. Control ewes (n = 7) were injected i.v. with 9 ml bovine serum and treated ewes were injected with 9 ml bFF, twice daily from days 1 to 10 of the luteal phase (day 0 = oestrus). The ewes were injected with prostaglandin analogue on day 11 of the cycle to induce luteolysis and the gonadotrophin patterns were studied in blood sampled from these animals every 10 min for up to 72 h during the subsequent follicular phase. Following luteolysis (and the end of bFF treatment), LH pulse frequency increased rapidly in both groups and reached 1 pulse/h within 6 h. Thereafter, pulse frequency increased marginally and reached 1 pulse/50 min by the onset of the LH surge. This pattern was not affected by previous treatment with bFF. In the control ewes, the amplitude of the LH pulses did not change significantly following luteolysis or at any time during the follicular phase, while the levels of FSH declined slowly until the onset of the surge. In the treated ewes, on the other hand, there was an immediate increase in both LH pulse amplitude and the concentration of FSH immediately after the end of bFF treatment at luteolysis, and they remained above control levels for 24 and 16 h respectively. Plasma prolactin levels did not appear to change around the time of luteolysis but showed a marked and significant diurnal rhythm (nadir around noon and peak around midnight) in both groups. The concentrations of prolactin were significantly (P less than 0.001) lower and the preovulatory peak was delayed and reduced in the bFF-treated ewes relative to controls. The onset of oestrus was also significantly (P less than 0.01) delayed by bFF treatment, but the ovulation rates did not differ between the groups. Furthermore, comparisons within or between groups revealed no significant relationships between any of the variables of plasma LH secretion during the follicular phase and the subsequent ovulation rate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of small doses of bovine follicular fluid on the tonic secretion of gonadotrophins in the ewe

Previous work has shown that treatment of ewes with steroid-free bovine follicular fluid (bFF), a rich source of inhibin, partially inhibits the increase in mean plasma concentrations of LH induced by ovariectomy. The present experiment was designed to test the hypothesis that this effect was a reflection of reduced LH pulse amplitude which would only be expressed at high (pharmacological) doses of bFF. To do this, we assessed the dose-response to bFF of the secretion of FSH and LH pulses in intact and acutely ovariectomized ewes. In intact ewes, a low dose of bFF (0.2 ml s.c. every 8 h) had no detectable effect on the secretion of FSH, an intermediate dose (0.6 ml s.c. every 8 h) depressed FSH concentrations for about 24 h and a high dose (1.8 ml s.c. every 8 h) reduced FSH concentrations to undetectable levels. In ewes treated with 1.8 ml bFF, FSH concentrations also remained undetectable after ovariectomy and did not increase until treatment was withdrawn. In ewes treated with 0.6 ml bFF, FSH concentrations were maintained at normal intact levels for about 32 h following ovariectomy but then rose to normal ovariectomized levels. In ewes treated with 0.2 ml bFF, FSH concentrations increased immediately after ovariectomy but more slowly than in control ovariectomized ewes. Profiles of LH pulses were recorded after ovariectomy, during and after the withdrawal of bFF treatment. In ewes treated with the highest dose (1.8 ml s.c. every 8 h), mean LH levels and pulse amplitude were lower than in control ewes and increased significantly following withdrawal of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

The roles of inhibin and gonadotrophin-releasing hormone in the control of gonadotrophin secretion in the ewe

The respective roles and relative importance of ovarian inhibition and hypothalamic stimulation in the differential control of the secretion of FSH and LH were studied in the ewe. In the first experiment two groups of ten intact ewes were injected i.v. twice daily with 9 ml charcoal-extracted bovine follicular fluid (bFF), a preparation rich in inhibin (3.65 ku./ml), throughout the luteal phase of the oestrous cycle. Compared with the control ewes, this treatment significantly reduced pituitary and plasma FSH concentrations and increased the frequency and amplitude of the LH pulses, but did not affect pituitary LH concentrations. In a second experiment, five control and five bFF-treated ewes from experiment 1 were ovariectomized and the injection regime was altered to 2.5 ml s.c. every 8 h. This treatment was maintained for 21 days. In control ewes, plasma FSH concentrations rose significantly within 12 h and continued to rise for 3-4 days. Treatment with bFF abolished this increase and maintained plasma FSH concentrations below those observed in intact ewes. The rise in mean plasma LH concentrations evoked by ovariectomy was also partially inhibited in the bFF-treated ewes. The response to the gonadotrophin-releasing hormone (GnRH) agonist buserelin (5 micrograms i.v.) was measured 6, 12 and 18 days after ovariectomy. In control ewes the agonist consistently evoked large surges of both hormones but in bFF-treated ewes the FSH response was completely blocked and the initial phase of the LH response (the first 'pool') was greatly reduced. In experiment 3, six ewes were ovariectomized and passively immunized against GnRH 3 days after oestrus. The increase in plasma LH which normally follows ovariectomy was completely abolished and mean concentrations remained very low and did not change over the following 14 days. In contrast, mean FSH concentrations rose significantly within 12 h of ovariectomy and continued to rise until the third day, after which they fell gradually. Treating three of the ewes with bFF (2.5 ml s.c. every 8 h) 8 days after ovariectomy and immunization further reduced the FSH concentrations. When the ewes were injected repeatedly (200 ng i.v., hourly for 5 h) with [D-penicillamine-(But)6]-GnRH(1-9)nonapeptide-ethylamide, a synthetic GnRH analogue which does not bind to the antiserum, there was a rapid rise in the secretion of LH in both control and bFF-treated animals but, as with the responses to buserelin, the initial response was significantly lower in bFF-treated than in control ewes.(ABSTRACT TRUNCATED AT 400 WORDS)     

HORMONE PROFILE IN PREMENSTRUAL TENSION: EFFECTS OF BROMOCRIPTINE AND DIURETICS

Plasma levels of prolactin, FSH, LH, progesterone and 17-beta-oestradiol in twenty women with premenstrual tension were compared with those in twenty controls. The former group was studied also during treatment with bromocriptine. The mean prolactin level in the PMT group was lower in the follicular phase than in the luteal phase (P less than 0.01), but there was no difference between the PMT and control group in the luteal phase. No differences were found between the controls and the PMT group in FSH,LH, 17-beta-oestradiol and progesterone levels in the luteal phase. Bromocriptine suppressed prolactin concentrations (P less than 0.01), but had no effect on the FSH, LH, 17-B-oestradiol or progesterone levels.     

Active immunization of ewes against luteinizing hormone releasing hormone, and its effects on ovulation and gonadotrophin, prolactin and ovarian steroid secretion.

Three Scottish Blackface ewes were immunized against luteinizing hormone releasing hormone (LH-RH) conjugated to bovine serum albumin (BSA) and three control ewes were immunized against BSA alone. When the antibody titre to LH-RH became raised the treated animals failed to show oestrus or ovulate; they had significantly lower levels of plasma luteinizing hormone (LH) and higher levels of prolactin than the controls, whereas the levels of follicle-stimulating hormone (FSH) were unaltered. The integrity of the hypothalamus-pituitary-gonadal system of these animals was then challenged by the injection of a highly active analogue of LH-RH and by ovariectomy. An i.v. injection of 5 microgram D-serine-t-butyl 6 des-glycine-NH2 10 LH-RH ethylamide raised plasma LH and FSH. Ovariectomy caused an eight- and ninefold rise in plasma levels of LH and FSH respectively in controls, but failed to increase plasma levels of LH and FSH in the LH-RH-immunized ewes. Plasma prolactin concentrations in the LH-RH-immunized ewes were significantly reduced by ovariectomy.     

Ovarian secretion rates and peripheral concentrations of inhibin in normal and androstenedione-immune ewes with an autotransplanted ovary

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 F2 alpha (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 alpha 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 less than 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 less than 0.01) from day 4 to 14 of the luteal phase (59 +/- 5 and 110 +/- 7 ng/l for control and immune respectively). The ovarian secretion rate of immunoactive inhibin was greater (P less than 0.01) during the follicular phase than during the luteal phase in both groups of ewes, and peripheral concentrations of inhibin increased (P less than 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.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of gonadotrophin-releasing hormone agonist-induced suppression of LH and FSH on follicle growth and corpus luteum function in the ewe

Continuous infusion of a gonadotrophin-releasing hormone (GnRH) agonist (buserelin) by osmotic minipump from day 1 of the luteal phase in five Welsh ewes resulted in a sustained suppression of plasma concentrations of FSH which increased three- to eightfold within 2 days after the end of infusion 29 days later. Plasma concentrations of LH increased three- to eightfold over the first 5 days of infusion and then became basal and non-pulsatile until 1 day after the end of infusion. Duration of the luteal phase and plasma concentrations of progesterone were not significantly different in control and treated ewes. Pulses of LH in control ewes were followed by increases in concentrations of progesterone in samples collected at 10-min intervals for 7 h on days 10 and 14 of the luteal phase. However, progesterone was also released in a pulsatile manner in the absence of LH pulses in both control and GnRH agonist-treated ewes. After natural luteolysis, no ovulation or corpus luteum function occurred in treated ewes up to 15 days after the end of treatment on day 29, even though oestrus, indicating follicular development and oestrogen secretion, had occurred 8-11 days after treatment ended. After 30 days of infusion the ovaries of GnRH agonist-treated ewes contained no follicles greater than 2.5 mm in diameter. In follicles of 1-2 mm in diameter the basal and LH-stimulated production of oestradiol and testosterone in vitro were similar in both control and GnRH agonist-treated ewes, and a similar proportion of these follicles was oestrogenic (greater than 370 mol oestradiol per follicle) in GnRH agonist-treated and control ewes. These results show (1) that progesterone secretion by the corpus luteum of the ewe can be sustained in the presence of basal concentrations but absence of pulsatile secretion of LH, and progesterone is released in a pulsatile manner whether or not LH pulses are present, (2) that follicular development beyond 2.5 mm in diameter in the ewe is dependent upon adequate stimulation by both LH and FSH and (3) that the continuous infusion of GnRH agonist is a simple method for providing reproducible suppression of LH and FSH and follicular development in the ewe to allow the study of gonadotrophin action on the ovary in vivo.     

Longitudinal evaluation of the luteal phase and its transition into the follicular phase

The precise patterns of LH, FSH, and PRL secretion and their correlation with estradiol (E2) and progesterone (P) secretion during the entire luteal phase have not been elucidated. To analyze in detail the secretory patterns of these hormones we performed 29 consecutive studies in 5 healthy, regularly menstruating women throughout their luteal phase [days 0 (ovulation), 2, 6, 10, and 14] and subsequent early follicular phase (day 2F). During each study plasma LH, FSH, PRL, E2, and P were measured at 10-min intervals for 6 h. Both plasma LH concentrations and LH pulse frequency declined from days 0 to 10 and increased thereafter, whereas LH pulse amplitude continued to decline throughout the luteal and early follicular phases. Plasma FSH concentrations followed a pattern similar to that of LH; however, there was a larger increase in the FSH level on days 14 and 2F. Plasma PRL levels declined initially on day 2 and again on day 14. Regression analysis indicated a positive correlation between LH concentrations and LH pulse frequency (r = 0.715; P less than 0.001) and between PRL and E2 concentrations (r = 0.528; P less than 0.01). A negative correlation was found between plasma P concentrations and both LH concentrations (r = -0.521; P less than 0.01) and LH pulse frequency (r = -0.633; P less than 0.001) and between plasma E2 and FSH concentrations (r = -0.762; P less than 0.001). Thirty-six (65%) PRL pulses and only 11 (39%) FSH pulses coincided with LH pulses. There was no clear pulsatile pattern of secretion of either E2 or P. We conclude that 1) the plasma LH, FSH, PRL, E2, and P concentrations vary markedly throughout the luteal phase; 2) the plasma LH level is largely dependent on the frequency of LH pulses; 3) plasma P decreases plasma LH by reducing the frequency of LH pulses; 4) the remarkable synchrony between PRL pulses and LH pulses implies that their secretion may be regulated by a common neuroendocrine mechanism; and 5) the preferential increase in FSH during the late luteal phase may play an important role in follicular recruitment for the subsequent cycle.     

Corpus luteum insufficiency induced by a rapid gonadotropin-releasing hormone-induced gonadotropin secretion pattern in the follicular phase

The pulse frequency of LH and FSH (and by inference, GnRH) is a major determinant of the relative baseline plasma levels of LH and FSH. Luteal phase deficiency has been reported to be associated with increased gonadotropin pulse frequency and inadequate preovulatory follicular development. In this study we induced in normal women a supraphysiological gonadotropin pulse frequency in the follicular phase to determine its effect on follicular development and corpus luteum function. Specifically, we tested the hypothesis that a supraphysiological GnRH pulse frequency would result in deficient luteal phase production of progesterone. The subjects were six normal ovulatory women (age range, 23-35 yr). They were initially studied during a control cycle (cycle 1). Then, 25 ng/kg GnRH was administered iv every 30 min from the early follicular phase of the next cycle (cycle 2) until ovulation occurred. GnRH administration resulted in increased follicular phase plasma LH and FSH levels and LH to FSH ratios, multiple preovulatory follicles (mean, 2.8) with increased mean integrated estradiol [1302 (pg/mL)day (cycle 1) vs. 2550 (pg/mL)day (cycle 2); P less than 0.05; 4780 vs. 9360 (pmol/L)day, Systeme International units], spontaneous ovulation, decreased luteal phase plasma immunoreactive and bioactive LH levels, decreased luteal phase length [13.5 days (cycle 1) vs. 8.8 days (cycle 2); P less than 0.05], and decreased mean integrated progesterone secretion [152 (ng/mL)day (cycle 1) vs. 66 (ng/mL)day (cycle 2); P less than 0.01; 482 vs. 209 (nmol/L)day, Systeme International units]. We conclude that high frequency LH and FSH secretion during the follicular phase can induce inadequate progesterone secretion during the subsequent luteal phase, and we infer that the pathophysiological basis for this induced luteal phase deficiency is decreased LH support of corpus luteum function.     

Anastomosis of the ovarian vein to the hepatic portal vein in sheep induces ovarian hyperstimulation associated with increased LH pulsatility, but only in the absence of the contralateral ovary

In this study, two experiments were performed, the first of which examined the ovarian response in ewes that were subject to unilateral ovariectomy (ULO) at different intervals (0-14 days) after surgical anastomosis (AN) of the ovarian vein to the mesenteric vein (n=7 ewes), or sham operation (SO; n=4 ewes). Hypertrophy and development of multiple follicular and luteal structures on AN ovaries were observed after ULO, while SO ovaries remained of normal size and appearance after ULO. The second experiment involving 11 ewes (five AN; six SO) aimed to clarify the mechanism by which AN following ULO-induced ovarian hypertrophy and increased follicle development. The results confirmed that there were more large (>5 mm) follicles on AN compared with SO ovaries; however, their rate of atresia was similar. Oestradiol and progesterone concentrations in follicular fluid of class 1 follicles (5-9 mm) were higher in AN ovaries than those in control follicles of the same size collected in the late follicular phase of an induced oestrous cycle. In AN ewes, intrafollicular progesterone concentrations increased while follicular aromatase activity and intrafollicular oestradiol, inhibin A, follistatin and activin A concentrations all decreased as follicle size increased. Oestradiol and progesterone concentrations were substantially higher in ovarian venous blood than in hepatic venous blood, both in AN and SO ewes, whereas inhibin A levels were not significantly modified by passage through the liver in either group. Mean plasma LH concentration, and LH pulse frequency and amplitude increased markedly after AN but were not affected by SO. Plasma FSH showed only a small transient increase after AN, presumably due to the maintenance of inhibin feedback. Injection of prostaglandin F(2)(alpha) 4 days later did not further modify LH or FSH secretion in either group. Full ovariectomy (FO) 9-14 days after AN or SO increased LH secretion markedly in SO ewes but to a lesser degree in AN ewes; FO induced a large and rapid increase in FSH levels in both groups. In conclusion, AN of the ovary to the liver via the mesenteric vein provides a useful model for studying the feedback between the ovary and the hypothalamo-pituitary system and the mechanisms controlling follicle development. The present results indicate that the pattern of LH secretion is an important factor controlling the terminal phase of follicle development in the ewe.     

Suppression of ovine plasma FSH by bovine follicular fluid: neutralization by plasma from ewes immunized against an inhibin-enriched preparation from bovine follicular fluid

Adult Merino ewes were immunized against an inhibin-enriched preparation (bFFI) obtained by affinity chromatography of bovine follicular fluid (bFF). Plasma was obtained in early luteal phase from these ewes and from control ewes immunized against bovine serum albumin. Ten months after ovariectomy the plasma concentration of FSH, but not LH, in control ewes was decreased by four s.c. injections of 8 ml bFFI (17,500 units inhibin/injection). There was no decrease in plasma concentrations of FSH or LH in immunized ewes with the same dose of bFFI. In a second study with long-term ovariectomized ewes, four injections of 20 ml plasma from the immunized ewes significantly reduced the decrease in FSH concentration caused by four injections of steroid-free bFF (2500 units inhibin/injection) in comparison with similar ewes injected with plasma from control ewes. These results show that the plasma of ewes immunized against bFFI contains substances, presumably antibodies, which neutralize the FSH-suppressive effects of bFF and bFFI in ovariectomized ewes.     

Effect of basal and pulsatile LH release on FSH-stimulated follicle growth in ewes chronically treated with gonadotrophin-releasing hormone agonist

Ewes chronically treated with gonadotrophin-releasing hormone (GnRH) agonist were used to investigate the importance of the peripheral concentration of LH in FSH-stimulated follicular development. Twenty-four Welsh Mountain ewes were treated with two agonist implants containing 3.3 mg buserelin. During week 6 of treatment all the ewes were given a 72-h continuous infusion of ovine FSH alone (3 micrograms/h) or FSH with large (7.5 micrograms)- or small (2.5 micrograms) amplitude pulses of ovine LH delivered at 4-hourly intervals. The importance of baseline LH throughout the FSH infusion was evaluated in six animals which were treated with a specific antiserum against bovine LH (LH-AS) 15-20 h before the start of FSH treatment. In the absence of LH-AS, infusion of FSH alone or with large or small pulses of LH stimulated the development of a normal number of small follicles (less than or equal to 2.5 mm in diameter) and large follicles (greater than 2.5 mm in diameter). These follicles had normal diameter and steroid secretion compared with control ewes on day 8 of the luteal phase. In contrast, the animals pretreated with LH-AS developed no follicles greater than 2.0 mm in diameter but the number of small follicles per ewe was significantly (P less than 0.05) increased. These results support the hypothesis that FSH in the absence of pulsatile LH release stimulates preovulatory follicular development in ewes treated with GnRH agonist. The follicular response to LH pulses of different amplitude is dependent on both the stage of development of the follicle and the peripheral concentration of FSH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steroid control of gonadotropin secretion and ovarian function in heifers

The failure of anti-steroid treatments to induce multiple ovulations in cattle in a repeatable way, prompted us to examine the role of the gonadal steroids in the control of gonadotropin secretion in this species. A better understanding of the control of gonadotropin secretion in the cow would assist in the development of treatments to control prolificacy. Groups of six heifers were implanted with one of three sizes of estradiol (E2) implant on day 9 of a synchronized estrous cycle, and five control heifers received empty implants. All heifers were ovariectomized during the luteal phase of the subsequent estrous cycle and given progesterone-releasing intravaginal devices (PRID). Blood samples were taken every 10 min for 12 h with PRIDs and for 6 h after PRID withdrawal, for the measurement of LH and FSH concentrations. The two larger implant sizes (increasing E2 concentrations to 7.4 and 18.7 pg/ml plasma during the luteal phase of the cycle) decreased ovulation rate, the number of large follicles, and luteal weight. After ovariectomy, the three implant sizes produced E2 concentrations comparable with those during the luteal and follicular phases of the estrous cycle and at estrus (1.5, 4.4, and 10.5 pg/ml, respectively). E2 alone decreased mean LH and FSH concentrations and LH pulse amplitude, whereas progesterone alone reduced mean gonadotropin concentrations and LH pulse frequency. Only in the presence of progesterone did E2 decrease LH pulse frequency. Steroid concentrations which mimicked those of the luteal and follicular phases of the cycle produced luteal- and follicular-phase patterns of LH and FSH secretion. These results confirm that E2 and progesterone are important regulators of gonadotropin secretion in cattle, and question the role of inhibin in this respect.     

Effects of long-term treatment with LH on induction of cyclic ovarian activity in seasonally anoestrous ewes

The aim of the present study was to establish whether cyclic ovarian activity could be induced and then maintained in anoestrous Romney ewes by the long-term administration of regular intravenous pulses of LH (10 micrograms ovine LH i.v. once every 1 or 2 h for 29-91 days). The LH pulse regimen was designed to generate plasma profiles of LH that were comparable to those experienced during the luteal and follicular phases of the oestrous cycle. The results showed that the LH treatments were capable of inducing cyclic ovarian activity, as assessed from the concentrations of progesterone in plasma, but that the treatments were inadequate for sustaining cyclic activity beyond two consecutive progestational phases. After 35-56 days of treatment, the plasma concentrations of FSH declined significantly (P less than 0.05) relative to those in the untreated animals. These data suggest that FSH supplementation as well as LH might be required for the long-term maintenance of cyclic ovarian activity in seasonally anoestrous ewes.     

Relationship between gonadotrophin subunit gene expression, gonadotrophin-releasing hormone receptor content and pituitary and plasma gonadotrophin concentrations during the rebound release of FSH after treatment of ewes with bovine follicular fluid during the luteal phase of the cycle.

The modulation of FSH secretion at the beginning and middle of the follicular phase of the cycle represents the key event in the growth and selection of the preovulatory follicle. However, the mechanisms that operate within the pituitary gland to control the increased release of FSH and its subsequent inhibition in vivo remain unclear. Treatment of ewes with bovine follicular fluid (bFF) during the luteal phase has been previously shown to suppress the plasma concentrations of FSH and, following cessation of treatment on day 11, a rebound release of FSH occurs on days 12 and 13. When luteal regression is induced on day 12, this hypersecretion of FSH results in an increase in follicle growth and ovulation rate. To investigate the mechanisms involved in the control of FSH secretion, ewes were treated with twice daily s.c. injections of 5 ml bFF on days 3-11 of the oestrous cycle and luteal regression was induced on day 12 with prostaglandin (PG). The treated ewes and their controls were then killed on day 11 (luteal), or 16 or 32 h after PG and their pituitaries removed and halved. One half was analysed for gonadotrophin and gonadotrophin-releasing hormone (GnRH) receptor content. Total pituitary RNA was extracted from the other half and subjected to Northern analysis using probes for FSH-beta, LH-beta and common alpha subunit. Frequent blood samples were taken and assayed for gonadotrophins. FSH secretion was significantly (P less than 0.01) reduced during bFF treatment throughout the luteal phase and then significantly (P less than 0.01) increased after cessation of treatment, with maximum secretion being reached 18-22h after PG, and then declining towards control values by 32h after PG. A similar pattern of LH secretion was seen after bFF treatment. Pituitary FSH content was significantly (P less than 0.05) reduced by bFF treatment at all stages of the cycle. No difference in the pituitary LH content was seen. The increase in GnRH receptor content after PG in the controls was delayed in the treated animals. Analysis of pituitary mRNA levels revealed that bFF treatment significantly (P less than 0.01) reduced FSH-beta mRNA levels in the luteal phase. Increased levels of FSH-beta, LH-beta and alpha subunit mRNA were seen 16h after PG in the bFF-treated animals, at the time when FSH and LH secretion from the pituitary was near maximum.(ABSTRACT TRUNCATED AT 400 WORDS)     

GONADOTROPHIN SECRETION IN THE SECOND HALF OF THE MENSTRUAL CYCLE: A COMPARISON OF WOMEN WITH NORMAL CYCLES, LUTEAL PHASE DEFECTS AND DISTURBED FOLLICULAR DEVELOPMENT

In order to study the relationship between episodic gonadotrophin secretion and alterations of ovarian hormone secretion, we examined women with normal menstrual cycles (n = 26), luteal phase defects (n = 10) or disturbed follicular oestradiol secretion (n = 8) as established by daily (except weekends) determinations of oestradiol and progesterone. Pulsatile gonadotrophin secretion was studied during the luteal phase or the second half of the menstrual cycle sampling at 15 min intervals for 12 h. LH and FSH mean concentrations and LH pulse frequency were significantly (P less than 0.01) increased in the group with disturbed follicular development in the presence of decreased oestradiol (E2) and progesterone (P4) levels. In women with luteal phase defects mean LH and FSH concentrations and pulsatile LH secretion showed a nonsignificant trend to lower values in the presence of significantly decreased P4 concentrations during the luteal phase.