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Leptin, the metabolic fat hormone, has been shown to have effects on reproduction in mice and to modulate steroid production by cultured ovarian somatic cells in a number of species. However, a direct role of leptin on normal ovarian function in vivo has not been shown. In this paper the effect of passive immunisation against leptin (experiment 1; 20 ml antiserum or non-immune plasma i.v.; n = 6/treatment) and direct ovarian infusion of leptin (experiment 2; 0, 2 or 20 mug recombinant ovine leptin; n = 4/treatment) during the early follicular phase was investigated in sheep with ovarian autotransplants, which allow recovery of ovarian venous blood and regular non-invasive scanning of the ovary. Passive immunisation against leptin resulted in an acute increase (P < 0.05) in ovarian oestradiol secretion but had no effect on gonadotrophin concentrations, ovulation or subsequent luteal function. Conversely, direct ovarian arterial infusion of the low dose of leptin resulted in an acute decline (P < 0.05) in ovarian oestradiol secretion whereas the high dose, which resulted in supra-physiological leptin concentrations, had no effect on oestradiol production compared with the controls. Neither dose of leptin had any effect on gonadotrophin concentrations or ovulation but both doses resulted in an increase (P < 0.05) in progesterone concentrations over the subsequent luteal phase. In conclusion, together these data provide strong in vivo evidence that leptin can modulate ovarian steroidogenesis directly and acutely in ruminants and suggest that leptin is an alternate regulatory system whereby nutritional status can regulate reproductive activity.

The bone morphogenetic proteins (BMPs) have been implicated in the paracrine regulation of ovarian follicular development. In this study, we investigated the expression of the BMP receptors (BMPRs) in sheep ovaries by immunohistochemistry and the effect of BMP2, a natural ligand for these receptors, on granulosa cells cultured in vitro. Ovaries from cyclic ewes were fixed, embedded in paraffin wax and cut into sections. The sections were rehydrated, submitted to microwave antigen retrieval and treated with polyclonal antibodies against BMPR1A, BMPR1B and BMPR2. Strong immunostaining for all three receptors was observed in the granulosa cell layer of follicles from the primary to late antral stages of development. Staining was also present in the oocyte, corpus luteum, ovarian surface epithelium and, to a lesser extent, the theca layer of antral follicles. For functional studies, granulosa cells were obtained from immature follicles 1-3 mm in diameter. The cells were cultured for 6 days in serum-free medium containing 1 ng oFSH-20 ml(-1) in the presence of 0, 3, 10 or 30 ng ml(-1) human recombinant BMP2. The medium was replaced every 2 days and oestradiol and inhibin A concentrations were measured in the spent medium. In the absence of BMP2, oestradiol and inhibin A production increased as the granulosa cells differentiated in vitro. The addition of the highest dose of BMP2 enhanced oestradiol production (P < 0.05) without affecting the proliferation of the cells. It is concluded that BMP receptors are present in sheep ovaries and that BMPs may have a role in the differentiation of granulosa cells by enhancing the action of FSH.

The human endometrium displays characteristic features, both structural and functional, across the menstrual cycle. It is the sex steroid hormones, oestrogen and progesterone, that drive the endometrium through the different phases of the cycle. Oestrogen and progesterone act sequentially to regulate cellular concentrations of their respective receptors, this interaction initiates gene transcription. Thereafter a cascade of local events prepares the endometrium for implantation, but in the absence of pregnancy, progesterone withdrawal leads to menstruation and cyclic repair. Withdrawal of progesterone from an oestrogen-progesterone primed endometrium is the initiating event for the cascade of molecular and cellular interactions that result in menstruation. Progesterone withdrawal first affects cells with progesterone receptors. Early events in the menstrual process are vasoconstriction and cytokine up-regulation. The activation of lytic mechanisms is a later event and involves cells that may lack progesterone receptors, for example, uterine leucocytes and epithelial cells. Hence progesterone withdrawal results in a local increase of inflammatory mediators and the enzymes responsible for tissue breakdown. The total complex of local factors implicated in normal menstrual and aberrant menstrual bleeding are yet to be fully defined.

Objective: To investigate the effects of a hormone replacement therapy regime of continuous oestrogen and interrupted progestogen, administered transdermally, on the endometrium of postmenopausal women, the pattern of bleeding and relief of menopausal symptoms., Design: Volunteer pilot study of up to six months duration involving weekly application of an oestrogen-only skin patch releasing 50 microg oestradiol per day interspersed with a combined oestrogen and progestogen patch releasing 50 microg oestradiol and 250 microg norethisterone acetate per day for three days. Transvaginal ultrasound measurements of endometrial thickness and endometrial biopsies were performed in the third month of treatment at the end of both an oestrogen-only phase of treatment and a combined oestrogen-progestogen phase., Setting: Specialist community menopause clinic, Dean Terrace Centre, Edinburgh., Participants: Fifteen healthy postmenopausal women., Main Outcome Measures: Effect of treatment on endometrial histology, the immunolocalisation of oestrogen and progesterone receptors and the cell proliferation marker Ki 67 after three months of treatment and the proportion of women without bleeding at six months., Results: Treatment provided relief of hot flushes and by the sixth month of study 10 of the 14 women who completed treatment had no vaginal bleeding (71%). No endometrial hyperplasia or atypical changes were observed in biopsies and ultrasound measurements of endometrial thickness demonstrated a thin endometrium. Reduced immunostaining for Ki 67 was observed in endometrium from the combined phase of treatment compared with the oestrogen-only phase, consistent with a progestogenic-antagonism of proliferation. Exposure to progestogen did not suppress steroid receptors as similar immunostaining was observed in both treatment phases., Conclusions: Continuous oestrogen and interrupted progestogen administered transdermally offers promise as a novel bleed-free hormone replacement therapy for postmenopausal women.

In this study, we investigated follicular dynamics and ovarian steroid secretion during the follicular and early luteal phases of the estrous cycle in sheep. Six Finn-Merino ewes with ovarian autotransplanted ovaries were monitored for 10 days during the follicular phase and subsequent early luteal phase after luteal regression was induced with cloprostenol (a potent analogue of prostaglandin F2alpha). Over this period, follicular diameter was measured by serial ultrasound scans, and the concentration of gonadotropins and steroids in ovarian venous blood was measured at intervals of 6-12 h. All animals had an LH surge (Day 0) 59 +/- 4.7 h after injection of cloprostenol. The ovulatory follicles were derived mainly from large antral follicles present at the time of injection of cloprostenol (5.1 +/- 0.4 mm; mean +/- SEM, n = 6), although in some animals recruitment of additional small follicles was observed after luteolysis. The concentration of FSH decreased during the follicular phase and peaked synchronously with the LH surge, while estradiol and androstenedione concentrations in ovarian venous plasma increased progressively from luteal regression to a maximum at the LH surge. The rise in concentration of FSH on Day 1 was followed by the growth of a new cohort of follicles. Follicular size and ovarian steroid secretion increased in a linear fashion from Day 1 to Day 3, with ovarian steroid secretion reaching a maximum when the first wave of luteal phase follicles achieved a diameter of 5 mm or more. On Day 4, steroid secretion began to decline without significant changes in follicular diameter, and a second wave of follicles emerged. We conclude that 1) the preovulatory follicles are usually derived from the large follicle population present at the time of luteal regression, but the sheep has the ability to promote smaller follicles if required; and 2) the second peak of FSH stimulates the development of large estrogenic follicles during the early luteal phase, but the period of functional dominance is shorter than the period of morphological dominance.

It has been demonstrated previously that administration of the antiprogestin mifepristone (RU 486; 1-5 mg daily) inhibits or delays both the pre-ovulatory luteinizing hormone (LH) surge and ovulation. To investigate this mechanism, dynamic tests of pituitary ovarian function were performed in six healthy women before and during the administration of mifepristone (2 mg daily for 30 days). On day 9 of the control and treatment cycles, samples of blood were collected every 15 min over 12 h for measurement of LH concentration. After 10 h, the responsiveness of the pituitary was tested by the i.v. injection of 10 micrograms of gonadotrophin-releasing hormone (GnRH). On day 10 of the control and treatment cycles, two patches releasing 200 micrograms/day of oestradiol were applied to skin on the abdomen for 3 days. Blood was collected at 24, 48, 59, 72, 81 and 96 h after application of the oestrogen patches for the measurement of gonadotrophin and ovarian hormone concentrations. Follicular development continued in all women during their treatment with mifepristone, and ovulation was suppressed (four women) or delayed (two women). There was no significant difference in the basal concentration of LH between the control and treatment cycles (mean +/- SE; 5.5 +/- 0.4 versus 7.7 +/- 0.4 IU/l respectively), or in the frequency (interpulse interval, 101 +/- 12 versus 105 +/- 13 min respectively) and the amplitude (2.1 +/- 0.4 versus 2.6 +/- 0.4 IU/l respectively) of LH pulses. The response to GnRH was similar. On day 10, the basal concentrations of LH, follicle-stimulating hormone (FSH), prolactin, oestradiol and progesterone and the diameter of the dominant follicle (15.7 +/- 1.8 versus 13.3 +/- 1.9 mm) were similar during control and treatment cycles. In control cycles, there were significant increases in the concentrations of LH and FSH within 72 h of application of the oestrogen patches. During treatment cycles, concentrations of FSH and LH remained low, and were significantly lower than the values observed during control cycles (P < 0.006). We conclude that the antiprogestin mifepristone disrupts ovulation by inhibiting the positive feedback effect of oestrogens and, hence, prevents or delays the generation of a pre-ovulatory LH surge.

The relationship between expression of inhibin mRNA and ovarian secretion of estradiol (E2) and immunoactive inhibin was investigated at midluteal phase and throughout the follicular phase of the sheep estrous cycle. At laparotomy, timed samples of ovarian blood were collected and ovaries were removed from 39 Scottish Blackface ewes (ovulation rate 1.3 +/- 0.1) on Day 10 of the luteal phase or 24, 48, 60, 72, or 84 h after injection of cloprostenol (PG; 100 micrograms) on Days 10-12. Ovaries were removed and fixed for in situ hybridization using 35S-labeled antisense riboprobes transcribed from inhibin alpha, beta A, and beta B cDNAs. LH, E2, and inhibin concentrations were determined by RIA. On the basis of peripheral LH levels and the presence of estrogen-active follicles (E-A; > or = 3 mm in diameter secreting > 1 ng/min E2) or recent ovulations, animals were grouped as follows: presurge (24 or 48 h post-PG; LH < 5 ng/ml; n = 7), midsurge (with E-A; LH > 5 ng/ml; n = 6), late surge (large follicle not E-A; LH > 5 ng/ml; n = 4), postsurge (large follicle not E-A; LH < 5 ng/ml; n = 7), and postovulation (n = 10). As expected, E2 secretion by the "active" ovary (containing preovulatory follicle) tended to increase with follicular development such that secretion was maximal at midsurge and then declined. E2 secretion by the "inactive" ovary was low at all stages. Immunoactive inhibin, in contrast, was secreted in substantial quantities by both ovaries, although secretion from active ovaries was higher at all stages (p < 0.05). Effects of stage on secretion were not significant, but immunoactive inhibin secretion from active ovaries was high in postsurge animals when E2 secretion was very low. Hybridization for inhibin mRNA was specific for granulosa cells of antral follicles. While most sheep in the luteal (4 of 5), presurge (2 of 3), and midsurge groups (5 of 5) had at least one inhibin-positive large follicle (expressing both alpha- and beta-subunit mRNA), none were present between the LH surge and ovulation (late and postsurge groups). Inhibin mRNA was undetectable in midcycle CL, but 4 of 10 recent ovulations hybridized weakly with the alpha probe and one very weakly with the beta A probe. The mean number of inhibin-positive large follicles per animal (in those having at least one) was 1.3 +/- 0.15 (n = 15 ewes).(ABSTRACT TRUNCATED AT 400 WORDS)

Objective: The objective of this study was to investigate serial changes in the plasma concentration of inhibin in both the very early days of pregnancy following implantation and in late pregnancy. The timing of the changes in inhibin concentration relative to changes in the concentrations of other hormones of pregnancy was also investigated., Design: Serial observations of the peripheral concentrations of inhibin and other hormones in two groups of healthy volunteers in (a) early pregnancy and (b) late pregnancy., Patients: (a) Four healthy women recruited on cessation of contraception prior to conception. (b) Nine healthy women recruited at the antenatal clinic., Measurements: In the early pregnancy subjects, the concentrations of inhibin, progesterone, oestradiol and hCG were measured in plasma samples obtained three times per week from day 8 to day 10 of each menstrual cycle until 11 weeks after the last menstrual period in the conception cycle. In the late pregnancy subjects, plasma samples were obtained at 4-week intervals from 12 weeks until term., Results: The concentration of inhibin, progesterone and oestradiol in conception cycles were similar to those in the preceding cycles until the mid/to late-luteal phase of the cycle when hCG was first measureable. By day 12 of the luteal phase the concentration of inhibin was significantly higher in the pregnancy cycle than in the non-pregnancy cycle (P < 0.05) and progressively increased after the time of the missed menstrual period. The concentration of inhibin reached a peak (513.0 U/l, CI 442.1-595.3) by day 47 when the concentration of hCG was maximal. In early pregnancy the concentration of inhibin was correlated with that of hCG (r = 0.361; P < 0.01) as well as progesterone (r = 0.584, P < 0.001) and oestradiol (r = 0.602, P < 0.001). After 12 weeks there was no significant correlation between hCG and inhibin although significant correlations persisted with progesterone (r = 0.553, P < 0.001) and oestradiol (r = 0.361, P < 0.01)., Conclusions: The corpus luteum makes a significant contribution to the production of inhibin in early pregnancy while after 12 weeks the placenta is the major source.

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 1-26 alpha peptide fragment of porcine inhibin and/or oestradiol-17 beta. 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 less than 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 less than 0.001) increase after ovariectomy which was not affected by treatment with progesterone alone. Treatment with inhibin or oestradiol alone caused a significant (P less than 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 less than 0.01) rise in the peripheral concentration of FSH, while immunization against the two hormones in combination resulted in a significantly (P less than 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.

In the sheep both FSH and LH are necessary for development of large antral follicles. The secretion of FSH is controlled by the negative feedback effect of two ovarian hormones, oestradiol and inhibin, acting at the level of the anterior pituitary. Both are derived from the granulosa cells of large antral follicles which are present in sheep ovaries throughout the oestrous cycle. FSH stimulates growth and mitosis and so the fully differentiated granulosa cells of the large preovulatory follicles acquire receptors for LH, have maximal aromatase activity and produce large amounts of inhibin. The number of these large antral follicles (oestrogenic) which have the potential for ovulation corresponds to the ovulation rate specific for each particular breed of sheep. Over 90% of the oestradiol secreted by the ovaries is derived from these chosen follicles. In contrast, inhibin (and androstenedione) is also secreted by large antral follicles which have lost or not yet acquired maximal aromatase activity. The secretion of oestradiol by the preovulatory follicle(s) is dependent on the supply of androgen precursors produced by the theca which is stimulated by LH. When the concentration of progesterone falls at the end of the luteal phase the increased secretion of LH stimulates the progressive increase in secretion of oestradiol which occurs during the follicular phase. At this stage of the cycle there is a modest inconsistent rise in inhibin, the secretion of which is not stimulated by LH but is related to the increase in the number of large antral follicles. It is suggested that inhibin with its long half-life sets the overall level of negative feedback while oestradiol is responsible for the day-to-day fluctuations in the concentration of FSH which determines the number of ovulatory follicles. This dual control of FSH is adapted to monitor both the total number of large antral follicles in the ovaries (inhibin) and the number which are selected for ovulation (oestradiol).

An experiment was conducted to examine the effect of steroid-free ovine follicular fluid (oFF) on ovarian hormone secretion. Eight Merino x 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 micrograms, 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 less than 0.01) with concentrations remaining increased (P less than 0.05) until 56 h after cloprostenol (32 h after the first oFF injection). Following oFF injection there was a profound (100%; P less than 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 less than 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 less than 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.(ABSTRACT TRUNCATED AT 400 WORDS)

An experiment was conducted in order to determine the pattern of, and the relationships between, the secretion of inhibin, estradiol, and androstenedione by the ovary and the concentration of LH, FSH, and PRL during the estrous cycle of sheep. The estrous cycles of 6 Finn-Merino ewes in which the left ovary had been autotransplanted to the neck were synchronized by two injections of cloprostenol (100 micrograms im) a potent analog of prostaglandin F2 alpha (PG) given 14 days apart. The ewes had ovarian and jugular venous blood samples taken at four hourly intervals from 42 h before the second PG injection until day 6 of the following cycle. All animals responded to PG with the preovulatory LH surge occurring within 58 +/- 2 h (mean +/- SEM). The concentration of FSH in jugular venous plasma fell (P less than 0.001) after the induction of luteolysis and then exhibited 3 peaks, the first coincident with the LH surge, the second on day 1, and the third on day 6. After injection of PG the secretion rates of inhibin, estradiol, and androstenedione increased (P less than 0.05) within 4-8 h. After this increase in the early follicular phase the secretion rate of estradiol continued to rise until the time of the LH surge (P less than 0.001). Although the secretion of androstenedione and inhibin increased in the 36 h before the LH surge the magnitude of this rise was less marked than for estradiol and was not statistically significant. Within 4-8 h of the start of the LH surge the secretion of estradiol and androstenedione declined rapidly reaching barely detectable levels within 16 h (P less than 0.001). In contrast the secretion of inhibin increased after the LH surge reaching a broad peak (P less than 0.05) of approximately 16-h duration, coincident with the second peak of FSH. From days 2-6 mean secretion of inhibin remained relatively stable at 2-6 ng/min although considerable variation was observed in individual profiles. The rate of estradiol secretion increased steadily from its nadir on day 1 to a broad peak centered around day 3 (3-6 ng/min, P less than 0.001) followed by a decline until by day 6 the estradiol secretion rate was less than 1 ng/min (P less than 0.01). The secretory profile for PRL showed a close relationship with estradiol secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

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 1-26 alpha peptide fragment of porcine inhibin, antiserum to oestradiol-17 beta, 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 less than 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 less than 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 less than 0.05) shorter in the group immunized against inhibin alone (17.0 +/- 0.5 h).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of sustained high plasma levels of prolactin, induced by repeated 2-h i.v. injections of thyrotrophin-releasing hormone (TRH; 20 micrograms), on ovarian oestradiol secretion and plasma levels of LH and FSH was investigated during the preovulatory period in the ewe. Plasma levels of progesterone declined at the same rate after prostaglandin-induced luteal regression in control and TRH-treated ewes. However, TRH treatment resulted in a significant increase in plasma levels of LH and FSH compared to controls from 12 h after luteal regression until 5 to 6 h before the start of the preovulatory surge of LH. In spite of this, and a similar increase in pulse frequency of LH in control and TRH-treated ewes, ovarian oestradiol secretion was significantly suppressed in TRH-treated ewes compared to that in control ewes. The preovulatory surge of LH and FSH, the second FSH peak and subsequent luteal function in terms of plasma levels of progesterone were not significantly different between control and TRH-treated ewes. These results show that TRH treatment, presumably by maintaining elevated plasma levels of prolactin, results in suppression of oestradiol secretion by a direct effect on the ovary in the ewe.

Plasma samples were obtained at 8 hourly intervals around the preovulatory surge of LH in three groups of women with spontaneously ovulatory menstrual cycles, in order to clarify the hormonal events around the time of ovulation. In 21 of 25 women in whom samples were collected every 8 hours the start of the LH surge occurred between midnight and 0800. In 16 of these women the concentration of LH, FSH and progesterone was measured every 8 hours around the pre-ovulatory surge of LH. A progressive increase in progesterone started with the onset of the LH surge, with a transient fall after 32-40 h at a time coincident with that of ovulation. In 10 women oestradiol, androstenedione and prolactin were measured 8 hourly around the pre-ovulatory surge of LH beginning at 0800 h. Prolactin showed a sustained increase in levels beginning at the start of and lasting for the duration of the pre-ovulatory LH surge; oestradiol levels did not rise around this time, and declined by 24 h after the onset of the LH surge. These results suggest that (1) the pre-ovulatory LH surge begins between midnight and 0800 h in the majority of women, (2) luteinization of the granulosa cells within the pre-ovulatory follicle occurs in response to the LH surge, (3) the increase in prolactin at the time of the LH surge is not directly related to increasing levels of oestradiol but may be due to a decrease in hypothalamic inhibition of prolactin secretion which occurs coincident with the release of LHRH associated with the preovulatory LH surge.

The concentrations of oestrone and oestradiol as measured by a double isotope derivative method in fluid from Graafian follicles at different phases of the menstrual cycle were compared to the corresponding concentrations in ovarian and peripheral venous plasma collected simultaneously. In follicular fluid the concentration of oestradiol was much greater than that of oestrone. The concentrations of oestrogens were similar in fluid collected from small follicles at all phases of the cycle. The concentration of oestradiol in large follicles (greater than 1 cm diameter) was much higher in the mid-late follicular phase of the cycle (mean = 5390 plus or minus 1330 nmol/l) than in large or small follicles at other phases of the cycle. In the mid-late follicular phase of the cycle large follicles were invariably associated with high concentrations of oestradiol in venous plasma draining the corresponding ovary (mean 49 plus or minus 12 nmol/l). The mean ratio of oestradiol/oestrone in fluid from large follicles in which the concentration of oestradiol was greater than 1400 nmol/l (18.0 plus or minus 1.0, n = 10) was significantly higher than the ratio in corresponding samples of ovarian venous plasma (10.7 plus or minus 1.4, n = 11). These findings suggest that when the concentration of oestradiol in a Graafian follicle exceeds 3500 nmol/l it is likely to be functionally active.

The secretion rates of oestradiol, androstenedione and progesterone and the peripheral plasma concentration of LH were measured in 12 ewes with ovarian autotransplants before and after luteal regression induced by a single intramuscular injection of a synthetic prostaglandin (PG) analogue, 16-aryloxyprostaglandin F 2alpha (I.C.I. 80996). Luteal regression was followed by a fourfold rise in the basal concentration of LH and increased secretion of oestradiol. In five out of six ewes there was a discharge of LH with the peak occurring 36--78 h after the injection of the PG analogue. The secretion of oestradiol declined from 3-68 +/- 1-08 to 0-33 +/- 0-6 (S.E.M.) ng/min in the 24 h following the LH peak (P less than 0-001). In the remaining six ewes in which progesterone was implanted subcutaneously 24 h after the injection of PG analogue, follicular development was suppressed as indicated by the low secretion of oestradiol and androstenedione. The basal concentration of LH fell to values similar to those observed during the luteal phase after the implant of progesterone. The secretion of androstenedione followed a similar pattern to that of oestradiol in those ewes which showed presumptive evidence of ovulation. These results suggest that progesterone reinforces the negative feedback effects of oestrogen in the ewe.

The concentrations of FSH, oestradiol and androstenedione in the follicular fluid of normal and cystic human follicles were measured at different stages of the menstrual cycle. In addition, the number of granulosa cells in the follicles was determined. In follicles in which FSH was detectable, the concentration of oestradiol was greater than that of androstenedione, irrespective of the stage of the cycle. In contrast, in those follicles in which FSH was undetectable and in all cystic follicles irrespective of the level of FSH, the concentration of androstenedione was greater than that of oestradiol. In follicles containing FSH there was a highly significant linear correlation between the number of granulosa cells and the concentration of follicular oestradiol (P less than 0.001). It is suggested that in human ovaries up to 90% of the oestradiol in follicular fluid may originate from the granulosa cells.

17 beta-estradiol and progesterone were administered to post-menopausal women to determine their effects in vivo on the capacity of human endometrium to synthesize prostaglandins (PGs) F2 alpha and E. Basal amounts of PGF2 alpha and PGE synthesized by endometrium exposed to 17 beta-estradiol and progesterone were significantly higher than the levels produced by endometrium exposed to 17 beta-estradiol alone (p less than 0.02 for both PGs). Levels found in the former endometrium were broadly comparable to levels in secretory endometrium and in the latter to amounts found in proliferative endometrium of spontaneous, ovulatory cycles.

Passive immunization against oestrone and oestradiol increased the rate of testicular growth (P less than 0.01) without a statistically significant change in the concentrations of LH and FSH at the time of measurement. Although the concentration of testosterone was higher in the group with the largest testes, the difference was not statistically significant. Within groups, the concentration of FSH was correlated with the oestrone antibody titre (r = 0.5), suggesting that oestrone may have a particular role to play in the feedback control of FSH release in the ram; in addition, the concentration of testosterone was correlated with that of LH (r = 0.65). The data indicate a role of oestrogens in the control of testicular growth and are compatible with earlier reports that the testes of Merino lambs grow more quickly when gonadal feedback is reduced.

Ewes with cervical ovarian autotransplants were studied after PGF-2alpha-induced luteal regression in November and February (mid- and late-breeding season) and June (anoestrum). Progesteron and oestradiol-17beta secretion rates and LH concentrations were determined in serial ovarian venous blood samples and the ewes were frequently tested for oestrus. All 4 ewes in November and 3 of the 4 ewes in February exhibited oestrus and endocrine changes indicative of ovulation. The remaining ewe in February and the three ewes in June failed to show elevated oestradiol-17beta secretion rates after luteal regression, indicating the absence of follicles in the final stages of maturation. The preovulatory rise in the oestradiol-17beta secretion rate, the LH surge and the display of oestrus all occurred earlier, with respect to the PGF-2alpha infusion, in November than in February, suggesting a greater stimulation of folliculogenesis, and therefore a greater availability of maturing follicles, in November than in February.

Immunoneutralization of LH releasing hormone (LHRH) by the i.v. administration of LHRH antiserum in the preovulatory period in the ewe resulted in a prompt abolition of the pulsatile secretion of LH without alteration of basal plasma levels of LH. This resulted in an immediate cessation of the pulsatile secretion of ovarian oestradiol, with no change in the secretion rate of androstenedione or plasma levels of FSH. The ovarian secretion rate of testosterone increased significantly. Twenty to 22h after the injection of LHRH antiserum, pulsatile secretion of LH and ovarian oestradiol and androgen secretion remained suppressed while plasma levels of FSH had increased significantly. The bolus injection of ovine LH (10 micrograms, i.v.) at this time caused a significant increase in oestradiol, androstenedione and testosterone, these responses being inversely related to the titre of LHRH antiserum in the plasma at this time. These results show that (1) the secretion of oestradiol from the preovulatory sheep follicle is entirely dependent on the pulsatile secretion of LH and (2) since the secretion of ovarian androgens was not inhibited, LH may exert a short-term control over the aromatase enzyme system controlling the conversion of androgen to oestrogen in the granulosa cell of the preovulatory sheep follicle.

The secretion of oestradiol and androstenedione was investigated in sheep by cannulating both ovarian veins in situ. The mean ovarian blood flow (ml/min +/- SE) was significantly higher on the side containing the corpus luteum (7.67 +/- 1.36) than on the contralateral side (4.93 +/- 0.85). The mean secretion (ng/min +/- SE) of oestradiol (1.09 +/- 0.36) and androstenedione (1.52 +/- 0.16) was higher on the side containing at least one large follicle (diameter greater than 4 mm) than on the contralateral side (0.19 +/- 0.05 and 0.62 +/- 0.21, respectively). The secretion of oestradiol was unchanged following enucleation of the corpus luteum but fell to very low levels (0.062 +/- 0.022 ng/min) after enucleation of follicles diameter greater than 4 mm. The secretion of androstenedione fell significantly after enucleation of both corpus luteum and largefollicles. These results suggest that during the luteal phase in the sheep oestradiol is secreted virtually exclusively by the largest non-atretic Graafian follicle. Androstenedione in contrast arises from both follicle, corpus luteum and possibly stroma.

The peripheral concentration of LH and the secretion rates of estradiol-17beta and androstenedione were measured every 10 min for 4 h during anestrus in six ewes with utero-ovarian autotransplants. The basal concentration of LH was 0.45 +/- 0.06 ng//ml (NIH LH S14; mean +/- SEM), with pulses of LH (6.0 +/- 0.3 ng/ml; N = 5) occurring with an average frequency of one per 5 h. The basal secretion rate of estradiol-17beta was 0.5--1.3 ng/min. Each pulse of LH was followed by a rise in the secretion of estradiol-17beta. Maximum secretion of estradiol-17beta was 5.18 +/- 0.72 ng/min and was reached 25 min after LH discharge. Basal secretion of androstenedione was higher than estradiol-17beta at about 1.5 to 3.4 ng/min, and increased to a maximum 26.8 +/- 6.6 ng/min 25 min after LH discharge. Steroid secretion was not maintained at maximum rates and had returned to basal levels 2 h after LH discharge. The data show that the ovary of the anestrous sheep is capable of secreting steroids following pulses of endogenous LH. These results strongly suggest that as in the luteal phase of the cycle, the quantity of estradiol and androstenedione secreted by the ovary is related to the frequency of episodic pulses of LH.

Endometrium collected from 18 normal women at various stages of the menstrual cycle was maintained in organ culture for periods of 6 days. Both prostaglandin F2 alpha (PGF2 alpha) and PGE were detected in medium from late proliferative (days 10-14; n = 13) and late secretory (days 20-26; n = 5) endometria cultured in the absence of exogenous steroids. During the first 48 h in culture, the output of PGF2 alpha by secretory endometrium was significantly greater than that of proliferative tissue. The addition of 17 beta-estradiol to the culture medium at doses ranging from 1.0-100.0 ng/ml induced a significant increase in the output of PGF2 alpha by secretory but not proliferative endometria. The addition of progesterone at doses ranging from 5.0-500.0 ng/ml significantly reduced the output of PGF2 alpha and PGE by both proliferative and secretory endometria and prevented the 17 beta-estradiol-stimulated increase in PGF2 alpha output by secretory tissue.

Blood was collected every 8 h in five women from Day 10 of the menstrual cycle for 5-7 d for the measurement of LH, oestradiol and progesterone. In all women the LH surge started between 2400 and 0800 h before there was any significant decline in the concentration of oestradiol. In order to investigate the pulsatile secretion of LH and estradiol during the mid-cycle surge, blood samples were collected every 5 min for up to 5 h. Immediately before and during the LH surge there were numerous episodic pulses of LH with an interpulse interval (44 +/- 7 min) very similar to that observed in two women with hypergonadotrophic hypogonadism (48 min). The concentration of oestradiol fluctuated in a similar manner although it was not always possible to relate each pulse of oestradiol to a corresponding pulse of LH. The mid-cycle surge of LH is characterized by frequent pulses of high amplitude. These results are compatible with the view that the positive feedback effect of oestradiol is due not only to enhanced sensitivity of the anterior pituitary to LHRH but also to the high frequency of LHRH pulses released from the hypothalamus.

Pituitary-ovarian relationships were studied in seven lactating women by measuring the basal plasma concentrations of pituitary and ovarian hormones and their responses to an estrogen provocation test at 7, 30, and 100 days after delivery. The results were compared to a similar group of seven women who did not breast feed. The first ovulation occurred in five of the nonlactating women beteen 43-87 days after delivery, as judged by the urinary excretion of total estrogen and pregnanediol. In all lactating women, ovarian cyclicity was suppressed for at least 150 days after delivery or until weaning. The basal concentration of PRL in lactating women was significantly higher than in the nonlactating women at all three times measured. At 30 and 100 days, the concentration of estradiol was significantly lower in the lactating women, although the basal concentrations of FSH were similar in the two groups. After an injection of 1 mg estradiol benzoate, the concentrations of FSH and LH in plasma were suppressed to a greater extent in lactating than in nonlactating women. In addition, fewer of the lactating group (one of seven and none of seven at 30 and 100 days, respectively) than the nonlactating group (two of seven and five of seven) subsequently showed a rise in the concentration of LH 58--96 h after the estrogen injection (positive feedback). These results suggest that during lactation the hypothalamic-pituitary system is more sensitive to the negative feedback and relatively insensitive to the positive feedback of estrogen.

OBJECTIVE Polycystic ovary syndrome (PCOS) is characterized by the presence of numerous small antral follicles arrested at a diameter of less than 10 mm. As development of large antral follicle(s) can be stimulated by the administration of FSH, it has been suggested that the arrest of follicle development is due to a relative lack of or resistance to FSH. We have measured the concentration of FSH and LH and the ovarian hormones oestradiol, inhibin A and B, in relation to the dynamics of follicular development in women with PCOS under basal conditions and during treatment with FSH. DESIGN The concentrations of FSH, LH, oestradiol, inhibin A and B were measured in women with PCOS who were untreated (n = 9) or following progesterone-induced menses (10 cycles in six women) and control women on day 3 of a normal cycle (n = 10). Serial measurements of the same hormones were made during induction of ovulation with low dose of exogenous FSH in the women with PCOS who had been treated with progesterone. The dynamics of follicle development were measured by serial pelvic ultrasound examinations during treatment with FSH in PCOS and in the follicular phase of control cycles. PATIENTS Nine anovulatory untreated women with PCOS were compared with 10 normal women and six women with PCOS undergoing FSH treatment. RESULTS The concentrations of inhibin B and LH were higher in both groups of women with PCOS than in normal control women on day 3. In untreated women with PCOS the concentration of inhibin A was also significantly elevated compared to control women (P < 0.01) but not in women pretreated with progesterone. Basal concentrations of oestradiol were also significantly higher in women with PCOS than in the early follicular phase of the normal cycle. There were no significant differences in the concentration of FSH between the groups. Treatment of women with PCOS with low doses of FSH stimulated the development of a single dominant follicle which had an identical rate of... [ABSTRACT FROM AUTHOR]