Luteinizing hormone-beta mRNA levels are regulated primarily by gonadotropin-releasing hormone and not by negative estrogen feedback on the pituitary

Long-term effects of gonadotropin-releasing hormone (GnRH) and/or estrogen on pituitary mRNA levels for the beta-subunit of luteinizing hormone (LH-beta) were determined in anterior pituitary glands from ovariectomized (OVX) ewes. The relative roles of these two factors were assessed by studying hypothalamopituitary disconnected (HPD) ewes with appropriate hormonal treatments. Levels of LH-beta mRNA were increased by ovariectomy and substantially reduced by HPD. Treatment of OVX-HPD ewes with pulses of GnRH (250 ng each 2 h) for 1 week restored LH-beta mRNA levels to OVX levels, whereas treatment with estrogen alone did not alter the low levels found in OVX-HPD ewes. Combined GnRH and estrogen treatment for one week produced LH-beta mRNA levels that were similar to those found in OVX-HPD ewes given GnRH alone; plasma LH pulse amplitudes were also similar in these two groups. From these data we conclude that the long-term negative feedback effect of estrogen to reduce LH secretion is due to a primary inhibition of GnRH secretion and is not a pituitary effect of estrogen. Long-term regulation of LH-beta mRNA is thus primarily regulated by GnRH.     

Long-term negative feedback effects of oestrogen and progesterone on the pituitary gland of the long-term ovariectomized ewe

The effects of long-term treatment with physiological doses of oestradiol or oestradiol plus progesterone on plasma gonadotrophin levels and pituitary content of LH and gonadotrophin-releasing hormone (GnRH) receptors were studied in ovariectomized-hypothalamo-pituitary disconnected ewes given 250 ng pulses of GnRH every 2 h (i.v.). A pilot experiment showed that 3 cm long Silastic implants (s.c.) reduced both LH pulse frequency and pulse amplitude in long-term (greater than 6 months) ovariectomized ewes. The main experiment was conducted over 3 weeks in ovariectomized-hypothalamo-pituitary disconnected ewes that had received pulsatile GnRH replacement for 1 week after pituitary surgery. Group 1 (n = 5) received GnRH pulses alone throughout the study. Group 2 (n = 6) received oestradiol in week 2 and oestradiol plus progesterone in week 3 and in group 3 (n = 6) the steroid treatments were reversed. Oestradiol reduced (P less than 0.05) the mean (+/- S.E.M.) amplitude of LH in pulses in group 2 (from 8.2 +/- 1.6 to 5.0 +/- 0.5 micrograms/l) and group 3 (from 11.6 +/- 1.2 to 9.3 +/- 1.0 micrograms 1): an additional effect of progesterone was seen in group 2 but not group 3. The amplitudes of the LH pulses did not change in the control ewes. Plasma concentrations of FSH were reduced by approximately 50% by the oestradiol treatments with no additional effects of progesterone. There was no effect of steroidal treatment on pituitary content of LH or pituitary levels of GnRH receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects on plasma luteinizing hormone and follicle-stimulating hormone of varying the frequency and amplitude of gonadotropin-releasing hormone pulses in ovariectomized ewes with hypothalamo-pituitary disconnection

The effects on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion of various regimens of pulsatile gonadotropin-releasing hormone (GnRH) replacement were examined in ovariectomized (OVX) ewes after hypothalamo-pituitary disconnection (HPD). Hourly pulses of 500 ng GnRH restored gonadotropin secretion in OVX-HPD sheep. Replacement beginning 2 days after HPD gave consistent responses of LH and FSH within a week. Replacement beginning 61-96 days after HPD caused more gradual re-establishment of LH and FSH secretion with LH responses appearing immediately and FSH responses appearing 2 weeks later. When hourly GnRH pulses were increased in amplitudes from 250 to 500 ng the plasma LH baseline, peak values and pulse amplitudes were increased. There was no significant change in plasma FSH levels over 10 pulses at the higher dose. Decreases in GnRH pulse frequency led to increases in LH pulse amplitude and decreases in plasma LH baseline. In contrast, immediately after a change from a 2-hourly to an hourly mode, an increase in LH baseline occurred without an immediate reduction in LH pulse amplitude. Mean plasma FSH concentrations increased when the frequency was reduced from hourly to 2-hourly or 4-hourly. However, a change from 4-hourly to hourly pulses did not reduce FSH values within 7 days. It is concluded that changes in the pattern of LH secretion observed during the ovine estrous cycle could be accounted for, in part, by changes in GnRH pulse frequency.     

Morphine decreases LH secretion in ovariectomized ewes only after steroid priming and not by direct pituitary action

To determine whether opiates directly modulate pituitary LH secretion in vivo, morphine was administered to hypothalamo-pituitary-disconnected (HPD) ewes which were receiving exogenous pulses of GnRH. To define the steroidal background which is permissive to a morphine-induced decrease in LH secretion, ovariectomized (OVX) ewes were treated as follows in groups of four: group 1, no implant; group 2, small 17 beta-estradiol (E2) (1 cm long x 0.33 diameter) and progesterone (P) implants; group 3, medium E2 (1 cm long x 0.46 diameter) and P implants, and group 4, medium E2 implants. Jugular blood samples were taken at 10-min intervals for 9 h, during which there was a 3-hour pretreatment period, a 3-hour treatment period when the sheep were given six intravenous injections of 10 mg morphine every 30 min, and a 3-hour run-off period. Morphine inhibited the mean plasma concentrations of LH and LH pulse frequency in group 3 only, and in 2/4 ewes in this group LH secretion was abolished and did not return to a pulsatile mode during the 3-hour run-off sampling period. In a second experiment designed to test the pituitary action of morphine, OVX-HPD ewes were primed with medium E2 and P implants and were given hourly pulses of 250 ng GnRH intravenously. Jugular blood samples were taken around each GnRH pulse over an 8-hour period. The first three pulses served as a control sampling period, after which the sheep were treated with morphine (six intravenous injections of 10 mg morphine every 30 min).(ABSTRACT TRUNCATED AT 250 WORDS)     

Progesterone blocks the estradiol-induced gonadotropin discharge in the ewe by inhibiting the surge of gonadotropin-releasing hormone.

Previous studies indicate an elevation of circulating progesterone blocks the positive feedback effect of a rise in circulating estradiol. This explains the absence of gonadotropin surges in the luteal phase of the menstrual or estrous cycle despite occasional rises in circulating estradiol to a concentration sufficient for surge induction. Recent studies demonstrate estradiol initiates the LH surge in sheep by inducing a large surge of GnRH secretion, measurable in the hypophyseal portal vasculature. We tested the hypothesis that progesterone blocks the estradiol-induced surge of LH and FSH in sheep by preventing this GnRH surge. Adult Suffolk ewes were ovariectomized, treated with Silastic implants to produce and maintain midluteal phase concentrations of circulating estradiol and progesterone, and an apparatus was surgically installed for sampling of pituitary portal blood. One week later the ewes were allocated to two groups: a surge-induction group (n = 5) in which the progesterone implants were removed to simulate luteolysis, and a surge-block group (n = 5) subjected to a sham implant removal such that the elevation in progesterone was maintained. Sixteen hours after progesterone-implant removal (or sham removal), all animals were treated with additional estradiol implants to produce a rise in circulating estradiol as seen in the follicular phase of the estrous cycle. Hourly samples of pituitary portal and jugular blood were obtained for 24 h, spanning the time of the expected hormone surges, after which an iv bolus of GnRH was injected to test for pituitary responsiveness to the releasing hormone. All animals in the surge-induction group exhibited vigorous surges of GnRH, LH, and FSH, but failed to show a rise in gonadotropin secretion in response to the GnRH challenge given within hours of termination of the gonadotropin surges. The surges of GnRH, LH, and FSH were blocked in all animals in which elevated levels of progesterone were maintained. These animals in the surge-block group, however, did secrete LH in response to the GnRH challenge. We conclude progesterone blocks the estradiol-induced gonadotropin discharge in the ewe by acting centrally to inhibit the surge of GnRH secreted into the hypophyseal portal vasculature.     

Effect of level of food intake of ewes on the secretion of LH and FSH and on the pituitary response to gonadotrophin-releasing hormone in ovariectomized ewes

The effect of level of food intake on LH and FSH profiles and pituitary sensitivity to gonadotrophin-releasing hormone (GnRH) was investigated in two groups of 12 ovariectomized ewes. Ewes with a high intake (group H) had a mean daily intake (+/- S.E.M.) of 1.99 +/- 0.075 kg dry matter (DM)/head per day while ewes with a moderate intake (group M) consumed a mean of 1.02 +/- 0.021 kg DM/head per day. Ovaries were surgically removed from six ewes of each group on day 11 of the luteal phase and from the remainder 30 h after an injection of 100 micrograms prostaglandin analogue given on day 11 to induce luteolysis. During both the luteal phase and the follicular phase, mean LH and FSH concentrations and LH pulse frequencies and amplitudes were unaffected by the level of intake but mean plasma prolactin concentrations were higher (P less than 0.05) in group H than in group M ewes in the follicular phase. Mean LH and FSH concentrations at day 2 after ovariectomy were unaffected by treatment while mean prolactin concentrations were higher (P less than 0.05) in group H than in group M ewes. At day 7 after ovariectomy, mean LH and FSH concentrations were lower (P less than 0.05) in group H than in group M ewes although mean LH pulse frequencies and pulse amplitudes were not significantly affected by the level of intake at either time.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circulating half-lives of follicle-stimulating hormone and luteinizing hormone in pituitary extracts and isoform fractions of ovariectomized and intact ewes

Previous studies have shown that the circulating half-life (t 1/2) of serum FSH in ewes after hypophysectomy (HPX) increased 10-fold after ovariectomy (OVEX). The basis for this difference was examined in this study by determining the circulating half-life of serum FSH and LH in HPX ewes after administration of pituitary extracts and gonadotropin isoform fractions. High-speed supernatants of pituitaries from gonadal-intact and OVEX ewes were fractionated by electrofocusing in sucrose gradients and based on the pI distribution of FSH and LH divided into four pools, pH 4.3-4.8, 4.8-5.55, 5.8-6.7, and 6.7-10. These extracts were administered by iv bolus injection to HPX gonadal-intact ewes and blood samples collected between 15-1000 min later. The clearance pattern for both serum FSH and LH was heterogenous, indicative of a major rapid and a minor slow dissociating component. A significant (P less than 0.05) difference in circulating half-lives (rapid component) was observed between pituitary extracts from intact and OVEX ewes for FSH (t 1/2 = 32.8 +/- 8.6 min vs. 89.9 +/- 32.3 min) but not LH (31.3 +/- 9.2 min vs. 39.3 +/- 6.1 min, respectively), whereas no significant difference was observed between the corresponding FSH or LH isoform preparations. To establish if the difference in circulating half-lives obtained after HPX and bolus iv injection was due to mode of delivery, an extract of pituitaries from OVEX ewes was infused for 12 h into HPX sheep and the t 1/2 values determined after cessation of treatment and compared to those after a bolus injection. The clearance of both FSH and LH from plasma after infusion was significantly prolonged than after a bolus injection. It is concluded that the difference in circulating half-lives of FSH between pituitary extracts from intact and OVEX ewes after bolus administration is due to a difference in pituitary FSH composition. However, the prolonged clearance with infusion compared to bolus administration suggests that extrapituitary factors are also responsible.     

Immunocytochemical localization of beta endorphin and gonadal steroid regulation of proopiomelanocortin messenger ribonucleic acid in the ewe.

In the ewe, estradiol and progesterone inhibit luteinizing hormone (LH) secretion during the breeding season. Endogenous opioid peptides (EOP) are also inhibitory to LH secretion, and both estrogen and progesterone have been reported to enhance EOP inhibition of LH release. Which EOP are involved in this inhibition is unclear. In this study, we concentrated on beta-endorphin because evidence for its ability to inhibit LH secretion exists in ewes. We first studied the distribution of beta-endorphin-immunoreactive neurons in 4 cycling ewes using immunocytochemistry. Cell bodies were found only within the medial basal hypothalamus (MBH) and were concentrated in arcuate nucleus and mammillary recess of the third ventricle, with a few in the median eminence. Extensive fiber tracts were seen in preoptic area (POA) and median eminence. We next tested the hypothesis that gonadal steroids increase the synthesis of EOP by measuring levels of mRNA for proopiomelanocortin (POMC), the precursor to beta-endorphin. Ovariectomized ewes were treated with no steroids (n = 7) or given subcutaneous Silastic implants containing either estradiol (n = 6) or progesterone (n = 6). After 4 days of treatment, EOP inhibition of LH secretion was measured by determining the LH response to WIN 44,441-3 (WIN), an EOP antagonist. LH pulse frequency and pulse amplitude were determined in blood samples collected at 12-min intervals for 3 h before and after intravenous administration of 12.5 mg WIN. WIN injection increased (p < 0.01) the LH pulse-frequency only in progesterone-treated and pulse amplitude only in estradiol-treated ewes. After blood sampling, the ewes were killed, and POA, MBH, and pituitary gland were removed. Total RNA was extracted from these tissues and dot blotted onto nitrocellulose membranes for hybridization with a DNA probe complementary to the POMC mRNA. The resulting autoradiographs were quantified densitometrically. Levels of POMC mRNA in the MBH were increased (p < 0.01) by both estradiol and progesterone as compared with the no steroid group. There was no detectable POMC mRNA in the POA. These results suggest that estrogen and progesterone enhance EOP inhibition of LH secretion by increasing POMC mRNA levels and thus synthesis of beta-endorphin.     

Increased gonadotropin-releasing hormone pulse frequency associated with estrogen-induced luteinizing hormone surges in ovariectomized ewes

Hypophyseal portal blood samples were taken from ovariectomized (OVX) ewes given 50 micrograms estradiol benzoate. This estrogen treatment elicited a biphasic alteration (decrease then increase) in LH secretion. During the negative feedback phase, pulsatile GnRH secretion continued; at this time the interpulse interval for the GnRH pulses (49.5 +/- 5.7 min, mean +/- SE, n = 6) was similar to that in 7 control OVX ewes (53.4 +/- 8.7 min). During the positive feedback phase the GnRH interpulse interval (26.8 +/- 9.8 min; n = 6) was significantly (P less than 0.05) less than in the controls. In 3/7 cases the GnRH pulse frequency in OVX controls was within the range observed for estrogen-treated sheep during the positive feedback phase. These data suggest that, in most cases, the LH surge that can be induced by estrogen in OVX ewes, is associated with an increased GnRH pulse frequency. In some animals the inherent GnRH pulse frequency may already be at a rate that is high enough to permit an LH surge by action of estrogen on the pituitary. In general, the mean concentrations of GnRH in portal blood during the LH surge were higher than those in untreated animals, suggesting an overall increase in GnRH output during the LH surge. Pulsatile GnRH secretion continues throughout the early negative feedback phase, suggesting that the predominant effect of estrogen at this time is at the pituitary level.     

Suppression of the secretion of luteinizing hormone due to isolation/restraint stress in gonadectomised rams and ewes is influenced by sex steroids

In this study we used an isolation/restraint stress to test the hypothesis that stress will affect the secretion of LH differently in gonadectomised rams and ewes treated with different combinations of sex steroids. Romney Marsh sheep were gonadectomised two weeks prior to these experiments. In the first experiment male and female sheep were treated with vehicle or different sex steroids for 7 days prior to the application of the isolation/restraint stress. Male sheep received either i.m. oil (control rams) or 6 mg testosterone propionate injections every 12 h. Female sheep were given empty s.c. implants (control ewes), or 2x1 cm s.c. implants containing oestradiol, or an intravaginal controlled internal drug release device containing 0.3 g progesterone, or the combination of oestradiol and progesterone. There were four animals in each group. On the day of application of the isolation/restraint stress, blood samples were collected every 10 min for 16 h for the subsequent measurement of plasma LH and cortisol concentrations. After 8 h the stress was applied for 4 h. Two weeks later, blood samples were collected for a further 16 h from the control rams and ewes, but on this day no stress was imposed. In the second experiment, separate control gonadectomised rams and ewes (n=4/group) were studied for 7 h on 3 consecutive days, when separate treatments were applied. On day 1, the animals received no treatment; on day 2, isolation/restraint stress was applied after 3 h; and on day 3, an i. v. injection of 2 microg/kg ACTH1-24 was given after 3 h. On each day, blood samples were collected every 10 min and the LH response to the i.v. injection of 500 ng GnRH administered after 5 h of sampling was measured. In Experiment 1, the secretion of LH was suppressed during isolation/restraint in all groups but the parameters of LH secretion (LH pulse frequency and amplitude) that were affected varied between groups. In control rams, LH pulse amplitude, and not frequency, was decreased during isolation/restraint whereas in rams treated with testosterone propionate the stressor reduced pulse frequency and not amplitude. In control ewes, isolation/restraint decreased LH pulse frequency but not amplitude. Isolation/restraint reduced both LH pulse frequency and amplitude in ewes treated with oestradiol, LH pulse frequency in ewes treated with progesterone and only LH pulse amplitude in ewes treated with both oestradiol and progesterone. There was no change in LH secretion during the day of no stress. Plasma concentrations of cortisol were higher during isolation/restraint than on the day of no stress. On the day of isolation/restraint maximal concentrations of cortisol were observed during the application of the stressor but there were no differences between groups in the magnitude of this response. In Experiment 2, isolation/restraint reduced the LH response to GnRH in rams but not ewes and ACTH reduced the LH response to GnRH both in rams and ewes. Our results show that the mechanism(s) by which isolation/restraint stress suppresses LH secretion in sheep is influenced by sex steroids. The predominance of particular sex steroids in the circulation may affect the extent to which stress inhibits the secretion of GnRH from the hypothalamus and/or the responsiveness of the pituitary gland to the actions of GnRH. There are also differences between the sexes in the effects of stress on LH secretion that are independent of the sex steroids.     

Suppression of LH response to gonadotrophin-releasing hormone or oestradiol by ACTH(1-24) treatment in anoestrous ewes

Stress is known to result in lowered female reproductive efficiency. The objective of this study was to examine how increased pituitary-adrenal activity may influence gonadotrophin release in anoestrous ewes. Various doses (0.06-1.0 mg) of a synthetic adrenocorticotrophic hormone (ACTH(1-24)) preparation were injected into ewes 30 min or 3 h before an i.v. injection of 500 ng gonadotrophin-releasing hormone (GnRH). The LH response to GnRH given 30 min after ACTH(1-24) was similar to that after GnRH alone, whereas the response 3 h after ACTH(1-24) was significantly lower, irrespective of the dose of ACTH(1-24). At 30 min and 3 h after ACTH(1-24) the concentrations of cortisol exceeded 50 nmol/l compared with baseline values of less than 10 nmol/l. The effect of ACTH(1-24) on oestradiol-induced LH release was also examined. Those ewes receiving 0.8 mg ACTH(1-24) depot and 50 micrograms oestradiol benzoate simultaneously had a preovulatory-type increase in LH 14-20 h later, similar to when oestradiol benzoate was given alone. None of the ewes receiving an additional 0.8 mg ACTH(1-24) depot 10 h after oestradiol benzoate had increases in LH concentration. The cortisol concentrations in all ewes receiving either one or two injections of ACTH(1-24) were greater than 35 nmol/l at 10 h after the oestradiol injection. However, concentrations of progesterone increased from 0.9 +/- 0.3 (S.E.M.) nmol/l at the time of the second ACTH(1-24) injection to 2.1 +/- 0.3 nmol/l after 2 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

LH, FSH and progesterone level in the blood plasma of women with normal menstrual cycle]

Luteinizing hormone (LH), folliculostimulating hormone (FSH) and progesterone were studied by the radioimmunoassay in a group of 10 women throughout a normal menstrual cycle. The LH and the FSH peak occurs at the middle of the cycle. At the early follicular phase the FSH level was higher than that of the LH. During the follicular and the lutein phases the plasma progesterone level was 0.65 +/- 0.12 ng/ml and 12.4 +/- 2.3 ng/ml, respectively. The laboratory criteria of the normal ovular cycle are: a) a mid-cycle LH peak; b) plasma progesterone level at the lutein phase were 10-15 times greater than at the follicular one; c) progesterone peak at the mid-lutein phase; d) the duration of the lutein phase of 12-15 days.     

Effects of tamoxifen on concentrations of luteinizing hormone and follicle-stimulating hormone in the plasma of ovariectomized ewes

The effects of tamoxifen on peripheral plasma concentrations of gonadotrophins were studied in ovariectomized ewes. First, ovariectomized ewes were injected (i.m.) with 10 mg tamoxifen citrate/day for 4 days which caused a significant reduction in plasma LH concentrations within 4 days and plasma FSH concentrations within 1 day of the commencement of treatment. Further groups of ovariectomized ewes were then injected (i.m.) with two injections of 10 mg tamoxifen citrate 6 h apart or 20 micrograms oestradiol benzoate (OB) or tamoxifen citrate plus OB or oil. Tamoxifen treatment caused a reduction in plasma LH and FSH concentrations within 6 h. In four of our ewes receiving OB, a surge in LH secretion was observed; a similar response was observed in two out of four ewes given the combination of tamoxifen citrate and OB. No LH surge was seen in ovariectomized ewes given tamoxifen alone. These results show that tamoxifen reduces plasma gonadotrophin levels in ovariectomized ewes suggesting it is an oestrogen agonist in the sheep pituitary gland. A partial oestrogen antagonist action of tamoxifen is similarly suggested by its ability to block the oestrogen-induced LH surge in some ovariectomized ewes. Since tamoxifen consistently lowers plasma gonadotrophin levels in ovariectomized ewes this could result from action via oestrogen receptors or by central nervous system, non-oestrogen receptor-mediated effects.     

Hypothalamic regulation of cyclic ovulation: evidence that the increase in gonadotropin-releasing hormone pulse frequency during the follicular phase reflects the gradual loss of the restraining effects of progesterone

The luteal-follicular transition is characterized by decreasing plasma levels of E(2), progesterone (P), and inhibin A, with concomitant increases in FSH and LH levels. LH (and by inference GnRH) pulse frequency increases from 1 pulse every 3-4 h during the luteal phase to approximately 1 pulse/h at the midcycle LH surge. To examine the regulation of GnRH pulse frequency, we gave 10 normally cycling women transdermal E(2) and oral P to produce midluteal levels [364 +/- 65.0 pmol/liter (99 +/- 18 pg/ml) and 29.7 +/- 6.8 nmol/liter (9.3 +/- 2.1 ng/ml), respectively] for 10 d after the LH surge (d 0). P was then discontinued, and E(2) was given alone for 3 additional wk. Pulsatile LH secretion and follicular size were assessed on d 10, 17, 24, and 31. Results are presented as the mean +/- SEM. LH pulse frequency was 3.1 +/- 0.5 pulses/12 h after 10 d of E(2) and P, and remained low on d 17 when P had fallen below 1.6 nmol/liter (<0.5 ng/ml). In the continued presence of midluteal levels of E(2) [ approximately 360 pmol/liter (100 pg/ml)], LH pulse frequency increased on d 24 and 31 to 5.5 +/- 0.9 and 5.8 +/- 0.5 pulses/12 h, respectively, whereas pulse amplitude remained unchanged. FSH increased 2-fold, but follicular size did not change. These results are consistent with E(2) potentiating the effects of low concentrations of P on the GnRH pulse generator for at least 7 d, after which pulse frequency increases despite maintenance of E(2) levels. This supports the hypothesis that the increasing GnRH pulse frequency throughout the follicular phase reflects the gradual loss of the inhibitory actions of low concentrations of P.     

Effects of bovine follicular fluid on gonadotrophin secretion in intact and chronically ovariectomized ewes before and after desensitization of pituitary gonadotrophs to gonadotrophin-releasing hormone

Intact and chronically ovariectomized ewes were treated for 4 days with charcoal-treated bovine follicular fluid (FF) or charcoal-treated bovine serum during the late-anoestrous period, and the effects on basal and gonadotrophin-releasing hormone (GnRH)-induced secretion of LH and FSH observed. Subsequently, ewes received s.c. implants containing a sustained-release formulation of a potent GnRH agonist D-Ser(But)6-Azgly10-LHRH (ICI 118630) to desensitize pituitary gonadotrophs to hypothalamic stimulation, and the effects of bovine FF and bovine serum were re-assessed 2 weeks later. Chronic exposure (for 2-3 weeks) to ICI 118630 significantly reduced basal levels of LH and FSH in both intact and ovariectomized ewes and completely abolished both spontaneous LH pulses as well as exogenous GnRH-induced acute increases in plasma LH and FSH levels. Treatment with bovine FF significantly reduced plasma FSH levels, but not LH levels, in both intact and ovariectomized ewes before and after chronic exposure to ICI 118630. In intact ewes before exposure to ICI 118630, treatment with bovine FF actually enhanced pulsatile LH secretion and raised mean plasma LH levels by 240% (P less than 0.05). No such stimulatory effect of bovine FF on LH secretion was observed in intact ewes exposed to ICI 118630 or in ovariectomized ewes before or after exposure to ICI 118630, suggesting that the effect probably involved an alteration in ovarian steroid feedback affecting hypothalamic GnRH output. Treatment with bovine FF did not significantly affect the magnitude of GnRH-induced surges of LH or of FSH observed in either intact or ovariectomized ewes before exposure to ICI 118630.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreased follicular phase gonadotropin secretion is associated with impaired estradiol and progesterone secretion during the follicular and luteal phases in normally menstruating women

We tested the hypothesis that disturbed follicular development and disturbed luteal progesterone (P4) secretion are associated with reduced gonadotropin secretion in the early follicular phase by measuring pulsatile LH and FSH secretion at that time in 53 normally menstruating women. Three groups of women were identified on the basis of serum sex steroid concentrations (measured daily throughout the cycle) and luteal phase length. Group A (n = 27) had normal ovarian hormone secretion with peak serum estradiol (E2) concentrations of 440 pmol/L or more, peak serum P4 concentrations of 19 nmol/L or more, and luteal phase length of 9 days or more. Group B (n = 16) had normal peak serum E2 values, but peak serum P4 values less than 19 nmol/L and/or luteal phase length less than 9 days. Group C (n = 10) had peak serum E2 values below 440 pmol/L. Risk factors for the disturbances found in groups B and C were exercise and/or intermittent dieting. Compared to group A, both groups B and C had reduced mean serum LH concentrations (3.1 +/- 1.5 vs. 2.3 +/- 1.4 and 2.0 +/- 1.0 IU/L; P less than 0.05) and reduced LH pulse frequencies (5.2 +/- 2.1 vs. 3.5 +/- 1.8 and 3.3 +/- 2.3 pulses/12 h; P less than 0.02). LH amplitude was similar in all 3 groups. Mean serum FSH concentrations were slightly but not significantly lower in group C. We conclude that reduced gonadotropin secretion during the follicular phase may indeed affect E2 and P4 secretion at later stages of the menstrual cycle. The patterns of alteration associated with disturbed E2 and P4 secretion in normally menstruating women are similar to those that occur in women with hypothalamic amenorrhea.     

Pulsatile luteinizing hormone and follicle-stimulating hormone secretion and gonadotropin subunit mRNA levels in the ovariectomized GPR-4 transgenic rat

Genetic targeting of the cAMP-specific phosphodiesterase 4D1 (PDE4D1) to gonadotropin-releasing hormone (GnRH) neurons in the GPR-4 transgenic rat resulted in decreased luteinizing hormone (LH) pulse frequency in castrated female and male rats. A similar decrease in the intrinsic GnRH pulse frequency was observed in GT1 GnRH cells expressing the PDE4D1 phosphodiesterase. We have extended these findings in ovariectomized (OVX) GPR-4 rats by asking what effect transgene expression had on pulsatile LH and follicle-stimulating hormone (FSH) secretion, plasma and pituitary levels of LH and FSH, and levels of the alpha-glycoprotein hormone subunit (alpha-GSU), LH-beta and FSH-beta subunit mRNAs. In OVX GPR-4 rats the LH pulse frequency but not pulse amplitude was decreased by 50% compared to wild-type littermate controls. Assaying the same samples for FSH, the FSH pulse frequency and amplitude were unchanged. The plasma and anterior pituitary levels of LH in the GPR-4 rats were significantly decreased by approximately 45%, while the plasma but not anterior pituitary level of FSH was significantly decreased by 25%. As measured by real-time RT-PCR, the mRNA levels for the alpha-GSU in the GPR-4 rats were significantly decreased by 41%, the LH-beta subunit by 38% and the FSH-beta subunit by 28%. We conclude that in the castrated female GPR-4 rats the decreased GnRH pulse frequency results in decreased levels of LH and FSH and in the alpha- and beta-subunit mRNA levels.     

The effect of estrogen on episodic secretory patterns of luteinizing hormone-releasing hormone (LHRH) and luteinizing hormone (LH) in hypergonadotropic hypogonadism

The secretory dynamics of plasma luteinizing hormone-releasing hormone (LHRH) and serum luteinizing hormone (LH) were studied in three hypogonadal women before and after chronic administration of mestranol. Blood samples were obtained through an indwelling iv line every 15 min over 3 hours, and plasma levels of LHRH and LH were measured by radioimmunoassay. LHRH and LH pulses were defined as rising from nadir to peak that exceed 2 times the intraassay coefficient of variation. All patients showed pulsatile LHRH and LH release before mestranol administration. The mean LH levels (89 +/- 20 mIU/ml) and pulse amplitude (33 +/- 14 mIU/ml) were significantly reduced after mestranol administration. On the other hand, the mean LHRH levels (1.87 +/- 0.49 pg/ml) and pulse amplitude (0.92 +/- 0.41 pg/ml) did not change significantly after mestranol administration. Pulse frequency (2 approximately 3 times/3 hrs) of LHRH and LH did not change after mestranol administration. These data show that the chronic administration of estrogen to such patients cause a decrease in mean LH levels and amplitude of LH pulse without a decrease of pulsatile LHRH secretions. These results suggest that the chronic negative feedback action of estrogen on episodic LH release in women may be at the level of the pituitary gland and estrogen may change the pituitary sensitivity to LHRH.     

Isoforms and half-life of FSH from sheep with different reproductive states

The glycoprotein hormone FSH comes in many different isoforms. In humans and rats the charges of the FSH isoforms vary with reproductive state and these affect the half-life of FSH in plasma. In this study we examined the charge heterogeneity of FSH in pituitary extracts from sheep with different reproductive states. Also the half-life of clearance of pituitary FSH from the different reproductive states was determined in mice. Pituitaries were collected from: anoestrous, luteal phase, follicular phase, early-pregnant and late-pregnant ewes, ewe lambs, ram lambs, rams during the breeding and non-breeding seasons and wethers (5 per group). After extraction, FSH isoforms were fractionated by HPLC anion exchange chromatography. The volume at which half of the FSH had eluted from the ion exchange column was determined (HP(50)). It was found that FSH isoforms from ewes (HP(50)=96.7+/- 1.3 ml (s.e.m. )) eluted later (P<0.01) than those from rams (HP(50)=82.3+/-1.3 ml) indicating that FSH isoforms in the ewes were more acidic than those from rams. There was a seasonal difference in ewes, with ewes in anoestrus (HP(50)=101.6+/-2.6 ml) having more-acidic (P<0.01) FSH isoforms than the ewes during the oestrous cycle (HP(50)=95.3+/-0.7 ml). There was an effect of age, with the FSH isoforms from cycling ewes (HP(50)=95.3+/- 0.7 ml) being more acidic (P<0.01) than those from ewe lambs (HP(50)=88.3+/-1.9 ml). There was an effect of pregnancy, with late-pregnant ewes (HP(50)=107.3+/- 1.6 ml) having more-acidic FSH isoforms (P<0.05) than those from anoestrous ewes (HP(50)=101.6+/-2.6 ml) and there was an effect of castration with the breeding season rams (HP(50)=80.7+/-1.4 ml) having more-acidic (P<0.05) FSH isoforms than wethers (HP(50)=74.0+/-0.5 ml). The half-life of pituitary FSH from animals in the different reproductive states was found to be negatively correlated with HP(50) (r(2)=0.56, P<0.01). The FSH isoforms from wethers were the least acidic and had the longest half-lives. Collectively, these findings show that in sheep, age, sex and reproductive state are all factors which influence the forms of FSH that are extracted from the pituitary gland. Moreover, these results demonstrate that FSH from sheep with the most-acidic FSH isoforms have the shortest half-life in plasma.     

Effects of oestradiol and progesterone on pulsatile secretion of luteinizing hormone in ovariectomized ewes

The effect of treatment with oestradiol, progesterone, a combination of the two steroids or no steroids on pulsatile release of luteinizing hormone (LH) was examined in ovariectomized ewes. Beginning 3 days after ovariectomy, 5 ewes were assigned to each of the following treatment groups: 0.7 mg oestradiol, 16 mg progesterone, 0.7 mg oestradiol plus 16 mg progesterone or no steroid. All treatments were administered twice daily for 3 weeks in a 0.5 ml injection of ethanol given sc. After 2 weeks of treatment and 1, 4, 8, 16 and 32 days after the treatment period ended, blood samples were obtained from all ewes at 10-min intervals for a 6-h period. At the end of the 6-h period, 100 micrograms gonadotrophin-releasing hormone (GnRH) was injected iv and blood samples were collected at 15 min intervals for an additional 5 h to estimate the relative pituitary content of LH. Ovariectomized ewes receiving no steroid presented regular pulses of LH at frequency of four to five pulses during a 6-h sampling period. Treatment with progesterone alone decreased the frequency of pulsatile release of LH to approximately 1 pulse/6 h, but did not affect the amplitudes of the pulses of LH. Recovery of pulsatile release of LH to a frequency of four or five pulses of LH in a 6-h period was complete between 16 ewes. Oestradiol, administered alone or with progesterone, resulted in a decrease in both the frequency and the amplitude of pulses of LH compared to control ewes and a decrease in GnRH-induced release of LH.(ABSTRACT TRUNCATED AT 250 WORDS)