Rapid recovery of estrogen-induced pituitary gonadotropin surges after luteectomy in rhesus monkeys

In the presence of a functional corpus luteum, positive estrogen feedback on the surge modes of gonadotropin secretion is blocked in rhesus monkeys. We investigated the effects of luteectomy (Lx) on the time required for recovery of pituitary responsiveness (LH/FSH surges) to positive estrogen feedback. Estradiol-17 beta-3- benzoate (EB, 50 microgram/kg sc) was given: 1) 24th prior to, 2) the day of, or 3) 24 h after luteal ablation. Daily measurements of serum follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol-17 beta (e2) and progesterone (P) were made on each monkey for 5 days. Serum P fell to undetectable levels within 24 h after Lx, whereas E2 levels in circulation peaked within 24h after injection of EB. Among early follicular phase monkeys, this EB treatment results in typical midcycle type LH/FSH surges within 48h. Lx alone was not soon followed by significant changes in pituitary gonadotropin secretion. When circulating P levels were undetectable the pituitary responded fully to EB; that is, typical midcycle type FSH/LH surges occurred. When serum P was in the midst of declining after Lx, gonadotropin surges were present, but attenuated. However, when P levels remained elevated for more than 24 h after EB injection, the surge modes of FSH/LH secretion remained fully blocked. These results demonstrate that the suppressive influence of luteal secretions (principally progesterone) on positive estrogen feedback regulation of the surge modes of pituitary gonadotropin secretion is quite transient in these primates.     

Naloxone enhances LH but not FSH release during various phases of the estrous cycle in the ewe

Suffolk x whiteface ewes were infused with 0.5 mg/kg/hr naloxone hydrochloride (NAL) for 6 hrs during the early, mid and late luteal and early follicular phases of the estrous cycle. Basal serum luteinizing hormone (LH) concentration was increased by NAL during each trial in the luteal phase and LH pulse amplitude was proportionately increased by 158%, 164% and 350% during the early luteal, mid luteal and early follicular phases, respectively. The apparent NAL induced increase (92%) in LH pulse amplitude during the late luteal phase was not significant. NAL only affected LH pulse frequency during the early follicular phase, when it was decreased. Mean serum follicle stimulating hormone (FSH) concentration was not affected by NAL. The results of this study indicate that endogenous opioid peptides (EOPs) may partially mediate the suppressive influence of estradiol-17 beta (E2) on LH pulse amplitude and also the stimulatory effect of E2 on LH pulse frequency in the early follicular phase. The data may suggest that NAL enhances the amplitude of pulses of gonadotropin releasing hormone (GnRH) by counteracting E2 inhibitory effects on LH release at the level of the pituitary. Alternately, some component of E2 feedback may be an EOP mediated component at the level of the hypothalamus.     

Differential effects of aging on estrogen negative and positive feedback

Recent studies have demonstrated an age-related decline in gonadotropins and a decrease in pituitary responsiveness to GnRH, indicating that aging influences the neuroendocrine components of the female reproductive axis independently of changes in ovarian function. To determine whether aging might also affect the luteinizing hormone (LH) negative and positive feedback responses to gonadal steroids, we administered a controlled, graded sex steroid infusion to 11 younger (45-56 yr) and nine older (70-80 yr) postmenopausal women (PMW) in whom endogenous ovarian steroids and peptides are uniformly low. The doses of estradiol (E(2)) and progesterone (P) were chosen to mimic levels across the normal follicular phase and have been shown previously to induce negative followed by positive feedback on LH. Similar E(2) and P levels were achieved in younger and older PMW (P = 0.4 and 0.3, respectively) and produced a biphasic LH response in all subjects. The early decline in LH to 53% of baseline was not different in older vs. younger PMW. However, the positive feedback effect was attenuated in older compared with younger PMW (peak LH 144.4 ± 19.5 vs. 226.8 ± 22.3 IU/l, respectively, P = 0.01). In conclusion, these studies in PMW demonstrate preservation of short-term steroid negative and positive feedback in response to exogenous E(2) and P with aging. Attenuation of positive feedback in older compared with younger PMW is consistent with previous reports of declining GnRH responsiveness with aging.     

Site of negative feedback action of estrogen on gonadotropin secretion in normal women

We have reported that iv administration of conjugated estrogens results in no significant change in the plasma LH-RH level during the negative feedback phase of LH, suggesting that estrogen does not suppress LH by decreasing hypothalamic LH-RH. To determine the site of estrogen action during the negative feedback phase, we studied the pituitary response to a small amount of LH-RH after estrogen administration in normal cyclic women in the mid-follicular phase. The pituitary responses to an iv bolus of 2.5 micrograms of synthetic LH-RH were evaluated by measuring serum LH and FSH 2 h before and 8 h after administration of 20 mg of conjugated estrogens (Premarin). The mean levels of serum LH and FSH were significantly (p less than 0.05) decreased 8 h after the injection. The peak responses of LH and FSH to LH-RH were also significantly (p less than 0.05) reduced after Premarin administration. These findings suggest that the negative feedback effect of estrogen on gonadotropin secretion is caused by its direct suppression on the pituitary response to LH-RH.     

Membrane-initiated actions of estradiol (E2) in the regulation of LH secretion in ovariectomized (OVX) ewes

Background  

We demonstrated that E2 conjugated to BSA (E2BSA) induces a rapid membrane-initiated inhibition of LH secretion followed hours later by a slight increase in LH secretion. Whether these actions of E2BSA are restricted to the pituitary gland and whether the membrane-initiated pathway of E2BSA contributes to the up-regulation of the number of GnRH receptors during the positive feedback effect of E2 were evaluated here. We have shown that the suppression of LH secretion induced by E2 and E2BSA is the result of a decreased responsiveness of the pituitary gland to GnRH. In this study we further tested the ability of E2BSA to decrease the responsiveness of the pituitary gland to GnRH under the paradigm of the preovulatory surge of LH induced by E2.     

Differential modulation of gonadotropin secretion by selective estrogen receptor 1 and estrogen receptor 2 agonists in ovariectomized ewes

The objectives of this study were to determine whether activation of estrogen receptor 1 (ESR1; also known as ERalpha), or estrogen receptor 2 (ESR2; also known as ERbeta), or both are required to: 1) acutely inhibit secretion of LH, 2) induce the preovulatory-like surge of LH, and 3) inhibit secretion of FSH in ovariectomized (OVX) ewes. OVX ewes (n = 6) were administered intramuscularly 25 micrograms estradiol (E2), 12 mg propylpyrazoletriol (PPT; a subtype-selective ESR1 agonist), 21 mg diaprylpropionitrile (DPN; a subtype-selective ESR2 agonist), or PPT + DPN. Like E2, administration of PPT, DPN, or combination of the two rapidly decreased (P < 0.05) secretion of LH. Each agonist induced a gradual, prolonged rise in secretion of LH after the initial inhibition, but neither agonist alone nor the combined agonists was able to induce a "normal" preovulatory-like surge of LH similar to that induced by E2. Compared with E2-treated ewes, the beginning of the increase in secretion of LH occurred earlier (P < 0.01) in DPN-treated ewes, later (P < 0.05) in PPT-treated ewes, and at a similar interval in ewes receiving the combined agonist treatment. Like E2, PPT decreased (P < 0.05) secretion of FSH, but the duration of suppression was much longer in PPT-treated ewes. DPN did not alter secretion of FSH in this study. Modulation of the number of GnRH receptors by PPT and DPN was examined in primary cultures of ovine pituitary cells. In our hands, both PPT and DPN increased the number of GnRH receptors, but the dose of DPN required to stimulate synthesis of GnRH receptors was 10 times higher than that of PPT. We conclude that in OVX ewes: 1) ESR1 and ESR2 mediate the negative feedback of E2 on secretion of LH at the level of the pituitary gland, 2) ESR1 and ESR2 do not synergize or antagonize the effects of each other; however, they do interact to synchronize the beginning of the stimulatory effect of E2 on secretion of LH, 3) ESR1 and ESR2 may mediate at least partially the positive feedback of E2 on LH secretion by increasing the number of GnRH receptors, and 4) only ESR1 appears to be involved in the negative feedback of E2 on secretion of FSH.     

The influences of GnRH, oxytocin and vasoactive intestinal peptide on LH and PRL secretion by porcine pituitary cells in vitro.

The aim of the present study was to evaluate the possible direct effects of GnRH, oxytocin (OT) and vasoactive intestinal peptide (VIP) on the release of LH and PRL by dispersed porcine anterior pituitary cells. Pituitary glands were obtained from mature gilts, which were ovariectomized (OVX) one month before slaughter. Gilts randomly assigned to one of the four groups were treated: in Group 1 (n = 8) with 1 ml/100 kg b.w. corn oil (placebo); in Group 2 (n = 8) and Group 3 (n = 8) with estradiol benzoate (EB) at the dose 2.5 mg/100 kg b.w., respectively, 30-36 h and 60-66 h before slaughter; and in Group 4 (n = 9) with progesterone (P4) at the dose 120 mg/ 100 kg b.w. for five consecutive days before slaughter. In gilts of Group 2 and Group 3 treatments with EB have induced the negative and positive feedback in LH secretion, respectively. Isolated anterior pituitary cells (10(6)/well) were cultured in McCoy's 5a medium with horse serum and fetal calf serum for 3 days at 37 degrees C under the atmosphere of 95% air and 5% CO2. Subsequently, the culture plates were rinsed with fresh McCoy's 5A medium and the cells were incubated for 3.5 h at 37 degrees C in the same medium containing one of the following agents: GnRH (100 ng/ml), OT (10-1000 nM) or VIP (1-100 nM). The addition of GnRH to cultured pituitary cells resulted in marked increases in LH release (p < 0.001) in all experimental groups. In the presence of OT and VIP we noted significant increases (p < 0.001) in LH secretion by pituitary cells derived from gilts representing the positive feedback phase (Group 3). In contrast, OT and VIP were without any effect on LH release in Group 1 (placebo) and Group 2 (the negative feedback). Pituitary cells obtained from OVX gilts primed with P4 produced significantly higher amounts (p < 0.001) of LH only after an addition of 100 nM OT. Neuropeptide GnRH did not affect PRL secretion by pituitary cells obtained from gilts of all experimental groups. Oxytocin also failed to alter PRL secretion in Group 1 and Group 2. However, pituitary cells from animals primed with EB 60-66 h before slaughter and P4 produced markedly increased amounts of PRL in the presence of OT. Neuropeptide VIP stimulated PRL release from pituitary cells of OVX gilts primed with EB (Groups 2 and 3) or P4. In contrast, in OVX gilts primed with placebo, VIP was without any effect on PRL secretion. In conclusion, the results of our in vitro studies confirmed the stimulatory effect of GnRH on LH secretion by porcine pituitary cells and also suggest a participation of OT and VIP in modulation of LH and PRL secretion at the pituitary level in a way dependent on hormonal status of animals.     

Effects of an opioid antagonist on pulsatile luteinizing hormone secretion in the ewe vary with changes in steroid negative feedback

In ewes during the breeding season, estradiol (E) and progesterone (P) synergistically regulate pulsatile luteinizing hormone (LH) secretion. E primarily inhibits LH pulse amplitude and P inhibits LH pulse frequency. To determine if endogenous opioid peptides (EOP) mediate these negative feedback effects, we administered the long-acting opioid antagonist WIN 44,441-3 (WIN) to intact ewes during the luteal and follicular phases of the estrous cycle and to ovariectomized ewes treated with no steroids, E, P, or E plus P. Steroid levels were maintained at levels seen during the estrous cycle by Silastic implants placed shortly after surgery. WIN increased LH pulse frequency, but not amplitude, in luteal phase ewes. In contrast, during the follicular phase, LH pulse amplitude was increased by WIN and pulse frequency was unchanged. Neither LH pulse frequency nor pulse amplitude was affected by WIN in long-term ovariectomized ewes untreated with steroids. In contrast, WIN slightly increased LH pulse frequency in short-term ovariectomized ewes. WIN also increased LH pulse frequency in ovariectomized ewes treated with P or E plus P. WIN did not affect pulse frequency but did increase LH pulse amplitude in E-treated ewes. These results support the hypothesis that EOP participate in the negative feedback effects of E and P on pulsatile LH secretion during the breeding season and that the inhibitory effects of EOP may persist for some time after ovariectomy.     

Luteinizing hormone secretion as affected by hypophyseal stalk transection and estradiol-17beta in ovariectomized gilts

The objectives were to determine hypothalamic regulation of pulsatile luteinizing hormone (LH) secretion in female pigs and the biphasic feedback actions of estradiol-17beta (E(2)-17beta). In the first study, the minimum effective dosage of E(2)-17beta that would induce estrus in ovariectomized gilts was determined to be 20microg/kg body weight. In the second study, ovariectomized gilts were assigned randomly on day 0 to treatments: (a) hypophyseal stalk transection (HST), (b) cranial sham-operated control (SOC), and (c) unoperated control (UOC). On day 3, gilts from each group received a single i.m. injection of either E(2)-17beta (20microg/kg body weight) or sesame oil. Blood was collected from an indwelling jugular cannula at 15min intervals for 3h before (day -2) and after treatment (day 2) from HST, SOC and UOC gilts. On day 3, blood was collected at 2h intervals for 12h after E(2)-17beta or sesame oil injection and at 4h intervals thereafter for 108h. Pulsatile LH secretion in all gilts 2 days after ovariectomy exhibited a frequency of 0.9+/-0.06peaks/h, amplitude of 1.3+/-0.13ng/ml, baseline of 0.8+/-0.07. Serum LH concentrations from SOC and UOC gilts were similar on day 2 and profiles did not differ from those on day -2. In HST gilts pulsatile LH release was abolished and mean LH concentration decreased compared with controls (0 versus 0.9+/-0. 06peaks/h and 0.77+/-0.03 versus 1.07+/-0.07ng/ml, respectively; P<0. 05). E(2)-17beta or sesame oil did not affect serum LH concentration in HST gilts, and LH remained constant throughout 120h (0.7+/-0. 07ng/ml). In SOC and UOC control gilts, E(2)-17beta induced a 60% decrease (P<0.05) in LH concentration within 12h, and LH remained low until 48h, then increased to peak values (P<0.05) by 72h, followed by a gradual decline to 120h. Although pituitary weight decreased 31% in HST gilts compared with controls (228 versus 332mg, P<0.05), an abundance of normal basophils was evident in coronal sections of the adenohypophysis of HST comparable to that seen in control gilts. The third and fourth studies determined that hourly i. v. infusions of LHRH (2microg) and a second injection of E(2)-17beta 48h after the first had no effect on the positive feedback action of estrogen in UOC. However, in HST gilts that received LHRH hourly, the first injection of E(2)-17beta decreased (P<0.05) plasma LH concentrations while the second injection of E(2)-17beta failed to induce a positive response to estrogen. These results indicate that both pulsatile LH secretion and the biphasic feedback action of E(2)-17beta on LH secretion depend on hypothalamic regulatory mechanisms in the gilts. The isolated pituitary of HST gilts is capable of autonomous secretion of LH; E(2)-17beta will elicit direct negative feedback action on the isolated pituitary gland if the gonadotropes are supported by exogenous LHRH, but E(2)-17beta at high concentrations will not induce positive feedback in isolated pituitaries. Thus, the direct effect of E(2)-17beta on the pituitary of monkeys cannot be mimicked in pigs.     

Plasma LH and FSH in ewes that were either fertile or infertile after long-term grazing of oestrogenic pasture

The levels of plasma LH and FSH were measured in serial blood samples taken at 15-min intervals for 6 h from ewes that had remained fertile after grazing oestrogenic pasture (clover-fertile ewes), from ewes that were permanently affected by clover disease (clover-infertile ewes) and from normal ewes. Two flocks of ewes from different locations were studied. In flock 1, tonic LH secretion (total area under the curve of LH concentration versus time, 1 area unit = 1 ng ml-1 x 1 h) was significantly (P < 0.05) greater in clover-infertile ewes (10.4 area units) during anoestrus than in ewes that had remained fertile after prolonged grazing of oestrogenic clover (5.4 area units). Tonic LH and FSH secretions during the bleeding season and FSH secretion during anoestrus were not significantly different. In flock 2, LH levels during the breeding season were significantly (P < 0.05) elevated in clover-infertile ewes (10.9 area units) compared to normal ewes (5.4 area units) that had never grazed oestrogenic clover. LH secretion in clover-infertile ewes (7.8 area units) was intermediate to that found in infertile and control ewes. Concentrations of FSH, progesterone and ovarian vein oestradiol-17 beta (E2) during the breeding season were similar in the three groups. In another experiment, the positive feedback release of LH following administration of E2 (12.5, 25 or 50 micrograms per ewe) was measured in anoestrous ewes of flock 2. Significantly (P < 0.01) more clover-infertile ewes demonstrated a positive feedback effect than control ewes when given 12.5 micrograms E2 but not when given higher doses. The elevation of LH secretion in permanently affected clover-infertile ewes is inconsistent with the hypothesis that the hypothalamo-pituitary axis of these ewes is less responsive to the negative feedback effect of oestrogen. Furthermore, the patency of the positive feedback loop is consistent with the ability to ovulate.     

Effect of neuropeptide Y on GnRH-induced LH release from bovine anterior pituitary cell cultures derived from heifers in a follicular,luteal or ovariectomized state

Objectives were to determine if neuropeptide Y (NPY) had direct effects GnRH induced secretion of LH from the anterior pituitary gland, and if endogenous steroids modulated the effect of NPY. To accomplish these objectives, 15 Hereford heifers were assigned to one of three ovarian status groups: follicular, luteal, or ovariectomized. One animal from each of the three ovarian status groups was slaughtered on each of 5 days and anterior pituitary gland harvested. Anterior pituitary gland cells within ovarian status were equally distributed and randomly assigned to one of three cell culture treatments: no NPY or GnRH (control), 10 nM GnRH, or 100 nM NPY+10 nM GnRH. Anterior pituitary cell cultures were incubated with or without NPY for 4 h and further incubated for an additional 2 h with or without GnRH and supernatant collected for quantification of LH. Treatment of anterior pituitary cell cultures with GnRH or GnRH+NPY did not affect LH release in cultures obtained from follicular (S.E.=5%; P=0.58) or ovariectomized (S.E.=7%; P=0.22) heifers. Both GnRH and GnRH+NPY increased LH release from anterior pituitary cell cultures from heifers in the luteal phase (S.E.=14%; P < or = 0.05) compared to control cultures. Cultures from luteal phase heifers treated with GnRH did not differ from those treated with GnRH+NPY (P=0.34). These data provide evidence to suggest that effects of NPY on LH release may occur primarily at the level of the hypothalamus.     

Age-dependent changes in the hypothalamo-pituitary-ovarian axis of the laying hen.

The functional integrity of the components of the hypothalamo-pituitary-ovarian axis was examined in young and old laying hens. Ovarian function was tested by measuring the amount of progesterone released in response to an injection of LH, and pituitary function was investigated by measuring the increase in the plasma LH level after an injection of LH-RH. There were no differences between young and old birds in the response of the pituitary gland or the ovary to these stimuli. Hypothalamic function was investigated by studying the positive feedback action of a standard dose of progesterone on LH release; the positive feedback response was smaller (P less than 0.05) in old hens. It is suggested that the fall in the rate of lay in hens towards the end of their laying year is caused partly by a decrease in the response of the LH-positive feedback mechanism to progesterone.     

Interactions between inhibin, oestradiol and progesterone in the control of gonadotrophin secretion in the ewe

Experiments were carried out to test the hypothesis that inhibin and oestradiol act synergistically to inhibit the secretion of FSH, to test for effects of progesterone, and to compare the FSH and LH responses to ovarian feedback. In Exp. 1, with 11 ovariectomized and 12 intact Romanov ewes during the anoestrous season, doses of oestradiol (administered by means of subcutaneous implants) that restored normal LH pulse frequencies were insufficient to restore normal concentrations of FSH. In Exp. 2, with 48 ovariectomized Welsh Mountain ewes during the breeding season, a factorial design with 4 ewes per cell was used to assess the responses in LH and FSH to 3 doses of oestradiol (s.c. implants) and 4 doses of bovine follicular fluid ('inhibin', 0.2-1.6 ml s.c. every 8 h). This was done initially in the absence of progesterone and then after 7 days of treatment with progesterone (s.c. implants). Analysis of variance revealed a significant synergistic interaction between oestradiol and inhibin on the plasma concentrations of FSH. Progesterone had little effect. In contrast, there was a significant synergistic interaction between oestradiol and progesterone on the concentrations of LH. 'Inhibin' also inhibited LH secretion but this effect was independent of the two steroids. We conclude that there are basic differences in the way that ovarian feedback acts to control the secretion of LH and FSH in the ewe. FSH secretion appears to be primarily controlled by the synergistic action of oestradiol and inhibin on the anterior pituitary gland, while the secretion of LH is inhibited during the follicular phase by an effect of oestrogen at pituitary level and during the luteal phase by the synergistic action of oestradiol and progesterone at the hypothalamic level. Inhibin, or another non-steroidal factor in follicular fluid, may also play a minor role in the control of LH secretion.     

A phase plane graph based model of the ovulatory cycle lacking the "positive feedback" phenomenon

ABSTRACT: When hormones during the ovulatory cycle are shown in phase plane graphs, reported FSH and estrogen values form a specific pattern that resembles the leaning "&" symbol, while LH and progesterone (Pg) values form a "boomerang" shape. Graphs in this paper were made using data reported by Stricker et al. [Clin Chem Lab Med 2006;44:883-887]. These patterns were used to construct a simplistic model of the ovulatory cycle without the conventional "positive feedback" phenomenon. The model is based on few well-established relations: - hypothalamic GnRH secretion is increased under estrogen exposure during two weeks that start before the ovulatory surge and lasts till lutheolysis. - the pituitary GnRH receptors are so prone to downregulation through ligand binding that this must be important for their function. - in several estrogen target tissue progesterone receptor (PgR) expression depends on previous estrogen binding to functional estrogen receptors (ER), while Pg binding to the expressed PgRs reduces both ER and PgR expression. Some key features of the presented model are here listed: - High GnRH secretion induced by the recovered estrogen exposure starts in the late follicular phase and lasts till lutheolysis. The LH and FSH surges start due to combination of accumulated pituitary GnRH receptors and increased GnRH secretion. The surges quickly end due to partial downregulation of the pituitary GnRH receptors (64% reduction of the follicular phase pituitary GnRH receptors is needed to explain the reported LH drop after the surge). A strong increase in the lutheal Pg blood level, despite modest decline in LH levels, is explained as delayed expression of pituitary PgRs. Postponed pituitary PgRs expression enforces a negative feedback loop between Pg levels and LH secretions not before the mid lutheal phase. - Lutheolysis is explained as a consequence of Pg binding to hypothalamic and pituitary PgRs that reduces local ER expression. When hypothalamic sensitivity to estrogen is diminished due to lack of local ERs, hypothalamus switches back to the low GnRH secretion rate, leading to low secretion of gonadotropins and to lutheolysis. During low GnRH secretion rates, previously downregulated pituitary GnRH receptors recover to normal levels and thus allow the next cycle.     

Regulation of LH beta subunit mRNA in the sheep pituitary gland during different feedback states of estradiol

The feedback effects of gonadal steroids on the amounts of in vitro translated luteinizing hormone (LH) beta subunit were examined using cell-free assays. These amounts were then correlated with serum and pituitary concentrations during various feedback states. RNA was prepared, translated and products identified by immunoprecipitation and gel electrophoresis. The amounts of beta subunit varied in a pattern similar to that observed for alpha subunit. In ovariectomized ewes, the amounts of beta were 2–3X those seen in negative feedback groups and slightly more than those seen in animals exhibiting an LH surge. The pituitary LH concentration in ovariectomized ewes was also higher than those seen in the other groups, however, the serum concentrations in the positive feedback group were the highest of all groups. These results provide evidence for: 1) a separate, but coordinate, control of gonadotropin subunit synthesis; and 2) a contribution of subunit synthesis to the effects of positive and negative steroid feedback on pituitary LH amounts.     

Direct pituitary effects of estradiol and progesterone on luteinizing hormone release, stores, and subunit messenger ribonucleic acids

To determine the direct, chronic actions of progesterone (P4) and estrogen (estradiol, E2) on anterior pituitary synthesis and release of LH, 24 western range ewes underwent hypothalamic-pituitary disconnection (HPD) and ovariectomy (OVX) during the breeding season and were pulsed with exogenous GnRH with or without steroid replacement. Sequential blood samples were collected before infusion of GnRH and on Days 7 and 14 of GnRH infusion. Silastic capsules of P4 and/or E2 were implanted s.c. on Day 7 and remained in place throughout the experiment. Control ewes received only GnRH infusion. Blood sampling was centered around three exogenous GnRH pulses. After the final blood sampling, pituitaries were collected and stored at -70 degrees C. Concentrations of LH in serum and pituitaries were determined by RIA. Relative concentrations of LH subunit mRNAs were determined by Fast Blot analysis. Simultaneous implantation of P4 and E2 lowered LH pulse amplitude 70% and mean serum levels 30% compared with controls. Neither steroid alone affected LH release. E2 alone or in combination with P4 lowered LH-beta subunit mRNA concentrations 40% compared with controls while alpha-subunit levels were unchanged. Only E2 alone altered the pituitary content of LH, causing a 60% decrease. We conclude that the combination of P4 and E2 is necessary for inhibition of GnRH-stimulated LH secretion. E2 inhibits GnRH-stimulated LH-beta subunit mRNA concentrations but does not affect alpha-subunit mRNA concentrations. The control of pituitary LH content by P4 and E2 is the result of changes in both LH-beta subunit mRNA concentrations and LH secretion.     

Estradiol promotes pituitary cell proliferation and gonadotroph differentiation at different doses and with different mechanisms in chick embryo

Estrogens are known to play a role in the feedback regulation of pituitary gonadotropin secretion in adults. However, it is still unknown whether estrogens are involved in promoting pituitary development. In this study, we selected chick embryo as the animal model and microinjected different doses of estradiol (E2) at stage E27–28, which was when endogenous E2 was not detected. First, the results demonstrated that E2 at different doses promoted pituitary cell proliferation and gonadotroph differentiation. Lower doses of E2 had a more significant effect on cell proliferation, while higher doses of E2 were required for luteinizing hormone (LH) secreting cell differentiation. Furthermore, the levels of early growth response protein 1 (Egr-1) and GATA2 mRNAs were also elevated with E2 treatment at a higher dose than that required to increase the level of proliferating cell nuclear antigen (PCNA) in vitro. To investigate whether estrogen receptors (ERs) mediate these effects of estradiol, the ER antagonist ICI 182,780 was added, and the results showed that ICI 182,780 did not modify the enhancing effects of E2 on cell proliferation; however, it inhibited the stimulatory effect of E2 on LH secreting cell differentiation. These results suggest that E2 at different doses promotes pituitary cell proliferation and gonadotroph differentiation with different mechanisms. Our results are important to further understanding of the physiological and pharmacological functions and related mechanisms of estrogens and their receptors, although the related mechanism need to be elucidated in future studies.     

Responses of luteinizing hormone (LH) and follicle-stimulating hormone levels to exogenous gonadotropin-releasing hormone during the estrogen-induced LH surge in the ovariectomized rhesus monkey

Experiments were performed to study the responsiveness of the pituitary to gonadotropin-releasing hormone (GnRH) during the dynamic changes in gonadotropin secretion associated with the estrogen-induced luteinizing hormone (LH) surge in the ovariectomized (OVX) rhesus monkey. Silastic capsules filled with estradiol-17-beta were implanted subcutaneously in ovariectomized rhesus monkeys, resulting in an initial lowering of circulating LH and follicle-stimulating hormone (FSH) concentrations followed by an LH-FSH surge. GnRH was injected intravenously just before estrogen implantation, during the negative feedback response and during the rising, the peak, and the declining phases of the LH surge. The LH and FSH responses during the negative feedback phase were as large as those before estrogen treatment (control responses). During the rising phase of the LH surge, the acute response to GnRH injection did not differ significantly from the control response, but the responses 60 and 120 min after injection were somewhat increased. During the declining phase of the LH surge, the pituitary was not responsive to exogenous GnRH, although LH probably continued to be secreted at this time since the LH surge decreased more slowly than predicted by the normal rate of disappearance of LH in the monkey. We conclude that an increased duration of response to GnRH may be an important part of the mechanism by which estrogen induces the LH surge, but we do not see evidence of increased sensitivity of the pituitary to GnRH as an acute releasing factor at that time.     

Dynamic changes in the gonadotrope cell subpopulations during an estradiol-induced surge in the ewe

Whether estradiol targets a subpopulation of gonadotrope cells was investigated in this study. Ovariectomized ewes (OVX) or OVX ewes immunized against GnRH and treated with hourly pulses of GnRH analogue (OVX-IMG) were killed at 6, 12, 16, and 24 h after administration of 50 microg of 17beta-estradiol (E(2)). Control ewes received no E(2) treatment. In OVX or OVX-IMG ewes killed 6 h after E(2) injection, a decrease in gonadotropin plasma levels was observed compared with non-E(2)-treated ewes. In contrast, a surge in gonadotropin plasma concentrations occurred in ewes killed 16 h after injection. The percentage of total immunoreactive gonadotrope cells among the pituitary cells was lower in E(2)-treated ewes compared with nontreated animals. The proportion of monohormonal LH cells was constant throughout the experiment, except at the surge peak, where it was enhanced. In the OVX ewes, the proportion of bihormonal LH/FSH cells was lower in the E(2)-treated ewes compared to the nontreated ewes (P: < 0.001), with a more pronounced decrease 16 h after E(2) injection. A slight increase occurred 12 h after E(2) injection compared with 6 h after injection (P: < 0.05). A similar pattern was observed in the OVX-IMG ewes, except at 12 h after E(2) injection, when no increase occurred. In both OVX and OVX-IMG ewes, injection of E(2) decreased FSHbeta mRNA expression but did not alter the relative levels of LHbeta mRNA. These data suggest that the negative feedback of E(2) on LH and FSH secretion mainly targets the bihormonal cells and occurs, at least in part, directly at the pituitary level. During the gonadotropin surge, the sustained FSH release from the bihormonal cells would induce a switch from bihormonal cells to monohormonal LH cells by depleting these cells of FSH.     

Prolactin and LH release in response to LH-RH and TRH in ewes during dioestrus, pregnancy and post partum

With advancing pregnancy in the ewe there was a marked decline in plasma LH concentrations and pituitary LH-RH responsiveness (integrated LH release) and a marked increase in plasma prolactin values and pituitary TRH responsiveness (integrated prolactin release). In lactating ewes plasma LH levels and pituitary LH-RH responsiveness had returned to values found in the luteal phase of the normal cycle by 21 days post partum, whereas at 42 days post partum prolactin levels were still high. No interaction between TRH and LH-RH on prolactin and LH release in dioestrous ewes was detected. In non-pregnant ewes plasma prolactin levels were significantly higher in June than in January but TRH responsiveness was similar. It is concluded that, in sheep, pituitary LH secretion recovers more rapidly from the chronic negative feedback effect of oestrogens and progesterone in pregnancy than prolactin secretion recovers from the chronic positive feedback effects of oestrogens. This finding may be a contributory factor in the resistance to resumption of breeding activity.