Interactions between 17 beta-estradiol and the hypothalamo-pituitary beta-endorphin system in the regulation of the cyclic LH secretion

This paper further substantiates the physiological role of beta-endorphin (beta-END) in the control of the cyclic LH secretion and provides new data on the interactions between 17 beta-estradiol (17 beta-E2) and beta-END at both the hypothalamic and pituitary levels. At the hypothalamic level, during the estrous cycle in rats, beta-END concentrations were highest on diestrus I in the arcuate nucleus, median preoptic area and median eminence and lowest at the time of the preovulatory 17 beta-E2 surge on proestrus, before the subsequent preovulatory hypothalamic GnRH and plasma LH surges. Data obtained in ovariectomized 17 beta-E2-treated ewes support the direct involvement of 17 beta-E2 in changes in beta-END and GnRH concentrations in these hypothalamic areas. At the anterior pituitary level, in vitro results obtained using anterior pituitaries from the proestrus morning cycling female rat have shown that 17 beta-E2 strongly suppresses beta-END secretion and that GnRH stimulates the release of beta-END. Furthermore, marked fluctuations were observed for plasma beta-END throughout the menstrual cycle in the woman. Low beta-END concentrations were observed in the period preceding the LH preovulatory surge. Taken together, these results show that: (1) decreases in hypothalamic beta-END concentrations, which are controlled at least by circulating levels of 17 beta-E2, modulate GnRH synthesis and/or release and contribute to the mechanisms which initiate the LH surge; (2) anterior pituitary beta-END might be involved in the mechanisms which terminate the LH surge.     

Dose-dependent inhibition of the preovulatory surges of gonadotropins and prolactin by the antiestrogen CI-628: possible sites of action

The present series of experiments was conducted in an attempt to correlate previously reported dose-dependent and site-selective inhibitory effects of an antiestrogen, CI-628, on 17 beta-estradiol (E2)-receptor interactions in the anterior pituitary gland (AP) and hypothalamus with its effects on the preovulatory surges of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin. The effects of CI-628 on the response of the AP to luteinizing hormone-releasing hormone (LHRH) and thyrotropin-releasing hormone (TRH) also were examined. In the first study, rats exhibiting 4-day estrous cycles were injected with various doses (0.02, 0.20, 2.0, and 20 mg/kg) of CI-628 or vehicle at 0900 h on diestrus-2 and proestrus. The preovulatory LH surge and both preovulatory and secondary FSH surges were marginally affected by 0.02 mg/kg CI-628, but were completely abolished by higher doses. In contrast, a dose of 0.20 mg/kg only delayed the prolactin surge; however, higher doses were effective in extinguishing cyclic prolactin release. In a second experiment, CI-628 in rats treated on diestrus-2 and proestrus exerted a dose-dependent suppression of the AP LH response to an initial injection of LHRH on proestrous afternoon in rats whose endogenous LH surges were blocked by phenobarbital. However, AP LH responses to a second LHRH injection to assess the self-priming capacity of LHRH were attenuated only in rats given 0.20, 2.0, and 20 mg/kg CI-628. Contrastingly, the AP prolactin response to TRH was suppressed only in rats given 0.20 mg/kg CI-628.(ABSTRACT TRUNCATED AT 250 WORDS)     

An in vitro study of LH release, synthesis and heterogeneity in pituitaries from proestrous and short-term ovariectomized rats

It is known that acute ovariectomy (OVX) greatly attenuates the pituitary luteinizing hormone (LH) response to gonadotropin-releasing hormone (GnRH) in vitro. The present study evaluated possible quantitative and/or qualitative differences in the biosynthesis and secretion of LH in pituitaries from proestrous and acutely (72 h) OVX rats. Paired anterior pituitary glands were incubated for 4 h in a medium containing +/- 10 nM GnRH. Pituitary and secreted LH were measured by radioimmunoassay with differences in total LH (tissue plus medium) +/- GnRH being indicative of GnRH-stimulated LH synthesis. Qualitative changes in LH were evaluated by isoelectrofocusing (IEF). The results show that the major form of LH stored in and released from the pituitaries consisted of LH molecules with an isoelectric point (pI) in the alkaline pH range (alkaline LH), and a lesser amount (approximately 30%) of LH molecules in the acidic pH range (acidic LH). The ratio of alkaline/acidic LH observed in the pituitary and medium was similar in the proestrous and OVX groups, although the amount of alkaline and acidic LH release in response to GnRH was 2-3 times greater in the proestrous group. In both groups, the alkaline/acidic LH ratio of secreted LH was higher in the presence of GnRH than in its absence. Alkaline LH synthesis was increased by GnRH in both groups, with the response being greater in the proestrous than in the OVX group; GnRH-stimulated acidic LH synthesis was observed only in the proestrous group. In both groups, the amount of LH synthesized was about 60% of the amount released, which suggests that LH synthesis does not fully account for differences in GnRH-stimulated LH release. Treatment of pituitary extracts with neuraminidase decreased acidic LH, and proportionately increased alkaline LH. These results suggest that the quality of LH stored in and secreted from pituitaries of proestrous and OVX rats is similar, and that there is a preferential release of the major alkaline LH isoform in response to GnRH. The ovarian steroid environment, presumably estradiol, proportionately increases the amount of alkaline and acidic LH released, and differentially affects the amounts of the various isoforms synthesized in response to GnRH. The charge heterogeneity of alkaline and acidic LH may be related to the sialic acid content of the LH molecule.     

Heme oxygenase in the rat anterior pituitary: immunohistochemical localization and possible role in gonadotropin and prolactin secretion

The objectives of this study were to determine if heme oxygenase (HO), which catalyzes the degradation of heme and the formation of carbon monoxide (CO), is localized in the rat anterior pituitary and, if so, to determine if hemin (a substrate for HO) or chromium mesoporphyrin (CrMP) (an inhibitor of HO), alter pituitary gonadotropin and prolactin secretion. For localization of HO, sections of anterior pituitaries obtained from mature Holtzman Sprague-Dawley rats in different stages of the estrous cycle were immunostained for two of the HO isoforms, HO-1 and HO-2. The immunostaining for the inducible HO isoform (HO-1) was limited to discrete populations of pituitary cells, whereas the constitutive isoform (HO-2) had a more widespread distribution. The afternoon surge of leutinizing hormone (LH) in the plasma of ovariectomized, estradiol-treated rats was advanced by 2 hr after 7 days of treatment with CrMP (4 micro M/kg), and this effect was reversed when hemin (30 micro M/kg) was co-administered with CrMP. The afternoon follicle-stimulating hormone (FSH) surge was not affected by either treatment. In contrast, the afternoon prolactin (PRL) surge was completely blocked or delayed by CrMP treatment, and this effect was not reversed by hemin. In vitro perifusion of pituitary explants with CrMP also significantly reduced PRL release compared with secretion from untreated explants. In vitro gonadotropin-releasing hormone (GnRH)-stimulated FSH secretion was significantly increased from pituitary explants of ovariectomized, estradiol-treated rats treated in vivo with hemin but was unaffected by CrMP treatment, whereas GnRH-stimulated LH release was not affected by hemin but was increased by CrMP treatment. In conclusion, this study demonstrates that HO exists in the rat anterior pituitary gland, and that a substrate and an inhibitor of this enzyme alter the secretion of gonadotropins and PRL.     

Changes in pituitary responsiveness during the ovulatory cycle of the Japanese quail, in vitro

Anterior pituitary glands from ovulating Japanese quail (Coturnix coturnix) were used to investigate variation in sensitivity to chicken luteinizing hormone-releasing hormone (cLHRH I; Gln8-LHRH). Grouping the pituitaries by ovulatory stage provided preliminary evidence of changes in sensitivity to LHRH during the ovulatory cycle. Pituitaries taken from quail before the preovulatory LH surge were responsive to cLHRH I, while pituitaries from the other times of the cycle showed minimal response to cLHRH I. Female pituitary glands release less LH than those of males. These data indicate a change in sensitivity to LHRH in the female quail that may be due to changes in gonadal steroids or the pool of releaseable LH from the pituitary.     

Binding studies of substance P anterior pituitary binding sites: changes in substance P binding sites during the rat estrous cycle

Previous studies have shown that substance P (SP), an undecapeptide widely distributed in the gastrointestinal tract and in the peripheral and central nervous system, is a putative regulatory peptide involved in the control of reproductive function. Specifically, SP inhibited, at the anterior pituitary (AP) level, the stimulatory action of a physiological concentration (10(-8) M) of Gonadotropin Releasing Hormone (GnRH) on the release of the luteinizing hormone (LH). In the present work, we have demonstrated the presence of specific SP binding sites in the AP and related changes in the number of these sites to GnRH receptor number, hypothalamic SP and GnRH content and LH secretion during the rat estrous cycle. High affinity saturable SP binding sites (Kd, 1.5 approximately equal to 10 nM) were demonstrated in AP membranes using [3H]-SP or a novel analog, [125I]-(D-Tyr0, NorLeu11)SP. The binding affinity of SP fragments decreased with progressive removal of amino acid residues from N or C termini of the molecule. Other neuropeptides had low affinity for the SP binding sites. During the rat estrous cycle, SP and GnRH binding capacity of the anterior pituitary were inversely related. At the time of the proestrous LH surge, the AP binding capacity was low for GnRH but high for SP. The highest content of SP in the hypothalamus were recorded during the afternoon of proestrus when hypothalamic GnRH levels were lowest and the preovulatory surge occurred. These studies have established the presence of high affinity specific binding sites for SP in the AP which alter during the estrous cycle in a manner appropriate for mediating the direct inhibitory effects of SP on LH release in vitro.     

A change in basal FSH release accompanies the onset of the second or selective phase of increased serum FSH in the cyclic rat

Experiments were undertaken to investigate if changes occur at the level of the anterior pituitary gland to result in selective follicle-stimulating hormone (FSH) release during late proestrus in the cyclic rat. At 1200 h proestrus, prior to the preovulatory luteinizing hormone (LH) surge in serum and the accompanying first phase of FSH release, serum LH and FSH concentrations were low. At 2400 h proestrus, after the LH surge and shortly after the onset of the second or selective phase of FSH release, serum LH was low, serum FSH was elevated about 4-fold, pituitary LH concentration was decreased about one-half and pituitary FSH concentration was not significantly decreased. During a two hour $\text{in}$$\text{vitro}$ incubation, pituitaries collected at 2400 h released nearly two-thirds less LH and 2.5 times more FSH than did pituitaries collected at 1200 h. Addition of luteinizing hormone releasing hormone (LHRH) to the incubations caused increased pituitary LH and FSH release. However, the LH and FSH increments due to LHRH in the 2400 h pituitaries were not different from those in the 1200 h pituitaries. The results indicate that a change occurs in the rat anterior pituitary gland during the period of the LH surge and first phase of FSH release which results in a selective increase in the basal FSH secretory rate. It is suggested that this change is primarily responsible for the selective increase in serum FSH which occurs during the second phase of FSH release.     

Pituitary gonadotropin-releasing hormone receptor content in rats with polycystic ovaries

A single injection of estradiol valerate (EV) induces, after a lag period of 4-6 wk, a chronic anovulatory polycystic ovarian (PCO) condition in adult rats. This condition is associated with a selective compromise of luteinizing hormone (LH) release and/or synthesis reflected in low basal serum LH concentrations, decreased pituitary content of LH, and decreased gonadotropin-releasing hormone (GnRH)-stimulated LH secretion. The present study was undertaken to determine to what extent the aberrant LH release in rats with PCO could be related to alterations in pituitary content of GnRH receptors. Pituitary GnRH-receptor content was assessed by the evaluation of saturation binding of a GnRH analog, [125I]-D-Ala6-des-Gly10-GnRH, to pituitary membrane preparations. The receptor content of pituitaries from rats with PCO was compared to that obtained from intact animals at estrus and diestrus. Receptor levels in ovariectomized normal rats and rats with PCO were also assessed. The pituitary GnRH receptor content in PCO rats was similar to that observed in normal controls at estrus and was significantly lower than that for rats at diestrus. Although a twofold increase in pituitary GnRH receptor content was observed at 28 days following the castration of control rats, GnRH receptor content in the pituitaries of PCO rats, at 28 days following ovariectomy, remained unchanged. Although, castration-induced elevations in mean serum LH and follicle-stimulating hormone (FSH) concentrations were observed in both the PCO and control animals, the rise in both gonadotropins was significantly attenuated in the PCO-castrates when compared to the ovariectomized controls. Since GnRH is a major factor in the regulation of pituitary GnRH receptor content, these findings suggest that hypothalamic GnRH release is impaired in rats with PCO and that this impairment is independent of any influences from the polycystic ovaries.     

A secondary surge of prolactin on the estrus afternoon

It has been described that throughout the estrous cycle of the rat, plasma prolactin (PRL) is basal except on proestrus afternoon when a preovulatory surge occurs. However, there have been controversies about PRL levels on the estrus day. Thus, the aim of this study was to evaluate the existence of a secondary surge of PRL on estrus afternoon and correlate it with plasma estradiol levels. The jugular vein of cycling rats was cannulated at 14:00 h on proestrus and a blood sample was withdrawn at 17:00 h for plasma LH measurement and determination of the preovulatory LH surge occurrence. In order to exclude the regular cycling rats that do not present the gonadotropins preovulatory surge and do not ovulate, only rats showing the LH surge on proestrus were considered in this study. Blood samples were collected hourly during estrus from midnight to 9:00 h (group 1) and from 10:00 to 18:00 h (group 2). In group 1, PRL showed a descending profile from midnight to 9:00 h, whereas the estradiol concentrations were constant. In group 2, a secondary surge of PRL was observed in 20 of 25 (80%) rats and plasma estradiol remained constant, but was higher in animals with the PRL surge. Thus the present data suggest the occurrence of a secondary surge of PRL in the afternoon of estrus that seems to be related to plasma estradiol levels of estrus day, which might exert only a permissive role in this surge generation.     

Subpopulations of luteinizing hormone (LH) possessing various ratios of bioactivity to immunoreactivity in the female rat pituitary glands and their changes during the estrous cycle

Twenty-four adult female Sprague-Dawley rats (3 from each of 8 litters), showing 4-day cycles, were used in the present study. Aqueous extracts of pools of 6 pituitary glands in each cycle date were fractionated with a column isoelectrofocusing (IEF) technique, pH range of 3.5-10. Biological and immunological LH activities were determined by an in vitro bioassay and a radioimmunoassay, respectively, in the original aqueous extracts of the pituitary glands and in the fractions separated by IEF. Pituitary content of LH was the highest in the proestrus before the preovulatory LH surge (1243.7 +/- 67.8 micrograms NIAMDD rat LH-RP-1/pituitary gland for the biological activity). In the estrus, after the LH surge, it was reduced to 688.9 +/- 51.2 micrograms/pituitary gland. The decreased pituitary content was recovered to the level in the proestrus during the metestrus and the diestrus (1047.0 +/- 53.8 and 1173.0 +/- 58.5 micrograms/pituitary gland, respectively). Rat LH in the pituitary aqueous extracts was separated into multiple subpopulations in terms of pI values by IEF; i.e. Subpopulations A (pI = 10.3), B (9.3), C (9.0), D (8.7), E (8.3), F (neutral LH), and G (acidic LH). Among them the most predominant one was Subpopulation A throughout the estrous cycle. Subpopulations A, B and C exhibited statistically significant cyclic changes as was observed in the pituitary LH content, whereas the remaining ones stayed at constant levels during the cycle. The highest ratio of biological to immunological LH activities (B/I ratio) was obtained in Subpopulation A (6.41), followed by G, C and B (5.15, 4.24 and 3.99, respectively). Depressed B/I ratios were revealed in D, E and F (2.59, 1.86 and 3.07, respectively). High alkaline LH subpopulations, i.e. A, B and C, preserving high biological potency and showing cyclic changes during the estrous cycle, seem to be the releasable types of the hormone and to be mainly discharged for the preovulatory LH surge. Although characteristic features of other types of the hormone are not known, it is possible that one of them, presumably the acidic LH, might be the newly-synthesized type of the hormone, which might attain releasability by certain molecular modifications involving a shift in the pI value.     

Decline in circulating estradiol during the periovulatory period is correlated with decreases in estradiol and androgen, and in messenger RNA for p450 aromatase and p450 17alpha-hydroxylase, in bovine preovulatory follicles

The preovulatory surge of gonadotropins induces meiotic maturation of the oocyte, the follicular/luteal phase shift in hormone production, and ovulation. This complex and rapid series of developmental changes is difficult to study in large mammals, such as primates and ruminants, because variability in the length of individual reproductive cycles makes it virtually impossible to predict the time of the LH surge. We have validated an experimental model for inducing the LH surge and ovulation in cattle and used it to study the sequence of changes in hormone secretion and some of the mechanisms of these changes. Luteolysis and a follicular phase were induced by injection of prostaglandin F(2alpha); injection of a GnRH analogue 36 h later induced an LH surge and ovulation. The LH surge peaked 2 h after GnRH and ovulation followed 22-31 h after the surge, consistent with the periovulatory interval in natural cycles. The ensuing luteal phase was normal, both in length and in concentrations of circulating progesterone. In experiment I, the uteroovarian effluent was collected, via cannulation of the vena cava, at frequent intervals relative to GnRH injection. Circulating estradiol declined progressively after GnRH, reaching a nadir by 8-10 h before ovulation, whereas concentrations of androstenedione and testosterone remained constant. In experiment II, preovulatory follicles were obtained at 0, 3.5, 6, 12, 18, or 24 h after GNRH: Concentrations of androgens and estradiol were measured in follicular fluid and medium from cultures of follicle wall (theca + granulosa cells); steady-state levels of mRNA for 17alpha-hydroxylase (17alphaOH) and P450 aromatase were measured in follicular tissue. Shortly after the LH surge (3.5 h post-GnRH) there was an acute increase in the capacity of follicular tissue to secrete androstenedione, but not estradiol, in vitro. Thereafter, both androgens and estradiol declined, both in follicular fluid and in medium collected from cultures of follicle wall. Levels of mRNA for 17alphaOH and aromatase in follicle wall decreased significantly by 6 h after GnRH, suggesting that declining levels of these enzymes underlie the decreases in steroid production by follicular cells. These results show that in cattle the preovulatory decrease in follicular estradiol production is mediated by redundant mechanisms, because androgen production and the capacity of granulosa cells to convert androgens to estradiol decline coordinately, in concert with decreases in mRNA for 17alphaOH and P450 aromatase.     

No evidence for pituitary priming to gonadotropin-releasing hormone in relation to luteinizing hormone (LH) secretion prior to the preovulatory LH surge in ewes

The purpose of this study was to determine the occurrence of and the regulatory mechanisms involved in priming of the pituitary to GnRH before the preovulatory LH surge in sheep. Experiment 1: Forty-two ewes had progestagen devices removed after 14 days and were assigned to luteal (Lut) or follicular (Foll) groups. Fifteen days later, blood sampling was initiated either immediately or 36 h after induced luteolysis in groups Lut and Foll, respectively. After 4 h, ewes were administered either saline (n = 5) or 250 ng (n = 8) or 10 microg (n = 8) of GnRH. Five ewes per treatment group were killed 1 h later, while remaining animals were blood sampled for a further 7 h. Experiment 2: Eighteen ewes were allocated to Lut and Foll groups (described above). Blood samples were collected from 2 h before GnRH (10 microg) treatment until 7 h after. Despite up-regulated GnRH-R mRNA levels in Foll ewes, pituitary content and plasma levels of LH and LHbeta mRNA levels were similar between groups. Mean FSHbeta mRNA and plasma FSH levels were elevated in Lut ewes but declined after GnRH treatment. Inversely, plasma estradiol and inhibin-A concentrations were higher in Foll ewes and declined after GnRH treatment. Fewer LH(+ve)/secretogranin II(-ve) (SgII(-ve)) granules were present in gonadotropes of Foll ewes, coincident with increased basal LH levels. Fewer smaller sized granules were present after GnRH treatment. In conclusion, there was no evidence of self-priming before onset of the preovulatory LH surge. Constitutive release of LH(+ve)/SgII(-ve) granules may maintain basal LH levels while smaller sized, presumably mature granules may be preferentially released after GnRH stimulation.     

Daily variations in the sensitivity of proestrous LH surge in the inhibitory effect of intraventricular injection of 5-HT or GABA in rats.

Intraventricular injection of 5-hydroxytryptamine (5-HT) into female rats at 11:00 h on the day of proestrus inhibited the preovulatory surge of luteinizing hormone (LH) and ovulation. A similar response was observed after the activation of the serotonergic system by stimulation of the median raphe nucleus. A diurnal rhythm of these responses was observed. In rats acclimated to a 14-h:10-h light:dark cycle the potency of 5-HT to inhibit the LH surge and ovulation was 2.06 and 2.3 times greater, respectively, when injected at 11:00 h than at 13:00 h. Also stimulation of the median raphe nucleus at 11:00 h was significantly more effective in inhibiting these parameters than stimulation at 13:00 h. Similarly, the ability of gamma-amino-butyric acid (GABA) to inhibit the preovulatory LH surge and ovulation was greater in rats injected in the morning than in the afternoon. The results of this study indicate that during proestrus the sensitivity of 5-HT and GABA to induce inhibition of preovulatory LH release and ovulation shows daily variations with maximal effect before the critical period.     

Induced ovulation and changes in pituitary responsiveness to continuous infusion of gonadotropin-releasing hormone during the ovarian cycle in the bullfrog, Rana catesbeiana

Chronic (2-4 days) constant-rate infusions of mammalian gonadotropin releasing hormone (GnRH) were performed in female bullfrogs, Rana catesbeiana. The magnitude and temporal relationship of profiles of plasma follicle-stimulating hormone (FSH), luteinizing hormone (LH) and sex steroids [testosterone (T), estradiol-17 beta (E2) and progesterone (P)] during GnRH infusion were dependent on ovarian stage. However, in all females, the same biphasic increase in plasma gonadotropins was apparent and initial elevations in gonadotropins were accompanied by correlated increments in plasma T and E2. Complete pituitary "desensitization" to chronic GnRH infusion was not observed. Females in early follicular stages were relatively unresponsive to infusions of 1.0-10.0 micrograms/h GnRH; elevations in plasma LH were marginal and FSH was unchanged. Females with fully developed (preovulatory) ovaries were more responsive: infusion of 1.0 micrograms/h GnRH produced significant elevations in plasma LH by 2 h followed by even larger increases ("surges") after 12 h. This LH "surge" was preceded by a decline in plasma T and E2 and was accompanied by abrupt elevations in plasma P and by ovulation. Postovulatory females showed a more gradual and smaller increase in plasma LH. Infusion of GnRH in the female bullfrog establishes a clear relationship between pituitary responsiveness and the ovarian cycle not evident from acute GnRH injection; GnRH was most effective immediately before ovulation. These data are also the first to detail periovulatory changes in plasma gonadotropins and ovarian steroids in an amphibian.     

Differential regulation of gonadotropin subunit messenger ribonucleic acids by gonadotropin-releasing hormone pulse frequency in ewes

Changes in the frequency of GnRH and LH pulses have been shown to occur between the luteal and preovulatory periods in the ovine estrous cycle. We examined the effect of these different frequencies of GnRH pulses on pituitary concentrations of LH and FSH subunit mRNAs. Eighteen ovariectomized ewes were implanted with progesterone to eliminate endogenous GnRH release during the nonbreeding season. These animals then received 3 ng/kg body weight GnRH in frequencies of once every 4, 1, or 0.5 h for 4 days. These frequencies represent those observed during the luteal and follicular phases, and the preovulatory LH and FSH surge of the ovine estrous cycle, respectively. On day 4, the ewes were killed and their anterior pituitary glands were removed for measurements of pituitary LH, FSH, and their subunit mRNAs. Pituitary content of LH and FSH, as assessed by RIA, did not change (P greater than 0.10) in response to the three different GnRH pulse frequencies. However, subunit mRNA concentrations, assessed by solution hybridization assays and expressed as femtomoles per mg total RNA, did change as a result of different GnRH frequencies. alpha mRNA concentrations were higher (P less than 0.05) when the GnRH pulse frequency was 1/0.5 h and 1 h, whereas LH beta and FSH beta mRNA concentrations were maximal (P less than 0.05) only at a pulse frequency of 1/h. Additionally, pituitary LH and FSH secretory response to GnRH on day 4 was maximal (P = 0.05) when the pulse infusion was 1/h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Peptides interact in gonadotrophin regulation

The regulation of luteinizing hormone (LH) activity is vital to normal reproductive functioning of the female. Although gonadotrophin-releasing hormone (GnRH) has a prominent role in the regulation of LH it is now believed that other peptides are also involved. Among these peptides is oxytocin. The addition of oxytocin to cultures of pituitary cells from female rats elicited a concentration-dependent secretion of LH. This secretion was enhanced in an oestrogenised environment and was inhibited by progesterone and testosterone. Oxytocin administered to female rats at pro-oestrus advanced the endogenous LH surge that occurs on the evening of pro-oestrus. Conversely oxytocin receptor antagonist suppressed the production of the LH surge in a dose-dependent manner, indicating that endogenous oxytocin is a crucial component of LH regulation. In the human female, oxytocin administered during the late follicular phase advanced the onset of the midcycle LH surge. Oxytocin added to rat pituitary cells in vitro induced LH synthesis. Furthermore rats administered oxytocin on pro-oestrus had higher LH pituitary content following development of the LH surge than did rats administered saline. Thus oxytocin promoted synthesis and replacement in the pituitary of LH released into the circulation. Incubation of pituitary pieces with oxytocin plus GnRH induced secretion of amounts of LH greater than the sum of the amounts released by oxytocin and GnRH separately. Additionally the increased LH levels observed in the peripheral circulation of pentobarbitone-anaesthetised rats administered GnRH were enhanced if the rats received oxytocin prior to the GnRH. Thus oxytocin synergised with GnRH in stimulating LH release. Addition of diBucAMP reduced the oxytocin-mediated augmentation and dideoxyadenosine enhanced the augmentation, suggesting that oxytocin worked most efficiently in a milieu low in cAMP activity. The use of a cell immunoblot assay revealed that individual cells responded differently to oxytocin and to GnRH and that the two peptides could act on the same cell. Perifusion studies performed on hemipituitaries demonstrated that a LH response could be determined by the presence of three peptides, oxytocin, neuropeptide Y and GnRH. Hence oxytocin is potentially involved also in multiple interactions during the process of LH regulation. LH regulation is therefore apparently the result of a community of peptides acting in a co-operative network.     

Interrelationship between estrogen and thyroxine on the release of luteinizing hormone and gonadotropin-releasing hormone in vitro

The present experiments were designed to study the interaction between estradiol benzoate (EB) and thyroxine (T4) given in vivo on the responsiveness of pituitary luteinizing hormone (LH) to gonadotropin-releasing hormone (GnRH) and the release of GnRH in vitro. Ovariectomized-thyroidectomized (Ovx-Tx) rats were injected s.c. with saline or T4 (2 micrograms/100 g b.wt), and oil or EB (0.1 microgram) once daily for 40 days following a 2 x 2 factorial design. All animals were then decapitated and blood samples were collected. Anterior pituitaries (APs) were incubated in vitro with and without 0.1 ng GnRH at 37 degrees C for 4 h. Mediobasal hypothalami (MBHs) were excised and then incubated with and without APs from Ovx donor rats. Concentrations of LH and GnRH in the medium and that of LH in the serum were measured by radioimmunoassay. The LH level in media containing MBHs and donor APs was used as the index of bioactive GnRH release. In Ovx-Tx rats, T4 injections reduced the serum LH concentration, the pituitary LH response to GnRH, and the bioactive as well as the immunoreactive GnRH release. The serum LH levels and the spontaneous as well as the GnRH-stimulated release of LH in vitro were suppressed in Ovx-Tx rats following administration of EB. By contrast, the serum LH concentration, as well as pituitary LH response to GnRH and GnRH release in vitro, were higher in the group treated with both T4 and EB than in that treated with saline and EB. These results suggest that the differential changes in the LH secretion after thyroidectomy of Ovx versus non-Ovx rats are due to an antagonistic effect between T4 and estrogen on the response of pituitary LH to GnRH, and the release of GnRH.     

Interaction between ovarian and adrenal steroids in the regulation of gonadotropin secretion

Recent work from our laboratory suggests that a complex interaction exists between ovarian and adrenal steroids in the regulation of preovulatory gonadotropin secretion. Ovarian estradiol serves to set the neutral trigger for the preovulatory gonadotropin surge, while progesterone from both the adrenal and the ovary serves to (1) initiate, (2) synchronize, (3) potentiate and (4) limit the preovulatory LH surge to a single day. Administration of RU486 or the progesterone synthesis inhibitor, trilostane, on proestrous morning attenuated the preovulatory LH surge. Adrenal progesterone appears to play a role in potentiating the LH surge since RU486 still effectively decreased the LH surge even in animals ovariectomized at 0800 h on proestrus. The administration of ACTH to estrogen-primed ovariectomized (ovx) immature rats caused a LH and FSH surge 6 h later, demonstrating that upon proper stimulation, the adrenal can induce gonadotropin surges. The effect was specific for ACTH, required estrogen priming, and was blocked by adrenalectomy or RU486, but not by ovariectomy. Certain corticosteroids, most notably deoxycorticosterone and triamcinolone acetonide, were found to possess "progestin-like" activity in the induction of LH and FSH surges in estrogen-primed ovx rats. In contrast, corticosterone and dexamethasone caused a preferential release of FSH, but not LH. Progesterone-induced surges of LH and FSH appear to require an intact N-methyl-D-aspartate (NMDA) neurotransmission line, since administration of the NMDA receptor antagonist, MK801, blocked the ability of progesterone to induce LH and FSH surges. Similarly, NMDA neurotransmission appears to be a critical component in the expression of the preovulatory gonadotropin surge since administration of MK801 during the critical period significantly diminished the LH and PRL surge in the cycling adult rat. FSH levels were lowered by MK801 treatment, but the effect was not statistically significant. The progesterone-induced gonadotropin surge appears to also involve mediation through NPY and catecholamine systems. Immediately preceding the onset of the LH and FSH surge in progesterone-treated estrogen-primed ovx. rats, there was a significant elevation of MBH and POA GnRH and NPY levels, which was followed by a significant fall at the onset of the LH surge. The effect of progesterone on inducing LH and FSH surges also appears to involve alpha 1 and alpha 2 adrenergic neuron activation since prazosin and yohimbine (alpha 1 and 2 blockers, respectively) but not propranolol (a beta-blocker) abolished the ability of progesterone to induce LH and FSH surges. Progesterone also caused a dose-dependent decrease in occupied nuclear estradiol receptors in the pituitary.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.