Modulation of endogenous opioid influence on luteinizing hormone secretion by progesterone and estrogen

Studies were undertaken using the opiate receptor antagonist naloxone (NAL) to evaluate the relative influence of endogenous opioid peptides (EOP) on LH secretion in cycling and ovariectomized, steroid-treated adult rats. Intact animals received NAL (2 mg/kg, sc) or saline (control vehicle) at 0800 and 1400 h on estrus, 0800 h on diestrus day 1,2000 h on diestrus day 2, and before (at 0800, 1200, and 1400 h) and during the preovulatory LH surge (at 1600 and 1800 h) on proestrus. NAL stimulated LH release by 2- to 3-fold at all stages of the estrous cycle, including during the proestrous gonadotropin surge. Ovariectomized rats were treated with estradiol benzoate (EB; 7.5 micrograms/rat, sc) and 2 days later received NAL (2 mg/kg, sc) or saline at 1000, 1200, 1400, 1600, and 2000 h. NAL induced a relatively small (44-73%), but significant, increase in LH release before (1000, 1200, and 1400 h), during (1600 h), and after (2000 h) the afternoon LH rise. While progesterone (P) treatment (5 mg, sc, day 2 at 1000 h) of EB-primed ovariectomized rats augmented NAL-induced LH release before the LH surge (1200 h), it abolished the LH secretory response to NAL during the LH surge (1400, 1600, and 1800 h). The NAL-induced LH response returned after the LH surge at 2000 h. Likewise, administration of P on proestrus morning (0900 h) abolished the LH secretory response to NAL during the LH surge. These studies indicate that central opioid neurons participate in the tonic inhibition of LH secretion at all stages of the estrous cycle of the rat. The ability of exogenous P to advance and amplify the LH surge on proestrus and in EB-primed ovariectomized rats appears to result in part from a reduction in the EOP inhibitory influence on LH secretion and may indicate a role for EOP in mediating the stimulatory effects of endogenous steroids on LH secretion in the female rat.     

Alpha and luteinizing hormone beta subunit messenger ribonucleic acids during the rat estrous cycle

Alpha and LH beta subunit mRNAs were measured in pituitaries of 4-day cycling rats during the estrous cycle. A two-fold increase in alpha mRNA occurred between 0800-2000 h on diestrus, but alpha mRNA concentrations were stable during other days of the cycle. LH beta mRNA concentrations were low during estrus and metestrus (11-16 pg cDNA bound/100 micrograms pituitary DNA), but were elevated (27-30 pg) between 0800-2000 h on diestrus. A second increase in LH beta mRNA was observed on the afternoon of proestrus, prior to the onset of the LH surge with maximum values (45 pg) coincident with peak LH secretion. LH beta mRNA concentrations declined rapidly and had fallen to basal values by midnight on proestrus. These data show that alpha and LH beta mRNAs change in a similar manner during metestrus, diestrus and estrus, suggesting coordinate regulation of alpha and LH beta gene expression at these times. During the LH surge, however, LH beta mRNA alone is increased, suggesting that the LH beta gene can be differentially expressed at times when maximum LH secretion is occurring.     

Increase in luteinizing hormone content occurs in cultured human fetal pituitary cells exposed to gonadotropin-releasing hormone

To investigate the mechanisms by which GnRH regulates LH production during intrauterine life, dispersed pituitary cells from second trimester human fetuses were cultured on extracellular matrix-coated plates for 48 h. Exposure of cells to 3 x 10(-10) mol/L GnRH for 1-48 h significantly increased cumulative LH secretion compared to that in respective controls (P less than 0.01). The rate of GnRH-stimulated LH release was accelerated during the first 6 h, after which it declined to a level similar to that of basal release. This phenomenon was associated with a decrease in the GnRH concentration of the medium. Exposure of cells to GnRH (3 x 10(-10) to 10(-6) mol/L) for 48 h induced a dose-dependent elevation of total LH which correlated with an increase in releasable, but not cellular, LH. Desensitization to GnRH (10(-7) mol/L) occurred when cells were cultured with pharmacological amounts of GnRH for 48 h. These results indicate that GnRH induces the increase in total and releasable LH in human fetal pituitary cells. These cells also appear to inactivate GnRH. Thus, GnRH may increase LH production in the human fetal pituitary and the pituitary receptor mechanism may be involved in GnRH action on LH release during intrauterine life.     

Progesterone secretion by granulosa cells from rats with four- or five-day estrous cycles: the development of responses to follicle-stimulating hormone, luteinizing hormone, and testosterone

Changes in granulosa cell sensitivity and responsiveness to gonadotropins during the rat estrous cycle were studied by measuring progesterone (P) secretion in vitro in response to treatment with increasing doses of LH or FSH (0, 0.1, 1, 10, 100 ng/ml). The effect of testosterone [(T); 0.5 microM] on response to gonadotropins was also examined. Granulosa cells were isolated from the largest ovarian follicles of rats with 4- and 5-day estrous cycles at 0800 h, 1400 h, and 2000 h on proestrus and on the preceding day of diestrus at 1200 h and 2000 h. In rats with 5-day cycles, granulosa cells were also obtained at 1200 h on the first day of diestrus. Fifty percent maximal P production and 50% effective dose (ED50-dose of gonadotropin which elicited 50% maximal P production) were calculated from dose-response curves for LH and FSH and were used as measures of responsivity and sensitivity to gonadotropins, respectively. Basal P secretion and 50% maximal P secretion increased progressively as cells were isolated at later stages of follicular growth in both 4- and 5-day cycles. In cells from 5-day rats, however, basal and gonadotropin-stimulated P secretion were higher on the second day of diestrus than in cells from 4-day rats. By proestrus responsiveness was equal. Granulosa cell sensitivity to FSH was constant during 4- and 5-day cycles, as indicated by a lack of change in the ED50. Granulosa cell sensitivity to LH was lower than sensitivity to FSH on diestrus of both 4- and 5-day cycles. However, by the morning of proestrus sensitivity to LH increased and was similar to that for FSH. T increased basal P production only slightly, but synergized with both LH and FSH to stimulate 2-fold increases in 50% maximal P production by granulosa cells isolated at all times except 2000 h on proestrus, after the endogenous LH surge. T had no effect on the sensitivity (ED50) of granulosa cells to LH or FSH. In summary, granulosa cell responsiveness to LH and FSH increased in parallel during the final stages of follicular growth, but increased sensitivity was noted only for LH. The development of granulosa cell capacity to secrete P appears to be more advanced in 5-day rats than in 4-day rats relative to the next estrus. Because T synergized with LH and FSH to increase P secretion without altering sensitivity to gonadotropins, it probably acts at a site distal to gonadotropin receptors.     

Changes in cellular levels of messenger ribonucleic acid encoding gonadotropin-releasing hormone in the anterior hypothalamus of female rats during the estrous cycle

Cellular levels of messenger RNA encoding GnRH were measured using quantitative in situ hybridization in coronal sections through the area of the organum vasculosum of the lamina terminalis of female rats examined at various times of the 4-day estrous cycle. GnRH mRNA levels were high on the morning of diestrus day 1, but declined throughout the day of diestrus day 2 to a nadir on the morning of proestrus. Although GnRH message levels were lowest on the morning of proestrus, they rose nearly two-fold by 1900h that evening and remained high during the day of estrus. These data support the hypothesis that GnRH synthesis is coupled to GnRH release, and indicate that GnRH biosynthesis is not stimulated on the morning of proestrus in preparation for the ovulatory surge release of GnRH and LH in the afternoon.     

Growth-associated protein-43 messenger ribonucleic acid expression in gonadotropin-releasing hormone neurons during the rat estrous cycle

We have shown previously at the ultrastructural level that morphological changes occur in the external zone of the median eminence allowing certain GnRH nerve terminals to contact the pericapillary space on the day of proestrus. The present study was designed to determine whether the intrinsic determinant of neuronal outgrowth, growth-associated protein-43 (GAP-43), was expressed in GnRH neurons of adult female rats, and whether its expression varied throughout the estrous cycle. To accomplish this, we perfusion-fixed groups of adult female rats at 0800 and 1600 h on diestrous day 2 (diestrous II), at 0800 h and 1600 h on proestrus, and at 0800 and 1600 h on estrus (n = 4 rats/group) and used double labeling in situ hybridization and quantification to compare the levels of GAP-43 messenger RNA (mRNA) in cells coexpressing GnRH mRNA. GnRH mRNA was detected with an antisense complementary RNA (cRNA) probe labeled with the hapten digoxigenin, whereas the GAP-43 cRNA probe was labeled with 35S and detected by autoradiography. In addition, GAP-43 protein was identified with immunohistochemistry in the median eminence. The results show that many GnRH neurons expressed GAP-43 mRNA and that GAP-43 protein was present in many GnRH axon terminals in the outer layer of the median eminence. The number of GnRH neurons expressing GAP-43 mRNA was significantly higher on proestrus (64 +/- 5%) than on diestrous II (40 +/- 2%; P < 0.001) or on estrus (45 +/- 8%; P < 0.05), and the GAP-43 mRNA levels in GnRH neurons also varied as a function of time of death during the estrous cycle. The GAP-43 mRNA levels in GnRH neurons were higher on proestrus and estrus than on diestrous II (P < 0.05). These data show that 1) GAP-43 is expressed in adult GnRH neurons; 2) GAP-43 mRNA expression in GnRH neurons fluctuates during the estrous cycle; and 3) GAP-43 mRNA content in GnRH neurons is highest on the day of proestrus, before and during the onset of the LH surge. These observations suggest that the increased GAP-43 mRNA expression in GnRH neurons on the day of proestrus could promote the outgrowth of GnRH axon terminals to establish direct neurovascular contacts in the external zone of the median eminence and thus facilitate GnRH release into the pituitary portal blood.     

Effect of pulsatile gonadotropin-releasing hormone on the release of luteinizing hormone and follicle-stimulating hormone in vitro by anterior pituitaries from lactating and cycling rats

The ability of pituitaries from lactating animals to secrete LH and FSH in response to gonadotropin-releasing hormone (GnRH) was studied in vitro using a pituitary incubation system. Hemipituitaries were exposed to GnRH for 6 min during each hour of incubation. LH release by anterior pituitaries (APs) from day 5 postpartum rats nursing eight pups, in response to pulsatile exposure to GnRH, was significantly less than that released by APs from diestrous cycling females. Even though the amount of LH released by APs increased as lactation progressed, LH release by APs from day 15 postpartum rats nursing eight pups was still less than LH release by APs from diestrous females. In contrast pituitaries from lactating females nursing two pups released amounts of LH similar to that released by pituitaries from diestrous females, whereas females deprived of their litters for 48 h showed a greater response than diestrous females. Generally, there was a good quantitative relationship between the amount of LH released in vitro and plasma LH concentrations for all the intact groups studied. The ability of lactation to suppress the postcastration rise in serum LH also was demonstrated in vitro as pituitaries from ovariectomized or intact females nursing eight pups released similar amounts of LH on days 5 and 10 postpartum. However, by day 15 postpartum, even though serum LH concentrations were still very low, pituitaries from ovariectomized lactating females released LH in vitro at a rate similar to pituitaries from nonlactating rats. Serum FSH concentrations were not suppressed but similar in intact and cycling females. Also, the total amount of FSH released in vitro in response to GnRH by pituitaries from lactating and cycling females did not differ significantly, even though LH release differed greatly among these groups of animals. However, the patterns of GnRH-stimulating FSH secretion differed among intact lactating, ovariectomized lactating, and nonlactating females. Pituitary LH concentrations were similar on day 5 postpartum and diestrus and on day 15 postpartum and proestrus. Pituitary FSH concentrations on day 5 postpartum were similar to those during diestrus and proestrus and had increased 2-3 times by day 15 postpartum. Generally, there was no correlation between the amount of LH or FSH released by pituitaries in response to GnRH and pituitary gonadotropin content. In summary, the inability of pituitaries from lactating rats to respond adequately to large doses of GnRH in vitro suggests that the suckling stimulus indirectly suppresses pituitary responsiveness to GnRH. This suppression differentially affects basal LH secretion, but not basal FSH secretion, and may be the direct result of inadequate GnRH stimulation in vivo.     

Cytological factors that support nonparallel secretion of luteinizing hormone and follicle-stimulating hormone during the estrous cycle

Gonadotropes from cycling female rats were studied to investigate possible mechanisms for the nonparallel release of LH and FSH. The percentages of total gonadotropes increased from 14% during estrus (E) to 18% by diestrous day 2. More of these cells became multihormonal on the morning of proestrus (P; from 46% during diestrus to 69%). Since LH-containing cells increased from 7% at E to 13.3% during early proestrus, this suggests that monohormonal FSH cells may have contributed by synthesizing LH. Gonadotrope cell areas were greatest just before the LH surge (P 1600 h). Microdensitometric measurements demonstrated that the amount and density of immunoperoxidase stain for either gonadotropin subunit were highest during the midafternoon of P. Interestingly, the amount of stain for LH continued to increase during the LH surge, suggesting that the stain had detected newly synthesized LH beta. At the same time, the average density of the LH beta stain decreased. In contrast, the amount, concentration, and density of stain for FSH beta increased during the afternoon of P and decreased during late P and early E. The percentages of granules that contained immunogold stains for only LH or FSH (monohormonal granules) at P 1600-P 1700 h were 3-4 times higher than those in diestrous rats. The percentages of monohormonal LH granules declined during the proestrous surge, whereas percentages of monohormonal FSH granules declined during the first rise (P 1900 h) and after the second rise in serum FSH (E 0800 h). Finally, the average number of gold particles per micron 2 granule area rose over the value in diestrous rats during P 1600-P 1700 h. These studies suggest that multihormonal gonadotropes support nonparallel gonadotropin release by changing the rate of subunit packaging and transit in the Golgi complex.     

Progesterone modulation of gonadotropin secretion by dispersed rat pituitary cells in culture. I. Basal and gonadotropin-releasing hormone-stimulated luteinizing hormone release

Dispersed, estradiol-treated, rat pituitary cells were cultured to characterize the influences of a physiologic concentration of progesterone (P, 10(-7) M) on gonadotroph responsiveness to gonadotropin-releasing hormone (GnRH). Acute (less than 6 h) P treatment enhanced and chronic (greater than 12 h) treatment suppressed both basal and GnRH-stimulated luteinizing hormone (LH) release. This modulation took place without any change in intracellular LH stores, indicating that the secretory changes are not attributable to changes in LH synthesis, and were not accompanied by similar alterations in basal or thyrotropin-releasing hormone-stimulated prolactin secretion. Moreover, the timing of these responses was fixed since a 10-fold lower P concentration produced only smaller and briefer alterations in LH release. Analyses of the temporal characteristics of effective P stimuli indicated that a brief 6 h exposure to P inhibited GnRH-stimulated LH secretion 18 h later. In contrast, P's acute actions rapidly dissipated following removal of the steroid from the culture medium. Finally, P-induced enhancement and suppression of GnRH-stimulated LH release could be blocked by appropriately timed treatments with protein synthesis inhibitors. Our findings are consistent with the hypothesis that P influences gonadotroph secretory function via the production of specific proteins.     

Effect of naloxone on luteinizing hormone, follicle-stimulating hormone, and prolactin secretion in the different phases of the estrous cycle

It is becoming increasingly clear that the effects exerted by opioid agonists and antagonists on the release of gonadotropins and of PRL may vary according to the endocrine milieu. To investigate this issue further, female rats with a regular 4-day estrous cycle have been injected sc with the opioid antagonist naloxone at different hours of the day, during each of the various days of the estrous cycle. The animals were killed 20 min after the sc administration of naloxone (2.5 mg/kg dissolved in 0.9% saline solution) at 1000 and 1600 h on the first and second day of diestrus and at 1000, 1200, 1400, 1600, 1800, and 2000 h on proestrus and estrus. Animals were killed by decapitation, and trunk blood was collected and assayed for LH, FSH, and PRL. The data obtained from naloxone-treated animals were compared to those derived from controls injected sc with 0.9% saline solution and killed at the same time intervals. The sc injections of naloxone stimulated LH release in every phase of the estrous cycle; however the magnitude of the responses was highly variable. Increases of the order of 700-1.000% were observed during the 2 days of diestrus, at 1000 and 1400 h of the day of proestrus, and at 1600, 1800, and 2000 h of the day of estrus. Much higher responses (of the order of 2.700-3.300%) were observed at 1600 h of the day of proestrus and at 1000, 1200, and 1400 h of the day of estrus. The LH response to naloxone appeared to be obliterated at 1800 and 2000 h of the day of proestrus. Serum levels of FSH and PRL were not affected by the treatment at any of the time intervals considered. These findings suggest that, in normally cycling adult female rats, naloxone exerts a stimulatory effect on LH release during each day of the estrous cycle; that the stimulatory effect of naloxone is minimal at the time of the spontaneous proestrous LH surge; and that the effect of naloxone on LH release is, on the contrary, maximal just before the spontaneous proestrous LH surge and up to 1400 h of the day of estrus. The observation that naloxone does not affect FSH and PRL release underlines once more that the central mechanisms controlling LH, FSH, and PRL secretion are different.     

Luteinizing hormone synthesis in cultured fetal human pituitary cells exposed to gonadotropin-releasing hormone.

To investigate whether GnRH regulates LH synthesis during human development, pituitary cells from second trimester fetuses were incubated with [35S]methionine ([35S]met) and [3H]glucosamine ([3H]gln) for 48 h with 0, 10(-9), and 10(-7) mol/L GnRH. Immunoassayable (i) LH was measured in media and cellular lysates, and dual label scintillation analysis was used to quantitate incorporation of radiolabeled precursors into cells, trichloroacetic acid-precipitable proteins, and immunoprecipitated LH subjected to electrophoresis. Exposure of cells to GnRH did not affect cellular uptake or incorporation of precursors into proteins, but specifically increased total (secreted plus cellular) LH synthesis. Both GnRH concentrations significantly increased iLH release and enhanced secreted and cellular [3H]gln-LH. The secretion of [35S] met-LH was stimulated only by 10(-7) mol/L GnRH. The proportion of newly synthesized LH that was secreted and the 3H/35S ratio of secreted and cellular LH were uninfluenced by GnRH. Although basal LH synthesis was not sex dependent, total iLH content and GnRH-stimulated LH translation were greater in cells from females than in those from males. Therefore, GnRH regulates LH synthesis by second trimester fetal human gonadotrophs without influencing the proportion of total radiolabeled LH that is secreted. The existence of a sex difference in total iLH content and GnRH-stimulated LH translation is consistent with the sexual dimorphism in pituitary LH content occurring during human development.     

Estrogen regulates the gonadotropin-releasing hormone-stimulated secretion of biologically active luteinizing hormone

Estrogen produces time-dependent bidirectional effects on the GnRH-stimulated release of immunoactive LH in various species. To examine estrogen's regulation of biologically active LH secretion in response to pulsatile stimulation by GnRH, we studied estrogen-deficient postmenopausal women basally and during treatment with diethlystilbesterol (DES; 1 mg, orally, daily). Basal and GnRH-stimulated plasma concentrations of bioactive LH were assayed by the in vitro rat interstitial cell testosterone bioassay. GnRH-promoted LH secretory bursts in response to two consecutive stimuli were quantitated by multiple parameter deconvolution analysis. Basal half-lives of LH averaged 171 +/- 17 min (immunoactive) and 223 +/- 10 min (bioactive). Analysis of variance revealed a significant decrease in mean basal plasma bioactive LH concentrations on days 10 and 30 of DES treatment. Mean serum immunoactive LH concentrations fell similarly. DES significantly increased the half-life of immunoactive LH (days 5 and 10), but did not change that of bioactive LH. GnRH self-priming of bioactive LH secretion (increased LH secretory peak 2 compared to peak 1) was demonstrated, with a maximal value on day 10 of DES treatment. In addition, the ratio of the mass of bioactive to immunoactive LH secreted in response to the first GnRH pulse was significantly enhanced by estrogen on day 5, whereas that after the second pulse of GnRH was significantly suppressed on day 30 of DES. The self-priming action of GnRH on bioactive LH release evident in the presence of oral DES was corroborated in a separate group of six women, who were treated for 30 days with 17 beta-estradiol via an intravaginally placed Silastic ring. In conclusion, we infer that estrogen exerts a highly selective effect on the gonadotroph secretory process, such that successive GnRH stimuli result in an increase in the maximal rate and mass of secretion of biologically active LH.     

Follicle-stimulating hormone beta subunit messenger ribonucleic acid concentrations during the rat estrous cycle

Serum follicle-stimulating hormone (FSH), pituitary FSH content and FSH beta subunit mRNA concentrations were measured at 1 to 3h intervals throughout the 4 day estrous cycle in rats. Serum FSH was stable (range 200-320 ng/ml) apart from the biphasic proestrus surge (5 fold elevation) which was present from 1800 h of proestrus through 0800 h on estrus. Basal FSH beta mRNA concentrations from late metestrus through the afternoon of proestrus were 0.10 +/- 0.04 f mol cDNA bound/100 micrograms pituitary DNA. The major increase in FSH beta mRNA began at 2000 h on proestrus, 2 h after the initial rise in serum FSH and peak mRNA concentrations (0.43 +/- 0.08 f mol cDNA bound) occurred at 0200 h on estrus. FSH beta subunit mRNA concentrations were again increased at 2300 h on estrus (peak 0.24 f mol cDNA bound) and remained elevated through 1700 h on metestrus. Pituitary FSH content was transiently increased during metestrus and diestrus, but was elevated at 1000 h through 1900 h on proestrus (peak 5-fold increase). FSH content fell rapidly at 2000 h and remained low until 1400 h on estrus when values again rose. These data show that FSH beta mRNA is increased 4-5 fold during the proestrus FSH surge, and a smaller increase occurs on metestrus in the absence of elevated FSH secretion. The increased concentrations of FSH beta mRNA occurred at different times to the previously reported changes in alpha and LH beta mRNAs. Therefore, the data suggest that different mechanisms are involved in the regulation of LH and FSH beta subunit gene expression during the 4-day estrous cycle in rats.     

Changes in the pulsatile pattern of luteinizing hormone secretion during the rat estrous cycle

To ascertain whether changes in the pattern of GnRH release from the hypothalmus occur during the 4-day rat estrous cycle, the pattern of LH release was characterized on each day of the estrous cycle, and the results were interpreted in light of the changes in pituitary responsiveness to GnRH previously described by this laboratory to occur during this time. Blood samples were taken from intact, freely moving rats via venous catheters at 6- to 10-min intervals for 3-4 h. LH pulse height and LH interpulse interval were quantified on each day of the cycle, and the transition on the afternoon of proestrus from tonic LH release to the preovulatory LH surge was detailed. The effects on the pattern of LH release during estrus of small doses of GnRH (0.4 ng) and the continuous infusion of the opioid antagonist naloxone were also examined. Plasma LH concentrations (NIAMDD rat LH-RP-1) were determined with a highly sensitive LH RIA. LH pulses were identified using the PULSAR algorithim. The LH interpulse intervals of 46 +/- 2 min on diestrous-1 day, 49 +/- 4 min on diestrous day 2, and 60 +/- 8 min on proestrus immediately before the LH surge were not significantly different. There were no changes immediately preceding the preovulatory LH surge on the afternoon of proestrus in either the LH interpulse interval or the LH pulse height. Instead, the transition from tonic LH secretion to the preovulatory LH surge was found to occur abruptly. These data suggest that an abrupt increase in GnRH secretion during the afternoon of proestrus initiates the dramatic rise in LH concentrations. The pattern of LH secretion during the day of estrus differed significantly from that on the other days of the cycle in that no LH pulses were observed. However, the administration of small pulses of GnRH elicited physiological elevations in LH release. Furthermore, the continuous infusion of naloxone to estrous rats immediately stimulated a pulsatile pattern of LH secretion, with a LH interpulse of 56 +/- 4 min. These data indicate that the absence of LH pulses during estrus may result from a deficit in GnRH release. Similar modifications in GnRH release during the other days of the cycle were inferred from the observed changes in LH pulse heights. The LH pulse height of 21 +/- 3 ng/ml on diestrous day 2 was significantly less than the LH pulse height of 41 +/- 4 ng/ml on diestrous day 1 or 35 +/- 4 ng/ml on proestrus before the surge.(ABSTRACT TRUNCATED AT 400 WORDS)     

Follicle-stimulating hormone beta subunit messenger ribonucleic acid concentrations during the rat estrous cycle

Serum follicle-stimulating hormone (FSH), pituitary FSH content and FSH beta subunit mRNA concentrations were measured at 1 to 3h intervals throughout the 4 day estrous cycle in rats. Serum FSH was stable (range 200-320 ng/ml) apart from the biphasic proestrus surge (5 fold elevation) which was present from 1800h of proestrus through 0800 h on estrus. Basal FSH beta mRNA concentrations from late metestrus through the afternoon of proestrus were 0.10 +/- 0.04 f mol cDNA bound/100 micrograms pituitary DNA. The major increase in FSH beta mRNA began at 2000 h on proestrus, 2 h after the initial rise in serum FSH and peak mRNA concentrations (0.43 +/- 0.08 f mol cDNA bound) occurred at 0200 h on estrus. FSH beta subunit mRNA concentrations were again increased at 2300 h on estrus (peak 0.24 f mol cDNA bound) and remained elevated through 1700 h on metestrus. Pituitary FSH content was transiently increased during metestrus and diestrus, but was elevated at 1000 h through 1900 h on proestrus (peak 5-fold increase). FSH content fell rapidly at 2000h and remained low until 1400 h on estrus when values again rose. These data show that FSH beta mRNA is increased 4-5 fold during the proestrus FSH surge, and a smaller increase occurs on metestrus in the absence of elevated FSH secretion. The increased concentrations of FSH beta mRNA occurred at different times to the previously reported changes in alpha and LH beta mRNAs. Therefore, the data suggest that different mechanisms are involved in the regulation of LH and FSH beta subunit gene expression during the 4-day estrous cycle in rats.     

The nature of the gonadotropin-releasing hormone stimulus-luteinizing hormone secretory response of human gonadotrophs in vivo

To examine the stimulus-secretion response of human pituitary gonadotrophs in vivo, we applied a new multiple parameter deconvolution technique to analyze (1) exogenous GnRH-stimulated LH secretory responses in 10 men with isolated hypogonadotropic hypogonadism (IHH), and (2) endogenous and exogenous GnRH-stimulated LH secretory responses in 8 normal men. The GnRH-deficient men were given 4 bolus doses of synthetic GnRH (7.5, 25, 75, and 250 ng/kg) iv at 2-h intervals in randomized order after long term pulsatile GnRH administration. The normal men were studied by sampling blood at 10-min intervals for 12 h basally and after 2 consecutive 10-micrograms iv GnRH doses. The serum LH peaks in both groups were subjected to quantitative deconvolution to resolve underlying LH secretory and clearance rates simultaneously. Such analyses revealed that exogenous GnRH-induced LH secretory episodes in GnRH-deficient men with IHH could be modeled as algebraically Gaussian distributions of instantaneous LH secretory rates with a mean half-duration of 14 +/- 2 min. The simultaneously resolved half-life of endogenous LH disappearance was 71 +/- 5 min. The log dose-response relationship for GnRH dose vs. maximal LH secretory rate or vs. calculated mass of LH released per secretory burst was linear. In contrast, varying GnRH doses did not alter the duration of LH secretory bursts, the half-time of LH disappearance, or the latency of LH secretory bursts after iv GnRH injections (viz. 7.6 min). Deconvolution analysis of the spontaneous (endogenous GnRH-stimulated) LH peaks in normal men revealed a mean half-duration of secretory bursts of 9.9 +/- 1.5 min, and a mean half-time of endogenous LH disappearance of 76 +/- 5 min. These values were not significantly different from those in the GnRH-treated normal or GnRH-deficient men. In summary, deconvolution analysis of LH release in men with IHH revealed a significant linear relationship between iv doses of pulsed GnRH and computer-resolved LH secretory rate and/or the mass of LH released per secretory event. In contrast, varying doses of GnRH did not alter the lag time between the GnRH stimulus and the LH secretory burst, the duration of LH secretion, or the calculated half-life of the LH released. We conclude that GnRH exerts dose-dependent effects on specific attributes of the secretory response of human gonadotrophs in vivo.     

Effects of glucocorticoids on responsiveness of luteinizing hormone and follicle-stimulating hormone to gonadotropin-releasing hormone by male rat pituitary cells in vitro

To determine if the inhibitory effects of glucocorticoids on GnRH-stimulated secretion of LH observed in male rats in vivo are exerted directly on the pituitary, dispersed pituitary cells from adult male rats were treated with 60 or 600 ng/ml cortisol (F) or corticosterone (B) during one or two 48-h incubations. Control cells received no glucocorticoids. During the second 48 h, some cells from each group were treated with GnRH (2.4 X 10(-11)-6.2 X 10(-8) M). Concentrations of LH and FSH in media and cells were measured by RIA. Treatment with steroids had no effect on basal secretion or maximal GnRH-stimulated secretion of LH, or on maximal secretion of FSH. Treatment with 600 ng/ml B for 96 h increased basal secretion of FSH relative to controls. All treatments with glucocorticoids increased the slopes of the GnRH dose-response curves for both LH and FSH, cell content of LH, total (cells + medium) LH, and total FSH. Incubation with 6 micrograms/ml F or B or 60 ng/ml dexamethasone gave similar results. Decreasing the time period of the second incubation to 6 h results in no significant differences between control cells and cells treated with B or F. These results show that glucocorticoids have different effects in vivo and in vitro, suggesting that inhibitory effects of glucocorticoids on secretion of LH in vivo may not be exerted directly on the pituitary but are exerted elsewhere, perhaps by altered hypothalamic secretion of GnRH. Also, these results show that male and female pituitaries in vitro respond differently to glucocorticoids.     

Development of gonadotrope desensitization to gonadotropin-releasing hormone (GnRH) and recovery are not coupled to inositol phosphate production or GnRH receptor number.

After initial GnRH pretreatment (10 nM, 5 h), subsequent GnRH-stimulated LH release from the gonadotrope was diminished (1 microM GnRH stimulated release of 36.4 +/- 1.4% total cellular LH over 3 h in cells initially pretreated with medium alone compared to 27.4 +/- 1.2% in GnRH-pretreated cells); however, inositol phosphate (IP) production in response to the releasing hormone remained unaffected (1 microM GnRH provoked IP accumulation of 161 +/- 9% above basal levels after 45 min in control cells and 162 +/- 11% in GnRH-pretreated cells). Pretreatment of pituitary cell cultures with NaF (a guanyl nucleotide binding protein activator, 10 mM, 3 h) also decreased subsequent GnRH-stimulated LH release, and in addition, provoked a decrease in GnRH receptor number, an increase in GnRH receptor affinity, reduction of GnRH-stimulated IP production to basal levels, and an increase in the amount of LH released in response to stimulation with the calcium ionophore A23187. In order to determine if the changes in LH release were a result of decreased IP production and/or decreased GnRH receptor binding, the time course of recovery to control levels of these processes was assessed. GnRH receptor binding continued to decrease after NaF pretreatment, reaching a nadir (62% of control) at 6 h after the pretreatment period and recovering at 48 h (90% of control). In contrast, GnRH-provoked IP accumulation did not return to control levels even after 48 h of recovery after NaF pretreatment (1 microM GnRH-stimulated IP accumulation in NaF-pretreated cells was 57% compared to control cells after 48 h of recovery). GnRH-stimulated LH release was inhibited immediately after NaF pretreatment (1 microM GnRH-stimulated LH release in NaF-pretreated cells was 65% of control levels). Cells began to recover within 3 h (80% of control) and were almost completely recovered by 6 h (90% of control). A23187-provoked LH release was enhanced immediately after NaF pretreatment (30 microM A23187-stimulated LH release in NaF-pretreated cells was 170% of control levels). Responsiveness to ionophore was 133% of control by 0.5 h, and complete recovery was measured within 1 h (100% of control). Furthermore, both NaF and GnRH pretreatment still provoked a decrease in gonadotrope responsiveness when IP production was inhibited by the phospholipase C inhibitor U-73122. The results suggest that the development of gonadotrope desensitization (by either NaF or GnRH pretreatment) can be uncoupled from changes in IP production.(ABSTRACT TRUNCATED AT 400 WORDS)     

Estradiol secretion by granulosa cells from rats with four- or five-day estrous cycles: the development of responses to follicle-stimulating hormone versus luteinizing hormone

The relative importance of LH vs. FSH in stimulating estradiol secretion by granulosa cells during follicular development was assessed in Sprague-Dawley rats with regular 4- or 5-day estrous cycles. At various times during diestrus (D-1200 h, D-2000 h) and proestrus (P-0800 h, P-1400 h, P-2000 h) granulosa cells were isolated from the presumptive preovulatory follicles. The cells were cultured with 0.5 microM testosterone and various doses of LH or FSH (0, 0.1, 1, 10, or 100 ng/ml). Media were collected and replaced daily for 3 days and measured for estradiol by RIA. Estradiol secretion in the absence of gonadotropins (endogenous aromatase activity) increased progressively with stage of the cycle. At earlier times of cell isolation (D-1200, D-2000, P-0800) secretion by cells from 5-day rats was greater relative to 4-day rats. Since estradiol production in the absence of gonadotropins declined progressively over each 3-day culture period, effects of the gonadotropins were most evident on the third day of culture when endogenous aromatase activity was low. On this day FSH consistently increased estradiol secretion above control levels. Sensitivity to FSH, as measured by the 50% maximally effective dose (ED50; approximately 1-3 ng FSH/ml), did not vary with cycle type or stage of the cycle and the response to FSH, in terms of 50% maximal estradiol secretion, was also relatively constant. In contrast, the effects of LH varied with cycle type and time of cell isolation. As follicles developed, cells from both 4- and 5-day rats became more sensitive to LH, as evidenced by a decline in the ED50 from approximately 32 ng LH/ml to approximately 3 ng/ml. This increase in sensitivity to LH is consistent with previous reports that the number of LH receptors on granulosa cells increases progressively during the final stages of follicular development. However, the increase in sensitivity to LH occurred earlier in cells from 5-day rats, relative to the expected next estrus (later, relative to the preceding estrus). Responsiveness to LH (50% maximal estradiol production) was relatively constant, except that it was lower in cells from 4-day rats isolated at D-1200. These results indicate that the LH receptors acquired by granulosa cells during diestrus and proestrus are functionally linked with aromatase activity and may, therefore, be important to the production of estradiol levels sufficient to elicit the LH surge.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential gonadotropin-releasing hormone (GnRH) and GnRH receptor messenger ribonucleic acid expression patterns in different tissues of the female rat across the estrous cycle

GnRH, the main regulator of reproduction, is produced in a variety of tissues outside of the hypothalamus, its main site of synthesis and release. We aimed to determine whether GnRH produced in the female rat pituitary and ovaries is involved in the processes leading to ovulation. We studied the expression patterns of GnRH and GnRH receptor (GnRH-R) in the same animals throughout the estrous cycle using real-time PCR. Hypothalamic levels of GnRH mRNA were highest at 1700 h on proestrus, preceding the preovulatory LH surge. No significant changes in the level of hypothalamic GnRH-R mRNA were detected, although fluctuations during the day of proestrus are evident. High pituitary GnRH mRNA was detected during the day of estrus, in the morning of diestrus 1, and at noon on proestrus. Pituitary GnRH-R displayed a similar pattern of expression, except on estrus, when its mRNA levels declined. Ovarian GnRH mRNA levels increased in the morning of diestrus 1 and early afternoon of proestrus. Here, too, GnRH-R displayed a somewhat similar pattern of expression to that of its ligand. To the best of our knowledge, this is the first demonstration of a GnRH expression pattern in the pituitary and ovary of any species. The different timings of the GnRH peaks in the three tissues imply differential tissue-specific regulation. We believe that the GnRH produced in the anterior pituitary and ovary could play a physiological role in the preparation of these organs for the midcycle gonadotropin surge and ovulation, respectively, possibly via local GnRH-gonadotropin axes.