The frequency of gonadotropin-releasing-hormone stimulation differentially regulates gonadotropin subunit messenger ribonucleic acid expression

The hypothalamic decapeptide GnRH is known to regulate the synthesis and secretion of LH and FSH by pituitary gonadotrope cells. The frequency of pulsatile GnRH secretion changes and LH and FSH are differentially secreted in various physiological situations. To investigate the potential role of altered frequency of GnRH stimulation in regulating differential secretion of LH and FSH, we examined the effects of GnRH frequency on expression of the alpha, LH beta, and FSH beta genes. GnRH pulses (25 ng/pulse) were administered to castrate testosterone-replaced rats at intervals of 8-480 min to cover the range of physiological pulsatile GnRH secretion. Fast frequency GnRH pulses (8-min pulse intervals) increased alpha-subunit mRNA concentrations 3-fold above those in saline-pulsed controls (controls, 1.01 fmol cDNA bound/100 micrograms pituitary DNA) and LH beta mRNA by 50% (controls, 0.18 fmol cDNA bound), but FSH beta mRNA was unchanged (controls, 0.38 fmol cDNA bound). GnRH pulses given every 30 min increased all three subunit mRNAs (alpha, 3-fold, LHbeta, 2-fold; FSH beta, 2-fold), and acute LH release and serum FSH concentrations were maximal after this frequency. Slower frequency GnRH stimuli (120- to 480-min pulse intervals) did not change alpha and LH beta mRNA levels, but increased FSH beta mRNA 2- to 2.5-fold, and FSH secretion was maintained. Equalization of the total dose of GnRH given at different intervals over 24 h confirmed the frequency dependence of subunit mRNA expression. Fast frequency GnRH stimuli (8 min) increased alpha mRNA 1.5- to 2.5-fold, while the same total GnRH doses were ineffective when given at slow frequency (480 min). Similarly, LH beta mRNA was only increased by GnRH pulses given at 8-min intervals. In contrast, FSH beta mRNA increased 2-fold after pulses given every 480 min, and the 8-min pulse interval was ineffective. The data show that the frequency of GnRH stimulation can differentially regulate gonadotropin subunit mRNA expression and may be a mechanism that enables a single GnRH peptide to selectively regulate gonadotropin subunit gene expression and hormone secretion.     

Differential regulation of gonadotropin subunit gene expression by gonadotropin-releasing hormone pulse amplitude in female rats

The role of GnRH in regulating gonadotropin subunit gene expression was examined in adult female rats. Animals were ovariectomized, estradiol implants inserted sc, and jugular cannulae placed into the right atria. On the next day, animals were given GnRH pulses (saline to controls) every 30 min for up to 48 h and alpha, LH beta, and FSH beta mRNA levels measured by hybridization to cDNA probes. To determine the effects of GnRH treatment duration, rats received GnRH pulses (25 ng at 30-min intervals) for 6, 12, 24, and 48 h. FSH beta mRNA was increased (by 92%) after 6 h of pulses and remained elevated through 48 h. alpha mRNA was not increased until 12 h (27% increase) and rose further (57%) by 48 h. LH beta mRNA levels were only transiently increased at 12 h (67%) and values were not different from saline controls after 24 or 48 h. To examine whether the rise in serum PRL which is characteristic of the ovariectomized-estradiol animal model was responsible for the decrease in LH beta mRNA responsiveness to GnRH over longer durations, studies were repeated in bromocriptine-treated animals (0.6 mg sc, twice daily). The results showed similar response patterns for all three subunit mRNAs including the decrease in LH beta after 48 h. A third experiment examined the effect of varying GnRH pulse amplitude (0.5-250 ng/pulse at 30-min intervals) over 12 h. alpha mRNA levels were increased by all GnRH doses greater than 5 ng with maximum responses after 250 ng pulses. LH and FSH beta mRNAs were both elevated by GnRH pulse doses of 0.5-25 ng (P less than 0.05 vs. saline controls). Maximal increases (2-fold) were seen after 5 ng pulses for LH beta and after 15-ng pulses for FSH beta mRNA. These results show that pulsatile GnRH increases FSH beta mRNA more rapidly than alpha or LH beta mRNAs in female rats. In addition, high amplitude GnRH pulses increase only alpha mRNA, whereas both LH beta and FSH beta mRNAs show maximum responses to lower doses. The data suggest that alterations in the amplitude of the GnRH pulsatile signal can exert differential effects on gonadotropin gene expression.     

Testosterone differentially modulates gonadotropin subunit messenger ribonucleic acid responses to gonadotropin-releasing hormone pulse amplitude.

Testosterone (T) inhibits GnRH secretion and can also modulate the effects of GnRH on gonadotropin synthesis and secretion. To assess the effect of T on GnRH stimulation of alpha, LH beta, and FSH beta mRNA expression, we replaced T at three levels to reproduce low (1.5 +/- 0.5 ng/ml), medium (3.5 +/- 0.3 ng/ml), and high (6.2 +/- 0.6 ng/ml) physiological plasma concentrations. Additionally, as peripheral conversion to dihydrotestosterone (DHT) or estradiol (E2) may mediate T action, the effects of GnRH pulses in the presence of DHT and E2 were also studied. Male rats were castrated, and steroids were replaced via implants containing either T (three doses) or DHT or E2 (two doses each). GnRH pulses (10-250 ng/pulse) were administered iv at 30-min intervals for 48 h. Pituitary subunit mRNA concentrations, gonadotropin content, and LH and FSH secretion were determined. The patterns of alpha, LH beta, and FSH beta mRNA responses to increasing GnRH pulse amplitude were similar at all concentrations of plasma T. Alpha mRNA concentrations were increased 2- to 4-fold by GnRH pulses. At the same plasma T concentration, all doses of GnRH produced similar increases in alpha mRNA, but the response tended to be lower at the higher (6.2 ng/ml) levels of T. LH beta mRNA showed a clear dependence on GnRH pulse amplitude, with the maximum responses (2- to 3-fold) occurring after 10- to 25-ng GnRH pulses. At the higher (3.5 and 6.2 ng/ml) T concentrations, the dose-response curve was shifted to the left. The lowest GnRH pulse dose (10 ng) produced maximum responses, and LH beta mRNA increments in response to the higher GnRH doses were suppressed. FSH beta mRNA concentrations were increased by T in saline-pulsed controls. FSH beta mRNA responses were similar (2- to 3-fold) after all GnRH doses and at all concentrations of T. Increasing GnRH pulse doses reduced the pituitary content of both LH and FSH at all levels of T. Acute LH secretion was maximal after 10- and 25-ng pulses of GnRH when plasma T was low, but increased progressively with GnRH dose at the highest plasma T concentrations. Plasma FSH did not show any differential responsiveness to GnRH pulse dose or to increasing plasma T. Thus, LH synthesis and secretion are affected more than those of FSH by changing plasma concentrations of T. T may modulate posttranslational events in LH secretion. The higher GnRH doses effected LH release without increasing LH beta mRNA in the presence of higher physiological concentrations of T.(ABSTRACT TRUNCATED AT 400 WORDS)     

Gonadotrophin-releasing hormone regulation of gonadotrophin subunit gene expression: studies in tri-iodothyronine-suppressed rats

We have previously shown that a pulsatile gonadotrophin-releasing hormone (GnRH) stimulus can increase steady-state levels of alpha and LH-beta subunit mRNAs in the male rat pituitary. Since alpha subunit is produced in both thyrotroph and gonadotroph cells, the effect of GnRH specifically on gonadotroph alpha gene expression is uncertain. To address this tissue, adult male rats were given injections of tri-iodothyronine (T3; 20 micrograms/100 g body wt, i.p.) daily for 8 days (day 8 = day of death) in order to decrease thyrotroph alpha mRNA levels (+T3 group). Saline injections (i.p.) were given to control animals (-T3group). Three days before GnRH administration, the animals were castrated and testosterone implants inserted s.c., to inhibit endogenous GnRH secretion. GnRH pulses (25 ng/pulse; 30-min interval) were given to freely moving animals (saline pulses to controls) via an atrial cannula for 12,24 or 48 h. Serum LH and FSH were measured before and 20 min after the last GnRH pulse. Pituitary RNA was extracted and alpha, LH-beta, FSH-beta and prolactin mRNA levels were determined by dotblot hybridization using 32P-labelled cDNA probes. Castration and testosterone replacement reduced alpha and LH-beta mRNA levels by 30 and 40% respectively, compared with levels in untreated intact males, but did not decrease FSH-beta concentrations. T3 administration further decreased alpha mRNA to 30% of values seen in intact males, but LH-beta mRNA levels were unchanged. FSH-beta mRNA concentrations were decreased by 23% in T3-treated rats (P less than 0.05 vs intact controls).(ABSTRACT TRUNCATED AT 250 WORDS)     

A mechanism for the differential regulation of gonadotropin subunit gene expression by gonadotropin-releasing hormone.

The hypothalamic hormone gonadotropin-releasing hormone (GnRH) is released in a pulsatile fashion, with its frequency varying throughout the reproductive cycle. Varying pulse frequencies and amplitudes differentially regulate the biosynthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by pituitary gonadotropes. The mechanism by which this occurs remains a major question in reproductive physiology. Previous studies have been limited by lack of available cell lines that express the LH and FSH subunit genes and respond to GnRH. We have overcome this limitation by transfecting the rat pituitary GH3 cell line with rat GnRH receptor (GnRHR) cDNA driven by a heterologous promoter. These cells, when cotransfected with regulatory regions of the common alpha, LH beta, or FSH beta subunit gene fused to a luciferase reporter gene, respond to GnRH with an increase in luciferase activity. Using this model, we demonstrate that different cell surface densities of the GnRHR result in the differential regulation of LH and FSH subunit gene expression by GnRH. This suggests that the differential regulation of gonadotropin subunit gene expression by GnRH observed in vivo in rats may, in turn, be mediated by varying gonadotrope cell surface GnRHR concentrations. This provides a physiologic mechanism by which a single ligand can act through a single receptor to regulate differentially the production of two hormones in the same cell.     

Differential regulation of gonadotropin subunit gene promoter activity by pulsatile gonadotropin-releasing hormone (GnRH) in perifused L beta T2 cells: role of GnRH receptor concentration

The pulsatile release of GnRH by the hypothalamus is required to stimulate the pituitary-gonadal axis, and variations in GnRH pulse frequency are associated with differential synthesis and release of LH and FSH by pituitary gonadotropes. How gonadotropes differentiate between GnRH pulse frequencies and subsequently differentially regulate the expression of the LH beta and FSH beta genes remains to be determined. In the present study, using a perifusion system that allows us to replicate the GnRH pulsatility occurring in vivo, we have systematically characterized the effects of varying GnRH pulse frequencies on LH beta, FSH beta, alpha, and GnRH receptor (GnRHR) gene promoter stimulation in L beta T2 cells. We demonstrate that LH beta gene promoter activity is stimulated to the greatest extent at higher GnRH pulse frequencies, whereas the FSH beta gene promoter is preferentially stimulated at lower GnRH pulse frequencies, reflecting previous observations in primary rat pituitary cells in vivo and in vitro. By measuring GnRH binding, we demonstrate that cell-surface GnRHR number is increased at higher frequencies of pulsatile GnRH and that this increase precedes the differential regulation of LH beta and FSH beta gene promoter activity. To test the role of GnRHR number in mediating the differential effects of pulsatile GnRH, the rat GnRHR was overexpressed in L beta T2 cells, and the response to pulsatile GnRH was again assessed. Interestingly, although overexpression of GnRHR had no effect on the frequency-dependent regulation of LH beta, the induction of FSH beta gene promoter activity by pulsatile GnRH was reduced, and frequency dependence was abrogated. Our results demonstrate that L beta T2 cells represent a suitable model for the study of the differential regulation of gonadotropin subunit gene expression by pulsatile GnRH. Furthermore, our studies indicate that cell-surface GnRHR density is a critical mediator of this differential regulation.     

Gonadotrophin-releasing hormone modulation of gonadotrophins in the ewe: evidence for differential effects on gene expression and hormone secretion.

While the regulation of gonadotrophin secretion by gonadotrophin-releasing hormone (GnRH) has been well documented in both rats and sheep, its role in the synthesis of gonadotrophin subunits remains unclear. We have investigated the effects of the specific inhibition of GnRH by a GnRH agonist on the expression of gonadotrophin subunit genes and the subsequent storage and release of both intact hormones and free alpha subunit. Treatment with GnRH agonist for 6 weeks abolished pulsatile LH secretion, reduced plasma concentrations of FSH and prevented GnRH-induced release of LH and FSH. This was associated with a reduction of pituitary LH-beta mRNA and FSH-beta mRNA levels (to 5 and 30% of luteal control values respectively), but not alpha mRNA which was significantly increased (75% above controls). While there was a small decrease in the pituitary content of FSH (30% of controls), there was a drastic reduction in LH pituitary content (3% of controls). In contrast to the observed rise in alpha mRNA, there was a decrease in free alpha subunit in both the pituitary and plasma (to 30 and 80% of control levels). These results suggest that, while GnRH positively regulates the expression of both gonadotrophin beta-subunit genes, it can, under certain circumstances, negatively regulate alpha-subunit gene expression. Despite the complete absence of LH and FSH in response to GnRH, there remained a basal level of beta-subunit gene expression and only a modest reduction (50%) in the plasma levels of both FSH and LH, suggesting that there is a basal secretory pathway.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of recombinant human inhibin and testosterone on gonadotropin secretion and subunit mRNA in superfused male rat pituitary cell cultures stimulated with pulsatile gonadotropin-releasing hormone.

Effects of recombinant human inhibin (rh inhibin) and testosterone on follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secretion and mRNA levels of gonadotropin subunits were investigated in superfused male rat pituitary cell cultures. During superfusion, the cells were stimulated with gonadotropin-releasing hormone (GnRH) pulses (10 nM, 6 min/h) and exposed to rh inhibin (2 ng/ml) and/or testosterone (10 nM) for up to 20 h. The concentrations of FSH and LH were measured in effluent media by radioimmunoassay (RIA), and subunit mRNAs were determined by Northern blot hybridizations using rat FSH beta, LH beta and alpha genomic and cDNA probes. Rh inhibin suppressed the secretion of FSH (30-40% of control) and the secretion of LH to 50-60% of control, but inhibited only FSH beta mRNA (to non-detectable levels). Testosterone alone suppressed the release of LH to 50% of control, whereas FSH release was increased to 130-160% (P less than 0.05) of control. This increase was due to higher interpulse values without significant changes in the pulse amplitude. Also FSH beta mRNA level was increased (1.5-fold, P less than 0.05) but only after 17-20 h of treatment. On the other hand, testosterone had no effect on LH beta and alpha subunit mRNA levels. Testosterone in combination with rh inhibin showed an inhibitory effect on LH beta mRNA; however, the pattern of LH release was not significantly different from that observed with rh inhibin or testosterone alone. Combined effects of testosterone and rh inhibin on FSH secretion and FSH beta mRNA were similar to those observed with rh inhibin alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of progesterone on pulsatile luteinizing hormone (LH) secretion and LH subunit messenger ribonucleic acid during lactation in the rat

In ovarian-intact lactating rats, removal of the suckling stimulus leads to restoration of pituitary LH beta mRNA levels and pulsatile LH secretion after 72 h, which correlates with a sharp decrease in plasma progesterone concentrations to basal levels. In contrast, in ovariectomized lactating rats, the increase in pituitary LH function is observed by 24 h after pup removal. To determine if progesterone secretion from the ovary participates in the delayed recovery of LH secretion, we treated lactating rats with the progesterone antagonist RU 486 and determined the effects on the time course of recovery of pulsatile LH secretion and LH subunit mRNA after pup removal and on pituitary responsiveness to GnRH. In ovarian-intact lactating rats treated with RU 486, pulsatile LH secretion was observed in about 40% of the rats within 24 h after pup removal (LH interpulse interval, 43.7 +/- 8.3 min) and in about 90% of the rats within 48 h after pup removal (LH interpulse interval, 46.1 +/- 3.6 min). The mean plasma LH level in the RU 486-treated rats was 10.1 +/- 2.2 ng/ml 24 h after removal of pups (control, less than 5 ng/ml) and had increased to 35.1 +/- 6.4 ng/ml 48 h after pup removal (control, 9.1 +/- 2.5 ng/ml). However, RU 486 treatment had no significant effect on LH mRNA subunit levels. To determine whether progesterone acts at the pituitary to block GnRH stimulation of LH secretion, we tested the effects of RU 486 on LH secretion in response to 2- and 5-ng pulses of GnRH. Pituitary responsiveness was tested 24 h after pup removal. We found that both doses of GnRH were effective in stimulating pulsatile LH secretion, and treatment with RU 486 had no significant effect on this response. We conclude from these studies that progesterone secretion from the ovary contributes to the inhibition of LH secretion that occurs after pup removal, since antagonizing progesterone's action resulted in an earlier restoration of pulsatile LH secretion. The increase in LH secretion occurred in the absence of any significant changes in responsiveness of the pituitary to GnRH stimulation or in LH subunit mRNA levels. Therefore, the primary site of action of progesterone would appear to be at the hypothalamus to suppress pulsatile GnRH secretion.     

The interaction of gonadotropin-releasing hormone and estradiol on luteinizing hormone and prolactin gene expression in female hypogonadal (hpg) mice

We have investigated the interaction of estrogen with GnRH on the regulation of LH subunit mRNA in female hypogonadal (hpg) mice receiving constant frequency and amplitude pulsatile GnRH treatment for up to 18 days. The level of cytosolic common alpha mRNA in female hpg mouse pituitaries was 45 +/- 6% of normal female littermate values, and treatment with pulsatile GnRH increased alpha mRNA to 40% above the normal value at 24 h and 2-4 times normal at 7 and 12 days (P less than 0.001); by 18 days levels had returned to those of untreated hpg controls. Concurrent treatment with estradiol (E2) did not affect those changes. However, in ovariectomized hpg mice the 2- to 4-fold rise in alpha mRNA was sustained for 18 days with GnRH treatment. E2 treatment alone for 7 and 12 days doubled alpha mRNA. LH beta mRNA levels in untreated female hpg mice were between 5-10% of normal values. Levels increased significantly (77 +/- 6.4%) 24 h after GnRH treatment and were normal at 7, 12, and 18 days. E2 together with GnRH did not affect the LH beta mRNA increase at 12 days, but reduced it to 45% of normal at 18 days. Ovariectomy did not alter the LH beta mRNA response to GnRH treatment, and E2 treatment alone did not increase LH beta mRNA. Serum LH concentrations were normalized by GnRH treatment at all times and did not increase in ovariectomized animals. LH release was prevented when E2 was combined with GnRH. Pituitary LH content in hpg mice was 20% of normal and increased gradually with GnRH treatment. Neither concurrent treatment with E2 nor ovariectomy affected the GnRH-induced synthesis of LH. PRL mRNA levels were 30-40% of normal littermate values in untreated female hpg mice, and pulsatile GnRH increased these to 70-80% of normal. E2 alone raised PRL mRNA slightly above normal values, although together with GnRH this rise was attenuated by about 40%. Pulsatile GnRH treatment of ovariectomized hpg mice did not increase PRL mRNA. E2 increased pituitary PRL content, and GnRH did not attenuate this aspect of E2 action. Serum PRL levels rose with E2 treatment at 7 and 12 days, and concurrent GnRH treatment prevented the rise at 12 days. We conclude the following: 1) The stimulatory action of pulsatile GnRH on the expression of both common alpha and LH beta mRNA is rapid (less than 24 h).(ABSTRACT TRUNCATED AT 400 WORDS)     

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

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

Effect of gonadotropin-releasing hormone pulse frequency on gonadotropin secretion and subunit messenger ribonucleic acids in perifused pituitary cells.

Slow frequency GnRH pulses have been proposed to preferentially increase circulating FSH levels by increasing FSH synthesis and pulsatile release. Examination of this proposal using various in vivo models, however, has produced conflicting results. To examine directly the effects of GnRH pulse frequency on the pituitary, we compared the effects of 2.5-nM GnRH pulses administered every 1 h or every 4 h vs. no GnRH, using perifused rat pituitary cells. FSH secretion (total area under the response curve) was 2-fold greater (P less than 0.01) with every hour than with every 4 h GnRH pulses. This difference resulted from the increased number of GnRH pulses and increased (P less than 0.05) interpulse FSH secretion, whereas FSH pulse amplitude was unchanged. FSH beta mRNA levels at the completion of the 11-h perifusion were increased 4.5-fold by GnRH every h (P less than 0.01) and 3.3-fold by GnRH every 4 h (P less than 0.05) above levels in untreated cells. FSH beta mRNA levels were greater (P less than 0.05) at the faster GnRH pulse frequency. Because more frequent stimulation delivered more GnRH during the study, cells were next stimulated with 2.5 nM GnRH every 1 h for nine pulses, 7.5 nM GnRH every 4 h for three pulses to equalize the GnRH dose, or 2.5 nM GnRH every 4 h for three pulses. Interpulse FSH secretion and FSH beta mRNA levels were again greater (P less than 0.05) with every hour than every 4 h GnRH pulses. Interpulse LH secretion, FSH and LH pulse amplitude, and LH beta and alpha-subunit mRNA levels were not different between the groups. GnRH doses of 0.1-10 nM every hour increased FSH and LH pulsatile secretion dose-dependently, but FSH beta, LH beta, and alpha-subunit mRNA levels were similar. In conclusion, our data reveal that reducing the frequency of GnRH pulses from every hour to every 4 h reduces both FSH beta mRNA levels and FSH interpulse secretion, but does not change GnRH-stimulated FSH pulsatile release. We suggest that the finding by others that slow frequency GnRH pulses increase circulating FSH levels under certain experimental conditions in vivo may instead be explained by complex hormonal interactions or changes in FSH clearance.     

Gonadotropin-releasing hormone desensitization preferentially inhibits expression of the luteinizing hormone beta-subunit gene in vivo

In this study we investigated changes in steady state cytoplasmic mRNA levels for LH subunits in pituitaries of male rats desensitized by continuous infusion of GnRH in vivo. Seven days of GnRH infusion (340 micrograms/day) reduced (P less than 0.01) LH beta mRNA levels in intact adult male rats and prevented the LH beta mRNA rise observed after castration. In contrast, common alpha mRNA doubled (P less than 0.05) in intact rats, and the elevated alpha mRNA after 7 days castration was unchanged. Serum and pituitary LH levels were suppressed below values of intact controls. Fourteen days of GnRH infusion (290 micrograms/day) further reduced LH beta mRNA levels in both intact and castrated male rat pituitaries. alpha mRNA levels in intact rat pituitaries were unchanged by 14 days of GnRH infusion, while in castrated rats there was a 23% (P less than 0.05) decrease, though values were still twice those of intact controls. As at 7 days, serum and pituitary LH were suppressed. Infusion of a superagonist analog (Buserelin) at a dose of 14 micrograms/day for 28 days reduced LH beta mRNA to 15% of intact control values in both castrated and intact rats. Common alpha mRNA was significantly (P less than 0.05) increased in intact rats and reduced by 13% (P less than 0.05) in castrates by superagonist infusion. These results were similar to those produced by 20- to 30-fold higher doses of native GnRH. GnRH and agonist analog effects were specific since no changes were observed in other mRNA species (GH, PRL, actin). These results indicate that in GnRH-desensitized gonadotropes LH beta gene expression is inhibited, and this may largely explain the reduced LH biosynthesis. However, there is a differential effect of continuous GnRH or agonist analog treatment on LH subunit gene expression, with a time-dependent stimulation of common alpha gene expression in intact rats. This may be caused by a stimulatory interaction between GnRH and progestagens at the level of the gonadotrope. Thus, common alpha gene expression is less tightly coupled than that of LH beta to GnRH action.     

Expression of alpha-subunit and luteinizing hormone (LH) beta messenger ribonucleic acid in the rat during lactation and after pup removal: relationship to pituitary gonadotropin-releasing hormone receptors and pulsatile LH secretion

Pituitary GnRH receptor (GnRH-R) levels and LH secretion are suppressed in the lactating rat. To determine if LH synthesis is also inhibited, we have measured LH subunit mRNA levels in the pituitary of lactating rats. We have also examined the temporal relationship among restoration of GnRH-R, LH secretion, and LH synthesis after withdrawing the sensory stimulus of suckling. Pituitary alpha-subunit and LH beta mRNA levels were sharply reduced on day 10 of lactation in both intact and ovariectomized (OVX) animals compared with those in cycling diestrous rats or OVX controls. Removal of the suckling stimulus from OVX animals led to significant increases in alpha-subunit and LH beta mRNA levels by 24 h. Upon removal of the suckling stimulus from intact rats, alpha-subunit mRNA levels were restored by 48 h, but LH beta mRNA levels did not return to diestrous levels until 72 h. Pituitary GnRH-R levels were clearly up-regulated within 1 day after pup removal. Some LH pulses were observed by 48 h, but consistent plasma LH pulses were not detected until 72 h. When pulsatile GnRH was administered during the 24 h after pup removal from intact rats, the regimen of pulsatile GnRH was successful in inducing LH secretion; however, the restoration of pulsatile LH was not accompanied by increases in alpha-subunit and LH beta mRNA levels. The present studies provide further evidence to support the hypothesis that during lactation, the suppression of pituitary gonadotroph function is mainly due to the loss of hypothalamic GnRH secretion. Our data also show that 1) the restoration of GnRH-R alone is not sufficient to activate LH subunit mRNA and LH secretion; 2) the normal restoration of pulsatile LH secretion and increases in LH subunit mRNA are temporally correlated, as increases in LH secretion appear to precede increases in LH subunit mRNA; and 3) the restoration of pituitary LH subunit mRNA levels and pulsatile LH secretion took longer in the intact rat than in the OVX rat, suggesting that ovarian steroids may play a role in the inhibitory effect of lactation.     

Gonadal regulation of gonadotropin subunit gene expression: evidence for regulation of follicle-stimulating hormone-beta messenger ribonucleic acid by nonsteroidal hormones in female rats

Gonadectomy results in a rise in gonadotropin secretion and subunit gene expression, although the relative contributions of declining gonadal hormones or increasing hypothalamic GnRH secretion are uncertain. To further delineate the roles of the hypothalamus and gonads in regulation of gonadotropin gene expression, male and female rats were castrated and gonadotropin subunit messenger RNA (mRNA) concentrations measured 2, 7, 14, or 21 days (d) later. In males, FSH beta mRNA was maximal (2-fold increase) by 7 d while peak levels of alpha (3-fold) and LH beta (3-fold) were seen by 14 d. Testosterone (T) replacement restored all three subunit mRNA concentrations to intact values. In females, FSH beta mRNA also reached plateau levels (8-fold increase) earlier than alpha (3-fold) or LH beta (11-fold). When female rats ovariectomized 7 days earlier were given estradiol (E2) and progesterone (P) implants for up to 14 d, suppression of alpha and LH beta to intact levels was observed. However, FSH beta mRNA concentrations only decreased to 67% of castrate values, and remained 2- to 3-fold higher than levels in intact female rats. Female rats were also given E2 replacement at the time of ovariectomy. LH beta mRNA was maintained at intact levels for 14 days while alpha and FSH beta showed partial castration responses (2-fold and 3-fold, respectively). Finally, to determine whether E2 and P regulate gonadotropin subunit expression directly or by reducing GnRH secretion, female rats were ovariectomized and immediately replaced with E2, P, or E2 + P in the presence or absence of a GnRH antagonist (A) for 2 d. alpha mRNA was increased (2-fold) by E2 but not by E2 + A suggesting that E2 requires the presence of GnRH to increase alpha mRNA. P alone was ineffective, but both E2 and A prevented the LH beta mRNA response to ovariectomy. The effects of E2 and A were not additive, suggesting that E suppresses LH beta mRNA by inhibiting the increase in GnRH secretion. In contrast, the FSH beta mRNA response to ovariectomy was only partially suppressed by E2, E2 + P, or E2 + P + A. These data indicate that in castrate males, replacement of T suppresses all three subunit mRNAs to intact levels. However, replacement of E2 to ovariectomized females did not prevent the increase in alpha and FSH beta mRNAs. In female rats, LH beta mRNA is predominantly regulated by GnRH. alpha mRNA expression is also mainly regulated by GnRH, and E2 appears to augment GnRH action on alpha mRNA expression.(ABSTRACT TRUNCATED AT 400 WORDS)     

Gonadotropin-releasing hormone is required for enhanced luteinizing hormone subunit gene expression in vivo

Pre- and postcastration changes in LH beta and common alpha mRNAs were correlated with pituitary and serum LH levels in two different species after abolition of pituitary stimulation by GnRH. A GnRH antagonist (GnRH-ANT) was used to block gonadotroph GnRH receptors in male rats, and a GnRH antiserum (GnRH-AS) was used to inhibit GnRH stimulation of female and male mouse and male rat pituitaries. The postcastration increases in LH beta and common alpha mRNA levels (2- and 3.5-fold, respectively) were abolished in male rats after 7 days of continuous GnRH-ANT infusion. The postcastration increases in LH beta and common alpha mRNA in female (1.9- and 2.2-fold respectively) and male mice (1.4- and 3.6-fold, respectively) were also prevented after daily sc injection of GnRH-AS, as were the rises in LH beta (3-fold) and common alpha (4-fold) in castrated male rats. The pituitary LH content (postgonadectomy) was no different from intact control levels in all experimental animals regardless of treatment, while the increase in serum LH concentration in rats (7- and 8-fold) and in female (4.8-fold) and male mice (9.8-fold) was prevented by both GnRH-ANT and GnRH-AS administration. In intact rats treated with GnRH-ANT the LH beta mRNA level decreased (57%) while the common alpha mRNA level was unaffected after 7 days. Neither pituitary nor serum LH levels were altered in intact rats or mice after appropriate treatments. We conclude that endogenous GnRH is required for the postcastration rise of both LH beta and common alpha-subunit mRNA levels in rats and mice.     

Alpha and luteinizing hormone beta messenger ribonucleic acid (RNA) of male and female rats after castration: quantitation using an optimized RNA dot blot hybridization assay

In this study we examined the changes in alpha and LH beta mRNAs in anterior pituitaries of male and female rats after castration. mRNA concentrations were measured by an optimized RNA dot blot hybridization assay. Rat alpha and LH beta cDNAs were nick-translated to specific activities of 2-5 X 10(8) cpm/micrograms and were used as hybridization probes. The total RNA per assay, RNA per dot, and saturating amounts of probe were optimized. The intra- and interassay coefficients of variation were 5% and 28%, respectively. Both alpha and LH beta mRNA concentrations increased after castration, but marked differences were observed in the kinetics of responses in male and female rats. In males, alpha and LH beta mRNAs were increased by 24 h postcastration (by 25% and 38%, respectively), and 4- to 5-fold increases over intact controls were evident by 18 days. Alpha mRNA rose rapidly and had doubled by 2 days, whereas LH beta mRNA concentrations showed a similar increase by 6-7 days postcastration. The slower rise in LH beta mRNA was associated with a transient decline in serum and pituitary LH concentrations between 2 and 6 days after castration. In female rats, alpha mRNA increased more slowly. Alpha concentrations had doubled by 10 days, while a similar increase in LH beta mRNA occurred 7 days after castration. Thereafter, both subunit mRNAs continued to rise, and by day 20 alpha mRNA was increased 5-fold and LH beta mRNA 16-fold over values in intact females. Serum and pituitary LH concentrations rose gradually, and both were increased by 7-10 days after castration. The increase in serum and pituitary LH followed a time course similar to that of the progressive rise in LH beta mRNA concentrations. These data show that an increase in steady state LH subunit mRNA concentrations is one of the mechanisms involved in increased gonadotropin biosynthesis and secretion after castration. The kinetics of LH subunit mRNA and LH secretory responses are different in male and female rats and suggest that the concentration of LH beta mRNA may be a limiting factor in LH secretion.     

Synergistic effects of salmon gonadotropin-releasing hormone and estradiol-17beta on gonadotropin subunit gene expression and release in masu salmon pituitary cells in vitro

Effects of salmon gonadotropin-releasing hormone (sGnRH) and estradiol-17beta (E2) on gene expression and release of gonadotropins (GTHs) were examined in masu salmon (Oncorhynchus masou) using primary pituitary cell cultures at three reproductive stages, initiation of sexual maturation in May, pre-spawning in July, and spawning in September. Amounts of GTH subunit mRNAs were determined by real-time polymerase chain reaction, and levels of GTH released in the medium were determined by RIA. In control cells, the amounts of three GTH subunit mRNAs (alpha2, FSHbeta, and LHbeta) peaked in July prior to spawning. FSH release spontaneously increased with gonadal maturation and peaked in September, whereas LH release remained low until July and extensively increased in September. Addition of E2 to the culture extensively increased the amounts of LHbeta mRNA in May and July in both sexes. It also increased the alpha2 mRNA in July in the females. In contrast, sGnRH alone did not have any significant effects on the amounts of three GTH subunit mRNAs at all stages, except for the elevation of alpha2 and FSHbeta mRNAs in July in the females. Nevertheless, synergistic effects by sGnRH and E2 were evident for all three GTH subunit mRNAs. In May, sGnRH in combination with E2 synergistically increased the amounts of LHbeta mRNA in the males and alpha2 mRNA in the females. However, in July the combination suppressed the amounts of alpha2 and FSHbeta mRNAs in the females. sGnRH alone stimulated LH release at all stages in both sexes, and the release was synergistically enhanced by E2. Synergistic stimulation of FSH release was also observed in May and July in both sexes. These results indicate that a functional interaction of sGnRH with E2 is differently involved in synthesis and release of GTH. The synergistic interaction modulates GTH synthesis differentially, depending on subunit, stage, and gender, whereas it potentiates the activity of GnRH to release GTH in any situation.