FSH Beta mRNA Levels Research Articles

Gonadotropin-releasing hormone-induced accumulation of follicle-stimulating hormone beta-subunit messenger ribonucleic acid in adenohypophysial cells developing in an ectopic position.

We investigated the influence of LHRH on the accumulation of FSH beta messenger RNA (mRNA) in anterior pituitary glands removed from hamster pups less than 36 h old and transplanted beneath the renal capsules of adult male hamsters (hosts). Three experiments were performed in which some hosts were injected sc with LHRH (1 microgram/injection) and others were injected with vehicle. Injections were begun in the afternoon of the day of transplantation (day 1) and were given at 0800 and 1700 h for 6 days and at 0800 h on the eighth day. An additional experiment was performed in which adult male hamsters not bearing allografts were injected with the same regimen of LHRH or vehicle. The hamsters were decapitated on the eighth day of the study, 2 h after the last injection. The allografts, adenohypophyses of the hosts, adenohypophyses of hamsters without allografts, and adenohypophyses of normal adult male rats were removed and frozen on dry ice immediately. Additionally, adenohypophyses were collected from hamster pups less than 36 h old and 8 and 15 days of age. Total RNAs from some pooled specimens were electrophoresed on a formaldehyde-agarose gel. After transfer to Nytran, the RNAs were hybridized sequentially to complementary DNAs for rat FSH beta and hamster beta-actin. The rat FSH beta complementary DNA probe hybridized to a single RNA (approximately 1.7 Kb) in rat adenohypophyses. It predominantly hybridized to RNA of approximately 1.7 Kb from hamster adenohypophyses. Sometimes it hybridized to RNAs ranging in size from 0.5 Kb to 1.7 kb. The hybridization signals for all samples obtained from dot blot analyses were quantitated and normalized to the signals for beta-actin. The hybridization signals obtained from adenohypophyses of hamsters of different ages increased from 36 h of age to adulthood. The hybridization signal obtained from adenohypophyses of hamsters less than 36 h old (the same age as the donor hamsters) was similar to the hybridization signal obtained from allografts in vehicle-treated hamsters. The relative levels of FSH beta mRNA in allografts of LHRH-treated hosts were: 1) greater than the relative levels in adenohypophyses of hamsters less than 36 h old (P less than 0.05) and in allografts in vehicle-treated hamsters (P less than 0.05), 2) greater than the relative levels in adenohypophyses of 8-day-old hamsters (P less than 0.05), and 3) not different compared to the relative levels in adenohypophyses of 15-day-old hamsters and adult male hamsters.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of FSH beta messenger ribonucleic acid levels in the rat by endogenous inhibin [corrected and republished article originally printed in Endocrinology 1991 Nov;129(5):2802-4

This study investigated the role of endogenous inhibin in regulating FSH beta mRNA levels subsequent to the gonadotropin surge in the immature, estradiol (E2)-treated female rat. Rats which undergo FSH surges on day 29 have low to undetectable levels of FSH beta mRNA at 0900 h on day 30, whereas those treated simultaneously with E2 and progesterone (P) implants to block these surges have considerably higher levels of FSH beta mRNA. In view of the profound inhibitory effect of inhibin on FSH beta mRNA, we examined the possibility that increased inhibin secretion is responsible for the decline in FSH beta mRNA levels on the morning after the FSH surge by immunoneutralization of endogenous inhibin. Twenty-eight day-old rats which received E2 and blank (B1) or P implants were injected iv with 0.4 ml of a potent anti-rat inhibin serum (anti I alpha, prepared in sheep against rat inhibin alpha (1-26)-Tyr27 coupled to human alpha-globulins) or normal sheep serum at 1700 to 1830 h on day 29 and were killed at 0900 h on day 30. Animals which received the inhibin antiserum showed significantly (P less than 0.001) elevated serum FSH levels (22.9 +/- 1.9 ng/ml [E2 + B1] and 17.1 +/- 0.6 ng/ml [E2 + P]) compared to those which received normal serum (4.4 +/- 0.1 [E2 + B1] and 4.2 +/- 0.1 [E2 + P]). Serum LH was undetectable (less than 0.6 ng/ml) in all groups. Free glycoprotein alpha-subunit was also increased (P less than 0.001) by antiserum to inhibin in E2 + B1-treated rats but was significantly suppressed by P after injection of either normal serum or anti I alpha. Total pituitary RNA was extracted and hybridized to cDNA probes for rat FSH beta, LH beta, and the common alpha-subunit by Northern blot analysis; RNA levels were normalized with beta-actin or cyclophilin probes. As expected, in rats which received normal serum, FSH beta mRNA levels were about 4-fold higher after treatment with E2 + P implants than after treatment with E2 + B1 implants. However, injection with anti-inhibin serum resulted in a striking elevation of FSH beta mRNA levels: 13-fold in animals treated with E2 + B1 implants and 5-fold in animals treated with E2 + P implants. There were no significant differences in levels of LH beta or alpha-subunit mRNAs between rats which received anti-inhibin or normal serum although there was a 30-40% decrease in alpha mRNA after P treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of FSH beta messenger ribonucleic acid levels in the rat by endogenous inhibin.

This study investigated the role of endogenous inhibin in regulating FSH beta mRNA levels subsequent to the gonadotropin surge in the immature, estradiol (E2)-treated female rat. Rats which undergo FSH surges on day 29 have low to undetectable levels of FSH beta mRNA at 0900 h on day 30, whereas those treated simultaneously with E2 and progesterone (P) implants to block these surges have considerably higher levels of FSH beta mRNA. In view of the profound inhibitory effect of inhibin on FSH beta mRNA, we examined the possibility that increased inhibin secretion is responsible for the decline in FSH beta mRNA levels on the morning after the FSH surge by immunoneutralization of endogenous inhibin. Twenty-eight day-old rats which received E2 and blank (B1) or P implants were injected iv with 0.4 ml of a potent anti-rat inhibin serum (anti I alpha, prepared in sheep against rat inhibin alpha (1-26)-Tyr27 coupled to human alpha-globulins) or normal sheep serum at 1700 to 1830 h on day 29 and were killed at 0900 h on day 30. Animals which received the inhibin antiserum showed significantly (P less than 0.001) elevated serum FSH levels (22.9 +/- 1.9 ng/ml [E2 + B1] and 17.1 +/- 0.6 ng/ml [E2 + P]) compared to those which received normal serum (4.4 +/- 0.1 [E2 + B1] and 4.2 +/- 0.1 [E2 + P]). Serum LH was undetectable (less than 0.6 ng/ml) in all groups. Free glycoprotein alpha-subunit was also increased (P less than 0.001) by antiserum to inhibin in E2 + B1-treated rats but was significantly suppressed by P after injection of either normal serum or anti I alpha. Total pituitary RNA was extracted and hybridized to cDNA probes for rat FSH beta, LH beta, and the common alpha-subunit by Northern blot analysis; RNA levels were normalized with beta-actin or cyclophilin probes. As expected, in rats which received normal serum, FSH beta mRNA levels were about 4-fold higher after treatment with E2 + P implants than after treatment with E2 + B1 implants. However, injection with anti-inhibin serum resulted in a striking elevation of FSH beta mRNA levels: 13-fold in animals treated with E2 + B1 implants and 5-fold in animals treated with E2 + P implants. There were no significant differences in levels of LH beta or alpha-subunit mRNAs between rats which received anti-inhibin or normal serum although there was a 30-40% decrease in alpha mRNA after P treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

In VivoRegulation of FSH Synthesis by Inhibin and Activin

The effect of a single sc injection of the gonadal peptide, recombinant human activin A (rhActivin A), on gonadotropin synthesis and secretion was examined in adult and immature male and female rats and the effect of recombinant human inhibin A (rhInhibin A) was examined in adult male rats. Pituitary FSH beta, LH beta and alpha messenger RNA (mRNA) levels were determined by blot hybridization. Trunk blood was collected to measure serum FSH levels. Treatment with rhInhibin A (100 micrograms/kg) resulted in a decrease in FSH beta mRNA to 2% of controls levels 6 h after injection. FSH beta mRNA levels started to rebound at 10 h, but were still significantly lower than vehicle-treated controls. Serum FSH levels were significantly reduced at 2 h and were reduced further at 6 and 10 h. There were no significant changes in alpha and LH beta mRNA levels. RhActivin A, at the highest dose (500 micrograms/kg), in immature male rats had only a modest effect (1.2- and 1.3-fold increase) on FSH beta mRNA levels and FSH secretion, respectively, at 2 h. No increase in FSH synthesis and FSH secretion was observed in adult male rats. In contrast, both immature and adult-ovariectomized E2 implanted females showed a robust response to rhActivin A. In immature females, 2 h after rhActivin A (100 and 500 micrograms/kg) administration, FSH beta mRNA levels were elevated 2.0- and 2.2-fold. At this time serum FSH was also elevated. At 6 and 10 h rhActivin A significantly reduced FSH beta mRNA levels from vehicle-treated controls. In contrast, FSH secretion was elevated at 6 h and returned to baseline at 10 h. Administration of rhActivin A (500 micrograms/kg) to adult, ovariectomized-E2 females resulted in a significant increase in FSH beta mRNA levels and FSH secretion at 2 and 6 h. There were no significant changes in alpha and LH beta mRNA levels in either males or females. Thus, these in vivo studies have shown that rhInhibin A can inhibit FSH beta mRNA levels and FSH secretion in the adult male rat. RhActivin A stimulates FSH synthesis and secretion in the immature and adult ovariectomized-E2 females, but has little or no effect in immature and adult males. Hence, there is a sexual dimorphic response to rhActivin A in vivo in the rat.

Estradiol Negatively Regulates Secretogranin II and Chromogranin A Messenger Ribonucleic Acid Levels in the Female Rat Pituitary but Not in the Adrenal

Estradiol (E2) effects on the pituitary and adrenal secretogranin II (SgII) and chromogranin A (CgA) proteins and mRNA levels were analyzed in the adult female rat. Animals were ovariectomized or sham-operated for 2 weeks and then daily injected with various doses of 17 beta-E2 (from 5-100 micrograms) for the following week. SgII and CgA levels were determined by Western blot analysis using two specific antisera. Messenger RNA (mRNA) levels were measured by RNA slot blot analysis using specific cDNA probes. Simultaneously, pituitary LH content and gonadotropin subunit (LH beta, FSH beta, alpha) mRNAs were quantified. Ovariectomy promoted a significant increase in pituitary SgII and CgA proteins (2-fold vs. sham-operated animals, P less than 0.01) and a concomitant rise in their mRNA levels (2.5-fold and 4.5-fold for SgII mRNA and CgA mRNA, respectively, P less than 0.01). In the same animals LH beta, FSH beta, and alpha-subunit mRNA levels increased by 20-, 12-, and 6-fold, respectively. Estrogen replacement resulted in a parallel decrease of CgA and LH beta mRNA to the control values, starting from the lowest steroid dose (5 micrograms). The SgII mRNA decrease was initiated only with a higher concentration of E2 (10 micrograms), as was that of alpha-subunit mRNA; yet, the SgII mRNA level remained significantly higher than in the control pituitary, even with the highest steroid dose (P less than 0.05) at variance with the alpha-mRNA level. Concerning protein concentration, the postcastration increase in CgA was fully reverted with 10 micrograms E2 while that of SgII remained unaffected, as was the pituitary LH content. In the adrenal gland, neither the ovariectomy nor the E2 therapy altered significantly the SgII or CgA protein and mRNA concentrations. We conclude that, in rats, 1) ovarian factors regulate the pituitary SgII and CgA protein and mRNA steady-state levels while such factors are inefficient in the adrenal gland, 2) CgA and LH beta mRNAs exhibit the same sensitivity to E2 while SgII and alpha-subunit mRNAs appear less sensitive, and 3) SgII and LH pituitary contents present a similar pattern of variations when the estrogenic status of the animal is modified.

Activin Stabilizes Follicle-Stimulating Hormone-Beta Messenger Ribonucleic Acid Levels

Activin, a gonadal peptide, stimulates FSH secretion in association with an increase in FSH beta messenger RNA (mRNA) levels at the level of the anterior pituitary gland. The goal of these studies was to determine whether the effects of recombinant human activin A (rhActivin A) are exerted at the post-transcriptional level by affecting the stability of FSH beta mRNA. We determined the apparent half-life of FSH beta mRNA in the presence and absence of rhActivin A using actinomycin D. The anterior pituitary glands from adult male rats were isolated and dispersed enzymatically. Cells were preincubated in the presence of rhActivin A for 24 h to increase FSH beta mRNA levels. Actinomycin D was then added and the cells were incubated for a subsequent 4, 6, 8, 12, and 24 h in the presence or absence of rhActivin A. As reported earlier, the addition of rhActivin A caused parallel increases in FSH secretion and FSH beta mRNA levels, while having no effect on alpha or LH beta mRNA levels. Actinomycin D treatment decreased FSH beta mRNA to 49, 39, and 16% of control levels at the 4, 6, and 8 h time points, respectively. In contrast, when actinomycin D was added in the presence of rhActivin A FSH beta mRNA was reduced to 80, 58, and 42% of control levels at the 4, 6, and 8 h time points, respectively. Using the least squares method of analysis, the apparent half-lives of FSH beta mRNA under these two conditions were calculated. In the presence of actinomycin D, the half-life of FSH beta mRNA was 3.1 h. The addition of activin significantly increased the half-life to 6.5 h. These results suggest that activin A stimulates FSH beta mRNA levels, at least in part, at the posttranscriptional level by increasing the stability of FSH beta mRNA.

The Effects of Activin on Follicle-Stimulating Hormone Secretion and Biosynthesis in Human Glycoprotein Hormone-Producing Pituitary Adenomas*

The effects of activin on pituitary FSH biosynthesis have been previously characterized using primary rat pituitary cultures; however, little is known of the effects of activin on FSH biosynthesis and secretion in human pituitary tissue. Production of intact glycoprotein hormones and free subunits is increasingly recognized in pituitary tumors; however, the regulation of gonadotropins in such tumors has not been addressed. We have investigated the effects of human recombinant activin on glycoprotein hormone biosynthesis and secretion in primary cultures of 12 human glycoprotein hormone-producing pituitary adenomas and compared this with the effects of activin in normal rat anterior pituitary cells. In 33% of the human pituitary tumors studied, significant (P less than 0.05) increases in FSH beta secretion occurred in response to incubation with 20 ng/mL activin for 24 h (19-287% stimulation), without changes in the production of intact FSH. A Northern analysis performed on cells derived from one tumor indicated that FSH beta mRNA levels increased 350% after activin treatments; however, FSH secretion did not parallel the mRNA changes. None of the human glycoprotein hormone-producing tumors significantly increased FSH secretion in response to activin. To validate the biological activity of recombinant human activin-A and to confirm time and dose conditions for the human tumor cultures, we also examined its ability to stimulate FSH production in rat pituitary cultures. Activin (20 ng/mL) added to the culture medium significantly increased FSH secretion and steady state levels of FSH beta mRNA after 24 h. These data indicate that some glycoprotein hormone-producing pituitary tumors treated with purified activin have discordant responses of intact gonadotropins and free subunit responses. In contrast to responses in normal rat gonadotrophs, FSH beta biosynthetic pathways may be uncoupled from intact FSH secretion in a subset of glycoprotein hormone-producing pituitary adenomas.

Dexamethasone Alters Responses of Pituitary Gonadotropin-Releasing Hormone (GnRH) Receptors, Gonadotropin Subunit Messenger Ribonucleic Acids, and Gonadotropins to Pulsatile GnRH in Male Rats*

Dexamethasone (Dex), when administered in high doses, has been shown to suppress spontaneous and GnRH-induced gonadotropin secretion, but the level and the mechanism(s) of this effect are unknown. We administered Dex to castrate testosterone-replaced male rats to determine if gonadotropin gene expression is affected and whether Dex differentially influences GnRH-modulated parameters of gonadotrope function: induction of GnRH receptors (GnRH-R) and gonadotropin synthesis and secretion. GnRH was given iv at 25 ng/pulse at 8, 30, and 120 min intervals for 48 h. Rapid GnRH injection frequency preferentially increased alpha and LH-beta messenger RNA (mRNA) responses to GnRH as well as LH secretion. Slower GnRH injection frequencies were required to increase levels of GnRH-R, FSH-beta mRNA, and FSH secretion. Dex selectively inhibited the serum LH, alpha, and LH-beta mRNA responses to GnRH, but not the serum FSH or FSH-beta mRNA responses. Additionally, it augmented the GnRH-induced increase in GnRH-R. We conclude: 1) induction of GnRH-R, gonadotropin synthesis, and secretion require different modes of GnRH stimulation; 2) Dex acts directly on the gonadotrope to differentially modulate GnRH-induced increases in GnRH-R levels, gonadotropin gene expression, and gonadotropin secretion; and 3) GnRH effects upon induction of GnRH-R, LH, and FSH synthesis and secretion are likely to be mediated via different cellular pathways.

Comparison of the Effects of Cycloheximide and Inhibin on the Gonadotropin Subunit Messenger Ribonucleic Acids*

Cycloheximide (CHX) has been shown to mimic the action of inhibin on gonadotropin secretion by pituitary cell cultures. We showed previously that suppression of FSH secretion by inhibin is associated with a rapid and profound suppression of FSH beta mRNA levels. The present study was designed to examine the mechanism of action of CHX and to determine whether inhibin's actions involve new proteins synthesis. Pituitary cell cultures were treated with control medium or medium containing inhibin, CHX, or inhibin plus CHX for 2 or 6 h. At 6 h, secretion of FSH was decreased by inhibin (72% of control), CHX (58% of control), and the combined inhibitors (56% of control). LH secretion was not significantly changed, while that of free alpha-subunit was reduced only by CHX (68% of control). Levels of FSH beta, LH beta, and alpha-subunit mRNAs were measured by Northern analysis. At 2 h inhibin decreased FSH beta mRNA to 49% of the control value. CHX alone had no effect, while CHX plus inhibin produced intermediate levels (77% of control). By 6 h, however, inhibin and CHX each decreased FSH beta mRNA to very low levels (12% and 15% of control, respectively), and in cultures treated with both inhibin and CHX, this RNA was barely detectable. To determine the reversibility of the effects of these inhibitors, cells were incubated with fresh control medium after 6 h. Secretion of FSH and free alpha-subunit remained suppressed 4 h later; recovery was complete by 16 h in inhibin treated cultures. FSH beta mRNA returned to control levels by 4 h in inhibin-treated and by 16 h in CHX-treated cultures. Levels of LH beta and alpha-subunit mRNA were comparable to control values at all times. In conclusion, 1) CHX, like inhibin, suppresses FSH beta mRNA levels, although its actions are less rapid and less rapidly reversible; 2) inhibin requires ongoing protein synthesis for full expression of its inhibitory effects; 3) the synthesis and secretion of LH are much less sensitive to inhibition by either inhibin or CHX than are the synthesis and secretion of FSH; and 4) secretion of free alpha-subunit involves a labile protein(s).

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.

Gonadotropin Subunit Messenger RNA Concentrations after Blockade of Gonadotropin-Releasing Hormone Action: Testosterone Selectively Increases Follicle-Stimulating Hormone β-Subunit Messenger RNA by Posttranscriptional Mechanisms

Regulation of gonadotropin gene expression by sex steroids may occur via direct effects on the pituitary and/or indirect effects of steroids mediated through hypothalamic GnRH. We aimed to define the effects of testosterone (T) on alpha, LH beta, and FSH beta mRNA expression in the male rat after blockade of GnRH action on the gonadotrope. A water-soluble GnRH antagonist was administered iv to castrate male rats (increased endogenous GnRH secretion) and to castrate T-replaced rats in which gonadotropin subunit mRNAs had been increased by prior treatment with exogenous GnRH pulses. In castrate male rats, GnRH antagonist resulted in a fall in all three subunit mRNAs. Alpha and LH beta declined at slower rates (half-disappearance after 50 and 65 h, respectively), and neither fell to values present in intact rats over 84 h. In contrast, FSH beta mRNA declined more rapidly, with a half-disappearance after 20 h. In castrate T-replaced rats, alpha mRNA declined at a rate similar to that in castrates (half-disappearance after 50 h). LH beta declined more slowly, and the rate of FSH beta decline was markedly prolonged in the presence of T (half-disappearance time increased from 20 to 50 h). These results suggest that T exerts direct effects on FSH beta transcription or mRNA stability which are independent of GnRH action. To assess these possibilities, a long-acting GnRH antagonist (Detirelix) was administered to castrate male rats, which also received T or sham implants 4 days after castration. FSH beta mRNA levels fell during the 4 days of Detirelix alone, but the addition of T on day 4 resulted in a 2-fold rise in FSH beta mRNA, restoring FSH beta mRNA to levels present in intact rats. Serum FSH closely paralleled FSH beta mRNA concentrations. Alpha mRNA was reduced by 25%, and LH beta mRNA concentrations were unchanged in the presence of T. The rate of alpha mRNA transcription was markedly reduced and that of LH beta tended to fall in T-treated rats, but T had no significant effect on the FSH beta transcription rate. Thus, the action of T to increase concentrations of cytosolic FSH beta mRNA appears to be exerted at a posttranscriptional level, possibly via effects of T on FSH beta mRNA stability. This may represent a mechanism by which T can effect differential regulation of gonadotropin subunit mRNA concentrations.

Effects of Progesterone on the Estradiol-Induced Follicle-Stimulating Hormone (FSH) Surge and FSHβMessenger Ribonucleic Acid in the Rat*

This study was designed to investigate the effects of progesterone on the estradiol (E2)-induced FSH surge and FSH beta messenger RNA (mRNA) using immature rat models developed previously to demonstrate inhibition or facilitation of the LH surge by progesterone. Twenty-eight day-old rats that received E2 implants at 0900 h had FSH surges about 1700 h on day 29 (32 h). In rats treated with E2 alone, serum FSH was 15.1 +/- 1.6 ng/ml at this time, while in those animals treated concurrently with E2 and progesterone, serum FSH was significantly suppressed (8.3 +/- 0.7 ng/ml, P less than 0.001). For demonstration of progesterone facilitation, rats were primed for 24 h with E2 before progesterone treatment. This led to premature and enhanced FSH secretion: at 1400 h on day 29 serum FSH was 45.5 +/- 2.7 ng/ml compared to 6.4 +/- 0.5 ng/ml in rats treated with E2 alone. To examine the effects of these dual actions of progesterone on FSH synthesis, steady state concentrations of FSH beta mRNA were measured by Northern analysis. FSH beta mRNA generally increased in parallel with FSH release. Levels of this mRNA were about 1.5-fold higher in rats undergoing E2-induced FSH surges than in rats in which the surge was blocked by progesterone. Also, at the onset of the progesterone-facilitated FSH surge, FSH beta mRNA was about 5-fold higher in animals treated with E2 and progesterone than in those treated with E2 only. On the morning after the FSH surge (48 h after E2 treatment) FSH beta mRNA was low to undetectable. In contrast, levels of FSH beta mRNA were 7- to 8-fold higher at this time in rats in which the surge was blocked by progesterone. Serum inhibin concentrations were significantly elevated (P less than 0.05) in animals treated with E2 alone for 32 h (3077 +/- 260 fmol/ml) or 48 h (2344 +/- 148 fmol/ml) compared to those treated with E2 and progesterone in the inhibition paradigm (2469 +/- 106, 1896 +/- 114 fmol/ml, respectively). After 32 h of E2 treatment in the facilitation paradigm, serum inhibin was comparable (P greater than 0.2) in rats treated for 8 h with blank implants (2592 +/- 168 fmol/ml) and those treated for 8 h with progesterone (2720 +/- 188 fmol/ml).(ABSTRACT TRUNCATED AT 400 WORDS)

Castration Induces Time-Dependent Changes in the Follicle-Stimulating Hormone β-Subunit Messenger Ribonucleic Acid-Containing Gonadotrope Cell Population*

We have previously observed 3- to 10-fold increases in pituitary LH beta-subunit mRNA levels in the rat 28 days after castration. These changes correlate with increases in percentages and areas of cells that bear the LH beta mRNA and with the amount of label for mRNA per cell. In contrast, FSH beta mRNA levels increase 2.5- to 4-fold 7-14 days after castration, decline to near-intact levels 28 days postcastration, and rise 4.5-fold by 96 days postcastration. The purpose of this study was to determine morphological correlates of these changes in FSH beta mRNA levels. Dispersed pituitary cells from intact and castrated rats were analyzed for FSH beta and LH beta mRNAs and protein by in situ hybridization techniques and immunocytochemistry, respectively. In intact animals over 79% of pituitary cells labeled for FSH beta mRNA were small (area less than 150 microns 2). However, 7 days after castration, the average area of labeled cells increased 4-fold (80% were over 200 microns 2 in area), without a significant change in percentages of FSH beta mRNA-containing cells. The amount of mRNA per cell (as measured by area of label per cell) increased 6-fold. Fourteen days after castration, the average area of cells containing FSH beta mRNA decreased to 2 times that in intact rats (48% were greater than 200 microns 2). The percentage of labeled cells increased from 11% (intact) to 20%. Furthermore, the dual labeling studies showed that 37% of these FSH cells were monohormonal (detected by FSH beta mRNA, but not LH beta antigen) compared with 23% in intact rats. At this same time, the FSH cells exhibited a decrease in the amount of mRNA per cell. In 21- to 84-day castrates, average areas of FSH beta mRNA remained at 2-2.5 times the areas of cells from intact rats. In addition, 21 days after surgery the percentages of labeled cells and amount of FSH beta mRNA per cell declined to those in intact rats. A greater proportion was multihormonal (only 15% expressed FSH beta mRNA but not LH beta antigens). At 84 days there were 2-fold increases in the percentages of labeled cells and the density of label, which correlate with the recovery in mRNA levels assayed at 96 days. Thus, factors that contribute to the early rise in FSH beta mRNA include increases in the amount of mRNA per cell, which coincides with increased cell area.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibin secretion during the rat estrous cycle: Relationships to FSH secretion and FSH beta subunit mRNA concentrations

Serum inhibin and FSH and FSH beta subunit mRNA levels were measured at 3h intervals throughout the 4 day estrous cycle in female rats and hourly between 1000 and 2400 h of proestrus. On proestrus, serum inhibin concentrations fell during the late morning-early afternoon, then increased transiently during the late afternoon gonadotropin surges. Inhibin levels decreased during the late evening of proestrus, coincident with the FSH surge-related rise in FSH beta mRNA levels. Serum inhibin remained relatively stable during estrus and early metestrus, but rose during the late evening of metestrus and remained elevated until early diestrus. FSH beta mRNA levels were elevated on late estrus and early metestrus and declined during the evening of metestrus as serum inhibin levels increased. These data show that concetrations of serum inhibin change during the estrous cycle and that a general inverse relationship exist between serum inhibin and FSH levels and FSH beta mRNA concentrations in the pituitary. This suggests that inhibin may inhibit FSH beta gene expression and FSH secretion during the 4 day cycle in female rats.

Inhibin, Activin, and Follistatin: Regulation of Follicle-Stimulating Hormone Messenger Ribonucleic Acid Levels

Primary pituitary cell cultures derived from adult male rats were used to explore the direct effects of purified porcine inhibin and follistatin, and recombinant human activin A on FSH beta, as well as LH beta and alpha-subunit mRNA levels. Subunit mRNAs were determined by blot hybridization using alpha, LH beta, and FSH beta cDNA and genomic fragments. Treatment with inhibin for 72 h significantly suppressed alpha and FSH beta mRNA levels with parallel changes in FSH secretion. No change in LH beta mRNA levels was observed. A decrease in FSH beta mRNA to undetectable levels was seen 4 h after inhibin administration. Recombinant human Activin A caused dose-dependent and parallel increases in FSH beta mRNA levels and FSH secretion. This increase was evident at 4 h after activin administration and maintained at longer times. alpha and LH beta mRNA levels remained unchanged. Follistatin addition to cultures for 72 h significantly reduced FSH beta mRNA levels. In a time-course experiment, a reduction in FSH beta mRNA to undetectable levels was observed 24 h after follistatin administration. There were no changes in alpha or LH beta mRNA levels. These data demonstrate that the actions of these gonadal peptides on FSH secretion may be accounted for, at least in part at the level of biosynthesis, by reductions in FSH beta mRNA levels directly at the level of the anterior pituitary gland.

Gonadotropin-Releasing Hormone Regulates Follicle- Stimulating Hormone β-Subunit Gene Expression in the Male Rat

Since the role of GnRH in the control of FSH release and synthesis is controversial, we have examined the effect of elimination of GnRH action on gonadotropes on FSH beta gene expression, FSH release, and synthesis. GnRH stimulation of the pituitary was abolished by continuous infusion of either a GnRH antagonist or a GnRH antiserum. We also examined the effects of gonadotrope desensitization, using a continuous infusion of GnRH or GnRH agonist analog. FSH beta mRNA levels were determined by dot blot hybridization using rat FSH beta cDNA, and changes were related to pituitary and serum FSH concentrations. FSH beta mRNA levels increased after orchidectomy and correlated well with serum FSH concentrations. Overall FSH synthesis was increased after castration, as judged by elevated serum FSH and unchanged pituitary FSH content. In orchidectomized rats, continuous GnRH antagonist infusion prevented the postcastration rise in FSH beta mRNA levels and serum FSH. Pituitary FSH content was reduced at 7 days, but not at 14 days. In intact rats, GnRH antagonist infusion for 7 days had no effect on FSH beta mRNA levels, but after 14 days, there was a 33% reduction, and serum FSH was suppressed. Pituitary FSH content was decreased after GnRH antagonist treatment for 7 or 14 days. Daily injection of GnRH antiserum for 6 days abolished the increases in FSH beta mRNA levels and serum FSH in orchidectomized rats, but pituitary FSH content was unaffected. In intact rats, GnRH antiserum treatment reduced FSH beta mRNA levels by 38%, suppressed serum FSH, and decreased pituitary FSH content. When gonadotropes were desensitized by a continuous infusion of GnRH for 14 days or GnRH agonist analog for 28 days, FSH beta mRNA levels and pituitary FSH content were markedly reduced, and serum FSH was suppressed to undetectable levels. We concluded that 1) endogenous GnRH is required for the maintenance of FSH beta mRNA levels in both intact and orchidectomized rats; 2) FSH beta mRNA levels are coupled to the level of FSH biosynthesis, indicating the physiological importance of this pretranslational regulation; 3) desensitization is more effective at inhibiting FSH beta gene expression and FSH synthesis than preventing gonadotrope stimulation using the GnRH antagonist or antiserum; and 4) the actions of GnRH on FSH beta mRNA levels are paralleled by its effects on LH beta mRNA levels, suggesting that GnRH provides a common primary stimulus for the induction of both beta-subunit genes in vivo. These data provide further evidence for the crucial stimulatory role of GnRH in the control of FSH synthesis.

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)

The effect of altered thyroid status on pituitary hormone messenger ribonucleic acid concentrations in the rat.

To study the effects of altered thyroid status on pretranslational control of pituitary hormones, adult male rats were given propylthiouracil for 6 weeks and underwent the following studies. 1) Rats were injected with T3 at 10 micrograms/100 g BW daily for 10 days. 2) Rats were given T3 injections at 0, 0.01, 0.1, 1.0, or 10 micrograms/100 g BW for 10 days. 3) Rats were killed 0, 1, 6, or 24 h after a single injection of T3 at 10 micrograms/100 g BW or after 5 or 10 days of daily T3 injections. Pituitary mRNA concentrations of TSH beta, alpha-subunit, PRL, GH, POMC, FSH beta, and LH beta were determined for individual animals. Marked increases in TSH beta and alpha-subunit mRNAs occurred after PTU treatment, and these changes were reversed by 1.0 microgram/100 g BW T3 and within 24 h of a single T3 injection of 10 micrograms/100 g BW. Further increases in the dose or time course of T3 administration led to a relatively greater suppression of TSH beta mRNA levels than alpha-subunit mRNA levels. In contrast, GH and PRL mRNA levels were low in hypothyroid animals, and both rose toward control levels with 0.1 microgram/100 g BW T3 and by 24 h after a single T3 dose. Induction of hyperthyroidism did not further increase GH mRNA levels above control, but increased PRL mRNA levels 2-fold over control. No changes were seen in FSH beta, LH beta, or POMC mRNA levels with any treatment. Thus, studies of altered thyroid status in the rat reveal dose-response and time-course variability in the pretranslational control of TSH beta, alpha-subunit, GH, and PRL by thyroid hormone.

Regulation of Follicle-Stimulating Hormone β and Common α-Subunit Messenger Ribonucleic Acid by Gonadotropin-Releasing Hormone and Estrogen in the Sheep Pituitary

The effect of gonadotropin-releasing hormone (GnRH) and/or estradiol (E2) on pituitary messenger ribonucleic acid (mRNA) levels of luteinizing hormone beta (LH beta), follicle-stimulating hormone beta (FSH beta) and the common alpha-subunit were determined in anterior pituitary glands from ovariectomized (OVX) ewes. Hypothalamo-pituitary disconnected (HPD) ewes receiving appropriate hormonal treatment were used to assess the relative roles of GnRH and E2 in directly regulating FSH beta and alpha-subunit mRNA levels. Levels of LH beta mRNA were increased in OVX animals compared with intact controls, and E2 treatment of OVX animals significantly reduced mRNA levels of LH beta and FSH beta. HPD substantially reduced FSH beta and alpha-subunit mRNA levels. Treatment of OVX/HPD animals with pulses of GnRH (250 ng/2 h) for 1 week restored FSH beta and alpha-subunit mRNA to OVX levels. Combined GnRH and E2 treatment significantly lowered FSH beta mRNA levels, but resulted in a rise in alpha-subunit mRNA levels. Treatment of OVX/HPD ewes with E2 alone had no effect on FSH beta and alpha-subunit mRNA levels. These findings indicate that E2 acts directly on the pituitary to negatively regulate FSH beta mRNA levels, and to positively regulate alpha-subunit mRNA levels in the presence of GnRH.

Gonadotropin-Releasing Hormone-Dependent Regulation of Gonadotropin Subunit Messenger Ribonucleic Acid Levels in the Rat*

The Nal-Glu GnRH antagonist (GnRHA) was given to castrate male and female rats 7 days after gonadectomy to assess the impact of selective GnRH inhibition on the steady state mRNA levels of FSH beta, LH beta, and alpha-subunit and serum levels of FSH and LH. A low dose of GnRHA (125 micrograms/kg.day) given to female rats for 1, 3, or 7 days resulted in suppression of serum FSH and LH levels by 7 days to 50% and 40%, respectively, of ovariectomized control values. LH beta mRNA levels decreased in a time-dependent manner, so that by 7 days, LH beta mRNA levels were less than those in intact controls. There were significant but less dramatic declines in alpha and FSH beta mRNA levels. A higher dose of GnRHA (500 micrograms/kg.day) for 7 or 14 days administered to castrate male or female rats resulted in inhibition of serum LH and FSH to or below levels in intact controls. At this dose, all three gonadotropin subunit mRNA levels fell from castrate values toward or below those in intact controls. Thus, although low dose GnRHA administration suppressed LH beta mRNA more than FSH beta mRNA levels, high dose GnRHA treatment resulted in equal suppression of all three gonadotropin subunits. No stimulatory effects on alpha-subunit mRNA levels were observed with either dose of GnRHA. We conclude that the pretranslational control of gonadotropin subunit biosynthesis is GnRH dependent. Adequate dose and length of administration of the potent Nal-Glu GnRHA results in suppression of both the serum gonadotropins FSH and LH and the mRNAs for FSH beta, LH beta, and alpha-subunit in female and male rats.