Selective failure of androgens to regulate follicle stimulating hormone beta messenger ribonucleic acid levels in the male rat

FSH beta, as well as LH beta, and alpha-subunit mRNA levels were examined in the pituitary glands of male rats after sex steroid replacement at various times (7, 28, or 90 days) after orchiectomy. Testosterone propionate, dihydrotestosterone propionate, or 17 beta-estradiol benzoate (E) were administered daily for 7 days before killing, to assess the role of different gonadal steroids on gonadotropin subunit mRNA levels. Subunit mRNAs were determined by blot hybridization using rat FSH beta genomic DNA, and alpha and LH beta cDNAs. At all time points, alpha and LH beta mRNAs increased after gonadectomy and fell toward normal levels with either androgen or estrogen replacement. FSH beta mRNA levels increased variably postcastration: 4-fold at 7 days, 2-fold at 28 days, and 4- to 5-fold at 90 days. Although E replacement uniformly suppressed FSH beta mRNAs, neither testosterone propionate nor dihydrotestosterone propionate administration suppressed FSH beta mRNA levels at any time point after orchiectomy. These data demonstrate that there is a relative lack of negative regulation of FSH beta mRNA levels by androgens in a paradigm in which E administration results in marked negative regulation of FSH beta mRNA levels. Thus, in the male rat, estrogens negatively regulate all three gonadotropin subunit mRNA levels while androgens negative regulate LH beta and alpha-subunit but fail to suppress FSH beta mRNAs.     

Divergent regulation of gonadotropin subunit mRNA levels by androgens in the female rat.

To examine the effects of gonadal steroids on the pretranslational regulation of the gonadotropin subunits in the female, adult female rats, beginning 7 or 28 days after ovariectomy, received daily injections of testosterone propionate (T), dihydrotestosterone propionate (D), or estradiol benzoate (E) for 7 days. Intact cycling females and ovariectomized rats that received vehicle served as controls. Serum was obtained for LH and FSH levels to assess changes in gonadotropin secretion. Total RNA from individual rats was recovered and analyzed by blot hybridization with specific radiolabeled cDNA probes for the alpha, LH beta, and FSH beta subunits. Autoradiographic bands were quantitated and standardized to mRNA levels in the intact animals. Ovariectomy resulted in a rise in serum gonadotropin levels and all three gonadotropin subunit mRNA levels. Estrogen replacement resulted in suppression of alpha, LH beta, and FSH beta mRNAs whether given at 7 or 28 days after ovariectomy. In contrast, whereas androgen replacement decreased alpha and LH beta mRNAs, D or T did not consistently suppress FSH beta mRNAs. We conclude that chronic estrogen administration to the castrated female rat uniformly suppresses all three gonadotropin subunit mRNA levels. In female rats, as in male rats, chronic androgen administration fails to negatively regulate FSH beta mRNAs.     

Sequence analysis and expressional regulation of messenger RNAs encoding beta subunits of follicle-stimulating hormone and luteinizing hormone in the red-bellied newt, Cynops pyrrhogaster

Two distinct cDNAs encoding beta subunits of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were cloned from the cDNA library constructed for the pituitary of the red-bellied newt, Cynops pyrrhogaster, and sequenced. The newt FSHbeta and LHbeta cDNAs encode polypeptides of 129 and 131 amino acids, including signal peptides of 20 and 19 amino acids, respectively. The number and position of cysteine and N-glycosylation in each of the beta subunits of FSH and LH, which are considered essential for assembly of the alpha subunit, are well conserved between the newt and other tetrapods. The high homology (41.6%) between the beta subunits of newt FSH and LH imply less specificity of FSH and LH in gonadal function. One cDNA encoding the common polypeptide chain alpha subunit of FSH and LH was also isolated from the newt pituitary gland. The mRNAs of FSHbeta, LHbeta, and the alpha subunit were expressed only in the pituitary gland among various newt tissues. Double-staining with in situ hybridization and immunohistochemistry revealed coexpression of FSHbeta and LHbeta in the same newt pituitary cells. Ovariectomy induced a significant increase in FSHbeta mRNA levels, but there was no significant change in LHbeta or alpha subunit mRNA levels compared with those in control animals. Taken together, these data suggest that two kinds of gonadotropins, namely FSH and LH, are expressed in the same gonadotropin-producing cells in the pars distalis of the newt as well as in other tetrapods and that the expression of FSHbeta is negatively regulated by the ovaries.     

Differential regulation of pituitary gonadotropin subunit messenger ribonucleic acid levels in photostimulated Siberian hamsters

FSH levels begin to rise 3-5 days after male Siberian hamsters are transferred from inhibitory short photoperiods to stimulatory long photoperiods. In contrast, LH levels do not increase for several weeks. This differential pattern of FSH and LH secretion represents one of the most profound in vivo examples of differential regulation of the gonadotropins. The present study was undertaken to characterize the molecular mechanisms controlling differential FSH and LH synthesis and secretion in photostimulated Siberian hamsters. First, we cloned species-specific cDNAs for the three gonadotropin subunits: the common alpha subunit and the unique FSHbeta and LHbeta subunits. All three subunits share high nucleotide and predicted amino acid sequence identity with the orthologous cDNAs from rats. We then used these new molecular probes to examine the gonadotropin subunit mRNA levels from pituitaries of short-day male hamsters transferred to long days for 2, 5, 7, 10, 15, or 20 days. Short-day (SD) and long-day (LD) controls remained in short and long days, respectively, from the time of weaning. We measured serum FSH and LH levels by RIA. FSHbeta, LHbeta, and alpha subunit mRNA levels were measured from individual pituitaries using a microlysate ribonuclease protection assay. Serum FSH and pituitary FSHbeta mRNA levels changed similarly following long-day transfer. Both were significantly elevated after five long days (2.3- and 3.6-fold, respectively; P < 0.02) and declined thereafter, but they remained above SD control values through 20 long days. Alpha subunit mRNA levels also increased significantly relative to SD control values (maximum 2-fold increase after seven long days; P < 0.03), although to a lesser extent than FSHbeta. Neither serum LH nor pituitary LHbeta mRNA levels changed significantly following long-day transfer. The results indicate that long-day-associated increases in serum FSH levels in Siberian hamsters reflect an underlying increase in pituitary FSHbeta and alpha subunit mRNA accumulation.     

Regulation of alpha and luteinizing hormone beta subunit messenger ribonucleic acids during stimulatory and downregulatory phases of gonadotropin-releasing hormone action

Decreased gonadotropin responsiveness (downregulation) to gonadotropin-releasing hormone (GnRH) following chronic in vivo and in vitro exposure to GnRH or its agonist (GnRH-A) has been previously reported. In the present studies, changes in LH subunit mRNAs in rat pituitary monolayer culture during stimulatory and down regulatory phases of GnRH action are described. Rat pituitary cells in culture, pretreated with medium alone or GnRH-A (10(-6) M) for 48 h were extensively washed and treated with graded concentrations of GnRH [10(-9) to 10(-7)] for 4 h. Medium was assayed for luteinizing hormone (LH) immunoreactivity, and total cytoplasmic RNAs were extracted by the hot phenol-guanidinium isothiocyanate method. Subunit-specific mRNAs were quantified by dot-hybridization assay using 32P-labeled subunit-specific cDNA probes. Cells pretreated with medium alone showed a dose-dependent increase in medium LH immunoreactivity, but the alpha and LH beta mRNAs showed no change over the 4-h period. Cells pretreated with GnRH-A showed no significant increase in medium LH with GnRH treatment, thus demonstrating that the cells had been desensitized by prior GnRH-A treatment. Alpha and LH beta subunit mRNAs of cells pretreated with GnRH-A did not show any significant change with further GnRH treatment. In subsequent experiments, cells were incubated with medium alone or 10(-7) M GnRH for 4, 8, or 24 h. GnRH failed to increase subunit mRNAs after 4 and 8 h incubation; after 24 h, alpha subunit mRNA showed a modest but significant increase and beta subunit mRNA showed a modest decrease compared to controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of inhibin from primate Sertoli cells and GnRH on gonadotropin subunit mRNA in rat pituitary cell cultures

Partially purified inhibin from primate Sertoli cell culture medium (pSCl) suppresses both LH and FSH secretion from cultured rat pituitary cells stimulated with GnRH. To examine the mechanism of action of pSCl, we have measured steady state levels of mRNAs for the gonadotropin subunits in pituitary cell cultures exposed to 10 nM GnRH for 6 h in control or pSCl-containing medium (short term) and after 72-h pretreatment with pSCl or control medium (long term). Messenger RNA levels were determined by Northern analysis using specific cDNA probes for rat FSH beta, LH beta, and the common alpha-subunit. In the long term experiments, pSCl inhibited GnRH-stimulated release of FSH (47.4 +/- 3.3% of control), LH (69.2 +/- 2.3%), and free glycoprotein alpha-subunit (74.2 +/- 4.5%), and intracellular FSH declined to 88.4 +/- 3.5% of control. Concentrations of the subunit mRNAs were all decreased: FSH beta to 54.4 +/- 5.0%, LH beta to 79.6 +/- 9.4%, and alpha to 70.8 +/- 8.7% of control. In the short-term experiments, pSCl also suppressed FSH, LH, and alpha-subunit secretion to 75.9 +/- 3.6%, 79.5 +/- 2.1%, and 90.9 +/- 1.8% of control, respectively. Intracellular LH and alpha-subunit levels were significantly increased in cells treated for 6 h with GnRH and pSCl (155 +/- 18%, 145 +/- 14% of control), while FSH was comparable to control. After 6 h, pSCl selectively reduced the level of mRNA for FSH beta (56.5 +/- 5.8% of control).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Stimulatory effects of insulin-like growth factor 1 on expression of gonadotropin subunit genes and release of follicle-stimulating hormone and luteinizing hormone in masu salmon pituitary cells early in gametogenesis

Insulin-like growth factor-I (IGF-I) has been shown to be involved in pubertal activation of gonadotropin (GTH) secretion. The aim of this study was to determine if IGF-I directly stimulates synthesis and release of GTH at an early stage of gametogenesis. The effects of IGF-I on expression of genes encoding glycoprotein alpha (GPalpha), follicle-stimulating hormone (FSH) beta, and luteinizing hormone (LH) beta subunits and release of FSH and LH were examined using primary pituitary cells of masu salmon at three reproductive stages: early gametogenesis, maturing stage, and spawning. IGF-I alone or IGF-I + salmon GnRH (sGnRH) were added to the primary pituitary cell cultures. Amounts of GPalpha, FSHbeta, and LHbeta mRNAs were determined by real-time PCR. Plasma and medium levels of FSH and LH were determined by RIA. In males, IGF-I increased the amounts of all three subunit mRNAs early in gametogenesis in a dose-dependent manner, but not in the later stages. In females, IGF-I stimulated release of FSH and LH early in gametogenesis, whereas no stimulatory effects on the subunit mRNA levels were observed at any stage. IGF-I + sGnRH stimulated release of FSH and LH at all stages in both sexes, but had different effects on the subunit mRNA levels depending on subunit and stage. The present results suggest that IGF-I itself directly stimulates synthesis and release of GTH early in gametogenesis in masu salmon, possibly acting as a metabolic signal that triggers the onset of puberty.     

Progesterone does not affect the amount of mRNA for gonadotropins in the anterior pituitary gland of ovariectomized ewes

To evaluate the effect of progesterone on the synthesis and secretion of gonadotropins, ovariectomized ewes either were treated with progesterone (n = 5) for 3 wk or served as controls (n = 5) during the anestrous season. After treatment for 3 wk, blood samples were collected from progesterone-treated and ovariectomized ewes. After collection of blood samples, hypothalamic and hypophyseal tissues were collected from all ewes. Half of each pituitary was used to determine the content of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and the number of receptors for gonadotropin-releasing hormone (GnRH). The amounts of mRNA for LH beta subunit, FSH beta subunit, alpha subunit, growth hormone, and prolactin were measured in the other half of each pituitary. Treatment with progesterone reduced mean serum concentrations of LH (p less than 0.001) but ot FSH (p greater than 0.05). Further, progesterone decreased (p less than 0.05) the total number of pulses of LH. We were unable to detect pulsatile release of FSH. Hypothalamic content of GnRH, number of receptors for GnRH, pituitary content of gonadotropins and mRNA for LH beta subunit, FSH beta subunit, alpha subunit, growth hormone, and prolactin were not affected (p greater than 0.05) by treatment with progesterone. Thus, after treatment with progesterone, serum concentrations of LH (but not FSH) are decreased. This effect, however, is not due to a decrease in the steady-state amount of mRNA for LH beta or alpha subunits.     

Effects of starvation and refeeding on gonadotropin and thyrotropin subunit mRNAs in male Japanese quail

The contents of mRNAs encoding LH beta-, FSH beta-, TSH beta- and common alpha-subunit precursor molecules were measured in food-deprived and subsequently re-fed male Japanese quail. Pituitary LH beta, FSH beta and common alpha mRNA levels were decreased by starvation, and increased to the control levels by re-feeding. The rates of decreases of LH beta and common alpha mRNA levels were greater the corresponding rate for FSH beta levels. Pituitary TSH beta mRNA levels were not decreased by starvation, but increased transitorily by re-feeding. Plasma LH and triiodothyronine levels were decreased by starvation, and then increased to control levels by re-feeding, while plasma FSH and thyroxine levels did not show significant changes. Plasma LH and FSH levels showed positive correlations with pituitary common alpha and FSH beta mRNA levels, respectively, while plasma thyroxine levels showed a negative correlation with TSH beta mRNA levels. Hepatic weight was decreased slightly but significantly by starvation, and then showed a remarkable rebound after re-feeding was started. These results suggest that LH synthesis and secretion are more sensitive to starvation than FSH synthesis and secretion in Japanese quail, and that LH production recovered to initial levels within several days when birds were fully fed. Also, there is a possibility that the synthesis of TSH is accelerated transitorily by re-feeding. Furthermore, these results showed that there are different relationships between the plasma levels of LH, FSH, and TSH and the various hormone subunit mRNA levels. The remarkable change in hepatic weight leads us to assume that hepatic thyroid hormone metabolism is affected by starvation and re-feeding.     

Effects of estradiol on gonadotropin subunit messenger ribonucleic acid amounts during an induced gonadotropin surge in anestrous ewes

The preovulatory gonadotropin surge in the sheep was recently characterized by a divergent pattern of LH beta and FSH beta mRNAs immediately preceding this event. It is not clear whether this pattern is due to estradiol (E2), inhibin or other effectors. In this study, to determine if E2 may be involved in the divergent beta mRNA patterns seen during the surge, gonadotropin surges were induced in anestrous ewes (An) by E2 (An + E2) and several parameters were then measured. These included the amounts of alpha, LH beta, and FSH beta mRNAs, as assessed by solution hybridization assays, plus pituitary and serum gonadotropin concentrations. The values were compared with those observed in control, An ewes, to assess the effect of E2. The E2 treatment resulted in LH and FSH surges that appeared to be similar to the normal surges seen during the breeding season. Concomitantly, the E2 treatment lowered pituitary concentrations of FSH (P less than 0.05), while LH amounts did not change. Although the effect of E2 on gonadotropin subunit mRNA amounts varied depending upon the individual subunit, the changes that were observed paralleled changes reported during the preovulatory surge of the cycle. Specifically, alpha mRNA amounts increased significantly (P less than 0.001) while FSH beta mRNA amounts fell dramatically (P less than 0.001). Moreover, LH beta mRNA amounts were slightly increased, although not significantly by E2. These results demonstrate that E2 effects changes in the amounts of the gonadotropin subunit mRNAs during an induced gonadotropin surge in An ewes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and sequence analysis of FSH and LH in the giant panda (Ailuropoda melanoleuca)

The giant panda (Ailuropoda melanoleuca) is an endangered species and indigenous to China. It has been proposed that it has a highly specialized reproductive pattern with low fecundity, but little is known about its basic reproductive biology at the molecular level. In this report the genes encoding gonadotropin subunits alpha, follicle-stimulating hormone (FSH) beta and luteinizing hormone (LH) beta of the giant panda were amplified for the first time by RT-PCR from pituitary total RNA, and were cloned, sequenced and analyzed. The results revealed that the open reading region (ORF) of gonadotropin subunits alpha, FSH beta and LH beta are 363, 390 and 426 bp long, respectively. They displayed a reasonably high degree (74-94, 85-93, 75-91%, for alpha, FSH beta and LH beta subunits, respectively) of identity when deduced amino acids were compared with homologous sequences from partial available mammals including human, cattle, sheep, pig, rat, mouse. Three distinct differences were found at the site of 59 aa of the alpha subunit and 55 aa, 68 aa of FSH beta subunit. Our results provide an insight into understanding the mechanism of reproduction regulation and genetic characteristics of giant panda which will make an actual contribution to its conservation. In addition they lay a foundation for a further study towards producing recombinant panda FSH and LH which can be used in artificial breeding aimed to increase its captive reproductive efficiency.     

Analysis of androgen action on pituitary gonadotropin and prolactin secretion in ewes

To study the role of androgens in the control of gonadotropin and prolactin secretion in ther ewe, we have characterized androgen receptors in pituitary cytosol, and investigated the effect of androgens on pituitary hormone release in vivo and in vitro. High affinity, low capacity receptors, with an affinity for methyltrienolone (R1881) greater than 5 alpha-dihydrotestosterone (5 alpha-DHT) greater than testosterone (T) much greater than androstenedione (A4), estradiol-17 beta (E2) and progesterone (P), were identified in pituitary cytosol. Addition of 1 nM 5 alpha-DHT, but not A4, inhibited luteinizing hormone (LH) release from pituitary cells in vitro, induced by 10(10) to 10(-7) M luteinizing hormone releasing hormone (LHRH). The release of follicle-stimulating hormone (FSH) with 10(-9) M LHRH was inhibited when cells were incubated with 1 nM 5 alpha-DHT. 5 alpha-DHT had no effect when higher or lower doses of LHRH were used. In ovariectomized ewes, neither an i.v. injection of 1 mg, nor intracarotid injections of up to 1 mg, 5 alpha-DHT affected plasma LH, FSH or prolactin levels, despite dose-related increases in plasma 5 alpha-DHT levels. Daily or twice daily i.m. injections of 5 mg 5 alpha-DHT in oil did not affect LH or FSH levels, but daily injections of 20 mg significantly reduced plasma LH levels within 4 days and plasma FSH levels within 6 days. Thus, despite the presence of androgen receptors in the ewe pituitary, we conclude that androgens per se are of minimal importance in the regulation of pituitary LH, FSH and prolactin secretion in the ewe. The low binding affinity of A4 and the lack of its effect on hormone secretion in vitro suggests that A4 may act as an estrogen precursor rather than an androgenic hormone. The function of the pituitary androgen receptor remains to be established.     

Early maturity in the male striped bass, Morone saxatilis: follicle-stimulating hormone and luteinizing hormone gene expression and their regulation by gonadotropin-releasing hormone analogue and testosterone

Striped bass are seasonal breeding fish, spawning once a year during the spring. All 3-yr-old males are sexually mature; however, 60-64% of the fish mature earlier as 1- or 2-yr-old animals. The endocrine basis underlying early maturity in 2-yr-old males was studied at the molecular level by monitoring changes in pituitary beta FSH and beta LH mRNA levels by ribonuclease protection assay, and correlating these changes to stages of testicular development. In maturing males, the mRNA levels of beta FSH were elevated during early spermatogenesis, whereas beta LH mRNA levels peaked during spermiation. The appearance of spermatozoa in the testis was associated with a decrease in beta FSH mRNA and a rise in beta LH mRNA abundance. Immature males had lower levels of beta LH mRNA than maturing males, but there were no differences in beta FSH mRNA levels between immature and maturing males. The regulation of gonadotropin gene expression in 2-yr-old males was studied by the chronic administration of GnRH analogue (GnRHa) and testosterone (T), with or without pimozide (P) supplementation. In immature males, the combination of T and GnRHa stimulated a three- to fivefold increase in beta FSH and beta LH mRNA levels, but the same treatment had no effect on gonadotropin gene expression in maturing males. In addition, the coadministration of P to immature males suppressed the stimulatory effect of GnRHa and T on beta FSH and beta LH mRNA levels, suggesting that dopamine may have a novel role in regulating gonadotropin gene expression.     

Plasma and pituitary concentrations of gonadotropins (FSH and LH) in minke whales (Balaenoptera acutorostrata) during the feeding season

This study investigated plasma and pituitary concentrations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and steroid hormones (progesterone: P4, testosterone:T, estradiol-17beta: E2) by enzyme-immunoassay (EIA) in minke whales (Balaenoptera acutorostrata) captured during the feeding season (December to March) in the Antarctic Ocean. Plasma FSH and LH levels in female minke whales were higher (P <0.05) than in male whales. Although the pituitary weight was not significantly different between male and female whales, pituitary FSH and LH levels were higher in females than in males (P<0.01) and mature whales than immature whales (P<0.05). Plasma levels of FSH, T and E2 were not significantly different between immature and mature male whales, but plasma LH and pituitary FSH and LH levels were higher (P<0.05) in mature than in immature whales. In both immature and mature whales regardless of gender, pituitary FSH and LH levels were correlated significantly (r=0.69: P<0.01). In mature male whales, plasma T and E2 levels (r=0.60: P<0.01), and testis weight and plasma T levels (r=0.46: P <0.05) were correlated. In immature female whales, plasma FSH and LH levels were highly correlated (r=0.68: P<0.001), but were not for mature female whales. The results show that gender and maturity influence gonadal and pituitary function of minke whales during the feeding season.     

Metformin decreases GnRH- and activin-induced gonadotropin secretion in rat pituitary cells: potential involvement of adenosine 5' monophosphate-activated protein kinase (PRKA)

Metformin is an insulin sensitizer molecule used for the treatment of infertility in women with polycystic ovary syndrome and insulin resistance. It modulates the reproductive axis, affecting the release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH). However, metformin's mechanism of action in pituitary gonadotropin-secreting cells remains unclear. Adenosine 5' monophosphate-activated protein kinase (PRKA) is involved in metformin action in various cell types. Here, we investigated the effects of metformin on gonadotropin secretion in response to activin and GnRH in primary rat pituitary cells (PRP), and studied PRKA in rat pituitary. In PRP, metformin (10 mM) reduced LH and follicle-stimulating hormone (FSH) secretion induced by GnRH (10(-8) M, 3 h), FSH secretion, and mRNA FSHbeta subunit expression induced by activin (10(-8) M, 12 or 24 h). The different subunits of PRKA are expressed in pituitary. In particular, PRKAA1 is detected mainly in gonadotrophs and thyrotrophs, is less abundant in lactotrophs and somatotrophs, and is undetectable in corticotrophs. In PRP, metformin increased phosphorylation of both PRKA and acetyl-CoA carboxylase. Metformin decreased activin-induced SMAD2 phosphorylation and GnRH-induced mitogen-activated protein kinase (MAPK) 3/1 (ERK1/2) phosphorylation. The PRKA inhibitor compound C abolished the effects of metformin on gonadotropin release induced by GnRH and on FSH secretion and Fshb mRNA induced by activin. The adenovirus-mediated production of dominant negative PRKA abolished the effects of metformin on the FSHbeta subunit mRNA and SMAD2 phosphorylation induced by activin and on the MAPK3/1 phosphorylation induced by GnRH. Thus, in rat pituitary cells, metformin decreases gonadotropin secretion and MAPK3/1 phosphorylation induced by GnRH and FSH release, FSHbeta subunit expression, and SMAD2 phosphorylation induced by activin through PRKA activation.     

Divergent changes in the concentrations of gonadotropin beta-subunit messenger ribonucleic acid during the estrous cycle of sheep

Cyclic changes in the production of the pituitary gonadotrophic hormones, LH and FSH are essential events in the maintenance of the reproductive system of female mammals. While studies have examined changes in the secretion of LH and FSH during the estrous cycle and demonstrated the importance of these hormones in regulation of ovarian development and gametogenesis, considerably less is known concerning the regulation of the biosynthesis of these hormones. Although initial studies have examined changes in LH subunit mRNA concentrations during the rat and ovine estrous cycles, no information concerning the physiological regulation of FSH beta mRNA concentrations has been available. In the present study we have examined the relationship between pituitary concentrations of LH and FSH subunit mRNAs and the serum concentrations of these gonadotropins. The results demonstrate a very different pattern of change for FSH beta subunit mRNA than that observed for alpha and LH beta subunit mRNAs. In fact, FSH beta mRNA concentration decline substantially during the preovulatory period, reaching minimal values at a time when alpha and LH beta mRNA levels are near maximal. Furthermore, this decline in FSH beta mRNA amounts occurs when serum FSH concentrations are maximal. Thus, FSH beta mRNA concentrations follow a very different pattern than that of serum FSH. In contrast, LH beta mRNA and serum LH concentrations tend to increase at the same time. These findings provide evidence that concentrations of LH beta and FSH beta mRNAs are likely regulated by different mechanisms.     

Detection of luteinizing hormone beta messenger ribonucleic acid (RNA) in individual gonadotropes after castration: use of a new in situ hybridization method with a photobiotinylated complementary RNA probe

Patterns of gonadotropin storage in individual gonadotropes change with alterations in the physiological state. After castration in the male rat, there is a 2.5-fold increase in the percentage of gonadotropes and an increase in the proportion of gonadotropes storing both LH and FSH. In addition, there are 6- to 8-fold increases in the pituitary concentrations of LH beta subunit mRNAs. In order to determine whether these changes are due to increases in the number of gonadotropes containing subunit mRNA, or the amount of mRNA per cell or both, an in situ hybridization technique using a photobiotinylated rat LH beta cRNA probe (bio-LH beta-cRNA) was applied to detect LH beta mRNA in fixed whole rat pituitary cells from intact or castrated rats. After hybridization, the bio-LH beta-cRNA was localized with either avidin-biotin peroxidase complex or the fluorescent streptavidin phycoprobe methods. The cells containing LH beta mRNA were then counted and the amount of mRNA per cell was measured by video microdensitometry. Ten percent of the anterior pituitary cells from intact animals contained LH beta mRNA. After castration (2-4 weeks) this percentage rose to 19-24.5%. Image and microdensitometric analyses showed that castration produced a 1.9-fold increase in the amount of LH beta mRNA per cell, and a 2.2-fold increase in the area of cells containing LH beta mRNA. Hence, castration resulted in an increase in the level of LH beta mRNA per cell as well as the number of LH beta mRNA-containing cells. When in situ hybridization was followed by immunocytochemistry in cells from intact rats, 83% of gonadotropes that stained for LH beta and 80% of gonadotropes that stained for FSH beta contained LH beta mRNA whereas after castration 99% of LH-storing and 93% of FSH-storing cells contained LH beta mRNA. This new in situ hybridization protocol is rapid and allows quantification of mRNA within individual gonadotropes. In addition, since the hybridization protocol does not apparently alter the gonadotropin antigens, the hormone content of the same gonadotrope may be defined by immunocytochemistry.