Pituitary adenylate cyclase activating polypeptide messenger RNA in the paraventricular nucleus and anterior pituitary during the rat estrous cycle

The neuropeptide pituitary adenylate cyclase activating polypeptide (ADCYAP 1, or PACAP) has been demonstrated to enhance gonadotropin-releasing hormone (GnRH)-induced gonadotropin secretion and regulate gonadotropin subunit gene expression in cultures of anterior pituitary cells. In the present study, we used in situ hybridization and real-time polymerase chain reaction to examine the expression of Pacap mRNA within the paraventricular nucleus (PVN) and anterior pituitary throughout the estrous cycle of the rat. Levels of luteinizing hormone in serum and pituitary gonadotropin subunit mRNAs were evaluated and displayed cyclic fluctuations similar to those reported previously. Pacap mRNA expression in the PVN and pituitary varied significantly during the estrous cycle, with the greatest changes occurring on the day of proestrus. Pacap mRNA levels in the PVN declined significantly on the morning of diestrus. During proestrus, PVN Pacap mRNA levels significantly increased 3 h before the gonadotropin surge and then declined. Pituitary expression of Pacap mRNA also varied on the afternoon of proestrus with a moderate decline at the time of the gonadotropin surge and a significant increase later in the evening. Expression of the mRNA species encoding the 288 amino acid form of follistatin increased significantly following the rise in pituitary Pacap mRNA, at the termination of the secondary surge in follicle-stimulating hormone beta (Fshb) gene expression. These results suggest that PACAP is involved in events before and following the gonadotropin surge, perhaps through increased gonadotroph sensitivity to GnRH and suppression of Fshb subunit expression through increased follistatin, as previously observed in vitro.     

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.     

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.     

Activin modulates differential effects of estradiol on synthesis and secretion of follicle-stimulating hormone in ovine pituitary cells

In several physiological paradigms, secretion of FSH and LH are not coordinately regulated. Because these hormones appear to be produced by a single cell type in the anterior pituitary gland, their discordant regulation must be related to differential intracellular responses to various stimuli. Estradiol-17beta (estradiol) has been shown to influence secretion of both FSH and LH and some of its effects are mediated directly on the gonadotrope. Changes in expression of intrapituitary factors such as activin and follistatin may mediate effects of estradiol and account for discordant patterns of FSH and LH. The aims of this study were 1) to determine if estradiol alters expression of genes encoding activin, follistatin, or both in ovine pituitary cells; and 2) to observe the effects of immunoneutralizing activin B in vitro on gonadotropin secretion. Pituitary cells from five ewes in the anestrous season were cultured for 24 h with estradiol (0.01 or 1.0 nM). Estradiol reduced basal secretion of FSH in a dose-dependent manner (P: < 0.001) and simultaneously increased basal secretion of LH (P: < 0.001). Decreased secretion of FSH in estradiol-treated cultures was accompanied by suppressed levels of FSHbeta subunit mRNA (P: < 0.001). Amounts of mRNA for activin beta(B) were reduced in a dose-dependent manner by estradiol (27% +/- 4.9% at 0.01 nM, P: < 0.02; and 46% +/- 3.9% at 1.0 nM, P: < 0.002). In contrast, mRNA for follistatin was not affected by treatment with estradiol. Treatment of pituitary cells with an antibody to activin B reduced secretion of FSH by 50% (P: < 0.01) without influencing secretion of LH. These data lead us to conclude that discordant secretion of gonadotropins can be induced by estradiol acting directly at the pituitary level. The inhibitory effect of estradiol on FSH secretion may be mediated indirectly through decreased pituitary expression of the activin gene.     

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.     

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.     

Regulation of gonadotropin subunit genes in tilapia

A steroidogenic tilapia gonadotropin (taGtH=LH) was purified from pituitaries of hybrid tilapia (Oreochromis niloticus x O. aureus) and a homologous RIA was established. This RIA enabled the study of the endocrine regulation of GtH release, the transduction pathways involved in its secretion and its profile during the spawning cycle. Discrepancies between steroid and taGtH peaks during the cycle led to the conclusion that an additional gonadotropin similar to salmonid FSH operates early in the cycle. In order to identify this hormone and to study the endocrine control of synthesis of all gonadotropin (GtH) subunits, a molecular approach was taken. The cDNA sequences and the entire gene sequences encoding the FSHbeta and LHbeta subunits, as well as an incomplete sequence of the glycoprotein hormone alpha subunit (GPalpha), were cloned. Salmon gonadotropin-releasing hormone (sGnRH) elevated mRNA steady-state levels of all three GtH subunits in cultured pituitary cells. Pituitary adenylate cyclase-activating polypeptide (PACAP) and neuropeptide Y (NPY) also stimulated the expression of these subunits and potentiated the effect of GnRH, except that NPY did not affect FSHbeta. The GnRH and NPY effects were found to be mediated mainly through protein kinase C (PKC), while protein kinase A (PKA) cascade was involved to a lesser extent. Mitogen-activated protein kinase (MAPK) cascade takes part in mediating GnRH effects, possibly via PKC. Testosterone (T) and estradiol (E2), but not 11-ketotestosterone (KT), are able to elevate GPalpha and LHbeta mRNAs in pituitary cells of early maturing or regressing males. Low levels of T exposure are associated with elevated FSHbeta mRNA in cells of mature fish, while higher levels suppress it, but elevate LHbeta mRNA. In vivo observations also showed the association of low T levels with increased FSHbeta mRNA and high T levels with elevated LHbeta mRNA. In accordance with these findings, analysis of LHbeta and FSHbeta 5' gene-flanking regions revealed on both gene promoters a GtH-specific element (GSE), half site estrogen response elements (ERE), cAMP response element (CRE) and AP1. In vitro experiments showed that recombinant human activin-A leads to higher levels of GPalpha, FSHbeta and LHbeta mRNAs in pituitary cell culture. Porcine inhibin marginally decreased the mRNA levels of GPalpha and FSHbeta, but at a low level (1 ng/ml) it stimulated that of LHbeta. These results shed some light on certain hypothalamic and gonadal hormones regulating the expression of GtH subunit genes in tilapia. In addition, they provide evidence for their differential regulation, and insight into their mode of action.     

Androgens positively regulate follicle-stimulating hormone beta-subunit mRNA levels in rat pituitary cells

We have shown previously that androgens negatively regulate LH alpha and beta-subunit mRNA levels, but have little or no effect on FSH beta mRNA levels in rats in vivo. In contrast, estrogen negatively regulates all three gonadotropin subunit mRNA levels in vivo. We have examined the effects of these sex steroids on gonadotropin subunit synthesis directly at the level of the pituitary gland by using cultured rat pituitary cells. Adult female and male rat pituitaries were dissected, dispersed enzymatically, and maintained in culture for 2 days. At that time, cells were treated for varying lengths of time with either medium alone or sex-steroid hormone treatments (estradiol or testosterone). Dose-response and time-course experiments were performed. Cells were then harvested and total RNA was extracted. Gonadotropin subunit mRNA levels were assessed by blot hybridization techniques. Sex-steroid hormones were added to achieve final concentrations ranging from 10(-12) to 10(-6) M for dose response experiments and 10(-8) M for time-course experiments. Testosterone treatment (10(-8) M) increased FSH beta mRNA levels 3-fold in females (P less than 0.01) and males (P less than 0.05), but had no effect on alpha or LH beta mRNA levels in either sex. Dose-related increases in FSH beta mRNA levels with increasing concentrations of testosterone were observed in both female and male pituitary cell cultures. Time-course studies revealed that the testosterone-stimulated increases in FSH beta mRNA levels are statistically significant by 12 h and 6 h after hormone addition in female and male cultures, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Weaning and the developmental changes in follicle-stimulating hormone, pituitary adenylate cyclase-activating polypeptide, and inhibin B in the male rat

Pituitary Fshb concentrations increase markedly and selectively beginning on Postnatal Day 20 in the male rat. To evaluate the factors potentially responsible for this rise in FSH, we adjusted the time of weaning, which is generally also on Day 20. Male rat pups were provided nutrients by suckling only and were weaned to laboratory chow earlier (Day 17) or later (Day 23) than normally performed in animal facilities (Day 20). Between ages 17 and 29 days, significant increases were seen in serum LH (1.4-fold) and FSH (2.4-fold) levels; pituitary expression of Lhb (5.4-fold), Fshb (21.3-fold), and inhibin beta B (Inhbb, 2.26-fold) mRNAs; and testicular expression of Inhbb (10-fold) mRNA. Concurrently, significant decreases occurred in serum inhibin B levels (1.8-fold); pituitary adenylate cyclase-activating polypeptide (Adcyap1, 4.2-fold), total follistatin (Fst, 3.5-fold), and Fst isoform 288 (5.6-fold) mRNAs; and testicular expression of inhibin beta A (8.2-fold) mRNA. Early weaning significantly increased serum FSH but not LH and increased pituitary expression of Fshb and GnRH receptor (Gnrhr) mRNAs but not Lhb. Early weaning also significantly decreased serum inhibin B but increased testicular expression of the Inhbb subunit. Early weaning also caused pituitary expression of Fst and Adcyap1 to decline earlier than in the control group. Immediately after weaning, growth accelerated substantially, and the time of weaning produced significant and differential effects on circulating leptin levels that were not related to indices of FSH production. From these observations, we propose the novel hypothesis that the increase in growth rate subsequent to weaning signals circulating inhibin B levels to fall and pituitary Adcyap1 and consequently Fst expression to decrease, and that these events together facilitate the rise in Fshb and Gnrhr expression by increasing pituitary activin signaling.     

Involvement of activin/BMP system in development of human pituitary gonadotropinomas and nonfunctioning adenomas

Roles of activin/bone morphogenetic protein (BMP) system in the pathogenesis of human pituitary adenoma remain unknown although these factors stimulate follicle-stimulating hormone (FSH) secretion in the normal pituitary. Here we demonstrated that type-I and -II subunit mRNAs of activin/BMP receptors are expressed in Pit-1-negative FSH-producing (FSH-oma) and nonfunctioning pituitary adenomas (NF-oma). Basal levels of serum FSH standardized by luteinizing hormone (LH) were markedly high in FSH-omas in contrast to NF-omas. However, gonadotropin-releasing hormone (GnRH)-induced increment of FSH standardized by that of LH was not changed in FSH-omas, suggesting that imbalanced FSH secretion by FSH-oma is not attributable to GnRH regardless of the expression of GnRH receptor. Although activin betaA subunit was detected in neither adenoma, the betaB subunit was expressed highly in FSH-omas and, to lesser extent, in NF-omas. As for BMPs, BMP-6 and -7 were detected in NF-omas while BMP-4 and -15 were not detected in either type of adenoma. In the presence of pituitary activin/BMP system, the levels of co-expressing follistatin mRNA in the tumors were reduced in FSH-oma compared with NF-oma, suggesting that endogenous follistatin is involved in FSH overproduction through inhibition of activin/BMP system independently of GnRH.     

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)     

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 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.     

Gonadotropin-releasing hormone stimulates annexin 5 messenger ribonucleic acid expression in the anterior pituitary cells

We previously reported that annexin 5 is found specifically in gonadotropes and that the expression is dramatically enhanced after ovariectomy. In the present study, the expression of annexin 5 was examined in the primary culture of rat anterior pituitary cells using semiquantitative RT-PCR to determine if it is under the direct control of gonadotropin-releasing hormone (GnRH). Continuous administration of GnRH analog for 1 h enhanced the expression of both FSH beta subunit and annexin 5 mRNA. The expression of annexin 5 mRNA was also augmented by phorbol 12-myristate 13-acetate but not by forskolin. Administration of recombinant rat annexin 5 to the culture increased LH beta mRNA expression. These data clearly demonstrate that the expression of annexin 5 mRNA is directly controlled by GnRH and suggest that annexin 5 is involved in mediating GnRH action in the pituitary gland.     

Co-localization of LH beta and FSH beta mRNAS in the porcine anterior pituitary by in situ hybridization with biotinylated probes

The localizations of mRNAs encoding LH beta and FSH beta in porcine pituitary were investigated by in situ hybridization technique. Biotinylated porcine LH beta and FSH beta cDNA probes were used on frozen sections of paraformaldehyde-fixed pituitary specimens. Hybridizations to both mRNAs were observed specifically in cytoplasm with unstained nuclei. Furthermore, cells hybridized for LH beta mRNA were demonstrated to be identical to those for FSH beta mRNA. This study provided the first morphological evidence that both gonadotropin beta genes are expressed in the same cell.