LHbeta mRNA Research Articles

The differential secretion of FSH and LH: regulation through genes, feedback and packaging

While the role of oestradiol and progesterone in the control of GnRH pulsatile secretion and generation of the preovulatory GnRH surge to induce release of the LH surge has been fully investigated, less attention has been given to changes in the pituitary gland that may sensitize gonadotrophs to switch from pulsatile release to surge release of LH, in particular. Furthermore, in the follicular phase while pulsatile secretion of LH is maximal, FSH secretion is reduced, yet both hormones are produced by the same gonadotrophs. The mechanisms whereby this differential release can occur are still unclear. The main regulator of FSH secretion is through the negative feedback effects of oestradiol and inhibin, which directly affect FSHbeta mRNA content and subsequent synthesis of FSH. FSH is then released predominantly via a constitutive pathway and the amount released is closely related to the rate of synthesis. In contrast, while basal LH secretion occurs via a constitutive pathway, the principal release of LH through pulsatile secretion is through the regulated pathway with GnRH stimulating the release of pre-synthesized LH contained in storage granules without significant changes in LHbeta mRNA. Secretogranin II (SgII) is associated with LH in these electron-dense storage granules and LH-SgII granules appear to be the principal form of granule released in response to GnRH through the regulated pathway. At the time of the preovulatory LH surge, granule movement to the gonadotrope cell membrane abutting a capillary, polarization, appears to play an important part in the priming mechanism for release of LH during the preovulatory LH surge in response to the GnRH surge. As there appears to be limited or no gonadotroph cell division in the adult pituitary gland, each gonadotroph passes through this synthesis and secretion pathway repeatedly through successive oestrous cycles. Packaging of LH and FSH into different secretory granules within the same cell is thus pivotal for the differential secretion of these gonadotrophins.

Role of GnRH in the ontogeny and regulation of the fetal hypothalamo-pituitary-gonadal axis in sheep

Adult reproductive ability is to a large extent determined by the appropriate development of the reproductive axis during fetal life. Studies have investigated the role of the fetal hypothalamus in the ontogeny and regulation of pituitary gonadal function during fetal development in sheep. Using immunocytochemistry, we examined the ontogeny of gonadotroph development in the pituitary of female sheep fetuses. At day 70 of gestation (term = 145 days), only immunopositive LH beta cells were present. The number and intensity of staining of these LH beta cells had increased by day 100 but had declined again by day 130. Immunopositive alpha-subunit and FSH beta cells appeared at day 100 of gestation and had further increased in number and staining intensity by day 130 of gestation. Treatment of fetuses with the GnRH agonist buserelin resulted in desensitization of the fetal pituitary gonadotrophs, inhibition of pituitary LH beta and FSH beta mRNA expression and a reduction in the number of immunopositive gonadotrophin-containing cells. Pulsatile GnRH treatment resulted in pituitary-gonadal activation and an increase in LH, FSH and testosterone secretion in males. Thus, the synthesis and secretion of the gonadotrophins during fetal development is critically dependent on the secretion of GnRH from the fetal hypothalamus. Inhibition of fetal gonadotrophins by buserelin treatment from day 70 of gestation resulted in a 40% reduction in the size of the fetal testis at birth, and there were no effects on the fetal ovaries. This reduction in testis size was due to a 45% reduction in the number of Sertoli cells. However, when buserelin was given between day 70 and day 110 of gestation, there were no effects on testis size or morphological development of the testis, suggesting that gonadotrophins regulate testicular development during a 'critical window' late in gestation. Taken together, these studies provide convincing evidence that GnRH plays a central role in the ontogeny and regulation of pituitary-gonadal function during fetal life.

Effect and molecular regulatory mechanism of monochromatic light colors on the egg-laying performance of Yangzhou geese

Photoperiodic control is essential for manipulating the reproductive performance of avian species. This study was conducted to assess the neuroendocrine mechanisms that regulate reproductive functions of Yangzhou geese when there are different monochromatic light colors from light emitter diode (LED) sources. A flock of geese was divided into four groups with white, red, blue, and green light treatments being imposed. The results indicated that peak laying rates and reproductive performance were greater in geese treated with white or red as compared with blue or green light treatments. The fertilization rate of eggs and hatchability of fertilized eggs were greater with the white or red as compared with blue or green light treatments. There was a greater abundance of OPN5, Dio2, c-Fos, and GnRH-I mRNA in the hypothalamus earlier in the treatment period and abundances of these hypothalamic factors were greater with the white or red light treatments. Abundances of pituitary LH beta and FSH beta mRNA increased at a lesser rate with the blue or green light treatments and were in greater abundances with the white or red light treatments. The lighting regimen also resulted in photo-refractoriness with there being greater abundances of GnIH, VIP, and PRL mRNA with the use of white or red light treatments. The results indicate that the use of white or red monochromatic lights while imposing a long photoperiod of 11 h daily could result in sustaining functions of the reproductive system of Yangzhou geese for considerably longer times, thus, resulting in greater egg-laying performance.

Gonadotropins and Their Receptors in the Hermaphroditic Ricefield Eel Monopterus albus: cDNA Cloning and mRNA Expression.

The ricefield eel is a protogynous hermaphroditic teleost fish that changes sex from functional female, through an intersexual stage, to the functional male phase during its life cycle, and a good model organism to study sex determination and differentiation of vertebrates. Previous studies suggested that HPG axis may play important roles in the initiation of sex change in ricefield eel; however, the gonadotropins and their cognate receptors remains to be elucidated in this fish. By using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), cDNAs encoding Cgalpha, Lhbeta, Fshbeta, Fsh receptor (Fshr), and Lh receptor (Lhr) of ricefield eel were cloned in the present study. Homology analysis showed that ricefield eel Cgalpha, Lhbeta, Fshbeta, Fshr, and Lhr shared highest identities with their counterparts in perciform fish,which conforms to the lineage relationship. A lhr pseudogene was identified from the genome of ricefield eel, and what is interesting is that phylogenetic analysis categorized teleost lhrs into two clusters, and ricefield eel lhr gene and pseudogene are grouped into one of the two clusters, respectively, which suggests that either one of the duplicated lhr genes became a pseudogene during evolution of different teleost lineages. During ovarian development, realtime PCR analysis showed an increasing trend of fshbeta mRNA levels in the pituitary from previtellogenesis to vitellogenesis, but then a decreasing trend toward maturation. As for lhbeta mRNA levels in the pituitary, a continuous increasing trend was observed from previtellogenesis to maturation. The ovarian fshr mRNA levels were significantly increased from previtellogenesis to early vitellogenesis, but then decreased significantly thereafter. However, the ovarian lhr mRNA levels were increased significantly from vitellogenesis to the mature stage. The above results may suggest that Fsh signal pathway is more important for vitellogenesis, whereas Lh signal pathway more important for oocyte maturation in ricefield eel. During sex change from female through an intersexual stage to male, fshr and lhr showed lower expression levels in transitional gonads than either ovary or testis. In the pituitary, fshbeta and lhbeta mRNA levels were significantly lower during the intersexual and male stages than the female stage. However, lhbeta /fshbeta mRNA ratios were increased significantly during sex change, suggesting that the changes in the ratio of LHbeta to FSHbeta may be important for the gonadal differentiation of ricefield eel. The present study provided a foundation for further elucidation of the roles of HPG axis in the gonadal development and sex change of ricefield eel. This research was supported by Natural Science Foundation of China (30970359, 30771651) and National High Technology Research and Development Program of China (863 Program) (No.2008AA09Z406). (poster)

The expression of pituitary FSHβ and LHβ mRNA and gonadal FSH and LH receptor mRNA in the chicken embryo

In avian species, synthesis of sex steroids by embryonic gonads is regulated by luteinizing hormone (LH) and follicle-stimulating hormone (FSH). In order to elucidate the role of the two gonadotropins in gonadal axis development during the second half of chicken embryogenesis, pituitary expression of LH beta subunit (LHbeta) and FSH beta subunit (FSHbeta) mRNAs as well as gonadal expression of LH and FSH receptor (LHR and FSHR) mRNAs were determined on days 11 (E11) and 17 (E17) of embryonic development and after hatching (D1). In the pituitary of the female embryo, the gene expression of FSHbeta was the lowest on E11 and increased on E17. In the male pituitary, the expression of FSHbeta did not differ among the studied days. The FSHbeta mRNA expression on E11 was higher in the male than in the female pituitary gland. The expression of LHbeta mRNA in the female pituitary increased on D1 in comparison to E11. In the male pituitary gland, the expression of LHbeta gene was relatively constant. The expression of mRNA encoding FSHR in the ovary increased on E17, while in testes it did not differ among the studied days. There were no significant alterations in LHR gene expression in the ovary or in the testes in the examined period however, the gene expression on E17 was higher in the ovary than in the testes. We observed positive correlations between the pituitary FSHbeta mRNA expression and ovarian expression of FSHR mRNA (r = 0.63; p<0.01) as well as between LHbeta mRNA and LHR mRNA in the testes (r=0.65; p<0.01). The reported alterations in gene expression of FSHbeta, LHbeta and their receptors between sexes and among the stages of embryonic development indicate time- and sex-dependent action of gonadotropins in gonads of chicken embryos.

Intra-Ovarian Production of GnRH-1, FSHbeta and LHbeta mRNA in Siberian Hamsters (Phodopus sungorus) During the Estrous Cycle and Photoperiod-Induced Regression/Recrudescence.

Intra-Ovarian Production of GnRH-1, FSHbeta and LHbeta mRNA in Siberian Hamsters (Phodopus sungorus) During the Estrous Cycle and Photoperiod-Induced Regression/Recrudescence.

Quantification of gonadotropin subunits GPα, FSHβ, and LHβ mRNA expression from Atlantic cod (Gadus morhua) throughout a reproductive cycle

To elucidate the role of the gonadotropins in Atlantic cod (Gadus morhua), complete coding sequences with partially or fully un-translated regions for the three subunits GPalpha, FSHbeta, and LHbeta were determined. The sequences of the corresponding genomic loci were also determined, allowing the design of mRNA-targeting quantitative PCR assays. Relative expression was analyzed during a complete seasonal sexual maturation cycle in Atlantic cod females. Increasing levels of lhbeta mRNA were observed during gonadal growth, peaking at spawning in February-March which corresponds to maximum gonadosomatic index. In contrast, both gpalpha and fshbeta gradually increased to a peak in December, two months before spawning started, and decreased in January just prior to spawning. Both mRNAs increased again and remained high during the spawning season, with a decline at the end of the spawning period, a further decrease in spent females, followed by a new gradual increase concurrent with the start of the next reproductive cycle. In addition to its role in vitellogenesis prior to spawning, FSH seems to have additional functions during the spawning period, possibly related to vitellogenesis that runs in parallel with final oocyte maturation and ovulation of the multiple batch spawner Atlantic cod.

Hormonal Regulation of GnRH and LHβ mRNA Expression in Cultured Rat Granulosa Cells

We have recently demonstrated that the rat ovary expresses LHbeta, FSHbeta, and the common alpha subunit mRNA. In the present report, we studied the regulation of LHbeta and of gonadotropin-releasing hormone (GnRH) mRNA expression in granulosa cells that were isolated from immature rats treated with either estrogen or pregnant mare serum gonadotropin (PMSG). In both cell types, GnRH agonist treatment resulted in a decrease in LHbeta mRNA expression. However, only in cells derived from PMSG-treated rats, GnRH treatment produced an increase in GnRH mRNA expression. A markedly increased GnRH mRNA expression was also obtained in granulosa cells derived from PMSG-primed rats in response to LH. In addition, FSH reduced the expression of LHbeta mRNA in granulosa cells from estrogen-primed rats. These results suggest that the expression of LHbeta in the ovary is regulated by locally produced GnRH and by FSH from either the ovary or the pituitary.

Molecular cloning, characterization and expression of FSH and LH beta subunits from grass carp (Ctenopharyngodon idella)

Follicle-stimulating hormone beta subunit (FSHbeta) and luteinizing hormone beta subunit (LHbeta) have been cloned and characterized from the pituitary of grass carp (Ctenopharyngodon idella). The full length of FSHbeta and LHbeta cDNA was 393 bp and 441 bp, with open reading frame encoding proteins of 130 and 146 amino acids, respectively. Phylogenetic analysis of the proteins of FSHbeta and LHbeta showed a high homology with other fishes. Homology analysis also indicated that LHbeta has higher conservation than FSHbeta. The expression analysis of grass carp FSHbeta and LHbeta by RT-PCR suggested that they were only expressed in the pituitary. Real-time quantitative PCR protocols were developed and validated to measure FSHbeta and LHbeta mRNAs during ovarian development. The FSHbeta and LHbeta mRNA level was very low in the pituitaries of early-pubertal fish and significantly increased during the ovulation period. These results suggested that in grass carp the gonadotropins synthesized synchronously in order for asynchronous oogenesis to take place.

Gonadotropin Releasing Hormone (GnRH) Enhances Annexin A5 mRNA Expression through Mitogen Activated Protein Kinase (MAPK) in L.BETA.T2 Pituitary Gonadotrope Cells

The mechanism by which GnRH stimulates annexin A5 expression was examined with LbetaT2 gonadotrope cells. Continuous stimulation with GnRH analog (GnRHa, Des-Gly10 [Pro9]-GnRH ethylamide) transiently elevated LHbeta mRNA expression while maintaining annexin A5 mRNA at high levels for 24 h. GnRH antagonist blocked the effect of GnRHa on annexin A5. While 12-O-tetradecanoyl-phorbol-13 acetate, a protein kinase C activator, increased the expression of annexin A5 mRNA, bisindolylmaleimide, an inhibitor of protein kinase C, suppressed GnRHa-stimulated expression of annexin A5 and LHbeta mRNA. GnRHa stimulation of LHbeta mRNA was inhibited to a greater extent than annexin A5 by a calcium chelator BAPTA/AM. Although a calcium ionophore ionomycin stimulated the expression of both genes, only LHbeta was down-regulated. The MAPK kinase inhibitor PD98059 inhibited GnRHa induction of annexin A5 but not LHbeta mRNA. EGF stimulated the expression of annexin A5 mRNA but caused only a transient effect on LHbeta mRNA expression. These results indicate that GnRH stimulation of signaling pathway for annexin A5 mRNA expression is distinct from that of LHbeta mRNA and dependent more on MAPK.

Expression of gonadotropin and gonadotropin receptor genes during early sexual maturation in male Atlantic salmon parr

Atlantic salmon males may mature already as small parr in freshwater. Sexual maturation in teleosts as in vertebrates is characterized by the activation of the brain-pituitary-gonad axis. The endocrine regulation of early puberty is still not well understood. In the present study, one-summer-old male Atlantic salmon parr were sampled regularly from December several months prior to the beginning of spermatogenesis until spawning in October. Pituitary expression levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) beta subunit genes were analyzed in parallel with testis expression of FSH receptor (FSHR) and LH receptor (LHR) genes by RT-PCR and plasma 11-ketostestosterone (11-KT) was measured. Expression levels of FSHbeta, low during winter and spring started to increase prior to the onset of gonadal growth at the end of May while LHbeta mRNA levels were hardly detectable. Both gonadotropin receptor genes were expressed in immature testis with FSHR transcripts being more abundant (8-fold). FSHR transcript levels increased in parallel to FSHbeta levels from early spermatogenesis onwards, while LHR mRNA started to increase prior to any large changes in LHbeta expression. Both transcript levels of LHbeta and LHR were highest during spermiation. Plasma 11-KT increased at the beginning of spermatogenesis reaching highest levels at spermiogenesis suggesting a possible role of FSH in inducing 11-KT production during early spermatogenesis while LH stimulates via its specific receptor 11-KT production at spermiogenesis. The commitment into sexual maturation appears to be dependant on both the presence of FSHR in immature testis and the increase of FSH expression.

Paracrine Control of Gonadotrophs

There is increasing evidence for communication among pituitary cells. Hormone-producing pituitary cells may communicate with each other and with folliculostellate cells. The latter cells surround pituitary hormone-producing cells and are connected by tight junctions to form a network that allows for their coordinated function. Folliculostellate cells are targets of cytokines, peptides, and steroid hormones, and produce growth factors and cytokines, including follistatin, the dynamic regulator of follicle-stimulating hormone (FSH) production that binds activin, and limits activin signaling. Pituitary adenylate cyclase-activating peptide (PACAP) and its receptor are found in folliculostellate cells in which they stimulate transcription of the follistatin gene through cyclic adenosine monophosphate/protein kinase A (PKA) signaling. When PACAP increases, follistatin levels increase, and FSH-beta mRNA is reduced. PACAP also activates gonadotrophs to stimulate transcription of the gonadotropin alpha-subunit gene and lengthen the LH-beta mRNA, presumably to prolong it half-life, and increases responsiveness to GnRH. Accordingly, PACAP differentially regulates FSH and LH, and may prove to be a key player in reproduction through a novel paracrine mechanism.

ESTROGEN RESPONSE ELEMENT DEPENDENT AND INDEPENDENT ESTROGEN RECEPTOR ALPHA SIGNALING PATHWAYS DIFFERENTIALLY REGULATE PITUITARY GONADOTROPIN TRANSCRIPTION AND SECRETION IN THE FEMALE MOUSE

Pituitary gonadotropins luteinizing hormone (LH) and follicle stimulating hormone (FSH) are both secreted by pituitary gonadotropes in response to gonadotropin-releasing hormone (GnRH). However, these gonadotropins are independently regulated throughout the estrous cycle to coordinate the timing of follicle development and ovulation. GnRH release is suppressed by estrogen negative feedback until, on the afternoon of proestrous, when estrogen positive feedback actions result in a GnRH and LH surge and the induction of ovulation. Subsequently, a FSH surge occurs to stimulate the development of a new cohort of follicles until when inhibin, produced by follicles, feeds back to the pituitary to suppress further FSH production and release. In ovariectomized females, estrogen treatment is capable of suppressing LH and FSH. While known to require the estrogen receptor alpha (ERalpha), the molecular mechanisms of this suppression remain unclear. Classical ERalpha signaling occurs through binding to an estrogen response element (ERE) in DNA, whereas in nonclassical signaling, ERalpha participates in protein-protein interactions to alter the activity of transcription factors at non-ERE sites (i.e. NF-kB, AP-1, or Sp1). Previously, our lab generated a Nonclassical Estrogen Receptor Knock In (NERKI) mouse model using a mutant ERalpha that is unable to bind to ERE but retains EREindependent activity. Breeding the NERKI mouse with the ERalpha knock out model (ERKO), we are able to selectively restore only EREindependent signaling to the ERalpha null background (NERKI/ERKO) and genetically dissociate in vivo roles for the ERE-dependent and independent ERalpha signaling pathways. Using this model, we have recently shown the ERE-independent ERalpha signaling pathway as capable of mediating partial estrogen negative feedback of serum LH. In this study, the ERE-dependent and independent estrogen effects on serum FSH and pituitary gonadotropin LHbeta and FSHbeta subunit transcription are examined following ovariectomy. We find that estrogen mediated suppression of pituitary LHbeta mRNA and serum FSH require the EREdependent ERalpha pathway. In contrast, the ERE-independent ERalpha signaling pathway is capable of mediating partial estrogen suppression of FSHbeta mRNA transcription and serum LH. These results support a model where, in the absence of other ovarian factors, estrogen acting through the ERE-dependent ERalpha pathway suppresses both LH and FSH transcription and secretion, whereas estrogen acting through the ERE-independent pathway partially suppresses LH secretion and FSHbeta transcription but is permissive to LHbeta transcription and FSH secretion. These data suggest that the balance of ERE-dependent and independent ERalpha signaling pathways may enable estrogen to dynamically regulate the transcription and secretion of both pituitary gonadotropins LH and FSH across the estrous cycle. Supported by NIH/NICHD grant PO1 HD21921, CMBD Training Grant T32 GM008061, UVA Ligand Core- NICHD (SCCPRR) Grant U54-HD28934 (platform)

Secretoneurin stimulates goldfish pituitary luteinizing hormone production

Secretoneurin (SN), a 33-34 amino acid neuropeptide is derived from endoproteolysis of secretogranin II (SgII), a member protein of the chromogranin family. SN is widely distributed in various tissues of vertebrates especially in pituitary and hypothalamus, and is a potential new hormone. In vivo, i.p. injection of SN increased luteinizing hormone (LH) release in goldfish pretreated with the dopamine antagonist domperidone. In 6-h static incubation of goldfish pituitary fragments, 10 and 100 nM but not 1 nM concentrations of goldfish SN had a direct stimulatory effect to increase LH release by 2.3- and 1.5-fold (p<0.05), respectively. In addition, 500 nM SN induced a 2.6-fold increase in LHbeta subunit messenger RNA (mRNA) levels in pituitary fragments, regardless of whether LHbeta mRNA levels were expressed relative to 18S ribosomal RNA or beta-actin mRNA. We suggest that the stimulatory actions of SN on LH release may be a part of a paracrine or autocrine feedback loop in the pituitary.

Role of PKC in the regulation of gonadotropin subunit mRNA levels: interaction with two native forms of gonadotropin-releasing hormone

Gonadotropin-releasing hormone (GnRH) is an important regulator of reproduction in all vertebrates through its actions on the production and secretion of pituitary gonadotropin hormones (GtHs). Most vertebrate species express at least two GnRHs, including one form, designated chicken (c)GnRH-II or type II GnRH, which has been well conserved throughout evolution. The goldfish brain and pituitary contain salmon GnRH and cGnRH-II. In goldfish, GnRH-induced luteinizing hormone (LH) secretion involves PKC; however, whether PKC mediates GnRH stimulation of GtH subunit mRNA levels is unknown. In this study, we used inhibitors and activators of PKC to examine its possible involvement in GnRH-induced increases in GtH-alpha, follicle-stimulating hormone (FSH)-beta and LH-beta mRNA levels in primary cultures of dispersed goldfish pituitary cells. Treatment with PKC inhibitors calphostin C and GF109203X unmasked a basal repression of GtH subunit mRNA levels by PKC; both inhibitors increased GtH subunit mRNA levels in a dose-dependent manner. PKC activators, 12-O-tetradecanoylphorbol 13-acetate (TPA), and 1,2-dioctanoyl-sn-glycerol, stimulated GtH subunit mRNA levels, whereas an inactive phorbol ester (4-alpha-TPA) was without effect. Thus, a dual, inhibitory and stimulatory, influence for PKC in the regulation of GtH subunit mRNA levels is suggested. In contrast, PKC inhibitor- and activator-induced effects were, for the most part, additive to those of GnRH, suggesting that conventional and novel PKCs are unlikely to be involved in GnRH-stimulated increases in GtH subunit mRNA levels. Our data illustrate major differences in the signal transduction of GnRH effects on GtH secretion and gene expression in the goldfish pituitary.

Differentiation of Mouse Embryonic Stem Cells Into Gonadotrope-like Cells In Vitro

This research was conducted to investigate the potential of mouse embryonic stem (ES) cells to differentiate in vitro into gonadotropes. Undifferentiated ES cells were maintained on mitomycin C-inactivated fibroblasts in the presence of leukemia inhibitory factor (LIF). By a 5-day hanging drop culture devoid of them, ES cells were induced to form multidifferentiated structures called embryoid bodies (EBs). Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry were used to analyze gene expression of gonadotrope markers in EBs at different time points during the culture. Homeo box gene expressed in ES cells (Hesx1), LIM homeobox protein 3 (Lhx3), paired like homeodomain factor 1 (Prop1), GATA binding protein 2 (GATA2), follicle-stimulating hormone beta (FSHbeta), and luteinizing hormone beta (LHbeta) mRNAs were detected at day 6 EBs and maintained throughout the culture to day 56. FSHbeta and LHbeta proteins were expressed in EBs from day 6 onward. Immunofluorescent labeling of FSHbeta and LHbeta showed that specific staining was restricted to the cytoplasm of some differentiated EB cells. With the prolongation of EB culture, the number of positive cells increased significantly. Both monohormonal and bihormonal cells were present, mainly in clusters within EBs and sparsely distributed among the outermost cells surrounding the EBs. These results indicate that mouse ES cells can give rise to mature gonadotrope-like cells in EBs. It also shows that EBs may serve as a novel model system to study the development and function of gonadotropes.

Signal Transduction Mediating Gene Expression of SP1, LHβ-Subunit and GH in Response to GnRH or GHRH in the Postnatal and Fetal Porcine Anterior Pituitary in Vitro

To clarify signal transduction pathways mediating putative gene expression of transcription factor SP1 (selective promoter factor 1 or specificity protein 1) by GnRH or GHRH porcine anterior pituitary monolayer cultures were exposed for various time periods to GnRH, GHRH, activators of adenylate cyclase (AC) or proteinkinase C (PKC), and mRNA levels of SP1, LHbeta-subunit, and GH were determined by multiplex RT-PCR. In many experiments LH and GH release were measured as well for comparison. Another approach was to inactivate AC, PKC, or proteinkinase A (PKA) by specific inhibitors, MDL, GFX, and H89, respectively. Postnatally (4 weeks) SP1 mRNA level was maximally increased by GnRH, GHRH and both by activation of AC or PKC after 2 h of exposure. Two-hour stimulation of SP1 mRNA levels by dbcAMP was totally blocked by H89, while this inhibitor not clearly blocked GHRH stimulated SP1 mRNA levels. Stimulation of LHbeta mRNA by GnRH was suppressed by inactivation of AC or of PKC but not by inactivation of PKA. Inactivation of AC or PKA but not of PKC inhibited GHRH induced GH mRNA. Already at day 50 of fetal life (and likewise day 80) SP1 mRNA levels were stimulated by GHRH or activation of AC, but not by GnRH or activation of PKC. The results are consistent with the notion that SP1 plays an important role 1) in conferring GnRH responsiveness to the LHbeta-subunit gene by mediating the actions of both AC and PKC and 2) in conferring GHRH responsiveness to the GH gene through activation of the AC probably PKA pathway. Furthermore, the data are in line with the view that the GHRH/AC/SP1/GH pathway develops earlier during fetal life than the GnRH/PKC/SP1/LHbeta pathway.

GnRH Antagonist-induced Down-regulation of the mRNA Expression of Pituitary Receptors: Comparisons with GnRH Agonist Effects

In order to compare the mechanism for the down regulation of the mRNA expression of pituitary receptors induced by GnRH antagonist (GnRHant) to that by GnRH agonist (GnRHa), we examined the effects of GnRHant (Cetrorelix, 333 mug/kg/day), GnRHa (leuprolide depot, 333 microg/kg), and GnRHant combined with GnRHa on LH response to exogenous GnRH, pituitary LH content, LH beta subunit mRNA, and GnRH receptor (GnRH-R) mRNA levels at 2, 5, 24, 72 hours, and 7 days after the treatment in ovariectomized rats. GnRHant significantly decreased serum LH, the LH response of the pituitary to exogenous GnRH, and the pituitary LH content compared to the control treatment, though GnRHa significantly increased serum LH. GnRHant with GnRHa significantly diminished the GnRHa-induced flare-up phenomenon. GnRHant significantly decreased LH beta mRNA and GnRH-R mRNA levels, but the magnitude of the decrease in these mRNA levels by GnRHant was significantly less than those by GnRHa until 72 hours following treatment. Prolonged treatment of GnRHant caused a marked inhibition of LH beta mRNA and GnRH-R mRNA expression, similar to that caused by GnRHa. Combination treatment with GnRHa and GnRHant was demonstrated to decrease LH beta mRNA and GnRH-R mRNA levels as much as GnRHa alone and GnRHant alone over 7 days of the treatment. The present study showed differences between GnRHant and GnRHa treatment in the reduction of GnRH-R mRNA levels up to 72 hours after the treatment, and indicated that the suppression of GnRH-R mRNA by GnRHant was the maximal by GnRHa 7 days after the treatment because more profound suppression was not observed upon additional treatment with GnRHa. The findings in the present study support the hypothesis that the mechanism by which GnRHant leads to down-regulation of the mRNA expression of pituitary receptors is similar to that of GnRHa.

Differential regulation of gonadotropins and glycoprotein hormone α-subunit by IGF-I in anterior pituitary cells from male rats

IGF-I has been demonstrated to stimulate basal and GnRH-induced gonadotropin release. IGF-I also elicites alpha-subunit secretion in human pituitary tumor cells. The aims of this study were to evaluate both the effect of IGF-I on gonadotropin LH-beta and FSH-beta mRNA levels and glycoprotein alpha-subunit gene expression in cultured rat anterior pituitary cells. The exposure of pituitary cells to recombinant human IGF-I (rhlGF-I; 2 microg/ml) for 72 h markedly stimulated basal LH and FSH release whereas their mRNA levels remained unmodified. IGF-I elicited a-subunit release from pituitary cells (p < 0.01) and augmented its mRNA levels. Exposure to IGF-I consistently reduced GH release from pituitary cells. This study shows that the gonadotropin-releasing effects of IGF-I are not paralleled by changes in their mRNAs whereas IGF-I stimulates not only alpha-subunit release but also its mRNA levels. This study provides the first observation of alpha-subunit regulation by IGF-I in normal pituitary cells, where a differential regulation between release and synthesis for gonadotropin a-and 1-subunits is also shown.

No evidence for pituitary priming to gonadotropin-releasing hormone in relation to luteinizing hormone (LH) secretion prior to the preovulatory LH surge in ewes.

The purpose of this study was to determine the occurrence of and the regulatory mechanisms involved in priming of the pituitary to GnRH before the preovulatory LH surge in sheep. Experiment 1: Forty-two ewes had progestagen devices removed after 14 days and were assigned to luteal (Lut) or follicular (Foll) groups. Fifteen days later, blood sampling was initiated either immediately or 36 h after induced luteolysis in groups Lut and Foll, respectively. After 4 h, ewes were administered either saline (n = 5) or 250 ng (n = 8) or 10 microg (n = 8) of GnRH. Five ewes per treatment group were killed 1 h later, while remaining animals were blood sampled for a further 7 h. Experiment 2: Eighteen ewes were allocated to Lut and Foll groups (described above). Blood samples were collected from 2 h before GnRH (10 microg) treatment until 7 h after. Despite up-regulated GnRH-R mRNA levels in Foll ewes, pituitary content and plasma levels of LH and LHbeta mRNA levels were similar between groups. Mean FSHbeta mRNA and plasma FSH levels were elevated in Lut ewes but declined after GnRH treatment. Inversely, plasma estradiol and inhibin-A concentrations were higher in Foll ewes and declined after GnRH treatment. Fewer LH(+ve)/secretogranin II(-ve) (SgII(-ve)) granules were present in gonadotropes of Foll ewes, coincident with increased basal LH levels. Fewer smaller sized granules were present after GnRH treatment. In conclusion, there was no evidence of self-priming before onset of the preovulatory LH surge. Constitutive release of LH(+ve)/SgII(-ve) granules may maintain basal LH levels while smaller sized, presumably mature granules may be preferentially released after GnRH stimulation.