The binding of GnRH to its receptor on pituitary gonadotropes leads to the targeting of a diverse array of signalling mediators. These mediators drive multiple signal transduction pathways, which in turn regulate a variety of cellular processes, including the biosynthesis and secretion of the gonadotropins LH and FSH. Advances in our understanding of the mechanisms and signalling pathways that are recruited to regulate gonadotrope function are continually being made. This review will focus on the recent demonstration that key mediators of the canonical Wnt signalling pathway are targeted by GnRH in gonadotropes, and that these may play essential roles in regulating the expression of many of the key players in gonadotrope biology, including the GnRH receptor and the gonadotropins.
Samantha Gardner, Emmanouil Stavrou, Patricia E Rischitor, Elena Faccenda and Adam J Pawson
R K Iles and T Chard
Although discovered in the early 1930s, much of the biochemistry and molecular biology of human chorionic gonadotrophin (hCG) has only recently been revealed. The sequence was described in 1973 (Bellisario et al. 1973; Carlsen et al. 1973), but the precise secondary and tertiary structures are still unknown. Only in the last 5 years has there been dramatic progress in the understanding of the molecular nature of this hormone. These studies shed light on many clinical aspects of the biology of hCG, including its association with non-trophoblastic epithelial cancers.
CG and the glycoprotein hormones
Chorionic gonadotrophin is a member of a group of four structurally homologous proteins commonly referred to as the glycoprotein hormones. As the name implies, the hormone protein chains are glycosylated; hCG contains approximately 30% carbohydrate by weight. The other members are luteinizing hormone (LH), follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH). CG differs from the other glycoprotein
E. L. Bittman, C. M. Hegarty, M. Q. Layden and J. A. Jonassen
Exposure to short daylengths arrests the oestrous cycle, provokes daily gonadotrophin surges and reduces the ability of exogenous oestradiol to trigger behavioural receptivity in golden hamsters. In order to examine neuroendocrine effects of photoperiod which might underlie these responses, ovariectomized hamsters were maintained under long or short photoperiods for 54 days before treatment with cholesterol or various doses of oestradiol-17β. Short days reduced the ability of low doses of oestrogen to prime hamsters for the induction of oestrus by progesterone. Upon repetition of oestrogen priming 2 weeks later, photoperiod was without significant influence on the concentrations of nuclear oestrogen receptors or cytosolic progestin receptors in a block of tissue containing the hypothalamus and preoptic area.
Oestradiol treatment provoked significant increases in serum concentrations of LH and prolactin in the afternoon, but photoperiod did not alter the positive-feedback efficacy of this gonadal steroid hormone. Adenohypophysial LH-β subunit and prolactin mRNAs were suppressed by short days in ovariectomized hamsters not treated with oestradiol. Oestradiol decreased expression of the LH-β subunit gene in both stimulatory and inhibitory photoperiods, but increased prolactin mRNA abundance in both long and short days. Photoperiod therefore exerts pronounced steroid-independent effects on phasic LH and prolactin secretion, but regulation of adenohypophysial abundance of LH-β subunit and prolactin mRNAs by oestradiol is not markedly influenced by daylength. Photoperiodic regulation of the priming effects of oestradiol on behavioural receptivity may result from modulation of events occurring subsequent to steroid—receptor interactions, or involve changes in receptor populations not detectable by the present methods.
PJ Chedrese, MR Rodway, CL Swan and C Gillio-Meina
We report the establishment and preliminary characterization of a stable steroidogenic granulosa cell line, JC-410. This cell line was obtained by spontaneous immortalization of a primary culture of porcine granulosa cells. Cultured JC-410 cells produced less progesterone than granulosa cells in primary culture. Progesterone synthesis by JC-410 cells was approximately 10% and 1% of the amount produced by granulosa cells from small and medium sized follicles, respectively. Although FSH and LH did not change progesterone levels in cultured JC-410 cells, forskolin and cholera toxin induced a 2.6- and 2.75-fold increase, respectively, versus control. The JC-410 cells responded to 0.1, 1 and 5 mM cAMP with an increase in progesterone synthesis of 2.5-, 28- and 49-fold versus control, respectively, after a 24 h incubation. No detectable levels of estradiol-17beta were found in JC-410 cells after 48 h in culture. However, addition of 0.01, 0.1 and 1 microM androstenedione elevated the levels of estradiol-17beta to 0.028, 0.3 and 1.21 pg/microg protein, respectively. The level of expression of 3betaHSD, aromatase and P450scc genes in JC-410 cells is of similar magnitude to the level of expression in granulosa cells in primary culture. The JC410 cells have been maintained in culture for more than one year during which their population doubled over 100 times. We conclude that JC-410 is a stable cell line that lost responsiveness to the gonadotropins during the process of immortalization, but retained its steroid biosynthetic capability and the expression of key steroidogenic genes. These characteristics may reflect features of cells arrested in an early stage of granulosa cell differentiation.
J. S. Fleming, D. J. Tisdall, P. J. Greenwood, N. L. Hudson, D. A. Heath and K. P. McNatty
Ovine cDNA probes for the α and βA inhibin subunits and for follistatin were used to investigate the mRNA species for these hormones in ovaries obtained during the luteal phase of the oestrous cycle, from Booroola ewes which were homozygous carriers (BB) or non-carriers (++) of the FecB gene. BB ewes had significantly higher concentrations of peripheral FSH and LH immunoreactivity than ++ ewes, but the peripheral inhibin immunoreactivity and ovarian inhibin and progesterone secretion rates were not significantly different between genotypes. No gene-specific differences in the number or size of mRNA transcripts detected by Northern blotting were noted for any of these genes. A single α inhibin mRNA species at 1.5 kb was observed in the follicle RNA from ++ and BB ovaries. Low amounts of α inhibin hybridization were discerned occasionally in + + and BB stroma and also in BB, but not in ++, corpora lutea. The βA inhibin gene was expressed only in the follicles from both ++ and BB ovaries. At least three βA inhibin transcripts were observed; one at 7.5kb and at least two between 1.4 and 5.0kb. The follistatin cDNA probe detected two major transcripts at 2.7 and 1.5 kb and a minor band at 0.5 kb in both follicle and corpora lutea RNA. Densitometry of the Northern blots revealed no significant gene-specific differences in the levels of α inhibin and follistatin gene mRNA transcripts. However, significantly greater amounts of total βA inhibin hybridization were detected in follicle RNA from BB compared with ++ ovaries (P<0.001) and this FecB-specific difference appeared to be associated with the 7.5 kb transcript. We conclude that the Booroola FecB gene does not influence the synthesis of the α inhibin subunit or follistatin during the luteal phase of the oestrous cycle, but may affect inhibin or activin synthesis in the ovaries of FecB carriers, by increasing the transcription or stability of the βA inhibin mRNA species.
T. Hirai, H. Takikawa and Y. Kato
To elucidate the structure and control of expression of the porcine FSH-β subunit gene, two genomic clones were isolated and the entire gene structure was determined to the extent of 10 kb, consisting of 6 kb of the 5′-flanking region and 4 kb of the transcriptional unit. The porcine FSH-β gene consisted of three exons the same as the human and bovine genes, but the positions of both splicing sites of porcine intron-1 were unique. It is known that the synthesis of FSH is regulated by gonadal steroids, gonadotrophin-releasing hormone (GnRH) and inhibin. However, the consensus steroid-responsive element was unexpectedly absent in the 5′-flanking region of 6 kb. On the other hand, the potential binding sites for activator protein-1 (AP1) and AP2, which might be stimulated by the GnRH—protein kinase C cascade, were present at seven and five positions respectively. An imperfect cyclic AMP-responsive element was also present. Southern blot analyses, using the cDNA and genomic fragments as probes, gave smear patterns suggesting the presence of repetitive sequences in the porcine FSH-β gene. A survey of homology with the repetitive sequences revealed that short interspersed repeated sequences (SINES)-type non-viral retroposons were present with about 250 bp length repeats twice in the 5′-flanking region and once each in intron-1 and the 3′-flanking region. Other SINES-like sequences were also found in intron-1, exon-2 and exon-3. In comparison with the 5′-flanking sequences of the porcine α and LH-β genes, there were no significantly conserved regions, implying a lack of common modulation of the three subunit genes.
J T Lakkakorpi, E M Pietilä, J T Aatsinki and H J Rajaniemi
To elucidate the molecular mechanisms involved in the homologous regulation of LH/chorionic gonadotrophin (CG) receptors, the receptor and its mRNA levels were analysed in the same pseudopregnant rat ovarian samples after human (h)CG-induced down-regulation using a binding assay, ligand blotting, immunoblotting and Northern blotting together with the polymerase chain reaction (PCR). Treatment of the animals with 500 IU hCG resulted in a loss of 125I-labelled hCG binding and the 90 kDa receptor on the ligand and immunoblots within 12 and 24 h respectively, followed by a transient partial recovery on days 4 and 5, while a distinct decline occurred only on day 7 in the controls. Northern blots of total ovarian RNA, as probed with a 293 bp AvaI/HindIII fragment from the extracellular domain of PCR-generated full-length rat LH/CG receptor cDNA, revealed six major mRNAs of 7·0, 4·2, 2·8, 2·0, 1·4 and 1·1 kb. The 4·2 kb mRNA, which was the most abundant, possibly encodes the 90 kDa receptor, while the smaller species probably represent alternatively spliced forms of the LH/CG receptor pre-mRNA, as also supported by the finding that PCR produced three cDNA bands of 2·1, 2·0 and 1·8 kb when oligomers derived from the N and C termini of rat LH/CG receptor cDNA were used as primers and rat ovarian total RNA as a template. Treatment with hCG led to the down-regulation of all six mRNAs in a fashion parallel to the changes in receptor protein. No smaller receptor components capable of binding radiolabelled hCG or receptor antibody appeared on the ligand or immunoblots prior to or during down-regulation or the subsequent transient period of up-regulation, suggesting that the smaller mRNA species are translated in minute amounts in vivo or are not translated at all. Laser densitometric analysis of the Northern blots revealed that the amounts of the four smallest mRNA species increased during the period of down-regulation in relation to the 4·2 kb mRNA, and correspondingly decreased during the subsequent period of up-regulation, indicating changes in the alternative splicing of the primary transcript. The data suggest that hCG-induced transient down-regulation of the LH/CG receptor results in part from down-regulation of its mRNA levels, and that changes in alternative processing of the receptor pre-mRNA may play a regulatory role in the expression of functional LH/CG receptor during down- and up-regulation.
Charit Taneja, Sakshi Gera, Se-Min Kim, Jameel Iqbal, Tony Yuen and Mone Zaidi
FSH has a primary function in procreation, wherein it induces estrogen production in females and regulates spermatogenesis in males. However, in line with our discoveries over the past decade of non-unitary functions of pituitary hormones, we and others have described hitherto uncharacterized functions of FSH. Through high-affinity receptors, some of which are variants of the ovarian FSH receptor (FSHR), FSH regulates bone mass, adipose tissue function, energy metabolism, and cholesterol production in both sexes. These newly described actions of FSH may indeed be relevant to the pathogenesis of bone loss, dysregulated energy homeostasis, and disordered lipid metabolism that accompany the menopause in females and aging in both genders. We are therefore excited about the possibility of modulating circulating FSH levels toward a therapeutic benefit for a host of age-associated diseases, including osteoporosis, obesity and dyslipidemia, among other future possibilities.
Sébastien Legardinier, Martine Duonor-Cérutti, Gérard Devauchelle, Yves Combarnous and Claire Cahoreau
Equine luteinizing hormone (eLH) and chorionic gonadotropin (eCG) are composed of identical α and β polypeptide chains, but eCG subunits are much more heavily glycosylated and sialylated. Consequently, eCG exhibits a much longer half-life than eLH in blood. Recombinant eLH/CG, expressed in Sf9 and Mimic insect cells, were compared with one another and to the natural hormones eCG and eLH. Mimic cells are stably-transformed Sf9 cells, expressing five mammalian genes encoding glycosyltransferases involved in the synthesis of complex N-carbohydrate chains. Recombinant eLH/CG expressed in Mimic cells exhibited a higher apparent molecular weight (MW) than that expressed in Sf9 cells, suggesting that its N-glycosylation was, as expected, more complete. Nevertheless, the two recombinant eLH/CG exhibited lower MW than natural eCG from pregnant mare plasma. The two eLH/CG produced in Sf9 and Mimic cells were found to be active in in vitro LH and FSH bioassays, with potencies similar to those of eCG. By contrast, they exhibited no significant in vivo bioactivity, neither in the specific follicle-stimulating hormone (FSH) assay nor in the specific eCG assay. Although recombinant eLH/CG produced in Mimic cells bears more elaborate carbohydrate chains than recombinant eLH/CG from Sf9 cells, it exhibits no significant in vivo bioactivity, probably because of insufficient terminal sialylation of its carbohydrate chains, leading to its rapid removal from blood.
L Abdennebi, L Couture, D Grebert, E Pajot, R Salesse and JJ Remy
Follicle-stimulating hormone (FSH) via interaction with G-protein coupled specific receptors plays a central role in the control of gametogenesis in mammals of both sexes. In females, FSH is crucial for follicle growth, follicle maturation and ovulation. FSH receptors, together with luteinizing hormone-chorionic gonadotropin and thyrotropin receptors belong to a subfamily of structurally related receptors within the seven transmembrane receptor family. Among several other regions, the N-terminus of these receptors is believed to be responsible for important specific hormone-receptor contact sites. Recombinant filamentous phages displaying at their surface three overlapping N-terminal decapeptides of the FSH receptor, peptides A18-27, B25-34 and C29-38 were constructed. Ewes and female mice were immunized against the three FSH receptor (FSHR) recombinant phages. Immunoglobulins purified from immunized animals were analyzed for their biochemical properties on a Chinese hamster ovary cell line expressing the porcine FSH receptor. AntiA and antiB immunoglobulins (IgGs) behave as antagonists for 125I-FSH binding and for FSH-dependent cAMP production, while antiC IgGs did not compete for hormone binding. By contrast, antibodies against the C29-38 peptide displayed FSH agonist activity and stimulated the FSH receptor, whereas antiA and antiB IgGs did not. Furthermore, when the FSHR phages were used as peptidic vaccines, they induced a reversible inhibition of ovulation rate in ewes, and impaired fertility in female mice.