Steroidogenic acute regulatory protein and luteinizing hormone are required for normal ovarian steroidogenesis and oocyte maturation in zebrafish†.

In recent studies, luteinizing hormone (LH) was reported to play important roles in oocyte maturation. However, the mechanism by which LH signaling, especially regarding the steroidogenesis process, affects oocyte maturation has not been clarified. In this study, zebrafish models with a functional deficiency in luteinizing hormone beta (Lhb) or steroidogenic acute regulatory protein (Star), an enzyme that promotes the transport of cholesterol into the inner mitochondrial membrane for maturation-induced hormone (MIH) production, were generated using transcription activator-like effector nucleases (TALENs). Similar phenotypes of the maturation-arrested oocytes in both female mutants have been observed. The levels of MIH in the oocytes of the female mutants were clearly decreased in both the lhb and star knockout zebrafish. The expression of star was dramatically down-regulated in the lhb mutant follicles and was clearly promoted by forskolin and hCG in vitro. Furthermore, treatment with the MIH precursors, pregnenolone or progesterone, as well as with MIH itself rescued the maturation-arrested oocyte phenotypes in both lhb and star mutants. The plasma levels of other steroids, including testosterone, estradiol, and cortisol, were not affected in the lhb mutants, while the levels of gonad hormones testosterone and estradiol were significantly increased in the star mutants. The cortisol levels were decreased in the star mutants. Collectively, our results confirm that LH plays important roles in the initiation of MIH synthesis from cholesterol and maintains oocyte maturation in zebrafish, as well as provide evidence that Star might act downstream of LH signaling in steroidogenesis.

Role of urocortin 2 secreted by the pituitary in the stress-induced suppression of luteinizing hormone secretion in rats.

We have previously shown that urocortin 2 (Ucn 2), a member of the corticotropin-releasing factor (CRF) peptide family that binds to CRF type 2 receptor, is expressed in proopiomelanocortin (POMC) cells of rat pituitary and that its secretion and expression are increased by CRF in both the anterior and intermediate lobes and suppressed by glucocorticoids in the anterior lobe. We have also shown that Ucn 2 secreted by POMC cells acts on gonadotrophs expressing CRF type 2 receptors and inhibits the expression and secretion of gonadotropins. In the present study, we examined whether pituitary Ucn 2 is involved in stress-induced inhibition of gonadotropin secretion. A 90-min period of immobilization stress increased POMC mRNA expression without influencing Ucn 2 mRNA expression and suppressed luteinizing hormone (LH) ß-subunit mRNA expression in the anterior lobe and plasma LH levels, while it increased both POMC and Ucn 2 mRNA expression in the intermediate lobe of the pituitary. Pretreatment with anti-CRF IgG blocked immobilization-induced increases in plasma ACTH and corticosterone and in POMC mRNA expression in both pituitary lobes and Ucn 2 mRNA expression in the intermediate pituitary. It also blocked immobilization-induced suppression of plasma LH and LH ß-subunit mRNA expression. Pretreatment with anti-Ucn 2 IgG blocked immobilization-induced suppression of plasma LH and LH ß-subunit expression without affecting immobilization-induced ACTH and corticosterone release and POMC or Ucn 2 mRNA expression. These results suggest that CRF suppresses the secretion and expression of LH probably through pituitary Ucn 2 in stress.

Differential effects of GnRH and androgens on Cres mRNA and protein in male mouse anterior pituitary gonadotropes.

The Cres gene defines a new subgroup in the family 2 cystatins of cysteine protease inhibitors. However, unlike typical cystatins, CRES does not inhibit cysteine proteases but rather inhibits the serine protease prohormone convertase 2, an enzyme with roles in proprotein processing in the neuroendocrine system. Cres is expressed in the gonadotropes and colocalizes with LHbeta, suggesting a role in the regulation of gonadotrope secretion. Our present studies were carried out to examine the regulation of Cres mRNA and protein expression by GnRH and steroid hormones, thus providing clues regarding its role in gonadotropes. Castration profoundly reduced Cres mRNA, while replacement with estradiol (E(2)), testosterone (T), or dihydrotestosterone (DHT) further decreased Cres, suggesting negative regulation by GnRH or steroid hormones. The administration of Antide, a GnRH antagonist, resulted in a 3-fold increase in Cres mRNA, supporting a negative regulation by GnRH. Because all hormonal manipulations in vivo resulted in alterations in steroid hormones, organ culture was used to assess the effects of GnRH independent of steroids. Mouse pituitaries cultured in the absence of GnRH or steroids showed high Cres mRNA levels, while DHT or E(2) resulted in decreases of 25% and 68%, respectively. However, an 85% decrease in Cres mRNA occurred following the administration of GnRH, demonstrating that GnRH, and to a lesser degree E(2), negatively regulate Cres mRNA in gonadotropes. Examination of CRES protein by immunohistochemistry showed that levels were profoundly reduced following castration, while DHT and in part T, but not E(2), restored CRES levels. Castrated mice treated with Antide showed little effect. However, castrated mice treated with Antide + DHT showed a dramatic recovery of CRES, suggesting that androgens act directly at the level of the gonadotrope to regulate CRES protein. Together, our studies suggest that Cres mRNA and protein are low at peak gonadotrope secretory activity, possibly as a means to allow proprotein processing events to occur that are integral to gonadotrope function.

Mechanisms for pulsatile regulation of the gonadotropin subunit genes by GNRH1.

The frequency of gonadotropin-releasing hormone (GNRH1, or GnRH) pulses secreted from the hypothalamus determine the ratios of the gonadotropin subunit genes luteinizing hormone beta (Lhb), follicle-stimulating hormone beta (Fshb) and the common alpha-glycoprotein subunit gene (Cga) transcribed in the anterior pituitaries of mammals. Fshb is preferentially transcribed at slower GNRH1 pulse frequencies, whereas Lhb and Cga are preferentially transcribed at more rapid pulse frequencies. Producing the gonadotropins in the correct proportions is critical for normal fertility. Currently, there is no definitive explanation for how GNRH1 pulses differentially activate gonadotropin subunit gene transcription. Several pathways may contribute to this regulation. For example, GNRH1-regulated GNRH1-receptor concentrations may lead to variable signaling pathway activation. Several signaling pathways are activated by GnRH, including mitogen-activated protein kinase, protein kinase C, calcium influx, and calcium-calmodulin kinase, and these may be preferentially regulated under certain conditions. In addition, some signaling proteins feed back to downregulate their own levels. Other arms of gonadotroph signaling appear to be regulated by synthesis, modification, and degradation of either transcription factors or regulatory proteins. Finally, the dynamic binding of proteins to the chromatin, and how that might be regulated by chromatin-modifying proteins, is addressed. Oscillations in expression, modification, and chromatin binding of the proteins involved in gonadotropin gene expression are likely a link between GNRH1 pulsatility and differential gonadotropin transcription.

Differentiation of mouse embryonic stem cells into gonadotrope-like cells in vitro.

OBJECTIVE: This research was conducted to investigate the potential of mouse embryonic stem (ES) cells to differentiate in vitro into gonadotropes. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

The LHbeta gene of several mammals embeds a carboxyl-terminal peptide-like sequence revealing a critical role for mucin oligosaccharides in the evolution of lutropin to chorionic gonadotropin in the animal phyla.

The expression of a previously untranslated carboxylterminal sequence is associated with the ancestral lutropin (LH) beta to the beta-subunit gene evolution of choriogonadotropins (CG). The peptide extension (denoted as CTP) is rich in mucin-type O-glycans and confers new hormonal properties on CG relative to the LH. Although the LHbeta gene is conserved among mammals and only a few frameshift mutations account for the extension, it is merely seen in primates and equids. Bioinformatics identified a CTP-like sequence that is encrypted in the LHbeta gene of several mammalian species but not in birds, amphibians, or fish. We then examined whether or not decoding of the cryptic CTP in the bovine LHbeta gene (boCTP) would be sufficient to generate the LHbeta species of a ruminant with properties typical to the CGbeta subunit. The mutated bovine LHbeta-boCTP subunit was expressed and N-glycosylated in transfected Chinese hamster ovary cells. However, unlike human (h) CGbeta CTP, the cryptic boCTP was devoid of mucin O-glycans. This deficiency was further confirmed when the boCTP domain was substituted for the natural CTP in the human CGbeta subunit. Moreover, when expressed in polarized Madin-Darby canine kidney cells, this hCGbeta-boCTP chimera was secreted basolaterally rather than from the apical compartment, which is the route of the wild type hCGbeta subunit, a sorting function attributed to the O-glycans attached to the CTP. This result shows that the cryptic peptide does not orientate CG to the apical face of the placenta, to the maternal circulation as seen in primates. The absence of this function, which distinguishes CG from LH, provides an explanation as to why the LHbeta to CGbeta evolution did not occur in ruminants. We propose that in primates and equids, further natural mutations in the progenitor LHbeta gene resulted in the efficient O-glycosylation of the CTP, thus favoring the retention of an elongated reading frame.

Targeted disruption of luteinizing hormone beta-subunit leads to hypogonadism, defects in gonadal steroidogenesis, and infertility.

Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) act on gonadal cells to promote steroidogenesis and gametogenesis. Clarifying the in vivo roles of LH and FSH permits a feasible approach to contraception involving selective blockade of gonadotropin action. One way to address these physiologically important problems is to generate mice with an isolated LH deficiency and compare them with existing FSH loss-of-function mice. To model human reproductive disorders involving loss of LH function and to define LH-responsive genes, we produced knockout mice lacking the hormone-specific LHbeta-subunit. LHbeta-null mice are viable but demonstrate postnatal defects in gonadal growth and function resulting in infertility. Mutant males have decreased testes size, prominent Leydig cell hypoplasia, defects in expression of genes encoding steroid biosynthesis pathway enzymes, and reduced testosterone levels. Furthermore, spermatogenesis is blocked at the round spermatid stage, causing a total absence of the elongated spermatids. Mutant female mice are hypogonadal and demonstrate decreased levels of serum estradiol and progesterone. Ovarian histology demonstrates normal thecal layer, defects in folliculogenesis including many degenerating antral follicles, and absence of corpora lutea. The defects in both sexes are not secondary to aberrant FSH regulation, because FSH levels were unaffected in null mice. Finally, both male and female null mice can be pharmacologically rescued by exogenous human chorionic gonadotropin, indicating that LH-responsiveness of the target cells is not irreversibly lost. Thus, LHbeta null mice represent a model to study the consequences of an isolated deficiency of LH ligand in reproduction, while retaining normal LH-responsiveness in target cells.

Lack of association of the common immunologically anomalous LH with endometriosis.

BACKGROUND: Subfertile women with endometriosis have been reported to demonstrate impaired follicular growth, ovulatory dysfunction and disturbed LH patterns. In addition, abnormal LH and/or LH receptors have been linked with endometriosis-associated infertility. Carriers of a variant of the beta-subunit of luteinizing hormone (V-LH) are largely healthy; however, differences in their gonadal function such as alterations in gonadal steroidogenesis, ovarian reserve, pubertal development and predisposition to diseases such as infertility and polycystic ovarian disease have been found. METHODS AND RESULTS: To explore the possible relationship between endometriosis and V-LH, we examined its frequency in 230 women undergoing laparoscopic surgery for the investigation of infertility. For the entire study population, 185 (80.4%) were wild type; 42 (18.3%) were heterozygous; and three (1.3%) were homozygous for V-LH. No difference was found between women with (n = 85) and without (n = 145) endometriosis concerning the frequency of the type of LH. CONCLUSION: Our results do not support the hypothesis that the variant form of LH is associated with an altered risk of endometriosis in the population tested.