Effects of luteinizing hormone and human chorionic gonadotropin on corpus luteum cells in a spheroid cell culture system

The human corpus luteum (CL) is a highly vascularized, temporarily active endocrine gland and consists mainly of granulosa cells (GCs), theca cells (TCs), and endothelial cells (ECs). Its cyclic growth and development takes place under the influence of gonadotropic hormones. If pregnancy does occur, human chorionic gonadotropin (hCG) takes over the function of luteinizing hormone (LH) and, in contrast to LH, extends the functional life span of the CL. In this study, we investigated the effects of hCG and LH in a spheroidal cell culture model of CL development. Our data indicate that GCs secrete factors under the control of hCG that increase sprout formation of EC-spheroids. We demonstrate that the most prominent of these factors is VEGF-A. Furthermore, we found that both LH and hCG decrease sprout formation of GC-spheroids. After forming EC-GC coculture spheroids and consequently bringing GCs and ECs in close contact, sprouting increased under the influence of hCG, however not under LH. These experiments provide evidence for an hCG dependent functional switch in the GCs after coming in contact with ECs. Moreover, it demonstrates the considerably different effects of hCG and LH on GCs although their signaling is transmitted via the same receptor.     

Transiently elevated levels of c-fos and c-myc oncogene messenger ribonucleic acids in cultured murine Leydig tumor cells after addition of human chorionic gonadotropin

The gonadotropic hormones LH and human CG (hCG) normally function to stimulate steroidogenesis in testicular and ovarian cells through receptor-mediated activation of adenylate cyclase. These hormones are also important in regulating the development and growth of responsive cells. Such regulation requires tightly controlled gene expression. Herein we demonstrate that hCG induces increases in mRNAs encoding the competence oncogenes c-fos and c-myc in a murine Leydig cell tumor line (MA-10). When stimulated by hCG (40 ng/ml), the mRNA levels of both genes increase rapidly, peaking at 30 min for c-fos and 1 h for c-myc. Both mRNAs fall to near control levels by 3-6 h. This response to hCG is dose-dependent with half-maximal stimulation of these genes occurring at a concentration of 3 ng/ml, approximating the level required for 50% occupancy of the LH/hCG receptors and the ED50 for steroidogenesis. (Bu)2 cAMP (2 mM) elicits responses similar to those produced by hCG. The observation of oncogene control by the gonadotropin hCG provides further insight regarding the pathways by which such hormones may regulate steroidogenesis, growth, and differentiation of endocrine and neoplastic cells.     

The role of the adenylyl cyclase system in the regulation of corpus luteum function in the human and in nonhuman primates

We have reviewed the properties of luteinizing hormone/human chorionic gonadotropic (LH/hCG)-sensitive adenylyl cyclase (AC) of human corpus luteum (CL) and its regulation by several hormones and nonhormonal activators. We have also described the changes in enzyme activity in membrane preparations of human and cynomolgus monkey CL obtained at various stages of the menstrual cycle and pregnancy. The data have been analyzed with respect to the functional status of the luteal tissue and to the species differences among primate CL. In the menstrual cycle, luteal AC responsiveness to LH/hCG was detectable during the midluteal phase, but not during the late luteal phase or in the follicular phase of the following cycle. In addition, nonhormonal stimulation was high in CL obtained during the midluteal and late luteal phases, but declined drastically by the follicular phase of the next cycle. In early pregnancy, the enzyme was unresponsive to LH/hCG stimulation, yet its sensitivity to nonhormonal stimulation was similar, if not identical, to that of midluteal phase CL. Functional activity was also evident at the end of pregnancy. These results demonstrate that expression of AC activity in primate luteal membrane changes significantly with varying hormonal status under physiologic conditions. It is concluded that the AC system in luteal membranes is an effective model to study the mechanisms that regulate function and life span of the human and nonhuman primate CL.     

Human chorionic gonadotropin: Unknown about known

The last two decade discoveries shift the accent from the consideration of human chorionic gonadotropin (hCG) as a hormone that controls progesterone production by corpus luteum cells to a powerful paracrine regulator that, in the tandem with its hyperglycosylated analog (h-hCG), induces successful implantation and coordinated dialog between blastocyst and uterine tissues. The ability of hCG and h-hCG to interact with TSH receptor and TGF-beta-RII, respectively, significantly extend the spectrum of processes controlled by these molecules. Differences between intracellular pathways of signal transduction between hCG and LH mediated by the same receptor (LH/hCG-R) impugn the unity of their effector mechanisms, which was previously considered as obvious. The paracrine properties of hCG include the control of fusion of trophoblasts into syncytiotrophoblasts, angiogenesis, immunity regulation, and endometrium predisposition to implantation. Angiogenesis is associated with LH/hCG-R expressed on mural cells of uterine spiral arteries as well as induced secretion of soluble VEGF form by endometrial cells. hCG regulates the ratio between different types of T-helper cells in maternal organism at the initial gestation stage determining a high level of Th2 cells. hCG supports local immunotolerance, functioning as a chemoattractant for T-suppressors (T-Treg) and an apoptotic factor for T-lymphocytes. Endometrial susceptibility arises from the activation of osteopantin secretion and the decline of mucin secretion by epithelial cells. h-hCG affects the same tissues as hCG, functioning as a paracrine agent regulating multiple cascades of cytokines. h-hCG plays the key role in the trophoblast invasion into the uterine decidua as a result of gelatinase secretion by these cells. The degree of the angiogenic effect of h-hCG is compatible with that of hCG, but its signal transduction is mediated by the TGF-beta signal transduction pathway that stimulates mural cell proliferation. h-hCG acts as a mitogen on NK-cells and is able to activate them and direct to angiogenesis maintenance. In this article, we attempted to elucidate the most important discoveries about the role of hCG and its hyperglycosylated analog, both accomplished and still upcoming.     

Complete dissociation of gonadotropin receptor binding and signal transduction in mouse Leydig tumour cells. Obligatory role of glycosylation in hormone action.

Utilizing a clonal cell line of mouse testicular Leydig cells (MA-10 cells) the complete steroidogenic and other hormonal properties of chemically deglycosylated ovine lutropin (DG-LH) and human choriogonadotropin (DG-hCG) were evaluated. In these cells, with the LH receptor-steroidogenic mechanism tightly coupled and in which there are few, if any, spare receptors, both DG-LH and DG-hCG failed to elicit progesterone production, unlike fully glycosylated native LH and hCG. The receptor-binding activity of DG-LH and DG-hCG was 2-3 times that of LH and hCG in competition experiments with radiolabelled hormones. The typical phenomenon of rounding of MA-10 cells induced by LH and hCG was absent when cells were incubated with DG-LH or DG-hCG. This could be directly attributable to their failure to produce cyclic AMP as second messenger. DG-LH and DG-hCG inhibited cell shape changes and steroidogenesis caused by LH and hCG. The deglycosylated hormones were potent antagonists of the action of glycosylated hormones. Delaying DG-hCG (antagonist) addition for up to 1 h after initiation of hCG action was also very effective in preventing further activation of steroidogenesis. Similar effects were produced by addition of affinity-purified anti-hCG antibodies. In affinity cross-linking experiments, both hCG and DG-hCG bound to the same 90 kDa receptor. Studies with MA-10 cells thus provide unequivocal evidence that the presence of antennary sugars in LH and hCG (and perhaps in other similar hormones such as follicle-stimulating hormone and thyroid-stimulating hormone), is essential for signal transduction. Differences observed in the literature in other cellular systems may be attributed to differences in hormone-receptor-effector coupling.     

Luteal enzymes of the luteinizing hormone and beta-adrenergic signal transduction pathways in hypophysectomized rabbits do not require pituitary hormone support.

Experiments were conducted to determine whether continuous pituitary hormone support is required for expression of the LH and beta-adrenergic cAMP signal transduction pathways in rabbit CL during pseudopregnancy. Parameters of the LH and catecholamine cAMP signal transduction pathways in CL of estrogen-treated hypophysectomized rabbits were compared to those of pituitary-intact rabbits. Results showed that each of the parameters of the LH and beta-adrenergic cAMP signal transduction pathways was retained in CL taken from estrogen-treated pseudopregnant rabbits that had been hypophysectomized for as long as 13 days at levels not significantly different from those of estrogen-treated pituitary-intact rabbits. These included luteal basal, and LH-, epinephrine-, and fluoride-stimulated adenylyl cyclase activities; total luteal cAMP levels; the number and affinity of cAMP-dependent protein kinase regulatory subunit cAMP binding sites; binding activity of the type I and type II regulatory subunits; and the amount of catalytic subunit protein of cAMP-dependent protein kinase. We conclude that expression of the proteins of the cAMP signal pathway for LH and beta-adrenergic hormones in CL of estrogen-treated rabbits does not require pituitary hormone support.     

Regulation of LH/hCG receptor by gonadotropins in rat ovary

To investigate the regulation of the LH/hCG receptor gene by gonadotropins, we examined the effect of PMSG and hCG on the expression of LH/hCG receptor in immature rat ovary. Northern blot analysis of ovarian RNA revealed a major mRNA of 5400 nucleotides and minor species of 7500, 3600, 2300, and 1200 nucleotides, and PMSG treatment slightly increased the intensity of all LH/hCG receptor messengers. Subsequently, hCG treatment decreased the number of LH/hCG receptor by day 2 and mRNA levels by 12h after injection. The level of mRNA recovered and increased 5-fold of control by day 6, then returned to control levels by day 10, followed by slower decline in LH/hCG receptor in plasma membrane. These studies demonstrate that the effects of PMSG and hCG on the number of LH/hCG receptor are closely related to the actions of these hormones on LH/hCG receptor messenger levels.     

Induction of alpha-caveolin-1 (alphaCAV1) expression in bovine granulosa cells in response to an ovulatory dose of human chorionic gonadotropin

Caveolins are implicated in endocytosis, cholesterol trafficking and signal transduction. A cDNA fragment corresponding to caveolin-1 (CAV1) was identified in a mRNA profiling expression study in bovine granulosa cells (GC) following human chorionic gonadotropin (hCG)-induced ovulation. Thus, we have characterized CAV1 cDNA and studied its spatio-temporal expression pattern in bovine ovarian follicles. The full-length bovine alphaCAV1 cDNA was cloned and encodes a putative 22 kDa protein. Expression of alphaCAV1 was studied in bovine GC obtained from follicles at different developmental stages: small follicles (SF: 2-4 mm), dominant follicles (DF), ovulatory follicles (OF: 24 hr post-hCG), and corpus luteum (CL). Semiquantitative RT-PCR analysis showed a 6.5-fold increase in alphaCAV1 mRNA in GC of OF versus DF (P < 0.0001), whereas CAV2 mRNA was increased by only twofold (P < 0.0007). Temporal expression of alphaCAV1 mRNA from OF recovered at 0, 6, 12, 18, and 24 hr after hCG injection showed an 8.5-fold increase of alphaCAV1 mRNA after 24 hr compared to 0 hr (P < 0.0018) whereas no significant variation was detected for CAV2. Immunoblot demonstrated an initial increase in alphaCAV1 protein level 12 hr post-hCG, reaching a maximum at 24 hr. Immunohistochemical localization of CAV1 was observed in GC of OF isolated 18 and 24 hr after hCG injection, whereas no signal was detected in GC of DF and SF. The induction of alphaCAV1 in GC of OF suggests that alphaCAV1 likely contributes to control the increase in membrane signaling that occurs at the time of ovulation and luteinization.     

Membrane-initiated actions of thyroid hormones on the male reproductive system

The presence of specific nuclear receptors to thyroid hormones, described in prepubertal Sertoli cells, implies the existence of an early and critical influence of these hormones on testis development. Although the mechanism of action thyroid hormones has been classically established as a genomic action regulating testis development, our research group has demonstrated that these hormones exert several effects in Sertoli cells lacking nuclear receptor activation. These findings led to the identification of non-classical thyroid hormone binding elements in the plasma membrane of testicular cells. Through binding to these sites, thyroid hormones could exert nongenomic effects, including those on ion fluxes at the plasma membrane, on signal transduction via kinase pathways, on amino acid accumulation, on modulation of extracellular nucleotide levels and on vimentin cytoskeleton. The evidence of the participation of different K(+), Ca(2+) and Cl(-) channels in the mechanism of action of thyroid hormones, characterizes the plasma membrane as an important microenvironment able to coordinate strategic signal transduction pathways in rat testis. The physiological responses of the Sertoli cells to hormones are dependent on continuous cross-talking of different signal transduction pathways. Apparently, the choice of the signaling pathways to be activated after the interaction of the hormone with cell surface binding sites is directly related to the physiological action to be accomplished. Yet, the enormous complexity of the nongenomic actions of thyroid hormones implies that different specific binding sites located on the plasma membrane or in the cytosol are believed to initiate specific cell responses.     

Interspecies differences in the effects of HCG on testicular function among rodents

Adult mice, rats and hamsters were injected with 0 or 0.3 IU hCG/g BW, 24 h before sacrifice. Basal LH receptor concentration was highest in rats and lowest in hamsters (rats greater than mice greater than hamsters). Injection of hCG caused LH receptor down-regulation in rats and mice, and up-regulation in hamsters. Basal plasma progesterone was highest in hamsters and lowest in rats (hamsters greater than mice greater than rats), however, hCG increased plasma progesterone levels in mice and rats, but not in hamsters. Mice had much higher plasma and testicular testosterone levels than other species, but hCG did not induce a relatively more dramatic increase in any species. When testes fragments were incubated with 0 or 12.5 mIU hCG/ml for 4 h, hCG increased media progesterone levels in rats and control mice, but not in hamsters and hCG-injected mice. Also, hCG elevated media testosterone levels in control but not in hCG-injected animals. Furthermore, addition of hCG in vitro partially prevented the elevation of media testosterone induced by in vivo hCG. The present results indicate that the mechanisms for the transduction of the gonadotropic signal by the Leydig cells are species-defined.     

大鼠促性腺激素细胞内蛋白激酶C的改变对黄体生成素mRNA表达的影响

目的:分析大鼠黄体生成素(LH)表达的受体后信号转导机制。方法:促性腺激素(GTH)细胞内蛋白激酶C(PKC)兴奋或抑制后,用促性腺激素释放激素(GnRH)脉冲刺激,然后用实时荧光定量PCR方法测定细胞LH的β亚基(LHβ)mRNA的表达量,并与空白组比较。结果:LHβmRNA随着PKC活性的升高而显著升高,随着PKC活性的降低而显著降低。结论:GnRH脉冲刺激引起LHβmRNA表达,其受体后的信号转导是PKC-Ca2+途径。     

Conserved cAMP signaling cascades regulate fungal development and virulence

Two well characterized signal transduction cascades regulating fungal development and virulence are the MAP kinase and cAMP signaling cascades. Here we review the current state of knowledge on cAMP signaling cascades in fungi. While the processes regulated by cAMP signaling in fungi are as diverse as the fungi themselves, the components involved in signal transduction are remarkably conserved. Fungal cAMP signaling cascades are also quite versatile, which is apparent from the differential regulation of similar biological processes. In this review we compare and contrast cAMP signaling pathways that regulate development in the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe, and differentiation and virulence in the human pathogen Cryptococcus neoformans and the plant pathogen Ustilago maydis. We also present examples of interaction between the cAMP and MAP kinase signaling cascades in the regulation of fungal development and virulence.     

Functional states of the luteinizing hormone/choriogonadotropin-receptor complex in rat Leydig cells

Three classes of gonadotropins with different ratios of stimulating to binding activities (S/B ratio) in rat Leydig cells have been identified. An S/B ratio of 1 was observed for rat luteinizing hormone (LH), porcine LH, and equine choriogonadotropin (CG) (class I), whereas ovine and equine LH exhibited and S/B ratio of 10-20 (class II) and human CG (hCG) (class III) an S/B ratio of 60. We coined the term "superactivity" to designate this particular behavior. This phenomenon was further studied by comparing the competitive activities of porcine LH (pLH) and hCG in radioreceptor assays using rat Leydig cell membranes and either radiolabeled oLH or hCG as the tracer, in the presence or absence of 150 mM NaCl. At equilibrium, both native hormones were equipotent in competing with 125I-oLH binding, but hCG was 4-fold more potent than pLH when 125I-hCG was used. Moreover, the binding rates of both hormones were considerably diminished in the presence of NaCl, but hCG binding at equilibrium was not affected, whereas that of oLH was almost completely abolished. From these results and previous data on the binding and internalization of these hormones, we suggest the existence of two interconvertible functional states of the hormone-receptor complex: (formula; see text). The equilibrium constant k3/k4 would be extremely high for hCG and lower and lower for the hormones in class II and class I, respectively. The equilibrium constant k1/k2 would be the one affected by the presence of NaCl and seems to be similar for all the hormones tested. The normal activity or superactivity of gonadotropins would thus be primarily dependent on the equilibrium between HR1 and HR2.     

Effect of human chorionic gonadotropin derivatives on leydig cell function.

BACKGROUND: Several human chorionic gonadotropin (hCG) derivatives have been detected in healthy human subjects, indicating that they may play a role in cell function. These hCG derivatives include deglycosylated hCG, proteolytic digestion products of hCG and free alpha and beta subunits of the hormone. It is well documented that testicular Leydig cells are responsive to luteinising hormone (LH) or its analogue hCG. These hormones have high affinity for LH/hCG receptors on the plasma membrane. METHODS: We designed functional and binding studies to compare the effects of native hCG and several hCG derivatives on a rat Leydig cell system. The molecular weight of the hCG derivatives was determined by SDS-PAGE and the binding affinity to LH/hCG receptors was measured by a radioligand assay. In addition, their ability to produce testosterone, cyclic AMP and arachidonic acid release was also studied. RESULTS: These hCG derivatives, with the exception of the free beta subunit, were able to bind to LH/hCG plasma membrane receptors with different affinities than that of native hCG. In addition, hCG derivatives did not increase intracellular cAMP levels or arachidonic acid release. However, they did increase testosterone production. CONCLUSION: Taken together, the results of this study lead us to suggest that these hCG derivatives may regulate the action of the native hormone in Leydig cells and are, thus, molecules of physiological relevance.     

Actin polymerization processes in plant cells

Growing evidence shows that the actin cytoskeleton is a key effector of signal transduction, which controls and maintains the shape of plant cells, as well as playing roles in plant morphogenesis. Recently, several signaling pathways, including those triggered by hormones, Ca(2+), and cAMP, have been reported to be connected to the reorganization of the actin cytoskeleton. The molecular mechanisms involved in such signaling cascades are, however, largely unknown. The Arabidopsis genome sequence is a valuable tool for identifying some of the highly conserved molecules that are involved in such signaling cascades. Recent work has begun to unravel these complex pathways using a panoply of techniques, including genetic analysis, live-cell imaging of intracellular actin dynamics, in vivo localization of factors that are involved in the control of actin dynamics, and the biochemical characterization of how these factors function.