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Breast cancers (BCs) are the most common malignancies among women worldwide. Giving birth to a first child before 24 years of age decreases the BC risk by about half, when women reach menopausal years. The scientific evidence suggests that the actions of human chorionic gonadotropin (hCG) are responsible for this decrease. Human BC cells and tissues contain hCG/luteinizing hormone receptors. The activation of the receptors results in an increase in cell differentiation and apoptosis. Conversely, it decreases the cell proliferation, invasion, and survival. The hCG actions are primarily cyclic adenosine monophosphate/protein kinase A mediated, require the presence of receptors, and involve blocking the activation and nuclear translocation of the transcription factor, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). The women with a higher hCG levels during pregnancy tend to have a lower BC incidence and those with the receptor-positive tumors have a longer metastasis-free survival. The long-term benefits of pregnancy/hCG seem to come from permanent signature genomic imprinting and expression changes, which are characterized by low cell proliferation, increased efficiency of DNA repair mechanisms, cell differentiation, and cells resistance to carcinogenesis. These findings could provide clinical opportunities to use hCG for the prevention of BC in this modern era of increasing number of young women in our societies waiting longer than ever to have their first child. In addition, hCG may be useful to reduce and/or eliminate cellular targets of carcinogenic changes during an active ongoing disease.

Painful bladder syndrome/interstitial cystitis is a debilitating chronic bladder disease that primarily affects women. The disease is due to a damage of urothelial cell lining. As a result, potassium particles and other toxic substances in urine can leak into bladder mucosa, causing the symptoms of lower abdominal/pelvic discomfort, pain, increased urination frequency, urgency, nocturia, and so on, all of which can substantially reduce the quality of daily life. There are multiple symptom reliving therapies. Among them, only pentosan polysulfate sodium, sold under the brand name of Elmiron, has been approved for oral use by US Food and Drug Administration. It provides the relief after several months of use. Based on the scientific leads presented in this article, we propose that human chorionic gonadotropin has a therapeutic potential that is worth investigating for the treatment of this disease., (© The Author(s) 2016.)

Overactive bladder (OAB) is a common form of urinary incontinence, resulting from spontaneous and random contractions of the urinary bladder. The affected individuals have an uncontrollable urge to urinate and experience incontinence and nocturia, which can greatly reduce the quality of daily life. There are several drugs for the treatment, and all of them have serious side effects. The following findings suggested that human chorionic gonadotropin (hCG) has a therapeutic potential that is worth investigating for the treatment of OAB. The finding are (1) human detrusor muscle contains hCG receptors, (2) detrusor muscle becomes quiescent during pregnancy, (3) hCG can inhibit detrusor muscle contractions induced by cholinergic stimulation in rats, and (4) hCG can mimic the anticholinergic drug on detrusor muscle contractions., (© The Author(s) 2015.)

Preterm births are an expensive global health problem. Despite the basic science and clinical research advances to better understand and prevent preterm births, the rates are increasing. There are several therapeutic options. While some options such as progestins work for selected women, others such as magnesium sulfate can only be used for delaying births for 24 to 48 hours so that the patients can be treated with corticosteroids to promote fetal lung maturity. Based on the scientific and clinical evidence, we recommend testing human chorionic gonadotropin in a large multicenter, randomized, double-blind, and placebo-controlled clinical trials in women with active preterm labor and those with a previous history of preterm births. Human chorionic gonadotropin is not only inexpensive but also has not shown any side effects so far in the infants or in the mothers., (© The Author(s) 2015.)

Autoimmune diseases such as rheumatoid arthritis (RA) and Sjögren syndrome (SS) ameliorate during pregnancy, through dampening (immunotolerance) of the maternal immune system which protects the fetus from rejection. A large number of studies have shown that human chorionic gonadotropin (hCG) contributes to this tolerance. Studies on animal models have reaffirmed that hCG treatment mimics the benefits of pregnancy. Based on the scientific evidence, randomized clinical trials comparing hCG with current therapies and/or placebo are recommended for RA, SS, and for other autoimmune diseases such as, type 1 diabetes and ankylosing spondylitis, which also get better during pregnancy and hCG treatment seems to help., (© The Author(s) 2015.)

The scientific evidence suggests that Neisseria gonorrhoeae (NG) infects human fallopian tubes by molecular mimicry in which pathogens act like a ligand to bind to epithelial cell surface human chorionic gonadotrophin (hCG)/luteinizing hormone (LH) receptors. The hCG-like molecule has been identified as ribosomal protein L12 in NG coat surface. Human fallopian tube epithelial cells have been shown to contain functional hCG/LH receptors. As previously shown in human fallopian tube organ and cell culture studies, cellular invasion and infection can be prevented by exposing the cells to excess hCG, which would outnumber and outcompete NG for receptor binding. Based on these data, we suggest testing hCG in clinical trials on infected women., (© The Author(s) 2015.)

Problem: Successful embryonic implantation requires an appropriate communication network between the embryo and its near environment within the implantation site. Herein, we examined whether human chorionic gonadotropin (hCG), the major embryonic signal, targets endothelial cells and regulate their responsiveness to interleukin 1 (IL1), one of the earliest signals released by embryonic cells., Method of Study: Human microvascular endothelial cell proliferation and migration following exposure to various concentrations of hCG and/or IL1B for different time periods were analyzed by BrdU incorporation and wound healing assays. The expression of soluble (s) and membrane-bound (mb) IL1 receptors (IL1Rs), IL1R antagonist (IL1RN), luteinizing hormone/choriogonadotropin receptor (LHCGR), and IL8 was determined by real-time PCR, Western blot, and ELISA., Results: Cell proliferation and migration increased in response to IL1B and further in the presence of hCG. IL1B up-regulated both the signaling IL1R1 and the inhibitory IL1R2, while adding hCG further increased IL1R1 and significantly downregulated IL1R2. This translated into an increased secretion of IL8, which was inhibited in cells where IL1R2 was overexpressed., Conclusions: These findings reveal a new mechanism by which hCG may target endothelial cells to directly stimulate angiogenesis and favor embryonic growth., (© 2013 John Wiley & Sons A/S.)

The presence of the conceptus in uterine cavity necessitates an elaborate network of interactions between the implanting embryo and a receptive endometrial tissue. We believe that embryo-derived signals play an important role in the remodeling and the extension of endometrial receptivity period. Our previous studies provided original evidence that human Chorionic Gonadotropin (hCG) modulates and potentiates endometrial epithelial as well as stromal cell responsiveness to interleukin 1 (IL1), one of the earliest embryonic signals, which may represent a novel pathway by which the embryo favors its own implantation and growth within the maternal endometrial host. The present study was designed to gain a broader understanding of hCG impact on the modulation of endometrial cell receptivity, and in particular, cell responsiveness to IL1 and the acquisition of growth-promoting phenotype capable of receiving, sustaining, and promoting early and crucial steps of embryonic development. Our results showed significant changes in the expression of genes involved in cell proliferation, immune modulation, tissue remodeling, apoptotic and angiogenic processes. This points to a relevant impact of these embryonic signals on the receptivity of the maternal endometrium, its adaptation to the implanting embryo and the creation of an environment that is favorable for the implantation and the growth of this latter within a new and likely hostile host tissue. Interestingly our data further identified a complex interaction between IL1 and hCG, which, despite a synergistic action on several significant endometrial target genes, may encompass a tight control of endogenous IL1 and extends to other IL1 family members.

Deep functional changes occurring within the endometrium during implantation are orchestrated by embryonic and maternal signals. Human chorionic gonadotropin (hCG), a major embryonic signal, plays a critical role in the initiation and maintenance of pregnancy. Interleukin (IL) 1, one of the earliest embryonic signals, appears to exert a direct impact on the receptive endometrium and to induce major molecular changes that are essential for embryo implantation. Herein we investigate whether hCG can modulate endometrial stromal cell (ESC) receptivity to IL1 during the implantation window and assess the impact on angiogenesis in vitro. Primary cultures of ESCs from normal fertile women during the implantation window were treated for 24 h with different concentrations of hCG (0-100 ng/ml) and stimulated for 24 h with IL1B (0-0.1 ng/ml). IL1 receptors (IL1Rs), IL1R antagonist (IL1RA), and monocyte chemotactic protein (MCP) 1 were analyzed by real-time PCR, ELISA, and Western blotting. The angiogenic activity in vitro was studied using human microvascular endothelial cell line, scratch wound assay, and cell proliferation via BrdU incorporation into DNA. Human CG induced a dose-dependent imbalance in ESC receptivity to IL1 by significantly upregulating the functional signaling IL1R1 and concomitantly downregulating the decoy inhibitory IL1R2 and IL1RA upon subsequent exposure to IL1B. Prior exposure to hCG amplified MCP1 secretion by ESCs in response to IL1B and triggered the release of angiogenic activity in vitro in which MCP1 appeared to play a significant role. Overexpression of IL1R2 using cell transfection inhibited IL1 and hCG/IL1B-mediated MCP1 secretion. These findings suggest that hCG coordinates embryonic signal interaction with the maternal endometrium, and point to a new possible pathway by which it may promote embryonic growth.

The role of luteinizing hormone (LH) and human chorionic gonadotrophin hormone (hCG) in the regulation of normal reproductive functions in males and females is quite well established. Besides the use of hCG in the development of diagnostic immunoassays, it has been successfully used in the induction of final follicular maturation and ovulation in the assisted reproductive technologies. The basic and clinical research on the nongonadal actions of LH/hCG in the recent years has extended the potential of using these hormones in several clinical indications. Hereby we will analyze the advances in the LH/hCG research (briefly emphasizing the nongonadal research), which has the potential for multiple novel therapies in reproductive and the other areas of medicine.

Secretion of embryonic IL-1beta seems to be the first response of the blastocyst to receptive endometrium, inducing molecular changes that are essential for attachment of the blastocyst. Here, we report that human chorionic gonadotropin (hCG), a glycoprotein hormone that plays a critical role in the initiation and maintenance of pregnancy, markedly down-regulates the expression of the decoy inhibitory IL-1 receptor type II (IL-1R2) in human endometrial epithelial cells. Treatment with hCG resulted in a dose-dependent decrease in IL-1R2 soluble and membrane bound protein forms and mRNA steady-state levels, whereas no significant effect on the expression of the activating IL-1 receptor type I (IL-1R1) was seen. Cell infection with the wild-type human LH/chorionic gonadotropin receptor corroborated the aforementioned data, whereas cell infection with the constitutively activated LH chorionic gonadotropin receptor led to similar effects on IL-1R2 and IL-1R1 expression without hCG treatment. Cloning of human IL-1R2 gene promoter in the pGL3 luciferase reporter vector and transient transfection experiments further showed a significant dose-dependent diminution of IL-1R2 promoter activity in response to hCG. These data suggest that hCG, by down-regulating the expression of IL-1R2, a potent and specific inhibitor of IL-1, without affecting the expression of the functional activating IL-1R1, diminishes the ability of endometrial epithelial cells to counterbalance the local effects of IL-1, making these cells probably more responsive to the cytokine. In view of IL-1's role as an embryonic signal, these data reveal a new mechanism by which hCG sustains human pregnancy and promotes embryonic growth.

The past and present published studies reaffirm that nongonadal LH and hCG actions are real and here to stay. These actions have led to a better understanding of the biology of the hormones and more importantly begin to pave the way for novel therapies in reproductive medicine and in other areas.

We tested the hypothesis that hCG can upregulate human trophoblast indoleamine 2, 3-dioxygenase (INDO), which catalyzes the breakdown of tryptophan in villous circulation. The results revealed that it can. Treatment of human trophoblasts with hCG resulted in a time and dose dependent increase in INDO mRNA and protein levels and its enzyme activity. The hCG effect was hormone specific and required the dimer conformation of hCG. The hCG effect required its receptors and was mediated by a cAMP dependent, but protein kinase A independent, mitogen-activated protein kinase 3/1 (MAPK3/1) signaling mechanism. In summary, the present data demonstrate a novel hCG effect on human placental INDO, which probably plays a key role at maternal fetal interface in preventing fetal rejection.

Angiogenesis and vascular remodeling are crucial processes in tumor invasion and metastasis as well as in embryo implantation and normal development of the placenta. We have previously shown that hCG expressed in trophoblast and various malignant tumors promotes cellular motility and that uterine endothelium expresses hCG/LH receptor in vivo. In this study hCG was proposed to promote angiogenesis. A three-dimensional in vitro angiogenesis system consisting of uterine microvascular endothelial cells seeded on microcarriers and entrapped in a fibrin matrix was used to study the influence of hCG on neovascularization. Physiological concentrations of hCG (5-50,000 mU/ml) significantly increased in vitro capillary formation (up to 2.5-fold) and migration of endothelial cells in a Boyden chamber assay (up to 3.6-fold) in a dose-dependent manner, whereas hCG had no effect on cell proliferation. In vivo, hCG induced neovascularization in the chicken chorioallantoic membrane assay comparable to the activity of vascular endothelial growth factor. hCG-secreting tumors (choriocarcinoma, endometrium, and ovarian carcinoma) promoted in vitro neovascularization (up to 3-fold), whereas hCG-neutralizing antibody, pertussis toxin (G protein inhibitor), or GRGDTP peptide (integrin antagonist), respectively, abolished both tumor- and hCG-induced capillary sprout formation. Our data indicate a novel function for hCG in uterine adaptation to early pregnancy as well as in tumor development and underline the importance of hCG as an as yet unrecognized angiogenic factor.

Luteinizing hormone (LH) and its homologue, human chorionic gonadotropin (hCG), are able to elicit multiple effects in the central nervous system (CNS) through binding to their receptors. Specific receptors for LH/hCG have been identified in the hippocampus, dentate gyrus, hypothalamus, cortex, brain stem, area postrema, cerebellum, choroid plexus, ependymal cells, glial cells, neural retina, pituitary gland, and neuron processes of the spinal cord. Neurotropic effects of LH and hCG have been demonstrated in fetal rat brain, where the expression of LH/hCG receptors is developmentally regulated. Administration of hCG has been found to be beneficial in restoration of transected spinal cord function in rats. In adult rat brain, LH and hCG are involved in the feedback regulation of synthesis and secretion of gonadotropin-releasing hormone (GnRH) in the hypothalamus and LH in the pituitary gland. LH and hCG also induce several behavioral and other changes that are associated with the hippocampus, which contains the highest density of LH/hCG receptors. Many of the behavioral changes induced by hCG in rats parallel those in pregnant women. Some of these behavioral effects are correlated with changes of eicosanoid metabolism induced by LH and hCG in the brain. The LH/hCG receptors present in the choroid plexus, brain vessels, and perihypophyseal vascular complex may be involved in the modulation of transport of LH, hCG, and GnRH into the CNS. Thus, the CNS is one of the specific target tissues for LH and hCG, by which LH/hCG act as pleiotropic hormones that regulate several reproduction-related as well as reproduction-nonrelated functions in the CNS.

The role of human chorionic gonadatropin (hCG) in the maintenance of early pregnancy is well known. Recent data suggests that hCG may play a role in the maintenance of the later stages of pregnancy as well, by directly and indirectly promoting uterine quiescence. If hCG acts as an endogenous tocolytic in normal pregnancy, then it may be an ideal candidate for therapy of preterm labor as well. We present compelling in vitro as well as in vivo data, which support the role of hCG in the maintenance of normal uterine quiescence. Additionally, we will present in vivo and in vitro data that confirms the ability of hCG to directly promote relaxation of uterine contractions. This review provides a basis for future study of the use of hCG in clinical obstetrics. Given the limited effectiveness of tocolytic therapies available at the time, hCG may provide a promising pharmacological approach to the pervasive problem of preterm labor in human pregnancy. While further work is needed, initial data strongly support this novel use of hCG in clinical obstetrics.

Even though there were hints, it was not until 1986 that a number of laboratories worldwide began to demonstrate unequivocally the presence and functions of luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptors in various female and male nongonadal tissues. There was no species specificity but there was tissue specificity in the nongonadal LH/hCG receptor distribution. Nongonadal receptor levels were lower but they were regulated and processed and used signaling mechanisms similarly to gonadal receptors. Although still greater understanding is needed, gains made in the last decade demonstrate that nongonadal actions of LH/hCG are physiologically important and may have relevance to better understanding several diseases and their treatment. A recently developed LH receptor knockout model has begun to reaffirm and extend the importance of nongonadal LH signaling in the body.

Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) share a common receptor. LH/hCG receptors, located in the vascular smooth muscle and endothelial cells of uterine blood vessels, are most numerous in smaller intramyometrial vessels and are cyclic in nature. There is a correlation between hCG levels and decreased uterine vascular resistance in humans, and in pseudopregnant rats, hCG decreases uterine blood flow. We found that systemic administration of hCG to cycling rats reduced uterine blood flow within 20 minutes on all days of the estrous cycle when flow was measured via the radioactive microsphere method. This effect was absent in ovariectomized rats. To determine the response to hCG at the microvascular level, we measured uterine arteriolar diameters in vivo via videomicroscopy after direct application or injection of hCG in rats on diestrus-1, diestrus-2, and proestrus. When hCG was suffused over the uterus (20 IU/60 mL), the uterine arterioles in diestrus (1 and 2) rats dilated but in proestrus rats were constricted. Neither response was altered in animals whose ovaries were removed 3 hours previously. An intraperitoneal injection of hCG (50 IU) caused uterine arteriolar constriction in diestrus-2 and proestrus animals but no change in diestrus-1. In ovariectomized rats, uterine arteriolar constriction following hCG injection was absent. Thus, the response of the uterine vasculature is not only cycle day dependent but also dependent on the route of administration. The effect of hCG on uterine blood flow in the rat may be a direct action of hCG, or it could be secondary to the release of other vasoactive substances known to be released by hCG. hCG might also alter blood flow by inducing angiogenesis.

Recent studies found that human and animal breast tissues and human breast cell lines contain low levels of receptors that bind hCG and its structural and functional homologue, LH. Those gonadotropins exert numerous anticancer effects in breast cancer models and cells, which might explain decreased breast cancer incidence in women who complete full-term pregnancies at a young age. The new findings also imply that premature chronic elevations of LH levels might contribute to decreased breast cancer incidence in women with early menopause, and elevated LH levels might contribute to a better prognosis after ovariectomy. Those findings predict that breast cancer risk might be reduced by early hCG treatment of women who plan to delay their first pregnancies; prophylactic hCG treatment might help women with family histories of breast cancer or oncogene mutations that predict breast cancer; and better prognoses might result when hCG is administered to breast cancer tissue.

Objective: The purpose of this study was to test the capability of human chorionic gonadotropin to inhibit prostaglandin-induced preterm delivery in a murine model., Study Design: A preterm delivery model was developed by using intraperitoneal injection of 20 microgram of prostaglandin F(2)(alpha) to induce preterm labor in C3H/HeN inbred mice. Mice were then pretreated with human chorionic gonadotropin 4 hours before administration of prostaglandin F(2)(alpha), and time to delivery of the first pup was recorded. After initial promising results, mice were then given increasing intraperitoneal doses of human chorionic gonadotropin (100 IU, 250 IU, or 1000 IU or sodium chloride solution vehicle) 4 hours after administration of prostaglandin F(2)(alpha). The specificity of the human chorionic gonadotropin effect was assessed by treating mice with whole human chorionic gonadotropin, an equal mass dose of the beta-subunit or the alpha-subunit of human chorionic gonadotropin, or an equal mass dose of luteinizing hormone 4 hours after administration of prostaglandin F(2)(alpha). Delivery times between groups were compared by using the Mann-Whitney U test and the log-rank test. Survival estimates were computed by using the Kaplan-Meier method., Results: Pilot studies in 52 mice confirmed that a single intraperitoneal injection of 20 microgram of prostaglandin F(2)(alpha) on day 16 (80% gestation) consistently induced preterm delivery compared with the effect of sodium chloride solution on control mice (prostaglandin F(2)(alpha), 19.3 +/- 2.9 hours; sodium chloride solution, 53.5 +/- 13.6 hours; P <.0001). Mice pretreated with human chorionic gonadotropin (1000 IU) demonstrated significant delays in delivery times compared with the prostaglandin-only group (prostaglandin F(2)(alpha) only, 21.9 +/- 2. 0 hours; human chorionic gonadotropin pretreatment plus prostaglandin F(2)(alpha), 48.5 +/- 20 hours; P <.0001; n = 17). Mice treated with human chorionic gonadotropin (100 IU, 250 IU, 1000 IU) 4 hours after administration of prostaglandin F(2)(alpha) demonstrated significant dose-dependent inhibition of preterm delivery compared with the prostaglandin-only group (P <.00005; n = 34). Mice treated with the alpha-subunit or the beta-subunit of human chorionic gonadotropin after prostaglandin administration did not demonstrate delays in delivery times (P =.46; n = 27). Administration of luteinizing hormone delayed delivery compared with the effect of prostaglandin F(2)(alpha) on control animals (P <.05; n = 17); however, the effect was less pronounced than that seen with a mass equivalent of human chorionic gonadotropin., Conclusions: Human chorionic gonadotropin exhibits potent inhibition of prostaglandin-induced preterm delivery in mice. The effect is dose-dependent, and whole human chorionic gonadotropin is required to elicit inhibition. Further studies are needed to determine the safety and efficacy of human chorionic gonadotropin as a potential therapy for preterm labor inhibition in human pregnancy.

Endometrial glands contain higher levels of LH/hCG receptors than other cells in the human uterus. The present study investigated their functional importance. Northern and Western blotting and covalent receptor cross-linking demonstrated that human endometrial gland epithelial cells in culture contained multiple LH/hCG receptor transcripts and an 80-kDa receptor protein that can bind [125I]hCG in a hormone-specific manner. Culturing cells with highly purified hCG resulted in a time- and dose-dependent increase in steady state levels of cyclooxygenase-2 (COX-2) messenger ribonucleic acid and protein and the secretion of PGE2. Although human LH could mimic hCG, FSH, TSH, and alpha- or beta-subunits of hCG had no effect on COX-2 protein levels. Studies on signaling revealed that treatment of cells with hCG resulted in an increase in cAMP levels and protein kinase A (PKA) activity. Inhibition of PKA activity by cotreatment with isoquinoline-sulfonamide (H-89) prevented hCG from increasing COX-2 protein levels. Treatment with 8-bromo-cAMP mimicked the effect of hCG, and cotreatment with a selective inhibitor of type I PKA, 8-chloro-cAMP, prevented 8-bromo-cAMP and hCG from increasing COX-2 protein levels. The requirement of receptors for LH/hCG action was investigated by 24-h treatment of human endometrial gland epithelial cells with 21-mer phosphorothioate oligodeoxynucleotides (ODNs) synthesized from human receptor sequence. Treatment with 2 micromol/L antisense, but not sense, ODN resulted in a dramatic reduction in LH/hCG receptor protein levels. hCG was unable to increase COX-2 protein, PGE2, and cAMP levels in an antisense, but not in sense, ODN-treated cells. In summary, we conclude that hCG and LH treatment can increase expression of the COX-2 gene in human endometrial gland epithelial cells. The effect was time and dose dependent, hormone specific, and mediated by the cAMP/type I protein kinase A signaling pathway. The hCG actions require a normal complement of its receptors in cells. These hCG and LH effects may be another action of these hormones in human endometrium that is important for implantation of the blastocyst and continuation of pregnancy.

The existence of the hormone passage from venous blood into arterial blood in the area of the perihypophyseal vascular complex has been demonstrated in some mammals, but its mechanism has not been defined. To study the regulatory mechanism we infused hCG into perihypophyseal cavernous sinus of ovariectomized, conscious ewes to test if the hCG would affect putative LH/hCG receptors and inhibit counter-current transfer of GnRH from the venous cavernous sinus to the arterial carotid rete. The latter study was done on an isolated head model. Ewes were ovariectomized in mid-anestrus and, after 4 to 5 wk were used in the experiments. On the day of experiment ewes were treated intramuscularly with estradiol benzoate or oil vehicle, and 18 to 20 h later were infused either with a multielectrolyte solution or hCG for 2 h via the venae angularis oculi. Immediately thereafter the ewes were anesthetized and exanguinated, and subsequently decapitated. The isolated head was perfused with Dextran in multielectrolyte. The 125I-GnRH was infused into the cavernous sinus via the venae angularis oculi for 5 min; contemporaneous samples were taken from the carotid rete and both jugular veins at 1-min intervals. Transfer of 125I-GnRH from the cavernous sinus to the carotid rete was inhibited by hCG in ewes pretreated with estradiol benzoate but not with oil (P<0.005). We collected tissue samples from the vascular complex of the cavernous sinus and carotid rete of cyclic ewes to determine the presence of LH/hCG receptors. In situ hybridization showed the presence of LH/hCG receptor mRNA transcripts in the walls of both arterial and venous compartments of the cavernous sinus-carotid rete complex, and immunohistochemistry revealed the presence of receptor proteins. These novel findings confirm previously obtained data suggesting that LH is a modulatory factor for the counter-current transfer of neuropeptides from the venous blood of the cavernous sinus to the arterial blood supplying the brain and hypophysis. The LH could modulate 125I-GnRH transfer acting directly on the vascular smooth muscle.

Human endometrial stromal cells contain luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptors and treatment with highly purified hCG results in an up-regulation of cyclooxygenase-2 (COX-2) gene expression and increased production of prostaglandin (PG) E2. Since PGE2 promotes the differentiation of endometrial stromal cells into decidua, we tested the hypothesis that LH and hCG themselves may promote this process. The results revealed that these hormones can promote morphological as well as functional differentiation. While their action on morphological differentiation did not require the presence of estradiol (E2) and progesterone (P4), they did require them for the functional differentiation. The hCG effect was mimicked by LH, but not by follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) or alpha and beta subunits of hCG, suggesting that the hCG action was hormone specific and requires the conformation of native hormone. The hCG treatment also increased the steady state PRL mRNA levels. This increase was due to an increase in the transcription rate of the gene rather than a decrease in the degradation of PRL transcripts. In summary, we conclude that hCG and LH can increase the morphological as well as functional differentiation of human endometrial stromal cells into decidua. This is one of the newly discovered actions of LH and hCG that may be important for the implantation of blastocyst and maintenance of pregnancy.

The present study tested the hypothesis that LH/hCG may regulate the type 2 5 alpha-reductase and androgen receptor protein levels in skin. The skin samples obtained from women undergoing abdominal laparotomy or abdominoplasty were incubated in the presence or absence of hCG. Western blotting was then performed to determine the response of type 2 5 alpha-reductase and androgen receptors. The results demonstrated that treatment with hCG resulted in a significant time- and dose-dependent, although modest, decrease in 5 alpha-reductase and androgen receptor levels compared to the controls. These effects were mimicked by LH, but not by other hormones in the glycoprotein hormone family, including alpha- and beta-subunits of hCG. Although the biological and clinical importance of this regulation remains to be determined, these findings reaffirm that human skin is among the nongonadal tissues that respond to LH and hCG treatment.

We investigated MCF-7 and MDA-MB-231 human breast cancer cell lines and immortalized mammary epithelial HBL-100 cells for the presence of functional LH/hCG receptors. The results revealed that all three breast cell lines contain LH/hCG receptor mRNA transcripts and receptor proteins that can bind 125I-hCG. The MCF-7 cells, however, contain higher levels than the others. Culturing MCF-7 cells with highly purified hCG resulted in a dose- and time-dependent significant decrease in steady-state estradiol receptor mRNA and protein levels as compared to controls, with the maximal decrease occurring after 4 h of culture with 10 ng/ml hCG. The studies on cell growth demonstrated that hCG treatment in the presence of minimal or no fetal bovine serum had a time-dependent significant inhibitory effect on MCF-7 and HBL-100, but not on MDA-MB-231 cells. In summary, our results demonstrate that human breast cell lines contain functional LH/hCG receptors. The hCG effects in MCF-7 cells are consistent with a premise that hCG protects women against breast cancer.

The alphaT3-1 cells are immortalized anterior pituitary gonadotropes which express gonadotropin-alpha subunit gene. These cells contain receptors for gonadotropin releasing hormone (GnRH) as well as for luteinizing hormone (LH) which can also bind human choriogonadotropin (hCG). Like GnRH, LH and hCG can upregulate the expression of gonadotropin-alpha subunit gene. While 0.1-1.0 ng/ml hCG can upregulate, higher concentrations have no effect. However, these higher hCG concentrations can act in a synergistic manner with GnRH to increase the steady state mRNA and protein levels of gonadotropin-alpha subunit. The synergism between hCG and GnRH was mimicked by LH but not by other hormones in the glycoprotein hormone family or alpha or beta subunits of hCG, suggesting that the synergism is hormone specific and requires the conformation of native hormone. The hCG induced increase in gonadotropin-alpha subunit mRNA levels was due to a significant increase in the half-life of gonadotropin-alpha subunit transcripts from 7.8 +/- 1.0 h in the controls to 16.5 +/- 3.8 h after treatment with hCG. The GnRH induced increase in gonadotropin-alpha subunit mRNA levels was due to both a significant increase in the half-life to 26.2 +/- 3.0 h as well as a significant increase in the transcription rate of the gene (159.0 +/- 7.7% of the control). A greater increase in gonadotropin-alpha subunit mRNA levels following a combined treatment with GnRH and hCG was due to a further increase in half-life to 37.6 +/- 3.1 h as well as a greater increase in the transcription rate of the gene (295.1 +/- 24.2% of the control) as compared to the treatment with GnRH alone. In summary, we conclude that LH and hCG can independently and synergistically act with GnRH to increase the expression of gonadotropin-alpha subunit gene by transcriptional as well as by post-transcriptional mechanisms in alphaT3-1 cells. These effects may be important for the increase of LH levels during the preovulatory surge.

We investigated the cis-acting elements and trans-acting proteins required for the transcriptional inhibition of the gonadotropin-releasing hormone (GnRH) gene by human chorionic gonadotropin (hCG) in GT1-7 neurons. Transient transfection of GT1-7 neurons with the 5'-flanking region of the rat GnRH gene-luciferase fusion constructs revealed that a 53-base pair (bp) sequence between -126 and -73 bp is required for the hCG inhibition. Nuclear extracts from GT1-7 neurons contained 110- and 95-kDa proteins that formed two complexes with the 53-bp sequence. These proteins are not related to Fos, cAMP response element-binding protein, Oct-1, or progesterone receptors, and hCG treatment selectively increased the 95-kDa protein. DNase I footprinting with GT1-7 cell nuclear extracts protected the -99 to -79-bp region, which contained a so-called imperfect AP-1 site (-99 to -94 bp) and two AT-rich palindromic sequences (-91 to -87 bp and -85 to -81 bp). The mutagenesis of the AT-rich regions, but not the AP-1 site, resulted in a loss of DNA binding of the 95-kDa protein and the inhibitory effect of hCG. In summary, our results are consistent with hCG inducing a 95-kDa trans-acting protein, which binds to -91- to -81-bp AT-rich sequences in the 5'-flanking region to inhibit the transcription of the GnRH gene.

Objective: Our purpose was to investigate whether uterine microvascular responses to human chorionic gonadotropin application depend on route of administration and estrous cycle day., Study Design: One uterine horn was exteriorized in pentobarbital-anesthetized cycling and ovariectomized rats and superfused with Krebs solution Uterine arterioles (64 +/- 2.1 microns) were viewed by videomicroscopy. Diameters were measured during a 20-minute baseline period and for 60 minutes during human chorionic gonadotropin suffusion (20 IU/60 ml) or 60 minutes after intraperitoneal injection of 50 IU of human chorionic gonadotropin. Papaverine (100 mumol/L) suffusion maximally dilated the uterine arterioles (80 +/- 2.6 microns)., Results: Suffusion of human chorionic gonadotropin-dilated arterioles on diestrus-1 (122% +/- 2% baseline) and diestrus-2 (118% +/- 4% baseline) but constricted arterioles on proestrus (78% +/- 7% baseline). Intraperitoneal injection of human chorionic gonadotropin resulted in arteriolar constriction on diestrus-2 (76% +/- 5% baseline) and proestrus (82% +/- 3% baseline). Ovariectomy eliminated the effects of injected but not suffused human chorionic gonadotropin. All results are significant at p < 0.05., Conclusions: Results indicate estrous cycle day-dependent direct and indirect effects of human chorionic gonadotropin on the resistance of uterine arterioles.

The present study investigated the regulation of cyclooxygenase-2 (COX-2) gene by human CG (hCG) in mucosal cells from human fallopian tubes. The mucosal cells contained a major [4.3 kilobase (kb)] and several minor (3.6, 2.4, and 1. 8 kb) messenger RNA (mRNA) transcripts of LH/hCG receptors and also an 80-kDa receptor protein. The receptor protein can bind 125I-hCG. Culturing mucosal cells with increasing concentrations of highly purified hCG resulted in a dose- and time-dependent increase in steady-state levels of a 4.4-kb mRNA transcript and 72-kDa protein of COX-2. Whereas hLH and hCG could mimic each other in increasing COX-2 protein levels, FSH, TSH, PRL, and isolated alpha- and beta-subunits of hCG had no effect, suggesting that the hCG effect is hormone specific and requires the conformation of native hormone. Culturing mucosal cells with increasing concentrations of hCG also resulted in a dose-dependent increase in media PGE2, levels, suggesting that the COX-2 protein increased by hCG is catalytically active. To determine the molecular mechanism of hCG action responsible for increasing the steady-state COX-2 mRNA levels, we measured the transcription rate of the COX-2 gene by nuclear run-on assay and the stability of its transcripts by an actinomycin D blocking method. The results showed that although hCG treatment had no effect on the transcription rate of COX-2 gene, it significantly increased the stability of COX-2 transcripts from 3.7 h in the control to 7.3 h after treatment. In summary, we conclude that tubal mucosal cells contain LH/hCG receptor transcripts and the receptor protein that can bind hCG. Culturing these cells with exogenous hCG and LH can up-regulate the expression of COX-2 gene by increasing the stability of transcripts. Through this up-regulation, LH and hCG may influence tubal functions that are important for early pregnancy in women.

Human endometrial stromal cells contain human CG (hCG)/LH receptors and in vitro, hCG/LH can promote stromal cells differentiation into decidua. In the present study, we tested the hypothesis that the treatment of stromal cells with exogenous hCG/LH to promote in up-regulation of cyclooxygenase-2 (COX-2) gene expression. The stromal cells from proliferative phase endometria were cultured for 10 days with 10 ng/ml estradiol and 100 ng/ml progesterone in the presence or absence of increasing concentrations of highly purified hCG. Northern blotting demonstrated that the cells contained a 4.4-kb COX-2 messenger RNA transcript whose levels significantly increased after treatment with hCG. Western blotting showed that the cells contained a 72-kDa COX-2 protein which also significantly increased after treatment with hCG. The effect of hCG on COX-2 messenger RNA and protein was seen at 10 ng/ml and higher concentrations sustained the increased levels. Although human LH mimicked hCG, human FSH, TSH, and isolated alpha- and beta-subunits of hCG had no effect on COX-2 protein levels suggesting that the hCG effect is hormone specific and requires the conformation of native hormone. The effect of hCG on COX-2 protein paralleled the increase in media prostaglandin E2 levels indicating that the increased COX-2 gene and half-life of its transcripts to determine the molecular mechanism of hCG action. The results showed that hCG treatment had no significant effect on transcription rate of the COX-2 gene. On the other hand, treatment with hCG significantly increased the half-life of the COX-2 transcripts from 2.6 h in the control to 6.7 h after treatment. In summary, we conclude that treatment of human endometrial stromal cells with exogenous hCG to promote their differentiation into decidua results in an up-regulation of COX-2 gene expression by increasing the stability of the transcripts.

Previous studies have shown that human CG (hCG) can down-regulate the expression of GnRH gene in GT1-7 neurons and that these neurons contain GnRH receptors and a self-stimulatory mechanism in the synthesis and release of GnRH. These findings have led us to hypothesize that hCG may down-regulate GnRH receptors to disrupt the self-stimulatory mechanism. To test this hypothesis, we cultured GT1-7 neurons in the presence or absence of an optimal concentration of highly purified hCG (100 ng/ml) and then measured steady-state levels of GnRH receptor messenger RNA (mRNA) transcripts and protein. Northern blotting demonstrated that GT1-7 neurons contain a major 5.5-kb and minor 2.4-kb and 1.6-kb GnRH receptor mRNA transcripts. Ligand blotting showed that GT1-7 neurons also contain 53-kDa and 43-kDa GnRH receptor proteins. Culturing with hCG resulted in a significant decrease in steady-state levels of GnRH receptor mRNA transcripts by 12 h and GnRH receptor proteins by 6 and 12 h, followed by a return to the controls by 24 h. The treatment with hCG had no obvious effect on the transcription rate of GnRH receptor gene. The hCG treatment, however, significantly decreased the half-life of GnRH receptor mRNA transcripts from 27 h to 16 h. In summary, we conclude that treatment with hCG can down-regulate the expression of GnRH receptor gene by decreasing the stability of transcripts in GT1-7 neurons. By down-regulating GnRH receptors, hCG may disrupt the self-stimulatory mechanism in GnRH synthesis.

The present study characterized hCG/LH receptors from messenger ribonucleic acid (mRNA) to protein and whether exogenous hCG can bind and regulate the expression of the cyclooxygenase-1 (COX-1) gene in human fetal membranes from term pregnancy. Northern blotting showed that fetal membranes contain 6.0, 4.4, 2.4, and 1.4 kilobases of hCG/LH receptor mRNA transcripts. In situ hybridization revealed that amnion, chorion, and decidua contain receptor transcripts. Western immunoblotting and immunocytochemistry showed that amnion, chorion, and decidua also contain an 80-kDa receptor protein. Ligand blotting demonstrated that the 80-kDa receptor protein in fetal membranes can bind [125I]hCG, and this binding was inhibited by excess unlabeled hCG. Treatment of fetal membranes with highly purified hCG resulted in a dose- and time-dependent increase in immunoreactive COX-1 protein. The response of hCG was seen in all layers of fetal membranes. The treatment with hCG also resulted in an increase in steady state COX-1 mRNA levels. The action of hCG was prevented by cotreatment with H-89, an inhibitor of protein kinase A, but not by calphostin or lavendustin, which inhibit protein kinase C and tyrosine kinase, respectively. In summary, human fetal membranes contain hCG receptor transcripts and receptor protein that can bind hCG and up-regulate the expression of COX-1 gene.

Human myometrial smooth muscle cells contain receptors for human chorionic gonadotropin (hCG)/luteinizing hormone (LH). Exogenous hCG and LH can cause a modest hyperplasia in myometrial smooth muscle cells in culture. This response is lost after about the third subculture of the cells. The present study investigated whether the loss of hCG response could be restored by co-culturing with human follicle stimulating hormone (FSH). The results showed that co-culturing with FSH can indeed restore a modest mitogenic response of hCG. However, FSH alone was not mitogenic. The FSH restoration of hCG response can be blocked by antibodies to FSH or hCG but not by non-specific rabbit IgG. The FSH treatment resulted in an increase of steady state levels of hCG/LH receptor mRNA and protein in myometrial smooth muscle cells. Since the FSH actions could be receptor mediated, we investigated the presence of FSH receptor mRNA transcripts and protein in freshly dispersed myometrial smooth muscle cells. Northern blotting demonstrated that myometrial smooth muscle cells, just as rat ovary, a classical target of FSH action, contain multiple FSH receptor mRNA transcripts. Western immunoblotting demonstrated that myometrial smooth muscle cells also contain a 60 kDA FSH receptor protein just as rat ovary and human granulosa cells used as positive control tissues. The immunocytochemistry also demonstrated that myometrial smooth muscle cells, as rat ovary and human granulosa cells, contain FSH receptor immunostaining. In summary, it is novel that FSH could restore the mitogenic response of hCG in human myometrial smooth muscle cells and these cells contain FSH receptors. These findings may have functional implications for direct regulation of human myometrium not only by hCG/LH but also by FSH.

Recent characterization of luteinizing hormone++ (LH)/human chorionic gonadotropin (hCG) receptors in uterine vascular tissue, evidence that expression of these receptors is cyclic in nature, and demonstration of a correlation between hCG level and uterine vascular resistance lead us to investigate the effect of hCG administration on blood flow in reproductive organs of cycling and ovariectomized Sprague-Dawley rats. Blood flow (ml/min/g dry wt/cardiac output +/- SEM) was determined by microsphere spectroscopy (57Co, 113Sn, 95Nb, 141Ce). Baseline uterine (0.5842 +/- 0.1037) and cervical (0.7785 +/- 0.1199) blood flows were greater in diestrus-2 rats than in every other group. Diestrus-2 (0.4530 +/- 0.0584) and estrus (0.4692 +/- 0.0848) rats had greater baseline ovarian blood flow than proestrus rats (0.2521 +/- 0.0279). A single intraperitoneal injection of 50 IU hCG on each day of the 4-day estrus cycle decreased uterine flow by more than 30% within 20 min (P<0.05), but did not alter uterine flow in ovariectomized rats. This dose of hCG also decreased ovarian flow in diestrus-2 rats (0.5219 +/- 0.0857 to 0.4207 +/- 0.0753), decreased liver flow in diestrus-2 (0.0282 +/- 0.0060 to 0.0231 +/- 0.0051) and estrus (0.0301 +/- 0.0029 to 0.0203 +/- 0.0038 rats, and increased liver flow in ovariectomized rats (0.0279 +/- 0.0054 to 0.0325 +/- 0.0050). Injection of 0.10 IU hCG did not alter blood flow to reproductive organs in any group, but decreased liver flow in estrus rats (0.0469 +/- 0.0121 to 0.0326 +/- 0.0088). Neither dose of hCG altered cervical, kidney, or skeletal muscle flow in any group. Our results indicate an organ specific, dose-dependent blood flow response to hCG in cycling rats, which appears, in the case of uterine flow, to be attenuated by removal of the ovaries. The present findings suggest high doses of hCG given clinically may decrease uterine flow and potentially lead to implantation failure.

We have discussed recent new findings on the presence and function of hCG/LH receptors in non-gonadal tissues with particular emphasis on the placenta. These findings support the hypothesis that hCG plays a broader role throughout pregnancy than previously believed. Since hCG is widely used in reproductive medicine, the possible impact of hCG treatment on non-gonadal tissues must be considered. In addition, the possible consequences of a relative excess or deficiency in hCG production or possible structural and functional defects in hCG and/or in its hCG receptors during pregnancy should be realized.

We recently demonstrated that immortalized GT1-7 neurons co-express luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptor and gonadotropin releasing hormone (GnRH) genes. Treatment of GT1-7 neurons with LH/hCG resulted in a transcriptional inhibition of GnRH gene. In the present study, we investigated the signaling and transacting factors involved in the action of hCG. Eight-bromo-cyclic AMP can mimic the down-regulating action of hCG on GnRH mRNA levels. H-89, a protein kinase (PK) A inhibitor, but not bisindolylmaleimide, a PKC inhibitor, blocked the down- regulating actions of hCG as well as of 8-bromocyclic AMP. Treatment with the PKA inhibitor alone modestly decreased GnRH mRNA levels suggesting that PKA signaling also controls the basal expression of the GnRH gene. The direct measurement of PK activities revealed that hCG treatment of GT1-7 neurons increased the PKA but not the PKC activity. New protein synthesis is required for the down-regulating action of hCG on GnRH mRNA levels. Since some of the new proteins could be nuclear transcription or transacting factors, we investigated the effects of hCG on cyclic AMP response element binding protein (CREB), c-Fos and c-Jun protein levels. Treatment of GT1-7 neurons with hCG resulted in an increase of 43 kDa phosphorylated CREB, 50 kDa c-Fos and 40 kDa c-Jun proteins compared to the corresponding controls. The kinetics of increases were different and in all cases the increases of the proteins preceded the decrease of GnRH mRNA levels. In summary, PKA signaling and transacting factors such as CREB, Fos and Jun are probably involved in transcriptional inhibition of GnRH gene by hCG in GT1-7 neurons.

Our recent demonstration of receptors for luteinizing hormone (LH)/human chorionic gonadotropin (hCG) in rat brain with the highest density within the hippocampus and dentate gyrus suggests novel functional roles for gonadotropic hormones within the brain. The present study investigated whether 125I-hCG can cross the blood-brain barrier and reach hippocampus and the possible role of hCG in the regulation of several behavioral activities associated with the hippocampal formation in the rat. About 1/100th of peripherally injected 125I-hCG crossed the blood-brain barrier in an intact form and was found in cerebrospinal fluid and in hippocampus. Intraperitoneal (IP) or intracerebroventricular (ICV) injections of highly purified hCG on the morning of proestrus of cycling female rats resulted in changes in several hippocampus-associated behaviors. hCG-treated animals were generally less active and showed less exploratory behavior as compared to saline-injected control animals. There was no difference, however, in latency to enter the open field between hCG-treated and control animals. Taste neophobia was dramatically decreased following IP as well as ICV injection of hCG. No differences were found in the memory component of T-maze performance; however, the hCG-treated rats exhibited decreased stereotypic behavior. In summary, hCG can cross the blood-brain barrier, and peripheral or central administration of hCG affects several hippocampus-associated behaviors suggesting that hippocampal LH/hCG receptors are most likely involved in mediating these effects. Some of the observed behavioral changes have parallels in pregnant women.

Our laboratory previously demonstrated that the human myometrium contains functional hCG/LH receptors. The present study investigated whether hCG can directly regulate oxytocin-stimulated human myometrial contractions. Uterine specimens were obtained from 30- to 40-yr-old women undergoing hysterectomy for leiomyomata, metrorrhagia, or prolapse. Myometrial strips from the lower uterine segment were primed for 24 h with 2.2 nmol/L estradiol. Then, the slices were incubated for 4 h at 37 C with or without 10 nmol/L hCG and stimulated with 1 mumol/L oxytocin, and the contractions were measured. The results showed that hCG inhibited the amplitude while paradoxically increasing the frequency of contractions. The effect of hCG was seen in proliferative, but not secretory, phase myometrial specimens. hCG had no effect on rat hepatic portal vein smooth muscle contractions, suggesting that the hCG action was tissue specific. Oxytocin treatment of human myometrial smooth muscle cells resulted in a dose-dependent increase in intracellular free Ca2+ levels. Pretreatment with hCG resulted in an attenuation of the oxytocin response, suggesting that the action of hCG was mediated by decreasing intracellular free Ca2+ levels. In summary, our results demonstrate that hCG can directly inhibit the amplitude of oxytocin-stimulated contractions of human myometria from the proliferative phase of the cycle. The hCG action is tissue specific and appears to be mediated by decreasing intracellular free Ca2+ levels in myometrial smooth muscle cells.

Human myometrium contains hCG/LH receptors. There are fewer of these receptors during labor compared to no labor at preterm or term deliveries. Exogenous hCG can directly inhibit oxytocin-stimulated human myometrial contractions. These findings suggest that hCG may directly maintain myometrial quiescence during pregnancy. As maintenance of uterine quiescence may involve down-regulation of myometrial gap junctions, we investigated the effect of hCG on connexin-43 (CX-43) gene expression from RNA to protein and morphological gap junctions. The addition of 5 or 10 nM highly purified hCG to subconfluent cultures of pregnant myometrial smooth muscle cells resulted in a significant decrease in CX-43 protein levels. Higher hCG concentrations (100 and 1000 nM), however, had no effect. The maximal effect of hCG was seen at 4-8 h of culture, followed by recovery after a longer duration of culture. hCG treatment also concomitantly decreased CX-43 messenger RNA and morphological gap junctions. The hCG effect on CX-43 protein levels is hormone specific and mediated by protein kinase-A signaling. Estradiol and oxytocin increased, whereas progesterone decreased, CX-43 protein levels and morphological gap junctions. The oxytocin-induced increase was reversed by cotreatment with hCG. Although RU 486 alone had no effect on CX-43 protein levels, it prevented the down-regulating action of hCG and progesterone. In summary, our results demonstrate that hCG can directly decrease CX-43 messenger RNA, protein, and morphological gap junctions in cultured pregnant human myometrial smooth muscle cells. The hCG action is concentration and time dependent, hormone specific, and mediated by protein kinase-A signaling and appears to involve progesterone receptors. These data lend support to the concept that hCG could be one of the hormones responsible for maintaining uterine quiescence by down-regulating myometrial gap junctions during pregnancy.

Epidermal growth factor (EGF) stimulates the secretion of hCG in choriocarcinoma cells. However, the molecular mechanisms involved in this EGF action have never previously been investigated. The present study investigated them as well as EGF regulation of the hCG/LH (LH) receptor gene in JEG-3 human choriocarcinoma cells. The JEG-3 cells contain multiple EGF receptor messenger RNA (mRNA) transcripts and a single 170-kilodalton immunoreactive receptor protein. The human EGF can bind to the receptor protein and stimulate the receptor autophosphorylation as well as the phosphorylation of four other membrane proteins. Culturing JEG-3 cells with recombinant human EGF resulted in a dose- and time-dependent increase in hCG secretion. The maximal effect was seen at 100 ng/ml EGF, with a time lag of about 5 h. Tyrosine kinase, but not protein kinase-C or protein kinase-A, signaling was involved in the EGF action to increase hCG secretion. The EGF-induced increase in hCG secretion was not due to an increase in cell number or differentiation into multinuclear syncytia. EGF treatment resulted in a dose- and time-dependent increase in steady state levels of hCG alpha and hCG beta mRNAs. This increase was due to the stabilization of subunit mRNA transcripts. The increase in subunit mRNAs preceded the increase in hCG secretion. The EGF treatment resulted in a dose- and time-dependent decrease in steady state levels of the hCG/LH receptor mRNA transcripts. The decrease was due to a transcriptional inhibition of receptor gene. EGF treatment paradoxically stabilized hCG/LH receptor protein. In summary, EGF treatment up-regulates hCG subunits gene expression and down-regulates hCG/LH receptor mRNAs involving transcriptional and posttranscriptional mechanisms in JEG-3 human choriocarcinoma cells.

We investigated the possible effects of human chorionic gonadotropin (hCG) on sleep-wake phases and other associated behaviors controlled by the medial preoptic area, cerebral cortex and hippocampus. Chronic epidural electroencephalographic (EEG) and temporal muscle electromyographic (EMG) electrodes were placed in cycling female rats. After a week of recovery, rats were injected intraperitoneally at 3.00 pm on the day of proestrus with either saline or highly purified hCG or indomethacin or hCG plus indomethacin. Three hours after injection, EEG, EMG and behavioral activities were recorded for 3.5 h. The administration of hCG increased high and low amplitude sleep, resting phase and decreased active awake phase, walking, sniffing and chewing as compared to the controls. While the administration of indomethacin alone had no effect, coadministration inhibited hCG effects. Medial preoptic area, cerebral cortex and hippocampus contain immunostaining for LH/hCG receptors. The administration of hCG resulted in an increase of immunoreactive PGD2 and a decrease of PGE2 in median preoptic area, cerebral cortex and hippocampus as compared to the controls. In summary, hCG administration affects sleep-wake phases and other associated behaviors in rats which can collectively be described as decreased activity. These effects are probably mediated by increasing PGD2 and decreasing PGE2 in areas of brain which control these activities. The above findings may be relevant to pregnant women who experience decreased activity when hCG is present in the circulation and cerebrospinal fluid.

We recently demonstrated that rat preoptic area and anterior hypothalamus, sites of GnRH neurons, contain receptors for LH/hCG. We investigated in the present study whether LH/hCG receptor and GnRH genes are coexpressed in the same neurons and whether LH/hCG can directly regulate GnRH gene expression in immortalized hypothalamic GT1-7 neurons. The immunostaining for both LH/hCG receptors and GnRH are present in the same neurons in rat preoptic area and the GT1-7 neurons. The reverse transcription-nested polymerase chain reaction generated an expected 255-basepair LH/hCG receptor fragment in GT1-7 neurons. Northern blotting showed the presence of a major 1.8-kilobase and minor 2.6- and 4.3-kilobase receptor transcripts. Immunoblotting detected an 80-kilodalton receptor protein. Covalent receptor cross-linking studies showed that [125I]hCG binds to an 80-kilodalton protein with a specificity expected of LH/hCG receptors. Scatchard plot analysis demonstrated that GT1-7 neurons contain a single class of high affinity (Kd = 3.8 x 10(-11) M) and low capacity (5000 sites/neuron) LH/hCG receptors. Culturing GT1-7 neurons with highly purified hCG resulted in a dose- and time-dependent decrease in steady state GnRH, but not glyceraldehyde-3-phosphate dehydrogenase, messenger RNA (mRNA) levels. Human and rat LH, but not hCG alpha or -beta, FSH, or TSH, mimicked the down-regulating action of hCG on GnRH mRNA levels. Pretreatment of GT1-7 neurons with LH/hCG receptor antisense, but not sense, phosphorothioate oligodeoxynucleotides for 48 h resulted in decreases in [125I]hCG binding and the GnRH mRNA response to exogenous hCG. The half-life of GnRH mRNA transcripts, as determined by blocking transcription by actinomycin-D, was 32.5 +/- 2.5 h. This half-life was virtually unchanged by treatment with 100 ng/ml hCG (30.5 +/- 3.5 h). Treatment of GT1-7 neurons with 100 ng/ml hCG resulted in a dramatic decrease in nuclear run-on transcription of GnRH, but not beta-actin, gene compared to that in the controls. The same hCG concentrations and time points that decreased steady state GnRH mRNA levels also decreased cellular GnRH protein levels. Paradoxically, hCG stimulated the secretion of preexisting GnRH until the levels were depleted. In summary, GnRH neurons in the rat preoptic area and GT1-7 neurons coexpress LH/hCG receptor gene. Treatment of GT1-7 neurons with LH/hCG results in a decrease in steady state GnRH mRNA levels. This decrease is dose and time dependent and hormone specific, and requires the presence of cellular LH/hCG receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Human myometrium contains receptors for hCG/human LH (hLH). This suggested the possibility that hCG and hLH might regulate human myometrium, which has not previously been considered a direct target of gonadotropin regulation. To investigate such a possibility, highly pure and viable smooth muscle cells were isolated from nonpregnant human myometrium and cultured as monolayers. The cells contained hCG/LH receptor mRNA transcripts and a 50-kDa immunoreactive protein that can bind 125I-hCG in a ligand-specific manner. The presence of hCG during culture resulted in a significant increase of myometrial smooth muscle cell density. The hCG effect was time- and concentration-dependent and was mimicked by hLH but not by human FSH or human FSH or human thyroid-stimulating hormone. Human CG also greatly increased the size of a subpopulation of myometrial smooth muscle cells without affecting their chromosomal ploidy. Antibodies to hCG/LH receptors and hCG blocked hCG effects. Human prolactin and growth hormone, which do not bind to hCG/LH receptors, also increased the myometrial smooth muscle cell density. A protein kinase A inhibitor (H-89) blocked hCG response whereas calphostin (a protein kinase C inhibitor) and lavendustin A (a tyrosine kinase inhibitor) had no effect on hCG response, suggesting that a cAMP/protein kinase A signaling mechanism is involved in hCG action. Eicosanoids from cyclooxygenase and 5-lipoxygenase pathways of arachidonic acid metabolism are probably not involved, because the inhibitors of these enzymes had no effect on hCG response. While progesterone and estradiol could not mimic or modify hCG action, epidermal growth factor did mimic hCG in increasing myometrial smooth muscle cell density.(ABSTRACT TRUNCATED AT 250 WORDS)

Human umbilical cord contains two arteries and a vein surrounded by Wharton's jelly with amnion covering the exterior surface. The cord blood and amniotic fluid contain human CG (hCG). Whether hCG can directly regulate cord functions is unknown. We now report that human umbilical cords contain a major 4.4-kilobase and minor 2.6- and 1.8-kilobase hCG/LH receptor messenger RNA transcripts. The cords also contain a 50-kilodalton immunoreactive receptor protein which can bind hCG and LH, but not hFSH or hTSH. Rat testis used as a positive tissue control contained the same major and minor receptor transcripts and an 80-kilodalton receptor protein which can bind [125I]hCG. Rat liver used as a negative control contained neither receptor transcripts nor receptor protein. The smooth muscle and endothelial cells of umbilical arteries and vein, umbilical amnion, and cells in Wharton's jelly contain the receptor transcripts and receptor protein which can bind [125I]hCG. The receptor expression was higher in umbilical vessels closer to the baby and decreased toward placenta, becoming barely detectable once the vessels were inside the placental tissue. In vitro treatment of umbilical cords with highly purified hCG resulted in an increase of immunoreactive cyclooxygenase-1, cyclooxygenase-2, prostacyclin synthase, and 6-keto-prostaglandin F1 alpha, little change in thromboxane A2 synthase and a decrease of prostaglandin E2 and thromboxane B2 as compared to the controls, indicating that the cord receptors are functional. In summary, these novel findings suggest that hCG present in cord blood and amniotic fluid may directly regulate the vascular tone and quite possibly other functions of human umbilical cord.

Term pregnancy human placenta contains hCG/LH receptor mRNA transcripts and immunoreactive receptor protein. Both the receptor transcripts and receptor proteins are present only in trophoblasts. These findings led us to investigate whether hCG can regulate its own synthesis in term pregnancy human placenta. Treatment of placental tissue in static cultures or in a dynamic superfusion system with increasing concentrations of highly purified hCG provoked a biphasic effect on the steady state hCG subunit mRNA levels. Although low concentrations of hCG (< 200 mIU/ml) were not effective, moderate concentrations (200-1000 mIU/ml) increased, and high concentrations (> or = 5000 mIU/ml) either had no effect or actually decreased mRNA levels relative to the control values. This response was specific, because none of the hCG concentrations tested had any effect on glyceraldehyde-3-phosphate dehydrogenase or beta-actin mRNA levels. The effects of hCG on steady state hCG subunit mRNA levels were paralleled by corresponding changes in tissue hCG protein levels. Endogenous hCG appears to down-regulate alpha-subunit mRNA levels and hCG secretion. The hCG effect is probably receptor mediated, because a receptor antagonist, deglycosylated hCG, partially antagonized the hCG action. Treatment with exogenous hCG also down-regulated its own receptor mRNA and receptor protein levels. hCG regulation of its alpha-subunit and receptor levels involved both transcriptional as well as posttranscriptional mechanisms. In summary, this is the first demonstration of hCG regulating its own synthesis in term pregnancy human placenta. The findings of this study could offer a potential molecular explanation for the profile of hCG levels in normal pregnant women.

The human uterus, including its blood vessels, contains hCG/human LH receptors. We now demonstrate that human fallopian tubes also contain a 4.4-kilobase hCG/LH receptor mRNA transcript and an 80-kilodalton immunoreactive protein that can bind [125I]hCG. Tubal mucosa contain more receptor transcripts, receptor protein, and [125I] hCG binding than the tubal smooth muscle or blood vessels. Human fallopian tubes also contain hCG protein and a 0.6-kilobase hCG alpha mRNA transcript. However, very little hCG is found in tubal cell layers other than mucosa. Ampullary segments contain more hCG/LH receptors and hCG than isthmus. Secretory phase tubes contain more than proliferative phase, postpartum, or postmenopause tubes. Incubation with highly purified hCG resulted in an increase in catalytically active 5-lipoxygenase, cyclooxygenase-1, and cyclooxygenase-2 enzymes in tubal tissues. In summary, human fallopian tubes, which have never previously been considered a direct target of hCG/LH action, express functional hCG/LH receptor gene as well as the gene of its ligand. These novel findings suggest numerous possibilities of both physiological and pathological importance in human fallopian tubes.

Our laboratory previously demonstrated that cytotrophoblasts and syncytiotrophoblasts in human placental tissue contain hCG/LH receptors. From this finding, we postulated that one role of hCG might be to promote the differentiation of cytotrophoblasts into syncytiotrophoblasts. To test this postulate, cytotrophoblasts were isolated from human term pregnancy placentas and cultured with and without increasing concentrations of highly purified hCG. The results showed that hCG had a biphasic effect on 1) the aggregation of cells without intervening plasma membranes; 2) the expression of cadherin, a cell adhesion receptor that facilitates cellular aggregation; 3) the expression of hCG/LH receptor gene; and 4) the expression of three different hormonal markers of differentiation. The hCG effects were time and dose dependent and hormone specific. The addition of excess polyclonal hCG antibody, but not normal rabbit serum or nonspecific antirabbit immunoglobulin G, decreased basal responses as well as those to exogenous hCG. The polyclonal hCG/LH receptor antibody increased differentiation and dramatically stimulated hCG secretion in the presence or absence of exogenous hCG. (Bu)2cAMP mimicked the actions of hCG. H-89, a protein kinase-A inhibitor, decreased basal as well as exogenous hCG responses. Calphostin, a protein kinase-C inhibitor and lavendustin, a tyrosine kinase inhibitor, on the other hand, had no effect. In summary, it is novel that hCG made in human placenta can regulate the differentiation of cytotrophoblasts, which make little hCG, into syncytiotrophoblasts, which make considerable amounts of hCG.