Ch.V. Rao

Peripheral and intracerebroventricular administration of human chorionic gonadotropin alters several hippocampus-associated behaviors in cycling female rats.

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.

Treatment of human endometrial stromal cells with chorionic gonadotropin promotes their morphological and functional differentiation into decidua

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.

Multiple novel roles of luteinizing hormone

LH is a heterodimeric glycoprotein hormone released from the anterior pituitary gland in response to GnRH stimulation from the hypothalamus. It is structurally and functionally related to hCG, which is primarily secreted by syncytiotrophoblasts in the human placenta. Both hormones signal through the same receptor, which is a transmembrane glycoprotein that belongs to the G-protein coupled receptor superfamily. Because hCG has a longer circulatory half-life, binds to the receptor with a higher affinity, is readily available, and is easy to radioiodinate, it is preferred for clinical and research use.

Consequences of targeted inactivation of LH receptors

The inactivation of luteinizing hormone (LH) receptors was neither lethal nor it had any effect on sex differentiation. However, it dramatically reduced the growth and development of gonads and the reproductive tract. As a result, both female and male animals were infertile. Serum LH levels were dramatically elevated, follicle stimulating hormone (FSH) levels moderately elevated in both sexes, estradiol and progesterone levels partially decreased in females, testosterone levels dramatically decreased and estradiol levels moderately increased in males. The knockout of LH receptors had no effect on gonadal FSH receptors in both sexes, progesterone receptors in females and androgen receptors in males. However, estrogen receptor ERalpha and steroidogenic acute regulatory protein decreased and ERbeta increased in both sexes. cDNA expression array analyses revealed that testes were affected more than ovaries and more genes showed an increase rather than a decrease in testes. The affected genes came from many unexpected families. Both null females and males had a decreased density of femur and became obese with age. The ovarian failure in knockout animals could not be reversed by estradiol/progesterone replacement therapy or by PMSG and human chorionic gonadotropin (hCG) injections. Although, testosterone replacement therapy of 30-60-day old null males partially improved spermatogenesis, the animals still remained infertile. A single testosterone injection on postnatal day 1 followed by 21-45-day testosterone replacement therapy beginning at 30 days of age, however, restored fertility. Studies showed that uterus of null animals could not initiate pregnancy even though the size and morphology were greatly improved by estradiol and progesterone replacement therapy. In general, non-gonadal phenotypes in null females and males were not completely reversed by hormone replacement therapy, suggesting that LH signaling could be important for their function. Heterozygous animals were indistinguishable from wild-type animals at 60 days of age. However, as they grew to about 1 year of age, they began to stop cycling, some became extremely obese, showed a decreased density of femur and all animals developed endometrial tumors with a cancer histology. LH receptor-knockout animals will be useful in advancing our present understanding on the importance of classical as well as non-classical actions of LH in the body, in advancing novel therapeutic uses of hCG, and in better understanding and rationalizing the consequences of inactivating type human LH receptor mutations.

The past, present and future of nongonadal LH/hCG actions in reproductive biology and medicine

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.

Messenger ribonucleic acid expression of LH/hCG receptor gene in human ovarian carcinomas

The mRNA expression of luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptors was analysed by the RT-nested PCR method in five normal ovarian tissues, 62 ovarian tumours (5 benign, 7 borderline and 43 malignant epithelial tumours, 3 sex cord-stromal tumours and 4 germ cell tumours) and in 2 ovarian cancer cell lines. In normal ovaries, two cDNA fragments of different sizes were detected using primers designed to amplify a region including exon 9. Sequencing revealed that the larger fragment was derived from a full-length receptor, while the smaller fragment was a splice variant lacking exon 9. In ovarian tumours, the larger fragment of LH/hCG receptors was detected in 40% of the epithelial ovarian carcinomas, none of the germ cell tumours, all of the sex cord-stromal tumours and one of the 2 ovarian cancer cell lines. Immunohistochemistry confirmed the localisation of LH/hCG receptor protein in the tumour cells which correlated with mRNA expression. Patients with full-length LH/hCG receptors in carcinomas showed a better prognosis compared with those without the receptors.

Lymphocytes from pregnant women express human chorionic gonadotropin/luteinizing hormone receptor gene

Previous studies have indicated that human chorionic gonadotropin (hCG) has immunoregulatory properties and alters the functions of human lymphocytes. However, it has not been determined whether the gene encoding the receptors for hCG/luteinizing hormone (LH) is expressed in human lymphocytes. Total peripheral mononuclear lymphocytes isolated from blood samples of pregnant women by Ficoll-Paque gradient centrifugation contained mRNA transcripts encoding the hCG/LH receptors and a 50 kDa receptor protein which can bind 125I-hCG. T-Lymphocytes isolated from total mononuclear cell fractions also contained these receptor mRNA transcripts as well as the receptor protein. The levels of receptor transcripts and receptor protein are lower in lymphocytes than in ovarian tissue. These findings suggest that the immunoregulatory actions of hCG are probably mediated by specific receptors in T-lymphocytes from pregnant women.

Binding of 125I-epidermal growth factor in human uterus

SummaryQuantitative light-microscopic autoradiography was used on five human uteri at two different phases of the menstrual cycle to ascertain the cell types with binding sites for epidermal growth factor (EGF). The results revealed that stromal cells, glandular epithelium of endometrium, elongated and circular muscle cells of myometrium, smooth muscle and endothelial cells of arterioles in the basal endometrium and myometrium contained numerous silver grains following incubation with 125I-EGF. Coincubation with 100-fold excess unlabeled EGF resulted in a complete disappearance of silver grains from all cell types. Quantitative grain analysis indicated that stromal cells contained the highest number of EGF-binding sites (P<0.05) with no significant differences among the others (P>0.05). There was no cyclic variation of EGF-binding to any of the uterine cell types. The present data demonstrate that all the cell types of human uterus, including arterioles, contain EGF-binding sites. This suggests that all the cells in human uterus subserving different functions are targets of EGF action.

Receptors for prostaglandins and gonadotropins in the cell membranes of bovine corpus luteum

Abstract The cell membranes exhibited specific binding to 3 H-prostaglandin E 1 ( 3 H-PGE 1 ) and 125 I-human chorionic gonadotropin ( 125 I-HCG). Unlabeled PGE 1 ,PGE 2 (1.4 × 10 −7 M), PGF 1α and PGF 2α (1.4 × 10 −5 M) decreased 3 H-PGE 1 binding by more than 80%. The binding of 125 I-HCG was completely inhibited by 5 × 10 −8 M unlabeled HCG. However, the unlabeled PGE 1 (1.15 × 10 −6 M) and HCG (8.4 × 10 −7 M) had no effect on 125 I-HCG and 3 H-PGE 1 binding respectively. A PG antagonist, 7-oxa-13-prostynoic acid, inhibited only 3 H-PGE 1 binding but not 125 I-HCG binding. These results suggest the presence of specific receptors for PGE 1 and HCG in the cell membranes and that the binding occurs either at two different sites on the same receptor or that each binds to a “different” receptor molecule.

Functional Luteinizing Hormone/Chorionic Gonadotropin Receptors in Human Adrenal Cortical H295R Cells

Abstract Previous studies have suggested that activation of normal human adrenal and adrenal tumor luteinizing hormone (LH)/chorionic gonadotropin (hCG) receptors results in an increased secretion of steroid hormones. Since it is not feasible to test this suggestion on normal human adrenal cells, we used human adrenal cortical carcinoma H295R cells, which are similar in some respects to normal adrenal cortical cells. These cells contained LH/hCG receptor transcripts and receptor protein that can bind 125I-hCG in a hormone-specific manner. Culturing the cells with highly purified hCG resulted in a time- and dose-dependent significant increase in dehydroepiandrosterone sulfate (DHEAS) secretion as compared with the controls. The DHEAS response was hormone as well as steroid specific. Since hCG treatment did not increase DHEA secretion, we suspected that the hCG might increase DHEA sulfotransferase (ST). Consistent with this possibility, hCG treatment increased steady-state DHEA-ST mRNA levels. The hCG effects require its receptors, as inhibition of their synthesis by treatment with antisense phosphorothioate oligodeoxynucleotides (ODN) made from the LH/hCG receptor sequence resulted in loss of DHEA-ST and DHEAS responses. The findings that 1) hCG treatment increased cAMP levels and activated protein kinase A (PKA), 2) 8-bromo cAMP mimicked hCG, and 3) blocking PKA activation prevented hCG as well as 8-bromo cAMP from increasing both DHEA-ST mRNA and DHEAS levels suggested that cAMP/PKA signaling was involved in the hCG actions. In conclusion, H295R cells contain LH/hCG receptors, which are coupled to increasing DHEAS secretion through upregulating the ST enzyme mRNA level. This action is mediated by the cAMP/PKA signaling pathway. These findings support the concept that adrenal function in normal and pathological conditions could be influenced by LH and hCG.