A. Jagannadha Rao

Effect of deprival of LH on Leydig cell proliferation: involvement of PCNA, cyclin D3 and IGF-1.

The levels of proliferating cell nuclear antigen (PCNA) and cyclin D3 which are known markers of cellular proliferation were monitored by immunoblotting in progenitor Leydig cells (PLC), immature Leydig cells (ILC) and adult Leydig cells (ALC) isolated from 21, 35 and 90 day old rats, respectively which represent the Leydig cells at different stages of development. The levels of PCNA and cyclin D3 were highest in PLC, intermediate in ILC and lowest in ALC. Following administration of an antiserum to LH to deprive endogenous LH in 21 day old rats, a significant decrease in the levels of PCNA and Cyclin D3 were observed suggesting the involvement of Lutenizing hormone (LH) in PLC proliferation. In support of this observation, Bromodeoxyuridine (BrdU) incorporation was highest in PLC when compared with ILC and ALC, and administration of LH antiserum to 21 day old rats led to a total absence of BrdU incorporation by the isolated PLC. Also, there was a decrease in the level of IGF-1 and IGF-1 receptor mRNA levels by 55 and 35%, respectively as assessed by semi-quantitative RT-PCR. In addition, the PLC isolated from rats deprived of endogenous LH incorporated much less BrdU following addition of IGF-1. These results, which are obtained using an in vivo model system establish that LH has a very important role in Leydig cell proliferation in immature rats.

Induction of infertility in adult male bonnet monkeys by immunization with phage-expressed peptides of the extracellular domain of FSH receptor.

Active immunization of proven fertile adult male bonnet monkeys (Macaca radiata) with phage-expressed follicle-stimulating hormone receptor (FSHR)-specific peptides from the extracellular domain resulted in a progressive drop in sperm count with all animals becoming azoospermic by day 100. However, serum testosterone concentrations were unaltered during the entire course of study and animals exhibited normal mating behaviour. Breeding studies with proven fertile female monkeys revealed that all the immunized males were infertile. Following interruption of immunization on day 225, sperm counts returned to normal with restoration of fertility. These results indicate that infertility can be induced in adult male monkeys by interfering with the action of FSH using specific peptides of the extracellular domain of FSHR as antigens, without the risk of producing cross-reacting antibodies to the other glycoprotein hormones.

Regulation of FSH receptor, PKIβ, IL-6 and calcium mobilization: Possible mediators of differential action of FSH

Sertoli cells support the development of germ cells by providing a microenvironment in the seminiferous tubules. FSH stimulates Sertoli cell proliferation only during neonatal period till day 18 in the immature rat whereas FSH regulates only functional parameters in the adult rat Sertoli cells. This suggests that FSH exerts differential action in immature and adult Sertoli cells. In an attempt to elucidate the mechanism by which FSH exerts the differential effects, we have carried out both in vivo and in vitro studies using Sertoli cells isolated from immature (7-10 days old) and adult (90 days old) rats. The differential role of FSH was studied at the receptor as well as at the signaling level. Monitoring the level of expression of FSH receptor by RTPCR and northern blot analysis revealed that the expression was more in immature Sertoli cells. Furthermore, it was found that FSH up (1.8-fold) regulates its receptor level only in the immature Sertoli cells and not in the adult. Results also revealed that PKIbeta and calcium, which are the downstream signaling molecules, are involved in FSH regulated Sertoli cells proliferation. It was also observed that FSH up (1.4-fold) regulates the levels of expression of IL-6 mRNA only in the immature rat Sertoli cells suggesting the possibility of its involvement in FSH regulated Sertoli cell proliferation.

Hormonal regulation of leydig cell proliferation and differentiation in rodent testis : a dynamic interplay between gonadotrophins and testicular factors

Studies over the last few decades have documented that LH is the principal regulator of Leydig cell function. Recent studies indicate that locally produced intratesticular factors are equally important in modulating Leydig cell development and function. In the present review, results of studies on Leydig development and function with rodent models, in conjunction with recent advances in our understanding, are discussed. Studies on Leydig cell development revealed that there are two different waves of proliferation: the first one is independent of LH and the other is dependent on LH. In addition to LH, FSH plays a major role in Leydig cell development and function by modulating the production of Sertoli cell-derived factors. Studies directed towards understanding the oestrogen-mediated inhibition of Leydig cell proliferation revealed that collagen IV-mediated signalling is involved in Leydig cell proliferation and 17beta-oestradiol inhibits this event. Leydig cell proliferation and differentiation is associated with changes in gene expression. Research in this area has identified several genes that are involved in Leydig cell proliferation and differentiation; the possible role of these genes in the context of Leydig cell development are discussed in this review.

Role of estrogen in regulation of cellular differentiation: a study using human placental and rat Leydig cells.

Estrogen classically is recognized as a growth-promoting hormone. Recent evidence suggests that estrogens are also involved in a wide variety of cellular and physiological functions involving the central nervous system, immune system, cardiovascular system and bone homeostasis. Our studies in cytotrophoblasts and BeWo cells, demonstrated that 17beta-estradiol induces terminal differentiation of placental trophoblasts directly and this differentiation is coupled with an increased production of TGFbeta1, which, in turn, affects telomerase activity and telomerase associated components at the level of hTERT. Furthermore, using rats treated in vivo with either EDS or estradiol and in vitro Leydig cell cultures, we proposed that 17beta-estradiol mediated down-regulation of collagen IV alpha4 expression could be one of the possible mechanisms for the inhibition of progenitor Leydig cell proliferation. In this review, we summarize the results from both the model systems, the human placental cytotrophoblast and rat Leydig cells to conclude that 17beta-estradiol has a unique stage-specific role in differentiation.

Regulation of growth and function of the human placenta

The human placenta is a tumor-like tissue in which highly proliferative, migratory, and invasive extra-villous trophoblast cells, migrate and invade the uterus and its vasculature, to provide a vital link between the mother and the developing fetus. In the present article, we review our studies on a series of experiments, designed to identify molecular events responsible for the phenotypic changes during placental growth. Our observations illustrate that the human placenta is endowed with the biochemical machinery to proliferate indefinitely throughout gestation, yet, there are intrinsic mechanisms that effectively circumscribe the extent and duration of trophoblast proliferation. The placenta combines in itself the unique ability to produce a wide variety of protein, peptide and steroid hormones, but intricately interwoven in this process, is also the remarkable capacity to simultaneously regulate their synthesis and secretion. The placenta therefore represents an autonomous or a self-sufficient unit capable of modulating its own growth and function, while assisting the developing fetus until it is capable of independent existence.

Regulation of progresterone biosynthesis in the human placenta by estradiol 17β and progesterone

Ex vivo addition of estradiol 17β to first trimester or term human placental minces caused a significant increase in the quantity of progesterone produced. Addition of an aromatase inhibitor, CGS 16949 A, or the estrogen receptor antagonist, ICI 182780, significantly inhibited progesterone production confirming the role of estradiol 17β in the regulation of progesterone synthesis in human placenta. RU 486 and ZK 98299, which are antagonists of progesterone receptor, significantly modulated progesterone synthesis in the human placenta but exhibited paradoxical effects on the first trimester and term placenta. We conclude that progesterone synthesis in the human placenta is regulated by estradiol 17β and progesterone. This is the first report providing evidence for autoregulation of progesterone synthesis in the human placenta.

Regulation of low density lipoprotein receptor mRNA levels by estradiol 17 beta and chorionic gonadotropin in human placenta

Inhibition of synthesis of estradiol 17β by the addition of inhibitors of aromatase, a key enzyme in the biosynthesis of estradiol 17β, or addition of tamoxifen - an estrogen receptor antagonist, to human placental minces resulted in an increase in the level of LDL-receptor mRNA. This increase could be blocked by the simultaneous addition of estradiol 17β. A concentration dependent effect of estradiol 17β on the level of LDL-receptor mRNA was seen both in first trimester, and term placenta. Addition of human chorionic gonadotropin (hCG) to term placental minces also increased the LDL-receptor mRNA levels. When hCG and cycloheximide were added together, an additive effect was observed. The results obtained in this study suggest that the LDL-receptor mRNA levels in the human placenta are regulated by estradiol 17β and hCG.

Proteomic profiling of forskolin-induced differentiated BeWo cells: an in-vitro model of cytotrophoblast differentiation

Placental trophoblastic differentiation is characterized by the fusion of monolayer cytotrophoblasts into syncytiotrophoblasts. During this process of differentiation, several morphological and biochemical changes are known to occur, and this model has been employed to investigate the changes that occur at the gene and protein level during differentiation. Using the sensitive technique of proteomics [two-dimensional gel electrophoresis (2DGE)], changes in protein profile were evaluated in the control and forskolin-induced differentiated cells of trophoblastic choriocarcinoma BeWo cell line. Several proteins were differentially expressed in control and differentiated cells. Four major proteins were up-regulated as assessed by silver staining, and were further characterized as c-h-ras p 21 (phosphorylated), retinoblastoma susceptibility protein 1 and integrase interactor protein 1. These proteins are known to play an important role in growth arrest of cells, and thus may play a role in initiating the process of differentiation.

Evaluation of the role of FSH in regulation of Leydig cell function during different stages of its differentiation

The role of follicle stimulating hormone (FSH) in Leydig cell function was evaluated by a passive neutralization approach at different stages of Leydig cell development. Neutralization of endogenous FSH in neonatal rats (10-day-old) resulted in reduction of testes weight, however the testicular testosterone levels and in vitro testosterone production by purified Leydig cells were elevated. Administration of FSH antiserum to immature (25-28-day-old) and adult (90-day-old) rats did not have any effect on testes weight, serum testosterone and testicular testosterone. Interestingly, there was a significant reduction in testosterone production by isolated Leydig cells under hCG stimulated and 22-R-hydroxycholesterol (22-R-OH CHOL) saturated conditions. In support of this observation administration of recombinant FSH to immature and adult rats resulted in significant increase in testosterone production by Leydig cells following incubations in presence of hCG and saturating concentrations of 22-R-OH CHOL, although there was no change in serum and testicular testosterone levels. The role of FSH in immature rats was also confirmed employing FSH receptor antiserum which was raised against the unique domains of FSH receptor. RT-PCR analysis revealed a significant reduction in the mRNA levels of StAR and IGF-1 following blockade of FSH action by FSH receptor antiserum. The results of our studies suggest a stage specific function for FSH in regulation of Leydig cell development by modulating the LH responsiveness and steroidogenesis.