Age-related changes in gene expression patterns of immature and aged rat primordial follicles.

Women are born with millions of primordial follicles which gradually decrease with increasing age and this irreversible supply of follicles completely exhausts at menopause. The fertility capacity of women diminishes in parallel with aging. The mechanisms for reproductive aging are not fully understood. We have observed a decline in Brca1 mediated DNA repair in aging rat primordial follicles. To further understand the age-related molecular changes, we performed microarray gene expression analysis using total RNA extracted from immature (18 to 20 day old) and aged (400 to 450 day old) rat primordial follicles. The results of current microarray study revealed that there were 1,011 (>1.5 fold, p<0.05) genes differentially expressed between two groups in which 422 genes were up-regulated and 589 genes were down-regulated in aged rat primordial follicles compared to immature primordial follicles. The gene ontology and pathway analysis of differentially expressed genes revealed a critical biological function such as cell cycle, oocyte meiosis, chromosomal stability, transcriptional activity, DNA replication, and DNA repair were affected by age. This considerable difference in gene expression profiles may have an adverse influence on oocyte quality. Our data provide information on the processes that may contribute to aging and age-related decline in fertility.

Comparative proteomic analysis of primordial follicles from ovaries of immature and aged rats.

Age related decline in reproductive performance in women is well documented and apoptosis has been considered as one of the reasons for the decline of primordial follicle reserve. Recently we observed a decline in the efficiency of DNA repair ability in aged rat primordial follicles as demonstrated by decreased mRNA levels of DNA repair genes BRCA1 and H2AX. In the present study, a two-dimensional electrophoresis (2DE) proteomic approach was employed to identify differentially expressed proteins in primordial follicles isolated from ovaries of immature (∼20 days) and aged (∼400-450 days) rats. Using MALDI-TOF/TOF MS, we identified 13 differentially expressed proteins (p < 0.05) which included seven up-regulated and six down-regulated proteins in aged primordial follicles. These proteins are involved in a wide range of biological functions including apoptosis, DNA repair, and the immune system. Interestingly, the differentially expressed proteins such as FIGNL1 (DNA repair) and BOK (apoptotic protein) have not been previously reported in the rat primordial follicles and these proteins can be related to some common features of ovarian aging such as loss of follicle reserve and genome integrity. The quantitative differences of two important proteins BOK and FIGNL1 observed by the proteomic analysis were correlated with the transcript levels, as determined by semi-quantitative RT-PCR. Our results improve the current knowledge about protein factors associated with molecular changes in rat primordial follicles as a function of aging and our understanding of the proteomic processes involved in degenerative changes observed in aging primordial follicles.

Expression of thyroid-stimulating hormone receptor, octamer-binding transcription factor 4, and intracisternal A particle-promoted polypeptide in human breast cancer tissues.

UNLABELLED: Abstract Background: Thyroid-stimulating hormone receptor (TSHR) is one of the members of glycoprotein hormone receptor family; activation of TSHR by thyroid-stimulating hormone (TSH) regulates thyroid function, proliferation, and differentiation. The other family members of glycoprotein hormone receptors, such as leutinizing hormone receptor (LHR), human chorionic gonadotropin (hCG), and follicle-stimulating hormone (FSH) are known to be expressed in nonendocrine tissues including human breast cancer and regulate proliferation and differentiation. The involvement of thyroid hormones in the growth and differentiation of normal breast tissue is well documented. However, the presence of TSHR in breast cancer has not been demonstrated. The aim of the present study was to establish the expression pattern of TSHR along with transcription factors, such as octamer 4 (OCT4) and intracisternal A particle-promoted polypeptide (IPP) in human breast tumor. MATERIALS AND METHODS: For this study, patients with stages I-III breast cancers and adjacent noncancerous tissues were prospectively accrued and analyzed. We employed semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis to determine the expression levels of TSHR in normal and human breast cancer tissues. RESULTS: The results indicated that a significant increase in TSHR expression was observed in tumor tissues compared to normal breast tissues. RT-PCR analysis of OCT4 and IPP also revealed a significant increase in breast tumor tissues over the controls. CONCLUSIONS: To our knowledge, this is the first report demonstrating expression of TSHR and IPP in normal breast and breast tumors. The expression of TSHR, IPP, and OCT4 increased in the human breast tumor samples over the noncancer tissues. However, further studies are needed to establish an unequivocal role for TSHR in breast tumor progression.

Oestrogenic regulation and differential expression of WNT4 in the bonnet monkey and rodent epididymis.

A role for oestrogen in regulating fluid reabsorption in the monkey epididymis was recently demonstrated. Here, these studies are extended to identify potential oestrogen-regulated proteins in the cauda region of monkey epididymis treated with vehicle and oestrogen receptor antagonist (ICI 182780). Two-dimensional electrophoretic analysis was used to identify the proteins. The results indicated down-regulation of WNT4 in the ICI-182780-treated monkey cauda. In addition, the Wnt4 mRNA concentration was also reduced in the caput regions of ICI-182780- treated rats and oestrogen receptor knockout mice. WNT4 is a key regulator of gonadal differentiation in humans and mice and plays a pivotal role in early mouse embryogenesis. The results of the present study establish the presence of WNT4 in the monkey epididymis and its regulation by oestrogen, and suggest a role for WNT4 in maintaining epididymal homeostasis.

Effect of deprivation of endogenous follicle stimulating hormone on rat epididymis: a histological evaluation.

The growth and function of the epididymis are regulated by testosterone produced by Leydig cells in the testes. In the present study it was observed that neutralization of endogenous follicle stimulating hormone (FSH) in immature rats using a highly specific antiserum to ovine FSH resulted in changes in the histology of the epididymis along with a decrease (50-60%) in its weight compared with the normal serum-treated controls. These changes were observed in both rat and monkey epididymis without any decrease in serum testosterone, on which epididymis is known to be dependent. A detailed study was therefore carried out on the effects of deprivation of FSH or testosterone on the histology of epididymis. The changes in epididymal histology following FSH deprivation included a decrease in the size of the tubule lumen in the rat as well as in the adult male bonnet monkey in which the antiserum against ovine FSH was raised. Intensive vacuolization and uneven surface of the luminal epithelium was also observed. In contrast, the effect of deprivation of testosterone support by way of administration of LH antiserum or fiutamide resulted in a decrease in the size of the lumen and degenerative changes. These results suggest that cauda epididymidis is a target for FSH action.

Delineating the role of estrogen in regulating epididymal gene expression.

Maturation of sperm within the epididymis is a pre-requisite for fertilization in mammals. Epididymal function is controlled by a complex array of hormones and growth factors. While testosterone is the primary stimulus for epididymal development and sperm maturation, the importance of estrogen effects on efferent ductules has been increasingly recognized, and points to a need to clarify the role of estrogen receptor-mediated action in the epididymis. Estrogens modulate the expression of genes involved in fluid absorption in the efferent ductules and the epididymis. The present review highlights the role of estrogen in regulation of epididymal gene expression.

Establishment of the need for oestrogen during implantation in non-human primates.

Deprivation of oestrogen during post-ovulatory mated cycles in proven fertile female bonnet monkeys by tamoxifen, aromatase inhibitor or oestrogen antiserum resulted in inhibition of pregnancy establishment in all three groups of animals. However, more than 85% of the animals became pregnant within three exposures to proven fertile males in the control group. These results suggest the requirement for oestrogen in pregnancy establishment in primates. Based on this conclusion, it is suggested that use of a suitable and potent anti-oestrogenic compound can be exploited as an alternative approach to contraception.

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.

Demonstration of follicle-stimulating hormone receptor in cauda epididymis of rat.

FSH receptor has been shown to be specifically expressed only in the Sertoli cells in males. In one of our studies that consisted of deprival of endogenous FSH in immature rats and adult bonnet monkeys, atrophy of the epididymis was observed, cauda region being the most affected. Although epididymis is an androgen-dependent tissue, the changes in histology of the cauda region were observed without any associated change in the levels of testosterone in FSH-deprived animals. Considering this, it was of interest to evaluate the possibility of epididymis being a direct target for FSH action. In the present study, we have examined the expression of FSH receptor in the epididymis of rat and monkey. In the cauda region of rat epididymis, FSH receptor expression was demonstrated by RT-PCR and Northern and Western blot analyses. FSH receptor was found to be functional as observed by its ability to bind 125IoFSH, by an increase in cAMP production, and by BrdU incorporation following addition of FSH under in vitro conditions. These results suggest the possibility of a role for FSH in regulating the growth of the epididymis.

Regulation of FSH receptor, PKIbeta, 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.

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.

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.

Effects of ICI 182780 on estrogen receptor expression, fluid absorption and sperm motility in the epididymis of the bonnet monkey.

BACKGROUND: The importance of estrogen in regulation of fluid absorption and sperm maturation in the rodent epididymis has been established from studies on estrogen receptor-alpha knockout mice. However, functional studies on the role of estrogen in primate epididymis have been few. The main objective of this study was therefore to extend these observations and systematically analyze the presence and function of estrogen receptors in modulating the function of the primate epididymis, using the bonnet monkey (Macaca radiata) as a model system. METHODS: A steroidal estrogen receptor (ER) antagonist, ICI 182780 (ICI), was administered to adult male bonnet monkeys via mini-osmotic pumps for a duration of 30 to 180 days. The expression of key estrogen-regulated genes (ER-alpha, Na-K ATPase alpha-1 and Aquaporin-1) was examined at specific time points. Further, the effect of ICI in modulating fluid reabsorption in efferent ductules was monitored, and critical sperm-maturation parameters were also analyzed. RESULTS: Our studies in the bonnet monkey revealed that both ER-alpha and ER-beta were expressed in all the three regions of the epididymis. We observed an increase in ER-alpha mRNA and protein in the caput of ICI-treated monkeys. Steady state mRNA levels of the water-channel protein, Aquaporin-1, was significantly lower in the caput of ICI-treated monkeys compared to controls, whereas the mRNA levels of Na-K ATPase alpha-1 remained unchanged. In vitro incubation of efferent ductules with ICI resulted in two-fold increase in tubular diameter, indicating affected fluid reabsorption capacity. Furthermore, sperm from ICI-treated monkeys were immotile. CONCLUSION: Taken together, our results point to an integral role for estrogen in modulating the functions of the bonnet monkey epididymis. This study also demonstrates possible differences in the epididymal physiology of rodents and non-human primates, and thus underscores the significance of reports such as these, that examine the physiology of non-human primates (as opposed to rodents), in an attempt to understand similar events in the human.

Collagen IV-mediated signalling is involved in progenitor Leydig cell proliferation.

In rats, during postnatal Leydig cell development, the progenitor Leydig cells (PLC) proliferate actively during days 14-21 of postnatal life. Luteinizing hormone (LH) is known to stimulate Leydig cell proliferation and oestradiol 17beta inhibits this process. In order to identify the molecules involved in Leydig cell proliferation, differentially expressed genes in proliferating and non-proliferating PLC isolated from vehicle and oestradiol 17beta-treated rats respectively, were analysed by differential display reverse transcription polymerase chain reaction (DD-RT-PCR). Results revealed that the expression of collagen IV alpha4 (Col IV alpha4), a subunit of extracellular matrix (ECM) protein collagen IV, was down regulated in PLC isolated from oestradiol 17beta-treated rats. Studies on stage specific expression of Col IV alpha4 during Leydig cell development revealed that this transcript is abundantly expressed at the stage where Leydig cell proliferation is maximal and the expression of this transcript decreased during differentiation of Leydig cells, which is associated with loss of proliferation. These observations suggest that Col IV alpha4 is important for PLC proliferation. Stimulation of PLC proliferation in vitro in the presence collagen IV provides additional support for the conclusion that collagen IV-mediated signalling is involved in PLC proliferation. Further studies revealed that active forms of focal adhesion kinase (FAK) and mitogen activated protein kinase 1/2 (MAPK 1/2), the intracellular signalling molecules that are known to mediate ECM protein signalling are present only in proliferating forms of Leydig cells and are absent in non-proliferating Leydig cells. These results suggest that collagen IV-mediated signalling is involved in PLC proliferation.

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.

Trophoblast 'pseudo-tumorigenesis': significance and contributory factors.

Trophoblast cells of the human placenta proliferate, migrate, and invade the pregnant uterus and its vasculature in order to nourish the developing fetus, in a way that is imitated by malignant tumors. Many similarities exist between embryo implantation and the growth of cancer cells. We begin this article by reviewing decades of studies that have helped unearth the mechanisms that contribute to the tumor-like phenotype of human trophoblast cells. Interestingly, these attributes are only transient in nature, with stringent spatial and temporal confines. The importance of intrinsic molecular controls that effectively circumscribe the extent and duration of trophoblast incursion, becomes increasingly evident in abnormal pregnancies that are characterized by aberrant trophoblast proliferation/invasion. We summarize and discuss the significance of abnormalities in these regulatory mechanisms, and finally, speculate about the use of human trophoblastic cells as model systems for the study of a variety of cellular processes. While on one hand, human placental cells are bestowed with a capacity to proliferate indefinitely and invade extensively, on the other, these cells are also replete with mechanisms to regulate these tumor-like attributes and eventually progress to a senescent apoptotic state. This is therefore, a 'well-behaved' tumor. The comparison in the present review is between the invasive cytotrophoblastic cell type and the tumor cell type.

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.

Changes in T-plastin expression with human trophoblast differentiation.

During the first trimester of pregnancy, the human placenta is an actively dividing and highly invasive tumour-like tissue, while near term, it represents a fully developed, non-invasive unit. In order to understand the molecular basis of this marked difference in the placental phenotypes, an approach based on a differential display-reverse transcription-polymerase chain reaction (DD-RT-PCR) was adopted to analyse changes in gene expression, using total RNA isolated from first-trimester and term placental villi. Using this approach, T-plastin was initially identified as being differentially expressed in the human first-trimester placenta. T-plastin is an actin-bundling protein and is known to be highly expressed in actively dividing cells and up-regulated in several carcinomas. Using a homogenous population of cytotrophoblasts and syncytiotrophoblasts isolated from human placentae, the present authors demonstrate the differential expression of T-plastin in cytotrophoblasts compared with the terminally differentiated syncytiotrophoblasts. The down-regulation of T-plastin expression is further demonstrated in human trophoblastic BeWo cells induced to differentiate using transforming growth factor (TGF)beta1, a growth factor known for its anti-proliferative and anti-invasive response in placental cells. These studies suggest that expression of T-plastin in the placental context may indeed be associated with the enhanced replicative potential of placental trophoblasts.

Effect of active and passive immunization of male and female rats with a recombinantly expressed bonnet monkey pituitary GnRH receptor fragment.

Gonadotropin releasing hormone (GnRH) exerts its action by binding to the specific receptor which belongs to the family of G-protein coupled receptors that are characterized by the presence of seven transmembrane domains linked together by extracellular and intracellular loops. A fragment of the pituitary receptor of the bonnet monkey (Macaca radiata) corresponding to amino acids 164-266 was cloned and expressed in Escherichia coli. This was used to raise antibodies to the receptor in rabbits. Active and passive immunization studies in both male and female rats were carried out using, both the 'overexpressed' fragment, as well as antibodies raised to the receptor fragment. Both active, as well as passive immunization in the male rats brought about an agonistic effect in terms of increase in serum LH level, as well as increase in serum and testicular testosterone levels. However, in the female rats, active immunization with the receptor fragment did not have any effect on the gonadal steroid levels but had a selective effect on the uterine morphology.