Identification of intracisternal A-particle like element as an FSH regulated transcript in immature rat sertoli cells

Sertoli cells under the influence of Follicle Stimulating Hormone are known to provide a microenvironment for the growth and differentiation of germ cells. Follicle Stimulating Hormone regulates proliferation of Sertoli cells only during the early neonatal period and during adult period it regulates functional parameters like Transferrin, Androgen binding protein and other factors needed for germ cell differentiation and how this is achieved is not known. In an attempt to understand the differential action of Follicle Stimulating Hormone on the Sertoli cells, we employed the approach of neutralization of endogenous Follicle Stimulating Hormone in neonatal rats for a very limited period and study the effect on the regulation of gene expression by Differential Display RT-PCR analysis. One of the transcripts was found to be down regulated following neutralization of endogenous Follicle Stimulating Hormone in the neonatal rats and it shared identity with Intracisternal A particle element, an endogenous retrovirus. Often integration of Intracisternal A particle into host genes and its regulation result in growth factor independence suggesting a role in proliferation. Interestingly, the expression of Intracisternal A particle transcript is not regulated by Follicle Stimulating Hormone in the adult rat Sertoli cells suggesting a possible reason for stimulation of proliferation of Sertoli cells by Follicle Stimulating Hormone only in the neonatal rats

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

Shh Signaling from the Nucleus Pulposus Is Required for the Postnatal Growth and Differentiation of the Mouse Intervertebral Disc

Intervertebral discs (IVD) are essential components of the vertebral column. They maintain separation, and provide shock absorbing buffers, between adjacent vertebrae, while also allowing movements between them. Each IVD consists of a central semi-liquid nucleus pulposus (NP) surrounded by a multi-layered fibrocartilagenous annulus fibrosus (AF). Although the IVDs grow and differentiate after birth along with the vertebral column, little is known about the mechanism of this. Understanding the signals that control normal IVD growth and differentiation would also provide potential therapies for degenerative disc disease, which is the major cause of lower back pain and affects a large proportion of the population. In this work, we show that during postnatal growth of the mouse, Sonic hedgehog (Shh) signaling from the NP cells controls many aspects of growth and differentiation of both the NP cells themselves and of the surrounding AF, and that it acts, at least partly, by regulating other signaling pathways in the NP and AF. Recent studies have shown that the NP cells arise from the embryonic notochord, which acts as a major signaling center in the embryo. This work shows that this notochord-derived tissue continues to carry out a major signaling function in the postnatal body and that the IVDs are signaling centers, in addition to their already known functions in the mechanics of vertebral column function

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 (125)IoFSH,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, PKIβ\beta, 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 PKI$\beta$ 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

Microarray analysis of hyperthyroid and euthyroid rat sertoli cells.

Thyroid hormone is known to play an important role in the regulation of prepubertal rat testes development and function with specific influence on the differentiation of Sertoli cells. Hypothyroidism results in continued proliferation of Sertoli cells beyond their normal period of cell proliferation arrest, whereas hyperthyroidism causes premature cell cycle arrest. In order to explore the in vivo mechanism of thyroid hormone action on proliferation and differentiation of Sertoli cell, global gene profiling was carried out using the hyperthyroid rat model system. Hyperthyroidism in rats was established by injecting T3 to pups (i.p. 100 g/ kg bw/ day) immediately after birth. The euthyroidic and hyperthyroidic rats were sacrificed on 13th day. A significant decrease in BrdU incorporation by the hyperthyroidic rat Sertoli cell indicated arrest of proliferation. Total RNA was isolated from both the group of Sertoli cells and microarray analysis was carried out using Rat oligo v 3.0 slides obtained from University of Cincinnati Medical Centre, Cincinnati, Ohio. Image Analysis was performed using Genepix Pro software. The results revealed upregulation of markers which are specific for mature Sertoli cells. It was observed that there was more that three-fold increase in the expression of clusterin, eppin, androgen binding protein, cystatin TE, cystatin C, and estradiol 17- dehydrogenase and down regulation of early growth response protein 2, collagen 1 (XII) and plaminogen activator inhibitor 1in Sertoli cells following hyperthyroidism. RTPCR analysis was carried out to confirm the regulation of a few markers. The results suggest that following neonatal hyperthyroidism there is a premature differentiation of the Sertoli cells and the cells behave as mature cells by upregulation of the functional markers. (Supported by CONRAD and MELLON foundation, USA, CSIR, ICMR, DST and DBT Govt. of India and CSIR Emeritus Scientist Fellowship.

Formation of the sacrum requires down-regulation of sonic hedgehog signaling in the sacral intervertebral discs

In humans, the sacrum forms an important component of the pelvic arch, and it transfers the weight of the body to the lower limbs. The sacrum is formed by collapse of the intervertebral discs (IVDs) between the five sacral vertebrae during childhood, and their fusion to form a single bone. We show that collapse of the sacral discs in the mouse is associated with the down-regulation of sonic hedgehog (SHH) signaling in the nucleus pulposus (NP) of the disc, and many aspects of this phenotype can be reversed by experimental postnatal activation of hedgehog (HH) signaling. We have previously shown that SHH signaling is essential for the normal postnatal growth and differentiation of intervertebral discs elsewhere in the spine, and that loss of SHH signaling leads to pathological disc degeneration, a very common disorder of aging. Thus, loss of SHH is pathological in one region of the spine but part of normal development in another

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 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 flutamide 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

Shh is the key regulator of other major cell signaling pathways.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035944#pone-0035944-g010" target="_blank">Figure 10</a> shows that cyclopamine treatment in vitro, and targeting of Shh in vivo both cause alterations in activities of other signaling pathways. (A) and (B) show increases in BMP signaling and (C) shows the graph for the intensity of immunostaining from both the experiments. (D) and (E) show decreases in TGFβ signaling represented by graph plotted for the intensity of immunostanining (E). (F) shows an increase in canonical Wnt signaling. In (A)–(D), red  =  expression of the specific protein. In E, blue  =  β-gal positive cells, pink  =  nuclei stained with nuclear fast red.</p