Mammalian testis has a reserve of germ cell progenitors exhibiting multipotency/ pluripotency.Spermatogonial stem cells (SSCs) are descendants of the primordial germ cells (PGCs), which migrate from extraembryonic sites to colonize the gonadal ridge early during embryonic life. The continuation of the spermatogenic process throughout life relies on a proper regulation of self-renewal and differentiation of germline testis stem cells, the spermatogonial stem cells. These are single cells situated on the basal membrane of the seminiferous epithelium. Only 0.03% of all germ cells are SSCs. Several lines of evidence have suggested extensive proliferation activity and pluripotency of germline stem cells, including spermatogonial stem cells. We are evaluating the gene expression profiles in these cells to identify the mechanisms that allow germline stem cells to maintain their stemness. We are also attempting to define the genome/proteome reprogramming that would initiate immediate early signs of differentiation in these cells.

Human autoimmune regulator gene (q21.1) is coded by a 1600 bp message derived from 12339 base pair chromosomal segment and is coded by 16 exonic units. While two alternate splice forms are detected experimentally, alternative splicing database has shown the possible presence of five splice variants. Wild type AIRE protein consists of 545 amino acids with an estimated molecular weight of 57727 Da. AIRE is expressed heavily in thymic medulla and immune-related cells in peripheral circulation. Human and mice testes also express AIRE in substantially high quantities, and the expression is restricted to germ cell lineage. The main function of AIRE in thymus is thought to be to accomplish deletion of autoreactive T-cells through thymic education. Its function in testis is obscure. In this context, my objectives are: (1) evaluate AIRE expression in testicular germ cells in relation to their developmental status; (2) identify AIRE-interactome in germ cells; and (3) generate AIRE knock-down and knock-out models to evaluate the impact of an Aire-null background on germ cell development and differentiation.

MicroRNAs (miRNAs) are small noncoding RNAs that have emerged as important regulators of gene expression at both transcriptional and posttranscriptional levels. Hundreds of miRNAs are expressed in mammals; however, their functions are just starting to be uncovered. MicroRNAs are processed from a long hairpin mRNA transcript, down to a approximately 23-nucleotide duplex by a Dicer1-dependent mechanism. Removal of dicer-1 resulted in male infertility by blocking spermatogenesis. Mouse knockouts of Dicer1 are embryonic lethal before 7.5 days postcoitus indicating the importance of miRNA dependent mechanisms in early embryo development. My studies focus on miRNA dependent mechanisms in germ cell development, differentiation and fertilization.

We have detected aberrant expression of a transcriptional repressor in human males with spermatogenic disorder. The first objective is to evaluate the transcriptional and post-transcriptional regulation of this gene in germ cells. Post-translational modification of the protein also is suspected, which would be examined. Since this gene is abundantly expressed in spermatocytes, we anticipate that interference with the expression levels of this gene might display a testis phenotype that might give clue(s) into the role of this gene in spermatogenesis. We would undertake the characterization of expression profiles of this gene in a variety of male factor infertility disorder cases and would try to draw a correlation between the expression of this gene and spermatogenesis. Overexpression and silencing of this gene in spermatogonia and/or spermatocytes, evaluation of cell fate in these models and global profiling of gene expression in testicular germ cells from animal model deficient for this gene are also planned.

Retinoic acid has been demonstrated to be the main factor causing the differentiation of SSCs in the mammalian testis. Retinoic acid exerts its effect through many of the receptors present in Sertoli cells and germ cells. RA receptors are broadly catagorised as Retinoic Acid Receptors (RARs) and Rexinoid receptors (RXRs) and both types have been shown to be expressed in the mammalian testis. RARs are of three types, viz, RAR? RAR? and RAR?. RA-dependent events are believed to be mediated predominantly by RA receptor ? (RAR ?) in spermatogonia and by RAR? in Sertoli cells. Recent reports indicate that RAR? may function in Sertoli cells to promote the survival and development of early meiotic prophase spermatocytes, whereas RAR? in germ cells functions to increase the proliferation and differentiation of spermatogonia, prior to meiotic prophase. However, RAR?-dependent pathways operating in Sertoli cells which ultimately affect the germline lineage at various stages of their development are not clear. In this context, we aim to study the specific roles of retinoic acid receptors ? and ? in germ cell differentiation using in vitro culture systems and selective silencing of respective receptor genes.

Sperm development and differentiation in mammalian testis is a unique event. Seminiferous tubules in the testis have germ cell progenitors known as primordial germ cells (PGCs), which are pluripotent stem cells. These cells can divide horizontally to make their own copies, but also can differentiate into spermatogonia. Spermatogonia retain a high level of multipotency, and can divide horizontally to make their copies. Through unknown mechanisms operating at their cellular level, they can differentiate into spermatozoa. While hormonal and local regulations operating on them are implicated in their differentiation, very little is known about the factors that regulate the transformation of a spermatogonium into primary spermatocyte. Similarly, factors that regulate the differentiation of primary spermatocyte into secondary spermatocyte also are intriguing. I focus on molecular aspects of initiation of meiosis in spermatogenic cells.

The primary interaction between mammalian spermatozoa and oocyte is mediated by partially defined sperm components and four major components of zona pellucida (ZP1, ZP2, ZP3 and ZP4). My aim is to characterize the molecular interactions between ZP proteins and spermatozoa. Using ZP proteins and sperm membrane components tagged with appropriate fluorescent reporters, we envisage to study the physical responses of sperm membranes to ZP binding and the downstream signalling events which follow this binding.

Our laboratory has identified a cadherin on spermatozoa from healthy and fertile human males, which was absent or underexpressed in oligozoospermic human males. Though N-terminal sequence analysis yielded homology with E-cadherin due to the presence of conserved EC repeats in all types of cadherins, subsequent RT-PCR analysis of the message in the testis revealed that this differentially displayed protein is a member of protocadherins. My objectives are to conduct semiquantitative RT-PCR analysis of human testis-expressed protocadherin (htpCadherin) in germ line collected from semen, sequence the full length message of htpCadherin and detect any defect(s) within the coding region of this message in infertile (oligozoospermic and idiopathic) males. I would also clone the full length htpCadherin gene into pTYB1 vector, express and purify the recombinant protein for functional studies.

NPHP1 is a differentially displayed protein expressed in human testis and its absence is associated with spermatogenic insufficiency in men. This protein is coded by nphp1, and is known to be a cilial protein and acomponent of adherans junctions. However, the presence of multiple functional domains in this protein and it interacts with crk associated substrate (p130cas alias BCAR1), nephrocystin-4, Pyk2, tensin, filamin A, filamin B and PACS-1 makes it an important candidate molecule for further investigations. Nphp1 is expressed specifically in post-meiotic germ cells murine testis, while expression is absent in sertoli cells, interstitial leydig cells and pre-meiotic germ cells. The presence of Mad_BUB1_I (Mad3/BUB1 hoMad3/BUB1 homology region 1) and helix loop helix domain (HLH) in NPHP1 prompts us to speculate it involvement as a possible check point protein in cell division. We propose to investigate the role of NPHP1 in germ cell division, differentiation and sperm functioning.

TCTEX1 is a gene associated with male sterility in mouse and human, and is expressed heavily in the testis and moderately in the brain. Though it was originally thought to be a part of dyenin motor complex, recent studies have indicated its association with stemness in neural cell progenitors. Expression of TCTEX1 in brain is limited to Neural cell progenitors, and suppression of its expression leads neuronal stem cells into the differentiation pathway. A 100-fold higher level of expression of TCTEX1 in adult testis when compared with other tissues, and the remarkable link between aberrant expression of TCTEX1 and impaired spermatogenesis in mouse and human prompt us to hypothesize that TCTEX1 might dictate stemness in germ cell progenitors, and that the absence of sufficient quantities of quality germ cell progenitors could be the root cause of spermatogenic defects in TCTEX1-defective animals and patients. This study is outlined to address whether TCTEX1 has any role to play in deciding on the stemness of germ cell progenitors in mouse testis.