Mammalian spermatozoa undergo a vast array of structural reorganizations duirng their post-testicular developmental processes including matucation, capacitation, acrosome reaction and sperm-oocyte binding. The lipophilic domains of the spermatozoa have been shown to display altered viscosity, polarity, molecular ordering and lateral diffusion patterns. With increase in the local fluidity with progressive sperm development, the mobility of proteins appeared to be restricted, suggesting the presence of membrane sub-domains in spermatozoa, in which specific lipids and proteins are assembled to carry out specific functions. If this is true, the spermatozoa might have well-defined lipid rafts. I am investigate the structural and functional aspects of membrane lipid rafts in spermatozoa during their development in the testis, maturation in the epididymis, capacitation, acrosome reacation and oocyte interaction phases.

NADPH-Oxidase (NOX), the functional equivalent of phagocytic oxidase (PHOX) is expressed in various tissues. We have identified a testis-specific NOX that appears to be a calcium-dependent enzyme. However, the molecular architecture of human testicular NOX (htNOX) and its regulation is not yet known. We are evaluating the nature of this multicomponent enzyme, and are trying to define its functional performance in relation to the possible assembly probabilities, especially with p22PHOX subunit. I would also address the downstream signaling events including MAPK activation, followed by the activation of downstream pathways including extracellular signal-regulated kinases ERK1/ERK2, c-Jun N-terminal kinases (JNK), Toll-like receptor 4-dependent NF-kappaB activation and their relevance in relation to sperm maturation, sperm activation and fertilization.

Two percent of human males are infertile because of severe defects in sperm production. In the clinical cases, spermatogenic arrest is an interruption of germ cell differentiation that may result in either oligozoospermia or azoospermia and can be diagnosed via testicular biopsy. Although spermatogenesis must require many gene products, mutation or absence of the gene expressed at different development levels of spermatogenesis may lead to spermatogenic arrest and infertility. Identification of new genes specifically involved in spermatogenesis and analysis of the phenotypes could provide both insight into this developmental process and a more rational basis for treatment of male infertility. Using differential display proteomics followed by genomic assays and molecular modeling, we have identified a few testis-specific genes that may regulate cell cycle in germ cells. We aim to clone, express and characterize a testis-experssed Cyclin-like protein (CLP-1). We would also attempt to examine whether there are defects in the CDS of CLP-1 gene associated with male subfertility.

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.

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.

Stem cell-based therapy is a promising approach for combating degenerative diseases. Adult stem cells with the pluripotency of embryonic stem cells (ESCs) would be an ideal cell source. Human primordial germ cells (PGCs) could be a source of pluripotent stem cells. In order to culture spermatogonial stem cells (SSCs) in vitro, researchers have to overcome some of the obstacles such as the low number of stem cells in the testis, absence of specific markers to identify SSCs and difficulties in keeping the SSCs alive in culture. Whereas rare A (single) spermatogonia comprise the rodent SSC pool, primate spermatogenesis arises from more abundant A (dark) and A (pale) spermatogonia, and the identity of the stem cell is subject to debate. In this context, it is important to develop protocols to harvest stem cells from testis and identify molecular markers that would indicate the “stemness” of SSCs. My research aims to evolve a robust protocol for stem cell isolation from testicular cell suspensions.

Using differential display proteome analysis in 11 human males with non-obstructive oligozoospermia versus 36 fertile males, we identified a sperm protein differentially displayed in fertile males, but absent in oligozoospermic human subjects. N-terminal sequence analysis of this protein identified it as a Tyramine Receptor Like Protein (TRLP), which is predictably a G-protein coupled receptor. We have been able to amplify the gene coding for this protein using a degenerate PCR approach using normal testis cDNA. Since preliminary results indicate a correlation between the presence of TRLP and sperm competence, we propose a full characterization of this protein including the isolation and purification of TRLP, Mass-spectroscopy analysis to determine the protein sequence and modifications, cloning the full length CDS into an expression vector and production of recombinant protein, production of anti-TRLP antibodies and analysis of expression levels of TRLP at various stages of germ cell maturation. The findings may contribute immensely towards understanding the molecular aspects of human male factor infertility. Also, it may give us some lead to device novel therapeutic agents that could block sperm-oocyte interaction.

Spermatogonial stem cells are rare (=1 in 3000 adult testis cells), just like any other adult stem cells. They are believed to be superior in genome quality due to evolutionary pressure, which makes it an attractive theme to try to harvest and transdifferentiate them into somatic lineages of interest. Literature argues that germline stem cells are negative for Kit tyrosine kinase (Kit) and stem cell antigen 1 (Sca-1), which are remarkable differences when compared with somatic stem cell counterparts. Further, spermatogonial stem cells appear to be positive for Thy-1 and alpha6-integrin. I propose to use a combination of surface markers to harvest germline stem cells from neonatal and adult testis, and to evolve protocols to transdifferentiate them into cells of ectodermal, mesodermal and endodermal lineages.