Germline stem cell maintenance, division and differentiation in testis

Germline stem cells (GSCs) are lineage of Primordial Germ Cells (PGCs) which may have retained the full level of pluripotency as that of the PGCs, or have lost a part of it to qualify to be a multipotent cell line. Mammalian testis is a place orchestrating one among the very fast cell division patterns in the entire body, amounting to the production of 1000 spermatozoa per heart beat in an adult human being. Despite this pace of division, germ cells manifest minimal loss of genome integrity. Our objectives include characterization of GSCs, harvesting and manipulation of GSCs to generate cells of interest and to understand mechanisms involved in GSC maintenance and differentiation.

Factors regulating meiotic drive in testis

Meiosis occurs in germ cells in the testis and ovary. Somatic cells are incapable of dividing through meiosis. In tesist, enormous number of cells undergo meiosis in a precisely synchronized fashion. We are addressing the molecular control of the processes associated with germ cells differentiation into spermatogonia, primary spermatocytes and ultimately into haploid secondary spermatocytes. We use differential display genomic and proteomic approaches to understand alterations in the levels of expression of a large subset of testis-specific proteins. Special focus is given to differential proteome of spermatozoa from fertile human males and subfertile human males with spermatogenic impairment. We also monitor differential proteome of primary spermatocytes and secondary spermatocytes to define meiosis-associated molecular events.

Critical molecules regulating biomembrane structure and cell fusion during fertilization

While it is hard to fuse two cells even from the same individual, fertilization poses a natural mechanism through which two haploid cells from two different individuals interact in a highly species-specific manner to yield a single diploid cell. During fertilization, the membrane of spermatozoon fuses with the membrane of oocyte with the highest level of specificity that one could think of. We work on the molecular aspects of cell-cell recognition, membrane-membrane interaction and the physical aspects of membrane structure that aids in fusion events. We have identified a few “critical molecules” involved in gamete interaction, and the absence of any one of these key players has been shown to have strong correlation with compromised gamete functioning. A few of these critical molecules are associated with sperm membrane RAFTS. In this context, we are evaluating the role of RAFTS and RAFT-associated proteins (RAPs) in determining the ability of spermatozoa to recognize, interact and fuse with oocytes.

Cellular Deprogramming during fertilization

Spermatozoa and oocyte are terminally differentiated cells with no “future” if they do not fuse. The fusion of these two cells produces a single cell, which is the storehouse of the entire pluripotency that would be required for the generation of an entire organism with extremely complex and diversified system. Oocyte cytoplasm might reprogram gene expression from male nucleus, and the spermatozoon (both its nucleus and non-nuclear components) might redefine gene expression in the oocyte. The role of sperm components in defining the division pattern in early embryos is obscure. In collaboration with Dr. Malini Laloraya, we address the intricacies of cellular deprogramming as an immediate early event during fertilization.

Sponsored Grants

Completed

1. Insect pest management through secondary plant metabolites: control of forest pest and mosquitoes (Funded by MP Council of Science and Technology, Bhopal, India). Duration: 1988-1991
2. Studies on the isolation, purification and structural and functional characterization of Fc receptors from mammalian sperm (funded by Council of Scientific and Industrial Research, New Delhi, India). Duration: 1990-1992
3. Free radical modulation of the physico-chemical architecture of mammalian sperm membranes and the molecular aspects of sperm-zona pellucida interaction (funded by Department of Science and Techology, New Delhi, India). Duration: 1992-1995
4. Active oxygen intermediates in female reproduction and the physico-chemical aspects of membrane configuration as a consequence of oxidative mechanisms at implantation (funded by Department of Science and Techology, New Delhi, India). Duration: 1992-1995
5. Isolation, purification and characterization of a transiently appearing endometrial adhesion molecule (EAM) in the mouse (funded by Department of Atomic Energy, Bombay, India). Duration: 1992-1995
6. Biophysical and immunological aspects of the perturbations in post-vasectomy spermatozoa and its relation in irreversibility of fertility (funded by Council of Scientific and Industrial Research, New Delhi). Duration: 1996-2000
7. Characterization of A/V CAM from human spermatozoa and its involvement in normal human fertility (funded by Department of Biotechnology, New Delhi, India). Duration: 2000-2004. Charactrization of 54 kilodalton uterine protein and its involvement in early pregnancy (funded by University Grants Commission, New Delhi, India). Duration: 2002-2005
8. Identification of superoxide generating machinery in mammalian spermatozoa (funded by Department of Science and Technology, New Delhi, India). Duration: 2003-2007.Mechanism of activation of NADPH Oxidase in blue-light signal transduction reactions in the coleoptile tip (Department of Science and Technology, New Delhi, India). 2004-2008
9. Evaluation of the expression of a sterility locus gene TCTEX-1 and its relation with protein trafficking in spermatozoa from infertile and fertile human males (BRNS, Bombay). Duration: 2006-2009.
10. Characterization of RAFTS and RAFT-associated proteins of spermatozoa during capacitation and acrosome reaction. (Department of Biotechnology, New Delhi). 2006-2009
11. Cloning, expression and semiquantitative analysis of protocadherins in human germ cells (CSIR, New Delhi) 2006-2009
12. Sperm proteome profiling in human male infertility (DBT-National Bioscience Award Project) 2007-2010
13. Cloning, expression and functional characterization of a cyclin-like protein (CLP-1) from human germ cells (DST, New Delhi) 2008-2011
14. Charcterization of testis-specific tyramine receptor like protein (TRLP) (KSCSTE, Trivandrum) 2008-2011
15. Isolation of germline stem cells from adult mouse testis and evaluation of their pluripotency (Department of Biotechnology, New Delhi) 2010-2011

1. Structural and phosphoproteome analysis of mouse sperm membrane rafts during their interaction with zona pellucida protein (Department of Biotechnology, New Delhi), 2011-2014
2. Association between stemness and TCTEX1 expression in testicular germ cells from adult mouse. (BRNS, Mumbai), 2011-2014