Principal Investigator :    Chandrima Shaha

Project Associates/Assistants
Bettina Sikha Mukherjee
G Sudhandiran
R Pal

PhD students
A V S Kondala Rao
T Hemchand
R Nair
Promila

Collaborators
S Mukherjee, ICGEB, New Delhi
Dinakar M Salunke

The primary theme of the above project can be divided into two distinct aspects. The first objective is to carry out studies to unravel certain aspects the mechanisms of cell-cell interactions during gamete recognition. The second objective is to further the understanding of the mechanism of cell death in a multicellular model, the mammalian male germ cells and a unicellular eukaryote - the L. donovani, the causative agent of Kala-azar.

1.        Mechanisms of male germ cell growth and survival

a.     Functional role of GSTs in germ cells

Phase II detoxification enzymes, the glutathione S-transferases (GSTs) of 24 kDa molecular mass are known to be cytosolic enzymes. A major finding from this Laboratory was the discovery of the important role of the enzyme glutathione S-transferase in reproduction related events over the past years. Studies conducted during the reporting period shows that multiple GST isoforms that are 24 kDa in size are present on the extracellular side of the plasma membrane of rat male germ cells. The GST activity of male germ cell plasma membranes is several folds higher than somatic cell plasma membrane GST activity. Isoform composition of the germ cell plasma membrane and the cytosolic pool differ, GST-M5 and GST-Pi being absent on the plasma membranes. The molecular masses of the common isoforms are comparable between the two pools and both pools show GST and GSH peroxidase activity.

After it was established during the last reporting period that sperm surface GSTs helped in binding to the zona pellucida of the oocyte, studies were conducted to determine how sperm surface GSTs functioned as detoxification enzymes. The data show that under oxidative stress the sperm can use extracellular GSH to prevent membrane damage like peroxidation of lipids. We show that extracellular GSH present in the reproductive tract fluids can be used to its advantage by the sperm to prevent oxidative injury.

b.     Male germ cell apoptosis

Continuing our studies on the effects of exposure of germ cells to environmental toxicants, we used 2, 4 hexanedione that is used in the paint and enamel industry to check its effect on germ cells. This toxin interferes with germ cell survival and initiates apoptosis by about 5 weeks of exposure by the oral route. Caspase 8 activity was evident indicating that Fas Fas-ligand pathway was involved.

Our studies on the effects of estrogens on germ cells, we found that estrogens could induce increased germ cell apoptosis. However, this apoptotic pathway was using both the Fas ligand and the cytochrome c to bring about cell death. There was an upregulation of Fas-ligand and caspase 8 activity and cytochrome c was detectable in the cytosol while p53 in the nuclei remained unchanged. The Fas-ligand upregulation occurred in the germ cells rather than the Sertoli cells. There was a translocation of the bax protein from the cytosol to the nucleus. This study has for the first time identified the pathway used by estrogens to bring about germ cell apoptosis.

It is known that exposure to high altitude causes disturbances in spermatogenesis. Effects of hypobaric hypoxia on male germ cells show that high altitude interferes with germ cell survival but the most pronounced effect on germ cell viability occurs during the period when they are brought down to sea-level. This shows that during re-acclimatizing period, the damage to testicular tissue is most pronounced.

2.     Cell death in single celled organisms

Studies during the reporting period shows that upon activation of death response by H2O2, a dose and time dependent loss of mitochondrial membrane potential (Dym) occurs. This loss is accompanied by a depletion of cellular glutathione but cardiolipin content or thiol oxidation status remain unchanged. A tight link exists between changes in cytosolic Ca2+ homeostasis and collapse of Dym, but the dissipation of Dym is independent of elevation of cytosolic Na+ and mitochondrial Ca2+. It is further demonstrated that the increase in cytosolic Ca2+ is an additive result of release of Ca2+ from intracellular stores as well as by influx of extracellular Ca2+ through non-selective cation channels. Mitochondrial changes do not involve opening of the mitochondrial transition pore as cyclosporin A is unable to prevent Dym loss. These findings reveal the importance of non-selective cation channels in altering oxidative stress induced changes in intracellular Ca2+ homeostasis that trigger downstream signaling cascades leading to apoptosis-like death in L. donovani promastigotes.

Publications

Original peer-reviewed articles

1.    Hemachand T, Gopalakrishnan B, Salunke DM, Totey SM and Shaha C (2002) Sperm plasma membrane associated glutathione S-transferases as gamete recognition molecules. J Cell Sci (in press).

2.     Kondala Rao AVS and Shaha C (2001) Multiple glutathione S-transferase isoforms are present on male germ cell plasma membrane. FEBS Lett 507:174-180.

3.    Das M, Mukherjee SB and Shaha C (2001) Hydrogen peroxide induces apoptosis-like death in Leishmania donovani promastigotes. J Cell Sci 114:2461-2469.

Reviews/Proceedings

1.     Shaha C (2002) Nitric oxide and inflammation. The Natl Med J of India (Eds Salvemini D, Billar TR, and Vodovotz Y), Birkhauser Virlag, Basel, 15:48-50.