Characterization of proteins important for fertility and cell death

Project Associates / Assistants
Bettina Sikha Mukherjee
G Sudhandiran
A Mehta
R Pal

Ph D Students
R Nair
J Iyer

The broad research interest of the laboratory is to understand the regulation of cell-cell interactions and mechanisms of cell death. Current projects explore pathways related to the survival of the male germ cells with special emphasis on processes leading to cellular apoptosis. The other major focus of this research program exploits a unicellular model, the protozoan parasite to answer questions related to the importance of apoptosis in parasite survival and host-parasite interactions. The current studies on male germ cell biology were to evaluate the role of sperm surface glutathione S-transferases as defensive enzymes in the context of the special environment that the spermatozoa survive in. In addition, the preferred pathways adopted by germ cells in the event of environmental stress were evaluated for environmental estrogens and a herbicide, 2, 4 exanedione. For biology of cell survival in protozoan parasites, the aim was to study the mechanisms of induction of apoptotic cell death by oxidative stress.

1.         Biology of male germ cell survival and function

The significant role that estrogens play in spermatogenesis has opened up an exciting area of research in male reproductive biology. The realization that estrogens are essential for proper maintenance of spermatogenesis, as well as growing evidence pointing to the deleterious effects of estrogen-like chemicals on male reproductive health, has made it imperative to dissect the role estrogens play in the male. Using a model estrogen, diethylstilbestrol (DES) to induce spermatogenic cell apoptosis in vivo in the male rat, we provide a new insight into an estrogen dependent regulation of the Fas-FasL system specifically in spermatogenic cells.

DES, a potent estrogenic analogue is known to disrupt spermatogenesis by the suppression of pituitary gonadotropins which leads to the inhibition of testosterone production resulting in insufficient circulating and intratesticular concentrations of the hormone. In our studies, DES induced abnormal spermatogenesis, paralleled by a loss in testicular weight. Data showed nucleosomal DNA laddering, increase in TUNEL positive cells and changes in the ploidy of spermatogenic cells upon DES administration, clearly establishing an apoptotic nature of spermatogenic cell death, particularly in the haploid cell type. We found a distinct increase in Fas-FasL expression in spermatogenic cells upon exposure to DES. This increase was confined to the spermatid population and correlated with increased apoptosis seen in the haploid cells. It is generally accepted that in the testis, FasL is expressed in Sertoli cells which precipitates cell death by engaging its cognate receptors on spermatogenic cells. Our studies revealed a clear up-regulation of FasL in spermatogenic cells and not in Sertoli cells within 24 h of DES treatment as determined by Western blots and RT-PCR with FasL specific antibodies and probes respectively. Interestingly, in isolated spermatogenic cells from DES treated animals on day 1, FasL and Fas showed differential localization. While Fas was distributed evenly throughout the cell, FasL staining was associated with vesicular bodies. Testosterone supplementation was able to prevent DES induced Fas-FasL upregulation and apoptosis in the spermatogenic cells showing that estrogen provoked apoptosis by blocking testosterone. DES induced germ cell apoptosis did not occur in Fas deficient lpr mice demonstrating that Fas expression was an absolute requirement for estrogen induced apoptosis of germ cells. One other important finding was that spermatogenic cells are type II cells, as the increase in Fas-FasL expression in the spermatogenic cells was followed by the cleavage of caspase-8 to its active form, following which Bax translocated to the mitochondria and precipitated the release of cytochrome c. Subsequent to this, activation of caspase-9 occurred that in turn activated caspase-3 leading to the cleavage of poly(ADP-ribose) polymerase.

Taken together, the data essentially illustrates the importance of the Fas-FasL system in spermatogenic cell death in the event of estrogen exposure. Furthermore, these experiments establishes germ cells as type II cells that are able to utilize the extrinsic and intrinsic apoptotic pathways, the link between the two pathways being the proapoptotic protein Bax in the event of estrogen exposure. We also postulate that normal testicular homeostasis may involve the Fas-FasL system regulated by estrogens to maintain proper spermatogenic cell number.

2.         Biology of cell survival in protozoan parasites

Apoptosis, that is crucial for embryogenesis, tissue homeostasis and disease control in metazoans has also been demonstrated in unicellular organisms including yeast and protozoan parasites (J Cell Sci (2001) 114: 2461-2469) The protozoan parasites have a digenic life cycle, residing as flagellated extracellular promastigotes in the gut of the insect vector or as obligatory intracellular amastigotes found in the parasitophorous vacuoles of mammalian macrophages. The capability of the obligate intracellular parasites like Leishmania donovani to survive within the host cell parasitophorous vacuoles as nonmotile amastigotes determine disease pathogenesis, but the mechanism of elimination of the parasites from these vacuoles are not well understood. By using the anti-leishmanial drug potassium antimony tartrate, we demonstrate that upon drug exposure, intracellular Leishmania donovani amastigotes undergo apoptotic death characterized by nuclear DNA fragmentation and externalization of phosphatidylserine. Changes upstream of DNA fragmentation included generation of reactive oxygen species like superoxide, nitric oxide and hydrogen peroxide that were primarily concentrated in the parasitophorous vacuoles. In the presence of antioxidants like N-acetylcysteine or Mn(III) tetrakis (4-benzoic acid) porphyrin chloride, or an inhibitor of inducible nitric oxide synthase, a diminution of reactive oxygen species generation and improvement of amastigote survival was observed suggesting a close link between drug induced oxidative stress and amastigote death. Changes downstream to reactive oxygen species increase involved elevation of intracellular Ca²⁺ concentrations in both the parasite and the host that was preventable by antioxidants. This influx of Ca²⁺ was preceded by a fall in the amastigote mitochondrial membrane potential. Therefore, these data project the importance of cellular Ca²⁺ as an important modulator of antimonial efficacy and lends credence to the suggestion that within the host cell, apoptosis is the preferred mode of death for the parasites.

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 115:2053-2065 (*in press last year, since published).

2.     Kondala Rao AVS and Shaha C (2002) N-acetylcysteine prevents MAA induced male germ cell apoptosis: role of glutathione and cytochrome c. FEBS Lett 527:133-137.

3.     Mukherjee SB, Das M, Sudhandiran G and Shaha C (2002) Increase in cytosolic ca2+ levels through the activation of non-selective cation channels induced by oxidative causes mitochondrial depolarization leading to apoptosis-like death in Leishmania donovani promastigotes. J Biol Chem 277:24717-24727.

4.     Nair R and Shaha C (2003) Diethylstilbestrol induces rat spermatogenic cell apoptosis in vivo through increased expression of spermatogenic cell Fas/FasL system. J Biol Chem 278:6470-6481.

5.     Hemachand T and Shaha C (2003) Functional role of glutathionen S-transferases and extracellular glutathione in the haploid spermatozoa under oxidative stress. FEBS Lett 538:14‑18.