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Characterization of reproductive tract proteins important for fertility |
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Principal Investigator : Chandrima Shaha Project Associates/ Assistants Ph D Students Collaborators The primary objective of this project is to understand
the mechanisms of development of male germ cells and the functional role of
sperm surface proteins in sperm maturation and fertilization. One of the major
events in the testis is male germ cell apoptosis that is pivotal in
maintaining proper testicular homeostasis. Deciphering the pathways of male
germ cell apoptosis under different situations form the first component of
this project. The emphasis in the second part is to unravel mechanisms of
cell-cell interactions during gamete recognition and fertilization. As a
spin-off of the above project we have initiated studies on apoptosis related
phenomenon in a single celled eukaryote - the Leishmania donovani that share
similar sensitivity to microtubule and mitochondrial inhibitors and the
deficiency in defensive enzymes with that of the spermatozoa. The aim of this
project is to understand cell death in single celled eukaryotes to trace the
evolution of the metazoan apoptotic process. A. Mechanisms of germ cell growth and survival a. Defense mechanisms in rat testicular germ cells that regulate germ cell apoptosis Following up on the last report that germ cells were sensitive to
oxidative stress induced by H2O2 and that germ cell GSTs were a part of the adaptive response of germ
cells to oxidative stress, we analyzed the role of different pools of GSTs
that are present in the germ cells in terms of detoxification of lipid
hydroperoxides. Two pools of germ cell GSTs were detected and purified. One
was a plasma membrane pool and another a cytosolic pool. Using a cell-free
system, we compared the ability of plasma membrane and cytosolic GSTs to
salvage products of lipid peroxidation that are generated by H2O2.
Cytosolic GSTs could salvage 70% of the lipid peroxidation products formed and
were comparatively more effective in salvaging these products as compared to
plasma membrane GSTs. Our earlier findings as detailed above provided evidence that
oxidative stress to germ cells led to apoptosis. Using a product of the paint
industry that is a known germ cell toxin and considered as an environmental
pollutant, we attempted to find out the mechanism of toxin induced germ cell
death. Methoxyacetic acid (MAA) was used as a model toxin and after oral
administration, testis was checked for cell death. There was a gradual
increase in the number of apoptotic pachytene spermatocytes over a period of
24 h as revealed by terminal deoxyribonucleotide transferase (TdT)-mediated
dUTP nick end labelling (TUNEL). After administration of the toxin, within a
period of 6 h no free radical generation could be detected. However, there was
a gradual drop in the intracellular glutathione (GSH) content that started
around 2 h and reached nadir at 8 h. This reduction in GSH content may have
generated severe oxidative stress on the cells by depleting them of an
important thiol and rendering some of the detoxification enzymes
non-functional. A hallmark of metazoan apoptosis is the cleavage of poly (ADP)
ribosyl polymerase (PARP), that is a DNA repair enzyme. Cleavage of this
protein was visible by 12 h after treatment with MAA. Arguably, if there is a
PARP cleavage at 12 h there should be caspase activation at earlier hours, as
PARP is a substrate for caspase-3. Both caspase-9 and caspase-3 activity was
detected at 4 and 8 h. Since caspase-9 was activated, it is clear that the
pathway of apoptosis is through the release of cytochrome c from the
mitochondria and not through the Fas Fas-ligand pathway that is the most
common pathway followed during germ cell death. Hence it can be inferred that
germ cell toxins may initiate a different apoptotic pathway in comparison to
the one that occur in response to developmental cues. b. Functional role of GSTs in sperm Consequent to earlier studies demonstrating the binding of sperm GSTs
to the zona pellucida, the mechanism of attachment of the GSTs on sperm
surface was worked out and it was found that the GSTs were attached to the
sperm surface by non-covalent peripheral attachments. Sperm GSTs (GST-Pi and
Mu) were able to bind to solubilized zona pellucida with GST-Pi showing
greater affinity for binding. Metabolic labeling studies in germ cells showed
that 10% of the total cellular GSTs were transported to the membranes while
90% remained as cytosolic pool. Attempts were made to determine if any
membrane targeting sequence was present in the GST-Pi gene and the endeavor
was unsuccessful. c. Mechanistic
studies on male germ cell apoptosis: implications of estrogen analogue
exposure With the reports of estrogens and compounds with estrogenic activity
as environmental pollutants that lead to defects of spermatogenesis, we have
initiated this project to study the changes induced in germ cells after
estrogen exposure. Different doses of estrogen analogue diethylstilbestrol
were administered to rats and the rate of apoptosis was established. It was
found that the rate of apoptosis was directly related to the dose of estrogen
applied. Currently, the mechanism of induction of gem cell death by the
estrogens is being investigated, as it is believed that estrogens may act
either as oxidants or antioxidants depending on the cell type. d. Mechanistic
studies on high altitude hypoxia induced germ cell spermatozoal apoptosis,
dysfunction and possible treatments: study in cells from mice This project was initiated to study the effects of high altitude
exposure on spermatogenesis. The aim is to establish if intermittent or
chronic exposure to high altitude conditions cause alterations in
spermatogenesis, and if so if there can be a mechanism to reduce this. Chronic
exposure for 7 days to high altitude conditions show increased germ cell
apoptosis. It is planned to study if mice from high altitude areas are more
resistant to hypoxic conditions as compared to mice from the plains, and if so
is there a difference in the early response in the apoptotic pathways. B. Cell death in single celled organisms Cell death in Leishmania donovani promastigotes was studied
using oxidative stress as an inducer of cell death, as during phagocytosis by
the host macrophages considerable oxidative stress is generated. Oxidative
stress results in a pattern of death in Leishmania donovani
promastigotes that demonstrates many features of metazoan apoptosis and is
dependent on the degree of the stress generated. Features of metazoan
apoptosis, like nuclear condensation, DNA fragmentation with accompanying DNA
ladder formation was observed. Caspase-like protease activity increased after
the cells were exposed to oxidative stress and specific caspase inhibitors
reduced the number of cells showing apoptosis-like features. Cleavage of a
poly (ADP) ribose polymerase like protein also occurs that is a substrate for
caspases. Increase in the accumulation of thiobarbituric acid reactive
substances followed by a reduction in intracellular reduced glutathione is
also detectable. In addition, exogenous lipid hydroperoxide intermediates
precipitate an apoptosis-like death. Taken together, the above data show for
the first time that in kinetoplastid parasites a distinct pathway for
apoptosis-like death exists. Publications Original peer-reviewed articles 1. Kondala Rao AVS and Shaha C (2000) Role of glutathione S-transferases in oxidative stress-induced male germ cell apoptosis. Free Radic Biol Med 29:1015-1027. |