UBC Theses and Dissertations
Demonstration of GnRH-R in bovine uterus and oviducts and GnRH agonist (buserelin) induced in vitro regulation of steroid hormone receptors and apoptosis in bovine endometrium Singh, Ravinder
The presence of GnRH-R in bovine uterus and oviducts and the local modulatory role of the GnRH, GnRH-R system in the uterine physiology is not known. This dissertation reports studies designed to determine the presence of GnRH-R in the bovine uterus and oviducts and further studied GnRH agonist, buserelin induced steroid hormone receptor regulation (ERα, ERβ and PR) and apoptosis in the follicular and luteal phase bovine endometrium. RT-PCR and immunoblotting revealed GnRH-R mRNA (920 bp) and protein (60 kD), respectively in both the follicular and luteal phase endometrium and oviducts. Immunohistochemistry further localized GnRH-R to the endometrial and oviductal epithelial cells. In the second experiment (Chapter 3) RT-PCR studies on in vitro endometrial explant cultures showed that the GnRH, agonist, buserelin (200 ng/mL) had a stimulatory response (P≤ 0.05) on ERα mRNA in the luteal phase endometrium after 6 h of treatment. The PR mRNA levels were not changed in these experiments. The last experiment (chapter 4) investigated the effect of buserelin on endometrial apoptosis at the molecular (Bax, Bcl-2, caspase3, Fas mRNA) and cellular level. This part used endometrial explant culture, RT PCR, endometrial epithelial cell culture, immunofluoroscence and apoptotic assay. Buserelin induced an apoptotic response (P ≤ 0.05) in the follicular phase endometrium by stimulating Bax (200 ng/mL) and caspase3 mRNA (200, 500 ng/mL) after 6 h of treatment. The Bcl-2 and Fas mRNA expressions remained unchanged. The buserelin (200 ng/mL) induced epithelial cell apoptosis in the follicular phase endometrium at the cellular level, when the cells were treated for 6 h and further allowed to grow for 24 h. The GnRH antagonist-antide did not induce similar effects on ERα mRNA and apoptosis in these experiments. In conclusion, this study a) demonstrated GnRH-R in the follicular and luteal phase bovine endometrium and oviducts, b) elucidated that GnRH agonist, buserelin up-regulates ERα mRNA in the luteal phase endometrium and, c) induced apoptosis in the follicular phase endometrium. These findings indicate that GnRH supports reproductive process at the endometrial level. These are new findings in the field of reproductive biology. The local modulatory role of GnRH, GnRH-R system in oviducts needs further investigation.
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