Functional Characterization of a Novel Receptor Mouse Guanylyl Cyclase G （mGC-G） in Reproduction System
| Autor: | Yi-Yun Ciou, 邱逸雲 |
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| Rok vydání: | 2006 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 94 We recently identified a novel testis-enriched receptor guanylyl cyclase (GC) in the mouse, designated mGC-G. To further investigate its protein expression and function, we generated a neutralizing antibody specifically against the extracellular domain of this receptor. Reverse transcriptase (RT)-PCR and immunohistochemical analyses show that mGC-G is predominantly expressed from round spermatids to spermatozoa in mouse testis at both mRNA and protein levels. In female genital tract, mGC-G also has a significant expression in granulosa cell and zona pellucida in the ovary by immunohistochemistry. Flow cytometry and confocal immunofluorescence reveal that mGC-G is a cell-surface protein restricted to the plasma membrane overlying the acrosome and the midpiece of the flagellum in mature sperm. Interestingly, Western-blot analysis demonstrates that testicular mGC-G harbors an apparent molecular mass of approximately 180 kDa, but is subject to a limited proteolysis during epididymal sperm transport, resulting in a smaller fragment tethered on mature sperm surface. By utilizing Fluo-3 cytometrical analysis and computer-assisted sperm assay, we found that albumin-induced elevation of sperm [Ca2+]i level, protein tyrosine phosphorylation associated with capacitation and progressive motility are markedly reduced by pre-incubation of the anti-mGC-G neutralizing antibody. To further characterize mGC-G fuction in vivo, we generate mGC-G knock-out mice. Preliminary results show that the fertility of mGC-G null mice is reduced both in vivo and in vitro. Together, this study provides evidence that mGC-G is proteolytically modified in mature sperm membrane and suggests that mGC-G-mediated signaling may play a critical role in gamete/reproductive biology. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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