Localization and Characterization of an Orphan Receptor, Guanylyl Cyclase-G, in Mouse Testis and Sperm

• Published in: Endocrinology (Philadelphia) 2006-10, Vol.147 (10), p.4792-4800
• Main Authors: Huang, Yen-Hua, Wei, Chih-Chun, Su, Yueh-Hsing, Wu, Bo-Tsung, Ciou, Yi-Yun, Tu, Cheng-Fen, Cooper, Trevor G, Yeung, Ching-Hei, Chu, Sin-Tak, Tsai, Ming-Tzu, Yang, Ruey-Bing
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; Antibodies, Blocking - pharmacology; Biological and medical sciences; Blotting, Western; Calcium (intracellular); Calcium ions; Capacitation; Cell surface; Flagella; Flow Cytometry; Fluo-3; Fundamental and applied biological sciences. Psychology; Gene expression; Guanylate cyclase; Guanylate Cyclase - antagonists & inhibitors; Guanylate Cyclase - genetics; Guanylate Cyclase - physiology; Immunofluorescence; Immunoglobulins - metabolism; Intracellular signalling; Localization; Male; Membrane Proteins - antagonists & inhibitors; Membrane Proteins - genetics; Membrane Proteins - physiology; Membranes; Mice; mRNA; Neutralizing; Phosphorylation; Proteins; Proteolysis; Receptors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - biosynthesis; Serum albumin; Sperm; Sperm Motility - physiology; Spermatids; Spermatozoa; Spermatozoa - metabolism; Testes; Testis - cytology; Testis - metabolism; Tyrosine; Tyrosine - metabolism; Vertebrates: endocrinology
• ISSN: 0013-7227; 1945-7170
• DOI: 10.1210/en.2005-1476

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. RT-PCR and immunohistochemical analyses show that mGC-G is predominantly expressed from round spermatids to spermatozoa in mouse testis at both the mRNA and protein levels. Flow cytometry and confocal immunofluorescence reveal that mGC-G is a cell surface protein restricted to the plasma membrane overlying the acrosome and midpiece of the flagellum in mature sperm. Interestingly, Western blot analysis demonstrates that testicular mGC-G is approximately 180 kDa but is subject to limited proteolysis during epididymal sperm transport, resulting in a smaller fragment tethered on the mature sperm surface. On Fluo-3 cytometrical analysis and computer-assisted sperm assay, we found that serum albumin-induced elevation of sperm intracellular Ca2+ concentration, protein tyrosine phosphorylation, and progressive motility associated with capacitation are markedly reduced by preincubation of the anti-mGC-G neutralizing antibody. Together, these results indicate that mGC-G is proteolytically modified in mature sperm membrane and suggest that mGC-G-mediated signaling may play a critical role in gamete/reproductive biology.