The Role Of Natriuretic Peptide Receptor 2 In Sperm Chemotaxis

Natriuretic peptide receptor 2 (NPR2) is essential for maintaining meiotic arrest in mouse oocytes residing in antral follicles. This study determined the expression of NPR2 in mouse sperm and the function on sperm chemotaxis. The inactivation of NPR2 was also studied by cultured cumulus-oocyte complexes (COCs).In the mammals, several million spermatozoa inseminated rapidly reach the storage site in the isthmus of the oviduct with reduced motility after mating, but only a few spermatozoa recover their motility and swim chemotactically from the storage to the fertilization site of ampulla when ovulation occurs. This phenomenon usually called sperm chemotaxis. To test the location of NPR2 on spermatozoon, we used immunoprecipitation followed by western blotting and found that NPR2 was expressed predominantly in sperm flagellum. We used fluorescent labeled ligand NPPC (FAM-NPPC) to bind NPR2, the green fluorescence, representing NPPC (natriuretic peptide type C) binding site, was observed at the flagellar midpiece. Furthermore, the NPPC in mouse ampulla was expressed highly after ovulation and the protein of NPPC was expressed in the mucous layer cytoplasm of the ampulla. NPPC also increased spermatozoa kinetic parameters of VAP, VSL, and progressive motility. To study the effect of NPPC on sperm chemotaxis, we carried out capillary assay as previous reports in vitro. The number of spermatozoa in the capillary was significantly increased in the presence of 1 nM NPPC. Intracellular cGMP levels of spermatozoa also increased in response to NPPC, and 8-Br-cGMP mimicked NPPC-mediated sperm chemotaxis, suggesting that NPPC induces sperm chemotaxis by NPR2-produced cGMP signaling pathway. NPPC elevated intracellular Ca2+ levels of spermatozoa. And the elevation of Ca2+ was observed initially in the midpiece region of the spermatozoon, but immediately spread toward the head with a mean latency of two fames (2 s). NPPC could not induce accumulation of spermatozoa from Npr2cn-2J/Npr2cn-2J mutant mice. The rate of two-cell embryo was 77.7±3.4% in spermatozoa from heterozygous males, but dramatically decreased to 5.4±2.2% in that from mutant males by artificial insemination. Therefore, NPR2 expressed in the sperm midpiece, by binding to NPPC, guides spermatozoa toward oocytes residing in the fertilization site, which is critical for ovulation and fertilization synchrony, and for normal fertility.Furthermore, we investigated the inactivation of NPR2 in cultured COCs. In preovulatory ovarian follicles, the oocyte is maintained in meiotic prophase arrest by NPPC and its receptor NPR2. Results showed that EGF could elevate intracellular calcium concentrations of cumulus cells and decrease NPR2 guanylyl cyclase activity. And calcium-elevating reagents ionomycin and sphingosine-1-phosphate mimicked the effects of EGF on oocyte maturation. These results suggest that EGF receptor signaling inhibits NPR2 activity in cumulus cells by elevating calcium concentrations and promotes meiotic resumption.Therefore, NPR2 in the sperm midpiece, by binding to NPPC, increases intracellular cGMP and Ca2+ levels, which is critical for sperm chemotaxis and normal fertility. Furthermore, the intracellular calcium elevation could lead to inactivation of NPR2. NPPC can also be exploited as a diagnostic tool to assess sperm quality.These processes can potentially be used as a means of contraception by interfering with the normal process of fertilization.