MP66-01 LOSS OF RA-GEF-2 (RAPGEF6) IN MOUSE CAUSES ALTERED LOCALIZATION OF N-CADHERIN AND CAN CAUSE MALE INFERTILITY

INTRODUCTION AND OBJECTIVES: Rap1 is a member of the Ras family small GTPases and activated by an array of guanine nucleotide exchange factors (GEFs) downstream of various extracellular stimuli. We discovered RA-GEF-2 (also called Rapgef6) as a specific GEF for Rap1 and analyzed its physiological functions through generation of mice carrying homozygous deletion of the gene encoding it [RA-GEF-2 knockout (KO) mice]. We have found that these mice exhibit prominent male infertility and analyzed the mechanism underlying RA-GEF-2’s function in the process of spermatogenesis. METHODS: To assess the fertility of RA-GEF-2 KO mice, we mated male RA-GEF-2 KOmice with female wild-type mice for 72 hours. Threeweeks later, we counted the number of fetuses.We also carried out measurement of the pituitary hormones [i. e., luteinizing hormone (LH), follicle stimulatinghormone (FSH)and testosterone] byELISAassay.The concentration and motility of epididymal sperms were measured with Makler Chamber and sperm morphology was analyzed by scanning electron microscopy. We also identified the expression of RA-GEF-2 in testis using real-timePCRandWesternblotting. Furthermore,weclarified the localization of RA-GEF-2 in testis by immunohistochemistry. We found that RA-GEF-2 was located on the apical junction, so we further assessed the expression of N-cadherin by Western blotting and immunohistochemistry. RESULTS: The pregnancy rate and the number of fetuses were low after mating with male RA-GEF-2 KO mice. Although the body weight and pituitary hormones levels showed no difference between KO and wild-type mice, the weight of testis was significantly low in KO mice. In addition, the concentration and motility of epididymal sperms were decreased. Furthermore, malformed sperms were ubiquitously observed in KO mice. Histological examination showed marked atrophy of the testis and hypospermatogenesis in KO mice. Because RA-GEF-2 was expressed on the luminal side of seminiferous tubules, it could be related to the apical ectoplasmic specialization (ES) that maintains the polarity of the spermatids and confers adhesion to Sertoli cells in the semiferous epithelium. We found that loss of RA-GEF-2 altered the expression of N-cadherin which was thought to be a marker of Sertoli cell. CONCLUSIONS: RA-GEF-2 KO mice exhibited marked testicular atrophy and a severe defect in mature sperm formation. RA-GEF-2 may function as a regulator of N-cadrin during the process of spermatogenesis and that might be one of the main causes of male infertility in RA-GEF-2 knockout mice.