Functional study of an acrosome-expressing gene, VAD1.2 in mice spermatogenesis

Abstract

Acrosome is a sac-like structure located at the head of spermatozoa, and its formation is regulated by a cascade of genes. VAD1.2 is an evolutionary-conserved acrosome-expressing protein 2 (AEP2) and the VAD1.2 mRNA is up-regulated on retinol-treated vitamin-A deficient rat testis. VAD1.2 transcript appears on day 32 in the postnatal rat testis and the protein is found at the acrosomal region in round spermatids and elongated spermatids. Yet, the functional role of VAD1.2 in spermatogenesis remains unknown. In the present study, the conditional targeting vector for VAD1.2 gene was constructed by DNA recombination technique in which the exons 2-5 of VAD1.2 was flanked by two LoxP sites. The targeting vector was linearized and electroporated into R1 ES cells. After selection with G418, one out of 192 clones was screened positively by 5'- and 3'-external probes with Southern blotting. The targeted ES cells which had normal karyotyping, were injected into C57BL/6 blastocysts, and 7 male chimeras were produced. Four chimeras after crossing with C57BL/6J mice showed germline transmission as detected by PCR analysis. Our preliminary data showed that crossing of the VAD1.2flox/+ mice with Sycp-Cre (expressing Cre at spermatocytes) transgenic mice produced heterozygous VAD1.2 mice with reduced sperm count in caudal epididymis (about 25% less than wild type) and reduced percentage of progressive motile spermatozoa (around 15% reduction). Our current findings suggested that VAD1.2 may play important role(s) in spermatogenesis and the VAD1.2 conditional knockout mice may help to delineate how VAD1.2 affects spermatogenesis and leading to infertility. [This work is supported in part by a CRCG grant to KFL.]