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Spermatogenesis is a complicated process involving mitotically proliferating spermatogonial cells, meiotically dividing spermatocytes, and spermatid going through maturation into spermatozoa. The post-translational modifications of proteins play important roles in this biological process. <i>S</i>-palmitoylation is one type of protein modifications catalyzed by zinc finger Asp-His-His-Cys (ZDHHC)-family palmitoyl <i>S</i>-acyltransferases. There are 23 mammalian ZDHHCs that have been identified in mouse. Among them, <i>Zdhhc19</i> is highly expressed in adult testis. However, the in vivo function of <i>Zdhhc19</i> in mouse spermatogenesis and fertility remains unknown. In this study, we knocked out the <i>Zdhhc19</i> gene by generating a 2609 bp deletion from exon 3 to exon 6 in mice. No differences were found in testis morphology and testis/body weight ratios upon <i>Zdhhc19</i> deletion. Spermatogenesis was not disrupted in <i>Zdhhc19</i> knockout mice, in which properly developed TRA98+ germ cells, SYCP3+ spermatocytes, and TNP1+ spermatids/spermatozoa were detected in seminiferous tubules. Nevertheless, <i>Zdhhc19</i> knockout mice were male infertile. <i>Zdhhc19</i> deficient spermatozoa exhibited multiple defects including abnormal morphology of sperm tails and heads, decreased motility, and disturbed acrosome reaction. All of these led to the inability of <i>Zdhhc19</i> mutant sperm to fertilize oocytes in IVF assays. Taken together, our results support the fact that <i>Zdhhc19</i> is a testis enriched gene dispensable for spermatogenesis, but is essential for sperm functions in mice.