Phenotypic analysis of Tspyl1 knockout mice

Abstract

Testis-specific protein, Y-encoded like 1 (TSPYL1) belongs to the nucleosome assembly protein superfamily. TSYPL1 has been associated with sudden infant death with dysgenesis of testes syndrome and male infertility. Recent studies have also shown that TSPYL1 transcriptionally regulates the expression of multiple cytochrome P450 genes and SLC6A4 in cell lines. However, the biological role of TSPYL1 remains elusive. To explore the biological importance of TSPYL1, our lab has previously generated two mutant mouse lines on the CBA/Ca mixed C57BL/6N genetic background (named Δ13 and Δ23) and one mouse line on an inbreed C57BL/6N background (named Δ463). Preliminary data revealed that these knockout (KO) mice suffered from premature death. Surviving KO mice attained normal size, but they rarely reproduced. Here I investigated the possible functions of TSPYL1 in cell proliferation and homeostasis using littermate wild-type (WT) and KO mouse embryonic fibroblasts (MEFs). Tspyl1 KO MEFs showed an earlier replication arrest compared with WT MEFs. Cell proliferation was arrested at passage 5 in KO MEFs compared to passage 6 in WT MEFs. Moreover, KO MEFs had significantly increased percentages of cell death when counted at 8, 24 and 48 hours of amino acid starvation. Western analysis revealed that the LC3-II level and cleaved caspase-3 level were already increased in KO compared to WT MEFs at 8 hours of starvation, indicating an increase in autophagy and apoptotic activity. The data suggested that TSPYL1 is important in regulating cell proliferation and autophagy. Both male and female KO mice were infertile and we focused on males here. To investigate the causes of infertility in Tspyl1 KO male mice, I examined the histology of testes, sperm motility and mating behavior. Expression of steroidogenic genes in the testes and the level of serum testosterone were also measured. Even though the transcript levels of steroidogenic genes (StAR, Cyp11a1, Cyp17a1, Hsd3b6, and Hsd17b3) were significantly reduced in KO testes, testes histology revealed complete spermatogenesis and the serum testosterone level of KO males remained within the normal range. Sperms collected from cauda epididymis of KO males had impaired sperm motility, with a significantly greater percentage of immotile sperms. To account for the fact that KO male mice seldom sire offspring, mating tests were performed. KO males were severely impaired in mating behavior and rarely plugged receptive females. Taken together, the data suggest that TSPYL1 is crucial in male fertility. To conclude, my study illustrated that TSPYL1 is involved in cell proliferation and autophagy. In mice, TSPYL1 is critical in sperm maturation and mating behavior.