β-1,4-Galactosyltransferase Expression during Spermatogenesis: Stage-Specific Regulation by t Alleles and Uniform Distribution in +-Spermatids and t-Spermatids

Abstract

In the mouse, gamete recognition is mediated in part by the binding of sperm surface β1,4-galactosyltransferase (GalTase) to specific oligosaccharide residues on the zona pellucida glycoprotein ZP3 (D. J. Miller, M. B. Macek, and B. D. Shur. Nature 357, 589-593, 1992). The expression of GalTase on the sperm surface is regulated by alleles within the distal segment of the T/t complex and results in a haploid-specific increase in GalTase expression on spermatids and sperm from t-bearing males, suggesting that differences in sperm GalTase activity may contribute to t-sperm transmission ratio distortion (B. D. Shur and N.F. Scully. Genet. Res. Camb. 55, 177-181, 1990). In this study, we characterized the expression of GalTase RNA during wild-type and T/t-mutant spermatogenesis and analyzed the potential role of GalTase in transmission ratio distortion. Using northern blot analysis, S1 nuclease protection assays, and in situ hybridization, it was found that spormatogenic cells predominantly express the long form of the GalTase RNA, which encodes the GalTase protein that is preferentially targeted to the cell surface in somatic cells. In wild-type testes, GalTase RNA accumulates during the maturation of primary spermatocytes, reaches peak levels prior to meiosis, and decreases at meiosis. GalTase RNA accumulates to similar levels during the maturation of +/ t and t/t primary spermatocytes, but unlike wild-type, the level of GalTase RNA in t-bearing spermatocytes remains elevated during meiotic division. Consequently, spermatids in t-mutant testes inherit higher levels of GalTase RNA than do wildtype spermatids, which likely accounts for the haploid-specific increase in surface GalTase activity characteristic of spermatids from t-bearing mice. The functional significance of the increased GalTase activity during t-sperm transmission ratio distortion was determined by examining the distribution of GalTase RNA and surface GalTase protein in haploid spermatids from heterozygous +/ t males. Result show that + and t spermatids have similar levels of GalTase RNA assayed by quantitative in situ hybridization and similar levels of surface GalTase protein assayed by PCR genotyping of spermatids separated by fluorescence-activated cell sorting. These results indicate that although the expression of GalTase is regulated by alleles within the distal segment of the T/t complex, transmission ratio distortion in +/ t mice is not likely due to haploid-specific differences in sperm surface GalTase activity.