175 Testis-Specific Protein Y Encoded Copy Number Variation in In Vitro-Produced Bovine Early Embryos

Abstract

Testis-specific protein Y (TSPY) is one of the genes located in the male-specific region of the Y chromosome involved in male reproduction, particularly in spermatogenesis. It has been shown to have different copy number (CN) in different species, in different individuals within a species and in the same family (i.e. among brothers from the same father). Previous studies from our laboratory in the bovine species showed that different TSPY CN was correlated with fertility, as bulls with higher CN showed higher fertility rate. In this study, we investigated development rate in in vitro-produced (IVP) bovine early embryos produced by sex-sorted semen (X and Y) from 3 bulls (1, 2, and 3) and TSPY copy number variation (CNV) in the male blastocysts. The IVP embryos by the 3 different types of semen (sub-divided: 1X, 1Y, 2X, 2Y, 3X, 3Y) and parthenotes (PART) for the negative control were assessed for developmental rates (shown in Table 1 below) and individually collected at each cell stage from zygote to blastocyst to extract DNA for TSPY CN analysis. The DNA from individual blastocysts was treated with a restriction enzyme for relative quantification of TSPY CN using digital droplet PCR (ddPCR) with SRY, known to have only one copy in bovine, as a reference gene. Developmental data showed that Y-carrying semen had higher rates both in cleavage and blastocyst compared with X-carrying semen from the same bull. Particularly, bull 3 (both 3X and 3Y) had the highest developmental rates compared with the other 2 types of bulls. TSPY was detected in male blastocyst, where both parthenotes and female embryos were negative. Relative CN of TSPY versus SRY from blastocysts produced from bulls 1, 2, and 3 averaged to 87.5, 55, and 20, respectively. Interestingly, the CN among brother blastocysts showed a variation of less than 7.5, where bull-to-bull variation was approximately from 20 to 60 copies. Our results show that TSPY CN in the embryo varies according to the sire and among brother blastocysts. Moreover, TSPY CNV among brother blastocysts allows us to speculate that individually different recombination events occurred in meiosis during spermatogenesis.

Table 1.  Embryo development rate by semen type

This research was funded by the Canada Research Chair’s program, Natural Sciences and Engineering Research Council of Canada and the Ontario Veterinary College.