Testis-Specific Protein Y-Encoded (TSPY) Is Required for Male Early Embryo Development in Bos taurus

TSPY is a highly conserved multi-copy gene with copy number variation (CNV) among species, populations, individuals and within families. TSPY has been shown to be involved in male development and fertility. However, information on TSPY in embryonic preimplantation stages is lacking. This study aims to determine whether TSPY CNV plays a role in male early development. Using sex-sorted semen from three different bulls, male embryo groups referred to as 1Y, 2Y and 3Y, were produced by in vitro fertilization (IVF). Developmental competency was assessed by cleavage and blastocyst rates. Embryos at different developmental stages were analyzed for TSPY CN, mRNA and protein levels. Furthermore, TSPY RNA knockdown was performed and embryos were assessed as per above. Development competency was only significantly different at the blastocyst stage, with 3Y being the highest. TSPY CNV and transcripts were detected in the range of 20-75 CN for 1Y, 20-65 CN for 2Y and 20-150 CN for 3Y, with corresponding averages of 30.2 ± 2.5, 33.0 ± 2.4 and 82.3 ± 3.6 copies, respectively. TSPY transcripts exhibited an inverse logarithmic pattern, with 3Y showing significantly higher TSPY. TSPY proteins, detected only in blastocysts, were not significantly different among groups. TSPY knockdown resulted in a significant TSPY depletion (p < 0.05), with no development observed after the eight-cell stage in male embryos, suggesting that TSPY is required for male embryo development.