3 TESTIS-SPECIFIC PROTEIN, Y-ENCODED (TSPY) COPY NUMBER AND EXPRESSION IN BOVINE BLASTOCYSTS

Abstract

Testis-specific protein, Y-encoded (TSPY) is a gene found on the Y chromosome that, like many genes on the Y chromosome of mammalian species, has multiple copies. Humans have between 20–60 copies, whereas cattle can have up to 200 copies. Genomic copy number of TSPY is of interest because it has been linked to fertility. In previous studies, enormous bull-to-bull variation in TSPY copy number has been found (Hamilton et al. 2009 Sex. Dev. 3, 205–213). The aims of this study were to a) examine the copy number of TSPY in brother embryos to see if variation exists among embryos of a single generation and b) to determine if TSPY mRNA (mRNA) is expressed in the early bovine embryo since there is evidence from transgenic mice studies that human TSPY may play a role in development. 80 Holstein blastocysts were produced using standard in vitro fertilization techniques with the semen of a single bull. Individual blastocysts (n = 50) were lysed and quantitative polymerase chain reaction (qPCR) was used to sex the embryos and to measure TSPY copy number in the embryos that were found to be male. The male-specific TSPY and SRY (sex determining region Y) genes were measured in order to sex the embryos along with ZAR1 (zygote arrest protein 1), as an autosomal control. TSPY copy number was measured with a modified 2ΔΔCT method (Hamilton et al. 2009 Sex. Dev. 3, 205–213) and SRY as the single copy reference gene. Messenger RNA was extracted from two pools of blastocysts (n = 20, n = 10) and reverse transcribed into cDNA. The presence of TSPY mRNA in these pools was determined with reverse transcription PCR (RT–PCR) along with the reference gene GAPDH, following standard protocols. Briefly, two sets of TSPY specific primers and one set of GAPDH specific primers were used to amplify the target mRNAs in both pools of embryos using AmpliTaq Gold DNA polymerase (Applied Biosystems) and an annealing temperature of 60°C. PCR products were visualised on a 2% gel using gel electrophoresis and then sequenced to verify specificity of the primers. The results of the embryo sexing experiment showed a 50:50 sex ratio (25 males, 25 females) in the group of individual embryos. TSPY copy number was found to vary significantly (using a one-way ANOVA, P < 0.0001) among brother embryos produced from the semen of the same Hostein bull (n = 25) and ranged from 21.9 ± 7.1 to 159.0 ± 14.0 copies, with an average of 65.9 ± 6.3 copies. Furthermore, the results of the expression experiment showed that TSPY mRNA transcripts were present in both pools of blastocysts. These results provide the first evidence that TSPY is expressed in bovine blastocysts and that copy number can vary within a single generation. This data suggests that the bovine Y chromosome is a dynamic chromosome that is not clonally inherited but can vary its genetic composition within a single generation. The fact that TSPY transcripts were found in the early embryo suggests that it might have another function other than simply a role in spermatogenesis.