Testis-specific protein Y-encoded copy number is correlated to its expression and the field fertility of Canadian Holstein bulls.

Testis-specific protein Y-encoded (TSPY) is present in varying copy number in both human (20-76 copies) and cattle (37-200 copies), and some studies have linked this variation to semen quality in men. The purpose of this study was to determine if TSPY copy number is associated with fertility in bulls by using adjusted non-return rates, a commonly used measure of field fertility in Canada. In addition, we investigated the associations between TSPY copy number and its expression as well as specific semen parameters, such as average sperm concentration, sperm count, ejaculate volume, and motility. In 2 independent trials, TSPY copy number was shown to be positively correlated to adjusted non-return rates (trial #1: Spearman r = 0.34, p < 0.05; trial #2: Spearman r = 0.77, p < 0.01). Furthermore, TSPY copy number was inversely correlated to TSPY mRNA expression in the testis (Pearson r = -0.71, p < 0.0001). There were no correlations of TSPY copy number or expression with the semen parameters measured. Therefore, TSPY copy number might represent a potential marker of bull fertility, but its mechanism does not appear to be directly related to the semen characteristics analyzed as part of this study.

The effects of substituting glassware for plasticware and the use of an ethanol vector on oocyte maturation in vitro.

The intent of this study was to evaluate specific technical aspects of in vitro oocyte maturation (IVM), which included container material and solvent delivery vector. Oocytes were matured in oil-free, open-well systems contained in either plastic or glass dishes and compared to control oocytes matured in media droplets on plastic dishes overlaid with mineral oil. Open-well experiments were repeated with ethanol in a quantity sufficient for delivery of nonmiscible compounds. Cleavage rates were significantly decreased in the glassware system when compared to controls. The plasticware open-well system did not differ from either the controls or the glassware groups. Cleavage in glassware with ethanol was significantly lower than controls or plasticware with ethanol. Blastocyst rates were only decreased in the glassware-ethanol treatment when compared to plasticware-ethanol treatment. Cell counts and percentage of TUNEL-positive cells did not differ significantly. Unexpectedly, sex ratio was significantly decreased (34% male) from the expected value of 50% male in the glassware group with added ethanol. The current study demonstrates the sensitivity of IVM to subtle technical changes, resulting in significant developmental consequences.

A novel approach to sexing bovine blastocysts using male-specific gene expression.

When examining gene expression profiles for the purposes of assessing embryo quality, it is imperative that sex be considered, because many embryonic transcripts have sex-related expression patterns. The objective of this study was to systematically examine eight Y chromosome linked genes (DDX3Y, EIF1AY, HSFY, SRY, TSPY, USP9Y, ZFY, and ZRSR2Y) to characterize their expression in bovine blastocysts and to examine the usefulness of this expression for the purpose of RNA-based embryo sexing. In order to examine the expression of these genes, pools of blastocysts (groups of 10 and 20) as well as single embryos (N = 50) were analyzed with reverse transcriptase polymerase chain reaction (RT-PCR) and reverse transcriptase quantitative PCR (RT-qPCR). Of the 50 single embryos, 32 were concurrently sexed with DNA-based methods. Transcripts of DDX3Y, EIF1AY, TSPY, USP9Y, ZFY and ZRSR2Y were detected in the pooled and single blastocysts, but no transcripts were detected for HSFY or SRY. After performing DNA-based sexing experiments, we concluded that this expression was restricted to the male embryos. The consistency of the expression varied according to the gene as well as the specific primer set. Three genes were expressed in the full set of male embryos, DDX3Y, USP9Y, and ZRSR2Y and therefore represent good candidates for RNA-based sexing methods.

Copy number variation of testis-specific protein, Y-encoded (TSPY) in 14 different breeds of cattle (Bos taurus).

Multi-copied gene families are prevalent in mammalian genomes, especially within the Y chromosome. Testis specific protein Y-encoded (TSPY) is present in variable copy number in many mammalian species. Previous studies have estimated that TSPY ranges from 50-200 copies in cattle. To examine TSPY localization on the Y chromosome we employed fluorescence in situ hybridization (FISH) and fiber-FISH. The results show a strong signal on the short arm of the Y chromosome (Yp). To investigate TSPY copy number we used relative real-time polymerase chain reaction (PCR) to analyze the DNA of 14 different cattle breeds. Variation both within and between breeds was observed. All breeds show significant variation in TSPY copy number among individual members. Brown Swiss (161 copies, CI = 133-195) had higher average levels of TSPY and Western Fjord Cattle (63 copies, CI = 45-86) had lower levels than some breeds. Overall, however, most breeds had a similar average TSPY copy number. The pooled average was 94 copies (CI = 88-100). The significance of the TSPY array remains uncertain, but as the function of TSPY is unraveled the purpose of the array may become clearer.