De novo transcription of thyroid hormone receptors is essential for early bovine embryo development in vitro.

Thyroid hormone receptor (THR) α and THRß mediate the genomic action of thyroid hormones (THs) that affect bovine embryo development. However, little is known about THRs in the preimplantation embryo. The aim of the present study was to investigate the importance of THRs in in vitro preimplantation bovine embryos. THR transcripts and protein levels were detected in developing preimplantation embryos up to the blastocyst stage. Embryonic transcription of THRs was inhibited by α-amanitin supplementation, and both maternal and embryonic transcription were knocked down by short interference (si) RNA microinjection. In the control group, mRNA and protein levels of THRs increased after fertilisation. In contrast, in both the transcription inhibition and knockdown groups there were significant (P<0.05) decreases in mRNA expression of THRs from the 2-cell stage onwards. However, protein levels of THRs were not altered at 2-cell stage, although they did exhibit a significant (P<0.05) decrease from the 4-cell stage. Moreover, inhibition of de novo transcripts of THRs using siRNA led to a significant (P<0.01) decrease in the developmental rate and cell number, as well as inducing a change in embryo morphology. In conclusion, THRs are transcribed soon after fertilisation, before major activation of the embryonic genome, and they are essential for bovine embryo development in vitro.

Enrichment of Y-chromosome-bearing bull spermatozoa by swim-up through a column.

With the advancement of assisted reproductive biotechnologies, preselecting the sex of offspring has become an important goal for cattle and other livestock breeding as well as for research. The aim of this study was to investigate the feasibility of producing enriched pools of X- or Y-chromosome-bearing sperm by vertical swim-up through a long, narrow column. Sperm recovered from the top portion of the column was predominantly Y-bearing (60%, p < 0.05), which were capable of fertilizing matured oocytes and produce significantly more male embryos compared with standard swim-up protocol.

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.