Dramatic reprogramming of gene expression occurs during embryonic genome activation (EGA), an essential event initiating as early as the 1-cell zygotic stage in the bovine and increasing gradually as embryonic development advances. It is this reprogramming of gene expression that sets the stage for later development. Expression of embryonic genes is altered in different culture conditions and this may influence developmental potential both during pre-implantation and during fetal development. The objective of this study was to define some most commonly used embryo culture media (KSOMaa, CR1aa, and SOFaa) based on their ability to support embryonic development to the blastocyst stage, mean cell number, percentages of apoptotic cells, and the expression patterns of a panel of developmentally important genes. Oocytes with several layers of cumulus cells obtained from an abattoir were matured in TCM 199 (supplemented with 0.25 mM pyruvate, 0.5 μg/mL FSH, 5 μg/mL LH, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% FCS) for 24 h and in vitro-fertilized (Day 0) using frozen bull semen. Presumptive zygotes were transferred into three different media (KSOMaa, CR1aa, and SOFaa) 16–18 h post-insemination, supplemented with 10% FCS on Day 4, and cultured until Day 8 at which time they were fixed or frozen for further analysis. Mean cell numbers and percentages of apoptotic cells in blastocysts were determined using terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL). Real-time quantitative PCR was performed to assess gene transcripts of glucose transporter-1 (Glut-1), heat shock protein 70.1 (Hsp70), interferon-tau (IF-tau), insulin-like growth factor II receptor (Igf-2r), desmosomal glycoprotein desmocollin III (DcIII), and DNA methyltransferase 3a (Dnmt3a). Gene expression data were analyzed relative to transcripts of housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (Gapdh). In three separate trials, a total of 538, 518, and 503 oocytes were used for KSOMaa, CR1aa, and SOFaa groups, respectively. Cleavage rates were 79.2%, 77.5%, and 80.2%; and rates of development to the blastocyst stage were 22.2%, 23.4%, and 32.9% for KSOMaa, CR1aa, and SOFaa groups, respectively. The blastocyst rate of the SOFaa group was significantly higher than those of the KSOMaa and CR1aa groups (P < 0.05). Mean cell numbers were 109.3, 101.0, and 114.0; and the percentages of apoptotic cell numbers per blastocyst were 1.25, 1.91, and 1.87 for KSOMaa, CR1aa, and SOFaa groups, respectively. There was no difference among groups in terms of mean cell numbers and percentages of apoptotic cells per blastocyst. The expressions of Glut-1 and DcIII genes did not differ among the groups. However, expressions of Hsp70, IF-tau, and Dnmt3a genes were all significantly up-regulated in the CR1aa group as compared to the SOFaa and KSOMaa groups (P < 0.05). In conclusion, SOFaa supports higher development to the blastocyst stage than KSOMaa and CR1aa, and culture conditions influence gene expression.
Rat spermatogenesis in vitro traced by quantitative flow cytometry.