Alkaline phosphatase isozyme expression in preattachment bovine embryos
The majority of loss of bovine embryos occurs during preattachment development, possibly due to faulty gene expression from the embryonic genome. Markers for successful transfer of control of development from maternal to embryonic genome are lacking in cattle. The mouse is the most extensively studied mammalian embryo model, and it was found that alkaline phosphatases (AP) are expressed differentially during embryogenesis, suggesting that APs would be a good model for examining embryonic genome transcription during bovine embryogenesis. Bovine embryos were produced in vitro and in vivo, mRNA expression was determined using reverse transcription and polymerase chain reaction and protein was detected by an azo-dye coupling technique. Tissue specific AP 2 (TSAP2) mRNA was detected at the 4-cell stage in vitro and morula stage in vivo. Tissue specific AP 3 (TSAP3) mRNA was detected at the 8-cell stage in vitro and 16-cell stage in vivo. Tissue non-specific AP (TNAP) was detected only in late blastocysts, and intestinal AP (IAP) only sporadically in late blastocysts in vitro. A cyclic pattern of transcription was detected for TSAP2 and TSAP3. Examination with cell-cycle inhibitors, which act by disrupting microtubules of embryos, affected expression, while DNA polymerase inhibition did not, suggesting that these transcripts are sensitive to microtubule status for detection, expression or storage. Embryos produced in vivo showed very low AP activity until the blastocyst stage, when activity was found only on trophectoderm cells. Embryos produced in vitro showed AP protein localized to areas of blastomere contact during early stages, then only on trophectoderm cells at the blastocyst stage. Alkaline phosphatase activity is due to maternal TNAP protein up to the 16-cell stage, with a switch to embryonic genome derived TSAP during later preattachment development. The differences in expression of TSAP2 and TSAP3 between in vitro and in vivo produced embryos and after treatment with microtubule inhibiting drugs suggest that culture does not accurately mimic the in vivo environment, and that in vitro manipulations cause changes in gene expression.