A Major Source of Methyl Groups in Blastocysts May Be Betaine Stored by Pre-Implantation Embryos until Betaine Homocysteine Methyltransferase (BHMT) Is Transiently Expressed at High Levels in the Inner Cell Mass.

Methylation reactions, mediated by methyltransferases, are vital to many processes in cells. DNA methylation, which epigenetically regulates gene expression, is of particular interest during early embryogenesis, since it is responsible for maintaining the methylation of imprinted genes during preimplantation (PI) embryogenesis, and for the global de novo DNA methylation that occurs in blastocysts around implantation. All methylation processes use S-adenosylmethionine (SAM) as the universal methyl group donor. SAM is synthesized via two pathways. The well-known ubiquitous mechanism depends on folate and vitamin B12. The other uses betaine (glycine betaine; N',N',N'-trimethylglycine) as the methyl donor, catalyzed by Betaine-Homocysteine Methyltransferase (BHMT, EC 2.1.1.5). SAM synthesis from betaine via BHMT is generally considered to be liver-specific. We have recently shown that betaine is accumulated by 1- and 2-cell mouse embryos via the SIT1 (Slc6a9) transporter. One role of the accumulated betaine is in cell volume regulation at these stages. Here, we explore the hypothesis that betaine may also be stored for subsequent use as a methyl donor in PI embryos. Measurements of the endogenous betaine content of in vivo-collected PI embryos using a fluorescence-based HPLC method, and in vitro studies using 3H-labeled betaine, each indicated that betaine remains present until the blastocyst stage. For betaine to be a methyl donor, BHMT must be expressed and active. Quantitative RT-PCT (qRT-PCR) showed that BHMT mRNA was expressed from the 4-cell through blastocyst stages. Expression peaked with very high transcript levels at the morula stage, decreasing sharply in blastocysts. We next determined whether BHMT protein was expressed. Immunofluorescence (IF) using a polyclonal anti-BHMT antibody revealed a dramatic increase in BHMT protein in the inner cell masses of blastocysts. In addition, blastocysts produced from embryos cultured with an antisense morpholino targeted against BHMT exhibited a much-reduced IF level, while a control morpholino had no effect. Western blots confirmed the presence of a band of the expected size for BHMT in blastocysts but not 2-cell embryos or morulae. Because of the substantial decrease in mRNA at the blastocyst stage, we hypothesized that BHMT protein expression might be transient. We thus further cultured blastocysts on fibronectin with serum, which allows trophectoderm to adhere, spread, and proliferate while maintaining a compact epiblast, mimicking early implantation. BHMT protein decreased in 24 hours and was detectable by IF for only for 48 hours after blastocyst hatching, indicating that BHMT expression may be restricted to blastocysts in early embryogenesis. To directly demonstrate BHMT activity in blastocysts, we developed a microassay that revealed high BHMT activity in blastocysts but none in morulae. Specificity was confirmed using CBHcy, a specific BHMT inhibitor. Furthermore, BHMT activity was reduced nearly to background in embryos cultured with the anti-BHMT morpholino, while the control morpholino had no effect. In summary, these results imply that betaine, accumulated by the 2-cell stage, may be stored by PI embryos and utilized to produce a methyl pool via BHMT in the blastocyst. The role of this methyl pool, and the consequences of disrupting BHMT activity in blastocysts, remains to be determined. Supported by Canadian Institutes of Health Research operating grant MOP74515. (platform)

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