267 CLASSICAL AND NON-CLASSICAL MAJOR HISTOCOMPATABILITY COMPLEX CLASS I GENE EXPRESSION IN IN VITRO-DERIVED BOVINE EMBRYOS

Abstract

The major histocompatibility complex class I (MHC-I) genes have been shown to play a role in the establishment of pregnancy in humans and mice. In particular, the secretion of the soluble isoform of the human non-classical HLA-G gene during in vitro embryo culture has been linked to higher pregnancy rates following embryo transfer. In contrast to the situation in humans, knowledge of the bovine MHC-I is quite limited. The objective of the current study was to characterize MHC-I gene expression during bovine pre-implantation development in vitro. Immature bovine oocytes were matured, fertilized, and cultured in vitro. Three replicate pools of 20 samples were collected at immature and mature oocyte, presumptive zygote, 2–4 cell, 8–16 cell, morula, blastocyst, and hatched blastocyst stages. Relative MHC-I mRNA expression levels were quantified using quantitative real-time PCR across pre-implantation development with a generic primer pair for global classical and non-classical MHC-I gene expression and primer pairs for two known non-classical MHC-I genes, N*50001 and N*50101 (NCBI accession numbers: X80936 and AY188807), cDNA sequencing, and wholemount immunocytochemistry with a Pan MHC-I-protein antibody (ILA88). MHC-I mRNA transcripts amplified by the generic primers were detected in greatest abundance in the oocyte and cumulus cells of immature oocytes (P < 0.05); following maturation, the expression levels decreased by 5-fold but remained stable until the 2- to 4-cell stage. Levels remained low until the morula and blastocyst stages when a small increase in relative expression was detected. Sequence analysis of oocyte and blastocyst PCR products amplified by the generic primers was carried out. The generic primers amplified a total of 6 discrete sequences, which are listed on the bovine MHC database (http://www.ebi.ac.uk/ipd/mhc/bola): (i) N*02601, (ii) N*02301, (iii) N*01301, (iv) N*04001, (v) N*01601, and (vi) non-classical N*5003. The mRNA expression pattern of Gene X differed from the global MHC-I mRNA profile in that mRNA abundance was highest in the matured oocytes (P < 0.05), after which abundance decreased 5-fold in the zygote and was not detected in the subsequent embryo stages until the morula and blastocyst stages. Sequence analysis confirmed that the PCR products were Gene X. Relative HD15 mRNA expression was highest in immature oocytes (P < 0.05); however, there was a steep decrease in abundance following oocyte maturation (P < 0.05), followed by a transient increase at the presumptive zygote stage (P < 0.05). Thereafter, expression levels remained low up to the morula and blastocyst stages. Sequence analysis confirmed that the PCR products were HD15. MHC-I protein expression was detected in immature and mature oocytes and their surrounding granulosa cells, in 2- and 4-cell embryos, and in blastocysts. In conclusion, these data demonstrate that classical and non-classical MHC-I genes are expressed in bovine oocytes and embryos at both the mRNA and the protein levels, and the expression pattern is gene- and stage-specific.