89 METHYLATION STATUS OF IGF2/H19 DMR3 REGION AFFECTS IN VITRO BLASTOCYST PRODUCTION IN GOAT (CAPRA HIRCUS)

Abstract

Parthenogenesis has been observed in lower animals but no known instance has been reported in mammals because both maternal and paternal genomes are a fundamental prerequisite for embryogenesis. A major reason for developmental failure of uniparental zygotes is expression of certain genes in a parent-of-origin-specific manner, i.e. genomic imprinting of genes. Out of many imprinted genes identified so far, IGF2/H19 have been extensively studied and known to play an important role in fetal and placental development. Gene IGF2 is expressed by the paternal allele, H19 is transcribed from the maternal allele, and the reciprocal expression of both genes is regulated by the DMR3 region placed upstream of the H19 gene. In the present study we compared the methylation status of IGF2/H19 DMR in parthenogenetic activated (PA) and IVF goat (Capra hircus) blastocyst through bisulphite sequencing. For this, immature oocytes of usable quality were subjected to in vitro maturation and subsequently used for embryo production through parthenogenesis (n = 993) (by calcium ionophore and 6-DMAP activation) and IVF (n = 1096). It was found that embryo production rate at all the embryonic stages (2-cell, 4-cell, 8–16-cell, morula, and blastocyst) was significantly higher (P < 0.05) in parthenogenesis (74.66 ± 3.35%, 61.90 ± 2.73%, 47.83 ± 2.95%, 38.13 ± 5.28%, and 21.11 ± 2.51%, respectively) as compared with IVF (55.21 ± 2.02%, 38.12 ± 2.48%, 28.53 ± 1.67%, 21.57 ± 1.59%, and 8.23 ± 1.02%, respectively). When blastocysts (n = 6 each) were subjected to TUNEL, it was found that PA blastocyst showed significantly higher (P < 0.05) total cell number (217.83 ± 18.80 v. 159.67 ± 13.94) and significantly low (P < 0.05) apoptotic index (2.04 ± 0.25 v. 4.03 ± 0.29) as compared with IVF blastocysts. For the methylation pattern study, we analysed 17 CpG sites on the DMR3 region of the IGF2/H19 gene. Variable methylation pattern was observed within these CpG sites in different clones (n = 15) of PA and IVF blastocyst. The DMR3 region of the IGF2/H19 gene was significantly hypermethylated (P < 0.05) in PA blastocysts as compared with IVF blastocysts (80.39 ± 2.96, 32.55 ± 4.37, respectively), which suggests higher expression of IGF2 in parthenotes. The result suggests IGF2 might play different roles in different species; the same expression pattern of IGF2 is observed in ovine, but a contrary result is found in porcine species. Our results signify the hypermethylation of IGF2/H19 DMR3, which leads to higher expression of IGF2 to support embryonic development at the blastocyst stage.

This work was supported by the NFBSFARA Project on Parthenogenetic Goat (CA-4002), New Delhi, India.