title: Quantitative mRNA expression in ovine blastocysts produced from X- and Y-chromosome bearing sperm, both in vitro and in vivo.
abstract: Artificial insemination (AI) of sex-sorted sperm results in decreased fertility, compared with non-sorted sperm, in most species. However, this has not been the case in sheep, where the low-dose AI of sex-sorted ram sperm produced similar, if not superior, fertility to non-sorted controls. The aim of the present study was to determine the impact of sex-sorting technology on ovine embryo gene expression following embryo production in vivo and in vitro. After semen collection, ejaculates were split and either sex-sorted by flow cytometry and frozen, or diluted and frozen. Embryos were produced in vivo by inseminating superovulated ewes with either X- or Y-chromosome enriched sperm, or non-sorted control sperm, and collected by uterine flushing on Day 6 after AI. Embryos were produced in vitro using the same sperm treatments and cultured in vitro for 6 d. The relative abundance of selected gene transcripts was measured in high-grade blastocysts, defined by morphological assessment, using RT-qPCR. The mRNA expression of DNMT3A and SUV39H1 was upregulated in embryos cultured in vitro, compared to those cultured in vivo (DNMT3A: 3.61 ± 1.08 vs 1.99 ± 0.15; SUV39H1: 1.88 ± 0.11 vs 0.88 ± 0.07; mean ± SEM; P < 0.05). Both G6PD and SLC2A3 transcripts were reduced in embryos produced from sex-sorted sperm, in vivo (SLC2A3: 0.23 ± 0.03 vs 0.64 ± 0.10; G6PD: 0.32 ± 0.04 vs 1.01 ± 0.16; P < 0.05). The expression of DNMT3A was up-regulated in male (3.85 ± 0.31), compared to female embryos (2.34 ± 0.15; P < 0.05). This study contributes to the growing body of evidence citing aberrant patterns of gene expression resulting from in vitro culture. Whereas the process of sex-sorting altered the expression of several of the genes examined, no effect on embryo development was detected.
authors.affiliation: Faculty of Veterinary Science, The University of Sydney, NSW 2006, Australia. k.beilby@usyd.edu.au
authors.forename: K H
authors.initials: KH
authors.lastname: Beilby
authors.forename: S P
authors.initials: SP
authors.lastname: de Graaf
authors.forename: G
authors.initials: G
authors.lastname: Evans
authors.forename: W M C
authors.initials: WM
authors.lastname: Maxwell
authors.forename: S
authors.initials: S
authors.lastname: Wilkening
authors.forename: C
authors.initials: C
authors.lastname: Wrenzycki
authors.forename: C G
authors.initials: CG
authors.lastname: Grupen
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry / Q000662:veterinary; D020869:Gene Expression Profiling; D007315:Insemination, Artificial / Q000662:veterinary; D008297:Male; D012333:RNA, Messenger / Q000378:metabolism*; D012732:Sex Determination Analysis / Q000662:veterinary; D012756:Sheep / Q000196:embryology / Q000235:genetics*; D013094:Spermatozoa / Q000378:metabolism*; D014960:X Chromosome; D014998:Y Chromosome
chemical_list: D012333:RNA, Messenger