Novel transcripts and alternatively spliced genes are associated with early development in bovine embryos

Abstract

Infertility in cattle is a major concern of farmers worldwide and despite the enormous improvements in assisted reproduction technologies, the success rates of pregnancies are still low. Embryonic loss is considered one of the main factors of infertility in cattle. As such, the identification of genetic markers for embryo quality and development can help elucidate the molecular mechanisms involved in the formation of embryos with the highest developmental potential. In a previous study, using nextgeneration RNA sequencing, we identified novel transcripts and alternatively spliced genes that were associated with embryo quality. The objectives of this study were to characterize these transcripts and validate their expression in new biological replications of embryos using quantitative real-time PCR. Two types of embryos differing in morphological and developmental statuses (blastocysts and degenerate embryos) were produced using in vitro fertilization. Quantitative expression of eight novel transcripts revealed a range of 2.5-to 90-fold difference in expression between degenerate embryos and blastocysts. Some of these novel transcripts showed sequence similarity to human and cattle genes known to affect differentiation, growth and development. In addition, expression analysis of alternative splicing isoforms of five genes (MYL6, NOP10, RNF187, RPS24 and RPS28) revealed significant differential expression of these isoforms in the different embryo types. Thus, results of this study suggest that novel transcripts and alternatively spliced genes, found to be differentially expressed between blastocysts and degenerate embryos, can be used as markers for blastocyst formation and development.Keywords: embryo, fertility, gene expression, novel transcripts, alternative splicing ImplicationsOver the last 40 to 50 years there has been a significant decline in reproductive performance of dairy cattle, due in part to low fertilization rates and early embryonic loss. Although genetics plays an important role in embryo development, the identification of genes associated with embryo quality and pregnancy success has been a serious challenge. In this study, we characterized several genes that showed altered expression in preimplantation embryos that do not develop normally to the blastocyst stage. These genes can be used as candidate markers for embryo quality in cattle. IntroductionIn the last few decades, production of embryos by in vitro fertilization (IVF) has become a common practice for the treatment of infertility in both humans and cattle, and although IVF procedures are considered to be a true revolution in the field of fertility, the success rate of IVF pregnancies is still low. For example, in humans, , 8 out of 10 transferred embryos fail to implant successfully (Bromer and Seli, 2008), most likely because of the inability to identify embryos with the best development potential. In cattle, there has been a significant decline in fertility over the past few decades (Dobson et al., 2007;Leroy et al., 2008) because of low...

Characterization of microRNA in bovine in vitro culture media associated with embryo quality and development

“…The GAPDH gene was chosen as an internal control following the stability test described by Vandesompele et al (2002). In our previous studies of embryo samples (Huang et al, 2010;Zhang et al, 2012;Driver et al, 2013), the expression of GAPDH showed the smallest relative stability value M in comparison to RPLP0 (ribosomal protein, large, P0) and ACTB (actin, β). The M value is the average of the pair-wise variation when compared with the other control genes across the examined samples (Vandensompele et al, 2002).…”

Male fertility status is associated with DNA methylation signatures in sperm and transcriptomic profiles of bovine preimplantation embryos

“…Primers for qRT-PCR reactions were designed to span exon-exon junctions to minimize amplification of genomic DNA, where the sequences are listed in Additional file 1: Table S1. The reference gene, glyceraldehyde 3-phosphate dehydrogenase ( GAPDH) , was used as in our previous work [17, 23, 24] it was found to be the most stable in blastocyst embryos following a stability test described by Vandensompele et al [25]. Primers and cDNA were combined with a SYBR green mastermix (iQSYBR Green Supermix kit; Bio-Rad Laboratories, CA) and reactions were carried out using a BioRad iCycler.…”

“…The program generates a matrix with the consensus peaks; which have been determined from a “minimum overlap” of 3 (the number of replications in the experiment). After setting a contrast between conditions, DiffBind runs an edgeR analysis, which is an empirical Bayes method [24]. For normalization, the method trimmed mean of M-values (TMM) that subtracts the controls reads and considers the effective library size (reads in peaks), was applied.…”

miRNAs and Their Gene Targets—A Clue to Differentiate Pregnancies with Small for Gestational Age Newborns, Intrauterine Growth Restriction, and Preeclampsia

“…These results are consistent with previous reports on the identification of genes associated with blastocyst formation [ 61 ], among which was myosin light chain 6 (MYL6) gene. Two transcripts of the MYL6 gene, as a result of alternative splicing, showed a difference in expression in degenerate embryos compared with blastocysts [ 62 ]. MYL6 is an essential light chain of myosin II, which is an actin-binding protein expressed in smooth muscle and nonmuscle tissues.…”