Small RNA-seq analysis of single porcine blastocysts revealed that maternal estradiol-17beta exposure does not affect miRNA isoform (isomiR) expression

Abstract

BackgroundThe expression of microRNAs (miRNAs) is essential for the proper development of the mammalian embryo. A maternal exposure to endocrine disrupting chemicals during preimplantation bears the potential for transgenerational inheritance of disease through the epigenetic perturbation of the developing embryo. A comprehensive assembly of embryo-specific miRNAs and respective isoforms (isomiR) is lacking to date. We aimed at revealing the sex-specific miRNA expression profile of single porcine blastocysts developing in gilts orally exposed to exogenous estradiol-17 (E2). Therefore we analyzed the miRNA profile specifically focusing on isomiRs and potentially embryo-specific miRNAs.ResultsDeep sequencing of small RNA (small RNA-seq) result in the detection of miRNA sequences mapping to known and predicted porcine miRNAs as well as novel miRNAs highly conserved in human and cattle. A set of highly abundant miRNAs and a large number of rarely expressed miRNAs were identified by using a small RNA analysis pipeline, which was integrated into a novel Galaxy workflow specifically benefits incompletely annotated species. In particular, orthologue species information increased the total number of annotated miRNAs, while mapping to other non-coding RNAs avoided falsely annotated miRNAs. Neither the low nor the high dose of E2 treatment (10 and 1000 µ E2/kg body weight daily, respectively) affected the miRNA profile in blastocysts despite the distinct differential mRNA expression and DNA methylation found in previous studies. The high number of generated sequence reads enabled a comprehensive analysis of the isomiR repertoire showing various templated and non-templated modifications. Furthermore, potentially blastocyst-specific miRNAs were identified.ConclusionsIn pre-implantation embryos, numerous distinct isomiRs were discovered indicating a high complexity of miRNA expression. Neither the sex of the embryo nor a maternal E2 exposure affected the miRNA expression profile of developing porcine blastocysts. The adaptation to the continuous duration of the E2 treatment might explain the lack of an effect.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4954-9) contains supplementary material, which is available to authorized users.

Sex Determination Using RNA-Sequencing Analyses in Early Prenatal Pig Development

“…Several studies have also used Y chromosome-linked genes to determine the sex of pigs at early development stages [13,18,19], however, all of them have applied DNA-based approaches. The Y chromosome harbors genes that are essential for testis development and function, such as the master gene for testis determination (SRY), which is expressed during a critical period of embryonic development, and other genes that are important for spermatogenic function, as the ZFY, EIF1AY and KDM5D.…”

“…Several studies have demonstrated how to identify the conceptuses sex at early stages of development using molecular techniques, as PCR (polymerase chain reaction) from Y chromosome-linked genes [13,[18][19][20]. The PCR provides sensitive, precise, rapid, and reliable results [20].…”

MiRNAs in the Peri-Implantation Period: Contribution to Embryo–Maternal Communication in Pigs

“…1 EVs-extracellular vesicles; 2 ULFs-uterine luminal flushings; 3 CM-conditioned media Intriguingly, porcine endometria [23] as well as blastocysts [38] are abundant not only in canonical miRNAs but also isomiRs-widely reported variants of miRNAs-which have been proven to work cooperatively [43]. Many miRNAs can be co-expressed with their isomiRs and/or other members of the family (e.g., 23a/b, 148a/b in the porcine endometrium), resulting in wider or stronger biological effects.…”

Small RNA-seq analysis of exosomes from porcine uterine luminal fluid during peri-implantation

“…A previous study has shown that highly expressed miRNAs may play vital roles in the regulation of embryo implantation [30]. The expression of top 20 most highly expressed known miRNAs in ULF exosomes was compared with that in endometrium [31] and embryo [29] collected on D10. Among these miRNAs, ssc-miR-21-5p was the most abundant in all libraries.…”

“…To determine whether the expression of miRNAs in porcine ULF exosomes is related to that of miRNAs in endometrium and embryos during peri-implantation period, the expression of top 20 highly expressed known miRNAs in ULF exosomes was compared with that in embryos and endometrium at D10 [29]. The results indicated that most of these miRNAs were expressed in both the embryo and endometrium (Additional file 6: Table S5).…”