Assisted reproductive technology (ART) has led to the birth of millions of babies. In cattle, thousands of embryos are produced annually. However, since the introduction and widespread use of ART, negative effects on embryos and offspring are starting to emerge. Knowledge so far, mostly provided by animal models, indicates that suboptimal conditions during ART can affect embryo viability and quality, and may induce embryonic stress responses. These stress responses take the form of severe gene expression alterations or modifications in critical epigenetic marks established during early developmental stages that can persist after birth. Unfortunately, while developmental plasticity allows the embryo to survive these stressful conditions, such insult may lead to adult health problems and to long-term effects on offspring that could be transmitted to subsequent generations. In this review, we describe how in mice, livestock, and humans, besides affecting the development of the embryo itself, ART stressors may also have significant repercussions on offspring health and physiology. Finally, we argue the case that better control of stressors during ART will help improve embryo quality and offspring health. K E Y W O R D S assisted reproductive technologies, bovine, embryo, human, mouse, stress

Most current knowledge of sex determination in mammals has emerged from mouse and human studies. To investigate the molecular regulation of the sex determination process in cattle, we used an RNA sequencing strategy to analyze the transcriptome landscape of male and female bovine fetal gonads collected in vivo at key developmental stages: before, during, and after SRY gene activation on fetal days D35 (bipotential gonad formation), D39 (peak SRY expression), and D43 (early gonad differentiation). Differentially expressed genes (DEGs) were identified in male vs. female germinal ridges and among group genes showing similar expression profiles during the three periods. There were 143, 96, and 658 DEG between males and female fetuses at D35, D39, and D43, respectively. On D35, genes upregulated in females were enriched in translation, nuclear export, RNA localization, and mRNA splicing events, whereas those upregulated in males were enriched in cell proliferation regulation and male sex determination terms. In time-course experiments, 767 DEGs in males and 545 DEGs in females were identified between D35 vs. D39, and 3157 DEGs in males and 2008 in females were identified between D39 vs. D43. Results highlight unique aspects of sex determination in cattle, such as the expression of several Y chromosome genes (absent in mice and humans) before SRY expression and an abrupt increase in the nuclear expression of SOX10 (instead of SOX9 expression in the Sertoli cell cytoplasm as observed in mice) during male determination and early differentiation.

BackgroundAlternative splicing (AS) may play an important role in gonadal sex determination (GSD) in mammals. The present study was designed to identify differentially expressed isoforms and AS modifications accompanying GSD in mice.ResultsUsing deep RNA-sequencing, we performed a transcriptional analysis of XX and XY gonads during sex determination on embryonic days 11 (E11) and 12 (E12). Analysis of differentially expressed genes (DEG) identified hundreds of genes related to GSD and early sex differentiation that may represent good candidates for sex reversal. Expression at time point E11 in males was significantly enriched in RNA splicing and mRNA processing Gene Ontology terms. Differentially expressed isoform analysis identified hundreds of specific isoforms related to GSD, many of which showed no differences in the DEG analysis. Hundreds of AS events were identified as modified at E11 and E12. Female E11 gonads featured sex-biased upregulation of intron retention (in genes related to regulation of transcription, protein phosphorylation, protein transport and mRNA splicing) and exon skipping (in genes related to chromatin repression) suggesting AS as a post-transcription mechanism that controls sex determination of the bipotential fetal gonad.ConclusionOur data suggests an important role of splicing regulatory mechanisms for sex determination in mice.Electronic supplementary materialThe online version of this article (10.1186/s12864-019-5572-x) contains supplementary material, which is available to authorized users.

Minor splicing plays an important role in vertebrate development. Zrsr1 and Zrsr2 paralog genes have essential roles in alternative splicing, mainly participating in the recognition of minor (U12) introns. To further explore their roles during early embryo development, we produced Zrsr1mu and Zrsr2mu mutant mice, containing truncating mutations within the second zinc finger domain. Both homozygous mutant mice were viable with a normal lifespan. When we crossed a homozygous Zrsr2mu/mu female with Zrsr1mu/mu male, the double heterozygotes were non-viable, giving rise to embryos that stopped developing mainly between the 2- and 4-cell stages, just after zygotic gene activation. RNA-seq analysis of Zrsr1/2mu 2-cell embryos showed altered gene and isoform expression of thousands of genes enriched in gene ontology terms and biological pathways related to ribosome, RNA transport, spliceosome, and essential zygotic gene activation steps. Alternative splicing was analyzed, showing a significant increase in intron retention in both U2 and U12 intron-containing genes related to cell cycle and mitotic nuclear division. Remarkably, both Zrsr1 and Zrsr2 were required for the conversion of mouse-induced pluripotent stem cells into 2C-like cells. According to our results, Zrsr1 or Zrsr2 are necessary for ZGA and both are indispensable for the conversion of induced pluripotent stem cells into 2C-like cells.

TRRAP, PIK3CB), which are known to play a key role in the regulation of biological processes that have high metabolic demand and are related to cell growth and regeneration, metabolism, and immune function. The genes identified and their associated functional variants may serve as candidate genetic markers and can be implemented into breeding programs to help improve the selection for feed efficiency in dairy cattle.