Differential alternative splicing in brain regions of rats selected for aggressive behavior

Profiles of alternative mRNA isoforms have been determined in three brain regions of rats from an aggressive and a tame line selected for 74 generations. Among 2319 genes with alternatively spliced exons, approximately 84% were confirmed by analyzing public databases. Based on Gene Ontology-guided clustering of alternatively spliced genes, it has been found that the sample was enriched in synapse-specific genes (FDR < 10^–17). Patterns of gene expression in the brains of animals with genetically determined high or low aggression were more frequently found to differ in the use of alternatively spliced exons than in animals environmentally conditioned for increased or lowered propensity to aggression. For the Adcyap1r1 gene, five alternatively spliced mRNA isoforms have been represented differentially in aggressive animals. A detailed analysis of the gene that encodes glutamate ionotropic receptor NMDA type subunit 1 (Grin1) has confirmed significant differences in the levels of its alternatively spliced isoforms in certain brain regions of tame and aggressive rats. These differences may affect the behavior in rats genetically selected for aggression levels.     

Knockdown of CDKN1C (p57kip2) and PHLDA2 Results in Developmental Changes in Bovine Pre-implantation Embryos

Imprinted genes have been implicated in early embryonic, placental, and neonatal development and alterations in expression levels of these genes can lead to growth abnormalities and embryonic lethality. However, little is known about the functions of bovine imprinted genes during the pre-implantation period. Therefore, the objective of this study was to assess the influence of altered expression of imprinted genes on developmental progress of embryos using small interfering RNA (siRNA). Expression levels of 18 imprinted genes (MAGEL2, UBE3A, IGF2R, NAP1L5, TSSC4, PEG3, NDN, CDKN1C, PHLDA2, MKRN3, USP29, NNAT, PEG10, RTL1, IGF2, H19, MIM1, and XIST) were compared between embryos reaching the blastocyst stage and growth-arrested embryos (degenerates) using quantitative real-time PCR (qRT-PCR). Ten genes were found to be differentially expressed between blastocysts and degenerates. The CDKN1C gene showed the highest upregulation in blastocysts whereas PHLDA2 was highly expressed in degenerates. To assess whether the observed differential gene expression was causative or resultant of embryo degeneration, these genes were selected for functional analysis using siRNA. Injection of siRNA specific to PHLDA2 into one-cell zygotes resulted in a substantial increase in blastocyst development, whereas injection of CDKN1C-specific siRNA resulted in a 45% reduction (P = 0.0006) in blastocyst development. RNA-Seq analysis of CDKN1C-siRNA-injected vs. non-injected embryos revealed 51 differentially expressed genes with functions in apoptosis, lipid metabolism, differentiation, and cell cycle regulation. Gene ontology analysis revealed nine pathways related to cell signaling, metabolism, and nucleic acid processing. Overall, our results show that proper expression levels of the imprinted genes CDKN1C and PHLDA2 are critical for embryo development, which suggests that these genes can be used as markers for normal blastocyst formation.     

Follistatin supplementation during in vitro embryo culture improves developmental competence of bovine embryos produced using sex-sorted semen

Using sex-sorted semen to produce offspring of desired sex is associated with reduced developmental competence in vitro and lower fertility rates in vivo. The objectives of the present study were to investigate the effects of exogenous follistatin supplementation on the developmental competence of bovine embryos produced with sex-sorted semen and possible link between TGF-β regulated pathways and embryotrophic actions of follistatin. Effects of follistatin on expression of cell lineage markers (CDX2 and Nanog) and downstream targets of SMAD signaling (CTGF, ID1, ID2 and ID3) and AKT phosphorylation were investigated. Follistatin was supplemented during the initial 72?h of embryo culture. Exogenous follistatin restored the in vitro developmental competence of embryos produced with sex-sorted semen to the levels of control embryos produced with unsorted semen, and comparable results were obtained using sorted semen from three different bulls. The mRNA abundance for SMAD signaling downstream target genes, CTGF (SMAD 2/3 pathway) and ID2 (SMAD 1/5 pathway), was lower in blastocysts produced using sex-sorted versus unsorted semen, but mRNA levels for CDX2, NANOG, ID1 and ID3 were similar in both groups. Follistatin supplementation restored CTGF and ID2 mRNA in blastocysts produced using sex-sorted semen to levels of control embryos. Moreover, levels of phosphorylated (p)AKT (Ser-473 and Thr-308) were similar in embryos derived from sex-sorted and unsorted semen, but follistatin treatment increased pAKT levels in both groups. Taken together, results demonstrated that follistatin improves in vitro development of embryos produced with sex-sorted semen and such effects are associated with enhanced indices of SMAD signaling.