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Accumulated evidence indicates that sperm-borne small RNA plays a crucial role in embryonic development, especially the absence of the sperm-borne small RNA might be a major cause of the abnormal development of cloned embryos. In this study, we found that sperm-borne small RNA can affect abnormal pronuclear-like structures, postpone the timing of first embryo cleavage and enhance developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos. In addition, the supplementation of sperm-borne small RNA can significantly increase live birth rates and decrease the birth weights of cloned offspring. To investigate the underlying mechanisms, the levels of α-tubulin K40 acetylation (Ac α-tubulin K40) and histone H3 lysine 9 trimethylation (H3K9me3) during early embryo development were investigated in SCNT embryos with sperm-borne small RNA supplementation (termed as T-NT), compared to those normal SCNT embryos and embryos obtained from standard IVF. The results showed that sperm-borne small RNA can significantly decrease the H3K9me3 levels at the pronuclear and two-cell stages, while significantly increase Ac α-tubulin K40 levels at anaphase and telophase of bovine SCNT embryos during the first cleavage. Collectively, our study for the first time demonstrates that sperm-borne small RNA plays a crucial role in the developmental competence of SCNT embryos by regulating H3K9me3 and Ac α-tubulin K40. Further studies will be required to determine how sperm small RNA regulate the H3K9me3 and Acα-tubulin K40. Our study suggests that the supplementation of sperm-borne small RNA is a potential application to improve the cloning efficiency.

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Cadmium (Cd) is a toxic heavy metal widely found in the environment. Cd is also a potential neurotoxicant, and its exposure is associated with impairment of cognitive function. However, the underlying mechanisms by which Cd induces neurotoxicity are unclear. In this study, we investigated the in vitro effect of Cd on primary murine neural stem/progenitor cells (mNS/PCs) isolated from the subventricular zone. Our results show that Cd exposure leads to mNS/PCs G1/S arrest, promotes cell apoptosis, and inhibits cell proliferation. In addition, Cd increases intracellular and mitochondrial reactive oxygen species (ROS) that activates mitochondrial oxidative stress, decreases ATP production, and increases mitochondrial proton leak and glycolysis rate in a dose‐dependent manner. Furthermore, Cd exposure decreases phosphorylation of protein kinase B (AKT) and glycogen synthase kinase‐3 beta (GSK3β) in mNS/PCs. In addition, pretreatment mNS/PCs with MitoTEMPO, a mitochondrial‐targeted antioxidant, improves mitochondrial morphology and functions and attenuates Cd‐induced inhibition of mNS/PCs proliferation. It also effectively reverses Cd‐induced changes of phosphorylation of AKT and the expression of β‐catenin and its downstream genes. Taken together, our data suggested that AKT/GSK3β/β‐catenin signaling pathway is involved in Cd‐induced mNS/PCs proliferation inhibition via MitoROS‐dependent pattern.

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Mature sperm contain both coding and non-coding RNAs, which can be delivered into an oocyte with the sperm at fertilization. Accumulating evidences show that these sperm-borne RNAs play crucial roles in epigenetic reprogramming, cytoskeleton remodeling, embryonic development, and offspring phenotype. Almost total lack of sperm-borne RNAs is regarded as one of the key factors that leads to the abnormal development of SCNT embryo. Bta-miR-183 was found to be highly expressed in bovine sperm, and can be delivered into oocytes during fertilization in our previous study, and in this study, EZR was confirmed as a target gene of bta-miR-183 in early embryos by bioinformatics, luciferase, and gain-and loss-of-function experiments. Scanning electron microscopy showed that the density of microvilli on the surface of somatic cell nuclear transfer (SCNT) embryos was significantly higher than that on in vitro fertilized (IVF) embryos and was significantly reduced by injection of bta-miR-183 mimic. EZR-siRNA injected into SCNT embryos had a similar effect. This indicated that the lack of bta-miR-183 might lead to abnormal changes in microvilli by down-regulating ezrin protein. In addition, Gain-of-function studies showed that bta-miR-183 significantly improved developmental competence of SCNT embryo in terms of cleavage (76.63% vs 64.32%, p<0.05), blastocyst formation (43.75% vs 28.26%, p<0.05), apoptotic index (5.21% vs 12.64%, p<0.05), and the trophoblast ratio (32.65% vs 25.58%, p<0.05) in day 7 blastocysts. Thus, the present study indicated that bta-miR-183 might play crucial roles in the formation of microvilli and embryo development by regulating expression of EZR mRNA.

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