The knock-in efficiency is very important to manipulate gene editing in the transgenic domestic animal. Recently, it is reported that transiently loosen nucleosome folding of transcriptionally inactive chromatin might have potential tp enhance the homologouse recombination efficiency. Histone deacetylases (HDAC) are a class of enzymes that remove acetyl groups from an amino acid on a histone. This is important because DNA is wrapped around histones, and DNA expression is regulated by acetylation and de-acetylation. In this study, Mac-T cell were treated with 10uM VPA (valproic acid, HDAC inhibitor) for 24 h and transfected with Knock-in vector and TALEN at targeting of β-casein gene. After 3 day of transfection, knock-in efficiency was confirmed by PCR. The level of HDAC2 protein in Mac-T cells was decreased by VPA treatment. The knock-in efficiency in the Mac-T cell with treated HDAC inhibitor was higher than cell not treated HDAC inhibitor. These results indicated that chromatin modification by HDAC inhibitor enhances homologous recombination efficiency in the Mac-T cell.

In general, the shape of cumulus-oocyte-complexes (COCs) at germinal vesicle (GV) stage is important roles on meiotic maturation of porcine oocyte during in vitro maturation (IVM). Then, mitochondria produce reactive oxygen species (ROS) such as superoxide from electron transport system during oocyte maturation. ROS levels on oocytes are regulated by various antioxidant enzymes in cumulus cells (CCs). However, the effect of mitochondria derived superoxide production from CCs during IVM of porcine oocyte has not been reported. Firstly, we divided groups according to large number of CCs (Grade 1: G1) and small number of CCs (Grade 2: G2). Then, we counted cumulus cells of G1 and G2 oocyte by using haemocytometer. The oocyte maturation rate was significant decreased (p < 0.05) in G2 oocytes than that of G1 oocytes. We measured mitochondria derived superoxide in G1 and G2 COCs by using Mito-SOX staining. Mitochondrial superoxide was higher in G2 COCs than G1 COCs. Then, the mRNA expression levels of antioxidant enzymes (SOD1, SOD2 and PRDX3) in G2 COCs were decreased compared to G1 COCs. To reduce mitochondria derived superoxide, we used Mito-TEMPO as mitochondrial superoxide scavenger. Oocyte maturation rates in both G1 and G2 groups treated with Mito-TEMPO were increased than that of non-treated groups. Mitochondrial superoxide was lower in G1 and G2 groups treated with Mito-TEMPO than that of non-treatment groups. The mRNA expression levels of antioxidant enzymes in G1 and G2 COCs treated with Mito-TEMPO were increased compared to non-treated groups. Based on these findings, we suggest that reduction of mitochondria derived superoxide by Mito-TEMPO assists maturation competence in porcine oocytes.

Bisphenol A (BPA), an endocrine-disrupting chemical, has received tremendous attention in the past few decades because of its detrimental health effects. Growing evidence supports that BPA is capable to alter the reproductive performance of the exposed individual. In spermatozoa, it has been reported that BPA increased oxidative stress by the overproduction of reactive oxygen species (ROS), subsequently affects the sperm function, biochemical properties, and fertility. Since antioxidants minimize cellular oxidative stress, therefore may have protective effects against BPA-induced stress. In the present study, we incubated mice spermatozoa for 6 h in a condition that support in vitro fertilization. The sperm incubation media was additionally supplemented with either BPA or BPA together with antioxidants, such as glutathione, vitamin C, and vitamin E. Our results showed that antioxidant significantly decreased the production of ROS that subsequently supports motility and acrosomal integrity of BPA-exposed spermatozoa. Particularly, glutathione and vitamin E inhibit protein kinase-A dependent phosphorylation of sperm proteins subsequently prevented precocious acrosome reaction. In addition, both antioxidants were found to restore fertilization and early embryo development potentiality of BPA-exposed spermatozoa. Therefore, we conclude that antioxidants minimize oxidative stress in spermatozoa in a BPA containing micro-environment, thus avoiding BPA-mediated harmful consequences. The current finding has both theoretical and clinical significance for developing potential remedies of the BPA toxicity.

Mitochondrion is an organelle for regulating calcium (Ca2+) homeostasis. Mitochondrial Ca2+ plays important roles on oocyte maturation, fertilization and embryonic development for ATP production. Low quality oocytes have mitochondrial dysfunction, which lead to overloaded Ca2+ in mitochondria. Recently, Rhod-2 is well known as a mitochondrial derived Ca2+ indicator. However, the changes of Rhod-2 in matured or fertilized porcine oocytes have not been reported. Therefore, the aim of study was to identify the effects of mitochondrial Ca2+ using Rhod-2 on quality assessment of matured oocyte and zygotes in pigs. Thus, we classified two groups (group 1: G1, compact COCs and group 2: G2, uncompact COCs) according to differences of cumulus cells amount and cytoplasm morphology in germinal vesicle (GV) stage of porcine COCs. Therefore, we investigated number of Rhod-2 spots in matured and fertilized oocytes from G1 and G2 groups. The Rhod-2 spot numbers were separated into four parts; n<10, 10≤ n < 20, 20 ≤ n < 30, and 30 < n. The Rhod-2 spots number of G2 group had greater than G1 group in part of 20 ≤ n. Additionally, we investigate mean number of Rhod-2 spots from G1 and G2 groups in matured and fertilized oocytes. As a result, we confirmed that average number of Rhod-2 spots in G2 group increased than that of G2 group. Finally, we also measured the Rhod-2 intensity in matured and fertilized oocytes of G1 and G2 groups. Interestingly, the Rhod-2 intensity in G2 group was higher than that of G1 group. (oocyte: p < 0.001 and fertilized oocyte: p < 0.05). These results demonstrated that changes in Rhod-2 spots and intensity were increased in low quality of matured and fertilized oocytes. Therefore, our results suggest that the differences in mitochondrial calcium level are associated with morphological quality of porcine COCs.

The deleted in azoospermia like (DAZL) gene has been identified in many vertebrate species. DAZL shows high homology with deleted in azoospermia (DAZ) genes that identified only in humans, great apes and Old World monkeys, and boule homolog (BOLL) that identified in many vertebrate species. These genes encode RNA binding proteins (RBP), which regulate the post-transcriptional functions of several genes. In humans, DAZ copies are linked to Y chromosome, while DAZL and BOLL are linked to chromosomes 3 and 2, respectively. DAZ copies has been reported to express in prenatal and postnatal germ cells, particularly in the premeiotic spermatogonia. BOLL has been reported to express during the meiotic G2/M transition in germ cells. DAZL has been reported to express in all stages of germ cells. Compared to humans and mice, the detailed functionalities of DAZL is not clear in many vertebrate species. In our studies, we use chickens as an animal model to examine the expression profiling of DAZL gene in germ cells right from the early embryonic development to the adult. Also, we are studying the effects of small interfering RNA (siRNA) mediated knockdown of DAZL and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR associated protein 9 (CRISPR/Cas9) mediated knockout of DAZL in the chicken primordial germ cells (PGCs). In the chicken, DAZL is linked to chromosome 2 (2p1.3-p1.2), and encodes a 289 amino acids protein. By in situ hybridization, we detected a strong expression of DAZL in the germ plasm of chicken oocytes. Later, the expression of DAZL was strongly detected in all stages of intrauterine development and post-ovipositional development especially in the PGC specifying cells. Moreover, the expression of DAZL was strong and constant in the male and female germ cells until adult stage. The siRNA mediated knockdown of DAZL significantly reduced the PGCs proliferation and increased the apoptosis in vitro. We examined the knockout efficiency of DAZL using CRISPR/Cas9 technique in chicken DF1 fibroblast cell line, prior to test in the PGCs. The results of T7 endonuclease I (T7E1) assay and subsequent sequencing indicates clear mutations on the DAZL gene in DF1 cells, and the method could be applicable to cause mutations on the DAZL gene in PGCs. In conclusion, chicken DAZL express in all stages of germ cells as a germ line marker, and alteration in the gene expression causes germ cells impairment.