Clonally derived chicken primordial germ cell lines maintain biological characteristics and proliferative potential in long-term culture.

Chicken primordial germ cells (PGCs) are important cells with significant implications in preserving genetic resources, chicken breeding and production, and basic research on genetics and development. Currently, chicken PGCs can be cultured long-term in vitro to produce single-cell clones. However, systematic exploration of the cellular characteristics of these single-cell clonal lines has yet to be conducted. In this study, single-cell clonal lines were established from male and female PGCs of Rugao Yellow Chicken and Shouguang Black Chicken, respectively, using a micropipette-based method for single-cell isolation and culture. Analysis of glycogen granule staining, mRNA expression of pluripotency marker genes (POUV, SOX2, NANOG), germ cell marker genes (DAZL, CVH), and SSEA-1, EMA-1, SOX2, C-KIT, and CVH protein expression showed positive results, indicating that PGCs maintain normal cellular properties after single-cell cloning. Furthermore, tests on proliferation ability and gene expression levels in PGC single-cell clonal lines showed high expression of the pluripotency-related genes and TERT compared to control PGCs, and PGC single-cell clonal lines demonstrated higher proliferation ability. Finally, green fluorescent protein (GFP)-PGC single-cell clonal lines were established, and it was found that these single-cell clonal lines could still migrate into the gonads of recipients, suggesting their potential for germ-line transmission. This study systematically validated the normal cellular characteristics of PGC single-cell clonal lines, indicating that they could be applied in genetic modification research on chickens.

Epigallocatechin-3-gallate protects porcine oocytes against post-ovulatory aging through inhibition of oxidative stress.

Increased levels of oxidative stress are major factors that drive the process of post-ovulatory oocyte aging. Epigallocatechin-3-gallate (EGCG), which accounts for up to 50% of the catechins, possesses versatile biological functions, including preventing or treating diabetes, cancer, and heart diseases. The aim of this study was to explore whether EGCG can delay porcine oocyte aging by preventing oxidative stress. Metaphase II (MII) oocytes were cultured for 48 h with different concentrations of EGCG (0-100 µM) in vitro as a post-ovulatory aging model. An optimal concentration of 5 µM EGCG maintained oocyte morphology and developmental competence during aging. The oocytes were randomly divided into five groups: fresh, 24 h control, 24 h EGCG, 48 h control, and 48 h EGCG. The results suggest that EGCG significantly prevents aging-induced oxidative stress, glutathione (GSH) reduction, apoptosis, and autophagy. Moreover, mitochondria DNA copy number was decreased, and the number of active mitochondria and adenosine triphosphate (ATP) levels significantly increased by supplementation with EGCG. Thus, EGCG has a preventive role against aging in porcine post-ovulatory oocytes due to its ability to inhibit oxidative stress and promote mitochondrial biogenesis.

LncRNA TCONS_00041002 improves neurological outcomes in neonatal rats with hypoxic-ischemic encephalopathy by inhibiting apoptosis and promoting neuron survival.

It has been reported that Neonatal hypoxic-ischemic encephalopathy (HIE) could induce apoptosis in neonates and result in cognitive and sensory impairments, which are associated with poor developmental outcomes. Despite the improvement in neonatology, there is still no clinically effective treatment for HIE presently. Long non-coding RNAs (lncRNAs) play important roles in cellular homeostasis. Nevertheless, their effects in developing rat brains with HI is little known. Here, we established HIE model in neonate rats and explored the expression and function of lncRNAs in HI, and found the expression of 19 lncRNAs was remarkably changed in the brains of HI rats, compared to the sham group. Among them, three lncRNAs (TCONS_00041002, TCONS_00070547, TCONS_00045572) were enriched in the apoptotic process via gene ontology (GO) and pathway analysis, which were selected for the further qRT-PCR verification. Through lentivirus-mediated overexpression of these three lncRNAs, we found that overexpression of TCONS_00041002 attenuated the cell apoptosis, and increased the vitality of neurons after oxygen-glucose deprivation (OGD), therefore reduced the brain infarction and further promoted the neuron survival as well as improved the neurological disorders in the rats subjected to HIE. What's more, ceRNA network prediction and co-expression verification showed that the expression of TCONS_00041002 was positively associated with Foxe1, Pawr and Nfkbiz. Altogether, this study has exhibited that lncRNA TCONS_00041002 participates in the cell apoptosis and neuronal survival of HIE and represents a potential new target for the treatment of HIE.

PINK1 regulates mitochondrial morphology via promoting mitochondrial fission in porcine preimplantation embryos.

Phosphatase and tensin homolog-induced kinase 1 (PINK1) on the outer membranes of impaired mitochondria promotes mitophagy and regulates mitochondrial morphology. Mammalian oocytes and early embryos are mitochondria rich, but mitochondrial dynamics during preimplantation embryo development is not well-studied. To investigate whether PINK1 is required for mitochondrial dynamics in porcine preimplantation embryos, gene knockdown and inhibitors were used, and mitochondrial dynamics were observed by transmission electron microscopy. PINK1 knockdown significantly impaired blastocyst formation and quality, induced mitochondrial elongation and swelling, and reduced mitochondrial DNA copy number. PINK1 knockdown-induced mitochondrial elongation caused mitochondrial dysfunction, oxidative stress, and ATP deficiency, significantly increasing autophagy and apoptosis. Profission dynamin-related protein 1 overexpression prevented PINK1 knockdown-induced impairment of embryo development, mitochondrial elongation, and dysfunction. Thus, PINK1 promotes mitochondrial fission in porcine preimplantation embryos.-Niu, Y.-J., Nie, Z.-W., Shin, K.-T., Zhou, W., Cui, X.-S. PINK1 regulates mitochondrial morphology via promoting mitochondrial fission in porcine preimplantation embryos.

Spindlin1 alters the metaphase to anaphase transition in meiosis I through regulation of BUB3 expression in porcine oocytes.

Spindlin 1 (SPIN1), which contains Tudor-like domains, regulates maternal transcripts via interaction with a messenger RNA (mRNA)-binding protein. SPIN1 is involved in tumorigenesis in somatic cells and is highly expressed in cancer cells. Nevertheless, the role of SPIN1 in porcine oocyte maturation remains totally unknown. To explore the function of SPIN1 in porcine oocyte maturation, knockdown, and overexpression techniques were used. SPIN1 mRNA was identified in maternal stages ranging from GV to MII. SPIN1 was localized in the cytoplasm and to chromosomes during meiosis. SPIN1 knockdown accelerated first polar body extrusion. Oocytes with overexpressed SPIN1 were arrested at the MI stage. SPIN1 depletion caused meiotic spindle defects and chromosome instability. The BUB3 signal was investigated, confirming that SPIN1 affects the stability of Bub3 mRNA as well as BUB3 expression. Further, overexpression of SPIN1 inhibited the degradation and regulation of G2/mitotic-specific cyclin-B1. In summation, SPIN1 regulates the meiotic cell cycle by modulating the activation of the spindle assembly checkpoint.

TIP60 contributes to porcine embryonic development by regulating DNA damage response.

The acetyltransferase TIP60 (also known as Kat5) is a member of the MYST family of histone acetyltransferases and was initially identified as a cellular protein. TIP60 acetylates histone and non-histone proteins and is involved in diverse biological processes, including apoptosis, cell cycle, and DNA damage responses. In this study, a specific inhibitor of TIP60 was used to detect the function of TIP60 in porcine parthenogenetic embryos. The results showed that TIP60 inhibition impaired porcine parthenogenetic embryonic development. The mechanism of TIP60 was also determined. We found that the TIP60 inhibition impaired embryonic development by ROS induced DNA damage, as demonstrated by the number of γH2A in the nuclei. TIP60 inhibition triggered DNA damage through the regulation of p53-p21 pathway and TIP60 played a role in DNA repair. TIP60 inhibition decreased the efficiency of DNA repair by regulating 53BP1-dependent repair after DNA damage. Inhibition of TIP60 also increased the adaptive response, autophagy, by modulating LC3. Therefore, TIP60 plays a role in early porcine parthenogenetic embryonic development by regulating DNA damage and repair.

Sodium fluoride exposure exerts toxic effects on porcine oocyte maturation.

Excessive long-term fluoride intake is associated with several health problems, including infertility. However, limited information is available on the toxic effects of fluoride exposure on the female reproductive system, especially oocyte maturation. In this study, we investigated the toxic effect of sodium fluoride (NaF) exposure on porcine oocyte maturation and its possible underlying mechanisms. Our results showed that NaF exposure during porcine oocyte maturation inhibited cumulus cell expansion and impaired polar body extrusion. Cell cycle analysis showed that NaF exposure blocked meiotic resumption, disturbed spindle dynamics, disrupted chromosome separation, and increased aneuploidy in porcine oocytes. Moreover, NaF exposure disturbed mitochondrial function, triggered DNA damage response, and induced early apoptosis in porcine oocytes. NaF exposure also induced oxidative stress, decreased GSH level, and increased cathepsin B activity in and impaired the further development potential of porcine oocytes, as indicated by a decrease in blastocyst formation rate, increase in apoptosis, and inhibition of cell proliferation. Together, these results indicate that NaF exposure impairs the maturation capacity of porcine oocytes by inhibiting cumulus cell expansion, disturbing cytoskeletal dynamics, and blocking nuclear and cytoplasmic maturation, thus decreasing the quality and affecting the subsequent embryonic development potential of porcine oocytes.