Preserving Porcine Genetics: A Simple and Effective Method for On-Site Cryopreservation of Ear Tissue Using Direct Cover Vitrification.

Cell cryopreservation is widely used for porcine genetic conservation; however, isolating and freezing primary cells in farms without adequate experimental equipment and environment poses a significant challenge. Therefore, it is necessary to establish a quick and simple method to freeze tissues on-site, which can be used for deriving primary fibroblasts when needed to achieve porcine genetic conservation. In this study, we explored a suitable approach for porcine ear tissue cryopreservation. The porcine ear tissues were cut into strips and frozen by direct cover vitrification (DCV) in the cryoprotectant solution with 15% EG, 15% DMSO and 0.1 M trehalose. Histological analysis and ultrastructural evaluation revealed that thawed tissues had normal tissue structure. More importantly, viable fibroblasts could be derived from these tissues frozen in liquid nitrogen for up to 6 months. Cells derived from thawed tissues did not show any cell apoptosis, had normal karyotypes and could be used for nuclear transfer. These results suggest that this quick and simple ear tissue cryopreservation method can be applied for porcine genetic conservation, especially in the face of a deadly emerging disease in pigs.

Impact of diethylhexyl phthalate on gene expression and development of mammary glands of pregnant mouse.

The widely used diethylhexyl phthalate (DEHP) is a known endocrine disruptor that causes persistent alterations in the structure and function of female reproductive system, including ovaries, uterus and oviducts. To explore the molecular mechanism of the effect of DEHP on the development of mammary glands, we investigated the cell cycle, growth, proliferation and gene expression of mammary gland cells of pregnant mice exposed to DEHP. It was demonstrated, for the first time, that the mammary gland cells of pregnant mice treated with DEHP for 0.5-3.5 days post-coitum had increased proliferation, growth rate and number of cells in the G2/S phase. The expression of cell proliferation-related genes was significantly altered after short time and low-dose DEHP treatment of mammary gland cells in vivo and in vitro. These findings showed adverse effects of DEHP on mammary gland cells in pregnant mice.

ATG7-mediated autophagy facilitates embryonic stem cell exit from naive pluripotency and marks commitment to differentiation.

Macroautophagy/autophagy is a conserved cellular mechanism to degrade unneeded cytoplasmic proteins and organelles to recycle their components, and it is critical for embryonic stem cell (ESC) self-renewal and somatic cell reprogramming. Whereas autophagy is essential for early development of embryos, no information exists regarding its functions during the transition from naive-to-primed pluripotency. Here, by using an in vitro transition model of ESCs to epiblast-like cells (EpiLCs), we find that dynamic changes in ATG7-dependent autophagy are critical for the naive-to-primed transition, and are also necessary for germline specification. RNA-seq and ATAC-seq profiling reveal that NANOG acts as a barrier to prevent pluripotency transition, and autophagy-dependent NANOG degradation is important for dismantling the naive pluripotency expression program through decommissioning of naive-associated active enhancers. Mechanistically, we found that autophagy receptor protein SQSTM1/p62 translocated into the nucleus during the pluripotency transition period and is preferentially associated with K63 ubiquitinated NANOG for selective protein degradation. In vivo, loss of autophagy by ATG7 depletion disrupts peri-implantation development and causes increased chromatin association of NANOG, which affects neuronal differentiation by competitively binding to OTX2-specific neuroectodermal development-associated regions. Taken together, our findings reveal that autophagy-dependent degradation of NANOG plays a critical role in regulating exit from the naive state and marks distinct cell fate allocation during lineage specification.Abbreviations: 3-MA: 3-methyladenine; EpiLC: epiblast-like cell; ESC: embryonic stem cell; PGC: primordial germ cell.

Defective chromatin architectures in embryonic stem cells derived from somatic cell nuclear transfer impair their differentiation potentials.

Nuclear transfer embryonic stem cells (ntESCs) hold enormous promise for individual-specific regenerative medicine. However, the chromatin states of ntESCs remain poorly characterized. In this study, we employed ATAC-seq and Hi-C techniques to explore the chromatin accessibility and three-dimensional (3D) genome organization of ntESCs. The results show that the chromatin accessibility and genome structures of somatic cells are re-arranged to ESC-like states overall in ntESCs, including compartments, topologically associating domains (TADs) and chromatin loops. However, compared to fertilized ESCs (fESCs), ntESCs show some abnormal openness and structures that have not been reprogrammed completely, which impair the differentiation potential of ntESCs. The histone modification H3K9me3 may be involved in abnormal structures in ntESCs, including incorrect compartment switches and incomplete TAD rebuilding. Moreover, ntESCs and iPSCs show high similarity in 3D genome structures, while a few differences are detected due to different somatic cell origins and reprogramming mechanisms. Through systematic analyses, our study provides a global view of chromatin accessibility and 3D genome organization in ntESCs, which can further facilitate the understanding of the similarities and differences between ntESCs and fESCs.