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Vascular smooth muscle cells (VSMCs) are an important source of foam cells in atherosclerosis. The mechanism for VSMC-derived foam cell formation is, however, poorly understood. Here, we demonstrate that the P2RY12/P2Y12 receptor is important in regulating macroautophagy/autophagy and VSMC-derived foam cell formation in advanced atherosclerosis. Inhibition of the P2RY12 receptor ameliorated lipid accumulation and VSMC-derived foam cell formation in high-fat diet-fed apoe(-/-) mice (atherosclerosis model) independent of LDL-c levels. Activation of the P2RY12 receptor blocked cholesterol efflux via PI3K-AKT, while genetic knockdown or pharmacological inhibition of the P2RY12 receptor inhibited this effect in VSMCs. Phosphoproteomic analysis showed that the P2RY12 receptor regulated the autophagy pathway in VSMCs. Additionally, activation of the P2RY12 receptor inhibited MAP1LC3/LC3 maturation, SQSTM1 degradation, and autophagosome formation in VSMCs. Genetic knockdown of the essential autophagy gene Atg5 significantly attenuated P2RY12 receptor inhibitor-induced cholesterol efflux in VSMCs. Furthermore, activation of the P2RY12 receptor led to the activation of MTOR through PI3K-AKT in VSMCs, whereas blocking MTOR activity (rapamycin) or reducing MTOR expression reversed the inhibition of cholesterol efflux mediated by the P2RY12 receptor in VSMCs. In vivo, inhibition of the P2RY12 receptor promoted autophagy of VSMCs through PI3K-AKT-MTOR in advanced atherosclerosis in apoe(-/-) mice, which could be impeded by an autophagy inhibitor (chloroquine). Therefore, we conclude that activation of the P2RY12 receptor decreases cholesterol efflux and promotes VSMC-derived foam cell formation by blocking autophagy in advanced atherosclerosis. Our study thus suggests that the P2RY12 receptor is a therapeutic target for treating atherosclerosis. Abbreviations: 2-MeSAMP: 2-methylthioadenosine 5′-monophosphate; 8-CPT-cAMP: 8-(4-chlorophenylthio)-adenosine-3ʹ,5ʹ-cyclic-monophosphate; ABCA1: ATP binding cassette subfamily A member 1; ABCG1: ATP binding cassette subfamily G member 1; ACTB: actin beta; ADPβs: adenosine 5′-(alpha, beta-methylene) diphosphate; ALs: autolysosomes; AMPK: AMP-activated protein kinase; APOA1: apolipoprotein A1; APs: autophagosomes; ATG5: autophagy related 5; ATV: atorvastatin; AVs: autophagic vacuoles; CD: chow diet; CDL: clopidogrel; CQ: chloroquine; DAPI: 4ʹ,6-diamidino-2-phenylindole; dbcAMP: dibutyryl-cAMP; DIL-oxLDL: dioctadecyl-3,3,3,3-tetramethylin docarbocyanine-oxLDL; EIF4EBP1/4E-BP1: eukaryotic translation initiation factor 4E binding protein 1; EVG: elastic van gieson; HE: hematoxylin-eosin; HDL: high-density lipoprotein; HFD: high-fat diet; KEGG: Kyoto Encyclopedia of Genes and Genomes; LDL-c: low-density lipoprotein cholesterol; LDs: lipid droplets; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; Masson: masson trichrome; MCPT: maximal carotid plaque thickness; MK2206: MK-2206 2HCL; NBD-cholesterol: 22-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl] amino)-23,24-bisnor-5-cholen-3β-ol; OLR1/LOX-1: oxidized low density lipoprotein receptor 1; ORO: oil Red O; ox-LDL: oxidized low-density lipoprotein; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TIC: ticagrelor; ULK1: unc-51 like autophagy activating kinase 1; VSMCs: vascular smooth muscle cells

The pluripotency gene regulatory network of porcine induced pluripotent stem cells(piPSCs), especially in epigenetics, remains elusive. To determine the biological function of epigenetics, we cultured piPSCs in different culture conditions. We found that activation of pluripotent gene- and pluripotency-related pathways requires the erasure of H3K9 methylation modification which was further influenced by mouse embryonic fibroblast (MEF) served feeder. By dissecting the dynamic change of H3K9 methylation during loss of pluripotency, we demonstrated that the H3K9 demethylases KDM3A and KDM3B regulated global H3K9me2/me3 level and that their co-depletion led to the collapse of the pluripotency gene regulatory network. Immunoprecipitation-mass spectrometry (IP-MS) provided evidence that KDM3A and KDM3B formed a complex to perform H3K9 demethylation. The genome-wide regulation analysis revealed that OCT4 (O) and SOX2 (S), the core pluripotency transcriptional activators, maintained the pluripotent state of piPSCs depending on the H3K9 hypomethylation. Further investigation revealed that O/S cooperating with histone demethylase complex containing KDM3A and KDM3B promoted pluripotency genes expression to maintain the pluripotent state of piPSCs. Together, these data offer a unique insight into the epigenetic pluripotency network of piPSCs.

Reelin plays important roles in brain development. Reeler mutant mice that lack the protein reelin (RELN) suffer from cell type- and region-dependent changes in their neocortical layers, and adult reeler mutant mice have dilated seminiferous tubules. Meanwhile, the mechanism by which Reelin regulates the spermatogenic cell development in mice and their reproductive abilities remains unclear. In the present study, we used reeler mutant mice to investigate the effects of Reelin on reproduction in mice. The results indicated variations in sex hormone expression among the reeler mice, indicating that they produce few offspring and their spermatogenic cells are irregularly developed. Moreover, glial cell line-derived neurotrophic factor (GDNF)/GDNF family receptor alpha 1, Ras/extracellular regulated protein kinases (ERK), and promyelocytic leukemia zinc finger (PLZF)/chemokine (C-X-C motif) receptor 4 (CXCR4) serve as potential regulatory pathways that respond to the changes in sertoli cells and the niche of male germ cells. Our findings provided valuable insights into the role of reeler in the reproductive abilities of male mice and development of their spermatogonia stem cells.

Somatic cloning has had a significant impact on the life sciences and is important in a variety of processes, including medical research and animal production. However, the application of somatic cloning has been limited due to its low success rate. Therefore, potential epigenetic variations between cloned and donor animals are still unclear. DNA methylation, one of the factors which is responsible for phenotypic differences in animals, is a commonly researched topic in epigenetic studies of mammals. To investigate the epigenetic variations between cloned and donor animals, we selected blood and ear fibroblasts of a donor pig and a cloned pig to perform whole-genome bisulfite sequencing (WGBS). A total of 215 and 707 differential methylation genes (DMGs) were identified in blood and ear fibroblasts, respectively. Functional annotation revealed that DMGs are enriched in many pathways, including T/B or natural killer (NK) cell differentiation, oocyte maturation, embryonic development, and reproductive hormone secretion. Moreover, 22 DMGs in the blood and 75 in the ear were associated with immune responses (e.g., CD244, CDK6, CD5, CD2, CD83, and CDC7). We also found that 18 DMGs in blood and 53 in ear fibroblasts were involved in reproduction. Understanding the expression patterns of DMGs, especially in relation to immune responses and reproduction, will reveal insights that will aid the advancement of future somatic cloning techniques in swine.

Background/Aims: Busulfan is commonly used for cancer chemotherapy. Although it has the advantage of increasing the survival rate of patients, it can cause male infertility via damaging the testes and reducing sperm counts. Therefore, the underlying mechanism should be explored, and new agents should be developed to protect the male reproductive system from busulfan-induced damage. Endoplasmic reticulum stress (ERS) is considered a key contributor to numerous pathologies. Despite several studies linking ERS to toxicants, studies have yet to determine whether ERS is a contributing factor to busulfan-induced testicular damage. Melatonin is a well-known broad-spectrum antioxidant, anti-inflammatory and antitumour agent, but the effects of melatonin on busulfan-induced ERS in mouse testes damage are less documented. Methods: The effects of melatonin were measured by immunofluorescence staining, Western blot, qRT-PCR analysis and flow cytometry assay. The underlying mechanism was investigated by measuring ERS. Results: We found that ERS was strongly activated in mouse testes (in vivo) and the C18-4 cell line (in vitro) after busulfan administration. ERS-related apoptosis proteins such as caspase-12, CHOP and caspase-3 were activated, and the expression of apoptotic proteins such as P53 and PUMA were upregulated. Furthermore, we investigated whether melatonin reduced the extent of damage to mouse testes and improved the survival rates of busulfan-treated mice. When exploring the underlying mechanisms, we found melatonin could counteract ERS by decreasing the expression levels of the ERS markers GRP78, ATF6, pIRE1 and XBP1 in mouse testes and mouse SSCs (C18-4 cells). Moreover, it blocked the activation of ERS-related apoptosis proteins caspase-12, CHOP and caspase-3 and suppressed P53 and PUMA expression stimulated by busulfan both in vivo and in vitro. Conclusion: Our results demonstrate that ERS is an important mediator for busulfan-induced apoptosis. The attenuation of ERS by melatonin can prevent busulfan-treated SSCs apoptosis and protect busulfan-treated testes from damage. Thus, this study suggests that melatonin may alleviate the side effects of busulfan for male patients during clinical treatment.

Diabetes mellitus affects a large number of men of reproductive age and it usually leads to serious reproductive disorders. However, the underlying mechanisms and specific therapies still remain largely unknown. We observed Leydig cell loss in the testes of diabetic mice. Continuous high glycemic status of testes stimulated expression of Caspase12, Grp78, and Chop, the three ERS response factors; this might induce cell cycle arrest and apoptosis of Leydig cells in response to ERS. In these diabetic mouse models, melatonin alleviated apoptosis of testicular stromal cell induced by ERS, and promoted SSCs self-renewal by recovering Leydig cells secretion of CSF1 after 8 weeks of treatment. To explore the relationship between CSF-1 and ERS in Leydig cells, we treated Leydig tumor cell line with an activator Tuniamycin and an inhibitor 4-Phenylbutyrate of ERS. Our data showed that the CSF-1 expression in mouse Leydig cell lines decreased six-fold while reversely increasing five-fold in the 4-Phenylbutyrate-treated group. Thus, melatonin likely alleviates the loss of Leydig cells in diabetic testes and provides a healthier niche for SSCs to self-renew and continually provide healthy sperm for male fertility.

Objectives To date, many efforts have been made to establish porcine embryonic stem (pES) cells without success. Extraembryonic endoderm (XEN) cells can self‐renew and differentiate into the visceral endoderm and parietal endoderm. XEN cells are derived from the primitive endoderm of the inner cell mass of blastocysts and may be an intermediate state in cell reprogramming. Materials and methods Porcine XEN cells (pXENCs) were generated from porcine pluripotent stem cells (pPSCs) and were characterized by RNA sequencing and immunofluorescence analyses. The developmental potential of pXENCs was investigated in chimeric mouse embryos. Results Porcine XEN cells derived from porcine pPSCs were successfully expanded in N2B27 medium supplemented with bFGF for least 30 passages. RNA sequencing and immunofluorescence analyses showed that pXENCs expressed the murine and canine XEN markers Gata6, Gata4, Sox17 and Pdgfra but not the pluripotent markers Oct4, Sox2 and TE marker Cdx2. Moreover, these cells contributed to the XEN when injected into four‐cell stage mouse embryos. Supplementation with Chir99021 and SB431542 promoted the pluripotency of the pXENCs. Conclusions We successfully derived pXENCs and showed that supplementation with Chir99021 and SB431542 confer them with pluripotency. Our results provide a new resource for investigating the reprogramming mechanism of porcine‐induced pluripotent stem cells.