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Heat stress is known to negatively impact the reproductive process of livestock, which inevitably leads to a decline in animal fertility. Nuclear factor E2-related factor 2 (Nrf2) is an inducible transcription factor, which is essential for maintaining redox signal transmission against oxidative stress. However, there is no reliable research on the response mechanism of Sertoli Cells (SCs) against heat stress and the activation of Nrf2 when SCs are exposed to heat stress. Here, we used primary mouse SCs and SCs line TM4, along with Nrf2 specific inhibitor to determine the reaction mechanism of SCs to maintain intracellular redox homeostasis and self-survival by activating Nrf2. We found that acute heat stress only affected the vitality of SCs and the expression of functional molecules (tight junction-associated proteins and lactate dehydrogenase A [LDHA]) but did not cause cell apoptosis. When Nrf2 was inhibited, more cell death occurred in TM4 cells post heat stress treatment, along with a greater decrease in cell viability and a significant increase in intracellular ROS levels. Our study clarified for the first time the protective effect of Nrf2 activation on heat stress-induced SCs damage. It explained the possible reasons or mechanisms involved in the survival of SCs, the critical protective cells in the testis, which were not affected by heat stress. This study further improved the response mechanism of SCs in the reproductive injury caused by a high-temperature environment.

Adipose-derived mesenchymal stem cells have been used to treat acute kidney injury (AKI). The role of endoplasmic reticulum (ER) stress in AKI treatment with canine adipose-derived mesenchymal stem cells (cADSCs) remains unknown. This study intended to investigate the therapeutic effects of cADSCs cultured in different media on AKI in mice and dogs and reveal the role of ER stress in this process. The mice were divided into two branches: a control group and a gentamicin induced group (this group treated with low-serum ADSC or high-serum ADSC or 4-phenylbutyric acid (4-PBA)). The dogs were divided into control, model, and cell-injected groups. To suppress ER stress, mice were simultaneously treated with 4-PBA. The results showed there were improvements in renal function and tissue damage and a corresponding decrease in ER stress in the kidneys of the mice that received cell injection. However, the cells cultured with 2% FBS showed a better growth state and resulted in lower ER stress levels in treated kidneys. In the 4-PBA-treated group, ER stress was suppressed, and there was corresponding kidney injury recovery. Similarly, both kidney damage and ER stress were alleviated after AKI dogs were injected with the cells. Our findings reveal that both allogeneic and xenogeneic cADSCs were effective treatments for AKI by inhibiting ER stress. These results also provide evidence for a new clinical therapy for acute renal disease in pets.

Pregnancy loss (PL) is one of the common complications that women can experience during pregnancy, with an occurrence rate of 1 to 5%. The potential causes of pregnancy loss are unclear, with no effective treatment modalities being available. It has been previously reported that the level of miR-125b was significantly increased in placentas of PL patients. However, the role of miR-125b in the development of PL still remains unknown. In the current study, an miR-125b placenta-specific over-expression model was constructed by lentiviral transfecting zona-free mouse embryos followed by embryo transfer. On gestation day 15, it was observed that the placenta was significantly smaller in the miR-125b placenta-specific overexpression group than the control group. Additionally, the abortion rate of the miR-125b placenta-specific overexpression group was markedly higher than in the control group. The blood vessel diameter was larger in the miR-125b-overexpressing specific placenta. In addition, miR-125b-overexpressing HTR8 and JEG3 cell lines were also generated to analyze the migration and invasion ability of trophoblasts. The results showed that miR-125b overexpression significantly suppressed the migration and invasion ability of HTR8 and JEG3 cells. Overall, our results demonstrated that miR-125b can affect embryo implantation through modulating placenta angiogenesis and trophoblast cell invasion capacity that can lead to PL.

Normal spermatogenic processes require the scrotal temperature to be lower than that of the body as excessive heat affects spermatogenesis in the testes, reduces sperm quality and quantity, and even causes infertility. Endoplasmic reticulum stress (ERS) is a crucial factor in many pathologies. Although several studies have linked ERS to heat stress, researchers have not yet determined which ERS signaling pathways contribute to heat-induced testicular damage. Melatonin activates antioxidant enzymes, scavenges free radicals, and protects the testes from inflammation; however, few studies have reported on the influence of melatonin on heat-induced testicular damage. Using a murine model of testicular hyperthermia, we observed that heat stress causes both ERS and apoptosis in the testes, especially in the spermatocytes. These observations were confirmed using the mouse spermatocyte cell line GC2, where the睾丸是精子发生的场所，哺乳动物的睾丸位于体腔外的阴囊中，成年哺乳动物包括人类，睾丸的正常温度均比身体核心温度要低。如果外界环境温度过高会影响睾丸中的精子生成，降低精子的质量和数量，甚至导致不育。当机体受到外界刺激时，内质网内会产生大量错误折叠或未折叠蛋白，使得内质网对蛋白的加工能力与载量失衡，从而导致内质网功能紊乱，即发生内质网应激（endoplasmic reticulum stress，ERS），ERS被认为是导致大量病理的关键因素。目前已有研究报道称热应激会引发ERS，但哪条ERS信号通路在热应激诱导的睾丸损伤中发挥主要作用尚不明确。褪黑激素（褪黑素）是一种具有抗氧化、抗凋亡能力的神经内分泌激素，它的抗氧化活性有助于防止应激、炎症引起的睾丸损伤，然而，褪黑素能否能缓解热应激所致的睾丸损伤尚未见报道。该研究通过建立小鼠睾丸热应激模型，并利用组织学、分子生物学检测发现，热应激会导致睾丸曲细精管内的生殖细胞数量减少、生精上皮紊乱及凋亡的产生，TUNEL染色结果显示凋亡主要发生在睾丸精母细胞中，通过实时荧光定量PCR（qRT-PCR）和免疫荧光技术发现热应激也会刺激小鼠睾丸发生ERS。在随后体外（GC2细胞系）试验中，我们通过qRT-PCR和Western blotting从RNA与蛋白水平进一步证实了上述结果，并发现ATF6和PERK信号通路在热诱导后的细胞中均被激活，而IRE1信号通路在该过程变化不明显。通过si-RNA干扰上述基因均可有效减少由热应激引起的精母细胞损伤。在此基础上继续探索褪黑素对热应激引起的精母细胞凋亡和ERS的影响，通过免疫荧光、流式细胞术、Western blotting等实验，发现褪黑素能通过褪黑素受体（MT1/MT2）抑制Atf6、Perk和凋亡相关基因Caspase12、Caspase3及Chop的表达。最后通过小鼠体内试验证实褪黑素可以缓解热应激引起的曲细精管生殖细胞数量减少和精母细胞凋亡。综上，该研究表明热应激能引起睾丸损伤并诱导精母细胞的ERS与凋亡的发生，褪黑素可以通过ATF6和PERK信号通路缓解热应激引起的精母细胞的凋亡，并对损伤的睾丸起到修复作用。该研究证明褪黑素可在一定程度上预防高温引起的精母细胞凋亡，并保护小鼠睾丸免受热应激的损害。.

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.

To facilitate temperature adjustments, the testicles are located outside the body cavity. In most mammals, the temperature of the testes is lower than the body temperature to ensure the normal progression of spermatogenesis. Rising temperatures affect spermatogenesis and eventually lead to a decline in male fertility or even infertility. However, the testes are composed of different cell types, including spermatogonial stem cells (SSCs), spermatocytes, spermatozoa, Leydig cells, and Sertoli cells, which have different cellular responses to heat stress. Recent studies have shown that using different drugs can relieve heat stress-induced reproductive damage by regulating different signaling pathways. Here, we review the mechanisms by which heat stress damages different cells in testes and possible treatments.

The pluripotency gene regulatory network of porcine-induced pluripotent stem cells (piPSCs), especially in epigenetics, remains elusive. To determine this 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.SummaryErasure of H3K9 methylation in porcine pluripotent stem cells depends on the complex of transcription factors OCT4/SOX2 and histone demethylase KDM3A/KDM3B.

Orchitis is one of the leading causes of male animal infertility and is associated with inflammatory reactions caused by the bacterium. It has been reported that there is a mutual coupling effect between endoplasmic reticulum stress (ERS) and inflammatory response. Our studies showed that lipopolysaccharide (LPS) could cause testicular damages, apoptosis, ERS, and inflammatory responses in spermatogonial stem cells (SSCs); ERS-related apoptosis proteins were activated and the expression of ERS genes was significantly upregulated; meanwhile, the expression of Toll-like receptor 4 and inflammation factors was apparently increased with LPS treatment. Moreover, melatonin (MEL) could rescue testicular damage, and significantly inhibited the expression of ERS-related apoptosis genes, ERS markers, and inflammatory factors in SSCs and MEL played repairing and anti-infection roles in LPS-induced testicular damage. Therefore, MEL may be used as a drug to prevent and control bacterial infections in male reproductive systems. However, the specific molecular mechanism of MEL to resist ERS and inflammatory response remains to be further studied.

Porcine pancreatic stem cells (pPSCs) can be induced to insulin-secreting cells and therefore considered the most promising seeding cells for curing human diabetes in future. However, insufficient pPSCs number is one of the bottleneck problems before its clinical application. SerpinB1 is a serine protease inhibitor in neutrophils and can directly promote the proliferation of β cells. Whether SerpinB1 is involved in pPSC proliferation and differentiation remains unknown. The effects of SerpinB1 on pPSCs proliferation were measured by Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine, qRT-PCR, western blot, and flow cytometry assays. We found that pPSCs did not efficiently reach the S phase when SerpinB1 expression was knocked down with short hairpin RNA (sh-SerpinB1), the expression of Cyclin D1, CDK-2, and PCNA also decreased. Meanwhile, cell viability and proliferation ability were both declined. Further analyses showed that the expression level of phosphorylated STAT3/STAT3was downregulated, along with an upregulation of p53 and p21. We used a two-step induction method to induce pPSCs to insulin-secreting cells and found that SerpinB1 expression in insulin-secreting cells was higher than in pPSCs. Meanwhile, the protein expression level of phosphorylated STAT3/STAT3 was increased while p53 and p21 was decreased in induced insulin-secreting cells in comparison with control cells. The insulin-secreting cells derived from the sh-SerpinB1 cells secreted less insulin and showed poor sensitivity to high glucose than control group. However, the insulin-secreting cells derived from the ov-SerpinB1 cells has a quite contrary tendency. In conclusion, this study demonstrates that SerpinB1 promotes the proliferation of pPSCs through the STAT3 signaling pathway, and SerpinB1 is a key factor for maintaining the viability of pPSCs during the transition to insulin-secreting cells.

Porcine pancreatic stem cells (pPSCs) can be induced to differentiate into insulin-producing cells in vitro and thus serve as a major cells source for β-cell regeneration. However, this application is limited by the weak cell proliferation ability and low insulin induction efficiency. In this study, we explored the role of folic acid in the proliferation of pPSCs and the formation of insulin-secreting cells. We found that FA-treated pPSCs cells had a high EDU positive rate, and the proliferation marker molecules PCNA, CyclinD1 and c-Myc were up-regulated, while the expression of folate receptor α (FOLRα) was up-regulated. In further research, interference FOLRα or adding canonical Wnt signaling pathway or ERK signaling pathway inhibitors could significantly inhibit the effect of FA on pPSCs proliferation. Meanwhile, during the differentiation of pPSCs into insulin-secreting cells, we found that the maturation marker genes Insulin, NKX6.1, MafA, and NeuroD1 was upregulated in insulin-secreting cell masses differentiationed from pPSCs after FA treatment, and the functional molecules Insulin and C-peptide were increased, the ability to secrete insulin in response to high glucose was also increased. With the addition of Wnt and ERK signaling pathway inhibitors, the pro-differentiation effect of FA was weakened. In conclusion, FA promotes the proliferation of pPSCs by binding to folate receptor α (FOLRα) and increase the efficiency of directed differentiation of pPSCs into insulin-producing cells by regulating canonical Wnt and ERK signaling pathway. This study lays theoretical foundation for solving the bottleneck in the treatment of diabetes with stem cell transplantation in future.