YAP regulates porcine skin-derived stem cells self-renewal partly by repressing Wnt/ß-catenin signaling pathway.

Skin-derived stem cells (SDSCs) are a class of adult stem cells (ASCs) that have the ability to self-renew and differentiate. The regulation mechanisms involved in the differentiation of SDSCs are a hot topic. In this paper, we explore the link between the transcriptional regulator yes-associated protein (YAP) and the fate of porcine SDSCs (pSDSCs). We found that lysophosphatidylcholine (LPC) activates YAP, promotes pSDSCs pluripotency, and counteracts transdifferentiation of pSDSCs into porcine primordial germ cell-like cells (pPGCLCs). YAP promotes the pluripotent state of pSDSCs by maintaining the high expression of the pluripotency genes Oct4 and Sox2. The overexpression of YAP prevented the differentiation of pSDSCs, and the depletion of YAP by small interfering RNA (siRNAs) suppressed the self-renewal of pSDSCs. In addition, we found that YAP regulates the fate of pSDSCs through a mechanism related to the Wnt/ß-catenin signaling pathway. When an activator of the Wnt/ß-catenin signaling pathway, CHIR99021, was added to pSDSCs overexpressing YAP, the ability of pSDSCs to differentiate was partially restored. Conversely, when XAV939, an inhibitor of the Wnt/ß-catenin signaling pathway, was added to YAP knockdown pSDSCs a higher self-renewal ability resulted. Taken together, our results suggested that YAP and the Wnt/ß-catenin signaling pathway interact to regulate the fate of pSDSCs.

The proliferation role of LH on porcine primordial germ cell-like cells (pPGCLCs) through ceRNA network construction.

BACKGROUND: The transdifferentiation of skin-derived stem cells (SDSCs) into primordial germ cell-like cells (PGCLCs) is one of the major breakthroughs in the field of stem cells research in recent years. This technology provides a new theoretical basis for the treatment of human infertility. However, the transdifferentiation efficiency of SDSCs to PGCLCs is very low, and scientists are still exploring ways to improve this efficiency or promote the proliferation of PGCLCs. This study aims to investigate the molecular mechanism of luteinising hormone (LH) to enhance porcine PGCLCs (pPGCLCs) proliferation. RESULTS: In this study, we dissected the proliferation regulatory network of pPGCLCs by whole transcriptome sequencing, and the results showed that the pituitary-secreted reproductive hormone LH significantly promoted the proliferation of pPGCLCs. We combined whole transcriptome sequencing and related validation experiments to explore the mechanism of LH on the proliferation of pPGCLCs, and found that LH could affect the expression of Hippo signalling pathway-related mRNAs, miRNAs and lncRNAs in pPGCLCs. CONCLUSIONS: For the first time, we found that LH promotes pPGCLCs proliferation through the competing endogenous RNA (ceRNA) regulatory networks and Hippo signalling pathway. This finding may help to elucidate the molecular mechanism by which LH promotes pPGCLCs proliferation.

Di (2-ethylhexyl) phthalate impairs primordial follicle assembly by increasing PDE3A expression in oocytes.

It is known that Di (2-ethylhexyl) phthalate (DEHP) may impact mammalian reproduction and that in females one target of the drug's action is follicle assembly. Here we revisited the phthalate's action on the ovary and from bioinformatics analyses of the transcriptome performed on newborn mouse ovaries exposed in vitro to DEHP, up-regulation of PDE3A, as one of the most important alterations caused by DEHP on early folliculogenesis, was identified. We obtained some evidence suggesting that the decrease of cAMP level in oocytes and the parallel decrease of PKA expression, consequent on the PDE3A increase, were a major cause of the reduction of follicle assembly in the DEHP-exposed ovaries. In fact, Pde3a RNAi on cultured ovaries reducing cAMP and PKA decrease counteracted the primordial follicle assembly impairment caused by the compound. Moreover, RNAi normalized the level of Kit, Nobox, Figla mRNA and GDF9, BMP15, CX37, γH2AX proteins in oocytes, and KitL transcripts in granulosa cells as well as their proliferation rate altered by DEHP exposure. Taken together, these results identify PDE3A as a new critical target of the deleterious effects of DEHP on early oogenesis in mammals and highlight cAMP-dependent pathways as major regulators of oocyte and granulosa cell activities crucial for follicle assembly. Moreover, we suggest that the level of intracellular cAMP in the oocytes may be an important determinant for their capability to repair DNA lesions caused by DNA damaging compounds including DEHP.

Identification of oxidative stress-related Xdh gene as a di(2-ethylhexyl)phthalate (DEHP) target and the use of melatonin to alleviate the DEHP-induced impairments in newborn mouse ovaries.

This study, using an in vitro ovary culture model, investigates the mechanisms through which di(2-ethylhexyl)phthalate (DEHP) impairs germ cell cyst breakdown and primordial follicle assembly. The results indicate the latter effects exerted by 10 or 100 µmol/L DEHP in cultured newborn ovaries were associated with increased levels of reactive oxygen species (ROS) and apoptosis. Based on a transcriptome analysis, we found the expression of the oxidative stress-related gene Xdh (xanthine dehydrogenase) was significantly upregulated in DEHP-cultured ovaries. Two treatments, namely Xdh RNAi or the addition of melatonin to the ovary culture, inhibited the increase in Xdh expression and ROS levels caused by DEHP and, at the same time, reduced apoptosis and the impairment of primordial follicle assembly in the treated ovaries. Together, the results identify Xdh gene as one of the major targets of DEHP in newborn ovaries and that the consequent increased level of ROS is possibly responsible for the increment of apoptosis and primordial follicle assembly impairment. At the same time, they highlight that melatonin alleviates the effects of DEHP as with other endocrine-disrupting compounds on the ovary.

RA promotes proliferation of primordial germ cell-like cells differentiated from porcine skin-derived stem cells.

Previous studies have shown that primordial germ cell-like cells (PGCLCs) can be obtained from human, porcine and mouse skin-derived stem cells (SDSCs). In this paper, we found retinoic acid (RA), the active derivative of vitamin A, accelerated the growth of porcine primordial germ cells (pPGCs) and porcine PGCLCs (pPGCLCs) which were derived from porcine SDSCs (pSDSCs). Moreover, flow cytometry results revealed that the proliferation promoting effect of RA was attenuated by U0126, a specific inhibitor of extracellular signal-regulated kinase (ERK). Western blot analysis showed the protein level of ERK, phosphorylated ERK, cyclin D1 (CCND1), and cyclin-dependent kinase 2 (CDK2) increased after stimulation with RA, and this effect could also be abolished by U0126. Our data revealed that ablation of ERK expression by U0126 should significantly decrease proliferation of pPGCLCS. This reduction was because CCND1 and CDK2 proteins level decrease and subsequently the pPGCLCs were arrested in the G0/G1 phase. In addition, we also confirmed RA indeed promoted the proliferation of pPGCs isolated from porcine fetal genital ridges in vitro. Furthermore, our data indicated that DNA methylation pattern were changed in pPGCLCs and this pattern were more similar to pPGCs.