Dmrt1 regulates the immune response by repressing the TLR4 signaling pathway in goat male germline stem cells.

Double sex and mab-3-related transcription factor 1 (Dmrt1), which is expressed in goat male germline stem cells (mGSCs) and Sertoli cells, is one of the most conserved transcription factors involved in sex determination. In this study, we highlighted the role of Dmrt1 in balancing the innate immune response in goat mGSCs. Dmrt1 recruited promyelocytic leukemia zinc finger (Plzf), also known as zinc finger and BTB domain-containing protein 16 (Zbtb16), to repress the Toll-like receptor 4 (TLR4)-dependent inflammatory signaling pathway and nuclear factor (NF)-κB. Knockdown of Dmrt1 in seminiferous tubules resulted in widespread degeneration of germ and somatic cells, while the expression of proinflammatory factors were significantly enhanced. We also demonstrated that Dmrt1 stimulated proliferation of mGSCs, but repressed apoptosis caused by the immune response. Thus, Dmrt1 is sufficient to reduce inflammation in the testes, thereby establishing the stability of spermatogenesis and the testicular microenvironment.

Characterization of porcine extraembryonic endoderm cells.

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.

Canonical Wnt signaling pathway contributes to the proliferation and survival in porcine pancreatic stem cells (PSCs).

Pancreatic stem cells (PSCs) transplantation is a potential therapeutic approach to type 1 diabetes mellitus (D1M). However, before clinical use, there are some major hurdles to be faced that need to be comprehensively considered and given some potential solutions in vitro. Human PSCs are difficult to obtain and have a short replicative senescence. As an alternative, we instead established porcine PSCs; as insulin is highly conserved and physiological glucose levels are similar between human and porcine. In order to solve the problems during transplantation therapy, such as the need for an enormous amount of PSCs and good cell survival in overactive autoimmunity induced by reactive oxygen cpecies (ROS) in D1M patients, we utilized Wnt3a overexpression to activate the canonical Wnt signaling pathway in PSCs. We found that the expression of proliferation genes, such as c-Myc, was up-regulated as the downstream of ß-catenin, which promoted the PSCs proliferation and made cell numbers to meet the transplantation needs. We also showed that activation of the Wnt pathway made cells more readily tolerate ROS-caused mitochondria injury and cell apoptosis, thus making cells survive in autoimmune patients. The present study provides a theoretical basis for cell transplantation therapy of diabetes.

Reconstruction of damaged corneal epithelium using Venus-labeled limbal epithelial stem cells and tracking of surviving donor cells.

Limbal epithelial stem cells are responsible for the self-renewal and replenishment of the corneal epithelium. Although it is possible to repair the ocular surface using limbal stem cell transplantation, the mechanisms behind this therapy are unclear. To investigate the distribution of surviving donor cells in a reconstructed corneal epithelium, we screened a Venus-labeled limbal stem cell strain in goats. Cells were cultivated on denuded human amniotic membrane for 21 days to produce Venus-labeled corneal epithelial sheets. The Venus-labeled corneal epithelial sheets were transplanted to goat models of limbal stem cell deficiency. At 3 months post-surgery, the damaged corneal epithelia were obviously improved in the transplanted group compared with the non-transplanted control, with the donor cells still residing in the reconstructed ocular surface epithelium. Using Venus as a marker, our results indicated that the location and survival of donor cells varied, depending on the corneal epithelial region. Additionally, immunofluorescent staining of the reconstructed corneal epithelium demonstrated that many P63(+) cells were unevenly distributed among basal and suprabasal epithelial layers. Our study provides a new model, and reveals some of the mechanisms involved in corneal epithelial cell regeneration research.

Expression of FGF4 mRNA is mediated by mating behaviours in mice.

In mammals, breeding is preceded by species-specific mating behaviours. In this study, we investigated whether parthenogenetic embryo quality could be improved by mating behaviours in mice. To investigate this hypothesis, female mice were mated with vasectomized Kunming white male mice after superovulation. Oocytes were collected and counted at 16 h after superovulation. The oocytes were then artificially activated by medium containing 10 mM strontium chloride and 5 µg/ml cytochalasin B. Blastocysts were obtained by cultivating activated oocytes in vitro. Expression levels of reprogramming transcription factors (i.e. Oct4, Sox2, Klf4 and c-Myc) in oocytes, apoptosis-related genes (i.e. Bax, Bcl2 and c-Myc) in cumulus cells and pluripotency-related transcription factors (i.e. Oct4, Nanog and FGF4) in blastocysts were analysed in samples collected from mated and unmated mice. Additionally, developmental competence of parthenogenetic embryos was used to assess following fibroblast growth factor 4 (FGF4) treatment. The results showed that the formation rate of blastocysts in unmated mice was significantly higher than that in mated mice (p < 0.05). Embryo development was primarily blocked at the eight-cell stage in mated mice; however, the blastocyst formation rate did not differ significantly between groups after the addition of 25 ng/ml FGF4 to the medium at the four-cell stage (p > 0.05). Moreover, the expression of the reprogramming factor Sox2 was significantly different in oocytes collected from mated versus unmated mice. Taken together, our results demonstrated that mating behaviours influenced embryonic development in vitro by decreasing FGF4 expression.

[Nuclear transfer and therapeutic cloning].

Nuclear transfer and therapeutic cloning have widespread and attractive prospects in animal agriculture and biomedical applications. We reviewed that the quality of oocytes and nuclear reprogramming of somatic donor cells were the main reasons of the common abnormalities in cloned animals and the low efficiency of cloning and showed the problems and outlets in therapeutic cloning, such as some basic problems in nuclear transfer affected clinical applications of therapeutic cloning. Study on isolation and culture of nuclear transfer embryonic stem (ntES) cells and specific differentiation of ntES cells into important functional cells should be emphasized and could enhance the efficiency. Adult stem cells could help to cure some great diseases, but could not replace therapeutic cloning. Ethics also impeded the development of therapeutic cloning. It is necessary to improve many techniques and reinforce the research of some basic theories, then somatic nuclear transfer and therapeutic cloning may apply to agriculture reproduction and benefit to human life better.