AR regulates porcine immature Sertoli cell growth via binding to RNF4 and miR-124a

Sertoli cells are the only somatic cells in the seminiferous epithelium which directly contact with germ cells. Sertoli cells exhibit polarized alignment at the basal membrane of seminiferous tubules to maintain the microenvironment for growth and development of germ cells, and therefore play a crucial role in spermatogenesis. Androgens exert their action through androgen receptor (AR) and AR signalling in the testis is essential for maintenance of spermatogonial numbers, blood–testis barrier integrity, completion of meiosis, adhesion of spermatids and spermiation. In the present study, we demonstrated that AR gene could promote the proliferation of immature porcine Sertoli cells (ST cells) and the cell cycle procession, and accelerate the transition from G1 phase into S phase in ST cells. Meanwhile, miR-124a could affect the proliferation and cell cycle procession of ST cells by targeting 3′-UTR of AR gene. Furthermore, AR bound to the RNF4 via AR DNA-binding domain (DBD) and we verified that RNF4 was necessary for AR to regulate the growth of ST cells. Above all, this study suggests that AR regulates ST cell growth via binding to RNF4 and miR-124a, which may help us to further understand the function of AR in spermatogenesis.     

The influence of progesterone and prostaglandin F2α on decorin and the adhesion of caruncular epithelial cells of bovine placenta at early-mid pregnancy—Part II

One of the most important processes determining the proper course of gestation and its physiological termination in cows is the adhesion of epithelial cells allowing for direct contact of maternal and foetal parts of the placenta. Throughout pregnancy, placental cells are under strict hormonal control, which among others regulates the concentration and activity of specific proteins participating in the extracellular matrix remodelling of foetal membranes. The aim of the study was to evaluate the influence of progesterone and prostaglandin F_2α on the adhesion of epithelial cells at early-mid pregnancy in cows. Additionally, the impact of selected hormones on anti-adhesive properties of decorin was evaluated. Caruncular epithelial cells were isolated from healthy cows during pregnancy, immediately after slaughter. Primary cell cultures derived from the 2nd and 4th month of gestation were used in the experiments. The viability of cells was assessed by MTT assay. The adhesion of cells to fibronectin was measured spectrophotometrically. The activity of metalloproteinases was confirmed by the metalloproteinase assay. Progesterone (10^–5 and 10^–7 mol/L) and prostaglandin F_2α (10^–4_, 10^–5 and 10^–7 mol/L) increased the viability of bovine caruncular epithelial cells in the 2nd month of pregnancy. The treatment with prostaglandin F_2α significantly reduced the number of adherent cells from the 2nd month of gestation at the doses of 10^–4 and 10^–5 mol/L. Both progesterone and prostaglandin F_2α were shown to have an effect of decorin resulting in both a decrease in metalloproteinase activity and an increase in adhesion of cells to fibronectin.     

Production and Identification of Clone Sheep Using Bone Marrow Mesenchymal Stem Cells as Donor Cell

In order to obtain the cloned sheep using bone marrow mesenchymal stem cells（BMSCs）as the donor cells,the BMSCs of sheep were chosen and reconstructed embryos were built to transfer.10 published microsatellite markers were chosen,and the DNA samples from clone sheep,donor cells and surrogate ewes were amplified,and the relationship of father-son（RCP）was analyzed using the Quantity One for genotyping.The results showed that the reconstructed embryos were successfully built for electric fusion using sheep BMSCs as nuclear donor,and making nuclear transplantation into enucleated mature oocytes of which the fusion rate was 80.62%.20 surrogate ewe were chosen to be implanted with the reconstructed embryos at morula stage by implant surgery,and 5 lambs were born and only 3 were survived.The genotype of cloned sheep was in line with the dornor cell and the RCP were more than 99.999%.In conclusion,the first clone sheep were obtained successfully by using BMSCs as a nuclear donor in this experiment.     

Effects of Donor Cells Treated with Egg Extracts on the Development Competence of Cloned Embryos

Japanese Balck cattle fetal fibroblasts (JBCFF) were induced with Xenopus leavis egg extracts and somatic cell nuclear transfer (SCNT) was carried out with the reprogrammed JBCFF as donor cells in order to investigate their effects on SCNT efficiency.Three samples of egg extracts were acquired from different Xenopus laevis.The protein contents and kinds in extracts were evaluated with BCA Protein Quantification Kit and SDS-PAGE.Concentration of Digitonin to permeabilize JBCFF was optimized and assessed with PI staining.Reprogrammed cells treated with egg extract were used as donor in SCNT.Additionally the reconstructed embryos were activated with ionomycin+6-DMAP and A23187+6-DMAP to compare their effects on the development competence.The protein contents of extracts samples were 56.2255,64.6570 and 71.2158 μg/mL,respectively,the each extract had the same composition about 40-55 and 70-100 ku.The optimal concentration of Digitonin was 7 μg/mL and the permeabilization rate was 55.44%.After extracts treatment and continuous culture for 6-7 d,JBCFF formed well-defined colony structures.No significant composition difference was found in rates of fusion (92.83% vs 96.04%),cleavage (89.64% vs 89.78%) and blastocyst formation (24.06% vs 23.12%) of cloned embryos when the colony cells and JBCFF without extracts treatment were used as donor cells (P>0.05).Similarly,the two activation methods had no significant effect on the developmental competence of cloned embryos (cleavage rate 92.16% vs 92.28%,blastocyst rate 23.21% vs 24.18%).Conclusively,Xenopus leavis,egg extracts could induce JBCFF reprogramming to a low differentiated state.However donor cells with reprogramming partially could not improve the development of cloned embryos and its mechanism requires further research.     

Analysis on Differential Expression Genes in Porcine Aortic Vascular Endothelial Cells Induced by Apolipoprotein C Ⅲ

The aim of this study was to investigate the differential expression genes induced by ApoCⅢ,and study the function of ApoCⅢ.Porcine aortic vascular endothelial cells were successfully isolated using enzyme digestion,and then screened the differential expression genes induced by ApoCⅢ using the Solexa high-throughput sequencing technology.The results showed 647 differential expression genes,including 390 up-regulated genes and 257 down-regulated genes.The qRT-PCR results verified that the gene expression results from Solexa sequencing data were reliable.GO and Pathway analysis showed that the function of differential expression genes were related to immune response,cell apoptosis and death.These findings suggested that ApoCⅢ affected the physiological function of porcine aortic endothelial cells by the molecular pathways of inflammation,cell adhesion and apoptosis,which provided a theoretical basis for further understanding the molecular mechanisms of atherosclerosis caused by ApoCⅢ.     

Effect of Vitamin E on Bovine Granulosa Cells Apoptosis and Proliferation through Cx43

The aim of this study was to determine the effect of vitamin E on Cx43,the mechanism and function of vitamin E on bovine granulosa cells apoptosis and proliferation.In this study,granulosa cells were isolated from bovine ovary and cultivated in vitro by adding different concentration of vitamin E (0,25,50,100,200 and 500 μmol/L) for 24 h.After cultured,apoptotic cells were detected by FCM,mRNA expression levels of BCL2/BAX、P53 and Cx43 genes were determined by Real-time PCR and cell proliferation was detected by CCK8.The results showed that compared to control group,100 μmol/L vitamin E could significantly inhibit the apoptosis of granulosa cells (P<0.05).Real-time PCR detection results showed that vitamin E significantly changed the mRNA expression levels of BCL2/BAX,P53,Cx43 genes (P<0.05).Vitamin E could significantly improve granulosa cells proliferation when granulosa cells were treated for 24 and 36 h (P<0.05).The results provided a theoretical basis on further analysis for studing the influence mechanism of vitamin E on oocytes development and maturity,and improvement of female animal reproduction by influencing granulosa cells proliferation and apoptosis.     

应用分子靶点药物治疗犬肥大细胞瘤的研究进展

犬肥大细胞瘤因其恶性率和转移率都较高,并且常规化疗药物副作用较强,患犬生存期和生存质量显著下降,因此急需新型的药物进行治疗。近几年,治疗肿瘤的分子靶点药物凭借其疗效好、副作用低的特点,受到越来越广泛的关注,特别是在治疗犬肥大细胞瘤的应用中效果显著。论文重点介绍了欧美小动物临床最常用的伊马替尼、托赛拉尼、马赛替尼这3种以异常KIT为靶点的药物及其药物作用机理及药效,最后介绍了由c-kit基因二次突变所导致的耐药问题及其预防措施,期望能对我国小动物临床医师和科研工作者有所启发。     

Lymphangiosarcoma in 12 dogs: a case series (1998–2013)

Lymphangiosarcoma (LAS) is an uncommon malignant neoplasia arising from lymphatic endothelium; little information exists regarding therapy. Single institutional retrospective review for canine LAS histopathology diagnoses over a 15‐year period yielded 12 dogs. Ten dogs were presented for a mass and/or swelling at cervical, trunk or limb regions. Prior to diagnosis, 10 dogs received empiric wound therapy. Cytology performed in 10 consisted of mild inflammation. Survival ranged from 60, 168 and 876 days for three dogs with palliation; 90 days with prednisone in one; 182 days with chemotherapy in one; 240, 267, 487, 630 and 941 days for five receiving surgery; and 574 days for one receiving surgery, radiation and chemotherapy. One dog is alive with recurrence at 243 days following surgery and carboplatin chemotherapy. Clinical improvement existed in LAS dogs receiving multimodal therapies. Early tissue biopsies are recommended for progressive oedematous lesions of unknown origin.