B Huang

In Vitro Culture and Differentiation of Buffalo (Bubalus bubalis) Spermatogonia

The objective of this study was to develop a culture system which could support buffalo spermatogonia differentiation into spermatids in vitro. Testes from 3- to 5-month-old buffaloes were decapsulated and seminiferous tubules were enzymatically dissociated to recover spermatogonia and sertoli cells. The cells were cultured in modified Dulbecco modified Eagle medium supplemented with different concentrations of foetal bovine serum, retinol, testosterone for 2 months at 37 degrees C. Spermatogonia and sertoli cells were identified with an antibody against c-kit or GATA4, respectively. The viability of spermatogonia in the media supplemented with different concentrations of serum was all significantly higher (p < 0.05) compared with that in the medium without serum. A-paired or A-aligned spermatogonia and spermatogonial colonies (AP-positive) were observed after 7-10 days of culture and spermatid-like cells with a flagellum (6-8 microm) appeared after 30 days of culture. For cultured conditions, retinol could not significantly promote the formation of spermatid-like cells (p > 0.05), whereas supplementation of testosterone could significantly promote (p < 0.05) the formation of spermatid-like cells after 41 days of culture. The expression of the spermatid-specific marker gene (PRM2) was identified after 30 days of culture by RT-PCR. Yet, the transition protein 1 (TP1, a haploid makers) was not detected. Meanwhile, spermatids developed in vitro were also confirmed by Raman spectroscopy. These results suggest that buffalo spermatogonia could differentiate into spermatids in vitro based on the analysis of their morphology, PRM2 expression and Raman spectroscopy. Yet, the normality of the spermatid-like cells was not supported by TP1 expression.

Isolation and characterization of EG-like cells from Chinese swamp buffalo (Bubalus bubalis).

There have been few studies done on the isolation and characterization of Chinese swamp buffalo embryonic germ cells (EG cells). Here, we first report on EG‐like cells isolated from Chinese swamp buffalo fetuses. The results showed the cells grew in large, multilayered colonies, which were densely packed with an obvious border resembling mouse embryonic stem cells (ES cells) and EG cells. The buffalo EG‐like cells expressed AP, SSEA‐1, SSEA‐3, SSEA‐4 and OCT‐4. By RT—PCR, we found that undifferentiated swamp buffalo EG‐like cells expressed the OCT‐4, NANOG, SOX2, FOXD3, GP130, STAT3, and HEB gene mRNA, but not Fgf4. When these cells were cultured for more than 2 weeks without passage, they could differentiate into several types of cells including fibroblast‐like, neuron‐like, smooth muscle‐like, and epithelial‐like cells. Some cells formed simple embryoid bodies (EBs) and cystic EBs by suspension culture. By RT—PCR, we found cystic EBs expressed FOXD3, GP130, STAT3 and HEB gene mRNA, but not OCT‐4, NANOG, and SOX2 gene mRNA, which could be detected in undifferentiated buffalo EG‐like cells. At the same time, the expression of KERATIN‐14 (Endoderm), GATA4, ACTA2 (Mesoderm) and TUBB3 (Ectoderm) gene mRNA were also detected in cystic EBs. The results suggested that these cells were capable of forming three germ layers in in vitro differentiation. The expression of OCT‐4, NANOG and SOX2 might be essential for Chinese swamp buffalo EG‐like cells in a pluripotent state. During the isolation and culture of Chinese swamp buffalo EG‐like cells, we found the fetuses that were at 30–80 days post‐coitus were more efficient than others; and the mechanical method was better than trypsin digestion. The maximal passage of the mechanical method was eight, but the trypsin digestion was just three passages. So it seemed like that the buffalo EG‐like cells were sensitive to trypsin. In summary, we were the first to isolate and characterize Chinese swamp buffalo EG‐like cells that had morphology and characterization similar to those of established EG/EG‐like cells in mouse and human.

Suberoylanilide hydroxamic acid, a novel histone deacetylase inhibitor, improves the development and acetylation level of miniature porcine handmade cloning embryos

Histone deacetylase inhibitors (HDACis) can change the histone acetylation and significantly enhance the developmental competence of the pre-implantation SCNT embryo. To select a proper histone deacetylase inhibitor to improve the success rate and potentially developmental ability of handmade cloning (HMC) embryos of miniature porcine, we compared the effect of two histone deacetylase inhibitors (SAHA vs. VPA) on HMC embryo development, their histone acetylation level and the expression level of relevant genes. The blastocyst rate and number of blastocyst cells of HMC embryos treated with SAHA (SAHA-HMC) or VPA (VPA-HMC) were significantly higher than those of control (Control-HMC), respectively, but there were no significant difference between SAHA-HMC and VPA-HMC groups. In addition, the acetylation level (AcH4K8) of Control-HMC and VPA-HMC embryos at the blastocyst stage, respectively, was significantly lower than that of in vitro fertilized (IVF) and SAHA-HMC embryos. However, the acetylation H4K8 of the blastocysts had no significant difference between SAHA-HMC and the IVF groups. The SAHA-HMC blastocysts indicated comparative expression levels of Oct4 and HDAC1 (histone deacetyltransferase gene) with those of IVF blastocysts. In contrast, the expression levels of Oct4 were lower and those of HDAC1 were higher in the VPA-HMC and Control-HMC blastocysts, respectively, compared to those of the IVF blastocysts. Our results demonstrated that the HMC embryos treated by SAHA could promote the pre-implantation development and increase the levels of histone H4K8 acetylation and the expression of the OCT4 gene, yet decrease the expression of the HDAC1 gene to the comparable level of the IVF embryos. Our results proved that SAHA may be a better histone deacetylase inhibitor for porcine HMC compared to VPA, and furthermore, it may indicate that SAHA can effectively correct the abnormal histone acetylation during the HMC embryo development and subsequently improve the full-term developmental potential of the HMC embryos after embryo transplantation.

Exogenous Estradiol Benzoate Induces Spermatogenesis Disorder through Influencing Apoptosis and Oestrogen Receptor Signalling Pathway

As the exact role for exogenous oestrogen in spermatogenesis is not fully understood, the aim of this study was to investigate the effect of estradiol benzoate (EB) exposure to male mice on their spermatogenesis and fertility. Sixty male mice aged 4 weeks were randomly divided into three groups, including a control group and two treatment groups. The mice of the control group were injected with 250 μl paraffin oil only by every other day subcutaneous injection for 4 weeks. Meantime, the mice of the treatment groups were injected with EB at the concentration of 5 or 10 mg/kg, respectively. Results showed that EB slowed down the body weight gains and generated testicular atrophy with spermatogenesis disorder compared with that of the control mice, and consequently induced their infertility. Moreover, the number of TUNEL-positive cells in the testis of EB-treated mice was significantly increased with the EB concentration rise. In comparison with controls, the mRNA expression level of pro-apoptosis factors (Fas, TNF, Cytochrome C, Apaf1, Chop, Caspase-3, Caspase-8, Caspase-9 and Caspase-12) and key genes in oestrogen receptor (ER) signalling pathway (ER α, ER β, Erk1/2, Hsp90 and DAX-1) were upregulated in the testes of the treatment groups. Furthermore, Western blotting results proved the protein expression level of Fas, TNF, Cytochrome C, Chop, Caspase-3, cleaved Caspase-3, Caspase-9, Erk1/2 and Hsp90 were upregulated, and the phosphorylation level of Erk1/2 was also increased. These results indicate that EB may impair spermatogenesis through influencing the apoptosis and ER signalling pathway.

Qualitative Analyses of Protein Phosphorylation in Bovine Pluripotent Stem Cells Generated from Embryonic Fibroblasts.

Pluripotent stem cells (PSCs) generated from somatic cells via ectopic expression of specific transcription factors provide an unlimited cell resource for regenerative medicine and transgenic breeding. Here, we describe the successful generation of bovine induced PSCs (biPSCs) from foetal fibroblasts by lentivirus-mediated delivery of bovine pluripotency reprogramming factors (PRFs) OCT3/4, SOX2, KLF4, c-MYC, NANOG and LIN28. The generated biPSCs resembled embryonic stem cells (ESCs) in their gene expression profiles, self-renewal capabilities and proliferation, as well as maintenance of a normal karyotype and differentiation into diverse cell types of all three germ layers both in vitro and in vivo. Qualitative phosphoproteomics of biPSCs revealed a large number of phosphorylated proteins, which might be related to the control of biPSCs status. The successful generation of biPSCs and the analysis of their phosphoproteome would further our understanding of the epigenetic mechanisms underlying iPSC pluripotency, thus promoting their application in bovine transgenic breeding and marking avenues for future research.

The modification of mitochondrial energy metabolism and histone of goat somatic cells under small molecules compounds induction

In recent years, induced pluripotent stem cells (iPSCs) technique is able to allow us to generate pluripotency from somatic cells in vitro through the over expression of several transcription factors. Normally, viral vectors and transcription factors are commonly used on iPSC technique, which could cause many barriers on further application. In this study, we attempt to process a new method to obtain pluripotency from goat somatic cells in vitro under fully chemically defined condition. The results showed that chemically induced pluripotent stem cells-like cells (CiPSC-like cells) colonies were generated from goat ear fibroblasts by fully small-molecule compounds. Those three dimensions colonies were similar with mouse iPSCs in morphology and had strong positive alkaline phosphatase (AP) activity and expressed pluripotency related genes OCT4, SOX2, NANOG, CDH1, TDGF, GDF3, DAX1, REX1, which determined by RT-PCR. Those colonies could also differentiate into different cell types derived from three germ layers proved by RT-PCR and immunofluorescence assays. The expression of glycolysis-related genes about PGAM1, KPYM2 and HXK2 in CiPSC-like colonies formation groups was significantly higher than their parental fibroblasts, but not in the non-CiPSC-like colonies formation group. The expression of histone acetylation and methylation-related genes, HAT1 and SMYD3, was not significantly up-regulated within different groups compared to their parental fibroblasts, respectively. Yet, the expression of histone methylation-related gene, KDM5B, was significantly up-regulated on the cells from non-colonies formation group compared to parental fibroblasts, but the expression of KDM5B of the cells from CiPSC-like cell colonies was not significantly difference compared to that of parental fibroblasts. In conclusion, this is the first report that CiPSC-like cells could be generated in vitro from goat rather than just mouse under fully chemically defined condition. The generation of CiPSC-like colonies may be depended on the correct modification of energy metabolism and histone epigenetic during the reprogramming, rather than just the over-expression of those pluripotency-related genes. This study will strongly support us to further establish the stable goat CiPSC lines without any integration of exogenous genes.