Fugui Fang

Characterization of Bovine Induced Pluripotent Stem Cells by Lentiviral Transduction of Reprogramming Factor Fusion Proteins

Pluripotent stem cells from domesticated animals have potential applications in transgenic breeding. Here, we describe induced pluripotent stem (iPS) cells derived from bovine fetal fibroblasts by lentiviral transduction of Oct4, Sox2, Klf4 and c-Myc defined-factor fusion proteins. Bovine iPS cells showed typical colony morphology, normal karyotypes, stained positively for alkaline phosphatase (AP) and expressed Oct4, Nanog and SSEA1. The CpG in the promoter regions of Oct4 and Nanog were highly unmethylated in bovine iPS cells compared to the fibroblasts. The cells were able to differentiate into cell types of all three germ layers in vitro and in vivo. In addition, these cells were induced into female germ cells under defined culture conditions and expressed early and late female germ cell-specific genes Vasa, Dazl, Gdf9, Nobox, Zp2, and Zp3. Our data suggest that bovine iPS cells were generated from bovine fetal fibroblasts with defined-factor fusion proteins mediated by lentivirus and have potential applications in bovine transgenic breeding and gene-modified animals.

Effects of l‐Ascorbic Acid, α‐Tocopherol and Co‐culture on In Vitro Developmental Potential of Porcine Cumulus Cells Free Oocytes

The maturation and developmental potential on cumulus-cell-free oocytes is of great importance theoretically and practically. The present study was to investigate the effects of l-ascorbic acid, alpha-tocopherol and co-culture on in vitro developmental potential of porcine denuded oocytes (DOs). Porcine DOs were cultured in maturation medium supplemented with vitamin C (0, 50, 100, 250, 500, 750 microM) and vitamin E (0, 10, 20, 50, 100, 250 microm), respectively. And they were also co-cultured with dispersed cumulus cells (group CCscoculture), intact cumulus cells oocyte complexes (COCs) (group COCscoculture), and COCs whose oocytes were removed (group OOXcoculture), respectively. After 44 h incubation, the maturation rates, cleavage rates and blastocyst rates after parthenogenetic activation in three experiments mentioned above were collected and analysed, respectively. L-Ascorbic acid promoted porcine DOs in vitro maturation and blastocyt development after parthenogenetic activation while alpha-tocopherol did not increase the in vitro maturation rates, but improved the blastocyst rate. None of the three co-culture manner promoted the in vitro maturation and the cleavage of porcine DOs after parthenogenetic activation, but all the co-culture manners improved the blastocyst rates. Both Vitamin C and E enhance the in vitro developmental potential of porcine DOs. Co-culture increases the developmental potential of porcine DOs.

Active immunization with recombinant GnRH fusion protein in boars reduces both testicular development and mRNA expression levels of GnRH receptor in pituitary

Immunization using recombinant maltose binding protein-gonadotropin releasing hormone (MBP-GnRH6) altered both testicular development and transcription of the pituitary GnRH receptor (GnRHR) gene in boars. Scrotal measurement and blood samples were taken at 4-week interval after immunization at 9 weeks of age. The concentrations of testosterone and anti-GnRH antibodies in serum were determined by radioimmunoassay and enzyme-linked immunosorbent assay, respectively. The results showed that active immunization with MBP-GnRH6 increased the serum concentration of anti-GnRH antibodies (P<0.05) and reduced the serum concentration of testosterone (P<0.05) as compared with MBP controls. At 25 weeks of age, boars were sacrificed and testes were evaluated histologically. Testicular development was suppressed in the MBP-GnRH6 immunized animals as compared with MBP immunized boars. MBP-GnRH6 immunized pigs exhibited mounting behavior 4 weeks later than MBP immunized boars. No mature spermatozoa were observed from MBP-GnRH6 immunized animals. By real-time quantitative PCR analysis, the amount of GnRHR mRNA in the pituitary tissue was found to be significantly lower in MBP-GnRH6 immunized animals than in controls (P<0.05). These data demonstrate that recombinant MBP-GnRH6 was effective in immunological castration in boars.

Role of ghrelin on testosterone secretion and the mRNA expression of androgen receptors in adult rat testis

The present study was designed to determine the effects of ghrelin on in vivo and in vitro secretion of testosterone (T) and the expression of androgen receptor (AR) mRNA in the adult rat testis. The distribution of growth hormone secretagogue receptors (GHS-R1a) in the testis was also investigated. GHS-R1a immunoreactivity presented mainly in Sertoli and Leydig cells, primary spermatocytes, and secondary spermatocytes. Adult rats that were intracerebroventricularly (i.c.v.) administrated different dosages (1 nmol and 3 nmol) of ghrelin could significantly inhibit the secretion of T. The experession of AR mRNA in the testis was also notably reduced with 3 nmol ghrelin. Additionaly, in vitro exposure of the Leydig cells to increasing concentrations of ghrelin resulted in no obvious changes of T secretion in the culture media and AR mRNA expression of Leydig cells. Overall, our data demonstrate that the i.c.v. injection of ghrelin plays a physiological role in T secretion and AR mRNA expression in the testis, further confirming the reproductive role of ghrelin.

Genome-Wide Dynamic Profiling of Histone Methylation during Nuclear Transfer-Mediated Porcine Somatic Cell Reprogramming

The low full-term developmental efficiency of porcine somatic cell nuclear transfer (SCNT) embryos is mainly attributed to imperfect epigenetic reprogramming in the early embryos. However, dynamic expression patterns of histone methylation involved in epigenetic reprogramming progression during porcine SCNT embryo early development remain to be unknown. In this study, we characterized and compared the expression patterns of multiple histone methylation markers including transcriptionally repressive (H3K9me2, H3K9me3, H3K27me2, H3K27me3, H4K20me2 and H4K20me3) and active modifications (H3K4me2, H3K4me3, H3K36me2, H3K36me3, H3K79me2 and H3K79me3) in SCNT early embryos from different developmental stages with that from in vitro fertilization (IVF) counterparts. We found that the expression level of H3K9me2, H3K9me3 and H4K20me3 of SCNT embryos from 1-cell to 4-cell stages was significantly higher than that in the IVF embryos. We also detected a symmetric distribution pattern of H3K9me2 between inner cell mass (ICM) and trophectoderm (TE) in SCNT blastocysts. The expression level of H3K9me2 in both lineages from SCNT expanded blastocyst onwards was significantly higher than that in IVF counterparts. The expression level of H4K20me2 was significantly lower in SCNT embryos from morula to blastocyst stage compared with IVF embryos. However, no aberrant dynamic reprogramming of H3K27me2/3 occurred during early developmental stages of SCNT embryos. The expression of H3K4me3 was higher in SCNT embryos at 4-cell stage than that of IVF embryos. H3K4me2 expression in SCNT embryos from 8-cell stage to blastocyst stage was lower than that in the IVF embryos. Dynamic patterns of other active histone methylation markers were similar between SCNT and IVF embryos. Taken together, histone methylation exhibited developmentally stage-specific abnormal expression patterns in porcine SCNT early embryos.

Dynamic changes of histone H3 lysine 27 acetylation in pre-implantational pig embryos derived from somatic cell nuclear transfer

Histone H3 lysine 27 acetylation (H3K27ac) is an active epigenetic modification which has been revealed to be associated with active gene expression. It was hypothesized that H3K27ac might also participate in the porcine somatic reprogramming process during early development of SCNT-derived embryos. The spatial and temporal expression profiles of H3K27ac were investigated at different developmental stages in SCNT embryos compared with in vitro fertilization (IVF) and parthenogenetic activation (PA) counterparts. Specifically, results showed that amounts of H3K27ac gradually decreased from the earliest pronuclear stage to 8-cell stage, corresponding to the major embryonic genome activation (EGA), followed by re-acetylation of H3K27 from the morula stage onwards accompanying the first cell lineage specification in IVF embryos. Similar dynamic patterns of H3K27ac signal was observed at all developmental stages of porcine SCNT and PA embryos except for the hatched stage in which amounts of H3K27ac in SCNT and PA embryos was slightly less than that in IVF counterparts. Moreover, the gradual decrease of H3K27ac before EGA was demonstrated to be an active process independent of DNA replication, RNA and protein synthesis. The expression of HDAC1, HDAC2, MBD3 and CBP genes were well correlated with the dynamic changes of H3K27ac mark. Overall, these results indicate that H3K27ac is only defective in late SCNT blastocysts, and that the dynamic changes of this marker might also underlie the EGA and initial cell lineage specification during early embryo development.

Follicle growth and oocyte development after ovary transplantation into back muscle of immune-intact adult castrated male mice

Ovary grafting is not only a method of investigating follicle and oocyte development, but also a useful model to explore the possibility of the re-establishment of the reproductive axis in male-to-female sexual reversal. This study investigated ovary survival and follicle development after mouse ovaries were transplanted into immune-intact castrated male mice. Ten-day-old mouse ovaries were transplanted into the back muscle of adult outbred castrated male mice treated with immunosuppressants. Twenty-two days later, the ovary structure and the number of follicles present was examined by hematoxylin and eosin staining. The oocytes were harvested, and then used for in vitro maturation (IVM) and IVF. The results showed that primordial and antral follicles were mainly found in the grafts, and there were obvious differences compared with 32-day-old fresh ovaries (P<0.05). Embryos were derived from collected oocytes after IVM and IVF with a 72.4% cleavage rate and 7.9% blastocyst rate; 12 live pups were generated by embryo transfer. The hormone assay showed that plasma concentrations of both estrogen and progesterone increased after ovarian transplantation (P<0.01). In conclusion, immune-intact adult castrated male mice can support ovary survival and further development of follicles with endocrine function after ovarian transplantation.

Screening and evaluating of long noncoding RNAs in the puberty of goats

BackgroundLong noncoding RNAs (lncRNAs) are involved in regulating animal development, however, their function in the onset of puberty in goats remain largely unexplored. To identify the genes controlling the regulation of puberty in goats, we measured lncRNA and mRNA expression levels from the hypothalamus.ResultsWe applied RNA sequencing analysis to examine the hypothalamus of pubertal (case; n = 3) and prepubertal (control; n = 3) goats. Our results showed 2943 predicted lncRNAs, including 2012 differentially expressed lncRNAs, which corresponded to 5412 target genes. We also investigated the role of lncRNAs that act cis and trans to the target genes and found a number of lncRNAs involved in the regulation of puberty and reproduction, as well as several pathways related to these processes. For example, oxytocin signaling pathway, sterol biosynthetic process, and pheromone receptor activity signaling pathway were enriched as Kyoto Encyclopedia of Genes and Genomes (KEGG) or gene ontology (GO) analyses showed.ConclusionOur results clearly demonstrate that lncRNAs play an important role in regulating puberty in goats. However, further research is needed to explore the functions of lncRNAs and their predicted targets to provide a detailed expression profile of lncRNAs on goat puberty.

Effect of vitamin C on growth of caprine spermatogonial stem cells in vitro.

The genetic manipulation of spermatogonial stem cells (SSCs) can be used for the production of transgenic animals in a wide range of species. However, this technology is limited by the absence of an ideal culture system in which SSCs can be maintained and proliferated, especially in domestic animals like the goat. The aim of this study therefore was to investigate whether the addition of vitamin C (Vc) in cell culture influences the growth of caprine SSCs. Various concentrations of Vc (0, 5, 10, 25, 40, and 50 μg/mL(-1)) were added to SSC culture media, and their effect on morphology and alkaline phosphatase activity was studied. The number of caprine SSC colonies and area covered by them were measured at 10 days of culture. The expression of various germ cell and somatic cell markers such as VASA, integrins, Oct-4, GATA-4, α-SMA, vimentin, and Thy-1 was studied to identify the proliferated cells using immunostaining analyses. Further, the intracellular reactive oxygen species (ROS) level was measured at the 3rd, 6th, and 9th day after culture, and expression of Bax, Bcl-2, and P53, factors involved in the regulation of apoptosis, were analyzed on the 7th day after culture using reverse transcription polymerase chain reaction and quantitative real-time polymerase chain reaction. The results showed that the SSCs formed compact colonies and had unclear borders in the different Vc-supplemented groups at 10 days, and there were no major morphologic differences between the groups. The number and area of colonies were both the highest in the 40 μg/mL(-1) Vc group. Differential expression of markers for germ cells, undifferentiated spermatogonia, and testis somatic cells was observed. Cultured germ cell clumps were found to have alkaline phosphatase activity regardless of the Vc dose. The number of Thy-1- and Oct-4-positive cells was the most in the 40 μg/mL(-1) Vc group. Moreover, the level of ROS was dependent on the Vc dose and culture time. The Vc dose 40 μg/mL(-1) was found to be optimum with regard to decreasing ROS generation, and increasing the expression of the antiapoptotic gene Bcl-2 and decreasing the expression of the proapoptotic genes Bax and P53. In conclusion, the addition of 40 μg/mL(-1) Vc can maintain a certain physiological level of ROS, trigger the expression of the antiapoptosis gene Bcl-2, suppress the proapoptotic gene P53 and Bax pathway, and further promote the proliferation of caprine SSCs in vitro.

Effects of vitamin C on characteristics retaining of in vitro-cultured mouse adipose-derived stem cells

Adipose-derived stem cells (ADSCs), a subset of mesenchymal stem cells, have promising potential for regenerative medicine applications. However, the efficient culture of mouse adipose-derived stem cells (mADSCs) is complicated or impracticable and many properties of mADSCs are still unknown. Here, we report that vitamin C (Vc) is available for the long-term culture of mADSCs in vitro. Compared with that cultured without Vc, mADSCs growing in Vc-added media proliferate faster. The occurrence of replicative senescence and transformation of Vc-treated mADSCs is also postponed. Vc also enhanced the secretory activity of collagen and adipogenic differentiation ability of mADSCs. Moreover, the expression of CD44, Sca-1, and CD105 is higher in Vc-treated mADSCs than nontreated ones. We also found that genes related to proliferation, secretion, and pluripotency are all promoted in Vc-treated mADSCs. However, the adipogenesis ability and expression of CD44, Sca-1, and CD105 decreased when passage increased from very low passages, in parallel to the downregulation of closed-related gene expression. Our results indicate that Vc is essential for the maintenance of original properties of mADSCs in vitro; additional insights into the function of Vc on mADSCs are provided. Furthermore, as the passage increased in six passages, the characteristics of mADSCs with Vc addition were also revealed.