Yuanliang Zhang

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.

Effects of cumulus cells on rabbit oocyte in vitro maturation.

Cumulus cells (CCs) are of great importance in oocyte development and maturation in many species, but detailed influence of CCs has not been extensively examined, especially on rabbit. The present study was designed to investigate the effects of CCs and the elongation of in vitro maturation (IVM) time on rabbit oocyte nuclear and ooplasmic maturation and survival. Cumulus oocyte complexes (COCs) and naked oocytes (NOs) were recovered directly from rabbits super-ovulated with eCG. Corona-enclosed oocytes (COs) and denuded oocytes (DOs) were obtained from COCs after removing a part or whole of CCs. The oocytes were cultured in the following seven groups. (i) Cumulus cell enclosed oocytes (CEOs) were cultured alone (CEOs); (ii) COs were cultured alone (COs); (iii) DOs were cultured alone (DOs); (iv) NOs were cultured alone; (v) DOs were co-cultured with COCs [DOs(COCs)]; (vi) DOs were co-cultured with CCs [DOs(CCs)]; (vii) NOs were co-cultured with CCs [NOs(CCs)]. After the oocytes were cultured for 24 and 30 h, the nuclear maturation was evaluated by first polar body (PB1) extrusion while the ooplasmic maturation was evaluated by the cleavage rate after parthenogenetic activation. The results showed that the nuclear maturation rate of CEOs, COs, DOs(COCs) and DOs(CCs) after 24 h incubation were significantly different from each other (p < or = 0.05), the rate of DOs(CCs) was similar to that of DOs (p > or = 0.05). The cleavage rates in the first two groups were significantly higher than those of the others (p < 0.05). For oocytes cultured for 30 h, the nuclear maturation rates were significantly different for each culture model (p < 0.05). The cleavage rates in first two groups were significantly higher than those of others (p < 0.05). Both the nuclear and cleavage rates significantly increased when the culture time of DOs(COCs) was prolonged from 24 to 30 h. DOs(CCs) nuclear maturation was significantly improved when the culture time was prolonged from 24 to 30 h, but the ooplasmic maturation was not. Few NOs incubated with or without CCs accomplished nuclear maturation (approximately 2% both), even when the culture time was prolonged from 24 to 30 h. The oocyte degeneration rates were significantly different for each culture model after both 24 and 30 h incubation (p < or = 0.05). There was no significant difference in oocyte degeneration in the same groups between 24 and 30 h incubation (p > 0.05). The results suggest that rabbit CCs affect oocyte nuclear and ooplasmic maturation, and their survival. The prolongation of the culture time of rabbit oocyte from 24 to 30 h improves the nuclear and ooplasmic maturation differently in the present system. Rabbit oocytes free of CCs, especially NOs, show weak meiotic resumption potential and compromised viability, which cannot be improved by co-culture with dispersed CCs. The degeneration mostly happens at early time of IVM.

Effect of Epigallocatechin-3-Gallate on the In Vitro Developmental Potential of Porcine Oocytes and Embryos Obtained Parthenogenetically and By Somatic Cell Nuclear Transfer

The present study aimed to investigate the effects of epigallocatechin-3-gallate (EGCG) on the in vitro development of porcine oocytes, parthenogenetic activation embryos (PA), and somatic cell nuclear transfer (SCNT) embryos. In Experiment 1, 0 (control), 10, 30, and 50 μg/mL EGCG were added to in vitro maturation (IVM) medium to explore the effect of EGCG on IVM of pig oocytes. The matured oocytes were then used to produce PA and SCNT embryos. Either for nuclear maturation of oocytes or for the rates of cleavage and blastocyst of PA and SCNT embryos, no significant difference was found among all groups. However, the total cell number per cloned blastocyst was significantly lower in blastocysts derived from oocytes matured in 50 μg mL EGCG (P<0.05) as compared with the other groups. In Experiment 2, we cultured pig SCNT and PA embryos in medium containing various concentrations of EGCG to examine the effect of EGCG on preimplantation development. The cleavage and blastocyst rates and the total cell number per blastocyst did not significantly differ between PA and SCNT embryos among all groups. However, the reactive oxygen species level was significantly lower in the PA embryos cultured in 10 μg mL EGCG than the other groups (P<0.05). Our results suggest that high doses of EGCG in IVM are harmful to the oocytes as evidenced by the decreased quality of SCNT embryos, and EGCG has no beneficial effects on in vitro development of pig cloned embryos.

Characterization and analysis of differentially expressed microRNAs in hircine ovaries during the follicular and luteal phases

Ovarian activity, which is mainly controlled by follicle-stimulating hormone and luteinizing hormone, is vital to successful reproduction and maintaining reproductive efficiency in livestock. To determine if the regulation of follicular-luteal transition occurs at the post-transcriptional level in hircine ovaries, the expression patterns of small RNAs in the ovarian tissues of Anhui white goats in the follicular and luteal phases were analyzed using Solexa sequencing. In total, 1039 miRNAs were co-expressed in the two libraries, and 278 and 469 miRNAs were specifically expressed in the hircine ovaries during the follicular and luteal phases, respectively. A total of 43 potential novel miRNAs were predicted in the two libraries. GO annotation and KEGG pathway analysis were applied to analyze the target genes of all miRNAs predicted in the two libraries. The highly and differentially expressed miRNAs included miR-26-5p, miR-145-5p, miR-145, miR-145a-5p, miR-125a-5p, miR-320d, and miR-320c, which may participate in follicular-luteal transition. Five co-expressed miRNAs, of which 2 were differentially expressed between the two libraries, were randomly selected to validate the expression pattern using RT-PCR, and the results were consistent with the Solexa sequencing data. Our present results help to clarify the roles of miRNAs in the regulation of follicular-luteal transition in goat ovaries, which may further enhance the reproductive efficiency of commercially important animals in the future.

Methylation pattern variation between goats and rats during the onset of puberty

Puberty is initiated by increased pulsatile gonadotropin-releasing hormone (GnRH) release from the hypothalamus. Epigenetic repression is thought to play a crucial role in the initiation of puberty, although the existence of analogous changes in methylation patterns across species is unclear. We analysed mRNA expression of DNA methyltransferases (DNMTs) and methyl-binding proteins (MBPs) in goats and rats by quantitative real-time PCR (qRT-PCR). DNA methylation profiles of hypothalamic were determined at the pre-pubertal and pubertal stages by bisulphite sequencing. In this study, expression of DNMTs and MBPs mRNA showed different patterns in goats and rats. Global methylation variation was low in goats and rats, and the profile remained stable during puberty. Gene ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis revealed the involvement of 62 pathways in puberty in goats and rats including reproduction, type I diabetes mellitus and GnRH signalling pathways and found that Edn3, PTPRN2 and GRID1 showed different methylation patterns during puberty in goats and rats and similar variation patterns for Edn3 and PTPRN2 were showed. These indicated that Edn3 and PTPRN2 would play a role in the timing of puberty. This study provides evidence of the epigenetic control of puberty.

Validation of a recombinant human bactericidal/permeability-increasing protein (hBPI) expression vector using murine mammary gland tumor cells and the early development of hBPI transgenic goat embryos

Human bactericidal/permeability-increasing protein (hBPI) is the only antibacterial peptide which acts against both gram-negative bacteria and neutralizes endotoxins in human polymorphonuclear neutrophils; therefore, hBPI is of great value in clinical applications. In the study, we constructed a hBPI expression vector (pBC1-Loxp-Neo-Loxp-hBPI) containing the full-length hBPI coding sequence which could be specifically expressed in the mammary gland. To validate the function of the vector, in vitro cultured C127 (mouse mammary Carcinoma Cells) were transfected with the vector, and the transgenic cell clones were selected to express hBPI by hormone induction. The mRNA and protein expression of hBPI showed that the constructed vector was effective and suitable for future application in producing mammary gland bioreactor. Then, female and male goat fibroblasts were transfected with the vector, and two male and two female transgenic clonal cell lines were obtained. Using the transgenic cell lines as nuclear donors for somatic cell nuclear transfer, the reconstructed goat embryos produced from all four clones could develop to blastocysts in vitro. In conclusion, we constructed and validated an efficient mammary gland-specific hBPI expression vector, pBC1-Loxp-Neo-Loxp-hBPI, and transgenic hBPI goat embryos were successfully produced, laying foundations for future production of recombinant hBPI in goat mammary gland.

316 INDUCTION OF PIG INDUCED PLURIPOTENT STEM CELLS BY RECOMBINANT PROTEINS ENCODED BY DEFINED FACTORS

Pluripotent cells derived from any differentiated cell type through ectopic expression of transcription factors were designated as induced pluripotent stem (iPS) cells, exhibiting similar morphology and growth properties to embryonic stem (ES) cells besides expressing ES cell marker. Because iPS have the ability to differentiate into all types of cells, iPS cell technology is thought to have enormous potential for generating disease models, drug screening, toxicology, and regenerative medicine. However, for virus-mediated transfection of defined factors, the exogenous genes generally would be randomly inserted into the target cell’s genome, possibly bringing the potential hazard of insertional mutagenesis. Therefore, it is necessary to seek some new methods to induce somatic cell reprogramming without viruses. With instruction from the work of Zhou et al. (2009 Cell Stem Cell 4, 381–384), in the present study we attempted to use defined factors recombinant proteins-carried cell-penetrating peptide for the generation of porcine iPS cells, which would be a benefit for safe applications of iPS cells. Defined factors genes were amplified by PCR with specific primers of 9 arginines (R9) from recombinant plasmid pLL-hOCT4/pSox2/pMyc/pKlf4-EGFP (Yin et al. 2010 Prog. Biochem. Biophys. 37, 607–612) and inserted into prokaryotic expression vector pET-28a-EGFP. After DNA sequencing confirmation, the 4 recombinant plasmids were then transformed into BL21 strains, respectively. After IPTG induction, hOCT4/pSox2/pMyc/pKlf4-R9-EGFP fusion proteins were purified using Novagen His-Bind kit and confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, respectively. Then, defined factors recombinant proteins were added into pig fetal fibroblasts (PFF) medium every 48 h to establish pig iPS cells. The results showed that purified hOCT4/pSox2/pMyc/pKlf4-R9-EGFP fusion proteins could enter into PFF efficiently, and most of them were located in nuclei. The PPF were subcultured in stem cell medium condition and treated with defined factors recombinant proteins for 6 cycles simultaneously; the clear-cut cell colonies were gradually derived. These cells had large translucent nuclei and a high nucleo:cytoplasmic ratio and were positive for AP, Oct4, and Nanog. Detailed characterisation of such induced cells is ongoing. This research would provide new ideas for the induction of porcine somatic cell reprogramming. Y. H. Zhang and H. G. Cao contributed equally. This work was supported by NSFC (30700574 30800784/c120103) and 973 (2009CB941004).