Growth Factors Enhance The Development Potential Of Yak Embryos Produced By IVF And SCNT, The Adaptation To Cryopreservation Of Oocytes

Yaks(Bos grunniens) is an important domestic animal that survives on Qinghai-Tibet Plateau. Improving reproductive performance of yak via assisted reproductive technology plays key roles in animal husbandry of Silk Road Economic Belt in China. The aims of this study were to enhance the quality of yak embryos produced in vitro, understand the functions of growth factors on yak embryo and its effects on apoptosis, optimize vitrification techniques of yak oocytes and embryos. The experiments and results in this study as follows:(1) The expression of EGF and EGFR in immature and mature COCs was detected by the methods of Real-time PCR and immunofluorescence at m RNA and protein levels. The effects of EGF on rate of maturation, cleavage and blastocyst of yak oocytes and embryos were compared among different groups. The genes related to apoptosis were also detected. It is interesting to note that genes and protein of EGF and EGFR could be detected all stages of oocyte and embryo, the levels of EGF and EGFR in mature oocyte and blastocyst were higher than which in other stages, andmore the levels of EGFR were higher than EGF. The rate of maturation, developmental competence of embryo and quality of blastocyst were enhanced significantly by EGF, the optimal concentration was 100 ng·m L-1(p＜0.05). The level of Bax gene in mature oocyte treated with 100 ng·m L-1 EGF were significantly lower than in other groups(p＜0.05), however, the level of Baxi gene in mature oocyte treated with 100 ng·m L-1 EGF were significantly higher than in other groups(p＜0.05). The expression of HSP70 and Survivin in blastocyst were also significantly enhanced by 100 ng·m L-1 EGF(p＜0.05). The results indicated that EGF and EGFR were important autocrine or paracrine growth factors during yak COCs in vitro maturation(IVM) or development of embryo, it could enhance developmental competence of yak oocytes and embryos by regulated the expression of gene related to apoptosis, such as Bax, Baxi, HSP70 and Survivin.(2) The expression of IGF-1 and IGF-1R in immature and mature COCs was detected by the methods of Real-time PCR, WB and immunofluorescence at m RNA and protein levels. The effects of IGF-1 on apoptosis rate of cumulus cell, rate of maturation, cleavage and blastocyst of yak oocytes and embryos were compared among different groups. The results show that genes and protein of IGF-1 and IGF-1R could be detected all stages of oocyte and embryo, the levels of IGF-1 and IGF-1R in mature oocyte and blastocyst were higher than which in other stages, andmore the levels of IGF-1R were higher than IGF-1. The rate of maturation, developmental competence of embryo and quality of blastocyst were enhanced significantly by IGF-1, the optimal concentration was 100 ng·m L-1(p＜0.05). The apoptosis rate of cumulus cell were reduced by IGF-1, apoptosis rate of cumulus cell and levels of Bax were lowest in groups with 100 ng·m L-1(p＜0.05), while the levels of Bax and Bcl-2 were highest. Thus, it conclude that expressions of HSP70, Bax and Bcl-2 on yak cumulus cells were regulated by IGF- 1, and the apoptosis rate of cumulus cells was also reduced, and IGF-1 as an important growth factors during development of yak pre-implantation embryos, which could enhance developmental competence of yak embryos.(3) The testicular tissues were collected from male yak at 6 and 24 months old, the expression of EGF and EGFR in yak testicular tissue was determined by Real-time PCR, WB and IHC. Frozen-thawed yak spermatozoa were incubated at 38°C for 1 hour in Sp-TALP for sperm culture with different concentrations(0, 50, 100, and 200 ng·m L-1) of IGF-1, the sperm motility and oocyte cleavage rates after insemination were measured, the expression of Bax and Bcl-2 was examined Real-time PCR, WB for the messenger RNA and protein levels. The results show that the expression quantity of EGF and EGFR at 24 months of age was higher than at 6 months(p < 0.05), and the increase rate of EGFR on m RNA and protein levels was higher than the increase rate EGF during post-natal testes development. At 24 month of age, both the EGF and EGFR could be detected in Leydig cells, peritubular myoid cells, sertoli cells and germ cells of the yak testes. IGF-1 improved yak spermatozoa motility and the cleavage rate of oocytes; these improvements were highest in the 100 ng·m L-1 IGF-1 group, the expression level of Bax was downregulated by IGF-1, whereas Bcl-2 was upregulated Both messenger RNA and Bax proteins were lowest in groups with 100 ng·m L-1 IGF-1, where the Bcl-2 was the highest. In conclusion, the findings in present studies suggest that growth factors regulate yak spermatogenesis during testes development, the sperm motility were also regulated by growth factors, and improvements in yak spermatozoa motility and the oocyte cleavage may be a result of the reduction of spermatozoa apoptosis rates by modulating the expression of Bax and Bcl-2.(4) Yak cumulus oocyte complexes(COCs) were aspirated from the antral follicles of ovaries and cultured with different concentrations of recombinant human BMP6 in oocyte maturation medium. Following maturation, we used the metaphase II(MII) oocytes for SCNT and these were cultured in vitro. The development of blastocyst and cell numbers were detected on day 8. The apoptosis and histone modifications of yak cloned blastocysts were evaluated by detecting the expression of relevant genes and proteins(Bax, Bcl-2, H3K9 ac, H3K18 ac, and H3K9me3) using relative quantitative RT-PCR or immunofluorescence. Presence of 100 ng·m L-1 BMP6 significantly enhanced the oocyte maturation ratios, cleavage rates, and blastocyst formation rates of cloned yak embryos. The total blastocyst, inner cell mass cell numbers, and ratio of ICM:TE were also enhanced(p < 0.05). The ratio of Bax to Bcl-2 gene was lowest in the SCNT + BMP6 groups(p < 0.05). The H3K9 ac and H3K18 ac levels were increased in SCNT + BMP6 groups(p < 0.05), whereas the H3K9me3 level was decreased and the differences in blastocysts were not significance(p > 0.05). The results in this study demonstrate that addition of oocyte maturation medium with recombinant BMP6 enhances yak oocyte developmental potential and the subsequent developmental competence of SCNT embryos, and provides evidence that BMP6 is an important determinant of mammalian oocyte developmental reprogramming.(5) Yak COCs were randomly allocated into three groups:(a) controls,(b) CT vitrification, and(c) SSV vitrification, and vitrified in vitro maturated and fertilized. The percentages of nuclear maturation and in vitro development were evaluated. The m RNA and protein expression levels of HSP90 were evaluated using Real-time PCR and WB at various stages: matured oocytes, 2-8 cells embryos and blastocysts. Yak immature yak oocytes were divided into four groups, and IVM supplemented with 0, 50, 100 and 200 ng·m L-1 IGF-1 was evaluated; the m RNA and protein expression levels of CIRP in mature oocytes in the four groups were evaluated, and the mature yak oocytes in the four groups were cryopreserved using the Cryotop(CT) method, followed by chemical activation and in vitro culture for two days and eight days to determine cleavage, blastocyst rates, and total cell number in the blastocysts. No difference was found in the percentages of nuclear maturation, cleavage or blastocyst in the two vitrified groups; however, the rates of maturation were significantly lower than those in the control group(p < 0.05). The HSP90 expression level in the matured oocytes and 2-8 cell embryos of the control group was significantly higher than that in the two vitrified groups;(p < 0.05) there was no significant difference in the blastocysts in the three groups, and the blastocysts rates and total cell number in the blastocysts were also similar. The expression of CIRP in the matured oocytes was up-regulated in the IGF-1 groups and was highest expression was observed in the 100 ng·m L-1 IGF-1 treatment group, the rates of cleavage and blastocyst were also highest in the 100 ng·m L-1 IGF-1 treatment group, while there was no significant difference in the total cell number per blastocysts. Thus, it conclude that CT and SSV perform equally in the vitrification of immature yak oocytes during the process of cryopreservation, and their influence on oocytes mainly occured from the maturation to cleavage stages. The HSP90 levels in the blastocysts of the vitrified groups increased is associated with the developmental competence of the embryo. The enhancement in developmental competence of mature yak vitrified-warmed oocytes after the addition of IGF-1 during IVM might result from the regulation of CIRP expression in mature yak oocytes prior to vitrification.