Jiangfeng Fan

Morphometric analysis of yak placentomes during gestation.

To study the morphological characteristics of placentomes during gestation, thirty‐one yaks were used to determine anatomical and histological data of placentomes by morphometric method. The results showed that there were no significant changes in the total number of yak placentomes during gestation, but remarkably fewer placentomes in the non‐pregnant horn compared with the pregnant horn. The mean size of placentomes increased significantly from <61 days to 181–210 days and decreased in 211+ days. The development of placentomes in the pregnant horn were obviously different from that of the non‐pregnant horn except <61 days, which were longer, wider, and thicker. The volume density of fetal villi increased during gestation, with a concomitant reduction in the volume density of caruncular endometrium. However, there was no significant change in the surface density of fetal villi. The volume density of binucleate cell increased in prophase and then decreased. No apparent changes in the volume densities of maternal and fetal pyknotic cell occurred throughout gestation. There was a significant increase in the average total volume of yak placentomes from 61–90 days to 181–210 days and a significant decrease in 211+ days. The same patterns were showed in the average total volumes of caruncular endometrium and binucleate cell as well as the mean total surface area of fetal villi. However, the mean total volumes of fetal villi, maternal and fetal pyknotic cell increased constantly with increasing gestation time. Three types of placentome shape were identified in yak. The results showed that the maternal‐fetal contact area of yak was enlarged by increasing the placentome size as well as the total volume and surface area of placentome component during pregnancy, so placental function could be enhanced further to meet the needs of fetal development. Anat Rec, 2010.

Insulinlike growth factor I improves yak (Bos grunniens) spermatozoa motility and the oocyte cleavage rate by modulating the expression of Bax and Bcl-2

The aim of our present study was to examine the effects of insulinlike growth factor 1 (IGF-1) on yak sperm motility during in vitro capacitation and the relationship between the effects of IGF-1 on yak sperm motility and apoptosis was evaluated. Frozen-thawed yak spermatozoa were incubated at 38 °C for 1 hour in Tyrodes bicarbonate-buffered medium for sperm culture (Sp-TALP) with different concentrations (0, 50, 100, and 200 ng/mL) of IGF-1. In every treatment, the sperm motility was measured by a computer-assisted sperm analyzer system. The fertilizing ability of spermatozoa was evaluated on the basis of oocyte cleavage rate after insemination. The expression of Bax and Bcl-2 was examined by real-time polymerase chain reaction and Western blot for the messenger RNA and protein levels. It is interesting to note that IGF-1 improved yak spermatozoa motility and the cleavage rate of oocytes; these improvements were highest in the 100 ng/mL IGF-1 group, followed by the 200 ng/mL and 50 ng/mL groups, with the lowest improvements in motility and cleavage rates in groups without IGF-1. 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/mL IGF-1, where the Bcl-2 was the highest. Bax expression in the groups with IGF-1 was lower than that in the group without IGF-1, and Bcl-2 expression was higher in groups with IGF-1 than that in the group without IGF-1. In conclusion, this research reports that improvements in yak spermatozoa motility and the oocyte cleavage rate after the addition of IGF-I may be a result of the reduction of spermatozoa apoptosis rates by modulating the expression of Bax and Bcl-2.

Developmental competence of mature yak vitrified-warmed oocytes is enhanced by IGF-I via modulation of CIRP during in vitro maturation.

The objective of this study was to investigate whether developmental competence of mature vitrified-warmed yak (Bos grunniens) oocytes can be enhanced by supplemented insulin-like growth factor I (IGF-1) during in vitro maturation (IVM), and its relationship with the expression of cold-inducible RNA-binding protein (CIRP). In experiment 1, immature yak oocytes were divided into four groups, and IVM supplemented with 0, 50, 100 and 200 ng/mL IGF-1 was evaluated; the mRNA and protein expression levels of CIRP in mature oocytes in the four groups were evaluated using quantitative real-time PCR and western blotting analyses. In experiment 2, 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. Mature yak oocytes without vitrification served as a control group. The outcomes were as following: (1) 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/mL IGF-1 treatment group. (2) In the vitrified-warmed groups, the rates of cleavage and blastocyst were also highest in the 100 ng/mL IGF-1 treatment group (81.04 ± 1.06%% and 32.16 ± 1.01%), which were close to the rates observed in groups without vitrification (83.25 ± 0.85% and 32.54 ± 0.34%). The rates of cleavage and blastocyst in the other vitrified-warmed groups were 70.92 ± 1.32% and 27.33 ± 1.31% (0 ng/mL); 72.73 ± 0.74% and 29.41 ± 0.84% (50 ng/mL); 72.43 ± 0.61% and 27.61 ± 0.59% (200 ng/mL), respectively. There was no significant difference in the total cell number per blastocysts between the vitrified-warmed groups and group without vitrification. Thus, we conclude that 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.

Association of heat shock protein 90 with the developmental competence of immature oocytes following Cryotop and solid surface vitrification in yaks (Bos grunniens)

The correlation between the 90 kDa heat-shock protein (HSP90) and the developmental competence of yak (Bos grunniens) oocytes following the process of vitrification has not been studied clearly. In the present study, we compare the efficacies of Cryotop (CT) and solid surface vitrification (SSV) methods for the cryopreservation of immature yak oocytes. Yak cumulus oocyte complexes were randomly allocated into three groups: (1) controls, (2) CT vitrification, and (3) SSV vitrification. Oocytes were vitrified and in vitro maturated and fertilized. The percentages of nuclear maturation and in vitro development were evaluated. The vitrified-warmed oocytes were evaluated for mRNA and protein expression levels of HSP90 using quantitative real-time PCR and western blotting at various stages: matured oocytes, 2-8 cells embryos and 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. Among the three groups, the maturation rates in CT: 51.14±0.86% and SSV: 50.82±1.34% were less than those of the controls: 69.65±1.13%; the cleavage rates in CT: 39.16±1.01% and SSV: 39.08±0.92%, were less than those of the controls: 58.14±0.76%; but the blastocysts rates and total cell number in the blastocysts were similar: CT: 32.20±0.73% and 104.6±3.72; SSV: 32.35±0.81% and 102.4±1.34; and controls: 34.38±1.32% and 103.8±4.13, respectively. 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; there was not significant difference in the blastocysts in the three groups. We thus 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.

Recombinant Human Bone Morphogenetic Protein 6 Enhances Oocyte Reprogramming Potential and Subsequent Development of the Cloned Yak Embryos

This study investigated the effects of bone morphogenetic protein 6 (BMP6) supplementation in the medium during in vitro maturation (IVM) on the developmental potential of oocytes and in the subsequent development of cloned yak embryos. Cumulus-oocyte complexes (COCs) were aspirated from the antral follicles of yak ovaries and cultured with different concentrations of recombinant human BMP6 in oocyte maturation medium. Following maturation, the metaphase II (MII) oocytes were used for somatic cell nuclear transfer (SCNT), and these were cultured in vitro. The development of blastocysts 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, H3K9ac, H3K18ac, and H3K9me3) using relative quantitative RT-PCR or immunofluorescence. The presence of 100 ng/mL BMP6 significantly enhanced the oocyte maturation ratios (66.12 ± 2.04% vs. 73.11 ± 1.38%), cleavage rates (69.40 ± 1.03% vs. 78.16 ± 0.93%), and blastocyst formation rates (20.63 ± 1.32% vs. 28.16 ± 1.67%) of cloned yak embryos. The total blastocysts (85.24 ± 3.12 vs. 103.36 ± 5.28), inner cell mass (ICM) cell numbers (19.59 ± 2.17 vs. 32.20 ± 2.61), and ratio of ICM to trophectoderm (TE) (22.93 ± 1.43% vs. 31.21 ± 1.62%) were also enhanced (p < 0.05). The ratio of the Bax to the Bcl-2 gene was lowest in the SCNT + BMP6 groups (p < 0.05). The H3K9ac and H3K18ac levels were increased in SCNT + BMP6 groups (p < 0.05), whereas the H3K9me3 level was decreased; the differences in blastocysts were not significant (p > 0.05). These study results 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.

Effects of estradiol and progesterone on secretion of epidermal growth factor and insulin-like growth factor-1 in cultured yak endometrial epithelial cells

We isolated and purified yak (Bos grunniens) endometrial epithelial cells and assayed different concentrations of estradiol (E2) and progesterone (P4) with respect to secretion of epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1). Uterine epithelia were confirmed with Feulgen staining, karyotype analysis, and immunohistochemistry. Then, cells were treated with E2 and P4 and cultured in serum-free medium for 24 h. EGF and IGF-1 were measured with immunofluorescence, Real-time PCR (RT-PCR), Western blot (WB), and ELISA. When E2 and P4 were applied separately, the expression of EGF and IGF-1 did not change significantly, and EGF and IGF-1 gene and protein expression and epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor (IGFR) gene expression were significantly increased when both hormones were combined. When the hormones were used singly, the optimal concentration of E2 was 10 ng/mL and of P4 was 100 ng/mL. When combined, the optimal E2 concentration was 10 ng/mL and P4 was 10 ng/mL. Thus, E2 and P4 can modulate expression of EGF and IGF-1 in endometrial epithelial cells at the morphological, gene, protein, and exocrine level, which is of great significance to improve the rate of yak embryo implantation and reproduction.