Junfeng He

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.

Histochemical and ultrastructural observations of respiratory epithelium and gland in yak (Bos grunniens).

Submucous glands and epithelial mucous cells of yak (Bos grunniens) respiratory tract have been studied by a variety of histochemical methods and transmission electron microscopy for differentiating and characterizing serous and mucous cells. By light microscopy, the distribution, numbers of mucous cells, volume of mucous glands (Reid index), and the ratio of mucous cell to serous cell in the bronchial tree were measured with different staining. Histochemically, a majority of mucous cells, presented in the surface epithelium of bronchi and glands, secreted neutral and acid mucosubstances, only a few sulfated mucosubstances were present. No mucus‐producing cells were observed from the terminal to respiratory bronchiolar level. Ultrastructurally, serous cells in glands of the lamina propria had two distinct forms: one type filled with many round dense secretory granules, plentiful RER and few other organelles, similar to other animals; the other type contained some oval mitochondrial and distended RER, the granules resembled the former. The mucous cells in gland were similar to that of epithelium, which containing abundant secretory granules with an eccentric core. The mucous cells of the surface epithelium differ from other animals in the structure and histochemistry of their secretory granules. Analysis of the size and distribution of the secretory granules and other organelles of serous cells suggested that differences represent different phases of a secretory cycle, not various populations of cell or granules. Anat Rec 293:1259–1269, 2010.

Morphological Analysis of the Lung of Neonatal Yak

With 20 figures and 5 tables

Cloning of HSP90, expression and localization of HSP70/90 in different tissues including lactating/non-lactating yak (Bos grunniens) breast tissue

The aim of this study is to investigate the expression and localization of HSP70/90 in different tissues and explore the regulation effects of HSP70/90 at lactation period of female yaks. HSP90 mRNA was cloned from the heart samples of female yaks, Quantitative real-time (qRT-PCR), Western blotting (WB), immunohistochemistry and immunofluorescence assays were utilized to analyze the expressions of HSP70/90 mRNA and protein in different tissues. Sequence analysis showed that HSP90 is a conserved molecular chaperone of female yaks. The qRT-PCR, WB results showed that the expressions of HSP70/90 mRNA and protein were significantly different in different tissues, and 3-fold higher expression during the lactation period than the non-lactation period of breast tissue (P < 0.01). Immunohistochemistry and immunofluorescence assays results showed that HSP70/90 were located in the cardiac muscle cells, cerebellar medulla, theca cells lining at the reproductive system, and the mammary epithelia of the breasts. In addition, the expression level of HSP70 was higher than those of HSP90 in all examined tissues. Therefore, our results strongly suggest that the expression and localization of HSP70/90 could provide significant evidence to further research in tissue specific expression, and lactation function of female yaks.

Segmentation features and structural organization of the intrapulmonary artery of the yak

This study aims to systematically investigate intrapulmonary artery segmentation, blood vessel wall characteristics and structure organization, and the interrelation between intrapulmonary artery structure and plateau hypoxia adaptation in yak. The normal intrapulmonary artery structure of the yak had been studied using histological methods and transmission electron microscopy. The intrapulmonary artery of the yak was also examined using morphometric analysis and angiography. Results showed that the elastic intrapulmonary artery is divided into two types, namely, classical and transitional elastic segments. The muscular intrapulmonary artery is divided into three types, namely, transitional, classical muscular, and muscular arteriole segments. In the transitional elastic artery, elastic fibers and smooth muscles are linked through three models of ends, lateral branches, and branch tops. Two phenomena are possible for the transition from the elastic intrapulmonary artery to the muscular artery. One phenomenon postulates that a less elastic membrane is first increased and then suddenly decreased, and another supposes that the elastic membrane is gradually reduced and assembled in one to two layers before entering the transitional muscular artery. The smooth muscle of the intrapulmonary artery tunica media had more apophysis; it was physically connected with elastic membrane or fiber and composed of functionally resilient unit of the intrapulmonary arterial wall. Glycogenosomes increased in the muscular intrapulmonary artery smooth muscle cells. It exist one to two layers intact smooth muscle in intrapulmonary arteriole, the presence of intact smooth muscle in the intrapulmonary arteriole of the yak is a kind of structure adaptation to low‐oxygen environment. Anat Rec, 296:1775–1788, 2013.

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.

Regulation by HSP70/90 in the different tissues and testis development of male cattle (Cattle-yak and Yak)

HSP70/90 play important role in testis develop and spermatozoa regulation, but the contact of HSP70/90 with infertility in cattle is unclear. Here, we focus on male cattle-yak and yak, which to investigate the expression and localization of HSP70/90 in different tissues, and explore the influence of HSP70/90 to infertility. In our study, a total of 54 cattle (24 cattle-yak and 30 yak) were examined. The HSP90 mRNA of cattle-yak was cloned first and found amino acid variation in HSP90, which led to difference at protein spatial structure compare with yak. To investigate whether the expression of HSP70/90 mRNA and protein are different in cattle-yak and yak, we used real-time quantitative PCR (qRT-PCR) and Western blot (WB) to examine them. We found that the expression level of HSP70/90 mRNA and protein are disparity in different tissues and testis development stages, and obviously high expression was observed in testicle during juvenile and adult, Moreover, it‘s interestingly in which the HSP70 expression is significant high in yak whereas HSP90 in cattle-yak (P<0.01). On this bases, we detect the location of HSP70/90 in testis by immunohistochemical (IHC) and immunofluorescence (IF), the results demonstrate that HSP70/90 were located in the epithelial cells, spermatogenic cells and mesenchymal cells. In summary, our study proved the expression of HSP70/90 are different in tissues, and the expression of HSP90 is obviously high in testis of cattle-yak, which propose that the infertility of cattle-yak may cause from up-regulating of HSP90.

Regulation by Hsp27/P53 in testis development and sperm apoptosis of male cattle (cattle-yak and yak): LIU et al.

Heat shock protein 27 (Hsp27)/protein 53 (P53) plays an important role in testis development and spermatozoa regulation, but the relationship between Hsp27/P53 and infertility in cattle is unclear. Here, we focus on male cattle‐yak and yak to investigate the expression and localization of Hsp27/P53 in testis tissues and to explore the influence of Hsp27/P53 on infertility. In our study, a total of 54 cattle (24 cattle‐yak and 30 yak) were examined. The Hsp27 and P53 messenger RNA (mRNA) of cattle‐yak were cloned, and amino acid variations in Hsp27 and P53 were found; the variations led to differences in the protein spatial structure compared with yak. We used real‐time quantitative polymerase chain reaction and western blot to investigate whether the expression of Hsp27/P53 mRNA and protein was different in cattle‐yak and yak. We found that the expression levels of Hsp27/P53 mRNA and protein were different in the testis developmental stages and the highest expression was observed in testicles during adulthood. Moreover, the Hsp27 expression was significantly higher in yak, whereas P53 expression was higher in cattle‐yak (p < 0.01). On this basis, we detected the location of Hsp27/P53 in the testis by immunohistochemistry and immunofluorescence. The results demonstrated that Hsp27 was located in spermatogenic cells at different developmental stages and mesenchymal cells of the yak testicles. However, P53 was located in the primary spermatocyte and interstitial cells of the cattle‐yak testicles. In summary, our study proved that the expression of Hsp27/P53 differed across the testis developmental stages and the expression of P53 was higher in the testis of cattle‐yak, which suggested that the infertility of cattle‐yak may be caused by the upregulation of P53.