Penggang Liu

Changes in the Anatomic and Microscopic Structure and the Expression of HIF-1α and VEGF of the Yak Heart with Aging and Hypoxia.

The study aimed to identify the changes of anatomic and microscopic structure and the expression and localization of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) in the myocardium and coronary artery of the yak heart adapted to chronic hypoxia with aging. Thirty-two yaks (1 day, 6 months, 1 year, 2 years, and 5 year old) were included, and immunoelectronmicroscopy, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) were used. Right ventricular hypertrophy was not present in yaks with aging. There was no intima thickening phenomenon in the coronary artery. The ultrastructure of myofibrils, mitochondria, and collagen fibers and the diameter and quantity of collagen changed significantly with aging. The enzymatic activity of complexes I, II, and V increased with age. Immunogold labeling showed the localization of HIF-1α protein in the cytoplasm and nuclei of endothelial cells and cytoplasm of cardiac muscle cells, and VEGF protein in the nuclei and perinuclei areas of smooth muscle cells of coronary artery, and in the cytoplasm and nuclei of endothelial cells. ELISA results showed that HIF-1α secretion significantly increased in the myocardium and coronary artery from an age of 1 day to 2 years of yaks and decreased in old yaks. However, VEGF protein always increased with aging. The findings of this study suggest that 6 months is a key age of yak before which there are some adaptive changes to deal with low-oxygen environment, and there is a maturation of the yak heart from the age of 6 months to 2 years.

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.

The histological characteristics, age-related thickness change of skin, and expression of the HSPs in the skin during hair cycle in yak (Bos grunniens)

Objective This experiment was conducted to study the histological characteristics, age-related thickness changes, and expression of HSPs in the skin of yak. Methods A total of 20 yaks (10 males and 10 females) were used. Different regions of the normal skin of three different ages (newborn, half-year-old and adult) of yaks were harvested for histological study and thickness measurement. Biopsy samples were taken from the scapula regions of the skin from the same five approximately 1-year-old yaks during the hair cycle (telogen, anagen and catagen). RT-PCR, western blot and immunohistochemistry methods using the mRNA and protein levels were used to detect the expression of HSP27, HSP70 and HSP90. RT-PCR method was used to detect the mRNA expression of CGI-58 and KDF1. The IPP6.0 software was used to analyze the immunohistochemistry and measure the thickness of the skin. Results The general histological structure of hairy yak skin was similar to other domestic mammals. The unique features included prominent cutaneous vascular plexuses, underdeveloped sweat glands, a large number of nasolabial glands in the nasolabial plate, and hair follicle groups composed of one primary follicle and several secondary follicles. The skin, epidermis and dermis thickness did vary significantly between different body regions and different ages. The thickness of the skin, epidermis and dermis increased from newborn to adult in yaks. Yak skin thickness decreased from dorsally to ventrally on the trunk. The skin on the lateral surface was thicker than the skin on the medial surface on the limbs. HSP27, HSP70 and HSP90 showed different expression patterns during the hair cycle using RT-PCR, western blot and immunohistochemistry methods. The expression of HSP27 mRNA and protein in the anagen stage was the highest, followed by the catagen stage, and the expression in the telogen stage was the lowest. The expression of HSP70 mRNA and protein in the telogen stage was the highest, followed by the anagen stage, and the expression in the catagen stage was the lowest. The expression of HSP90 mRNA and protein in the anagen stage was the highest, followed by the telogen stage, and the expression in the catagen stage was the lowest. HSPs were mainly expressed in the outer root sheath of hair follicle during the hair cycle, also expressed in epidermis, sebaceous gland and sweat gland in the skin of Yak. The expression of CGI-58 mRNA in the anagen stage was the highest, followed by the catagen stage, and the expression in the telogen stage was the lowest. The expression of KDF1 mRNA in the telogen stage was the highest, followed by the catagen stage, and the expression in the anagen stage was the lowest. Meaning In this study, we examined and fully described the histology of normal skin in Yak and measured the skin thickness of different ages and different regions in Yak. These data may be useful to better understand and appreciate the adaptability features of yak skin. Our investigation reports the expression patterns of HSPs in yak skin for the first time. The different expression pattern of HSPs during the hair cycle suggests they may play different roles in yak hair follicle biology.

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.

Survivin expression and localization in different organs of yaks (Bos grunniens)

Yaks (Bos grunniens) have special physiological structures that help them adapt to high-altitude environments. Survivin is actively studied in cancer tissues, but less in normal tissues. Therefore, the aim of the present study was to analysis the relationship between survivin expression and apoptosis rate in yaks. A partial gene sequence of survivin was cloned and characterized using bioinformatics. The expression of survivin was investigated using real-time quantitative PCR (RT-qPCR) and western blot (WB) analysis and localized using immunohistochemistry (IHC). The results revealed that in normal physiological organs, survivin is mainly expressed in cytoplasm and its expression was up-regulated with age. Its expression in heart and liver was higher than in other organs, such as spleen, lung, brain, kidney, and testis. It is noteworthy that the expression of survivin in spleen is differed from that in other organs. Therefore, we selected immune organs (lymph node, thymus and spleen) to investigate the relationship between survivin expression and apoptosis. Caspase-3 was used as a reference. Within the same age group, the expression of survivin was the highest in the spleen, but that of caspase-3 was the highest in the lymph node (P < 0.01). Furthermore, the IHC analysis revealed that survivin and caspase-3 are expressed in the same location (mainly in the cytoplasm, Hassalls corpuscles, the medulla of the lymph node, the red pulp and marginal zone of the spleen. More importantly, survivin expression was down-regulated with age in immune organs, and the opposite trend was observed for caspase-3 expression (P < 0.01). The results proved that the expression of survivin and caspase-3 is down- and up-regulated with age, respectively, suggesting that survivin and caspase-3 might coordinating and participating in slowing down the rate of apoptosis rate in immune organs of healthy yak.

Yak (Bos grunions) tonsils: Morphological description and expression of IgA and IgG: Expression of IgA and IgG and morphology of yak tonsils

This study aimed to describe the morphology, expression of IgA and IgG in adult yak tonsils. The 12 clinically healthy yak tonsils [3‐ to 6‐year old, n = 12] were examined for morphology using light, and transmission electron microscopes. Expression of IgA and IgG was measured by qRT‐PCR, ELISA, and immunohistochemistry. The results showed that the palatine tonsil, the tonsil of the soft palate, and the lingual tonsil were oropharyngeal tonsils. The stratified squamous epithelia covering them had a thick underlying layer of connective tissue and their crypts were heavily infiltrated by lymphocytes. The pharyngeal tonsil and the tubal tonsil were nasopharyngeal tonsils. The epithelia of them was predominantly pseudostratified columnar ciliary epithelium, which were loosely arranged with a number of desmosomes or intermediate junctions variably connecting them. The expression levels of IgA and IgG mRNA and protein from high to low was in the pharyngeal tonsil, palatine tonsil, tonsil of the soft palate, lingual tonsil, and tubal tonsil, respectively. Interestingly, the expression of IgG was very significantly higher than that of IgA in yak tonsils (P < 0.01). Both the IgA and IgG ASCs were distributed in the subepithelial areas of the non‐reticular crypt epithelium, especially areas of pseudostratified columnar ciliary epithelium, the reticular crypt epithelium, lymphoid follicles, interfollicular areas, and with some of the positive cells aggregating around the glands. The results indicated that the tonsils were not only typical secondary lymphoid organs but also lymphoepithelial structures. IgG could be a significant component of mucosal immune responses in yak tonsils. Anat Rec, 302:999–1009, 2019.

Expression Characteristics of BMP2, BMPR-IA and Noggin in Different Stages of Hair Follicle in Yak Skin

Bone morphogenetic protein 2 (BMP2), BMP receptor-IA (BMPR-IA), and the BMP2 antagonist Noggin are important proteins involved in regulating the hair follicle (HF) cycle in skin. In order to explore the expression profiles of BMP2, BMPR-IA, and Noggin in the HF cycle of yak skin, we collected adult yak skin in the telogen, proanagen, and midanagen phases of HFs and evaluated gene and protein expression by real-time quantitative polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry. qRT-PCR and western blotting results showed that BMP2 and BMPR-IA expression levels were highest in the telogen of HFs and higher than that of Noggin in the same phase. The expression of Noggin was significantly higher in proanagen and midanagen phases of HFs than in the telogen phase, with the highest expression observed in the proanagen phase. Moreover, the expression of Noggin in the proanagen phase was significantly higher than those of BMP2 and BMPR-IA during the same phase. Immunohistochemistry results showed that BMP2, BMPR-IA, and Noggin were expressed in the skin epidermis, sweat glands, sebaceous glands, HF outer root sheath, and hair matrix. In summary, the characteristic expression profiles of BMP2, BMPR-IA, and Noggin suggested that BMP2 and BMPR-IA had inhibitory effects on the growth of HFs in yaks, whereas Noggin promoted the growth of yak HFs, mainly by affecting skin epithelial cell activity. These results provide a basis for further studies of HF development and cycle transition in yak skin.

Characteristics of yak platelet derived growth factors-alpha gene and its expression in brain tissues

BACKGROUND Platelet derived growth factors (PDGFs) are key components of autocrine and paracrine signalling, both of which play important roles in mammalian developmental processes. PDGF expression levels also relate to oxygen levels. The characteristics of yak PDGFs, which are indigenous to hypoxic environments, have not been clearly described until the current study. MATERIALS AND METHODS We amplified the open reading frame encoding yak (Bos grunniens) platelet derived growth factor-alpha (PDGFA) from a yak skin tissue cDNA library by reverse transcriptase polymerase chain reaction (PCR) using specific primers and Sanger dideoxy sequencing. Expression of PDGFA mRNA in different portions of yak brain tissue (cerebrum, cerebellum, hippocampus, and spinal cord) was detected by quantitative real-time PCR (qRT-PCR). PDGFA protein expression levels and its location in different portions of the yak brain were evaluated by western blot and immunohistochemistry. RESULTS We obtained a yak PDGFA 755 bp cDNA gene fragment containing a 636 bp open reading frame, encoding 211 amino acids (GenBank: KU851801). Phylogenetic analysis shows yak PDGFA to be well conserved, having 98.1% DNA sequence identity to homologous Bubalus bubalus and Bos taurus PDGFA genes. However, 8 nucleotides in the yak DNA sequence and 4 amino acids in the yak protein sequence differ from the other two species. PDGFA is widely expressed in yak brain tissue, and furthermore, PDGFA expression in the cerebrum and cerebellum are higher than in the hippocampus and spinal cord (p > 0.05). PDGFA was observed by immunohistochemistry in glial cells of the cerebrum, cerebellum, and hippocampus, as well as in pyramidal cells of the cerebrum, and Purkinje cell bodies of the hippocampus, but not in glial cells of the spinal cord. CONCLUSIONS The PDGFA gene is well conserved in the animal kingdom; however, the yak PDGFA gene has unique characteristics and brain expression patterns specific to this high elevation species.

Cloning and expression of cold-inducible RNA binding protein in domestic yak (Bos grunniens)

Cold-inducible RNA binding protein (CIRP) is over-expressed during cold and many other stresses, and could regulate the adaptation to hypothermia. In the present investigation, the objective was to determine the expression of CIRP in adult yak heart, liver, spleen, lung, kidney, brain, ovary, testis and skin by relative quantitative real time polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry from mRNA and protein levels. The CIRP open reading frame encoding was cloned from the domestic yak brain. Results of RT-PCR and Western blot showed the high expression level of CIRP in the heart, kidney, brain, testis and skin, and the lower expression level of CIRP in the lung. Immunohistochemical staining showed CIRP was expressed in the nucleus of neuronal cells, spermatogonia, primary spermatocytes and epidermal cells, and in the cytoplasm of the residual tissues. These observations may provide new data to understand and further study the important role of CIRP protein in the plateau adaptation of the domestic yak on long-term evolution.