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.

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

Hypoxia-inducible factors (HIFs) play an important role in mediating the physiological response to low oxygen environments. However, whether the expression of HIFs changes with age is unknown. In the present study, the effect of aging on HIF-1α, HIF-2α, HIF-3α and VEGF expression in the heart and lung of 30 Tibetan sheep that were adapted to hypoxia was evaluated. The 30 sheep were subdivided into groups of 10 animals that were 1, 2 or 6 years of age. Immunohistochemistry for HIF-1α, HIF-2α, HIF-3α and VEGF revealed that the immunostaining intensity of VEGF protein in the heart and lung was significantly higher than the intensity of immunostaining against the HIFs (p < 0.05). HIF-1α and HIF-2α protein translocated into the nucleus of cardiac muscle cells. However, immunostaining for HIF-3α was restricted to the cytoplasm of the myocardial cells. Immunostaining for HIF-1α, HIF-2α, HIF-3α and VEGF was detected within alveolar macrophages. The concentration of HIF-1α and HIF-2α was higher in the lung of 1-year-old than 6-year-old sheep (p < 0.05). In contrast, HIF-3α and VEGF immunostaining was most prominent in the hearts of the oldest sheep. However, when RT-PCR was used to evaluate RNA within the tissues, the expression of all four studied genes was higher in the lung than in the heart in the 1-year-old sheep (p < 0.05). Furthermore, VEGF and HIF-3α gene expression was higher in the heart from 1-year old than 6-year old sheep (p < 0.05). However, in the lung, HIF-1α and HIF-2α gene expression was lower in 1-year old than 6-year old sheep (p < 0.05). We conclude that HIF-3α and VEGF may play be important in how the heart responds to hypoxia. Additionally, HIF-1α and HIF-2α may have a role in the adaptation of the lung to hypoxia. The expression of these proteins in alveolar macrophages suggests a potential role of these cells in the physiological response to hypoxia. These results are useful in understanding how age influences the hypoxia adaption mechanisms of the heart and lung and may help to better understand chronic mountain sickness that is commonly observed in Tibetan people living on the Qinghai-Tibetan plateau.

Association of Age with the Expression of Hypoxia-Inducible Factors HIF-1α, HIF-2α, HIF-3α and VEGF in Lung and Heart of Tibetan Sheep

Small-Tailed Han (STH) sheep are known for their high fecundity, but the survival of lambs is compromised and influences the commercial return from farming these sheep, with this being attributed in part to starvation from insufficient milk production by the ewes. In this study, the transcriptome profiles of the mammary gland of lactating and non-lactating STH ewes were investigated using paired-end RNA sequencing (RNA-Seq). An average of 14,447 genes were found to be expressed at peak-lactation in the STH sheep, while 15,146 genes were expressed in non-lactating ewes. A total of 4,003 differentially expressed genes (DEGs) were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the DEGs were associated with a wide range of cellular components, biological processes and metabolic pathways, including binding activities, signaling pathways, cellular structures, and immune responses. The most highly expressed genes at peak-lactation included CSN2, LGB, LALBA, CSN1S1, CSN1S2, and CSN3, and the 10 most highly expressed genes accounted for 61.37% of the total Reads Per Kilobase of transcript, per Million mapped reads (RPKM). The most highly expressed genes in the mammary gland of non-lactating ewes included IgG, THYMB4X, EEF1A1, IgA, and APOE, and the 10 most highly expressed genes accounted for only 12.97% of the total gene RPKM values. This suggests that the sheep mammary gland undergoes a substantial development in milk protein synthesis infrastructure and promotion of protein transportation during lactation.

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Comparison of the Transcriptome of the Ovine Mammary Gland in Lactating and Non-lactating Small-Tailed Han Sheep.

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.

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

Overexpression of genes associated with hypoxia in cattle adapted to Trans Himalayan region of Ladakh

Expression of Rad51 and the histo-morphological evaluation of testis of the sterile male cattle-yak

Yak milk is superior to common cow milk in nutrients including protein, fat and calories. However, the milk yield of the yak is very much lower compared with other dairy bovines. To understand the molecular mechanisms of lactogenesis, lactation and mammary gland development, mammary tissue samples were taken from five yaks during a dry period (DP, n = 3) and lactation period (LP, n = 2). Two types of cDNA sequence libraries that reflected the different physiological states of the mammary gland were constructed using RNA sequencing technology. After removing reads containing adapters, reads containing poly-N and low-quality reads from the raw data, 45,423,478 to 53,274,976 clean reads were obtained from these libraries. A total of 74.72% to 80.65% of the high-quality sequence reads were uniquely aligned to the BosGru v2.0 yak reference genome. Using the DESeq R package, 360 differentially expressed genes were detected between the two groups when the adjusted P value (padj < 0.05) was used as the cutoff value; this included 192 upregulated and 168 downregulated genes in the yak mammary gland tissue of the DP compared to the LP. A gene ontology analysis revealed that the most enriched GO terms were protein binding, multi-organism process, immune system and others. KEGG pathway analysis indicated that the differentially expressed genes were mostly enriched in Hippo signaling, insulin signaling, steroid biosynthesis and others. The analysis of the up- and downregulated genes provides important insights into the molecular events involved in lactogenesis, lactation and mammary gland development and will guide further research to enhance milk yield and optimize the constituents of yak milk.

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Transcriptional profiling of two different physiological states of the yak mammary gland using RNA sequencing

To explore the adaptive mechanism of yak's lung,the distribution of vascular endothelial growth factor(VEGF) in one-day-old yak's lung was observed by immunohistochemical method.The results showed bulk of VEGF was distributed in intrapulmonary conducting airways,mainly in Clara cell of epithelium and pulmonary artery's endothelium.Some of VEGF was distributed in ciliated cell and smooth muscle layer in bronchus and its branches,and no VEGF was distributed in epithelium of gas exchange area,but a few in alveolar wall.VEGF decreased with the decrease of pulmonary artery's caliber.These results indicated that hypoxia could stimulate lung to secrete VEGF in intrapulmonary conducting airways and pulmonary artery of one-day-old-yak,which affects the development of lung.It may be an important mechanism of yak to adapt hypoxia.

Distribution of vascular endothelial growth factor in plateau yak's lung

Summary Corpus luteum of adult female yak removed at early pregnancy stages were studied using 1ight and electron microscopes. During the pregnancy of 2-4 weeks, luteal cells were small and had no prominent morphological changes in cytoplasm. At this time, corpus luteum provided a rich source of microvascula and there was no or a few intravascular cellular debris. During the pregnancy of 5-8 weeks, luteal cells increased significantly with development of corpus luteum, many capillaries contained cellular debris, and thick walled vessels became numerous. There were spaces between oolemma and cytoplasm in luteum cells. Following the regressional changes, the granulose luteal cells enlarged, had round, oval or polyhedral morphology, and cytoplasm eosinophilia and nuclear chromatin aggregated. The ultrastructure of regressional granulose luteal cell followed as: nuclear decondensed, nuclear membrane wrinkled, all organelles denaturated, lipid droplets (LD) accumulated, smooth endoplasmic reticulum (SER) decreased or disappeared, rough endoplasmic reticulum (RER) increased and

Histological characteristics of corpus luteum in yak during early pregnancy

To investigate histological structure of coronary artery of animals living in plateau,the coronary artery and its main branches in heart of different age yak living at 3 500 m high altitude were observed by light microscopy.The wall of coronary artery branches in yak had obvious difference in composition,especially in the right coronary artery among different individuals.Within muscular coronary artery,there was no external elastic membrane,the tunica media was well-developed and mainly composed of smooth muscle cells,and most of them were arranged circularly.Near the tunica externa,bundles of longitudinal smooth muscle were observed and increased with the increase of age.The layer of subendothelial connective tissue was deficiency in muscular coronary artery of the yak.There were fractures presented in internal elastic membrane in 5-month-old yak,then the secondary internal elastic membrane gradually appeared in fracture in adult yak.The average values(r) of relative thickness of the intima were less than 0.1 in all age groups.No difference was observed in both sexes.These results showed that the histological structure of coronary artery was unique and greater changes occurred in yak with increased age,which might be an adaption to special pressure of hemodynamic.

Cui Yan

Papers

Expression of Rad51 and the histo-morphological evaluation of testis of the sterile male cattle-yak

Transcriptional profiling of two different physiological states of the yak mammary gland using RNA sequencing

Distribution of vascular endothelial growth factor in plateau yak's lung

Histological characteristics of corpus luteum in yak during early pregnancy

Histological characteristics of coronary artery of yak in different ages.