Differential expression and localization of immunity-related factors in main immune organs of yak.

The yak is an agricultural animal with strong disease resistance in Qinghai-Tibet Plateau. Immune organs are directly involved in the body's immune response and protect it from external aggression. In this study, we characterized and evaluated the main markers of interleukin (IL)-1ß, IL-17a, hypoxia inducer factor-1 (HIF-1)α, and heat shock protein 90 (HSP90) in the lymph nodes, spleen, thymus, and hemal nodes of adult yaks using network informatics, molecular cloning, immunohistochemistry, real-time quantitative polymerase chain reaction (RT-qPCR), and western blotting. We first cloned the IL-1ß and IL-17a mRNA of yaks. A significant feature was the higher IL-1ß and IL-17a expression in the lymph nodes than in the spleen, hemal nodes, and thymus. Immunohistochemistry and immunofluorescence revealed that IL-1ß and IL-17a cells were mainly located in the paracortex area of the lymph nodes and the T-cell-dependent area in the hemal nodes and spleen. Several HIF-1α proteins were detected in the cortex of the hemal nodes mantle, while HSP90 was detected in the lymphoid nodules of the hemal nodes and lymph nodes. This study sheds light on the relationship between the morphology and function of these organs and provides an important reference for studies on the participation of yak immune organs in immune responses.

TGF-ß and HSP70 profiles during transformation of yak hair follicles from the anagen to catagen stage.

Transforming growth factor-ß (TGF-ß) and heat shock protein 70 (HSP70) are important for the hair follicle (HF) cycle, but it is unclear whether they participate in HF regression in yak skin. In this study, we investigated the role of TGF-ß, TGF-ßRII, and HSP70 in the transition from anagen to catagen of HFs. The results showed that TGF-ß2 transcription was significantly higher than that of TGF-ß1 and TGF-ß3 in the same periods. Meanwhile, the expressions of TGF-ß2, TGF-ßRII, and caspase-3 were higher in the catagen phase than that in mid-anagen, and some TGF-ßRII-positive HF cells were terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-positive. Moreover, the HSP70 protein levels in mid-anagen were higher than those in late-anagen and catagen. These results suggested that TGF-ß2 plays a major role in catagen induction in yak HFs, which might be achieved via TGF-ßRII-mediated apoptosis in HF epithelial cells. In contrast, HSP70 might protect epithelial cells from apoptosis and ultimately inhibit HF regression. In conclusion, TGF-ß2 has positive effects, whereas HSP70 has negative effects, on catagen induction.

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.

Effects of PHD and HSP90 on erythropoietin production in yak (Bos grunniens) renal interstitial fibroblast-like cells under hypoxia.

Erythropoietin (EPO), a central protein of erythropoiesis, plays an important role during hypoxia adaptation and is regulated by hypoxia-inducible factor (HIF). However, there is no report on EPO-producing cells and their regulatory mechanisms in yak (Bos grunniens). To understand EPO production and regulation of yak, kidneys from different age of yak were collected and expression of EPO, hypoxia-inducible factor 1 alpha (HIF-1α), and hypoxia-inducible factor 2 alpha (HIF-2α) were detected. Then renal tubule epithelial cells (RTECs) and peritubular interstitial fibroblast-like (RIFs) cells were isolated and cultured to determine their EPO production abilities. Subsequently, the cells were treated with dimethyloxalylglycine (DMOG) and Geldanamycin (GA), which are inhibitors of prolyl-4-hydroxylase domain (PHD) and heat shock protein 90 (HSP90) respectively, and siRNAs of HIF-1α and HIF-2α to explore their effect on EPO production and regulation. The results showed that expressions of EPO, HIF-1α, and HIF-2α were different in the different age groups of yak. High DMOG concentration caused a corresponding increase in the levels of HIF-1α and HIF-2α in RIFs and RTECs, however, EPO levels increased in RIFs only and was not detected at any concentration in RTECs; suggesting that EPO was produced in RIFs. Upon treating RIFs with siRNAs of HIF-1α and HIF-2α, we found that EPO was regulated by PHD through HIF-2α. In addition, increasing GA concentration caused a decrease in expression of HSP90, HIF-1α, HIF-2α, and EPO in RIFs. In conclusion, these findings support our proposition that PHD regulates EPO via HIF-2α in yak RIFs, while HSP90 impelled EPO expression.

[Quantitative measurement of in vitro phagocytosis of apoptotic granulosa cells by monocytes in mice.].

To establish a method for quantitative measurement of phagocytosis, the phagocytic process of apoptotic granulosa cells by monocytes was imitated in vitro. Monocytes and granulosa cells were isolated from Kunming mice and cultured. Granulosa cells were induced to apoptosis by garlic, and then co-cultured with monocytes. At different time points (1 h, 2 h, 3 h, 4 h, 5 h), co-cultured cells were observed by microscope after Wright's staining. The results showed that at the beginning of morphological changes in apoptotic granulosa cells, monocytes captured the apoptotic cells. Meanwhile, the apoptosis of granulosa cells were progressing. Debris was found in phagocytic vacuole. At the point of 3 h after co-culture, the ratio of monocytes which attached to apoptotic granulosa cells to those which engulfed the apoptotic cells was close to one. Namely, half of monocytes were in the state of recognition and half were in the state of engulfment, and this time point was named as 'half phagocytic period'. Regression analysis showed that the equation of linear regression was y = -0.247x +1.644 (y represents Attachment/Engulfment ratio, x represents co-culture time), R(2)=0.912, F=31.095, P=0.011 (<0.05), T= -5.576, P=0.011 (<0.05). In conclusion, the present mode of phagocytosis in vitro can be used as a method to quantitatively assay some effective factors such as medicines which could enhance or restrain phagocytosis.

Yak (Bos grunniens) Tonsils: Morphological Description and Expression of IgA and IgG.

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. © 2018 Wiley Periodicals, Inc.

Pure cultures and characterization of yak Sertoli cells.

The culture of primary Sertoli cells has become an important resource in the study of their function. However, their use is limited because of contamination of isolated cells with other testicular cells, mainly germ cells. The aim was to establish technique to obtain pure yak Sertoli cells as well as to study the growth kinetics and biological characteristics of Sertoli cells in vitro. Two-step enzyme digestion was used to separate and culture yak Sertoli cells. Cultured using starvation method and the hypotonic treatment were also invented to get pure yak Sertoli cells. Furthermore, the purification of Yak Sertoli cells were identified according to their characteristics, such as bipolar corpuscular around the nucleus and expression of Fasl, in addition to their morphology. The average viability of the Sertoli cells was 97% before freezing and 94.5% after thawing, indicating that cryopreservation in liquid nitrogen had little influence on the viability of Sertoli cells. The growth tendency of yak Sertoli cells was similar to an S-shaped growth curve. Purified yak Sertoli cells frequently exhibited bipolar corpuscula in nucleus after Feulgen staining, and did have a positive reaction of Fasl by the immunocytochemical identification. After recovery chromosomal analysis of Sertoli cells had a normal chromosomal number of 60, comprising 29 pairs of autosomes and one pair of sex chromosomes. Assays for bacteria, fungi and mycoplasmas were negative. In conclusion, yak Sertoli cells have been successfully purified and cultured in vitro, and maintain stable biological characteristics after thawing. Therefore, it will not only preserve the genetic resources of yaks at the cellular level, but also provide valuable materials for transgenic research and feeder layer and nuclear donor cells in yak somatic cell cloning technology.

[Studies on isolation, purification and antibacterial activities of beta-defensin in Yak neutrophils].

AIM: To isolate and purify beta-defensin from the neutrophils of Yak. METHODS: The method of percoll gradient centrifugation was employed to purify neutrophils from Yak peripheral blood. The crude extraction from Yak neutrophils was isolated respectively through a way of extraction by 50 mL/L acetic acid, acid soluble extract of Yak neutrophils was obtained. Then it was further purified by Bio-Gel P-10 Polyacrylamide gel filtration and RP-HPLC, and 9 out of 22 apices showed antibacterial activity. 9 apices showing antibacterial activity were selected to be analyzed by mass spectrograph.The antibacterial activities of the extracts from every step were analyzed by agarose radial diffusion assay. RESULTS: The molecular weight of the purified Yak BNBD-1-3 was determined to be 4.2, 4.6 and 4.8 kDa by mass spectrograph. BNBD-1-3 from Yak neutrophils were able to effectively kill Escherichia coli, Staphylococcus aureus and Bacillus subtilis. CONCLUSION: Yak neutrophils have beta-defensin which have broad-spectrum antimicrobial activity, Yak defensin has an important part in innate immunity.