Ginsenoside Rg1 Alleviates Lipopolysaccharide-Induced Fibrosis of Endometrial Epithelial Cells in Dairy Cows by Inhibiting Reactive Oxygen Species-Activated NLRP3.

Abnormal function and the fibrosis of endometrium caused by endometritis in cows may lead to difficult embryo implantation and uterine cavity adhesions. Emerging evidence indicates that ginsenoside Rg1 can effectively resist inflammation and pathological fibrosis in different organs. It is hypothesized that ginsenoside Rg1 may possess the potential to mitigate endometrial fibrosis induced by lipopolysaccharides (LPS) in dairy cows. Herein, a model of LPS-stimulated fibrosis was constructed using bovine endometrial epithelial cell line (BEND) cells and ICR mice. Western blotting was used to detect the protein level, and reactive oxygen species (ROS) content was measured by means of DCFH-DA. The uterine tissue structure was stained with H&E and Masson staining. The murine endometrium was assessed for oxidative stress by detecting the concentration of MDA together with the activity of enzymatic antioxidants SOD and CAT. Ginsenoside Rg1 interfered with NLRP3 activation by reducing ROS generation. After the application of ROS inhibitor NAC and NLRP3 inhibitor MCC950, ginsenoside Rg1 could interfere in the ROS/NLRP3 inflammasome signaling pathway by suppressing the epithelial-mesenchymal transition (EMT) of BEND cells. Our in vivo data showed that ginsenoside Rg1 relieved endometrial fibrosis of the mouse model of LPS-induced endometritis by restraining the ROS/NLRP3 inflammasome signaling pathway. Ginsenoside Rg1 inhibits LPS-induced EMT progression in BEND cells probably by inhibiting the activation of ROS-NLRP3 inflammasome.

Expression Analysis of Molecular Chaperones Hsp70 and Hsp90 on Development and Metabolism of Different Organs and Testis in Cattle (Cattle-yak and Yak).

Hsp70 and Hsp90 play an important role in testis development and spermatogenesis regulation, but the exact connection between Hsp70 and Hsp90 and metabolic stress in cattle is unclear. Here, we focused on the male cattle−yak and yak, investigated the expression and localization of Hsp70 and Hsp90 in their tissues, and explored the influence of these factors on development and metabolism. In our study, a total of 54 cattle (24 cattle−yaks and 30 yaks; aged 1 day to 10 years) were examined. The Hsp90 mRNA of the cattle−yak was first cloned and compared with that of the yak, and variation in the amino acid sequence was found, which led to differences in protein spatial structure. Using real-time quantitative PCR (RT-qPCR) and Western blot (WB) techniques, we investigated whether the expression of Hsp70 and Hsp90 mRNA and protein are different in the cattle−yak and yak. We found a disparity in Hsp70 and Hsp90 mRNA and protein expression in different non-reproductive organs and in testicular tissues at different stages of development, while high expression was observed in the testes of both juveniles and adults. Moreover, it was intriguing to observe that Hsp70 expression was significantly high in the yak, whereas Hsp90 was high in the cattle−yak (p < 0.01). We also examined the location of Hsp70 and Hsp90 in the testis by immunohistochemical (IHC) and immunofluorescence (IF) techniques, and the results showed that Hsp70 and Hsp90 were positive in the epithelial cells, spermatogenic cells, and mesenchymal cells. In summary, our study proved that Hsp70 and Hsp90 expressions were different in different tissues (kidney, heart, cerebellum, liver, lung, spleen, and testis), and Hsp90 expression was high in the testis of the cattle−yak, suggesting that dysplasia of the cattle−yak may correlate with an over-metabolism of Hsp90.

The expression of melatonin receptors MT1 and MT2 is regulated by E2 in sheep oviduct.

Some of the functions of melatonin in mammals are exerted through its membrane receptors (MRs) and studies have shown that estradiol (E2) might play an important role in regulating the expression of these proteins in female reproductive organs. However, no reports have reported the expression of MRs in the sheep oviduct or whether they are regulated by E2. Thus, herein, we detected the localization of MT1 and MT2 in the sheep oviduct. Moreover, we also investigated the expression pattern of these markers in the ovulating and non-ovulating side of the oviduct in the sheep ampulla and isthmus. Immunohistochemistry analyses revealed that both MT1 and MT2 are mainly expressed on oviduct epithelial cells. Both real-time polymerase chain reaction (qPCR) and western blot analyses showed that MT1 and MT2 genes and proteins are highly expressed on the non-ovulating side of the oviduct ampulla, but not the ovulating side. However, regarding the oviduct isthmus, there were no significant differences between the ovulating and non-ovulating sides. In vitro, 10 ng/ml and 1 µg/ml of E2, as well as 1 µg/ml of E2 combined with 0.1 µg/ml, 1 µg/ml, and 10 µg/ml of ICI182780 (a non-selective estrogenreceptor antagonist), were used to treat oviduct epithelial cells. We found that E2 inhibited the expression of MT1 and MT2 in cultured oviduct cells. Moreover, the inhibitory effect was suppressed by ICI182780. In conclusion, it was demonstrated that MRs are present in the sheep oviduct, and that E2, via the ER pathway, regulates their expression in the oviduct.

DHT and E2 synthesis-related proteins and receptors expression in male yak skin during different hair follicle stages.

Dihydrotestosterone (DHT) and 17ß-estradiol (E2) are sex hormones that regulate human hair follicle (HF) growth and are produced by peripheral reduction and aromatization of testosterone. However, the expression patterns of DHT and E2 synthesis-related proteins and their receptors in male yak skin during different HF stages (telogen, anagen, and catagen) are unknown. In this study, we found that both 5α-red and androgen receptor (AR) were expressed in epithelial cells and AR was expressed in the dermal papilla. Additionally, the transcription level of 5α-red1 at different HF stages was significantly higher than that of 5α-red2 mRNA at the same stage; 5α-red1 and 5α-red2 proteins peaked during the anagen and telogen periods of HF, respectively. However, AR protein was only expressed in the skin during the anagen phase of HF. Aromatase and estrogen receptors (ERα and ERß) were expressed in cutaneous epithelial cells, whereas ERα and ERß were expressed in the dermal papilla; the transcription level of ERα in HFs at each stage was much higher than that of ERß. From the catagen to telogen phase, aromatase protein expression was down-regulated, while ERα protein expression was up-regulated. Based on our results, we speculate that 5α-red1 is essential for the synthesis of DHT in male yak skin epithelial cells and promotes the growth of HFs through AR. E2 synthesized by male yak skin epithelial cells may inhibit the growth of male yak skin HFs by ERα. These results provide a foundation for further study on the mechanism of hormone-regulated male yak skin HFs.

Expression of oestrogen receptor, androgen receptor and progesterone nuclear receptor in sheep uterus during the oestrous cycle.

Oestrogen, androgen and progesterone are involved in the regulation of uterine physiological functions, with the participation of the following proteins: oestrogen receptor (ER), androgen receptor (AR) and progesterone nuclear receptor (PGR). In this study, we used immunohistochemistry to detect the localization of ERα, ERß, AR and PGR in sheep uterus. Additionally, we used real-time polymerase chain reaction (RT-qPCR) and Western blot technique to analyse their expression profiles at different stages of sheep oestrous cycle in the endometrium and myometrium. Immunohistochemical analysis showed that ERα, ERß, AR and PGR were present in sheep uterus in oestrus, mainly in the uterine luminal epithelium, stroma, gland and myometrium. Real-time polymerase chain reaction results showed that in the endometrium, ERα expression level was highest in oestrus. ERß and PGR, instead, were highly expressed in pro-oestrus. In the myometrium, ERα was highly expressed in both oestrus and pro-oestrus, and ERß was highly expressed in oestrus and dioestrus. Progesterone nuclear receptor expression was highest in oestrus, followed by metoestrus. In the endometrium, both receptors ERα and ERß were abundant in pro-oestrus, while the maximum AR protein content was found in oestrus. At this stage of the oestrous cycle, PGR protein concentration in the myometrium was significantly lower than those observed in other stages. These results suggest that these receptors are important for sheep reproductive function, as their expression at mRNA and protein levels exhibits particular time- and tissue-specific profiles along the oestrous cycle.

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.

Dihydrotestosterone synthesis in the sheep corpus luteum and its potential mechanism in luteal regression.

Corpus luteum (CL) regression is a complex physiological process. Previous studies have shown that dihydrotestosterone (DHT) may be involved in regulating CL regression, but the mechanism is still unclear. In this study, we evaluated the localization of the two isoforms of DHT synthetase 5α-reductase (5α-red1 and 5α-red2) and androgen receptor (AR) in sheep CL, and investigated 5α-red1, 5α-red2, AR, and DHT levels at different luteal stages of CL (early, middle, and late phase) by immunohistochemistry, quantitative real-time polymerase chain reaction, and western blot analysis. Moreover, we cultured luteal cells from middle phase CL and treated them with different concentrations of DHT (10-10 -10 -6 M) and the AR antagonist flutamide (10 -5 M), to evaluate whether DHT is involved in the regulation of progesterone (P4) secretion and progesterone nuclear receptor (PGR) expression and whether these effects are regulated by the AR pathway. We also investigated the effects of DHT and flutamide on prostaglandin F2α (PGF2α) secretion and apoptotic gene and protein expression. Our results showed that 5α-red1, 5α-red2, and AR were expressed in the CL, and their expression and DHT levels were changed during the luteal phase. DHT was involved in mediating P4 and PGF2α secretion and PGR and apoptotic gene and protein expression. The effects of DHT on CL were at least partially regulated by the AR pathway. This study reveals the mechanism of action of DHT on sheep CL regression and lays the foundation for further exploration of androgen regulation of CL function.

Profile of melatonin and its receptors and synthesizing enzymes in cumulus-oocyte complexes of the developing sheep antral follicle-a potential estradiol-mediated mechanism.

BACKGROUND: Melatonin is an amine hormone that plays an important role in regulating mammalian reproduction. This study aimed to investigate the expression pattern of melatonin synthesis enzymes AANAT and HIOMT and melatonin receptors MT1 and MT2 in sheep cumulus-oocyte complexes (COCs) as well as the change of melatonin level in follicular fluid (FF) during antral follicle development. In this research, we also study the effect of ß-estradiol (E2) on MT1 and MT2 expression as well as melatonin synthesis in COCs so as to lay the foundation for further exploration of the regulation mechanism of melatonin synthesis in the ovary. METHODS: COCs and FF were collected from different size (large follicles (diameter ≥ 5 mm), medium follicles (diameter 2-5 mm), and small follicles (diameter ≤ 2 mm)) of antral follicles in sheep ovaries. To assess whether E2 regulates melatonin synthase and its receptors expression in sheep COCs and whether it is mediated through estrogen receptor (ER) pathway. The collected COCs were cultured in vitro for 24 h and then treat with 1 µM E2 and/or 1 µM ICI182780 (non-selective ER antagonist). The expression of AANAT, HIOMT, MT1 and MT2 mRNA and protein were determined by qRT-PCR and western blot. The melatonin level was determined by ELISA. RESULTS: The expression of AANAT, HIOMT, MT1 and MT2 were significantly higher expression in the COCs of small follicles than in those of large follicles (P < 0.05). However, the melatonin level was significantly higher in large follicle FF than in small follicle FF (P < 0.05). Further, the expression of AANAT, HIOMT, MT1, and MT2 and melatonin production were decreased by E2 treatment (P < 0.05), but when ICI182780 was added, the expression of AANAT, HIOMT, MT1, and MT2 and melatonin production recovered (P < 0.05). CONCLUSIONS: We suggest that sheep COCs can synthesize melatonin, but this ability is decreased with increasing follicle diameter. Furthermore, E2 play an important role in regulated the expression of MT1 and MT2 as well as melatonin synthesis in sheep COCs through the ER pathway.

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, Hassall's 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.

Expression of melatonin and its related synthase and membrane receptors in the oestrous corpus luteum and corpus luteum verum of sheep.

Melatonin is an important factor involved in regulating reproduction; it is synthesized enzymatically by the sequential action of melatonin-synthesizing enzymes, arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT), and exerts its biological functions mainly through receptor-mediated action. To evaluate the expression of melatonin, two melatonin-synthesizing enzymes (HIOMT and AANAT), and membrane receptors (MT1 and MT2) in oestrous corpus luteum (CL) and CL verum of sheep (Ovis aries), we performed ELISA, qRT-PCR, western blotting and immunohistochemistry. The quantitative results showed that melatonin, HIOMT and AANAT levels in the CL verum were significantly higher than those in oestrous CL (p < 0.05), whereas MT1 and MT2 exhibited no change between the oestrous CL and CL verum (p > 0.05); moreover, the localization results showed that HIOMT, AANAT, MT1 and MT2 were mainly expressed in large luteal cells (LLCs). In summary, the above results suggested that sheep CL has potential for the synthesis of melatonin; meanwhile, they also suggested that CL is one of the targets of melatonin. These results provide not only a basis for whether sheep CL can synthesize melatonin but also provide a reference for further study on the mechanism of melatonin in the CL.

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.

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.