Quantum dot fluorescent microsphere-based immunochromatographic strip for detecting PRRSV antibodies.

Porcine reproductive and respiratory syndrome (PRRS) is an immunosuppressive disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). Current vaccine prevention and treatment approaches for PRRS are not adequate, and commercial vaccines do not provide sufficient cross-immune protection. Therefore, establishing a precise, sensitive, simple, and rapid serological diagnostic approach for detecting PRRSV antibodies is crucial. The present study used quantum dot fluorescent microspheres (QDFM) as tracers, covalently linked to the PRRSV N protein, to develop an immunochromatography strip (ICS) for detecting PRRSV antibodies. Monoclonal antibodies against PRRSV nucleocapsid (N) and membrane (M) proteins were both coated on nitrocellulose membranes as control (C) and test (T) lines, respectively. QDFM ICS identified PRRSV antibodies under 10 min with high sensitivity and specificity. The specificity assay revealed no cross-reactivity with the other tested viruses. The sensitivity assay revealed that the minimum detection limit was 1.2 ng/mL when the maximum dilution was 1:2,048, comparable to the sensitivity of enzyme-linked immunosorbent assay (ELISA) kits. Moreover, compared to PRRSV ELISA antibody detection kits, the sensitivity, specificity, and accuracy of QDFM ICS after analyzing 189 clinical samples were 96.7%, 97.9%, and 97.4%, respectively. Notably, the test strips can be stored for up to 6 months at 4 °C and up to 4 months at room temperature (18-25 °C). In conclusion, QDFM ICS offers the advantages of rapid detection time, high specificity and sensitivity, and affordability, indicating its potential for on-site PRRS screening. KEY POINTS: • QDFM ICS is a novel method for on-site and in-lab detection of PRRSV antibodies • Its sensitivity, specificity, and accuracy are on par with commercial ELISA kits • QDFM ICS rapidly identifies PRRSV, aiding the swine industry address the evolving virus.

Heme Oxygenase-1 Regulates Zearalenone-Induced Oxidative Stress and Apoptosis in Sheep Follicular Granulosa Cells.

Zearalenone (ZEA) is a common non-steroidal estrogenic mycotoxin found in a range of animal feeds and poses a serious threat to the reproductive health of farm animals and humans. However, the mechanism underlying ZEA-induced reproductive toxicity in sheep remains unknown. Granulosa cells are crucial for egg maturation and the fertility of female sheep. In this study, we aimed to examine the impact of different ZEA concentrations on sheep follicular granulosa cells and to elucidate the potential molecular mechanism underlying ZEA-induced toxicity using transcriptome sequencing and molecular biological approaches. Treating primary sheep follicular granulosa cells with different concentrations of ZEA promoted the overproduction of reactive oxygen species (ROS), increased lipid peroxidation products, led to cellular oxidative stress, decreased antioxidant enzyme activities, and induced cell apoptosis. Using transcriptome approaches, 1395 differentially expressed genes were obtained from sheep follicular granulosa cells cultured in vitro after ZEA treatment. Among them, heme oxygenase-1 (HMOX1) was involved in 11 biological processes. The protein interaction network indicated interactions between HMOX1 and oxidative and apoptotic proteins. In addition, N-acetylcysteine pretreatment effectively reduced the ZEA-induced increase in the expression of HMOX1 and Caspase3 by eliminating ROS. Hence, we suggest that HMOX1 is a key differential gene involved in the regulation of ZEA-induced oxidative stress and apoptosis in follicular granulosa cells. These findings provide novel insights into the prevention and control of mycotoxins in livestock.

The effect of melatonin on sheep endometrial epithelial cell apoptosis through the receptor and non-receptor pathways.

Melatonin potentially regulates the female animal reproductive function, but its regulatory mechanism in the apoptosis of sheep endometrial epithelial cells (SEECs) remains to be elucidated. In the present study, immunofluorescence staining, western blotting, and quantitative real-time polymerase chain reaction were performed to detect the distribution of melatonin receptors (MT1 and MT2) in the uterus of sheep and the effect of melatonin via the receptor and non-receptor pathways on the apoptosis of SEECs in vitro. The results showed that melatonin inhibits the apoptosis of SEECs to varying degrees to regulate the expression of estrogen receptors (ERs) and progesterone receptors (PGR) via its interaction with MT1 and MT2. In addition, the ER antagonist partially relieved the inhibitory effect of melatonin on the apoptosis of SEECs, while the PGR antagonist did not. Thus, melatonin mediates endometrial epithelial apoptosis through the MT receptors and also by regulating estrogen function. This study provides evidence of the regulatory mechanism of melatonin on the physiological function of the sheep uterus.

Establishment of Bactrian Camel Induced Pluripotent Stem Cells and Prediction of Their Unique Pluripotency Genes.

Induced pluripotent stem cells (iPSCs) can differentiate into all types of cells and can be used in livestock for research on biological development, genetic breeding, and in vitro genetic resource conservation. The Bactrian camel is a large domestic animal that inhabits extreme environments and holds value in the treatment of various diseases and the development of the local economy. Therefore, we transferred four mouse genes (Oct4, Sox2, Klf4, and c-Myc) into Bactrian camel fetal fibroblasts (BCFFs) using retroviruses with a large host range to obtain Bactrian camel induced pluripotent stem cells (bciPSCs). They were comprehensively identified based on cell morphology, pluripotency gene and marker expression, chromosome number, transcriptome sequencing, and differentiation potential. The results showed the pluripotency of bciPSCs. However, unlike stem cells of other species, late formation of stem cell clones was observed; moreover, the immunofluorescence of SSEA1, SSEA3, and SSEA4 were positive, and teratoma formation took four months. These findings may be related to the extremely long gestation period and species specificity of Bactrian camels. By mining RNA sequence data, 85 potential unique pluripotent genes of Bactrian camels were predicted, which could be used as candidate genes for the production of bciPSC in the future. Among them, ASF1B, DTL, CDCA5, PROM1, CYTL1, NUP210, Epha3, and SYT13 are more attractive. In conclusion, we generated bciPSCs for the first time and obtained their transcriptome information, expanding the iPSC genetic information database and exploring the applicability of iPSCs in livestock. Our results can provide an experimental basis for Bactrian camel ESC establishment, developmental research, and genetic resource conservation.

Cloning and Molecular Characterization of HSL and Its Expression Pattern in HPG Axis and Testis during Different Stages in Bactrian Camel.

Hormone-sensitive lipase (HSL) is a key enzyme in animal fat metabolism and is involved in the rate-limiting step of catalyzing the decomposition of fat and cholesterol. It also plays an important regulatory role in maintaining seminiferous epithelial structure, androgen synthesis and primordial germ cell differentiation. We previously reported that HSL is involved the synthesis of steroids in Bactrian camels, although it is unclear what role it plays in testicular development. The present study was conducted to characterize the biological function and expression pattern of the HSL gene in the hypothalamic pituitary gonadal (HPG) axis and the development of testis in Bactrian camels. We analyzed cloning of the cDNA sequence of the HSL gene of Bactrian camels by RT-PCR, as well as the structural features of HSL proteins, using bioinformatics software, such as ProtParam, TMHMM, Signal P 4.1, SOPMA and MEGA 7.0. We used qRT-PCR, Western blotting and immunofluorescence staining to clarify the expression pattern of HSL in the HPG axis and testis of two-week-old (2W), two-year-old (2Y), four-year-old (4Y) and six-year-old (6Y) Bactrian camels. According to sequence analysis, the coding sequence (CDS) region of the HSL gene is 648 bp in length and encodes 204 amino acids. According to bioinformatics analysis, the nucleotide and amino acid sequence of Bactrian camel HSL are most similar to those of Camelus pacos and Camelusdromedarius, with the lowest sequence similarity with Mus musculus. In adult Bactrian camel HPG axis tissues, both HSL mRNA and protein expression were significantly higher in the testis than in other tissues (hypothalamus, pituitary and pineal tissues) (p < 0.05). The expression of mRNA in the testis increased with age and was the highest in six-year-old testis (p < 0.01). The protein expression levels of HSL in 2Y and 6Y testis were clearly higher than in 2W and 4Y testis tissues (p < 0.01). Immunofluorescence results indicate that the HSL protein was mainly localized in the germ cells, Sertoli cells and Leydig cells from Bactrian camel testis, and strong positive signals were detected in epididymal epithelial cells, basal cells, spermatocytes and smooth muscle cells, with partially expression in hypothalamic glial cells, pituitary suspensory cells and pineal cells. According to the results of gene ontology (GO) analysis enrichment, HSL indirectly regulates the anabolism of steroid hormones through interactions with various targets. Therefore, we conclude that the HSL gene may be associated with the development and reproduction of Bactrian camels in different stages of maturity, and these results will contribute to further understanding of the regulatory mechanisms of HSL in Bactrian camel reproduction.

A novel rapid detection of Senecavirus A using recombinase polymerase amplification (RPA) coupled with lateral flow (LF) dipstrip.

SENECAVIRUS A: (SVA), an emerging picornavirus, has been associated with vesicular disease and neonatal mortality in swine, posing a great threat to the global swine industry. Accurate diagnosis of SVA is crucial for the effective prevention and control disease. In the present study, a simple, rapid and accurate diagnostic assay was developed combining recombinase polymerase amplification and a lateral flow dipstrip (RPA-LF) to detect SVA infection. Using recombinant plasmid pMD19-T-VP1 DNA as a template, the RPA-LF optimal reaction conditions were incubated at 35 °C for 25 min, and the result was visualized directly on the dipstrip. The specificity assay showed no cross-reactivity with other tested viruses, and the sensitivity assay revealed the minimum detection limit was 15 copies/µl. Moreover, the RPA-LF method was successfully applied with viral cDNA as template to test clinical samples, with no significant difference being observed between RPA-LF and qRT-PCR. Hence, the established RPA-LF assay could be used as a potential optional rapid, reliable, sensitive and low-cost method for field diagnosis of SVA, especially in resource-limited regions.

Proteomic analysis of iTRAQ in melatonin-treated sheep epididymal epithelial cells.

During maturation, spermatozoa acquire motility and fertilizing capacity as they transit through the epididymis. Melatonin is a lipophilic hormone with multiple functions in regulating the fertility. Previous studies have shown that melatonin affected the capacitation or maturation of sperm in the epididymis. The aim of this study was to investigate the effects of melatonin on epididymal caput epithelial cells in sheep. In the study, we used iTRAQ labelling coupled with LC-MS/MS for quantitative identification of differentially expressed proteins in melatonin-treated sheep epididymal caput epithelial cells. We identified 69 differentially expressed protein; 41 were upregulated and 28 were downregulated in samples from sheep in melatonin treated. We validated the differential expression of a subset of these proteins using qPCR and Western blot. Gene ontology annotation identified that the differentially expressed proteins function in cellular processes and metabolic processes. Notably, five of the differentially expressed proteins as SOD1, COL1A1, PRM1, NQO2, and FN1 are involved in sperm migration and sperm maturation. KEGG enrichment analysis demonstrated significant enrichment in several cardiac-related pathways, such as "PI3K-Akt signaling pathway", "AGE-RAGE signaling pathway in diabetic complications", "ECM-receptor interaction", and "Ribosome". Our results suggest that candidate biomarker (SOD1, COL1A1, PRM1, NQO2, and FN1) discovery can aid in understanding sperm development and maturation in sheep. These results provide insights into the potential mechanisms of melatonin regulation of sperm maturation in epididymal caput epithelial cells.

RBP4 regulates androgen receptor expression and steroid synthesis in Sertoli cells from Bactrian camels.

Retinol-binding protein (RBP4) plays an important role in the transport and metabolism of retinol. In addition, RBP4 contributes to testicular homeostasis, including maintenance of spermatogenesis and synthesis of androgens that mediate their physiological functions through the androgen receptor. RBP4 in Sertoli cells regulates testosterone and dihydrotestosterone synthesis and secretion, although the mechanisms have yet to be revealed. In this study, we examined the expression and function of RBP4 in Sertoli cells isolated from Bactrian camels. qRT-PCR analysis of various Bactrian camel tissues revealed high expression of RBP4 in the testis and epididymis. To examine RBP4 function, Sertoli cells isolated from testes were transfected with an RBP4 overexpression plasmid or RBP4-targeting siRNA. RBP4 overexpression resulted in significant inhibition of transcription and translation of the steroidogenic enzymes 3ßHSD and SRD5A1 concomitant with a significant decrease in androgen receptor expression and dihydrotestosterone secretion. Conversely, RBP4 knockdown significantly increased the expression of 3ßHSD, SRD5A1 and androgen receptor and enhanced the secretion of dihydrotestosterone and testosterone. These data reveal a novel role for RBP4 in regulating steroid synthesis in Sertoli cells from Bactrian camels.

Melatonin protects sheep endometrial epithelial cells against lipopolysaccharide-induced inflammation in vitro.

Melatonin has known anti-inflammatory effects. Yet, how melatonin protects sheep endometrial epithelial cells from inflammation remains unknown. In this study, we investigated the melatonin synthetase AANAT and HIOMT and melatonin membrane receptors MT1 and MT2 distribution in sheep uterus. Using lipopolysaccharide (LPS)-stimulated sheep endometrial epithelial cells as an in vitro inflammation model. The results showed that melatonin attenuated the expression of inflammatory factors in a concentration-response manner. Melatonin also inhibited the LPS-stimulated phosphorylation of ERK1/2, JNK and NF-κB p65. This attenuation was partially blocked by luzindole (a non-specific MT1 and MT2 inhibitor) or 4P-PDOT (specific MT2 inhibitor). In addition, the above inhibition of melatonin was abolished by the PI3K/AKT pathway inhibitor LY294002. It was concluded that melatonin had an inhibitory effect on LPS-induced endometrial epithelial cell inflammation in sheep, which was mediated by the activation of the PI3K/AKT pathway via melatonin receptors.

Effect of dihydrotestosterone on melatonin secretion and the expression of melatonin receptors and apoptosis-related factors in sheep epididymides.

Melatonin (MEL) is involved in homeostasis of the epididymis lumen environment. Dihydrotestosterone (DHT) partakes in the development of gonads and organs in male animals. However, whether MEL secretion, the expression of its receptors, MT1 and MT2, and sheep epididymal epithelial cell apoptosis is regulated by DHT remains unclear. In this study, we used immunohistochemical staining to detect the distribution patterns of DHT synthetases [5α-reductase (5α-red)] and its androgen receptor (AR) in sheep epididymides. 5α-red1, 5α-red2 and AR were positively expressed in sperm, epididymal epithelial cells, and the smooth muscle cells of the caput, corpus and cauda regions of the epididymis. DHT concentration and the expression levels of 5α-red and AR in the caput, corpus and cauda regions were measured by enzyme-linked immunosorbent assay, liquid chromatography-mass spectrometry, real-time quantitative polymerase chain reaction and western blot analysis. DHT concentration in the caput was significantly higher than those in corpus and cauda, probably because of the high expression of 5α-red2 in the caput and secretion and transport of DHT by the testicles. DHT inhibited MEL secretion, the expression of its membrane receptors and MEL synthetases in cultured sheep epididymal epithelial cells in vitro. In addition, the Bax/Bcl-2 ratio, ACT CASP3 and caspase-3 mRNA expression were also decreased. The decreasing effect was partially reversed after flutamide treatment. Therefore, DHT regulates sheep epididymal function by influencing MEL expression and apoptosis-related factors. This study provides basic data for further research on the reproductive physiology of male animals.

CTH/H2S Regulates LPS-Induced Inflammation through IL-8 Signaling in MAC-T Cells.

Hydrogen sulfide (H2S), as an endogenous gaseous signaling molecule, plays an important role in the inflammatory process. Our previous study found that Cystathionine-γ-lyase (CTH) and H2S are correlated with the occurrence and development of Clinical Mastitis (CM) in Holstein cows. However, the functions and regulatory mechanisms of CTH/H2S are still unknown. In this study, the inflammatory mammary cell model based on the MAC-T cell line was established by Lipopolysaccharide (LPS)-induced manner to further explore the function and regulatory mechanism of CTH/H2S in cows with CM. In the inflammatory MAC-T cell, the CTH expression and H2S production were both repressed in an LPS-dose dependent manner, which demonstrated that CTH/H2S is related to the progression of inflammation. The inhibition of CTH/H2S using a selective CTH inhibitor, ß-cyano-l-Alanine (BCA), promoted LPS-induced inflammation response and the expression of inflammatory cytokines. However, this was reversed by the H2S donor NaHS, demonstrating that H2S can protect cells from inflammatory damage. Intriguingly, interleukin-8 (IL-8) showed an inverse expression pattern correlated with the H2S-mediated cell protection effect during the inflammation process, and the inhibition test using a selective IL-8 receptor antagonist, SB225002, showed that IL-8 signaling plays a critical role in mediating endogenous H2S synthesis, and CTH/H2S exerts its anti-inflammation via IL-8-mediated signaling. This study provided support for the prevention and treatment of CM and the development of a novel anti-inflammatory strategy.

Toll-like Receptor 2 Is Associated with the Immune Response, Apoptosis, and Angiogenesis in the Mammary Glands of Dairy Cows with Clinical Mastitis.

Toll-like receptor 2 (TLR2) plays a crucial role in bacterial recognition and the host immune response during infection. However, its function and downstream biological processes (BPs) in the mammary glands (MGs) of Holstein cows with clinical mastitis (CM) are not fully understood. This study aimed to comprehensively identify the BPs and differentially expressed proteins (DEPs) associated with the bacterial response and TLR2 using data-independent acquisition (DIA) proteomic data. A possible mechanism for the action of TLR2 was proposed, and the results suggested that the expression levels of TLR2 and caspase 8 (CASP8) were positively correlated with the apoptosis of MGs. The expression patterns of TLR2 and TEK receptor tyrosine kinase 2 (Tie2) were negatively correlated with angiogenesis. These results indicated that TLR2 might promote apoptosis in mammary epithelial cells (MECs) and vascular endothelial cells (VECs) via upregulation of CASP8 expression, and inhibition of angiogenesis in VECs via downregulation of Tie2 expression in dairy cows with CM. In conclusion, TLR2 is associated with inflammation, apoptosis, and angiogenesis in the MGs of dairy cows with bacteria-induced mastitis. These results contribute to a deeper understanding of the pathogenic mechanisms and provide the knowledge needed for developing the prevention and treatment of dairy mastitis.

Role of AURKA in the hypothalamus-pituitary-testicular axis in Tibetan sheep from Tianzhu.

The mitosis-associated protein aurora kinase A (AURKA) regulates the maturation of germ cells. We have previously reported using transcriptome analysis that AURKA is expressed in yak testes. Although Tibetan sheep possess an immense economic value, their reproductive rate is low. Herein, the expression and functions of AURKA in the hypothalamus-pituitary-testicular (HPT) axis in Tibetan sheep from Tianzhu were investigated. The cDNA sequence of sheep AURKA was cloned and bioinformatics techniques were used to predict its structure. Tissue expression of AURKA was determined by qPCR, immunoblotting, immunostaining, and immunohistochemistry. The AURKA coding sequence was found to be 1218 bp in length, encoding a 405-amino acid polypeptide chain. Furthermore, the highest sequence similarity of AURKA with the corresponding sequence in other species was seen in goat and cattle; the least degree of similarity was seen in the domestic cat. In addition, AURKA expression was elevated in the testes compared to that in the hypothalamus and pituitary (p < 0.01). Moreover, AURKA was mainly localized in the hypothalamic paraventricular nucleus (magnocellular), chromophobe cells of the pituitary, and spermatogenic cells of the testis. These results indicated that AURKA might participate in sheep reproductive regulation, thus providing a reference for the study of AURKA function in the reproductive process of Tibetan sheep from Tianzhu.

Transcriptomic analysis of gene expression in normal goat ovary and intersex goat gonad.

Intersexuality is a congenital reproductive disorder that usually occurs in hornless goats, hindering breeding of goats with hornless traits and the development of the goat industry. In this study, we aimed to identify differentially expressed genes in intersex and normal goat gonads by comparing gene transcription profiles of intersex and normal goat gonads. As intersex goats are genetically based on females, we chose female goats as controls. The goats in the control group and the experimental group were both over one-year old. We evaluated the anatomical characteristics of the reproductive organs of five intersex goats using histopathological methods. The gonads were found to be ovarian and testicular types. RNA-Seq technology was used to identify differentially expressed genes in gonads and normal goat ovary tissues. Transcription analysis results were verified by qPCR. The results showed that 2,748 DEGs were upregulated and 3,327 DEGs were downregulated in intersex ovaries unlike in controls, whereas 2006 DEGs were upregulated and 2032 DEGs were downregulated in the interstitial testes. Many of these genes play important roles in mammalian sex determination and sex differentiation, such as SOX9, WT1, GATA4, DMRT1, DHH, AMH, CYP19A1 and FST. We found that many DEGs are involved in biological developmental regulation by GO and KEGG enrichment analyses, and that most genes associated with the steroid synthesis pathway were downregulated. The DEGs identified in this study may be involved in the regulation of intersex goat sex determination and differentiation, and may increase our understanding of the molecular mechanisms of mammalian sex differentiation.

Follicle-stimulating hormone and luteinizing hormone regulate the synthesis mechanism of dihydrotestosterone in sheep granulosa cells.

Steroid hormones and receptors play important roles in female reproduction, and their expression patterns affect follicular growth and development. To examine the expression of dihydrotestosterone (DHT) synthases (5α-reductases (5α-red1 and 5α-red2)) and androgen receptor (AR) during follicular development, and the regulation of DHT signalling by follicle-stimulating hormone (FSH) and luteinizing hormone (LH), we have used enzyme-linked immunosorbent assays, quantitative real-time polymerase chain reaction, immunohistochemical staining and Western blotting to examine DHT synthesis in small (≤2 mm), medium (2-5 mm) and large (≥5 mm) sheep follicles. Expression of 5α-red1, 5α-red2 and AR was observed in ovine ovaries, and with the development of follicles, the expressions of 5α-red1 and 5α-red2 mRNA and protein increased, but the levels of AR mRNA, protein and DHT level decreased. In addition, granulosa cells were treated with FSH (0.01, 0.1 and 1 international unit (IU)/ml), LH (0.01, 0.1 and 1 IU/ml) and testosterone (T, 10-7  M) to evaluate the effects of FSH and LH on DHT and oestradiol (E2) synthesis and 5α-red1, 5α-red2 and AR expression. We found that FSH and LH upregulated 5α-red1 and 5α-red2 in sheep granulosa cells, but downregulated the concentration of DHT and expression of AR. Meanwhile, FSH and LH significantly upregulated the expression of aromatase (P450arom) and secretion of E2. This result indicates that although FSH and LH promote the expression of 5α-red1 and 5α-red2, T is not transformed into DHT, but E2. This study reveals the reason why DHT concentration is downregulated in large follicles and lays a foundation for further exploring the synthesis mechanism of DHT during follicular development.

Screening for reproductive biomarkers in Bactrian camel via iTRAQ analysis of proteomes.

Bactrian camel is an ancient and precious species of livestock; that is, unique resources exist in the desert and have important economic and scientific value. In recent years, the number of Bactrian camels has declined sharply. Due to its long reproductive cycle and seasonal oestrus, the mechanism of oestrus is unknown. To identify candidate biomarkers of reproduction, we performed a comprehensive proteomic analysis of serum from Bactrian camel in oestrus and non-oestrus, using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with tandem mass spectrometry. We identified 359 proteins, of which 32 were differentially expressed: 11 were up-regulated and 21 were down-regulated in samples from camels in oestrus. We validated the differential expression of a subset of these proteins using qPCR and Western blot. Gene ontology annotation identified that the differentially expressed proteins function in cellular processes, metabolic processes and immune system processes. Notably, five of the differentially expressed proteins, PCGF5, histone H1.2, RBP4, FOLR1 and ANTXR2, are involved in reproductive regulatory processes in other animals. KEGG enrichment analysis demonstrated significant enrichment in several cardiac-related pathways, such as 'dilated cardiomyopathy', 'hypertrophic cardiomyopathy', 'cardiac muscle contraction' and 'adrenergic signalling in cardiomyopathy'. Our results suggest that candidate biomarker (PCGF5, histone H1.2, RBP4, FOLR1 and ANTXR2) discovery can aid in understanding reproduction in Bactrian camels. We conclude that the profiling of serum proteomes, followed by the measurement of selected proteins using more targeted methods, offers a promising approach for studying mechanisms of oestrus.

Androgen receptor, aromatase, oestrogen receptor α/ß and G protein-coupled receptor 30 expression in the testes and epididymides of adult sheep.

The androgen receptor (AR) plays a key role in reproduction, and aromatase (P450arom), nuclear oestrogen receptors (ERs) α and ß, and G protein-coupled receptor 30 (GPR30) are important for testicular and epididymal cell proliferation and development. In the study, we have investigated the expression and localization of AR, P450arom, ERα, ERß and GPR30 in testes and epididymides of sexually mature sheep by quantitative reverse transcription-polymerase chain reaction, Western blotting and immunohistochemistry. The results demonstrate that the AR, P450arom and ERα levels in the caput and corpus epididymis were significantly lower than those in the testis and cauda epididymis (p < .05), the ERß level in the testis was significantly higher than in the caput, corpus and cauda epididymis (p < .05), and the GPR30 level in the caput epididymis was significantly lower than in the testis and corpus and cauda epididymis (p < .05). These receptors were mainly detected in epididymal epithelial, basal, smooth muscle, Sertoli and Leydig cells, as well as in spermatozoa. Taken together, the results suggest that sheep epididymides and testes have the potential for estradiol synthesis and are the targets of both androgens and estradiol. These results provide a foundation for further studies on the mechanisms of androgens and estradiol signalling in the testes and epididymides of sheep.

Cloning, Molecular Characterization and Expression Patterns of DMRTC2 Implicated in Germ Cell Development of Male Tibetan Sheep.

The double sex and mab-3-related transcription factors like family C2 (DMRTC2) gene is indispensable for mammalian testicular function and spermatogenesis. Despite its importance, what expression and roles of DMRTC2 possesses and how it regulates the testicular development and spermatogenesis in sheep, especially in Tibetan sheep, remains largely unknown. In this study, DMRTC2 cDNA from testes of Tibetan sheep was firstly cloned by the RT-PCR method, and its molecular characterization was identified. Subsequently, the expression and localization patterns of DMRTC2 were evaluated by quantitative real-time PCR (qPCR), Western blot, and immunofluorescence. The cloning and sequence analysis showed that the Tibetan sheep DMRTC2 cDNA fragment contained 1113 bp open reading frame (ORF) capable of encoding 370 amino acids, and displayed high identities with some other mammals, which shared an identical DM domain sequence of 47 amino acids ranged from residues 38 to 84. qPCR and Western blot results showed that DMRTC2 was expressed in testes throughout the development stages while not in epididymides (caput, corpus, and cauda), with higher mRNA and protein abundance in Tibetan sheep testes of one- and three-year-old (post-puberty) compared with that of three-month-old (pre-puberty). Immunofluorescence results revealed that immune staining for DMRTC2 protein was observed in spermatids and spermatogonia from post-puberty Tibetan sheep testes, and gonocytes from pre-puberty Tibetan sheep testes. Together, these results demonstrated, for the first time, in sheep, that DMRTC2, as a highly conserved gene in mammals, is essential for sheep spermatogenesis by regulating the proliferation or differentiation of gonocytes and development of spermatids in ram testes at different stages of maturity.

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.

Yak OXGR1 promotes fibroblast proliferation via the PI3K/AKT pathways.

Oxoglutarate receptor 1 (OXGR1), as one of the intermediates in G protein-coupled receptors (GPCRs), plays a crucial role in the citric acid cycle receptor of α-ketoglutarate and metabolism. GPCR can control the cell proliferation by regulating the downstream signaling of G protein signaling pathways. The PI3K/AKT pathway transmits the downstream signals of GPCRs and receptor tyrosine kinases. However, the specific role of OXGR1 promoting cell proliferation and differentiation are still unknown. In current study, the over-expression vector and knockdown sequence of yak OXGR1 were transfected into yak fibroblasts, and the effects were detected by a series of assays. The results revealed that OXGR1 expression in yak lung parenchyma tissue was significantly higher than that of other tissues. In yak fibroblasts, the upregulated expression of OXGR1 resulted in activating the PIK3CG (downstream signal) of the PI3K/AKT1 pathway that can upregulated the expression of proliferation genes ( CDK1, PCNA, and CyclinD1) and promote cell proliferation. Conversely, the downregulated expression of OXGR1 inhibited cell proliferation via PI3K/AKT1 pathway. Cell cycle and cell proliferation assays demonstrated that over-expression of OXGR1 can enhanced the DNA synthesis and promoted yak fibroblasts proliferation. While the conversely, knockdown of OXGR1 can decreased DNA synthesis and inhibited cell proliferation. These results illustrated that changes of OXGR1 expression can trigger the fibroblasts proliferation via PI3K/AKT signaling pathway, which indicating that OXGR1 is a novel regulator for cell proliferation and differentiation. Furthermore, these results provide evidence supporting the functional role of GPCRs-PI3K-AKT1 and OXGR1 in cell proliferation.