Molecular characteristics and regulatory role of insulin-like growth factor 1 gene in testicular Leydig cells of Tibetan sheep.

This study aimed to analyze the molecular characteristics of insulin-like growth factor 1 (IGF1) gene in the testes of Tibetan sheep and its role in the testosterone synthesis and cell development. First, we cloned IGF1 gene for bioinformatics analysis, and the primary Leydig cells (LCs) of Tibetan sheep were isolated to explore its effect on the proliferation, apoptosis and function of LCs. Finally, the specific regulatory mechanism of IGF1 on LCs was analyzed by transcriptome sequencing. Results showed that overexpression of IGF1 increased the proliferation rate and decreased apoptosis of LCs. In addition, overexpression of IGF1 altered expression of genes related to testosterone synthesis and transformation and significantly increased amount of the final product testosterone. Mechanistically, IGF1 stimulated the expression of the proliferating cell nuclear antigen and IGF1R and promoted the proliferation of LCs via the PI3K/Akt signaling pathway. Collectively, what should be clear from the results reported here is that IGF1 might play roles in the proliferation or differentiation and testosterone synthesis of LCs. These findings add to our understanding on the regulation of testosterone synthesis in sheep and other mammals.

Quercetin ameliorates oxidative stress-induced apoptosis of granulosa cells in dairy cow follicular cysts by activating autophagy via the SIRT1/ROS/AMPK signaling pathway.

BACKGROUND: Follicular cysts contribute significantly to reproductive loss in high-yield dairy cows. This results from the death of follicular granulosa cells (GCs) caused by oxidative stress. Quercetin is known to have significant antioxidant and anti-apoptotic effects. However, the effect of quercetin on follicular cysts has yet been elucidated. Therefore, this study aimed to explore the anti-oxidant and anti-apoptosis effects and potential molecular mechanisms of quercetin in H2O2-induced primary cow GCs and 3-nitropropionic acid (3-NPA)-induced mouse model of oxidative stress and thus treat ovarian cysts in dairy cows. RESULTS: In this study, compared with estrus cows, cows with follicular cysts showed heightened levels of oxidative stress and increased follicular cell apoptosis, while autophagy levels were reduced. A model of oxidative stress was induced in vitro by H2O2 and showed significant increases in apoptosis together with reduced autophagy. These effects were significantly ameliorated by quercetin. Effects similar to those of quercetin were observed after treatment of cells with the reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC). Further investigations using chloroquine (autophagy inhibitor), rapamycin (autophagy activator), selisistat (SIRT1 inhibitor), and compound C (AMPK inhibitor) showed that chloroquine counteracted the effects of quercetin on oxidative stress-induced apoptosis, while rapamycin had the same effect as quercetin. In addition, the SIRT1/AMPK pathway inhibitors antagonized quercetin-mediated mitigation of the effects of oxidative stress on increased apoptosis and reduced autophagy. Consistent with the results in vitro, in mouse ovarian oxidative stress model induced by 3-NPA, quercetin activated autophagy through the SIRT1/AMPK signaling pathway, while alleviating oxidative stress damage and inhibiting apoptosis in mouse ovaries. CONCLUSIONS: These findings indicate that quercetin can inhibit apoptosis in GCs and restore ovarian function by activating autophagy through the SIRT1/ROS/AMPK signaling pathway, suggesting a new direction for the treatment of ovarian follicular cysts in high-yield dairy cows.

Berberine-doped montmorillonite nanosheet for photoenhanced antibacterial therapy and wound healing.

Bacterial infections pose a substantial threat to human health, particularly with the emergence of antibiotic-resistant strains. Therefore, it is essential to develop novel approaches for the efficient treatment of bacterial diseases. This study presents a therapeutic approach involving BBR@MMT nanosheets (NSs), wherein montmorillonite (MMT) was loaded with berberine (BBR) through an ion intercalation reaction to sterilize and promote wound healing. BBR@MMT exhibits nano-enzymatic-like catalytic activity, is easy to synthesize, and requires low reaction conditions. This nanocomplex showed photodynamic properties and superoxide dismutase (SOD) activity. The in vitro experiments indicated that BBR@MMT was able to effectively inhibit the growth of Gram-positive bacteria (S. aureus) and Gram-negative bacteria (E. coli) through the production of ROS when exposed to white light. Meanwhile, BBR@MMT inhibited the secretion of pro-inflammatory factors and scavenged free radicals via its SOD-like activity. In vivo results showed that BBR@MMT NSs were capable of effectively promoting the wound-healing process in infected mice under white light irradiation. Hence, it can be concluded that photodynamic therapy based on BBR@MMT NSs with nano-enzymatic activity has the potential to be used in treating infections and tissue repair associated with drug-resistant microorganisms.

Untargeted Metabolomic Analysis Reveals Plasma Differences between Mares with Endometritis and Healthy Ones.

The aim of this study was to explore alterations in plasma metabolites among mares afflicted with endometritis. Mares were divided into two groups, namely, the equine endometritis group (n = 8) and the healthy control group (n = 8), which included four pregnant and four non-pregnant mares, using a combination of clinical assessment and laboratory confirmation. Plasma samples from both groups of mares were analyzed through untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics. A total of 28 differentially abundant metabolites were identified by screening and identifying differentially abundant metabolites and analyzing the pathway enrichment of differentially. Ten metabolites were identified as potential biomarkers for the diagnosis of endometritis in mares. Among them, seven exhibited a decrease in the endometritis groups, including hexadecanedioic acid, oleoyl ethanolamide (OEA), [fahydroxy(18:0)]12_13-dihydroxy-9z-octa (12,13-diHOME), deoxycholic acid 3-glucuronide (DCA-3G), 2-oxindole, and (+/-)9-HPODE, and 13(S)-HOTRE. On the other hand, three metabolites, adenosine 5'-monophosphate (AMP), 5-hydroxy-dl-tryptophan (5-HTP), and l-formylkynurenine, demonstrated an increase. These substances primarily participate in the metabolism of tryptophan and linolenic acid, as well as fat and energy. In conclusion, metabolomics revealed differentially abundant metabolite changes in patients with mare endometritis. These specific metabolites can be used as potential biomarkers for the non-invasive diagnosis of mare endometritis.

Exploring the Mechanism of H2S Synthesis in Male Bactrian Camel Poll Glands Based on Data Independent Acquisition Proteomics and Non-Targeted Metabolomics.

During estrus, the poll glands of male Bactrian Camels (Camelus Bactrianus) become slightly raised, exuding a large amount of pale yellow watery secretion with a characteristic odor that may contain hydrogen sulfide (H2S). However, whether H2S can be synthesized in the poll glands of male Bactrian Camels and its role in inducing camel estrus remains unclear. This study aimed to identify differentially expressed proteins (DEPs) and signaling pathways in the poll gland tissues of male Bactrian Camels using data independent acquisition (DIA) proteomics. Additionally, gas chromatography-mass spectrometry (GC-MS) was performed to identify differentially expressed metabolites (DEMs) in the neck hair containing secretions during estrus in male Bactrian Camels, to explore the specific expression patterns and mechanisms in the poll glands of camels during estrus. The results showed that cystathionine-γ-lyase (CTH) and cystathionine-ß-synthase (CBS), which are closely related to H2S synthesis in camel poll glands during estrus, were mainly enriched in glycine, serine, and threonine metabolism, amino acid biosynthesis, and metabolic pathways. In addition, both enzymes were widely distributed and highly expressed in the acinar cells of poll gland tissues in camels during estrus. Meanwhile, the neck hair secretion contains high levels of amino acids, especially glycine, serine, threonine, and cystathionine, which are precursors for H2S biosynthesis. These results demonstrate that the poll glands of male Bactrian Camels can synthesize and secrete H2S during estrus. This study provides a basis for exploring the function and mechanism of H2S in the estrus of Bactrian Camels.

Caveolin 1 Regulates the Tight Junctions between Sertoli Cells and Promotes the Integrity of Blood-Testis Barrier in Yak via the FAK/ERK Signaling Pathway.

Yaks, a valuable livestock species endemic to China's Tibetan plateau, have a low reproductive rate. Cryptorchidism is believed to be one of the leading causes of infertility in male yaks. In this study, we compared the morphology of the normal testis of the yak with that of the cryptorchidism, and found dysplasia of the seminiferous tubules, impaired tightness of the Sertoli cells, and a disruption of the integrity of the blood-testis barrier (BTB) in the cryptorchidism. Previous studies have shown that CAV1 significantly contributes to the regulation of cell tight junctions and spermatogenesis. Therefore, we hypothesize that CAV1 may play a regulatory role in tight junctions and BTB in Yaks Sertoli cells, thereby influencing the development of cryptorchidism. Additional analysis using immunofluorescence, qRT-PCR, and Western blotting confirmed that CAV1 expression is up-regulated in yak cryptorchidism. CAV1 over-expression plasmids and small RNA interference sequences were then transfected in vitro into yak Sertoli cells. It was furthermore found that CAV1 has a positive regulatory effect on tight junctions and BTB integrity, and that this regulatory effect is achieved through the FAK/ERK signaling pathway. Taken together, our findings, the first application of CAV1 to yak cryptorchidism, provide new insights into the molecular mechanisms of cell tight junctions and BTB. This paper suggests that CAV1 could be used as a potential therapeutic target for yak cryptorchidism and may provide insight for future investigations into the occurrence of cryptorchidism, the maintenance of a normal physiological environment for spermatogenesis and male reproductive physiology in the yak.

Gas/Liquid Chromatography-Mass Spectrometry Analysis of Key Functional Substances Regulating Poll Gland Secretion in Male Camels during Seasonal Estrus.

Increased poll gland secretion is a major characteristic and indicator of estrus in male Bactrian camels; however, research on these poll glands and their secretion is extremely rare. In this study, we determine the chemical composition of poll gland secretions and identify the key functional substances that regulate seasonal estrus in male camels. A GC/LC-MS dual platform was used to analyze ventral hair (control) and neck mane samples containing poll gland secretions from male Bactrian camels during estrus. Multidimensional and single-dimensional analyses were used to screen differentially expressed metabolites (DEMs) between groups. Functional prediction of enriched metabolites was performed using a Human Metabolome Database comparison and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, which were then compared with a behavioral analysis of male Bactrian camels in estrus. A total of 1172 DEMs and 34 differential metabolic pathways were identified. One metabolite group was found to relate to steroid synthesis and metabolism, and another metabolite group was associated with neural metabolism. Therefore, we speculate that steroids and neurochemicals jointly regulate estrous behavior in male Bactrian camels, thus providing theoretical insights into the development and function of poll glands in Bactrian camels.

Expression of FMD virus-like particles in yeast Hansenula polymorpha and immunogenicity of combine with CpG and aluminum adjuvant.

BACKGROUND: Inactivated vaccines are limited in preventing foot-and-mouth disease (FMD) due to safety problems. Recombinant virus-like particles (VLPs) are an excellent candidate for a novel vaccine for preventing FMD, given that VLPs have similar immunogenicity as natural viruses and are replication- and infection-incompetent. OBJECTIVES: The 3C protease and P1 polyprotein of type O FMD virus (FDMV) was expressed in yeast Hansenula polymorpha to generate self-resembling VLPs, and the potential of recombinant VLPs as an FMD vaccine was evaluated. METHODS: BALB/c mice were immunized with recombinant purified VLPs using CpG oligodeoxynucleotide and aluminum hydroxide gel as an adjuvant. Cytokines and lymphocytes from serum and spleen were analyzed by enzyme-linked immunosorbent assay, enzyme-linked immunospot assay, and flow cytometry. RESULTS: The VLPs of FMD were purified successfully from yeast protein with a diameter of approximately 25 nm. The immunization of mice showed that animals produced high levels of FMDV antibodies and a higher level of antibodies for a longer time. In addition, higher levels of interferon-γ and CD4+ T cells were observed in mice immunized with VLPs. CONCLUSIONS: The expression of VLPs of FMD in H. polymorpha provides a novel strategy for the generation of the FMDV vaccine.

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.

Melatonin regulates dihydrotestosterone formation via its membrane receptor in the epididymal epithelial cells of sheep.

Both melatonin and androgen, which affect sperm fertility, are the important factors in epididymis of male animal. In the present study, we confirmed that melatonin regulates the formation of dihydrotestosterone (DHT) in sheep epididymides. Here, we investigated the localization and the expression levels of melatonin keys synthases AANAT and HIOMT, membrane receptors MT1 and MT2, and nuclear receptor RORα in sheep epididymides and testes. We also cultured epididymal epithelial cells and treated them with different concentrations of melatonin (10-11-10-7 M) and luzindole (10-5 M) and 4P-PDOT (10-5 M) to investigate whether melatonin is involved in the regulation of DHT formation and whether these effects are mediated through its receptor pathways. The results showed that AANAT, HIOMT, MT1, MT2, and RORα were differentially expressed between sheep epididymides and testes. In addition, melatonin is involved in mediating the formation of DHT in epididymal epithelial cells, and its influence on DHT is at least partially regulated by the melatonin receptor pathway. Our findings showed that melatonin regulates the functions of the testes and epididymides through an autocrine mechanism and regulates the formation of androgen in sheep epididymides via the receptor pathway. These results provide a basis for further exploring the regulatory mechanisms of melatonin in animal reproduction.

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.

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.

Regulatory role of melatonin on epidermal growth factor receptor, Type I collagen α1 chain, and caveolin 1 in granulosa cells of sheep antral follicles.

We investigated the expression of epidermal growth factor receptor (EGFR), Type I collagen α1 chain (COL1A1), and caveolin 1 (CAV1) during follicular development and examined the regulatory role of melatonin (MLT) on EGFR, COL1A1, and CAV1 in sheep antral ovaries. The expression was detected in granulosa and theca cells by immunohistochemistry. Quantitative real-time polymerase chain reaction and Western blotting were used to examine the expression levels of EGFR, COL1A1, and CAV1 in small (≤2 mm), medium (2-5 mm), and large (≥5 mm) follicles. The mRNA and protein levels of EGFR, COL1A1, and CAV1 were found to be the highest in large follicles. Furthermore, cultured granulosa cells were treated with MLT (10-7 -10-11  M), luzindole (nonselective MT1 and MT2 receptor antagonist, 10-7  M), and 4-phenyl-2-propanamide tetraldehyde (4P-PDOT, MT2 selective antagonist, 10-7  M) to detect the regulatory role of MLT on EGFR, COL1A1, and CAV1. Results indicated COL1A1 and CAV1 were at least partially regulated by MLT through MT1 and MT2 pathways, whereas EGFR was not. This study provided a reference for further studies on MLT regulatory role on EGFR, COL1A1, and CAV1 during sheep follicular development and elucidated the physiological mechanism of MLT regulator production.

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.

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.

Sulfur Amino Acid Metabolism and the Role of Endogenous Cystathionine-γ-lyase/H2S in Holstein Cows with Clinical Mastitis.

H2S plays an important role in various inflammatory diseases. However, the role of H2S and synthetic enzymes in Holstein cows with CM is unknown. The aim of this study was to identify DEPs associated with sulfide metabolism and further investigate their roles in dairy cows with CM. From 3739 DEPs generated by data-independent acquisition proteomics, we identified a total of 17 DEPs included in 44 GO terms and five KEGG pathways related to sulfide metabolism, including CTH and cystathionine-ß-synthase (CBS). Immunohistochemical and immunofluorescence staining results showed that CTH and CBS proteins were present mainly in the cytoplasm of mammary epithelial cells. Endogenous H2S production in the serum of the CM group was significantly lower than that of the healthy Holstein cows. CTH and CBS mRNA and protein levels in the mammary glands of the CM group were significantly downregulated compared to those of the healthy group. These results indicate that CTH and H2S were correlated with the occurrence and development of CM in Holstein cows, which provides important insights into the function and regulatory mechanism of CTH/H2S in Holstein cows.

Exploring the Action Mechanism of the Active Ingredient of Quercetin in Ligustrum lucidum on the Mouse Mastitis Model Based on Network Pharmacology and Molecular Biology Validation.

Aim: The aim of this study is to explore the mechanism of action of quercetin, the main active anti-inflammatory component of Ligustrum lucidum, in the prevention and treatment of mastitis. Methods: Prediction of the main active ingredients and key anti-inflammatory targets of Ligustrum lucidum using a network pharmacology platform and molecular biology validation of the results. Observation of histopathological changes in the mouse mammary gland by hematoxylin-eosin staining(H&E) method, quantitative real-time PCR(qPCR), and Western blot (WB) to detect the expression levels of relevant inflammatory factors mRNA and protein. Results: A total of 7 active ingredients and 42 key targets were obtained from the network pharmacological analysis of Ligustrum lucidum, with quercetin as the main core ingredient and tumor necrosis factor(TNF), serine threonine protein kinase1(AKT1), and interleukin6(IL6) as the core targets; H&E results showed that pathological changes were reduced to different degrees in the dose group compared to the model group. The qPCR results showed that the relative expression of TNF and IL6 mRNA in the high dose group on day 3 and the high and medium dose groups on day 7 were not significantly different compared with the blank group (P > 0.05), and the difference between the dose groups on day 5 was significant (P < 0.05). WB results showed that the difference in nuclear factor kappa-B(NF-κB) protein expression in the medium and low dose groups on day 7 was significant compared with the blank group (P < 0.05), the difference in 5 and 7 days, significant differences in AKT1 protein expression between the middle and low dose groups (P < 0.05), nonsignificant differences in the TNF protein expression between the high dose groups on day 7 (P > 0.05), and significant differences in the IL6 protein expression between the middle and low dose groups on days 3 and 7 (P < 0.05). Conclusion: Quercetin, the main active ingredient of Ligustrum lucidum, may act in the prevention and treatment of mastitis by inhibiting the expression of inflammatory factors in phosphoinositol 3-kinase(PI3K)-AKT and NF-κB signaling pathways and showa a significant dose-dependent effect. This study provides theoretical basis and clues for the control of mastitis in dairy cows.

Expression of dihydrotestosterone synthases and androgen receptor in sheep oviduct ampulla and its regulation by estradiol and progesterone.

Oviduct ampulla plays an important role in steroid hormone-regulated sperm-oocyte binding in female animals. Although studies have shown that androgen receptor are expressed in many species oviduct, the interaction among androgen receptor (AR), estradiol (E2) and progesterone (P4) in the sheep oviduct have rarely been reported. In this study, we evaluated the localization of two isoforms of dihydrotestosterone (DHT) sythetase enzymes 5α-reductase (5α-red1, 5α-red2) and AR in sheep oviduct ampulla by immunohistochemistry and immunofluorescence. Results showed that they were all distributed in oviduct epithelium layer. In epithelial cells, 5α-red1, 5α-red2 were expressed in cytoplast and nuclear, but AR were stained in nuclear. We also investigated their expression pattern in the sheep oviduct ampulla at different development stages of follicles (Large follicles stage; hemorrhagium, luteum and albicans of corpus stage) by molecular experiments. We found that 5α-red1, 5α-red2 and AR mRNA abundance and protein were expressed highest in corpus albicans stage and lowest in corpus hemorrhagium stage. In vitro, when sheep oviduct ampulla epithelial cells (SOAECs) were cultured and treated with different concentrations of E2/P4 (10-9-10-6 M), we found that E2 inhibited the expression of AR mRNA and protein, while P4 promoted this expression. In addition, when the SOAECs were treated with E2 (10-8 M) and/or its non-selective inhibitor ICI182780 (10-7 M) as well as with P4 (10-6 M) and/or its non-specific inhibitor RU486 (10-5 M), we found that E2 and P4 inhibited and promoted the expression of AR mRNA and proteins, respectively, via their nuclear receptor pathways. This study provides a basic insight for the further research of oviduct epithelium physiological function closely related to androgen.

ß-Estradiol inhibits melatonin synthesis and melatonin receptor expression in sheep granulosa cells.

Melatonin, an important regulator of mammalian reproduction, is mainly produced in the pineal gland, and granulosa cells (GCs), the main mammalian ovarian secretory cells, synthesize melatonin and express melatonin receptors (MRs) MT1 and MT2. However, studies on melatonin regulation in GCs are lacking in sheep. In this study, we explored the effects of ß-estradiol (E2) on melatonin production and MR expression in GCs. We cultured sheep GCs to analyze the expression of the melatonin rate-limiting enzymes AANAT and HIOMT and the effects of E2 on AANAT, HIOMT, and MR expression and melatonin synthesis. To determine whether estrogen receptors (ERs) mediated E2 action on melatonin secretion and MR expression, we assessed ERA and ERB expression in GCs and observed whether ER antagonists counterbalanced the effects of E2. GCs expressed AANAT and HIOMT mRNA, indicating that they transformed exogenous serotonin into melatonin. E2 inhibited melatonin production by downregulating AANAT, HIOMT, and MRs. GCs expressed ERA and ERB; ERA/ERB inhibitors abolished E2-mediated inhibition of melatonin secretion and MR expression. PHTPP upregulated melatonin secretion and MT1 expression in E2-treated GCs, but did not significantly affect AANAT and MT2 expression. In conclusion, melatonin secretion in GCs was inhibited by E2 through an ERA- and ERB-mediated process.

LncRNA GAS5 Competitively Combined With miR-21 Regulates PTEN and Influences EMT of Peritoneal Mesothelial Cells via Wnt/ß-Catenin Signaling Pathway.

OBJECTIVE: Epithelial-mesenchymal transition (EMT) is an important factor leading to peritoneal fibrosis (PF) in end-stage renal disease (ESRD) patients. The current research aimed to evaluate the effect of long non-coding RNA growth arrest-specific 5 (lncRNA GAS5) in human peritoneal mesothelial cells (HPMCs) EMT and explore the potential molecular mechanisms. MATERIALS AND METHODS: HPMCs were cultured under control conditions or with high glucose (HG). The cells were then treated with lncRNA GAS5, lncRNA GAS5 siRNA, with or without miR-21 inhibitor and PTEN transfection. Expression of lncRNA GAS5, miR-21, α-SMA, Vimentin, E-cadherin, phosphatase and tensin homolog deleted on chromosome ten (PTEN), Wnt3a, and ß-catenin were measured by real time PCR and Western blotting. Bioinformatics analyses were used to test the specific binding sites between the 3' UTR of the PTEN gene, miR-21, and lncRNA GAS5. Rescue experiments were performed to confirm the lncRNA GAS5/miR-21/PTEN axis in HPMC EMT. RESULTS: We found that HG-induced EMT decreased lncRNA GAS5 and that overexpression of lncRNA GAS5 can attenuate EMT in HPMCs. In addition, lncRNA GAS5 regulated HG-induced EMT through miR-21/PTEN. Cotransfection of miR-21 inhibitors remarkably increased PTEN expression and attenuated EMT in lncRNA GAS5 knockdown HPMCs. Moreover, rescue experiments showed that overexpression of PTEN attenuated the EMT effects of lncRNA GAS5 siRNA in HPMCs. We also confirmed that the Wnt/ß-catenin pathway was stimulated in lncRNA GAS5/miR-21/PTEN-mediated EMT. CONCLUSION: Our research showed that lncRNA GAS5 competitively combined with miR-21 to regulate PTEN expression and influence EMT of HPMCs via the Wnt/ß-catenin signaling pathway. This study provides novel evidence that lncRNA GAS5 may be a potential therapeutic target for HPMC EMT.