Beneficial effects of melatonin on boar sperm motility and kinematics are mediated by MT1 receptor.

Melatonin, a hormone synthesized in various tissues, plays a crucial role in modulating sperm characteristics, yet its protective function on boar sperm remains poorly understood. This study aimed to investigate the expression and localization of melatonin-related proteins (AANAT, ASMT, MT1, MT2, and NQO2) in pig tissues, assess the impact of melatonin on pig sperm motility parameters and quality, and elucidate the underlying molecular mechanisms. Our results revealed widespread expression of AANAT, ASMT, MT1, MT2, and NQO2 proteins in pig tissues, particularly in the testis. Specific localization patterns were observed in Leydig cells, reproductive epithelium, and columnar epithelium cells in the testis and cauda epididymis. Additionally, melatonin membrane receptors MT1 and MT2 were detected in boar sperm. Melatonin treatment significantly enhanced boar sperm motility parameters and quality, particularly with 10 nM melatonin treatment. Inhibition of the MT1 receptor, but not the MT2 receptor, resulted in decreased sperm motility, highlighting the pivotal role of the MT1 receptor in mediating melatonin's effects on boar sperm. Metabolomic analysis revealed significant alterations in sperm metabolites following melatonin supplementation, particularly in amino acid metabolism. Overall, our findings provide comprehensive insights into melatonin's mechanisms in improving boar sperm quality, suggesting its potential as a therapeutic agent for enhancing male fertility.

Histone Lactylation Is Involved in Mouse Oocyte Maturation and Embryo Development.

Numerous post-translational modifications are involved in oocyte maturation and embryo development. Recently, lactylation has emerged as a novel epigenetic modification implicated in the regulation of diverse cellular processes. However, it remains unclear whether lactylation occurs during oocyte maturation and embryo development processes. Herein, the lysine lactylation (Kla) modifications were determined during mouse oocyte maturation and early embryo development by immunofluorescence staining. Exogenous lactate was supplemented to explore the consequences of modulating histone lactylation levels on oocyte maturation and embryo development processes by transcriptomics. Results demonstrated that lactylated proteins are widely present in mice with tissue- and cell-specific distribution. During mouse oocyte maturation, immunofluorescence for H3K9la, H3K14la, H4K8la, and H4K12la was most intense at the germinal vesicle (GV) stage and subsequently weakened or disappeared. Further, supplementing the culture medium with 10 mM sodium lactate elevated both the oocyte maturation rate and the histone Kla levels in GV oocytes, and there were substantial increases in Kla levels in metaphase II (MII) oocytes. It altered the transcription of molecules involved in oxidative phosphorylation. Moreover, histone lactylation levels changed dynamically during mouse early embryogenesis. Sodium lactate at 10 mM enhanced early embryo development and significantly increased lactylation, while impacting glycolytic gene transcription. This study reveals the roles of lactylation during oocyte maturation and embryo development, providing new insights to improving oocyte maturation and embryo quality.

Protective effects of l-cysteine and N-acetyl-l-cysteine on boar sperm quality during hypothermic liquid storage with bovine serum albumin as a protectant.

Hypothermic liquid storage at 4-5 °C has emerged as a novel approach for preserving boar semen, offering innovative possibilities for semen preservation. However, this method also presents challenges, including cold shock and excessive reactive oxygen species (ROS) production. Therefore, reducing oxidative damage induced by low temperatures becomes essential while supplementing appropriate protectants. In this study, we investigated the efficacy of Bovine Serum Albumin (BSA) compared to Polyvinylpyrrolidone (PVP) and Skim Milk Powder (SMP) in maintaining boar sperm motility and progressive motility using computer-assisted sperm analysis (CASA). Among the tested concentrations, 4 g/L of BSA exhibited the best protective effect. Subsequently, we supplemented different concentrations of l-cysteine (LC) and N-acetyl-l-cysteine (NAC) as additives in the presence of BSA as a protectant. Our results demonstrated that 1 mmol/L of LC and 0.5 mmol/L of NAC exhibited superior protection of sperm quality compared to other concentrations. Furthermore, the 1 mmol/L LC and 0.5 mmol/L NAC groups showed significantly improved plasma membrane integrity and acrosome integrity compared to the control group. These groups also exhibited enhanced antioxidant capacity, evidenced by increased mitochondrial membrane potential (MMP), ATP production, total superoxide dismutase (T-SOD) activity, total antioxidant capacity (T-AOC), glutathione (GSH), glutathione peroxidase (GSH-PX), and GPX-4 levels. Additionally, they demonstrated decreased reactive oxygen species (ROS) and malondialdehyde (MDA) levels, as well as reduced oxidized glutathione (GSSG) and glutathione reductase (GR) levels. Furthermore, LC and NAC treatment enhanced AMP-activated protein kinase (AMPK) phosphorylation. However, inhibiting AMPK using compound C did not inhibit the protective effects of LC and NAC on low-temperature preserved boar sperm. These findings suggest that 4 g/L BSA can serve as an effective protectant for hypothermic liquid storage of boar semen. Additionally, LC and NAC supplementation reduces oxidative damage by enhancing antioxidant capacity rather than through AMPK-mediated ATP supplementation. These results contribute to advancing the application of LC and NAC in hypothermic liquid storage of boar semen.

NLRP9 involved in antiviral innate immunity via binding VIM in IPEC-J2 cells.

BACKGROUND: Nucleotide-binding oligomerization domain (NOD)-like receptors with a pyrin domain (PYD)-containing protein 9 (NLRP9) was the first nucleotide-binding region receptor (NLR) proposed to be expressed and function only in the reproductive system. Recent evidence suggests that NLRP9 is also capable of playing a role in infectious and inflammatory diseases. RESULTS AND CONCLUSIONS: In this study, we examined the expression of NLRP9 in various tissues of piglets and IPEC-J2 cells. The results showed that high expression of NLRP9 mRNA and protein were detected in both intestine of piglets and IPEC-J2 cells. Both LPS and poly I:C significantly up-regulated NLRP9 protein levels in the IPEC-J2 cells. Besides, poly I:C upregulated the level of transcriptional elements NF-κB, IRF3, IRF7, ISG15, ISG56, OAS1, and IFNB1. Furthermore, interference with the NLRP9 gene in the presence of poly I:C strongly downregulated the expression of all the above genes. Moreover, we demonstrated for the first time that NLRP9 acts in combination with VIM (Vimentin). These results suggested that NLRP9 may participate in the antiviral innate immune by binding to VIM in the porcine intestine. The findings provide preliminary insights into the molecular mechanisms involved in the regulation of mucosal immunity in the porcine intestine by NLRP9.

Qualitative lysine crotonylation and 2-hydroxyisobutyrylation analysis in the ovarian tissue proteome of piglets.

Ovarian function influences diverse aspects of fertility and reproductive lifespan by regulating oocyte supply and hormone secretion. Lysine crotonylation (Kcr) and lysine 2-hydroxyisobutyryllysine (Khib) are newly identified post-translational modifications and function as regulators of transactivation in mammals. In this study, we investigated protein post-translational Kcr and 2-hydroxyisobutyrylation in the ovarian tissues of piglets. A total of 653 overlapping proteins among differentially modified proteins were identified for both crotonylation and 2-hydroxyisobutyrylation. Gene Ontology enrichment analysis indicated that 653 DMPs were significantly enriched in nucleosome organization, chromatin assembly, DNA packaging, peptide biosynthetic process and peptide metabolic process. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed enrichment in proteasome, ribosome, fatty acid elongation, pyruvate metabolism and pentose phosphate pathway. Fifteen DMPs were identified in the proteasome pathway, of which PSMC6 and PSMB7 were the core proteins. In addition, the significant changes in Kcr and Khib in the complex subunits of the proteasome may be involved in cell cycle processes during oocyte development. Forty-four DMPs with both Kcr and Khib modifications were related to the ribosome pathway. The regulated ribosome pathway may indicate that Kcr and Khib comodified proteins participate in protein synthesis during oocyte development. Western blot and immunofluorescence staining results supported the reliability of the sequencing results. Our results may provide a valuable resource to help illuminate the roles of Kcr and Khib in ovarian development and may serve as new tools to better control diseases.

Electroacupuncture relieves visceral hypersensitivity through modulation of the endogenous cannabinoid system.

BACKGROUND: Electroacupuncture (EA) can effectively relieve visceral hypersensitivity (VH). However, its mechanisms are still unclear. OBJECTIVE: To investigate the impact of EA on VH caused by ileitis, and whether EA relieves VH by modulating the endogenous cannabinoid system (ECS). METHODS: Thirty male native goats were randomly divided into a saline-treated control group (Saline, n = 9) and three 2,4,6-trinitro-benzenesulfonic acid (TNBS)-treated VH model groups that underwent injection of TNBS into the ileal wall to induce VH and remained untreated (TNBS, n = 9) or received six sessions of EA (for 30 min every 3 days) (TNBS + EA, n = 6) or sham acupuncture (TNBS + Sham, n = 6). The visceromotor response (VMR) to colorectal distention (CRD) was measured after each EA treatment. Three goats in the Saline/TNBS groups were euthanized after 7 days for histopathological examination; the remaining 24 (n = 6/group) underwent sampling of the ileal wall, T11 spinal cord and brain nuclei/areas related to visceral regulation and ascending pain modulation system on day 22. Expression of cannabinoid receptor 1 (CB1R), fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) was detected by immunohistochemistry. RESULTS: VMR to CRD was greater in TNBS-treated goats than in saline-treated goats (p < 0.01) from day 7 to 22. After day 7, EA-treated goats showed a decreased (p < 0.05) VMR compared with untreated TNBS-exposed goats. TNBS treatment decreased CB1R and increased FAAH and MAGL expression in the ileum and related nuclei/areas; this was reversed by EA. CONCLUSION: EA ameliorates VH, probably by regulating the ECS in the intestine and nuclei/areas related to visceral regulation and descending pain modulation systems.

Regulation of Tight Junctions by Sex Hormones in Goat Mammary Epithelial Cells.

The sex hormones of estrogen and progesterone (P4) play a vital role in mammary gland development and milk lactation in ruminants. The tight junction (TJ) between adjacent secretory epithelial cells is instrumental in establishing the mammary blood-milk barrier. However, whether estrogen and P4 exert their effect on mammary function via regulating TJ remain unclear. Here, to clarify the role of 17-ß estradiol (E2) and P4 in the regulation of TJ in goat mammary gland, we first explored the relationships between the concentrations of E2, P4, and the protein expression of claudin-1, claudin-3, occludin, and ZO-1 during the mammary gland development in goat. Then, we further explored the mRNA and protein expression of claudin-1, claudin-3, occludin, and ZO-1 in the goat mammary epithelial cells (GMECs) in vitro under different concentrations of E2 and P4. The results demonstrated that the protein expression of claudin-1 decreased, but occludin and ZO-1 increased with the decline in E2 and P4 during the transition from pregnancy to lactation. In the in vitro studies, E2 exerted a positive effect on the mRNA expression of claudin-1, and accelerated the proteins' expression of claudin-1 and ZO-1 in GMECs; P4 upregulated the mRNA expression of claudin-1, claudin-3, occludin, and ZO-1, and also improved the protein expression of claudin-1, claudin-3, and ZO-1 in the GMECs. The results demonstrated that E2 and P4 play an important role in regulating the expression of the mammary TJ components, which may ultimately affect the mammary gland development and milk lactation.

Polystyrene micro- and nano-particle coexposure injures fetal thalamus by inducing ROS-mediated cell apoptosis.

The adverse effects of plastic on adult animal and human health have been receiving increasing attention. However, its potential toxicity to fetuses has not been fully elucidated. Herein, biodistribution of polystyrene (PS) particles was determined after the maternal mice were orally given PS micro- and/or nano-particles with and without surface modifications during gestational days 1 to 17. The results showed that PS microplastics (MPs) and nanoparticles (NPs) mainly emerged in the alimentary tract, brain, uterus, and placenta in maternal mice, and only the latter infiltrated into the fetal thalamus. PS NPs and carboxyl-modified NPs induced differentially expressed genes mainly enriched in oxidative phosphorylation and GABAergic synapse. Maternal administration of PS particles during gestation led to anxiety-like behavior of the progenies and their γ-aminobutyric acid (GABA) reduction in the prefrontal cortex and amygdala at Week 8. N-Acetylcysteine (NAC), an antioxidant, alleviated PS particles-induced oxidative injury in the fetal brain and rescued the anxiety-like behavior of the progenies. Additionally, PS nanoparticles caused excessive ROS and apoptosis in neuronal cell lines, which were prevented by glutathione supplementation. These results suggested that PS particles produced a negative effect on fetuses by inducing oxidative injury and suppressing GABA synthesis in their brain. The findings contribute to estimating the risk for PS particles to human and animal health.

Baicalin alleviates endometrial inflammatory injury through regulation of tight junction proteins.

Endometritis is the foremost reason for reduced reproductive performance, which impedes the establishment of pregnancy in ruminants. Baicalin is extensively acknowledged as a tocolytic drug. However, the preventive effect of baicalin on endometrial inflammatory injury remains unclear. The present study aimed to determine the potential benefits of baicalin on endometrial inflammatory injury in animal and cellular models. The results showed that baicalin alleviated the impairment of tight junctions (TJs) and inflammation in the endometrium induced by LPS treatment. Baicalin increased claudin 3 (CLDN3) and tight junction protein 1 (TJP1) levels in a dose-dependent manner in endometrial epithelial cells (EECs) accompanied by autophagy activation with or without LPS treatment. Immunofluorescence staining revealed that baicalin pretreatment prompted MAP1LC3B-positive structures to surround TJ proteins in the cytoplasm and decreased the abnormal aggregation of CLDN3 and TJP1 in the cytosol of EECs. Activation or blockage of autophagy using pharmacologic methods affected the redistribution of TJ proteins by baicalin pretreatment with LPS treatment. The role of autophagy in the modulation of TJ proteins was also confirmed by ATG7 and TFEB overexpression, as evidenced by accelerated redistribution of CLDN3 and TJP1 from the EEC cytosol to the membrane and a loss of membranous CLDN2 in EECs. These data demonstrate that baicalin influences the redistribution of TJ proteins to maintain the barrier function during LPS-induced endometrial inflammatory injury by regulating autophagy and provides a new therapeutic to potentially prevent embryo loss and endometritis.

Mixed polysaccharides derived from shiitake mushroom, Poriacocos, Ginger, and Tangerine peel prevent the H1N1 virus infections in mice.

The pandemic influenza A (H1N1) virus spread globally and posed one of the most serious global public health challenges. The traditional Chinese medicine is served as a complementary treatment strategy with vaccine immunization. Here, we demonstrated that the mixed polysaccharides (MPs) derived from shiitake mushroom, poriacocos, ginger, and tyangerine peel prevent the H1N1 virus infections in mice. MP pretreatment attenuated H1N1 virus-induced weight loss, clinical symptoms, and death. The lymphocytes detection results showed that the CD3+, CD19+, and CD25+ cell proportions were upregulated in thymus under MP pretreatment. Besides, MP pretreatment reduced the inflammatory cell infiltration and increased the cell proportions of CD19+, CD25+, and CD278+ in lung. However, MP treatment have no effective therapeutic effect after H1N1 virus challenge. The current study suggested that pretreatment with MPs could attenuate H1N1 virus-induced lung injury and upregulate humoral and cellular immune responses in nonimmunized mice.

Gastrodin Improves Nonalcoholic Fatty Liver Disease Through Activation of the Adenosine Monophosphate-Activated Protein Kinase Signaling Pathway.

BACKGROUND AND AIMS: NASH is currently one of the most common causes of liver transplantation and hepatocellular carcinoma. Thus far, there is still no effective pharmacological therapy for this disease. Recently, Gastrodin has demonstrated hepatoprotective effects in a variety of liver diseases. The aim of this study is to investigate the function of Gastrodin in NASH. APPROACH AND RESULTS: In our study, Gastrodin showed potent therapeutic effects on NASH both in vivo and in vitro. In high-fat diet or high-fat and high-cholesterol diet-fed mice, the liver weight, hepatic and serum triglyceride and cholesterol contents, and serum alanine aminotransferase and aspartate aminotransferase activity levels were markedly reduced by Gastrodin treatment as compared with the corresponding vehicle groups. Notably, Gastrodin showed minimal effects on the function and histological characteristics of other major organs in mice. We further examined the effects of Gastrodin on lipid accumulation in primary mouse hepatocytes and human hepatocyte cell line and observed that Gastrodin showed a significant decrease in lipid accumulation and inflammatory response in hepatocytes under metabolic stress. Furthermore, RNA-sequencing analysis systemically indicated that Gastrodin suppressed the pathway and key regulators related to lipid accumulation, inflammation, and fibrosis in the pathogenesis of NASH. Mechanistically, we found that Gastrodin protected against NASH by activating the adenosine monophosphate-activated protein kinase (AMPK) pathway, which was supported by the result that the AMPK inhibitor Compound C or AMPK knockdown blocked the Gastrodin-mediated hepatoprotective effect. CONCLUSIONS: Gastrodin attenuates steatohepatitis by activating the AMPK pathway and represents a therapeutic for the treatment of NASH.

Essential Role of CRIM1 on Endometrial Receptivity in Goat.

In domestic ruminants, endometrial receptivity is related to successful pregnancy and economic efficiency. Despite several molecules having been reported in the past regarding endometrial receptivity regulation, much regarding the mechanism of endometrial receptivity regulation remains unknown due to the complex nature of the trait. In this work, we demonstrated that the cysteine-rich transmembrane bone morphogenetic protein (BMP) regulator 1 (CRIM1) served as a novel regulator in the regulation of goat endometrial receptivity in vitro. Our results showed that hormones and IFN-τ increased the expression of CRIM1 in goat endometrial epithelial cells (EECs). Knockdown of CRIM1 via specific shRNA hindered cell proliferation, cell adhesion and prostaglandins (PGs) secretion and thus derailed normal endometrial receptivity. We further confirmed that receptivity defect phenotypes due to CRIM1 interference were restored by ATG7 overexpression in EECs while a loss of ATG7 further impaired receptivity phenotypes. Moreover, our results showed that changing the expression of ATG7 affected the reactive oxygen species (ROS) production. Moreover, mR-143-5p was shown to be a potential upstream factor of CRIM1-regulated endometrial receptivity in EECs. Overall, these results suggest that CRIM1, as the downstream target of miR-143-5p, has effects on ATG7-dependent autophagy, regulating cell proliferation, cell adhesion and PG secretion, and provides a new target for the diagnosis and treatment of early pregnancy failure and for improving the success rates of artificial reproduction.

Luman/CREB3 knock-down inhibit hCG induced MLTC-1 apoptosis.

Luman has been reported to be involved in the formation of COP II-mediated transport vesicles that affect protein transportation and secretion. Western blotting, immunohistochemistry, immunofluorescence, and RT-qPCR indicated that Luman is widely expressed in the male mouse reproductive system. In sperm, Luman was mainly located in the sperm tail, and the expression level increased with sperm maturity. In the testis, Luman was located in Leydig cells. In MLTC-1, a high-concentration hCG treatment significantly increased GRP78, ATF6, p-IRE1, and p-EIF2S1 expression but had no effect on Luman expression. To investigate the role of Luman in hCG-induced ER stress (ERS), experiments were conducted to examine the consequences of short hairpin RNA (shRNA)-mediated Luman knockdown in MLTC-1 cells. Luman knockdown decreased the percentage of S phase cells and up-regulated Cyclin A1, Cyclin B1, and Cyclin D2 expression. ELISA and WB results showed that with Luman knockdown, Cyp11a1, p-IRE1, and p-EIF2S1 expression and testosterone secretion were significantly increased, while GRP78 and CHOP expression were decreased. Flow cytometry results showed that Luman knockdown reduced MLTC-1 cell apoptosis. RT-qPCR and WB results showed that Luman knockdown significantly up-regulated BCL-2 expression and decreased Caspase-3 and BAX expression. These data suggest that Luman is widely expressed in the male mouse reproductive system. In MLTC-1 cells, Luman knockdown up-regulated p-IRE1, p-EIF2S1, and BCL-2 expression and decreased GRP78, CHOP, BAX, and Caspase-3 expression. We propose that Luman knockdown reduces cell apoptosis through the ERS pathway, thereby promoting cell survival and testosterone secretion. These findings provide new insights into the role of Luman in hCG-induced ERS.

BCL2L15 Depletion Inhibits Endometrial Receptivity via the STAT1 Signaling Pathway.

In domestic ruminants, endometrial receptivity is critical for a successful pregnancy and economic efficiency. Although the endometrium undergoes major cellular changes during peri-implantation, the precise mechanisms regulating goat endometrial receptivity remain unknown. In this study, we investigated the functional roles and signal transduction of the B-cell lymphoma 2 (Bcl-2)-like protein 15 (BCL2L15) in the regulation of endometrial receptivity in vitro. Our results showed that BCL2L15 was up-regulated in goat endometrial epithelial cells (EECs) under progesterone (P4), estradiol (E2), and interferon-tau (IFN-τ) treatments. Our knockdown of BCL2L15 by specific shRNA that significantly hampered endometrial receptivity. In the absence of BCL2L15, the signal transducer and activator of transcription (STAT)1 and STAT3 pathway were activated. Additionally, pretreatment with the STAT1 inhibitor, fludarabine, restored the effect of silencing BCL2L15 on the endometrial receptivity, but not the STAT3 inhibitor Stattic. Overall, these results suggested that BCL2L15 is the key regulator of endometrial receptivity in goats, regulating the endometrial receptivity through the STAT1 pathway. Understanding the function of BCL2L15-STAT1 in endometrial receptivity is important to the exploration of new targets for the diagnosis and treatment of early pregnancy failure, and improving the success rates for artificial reproduction.

Endoplasmic reticulum stress is involved in lipopolysaccharide-induced inflammatory response and apoptosis in goat endometrial stromal cells.

Endoplasmic reticulum (ER) stress is involved in regulating cell metabolism, apoptosis, autophagy, and survival. However, there is not enough information about the role of ER stress in lipopolysaccharide (LPS)-induced apoptosis and inflammatory cytokine secretion in the uterus. In this study, we found that LPS induced apoptosis and inflammation in goat endometrial stromal cells (ESCs). LPS treatment inhibited cell viability and cell proliferation. In addition, the genes associated with proliferation, such as proliferating cell nuclear antigen and MKI67, were affected by LPS treatment. Moreover, LPS increased the secretion of interleukin (IL)-1ß and IL-8, promoting the levels of MYD88, caspase1, and TRL4. The 4-phenylbutyric acid pretreatment inhibited the expression of unfolded protein response proteins and the secretion of inflammatory cytokines in LPS-treated cells. However, blockage of inositol-requiring enzyme 1 and activating transcription factor 6 did not significantly reduce apoptosis and inflammatory cytokine secretion. Collectively, ER stress involved in LPS-induced apoptosis and inflammatory cytokine increased in goat ESCs. This study provides new insight into the function of ER stress in the pathological process.

COPS5 negatively regulates goat endometrial function via the ERN1 and mTOR-autophagy pathways during early pregnancy.

In ruminant, adequate endometrial function is a major factor affecting implantation and economic efficiency. However, the precise mechanisms regulating goat endometrial function during the peri-implantation period of pregnancy are still unclear. Here, we investigated the functional role and signal transduction of the fifth component of the constitutive photomorphogenic-9 signalosome (COPS5) in the regulation of endometrial function in endometrial epithelial cells (EECs). Our results showed that hormones decreased COPS5 expression, and COPS5-mediated regulation of endometrial function. We also found that knockdown of COPS5 hindered EECs proliferation by the G1-phase cell cycle arrest. Hormones affected the activity of COPS5 through hormones receptors, while feedback from the expression of COPS5 regulated the transcription of the receptor. Moreover, knockdown of endoplasmic reticulum (ER) to nucleus signaling 1 (ERN1) via si-ERN1 partly inhibited endometrial function in shCOPS5 EECs. In addition, blocking the mTOR pathway by rapamycin promoted endometrial function in si-ERN1-transfected shCOPS5 EECs. Overall, these results suggest that COPS5 negatively regulates goat endometrial function via the ERN1 and mTOR-autophagy pathways and provide new insights into the mechanistic pathways of COPS5 during female reproductive development.

Inhibition of Luman/CREB3 expression leads to the upregulation of testosterone synthesis in mouse Leydig cells.

Luman, also known as cAMP-response element-binding protein 3, is an endoplasmic reticulum stress-related protein that has been identified as a novel transcriptional coregulator of a variety of nuclear receptors. Herein, immunohistochemistry results showed that Luman was specifically expressed in mouse Leydig cells in an age-dependent increase manner, from prepuberty to sexual maturation. Luman was not detected in Sertoli cells within the seminiferous tubules at any developmental period. The immunofluorescent experiment indicated that Luman was mainly located within the cytoplasm of murine Leydig tumor cells (MLTC-1) and primary Leydig cells (PLCs). To investigate the physiological function of Luman, experiments were conducted to examine the consequences of short hairpin RNA- and small interfering RNA-mediated Luman knock-down in MLTC-1 and PLCs, respectively. Luman knock-down significantly upregulated the expression of steroidogenic acute regulatory, cytochrome P450 cholesterol side-chain cleavage enzymes, 3ß-hydroxysteroid dehydrogenase, and 17-α-hydroxylase/C17-20 lyase in MLTC-1 cells and PLCs. Luman knock-down caused an increase in human chorionic gonadotropin-stimulated testosterone production in vitro and in vivo. The nuclear receptors SF-1 and Nur-77 were significantly increased upon Luman knock-down in MLTC-1. By contrast, the level of the nuclear receptor SHP decreased. Luciferase reporter assay results demonstrated that Luman knock-down upregulated the activity of SF-1 and Nur-77 promoters. These data suggested that Luman expressed in mouse Leydig cells in an age-dependent increase manner. Luman knock-down upregulated the activity of SF-1 and Nur-77 promoters, which lead to the increase of testosterone synthesis and steroidogenesis genes expression. In conclusion, these findings provide us with new insights into the role Luman played in male reproduction.

Interferon-τ regulates prostaglandin release in goat endometrial stromal cells via JAB1 - unfolded protein response pathway.

Prostaglandins (PGs) are major products of the uterine endometrium, and they are critical for recognition of pregnancy in ruminants. During the peri-implantation period of pregnancy, interferon tau (IFN-τ) plays an important role in the regulation of endometrial PGs synthesis, but the underlying mechanisms remain poorly understood. In this work, the results demonstrated that IFN-τ increased the PGE2/PGF2α ratio, up-regulated the expression of JAB1 and activated the unfolded protein response (UPR). Knockdown of JAB1 reduced the PGE2/PGF2α ratio and inhibited the expression of UPR markers in endometrial stromal cells (ESCs) under IFN-τ treatment. Pre-treatment with endoplasmic reticulum (ER) stress activator thapsigargin (Tg) activated UPR and restored the PGE2/PGF2α ratio in shJAB1 groups under IFN-τ treatment. In conclusion, our results indicated that IFN-τ regulated the PGE2/PGF2α ratio via cooperation between JAB1 and UPR, and the reduction of JAB1 led to the down-regulation of the PGE2/PGF2α ratio, which inhibits UPR, and thus is harmful to early pregnancy. Activation of UPR could restore JAB1 reduction, resulting in a reduced PGE2/PGF2α ratio. These findings extend our understanding and may provide new insights into the mechanism of IFN-τ regulation of PG secretion in ESCs and the biological functions of JAB1 and UPR.

CREB3 regulatory factor -mTOR-autophagy regulates goat endometrial function during early pregnancy.

In domestic ruminants, a receptive endometrium is crucial for successful pregnancy. Although many essential molecular modulators and pathways have been identified during early pregnancy, the precise mechanisms regulating goat endometrial function remains largely unknown. Here, we describe a novel regulator during early pregnancy, whereby hormones increased CREB3 regulatory factor (CREBRF) expression and act as a potential activator of autophagy in endometrial epithelial cells (EECs) via the mTOR pathway. Our results showed that knockdown of CREBRF via shCREBRF hampered EECs proliferation by S-phase cell cycle arrest and significantly inhibited endometrial function. We also reported that CREBRF-mTOR-autophagy pathway plays a vital role in regulating endometrial function, with a blockade of the mTOR by rapamycin demonstrating the regulatory function on prostaglandin (PGs) secretion and cell attachment in EECs. Moreover, chloroquine pretreatment also proved the above conclusion. Collectively, our findings provide new insight into the molecular mechanisms of goat endometrial function and indicate that the CREBRF-mTOR-autophagy pathway plays a central role in PGs secretion and cell attachment.

Hormone regulates endometrial function via cooperation of endoplasmic reticulum stress and mTOR-autophagy.

In ruminant, the receptive endometrium and the elongation of the hatched blastocyst are required to complete the process of implantation. However, the mechanisms regulating goat endometrial function during the peri-implantation period of pregnancy are still unclear. In this study, EECs were treated with progesterone, estradiol, and interferon-tau (IFNT). We have found that endoplasmic reticulum (ER) stress was activated under hormones treatment. To identify the cellular mechanism of regulation of endometrial function, we investigated the effect of ER stress activator thapsigargin (TG) and inhibitor 4 phenyl butyric acid (4-PBA) on EECs. We found that TG, which activated the three branches of UPR, increased the expression of genes associated with promoting conceptus elongation and cellular attachment, significantly up-regulated the spheroid attachment rate and PGE2 /PGF2α ratio. 4-PBA pre-treatment inhibited UPR and inhibited promoting conceptus elongation and cellular attachment related genes, but the spheroid attachment rate and PGE2 /PGF2α ratio were not changed significantly. Moreover, knockdown of ATF6 via shATF6 promoted the conceptus elongation related genes, but increased the dissolution of the corpus luteum. Besides, blocking ATF6 attenuated autophagy by activating mammalian target of rapamycin (mTOR) pathway. Moreover, rapamycin (mTOR inhibitor) pre-treatment inhibited the expression of promoting conceptus elongation and increased PGE2 /PGF2α ratio. Taken together, our study indicated that physiological level of ER stress may contribute to early pregnancy success, and ATF6 signaling pathway cooperated with autophagy to regulate endometrial function by modulating mTOR pathway.