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Blood-testis barrier (BTB) made of concomitant junction apparatus between Sertoli cells (SCs) is crucial for spermatogenesis. The tight junction (TJ) function is impaired in SCs with age, exhibiting an intimate relationship to testicular dysfunction induced by age. In this study, compared with those in young boars, TJ proteins (i.e., Occludin, ZO-1, and plus Claudin-11) were discovered to have reduced expressions in testes, and spermatogenesis ability declined in old boars. An in vitro age model for D-gal-treated porcine SCs was established, the performance of Curcumin as a natural antioxidant and anti-inflammatory compound in affecting the TJ function of SCs was appraised, and related molecular mechanisms were exploited. The results manifested that 40 g/L D-gal downregulated ZO-1, Claudin-11, and Occludin in terms of the expression in SCs, whereas Curcumin restored such expressions in D-gal-treated SCs. Using the AMPK and SIRT3 inhibiters demonstrated that activation of the AMPK/SIRT3 pathway was associated with Curcumin, which not only rescued the expression of ZO-1, Occludin, Claudin-11, and SOD2 but also inhibited the production of mtROS and ROS and the activation of NLRP3 inflammasome and release of IL-1β in D-gal-treated SCs. Furthermore, with mtROS scavenger (mito-TEMPO), NLRP3 inhibitor (MCC950) plus IL-1Ra treatment ameliorated D-gal-caused TJ protein decline in SCs. In vivo data also showed that Curcumin alleviated TJ impairment in murine testes, improved D-gal-triggered spermatogenesis ability, and inactivated the NLRP3 inflammasome by virtue of the AMPK/SIRT3/mtROS/SOD2 signal transduction pathway. Given the above findings, a novel mechanism where Curcumin modulates BTB function to improve spermatogenesis ability in age-related male reproductive disorder is characterized.

Endometritis is a common disease affecting fertility in cows during the perinatal period, which disturbs the molecular milieu of the uterine environment and impairs embryo development and implantation. Exosomes are important extracellular components that transmit a variety of micro RNAs (miRNAs), which perform key regulatory functions. In this study, we investigated plasma exosomal miRNAs from cows with endometritis and from cultured endometrial epithelial cells (EECs) challenged with lipopolysaccharide (LPS) to explore the role of EEC-derived exosomes and their miRNAs in bovine endometritis. Plasma exosomes were collected from nine healthy dairy cows and nine dairy cows with endometritis, and culture supernatant exosomes were isolated from EECs challenged with or without LPS. Exosomal RNA was extracted using commercial kits and miRNA profiles were generated using RNA-seq. We found that miR-218 was differentially expressed in EECs under conditions of endometrial inflammation. Inhibition studies suggested that reduced levels of miR-218 in EEC-derived exosomes when transferred into placental trophoblast cells impaired embryonic development and decreased placental trophoblast cell migration by targeting secreted frizzled related protein 2. We propose that exosomal miR-218 secreted from EECs acts as a driver of embryonic development and differentiation. In addition, exosomal miR-218 may provide a valuable diagnostic marker for bovine endometritis.

The objective of this study was to investigate differences in mRNA expression between fresh and frozen–thawed sperm in roosters. In trial 1, gene expression profiles were measured using microarray with Affymetrix GeneChip Chicken Genome Arrays. The results showed that 2,115 genes were differentially expressed between the 2 groups. Among these genes, 2,086 were significantly downregulated and 29 were significantly upregulated in the frozen–thawed sperm group. Gene Ontology (GO) analysis showed that more than 1,000 differentially expressed genes (DEG) of all significantly regulated genes were involved in GO terms including biological processes, molecular function, and cellular component. Kyoto Encyclopedia of Genes and Genomes analysis showed that DEG were significantly (P

Cryptorchidism, as a common congenital disease of canine testes, is mainly caused by factors leading to endocrine abnormalities in testes and infertility in a heat stress and hypoxia microenvironment. Moreover, heat stress and hypoxia, as critical microenvironmental factors, promote epithelial-mesenchymal transition (EMT), which occurs during adult tissue remodelling responses including carcinogenesis and fibrosis and is the main cause of testicular tumours. In this study, we found by haematoxylin-eosin staining that the canine cryptorchid tissue produced a lot of collagen fibres. Also, the quantitative PCR and Western blot results showed that the mRNA and protein levels of the heat stress makers HSP70 and HO-1 and the hypoxia maker HIF-1α are significant higher compared with normal testes. Moreover, we found the expression levels of TGF-βs and its two receptors TGF-βRI and TGF-βRII increased in case of cryptorchidism. From the study in vitro, we found both heat stress and COCl

Background Natural astaxanthin (ASTA) has strong antioxidant properties and has been widely used as a health product to improve human health. However, the effects of ASTA on the reproductive performance of aging roosters have been poorly studied. We aimed to investigate the effects of dietary ASTA on semen quality and antioxidant capacity in aging roosters and to explore the potential mechanism of semen quality change via anti-oxidation defense system. Methods In the present study, 96 53-week-old Jinghong No. 1 layer breeder roosters were fed a corn-soybean meal basal diet containing 0, 25, 50, or 100 mg/kg ASTA for 6 weeks. Results Semen quality in the ASTA groups remarkably improved than that in the control group, and antioxidant activities, the abilities to scavenge hydroxyl radicals and superoxide anions, increased gradually with ASTA addition (P P Conclusions Collectively, these results demonstrate that dietary ASTA may improve semen quality by increasing antioxidant enzyme activities and the ability to scavenge hydroxyl radicals, which may be related to upregulation of the MAPK/Nrf2 pathway.

Description, Evolutionary analyses reveal conservation and divergence of epigenetic transition during early development across mammals., While mouse remains the most popular model, the conservation of parental-to-embryonic epigenetic transition across mammals is poorly defined. Through analysis of oocytes and early embryos in human, bovine, porcine, rat, and mouse, we revealed remarkable species-specific innovations as no single animal model fully recapitulates the human epigenetic transition. In rodent oocytes, transcription-dependent DNA methylation allows methylation of maternal imprints but not intergenic paternal imprints. Unexpectedly, prevalent DNA hypermethylation, paralleled by H3K36me2/3, also occurs in nontranscribed regions in porcine and bovine oocytes, except for megabase-long “CpG continents (CGCs)” where imprinting control regions preferentially reside. Broad H3K4me3 and H3K27me3 domains exist in nonhuman oocytes, yet only rodent H3K27me3 survives beyond genome activation. Coincidently, regulatory elements preferentially evade H3K27me3 in rodent oocytes, and failure to do so causes aberrant embryonic gene repression. Hence, the diverse mammalian innovations of parental-to-embryonic transition center on a delicate “to-methylate-or-not” balance in establishing imprints while protecting other regulatory regions.

Vascular endothelial growth factor (VEGF) plays a pivotal role in angiogenesis in ovaries, particularly during follicular development and ovulation. Interleukin-6 (IL-6) is one of the major pro-inflammatory factors that are involved in the angiogenesis process physiologically and pathologically. Previous studies have shown that 17β-estradiol (E2) stimulates VEGF expression by upregulating hypoxia-inducible factor 1α (HIF-1α) in many cell types, and the high level of E2 causes an inflammatory-like microenvironment before ovulation. However, whether IL-6 signaling is involved in E2-regulating VEGF expression in swine granulosa cells (GCs) is still unknown. In this study, we found the estrogen membrane receptor, G-protein-coupled estrogen receptor 1 (GPER), was expressed in swine GCs, and the expression level of GPER, HIF-1α, and VEGF increased with follicular development. In vitro study showed that E2, ICI182780, and GPER agonist (G1) promoted the expressions of HIF-1α and VEGF in swine GCs, while GPER antagonist (G15) inhibited the stimulating effect of E2 and G1. Meanwhile, G15 inhibited the stimulating effect of E2 and G1 on IL-6 mRNA expression and secretion. Furthermore, IL-6 antibody and AG490 (JAK2/STAT3 inhibitor) attenuated G1-induced HIF-1α and VEGF expression. In conclusion, this study revealed how estrogen-induced HIF-1α and VEGF expressions in swine GCs are mediated through GPER-derived IL-6 secretion leading to JAK2/STAT3 activation.

The hypoxic microenvironment of cryptorchidism is an important factor to induce the impairment of the structure and function of Sertoli cells and thus lead to spermatogenesis loss or tumorigenesis. Dihydrotestosterone (DHT), as a potent nonaromatizable 5α-reduced androgen, has both positive and negative effect on pathological fibrosis process. However, it is still unknown whether DHT can regulate hypoxia-induced fibrosis of Sertoli cells. Herein, in this study, we evaluate the DHT level, two 5α-reductase isoforms, 5α-red1 and 5α-red2, as well as HIF-1α expression pattern in canine cryptorchidism and contralateral normal testis. Results showed that the abdominal testes presented low DHT levels and 5α-red1 and 5α-red2 expression, while significantly higher HIF-1α expression and ECM production compared with the scrotum. Moreover, we established a hypoxia-induced fibrosis model in canine Sertoli cells induced by cobalt chloride (CoCl

In vitro fertilization (IVF) for ovine embryos is strongly associated with low pregnancy rates and negative effects on embryonic and postnatal development, but the mechanisms remain unclear. We aimed to identify genes essential for successful implantation of IVF embryos in sheep by species-specific microarray analysis. Differential gene expression analysis between caruncles and intercaruncle sites of the ovine uterus with in vivo (IVV) -produced embryos on day 10 of pregnancy found 43 differentially expressed genes (DEGs) with at least a 2-fold change, such as coiled-coil domain-containing 152 (CCDC152) and pregnancy-associated plasma protein A (PAPPA). Gene ontology analysis revealed these DEGs were related to cell proliferation, the immune system process, localization and response to stimulus. Moreover, we identified 93 DEGs in endometrial caruncles with IVV- versus in vitro (IVT)-produced embryos on days 12, 14 and 16 of pregnancy, including prostaglandin-endoperoxide synthase 2 (PTGS2) and cubilin (CUBN). GO analysis revealed that these DEGs were related to cell adhesion, cell proliferation, embryo implantation, embryonic morphogenesis, the immune system process and localization. Two DEGs (PAPPA and RSAD2) in caruncle versus intercaruncle sites at day 10 were also differentially expressed between the IVV and IVT groups at the three implantation stages. Self-organizing feature map cluster analysis of transcript profiles during embryo implantation revealed different gene expression patterns between the IVV and IVT groups. In conclusion, this study identified many genes that may be associated with mechanisms underlying IVF-induced ovine embryo implantation failure during the peri-implantation period, and such genes provide potential candidates for further study.

Fat deposition is very important in pig production, and its mechanism is not clearly understood. MicroRNAs (miRNAs) play critical roles in fat deposition and energy metabolism. In the current study, we investigated the mRNA and miRNA transcriptome in the livers of Landrace pigs with extreme backfat thickness to explore miRNA-mRNA regulatory networks related to lipid deposition and metabolism. A comparative analysis of liver mRNA and miRNA transcriptomes from pigs (four pigs per group) with extreme backfat thickness was performed. We identified differentially expressed genes from RNA-seq data using a Cufflinks pipeline. Seventy-one differentially expressed genes (DEGs), including twenty-eight well annotated on the porcine reference genome genes, were found. The upregulation genes in pigs with higher backfat thickness were mainly involved in fatty acid synthesis, and included fatty acid synthase (FASN), glucokinase (GCK), phosphoglycerate dehydrogenase (PHGDH), and apolipoprotein A4 (APOA4). Cytochrome P450, family 2, subfamily J, polypeptide 34 (CYP2J34) was lower expressed in pigs with high backfat thickness, and is involved in the oxidation of arachidonic acid. Moreover, 13 differentially expressed miRNAs were identified. Seven miRNAs were associated with fatty acid synthesis, lipid metabolism, and adipogenic differentiation. Based on comprehensive analysis of the transcriptome of both mRNAs and miRNAs, an important regulatory network, in which six DEGs could be regulated by differentially expressed miRNAs, was established for fat deposition. The negative correlate in the regulatory network including, miR-545-5p and GRAMD3, miR-338 and FASN, and miR-127, miR-146b, miR-34c, miR-144 and THBS1 indicate that direct suppressive regulation may be involved in lipid deposition and energy metabolism. Based on liver mRNA and miRNA transcriptomes from pigs with extreme backfat thickness, we identified 28 differentially expressed genes and 13 differentially expressed miRNAs, and established an important miRNA-mRNA regulatory network. This study provides new insights into the molecular mechanisms that determine fat deposition in pigs.

Fatness traits are important in pigs because of their implications for fattening efficiency, meat quality, reproductive performance and immunity. Songliao black pigs and Landrace pigs show important differences in production and meat quality traits, including fatness and muscle growth. Therefore, we used a high-throughput massively parallel RNA-seq approach to identify genes differentially expressed in backfat tissue between these two breeds (six pigs in each). An average of 37.87 million reads were obtained from the 12 samples. After statistical analysis of gene expression data by edgeR, a total of 877 differentially expressed genes were detected between the two pig breeds, 205 with higher expression and 672 with lower expression in Songliao pigs. Candidate genes (LCN2, CES3, DGKB, OLR1, LEP, PGM1, PCK1, ACACB, FADS1, FADS2, MOGAT2, SREBF1, PPARGC1B) with known effects on fatness traits were included among the DEGs. A total of 1071 lncRNAs were identified, and 85 of these lncRNAs were differentially expressed, including 53 up-regulated and 32 down-regulated lncRNAs, respectively. The differentially expressed genes and lncRNAs involved in glucagon signaling pathway, glycolysis/gluconeogenesis, insulin signaling pathway, MAPK signaling pathway and so on. Integrated analysis potential trans-regulating or cis-regulating relation between DEGs and DE lncRNAs, suggested lncRNA MSTRG.2479.1 might regulate the expressed level of VLDLR affecting porcine fat metabolism. These results provide a number of candidate genes and lncRNAs potentially involved in porcine fat deposition and provide a basis for future research on the molecular mechanisms underlying in fat deposition.

Omega-3/n-3 polyunsaturated fatty acids (ω-3/n-3 PUFAs) play an important role in male reproductive function. The present study was designed to evaluate the effects of linseed oil (LO) as a source of α-linolenic acid (ALA, n-3 PUFA) on semen quality, plasma reproductive hormone and expression of key enzyme and protein related to steroidogenesis in aging layer breeder roosters. Ninety-six 57-wk-old Nongda No.3 layer breeder roosters were randomly assigned into one of four dietary treatments. All birds were fed a basal diet for 1wk and then assigned to a corn-soybean meal-based diet containing 0, 1, 2, 4% LO for 4 wk. After feeding trial, the roosters were slaughtered and investigated. The results showed that semen volume was dramatically increased relative to the other treatments in 2% LO group (P 0.05). With an increase in dietary LO, semen concentration, sperm viability, sperm motility and total sperm count increased linearly (P 0.05). Plasma follicle-stimulating hormone (FSH) level increased gradually and reached a maximum when 4% LO was fed (P 0.01). Plasma luteinizing hormone (LH) levels in 1% and 2% LO group were improved significantly (P 0.05) relative to the control group. Plasma testosterone (T) levels were remarkably improved compared with the control when birds were fed 2% and 4% LO (P 0.05). A significant increase of steroidogenic acute regulatory protein (StAR) mRNA expression in 2% and 4% LO group was observed relative to the control group (P 0.05). An increase in dietary LO supplementation from 1% to 4% markedly enhanced (P 0.05) the mRNA expression of cholesterol side-chain cleavage enzyme (P450scc) compared to the control. A significant increase (P 0.05) in the Steroidogenic Factor 1 (SF-1) mRNA levels was observed in the 2% and 4% LO-added groups. SF-1 protein expression was markedly increased by adding LO in diets (P 0.05), and reached a maximum in 2% LO group. In conclusion, the results above suggest that dietary LO may improve semen quality by increasing the T hormone secretion, which may be related to higher StAR and P450scc mRNA expression and SF-1 expression. These findings provide a potential for using LO to attenuate the age-related sub-fertility in commercial layer breeder roosters.

Background: Interferon-tau (IFNτ), as an antiluteolytic factor secreted by trophoderm during the pregnancy of ruminants, actually functions by activating the IFNτ receptor 1 (IFNAR1) and IFNτ receptor 2 (IFNAR2). However, it has not been clearly understood how IFNτ-IFNAR cascade regulation processes between the embryo and uterine epithelial cells in ruminants. Methods: CRISPR/Cas9 technology was used to compare the differences of IFNτ secretion in In vitro fertilization(IVF), parthenogenetic activation(PA) and somatic cell nuclear transfer (SCNT) and their in vitro co-culture effect with uterine endometrial epithelial cell with those in vivo ones in this study.Results: In this study, we found the expression and location of IFNτ in the blastocysts from different sources. IFNτ, IFNAR1 and IFNAR2 were all located in the trophoblast cells of the blastocyst. However, the fluorescence intensity of IFNAR1 was consistent with that of IFNτ. Antagonizing the expressions of IFNAR1 and IFNAR2 in embryos and co-culture with EEC reduced the expressions of Integrin αv β3, WNT7A, and ISG15 in EECs. Knocking out IFNAR1 and IFNAR2 reduce the expressions of Integrin αv β3 and WNT7A in EECs, the deletion of IFNAR2 gene has a greater impact than that of IFNAR1 gene. IFNAR1-/IFNAR2+ and IFNAR1+/IFNAR2- EECs were co-cultured with IVF embryos, the expression of Integrin αv β3 was inhibited, and the inhibition of IFNAR1+/IFNAR2- was much stronger, and the expression of WNT7A was not inhibited. The expressions of Integrin αv β3 and WNT7A did not change significantly after IFNAR1-/IFNAR2+ and IFNAR1+/IFNAR2- co-culture with PA embryos. Conclusions: All of these results strongly suggest that specific activation of embryonic IFNAR1 and endometrial IFNAR2 induced by embryonic IFNτ directs normal uterine preparation for bovine early implantation.

Background Inadequate feto-maternal interaction will directly lead to the failures of pregnancy and bring serious damage to dairy cows. Exosomes are widely involved in endometrial matrix remodeling, immune function changes, placental development, and other processes of embryo implantation and pregnancy of dairy cows. However, the role of placental trophoblast cells derived exosomes is still unclear in regulating the receptivity of endometrial cells and facilitating the interaction between mother and fetus. Methods In this study, bovine trophoblast cells (BTCs) were obtained from bovine placenta and immortalized through the transfection of telomerase reverse transcriptase (TERT) gene. After that, the effect of trophoblast derived exosomes (TDEs) on endometrial receptivity in endometrial epithelial cells (EECs) was detected and the mechanism explored that TDEs and their proteins participated in feto-maternal interaction during bovine pregnancy. EECs were co-cultured with the exosomes derived from progesterone (P4) and treated with BTCs. Results Immortalized BTCs still possessed the basic and key properties of primary BTCs without showing a neoplastic transformation sign. Exosomes derived from P4 and treated with BTCs enhanced the expression of endometrial receptivity factors in EECs by changing the extracellular environment, metabolism and redox balance in EECs with proteome alignment, compared with those untreated according to the DIA quantitation analysis. Conclusions Our study found that trophoblast derived exosomal proteins are one of the most critical elements in feto-maternal interaction and their changes act as a key signal in altering endometrial receptivity and provided a potential target for improving fertility.

The objective of this study was to evaluate the effects of natural astaxanthin (ASTA) from Haematococcus pluvialis on production performance, egg quality, antioxidant enzyme activity, free radical scavenging ability, and gene expression of antioxidant enzymes in laying hens. Nongda No. 3 laying hens (n = 450) were randomly allocated to 1 of 5 dietary treatments. Each treatment had 6 replicates of 15 hens each. All birds were assigned to a corn-soybean meal-based diet containing 0, 20, 40, 80, or 160 mg/kg ASTA for 4 wk. With increasing dietary ASTA, no significant effects were observed on egg weight, feed consumption, feed efficiency, laying rate, Haugh unit, or eggshell strength. Yolk color darkened linearly with increasing dose of ASTA (P

Background: Natural astaxanthin (ASTA) has strong antioxidant properties and has been widely used as a health product to improve human health. However, the effects of ASTA on the reproductive performance of aging roosters have been poorly studied. We aimed to investigate the effects of dietary ASTA on semen quality and antioxidant capacity in aging roosters and to explore the potential mechanism of semen quality change via anti-oxidation defense system.Methods: In the present study, 96 53-week-old Jinghong No. 1 layer breeder roosters were fed a corn-soybean meal basal diet containing ASTA at 0, 25, 50, or 100 mg/kg for 6 week. Results: Semen quality in the ASTA groups remarkably improved as compared to those in the control group and antioxidant activities, the abilities to scavenge hydroxyl radicals and superoxide anions increased gradually with ASTA addition (P < 0.05). In addition, the mRNA levels of antioxidant enzymes, the mRNA and protein levels of the mitogen-activated protein kinase (MAPK), nuclear factor-erythroid 2-related factor 2 (Nrf2) were markedly increased in the 50-100 mg/kg ASTA group (P < 0.05). Conclusions: Collectively, these results demonstrate that dietary ASTA may improve semen quality by increasing antioxidant enzyme activities, and the ability to scavenge hydroxyl radicals, which may be related to up-regulation of the MAPK/Nrf2 pathway.

Skeletal muscle development is a complex biological process involving multiple key genes, signaling pathways and noncoding RNAs, including microRNAs and circular RNAs (circRNAs). However, the regulatory relationship among them is so complicated that it has not yet been fully elucidated. In this study, we found that miR-7 inhibited C2C12 cell proliferation and differentiation by targeting transcription factor 12 (TCF12). circHIPK3 acted as a competing endogenous RNA, and its overexpression effectively reversed the regulation of miR-7 on C2C12 cell proliferation and differentiation by increasing TCF12 expression. Taken together, our findings provide evidence that circHIPK3 regulates skeletal muscle development through the miR-7/TCF12 pathway. This study provides a scientific basis for further research on skeletal muscle development at the circRNA level.

The objective of this study was to evaluate the effects of different levels of dietary natural astaxanthin (ASTA) (from the microalga Haematococcus pluvialis) and storage at 4°C and 25°C on the quality of eggs from laying hens. Nongda No. 3 laying hens (n = 450) were randomly allocated to 1 of 5 dietary treatments. Each treatment had 6 replicates of 15 hens each. All birds were assigned to a corn-soybean meal-based diet containing 0, 20, 40, 80, or 160 mg/kg natural ASTA for 4 wk. A total of 540 eggs were collected at the end of the 4-week feeding trial. Sixty fresh eggs were collected and measured for egg quality within 24 h after collection. The other 480 eggs were used in a factorial arrangement with 5 dietary ASTA levels, 4 storage times, and 2 storage temperatures. During the 8-week storage period at 4°C and 25°C, egg quality measurements were performed every 2 wk on 12 eggs per treatment. No significant effects (P > 0.05) on yolk index, yolk pH, Haugh units, weight loss, or eggshell strength were observed with increasing concentrations of dietary ASTA. Yolk color darkened linearly with increasing dose of ASTA (P < 0.05). During storage of eggs, yolk index and Haugh units decreased significantly (P < 0.05), whereas yolk pH and weight loss increased (P < 0.05). An interaction was observed between dietary ASTA level and storage time on yolk index, yolk color, and Haugh units (P < 0.05). These results demonstrated that dietary ASTA from H. pluvialis delayed the decrease in yolk index and yolk color during storage at 4°C and 25°C. Therefore, we speculate that there may be a combined effect of dietary ASTA level and storage time on egg internal quality; this information may provide additional options by which to extend the storage time of eggs.

The cryopreservation of mammalian embryos is an important technology in embryo engineering. The discovery and application of the embryo's own high freezing resistance factors are the main methods to improve the utilization of mammalian embryos in cryopreservation. Cathepsin L gene expression in the frozen and thawed dormant embryos displayed a significant difference from those normal hatched ones. The aim of the present study was to dig out the potential role of Cathepsin L in anti-freezing capacity of murine blastocysts by investigating the location and expression of Cathepsin L in frozen and thawed both activated and dormant hatching blastocysts. Different concentrations of Cathepsin L recombinant protein and E-64d were then respectively added into the embryo cryoprotectant and pre-cryo culture medium. Our results found that down-regulation of Cathepsin L improves the freezing resistance of murine normal hatching embryos by reducing apoptosis. Cathepsin L inhibitors can be used to improve the efficiency of cryopreservation and recovery of blastocysts in vitro. Our study provides a theoretical basis for the further development and application of Cathepsin L.

Uterine prostaglandins F2α (PGF2α) is essential for implantation, initiation of luteolysis and delivery. Previous studies have demonstrated that the expression of Cyclooxygenase-2 (COX-2), an enzyme limiting PGF2α rate, is regulated by steroid hormones, and also dihydrotestosterone (DHT) may be involved in regulating COX-2 expression both positively and negatively. However, it remains unclear how whether DHT regulates COX-2 expression and consequent PGF2α release in bovine endometrial epithelial cells (EECs). 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 bovine endometria by immunohistochemistry, and investigated 5α-red1, 5α-red2, AR, and DHT levels at the different stages of endometria (follicle, early-, mid-, and late-pregnancy phases). The results showed that 5α-red1, 5α-red2 and AR all were expressed in endometria, and their expressions and the level of DHT significantly increased in the late-pregnancy phase compared with the mid-pregnancy phase. Moreover, we cultured EECs from the mid-pregnancy phase and the in vitro study showed that DHT dose-dependently increased COX-2 expression and PGF2a release, but AR antagonist (flutamide) inhibited the stimulating effect via DHT. In addition, the DHT-induced COX-2 expression and PGF2α release were subjected to the regulation of both EGFR/PI3K/Akt/NFkB signaling as the inhibitors of EGFR (AG1478) and PI3K/Akt (LY294002) and NFkB (QNZ) attenuated the DHT mediated effect. Taken together, the results demonstrated that DHT-induced COX-2 expression and consequent PGF2α release in bovine EECs were mediated through AR-derived EGFR transactivation and PI3K/Akt cascade leading to NFkB activation.

The reproductive efficiency of roosters is an important trait in poultry production; however, the molecular mechanisms underlying this trait are not clearly understood. Here, we compared the mRNA and microRNA (miRNA) transcriptomes of testis from roosters with divergent sperm motility. A total of 302 differentially expressed genes (DEGs), including 182 upregulated genes and 120 downregulated genes, were identified in high sperm motility groups compared with low sperm motility groups. A subset of these DEGs related to steroid hormone biosynthesis and thus could be important for spermatogenesis. Additionally, we detected 13 differentially expressed miRNAs (DEMs) between two groups, and target gene prediction indicated seven of these could be associated with spermatogenesis. Based on a comprehensive analysis of these transcriptomes, miRNA-mRNA interaction networks were constructed. Six DEGs were predicted to be regulated by DEMs. Subsequently, we validated the negative regulation of family with sequence similarity 84, member A (FAM84A) by miR-215 using a dual-luciferase reporter system. These results provide new insights into the molecular profile of the testis and identify genes that may determine sperm motility in chickens.

Summary As an inflammation of the endometrium, endometritis can affect fertility and lead to serious economic losses in the dairy industry. Widely found in various tissues and body fluids, exosomes and exosome micro (mi)RNAs have been shown to play an important regulatory role in the immune responses. As one of differentially expressed exosome miRNAs, miR‐218 is involved in the pathogenesis of bovine endometritis. The mechanisms of miR‐218 in regulating the release of cytokines and chemokines in endometritis, however, are poorly understood. Exosomes were isolated from bovine uterine cavity fluid and verified by transmission electron microscopy. An in vitro lipopolysaccharide‐treated cell model for bovine endometritis was then established to evaluate the correlation between exosome‐derived miR‐218 and the immune responses. We demonstrated that exosomes could be used to deliver miR‐218 from endometrial epithelial cells (EECs) into the uterine microenvironment and adjacent recipient cells to modulate local immune responses. miR‐218 packaged in the exosomes secreted from EECs acts as an inhibitor by blocking immune factors such as interleukin (IL)‐6, IL‐1β, tumour necrosis factor‐α, the chemokines macrophage inflammatory genes (MIP)‐1α and MIP‐1β to maintain the immune balance in the uterus. However, uterine inflammation altered the immunoregulatory mechanism of exosome miR‐218. MiR‐218 is a potential biomarker for the detection of endometritis. Our findings also revealed a new mechanism for the development of endometritis in cows., Exosomes could be used to deliver miR‐218 from endometrial epithelial cells (EECs) into uterine microenvironment and adjacent recipient cells to modulate local immune responses. MiR‐218 packaged in exosomes secreted from EECs acts as an inflammatory inhibitor by blocking immune factors and chemokines. MiR‐218 is a potential biomarker for monitoring the pathogenesis of bovine endometritis.

As a common disease of cows occurring during their perinatal period, endometritis is known to affect fertility. At present, the studies on endometritis mainly focus on preventing microbial invasion. However, the mechanism that uterine inflammation affects embryo activity and implantation is unclear. Mainly containing lipids, proteins, mRNAs, and microRNAs, exosomes widely exist in various tissues and body fluids. Exosome extractions were used by commercial kits and confirmed through morphological examinations and Western blot. After exosomes’ mRNA profiles were generated using RNA sequencing, it was investigated how uterine cavity fluid exosomes affect the developmental competence of in vitro fertilization (IVF) embryos in case of endometritis. In this study, the isolated exosomes were spherical particles with a diameter of 30–150 nm according to the transmission electron microscopy. Identified with Western blotting, positive CD63 and CD9 expressions showed that the isolated exosomes could be used for the subsequent tests. We found 118 differentially expressed miRNAs in the exosomes of the uterine cavity fluid in healthy cows and those with endometritis, among which, 52 miRNAs were down regulated and 66 up regulated. Furthermore, the qRT-PCR results confirmed the up-regulation of three miRNAs and down-regulation of six miRNAs, which were consistent with the deep sequencing results. IVF embryos co-incubated with the endometritis exosomes significantly decreased the blastocyst formation rate in comparison with those co-incubated with the healthy exosomes (21.84 + 3.17 vs. 32.37 + 2.69). Therefore, exosome miRNAs may be a cause of infertility in cows with endometritis.

G-protein-coupled receptor 39 (GPR39) plays a role in cellular and physiological processes, including insulin secretion, cell death inhibition, wound healing, and obesity. Increasing evidence suggests that GPR39 is potently stimulated by zinc ions (Zn2+) and is therefore considered a putative Zn2+ receptor. Given the importance of Zn2+ in the reproductive system, we proposed that GPR39 might have a functional role in the reproductive system. However, the localization of GPR39 in the reproductive system remains unknown. Here, we used mice expressing a Gpr39 promoter-driven LacZ reporter system to detect Gpr39 expression in the reproductive system at different phases of the estrous cycle and found an interesting region-specific distribution of Gpr39 in the mouse oviduct epithelium, with strong expression at the ampulla and weak expression at the isthmus, which was consistent with the results using reverse transcription polymerase chain reaction and immunofluorescence. Moreover, using ZnSeAMG staining, we found that Zn2+, the putative ligand of GPR39, also found a distribution similar to GPR39 expression, suggesting that their potential interaction mediates fertilization and embryo transportation.

The objective of this study consisting of 2 trials was to investigate the antioxidant role of conjugated linoleic acid (CLA) isomers (c9, t11-CLA and t10, c12-CLA) and the underlying mechanism by which they act in modulating redox status in a primary laying hen hepatocyte culture. In trial 1, the cytotoxicity of CLA isomers or linoleic acid (LA) (0, 25, 50, 100, 200, 400, 800 μmol/L) was evaluated by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay. The concentration of CLA isomers or LA (25, 50, 100 μmol/L) for proper antioxidant activity was evaluated by measuring the antioxidant enzyme activity. In trial 2, there were 5 groups: control group, cells were untreated; H2O2 group, cells were exposed to 4 mmol/L H2O2 for 2 h; c9, t11 or t10, c12 or LA group, cells were treated with c9, t11-CLA or t10, c12-CLA or LA for 24 h and then exposed to 4 mmol/L H2O2 for 2 h. Trial 1 showed that the non-toxic dose range for CLA isomers was 0 to 200 μmol/L. The optimum concentration of c9, t11-CLA and t10, c12-CLA for trial 2 was 100 μmol/L. In trial 2, pretreatment with t10, c12-CLA but not c9, t11-CLA attenuated the increase in reactive oxygen species (ROS) compared to hydrogen peroxide (H2O2) group (P < 0.05). t10, c12-CLA elevated the superoxide dismutase (SOD) and catalase (CAT) activities compared with the H2O2 group (P < 0.05). In addition, t10, c12-CLA up-regulated the mRNA expression of nuclear factor E2-related factor-2 (Nrf2) as well as its target genes, Cu-Zn superoxide dismutase (SOD1) and CAT (P < 0.05). Pretreatment with t10, c12-CLA but not c9, t11-CLA decreased Nrf2 protein expression in the cytoplasm and increased Nrf2 protein expression in the nucleus compared with the H2O2 group (P < 0.05). The results indicate that t10, c12-CLA exhibits a stronger antioxidant capacity than c9, t11-CLA in primary cultured laying hen hepatocytes. t10, c12-CLA increases the activity and mRNA expression of antioxidant enzymes via facilitating nuclear translocation of Nrf2.

Mucin 1 (MUC1), a cell surface glycoprotein, is expressed mainly in the endometrial luminal epithelium (LE) and glandular epithelium (GE) of the endometrium in many mammalian species including mice, rats, pigs, sheep, horses and humans during various stages of a menstrual or oestrous cycle, where it plays an important role in embryo implantation and placentation. However, the expression and function of MUC1 in uterine tissues during early pregnancy in sheep after artificially-induced oestrous is not known. Therefore, we investigated the expression and function of MUC1 in the early pregnant and non-pregnant uterine tissues of sheep with natural oestrous or artificially-induced oestrous on days 10, 12, 14, 16 and 18 of the cycle by in situ hybridization, quantitative real-time polymerase chain reaction, immunohistochemical staining and western blotting methods. According to our results, MUC1 mRNA and protein expression increased initially but then decreased from days 10-18, peaking on day 14 in the uterine tissues of non-pregnant ewes after both natural and artificially-induced oestrous. MUC1 protein localisation was observed in the LE on days 10, 12 and 14 and in the GE on days 16 and 18. In contrast, MUC1 mRNA and protein expression increased on days 10 and 12, decreased on day 14, but increased again on days 16 and 18 in the uterine tissues of pregnant ewes both in natural oestrous and in artificially-induced oestrous. Additionally, the MUC1 mRNA and protein expression levels in the uterine tissues of the early pregnant sheep were significantly lower than those in the non-pregnant sheep on days 10, 14, and 18, except on day 16(P 0.01). Enhancing MUC1 protein expression with oestrogen or/and progesterone decreased the blastocyst adhesion rate when blastocysts were co-cultured with endometrial epithelial cells (EECs), while inhibiting MUC1 protein expression with IFN-τ increased the blastocyst adhesion rate when the blastocysts were co-cultured with EECs. Compared with the ewes undergoing natural oestrus, the expression trend and regulation of MUC1 did not change, and the MUC1 expression levels only increased under artificial oestrus conditions. Our data provide important information for improving the conception rate in sheep undergoing artificially-induced oestrus and offer some reference points relating to embryo transfer, oestrus synchronisation and superovulation.

It is well established that trophoblasts play a crucial role in pregnancy establishment and maintenance through production of various biological substances. In this regard, Wnt signaling is an important regulator of embryo implantation and placentation in various species. However, the role of the Wnt signaling pathway during bovine placental development has remained largely unknown. Employing multiple approaches, we herein found that Wnt2 mRNA was more abundant in cotyledon tissues compared with caruncle tissues, whereas Wnt5b mRNA was more abundant in caruncle tissues compared with cotyledon tissues. Moreover, the Wnt receptor Fzd4 was detected in caruncle epithelial cells and binucleate trophoblasts, but not in uninucleate trophoblasts. In addition, β-catenin, an integral cell-cell adhesion adaptor protein as well as transcriptional co-regulator of Wnt canonical pathway, was spatiotemporally expressed in bovine trophoblasts, with high levels of cellular accumulation and nuclear translocation, particularly in binucleate trophoblasts. Lymphoid enhancer factor-1 mRNA was more abundant in caruncle tissues compared with cotyledon tissues, which was well correlated with the expression profile of Dickkopf-1, a secreted antagonist of the canonical Wnt signaling pathway. These results provided new evidence that precisely regulated canonical Wnt activation may have a very important physiological role during fetal-maternal recognition and pregnancy maintenance in cattle.

Embryonic cryopreservation has a relatively low survival rate because of cytoskeletal damage. However, molecular anti-freezing mechanisms have been largely unexplored. This study investigated the significance of RhoA, involved in embryonic development, and the Rho/RhoA-associated kinase (ROCK) signalling pathway in cryopreservation. The anti-freezing mechanism in murine dormant embryos, compared with normal blastocysts, was assessed by combining molecular, physiological and pharmacological approaches. Real-time PCR and western blotting experiments showed high RhoA expression in cryo-dormant and dormant embryos. RhoA GTPases were overexpressed on the surface of trophectoderm cells in dormant embryos. Treatment with Y-27632, a ROCK antagonist, decreased survival of both normal and dormant blastocysts, while recombinant RhoA protein remarkably increased survival, after freeze–thawing, of normal hatched blastocysts. Our findings elucidated the molecular mechanism of anti-freezing, involving RhoA phosphorylation, meditated by the Rho/ROCK signalling pathway, in hatched and diapaused murine blastocysts. In addition, evidence for a potentially protective additive suggests a new method for improving the anti-freezing potential of mammalian embryos, without protecting the zona pellucida.

Average daily gain (ADG) is the most economically important trait in beef cattle industry. Using genome-wide association study (GWAS) approaches, previous studies have identified several causal variants within the PLAG1, NCAPG and LCORL genes for ADG in cattle. Multi-strategy GWASs were implemented in this study to improve detection and to explore the causal genes and regions. In this study, we conducted GWASs based on the genotypes of 1,173 Simmental cattle. In the SNP-based GWAS, the most significant SNPs (rs109303784 and rs110058857, P = 1.78 × 10−7) were identified in the NCAPG intron on BTA6 and explained 4.01% of the phenotypic variance, and the independent and significant SNP (rs110406669, P = 5.18 × 10−6) explained 3.32% of the phenotypic variance. Similarly, in the haplotype-based GWAS, the most significant haplotype block, Hap-6-N1416 (P = 2.56 × 10−8), spanned 12.7 kb on BTA6 and explained 4.85% of the phenotypic variance. Also, in the gene-based GWAS, seven significant genes were obtained which included DCAF16 and NCAPG. Moreover, analysis of the transcript levels confirmed that transcripts abundance of NCAPG (P = 0.046) and DCAF16 (P = 0.046) were significantly correlated with the ADG trait. Overall, our results from the multi-strategy GWASs revealed the DCAF16-NCAPG region to be a susceptibility locus for ADG in cattle.

Somatic cell nuclear transfer (SCNT) is frequently used to produce transgenic cloned livestock, but it is still associated with low success rates. To our knowledge, we are the first to report successful production of transgenic cattle that overexpress bovine adipocyte-type fatty acid binding proteins (A-FABPs) with the aid of SCNT. Intragenomic integration of additional A-FABP gene copies has been found to be positively correlated with the intramuscular fat content in different farm livestock species. First, we optimized the cloning parameters to produce bovine embryos integrated with A-FABP by SCNT, such as applied voltage field strength and pulse duration for electrofusion, morphology and size of donor cells, and number of donor cells passages. Then, bovine fibroblast cells from Qinchuan cattle were transfected with A-FABP and used as donor cells for SCNT. Hybrids of Simmental and Luxi local cattle were selected as the recipient females for A-FABP transgenic SCNT-derived embryos. The results showed that a field strength of 2.5 kV/cm with two 10-μs duration electrical pulses was ideal for electrofusion, and 4–6th generation circular smooth type donor cells with diameters of 15–25 μm were optimal for producing transgenic bovine embryos by SCNT, and resulted in higher fusion (80%), cleavage (73%), and blastocyst (27%) rates. In addition, we obtained two transgenic cloned calves that expressed additional bovine A-FABP gene copies, as detected by PCR-amplified cDNA sequencing. We proposed a set of optimal protocols to produce transgenic SCNT-derived cattle with intragenomic integration of ectopic A-FABP-inherited exon sequences.

The molecular mechanism regulated by microRNAs (miRNAs) that underlies postnatal hypertrophy of skeletal muscle is complex and remains unclear. Here, the miRNAomes of longissimus dorsi muscle collected at five postnatal stages (60, 120, 150, 180, and 210 days after birth) from Large White (commercial breed) and Min pigs (indigenous breed of China) were analyzed by Illumina sequencing. We identified 734 miRNAs comprising 308 annotated miRNAs and 426 novel miRNAs, of which 307 could be considered pig-specific. Comparative analysis between two breeds suggested that 60 and 120 days after birth were important stages for skeletal muscle hypertrophy and intramuscular fat accumulation. A total of 263 miRNAs were significantly differentially expressed between two breeds at one or more developmental stages. In addition, the differentially expressed miRNAs between every two adjacent developmental stages in each breed were determined. Notably, ssc-miR-204 was significantly more highly expressed in Min pig skeletal muscle at all postnatal stages compared with its expression in Large White pig skeletal muscle. Based on gene ontology and KEGG pathway analyses of its predicted target genes, we concluded that ssc-miR-204 may exert an impact on postnatal hypertrophy of skeletal muscle by regulating myoblast proliferation. The results of this study will help in elucidating the mechanism underlying postnatal hypertrophy of skeletal muscle modulated by miRNAs, which could provide valuable information for improvement of pork quality and human myopathy.

Epidermal growth factor (EGF) has been shown to facilitate the in vitro maturation of sheep oocytes, and enhance embryo’s capability for further development. However, such kind of molecular mechanism has not yet been elucidated. In the present study, we investigated the effect of EGF-mediated mitogen-activated protein kinases 3 and 1 (MAPK3/1) pathway on in vitro maturation of sheep oocytes. U0126, a specific inhibitor of MEK (MAPK kinase), was added into the maturation culture medium to block the EGF-mediated MAPK3/1 pathway with different doses. Then, the nuclear maturation of sheep oocytes was examined. Additionally, the effect of EGF-mediated MAPK3/1 on cytoplasmic maturation was examined though in vitro fertilization and embryonic development. The rate of germinal vesicle breakdown (GVBD) after 6 h of culture with 10−4 mol/l of U0126 (50.4%) was significantly decreased compared with control (67.2%, p < 0.05), and the first polation body (PB1) extrusion rate after 22 h of culture in drug treatment was also significantly inhibited compared with control (28.6% vs. 48.4%, p < 0.05). However, 10−6 mol/l U0126 had slight effect on oocyte nuclear maturation. The normal distribution rate of α-tubulin in the oocytes after 22 h of in vitro maturation was significantly decreased in the 10−4 mol/l U0126 group (54%) compared with control (68%, p < 0.05). After in vitro fertilization, the cleavage rate in drug treatments (56.8% in 10−6 mol/l U0126 group and 42.6% in 10−4 mol/l U0126 group) was significantly decreased compared with control (72.3%, p < 0.01). The blastocyst rate in 10−4 mol/l U0126 group (17.6%) was also significantly decreased compared with control (29.9%, p < 0.05). Collectively, these results suggest that EGF-mediated MAPK3/1 pathway is conducive to in vitro maturation of sheep oocytes.