Melatonin Inhibits Testosterone Synthesis in Rooster Leydig Cells by Targeting CXCL14 through miR-7481-3p.

Melatonin has been proved to be involved in testosterone synthesis, but whether melatonin participates in testosterone synthesis by regulating miRNA in Leydig cells is still unclear. The purpose of this study is to clarify the mechanism of melatonin on Leydig cells testosterone synthesis from the perspective of miRNA. Our results showed that melatonin could significantly inhibit testosterone synthesis in rooster Leydig cells. miR-7481-3p and CXCL14 were selected as the target of melatonin based on RNA-seq and miRNA sequencing. The results of dual-luciferase reporter assays showed that miR-7481-3p targeted the 3'-UTR of CXCL14. The overexpression of miR-7481-3p significantly inhibited the expression of CXCL14 and restored the inhibitory role of melatonin testosterone synthesis and the expression of StAR, CYP11A1, and 3ß-HSD in rooster Leydig cells. Similarly, interference with CXCL14 could reverse the inhibitory effect of melatonin on the level of testosterone synthesis and the expression of StAR, CYP11A1, and 3ß-HSD in rooster Leydig cells. The RNA-seq results showed that melatonin could activate the PI3K/AKT signal pathway. Interference with CXCL14 significantly inhibited the phosphorylation level of PI3K and AKT, and the inhibited PI3K/AKT signal pathway could reverse the inhibitory effect of CXCL14 on testosterone synthesis and the expression of StAR, CYP11A1 and 3ß-HSD in rooster Leydig cells. Our results indicated that melatonin inhibits testosterone synthesis by targeting miR-7481-3p/CXCL14 and inhibiting the PI3K/AKT pathway.

Melatonin inhibits testosterone synthesis in Roosters Leydig cells by regulating lipolysis of lipid droplets.

Leydig cells are important component of testis cells, which can synthesize testosterone with free cholesterol derived from lipid droplets (LDs). It is well known that melatonin could regulate synthesis of testosterone. However, it is still unclear whether melatonin participates in the synthesis of testosterone by regulating the lipolysis of LDs in Leydig cells. The purpose of this study was to elucidate the effect of melatonin on synthesis of testosterone in roosters Leydig cells by regulating lipolysis of LDs. The results showed that melatonin decreased synthesis of testosterone and intracellular free cholesterol in roosters Leydig cells. Exogenous addition of 22-OH-Cholesterol counteracted the inhibitory effect of melatonin on synthesis of testosterone. Furthermore, melatonin increased the LDs content and expression of perilipin 1 (PLIN1), and decreased expression of hormone-sensitive lipase (HSL) and triacylglycerol hydrolase (ATGL) in roosters Leydig cells. In addition, silencing PLIN1 reversed the inhibitory effect of melatonin on synthesis of testosterone in roosters Leydig cells by increasing free cholesterol content and expression of HSL and ATGL, and decreasing the lipid droplet content. Activation of cAMP/PKA pathway by using the pathway activators Forskolin and 8-Bromo-cAMP attenuated the inhibitory effect of melatonin on synthesis of testosterone accompanied by increasing level of free cholesterol content and expression of HSL and ATGL, and decreasing level of lipid droplet content and expression of PLIN1 in roosters Leydig cells. These results suggested that melatonin could inhibit the synthesis of testosterone in roosters Leydig cells by reducing the content of intracellular free cholesterol in which expression of PLIN1 and cAMP/PKA pathway were inhibited to reduce the lipolysis of LDs.

Quercetin Reduces Inflammation and Protects Gut Microbiota in Broilers.

The aim of this study was to investigate the effects of quercetin on inflammatory response and intestinal microflora in broiler chicken jejuna. A total of 120 broiler chickens were allocated into 3 groups: saline-challenged broilers fed a basal diet (CTR group), lipopolysaccharide (LPS)-challenged broilers fed a basal diet (L group) and LPS-challenged broilers fed a basal diet supplemented with 200 mg/kg quercetin (LQ group). Our results showed that LPS significantly increased expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-8, interferon (IFN)-γ, toll-like receptor (TLR)-4, Bax, Caspase-3 and diamine oxidase activity (DAO), and decreased expression of zona occludens-1 (ZO-1), Occludin and Bcl-2 in the jejunum, while dietary quercetin prevented the adverse effects of LPS injection. LPS injection significantly decreased the number of Actinobacteria, Armatimonadetes and Fibrobacteriae at the phylum level when compared to the CTR group. Additionally, at genus level, compared with the CTR group, the abundance of Halomonas, Micromonospora, Nitriliruptor, Peptococcus, Rubellimicrobium, Rubrobacter and Slaclda in L group was significantly decreased, while dietary quercetin restored the numbers of these bacteria. In conclusion, our results demonstrated that dietary quercetin could alleviate inflammatory responses of broiler chickens accompanied by modulating jejunum microflora.

Kisspeptin-10 Promotes Progesterone Synthesis in Bovine Ovarian Granulosa Cells via Downregulation of microRNA-1246.

The objective of this study was to clarify the effect of kisspeptin-10 (kp-10) on the synthesis of progesterone (P4) in bovine granulosa cells (BGCs) and its mechanisms via microRNA 1246 (miR-1246). According to the results, we found that treating with kp-10 for 24 h could increase P4 level, the mRNA expression of the steroidogenesis-related gene steroidogenic acute regulatory protein (StAR), free cholesterol content, and decrease miR-1246 expression in BGCs. Overexpression of miR-1246 significantly inhibited P4 synthesis, StAR mRNA expression, and free cholesterol content in BGCs, whereas underexpression of miR-1246 significantly reversed this effect in BGCs. Additionally, overexpression of miR-1246 counteracted the accelerative effect of kp-10 on P4 synthesis, StAR mRNA expression, and free cholesterol content in BGCs. Conversely, underexpression of miR-1246 enhanced the accelerative effect of kp-10 on P4 synthesis, StAR mRNA expression, and free cholesterol content in BGCs. Meanwhile, results of dual-luciferase reporter assays indicated that miR-1246 targeted the 3'UTR of StAR in BGCs. These results demonstrated that kp-10 induced P4 synthesis in BGCs by promoting free cholesterol transport via regulating expression of miR-1246/StAR.

Untargeted LC-MS-based metabonomic analysis of the effect of photoperiod on the testes of broiler roosters.

Photoperiod is an important factor that stimulates the reproductive performance of broiler breeder roosters. However, the mechanism by which photoperiod affects the reproductive performance of broiler breeder roosters has not been fully studied. To study the effects of different photoperiods on the reproductive performance of broiler breeder roosters, 120 Arbor Acres broiler breeder roosters aged 20 weeks were randomly assigned to three groups (n = 40), and the three groups were treated with different photoperiod regimes: control (CTR; 12.5 h of light and 11.5 h of dark, 12.5 L: 11.5 D), short day (SD; 16 L: 8 D) and long day (LD; 8 L: 16 D). Serum and testes were collected after 4 weeks of feeding, and testosterone-related indices were detected. We found that testosterone synthesis in the testes of broiler roosters was boosted with prolonged of photoperiod. Subsequently, metabonomics was used to identify the differential endogenous metabolites that may affect the function of the testes in breeder roosters. We found compared with other groups, the concentrations of creatine, uridine monophosphate, phosphoribosyl pyrophosphate, dCMP, α-D-glucose and citric acid in the SD group decreased significantly (p < 0.05), and glyoxylic acid, D-ribose 5-phosphate, deoxyuridine and orotic acid in the SD group increased significantly (p < 0.05), while the CTR group and LD group showed no significant difference (p > 0.05). The concentrations of linoleic acid and α-linolenic acid in the LD group were increased significantly (p < 0.05) than those in the CTR and SD groups. Compared with the CTR group, the concentrations of histamine in the SD and LD groups were significant increased (p < 0.05). The 13 of the different metabolites could be used as candidate biomarkers for different photoperiods affecting testosterone synthesis, may be used to molecular breeding of high reproductive performance broiler roosters.

microRNA-10b promotes the apoptosis of bovine ovarian granulosa cells by targeting plasminogen activator inhibitor-1.

Granulosa cells (GCs) are essential somatic cells in the ovaries, and apoptosis of GCs causes follicular atresia. microRNA-10b (miR-10b) is pivotal for cell apoptosis. However, currently, little is known about the role of miR-10b in bovine ovarian GCs (BGCs). In this study, the effect of miR-10b on the apoptosis of BGCs was investigated. Our results showed that the overexpression of miR-10b could increase the apoptosis rate of BGCs, which is associated with the increased expression of Caspase-3 and decreased expression ratio of Bcl-2/Bax (P < 0.05). Furthermore, plasminogen activator inhibitor-1 (PAI-1) was confirmed to be a validated target of miR-10b in BGCs using dual-luciferase reporter analysis, and transfection of miR-10b mimics decreased the expression of PAI-1 (P < 0.05). In addition, overexpression of PAI-1 significantly inhibited BGC apoptosis (P < 0.05), and PAI-1 could alleviate BGC apoptosis induced by miR-10b (P < 0.05). Subsequently, phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) was found to be the downstream pathway of PAI-1 by RNA-Seq analysis and verified by Western blot. Finally, a PI3K/AKT inhibitor (Miltefosine) was used to inhibit the PI3K/AKT pathway, which reversed the inhibitory effect of PAI-1 on the apoptosis of BGCs (P < 0.05), and enhanced the promotion effect of miR-10b on the apoptosis of BGCs (P < 0.05). Our results indicated that miR-10b promotes BGC apoptosis by targeting PAI-1 to regulate the PI3K/AKT pathway.

Photoperiodic effect on the testicular transcriptome in broiler roosters.

Information about the effects of photoperiod on the testicular transcriptome of broiler roosters is limited. The aim of the present study was to explore the effect of different photoperiodic regimes on gene expression in the testes of broiler breeder roosters. One hundred and twenty Arbor Acres broiler breeder roosters aged 20 weeks were assigned to one of three groups (n = 40) and subjected to different photoperiodic regimes: control (CTR; 12.5 L:11.5 D), short day (SD; 8 L:16 D) and long day (LD; 16 L:8 D). After 4 weeks, the testes of 10 randomly selected birds from each group were dissected, sliced and haematoxylin-eosin stained. The testicular transcriptome of roosters from the SD and LD groups was determined by RNA sequencing (RNA-Seq), and the results were confirmed using quantitative real-time PCR. The seminiferous tubule area and sperm count increased significantly with the prolongation of photoperiod (p < .01). Additionally, the RNA-Seq results indicated that 387 genes were upregulated and 1,052 genes were downregulated in the LD group compared with those in the SD group. Several crucial genes involved in rooster testicular development and reproduction were also screened, including heat shock proteins 90, extracellular regulated protein kinases 1, phosphatidylinositol 3-kinase, adenosine 5'-monophosphate -activated protein kinase, BCL-6 and Smad3. The differentially expressed genes were enriched in the mammalian targets of rapamycin (mTOR), forkhead box (FoxO), transforming growth factor beta (TGF-ß) and insulin signalling pathway. In conclusion, a 16 hr photoperiod for 4 weeks increased the seminiferous tubule duct area and promoted spermatogenesis in the rooster's testicles, and the mTOR, FoxO, TGF-ß and insulin signalling pathways may be involved.