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

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

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

Abstract: Melatonin is an important factor involved in regulating reproduction; it is synthesized enzymatically by the sequential action of melatonin‐synthesizing enzymes, arylalkylamine N‐acetyltransferase (AANAT) and hydroxyindole‐O‐methyltransferase (HIOMT), and exerts its biological functions mainly through receptor‐mediated action. To evaluate the expression of melatonin, two melatonin‐synthesizing enzymes (HIOMT and AANAT), and membrane receptors (MT1 and MT2) in oestrous corpus luteum (CL) and CL verum of sheep (Ovis aries), we performed ELISA, qRT‐PCR, western blotting and immunohistochemistry. The quantitative results showed that melatonin, HIOMT and AANAT levels in the CL verum were significantly higher than those in oestrous CL (p < 0.05), whereas MT1 and MT2 exhibited no change between the oestrous CL and CL verum (p > 0.05); moreover, the localization results showed that HIOMT, AANAT, MT1 and MT2 were mainly expressed in large luteal cells (LLCs). In summary, the above results suggested that sheep CL has potential for the synthesis of melatonin; meanwhile, they also suggested that CL is one of the targets of melatonin. These results provide not only a basis for whether sheep CL can synthesize melatonin but also provide a reference for further study on the mechanism of melatonin in the CL. [ABSTRACT FROM AUTHOR]