BackgroundMelatonin is an amine hormone that plays an important role in regulating mammalian reproduction. This study aimed to investigate the expression pattern of melatonin synthesis enzymes AANAT and HIOMT and melatonin receptors MT1 and MT2 in sheep cumulus–oocyte complexes (COCs) as well as the change of melatonin level in follicular fluid (FF) during antral follicle development. In this research, we also study the effect of β-estradiol (E2) on MT1 and MT2 expression as well as melatonin synthesis in COCs so as to lay the foundation for further exploration of the regulation mechanism of melatonin synthesis in the ovary.MethodsCOCs and FF were collected from different size (large follicles (diameter ≥ 5 mm), medium follicles (diameter 2–5 mm), and small follicles (diameter ≤ 2 mm)) of antral follicles in sheep ovaries. To assess whether E2 regulates melatonin synthase and its receptors expression in sheep COCs and whether it is mediated through estrogen receptor (ER) pathway. The collected COCs were cultured in vitro for 24 h and then treat with 1 μM E2 and/or 1 μM ICI182780 (non-selective ER antagonist). The expression of AANAT, HIOMT, MT1 and MT2 mRNA and protein were determined by qRT-PCR and western blot. The melatonin level was determined by ELISA.ResultsThe expression of AANAT, HIOMT, MT1 and MT2 were significantly higher expression in the COCs of small follicles than in those of large follicles (P < 0.05). However, the melatonin level was significantly higher in large follicle FF than in small follicle FF (P < 0.05). Further, the expression of AANAT, HIOMT, MT1, and MT2 and melatonin production were decreased by E2 treatment (P < 0.05), but when ICI182780 was added, the expression of AANAT, HIOMT, MT1, and MT2 and melatonin production recovered (P < 0.05).ConclusionsWe suggest that sheep COCs can synthesize melatonin, but this ability is decreased with increasing follicle diameter. Furthermore, E2 play an important role in regulated the expression of MT1 and MT2 as well as melatonin synthesis in sheep COCs through the ER pathway.
Toll-like receptor 2 (TLR2) plays a crucial role in bacterial recognition and the host immune response during infection. However, its function and downstream biological processes (BPs) in the mammary glands (MGs) of Holstein cows with clinical mastitis (CM) are not fully understood. This study aimed to comprehensively identify the BPs and differentially expressed proteins (DEPs) associated with the bacterial response and TLR2 using data-independent acquisition (DIA) proteomic data. A possible mechanism for the action of TLR2 was proposed, and the results suggested that the expression levels of TLR2 and caspase 8 (CASP8) were positively correlated with the apoptosis of MGs. The expression patterns of TLR2 and TEK receptor tyrosine kinase 2 (Tie2) were negatively correlated with angiogenesis. These results indicated that TLR2 might promote apoptosis in mammary epithelial cells (MECs) and vascular endothelial cells (VECs) via upregulation of CASP8 expression, and inhibition of angiogenesis in VECs via downregulation of Tie2 expression in dairy cows with CM. In conclusion, TLR2 is associated with inflammation, apoptosis, and angiogenesis in the MGs of dairy cows with bacteria-induced mastitis. These results contribute to a deeper understanding of the pathogenic mechanisms and provide the knowledge needed for developing the prevention and treatment of dairy mastitis.
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.
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