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.

The link between tendon stem/progenitor cells (TSPCs) senescence and tendon aging has been well recognized. However, the cellular and molecular mechanisms of TSPCs senescence are still not fully understood. In present study, we investigated the role of Aquaporin 1 (AQP1) in TSPCs senescence. We showed that AQP1 expression declines with age during tendon aging. In aged TSPCs, overexpression of AQP1 significantly attenuated TSPCs senescence. In addition, AQP1 overexpression also restored the age-related dysfunction of self-renewal, migration and tenogenic differentiation. Furthermore, we demonstrated that the JAK-STAT signaling pathway is activated in aged TSPCs, and AQP1 overexpression inhibited the JAK-STAT signaling pathway activation which indicated that AQP1 attenuates senescence and age-related dysfunction of TSPCs through the repression of JAK−STAT signaling pathway. Taken together, our findings demonstrated the critical role of AQP1 in the regulation of TSPCs senescence and provided a novel target for antagonizing tendon aging.

Diminished regeneration or healing capacity of tendon occurs during aging. It has been well demonstrated that tendon stem/progenitor cells (TSPCs) play a vital role in tendon maintenance and repair. Here, we identified an accumulation of senescent TSPCs in tendon tissue with aging. In aged TSPCs, the activity of JAK-STAT signaling pathway was increased. Besides, genetic knockdown of JAK2 or STAT3 significantly attenuated TSPC senescence in aged TSPCs. Pharmacological inhibition of JAK-STAT signaling pathway with AG490 similarly attenuated cellular senescence and senescence-associated secretory phenotype (SASP) of aged TSPCs. In addition, inhibition of JAK-STAT signaling pathway also restored the age-related dysfunctions of TSPCs, including self-renewal, migration, actin dynamics, and stemness. Together, our findings reveal the critical role of JAK-STAT signaling pathway in the regulation of TSPC aging and suggest an ideal therapeutic target for the age-related tendon disorders.

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 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 < 0.05). Glutathione peroxidase activity was improved in the kidney with dietary ASTA at levels of 40 mg/kg. Total superoxide dismutase ( SOD ) was significantly increased in the liver, kidney, and plasma with dietary ASTA supplementation at 40 mg/kg. With increasing dietary ASTA, the scavenging abilities of hydroxyl radicals and superoxide anions were linearly increased ( P < 0.05), and the malondialdehyde content decreased linearly ( P < 0.05). Compared with the control group, mRNA expression of Cu-Zn SOD ( SOD1 ), Mn SOD ( SOD2 ), and nuclear factor E2-related factor 2 ( NRF2 ) in the liver and kidney was significantly increased in the 40 mg/kg ASTA group ( P < 0.05). The level of GPX4 mRNA in the liver and kidney was significantly increased with ASTA supplementation at 40 and 80 mg/kg ( P < 0.05). The results demonstrate that dietary ASTA improves free radical scavenging ability and antioxidant enzyme activity, which may be related in part to the upregulated mRNA expression of genes encoding antioxidant enzymes and NRF2 .

Oestrogen, androgen and progesterone are involved in the regulation of uterine physiological functions, with the participation of the following proteins: oestrogen receptor (ER), androgen receptor (AR) and progesterone nuclear receptor (PGR). In this study, we used immunohistochemistry to detect the localization of ERα, ERβ, AR and PGR in sheep uterus. Additionally, we used real‐time polymerase chain reaction (RT‐qPCR) and Western blot technique to analyse their expression profiles at different stages of sheep oestrous cycle in the endometrium and myometrium. Immunohistochemical analysis showed that ERα, ERβ, AR and PGR were present in sheep uterus in oestrus, mainly in the uterine luminal epithelium, stroma, gland and myometrium. Real‐time polymerase chain reaction results showed that in the endometrium, ERα expression level was highest in oestrus. ERβ and PGR, instead, were highly expressed in pro‐oestrus. In the myometrium, ERα was highly expressed in both oestrus and pro‐oestrus, and ERβ was highly expressed in oestrus and dioestrus. Progesterone nuclear receptor expression was highest in oestrus, followed by metoestrus. In the endometrium, both receptors ERα and ERβ were abundant in pro‐oestrus, while the maximum AR protein content was found in oestrus. At this stage of the oestrous cycle, PGR protein concentration in the myometrium was significantly lower than those observed in other stages. These results suggest that these receptors are important for sheep reproductive function, as their expression at mRNA and protein levels exhibits particular time‐ and tissue‐specific profiles along the oestrous cycle.

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.

Inadequate fetomaternal interactions could directly lead to pregnancy failure in dairy cows. Exosomes are widely involved in endometrial matrix remodeling, immune function changes, placental development, and other processes of embryo implantation and pregnancy in dairy cows. However, the role of exosomes derived from placental trophoblast cells in regulating the receptivity of endometrial cells and facilitating fetomaternal interaction remains unclear. In this study, bovine trophoblast cells (BTCs) were obtained from bovine placenta and immortalized by transfection with telomerase reverse transcriptase (TERT). Immortalized BTCs still possess the basic and key properties of primary BTCs without exhibiting any neoplastic transformation signs.Subsequently, the effect of trophoblast-derived exosomes (TDEs) on endometrial receptivity in endometrial epithelial cells (EECs) was determined, and the mechanism whereby TDEs and their proteins participate in the fetomaternal interaction during bovine pregnancy were explored. EECs were co-cultured with the exosomes derived from BTCs treated with progesterone (P4). Such treatment enhanced the expression of the endometrial receptivity factors, integrin αv, β3, Wnt7a, and MUC1 by changing the extracellular environment, metabolism, and redox balance in EECs via proteome alignment, compared with no treatment according to the DIA quantitation analysis.Our study demonstrated that trophoblast-derived exosome proteins are one of the most critical elements in fetomaternal interaction, and their changes may act as a key signal in altering endometrial receptivity and provide a potential target for improving fertility.