Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Ameliorate Aging-Induced BTB Impairment in Porcine Testes by Activating Autophagy and Inhibiting ROS/NLRP3 Inflammasomes via the AMPK/mTOR Signaling Pathway.

As a pivotal player in spermatogenesis, the blood-testis barrier (BTB) made from junction apparatus coexisting in Sertoli cells (SCs) is impaired with an increase in age and ultimately induces spermatogenic dysfunction or even infertility. It has been corroborated that bone marrow mesenchymal stem cell (BMSC) transplantation can efficiently repair and regenerate the testicular function. As vital mediators of cell-to-cell communication, MSC-derived exosomes (Exos) can directly serve as therapeutic agents for tissue repair and regeneration. However, the therapeutic value of BMSC-Exos in aging-induced BTB damage remains to be confirmed. In this study, we explored that the old porcine testes had defective autophagy, which aggravated BTB disruption in SCs. BMSC-Exos could decrease ROS production and NLRP3 inflammasome activation but enhanced autophagy and tight junction (TJ) function in D-gal-triggered aging porcine SCs and mouse model testes, according to in vitro and in vivo experiments. Furthermore, rapamycin, NAC, MCC950, and IL-1Ra restored the TJ function in D-gal-stimulated aging porcine SCs, while BMSC-Exos' stimulatory effect on TJ function was inhibited by chloroquine. Moreover, the treatment with BMSC-Exos enhanced autophagy in D-gal-induced aging porcine SCs by means of the AMPK/mTOR signal transduction pathway. These findings uncovered through the present study that BMSC-Exos can enhance the BTB function in aging testes by improving autophagy via the AMPK/mTOR signaling pathway, thereby suppressing ROS production and NLRP3 inflammasome activation.

17ß-Estradiol mediates TGFBR3/Smad2/3 signaling to attenuate the fibrosis of TGF-ß1-induced bovine endometrial epithelial cells via GPER.

Abnormal function and fibrosis of endometrium caused by cows' endometritis pose difficult implantation of embryos and uterine cavity adhesions. 17ß-Estradiol (E2) serves as the most effective aromatized estrogen, and its synthetase and receptors have been detected in the endometrium. Studies have demonstrated the positive role of estrogen in combating pathological fibrosis in diverse diseases. However, it is still unknown whether E2 regulates endometrium fibrosis in bovine endometritis. Herein, we evaluated the expression patterns of transforming growth factor-ß1 (TGF-ß1), epithelial-mesenchymal transformation (EMT)-related proteins (α-SMA, vimentin N-cadherin and E-cadherin), cytochrome P450 19A1 (CYP19A1), and G protein-coupled estrogen receptor (GPER) in bovine healthy endometrium and Inflammatory endometrium. Our data showed that the inflamed endometrium presented low CYP19A1 and GPER expression, and significantly higher EMT process versus the normal tissue. Moreover, we established a TGF-ß1-induced fibrosis model in BEND cells, and found that E2 inhibited the EMT process of BEND cells in a dose-dependent manner. The anti-fibrotic effect of E2 was blocked by the GPER inhibitor G15, but not the estrogen nuclear receptors (ERs) inhibitor ICI182780. Moreover, the GPER agonist G1 inhibited fibrosis and Smad2/3 phosphorylation but increased the expression of TGFBR3 in BEND cells. Transfection with TGFBR3 small interfering RNA blocked the effect of G1 on fibrosis of BEND cells and upregulated the expression of P-Smad2/3. Our in vivo data also showed that E2 and G1 affected uterus fibrosis in mice endometritis model caused by LPS, which was associated with the inhibition of TGFBR3/Smad2/3 signaling. In conclusion, our data implied that E2 alleviates the fibrosis of TGF-ß1-induced BEND cells, which is associated with the GPER mediation of TGFBR3/Smad2/3 signaling.

Dihydrotestosterone mediates the inflammation effect under lipopolysaccharides in bovine endometrial epithelial cells via AR blockading TLR4/MyD88 signaling pathway.

Dihydrotestosterone (DHT) is a potent nonaromatizable 5α-reduced androgen with both positive and negative effect on inflammation process. However, it remains unknown whether DHT can regulate Lipopolysaccharides (LPS)-induced inflammation in bovine endometrial epithelial cells (bEECs). Here, we demonstrated that the DHT biosynthesis ability and androgen receptors (AR) expression is defective in bovine endometrial with endometritis, which aggravates endometrial inflammation. In vitro study, we established a LPS-induced inflammation model in bEECs, and found that DHT inhibited the TLR4 and MyD88 protein as well as TNF-α, IL-1ß, and IL-6 mRNA of bEECs in a dose-dependent manner. Moreover, the anti-inflammation effect of DHT was blocked by AR inhibitor flutamide. Together, we demonstrated that supplementing DHT can alleviate the inflammation response of bEECs caused by LPS, which is associated with AR regulating the inhibition of TLR4/MyD88 signaling pathway.

Identification of Key Genes Affecting Flavor Formation in Beijing-You Chicken Meat by Transcriptome and Metabolome Analyses.

The flavor of chicken meat is influenced by muscle metabolites and regulatory genes and varies with age. In this study, the metabolomic and transcriptomic data of breast muscle at four developmental stages (days 1, 56, 98, and 120) of Beijing-You chickens (BJYs) were integrated and 310 significantly changed metabolites (SCMs) and 7,225 differentially expressed genes (DEGs) were identified. A Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that SCMs and DEGs were enriched in amino acid, lipid, and inosine monophosphate (IMP) metabolism pathways. Furthermore, genes highly associated with flavor amino acids, lipids, and IMP were identified by a weighted gene co-expression network analysis (WGCNA), including cystathionine ß-synthase (CBS), glycine amidinotransferase (GATM), glutamate decarboxylase 2 (GAD2), patatin-like phospholipasedomain containing 6 (PNPLA6), low-specificity L-threonine aldolase (ItaE), and adenylate monophosphate deaminase 1 (AMPD1) genes. A regulatory network related to the accumulation of key flavor components was constructed. In conclusion, this study provides new perspectives regarding the regulatory mechanisms of flavor metabolites in chicken meat during development.

3D-bioprinted double-crosslinked angiogenic alginate/chondroitin sulfate patch for diabetic wound healing.

Improving chronic wound healing remains a challenge in the clinical practice. In this study, we developed double-crosslinked angiogenic 3D-bioprinted patches for diabetic wound healing by the photocovalent crosslinking of vascular endothelial growth factor (VEGF) using ultraviolet (UV) irradiation. 3D printing technology can precisely customize the structure and composition of patches to meet different clinical requirements. The biological polysaccharide alginate and chondroitin sulfate methacryloyl were used as biomaterials to construct the biological patch, which could be crosslinked using calcium ion crosslinking and photocrosslinking, thereby improving its mechanical properties. More importantly, acrylylated VEGF could be easily and rapidly photocrosslinked under UV irradiation, which simplified the step of chemically coupling growth factors and prolonged VEGF release time. These characteristics suggest that 3D-bioprinted double-crosslinked angiogenic patches are ideal candidates for diabetic wound healing and other tissue engineering applications.

The Role and Application of Exosomes and Their Cargos in Reproductive Diseases: A Systematic Review.

In recent years, the incidence of the reproductive diseases is increasing year-by-year, leading to abortion or fetal arrest, which seriously affects the reproductive health of human beings and the reproductive efficiency of animals. Exosomes are phospholipid bilayer vesicles that are widely distributed in living organisms and released by the cells of various organs and tissues. Exosomes contain proteins, RNA, lipids, and other components and are important carriers of information transfer between cells, which play a variety of physiological and pathological regulatory functions. More and more studies have found that exosomes and their connotations play an important role in the diagnosis, prognosis and treatment of diseases. A systematic review was conducted in this manuscript and then highlights our knowledge about the diagnostic and therapeutic applications of exosomes to reproductive diseases, such as polycystic ovary syndrome (PCOS), endometriosis, premature ovarian failure (POF), preeclampsia, polycystic, endometrial cancer, cervical cancer, ovarian cancer, and prostate gland cancer.

Dietary supplementation with selenomethionine enhances antioxidant capacity and selenoprotein gene expression in layer breeder roosters.

This study's objective was to investigate the effects of dietary Se (in the form of selenomethionine) on the antioxidant activity and selenoprotein gene expressions in layer breeder roosters. One hundred and eighty, 36-wk-old Jingfen layer breeder roosters were randomly allocated to one of 5 dietary treatments (0, 0.25, 0.5, 1, or 2 mg/kg Se) for 6 wk on a corn-soybean meal-based diet. Antioxidant parameters and selenoprotein gene expressions were assessed at the end of the experiment. The results showed that Se supplementation significantly increased the activity of T-SOD, CAT, GSH-Px, and superoxide anion scavenging ability in plasma (P ≤ 0.05), and activities of T-SOD, CAT, GSH-Px, superoxide anion scavenging ability, and hydroxyl radical scavenging ability in the liver, kidney, and testis (P < 0.05). Moreover, MDA levels were significantly reduced in plasma, liver, kidney, and testis (P < 0.01), compared to the control group. Furthermore, the dietary administration of Se significantly increased TrxR2 and GPx4 mRNA levels in kidney and testis, and ID1 mRNA levels in liver and kidney. Most of the antioxidant parameters and selenoprotein-related gene expressions significantly increased, and MDA significantly decreased at dietary supplementation with 0.5 mg/kg Se. Whereas a higher dose of Se level (1 or 2 mg/kg) inhibited the activities of some of the antioxidant enzymes and selenoprotein-related gene expressions in selected tissues. In conclusion, dietary Se supplementation with 0.5 mg/kg significantly improved roosters' antioxidant status and selenoprotein-related gene expression in liver, kidney, and testis, while higher doses led to inhibit these; dietary Se might increase reproductive performance by enhancing their antioxidant status in roosters.

Specific activation of embryonic IFNAR1 and endometrial IFNAR2 induced by embryonic IFNτ directs normal uterine fate for bovine early implantation.

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. In this study, we found the expression and location of IFNτ in the bovine blastocysts from different production 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 endometrial epithelium cells (EECs) reduced the expressions of Integrin αv ß3, WNT7A, and ISG15 in EECs. Knocking out IFNAR1 and IFNAR2 reduce the expressions of Integrin αv ß3 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 ß3 was inhibited, and the inhibition of IFNAR1+/IFNAR2- was much stronger, and the expression of WNT7A was not inhibited. The expressions of Integrin αv ß3 and WNT7A did not change significantly after IFNAR1-/IFNAR2+ and IFNAR1+/IFNAR2- co-culture with PA embryos. 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.

Heat stress and hypoxia inhibit the secretion of androgens and induce epithelial-to-mesenchymal transition associated with activated TGF-ß/Smad signalling in canine cryptorchidism.

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-ßs and its two receptors TGF-ßRI and TGF-ßRII increased in case of cryptorchidism. From the study in vitro, we found both heat stress and COCl2 mimic hypoxia inhibited the secretion of testosterone (T) and androstenedione (A4) and promoted the expression of the EMT maker α-SMA and vimentin in Leydig cells, and also that heat stress and COCl2 stimulated with the TGF-ß signalling promoted the expression of TGF-ßs and its two type receptors and also the active phosphorylation of Smad2 and Smad3. The use of LY2109761, a receptor inhibitor of TGF-ßs/Smad signalling pathway, was associated with heat stress and COCl2 suppression of androgens' secretion and stimulated EMT in Leydig cells. These findings characterized a novel pathogenesis of cryptorchidism and provided a new idea for therapeutics.

Dihydrotestosterone through blockade of TGF-ß/Smad signaling mediates the anti-fibrosis effect under hypoxia in canine Sertoli cells.

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 (CoCl2), and found that DHT inhibited the fibrosis of Sertoli cells in a dose-dependent manner. Meanwhile, DHT interfered with the TGF-ß signaling by reducing the expression of TGF-ßRI and TGF-ßRII and inhibiting the expression and phosphorylation of Smad2 and Smad3, while flutamide (androgen receptor inhibitor) inhibited these effects of DHT. Furthermore, use of LY2109761 (TGF-ß receptor type I/II inhibitor) to interfere with the TGF-ß/Smad pathway showed a similar effect with DHT suppression of the fibrosis in Sertoli cells. Our research data demonstrated that cryptorchidism is located in a hypoxic and DHT deficiency microenvironment. Moreover, supplementing DHT can alleviate the fibrosis process of Sertoli cells caused by hypoxia, which is associated with AR regulating the inhibition of TGF-ß/Smad signaling.

Exosomes derived from placental trophoblast cells regulate endometrial epithelial receptivity in dairy cows during pregnancy.

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, ß3, 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.

GPER mediates the IL6/JAK2/STAT3 pathway involved in VEGF expression in swine ovary GCs.

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 17ß-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.

Dihydrotestosterone regulation of cyclooxygenase-2 expression in bovine endometrial epithelium cells by androgen receptor mediated EGFR/PI3K/Akt pathway.

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.

Angiogenic Microspheres for the Treatment of a Thin Endometrium.

The poor vascular development of an endometrium is the key cause of a thin endometrium due to the vascular endothelial growth factor (VEGF) decreasing in the glandular epithelium. Hence, inducing angiogenesis is an effective strategy for thin endometrium treatment in clinic. Herein, we developed a novel angiogenic hydrogel microsphere based on methacrylated hyaluronic acid (HAMA) loaded with VEGF for the treatment of a thin endometrium by a microfluidic electrospray technique. The generated HAMA microspheres with uniform size, porous structure, and satisfactory biocompatibility increased the drug-loading ability and controlled the drug-release rate by adjusting the hydrogel concentration. Besides, the HAMA microspheres loaded with VEGF showed satisfactory biocompatibility and promoted blood vessel formation in vitro. More importantly, the combination of HA and VEGF promoted new blood vessels and endometrial regeneration of a thin endometrium in vivo. Therefore, the combination of HA and VEGF would be conducive to the development of a drug-delivery microsphere with excellent biocompatibility and therapeutic effect for thin endometrium treatment and other biomedical applications.

Case Report: Occupation Radiation Disease, Skin Injury, and Leukemia After Accidental Radiation Exposure.

Objective: Follow-up observation of radiation accident in which a worker developed acute radiation disease and eventually died of leukemia. The case provided key practical information for the study on clinical effects of radiation on the health of workers. Case Presentation: We observed and followed-up the progression and effect of radiation exposure at various stages in a 28-year-old male patient. We examined the chromosomal morphology, white blood cell count, and sperm count. Laboratory tests for leukemia diagnosis and other clinical parameters were performed. Results: After the patient was irradiated, the white blood cell level decreased, the sperm count dropped to 0, and the libido completely disappeared. The patient's chromosome aberration cell rate and total chromosome aberration cell rate were 7.33 and 7.66%, respectively. Examination of leukemia diagnostic experiments revealed that abnormal cells accounted for 60%; bone marrow examination showed that prolymphocytes abnormally proliferated, accounting for 89%, and had positive extracellular iron staining. After the initial treatment, the patient's white blood cell level increased and was finally maintained at a normal level, the sperm count returned to normal levels, and libido was restored. The patient died of acute lymphoblastic leukemia 34 years after the exposure. Conclusion: More attention has been paid to the long-term effects of ionizing radiation-induced malignant tumors. The occupational protection of radiographic inspection workers should be strengthened to reduce and avoid occupational injuries to protect the health and safety of workers.

Dietary supplementation with natural astaxanthin from Haematococcus pluvialis improves antioxidant enzyme activity, free radical scavenging ability, and gene expression of antioxidant enzymes in laying hens.

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.

Exosomes from bovine endometrial epithelial cells ensure trophoblast cell development by miR-218 targeting secreted frizzled related protein 2.

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.

Effects of dietary supplementation of natural astaxanthin from Haematococcus pluvialis on antioxidant capacity, lipid metabolism, and accumulation in the egg yolk of laying hens.

The present study evaluated the effects of natural astaxanthin (ASTA) from Haematococcus pluvialis on the antioxidant capacity, lipid metabolism, and ASTA accumulation in the egg yolk of laying hens. Hy-Line Brown layers (n = 288, 50 wk old) were randomly assigned to 1 of 4 dietary treatment groups. Each group had 6 replicates of 12 hens each. All birds were given a corn-soybean meal-based diet containing 0, 25, 50, or 100 mg/kg ASTA for 6 wk. The results showed that the total antioxidant capacity, superoxide dismutase level, and glutathione peroxidase level in the plasma, livers, and egg yolks were significantly increased in the ASTA groups compared with those of the control group (P < 0.05), whereas the content of malondialdehyde linearly decreased (P < 0.05). The plasma levels of high-density and very-low-density lipoprotein cholesterol in the ASTA groups were significantly higher than those in the control group (P < 0.05). In addition, ASTA supplementation decreased low-density lipoprotein cholesterol and triglyceride plasma levels (P < 0.05). However, there were no significant differences in the other lipid metabolism parameters among the ASTA-supplemented groups relative to the control group except for an increase in high-density lipoprotein cholesterol in the liver. Compared with the control, dietary ASTA supplementation significantly increased the enrichment of ASTA in egg yolks at the end of week 2, 4, and 6 (P < 0.05). The mRNA expression of scavenger receptor class B type 1 (SCARB1) and very-low-density lipoprotein receptor (VLDLR) in the ASTA groups was markedly higher (P < 0.05) than that in the control group in the liver and ovaries, respectively. In conclusion, these results suggest that dietary ASTA enhances the antioxidant capacity and regulates lipid metabolism in laying hens. ASTA enrichment in egg yolks may be closely related to the upregulation of SCARB1 and VLDLR gene expression.

Exosome-derived uterine miR-218 isolated from cows with endometritis regulates the release of cytokines and chemokines.

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-1ß, tumour necrosis factor-α, the chemokines macrophage inflammatory genes (MIP)-1α and MIP-1ß 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.

Transcriptional analyses of endometrial caruncles in sheep with in vivo / in vitro produced embryos during the peri-implantation period.

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.