Antioxidant activity and metabolic regulation of sodium salicylate on goat sperm at low temperature.

OBJECTIVE: The purpose of this study was to explore the effect of sodium salicylate (SS) on semen preservation and metabolic regulation in goats. METHODS: Under the condition of low temperature, SS was added to goat semen diluent to detect goat sperm motility, plasma membrane, acrosome, antioxidant capacity, mitochondrial membrane potential (MMP) and metabonomics. RESULTS: The results show that at the 8th day of low-temperature storage, the sperm motility of the 20 µM SS group was 66.64%, and the integrity rates of the plasma membrane and acrosome were both above 60%, significantly higher than those of the other groups. The activities of catalase and superoxide dismutase in the sperm of the 20 µM SS group were significantly higher than those of the control group, the contents of reactive oxygen species and malondialdehyde were significantly lower than those in the control group, the MMP was significantly higher than that in the control group, and the contents of Ca2+ and total cholesterol were significantly higher than those in the control group. Through metabonomics analysis, there were significant metabolic differences between the control group and the 20 µM SS group. Twenty of the most significant metabolic markers were screened, mainly involving five metabolic pathways, of which nicotinic acid and nicotinamide metabolic pathways were the most significant. CONCLUSION: The results indicate that SS can effectively improve the low-temperature preservation quality of goat sperm.

Roles of Y-27632 on sheep sperm metabolism.

To investigate the effect of Y-27632 on low-temperature metabolism of sheep sperm, different concentrations of Y-27632 were added to sheep semen at 4 °C in this experiment to detect indicators such as sperm motility, plasma membrane, acrosome, antioxidant performance, mitochondrial membrane potential (MMP), and metabolomics. The results showed that the addition of 20 µM Y-27632 significantly increased sperm motility, plasma membrane integrity rate, acrosome integrity rate, antioxidant capacity, MMP level, significantly increased sperm adenosine triphosphate (ATP) and total cholesterol content, and significantly reduced sperm Ca2+ content. In metabolomics analysis, compared with the control group, the 20 µM Y-27632 group screened 20 differential metabolites, mainly involved in five metabolic pathways, with the most significant difference in Histidine metabolism (P = 0.001). The results confirmed that Y-27632 significantly improved the quality of sheep sperm preservation under low-temperature conditions.

Sheep semen preservation and artificial insemination is an important reproductive technology that supports the large-scale and intensive development of the sheep farming industry. Under low-temperature condition, sperm metabolic activity slows down or pauses, energy consumption decreases, thereby prolonging sperm preservation time and motility. During the process of sperm preservation, sperm are susceptible to cold shock damage, which affects the quality of sperm preservation. Y-27632 is a rho-associated cooled-coil kinase (ROCK) inhibitor that competes with ATP to inhibit the kinase activity of ROCK-I and ROCK-II. However, the study of Y-27632 used in sheep semen preservation and its protective mechanism is less. In this study, we used the ROCK inhibitor Y-27632 and the ROCK activator arachidonic acid (AA) for low-temperature preservation of sheep semen and related metabolic regulation mechanisms. This experiment confirmed that Y-27632 played a significant protective role by regulating sperm metabolism and protecting sperm plasma membrane in sheep.

Metabolomics Analysis of Sodium Salicylate Improving the Preservation Quality of Ram Sperm.

The aim of this study was to investigate the effects of sodium salicylate (SS) on the preservation and metabolic regulation of sheep sperm. Under 4 °C low-temperature conditions, SS (at 10 µM, 20 µM, 30 µM, and 50 µM) was added to the semen diluent to detect sperm motility, plasma membrane, and acrosome integrity. Based on the selected optimal concentration of SS (20 µM), the effects of 20 µM of SS on sperms' antioxidant capacity and mitochondrial membrane potential (MMP) were evaluated, and metabolomics analysis was conducted. The results showed that on the 20th day of low-temperature storage, the sperm motility of the 20 µM SS group was 62.80%, and the activities of catalase (CAT) and superoxide dismutase (SOD) were significantly higher than those of the control group (p < 0.01). The content of Ca2+, reactive oxygen species (ROS), and malondialdehyde (MDA) were significantly lower than those of the control group (p < 0.01), and the total antioxidant capacity (T-AOC) was significantly higher than that of the control group (p < 0.05); mitochondrial activity and the total cholesterol (TC) content were significantly higher than those in the control group (p < 0.01). An ultrastructural examination showed that in the SS group, the sperm plasma membrane and acrosome were intact, the fibrous sheath and axoneme morphology of the outer dense fibers were normal, and the mitochondria were arranged neatly. In the control group, there was significant swelling of the sperm plasma membrane, rupture of the acrosome, and vacuolization of mitochondria. Using metabolomics analysis, 20 of the most significant differential metabolic markers were screened, mainly involving 6 metabolic pathways, with the amino acid biosynthesis pathway being the most abundant. In summary, 20 µM of SS significantly improved the preservation quality of sheep sperm under low-temperature conditions of 4 °C.

Detection and comparison of microRNAs in the caprine mammary gland tissues of colostrum and common milk stages.

BACKGROUND: MicroRNAs (miRNAs) have a great influence on various physiological functions. A lot of high-throughput sequencing (HTS) research on miRNAs has been executed in the caprine mammary gland at different lactation periods (common milk lactation and dry period), but little is known about differentially expressed miRNAs in the caprine mammary gland of colostrum and peak lactation periods. RESULT: This study identified 131 differentially expressed miRNAs (P < 0.05 and log2 colostrum normalized expression (NE)/peak lactation NE > 1 or log2 colostrum NE/peak lactation NE < -1), including 57 known miRNAs and 74 potential novel miRNAs in the colostrum and peak lactation libraries. In addition, compared with differentially expressed miRNAs in the peak lactation period, 45 miRNAs in the colostrum lactation period were remarkably upregulated, whereas 86 miRNAs were markedly downregulated (P < 0.05 and log2 colostrum NE/peak lactation NE > 1 or log2 colostrum NE/peak lactation NE < -1). The expressions of 10 randomly selected miRNAs was analyzed through stem-loop real-time quantitative PCR (RT-qPCR). Their expression patterns were the same with Solexa sequencing results. Pathway analysis suggested that oestrogen, endocrine, adipocytokine, oxytocin and MAPK signalling pathways act on the development of mammary gland and milk secretion importantly. In addition, the miRNA-target-network showed that the bta-miR-574 could influence the development of mammary gland and lactation by leptin receptor (LEPR), which was in the adipocytokine signalling pathway. Chr5_3880_mature regulated mammary gland development and lactation through Serine/threonine-protein phosphatase (PPP1CA), which was in the oxytocin signalling pathway. CONCLUSIONS: Our finding suggested that the profiles of miRNAs were related to the physiological functions of mammary gland in the colostrum and peak lactation periods. The biological features of these miRNAs may help to clarify the molecular mechanisms of lactation and the development of caprine mammary gland.

Plasticity of marrow mesenchymal stem cells from human first-trimester fetus: from single-cell clone to neuronal differentiation.

Recent results have shown that bone marrow mesenchymal stem cells (BMSCs) from human first-trimester abortus (hfBMSCs) are closer to embryonic stem cells and perform greater telomerase activity and faster propagation than mid- and late-prophase fetal and adult BMSCs. However, no research has been done on the plasticity of hfBMSCs into neuronal cells using single-cell cloned strains without cell contamination. In this study, we isolated five single cells from hfBMSCs and obtained five single-cell cloned strains, and investigated their biological property and neuronal differentiation potential. We found that four of the five strains showed similar expression profile of surface antigen markers to hfBMSCs, and most of them differentiated into neuron-like cells expressing Nestin, Pax6, Sox1, ß-III Tubulin, NF-L, and NSE under induction. One strain showed different expression profile of surface antigen markers from the four strains and hfBMSCs, and did not differentiate toward neuronal cells. We demonstrated for the first time that some of single-cell cloned strains from hfBMSCs can differentiate into nerve tissue-like cell clusters under induction in vitro, and that the plasticity of each single-cell cloned strain into neuronal cells is different.

Pancreatic islet-like clusters from bone marrow mesenchymal stem cells of human first-trimester abortus can cure streptozocin-induced mouse diabetes.

Bone marrow mesenchymal stem cells (BMSCs) have been reported to possess low immunogenicity and cause immunosuppression of recipients when allografted. They can differentiate into insulin-producing cells and may be a valuable source for islet formation. However, the extremely low differentiating rate of adult BMSCs toward insulin-producing cells and the insufficient insulin secretion of the differentiated BMSCs in vitro prevent their clinical use in diabetes treatment. Little is known about the potential of cell replacement therapy with human BMSCs. Previously, we isolated and identified human first-trimester fetal BMSCs (hfBMSCs). Under a novel four-step induction procedure established in this study, the hfBMSCs effectively differentiated into functional pancreatic islet-like cell clusters that contained 62 ± 14% insulin-producing cells, expressed a broad gene profile related to pancreatic islet ß-cell development, and released high levels of insulin (2.245 ± 0.222 pmol/100 clusters per 30 min) and C-peptide (2.200 ± 0.468 pmol/100 clusters per 30 min) in response to 25 mmol/L glucose stimulus in vitro. The pancreatic islet-like cell clusters normalized the blood glucose level of diabetic model mice for at least 9 weeks when xenografted; blood glucose levels in these mice rose abnormally again when the grafts were removed. Examination of the grafts indicated that the transplanted cells survived in recipients and produced human insulin and C-peptide in situ. These results demonstrate that hfBMSCs derived from a human first-trimester abortus can differentiate into pancreatic islet-like cell clusters following an established four-step induction. The insulin-producing clusters present advantages in cell replacement therapy of type 1 diabetic model mice.

In vitro production of haploid sperm cells from male germ cells of foetal cattle.

The purpose of this study was to isolate the foetal cattle male germ cells (mGCs) and then induce them into sperm cells. The mGCs were purified and enriched by a two-step plating method based on the different adherence velocities of mGCs and somatic cells. The percentage of the vasa and the c-kit positive cells were 95.34+/-2.25% and 53.3+/-1.03% by using flow cytometry analysis (FCA), respectively. In feeder-free culture system, the half-suspending cells appeared and formed a 16-cell rosary in medium after the mGCs were cultured for 6-8 days. On immunocytochemical staining during the second passage, some single cells adhering to the plate appeared to be both Oct-4 and alpha6-integrin positive. During the third passage, the mGCs were induced for 48 h by retinol acid (RA) on Sertoli cell-feeder layer, followed by 5-7 days culture in an RA-free medium. Some elongated sperm-like cells appeared in the medium at this stage. We found that the most effective concentration of RA for the inducement was 10(-7)moll(-1) (P<0.01). The haploid cells in suspension were identified by FCA. The elongated sperm-like cells showed proacrosome-like structure and the flagellum with fibre construct under electron microscopy. The mRNA of outer dense fibre-3 (ODF-3) and transcription protein-1 (TP-1) could be detected in the suspended cells by using reverse transcription polymerase chain reaction (RT-PCR). About 23.1% bovine oocytes could be activated to perform cleavage by intracytoplasmic injection with the sperm-like cells, but embryos did not further develop. Our investigation further demonstrated that foetal cattle mGCs could be induced in vitro into haploid sperm in the short term.

Effect of 5-azacytidine induction duration on differentiation of human first-trimester fetal mesenchymal stem cells towards cardiomyocyte-like cells.

The aim of this study is to investigate effects of 5-azacytidine (5-aza) induction duration on differentiation of bone marrow mesenchymal stem cells (MSCs) from human first-trimester abortus (hfMSCs) towards cardiomyocyte-like cells. hfMSCs were stimulated with 10 micromol/l 5-aza for 24 h (group A), 48 h (group B) and 21 days (group C), respectively. During the induction, 30-40% of the cells gradually enlarged, elongated, connected with adjoining cells and formed myotube-like structures, branches and string-bead-like nuclei. Some of the cells congregated into cell clusters or strips. After the induction, numerous myofilaments in the cytoplasm and conjunction of intercalated disc-like structure between adjoining cells were observed. The induced cells expressed messenger ribonucleic acids (mRNAs) and proteins of myocardium-specific alpha-actin, sarcomeric beta-myocin heavy chain and troponin-T. The positive cell percentages for the three antigens in group C were each significantly higher than those antigens in group A and B (P<0.01) and the cell population doubling time (PDT) of group C was longer than those of group A and B (P<0.01). These indicate that 21-d induction with 10 micromol/l 5-aza slows down proliferation speed of hfMSCs but increases differentiation rate of hfMSCs into cardiomyocyte-like cells if compared with 24-48 h induction.

[Study on pluripotency and cultivation of ES-like cells derived from male germ stem cells of bovine fetuses].

Male germ stem cells (mGSCs), which is in testis after sex differentiation, derive from primordial germ cells. In this study, bovine mGSCs were isolated from testis of 20 weeks fetuses. Number of CD9 positive cells of the cells through two-steps adhering plates velocity different was 95.8% by flow cytometer. The carina-type cells clones and the plane-type cells clones appeared in co-cultured system. One cells lines had been successively maintained for 4 passages, and the cells clusters showed AKP positive staining. The cells clusters showed nest-shape in third passage showed SSEA1 and Oct-4 positive staining. These cells can also spontaneously differentiate into c-kit positive staining germ cells, and the cells were directional induced to formaactin positive staining cardiac-like cells cluster and NF positive staining neuron-like cells. The conclusion showed that male germ stem cells from 20 weeks bovine fetuses could be in vitro formed like embryonic stem cells.