Whole-genome resequencing of the native sheep provides insights into the microevolution and identifies genes associated with reproduction traits.

BACKGROUND: Sheep genomes undergo numerous genes losses, gains and mutation that generates genome variability among breeds of the same species after long time natural and artificial selection. However, the microevolution of native sheep in northwest China remains elusive. Our aim was to compare the genomes and relevant reproductive traits of four sheep breeds from different climatic environments, to unveil the selection challenges that this species cope with, and the microevolutionary differences in sheep genomes. Here, we resequenced the genomes of 4 representative sheep breeds in northwest China, including Kazakh sheep and Duolang sheep of native breeds, and Hu sheep and Suffolk sheep of exotic breeds with different reproductive characteristics. RESULTS: We found that these four breeds had a similar expansion experience from ~ 10,000 to 1,000,000 years ago. In the past 10,000 years, the selection intensity of the four breeds was inconsistent, resulting in differences in reproductive traits. We explored the sheep variome and selection signatures by FST and θπ. The genomic regions containing genes associated with different reproductive traits that may be potential targets for breeding and selection were detected. Furthermore, non-synonymous mutations in a set of plausible candidate genes and significant differences in their allele frequency distributions across breeds with different reproductive characteristics were found. We identified PAK1, CYP19A1 and PER1 as a likely causal gene for seasonal reproduction in native sheep through qPCR, Western blot and ELISA analyses. Also, the haplotype frequencies of 3 tested gene regions related to reproduction were significantly different among four sheep breeds. CONCLUSIONS: Our results provide insights into the microevolution of native sheep and valuable genomic information for identifying genes associated with important reproductive traits in sheep.

[Characteristics, Sources, and SOAP of VOCs During Winter in Jiyuan].

Haze pollution events often occur in the heavy industry city of Jiyuan. Volatile organic compounds (VOCs) are the precursors of secondary organic aerosol (SOA), which accounts for 15%-20% of particulate matter (PM2.5). Here, PM2.5, O3, VOCs, and trace gases were monitored by using online instruments in Jiyuan from December 1st to 31st. The characteristics, sources, and secondary organic aerosol potential (SOAP) of VOCs were analyzed. The mean concentrations of TVOC were (54.3±27.5)×10-9. Alkanes, halocarbons, and alkynes were the predominant VOCs. The positive matrix factorization model was used to identify and apportion VOCs sources. Eight major sources of VOCs were identified, which included liquefied petroleum gas (LPG) or natural gas (NG), the polyvinyl chloride (PVC) industry, vehicular exhaust, the coking industry, solvent usage, industry, technological process, and fuel evaporation. The SOAP of aromatics was the largest. Among them, BTEXs were the dominant contributors to SOAP.

Insights into site-specific influences of emission sources on accumulation of heavy metal(loid)s in soils by wheat grains.

Excessive accumulation of heavy metal(loid)s in agricultural environment usually originates from anthropogenic activities. Both large diversities of emission sources and complexity of plant accumulation challenge the understanding of the site-specific effects of emission sources on heavy metal(loid)s in wheat grains. Herein, both soil samples and wheat grain samples (n = 80) were collected from the farmland of Jiyuan City, China. Soil and grain burdens of heavy metal(loid)s were determined by inductively coupled plasma mass spectrometry (ICP-MS) and/or X-ray fluorescence spectrometry (XRF). The quotients (Q) were developed to indicate relative impacts of industrial plants and traffic to soil sites. Principal component analysis-absolute principal component scores-multivariate linear regression (PCA-APCS-MLR) analysis was conducted to reveal the source contributions to heavy metal(loid)s in grains, considering Q values, soil, and wheat grain data. Results showed that contributions of main sources and factors drastically varied with soil sites, and usually overlapped to different extents. For grain Cd and grain Pb, natural soil silicate (0.066/0.104 mg/kg) and iron-bearing minerals (- 0.044/ - 0.174 mg/kg) contributed to high extents, while metal smelting activities (0.018/0.019 mg/kg) and agronomic activities (- 0.017/ - 0.019 mg/kg) unexpectedly posed low or moderate contributions. The pH-mediated availability of soil Cd (0.035 mg/kg) and the sand-dust weather (0.028 mg/kg) also made considerable contributions to grain Cd. For grain As, both natural soil iron-bearing (- 0.048 mg/kg) and silicate minerals (- 0.013 mg/kg) made negative contributions. The results benefit to the decision-making of pollution remediation of farmland soils in the regional scales.

Polymorphisms and association of GRM1, GNAQ and HCRTR1 genes with seasonal reproduction and litter size in three sheep breeds.

Litter size is one of the important economic traits of livestock. Seasonal oestrus, ovulation and lambing of sheep have severely restricted the development of sheep farming in Xinjiang, China. The purpose of this study was to investigate the polymorphisms and genetic correlation between GRM1, GNAQ and HCRTR1 genes and the seasonal reproduction and litter size in three sheep breeds. The DNA mixed pool sequencing and PCR-SSCP methods were used to detect single nucleotide polymorphisms (SNPs) of GRM1, GNAQ and HCRTR1 genes in seasonal oestrous (Kazakh and Chinese Merino [Xinjiang Junken type]) and perennial oestrous (Hu) sheep breeds. The association between genetic polymorphism and litter size was also analysed. The results showed that T945C in exon 2 of GRM1 gene, C589T in exon 2 of HCRTR1 gene and A191G in exon 2 of GNAQ gene were identified by Sanger sequencing, and three genotypes were existed in each SNP site, which all belonged to the synonymous mutation. GRM1 (CC), GNAQ (GA) and HCRTR1 (TC) were the dominant genotypes of seasonal reproduction and litter size in Kazakh sheep and Chinese Merino sheep, respectively, while, in perennial oestrous Hu sheep populations, the dominant genotypes were GRM1 (TC), GNAQ (GA) and HCRTR1 (TC), respectively, and association analysis also confirmed the results. The above results implied that GRM1, GNAQ and HCRTR1 genes are significantly associated with lambing traits in Kazakh, Chinese Merino and Hu sheep. Among them, the locus of GRM1 (T945C), GNAQ (A191G) and HCRTR1 (C589T) might be considered as a potential molecular marker, which controls seasonal reproduction and litter size in sheep.

Targeting GNAQ in hypothalamic nerve cells to regulate seasonal estrus in sheep.

Kazakh sheep are typical seasonal estrus animals. Their reproductive system regulation mainly involves the complex regulation of the hypothalamic-pituitary-gonadal axis (HPGA), which is also closely related to reproductive hormone secretion. Gonadotropin-releasing hormone (GnRH), synthesized and secreted by the hypothalamus, is the key to controlling sheep reproductive activity. We studied how GNAQ (G protein subunit alpha q) regulates estrus in sheep by controlling GnRH expression and secretion. We used hypothalamic nerve cells as the research model. GNAQ overexpression and RNA interference vectors were constructed and transfected into the hypothalamic nerve cells of fetal Kazakh sheep. qPCR, western blotting, and enzyme-linked immunosorbent assay were used to detect GNAQ gene expression in Kazakh ewe tissues and analyze its regulatory effect on GnRH expression in the hypothalamic nerve cells. The fetal sheep hypothalamic nerve cells were successfully isolated and cultured. qPCR and cell immunofluorescence showed that the purity of positive cells was >95%. The tissue expression profile showed that there were different degrees of GNAQ gene expression in the Kazakh ewe tissue. Expression levels were relatively higher in the hypothalamus, pituitary, brain, and uterine tissues. When GNAQ expression was downregulated in the hypothalamic nerve cells, the upstream genes KISS1 (kisspeptin), GPR54 (KISS1 receptor), and ER (estrogen receptor) were all upregulated, as were the downstream genes PLCB1 (phospholipase C beta 1), PRKCB (protein kinase C beta), and GNRH. At the same time, GnRH secretion levels were also upregulated. GNAQ regulated its downstream gene PLCB1 in the hypothalamic nerve cells, and directly regulated GnRH expression and secretion through the calcium and PRKC signaling pathways. GNAQ also regulated kisspeptin expression, subsequently regulating GnRH expression and secretion indirectly through the kisspeptin-GPR54 signaling pathway. Our results are of great importance for improving the reproductive performance of seasonal-estrus sheep.

Changeable effects of coexisting heavy metals on transfer of cadmium from soils to wheat grains.

Cadmium (Cd) and other heavy metals usually coexist in soils. Effects of coexisting heavy metals on the accumulation and transfer of Cd in field soils by wheat remain poorly understood. Here we revealed changeable effects of coexisting Pb, Zn and Cu on the Cd transfer from soils to wheat grains. Soil burdens of Cd were found to exhibit positive correlations (r = 0.459-0.946) with those of coexisting Pb, Zn and Cu (particularly Pb). Effects of three coexisting metals on to the uptake of Cd by wheat varied in the directions and/or extents with types of metals and transfer processes of Cd. Coexisting Zn inhibited the uptake of Cd by wheat grains to higher extent than Pb and Cu. Soil Zn, along with soil Cd, soil pH and soil Ca, was used to construct the predictive model of grain Cd (R2 = 0.868). External verifications of the model on 572 datasets of large representation performed well. The predictive accuracy was about 54%, 73% and 89% for a factor of 1, 2 and 5 above and below the ideal fit, respectively. This finding has practical interest in risk assessments and remediation measures of Cd-contaminated soil sites in regional scales.

Comparative Analysis and Identification of Differentially Expressed microRNAs in the Hypothalamus of Kazakh Sheep Exposed to Different Photoperiod Conditions.

MicroRNAs (miRNA) plays an important role in several mammalian biological regulatory processes by post-transcriptionally regulating gene expression. However, there is little information on the miRNAs involved in the photoperiodism pathway that controls seasonal activity. To enhance our knowledge on the effect of different photoperiod conditions on miRNA, we divided Kazakh sheep into two groups: one exposed to a long photoperiod (LP, 16L:8D) and another with exposed to a short photoperiod (SP, 8L:16D) under supplemental feeding conditions. Further we compared the related miRNAs and target genes between the two groups. Fifteen differentially expressed miRNAs were identified, which were associated with 310 regulatory pathways covering photoperiodism, reproductive hormones, and nutrition. The miR-136-GNAQ pair was selected and validated as a differentially expressed, and a dual-luciferase reporter assay showed that the negative feedback loop existed between them. Examination of the expression profile revealed that the GNAQ expression was low in the estrous females both under LP and SP conditions, but high expression of GNAQ was observed in the anestrous females under LP conditions. Moreover, functional analysis revealed that KISS1 and GnRH expression was upregulated when GNAQ expression was downregulated in the hypothalamic cells, whereas DIO2 and TSHB expression was downregulated. Thus, miR-136-GNAQ might act as a switch in the regulation of seasonal estrus under different photoperiod conditions. These findings further enrich our understanding of the relationship between miRNAs and seasonal regulation of reproductive activity. Furthermore, our study provides novel insights into the miRNA-mediated regulatory mechanisms for overcoming photoinhibition in the seasonally breeding mammals, such as Kazakh sheep.

Corrigendum: Brucella-Induced Downregulation of lncRNA Gm28309 Triggers Macrophages Inflammatory Response Through the miR-3068-5p/NF-κB Pathway.

[This corrects the article DOI: 10.3389/fimmu.2020.581517.].

Identification and validation of key miRNAs and miRNA-mRNA regulatory network associated with uterine involution in postpartum Kazakh sheep.

MicroRNAs (miRNAs) are widely expressed in different mammalian tissues and exert their biological effects through corresponding target genes. miRNA target genes can be rapidly and efficiently identified and screened by combining bioinformatics prediction and experimental validation. To investigate the possible molecular regulatory mechanisms involving miRNAs during uterine involution in postpartum ewes, we used Illumina HiSeq sequencing technology to screen for the number and characteristics of miRNAs in faster uterine involution and normal uterine involution group. A total of 118 differentially expressed miRNAs, including 33 known miRNAs and 85 new miRNAs, were identified in the hypothalamic library, whereas 54 miRNAs, including 5 known miRNAs and 49 new miRNAs, were identified in the uterine library. Screening with four types of gene prediction software revealed 73 target genes associated with uterine involution, and subsequently, GO annotation and KEGG pathway analysis were performed. The results showed that, in the hypothalamic-uterine axis, uterine involution in postpartum ewes might primarily involve two miRNA-target gene pairs, namely, miRNA-200a-PTEN and miRNA-133-FGFR1, which can participate in GnRH signal transduction in the upstream hypothalamus and in the remodeling process at the downstream uterus, through the PI3K-AKT signaling pathway to influence the recovery of the morphology and functions of the uterus during the postpartum period in sheep. Therefore, identification of differentially expressed miRNAs in this study fills a gap in the research related to miRNAs in uterine involution in postpartum ewes and provides an important reference point for a comprehensive understanding of the molecular mechanisms underlying the regulation of postpartum uterine involution in female livestock.

Comparative analysis of differentially expressed miRNAs related to uterine involution in the ovine ovary and uterus.

To examine the possible miRNA molecular regulatory mechanisms during maternal uterine involution after delivery, we selected ovary and uterus tissues that are structurally connected as experimental materials. We employed Illumina HiSeq sequencing to screen and analyze the quantity and characteristics of miRNA in postpartum ewes in the methylergometrine-treated group and physiological saline control group. Results showed that 16 miRNAs were identified in the ovary libraries, including 4 known miRNAs and 12 novel miRNAs. In the uterus libraries, 54 miRNAs were identified, which included 5 known miRNAs and 49 novel miRNAs. At the same time, target gene prediction, GO annotation, and KEGG signaling pathway enrichment analysis were employed. We found that maternal uterine involution after delivery may involve two miRNA-target gene pairs, i.e., miRNA-200a-ZEB1 and YAP1. The YAP1/Hippo signaling pathway is used to construct an ovary-uterine axial regulatory mechanism to regulate the restoration of postpartum maternal uterine morphology and function. In view of this, the identification of miRNAs with significant differences in this study fills a gap in research on miRNAs associated with regulation of postpartum uterine recovery in ewes and provided an important reference for comprehensive understanding and in-depth research on the regulatory molecular network mechanism for postpartum uterine involution in small ruminants.

miR-509-5p anti-infection response for mycoplasma pneumonia in sheep by targeting NF-κB pathway.

MicroRNAs play a key role in Mannan-binding lectin-mediated resistance to Mycoplasma ovipneumoniae pneumonia, by regulating the translation of mRNAs of target genes, thereby regulating the immune response. Additionally, TRAF6 is a key molecule in Toll-like receptor signal transduction, which mediates inflammation and apoptosis signaling pathways and is widely involved in inflammation and immune response. While the molecular regulation mechanism has not been reported. In this study, we screened differentially expressed miRNAs and genes of Anti-infection for M. pneumonia on Sheep, through relevant bioinformatics analysis. Further, the effect of differential expression of NF-κB signaling pathway related genes on the molecular mechanism of M. pneumonia was detected. We used miRNA-mRNA integrated analysed, the target gene TRAF6 of miR-509-5p was selected. TRAF6 dual luciferase reporter vector was co-transfected into HEK 293T cells and primary sheep respiratory mucosal epithelial cells to detect changes in luciferase activity. qRT-PCR was used to analyze the effect of miR-509-5p on the expression and regulation of TRAF6 and other genes related to the NF-κB signaling pathway. The result confirmed that TRAF6 was a target gene of miR-509-5p. Compared with miR-509-5p-NC group, the luciferase activity of miR-509-5p group was significantly down-regulated (P < 0.01). Further, in sheep respiratory mucosal epithelial cells, miR-509-5p mimic could significantly down-regulate the fold change value of TRAF6 (P < 0.01). On the contrary, miR-509-5p-inhibitor up-regulated the fold change value of TRAF6 (P < 0.05). Interestingly, the expression levels of other genes were different. Among them, miR-509-5p mimic significantly up-regulated TLR4 and IRAK4 (P < 0.05), significantly down-regulated TAK1 (P < 0.05) and NF-κB (P < 0.01). miR-509-5p-inhibitor significantly up-regulated NF-κB (P < 0.05) and TAK1 (P < 0.01). miR-509-5p targets TRAF6 to affect the expression of downstream genes, which negatively regulates the NF-κB pathway, thereby affecting the inflammatory response.

Thiol-functionalized nano-silica for in-situ remediation of Pb, Cd, Cu contaminated soils and improving soil environment.

Heavy metal contamination has been threatening the health of human beings. To decrease the bio-toxicity of heavy metals, a thiol-functionalized nano-silica (SiO2-SH) was adopted to remediate the soil contaminated by lead (Pb), cadmium (Cd) and copper (Cu). The remediation effect of SiO2-SH on contaminated soils was investigated by the uptake of the heavy metals into lettuce and pakchoi in pot experiment. The bio-toxicity of the SiO2-SH was evaluated, and its immobilization mechanisms were proposed by the fraction distribution of Cd, Pb and Cu. It was found that the SiO2-SH can significantly reduce the uptake of Cd, Pb, Cu into pakchoi by 92.02%, 68.03%, 76.34% and into lettuce by 89.81%, 43.41%, 5.76%, respectively. The chemical species analyses of Cd, Pb, Cu indicate SiO2-SH can transform the heavy metal in acid soluble states into reducible fraction and oxidizable fraction, thereby inhibiting the extraction of heavy metals into soil solution. The concentrations of microbial biomass carbon, organic matter, and cation exchange capacity of the soil increased while the soil bulk density decreased after remediation. Those changes demonstrate that SiO2-SH not only has no bio-toxic impact on the soil environment but also improves the soil environment, which proves the prepared SiO2-SH is environmental-friendly. The SiO2-SH could be a promising amendment for heavy metal contaminated soils.

Comparative profiling of the resistance of different genotypes of mannose-binding lectin to Mycoplasma pneumoniae infection in Chinese Merino sheep based on high-throughput sequencing technology.

Mannose-binding lectin (MBL) glycoproteins in blood can selectively recognise lectins on the surface of bacteria, and play an important role in natural immunity. Micro RNAs (miRNAs) are key molecules that regulate gene expression at the post-transcriptional level in vivo, and their pathways are specific and effective. Previous studies indicate that small RNAs such as miRNAs perform regulatory roles in immunology. Herein, we investigated differential expression of miRNAs during MBL protein immunotherapy in sheep following treatment with different MBL genotypes (resistant and susceptible), and identified miRNAs linked to different target genes and pathways. RNA was extracted from liver tissue of resistant and susceptible sheep, miRNAs were identified by high-throughput sequencing, and differentially expressed miRNAs were analysed by SOAP to predict target genes and biological pathways. Results: Some miRNAs (oar-mir-143, oar-mir-10b, oar-mir-382, oar-mir-432 and oar-mir-379) were up-regulated, while others were down-regulated. GPATCH3 and DNAJC5 were predicted target genes of oar-mir-379, DMRT1 and GATA4 were linked to oar-mir-382, and oar-mir-432 was associated with STAT2, DMRT1 and ATG16L1. Identification of miRNAs differentially expressed in resistant and susceptible sheep may expand our understanding of miRNAs in immune regulation, and the role of MBL in innate immunity.

Characterization of muscle development and gene expression in early embryos of chicken, quail, and their hybrids.

Myogenesis is a complex, regulated process that involves myoblast proliferation, migration, adhesion, and fusion into myotubes. To investigate early development of embryonic muscles and the expression of regulatory genes during myogenesis in chicken, quail and their hybrids, meat-breeding cocks and egg-breeding cocks were selected as male parents, quails were used as female parents. Their offspring were meat and egg hybrids via Artificial insemination. We measured expression of MUSTN1, IGF-1, and PDK4 using qRT-PCR. We examined muscle fiber diameter using scanning electron microscopy. The results showed that muscle development was two days slower in chicken, egg hybrid, and meat hybrid than in quail. Muscle fiber spacing was the largest in chicken, followed by meat hybrid, egg hybrid, and quail. A similar trend was obtained for muscle fiber diameter. Additionally, muscle fiber diameter increased with embryogenesis. The sarcomere was present on day 17 of incubation in quail, but not in the other species. MUSTN1 could up-regulated IGF-1 by activating PI3K/Akt. IGF-1 expression was consistent with myoblast proliferation and myotube fusion. PDK4 was expressed from E7 to E17. The first peak was reached on E10, egg hybrid and meat hybrid reached their peak at E15. PDK4 is involved in the early proliferation and differentiation of muscle, thereby affecting muscle growth and development. Our findings demonstrated that MUSTN1, IGF-1 and PDK4 genes are expressed to varying levels in breast muscle of chicken, quail, egg hybrid and meat hybrid during the embryonic period. Interestingly, with increasing embryonic age, muscle development was approximately 48 h faster in quail than in other species. We speculated that MUSTN1, IGF-1 and PDK4 genes may be the main candidate genes that cause differences in poultry muscle traits, but the molecular regulation mechanisms need to be further studied. Our findings shed some light on the avian embryo muscle formation and molecular breeding of poultry muscle traits, which provide theoretical basis for poultry breeding.

Brucella-Induced Downregulation of lncRNA Gm28309 Triggers Macrophages Inflammatory Response Through the miR-3068-5p/NF-κB Pathway.

Objectives: The underlying mechanism of the inflammatory response against Brucellosis caused by Brucella remains poorly understood. This study aimed to determine the role of long non-coding RNAs (lncRNAs) in regulating of inflammatory and anti-Brucella responses. Materials and methods: Microarray analysis was performed to detect differentially expressed lncRNAs in THP-1 cells infected with an S2308 Brucella strain. The candidate lncRNAs were screened using bioinformatic analysis and siRNAs; bioinformatic prediction and luciferase reporter assay were also conducted, while inflammatory responses was assessed using RT-qPCR, western blot, immunofluorescence, ELISA, HE, and immunohistochemistry. Results: The lncRNA Gm28309 was identified to be involved in regulating inflammation induced by Brucella. Gm28309, localized in the cytoplasm, was down-expressed in RAW264.7 cells infected with S2308. Overexpression of Gm28309 or inhibition of miR-3068-5p repressed p65 phosphorylation and reduced NLRP3 inflammasome and IL-1ß and IL-18 secretion. Mechanistically, Gm28309 acted as a ceRNA of miR-3068-5p to activate NF-κB pathway by targeting κB-Ras2, an inhibitor of NF-κB signaling. Moreover, the number of intracellular Brucella was higher when Gm28309 was overexpressed or when miR-3068-5p or p65 was inhibited. However, these effects were reversed by the miR-3068-5p mimic. Conclusions: Our study demonstrates, for the first time, that LncRNAs are involved in regulating immune responses during Brucella infection, and Gm28309, an lncRNA, plays a crucial role in activating NF-κB/NLRP3 inflammasome signaling pathway.

Protective Effect of Resveratrol Improves Systemic Inflammation Responses in LPS-Injected Lambs.

Highly intensive livestock production often causes immune stress to animals, which makes them more susceptible to infections. The aim of this study was to examine whether resveratrol (Res) alleviates inflammation in lambs. In Experiment 1, 16 male lambs were injected with lipopolysaccharides (LPS) at an initial dose of 0.25, 1.25, and 2.5 µg/kg body weight (BW) for 9 days. Average daily gain and blood parameters were measured and clinical symptoms were recorded. In Experiment 2, 20 male lambs were injected intravenously with LPS (0 mg/kg) + Res (0 mg), LPS (2.5 µg /kg) + Res (0 mg, 82.5 mg, 165 mg, 330 mg), 4 h after LPS injection. Jugular blood was collected from each lamb to determine white blood cell (WBC) counts and the expression of inflammatory genes. In Experiment 1, all LPS-treated lambs showed clinical signs of sickness including rhinorrhea, lethargy, and shivering, and systemic inflammatory responses of increased inflammatory genes levels and cortisol concentration. The lambs had increased respiratory and heart rates and rectal temperature and decreased average daily gain and feed intake. In Experiment 2, resveratrol significantly reduced WBCs and the expression levels of several genes associated with inflammation response (TLR4, NF-κB, c-jun) and inhibited the signaling cascades of NF-κB and MAPKs by down-regulating the expression levels of inflammatory cytokines (IL-1ß, IL-4, IL-6, TNF-α, IFN-γ) induced by LPS. Resveratrol attenuated the LPS-evoked inflammatory responses in lambs by suppressing expression levels of inflammatory cytokines, and blocking NF-κB and MAPK signaling pathways.

The effect of GNAQ methylation on GnRH secretion in sheep hypothalamic neurons.

Kazakh sheep are seasonal estrous animals, and gonadotropin-releasing hormone (GnRH) is the key to fertility regulation. The nutritional level has a certain regulatory effect on estrous, and vitamin B folate plays a role in DNA methylation, directly participating in the process. The goal of this study was to determine whether folate is involved in GnAQ methylation and its effect on GnRH secretion. The hypothalamic neurons of Kazakh fetal sheep were treated with folate at concentrations of 0 mg/mL, 4 mg/mL, 40 mg/mL, and 80 mg/mL. GnAQ promoter methylation, DNMT1, GnAQ expression, and GnRH secretion following treatment with different concentrations of folate were analyzed. One CpG site was methylated in the GNAQ promoter with 40 mg/mL folic acid, and no CpG methylation was found in the other groups. GnAQ expression was related to folate concentration and showed a trend of increasing first and then decreasing. The GnRH expression level in the 40 mg/mL folate group was significantly higher than in the other three groups ( P < .05). These results demonstrate that the appropriate folate concentration promoted GANQ promoter methylation, which in turn affected GnRH secretion.

Simultaneous isolation of mesenchymal stem cells and endothelial progenitor cells derived from murine bone marrow.

Mesenchymal stem or stromal cells (MSCs) are identified as sources of pluripotent stem cells with varying degrees of plasticity. Endothelial progenitor cells (EPCs) originate from either bone marrow (BM) or peripheral blood and can mature into cells that line the lumen of blood vessels. MSC and EPC therapies exhibit promising results in a variety of diseases. The current study described the simultaneous isolation of EPCs and MSCs from murine BM using a straightforward approach. The method is based on differences in attachment time and trypsin sensitivity of MSCs and EPCs. The proposed method revealed characteristics of isolated cells. Isolated MSCs were positive for cell surface markers, cluster of differentiation (CD)29, CD44 and stem cell antigen-1 (Sca-1), and negative for hematopoietic surface markers, CD45 and CD11b. Isolated EPCs were positive for Sca-1 and vascular endothelial growth factor receptor 2 and CD133. The results indicate that the proposed method ensured simultaneous isolation of homogenous populations of MSCs and EPCs from murine BM.

Comparative profiling of differentially expressed microRNAs in estrous ovaries of Kazakh sheep in different seasons.

Seasonal estrus is a critical limiting factor for animal fecundity. However, estrus occurs in some seasonally estrous sheep in the non-breeding season, and this phenomenon involves changes in ovarian biology. Previous studies indicated that small RNAs, such as microRNAs (miRNAs), play important regulatory roles in ovarian biology. Differentially expressed miRNAs in the ovaries of estrous sheep were identified using Solexa sequencing technology. A total of 423 known miRNAs were identified in ovaries of estrous sheep in the breeding season and non-breeding season. In the comparison of these two groups, 48 miRNAs were identified that were differentially expressed between the two groups (including 5 up-regulated and 43 down-regulated miRNAs). KEGG pathway analysis revealed that the target genes of some differentially expressed miRNAs were involved in pathways related to reproductive hormone signaling and follicular development. Furthermore, the levels of estradiol (E2), progesterone (P4), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were lower in anestrus sheep than in sheep during the breeding season. Upon combining the pathway enrichment analysis, target gene expression and hormone measurement results, we suggest that these differentially expressed miRNAs might influence ovarian activity in the non-breeding season by regulating the above pathways. The identification of miRNAs that are differentially expressed between ovines in the breeding season and non-breeding season will contribute to our understanding of the role of miRNAs in estrus regulation, and these data may provide a basis for regulating estrus in sheep during the non-breeding season.

Identification and analysis of microRNAs-mRNAs pairs associated with nutritional status in seasonal sheep.

Given the important role of nutritional status for reproductive performance, we aimed to explore the potential microRNA (miRNA)-mRNA pairs and their regulatory roles associated with nutritional status in seasonal reproducing sheep. Individual ewes were treated with and without 0.3 kg/day concentrates, and the body condition score, estrus rate, and related miRNAs and target genes were compared. A total of 261 differentially expressed miRNAs were identified, including 148 hypothalamus-expressed miRNAs and 113 ovary-expressed miRNAs, and 349 target genes were predicted to be associated with nutritional status and seasonal reproduction in sheep. Ultimately, the miR-200b-GNAQ pair was screened and validated as differentially expressed, and a dual luciferase reporter assay showed that miR-200b could bind to the 3'-untranslated region of GNAQ to mediate the hypothalamic-pituitary-ovarian axis. Thus, miR-200b and its target gene GNAQ likely represent an important negative feedback loop, providing a link between nutritional status and seasonal reproduction in sheep toward enhancing reproductive performance and productivity.