Dissecting the initiation of female meiosis in the mouse at single-cell resolution.

Meiosis is one of the most finely orchestrated events during gametogenesis with distinct developmental patterns in males and females. However, the molecular mechanisms involved in this process remain not well known. Here, we report detailed transcriptome analyses of cell populations present in the mouse female gonadal ridges (E11.5) and the embryonic ovaries from E12.5 to E14.5 using single-cell RNA sequencing (scRNA seq). These periods correspond with the initiation and progression of meiosis throughout the first stage of prophase I. We identified 13 transcriptionally distinct cell populations and 7 transcriptionally distinct germ cell subclusters that correspond to mitotic (3 clusters) and meiotic (4 clusters) germ cells. By analysing cluster-specific gene expression profiles, we found four cell clusters correspond to different cell stages en route to meiosis and characterized their detailed transcriptome dynamics. Our scRNA seq analysis here represents a new important resource for deciphering the molecular pathways driving female meiosis initiation.

[Discussion on research idea of quality marker of Salvia miltiorrhiza based on biosynthetic pathway of tanshinone compounds].

Based on the theory of Q-marker, the hairy root of Salvia miltiorrhiza and S. miltiorrhiza in many provinces were studied. The relative expressions of SmCPS, SmKSL and CYP76AH1 genes in hairy roots were detected by real-time fluorescence quantitative PCR and the contents of tanshinoneⅡ_A, cryptotanshinone, tanshinoneⅠ, 1,2-dihydrotanshinone, ferruginol and miltiradiene were detected by UPLC and GC-MS, respectively. Statistical analysis shows as fllows: in the hairy root of S. miltiorrhiza, the content of miltiradiene and ferruginol is positively correlated with the content of tanshinone compounds in the downstream, and the relative expression of important genes in the biosynthetic pathway of tanshinone can reflect the content of tanshinone compounds to a certain extent; in many provinces of S. miltiorrhiza, the content of ferruginol and tanshinone compounds can also be found that there is a positive correlation between the contents. Based on the biosynthetic pathway of tanshinone compounds, which is a special index component in S. miltiorrhiza, this study focused on the important relationship between the upstream gene, the middle intermediate compound and the downstream tanshinone compound content of the biosynthetic pathway, and explored the possible research ideas of improving the quality marker system of S. miltiorrhiza, and then provided the possible research ideas for understanding and studying the quality marker of traditional Chinese medicine from the biosynthetic pathway.

Genomic Signatures of Selection Associated With Litter Size Trait in Jining Gray Goat.

Litter size (LS), an important economic trait in livestock, is so complicate that involves many aspects of reproduction, the underlying mechanism of which particularly in goat has always been scanty. To uncover the genetic basis of LS, the genomic sequence of Jining Gray goat groups (one famous breed for high prolificacy in China) with LS 1, 2, and 3 for firstborn was analyzed, obtaining 563.67 Gb sequence data and a total of 31,864,651 high-quality single nucleotide polymorphisms loci were identified. Particularly, the increased heterozygosity in higher LS groups, and large continuous homozygous segments associated with lower LS group had been uncovered. Through an integrated analysis of three popular methods for detecting selective sweeps (Fst, nucleotide diversity, and Tajima's D statistic), 111 selected regions and 42 genes associated with LS were scanned genome wide. The candidate genes with highest selective signatures included KIT, KCNH7, and KMT2E in LS2 and PAK1, PRKAA1, and SMAD9 in LS3 group, respectively. Meanwhile, functional terms of programmed cell death involved in cell development and regulation of insulin receptor signaling pathway were mostly enriched with 42 candidate genes, which also included reproduction related terms of steroid metabolic process and cellular response to hormone stimulus. In conclusion, our study identified novel candidate genes involving in regulation of LS in goat, which expand our understanding of genetic fundament of reproductive ability, and the novel insights regarding to LS would be potentially applied to improve reproductive performance.

Preparation, film fabrication and gas-sensitive responsive properties of MWCNTs@PS-b-HTPB5-b-PS conductive polymer nanocomposites.

Novel nanocomposites consisting of polystyrene-block-polybutadienyl polyhexamethylene dicarbamate-block-polystyrene (PS-b-HTPB5-b-PS) and multiwalled carbon nanotubes (MWCNTs) were designed and prepared via noncovalent interactions. Scanning electron microscopy and transmission electron microscopy observations showed that segregated networks of MWCNTs were formed due to the cladding of PS-b-HTPB5-b-PS, presenting a parallel-arranged topology of the MWCNTs in a continuous PS-b-HTPB5-b-PS phase, which improved the dispersibility of the MWCNTs. The nanocomposites were fabricated into vapor sensing elements to detect CH2Cl2 vapor in the environment, exhibiting excellent responsive sensitivity, reproducibility and a low limit of detection (LOD) of 1 ppm when exposed to CH2Cl2 vapor. The chain extension of HTPB overcame the fragility and improved the tenacity of the thin films, and the responsivity was optimized by adjusting the content of the MWCNTs and the length of the PS chains. The newly developed conductive composites can be applied as a promising vapor sensor to accurately monitor CH2Cl2 vapor in the environment.

Metagenomic analysis of gut microbiota alteration in a mouse model exposed to mycotoxin deoxynivalenol.

As one of the most prevalent contaminants in animal and human food, the deleterious effects of trichothecene mycotoxin deoxynivalenol (DON) warrant extensive investigation. Here, to assess the effects of DON exposure to the populations of gut microbiota, four-weeks-old mice were exposed to different doses (1.0 and 5.0 mg/kg) of DON every two days for 14 days. The contents of the cecum were then collected for DNA extraction and metagenomic shotgun sequencing, in order to detect alterations of the gut microbiota. We found that the average body weight and daily gain in the high dose DON treated group decreased. Metagenomic analysis demonstrated that the relative abundance of Firmicutes in the low and Bacteroidetes in the high dose groups increased compared to that in the untreated control group. Moreover, using gene calling and functional annotation, we found that large numbers of biosynthesis and degradation dependent populations were altered. As a result, metabolism pathways including sphingolipid, protein digestion/absorption, and lipoic acid pathways in the high dose DON exposed group dramatically fluctuated in comparison to the control and low dose groups. In addition, metagenomic binning identified ten microbiota genome drafts, with high levels of completeness, that further explain the DON-induced intestinal toxicity. Our findings suggested that DON exposure significantly impacted the microbiota community in the mouse, causing biosynthesis and degradation damage and metabolism pathway disorders.

Copy-number variation in goat genome sequence: A comparative analysis of the different litter size trait groups.

Copy number variation (CNV), as an important component of genomic structural variation (SV), plays essential roles in phenotypic variability, disease susceptibility and species evolution. To investigate whether critical CNVs exist in dairy goats with differing fecundity, we performed genome-wide sequencing of two populations of Laoshan dairy goats with large differences in litter size. After reference genome aligning, CNV calling, and annotation, we screened identified CNVs in the high-fecundity (HF) and low-fecundity (LF) groups to identify discrepant CNVs and their distribution within the genome. Prolactin-related protein 1 and 6 (PRP1 and PRP6), important factors regulating reproductive processes, were demonstrated to be duplicated in the HF group. In summary, based on the differences in CNVs between goats with differing litter sizes, it suggests CNVs may contribute to litter size in Laoshan dairy goats.

Effects of activin A on the transcriptome of mouse oogenesis in vitro.

From the previous research, it has been supported that activin A (ActA) is conducive to ovarian development in vitro. In the present paper, with the aim to identify the molecular pathways through which ActA can influence processes of the fetal and early postnatal oogenesis, we analyzed the transcriptome of embryonic ovaries (12.5 days postcoitum) in vitro cultured with or without ActA for 6 days, as well as the produced oocytes for 28 days, and further compared the gene expression profile with their in vivo counterparts. With the confirmation of designed test, we found that the addition of ActA to the ovary culture tended, generally, to align oocyte gene expression to the in vivo condition, in particular of a number of genes involved in meiosis and epigenetic modifications of histones. In particular, we identified DNA recombination during the oocyte meiotic prophase I and lysine trimethylation of the histone H3K27 during the oocyte growth phase as molecular pathways modulated by ActA.

Ochratoxin A Exposure Impairs Porcine Granulosa Cell Growth via the PI3K/AKT Signaling Pathway.

The mycotoxin ochratoxin A (OTA), a naturally occurring food contaminant, has a toxic effect on the growth and development of follicles in pigs. However, little is known regarding the specific toxic effects of OTA exposure on oocytes and granulosa cells (GCs). In this study, we cultured porcine ovarian GCs and exposed them to OTA in vitro in order to explore the mechanism causing the negative effects. Initially, it was found that OTA exposure inhibited cell viability in a time and dose dependent manner. We also showed that OTA exposure increased oxidative stress, decreased proliferation ratio, and increased apoptosis ratio in GCs. We revealed an important role for the PI3K/AKT signal pathway in GC proliferation and apoptosis by RNA-seq analysis. The results not only showed that OTA treatment significantly affected the expression of genes within the PI3K/AKT pathway but also demonstrated a concrete relationship between the PI3K/AKT pathway and GC cell proliferation and apoptosis. In conclusion, the results demonstrated that OTA exposure impaired porcine GC growth via the PI3K/AKT signaling pathway.

Di (2-ethylhexyl) Phthalate Exposure Impairs the microRNAs Expression Profile During Primordial Follicle Assembly.

This research was performed to estimate the potential effects of Di (2-ethylhexyl) phthalate (DEHP) on changes of ovarian miRNA expression profile during mouse primordial follicle assembly using miRNAs-seq analysis. The ovaries of newborn mice were collected and in vitro cultured with different concentration of DEHP for 72 h. Then they were prepared for miRNAs-seq analysis. The results indicated that DEHP exposure altered ovarian miRNA expression profile of newborn mice. Eighteen differentially expressed miRNAs were screened after 100 µM DEHP exposure. The target mRNAs of differentially expressed miRNAs were predicted and further analyzed through gene ontology (GO) enrichment analysis and pathway enrichment analysis. Our results showed that the differentially expressed miRNAs from DEHP exposure can regulate ovarian development by targeting mRNAs involved in MAPK, mTOR, FoxO signaling pathways. Three miRNAs of miR-32-5p, miR-19a-3p, and miR-141-3p were randomly selected from the differentially expressed miRNAs to quantify their expression level by miRNA qRT-PCR. The results of qRT-PCR and miRNA-seq were consistent. Considering one of its target gene PTEN of miR-19a-3p and the decreased level of pAKT and increased Bax/Bcl-2 under DEHP exposure, we speculated that the altered expression of miR-19a-3p by DEHP exposure affected mouse primordial follicle assembly via PI3K/AKT1/mTOR signaling pathway. Epigenetic changes are one of the most important targets of toxicant exposure. The effects of DEHP exposure on microRNA (one of the epigenetic regulators) expression profile were uncovered to enrich the research on relationship of epigenetics and toxicant exposure.

Phosphatidylcholine could protect the defect of zearalenone exposure on follicular development and oocyte maturation.

Zearalenone (ZEA) is a well-known exogenous endocrine disruptor and can lead to severe negative effects on the human and animal reproductive process. Using a follicle culture model, we have previously shown that ZEA exposure significantly affected the follicular development and antrum formation but the underlying mechanisms are not well known. Therefore, in this study, we explored the metabolomic changes of granulosa cell (GC) culture media with or without ZEA exposure. The results showed that ZEA significantly increased phosphatidylcholine or phosphatidyl ethanolamine adducts in culture medium. A comprehensive analysis with the metabolome data from follicular fluid of small and large antral follicles showed that lyso phosphatidylcholine (LPC) was accumulated during follicle growth, but was depleted by ZEA exposure. Exogenous supplement with LPC to the follicle growth media or oocyte maturation media can partly protect the defect of ZEA exposure on follicular antrum formation and oocyte maturation. Taken together, our results demonstrate that ZEA exposure hinders the follicular growth and exogenous LPC can practically protect the defect of ZEA on follicular development and oocyte maturation.

Zearalenone exposure elevated the expression of tumorigenesis genes in mouse ovarian granulosa cells.

Zearalenone (ZEA) is one of mycotoxins which are from corn, sorghum and wheat. As an estrogenic compound, ZEA mainly affects animal growth and reproduction with causing abnormal reproduction capability. Previous studies have shown that ZEA poses adverse effects on follicular development, but the mechanism of genetic toxicity of ZEA is not understood. The purpose of this study was to explore the effects of ZEA exposure on granulosa cells which play vital roles during follicular development. Mouse granulosa cells were exposed to 10 µM or 30 µM ZEA for 72 h in vitro, and the differences in gene expression patterns between control and ZEA exposures were analyzed by RNA-seq. The data demonstrated that 30 µM ZEA had a significant effect on the gene expression, especially ZEA exposure increased the expression of many genes related to different kinds of cancers and cancer related pathways like Hippo signaling pathway and the related genes, such as Ccnd1, Smad3, Tead3, Yap1 and Wwtr1. Furthermore, immunohistochemistry confirmed the increase in the protein levels of YAP1, WWTR1 and CCND1 in 30 µM ZEA exposure group. Collectively, this investigation indicated that ZEA exposure promoted the expression of tumorigenesis genes in mouse granulosa cells to.

Differentiation of sow and mouse ovarian granulosa cells exposed to zearalenone in vitro using RNA-seq gene expression.

Zearalenone (ZEA), a natural contaminant found in feed, has been shown to have a negative impact on domestic animal reproduction, particularly in pigs. There are species-specific differences in the ZEA-induced toxicity pattern. Here, we investigated the different biological effects of ZEA exposure on porcine and mouse granulosa cells, using RNA-seq analysis. We treated murine and porcine granulosa cells with 10 µM and 30 µM ZEA during 72 h of culturing, in vitro. The results showed that 10 µM ZEA exposure significantly altered mitosis associated genes in porcine granulosa cells, while the same treatment significantly altered the steroidogenesis associated genes in mouse granulosa cells. Exposure to 30 µM ZEA resulted in significantly up-regulated expression of inflammatory related genes in porcine granulosa cells as well as the cancer related genes in mouse granulosa cells. Similarly, 30 µM ZEA exposure significantly decreased the expression of tumor suppressor factors in the mouse granulosa cells. Furthermore, immunofluorescence, RT-qPCR as well as western-blot analysis verified the different expression of related genes in ZEA exposed porcine and mouse granulosa cells. Collectively, these results illustrate the presence of species differences with regards to ZEA effects between porcine and mouse ovarian granulosa cells, in vitro.

Mycotoxin zearalenone exposure impairs genomic stability of swine follicular granulosa cells in vitro.

Zearalenone (ZEA), a metabolite of Fusarium fungi, is commonly found on moldy grains. Because it can competitively combine to estrogen receptor to disrupt estrogenic signaling, it has been reported to have serious adverse effects on animal reproduction systems. In order to explore the genotoxic effects of ZEA exposure on ovarian somatic cells, porcine granulosa cells were exposed to 10 µM and 30 µM ZEA for 24 or 72 h in vitro. The results showed that ZEA exposure for 24 h remarkably reduced the proliferation of porcine granulosa cells in a dose-dependent manner as determined by MTT analysis and flow cytometry. Furthermore, exposure to ZEA for 72 h induced apoptosis, and RNA sequence analysis also revealed that the expression of apoptosis related genes were altered. RT-qPCR, immunofluorescence and western blot analysis further confirmed the expression of DNA damage and repair related genes (γ-H2AX, BRCA1, RAD51 and PRKDC) were increased in ZEA exposed granulosa cells. When the estrogen antagonist, tamoxifen, was added with ZEA in the culture medium, the DNA damage and repairment by ZEA returned to normal level. Collectively, these results illustrate that ZEA disrupts genome stability and inhibits growth of porcine granulosa cells via the estrogen receptors which may promote granulosa cell apoptosis when the DNA repair system is not enough to rescue this serious damage.

Analysis of the SNP loci around transcription start sites related to goat fecundity trait base on whole genome resequencing.

Genome flanking regions surrounding transcription start sites (TSSs) are critical for the regulation of gene expression, containing many translational regulatory elements. To investigate whether critical single nucleotide polymorphisms (SNPs) exist around TSSs in the dairy goat genome, we performed high throughput DNA sequencing to compare two dairy goat groups with discrepant litter sizes. After genome mapping, SNP calling, and annotation, we screened the SNPs within 2kb scales surrounding annotated TSSs in high fecundity (HF) and low fecundity (LF) groups, respectively. We attempted to identify distinct SNPs and motifs near the TSSs in both groups. The SNPs near the TSSs most were consistent; 318 new SNPs were uncovered in the HF group, of which 305 were heterozygote SNPs, 13 were homozygote SNPs, and majority of which were distributed on chromosome 2 and 29. After validation by Sanger sequencing we found that a SNP in CHI16: 27612330 C>A in the PSEN2 gene presented an A/A genotype in the HF group and an A/A or A/C genotype in the LF group. In conclusion, our study provides insightful information into the dairy goat genomic variations surrounding TSSs, which may contribute to enhanced litter size. Based on comparison studies of SNPs exist around transcription start sites between high fecundity group and low fecundity group. Our finding provides insights concerning the goat litter size phenotypic and will promote future goat breeding.

RNA-seq based gene expression analysis of ovarian granulosa cells exposed to zearalenone in vitro: significance to steroidogenesis.

Zearalenone (ZEA) is a natural contaminant of various food and feed products representing a significant problem worldwide. Since the occurrence of ZEA in grains and feeds is frequent, the present study was carried out to evaluate the possible effects of ZEA on steroid production and gene expression of porcine granulosa cells, using RNA-seq analysis. Porcine granulosa cells were administered 10 µM and 30 µM ZEA during 72 h of culture in vitro. Following ZEA treatment the gene expression profile of control and exposed granulosa cells was compared using RNA-seq analysis. The results showed that in the exposed granulosa cells ZEA significantly altered the transcript levels, particularly steroidogenesis associated genes. Compared with the control group, 10 µM and 30 µM ZEA treatment significantly increased the mRNA expression of EDN1, IER3, TGFß and BDNF genes and significantly reduced the mRNA expression of IGF-1 and SFRP2 genes. In particular, ZEA significantly decreased the expression of genes essential for estrogen synthesis including FSHR, CYP19A1 and HSD17ß in granulosa cells. Furthermore, Q-PCR and Western-blot analysis also confirmed reduced expression of these genes in ZEA exposed granulosa cells. These effects were associated with a significant reduction of 17ß-estradiol concentrations in the culture medium of granulosa cells. Collectively, these results demonstrated a concretely deleterious effect of ZEA exposure on the mRNA expression of steroidogenesis related genes and the production of steroid hormones in porcine ovarian granulosa cells in vitro.

Whole-genome scanning for the litter size trait associated genes and SNPs under selection in dairy goat (Capra hircus).

Dairy goats are one of the most utilized domesticated animals in China. Here, we selected extreme populations based on differential fecundity in two Laoshan dairy goat populations. Utilizing deep sequencing we have generated 68.7 and 57.8 giga base of sequencing data, and identified 12,458,711 and 12,423,128 SNPs in the low fecundity and high fecundity groups, respectively. Following selective sweep analyses, a number of loci and candidate genes in the two populations were scanned independently. The reproduction related genes CCNB2, AR, ADCY1, DNMT3B, SMAD2, AMHR2, ERBB2, FGFR1, MAP3K12 and THEM4 were specifically selected in the high fecundity group whereas KDM6A, TENM1, SWI5 and CYM were specifically selected in the low fecundity group. A sub-set of genes including SYCP2, SOX5 and POU3F4 were localized both in the high and low fecundity selection windows, suggesting that these particular genes experienced strong selection with lower genetic diversity. From the genome data, the rare nonsense mutations may not contribute to fecundity, whereas nonsynonymous SNPs likely play a predominant role. The nonsynonymous exonic SNPs in SETDB2 and CDH26 which were co-localized in the selected region may take part in fecundity traits. These observations bring us a new insights into the genetic variation influencing fecundity traits within dairy goats.