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A single-cell atlas of the single versus multiple parous Hu Sheep ovary

ABSTRACT: Follicular development is a highly coordinated process in Hu sheep. Follicle-cyclic recruitment, spatial displacement, follicle atresia, and ovulation are implicated events resulting from the somatic cells' release of molecular signals. Hu sheep is a high-quality sheep breed with high fecundity in China and is ideal for investigating high reproductive traits. In the current study, the sheep with lambing number ≥3 in three consecutive lambing records were assigned to the HLS group, and lambing number = 1 as the LLS group selected from the same farm with three consecutive lambings. Three randomly picked ewes were slaughtered within 12 h of estrus, and unilateral ovarian tissue was collected and analyzed by single-cell RNA sequencing in each group. A total of five types of somatic cells were identified, and corresponding expression profiles were mapped in the ovaries of the Hu sheep. Additionally, the results of the difference in ovary somatic cell expression profiles between HLS and LLS present that the differences between multiples vs. singleton Hu sheep were mainly clustered in the GCs. In addition, 4 granulosa cell subtypes were identified. GeneSwitches results revealed the opening of JPH1 expression and the closure of LOC101112291, which leads to different evolutionary directions of the granular cells. The expression levels of FTH1 and FTL in GCs of Hu sheep in the HLS group were significantly higher, which inhibited necroptosis and ferroptosis of mural-GCs from decreasing follicular atresia. This study constructed the cellular atlas of the ovary and revealed related biological characteristics at the cellular molecular level. It provides a theoretical basis for the mechanisms underlying the differences in ovulation numbers, which contributes to breeding high-fertility sheep and molecular genetics-based selection.

ORGANISM(S): Ovis aries

PROVIDER: GSE233801 | GEO | 2023/08/15

REPOSITORIES: GEO

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Project description:Background: Hu sheep, renowned for their prolificacy and economic value, have been introduced to Xinjiang for intensive farming operations. However, their suboptimal adaptation to local harsh environments limits growth efficiency and health management. In contrast, Duolang sheep, an indigenous Xinjiang breed, exhibit exceptional environmental resilience, largely attributed to their unique rumen microbiome enriched with fiber-degrading bacteria, which enhance roughage utilization and stress tolerance. To address Hu sheep’s adaptive challenges, this study applies Ligilactobacillus salivarius KS1018, a probiotic strain isolated from Duolang sheep, to Hu sheep, investigating its role in improving rumen microbial homeostasis, metabolic adaptation, and physiological resilience under Xinjiang’s environmental conditions.Results: Thirty-two male Hu lambs were allocated into four groups: Control (C) and three treatment groups receiving daily doses of 0.5×109 CFU/d (low-dose group, LLS, 1.0×109 CFU/d (medium-dose group, MLS), or 1.5×109 CFU/d (high-dose group, HLS) for 56 days. Although growth performance did not reveal significant enhancement, supplementation with the high dose of L. salivarius KS1018 (HLS group) significantly reduced ruminal NH3-N (p < 0.05) and increased total VFAs (p = 0.043), alongside elevated serum triglycerides (p = 0.021) and β-hydroxybutyrate (p < 0.05), indicating enhanced nitrogen utilization and ketogenesis. Rumen metabolomics revealed dose-dependent metabolic shifts: group HLS prioritized oxidative phosphorylation and pyrimidine metabolism (p < 0.001), while group MLS upregulated branched-chain amino acid biosynthesis (p < 0.01). Procrustes analysis confirmed strong microbiome-metabolome coordination (M2 = 0.56, p = 0.001), linking Lachnospira and Negativicutes enrichment to VFA production and redox homeostasis. Group HLS exhibited a decline in ruminal pH, suggesting a potential risk of subacute acidosis at higher doses.Conclusions: L. salivarius KS1018 optimizes rumen metabolism through microbial-host crosstalk, improving nitrogen efficiency and stress resilience in Hu sheep. The strain promoted ketogenesis and oxidative phosphorylation pathways, which alleviated oxidative stress and enhanced immune function under environmental challenges. Notably, the arid-adaptive genetic traits likely contribute to stabilizing rumen microbial networks, mimicking the superior roughage utilization capacity of its native Duolang sheep host. Our findings highlight the potential of L. salivarius KS1018 as a metabolic modulator in ruminant nutrition, redefining its role beyond traditional growth-centric applications.

Project description:Purpose: To investigate the differentially expressed genes in high litter size samples v/s low litter size samples of F4 inbred Swiss albino mice using RNA-Seq and to validate some identified differentially expressed genes using qRT-PCR. Methods: Intact ovarian tissue samples were obtained from six F4 female [from two groups i.e. three from high litter size group (HLS; n=3) and three from low litter size group (LLS; n=3)] inbred mice, after assessment of estrous phase of ovarian cycle by vaginal cytology smear technique (Byers et al., 2012). After sacrifice of mice, their intact ovaries were rapidly harvested and immediately preserve in RNAlater (Invitrogen). The QC passed libraries were subjected to RNA sequencing by use of HiSeq X10 of Illumina® platform. The paired ends (2 X 150 bp) reads were sequenced and about 3GB raw data were generated for both high and low groups, separately. The provided raw data were processed with trimgalore for quality purpose and kallisto pipeline to get transcript abundances. Tximport was used to generate gene counts and DESeq2 was performed on gene count data of RNA-Seq experiment to get DEGs. DEGs in HLS v/s LLS groups in Swiss albino mice were subjected to functional enrichment using bioinformatics tools, eg: Profiler and GSEA. The interaction of important DEGs having fold change ≥ ± 1.5 and Padj ˂ 0.05 was studied using Network Analyst 3.0. A total 5 DEGs identified through RNA-Seq in high and low litter size groups were validated by quantitative real time PCR (qRT-PCR). Results: All QC passed transcriptomic libraries were sequenced in paired end read sequencing platform and on an average 49.7 million reads of 150bp per sample were generated. All six samples produced a total of 44.7 GB of data, with an average Phred score of more than 35 per sample. All six samples yielded a total of 42.23 GB of trimmed data. The average mapping percentage was 78.5. The average GC percent and unique reads percent for clean data samples were 50 and 55.66, respectively. DESeq2 analysis yielded the 1018 genes under R environment with 5 % FDR. 347 genes were found to be up regulated, and about 671 genes were found to be down regulated out of 1018 genes. Based on functional enrichment analysis of 659 DEGs with fold change ≥ ± 1.5 and Padj ˂ 0.05, 166 significantly enriched gene ontology (GO) classes were identified. Out of 166 categories, 17, 109, 28, and 12 were significantly enriched in molecular function (MF), biological process (BP), cellular component (CC), KEGG, and REAC, respectively. Conclusion: In the present analysis of ovarian transcriptome, cell–cell signaling and metabolic related pathways were significantly enriched in HLS as compare to LLS group of F4 inbred Swiss albino mice. In these pathways, TP63, SLC16A10, APOD, S100A8, LRRK2, GPD1, CNR1, HDAC9, WNT11, LPIN1, SLC5A7, KLB, GNAI1, IRS2, TIMP4, PRKAR2B, SLC1A3, GSTZ1, FAH, CLYBL, SYNGR1, FAT4, CIDEA, ECHDC1, FMO5, GPT and ALDH1A1genes were significantly up-regulated in HLS group of inbred mice.

Project description:Background: Follicular growth and maturation in semi-synchronously spawning fish involve numerous cell signaling cascades and different molecular cascades are activated or inhibited during specific stages of oocyte development. The objectives of the current study were to identify molecular pathways and temporal gene expression patterns throughout a complete breeding cycle in female wild LMB to characterize the molecular sequence of events underlying follicle and ovary development. Methods: Microarray analysis was performed on eight morphologically distinct stages, from primary stages of oocyte growth to ovulation and atresia. Ovarian tissue histology, plasma vitellogenin, and sex steroids (E2 and T) were also measured to correlate molecular signaling cascades to higher levels of biological organization. Results: Global expression patterns revealed dramatic differences between early and late stages of ovarian follicle progression, with over 200 and 500 genes being differentially expressed during both ovulation and atresia respectively (p < 0.01). Time course analysis for all stages leading to ovulation and atresia identified increased expression of GABAA receptor subunits and peroxisome proliferator-activated receptor gamma during ovulation. Gene set enrichment analysis (GSEA) revealed that early stages of oocyte growth involved increases in pathways of natural killer cell and mast cell activation, as well as gap junction regulation. GSEA revealed that arachidonic acid metabolism was significantly up-regulated while CD2, fibronectin, and neuropeptides Y receptor signaling cascades were down-regulated at ovulation. Expression targets for LH signaling were decreased during vitellogenesis but increased at ovulation. GSEA revealed decreases in actin cytoskeleton regulation and receptor mediated signaling pathways involving TGF? and ephrin receptor regulation at atresia. Conclusions: This study offers new insight into the molecular pathways involved in vitellogenesis, ovulation and atresia in LMB and provides new hypotheses about the cellular pathways involved in oocyte growth and maturation. 31 microarrays on 8 unique stages of ovary development; developmental profile time course, development of LMB ovary

Project description:Background: Follicular growth and maturation in semi-synchronously spawning fish involve numerous cell signaling cascades and different molecular cascades are activated or inhibited during specific stages of oocyte development. The objectives of the current study were to identify molecular pathways and temporal gene expression patterns throughout a complete breeding cycle in female wild LMB to characterize the molecular sequence of events underlying follicle and ovary development. Methods: Microarray analysis was performed on eight morphologically distinct stages, from primary stages of oocyte growth to ovulation and atresia. Ovarian tissue histology, plasma vitellogenin, and sex steroids (E2 and T) were also measured to correlate molecular signaling cascades to higher levels of biological organization. Results: Global expression patterns revealed dramatic differences between early and late stages of ovarian follicle progression, with over 200 and 500 genes being differentially expressed during both ovulation and atresia respectively (p < 0.01). Time course analysis for all stages leading to ovulation and atresia identified increased expression of GABAA receptor subunits and peroxisome proliferator-activated receptor gamma during ovulation. Gene set enrichment analysis (GSEA) revealed that early stages of oocyte growth involved increases in pathways of natural killer cell and mast cell activation, as well as gap junction regulation. GSEA revealed that arachidonic acid metabolism was significantly up-regulated while CD2, fibronectin, and neuropeptides Y receptor signaling cascades were down-regulated at ovulation. Expression targets for LH signaling were decreased during vitellogenesis but increased at ovulation. GSEA revealed decreases in actin cytoskeleton regulation and receptor mediated signaling pathways involving TGFβ and ephrin receptor regulation at atresia. Conclusions: This study offers new insight into the molecular pathways involved in vitellogenesis, ovulation and atresia in LMB and provides new hypotheses about the cellular pathways involved in oocyte growth and maturation.

Project description:Background: In mammals, female fertility is determined by the outcome of follicular development (ovulation or atresia). Follicular atresia is a complex physiological process that results in the degeneration of oocytes from the ovary. However, the molecular mechanisms of oocyte degeneration and key protein markers of follicular atresia remain unclear. In this study, we explored the complex transcriptional regulatory mechanisms and protein profiles in oocytes and follicular fluid in atretic follicle stages using single-cell RNA sequencing and tandem mass tag proteomics. Results: First, through paired analysis of different follicle development stages, we identified 175 atresia-specific genes and eight candidate oocyte-secreted factors, including PKG1, YTHDF2, and MYC. Meanwhile, we also characterized unique features of the oocyte transcriptional landscape in the atretic follicle stage that displayed cell death-related transcriptional changes and mechanisms, such as autophagy (TBK1 and IRS4), necroptosis (PKR), and apoptosis (MARCKS). Moreover, we identified atresia-specific genes, namely FTH1, TF, and ACSL4, which may participate in regulation of oocyte ferroptosis in atretic follicles through a series of mechanisms including ferritinophagy, ferritin transport, and lipid metabolism. Additionally, we uncovered 333 differentially expressed proteins that may coordinate follicular atresia and revealed key pathways, such as negative regulation of angiogenesis, metabolic pathways, and transcription and mRNA splicing, that lead to oocyte degeneration. Finally, by combining transcriptome and proteomics analyses, we identified two oocyte-secreted biomarkers, PGK1 and ANGPT2, that may be associated with follicular atresia. Conclusions: In conclusion, our work provides a comprehensive characterization of oocyte degeneration in ovine atretic follicles, which provides a basis for establishing an oocyte quality evaluation system and understanding the mechanism of follicular atresia in sheep, as well as an important reference for in vitro production of embryos.

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A single-cell atlas of the single versus multiple parous Hu Sheep ovary

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