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.

Project description:Running title: More insights into ovulation Purpose:Ovulation is the process by which mature follicles release fertilized oocytes under the stimulation of gonadotropin. This involves the precise interaction and destiny of various cells in the follicle. Granulosa cells (GCs) are the only cells that recognize and respond to ovulation LH (or HCG) signals. Therefore, the classification and deep interpretation of genes in GCs under LH surge stimulation is the key to understand ovulation. Methods: In this study, RNA-seq, Q-PCR, Western blot, gene knockout, gene knockout and other experimental techniques were used to conduct trend and classification analysis of the genes regulated by ovulation signal of LH at the cellular and individual levels. Genes that may be involved in ovulation were screened, identified and were selected for functional verification. Results:(1) Firstly, 7104 and 347 mRNA and Long noncoding RNA (LncRNA) that may be involved in ovulation activity were screened at the gene level by transcriptome sequencing technology, respectively. According to gene expression, they were divided into activated, inhibited and transient activated gene expression patterns. 44 secreted proteins, 62 transcription factors, 22 periodic expression characteristic genes, 10 ovulation process unknown genes, 16 LncRNA-target gene cis pair combinations and 41 highly expressed LncRNA were identified and listed in the three models;(2) The functions of LncRNA Gm12840 and Gm20186 were studied at the cellular level. The results showed that knockdown Gm12840/20186 not only inhibited the proliferation and hormone synthesis of GCs, but also induced cell apoptosis;(3) The function of LncRNA Gm12840 was further studied in mice, and Gm12840 knockout mice were constructed by CRISPR/CAS9 technology: Results It was found that Gm12840 knockout significantly increased the number of antral follicles and ovulation, and significantly inhibited the number of atretic follicles and antral follicles not discharged after ovulation. Gm12840 knockout significantly increased litter size at 2-3 months of age, but also significantly inhibited litter size at 7-9 months of age. Meanwhile, Gm12840 knockout also significantly inhibited the number of follicles growing in the ovary of 11-month-old mice;(4) Finally, transcriptomic sequencing analysis of wilt-type and Gm12840-/- mouse GCs showed 349 genes were differentially expressed. KEGG enrichment analysis and WB results showed that Gm12840 knockout enhanced mTOR and MAPK signals. Conclusions:The above results screened and identified genes with potential research value during ovulation process, and took LncRNA Gm12840 as an example to verify their functions at the cell and individual levels, providing reference for further revealing the ovulation process.

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

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