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.

Project description:Ovulation refers to the process when the ovarian surface-facing wall of a preovulatory follicle ruptures and releases the cumulus oocyte complex (COC) into the oviduct or fallopian tube in response to hormonal cues. In parallel, the unruptured wall of the follicle within the ovary transitions to becoming a progesterone-producing corpus luteum. Ovulation is essential for fertilization and eventual pregnancy. Disruption of ovulation, whether purposefully through contraceptive intervention or idiopathically in cases of anovulatory infertility, has translational implications for human health. Importantly, key processes of ovulation, including follicle rupture and luteinization, are recapitulated in models of ex vivo ovulation despite the absence of an intact hypothalamic-pituitary-gonadal axis and intra-ovarian cues. In our study, we used an ex vivo ovulation model to identify functional and molecular differences between distinct regions of the follicle wall, which we refer to as the ruptured and unruptured sides. We observed that the unruptured side of the follicle wall exhibits hallmarks of luteinization after ovulation while the ruptured side exhibits signs of cell death. RNA-sequencing of these specific follicle regions revealed 2,099 differentially expressed genes between follicle sides without hCG exposure and 1,673 between follicle sides 12 hours post-hCG, which were further validated in vivo. We found enriched pathways that recapitulate known ovulation biology, including oxidative stress on the ruptured side and angiogenesis on the unruptured side. We also identified previously unappreciated pathways that may play an important role in ovulation, such as amino acid transport and Jag-Notch signaling on the ruptured side, as well as metal ion processing and IL-11 signaling on the unruptured side. Ultimately our studies demonstrate that our ex vivo model recapitulates known in vivo ovarian biology, identifies pathways that may be novel regulators of ovulation and luteinization, and may have future translational applications for the study of ovulatory disorders and the development of novel non-hormonal contraceptives.

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Integrated analysis of transcriptome and histone modifications in granulosa cells during ovulation in female mice [ChIP-seq]

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