Project description:The rupture of ovarian follicles during ovulation is a crucial and intricate process essential for procreation, yet the molecular mechanisms behind this process are not fully understood. Here, we use high-resolution spatial transcriptomics to reveal the spatiotemporal regulation of cell-type-specific molecular programs driving follicle maturation and rupture during hormone-induced ovulation.
Project description:The process of ovulation includes oocyte meiotic maturation, follicle rupture and transformation of the follicle into a corpus luteum. These events are initiated by the midcycle surge of gonadotropins and require the coordinated regulation of thousands of genes. The aim of the study was to monitor the changes in granulosa cell gene expression across five different time points during the first 36 hours of ovulation until follicle rupture, in order to increase our understanding of the events of human ovulation. We conducted a prospective cohort study including women undergoing ovarian stimulation for fertility treatment. Women were treated in a standard antagonist protocol with individually dosed human menopausal gonadotropin (hMG) or recombinant follicle stimulating hormone (rFSH). Ovulation was induced with either recombinant hCG (rhCG) or gonadotropin releasing hormone agonist (GNRHa). The granulosa cells were collected by transvaginal ultrasound-guided follicle puncture of one follicle at two specific time points during ovulation (repeated measurements), and the study covered a total of five time points: before ovulation induction (OI), 12, 17, 32 and 36 hours after OI.
Project description:The effects of exogenous hormones used for estrus synchronization, and ovarian hyper stimulation on cumulus oocyte complexes (COCs) gene expression in sexually mature rats were determined using microarrays. Gene expression in COCs collected from GnRH (Gtrt), GnRH+eCG (G+Etrt), and GnRH+eCG+hCG (G+E+Htrt) treatments were compared to COCs from naturally cycling (NC) rats. There was no significant difference in gene expression among NC, Gtrt and G+Etrt. Over 2600 genes were significantly different between NC and G+E+Htrt (P<0.05). Genes encoding proteins that are involved in prostaglandin synthesis (Ptgs2, Pla2g4a, Runx1); cholesterol biosynthesis (Hmgcr, Sc4mol, Dhcr24); receptors that allow cholesterol uptake (Ldlr, Scarb1); regulate progesterone synthesis (Star); inactivate estrogen (Sult1e1); and downstream effectors of LH signal (Pgr, Cebpb, Creb3l1, Areg, Ereg, Adamts1) were upregulated in G+E+Htrt. Genes encoding proteins that are involved in DNA replication (Ccne2, Orc5l, Rad50, Mcm6); reproductive developmental process and granulosa cell expansion (Gdf9, Bmp15, Amh, Amhr2, Bmpr1b, Tgfb2, Foxl2, Pde3a, Esr2, Fshr, Ybx2, Ccnd2, Ccnb1ip1, Zp3); maternal effect genes that are important for embryo development (Zar1, Npm2, Nlrp5, Dnmt1, H1foo, Zfp57); amino acid degradation and ketogenesis (Hmgcs2 , Cpt1b) were downregulated in G+E+Htrt. These results on rat model show that hormones used for estrus synchronization (Gtrt) and ovarian hyper stimulation (G+Etrt) had minimal effects on gene expression. However, induction of ovulation (G+E+Htrt) caused major changes in gene expression of rat COCs. This study provides comprehensive information about regulated genes during late follicle development and ovulation induction. Four experimental groups were used and there were 6 animals in each group. Total of 24 arrays were used. Only raw data used in publication.
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.
Project description:Follicles of polycystic ovaries (PCO) often become arrested in early antral stages at around 3 to 11 mm in diameter. The condition disturbs dominant follicle selection and may result in altered ovulation and anovulation. During the growth and development of human follicles, the follicular fluid (FF) constitutes the avascular microenvironment in which the oocyte develops and acts as a vehicle for hormone signaling between cues from circulation and follicular cells. Previous proteomics studies performed on FF from women with polycystic ovarian syndrome (PCOS) have revealed information on the protein changes associated with the pathophysiology of this disorder. However, these studies have been conducted on FF samples obtained in connection with oocyte pick-up during ovarian stimulation right at the time of ovulation and are limited to follicular conditions during the follicular phase of the menstrual cycle. This study aimed to detect proteomic alterations in FF from human small antral follicles (hSAF) obtained from women with PCO as compared to normal women.
Project description:Vitrification is a method for long-term biological sample cryopreservation without causing intra- and extra-cellular ice formation. We recently established a novel closed vitrification system to cryopreserve mouse ovarian follicles. Using the 3D alginate hydrogel encapsulated in vitro follicle growth (eIVFG) method, we have demonstrated that compared to freshly-harvested follicles, vitrified follicles have normal follicle and oocyte reproductive outcomes. Our recent study further demonstrated the faithful preservation of molecular signatures of follicle-stimulating hormone (FSH)-stimulated follicle maturation in vitrified follicles. However, it is unknown whether ovulation, another crucial gonadotropin-dependent follicular event, and involved ovulatory gene regulatory pathways are well conserved in vitrified follicles. Fresh and vitrified follicles grown for 8 days by eIVFG were collected at 0, 1, 4 and 8-hour post human chorionic gonadotropin (hCG) treatment for the single-follicle RNA sequencing. Principal component analysis (PCA) and Pearson’s correlation analysis revealed that vitrified follicles have similar transcriptomic profiles to fresh follicles. Furthermore, the expression of several genes essential for ovulation were comparable between vitrified and fresh follicles. In summary, these results demonstrate that our closed vitrification system preserves follicular transcriptomic dynamics during ex vivo ovulation. Together with our previous findings that vitrification preserves FSH-stimulated follicle maturation, the integration of vitrification of immature follicles and eIVFG has a great potential to serve as an additional fertilization preservation approach for young cancer patients and endangerers species. Moreover, follicle vitrification enables a high-content ovarian follicle biobank, which can greatly improve the throughput of eIVFG for studying ovulation biology, anovulatory disease, toxicology, and novel contraceptive drug development targeting ovulation.
Project description:Ovulation requires sequential molecular events and structural remodeling in the ovarian follicle for the successful release of a mature oocyte capable of being fertilised. Critical to this process is progesterone receptor (PGR), a transcription factor highly yet transiently expressed in granulosa cells of preovulatory follicles. Progesterone receptor knockout (PRKO) mice are anovulatory, with a specific and complete defect in follicle rupture. Therefore, this model was used to examine the critical molecular and biochemical mechanisms necessary for successful ovulation. We used microarrays to identify putative PGR-regulated genes in granulosa cells at a time when PGR expression is maximal (eCG + 8h hCG) and the preovulatory follicle is undergoing the final changes necessary for successful ovulation.
Project description:The effects of exogenous hormones used for estrus synchronization, and ovarian hyper stimulation on cumulus oocyte complexes (COCs) gene expression in sexually mature rats were determined using microarrays. Gene expression in COCs collected from GnRH (Gtrt), GnRH+eCG (G+Etrt), and GnRH+eCG+hCG (G+E+Htrt) treatments were compared to COCs from naturally cycling (NC) rats. There was no significant difference in gene expression among NC, Gtrt and G+Etrt. Over 2600 genes were significantly different between NC and G+E+Htrt (P<0.05). Genes encoding proteins that are involved in prostaglandin synthesis (Ptgs2, Pla2g4a, Runx1); cholesterol biosynthesis (Hmgcr, Sc4mol, Dhcr24); receptors that allow cholesterol uptake (Ldlr, Scarb1); regulate progesterone synthesis (Star); inactivate estrogen (Sult1e1); and downstream effectors of LH signal (Pgr, Cebpb, Creb3l1, Areg, Ereg, Adamts1) were upregulated in G+E+Htrt. Genes encoding proteins that are involved in DNA replication (Ccne2, Orc5l, Rad50, Mcm6); reproductive developmental process and granulosa cell expansion (Gdf9, Bmp15, Amh, Amhr2, Bmpr1b, Tgfb2, Foxl2, Pde3a, Esr2, Fshr, Ybx2, Ccnd2, Ccnb1ip1, Zp3); maternal effect genes that are important for embryo development (Zar1, Npm2, Nlrp5, Dnmt1, H1foo, Zfp57); amino acid degradation and ketogenesis (Hmgcs2 , Cpt1b) were downregulated in G+E+Htrt. These results on rat model show that hormones used for estrus synchronization (Gtrt) and ovarian hyper stimulation (G+Etrt) had minimal effects on gene expression. However, induction of ovulation (G+E+Htrt) caused major changes in gene expression of rat COCs. This study provides comprehensive information about regulated genes during late follicle development and ovulation induction.
Project description:In the ovary, follicular growth and maturation are complicated processes that involve a series of morphological and physiological changes in oocytes and somatic cells leading to ovulation and luteinization, essential processes for fertility in females. Given the complexity of ovulation, characterization of genome-wide regulatory elements is essential to understand the mechanisms governing the expression of specific genes in the rapidly differentiating follicle. We therefore employed a systems biology approach to determine global transcriptional mechanisms during the early stages of the ovulatory process. We demonstrate that, following the hormonal signal that initiates ovulation, granulosa cells undergo major modification of distal regulatory elements which coincides with cistrome reprogramming of the indispensable orphan nuclear receptor, liver receptor homolog-1 (LRH-1). This cistromic reorganization correlates with the extensive changes in gene expression in ovarian granulosa cells, leading to ovulation. Together, our study yields a transcriptional map of unprecedented detail, delineating ovarian cell differentiation during the initiation of ovulation. It further demonstrates the power of complementary global approaches to accurately characterize molecular events that direct the multiple and rapid changes that characterize reproductive processes.