Project description:The fallopian tube transports the gametes to the fertilization site and delivers the embryo to the uterus at the optimal time for implantation. Progesterone and the classical progesterone receptor (PGR) are known to be involved in regulating both tubal ciliary beating and muscular contractions, possibly involving both genomic and non-genomic actions. To provide more clues on the mechanisms involved, we investigated the effect of progesterone on gene expression in mice fallopian tubes in vitro at early (20 min) and later (2 h, 8 h) time-points using microarray and/or quantitative PCR. In parallel, oocyte cumulus complex transport was investigated in ovulating mice injected with one of the PGR antagonists, Org 31710 or CDB2194. Microarray analyses did not reveal any apparently regulated genes 20 min after progesterone treatment, in agreement with a proposed non-genomic action of progesterone controlling ciliary beating. After 2 h, 11 genes were significantly up-regulated. Analyses by quantitative PCR at 2 h and 8 h showed a consistent up-regulation of endothelin 1 (Edn1) and a down-regulation of its receptor Ednra by progesterone. We also show that treatment with progesterone receptor antagonist before ovulation accelerates the transport of the oocyte cumulus complex. This is the first study showing that progesterone regulates Edn1 and Ednra in the fallopian tube. Together with previous studies on endothelin-mediated effects on muscular contractions in the fallopian tube, the results from this study suggest that endothelin is a mediator of the progesterone-controlled effects on muscular contraction, and eventually gamete transport, in the fallopian tube. 16 pooled samples from mice fallopian tubes were exposed in vitro to progesterone for up to 2 hours.
Project description:The fallopian tube transports the gametes to the fertilization site and delivers the embryo to the uterus at the optimal time for implantation. Progesterone and the classical progesterone receptor (PGR) are known to be involved in regulating both tubal ciliary beating and muscular contractions, possibly involving both genomic and non-genomic actions. To provide more clues on the mechanisms involved, we investigated the effect of progesterone on gene expression in mice fallopian tubes in vitro at early (20 min) and later (2 h, 8 h) time-points using microarray and/or quantitative PCR. In parallel, oocyte cumulus complex transport was investigated in ovulating mice injected with one of the PGR antagonists, Org 31710 or CDB2194. Microarray analyses did not reveal any apparently regulated genes 20 min after progesterone treatment, in agreement with a proposed non-genomic action of progesterone controlling ciliary beating. After 2 h, 11 genes were significantly up-regulated. Analyses by quantitative PCR at 2 h and 8 h showed a consistent up-regulation of endothelin 1 (Edn1) and a down-regulation of its receptor Ednra by progesterone. We also show that treatment with progesterone receptor antagonist before ovulation accelerates the transport of the oocyte cumulus complex. This is the first study showing that progesterone regulates Edn1 and Ednra in the fallopian tube. Together with previous studies on endothelin-mediated effects on muscular contractions in the fallopian tube, the results from this study suggest that endothelin is a mediator of the progesterone-controlled effects on muscular contraction, and eventually gamete transport, in the fallopian tube.
Project description:Progesterone (P4) is predicted to act as a negative regulatory hormone for oocyte maturation events; however, its local effects during follicular development remain poorly understood in bovine. The complex process of oocyte meiosis progression is dependent on cellular communication among follicular cells. Besides, the breakdown of this communication, mainly between cumulus cells and oocyte, through the retraction of cumulus projections connecting these cells can impact oocyte maturation. In our study, we observed that ipsilateral follicles containing high intrafollicular P4 concentrations enhance the expression of proteins in follicular small extracellular vesicles predicted to be involved in the retraction of membrane projections based on actin filaments, such transzonal projections (TZPs). Additionally, we found that contralateral follicles containing low intrafollicular P4 concentrations are predicted to regulate cellular communication pathways inducing the maintenance of membrane projections in cumulus cells. Our functional analysis confirmed that supplementation of cumulus-oocyte complexes during in vitro maturation with P4 at concentration similar to ipsilateral follicles reduces the number of TZPs. In summary, our study underscores a direct association between P4 concentration and cumulus-oocyte interaction, with potential consequences for the acquisition of oocyte competence.
Project description:The fallopian tubes are essential to several physiological and pathological processes from pregnancy to ovarian cancer. However, current in vitro models to study their pathophysiology were validated using two-dimensional (2D) tissue sections and measuring the organoid response to female hormone stimulation. These analyses overlook the 3D heterogeneity of the tissue and the fallopian tube mechanical function (transport of an oocyte). We developed a multi-compartment organoid model of the human fallopian tube that was meticulously tuned to reflect the compartmentalization and heterogeneity of the tissue composition. We validated the proteome, cilia-driven transport function, and architectural accuracy of this organoid through a novel platform wherein organoids are quantitatively compared to a 3D single-cell resolution reference map of a healthy, transplantation-quality human fallopian tube. This organoid model was precision-engineered using our iterative platform to resemble the cellular and extracellular proteome, biological function, and 3D microanatomy of the human fallopian tube.
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. Although PGR is not expressed in the cumulus cells or oocyte of the preovulatory cumulus oocyte complex (COC), it is well known that the COC responds to the cascade of gene expression changes that occurs in preovulatory granulosa cells. We used microarrays to identify putative ‘ovulation’ genes in preovulatory COCs at a time when PGR expression is maximal in granulosa cells (eCG + 8h hCG) and the preovulatory COC and follicle are undergoing the final changes necessary for successful ovulation.
Project description:Cumulus oophorus cells play an essential role in oocyte development. CBX4 is a member of the Polycomb complex, which plays a role in regulating cellular differentiation. We used siRNA mediated knockdown of Cbx4 expression followed by Affymetrix array analysis to evaluate the role of CBX4 in cumulus cell differentiated state.
Project description:We sought to identify the potential specific roles of the MTOR signalling in cumulus cells by comparing the transcriptomes of the Control (treated with the DMSO vehicle) and MTOR-specific inhibitor Torin 1(5uM)-treated cumulus-oocyte complexes that were cultured for 16 hours. We compared the transcriptomes between DMSO- and Torin 1- treated cumulus-oocyte complexes. 3 individual cumulus-oocyte complex samples of DMSO- and Torin1-treated were collected. Comparison was done between DMSO- and Torin1-treated groups.
Project description:Purpose: Extracellular vesicles (EVs) are nanoparticles that can be secreted by different cells, including cells found within the ovarian follicle. Currently, EVs are considered an important form of intercellular communication, since they carry biological contents. The goal of this study was to survey the effects of small EVs from follicles at different estrous cycle stage in bovine cumulus cells. Methods: We used an established model to obtain follicular fluid (FF) at early and late estrous cycle stage according to corpus luteum appearance, corresponding to low and high progesterone (P4) levels, respectively. We collected FF from 3-6 mm follicles and isolated small EVs, which were used as a supplement during in vitro maturation (IVM). Cumulus cells were collected from cumulus-oocyte complexes (COCs) pools and the RNAs were obtained and subjected to RNA sequencing. Results: The results showed that small EVs from different estrous cycle stage are capable to affect transcripts in cumulus cells and modulate different pathways and biological processes related to oocyte maturation, ovulation and immune response. Conclusion: This study demonstrated that small EVs from low and high P4 group impact the RNA profile in cumulus cells after 9 hours of in vitro maturation.
Project description:The oocyte forms a complex with their somatic cumulus cells within the follicle throughout the preovulatory maturation steps. Cumulus cells support their oocyte not only through mechanical protection but also with a close bidirectional exchange of metabolites. Analysis of the oocytes cumulus gives the opportunity to explore non-invasively oocytal well-being and quality. In vitro maturation (IVM) is the first rate-limiting step in in vitro embryo production. Analysis of protein expression in cumulus cells around this critical step helps to explore the impact of maturation conditions and to examine an influence on maturational competence of the oocyte. The goal of this study was the comparison of the cumulus proteome of oocytes with and without maturational competence matured under in vivo and in vitro conditions. Therefore twenty cumulus samples corresponding to single oocytes were analysed. Half of the samples were matured in vivo and the other half in vitro. For each maturation group, cumulus from oocytes matured successfully (SM; n=5) and failed to mature (FM; n=5) were analysed.