Oocyte-somatic Cell Interactions Research Articles

Oocyte-somatic cell interactions in the human ovary-novel role of bone morphogenetic proteins and growth differentiation factors.

BACKGROUNDInitially identified for their capability to induce heterotopic bone formation, bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor β superfamily. Using cellular and molecular genetic approaches, recent studies have implicated intra-ovarian BMPs as potent regulators of ovarian follicular function. The bi-directional communication of oocytes and the surrounding somatic cells is mandatory for normal follicle development and oocyte maturation. This review summarizes the current knowledge on the physiological role and molecular determinants of these ovarian regulatory factors within the human germline-somatic regulatory loop.OBJECTIVE AND RATIONALEThe regulation of ovarian function remains poorly characterized in humans because, while the fundamental process of follicular development and oocyte maturation is highly similar across species, most information on the regulation of ovarian function is obtained from studies using rodent models. Thus, this review focuses on the studies that used human biological materials to gain knowledge about human ovarian biology and disorders and to develop strategies for preventing, diagnosing and treating these abnormalities.SEARCH METHODSRelevant English-language publications describing the roles of BMPs or growth differentiation factors (GDFs) in human ovarian biology and phenotypes were comprehensively searched using PubMed and the Google Scholar database. The publications included those published since the initial identification of BMPs in the mammalian ovary in 1999 through July 2016.OUTCOMESStudies using human biological materials have revealed the expression of BMPs, GDFs and their putative receptors as well as their molecular signaling in the fundamental cells (oocyte, cumulus/granulosa cells (GCs) and theca/stroma cells) of the ovarian follicles throughout follicle development. With the availability of recombinant human BMPs/GDFs and the development of immortalized human cell lines, functional studies have demonstrated the physiological role of intra-ovarian BMPs/GDFs in all aspects of ovarian functions, from follicle development to steroidogenesis, cell–cell communication, oocyte maturation, ovulation and luteal function. Furthermore, there is crosstalk between these potent ovarian regulators and the endocrine signaling system. Dysregulation or naturally occurring mutations within the BMP system may lead to several female reproductive diseases. The latest development of recombinant BMPs, synthetic BMP inhibitors, gene therapy and tools for BMP-ligand sequestration has made the BMP pathway a potential therapeutic target in certain human fertility disorders; however, further clinical trials are needed. Recent studies have indicated that GDF8 is an intra-ovarian factor that may play a novel role in regulating ovarian functions in the human ovary.WIDER IMPLICATIONSIntra-ovarian BMPs/GDFs are critical regulators of folliculogenesis and human ovarian functions. Any dysregulation or variations in these ligands or their receptors may affect the related intracellular signaling and influence ovarian functions, which accounts for several reproductive pathologies and infertility. Understanding the normal and pathological roles of intra-ovarian BMPs/GDFs, especially as related to GC functions and follicular fluid levels, will inform innovative approaches to fertility regulation and improve the diagnosis and treatment of ovarian disorders.

Oocyte-Somatic Cell Interactions and Ovulation Rate: Effects on Oocyte Quality and Embryo Yield

The use of exogenous gonadotropins to increase oocyte yields for better pregnancy outcomes remains unpredictable and inefficient. The ­objectives of this review are to address two questions concerning this issue: (1) are there any alternatives for improving consistency in oocyte yields? (2) is it possible to develop a molecular diagnostic test for oocytes with blastocyst potential? Studies in sheep with a heterozygous inactivating mutation in the oocyte-derived growth factor, bone morphogenetic protein 15 (BMP15), report increased oocyte yields and significantly more offspring. Moreover, partial immuno-neutralization of BMP15 bioactivity increases oocyte yield without modifying gonadotropin or ovarian steroid secretion. Therefore, it is hypothesized that the development of BMP15 antagonists may prove them to be suitable alternatives to exogenous gonadotropins. Recent evidence from analyses of candidate gene expression in human cumulus cells separated from individual oocytes before IVF indicates a potential non-invasive molecular screen for oocytes with improved blastocyst and live-birth outcomes.

Revisiting oocyte-somatic cell interactions: in search of novel intrafollicular predictors and regulators of oocyte developmental competence.

Prediction and improvement of oocyte competence are two critical issues in assisted reproductive technology to improve infertility therapy. The lack of reliable and objective predictors of oocyte developmental competence for oocyte/embryo selection during in vitro fertilization hampers the effectiveness of this technology. Likewise, the low pregnancy rate resulting from in vitro maturation of human oocytes represents a major obstacle for its clinical application. Oocyte competence is progressively acquired during follicular development, and the oocyte plays a dominant role in regulating granulosa cell functions and maintaining the microenvironment appropriate for the development of its competence. Hence, granulosa cell functions are reflective of oocyte competence, and molecular markers of granulosa cells are potentially reliable predictors of oocyte quality. With the advent of the functional genomics era, the transcriptome of granulosa cells has been extensively characterized. Experimental data supporting granulosa cell markers as predictors of oocyte competence are now emerging in both animal models and humans. Future efforts should focus on integrating granulosa cell genetic markers as parameters for oocyte/embryo selection. Moreover, novel in vitro evidence highlights the effectiveness of exogenous oocyte-secreted factors in promoting oocyte developmental competence in animal models. The challenge in evaluating the effect of oocyte-secreted factors on oocyte quality in a clinical setting is to standardize the various preparations of these recombinant proteins and decipher their complex interactions/cooperativity within the germline-somatic cell regulatory loop.

Effects of in vitro maturation on gene expression in rhesus monkey oocytes

In vitro oocyte maturation (IVM) holds great promise as a tool for enhancing clinical treatment of infertility, enhancing availability of nonhuman primates for development of disease models, and facilitating endangered species preservation. However, IVM outcomes have remained significantly below the success rates obtained with in vivo matured (VVM) oocytes from humans and nonhuman primates. A cDNA array-based analysis is presented, comparing the transcriptomes of VVM oocytes with IVM oocytes. We observe a small set of just 59 mRNAs that are differentially expressed between the two cell types. These mRNAs are related to cellular homeostasis, cell-cell interactions including growth factor and hormone stimulation and cell adhesion, and other functions such as mRNA stability and translation. Additionally, we observe in IVM oocytes overexpression of PLAGL1 and MEST, two maternally imprinted genes, indicating a possible interruption or loss of correct epigenetic programming. These results indicate that, under certain IVM conditions, oocytes that are molecularly highly similar to VVM oocytes can be obtained; however, the interruption of normal oocyte-somatic cell interactions during the final hours of oocyte maturation may preclude the establishment of full developmental competence.

Effect of a partially purified 30.1 kDa ovine follicular fluid protein on ovine follicle and ovarian somatic cell growth, and oocyte maturation in vitro

Regulation of folliculogenesis and oocyte-somatic cell interactions in the ovarian follicles is under the control of gonadotrophins and various local factors. In the present study, an attempt was made to isolate and examine the biological activities of ovarian follicular fluid protein(s) in sheep in vitro. Follicular fluids aspirated from ovarian follicles of slaughterhouse-derived ovaries were made cell free by centrifugation (5000 g for 30 min) and steroid free by charcoal treatment. The follicular fluid was then subjected to ammonium sulphate precipitation and gel filtration chromatography using G-75 Sephadex. Protein detection was performed using a UV spectrophotometer at 280 nm. The 35-50% fraction yielded a detectable peak and a protein of 30.1 kDa as examined by SDS-PAGE. The effect of increasing doses of the 30.1 kDa ovine follicular fluid protein (oFFP) was tested at different doses on pre-antral and antral follicle growth; cumulus cell expansion; oocyte maturation; changes in protein, calcium and phosphorus levels of oocytes after culture in media containing different levels of isolated protein; mural granulosa cell, polar granulosa cell (cumulus cell), oviductal epithelial cell monolayer formation and granulosa cell proliferation in vitro. The oFFP significantly inhibited antral follicle growth, cumulus expansion, oocyte maturation and somatic cell growth in vitro in a dose-dependent manner. The oFFP did not have a significant effect on the pre-antral follicle growth in vitro. The protein, calcium and phosphorus contents of oocytes were found to decrease in oocytes cultured in maturation medium containing the oFFP. The present study demonstrates a follicular fluid factor regulating folliculogenesis and oocyte maturation in sheep.

Glycolytic enzyme expression in human granulosa cells

To examine the differences in specific protein expression between mural and cumulus granulosa cells following 24-hour in vitro culture. Laboratory study. University Hospital. Human granulosa cells were collected at the time of egg collection during assisted reproduction. Cumulus cells associated with the oocyte were separated from mural cells from the periphery of the follicle before in vitro culture for 24 hours. Cells were then lysed and subjected to two-dimensional gel electrophoresis. Given that cumulus (cGC) and mural granulosa cells (mGC) differentiate from a single layer, it is likely that phenotypic differences between them may reflect specific molecular processes and structural adaptations. Computer-assisted analysis using dedicated software enabled the presence, absence, or relative volume of each individual protein spot to be estimated. Differentially expressed spots were identified using tandem mass spectrometry. The mean number of separate protein spots detected in mGC gels was 1,105 +/- 146, and in cGC it was 887 +/- 236, although there was no statistically significant difference between the two. Five enzymes of the glycolytic pathway were never expressed in cGC after 24 hours in vitro; these were triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase 1, and two isoforms of alpha enolase. These are the first data collected in humans consistent with a recent demonstration that isolated murine cGC cultured in vitro exhibit decreased expression of mRNA encoding glycolytic enzymes, and support the suggestion that some factor or factors secreted by the oocyte may be responsible for the maintenance of glycolysis in the adjacent cGC.

Society for Reproductive Biology Founders' Lecture 2007. Insights into germ cell biology: from the bench to the clinic

The germline is unique among tissues in being the only lineage that is transmitted through generations. The gonadal somatic cells that interact with male and female germ cells are equally important for their juxtacrine and paracrine signalling pathways that lead to the formation of functionally mature gametes and healthy progeny. The present review summarises exciting new studies that our group and others have achieved at the frontier of male and female germ cell biology and in studying transforming growth factor-beta signalling pathways in oocyte-somatic cell interactions and gonadal growth and differentiation. In the process, we have produced over 70 transgenic and knockout models to study reproduction in vivo. These models have helped us identify novel and unexplored areas of germ cell biology and translate this work into the fertility clinic.

Oocyte–follicle cell interactions during ovarian follicle development, as seen by high resolution scanning and transmission electron microscopy in humans

The aim of this article is to summarize and update, through an integrated analysis by transmission electron microscopy (TEM) and high resolution scanning electron microscopy (SEM) after osmium-dimethyl sulfoxide-osmium (ODO) maceration, the studies of our research group on the morphodynamics of oocyte-follicle cell associations during follicle development in humans. In resting oocytes, follicular cells project few and short cytoplasmic processes in the perioocytic space. They often form bulbous terminals very close to the oolemma where zonulae adherentes, maculae adherentes, and gap junctions are present. The oolemma mostly appears smooth with short and scanty microvilli. In early growing follicles, follicular cell projections appear as (a) long and tortuous microvilli or (b) large and short extensions. The oolemma shows numerous short microvilli. By TEM, long and thin follicular "intraooplasmic processes" have been seen to penetrate deeply into some oolemma invaginations. In macerated samples, they are observed by SEM to come very close to the nucleus and contact different oocyte organelles. These processes are more likely involved in early oocyte growth. In late growing follicles, oocyte-somatic cell interactions-now established through the interposition of the zona pellucida (ZP)-preserve the general features of early growth stage, with the exceptions of "intraooplasmic processes," which are no more present. In mature follicles subjected to a long ODO maceration, corona cells appear to contact the oocyte through an apical plume of numerous very long "curly hair-like microvilli." Corona cell microvilli, quite likely provide a sort of cytoplasmic skeleton for the ZP and they are possibly involved in (a) release of nutrients or removal catabolites to/from oocyte and vice versa and (b) transfer of substances to build up ZP. In conclusion, among oocyte and somatic cells a structural and functional association is revealed. This association, certainly highly dynamic in vivo, plays a key role in regulating the healthy folliculogenesis to assure a correct and timed oocyte maturation and ovulation.

Effects of chilling on structural aspects of early preantral mouse follicles.

Chilling injury is one of the major limiting factors for achieving optimal cryopreservation of gametes. This study aimed to determine potential chilling-induced damage on several structural aspects of early preantral mouse follicles. Mechanically isolated intact early preantral follicles (type 3b-4) were exposed to 0 degrees C for 1, 5, 10, or 30 min. Control and chilled follicles were analyzed by confocal microscopy after staining for tubulin, F-actin, and chromatin, and by electron microscopy. Chilling for only 1 min was sufficient to cause depolymerization of microtubules in the oocyte and the surrounding granulosa cell layer as evidenced by a substantial decrease in fluorescence intensity after antitubulin labeling. Cooling for longer periods caused alterations in microtubule organization in the follicle-enclosed oocyte. These alterations included the loss of interphase microtubules, concomitant with the formation of perinuclear or cortical microtubule asters and sometimes a complete disappearance of microtubules. The extent of microtubule modification was related to the time of chilling, but was fully reversible after rewarming follicles at 37 degrees C for 1 h. Chilling had only minor effects on the actin-containing elements located predominantly in the oocyte cortex and the transzonal projections. Ultrastructural analysis confirmed that oocyte-somatic cell interactions were present. There was no influence on the chromatin configuration within the follicle-enclosed oocyte. These results indicate that mouse follicles are relatively tolerant to direct chilling injury and, as a consequence, are able to withstand the cooling-warming steps during conventional cryopreservation procedures.

Interactions between the oocyte and surrounding somatic cells in follicular development: lessons from in vitro culture.

Mammalian oogenesis occurs concomitantly with folliculogenesis in a coordinated manner in the ovaries. In vitro growth (IVG) culture systems of the oocytes have been developed as a new technology for utilizing incompetent oocytes in the ovary as a source of mature oocytes as well as for studying oogenesis, folliculogenesis, and oocyte-somatic cell interactions. The results of IVG experiments have suggested that direct association of oocytes and surrounding granulosa cells supports oocyte viability and growth through the gap junctions, which are efficient conduits for low molecular weight substances. It has been revealed that granulosa cells metabolize some molecules which are in turn transported into the oocytes. IVG systems have also provided evidence that FSH promotes the development of follicles at secondary or later stages by its stimulation of proliferation and differentiation of granulosa cells, and perhaps by its anti-apoptotic effects. In addition, interactions between granulosa cell-derived KIT ligands and oocyte KIT receptors have been suggested as initiating oocyte growth and follicular development. Furthermore, recent findings suggest there are growth factors derived from oocytes such as GDF-9 and BMP-15. With such factors, oocytes participate in follicular development by regulating the differentiation of surrounding somatic cells. These bidirectional communications between oocytes and somatic cells are important for oocyte growth and follicular development. IVG systems should provide further information regarding oogenesis and folliculogenesis in the ovary.

Development of mouse and rat oocytes in chimeric reaggregated ovaries after interspecific exchange of somatic and germ cell components.

The germ cell and somatic cell compartments of newborn rat and mouse ovaries, which contain only primordial stage follicles, were completely exchanged and reaggregated to produce xenogeneic chimeric ovaries. The reaggregated ovaries were grafted beneath the renal capsules of ovariectomized SCID mice to develop for periods up to 21 days. Xenogeneic follicles developed with essentially normal morphological characteristics. Both rat and mouse oocytes with species-specific characteristics grew within follicles that were composed of somatic cells exclusively of the alternative species. Rat oocytes grown in mouse follicles became competent to resume meiosis, and progressed to metaphase II when they were removed from follicles and cultured. In addition, mouse oocytes grown in rat follicles underwent fertilization and preimplantation development in vitro, and developed to term after embryos were transferred to pseudopregnant mouse foster mothers. Therefore, despite an estimated 11 million years of divergent evolution, oocytes and somatic cells of rat and mouse ovaries can be exchanged and can produce functional oocytes. It is concluded that factors involved in oocyte-somatic cell interactions necessary to support oocyte development and appropriate differentiation of the oocyte-associated granulosa cells are conserved between rats and mice. Moreover, although granulosa cells play important roles in oocyte development, the development of species-specific characteristics of oocytes occurs without apparent modification by a xenogeneic follicular environment.

Growth differentiation factor-9 stimulates progesterone synthesis in granulosa cells via a prostaglandin E2/EP2 receptor pathway.

Growth differentiation factor-9 (GDF-9), an oocyte-secreted member of the transforming growth factor beta superfamily, progesterone receptor, cyclooxygenase 2 (Cox2; Ptgs2), and the EP2 prostaglandin E(2) (PGE(2)) receptor (EP2; Ptgerep2) are required for fertility in female but not male mice. To define the interrelationship of these factors, we used a preovulatory granulosa cell culture system in which we added recombinant GDF-9, prostaglandins, prostaglandin receptor agonists, or cyclooxygenase inhibitors. GDF-9 stimulated Cox2 mRNA within 2 h, and PGE(2) within 6 h; however, progesterone was not increased until 12 h after addition of GDF-9. This suggested that Cox2 is a direct downstream target of GDF-9 but that progesterone synthesis required an intermediate. To determine whether prostaglandin synthesis was required for progesterone production, we analyzed the effects of PGE(2) and cyclooxygenase inhibitors on this process. PGE(2) can stimulate progesterone synthesis by itself, although less effectively than GDF-9 (3-fold vs. 6-fold increase over 24 h, respectively). Furthermore, indomethacin or NS-398, inhibitors of Cox2, block basal and GDF-9-stimulated progesterone synthesis. However, addition of PGE(2) to cultures containing both GDF-9 and NS-398 overrides the NS-398 block in progesterone synthesis. To further define the PGE(2)-dependent pathway, we show that butaprost, a specific EP2 agonist, stimulates progesterone synthesis and overrides the NS-398 block. In addition, GDF-9 stimulates EP2 mRNA synthesis by a prostaglandin- and progesterone-independent pathway. Thus, GDF-9 induces an EP2 signal transduction pathway which appears to be required for progesterone synthesis in cumulus granulosa cells. These studies further demonstrate the importance of oocyte-somatic cell interactions in female reproduction.

Paracrine actions of growth differentiation factor-9 in the mammalian ovary.

Although the transforming growth factor-beta (TGF-beta) superfamily is the largest family of secreted growth factors, surprisingly few downstream target genes in their signaling pathways have been identified. Likewise, the identities of oocyte-derived secreted factors, which regulate important oocyte-somatic cell interactions, remain largely unknown. For example, oocytes are known to secrete paracrine growth factor(s) which are necessary for cumulus expansion, induction of hyaluronic acid synthesis, and suppression of LH receptor (LHR) mRNA synthesis. Our previous studies demonstrated that absence of the TGF-beta family member, growth differentiation factor-9 (GDF-9), blocks ovarian folliculogenesis at the primary follicle stage leading to infertility. In the present study, we demonstrate that mouse GDF-9 protein is expressed in all oocytes beginning at the type 3a follicle stage including antral follicles. To explore the biological functions of GDF-9 in the later stages of folliculogenesis and cumulus expansion, we produced mature, glycosylated, recombinant mouse GDF-9 using a Chinese hamster ovary cell expression system. A granulosa cell culture system was established to determine the role of GDF-9 in the regulation of several key ovarian gene products using semiquantitative RT-PCR. We find that recombinant GDF-9 induces hyaluronan synthase 2 (HAS2), cyclooxygenase 2 (COX-2), and steroidogenic acute regulator protein (StAR) mRNA synthesis but suppresses urokinase plasminogen activator (uPA) and LHR mRNA synthesis. Consistent with the induction of StAR mRNA by GDF-9, recombinant GDF-9 increases granulosa cell progesterone synthesis in the absence of FSH. Since induction of HAS2 and suppression of the protease uPA in cumulus cells are key events in the production of the hyaluronic acid-rich extracellular matrix which is produced during cumulus expansion, we determined whether GDF-9 could mimic this process. Using oocytectomized cumulus cell-oocyte complexes, we show that recombinant GDF-9 induces cumulus expansion in vitro. These studies demonstrate that GDF-9 can bind to receptors on granulosa cells to regulate the expression of a number of gene products. Thus, in addition to playing a critical function as a growth and differentiation factor during early folliculogenesis, GDF-9 functions as an oocyte-secreted paracrine factor to regulate several key granulosa cell enzymes involved in cumulus expansion and maintenance of an optimal oocyte microenvironment, processes which are essential for normal ovulation, fertilization, and female reproduction.