Neighboring Granulosa Cells Research Articles

Deciphering Cellular Heterogeneity and Communication Patterns in Porcine Antral Follicles by Single-Cell RNA Sequencing.

The antral follicle stage is a critical period in mammalian oocyte maturation, marked by complex interactions between oocyte development and neighboring granulosa cells. Understanding the heterogeneity and communication patterns of granulosa cells within antral follicles is crucial for deciphering their roles in follicle development and oocyte maturation. Here, we employed single-cell RNA-sequencing to explore the molecular and cellular characteristics of porcine antral follicles. Our analysis revealed distinct subpopulations within mural and cumulus granulosa cells, indicating diverse cellular states and functions within the follicles. Functional enrichment analysis unveiled the involvement of specific subpopulations in steroid biosynthesis, cumulus expansion, and cellular communication. Moreover, comparing mature and less mature follicles highlighted differences in cell distribution and functions, indicating developmental-specific variations. Our findings shed light on the intricate cellular heterogeneity and communication network within porcine antral follicles, providing valuable insights into the regulation of follicle development and oocyte maturation in pigs. These results hold promise for improving pig reproductive efficiency and advancing human reproductive medicine.

R-spondin2 signaling is required for oocyte-driven intercellular communication and follicular growth

R-spondin2 (RSPO2) is a member of the R-spondin family, which are secreted activators of the WNT/β-catenin (CTNNB1) signaling pathway. In the mouse postnatal ovary, WNT/CTNNB1 signaling is active in the oocyte and in the neighboring supporting cells, the granulosa cells. Although the role of Rspo2 has been previously studied using in vitro experiments, the results are conflicting and the in vivo ovarian function of Rspo2 remains unclear. In the present study, we found that RSPO2/Rspo2 expression is restricted to the oocyte of developing follicles in both human and mouse ovaries from the beginning of the follicular growth. In mice, genetic deletion of Rspo2 does not impair oocyte growth, but instead prevents cell cycle progression of neighboring granulosa cells, thus resulting in an arrest of follicular growth. We further show this cell cycle arrest to be independent of growth promoting GDF9 signaling, but rather associated with a downregulation of WNT/CTNNB1 signaling in granulosa cells. To confirm the contribution of WNT/CTNNB1 signaling in granulosa cell proliferation, we induced cell type specific deletion of Ctnnb1 postnatally. Strikingly, follicles lacking Ctnnb1 failed to develop beyond the primary stage. These results show that RSPO2 acts in a paracrine manner to sustain granulosa cell proliferation in early developing follicles. Taken together, our data demonstrate that the activation of WNT/CTNNB1 signaling by RSPO2 is essential for oocyte-granulosa cell interactions that drive maturation of the ovarian follicles and eventually female fertility.

Recombinant BMP4 and BMP7 increase activin A production by up-regulating inhibin βA subunit and furin expression in human granulosa-lutein cells.

Granulosa cell-derived activins play important roles in the regulation of ovarian functions. To date, there is limited information pertaining to the intracellular regulation, assembly, and secretion of endogenous activin A in human granulosa cells. The aim of this study was to examine the effects of BMP4 and BMP7 on furin expression and activin A production as well as the underlying mechanisms of action in human granulosa cells. An established immortalized human granulosa cell line (SVOG) and primary granulosa-lutein cells were used as study models. Expression of inhibin subunits and furin as well as activin A accumulation were examined after exposure to recombinant human BMP4 or BMP7. A BMP type I receptor inhibitor (dorsomorphin), a furin inhibitor (Decanoyl-Arg-Val-Lys-Arg-chloromethylketone), and small interfering RNAs targeting SMAD4 and furin were used to verify the specificity of the effects and investigate potential mechanisms. The study was conducted in an academic center. Specific mRNA and protein levels were examined using real time qPCR and Western blot. Activin A levels were measured using enzyme immunoassay. Treatment with bone morphogenetic protein (BMP) 4 and BMP7 significantly increased furin mRNA and protein, inhibin βA mRNA, and activin A accumulation. Pre-treatment with dorsomorphin or SMAD4 knockdown reversed the stimulatory effects of BMP4 and BMP7 on furin and inhibin βA expression. In addition, furin knockdown or pre-treatment with a furin inhibitor attenuated the BMP4- and BMP7-induced accumulation of activin A. Recombinant BMP4 and BMP7 increase the production of bioactive mature activin A by up-regulating both the production and proteolytic processing of inhibin βA subunit in human granulosa cells. The enhancement of inhibin βA subunit processing is attributable to a SMAD-dependent up-regulation of its proprotein convertase, furin. These findings provide a potential mechanism by which theca cells can regulate neighboring granulosa cells in the ovary.

Transcriptome profiling of the theca interna from bovine ovarian follicles during atresia.

The theca interna is a specialized stromal layer that envelops each growing ovarian follicle. It contains capillaries, fibroblasts, immune cells and the steroidogenic cells that synthesize androgens for conversion to estradiol by the neighboring granulosa cells. During reproductive life only a small number of follicles will grow to a sufficient size to ovulate, whereas the majority of follicles will undergo regression/atresia and phagocytosis by macrophages. To identify genes which are differentially regulated in the theca interna during follicular atresia, we undertook transcriptome profiling of the theca interna from healthy (n = 10) and antral atretic (n = 5) bovine follicles at early antral stages (<5 mm). Principal Component Analyses and hierarchical classification of the signal intensity plots for the arrays showed primary clustering into two groups, healthy and atretic. A total of 543 probe sets were differentially expressed between the atretic and healthy theca interna. Further analyses of these genes by Ingenuity Pathway Analysis and Gene Ontology Enrichment Analysis Toolkit software found most of the genes being expressed were related to cytokines, hormones and receptors as well as the cell cycle and DNA replication. Cell cycle genes which encode components of the replicating chromosome complex and mitotic spindle were down-regulated in atretic theca interna, whereas stress response and inflammation-related genes such as TP53, IKBKB and TGFB1 were up-regulated. In addition to cell cycle regulators, upstream regulators that were predicted to be inhibited included Retinoblastoma 1, E2 transcription factor 1, and hepatocyte growth factor. Our study suggests that during antral atresia of small follicles in the theca interna, arrest of cell cycle and DNA replication occurs rather than up- regulation of apoptosis-associated genes as occurs in granulosa cells.

Oocyte-Expressed Interleukin 7 Suppresses Granulosa Cell Apoptosis and Promotes Oocyte Maturation in Rats

Development of ovarian follicles is regulated by pituitary-derived gonadotropins together with local ovarian paracrine factors. Based on DNA microarray data, we performed RT-PCR and immunostaining to demonstrate the expression of interleukin 7 transcripts in oocytes of preantral, antral, and preovulatory follicles in rats. We also found the expression of interleukin 7 receptor and the coreceptor interleukin 2 receptor gamma in granulosa cells, cumulus cells, and preovulatory oocytes. In cultured rat granulosa cells obtained from early antral and preovulatory follicles, treatment with interleukin 7 stimulated the phosphorylation of AKT, glycogen synthase kinase (GSK3B), and STAT5 proteins in a time- and dose-dependent manner. Furthermore, measurement of mitochondrial reductase activity indicated that treatment with interleukin 7, similar to gonadotropins, increased the number of viable granulosa cells during a 24-h culture period. Furthermore, monitoring of the activities of apoptotic enzymes (caspase 3/7) indicated that treatment with interleukin 7 suppressed apoptosis of cultured granulosa cells from both antral and preovulatory follicles following serum withdrawal. The apoptosis-suppressing actions of interleukin 7 were blocked by an inhibitor of the phosphoinositol-3-kinase (PIK3)/AKT pathway. Furthermore, treatment of cultured preovulatory follicles with interleukin 7, like treatment with human chorionic gonadotropin, induced germinal vesicle breakdown of oocytes. The stimulatory effect of interleukin 7 was also blocked by inhibitors of the PIK3/AKT pathway. The present findings suggest that oocyte-derived interleukin 7 could act on neighboring granulosa cells as a survival factor and promote the nuclear maturation of preovulatory oocytes through activation of the PIK3/AKT pathway.

Phagocytosis mechanism of apoptotic granulosa cells regulated by milk-fat globule-EGF factor 8

In the process of ovary sexual maturation, most immature ovarian follicles degrade into atretic follicles accompanied by apoptosis in granulosa cells. Macrophages can recognize apoptotic cells through specific binding with phosphatidylserine (PS), exposed on the surface of apoptotic cells, which is mediated by milk-fat globule-EGF factor 8 (MFG-E8). In the present research, we examined the involvement of the MFG-E8-dependent phagocytosis system in the atretic follicles of developing mouse ovaries. The number of atretic follicles and DNA-fragmented granulosa cells significantly increased in B6C3F1 mice during 2 to 6 weeks. Chromatin-condensed granulosa cells were engulfed by macrophages, which existed in the stroma or atretic follicles, or by neighboring normal granulosa cells. MFG-E8 mRNA increased in ovaries during 2 to 6 weeks, and immunoreactivity of MFG-E8 was detected at the surface of apoptotic cells existing around the antrum. Immunoelectron microscopic study revealed MFG-E8-positive signals on the membrane of apoptotic cells near macrophages, but apoptotic cells engulfed by neighboring granulosa cells showed few signals. Anti-Fas antibody elevated the annexin-V-positive reaction in isolated granulosa cells from 3-week-old mouse ovaries. MFG-E8 seems to act on the phagocytosis of apoptotic granulosa cells via macrophages and contribute to the regression process of atretic follicles.

Ultrastructural characteristics of human granulosa cells in a coculture system for in vitro fertilization

The use of somatic cells for cocultures during in vitro fertilization (IVF) is currently finalized to obtain a higher number of healthy and viable embryos with a high potential of implantation. Among the different cell lines that can be used as feeder cells for cocultures, granulosa cells (GCs) are autologous cells, safe and easy to recover. The aim of the present study was to analyze the fine structure of human GCs used in a coculture system to evaluate, from a morphodynamic point of view, their role in supporting embryo development. GCs were collected during oocyte pick-up, 36 h after human chorionic gonadotropin administration, from patients undergoing IVF procedures, who had given their informed consent to be included in this protocol. After coculture, GCs were fixed and processed for light microscopy (LM) and transmission electron microscopy (TEM). By LM, GCs appeared as clusters of loosely packed cells, irregularly rounded or polyhedral in shape, varying in diameter from 18 to 25 microm. Mitotic cells, as well as regressing elements (with pyknotic nuclei or dense cytoplasm) and cell fragments were occasionally observed. By TEM, the plasma membrane was irregular due to the presence of cytoplasmic evaginations. Linear and annular gap junctions between neighboring GCs were found. GC nuclei, rounded and eccentrically located, contained finely dispersed chromatin, one (often two) prominent nucleoli and, infrequently, peripheral patches of heterochromatin. Numerous organelles populated the GC cytoplasm, among them, mitochondria were rod-shaped or elongated, usually provided with tubular-vesicular cristae but occasionally showing atypical, longitudinally oriented cristae. Membranes of smooth endoplasmic reticulum, Golgi stacks and vesicles, secretory-like granules, cisternae of rough endoplasmic reticulum (RER), free ribosomes and polysomes, lysosomal-like bodies, microfilaments, and lipid droplets were also seen in the GC cytoplasm. In most cells, RER was scarcely represented and numerous lipid droplets filled the perinuclear space. On the contrary, some GCs contained an abundant RER and rare lipid droplets scattered in the cytoplasm. In conclusion, our data demonstrated the presence, in a coculture system, of GCs provided with ultrastructural characteristics typical of healthy, metabolically active, mostly steroidogenic cells. Protein-synthetic cells have also been detected. These data, evaluated at the light of biochemical and clinical studies, sustain the capability of human GCs cocultures to positively affect early embryo development in vitro by the secretion of steroids and proteins, putative "embryotrophic" factors.

Oocyte–somatic cell interactions during follicle development in mammals

Our current perspectives on the relationship between the oocyte and its surrounding somatic cells are changing as we gain a greater understanding of factors regulating folliculogenesis. It is now widely accepted that the oocyte plays a very active role in promoting follicle growth and directing granulosa cell differentiation. The oocyte achieves this, in part, by secreting soluble paracrine growth factors that act on its neighboring granulosa cells, which in turn regulate oocyte development. In preantral follicles, the oocyte directs granulosa cells to regulate oocyte growth, and oocytes may also directly drive follicle growth. In antral follicles, the oocyte governs the behaviour of cells in its immediate vicinity, thereby actively regulating its own microenvironment. As such, the oocyte establishes and maintains the distinct cumulus lineage of granulosa cells. This oocyte–cumulus cell interaction, in general, prevents luteinization of cumulus cells by promoting growth, regulating steroidogenesis and inhibin synthesis, and suppressing luteinizing hormone receptor expression. Conversely, mural granulosa cells in antral follicles, which have no direct physical contact with the oocyte and, presumably, experience a more diffuse concentration of oocyte-secreted factors, proceed to a different phenotype. In the ovulating follicle, oocyte-secreted factors also play vital roles in enabling cumulus cell expansion and regulating extracellular matrix stability, thus facilitating ovulation. The identities of these oocyte-secreted growth factors regulating such key ovarian functions remain unknown, although growth differentiation factor-9 (GDF-9), GDF-9B and/or bone morphogenetic protein-6 (BMP-6) are likely candidate molecules, probably forming complex local interactions with other related members of the transforming growth factor-β (TGF-β) superfamily. Elucidating the nature of oocyte–somatic cell interactions at the various stages of follicle development will have important implications for our understanding of factors regulating folliculogenesis, ovulation rate and fecundity.

Subcellular distribution of ZP1, ZP2, and ZP3 glycoproteins during folliculogenesis and demonstration of their topographical disposition within the zona matrix of mouse ovarian oocytes.

The zona pellucida (ZP) is an extracellular coat synthesized and secreted by the oocyte during follicular development and surrounding the plasma membrane of mammalian eggs. To date, the mechanism of synthesis and secretion, mode of assembly, and intracellular trafficking of the ZP glycoproteins have not been fully elucidated. Using antibodies against mouse ZP1, ZP2, and ZP3 in conjunction with the protein A-gold technique, we have shown an association of immunolabeling with the Golgi apparatus, secretory granules, and a complex structure called vesicular aggregate, respectively, in mouse ovarian follicles. In contrast, the neighboring granulosa cells were not reactive to any of the three antibodies used. Immunolabeling of ZP1, ZP2, and ZP3 was detected throughout the entire thickness of the ZP, irrespective of the developmental stage of ovarian follicles. Double and triple immunolocalization studies, using antibodies tagged directly to different sizes of gold particles, revealed an asymmetric spatial distribution of the three ZP glycoproteins in the zona matrix at various stages of follicular development. All three glycoproteins were specifically localized over small patches of darkly stained flocculent substance dispersed throughout the zona matrix. Very often, ZP1, ZP2, and ZP3 were found in close association. These results confirm findings from previous studies demonstrating that ovarian oocytes and not granulosa cells are the only source for mouse ZP glycoproteins. In addition, results from our morphological and immunocytochemical experiments suggest that the vesicular aggregates in the ooplasm are likely to serve as an intermediary in the synthesis and secretion of ZP glycoproteins. The stoichiometric disposition of ZP1, ZP2, and ZP3 in the zona matrix as revealed by double and triple immunolocalization studies provide further insight into some of the unanswered questions pertinent to the current model of mouse ZP structure proposed by the Wassarman group.

Species-specific Differences in Apoptotic Cell Localization in Granulosa and Theca Interna Cells during Follicular Atresia in Porcine and Bovine Ovaries.

Species-specific differences in the process of apoptosis in granulosa and theca layers during follicular atresia in porcine and bovine ovaries were investigated by in situ DNA 3’end-labeling (TUNEL) at the level of individual cells. In porcine ovaries, granulosa cells located on the inner surface of the granulosa layer appeared to undergo apoptosis at the first stage, followed by neighboring granulosa cells. In the contrast, granulosa cells located on the middle region appeared to undergo apoptosis at the first stage in bovine ovaries. In both porcine and bovine ovaries, detachment and degeneration of the granulosa cell layer and fragmentation of basement membrane occurred in the follicles at advanced stage of atresia. In the ovaries of both species, theca interna cells were still intact at the stage of early atresia and then apoptotic cells appeared in the late to final stages of atresia, but no apoptotic cells were observed in theca externa layers during follicular atresia. The present findings indicated that apoptosis occurring in granulosa cells is an initial symptom of follicular atresia in the ovaries of both species, and that the apoptosis inducing factor(s) and/or survival factor(s) for the granulosa cells may be different between porcine and bovine ovaries.

Interactions of Apoptosis and Extracellular Matrices in Granulosa Cells of Atretic Follicles in Porcine Ovaries.

The process of apoptosis of follicular granulosa cells in porcine ovaries during follicular atresia was investigated by in situ DNA 3'end-labeling at the level of individual cells. Histochemical changes in the follicular basement membrane (BM) were visualized by immunofluorescent staining of BM extracellular matrix (ECM) components, i.e. type IV collagen, laminin and fibronectin. At the first stage, granulosa cells located on the inner surface of the granulosa layer appeared to undergo apoptosis, followed by neighboring granulosa cells. No apoptotic granulosa cells making tight contact with intact BM were observed. Detachment and degeneration of the granulosa cell layer and fragmentation of BM occurred in follicles at the advanced stage of atresia. Finally, intermittent structures of BM and subsequent invasion of macrophages and fibroblasts were observed. Therefore we concluded that granulosa cell apoptosis is an initial symptom of follicular atresia in the porcine ovaries, and the degradation of BM follows granulosa cell apoptosis in the pig. Our results suggest that ECM components of follicular BM act as survival factors on follicular granulosa cells in porcine ovaries.

Ultrastructural Changes in Granulosa Cells in Porcine Antral Follicles Undergoing Atresia Indicate Apoptotic Cell Death.

Ultrastructural changes in follicular granulosa cells during atresia were examined in pig ovaries. No apoptotic cells were observed in healthy follicles. However, in follicles in the early stage of atresia apoptosis occurred in granulosa cells located on the inner surface of the follicular wall. In these apoptotic granulosa cells, nuclear condensation with large clumps of chromatin and cytoplasmic condensation but with retention of the integrity of organelles were observed by electron microscopy. As atresia progressed, pyknotic granulosa cells and apoptotic bodies increased in number in the follicular wall. In follicles in the late stage of atresia, apoptotic bodies were suspended in the follicular antrum. Concurrently, cell debris without distinct membranes but with condensed chromatin, degenerated apoptotic bodies, occurred in follicles. The apoptotic bodies and cell debris were occasionally surrounded and phagocytosed by neighboring granulosa cells. In follicles in the advanced stage of atresia, apoptotic bodies and cell debris were fed by macrophages.

Ultrastructural changes in micropylar cells and formation of the micropyle during oogenesis in the medaka Oryzias latipes

Ultrastructural changes in differentiating micropylar cells and in the micropyle occur during oogenesis in the medaka, Oryzias latipes. A micropylar cell is not detectable in previtellogenic ovarian follicles. In the early vitellogenic phase, the micropylar cell becomes differentiated from neighboring granulosa cells by its electrondense cytoplasm. The micropylar cell in this phase characteristically displays an increase in rough endoplasmic reticulum, Golgi complexes, and tonofilaments around the nucleus. By the late vitellogenic phase, the enlarged micropylar cell extends a broad cytoplasmic process to the oocyte surface. A conspicuous feature of the process is a large bundle of microtubules oriented perpendicular to the oocyte surface. The inner surface of the micropylar canal has a spiral structure and is covered with the outermost layer of the chorion. In the postvitellogenic phase, the main cell body possesses many tonofilaments, mitochondria, Golgi complexes, and rough endoplasmic reticulum, and the winding cytoplasmic process contains a twisted large bundle of microtubules with a bundle of tonofilaments as its core. The spiral structure of the micropylar vestibule and the micropylar canal reflects the twisting associated with elongation of fibrous bundles of the micropylar cell anchored on the chorion at the animal pole of the oocyte.

Fine structure of the micropylar cell and its change during oocyte maturation in the chum salmon, Oncorhynchus keta.

In the ovarian follicle, the micropylar cell (MPC) is distinguished from neighboring granulosa cells by its larger cell size and its thick cytoplasmic process. The micropylar cell body fits into a shallow depression (micropylar vestibule) on the outer surface of the egg envelope; its process extends through the micropylar canal, which extends from the bottom of the vestibule through the full thickness of the zona pellucida interna. At its distal end, the cell process expands into a bulb which fits into an indentation of the ooplasmic surface immediately beneath the inner opening of the micropylar canal. Intermediate and desmosomelike junctions establish an intimate association between MPC process and oocyte. Various kinds of organelles and inclusions in the MPC show a characteristic pattern of cytoplasmic distribution; rough endoplasmic reticulum with markedly dilated cisternae is found exclusively in the main cell body, while microtubules and thin filaments are observed in the cytoplasmic process. Immediately before or during the breakdown of the germinal vesicle in the intrafollicular oocyte, the cytoplasmic process of the MPC gradually decreases in length and begins to withdraw from the micropylar canal. At the same time, the ooplasmic surface protrudes outward to form a papilla in the canal. The intimate MPC-oocyte association disappears during formation of the ooplasmic papilla. Hydration of the oocyte apparently occurs at the final stage of maturation and probably participates in papilla formation. Although the MPC undergoes degenerative changes as ovulation draws near, it remains attached to the inner surface of the granulosa cell layer even after its association with the oocyte has completely disappeared. We speculate that the micropyle develops during fish oogenesis through the combined activity of the MPC and neighboring granulosa cells. It appears that the cell body of the micropylar cell and nearby granulosa cells exert mechanical pressure on the external surface of the growing oocyte and thus participate in formation of the micropylar vestibule. The cytoplasmic process of the MPC evidently forms a passive barrier to deposition of material for the egg envelope in the animal pole, thereby resulting in formation of the micropylar canal.