The ontogeny of mullerian inhibiting substance in granulosa cells of the bovine ovarian follicle

Mullerian Inhibiting Substance (MIS) previously detected in the Sertoli cells of the calf testis has been localized in the granulosa cells of the ovarian Graafian follicle by using an immunoperoxidase technique and a monoclonal antibody (IG8) to MIS that almost completely blocks its biological activity. The immunoperoxidase technique (avidin-biotin complex method) demonstrated specific localization of MIS in the cytoplasm of the ovarian granulosa cells in the bovine Graafian follicles over a wide age span, i.e. one day, one week, three months, two-and-a-half years and five years. The presence of MIS in the ovary implies a function that is as yet unknown.     

Basal lamina of avian ovarian follicle: influence on morphology of granulosa cells in-vitro

Experiments were conducted to determine the influence of basal lamina on the morphology of ovarian granulosa cells in vitro. Pure and intact basal lamina was isolated from the large preovulatory follicles of the chicken ovary and designated basal lamina of avian ovarian follicle (BLAOF). Examination of the isolated basal lamina with electron microscope revealed an ultrastructure that is similar to that of basal lamina in the intact ovarian follicle. Pieces of the intact basal lamina were attached to the bottom of 32 mm culture dishes (BLAOF-coated dishes) in which differentiated granulosa cells isolated from the largest preovulatory follicle or undifferentiated granulosa cells isolated from immature small yellow chicken ovarian follicles were cultured; uncoated dishes served as controls. Granulosa cells incubated on intact basal lamina assumed spherical shape, whereas granulosa cells incubated directly on plastic in control dishes became highly flattened. Interestingly, granulosa cells that attached to plastic close to BLAOF (in BLAOF-containing dishes) became rounded. The storage of BLAOF-coated culture dishes at 4°C for 2 years had no apparent effect on its ability of the matrix material to induce changes in granulosa cell shape. Some components of the basal lamina could be solubilized with guanidine–HCl alone (fraction 1; 90–95% of total protein in BLAOF) with the remaining components solubilized with β-mercaptoethanol containing guanidine–HCl (fraction 2; 5–10% of total protein in BLAOF). Differentiated and undifferentiated chicken granulosa cells became rounded when incubated in fraction 1-pre-coated wells; whereas those incubated directly on plastic in control wells were flattened. Similarly, when fraction 1 of solubilized basal lamina was added as liquid to incubation mixture, it caused both differentiated and undifferentiated granulosa cells to assume spherical shapes. The storage of fraction 1-coated culture dishes at 4°C for 12 or more months had no apparent effect on its ability to influence granulosa cell shape. Fraction 1-induced changes in granulosa cell shape were similar to those observed for complete and intact basal lamina (BLAOF). These findings demonstrate that intact homologous basal lamina (BLAOF) or its solubilized (fluidized) form can induce normal (in vivo) morphology in granulosa cells. It is suggested that BLAOF or its solubilized form can be used to culture cells in experiments designed to examine the influence of the natural basal lamina microenvironment on cellular behavior and function.     

Basal lamina of ovarian follicle regulates an inward Cl- current in differentiated granulosa cells

Patch-clampexperiments were conducted to study the effects of basal lamina(basement membrane) of preovulatory chicken ovarian follicle onmembrane currents in differentiated chicken granulosa cells in ahomologous system. The membrane capacitance (measure of total membranearea) was smaller in cells cultured on intact basal lamina than that ofcontrol cells. The granulosa cells expressed outward and two inwardcurrents. A small fraction of the cells (3%) expressed only atransient fast-activating and -inactivating inward current carried byCa²⁺. The majority of the cells, however, expressed aslowly activating and inactivating inward current (carried byCl) that was superimposed on the transientCa²⁺ current. All cells expressed an outward currentcharacteristic of the delayed-rectifier K⁺ current. Theremoval of extracellular Ca²⁺ led to elimination of theslow inward Cl current, indicating that it is aCa²⁺-dependent Cl current. Both peakamplitude and current density of the inward Cl currentwere significantly lower in cells cultured on freshly isolated intactbasal lamina (or basal lamina stored at 4°C for 12 mo) than those ofcontrol cells; however, basal lamina had no significant effect on thedensity of the outward current. Similar to the observations made forintact basal lamina, solubilized basal lamina suppressed the inwardCl current in differentiated granulosa cells. These datashow that homologous basal lamina modulates aCa²⁺-dependent Cl current in differentiatedgranulosa cells. These findings provide a partial explanation for themechanisms that subserve the reported effects of basal lamina (basementmembrane) on the metabolic functions of differentiated granulosa cells.

     

人卵巢颗粒细胞的体外培养方法探讨

目的建立人卵巢颗粒细胞分离纯化、体外培养的有效方法。方法收集体外受精—胚胎移植(IVF-ET)穿卵时的卵泡液,用胰蛋白酶消化法及密度梯度离心法分离纯化颗粒细胞并用不同培养基进行培养。结果用体积分数为50%的Percoll细胞分离液分离,DMEM/F12或McCoy’5a液体培养基进行培养,细胞纯度高,存活率高,后续生长良好。结论建立了人卵巢颗粒细胞体外培养的稳定模型,为颗粒细胞的体外研究奠定良好的基础。     

Filaments and microtubules in the cytoplasm of the granulosa cells from the human follicle

Summary The cytoplasm of granulosa cells in human primordial follicles from normal women shows a system of filaments and microtubules. Filaments about 70–150 Å in diameter and several m in length can be seen as bundles or irregularly distributed. Microtubules about 200 Å in diameter are predominantly oriented in paranuclear regions. The relationships between cytoplasmic filaments and microtubules in granulosa cells and those of Sertoli cells are briefly discussed.     

Fate of the granulosa cells in the hen's follicle

Summary In an early growing follicle the single row of granulosa cells are in apposition — they have abundant Golgi substance and granular endoplasmic reticulum. During growth the granulosa cells become separated by wide intercellular spaces filled with perivitelline substance. The granulosa cells of the mature follicle immediately preceding ovulation have an agranular reticulum and numerous lipid droplets. Granulosa cells can be identified up to 72 h after ovulation. During the 72 h the cytoplasm becomes progressively replaced by lipid. The initial change in intracellular structure may indicate a shift in function towards production of progesterone followed by fatty degeneration preceding their final disintegration.     

Presence and release of immunoreactive atrial natriuretic peptide in granulosa cells of the pig ovarian follicle.

Atrial natriuretic peptide (ANP) has been reported to be locally synthesized in the ovary although its physiological roles are still unknown. To define the origin of ovarian ANP, we demonstrated the presence and release of immunoreactive (ir) ANP in pig granulosa cells and characterized its biochemical properties. Serial dilution curves made with the extracts of pig granulosa cells, their perfusates and follicular fluid were paralleled to the standard curve of ANP. The amount of irANP in the granulosa cell was 2 fg/cell. The total amount of irANP in granulosa cells significantly correlated with the levels of irANP in follicular fluid. Additionally, the total content of irANP in the follicle negatively correlated with the follicular size. On reverse phase HPLC, the major form of irANP in granulosa cells and follicular fluid was high molecular weight but that in perfusate was low molecular weight. In Northern blot analysis, ANP mRNA was detected in the pig granulosa cells. Immunohistochemistry showed ANP prohormone location in granulosa cells of rat ovary. These data strongly suggest that the granulosa cells synthesize and secrete ANP.     

mTOR controls ovarian follicle growth by regulating granulosa cell proliferation

We have shown that inhibition of mTOR in granulosa cells and ovarian follicles results in compromised granulosa proliferation and reduced follicle growth. Further analysis here using spontaneously immortalized rat granulosa cells has revealed that mTOR pathway activity is enhanced during M-phase of the cell cycle. mTOR specific phosphorylation of p70S6 kinase and 4E-BP, and expression of Raptor are all enhanced during M-phase. The predominant effect of mTOR inhibition by the specific inhibitor Rapamycin (RAP) was a dose-responsive arrest in the G1 cell cycle stage. The fraction of granulosa cells that continued to divide in the presence of RAP exhibited a dose-dependent increase in aberrant mitotic figures known as anaphase bridges. Strikingly, estradiol consistently decreased the incidence of aberrant mitotic figures. In mice treated with RAP, the mitotic index was reduced compared to controls, and a similar increase in aberrant mitotic events was noted. RAP injected during a superovulation regime resulted in a dose-dependent reduction in the numbers of eggs ovulated. Implications for the real-time regulation of follicle growth and dominance, including the consequences of increased numbers of aneuploid granulosa cells, are discussed.     

Impaired follicle development and infertility in female mice lacking steroidogenic factor 1 in ovarian granulosa cells

The nuclear receptor steroidogenic factor 1 (SF-1 [officially designated NR5A1]) is essential for fetal gonadal development, but its roles in postnatal ovarian function are less well defined. Herein, we have extended our analyses of knockout (KO) mice with markedly decreased SF-1 expression in granulosa cells. As described, these SF-1 KO mice had hypoplastic ovaries that contained a decreased number of follicles and lacked corpora lutea. In the present study, we showed that SF-1 KO mice exhibited abnormal estrous cycles, were infertile, and released significantly fewer oocytes in response to a standard superovulation regimen. Moreover, they had blunted induction of plasma estradiol in response to gonadotropins. The granulosa cell-specific SF-1 KO also significantly affected ovarian expression of putative SF-1 target genes. Consistent with their decreased follicle number, these mice had reduced ovarian expression of anti-müllerian hormone (Amh), which correlates with the reserve pool of ovarian follicles, as well as decreased gonadotropin-induced ovarian expression of aromatase (Cyp19a1) and cyclin D2 (Ccnd2). In contrast, perhaps because of their abnormal cyclicity, SF-1 KO ovaries had higher basal expression of inhibin-alpha. They also had decreased immunoreactivity for genes related to proliferation (Ccnd2 and Mki67 [also known as Ki67]) and increased expression of Cdkn1b, also known as p27, which inhibits cyclin-dependent kinases, arguing for a role of SF-1 in granulosa cell proliferation. These findings demonstrate that SF-1 has a key role in female reproduction via essential actions in granulosa cells.     

Phosphodiesterase regulation is critical for the differentiation and pattern of gene expression in granulosa cells of the ovarian follicle

Feedback regulations are integral components of the cAMP signaling required for most cellular processes, including gene expression and cell differentiation. Here, we provide evidence that one of these feedback regulations involving the cyclic nucleotide phosphodiesterase PDE4D plays a critical role in cAMP signaling during the differentiation of granulosa cells of the ovarian follicle. Gonadotropins induce PDE4D mRNA and increase the cAMP hydrolyzing activity in granulosa cells, demonstrating that a feedback regulation of cAMP is operating in granulosa cells in vivo. Inactivation of the PDE4D by homologous recombination is associated with an altered pattern of cAMP accumulation induced by the gonadotropin LH/human chorionic gonadotropin (hCG), impaired female fertility, and a markedly decreased ovulation rate. In spite of a disruption of the cAMP response, LH/hCG induced P450 side chain cleavage expression and steroidogenesis in a manner similar to wild-type controls. Morphological examination of the ovary of PDE4D-/- mice indicated luteinization of antral follicles with entrapped oocytes. Consistent with the morphological finding of unruptured follicles, LH/hCG induction of genes involved in ovulation, including cyclooxygenase-2, progesterone receptor, and the downstream genes, is markedly decreased in the PDE4D-/- ovaries. These data demonstrate that PDE4D regulation plays a critical role in gonadotropin mechanism of action and suggest that the intensity and duration of the cAMP signal defines the pattern of gene expression during the differentiation of granulosa cells.     

A novel set of structures within the elasmobranch,ovarian follicle

Elasmobranch fishes produce some of the largest oocytes known, exceeding 10 cm in diameter. Using various microscopy techniques we investigated the structural adaptations which facilitate the production of these large egg cells in three species of shark: the Atlantic sharpnose shark, Rhizoprionodon terraenovae, dusky smoothound, Mustelus canis and the little gulper shark, Centrophorus uyato. The ovarian follicle of elasmobranchs follows the typical vertebrate pattern, with one notable exception; the zona pellucida reaches extreme widths, over 70 μm, during early oogenesis. Contact between the follicle cells and the oocyte across the zona pellucida is necessary for oogenesis. We describe here a novel set of large, tube‐like structures, which we named follicle cell processes that bridge this gap. The follicle cell processes are more robust than the microvilli associated with the follicle cells and the oocyte plasma membrane and much longer. During early oogenesis the follicle increases in size relatively quickly resulting in a wide zona pellucida. At this stage the follicle cell processes appear taut, uniform and radially oriented. As oogenesis continues the zona pellucida narrows and the follicle cell processes change their orientation, appearing to wrap around the oocyte. The presence of the contractile protein actin within the follicle cell processes and their change in orientation may well be an adaptation for maintaining the integrity of these large oocytes. The follicle cell processes also contain electron dense material, identical to material found within the follicle cells, suggesting a role in the transport of metabolites to the developing oocyte. J. Morphol., 2011. © 2011 Wiley‐Liss, Inc.     

Tube-like structures within the ovarian follicle of Hydrolagus colliei indicate the early origins of follicle cell processes

Follicle cell processes (FCP) are actin-based, tube-like structures that connect the developing oocyte to the follicle cells throughout oogenesis. They were first described in Selachians (sharks) where their suggested roles were facilitating the transport of metabolites to the developing oocyte and providing structural support to the large egg cells of sharks, an early stage in the evolution of viviparity. Subsequent studies found that FCP are absent in Rajiformes (skates), suggesting that FCP may have been novel structures specific to the sharks. Here, FCP in Hydrolagus colliei, a Chimaeriform, were described. The FCP of H. colliei differ morphologically from those previously described in sharks, but as they also contain actin, they presumably play similar roles provisioning the developing oocyte and providing structural support. The presence of FCP in the order Chimaeriformes suggests that their origin predates the split of the elasmobranchs and the holocephalans.     

Cloning and sequence analysis of a cDNA encoding poly-ubiquitin in human ovarian granulosa cells

A cDNA clone, pHGR21 encoding poly-ubiquitin, was isolated from a human ovarian granulosa cDNA library. This clone contained three complete, and part of a fourth, ubiquitin coding sequence joined head to tail with no spacer sequences. Northern analysis employing a restriction fragment comprising a complete ubiquitin coding unit indicated the existence of two mRNA species of 1.1kb and 2.8kb. Sequence comparison of pHGR21 with the known two human ubiquitin genes revealed differences to the human ubiquitin-3 repeat gene but significant homology to the human ubiquitin-9 repeat gene. The untranslated 3'-region and the adjacent ubiquitin coding repeat were found to be identical to that of the human ubiquitin-9 repeat gene. The other 3 ubiquitin coding repeats were of close homology to the fourth ubiquitin coding repeat of the human ubiquitin-9 repeat gene. These findings suggest the existence of yet another human poly-ubiquitin gene.     

Orotic acid induces apoptotic death in ovarian adult granulosa tumour cells and increases mitochondrial activity in normal ovarian granulosa cells

Orotic acid (OA) is a natural product that acts as a precursor in the pyrimidine nucleotide biosynthesis pathway. Most studies concerning administration of OA focus on its therapeutic effects; however, its effect on tumours is unclear. We aimed to determine whether treatment with OA influences the viability and apoptosis of normal (HGrC1) and tumour-derived (KGN) human ovarian granulosa cells. The effects of OA (10–250 μM) on viability and apoptosis of both cell lines were determined by using alamarBlue and assessing caspase-3/7 activity, respectively. Annexin V binding and loss of membrane integrity were evaluated in KGN cells. The cell cycle and proliferation of HGrC1 cells were assessed by performing flow cytometric and DNA content analyses, respectively. The influence of OA (10 and 100 μM) on cell cycle- and apoptosis-related gene expression was assessed by RT-qPCR in both cell lines. Mitochondrial activity was analysed by JC-1 staining in HGrC1 cells. In KGN cells, OA reduced viability and increased caspase-3/7 activity, but did not affect mRNA expression of Caspase 3, BAX, and BCL2. OA enhanced proliferation and mitochondrial activity in HGrC1 cells without activating apoptosis. This study demonstrates that the anti-cancer properties of OA in ovarian granulosa tumour cells are not related to changes in apoptosis-associated gene expression, but to increased caspase-3/7 activity. Thus, OA is a promising therapeutic agent for ovarian granulosa tumours. Further, our results suggest that differences in basal expression of cell cycle- and apoptosis-related genes between the two cell lines are responsible for their different responses to OA.