Subpopulations of granulosa cells within the human ovarian follicle

Human follicular cells were separated according to their isopycnic densities. Three populations were isolated and identified in terms of their secretion of progesterone and oestradiol. Cells in the least dense population secreted approximately 60% as much progesterone and 20% as much oestradiol per cell as did cells in the two denser bands. It is proposed that cumulus cells compose the least dense band and that another band may be made up of antral cells.     

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.     

Interleukin-1 inhibits follicle stimulating hormone-induced differentiation in rat granulosa cells in vitro

Increasing evidence suggests that factors secreted from cells of the immune system can affect endocrine function. In this report we show that the monokine, interleukin-1, inhibits follicle stimulating hormone-induced development of luteinizing hormone receptors and reduces progesterone secreted from cultured rat granulosa cells. These effects of interleukin-1 were observed in the physiological range of 10(-9) M. The ability of sex steroids to influence the immune response together with our results support the hypothesis that there is a bidirectional communication network which links the immune and reproductive endocrine systems.     

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.

     

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.     

Lipin-1 expression is critical for keratinocyte differentiation

Lipin-1 is an Mg²⁺-dependent phosphatidate phosphatase that facilitates the dephosphorylation of phosphatidic acid to generate diacylglycerol. Little is known about the expression and function of lipin-1 in normal human epidermal keratinocytes (NHEKs). Here, we demonstrate that lipin-1 is present in basal and spinous layers of the normal human epidermis, and lipin-1 expression is gradually downregulated during NHEK differentiation. Interestingly, lipin-1 knockdown (KD) inhibited keratinocyte differentiation and caused G1 arrest by upregulating p21 expression. Cell cycle arrest by p21 is required for commitment of keratinocytes to differentiation, but must be downregulated for the progress of keratinocyte differentiation. Therefore, reduced keratinocyte differentiation results from sustained upregulation of p21 by lipin-1 KD. Lipin-1 KD also decreased the phosphorylation/activation of protein kinase C (PKC)α, whereas lipin-1 overexpression increased PKCα phosphorylation. Treatment with PKCα inhibitors, like lipin-1 KD, stimulated p21 expression, while lipin-1 overexpression reduced p21 expression, implicating PKCα in lipin-1-induced regulation of p21 expression. Taken together, these results suggest that lipin-1-mediated downregulation of p21 is critical for the progress of keratinocyte differentiation after the initial commitment of keratinocytes to differentiation induced by p21, and that PKCα is involved in p21 expression regulation by lipin-1.     

排卵前期卵泡颗粒细胞端粒酶的表达及其影响因素

用端粒酶重复扩增酶联免疫吸附分析法（telomeric repeat amplification protocol-enzyme linked immunoadsordent assay,TRAP-ELISA）观察体外培养的大鼠排卵前期卵巢颗粒细胞中端粒酶活性的表达及其影响因素,并用放射免疫分析法（radioimmunoassay,RIA）同步测定培养液中雌二醇（estradiol,E2）、孕西阿（progesterone,P0）含量的变化及MTT（四甲基偶氮唑盐）法测定颗粒细胞增殖指数,分析颗粒细胞中端粒酶活性的表达以及端粒酶活性表达的影响因素。本实验中大鼠排卵前期卵巢颗粒细胞中有端粒酶活性表达,且在人绒毛膜促性腺激素（human chorionic gonadotropin,HCG）、卵泡刺激素（follicle-stimu1ating hormone,FSH）、二丁酰环磷腺苷（dbcAMP）及维拉帕米（verapamil）作用下活性明显升高,而在反义c-myb作用下活性明显降低。RIA测定培养液中雌激素及孕激素含量发现,在verapamil及FSH作用下E2与P0分泌量明显升高,在dbcAMP及HCG作用下分泌量无明显改变,而在反义c-myb作用下分泌量明显降低,在不同作用因素下的端粒酶活性与它相对应的E2及P0分泌量无相关性。MTT法测定显示,反义hTERT能明显抑制颗粒细胞的增殖。由此可以证实,排卵前期卵巢的颗粒细胞中表达有端粒酶活性,其活性受FSH、HCG、verapamil、dbcAMP及癌基因的影响,并且端粒酶活性与颗粒细胞增殖功能相关。     

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.     

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.     

Inverse expression of peroxisomes and connexin-43 in the granulosa cells of the quail follicle.

Studying the regulation of peroxisome (Px) expression could improve our understanding of human peroxisomal disorders. The granulosa of the largest preovulatory quail follicles proved to be a relevant model because (a) Px expression changes according to the follicular maturation stage and (b) Px expression varies regionally according to the distance of the granulosa relative to the germinal disc region containing the female gamete (oocyte). The question was asked whether Px expression is related to the extent of metabolic cell coupling and whether zonal Px variation is causally related to oocytal factors. This was evaluated by the presence of catalase and Cx-43 (marker proteins for peroxisomes and gap junctions, respectively) and by in vitro experiments with granulosa explants. The data obtained show that the expression of Cx-43 and Px is inversely correlated both temporally and spatially. Uncoupling of gap junctions results in an upregulation of alpha-catalase immunofluorescence. This is in agreement with reports that gap junctions are often negatively affected by Px proliferators. The zonal gradient in Px expression appears to be imposed by the oocyte, as is the case for steroidogenesis and proliferative capacity in the granulosa epithelium. (J Histochem Cytochem 48:167-177, 2000)     

Gonadotropin-induced differentiation of granulosa cells is associated with the co-ordinated regulation of cytoskeletal proteins involved in cell-contact formation

The gonadotropin-induced differentiation of granulosa cells in culture was studied, with particular attention being given to the organization and expression of cytoskeletal proteins involved in the formation of cell contacts, as well as to progesterone production. Gonadotropin-treated granulosa cells formed clusters of spherical cells containing few vinculin-containing focal contacts, exhibited a diffuse distribution of actin, and had few adherens junctions but more gap junctions than cells grown without the hormone. In gonadotropin-treated cells, the levels of synthesis of the cytoskeletal proteins, vinculin, alpha-actinin, and actin, were dramatically reduced, but the synthesis of the tubulins and vimentin was unaffected. Decreased levels of synthesis of these cytoskeletal proteins were also observed in an in vitro translation assay using poly(A)+ RNA from gonadotropin-treated cells. The hybridization of cytoplasmic RNA with cloned actin and vimentin cDNAs revealed a marked decrease in actin-RNA levels, but no change in vimentin-RNA levels in these cells. Such alterations in cytoskeletal-protein expression were also observed in cells treated with compounds that cause elevated cellular cAMP levels by acting at a stage beyond gonadotropin receptor stimulation. Furthermore, by keeping the cells in a spherical configuration in suspension culture, or by treating the cells with cytochalasin B, similar changes in the synthesis of these cytoskeletal proteins were observed. During this process, there was a concomitant increased in the production of progesterone (although to a much lesser extent in suspension culture) that occurred in parallel with the appearance of large mitochondria with lamellar-tubular cristae and a well-developed smooth endoplasmic reticulum, these features being characteristic of granulosa-lutein cells in vivo. Our results suggest that changes in cell shape and contact, together with the regulation of cytoskeletal elements that determine cellular morphogenesis, are part of the gonadotropin-controlled differentiation program in granulosa cells and may also occur during the maturation of these cells in vivo.     

The microfilament pattern in the somatic follicle cells of mid-vitellogenic ovarian follicles of Drosophila

The microfilament pattern in the somatic follicle cells of mid-vitellogenic stage 9 to 11 follicles of Drosophila was analyzed by staining F-actin with fluorescence-labeled phalloidin. During the analyzed stages of oogenesis, the follicular epithelium differentiates morphologically and functionally. These changes are also reflected at the organization of the microfilaments. At stage 10, they show no preferred orientation in the very thin follicle cells covering the nurse cells. In contrast, the microfilaments in the basal part of the columnar follicle cells covering the oocyte become organized in parallel bundles oriented perpendicular to the long axis of the follicle. During stages 10B/11 this organization is maintained at the nurse cell/oocyte border but becomes more sloppy towards the posterior pole of the follicle. The basal part of the follicle cells containing the microfilament bundles adheres so tightly to the basement membrane that this acellular layer cannot be separated mechanically from the epithelium. Indirect evidence from inhibition studies with cytochalasins and the effects of collagenase or pronase E added to the culture medium suggest that the microfilament bundles may promote increased adhesiveness of the follicle cells to the basement membrane. The possible functional implications of the microfilaments and their orientation are discussed.     

MYCN gene expression is required for the onset of the differentiation programme in neuroblastoma cells

Neuroblastoma is an embryonic tumour of the sympathetic nervous system and is one of the most common cancers in childhood. A high differentiation stage has been associated with a favourable outcome; however, the mechanisms governing neuroblastoma cell differentiation are not completely understood. The MYCN gene is considered the hallmark of neuroblastoma. Even though it has been reported that MYCN has a role during embryonic development, it is needed its decrease so that differentiation can be completed. We aimed to better define the role of MYCN in the differentiation processes, particularly during the early stages. Considering the ability of MYCN to regulate non-coding RNAs, our hypothesis was that N-Myc protein might be necessary to activate differentiation (mimicking embryonic development events) by regulating miRNAs critical for this process. We show that MYCN expression increased in embryonic cortical neural precursor cells at an early stage after differentiation induction. To investigate our hypothesis, we used human neuroblastoma cell lines. In LAN-5 neuroblastoma cells, MYCN was upregulated after 2 days of differentiation induction before its expected downregulation. Positive modulation of various differentiation markers was associated with the increased MYCN expression. Similarly, MYCN silencing inhibited such differentiation, leading to negative modulation of various differentiation markers. Furthermore, MYCN gene overexpression in the poorly differentiating neuroblastoma cell line SK-N-AS restored the ability of such cells to differentiate. We identified three key miRNAs, which could regulate the onset of differentiation programme in the neuroblastoma cells in which we modulated MYCN. Interestingly, these effects were accompanied by changes in the apoptotic compartment evaluated both as expression of apoptosis-related genes and as fraction of apoptotic cells. Therefore, our idea is that MYCN is necessary during the activation of neuroblastoma differentiation to induce apoptosis in cells that are not committed to differentiate.