Multigenic and imprinting control of ovarian granulosa cell tumorigenesis in mice

Spontaneous juvenile ovarian granulosa cell (GC) tumors that occur in young girls are similar to GC carcinomas that develop in SWR-derived inbred mice. We analyzed female offspring from a series of matings among SWR and SJL inbred mice for chromosomal loci underlying tumor susceptibility. Intercross F2 female mice were produced by reciprocal matings of (SWR x SJL)F1 and (SJL x SWR)F1 parents. Tumorigenesis in these F2 mice as well as in SWXJ recombinant inbred and congenic strains of mice derived from SWR and SJL showed significant (P < 0.001) association with Gct1, a dominant susceptibility locus on chromosome (CHR) 4 and with Gct2 on CHR 12. Suggestive (P < 0.01) association was found with Gct3 on CHR 15. A fourth susceptibility locus, Gct4 on CHR X, was demonstrated with a strong parent-of-origin effect associated with the paternal genotype. Imprinting and complex interactions among these four loci combine to establish the probability for GC tumorigenesis in this mouse model.     

Monoclonal antibodies against pig ovarian follicular granulosa cells induce apoptotic cell death in cultured granulosa cells

Two monoclonal antibodies capable of inducing granulosa cell apoptosis were produced against granulosa cells prepared from antral follicles of pig ovaries. The healthy follicles, 4-5 mm in diameter, were dissected from the ovaries of gilts, and then granulosa cells were isolated. BALB/c female mice were immunized with the isolated granulosa cells. Antibodies against the granulosa cells were detected by immunofluorescent staining using frozen ovarian sections. The isolated spleen cells prepared from immunized mice producing antibodies against the granulosa cells were fused with Sp2/O-Ag 14 mouse myeloma cells by standard hybridization techniques. Two hybridoma clones, PFG-1 and PFG-2, which produced specific IgM antibodies against granulosa cells were selected. Western blotting analysis revealed that PFG-1 and PFG-2 antibodies specifically recognized cell-membrane proteins with molecular weights of 55 and 70 kD and isoelectric points of 5.9 and 5.4, respectively. The monoclonal antibodies immunohistochemically reacted with granulosa cells of healthy follicles. When the isolated granulosa cells prepared from healthy follicles were cultured in medium containing 0.1 or 10 micrograms/m/PFG-1 or PFG-2 antibodies, respectively, the cells underwent apoptosis as determined by nuclear morphology, DNA electrophoresis and flow cytometric analysis. In conclusion, these two monoclonal antibodies against granulosa cells have cell-killing activity in cultured granulosa cells.     

Cyclins: relevance of subcellular localization in cell cycle control

OBJECTIVE: To develop a filter utilizing mathematical theory to extract the skeletal patterns of trabecular bone. METHODS: Studies of morphology in the extraction of patterns of calcification in mammograms provided the theoretical framework. Using these studies as a basis, a morphological filter was applied to extract skeletal patterns from digital images of trabecular bone. Sequential images (subset) were combined in a structured fashion to create an aggregate (sumset) which compared with the original images, skeleton and line skeleton images. RESULTS: Binary images of the skeletal patterns in continuous, round and mesh-like forms were obtained from the original images by processing with the skeleton operation using a disc-shaped single structuring element. The line skeleton operation using line structuring elements with constant directions allowed the extraction of linear and discontinuous patterns. Both the skeleton and line skeleton operations extracted binary subset images which depicted skeletal patterns correlating with the operation sequence. CONCLUSIONS: Modification of the morphological filter enhanced the extraction of skeletal characteristics of trabecular bone. A morphological filter may be a useful adjunct in computer-aided structural analysis of bone.     

Measurement of contribution from intracellular cysteine to sulfate in phosphoadenosine phosphosulfate in rat ovarian granulosa cells

Synthesis of the large dermatan sulfate (DS) proteoglycan by rat ovarian granulosa cells was studied using metabolic radiolabel precursors in culture media with varying concentrations of environmental sulfate (20-800 microM) and cysteine (130 and 650 microM). Experiments using [3H]glucosamine and [35S]sulfate showed that the average size of the DS chains and the rate of DS proteoglycan synthesis were independent of the sulfate and cysteine concentrations in the medium. Experiments with [35S]cysteine were then used to determine the contribution that metabolic conversion of cysteine sulfur to sulfate makes to the 3'-phosphoadenosine 5'-phosphosulfate (PAPS) pool which provides the substrate for sulfoester formation in DS synthesis. When 35S in cysteine is metabolized into [35S]PAPS, the specific activity is reduced from that of the [35S]cysteine pool, by dilution with other sulfur sources such as extracellular sulfate, and this dilution factor directly reflects the contribution of cysteine to the PAPS pool. The decreases of 35S specific activity were measured under various sulfate-depleted and cysteine-supplemented conditions by comparing the specific activity of [35S]sulfate ester in the DS chains with that of [35S]cysteine residues in the core protein of the DS proteoglycan. The contribution of sulfur in cysteine to the intracellular PAPS pool was 0.03% in culture medium with normal sulfate (800 microM). Depleted environmental sulfate (20 microM) and increased cysteine supply (650 microM) only increased the sulfur contribution from cysteine to PAPS up to 0.74 and 1.5%, respectively, even though the DS chains were greatly undersulfated (55 and 82% of the control value). Thus, the source of sulfur in the intracellular pool of PAPS was mainly derived from environmental sulfate, and the contribution from cysteine was minimal in these cells.     

Alterations in the cell cycle of mouse cumulus granulosa cells during expansion and mucification in vivo and in vitro

The cell cycle characteristics of mouse cumulus granulosa cells were determined before, during and following their expansion and mucification in vivo and in vitro. Cumulus-oocyte complexes (COC) were recovered from ovarian follicles or oviducts of prepubertal mice previously injected with pregnant mare serum gonadotrophin (PMSG) or a mixture of PMSG and human chorionic gonadotrophin (PMSG+hCG) to synchronize follicle differentiation and ovulation. Cell cycle parameters were determined by monitoring DNA content of cumulus cell nuclei, collected under rigorously controlled conditions, by flow cytometry. The proportion of cumulus cells in three cell cycle-related populations (G0/G1; S; G2/M) was calculated before and after exposure to various experimental conditions in vivo or in vitro. About 30% of cumulus cells recovered from undifferentiated (compact) COC isolated 43-45 h after PMSG injections were in S phase and 63% were in G0/G1 (2C DNA content). Less than 10% of the cells were in the G2/M population. Cell cycle profiles of cumulus cells recovered from mucified COC (oviducal) after PMSG+hCG-induced ovulation varied markedly from those collected before hCG injection and were characterized by the relative absence of S-phase cells and an increased proportion of cells in G0/G1. Cell cycle profiles of cumulus cells collected from mucified COC recovered from mouse ovarian follicles before ovulation (9-10 h after hCG) were also characterized by loss of S-phase cells and an increased G0/G1 population. Results suggest that changes in cell cycle parameters in vivo are primarily mediated in response to physiological changes that occur in the intrafollicular environment initiated by the ovulatory stimulus. A similar lack of S-phase cells was observed in mucified cumulus cells collected 24 h after exposure in vitro of compact COC to dibutyryl cyclic adenosine monophosphate (DBcAMP), follicle-stimulating hormone or epidermal growth factor (EGF). Additionally, the proportion of cumulus cells in G2/M was enhanced in COC exposed to DBcAMP, suggesting that cell division was inhibited under these conditions. Thus, both the G1-->S-phase and G2-->M-phase transitions in the cell cycle appear to be amenable to physiological regulation. Time course studies revealed dose-dependent changes in morphology occurred within 6 h of exposure in vitro of COC to EGF or DBcAMP. Results suggest that the disappearance of the S-phase population is a consequence of a decline in the number of cells beginning DNA synthesis and exit of cells from the S phase following completion of DNA synthesis. Furthermore, loss of proliferative activity in cumulus cells appears to be closely associated with COC expansion and mucification, whether induced under physiological conditions in vivo or in response to a range of hormonal stimuli in vitro. The observations indicate that several signal-transducing pathways mediate changes in cell cycle parameters during cumulus cell differentiation.     

Acquisition of chemoresistance in a human ovarian carcinoma cell is linked to a defect in cell cycle control

Chemoresistance is a major concern in cancer erradication; it involves various mechanisms, including defects in the apoptosis program induced by anticancer drugs. In order to further explore the mechanisms underlying the development of chemoresistance in ovarian carcinoma after cisplatin treatment, we established an in vitro model, mimicking a clinical protocol of administration of cisplatin. Therefore, IGROV1 ovarian carcinoma cells were exposed for 2 hr to the drug and allowed to recover for several weeks; this way of exposure was reiterated with escalating doses. We followed changes in cytotoxicity of the drug, cell cycle kinetics and long-term survival of cells after cisplatin treatment, and found that resistance to cisplatin was not associated with altered apoptosis pathway, since both cisplatin sensitive and resistant cells underwent apoptosis in a similar way. Acquisition of resistance to cisplatin was associated with the ability of the treated cells to progress through the cell cycle beyond the G1/S checkpoint; although most cells died by apoptosis, a few surviving cells proliferated and recolonized the cultures. Compared to sensitive cells, the chemoresistant variants were able to override the G1/S checkpoint whatever the dose, and the recurrent cells recolonized the cultures much faster. Analysis of alterations in gene expression suggests that the defect in cell cycle regulation could take place at the level of the cdk inhibitor p21(CIP1/WAF1).     

Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells

DNA replication in eukaryotic cells is restricted to the S-phase of the cell cycle. In a cell-free replication model system, using SV40 origin-containing DNA, extracts from G1 cells are inefficient in supporting DNA replication. We have undertaken a detailed analysis of the subcellular localization of replication proteins and cell cycle regulators to determine when these proteins are present in the nucleus and therefore available for DNA replication. Cyclin A and cdk2 have been implicated in regulating DNA replication, and may be responsible for activating components of the DNA replication initiation complex on entry into S-phase. G1 cell extracts used for in vitro replication contain the replication proteins RPA (the eukaryotic single-stranded DNA binding protein) and DNA polymerase alpha as well as cdk2, but lack cyclin A. On localizing these components in G1 cells we find that both RPA and DNA polymerase alpha are present as nuclear proteins, while cdk2 is primarily cytoplasmic and there is no detectable cyclin A. An apparent change in the distribution of these proteins occurs as the cell enters S-phase. Cyclin A becomes abundant and both cyclin A and cdk2 become localized to the nucleus in S-phase. In contrast, the RPA-34 and RPA-70 subunits of RPA, which are already nuclear, undergo a transition from the uniform nuclear distribution observed during G1, and now display a distinct punctate nuclear pattern. The initiation of DNA replication therefore most likely occurs by modification and activation of these replication initiation proteins rather than by their recruitment to the nuclear compartment.     

Quantitation of nexus junctions in the granulosa cell layer of rabbit ovarian follicles during ovulation

Electron microscopy was used in a semi-quantitative study to determine changes in the abundance and size of surface nexuses and changes in the abundance of interiorized nexuses in growing and mature ovarian follicles during the ovulatory process. Mature follicles contain larger granulosa cells than follicles in the early stage of antral formation. Also, the granulosa cells of mature follicles have a slightly greater number of surface nexuses (without a change in nexus length), and more interiorized nexuses, compared to immature follicles. As a mature follicle approaches rupture, there is an appreciable decrease in the number of surface nexuses per granulosa cell. There is also a slight reduction in the number of interiorized nexuses at this time. It is concluded that this decrease in both surface nexuses and interiorized nexuses may be a consequence of ovulatory changes during which the rate of granulosa cell division is greater than the rate of formation of new nexuses. Additionally, the disruption to cell-to-cell cohesion during the ovulatory process appears to be independent of the interiorization of surface nexuses.     

Inhibition of ovarian follicular development associated with a decrease in luteinizing hormone levels during the estrous cycle of the rat

The orientation of microtubules (MTs) was examined in epidermal cells of azuki bean (Vigna angular is Ohwi et Ohashi) epicotyls. The orientation of MTs adjacent to the outer tangential wall of the cells, which has a crossed polylamellate structure with lamellae of longitudinal cellulose microfibrils alternating with lamellae of transverse cellulose microfibrils, differed from one cell to another. Treatment with an auxin-free solution caused the accumulation of cells with longitudinal MTs and subsequent treatment with a solution that contained auxin resulted in the accumulation of cells with transverse MTs, showing that sequential treatments with auxin-free and auxin-containing solutions can synchronize the reorientation of MTs. The MTs, once reoriented from longitudinal to transverse, returned to longitudinal and then back to transverse once again, the duration of the cycle being about 6h. Gibberellic acid, known to increase the percentage of cells with transverse MTs, promoted reorientation of MTs from longitudinal to transverse and inhibited that from transverse to longitudinal. Cytochalasin D, an agent that disrupts actin filaments, speeded up the reorientation from transverse to longitudinal and slowed down that from longitudinal to transverse. It caused an increase in the percentage of cells with MTs in mixed orientation, and the percentage of such cells was highest when the percentage of cells with longitudinal MTs was decreasing and that of cells with transverse MTs was increasing.     

Immunohistochemical staining of ovarian granulosa cell tumors with monoclonal antibody against inhibin

Inhibin is a peptide hormone produced by ovarian granulosa cells and by granulosa cell tumors. Serum inhibin measurements have been used as a biochemical marker of the presence or progression of ovarian granulosa cell tumors and their metastases. In the current study, an antibody against the alpha-subunit of human inhibin was used to stain 16 cases of ovarian adult granulosa cell tumors, 15 cases of other ovarian sex cord-stromal tumors, and 51 cases of a range of ovarian and extraovarian neoplasms, many of which may mimic granulosa cell tumor. There was diffuse strong cytoplasmic staining of all cases of adult granulosa cell tumor. Diffuse positive staining also was observed in all Leydig cell tumors, and there was focal staining in a proportion of fibrothecomas. There was focal weak staining of one case of ovarian clear cell carcinoma but no staining of other ovarian and extraovarian neoplasms. Immunohistochemical staining with antibodies against inhibin is of value in the diagnosis of granulosa cell tumor and in the distinction of this neoplasm from others that may mimic it. The antibody also may be useful for the confirmation of late metastasis of granulosa cell tumor, especially when the previous history is not known.     

The supraependymal cells of the rat hypothalamus: changes in their morphology and cell number during the ovarian cycle

The number of SEC in the hypothalamus of the rat change during the ovarian cycle (5-8 cells in oestrus, 100 cells in dioestrus per ventricular surface). The changes in the number as well the morphology of the SEC support the hypothesis that they are of mesenchymal nature.     

PTHrP and cell division: expression and localization of PTHrP in a keratinocyte cell line (HaCaT) during the cell cycle

Parathyroid hormone-related protein (PTHrP) is highly expressed in normal skin keratinocytes, and its involvement in growth and differentiation processes in these cells has been implicated by several lines of evidence which include the use of antisense PTHrP (Kaiser et al., 1994, Mol. Endocrinol., 8:139-147). In this study, we have investigated whether PTHrP expression and its subcellular localization is linked to cell cycle progression in a human keratinocyte cell line (HaCat), which constitutively expresses and secretes PTHrP. PTHrP mRNA and immunoreactive PTHrP were assessed in asynchronous dividing cells and in cells blocked at G1 or G2 + M phases of the cell cycle using several different protocols. The response of PTHrP mRNA expression was examined following readdition of serum in the continued presence of cycle blockers, and after release from cell cycle block, or from cell synchronization by serum deprivation. PTHrP expression was greatest in actively dividing cells when cells were in S and G2 + M phases of the cell cycle and were lowest in quiescent G1 cells. Most notable were the high levels of PTHrP mRNA and protein in cells at G2 + M phase of the cell cycle at division. Furthermore, PTHrP was localized to the nucleolus in quiescent cells, but redistributed to the cytoplasm when cells were actively dividing. Taken together, these results support a role for PTHrP in cell division in keratinocytes. In asynchronously growing cells, PTHrP expression fell as cells became confluent at a time when cell growth is inhibited and cells begin to differentiate. Mitogen stimulation of HaCaT cells resulted in a rapid increase in PTHrP mRNA expression, but was dependent upon cells being in the G1 phase of the cell cycle. Cells blocked in G1 responded to mitogen both in the continued presence of aphidicolin or when released from block. Cells blocked at G2 + M with colcemid expressed high levels of PTHrP mRNA and protein, and PTHrP mRNA did not respond further to mitogen in the continued presence of blocker. However, in cells released from block at G2 + M by addition of serum, an increase in PTHrP expression was seen coincident with the progression of cells into G1. In contrast, in a squamous cancer cell line (COLO16), basal PTHrP expression was high and was not altered during the cell cycle or by cell cycle block, consistent with association of its dysregulated expression in malignant cells. The results of this study suggest that PTHrP may have two roles in the cell cycle; one in G1 in response to mitogen, and a second at cell division when its expression is high and it is relocated from the nucleolus to the cytoplasm.     

The eleven stages of the cell cycle, with emphasis on the changes in chromosomes and nucleoli during interphase and mitosis

Since we had subdivided the cell cycle into 11 stages--four for mitosis and seven for the interphase--and since we had experience in detecting DNA in the electron microscope (EN) by the osmium-amine procedure of Cogliati and Gauthier (Compt. Rend. Acad. Sci., 1973;276:3041-3044), we combined the two approaches for the analysis of DNA-containing structures at all stages of the cell cycle. Thin Epon sections of formaldehyde-fixed mouse duodenum were stained by osmium-amine for electron microscopic examination of the stages in the 12.3-hr long cell cycle of mouse duodenal crypt columnar cells. In addition, semi-thin Lowicryl sections of mouse duodenal crypts and cultured rat kidney cells were stained with the DNA-specific Hoechst 33258 dye and examined in the fluorescence microscope. The DNA detected by osmium-amine is in the form of nucleofilaments, seen at high magnification as long rows of 11 nm-wide rings (consisting of stained DNA encircling unstained histones). At all stages of the cycle as well as in nondividing cells, nucleofilaments are of three types: 'free,' 'attached' to chromatin accumulations, and 'compacted' in all chromatin accumulations, the form of dense spirals within. At stage I of the cycle, besides free and attached nucleofilaments, compacted ones are observed in the three heterochromatin forms (peripheral, nucleolus-associated, clumped). Soon after the S phase begins, chromatin 'aggregates' appear, which are small at stage II, mid-sized at stage III, and large at stage IV. Chromatin 'bulges' also appear at stage III and enlarge at stage IV, while heterochromatins disappear. At stage V, aggregates and bulges accrete into 'chromomeres,' a process responsible for the apparent chromosome condensation observed at prophase. The chromomeres gradually line up in rows and, at stage VIa (prometaphase), approach one another within each row and coalesce to build up the metaphase chromosomes which are fully formed at stage VIb (metaphase). Daughter chromosomes arising at stage VII (anaphase) are eventually packed into a chromosomal mass at each pole of the cell. During stage VIII (telophase), the chromosomal mass is split into large chunks. In the course of the G1 phase, the chunks thin out to give rise to irregular 'bands' at stage IX, the bands are then cleaved into central and peripheral fragments at stage X, and finally the central fragments are replaced by free nucleofilaments and clumps at stage XI, while the peripheral fragments are replaced by peripheral heterochromatin. The "nucleoli" at stages I-III are associated with stained heterochromatin but otherwise appear as unstained lucent areas, except for weakly stained patches composed of histone-free DNA filaments. During stage IV, nucleoli lose patches and associated heterochromatin, while weakly lucent, pale vesicles appear within nucleoli and in the nucleoplasm. By the end of substage VIa, nucleoli generally disappear, while pale vesicles persist around the chromosomes appearing at substage VIb. At stages VIII and IX, the vesicles seem to become strongly lucent and, at stages IX and X, they associate and fuse to yield homogeneous lucent areas, the 'prenucleolar bodies,' which include histone-free DNA patches. During stage XI, groups of these bodies associate to give rise to nucleoli. In conclusion, the cell cycle DNA changes can be classified into 4 broad periods (Fig. 6): 1) Stage I is a 2-hr long interphase "pause," during which the stained DNA shows no signs of either chromosome condensation or decondensation, while the overall nuclear pattern is similar to that in nondividing cell nuclei. Nucleoli are fully developed. 2) From stage II to VIa, the "chromosome condensation" period extends over about 7 hr, during which the events are interpreted as follows. Throughout the S phase (stages II-IV), newly-synthesized segments of nucleofilaments approach one another, adhere and thus build aggregates and later bulges on nuclear matrix sites. (ABSTRACT TRUNCATED)     

Expression of the cell cycle inhibitor p27KIP1 is a new prognostic marker associated with survival in epithelial ovarian tumors

This case-control study was designed to identify factors associated with long-term survival. We examined two groups of patients with epithelial ovarian cancer, one group of long-term survivors (> 5 years) and one group of short-term survivors (< 2 years), for levels of expression of p53 and p27KIP1 proteins (as both proteins have been shown to be independent prognostic markers in tumors other than ovary) and the relationship with patient survival. Our findings show that p27KIP1 expression, in contrast to p53 expression, is positively associated with long-term survival in univariate analysis (P = 0.001), in analyses stratified by residual disease (P = 0.02) or performance status (P = 0.02), the two strongest prognostic factors for ovarian cancer, as well as multivariate analysis (P = 0.002) adjusting simultaneously for age, tumor stage, residual disease, performance status, and grade of differentiation. Therefore, immunostaining for levels of p27KIP1 expression may have potential as a new prognostic factor in the management of ovarian cancer.     

The role of apoptosis of granulosa cells in follicular atresia

Apoptosis plays an important role in the process of morphogenesis and embryogenesis. Its increase or inhibition is an etiopathological factor in many different diseases. It has recently been shown that apoptosis of granulosa cells is one of the main mechanisms responsible for follicular atresia. There are many other factors influencing the process of granulosa cells apoptosis, among them the most important are: RnGH, FSH, LH, sex hormones (estrogens and androgens), growth factors and their receptors (EGF/TGF-alpha, FGF, IGF-1) and cytokines (e.g. TNF-alpha). The article presents data concerning the regulatory mechanisms of granulosa cells apoptosis in the ovary.     

Proliferative index of human luteinized granulosa cells varies as a function of ovarian reserve

OBJECTIVE: We examined whether the proliferative index of luteinized granulosa cells, as determined by flow cytometry, varied as a function of a woman's ovarian reserve, as reserve, as reflected by follicular-phase day 3 serum follicle-stimulating hormone. STUDY DESIGN: This prospective cohort study consisted of 19 women of similar chronologic age preparing for in vitro fertilization-embryo who met specific day 3 serum follicle-stimulating hormone criteria. The "low follicle-stimulating hormone" group consisted of 11 women with day 3 serum follicle-stimulating hormone levels < or = 6 IU/L. The "high follicle-stimulating hormone" group consisted of eight women with day 3 serum follicle-stimulating hormone levels > or = 18 IU/L. A total of 56 preovulatory follicles containing > or = 10(4) luteinized granulosa cells were examined by flow cytometry. The low follicle-stimulating hormone group was compared with the high follicle-stimulating hormone group to examine proliferative index as a function of serum day 3 follicle-stimulating hormone levels. RESULTS: The low follicle-stimulating hormone group had a greater proliferative index (11.1% +/- 0.4%) than did the high follicle-stimulating hormone group (8.3% +/- 0.6%), p < 0.001). This study demonstrates that in spite of the same chronologic age, luteinized granulosa cells from preovulatory follicles of women with day 3 serum follicle-stimulating hormone levels > or = 18 IU/L have a 25% decreased proliferative index compared with luteinized granulosa cells from women with day 3 serum follicle-stimulating hormone levels < or = 6. CONCLUSIONS: This suggests that granulosa cell proliferation is influenced by ovarian reserve and may explain in part the more favorable response to ovulation induction protocols that younger women demonstrate compared with women of more advanced reproductive age.     

Dynamic organisation of intermediate filaments and associated proteins during the cell cycle

Two black yeast isolates from plants from the Canary Islands (Spain) are described and illustrated. Absence of Woronin bodies at simple septal pores, local coralloid terminal hyphal cells, indeterminate thallus maturation, the presence of budding cells and local conversion to meristematic growth all indicate a relationship to the Dothideaceae (Dothideales, Ascomycota). Morphological properties were consistent with the genus Hormonema Lagerberg & Melin, as defined by presence of percurrent conidiogenous loci alongside undifferentiated hyphae, and results of PCR-ribotyping supported this classification. The isolates were judged to belong to a hitherto undescribed species, characterized in particular by curved conidia soon developing transverse septa. The physiological profile of this species is also described.