A Retrospective Analysis of Clinicopathological Features in 117 Isolated Maxillary Sinus Pathologies in a Tertiary Care Hospital.

Diagnosis of maxillary sinus pathologies is challenging. Herewith we describe the clinicopathological features in isolated maxillary sinus lesions in tertiary care hospital in Goa, India. The retrospective study included patients treated between 2017 and 2022, of all age groups and gender, who underwent either a biopsy or surgery, providing a histopathological diagnosis. Of the 117 pathologies, 88 (75.2%) were non-neoplastic. The overall frequency of pathologies were polyp in 40.2%, fungal lesions (18.8%), malignancy (13.7%), chronic rhinosinusitis (11.9%) and inverted papilloma (10.3%). There were 71 men (60.7%) and 46 women (39.3%). There were 10 patients (8.5%) below 20 years of age, of which 8 patients (80%) had non-neoplastic pathology. Common comorbidities were diabetes and hypertension, while symptoms were nasal blockage (75.2%), nasal discharge (47%) and ocular redness (16.2%). Each pathology was evaluated for demography, side of lesion, comorbidity, and symptoms. Most isolated maxillary sinus pathologies were benign lesions. However, a strong clinical suspicion and histopathological confirmation is needed for all lesions in all age groups due to a risk of malignancy.

Influence of protein supplements on the secretion of leukaemia inhibitory factor by mitomycin-pretreated Vero cells: possible application to the in vitro production of bovine blastocysts with high cryotolerance.

A co-culture system for bovine embryos using mitomycin-treated Vero cells and serum-supplemented modified synthetic oviduct fluid (mSOF) supports the development of in vitro maturation and fertilization-derived oocytes to hatched blastocysts. In this system, it has been suggested that one contribution made by the co-culture cells to embryo development is production of the cytokine leukaemia inhibitory factor (LIF). However, there are concerns about exposure of early embryos to serum due to its incompatibility with embryo cryosurvival. In this study, the influence of two protein supplements (synthetic serum substitute (SSS), a lipid-free human serum-derived product) and oestrous cow serum (ECS)) on Vero cell LIF secretion was compared, with the aim of designing a co-culture system that is supportive of bovine embryo cryopreservation. Vero cells cultured for 72 h in medium 199 + 5% fetal bovine serum (FBS) (recommended maintenance medium for this cell line) secreted detectable amounts of LIF (13.1 +/- 0.9 pg LIF per 10(5) cells). Culture in mSOF, the medium routinely used in this laboratory for embryo culture, also supported LIF secretion in Vero cells. However, the amount of LIF was tenfold higher (24.7 +/- 6.2 pg LIF per 10(5) cells; P < 0.05) when mSOF was supplemented with 10% (v/v) ECS compared with supplementation with 2% (v/v) SSS. Results of a second series of experiments in which supplementation with each protein was normalized to 10% revealed similar differences in LIF secretion, indicating that LIF secretion was affected by the type, not the amount, of protein. Time course analysis revealed stepwise increases (P < 0.05) in cumulative LIF secretion with every 24 h of culture in mSOF + either SSS or ECS. In terms of embryo development and post-cryopreservation viability, medium supplementation with 2% (v/v) SSS alone versus the two-step system of 2% (v/v) SSS (days 1-4) + 10% (v/v) ECS (days 4-10) had no influence (P > 0.05) on the ability of bovine blastocysts to hatch, with or without intervening cryostorage. However, the rate of blastocyst formation (expressed as the percentage of cleaved embryos) was only 27% in the presence of 2% (v/v) SSS, and increased almost twofold (P < 0.05) when ECS was added beginning on day 4 of co-culture. In summary, Vero cell LIF secretion was increased markedly by ECS. A two-step system of medium supplementation, in which embryos are exposed to ECS beginning on day 4 of in vitro development combined high rates of blastocyst formation with cryotolerance. This effect may be a result of limiting embryo exposure to serum-derived lipid until after the eight-cell stage and providing an increase in LIF during the critical developmental stages of compaction and cavitation.

Induction of apoptosis in equine chorionic gonadotropin (eCG)-primed rat ovaries by anti-eCG antibody.

We have established a model to examine the early events of apoptosis in small antral follicles in vivo. Immature female rats injected with 15 IU eCG, and subsequently (24 h later) with an anti-eCG antibody to induce gonadotropin withdrawal, displayed a significantly lower ovarian weight and increased incidence of follicular atresia and granulosa cell death, especially in small- to medium-sized follicles. Evidence of apoptosis was apparent in a significantly higher proportion of granulosa cells from antibody-treated rats, which exhibited membrane blebbing, nuclear and cytoplasmic condensation, fragmentation, and phagocytosis. In addition, there was a loss of the regular organization of the lamina densa in the follicular basement membrane. Degradation of DNA was consistently found by 24 h in the antibody-treated group, and ladders could be observed as early as 1 h after treatment. Although cell death was observed after antibody treatment in some larger antral follicles, the occurrence of apoptosis was less frequent. These results demonstrate that gonadotropin withdrawal in vivo rapidly induces apoptosis in small- to medium-sized antral follicles at the critical stage of development when atresia is commonly observed, suggesting that this model is ideal for studying apoptosis in the ovary.

Evaluation of mitomycin-treated vero cells as a co-culture system for IVM/IVF-derived bovine embryos.

Support of the in vitro development of IVM/IVF-derived bovine embryos by Vero cells was evaluated by comparing the following treatment groups: 1) proliferating (Unt-Vero) vs nonproliferating (Mit-Vero) cells; 2) supplementation of medium with estrous cow serum (ECS) vs bovine serum albumin (BSA); 3) Mit-Vero cells vs bovine oviduct epithelial cells (BOECs); and 4) addition of leukemia inhibitory factor (LIF) to Mit-Vero cell co-cultures at Day 1 vs Day 4. Mit-Vero cells stimulated higher rates of blastocysts (Day 7, 40 vs 27%) and hatched blastocyst (Day 10, 38 vs 12%) formation than Unt-Vero cells. These rates were comparable to those obtained with BOECs; blastocyst hatching was slightly higher following co-culture with Mit-Vero cells (36%) than BOECs (29%). Blastocyst formation was similar in ECS- vs BSA-supplemented medium; however, hatching was greatest (37%) during co-culture in medium +10% ECS. While the addition of LIF throughout the co-culture period was ineffective, addition of the cytokine beginning at Day 4 slightly increased blastocyst formation rates. Evaluation of LIF secretion using ELISA revealed detectable levels of the cytokine in Mit-Vero-conditioned medium (50 pg/10(5) cells); this may explain the minimal influence of exogenous LIF during embryo co-culture. Mit-Vero cells provided comparable support of bovine embryo development when used even up to 2 wk after establishment as monolayers. In conclusion, Mit-Vero cells provide a readily-available, safe and easy-to-use co-culture method which is at least as supporting of bovine embryo development as BOECs. One contribution of these cells may be secretion of the cytokine LIF.

Expression of urokinase-type plasminogen activator and its receptor during ovarian follicular development.

Although tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) are believed to be involved in the biochemical cascade leading to extracellular matrix degradation during ovulation, the presence and possible role of urokinase-type PA (uPA) and its receptor (uPAR) in follicular wall remodeling during follicular development are poorly understood. In the current studies, we have examined their presence in the rat ovary and compared the changes in both uPA and uPAR expression with those of tPA and PAI-1 during follicular growth in vivo. The presence of these proteins in various follicular cells at different stages of maturation was evaluated by immunolocalization and ELISA. Abundance of respective messenger RNA in granulosa cells from preantrallearly antral, midantral and preovulatory follicles and the residual ovaries was determined by Northern blot analysis. Whereas uPA transcript and protein levels were highest at the earliest stage of follicular growth examined and decreased markedly before the expected time of ovulation, the opposite was true for uPAR. In addition, tPA and PAI-1 messenger RNA abundance and protein contents were low in both granulosa and residual ovarian tissue during early follicular development but increased thereafter, reaching highest levels at the preovulatory period. These findings demonstrate for the first time the presence of uPAR in ovarian follicles and its developmental expression. The coincidental rise in uPAR and PAI-1 proteins during the preovulatory period may be important for the regulation of extracellular matrix remodelling before ovulation. The reciprocal expression of uPA and tPA during follicular development are consistent with the notion that these proteases have different biological functions in the ovary, i.e. tPA is involved in follicular wall remodelling before ovulation whereas uPA is important in extracellular matrix degradation during cell proliferation and migration that accompany follicle growth.

TGF-beta 1 regulation of laminin secretion by F9-derived primitive endoderm cells: a potential role in cell migration within the mouse blastocyst.

The first differentiation event occurring in the preimplantation mouse blastocyst is the formation of inner cell mass (ICM) derived primitive endoderm (PrE) cells, some of which migrate over the inner surface of the trophectoderm to establish the parietal endoderm layer. Reichert's membrane, a basement membrane located between the trophectoderm and the emerging endodermal layer, is implicated in this migration. In this study, F9 murine embryonal carcinoma cells were used as an in vitro model for embryonic endoderm to investigate the regulation of laminin secretion by these cells during their differentiation under serum-free culture conditions. The formation of PrE-like cells was induced using retinoic acid, and the cells were then cultured with dibutyryl cAMP (dbcAMP), forskolin, or transforming growth factor (TGF) beta 1 or beta 2, and the levels of secreted laminin were measured using ELISA. dbcAMP and forskolin stimulated (p < 0.01) laminin secretion, whereas TGF-beta 1 decreased (p < 0.01) the secretion of laminin by PrE-like cells during a 72-h culture period (without influencing laminin deposition by these cells). In contrast, TGF-beta 2 did not significantly influence (p > 0.05) laminin secretion. The results of in vitro migration experiments using mouse ICMs prepared by immunosurgery indicated that, unlike fibronectin, neither laminin nor type IV collagen supported the outward movement of differentiating PrE-like cells. These findings support a potential role for TGF-beta 1 in influencing the establishment of the parietal endoderm cell layer within the mouse blastocyst by reducing the extent of laminin deposition in Reichert's membrane during endoderm cell migration.

Influence of extracellular matrix gradients on the haptotactic migration of F9 embryocarcinoma-derived primitive and parietal endoderm-like cells.

The establishment of the parietal endoderm (PE) layer within the rodent blastocyst requires extensive outward migration of inner cell mass (ICM)-derived primitive endoderm (PrE) cells over the blastocoelic surface of the trophectoderm. While a role for Reichert's membrane in this process has been proposed, the identity of the basement membrane component(s) involved and the mechanism(s) by which they might promote and/or guide this movement remain to be determined. This study was a comparison of the gradient-associated influences of three components of Reichert's membrane--fibronectin, laminin, and collagen IV--on the migration of embryonic endoderm-like cells obtained from the F9 embryocarcinoma cell line. Gradients (positive or negative) or even coatings of each of these glycoproteins were established across polycarbonate filters; the filters were then placed in blind-well chambers. F9 cells, either undifferentiated or pretreated with retinoic acid (RA) alone or RA+dibutyryl cAMP to resemble PrE and PE, respectively, were then loaded into the upper chamber compartments. At the end of the migration assay (4-24 h), the number of cells that had moved through the filter pores and attached to the lower filter surface was determined for each treatment group. While even coatings of either fibronectin or laminin supported some cell migration through the filters, these levels were increased 13- and 20-fold (fibronectin and laminin, respectively) by application of the glycoproteins as positive gradients. In contrast, no migration occurred in response to negative gradients of either fibronectin or laminin.(ABSTRACT TRUNCATED AT 250 WORDS)

Extracellular matrix composition and resilience: two parameters that influence the in vitro migration and morphology of rat inner cell mass-derived cells.

Parietal endodermal (PE) migration along rat trophectodermal (TE) cells coincides with the deposition of Reichert's membrane between these two cell layers. In this study, we compared the influences of fibronectin and laminin, two components of Reichert's membrane, on the migration and replication of PE-like cells from cultured rat inner cell masses (ICMs). We also explored the role of substrate nondeformability by comparing cell translocation on gels versus coatings of Matrigel (a tumor cell-derived basement membrane preparation) or of collagen. ICMs, isolated by immunosurgery from Day 5 blastocysts, were cultured on coatings of collagen IV, laminin, fibronectin, collagen I, or Matrigel, or on gels of the latter two substrates. Minimal laminin or fibronectin coating concentrations of 2.5 micrograms/ml were required for ICM attachment and cell migration. Migration was similar during the first 48 h of culture on fibronectin and on laminin; however, by 72 h, the extent of cell translocation on fibronectin was greater (1.5- to 2-fold) than that measured on laminin. Fibronectin-cultured ICM-derived cell clusters also contained 1.5- to 2-fold more cells than those on laminin. Migration did not occur on undiluted gels of Matrigel but was supported by diluted (1:10 and 1:20) Matrigel coatings. Similarly, cell migration on coatings of collagen IV reached almost 3-fold that measured on collagen I gels. Most of the cells migrating on fibronectin or collagen (I or IV) were flattened and elongated. In contrast, a high proportion of the cells migrating on laminin or Matrigel coatings were tall and rounded, with thin cytoplasmic extensions. Fibronectin- and collagen IV-cultured cells stained strongly for both collagen IV and laminin, but contained no fibronectin. In contrast, laminin-cultured cells contained fibronectin but were less immunoreactive for laminin and collagen IV. These findings indicate that substrate composition and resilience influence the in vitro migration and morphology of ICM-derived PE-like cells. A role for the TE cells in anchoring Reichert's membrane during development of the PE cell layer within the blastocyst is postulated. Furthermore, the sensitivity of cell morphology and differentiation to individual basement membrane components provides a potential key mechanism whereby an emerging basement membrane can regulate cell migration and differentiation, two fundamental processes that occur throughout embryonic development.

Fibronectin production by chicken granulosa cells in vitro: effect of follicular development.

In vitro studies were conducted to investigate the role of chicken ovarian granulosa cells in the production of fibronectin, a component of the basal lamina of ovarian follicles. Collagenase dispersed granulosa cells obtained from the first (F1; about 35 mm in diameter) and third (F3; 15-20 mm in diameter) largest preovulatory follicles, as well as from a pool of small yellow follicles (SF; 6-10 mm in diameter), were incubated in serum-free medium-199 for 24 to 96 h in the absence and presence of luteinizing hormone (LH) or forskolin. Fibronectin secreted in the medium was quantitated by enzyme linked immunosorbent assay. Basal fibronectin production (which increased with the duration of incubation) was significantly greater (p < 0.001) in granulosa cells derived from mature follicle (F1) than in F3 or SF cells. Both LH and forskolin stimulated fibronectin production in SF and F3 cells in a dose-dependent manner; however, they were without effect in F1 cells. The magnitude of increase in fibronectin production elicited by LH or forskolin was greater in SF cells than in F3 cells. The cytoplasm of cultured granulosa cells taken at all stages of follicular development stained positively for fibronectin. These findings indicate that chicken granulosa cells produce fibronectin. This ability is acquired early in follicular development and the stimulatory effect of the gonadotropin (LH) diminished as the follicle approached ovulation.

The influence of extracellular matrix components on the proliferation and migration of inner cell mass-derived parietal endodermal cells.

Reichert's membrane is a basement membrane deposited on the inner surfaces of rat and mouse trophectodermal (TE) cells beginning at the blastocyst stage of embryonic development that may play a role in the migration of the parietal endodermal (PE) cells to form an inner lining to the TE. The abilities of various glycoproteins present in Reichert's membrane to support PE cell migration and replication in vitro were examined by isolating inner cell masses (ICMs) from Day 5 rat blastocysts (Day 1 = day of vaginal plug) and culturing them (24-72 h) either on surfaces that had been precoated with collagen IV, fibronectin, or laminin or on thin (1-2-mm) gels of Matrigel (a tumor cell-derived basement membrane preparation) or type I collagen. Time-dependent changes in the area occupied by each ICM on the culture surface and the number of migrating cells per ICM were quantified by morphometric analysis. Type IV collagen, the basement membrane-specific collagen, supported ICM attachment and the outward migration (overall increase of approx. 60-fold in mean ICM area occupied on the culture surface) and proliferation (cell doublings following every 24 h of culture) of laminin-containing PE-like cells. These effects were not altered by the inclusion of exogenous fibronectin or laminin in the culture medium. Collagen IV coating concentrations as low as 0.16 micrograms/ml supported PE cell attachment and migration, and maximal responses were seen with a coating concentration of 0.63 micrograms/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunolocalization of fibronectin and laminin within rat blastocysts cultured under serum-free conditions.

Blastocysts (approximately 50 per female) were collected on Day 5 of gestation from immature Sprague-Dawley rats superovulated using FSH/hCG-loaded mini-osmotic pumps and a single injection of the LHRH analogue, des-gly10 (D-ala6)-LHRH-ethylamide. The cytoplasmic distribution of fibronectin and laminin was determined by immunofluorescence within these blastocysts, either immediately following their isolation or after they had been cultured in serum-free medium for 48-96 h (to allow trophectodermal cell attachment and outgrowth). In addition, inner cell masses (ICMs; isolated by immunosurgery) were cultured under serum-free conditions and immunofluorescently stained for the presence of the two adhesive glycoproteins. Within the freshly isolated blastocysts, positive immunostaining was obtained only for fibronectin and this was associated with the trophectodermal layer. After 48-96 h of culture, the cytoplasm of all trophectodermal cells contained both fibronectin (organized as a slightly granular network) and laminin (the staining pattern was distinctly punctate and perinuclear concentrations of immunoreactivity were evident). ICM-cells stained intensely for the presence of laminin at 48, 72 and 96 h of culture, but appeared to contain little to no fibronectin. While further studies using serum-free culture are needed to define the hormonal regulation of this process, these findings support a role for early gestation rat trophectodermal cells, in addition to the established involvement of ICM-derived parietal endodermal cells, in the synthesis of extracellular matrix components found in Reichert's membrane. The appearance of trophectoderm-associated fibronectin in freshly isolated blastocysts before the establishment of the parietal endoderm layer may implicate this glycoprotein in the provision of a substrate for the migration of these cells as they form an endodermal lining to the blastocoele.

Secretion of fibronectin by rat granulosa cells occurs primarily during early follicular development.

Rats were pretreated with oestradiol-17 beta or PMSG, treatments producing mainly preantral and antral follicles respectively. The granulosa cells from these two treatments, E2- and PMSG-cells respectively, were cultured for 2 successive 24-h periods. Basal progesterone secretion, stimulated 3- to 5-fold by FSH, was almost 8-fold higher by PMSG-cells than by E2-cells at 24 and 48 h. Similarly, PMSG-cells secreted 16-fold more 20 alpha-dihydroprogesterone than did E2-cells, in the absence of FSH, and 6- to 10-fold more of the 20 alpha-reduced metabolite in the presence of FSH. In contrast, fibronectin secretion by PMSG-cells was only about 60% and 30% that by E2-cells at 24 and 48 h, respectively. Fibronectin secretion by E2- and PMSG-cells was reduced in the presence of FSH at 24 and at 48 h of culture. While cells in all treatment groups underwent spreading during culture to become elongated and irregular in outline, elevated fibronectin secretion in vitro was accompanied by enhanced cellular spreading. At 24 and 48 h of culture respectively, the mean area occupied by E2-cells on the culture surface was x 2 and x 1.6 that by PMSG-cells. Coincident with its inhibitory effect on fibronectin secretion, FSH reduced the mean area occupied by E2- and PMSG-cells on the culture surface at both time intervals. These findings suggest that granulosa cell fibronectin secretion is a feature of early follicular development. It may be that the secretion of this adhesive glycoprotein by granulosa cells provides a pool of fibronectin which is used for basement membrane deposition during follicular growth.

A review of the proliferative capacity of major salivary glands and the relationship to current concepts of neoplasia in salivary glands.

The classification of salivary gland tumors relies heavily on histogenetic postulates. One of these, the semipluripotential reserve cell theory, suggests that certain reserve cells in specific segments of the duct system of major and minor salivary glands are critical to the development of neoplasms in these glands. However, direct evidence in support of this hypothesis is unavailable. This survey of proliferative capacity in normal salivary gland is based on a review of data in the literature, our observations of DNA synthetic and mitotic activity in developing rat and human salivary gland, and autoradiographic studies of induced cell proliferation in rat salivary gland. Autoradiography of neonatal rat salivary gland after tritiated thymidine administration, and electron microscopy of these tissues, reveals that as well as duct basal cells, luminal cells at all levels of the duct system and even acinar cells are capable of DNA synthesis and mitosis. Indeed, in such studies, more luminal than basal cells are seen in mitosis. In adult rat salivary gland induced to undergo hyperplasia, more acinar cells than intercalated duct cells are in the S phase of the cell cycle. However, cycling cells were observed even in striated ducts and, importantly, both basal and luminal cells of major interlobular excretory ducts are also labeled. Similar findings are present in fetal and adult human salivary glands. From such observations, it is evident that dividing cells are not limited to basal cells of excretory ducts and luminal cells of intercalated ducts, so that there is no support for the semipluripotential bicellular reserve cell hypothesis. However, there is considerable evidence for a multicellular theory of tumor histogenesis; that is, any of the multiplicity of cell types in normal salivary gland have the potential to give use to any of the various types of tumor occurring in this organ.

Culture of granulosa cells in collagen gels: the influence of cell shape on steroidogenesis.

The gonadotropic regulation of granulosa cells steroidogenesis in vitro has been shown to be accompanied by cellular rounding. In this study, the possible relationship between cell shape, microtubules, and granulosa cell steroidogenesis in vitro was further explored by culturing (24 h) granulosa cells obtained from antral follicles of pregnant mare's serum gonadotropin-treated rats in either Eagles's Minimum Essential Medium alone (MEM-cells) or in collagen gels (GEL-cells) in the absence or presence of colchicine, a microtubule-depolymerizing agent previously shown to inhibit cell-spreading in vitro. Cellular morphology was assessed by electron microscopy and compared with that seen in vivo. In addition, the influence of the various culture conditions on progesterone and 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-OH-progesterone) secretion was determined by specific radioimmunoassays. Whereas the majority of granulosa cells in sections of antral follicles appeared rounded in shape, cells cultured in MEM underwent considerable spreading and assumed a variety of shapes at the end of 24 h of culture. GEL-cells, on the other hand, remained rounded and had cellular diameters only slightly larger than those observed in vivo. They also secreted more progesterone (almost 3-fold) and less 20 alpha-OH-progesterone (0.6-fold) than MEM-cells. Colchicine increased the secretion of progesterone (1.6-fold) and 20 alpha-OH-progesterone (1.8-fold) comparably in MEM-cells but had no influence on the secretion of either progestin by GEL-cells. Hence, although colchicine-stimulated progestin secretion by granulosa cell monolayers appeared to reflect increased metabolism of substrate-possibly due to a closer association between lipid droplets and mitochondria, the elevated secretion of progesterone by GEL-cells may have been largely due to a shift in the equilibrium between progesterone and its inactive 20 alpha-reduced metabolite. The high ratio of 20 alpha-OH-progesterone to progesterone secretion seen in MEM-cultured cells may be an adaptation of granulosa cell metabolism to culture as monolayers on plastic or glass surfaces. The morphology of GEL-rather than MEM-cells resembled closely that seen in vivo. This culture method may represent a more physiologic approach to the maintenance of granulosa and other steroidogenic cells in vitro and provide a more appropriate means of assessing cytoskeletal function in the regulation of steroid hormone production.

Microfilaments and FSH stimulation of rat granulosa cell steroidogenesis in vitro.

The secretion of progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-dihydroprogesterone) by granulosa cells from 30-day-old rats pretreated with PMSG (4 i.u.; i.p.) was significantly increased in a time- and concentration-dependent manner by FSH or cytochalasin B. Whereas FSH markedly stimulated progestagen secretion during 3 h of incubation, a significant enhancement of the steroidogenic response was not noted until 12 h of exposure to the inhibitor in vitro. Although cytochalasin B also enhanced the submaximal stimulation of progestagen production by FSH (15 ng/ml), it was ineffective in the presence of maximal stimulatory concentration of the gonadotrophin (150 ng/nl). With increasing concentrations of cytochalasin B, the ability of FSH to further stimulate progestagen secretion was progressively reduced. Granulosa cells cultured in medium alone contained a prominent cytoplasmic array of microfilaments which was markedly reduced by FSH or cytochalasin B. FSH and, to a greater extent, cytochalasin B elicited concentration-dependent reductions in the mean area occupied by the cells on the culture surface, the contour index (a size-independent representation of cell profile irregularity) and cell perimeter, indicating that the cells underwent less spreading and were more spherical and regular in outline in the presence of either agent. The FSH-induced reductions in the three shape-related parameters were augmented by cytochalasin B although the influence of the FSH on the mean area and perimeter was progressively reduced in the presence of higher concentrations of cytochalasin B.(ABSTRACT TRUNCATED AT 250 WORDS)

The cytoskeleton and rat granulosa cell steroidogenesis: possible involvement of microtubules and microfilaments.

The participation of both microtubules and microfilaments in granulosa cell steroidogenesis was assessed by monitoring the effects of colchicine (0-250 microM) and/or cytochalasin B (0-10 micrograms/ml) or dihydrocytochalasin B (0-2.0 micrograms/ml) on cellular morphology and production of progestins during 24 h of culture. Both colchicine and the cytochalasins increased granulosa cell production of progesterone and of 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-OH-progesterone) in a dose-dependent manner. The largest increase in steroidogenesis (about 2- to 3-fold) was observed at 4-250 microM colchicine and at 2-10 micrograms/ml cytochalasin. Those concentrations of the inhibitors of microtubule or microfilament polymerization that stimulated basal progestin production also markedly influenced cell spreading. Whereas cells cultured for 24 h in medium alone became very flattened with numerous cytoplasmic extensions, those cultured with colchicine (0.2-250 microM) or cytochalasin (0.4-2 micrograms/ml) were much less spread and progressively became more rounded and regular in outline. These changes in cell morphology were reflected by decreases in the mean area occupied by the cells on the culture surface of up to 60-65% and reductions in mean contour index values from 5.7 +/- 0.1 (control) to 3.9 +/- 0.1 (250 microM colchicine), 4.2 +/- 0.1 (2 micrograms/ml cytochalasin B), or 4.1 +/- 0.1 (2 micrograms/ml dihydrocytochalasin B). Cultures containing both colchicine and cytochalasin B exhibited a greater steroidogenic response than that elicited by either inhibitor alone. For example, granulosa cell progesterone production was stimulated almost 2-fold by 4 microM colchicine or 2 microM/ml cytochalasin B, but 5.5-fold by 4 microM colchicine plus 2 micrograms/ml cytochalasin B.(ABSTRACT TRUNCATED AT 250 WORDS)

Microtubules and the calcium-dependent regulation of rat granulosa cell steroidogenesis.

The possible relationship between calcium and microtubules in the regulation of granulosa cell steroidogenesis was assessed by using agents known to alter microtubule-tubulin equilibrium together with the ionophore A23187, an antibiotic that facilitates the movement of calcium across plasma membranes. Using immunofluorescence and morphometric analysis, we determined alterations in microtubule organization and overall cell shape, respectively, in response to ionophore-stimulated production of progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-progesterone) during 24 h of culture. In addition, the influences of colchicine and nocodazole, two agents known to induce microtubule depolymerization, and of taxol, an agent that stabilizes tubulin polymers, on calcium-dependent regulation of granulosa cell progestin production in vitro were examined. Cells cultured as controls were flattened, highly irregular in outline, and associated with a complexly organized, well-spread cytoplasmic network of microtubules. In contrast, those maintained in the presence of increasing concentrations of ionophore were progressively more circular and smooth in outline, occupied less area on the growth surface, and contained cytoplasmic arrays of microtubules considerably less extensive than those of the controls and occupying areas defined by the more regular cellular perimeters. While progestin production in the absence or presence of a submaximally stimulatory concentration of A23187 was increased by both colchicine and nocodazole, the microtubule-depolymerizing agents had little to no effect on the production of the steroids by granulosa cells maximally stimulated by the ionophore. However, both basal and ionophore-induced progestin production were unaltered by taxol except at a concentration of 10 microM in the presence of 0.25 micrograms/ml A23187.(ABSTRACT TRUNCATED AT 250 WORDS)

Microtubules and the gonadotropic regulation of granulosa cell steroidogenesis.

The involvement of microtubules in the gonadotropic regulation of granulosa cell steroidogenesis was assessed at the preantral (E2-cells) and antral (PMS-cells) stages of follicular development. The influence of agents that alter microtubule-tubulin equilibrium on basal and FSH-stimulated progesterone production was determined in vitro and compared with that on microtubule integrity and organization using immunofluorescence. Basal and FSH-stimulated progesterone production was approximately 2-fold higher in PMS-cells than in E2 cells. Colchicine and nocodazole, two agents that depolymerize microtubules, significantly stimulated progesterone and 20 alpha-hydroxypregn-4-en-3-one production in PMS-cells. Although progesterone production by E2-cells was increased by nocodazole, the amount produced was considerably less than that produced by PMS-cells. FSH-stimulated progesterone biosynthesis was reduced by colchicine and nocodazole in both cell types. Taxol, an agent that stabilizes microtubules, markedly reduced FSH-stimulated progesterone production in both E2- and PMS-cells, but failed to exert a comparable effect on basal steroid production. A close association existed between the concentrations of colchicine, nocodazole, and taxol that altered basal and/or FSH-stimulated steroidogenesis and those that affected microtubule organization and/or distribution. Whereas granulosa cells appeared flattened with numerous cytoplasmic processes after 24 h of culture in medium alone, they were almost spherical and devoid of projections after culture with these agents. FSH-stimulated cells also occupied less area than controls, although cytoplasmic processes were present. These findings indicate an involvement of microtubules in the regulation of granulosa cell steroidogenesis. It is proposed that one of their roles is to facilitate the movement of cholesterol from lipid droplets to mitochondria, possibly by bringing these cellular inclusions closer together.

The early development of the sheep trophoblast and the involvement of cell death.

During the period of rapid elongation prior to the initiation of placental attachment (days 12-16 of gestation), the ovine blastocyst consists of a single layer (primarily) of roughly cuboidal trophoblastic cells with an inner lining of flattened endodermal cells. Well-developed spot desmosomes link the adjacent cell borders in both the trophoblastic and endodermal layers. The trophoblastic cells contain acid phosphatase-positive, lysosomelike organelles, the mean diameter of which increases greatly between days 12 and 16 and whose contents vary during development. Also during the developmental period studied, trophoblast cells accumulate lipid; and periodic acid-Schiff-positive binucleate cells appear within the trophoblast layer. A consistent observation throughout the 5 days of rapid growth and differentiation of the blastocyst was the death and disintegration of some trophoblast cells. These disintegrating cells are usually singly dispersed within the trophoblast, although occasionally groups of four or five are observed. The cell death may indicate overall remodelling of the blastocyst, or the cells may represent genetically deficient cells which are unable to respond to the appropriate signal to differentiate.

Calcium-dependent regulation of progesterone production by isolated rat granulosa cells: effects of the calcium ionophore A23187, prostaglandin E2, dl-isoproterenol and cholera toxin.

The role of calcium in the regulation of ovarian steroidogenesis was investigated in granulosa cells from estradiol-treated immature rats. Incubation of granulosa cells with various calcium channel blockers (verapamil, cobalt or manganese) and a calcium chelator (EGTA) resulted in marked decreases in progesterone production in response to follicle-stimulating hormone (FSH), cholera toxin, prostaglandin E2, dl-isoproterenol and dibutyryl cyclic AMP (Bt2cAMP). Cyclic AMP production, however, was unaffected by treatment with EGTA and verapamil at concentrations which attenuated steroidogenesis (0.1-1.0 mM and 125 microM, respectively). Two inhibitors of the calcium-dependent regulatory protein, calmodulin [trifluoperazine, 40 microM and 1[bis-(p-chlorophenyl)methyl] 3-[2,4-dichloro-beta-(2,4- dichlorobenzyloxy )-phenethyl]imidazolium chloride, ( R24571 ) 20 microM] significantly inhibited both cyclic AMP and progesterone production elicited by these stimulatory agents. Over the concentration range of 62.5 ng/ml-1.0 micrograms/ml, the calcium ionophore A23187 increased basal progesterone production in a dose-dependent manner, with half-maximal stimulation at approximately 0.14 microgram/ml. Maximal steroidogenic response to the calcium ionophore (1 microgram/ml) however, was only 50% of that evoked by FSH (0.33 microgram/ml). A23187 (0.5 microgram/ml) significantly enhanced progesterone production stimulated by a low concentration of FSH (0.025 microgram/ml) but failed to potentiate the maximally stimulatory action of the gonadotropin (0.33 microgram/ml). These findings support our earlier suggestion that the calcium-calmodulin system plays a central role in the gonadotropic regulation of ovarian steroidogenesis and suggest that a transmembrane flux of extracellular calcium may be an important and common step in the mechanism of stimulation of granulosa cell progesterone production.