Effects of Cycloheximide,Aminoglutethimide, Cytochalasin B,and Colchicine on Ovulation and the Ultrastructure of the Ovarian Follicle Wall in the Stellate Sturgeon Acipenser stellatus Pall

The inhibitor of protein synthesis cycloheximide, inhibitor of steroidogenesis aminoglutethimide, and inhibitor of prostaglandin synthesis indomethacin, as well as the drugs affecting the cell cytoskeleton, such as cytochalasin B and colchicine, were used for studying the mechanisms of ovulation in the stellate sturgeon Acipenser stellatus Pall. Follicles were isolated from the body cavity within certain time intervals after the injection of pituitary suspension to a female and cultivated in media with the inhibitors. In the case of follicles isolated in the middle of the period from hormonal injection until ovulation, cycloheximide, cytochalasin B, and aminoglutethimide suppressed ovulation most effectively, while in the case of oocytes isolated during the last quarter of this period, aminoglutethimide and cytochalasin B were the most effective. It was shown using TEM and SEM that cycloheximide suppressed all processes related to the preparation for ovulation, except the initial ones: contraction of follicle cells and their processes and secondary flattening of these cells. In the presence of aminoglutethimide, the follicle cells underwent pathological changes. Incubation in the media containing indomethacin and colchicine prevented degradation of the outer theca layer at the follicle apex. In the presence of cytochalasin B affecting the cytoskeleton, the drawing of follicle cell processes from the jelly coat channels was blocked, the outer theca cells were strongly contracted, but the cell layer integrity was affected and it was divided in separate fragments. A relationship is discussed between the metabolic processes and morphological changes that lead to ovulation. It was proposed on the basis of the present and previous data that the preovulatory preparation of the follicle tissues comprises two contractile and two apoptotic processes distinctly coordinated in time and space.     

Differential effects of indomethacin on the sheep ovary: Prostaglandin biosynthesis, intracellular calcium, apoptosis, and ovulation

Cells of the apical wall of the dominant follicle and contiguous ovarian surface epithelium become apoptotic with the approach of ovulation in the sheep. It was hypothesized that indomethacin, an established inhibitor of prostaglandin biosynthesis and ovulation, would protect apical ovarian cells from programmed death. The anovulatory potencies of two systemic doses of indomethacin (200 and 800 mg) were tested in gonadotropin-stimulated ewes. A complete blockade of ovulation occurred at the higher dose of indomethacin. Ovulation was not inhibited by 200 mg indomethacin. Both doses of drug suppressed follicular prostaglandin production below pregonadotropin levels. Immunofluorescence detection of digoxigenin end-labeled (fragmented) DNA was used as a marker of apoptosis among ovarian surface epithelial and granulosa cells recovered from the apical hemisphere of preovulatory ovine follicles. Cellular DNA fragmentation was averted in animals given 800 mg indomethacin, whereas apoptosis ensued after 200 mg. A sustained increase in cytosolic calcium is generally a prerequisite to apoptotic DNA fragmentation and cell death. Indeed, intracellular calcium, detected by fluorescence of fura-2, was elevated in ovarian cells of animals destined to ovulate (controls, 200 mg indomethacin) in comparison to (safeguarded) cells of anovulatory ewes (800 mg indomethacin). These observations provide circumstantial evidence that apical ovarian cell degeneration by calcium-mediated apoptosis is a determinant of follicular instability and rupture, but that these events are unrelated to the gonadotropin-induced rise in prostanoid production characteristic of preovulatory follicles.     

Ovarian expression and regulation of the stromelysins during the periovulatory period in the human and the rat

The matrix metalloproteinases (MMPs) are postulated to facilitate follicular rupture. In the present study, expression of the stromelysins (MMP3, MMP10, MMP11) was analyzed in the periovulatory human and rat ovary. Human granulosa and theca cells were collected from the dominant follicle at various times after human chorionic gonadotropin (hCG). Intact rat ovaries, granulosa cells, and residual tissue (tissue remaining after granulosa cell collection) were isolated from equine CG (eCG)-hCG-primed animals. Mmp10 mRNA was highly induced in human granulosa and theca cells and intact rat ovaries, granulosa cells, and residual tissue. Localization of MMP10 to granulosa and theca cells in both human and rat ovarian follicles was confirmed by immunohistochemistry. Mmp3 mRNA was unchanged in human cells and rat granulosa cells, but increased in intact rat ovaries and residual tissue. Mmp11 mRNA decreased following hCG treatment in human granulosa and theca cells as well as rat granulosa cells. Regulation of Mmp10 in cultured rat granulosa cells revealed that the EGF inhibitor AG1478 and the progesterone receptor antagonist RU486 suppressed the induction of Mmp10 mRNA, whereas the prostaglandin inhibitor NS398 had no effect. Studies on the Mmp10 promoter demonstrated that forskolin plus PMA stimulated promoter activity, which was dependent upon a proximal AP1 site. In conclusion, there are divergent patterns of stromelysin expression associated with ovulation, with a marked induction of Mmp10 mRNA and a decrease in Mmp11 mRNA, yet a species-dependent pattern on Mmp3 mRNA expression. The induction of Mmp10 expression suggests an important role for this MMP in the follicular changes associated with ovulation and subsequent luteinization.     

Neutrophil F-actin and myosin but not microtubules functionally regulate transepithelial migration induced by interleukin 8 across a cultured intestinal epithelial monolayer.

The role of the polymorphonuclear leukocyte (PMN) cytoskeleton during the transmigration across colonic epithelial cells is not very well understood. In order to study the role of different components of the PMN cytoskeleton during transepithelial migration across a colonic epithelial cell monolayer (T84), PMN were preincubated with drugs affecting either the actin cytoskeleton (cytochalasin B, iota toxin of Clostridium perfringens, and phalloidin) or the microtubules (colchicine and taxol). The role of PMN myosin during transepithelial migration was investigated using the inhibitor 2,3-butanedione monoxime (BDM) and DC3B toxin. PMN intracellular Ca2+, during neutrophil adhesion and translocation across the epithelium, was assessed by the Ca2+ chelator 1, 2bis-(2-aminophenoxy)-ethane-N,N,N', N'-tetra-acetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM). Transmigration of PMN was initiated by applying either interleukin-8 or formyl-met-leu-phe (fMLP). While colchicine and taxol preexposure did not influence PMN transepithelial migration, treatment with cytochalasin B, iota toxin, phalloidin, BDM, DC3B toxin and BAPTA-AM greatly diminished migration of PMN across T84 monolayers. Similarly, cell-cell contacts established between PMN and epithelial cells during the transmigration were diminished after treatment of PMN with iota toxin or cytochalasin B. These data show that the neutrophil actin cytokeleton and myosin, but not the microtubules, evoke a Ca2+ -dependent motility that facilitates migration across the colonic epithelial barrier.     

Cytoskeleton elements mediate the inhibition of the (Na++K+)atpase activity by PKC in Rhodnius prolixus malpighian tubules during hyperosmotic shock

In a previous paper, we observed that the specific activity of (Na++K+)ATPase of the isolated Malpighian tubules from Rhodnius prolixus is inhibited by protein kinase C (PKC) during hyperosmotic shock [Arenstein et al., J Membr Biol 146:47-57 [1995]; Caruso-Neves et al., Z Naturforsch 53c:911-917 [1998]). In the present paper, we study the involvement of the cytoskeleton in this process using isolated Malpighian tubules of Rhodnius prolixus. We observed that pre-incubation of the Malpighian tubule cells in hyperosmotic media decreases the specific activity of (Na++K+)ATPase by 90%. This effect was completely reversed when colchicine, which disrupts microtubules, or cytochalasin B, an inhibitor of actin microfilament polymerization, were added to the media in a dose-dependent manner. The maximal reversion was obtained with colchicine 7.0 microM or cytochalasin B 5.0 microM. The simultaneous addition of sphingosine 50 ng/mL, an inhibitor of PKC, to 10 microM colchicine or 5 microM cytochalasin B, in hyperosmotic media, did not change the stimulatory effect of these drugs on the specific activity of (Na++K+)ATPase. On the other hand, the co-incubation of TPA 20 ng/mL, an activator of PKC, to colchicine or cytochalasin B within hyperosmotic media, abolished the stimulatory effect of these drugs on the specific activity of (Na++K+)ATPase to a similar extent as hyperosmotic shock. These results suggest that inhibition of the (Na++K+)ATPase of the isolated Malpighian tubules from Rhodnius prolixus by PKC during hyperosmotic shock is mediated by cytoskeletal elements.     

Effects of cytochalasin B and enucleation on EGF receptor down regulation

The loss of epidermal growth factor (EGF) binding activity on cultured murine 3T3 cells exposed to EGF (EGF receptor down regulation) was determined in colchicine treated cells, cytochalasin B treated cells, and untreated cells. Neither colchicine nor cytochalasin B altered the affinity of the receptor for EGF, but colchicine decreased maximal EGF binding activity by 20%. The maximal extent of EGF receptor down regulation was similar in colchicine treated cells and cytochalasin B treated cells, but the rate of receptor down regulation was higher in cytochalasin B treated cells. Cytoplasts produced by subjecting cytochalasin B treated cells adhering to the substratum to centrifugal force responded to EGF with nearly normal down regulation kinetics. The results suggest that the cytoskeleton is not obligatorily involved in EGF-induced EGF receptor down regulation.     

Electrical Conductance of Cell-to-Cell Junctions and the Cytoskeleton of Plant Cells

In the submerged trichomes of floating-moss (Salvinia auriculataAubl.) and the roots of the higher water plant Trianea bogotensisKarst., the dependence of the electrical resistance of intercellular junctions on the presence of the agents that destroy microfilaments (cytochalasin B) and microtubules (colchicine) was investigated using the microelectrode technique. The resistance of the junctions (R _c) was estimated taking into account the input resistance and the coefficient of intercellular electrical communication. Should the cells be connected via symplast, R _cwill describe the resistance of plasmodesmata. Cytochalasin B (3–30 g/ml) reversibly changed R _cduring the first minutes after application. The extent of the change depended on the concentration of the inhibitor; its character of action depended on the initial strength of intercellular communication. When the initial conductance of the contact was high, cytochalasin B elevated the resistance; when it was low, the inhibitor decreased it. In all the experiments, cytochalasin B reduced the input resistance (R _i) that suggests the dependence of plasma membrane resistance on actin cytoskeleton. The effect of colchicine (0.1–1.0 mM) on R _iand R _cwas observed only when the cellular membrane was hyperpolarized or after a prolonged action of the inhibitor (for about 0.5 h). It was concluded that the electrical conductance of plasmodesmata and plasma membrane depended on the state of actin cytoskeleton. A complex and probably mediated interaction of microtubules with the processes affecting these characteristics of the cells was suggested.     

Morphological Differentiation of the Microvasculature During Follicular Development, Ovulation and Luteinization of Mouse Ovaries

The chronological changes of the microvasculature during follicular development, ovulation and luteinization of mouse ovaries were examined by observation of serial histological sections, lectin angiographs and resin-corrosion casts. Graafian follicles possessing oocytes with germinal vesicles were surrounded by a few layers of basket-like capillary wreath adjacent to the follicular basement membrane. Just before ovulation 11–12 hr after hCG administration, some theca cells differentiated into hypertrophic cells, and the follicular basement membrane underwent fragmentation. Then the capillaries within the theca interna became dilated, and hyperpermeable and appeared to be injured. The capillary wreath extended into the follicle via the hypertrophied theca interna. After ovulation, the follicular wall became markedly edematous. Capillary branches invaded the granulosa cell layer of the ruptured follicle from the region of extravasation to form an intricate capillary network. The capillary network occupied the whole corpus luteum until 24 hr after hCG administration.     

Effect of cytoskeleton disruptors on L-type Ca channel distribution in rat ventricular myocytes

The cytoskeleton plays an important role in many aspects of cardiac cell function, including protein trafficking. However, the role of the cytoskeleton in determining Ca channel location in cardiac myocytes is unknown. In the present study we therefore investigated the effect of the cytoskeletal disruptors cytochalasin D, latrunculin, nocadazole and colchicine on the distribution of Ca channels in rat ventricular myocytes during culture for up to 96 h. During culture in the absence of these agents, cell edges became rounded, t-tubule density decreased, and the normal transverse distribution of the alpha1 (pore-forming) subunit of the L-type Ca channel became more punctate and peri-nuclear; these changes were associated with loss of synchronous Ca release in response to electrical stimulation. Disruption of tubulin using nocadazole or colchicine or sequestration of monomeric actin by latrunculin had no effect on these changes. In contrast, cytochalasin D inhibited these changes: cell shape, t-tubule density, transverse Ca channel staining and synchronous Ca release were maintained during culture. The protein synthesis inhibitor cycloheximide had similar effects to cytochalasin. These data suggest that cytochalasin stabilizes actin in adult ventricular myocytes in culture, thus stabilizing cell structure and function, and that actin is important in trafficking L-type Ca channels from the peri-nuclear region to the t-tubules, where they are normally located and provide the trigger for Ca release.     

Enhanced invasion of Tyzzer's organism into cultured mouse hepatocytes by cytochalasin D

The effects of cytoskeleton inhibitors on the invasion of Tyzzer's organism, an obligate intracellular bacterium, into cultured mouse hepatocytes were examined by double immunofluorescence observation and plaque assay. The two techniques gave comparable results. Invasion of bacteria was significantly enhanced by cytochalasin D, a microfilament disrupting drug, and markedly suppressed by vinblastine, a microtubule inhibitor. Another microtubule inhibitor, colchicine, did not show any substantial effect. However, the cytoskeletal system of cultured mouse hepatocytes was sensitive to these three drugs, as judged by inhibition of FITC-dextran uptake. These results suggest that Tyzzer's organisms invade host cells by a unique mechanism that is suppressed by the normal functions of host cell microfilaments.     

COLLAGEN SYNTHESIS AND CELL GROWTH IN CHICK EMBRYO FIBROBLASTS: INFLUENCE OF COLCHICINE,CYTOCHALASIN B AND CONCANAVALIN A

Culturing of chick embryo fibroblasts in the presence of colchicine or cytochalasin B with and without concanavalin A (Con A) demonstrated that colchicine induces greater neosynthesis of endocellular type I collagen, whereas cytochalasin B boosts secretion. The effects are modified by the addition of Con A, which increases α₂more than a1 chain production.³H-thymidine incorporation is unaffected by cytochalasin B, but stimulated by colchicine. Con A neutralizes the stimulatory action of colchicine. It would therefore seem that Con A exerts transmembrane control of effects induced by colchicine and cytochalasin B by binding to cell surface receptors and so triggering rearrangement of the cytoskeleton.     

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)     

Effect of treatment of estrous ewes with indomethacin on the distribution of ovarian blood to the periovulatory follicle

The level of prostaglandin (PG) F2 alpha increases within the wall of the ovine follicle pending ovulation. Coincidently, the quantity of ovarian blood distributed to the follicular wall progressively declines. A potential cause(PGF2 alpha)-and-effect (impaired follicular blood supply) relationship was considered. At an early stage of estrus, ewes were injected systemically either with vehicle or indomethacin (an inhibitor of biosynthesis of prostaglandins). Abdominal laparotomies were carried out and the ovaries examined near the expected time of ovulation. The ovary containing the largest follicle or an ovulation point was perfused with radioactive microspheres via the ovarian artery. The periovulatory follicle was isolated from the ovary and the content of radioactivity monitored with respect to that of the whole ovary. Follicular tissue was analyzed for PGF2 alpha. Treatment with the drug was associated with: 1) failure of follicular rupture; 2) follicular hyperemia and edema; and 3) suppressed synthesis of PGF2 alpha. A reduction of the supply of ovarian blood reaching the preovulatory follicle, and a mediatory task of follicular prostaglandins in this process, could be a critical determinant of ovulation.     

Preovulatory changes in the perifollicular capillary network in the rat: role of eicosanoids.

The aim of this study was to evaluate morphometrically the influence of ovulation-inhibiting doses of indomethacin, an inhibitor of the cyclooxygenase pathway, and esculetin and caffeic acid, inhibitors of the lipoxygenase pathway, on the dilatation of the perifollicular capillary network in the theca interna. The development of the perifollicular capillary network as a function of follicular size and the changes in the vascular lumen were examined by light microscopy on a series of semithin cross sections of rat ovaries. The number of capillaries in the theca interna increased linearly with increasing follicle diameter. Thus, the relative number of capillaries in the theca interna supplying the avascular stratum granulosum remained constant. This indicates that follicular function is not regulated through changes in the number of capillaries in the theca interna. After hCG injection, an increase in the capillary area could be observed in follicles having a diameter of more than 600 microns. Indomethacin administration increased the capillary area of the ovulatory follicles as compared to the untreated side only at 6 h after treatment. By contrast, treatment with inhibitors of lipoxygenase resulted in a significant decrease in the capillary area of large follicles at all times examined (3, 6, and 9 h after hCG injection). Nevertheless, since both types of eicosanoid inhibitors suppressed follicle rupture, in spite of their opposing actions on the capillary area, it seems unlikely that their action on ovulation is primarily due to their effect on this parameter.     

Characterization of cellular and vascular changes in equine follicles during hCG-induced ovulation

In contrast to other species, the histology of the equine follicle during ovulation has not been described. Preovulatory follicles were isolated during oestrus at 0, 12, 24, 30, 33, 36 and 39 h (n = 5-6 follicles per time point) after an ovulatory dose of hCG to characterize the cellular and vascular changes associated with ovulation in mares. Pieces of follicle wall were formalin-fixed and processed for light microscopy to evaluate the general follicular morphology and quantify selected parameters. Marked changes were observed in the histology of equine follicles in the hours before ovulation. The thickness of the granulosa cell layer doubled between 0 and 39 h after hCG (77.8 +/- 4.8 versus 158.8 +/- 4.8 microns, respectively; P < 0.01). This expansion was caused primarily by a pronounced accumulation of acid mucosubstances between granulosa cells, which was first detected at 12 h after hCG and peaked at 36-39 h. In contrast, a significant thinning of the theca interna was observed after hCG treatment. Fewer cell layers were present; theca interna cells appeared smaller than before hCG; and the presence of occasional pyknotic cells was noted at 36 and 39 h after hCG. In addition, the theca layers were invaded by numerous eosinophils. No eosinophils were observed in preovulatory follicles isolated between 0 and 24 h after hCG, but the number increased to 14.0 +/- 0.8 and 5.6 +/- 0.3 eosinophils per field (x 400) in theca interna and theca externa, respectively, 39 h after hCG treatment (P < 0.01). Severe oedema, hyperaemia and haemorrhages, and significant increases in the number of blood vessels in theca interna and externa were observed at 33, 36 and 39 h after hCG. This study provides the first in-depth characterization of the sequential cellular and vascular changes that occur in equine follicles before ovulation.     

Effects of colchicine- and cytochalasin B-treatment on the intracellular distribution of yolk droplets, lipid bodies, and Golgi apparatus of the chick neuroepithelial cells

Treatment with colchicine (antimicrotubular agent) and cytochalasin B (antimicrofilamentous agent) has been used to investigate the possible role played by the cytoskeleton in the maintenance of intracellular distribution of yolk droplets, lipid bodies, and Golgi apparatus of the chick neuroepithelial cells. On the one hand, embryos treated with colchicine showed modifications in their distribution patterns of yolk droplets and lipid bodies, which suggests the involvement of the microtubular integrity of neuroepithelial cells in the maintenance of normal distribution patterns. On the other hand, the close relationships between vitelline and lipid inclusions and Golgi apparatus observed in untreated embryos seems to be kept in the embryos treated with colchicine and cytochalasin B. Moreover, from the effects of colchicine on Golgi apparatus position a possible functional role for the microtubular system in the maintenance of Golgi apparatus polarity in the chick neuroepithelial cells can be proposed. The results provided here constitute new information about the cellular mechanisms involved in chick neurulation.     

In vitro ovulation of trout oocytes : Effect of prostaglandins on smooth muscle-like cells of the theca

Ovulation (active expulsion of oocyte from the mature follicle) of trout follicles matured can be induced by adding PGF_2α at doses of 1 and 5 μg/ml. PGE₂ is ineffective.The induction of ovulation by PGF_2α is inhibited in a calcium free medium or by inhibitors of calcium influx, particularly by Mn⁺⁺ and La⁺⁺⁺, suggesting that ovulation process implies active contraction of the smooth muscle cells of the theca.A significant but partial inhibition is also observed with cytochalasin B (1 and 5 μg/ml) demonstrating that contraction of other cell types than muscle, containing actin-like filaments, may also participate in the process.     

Development and fate of the postovulatory follicle complex,postovulatory follicle,and observations on folliculogenesis and oocyte atresia in ovulated common snook,Centropomus undecimalis (Bloch, 1792)

The common snook, Centropomus undecimalis , was induced to ovulate using a time‐release, GnRH analogue. Ovulation occurred the afternoon or evening the day after hormone administration. The time of ovulation was established within half an hour. At ovulation, three fish per time‐group were divided into 0, 6, 12, 18 hr and one thru five days post‐ovulation to study changes in the postovulatory follicle complex (POC). Histology of the ovaries revealed changes in the POC, postovulatory follicle (POF) and oocyte atresia through five days post‐ovulation. Within 24 hr, nuclei of the POF cells lost their initial spherical or oval configuration, and by four days the basement membrane within the POC had fragmented. There was a temporal separation between ovulation and post‐ovulation folliculogenesis; that is, in that the formation of new follicles commenced within the germinal epithelium between 12–48 hrs after ovulation. Morphology of the POC was best revealed with the reticulin stain; it is composed of the POF and postovulatory theca (POT). These are separated by a basement membrane, reflecting the origin of a follicle from a germinal epithelium while the theca is derived from stroma. The POF is composed of the former follicle cells that surrounded and contacted the oocyte during its development; the follicle is composed of the oocyte and its surrounding follicle cells. The POC is composed of a prominent basement membrane separating the POT from the POF. The reticulin stain clearly defines compartmentation in the ovary and supports redefinition of the POF as the follicle cells that formerly surrounded the oocyte prior to ovulation. J. Morphol. 278:547–562, 2017. © 2017 Wiley Periodicals, Inc.