Gonadotropin-induced accumulation of 4,4-dimethylsterols in mouse ovaries and its temporal relation to meiosis.
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The resumption of oocyte meiosis is triggered by a number of 4,4-dimethylsterols termed meiosis-activating sterols (MAS). The levels of meiosis active (follicular fluid [FF]-MAS and bull testes [T]-MAS) and inactive (lanosterol) 4,4-dimethylsterols, free cholesterol, and progesterone were determined in gonadotropin-primed prepubertal mouse ovaries in vivo by high-performance liquid chromatography. Ovaries responded to an ovulatory stimulation by increasing their content of 4,4-dimethylsterols but not of free cholesterol. The ovarian 4,4-dimethylsterol response was followed with regard to time and dose-response to the gonadotropins and the resumption of meiosis was evaluated using histologic sections. All 4,4-dimethylsterols accumulated in a time-dependent manner in gonadotropin-primed mice after a subsequent stimulation with hCG. The peak of 4,4-dimethylsterol accumulation appeared postmeiotically but coincided roughly with ovulation, and the resumption of meiosis was triggered when the intraovarian level of MAS was <20% of its maximum. The ovarian accumulation of progesterone preceded the 4,4-dimethylsterol accumulation. The FF-MAS accumulation displayed a dose-response maximum with respect to hCG, and a variation of the follicular priming regime revealed that, in contrast to progesterone production, 4,4-dimethylsterol accumulation is dependent on previous follicular growth beyond the gonadotropin-dependent stage. The FF-MAS was not liberated from esterified stores during the accumulatory response and appeared to be synthesized de novo from a precursor (or precursors) metabolically upstream to lanosterol. The data remain inconsistent with a model in which MAS is regarded as the physiological trigger of meiosis. The 4,4-dimethylsterol accumulation is suggested to influence maturation processes by affecting membrane sterol composition. (+info)
Resumption of meiosis induced by meiosis-activating sterol has a different signal transduction pathway than spontaneous resumption of meiosis in denuded mouse oocytes cultured in vitro.
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The sterol 4,4-dimethyl-5-cholesta-8,14,24-trien-3-ol (follicular fluid meiosis-activating sterol [FF-MAS]) isolated from human follicular fluid induces resumption of meiosis in mouse oocytes cultured in vitro. The purpose of this study was to examine the hypothesis that differential signal transduction mechanisms exist for FF-MAS-induced and spontaneous in vitro resumption of meiosis in mouse oocytes. Mouse oocytes were dissected from ovaries originating from mice primed with FSH 48 h before oocyte collection. Mechanically denuded germinal vesicle (GV) oocytes were in vitro matured in medium supplemented with hypoxanthine and FF-MAS or allowed to mature spontaneously; both groups were exposed to individual compounds known to inhibit specific targets in the cell. After 20-22 h of in vitro maturation, resumption of meiosis was assessed as the frequency of oocytes in GV breakdown (GVBD) stage. Pertussis toxin (2.5 microg/ml) did not influence resumption of meiosis in either group. Dibutyryl cyclic GMP (320 microM) inhibited FF-MAS-induced GVBD, but not spontaneous GVBD, whereas the subtype 5 phosphodiesterase-inhibitor zaprinast (50 microM) inhibited GVBD in both groups. Microinjection of the catalytic subunit of cAMP-dependent protein kinase into oocytes inhibited spontaneous GVBD, but not FF-MAS-induced GVBD. An inhibitor of cytoplasmic polyadenylation, cordycepin (80 microM), inhibited or retarded spontaneous GVBD to a further extent than it did FF-MAS-induced GVBD. Spontaneous GVBD was more sensitive to the histone H1 kinase-inhibitor olomoucine (250 microM) than was FF-MAS-induced GVBD. Addition of the mitogen-activated protein kinase (MAPK)-inhibitor PD 98059 (50 microM), phospholipase C-inhibitor U-73122 (10 microM), p21(ras)-inhibitor lovastatine (250 microM), and the src-like kinase inhibitor PP2 (20 microg/ml) inhibited FF-MAS-induced GVBD, but not spontaneous GVBD. Both MAPKs, extracellular regulated kinase (ERK) 1 and ERK2, were phosphorylated under FF-MAS-induced meiotic resumption, in contrast to spontaneous meiotic resumption, in which ERK1 and ERK2 phosphorylation occurred 2 h after GVBD. In the present study, we show that FF-MAS acts through an MAPK-dependent pathway, and we suggest that src-like kinase, p21(ras), and phosphoinositide signaling lie upstream of MAPK in the FF-MAS-activated signaling pathway. Clearly, striking pathway differences are present between spontaneous versus FF-MAS-induced meiotic resumption. (+info)
A new and superior adrenal scanning agent, NP-59.
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The first synthesis of 131I-19-iodocholesterol had a 10-25% radiochemical impurity that was not iodide ion. This impurity has been identified as 6beta-131I-iodomethyl-19-nor cholest 5(10)-en-3beta-ol (NP-59) and has been synthesized. Tissue distribution studies with 131I-NP-59 in rats and dogs revealed a higher adrenal uptake and adrenal-to-tissue ratios compared to 131I-19-iodocholesterol, probably less in vivo deiodination, and superior adrenal images. A high uptake was seen in the adrenal medulla in addition to that in the cortex. Iodine-131-NP-59 is being evaluated for the early detection of adrenal-cortical disorders and as a potential scanning agent for detecting structural abnormalities of the adrenal medulla. (+info)
Microtubule involvement in the intracellular dynamics for gene transfection mediated by cationic liposomes.
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The effects of microtubule polymerization on liposome-mediated gene transfection were investigated by confocal laser scanning microscopy in target living cells. Both nocodazole and taxol apparently increased the efficiency of gene transfection. Lipofection with fluorescence-labeled cationic liposomes in a COS-7 cell expressing yellow fluorescent protein (YFP)-tagged tubulin revealed that the liposomes were transported along microtubules to lysosomes which are colocalized with the microtubule organizing center (MTOC). Nocodazole disrupted microtubules and produced a uniform distribution of YFP-tagged tubulin in the cytoplasm. Under these conditions, both liposomes and lysosomes were scattered throughout the cytoplasm and they did not colocalize. In the presence of taxol, microtubules were stabilized and several focal regions, like the MTOC, were formed. Lysosomes resided around the nucleus, while liposomes were trapped in microtubules. Under these conditions, neither liposomes nor DNA colocalized with lysosomes. These results demonstrated that the liposome-DNA complexes are transported to lysosomes by a microtubule-mediated pathway, and the effects of nocodazole and taxol on transfection efficiency can be explained by failure of the transport of the liposome-DNA complexes to lysosomes where DNAs are degraded. (+info)
Sterol effects and sites of sterol accumulation in Caenorhabditis elegans: developmental requirement for 4alpha-methyl sterols.
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Caenorhabditis elegans requires sterol, usually supplied as cholesterol, but this is enzymatically modified, and different sterols can substitute. Sterol deprivation decreased brood size and adult growth in the first generation, and completely, reversibly, arrested growth as larvae in the second. After one generation of sterol deprivation, 10 ng/ml cholesterol allowed delayed laying of a few eggs, but full growth required 300 ng/ml. C. elegans synthesizes two unusual 4alpha-methyl sterols (4MSs), but each 4MS supported only limited growth as the sole sterol. However, addition of only 10 ng of cholesterol to 1,000 ng of 4MS restored full growth and egg-laying, suggesting that both a 4MS and an unmethylated sterol are required for development. Filipin stained sterols in only a few specific cells: the excretory gland cell, two amphid socket cells, two phasmid socket cells and, in males, spicule socket cells. Sterols were also present in the pharynx and in the intestine of feeding animals in a proximal-to-distal gradient. This non-random sterol distribution, the low concentration requirements, and the effects of 4MSs argues that sterols are unlikely to be used for bulk structural modification of cell membranes, but may be required as hormone precursors and/or developmental effectors. (+info)
Physiology of meiosis-activating sterol: endogenous formation and mode of action.
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BACKGROUND: In the context of mammalian oocyte maturation, it has been suggested that intermediates of cholesterol biosynthesis may represent the physiological signal that instructs the oocyte to reinitiate meiosis. METHODS: Endogenous levels of follicular fluid meiosis-activating sterol (FF-MAS) were monitored in rabbit ovarian tissue, and the influence of exogenous gonadotrophins on sterol formation was assessed. The involvement of cAMP in FF-MAS-induced versus spontaneous oocyte maturation in vitro in mice was also investigated, as was the direct microinjection of FF-MAS into mouse oocytes. RESULTS: Levels of FF-MAS in rabbit ovaries were significantly elevated 1 h after hCG/LH induction and remained so for 4 and 12 h after induction. In naked oocytes undergoing spontaneous maturation, a significant decrease in cAMP was detected after 30 min of culture. However, FF-MAS-mediated induction of oocyte maturation in hypoxanthine-arrested naked oocytes was not associated with any detectable decrease in intracellular cAMP levels. Microinjected FF-MAS failed to induce any noticeable meiosis. CONCLUSIONS: A rapid increase in FF-MAS level occurred in vivo in the rabbit ovary in response to LH, and clear differences were seen in the cAMP pattern during spontaneous and induced oocyte maturation in mice. (+info)
Effects on M5076-hepatic metastasis of retinoic acid and N-(4-hydroxyphenyl) retinamide, fenretinide entrapped in SG-liposomes.
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Retinoic acid (RA), a potent inducer of cell differentiation, and N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide), a potent inducer of apoptosis, are well known as anticancer agents that are administered orally to patients for leukemia, breast and prostate cancer, respectively. However, it has not been studied whether both retinoids are effective on metastatic cancer. In mice implanted with M5076 cells, murine reticulum cell sarcoma survival times were prolonged by i.v. treatment of RA and 4-HPR entrapped in liposomes containing soybean-derived sterylglucoside mixture (SG), which accumulates in liver. In contrast, free RA and 4-HPR were inactive. These results indicate that RA and 4-HPR in SG-liposomes exhibit anticancer efficacy on metastatic cancers, and may have great potential for clinical use in the treatment of various cancers. (+info)
Meiosis-activating sterol protects oocytes from precocious chromosome segregation.
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BACKGROUND: Follicular fluid meiosis-activating sterol (FF-MAS) overcomes hypoxanthine (HX)-mediated meiotic arrest in mammalian oocytes. METHODS: In order to determine whether chromosome segregation was normal in oocytes matured in FF-MAS, the development, chromosomal constitution and chromosome alignment was analysed in spontaneously matured as well as HX-arrested mouse oocytes cultured in the absence or presence of FF-MAS. RESULTS: FF-MAS-induced meiotic maturation was significantly less effective compared with spontaneous maturation in supporting cytokinesis ( approximately 40 and approximately 90% polar body formation respectively). The majority of oocytes stimulated by FF-MAS to overcome the HX block developed to metaphase II (MII), but 23.4% of meiosis II oocytes were diploid. Chromosomes were well aligned on the spindle, and hyperploidy was low in spontaneously matured oocytes and HX-arrested oocytes cultured with or without FF-MAS. Unexpectedly, almost 40% of spontaneously matured MII oocytes contained chromatids/monads. Precocious loss of chromatid cohesion was significantly reduced in spontaneously matured as well as HX-arrested oocytes cultured in the presence of FF-MAS but not lanosterol. CONCLUSIONS: FF-MAS induces full nuclear maturation to MII, and chromosomes segregate with high fidelity. However, in delayed FF-MAS-stimulated meiotic maturation, anaphase I may occur in the absence of cytokinesis. FF-MAS appears to protect mammalian oocytes from precocious chromatid segregation. (+info)