Tag: Progesterone

DISCUSSION

Me-p-CD has been used extensively as a pharmacological tool to deplete cellular cholesterol, disrupt cholesterolrich membrane domains, and investigate coincident changes in signaling phenomena and cellular responses. In cultured mammalian cells, 5-100 mM cyclodextrin has been used, and longer exposure to high concentrations is sometimes toxic to cells. Therefore, the toxic response observed in some groups of frog oocytes in the present study was neither unexpected nor unusual. Lower doses of cyclo-dextrin exert maximal effects in mammalian cell lines with various time lines for effective cholesterol depletion and consequent effects. After long-term (24 h) labeling of mouse L-cell fibroblasts with [3H]cholesterol, 90% of cellular label was released after 8 h of incubation in 10 mM Me-p-CD. After short-term (15 min) labeling of HEp-2 cells, 90% of label was removed after 15 min of treatment with 15 mM Me-p-CD, and after long-term (20 h) labeling of HEp-2 cells with [3H]cholesterol, 50% of label was removed by 15 mM Me-p-CD after 15 min and 70% was removed after 60 min. In contrast, when the frog oocyte surface pools of cholesterol were labeled by 30 min of exposure to [3H]cholesterol, only 50% of cell-associated counts was removed after 15 min of incubation with 50 mM Me-p-CD, and approximately 70% of label was removed after 90 min of incubation (Fig. 4A, left). Following long-term loading, only 30% of label was removed by 50 mM Me-p-CD after 1 h and only 50% was removed after 9 h (Fig. 4B, left).

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To test the effect of cholesterol depletion on the time course of progesterone-induced GVBD, groups of 25-30 oocytes were incubated in increasing concentrations of Me-p-CD in Ringers-HCO3 at room temperature for 4 h, washed, and incubated with 1.5 |xM progesterone. Treatment of oocytes with concentrations of Me-p-CD <5 mM had no apparent effect on the time course of GVBD (data not shown). Treatment of oocytes with 5, 25, or 50 mM Me-p-CD accelerated the GVBD response in a dose-dependent fashion. The GVBD50 (time required for 50% of oocytes to display white spot formation) was approximately 5.5 h in a group of oocytes treated with progesterone alone, and the GVBD50 was accelerated to approximately 2 h after pretreatment of oocytes with 50 mM Me-p-CD (Fig. 1A). To combine GVBD data from different experiments, GVBD50 values in the absence of Me-p-CD were each normalized to 1, and effects of increasing concentrations of Me-p-CD were expressed as the GVBD50 in the presence of drug divided by the GVBD50 in the absence of drug. The GVBD response was accelerated by increasing concentrations of cyclodextrin; 50 mM Me-p-CD reduced the observed GVBD50 ratio to 0.42 ± 0.10 (Fig. 1B). Thus, treatment of oocytes with 50 mM Me-p-CD for 4 h before transfer to progesterone resulted in a GVBD response that was more than twice as fast as that observed for cells that were not pretreated with Me-p-CD. This effect was apparently due to drug action on the oocyte. Incubation of oocytes in 50 |xg/ml of freshly prepared pronase E (type XXV; Sigma) in oocyte Ringers for 5 min at room temperature with gentle swirling and then washing with 10 mg/ml insulin-free BSA in oocyte Ringers to damage/remove follicle cells did not block Me-p-CD action but rather accelerated the GVBD response in cells incubated in the absence or presence of Me-p-CD before progesterone treatment (data not shown). Treatment of oocytes for 5 h with 50 |xM lovastatin, an inhibitor of cholesterol synthesis, did not increase the sensitivity of oocytes to Me-p-CD (data not shown).

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METHODS

Experimental Animals and Oocyte Isolation

Mature Xenopus laevis females (Xenopus I, Dexter, MI) were housed in aquatic tanks in a temperature controlled room at 16°C, fed ground beef twice weekly, and maintained on daily cycles of 14L:10D. Frogs were primed by injection of 35 IU of eCG (PmSg; Calbiochem, La Jolla, CA) into the dorsal lymph sac 3-7 days before surgical removal of ovary. Prior to surgery, frogs were anesthetized by partial immersion in 200 ml of solution containing 0.12% tricaine (3-aminobenzoic acid ethyl ester; Sigma, St. Louis, MO) in 25 mM Hepes (Calbiochem), pH 7.0. Pieces of ovary were stored at room temperature in oocyte Ringers (83 mM NaCl, 1 mM KCl, 1 mM MgCl2, 0.5 mM CaCl2, 10 mM Hepes, pH 7.9). Oocytes (stages V or VI according to Dumont ) were manually dissected using watchmaker’s forceps under a stereomicroscope and stored in oocyte Ringers at room temperature until used for drug or hormone treatment.

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INTRODUCTION

Accumulating evidence supports a role for low-density membrane (LDM) microdomains (rafts and caveolae) in cell signaling and in transmembrane and transcellular processing (for reviews, see ). Cholesterol-enriched LDM containing immunodetectable caveolin-like protein has been recovered from Xenopus laevis oocytes. Comparison of past observations in the oocyte system with more recent evidence in other cells suggests the intriguing possibility that LDM might be involved in triggering oocyte maturation. Isolated amphibian oocytes can be induced to mature (reinitiate the meiotic cell cycle) in vitro in response to treatment with the presumed natural steroid progesterone or with insulin or insulin-like growth factor 1. One early oocyte response to treatment with inducing hormone is a decrease in intracellular cAMP that can be accounted for at least in part by inhibition of adenylyl cyclase and stimulation of phosphodiesterase type III. Inhibition of adenylyl cyclase by progesterone does not involve pertussis toxin-sensitive Gai subunits of the heterotrimeric G protein complex, is correlated with slowing of guanine nucleotide exchange, and shares certain features with P site agonist action. In other cell systems, P site adenosine action may be associated with caveolae.

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Progesterone and zona glycoprotein (ZP3) can induce the AR but do not exert their effects through the same receptor, as far as the receptor-mediated ions fluxes are concerned. Despite the molecular dissimilarity of progesterone and ZP3, each of them can activate at least two different types of receptors responsible for Ca2+ channels—onereactingrapidly,andthusprobablyactivateddi-rectly by the inducer, and the other responding after some delay and probably regulated by the inducer indirectly tia a signaling cascade. The nature of the stallion sperm progesterone receptor as well as the putative Ca2+ channel involved in signaling remains to be elucidated. In human spermatozoa, the molecular mass recognized by C-262 was 50-52 kDa protein, which is different from that of the A and B isoforms of human intracellular progesterone receptor, 94 and 120 kDa, respectively. A specific membrane protein with affinity for progesterone has been isolated from rat brain that has an estimated molecular mass of 40-50 kDa. flovent inhaler

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DISCUSSION(7)

C-262- and P-BSA-conjugates have affinity only for the native, not for the denatured progesterone receptor of human and sperm samples. Therefore, we believe that identification of the progesterone receptor is not possible with Western blots of SDS-PAGE-separated sperm protein samples (although similar results were obtained on Western blots of SDS-PAGE human sperm protein samples as described by Sabeur et al. ). Another line of evidence for the presence of a progesterone receptor is the fact that for P-BSA-FITC and for C-262/RAM-FITC, 1) fluorescent labeling patterns (Fig. 1) and relative proportions of sperm cell populations with one type of labeling (Fig. 4) were identical and 2) ultrastructural localization was identical(Fig. 3). buy birth control online

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These non-viable spermatozoa remained P-BSA-FITC positive, probably due to postacrosomal cytosolic staining (see Figs. 1C and 3B). Although a slightly higher proportion of P-BSA-FITC spermatozoa was determined with the flow cytometer in comparison with the fluorescence microscopy observation, the differences were smaller than 3-6%. It should be mentioned that P-BSA-FITC-stained sperm samples were analyzed as fixed samples for the fluorescence microscopy but were not fixed for analysis with the flow cytometer. buy flovent inhaler

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