Cytochemical and Biochemical Studies on Progesterone—Induced Maturation in Amphibian Oocytes

Baltus, Elyane; Brachet, Jean; Hanocq-Quertier, J.; Hubert, E.

doi:10.1111/j.1432-0436.1973.tb00108.x

Cited by 54 publications

(22 citation statements)

References 29 publications

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“…Data from Table 2 confirm moreover that cycloheximide is as a potent inhibitor of meiosis reinitiation in Ambystoma as it has been previously described for Xenopus (2,3,4,12,20,27) and for Rana (17,23). They also demonstrated this effect to be reversible by simple washing without additional progesterone exposure as previously shown for Xenopus (2) and for Rana (19). True maturations were Essentially identical results were obtained with cycloheximide.…”

Section: Fromsupporting

confidence: 62%

Propionate and Cycloheximide Reversibly Block Progesterone Induced Calcium Surge in Ambystoma Mexicanum Oocytes

Vilain¹,

Moreau²,

Galle³

1980

Dev Growth Differ

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The protoprotein aequorin was used in order to monitor CaZ+ transients in conditions where progesterone induced maturation was reversibly inhibited. Propionate but not isethionate CI-free medium impaired both meiosis reinitiation and the Caz+ transient, unless oocytes were returned to normal CI-containing medium. Similar results were obtained with the protein synthesis inhibitor cycloheximide. In both cases, the incidence of germinal vesicle breakdown (GVBD) and the time schedule relating it to the Ca2+ surge appeared not very different from that found from control oocytes. The evidence suggests that both treatments act on the initial step by which progesterone triggers the intracellular CaZ+ release needed for maturation promoting factor (MPF) elaboration. No definitive conclusion can be reached however from these experiments concerning the need for protein synthesis during meiosis reinitiation.We found recently, using Ca2+ sensitive electrodes and the photoproteins aequorin and obelin that treatment of anuran and urodele oocytes with progesterone and some of its mimetics triggers an early Ca2+ transient (16). We considered this CaZ+ transient as an essential step for release of the meiotic block since it never occurred in progesterone irresponsive oocytes or in oocytes simultaneously treated with 10 mM of either theophylline or procaine. In this work, aequorin-injected oocytes were used in order to demonstrate the effect of two different inhibitory treatments on both meiosis reinitiation and the Ca2+ surge. The first treatment consisted of using C1-free medium since data from ROBINSON (1 8) suggested that progesterone might indirectly affect a Ca2+-dependent chloride permeability of the oocyte. In these condig tions, we found that propionate but not isethionate-chloride exchange impaired meiosis reinitiation, unless oocytes were washed and returned to normal Cl-containing medium. These effects were found to coincide with the inhibition and recovery of the progesterone induced Ca2+ surge. Identical results were obtained with the protein synthesis inhibitor cycloheximide which has been shown to inhibit reversibly meiosis reinitiation both in Xenopus (2) and Rana oocytes (19). These data strengthen the idea that Ca2+ surge is an important step in meiosis reinitiation and that most inhibitors act at this level, blocking the production of the maturation promoting factor. MATERIALS AND METHODSFully-grown oocytes (1.8 to 2.0 mm in diameter) were isolated from ovarian tissues excised from matare

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Section: Fromsupporting

confidence: 62%

Propionate and Cycloheximide Reversibly Block Progesterone Induced Calcium Surge in Ambystoma Mexicanum Oocytes

Vilain¹,

Moreau²,

Galle³

1980

Dev Growth Differ

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“…Although we are still far from understanding the molecular mechanism that is responsive to these various stimuli, the work of several research groups has provided evidence that de novo protein synthesis, protein phosphorylation, and calcium ions play important roles in the early steps of the maturation process (6)(7)(8)(9)(10). Recent evidence has also pointed to the participation of cyclic AMP (cAMP) in amphibian oocyte maturation induced by progesterone.…”

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confidence: 99%

Amphibian oocyte maturation and protein synthesis: Related inhibition by cyclic AMP, theophylline, and papaverine

Bravo

Otero

Allende

et al. 1978

Proc. Natl. Acad. Sci. U.S.A.

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Two inhibitors of cyclic AMP phosphodiesterase (3':5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17), theophylline and papaverine, inhibit the maturation of Xenopus laevis oocytes induced by four different stimuli: human chorionic gonadotropin, progesterone, testosterone, and lanthanum ions. Addition of 1 mM cyclic AMP to the medium delays maturation by approximately 2 hr. Papaverine, theophylline, and cyclic AMP inhibit amino acid incorporation into oocyte proteins by 50% or more but do not inhibit amino acid uptake. The capacity of theophylline to block maturation and protein synthesis is reversed in a parallel fashion by addition of 1-5 mM calcium ion to the medium. Addition of papaverine, theophylline, and cycloheximide to oocytes at different times after hormonal treatment shows that the step sensitive to blockage by the three drugs is coincident and precedes germinal vesicle breakdown by about 1.5 hr. The maturation of full-grown oocytes is a complex process that involves meiotic cell division and prepares the cell for fertilization. Much attention has been devoted to the study of the maturation of amphibian oocytes because these cells can be obtained in large amounts from a single ovary, they are big enough to be microinjected, and they can be induced to mature in vitro.One of the puzzling aspects of the maturation process is that its onset can be triggered by such different stimuli as the hormones human chorionic gonadotropin (hCG), progesterone, and testosterone and by nonphysiological reagents such as ionophore A 23187, organomercurial compounds, and 10 mM lanthanum ions (1-5).Although we are still far from understanding the molecular mechanism that is responsive to these various stimuli, the work of several research groups has provided evidence that de novo protein synthesis, protein phosphorylation, and calcium ions play important roles in the early steps of the maturation process (6-10). Recent evidence has also pointed to the participation of cyclic AMP (cAMP) In this report we show that theophylline and papaverine, two well-known cAMP phosphodiesterase (3':5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17) inhibitors, are potent inhibitors of oocyte maturation induced by four different stimuli: hCG, progesterone, testosterone, and lanthanum ions. These drugs, which inhibit endogenous oocyte cAMP phosphodiesterase (13), do not cause any measurable increase in the overall cAMP concentration of these cells. However, theophylline or papaverine treatment of oocytes eliminates the increase of Ca2+ uptake induced by progesterone and hCG and results in a very significant inhibition of protein synthesis in these cells. cAMP itself, added to the oocyte incubation medium, also causes an inhibition of protein synthesis and delays the onset of hormone-induced maturation. Several characteristics of the effect of theophylline, papaverine, and cAMP in blocking oocyte maturation seem to indicate that this effect is due to their inhibition of cellular protein synthesis.MATERIALS AND METHODS X. laevis wer...

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“…As described in a previous paper (1), DNA could be extracted from the yolk platelets isolated from whole ovaries of Xenopus laevis. Since it was likely that the yolk fraction was contaminated with nuclear DNA from the follicle cells in these earlier experiments, we now worked with oocytes that have been freed from their follicle cells in two differentways: (i) manual removal of follicle cells after a short treatment of the oocytes with chemical agents, such as Pronase (0.15%) or sodium dodecyl sulfate (0.3%); (ii) in vitro ovulation of the oocytes in the presence of hormones.…”

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Characterization of Yolk DNA from Xenopus laevis Oocytes Ovulated In Vitro

Hanocq

Kirsch‐Volders

Hanocq-Quertier

et al. 1972

Proc. Natl. Acad. Sci. U.S.A.

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High molecular weight DNA was isolated from the yolk platelets of Xenopus laevis oocytes ovulated in vitro. Yolk DNA has the same buoyant density in CsCl gradients as mitochondrial and nuclear DNAs, and, like them, it is double-stranded. Yolk DNA behaves like nuclear DNA, and not like mitochondrial DNA, upon renaturation after denaturation. The molecules are always linear. Cytochemical and biochemical controls preclude the possibility that the yolk DNA might be contaminated by nuclear or mitochondrial DNA. We conclude that the yolk DNA is an endogenous component of the yolk platelets.As described in a previous paper (1), DNA could be extracted from the yolk platelets isolated from whole ovaries of Xenopus laevis. Since it was likely that the yolk fraction was contaminated with nuclear DNA from the follicle cells in these earlier experiments, we now worked with oocytes that have been freed from their follicle cells in two differentways: (i) manual removal of follicle cells after a short treatment of the oocytes with chemical agents, such as Pronase (0.15%) or sodium dodecyl sulfate (0.3%); (ii) in vitro ovulation of the oocytes in the presence of hormones. In both cases, high molecular weight DNA could be extracted from the yolk platelets; the physicochemical properties of yolk DNA from oocytes ovulated in vitro will be described. Our experimental data exclude beyond reasonable doubt the possible contamination of yolk DNA by either nuclear or mitochondrial DNA arising from follicular or other ovarian contaminating cells. We shall discuss the extraction of yolk DNA from unfertilized eggs of Xenopus and Ambystoma mexicanum. MATERIALS AND METHODSIn Vitro Ovulation. Ovaries were removed from adult Xenopus laevis, and the membranes of the ovarian follicles were torn apart. (ViPyr). (pH 7.0); 10 ml of medium were used for each 1000 oocytes. The homogenate was layered on an equal volume of 1 M sucrose-5% (ViPyr). and centrifuged for 20 min at 500 X g. The pellet was suspended in 0.25 M sucrose-5% (ViPyr). and centrifuged again in the same way. Electron microscopic controls showed that contamination of the yolk platelets by pigment and mitochondria was negligible.Mitochondria were isolated by Dawid's method (4). Removal of the Jelly of Unfertilized Shed Eggs. For Xenopus, a cysteine-papain mixture at pH 7.8 was used (4). For Ambystoma, the jelly was removed with forceps without any chemical treatment. Both types of eggs were maintained in 0.1 X Holtfreter's medium (5).DNA Determination. Instead of the fluorometric method (6), we used a modification of Keck's indol method (7): 75 oocytes were homogenized in 2 ml of 0.25 M sucrose-0.03 M Tris HCl (pH 7.4)-0.1 M EDTA; 2 ml of cold 10% Cls-CCOOH were added; after 15 min, the pellet was centrifuged and washed with cold 5% Cl3CCOOH. Lipids were removed by single successive washings with 94% alcohol at 40, 94% alcohol at 500, n-butyl alcohol at 500, alcohol-ether (3: 1) at 500, and ether. The pellet was extracted for 20 min at 1000 with 600 ,l of 5% Cl3CCOOH and centri...

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Cytochemical and Biochemical Studies on Progesterone—Induced Maturation in Amphibian Oocytes

Cited by 54 publications

References 29 publications

Propionate and Cycloheximide Reversibly Block Progesterone Induced Calcium Surge in Ambystoma Mexicanum Oocytes

Propionate and Cycloheximide Reversibly Block Progesterone Induced Calcium Surge in Ambystoma Mexicanum Oocytes

Amphibian oocyte maturation and protein synthesis: Related inhibition by cyclic AMP, theophylline, and papaverine

Characterization of Yolk DNA from Xenopus laevis Oocytes Ovulated In Vitro

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