Induction of Maturation (Meiosis) in Xenopus laevis Oocytes by

Transcription

1 Proc. Nat. Acad. Sci. USA Vol. 72, No. 4, , April 1975 Induction of Maturation (Meiosis) in Xenopus laevis Oocytes by Three Organomercurials* (progesterone/p-hydroxymercuribenzoate/p-hydroxymercuriphenylsulfonate/mersalyl/ maturation promotion factor) J. BRACHET, E. BALTUS, A. DE SCHUTTER-PAYS, J. HANOCQ-QUERTIER, E. HUBERT, AND G. STEINERT Laboratory of Molecular Cytology and Embryology, Department of Molecular Biology, Free University of Brussels, Rhode-St-Genbse, Belgium; Laboratorio di Embriologia Molecolare, CNR, Arco Felice, Napoli, Italy Contributed by Jean Brachet, January 3, 1975 ABSTRACT Three organomercurials, p-hydroxymercuribenzoate, p-hydroxymercuriphenylsulfonate, and mersalyl, induce maturation (meiosis) in a large percentage (2-1%) of Xenopus laevis oocytes. Maturation takes place even when the follicle cells which surround the oocytes have been withdrawn. Organomercurial- and progesterone-induced maturations have many features in common: they do not occur when the inducer is injected into the oocytes, they require the presence of Ca++ in the medium, they are inhibited by cycloheximide but not by actinomycin D. In both cases, the maturation producing factor and the pseudomaturation inducing factor are produced. Organomercurial-treated oocytes react normally to activating stimuli; their protein synthesis increases, but uptake of amino acids is strongly inhibited. Progesterone and p-hydroxymercuriphenylsulfonate act synergically in inducing maturation. The main difference between the two agents is that p-hydroxymercuriphenylsulfonate must act for several hours, whereas, short contact with progesterone is sufficient to induce maturation. Maturation (breakdown of the germinal vesicle and first meiotic division) is easily induced by treating amphibian oocytes with various steroid hormones (1-5). There are good reasons to believe that the physiologically active hormone is progesterone or a closely related steroid; progesterone is probably synthesized in response to pituitary stimulation (2, 3) by the follicle cells which surround the oocytes. Progesterone is inactive if it is injected directly into the oocyte (1, 5); it probably reacts, in an unknown way, with constituents of the plasma membrane. Once it has crossed the plasma membrane, progesterone binds to a specific receptor associated with the melanosomes (5, 6). Progesterone does not by itself induce germinal vesicle breakdown, but produces the appearance, in the cytoplasm, of a "maturation promoting factor" (MPF); injection of endoplasm taken from progesterone-treated oocytes induces maturation in recipient oocytes (7-11). MPF, which is probably a Abbreviations: MPF, "maturation promoting factor"; PIF, "pseudomaturation inducing factor". In this paper the abbreviations PHMB and PHMPS have been used for p-hydroxymercuribenzoate and p-hydroxymercuriphenylsulfonate respectively since at the ph used (neutral), the Cl- of p-chloromercuribenzoic acid (PCMB) and of p-chloromercuribenzenesulfonate (PCMBS) is replaced by OH-. * This paper is dedicated to the memory of Louis Rapkine, who discovered the importance of -SH groups for early embryonic development. protein, becomes detectable in progesterone-stimulated oocytes a few hours before germinal vesicle breakdown (11). Injection of a homogenate from progesterone-treated oocytes induces, in recipient oocytes of Xenopus laevis, an abortive and highly abnormal maturation that we have called pseudomaturation (4, 12, 13); the homogenate thus contains, besides MPF, a "pseudomaturation inducing factor" (PIF). While MPF is localized in the endoplasm and stimulates protein synthesis in injected recipient oocytes, PIF is located in the cortical layers of the donor oocytes and is a powerful inhibitor of protein synthesis (4, 12). Maturation is a very exceptional event in amphibian oocytes which have not been treated with steroid hormones; among thousands of cytologically analyzed oocytes, we found only three sporadic cases of germinal vesicle breakdown in control oocytes after treatment with respectively, x-rays, hydroxyurea, and a-amanitin (4, 14). It was, therefore, a great surprise when one of us (A. De Sc.-P.) found, in the course of experiments where the effects of inhibitors of -SH groups on progesterone-induced maturation were studied, that three organomercurials induced germinal vesicle breakdown in a high percentage of control oocytes. They are p-chloromercuribenzoate or more correctly p-hydroxymercuribenzoate (PHMB), p-chloromercuribenzenesulfonate or more correctly p-hydroxymercuriphenylsulfonate (PHMPS) and, as found in two preliminary experiments, mersalyl (Salyrgan, o-{ [3-(hydroxymercuri)-2-methoxypropyl]carbamoyl} phenoxyacetic acid sodium salt. The purpose of this communication is to present the results of a preliminary analysis of this unexpected finding. MATERIAL AND METHODS Females of X. laevis were obtained from a dealer (South African Snake Farm, Fish Hoek, Cape Town) or from our own colony. Full grown (stage 6) oocytes were isolated by manual dissection or by collagenase (Worthington,.1%) digestion for4or 12 hr. PHMPS was used in the majority of the experiments because of its greater solubility. The organomercurials were dissolved in Ringer's or in Wallace's medium (15). This - medium which is buffered at ph 7. with 4. mm of Tris HCl, is greatly superior to Ringer's solution so far as survival of organomercurial-treated eggs is concerned. Living oocytes were observed under a dissecting microscope; a number of oocytes were fixed with Zenker for cyto- 1574

2 TABLE 1. Percentages of maturation in combined treatments with progesterone (Pg) and PHMPS \ O~~~~~~ P9 Agg/ml g/ml Al/m, Agml PHMPS X 11M M X 1^M M M 1 logical analysis (12) or with glutaraldehyde for electron microscopy (13). Several methods (2, 3) have been used in order to remove the follicle cells: collagenase digestion for 12 hr or more, Pronase digestion (5,ug/ml for 5 min), treatment with Ca++ free Wallace's medium containing 1 mm EDTA. At the end of the treatment, the remnants of the follicular epithelium were removed with watchmaker's forceps and the oocytes were thoroughly washed in Wallace's medium. Preparation and micro-injection of MPF and PIF have already been described (4, 12). Oocytes were activated, after maturation, by pricking or by treatment with the Ca-ionophore A (16, 17). For measurements of protein synthesis, samples of eight oocytes were treated, in quadruplicate, for 1 hr with 1,uCi/ml of [3H ]phenylalanine; this radioactive pulse was given at various time intervals after treatment with PHMPS or progesterone. After extensive washings in Wallace's medium O the oocytes were homogenized in 1 ml of.15 M NaCl. An aliquot of the homogenate served for measurement of total radioactivity; the rest was precipitated with 5% trichloroacetic acid, washed twice with 5% trichloroacetic acid containing.1% cold phenylalanine, dissolved in Soluene, and counted in an Omnifluor-toluene mixture. In other experiments, 5 nl of [8H]- phenylalanine (1 mci/ml) were injected into three groups of four oocytes which had been treated, for various lengths of time, with progesterone or PHMPS; the injected oocytes were processed as described above. Binding of [14C]PHMB or [2'OHg]PHMB to whole oocytes was studied as follows: groups of five isolated oocytes were incubated, during various lengths of time, in.1 mm [23Hg]- PHMB (NEN, 76.1 mci/g) or [14C]PHMB (ICN, 5 MCi in 1.67 mg), washed quickly twice in Wallace's medium, dissolved in 1 ml of Soluene 35, and counted in the presence of Omnifluor-toluene. RESULTS 1. Biological observations (a) Concentration of Organomercurials, Kinetics. In all experiments (more than 2) a high percentage (between 2 and 1%) of maturations was obtained. The highest percentages were obtained with oocytes which also responded well to progesterone stimulation. Best results were obtained with.3 and.1 mm PHMPS and with.1 mm PHMB. Treatment of the oocytes with the organomercurials, in contrast to progesterone, must be long: it should last at least 4-5 hr. We are now using routinely continuous treatments with.1 mm PHMB or PHMPS in Wallace's medium. After about 4 hr of treatment, a large grey or yellowish spot becomes visible at the animal pole: it corresponds to the 2 v tx A.,'.-. W *, * z,, _; x -,- Organomercurials Induce Maturation in Oocytes 1575 _,;.; ; Xr b a+; D-!_ * it ':,;... f..: '-.. * Me,,,,, \ x *.s,^n-*4>.,'' '^ *Xs, so :,.*/., A:> me r <S B.S., s SC r*; ^ i; ;^. * d > * o -?. _ Or._ i w 4,, FIG. 1. The germinal vesicle remains intact 15 hr after injection of 1 mm PHMPS (5 nl) into an oocyte. (Unna: 9X). swollen, but still intact germinal vesicle. Its breakdown is an extremely fast process, which takes place within a few minutes (usually after 6-7 hr of treatment with the organomercurials). A few hours later, part of the oocytes present a maturation spot like progesterone treated oocytes; others display a mottled pigmentation at the animal pole. As in the case of progesterone, there is a marked variability in the response of oocytes taken from different females. There is a general parallelism in the response of the oocytes towards organomercurials and progesterone (percentage of maturations, speed of germinal vesicle breakdown). (b) Effect of Follicle Cells Withdrawal. The organomercurials might act by releasing progesterone present in follicle cells. It was thus important to establish whether they would still be active on oocytes deprived of their follicle cells. We found that the various treatments described in Material and Methods have no effect on PHMB or PHMPS induced maturation, whereas, they markedly decrease or suppress the response of the oocytes to pituitary hormone (1 crushed Xenopus pituitary in 2 ml of Wallace's medium ). Furthermore, 1% of the oocytes which had not reacted to pituitary extract after 15 hr underwent maturation if they were then treated with.1 mm PHMPS. We conclude that the presence of the follicle cells is not required for organomercurial induced maturation and that PHMB or PHMPS must act directly on the oocyte. (c) Injection of PHMPS. In four different experiments, 5 nl of PHMPS (1 mm, 5 mm, and 1 mm) were injected into large Xenopus oocytes; no case of maturation and only one of pseudomaturation were observed among 52 oocytes which were analyzed cytologically (Fig. 1). It can be concluded that, like progesterone, PHMPS must act on the cell membrane and that it is inactive by injection. (d) Combined Effects of PHMPS and Progesterone. Pretreatment for 1 hr with.1 mm PHMPS or injection of.2 /Ag of PHMPS does not prevent progesterone-induced maturation. In another experiment, oocytes have been treated with various mixtures of PHMPS and progesterone; as shown in Table 1, low concentrations of PHMPS and progesterone exert synergistic effects on maturation. (e) Requirement for Ca++, Effects of Cycloheximide and Actinomycin D. Progesterone-induced maturation requires the s

3 ~~~~~~~~~~ 1576 Cell Biology: Brachet et al. IL 4,wi' 1.~~~ //e's<#~~~.'.'.t 4.4 V ineto o nolm(5n)frma; ~. oyewihhdudr FIG. 2. Normal maturation spindle in an oocyte (15 hr) after injection of endoplasm (5 nl) from an oocyte which had undergone PHMPS-induced maturation. (Feulgen: 12X). presence of Ca++ in the medium (1, 18); it is inhibited by cycloheximide, but not by actinomycin D (4, 11). Likewise, PHMB and PHMPS no longer induce maturation in Wallace's medium without Ca++, or in the presence of cycloheximide (1,ug/ml), whereas, actinomycin D (1,ug/ml) has no effect. (f) Effects of Other -SH Reagents. In contrast to PHMB and PHMPS, iodoacetamide (.1 mm), HgCl2, (from.1 to.1 mm), N-ethylmaleimide (.1 and 1 mm) and dithio- &.a- '!uv.:''2a~~~~~~~~~~~- ~ nitrosobenzene (1 and 2 mm) do not induce maturation in X. laevis oocytes. Addition to PHMPS (.1 mm) of.1 mm cysteine, mercaptoethanol, or dithiothreitol completely prevents germinal vesicle breakdown. (g) Production of MPF and PIF. Injection of endoplasm taken from oocytes which have undergone maturation by PHMPS treatment regularly induces germinal vesicle breakdown in injected recipient oocytes (Fig. 2), provided that the latter are kept under ph 8.. Pseudomaturation can be found as an exception among such injected oocytes; it is probably due to the fact that the PHMPS-treated oocytes become very soft and that separation of the endoplasm from the cortex is, therefore, difficult. Injection of a homogenate from PHMPStreated oocytes induced 1% pseudomaturation while an in vitro treatment of the PIF-containing homogenate with.1 mm PHMPS did not inhibit its pseudomaturation activity. (h) Artificial Activation. Pricking or treatment with the Caionophore A induce activation in oocytes which have undergone PHMPS-induced maturation (two experiments). Activation (as indicated by the rotation of the oocytes in their envelopes) is much faster after A than after pricking. The ionophore produces a quick and profound contraction of the pigmented half of the egg. The activated oocytes did not divide and soon underwent cytolysis. 2. Cytological observations Only two cases of incipient germinal vesicle breakdown have been seen among 48 cytologically studied oocytes which had undergone maturation after organomercurial treatment. The rupture of the nuclear envelope in the oocytes of Fig. 3 is more "explosive" than in progesterone-treated oocytes; it breaks down simultaneously in several places. The disappearance of the nucleoli, the condensation of the chromosomes, the formation of the spindle and its migration towards the animal pole are all unusually fast processes. As a result of this acceleration, polar bodies are often already expulsed 7 hr after the beginning of organomercurial treatment (Fig. 4).. The structure and ultrastructure of the oocytes which showed a normal maturation spot are identical with those of the progesterone controls (19). However, the oocytes which displayed mottled pigmentation at the animal pole have an abnormal structure; the pigment penetrates deeply towards the center of the oocyte, the basophilic material is irregularly CAL -.% # op. a *a r O' 1 ti :WL~~~~~~...4.,v t. A.. ~ ~ ~. -Jr - t. I * *,. W I FIG. 3. Germinal vesicle breakdown after a 4 hr treatment with 1 mm PHMPS in Wallace's medium. (Unna: 84 X). FIG. 4. Expulsion of first polar body after 6 hr of treatment with 1 mm PHMPS in Wallace's medium. (Feulgen: 84X).

4 t / w x. >, Organomercurials Induce Maturation in Oocytes ,* s,. S s. Rr '.,... eb, ;*r2xx 't ; - *,{. *>^< w < rx >.C IN-C IO.- I.. 1: " '. O 'I 5 x 4 E 3.,'/.,. PHMPS _- -~, Progesterone FIG. 5. Activation, with production of asters and nuclear multiplication after 17 hr of treatment with 1 mm PHMPS in Wallace's medium. (Feulgen: 84X). scattered and tends to form cytasters. In one case, an oocyte had spontaneously undergone activation and contained several nuclei and asters (Fig. 5). 3. Preliminary biochemical observations As shown in Table 2, there is an apparent decrease in protein synthesis when [3Hjphenylalanine is added to PHMPStreated oocytes; but this decrease is entirely due to a dramatic inhibition of amino-acid uptake (94% after 6-7 hr, Table 2). If, as was done in two experiments, [3H phenylalanine is injected into PHMPS-treated oocytes, a marked stimulation of protein synthesis can be observed (Fig. 6). TABLE 2. Incorporation of [3H]phenylalanine (1 jusi/ml) into oocytes treated continuously with progesterone (1 gsg/ml) or PCMBS (1-4M) in Wallace's medium, ph 7. Trichloroacetic Time of pulse Incorporation acid-soluble after beginning cpm/8 % of cpm/8 % of of experiment oocytes controls oocytes controls Control -1 hr Progesterone PHMPS Control 1-2 hr Progesterone PHMPS Control 2-3 hr Progesterone PHMPS Control 4-5 hr Progesterone PHMPS Control 6-7 hr Progesterone PHMPS Control hr Progesterone PHMPS The oocytes were incubated for 1 hr in [3Hiphenylalanine at various times after the beginning of PHMPS treatment. Each value is the mean of four measurements on eight oocytes. 2 1' -_ (hours) 7 - *Control FIG. 6. Incorporation of [3H]phenylalanine into oocytes treated continuously with progesterone (1 Ag/ml) or PHMPS (1-4 M) in Wallace's medium, ph 7.. Each oocyte was injected with 5 nl of [3H]phenylalanine (1 mci/ml) and the incorporation into the trichloroacetic acid-insoluble fraction was estimated 3 min after the injection. Each point is the mean value for three batches of eight oocytes. The binding of radioactive PHMB to intact oocytes has been studied in preliminary experiments: it is linear during 3-4 hr; after a plateau, an increase in radioactivity occurs at the approximate time of germinal vesicle breakdown (Fig. 7). Between 4 and 7% of the radioactivity is removed if the labeled oocytes are washed and treated for 1 hr with 5 mm cysteine. DISCUSSION It is still too early to propose a concrete explanation for the unexpected induction of maturation by organomercurials x E GVBD (hours) FIG. 7. Binding of [23Hg]PHMB (.1 mm, 76.1 mci/g) to groups of five oocytes. Average of three different experiments. GVBD, germinal vesicle breakdown.

5 1578 Cell Biology: Brachet et al. An interaction with the cell membrane is strongly suggested by the following facts: maturation is not induced when the organomercurials are injected into the oocytes; amino acid uptake is very strongly inhibited; -SH reagents which penetrate more easily in the cells do not induce maturation. On the other hand, the necessity for a long treatment with organomercurials suggests that they may have to penetrate the oocytes in order to induce maturation or that they must react with sluggish -SH groups of the membrane proteins. The linear portion of the [14C]PHMB binding curve (Fig. 7) corresponds to the length of time generally required for the induction of maturation. There are many similarities between progesterone- and organomercurial-induced maturation: both progesterone and organomercurials are inactive in injection; in both cases, follicle cells can be removed without affecting the results; the presence of Ca++ and protein synthesis, but not of RNA synthesis, are required. In both cases, the oocytes can respond to activating stimuli and undergo a stimulation of protein synthesis. Most important of all, probably, the organomercurials induce, like progesterone, the formation of MPF. The main difference between the two systems lies in their kinetics; a much longer treatment is needed in the case of organomercurials than in that of progesterone. But, after germinal vesicle breakdown, expulsion of the first polar body is often speeded up in organomercurial-treated oocytes. PHMB and PHMPS are, as expected, more toxic than progesterone as shown by a larger proportion of oocytes with a "mottled" pigmentation and by fast cytolysis after activation. The possibility that PHMB and PHMPS act by releasing progesterone present in the follicle cells should not be dismissed lightly, since organomercurials have been shown to be powerful insulinogogues in the pancreas (2). All of our results plead against this hypothesis, but cytological proof that the follicle cells have been adequately removed is still missing. We do not know how general a phenomenon induction by organomercurials might be; in two experiments, we have obtained germinal vesicle breakdown in Ambystoma mexicanum (axolotl) oocytes after continuous treatment with.1 mm PHMPS. The situation is probably different in starfish oocytes, where PH1MB inhibits 1-methyladenine-induced maturation whereas dithiols induce it (21). Despite our present ignorance of the mechanisms by which maturation is induced by organomercurials, these substances provide an entirely new tool, for analyzing, at the molecular level (in particular MPF production), a fundamental biological process. E. B., Maitre de Recherches du Fonds National de la Recherche scientifique. 1. Smith, L. D. & Ecker, R. E. (197) Curr. Top. Develop. Biol. 5, Schuetz, A. W. (1972) in "Oogenesis," eds. Biggers, J. D. & Schuetz, A. W. (Univ. Park Press, Baltimore, Md.), pp Schuetz, A. W. (1974) Biol. Reprod. 1, Brachet, J., Baltus, E., Hanocq, F., Hanocq-Quertier, J., Hubert, E., Iacobelli, S. & Steinert, G. (1974) Mol. Cell. Biochem. 3, Iacobelli, S., Hanocq, J., Baltus, E. & Brachet, J. (1974) Differentiation 2, Ozon, R. & Bell6, R. (1973) Biochim. Biophys. Ada -33, Smith, L. D. & Ecker, R. E. (1971) Develop. Biol. 25, Masui, Y. & Markert, C. L. (1971) J. Exp. Zool. 177, Schorderet-Slatkine, S. (1971) Cell Differentiation 1, Schorderet-Slatkine, S. & Drury, K. C. (1973) Cell Differenti ation 2, Reynhout, J. K. & Smith, L. D. (1974) Develop. Biol. 38, Baltus, E., Brachet, J., Hanocq-Quertier, J. & Hubert, E. 1973) Differentiation 1, Steinert, G., Baltus, E., Hanocq-Quertier, J. & Brachet, J. (1974) J. Ultrastruct. Res. 49, Hanocq, F., De Schutter, A., Hubert, E. & Brachet, J. (1974) Differentiation 2, Wallace, R. A., Jared, D. W., Dumont, J. N. & Sega, M. W. (1973) J. Exp. Zool. 184, Steinhardt, R. A., Epel, D., Carroll, E. J. & Yanagimachi, R. (1974) Nature 252, Hanocq, J., Baltus, E. & Steinert, G. (1974) C.R.H. Acad. Sci Merriam, R. W. (1971) Exp. Cell Res. 68, Brachet, J., Van Gansen, P. & Hanocq, F. (197) Develop. Biol. 21, Hellman, B., Lernmark, A., Sehlin, J., Loderberg, M. & Taljedal, I. B. (1973)-Arch. Biochem. Biophys. 158, Kinoshito, T. & Kanatani, H. (1973) Exp. Cell Res. 83,