J. C ell Set. aa, 531-545 (1976) 531

Printed in Great Britain

MEIOTIC MATURATION OF MOUSE OOCYTES

IN VITRO: INHIBITION OF MATURATION

AT SPECIFIC STAGES OF NUCLEAR

PROGRESSION

P. M. WASSARMAN*, W. J. JOSEFOWICZAND G. E. LETOURNEAUThe Department of Biological Chemistry and Laboratory of Human Reproductionand Reproductive Biology, Harvard Medical School, Boston, Massachusetts 02115,U.S.A.

SUMMARYIn vitro studies of meiotic maturation of mouse oocytes have been carried out in the presence

of several drugs. The individual steps of nuclear progression, including dissolution of thenuclear (germinal vesicle) membrane, condensation of dictyate chromatin into compact biva-lents, formation of the first metaphase spindle, and extrusion of the first polar body, are eachsusceptible to one or more of these drugs.

Germinal vesicle breakdown, the initial morphological feature characteristic of meioticmaturation, is inhibited by dibutyryl cyclic AMP. However, even in the presence of dibutyrylcyclic AMP, the nuclear membrane becomes extremely convoluted and condensation ofchromatin is initiated but aborts at a stage short of compact bivalents. Germinal vesicle break-down and chromatin condensation take place in an apparently normal manner in the presenceof puromycin, Colcemid, or cytochalasin B. Nuclear progression is blocked at the circularbivalent stage when oocytes are cultured continuously in the presence of puromycin or Colce-mid, whereas oocytes cultured in the presence of cytochalasin B proceed to the first meioticmetaphase, form an apparently normal spindle, and arrest. Emission of a polar body is inhibitedby all of these drugs. The inhibitory effects of these drugs on meiotic maturation are reversibleto varying degrees dependent upon the duration of exposure to the drug and upon the nature ofthe drug.

These studies suggest that dissolution of the mouse oocyte's germinal vesicle and condensa-tion of chromatin are not dependent upon concomitant protein synthesis or upon microtubules.On the other hand, the complete condensation of chromatin into compact bivalents apparentlyrequires breakdown of the germinal vesicle. Failure of homologous chromosomes to separateafter normal alignment on the meiotic spindle in the presence of cytochalasin B suggest thatmicrofilaments may be involved in nuclear progression at this stage of maturation. Cytokinesis,in the form of polar body formation, is blocked when any one of the earlier events of maturationfails to take place.

INTRODUCTION

During the process of oogenesis, oocytes of many animal species undergo meioticarrest prior to the completion of chromosomal reduction and it is in this state that theyundergo tremendous growth. The length of time that oocytes remain in this arrested

• To whom enquiries and reprint requests should be directed.35-2

532 P. M. Wassarman, W. J. Josefowicz and G. E. Letourneau

state and the nature of the stimulus which reinitiates meiosis are species-dependent(Baker, 1972a; Schuetz, 1974; Smith, 1975).

In the mouse, nearly all oocytes have arrested at the diplotene (dictyate) stage ofprophase of the first meiotic division by 5 days post partum and they remain in dictyateuntil just prior to ovulation, a period extending from several weeks to more than ayear. The resumption of meiosis can be mediated by a hormonal stimulus in vivo(Baker, 19726) or by the release of oocytes from their ovarian follicles into a suitableculture medium in vitro (Biggers, Whittingham & Donahue, 1967; Donahue, 1968;Sorensen, 1973; Wassarman & Letourneau, 1976a). The oocytes undergo nuclearprogression from dictyate to metaphase II and remain at this stage of meiosis in theoviduct, or in culture, until fertilization or parthenogenetic activation takes place. Theperiod of time during which meiosis progresses from dictyate to metaphase II istermed the period of 'meiotic maturation'. Mouse oocytes matured and fertilized invitro have produced viable foetuses following transplantation to the uteri of fostermothers (Cross & Brinster, 1970).

During the period of meiotic maturation, mouse oocytes undergo a series of welldefined morphological and physiological changes in preparation for fertilization andembryogenesis. This process which takes approximately 12 h to complete in vitro, ischaracterized by dissolution of the nuclear (germinal vesicle) membrane, condensa-tion of diffuse dictyate chromatin into distinct bivalents, separation of homologouschromosomes and emission of the first polar body, and arrest of meiotic progressionat metaphase II.

We have examined the effects of several drugs on meiotic maturation of mouseoocytes in vitro in an attempt to block reversibly the process at discrete stages prior tothe second meiotic metaphase. It is the purpose of this paper to provide a descriptiveaccount of meiotic maturation of mouse oocytes in vitro in the presence of dibutyrylcyclic AMP, puromycin, Colcemid, or cytochalasin B.

MATERIALS AND METHODSOocytes were obtained by puncturing ovaries from adult (8—12 weeks of age) female Swiss

mice (CD-i, Charles River Laboratories) (Donahue, 1968). Oocytes containing an intact germi-nal vesicle and free of cumulus cells were harvested and washed in culture medium (Biggers,1971). Cell culture was carried out in plastic dishes (Falcon) in 50- or 100-//.I drops of mediumunder paraffin oil at 37 °C in an atmosphere of 5 % COS in air. Stock solutions of dibutyrylcyclic AMP (N', O*'-dibutyryl adenosine 3',s'-cyclic monophosphate, dbcAMP) (Sigma),puromycin (Sigma), and Colcemid (Calbiochem) were stored frozen. Cytochalasin B (CCB)(Calbiochem) was made up in dimethylsulphoxide and stored frozen.

Air-dried chromosome spreads were prepared essentially by Tarkowski's (1966) procedure.Chromosomes were stained with Giemsa (Harleco no. 620) at a 1 :5o dilution with o-i Mphosphate buffer, pH 6-8, for 30 min. Light microscopy was performed on isolated, unfixedoocytes and on fixed preparations using a Zeiss Photomicroscope II equipped with Nomarskidifferential-interference optics.

For electron microscopy, oocytes were fixed in 3 % glutaraldehyde in o-1 M cocodylatebuffer, pH 7-4, for 1 h at 4 °C and then postfixed in 1 % osmium tetroxide in the same bufferfor 1 h at 4 °C. The oocytes were rinsed in buffer, transferred to 30 % ethanol for 5 min,stained with 3 % uranyl acetate (in 30% ethanol) for 15 min, and dehydrated through a series

Inhibition of meiotic maturation 533of ethanols. The specimens were then infiltrated with epoxy propane and embedded in Epon812. Thin sections were stained with methanolic uranyl acetate (Watson, 1958) followed bylead citrate (Reynolds, 1963) and were examined in a Philips 200 electron microscope.

RESULTS

Meiotic maturation in vitro

Meiotic maturation takes place spontaneously when mouse oocytes are releasedfrom their ovarian follicles into a suitable culture medium. This process, which takesapproximately 12 h to complete in vitro, is characterized by dissolution of the nuclear

Fig. 1 A-E. Photomicrographs of mouse oocytes during culture in vitro and of Giemsa-stained chromosome spreads from mouse oocytes during condensation of dictyatechromatin into compact bivalents in vitro. Shown are oocytes in dictyate and meta-phase II of meiosis (A and B, respectively) (Nomarski differential-interference optics)and oocyte chromosomes at various stages of condensation (C-E). A, B, X 225 approx.;c-E, x 1400 approx.

(germinal vesicle, GV) membrane, condensation of diffuse dictyate chromatin intodistinct bivalents, separation of the homologous chromosomes and emission of thefirst polar body, and arrest of meiotic progression at metaphase II. Some of theseevents are shown in Fig. 1. Under the experimental conditions used in this study,approximately 85 % of the oocytes placed in culture underwent GV breakdown within4 h and, of these, approximately 80% subsequently emitted polar bodies (Fig. 2).

534 P. M. Wassarman, W. jf. Josefowicz and G. E. Letourneau

Effect of dibutyryl cyclic AMP on maturation in vitro

Dibutyryl cyclic AMP, at a concentration of ioo /tg/ml, is an effective inhibitor ofGV breakdown in mouse oocytes. Less than 5 % of the oocytes placed in cultureresume meiosis, as evidenced by the retention of an intact GV, even after 16 h (Fig. 2).The inhibitory effect of dbcAMP is reversible although the degree of reversibility

100r

toQ_

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O

Medium dbcAMP Puromycin Colcemid CCB

Fig. 2. Analysis of the effects of several drugs on meiotic maturation of mouse oocytesin vitro. Ocytes were cultured for 16 h in medium alone or in medium containingdbcAMP (100 //g/ml), puromycin (10 fig/m\), Colcemid (10 //g/ml), or CCB (5 /tg/ml)and the percentages of oocytes which retained a GV (closed bar), underwent GVBD(open bar), or emitted a polar body (PB, cross-hatched bar) were determined at the endof the culture period. The data shown for each treatment represent the average of aminimum of 3 replicates, with a total of 100 or more oocytes.

decreases over extended periods of culture (Fig. 3). Light-microscopic examinationof Giemsa-stained chromosome spreads prepared from mouse oocytes cultured in thepresence of dbcAMP shows that chromosome condensation is initiated in theseoocytes, but aborts prior to the formation of compact bivalents. The stage at whichchromosome condensation aborts is seen transiently during nuclear progression ofoocytes cultured in medium without the drug (Fig. 4). Furthermore, electron-micro-scopic examination of fixed and sectioned oocytes cultured in the presence of dbcAMPshowed that the nuclear envelope becomes very convoluted, which is usually thefirst morphological indication of the onset of meiotic maturation, but still retainsmany nuclear pores (Fig. 5).

Inhibition of meiotic maturation 535

100 r

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40

20

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Fig. 3. Analysis of the degree of reversibility of the inhibition of meiotic maturationof mouse oocytes in vitro by several drugs. Oocytes were cultured for 5 h in thepresence of dbcAMP (100 /ig/ml), puromycin (10 fig/ml), Colcemid (10 /ig/ml), orCCB (5 /ig/ml), B-E respectively, and were then transferred to medium containing thesame drug (closed bar) or to medium alone (open bar) for an additional 12 h of cul-ture. The percentage of polar body formation was determined at the end of the cul-ture period and compared with oocytes cultured continuously in medium alone (A).The data shown for each treatment represent the average of a minimum of 2 replicates,with a total of 60 or more oocytes.

Effect of puromycin on maturation in vitro

Puromycin, at a concentration of 10 /tg/ml, is an effective inhibitor of meioticmaturation of mouse oocytes (Fig. 2) and dramatically reduces protein synthesis inthese oocytes (Fig. 6). However, GV breakdown and chromosome condensation takeplace in a normal manner, despite the effect on protein synthesis, with meiotic arrestoccurring at the circular bivalent stage (Fig. 7). The inhibitory effect of puromycin iscompletely reversible, even after exposure of oocytes to the drug for 5 h in culture(Fig- 3)-

Effect of Colcemid on maturation in vitro

Colcemid, at a concentration of 10 /ig/ml, a dose sufficient to prevent formation of ametaphase I spindle, inhibits meiotic maturation at the circular bivalent stage (Fig. 2).As with puromycin, GV breakdown and chromosome condensation take place in anormal manner (Fig. 7) and the inhibitory effect is reversible (Fig. 3).

536 P. M. Wassarman, W. J. Josefowicz and G. E. Letoumeau

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Inhibition of meiotic maturation 537

Effect of cytochalasin B on maturation in vitro

Eighty per cent or more of mouse oocytes cultured in the presence of CCB (5 /<g/ml)resume meiosis, undergo nuclear progression to metaphase I with the formation of aspindle, and then arrest without emitting the first polar body (Fig. 2). Light- andelectron-microscopic examination of CCB-treated eggs shows that meiosis aborts at astage which is seen transiently with oocytes cultured in medium without the drug(Fig. 8). The metaphase I spindle appears to be quite normal in configuration and iscompletely surrounded by a dense area composed of mitochondria, vacuoles andgranules.

DISCUSSION

The process of meiotic maturation of mouse oocytes consists of a series of well de-fined morphological changes, beginning with GV breakdown and chromosome con-densation and ending with alignment of chromosomes on the second metaphasespindle (Donahue, 1968; Sorensen, 1973). However, the question of the regulation ofmammalian oocyte maturation is quite perplexing in so far as oocytes released fromlarge ovarian follicles undergo meiotic maturation 'spontaneously' when cultured invitro in a suitable medium. This may be compared with in vitro oocyte maturation inthe starfish and in the frog which requires the interaction of a diffusible molecule(i-methyladenine and progesterone, respectively), probably produced by the folliclecells, with the oocyte's plasmalemma (Smith & Ecker, 1969; Schuetz, 1974; Smith,1975; Kanatani, 1975). Recent studies in our laboratory have shown that, in thejuvenile mouse (up to 21 days post partum), there is a direct relationship between thesize of isolated oocytes and their ability to undergo meiotic maturation in vitro (Soren-sen & Wassarman, 1976). These results suggest that the ability to resume meiosis('meiotic competence') is acquired at a specific stage of oocyte growth in the juvenilemouse and that, perhaps, the acquisition of meiotic competence is associated with achange in the oocyte's biochemical capabilities. It is in this context that we have begunto study the sequence of events which constitutes meiotic maturation in the mam-malian oocyte.

The results reported here have confirmed and extended previous observations onthe effect of dbcAMP on meiotic maturation of mouse oocytes in vitro (Stern &Wassarman, 1973, 1974; Cho, Stern & Biggers, 1974; Wassarman & Turner, 1976).The fact that dbcAMP reversibly inhibits the onset of nuclear progression in isolatedmouse oocytes may be related to the proposed regulatory role for cAMP during themitotic cell cycle (Burger, Bombik, Breckenridge & Sheppard, 1972; Willingham,Johnson & Pastan, 1972). In this regard it has been shown that elevated cAMP levelsinhibit cell multiplication (Johnson & Pastan, 1972; Bombik & Burger, 1973), that

Fig. 4A-C. Giemsa-8tained chromosome spreads from mouse oocytes cultured invitro. Chromosome spreads were prepared from oocytes immediately after isolation(A), after 1 h of culture in medium alone (B), or after 16 h of culture in medium con-taining dbcAMP (ioo /ig/ml) (c). x 830 approx.

P. M. Wassarman, W. J. Josefowicz and G. E. Letourneau

Inhibition of meiotic maturation 539

ioor

80

S 60&•o

20

I

0 1 2 3 4Time, h

Fig. 6. The effect of puromycin on protein synthesis in mouse oocytes during culturein vitro. Incorporation of PH]valine into TCA-insoluble material was examined in thepresence (O—O) and absence ( • — • ) of io fig/ml puromycin as previously de-scribed (Stem & Wassarman, 1974). The data points shown represent the mean valuesof 3 replicates.

the concentration of cAMP is lower in fast-growing than in slow-growing cells (Shep-pard, 1971), that lowered cAMP levels occur during G1 and mitosis relative to S-phase (Willingham et al. 1972; Burger et al. 1972) and that exogenously supplieddbcAMP results in termination of the cell cycle in G1-phase (Zimmerman & Raska,1972; Willingham et al. 1972; Kaukel, Fuhrmann & Hilz, 1972). It is tempting tosuggest that fluctuations in cAMP levels may play an analogous role in the mammalianoocyte, with elevated intracellular levels of cAMP maintaining mouse oocytes in dic-tyate of the first meiotic prophase from several weeks to more than a year prior toundergoing ovulation or atresia. Since cAMP is believed to influence cellular pro-cesses by activation of protein kinase(s) (Bitensky & Gorman, 1973) it is of interest tonote that the appearance of a new cAMP-dependent protein kinase has been detectedfollowing progesterone-induced meiotic maturation of amphibian oocytes (Wiblet,1974). On the other hand, Smith (1975) has reported that neither cAMP, its dibutyrylanalogue, nor theophylline, either singly or in combination, mimicked the effects ofprogesterone on amphibian oocytes, regardless of whether they were injected into theoocytes or were present in the culture medium. Although the molecular basis of theinhibitory effect of dbcAMP on meiotic maturation of mouse oocytes is not clear at

Fig. 5 A, B. Electron micrographs illustrating the state of the GV membrane follow-ing oocyte culture in the presense of dbcAMP. Shown are thin sections of oocytesfixed immediately after isolation (A) and after 16 h of culture in the presence ofdbcAMP (100 /*g/ml) (B). X 5000 approx.

540 P. M. Wassarman, W. J. Josefowicz and G. E. Letourneau

present, it appears likely that it interferes primarily with one or more of the eventsleading to GV dissolution, e.g. activation of (a) specific enzyme (s) involved in de-grading the GV membrane. Our observations indicate that the nuclear membranebecomes convoluted and that chromosome condensation is initiated in oocytes evenin the presence of dbcAMP; however, condensation aborts prematurely. This pheno-menon is probably a direct consequence of the failure of the GV to break down. It hasbeen reported previously that much of the chromatin condensation during meiotic

7A B.

Fig. 7 A—C. Giemsa-8tained chromosome squashes prepared from mouse oocytescultured in vitro in the presence or absence of drugs. Shown are chromosomes fromoocytes cultured for 16 h in the presence of puromycin (10 /tg/ml) (A) or Colcemid(10 fig/m\) (B) and for 6 h in medium alone (c).

maturation of mouse oocytes takes place along the nuclear envelope, suggesting thatthe chromosomes are actually attached to it (Calarco, Donahue & Szollosi, 1972;Szollosi, Calarco & Donahue, 1972; Calarco, 1972). Accordingly, it may be inferredfrom these observations that nuclear progression, in the form of chromosome conden-sation, is initiated in the presence of dbcAMP but is not completed due to the main-tenance of an intact nuclear envelope.

It is well documented that protein synthesis is required for the successful comple-tion of meiotic maturation of oocytes from a variety of species, including echino-derms (Brachet & Steinert, 1967; Zampetti-Bosseler, Huez & Brachet, 1973; Houk &Epel, 1974), amphibians (Dettlaff, Nikitina & Stroeva, 1964; Dettlaff, 1966; Brachet,1967; Schuetz, 1967; Smith & Ecker, 1969; Merriam, 1972; Brachet et al. 1974), andmammals (Jagiello, 1968; Stern, Rayyis & Kennedy, 1972; Wassarman & Letourneau,1976). Extensive in vitro studies carried out by Smith & Ecker (1969), using oocytesisolated from Rana pipiens, show that there are changes in both the rate of proteinsynthesis and the nature of proteins synthesized during progesterone-induced meioticmaturation (see also Smith & Ecker, 1969; Smith, 1975). Furthermore, in the

Inhibition of meiotic maturation

Fig. 8A-C. Light and electron, micrographs of mouse oocytes cultured in vitro in thepresence of CCB. Shown are photomicrographs of mouse oocytes cultured for 16 hin the presence of CCB (5 /tg/ml) using Nomarski differential-interference optics(A, B) and an electron micrograph of a thin section from an oocyte cultured under thesame conditions (c). A, X 550; B, X I IOO; C, X 5500, approx.

amphibian oocyte, both GV breakdown and the appearance of' maturation-promotingfactor' during meiotic maturation are dependent upon continued protein synthesis(Schuetz, 1967; Smith & Ecker, 1969; Wasserman & Masui, 1976). In this connexion,Ziegler & Masui (1973) have shown that puromycin blocks chromosome condensa-tion in brain nuclei injected into amphibian oocytes arrested at metaphase I. On theother hand, GV breakdown and chromosome condensation during maturation of themouse oocyte do not appear to be dependent upon concomitant protein synthesis (or

542 P- M. Wassarman, W. J. Josefowicz and G. E. Letourneau

RNA synthesis; Wassarman & Letourneau, 19766 and unpublished results). Thepresence of puromycin (or cycloheximide) has no effect on the rate of resumption ofmeiosis, only on the extent of maturation, since nuclear progression stops at the circularbivalent stage. These results suggest that, unlike amphibian oocytes, the fully-grownmouse oocyte is poised to resume meiosis, such that no new proteins need be synthesizedfor GV breakdown and chromosome condensation to take place spontaneously in vitro.Previous studies have shown that dbcAMP has no effect on the apparent rate ofprotein synthesis or on the spectrum of proteins synthesized by the mouse oocyteduring culture in vitro (Stern & Wassarman, 1973, 1974). In this context, it should bepointed out that Lindner et al. (1974) have reported that de novo protein synthesis isrequired in isolated rat follicles for the initiation of oocyte maturation during the first2 h of LH action. It is not clear whether the protein(s) essential for LH-inducedmeiosis in follicle-enclosed oocytes is synthesized by the oocyte itself or by some otherfollicular component. These results can be reconciled with those presented in thisreport if it is assumed that protein synthesis is required for an event which is obviatedby physical removal of the oocyte from its surrounding follicle.

dbcAMP Puromycin

Colcemid

Dictyate Circularbivalent

CCB

Metaphaie Metaphrase II

Fig. 9. Schematic representation of the inhibitory effects of several drugs on meioticmaturation of mouse oocytes in vitro.

The first morphological indication of incipient oocyte maturation appears to be theincreased undulatory activity of the nuclear envelope which precedes GV breakdown(Sorensen, 1973). During this period a number of microtubules are located around theoocyte's GV and some actually project perpendicularly toward the nuclear envelopeas it becomes convoluted (Szollosi et al. 1972). Since in some instances, folding of thenuclear envelope during mitosis has been directly attributed to impinging micro-tubules (Bajer & Mole-Bajer, 1969) it was of interest to find that GV dissolution andchromosome condensation took place in a normal manner in the presence of Colcemid,a drug which disaggregates spindle microtubules, at concentrations sufficient toprevent spindle formation. These observations suggest that microtubules do not playan essential role in the initial phases of meiotic maturation of mouse oocytes.

Cytochalasin B, a fungal alkaloid first described by Carter (1967), has many diverseeffects on cultured cells, ranging from inhibition of morphogenetic and movementprocesses to interference with hormone-mediated cellular responses (Carter, 1972).In addition, CCB induces a variety of cultured mammalian cells to segregate the cell'snucleus into an evagination of the plasma membrane which occasionally breaks awayfrom the cell body (Poste, 1973). CCB was orginally thought to interact specificallywith contractile components of the cell, affecting various aspects of cellular motility;

Inhibition of meiotic maturation 543

however, this interpretation, has been the subject of some controversy (Estensen,Rosenberg & Sheridan, 1971; Forer, Emmersen & Behnke, 1972; Pollard & Weihing,1974). The results of several studies of cytokinesis in maiine, amphibian and mam-malian eggs indicate that CCB interferes with normal cleavage; however, while incertain instances it can be correlated with disruption or disorganization of arrays ofmicrofilaments associated with the contractile ring, in others it cannot (Schroeder,1970, 1972; Bluemink, 1971a, b; Snow, 1973). In a study of the effects of CCB onmorphogenetic events in the fertilized egg from the surf clam, Spisula solidissima,Longo (1972) found that the drug prevented the formation of a polar body; this wasattributed to the failure of a cleavage furrow to form in the presence of CCB. On theother hand, CCB did not interfere with migration of the metaphase spindle from itscentral position to the cell's cortex. In the experiments reported here, CCB appearedto prevent not only polar body formation, but migration of the meiotic spindle of themouse oocyte to a position in the cell's cortex as well. These results suggest that thecytoskeletal apparatus of the mouse oocyte is involved in movement of the spindlefrom its central position to the cortex.

The effects of dbcAMP, puromycin, Colcemid, and CCB on meiotic maturationof mouse oocytes in vitro are summarized in Fig. 9. The results presented here haveshed light on the molecular basis of meiotic maturation in the mammal and indicatethat these reversible inhibitors will prove useful in further biochemical studies of thisprocess.

This research was supported by grants awarded to P.M.W. by The National Institute ofChild Health and Human Development (HD-06916) and The National Science Foundation(GB-43528).

A preliminary account of some of this work was reported at the Annual Meeting of the TissueCulture Association as part of a Symposium on Differentiation of The Haploid Cell, Montreal,Canada, June 1975 (Wassarman, 1974).

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(Received 18 May 1976)36 CEL 22