J. Cell Sci. 30, 251-264 (1978) 251Printed in Great Britain © Company of Biologists Limited igjS

MEIOTIC MATURATION OF MOUSE OOCYTES

IN VITRO: PROTEIN SYNTHESIS IN NUCLEATE

AND ANUCLEATE OOCYTE FRAGMENTS

RICHARD M. SCHULTZ, GAIL E. LETOURNEAUAND PAUL M. WASSARMAN*Department of Biological Chemistry and Laboratory of Human Reproduction andReproductive Biology, Harvard Medical School, 45 Shattnck Street, Boston,Massachusetts 02115, U.S.A.

SUMMARY

Nucleate and anucleate fragments of mouse oocytes have been isolated following treatmentof fully grown, oocytes with cytochalasin B. The nucleate oocyte fragments resume meiosisin vitro, progressing from dictyate of the first meiotic prophase to metaphase II ('meioticmaturation'), and exhibit all of the changes in protein synthesis normally associated withmeiotic maturation of mouse oocytes. The anucleate oocyte fragments also undergo certainof the changes in protein synthesis associated with meiotic maturation, despite the absence ofnuclear progression. These results suggest that the acquisition of meiotic competence (i.e.the ability to undergo meiotic maturation) during growth of the mammalian oocyte is due tochanges in the quality, rather than the quantity, of cytoplasm and that the reprogrammingof protein synthesis during meiotic maturation is directed by RNA templates already presentin the cytoplasm. The behaviour of anucleate oocyte fragments is discussed in terms of theproposed role for nucleoplasm in the initiation of changes in protein synthesis during meioticmaturation of mouse oocytes.

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 thatthey undergo tremendous growth. The length of time that oocytes remain in thisarrested 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') stageof prophase of the first meiotic division by 5 days post partum and they remain indictyate until just prior to ovulation, a period extending from several weeks to morethan a year. The resumption of meiosis can be mediated by a hormonal stimulusin vivo (Baker, 19726) or by the release of oocytes from their ovarian follicles intoa suitable culture medium (Biggers, Whittingham & Donahue, 1967; Donahue, 1968;Sorensen, 1973; Wassarman & Letourneau, 1976). 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.The period of time during which meiosis progresses from dictyate to metaphase II

* To whom enquiries and reprint requests should be directed.

252 R. M. Schultz, G. E. Letourneau and P. M. Wassannan

is termed the period of ' meiotic maturation.' The process of meiotic maturation ischaracterized by dissolution of the nuclear (germinal vesicle) membrane, condensationof diffuse dictyate chromatin into distinct bivalents, separation of homologouschromosomes and emission of the first polar body, and arrest at metaphase II. Mouseoocytes matured and fertilized in vitro have developed into viable foetuses followingtransplantation to the uteri of foster mothers (Cross & Brinster, 1970).

Meiotic maturation of mouse oocytes in vitro is accompanied by marked changesin the pattern of proteins synthesized by the oocyte, with virtually all of the changesoccurring after the breakdown of the germinal vesicle (Schultz & Wassarman, 1977a,b). This reprogramming does not take place in oocytes which fail to undergo germinalvesicle breakdown spontaneously or in oocytes arrested at the germinal vesicle stageby dibutyryl cyclic AMP. These and other observations led us to suggest that it isnucleoplasmic factors released into the oocyte's cytoplasm which trigger the changesin protein synthesis associated with meiotic maturation. Although the nature of thenucleoplasmic factors is not known, it is likely that they originate from the cytoplasmand are concentrated and stored in the germinal vesicle. In this connexion, it is welldocumented that the germinal vesicle serves as a reservoir in which specific proteinsand other macromolecules are sequestered during oogenesis (see Discussion).Apparently, as long as these factors reside within the germinal vesicle the changesin protein synthesis associated with the resumption of meiosis do not occur.

Our model for the reprogramming of protein synthesis during meiotic maturationpredicts that physically enucleated oocytes should manifest this reprogramming ifthe factors accumulate to critical levels in the cytoplasm. In this report, we describethe results of experiments that are consistent with our model. These experimentsemployed high-resolution 2-dimensional electrophoretic analysis of proteins syn-thesized in nucleate and anucleate fragments of fully grown mouse oocytes.

We have shown previously that oocytes isolated from mice younger than 15 daysof age fail to undergo spontaneous meiotic maturation in vitro, whereas oocytesisolated from mice 15 days of age or older resume meiosis at a frequency whichincreases with the age of the donor mice (Sorensen & Wassarman, 1976; Schultz& Wassarman, 1977a). Since the average mean diameter of the isolated oocytes in-creased linearly with the age of the donor mice we suggested that the ability toresume meiosis ('meiotic competence') was probably achieved at a specific stage ofoocyte growth. In this report, we have determined the ability of nucleate fragmentsderived from fully grown oocytes to undergo meiotic maturation. The latter is ofparticular interest with respect to the control of meiosis, since some of the nucleatefragments are smaller than meiotically incompetent oocytes isolated from juvenilemice.

MATERIALS AND METHODS

Oocyte collection and culture

Fully grown oocytes were obtained from adult (8-12 weeks of age), randomly bred, femaleSwiss albino mice (CD-i, Charles River Laboratories) by puncturing ovaries with fine steelneedles under a dissecting microscope, essentially as described by Donahue (1968) and Rafferty

Meiotic maturation of mouse oocytes 253

(1970). Oocytes containing an intact GV and free of cumulus cells were harvested using amouth-operated micropipette and washed in culture medium (Biggers, 1971) containing100 /<g/ml Ar°,O2'-dibutyryl adenosine 3,5'-cyclic monophosphate (Bt2cAMP, Sigma) (Stern& Wassarman, 1974). Cell culture was carried out in either plastic dishes (Falcon) or embryo-logical watch glasses in 50-250 /tl of medium under paraffin oil at 37 °C in a humidifiedatmosphere of 5 % CO2 in air.

Growing oocytes were obtained from juvenile Swiss mice, i.e. less than 21 days of age,using essentially the method described by Mangia & Epstein (1975). Ovaries dissected fromanimals killed by cervical dislocation were first washed thoroughly with culture mediumcontaining Bt2cAMP (100 /tg/ml) and then incubated at 37 °C for 15-20 min in medium con-taining Bt2cAMP (100 /tg/ml) and 0-5 mg/ml of testicular hyaluronidase (type V, Sigma), 100units of collagenase (type III, Sigma), and 05 mg/ml of egg white lysozyme (grade I, Sigma).After incubation, the ovaries were gently punctured using fine steel needles under a dissectingmicroscope and oocytes containing an intact GV and free of cumulus cells were harvestedusing a mouth-operated micropipette. The oocytes harvested from each ovary were transferredimmediately to fresh medium containing Bt2cAMP (100/tg/ml) and were washed thoroughlyto eliminate further contact with the enzyme mixture.

Air-dried chromosome spreads were prepared essentially by Tarkowski's procedure (1966).Chromosomes were stained with Giemsa (Harleco No. 620) at a 1 : 50 dilution with o-i Mphosphate buffer, pH 68, for 30 min. Light microscopy was performed on isolated, unfixedoocytes and on fixed preparations using a Zeiss Photomicroscope II equipped with Nomarskidifferential-interference optics.

Preparation of nucleate and anucleate fragments of fully grown oocytes

Mouse oocytes are induced by cytochalasin B (CCB) in vitro to divide into 2 separable com-partments. This process is called ' pseudocleavage' and is dependent upon the presence of anintact GV and zona pellucida (a glycoprotein envelope surrounding the oocyte) (Wassarman,Albertini, Josefowicz & Letourneau, 1976a; Wassarman, Josefowicz & Letourneau, 19766;Wassarman et al. 1977). The compartments resulting from pseudocleavage may be mechanicallyseparated from one another and cultured in vitro. We have taken advantage of this phenomenonto prepare nucleate and anucleate fragments of fully grown mouse oocytes. A schematicrepresentation of the method used to obtain these fragments is presented in Fig. 1.

Oocytes were collected from adult mice as described above. They were cultured in thepresence of Bt2cAMP (100 /tg/ml), which reversibly inhibits GV breakdown (Stern & Wassar-man, 1973; Cho, Stern & Biggers, 1974; Wassarman & Turner, 1976; Wassarman et al.19766) and CCB (5/tg/ml) for 3 h. The oocytes were then transferred as a group to freshmedium containing Pronase (0-5 mg/ml), CCB (5 /fg/ml), and Bt2cAMP (100/tg/ml) in orderto remove the zona pellucida. As soon as the zona pellucida could no longer be visualizedmicroscopically (approximately 10 min at room temperature) the denuded oocytes weretransferred into medium containing Bt2cAMP and CCB, as above. These transfers wereperformed with drawn-out micropipettes having a bore size significantly larger than the dia-meter of the oocytes; this procedure resulted in minimal loss of pseudocleaved oocytes. After3 h the population of pseudocleaved oocytes was heterogeneous with respect to compartmentsize (Fig. 1). The separation of oocyte compartments from one another was accomplished bygentle pipetting using a mouth-operated micropipette possessing a bore size slightly smallerthan the diameter of a compartment. Each pseudocleaved oocyte was processed individuallyso as to account for both the nucleate and anucleate compartment. It was found that nucleatefragments were significantly more fragile than anucleate fragments. Nucleate and anucleatefragments were then cultured separately in plain medium.

Two -dimensional gel electrophoresis

Oocyte proteins labelled with [35S]methionine were prepared for electrophoresis in thefollowing manner. Oocytes cultured in the presence of [35S]methionine (200 /tCi/ml, NewEngland Nuclear) were washed thoroughly and harvested in 2 /tl of medium. Eight microlitres

17 CEL 30

254 R. M. Schultz, G. E. Letourneau and P. M. Wassarman

of o-oi M Tris-HCl, pH 7-4, containing 50/tg/ml RNase (Sigma) were added and the oocyteswere frozen and thawed 3 times and incubated on ice for 5-10 min. The solution was thenbrought to 95 M in urea, 17 /tl of 'lysis buffer' (O'Farrell, 1975) were added, a 2-//1 aliquotof the solution was assayed by liquid scintillation counting, and the remainder was subjectedto isoelectric focusing and discontinuous SDS gel electrophoresis as described by O'Farrell(I97S)- Gels were processed for fluorography according to the procedure described by Bonner& Laskey (1974).

BtocAMP+CCBPronase

icropipette

Fig. 1. Schematic representation of the preparation of nucleate and anucleate frag-ments of fully grown mouse oocytes. Details of the procedure are presented in theResults. Photomicrographs of fully grown oocytes and pseudocleaved oocytes weretaken using Nomarski differential-interference optics.

RESULTS

Meiotic maturation in mouse oocytes in vitro

Meiotic maturation takes place spontaneously when oocytes from adult mice(3 weeks of age or older) are released from their ovarian follicles into a suitable culturemedium (Fig. 2). This process takes approximately 15 h to complete in vitro. Thetime sequence of meiotic maturation in vitro can be approximated as follows: germinalvesicle (GV) breakdown takes place within 1-5 h, metaphase I is reached in 5-10 h

Meiotic maturation of mouse oocytes a,jj

and metaphase II is reached in 10-16 h. Under the experimental conditions used inthis study, approximately 80% of the oocytes placed in culture underwent GVbreakdown within 3 h and, of these, approximately 70% subsequently emitted firstpolar bodies.

Fig. 2. Photomicrographs of fully grown mouse oocytes (A) and nucleate mouseoocyte fragments (B) which have undergone meiotic maturation in vitro. Lightmicroscopy was performed on unfixed preparations using Nomarski differential-interference optics. The oocytes and oocyte fragments are arrested in metaphaseII of meiosis (confirmed by stained chromosome spreads). The position of the meta-phase II spindle apparatus is visible in several of the oocytes and oocyte fragments(arrows). It was noted that the polar bodies associated with oocyte fragments tendto be smaller than those associated with oocytes. pb, polar body; zp, zona pellucida.

Meiotic maturation of nucleate oocyte fragments

As the mouse oocyte grows in vivo the volume of the GV increases from approxi-mately 0-9 pi in the primordial oocyte (20 /tra diameter) to approximately 8-2 pi in thefully grown oocyte (80 /6m). Concomitantly, the volume of cytoplasm increases fromapproximately 3-3 pi to about 250 pi. Therefore, the growth of the oocyte results ina dramatic change in the ratio of cytoplasm to nucleoplasm, increasing from 3 :1 inthe primordial oocyte to 35 : 1 in the fully grown oocyte. The acquisition of meioticcompetence (i.e. the ability to undergo meiotic maturation) is apparently achieved ata particular stage of oocyte growth (Sorensen & Wassarman, 1976; Schultz & Wassar-man, 1977a). These observations suggest that the induction of meiotic maturationcould be controlled, not by a change in the composition of the cytoplasm duringgrowth, but rather by the amount of cytoplasm relative to nucleoplasm. In order totest this possibility, we have used the nucleate fragments isolated from fully grownoocytes which were smaller in diameter than meiotically incompetent oocytes isolatedfrom juvenile mice.

The nucleate fragments were isolated as described above and their diameters(exclusive of zona pellucida) were rneasured with an ocular micrometer attached to an

17-2

256 R. M. Schultz, G. E. Letourneau and P. M. Wassarman

inverted microscope. The fragments were then cultured overnight and scored micro-scopically for GV breakdown and polar body emission. The results of these experi-ments are presented in Table 1 and representative fragments are shown in Fig. 2.It is clear that the nucleate fragments are capable of resuming meiosis at a relativelyhigh frequency and in most cases proceed to metaphase II. Furthermore, the time-course of meiotic maturation is similar to that observed with untreated fully grown

Table 1. Meiotic maturation of mouse oocytes andnucleate fragments of mouse oocytes*

Sample % GV breakdown % Polar bodies

Fully grown oocytes (>8o/tm)f >8s >6oGrowing oocytes (< 60 fim) < 5 oNucleate fragments (< 60 fim) > 50 > 40* In each case 50 or more oocytes or fragments were examined,f Oocyte or fragment diameter in parentheses.

oocytes. The relationship between diameter and meiotic competence is shown inFig. 3 for both nucleate fragments derived from fully grown oocytes and oocytesisolated from juvenile mice. It is apparent that, while juvenile oocytes smaller thanapproximately 60 /tm in diameter fail to resume meiosis in vitro, nucleate fragmentsas small as 40 /«m undergo meiotic maturation; the latter fragments possess less than10% of the cytoplasmic volume of a fully grown oocyte. In fact, the cytoplasmicto nuclear volume ratio for fragments 40 /tm in diameter is approximately 3 : 1 ,similar to that for primordial oocytes. Therefore the initiation of meiotic maturationis apparently not controlled by a change in the ratio of cytoplasm to nucleoplasm.

Protein synthesis in nucleate and anucleate oocyte fragments

Two-dimensional gel electrophoresis and fluorography were carried out on[35S]methionine-labelled proteins from (i) fully grown oocytes with a GV, (ii) fullygrown oocytes which had undergone GV breakdown, (iii) nucleate fragments whichhad undergone GV breakdown, and (iv) anucleate fragments. Regions taken fromfluorograms of these gels are shown in Fig. 4B. It is clear that those changes in proteinsynthesis which take place during meiotic maturation of fully grown oocytes alsooccur during meiotic maturation of nucleate fragments. Furthermore, many of thechanges in protein synthesis also occur in the anucleate fragments in the absenceof nuclear progression., Previous observations led us to suggest that the oocyte's GV contains factorswhich initiate changes in the pattern of protein synthesis during meiotic maturation(Schultz & Wassarman, 1977a, b). Consequently, the results obtained with anucleatefragments could be attributed to nucleoplasmic contamination. We feel that thispossibility is unlikely, however, since for each anucleate fragment used in theseexperiments a corresponding nucleate fragment was isolated from the same oocyte. Inaddition, the results of the following 2 experiments, designed to detect drug-inducedleakage of nucleoplasmic material during preparation of the fragments, proved

Meiotic maturation of mouse oocytes 257

negative, (i) Fluorograms of [35S]methionine-labelled proteins from oocytes culturedovernight in the presence of Bt2cAMP (ioo/tg/ml), to prevent GV breakdown,showed none of the changes in protein synthesis characteristic of meiotic maturation.However, all of the changes in protein synthesis were observed when GV breakdownwas permitted to take place prior to culture of the oocytes overnight in the presence ofBt2cAMP and [35S]methionine. (ii) Fluorograms of [35S]methionine-labelled proteins

GVBD

GV

GVBD

GV

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1 4- i*1

30 40 50 60 70 80Diameter, /<m

Fig. 3. Meiotic maturation of growing mouse oocytes (A) and nucleate fragments (B)of fully grown mouse oocytes in vitro. Oocytes at various stages of growth wereisolated from juvenile mice'as described in Experimental procedures. Nucleatefragments were obtained from fully grown oocytes isolated from adult mice asdescribed in Results. Diameters were measured with an ocular micrometer attachedto an inverted microscope. Oocytes and fragments were cultured separately over-night and scored for GV breakdown microscopically. Each point, scored as GVor GV breakdown,(GVBD), represents a single oocyte or oocyte fragments of a

' given diameter.

from oocytes cultured 3 h in the presence of Bt2cAMP (100 /<g/ml) and CCB (5 /tg/ml),to induce pseudocleavage, and then overnight in medium containing Bt2cAMP(ioo/tg/ml) and [35S]methionine, showed none of the changes in protein synthesischaracteristic of meiotic maturation.

DISCUSSION

During each reproductive cycle, a small fraction of the population of primordialoocytes in the mouse ovary grow in volume nearly 70-fold. We have shown previouslythat the ability to resume meiosis (meiotic competence) is acquired at a specific stageof oocyte growth in the juvenile mouse; oocytes smaller than approximately 60 /tmin diameter fail to resume meiosis in vitro (Sorensen & Wassarman, 1976). The data

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Meiotic maturation of mouse oocytes

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260 R. M. Schultz, G. E. Letourneau and P. M. Wassarman

reported here for nucleate oocyte fragments 60 /.im. or less in diameter suggest that achange in the quality of oocyte cytoplasm, rather than the quantity of cytoplasm, isresponsible for the acquisition of meiotic competence during oocyte growth. In thisconnexion, we found that oocyte growth is accompanied by significant qualitativechanges in the types of proteins synthesized (Schultz & Wassarman, 1977ft; Schultz& Wassarman, unpublished results). Since protein synthesis is not required for thereinitiation of meiosis (i.e. GV dissolution and chromosome condensation) of mouseoocytes in vitro (Wassarman et al. 19766; Schultz & Wassarman, 1977a), it seemslikely that those proteins involved in the early events of meiotic maturation aresynthesized during growth of the oocyte. It is tempting to suggest that the acquisitionof meiotic competence is related to the onset of synthesis of these proteins at a specificstage of oocyte growth.

It is well documented that protein synthesis is required for the successful completionof meiotic maturation (i.e. nuclear progression to metaphase II) of oocytes from avariety of species, including echinoderms, amphibians, and mammals. Extensivein vitro studies carried out by Smith & Ecker (1969), and Ecker & Smith (1971) usingoocytes isolated from Rana pipiens, have shown that there are changes in both therate of protein synthesis and the nature of the proteins synthesized during progesterone-induced meiotic maturation. Furthermore, these investigators provided strongevidenceto support the contention that, in the amphibian oocyte, the morphological eventsassociated with meiotic maturation are under cytoplasmic, not nuclear, control. Inorder to test the hypothesis that the programme of protein synthesis which functionsduring meiotic maturation is directed by RNA templates already present in theoocyte's cytoplasm, Smith & Ecker compared the rate of protein synthesis and thenature of the proteins synthesized in nucleated oocytes, with enucleated oocytes,after exposure to progesterone in vitro. They found that those qualitative and quanti-

Fig. 4. High resolution 2-dimensional gel electrophoresis of [36S]methionine-labelledfully grown mouse oocytes or fragments of fully grown mouse oocytes. The oocytesand fragments were collected, labelled with [34S]methionine for approximately 20 h,and processed for electrophoresis as described in Experimental procedures. Thesamples, contained in approximately 20 fi\, were focused on 5 % polyacrylamide gelscontaining 2% pH 3-10 Ampholines for 16 h at 400 V and finally for 1 h at 800 V.The focusing gels were equilibrated with SDS sample buffer (O'Farrell, 1975) for 30min and subjected to SDS-polyacrylamide gel electrophoresis at 20 mA per gel atroom temperature. The SDS separating gel was 12-5 cm long and 15 mm thick; thestacking gel was 2-5 cm long. The separating gel was a continuous 9-15 % acrylamidegradient (exponential) and the total volume of the gel was 28 ml. The gels weredeveloped by fluorography (Bonner & Laskey, 1974). A, Fluorogramme of 2-dimen-sional electrophoretic analysis of oocytes which had undergone GV breakdownduring culture. Regions of the fluorogrammes designated I, II, and III are indicated.B, regions I, II, and III from fluorogrammes of 2-dimensional electrophoreticanalyses of: (i) oocytes which retained a GV during culture in the presence ofBt2cAMP (100 fig/m\) (5 x io5 cpm/gel); (ii) oocytes which underwent meiotic matura-tion to metaphase II during culture in plain medium (5 x io5 cpm/gel); (iii) nucleateoocyte fragments which underwent GV breakdown during culture in plain medium(6 x 1 o4 cpm/gel); and (iv) anucleate oocyte fragments cultured in plain medium(6 x io4 cpm/gel). The gels were exposed for a length of time calculated empiricallyaccording to the formula: 27 x ios/cpm = h of exposure.

Meiotic maturation of mouse oocytes 261

tative changes in protein synthesis which normally take place in the amphibianoocyte following stimulation with progesterone, also take place in enucleated oocytes.Based on the results of their experiments these investigators concluded that themixing of the amphibian oocyte's nucleoplasm and cytoplasm is not essential for thesuccessful completion of meiotic maturation or, for that matter, for the activationof the programme of protein synthesis which accompanies nuclear progression.

In this report, we have shown that many of the changes in protein synthesis norm-ally associated with meiotic maturation of mouse oocytes also occur in anucleateoocyte fragments in the absence of nuclear progression. This result is very reminiscentof that obtained with progesterone-treated enucleated amphibian oocytes (see above)and strongly suggests that the reprogramming of protein synthesis during meioticmaturation of mouse oocytes is directed by RNA templates already present in thecytoplasm. Experiments carried out with mouse oocytes cultured in the presence ofinhibitors of RNA synthesis, such as a-amanitin, actinomycin D, or ethidiumbromide, lead to a similar conclusion, but are certainly less reliable due to the probableside-effects of these drugs (Jagiello, 1968; Golbus & Stein, 1976; Wassarman &Letourneau, unpublished results). The results obtained with anucleate oocytefragments also confirm our previous conclusion that virtually all of the changes inprotein synthesis which take place during meiotic maturation are not dependentupon the occurrence of such morphological events as spindle formation and/or polarbody emission (Schultz & Wassarman, 1977a, b).

We have shown previously that the failure of the mouse oocyte's GV to breakdown, either in medium alone or in the presence of Bt2cAMP, prevents all of thechanges in protein synthesis which normally accompany meiotic maturation (Schultz& Wassarman, 1977a, b). Accordingly, we suggested that the initiation of thosechanges in protein synthesis which characterize meiotic maturation of mammalianoocytes is dependent upon the mixing of the oocyte's nucleoplasm and cytoplasm.Such a proposal is in apparent contradiction with the results of experiments carriedout with enucleated amphibian oocytes (Smith & Ecker, 1969; Ecker & Smith, 1971)and with anucleate mouse oocyte fragments, as reported here. However, we feel thatthese results are consistent with our model for the reprogramming of protein synthesisduring meiotic maturation if one assumes that the nucleoplasmic factors responsiblefor the changes in protein synthesis are derived from the cytoplasm and stored in theGV. As long as these factors accumulate within the GV and not in the cytoplasm,the changes in protein synthesis associated with the resumption of meiosis do notoccur. The absence of a GV, in either physically enucleated oocytes or anucleateoocyte fragments, could permit the accumulation of these factors to critical levelsin the cytoplasm. In this connexion, it is well documented that the GV serves as areservoir in which specific proteins and other macromolecules are sequestered duringoogenesis. For example, the intracellular distribution of proteins synthesized duringprogesterone-stimulated meiotic maturation of Rana pipiens oocytes was examinedby Ecker & Smith (1971). Autoradiographs of sections of oocytes which had beeninjected with [3H]leucine revealed an accumulation of grains within the GV whichincreased with time; 1 h after injection the GV contained about twice as many

262 R. M. Schultz, G. E. Letourneau and P. M. Wassarman

grains per unit volume as did the cytoplasm and by 20 h the ratio increased to aboutsix. Similarly, Wassarman & Letourneau (1976) found a 3- to 4-fold concentration ofgrains, on a per unit volume basis, over the GV of mouse oocytes during a 3-hlabelling period with [3H]lysine. There is also evidence to suggest that only certaintypes of proteins are concentrated in the GV. For example, Gurdon (1970) foundthat iodinated histones, but not iodinated BSA, were rapidly accumulated in the GVafter injection into amphibian oocytes. Twenty-four hours after injection, histoneswere at least 100 times more concentrated in the GV than in the cytoplasm, whereas,iodinated BSA was less concentrated in the GV than in the cytoplasm of the oocyte.It was calculated that the GV of injected oocytes contained at least 500 times theirnormal content of histones. More recent work by Bonner (1975 a, b) has shown thatthe pronounced accumulation of histones in the GV of amphibian oocytes is notseen with other proteins of comparable size and isoelectric points, such as lysozyme,strongly suggesting a high degree of specificity for the proteins derived from thecytoplasm and concentrated within the GV. In this connexion, a variety of biologicalactivities and factors have been identified in the oocyte's GV, many of which arerequired for normal cleavage and early development (Smith, 1975), and recent analysesof isolated GVs indicate the great diversity of proteins present (Merriam & Hill,1976). It seems probable that, when the GV breaks down at the beginning of meioticmaturation, nucleoplasmic proteins mix with cytoplasm in either an active orpotentially active state and play a role in the latter stages of meiotic maturation andin early embryogenesis.

In view of the evidence discussed above, we believe that the results obtained withprogesterone-stimulated enucleated amphibian oocytes and with anucleate mouseoocyte fragments are consistent with a role for nucleoplasm in the initiation of changesin protein synthesis during meiotic maturation. We suggest that these changes inprotein synthesis take place in the absence of a GV due to the accumulation of factorswhich would normally be sequestered in the oocyte's GV until the onset of meioticmaturation.

We are grateful to the members of our laboratory for willing assistance and constructivecriticism during this research. The research was supported by grants awarded to P. M. W.by The National Institute of Child Health, and Human Development and The NationalScience Foundation. R.M.S. is a Postdoctoral Fellow of The Rockefeller Foundation.

REFERENCES

BAKER, T. G. (1972a). Oogenesis and ovulation. In Reproduction in Mammals, vol. 1, GermCells and Fertilization (ed. C. R. Austin & R. V. Short), pp. 14-45. Cambridge UniversityPress.

BAKER, T. G. (1972ft). Oogenesis and ovarian development. In Reproductive Biology (ed. H.Balin & S. Glasser), pp. 389-437. Amsterdam: Excerpta medica.

BIGGERS, J. D. (1971). New observations on the nutrition of the mammalian oocyte and thepreimplantation embryo. In The Biology of Blastocyst (ed. R. J. Blandau), pp. 319-332.University of Chicago Press.

BIGGERS, J. D., WHITTINGHAM, D. G. & DONAHUE, R. P. (1967). The pattern of energymetabolism in the mouse oocyte and zygote. Proc. natn. Acad. Sci. U.S.A. 58, 560-567.

Meiotic maturation of mouse oocytes 263

BONNER, W. M. & LASKEY, R. A. (1974). A film detection method for tritium-labelled proteinsand nucleic acids in polyacrylamide gels. Eur.J. Biochem. 46, 83-88.

BONNER, W. M. (1975 a). Protein migration into nuclei. I. Frog oocyte nuclei in vivo accumulatemicroinjected histones, allow entry to small proteins, and exclude large proteins. J. CellBiol. 64, 421-430.

BONNER, W. M. (19756). Protein migration into nuclei. II . Frog oocyte nuclei accumulate aclass of microinjected oocyte nuclear proteins and exclude a class of microinjected oocytecytoplasmic proteins. X Cell Biol. 64, 434-437.

CHO, W. K., STERN, S. & BIGGERS, S. D. (1974). Inhibitory effect of dibutyryl cAMP onmouse oocyte maturation in vitro. J. exp. Zool. 187, 383-386.

CROSS, P. C. & BRINSTER, R. L. (1970). In vitro development of mouse oocytes. Biol. Reprod.3, 298-307.

DONAHUE, R. P. (1968). Maturation of the mouse egg in vitro. I. Sequence and timing ofnuclear progression. J. exp. Zool. 169, 237-250.

ECKER, R. E. & SMITH, L. D. (1971). The nature and fate of Rana pipiens proteins synthesizedduring maturation and early cleavage. Devi Biol. 24, 559-576.

GOLBUS, M. S. & STEIN, M. P. (1976). Qualitative patterns of protein synthesis in the mouseoocyte. J. exp. Zool. 198, 337-342.

GURDON, J. B. (1970). Nuclear transplantation and control of gene activity in animal develop-ment. Proc. R. Soc. B 176, 303-314.

JAGIELLO, G. M. (1968). Meiosis and inhibition of ovulation in mouse eggs treated with actino-mycin D. J. Cell Biol. 42, 571-574.

MANGIA, F. & EPSTEIN, C. J. (1975). Biochemical studies of growing mouse oocytes: pre-paration of oocytes and analysis of glucose-6-phosphate dehydrogenase and lactate dehydro-genase activities. Devi Biol. 45, 211—220.

MERRIAM, R. W. & HILL, R. J. (1976). The germinal vesicle nucleus of Xenopus laevis oocytesas a selective storage receptacle for proteins. J. Cell Biol. 69, 659—668.

O'FARRELL, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. J. biol.Chem. 250, 4007-4021.

RAFFERTY, K. A. (1970). Methods in Experimental Embryology of the Mouse. Baltimore: TheJohns Hopkins Press.

SCHUETZ, A. W. (1974). Role of hormones in oocyte maturation. Biol. Reprod. 10, 150-178.SCHULTZ, R. M. & WASSARMAN, P. M. (1977a). Biochemical studies of mammalian oogenesis:

Protein synthesis during oocyte growth and meiotic maturation in the mouse, jf. Cell Sci.24, 167-194.

SCHULTZ, R. M. & WASSARMAN, P. M. (19776). Specific changes in the pattern of proteinsynthesis during meiotic maturation of mammalian oocytes in vitro. Proc. natn. Acad. Sci.U.S.A. 74, 538-541.

SMITH, L. D. (1975). Molecular events during oocyte maturation. In The Biochemistry ofAnimal Development (ed. R. Weber), pp. 1-46. New York: Academic Press.

SMITH, L. D. & ECKER, R. E. (1969). Role of oocyte nucleus in physiological maturation inRana pipiens. Devi Biol. 19, 281-309.

SORENSEN, R. A. (1973). Cinemicrography of mouse oocyte maturation utilizing Nomarskidifferential-interference microscopy. Am. J. Anat. 136, 265-276.

SORENSEN, R. A. & WASSARMAN, P. M. (1976). Relationship between growth and meioticmaturation of the mouse oocyte. Devi Biol. 50, 531—536.

STERN, S. & WASSARMAN, P. M. (1974). Protein synthesis during meiotic maturation of themammalian oocyte.^. Cell Biol. 59, 335a.

TARKOWSKI, A. K. (1966). An air-drying method for chromosome preparation from mouseeggs. Cytogenetics 5, 394-400.

WASSARMAN, P. M., ALBERTINI, D. A., JOSEFOWICZ, W. J. & LETOURNEAU, G. E. (1976a).Cytochalasin B-induced pseudo-cleavage of mouse oocytes in vitro: asymmetric localizationof mitochondria and microvilli associated with a stage-specific response. J. Cell Sci. 31,523-535-

WASSARMAN, P. M., JOSEFOWICZ, W. J. & LETOURNEAU, G. E. (19766). Meiotic maturation ofmouse oocytes in vitro: inhibition of maturation at specific stages of nuclear progression.J. Cell Sci. 22, 531-545-

264 R. M. Schultz, G. E. Letourneau and P. M. Wassartnan

WASSARMAN, P. M. & LETOURNEAU, G. E. (1976). Meiotic maturation of mouse oocytes htvitro: association of newly synthesized proteins with condensing chromosomes.^. Cell Sci.20, S49-568.

WASSARMAN, P. M. & TURNER, P. E. (1976). Effect of dithiothreitol on meiotic maturation ofmouse oocytes in vitro: dependence of the effect on iV°,O2-dibutyryl adenosine 3',s'-cyclicmonophosphate. J. exp. Zool. 196, 183-187.

WASSARMAN, P. M., UKENA, T. E., JOSEFOWICZ, W. J., LETOURNEAU, G. E. & KARNOVSKY,M. F. (1977). Cytochalasin B-induced pseudocleavage of mouse oocytes invitro II. Studies ofthe mechanism and morphological consequences of pseudocleavage. J. Cell Sci. 26, 323-337.

(Received 15 March 1977)