Proc. Natl. Acad. Sci. USAVol. 75, No. 11, pp. 5534-5538, November 1978Cell Biology

Long-term growth and differentiation of Xenopus oocytes in adefined medium

(oocyte growth/oocyte maturation/insulin/vitellogenin)

ROBIN A. WALLACE AND ZIVA MISULOVINBiology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830

Communicated by W. L. Russell, August 28,1978

ABSTRACT Xenopus laevis oocytes over a size range of0.15-0.78 mm3 were dissected from their follicles and culturedin a defined medium for up to 28 days. Oocytes grew at averagerates of 0.021 mm3-day-l in the absence of insulin and 0.030mm3 day-l in the presence of insulin. The latter average growthrate corresponds to the fastest growth rate reported to date foroocytes in vivo. Oocytes grown in vitro can reach a size of atleast 1.43 mm3, which is larger than the maximum size generallyfound in vivo. During growth in vitro, oocytes also acquire botha normal pigment pattern and, once they reach about 0.7 mm3,the ability to undergo complete maturation as a response toexternally applied progesterone. These results show that Xen-opus oocytes freed of their follicular investments are able togrow and differentiate in vitro.

The growing oocyte from any animal has traditionally been themost difficult cell type to sustain in a physiologically normalcondition both in vivo and in vitro. In nonmammalian verte-brates, experimental or environmental perturbation of femalescarrying vitellogenic oocytes has frequently led to atresia of theoocytes (1). Likewise, early attempts to develop organ cultureprocedures for amphibian ovaries, although successful for mostcell types present in the ovary, failed to maintain yolk-con-taining oocytes, which were the first and sometimes only celltype to degenerate in vitro (2, 3).More recently, several researchers have at least been able to

maintain Xenopus laevis oocytes in vitro for extended periods.Stage IV/V [all oocyte stages are those described by Dumont(4)] and Stage VI oocytes were cultured either within theirfollicles (5) or after removal of investing follicular layers (6, 7);several physiological criteria indicated that the oocytes remainrelatively healthy for 2-4 weeks. However, no growth was re-ported for such oocytes.Numerous studies have established that oocytes of non-

mammalian vertebrates grow primarily by sequestering theyolk-protein precursor, vitellogenin, from the maternalbloodstream (8). Normally, vitellogenin enters the capillarynetwork within the theca of the follicle and from there hasaccess to the oocyte (9, 10); it cannot, however, penetrate theouter surface epithelium of the follicle (11). These consider-ations have led us to conclude that X. laevis oocytes can begrown in vitro only when they are removed from their follicularinvestments and are placed in a nutritionally adequate andosmotically appropriate medium containing vitellogenin. Wehave therefore developed a culture procedure for growing suchoocytes based on this premise (12). We report here on thegrowth and differentiation of X. laetvs oocytes individuallycultured in the defined medium we have developed.

MATERIALS AND METHODSOocyte Culture. Females stimulated with human chorionic

gonadotropin (hCG) were given 1000 international units (IU)of hCG 2-3 days prior to laparotomy; unstimulated femalesreceived no hCG. A part of the ovary was removed from eachfemale and placed in solution O-R2(13). Qocytes within anarrow size range (10.05-mm diameter) were manually dis-sected from their follicles and individually placed in 50-100,gl of medium containing 50% Liebovitz L-15 medium, 5 mgof vitellogenin per ml, 1 mM L-glutamine, 15 mM Hepes-NaOH buffer, and the antibiotics gentamycin (100 ,ug-ml-1)and nystatin (50 units ml-1). The final pH of the medium was7.8. Crystalline porcine insulin (1 Ag-ml-m; 25 units -mg-1; lot615-063-10 kindly provided by Ronald Chance, Eli Lilly Re-search Laboratories) was also included unless specified other-wise. Oocytes were then cultured in a dark, humidifiedchamber at 20'C. Complete details including the isolation ofvitellogenin are specified elsewhere (12).Other Procedures. Oocyte diameters were measured with

an ocular micrometer in at least two directions (at 900) in casethey were not strictly spherical. Very large oocytes tended toflatten somewhat; in such cases three diameters were measuredby appropriate propping and averaged. The oocyte volumesindicated throughout are based on these diameter measure-ments. [3H]Vitellogenin incorporation was measured as de-scribed elsewhere (12). We determined protein by individuallydissolving oocytes in a sodium dodecyl sulfate/diothioerythritolsolution* and placing the protein solution (plus washings) onindividual 2.3-cm-diameter discs punched out from Whatman42 paper. Discs were processed with ice-cold 10% trichloroaceticacid, alcohol/ether (3:1), and ether (14), and the processed discswere used for protein determination (15). For a protein stan-dard, yolk platelets were isolated (16) from X. laevis oocytes,washed in distilled H20, and extracted with alcohol/ether (3:1)and ether. The dried powder was dissolved in sodium dodecylsulfate/dithioerythritol, placed on discs, and processed as above.Responsivity to progesterone was assessed by incubating oocytesfor approximately 20 hr in media containing 1 jug of proges-terone per ml. Ooctyes were then punctured with a fine probeat the animal pole and gently squeezed at the equator with aforceps; extrusion of a germinal vesicle from the wound indi-cated that germinal vesicle breakdown (GVBD) had not oc-curred. This procedure proved more reliable than scoring thetransient appearance of a large "white spot" (17). Progester-one-treated oocytes were also placed in Smith fixative (18),embedded in paraffin, sectioned at 5 my, and stained withMayer haemalum for light microscopic examination.

Abbreviations: hCG, human chorionic gonadotropin; GVBD, germinalvesicle breakdown.* Wallace, R. A. & Hollinger, T. G. (1978) Exp. Cell Res., in press.

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The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked "ad-vertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

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Proc. Nati. Acad. Sci. USA 75 (1978) 5535

RESULTSOocyte Growth. Oocytes ranging in volume from 0.15 to

0.78 mm3 (diameter = 0.66-1.14 mm) were isolated from a totalnine different unstimulated or hCG-stimulated females and

placed in culture both with and without insulin. Their increasein size was then measured over 14-28 days (Table 1). Completedata for eight different groups are provided in Fig. 1. The useof oocyte size as an indication of oocyte growth was validatedby measuring the relationship between oocyte size and proteincontent both in Vivo (freshly dissected oocytes) and after culturein vitro for at least 5 days. These two sets of data were essentiallysuperimposable (Fig. 2; A protein content - A mm-3 = 326 and329,ug-mm-3, respectively).

In the absence of insulin, oocytes grew at an average rate of0.021 + 0.005 mm3-day-'; in the presence of insulin the ob-served growth rate for all oocytes was 0.030 + 0.007 mm3.day-1(Table 1). When oocytes of similar size from six different fe-males were divided into two groups and cultured with orwithout insulin, those cultured in the presence of insulin werefound to grow at a rate that was 136 + 22% that of controls. Nosignificant differences in growth rates were found betweenoocytes isolated from unstimulated females and those fromhCG-stimulated females.The data provided in Fig. 1 indicate that the growth of oo-

cytes in vitro is progressive with time and that oocytes withina size range of 0. 15-0.78 mm3 grow at about the same rate invitro. This was explored at a greater level of resolution bymeasuring the rate of [3H]vitellogenin incorporation for threedifferent groups of oocytes during culture with insulin: 0.24-mm3 oocytes from frog 17, 0.48-mm3 oocytes from frog 21, and0.78-mm3 oocytes from frog 23. The first two groups of oocytesgrew at an average rate of 0.024 mm3.day-I and the third, ata rate of 0.038 mm3-day-I (Table 1). This range in growth ratewas expected, because oocytes were obtained from "wild-type"rather than genetically similar animals. The two growth ratescorrespond to average protein increments of 329 and 521 ng-hr-1, respectively, based on the data provided in Fig. 2. Theseoverall rates of protein increment correlate well with the av-erage rates of vitellogenin incorporation observed for the threegroups of oocytes during their growth in vitro (Fig. 3). The dataprovided in Fig. 3 indicate further that a maximal rate of[3H]vitellogenin incorporation appears to occur among oocytes

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FIG. 1. Growth of X. laevis oocytes with time in vitro. Examplesare provided of eight groups cultured either in the presence (closedsymbol) or absence (open symbol) of 1 ,ggml-l insulin. Each pointrepresents the average volume ISD for 8-10 oocytes. At various times,oocytes were also placed for --20 hr in 1 ,g of progesterone per ml; thenumbers in parentheses indicate the fraction which underwentGVBDas a response to progesterone treatment. Frog: 17, circle; 18, diamond;21, square; 22, reverse triangle; 23, triangle.

with a volume of 0.6-0.8 mm3 (diameter = approx. 1.05-1.15mm), which is similar to what has been observed in vivo (un-published observations).Oocyte Differentation. The smallest oocytes placed in cul-

ture (0.15 mm3 from fkog 17) were barely pigmented at thebeginning of the culture period. With time, they became fullypigmented over their entire surface, and subsequently thepigment migrated to the animal half of the oocyte, as occurs

Table 1. Average growth rates for various sizes of ooctyes in vitro*

Without insulin With insulinFrog Days Vo Vf AV-day-' Vo Vf AV-day-1 AV with insulinno. cultured (mm3) (mm3) (mm3) (mm3) (mm3) (mm3) AV without insulin

Oocytes obtained from unstimulated females16 14 0.47 0.81 0.024 0.48 1.01 0.038 1.5819 14 0.48 0.84 0.026 0.43 0.86 0.031 1.1924 15 - 0.28 0.70 0.028Average 0.025 I 0.001 0.032 + 0.005

Oocytes obtained from hCG-stimulated females17 27 - 0.15 0.73 0.021t17 28 0.24- 0.91 0.024t17 14 0.44 0.81 0.026 0.43 0.89 0.033 1.2718 24 0.56 1.43 0.036t20 18 0.56 0.86 0.017 0.56 1.01 0.020 1.1821 22 0.48 0.78 0.014t 0.48 1.00 0.024t 1.7122 25 0.33 0.82 0.020t 0.33 0.95 0.025t 1.2523 17 0.78 1.43 0.038tAverage 0.019 ± 0.005 0.028 ± 0.007± indicates SD.

* Vo = volume after 24 hr of culture; Vf = volume on day of termination.t Complete data plotted in Fig. 1.

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5536 Cell Biology: Wallace and Misulovin

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FIG. 2. The relationship between protein content and oocyte sizein vivo (@) and in vitro (0). Each point represents the average of 8-10oocytes; five females were used as oocyte donors in each case. Forprotein content in vivo, groups of oocytes were dissected from theovary and immediately used for determinations; a size range wasmeasured extending from a diameter of 0.45 mm [the smallest oocytesundergoing pinocytosis and hence yolk deposition (4); smaller oocytespresumably grow by some other mechanism] up to a diameter of 1.28mm (the largest oocytes found). For protein content in vitro, mea-surements were made on groups of oocytes cultured for 5-28 days. Thesolid and dashed lines represent the least-squares fit to the data foroocytes in vivo and in vitro, respectively (slopes = 326 and 329 Mlg-mm-3; Y-intercepts = -23 and -31 ng-oocyte-1).

in vivo (4). After 28 days they had reached an average size of0.73 mm3 (Table 1), and about half underwent GVBD in re-sponse to added progesterone (Fig. 1). Larger oocytes placedin culture were already pigmented. The subsequent migrationand appearance of pigment seemed normal in every respect,and most oocytes also eventually underwent GVBD in responseto added progesterone.The response to progesterone was explored in greater detail

because it could be more readily quantitated than the processof pigmentation. In general, we found that oocytes with a vol-ume less than 0.6 mm3 (diameter = 1.05 mm) did not undergoGVBD in the presence of progesterone when initially placedin culture, whereas the largest oocytes examined (diameter =1.14 mm) did (Fig. 1). All groups of oocytes eventually becameresponsive to progesterone once they reached a volume of about0.7 mm3 (diameter = 1.1 mm) (Fig. 1). The fact that this ap-peared to be the case for oocytes cultured either with or withoutinsulin (Fig. 1) indicates that size rather than the presence ofinsulin was the critical determinant. Progesterone-treated oo-cytes that appeared to have undergone GVBD were also fixedand prepared for light microscopy. An examination of serialsections revealed in every case the presence of a metaphase

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FIG. 3. [3HJVitellogenin incorporation as a function of oocyte sizefor three groups of oocytes grown in vitro. Cultured oocytes from frogs17 (0), 21 (o), and 23 (&) were periodically measured (see Fig. 1) and[3H]vitellogenin incorporation was simultaneously determined. Eachpoint represents the average [3Hjvitellogenin incorporation i SD for8-10 oocytes. The average protein increment for oocytes from frogs17 and 21 was 329 ng-hr-1 and for those from frog 23 was 521 ng-hr-1as derived from the rate of growth calculated in Table 1 (0.024 and0.038 mm3.day-1, respectively) and the relationship between volumeand protein content provided in Fig. 2 for oocytes cultured in vitro(329 ,g-mm-3); these values are indicated by the horizontal dottedlines.

spindle together with a polar body, several examples of whichare provided in Fig. 4. As also observed in Fig. 4, only an oc-casional follicle cell was attached to the vitelline membrane.

DISCUSSIONThe volume increments we have measured in vitro (Table 1,Fig. 1) appear to be true reflections of oocyte growth rather thanhydration, because (a) oocyte volume and protein content haveessentially the same relationship in vivo and in vitro (Fig. 2)and (b) observed growth rates correlate well with overall vi-tellogenin incorporation (Fig. 3). The latter process is primarilyresponsible for oocyte growth because growing oocytes incor-porate per hour at least eight times more vitellogenin than thetotal protein that they synthesize (19). This discrepancy isfurther enhanced by the turnover of endogenously synthesizedprotein, whereas sequestered vitellogenin does not undergoturnover (unpublished observations; ref. 19). The addition ofvitellogenin to our culture medium is thus the most likely reasonwe have been able to obtain oocyte growth in vitro, in contrastto previous experiences with long-term culture of defolliculatedoocytes (6, 7).

In the intact, unstimulated X. laevs female, oocytes havebeen estimated to increase from 0.4 to 0.8 mm in diameter in4-8 months (20), which corresponds to an average volume in-crement of 0.001-0.002 mm3-day'1. More recently, growthrates of oocytes in vivo ranging from 0.6 to 1.2 mm diameterwere measured, and volume increments of 0.004-0.014mm3.day-1 for oocytes from unstimulated females and0.011-0.027 mm3-dayf' for those from hCG-stimulated femaleswere found (unpublished data). In a more extreme case, Scheer(21) removed most of an ovary from each of several females,

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Proc. Natl. Acad. Sci. USA 75 (1978) 5537

S f p

FIG. 4. Light micrographs of eggs derived from oocytes grown invitro. Oocytes with an average volume of 0.56 mm3 (unresponsive toprogesterone) from frog 20 were grown for 14 days in vitro (averagevolume = 0.90 mm3) and then incubated overnight in medium con-taining 1 jig of progesterone per ml. Progesterone treatment convertedthe oocytes into eggs as revealed by the simultaneous presence of asecond meiotic metaphase spindle and a first polar body. (A-D) Fourexamples of sections in which portions of the second meiotic meta-phase spindle and first polar body were found in the same section; (Eand F) two examples in which the spindle and polar body were foundin separate sections. s, Second meiotic metaphase spindle; p, polarbody; f, follicle cell. (X400.)

leaving behind only "small oocytes adhering to the rest of themesovarium"; the females were then stimulated with hCG, andthe oocyte diameters were periodically measured. We havetaken Scheer's data for the steepest portion of his growth curve(diameter increase from 0.48 to 1.10 mm), converted the di-ameter measurements to volume measurements, and drawn,by least-squares analysis, a line to fit the points; the slope of thisline was 0.032 mm3-day-'. The average growth rates we haveobserved in vitro in the presence of insulin (0.028-0.032mm3.day-l, Table 1) correspond to this rate observed for oo-cytes in vivo. Note that our culture medium contains vitello-genin at a concentration (5.0 mg-ml-l) that essentially saturatessequestration by the oocyte [Km = 0.7 mg-ml-l (22)].The largest oocytes in laboratory-maintained X. Levis have

been reported to have a diameter between 1.3 and 1.4mm (23,24); in most females, the largest oocytes are somewhat smaller

(4). On two occasions (Table 1, frogs 18 and 23) we found thatoocytes had grown to a diameter of 1.4 mm (volume = 1.43mm3) by the time the culture was terminated. Growth curvesfor these two groups of oocytes did not indicate that a sizeplateau had been reached by this time (Fig. 1). The proteincontent of one group of these 1.4-mm oocytes was measuredand found to correlate with size in a manner similar to otheroocyte groups (Fig. 2, upper right-hand point). Thus, oocytesin vitro can grow at least as large as, if not larger than, thosenormally found in vivo.

Both the relatively rapid growth rate and the large size thatoocytes achieve in vitro suggest that oocyte growth and ultimatesize in vivo may be regulated within the ovary by nutrient(specifically vitellogenin) availability. This notion is furtherreinforced by the observation that although oocytes grow moreslowly in unstimulated females than in hCG-stimulated females,they grow at essentially identical rates when removed from theanimal and placed for 14-28 days in a nutritionally adequatemedium containing a saturating concentration of vitellogenin(Table 1). However, several observations have also been madewhich tend not to support this postulate: (a) oocytes isolatedfrom females injected with hCG 2-3 days previously have atransiently higher rate of vitellogenin incorporation (12), (b)oocytes from unstimulated females increase their rate of vi-tellogenin incorporation when incubated with hCG for 48-60hr (12, 25), and (c) oocytes in vitro do not incorporate vitello-genin at a uniform rate throughout their growth (Fig. 3). Themodulation of both vitellogenin incorporation and growth rateby insulin (ref. 12; Table 1; Fig. 1) may reflect a quantitativechange in oocyte metabolism caused by the presence of thishormone rather than a process related to vitellogenin avail-ability.

Recently, Eppig (26) was able to obtain a significant increasein the size of apparently normal mouse oocytes grown for 7 dayseither within their follicles or in organ culture. Isolated oocytesfailed to grow, however, despite various attempts at cellular orhormonal supplementation and Eppig thus concluded that "anassociation of granulosa cells and oocytes was necessary foroocyte growth." Unlike mouse oocytes, those of lower verte-brates normally grow to a relatively enormous size primarilyby sequestering an external yolk precursor (8) so that strictcomparisons cannot be made. We have nevertheless found thatX. laevis oocytes can both substantially increase in size anddifferentiate over a 2- to 4-week period (Table 1; Fig. 1) whileretaining an apparently normal morphology and physiology(ref. 12; Fig. 4). When X. laevis oocytes are freshly dissectedfrom their follicles, they are usually still invested by a singlelayer of flattened follicle cells (27) which tend to hinder thepassage of vitellogenin from the medium to the oocyte in thoseregions where they remain closely applied to the surface (seefigure 2b in ref. 28). However, we have observed that the folliclecells normally bunch up together or slough off the oocyte sur-face after only a few days in culture, thus completely exposingthe oocyte surface, as has been previously reported (28). Onlyan occasional follicle cell remains attached to the oocyte for anylength of time (Fig. 4). Under such circumstances, vitellogeninis maximally accessible to the oocyte and optimal growth canoccur, apparently regardless of the hormonal history of theoocyte donor (ref. 12; Table 1). In the intact animal, vitellogeninnormally reaches the growing oocyte after passing throughchannels between the follicle cells (9). These cells are generallythought to be the target of gonadotropins in amphibians (25,29-34); as a corollary notion, gonadotropins may thus regulateoocyte growth in vivo by causing the follicle cells to open orclose these channels.The smallest oocytes isolated in our experiments had an av-

erage volume of 0.15 mm3 and were barely pigmented. After

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5538 Cell Biology: Wallace and Misulovin

28 days in culture these oocytes reached an average volume of0.73 mm3 (Table 1) and acquired a normal pigment pattern,and about half responded to added preogesterone by under-going GVBD (Fig. 1). Data for other groups indicated thatoocytes consistently acqqired an ability to respond to proges-terone once they reached a size of about 0.7 mm3 (diameter =1.1 mm), both in the presence and in the absence of insulin (Fig.1), and that the response included not only GVBD but thecomplete transition to an egg, because both metaphase spindlesand polar bodies were found to be produced by progesteronetreatment (Fig. 4). Thus, at least two aspects of oocyte differ-entiation were observed for oocytes grown in vitro. However,our quantitative data collected for the response to 1 ,ug of pro-gesterone per ml (Fig. 1) does not exactly correlate with pre-viously published observations (24). In only one case (Fig. 1, frog22) was a positive response (four out of nine) noted for oocyteswith a diameter less than 1.1 mm, and also in one case (Fig. 1,frog 21) not all oocytes (seven out of ten) responded to a 20-hrexposure to 1 ,tg of progesterone per ml even after reaching adiameter of 1.2 mm. In contrast, Reynhout et al. (24) noted thatfreshly dissected oocytes with diameters ranging from 0.90-0.99, 1.00-1.09, and 1.10-1.19 mm underwent an average of22, 77, and 98% GVBQ), respectively, when scored after a 12-hrexposure to 0.1 ,ug of progesterone per ml. Thus, our culturedoocytes seem to be somewhat more refractory to progesteronetreatment than freshly dissected oocytes. This may be due tosome inadequacy of our culture method, or it is possible thatcontinuous exposure of oocytes to environmental steroids re-duces the threshold of response to progesterone. Ovarian fol-licles in X. laevis produce progesterone (32) as well as othersteroids (33, 34); therefore, freshly dissected oocytes may bemore sensitized to externally applied progesterone than arethose removed from the ovary and incubated in the absence ofsteroids for 1-4 weeks.Our overall conclusions from the results reported here are:

(a) X. laevis oocytes.can be grown in vitro at a rate comparableto the fastest rate yet reported for oocytes in vivo, (b) vitello-genin accessibility may be the major determinant of oocytegrowth, (c) differentiation of oocytes also can be achieved invitro, and (d) although vitellogenin incorporation is undoubt-edly related to oocyte growth, it remains to be demonstratedwhether it is also related to oocyte differentiation. The resultspresented here and in a report describing the development ofour culture procedure (12) establish that oocytes are able togrow and differentiate in vitro when they are substantially freeof associated follicular materials. Our procedure relies upon adefined culture medium, so that it now also appears possiblein future experimentation to obtain more definite answers toquestions concerning the regulation of X. laevis oocyte growthand differentiation.

This research was supported by the Division of Biomedical andEnvironmental Research, U.S. Department of Energy, under contractW-7405-eng-26 with the Union Carbide Corporation.

1. Barr, W. A. (1968) in Perspectives in Endocrinology, eds. Bar-rington, E. J. W. & Jorgensen, C. B. (Academic, New York), pp.163-237.

2. Foote, C. L. & Foote, F. M. (1957) Anat. Rec. 127, 145(abstr.).

3. Foote, C. L. & Foote, F. M. (1958) Anat. Rec. 130,553-565.4. Dumont, J. N. (1972) J. Morphol. 136, 153-179.5. Gurdon, J. B., De Robertis, E. M. & Partington, G. (1976) Nature

(London)-260, 116-120.6. Eppig, J. J. & Dumont, J. N. (1976) In Vitro 12, 418-427.7. Eppig, J. J. & Steckman, M. L. (1976) In Vitro 12, 173-179.8. Wallace, R. A. (1978) in The Vertebrate Ovary: Comparative

Biology and Evolution, ed. Jones, R. E. (Plenum, New York), pp.469-502.

9. Dumont, J. N. (1978) J. Exp. Zool. 204, 193-217.10. Dumont, J. N. & Brummett, A. R. (1978) J. Morphol. 155,73-

97.11. Wallace, R. A., Jared, D. W. & Nelson, B. L. (1970) J. Exp. Zool.

175,259-269.12. Wallace, R. A., Misulovin, Z., Jared, D. W. & Wiley, H. S. (1978)

Gamete Res., in press.13. Wallace, R. A., Jared, D. W., Dumont, J. N. & Sega, M. W. (1973)

J. Exp. Zool. 184,321-333.14. Mans, R. J. & Novelli, G. D. (1961) Arch. Biochem. Biophys. 94,

48-53.15. Bramhall, S., Noack, N., Wu, M. & Loewenberg, J. R. (1969)

Anal.. Biochem. 31, 146-148.16. Wallace, R. A. & Karasaki, S. (1963) J. Cell Biol. 18, 153-166.17. Merriam, R. W. (1972) J. Exp. Zool. 180,421-426.18. Davidson, M.H. (1932) Turtox News 10, 203-204.19. Wallace, R. A., Nickol, J. M., Ho, T. & Jared, D. W. (1972) Dev.

Biol. 29, 255-272.20. Davidson, E. H. (1968) Gene Activity in Early Development

(Academic, New York).21. Scheer, U. (1973) Dev. Biol. 30, 13-28.22. Wallace, R. A. & Jared, D. W. (1976) J. Cell Biol. 69, 345-

35L23. Wallace, R. A. & Steinhardt, R. A. (1977) Dev. Biol. 57, 305-

316.24. Reynhout, J. K., Taddei, C., Smith, L-D. & LaMarca, M. J. (1975)

Dev. Biol. 44,375-379.25. Wiley, H. S. & Dumont, J. N. (1978) Biol. Reprod. 18, 762-

771.26. Eppig, J. J. (1977) Dev. Biol. 60, 371-388.27. Jared, D. W. & Wallace, R. A. (1969) Exp. Cell Res. 57, 454-

457.28. Wallace, R. A., Ho, T., Salter, D. W. & Jared, D. W. (1973) Exp.

Cell Res. 82, 287-295.29. Masui, Y. (1967) J. Exp. Zool. 166,365-376.30. Schuetz, A. W. (1967) Proc. Soc. Exp. Biol. Med. 174, 1307-

1310.31. Smith, L. D., Ecker, R. E. & Subtelny, S. (1968) Dev. Biol. 17,

627-643.32. Fortune, J. E., Concannon, P. W. & Hansel, W. (1975) Biol.

Reprod. 13, 561-567.33. Redshaw, M. R. & Nicholls, T. J. (1971) Gen. Comp. Endocrinol.

16,85-96.34. Mulner, O., Thibier, C. & Ozon, R. (1978) Gen. Comp. Endo-

crinol. 34, 287-295.

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