Proc. Nati. Acad. Sci. USAVol. 83, pp. 6883-6886, September 1986Developmental Biology

Identification of a paternal developmental effect on the cytoplasmof one-cell-stage mouse embryos

(male genome/egg cytoplasm/embryonic mortality/nuclear transplantation/DDK mice)

J. P. RENARD AND CH. BABINETUnite de Genetique des Mammiferes, Institut Pasteur, and Laboratoire Associd, Centre National de la Recherche Scientifique 040361, 25 rue du Docteur Roux,75724 Paris Cedex 15, France

Communicated by Frangois Jacob, May 19, 1986

ABSTRACT Matings of female DDK mice with males ofthe BALB/c strain are sterile, whereas reciprocal crosses arenormally fertile. We used nuclear transplantation between thehybrid eggs of these two strains to investigate the basis of thiseffect. We demonstrate that the observed sterility results fromearly embryonic mortality, that the mortality is due to amodification of the egg cytoplasm, and that the modification ismediated by the male pronucleus. Once established, thismodification may affect female pronuclei of unrelated genotypesuch as C57BL/6. These results support the notion that aproduct derived from the male genome acts at the pronuclearstage and can affect later stages of embryonic development.

When female DDK mice are mated to males of other strains,they generally exhibit low fertility, whereas intrastrainmatings or reciprocal crosses (ofDDK males with females ofother strains) show normal fertility (1). This phenomenon,which is due to death occurring before or soon after implan-tation, stems from a particularity of the embryos themselvesand not from an adverse effect of the DDK uterine environ-ment (2). Genetic analysis (3) has suggested that four allelesat either one locus or two closely linked loci are involved inthe establishment of an incompatibility between the cyto-plasm of egg and a factor in the sperm. Here we examinedirectly whether such an interaction determines the appear-ance of this embryonic lethality. We performed reciprocalnuclear transplantations (4) between one-cell embryos pro-duced from matings involving DDK, BALB/c, and C57BL/6mice. Our results show that a BALB/c male pronucleus canmodify the DDK egg cytoplasm in a way that leads to earlyembryonic lethality.

MATERIALS AND METHODSEmbryo Isolation. Embryos were obtained from DDK,

BALB/c, and C57BL/6 strains of mice bred at the PasteurInstitute and maintained on a 12-hr light-dark cycle. Prepu-beral females (4-6 weeks old) were induced to superovulateby intraperitoneal injection of pregnant mare serumgonadotropin (Folligon-Intervet, 2.5 international units) fol-lowed by human chorionic gonadotropin (Chorulon-Intervet,5 international units) 44-49 hr later and then caged overnightwith males. One-cell-stage embryos were isolated 19-26 hrafter human chorionic gonadotropin injection from femaleswith vaginal plugs (day 1). The ampullary region of excisedoviducts was placed at 30°C in PB1 medium (5) containingbovine serum albumin (Sigma catalog no. A 7638) at 4 mg/mltogether with bovine hyaluronidase (Sigma) at 500 units/mland punctured with watchmaker forceps to release the clutchof eggs. After 3-5 min the cumulus cells dissociated and theeggs were washed by transfer through successive passages

(four to six) in fresh PB1 medium. After careful examinationunder a stereo microscope (x 80) fertilized eggs showing twopronuclei and polar body were pooled from several femalesof the same genotype.

In Vitro Culture. Embryos were cultured under paraffin oil(BDH) in 10-A.l drops of Whitten's medium (6) in an atmo-sphere of 5% CO2 in air at 370C. Before each micromanipula-tion, embryos were cultured for 5-20 min in Whitten'smedium supplemented with cytochalasin B and nocodazole(Sigma) at final concentrations of 1 pug/ml and 0.05 ,tg/ml,respectively (7).

Micromanipulation. Micromanipulations and the prepara-tion of microinstruments were performed essentially as de-scribed previously (4). All manipulations were performed usingLeitz micromanipulators and a fixed-stage Ergaval microscope(Zeiss, Jena, G.D.R.). Both groups of donor and recipient eggs(six to nine per group) were placed in PB1 medium supplement-ed with 0.1% bovine serum albumin containing the amount ofcytoskeletal inhibitors indicated above. An 18-gm beveled andtip-sharpened glass micropipette was used for enucleation andnuclear transfer. The reconstituted eggs were placed in Hanks'medium supplemented with 0.1% bovine serum albumin for 30min at 30'C, washed four times in Whitten's medium plus 0.4%bovine serum albumin, and cultured at 370C as described above.Control (nonmicromanipulated) embryos were exposed to thecytoskeletal inhibitors for the same period of time as themicromanipulated ones (1-2 hr).

Assessment of Embryonic Development. All the eggs thatcleaved at the two-cell stage (day 2) were transplanted intothe oviducts of immature (C57BL/6 x SJL)F1 females andrecovered 48 hr later (day 4) after perfusion of the oviductswith PB1 containing bovine serum albumin at 4 mg/ml. Thisprocedure allowed the eggs to bypass the in vitro "two-cellblock" (8). The recovered embryos were cultured up to 72 hrin vitro as indicated above and examined several times duringculture. For each group of embryos development was as-sessed by the proportion of compacted morulae that wereable (i) to cavitate into zona-enclosed blastocysts with ablastocoelic cavity expanding to more than half the totalvolume of the embryos or (ii) to hatch, which was consideredto have occurred when more than half of the volume of theblastocyst had escaped from the surrounding zona pellucida.

Statistics. The number of zona-enclosed and hatchedblastocysts resulting from in vitro development of morulaewas compared between groups by using ax2 contingency testwith Yate's correction in case of low number. All statisticalcomparisons were done on the pooled results of 5-11 repli-cates of each group.

RESULTSMorulae Derived from Matings ofDDK Females with Either

BALB/c or C57BL/6 Males Are Unable to Form NormalBlastocysts in Vitro. When mated with BALB/c males, DDK

6883

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6884 Developmental Biology: Renard and Babinet

Table 1. Developmental ability of control embryos obtained fromDDK, BALB/c, and C57BL/6 matings

No. (%) of compacted morulae

Developing, during 72 hrin vitro

Formed at day 4 Into IntoEmbryo from one-cell zona-enclosed hatchedgenotype* eggst blastocysts blastocysts

DDK-DDK 72/79 66 (91.7) 45 (62.5)BALB/c-BALB/c 51/57 44 (86.3) 32 (62.7)C57BL/6-C57BL/6 35/40 30 (85.7) 24 (68.6)BALB/c-DDK 116/137 103 (88.8) 74 (63.8)C57BL/6-DDK 40/48 38 (95.0) 32 (80.0)DDK-BALB/c 162/196 15 (9.3)t 8 (4.9)tDDK-C57BL/6 32/45 8 (25.0)t 5 (15.6)t*For all the strain combinations, the female genotype is designatedfirst and the male genotype second.tEmbryos were grown in vivo from day 2 to day 4 and subsequentlycultured in vitro.tSignificantly different from other groups: P < 0.001.

females are almost sterile: only 2 live young could beobtained from 27 females found with vaginal plugs (H.Condamine, personal communication). In contrast, DDKfemales are reported to exhibit only a semisterility withC57BL/6 males: 9 live young were obtained from 12 pregnantfemales examined (1). In both cases reciprocal crosses were

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normally fertile. We therefore used BALB/c mice as the malepartner in most of our experiments.When we examined the developmental ability of embryos

grown in vivo from the two-cell stage to the morula stage andsubsequently cultured in vitro, we found that embryosresulting from intrastrain matings of DDK mice developednormally to the blastocyst stage: no significant differenceswere found with embryos resulting from intrastrain matingsof BALB/c or C57BL/6 mice (Table 1). Development of F1embryos from interstrain crosses involving DDK mice wasmarkedly affected by the sex of the parental strain. WhenDDK males were mated with either BALB/c or C57BL/6females the proportion of morulae developing to zona en-closed or hatched blastocysts remained high (more than 85%and 60%, respectively). However, reciprocal crosses ofDDKfemales with either BALB/c or C57BL/6 males resulted inpoor development: morulae that appeared compacted at thebeginning of culture frequently exhibited drastic morpholog-ical alterations within less than 48 hr and degenerated rapidly(Fig. 1). With BALB/c males less than 10% of these embryoswere able to form a blastocyst and only 5% hatched out oftheir zona. Although less pronounced, the same effects alsooccurred with C57BL/6 males: only 25% of the resultingmorulae were able to form a blastocyst and 15% hatchedduring the culture period. These results made it possible touse blastocyst formation and hatching in vitro to assess theDDK defect in subsequent transplantation experiments.

Lethality of DDK Hybrid Embryos Is Not Due to a NuclearDefect. In the next series of experiments we checked the

C + s~~~~~~~~~~~~~~~~'0 .1

D

FIG. 1. Morphological appearance of in vitro cultured morulae derived from crosses of DDK and BALB/c strains. Fertilized eggs fromsuperovulated females were isolated 18-21 hr after mating (day 1), then cultured overnight at 37°C and transplanted at the two-cell stage (day2) into the oviducts of immature (C57BL/6 x SJL)Fj females. The embryos were recovered 48 hr later (day 4), cultured for up to 72 hr in vitro,and examined several times during the culture period. (A) Precompacted and compacted day 4 morulae obtained from BALB/c females fertilizedwith DDK male (BALB/c-DDK embryo). (B) Zona-enclosed and hatched BALB/c-DDK blastocysts obtained from the same morulae after 48hr of culture. Hatching was considered to have occurred when more than half of the volume of the blastocyst had escaped from the surroundingzona. (C) Compacted day 4 morulae obtained from DDK females, fertilized with BALB/c males (DDK-BALB/c embryo). (D) DegeneratedDDK-BALB/c embryos obtained from the same morulae after 48 hr of culture; embryos only rarely formed blastocysts and hatched in vitro;they usually exhibit drastic morphological alterations within less than 24 hr of culture, blastomeres being "extruded" from the resting part ofthe morulae (arrows), which cavitate abnormally (crosses). (Scale bar = 10 gm.)

Proc. Natl. Acad. Sci. USA 83 (1986)

Proc. Natl. Acad. Sci. USA 83 (1986) 6885

efficiency of the nuclear transfer method on one-cell-stageembryos obtained after matings of DDK mice. As seen inTable 2 (group A), transfers of DDK pronuclei into DDKcytoplasm resulted in embryos developing to blastocyst stageat a frequency comparable to that of unmanipulated embryos(Table 1, line 1). In both groups more than 80% of the morulaeformed blastocysts (respective values: 82.1% and 91.7%),and more than 60% hatched in vitro (respective values: 64.3%and 62.5%).We next asked if the developmental defect of DDK-

BALB/c eggs was due to an incompatibility between DDKfemale and BALB/c male genomes. Both pronuclei of DDKeggs fertilized with BALB/c males were transferred into a

BALB/c enucleated cytoplasm and subsequent developmentwas assessed. More than 70% of the resulting morulaecavitated into blastocysts and 55% hatched out of their zona(Table 2, group B). These developmental rates were notsignificantly different from those of the reconstructed DDKembryos indicated above. This result is evidence that nuclearinteraction per se at the one-cell stage is not sufficient to alterthe developmental potentiality of DDK eggs fertilized byBALB/c sperm.

Lethality of DDK Hybrid Embryos Occurs When MalePronucleus Has Formed. When both pronuclei of DDK eggsfertilized by BALB/c sperm were transferred into anenucleated DDK cytoplasm only 15% of the resultingmorulae were able to reach the blastocyst stage; moreover,they did not hatch in vitro (Table 2, group C). Such recon-stituted embryos thus behave like unoperated DDK eggsfertilized by BALB/c sperm (Table 1). However, whentransfers were performed with the pronuclei ofBALB/c eggsfertilized with DDK sperm (Table 2, group D) a high devel-opment rate was obtained: 80.9% of the resulting morulaedeveloped to the blastocyst stage and 57.1% hatched in vitro.These proportions were similar to those obtained with controlmorulae of the same nuclear constitution (Table 1, respec-

tively 88.8% and 63.8%). It thus appears that the presence ofa female pronucleus of an alien strain in an enucleated DDKegg does not preclude normal development; instead, lethalityof DDK hybrid embryos occurs when a sperm of an alienstrain has interacted with DDK cytoplasm to form a malepronucleus.DDK Cytoplasm Is Modified by Male Pronucleus of Alien

Strain. To seek evidence for modification of DDK eggcytoplasm by BALB/c sperm the following reconstructionexperiment was performed: both pronuclei ofBALB/c-DDKeggs were transferred into enucleated DDK-BALB/c eggs.

Results (Table 2, group E) show that only 35.7% of theresulting morulae formed blastocysts and 17.8% hatched invitro. The developmental rates of such reconstituted embryoswere significantly lower than those obtained after transfer ofthe same pronuclear combination into an enucleated DDK

egg previously fertilized by DDK sperm (Table 2, group D).In this case 80.9% of the morulae formed blastocysts (x2 =

8.17; P < 0.01) and 57.1% hatched in vitro (x2 = 6.52; P <0.02). These data show that the male pronucleus of an alienstrain exerts a detrimental effect on the ability of DDKcytoplasm to support normal development. This effect,however, is less pronounced than the one obtained withunoperated DDK-BALB/c eggs (Table 1), which only occa-

sionally developed to the blastocyst stage (9.3%, x2 = 12.41;P < 0.01) or hatched in vitro (4.9%, x2 = 4.42; P < 0.05).

Modified Cytoplasm of DDK-BALB/c Hybrid Eggs Exerts a

Detrimental Effect on Embryos with Unrelated MaternalGenotype. To see if the cytoplasmic modification induced bya BALB/c male pronucleus on DDK cytoplasm can alsoexert a detrimental effect on an unrelated maternal genotype,the following experiment was performed. The pronuclei ofC57BL/6 eggs fertilized with DDK sperm were transferred toenucleated DDK eggs that had previously been fertilized witheither BALB/c orDDK sperm. Results (Table 3) showed thatmorulae obtained from the latter group of reconstituted eggsdevelop normally into zona-enclosed blastocysts (92.0%) andhatched blastocysts (76.0%), whereas development ratesfrom the former group were significantly reduced: only 48.3%of the morula formed blastocysts (x2 = 11.91; P < 0.01) and13.7% hatched in vitro (x2 = 20.61; P < 0.001).

DISCUSSION

We have used the technique of nuclear transplantation toinvestigate the basis of the infertility of DDK females matedto males of other strains. Our data clearly indicate that someevents occurring after fertilization but during the one-cellstage underlie this apparent sterility. This event, whichinvolves interaction between the DDK egg cytoplasm and themale pronucleus of an alien strain, renders the cytoplasmincapable of supporting continued development. This impliesthat some male gene product necessary for further develop-ment is acting as early as during the one-cell stage.The fact that the modification of the cytoplasm of DDK

eggs can occur once the male pronucleus has formed (seeTable 2, group C) implies that sperm membrane interactionswith the egg at fertilization, which result in the formation ofa localized patch of membrane (9), are not directly involvedin the phenomenon. In our experiments, enucleation was

performed between 19 and 26 hr after injection of humanchorionic gonadotropin. This period is after male pronucleusformation, a process that involves dramatic and poorlyunderstood modifications of both the nuclear membrane andsperm chromatin (10-12), with a possible involvement ofnonnuclear components of the sperm (13). The experimentalmanipulations we have used take place around the time of

Table 2. Development of embryos reconstituted from eggs of DDK or BALB/c strains

No. (%) of morulaeDeveloping, during 72 hr

No. of eggs in vitroEggconstitution*_____

With fused Formed from Into IntoCytoplast- karyoplast/ Transferred Recovered the recovered zona-enclosed hatched

Group Pronuclei sperm total at day 2 at day 4 eggs blastocysts blastocysts

A DDK-DDK DDK-DDK 32/53 32 30 28 23 (82.1) 18 (64.3)B DDK-BALB/c BALB/c-DDK 44/63 39 37 29 21 (72.4) 16 (55.2)C DDK-BALB/c DDK-DDK 25/37 24 24 19 3 (15.8)t 0D BALB/c-DDK DDK-DDK 28/35 28 26 21 17 (80.9) 12 (57.1)E BALB/c-DDK DDK-BALB/c 41/59 37 30 28 10 (35.7)t 5 (17.8)t

*Egg constitution is defined by the genotype of donor female and male pronuclei, with the female given first, and by the genotype of recipientcytoplast with indication of the genotype of the sperm used for fertilization before enucleation.

tSignificantly different from other groups: P < 0.025.

Developmental Biology: Renard and Babinet

6886 Developmental Biology: Renard and Babinet

Table 3. Development of embryos derived from reconstituted eggs of the DDK strain after fertilization with DDK or BALB/c sperm:Comparison with control

No. (%) of compacted morulaeDeveloping, during 72 hr

No. of eggs in vitroEgg constitution* With fused Formed from Into Into

Cytoplast- karyoplast/ Transferred Recovered the recovered zona-enclosed hatchedGroup Pronuclei sperm total at day 2 at day 4 eggs blastocysts blastocystsReconstituted eggsA C57BL/6-DDK DDK-BALB/c 54/69 50 38 29 14 (48.3)t 4 (13.8)tB C57BL/6-DDK DDK-DDK 40/52 40 29 25 23 (92.0) 19 (76.0)

Control eggsC57BL/6-DDK 40 38 (95.0) 32 (80.0)DDK-C57BL/6 - 32 8 (25.0)t 5 (15.6)t

*Egg constitution is defined by the genotype of donor female and male pronuclei, with the female given first, and the genotype of recipientcytoplast with indication of the genotype of the sperm used for fertilization before enucleation.

tSignificantly different from other groups: P < 0.001.

DNA replication (14, 15) and are concomitant with changesthat occur normally in the polypeptide profiles of the cyto-plasm once the sperm has penetrated the egg (15). Each ofthese events represents the expression of a program neces-sary for future normal embryonic development (16) and maythus be suspected of participating directly in the studiedphenomenon.Recent investigations (17) have clearly demonstrated that

the paternal and maternal genomes have differential rolesduring embryogenesis; these differences may be expressedduring preimplantation development. Our data suggest thatsuch a paternal-maternal effect may appear just after fertil-ization and before the end of the first cell cycle. The natureof this effect remains unknown, but our data indicate that itinvolves some particular aspect of the nucleocytoplasmicinteractions occurring in the one-cell embryo. From this pointof view the DDK syndrome offers a unique opportunity tostudy such interactions.

Note. Recently Mann (18) has shown thatDDK egg-foreignsperm incompatibility in mice is not between the pronuclei,which confirms one of the results of our work.

We thank J. M. Grath and J. Mann for their generous gift of activeSendai virus and we are indebted to C. Hannoun for providingseveral efficient batches of inactivated Sendai preparations. Wethank A. Surani for cordial welcome and fruitful discussion, and H.Condamine for constant and stimulating attention all through our

experiments. We thank J. L. Guenet, J. Barra, D. Morello, P. Avner,and J. R. Sanes for their help and comments during the preparationof the manuscript and A. M. Salmon for constant and skillful

technical assistance. J.P.R. is "Charge de Recherches" at InstitutNational de la Recherche Agronomique (INRA).

1. Wakasugi, N., Tomita, T. & Kondo, K. (1967) J. Reprod.Fertil. 13, 41-50.

2. Wakasugi, N. (1973) J. Reprod. Fertil. 33, 283-291.3. Wakasugi, N. (1974) J. Reprod. Fertil. 41, 85-86.4. McGrath, J. & Solter, D. (1983) Science 220, 1300-1302.5. Whittingham, D. G. & Wales, R. G. (1969) Aust. J. Biol. Sci.

22, 1065-1072.6. Whitten, W. K. & Biggers, J. D. (1968) J. Reprod. Fertil. 17,

399-401.7. Surani, M. A. H., Barton, S. C. & Norris, M. L. (1984) Na-

ture (London) 308, 548-550.8. Whittingham, D. G. & Biggers, J. D. (1967) Nature (London)

213, 942-943.9. Gabel, C. A., Eddy, E. M. & Shapiro, B. M. (1979) Cell 18,

207-215.10. Yanagimachi, R. (1981) in Fertilization and Embryonic Devel-

opment in Vitro, eds. Mastroianni, L., Jr., & Biggers, J. D.(Academic, New York), pp.- 81-182.

11. Zirkin, B. R., Soucek, D. A., Chang, T. S. K. & Perreault,S. D. (1985) Gamete Res. 11, 349-365.

12. Maro, B., Johnson, M. H., Pickering, S. J. & Flach, G. (1985)J. Embryol. Exp. Morphol. 81, 211-237.

13. Szollosi, D. (1976) Curr. Top. Pathol., 9-27.14. Luthardt, F. W. & Donahue, R. P. (1973) Exp. Cell. Res. 82,

143-151.15. Howlett, S. K. & Bolton, V. N. (1985) J. Embryol. Exp.

Morphol. 87, 175-206.16. Johnson, M. H., McConnell, J. & Van Blerkom, J. (1984) J.

Embryol. Exp. Morphol. Suppl. 83, 917-231.17. Barton, S. C., Adams, C. A., Norris, M. L. & Surani,

M. A. H. (1985) J. Embryol. Exp. Morphol. 90, 267-285.18. Mann, J. R. (1986) J. Reprod. Fertil. 76, 779-781.

Proc. Natl. Acad. Sci. USA 83 (1986)