Effects of in-vitro fertilization, culture, freezing and transfer on the ability of mouse embryos to implant and survive

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1 Effects of in-vitro fertilization, culture, freezing and transfer on the ability of mouse embryos to implant and survive A. Massip, P. Van der Zwalmen, F. Puissant, M. Camus and F. Leroy Department of Obstetrics and Animal Reproduction, Faculty of Veterinary Medicine, 45 rue des Vétérinaires, 1070 Brussels, and *Free University of Brussels Medical School, Human Reproduction Research Unit and Laboratory of Embryology, 1000 Brussels, Belgium Summary. The survival after transfer of frozen\p=n-\thawedmouse blastocysts obtained from culture of in-vitro fertilized oocytes or 1- and 2-cell ova was compared. About 10% of transferred embryos developed to term in each group and there was no difference between embryos fertilized in vitro or in vivo. In addition to embryonic loss due to transfer, in-vitro cultivation and freezing reduced the proportion of fetuses considered viable at Day 15 of pregnancy (29\m=.\8 versus 50\m=.\7%and 26\m=.\3versus 50\m=.\7% respectively). When used together these procedures had an additive effect on fetal wastage (18\m=.\4 versus 50\m=.\7%). In-vitro culture also entailed a significant increase of resorbing implantation sites (10\m=.\2 versus 4\m=.3%). The re-expansion rate after freezing and thawing of blastocysts grown in vitro was paradoxically greater than that of blastocysts grown in vivo (85\m=.\8 versus 54\m=.\6%). Introduction Normal fetuses or young have been produced after transfer to recipient females of mouse blastocysts obtained from culture of in-vitro fertilized oocytes (Hoppe & Pitts, 1973; Kasai, Sugimoto & Toyoda, 1979), one-cell ova (Whitten & Biggers, 1968) and two-cell ova (Biggers, Moore & Whittingham, 1965). The birth of viable young after transfer of cultured and frozen embryos has also been reported for the mouse (Nakagata & Toyoda, 1980) and the rabbit (Schneider, Hahn & Sulzer, 1974); oocytes were fertilized in vitro and cultured to 2- or 4-cell stages. In such experiments the viability of embryos is low and should be compared with that of untreated embryos in order to assess losses due to transfer, culture, freezing or a combination of these procedures. The present paper deals with the survival after transfer of mouse blastocysts obtained from culture of in-vitro fertilized oocytes, one- and two-cell ova, followed by freezing and thawing. Source of oocytes and ova Materials and Methods Experiment!. Hybrid mice (Fl, C57BL CBA/Ca), 3-5 weeks of age, were induced to superovulate by intraperitoneal injections of 5 i.u. PMSG and 5 i.u. hcg given 48 h apart. These females were killed h after hcg injection for collection of oocytes from excised oviducts or mated 1984 Journals of Reproduction & Fertility Ltd

2 7 C, 25 C with OFl Swiss albino males (IFFA/CREDO, Brussels). One- and 2-cell ova were collected from the oviducts in Media T6 and PB1, respectively (Quinn, Barros & Whittingham, 1982), on the day of finding the vaginal plug (1-cell) or 24 h later (2-cell). Cumulus cells of 1-cell ova were dispersed by using hyaluronidase (Sigma, St Louis, MO, U.S.A.) in Medium T6 at 1 mg/ml. Experiment 2. For this study donor females treated and mated as in Exp. 1 were used in each replicate. Half of them were killed 24 h after finding the coital plug and 2-cell ova were flushed from excised oviducts with Medium PB1 to be cultured to the blastocyst stage. Blastocysts obtained from these cultures were transferred to foster mothers or frozen. The other donors were killed 2 days later and blastocysts developed in vivo were collected from uterine horns and then immediately transferred or frozen. In-vitro fertilization and culture offertilized eggs and embryos The procedures for fertilization in vitro were essentially those described by Whittingham (1968) and Hoppe & Pitts (1973). Spermatozoa were obtained by mincing the vasa deferentia and each cauda epididymidis from fertile Swiss Albino OFl males into Medium T6 (Quinn et al., 1982) containing 4 mg BSA (Sigma V)/ml under oil and allowed to disperse at 37 C for 30 min; spermatozoa were pipetted in a volume of µ into insemination wells containing the oocytes in 1 ml Medium T6 under oil (dimethyl polysiloxane : 5 10 "3 m2. sec ' viscosity : Sigma). " Fertilized ova were removed after 7-8 h and rinsed in fresh medium to remove adhering cumulus and corona cells. Ova fertilized in vivo or in vitro were cultivated for 72 h (2-cell ova) or 96 h (1-cell ova and oocytes fertilized in vitro) at 37 C, under an atmosphere of 5% C02, 5% 02 and 90% N2 in polystyrene culture tubes (12 75 mm, Falcon B-D) containing 1 ml Medium T6 supplemented with 10% heat-treated serum or 0-4% BSA (Sigma V). Fetal calf serum and BSA were mostly employed but various other sera (adult human, sheep, steer and human umbilical cord) were also used successfully in one experiment. The medium was covered by 1 ml dimethyl polysiloxane oil. Freezing, thawing and culture offrozen-thawed embryos Blastocysts developed in vivo or in vitro were washed in PBS and frozen in PBS containing 1-36 M-glycerol and 0-25 M-sucrose (Massip & Van der Zwalmen, 1982). The freezing procedure was as follows. (1) Embryos were pipetted directly into the cryoprotective solution. (2) After min equilibration at room temperature (~ 20 C), embryos were drawn into 100 µ freezing medium between two air bubbles in 0-25 ml French straws. The straws ~ were sealed with polyvinyl alcohol powder and placed vertically in the cooling chamber of a programmable biological freezer (Minicool, Air Liquide, Paris) precooled at 7 C. (3) After 5 min at the samples were seeded by touching the straws with forceps cooled in liquid nitrogen, and maintained for a further 5-10 min at seeding temperature. Further cooling was allowed to proceed at 0-3 C/min to before transfer and storage into liquid nitrogen (-196 C)for 3-53 days. The samples were thawed by gently shaking the straws in a water bath at 20 C until ice had disappeared. Embryos were transferred into watch glasses and washed in three changes of fresh PBS. They were then replaced for h in culture medium. The numbers of blastocysts showing normal expansion after this period were recorded. Embryo transfer In Exp. 1, nulliparous random-bred OFl Swiss albino females, 6-8 weeks old, were mated with vasectomized males of the same strain to be used as foster mothers.

3 In Exp. 2, nulliparous hybrid mice from the cross C57BL CBA/Ca were mated with fertile OFl males to provide recipient females. When 8 weeks old, the latter were caged with vasectomized OFl males. Blastocysts developed in vivo or in vitro and blastocysts that had re-expanded in vitro after freezing and thawing in each category were transferred to recipient females on the morning of Day 3 of pseudopregnancy ; 5-8 blastocysts were usually deposited surgically into each horn (McLaren & Michie, 1956). Recipient females in Exp. 1 were allowed to litter; those in Exp. 2 were killed and their uteri were examined on Day 15 of gestation. The numbers of normal live embryos and resorbing implantation sites were recorded. Numerical differences between groups were compared by 2 analysis. Yates' correction for low numbers was applied when comparing résorption rates. Results Experiment 1 : survival offrozen-thawed blastocysts obtained by culturing ova fertilized in vivo and in vitro At the end of the culture period the proportion of blastocysts in each group was 55-8% for oocytes fertilized in vitro, 51-3% for 1-cell ova and 79-9% for 2-cell ova. Survival rates of these blastocysts after freezing and thawing are given in Table 1. The percentage of re-expansion was around 80% and was not significantly different between groups. The number of recipients pregnant was greatest when 2-cell ova were used but the differences were not significant. The proportion of live young born did not differ significantly and was about 10%. Duration of gestation was normal. As expected, all young born developed a brown fur, thereby precluding faulty vasectomy, and were capable of normal reproduction when adult. Table 1. In-vitro and in-vivo development of mouse blastocysts frozen-thawed after culture of in-vitro fertilized oocytes, and 1 and 2-cell ova Initial stage No. of blastocysts frozenthawed No. (%) of blastocysts expanding after h in culture No. of blastocysts transferred No. of recipients No. (%) pregnant No. (%) of live young In-vitro fertilized oocytes 1-cell ova 2-cell ova (85-7) 113(78-5) 247 (82-9) (25-0) 4 (40-0) 8 (53-3) 16(11-7) 11 (10-7) 17 (9-1) Experiment 2: effects on survival of transfer procedure, culture in vitro and freezing The results of this investigation are shown in Table 2. There was no significant difference between numbers of recipient mice which became pregnant in the different groups. The highest rate of normal fetuses was predictably obtained after direct transfer from donors to recipients (Group I). An unexpected finding was that, after freezing and thawing, blastocysts obtained by culture (Group IV) re-expanded significantly more often than did those developing in vivo (Group III).

4 Table 2. Implantation and survival of blastocysts developed in vivo and in vitro and transferred to recipients directly or after cryopreservation Group No. No. transferred No. (%) of No. (%) of frozen- (re-expansion No. of No. normal résorption thawed rate) recipients pregnant fetuses sites I (developed in vivo) II (developed in vitro) III (developed in vivo and frozen-thawed) IV (developed in vitro and frozen-thawed) (541) 206 (85-8)* (50-7)t 61 (29-8)J 31 (26-3) 38 (18-4) 6 (4-3) 21 (10-2) 9 (7-6) 28 (13-6)11 Results were pooled from 6 replicates. * < 0001 compared with Group III. t < 0001 compared with Groups II, III and IV. Î < 001 compared with Group IV. < 005 compared with Group I. HP < 001 compared with Group I. Discussion The results of Exp. 1 demonstrate that mouse blastocysts obtained by in-vitro fertilization, or by culture of 1- and 2-cell ova, can be successfully frozen and thawed and that these embryos can develop to normal live young after transfer. Re-expansion rates after freezing and thawing and the results of transferring embryos fertilized in vitro are similar to those obtained for embryos fertilized in vivo. In spite of high rates of re-expansion of the frozen-thawed blastocysts ( %), the percentages of survival to live young after transfer were low ( %). Similar observations were reported by Nakagata & Toyoda (1980) using a different method of freezing. Low in-vitro viability (27%) was recorded by Shelton & Craft (1982) with in-vitro fertilized mouse embryos cultured to the 2-cell stage and subsequently frozen. There may be several reasons for the discrepancy between percentages of blastocysts able to re-expand and number of live young obtained after transfer. The method of transfer itself is known to entail variable loss of embryos according to operative skill. The strain of mice, cultivation in vitro, as well as freezing and thawing can probably each account for some reduction of implantation rates (Whittingham, 1976 ; Nakagata & Toyoda, 1980 ; Hahn & Schneider, 1982; Shelton & Craft, 1982). Therefore, additional experiments were performed to assess quantitatively the role of each of these possible factors. The data in Table 2 indicate that invitro cultivation and freezing both have a deleterious effect on blastocysts, even when morphologically normal embryos were selected for transfer to foster mothers. When combined, these two experimental procedures exert their detrimental influences in an additive manner. It is known that a culture period as short as 24 h after collecting cow blastocysts developed in vivo is enough to reduce the pregnancy rate after transfer (Renard, Heyman & Ozil, 1980). For mouse embryos grown in vitro, it has been shown that the rate of successful transfer is related to the dura tion of the culture period (Hahn & Schneider, 1982). Relevant to this discussion is the observation that, although proceeding at a cleavage rate initially as great as in the reproductive tract, mouse embryos developing in vitro slow down as they approach the blastocyst stage (Bowman & McLaren,

5 1970; Harlow & Quinn, 1979). In our work, however, only fully grown and expanded blastocysts were transferred in each of the four experimental groups. An ultrastructural comparative study of late blastocysts developed in vivo and in vitro was performed by McReynolds & Hadek (1972). It appears from their observations that differentiation of nuclear and cytoplasmic structures is less advanced in mouse embryos cultured in vitro. Heterochromatin is less abundant suggesting retarded programmed gene repression and nucleoli are less developed. In the cytoplasm, ribosomes are less numerous and mitochondria show fewer cristae per organelle. These differences indicate impaired metabolic ability which could be involved in the low percentage of healthy fetuses that we observed after transfer. In-vitro culture also entailed a higher percentage of résorptions after implantation. Biggers et al. (1965) have reported résorption rates as high as 50% under similar conditions. Hoppe & Coman (1983) have demonstrated a reduced survival in utero from transferred cultured mouse blastocysts compared with morulae, due to inadequate implantation. It cannot be concluded from our data whether freezing has the same effect after implantation or whether this difference is specific to embryos grown in vitro. Reduced survival of frozen-thawed mouse blastocysts after transfer has been previously documented (Whittingham, 1977). In the present work freezing also entailed a significant reduction in the rate of ongoing pregnancies obtained from blastocysts developed in vivo. The method which was used was originally developed to allow direct transfer after thawing and did not include stepwise dilution of the cryoprotectant (Massip & Van der Zwalmen, 1982). This factor may have contributed to the lowered rate of successful transfer after freezing. The endocrine status of foster mothers should also be taken into account since it has been found that a culture period of 20 h after thawing is more compatible with transfer of blastocysts at Day 4 than at Day 3 of pseudo pregnancy (Whittingham, 1977). In our hands, however, transfer at Day 4 of cultured blastocysts did not improve success rates. Blastocyst expansion has been used as an index of viability of embryos cultured after having been frozen and thawed. Our results indicate that this view might be mistaken for blastocysts grown in vitro. Blastocysts developed in vivo and frozen seemed to fare better after transfer than did corresponding blastocysts grown in vitro. However, the rate of re-expansion was much higher in the latter than in the former. Although no ultrastructural differences at the level of cellular membranes and junctions were described by McReynolds & Hadek (1972), it is probable that blastocysts grown in vitro display specific permeability changes allowing easier osmotic exchanges of the blastocoelic cavity after thawing. Transfer to foster mothers therefore remains the definitive test of success in experimental manipulation of mammalian embryos (Whittingham, 1981). We thank Professor J. Mulnard for advice and facilities, and Mrs Jeannine Deweze-Van Hoeck and W. Zwijsen for technical assistance. This work was supported by the Belgian I.R.S.I.A., F.N.R.S. and F.R.S.M. References Biggers, J.D., Moore, B.D. & Whittingham, D.G. (1965) Development of mouse embryos in vivo after cultiva tion from two cell ova to blastocysts in vitro. Nature, Lond. 206, Bowman, P. & McLaren, A. (1970) Cleavage rate of mouse embryos in vivo and in vitro. J. Embryol. exp. Morph. 24, Hahn, J. & Schneider, U. (1982) Embryo transfer in laboratory animals as a tool in reproductive research. Exp! Biol. Med. 7, Harlow, G.M. & Quinn, P. (1979) Foetal and placental growth in the mouse after pre-implantation development in vitro under oxygen concentrations of 5 and 20%. Ausi. J. biol. Sci. 32, Hoppe, P.C. & Coman, D.R. (1983) Reduced survival in ulero from transferred mouse blastocysts compared with morulae. Gamete Res. 7, Hoppe, P.C. & Pitts, S. (1973) Fertilization in vitro and development of mouse ova. Biol. Reprod. 8, Kasai, K., Sugimoto, M. & Toyoda, Y. (1979) Normal progeny produced by the parents derived from mouse eggs fertilized in vitro. Jap. J. Zootech. Sci. 50, Massip, A. & Van der Zwalmen, P. (1982) In vitro survival

6 of mouse embryos frozen in glycerol or glycerolsucrose. Cryo-Letters 3, 326, Abstr. McLaren, A. & Michie, D. (1956) Studies on the transfer of fertilized mouse eggs to uterine foster mothers. I. Factors affecting the implantation and survival of native and transferred eggs. J. exp. Biol. 33, McReynolds, H.D. & Hadek, R. (1972) A comparison of the fine structure of late mouse blastocysts developed in vivo and in vitro. J. exp. Zool. 182, Nakagata, N. & Toyoda, Y. (1980) Normal young after transfer of frozen-thawed 2-cell mouse embryos obtained by fertilization in vitro. Jap. J. Zootech. Sci. 51, Quinn, P., Barros, C. & Whittingham, D.G. (1982) Preservation of hamster oocytes to assay the fertiliz ing capacity of human spermatozoa. J. Reprod. Fert. 66, Renard, J.P., Heyman, Y. & Ozil, J.P. (1980) Importance of gestation losses after non-surgical transfer of cultured and non cultured bovine blastocysts. Vet. Ree. 107, Schneider, U., Hahn, J. & Sulzer, H. (1974) Erste ergebnisse der tiefgefrierkonservierung von mauseund kanincheneizellen. Dt. tierärztl. Wschr. 81, Shelton, K. & Craft, I. (1982) The successful cryopreservation of in vitro fertilized mouse embryos. Cryo- Letters 2, Whitten, W.K. & Biggers, J.D. (1968) Complete develop ment in vitro of the pre-implantation stages of the mouse in a simple chemically defined medium. J. Reprod. Fert. 17, Whittingham, D.G. (1968) Fertilization of mouse eggs in vitro. Nature, Lond. 220, Whittingham, D.G. (1976) Low temperature storage of mammalian embryos. In Basic Aspects of Freeze Preservation of Mouse Strains, pp Ed. O. Mühlbock. Gustav-Fisher Verlag, Stuttgart. Whittingham, D.G. (1977) Some factors affecting embryo storage in laboratory animals. In Freezing ofmamma lian Embryos (Ciba Fdn Symp. No. 52 (new series)), pp Eds K. Elliott & J. Whelan. Elsevier/ North Holland, Amsterdam. Whittingham, D.G. (1981) Viability assays. In Frozen Storage oflaboratory Animals, pp Ed. G. H. Zeilmaker. Gustav-Fisher Verlag, Stuttgart. Received 9 September 1983