Pseudopregnant Recipients Research Articles

Effect of hypotaurine on in vitro fertilization and production of term offspring from in vitro-fertilized ova of the Japanese field vole, Microtus montebelli.

The present study was proposed to establish new in vitro fertilization (IVF)-embryo transfer techniques for wild mammals. We demonstrated that IVF of the Japanese field vole, Microtus montebelli, is improved by hypotaurine, and that IVF vole embryos can develop to normal offspring after transfer to recipient females. The rate of IVF was significantly increased when 0.1 or 1 mM of hypotaurine was added during sperm preincubation and fertilization (p < 0.05). Preincubation of spermatozoa with 1 mM hypotaurine prior to oocyte insemination did not have a significant effect on fertilization rate (p < 0.01). At 96 h after insemination, most cultured embryos developed to the 2-cell stage (68-83%), but development to blastocysts was very low (0-3%) for all treatments. Although 43 living offspring were produced after transfer of IVF pronuclear embryos to recipients, the delivery rate of recipients was very low (21%). The litter size of pseudopregnant recipients that produced a litter was 4-11, and the rate of in vivo development of transferred embryos in the pregnant recipients was 41%. These results suggest that hypotaurine affects IVF rather than sperm preincubation and that IVF embryos can develop to normal offspring.

Prolonged development of normal and parthenogenetic postimplantation mouse embryos in vitro.

Parthenogenetic mammalian embryos show reduced placental development and do not develop beyond the 25-somite stage. But non-parthenogenetic embryos in culture, without a functional placenta, can develop to 40 somites or more. We have therefore examined the possibility that parthenogenetic embryos might also show prolonged development in culture. After parthenogenetic activation and diploidization, 23% of CBA and 56-58% of hybrid (CBAxC57BL/6) F1 mouse eggs developed in culture to blastocysts. When transferred to pseudopregnant recipients: 60% of the CBA blastocysts implanted and 26% of these developed to somite stage embryos; 71-72% of the hybrid blastocysts implanted and 11-17% of these developed to somite stage embryos. Improved development of postimplantation embryos explanted into culture at about the 15-20 somite stage was obtained by opening the visceral yolk sac (without exteriorizing the embryo). All the normal (non-parthenogenetic) embryos cultured in this way developed to more than 35 somites and many reached 45-55 somites. Under the same conditions, 11/17 diploid parthenogenetic CBA embryos developed in culture to more than 35 somites and 5 of these reached 45 somites; and 9/28 diploid parthenogenetic (CBAxC57BL/6) F1 embryos developed to 35 somites or more and 5 of these reached 45 somites. The size and protein content of the parthenogenetic embryos after culture was less than that of the normal embryos of equivalent stages. These results raise new possibilities for the analysis of parthenogenesis and genomic imprinting, including studies of the effects of adding to the culture medium specific growth factors and demethylating agents.

Trophic effects of myeloid leukaemia inhibitory factor (LIF) on mouse embryos.

Myeloid leukaemia inhibitory factor (LIF) is expressed at highest concentrations in the maternal endometrial glands at about the stage of blastocyst implantation. LIF is also expressed by the extraembryonic membranes of the early mouse embryo. Embryos of different ages were cultured with, or without, LIF, and embryo growth in vivo and in vitro was examined to determine whether LIF is important for embryo development. Supplementing embryo culture media with 1000 U recombinant human LIF ml-1 increased the number of eight-cell mouse embryos developing beyond the hatched blastocyst stage in vitro from 62.1% to 85.1% (P < 0.05). LIF significantly increased the number of embryos hatching (33.8% versus 7.65% for controls 96 h after hCG injection, P < 0.001), completely hatching (85.1% versus 62.1%, P < 0.05), and exhibiting trophoblast outgrowth (13.5% versus 0% 120 h after hCG treatment, 85.1% versus 47.0% 144 h after hCG treatment, P < 0.001) in vitro. LIF-treated embryos also displayed a significantly greater area of trophoblast outgrowth than did controls as early as day 5 in culture (P < 0.005). These data show that LIF enhances mouse eight-cell embryo development in vitro, as seen by the accelerated rate of embryo hatching and trophoblast outgrowth. In addition, enhanced embryo survival in vivo is shown, following the transfer of LIF-treated embryos into a pseudopregnant recipient female. Expression of mRNA encoding LIF was detected in endometrial cells cultured in monolayer from uteri of day 3 pregnant females, explaining the known embryotrophic effects of endometrial coculture. This expression was not enhanced significantly by treatment with oestradiol (3.7 x 10(-5) mol l-1) or progesterone (3.2 x 10(-6) mol l-1) or both hormones. These results indicate that LIF could have a dual action in early embryogenesis as an embryotrophin and as a factor required for embryo implantation. Multiple roles for LIF are consistent with the expression of this factor at embryonic, extraembryonic and maternal sites during early embryogenesis.

Effect of mitochondrial DNA (mtDNA) type on pregnancy rate after embryo transfer in rats

Because the mtDNA is maternally inherited, embryos resulting from matings or artificial inseminations have the same type of mtDNA as that of the mother. However, when embryos are transferred the mtDNA of the embryos may be different to that of the recipient and this may interfere with maternal recognition and the establishment of pregnancy. This study was done to determine whether differences in the mtDNA between embryos and recipients would influence the survival to term of transferred embryos.A total of 1,220 rat embryos were recovered from non-superovulated donors of known mtDNA type. The number and distribution of developmental stages of embryos collected from 51 rats of mtDNA type A (n = 595) were not different (P>0.05) from those collected from 50 rats of mtDNA type B (n = 625). The overall pregnancy rate after transfer of embryos to pseudopregnant rats was 54% ( 26 48 ). The pregnancy rate was not affected (P>0.05) by the type of mtDNA of the recipient or of the embryo, and the interaction between mtDNA type of embryos and recipients was also not significant (P>0.05). Embryonic survival to birth was low ( 78 622 , 12.5%) but was not affected (P>0.05) by the type of mtDNA of the recipient ( A = 28 250 ; B = 50 372 ) or of the embryo ( A = 41 306 ; B = 37 316 ). Survival of pups to weaning was affected by the type of mtDNA of the embryo (P < 0.01) but not by the type of mtDNA of the recipient (P>0.05) and the interaction between mtDNA type of embryos and recipients was also not significant (P>0.05). More pups (P < 0.005) derived from donor rats of mtDNA type A ( 34 41 ) survived to weaning age than pups from donor rats of mtDNA type B ( 18 37 ). These results indicate that differences in the type of mtDNA between embryos and recipients do not interfere with establishment of pregnancy in pseudopregnant recipients.

The developmental potential of mouse oocytes matured in serum-free culture conditions.

Studies were undertaken to identify serum-free conditions for the maturation of mouse oocytes in vitro. Oocytes were recovered from the antral follicles of juvenile mice 48 h after injection with gonadotrophin and allowed to resume meiosis in modified Ham's F-10 (mHF-10) medium unsupplemented or supplemented with bovine serum albumin (BSA), fetal calf serum, human pre-ovulatory serum, human follicular fluid or EDTA. They were inseminated 14-16 h later, scored for polar body extrusion after 4-6 h with spermatozoa, and transferred to protein-free mHF-10 for further development. In-vivo matured ova were inseminated and cultured in parallel as controls. Fertilization and development were scored as two cells 24 h after insemination and blastocysts 4 days following insemination respectively. Surprisingly, 41% of oocytes cultured in unsupplemented mHF-10 completed meiosis I, and of those, 50% fertilized; serum supplementation did not improve maturation or fertilization rates. Although the addition of human follicular fluid to the mHF-10 improved meiosis (69%) and fertilization (68% of eggs with polar bodies) to levels comparable with the in-vivo control eggs (79 and 66% respectively), BSA supplementation was equally beneficial. Blastocyst development varied, but within each maturation/fertilization group, the development from in-vitro matured eggs was comparable with embryos from in-vivo matured eggs. In addition, two out of eight 4-cell embryos from oocytes cultured in mHF-10 with BSA and EDTA gave rise to apparently normal pups following transfer to pseudopregnant recipients.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth of mouse pups derived from biopsied blastocysts

To test the hypothesis that removal of cells from the pre-embryo affects intrauterine growth, as reflected in birth weight or growth to weaning. ICR Swiss albino and C57Bl/6 mice served as embryo donors to provide two strains of distinct coat colors. Alternate strains were assigned to control treatment or blastocyst biopsy of trophectoderm. Embryos from both treatments were transferred to a different uterine horn within each pseudopregnant recipient. A total of 412 embryos (206 controls and 206 biopsied blastocysts) were transferred to 26 recipients, which resulted in the birth of 122 pups. There was no significant difference (P > .1) in birth weight or growth to weaning, defined as [[(weaning weight-birth weight)/birth weight)] x 100], between male and female pups. There was a significant difference (P < .01) between pups derived from ICR (birth weight 1.9 +/- 0.02 g; growth to weaning 544 +/- 14%; n = 72) versus C57Bl/6 (birth weight 1.43 +/- 0.03 g; growth to weaning 460 +/- 14%; n = 50) embryos. However, there was no significant difference (P > .1) in birth weight or growth to weaning for pups derived from biopsied embryos (birth weight 1.70 +/- 0.03 g; growth to weaning 502 +/- 14%; mean +/- standard error of the mean; n = 67) or control embryos (birth weight 1.71 +/- 0.05 g; growth to weaning 520 +/- 17%; n = 55). These data support the null hypothesis that embryo biopsy does not result in reduced birth weight or altered subsequent growth to weaning.

Sex-linked differences in developmental potential of single blastomeres from in vitro-fertilized 2-cell stage mouse embryos.

Single blastomeres were isolated from 2-cell mouse embryos and analyzed for their sex using the partially deleted Y chromosome as a marker. Sex identification of 83% of the embryos was achieved with conventionally Giemsa-stained chromosome preparations. The other half-embryos were cultured individually and were transferred to pseudopregnant recipient females. The implantation rate was significantly higher in the male half-embryos. Although 6% of the half-embryos were male. The male half-embryos contained more blastomeres in the blastocyst stage than female ones. These facts suggests that the potential for the development of male half-embryos into fetuses is different from that of female half-embryos.

Preimplantation development of tetraploid mouse embryo produced by cytochalasin B.

Tetraploid mouse embryos usually cease to develop early after implantation, though they can develop to blastocysts. To characterize the failure of development in detail, tetraploid mouse embryos at the preimplantation period were examined as to both their morphology and number of cells. The tetraploid embryos were produced by 12 hr treatment with cytochalasin B (CB) at the 2-cell stage of backcross of (C57BL/6 x C3H/He) F1 x C3H/He. The tetraploid embryos in the preimplantation period exhibited compaction at 72 hr after hCG injection and blastocyst formation at 96 hr, as well as diploid embryos, but the number of cells composing the embryos was significantly smaller than that in the diploid embryos. At the term 60-96 hr after hCG injection, mean cell cycles were 14.03 hr in the tetraploid embryos, but 12.02 hr in the diploid. When tetraploid embryos were transferred into the oviducts of pseudopregnant recipients immediately after CB treatment, the number of cells in tetraploid blastocysts was increased compared with the embryos cultured in vitro, though the number did not reach that of diploid embryos. These results suggested that compaction and blastocyst formation in preimplantation development of tetraploid embryos depended on the time after hCG injection, irrespective of the number of cells or the length of the cell cycle. The lengthening of the cell cycle in tetraploid embryos may be one of the causes of failure in postimplantation development.

Conversion of the genetic background of transgenic mice by in vitro fertilization.

We attempted to convert the genetic background of transgenic (Tg) mice in a short period of time by applying in vitro fertilization techniques. Tg mice were obtained by injecting human interleukin-2 (hIL-2) gene into the fertilized eggs of the C57BL/6N strain. These Tg mice were back-crossed 8 times to the inbred C3H/HeN strain using the hIL-2 gene as the genetic selection marker. In order to shorten the length of the back-crossing time, in vitro fertilization was performed with eggs collected from immature Tg females and spermatozoa from mature C3H/HeN males, and successfully fertilized eggs (2-cell stage) were transferred to pseudopregnant recipients to obtain the offspring of next generation. When no offspring were obtained through the procedures using immature Tg females, in vitro fertilization was performed with mature Tg males and mature C3H/HeN females to continue successive back-crosses. With this method, it was possible to perform 8 successive back-crosses in 18 months.

Developmental potential of microbiopsied marine blastocysts

To evaluate the frequency of mouse pups born following blastocyst biopsy and embryo transfer compared to nonbiopsied controls. ICR Swiss albino and C57B1/6 mice served as embryo donors. Pregnant mare serum gonadotropin treatment was followed 46-52 hours later by hCG, when donors were paired with fertile males. Mating was confirmed the following day and embryos were collected on the afternoon of day 4. After overnight culture, hatching trophoblast was excised by micromanipulation with a fine-pulled glass pipette. Embryos (206 controls, 206 biopsied) were transferred to 26 pseudopregnant recipients. Alternate mouse strains were used to identify pups born from control or biopsied embryos. The end point was percentage of pups born ((number born/number transferred) x 100), using angular transformation before analysis. There was no significant difference (P > .1) between percent live-born in control (27.7%) or biopsied (34.5%) embryos, nor were there any strain differences. These data support the hypothesis that the developmental potential of murine blastocysts is not affected adversely by the biopsy procedure.

The creation of mouse models for human diseases associated with reproductive disturbances by in vitro fertilization and embryo transfer.

The induction of ovulation by hormone treatment, preparation of fertilized eggs by in vitro fertilization and recovery of offspring by embryo transfer were studied in five strains of mutant mice: C57BL/6-dy/dy progressive muscular dystrophy model, C57BL/6-ob/ob obesity model, and BALB/c-rl/rl, BALB/c- shi/shi and C57BL/6-mld/mld motor ataxia models. The homozygotes of these mutant mice are all affected with the disease about 2 weeks after birth, followed by reproductive disturbances. Ovulation could be induced by injection with PMSG-hCG in the females. Sperm was obtained from the cauda epididymis of males and used for in vitro fertilization. The success rate of the in vitro fertilization was as low as 71.6% in C57BL/6-dy/dy mice, but was over 85% in the other strains. When 2-cell embryos obtained by in vitro fertilization were transferred to the oviducts of pseudopregnant recipients, offspring were obtained from 39.2-57.7% of the transferred embryos. These offspring developed the expected diseases about 2 weeks after birth, and it was confirmed that the disease characters were reliably reproduced. These results demonstrate that the experimental system of in vitro fertilization and embryo transfer enables production of the offspring homozygous for a mutant gene and use of them for experiments before the onset of the disease which has been impossible.

Nicotinamide, a component of complex culture media, inhibits mouse embryo development in vitro and reduces subsequent developmental potential after transfer

To determine the effects of B-group vitamins present in culture media on mouse embryo development in vitro and subsequent viability. Mouse zygotes were cultured in the presence of B-group vitamins. Embryo morphology and cell numbers were determined at 96 and 120 hours after hCG. Viability was assessed by transfer of embryos after 3 days of culture to pseudopregnant recipients. Resultant pregnancy rates (PRs) and fetal weights were determined. Supplementation of an amino acid-free medium with minimal essential medium (MEM) B-group vitamins significantly decreased embryo cleavage rates, whereas the inclusion of Ham's F-10 medium B-group vitamins significantly reduced both cleavage rates and morphological development. Subsequent experiments determined that nicotinamide (5 microM) significantly reduced blastocyst cell number, implantation rate, viable PR, and fetal weight. The data indicate that nicotinamide inhibits mouse embryo development in culture and reduces viability. Nicotinamide is present at high levels in Ham's F-10 and MEM media that are used routinely in human embryo culture. The role of vitamins in human embryo development in vitro warrants investigation.

Quick freezing of unfertilized mouse oocytes using ethylene glycol with sucrose or trehalose

Unfertilized mouse oocytes were frozen by directly plunging them into liquid nitrogen vapour after equilibration in a freezing medium containing 3 mol ethylene glycol l-1 with 0.25 mol sucrose or trehalose l-1 for 5-40 min. After thawing and dilution of the cryoprotectant, oocytes of normal morphology were inseminated in vitro and the effect of equilibration period on the rates of fertilization and development in vitro was examined. Regardless of the equilibration in the freezing medium, no significant difference was observed on the fertilization rate of frozen-thawed oocytes. However, higher fertilization and higher normal fertilization rates were obtained with equilibration in 3 mol ethylene glycol l-1 with either 0.25 mol sucrose l-1 or trehalose for 20 and 40 min than with 5 and 10 min equilibration. Development rates to two-cell embryos and expanded blastocysts of in vitro fertilized frozen-thawed oocytes that were equilibrated in the freezing medium for 20 and 40 min were significantly higher (P < 0.05 or P < 0.01) than with 5 min equilibration. Development in vivo was assessed by transferring blastocysts derived from unfertilized oocytes frozen by the optimum treatment (20 min equilibration in the freezing medium before freezing) into the uterine horns of day 3 pseudopregnant female recipients. The development rate of frozen-thawed oocytes to the blastocyst stage after insemination in vitro was significantly lower than that of the non-frozen control (P < 0.001). However, transfer of the blastocysts derived from frozen-thawed oocytes to the uterine horns of the recipients in fetal development and implantation rates similar to those of the control.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the reduction of cytoplasm of mouse 2-cell embryos on blastocele formation timing and developmental ability in vitro and in vivo

Experiments were conducted to test the hypothesis that the nucleo/cytoplasmic ratio of mouse embryos determines the time of blastocele formation. Half the volume of 2-cell stage embryos was removed from each blastomere by micropipette to alter the nucleo/cytoplasmic ratio. Reduced embryos whose nucleo/cytoplasmic ratio increased and non-treated control embryos were cultured in vitro to compare the timing of division to the 4-cell stage and blastocele formation. Reduced 2-cell embryos formed blastoceles significantly earlier than the controls (49.0 ±2.9 vs 52.2 ±6 h) and with fewer cells, although division into the 4-cell stage was significantly delayed (11.4 ±4.4 vs 9.0±2.4 h). The cell number of blastocysts 70 h after treatment and developmental ability of blastocysts after transfer to pseudopregnant recipients were the same for the reduced and control groups. The present study indicates that the nucleo/cytoplasmic ratio of embryos may possibly be an important factor that determines the time of blastocele formation.

Gene transfer efficiency during gestation and the influence of co-transfer of non-manipulated embryos on production of transgenic mice.

Litter size of DNA microinjected zygotes is lower than for non-manipulated zygotes. The rate of embryonic and fetal survival in early, mid and late gestation was determined to assess whether DNA integration was responsible for embryonic losses. Also, the effect of including non-microinjected embryos with injected embryos on pregnancy rate and transgenic pup production was determined. In Experiment 1, one-cell embryos from immature CD-1 mice were microinjected with a whey acidic protein promoter-human protein C gene construct. One hour after microinjection embryos were transferred to pseudopregnant recipients (45 transfers of 30 embryos each). Fifteen recipients were sacrificed on day 4, 12 and 18 of gestation and the embryos/fetuses analysed for the transgene. The percentage of embryos or fetuses that were positive for the transgene was not significantly different at any day. However, the number of viable embryos at day 4 was significantly greater than fetuses on days 12 or 18. In addition, a high degree of mosaicism was observed in day 18 fetuses and placentae recovered. In Experiment 2, one-cell embryos from CD-1 mice were microinjected and co-transferred with non-manipulated embryos (C57BL/6). Pregnancy rate and the total number of pups born were improved by addition of non-injected embryos. However, the number of transgenic mice produced was similar whether non-injected embryos were included or not. There were 32.2% (15/46) transgenic pups when 0 non-injected embryos were transferred compared with 15.1% (13/86) transgenic pups when 4 or 8 non-injected embryos were added to the transfers. In summary, a high degree of embryonic and fetal mortality occurs among microinjected embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of superovulatory response of mature outbred mice treated with FSH or PMSG and developmental potential of embryos produced

A superovulatory treatment for mice based on FSH administration was compared with a standard one based on PMSG. Our aim was to determine if a mean number of embryos recovered per donor could be increased and if in vitro or in vivo viability was affected by the hormonal treatment used. Thus, female Swiss mice were subjected to 2 superovulatory treatments, and the 1-cell and 2-cell stage embryos were cultured in 2 different media to the blastocyst stage or were transferred to pseudopregnant recipients. The data show that despite a lower mating percentage (52% with FSH vs 66% with PMSG), the FSH-treated mice provided twice the number of total embryos (53.4 vs 24.5) with a similar percentage of morphologically normal embryos (74% for FSH vs 69% for PMSG). We also found that in vitro culture results can be influenced by the source of gonadotropins depending on the culture medium used. A culture medium such as CZB which prevents the 2-cell block, provided the same developmental rates regardless of hormonal treatment used. However, with M-16 medium, which does not prevent this blockage, only 39% of the 2-cell FSH-derived embryos and 49% of the PMSG-derived 2-cell embryos developed into blastocysts (P<0.05). FSH-derived embryos resulted in a higher percentage of pregnant recipients (73 vs 56%) than PMSG-derived embryos, but the number of alive fetuses and the number of implantations per pregnant recipient was affected only by the kind of culture system used before transfer. The results show that FSH can provide very good superovulatory response in mice, thus reducing the number of donors needed for a given experiment and providing embryos of at least the same quality as those derived from the standard PMSG treatment.

Development of Whole and Demi-embryos of Mice in Culture and in Vivo after Supercooled Storage

Demi-embryos (produced by destroying 1 or 2 blastomeres of 2- or 4-cell embryos, respectively) and intact mouse embryos were cultured to the blastocyst stage, stored at - 5°C for 48 h, then cultured for 24 h and transferred into pseudopregnant recipients. Supercooled storage did not impair the developmental potential of whole or demi-embryos in vitro, nor was there a difference between whole and demi-embryos with respect to growth in vitro. Similarly, there was no effect of supercooling on development of intact or demi embryos after transfer into pseudopregnant recipient mice, but fewer recipients of demi-embryos remained pregnant ( P < 0.05). This was considered to be partly due to the lesser ability of demi-embryos to maintain luteal function and establish pregnancy.

Blastocyst's state of activity determines the "window" of implantation in the receptive mouse uterus.

The present investigation studied the influence of the blastocyst's state of activity on the "window" of implantation in the receptive uterus in the mouse. The receptive state of the uterus is defined as the limited time when the uterine milieu is favorable to blastocyst acceptance and implantation. In the mouse, implantation occurs on day 4 (day 1 = vaginal plug). Ovariectomy in the morning of day 4 prior to preimplantation estrogen secretion results in blastocyst dormancy and delayed implantation. These conditions are maintained by continued progesterone (P4) treatment but can be terminated with an injection of estrogen leading to blastocyst activation and subsequent implantation. Blastocyst transfers into intact pseudopregnant mice demonstrated that the window of implantation on day 4 remains open at least through 1800 h for normal day 4 blastocysts but only up to 1400 h for dormant blastocysts. These results suggested that the blastocyst's state of activity influenced the normally operative window of implantation in the receptive uterus. This finding was further confirmed by inducing conditions of delayed implantation in pregnant donors and pseudopregnant recipients. They were ovariectomized on the morning of day 4 and maintained with daily injections of P4 from days 5 to 7. On day 7, dormant blastocysts from P4-treated delayed donors were transferred into the uteri of P4-treated delayed pseudopregnant recipients at 1, 2, 4, or 8 h after an injection of 17 beta-estradiol (E2). Dormant blastocysts transferred into delayed recipients at 1 h after E2 treatment resulted in implantation in most of the animals as compared to complete failure of blastocysts to implant after transfer to P4-treated delayed recipients at 4 or 8 h after E2 treatment. However, implantation did occur in P4-treated delayed recipients at these later hours of E2 treatment when the P4-treated delayed donors also received E2 prior to blastocyst transfer. Furthermore, the majority of day 4 normal blastocysts implanted when transferred into P4-treated delayed recipients even at 16 h after E2 treatment. Interestingly, day 7 dormant blastocysts cultured for 8 or 24 h for in vitro activation failed to implant after transfer to P4-treated delayed pseudopregnant recipients at 4 ir 8 h after E2 treatment, although they did implant after transfer at 1 h after E2 treatment. As expected, normal day 4 blastocysts failed to implant after transfer to P4-treated delayed pseudopregnant recipients. Thus, these results establish that the blastocyst's state of activity alters the timing of implantation (window) in the receptive uterus. Thus, the window for successful implantation could be defined as a limited time span when the activated stage of the blastocyst is superimposed on the receptive state of the uterus. This window remains open for a shorter period for dormant blastocysts than for a normal or dormant blastocysts after E2 activation. Furthermore, dormant blastocysts, which apparently achieved metabolic activation in vitro, failed to attain the same status as blastocysts activated in utero by E2 for implantation into the receptive uterus. A key finding of this investigation is that E2 induces very rapidly, but transiently (1 h), a factor(s) in the P4-primed uterus that activates the dormant blastocysts for implantation in the receptive uterus.

Cryopreserved mouse embryos can successfully survive biopsy and refreezing.

We examined whether cryopreserved mouse eight-cell embryos could be thawed, biopsied, refrozen, thawed, and grown in vitro and in vivo in two sets of experiments. In the in vitro studies, the blastulation rate of cryopreserved embryos which had been thawed-->biopsied-->refrozen-->thawed-->grown 48 hr in vitro were compared with that of sham-operated (zona dissected) and untreated control embryos (twice frozen only). For the in vivo studies, five control embryos were transferred to one horn and five experimental embryos were transferred to the contralateral horn of day 3 pseudopregnant recipient female mice. Recipient mice were sacrificed on day 18. The day 2 blastulation rate was the same for the control, sham-operated, and biopsied embryos when examined in vitro. In the first set of in vivo studies, 42.7% of control and 39.7% of sham-operated embryos that had been transferred implanted, and most embryos progressed to day 18. In the second set, 45.6% (57/125) and 39.7% (49/125), respectively, of the transferred embryos progressed to day 18 fetuses. There were no significant differences in the rate of fetal development in the different groups. These studies demonstrate that cryopreserved mouse eight-cell embryos can successfully undergo thawing, biopsy, and refreezing. The results suggest that under certain conditions, it may be possible to utilize cryopreservation in strategies involving human genetic diagnosis in the preimplantation period.

High Rates of Survival and Fertilization of Mouse and Hamster Oocytes after Vitrification in Dimethylsulphoxide1

A high proportion (> 70%) of mouse and hamster oocytes exposed for 3-5 min to 1.5 M dimethylsulfoxide (DMSO) and washed briefly in 3.9 M DMSO before vitrification in 6.0 M DMSO appeared morphologically normal on recovery. Significantly fewer (< 46%) mouse oocytes appeared normal when the time of exposure to 1.5 M DMSO was reduced to 1 min or less. The rate of fertilization in vitro of vitrified oocytes was reduced compared to the rate for untreated controls (mouse: 79% vs. 94%; hamster: 73% vs. 87%). After removal of the zona pellucida, fertilization was similar in vitrified and control hamster oocytes inseminated with hamster (> 90%) or human (21% vs. 23%) sperm. Sperm nuclear decondensation and pronuclear formation appeared to be delayed in the cytoplasm of vitrified hamster oocytes. Seventy-nine percent of 2-cell-stage mouse embryos derived from vitrified oocytes implanted after transfer to pseudopregnant recipients, but only 40% developed to normal fetuses compared to 61% of controls. The reason for this high rate of postimplantation loss is unknown.