Variable expressivity of the tumour suppressor protein TRP53 in cryopreserved human blastocysts

In a mouse model, in vitro fertilization or extended embryo culture leads to the increased expression of TRP53 in susceptible embryos. Ablation of the TRP53 gene improved embryo viability indicating that increased expression of TRP53 is a cause of the reduction of embryo viability resulting from in vitro fertilization or embryo culture. This study investigates the status of TRP53 expression in human embryos produced by intracytoplasmic sperm injection. Following fertilization, embryos were cultured for 96 h and then cryopreserved. Immediately upon thawing they were fixed in formaldehyde and subjected to immunostaining for TRP53. Staining was visualized by confocal microscopy. Negative controls were incubated with isotype control immunoglobulin and showed negligible staining. All embryos showed TRP53 staining above negative controls. TRP53 staining was heterogenous within and between embryos. An embryo that showed retarded development showed high levels of TRP53 expression. A blastocyst that had a collapsed blastocoel also showed high levels of TRP53 compared to morphologically normal blastocysts. Most TRP53 staining was in the region of the nucleus. Morphologically normal blastocysts tended to show little nuclear accumulation of stain. However, some cells within these embryos had high levels of nuclear TRP53 expression. The results show that embryos have varying sensitivity to the stresses of production and culture in vitro, and this resulted in variable expressivity of TRP53.     

The effect of Trp53 gene-dosage and parent-of-origin of inheritance on mouse gamete and embryo function in vitro

The transformation-related protein 53 (TRP53) has a canonical role as the "guardian of the genome," serving to protect against the propagation of cells with genomic damage. Autocrine trophic signals act to block the accumulation of TRP53 in the normal preimplantation embryo. Culture of the early embryo at limiting dilutions in simple defined media limits autocrine signaling, resulting in the accumulation of TRP53. This TRP53 reduces the rate of development of embryos. In this study we show that deletion of the Trp53 gene improved development in vitro in a dose-dependent manner. Development to morphological blastocysts increased as the dose of Trp53 was reduced, and this was accompanied by a Trp53-dependent increase in the allocation of cells to the inner cell mass. The intermediate developmental response of heterozygous mice provides evidence for haploinsufficiency of this trait. This haploinsufficiency was evident irrespective of the parent-of-origin of the null allele; however, zygotes with paternal inheritance of the Trp53-null allele had better development in vitro than those with maternal inheritance. There was a beneficial effect of the Trp53-null allele on the number of oocytes released by Trp53(+/-) females, and heterozygous males produced higher fertilization rates than controls, although this was independent of the genotype of the fertilizing sperm. The study shows that ovulation induction or culture of embryos in limiting conditions creates conditions that favor the early development of embryos inheriting loss of Trp53 function. This occurs even in the heterozygous state, showing that the conditions provide a potential basis for accelerated accumulation of deleterious mutations within a population.     

Optimisation of in vitro culture conditions in B6CBF1 mouse embryos

This study was undertaken to investigate the effects of three media, volume and type of oil and frequency of observation on the in vitro development of mouse zygotes. B6CBF1 female mice (4 to 6 wk old) were superovulated using PMSG/hCG and mated with a proven fertile male of the same strain. Putative zygotes with polar bodies were collected from the oviducts of mated mice, 25-28 h after hCG injection, and were cultured in vitro. Embryo development was evaluated at either 96 h and 120 h or every 24 h for 120 h. The results obtained showed that the CZB medium was better than the KSOM and HCO3HTF media, and the use of 1 mL of paraffin oil was better than the use of 0.5 mL of paraffin oil. The effect of paraffin oil and mineral oil on embryo development was examined and the results indicated that the use of paraffin oil was better than the use of mineral oil. Repeated observations did not influence the proportion of embryos developing to blastocysts.     

Evaluation of mouse blastocyst implantation rate by morphology grading

The aim of our study is to observe the relationship between the blastocyst morphology and the implantation rate for mice. Mouse embryos obtained from the superovulated-ICR mice were cultured in vitro from 1-cell zygotes to blastocysts. Mouse blastocysts were then classified into 3 grades: grade I, small blastocysts; grade II, large blastocysts; grade III, hatching blastocysts. They were independently transferred into the uterus of recipient females mated with vasectomized male mice on 96 hours after the zygotes were cultured in vitro. The successful implantation was checked by injection of Chicago Sky Blue 6B on the second day after embryo transfer. Although there was no significant difference in the implantation rates between the grade III and grade II, grade I was significantly decreased, as compared with grade III. Grade I and grade II was also significantly decreased in both the diameter of blastocysts and cell number of inner cell mass (ICM) and trophectoderm (TE), as compared with grade III. These findings indicate that the expanded and hatching blastocyst selections for embryo transfer in in vitro fertilization were evaluated with the high implantation rate.     

Birth of piglets derived from porcine zygotes cultured in a chemically defined medium.

We evaluated the in vitro development of porcine zygotes that were cultured in a novel culture medium, porcine zygote medium (PZM), under different conditions and compared to in vivo development. The viability of these zygotes to full term after culture was also evaluated by embryo transfer to recipients. Porcine single-cell zygotes were collected from gilts on Day 2 after hCG injection. Culture of zygotes in PZM containing 3 mg/ml of BSA (PZM-3) produced better results in terms of proportion of Day 6 blastocysts, Day 8 hatching rate, and numbers of inner cell mass (ICM) cells and total cells in Day 8 embryos than that in North Carolina State University (NCSU)-23 medium. In culture with PZM-3, embryo development was optimized in an atmosphere of 5% CO2:5% O2:90% N2 compared to 5% CO2 in air. The ICM and total cell numbers in Day 6 embryos cultured in PZM-3 or in PZM-3 in which BSA was replaced with 3 mg/ml of polyvinyl alcohol (PZM-4) were also greater than those of NCSU-23 but less than those developed in vivo. However, no difference was found in the ratio of ICM to total cells among embryos developed in PZM-3, PZM-4, or in vivo. When the Day 6 embryos that developed in PZM-4 (99 embryos) or in vivo (100 embryos) were each transferred into six recipients, no difference was found in the farrowing rate (83.3% for both treatments) and in the number of piglets born (33 and 42 piglets, respectively). Our results indicate that porcine zygotes can develop into blastocysts in a chemically defined medium and to full term by transfer to recipients after culture.     

Culture of early stage ovine embryos to blastocyst enhances survival rate after cryopreservation

The current study assessed both the effects of in vitro culture and developmental stage of early stage in vivo produced ovine embryos on their ability to survive cryopreservation. Early stage embryos (n=226) were recovered from the oviduct, at different days of the early luteal phase, at three different developmental stages: 2- to 4-cell, 5- to 8-cell and 9- to 12-cell. For each stage, half of the embryos were cultured to the blastocyst stage and frozen thereafter (CF), while the remainder was frozen just after recovery (EF). A third experimental group (BF; n=43) included blastocysts obtained from the uterus and frozen immediately after recovery. Embryo viability post-thawing was determined by assessing their rate of development to the hatched blastocyst stage following in vitro culture. Culture negatively affected embryo viability, since survival rate was higher in blastocysts obtained from the uterus than in those from culture (83.7% versus 66.1%; P<0.05); also the cryosurvival of cultured embryos was lower when the timing of blastocyst formation was extended (P<0.01). However, survival following freezing-thawing of early developmental stages was significantly lower when compared to viability of their counterparts cultured to the blastocyst stage (23.1% versus 66.1%, P<0.0001). In conclusion, our results indicate that, despite the deleterious effects of culture per se, the culture of early in vivo produced ovine embryos to the blastocyst stage prior to be frozen improves their survival after thawing.     

Fetal development of mouse oocytes and zygotes cryopreserved in a nonconventional freezing medium

This study (1) analyzed fetal development of mouse embryos after oocyte cryopreservation in CJ2, a choline-based medium, (2) examined the effect of culture duration in vitro on subsequent fetal development, and (3) compared survival and fetal development of zygotes frozen in embryo transfer freeze medium (ETFM; sodium-based medium) or CJ2. Unfertilized oocytes and zygotes were cryopreserved using a slow-cooling protocol. After thawing, oocytes were inseminated after drilling a hole in their zona, cultured in vitro either to the two-cell or blastocyst stage, and transferred to the oviducts or uterine horns of recipient mice. In parallel experiments, frozen-thawed zygotes were similarly cultured and transferred. Implantation rates for transferred embryos were high (range 66-88%), regardless of whether they had been frozen as oocytes or zygotes and whether they had been transferred to the oviduct or uterus. However, fetal development was significantly higher when two-cell embryos were transferred. With blastocyst transfer, control embryos implanted and produced a greater proportion of fetuses than did oocytes frozen in CJ2, whereas transfer at the two-cell stage resulted in similar proportions of implantation sites and fetuses. Blastocyst transfer of zygotes cryopreserved in ETFM or CJ2 produced similar fetal development rates (23.6% vs 20.0%), but when frozen-thawed zygotes were transferred at the two-cell stage the fetal development rates were higher in the ETFM group (53.3%) than in the CJ2 group (32.0%). A high proportion (46.7%) of oocytes frozen in CJ2 in a nonprogrammable freezer and plunged at -20 degrees C developed into live offspring. This study shows that in the mouse (1) oocytes frozen in CJ2 can develop into viable fetuses, (2) prolonging culture in vitro has a detrimental effect on embryo transfer outcome, and (3) CJ2 offers no advantage for zygote cryopreservation.     

Developmental patterns of zygotes from transgenic female mice with elevated tissue glutathione

Zygotes were collected from transgenic mice overexpressing glutathione synthetase to test the hypothesis that such zygotes are more capable of developing in suboptimal culture media than zygotes from non-transgenic (control) mice. The effects of injection of donor mice with gamma-glutamylcysteinyl ethyl ester (gamma-GCE) on embryonic development were also investigated. In addition, the effects of genetic background (i.e., transgenic vs. non-transgenic) and injection with gamma-GCE on developmental capacity in kSOM were studied after exposure of zygotes to diamide (0.01 mM, for 30 min). When cultured in modified Medium 16 significantly more pronuclear ova from transgenic females reached the morula (M) and early blastocyst (EB) stages than embryos derived from control mice. Genetic background significantly affected the proportions of embryos reaching 4- to 16-cell, M and EB stages during culture in modified Whitten's medium (WM); more zygotes collected from transgenic than from control mice developed. The injection of experimental mice with gamma-GCE significantly increased proportions of zygotes developing to M and EB stages in WM. Following exposure to diamide and subsequent culture in kSOM significantly more zygotes collected from transgenic mice reached the 4- to 16-cell stages than those from control females; a significant positive effect on developmental capacity was also seen after injection of donor mice with gamma-GCE. When cultured in suboptimal conditions, zygotes derived from transgenic mice overexpressing glutathione synthetase were more capable of developing than zygotes of non-transgenic control females. Zygotes from the transgenic mice also exhibited greater capacity to withstand toxic exposure to diamide. Present data suggest that commonly known strain differences in preimplantational development in vitro may reflect differences in the synthesis and/or metabolism of glutathione. J. Exp. Zool. 286:173-180, 2000.     

Mouse zygote development in culture medium without protein in the presence of ethylenediaminetetraacetic acid

Development of zygotes from a hybrid-inbred (B6D2F1) and two random-bred (CD1 and CF1) strains of mice were compared after culture in several modifications of a simple, chemically defined medium based on Earle's Balanced Salts Solution. When cultured without the addition of protein or the chelating agent, ethylenediaminetetraacetic acid (EDTA), none of the zygotes reached the blastocyst stage. The addition of EDTA or protein significantly improved embryo development to blastocysts (p less than 0.05). The degree of improvement was dependent upon the strain of the female (85% or 91% for B6D2F1, 56% or 45% for CD1, and 19% or 28% for CF1, respectively). The addition of protein to the media in the presence of EDTA did not further improve embryo development. In all supportive conditions, zygotes from B6D2F1 females developed to blastocysts better than those from CD1 or CF1 females; embryos of the latter strain exhibited the lowest rates of development in vitro. Glycine and alanine (20 microM) partially substituted for EDTA; the decreased hybrid-inbred embryo development to blastocysts (20% and 26%, respectively) obtained in the presence of the amino acids suggested, however, that the stimulatory effect of EDTA on embryo development was other than as a source of fixed nitrogen. The rates of development observed with an alternate chelating agent, citric acid (less than or equal to 20% vs. 83% blastocysts, p less than 0.01), although better than the unsupplemented medium, were significantly less effective than EDTA-supplemented medium (83% blastocysts, p less than 0.01). The results of this study suggest that the protective effect of proteins in culture medium may be more important than their nutritive role.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of in vitro development and gene expression of in vivo- and IVM/IVF-derived porcine embryos after microinjection of foreign DNA

We compared the developmental ability and gene expression of in vivo- and IVM/IVF-derived porcine embryos following microinjection with SV40-LacZ. A total of 412 IVM/IVF-derived and 129 in vivo-collected zygotes was used to examine developmental ability and gene expression following DNA microinjection. When either DNA injected or noninjected zygotes were cultured for 4 d in NCSU 23 followed by 5 d in Eagle's minimal essential medium (EMEM), the percentages of zygotes developing to blastocysts and hatched blastocysts were higher (P < 0.05) compared with groups cultured in NCSU 23 alone. The percentages of injected embryos reaching the morula and blastocyst stages were significantly lower (P < 0.05) than that of noninjected control embryos whether in vivo or IVM/IVF derived. The percentages of morula and blastocyst stage embryos expressing the gene were higher in the in vivo-derived embryos than in IVM/IVF-derived embryos. A lower proportion of (67 to 77%) mosaicism was observed in the in vivo-derived embryos than in IVM/IVF (90 to 100%) derived embryos. The total cell number of blastocysts cultured in both NCSU 23 and EMEM media was significantly higher than that of blastocysts cultured continuously in NCSU 23. Our results suggest that this dual culture system enhanced embryo viability following microinjection of foreign DNA.     

Effects of the culture density of mouse zygotes on the development in vitro and in vivo

Different numbers of CD-1 mouse zygotes(1, 5, 10, 20, 40 and 60) were cultured in 10 mul M16 medium, in M16 medium+EDTA, in M16 dedium+SOD+thioredoxin, and in CZB medium, respectively. When the zygotes, regardless of the number, were cultured with M16, no blastocysts could be obtained. The suitable ratio of embryos to 1 mul of M16 medium+EDTA or M16 medium+SOD+thioredoxin was 1:1 or 2:1. Medium volume from 1 to 10 mul did not affect blastocyst development when the embryo density was 1:1. However, blastocysts obtained from zygotes cultured singly had fewer cell numbers and showed inferior development to live fetuses after transfer to recipients. When CZB medium was used, suitable embryo density was not clear. The ratio of embryos to volume of culture medium was shown to be an important factor for in vitro culture of mouse zygotes.     

Number and viability of embryos collected in vivo or from the excised uteri of slaughtered donor cows

Embryos were collected from superovulated donor cows on Day 7 of the cycle either in vivo by a standard nonsurgical method (A) or in vitro from the excised uterus after slaughter of the donor cow (B). In Method B, the time between slaughter and flushing of the uterus ranged from 0.5 to 4 hours. Flush yield was 5.6 +/- 5.0 and 8.4 +/- 5.3 embryos (P<0.01); the recovery rate was 0.6 +/- 0.4 and 0.8 +/- 0.3 (P<0.05) for Methods A and B, respectively. Method B resulted in more Grade 3 (P<0.001) and 4 embryos, while Method A resulted in more Grade 1 and 2 embryos. The correlation between the percentage of Grade 1 and 2 embryos and the time interval between slaughter of the donor cow and flushing the excised uterus was -0.42 for in vitro flushes. Viability of fresh and frozen-thawed embryos, as determined by in vitro culture of Grade 1 and 2 morulae and early blastocysts, was considerably lower for Method B than Method A. The percentage of embryos developing into expanded blastocysts was 100% (10 10 ) and 40% (4 10 ) for fresh embryos (P<0.01) and 52.7% (29 55 ) and 0% (0 25 ) for the frozen-thawed embryos (P<0.001) for Methods A and B, respectively. This reduction in viability might be the result of a postmortem pH decrease in the uterine fluids within the first 30 minutes from 7.0 to 5.8 and 6.0. Flushing of the uterus directly after slaughter (within 5 to 10 minutes) may prevent the possible detrimental effect of a low pH on the embryos.     

Beneficial effects of taurine on mouse zygotes developing in protein-free culture medium

The objectives of this study were to determine if mouse zygotes from outbred mice can develop in simple culture medium in the absence of bovine serum albumin (BSA), and if taurine can be used as a medium supplement to improve development. Zygotes from 2 stocks of outbred mice (CD-1 and CF-1) were cultured in simple embryo culture medium (TE medium) lacking BSA and with or without taurine (24 mM), or in regular TE medium with BSA. The presence of BSA had little or no effect on development, but development to post-blastocyst endpoints was enhanced when CD-1 zygotes were cultured in medium containing taurine. In addition, when CD-1 blastocysts were transferred to pseudopregnant animals, embryos cultured in the presence of taurine developed into fetuses more often than those cultured in medium without taurine, and their weights were higher than those of embryos cultured in regular TE medium with BSA. These beneficial effects of taurine do not appear to be the nonspecific effects of a fixed nitrogen source, because the addition of glycine to BSA-free TE medium did not have similar beneficial effects. It was concluded that mouse zygotes from outbred mice do not require BSA for their preimplantation development in culture and that the presence of taurine in preimplantation culture medium is beneficial not only for preimplantation development of the zygotes, but also for their post-blastocyst development.     

Intraspecific blastocyst reconstitution in mice using giant trophectodermal vesicles produced by multiple embryo aggregation

Giant trophectodermal (TE) vesicles, produced by aggregating multiple embryos, were evaluated as a means of enhancing the viability of reconstituted murine blastocysts. Previous studies have indicated that the development of chimeric conceptuses produced by multiple embryo aggregation is impaired, thus the initial objective here was to examine how this is affected by 1) differences in developmental rates of individual embryos comprising the aggregates, 2) lengths of in vitro culture, and 3) incompatibilities between maternal hosts and chimeric fetal allografts. The results indicate that the viability of aggregates was influenced by embryo genotype and length of in vitro culture. The average number of B6D2-F1 x BL/6 offspring resulting from aggregates cultured for 0.7 day was not significantly different (p greater than 0.05) from that obtained from either embryo singletons cultured for 0.7 and 1.7 days (2.9 +/- 0.3 and 3.9 +/- 0.4, respectively) or from nonmanipulated controls (3.2 +/- 0.2 and 4.3 +/- 0.3, respectively). Blastocyst reconstitution studies were then conducted using giant TE vesicles from B6D2-F1 x BL/6 mice and inner cell masses from C3H mice. The average number of C3H offspring produced (2.7 +/- 0.1) was not significantly different (p greater than 0.05) from that of B6D2-F1 x BL/6 embryo aggregates (3.2 +/- 0.6) or of nonmanipulated controls (4.3 +/- 0.3). The results demonstrate that giant TE vesicles can be effectively used for intraspecific blastocyst reconstitution in mice.     

Comparison of tetraploid blastocyst microinjection of outbred Crl:CD1(ICR), hybrid B6D2F1/Tac, and inbred C57BL/6NTac embryos for generation of mice derived from embryonic stem cells

Embryo electrofusion and tetraploid blastocyst microinjection is a modification of the traditional embryonic stem cell (ES cell)-based method to generate targeted mutant mice. Viability of tetraploid embryos is reportedly lower than with diploid embryos, with considerable interstrain variation. Here we assessed fetus and pup viability after ES cell microinjection of tetraploid blastocysts derived from outbred, hybrid, and inbred mice. Two-cell mouse embryos (C57BL/6NTac [B6], n = 788; B6D2F1/Tac [BDF1], n = 1871; Crl:CD1(ICR) [CD1], n = 1308) were electrofused; most resultant tetraploid blastocysts were injected with ES cells and surgically transferred into pseudopregnant recipient mice. Reproductive tracts were examined at midgestation for embryologic studies using B6 and BDF1 blastocysts; implantation sites and viable fetuses were counted. Pregnancies were carried to term for studies of targeted mutant mice using BDF1 and CD1 blastocysts, and pup yield was evaluated. Electrofusion rates of 2-cell embryos did not differ among B6, BDF1, and CD1 mice (overall mean, 92.8% +/- 5.4%). For embryologic studies, 244 B6 blastocysts were surgically transferred and 1 fetus was viable (0.41%), compared with 644 BDF1 blastocysts surgically transferred and 88 viable fetuses (13.7%). For targeted mutant mouse studies, 259 BDF1 blastocysts were surgically transferred yielding 10 pups (3.9%); 569 CD1 blastocysts yielded 44 pups (7.7%).     

In vivo culture of embryos in the immature mouse oviduct

In-vitro culture of mammalian preimplantation embryos is associated with subsequent decreased viability. This phenomenon is more pronounced with the domestic species embryos as culture conditions are at present unable to sustain cleavage of early preimplantation embryos for more than one or two cell divisions. In this study, the immature mouse oviduct is shown to be capable of supporting cleavage and morphological development of rabbit and porcine embryos. The immature mouse oviduct was shown to be comparable to in vitro culture as 76% and 60% of the transferred zygotes developed to the morula stage after 2 and 3 d respectively. The porcine zygotes, however, failed to develop beyond the 4-cell stage in either the immature mouse oviduct or in vitro. Porcine morula showed better tolerance of the oviduct environment and when recovered after 2 d contained an average of 64 cells, which was significantly more than in in vitro cultured morulae (40 cells). Early porcine blastocysts transferred to the mouse oviduct had over a two-fold increase in cell division (104 cells) over comparable blastocysts grown in vitro (57 cells). The immature mouse oviduct is, therefore, a potential surrogate environment for short-term storage of embryos of other species.