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Embryo |
The Graduate School of Natural Science and Technology,4 Okayama University, Okayama 700-8530, Japan
Faculty of Agriculture,5 Okayama University, Okayama 700-8530, Japan
College of Agriculture,6 Kochi University, Nankoku, Kochi 783-8502, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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63%) developed to term in reasonably synchronous recipients (Day 0 to -0.5) but not in more asynchronous ones (6%; Day -1). A majority of vitrified morulae also developed to term (52%–68%) in a wider range of recipients (Days 0 to -1), the greatest success occurring in recipients on Day -0.5. Similar proportions of vitrified and fresh 4-cell embryos, 8-cell embryos, and morulae developed to term when appropriate synchronization existed between embryo and recipient. Thus, vitrification of preimplantation-stage rat embryos does not appear to impair their developmental potential in vivo.
early development, embryo, oviduct, pregnancy, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rat embryos have been successfully cryopreserved at various developmental stages, such as the 1-cell [11, 12], 2-cell [13–15], 4-cell [13], 8-cell [13, 16–18], morula [19], and late-morula to early blastocyst [14] stages. In many cases, survival of the embryos was assessed by transfer to recipients, probably because the in vitro culture system for rat embryos was not as effective as that for mouse embryos. Consequently, reported survival rates have been variable but generally low.
In a recent study [20], we compared the survival of vitrified rat embryos, ranging from the 1-cell to the blastocyst stage, using an efficient culture system (in vitro) and a successful embryo-transfer technique (in vivo). Because very high proportions (94%–100%) of vitrified embryos developed in vitro, we concluded that the 4-cell, 8-cell, and morula stages are suitable for embryo cryopreservation; furthermore, the good developmental potential in vitro of these embryos led us to expect they would develop equally well in vivo. However, the in vivo survival rate of vitrified 4-cell embryos was relatively low (40%) and that of 8-cell embryos extremely low (4%), although similar poor results were obtained with fresh embryos (29% and 5%, respectively). In contrast, the in vivo survival rate of vitrified morulae (61%) was high and very similar to that of fresh embryos (70%). In that study, both fresh and vitrified 4- and 8-cell embryos were transferred into oviducts of pseudopregnant recipients that were at a point in pseudopregnancy 1 day earlier than the embryos (Day -1 of synchrony), whereas morulae were transferred to uterine horns of synchronous recipients (Day 0).
We hypothesized that it might be possible to improve the in vivo survival of vitrified embryos at all stages by adjusting the synchrony/asynchrony with respect to the recipient females and by altering the site of transfer. The present study was undertaken to test this hypothesis using rat embryos vitrified at the 4-cell, 8-cell, and morula stages.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Collection of Embryos
Outbred Wistar rats were bred in-house and kept in a room under a 14L:10D photoperiod (lights-on, 0600–2000 h; lights-off, 2000–0600 h). Mature female rats (age, 2–3 mo) at proestrus, as assessed from vaginal smears, were placed overnight with mature males. On the following morning (Day 1 of pregnancy), the females were examined for mating by the presence of a vaginal plug or spermatozoa in the vagina. Mated females were humanely killed by cervical dislocation at 2100–2200 h on Day 3 of pregnancy for 4-cell embryos, at 0800–0900 h on Day 4 for 8-cell embryos, and at 1800–2000 h on Day 4 for morulae. Embryos were recovered by flushing the excised oviducts and/or uterine horns with modified phosphate-buffered saline (PB1) [21].
Vitrification of Embryos
For vitrification, EFS40 was used [22]; this solution was 40% (v/v) ethylene glycol and 60% (v/v) PB1 medium containing 30% (w/v) Ficoll 70 (average molecular weight, 70 000; Amersham Pharmacia Biotech, Buckinghamshire, U.K.) and 0.5 M sucrose. Thus, the final concentrations of Ficoll 70 and sucrose in EFS40 were 18% (w/v) and 0.3 M, respectively.
Embryos were vitrified in EFS40 in 0.25-ml Cassou straws (IMV, L'Aigle, France) following the procedure described by Kasai et al. [22]. All the procedures were conducted in a room at 25°C. Before freezing, PB1 medium containing 0.5 M sucrose (S-PB1) was drawn up into a straw to a depth of 60 mm, followed by air (25–30 mm), EFS40 (5 mm), another volume of air (5 mm), and finally, more EFS40 (12 mm). Twelve to 14 embryos were transferred directly from PB1 medium into the larger volume of EFS40 in the straw, and the straw was sealed. After exposure of embryos to EFS40 for 30 sec, the straw was positioned in the liquid nitrogen vapor phase by placing it horizontally on a styrofoam boat (thickness, 1 cm) floating on the surface of the liquid nitrogen for at least 3 min in a Dewar vessel (inner diameter, 140 mm). The straw was then immersed in liquid nitrogen.
After being stored in liquid nitrogen for at least 1 day, each straw was kept in air for 10 sec and then immersed in water at 25°C. When the crystallized S-PB1 medium in the straw began to melt (after 7 sec), the straw was removed from the water and quickly wiped dry, and the contents of the straw were then expelled into a watch glass by flushing the straw with 0.8 ml of S-PB1 medium. After gently agitating the watch glass to promote mixing of the contents, the embryos were pipetted into fresh S-PB1 medium. Approximately 5 min after being flushed out, the embryos were transferred to fresh PB1 medium.
Embryo Transfer
The females were stimulated by inserting a glass rod connected to an electric vibrator into the vagina at 1930–2000 h on the day of proestrus (Day 0 of pregnancy) to induce pseudopregnancy. Transfers were then carried out on specified days of pseudopregnancy. Because we did not have access to animal rooms with different lighting schedules, it was only possible to adjust the synchrony by intervals of 24 h for investigations using fresh embryos. However, with vitrified embryos (the focus of the present study), it was possible to adjust the synchrony by smaller intervals. Morphologically normal vitrified embryos were recovered in PB1 medium as described above and transferred to pseudopregnant females without further culture.
Vitrified 4-cell embryos were transferred into oviducts at 0900 and 2100 h on Day 1, 2, or 3 of pseudopregnancy. The time of 2100 h on Day 3 of pseudopregnancy was designated as Day 0 of synchrony, because 4-cell embryos were collected at the same hour on Day 3 of pregnancy. As a control, uncultured fresh 4-cell embryos were transferred into oviducts at 2100 h on Day 1, 2, or 3 of pseudopregnancy. Vitrified 8-cell embryos were transferred into oviducts or uterine horns at 0800 and 2000 h on Day 3 or at 0800 h on Day 4 of pseudopregnancy (the latter time was Day 0 of synchrony for 8-cell embryos). As a control, uncultured fresh 8-cell embryos were transferred into oviducts or uterine horns at 0800 h on Day 3 or 4 of pseudopregnancy. Because the results in our previous study [20] were poor when either fresh or vitrified 8-cell embryos were transferred into oviducts on Day -1 of synchrony, those treatment conditions were not repeated in the present study. Vitrified morulae were transferred into uterine horns at 0600 and 1800 h on Day 3 or 4 of pseudopregnancy; the time of 1800 h on Day 4 of pseudopregnancy was designated as Day 0 of synchrony. As a control, uncultured fresh morulae were transferred into uterine horns at 1800 h on Day 3 or 4 of pseudopregnancy.
Six to seven embryos were transferred to each oviduct or uterine horn. Transfer of embryos into oviducts was conducted as described by Toyoda and Chang [23], except that a small drop of 0.1% epinephrine solution was put on the surface of the bursal membrane before cutting the membrane to prevent bleeding. Transfer into uterine horns was conducted as described by Miyoshi et al. [24]. After transfer, vaginal smears from the recipients were examined daily. Recipients showing proestrus or estrus were killed, and their uterine horns were examined for implantation sites. However, recipients that showed proestrus or estrus on the fourth or fifth day were considered to be nonpseudopregnant and were excluded from the present study. Pregnant females were allowed to give birth and were then killed. Females that had not delivered by Day 25 of pregnancy were killed, and their uterine horns were examined for resorption sites, implantation sites, and fetuses.
Statistical Analyses
The data were analyzed using chi-square tests unless the expected frequency was less than five, in which case the Fisher exact probability test was used.
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RESULTS |
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As shown in Table 1, when 4-cell embryos were transferred into oviducts of synchronous recipients (Day 0 of synchrony), none of the females became pregnant. Similarly, no recipients became pregnant after receiving vitrified embryos either on Day -0.5 (nearly synchronous) or on Day -2.5 (extremely asynchronous). However, when 4-cell embryos were transferred on Days -1 to -2 of synchrony, most females became pregnant. Although implantation rates in recipients on Day -2 were relatively high with both vitrified and fresh embryos, less than a quarter of the transferred embryos developed to term, and of these, significantly more (P < 0.05) fresh embryos than vitrified embryos completed development. In contrast, when transferred on Day -1, significantly more (P < 0.05) vitrified embryos than fresh embryos developed to term. Although more vitrified embryos developed to term when transferred on Day -1 to -1.5 than on Day -2, fresh embryos developed to term equally well when transferred on either Day -1 or Day -2.
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In Vivo Development of 8-Cell Embryos
As shown in Table 2, when 8-cell embryos were transferred to oviducts of recipients on Day 0 or Day -0.5, the pregnancy rates were low with both vitrified and fresh embryos. In contrast, when embryos were transferred to uterine horns on either Day 0 or Day -0.5, all the recipients became pregnant; in more asynchronous females (Day -1), fewer pregnancies occurred irrespective of embryo type.
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Thus, transfers to recipients on Day 0 to -0.5 were much more successful when vitrified embryos were transferred into uterine horns than into oviducts; transfers into more asynchronous recipients (Day -1) gave very poor results. Similarly, when fresh embryos were transferred into uterine horns of asynchronous recipients (Day -1), less than a third implanted and developed to term. In synchronous recipients, most embryos implanted, and approximately two thirds developed to term. Transfer into oviducts of synchronous recipients resulted in poor development.
In Vivo Development of Morulae
As shown in Table 3, when morulae were transferred into uterine horns on Days 0 to -1 of synchrony, all recipients became pregnant, and high rates of implantation and full-term development were obtained with both vitrified and fresh embryos. The highest rate of full-term development with vitrified embryos was obtained with transfer to slightly asynchronous females (Day -0.5); those results were significantly (P < 0.05) higher than those obtained after synchronous transfer and equivalent to those obtained from transfer of fresh embryos on Day -1. In contrast, when vitrified embryos were transferred to females on Day -1.5, two females failed to get pregnant, and implantation and full-term development was very poor in the remaining five females.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, when we varied the degree of synchrony between embryo and pseudopregnant recipient, reasonable results with vitrified 4-cell embryos were obtained only with asynchronous recipients (Day -1), with 52% developing to term. However, we think that this result may be slightly misleading, because only three recipients on Day -1 were used in the present study. In our earlier study [20], six recipients at this stage were used. Of those, five got pregnant, and 40% of embryos developed to term. Taken together, these results suggest that vitrified 4-cell embryos are only moderately able to develop to term. Surprisingly, the best results with fresh embryos (24%–33%) were either lower than or only just comparable to those obtained with vitrified embryos. This may reflect the fact that we could not adjust lighting conditions in the animal unit to vary the degree of synchrony for recipients of fresh embryos; thus, we could only adjust the synchrony by intervals of 24 h (Days -1 and -2) in experiments using fresh embryos. In contrast, with vitrified embryos we were able to assess developmental potential following transfer to recipients on Days -0.5, -1.5, and -2.5, as well as -1 and -2, of synchrony. Thus, survival of 4-cell rat embryos does not decrease significantly after vitrification, which is consistent with our recent demonstration that as much as 94% of 4-cell embryos vitrified by our method could develop into blastocysts in culture [20].
The proportion of vitrified 4-cell embryos that developed to term (34%–52%) (Table 1) may be adequate for practical use, but even higher success rates were obtained with vitrified 8-cell embryos (62%–63%) (Table 2) and morulae (58%–68%) (Table 3). For 8-cell embryos, transfer into oviducts was not effective, but transfer into uterine horns (especially of more synchronous recipients) was very effective. These results led us to evaluate transfer of vitrified 4-cell embryos into uterine horns of recipients on Day 0 to -0.5, but the in vivo survival rate was very low (unpublished observations). Therefore, we conclude that the lower in vivo survival of 4-cell embryos, whether cryopreserved or fresh, reflects something unusual about this developmental stage in the rat.
Higher proportions of transferred embryos can develop in vivo when recipients are either synchronous with or at a slightly earlier stage than the embryos. In both the present study and that of Han et al. [20], few (<15%) 8-cell embryos, either vitrified or fresh, developed to term when transferred into oviducts, regardless of the day of synchrony, indicating that the site of transfer is very important. Consistent with our results, Noyes and Dickmann [1] found that in vivo development of fresh embryos obtained 4 days after mating (possibly at the 8-cell to morula stage) was well supported when transferred into uterine horns, but not oviducts, of synchronous recipients. However, comparatively low rates (0–38%) of in vivo development of cryopreserved 8-cell rat embryos have been reported even after synchronous transfer into uterine horns [16–18]. In contrast to those earlier investigations, in the present study the majority (63%) of vitrified 8-cell embryos developed to term when transferred into uterine horns of recipients on Day 0 to -0.5 (Table 2), which is very similar to the results with fresh embryos (66%). Thus, the in vivo developmental potential of 8-cell embryos is not markedly reduced by vitrification using EFS40; indeed, 100% of vitrified and thawed 8-cell embryos had normal morphology and, thus, could be transferred [20]. When transferred into uterine horns of more asynchronous recipients (Day -1), however, only a minority of both vitrified and fresh 8-cell embryos developed to term (6%–21%). This may indicate that earlier in the cycle, the uterine environment is hostile to such embryos; results in the various earlier studies cited above suggest that in the uterine cycle, there are times when conditions within the uterus can either kill or prevent implantation of early embryos. In contrast, the majority of morulae (Table 3) developed to term following transfer to both synchronous (Day 0 to -0.5) and asynchronous (Day -1) recipients, suggesting that these older embryos are more flexible in their requirements for implantation and subsequent development.
Kasai et al. [19] reported that 0%–50% of rat morulae frozen-thawed in the presence of various cryoprotectants developed to term after transfer to synchronous recipients. In our previous study [20], when rat morulae were transferred to synchronous recipients, 61% of vitrified embryos developed to term, which is very similar to the 70% rate obtained with fresh embryos. In the present study, high proportions of vitrified morulae developed to term when transferred to either asynchronous (Day -0.5 to -1) or synchronous (Day 0) recipients, but the best results were obtained following transfer into slightly asynchronous recipients (Day -0.5). Thus, vitrified 8-cell embryos and morulae appear to have similar developmental potential. However, the morula may be a more convenient stage for embryo cryopreservation, because the synchrony requirements of the recipients appear to be less exacting.
In conclusion, although approximately half the vitrified 4-cell rat embryos developed to term in asynchronous recipients (Day -1), this does not appear to be the optimal stage for cryopreservation; even fresh embryos had a similarly modest developmental potential. On the other hand, high proportions (>60%) of vitrified 8-cell embryos and morulae developed to term when transferred into uterine horns of more synchronous recipients (Day 0 to -0.5) or slightly asynchronous recipients (Day -0.5 to -1), respectively. The proportions of vitrified 4-cell, 8-cell, and morula-stage embryos developing to term were all comparable with the results for fresh embryos as long as the recipient was at the appropriate stage of synchrony. We therefore conclude that the full-term developmental potential of rat embryos at these stages is not damaged by vitrification, although further experiments are needed to determine whether similar results can be obtained with other strains of rats.
Because the rat is a very important model for the study of human disease and a traditional experimental model system, it is vital to have genetic integrity and transportability of strains. The present data suggest that vitrified rat embryos may provide a valuable experimental tool that will allow investigators using the rat model system to achieve these requirements.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: FAX: 81 86 251 8388; kniwa{at}cc.okayama-u.ac.jp
3 Current address: Department of Animal Science, Jinju National University, Jinju 660-758, Korea
Received: 13 August 2003.
First decision: 30 August 2003.
Accepted: 7 October 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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