1 Nexia Biotechnologies Inc, Montreal, Canada, and 2 Department of Biotechnology, Institut fur Tierzucht und Tierverhalten (FAL), Mariensee, 31535 Neustadt, Germany
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Abstract |
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Key words: blastocyst/blastomere/extracellular matrix/inner cell mass/rabbit
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Introduction |
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The reproductive characteristics of the rabbit, including the easily controlled ovulation and the formation of the blastocyst at a higher cell number than in the mouse, make this species a good model for the study of embryology, developmental biology and genetic engineering (Yang and Foote, 1987). The preimplantation rabbit embryo undergoes a rapid series of cell divisions resulting in blastocysts with 128 cells by day 3 after fertilization. On day 6, the blastocyst already possesses ~8x104 cells (Lewis and Gregory, 1929
; Daniel, 1964
; Allison and Pardee, 1973
). By employing differential staining, the proportion of ICM to total cell numbers in rabbit embryos grown in vivo at days 3.54.0 has been calculated at 2130% and the proportion of ICM to TE cells at 2740% (Giles and Foote, 1995
). Rabbit 1-cell embryos have been successfully cultured to blastocysts in a variety of complex media, including largely defined conditions (Carney and Foote, 1991
; Li and Foote, 1993
; Li et al., 1993
). Extracellular matrix components, e.g. fibronectin (FN) and laminin are glycoproteins with a variety of physiological functions in cellular morphology, cytoskeletal organization, cellular adhesion as well as differentiation (Dziadek and Timpl, 1985
; Armant et al., 1986
). FN has been shown to promote attachment and proliferation of mouse blastomeres and blastocysts in vitro (Armant et al., 1986
; Wilton and Trounson, 1989
) and to enhance, in particular, blastocyst development of 1/8 and 1/16 porcine blastomeres (Saito and Niemann, 1991
). Rabbit single blastomere development in vitro has not been studied and characterized systematically.
The purpose of this study was: (i) to evaluate the in-vitro developmental potential of single rabbit blastomeres isolated from 4-, 8- and 16-cell embryos under various culture conditions; and (ii) to investigate the effects of FN precoated on the culture dishes on the in-vitro development of single blastomeres. The number and proportion of ICM and TE cells in rabbit blastocysts produced from single blastomeres of parent embryos at the 4-, 8- and 16-cell stages as well as intact control embryos was determined by employing a double-dye staining procedure.
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Materials and methods |
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Preparation of single blastomeres
The mucin coat and zona pellucida were removed by a short-term exposure in acidified (pH 2.5) PBS (510 s) followed by 1% pronase (12 min) treatment. Individual blastomeres were isolated by repeated pipetting the zona pellucida-free embryos in PBS plus 5% NBCS with a 50 µm fire-polished pipette. Blastomeres derived from 4-, 8- and 16-cell stage rabbit embryos were designated as 1/4, 1/8 and 1/16 blastomeres respectively. In experiment 2, at least 30% of the embryos were left intact with or without zona pellucida to serve as controls in parallel to the isolated blastomeres.
In-vitro culture of isolated blastomeres and control embryos
TCM 199 (M2520; Sigma, Deisenhofen, Germany), Medium RD and Medium CMRL-1066 (C 0422; Sigma), each supplemented with 10% fetal calf serum (FCS), bovine serum albumin (BSA, fraction V; Sigma) or polyvinylalcohol (PVA) were employed for culture of blastomeres and control embryos (Carney and Foote, 1991). Medium RD is a 1:1 (v/v) mixture of Roswell Park Memorial Institute (RPMI) 1640 medium (R-6504; Sigma) and Dulbecco's modified Eagle's medium (DMEM), (D 3656; Sigma). These complex media were chosen because, previously, it has been shown that they allow high development of one-cell rabbit embryos even under protein-free conditions (Carney and Foote, 1991
; Li and Foote, 1993
; Li et al., 1993
). These media differ in their relative concentrations of carbohydrates, amino acids and vitamins. RD-medium contains relatively high concentrations of glucose and inositol, while TCM has lower glucose concentrations and CMRL lacks any glucose supplementation. Intact embryos at the 4-cell, 8-cell and 16-cell stage, with and without zona pellucida, were cultured in parallel. For the second experiment, culture dishes were coated with FN (Fi 4759 Sigma; Cell Culture Reagents, from bovine plasma) according to a previously described method (Saito and Niemann, 1991
), with minor modifications. The surface of a 4-well dish (Greiner Labortechnik, Frickenhausen, Germany) was pre-coated by incubating 75 µl of a 25 µg/ml FN solution in Hanks' salt solution in each well at 37°C for 4 h. After aspiration of the FN solution from the wells, each well was washed twice with culture medium. In-vitro culture was carried out in 4-well dishes, either coated or uncoated with FN, each well containing 200 µl medium covered with paraffin oil, and in a humidified atmosphere of 5% CO2 in air at 37°C. Five blastomeres/embryos were cultivated per well and cultures were replicated 1015 times.
Evaluation of development and determination of embryo diameter
Cultures were observed after 24 h to monitor the cleavage rate. Isolated 1/4 blastomeres and 4-cell control embryos were cultured for 96 h while isolated 1/8 and 1/16 blastomeres and their intact controls were cultured for 72 h, to obtain a similar endpoint in all blastomere groups. All observations were carried out at x100150 magnification. After culture, the diameter of blastocysts was measured with an eyepiece micrometer at x100 magnification. Blastocysts were then fixed and stained to count the number of ICM and trophoblastic cells.
Differential staining procedure for ICM and TE cells of rabbit blastocysts derived from isolated blastomeres and control embryos
To generate goat anti-rabbit antiserum, spleen from an adult female rabbit was homogenized in PBS (24 g/25 ml) and 3 ml were injected s.c. to a male goat. For immunization, the antigen was emulsified with Freund's adjuvant. Booster injections were given 21, 35 and 49 days later and blood samples for antibody testing were taken 1012 days after the last booster injection. Serum was collected, heat inactivated (50°C, 30 min), and tested for biological efficacy. The procedure of immunosurgery (Solter and Knowles, 1975) and differential staining of ICM and TE cells as originally developed for the mouse (Handyside and Hunter, 1984
) and adapted for porcine embryos (Papaioannou and Ebert, 1988
), for porcine demi-embryos (Tao et al., 1995
) and for blastocysts derived from single porcine blastomeres (Eckert et al., 1997
), were modified for rabbit blastocysts of various origin. Initially, intact embryos were treated with 1% pronase in PBS for 1 min to remove the zona pellucida and the mucin coat. All zona pellucida-free embryos were then incubated in PBS supplemented with 10% goat anti-rabbit antiserum for 20 min at 37°C. Subsequently, embryos were briefly washed twice in warm PBS plus 5% NBCS, 10% guinea pig complement and 10 µg/ml propidium iodide for 15 min (control intact embryos) or 10 min (blastocysts from isolated blastomeres) at 37°C. Subsequently, they were washed quickly in cold PBS plus 10 µg/ml propidium iodide and in cold (0°C) bisbenzimide ethanol solution. Embryos were then fixed in cold bisbenzimide (10 µg/ml) ethanol solution overnight. Finally, embryos were observed under fluorescence light and then squashed on a slide and assessed under a Nikon fluorescence microscope with an excitation filter of 365 nm and a barrier filter of 410 nm. ICM cells could be recognized by the blue fluorescence of the bisbenzimide and TE cells by a red to pink fluorescence due to the accumulated propidium iodide (Figure 1
). Detection of at least one blue nucleus was taken as the presence of an ICM.
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Statistical analysis
Statistical analysis was performed using the software package Sigma Stat for Windows (Jandel Scientific, Erkrath, Germany). Development to the blastocyst stage at the end of culture and the number of embryos lacking ICM were analysed with 2 test followed by Yate's correction or Fisher's exact test (if necessary) and are presented as proportion of cultured embryos or proportion of stained blastocysts. The diameter of blastocysts, total cell number, number of ICM cells, and proportion of ICM to the total cell number are given as mean ± SEM. Analysis of variance (ANOVA) revealed significant interactions between embryonic stage, presence or absence of zona pellucida, and media with PVA, BSA or FCS. Significant differences were identified by the Tukey test; P < 0.05 was considered to be statistically significant.
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Results |
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Effects of different media with or without protein on in-vitro development of blastomeres isolated at 4-, 8- and 16-cell stages
The development of rabbit blastomeres, the total cell number and the diameter of blastocysts derived from blastomeres (1/4, 1/8 and 1/16) are presented in Tables I, II and III for in-vitro culture in different media (TCM, RD, CMRL) supplemented with either 10% FCS, 1.5% BSA or 0.1% PVA (defined medium). In 1/4 blastomeres, the development to the blastocyst stage was higher (P < 0.05) in all media supplemented with FCS or BSA than with PVA with one exception (RD + BSA compared with RD + PVA) (Table I
). The total number of cells and diameter of blastocysts derived in TCM supplemented with FCS were significantly higher (P < 0.05) than those with BSA or PVA. The total number of cells and diameter of blastocysts derived in CMRL supplemented with PVA were significantly lower (P < 0.05) than those cultured in CMRL supplemented with FCS or BSA.
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In 1/16 blastomeres, development to the blastocyst stage was significantly (P < 0.05) different between media TCM and RD supplemented with FCS, BSA or PVA with one exception (TCM + FCS versus TCM + BSA). The highest proportion of blastocysts was obtained in serum supplemented medium (Table III). The total number of cells and diameter of blastocysts derived from media TCM or RD supplemented with FCS were significantly higher (P < 0.05) than those with BSA or PVA with one exception (RD + FCS versus RD + BSA). There were no significant differences in development, diameter and total cell number of blastocysts in medium CMRL supplemented with either FCS, BSA or PVA. Interestingly, in defined medium (CMRL plus PVA) even 42.6% of 16-cell blastomeres developed to blastocysts (Table III
).
Effects of FN on development of rabbit intact embryos and isolated blastomeres
In all blastomere groups (1/4, 1/8 and 1/16), rates of blastocyst formation were higher (P < 0.05) in matrix-free groups than in FN-coated groups (Table IV). The cleavage rates after 24 h of in-vitro culture were similar among the different groups (8497% for 1/4 blastomeres, 7792% for 1/8 blastomeres, 8897% for 1/16 blastomeres). The diameter of the blastocysts (Figure 2
) derived from 1/4 blastomeres was greater (P < 0.05) in FN-coated groups than in matrix-free groups. In control intact embryos (4-, 8- and 16-cell stages), the average diameter of blastocysts derived from zona-free intact embryos was bigger (P < 0.05) than that from zona intact embryos in both matrix-free and FN-coated groups.
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Discussion |
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The present results indicate that in intact control embryos (4-, 8- and 16-cell stages), the number of ICM and total cells of blastocysts, as well as the diameter of blastocysts derived from zona-free intact embryos were greater than that from zona intact embryos. This is in agreement with previous findings for mouse and pig embryos (Tao et al., 1995; Suzuki et al., 1995
; Eckert et al., 1997
) indicating that the zona pellucida does not only act as a protective barrier but regulates cell division and trophectodermal outgrowth in a variety of different species. In the mouse, the zona pellucida affects preimplantation development by regulating the number of cell contacts of the blastomeres (Suzuki et al., 1995
).
Previously it has been shown that a high proportion of intact rabbit 1-cell embryos can develop to hatching blastocysts in protein-free culture medium (Carney and Foote, 1991; Li et al., 1993
; Li and Foote, 1993
). In contrast, we have shown that serum supplementation is superior to BSA or PVA (= protein-free conditions) for the development of isolated blastomeres. In the present study, the three basic culture media were found to support development of rabbit blastomeres equally well. The presence of vitamins and amino acids has previously been shown to be an essential requirement for rabbit embryo development (Kane, 1987
). The nutrient requirements of the rabbit preimplantation embryo are complex and differ significantly from those of the mouse embryo resembling more those of the human embryo. As in embryos from domestic species it has been demonstrated that pyruvate is the preferred substrate for human preimplantation embryos and glucose can even be inhibitory to early development (Bavister, 1995
; Gardner, 1998
). The nutrient requirements change upon embryonic genomic activation which has led to the development of sequential media that better mimic the physiological conditions in the uterine cavity and improve embryo development and viability in the mouse (Gardner and Lane, 1996
) and human (Gardner, 1994
). It remains to be investigated whether the development of isolated blastomeres can be similarly improved by employing sequential culture systems. Biopsy of the human embryo to collect blastomeres for preimplantation genetic diagnosis is facilitated by short-term incubation in Ca2+/Mg2+ free medium (Dumoulin et al., 1998
). This requires cryopreservation of the biopsied embryo until the diagnosis has been completed and the embryo can be reimplanted. However, current freezing protocols are not yet compatible with high survival of the biopsied mouse and human embryos (Ludwig et al., 1998
; Magli et al., 1999
). In contrast, the survival of frozenthawed bovine embryos was not impaired either after slitting the zona pellucida or after biopsy of a few blastomeres (Niemann et al., 1987
; Bondioli et al., 1989
).
The present culture system with groups of five blastomeres did not lead to reaggregation of the blastomeres and allowed high rates of development. Culture in groups has been shown to enhance development of murine and bovine embryos in an autocrine and/or paracrine manner (Paria and Dey, 1990; Keefer et al., 1994
). Cell division of isolated blastomeres decreased with increasing stage of the parent embryos reflecting a cell size-dependent control of cell division such that larger blastomeres (1/4) divide sooner than smaller blastomeres (1/8 and 1/16). The individual blastomeres may be programmed to cavitate after a predetermined number of cell divisions. The reason for the lower percentage of ICM to total cell number in blastomere-derived blastocysts in comparison with the control embryos could be a retarded cleavage in the ICM cells of blastomere-derived embryos (Erbach et al., 1994
) and/or a greater sensitivity to suboptimal in-vitro conditions (Iwasaki et al., 1990
; 1994
). It should be borne in mind, however, that identification of one blue ICM cell was taken as the criterion for the presence of an ICM. The minimum number of ICM cells required for an undisturbed fetal development in the rabbit is unknown. However, given the high proportion of ICM cells in in-vivo-derived blastocysts (40%, Giles and Foote, 1995), it is questionable whether blastocysts with such low ICM cell numbers will develop normally upon transfer to suitable recipients. In addition, an intact and thick enough mucin layer would be required for implantation to occur after uterine transfer of the blastocysts (Murakami and Imai, 1996
). The transfer of zona pellucida-enclosed single blastomeres from 2-, 4- and 8-cell rabbit embryos to the Fallopian tubes of recipient does resulted in pregnancies and normal offspring. In contrast, rabbit zona-free blastomeres and blastocysts failed to show implantation (Moore et al., 1968
; Rottmann and Lampeter, 1981
), whereas uterine transfer of zona-free porcine blastocysts derived from isolated blastomeres yielded pregnancies and normal piglets (Saito and Niemann, 1991
). However, rabbit blastomeres from morula stages (32 cells) lead to blastocysts and offspring upon transfer to enucleated oocytes, fusion of both components and subsequent transfer of the reconstituted blastocysts to recipients (Heyman et al., 1990
; Yang et al., 1992
). In addition, normally appearing blastocysts have been obtained following nuclear transfer of ICM cells, whereas no development was observed when TE cells were used as donors in nuclear transfer (Collas and Robl, 1991
).
The results of the present study indicate that the effects of FN on the proliferation of blastomeres decreased with progression of development and concomitant decreasing blastomere size. FN and collagen type IV are first detectable at the blastocyst stage of the mouse embryo, i.e. not earlier in development (Zetter and Martin, 1978; Richoux et al., 1989
). Later in development, extracellular matrices are present on the surface membranes of embryonic and trophoblastic tissues of more advanced embryonic stages (Cooper and MacQueen, 1983
; Kleiman et al., 1984
; Dziadek and Timpl, 1985
). FN-induced attachment and outgrowth of rabbit blastomeres is similar to that of their mouse counterparts (Wilton and Trounson, 1989
), but this was not detected during culture of porcine blastomeres (Saito and Niemann, 1991
; Eckert et al., 1997
). The origin of FN is probably not the cause of the different effects, as in the murine study (Wilton and Trounson, 1989
), FN from human plasma was employed, while both the porcine studies (Saito and Niemann, 1991
; Eckert et al., 1997
) and the current study were conducted using FN derived from bovine plasma. FN is capable of interactions with a great range of molecules and the various fibronectins share a common binding site. However, conformational differences may account for the divergent observations between mouse and rabbit, on the one hand, and pig, on the other (Hynes, 1990
). The formation of cell sheets has contributed to the lower blastocyst rates in FN-coated groups compared with the matrix-free groups. Speculatively, this formation of cell sheets might open the road towards generating rabbit stem cell lines by culture of single blastomeres.
In conclusion, the results of our experiments show that for culture of isolated rabbit blastomeres a variety of basic culture media is equally well suited. However, serum supplementation is superior to BSA or protein-free conditions. Although the developmental capacity of single blastomeres decreases with increasing cell stage of the parent embryos, at least up to the 16 cell stage single blastomeres of rabbit embryos are capable of forming blastocysts, even under defined culture conditions. FN-coating cannot further improve blastocyst yields from isolated blastomeres but promotes attachment and proliferation of rabbit blastomeres at the 4-cell-stage. However, the total cell number as well as the percentage of ICM to total cells in blastomere-derived blastocysts remain considerably lower than in intact embryos. Nevertheless, the developmental capacity of blastocysts derived from single blastomeres upon transfer to suitable recipients warrants further investigation. Furthermore, these findings may contribute to develop an efficient culture system for human blastomeres in an effort to study their regulatory potential.
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Acknowledgments |
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Notes |
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References |
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Submitted on June 16, 1999; accepted on November 19, 1999.