and 16-cell embryos and isolated blastomeres cultured in vit

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already possesses ~8104 cells (Lewis and Gregory, 1929;. Daniel, 1964; Allison and Pardee, 1973). By employing differential staining, the ... cysts in vitro (Armant et al., 1986; Wilton and Trounson,. 1989) and to enhance, in particular, ...
Human Reproduction vol.15 no.4 pp.881–889, 2000

Cellular characterization of blastocysts derived from rabbit 4-, 8- and 16-cell embryos and isolated blastomeres cultured in vitro

T.Tao1 and H.Niemann2,3 1Nexia

Biotechnologies Inc, Montreal, Canada, and 2Department of Biotechnology, Institut fur Tierzucht und Tierverhalten (FAL), Mariensee, 31535 Neustadt, Germany

3To

whom correspondence should be addressed

The purpose of this study was to investigate the developmental potential of isolated rabbit blastomeres under various culture conditions to gain insight into their ability to form the two cell lineages of a viable blastocyst. Intact embryos at the 4-cell, 8-cell, 16-cell stages and blastomeres isolated from 4-, 8- and 16-cell rabbit embryos (1/4, 1/8 or 1/16 blastomeres respectively) were cultured in drops of one of three different media, each supplemented with either fetal calf serum (FCS), bovine serum albumin (BSA) or polyvinyl alcohol (PVA). The effects of the extracellular matrix fibronectin (FN) on the development of isolated rabbit blastomeres were also investigated. Supplementation of the medium with FCS yielded a higher (P < 0.05) proportion of blastocysts than BSA or PVA, predominantly from 1/4 blastomeres. No major differences were found between the three basic culture media. In 1/4, 1/8 or 1/16 blastomeres, blastocyst formation rates were greater (P < 0.05) in groups cultured in matrix-free (54.5, 59.6 and 54.6% respectively) than in FN-coated groups (35.4, 46.0 and 26.1% respectively). Only in blastocysts derived from 1/4 blastomeres, were the numbers of inner cell mass (ICM) and total cells of blastocysts higher (P < 0.05) in FN-coated groups than in matrix-free groups (12.7 ⍨ 1.1 versus 8.5 ⍨ 0.7 ICM, 73.8 ⍨ 3.7 versus 57.8 ⍨ 3.3 total cells). The percentage of blastocysts derived from single blastomeres with ICM cells decreased with increasing cell stage of the parent embryos in FN-coated (93.6, 78.3 and 44.0%, respectively) as well as matrix-free groups (96.2, 69.3 and 55.2%). In FN-coated groups, after 96 h (1/4) or 72 h (1/8 and 1/16) of culture, ~20–30% of blastomeres did not develop into normal blastocysts but formed sheets with 30–50 cells attached to the bottom of the dishes. These results indicate that the development of rabbit blastomeres shares important characteristics with those from mouse and domestic species and may thus aid in developing an efficient culture system for blastomeres, derived from human embryos. Key words: blastocyst/blastomere/extracellular matrix/inner cell mass/rabbit

Introduction The developmental capacity of isolated blastomeres from mammalian embryos has been studied extensively, and has © European Society of Human Reproduction and Embryology

proved of great value in the investigation of cellular and regulatory aspects of development in laboratory animals, e.g. rat (Nicholas and Hall, 1942), mouse (Tarkowski, 1959; Rossant, 1976), rabbit (Moore et al., 1968), and later in domestic animals, e.g. sheep (Willadsen, 1979, 1980, 1982), cattle (Willadsen and Polge, 1981; Johnson et al., 1995) and pig (Willadsen, 1982; Saito and Niemann, 1991; Eckert et al., 1997). In the human, isolated blastomeres from cleavage stage embryos have been employed for preimplantation genetic diagnosis (Viville et al., 1998), for assessing the developmental potential of the parent embryos (Geber and Sampaio, 1999) or to gain insight into regulatory mechanisms in preimplantation development (Krussel et al., 1998). The developmental potential of a single blastomere from a mammalian embryo is usually determined by its ability to form a blastocyst with a visible inner cell mass (ICM) and a distinct trophoblast. It has been shown that isolated blastomeres from 2-cell mouse-, 4–8-cell rabbit, 4-cell bovine as well as 8-cell ovine and porcine embryos are capable of regular in-vivo development upon transfer to suitable recipients (Tarkowski and Wroblewska, 1967; Moore et al., 1968; Rossant, 1976; Saito and Niemann, 1991). However, fetal survival after transfer of embryos derived from blastomeres of cleavage stage embryos is poor and this may be attributed to a complete lack of, or an insufficient, ICM in a considerable proportion of the blastocysts. Microscopic observations frequently do not allow the proportion of ICM and trophectoderm (TE) cells to be determined in a precise manner. This can be achieved by the differential staining of ICM and TE in blastocysts during immunosurgery, using two different fluorescent dyes (Solter and Knowles, 1975; Handyside and Hunter, 1984). The proportion of ICM and TE has been identified in porcine blastocysts derived from either single blastomere culture or microsurgical embryo bisection by employing differential staining of the ICM and TE cells (Tao et al., 1995; Eckert et al., 1997). 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 ~8⫻104 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 3.5–4.0 has been calculated at 21–30% and the proportion of ICM to TE cells at 27–40% (Giles and Foote, 1995). Rabbit 1-cell embryos 881

T.Tao and H.Niemann

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 doubledye staining procedure.

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 10–15 times.

Materials and methods

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 ⫻100–150 magnification. After culture, the diameter of blastocysts was measured with an eyepiece micrometer at ⫻100 magnification. Blastocysts were then fixed and stained to count the number of ICM and trophoblastic cells.

Ovulation induction The animals were housed singly in cages, fed a concentrate and hay and kept at temperatures of 10–15°C. Ovulation was stimulated in 50 mature female New Zealand rabbits (4–6 kg bodyweight) with either 150 IU of pregnant mare’s serum gonadotrophin (PMSG) (Intergonan, Vemie, Germany) or with five s.c. injections of FSH (Follitropin V;Vetrepharm, Clonee, Co. Meath, Ireland) given 12 h apart (1.7 mg/rabbit). Donors were mated to fertile males 74–78 h after the first injection. Ovulation was induced by an i.v. injection of 75 IU human chorionic gonadotrophin (HCG) (Ovogest; HydroChemie, Munich, Germany) immediately after mating. The animals were killed in groups of two or three and 4-cell, 8-cell, and 16-cell embryos were flushed from the excised oviducts with phosphatebuffered saline (PBS) plus 1% heat-inactivated new-born calf serum (NBCS) 32, 40 and 48 h after mating respectively. Embryos were rinsed twice in PBS plus 5% NBCS, and morphologically intact embryos (intact blastomeres of equal size, intact zona pellucida and mucin coat) were randomly allocated to the experimental groups. Preparation of single blastomeres The mucin coat and zona pellucida were removed by a short-term exposure in acidified (pH 2.5) PBS (5–10 s) followed by 1% pronase (1–2 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%

882

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 (2–4 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 10–12 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)

Rabbit blastomere development

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.

Results

Figure 1. Differential staining picture of a blastocyst derived from an intact zona-free rabbit 8-cell embryo after 72 h of culture; inner cell mass and trophectodermal cell nuclei are coloured blue and pink respectively. Original magnification approx. ⫻350.

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. Experimental design Experiment 1 was employed to compare the development of isolated blastomeres in three different media. This experiment used a 3⫻3 factorial design comparing the effects of TCM 199, RD and CMRL1066, each supplemented with either 0.1% of the synthetic macromolecule PVA (defined medium), 1.5% BSA or 10% FCS, on the development of isolated blastomeres derived from 4-, 8- and 16-cell embryos. These concentrations were chosen based on previous studies employing intact rabbit embryos (Carney and Foote, 1991). In these experiments, total cell counts were made at the end of the culture period by staining embryos with the fluorescent bisbenzimidazole stain, Hoechst 33342 (Sigma) (Pursel et al., 1985). In experiment 2, we investigated the effects of FN coating of the culture dishes on the development of intact embryos with and without zona pellucida and blastomeres isolated from 4-, 8- and 16-cell parent embryos. The medium used was TCM-199 plus 10% FCS. The cell counts were made after differential staining of ICM and TE cells. The diameter of the resulting blastocysts was determined in both experiments. 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 χ2 test followed by Yate’s correction or Fisher’s exact test (if necessary) and are presented as

General observations A total of 1465 embryos were collected from the 50 stimulated donors (29.3 per donor), 1214 of them were considered as morphologically intact embryos at the 4-, 8- and 16-cell stages (459, 436 and 319 respectively). From these, 283, 238 and 166 of 4-cell, 8-cell and 16-cell embryos were used for isolation of blastomeres respectively. The remaining embryos were cultured as controls. A total of 1082, 1488 and 1644 1/4, 1/8 and 1/16 blastomeres were obtained. The isolation rates decreased with increasing cell stage of the parent embryo and accounted for 95.6% (1/4 blastomeres), 78.2% (1/8 blastomeres) and 61.9% (1/16 blastomeres) (P ⬍ 0.05). 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. In 1/8 blastomeres, development to the blastocyst stage was significantly higher (P ⬍ 0.05) in all media supplemented with FCS than with BSA or PVA. Similarly, the diameter was significantly greater in blastocysts cultured in TCM or RD supplemented with FCS as compared with all other groups. There were no significant differences in development to blastocysts between media supplemented with BSA or PVA (Table II). 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 883

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Table I. Development of rabbit 1/4 blastomeres after culture for 96 h in TCM 199, medium RD and medium CMRL each supplemented with fetal calf serum (FCS), bovine serum albumin (BSA) or polyvinyl alcohol (PVA) Medium

TCM ⫹ FCS TCM ⫹ BSA TCM ⫹ PVA RD ⫹ FCS RD ⫹ BSA RD ⫹ PVA CMRL ⫹ FCS CMRL ⫹ BSA CMRL ⫹ PVA

No. of blastomeres cultured

No. of blastomeres developed to BL 96 h

%

62 62 62 68 70 62 63 60 60

33 35 16 44 40 25 46 38 19

53.2a 56.5a 25.8b 64.7a 57.1ab 40.3b 73.0a 63.3a 31.7b

No. BL stained

No. of total cells Mean ⫾ SEM

33 35 16 44 40 25 45 37 19

85.6 51.5 35.6 64.1 68.2 53.3 79.5 84.2 40.5

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

6.8a 3.4b 4.2b 5.0ab 4.2ab 3.7b 6.4a 7.8a 3.3b

Diameter of blastocysts (µm) Mean ⫾ SEM 150.1 ⫾ 8.6a 117.8 ⫾ 4.4b 82.5 ⫾ 4.8c 123.3 ⫾ 5.9b 120.2 ⫾ 5.3b 104.2 ⫾ 4.4bc 134.6 ⫾ 6.5ab 125.5 ⫾ 6.7b 85.1 ⫾ 4.8c

in columns with different superscripts are significantly different (P ⬍ 0.05). BL ⫽ Blastocysts.

a,b,cNumbers

Table II. Development of rabbit 1/8 blastomeres after culture for 72 h in TCM 199, medium RD and medium CMRL each supplemented with fetal calf serum (FCS), bovine serum albumin (BSA) or polyvinyl alcohol (PVA) Medium

TCM ⫹ FCS TCM ⫹ BSA TCM ⫹ PVA RD ⫹ FCS RD ⫹ BSA RD ⫹ PVA CMRL ⫹ FCS CMRL ⫹ BSA CMRL ⫹ PVA

No. of blastocysts cultured

No. of blastomeres developed to BL 72 h

%

66 72 66 72 72 66 72 72 66

27 10 12 33 8 2 25 10 6

40.9a 13.9b 18.2b 45.8a 11.1b 3.0b 34.7a 13.9b 9.1b

No. BL stained

No. of total cells Mean ⫾ SEM

27 10 12 30 8 0 25 10 6

29.2 18.2 18.1 26.3 17.9 – 19.2 18.0 12.5

⫾ ⫾ ⫾ ⫾ ⫾

2.6a 2.0a 2.2a 1.5a 3.2a

⫾ 2.1a ⫾ 2.5a ⫾ 2.1b

Diameter of blastocysts (µm) Mean ⫾ SEM 103.0 88.8 81.3 98.8 50.0 – 89.1 72.6 87.7

⫾ ⫾ ⫾ ⫾ ⫾

4.4a 5.6b 6.3b 3.4ab 4.0c

⫾ 4.2b ⫾ 7.2b ⫾ 7.2b

in columns with different superscripts are significantly different (P ⬍ 0.05). BL ⫽ blastocysts.

a,b,cNumbers

Table III. Development of rabbit 1/16 blastomeres after culture for 72 h in TCM 199, medium RD and medium CMRL each supplemented fetal calf serum (FCS), bovine serum albumin (BSA) or polyvinyl alcohol (PVA) Medium

TCM ⫹ FCS TCM ⫹ BSA TCM ⫹ PVA RD ⫹ FCS RD ⫹ BSA RD ⫹ PVA CMRL ⫹ FCS CMRL ⫹ BSA CMRL ⫹ PVA

No. of blastomeres cultured

94 85 100 98 93 88 91 82 82

No. of blastomeres developed to BL 72 h

%

51 34 34 64 34 18 40 34 35

54.3a 40.0ab 34.0b 65.3a 36.6b 20.5c 44.0ab 41.5ab 42.6ab

No. BL stained

No. of total cells Mean ⫾ SEM

49 34 25 64 34 18 40 34 33

27.3 20.2 18.2 37.9 30.5 18.6 36.3 33.7 28.6

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

1.4a 2.0b 1.3b 2.0a 2.3a 1.4b 3.4a 4.2a 3.2a

Diameter of blastocysts (µm) Mean ⫾ SEM 113.0 ⫾ 3.1a 79.5 ⫾ 3.8b 75.7 ⫾ 4.2b 109.3 ⫾ 2.7a 91.3 ⫾ 3.0b 82.1 ⫾ 4.0b 101.3 ⫾ 5.0ab 86.8 ⫾ 3.9b 93.5 ⫾ 5.0b

in columns with different superscripts are significantly different (P ⬍ 0.05). BL ⫽ Blastocysts

a,b,cNumbers

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). 884

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

Rabbit blastomere development

Table IV. Effects of fibronectin (FN) on the development of rabbit blastomeres and intact embryos in vitro and the diameter of blastocysts derived from blastomeres and intact embryos Experimental groups Controls No matrix 4 C.ZP⫹ 4 C. ZP– FN-coated 4 C.ZP⫹ 4 C.ZP– No matrix 8 C.ZP⫹ 8 C. ZP– FN-coated 8 C.ZP⫹ 8 C. ZP– No matrix 16 C.ZP⫹ 16 C. ZP– FN-coated 16 C.ZP⫹ 16 C.ZP– Blastomeres 1/4 1/4 FN 1/8 1/8 FN 1/16 1/16 FN

No. cultured

No. of blastomeres developed to BL (%)

No. of BL measured

Diameter of BL (µm) Mean ⫾ SEM

44 45

38 (86.4)a 29 (64.4)b

36 29

150.2 ⫾ 4.4a 250.1 ⫾ 13.4b

43 44

32 (74.4)ab 31 (70.5)ab

32 31

165.0 ⫾ 9.2a 222.3 ⫾ 10.8b

68 54

62 (91.2)a 40 (74.1)b

56 37

166.0 ⫾ 2.2a 240.4 ⫾ 10.1b

38 38

34 (89.5)a 31 (81.6)ab

34 31

184.7 ⫾ 3.5a 250.0 ⫾ 12.6b

35 39

29 (82.9)ab 29 (74.4)b

29 29

182.8 ⫾ 6.1a 305.3 ⫾ 11.7b

43 36

41 (95.3)a 27 (75.0)b

40 27

191.9 ⫾ 6.7a 246.9 ⫾ 10.6b

242 271 453 411 355 476

132 (54.5)a 96 (35.4)b 270 (59.6)a 189 (46.0)b 194 (54.6)a 124 (26.1)b

113 96 188 169 166 118

129.7 140.2 101.6 107.5 99.3 94.5

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

4.4a 3.8b 1.9c 1.9c 1.8c 2.4c

in columns with different superscripts are significantly different (P ⬍ 0.05). Comparisons were made within blastomeres or within each stage of intact embryos. 4 C ⫽ 4-cell; 8 C⫽ 8-cell; 16 C ⫽ 16-cell embryos; BL ⫽ blastocyst. ZP⫹ and ZP– intact embryos were cultured with or without zona pellucida respectively.

a,b,cNumbers

Figure 2. Blastocysts derived from a rabbit 4-cell zona-free embryo (control) and five 1/4 blastomeres after 96 h in-vitro culture. Scale bar ⫽ 55 µm.

different groups (84–97% for 1/4 blastomeres, 77–92% for 1/8 blastomeres, 88–97% 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. The number of ICM cells in blastocysts derived from 1/4

and 1/8 blastomeres, and the number of total cells of blastocysts derived from 1/4 blastomeres were higher (P ⬍ 0.05) in FN-coated groups than in matrix-free groups (Table V). No significant difference was found for 1/16 blastomeres between the two groups. The number of ICM and total cells of blastocysts derived from zona-free control embryos was higher than that from zona-intact embryos in most groups (Table V). The proportion of ICM to total cell number in blastocysts derived from blastomeres was lower (P ⬍ 0.05) compared with intact embryos. The percentage of ICM to total cell number in blastocysts derived from 1/4 and 1/8 was higher (P ⬍ 0.05) than in those from 1/16 blastomeres. The percentage of blastocysts with ICM cells decreased with increasing developmental stage of the parent embryo in both FN-coated and matrix-free groups (P ⬍ 0.05) (Table VI). In FN-coated groups, after 96 h (1/4) or 72 h (1/8 and 1/16) of culture, ~20–30% of the blastomeres did not develop into normal blastocysts but formed sheets of ~30–50 cells that attached at the bottom of the dishes. Similar behaviour was noted for a few zona-free embryos (⬍10%). These attached cells continued to divide rapidly from days 4–12. Starting day 6, these cells formed colonies (Figure 3) with an embryonic stem celllike morphology (Shim et al., 1997). Discussion Results of the present study show that the capability for rapid cell division of the rabbit embryo is maintained in isolated 885

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Table V. Number and proportion of inner cell mass (ICM) and trophoblastic cells in blastocysts from isolated blastomeres after 96 h (1/4) or 72 h (1/8 and 1/16) of development in vitro Experimental groups

Embryos (controls) No. matrix 4 C.ZP⫹ 4 C.ZP– FN-coated 4 C.ZP⫹ 4 C.ZP– No matrix 8 C.ZP⫹ 8 C.ZP– FN-coated 8 C.ZP⫹ 8 C.ZP– No matrix 16 C.ZP⫹ 16 C.ZP– FN-coated 16 C.ZP⫹ 16 C.ZP– Blastomeres 1/4 1/4 FN 1/8 1/8 FN 1/16 1/16 FN

No. of blastocysts stained

No. of total cells Mean ⫾ SEM

No. and proportion of ICM Mean ⫾ SEM

% ⫾ SEM

30 30

254.7 ⫾ 17.3a 346.0 ⫾ 25.1b

75.7 ⫾ 6.6a 93.4 ⫾ 6.9a

9.6 ⫾ 1.7a 8.2 ⫾ 1.5a

30 30

271.0 ⫾ 17.2a 305.0 ⫾ 21.6a

71.1 ⫾ 5.3a 92.9 ⫾ 7.2a

5.8 ⫾ 1.5a 1.4 ⫾ 1.7b

32 38

265.1 ⫾ 16.2a 338.2 ⫾ 21.4b

108.5 ⫾ 7.7a 80.1 ⫾ 7.2b

41.6 ⫾ 2.1a 23.8 ⫾ 1.5b

36 34

283.2 ⫾ 13.1ab 339.1 ⫾ 24.9ab

94.0 ⫾ 6.4ab 107.3 ⫾ 8.4a

33.5 ⫾ 1.9a 33.1 ⫾ 1.7a

30 31

379.5 ⫾ 22.2a 478.8 ⫾ 20.7b

126.6 ⫾ 9.0a 133.1 ⫾ 12.3a

34.2 ⫾ 2.2a 27.7 ⫾ 2.5a

35 33

312.3 ⫾ 20.3a 385.7 ⫾ 26.1a

109.5 ⫾ 7.7a 115.5 ⫾ 9.2a

36.2 ⫾ 1.9a 30.0 ⫾ 1.3a

104 94 179 157 145 104

57.8 73.8 28.4 29.8 25.8 21.5

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

3.3a 3.7b 1.0c 1.1c 1.0c 1.1c

8.5 12.7 3.2 7.1 2.4 2.1

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.7a 1.1b 0.3c 0.7a 0.3c 0.2c

14.6 ⫾ 1.0ab 16.4 ⫾ 1.1a 11.3 ⫾ 1.0b 20.8 ⫾ 1.7a 8.1 ⫾ 0.9c 8.8 ⫾ 0.8c

in columns with different superscripts are significantly different (P ⬍ 0.05). Comparisons were made within blastomeres or within each stage of intact embryos. 4 C ⫽ 4-cell; 8 C ⫽ 8-cell; 16 C ⫽ 16-cell embryos. ZP⫹ and ZP– ⫽ intact embryos were cultured with or without zona pellucida respectively.

a,b,cNumbers

Table VI. Number and percentage of blastocysts (BL) with inner cell mass (ICM) from isolated blastomeres Blastomere-derived blastocysts

Total

Matrix-free Total BL with ICM (%)

FN-coated BL with ICM (%)

1/4 1/8 1/16

104 179 145

100 (96.2)a 124 (69.3)b 80 (55.2)c

88 (93.6)a 123 (78.3)b 44 (44.0)c

94 157 100

a,b,cNumbers

in columns with different superscripts are significantly different (P ⬍ 0.05).

blastomeres and that, at least, up to the 16-cell stage individual blastomeres are able to develop into apparently normal looking blastocysts in vitro. The rates of blastocysts derived from blastomeres isolated at different developmental stages were similar. The total number of cells in blastocysts derived from 1/4, 1/8 and 1/16 rabbit blastomeres corresponded to that of intact control embryos divided by 4, 8 or 16 respectively, which coincides with observations made for porcine blastomeres (Eckert et al., 1997). This indicates a similar developmental pattern at this early stage in embryos from rabbit and domestic species. The total number of cells in our study was similar to that of rabbit 1-cell stages recovered from superovulated donors and cultured for 96 or 108 h (Giles and Foote, 1995). Blastocysts from blastomeres of 4-cell parent embryos consistently possessed an inner cell mass showing that at this 886

Figure 3. After in-vitro culture for 6 days in a fibronectin-coated dish, 1/8 blastomeres attached to the bottom and formed colonies with embryonic stem-like morphology. Scale bar ⫽ 30 µm.

stage of development the rabbit blastomere still has the inherent potential to contribute equally well to TE and ICM. Under our experimental conditions the developmental capacity of blastomeres progressively decreased with increasing cell stage of the parent embryo. In 8–16 cell stages, positional effects may already become apparent. It is known that the mouse embryo proper should be derived from the inner cells of the early cleavage stage (McLaren, 1982). Recently it has been shown that the proteins leptin and STAT3 are likely to

Rabbit blastomere development

be involved in this early step of differentiation. The inner blastomeres of the mouse morula contained no or very little leptin/STAT3, while in the outer cells leptin/STAT3 was detected at high levels (Antczak and Van Blerkom, 1997). In freshly fertilized mouse eggs, however, no evidence for a preferential localization of factors involved in early differentiation has been found (Zernicka-Goetz, 1998). In the rabbit embryo, polarization as an indication of early differentiation was found to occur after the fifth cell cycle, e.g. with 38–66 cells (Ziomek et al., 1990). In early human embryos, the initiating steps for the formation of the two cell lines ICM and TE are observed at the 2–4-cell stage (Edwards and Beard, 1997). 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 (⫽ proteinfree 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 frozen–thawed 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 887

T.Tao and H.Niemann

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. Acknowledgements The authors thank Dr F.Klobasa for preparation of the antiserum; H.-H.Do¨ pke for his skilled technical assistance and Christine Weidemann for competent statistical analysis of the data. The financial support for T.Tao by the Federal Agriculture Ministry and the DFG (SFB 330) is gratefully acknowledged.

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