Cytogenetic analysis of diploidy in cloned bovine embryos using an improved air-dry karyotyping method

Theriogenology 63 (2005) 2434–2444 www.journals.elsevierhealth.com/periodicals/the

Cytogenetic analysis of diploidy in cloned bovine embryos using an improved air-dry karyotyping method Guang-Peng Li, Ying Liu, Kenneth L. White, Thomas D. Bunch* Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322, USA Received 4 February 2004; accepted 6 September 2004

Abstract Of the few published studies on the cytogenetic analyses of bovine nuclear transferred (NT) embryos, results differ between air-dry and fluorescent in situ hybridization (FISH) procedures. A modified air-dry procedure is reported in this study that provides more metaphase plates for analysis. Day 5 and Day 7 bovine NT embryos were cultured in colcemid-containing CR1aa for 10–12 or 16– 18 h, then treated in hypotonic sodium citrate for 3–5 min. The standard procedure of 5 h in colcemid and 15–20 min in hypotonic solution was the control. A much higher (P < 0.01) percent of mitotic nuclei was observed in the experimental groups. The 33 and 41% mitotic nuclei were obtained from 10 to 12 h and 16 to 18 h-colcemid-treated Day 5 embryos, respectively, which was higher (P < 0.001) than the control (15%). The mitotic nuclei in Day 7 NT embryos were 24% in 10– 12 h- and 28% in 16–18 h-colcemid-treated groups, which also was higher (P < 0.05) than the control (10%). The majority of analyzable embryos were diploid. Analyses of mixoploid embryos showed on average that 70% of the cells were diploid. Day 5 mixoploid embryos contained numerically higher polyploid cells than Day 7 embryos, although statistically there were no differences. We concluded that the modified air-dry method provided a larger source of mitotic nuclei for chromosome analyses of cloned bovine embryos. # 2004 Elsevier Inc. All rights reserved. Keywords: Chromosomes; Nuclear transfer; Embryos; Bovine

* Corresponding author. Tel.: +1 435 797 2148; fax: +1 435 797 2118. E-mail address: [email protected] (T.D. Bunch). 0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2004.09.056

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1. Introduction Successful somatic cell nuclear transfer (NT) has been achieved in domestic animals and rodents, as reported by the birth of offspring. The overall efficiency of this technique, however, remains low [1,2] or less than 2% [1]. A high frequency of early postimplantation developmental arrest and abortion exists, especially in cattle. Studies in cattle and sheep have shown that the most fetal loss occurs at a time of placental attachment between the first and second month of gestation [1,3,4]. The exact mechanism(s) contributing to losses are still unclear. Epigenetic alterations [5,6] and chromosomal abnormalities [7] may contribute to developmental failure. Cytogenetic analysis has established that chromosomal abnormalities in various forms contribute to reproductive failure ranging from embryonic to neonatal losses [8]. Data regarding the cytogenetic status of NT embryos are limited, and the results are confounded by differences between NT protocols, karyotyping methods, developmental stages, types of donor cells, donor cell-cytoplast cell cycle synchronization, and perhaps, species [8–12]. Of the few published studies on the cytogenetic analyses of bovine NT embryos, results differ between air-dry and fluorescent in situ hybridization (FISH) procedures. Diploid chromosome rates of NT bovine blastocysts have been approximately 80% using air-dry methods [13,14], whereas with FISH methods most NT embryos were mixoploid and polyploid [15]. Air-dry karyotype analysis has been limited due to low numbers of analyzable chromosome spreads. Furthermore, perhaps polyploid or mixoploid blastomeres are not at metaphase during the time of chromosome preparation, thereby skewing results of the air-dry method. Theoretically, prolonged colcemid treatment lets more cells enter metaphase, thereby resulting in more metaphase plates. In this study, we prolonged treatment time in colcemid-containing medium and shortened time in hypotonic solution, with the objective to produce more identifiable chromosomal plates of bovine cloned embryos.

2. Materials and methods 2.1. Chemicals Unless otherwise noted, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Each experiment included at least four replicates. 2.2. Oocyte maturation in vitro (IVM) Maturation of bovine oocytes was as described previously [10,11]. Briefly, bovine cumulus oocyte complexes (COC) were aspirated from 3 to 8 mm diameter follicles in ovaries obtained from a local abattoir. Only COC with a compact and a homogenous ooplasm were selected. The COC were matured in TCM 199 with Earle’s salts, Lglutamine, and sodium bicarbonate (Gibco Inc., Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, Utah, USA), 25 mg/mL gentamycin, 0.01U/mL FSH (NIH-FSH-S17), 0.01U/mL LH (USDA-bLH-6), and 1 mM penicillamine,

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hypotaurine and epinephrine (PHE) in 4-well plates with 0.5 mL medium and 30–50 oocytes per well at 39 8C in humidified 5% CO2 in air for 18–19 h. 2.3. Donor cell culture and preparation Donor cells were bovine cumulus cells. A primary cumulus cell line was established from six oocytes collected 20 h after the start of maturation. Cumulus cells were separated with 0.1% hyaluronidase (Type-1), washed several times in DME/F12 (1:1) (Gibco Inc., Grand Island, NY, USA), and cultured in DME/F12 supplemented with 10% FBS in 25 cm flasks at 37 8C under 5% CO2 in air. The cells were passaged when they were approximately 80% confluent. Cells from passage numbers 3–5 were used for nuclear transfer. Immediately before nuclear transfer, cells at 80% confluence were dissociated by trypsinization with 0.25% trypsin with EDTA solution (HyClone, Logan, Utah, USA). Small-sized (10–12 mm in diameter) cells that were smooth round shaped were used as donors. 2.4. Nuclear transfer, fusion, and activation After maturation, the cumulus cells were removed by vortexing COC in TL-hepes medium containing 100 IU/mL hyaluronidase. Enucleation was done with the assistance of colcemid as described [10,11,16]. Briefly, oocytes with the first polar bodies (Pb1) were cultured in TCM-199 supplemented with 0.2 mg/mL colcemid for 2 h. Oocytes with a membrane projection were transferred into TL-hepes medium containing 7.5 mg/mL cytochalasin B and then the membrane projection (site of maternal genetic material) and Pb1 were removed. Single cells were individually transferred to the perivitelline space of recipient cytoplasts. Fusions were done by one direct current pulse of 1.2 kV/cm for 25 ms with an Electro Cell Manipulator 2001 (BTX, San Diego, CA, USA) in 0.27 M mannitol, 0.1 mM CaCl2, 0.1 mM MgCl2, and 0.05% BSA. Fused couplets were activated at IVM 24–25 h with 5 mM ionomycin for 5 min, followed by treatment with 10 mg/mL cycloheximide in CR1aa plus 3% FBS for 5 h at 39 8C in 5% CO2 in air. 2.5. In vitro culture of NT embryos After activation, fused couplets were cultured under mineral oil in 30 mL droplets of CR1aa supplemented with 3% FBS with a monolayer of bovine cumulus cells for coculture at 39 8C in 5% CO2 in air. Day 5 morulae and Day 7 blastocysts were collected for chromosome preparation and analysis. 2.6. Chromosomal analysis and cell number count To obtain more metaphase plates for evaluation, three treatments were used to examine chromosomal complements of Day 5 morulae and Day 7 blastocysts. Treatment 1, embryos were cultured in 0.1 mg/mL of colcemid in CR1aa supplemented with 3% FBS for 10–12 h, or for 16–18 h in Treatment 2 at 39 8C under 5% CO2 in air. The embryos were then treated in 1% sodium citrate for 3–5 min. Treatment 3 (control) embryos were cultured in colcemidcontaining CR1aa for 5 h and in sodium citrate for 15–20 min. Embryos with approximately

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1–2 mL medium were transferred individually onto a clean glass slide. As soon as an embryo was on a slide, about 2–3 mL of fixative (methanol:acetic acid, 1:1) was dropped onto the embryo. Just before the first droplet of fixative dried, a second drop of fixative was added. If more than 1–2 mL medium was transferred onto a slide during transfer of an embryo, then some of the medium was gently removed with a mouth pipette. Otherwise, the embryo would continue to roll on the slide and would be usually lost when the fixative was added. After at least 24 h of drying (room temperature at 25 8C), the slides were stained with 1% Giemsa for 10–15 min. Chromosomes were examined at 1000 under oil, and the chomosome composition was determined for each embryo. Images were captured by digital camera with PIXERA Viewfinder Program (Pixera Corporation, Los Gatos, CA, USA) under a Zeiss microscope. The categories of embryos assigned based on chromosome composition were as follows: diploid (2n), triploid (3n), tetraploid (4n), and mixoploid (including 2n/3n, 2n/4n, and 2n/3n/4n). Embryos that did not show an interpretable metaphase spread due to gross overspreading or clumped chromosomes were not used. Chromosome spreads that were either too dispersed, one on top of another or with clumped chromosomes were excluded. 2.7. Statistical analysis Differences in blastocyst cell numbers between groups were analyzed by one-way ANOVA. The frequencies of mitotic cells in each category were compared using the x2 test. Differences of P < 0.05 were considered significant.

3. Results Two hundred seventeen embryos were examined, and 2954 metaphase plates were analyzed from a total 11,785 blastomeres (Table 1). 3.1. Frequency of mitotic cells in embryos Using the modified air-dry procedure, significantly more identifiable spreads were observed as shown in Fig. 1A. The 33% (420/1259) and 41% (700/1701) mitotic nuclei from 10 to 12 h and 16 to 18 h-colcemid-treated Day 5 embryos, respectively, and 24% (644/2690) and 28% (839/3027) mitotic nuclei in 10–12 h and 16–18 h-colcemid-treated Day 7 blastocysts, respectively, were obtained, which was greater (P < 0.01) than the control (Table 1). The frequency distribution of mitotic cells in embryos in experimental and control groups are shown in Fig. 2. There were 52% (37/71) and 60% (59/99) embryos whose mitotic nuclei were 30%, and over 30%, when treated in colcemid for 10–12 h and 16– 18 h, respectively. Sixteen percent of embryos treated in colcemid for 16–18 h had 60–80% (16/99) mitotic nuclei. 3.2. Chromosomal composition of embryos Of the analyzed metaphase plates in diploid and mixoploid embryos, 90.6% (2675/ 2954) cells were diploid (Table 1). The majority of the analyzed embryos (from 69.7 to

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Time in colcemid (h)

Embryos analyzed/total embryos

Total cell number (cells/embryo)

Metaphase plates analyzed

Percentages of mitotic nuclei

No. diploid metaphase plates (%)*

Day 5 embryos 5 (control) 10–12 16–18

13/15 38/42 55/61

419 (32.2  8.4) 1259 (33.1  9.5) 1701 (30.9  8.7)

63 420 700

15.0c 33.3b 41.1a

60 (95.2) 364 (86.7) 608 (86.8)

Day 7 embryos 5 (control) 10–12 16–18

34/37 33/33 44/47

2689 (78.8  14.7) 2690 (81.5  17.5) 3027 (69.8  21.2)

288 644 839

10.7c 23.9b 27.7b

268 (93.0) 591 (91.8) 784 (93.4)

2954

25.0b

2675 (90.6)

Total *

217/235

11,785

Percentages of Metaphase plates analyzed. Superscripts (a, b) and (b, c): P < 0.05; (a, c): P < 0.01.

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Table 1 Percentages of mitotic nuclei of bovine Day 5 and Day 7 nuclear transfer embryos prepared by an improved air-dry karyotyping method

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Fig. 1. Metaphase spreads after traditional (D) and improved protocols in colcemid for 10–12 h (C, F) or 16–18 h (A, B, E). (A) More metaphase plates were observed after treatment with colcemid for 16–18 h (100); (B) diploid (2n), triploid (3n), and tetraploid (4n) spreads in an embryo specimen (400); (C) 2n and 3n spreads in an embryo; and (D–F) 2n spreads (1000).

Fig. 2. Distribution of percent mitotic nuclei of bovine Day 5 and Day 7 nuclear transfer embryos after treatment with colcemid for 5 h, 10–12 h, and 16–18 h, respectively.

Table 2 Number of diploid Day 5 and Day 7 bovine nuclear transfer embryos resulted from an improved air-dry karyotyping method Time in colcemid (h)

No. of embryos analyzed

No. of 2n embryos (%)

Day 5 embryos 5h (control) 10–12 16–18

13 38 55

10 (76.9) 27 (71.1) 40 (72.7)

Day 7 embryos 5 (control) 10–12 16–18

34 33 44

27 (79.4) 23 (69.7) 35 (79.5)

217

162 (74.7)

Total

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Time in colcemid (h)

No. of non-2n embryos*

Total cell numbers

Metaphase plates analyzed

Composition of mitotic cells (%) 2n

3n

4n

Day 5 embryos 5 (control) 3 10–12 11 16–18 15

95 345 458

11 130 252

8(72.7) 74(56.9) 163(64.7)

1(9.0) 21(16.2) 46(18.3)

2(18.2) 35(26.9) 42(16.7)

Day 7 embryos 5 (control) 7 10–12 10 16–18 9

607 758 593

79 230 214

59(74.7) 177(76.9) 159(74.3)

12(15.2) 16(7.0) 35(16.4)

8(10.1) 37(16.1) 20(9.3)

2856

916

640(70.0)

131(14.3)

144(15.7)

Total *

55

Polyploid or mixoploid embryos.

Composition of polyploid or mixoploid embryos 6n

1(0.4)

1(0.1)

2n/3n

2n/4n

1 3 5

2 3 3

4 4

1 4 5

3 5 2

2 1 2

19(34.5)

18(32.7)

2n/3n/4n

13(23.6)

3n

1

4n

3n/4n/6n

1 1

1

2(3.6)

2(3.6)

1

2(3.6)

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Table 3 Chromosome composition of cells in embryos and composition of polyploid and mixoploid of bovine nuclear transfer Day 5 and Day 7 embryos

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79.5%) were diploid (Fig. 1D–F). Only 1.8% (4/217) of embryos were polyploidy, e.g., triploidy or tetraploidy. No chromosomal composition differences were observed among experimental groups and controls (Table 2). Of the 55 polyploid or mixoploid embryos from both experimental and control groups, 916 metaphase plates were analyzed out of 2856 cells. Most of the cells were diploid ranging from 56.9 to 76.9% in experimental groups, and 72.7 and 74.7% in controls. A majority of 2n/3n, 2n/4n, and 2n/3n/4n mixoploid cells were observed in mixoploid embryos (Fig. 1B and C). Day 5 polyploid or mixoploid embryos contained numerically higher polyploid cells than Day 7 embryos, but was not statistically different (Table 3). Seventy percent (640/916) of cells were diploid and 30% were polyploid in Day 5 and Day 7 embryos.

4. Discussion To increase the efficiency of air-dry chromosome analysis by obtaining more metaphase plates, an improved air-dry protocol was developed for this study. Routinely, embryos at morula and blastocyst stages are incubated in colcemid-containing medium for 5 h, and then treated in hypotonic solution for 15–30 min. This traditional air-dry procedure, however, only results in 10–13% mitotic nuclei in bovine in vitro produced (IVP) [12,17– 19], and NT [10,13,14], [this study] blastocysts. We modified the protocol by prolonging the colcemid treatment time to 10–12 h or 16–18 h, and shortening the hypotonic treatment to 3–5 min. As a result, a significantly higher number of mitotic nuclei were consistently obtained. In a preliminary experiment, we treated embryos in hypotonic solution for 8, 10, or 15 min. Chromosome overspreads and overlaps occurred in most embryos making it difficult to identify and score chromosome plates. Treatment from 3 to 5 min in hypotonic solution localizes chromosomes to very limited areas and makes scoring easier. From among 2989 metaphases in Treatment 1 and 2 (data not shown), 2603 (87%) metaphases were identifiable and analyzable. The remaining (13%) was overspreads or condensed together and had to be excluded from the data set. The most important step in the improved procedure is to ensure that blastomeres spread totally and with no overlap when the fixative is added. A maximum volume of 1– 2 mL medium containing the embryo should be dropped onto a clean slide. It is very important that an embryo neither rolls too far on the slide nor condenses or suddenly attaches to the slide as a dot when the fixative is added. The prolonged colcemid treatment of embryos probably makes the microtubule network disorganized and disconnects the intercellular matrix associated with the microtubules. By shortening the hypotonic treatment and carefully handling the fixative, blastomeres totally spread, with single metaphases localizing in small areas. The question may arise whether the high condensation of chromosomes due to prolonged colcemid treatment induces greater risks for errors when locating individual chromosomes. Embryonic cell division is not synchronous in morulae and blastocysts. Evidence from the traditional air-dry procedure where blastocysts were cultured in colcemid-containing medium for 5–6 h showed that only about 10 out of 100 cells were at metaphase [10–14,17–19]. Moreover, the chromosomes in different cells have different morphology such as size, length, and

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width. Based upon our experience, there is variation in chromosome morphology between plates within a single embryo including mouse [20], pig [21], rabbit [22], and bovine [10,11]. Even when using prolonged colcemid treatments as in this study, chromosomes in different cells still have some morphological differences at 1000 magnification. Even after prolonged exposure to colcemid, most mitotic cells have medium chromosome condensation due to their asynchronous divisions, and only about 15–20% of the mitotic cells had extreme condensation. When two mitotic cells were in close proximity, their size, and morphology were generally distinguishable. Cells that were totally overlapped and the chromosomes intermixed were not included in the data set. Therefore, the risk was minimal or unlikely for wrongly classifying the nuclei of overlapping plates as a tetraploid nucleus. We have observed that this improved method helps to analyze the multiples of whole sets of chromosomes, e.g., triploid or tetraploid embryos. We did not attempt to detect single chromosome abnormalities such as trisomies. Prolonged treatment with colcemid would render most chromosome spreads unsuitable for banding because of their contracted state. No significant differences were observed in the chromosomal composition between morula and blastocysts among the experimental groups and the controls. Our previous reported data [10,11] and data from other laboratories [13,14] used the traditional air-dry procedure and observed that about 80% of bovine cloned blastocysts were diploid. The present results obtained from the improved air-dry method showed that 74.7% bovine NT Day 5 and Day 7 embryos were diploid. In the mixoploid embryos, approximately 70% of the cells were diploid, and only 30% of the cells were 3n, 4n, or 6n. Although the improved method yielded more cells in metaphase, the percentage of diploid embryos was similar to the traditional protocol. A possible explanation for the similarity is that both diploid and polyploid cells have equal opportunities to enter into metaphase, although polyploid blastomeres often have a prolonged cell cycle due to the time needed for the replication of larger numbers of chromosomes [17]. The prolonged colcemid treatment increased both diploid and polyploid cells into metaphase. Conversely, the results may be indicative that cloned bovine embryos contain predominately diploid cells. Therefore, the high risk of abortion of cloned bovine embryos is probably not a chromosomal problem, but rather a molecular error problem such as epigenetic alterations [23–26], or the incomplete reprogramming of chromosome methylation [27,28]. A majority of the mixoploid blastocysts were 2n/3n, 2n/4n, and 2n/3n/4n, which was consistent with previous reports for NT embryos [10,13,14] and IVP embryos [12,17,18]. Mixoploid is a common phenomenon in embryos derived from IVP, NT, and even in vivo processes [10–14,17–19]. The mechanisms involved in the formation of polyploid cells in embryos are still unclear. For cloned embryos, the formation of polyploid cells probably results from the complex cloning procedure and status of donor cells and recipient oocytes, embryonic culture condition [10,11], and the combination of these factors [9,29,30]. Cellcycle incompatibility between the donor cell and cytoplast can also contribute to the disruption of normal numerical chromosome constitution [31]. In summary, the improved air-dry karyotyping method produced significantly higher mitotic nuclei and can be helpful in the cytogenetic analyses of chromosome composition of cloned bovine embryos. Our results confirmed that diploid cells were predominant in bovine cloned morula and blastocysts.

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Acknowledgement Published as Utah Agriculture Experiment Station Journal Paper Number 7588.

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