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Conditioned medium increases the polyploid cell composition of bovine somatic cell nuclear-transferred blastocysts

Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, Utah 84322, USA

Correspondence should be addressed to T D Bunch; Email: tombunch{at}cc.usu.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
The effects of bovine cumulus cell-conditioned medium on cloned bovine embryonic development and subsequent chromosome complement were examined using an air-dry procedure. Conditioned media were prepared using CR1aa supplemented with either fetal bovine serum (FBS) or bovine serum albumin (BSA). Nuclear-transferred embryos were reconstructed with nuclei from cumulus cells. Similar cleavage, morula, and blastocyst development was observed in conditioned media groups compared with the co-culture group. No differences (P > 0.05) were observed in the composition of blastocyst chromosomes after co-culture in different media, either with or without starvation of donor cells. The overall diploid blastocyst rate ranged from 75% to 84%. Chromosomal complement of blastocysts, however, was very different between conditioned medium and co-culture treatments. Overall incidence of chromosomal anomalies was 40% in conditioned medium, which was significantly higher (P < 0.001) than the co-culture group (20%). Moreover, a higher incidence (P < 0.05) of chromosomally abnormal blastocysts (41.5%) was observed after culture with FBS-containing conditioned medium than those cultured in BSA-containing conditioned medium (31.4%). No diploid improvement was observed after exchange of the culture system from conditioned medium to co-culture, or from co-culture to conditioned medium after the first 72 h of culture. The results of this study also indicated that the overall cell number was much lower (P < 0.01) in blastocysts with chromosomal abnormalities than those with a normal diploid state. We have concluded that medium conditioned with bovine cumulus cells increases the incidence of chromosomal anomalies in nuclear reconstructed embryos.

	Introduction

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Successful somatic cell nuclear transfer (NT) has now been achieved in several mammalian species as reported by the birth of offspring. The overall efficiency of the technique, however, remains low. In each animal species so far studied, less than 10% of transferred embryos typically survive to term (Hill 2002). A high frequency of early post-implantation developmental arrest and abortion exists, especially in cattle. Studies in cattle and sheep have shown the most significant period for fetal loss to be at the time of placental attachment, which occurs between the first and second month of gestation (Hill 2002). The exact mechanism(s) of these phenomena is still unclear. The NT procedures, i.e. removal of oocyte nuclear material, transfer of a differentiated cell into an enucleated cytoplast, fusion with electric pulse, chemical or electric pulse activation, and culture conditions, can all lead to potential cell cycle blockages. All of these artificial manipulations can potentially influence the chromosomal composition of the resulting embryos. Approximately half of all human spontaneous abortions in the first and second trimesters are chromosomally abnormal (Burgoyne et al. 1991). Only a few reports have been published on the chromosome analysis of bovine NT embryos (Mohamed & Takahashi 2000, Arat et al. 2002, Slimane-Bureau & King 2002, Booth et al. 2003, Slimane-Bureau et al. 2003).

Although bovine embryos can be cultured in vitro in simple media under defined conditions (Keskintepe et al. 1995, Takahashi et al. 1996), the addition of growth factors facilitates embryo development (Paria & Dey 1990, O’Neill 1997), which suggests that paracrine and/or autocrine pathways exist between helper cells and embryos. To mimic the in vivo environment, co-culture systems are widely used in the culture of in vivo or in vitro fertilized embryos (Van Langendoncket et al. 1996, Funston et al. 1997, Vanroose et al. 2001). Various studies have reported that co-culture improves the morphological development and implantation rate of exposed embryos. Numerous growth factors, receptors, and binding proteins are secreted into the medium by helper cells. Embryos have also been reported to secrete substances into their surrounding environment (Van Langendoncket et al. 1996, Izquierdo et al. 1999). Cell-free conditioned medium (CM) has been used in several laboratories in the culture of in vivo and in vitro fertilized embryos (Baranao et al. 1997, Funston et al. 1997, Ishiwata et al. 2000, Galli et al. 2003). Studies have shown that CM has similar effects on embryo development to those produced in various co-culture systems. It is thought that the CM system may eliminate partially, or completely, some of the in vitro culture artifacts, e.g. developmental blockage and fragmentation.

Currently, there are no reports on the chromosome complement of NT blastocysts derived from CM culture. This study was designed to determine: (1) the effects of medium conditioned by bovine cumulus cells on the subsequent development of NT bovine embryos and (2) the chromosome complements of NT blastocysts after CM culture.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Chemicals were purchased from Sigma Chemical Co. unless otherwise indicated.

Oocyte maturation in vitro (IVM)
Maturation of bovine oocytes was carried out according to routine procedures (Li et al. 2003). Briefly, bovine cumulus oocyte complexes (COCs) were aspirated from 3–8 mm diameter follicles from ovaries obtained from a local abattoir. Only COCs with a compact cumulus complex and a homogenous ooplasm were selected. The COCs were matured in TCM 199 with Earle’s salts, L-glutamine and sodium bicarbonate (Gibco Inc., Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA, 25 µg/ml gentamicin, 0.01 U/ml follicle-stimulating hormone (NIH-FSH-S17), 0.01 U/ml luteinizing hormone (USDA-bLH-6) and 1 µM penicillamine, hypotaurine and epinephrine (PHE) in four-well plates with 0.5 ml medium and 30–50 oocytes/per well under 39 °C in humidified 5% CO₂ in air for 18–19 h.

Donor cell culture and preparation
Bovine cumulus cells were used as nuclear donor cells. A primary cumulus cell line was established from six oocytes collected 18–20 h after the start of maturation. Cumulus cells were separated with 0.1% hyaluronidase (type-1), washed several times in Dulbecco’s modified Eagles’ medium (DMEM)/F12 (1:1) (Gibco Inc.), and cultured in DMEM/F12 supplemented with 10% FBS in 25 cm flasks at 37 °C under 5% CO₂ in air. The cells were passed at 80% or more confluence. They were starved by culture in 0.5% FBS for 3–5 days. The non-starved cells were cultured in normal medium with 10% serum, and the 80% confluent cells were used as donors. Immediately before NT, cells were dissociated by trypsinization with 0.25% trypsin with EDTA solution (HyClone). Small sized (10–12 µm) cells with a smooth round shape from both starved and non-starved groups were used as nuclear donor cells.

Preparation of CM
CM was prepared from fresh CR1aa exposed to cumulus cell cultures for 24 h. Twenty-four hours after passaging in DMEM/F12 + 10% FBS (cells in one flask were seeded into four 25 cm² flasks, once those cells reached 70%–80% confluence in 24 h), the flasks were washed twice with CR1aa, and then 3 ml fresh CR1aa supplemented with 3% FBS (serum-containing CM; SCM) or 0.3% fatty acid free bovine serum albumin (BSA) (BSA-containing CM; BCM) was added. The SCM and BCM were collected 24 h after seeding and subsequently filtered (0.2 µm) to remove cellular debris and stored at 4 °C for less than 2 weeks.

Enucleation and NT
After maturation, the cumulus cells were removed by vortexing COCs in TL-Hepes (Tyrode lactate Hepes buffered) medium containing 100 IU/ml hyaluronidase. Removal of chromosomes was carried out with the assistance of colcemid (Gibco) as described by Yin et al.(2002). Briefly, oocytes with first polar bodies were cultured into TCM 199 supplemented with 0.2 µg/ml colcemid for 2 h. The eggs with membrane protrusions were transferred into TL-Hepes medium containing 7.5 µg/ml cytochalasin B and 0.1 µg/ml colcemid and then the protrusion and the first polar body were removed by a beveled pipette. Single cells were individually transferred to the perivitelline space of the recipient cytoplasts.

Fusion and activation
Fusion was performed using one direct current pulse of 1.2 kV/cm for 25 µs by 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 eggs were activated 24–25 h after the start of IVM with 5 µM ionomycin for 5 min, followed by treatment with 10 µg/ml cycloheximide in CR1aa plus 3% FBS for 5 h at 38.5 °C in 5% CO₂ in air.

In vitro culture of NT embryos
After activation, NT embryos were cultured in 30 µl droplets of chemically defined medium (CDM) (Olson & Seidel 2000, Li et al. 2003), or CR1aa supplemented with 3% FBS and co-cultured with monolayer bovine cumulus cells in experiment 1, or cultured in SCM or BCM in experiments 2 and 3 overlaid with mineral oil at 39 °C in 5% CO₂ in air for 7 days. Cleavage, morula and blastocyst stages were evaluated at 48 h, day 5 and day 7 after activation (day 0) respectively.

Chromosomal analysis and cell number count
Day 7 blastocysts were prepared and examined for their cytogenetic composition and the number of nuclei contained within each embryo. Blastocysts were incubated in 0.05 µg/ml colcemid in culture medium for 5 h. The blastocysts were then treated with 1% trisodium citrate for 10–15 min, and transferred individually onto a clean glass slide and fixed (Li et al. 1998, 2003). Slides were stained with 1% Giemsa for 10 min. Chromosome spreads were examined at 1000 x under oil, and the chromosome composition was determined for each blastocyst. Images were captured by digital camera with PIXERA Viewfinder Program (Pixera Corporation, Los Gatos, CA, USA) under a Zeiss microscope. The categories for the chromosome composition were classified as follows: diploid, polyploid (tetraploid), mixoploid (including 1n/2n, 2n/3n, 2n/4n, 2n/3n/4n), and aneuploid. Only those embryos with over four chromosome spreads were scored and analyzed. Embryos that did not show an interpretable metaphase spread because of gross overspreading or clumped chromosomes were not classified.

Experimental design
Three experiments were designed. Experiment 1 examined the effect of different media on blastocyst development of NT embryos and their chromosomal compositions. Fused NT embryos from starved or non-starved donor cells were randomly cultured in CDM or CR1aa medium with co-culture. On day 7 of culture, blastocysts were prepared for chromosomal analysis. Because no differences in blastocyst development and chromosomal composition between starved and non-starved donor cells were observed in experiment 1, only non-starved cells were used in experiments 2 and 3.

Experiment 2 evaluated the effects of BCM and SCM on development and chromosomal composition of NT blastocysts. Fused NT embryos were randomly cultured in SCM and BCM, and CR1aa + 3% FBS with co-culture as control.

Experiment 3: because of the effect of CM on blastocyst chromosomal composition in experiment 2, this experiment was designed to test whether changes of the culture system from co-culture to SCM or from SCM to co-culture after the first 72 h of culture would improve blastocyst diploid complements. Fused NT embryos were co-cultured in CR1aa + 3% FBS for 72 h, then transferred into fresh SCM for further culture (from co-culture to SCM; CO–SCM), or embryos were cultured in SCM for the first 72 h, then transferred into the co-culture system up to day 7 (from SCM to co-culture; SCM–CO). Embryos that were cultured during the entire culture period in co-culture or in SCM for 7 days were used as the controls.

Statistical analysis
Differences in blastocyst cell numbers between groups were analyzed by one-way ANOVA. Differences in rates of blastocyst development and chromosome composition between experimental groups were analyzed using Student’s t-test. A probability of P < 0.05 was considered statistically significant.

	Results

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Experiment 1
A total of 349 fused oocytes was cultured in six replicates. As shown in Table 1, cleavage, morula and blastocyst rates were similar between starved and non-starved donor cells, or between CDM and CR1aa co-culture. Blastocyst development was approximately 30%. Of the blastocysts produced, 75, 84, and 80.7% were diploid in the starved, non-starved in CDM, and non-starved in the CR1aa co-culture groups respectively (Table 2). The anomalies in the three groups ranged from 16 to 25%. Mixoploids of 2n/3n, 2n/4n, and 2n/3n/4n, and 4n cells were observed among the anomalies.

View larger version (105K): [in this window] [in a new window]   Figure 1 Chromosome spreads of haploid (A, two groups of haploid chromosomes), aneuploid (B, 56 chromosomes), diploid (C and D), triploid (E), and tetraploid (F). (A), (B), (C), (E), and (F) from CM culture and (D) from co-culture. (B), (C), and (D) 1000 x and (A), (E), and (F) 400 x .

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

Results in this study showed that there were no differences in diploid blastocyst rates between CDM and CR1aa groups, or between starvation and non-starvation groups (Table 2). This suggests that culture medium and donor nuclear cell status do not affect chromosome composition at the blastocyst stage. This conclusion is in agreement with other studies where 82% (Mohamed & Takahashi 2000) and 87% (Booth et al. 2003) of NT blastocysts derived from cumulus cells, 78% from fetal fibroblasts and 88% from adult fibroblasts (Arat et al. 2002) were diploid. After culture with CM, however, a significantly higher incidence of chromosomal abnormalities was observed in SCM and BCM groups than in the co-culture group (Table 4). Normal chromosome complement rates did not improve, anomalies were still higher in SCM and changed groups than in co-culture alone in experiment 3 (Table 6). The results of this study suggest that factors leading to a higher incidence of chromosomal anomalies may be inherent within the CM.

Diploid chromosome rates of NT bovine blastocysts determined by karyotyping are approximately 80% (Mohamed & Takahashi 2000, Arat et al. 2002, this study). Similar results were also reported by using fluorescent in situ hybridization protocols (Booth et al. 2003, Slimane-Bureau et al. 2003). Mixoploids of 2n/3n, 2n/4n, and 2n/3n/4n, tetraploids, and aneuploids were observed in this study. A majority of the mixoploid blastocysts were diploid–tetraploid, which is consistent with previous reports for NT embryos (Mohamed & Takahashi 2000, Arat et al. 2002, Booth et al. 2003) and IVP embryos (Hare et al. 1980, Iwasaki et al. 1992, Mohamed & Takahashi 2000). A haploid/diploid blastocyst was observed in the SCM–CO culture group. Mixoploidy of 1n/2n is usually found in 2-cell (Slimane et al. 2000) and in 4- to 32-cell embryos (Iwasaki et al. 1992). It has been suggested that the 1n/2n mosaic results from the retained activity of a polar body or is a product of polyspermy (King et al. 1981, Iwasaki et al. 1992). The mechanism that gives rise to a haploid NT blastocyst is unclear.

Chromosomal analysis of bovine embryos has indicated an overall incidence of abnormalities of 10% in in vivo-derived embryos and 15–30% in IVP embryos (Kawarsky et al. 1996, Viuff et al. 2002). In another study, 20% of in vivo bovine blastocysts and 44% of IVP blastocysts had anomalies (Iwasaki et al. 1992). The rate of embryos with anomalies in whole blastocysts was significantly higher than in inner cell mass cells only, and most were 2n/4n (Iwasaki et al. 1992). In vivo, 2n/4n mixoploids have been observed in day 12–18 bovine blastocysts (Hare et al. 1980). Results from diploid–tetraploid chimeras have confirmed that even high proportions of tetraploid cells are tolerated, but the tetraploid cells are eliminated in the developing embryo and are preferentially allocated to the extra-embryonic membranes (James et al. 1995). The fact that live-born calves do not usually exhibit chromosomal abnormalities suggests that embryos or fetuses with chromosomal anomalies are eliminated prenatally (King 1990, Kawarsky et al. 1996).

The formation of mixoploid embryos is assumed to result from cytokinetic failure, blastomere fusion, endo-reduplication, or combinations of these events (King 1990). Tetraploid cells may arise through the fusion of two diploid blastomeres in a growing embryo (King et al. 1981) or from nuclear division without cytoplasmic division (Hare et al. 1980). Moreover, abnormalities imposed during in vitro culture of the embryo may cause abnormal chromosome segregation leading to mosaicism during cleavage, although Schumacher et al.(1998) concluded that physical factors associated with in vitro culture do not increase DNA ploidy abnormalities in rabbit embryos.

A high incidence of anomalies occurred in the CM groups in this study. The diploid complements were not improved even when a culture-change system was used. In a co-culture system, both the helper cell and the embryo contribute to a dynamic environment that results in increasing concentrations of growth factors and cytokines (Desai & Goldfarb 1998). The rates of diploid blastocysts under co-culture versus non-co-culture conditions (cultured in CR1aa+3% FCS, data not shown) were not different, but both were significantly higher than embryos in the CM group. The presence of serum in CM, that is SCM, caused more anomalies than BCM (Table 4). Coincidently, both studies reported a high incidence of chromosomal abnormalities in bovine embryos (32–45% in day 5 (Kawarsky et al. 1996), and 44% in days 6–8) (Iwasaki et al. 1992) cultured in a co-culture system with cumulus cells (Iwasaki et al. 1992) or oviductal epithelial cells (Kawarsky et al. 1996) with medium TCM 199. A lower incidence of chromosomal anomalies was reported when cloned embryos were cultured in simple media (Mohamed & Takahashi 2000, Arat et al. 2002). Because complex medium contains many components, as does serum, and particularly when the two are used together, it is difficult to identify factors that affect embryonic development and those factors that might alter the genetic composition of the embryo. It becomes even more complicated when a co-culture system is involved and more complex still when considering the potential effects of the NT procedure. All of these factors, particularly when used in an NT culture system, can affect chromosomal behavior and thus lead to chromosomal abnormalities.

The results of this study also showed that the rate of embryo development was significantly affected by the chromosomal complement. Embryos with chromosomal anomalies had decreased development, and cell number was significantly lower than diploid blastocysts in either CM or in co-culture (Tables 4 and 6). Morphologically poor blastocysts tended to have lower cell numbers and exhibited more chromosomal errors (King et al. 1987, Kawarsky et al. 1996).

In summary, CM has similar effects on the development of bovine NT embryos as the co-culture system. Cumulus cell CM may contribute to an increased incidence of blastocyst chromosomal abnormalities. In turn, chromosomal anomalies may lead to lower cloning efficiency and higher rates of embryonic, fetal, and perinatal mortality.

	Acknowledgements

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
This work was supported by the Utah Agriculture Experiment Station (7556).

	Footnotes

 
Received 22 August 2003
First decision 8 October 2003
Accepted 28 October 2003

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Li, G-P Articles by Bunch, T D Search for Related Content PubMed PubMed Citation Articles by Li, G-P Articles by Bunch, T D