Article Can Vero cell co-culture improve in-vitro maturation of bovine oocytes?

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1 RBMOnline - Vol 13. No Reproductive BioMedicine Online; on web 5 July 2006 Article Can Vero cell co-culture improve in-vitro maturation of bovine oocytes? Fariba Moulavi, has been working in the field of developmental biology since She is an active member of the bovine cloning project at the Royan Institute, Isfahan Division. The main research areas of the group with which she is working are bovine stem cells and cloning. Fariba Moulavi Fariba Moulavi, Sayyed Mortaza Hosseini, Saeid Kazemi Ashtiani, Abdolhossein Shahverdi, Mohammad Hossein Nasr-Esfahani 1 Royan Institute, Tehran, Iran 1 Correspondence: Department of Clinical and Experimental Embryology, Royan Institute, Tehran, Iran. Tel: ; Fax: ; info@royaninstitute.org Abstract This study was carried out to evaluate the effect of Vero cell co-culture on developmental competence of immature oocytes. Bovine cumulus oocyte complexes (COC) were matured in presence or absence of Vero cells. Matured oocytes were inseminated and cultured for up to 9 days. Cleavage percentages were recorded on day 2 after insemination and embryos were evaluated on a daily basis. Expanding/expanded and hatching/hatched blastocysts were used for cell number assay. Results indicated a significantly greater cleavage percentage in oocytes matured in presence of Vero cells than control (86% versus 76%, P 0.05). The percentages of advanced embryos appear to be greater on a daily basis in COC matured in presence of Vero cells compared with control. However, these differences were not significant. Blastocysts derived from COC matured in the presence of Vero cells had a significantly higher (P 0.05) number of inner cell mass, trophectoderm and total cell number in expanding/expanded (65.25, and respectively) and hatching/hatched (67.75, and 357.5) embryos in comparison to the control (42, 203.5, and 51.3, 265, respectively). Results confirm that co-culture of bovine COC during in-vitro maturation, enhances their ability for cleavage and for producing blastocysts with higher quality. Keywords: blastocyst quality, bovine oocytes, cleavage, Vero cells 404 Introduction Since the birth of the first calf derived from IVF in 1981 (Brackett et al., 1982), considerable progress has been made in the development of techniques for in-vitro production (IVP) of bovine embryos for both research and commercial purposes (Khurana and Niemann, 2000). However, the success rates in terms of blastocyst yield remain modest and range between 30 and 40% (Kreysing et al., 1997), which is still lower than that obtained from embryos produced in vivo (Krisher et al., 1999). Furthermore, the quality of IVP embryos is inferior to that of embryos produced in vivo as judged by morphology, increased susceptibility to cryo-injury and poor implantation and viability (Wright et al., 1995; Thompson et al., 1998). Various factors including the quality of oocyte, protein source, somatic cells, culture media, oxygen tension, number of embryos per culture unit (embryo density), and energy substrate, may affect preimplantation embryo quality and its competence in further development (Bavister, 1995; Khurana and Niemann, 2000; Abdoon et al., 2001). Co-cultures were advocated in laboratory animal production (Camous et al., 1984) and more importantly in human assisted reproduction techniques, until the establishment of sequential media (Gardner et al., 1998), due to the inadequacy of simple media to support embryo development beyond the cleaving stage and implantation (Myers et al., 1994; Menézo et al., 1995; Fong and Bongso, 1999). Numerous studies have demonstrated the beneficial effects of cellular monolayers of various somatic cell types on mammalian embryonic development in vitro (Ellington et al., 1990; Menézo et al., 1990; Rief et al., 2002). Suggested

2 benefits of co-culture include secretion of tropic factors such as nutrients, substrates, growth factors, and cytokines and removal of potentially toxic substances from culture medium by co-cultured cells (Joo et al., 2001; Kim et al., 2002). In this regard, many different somatic cell types have been used routinely for co-culture including granulosa cells, oviductal cells, uterine cells, fetal bovine endometrial fibroblasts and African green monkey kidney epithelial cells (Vero cells) (Maeda et al., 1996; Lee et al., 2001). All these feeder cells used in co-culture were able to improve in-vitro development of bovine zygotes compared with a medium devoid of cells (Menck et al., 1997). Vero cells, derived from African green monkey (Cercopithecus aethiops) kidney, have a common embryonic origin (mesoderm) with cells from the genital tract (Duszewska et al., 2000). Furthermore, they are very common, easily available cells giving an improved effect on embryo culture in vitro. Therefore co-culture using Vero cells has been widely utilized to enhance embryo viability and development (Pegoraro et al., 1998; Duszewaka et al., 2000). Although, the beneficial effects of co-culture on the quality of IVF derived embryos is well documented, nearly all of the reports pertain to the establishment of co-culture for development of zygotes to blastocyst stage and there are no reports on the influence of co-culture upon in-vitro maturation of bovine oocytes. So, due to the fundamental importance of optimized maturation of oocytes for subsequent normal embryo development in vitro, this study was conducted to compare the developmental competence of bovine oocytes matured in the presence or absence of Vero cell co-culture. Materials and methods Chemicals were purchased from Sigma chemical Co. (St. Louis, MO, USA) unless otherwise indicated. Culture media The media used for maturation of oocytes and development of zygotes in vitro were based on tissue culture medium (TCM) 199 supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS). For maturation, TCM 199 was enriched by routinely used hormones FSH (10 μg/ml), LH (10 μg/ml), 17-β-oestradiol (1 μg/ml). Establishment and maintenance of Vero cell monolayer After thawing the Vero cells, they were seeded at a concentration of /ml in 25-cm 2 flasks (Falcon) in the TCM 199 medium and cultured at 38.5 C, 5% CO 2 in humidified air. Upon confluency (3 4 days after culture), the medium and unattached cells were removed. After trypsinization with 0.25% trypsin EDTA, the harvested cells were used for further seeding and for maturation in 100 μl drops at a concentration of At least 2 h before maturation of the oocytes, the medium of drops containing Vero cells were exchanged with fresh equilibrated maturation medium. Oocyte recovery and in-vitro maturation Ovaries were collected at a local abattoir immediately after slaughter and transported to the laboratory in saline solution (0.9% NaCl) supplemented with penicillin G (100 IU/ml), streptomycin (100 μg/ml) at 30 C. At the laboratory, the cumulus oocyte complexe (COC) were aspirated from transparent follicles (2 8 mm in diameter). Only oocytes having a homogenous evenly granulated cytoplasm surrounded by a compact cumulus oophorus with more than three layers were used. Experimental design The selected COC were washed in HEPES-TCM 199 and maturation medium, TCM 199 supplemented with FSH (10 μg/ml), LH (10 μg/ml), 17-β-oestradiol (1 μg/ml), and were randomly divided into two groups: every five COC were placed in 100 μl maturation medium for 24 h in presence or absence (control) of Vero cell monolayer in 90% humidity at 38.6 C and 5% CO 2 in air in a C200 incubator (Labotect, Germany). This experiment was repeated in 10 replicates containing between COC in each group. Determination of maturation status At the end of maturation period, nuclear status of some matured oocytes was determined by 4,6-diamino-2-phenylindole (DAPI) staining as described by Izadyar et al. (1996). Briefly COC were denuded by vortexing for 3 7 min and then fixed for 15 min in 2.5% (w/v) glutaraldehyde, washed with phosphate-buffered saline (PBS), stained with 2.5% (w/v) DAPI, and mounted on glass slides. The nuclear status of the stained oocytes was assessed under a fluorescence microscope. The status of nuclear maturation of each oocyte was recorded as: GV (germinal vesicle: no maturation occurred); GVBD (germinal vesicle breakdown: the early stage of maturation); MI (metaphase one of the meiosis resumption: the middle stage of maturation); MII (metaphase two arrested oocyte: the fully nuclear matured oocyte), as described by Mori et al. (1988). Sperm preparation and IVF Commercially distributed frozen semen from two Holstein bulls with proven fertility was used throughout this study. For IVF, COC were washed twice and transferred in groups of per 200-μl drop of fertilization medium under mineral oil. The IVF medium consisted of Fert TALP (Parrish et al., 1986) supplemented with penicillamine (20 μm), hypotaurine (10 μm), epinephrine (1 μm) and heparin (0.56 μg/ml). After thawing semen in 37 C water, motile spermatozoa were obtained by swim-up procedure and were added to the fertilization drop in a final concentration of per ml. Spermatozoa and oocytes were co-incubated for 20 h at 38.6 C with 5% CO 2 in humidified air. 405

3 In-vitro culture Following fertilization, presumptive zygotes (note that in bovine embryos the two-pronuclear stage is not observed, so cleavage is presumed to indicate fertilization) derived from matured oocytes in presence or absence (control) of Vero cells were freed from loosened cumulus cells, washed and then the presumptive zygotes from both groups were cultured over Vero cells in TCM 199 plus 10% FCS at 90% humidity, 38.6 C in 5% CO 2 and 5% O 2. During the whole period of embryo culture, the embryos were transferred daily to the new culture dish containing established Vero cells. It has been suggested that Vero cells, once stressed by trypsinization, may release non-specific stress proteins that may provide beneficial effects for embryo viability (Kim et al., 2002). Embryo evaluation All presumptive zygotes were co-cultured at 90% humidity, 38.6 C, 5% CO 2 and 5% O 2 for a period of 8 9 days (day 0 = day of insemination). Embryos were evaluated daily and scored as cleaved, 4-, 8-, 8 16-cell, morula, compact, early/ expanded and hatching/hatched blastocyst. Differential staining of embryos At the end of the culture period (day 9), the number of cells in some fully developed blastocysts in both groups were determined by differential staining as described by Thouas et al. (2001). Briefly, blastocysts were first incubated in 500 μl of 1% Triton X-100 and 100 μg/ml propidium iodide for up to 30 s and then immediately transferred into 500 μl of 100% ethanol with 25 μg/ml bisbenzimide (Hoechst 33258) and stored at 4 C overnight. Fixed and stained embryos were then mounted on to a glass slide in a drop of glycerol, gently flattened with a cover slip and visualized for cell counting on a fluorescence microscope (excitation filter 460 nm for blue and 560 nm for red) (Figure 1). Statistical analysis The difference between groups was tested using t-test and chi-squared test as appropriate by means of SPSS software. Difference was considered to be significant at P Results Experiment 1: Comparison of chromatin status of oocytes matured in the presence of Vero cells with the control group. The results of chromatin assessment are detailed in Table 1. These results indicate no significant difference (P 0.05) in percentage of MII, GV, GVBD and MI of oocytes matured in presence or absence of Vero cells. Experiment 2: Developmental competence of oocytes matured in presence or absence of Vero cells: Table 2 represents the number and percentage of inseminated oocytes that have cleaved and progressed to the different stages of embryo development. For oocytes matured in presence of Vero cells, the average cleavage percentage on day 2 after insemination was significantly higher than the control (86.32 versus 76.32%, P 0.05). From day 3 onwards, the percentage of advanced developing embryos was higher in COC matured in presence of Vero cells compared with control group on a daily basis. However, these differences were not significant (Figure 2). At day 9, the percentages of total blastocysts and hatching/hatched blastocysts obtained in COC matured in presence of Vero cells were 43.08% and 19.88%, respectively, which is slightly, but not significantly, greater than the control (35.85% and 16.28% respectively). These results suggest that maturation of bovine oocytes in presence of Vero cells may improve their maturational competency for fertilization. Experiment 3: Effect of Vero cell coculture during maturation on inner cell mass, trophectoderm and total cell number of bovine embryos: The quality of expanding/expanded, hatching/hatched embryos yielded at day 9 in both groups was assessed by evaluating the total cell number (TCN) from the sum of inner cell mass (ICM) and trophectoderm (TE). As shown in Table 3, embryos that were obtained from oocytes matured in presence of Vero cells had a significantly (P 0.05) greater cell number for both ICM and TE compared with the control. Interestingly the ICM:TE ratio and the percentages of ICM:TCN in both expanding/expanded and hatching/hatched blastocysts (day 9) derived from maturation of COC in presence of Vero cells were also higher than in the corresponding controls. 406

4 Figure 1. Image of a day-9 bovine hatched blastocyst cultured over a Vero cell monolayer; the white arrow shows the inner cell mass (A); the same blastocyst stained differentially with Triton X-100/propidium iodide for trophectoderm and ethanol/hoechst for inner cell mass cells (B). Table 1. Nuclear status of oocytes matured in presence (treatment) or absence (control) of Vero cells after staining using triton X-100/4,6-diamino-2-phenyl-indole) solution (2 μg/ml). The results of three replicates are presented. GV = germinal vesicle, GVBD = germinal vesicle breakdown, MI = metaphase I, MII = metaphase II. Maturation method Nuclear status GV GVBD MI MII (%) (%) (%) (%) Presence of Vero cells Absence of Vero cells Values are not significantly different according to chi-squared test. Table 2. Percentage of cleaved embryos, total blastocysts and hatching/hatched blastocysts per cleaved embryos derived from cumulus oocyte complexes matured in presence or absence of Vero cells. Maturation method No. Cleaved embryos Total blastocyst/ Hatched presumptive (% ± SD) cleaved embryos blastocyst/ zygotes (% ± SD) cleaved embryos (% ± SD) Presence of Vero cells ± 9.01 a ± ± Absence of Vero cells ± b ± ± a,b Values differ significantly according to chi-squared test, P

5 Figure 2. Different stages of bovine embryo development following maturation of oocytes in presence or absence of Vero cell monolayer between days 2 to 9 after insemination. Bars with different superscripts within each developmental stage differ significantly (P 0.05). Table 3. Number of inner cell mass (ICM), trophectoderm (TE) and total cell number (TCN) in blastocysts derived from cumulus oocyte complexes matured in presence or absence of Vero cells on day 9 after insemination. Treatments Expanding/ Hatching/hatched expanded blastocysts blastocysts Vero cell Absence Presence Absence Presence No. blastocysts Total cell no ± 9.2 a ± 6.6 b ± 7.4 a ± 13 b No. ICM cells 42 ± 1.4 a ± 2.8 b 51.3 ± 2.08 a ± 2.75 b No. TE cells ± 7.8 a ± 5.4 b 265 ± 5.57 a ± 11.8 b ICM/TCN (%) ICM:TE ratio 1:4.84 1:3.43 1:5.16 1:4.28 a,b Values differ significantly according to chi-squared test, P ICM = inner cell mass; TE = trophectoderm; TCN = total cell number. 408

6 Discussion Monolayers of Vero cells have been used widely to enhance invitro development of early embryos from many species since they are easy to harvest and culture and have beneficial effects on embryo growth (Turner and Lenton, 1996). Furthermore, embryo co-culture has proven to be an appropriate system for production of embryos for commercially focused purposes (Rife et al., 2002). Embryonic stem cells are being also grown on layers of fibroblast cells that form the matrix upon which stem cells grow. In this regard, the first established human stem cells were cultured on mitotically inactivated mouse embryonic fibroblasts as feeder cells (Lysdahl et al., 2006). Such feeder cells provide an ideal environment for the growth and maintenance of human embryonic stem cells, because the feeder cells detoxify culture medium, secrete many unique proteins that facilitate cell growth and remodel the extracellular matrix (Rodriguez et al., 2006). A number of studies has been performed to evaluate the influence of co-culture system on nuclear and cytoplasmic maturation of primary oocytes to be used in IVF programme in some limited species such as equine (Li et al., 2001), canine (Hewitt and England, 1999) and human cumulus-free germinal vesicle oocytes (Janssenswillen et al., 1995). Accordingly, these authors proposed that co-culture of oocytes with somatic cells during maturation, improves their subsequent percentage of fertilization and embryo development in vitro. As far as is known, this is the first study that evaluates the effect of Vero cells as a co-culture system during in-vitro maturation of bovine oocytes. Briefly, COC were cultured in presence or absence of Vero cells for 24 h, and then presumptively fertilized oocytes were cultured over Vero cells in TCM. Therefore, the only difference between two groups was whether the maturation process was performed in presence or absence of a Vero cell monolayer. The results of this study indicate that maturation of oocytes in presence of Vero cells either improves their fertilization potential and/or improves the cleavage rate; thus the number of cleaved zygotes with respect to number of oocytes inseminated increases (Figure 2 and Table 2). The results of total cell counting in embryos derived from oocytes matured in presence of Vero cells were significantly higher compared with control. Additionally, the ICM/TE ratio of embryos in co-cultured oocytes was higher than control, which further indicates better quality of embryos produced in this group (Table 3). A significant increase in number of cleaved zygotes in treated groups, compared with control, may be related to the improved oocyte cytoplasmic or nuclear maturation. Since there was no difference in the rate of nuclear maturation between two groups, this may further highlight better cytoplasmic maturation and hence higher fertilization rate and developmental competence of oocytes matured in presence of Vero cells compared with control group. In this regard, Li et al. (2001) investigated the influence of co-culture during maturation of equine oocytes on their competence after intracytoplasmic sperm injection. Similarly, they showed that the proportion of MII stage oocytes after culture in TCM 199 alone or in co-culture does not differ significantly. They assumed that maturation of oocytes in presence of co-culture would provide an appropriate synchrony between nuclear and cytoplasmic maturation, which may result in better sperm oocyte interaction. Indeed, Balaban and Urman (2006) suggested that cytoplasmic properties of oocytes have a more detrimental effect on their fertilization and future development compared with extracytoplasmic factors (presence of intact polar body, perivitelline space, and dark zona pellucida) during fertilization and subsequent development. Therefore, a higher fertilization rate and better blastocyst quality could be attributed to the effect of co-culture conditioning on cytoplasmic maturity. Total cell number has been considered to be the most sensitive criterion for post-implantation developmental capacity of embryos produced in vitro (Brinsko et al., 1994). Since the results of cell counting in embryos derived from the oocytes matured in the presence of Vero cells were higher than control, thus implying maturation of oocyte in the presence of Vero cells could improve their future competence for fertilization and development. This confirms the belief that the kinetics of embryo development is different according to the culture system. By glancing at Figure 2, it is obvious that throughout the study there is a tendency in the treated group to produce more blastocysts than the control; however this difference was not significant. Despite the numerous confirmative reports about the benefits of Vero cell co-culture for embryo development in human and animals, the exact mechanism by which Vero cells exert their effects is still unclear. One of the supposed mechanisms by which Vero cells and other cell types used for co-cultures improve quality of embryo development is the ability of these cells to overcome developmental block (Chen et al., 1994); the arresting phase usually occurs during the initial periods of invitro development in almost all mammalian embryos (Bavister, 1995; Carnegie et al., 1999). The developmental block may be overcome by either secretion of embryotrophic factors and/or by neutralizing and detoxification of the culture medium by the cells (Schillaci et al., 1994). Therefore, the increased cleavage rate observed in this study may be due to such a mechanism. Surprisingly, Janssenswillen et al. (1995) showed that coculture with Vero cells improves the in-vitro maturation of human cumulus-free germinal vesicle stage oocytes. It is documented that Vero cells produce and release interleukin, platelet-derived growth factor, leukaemia inhibitory factor and insulin-like growth factor (IGF) (Joo et al., 2001). Also, it is well understood that bovine COC, like bovine embryos, have receptors for insulin, IGF-1, IGF-2R, and epidermal growth factor (Chen et al., 1994). The regulation of oocyte maturation by growth factors in animal species such as mice, rats, rabbits, pigs, and cattle (Lorenzo et al., 1994) has been described. IGF-1 stimulates maturation in Xenopus oocytes, enhances bovine oocyte maturation and fertilization in vitro (Herrler et al., 1992; Lorenzo et al., 1993), and promotes rabbit blastocyst development. Carneiro et al. (2001) also concluded that IGF-1 has an important role not only in the nuclear maturation of oocytes but also in cytoplasmic maturation. In these studies, nuclear and cytoplasmic maturation were measured by cleavage, parthenogenic cleavage and blastocyst formation (Carneiro et al., 2001). By looking through the literature it can be concluded that growth factors like IGF-1 and epidermal growth factor promote both 409

7 410 nuclear and cytoplasmic maturation in many species as well as bovine COC. Therefore, it is possible to conclude that one of the routes through which Vero cells may exert their stimulatory effects is via growth factors and maturation in presence of Vero cells might be considered as a suitable method for in-vitro maturation and production of competent embryos. Blastocyst cell count also reveals a better quality of embryo produced from oocytes matured in presence of Vero cells. The authors believe that initial priming of bovine immature oocytes with Vero cells triggers pathways that ultimately result in higher cell numbers in the inner cell mass rather than TE. Further study is necessary to clarify this. Finally, the results of this study suggest that the use of Vero cell co-culture for maturation of bovine oocytes gives encouraging results and demonstrates the beneficial influence of co-culture during oocyte maturation. Furthermore, it is of interest to evaluate the transferability, implantation and pregnancy rate following transfer of the embryos produced from oocytes matured in presence or absence of Vero cells. Acknowledgements This study was funded by the grant of Royan Institute of IRI. The authors would like to gratefully thank Dr A Vsough for his full support. References Abdoon ASS, Kandil OM, Otoi T, Suzuki T 2001 Influence of oocyte quality, culture media and gonadotropins on cleavage rate and development of in vitro fertilized buffalo embryos. Animal Reproduction Science 65, Balaban B, Urman B 2006 Effect of oocyte morphology on embryo development and implantation. Reproductive BioMedicine Online 12, Bavister BD 1995 Culture of preimplantation embryos: facts and artifacts. Human Reproduction Update 1, Brackett BG, Bousquet D, Boice ML et al Normal development following in vitro fertilization in the cow. Biology of Reproduction 27, Brinsko SP, Ball BA, Miller PG 1994 In vitro development of day 2 embyos obtained from young, fertile mares and aged, subfertile mares. Journal of Reproduction and Fertility 102, Camous S, Heyman Y, Meziou W, Menézo Y 1984 Cleavage beyond the block stage and survival after transfer of early bovine embryos cultured with trophoblastic vesicles. Journal of Reproduction and Fertility 72, Carnegie JA, Morgan JJ, McDiarmid N, Durnford R 1999 Influence of protein supplements on the secretion of leukemia inhibitory factor by mitomycin-pretreated Vero cells: possible application to the in vitro production of bovine blastocysts with high cryotolerance. Journal of Reproduction and Fertility 117, Carneiro G, Lorenzo P, Pimentel C et al Influence of insulin-like growth factor-i and its interaction with gonadotropins, estradiol, and fetal calf serum on in vitro maturation and parthenogenic development in equine oocytes. Biology of Reproduction 65, Chen HF, Ho HN, Chen SU et al Peptides extracted from Vero cell cultures overcomes the blastocyst block of mouse embryos in a serum-free medium. Journal of Assisted Reproduction and Genetics 11, Duszewska AM, Reklewski Z, Pienkowski M et al Development of bovine embryos on Vero/BRL cell monolayers (mixed coculture). Theriogenology 54, Ellington JE, Carney EW, Farrell PB et al Bovine 1 2 -cell embryo development using a single medium in three oviduct epithelial cell co-culture systems. Biology of Reproduction 43, Fong CY, Bongso A 1999 Comparison of human blastulation rates and total cell number in sequential culture media with or without coculture. Human Reproduction 14, Gardner DK, Schoolcraft WB, Wagley L et al A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Human Reproduction 13, Herrler A, Lucas-Hann A, Niemann A 1992 Effects of insulinlike growth factor-i on in vitro production of bovine embryos. Theriogenology 37, Hewitt DA, England GCW 1999 Synthetic oviductal fluid and oviductal cell co-culture for canine oocyte maturation in vitro. Animal Reproduction Science 55, Izadyar F, Colenbrander B, Bevers MM 1996 In vitro maturation of bovine oocytes in the presence of growth hormone accelerates nuclear maturation and promotes subsequent embryonic development. Molecular Reproductive Development 45, Janssenswillen C, Nagy ZP, Van Steirteghem A 1995 Maturation of human cumulus-free germinal vesicle-stage oocytes to metaphase II by co-culture with monolayer of Vero cells. Human Reproduction 10, Joo BS, Kim MK, Na YJ et al The mechanism of action of co-culture on embryo development in the mouse model: direct embryo-to-cell contact and the removal of deleterious components. Fertility and Sterility 72, Khurana NK, Niemann H 2000 Effects of oocyte quality, oxygen tension, embryo density, cumulus cells and energy substrates on cleavage and morula/blastocyst formation of bovine embryos. Theriogenology 54, Kim YB, Ahn SH, Chang DY et al Vero cell co-culture counteracts the detrimental effects of hydrosalpinx fluid on the development of mouse embryos in vitro. Journal of Korean Medical Science 17, Kreysing U, Nagai T, Niemann H 1997 Male dependent variability of fertilization and embryo development in two bovine in vitro fertilization systems and the effects of casein phosphopeptides (CPPs). Reproduction Fertility and Development 9, Krisher RL, Lane M, Bavister BD 1999 Developmental competence and metabolism of bovine embryos cultured in semi-defined and defined culture media. Biology of Reproduction 60, Lee YL, Xu JS, Chan ST et al Vero cells, but not oviductal cells, increase the hatching frequency and total cell count of mouse blastocysts partly by changing energy substrate concentrations in culture medium. Journal of Assisted Reproduction and 18, Li X, Morris HA, Allen WR 2001 Influence of co-culture during maturation on the developmental potential of equine oocytes fertilized by intracytoplasmic sperm injection (ICSI). Reproduction 121, Lorenzo PL, Illera MJ, Illera JC, Illera M 1994 Enhancement of cumulus expansion and nuclear maturation during bovine oocyte IVM with the addition of epidermal growth factor and insulin-like growth factor-1. Journal of Reproduction and Fertility 101, Lorenzo PL, Illera MJ, Sanchez J et al Bovine oocyte maturation and fertilization in vitro with growth factors and estrous cow serum. Theriogenology 39,262. Lysdahl H, Gabrielsen A, Minger SL et al Derivation and characterization of four new human embryonic stem cell lines: the Danish experience. Reproductive BioMedicine Online 12, Maeda J, Kotsuji F, Negami A et al In vitro development of bovine embryos in conditioned media from bovine granulosa cells and Vero cells cultured in exogenous protein and amino acidfree chemically defined human tubal fluid medium. Biology of Reproduction 54, Menck MC, Guyader-Joly C, Peynot N et al Beneficial effects of Vero cells for developing IVF bovine eggs in two different

8 co-culture systems. Reproduction Nutrition Development 37, Menézo YJ, Sakkas D, Janny L 1995 Co-culture of the early human embryo: factors affecting human blastocyst formation in vitro. Microscopic Research Technique 32, Menézo YJR, Guerin JF, Czyba JC 1990 Improvement of human early embryo development in vitro by co-culture on monolayers of Vero cells. Biology of Reproduction 42, Mori C, Hashimoto H, Hoshino K 1988 Fluoresence microscopy of nuclear DNA in oocytes and zygotes during in vitro fertilization and development of early embryos in mice. Biology of Reproduction 39, Myers MW, Broussard JR, Menézo Y et al Established cell lines and their in vitro conditioned media support bovine embryo development during in-vitro culture. Human Reproduction 9, Parrish JJ, Susko-Parrish JL, Liebfried-Rutledge ML et al Bovine in vitro fertilization with frozen-thawed semen. Theriogenology 25, Pegoraro LMC, Thuard JM, Delalleau N et al Comparison of sex ratio and cell number of IVM- IVF bovine blastocysts cocultured with bovine oviduct epithelial cells or with Vero cells. Theriogenology 49, Rief S, Sinowatz F, Stojkovic M et al Effect of a novel coculture system on development, metabolism and gene expression of bovine embryos produced in vitro. Reproduction 124, Rodriguez CI, Galan A, Valbuena D, Simon C 2006 Derivation of clinical-grade human embryonic stem cells. Reproductive BioMedicine Online 12, Schillaci R, Ciriminna R, Cefalu E 1994 Vero cell effect on in-vitro human blastocyst development: preliminary results. Human Reproduction 9, Thompson JG, Sherman ANM, Alen NW et al Total protein content and protein synthesis within pre-elongation stage bovine embryos. Molecular Reproductive Development 50, Thouas GA, Korfiatis NA, French AJ et al Simplified technique for differential staining of inner cell mass and trophectoderm cells of mouse and bovine blastocysts. Reproductive BioMedicine Online (web paper) 149, Turner K, Lenton EA 1996 The influence of Vero cell culture on human embryo development and chorionic gonadotrophin production in vitro. Human Reproduction 11, Wright RW Jr, Elington J 1995 Morphological and physiological differences between in vivo and in vitro produced preimplantation embryos from livestock species. Theriogenology 44, Received 12 December 2005; refereed 6 January 2006; accepted 31 May