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Bovine embryos cultured in serum-poor oviduct-conditioned medium need cooperation to reach the blastocyst stage
Theriogenology
42:445-453,
1994
BOVIhJE EMBRYOS CULTURED IN SERUM-POOR OVIDUCT-CONDITIONED MEDIUM NEED COOPERATION TO REACH THE BLASTOCYST STAGE L. Ferry,1 I’. Mermillod,2
A. Massipl
and F. Dessyls a
tuniversite catholique de Louvain, Unite des Sciences Veterinaires Place Croix du Sud 3, B - 1348 Louvain-la-Neuve, Belgium ZINRA, Station de Physiologie de la Reproduction F - 37380 Nouzilly, France Received for publication : August 24, 1993 Accepted:
5, 1994
ABSTRACT Immature bovine oocytes were matured and fertilized in vitro, and the resulting zygotes were cultured to the blastocyst stage in droplets of tissue culture medium 199 (TCM 199) conditioned by oviduct cells in the absence of serum. In Experiment 1, the effect of the number of zygotes in a constant culture volume was investigated by culturing 1, 4 or 40 zygotes in 40 ~1 of culture medium. The c1eavag.e rate was low with a single embryo (36%) but increased with the number of embryos, to reach 50% with 4 embryos/40 ~.rland 59% with 40 embryos/40 ~1. Blastocyst formation was nil with 1 embryo per 40 ~1, reaching 2.5 % with 4 embryos/40 l.tl and 18% with 40 embryos/40 ~1. The effect of the size of the drop was assessed in Experiment 2, the concentration of embryos remaining constant (I embryo/l ~1). The volumes tested were 10, 20, 30 and 40 ~1. Development into blastocysts increased gradually from 12% in the lO/lO group to 20% in the 40/40 group. Experiment 3 was designed to find a minimal droplet volume able to support the development of a single embryo to the blastocyst stage. The minimum tested volume was 5 ~1 and was not successful. These results show that bovine embryos cultured in oviduct-conditioned TCM 199 need to cooperate to reach the blastocyst stage. The mechanism of this cooperation is not known, but some autocrine/paracrine factors, probably growth factors, could promote embryo development as was demonstrated in mice. From Experiment 2 we can hypothesize that the surface/volume ratio of the droplets could play a role in the culture conditions by interfering with the exchanges between the culture medium and the surrounding environment. Key words : bovine, embryo, culture, serum-poor conditioned medium Acknowledgements This work was supported by a grant from Minis&e de 1’Agriculture de la Region Wallonne de Belgique and by EEC Biotechnology programme grant BI02-CT92-0067. We thank J.F. Beckers for the gift of p-FSH and p-LH; G. Gerard and L. Collette for statistical advice and M.-A. Mauclet for typing the manuscript. a Correspondence and reprint requests : Fax : +/32/10/47.37.17.
Copyright
0 1994 Butterworth-Heinemann
Theriogenology
446
INTRODUCTION The in vitro methodology for generating bovine embryos offers several advantages as for example 1) low cost production of a large number of embryos from abattoir material for research and commercial purposes; 2) development of new technologies in animal breeding such as cloning, gene transfer and the like; 3) circumventing the uncertainty of the reaction of donor cows after hormonal superovulation treatment, by using ultrasonographically guided oocyte retrieval from all available ovarian follicles. This last procedure could become an alternative to superovulation in specified donor cows (8,18). In such cases oocytes from individual animals have to be manipulated separately whatever their number. It has been shown in the mouse (1, 4, 16, 26) and hamster (20) and suspected in the ovine (19) and bovine (24) that culture conditions such as embryo density could influence embryonic Therefore we have undertaken a study on the effect of the development. embryo/medium volume ratio and of the size of the culture droplet on development in medium conditioned with oviduct cells without serum (12).
MATERIALS
AND METHODS
Oviduct Cell Culture and Medium Conditioning If not specified, all reagents used in this study were cell culture-tested grade reagents from Sigma. b Oviduct cell cultures were initiated as previously described by Eyestone and First (3). All the solutions and vessels were sterile. Cow oviducts were obtained from an abattoir without control of estrous cycle status of the donors. After dissection, the oviducts were briefly immersed in 70% ethanol and mucosa was gently scraped free using a microscope slide. Mucosal tissue was transferred to a conical tube containing 10 ml of TCM 199 supplemented with 10% heat-treated fetal calf serum (FCS).c Tissue was then washed twice in fresh medium and resuspended in 50 volumes of medium for seeding in 25-cm2 culture flasks; 6 ml were introduced in each f1ask.d After 4 d, medium was renewed and unattached cells were discarded. Confluence was reached 6 d after seeding. For conditioning, confluent cells were washed once with TCM 199 without FCS, and 6 ml of the same medium were added to each 25-cm2 flask. Because some serum factors would perhaps remain on the cell membranes and leaching of serum components from the cells into the medium cannot be avoided, this medium was called “serum-poor conditioned medium” instead of protein-free medium as was used in previous publications (12). Serum-poor conditioned-medium (SPCM) was collected every 2 d, centrifuged at 500 g for 10 min and stored refrigerated (4°C). After 3 harvests (6 d) from the same monolayer, the 3 collections were pooled and frozen (20°C) in l-ml aliquots. Conditioned medium was centrifuged prior embryo culture (2 min 13,000 g). b Sigma, St-Louis, MO, U.S.A c Gibco, Paisley, Scotland. d Nunc, Roskilde, Denmark.
447
Theriogenology
ln Vitro
Maturation
(IVM)
and Fertilization
(IVF)
The methods used for IVM and IVF were modifications of those described previously by Parrish et al. (17) and Sirard et al. (22). All cultures were performed at 39°C under 5% CO2 in water-saturated air. Qocvte collection and maturation. Oocytes were collected by aspiration of 2 to 5-mm follicles from slaughtered cow ovaries. Only those oocytes completely surrounded by compact cumulus cells were selected, as described by LeibfriedRutledge (lo), and were washed 4 times in Tyrode’s modified medium containing albumin, lactate and pyruvate (low bicarbonate TALP) according to Parrish et al. (17). Groups of up to 100 were transferred to 4-well plates d containing 500 ~1 medium (TCM 199 su plemented with 10% heat treated FCS, 1 mg 17b-estradiol ml-l, 5 pg p LH ml- P and 0.5 pg p-FSH ml-1 (p-LH and p-FSH were from Dr J.F. Beckers, LiPge, Belgium) for 24 h of maturation. krtilization. Mature oocytes were washed 4 times in TALP, and groups of 100 were transferred into 4-well plates containing 250 ~1 of fertilization medium per well (TALP containing 10 yg heparin ml-1 Na salt e , 167 U/m@. Motile spermatozoa were obtained by centrifugation of 500 ~1 of thawed semen on a Percoll f discontinuous density gradient (2 ml at 45/2 ml at 90%) for 30 min at 700 g. Living spermatozoa (at the bottom of the 90% Percoll fraction) were washed in TALP and pelleted by centrifugation at 100 g for 10 min. Spermatozoa were counted in a hemocytometer, diluted in the appropriate volume of TALP and 250 ~1 of the suspension were added to each fertilization well to obtain a final concentration of 2 x lo6 spermatozoa ml- 1. Plates were then incubated for 18 h. Embryo
Culture
Fertilized ova were washed in TCM 199 supplemented with 10 % of heat treated FCS. Cumulus cells were removed by vortex mixing at medium speed during 2 min. About 100 ova were mixed in 2 ml of PBS containing 0.25% trypsin. All the zygotes were then pooled and carefully observed before transfer to culture droplets. Only those completely denuded were selected. This selection is important in order to avoid the metabolite depletion of the small volume of medium by the fast growth of granulosa cells. They were washed in TCM 199 without FCS and cuItured in droplets of conditioned medium under mineral oil. Droplet volumes and number of zygotes in each droplet varied according to experimental procotols.
e Calbiochem, San Diego, CA, USA. f Pharmacia, Uppsala, Sweden.
Theriogenology
448
Experiments Three experiments were conducted and in each case the 5 to g-cell stage rate was assessed on Day 2 of culture (42 h post insemination), and the number of embryos reaching the expanded blastocyst stage was determined on Day 8 (210 h post insemination). In Experiment 1, the effect of the number of zygotes in a constant culture volume was investigated by culturing either 1, 4 or 40 zygotes in 40 ~1 of culture medium. In Experiment 2, the effect of droplet volume was examined, with the zygote concentration remaining constant (1 zygote/l ul). The droplet volumes were 10, 20, 30 and 40 ul. In Experiment 3, we attempted to find a minimal droplet volume able to support the development of a single embryo to the blastocyst stage. It was assumed that embryos produce some autocrine factors and that reducing the volume would reduce the dilution of these factors. A single zygote was cultured in a drop of 5, 10 or 40 ~1; the control consisted of 40 Zygotes/40 ul. Statistical
Analysis
using Chi-square The ANOVA for categorical data . _ (log transformed) r . . . analysis was performed to compare the trequencies ot the various development stages. The CATMOD procedure of SAS g was used to perform the analysis. Linear regression analysis was used to test the relation between droplet Percentages were transformed by arcsin volume and embryo development. (square root [data]) to normalize the distribution.
RESULTS The results of Experiment 1 are reported in Table 1. They clearly show an effect of the number of embryos in a constant culture volume on their rate of development. The cleavage rate was low with only a single embryo (36%), but it increased to 50% with 4 embryos/40 ul and to 59% with 40 embryos/40 ~1. the most significant effect was observed on blastocyst However, development : 0% with 1 embryo per 40 ~1, 2.5% with 4 embryos per 40 1.11and 18% with 40 zygotes per 40 ~1. A higher density of zygotes increased their rate of development to the blastocyst stage. Experiment 2 examines the effect of droplet volume on embryo development when zygote concentration remained constant (1 zygote/ul). The results are iven in Table 2. After 2 d of culture, some differences were observed using the X5 analysis on the number of 5 to g-cell embryos; however, no statistical correlation could be established with droplet volume. g SAS Institute Inc., Cary, NC, USA.
449
Theriogenology
Regression analysis revealed a significant regression slope (I? < 0.05) for the blastocyst rate. It seems that an increase in droplet volume allows for more embryos to reach the blastocyst stage. Table 1. Effect of embryo number on cleavage and blastocyst formation in a constant volume Total no. of No. of 5 to 8-cell zygotes embryos (%)
No. of expanded blastocysts (%)
No. of zygotes /
No. of
droplet volume (~1)
reP1icates
l/40
2
80
29 (36) a
0 (0) a
4/40
2
80
40 (50) a,b
2 (2.5) a
40/40
2
200
117 (59) b
36 (18) b
Gues Table 2
with different superscripts within a column are different
(X2; P ~0.05).
Effect of droplet volume at constant zygote concentration and blastocyst development
on cleavage
No. cd zygotes /
No. of
Total no.
droplet volume (@)
rePlicates
40/40
of zYgotes
No. of 5 to &cell embryos (%)
No. of expanded blastocysts (%)
4
320
147 (46) a
62 (20) c
30/30
2
120
79 (66) b
20 (17) c
20/20
2
80
42 (53) b
11 (14) c
lO/lO
4
190
89 (47) a
23 (12) c
mues with different superscripts within a column are different c Slope of the linear regression is different from zero (I’ <0.05).
(X2; P <0.05).
Experiment 3 was conducted to find the minimal drop size and culture volume for a single zygote to reach the blastocyst stage. A single zygote in 5, 10 or 4O/yl could not meet this objective (Table 3). Blastocysts could only be obtained in the control group. In Experiment 3, no difference was evidenced in the number of embryos reaching the 5 to 8-cell stage in the l/40 and 40/40 groups, in contrast with the results of Experiment ‘I.
Theriogenology
450
Table 3. Effect of droplet volume on development of single zygotes No. of zygotes / droplet volume (~1)
l/10
No. of replicates
Total no.
ofzygotes
No. of 5 to &cell embryos (%)
No. of expanded blastocysts (%)
2
13 (33) a
0 (0)a
2
19 (48) a
0 (0)a
81 (51) a
40 (25) b
2 40/40
2
160
a No differences were found in this column (X2; p >0.05). a! b Values with different superscripts within a column are different
(X2; P ~0.05).
DISCUSSION The results in Tables 1 and 3 demonstrate very clearly that single zygotes cannot develop to the blastocyst stage whatever the volume or size of the culture drop in our medium conditioned by oviduct cells without serum. When more than 1 zygote was present in the culture drop, blastocysts were obtained. Similar observations were reported in the mouse (1, 4, 16, 26) and hamster (20). In sheep and cattle, it was suggested that small droplets can induce cooperative interaction among embryos or that they influence the embryo environment in a positive manner (19, 24). This cooperative interaction could be mediated by autocrine and/or paracrine factors secreted by embryos themselves, and this secretion could depend on the number of embryos present per volume unit. These factors could interfere, during the first cleavage, with mechanisms to be used later by the embryo. Paria and Dey (16) obtained mouse blastocysts from 2-cell embryos cultured individually by adding EGF (epidermal growth factor) or TGFPl (transforming growth factor) to the culture medium. In the bovine, EGF stimulated hatching of singly cultured 8-cell embryos but did not significantly affect blastocyst development (7). In cattle, mRNAs for TGFa, TGFP2, PDGF, IGF-I and IGF-II as well as mRNAs for the receptors of IGF-I, IGF-II and PDGF are present in the embryo from the oocyte to the blastocyst stage (25). The use of EGF during maturation of oocytes in serum-free medium enhances the cleavage rate of the subsequently cultured embryos (2). The sequential addition of PDGF and TGFa to a serum-free culture medium promotes, respectively, development through the 8 to 16-cell block and blastulation (9). These results indicate that growth factors can be involved in bovine preimplantation embryo development and that some of these or other factors can be produced by the embryo itself, as is the case with human embryos (6). Early embryos are, therefore, capable of producing, have receptors for, and can respond to a number of growth factors, suggesting that autocrine or paracrine mechanisms are involved in early development (21).
Theriogenology
451
These reports have significant implications for an understanding of the control of early embryo development and for the improvement of culture of domestic animal embryos. In fact we have to mimic in vivo conditions where embryos develop within a small volume of fluid, which may allow endogenously produced growth factors to act effectively in a paracrine/autocrine manner. When oocytes are collected from individual cows, their number varies greatly (13), thus their developmental capacity could be low. To solve this problem some factors need to be investigated : 1) co-culturing with embryos of another species as, for example, with mouse embryos (11); 2) re-using the droplets in which embryos were cultured; however, after oviduct-conditioning and culturing of a first batch of embryos the medium would probably be depleted of some basic constituents like glutamine; 3) using very small droplets, with handling difficulties possibly arising; 4) identifying factors like autocrine/paracrine growth factors or enzymes, if they exist, which could be added to the medium. In Table 2, at constant embryo volume concentration (1 embryo/l ul), the yield of expanded blastocysts increased with the volume of the drop : larger volumes (40 ~1) seemed to be more favorable to the development of blastocysts than smaller volumes. Similar data were reported by Siiss et al. (23) and Palma et al. (15) for cattle embryos. Two hypothesis can be formulated to explain this phenomenon : 1) Physical contacts between embryos are needed for cooperation. A decrease in the absolute number of embryos would decrease in a geometric proportion with the probability of such contacts. In our laboratory, Grisart et al. (5) have analyzed the kinetics of development of bovine embryos by time-lapse cinematography. They have not observed any contact between the embryos, althoughan unusual proportion of them (23%) reached the expanded blastocyst stage. 2) .The increase of the surface/volume ratio could become detrimental to the embryos when decreasing the volume; increasing this ratio would increase the flow of the exchanges between the medium, the covering oil and the atmosphere. Some toxic constituents coming from oil could appear more quickly in the medium. Since we used a COz/air mixture, the concentration of 02 was in the range of 18 to 20% which is high and could perhaps induce some deleterious free radicals as was observed with mouse embryos (14). The use of pure nontoxic silicone oil or an 02 controlled atmosphere could perhaps lead to an answer to this question. The volume of oil was not adapted to the volume of the culture drop. So the depth of the cover layer decreased with the volume of the drop, and thus gaseous exchanges probably occurred more quickly in the large drops. In conclusion, bovine embryos cultured in SPCM need to cooperate to develop to the blastocyst stage in vitro, and it is important to define optimal conditions in embryo/volume density.
452
Theriogenology
REFERENCES 1.
2.
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13. 14. 15.
Canseco RS, Sparks AET, Pearson RE, Gwazdauskas FC. Embryo density and medium volume effects on early murine embryo development. J Assisted Reprod Genet 1992;9:454-457. Coskun S, Sanbuissho A, Lin YC, Rikihisa Y. Fertilizability and subsequent developmental ability of bovine oocytes matured in medium containing epidermal growth factor (EGF). Theriogenology 1991;36:485-494. Eyestone WH, First NL. Co-culture of early cattle embryos to the blastocyst stage with oviductal tissue or in conditioned medium. J Reprod Fertil 1989;85:715-720. Gardner DK, Lane M. Amino acids and ammonium regulate mouse embryo development in culture. Biol Reprod 1993;48:377-385. Grisart B, Massip A, Dessy F. Cinematographic analysis of bovine embryo development in serum-free conditioned medium. J Reprod Fertil 1994; In press Hemmings R, Granger L, Langlais J, Miron P, Falcone T, Guyda H. Human embryos produce transforming growth factors alpha activity and insulin-like growth factors II. Fertil Steril 1992;58:101-104. Keefer CL. Development of in vitro produced bovine embryos cultured individually in a simple medium : effect of TEGF and TGEbl. Theriogenology 1992;7:236 abstr. Kruip TAM, Pieterse MC, Van Beneden TH, Vos PLAM, Wurth YA, Taverne MAM. A new method for bovine embryo production: A potential alternative to superovulation. Vet Ret 1991;128:208-210, Larson RC, Ignotz GG, Currie WB. Platelet derived growth factor stimulates development of bovine embryos during the fourth cell cycle. Development 1992;115:821-826. Leibfried-Rutledge ML, Critser ES, Parrish JJ, First NL. In vitro maturation and fertilization of bovine oocytes. Theriogenology 1989;31:61-74. Li LY, Denniston RS , Godke RA. Enhanced development of IVF-derived bovine embryos by culturing with mouse embryos. Theriogenology 1993;39:259 abstr. Mermillod I?, Mourmeaux JL, Wils C, Massip A, Dessy F. Protein free oviduct conditioned medium supports complete bovine embryo development. Vet Ret 1992;130:13. Mermillod I’, Wils C, Massip A, Dessy, F. Collection of oocytes and production of blastocysts in vitro from individual, slaughtered cows. J Reprod Fertil 1992;96:717-723. Noda Y, Matsumoto H, Umaoka Y, Tatsumi K, Kishi J, Mori T. Involvement Mol Reprod Dev of superoxide radicals in the mouse two-cell block. 1991;28:356-360. Palma GA, Clement-Sengewald A, Berg U, Brem G. Role of the embryo number in the development of in vitro produced bovine embryos. Theriogenology 1992:37:271 abstr.
Theriogenology 16. Paris
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BC, Dey SK. Preimplantation embryo development in vitro : cooperative interactions among embryos and role of growth factors. Proc Natl Acad Sci USA 1990;87:4756-4760. Parrish JJ, Susko-Parrish JL, Leibfried-Rutledge ML, Critser ES, Eyestone WH, Bovine in vitro fertilization with frozen-thawed semen. First NL. Theriogenology 1986;25:591-600. Pieterse MC, Kappen KA, Kruip TAM, Taverne MAM. Aspiration of bovine ultrasound scanning of the ovaries. oocytes during transvaginal Theriogenology 1988;30: 751-762. Rexroad CE Jr, Powell AM. Co-culture of ovine ova with oviductal cells in medium 199. J Anim Sci 1988;66:947-953. Schini SA, Bavister BD. Development of golden hamster embryos through the two-cell block in chemically defined medium. J Exp Zoo1 1988;245: lll115. Schultz GA, Heyner S. Growth factors in preimplantation mammalian embryos. Oxford Reviews of Reproductive Biology, Ed. SR Milligan, Oxford Unia,ersity Press, U.K. 1993;15:43-82. Sirard MA, Parrish JJ, Ware,CB, Leibfried-Rutledge ML, First NL. The culture of bovine oocytes to obtain developmentally competent embryos. Biol Reprod 1988;39:546-552. Siiss U, Kassner J, Wuthrich K, Stranzinger G. Cumulus expansion, in vitro fertilization and embryonnic development after in vitro maturation of bovine oocytes in the presence of follicle stimulating or luteinizing hormone. Reprod Dom Anim 1990;25:3-13. Van Inzen WG, Kruip TAM, Weima SM. Use of conditioned medium for IVM-IVF bovine embryos in vitro culture systems. Theriogenology 1993;39: 236 abstr. Watson AJ, Hogan A, Hahnel A, Wiemer KE, Schultz GA. Expression of growth factor ligand and receptor genes in the preimplantation bovine embryo. Mol Reprod Dev 1992;1:87-95. Wiley LM, Yamami S, Van Muyden D. Effect of potassium concentration, type of protein supplement, and embryo density on mouse preimplantation development in vitro. Fertil Steril 1986;45: 111-119.
42:445-453,
1994
BOVIhJE EMBRYOS CULTURED IN SERUM-POOR OVIDUCT-CONDITIONED MEDIUM NEED COOPERATION TO REACH THE BLASTOCYST STAGE L. Ferry,1 I’. Mermillod,2
A. Massipl
and F. Dessyls a
tuniversite catholique de Louvain, Unite des Sciences Veterinaires Place Croix du Sud 3, B - 1348 Louvain-la-Neuve, Belgium ZINRA, Station de Physiologie de la Reproduction F - 37380 Nouzilly, France Received for publication : August 24, 1993 Accepted:
5, 1994
ABSTRACT Immature bovine oocytes were matured and fertilized in vitro, and the resulting zygotes were cultured to the blastocyst stage in droplets of tissue culture medium 199 (TCM 199) conditioned by oviduct cells in the absence of serum. In Experiment 1, the effect of the number of zygotes in a constant culture volume was investigated by culturing 1, 4 or 40 zygotes in 40 ~1 of culture medium. The c1eavag.e rate was low with a single embryo (36%) but increased with the number of embryos, to reach 50% with 4 embryos/40 ~.rland 59% with 40 embryos/40 ~1. Blastocyst formation was nil with 1 embryo per 40 ~1, reaching 2.5 % with 4 embryos/40 l.tl and 18% with 40 embryos/40 ~1. The effect of the size of the drop was assessed in Experiment 2, the concentration of embryos remaining constant (I embryo/l ~1). The volumes tested were 10, 20, 30 and 40 ~1. Development into blastocysts increased gradually from 12% in the lO/lO group to 20% in the 40/40 group. Experiment 3 was designed to find a minimal droplet volume able to support the development of a single embryo to the blastocyst stage. The minimum tested volume was 5 ~1 and was not successful. These results show that bovine embryos cultured in oviduct-conditioned TCM 199 need to cooperate to reach the blastocyst stage. The mechanism of this cooperation is not known, but some autocrine/paracrine factors, probably growth factors, could promote embryo development as was demonstrated in mice. From Experiment 2 we can hypothesize that the surface/volume ratio of the droplets could play a role in the culture conditions by interfering with the exchanges between the culture medium and the surrounding environment. Key words : bovine, embryo, culture, serum-poor conditioned medium Acknowledgements This work was supported by a grant from Minis&e de 1’Agriculture de la Region Wallonne de Belgique and by EEC Biotechnology programme grant BI02-CT92-0067. We thank J.F. Beckers for the gift of p-FSH and p-LH; G. Gerard and L. Collette for statistical advice and M.-A. Mauclet for typing the manuscript. a Correspondence and reprint requests : Fax : +/32/10/47.37.17.
Copyright
0 1994 Butterworth-Heinemann
Theriogenology
446
INTRODUCTION The in vitro methodology for generating bovine embryos offers several advantages as for example 1) low cost production of a large number of embryos from abattoir material for research and commercial purposes; 2) development of new technologies in animal breeding such as cloning, gene transfer and the like; 3) circumventing the uncertainty of the reaction of donor cows after hormonal superovulation treatment, by using ultrasonographically guided oocyte retrieval from all available ovarian follicles. This last procedure could become an alternative to superovulation in specified donor cows (8,18). In such cases oocytes from individual animals have to be manipulated separately whatever their number. It has been shown in the mouse (1, 4, 16, 26) and hamster (20) and suspected in the ovine (19) and bovine (24) that culture conditions such as embryo density could influence embryonic Therefore we have undertaken a study on the effect of the development. embryo/medium volume ratio and of the size of the culture droplet on development in medium conditioned with oviduct cells without serum (12).
MATERIALS
AND METHODS
Oviduct Cell Culture and Medium Conditioning If not specified, all reagents used in this study were cell culture-tested grade reagents from Sigma. b Oviduct cell cultures were initiated as previously described by Eyestone and First (3). All the solutions and vessels were sterile. Cow oviducts were obtained from an abattoir without control of estrous cycle status of the donors. After dissection, the oviducts were briefly immersed in 70% ethanol and mucosa was gently scraped free using a microscope slide. Mucosal tissue was transferred to a conical tube containing 10 ml of TCM 199 supplemented with 10% heat-treated fetal calf serum (FCS).c Tissue was then washed twice in fresh medium and resuspended in 50 volumes of medium for seeding in 25-cm2 culture flasks; 6 ml were introduced in each f1ask.d After 4 d, medium was renewed and unattached cells were discarded. Confluence was reached 6 d after seeding. For conditioning, confluent cells were washed once with TCM 199 without FCS, and 6 ml of the same medium were added to each 25-cm2 flask. Because some serum factors would perhaps remain on the cell membranes and leaching of serum components from the cells into the medium cannot be avoided, this medium was called “serum-poor conditioned medium” instead of protein-free medium as was used in previous publications (12). Serum-poor conditioned-medium (SPCM) was collected every 2 d, centrifuged at 500 g for 10 min and stored refrigerated (4°C). After 3 harvests (6 d) from the same monolayer, the 3 collections were pooled and frozen (20°C) in l-ml aliquots. Conditioned medium was centrifuged prior embryo culture (2 min 13,000 g). b Sigma, St-Louis, MO, U.S.A c Gibco, Paisley, Scotland. d Nunc, Roskilde, Denmark.
447
Theriogenology
ln Vitro
Maturation
(IVM)
and Fertilization
(IVF)
The methods used for IVM and IVF were modifications of those described previously by Parrish et al. (17) and Sirard et al. (22). All cultures were performed at 39°C under 5% CO2 in water-saturated air. Qocvte collection and maturation. Oocytes were collected by aspiration of 2 to 5-mm follicles from slaughtered cow ovaries. Only those oocytes completely surrounded by compact cumulus cells were selected, as described by LeibfriedRutledge (lo), and were washed 4 times in Tyrode’s modified medium containing albumin, lactate and pyruvate (low bicarbonate TALP) according to Parrish et al. (17). Groups of up to 100 were transferred to 4-well plates d containing 500 ~1 medium (TCM 199 su plemented with 10% heat treated FCS, 1 mg 17b-estradiol ml-l, 5 pg p LH ml- P and 0.5 pg p-FSH ml-1 (p-LH and p-FSH were from Dr J.F. Beckers, LiPge, Belgium) for 24 h of maturation. krtilization. Mature oocytes were washed 4 times in TALP, and groups of 100 were transferred into 4-well plates containing 250 ~1 of fertilization medium per well (TALP containing 10 yg heparin ml-1 Na salt e , 167 U/m@. Motile spermatozoa were obtained by centrifugation of 500 ~1 of thawed semen on a Percoll f discontinuous density gradient (2 ml at 45/2 ml at 90%) for 30 min at 700 g. Living spermatozoa (at the bottom of the 90% Percoll fraction) were washed in TALP and pelleted by centrifugation at 100 g for 10 min. Spermatozoa were counted in a hemocytometer, diluted in the appropriate volume of TALP and 250 ~1 of the suspension were added to each fertilization well to obtain a final concentration of 2 x lo6 spermatozoa ml- 1. Plates were then incubated for 18 h. Embryo
Culture
Fertilized ova were washed in TCM 199 supplemented with 10 % of heat treated FCS. Cumulus cells were removed by vortex mixing at medium speed during 2 min. About 100 ova were mixed in 2 ml of PBS containing 0.25% trypsin. All the zygotes were then pooled and carefully observed before transfer to culture droplets. Only those completely denuded were selected. This selection is important in order to avoid the metabolite depletion of the small volume of medium by the fast growth of granulosa cells. They were washed in TCM 199 without FCS and cuItured in droplets of conditioned medium under mineral oil. Droplet volumes and number of zygotes in each droplet varied according to experimental procotols.
e Calbiochem, San Diego, CA, USA. f Pharmacia, Uppsala, Sweden.
Theriogenology
448
Experiments Three experiments were conducted and in each case the 5 to g-cell stage rate was assessed on Day 2 of culture (42 h post insemination), and the number of embryos reaching the expanded blastocyst stage was determined on Day 8 (210 h post insemination). In Experiment 1, the effect of the number of zygotes in a constant culture volume was investigated by culturing either 1, 4 or 40 zygotes in 40 ~1 of culture medium. In Experiment 2, the effect of droplet volume was examined, with the zygote concentration remaining constant (1 zygote/l ul). The droplet volumes were 10, 20, 30 and 40 ul. In Experiment 3, we attempted to find a minimal droplet volume able to support the development of a single embryo to the blastocyst stage. It was assumed that embryos produce some autocrine factors and that reducing the volume would reduce the dilution of these factors. A single zygote was cultured in a drop of 5, 10 or 40 ~1; the control consisted of 40 Zygotes/40 ul. Statistical
Analysis
using Chi-square The ANOVA for categorical data . _ (log transformed) r . . . analysis was performed to compare the trequencies ot the various development stages. The CATMOD procedure of SAS g was used to perform the analysis. Linear regression analysis was used to test the relation between droplet Percentages were transformed by arcsin volume and embryo development. (square root [data]) to normalize the distribution.
RESULTS The results of Experiment 1 are reported in Table 1. They clearly show an effect of the number of embryos in a constant culture volume on their rate of development. The cleavage rate was low with only a single embryo (36%), but it increased to 50% with 4 embryos/40 ul and to 59% with 40 embryos/40 ~1. the most significant effect was observed on blastocyst However, development : 0% with 1 embryo per 40 ~1, 2.5% with 4 embryos per 40 1.11and 18% with 40 zygotes per 40 ~1. A higher density of zygotes increased their rate of development to the blastocyst stage. Experiment 2 examines the effect of droplet volume on embryo development when zygote concentration remained constant (1 zygote/ul). The results are iven in Table 2. After 2 d of culture, some differences were observed using the X5 analysis on the number of 5 to g-cell embryos; however, no statistical correlation could be established with droplet volume. g SAS Institute Inc., Cary, NC, USA.
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Theriogenology
Regression analysis revealed a significant regression slope (I? < 0.05) for the blastocyst rate. It seems that an increase in droplet volume allows for more embryos to reach the blastocyst stage. Table 1. Effect of embryo number on cleavage and blastocyst formation in a constant volume Total no. of No. of 5 to 8-cell zygotes embryos (%)
No. of expanded blastocysts (%)
No. of zygotes /
No. of
droplet volume (~1)
reP1icates
l/40
2
80
29 (36) a
0 (0) a
4/40
2
80
40 (50) a,b
2 (2.5) a
40/40
2
200
117 (59) b
36 (18) b
Gues Table 2
with different superscripts within a column are different
(X2; P ~0.05).
Effect of droplet volume at constant zygote concentration and blastocyst development
on cleavage
No. cd zygotes /
No. of
Total no.
droplet volume (@)
rePlicates
40/40
of zYgotes
No. of 5 to &cell embryos (%)
No. of expanded blastocysts (%)
4
320
147 (46) a
62 (20) c
30/30
2
120
79 (66) b
20 (17) c
20/20
2
80
42 (53) b
11 (14) c
lO/lO
4
190
89 (47) a
23 (12) c
mues with different superscripts within a column are different c Slope of the linear regression is different from zero (I’ <0.05).
(X2; P <0.05).
Experiment 3 was conducted to find the minimal drop size and culture volume for a single zygote to reach the blastocyst stage. A single zygote in 5, 10 or 4O/yl could not meet this objective (Table 3). Blastocysts could only be obtained in the control group. In Experiment 3, no difference was evidenced in the number of embryos reaching the 5 to 8-cell stage in the l/40 and 40/40 groups, in contrast with the results of Experiment ‘I.
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450
Table 3. Effect of droplet volume on development of single zygotes No. of zygotes / droplet volume (~1)
l/10
No. of replicates
Total no.
ofzygotes
No. of 5 to &cell embryos (%)
No. of expanded blastocysts (%)
2
13 (33) a
0 (0)a
2
19 (48) a
0 (0)a
81 (51) a
40 (25) b
2 40/40
2
160
a No differences were found in this column (X2; p >0.05). a! b Values with different superscripts within a column are different
(X2; P ~0.05).
DISCUSSION The results in Tables 1 and 3 demonstrate very clearly that single zygotes cannot develop to the blastocyst stage whatever the volume or size of the culture drop in our medium conditioned by oviduct cells without serum. When more than 1 zygote was present in the culture drop, blastocysts were obtained. Similar observations were reported in the mouse (1, 4, 16, 26) and hamster (20). In sheep and cattle, it was suggested that small droplets can induce cooperative interaction among embryos or that they influence the embryo environment in a positive manner (19, 24). This cooperative interaction could be mediated by autocrine and/or paracrine factors secreted by embryos themselves, and this secretion could depend on the number of embryos present per volume unit. These factors could interfere, during the first cleavage, with mechanisms to be used later by the embryo. Paria and Dey (16) obtained mouse blastocysts from 2-cell embryos cultured individually by adding EGF (epidermal growth factor) or TGFPl (transforming growth factor) to the culture medium. In the bovine, EGF stimulated hatching of singly cultured 8-cell embryos but did not significantly affect blastocyst development (7). In cattle, mRNAs for TGFa, TGFP2, PDGF, IGF-I and IGF-II as well as mRNAs for the receptors of IGF-I, IGF-II and PDGF are present in the embryo from the oocyte to the blastocyst stage (25). The use of EGF during maturation of oocytes in serum-free medium enhances the cleavage rate of the subsequently cultured embryos (2). The sequential addition of PDGF and TGFa to a serum-free culture medium promotes, respectively, development through the 8 to 16-cell block and blastulation (9). These results indicate that growth factors can be involved in bovine preimplantation embryo development and that some of these or other factors can be produced by the embryo itself, as is the case with human embryos (6). Early embryos are, therefore, capable of producing, have receptors for, and can respond to a number of growth factors, suggesting that autocrine or paracrine mechanisms are involved in early development (21).
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451
These reports have significant implications for an understanding of the control of early embryo development and for the improvement of culture of domestic animal embryos. In fact we have to mimic in vivo conditions where embryos develop within a small volume of fluid, which may allow endogenously produced growth factors to act effectively in a paracrine/autocrine manner. When oocytes are collected from individual cows, their number varies greatly (13), thus their developmental capacity could be low. To solve this problem some factors need to be investigated : 1) co-culturing with embryos of another species as, for example, with mouse embryos (11); 2) re-using the droplets in which embryos were cultured; however, after oviduct-conditioning and culturing of a first batch of embryos the medium would probably be depleted of some basic constituents like glutamine; 3) using very small droplets, with handling difficulties possibly arising; 4) identifying factors like autocrine/paracrine growth factors or enzymes, if they exist, which could be added to the medium. In Table 2, at constant embryo volume concentration (1 embryo/l ul), the yield of expanded blastocysts increased with the volume of the drop : larger volumes (40 ~1) seemed to be more favorable to the development of blastocysts than smaller volumes. Similar data were reported by Siiss et al. (23) and Palma et al. (15) for cattle embryos. Two hypothesis can be formulated to explain this phenomenon : 1) Physical contacts between embryos are needed for cooperation. A decrease in the absolute number of embryos would decrease in a geometric proportion with the probability of such contacts. In our laboratory, Grisart et al. (5) have analyzed the kinetics of development of bovine embryos by time-lapse cinematography. They have not observed any contact between the embryos, althoughan unusual proportion of them (23%) reached the expanded blastocyst stage. 2) .The increase of the surface/volume ratio could become detrimental to the embryos when decreasing the volume; increasing this ratio would increase the flow of the exchanges between the medium, the covering oil and the atmosphere. Some toxic constituents coming from oil could appear more quickly in the medium. Since we used a COz/air mixture, the concentration of 02 was in the range of 18 to 20% which is high and could perhaps induce some deleterious free radicals as was observed with mouse embryos (14). The use of pure nontoxic silicone oil or an 02 controlled atmosphere could perhaps lead to an answer to this question. The volume of oil was not adapted to the volume of the culture drop. So the depth of the cover layer decreased with the volume of the drop, and thus gaseous exchanges probably occurred more quickly in the large drops. In conclusion, bovine embryos cultured in SPCM need to cooperate to develop to the blastocyst stage in vitro, and it is important to define optimal conditions in embryo/volume density.
452
Theriogenology
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