- Email: [email protected]
Effects of co-culture, medium components and gas phase on in vitro culture of in vitro matured and in vitro fertilized bovine embryos
THERIOGENOLOGY
EFFECTS OF CO-CULTURE, MEDIUM COMPONENTS AND GAS PHASE ON IN VITRO CULTURE OF IN VITRO MATURED AND IN VITRO FERTILIZED BOVINE EMBRYOS H. Nakao and N. Nakatsuji Division of Developmental Biology Meiji Institute of Health Science 540 Naruda, Odawara 250 Japan Received
for publication: August '7, 1989 Accepted: January 5, 1990 ABSTRACT
To develop an in vitro culture system for bovine oocytes and early embryos, we examined the effects of co-culture of in vitro matured and in vitro fertilized embryos with trophoblastic vesicles and cumulus cells. We also studied the effects of culture medium components and oxygen gas pressure by modifying TCM-199 medium and using a gas-tight chamber. We found that co-culture with trophoblastic vesicles or cumulus cells promoted early embryos to develop beyond the eight-cell block; 17 to 19% of the initial oocytes developed to the morula stage. The effects of removing glucose and other energy sources from the medium, adding EDTA to the medium, reducing the concentration of serum, and reducing the oxygen gas pressure on the development of embryos were also examined. These modifications during the initial phase of co-culture greatly increased the rate of embryo development to the morula (36 to 38% of oocytes developed to morulae) and blastocyst stages. Key words: bovine eggs and early embryos, co-culture, in vitro fertilization Acknowledgments: The authors wish to thank Professor A. Iritani of Kyoto University for his critical reading of the manuscript. They also thank Mr. H. Sato and staff at Ibaraki-Chuo-Shokuniku-Kohsha and the veterinarians at the Kenhoku meat inspection station for their help with obtaining ovaries. They thank Mr. H. Suemori, who devised the culture method for trophoblastic vesicles, and Mr. I. Torihata, Miss Y. Endoh and Miss K. Okazaki for their technical assistance. This work was carried out as a project of the Embryo Transfer Research Group supported in part by a grant from the Ministry of Agriculture, Forestry and Fishery, Tokyo, Japan.
MARCH 1990 VOL. 33 NO. 3
591
THERIOGENOLOGY
INTRODUCTION In vitro culture of bovine oocytes and embryos up to the morula stage, at which nonsurgical transfer is possible, is a valuable technique for widespread application of embryo transfer, and also for various types of manipulation of early bovine embryos. We have therefore tried to develop an optimal system for the in vitro culture of in vitro matured and in vitro fertilized bovine embryos. In vitro fertilization of bovine oocytes was first reported by Iritani et al. (1). They isolated immature oocytes from the ovary and fertilized them after maturing them in vitro. Later, Brackett et al. (2) obtained offspring from the in vitro fertilized embryos which had been matured in vivo. Hanada et al. (3) first reported offspring from the in vitro matured and in vitro fertilized embryos. They used rabbit oviducts to allow for the development of the fertilized embryos to the morula stage. It is well known that fertilized bovine embryos stop development at the eight-cell stage when cultured in vitro. This phenomenon is called eight-cell block (2, 4, 5). There have been reports that the co-culture of in vivo fertilized early bovine embryos with oviduct epithelial cells (6) or trophoblastic cells (7) is effective in advancing development beyond the eight-cell block in vitro. Recently, Goto et al. (8) obtained offspring from blastocysts which had been co-cultured with cumulus cells. There have been numerous studies on the effects of various culture conditions on the development of early mouse embryos, and these conditions were recently tested on bovine oocytes and early embryos. Fukushima et al. (9) obtained embryo development beyond the eight-cell stage of in vitro fertilized embryos in lower (less than 10%) oxygen gas pressure. Sat0 et al. (10) observed beneficial effects on development of embryos when they removed glucose, Na-pyruvate and Na-acetate from the culture medium. Kajihara et al. (11) obtained similar beneficial effects by lowering serum concentration in the medium. In our study, we examined the effects of embryo co-culture with trophoblastic cells or cumulus cells, and of modifications of several components of the culture medium as well as the gas phase on the development of bovine embryos. MATERIALS
AND METHODS
Oocytes The ovaries of adult Holstein-Friesian cows and heifers were obtained from an abattoir, and were
592
MARCH 1990 VOL. 33 NO. 3
THERIOGENOLOGY transported in Ringer solution at 39°C supplemented with penicillin (1 x 106 units/ 1). Immature oocytes were sucked into a syringe with an 18-gauge needle from follicles having a diameter of 1 to 5 mm. They were then transferred into PBl solution (12) at 39°C. Oocytes with compact cumulus cells were selected under a binocular microscope. They were washed three times in PBl solution and three times in TCM-199 solution (13) and transferred to a petri dish (35 mm diameter) containing 2.5 ml of TCM-199 solution supplemented with 10% fetal calf serum (FCS). The oocytes were then incubated for 20 to 24 h at 39°C in an atnosphere of 5% CO2 and 95% air. Sperm Pretreatment Commercially distributed frozen semen collected from a single bull and stored in 0.5-ml straws was used in our experiments. Semen was thawed and spermatozoa were washed three times with Brackett and Oliphant (BO) medium (14), without bovine serum albumin (BSA), but supplemented with 10 mM caffeine. The sperm were then treated for 60 set with 0.1 FM ionophore A23187 diluted with an equal volume of BO medium containing 20 mg/ml BSA to induce capacitation. Thus prepared sperm suspension contained 15 to 20 X 106 cells /ml. Trophoblastic
Vesicles
Hatched and elongated blastocysts 12 to 14 d old were collected by flushing the uteri of superovulated and artificially inseminated Holstein-Friesian heifers. The blastocysts were washed with PBl solution and cut, excluding the embryonic disk portion, into small pieces (approximately 0.3 mm) with fine forceps. These pieces were cultured in a medium of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS at 39°C in an atmosphere of 5% CO2 and 95% air. The pieces were cultured for 1 to 2 ma as trophoblastic vesicles, and those having a diameter of 2 to 3 mm were used for co-culture. Insemination Droplets (0.1 ml each) of the sperm suspension were placed on the bottom of a petri dish and covered with liquid paraffin. Matured oocytes were washed twice with BO medium. Ten to 15 oocytes were transferred into the droplet and incubated at 39-C for 5 to 7 h. Experiment
1 - Effects of Co-culture
Fertilized embryos with cumulus cells were washed with TCM-199 supplemented with 10% FCS, and cultured in a tissue culture dish (35 mm diameter) containing 2.5 ml of the same
MARCH 1990 VOL. 33 NO. 3
593
THERIOGENOLOGY
medium at 39-C in an atmosphere of 5% CO2 and 95% air. The cumulus cells attached to the dish bottom and made a monolayer after 48 h of culture. The cumulus cell mass was removed from embryos at the two- to eight-cell stages. The apparently normal embryos were divided into the following five groups for further culture at 39°C in an atmosphere of 5% CO2 and 95% air in the medium of TCM-199 supplemented with 10% FCS and 1.25 mM Na-pyruvate. The culture medium was changed at every 48 h. Group 1 consisted of denuded Group 2 embryos were co-cultured with cumulus embryos. Denuded embryos were cultured on the cumulus cell cells. monolayer in a dish. Group 3 embryos were co-cultured with trophoblastic vesicles. Droplets (0.4 ml) of medium in a petri dish were covered with liquid paraffin, and 20 to 30 denuded embryos and two to three trophoblastic vesicles were cultured in each drop. Group 4 embryos were co-cultured with both cumulus cells and trophoblastic vesicles. Two or three trophoblastic vesicles were added to a dish prepared as in the experiment with Group 2. Group 5 embryos were co-cultured with bovine fibroblasts. Fibroblasts were obtained from lung tissues of a bovine fetus 120 d old. The fibroblasts were cultured as a monolayer in a culture dish (35 mm diameter), and denuded embryos were co-cultured in 2.5 ml of medium. Experiment
2 - Effects of Various Culture Conditions
Four components of the various culture conditions were investigated for their effects on the development of fertilized bovine embryos (Group 2) which had been co-cultured with cumulus cells as described in Experiment 1. Embryos in control groups were co-cultured in the standard medium described in Experiment 1, at 39'C in an atmosphere of 5% CO2 and 95% air. Group 1 fertilized embryos were transferred into culture dishes (35 mm diameter) with 2.5 ml of the culture medium. They were incubated at 39°C in an airtight chamber (Modular Incubator Chamber, Billups-Rothenberg Inc., Del Mar, CA) filled with a gas mixture of 5% Cop, 5% 02 and 90% Np. Group 2 fertilized embryos were cultured in the medium with 2% FCS. Group 3 fertilized embryos were cultured in the medium from which glucose, Na-pyruvate and Na-acetate were deleted during the 48 h period following insemination, and transferred to the standard medium for further culture. The culture medium for Group 4 embryos contained ethylenediamine-tetraacetic acid (EDTA) at a concentration of 0.1 mM. Fertilized embryos were cultured in this medium up to 72 h following insemination, and were then transferred into the standard medium. Experiment
3 - Combined
Effects of Culture Conditions
The combined effects of the modifications investigated in Experiments 1 and 2 were examined by comparing the
594
MARCH 1990 VOL. 33 NO. 3
THERIOGENOLOGY
following five groups: Group 1 embryos had the most favorable culture conditions but they were not co-cultured. The fertilized denuded embryos were treated as in Experiment 1. They were cultured in medium and gas pressure using a combination of treatments of Groups l-4 in Experiment 2; 2% FCS was added, glucose and other energy sources were removed, 0.1 mM EDTA was added, 02 gas pressure was reduced to 5%. The embryos were transferred into the medium with energy sources at 48 h, and then into medium without EDTA 72 h after insemination. Group 2 embryos were co-cultured with cumulus cells. Other culture conditions were the same as those in Group 1. Group 3 embryos were co-cultured with cumulus cells, but under the standard culture conditions (10% FCS, with energy sources, no EDTA, and 20% 02). Group 4 embryos were co-cultured with trophoblastic vesicles. All other culture conditions were the same as in Group 1. Group 5 embryos were co-cultured with trophoblastic vesicles but under standard culture conditions. RESULTS Experiment
1
Table 1 shows the results of Experiment 1. Co-culture of fertilized embryos with either trophoblastic vesicles or cumulus cells greatly improved their development beyond the eight-cell block to the morula stage compared with the controls (Group 1). Co-culture with both the trophoblastic vesicles and cumulus cells showed no further improvement. Co-culture with fibroblasts also showed no improvement. Table 1. -
-
Effects of co-culture with trophoblastic vesicles or cumulus cells on the development of bovine embryos a b Eight-cell Morula Blastocyst Oocyte embryo(%) f%) f%f
Group 1 26 (35.1) 5 ( 6.8) Group 2 1:: 32 (17.2) 61 (32.8) 224 Group 3 79 (35.3) 43 (19.2) Group 4 228 76 (33.3) 37 (16.21 Group 5 149 53 (35.6j 9 ( 6.0j a b E:xamined at 120 h after insemination. Examined after insemination. Experiment
1 14 23 22 2
( 1.4) ( 7.5) (10.3) ( 9.6) i 1.3j
at 168 h
2
Table 2 shows the results of Experiment 2. Reduced oxygen gas pressure from 20% to 5% improved the development of the embryos. Lowered concentration of FCS from 10 to 2% also improved their development. Removing energy sources,
PARCH
1990 VOL. 33 NO. 3
595
THERIOGENOLOGY
glucose, Na-pyruvate and Na-acetate from the culture medium during the first 48 h of culture resulted in a higher rate of embryo development to the morula and blastocyst stages. Addition of a small amount of EDTA (0.1 mM) also improved embryo development rates. Table 2.
Effects of medium components
Developmental
rate ("a) (No. embryos / Total oocytes) Blastocysts Morulae
Group 1 5% 20%
Oxygen 38 18
Group 2 2% 10%
Serum
Group 3 + Group 4 0.1 mM None Experiment
and gas pressure
(33 / 87) (12 / 65)
14 x
(12 / 07) ( 7 / 65)
(12 / 45) (13 / 76)
16 7
( 7 / 45) ( 5 / 76)
Energy sources 25 ( 9 / 36) 17 (13 / 76)
17 7
( 6 / 36) ( 5 / 76)
14 8
(23 /161) ( 7 / 65)
27 17
EDTA 29 18
(46 /161) (12 / 65)
3
Table 3 shows the results of Experiment 3. As shown in the results for Group 1, the combined modifications of the culture medium and gas pressure could not be substituted for the effects of co-culture. Most embryos stopped developing at eight-cell stage, and did not develop to the morula stage. Co-culture with trophoblastic vesicles or cumulus cells greatly improved embryo development. The percentage of embryos showing development beyond the eight-cell block was increased by the combined modification treatment when compared with the standard culture treatment. The culture conditions of Groups 2 and 4 appear to be optimal, with 36 to 38% of the embryos developing to the morula stage. Table 3. Combined Developmental Group Group Group Group Group
696
1 2 3 4 5
effects of modifications rate (%) Morulae
9 311 19 36 20
(5/ (62 (23 (44 (19
(No.
57) / 165) / 119) / 123) / 95)
and co-culture
embryos / Total oocytes) Blastocysts 4 14 9 11 9
(2/ (23 (11 (14 ( 9
57) / 165) / 119) / 123) / 95)
MARCH 1990 VOL. 33 NO. 3
THERIOGENOLOGY
DISCUSSION Effects of Co-culture Heyman et al. (7) and Camous et al. (15) reported that fertilized bovine embryos at the one- to eight-cell stages collected from pregnant cows developed to morulae efficiently when they were co-cultured with trophoblastic vesicles. In our s%udy, we tested the beneficial effects of trophoblastic vesicles on in vitro matured and in vitro fertilized embryos and obtained similar results. From their experiments using a medium supplemented with trophoblasts, Heyman et al. (7) also speculated that the beneficial developmental effects were due to peptides with molecular weights of less than 2,500. The molecular nature of factors produced by the trophoblastie cells is not yet understood. Goto et al. (8) and Kajihara et al. (11) reported that co-culture of in vitro fertilized bovine embryos with cumulus cells improved the development rate to morulae and biastocysts, It is not clear whether such an effect is caused by soluble factors secreted from the cumulus cells, or by some kind of cellular contacts. Allen et al. (16) showed beneficial effects on porcine embryo development of uterine epithelial cells. Kuzan et a:.. (17) reported that co-culture of bovine morulae with either uterine or testis fibroblasts improved development. They concluded that the contact of embryos with any feeder C
MARCH 1990 VOL. 33 NO. 3
597
THERIOGENOLOGY
stage. Kajihara et al. (11) studied effects of various serum concentrations in the culture medium on in vitro fertilized bovine embryos and found that 2.5% of the serum was optimal. Our results also show that 2% of FCS is better than 10% during the early phase of culture. Serum concentration of 10% is generally used in culture experiments with bovine oocytes and embryos (5, 20). However, lower concentrations may produce better results, except in cases where specific cells for co-culture require higher concentrations. Abramczuk (21) reported that addition of EDTA to the culture medium enabled one-cell stage mouse embryos to develop beyond the two-cell block, even when mouse strains such as the ICR were used. Our study also shows that the addition of EDTA improved the development rate of bovine embryos, although used alone it could not circumvent the eight-cell block. It is not clear how such a low concentration (0.1 mM) of EDTA affects embryo development. Combined Optimal Culture Conditions Experiments in our study showed that combined culture conditions could influence embryo development. Developmental rates increased to 36 to 38% when embryos were co-cultured with trophoblastic vesicles or cumulus cells. Our experiments also show that bovine embryos can not develop beyond the eight-cell block even when optimal culture conditions are present if no co-culture cells are used. Thus, even after the medium and gas pressure were variously modified, co-culture remained the prerequisite for a high rate of development of bovine embryos in vitro to the morula and blastocyst. stages. REFERENCES 1. Iritani, A. and Niwa, K. Capacitation of bull spermatozoa and fertilization in vitro of cattle follicular oocytes matured in culture. J. Reprod. Fertil. -50:119-121 (1977). 2. Brackett, B. G., Bousquet, D., Boice, M. L., Donawick, W. J., Evans, J. E. and Dressel, M. A. Normal development following in vitro fertilization in the cow. Biol. Reprod. -27:147-158 (1982). 3. Hanada, A., Suzuki, T. and Shioya, Y. Birth of calves originated from non-surgical transfer of blastocysts originated from in vitro fertilized oocytes matured in vitro (in Japanese). Proc. 78th Meet. Jpn. Sot. Zootech. Sci. :lR abstr. (1986).
MARCH 1990 VOL. 33 NO. 3
THERIOGENOLOGY
.!.Bondioli, K. R. and Wright, R. W., Jr. In vitro fertilization of bovine oocytes by spermatozoa capacitated in vitro. J. Anim. Sci. -57:1001-1005 (1983).
d
5 . Hanada, A. In vitro fertilization of the cow (in Japanese). Jpn. J. Anim. Reprod. -31:56-61 (19R5). 6 . Eyeston, W. H., Vignieri, J. and First, N. L. Co-culture of early bovine embryo with oviductal epithelium. Theriogenology -27:228 abstr. (1987). '?. Heyman, Y., Mdngzo, Y., Chesnk, P., Camous, S. and Garnier, V. In vitro cleavage of bovine and ovine early embryos: improved development using co-culture with trophoblastic vesicles. Theriogenology -27:59-68 (19S7). II . Goto, K., Kajihara, Y., Kosaka, S., Koba, M., Nakanishi, Y. and Ogawa, K. Pregnancies after co-culture of cumulus cells with bovine embryos derived from in-vitro fertilization of in-vitro matured follicular oocytes. J. Reprod. Fertil. -833753-758 (1988). 9, Fukushima, J., Tominaga, K. and Hataya, Y. Effects of oxygen concentration on maturation and development of the bovine eggs matured and fertilized in vitro (in Japanese). Proc. 74th Meet. Jpn. Sot. Anim. Reprod. :6R abstr. (19RS). 10. Sato, T., Shioya, Y., Yamada, H. and Kitamura, S. Effects of addition or deletion of glucose, Na-pyruvate, Na-acetate in the culture medium on development of the in vitro-fertilized bovine eggs into blastocysts (in Japanese). Proc. 74th Meet. Jpn. sot. Anim. Reprod. :63 abstr. (1998). 11. Kajihara, Y., Goto, K., Kosaka, S., Nakanishi, Y. and Ogawa, K. In vitro fertilization of bovine follicular oocytes and their development up to hatched blastocysts in vitro (in Japanese). Jpn. J. Anim. Reprod. -33: 173-180 (1987). 12. Whittingham, D. G. and Wales, R. G. Storage of two-cell mouse embryos in vitro. Austr. J. Biol. Sci. -22:1065- 1068 (1969). 13. Morgan, J. F. and Morton, H. J. Nutrition of animal cells in tissue culture. I. Initial studies on a Proc. Sot. exp, Biol. Med. 73:1-S synthetic medium. (1950).
MARCH 1990 VOL. 33 NO. 3
599
THERIOGENOLOGY
14. Brackett, B. G. and Oliphant, G. Capacitation of rabbit spermatozoa in vitro. Biol. Reprod. -12: 260-274 (1975). Y. 15. Camous, S., Heyman, Y., MGziou, W. and M&&zo, Cleavage beyond the block stage and survival after transfer of early bovine embryos cultured with trophoblastic vesicles. J. Reprod. Fertil. -72~479-485 (1984). 16. Allen, R. L. and Wright, R, W. Jr. In vitro development of porcine embryos in co-culture with endometrial cell monolayers or culture supernatants. J. Anim. Sci. -59:1657-1661 (1984). 17. Ruzan, F. B. and Wright, R. W., Jr. Observations on the development of bovine morulae on various cellular and noncellular substrata. J. Anim. Sci. -54:811-816 (19R2). 18. Whitten, W. K. Nutritional requirements for the Adv. culture of preimplantation embryos in vitro. Biosci. 5:129-139 (1971). 19. Quinn, P. and Harlow, G. M. The effect of oxygen on the development of preimplantation mouse embryos in vitro. J. Exp. Zool. 206:73-80 (1978). 20. Lambert, R. D., Sirard, M. A., Bernard, C., Beland, R., Rioux, J. E., Leclerc, P., Menard, D. P. and Bedoya, M. In vitro fertilization of bovine oocytes matured in vivo and collected at laparoscopy. Theriogenology -25: 117-133 (1986). 21. Abramczuk, J., Solter, D. and Koprowski, H. The beneficial effect of EDTA on development of mouse one-cell embryos in chemically defined medium. Dev. Biol. -61:378-383 (1977).
600
MARCH 1990 VOL. 33 NO. 3
EFFECTS OF CO-CULTURE, MEDIUM COMPONENTS AND GAS PHASE ON IN VITRO CULTURE OF IN VITRO MATURED AND IN VITRO FERTILIZED BOVINE EMBRYOS H. Nakao and N. Nakatsuji Division of Developmental Biology Meiji Institute of Health Science 540 Naruda, Odawara 250 Japan Received
for publication: August '7, 1989 Accepted: January 5, 1990 ABSTRACT
To develop an in vitro culture system for bovine oocytes and early embryos, we examined the effects of co-culture of in vitro matured and in vitro fertilized embryos with trophoblastic vesicles and cumulus cells. We also studied the effects of culture medium components and oxygen gas pressure by modifying TCM-199 medium and using a gas-tight chamber. We found that co-culture with trophoblastic vesicles or cumulus cells promoted early embryos to develop beyond the eight-cell block; 17 to 19% of the initial oocytes developed to the morula stage. The effects of removing glucose and other energy sources from the medium, adding EDTA to the medium, reducing the concentration of serum, and reducing the oxygen gas pressure on the development of embryos were also examined. These modifications during the initial phase of co-culture greatly increased the rate of embryo development to the morula (36 to 38% of oocytes developed to morulae) and blastocyst stages. Key words: bovine eggs and early embryos, co-culture, in vitro fertilization Acknowledgments: The authors wish to thank Professor A. Iritani of Kyoto University for his critical reading of the manuscript. They also thank Mr. H. Sato and staff at Ibaraki-Chuo-Shokuniku-Kohsha and the veterinarians at the Kenhoku meat inspection station for their help with obtaining ovaries. They thank Mr. H. Suemori, who devised the culture method for trophoblastic vesicles, and Mr. I. Torihata, Miss Y. Endoh and Miss K. Okazaki for their technical assistance. This work was carried out as a project of the Embryo Transfer Research Group supported in part by a grant from the Ministry of Agriculture, Forestry and Fishery, Tokyo, Japan.
MARCH 1990 VOL. 33 NO. 3
591
THERIOGENOLOGY
INTRODUCTION In vitro culture of bovine oocytes and embryos up to the morula stage, at which nonsurgical transfer is possible, is a valuable technique for widespread application of embryo transfer, and also for various types of manipulation of early bovine embryos. We have therefore tried to develop an optimal system for the in vitro culture of in vitro matured and in vitro fertilized bovine embryos. In vitro fertilization of bovine oocytes was first reported by Iritani et al. (1). They isolated immature oocytes from the ovary and fertilized them after maturing them in vitro. Later, Brackett et al. (2) obtained offspring from the in vitro fertilized embryos which had been matured in vivo. Hanada et al. (3) first reported offspring from the in vitro matured and in vitro fertilized embryos. They used rabbit oviducts to allow for the development of the fertilized embryos to the morula stage. It is well known that fertilized bovine embryos stop development at the eight-cell stage when cultured in vitro. This phenomenon is called eight-cell block (2, 4, 5). There have been reports that the co-culture of in vivo fertilized early bovine embryos with oviduct epithelial cells (6) or trophoblastic cells (7) is effective in advancing development beyond the eight-cell block in vitro. Recently, Goto et al. (8) obtained offspring from blastocysts which had been co-cultured with cumulus cells. There have been numerous studies on the effects of various culture conditions on the development of early mouse embryos, and these conditions were recently tested on bovine oocytes and early embryos. Fukushima et al. (9) obtained embryo development beyond the eight-cell stage of in vitro fertilized embryos in lower (less than 10%) oxygen gas pressure. Sat0 et al. (10) observed beneficial effects on development of embryos when they removed glucose, Na-pyruvate and Na-acetate from the culture medium. Kajihara et al. (11) obtained similar beneficial effects by lowering serum concentration in the medium. In our study, we examined the effects of embryo co-culture with trophoblastic cells or cumulus cells, and of modifications of several components of the culture medium as well as the gas phase on the development of bovine embryos. MATERIALS
AND METHODS
Oocytes The ovaries of adult Holstein-Friesian cows and heifers were obtained from an abattoir, and were
592
MARCH 1990 VOL. 33 NO. 3
THERIOGENOLOGY transported in Ringer solution at 39°C supplemented with penicillin (1 x 106 units/ 1). Immature oocytes were sucked into a syringe with an 18-gauge needle from follicles having a diameter of 1 to 5 mm. They were then transferred into PBl solution (12) at 39°C. Oocytes with compact cumulus cells were selected under a binocular microscope. They were washed three times in PBl solution and three times in TCM-199 solution (13) and transferred to a petri dish (35 mm diameter) containing 2.5 ml of TCM-199 solution supplemented with 10% fetal calf serum (FCS). The oocytes were then incubated for 20 to 24 h at 39°C in an atnosphere of 5% CO2 and 95% air. Sperm Pretreatment Commercially distributed frozen semen collected from a single bull and stored in 0.5-ml straws was used in our experiments. Semen was thawed and spermatozoa were washed three times with Brackett and Oliphant (BO) medium (14), without bovine serum albumin (BSA), but supplemented with 10 mM caffeine. The sperm were then treated for 60 set with 0.1 FM ionophore A23187 diluted with an equal volume of BO medium containing 20 mg/ml BSA to induce capacitation. Thus prepared sperm suspension contained 15 to 20 X 106 cells /ml. Trophoblastic
Vesicles
Hatched and elongated blastocysts 12 to 14 d old were collected by flushing the uteri of superovulated and artificially inseminated Holstein-Friesian heifers. The blastocysts were washed with PBl solution and cut, excluding the embryonic disk portion, into small pieces (approximately 0.3 mm) with fine forceps. These pieces were cultured in a medium of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS at 39°C in an atmosphere of 5% CO2 and 95% air. The pieces were cultured for 1 to 2 ma as trophoblastic vesicles, and those having a diameter of 2 to 3 mm were used for co-culture. Insemination Droplets (0.1 ml each) of the sperm suspension were placed on the bottom of a petri dish and covered with liquid paraffin. Matured oocytes were washed twice with BO medium. Ten to 15 oocytes were transferred into the droplet and incubated at 39-C for 5 to 7 h. Experiment
1 - Effects of Co-culture
Fertilized embryos with cumulus cells were washed with TCM-199 supplemented with 10% FCS, and cultured in a tissue culture dish (35 mm diameter) containing 2.5 ml of the same
MARCH 1990 VOL. 33 NO. 3
593
THERIOGENOLOGY
medium at 39-C in an atmosphere of 5% CO2 and 95% air. The cumulus cells attached to the dish bottom and made a monolayer after 48 h of culture. The cumulus cell mass was removed from embryos at the two- to eight-cell stages. The apparently normal embryos were divided into the following five groups for further culture at 39°C in an atmosphere of 5% CO2 and 95% air in the medium of TCM-199 supplemented with 10% FCS and 1.25 mM Na-pyruvate. The culture medium was changed at every 48 h. Group 1 consisted of denuded Group 2 embryos were co-cultured with cumulus embryos. Denuded embryos were cultured on the cumulus cell cells. monolayer in a dish. Group 3 embryos were co-cultured with trophoblastic vesicles. Droplets (0.4 ml) of medium in a petri dish were covered with liquid paraffin, and 20 to 30 denuded embryos and two to three trophoblastic vesicles were cultured in each drop. Group 4 embryos were co-cultured with both cumulus cells and trophoblastic vesicles. Two or three trophoblastic vesicles were added to a dish prepared as in the experiment with Group 2. Group 5 embryos were co-cultured with bovine fibroblasts. Fibroblasts were obtained from lung tissues of a bovine fetus 120 d old. The fibroblasts were cultured as a monolayer in a culture dish (35 mm diameter), and denuded embryos were co-cultured in 2.5 ml of medium. Experiment
2 - Effects of Various Culture Conditions
Four components of the various culture conditions were investigated for their effects on the development of fertilized bovine embryos (Group 2) which had been co-cultured with cumulus cells as described in Experiment 1. Embryos in control groups were co-cultured in the standard medium described in Experiment 1, at 39'C in an atmosphere of 5% CO2 and 95% air. Group 1 fertilized embryos were transferred into culture dishes (35 mm diameter) with 2.5 ml of the culture medium. They were incubated at 39°C in an airtight chamber (Modular Incubator Chamber, Billups-Rothenberg Inc., Del Mar, CA) filled with a gas mixture of 5% Cop, 5% 02 and 90% Np. Group 2 fertilized embryos were cultured in the medium with 2% FCS. Group 3 fertilized embryos were cultured in the medium from which glucose, Na-pyruvate and Na-acetate were deleted during the 48 h period following insemination, and transferred to the standard medium for further culture. The culture medium for Group 4 embryos contained ethylenediamine-tetraacetic acid (EDTA) at a concentration of 0.1 mM. Fertilized embryos were cultured in this medium up to 72 h following insemination, and were then transferred into the standard medium. Experiment
3 - Combined
Effects of Culture Conditions
The combined effects of the modifications investigated in Experiments 1 and 2 were examined by comparing the
594
MARCH 1990 VOL. 33 NO. 3
THERIOGENOLOGY
following five groups: Group 1 embryos had the most favorable culture conditions but they were not co-cultured. The fertilized denuded embryos were treated as in Experiment 1. They were cultured in medium and gas pressure using a combination of treatments of Groups l-4 in Experiment 2; 2% FCS was added, glucose and other energy sources were removed, 0.1 mM EDTA was added, 02 gas pressure was reduced to 5%. The embryos were transferred into the medium with energy sources at 48 h, and then into medium without EDTA 72 h after insemination. Group 2 embryos were co-cultured with cumulus cells. Other culture conditions were the same as those in Group 1. Group 3 embryos were co-cultured with cumulus cells, but under the standard culture conditions (10% FCS, with energy sources, no EDTA, and 20% 02). Group 4 embryos were co-cultured with trophoblastic vesicles. All other culture conditions were the same as in Group 1. Group 5 embryos were co-cultured with trophoblastic vesicles but under standard culture conditions. RESULTS Experiment
1
Table 1 shows the results of Experiment 1. Co-culture of fertilized embryos with either trophoblastic vesicles or cumulus cells greatly improved their development beyond the eight-cell block to the morula stage compared with the controls (Group 1). Co-culture with both the trophoblastic vesicles and cumulus cells showed no further improvement. Co-culture with fibroblasts also showed no improvement. Table 1. -
-
Effects of co-culture with trophoblastic vesicles or cumulus cells on the development of bovine embryos a b Eight-cell Morula Blastocyst Oocyte embryo(%) f%) f%f
Group 1 26 (35.1) 5 ( 6.8) Group 2 1:: 32 (17.2) 61 (32.8) 224 Group 3 79 (35.3) 43 (19.2) Group 4 228 76 (33.3) 37 (16.21 Group 5 149 53 (35.6j 9 ( 6.0j a b E:xamined at 120 h after insemination. Examined after insemination. Experiment
1 14 23 22 2
( 1.4) ( 7.5) (10.3) ( 9.6) i 1.3j
at 168 h
2
Table 2 shows the results of Experiment 2. Reduced oxygen gas pressure from 20% to 5% improved the development of the embryos. Lowered concentration of FCS from 10 to 2% also improved their development. Removing energy sources,
PARCH
1990 VOL. 33 NO. 3
595
THERIOGENOLOGY
glucose, Na-pyruvate and Na-acetate from the culture medium during the first 48 h of culture resulted in a higher rate of embryo development to the morula and blastocyst stages. Addition of a small amount of EDTA (0.1 mM) also improved embryo development rates. Table 2.
Effects of medium components
Developmental
rate ("a) (No. embryos / Total oocytes) Blastocysts Morulae
Group 1 5% 20%
Oxygen 38 18
Group 2 2% 10%
Serum
Group 3 + Group 4 0.1 mM None Experiment
and gas pressure
(33 / 87) (12 / 65)
14 x
(12 / 07) ( 7 / 65)
(12 / 45) (13 / 76)
16 7
( 7 / 45) ( 5 / 76)
Energy sources 25 ( 9 / 36) 17 (13 / 76)
17 7
( 6 / 36) ( 5 / 76)
14 8
(23 /161) ( 7 / 65)
27 17
EDTA 29 18
(46 /161) (12 / 65)
3
Table 3 shows the results of Experiment 3. As shown in the results for Group 1, the combined modifications of the culture medium and gas pressure could not be substituted for the effects of co-culture. Most embryos stopped developing at eight-cell stage, and did not develop to the morula stage. Co-culture with trophoblastic vesicles or cumulus cells greatly improved embryo development. The percentage of embryos showing development beyond the eight-cell block was increased by the combined modification treatment when compared with the standard culture treatment. The culture conditions of Groups 2 and 4 appear to be optimal, with 36 to 38% of the embryos developing to the morula stage. Table 3. Combined Developmental Group Group Group Group Group
696
1 2 3 4 5
effects of modifications rate (%) Morulae
9 311 19 36 20
(5/ (62 (23 (44 (19
(No.
57) / 165) / 119) / 123) / 95)
and co-culture
embryos / Total oocytes) Blastocysts 4 14 9 11 9
(2/ (23 (11 (14 ( 9
57) / 165) / 119) / 123) / 95)
MARCH 1990 VOL. 33 NO. 3
THERIOGENOLOGY
DISCUSSION Effects of Co-culture Heyman et al. (7) and Camous et al. (15) reported that fertilized bovine embryos at the one- to eight-cell stages collected from pregnant cows developed to morulae efficiently when they were co-cultured with trophoblastic vesicles. In our s%udy, we tested the beneficial effects of trophoblastic vesicles on in vitro matured and in vitro fertilized embryos and obtained similar results. From their experiments using a medium supplemented with trophoblasts, Heyman et al. (7) also speculated that the beneficial developmental effects were due to peptides with molecular weights of less than 2,500. The molecular nature of factors produced by the trophoblastie cells is not yet understood. Goto et al. (8) and Kajihara et al. (11) reported that co-culture of in vitro fertilized bovine embryos with cumulus cells improved the development rate to morulae and biastocysts, It is not clear whether such an effect is caused by soluble factors secreted from the cumulus cells, or by some kind of cellular contacts. Allen et al. (16) showed beneficial effects on porcine embryo development of uterine epithelial cells. Kuzan et a:.. (17) reported that co-culture of bovine morulae with either uterine or testis fibroblasts improved development. They concluded that the contact of embryos with any feeder C
MARCH 1990 VOL. 33 NO. 3
597
THERIOGENOLOGY
stage. Kajihara et al. (11) studied effects of various serum concentrations in the culture medium on in vitro fertilized bovine embryos and found that 2.5% of the serum was optimal. Our results also show that 2% of FCS is better than 10% during the early phase of culture. Serum concentration of 10% is generally used in culture experiments with bovine oocytes and embryos (5, 20). However, lower concentrations may produce better results, except in cases where specific cells for co-culture require higher concentrations. Abramczuk (21) reported that addition of EDTA to the culture medium enabled one-cell stage mouse embryos to develop beyond the two-cell block, even when mouse strains such as the ICR were used. Our study also shows that the addition of EDTA improved the development rate of bovine embryos, although used alone it could not circumvent the eight-cell block. It is not clear how such a low concentration (0.1 mM) of EDTA affects embryo development. Combined Optimal Culture Conditions Experiments in our study showed that combined culture conditions could influence embryo development. Developmental rates increased to 36 to 38% when embryos were co-cultured with trophoblastic vesicles or cumulus cells. Our experiments also show that bovine embryos can not develop beyond the eight-cell block even when optimal culture conditions are present if no co-culture cells are used. Thus, even after the medium and gas pressure were variously modified, co-culture remained the prerequisite for a high rate of development of bovine embryos in vitro to the morula and blastocyst. stages. REFERENCES 1. Iritani, A. and Niwa, K. Capacitation of bull spermatozoa and fertilization in vitro of cattle follicular oocytes matured in culture. J. Reprod. Fertil. -50:119-121 (1977). 2. Brackett, B. G., Bousquet, D., Boice, M. L., Donawick, W. J., Evans, J. E. and Dressel, M. A. Normal development following in vitro fertilization in the cow. Biol. Reprod. -27:147-158 (1982). 3. Hanada, A., Suzuki, T. and Shioya, Y. Birth of calves originated from non-surgical transfer of blastocysts originated from in vitro fertilized oocytes matured in vitro (in Japanese). Proc. 78th Meet. Jpn. Sot. Zootech. Sci. :lR abstr. (1986).
MARCH 1990 VOL. 33 NO. 3
THERIOGENOLOGY
.!.Bondioli, K. R. and Wright, R. W., Jr. In vitro fertilization of bovine oocytes by spermatozoa capacitated in vitro. J. Anim. Sci. -57:1001-1005 (1983).
d
5 . Hanada, A. In vitro fertilization of the cow (in Japanese). Jpn. J. Anim. Reprod. -31:56-61 (19R5). 6 . Eyeston, W. H., Vignieri, J. and First, N. L. Co-culture of early bovine embryo with oviductal epithelium. Theriogenology -27:228 abstr. (1987). '?. Heyman, Y., Mdngzo, Y., Chesnk, P., Camous, S. and Garnier, V. In vitro cleavage of bovine and ovine early embryos: improved development using co-culture with trophoblastic vesicles. Theriogenology -27:59-68 (19S7). II . Goto, K., Kajihara, Y., Kosaka, S., Koba, M., Nakanishi, Y. and Ogawa, K. Pregnancies after co-culture of cumulus cells with bovine embryos derived from in-vitro fertilization of in-vitro matured follicular oocytes. J. Reprod. Fertil. -833753-758 (1988). 9, Fukushima, J., Tominaga, K. and Hataya, Y. Effects of oxygen concentration on maturation and development of the bovine eggs matured and fertilized in vitro (in Japanese). Proc. 74th Meet. Jpn. Sot. Anim. Reprod. :6R abstr. (19RS). 10. Sato, T., Shioya, Y., Yamada, H. and Kitamura, S. Effects of addition or deletion of glucose, Na-pyruvate, Na-acetate in the culture medium on development of the in vitro-fertilized bovine eggs into blastocysts (in Japanese). Proc. 74th Meet. Jpn. sot. Anim. Reprod. :63 abstr. (1998). 11. Kajihara, Y., Goto, K., Kosaka, S., Nakanishi, Y. and Ogawa, K. In vitro fertilization of bovine follicular oocytes and their development up to hatched blastocysts in vitro (in Japanese). Jpn. J. Anim. Reprod. -33: 173-180 (1987). 12. Whittingham, D. G. and Wales, R. G. Storage of two-cell mouse embryos in vitro. Austr. J. Biol. Sci. -22:1065- 1068 (1969). 13. Morgan, J. F. and Morton, H. J. Nutrition of animal cells in tissue culture. I. Initial studies on a Proc. Sot. exp, Biol. Med. 73:1-S synthetic medium. (1950).
MARCH 1990 VOL. 33 NO. 3
599
THERIOGENOLOGY
14. Brackett, B. G. and Oliphant, G. Capacitation of rabbit spermatozoa in vitro. Biol. Reprod. -12: 260-274 (1975). Y. 15. Camous, S., Heyman, Y., MGziou, W. and M&&zo, Cleavage beyond the block stage and survival after transfer of early bovine embryos cultured with trophoblastic vesicles. J. Reprod. Fertil. -72~479-485 (1984). 16. Allen, R. L. and Wright, R, W. Jr. In vitro development of porcine embryos in co-culture with endometrial cell monolayers or culture supernatants. J. Anim. Sci. -59:1657-1661 (1984). 17. Ruzan, F. B. and Wright, R. W., Jr. Observations on the development of bovine morulae on various cellular and noncellular substrata. J. Anim. Sci. -54:811-816 (19R2). 18. Whitten, W. K. Nutritional requirements for the Adv. culture of preimplantation embryos in vitro. Biosci. 5:129-139 (1971). 19. Quinn, P. and Harlow, G. M. The effect of oxygen on the development of preimplantation mouse embryos in vitro. J. Exp. Zool. 206:73-80 (1978). 20. Lambert, R. D., Sirard, M. A., Bernard, C., Beland, R., Rioux, J. E., Leclerc, P., Menard, D. P. and Bedoya, M. In vitro fertilization of bovine oocytes matured in vivo and collected at laparoscopy. Theriogenology -25: 117-133 (1986). 21. Abramczuk, J., Solter, D. and Koprowski, H. The beneficial effect of EDTA on development of mouse one-cell embryos in chemically defined medium. Dev. Biol. -61:378-383 (1977).
600
MARCH 1990 VOL. 33 NO. 3