FERTILITY AND STERILITY
Vol. 58, No.1, July 1992
Copyright I[) 1992 The American Fertility Society
Printed on acid-free paper in U.S.A.
Human embryos produce transforming growth factors a activity and insulin-like growth factors II
Robert Hemmings, M.D.*t+ Jean Langlais, Ph.D.* Tommaso Falcone, M.D.*t
Louis Granger, M.D.* Pierre Miron, M.D.* Harvey Guyda, M.D.t
Institut de Medecine de la Reproduction de Montreal and McGill University, Montreal, Quebec, Canada
Objective: To assess whether growth factors are produced by early human embryos in culture. Design: We studied various growth factors in the culture media of human embryos (n = 6) cultured from days 3 to 8 after fertilization. Main Outcome Measures: Four growth factors were measured: Insulin growth factors I and II (I G F -I and I G F -I1), epidermal growth factor (EG F) and transforming growth factor 0' (TG F 0') activity. Results: Nonconditioned INRA Menezo B2 (Biomerieux, S.A., Paris, France) culture medium contained significant levels of TGFO' activity (5.2 ng/mL) and low levels ofIGF-I (1.02 ng/mL) and IGF -II (2.8 ng/mL), whereas EGF was below detection of our assay. With human embryo, the culture media contained lower TGFO' activity on days 3 and 4 after fertilization (2.5 ng/mL and 2.8 ng/mL, P < 0.05). From days 5 to 8 after fertilization, a significant increase in TGFO' activity and IGF-II was detected (TGFO' activity: day 5: 3.7 ng/mL; day 6: 4.4 ng/mL; day 7: 6.4 ng/mL; day 8: 8.4 ng/mL) (IGF-II: day 5: 3.4 ng/mL; day 6: 3.1 ng/mL; day 7: 4.1 ng/mL; day 8: 4.2 ng/mL). Epidermal growth factor was undetectable, and IGF-I did not vary significantly. Conclusion: Transforming growth factor 0' activity and IGF-II are produced by human embryos in culture at a time when they could playa role in morula to blastocyst transformation. Fertil Steril1992;58:101-4 Key Words: Preimplantation human embryo, transforming growth factor
Little is currently known about the role played by specific growth factors (GFs) during early human embryo development. Such knowledge might be helpful to establish optimal culture conditions for in vitro fertilization (IVF), which has been suboptimal to date and needs to be improved to grow better quality embryos (1). Although culture media without protein source have been used successfully in human IVF (2), a number of observations suggest that pro-
Received November 14, 1991; revised and accepted March 17, 1992. • Institut de Medicine de la Reproduction de Montreal. t Department of Obstetrics and Gynecology, McGill University. :j: Reprint requests: Robert Hemmings, M.D., Institut de Medicine de la Reproduction, 1100 Beaumont, number 305, MontRoyal, Quebec, Canada H3C 3P5.
Vol. 58, No.1, July 1992
0',
insulin growth factor
tein supplementation, including GFs, is important to improve embryonic development (3). In human fetal cord serum, growth-promoting substances were found in 1,000 to 5,000 molecular weight fraction, corresponding to the size of several GFs (4). Recently, several authors have reported improved human embryo development when cocultured with human ampullary tubal cells (3-5). In the pre implantation mouse embryo, messenger ribonucleic acid (mRNA) of a number of GFs was detected (6). Cooperative interaction has been observed by culturing mouse embryos in groups instead of singly (7). These interactions were reproduced by adding transforming growth factor a (TGFa) or epidermal growth factor (EGF) to the culture media of singly cultured embryos. Insulin-like growth factors (IGF-I and IGF-II) appear early in embryogenesis
Hemmings et al.
Human embryos produce TGFa and IGF-II
101
r
(8, 9), and insulin was found to stimulate protein synthesis in compacted eight-cell mouse embryos, blastocysts, and expended blastocysts (10). These observations suggest a role for GFs in the growth and differentiation of early mammalian embryos. The purpose of this study was to assess whether GFs factors are produced by early human embryos.
MATERIALS AND METHODS
Six human embryos were cultured with INRA Menezo B2 medium (B 2) (2) (Biomerieux, S.A., Paris, France) without maternal serum supplementation. These embryos were obtained from couples undergoing IVF who had elected not to freeze nontransferred embryos. Each embryo was cultured singly in 0.8 mL of B2 medium, which was changed every 24 hours. A sample of nonconditioned B2 was kept corresponding to each embryo culture medium sample. These samples were frozen at -70°C for later assay determination. Morphological development of each embryo was followed from days 3 to 8 postfertilization. All embryos (6) evolved toward the morula to blastocyst stage on days 5 to 8 postfertilization. Assays IGF-I and IGF-II
Insulin-like growth factor-I concentrations were determined by double-antibody nonequilibrium radioimmunoassay (RIA) as previously reported (11). All media samples were assayed in the same assay without extraction because the cultures were performed under serum-free conditions. Insulin-like growth factor- II concentrations were determined in the same assay, without extraction, by double-antibody RIA using a monoclonal antibody to IGF-II (Amano, Troy, VA) and the supplier's recommendations. The sensitivity of these assays is 0.25 ng/mL and 1.0 ng/mL for IGF-I and IGF-II, respectively, and there is no significant cross-reactivity for these two peptides in either assay.
Epidermal Growth Factor/TGFa Radioreceptor Assay
Because a specific RIA for TGFa (11) was no longer available to us, we elected to measure EGF / TGFa receptor activity using a human placental radioreceptor assay (RRA), as previously described (13). The sensitivity of this assay is 1.0 ng/mL for both human EGF and human TGFa because both cross-react equally with this receptor assay. However, because EGF and TGFa bind to the same receptor, the difference between EGF RRA and EGF RIA values was used to estimate TGFa activity in the culture media. These results are expressed as TGFa activity. Statistical Analysis
Data were expressed as means ± SEM and analyzed by ANOVA with a posterior comparison made by Student-Newman-Keuls and Duncan tests. Differences with P values < 0.05 were considered statistically significant. RESULTS
Insulin-like growth factor-I and IGF-II were present in low concentrations in nonconditioned INRA Menezo B2 medium. From days 3 to 8 after fertilization, the level of IGF-I did not change significantly in the human embryo culture media from the nonconditioned B2 medium, whereas IGF-II increased significantly from days 5 to 8 after fertilization, compared with day 3 after fertilization (Fig. 1).
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Epidermal Growth Factor-Radioimmunoassay
Epidermal growth factor concentrations were determined using a polyclonal antibody against human EGF raised in our laboratory and recombinant human EGF purchased from Collaborative Research Inc. (Natham, MA), as previously described (12). The sensitivity of this assay is 0.25 ng/mL, and TGFa does not cross-react. 102
Hemmings et al.
:::::
-
u..
!:2
I
• IGFI . - ' - - - - - i - - - - - i - - - - - i _____ • _____ •
• Significantly different from day 3 after fertliizC'ltion and pure 8 2 medium
Pure 82
3
4
5
6
7
8
Days After Fertilization
Figure 1 Concentrations of IGF-I and IGF-II (ng/mL ± SE) in nonconditioned culture media (B 2 ) and culture media of human embryos (n = 6) from days 3 to 8 after fertilization.
Human embryos produce TGFa and IGF-II
Fertility and Sterility
Epidermal growth factor, as measured by our RIA, was below the sensitivity of the assay in all samples measured. Activity of TGFa was found in high concentration in nonconditioned B2 medium (Fig. 2). When human embryos (days 3 and 4 after fertilization) were cultured in this medium, a significant decrease (P < 0.05) in TGFa activity was observed in all samples of culture medium. Subsequently, (days 5 to 8 after fertilization) a significant (P < 0.05) and progressive increase in TGFa activity, compared with day 3 after fertilization level, was observed (Fig. 2). DISCUSSION
These data represent the first evidence that human embryos, cultured in vitro, can produce TGFa activity and IGF-II. This production becomes significant from day 5 after fertilization, which corresponds with the morula to blastocyst transformation. Our observations also suggest that IGF-I and EGF are not secreted by early human embryos. These observations are compatible with the findings of Paria and Dey (7) who suggested that IGF-I has no effect on early mammalian embryo development. Our results suggest that human embryos can bind TGFa activity from the surrounding culture medium (B 2 in this instance) samples from days 3 and 4 after fertilization contained significantly lower TGFa activity than the nonconditioned B2 growth medium (Fig. 2). This ability of early human embryos to bind available growth factors in culture media might explain, in part, recent evidences that coculturing early pre implantation human embryos on different cell lines enhance embryo development (3-5). In bovine species, Ellington et al. (14) found that conditioned medium, cocultured with oviductal cells, was superior for supporting bovine embryo development, suggesting that GFs, released by cocultured cells, might diffuse into the embryo, crossing the zona pellucida. We found a relatively high level ofTGFa activity present in INRA Menezo B2 medium before exposure to cultured human embryos. Such a high level might be beneficial for cellular growth and maturation as suggested by Das et al. (15) who observed that addition to immature human oocytes of EGF, which binds to the same membrane receptors as TGFa, stimulated the cytoplasmic maturation of immature human oocytes. Despite the relatively small number of embryos cultured in this study, a significant increase in TGFa activity and IGF-II in culture medium was observed from days 3 to 5 after fertilization. This increase Vol. 58, No.1, July 1992
9 8
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* significantly different from nonconditioned 8 2 medium
* * significantly different from day 3 after fertilization non3 conditioned B2
4
5
6
7
8
Days After Fertilization
Figure 2 Concentration of TGFa activity (ng/mL ± SE) in nonconditioned culture media (B 2 ) and culture media of human embryos (n = 6) from days 3 to 8 after fertilization.
was progressive up to day 8 after fertilization, which was the maximum time of culture. This increase in TGFa activity and IGF-II was observed in each of the embryos cultured. Because no serum was added to the culture medium, these changes cannot be on the basis of serum variation in TGFa activity and IGF-II. The RIA used for EGF determination is very sensitive, and it did not detect EGF in any of the samples reported. Therefore, we believe that the measurement obtained by the RRA really represents TGFa activity. Transforming growth factor a has been detected in preimplantation mouse blastocysts (6). The expression of TGFa by mammalian blasto cysts could be linked to decidual tissue formation, an important aspect of the implantation process. For instance, rat decidual cells contain very high level of TGFa mRNA and an elevated number of EGF receptors (16). Therefore, TGFa could bind to the EGF receptor and function in an autocrine and/ or paracrine fashion to mediate decidualization. In addition, mouse embryos, at the morula and blastocyst stage, have been found to possess EGF receptors on the cell surface (7), suggesting a possible effect ofEGF and/or TGFa on preimplantation embryo and blastocyst functions. Paria and Dey (7) have clearly demonstrated that two-cell mouse embryos had inferior development to blastocysts and lower cell number per blastocyst when cultured singly, compared with cultured in groups of 5 or 10 embryos. This cooperative interaction among embryos could be reproduced by adding EGF and/or TGFa to singly cultured mouse embryos. Rappolee et al. (6), using single-cell mRNA phenotyping, have studied the expression of GF transcripts in preimplantation mouse embryo and have found that TGFa
Hemmings et al.
Human embryos produce TGFa and IGF-II
103
was expressed in mouse blastocysts. These studies support the concept that GFs produced by the embryo can stimulate preimplantation embryo development. Similarly, the recent demonstration by a number of authors (3, 5) that human preimplantation embryo development is significantly improved when cocultured with a number of cell lines, including human ampullary (3) cells, suggests that secretion from the reproductive tract of specific GFs might play an important role in embryo development. The importance of such GFs secretion may furthermore explain the significantly higher implantation rate reported with tubal embryo transfer than for IVF (17). Our findings of increased TGFa activity binding from the culture medium in early development (3 days after fertilization) suggest that the development of early preimplantation human embryos might be influenced by this TGFa activity. These findings also suggest that this TGFa activity might be important in the implantation process, possibly by stimulation of decidualization because it is produced in significant concentration at the time when the embryo reached the uterine cavity. The significant production of IGF-II by human embryos from day 5 postfertilization, observed in this study, suggests a possible role for IGF-II in early embryonic development. The different patterns observed in the secretion of IGF-I and IGF-II by human embryo suggest that the transcriptional control of the two genes encoding for the IGFs differs, as observed by Tricoli et al. (18). The effect of IGF-II on embryonic development remains to be determined. Acknowledgment. The authors are grateful to Ms. Nicole Dubuc, from the Institut de Medecine de la Reproduction de Montreal, for the preparation of this manuscript. REFERENCES 1. Trounson A. In: Wood C, Trounson A, editors. Clinical in vitro fertilization, selection of patients-patient management. Berlin-Heidelberg: Springer-Verlag, 1989;5:33-50. 2. Menezo Y, Testart J, Perrone D. Serum is not necessary in human in vitro fertilization, early embryo culture, and transfer. Fertil Steril 1984;42:750-5.
104
Hemmings et a1.
3. Bonsgo A, Ng SC, Sathamanthan AH, Pohlian N, Fauff M, Ratman SS. Improved quality of human embryos when cocultured with human ampullary cells. Hum Reprod 1989;4: 706-13. 4. Ogawa T, Ono T, Mars R. The effect of serum fractions on single-cell mouse embryos in vitro. J In Vitro Fert Embryo Transf 1987;4:153-8. 5. Menezo Y, Suerin VF, Czyba UC. Improvement of human early development in vitro by co-culture on monolayers of vero cells. Bioi Reprod 1990;42:301-6. 6. Rappolee DA, Brenner CA, Schultz R, Mark 0, Wesb Z. Developmental expression of PDGF, TGFa and TGF{:1 genes in preimplantation mouse embryos. Science 1988;241:1823-5. 7. Paria BC, Dey SK. Preimplantation embryo development in vitro: cooperative interactions among embryos and role of growth factors. Proc Natl Acad Sci USA 1990;87:4756-60. 8. Engstrom W, Bell KM, Schofield PN. Expression of the insulin like growth factor II gene in the developing chick limb. Cell Bioi Int Rep 1987;11:415-21. 9. Beck F, Samani NV, Pense how VD, Thorley B, Tregear GW, Cochlan VP. Histochemical localization of IGF I and II mRNA in the developing rat embryo. Development 1987;101: 175-84. 10. Harvey MB, Kaye PL. Insulin stimulates protein synthesis in compacted mouse embryo. Endocrinology 1988;122:1182-4. 11. Jesionowska H, Hemmings R, Guyda HJ, Posner BI. Determination of insulin and insulin-like growth factors (IGFs) in the ovarian circulation. Fertil Steril 1990;53:88-91. 12. Goodyer PR, Fata J, Mulligan L, Fischer D, Fagan R. Expression of transforming growth factor-a and epidermal growth factor receptor in human fetal kidneys. Mol Cell Endocrinol 1991;77:199-206. 13. Lai WH, Guyda JH. Epidermal growth factor receptors of human placental cells in culture; characterization and regulation. J Clin Endocrinol Metab 1984;58:344-52. 14. Ellington VE, Carney EW, Farrell CB, Simkin ME, Foote RH. Bovine 1-2-cell embryo development using a simple medium in the oviduct epithelial cell co-cultured systems. Bioi Reprod 1990;43:97~ 103. 15. Das K, Stout LE, Hensleigh HC, Tagatz GE, Phipps WR, Leung BS. Direct positive effect of epidermal growth factor on the cytoplasmic maturation of mouse and human oocytes. Fertil Steril 1991;55:1000-4. 16. Han VKM, Hunter ES, Pratt RM, Zendegui VG, Lee DC. Expression of rat transforming growth factor a mRNA during development occurs predominantly in the maternal decidua. Mol Cell Bioi 1987;7:2335-43. 17. Yovich JL, Yovich JM, Edirisinghe WR. The relative chance of pregnancy following tubal or uterine transfer procedures. Fertil Steril 1988;49:858-64. 18. Tricoli UV, Rail LB, Scott V, Bell GI, Shores TB. Localization of insulin-like growth factor genes to human chromosomes 11 and 12. Nature 1984;310:784-6.
Human embryos produce TGFa and IGF-II
Fertility and Sterility
r I
Vol. 58, No.1, July 1992
Copyright I[) 1992 The American Fertility Society
Printed on acid-free paper in U.S.A.
Human embryos produce transforming growth factors a activity and insulin-like growth factors II
Robert Hemmings, M.D.*t+ Jean Langlais, Ph.D.* Tommaso Falcone, M.D.*t
Louis Granger, M.D.* Pierre Miron, M.D.* Harvey Guyda, M.D.t
Institut de Medecine de la Reproduction de Montreal and McGill University, Montreal, Quebec, Canada
Objective: To assess whether growth factors are produced by early human embryos in culture. Design: We studied various growth factors in the culture media of human embryos (n = 6) cultured from days 3 to 8 after fertilization. Main Outcome Measures: Four growth factors were measured: Insulin growth factors I and II (I G F -I and I G F -I1), epidermal growth factor (EG F) and transforming growth factor 0' (TG F 0') activity. Results: Nonconditioned INRA Menezo B2 (Biomerieux, S.A., Paris, France) culture medium contained significant levels of TGFO' activity (5.2 ng/mL) and low levels ofIGF-I (1.02 ng/mL) and IGF -II (2.8 ng/mL), whereas EGF was below detection of our assay. With human embryo, the culture media contained lower TGFO' activity on days 3 and 4 after fertilization (2.5 ng/mL and 2.8 ng/mL, P < 0.05). From days 5 to 8 after fertilization, a significant increase in TGFO' activity and IGF-II was detected (TGFO' activity: day 5: 3.7 ng/mL; day 6: 4.4 ng/mL; day 7: 6.4 ng/mL; day 8: 8.4 ng/mL) (IGF-II: day 5: 3.4 ng/mL; day 6: 3.1 ng/mL; day 7: 4.1 ng/mL; day 8: 4.2 ng/mL). Epidermal growth factor was undetectable, and IGF-I did not vary significantly. Conclusion: Transforming growth factor 0' activity and IGF-II are produced by human embryos in culture at a time when they could playa role in morula to blastocyst transformation. Fertil Steril1992;58:101-4 Key Words: Preimplantation human embryo, transforming growth factor
Little is currently known about the role played by specific growth factors (GFs) during early human embryo development. Such knowledge might be helpful to establish optimal culture conditions for in vitro fertilization (IVF), which has been suboptimal to date and needs to be improved to grow better quality embryos (1). Although culture media without protein source have been used successfully in human IVF (2), a number of observations suggest that pro-
Received November 14, 1991; revised and accepted March 17, 1992. • Institut de Medicine de la Reproduction de Montreal. t Department of Obstetrics and Gynecology, McGill University. :j: Reprint requests: Robert Hemmings, M.D., Institut de Medicine de la Reproduction, 1100 Beaumont, number 305, MontRoyal, Quebec, Canada H3C 3P5.
Vol. 58, No.1, July 1992
0',
insulin growth factor
tein supplementation, including GFs, is important to improve embryonic development (3). In human fetal cord serum, growth-promoting substances were found in 1,000 to 5,000 molecular weight fraction, corresponding to the size of several GFs (4). Recently, several authors have reported improved human embryo development when cocultured with human ampullary tubal cells (3-5). In the pre implantation mouse embryo, messenger ribonucleic acid (mRNA) of a number of GFs was detected (6). Cooperative interaction has been observed by culturing mouse embryos in groups instead of singly (7). These interactions were reproduced by adding transforming growth factor a (TGFa) or epidermal growth factor (EGF) to the culture media of singly cultured embryos. Insulin-like growth factors (IGF-I and IGF-II) appear early in embryogenesis
Hemmings et al.
Human embryos produce TGFa and IGF-II
101
r
(8, 9), and insulin was found to stimulate protein synthesis in compacted eight-cell mouse embryos, blastocysts, and expended blastocysts (10). These observations suggest a role for GFs in the growth and differentiation of early mammalian embryos. The purpose of this study was to assess whether GFs factors are produced by early human embryos.
MATERIALS AND METHODS
Six human embryos were cultured with INRA Menezo B2 medium (B 2) (2) (Biomerieux, S.A., Paris, France) without maternal serum supplementation. These embryos were obtained from couples undergoing IVF who had elected not to freeze nontransferred embryos. Each embryo was cultured singly in 0.8 mL of B2 medium, which was changed every 24 hours. A sample of nonconditioned B2 was kept corresponding to each embryo culture medium sample. These samples were frozen at -70°C for later assay determination. Morphological development of each embryo was followed from days 3 to 8 postfertilization. All embryos (6) evolved toward the morula to blastocyst stage on days 5 to 8 postfertilization. Assays IGF-I and IGF-II
Insulin-like growth factor-I concentrations were determined by double-antibody nonequilibrium radioimmunoassay (RIA) as previously reported (11). All media samples were assayed in the same assay without extraction because the cultures were performed under serum-free conditions. Insulin-like growth factor- II concentrations were determined in the same assay, without extraction, by double-antibody RIA using a monoclonal antibody to IGF-II (Amano, Troy, VA) and the supplier's recommendations. The sensitivity of these assays is 0.25 ng/mL and 1.0 ng/mL for IGF-I and IGF-II, respectively, and there is no significant cross-reactivity for these two peptides in either assay.
Epidermal Growth Factor/TGFa Radioreceptor Assay
Because a specific RIA for TGFa (11) was no longer available to us, we elected to measure EGF / TGFa receptor activity using a human placental radioreceptor assay (RRA), as previously described (13). The sensitivity of this assay is 1.0 ng/mL for both human EGF and human TGFa because both cross-react equally with this receptor assay. However, because EGF and TGFa bind to the same receptor, the difference between EGF RRA and EGF RIA values was used to estimate TGFa activity in the culture media. These results are expressed as TGFa activity. Statistical Analysis
Data were expressed as means ± SEM and analyzed by ANOVA with a posterior comparison made by Student-Newman-Keuls and Duncan tests. Differences with P values < 0.05 were considered statistically significant. RESULTS
Insulin-like growth factor-I and IGF-II were present in low concentrations in nonconditioned INRA Menezo B2 medium. From days 3 to 8 after fertilization, the level of IGF-I did not change significantly in the human embryo culture media from the nonconditioned B2 medium, whereas IGF-II increased significantly from days 5 to 8 after fertilization, compared with day 3 after fertilization (Fig. 1).
3............... 0 ••••••.
4
6
:::r
....E
~
IGF II
••,/
.... ···········.... i/·/ 3
o
0.•••••••••..••• 6•••••••••••••••
iii
~
+'
2
u..
C1
Epidermal Growth Factor-Radioimmunoassay
Epidermal growth factor concentrations were determined using a polyclonal antibody against human EGF raised in our laboratory and recombinant human EGF purchased from Collaborative Research Inc. (Natham, MA), as previously described (12). The sensitivity of this assay is 0.25 ng/mL, and TGFa does not cross-react. 102
Hemmings et al.
:::::
-
u..
!:2
I
• IGFI . - ' - - - - - i - - - - - i - - - - - i _____ • _____ •
• Significantly different from day 3 after fertliizC'ltion and pure 8 2 medium
Pure 82
3
4
5
6
7
8
Days After Fertilization
Figure 1 Concentrations of IGF-I and IGF-II (ng/mL ± SE) in nonconditioned culture media (B 2 ) and culture media of human embryos (n = 6) from days 3 to 8 after fertilization.
Human embryos produce TGFa and IGF-II
Fertility and Sterility
Epidermal growth factor, as measured by our RIA, was below the sensitivity of the assay in all samples measured. Activity of TGFa was found in high concentration in nonconditioned B2 medium (Fig. 2). When human embryos (days 3 and 4 after fertilization) were cultured in this medium, a significant decrease (P < 0.05) in TGFa activity was observed in all samples of culture medium. Subsequently, (days 5 to 8 after fertilization) a significant (P < 0.05) and progressive increase in TGFa activity, compared with day 3 after fertilization level, was observed (Fig. 2). DISCUSSION
These data represent the first evidence that human embryos, cultured in vitro, can produce TGFa activity and IGF-II. This production becomes significant from day 5 after fertilization, which corresponds with the morula to blastocyst transformation. Our observations also suggest that IGF-I and EGF are not secreted by early human embryos. These observations are compatible with the findings of Paria and Dey (7) who suggested that IGF-I has no effect on early mammalian embryo development. Our results suggest that human embryos can bind TGFa activity from the surrounding culture medium (B 2 in this instance) samples from days 3 and 4 after fertilization contained significantly lower TGFa activity than the nonconditioned B2 growth medium (Fig. 2). This ability of early human embryos to bind available growth factors in culture media might explain, in part, recent evidences that coculturing early pre implantation human embryos on different cell lines enhance embryo development (3-5). In bovine species, Ellington et al. (14) found that conditioned medium, cocultured with oviductal cells, was superior for supporting bovine embryo development, suggesting that GFs, released by cocultured cells, might diffuse into the embryo, crossing the zona pellucida. We found a relatively high level ofTGFa activity present in INRA Menezo B2 medium before exposure to cultured human embryos. Such a high level might be beneficial for cellular growth and maturation as suggested by Das et al. (15) who observed that addition to immature human oocytes of EGF, which binds to the same membrane receptors as TGFa, stimulated the cytoplasmic maturation of immature human oocytes. Despite the relatively small number of embryos cultured in this study, a significant increase in TGFa activity and IGF-II in culture medium was observed from days 3 to 5 after fertilization. This increase Vol. 58, No.1, July 1992
9 8
::J" ....E
C'I
E. W
7 6
5
vi +,
4
II-
3
~
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--.
I
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* significantly different from nonconditioned 8 2 medium
* * significantly different from day 3 after fertilization non3 conditioned B2
4
5
6
7
8
Days After Fertilization
Figure 2 Concentration of TGFa activity (ng/mL ± SE) in nonconditioned culture media (B 2 ) and culture media of human embryos (n = 6) from days 3 to 8 after fertilization.
was progressive up to day 8 after fertilization, which was the maximum time of culture. This increase in TGFa activity and IGF-II was observed in each of the embryos cultured. Because no serum was added to the culture medium, these changes cannot be on the basis of serum variation in TGFa activity and IGF-II. The RIA used for EGF determination is very sensitive, and it did not detect EGF in any of the samples reported. Therefore, we believe that the measurement obtained by the RRA really represents TGFa activity. Transforming growth factor a has been detected in preimplantation mouse blastocysts (6). The expression of TGFa by mammalian blasto cysts could be linked to decidual tissue formation, an important aspect of the implantation process. For instance, rat decidual cells contain very high level of TGFa mRNA and an elevated number of EGF receptors (16). Therefore, TGFa could bind to the EGF receptor and function in an autocrine and/ or paracrine fashion to mediate decidualization. In addition, mouse embryos, at the morula and blastocyst stage, have been found to possess EGF receptors on the cell surface (7), suggesting a possible effect ofEGF and/or TGFa on preimplantation embryo and blastocyst functions. Paria and Dey (7) have clearly demonstrated that two-cell mouse embryos had inferior development to blastocysts and lower cell number per blastocyst when cultured singly, compared with cultured in groups of 5 or 10 embryos. This cooperative interaction among embryos could be reproduced by adding EGF and/or TGFa to singly cultured mouse embryos. Rappolee et al. (6), using single-cell mRNA phenotyping, have studied the expression of GF transcripts in preimplantation mouse embryo and have found that TGFa
Hemmings et al.
Human embryos produce TGFa and IGF-II
103
was expressed in mouse blastocysts. These studies support the concept that GFs produced by the embryo can stimulate preimplantation embryo development. Similarly, the recent demonstration by a number of authors (3, 5) that human preimplantation embryo development is significantly improved when cocultured with a number of cell lines, including human ampullary (3) cells, suggests that secretion from the reproductive tract of specific GFs might play an important role in embryo development. The importance of such GFs secretion may furthermore explain the significantly higher implantation rate reported with tubal embryo transfer than for IVF (17). Our findings of increased TGFa activity binding from the culture medium in early development (3 days after fertilization) suggest that the development of early preimplantation human embryos might be influenced by this TGFa activity. These findings also suggest that this TGFa activity might be important in the implantation process, possibly by stimulation of decidualization because it is produced in significant concentration at the time when the embryo reached the uterine cavity. The significant production of IGF-II by human embryos from day 5 postfertilization, observed in this study, suggests a possible role for IGF-II in early embryonic development. The different patterns observed in the secretion of IGF-I and IGF-II by human embryo suggest that the transcriptional control of the two genes encoding for the IGFs differs, as observed by Tricoli et al. (18). The effect of IGF-II on embryonic development remains to be determined. Acknowledgment. The authors are grateful to Ms. Nicole Dubuc, from the Institut de Medecine de la Reproduction de Montreal, for the preparation of this manuscript. REFERENCES 1. Trounson A. In: Wood C, Trounson A, editors. Clinical in vitro fertilization, selection of patients-patient management. Berlin-Heidelberg: Springer-Verlag, 1989;5:33-50. 2. Menezo Y, Testart J, Perrone D. Serum is not necessary in human in vitro fertilization, early embryo culture, and transfer. Fertil Steril 1984;42:750-5.
104
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Human embryos produce TGFa and IGF-II
Fertility and Sterility
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