In VitroCell. Dev.Biol.26:1043-1048, November1990 9 1990TissueCultureAssociation 0883-8364/90 $01.50+0.00
F U N C T I O N A L R O L E S OF I N S U L I N AND I N S U L I N L I K E G R O W T H F A C T O R S IN P R E I M P L A N T A T I O N MOUSE E M B R Y O D E V E L O P M E N T L. V. RAO, M. L. WIKARCZUK, ANDS.HEYNER' Department of Obstetrics and Gynecology, Albert Einstein Medical Center, Philadelphia, Pennsylvania 19141
{Received29 May 1990; accepted 19 June 1990)
SUMMARY Growth factors are known to play important roles in cellular proliferation and differentiation. However, little information is available concerning their roles in the earliest stages of mammalian development. The effect of physiologic levels of insulin, insulinlike growth factor-I, and insulinlike growth factor II (IGF-I and -II) on DNA, RNA, and protein synthesis in preimplantation stages of the mouse are described in this study. Quantitative studies of the incorporation of labeled thymidine, uridine, and methionine into trichloroacetic acid-insohtble material by different developmental stages of preimplantation mouse embryos labeled in vitro, indicate that physiologic levels of insulin stimulated DNA, RNA, and protein synthesis with significant effects observed first at the morula stage of development. In contrast, neither IGF-I nor IGF-II stimulated DNA, RNA, or protein synthesis to a significant degree under the same experimental conditions. These results suggest a functional role for insulin at the earliest stages of mammalian embryogenesis. Key words: insulin; insulinlike growth factor; embryo; metabolism.
INTRODUCTION
the morula stage of development. High resolution electron microscopy in conjunction with colloidal gold-labeled insulin has shown that the hormone is internalized by preimplantation mouse embryos by means of receptor-mediated endocytosis and concentration in coated pits {Heyner et al., 1989). Although it is clear that the receptors for insulin and IGFs are expressed during early mammalian development, the functional roles oi these growth factors remain to be elucidated. The demonstration of hormonal action in vivo requires the presence of hormone, its receptor and evidence of postbinding effects at physiologic concentrations. There is evidence that insulin stimulates the incorporation of [3H]leucine into compacted eight-cell stage mouse embryos, although the concentrations oI the hormone used were supraphysiological (Harvey and Kaye, 1988). At physiologic levels of the hormone, insulin stimulates both DNA and RNA synthesis in the mouse embryo, beginning at the morula stage of development {Heyner et al., 1989}. It is of considerable interest to monitor changes in the preimplantation embryo's ability to transport and incorporate macromolecular precursors. Such changes can be considered to reflect alterations in synthetic rates of specific molecules during early development. In the present study we report the effects of physiologic levels of insulin, IGF-I, and -II on DNA, RNA, and protein synthesis during preimplantation stages of development in the mouse.
Regular progression of preimplantation development depends on the expression of specific genes, and on a continuous supply of energy, hormones, and growth factors. Recent studies in a number of vertebrate and invertebrate embryos have suggested that growth factors and their receptors may play important roles in cell proliferation and differentiation (reviewed in Mattson et al., 1989). Among the large number of growth factors, insulin and insulinlike growth factor- I and -II ~IGF-I and -II) are considered to be potentially important developmental factors because of their roles in stimulating biological responses. The peptides of the insulin family affect membrane transport (Yu and Czech, 1984), enzymatic activities (Verspohl et al., 1984), cell growth in a number of cell lines in vitro and in vivo, as well as early postnatal growth {Alexandrides and Smith, 1989). Experimental evidence supports the involvement of insulin and the IGFs in early development. Insulin binding to the cells of the preimplantation mouse embryo was first demonstrated using indirect immunofluorescence by Rosenblum et al. {1986). Using autoradiographic methods, Mattson et al. (1988) detected the expression of specific receptors that bind insulin and IGFs as early as
To whom correspondence should be addressed at Dept. of Ob/Gyn, Rm. 311A, John Morgan University of Pennsylvania Medical Center, 36th and Hamilton Walk, Philadelphia, PA 19104. 1043
1044
RAO ET AL.
MATERIALS AND METHODS
70
Animals and embryo recovery. Female mice (CD-1; 6- to 8-wk old: Charles River Breeding Laboratories) were injected with 5 IU pregnant mare's serum gonadotropin (PMSG; Sigma Chemical Co., St. Louis, MO) and 48 hrs later with 5 IU Human Chorionic Gonadotropin (hCG; Sigma). A single female was placed with a singly caged male ICD-1; Charles River Breeding Laboratories) overnight, and the presence of a vaginal plug the following morning was taken as evidence that mating had occurred. Embryos of different developmental stages (two-, eight-cell, morula and blastocyst) were flushed from the reproductive tract using M-2 medium (Whittingham, 1971). Embryos were cultured for at least 1 h in medium M-16 (Whittingham, 1971) at 37~ C in an atmosphere of 5% COs in humidified air. Both culture media M2 and M16 contained 0.4% bovine serum albumin (BSA), that had been screened previously to ensure that it w a s insulin-free. Uptake
and
incorporation
of labeled precursors.
Groups of 15 to 20 embryos (two-, eight-cell, morula and blastocyst} were flushed from the reproductive tract and cultured for at least 1 h in medium M16 containing 0.4% BSA in an atmosphere of 5% COs in air at 37 ~ C. They were then incubated for another hour in medium containing 0.4% insulin-free BSA, with or without 40 ng/ml of insulin (Eli Lilly, Indianapolis, IN), IGF-I (AmGen, Thousand Oaks, CA), or IGF-II (Eli Lilly). Embryos were then transferred to M16 containing 1 mCi/ml labeled methionine (L-methionine 3sS, 1196 Ci/mmol, ICN, Cleveland, OH) or 50 /~Ci/ml of [3H]thymidine (methyl [3H]thymidine, 83 Ci/mmol, Amersham, Arlington Heights, IL) or [3H]uridine (5,6 [3H]uridine 40 Ci/mmol, Amersham) in the presence or absence of growth factors, for another 2 h. Embryos were washed extensively in ice-cold M2, and total uptake and incorporation of labeled precursors were measured after precipitation with trichloroacetic acid (TCA) using methods described previously ~Heyner et al., 1989). Rndiolabeling of embryonic proteins. Groups of 20 blastocyst-stage embryos were cultured for at least 1 h in
25000
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910
, 120
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T i m e in M i n u t e s
FIG. 2. Effect of length of incubation on net accumulation of labeled methionine in intact mouse blastocysts. medium M16 containing 0.4% insulin-free BSA in an atmosphere of 5% CO2 in air at 37 ~ C. They were then incubated for another hour in medium containing 0.4% insulin-free BSA, with or without 40 ng/ml of insulin. Embryos were then transferred to M16 containing 1 mCi/ml [3~S]methionine, in the presence or absence of insulin for another 2 h. Embryos were washed extensively in ice-cold M2, denatured, and reduced by heating in Laemmli's sample buffer iLaemmli, 1970), containing 0.1% dithiothreitol IDTT) at 95 ~ C for 3 min and then subjected to 7.5% one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGEL Radiolabeled 35S-proteins were detected by fluorography (Bonner and Laskey, 1974) at --70 ~ C using Kodak X-OMAT AR film. Fluorograms were scanned using a LKB Laser densitometer for the comparison of peaks. RESULTS
Net uptake of ~sS-methionine. The net uptake of 3SS-methionine into groups of intact mouse blastocysts was measured using an increasing concentration of labeled methionine (0.25, 0.5, 1, 2, 5 mCi/ml) for 2 hrs. The incorporation of labeled precursor into intact blastocysts increased in a dose-dependent manner, reached a peak value at 1 mCi/ml and decreased thereafter ~Fig. 1). The net accumulation of ~'Smethionine over time, at a concentration of 1 mCi/ml labeled precursor, was also measured in blastocysts (Fig. 2). Incorporation gradually increased for 2 h and then reached a plateau. The studies described above showed that pS]methionine was taken up by mouse blastocysts, and the rate of incorporation was maximum in vitro when blastocysts were incubated for 2 h with 1 mCi/ml labeled methionine at 37~ C in an atmosphere of 5% COs in humidified air. Effect of growth factors on [~S/methionine uptake and incorporation. Studies in a variety of systems have
1
2
3 mCi/ml
4
5
6
FIG. 1. Kinetics of uptake of labeled methionine into intact mouse blastocysts.
indicated that insulin and the IGFs activate the production of proteins that are required for cell proliferation, as well as for maintenance. Table 1 shows the results of [3sS]methionine uptake and incorporation in different preimplantation stages of development in the
INSULIN FAMILY PEPTIDES IN MURINE EMBRYOGENESIS
1045
TABLE 1 EFFECT OF GROWTH FACTORS ON INCORPORATION OF LABELED METHIONINE BY MOUSE PREIMPLANTATION EMBRYOS" Control Stage
40 ng/ml
Incorporation
Total U ptake
Incor~)ration
Total Uptake
TwoceU Eightcell Morula Blastocyst
3512.3 -+ 8091.6-+ 13337.3 -+ 21889.7 -+
319.2 416.8 548.8 1408.6
13027.0 -+ 1454.4 1,5663.0-+ 690.3 26364.6 -+ 2264.1 52440.0 -+ 6321.5
3492.0 + 8163.3 -+ 15743.0 -+ 30244.2 -+
274.3 359.9 1261.1 1838.6
12473.6 4- 1199.6 17376.6_ 1909.7 28470.0 4- 1670.9 55529.7 + 4399.1
Twocell Eight cell Morula Blastocyst
3619.0-+ 8397.3 + 13621.3 -+ 24297.0 -+
510.0 1108.1 475.4 1250.4
15175.0-+ 598.1 18445.3 -+ 2668.9 24091.0 -+ 2457.5 53695.2 -4- 8564.5
3576.0-48382.3 -+ 13934.6 -+ 23735.5 -+
145.1 1028.9 463.1 1041.8
12473.6--4-116.6 17820.0 -+ 1910.2 25428.6 -+ 1580.1 51118.7 -+ 7255.8
Twocell Eightcell Morula Blastocyst
3709.3--+ 297.2 7711.0-+ 693.7 11644.3 -+ 420.2 23657.2 ___2225.8
15053.0-+ 325.1 17099.0-+ 773.4 22384.0 -+ 1818.2 41849.0 -+ 2784.9
3694.0-+ 381.9 7499.6-4- 290.2 12030.0 -4- 629.6 23669.0 -+ 1133.5
15321.3-+ 1310.0 16999.3-+ 488.0 22937.6 _+ 1025.0 39250.0 -+ 2699.7
"10 to 15 Embryos were used for each determination, which was performed 3 to 4 times. The data are expressed as mean + the standard error of the mean. Incorporation refers to the number of acid-insoluble cpm per embryo. Total uptake refers to the number of both acid soluble and acid insoluble cpm per embryo.
presence or absence of physiologic levels of insulin, I G F - I , and I G F - I I . These results show that incubation of embryos in insulin and I G F s had no effect on total accumulation of labeled precursors in the intact embryo. Insulin at concentration of 40 n g / m l increased the incorporation of labeled methionine into the TCA-insoluble precipitate at the morula and blastocyst stages of development. There was an 18 and 38% increase, respectively, in methionine incorporation when morula and blastocyst-stage embryos were incubated for "2 h with physiologic levels of insulin (Fig. 3). On the other hand, I G F - I and I G F - I I at similar physiologic levels had no effect on labeled methionine incorporation. T h e results of one dimensional S D S - P A G E autoradiography (Fig. 4) also showed that there was an overall stimulation of protein synthesis at the blastocyst stage when embryos were incubated with physiologic levels of insulin. The fluorogram shows clearly that there is a
50 40 20 ku
o
-10
quantitative increase in labeled precursor uptake in insulin-treated embryos, as compared with the same number of embryos processed identically, but without
~- INSULIN I t,, I
2-CELL
G .|
8-CELL
& I
|
MORULABLASTOCYST
FIG. 3. Mean percentage increase in the incorporation of labeled methionine in the presence of insulin or insulinlike growth factors in different stages of early development.
FIG. 4. Fluorogram of radiolabeled blastocysts incubated with (40 ng/ml) or without insulin.
1046
q)
RAO ET AL.
31
120 ,,-:
100
9
! 8O
= ;
-
INSULIN IGF-1
/o /
.
60 I
FIG. 5. Densitometric analysis of the fluorograms shown in Fig. 4. Comparison of area under the peaks (2,3,9) of radiolaheled mouse blastocyst proteins, from embryos incubated in the presence or absence of insulin revealed ratios of 1.40:1, 1.50:1, and 1.46:1, respectively. insulin. Laser scanning densitometer analysis of the fluorograms revealed that incubation in the presence of insulin resulted in increased area under the peaks {Fig. 5). Comparison of the area under the three major peaks of both the groups resulted in the ratios of 1.40:1, 1.50:1, and 1.46:1, respectively.
Effect of growth factors on the incorporation [3H]thymidine and p H]uridine. The incubation
of
of different developmental stages in insulin or insulinlike growth factors had no effect on total accumulation of labeled uridine or thymidine in intact embryos. However, our earlier studies had shown that insulin at a concentration of 4 ng/ml increased significantly the incorporation of labeled thymidine and uridine into the TCA-insoluble precipitate at the morula and blastocyst stages of development (Heyner et al., 1989). In contrast, the present study revealed that IGF-I and -II had very little effect on the incorporation of labeled thymidine {Fig. 6) or uridine {Fig. 7), when morula and blastocyststage embryos were incubated for 2 h at a concentration of 40 ng/ml. There was an 9 and 12% increase, respectively, in uridine incorporation, and 9 and 10% increase, respectively, in thymidine incorporation when morula and blastocyst-stage embryos were incubated for 2 h in medium containing 40 ng/ml IGF-I. Similarly, incubation of morulae and blastocysts in medium containing 40 ng/ml IGFII resulted in 2 and 10% increases, respectively, in uridine
30 I
:"
INSULIN IGF-1
/
/o
IGF-
20
~ 20
~ll
I
I
|
2-CELL 8-CELL MORULABLASTOCYST FIG. 6. Mean percentage increase in the incorporation of labeled thymidine in the presence of insulin or insulinlike growth factors in different stages of early development.
2O I
I
I
I
2-CELL 8-CELL MORULABLASTOCYST FIG. 7. Mean percentage increase in the incorporation of labeled uridine in the presence of insulin and insulinlike growth factors in different stages of early development. incorporation. Thymidine incorporation increased 12 and 11%, respectively, for the morula and blastocyst stages under similar conditions. Effects of the IGFs on thymidine and uridine incorporation were not significantly different from controls at the P < 0.05% level. D ISCUSSION Insulin is an anabolie hormone that can increase the overall rate of protein synthesis and regulate the preferential expression of specific proteins. IGF-I and I G F - I I are two single-chain polypeptide hormones that are structurally related to each other and proinsulin (Blundell and Humbel, 1980L Peptides of the insulin family have been shown to stimulate protein synthesis and mitogenic responses in many cell types. Their role in growth control during early development is not known. The transition of a single cell zygote to a multicellular blastocyst, during early mammalian development, involves a series of specific changes which remain to be elucidated. Previous studies utilizing immunofluorescence (Rosenblurn et al., 1986), autoradiography (Mattson et al., 1988), and high resolution electron microscopy (Heyner et al., 1989) have shown that mouse preimplantation embryos express receptors for insulin and insulinlike growth factors in a developmentally regulated manner. Receptors could he detected beginning at the late eight-cell stage, and increased in density through the blastocyst stage. The present experiments were designed to investigate whether physiologic levels of insulin and IGFs affected the rate at which preimplantation mouse embryos incorporated macromolecular precursors from their environment in vitro. Preimplantation mouse embryos are capable of synthesizing nucleic acids (Piko, 1970; Piko and Clegg, 1982) and it is well known that changes in the pattern of protein synthesis occur during the preimplantation development of mouse embryos (Epstein and Smith, 1974). The studies described above showed that labeled methionine, uridine, and thymidine were taken up by two-, eight-cell, morula and blastocyststage embryos in vitro, and as development proceeded the rate of this process increased. This increased incorporation may reflect an increase in metabolic
INSULIN FAMILY PEPTIDES IN MURINE EMBRYOGENESIS activity as well as an increase in cell surface area. Incubation of embryos in insulin and I G F s at physiologic levels had no influence on total accumulation of precursors, showing that the growth factors did not affect the rate of macromolecular transport into intact embryos. However, insulin significantly increased the incorporation of labeled precursors into TCA-insoluble material in early embryos, indicating that this effect was specific for stimulation of maeromolecular synthesis. DNA, RNA, and protein synthesis were significantly increased when morula and blastocyst-stage embryos were incubated in physiologic levels of insulin. In contrast, I G F - I and I G F - I I did not stimulate incorporation of precursors under the same experimental conditions. A quantitative increase in labeled methionine uptake in insulin-treated blastocyst-stage embryos was also observed in onedimensional SDS-PAGE fluorograms (Fig. 4). Preliminary studies using high-resolution, two-dimensional gel eleetrophoresis of blastocysts incubated for 2 h in the presence or absence of physiologic levels of insulin have shown evidence of a generalized increase in the level of protein synthesis. In addition, there is evidence of some posttranslational modifications and expression of novel minor polypeptides (Rao, Heyner, Collins and Matsehiusky, unpublished observations). Results of other investigators support our findings; thus, Sellens et al., (1981) observed that in the absence o! serum, but in the presence of amino acids, there was only a modest increase in the protein content of cultured blastocysts, suggesting that in addition to an exogenous supply of amino acids other factors are required for optimal growth. Recently, Travers et al., {1989) examined the effect of hypoinsulinemia on rat embryonic growth and development in culture, and observed that addition of physiologic amounts of insulin to insulin-depleted serum restored growth levels to those of control embryos. Their in vitro experiments strongly suggest that the early mammalian embryo has an absolute requirement of insulin, even in the presence of insulinlike growth factors. Although insulin stimulated significant increases in DNA, RNA, and protein synthesis, it is known from a number of studies that insulin is not an absolute requirement for in vitro development to the blastocyst stage. However, embryos grown in vitro lag developmentally behind their in vivo counterparts (Bowman and McLaren, 1970). Further, embryo development in vitro cannot be evaluated merely by the morphologic development of the blastocyst, but rather by the maintenance of its subsequent developmental viability and establishment of pregnancy. The role of insulin may be to stimulate overall preimplantation development to ensure a successful implantation. The lack of an effect of I G F - I , is intriguing, particularly since the receptor for this peptide is expressed around the same developmental period as the insulin receptor. However, I G F - I has been postulated to act as a proliferative agent on cellsof the mesodermal lineage in mammals (Beck et ai., 1987), and it is entirely conceivable that the real importance of this peptide in early development occurs after implantation, during organogenesis. IGF-II ligand has been detected as early as the late two-ceU stage in the mouse ~Rappolee et al., 1990), and inasmuch as this peptide is produced in an autocrine manner, it is not surprising that
1047
addition of exogenous ligand does not have a significant effect on metabolism. On the other hand, preliminary studies suggest that when embryos are incubated in the presence of antisense, but not sense oligonucleotides for I G F - I I coding sequences, there is retardation in the rate of preimplantation development in vitro (Werb and Rappolee, 1990). Clearly, more studies are needed to reveal the precise roles played by peptides of the insulin family during the early stages of mammalian development. REFERENCES 1. Aiexandrides, T. K.; Smith, R. J. A novel fetal insulin-llke growth factor (IGF) I receptor. J. Biol. Chem. 264:12922-12930; 1989. 2. Beck, F.; Samani, N. J.; Peuschow, J. D., et al. Histocbemlcal localization of IGF-I and -II mRNA in the developing rat embryo. Development 101:175-184; 1987. 3. Blundell, T. L.; Humbel, R. E. Hormone families; pancreatic hormones and homologous growth factors. Nature 287:781-787; 1980. 4. Bonner, W. M.; Laskey, R. A. A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:83-88; 1974. 5. Bowman, P.; McLaren, A. Viability and growth of mouse embryos after in vitro culture and fusion. J. Embryol. Exp. Morphol. 23:693-704; 1970. 6. Epstein, C. J.; Smith, S. A. Electrophoretic analysis of proteins synthesized by preimplantation embryos. Dev. Biol. 40:233-244; 1974. 7. Harvey, M. B.; Kaye, P. L. Insulin stimulates protein synthesis in compacted mouse embryos. Endocrinology 122:1182-1184; 1988. 8. Heyner, S.; Rao, L. V.; Jarett, L., et al. Preimplantation mouse embryos internalize maternal insulin via receptor mediated endocytosis: patterns of uptake and functional correlations. Dev. Biol. 134:48-58; 1989. 9. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685; 1970. 10. Mattson, B. A.; Rosenblum, I. Y.; Smith, R. M., et al. Autoradiographlc evidence for insulin and insulin like growth factor binding to early mouse embryos. Diabetes 37:585-589; 1988. 11. Mattson, B. A.; Chambers, S. A.; de Pabio, F. Comparative aspects of the insulin and the insulin receptor throughout evolution. In: Rosenblum, I. Y.; Heyner, S., eds. Growth factors in mammalian development. Boca Raton, FL: CRC Press; 1989:47-70. 12. Piko, L. Synthesis of macromolecules in early mouse embryos cultured in vitro: RNA, DNA and a polysaccharide component. Dev. Biol. 21:257-279; 1970. 13. Piko, L.; Clegg, K. B. Quantitative changes in total RNA, total poly (A) and ribosomes in early mouse embryos. Dev. Biol. 89:362-378; 1982. 14. Rappolee, D. A.; Sturm, K. S.; Schultz, G. A., et al. The expression of growth factor ligands and receptors in preimplantation mouse embryos. In: Heyner, S.; Wiley, L. M., eds. Early embryo development and paracrine relationships. New York: Wiley-Liss; 1990:11-26. 15. Rosenblum, I. Y.; Mattson, B. A.; Heyner, S. Stage specific insulin binding in mouse preimplantation embryos. Dev. Biol. 116:261-263; 1986. 16. Sellens, M. H.; Stein, S.; Sherman, M. I. Protein and free amino acid content in preimplantation mouse embryos and in blastocysts under various culture conditions. J. Reprod. Fertil. 61:307-315; 1981. 17. Travers, J. P.; Pratten, M. K.; Beck, F. Effects of low insulin levels on rat embryonic growth and development. Diabetes 38:773-778; 1989. 18. Verspohl, E. J.; Roth, R. A.; Vigneri, R., et al. Dual regulation of glycogen metabolism by insulin and insulin like growth
1048
RAO ET AL.
factors in human hepatoma cells {Hep- G2). J. Clin. Invest. 74:1436-1443; 1984. 19. Werb, Z.; Rappolee, D. A. The expression and function of growth factors during early development of mouse embryos. J. Cell. Biochem. {Suppl)14E:48;1990.
20. Whittingham, D. G. Culture of mouse ova. J. Reprod. Fertil. ~Suppl).14:7-21; 1971. 21. Yu, K. T.; Czech, M. P. Tyrosine phosphorylation of the insulin receptor B-subunit aetivates the receptor associated tyrosine kinase activity. J. Biol. Chem. 259:5277-5286; 1984.
This work was supported by grant HD 23511 from the National Institutes of Health, Bethesda, MD.
F U N C T I O N A L R O L E S OF I N S U L I N AND I N S U L I N L I K E G R O W T H F A C T O R S IN P R E I M P L A N T A T I O N MOUSE E M B R Y O D E V E L O P M E N T L. V. RAO, M. L. WIKARCZUK, ANDS.HEYNER' Department of Obstetrics and Gynecology, Albert Einstein Medical Center, Philadelphia, Pennsylvania 19141
{Received29 May 1990; accepted 19 June 1990)
SUMMARY Growth factors are known to play important roles in cellular proliferation and differentiation. However, little information is available concerning their roles in the earliest stages of mammalian development. The effect of physiologic levels of insulin, insulinlike growth factor-I, and insulinlike growth factor II (IGF-I and -II) on DNA, RNA, and protein synthesis in preimplantation stages of the mouse are described in this study. Quantitative studies of the incorporation of labeled thymidine, uridine, and methionine into trichloroacetic acid-insohtble material by different developmental stages of preimplantation mouse embryos labeled in vitro, indicate that physiologic levels of insulin stimulated DNA, RNA, and protein synthesis with significant effects observed first at the morula stage of development. In contrast, neither IGF-I nor IGF-II stimulated DNA, RNA, or protein synthesis to a significant degree under the same experimental conditions. These results suggest a functional role for insulin at the earliest stages of mammalian embryogenesis. Key words: insulin; insulinlike growth factor; embryo; metabolism.
INTRODUCTION
the morula stage of development. High resolution electron microscopy in conjunction with colloidal gold-labeled insulin has shown that the hormone is internalized by preimplantation mouse embryos by means of receptor-mediated endocytosis and concentration in coated pits {Heyner et al., 1989). Although it is clear that the receptors for insulin and IGFs are expressed during early mammalian development, the functional roles oi these growth factors remain to be elucidated. The demonstration of hormonal action in vivo requires the presence of hormone, its receptor and evidence of postbinding effects at physiologic concentrations. There is evidence that insulin stimulates the incorporation of [3H]leucine into compacted eight-cell stage mouse embryos, although the concentrations oI the hormone used were supraphysiological (Harvey and Kaye, 1988). At physiologic levels of the hormone, insulin stimulates both DNA and RNA synthesis in the mouse embryo, beginning at the morula stage of development {Heyner et al., 1989}. It is of considerable interest to monitor changes in the preimplantation embryo's ability to transport and incorporate macromolecular precursors. Such changes can be considered to reflect alterations in synthetic rates of specific molecules during early development. In the present study we report the effects of physiologic levels of insulin, IGF-I, and -II on DNA, RNA, and protein synthesis during preimplantation stages of development in the mouse.
Regular progression of preimplantation development depends on the expression of specific genes, and on a continuous supply of energy, hormones, and growth factors. Recent studies in a number of vertebrate and invertebrate embryos have suggested that growth factors and their receptors may play important roles in cell proliferation and differentiation (reviewed in Mattson et al., 1989). Among the large number of growth factors, insulin and insulinlike growth factor- I and -II ~IGF-I and -II) are considered to be potentially important developmental factors because of their roles in stimulating biological responses. The peptides of the insulin family affect membrane transport (Yu and Czech, 1984), enzymatic activities (Verspohl et al., 1984), cell growth in a number of cell lines in vitro and in vivo, as well as early postnatal growth {Alexandrides and Smith, 1989). Experimental evidence supports the involvement of insulin and the IGFs in early development. Insulin binding to the cells of the preimplantation mouse embryo was first demonstrated using indirect immunofluorescence by Rosenblum et al. {1986). Using autoradiographic methods, Mattson et al. (1988) detected the expression of specific receptors that bind insulin and IGFs as early as
To whom correspondence should be addressed at Dept. of Ob/Gyn, Rm. 311A, John Morgan University of Pennsylvania Medical Center, 36th and Hamilton Walk, Philadelphia, PA 19104. 1043
1044
RAO ET AL.
MATERIALS AND METHODS
70
Animals and embryo recovery. Female mice (CD-1; 6- to 8-wk old: Charles River Breeding Laboratories) were injected with 5 IU pregnant mare's serum gonadotropin (PMSG; Sigma Chemical Co., St. Louis, MO) and 48 hrs later with 5 IU Human Chorionic Gonadotropin (hCG; Sigma). A single female was placed with a singly caged male ICD-1; Charles River Breeding Laboratories) overnight, and the presence of a vaginal plug the following morning was taken as evidence that mating had occurred. Embryos of different developmental stages (two-, eight-cell, morula and blastocyst) were flushed from the reproductive tract using M-2 medium (Whittingham, 1971). Embryos were cultured for at least 1 h in medium M-16 (Whittingham, 1971) at 37~ C in an atmosphere of 5% COs in humidified air. Both culture media M2 and M16 contained 0.4% bovine serum albumin (BSA), that had been screened previously to ensure that it w a s insulin-free. Uptake
and
incorporation
of labeled precursors.
Groups of 15 to 20 embryos (two-, eight-cell, morula and blastocyst} were flushed from the reproductive tract and cultured for at least 1 h in medium M16 containing 0.4% BSA in an atmosphere of 5% COs in air at 37 ~ C. They were then incubated for another hour in medium containing 0.4% insulin-free BSA, with or without 40 ng/ml of insulin (Eli Lilly, Indianapolis, IN), IGF-I (AmGen, Thousand Oaks, CA), or IGF-II (Eli Lilly). Embryos were then transferred to M16 containing 1 mCi/ml labeled methionine (L-methionine 3sS, 1196 Ci/mmol, ICN, Cleveland, OH) or 50 /~Ci/ml of [3H]thymidine (methyl [3H]thymidine, 83 Ci/mmol, Amersham, Arlington Heights, IL) or [3H]uridine (5,6 [3H]uridine 40 Ci/mmol, Amersham) in the presence or absence of growth factors, for another 2 h. Embryos were washed extensively in ice-cold M2, and total uptake and incorporation of labeled precursors were measured after precipitation with trichloroacetic acid (TCA) using methods described previously ~Heyner et al., 1989). Rndiolabeling of embryonic proteins. Groups of 20 blastocyst-stage embryos were cultured for at least 1 h in
25000
t ;~ 2oooo1 o
15000
g,oooo] 0
6O
o 30 2O
__=
I0
0
, 30
, 60
910
, 120
J 150
w 180
210
T i m e in M i n u t e s
FIG. 2. Effect of length of incubation on net accumulation of labeled methionine in intact mouse blastocysts. medium M16 containing 0.4% insulin-free BSA in an atmosphere of 5% CO2 in air at 37 ~ C. They were then incubated for another hour in medium containing 0.4% insulin-free BSA, with or without 40 ng/ml of insulin. Embryos were then transferred to M16 containing 1 mCi/ml [3~S]methionine, in the presence or absence of insulin for another 2 h. Embryos were washed extensively in ice-cold M2, denatured, and reduced by heating in Laemmli's sample buffer iLaemmli, 1970), containing 0.1% dithiothreitol IDTT) at 95 ~ C for 3 min and then subjected to 7.5% one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGEL Radiolabeled 35S-proteins were detected by fluorography (Bonner and Laskey, 1974) at --70 ~ C using Kodak X-OMAT AR film. Fluorograms were scanned using a LKB Laser densitometer for the comparison of peaks. RESULTS
Net uptake of ~sS-methionine. The net uptake of 3SS-methionine into groups of intact mouse blastocysts was measured using an increasing concentration of labeled methionine (0.25, 0.5, 1, 2, 5 mCi/ml) for 2 hrs. The incorporation of labeled precursor into intact blastocysts increased in a dose-dependent manner, reached a peak value at 1 mCi/ml and decreased thereafter ~Fig. 1). The net accumulation of ~'Smethionine over time, at a concentration of 1 mCi/ml labeled precursor, was also measured in blastocysts (Fig. 2). Incorporation gradually increased for 2 h and then reached a plateau. The studies described above showed that pS]methionine was taken up by mouse blastocysts, and the rate of incorporation was maximum in vitro when blastocysts were incubated for 2 h with 1 mCi/ml labeled methionine at 37~ C in an atmosphere of 5% COs in humidified air. Effect of growth factors on [~S/methionine uptake and incorporation. Studies in a variety of systems have
1
2
3 mCi/ml
4
5
6
FIG. 1. Kinetics of uptake of labeled methionine into intact mouse blastocysts.
indicated that insulin and the IGFs activate the production of proteins that are required for cell proliferation, as well as for maintenance. Table 1 shows the results of [3sS]methionine uptake and incorporation in different preimplantation stages of development in the
INSULIN FAMILY PEPTIDES IN MURINE EMBRYOGENESIS
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TABLE 1 EFFECT OF GROWTH FACTORS ON INCORPORATION OF LABELED METHIONINE BY MOUSE PREIMPLANTATION EMBRYOS" Control Stage
40 ng/ml
Incorporation
Total U ptake
Incor~)ration
Total Uptake
TwoceU Eightcell Morula Blastocyst
3512.3 -+ 8091.6-+ 13337.3 -+ 21889.7 -+
319.2 416.8 548.8 1408.6
13027.0 -+ 1454.4 1,5663.0-+ 690.3 26364.6 -+ 2264.1 52440.0 -+ 6321.5
3492.0 + 8163.3 -+ 15743.0 -+ 30244.2 -+
274.3 359.9 1261.1 1838.6
12473.6 4- 1199.6 17376.6_ 1909.7 28470.0 4- 1670.9 55529.7 + 4399.1
Twocell Eight cell Morula Blastocyst
3619.0-+ 8397.3 + 13621.3 -+ 24297.0 -+
510.0 1108.1 475.4 1250.4
15175.0-+ 598.1 18445.3 -+ 2668.9 24091.0 -+ 2457.5 53695.2 -4- 8564.5
3576.0-48382.3 -+ 13934.6 -+ 23735.5 -+
145.1 1028.9 463.1 1041.8
12473.6--4-116.6 17820.0 -+ 1910.2 25428.6 -+ 1580.1 51118.7 -+ 7255.8
Twocell Eightcell Morula Blastocyst
3709.3--+ 297.2 7711.0-+ 693.7 11644.3 -+ 420.2 23657.2 ___2225.8
15053.0-+ 325.1 17099.0-+ 773.4 22384.0 -+ 1818.2 41849.0 -+ 2784.9
3694.0-+ 381.9 7499.6-4- 290.2 12030.0 -4- 629.6 23669.0 -+ 1133.5
15321.3-+ 1310.0 16999.3-+ 488.0 22937.6 _+ 1025.0 39250.0 -+ 2699.7
"10 to 15 Embryos were used for each determination, which was performed 3 to 4 times. The data are expressed as mean + the standard error of the mean. Incorporation refers to the number of acid-insoluble cpm per embryo. Total uptake refers to the number of both acid soluble and acid insoluble cpm per embryo.
presence or absence of physiologic levels of insulin, I G F - I , and I G F - I I . These results show that incubation of embryos in insulin and I G F s had no effect on total accumulation of labeled precursors in the intact embryo. Insulin at concentration of 40 n g / m l increased the incorporation of labeled methionine into the TCA-insoluble precipitate at the morula and blastocyst stages of development. There was an 18 and 38% increase, respectively, in methionine incorporation when morula and blastocyst-stage embryos were incubated for "2 h with physiologic levels of insulin (Fig. 3). On the other hand, I G F - I and I G F - I I at similar physiologic levels had no effect on labeled methionine incorporation. T h e results of one dimensional S D S - P A G E autoradiography (Fig. 4) also showed that there was an overall stimulation of protein synthesis at the blastocyst stage when embryos were incubated with physiologic levels of insulin. The fluorogram shows clearly that there is a
50 40 20 ku
o
-10
quantitative increase in labeled precursor uptake in insulin-treated embryos, as compared with the same number of embryos processed identically, but without
~- INSULIN I t,, I
2-CELL
G .|
8-CELL
& I
|
MORULABLASTOCYST
FIG. 3. Mean percentage increase in the incorporation of labeled methionine in the presence of insulin or insulinlike growth factors in different stages of early development.
FIG. 4. Fluorogram of radiolabeled blastocysts incubated with (40 ng/ml) or without insulin.
1046
q)
RAO ET AL.
31
120 ,,-:
100
9
! 8O
= ;
-
INSULIN IGF-1
/o /
.
60 I
FIG. 5. Densitometric analysis of the fluorograms shown in Fig. 4. Comparison of area under the peaks (2,3,9) of radiolaheled mouse blastocyst proteins, from embryos incubated in the presence or absence of insulin revealed ratios of 1.40:1, 1.50:1, and 1.46:1, respectively. insulin. Laser scanning densitometer analysis of the fluorograms revealed that incubation in the presence of insulin resulted in increased area under the peaks {Fig. 5). Comparison of the area under the three major peaks of both the groups resulted in the ratios of 1.40:1, 1.50:1, and 1.46:1, respectively.
Effect of growth factors on the incorporation [3H]thymidine and p H]uridine. The incubation
of
of different developmental stages in insulin or insulinlike growth factors had no effect on total accumulation of labeled uridine or thymidine in intact embryos. However, our earlier studies had shown that insulin at a concentration of 4 ng/ml increased significantly the incorporation of labeled thymidine and uridine into the TCA-insoluble precipitate at the morula and blastocyst stages of development (Heyner et al., 1989). In contrast, the present study revealed that IGF-I and -II had very little effect on the incorporation of labeled thymidine {Fig. 6) or uridine {Fig. 7), when morula and blastocyststage embryos were incubated for 2 h at a concentration of 40 ng/ml. There was an 9 and 12% increase, respectively, in uridine incorporation, and 9 and 10% increase, respectively, in thymidine incorporation when morula and blastocyst-stage embryos were incubated for 2 h in medium containing 40 ng/ml IGF-I. Similarly, incubation of morulae and blastocysts in medium containing 40 ng/ml IGFII resulted in 2 and 10% increases, respectively, in uridine
30 I
:"
INSULIN IGF-1
/
/o
IGF-
20
~ 20
~ll
I
I
|
2-CELL 8-CELL MORULABLASTOCYST FIG. 6. Mean percentage increase in the incorporation of labeled thymidine in the presence of insulin or insulinlike growth factors in different stages of early development.
2O I
I
I
I
2-CELL 8-CELL MORULABLASTOCYST FIG. 7. Mean percentage increase in the incorporation of labeled uridine in the presence of insulin and insulinlike growth factors in different stages of early development. incorporation. Thymidine incorporation increased 12 and 11%, respectively, for the morula and blastocyst stages under similar conditions. Effects of the IGFs on thymidine and uridine incorporation were not significantly different from controls at the P < 0.05% level. D ISCUSSION Insulin is an anabolie hormone that can increase the overall rate of protein synthesis and regulate the preferential expression of specific proteins. IGF-I and I G F - I I are two single-chain polypeptide hormones that are structurally related to each other and proinsulin (Blundell and Humbel, 1980L Peptides of the insulin family have been shown to stimulate protein synthesis and mitogenic responses in many cell types. Their role in growth control during early development is not known. The transition of a single cell zygote to a multicellular blastocyst, during early mammalian development, involves a series of specific changes which remain to be elucidated. Previous studies utilizing immunofluorescence (Rosenblurn et al., 1986), autoradiography (Mattson et al., 1988), and high resolution electron microscopy (Heyner et al., 1989) have shown that mouse preimplantation embryos express receptors for insulin and insulinlike growth factors in a developmentally regulated manner. Receptors could he detected beginning at the late eight-cell stage, and increased in density through the blastocyst stage. The present experiments were designed to investigate whether physiologic levels of insulin and IGFs affected the rate at which preimplantation mouse embryos incorporated macromolecular precursors from their environment in vitro. Preimplantation mouse embryos are capable of synthesizing nucleic acids (Piko, 1970; Piko and Clegg, 1982) and it is well known that changes in the pattern of protein synthesis occur during the preimplantation development of mouse embryos (Epstein and Smith, 1974). The studies described above showed that labeled methionine, uridine, and thymidine were taken up by two-, eight-cell, morula and blastocyststage embryos in vitro, and as development proceeded the rate of this process increased. This increased incorporation may reflect an increase in metabolic
INSULIN FAMILY PEPTIDES IN MURINE EMBRYOGENESIS activity as well as an increase in cell surface area. Incubation of embryos in insulin and I G F s at physiologic levels had no influence on total accumulation of precursors, showing that the growth factors did not affect the rate of macromolecular transport into intact embryos. However, insulin significantly increased the incorporation of labeled precursors into TCA-insoluble material in early embryos, indicating that this effect was specific for stimulation of maeromolecular synthesis. DNA, RNA, and protein synthesis were significantly increased when morula and blastocyst-stage embryos were incubated in physiologic levels of insulin. In contrast, I G F - I and I G F - I I did not stimulate incorporation of precursors under the same experimental conditions. A quantitative increase in labeled methionine uptake in insulin-treated blastocyst-stage embryos was also observed in onedimensional SDS-PAGE fluorograms (Fig. 4). Preliminary studies using high-resolution, two-dimensional gel eleetrophoresis of blastocysts incubated for 2 h in the presence or absence of physiologic levels of insulin have shown evidence of a generalized increase in the level of protein synthesis. In addition, there is evidence of some posttranslational modifications and expression of novel minor polypeptides (Rao, Heyner, Collins and Matsehiusky, unpublished observations). Results of other investigators support our findings; thus, Sellens et al., (1981) observed that in the absence o! serum, but in the presence of amino acids, there was only a modest increase in the protein content of cultured blastocysts, suggesting that in addition to an exogenous supply of amino acids other factors are required for optimal growth. Recently, Travers et al., {1989) examined the effect of hypoinsulinemia on rat embryonic growth and development in culture, and observed that addition of physiologic amounts of insulin to insulin-depleted serum restored growth levels to those of control embryos. Their in vitro experiments strongly suggest that the early mammalian embryo has an absolute requirement of insulin, even in the presence of insulinlike growth factors. Although insulin stimulated significant increases in DNA, RNA, and protein synthesis, it is known from a number of studies that insulin is not an absolute requirement for in vitro development to the blastocyst stage. However, embryos grown in vitro lag developmentally behind their in vivo counterparts (Bowman and McLaren, 1970). Further, embryo development in vitro cannot be evaluated merely by the morphologic development of the blastocyst, but rather by the maintenance of its subsequent developmental viability and establishment of pregnancy. The role of insulin may be to stimulate overall preimplantation development to ensure a successful implantation. The lack of an effect of I G F - I , is intriguing, particularly since the receptor for this peptide is expressed around the same developmental period as the insulin receptor. However, I G F - I has been postulated to act as a proliferative agent on cellsof the mesodermal lineage in mammals (Beck et ai., 1987), and it is entirely conceivable that the real importance of this peptide in early development occurs after implantation, during organogenesis. IGF-II ligand has been detected as early as the late two-ceU stage in the mouse ~Rappolee et al., 1990), and inasmuch as this peptide is produced in an autocrine manner, it is not surprising that
1047
addition of exogenous ligand does not have a significant effect on metabolism. On the other hand, preliminary studies suggest that when embryos are incubated in the presence of antisense, but not sense oligonucleotides for I G F - I I coding sequences, there is retardation in the rate of preimplantation development in vitro (Werb and Rappolee, 1990). Clearly, more studies are needed to reveal the precise roles played by peptides of the insulin family during the early stages of mammalian development. REFERENCES 1. Aiexandrides, T. K.; Smith, R. J. A novel fetal insulin-llke growth factor (IGF) I receptor. J. Biol. Chem. 264:12922-12930; 1989. 2. Beck, F.; Samani, N. J.; Peuschow, J. D., et al. Histocbemlcal localization of IGF-I and -II mRNA in the developing rat embryo. Development 101:175-184; 1987. 3. Blundell, T. L.; Humbel, R. E. Hormone families; pancreatic hormones and homologous growth factors. Nature 287:781-787; 1980. 4. Bonner, W. M.; Laskey, R. A. A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:83-88; 1974. 5. Bowman, P.; McLaren, A. Viability and growth of mouse embryos after in vitro culture and fusion. J. Embryol. Exp. Morphol. 23:693-704; 1970. 6. Epstein, C. J.; Smith, S. A. Electrophoretic analysis of proteins synthesized by preimplantation embryos. Dev. Biol. 40:233-244; 1974. 7. Harvey, M. B.; Kaye, P. L. Insulin stimulates protein synthesis in compacted mouse embryos. Endocrinology 122:1182-1184; 1988. 8. Heyner, S.; Rao, L. V.; Jarett, L., et al. Preimplantation mouse embryos internalize maternal insulin via receptor mediated endocytosis: patterns of uptake and functional correlations. Dev. Biol. 134:48-58; 1989. 9. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685; 1970. 10. Mattson, B. A.; Rosenblum, I. Y.; Smith, R. M., et al. Autoradiographlc evidence for insulin and insulin like growth factor binding to early mouse embryos. Diabetes 37:585-589; 1988. 11. Mattson, B. A.; Chambers, S. A.; de Pabio, F. Comparative aspects of the insulin and the insulin receptor throughout evolution. In: Rosenblum, I. Y.; Heyner, S., eds. Growth factors in mammalian development. Boca Raton, FL: CRC Press; 1989:47-70. 12. Piko, L. Synthesis of macromolecules in early mouse embryos cultured in vitro: RNA, DNA and a polysaccharide component. Dev. Biol. 21:257-279; 1970. 13. Piko, L.; Clegg, K. B. Quantitative changes in total RNA, total poly (A) and ribosomes in early mouse embryos. Dev. Biol. 89:362-378; 1982. 14. Rappolee, D. A.; Sturm, K. S.; Schultz, G. A., et al. The expression of growth factor ligands and receptors in preimplantation mouse embryos. In: Heyner, S.; Wiley, L. M., eds. Early embryo development and paracrine relationships. New York: Wiley-Liss; 1990:11-26. 15. Rosenblum, I. Y.; Mattson, B. A.; Heyner, S. Stage specific insulin binding in mouse preimplantation embryos. Dev. Biol. 116:261-263; 1986. 16. Sellens, M. H.; Stein, S.; Sherman, M. I. Protein and free amino acid content in preimplantation mouse embryos and in blastocysts under various culture conditions. J. Reprod. Fertil. 61:307-315; 1981. 17. Travers, J. P.; Pratten, M. K.; Beck, F. Effects of low insulin levels on rat embryonic growth and development. Diabetes 38:773-778; 1989. 18. Verspohl, E. J.; Roth, R. A.; Vigneri, R., et al. Dual regulation of glycogen metabolism by insulin and insulin like growth
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factors in human hepatoma cells {Hep- G2). J. Clin. Invest. 74:1436-1443; 1984. 19. Werb, Z.; Rappolee, D. A. The expression and function of growth factors during early development of mouse embryos. J. Cell. Biochem. {Suppl)14E:48;1990.
20. Whittingham, D. G. Culture of mouse ova. J. Reprod. Fertil. ~Suppl).14:7-21; 1971. 21. Yu, K. T.; Czech, M. P. Tyrosine phosphorylation of the insulin receptor B-subunit aetivates the receptor associated tyrosine kinase activity. J. Biol. Chem. 259:5277-5286; 1984.
This work was supported by grant HD 23511 from the National Institutes of Health, Bethesda, MD.
