1 Department of Physiology and Pharmacology, The University of Queensland, Brisbane Qld, Queensland, 4072 and 2 Australian Quarantine Inspection Service, Canberra, Australia
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Abstract |
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Key words: fetus/growth/insulin/preimplantation embryo/protein
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Introduction |
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In the mouse, functional receptors for insulin and IGF-I are expressed at the 8-cell preimplantation stage, during compaction; well before the fetus develops (Harvey and Kaye, 1988, 1990
, 1991a
, 1992a
, b
; Mattson et al., 1988
; Heyner et al., 1989
; Telford et al., 1990a
,b
; Rappolee et al., 1992
; Smith et al., 1993
). From this stage, which marks the beginning of the embryonic differentiation leading to blastocyst formation (Gardner and Johnson, 1975
), many parameters of embryonic physiology may be regulated by exogenous insulin or IGF-I (for reviews, see Kaye et al., 1992; Schultz and Heyner, 1993; Kaye and Harvey, 1995; Kaye, 1997). Human oocytes express insulin and IGF receptors (Lighten et al., 1997
), which respond to IGF-I (Lighten et al., 1998
). These hormonally induced modifications to metabolism and cell physiology have the net effects of increasing mitotic rate, cell proliferation and morphogenesis. Preimplantation embryos do not synthesize insulin or its mRNA (Telford et al., 1990a
,b
; Kaye, 1997
; Lighten et al., 1997
) but have access to maternal insulin in vivo via oviduct and uterine fluids (Heyner et al., 1989
). Thus in-vitro preimplantation embryos would be deprived of this maternal hormone.
Morphological development and cell proliferation during preimplantation embryogenesis are retarded in vitro when insulin is not present (Bowman and Mclaren, 1970; Gardner and Kaye, 1991
). Insulin increased the rate of compaction and blastocyst formation in vitro and the resulting blastocysts contained about 25% more cells, all of which were located in the inner cell mass (Harvey and Kaye, 1990
). This suggests that the size of the pool of fetal progenitor cells in the mouse embryo is under control of maternal insulin. Since the proportion of cells in the inner cell mass is a determinant of fetal growth, it is likely that preimplantation access to maternal insulin is required for optimal fetal growth.
Maternal protein nutrition is another determinant of fetal growth (Koshy et al., 1975) which also affects preimplantation development (Caro and Trounson, 1984
; Gardner and Kaye, 1991
; Lane and Gardner, 1994
). In vivo, maternal serum protein is taken up by the morulae and blastocysts (Glass, 1963
), via endocytosis and in vitro the exogenous albumin is metabolized in lysosomes (Pemble and Kaye, 1986
; Dunglison et al., 1995
). Endocytic rate increases in the absence of protein (Dunglison and Kaye, 1995
) suggesting that metabolic demand regulates protein supply to the embryo through endocytosis. The importance of this maternal protein is emphasized by the observation that albumin increased cell proliferation and morphogenesis of preimplantation embryos in vitro (Gardner and Kaye, 1991
). This suggests that the maternal albumin present in oviductal and uterine secretions is a natural component of the preimplantation milieux, required for natural development.
Do the increased inner cell mass population, stimulated anabolism and developmental acceleration generated before implantation by maternal insulin or albumin propagate into fetal development? This hypothesis was tested in wild type embryos, by taking control of the preimplantation environment in vitro and then transferring them to recipient mice to complete gestation and assessing effects on fetal growth.
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Materials and methods |
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Model formulation and statistical analyses
Eight factors known to influence weight (weight modifying factors) were selected from studies of mice (McLaren and Michie, 1960; Barr et al., 1970
) and rats (Bruce and Norman, 1975
): the sex of the fetus (S); the uterine horn in which the fetus was found (H); the number of fetuses in the horn (nH); the number of fetuses in the litter (nL); the position in the horn (P); the age of the fetus since fertilization (A); the recipient effect (M) and the treatment (T). The equation describing the model is:
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The generalized linear interactive modelling system (GLIM; Nelder and Wedderburn, 1972) was used on the basis that modifying factors were linearly dependent. A nonorthogonal unbalanced analysis of variance (ANOVA) was used to analyse the results and describe the source of variation. In the analysis of the contribution of albumin to the variance in fetal mass (Table II), one degree of freedom was lost. Three concentrations of albumin were used: 0, 0.17 µmol/l and 15 µmol/l, but only two were tested in the ANOVA, because E20 fetal units from cultures supplemented with 0.17 µmol/l albumin were not examined (Table II
). These data were absorbed or described by modifying factors that precede them in the analysis (not shown). The variation attributed to albumin was obtained from the interactions between insulin and albumin and albumin and fetal stage (Table III
). Mean fetal mass and placental mass were obtained from the data and the standard error calculated from the variance. Protected Student's t-test, or Fisher's t-test examined differences in weight of fetal units within a data set where the variance comes from the ANOVA. Percentages were transformed to radians before analysis by Student's t-test, but untransformed data are reported in tables and figures. The skeletal development data were not normally distributed and could not be normalized by transformation, so these are described by the median, mode and range to describe the distribution of results. Also because of the non-normal distribution, the non-parametric MannWhitney U test was used to assess the effects of albumin and insulin supplementation of culture medium on the distribution of numbers of ossified vertebrae and bones of the lower limbs of the E19 fetus. Correlation between the fetal mass and the number of ossified bones was tested by Spearman's rank test.
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Results |
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Fetal age
Naturally, fetal mass increased approximately exponentially from E17 to E19, with about a 37% increase per day (doubling time: 3.71.6 days; Figure 1; Table I
). Embryos that were cultured grew more slowly, gaining about 28% increase in fetal mass between E19 and E20. However by E20 all fetuses from cultured embryos, irrespective of access to albumin or insulin had reached a mass of 1.6 g (Table II
). This was greater than the birth weight of naturally gestated embryos (1.542 ± 0.014; n = 129 pups, 8 litters) and also of E19 fetuses developing from in-vivo developed blastocysts directly transferred into day 4 recipient uteri (Table I
).
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Insulin increased fetal mass by 4.6% in the absence of protein (P < 0.001) and 4.1% in the presence of equimolar protein, but this latter difference was not statistically significant (probably because of the low number of embryos involved; Tables III and IV). There was no added benefit of combining 0.17 µmol/l insulin with 15 µmol/l albumin; the combination produced the same 45% increase as each supplement alone. In summary, insulin at 0.17 µmol/l was as effective as 100 times more albumin, causing a 46% increase in fetal mass (Figure 2
), but 0.17 µmol/l albumin was inhibitory to fetal growth. Insulin treatment stimulated fetal growth in the absence of albumin and possibly in the presence of equimolar albumin, but was no more beneficial than the 100-fold higher concentration of albumin.
Natural pregnancy versus embryo transfer, placental weight
Cultured blastocysts (E4) transferred to day 3 pseudopregnant uteri reached a fetal mass of about 1.6 g if allowed to gestate until E20 (Table II). This fetal mass was greater than that of fetuses arising from direct blastocyst (E4) transfer to day 3 pseudopregnant uteri (Table I
, P < 0.01), E19 naturally gestated fetuses (Table I
, P < 0.01) or birth weight (see above).
Thus the fetuses developing from blastocysts with access to insulin or 15 µmol/l albumin grew as rapidly as exactly comparable direct transfer blastocysts. Provision of the missing maternal factors, insulin and/or albumin had rescued the embryos from the trauma of culture in simple medium in vitro. Supplementation with albumin or insulin did not affect the placental mass but the extra day of gestation caused a 19% decrease in placental weight, E19 (all treatments): 0.16 ± 0.011, n = 222; E20 (all treatments): 0.13 ± 0.008, n = 58, P < 0.01.
Skeletal development
Although there was a correlation between the amount of ossification of the skeleton and fetal mass for embryos which developed with 0 or 0.17 µmol/l albumin, this link disappeared when the embryos were provided with 15 µmol/l albumin, which tended to reduce the amount of ossification, but not significantly (Table V). Despite the apparent increased skeletal formation in the fetuses developing from blastocysts provided with insulin (Figure 2
); provision of albumin or insulin alone did not affect ossification of the lower limbs or vertebrae. The patterns of ossification of most skeletal parameters were nearly symmetrical, with the mode slightly greater than the median, except in fetuses obtained from blastocysts provided with insulin (Table V
). The forelimbs of a few of these fetuses were extensively ossified, but there were also fetuses with little ossification. Insulin exposed blastocysts developed to fetuses with more extensively developed limbs than those which had been exposed to 15 µmol/l albumin (P < 0.05, MannWhitney U-test), but addition of albumin to insulin inhibited this stimulation (Table V
, P < 0.01; MannWhitney U-test).
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Discussion |
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Role of insulin
Provision of insulin to the preimplantation embryo enhanced subsequent fetal growth rate. This acceleration of fetal growth is likely to arise from the increased mitotic rate and morphological development of these blastocysts induced by insulin or albumin (Gardner and Kaye, 1991), because the increase in cells induced by insulin was solely allocated to the inner cell mass (Harvey and Kaye, 1990
). These inner cell mass cells are the progenitors of the fetus, whilst those of the trophectoderm contribute only to the placenta. Insulin stimulated inner cell mass proliferation and fetal growth, but not trophectoderm proliferation or placental growth, therefore fetal growth appears to relate to inner cell mass cell number or inner cell mass/trophectoderm cell number ratio and the rates of cell proliferation prior to implantation. Support for this proposal comes from studies of blastocysts from experimentally diabetic mice, which contained fewer cells and exhibited reduced metabolic rate (Beebe and Kaye, 1991
). A smaller inner cell mass population has been proposed to cause reduced stem cell population leading to the failure of embryogenesis in parthenogenetic embryos (Kaufman et al., 1977
, 1984
). The present data suggest that reduction in inner cell mass number leads to retarded fetal growth. All of the mammals which have been examined express the insulin receptor transcript by E3 at the latest, and in several the transcript is present in the oocyte (Kaye, 1997
). Yet none expresses insulin, which is available through maternal secretion into the fluids surrounding the embryo at this time (Heyner et al., 1989
). The expression of the insulin receptor, but not its ligand, in preimplantation embryos of diverse species, implies a critical role for maternal insulin in early embryonic development.
Insulin may act through the insulin receptor or the closely related type I IGF receptor. Insulin is effective on blastocysts from about 0.3 pmol/l with an EC50 about 1 pmol/l (Harvey and Kaye, 1988, 1990
). However, insulin may also bind to, and activate, the blastocyst type I IGF receptor from concentrations of about 5 nmol/l and EC50 of about 50 nmol/l (Harvey and Kaye, 1991b
, 1992b
). Thus the fetal growth stimulus from 0.17 µmol/l insulin may have occurred through binding to either or both of the key insulin binding receptors present on the preimplantation mouse embryo. Mice lacking the insulin receptor die shortly after birth (Accili et al., 1996
). Mice lacking the insulin receptor substrate 1 (IRS1) protein, substrate of the insulin and type 1 IGF receptor tyrosine kinases (Araki et al., 1994
) (which is present in wild type peri-implantation embryos; Puschek et al., 1998) survive and are severely growth retarded (Tamemoto et al., 1994
). This is similar to humans lacking the insulin receptor (Taylor, 1992
). These observations support mediation of insulin's stimulus by the embryonic insulin receptor. However, type I IGF receptor deficient mice also display fetal growth retardation (Baker et al., 1991; Liu et al., 1991) so either or both receptors might contribute to the fetal growth. Recently it has been suggested that insulin acts via embryonic type II IGF/mannose 6 phosphate receptors to regulate fetal growth in mice, on the basis of double deletion of the insulin receptor and the type I IGF receptor. These mice are severely growth retarded at E13.5 and exhibit delayed skeletal development (Louvi et al., 1997
). It is difficult to isolate preimplantation requirements from those of later fetal development in these mutant mouse models. Interestingly the present studies support a particular requirement for insulin in skeletal development, similar to the gene deletion model. The in-vitro approach using wild type embryos has enabled restriction of the insulin deficiency to the preimplantation phase (E2-E4), avoiding perturbation of prior gametogenesis and subsequent gestation in utero which may confound observations of genetically modified mice.
Insulin was active before compaction (E3) generating more undifferentiated cells with higher mitotic indices in uncompacted morulae (Gardner and Kaye, 1991). Albumin did not have this effect on proliferation of undifferentiated cells prior to compaction. Since provision of insulin and albumin to the preimplantation embryo has the same effect on fetal growth, it follows that the increased number of undifferentiated cells generated by insulin before compaction does not contribute to the fetal growth enhancement of insulin. Rather, the increase in differentiated inner cells produced by insulin during E3-E4 accelerates fetal growth supporting the conclusions above.
Albumin
Similar benefits to fetal growth arise from preimplantation embryonic access to 15 µmol/l albumin. This may not be surprising since the albumin used was a relatively crude preparation in which both negative (Caro and Trounson, 1984) and positive (Ueno et al., 1984
; Kane, 1985
) affectors of preimplantation development have been reported. There may be some mildly toxic component of the albumin fraction used, since 0.17 µmol/l albumin inhibited fetal growth and both concentrations inhibited the effects of insulin on skeletal development. However the beneficial effects of 15 µmol/l albumin may relate to protein nutrition (Koshy et al., 1975
). After compaction endocytosis is activated (Reeve, 1981
) and would provide significant amino acid nutrients by lysosomal degradation of maternal protein (Pemble and Kaye, 1986
; Dunglison and Kaye, 1993
, 1995
). It is therefore probable that the stimulation of preimplantation cell division and subsequent fetal growth caused by albumin arise from increased nutrient supply via endocytosis. Supporting this conclusion, supply of free amino acids also increased the developmental potential of blastocysts in vitro (Lane and Gardner, 1994
, 1998
). Interestingly, insulin may produce a similar anabolic metabolic/nutrient status by increasing protein synthesis (Harvey and Kaye, 1988
), endocytosis and lysosomal degradation of exogenous protein and inhibiting breakdown of endogenous protein (Dunglison and Kaye, 1993
; Dunglison et al., 1995
). Perhaps these anabolic changes have the same net effect on protein nutrition or metabolism in the preimplantation embryo as providing moderate concentrations of albumin, both resulting in optimal inner cell mass cell number and consequent fetal growth rates. This aspect is relevant to human in-vitro fertilization (IVF) procedures which are trending towards longer culture with media containing pooled serum albumin fractions. These data suggest that concentration of albumin should be maintained at least at 15 µmol/l to optimize subsequent fetal growth.
Placental growth and trophectoderm
Placental growth was not affected by preimplantation supply of either insulin or albumin. Yet this tissue develops from the trophectoderm, which like other cells of the blastocyst expresses insulin receptors. In addition to providing the transcytotic pathway for insulin to reach the inner cell mass (Heyner et al., 1989), the trophectoderm participates in the overall resetting of embryonic physiology induced by insulin through enhanced endocytic activity (Dunglison and Kaye, 1993
; Dunglison et al., 1995
) and protein anabolism (Kaye et al., 1992
). Thus the trophectoderm is important in the blastocyst's response to both insulin and albumin, by providing metabolic pathways to support the development of the inner cell mass and therefore the fetus.
The fetuses developing from direct synchronous transfer were of similar weight to those which gestated naturally, indicating that the traumas of anaesthesia and surgery do not impact on fetal growth as previously reported (Harlow and Quinn, 1979). Comparison of the natural fetuses with those arising from blastocysts (E4) transferred to day 3 uteri reveals the fetal growth retardation caused by asynchronous transfer possibly reflecting the maternal control of implantation (Psychoyos, 1973
).
The preimplantation effects of insulin that include accelerated morphological progression increase in the size of the inner cell mass cell population and protein anabolism, optimize blastocyst physiology for successful early implantation and embryogenesis leading to accelerated fetal growth. Fetal growth acceleration also arises from the increased protein nutrient supply provided by albumin, which produces similar effects on preimplantation embryo cell proliferation. The demonstrated roles of embryonic growth factors in control of invasive proteolytic activity required for implantation (Harvey et al., 1995), likely to be produced in greater amount from larger more advanced blastocysts developing in the presence of maternal insulin and albumin, support this hypothesis. Human embryos express insulin and IGF-I receptors and respond to IGF-I similarly to mouse embryos; it is therefore likely that inclusion of the easily obtained alternate ligand insulin in media for blastocyst culture may lead to improved outcomes in terms of fetal growth and subsequent morbidity for these procedures.
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Acknowledgments |
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Notes |
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References |
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Submitted on May 17, 1999; accepted on September 9, 1999.