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Endocrinology Vol. 142, No. 3 1309-1316
Copyright © 2001 by The Endocrine Society

ARTICLES

Insulin-Like Growth Factor I (IGF-I) and Long R3IGF-I Differently Affect Development and Messenger Ribonucleic Acid Abundance for IGF-Binding Proteins and Type I IGF Receptors in in Vitro Produced Bovine Embryos1

Katja Prelle2, Miodrag Stojkovic2, Katja Boxhammer, Jan Motlik, Dagmar Ewald, Georg J. Arnold and Eckhard Wolf

Institute of Molecular Animal Breeding (K.P., M.S., K.B., D.E., E.W.) and Laboratory of Molecular Biology (G.J.A.), Gene Center, Ludwig Maximilian University, 81377 Munich, Germany; and Institute of Animal Physiology and Genetics, Czech Academy of Sciences (J.M.), 27721 Libechov, Czech Republic

Address all correspondence and requests for reprints to: Prof. Dr. Eckhard Wolf, Institute of Molecular Animal Breeding, Gene Center, Ludwig Maximilian University, Feodor Lynen Strasse 25, 81377 Munich, Germany. E-mail: ewolf{at}lmb.uni-muenchen.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The insulin-like growth factor (IGF) system is a complex network, including ligands (IGF-I and -II), binding proteins (IGFBP-1 to -6), and receptors, of which the type I IGF receptor (IGF-I-R) is important for transmission of most biological effects of IGFs. As IGFs are secreted in large amounts by the female reproductive tract, it has been hypothesized that maternal IGFs may affect embryonic growth and differentiation in a fine-tuned manner, involving modulation of IGF effects by embryonic IGFBP and IGF-I-R expression. To address this point, we cultured in vitro produced bovine embryos in a chemically defined culture system in the presence (100 ng/ml) of recombinant human IGF-I, long R3IGF-I (LR3), or without IGF supplementation (control). The affinity of LR3 to IGFBPs measured by competition assays and Western ligand blots is at least 3 orders of magnitude lower than that of IGF-I. LR3 was most efficient in stimulating early embryonic cleavage, whereas further development was most potently supported by IGF-I. Total cell numbers of blastocysts were highest in the presence of LR3 (105 ± 4), followed by IGF-I (96 ± 5), and the control group (91 ± 3; P < 0.05). Differential cell staining of blastocysts revealed that these differences were mainly represented by trophectoderm cell numbers. Analysis of messenger RNA (mRNA) expression for IGFBPs and IGF-I-R was performed by RT-real-time PCR, using expression of the nonregulated housekeeping gene glyceraldehyde-3-phosphate dehydrogenase for normalization. Embryonic IGFBP-2 mRNA levels in the LR3 treatment group were 1.7-fold (P < 0.001) and 2.8-fold (P < 0.001) higher than those in the IGF-I and control groups, respectively. IGFBP-5 mRNA levels were about 2-fold (P < 0.001) elevated in both IGF treatment groups, with slightly (P < 0.05) higher levels in IGF-I- than in LR3-treated embryos. Similarly, IGFBP-3 mRNA abundance was increased (P < 0.05) in embryos from the IGF-I vs. the LR3 culture system. IGF-I-R mRNA levels were reduced by IGF-I (80% of control; P < 0.01), but increased by LR3 (1.3-fold vs. control; P < 0.001). These data show that the affinity for IGFBPs of IGF peptides is relevant for their effects on preimplantation embryos and affects different parameters, i.e. development, cell numbers, and mRNA expression for components of the IGF system, in different directions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE INSULIN-LIKE growth factor (IGF) system comprises an increasingly complex network of ligands (IGF-I and IGF-II), receptors [type I IGF receptor (IGF-I-R) and type II IGF/cation-independent mannose-6-phosphate receptor (IGF-II-R)], binding proteins (IGFBP-1 to -6 and IGFBP-related proteins), and specific proteases affecting their biological activity, i.e. regulation of cell growth and differentiation (for review, see Refs. 1, 2, 3). Components of the IGF system are widely expressed in the female reproductive tract (4, 5, 6, 7, 8, 9), with maximum expression in the oviduct of IGF-I, IGF-II, IGF-I-R, and IGFBP-3 messenger RNAs (mRNAs) in the period when gametes and embryos are in transit, eventually creating an optimum environment for early embryonic growth and metabolism (for review, see Ref. 10).

Furthermore, expression of components of the IGF system has been detected in preimplantation embryos from a variety of mammalian species, including mouse (11, 12), cattle (4, 13), and water buffalo (14). However, targeted inactivation of the genes coding for IGF-I (Igf1), IGF-II (Igf2), and IGF-I-R (Igf1r) in mice resulted in an obvious phenotype (fetal growth retardation) only in the second half of pregnancy (15, 16, 17, 18). These findings suggest that IGF-I and IGF-II are not absolutely required during early embryonic development, although detailed analyses for quantitative effects have not been conducted.

On the other hand, numerous studies demonstrated a positive effect of IGF supplementation of culture medium for early embryos from various species, including mouse (19, 20) and rabbit (21). For bovine embryos, variable effects of IGF supplementation have been described depending on the dose and the respective culture system used. Whereas Flood et al. (22) could not show an advantage of either long R3IGF-I (LR3) or IGF-II supplementation in bovine embryo culture, several other studies demonstrated a positive effect of IGF-I on the development of bovine (23, 24) or buffalo embryos (25) in serum-containing culture systems. In a serum-free, chemically defined culture medium, both insulin and IGF-I stimulated the development of in vitro produced bovine embryos to the morula stage on day 5 and increased cell numbers, but not the proportion of blastocysts (26). These effects could be blocked by the anti-IGF-I-R-antibody {alpha}IR-3, demonstrating that insulin and IGF-I act via the IGF-I-R on early bovine embryos (27).

In many biological systems, the bioavailability and effects of IGFs are modulated by IGFBPs, e.g. by competition with IGF receptors for IGFs (for review, see Ref. 2). Expression of mRNAs coding for IGFBP-2, -3, and -4 was detected by RT-PCR analysis of large pools (n > 50) of bovine embryos throughout development to blastocysts, whereas IGFBP-5 transcripts were only detectable in blastocysts. In contrast, IGFBP-1 and -6 mRNAs were not detectable in early bovine embryos (4). This observation invited the concept that the effects of maternally derived IGFs might be modulated by embryonic IGFBPs to support bovine preimplantation development (4). A recent study of horse embryos indicated that the horse conceptus secretes significant amounts of IGFBP-3 toward the surrounding capsule and speculated on a role for IGFBP-3 to support the developing conceptus (28). However, to date there are no experimental data demonstrating that expression of IGFBPs in preimplantation embryos is regulated by IGFs or that interactions between IGFs and IGFBPs in these early embryos are functionally relevant.

To address this point, we used IGF-I and the synthetic analog LR3, which is characterized by a more than 1000-fold reduced affinity for IGFBPs (29), as supplements for the culture of bovine embryos in a chemically defined system. In addition to proportions of embryonic development and cell numbers of blastocysts, we studied embryonic mRNA levels for IGFBP-2, -3, and -5 as well as IGF-I-R transcript levels. This was performed for the first time using sensitive RT-real-time PCR assays in small pools of two blastocysts. Our study demonstrates that the IGF peptides used affect embryonic development, cell numbers, and mRNA expression of components of the IGF system differently, which may be due to modulating effects of embryonic IGFBPs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
In vitro production of bovine embryos
Bovine embryos were produced as previously described (30) with some minor modifications. Briefly, follicles with a diameter of 2–8 mm were aspirated from the ovaries of slaughtered cows using a 20-gauge needle and a vacuum pressure of approximately 13 kPa. The cumulus-oocyte complexes (COCs) were collected in a 50-ml centrifuge tube and washed twice with preincubated (39 C; 5% CO2) tissue culture medium 199 (Life Technologies, Inc., Eggenstein, Germany) supplemented with 10% (vol/vol) heat-inactivated serum from cows at estrus. Only COCs with a complete dense cumulus and a dark, evenly granulated cytoplasm were selected for in vitro maturation. The COCs were washed in medium 199 supplemented with 10% estrous cow serum and 10 µg/ml bovine FSH and LH (Sioux, Sioux Center, IA) and matured in this medium for 24 h at 39 C in an atmosphere of 5% CO2 in air and maximum humidity. After maturation, the COCs were maintained in a Tyrode’s-albumin-lactate-pyruvate medium containing 6 mg/ml BSA, 10 µg/ml heparin (both from Sigma, Deisenhofen, Germany), and frozen/thawed semen (106 spermatozoa/ml) that had been subjected to a swim-up procedure. COCs were maintained in this medium for 18 h under the same conditions as those used for in vitro maturation. For in vitro culture, cumulus cells were removed from presumptive zygotes by vortexing (120 sec) and gentle pipetting. Then groups of 30–35 presumptive zygotes were washed three times and cultured in 400 µl synthetic oviduct fluid supplemented with 3 mg/ml polyvinyl alcohol (PVA; Sigma; control group) and with 100 ng/ml IGF-I or LR3 (Schützdeller, Tübingen, Germany) and covered with 400 µl equilibrated paraffin oil (Merck & Co., Darmstadt, Germany) for a period of 8 days. The cleavage rate (proportion of embryos at the five- to eight-cell stage) was evaluated 66 h postinsemination (hpi). The numbers of morulae and blastocysts were recorded 162 hpi, and total numbers of blastocysts were determined 186 hpi. For each experiment three replicates were performed.

Differential cell staining of blastocysts
Equal numbers of embryos recovered 186 hpi (18–22 expanded blastocysts/treatment group) were subjected to differential cell staining as described previously (31). Briefly, blastocysts were washed several times in Dulbecco’s PBS to remove excess protein. Blastocysts were treated with 0.5% (wt/vol) pronase (protease, type XXV; Sigma) in PBS for 3–5 min to dissolve the zona pellucida. Then, blastocysts were incubated in a 1:2 dilution in PBS of rabbit antiserum raised against recombinant bovine interferon-{tau} and other trophoblastic secretions (32) for 45 min at 39 C in a humidified atmosphere of 5% CO2 in air. Subsequently, embryos were washed five times in PBS warmed at 39 C and then incubated in PBS supplemented with 5% (vol/vol) guinea pig complement (ICN Biochemicals, Inc., Costa Mesa, CA) and 50 µg/ml propidium iodide (Sigma) for 45 min at 39 C in a humidified atmosphere of 5% CO2. After this step, blastocysts were washed again in PBS and then placed in cold absolute ethanol (Merck & Co., Ismaning, Germany) containing 25 µg/ml of the fluorochrome bisbenzimide (Riedel-de Haen AG, Hannover, Germany) for 30 min at 4 C. Finally, embryos were washed in absolute ethanol, mounted in undiluted glycerol (Merck & Co.), and squashed on a glass slide. The stained embryos were observed using a fluorescent microscope (Axiovert 135, Carl Zeiss, Jena, Germany) with a mercury HBO lamp under transmittance illumination, a UV excitation filter of 365 nm, and a barrier filter of 420 nm.

RT-real-time PCR analysis of embryonic IGFBP and IGF-I-R mRNA expression
Morphologically intact embryos generated in vitro and cultured in the different media were collected on day 8 as expanded blastocysts. After washing three times in PBS containing 0.1% PVA, they were stored in pools of two embryos at -80 C in a minimum volume (5 µl or less) of PBS and 0.1% PVA until use.

Total RNA was isolated according to a modified protocol of McDougall (personal communication) by adding 100 µl embryo extraction buffer [0.2 M NaCl, 0.025 M Tris (pH 7.4), 0.01 M EDTA in ribonuclease- and deoxyribonuclease (DNase)-free diethylpyrocarbonate-treated water], 75 µl chloroform-isoamyl alcohol (24:1), 75 µl RNA grade phenol, and 0.1 ng synthetic A oligonucleotides as a carrier to the frozen embryos, followed by vortexing for 30 sec to homogenize the samples. The homogenate was transferred to long (45 mm, diameter 4 mm) 250-µl centrifuge tubes (Milian, Geneva, Switzerland), facilitating the removal of the top phase later on, and were centrifuged (16,000 x g) at room temperature for 15 min. The aqueous (top) phase was transferred to a new tube, mixed with 100 µl chloroform/isoamyl alcohol (24:1), vortexed, and centrifuged as described above. The top phase was transferred to a 1.5-ml Eppendorf tube and gently mixed with 2 µl of the co-precipitant seeDNA (Amersham Pharmacia Biotech, Amsterdam, The Netherlands) and 0.1 vol equivalents of 3 M sodium acetate. After the addition of 2 vol equivalents 100% ethanol at room temperature, the mixture was vortexed briefly and incubated at room temperature for 2 min followed by 10 min on ice/water. After 30 min centrifugation at 4 C and 16,000 x g, the pellet was washed in 200 µl ice-cold 70% ethanol and again centrifuged as described above. The pellet was dried on ice, dissolved in DNase digestion solution [16 µl diethylpyrocarbonate-treated water, 2 µl 10 x DNase digestion buffer, and 2 µl DNase (10 IU/µl); Roche, Mannheim, Germany], and incubated at 37 C for 25 min to remove genomic DNA contamination. After enzyme inactivation at 65 C for 10 min, the solution was stored at -80 C. The absence of genomic DNA contamination was controlled by PCR using isolated RNA without RT as template.

For mRNA quantification, 10 µl RNA sample were incubated for 5 min at 94 C, vortexed for 5 sec, and chilled on ice/water. RT was performed in a total volume of 20 µl containing 50 mM Tris-HCl (pH 8.3); 75 mM KCl; 3 mM MgCl2; 10 mM dithiothreitol; 1 mM each of deoxy (d)-GTP, dATP, dTTP, and dCTP (MBI Fermentas, St. Leon-Rot, Germany); 600 ng/ml random hexamer primer (pN6; Roche); and 20 IU murine leukemia virus reverse transcriptase (Life Technologies, Inc.). The RT reaction was carried out at 40 C for 1 h, terminated by 5 min at 95 C, and placed on ice/water.

Quantification of mRNA abundance was performed by real-time PCR detection using an ABI PRISM 7700 sequence detector (PE Biosystems, Inc., Weiterstadt, Germany) and SybrGreen as a double stranded DNA-specific fluorescent dye. Amplification mixes (25 µl) contained 2 µl complementary DNA (cDNA) solution; 10 x SybrGreen PCR buffer; 200 µM dATP, dCTP, dGTP, and 400 µM dUTP; 3 mM MgCl2; 4 pM of each primer; 0.25 IU AmpErase uracil N-glycosylase; and 0.625 IU AmpliTaq Gold DNA polymerase (PE Biosystems, Inc.). Amplification primers (Table 1Go) were designed using the software Primer Express (PE Biosystems, Inc.).


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  		Table 1. Primers used for RT-real-time PCR

 
PCR was started with 2 min at 50 C for AmpErase activation and 10 min at 95 C for denaturation. The program continued with 40 cycles of 15 sec at 95 C and 60 sec at 62 C. Each assay included duplicates of cDNA primed separately for the four genes of interest, cDNA primed for the reference gene GAPDH, a no template control, and four dilutions of cDNA pooled from 16 embryos primed separately for each gene to calculate the corresponding amplification efficiency (E = 10-(1/b) - 1; b = regression coefficient). The parameter CT (threshold cycle) is defined as the cycle number at which fluorescence intensity exceeds a fixed threshold. Relative mRNA expression for each gene of interest (I) was calculated using the formula: (1 + E[I])-CT[I]/(1 + E[GAPDH])-CT[GAPDH]. These values obtained for the embryos treated with LR3 and IGF-I, respectively, were referenced to the corresponding values obtained for untreated embryos.

Eighteen pools of two embryos each, recovered from three independent IVP experiments, were analyzed per treatment group, and each mRNA was quantified for each gene simultaneously in one PCR run.

Statistical analysis
Cleavage rate (66 hpi), morula rate (168 hpi), and blastocyst rates (168 and 186 hpi) were evaluated by {chi}2 tests. Means of the cell numbers were compared using Mann-Whitney U test. Data for mRNA expression levels were analyzed using one-way ANOVA followed by least significant difference post-hoc test. Data are presented as the mean ± SEM. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of IGF-I and LR3 on embryonic development
The proportion of early cleavage (66 hpi) was significantly higher in the LR3 treatment group compared with the control system; the IGF-I group was intermediate (Table 2Go). At 162 hpi, the morula and morula/blastocyst rates were highest for embryos cultured in the presence of IGF-I, followed by LR3, and the control system. Significant (P < 0.05) differences were found between both IGF-supplemented groups and the control. At 186 hpi the proportion of blastocysts was significantly higher in the IGF-I than in the control system; the LR3 group was intermediate (Table 2Go).


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  		Table 2. Effect of supplementation with IGF-I variants of serum-free medium on the in vitro development of bovine embryos

 
Effects of IGF-I and LR3 on cell numbers of blastocysts
Cell numbers of blastocysts were highest in the LR3 group, followed by the IGF-I and control groups. Although significant differences between these groups were not found for inner cell mass (ICM) cell numbers, blastocysts produced in the presence of LR3 had significantly more trophectoderm (TE) cells and greater total cell numbers than control blastocysts (Table 3Go).


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  		Table 3. Effect of IGF-I variants on cell numbers of bovine blastocysts after in vitro culture in a serum-free system

 
Effects of IGF-I and LR3 on embryonic IGFBP-2, -3, and -5 and IGF-I-R mRNA expression
The abundance of mRNAs coding for different IGFBPs and for the IGF-I-R was calculated using the standard curve method, with determination of PCR amplification efficiency and normalization for GAPDH mRNA as the internal reference. Figure 1Go shows representative PCR amplification curves for GAPDH cDNAs from embryos of the different treatment groups (A) as well as the corresponding standard curve for calculation of PCR efficiency (B). CT values obtained for GAPDH mRNA were not different between treatment groups (Table 4Go). The expression of this housekeeping gene was assumed not to be affected by IGF-I or LR3 supplementation. Standard curves and PCR efficiencies (E) for all other genes investigated are shown in Fig. 2Go. Means and SEMs of CT values for IGFBP-2, -3, and -5, and IGF-I-R mRNA are shown in Table 4Go.



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  		Figure 1. Fig. 1. Quantification of GAPDH mRNAexpression by RT-real-time PCR. A, Representative amplification plots for GAPDH mRNA expression in doublets of blastocysts derived from the different experimental groups (red, IGF-I; green, LR3; yellow, control). B, Standard curve for calculation of the PCR amplification efficiency (E), performed as described in Materials and Methods. One blastocyst RNA unit (BRU) is equivalent to 1/16th of the total RNA amount extracted from a pool of 16 blastocysts.

 

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  		Table 4. CT values obtained for GAPDH; IGFBP-2, -3, and -5; and IGF-I-R mRNA expression in the different treatment groups

 


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  		Figure 2. Standard curves for calculation of the PCR amplification efficiencies (E) for IGFBP-2 (A), IGFBP-3 (B), IGFBP-5 (C), and IGF-I-R cDNAs (D). The calculation was performed as described in Materials and Methods. One blastocyst RNA unit (BRU) is equivalent to 1/16th of the total RNA amount extracted from a pool of 16 blastocysts.

 
The corresponding expression levels in IGF-I- or LR3-treated embryos were compared with the expression levels in control embryos cultured without IGF supplementation (Fig. 3Go). The greatest differences relative to control embryos were detected in mRNA levels of IGFBP-2 in LR3-cultured embryos (2.8-fold; P < 0.001), whereas IGF-I had a smaller effect on IGFBP-2 mRNA levels (1.6-fold; P < 0.01). The difference between the two IGF treatment groups was also significant (P < 0.001; Fig. 3AGo). Both IGF-I and LR3 showed a significant (P < 0.001) influence on IGFBP-5 mRNA abundance by increasing it 2.2- and 2.0-fold, respectively (Fig. 3CGo). Again the difference between IGF-I- and LR3-treated embryos was significant (P < 0.05). Similarly, IGFBP-3 mRNA levels were increased (P < 0.05) in embryos from the IGF-I vs. the LR3 culture system, both of which were not significantly different from the control group (Fig. 3BGo). Compared with the control group, the abundance of IGF-I-R mRNA was significantly increased (1.3-fold; P < 0.001) by LR3, but was significantly reduced (by 20%; P < 0.01) by IGF-I (Fig. 3DGo).



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  		Figure 3. Relative differences in mRNA abundance for IGFBP-2 (A), IGFBP-3 (B), IGFBP-5 (C), and IGF-I-R (D). Expression of IGFBP and IGF-I-R mRNA was normalized for GAPDH expression. The figure shows the means and SEMs. Significant differences are marked by asterisks: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study was performed to evaluate a potential role of IGF/IGFBP interactions for the support of embryonic growth by maternally derived IGF-I, as speculated in the first report demonstrating mRNA expression for various IGFBPs in preimplantation bovine embryos (4). For this purpose, we used recombinant human IGF-I and a synthetic IGF peptide, LR3, engineered for minimal binding to IGFBPs. IGF-I analogs with modified binding properties are important tools for studying the relevance of interactions with receptors and binding proteins for biological effects of IGF-I (33).

For our study we used LR3 in which the glutamate in position 3, which has been shown to be important for IGF/IGFBP interactions, was replaced by an arginine. In addition, this peptide carries a 13-amino acid N-terminal extension derived from methionyl porcine GH (29). LR3 was chosen rather than the naturally occurring IGF variant des(1, 2, 3)IGF-I, which is characterized by no detectable affinity for IGFBPs in rat plasma, but shows significant binding to IGFBPs from sheep plasma (34). Although binding to ovine plasma IGFBPs was even shown for LR3, this was markedly weaker than that observed with des(1, 2, 3)IGF-I. The competition strength of LR3 to displace radiolabeled IGF-I from IGFBPs in sheep plasma was more than 3 orders of magnitude lower than that of IGF-I (34). Thus, LR3 was the best choice for our experiment. Both IGF peptides were used at a concentration of 100 ng/ml, because this concentration had the greatest embryotrophic effect in previous studies (24, 25) and in our own pilot study in which we tested IGF-I at concentrations of 10, 50, and 100 ng/ml (data not shown).

Overall, the embryotrophic activity of the supplemented IGF peptides in protein-free medium was not marked. The proportions of development to blastocysts were 2-fold lower than those routinely obtained in culture systems involving serum, which is in line with other studies evaluating the effects of IGFs (22, 23, 24) or other growth factors, such as epidermal growth factor, acidic fibroblast growth factor, or platelet-derived growth factor (35) in bovine embryo culture. Nevertheless, the effects were significant for the proportion of early cleavage (LR3), development to morulae/blastocysts at 162 hpi (IGF-I and LR3), development to blastocysts at 186 hpi (IGF-I), and blastocyst cell numbers (LR3). Compared with the control system, LR3 was most effective in stimulating early embryonic development and cell numbers of blastocysts, whereas the proportion of blastocysts was greatest in the IGF-I treatment group. Interestingly, supplementation of culture medium with IGFs increased TE cell numbers of blastocysts (LR3, 21%; IGF-I, 9% vs. control) more than the numbers of ICM cells (LR3, 9%; IGF-I, 1% vs. control). The same tendency was also observed in a study using IGF-I for culturing buffalo embryos (25); however, in the latter study the effect of IGF-I on blastocyst cell numbers was much greater than that in our experiments. In contrast to these findings in bovine embryos, both IGF-I and IGF-II preferentially induced proliferation of ICM cells in mouse embryos (19, 20).

Although not statistically significant, our study showed a tendency of a greater potential of LR3 vs. IGF-I to increase both TE and ICM cell numbers of bovine blastocysts. These findings suggest that 1) an interaction with IGFBPs is not required for the mitogenic effect of IGF peptides on early embryos; and 2) the effect of IGF-I may be partially blocked by IGFBPs.

The most prominent finding concerning mRNA expression for components of the IGF system was 2.8- and 1.6-fold up-regulation of IGFBP-2 transcript levels in the LR3 and IGF-I treatment groups, respectively. It is known both from in vitro (36, 37, 38) and in vivo studies (39, 40) that IGF-I is capable of up-regulating IGFBP-2 expression in various cell types. Again, the greater effect of LR3 vs. IGF-I observed in the present study could be due to interference of embryonic IGFBPs.

The functional relevance of increased embryonic IGFBP-2 mRNA levels is still unclear. Overexpression of IGFBP-2 in transfected 293 human embryonic kidney cells reduced cell proliferation. The same effect was seen for IGF-responsive colon carcinoma cell lines. In both systems, the proliferation-inhibiting effect could be overcome by the addition of exogenous IGFs, as LR3 is markedly more effective than IGF-I (41). On the other hand, long-term overexpression of IGFBP-2 in transfected mouse adrenocortical tumor cells (Y-1) was associated with increased cell proliferation and tumorigenic potential. These effects were independent of exogenous IGFs (42). The relevance of IGFBP-2 actions in preimplantation embryonic development deserves further investigation. Targeted inactivation of the IGFBP-2 gene in mice caused only subtle phenotypic changes, which has been attributed to a potential functional compensation by other IGFBPs (43). Overexpression of IGFBP-2 in transgenic mice resulted in reduced body weight gain; however, this effect became obvious only after weaning (44, 45). These studies suggest that neither lack nor overexpression of IGFBP-2 have deleterious effects on embryonic development.

IGFBP-3 mRNA expression was only slightly affected by IGF-I or LR3 supplementation of the culture medium. Therefore, IGFs do not seem to play a role in the regulation of this IGFBP in early bovine embryonic development. Regulation of IGFBP-3 mRNA levels by IGF-I has, for example, been demonstrated in the liver of hypophysectomized rats (46). Interestingly, a recent IGF treatment study of adolescent monkeys showed that IGF-I, but not LR3, increases serum IGFBP-3 (47).

In contrast to IGFBP-3, IGFBP-5 mRNA expression was consistently up-regulated in the IGF treatment groups, with a slightly stronger effect of IGF-I than of LR3. For IGFBP-5, both inhibitory and stimulatory effects on IGF actions have been described (reviewed in Ref. 1). Potentiating effects of IGFBP-5 have been observed when its binding affinity for IGFs was lowered by association to extracellular matrix or by specific proteolysis, facilitating subsequent binding of IGF-I to its receptors. In addition to these IGF-dependent effects, IGF-independent actions, i.e. involving neither IGF binding nor activation or inhibition of the IGF-I-R, have been proposed for IGFBP-5 (48, 49). A novel putative IGFBP-5 receptor, a 420-kDa membrane protein, was recently purified from osteoblast cells and shown to be phosphorylated upon IGFBP-5 binding (50). Furthermore, IGFBP-5 has a functional nuclear localization sequence and is imported into the nucleus via the importin {beta}-subunit (51). The potential roles of IGFBP-5 effects on early embryonic development deserve further investigation, e.g. by IGFBP-5 supplementation of defined culture medium.

In addition to IGFBP mRNAs, the abundance of IGF-I-R transcripts in bovine blastocysts was influenced by IGF supplementation in the culture medium, in this case toward opposite directions by IGF-I (20% reduction) and LR3 (30% increase). The biological relevance of this observation is not clear, as IGF-I was most potent in stimulating blastocyst formation, whereas LR3 supplementation of the culture medium yielded the greatest cell numbers. We do not know whether the observed differences in IGF-I-R mRNA levels are reflected at the protein level. However, the lower affinity of LR3 vs. IGF-I for the IGF-I-R [shown, for example, in rat L6 myoblasts (29)] might be involved in the regulation of IGF-I-R mRNA expression by the used IGF peptides.

In summary, our study provides for the first time a direct comparison of effects of IGF-I and LR3 on development and gene expression of preimplantation bovine embryos. LR3 was more effective in stimulating early embryonic development and blastocyst cell numbers, whereas the proportion of development to blastocysts was highest in medium supplemented with IGF-I. Using real-time quantitative RT-PCR assays, we were for the first time able to determine the abundance of transcripts for IGFBPs and IGF-I-R in pools of only two blastocysts. These experiments demonstrate that the mRNA levels for components of the IGF system are regulated by exogenous IGF-I already in preimplantation embryos. Our study provides the first experimental data supporting the previous hypothesis (4) that preimplantation bovine embryos modulate effects of maternal IGFs by expression of IGFBPs and IGF-I-R.


   Acknowledgments
 
We thank Dr. Jens-Peter Sölch for helpful discussions and advice, Dr. Kathy McDougall for the RNA extraction protocol, and Petra Stojkovic for excellent technical assistance.


   Footnotes
 
1 This work was supported in part by grants from the Bayerische Forschungsstiftung (76/93) and from the Bayerisches Staatsministerium für Ernährung, Landwirtschaft und Forsten (A/99/8). Back

2 K.P. and M.S. contributed equally to this work. Back

Received October 2, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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