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a Department of Animal Science and
b Department of Food Animal and Equine Medicine, North Carolina State University, Raleigh, North Carolina 27695-7621
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The insulin-like growth factors (IGF-I and -II), along with their receptors (Type-I and -II) and binding proteins (IGFBPs), play key roles in regulating fetal growth. The distinct role for IGF-II in fetal growth has been well documented using genetically engineered mice (see [10] for review). Mice expressing decreased levels of IGF-I [11], IGF-II [12], and Type-I receptor [11] exhibit body weights that are 45% to 60% less than that of normal controls. In contrast, mutant mice expressing increased levels of IGF-II [13,14] exhibit body weights that are 125% to 135% that of normal mice. Therefore, alterations in the expression of members of the IGF family may be responsible for the deviated growth characteristics seen in fetuses and offspring originating from embryos produced using in vitro systems. The objective of this study was to assess the levels of mRNA for IGF-II in liver and skeletal muscle of fetuses originating from either in vivo or in vitro embryo production systems.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Holstein cows were synchronized by administration of two i.m. injections of 25 mg prostaglandin F2
(PGF2, Lutalyse; Upjohn Co., Kalamazoo, MI) 12 days apart. Between Days 10 and 13 of the estrous cycle (Day 0 = estrus), donor cows were superovulated with FSH administered in decreasing doses over a 4-day period (20 to 32 mg FSH-P, Schering-Plough, Piscataway, NJ; or 400 mg Folltropin, Vetrapharm Canada, London, ON). Estrus was induced by the administration of two i.m. injections of 25 mg of PGF2 on the morning and evening of the third day of FSH treatment. Donors were artificially inseminated 12 and 24 h after first detection of standing estrus. Thawed frozen semen from a single, proven Holstein bull was used. Embryos (multiple ovulations [MO]) were collected by nonsurgical uterine flushing on Day 7 (Day 0 = first detected estrus; [5]).
In Vitro Embryo Production
Reagents and media supplements used in these experiments were of tissue culture grade and were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated. Tissue culture medium (TCM-199 with Earle's salts) was obtained from Gibco Laboratories (Grand Island, NY). Fatty-acid free BSA was purchased from Boehringer-Mannheim (Indianapolis, IN). With the exception of in vitro fertilization medium, all in vitro culture media contained 50 µg/ml of gentamicin.
Ovaries from Holstein cows were obtained from a local abattoir. Cumulus-oocyte complexes were collected, matured, and fertilized as described by Farin and Farin [5]. Mature oocytes were fertilized with semen from the same Holstein bull used to produce embryos in vivo. At 18 to 20 h postinsemination (hpi), presumptive zygotes were washed six times in modified Tyrode's-lactate Hepes [15] and cultured in groups of 15 to 30 zygotes in either 1 ml of TCM-199 + 10% estrous cow serum (ECS; in vitro-produced with serum [IVPS]) or 1 ml of TCM-199 + 1% BSA (in vitro-produced with serum restriction [IVPSR]). At 72 hpi, IVPSR embryos were transferred into fresh TCM-199 + 10% ECS whereas IVPS embryos had fresh medium replaced. At 120 hpi, fresh TCM-199 + 10% ECS was replaced in both treatments. At 168 hpi, blastocyst-stage embryos were harvested and assigned a morphological quality grade [16]. On the basis of a previous study, rates of development to the blastocyst stage were similar between IVPS (28 ± 3%) and IVPSR (33 ± 3%) production systems [17].
Transfer of Embryos and Tissue Collection
Estrus was induced in crossbred Angus recipient heifers by the administration of two i.m. injections of 25 mg of PGF2 10 to 12 days apart. On Day 7 of the estrous cycle, single blastocyst-stage embryos produced in vitro or in vivo were transferred nonsurgically to the uterine horn ipsilateral to the ovary bearing the corpus luteum. Only Grade 1 (excellent) blastocysts were selected for transfer. At Day 70 of gestation (63 days after transfer), a total of 38 pregnant recipients (n = 14, 13, and 11 for MO, IVPS, and IVPSR, respectively) were slaughtered, and their reproductive tracts were collected and kept on ice until processed. Fetuses were removed from the reproductive tracts; physical measurements including fetal weight, liver weight, and placental weight were recorded, and samples of liver and skeletal muscle from individual fetuses were snap frozen in liquid nitrogen. In addition, samples of liver from a random-bred bovine fetus at Day 90 of gestation (estimated by crown-rump length, 130 mm) and an adult Holstein cow, obtained at the abattoir, were used as positive controls for each mRNA assay performed.
Whole-cell RNA (wcRNA) was extracted from liver and skeletal muscle tissues. Briefly, frozen tissue was removed from -80°C storage, weighed, placed in a mortar, covered with liquid nitrogen, and subsequently crushed to a fine powder. The powder was then briefly homogenized (Brinkmann Homogenizer PT 10/35; Westbury, NY) and dissociated in TriReagent using a w:v ratio of 100 mg tissue per milliliter reagent. WcRNA was extracted according to the manufacturer's protocol. Purified wcRNA was resuspended in diethyl pyrocarbonate-treated water, and concentration was determined by measuring the absorbance at 260 nm. The quality and integrity of the wcRNA were assessed using the ratio of absorbances at 260 and 280 nm and by visualization of ethidium bromide-stained 28S and 18S rRNA bands on 1% agarose gels.
RNase Protection Assay (RPA)
A cDNA for bovine 18S rRNA, subcloned into the SmaI site of pBS (±) plasmid, was generously provided by Dr. J.E. Fortune (Cornell University, Ithaca, NY) [18]. After linearization of the recombinant plasmid with EcoRI, a 32P-labeled antisense riboprobe of 170 base pairs (bp) was transcribed in vitro using T3 RNA polymerase and [-32P]CTP (3000 Ci/mmol; ICN, Costa Mesa, CA). For bovine IGF-II, a cDNA fragment was generated by using reverse transcription-polymerase chain reaction (RT-PCR). Briefly, bovine wcRNA was reverse transcribed using an oligo-dT primer and SuperScript AMV reverse transcriptase (Gibco Laboratories) under reaction conditions recommended by the manufacturer. After reverse transcription, PCR was performed for 40 cycles (30 sec 94°C, 1 min 55°C, 1 min 72°C) using forward and reverse primers based on the ovine IGF-II mRNA sequence (forward: 3'(5'-TCGTGCTGCTATGCTGCTTACC-3', reverse: 5'(5'-ACTGCTTCCAGGTGTCAGATTGG-3') [19]. A 470-bp amplicon was produced and was verified to be 100% similar to bovine IGF-II by dideoxy sequence analysis. This cDNA for bovine IGF-II was subcloned into the EcoRI site of a pSP70 plasmid (Promega, Madison, WI). A double-restriction digest of the plasmid was performed using PvuII and HpaI to release the insert and the T7 promoter sequences (Boehringer-Mannheim). This plasmid piece then was purified by means of GeneClean II (Bio 101, Vista, CA) and used to produce a 32P-labeled antisense riboprobe (470 bp) by in vitro transcription with T7 RNA polymerase and [-32P]CTP (3000 Ci/mmol).
The RPA used for analysis of IGF-II mRNA and 18S rRNA was adapted from Tian et al. [18]. Briefly, excess 32P-labeled antisense riboprobes (106 dpm) for bovine IGF-II and 18S were mixed with duplicate samples of 1 µg wcRNA. Hybridization was conducted for 16 h at 50°C in the presence of 80% formamide, 40 mM PIPES (pH = 6.4), 400 mM NaCl, and 1 mM EDTA. Unhybridized riboprobes and wcRNA were removed by digestion at 35°C for 30 min with 1 mg/ml RNase A and 20 000 U/ml RNase T1. RNases were inactivated by incubation for 30 min at 37°C in 0.4% SDS with 0.2 mg/ml proteinase K and removed by phenol/chloroform/isoamyl alcohol extraction. The protected fragments were ethanol precipitated for at least 1 h at -20°C and then analyzed by electrophoresis through a 5% denaturing PAGE gel containing 7 M urea. After electrophoresis, the intensities of the respective band fragments for IGF-II were quantified using phosphorimage analysis (ImageQuant software; Molecular Dynamics, Sunnyvale, CA). Signals were expressed as arbitrary units above background for each autoradiograph and were adjusted for RNA loading differences by expressing each message as a ratio to the 18S message. Glycogen was used as the control for background signals. For each tissue type, three RPAs were performed to analyze fetal samples from all treatments. Therefore, adult and Day 90 fetal liver wcRNA samples were included in each RPA to monitor assay-to-assay variability. The interassay coefficients of variation for all D90 fetal liver and adult liver samples assayed in each RPA were 22.4% ± 8.2 and 7.1% ± 2.3, respectively (mean ± SEM). The intraassay coefficients of variation for all liver samples assayed in each RPA were 15.7% ± 5.4, 15.9% ± 6.3, and 9.6% ± 2.8 (mean ± SEM).
Statistical Analysis
Relative levels of mRNA for IGF-II within each sample were calculated as the ratio of the band intensity for IGF-II to 18S rRNA and expressed as mean arbitrary units ± SEM. Relative levels of mRNA for IGF-II were log10 transformed when the raw data failed to pass a test of normality. Data for effect of treatment on IGF-II mRNA levels were analyzed using a General Linear Models procedure [20]. The final model included the effects of treatment, sex, assay, and all two-way interactions. When a significant F-statistic was encountered, treatment means were separated using a Duncan's Multiple Comparison test [20]. Data for assessment of IGF-II mRNA levels in Day 90 fetal liver and adult liver control samples were analyzed by using Mann-Whitney's Rank Sum test [20]. Spearman correlation coefficients were used to test the relationship between IGF-II mRNA in fetal liver or fetal skeletal muscle and fetal weight, liver weight, and placental weight [20].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A representative autoradiograph from an RPA is shown in Figure 1. Samples included wcRNA from fetuses at Day 70 of gestation, as well as liver wcRNA from an adult liver and from a fetus at Day 90 of gestation. The upper panel illustrates signals representing the 470-bp protected fragment for IGF-II mRNA; the lower panel illustrates the corresponding 170-bp protected fragment for 18S rRNA. Glycogen was used as a control to correct for background signals. As expected, liver from fetuses at Day 90 of gestation possessed 9-fold greater (P < 0.05) levels of IGF-II mRNA than adult liver (0.18 ± 0.11 and 0.02 ± 0.01, respectively). Because a significant (P = 0.003) sex-by-treatment interaction was found for levels of IGF-II mRNA in Day 70 fetal bovine liver, data for male and female fetuses are presented separately (Fig. 2A and B, respectively). In male fetuses originating from in vitro procedures (IVPS and IVPSR), levels of IGF-II mRNA were 1.8- to 2.4-fold greater (0.25 ± 0.07 and 0.33 ± 0.04, respectively) than those from in vivo procedures (MO: 0.14 ± 0.03; P = 0.04). For female fetuses, numerically greater levels of IGF-II mRNA were seen in fetuses originating from in vitro procedures (0.38 ± 0.04 and 0.34 ± 0.11 for IVPS and IVPSR, respectively) than in those originating in vivo (MO: 0.19 ± 0.07); however, these differences were not statistically significant. A scattergram of the relative IGF-II mRNA levels for the 38 individual liver samples analyzed is presented in Figure 3.
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Levels of IGF-II in Skeletal Muscle of Fetuses at Day 70 of Gestation
Because no sex-by-treatment effect was observed for levels of IGF-II in skeletal muscle, results for male and female fetuses were combined (Fig. 4). Skeletal muscle of fetuses from the MO group tended (P = 0.07) to have higher levels of IGF-II mRNA (0.07 ± 0.007) than that of the IVPSR group (0.04 ± 0.005). Skeletal muscle of fetuses from the IVPS group had intermediate levels of IGF-II mRNA (0.05 ± 0.009). No significant correlations were found between the level of liver or skeletal muscle IGF-II mRNA and fetal weight (r = 0.15 and 0.36, respectively), liver weight (r = 0.06 and -0.14, respectively), or placental weight (r = -0.23 and 0.10, respectively) across all treatments. However, a positive correlation (r = 0.49; P = 0.07) was found between levels of IGF-II mRNA in liver versus skeletal muscle in fetuses from the MO group (Fig. 5A). Interestingly, this correlation was absent in fetuses from the IVPS and IVPSR groups (P = 0.92; Fig. 5B and C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fetuses originating from in vitro-produced embryos cultured in a serum-restricted environment expressed lower levels of mRNA for IGF-II in skeletal muscle compared to those from the MO group. This may seem somewhat contradictory to our original hypothesis that alterations in the expression of members of the IGF-axis could contribute to fetal oversize later in gestation [5]. However, Magri and coworkers [23] demonstrated that IGF-I, -II, and insulin repressed endogenous expression of IGF-II in cultured rat and human muscle cells. Both IGF-I and -II acted primarily via the Type-I receptor to cause this negative feedback inhibition of IGF-II gene expression. Therefore, it is plausible that increased levels of IGFs from other tissues, such as fetal liver, could inhibit or reduce the expression of mRNA for IGF-II in fetal skeletal muscle. Although treatment with IGF-I or -II suppressed IGF-II mRNA and protein expression in muscle cells in the study by Magri and coworkers [23], these cells still underwent terminal myogenic differentiation in response to the added growth factors [23–25]. Interestingly, our study demonstrates that levels of mRNA for IGF-II in liver were positively correlated to levels found in skeletal muscle of MO fetuses. In contrast, for fetuses of in vitro origin (IVPS and IVPSR), this correlation was not present. This finding is consistent with our view that in vitro production of embryos may alter the normal relationships between tissues in their expression of IGF-related mRNAs.
It has been suggested that large offspring syndrome could result from the manipulation of the preimplanatation mammalian embryos during in vitro production [5,6]. Specific alleles involved in controlling fetal, placental, and/or neonatal growth, which are genomically imprinted, may be differentially expressed due the manipulations of the embryos [26]. The genes encoding IGF Type-II receptor (Igf2r) and the nontranslated mRNA, H19 (H19), demonstrate allele-specific patterns of expression during embryonic development. These allele-specific patterns are controlled by epigenetic modifications resulting from the methylation of specific DNA sites [27,28]. Genetically engineered mice that are null mutants for Igf2r or H19 are associated with fetal or neonatal overgrowth [13,14]. Type-II receptor down-regulates IGF-II activity by binding, internalizing, and degrading this growth factor whereas the H19 gene, which lies approximately 100 kilobases upstream of Igf2r in mice, indirectly controls the expression of IGF-II mRNA [14]. Thus, these imprinted genes are strongly implicated in the control of fetal, placental, and neonatal growth. It is highly likely that disruption of imprinting of Igf2r or H19 due to in vitro culture conditions could in turn alter the normal embryonic and fetal development. Interestingly, Razin and Shemer [29] demonstrated that methylation imprints are extensively remodeled after fertilization. Igf2 was methylated in mature oocytes and sperm, demethylated after fertilization, and then de novo methylated prior to the morula stage [29]. Thus, it is possible that in vitro culture, or in vitro fertilization, could substantially affect patterns of genetic imprinting established during early embryonic cleavage. In fact, in vitro culture has been shown to affect the abundance of mRNAs for IGF-I, -II, Type-1, and-2 receptors in murine embryos [30].
Although we chose to focus on IGF-II in the present study, it is clear that other members of the IGF family including IGF-I, Type-I or -II receptors, or the IGF binding proteins could be modified by in vitro culture conditions as well. In both rats and humans, IGF-II receptors were more abundant than IGF-I receptors in fetal muscle but at birth IGF-II receptors declined [31,32], suggesting a potentially important role for IGF-II receptor in muscle growth and differentiation.
In summary, our results demonstrate that in vitro production of bovine embryos affects subsequent expression of IGF-II mRNA in both fetal liver and fetal skeletal muscle as early as 63 days following embryo transfer. Furthermore, the positive relationship between levels of liver and skeletal muscle IGF-II mRNA present in control fetuses appears to be disrupted as a result of in vitro embryo production. Our data are consistent with the hypothesis that alterations in the expression of genes regulating fetal growth may occur as a result of in vitro culture and that disruption in the patterns of imprinting of these genes may also be affected. Future assessment of actual patterns of DNA methylation are needed to fully verify this proposed mechanism.
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FOOTNOTES |
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1 This research was supported by USDA Grant 9602482 and the North Carolina Agricultural Research Service. P.B was a National Sciences and Engineering Research Council of Canada Fellow. 
2 Correspondence: Charlotte E. Farin, Department of Animal Science, North Carolina State University, Raleigh, North Carolina 27695-7621. FAX: 919-515-7780; char_farin{at}ncsu.edu
Accepted: October 6, 1999.
Received: June 21, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-induced luteolysis in cattle. Biol Reprod 1994; 50:349–356.[Abstract]This article has been cited by other articles:
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  A. El-Sayed, M. Hoelker, F. Rings, D. Salilew, D. Jennen, E. Tholen, M.-A. Sirard, K. Schellander, and D. Tesfaye Large-scale transcriptional analysis of bovine embryo biopsies in relation to pregnancy success after transfer to recipients Physiol Genomics, December 13, 2006; 28(1): 84 - 96. [Abstract] [Full Text] [PDF]
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 T. P. Fleming, W. Y. Kwong, R. Porter, E. Ursell, I. Fesenko, A. Wilkins, D. J. Miller, A. J. Watkins, and J. J. Eckert The Embryo and Its Future Biol Reprod, October 1, 2004; 71(4): 1046 - 1054. [Abstract] [Full Text] [PDF]
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W. Shi, F. Dirim, E. Wolf, V. Zakhartchenko, and T. Haaf Methylation Reprogramming and Chromosomal Aneuploidy in In Vivo Fertilized and Cloned Rabbit Preimplantation Embryos Biol Reprod, July 1, 2004; 71(1): 340 - 347. [Abstract] [Full Text] [PDF]
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 C. E. Farin, P. W. Farin, and J. A. Piedrahita Development of fetuses from in vitro-produced and cloned bovine embryos J Anim Sci, January 1, 2004; 82(13_suppl): E53 - 62. [Abstract] [Full Text] [PDF]
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G. Lazzari, C. Wrenzycki, D. Herrmann, R. Duchi, T. Kruip, H. Niemann, and C. Galli Cellular and Molecular Deviations in Bovine In Vitro-Produced Embryos Are Related to the Large Offspring Syndrome Biol Reprod, September 1, 2002; 67(3): 767 - 775. [Abstract] [Full Text] [PDF]
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A. E. Crosier, C. E. Farin, K. F. Rodriguez, P. Blondin, J. E. Alexander, and P. W. Farin Development of Skeletal Muscle and Expression of Candidate Genes in Bovine Fetuses from Embryos Produced In Vivo or In Vitro Biol Reprod, August 1, 2002; 67(2): 401 - 408. [Abstract] [Full Text] [PDF]
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