233 EXPRESSION OF INSULIN-LIKE GROWTH FACTOR (IGF) MRNA IN BOVINE CONCEPTUSES FROM EMBRYOS PRODUCED IN VIVO OR IN VITRO
C.E. Farin A , J.E. Alexander A , K.F. Rodriguez A and P.W. Farin BA Department of Animal Science, North Carolina State University, Raleigh, NC, USA. email: Char_Farin@ncsu.edu;
B Department of Population Health and Pathobiology, North Carolina State University, Raleigh, NC, USA.
Reproduction, Fertility and Development 16(2) 237-238 https://doi.org/10.1071/RDv16n1Ab233
Submitted: 1 August 2003 Accepted: 1 October 2003 Published: 2 January 2004
Abstract
The objective of this study was to determine the effect of in vitro embryo production (IVP) on expression of mRNAs for IGF-1, IGF-2, IGF-1 receptor (IGF-1R), IGF-2R and GAPDH in bovine conceptuses at Day 17 of gestation. In vivo embryos (In vivo) were recovered from superovulated Holstein cows. For IVP, Holstein oocytes were matured, fertilized and then cultured in M199 with 10% serum (IVPS) or 1% BSA (IVPSR) to 72 hpi. All embryos were then transferred to M199 with 10% serum and cultured to 168 hpi. The same Holstein sire was used to produce all embryos. Single grade 1 blastocysts were transferred into recipient heifers. On day 10 after transfer, conceptuses were recovered, measured and stored at −80°C prior to extraction of whole cell (wc) RNA and genomic (g) DNA. wcRNA was assessed for quality based on A260/A280 ratio and visualization of 18S and 28S ribosomal RNA. Only complete (intact) conceptuses with at least 10 μg high-quality wcRNA recovered after extraction were included in the analysis (In vivo, n = 8; IVPS n = 8; IVPSR n = 7). Conceptus sex was determined by PCR analysis of gDNA. Semi-quantitative PCR assays were used to assess relative expression of all mRNAs. For each conceptus, 2 μg wcRNA were mixed with 3 pg rabbit globin mRNA, DNase-treated and reverse-transcribed using random hexamers. Conceptus cDNA samples (140 ng each for IGF-1, IGF-2 mRNAs;; 100 ng each for IGF-1R, IGF-2R, GAPDH and globin mRNAs) were assayed in duplicate within single assays. Relative mRNA expression was calculated as the ratio of band intensities representing the mRNA of interest to globin mRNA. All data were analyzed for effects of treatment (In vivo, IVPS, IVPSR or In vivo, IVP), sex, stage of blastocyst development at transfer (early, mid, expanded) and the interactions of treatment sex and treatment stage. Conceptus length (LSM ± SEM mm) did not differ with treatment (In vivo: 182 ± 55; IVPS: 257 ± 47; IVPSR: 310 ± 72). Expression of GAPDH mRNA was greater (P = 0.02) in female conceptuses (1.31 ± 0.11) than in males (0.96 ± 0.07) and did not differ (P = 0.14) between treatment groups (In vivo: 1.30 ± 0.11, IVPS: 1.00 ± 0.09, IVPSR: 1.10 ± 0.14). However, when the IVP groups were combined and compared to In vivo controls, expression of GAPDH mRNA was both greater (P = 0.002) in females (1.38 ± 0.10) than in males (0.94 ± 0.06) and differed (P = 0.03) with treatment (In vivo: 1.30 ± 0.10, IVP: 1.01 ± 0.07). Expression of IGF-1 and IGF-2 mRNA was not detected in the majority of conceptuses. Expression of IGF-1R mRNA was greater (P = 0.02) in female (1.19 ± 0.10) than in male conceptuses (0.87 ± 0.07) and differed (P = 0.02) with stage of blastocyst development at transfer (early: 0.79 ± 0.07; mid: 1.23 ± 0.13; expanded: 1.07 ± 0.11). There was no effect of treatment on expression of IGF-1R mRNA. Expression of IGF-2R mRNA differed (P = 0.03) with treatment (In vivo: 0.85 ± 0.10; IVPS: 0.76 ± 0.09; IVPSR: 0.33 ± 0.13). In conclusion, expression of both imprinted and non-imprinted genes is altered in conceptuses resulting from embryos produced in vitro. Supported by the North Carolina Agricultural Research Service.
