Expression of Insulin-Like Growth Factor Receptor mRNA in Bovine Conceptuses from Embryos produced In Vivo or In Vitro

 

C. E. Farin, P. W. Farin¹ and J. E. Alexander

 

¹Department of Population Health and Pathobiology,

North Carolina State University

 

Abstract

The objective of this study was to determine the effect of in vitro embryo production on the expression of mRNAs for insulin-like growth factor (IGF) ligands (IGF-1, IGF-2), IGF receptors (IGF-1R, IGF-2R), and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) in bovine conceptuses at Day 17 of gestation (10 d after transfer).   Embryos were produced in vivo using multiple-ovulation procedures (MO) or in vitro using serum (IVPS) or serum-restricted (IVPSR) culture systems.  MO embryos were recovered from superovulated Holstein cows on day 7 after estrus.  IVPS and IVPSR embryos were produced from oocytes aspirated from the ovaries of Holstein cows obtained at a local abattoir.  Following in vitro maturation and fertilization, presumptive zygotes were transferred with their cumulus investments to TCM-199 with 10% estrus cow serum (IVPS) or 1% BSA (IVPSR) and cultured until 72h post-insemination (hpi).  At 72 hpi embryos were transferred into TCM-199 with 10% estrus cow serum and culture was continued in the presence of cumulus cells until 168 hpi.  The same Holstein sire was used for production of embryos in vivo and in vitro.  Single grade 1 blastocysts were transferred nonsurgically into cross-bred Angus heifers on day 7 of the estrous cycle (estrus = d 0).  On day 10 after transfer, intact uteri were recovered at slaughter and conceptuses were recovered by uterine lavage.  Conceptuses were measured, snap frozen in liquid nitrogen and stored at -80C prior to extraction of whole cell (wc) RNA and genomic DNA. wcRNA, extracted from individual conceptuses, was quantified by measurement of UV absorbance at A260 and assessed for quality by evaluation of the A260/A280 ratio and visualization of 18S and 28S ribosomal RNA bands.  For inclusion in this study conceptuses (MO n=8; IVPS n=8; IVPSR n=7) must have been complete (intact) upon recovery and must have had at least 10 ug high-quality wcRNA recovered following extraction.  Conceptus sex was determined by PCR analysis of genomic DNA.  Semi-quantitative PCR assays were used to assess expression levels of mRNAs for IGF-1, IGF-2, IGF-1R, IGF-2R and GAPDH.  Briefly, for each conceptus a 2 ug sample of wcRNA was mixed with 3 pg rabbit alpha-globin mRNA, DNase-treated and reverse-transcribed using random hexamers.  A cDNA pool was then constructed from 40 ng aliquots of cDNA from all conceptuses and used to determine the linear phase of amplificiation for each primer pair representing each mRNA of interest.  No PCR products for IGF-1, -2, -1R, -2R or GAPDH were observed after amplification of cDNA derived from control samples containing only globin mRNA.  For assessment of IGF-1 and IGF-2 mRNA, duplicate 140 ng samples of conceptus cDNA were assessed in a single assay for each mRNA; for IGF-1R, IGF-2R, GAPDH and globin mRNAs, duplicate 100ng samples of conceptus cDNA were assessed in a single assay for each mRNA.  Amplification products were visualized on EtBr-stained agarose gels and band intensities were assessed using computer-assisted video image analysis.  The mean intra-assay coefficients of variation for sample duplicates ranged from 4%-8% across all assays.  For each conceptus, data were expressed as the ratio of the band intensities representing the mRNA of interest to that globin mRNA.  Data (LSMean±SEM) were analyzed by GLM procedures using models that included main effects of treatment (MO, IVPS, IVPSR or MO, IVP), sex, stage of blastocyst development at the time of transfer (early, mid, expanded), and the interactions of treatment*sex and treatment*stage.  Conceptus length (mm) did not differ significantly between the 3 treatment groups (MO: 182 ±55, IVPS: 257±47, IVPSR: 310±72).  However, when data for the IVP groups were combined and compared to MO controls, conceptus length tended to differ with treatment (MO: 182±48, IVP: 270±33; P=.15) and with stage of blastocyst development at the time of transfer (early: 172±38, mid: 208±59, expanded: 298±47; P=.14).  Relative expression of GAPDH mRNA was higher (P=.02) in female (1.31±.11) compared to male (0.96±.07) conceptuses and tended (P=.14) to differ with treatment (MO: 1.30±.11, IVPS: 1.00±.09, IVPSR: 1.10±0.14).  When data for the IVP groups were combined and compared to MO controls, relative expression of GAPDH mRNA was higher (P=.002) in female (1.38±.10) compared to male (0.94±.06) conceptuses and also differed (P=.03) with treatment (MO: 1.30±.10, IVP: 1.01±.07).  Expression of IGF-1 mRNA was below the limits of detection of the assay for the majority of conceptuses tested.  Similarly, expression of IGF-2 mRNA was below the limits of detection of the assay for 21 of the 23 conceptuses.   Relative expression of IGF-1R mRNA was higher (P=.02) in female (1.19±.10) compared to male (0.87±.07) conceptuses and differed (P=.02) with stage of blastocyst development at the time of transfer on day 7 (early: 0.79±.07, mid: 1.23±.13, expanded: 1.07±.11).  There was no effect of treatment on relative expression of mRNA for IGF-1R.  Relative expression of IGF-2R mRNA differed (P=.03) with treatment (MO: 0.85±.10, IVPS: 0.76±.09, IVPSR: 0.33±.13) but not with sex, stage of blastocyst development or any interactions. 

In summary, in conceptuses at Day 17 of gestation  expression of both IGF-1 and IGF-2 mRNAs was minimal, expression of IGF-1R mRNA was unaffected by  embryo production system but was significantly greater in female conceptuses and in conceptuses that resulted from the transfer of blastocysts at advanced stages of development,  expression of GAPDH mRNA was greatest in female conceptuses and in conceptuses from embryos produced in vivo, expression of IGF-2R was also greatest in conceptuses from embryos produced in vivo.  These observations support the conclusion that production of embryos in vitro is associated with alterations in the expression of both imprinted and non-imprinted genes as early as 10 days following transfer.

 

Supported by the North Carolina Agricultural Research Service.