Analysis of differential messenger RNA expression between bovine blastocysts produced in different culture systems: implications for blastocyst quality.
Using reverse transcriptase-amplified fragment length polymorphism (RT-AFLP) analysis of differential mRNA expression and semiquantitative reverse transcriptase-polymerase chain reaction, we compared mRNA expression in bovine blastocysts from 4 sources, known to differ in quality in terms of their ability to withstand cryopreservation: 1) in vitro culture in synthetic oviduct fluid of in vitro-matured (IVM)/in vitro fertilized (IVF) zygotes; 2) in vitro culture in TCM-199 supplemented with granulosa cells (coculture) of IVM/IVF zygotes; 3) in vivo culture in the ewe oviduct of IVM/IVF zygotes; or 4) superovulation, artificial insemination, and nonsurgical embryo recovery. Total mRNA was isolated from pools of blastocysts and reverse transcription was performed. Triplicate reactions from each sample were displayed, and only consistent banding variations were recorded. Using AFLP-differential display assay, we found that cDNA banding patterns are highly conserved between the 4 groups of blastocysts studied; however, there was a difference of 7% in bands either missing or expressed across the groups. Fifty bands were reamplified, and a sequence comparison search revealed similarity of 14 isolated fragments to ribosomal and mitochondrial genes, 16 matched to described cDNA, and 20 corresponded to unknown sequences that may represent novel genes. The study of 7 differentially expressed mRNAs known to be involved in developmental process in the embryo suggests roles for apoptosis, oxidative stress, gap junctions, and differentiation in the determination of embryo quality. The aberrant transcription patterns detected in in vitro-produced bovine embryos compared with those produced in vivo may explain their reduced quality in terms of viability after cryopreservation.
Rizos
D
D
Department of Animal Science and Production, University College Dublin, Lyons Research Farm, Newcastle, County Dublin, Ireland.
Lonergan
P
P
Boland
M P
MP
Arroyo-García
R
R
Pintado
B
B
de la Fuente
J
J
Gutiérrez-Adán
A
A
D000818:Animals; D001755:Blastocyst / Q000737:chemistry* / Q000502:physiology*; D002417:Cattle / Q000196:embryology*; D018920:Coculture Techniques; D018031:Connexin 43 / Q000235:genetics; D017630:Connexins / Q000235:genetics; D015925:Cryopreservation; D046508:Culture Techniques; D005187:Fallopian Tubes; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D015870:Gene Expression; D006131:Growth Inhibitors / Q000235:genetics; D007315:Insemination, Artificial / Q000662:veterinary; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D008222:Lymphokines / Q000235:genetics; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics; D011110:Polymorphism, Genetic; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger / Q000032:analysis*; D018121:Receptors, Cytokine / Q000235:genetics; D053606:Receptors, OSM-LIF; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050879:Sarcosine Oxidase; D012756:Sheep; D013480:Superovulation; D013482:Superoxide Dismutase / Q000235:genetics; D051028:bcl-2-Associated X Protein
D018031:Connexin 43; D017630:Connexins; D006131:Growth Inhibitors; D015850:Interleukin-6; C507837:LIF protein, human; C507840:LIFR protein, human; D053605:Leukemia Inhibitory Factor; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D008222:Lymphokines; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D018121:Receptors, Cytokine; D053606:Receptors, OSM-LIF; D051028:bcl-2-Associated X Protein; C068044:GJB3 protein, human; D013482:Superoxide Dismutase; D010089:Oxidoreductases, N-Demethylating; D050879:Sarcosine Oxidase