Global poly(A) mRNA expression profile measured in individual bovine oocytes and cleavage embryos.
The objective of this article was to estimate quantitative differences for GAPDH transcripts and poly(A) mRNA: (i) between oocytes collected from cumulus-oocyte complexes (COCs) qualified morphologically as grades A and B; (ii) between grade A oocytes before and after in vitro maturation (IVM); and (iii) among in vitro-produced embryos at different developmental stages. To achieve this objective a new approach was developed to estimate differences between poly(A) mRNA when using small samples. The approach consisted of full-length cDNA amplification (acDNA) monitored by real-time PCR, in which the cDNA from half of an oocyte or embryo was used as a template. The GAPDH gene was amplified as a reverse transcription control and samples that were not positive for GAPDH transcripts were discarded. The fold differences between two samples were estimated using delta Ct and statistical analysis and were obtained using the pairwise fixed reallocation randomization test. It was found that the oocytes recovered from grade B COCs had quantitatively less poly(A) mRNA (p<0.01) transcripts compared with grade A COCs (1 arbitrary unit expression rate). In the comparison with immature oocytes (1 arbitrary unit expression rate), the quantity of poly(A) mRNA did not change during IVM, but declined following IVF and varied with embryo culture (p<0.05). Amplification of cDNA by real-time PCR was an efficient method to estimate differences in the amount of poly(A) mRNA between oocytes and embryos. The results obtained from individual oocytes suggested an association between poly(A) mRNA abundance and different morphological qualities of oocytes from COCs. In addition, a poly(A) mRNA profile was characterized from oocytes undergoing IVM, fertilization and blastocyst heating.
Biase
F H
FH
Departamento de Ciências Básicas, Faculdade de Zootecnia e Engenharia de Alimentos--Universidade de São Paulo, Rua Duque de Caxias Norte 225, Pirassununga-SP, Brasil. meirellf@usp.br
Fonseca Merighe
G Krempel
GK
Santos Biase
W Karyna Freitas
WK
Martelli
L
L
Meirelles
F Vieira
FV
D000818:Animals; D001755:Blastocyst; D002417:Cattle; D002970:Cleavage Stage, Ovum; D004622:Embryo, Mammalian / Q000502:physiology*; D005260:Female; D005307:Fertilization in Vitro; D020869:Gene Expression Profiling; D008297:Male; D009865:Oocytes / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism
D012333:RNA, Messenger