325 RETINOID-DEPENDENT POLY(A) MRNA CONTENTS IN BOVINE OOCYTES PREMATURED AND/OR MATURED IN VITRO

Abstract

Retinoic acid (RA) can induce cell differentiation and plays a role in controlling events within the cell cycle, but little is known of RA post-transcriptional modifications in the oocyte. Bovine oocyte and cumulus cells express most of RA receptors, and the presence of 9-cis-RA during in vitro prematuration and maturation (IVM) improves oocyte developmental competence (Duque et al., 2002 Hum. Reprod. 17, 2706–2714; Hidalgo et al., 2003 Reproduction 125, 409–416). This work analyzes the mRNA stability in bovine oocytes during in vitro prematuration and/or maturation. Cumulus-oocyte complexes (COCs) were cultured in defined medium with polyvinyl alcohol (DM). Those COCs undergoing prematuration were cultured for 24 h in DM with 25 μM roscovitine. For IVM, COCs were cultured in DM containing pFSH, LH and E₂ for 24 h, and some prematured COCs were then allowed to mature. Incubations were made at 39°C in 5% CO₂ in air and high humidity. Within experiments, COCs were cultured with 5 nM 9-cis-RA, in 1% ethanol (both as a vehicle and as an inhibitor of endogenous RA synthesis), 3% ethanol, 5% ethanol and untreated. Groups of 10 COCs per treatment were cultured, and oocytes detached from cumulus cells were analyzed. Poly(A) mRNA quantification was based on the pyrophosphorylation property of the DNA polymerase (Klenow). ATP production was measured by luminometric assay as a function of numbers of poly(A) tails. Data (4 replicates) were analyzed by ANOVA and Duncan’s test (^v,x,y,zP < 0.01; ^a,bP < 0.05), and poly(A) mRNA (pg oocyte⁻¹) was expressed as LSM ± SE. After prematuration, poly(A) mRNA contents differed between 9-cis-RA (125.7 ± 4.8^x) and untreated (95.5 ± 4.8^y) oocytes, as compared to 1% ethanol (72.2 ± 4.8^z) and immature (71.5 ± 4.8^z) oocytes. After IVM, untreated oocytes (23.0 ± 2.2^v) showed the lowest poly(A) mRNA amount, and poly(A) mRNA in 9-cis-RA (36.2 ± 2.2^y) basically equalled that in 1% ethanol (35.2 ± 2.2^y), while 3% (44.5 ± 2.2^yz) and 5% ethanol (52.0 ± 2.2^z) increased poly(A) mRNA levels. All groups of matured oocytes showed poly(A) mRNA contents lower than in immature (71.5 ± 4.8^x). After prematuration + maturation, poly(A) mRNA values were 34.2 ± 2.2^v (untreated + untreated), 36.5 ± 2.2^v (9-cis-RA + untreated), 49.5 ± 2.2^xa (untreated + 9-cis-RA), 41.0 ± 2.2^vxb (9-cis-RA + 9-cis-RA) and 59.0 ± 2.2^y (untreated + 1% ethanol). Levels of poly(A) mRNA from prematured + matured oocytes were again lower than in immature (71.5 ± 4.8^x). Our study shows that beneficial effects of RA on the oocyte developmental competence can be represented in part as a gain in the quality of mRNAs stored. Grant support: Spanish Ministry of Science and Technology (AGL-2002-01175).