251 SEARCH FOR GENES INVOLVED IN DEVELOPMENTAL COMPETENCE IN MOUSE OOCYTES USING SUPPRESSION SUBTRACTIVE HYBRIDIZATION

Abstract

During the first month after birth, synchronous follicular growth occurs in the ovary of immature mice (first wave). Previously, we showed that mouse oocytes during the first wave were more competent developmentally in older females (Suzuki O et al., 2002 Theriogenology 57, 628 abst), although the numbers of mature oocytes did not differ with female age (17, 18, and 24 days old). In this study, we examined the genetic factors that affect the developmental competence of mouse oocytes during the first wave using suppression subtractive hybridization (SSH). Oocytes collected from 17- and 24-day-old B6D2F1 females (D17 and D24, respectively) without hormonal treatment were matured in Waymouth medium supplemented with pyruvate (0.23 mM), antibiotics, bovine fetuin (1 mg mL⁻¹), and polyvinylpyrrolidone (3 mg mL⁻¹). After 17-h culture at 37°C in an atmosphere of 5% CO₂, 5% O₂, and 90% N₂, total RNA was isolated from oocytes whose germinal vesicles had broken down (mature oocytes), separately, in three independent culture groups per age (each group contained oocytes from four animals) using Cell-to-cDNA Cell Lysis Buffer (Ambion, Austin, TX, USA). Some of the total RNA from each independent group was pooled by age (total of RNA from approximately 100 oocytes per age) and used for SSH. A SMART cDNA Synthesis Kit (Clontech, Palo Alto, CA, USA) was used to reverse-transcribe total RNA to cDNA. SSH was performed with a PCR-Select cDNA Subtraction Kit (Clontech). The subtracted PCR products were cloned into pGEM-T vector (Promega, Madison, WI, USA). Clones from the subtracted library (D24−D17) were sequenced and their identities were examined using the NCBI BLAST search. The differential expression of candidate genes preferentially expressed in mature D24 oocytes suggested by SSH was confirmed with cDNA transcribed separately in the three independent culture groups per age using real-time quantitative PCR with an ABI Prism 7900HT with TaqMan technology (Applied Biosystems, Foster City, CA, USA). Of 513 clones sequenced, the top six preferentially-expressed candidate genes in more developmentally-competent D24 oocytes were spindlin (20 clones), bmi-1 (4 clones), cyclin B1 (4 clones), E330034G19Rik (4 clones), Jagged1 (4 clones), and Ndfip2 (4 clones). The expression of spindlin in mature D24 oocytes (relative threshold cycle: −3.8 ± 0.7, mean ± SD) was confirmed to be approximately 11-fold higher than in D17 oocytes (−0.3 ± 1.5) when GAPDH was used as an internal control (P < 0.05, t-test). Quantitative analyses of mRNA expression of the remaining genes are now under way. Our results suggest that spindlin is one of the key factors leading to the acquisition of developmental competence in mouse oocytes during folliculogenesis. Supported by JSPS KAKENHI (No.145716000) and MHLW.