257 DIFFERENTIAL GENE EXPRESSION IN GERMINAL VESICLE AND METAPHASE II MOUSE OOCYTES.

Abstract

The present study was conducted to identify oocyte-specific genes according to developmental stage. We used an accurate Annealing Control Primer (ACP;; SeeGene, Seoul, Korea)-based GeneFishing technology. This new and innovative method has developed 120 ACPs so far to locate differentially expressed genes. This method is more specific and sensitive than differential display-PCR that is labor-intensive and results in a high degree of false positives. To identify genes differentially expressed in germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, fully grown GV-intact and MII oocytes were obtained. The addition of 0.2 mM IBMX in the collection medium inhibited GV breakdown. The mRNA samples were prepared from the same number of GV and MII oocytes using a Dynabeads mRNA DIRECT Kit. GeneFishing system utilizes a two-stage PCR to fish out authentic differentially expressed genes by regulating binding of each portion of ACP at a different stage of PCR. The differentially expressed bands were extracted and cloned into a TOPO TA cloning vector (Invitrogen, Carlsbad, CA, USA), sequenced, and analyzed by BLAST search. To confirm the differential expression of selected transcripts, RT-PCR and immunofluorescence staining were conducted. Using 12 different ACPs, we isolated 27 differentially expressed bands from GV and MII oocytes. BLAST results indicated that most of these bands had strong homology with known genes. We confirmed that pleckstrin homology, protein kinase D2 (PKD), and COP9 (constitutive photomorphogenic) homologs were GV-specific, while minichromosome maintenance deficient 2 mitotin (MCM2), malate dehydrogenase, soluble Mor2, growth arrest specific 6 (Gas-6), Bcl-2 homolog (Diva), and suppressor of cytokine signaling 4 were expressed highly in MII. Immunofluoresence staining results confirmed higher expression of Gas-6 protein in MII oocytes compared to GV oocytes. Immunohistochemistry results with ovarian tissues demonstrated that MCM2 protein was highly expressed in the nucleus of developing oocytes and granulosa cells, and Diva was highly expressed in the oocytes of primordial follicles. Immunofluoresence staining for these proteins is under investigation. The functions of many of these genes have been determined in systems other than gamete systems. Those data provide insight about their function in oocyte maturation. For example, PKD, COP9, and pleckstrin homology are genes in the axis of a newly identified PKD signaling system. Therefore, further investigation of the functions of each gene identified in this study will provide a basis for understanding the mechanism of oocyte maturation.