In the ovary, oocytes are arrested at the prophase of the first meiotic division. Shortly before ovulation, meiotic division resumes only to be arrested at the metaphase of the second division. A protein-serine/threonine kinase (cytostatic factor) is responsible for this arrest by inhibiting cyclin degradation and stabilizing maturation promoting factor during metaphase II. This cytostatic factor has been identified as the proto-oncogene c-mos. In this project, we cloned a 328 bp segment of the rhesus monkey c-mos using reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted from a rhesus ovary, reverse transcribed using a poly d(T) primer and subjected to polymerase chain reaction using sense (5'-CGG.TGT.TCC.TGT.GGC.CAT.AA -3') and antisense (5'-GAT.GTT.GTG.AAC.GGC.CTG.CT -3') primers derived from the published human c-mos cDNA sequence. After purification in NuSieve gel, the RT-PCR product was subcloned into pGEM-T vector and sequenced. This revealed a 97.3% homology with the human c-mos cDNA sequence. In situ hybridization detected c-mos mRNA predominantly in oocytes in the primate ovary. Mos mRNA is not expressed in primordial oocytes but high levels are present throughout oocyte growth and maturation including antral and preovulatory Graafian follicles. The next phase is to study the expression of c-mos mRNA in germinal vesicle- intact, MI, and MII oocytes as well as preimplantation embryos at the pronuclear and cleavage stages. To this end, we are developing a quantitative RT-PCR system by subcloning the c-mos cDNA in the pSP64(polyA) vector. This will allow us to generate a standard curve using the poly(A) tail of this vector to quantify the levels of c-mos mRNA in different stages of oocytes and preimplantation embryos. Finally, we will use a c-mos antibody to study the pattern of protein distribution and compare that to mRNA levels.
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