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[OR14-1] Fxna, a Novel Gene Differentially Expressed in the Rat Ovary at the Time of Folliculogenesis Is Required for Normal Ovarian Histogenesis.
Cecilia Garcia-Rudaz, Felix Luna, Gregory A Dissen, Thomas Scior, Neil D Rawlings, Francesco Galimi, Sergio R Ojeda. Div of Neuroscience, Oregon Natl Primate Res Ctr, Beaverton, OR; Instituto de Ciencias, Puebla, Mexico; the Sanger Inst, Hinxton, UK; the Salk Inst, San Diego, CA
In rodents, follicular formation is an event that occurs after birth. In recent years several factors required for follicular assembly and the growth of the newly formed follicles have been identified, and some of their roles have been characterized. We now describe the expression and potential functions of a novel gene identified by differential display in the neonatal rat ovary, and that we have named Fxna (GenBank Acc. No. NM_184050). The Fxna gene consists of 15 exons spanning 34,200 bp in rat chromosome 1. It encodes an mRNA of 5.1 Kb in length, and a protein of 898 amino acids. Fxna belongs to the M28 family of aminopeptidases (presumably EC 3.4.11.10). Computer modeling predicts a globular protein with a hydrophobic core containing a catalytic site endowed with a double zinc finger-domain, consistent with an involvement of Fxna in the enzymatic cleavage of prepro-proteins. Fxna expression is highest in the ovary, kidney and brain. In the ovary, Fxna mRNA levels (measured by RNase protection assay) become maximally elevated 48 h after birth, i.e.. during follicular assembly. Fxna mRNA is expressed in somatic cells of the ovary (mostly in granulosa cells), but not in oocytes. To determine the role that Fxna may play in ovarian development we used siRNA technology. Upon identification of an siRNA effective in knocking down Fxna expression in a rat embryonic kidney cell line, the cDNA encoding the most effective siRNA was placed under the control of the U6 RNA polymerase III promoter to generate short hairpin siRNAs. A third generation lentiviral vector, containing an enhanced green fluorescent protein (EGFP) reporter gene and the U6-siRNA producing cassette, was then used to deliver Fxna siRNA to the ovary. Newborn rat ovaries in organ culture were exposed for four days to 4 x 106 transforming units (TU)/ml of either the lentiviral vector alone or the vector producing Fxna siRNA. As determined by EGFP immunohistochemistry, most ovarian cells were infected. Histological analysis of the ovaries demonstrated widespread disorganization of follicular assembly, with many abnormal follicles containing more than one oocyte, and clusters of somatic cells not associated with any oocytes. These results suggest that Fxna is required for the correct organization of somatic cells and oocytes into discrete follicular structures.
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