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The Identification of Novel Ovarian Proteases Through the Use of Genomic and Bioinformatic Methodologies¹

Division of Reproductive Sciences,³ Oregon National Primate Research Center, Oregon Health & Science University West Campus, Beaverton, Oregon 97006 Departments of Medical Informatics⁴ and Obstetrics and Gynecology,⁵ Oregon Health & Science University, Portland, Oregon 97239

ABSTRACT

Proteolytic activities are essential for follicular growth, ovulation, as well as for luteal formation and regression. Using suppression subtractive hybridization (SSH), a novel mouse ovary-selective gene (termed protease serine 35, Prss35) was identified. Analysis of the mouse genome database using the Prss35 sequence led to the identification of a homologous protease (protease serine 23, Prss23). PRSS35 possesses general features that are characteristic of serine (Ser) proteases, but is unique in that the canonical Ser that defines this enzyme family is replaced by a threonine (Thr). In contrast, PRSS23 possesses the standard catalytic Ser typical for this family of proteases. As determined by real-time polymerase chain reaction (PCR), the Prss35 mRNA levels increased around the time of ovulation and remained elevated in the developing corpus luteum. Steroid ablation/replacement studies demonstrated progesterone-dependent regulation of Prss35 gene expression prior to follicle rupture. Prss35 gene expression was localized to the theca cells of pre-antral follicles, the theca and granulosa cells of pre-ovulatory and ovulatory follicles, as well as to the developing corpus luteum. In contrast, Prss23 mRNA levels decreased transiently after ovulation induction and again in the postovulatory period. Prss23 gene expression was noted primarily in the granulosa cells of the secondary/early antral follicles. PRSS35 and PRSS23 orthologs in the rat, human, rhesus macaque, chimpanzee, cattle, dog, and chicken were identified and found to be highly homologous to one another (75–99% homology). Collectively, these results suggest that the PRSS35 and PRSS23 genes have been conserved as critical ovarian proteases throughout the course of vertebrate evolution.

corpus luteum, follicle, granulosa cell, ovary, theca cell

¹Supported by NICHD HD42000, NICHD U54-18185, and NCRR RR00163. The following sequences have been submitted to Genbank: mouse putative serine protease 35 mRNA, complete cDNA: DQ223037; rhesus macaque putative serine protease 35 mRNA: DQ223038; and rhesus macaque putative serine protease 23 mRNA: DQ223039.

Correspondence: ² Jon D. Hennebold, Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health & Science University West Campus, 505 NW 185th Ave., Beaverton, OR 97006. FAX: 503 690 5563; e-mail: henneboj{at}ohsu.edu

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