Processing and Localization of ADAMTS-1 and Proteolytic Cleavage of Versican during Cumulus Matrix Expansion and Ovulation

Darryl L Russell,Kari M.H Doyle,Scott A Ochsner,John D Sandy,Joanne S Richards

doi:10.1074/jbc.m300519200

Abstract

ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs-1) is a member of the ADAMTS family of metalloproteases which, together with ADAMTS-4 and ADAMTS-5, has been shown to degrade members of the lectican family of proteoglycans. ADAMTS-1 mRNA is induced in granulosa cells of periovulatory follicles by the luteinizing hormone surge through a progesterone receptor-dependent mechanism. Female progesterone receptor knockout (PRKO) mice are infertile primarily due to ovulatory failure and lack the normal periovulatory induction of ADAMTS-1 mRNA. We therefore investigated the protein localization and function of ADAMTS-1 in ovulating ovaries. Antibodies against two specific peptide regions, the pro-domain and the metalloprotease domain of ADAMTS-1, were generated. Pro-ADAMTS-1 of 110 kDa was identified in mural granulosa cells and appears localized to cytoplasmic secretory vesicles. The mature (85-kDa pro-domain truncated) form accumulated in the extracellular matrix of the cumulus oocyte complex (COC) during the process of matrix expansion. Each form of ADAMTS-1 protein increased >10-fold after the ovulatory luteinizing hormone surge in wild-type but not PRKO mice. Versican is also localized selectively to the ovulating COC matrix and was found to be cleaved yielding a 70-kDa N-terminal fragment immunopositive for the neoepitope DPEAAE generated by ADAMTS-1 and ADAMTS-4 protease activity. This extracellular processing of versican was reduced in ADAMTS-1-deficient PRKO mouse ovaries. These observations suggest that one function of ADAMTS-1 in ovulation is to cleave versican in the expanded COC matrix and that the anovulatory phenotype of PRKO mice is at least partially due to loss of this function.

Highlights

ADAMTS-1 is a member of the ADAMTS family of metalloproteases which, together with ADAMTS-4 and ADAMTS-5, has been shown to degrade members of the lectican family of proteoglycans
ADAMTS-1 mRNA is induced in granulosa cells of periovulatory follicles by the luteinizing hormone surge through a progesterone receptor-dependent mechanism
Immunoreactive Precursor and Mature ADAMTS-1 Produced by Ovulating Granulosa Cells—Antibodies raised against the pro- and metalloprotease (MP)-domains of ADAMTS-1 were tested by Western blot using 20 ␮g of protein extracted from whole ovaries of wild-type C57BL/6 mice isolated after a time course of PMSG/hCG stimulation (Fig. 2A)

Summary

Introduction

ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs-1) is a member of the ADAMTS family of metalloproteases which, together with ADAMTS-4 and ADAMTS-5, has been shown to degrade members of the lectican family of proteoglycans. Versican is localized selectively to the ovulating COC matrix and was found to be cleaved yielding a 70-kDa N-terminal fragment immunopositive for the neoepitope DPEAAE generated by ADAMTS-1 and ADAMTS-4 protease activity This extracellular processing of versican was reduced in ADAMTS-1-deficient PRKO mouse ovaries. Based on the anovulatory phenotypes of mice with null mutations of cyclooxygenase-2 (COX-2) [7, 8], prostaglandin E2 receptor [9, 10], and bikunin [11], it is becoming evident that expansion of the matrix surrounding the cumulus cells and oocyte is essential for ovulation. The C-terminal globular domain of versican can interact with sulfated cell surface glycolipids [26], ECM glycoproteins such as tenascin-R [27, 28]

Methods

Results

Conclusion