Summary PTX 3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization

Fertilization of a mammalian oocyte requires a series of interactions between spermatozoa and the extracellular investments of the oocyte. In contrast to the zona pellucida, the cumulus oophorus had not received much attention until recently. However, findings reported in the past few years indicate that the cumulus matrix, though dispensable in vitro, plays a key role in the early events of in vivo fertilization (Hizaki et al., 1999; Zhuo et al., 2001; Fulop et al., 2003). This matrix is synthesized by cumulus cells a few hours before ovulation and, in the mouse, the combined action of gonadotropins and soluble oocyte factors is required for triggering this process (Buccione et al., 1990; Salustri et al., 1990b). The major component is hyaluronan (HA), a large polyanionic glycosaminoglycan responsible for viscoelastic properties and expansion of the cumulus oophorus (Salustri et al., 1992). In vitro and in vivo studies have shown that serumderived inter-α-trypsin inhibitor (IαI or ITI) is essential for HA-matrix assembly and mouse female fertility (Chen et al., 1992; Chen et al., 1996; Zhuo et al., 2001; Fulop et al., 2003). ΙαΙ consists of a small protein, named bikunin or light chain, with a chondroitin sulfate moiety that connects two additional proteins, named heavy chains. The heavy chains of ΙαΙ are covalently transferred to HA in the preovulatory follicle. It has been proposed that these polypeptides stabilize the HA-matrix by cross-linking HA molecules through covalent and ionic bonds. Another crucial component of the cumulus matrix is tumour necrosis factor α -induced protein 6 (TNFAIP6 or TSG6), a multifunctional protein usually associated with inflammation, which has the ability to specifically bind HA (Lee et al., 1992; Milner and Day, 2003). TNFAIP6 is synthesized by cumulus and granulosa cells in the preovulatory follicle (Fulop et al., 1997; Yoshioka et al., 2000), and T faip6deficient mice are unable to form stable cumulus matrix and are sterile (Fulop et al., 2003). Covalent transfer of heavy chains from ΙαΙ to HA does not occur in Tnfaip6–/– mice, indicating that TNFAIP6 is a key catalyst in this reaction. Oocytes, besides promoting cumulus matrix synthesis, inhibit hormone-induced proteolytic enzyme expression by mouse cumulus cells during matrix deposition, probably providing an additional mechanism for matrix stabilization (Canipari et al., 1995). Indeed, synthesis of cumulus matrix ceases at ovulation PTX3 is a prototypic long pentraxin that plays a nonredundant role in innate immunity against selected pathogens and in female fertility. Here, we report that the infertility of Ptx3 –/– mice is associated with severe abnormalities of the cumulus oophorus and failure of in vivo, but not in vitro, oocyte fertilization. PTX3 is produced by mouse cumulus cells during cumulus expansion and localizes in the matrix. PTX3 is expressed in the human cumulus oophorus as well. Cumuli from Ptx3–/– mice synthesize normal amounts of hyaluronan (HA), but are unable to organize it in a stable matrix. Exogenous PTX3 restores a normal cumulus phenotype. Incorporation in the matrix of interα-trypsin inhibitor is normal in Ptx3–/– cumuli. PTX3 does not interact directly with HA, but it binds the cumulus matrix hyaladherin tumor necrosis factor α-induced protein 6 (TNFAIP6, also known as TSG6) and thereby may form multimolecular complexes that can cross-link HA chains. Thus, PTX3 is a structural constituent of the cumulus oophorus extracellular matrix essential for female fertility.