Defect in SHAP-Hyaluronan Complex Causes Severe Female Infertility
2001; Elsevier BV; Volume: 276; Issue: 11 Linguagem: Inglês
10.1074/jbc.c000899200
ISSN1083-351X
AutoresLisheng Zhuo, Masahiko Yoneda, Ming Zhao, Wannarat Yingsung, Naoko Yoshida, Yasuo Kitagawa, Kumiko Kawamura, Toshiro Suzuki, Koji Kimata,
Tópico(s)Tendon Structure and Treatment
ResumoHyaluronan (HA) associates with proteins and proteoglycans to form the extracellular HA-rich matrices that significantly affect cellular behaviors. So far, only the heavy chains of the plasma inter-α-trypsin inhibitor (ITI) family, designated as SHAPs (serum-derivedhyaluronan-associated proteins), have been shown to bind covalently to HA. The physiological significance of such a unique covalent complex has been unknown but is of great interest, because HA and the ITI family are abundant in tissues and in plasma, respectively, and the SHAP-HA complex is formed wherever HA meets plasma. We abolished the formation of the SHAP-HA complex in mice by targeting the gene of bikunin, the light chain of the ITI family members, which is essential for their biosynthesis. As a consequence, the cumulus oophorus, an investing structure unique to the oocyte of higher mammals, had a defect in forming the extracellular HA-rich matrix during expansion. The ovulated oocytes were completely devoid of matrix and were unfertilized, leading to severe female infertility. Intraperitoneal administration of ITI, accompanied by the formation of the SHAP-HA complex, fully rescued the defects. We conclude that the SHAP-HA complex is a major component of the HA-rich matrix of the cumulus oophorus and is essential for fertilizationin vivo. Hyaluronan (HA) associates with proteins and proteoglycans to form the extracellular HA-rich matrices that significantly affect cellular behaviors. So far, only the heavy chains of the plasma inter-α-trypsin inhibitor (ITI) family, designated as SHAPs (serum-derivedhyaluronan-associated proteins), have been shown to bind covalently to HA. The physiological significance of such a unique covalent complex has been unknown but is of great interest, because HA and the ITI family are abundant in tissues and in plasma, respectively, and the SHAP-HA complex is formed wherever HA meets plasma. We abolished the formation of the SHAP-HA complex in mice by targeting the gene of bikunin, the light chain of the ITI family members, which is essential for their biosynthesis. As a consequence, the cumulus oophorus, an investing structure unique to the oocyte of higher mammals, had a defect in forming the extracellular HA-rich matrix during expansion. The ovulated oocytes were completely devoid of matrix and were unfertilized, leading to severe female infertility. Intraperitoneal administration of ITI, accompanied by the formation of the SHAP-HA complex, fully rescued the defects. We conclude that the SHAP-HA complex is a major component of the HA-rich matrix of the cumulus oophorus and is essential for fertilizationin vivo. hyaluronan α-1-microglobulin bikunin days post coitus heavy chain human chrionic gonadotropin inter-α-trypsin inhibitor pre-α-inhibitor urinary trypsin inhibitor kilobase embryonic cell polymerase chain reaction cumulus-oocyte complex chondroitin-4-sulfate Most cells undergoing active proliferation, differentiation, and locomotion in vitro and in vivo form extracellular hyaluronan (HA)1-rich matrices, which have been shown to play significant roles in many biological processes, such as tissue organization, morphogenesis, inflammation, and cancer metastasis (1Toole B.P. Hay E.D. Cell Biology of Extracellular Matrix. 2nd Ed. Plenum Press, New York1991: 305-341Crossref Google Scholar, 2Laurent T.C. Fraser J.R. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2053) Google Scholar, 3Knudson C.B. Knudson W. FASEB J. 1993; 7: 1233-1241Crossref PubMed Scopus (599) Google Scholar). We previously identified the covalent SHAP (serum-derivedhyaluronan-associated protein)-HA complex in the extracellular HA-rich matrix of cultured mouse dermal fibroblasts and found that the SHAPs are the heavy chains of the inter-α-trypsin inihibitor (ITI) family in serum supplemented to culture medium (4Yoneda M. Suzuki S. Kimata K. J. Biol. Chem. 1990; 265: 5247-5257Abstract Full Text PDF PubMed Google Scholar, 5Huang L. Yoneda M. Kimata K. J. Biol. Chem. 1993; 268: 26725-26730Abstract Full Text PDF PubMed Google Scholar, 6Zhao M. Yoneda M. Ohashi Y. Kurono S. Iwata H. Ohnuki Y. Kimata K. J. Biol. Chem. 1995; 270: 26657-26663Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar).The ITI family members are synthesized and assembled in liver and secreted into blood at high concentrations (0.15–0.5 mg/ml of plasma) (7Mizon C. Balduyck M. Albani D. Michalski C. Burnouf T. Mizon J. J. Immunol. Methods. 1996; 190: 61-70Crossref PubMed Scopus (31) Google Scholar). The members are composed of a common light chain, bikunin (Bik), and one or two of the three genetically different heavy chains (HC1, HC2, and HC3) (8Salier J.P. Rouet P. Raguenez G. Daveau M. Biochem. J. 1996; 315: 1-9Crossref PubMed Scopus (233) Google Scholar). Free bikunin in circulation is excreted rapidly into the urine (9Sugiki M. Sumi H. Maruyama M. Yoshida E. Mihara H. Enzyme. 1989; 42: 31-38Crossref PubMed Scopus (25) Google Scholar) where it is present as the urinary trypsin inhibitor (UTI) (10Proksch G.J. Lane J. Nordschow C.D. Clin. Biochem. 1973; 6: 200-206Crossref PubMed Scopus (27) Google Scholar). The serine residue at position 10 of bikunin contains anO-glycosidically linked chondroitin-4-sulfate (CS) chain (11Hochstrasser K. Schonberger O.L. Rossmanith I. Wachter E. Hoppe-Seyler's Z. Physiol. Chem. 1981; 362: 1357-1362Crossref PubMed Scopus (77) Google Scholar), to which the C terminal aspartate of a heavy chain is covalently bound via a unique ester bond (12Enghild J.J. Salvesen G. Hefta S.A. Thogersen I.B. Rutherfurd S. Pizzo S.V. J. Biol. Chem. 1991; 266: 747-751Abstract Full Text PDF PubMed Google Scholar). The SHAPs link to HA via an equivalent ester bond, suggesting that the formation of the SHAP-HA complex is a substitution reaction, namely HA replaces the CS of bikunin to link to a heavy chain (Fig.1) (6Zhao M. Yoneda M. Ohashi Y. Kurono S. Iwata H. Ohnuki Y. Kimata K. J. Biol. Chem. 1995; 270: 26657-26663Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Plasma includes an enzyme activity that catalyzes the reaction (13Yoneda M. Kida D. Iwata H. Zhao M. Kimata K. Connective Tissue. 1998; 30: 69-73Google Scholar). Therefore, we might expect that the SHAP-HA complex would be formed once plasma meets HA, thereby contributing to some physiological and pathological processes.This paper attempts to clarify not only the physiological function of the SHAP-HA complex but also that of the ITI family by eliminating their formation in mice in which the bikunin gene has been inactivated.DISCUSSIONThe present results assign a definite physiological function to the SHAP-HA complex, as well as the ITI family, in the process of ovulation and fertilization. It is now clear that the blood has roles in fertilization not only indirectly by transporting hormones and nutrients but also directly by participating in the construction of the expanded cumulus oophorus. The results also provide a new insight into the structure of the cumulus matrix and show directly the importance of the expanded cumulus oophorus in fertilization in vivo.The protease inhibitory activity of bikunin has drawn most of the research attention to the ITI family. A successful example is the clinical application of UTI to the treatment of acute pancreatitis and shock. The inhibitory activities of UTI to cancer metastasis (25Kobayashi H. Shinohara H. Fujie M. Gotoh J. Itoh M. Takeuchi K. Terao T. Int. J. Cancer. 1995; 63: 455-462Crossref PubMed Scopus (45) Google Scholar) and nephrolithiasis (15Atmani F. Lacour B. Drueke T. Daudon M. Urol. Res. 1993; 21: 61-66Crossref PubMed Scopus (51) Google Scholar) have also been reported. Here, we showed that bikunin is necessary for the formation of the SHAP-HA complex that is essential for fertilization. The finding identifies an important "SHAP-presenting" role for bikunin, i.e. activating (esterifying), transporting, and presenting the heavy chains to suitable recipients under suitable conditions, such as the newly synthesized HA in the expanding cumulus oophorus. Such a role is physiologically most important, because bikunin deficiency itself did not significantly impair ontogenesis. The fact that most bikunin in plasma is linked with the heavy chains (ITI and PαI) (8Salier J.P. Rouet P. Raguenez G. Daveau M. Biochem. J. 1996; 315: 1-9Crossref PubMed Scopus (233) Google Scholar), and the released bikunin in circulation is rapidly excreted into urine (9Sugiki M. Sumi H. Maruyama M. Yoshida E. Mihara H. Enzyme. 1989; 42: 31-38Crossref PubMed Scopus (25) Google Scholar), supports this notion.The influx of plasma into preovulatory follicles and the local formation of the SHAP-HA complex recalls similar situations in inflammatory sites, where cytokines stimulate local HA synthesis and induce capillary hyperpermeability to allow the efflux of plasma components. The SHAP-HA complex may also play roles in such inflammatory responses. Indeed, the SHAP-HA complex was found to accumulate significantly in the synovial fluid of patients suffering from rheumatoid arthritis (6Zhao M. Yoneda M. Ohashi Y. Kurono S. Iwata H. Ohnuki Y. Kimata K. J. Biol. Chem. 1995; 270: 26657-26663Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 26Kida D. Yoneda M. Miyaura S. Ishimaru T. Yoshida Y. Ito T. Ishiguro N. Iwata H. Kimata K. J. Rheumatol. 1999; 26: 1230-1238PubMed Google Scholar). Studies of the SHAP-HA complex will help us to understand the pathogenesis of such diseases.The molecular mechanism for the construction and metabolism of the HA-rich cumulus matrix is still largely unknown. Its HA-rich nature has been manifested by many studies (22Salustri A. Yanagishita M. Hascall V.C. J. Biol. Chem. 1989; 264: 13840-13847Abstract Full Text PDF PubMed Google Scholar, 27Chen L. Russell P.T. Larsen W.J. Mol. Reprod. Dev. 1993; 34: 87-93Crossref PubMed Scopus (233) Google Scholar, 28Talbot P. DiCarlantonio G. Dev. Biol. 1984; 103: 159-167Crossref PubMed Scopus (56) Google Scholar, 29Camaioni A. Hascall V.C. Yanagishita M. Salustri A. J. Biol. Chem. 1993; 268: 20473-20481Abstract Full Text PDF PubMed Google Scholar, 30Fulop C. Salustri A. Hascall V.C. Arch. Biochem. Biophys. 1997; 337: 261-266Crossref PubMed Scopus (131) Google Scholar). Here we demonstrate that the cumulus matrix contains the SHAP-HA complex as an essential component. Previous studies with scanning electron microscopy revealed many trypsin-sensitive granules along with the hyaluronan filament in the cumulus matrix (28Talbot P. DiCarlantonio G. Dev. Biol. 1984; 103: 159-167Crossref PubMed Scopus (56) Google Scholar). The N-terminal regions of the heavy chains of ITI have granule appearances (31Blom A.M. Mörgelin M. Oyen M. Jarvet J. Fries E. J. Biol. Chem. 1999; 274: 298-304Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Therefore, it is very likely that the granule-filament structure represents the SHAP-HA complex. Our findings also help explain the previous observations, which showed that ITI and PαI stabilized the HA-rich matrix of cultured cells (32Castillo G.M. Templeton D.M. FEBS Lett. 1993; 318: 292-296Crossref PubMed Scopus (49) Google Scholar, 33Blom A. Pertoft H. Fries E. J. Biol. Chem. 1995; 270: 9698-9701Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and in vitro cumulus expansion (34Chen L. Mao S.J.T. Larsen W.J. J. Biol. Chem. 1992; 267: 12380-12386Abstract Full Text PDF PubMed Google Scholar), and that HA oligomers interfered with ovulation in vivo(35Hess K.A. Chen L. Larsen W.J. Biol. Reprod. 1999; 61: 436-443Crossref PubMed Scopus (103) Google Scholar). The cumulus matrix might include other components, such as proteoglycans (36Camaioni A. Salustri A. Yanagishita M. Hascall V.C. Arch. Biochem. Biophys. 1996; 325: 190-198Crossref PubMed Scopus (66) Google Scholar, 37Eriksen G.V. Carlstedt I. Mörgelin M. Uldbjerg N. Malmström A.Y. Biochem. J. 1999; 340: 613-620Crossref PubMed Scopus (52) Google Scholar), TSG-6 (38Fulop C. Kamath R.V. Li Y. Otto J.M. Salustri A. Olsen B.R. Glant T.T. Hascall V.C. Gene. 1997; 202: 95-102Crossref PubMed Scopus (109) Google Scholar), and the link protein (39Kobayashi H. Sun G.W. Hirashima Y. Terao T. Endocrinology. 1999; 140: 3835-3842Crossref PubMed Google Scholar). The characterization of their interaction with the SHAP-HA complex is necessary for the completely delineation of the structure of cumulus matrix, for example the possible interaction between the SHAP and PG-M/versican (40Yoneda M. Zhao M. Zhuo L. Watanabe H. Yamada Y. Huang L. Nagasawa S. Nishimura H. Shinomura T. Isogai Z. Kimata K. Abatangelo G. Weigel P.H. New Frontiers in Medical Sciences: Redefining Hyaluronan. Elsevier Science Publishers B.V., Amsterdam2000: 21-30Google Scholar). The complete shedding of the cumulus cells in Bik−/− mice raised an interesting question about the role of SHAP in the anchoring of the cumulus oophorus matrix on the protein surface of the zona pellucida.We have clarified a molecular mechanism underlying a form of female infertility. It encourages us to survey spontaneous genetic mutations in the infertile women population to identify possible defects in ITI. So far no such case has been reported. However, a heritable null allele of the HC1 gene resulting from a deletion/frameshift has previously been identified (41Nakayashiki N. Ding M. Umetsu K. Yuasa I. Suzuki T. Aoki Y. Jpn. J. Hum. Genet. 1997; 42: 363-368Crossref PubMed Scopus (2) Google Scholar). Most cells undergoing active proliferation, differentiation, and locomotion in vitro and in vivo form extracellular hyaluronan (HA)1-rich matrices, which have been shown to play significant roles in many biological processes, such as tissue organization, morphogenesis, inflammation, and cancer metastasis (1Toole B.P. Hay E.D. Cell Biology of Extracellular Matrix. 2nd Ed. Plenum Press, New York1991: 305-341Crossref Google Scholar, 2Laurent T.C. Fraser J.R. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2053) Google Scholar, 3Knudson C.B. Knudson W. FASEB J. 1993; 7: 1233-1241Crossref PubMed Scopus (599) Google Scholar). We previously identified the covalent SHAP (serum-derivedhyaluronan-associated protein)-HA complex in the extracellular HA-rich matrix of cultured mouse dermal fibroblasts and found that the SHAPs are the heavy chains of the inter-α-trypsin inihibitor (ITI) family in serum supplemented to culture medium (4Yoneda M. Suzuki S. Kimata K. J. Biol. Chem. 1990; 265: 5247-5257Abstract Full Text PDF PubMed Google Scholar, 5Huang L. Yoneda M. Kimata K. J. Biol. Chem. 1993; 268: 26725-26730Abstract Full Text PDF PubMed Google Scholar, 6Zhao M. Yoneda M. Ohashi Y. Kurono S. Iwata H. Ohnuki Y. Kimata K. J. Biol. Chem. 1995; 270: 26657-26663Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). The ITI family members are synthesized and assembled in liver and secreted into blood at high concentrations (0.15–0.5 mg/ml of plasma) (7Mizon C. Balduyck M. Albani D. Michalski C. Burnouf T. Mizon J. J. Immunol. Methods. 1996; 190: 61-70Crossref PubMed Scopus (31) Google Scholar). The members are composed of a common light chain, bikunin (Bik), and one or two of the three genetically different heavy chains (HC1, HC2, and HC3) (8Salier J.P. Rouet P. Raguenez G. Daveau M. Biochem. J. 1996; 315: 1-9Crossref PubMed Scopus (233) Google Scholar). Free bikunin in circulation is excreted rapidly into the urine (9Sugiki M. Sumi H. Maruyama M. Yoshida E. Mihara H. Enzyme. 1989; 42: 31-38Crossref PubMed Scopus (25) Google Scholar) where it is present as the urinary trypsin inhibitor (UTI) (10Proksch G.J. Lane J. Nordschow C.D. Clin. Biochem. 1973; 6: 200-206Crossref PubMed Scopus (27) Google Scholar). The serine residue at position 10 of bikunin contains anO-glycosidically linked chondroitin-4-sulfate (CS) chain (11Hochstrasser K. Schonberger O.L. Rossmanith I. Wachter E. Hoppe-Seyler's Z. Physiol. Chem. 1981; 362: 1357-1362Crossref PubMed Scopus (77) Google Scholar), to which the C terminal aspartate of a heavy chain is covalently bound via a unique ester bond (12Enghild J.J. Salvesen G. Hefta S.A. Thogersen I.B. Rutherfurd S. Pizzo S.V. J. Biol. Chem. 1991; 266: 747-751Abstract Full Text PDF PubMed Google Scholar). The SHAPs link to HA via an equivalent ester bond, suggesting that the formation of the SHAP-HA complex is a substitution reaction, namely HA replaces the CS of bikunin to link to a heavy chain (Fig.1) (6Zhao M. Yoneda M. Ohashi Y. Kurono S. Iwata H. Ohnuki Y. Kimata K. J. Biol. Chem. 1995; 270: 26657-26663Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Plasma includes an enzyme activity that catalyzes the reaction (13Yoneda M. Kida D. Iwata H. Zhao M. Kimata K. Connective Tissue. 1998; 30: 69-73Google Scholar). Therefore, we might expect that the SHAP-HA complex would be formed once plasma meets HA, thereby contributing to some physiological and pathological processes. This paper attempts to clarify not only the physiological function of the SHAP-HA complex but also that of the ITI family by eliminating their formation in mice in which the bikunin gene has been inactivated. DISCUSSIONThe present results assign a definite physiological function to the SHAP-HA complex, as well as the ITI family, in the process of ovulation and fertilization. It is now clear that the blood has roles in fertilization not only indirectly by transporting hormones and nutrients but also directly by participating in the construction of the expanded cumulus oophorus. The results also provide a new insight into the structure of the cumulus matrix and show directly the importance of the expanded cumulus oophorus in fertilization in vivo.The protease inhibitory activity of bikunin has drawn most of the research attention to the ITI family. A successful example is the clinical application of UTI to the treatment of acute pancreatitis and shock. The inhibitory activities of UTI to cancer metastasis (25Kobayashi H. Shinohara H. Fujie M. Gotoh J. Itoh M. Takeuchi K. Terao T. Int. J. Cancer. 1995; 63: 455-462Crossref PubMed Scopus (45) Google Scholar) and nephrolithiasis (15Atmani F. Lacour B. Drueke T. Daudon M. Urol. Res. 1993; 21: 61-66Crossref PubMed Scopus (51) Google Scholar) have also been reported. Here, we showed that bikunin is necessary for the formation of the SHAP-HA complex that is essential for fertilization. The finding identifies an important "SHAP-presenting" role for bikunin, i.e. activating (esterifying), transporting, and presenting the heavy chains to suitable recipients under suitable conditions, such as the newly synthesized HA in the expanding cumulus oophorus. Such a role is physiologically most important, because bikunin deficiency itself did not significantly impair ontogenesis. The fact that most bikunin in plasma is linked with the heavy chains (ITI and PαI) (8Salier J.P. Rouet P. Raguenez G. Daveau M. Biochem. J. 1996; 315: 1-9Crossref PubMed Scopus (233) Google Scholar), and the released bikunin in circulation is rapidly excreted into urine (9Sugiki M. Sumi H. Maruyama M. Yoshida E. Mihara H. Enzyme. 1989; 42: 31-38Crossref PubMed Scopus (25) Google Scholar), supports this notion.The influx of plasma into preovulatory follicles and the local formation of the SHAP-HA complex recalls similar situations in inflammatory sites, where cytokines stimulate local HA synthesis and induce capillary hyperpermeability to allow the efflux of plasma components. The SHAP-HA complex may also play roles in such inflammatory responses. Indeed, the SHAP-HA complex was found to accumulate significantly in the synovial fluid of patients suffering from rheumatoid arthritis (6Zhao M. Yoneda M. Ohashi Y. Kurono S. Iwata H. Ohnuki Y. Kimata K. J. Biol. Chem. 1995; 270: 26657-26663Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 26Kida D. Yoneda M. Miyaura S. Ishimaru T. Yoshida Y. Ito T. Ishiguro N. Iwata H. Kimata K. J. Rheumatol. 1999; 26: 1230-1238PubMed Google Scholar). Studies of the SHAP-HA complex will help us to understand the pathogenesis of such diseases.The molecular mechanism for the construction and metabolism of the HA-rich cumulus matrix is still largely unknown. Its HA-rich nature has been manifested by many studies (22Salustri A. Yanagishita M. Hascall V.C. J. Biol. Chem. 1989; 264: 13840-13847Abstract Full Text PDF PubMed Google Scholar, 27Chen L. Russell P.T. Larsen W.J. Mol. Reprod. Dev. 1993; 34: 87-93Crossref PubMed Scopus (233) Google Scholar, 28Talbot P. DiCarlantonio G. Dev. Biol. 1984; 103: 159-167Crossref PubMed Scopus (56) Google Scholar, 29Camaioni A. Hascall V.C. Yanagishita M. Salustri A. J. Biol. Chem. 1993; 268: 20473-20481Abstract Full Text PDF PubMed Google Scholar, 30Fulop C. Salustri A. Hascall V.C. Arch. Biochem. Biophys. 1997; 337: 261-266Crossref PubMed Scopus (131) Google Scholar). Here we demonstrate that the cumulus matrix contains the SHAP-HA complex as an essential component. Previous studies with scanning electron microscopy revealed many trypsin-sensitive granules along with the hyaluronan filament in the cumulus matrix (28Talbot P. DiCarlantonio G. Dev. Biol. 1984; 103: 159-167Crossref PubMed Scopus (56) Google Scholar). The N-terminal regions of the heavy chains of ITI have granule appearances (31Blom A.M. Mörgelin M. Oyen M. Jarvet J. Fries E. J. Biol. Chem. 1999; 274: 298-304Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Therefore, it is very likely that the granule-filament structure represents the SHAP-HA complex. Our findings also help explain the previous observations, which showed that ITI and PαI stabilized the HA-rich matrix of cultured cells (32Castillo G.M. Templeton D.M. FEBS Lett. 1993; 318: 292-296Crossref PubMed Scopus (49) Google Scholar, 33Blom A. Pertoft H. Fries E. J. Biol. Chem. 1995; 270: 9698-9701Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and in vitro cumulus expansion (34Chen L. Mao S.J.T. Larsen W.J. J. Biol. Chem. 1992; 267: 12380-12386Abstract Full Text PDF PubMed Google Scholar), and that HA oligomers interfered with ovulation in vivo(35Hess K.A. Chen L. Larsen W.J. Biol. Reprod. 1999; 61: 436-443Crossref PubMed Scopus (103) Google Scholar). The cumulus matrix might include other components, such as proteoglycans (36Camaioni A. Salustri A. Yanagishita M. Hascall V.C. Arch. Biochem. Biophys. 1996; 325: 190-198Crossref PubMed Scopus (66) Google Scholar, 37Eriksen G.V. Carlstedt I. Mörgelin M. Uldbjerg N. Malmström A.Y. Biochem. J. 1999; 340: 613-620Crossref PubMed Scopus (52) Google Scholar), TSG-6 (38Fulop C. Kamath R.V. Li Y. Otto J.M. Salustri A. Olsen B.R. Glant T.T. Hascall V.C. Gene. 1997; 202: 95-102Crossref PubMed Scopus (109) Google Scholar), and the link protein (39Kobayashi H. Sun G.W. Hirashima Y. Terao T. Endocrinology. 1999; 140: 3835-3842Crossref PubMed Google Scholar). The characterization of their interaction with the SHAP-HA complex is necessary for the completely delineation of the structure of cumulus matrix, for example the possible interaction between the SHAP and PG-M/versican (40Yoneda M. Zhao M. Zhuo L. Watanabe H. Yamada Y. Huang L. Nagasawa S. Nishimura H. Shinomura T. Isogai Z. Kimata K. Abatangelo G. Weigel P.H. New Frontiers in Medical Sciences: Redefining Hyaluronan. Elsevier Science Publishers B.V., Amsterdam2000: 21-30Google Scholar). The complete shedding of the cumulus cells in Bik−/− mice raised an interesting question about the role of SHAP in the anchoring of the cumulus oophorus matrix on the protein surface of the zona pellucida.We have clarified a molecular mechanism underlying a form of female infertility. It encourages us to survey spontaneous genetic mutations in the infertile women population to identify possible defects in ITI. So far no such case has been reported. However, a heritable null allele of the HC1 gene resulting from a deletion/frameshift has previously been identified (41Nakayashiki N. Ding M. Umetsu K. Yuasa I. Suzuki T. Aoki Y. Jpn. J. Hum. Genet. 1997; 42: 363-368Crossref PubMed Scopus (2) Google Scholar). The present results assign a definite physiological function to the SHAP-HA complex, as well as the ITI family, in the process of ovulation and fertilization. It is now clear that the blood has roles in fertilization not only indirectly by transporting hormones and nutrients but also directly by participating in the construction of the expanded cumulus oophorus. The results also provide a new insight into the structure of the cumulus matrix and show directly the importance of the expanded cumulus oophorus in fertilization in vivo. The protease inhibitory activity of bikunin has drawn most of the research attention to the ITI family. A successful example is the clinical application of UTI to the treatment of acute pancreatitis and shock. The inhibitory activities of UTI to cancer metastasis (25Kobayashi H. Shinohara H. Fujie M. Gotoh J. Itoh M. Takeuchi K. Terao T. Int. J. Cancer. 1995; 63: 455-462Crossref PubMed Scopus (45) Google Scholar) and nephrolithiasis (15Atmani F. Lacour B. Drueke T. Daudon M. Urol. Res. 1993; 21: 61-66Crossref PubMed Scopus (51) Google Scholar) have also been reported. Here, we showed that bikunin is necessary for the formation of the SHAP-HA complex that is essential for fertilization. The finding identifies an important "SHAP-presenting" role for bikunin, i.e. activating (esterifying), transporting, and presenting the heavy chains to suitable recipients under suitable conditions, such as the newly synthesized HA in the expanding cumulus oophorus. Such a role is physiologically most important, because bikunin deficiency itself did not significantly impair ontogenesis. The fact that most bikunin in plasma is linked with the heavy chains (ITI and PαI) (8Salier J.P. Rouet P. Raguenez G. Daveau M. Biochem. J. 1996; 315: 1-9Crossref PubMed Scopus (233) Google Scholar), and the released bikunin in circulation is rapidly excreted into urine (9Sugiki M. Sumi H. Maruyama M. Yoshida E. Mihara H. Enzyme. 1989; 42: 31-38Crossref PubMed Scopus (25) Google Scholar), supports this notion. The influx of plasma into preovulatory follicles and the local formation of the SHAP-HA complex recalls similar situations in inflammatory sites, where cytokines stimulate local HA synthesis and induce capillary hyperpermeability to allow the efflux of plasma components. The SHAP-HA complex may also play roles in such inflammatory responses. Indeed, the SHAP-HA complex was found to accumulate significantly in the synovial fluid of patients suffering from rheumatoid arthritis (6Zhao M. Yoneda M. Ohashi Y. Kurono S. Iwata H. Ohnuki Y. Kimata K. J. Biol. Chem. 1995; 270: 26657-26663Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 26Kida D. Yoneda M. Miyaura S. Ishimaru T. Yoshida Y. Ito T. Ishiguro N. Iwata H. Kimata K. J. Rheumatol. 1999; 26: 1230-1238PubMed Google Scholar). Studies of the SHAP-HA complex will help us to understand the pathogenesis of such diseases. The molecular mechanism for the construction and metabolism of the HA-rich cumulus matrix is still largely unknown. Its HA-rich nature has been manifested by many studies (22Salustri A. Yanagishita M. Hascall V.C. J. Biol. Chem. 1989; 264: 13840-13847Abstract Full Text PDF PubMed Google Scholar, 27Chen L. Russell P.T. Larsen W.J. Mol. Reprod. Dev. 1993; 34: 87-93Crossref PubMed Scopus (233) Google Scholar, 28Talbot P. DiCarlantonio G. Dev. Biol. 1984; 103: 159-167Crossref PubMed Scopus (56) Google Scholar, 29Camaioni A. Hascall V.C. Yanagishita M. Salustri A. J. Biol. Chem. 1993; 268: 20473-20481Abstract Full Text PDF PubMed Google Scholar, 30Fulop C. Salustri A. Hascall V.C. Arch. Biochem. Biophys. 1997; 337: 261-266Crossref PubMed Scopus (131) Google Scholar). Here we demonstrate that the cumulus matrix contains the SHAP-HA complex as an essential component. Previous studies with scanning electron microscopy revealed many trypsin-sensitive granules along with the hyaluronan filament in the cumulus matrix (28Talbot P. DiCarlantonio G. Dev. Biol. 1984; 103: 159-167Crossref PubMed Scopus (56) Google Scholar). The N-terminal regions of the heavy chains of ITI have granule appearances (31Blom A.M. Mörgelin M. Oyen M. Jarvet J. Fries E. J. Biol. Chem. 1999; 274: 298-304Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Therefore, it is very likely that the granule-filament structure represents the SHAP-HA complex. Our findings also help explain the previous observations, which showed that ITI and PαI stabilized the HA-rich matrix of cultured cells (32Castillo G.M. Templeton D.M. 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It encourages us to survey spontaneous genetic mutations in the infertile women population to identify possible defects in ITI. So far no such case has been reported. However, a heritable null allele of the HC1 gene resulting from a deletion/frameshift has previously been identified (41Nakayashiki N. Ding M. Umetsu K. Yuasa I. Suzuki T. Aoki Y. Jpn. J. Hum. Genet. 1997; 42: 363-368Crossref PubMed Scopus (2) Google Scholar). We thank M. Hooper for the E14 ES cell line, H. Kondo for the STO/NHL feeder cell line, F. Azumi and M. Matsumoto for technical assistance in animal experiments, and A. Iida for technical assistance in histology; also, we thank A. Salustri and V. C. Hascall for kind advice and Drs. H. Watanabe and H. Suzuki for critical reading of this manuscript.
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