Inherited Proteoglycan Biosynthesis Defects-Current Laboratory Tools and Bikunin as a Promising Blood Biomarker.

Proteoglycans consist of proteins linked to sulfated glycosaminoglycan chains. They constitute a family of macromolecules mainly involved in the architecture of organs and tissues as major components of extracellular matrices. Some proteoglycans also act as signaling molecules involved in inflammatory response as well as cell proliferation, adhesion, and differentiation. Inborn errors of proteoglycan metabolism are a group of orphan diseases with severe and irreversible skeletal abnormalities associated with multiorgan impairments. Identifying the gene variants that cause these pathologies proves to be difficult because of unspecific clinical symptoms, hardly accessible functional laboratory tests, and a lack of convenient blood biomarkers. In this review, we summarize the molecular pathways of proteoglycan biosynthesis, the associated inherited syndromes, and the related biochemical screening techniques, and we focus especially on a circulating proteoglycan called bikunin and on its potential as a new biomarker of these diseases.

The roles of free iron, heme, haemoglobin, and the scavenger proteins haemopexin and alpha-1-microglobulin in preeclampsia and fetal growth restriction.

BACKGROUND: Preeclampsia (PE) is a complex pregnancy syndrome characterised by maternal hypertension and organ damage after 20 weeks of gestation and is associated with an increased risk of cardiovascular disease later in life. Extracellular haemoglobin (Hb) and its metabolites heme and iron are highly toxic molecules and several defence mechanisms have evolved to protect the tissue. OBJECTIVES: We will discuss the roles of free iron, heme, Hb, and the scavenger proteins haemopexin and alpha-1-microglobulin in pregnancies complicated by PE and fetal growth restriction (FGR). CONCLUSION: In PE, oxidative stress causes syncytiotrophoblast (STB) stress and increased shedding of placental STB-derived extracellular vesicles (STBEV). The level in maternal circulation correlates with the severity of hypertension and supports the involvement of STBEVs in causing maternal symptoms in PE. In PE and FGR, iron homeostasis is changed, and iron levels significantly correlate with the severity of the disease. The normal increase in plasma volume taking place during pregnancy is less for PE and FGR and therefore have a different impact on, for example, iron concentration, compared to normal pregnancy. Excess iron promotes ferroptosis is suggested to play a role in trophoblast stress and lipotoxicity. Non-erythroid α-globin regulates vasodilation through the endothelial nitric oxide synthase pathway, and hypoxia-induced α-globin expression in STBs in PE placentas is suggested to contribute to hypertension in PE. Underlying placental pathology in PE with and without FGR might be amplified by iron and heme overload causing oxidative stress and ferroptosis. As the placenta becomes stressed, the release of STBEVs increases and affects the maternal vasculature.

Midgut Laterality Is Driven by Hyaluronan on the Right.

For many years, biologists have focused on the role of Pitx2, expressed on the left side of developing embryos, in governing organ laterality. Here, we identify a different pathway during left-right asymmetry initiated by the right side of the embryo. Surprisingly, this conserved mechanism is orchestrated by the extracellular glycosaminoglycan, hyaluronan (HA) and is independent of Pitx2 on the left. Whereas HA is normally synthesized bilaterally as a simple polysaccharide, we show that covalent modification of HA by the enzyme Tsg6 on the right triggers distinct cell behavior necessary to drive the conserved midgut rotation and to pattern gut vasculature. HA disruption in chicken and Tsg6-/- mice results in failure to initiate midgut rotation and perturbs vascular development predisposing to midgut volvulus. Our study leads us to revise the current symmetry-breaking paradigm in vertebrates and demonstrates how enzymatic modification of HA matrices can execute the blueprint of organ laterality.

ITIH3 and ITIH4 polymorphisms and depressive symptoms during pregnancy in Japan: the Kyushu Okinawa Maternal and Child Health Study.

ITIH3 and ITIH4 are involved in the stabilization of the extracellular matrix. Several genome-wide association studies and case-control studies regarding psychiatric disorders have identified ITIH3 and ITIH4 single nucleotide polymorphisms (SNPs). The present case-control study examined the relationship between ITIH3 SNPs rs2535629 and rs736408 and ITIH4 SNPs rs3821831 and rs2239547 and depressive symptoms during pregnancy in Japan. Cases comprised 273 women with depressive symptoms during pregnancy defined as a Center for Epidemiological Studies Depression Scale (CES-D) score ≥ 16. Control subjects comprised 1176 women without depressive symptoms during pregnancy, according to the CES-D criteria, who had not been diagnosed with depression by a doctor. Adjustment was made for age, gestation at baseline, region of residence, the presence of children, family structure, smoking, employment, and education. Compared with the TT genotype of ITIH4 SNP rs2239547, the CC genotype was significantly related to a reduced risk of depressive symptoms during pregnancy: the adjusted odds ratio (95% CI) was 0.84 (0.63-1.11) for the TC genotype and 0.57 (0.36-0.91) for the CC genotype. ITIH3 SNPs rs2535629 and rs736408 and ITIH4 SNP rs3821831 were not related to depressive symptoms during pregnancy. The GCCT haplotype of rs2535629, rs736408, rs3821831, and rs2239547 was significantly positively associated with depressive symptoms during pregnancy. A significant interaction was found between rs2239547 and the presence of children. This is the first study to show significant associations of ITIH4 SNP rs2239547 and the GCCT haplotype with depressive symptoms during pregnancy. The effect of the presence of children might depend on rs2239547.

Organization of the expanded cumulus-extracellular matrix in preovulatory follicles: a role for inter-alpha-trypsin inhibitor.

It has been shown that following endogenous gonadotropin surge, oocyte-cumulus complexes (OCC) synthesize hyaluronan (HA) in a process called cumulus expansion. During this process, HA associates with proteins and proteoglycans to form the expanded HA-rich oocyte-cumulus extracellular matrix (ECM), where the heavy chains of the serum derived inter-α-trypsin inhibitor family (IαI) bind covalently to HA. No study has been performed on the occurrence and regulation of this process during oocyte maturation in species other than mouse and pig, although, the heavy chains (of IαI)-HA complex was purified from human amniotic membrane. The present review pointing out that: 1/ formation of expanded HA-rich oocyte-cumulus ECM is dependent on the presence of IαI molecules, 2/ the heavy chains of IαI molecules identified in the serum are covalently linked to HA during cumulus expansion in mouse and pig, 3/ the family of IαI molecules can freely cross the blood-follicle barrier, and the follicular fluid collected at any stage of folliculogenesis can be successfully used instead of serum to form expanded cumulus ECM in pig, and 4/ proteins of the IαI family can affect reproductive process by modulating the expression of a large number of cellular genes during a preovulatory period. Finally, this review provides clear evidence that IαI family members present in the serum or follicular fluid become responsible for cumulus expansion, as without these proteins, expanded cumulus HA-rich ECM is not formed and HA is released into medium.

Systemic differential gene regulation of the inter-α-trypsin inhibitor family in acute necrotizing pancreatitis in mice.

BACKGROUND: Therapy for systemic complications in severe necrotizing pancreatitis remains symptomatic owing to the unavailability of more specific therapeutic targets. We investigated the differential gene expression in typically affected organs in a mouse model of severe necrotizing pancreatitis. METHODS: Acute necrotizing pancreatitis was induced in mice by retrograde infusion of taurocholate into the common bile duct. Microarray hybridization was subsequently performed with mRNA isolated from the spleen, liver, intestine, and lungs. Additionally, quantitative real-time polymerase chain reaction was performed to confirm the microarray results. RESULTS: Severe necrotizing pancreatitis induced widespread changes in gene expression, affecting 27.20% of the genes tested in the spleen and 29.07% in the liver. Fewer genes were differentially regulated in the intestine (10.28%) and the lungs (10.75%). Only 10 genes were found to be upregulated in all 4 organs using microarray analysis. This upregulation in all organs was confirmed by quantitative real-time polymerase chain reaction for only 3 molecules. These molecules were lipocalin 2, insulin-like growth factor binding protein 1, and CD14. Additionally we observed significantly aberrant gene regulation of inter-α-trypsin inhibitor family members in several organs. CONCLUSIONS: Differential gene regulation in severe necrotizing pancreatitis is far more organ specific than anticipated, with only 3 molecules uniformly regulated systemically. The inter-α-trypsin inhibitor family of molecules appears to play a crucial biologic role in the systemic inflammatory response in acute pancreatitis. Finally, owing to its regulation and function, α1-microglobulin (or bikunin) may be a suitable predictive marker of the systemic inflammatory response syndrome in acute pancreatitis.

Role of serum-derived hyaluronan-associated protein-hyaluronan complex in ovarian cancer.

The objective of this study was to determine if the level of serum hyaluronan (HA), serum-derived HA-associated protein (SHAP)-HA complex, and urinary trypsin inhibitor (UTI) correlate with the clinical outcome of ovarian cancer patients. The relationship of metalloproteinase and its inhibitor with HA and the SHAP-HA complex was also examined. Serum and urine samples were obtained from 45 patients with ovarian cancer, 22 patients with benign ovarian tumors and 50 healthy women. Concentrations of serum HA and UTI were measured by an inhibitory sandwich enzyme-linked immunosorbent assay, and concentrations of the serum SHAP-HA complex were measured by a sandwich enzyme-linked immunosorbent assay. Concentrations of MMP-2, MMP-9 and TIMP-1 were measured by a one-step enzyme immunoassay. The levels of HA, SHAP-HA complex, MMP-9 and TIMP-1 were higher in the ovarian cancer group than in the benign ovarian tumor group. In ovarian cancer patients, the levels of HA, SHAP-HA complex and MMP-9 were higher in the stage III/IV group than in the stage I/II group, and the levels of SHAP-HA complex, MMP-9 and TIMP-1 were higher in the non-responder group than in the responder group. The serum concentration of SHAP-HA complex had a significant correlation with HA, MMP-9 and TIMP-1 in ovarian cancer patients. The patients with elevated SHAP-HA complex had a shorter disease-free survival compared with those with normal levels of SHAP-HA complex. The multiple regression analysis revealed that SHAP-HA complex is the significant independent variable for progression-free survival. The elevated level of SHAP-HA complex may indicate the prognosis of recurrence and reflect the tumor metastasis associated with MMP-9 in ovarian cancer patients.

Molecular structure and function analysis of bikunin on down-regulation of tumor necrosis factor-alpha expression in activated neutrophils.

OBJECTIVE: We performed a detailed molecular analysis of bikunin-mediated anti-inflammation (suppressive effect of cytokine release, MAP kinase activation, and nuclear translocation of NF-kB) using a truncated form of bikunin. MATERIALS AND METHODS: We obtained bikunin derivatives that contained O-glycoside-linked N-terminal glycopeptide (Bik-m1), N-glycoside-linked C-terminal tandem Kunitz domains (Bik-m2), bikunin lacking O-glycoside (Bik-c), asialo bikunin (Bik-a), bikunin lacking N-glycoside (Bik-n), and purified C-terminal Kunitz domain II (kII) of bikunin (HI-8). Enzyme-linked immunosorbent assay and Western blot were carried out to measure secreted TNF-alpha and MAP kinase activation. RESULTS: We examined the TNF-alpha secretion in control and lipopolysaccharide (LPS)-treated neutrophils and did not see any changes of its protein levels in the cells pretreated with Bik-m1, Bik-m2, Bik-c, or HI-8. In all of the derivatives tested, only the derivatives that lacked N-glycoside side chain showed a significant suppression of TNF-alpha secretion by LPS. Only a small (21 amino acids) deletion of the N-terminal portion of bikunin (which corresponds to Bik-m2) abolished its suppressing activity of TNF-alpha secretion, thus suggesting that the N-terminal 21 amino acids play a critical role in anti-inflammation. Bik-m1 alone failed to show anti-inflammatory response. Bikunin failed to inhibit ionomycin-induced phosphorylation of MAP kinases. CONCLUSION: These data allow us to conclude that the cytokine expression was inhibited only by the O-glycoside-linked core protein without the N-glycoside side chain. Our results also suggest a possible role of bikunin for receptor-dependent MAP kinase activation.

Inter-alpha-trypsin inhibitor attenuates complement activation and complement-induced lung injury.

Complement activation is a central component of inflammation and sepsis and can lead to significant tissue injury. Complement factors are serum proteins that work through a cascade of proteolytic reactions to amplify proinflammatory signals. Inter-alpha-trypsin inhibitor (IaI) is an abundant serum protease inhibitor that contains potential complement-binding domains, and has been shown to improve survival in animal sepsis models. We hypothesized that IaI can bind complement and inhibit complement activation, thus ameliorating complement-dependent inflammation. We evaluated this hypothesis with in vitro complement activation assays and in vivo in a murine model of complement-dependent lung injury. We found that IaI inhibited complement activation through the classical and alternative pathways, inhibited complement-dependent phagocytosis in vitro, and reduced complement-dependent lung injury in vivo. This novel function of IaI provides a mechanistic explanation for its observed salutary effects in sepsis and opens new possibilities for its use as a treatment agent in inflammatory diseases.

TSG-6 potentiates the antitissue kallikrein activity of inter-alpha-inhibitor through bikunin release.

TSG-6 (the protein product of TNF-stimulated gene-6), an inflammation-associated protein, forms covalent complexes with heavy chains (HCs) from inter-alpha-inhibitor and pre-alpha-inhibitor and associates noncovalently with their common bikunin chain, potentiating the antiplasmin activity of this serine protease inhibitor. We show that TSG-6 and TSG-6.HC complexes are present in bronchoalveolar lavage fluid from patients with asthma and increase after allergen challenge. Immunodetection demonstrated elevated TSG-6 in the airway tissue and secretions of smokers. Experiments conducted in vitro with purified components revealed that bikunin.HC complexes (byproducts of TSG-6.HC formation) release bikunin. Immunoprecipitation revealed that bikunin accounts for a significant proportion of tissue kallikrein inhibition in bronchoalveolar lavage after allergen challenge but not in baseline conditions, confirming that bikunin in its free state, but not when associated with HCs, is a relevant protease inhibitor in airway secretions. In primary cultures of differentiated human airway epithelial and submucosal gland cells, TSG-6 is induced by TNF-alpha and IL-1beta, which suggests that these cells are responsible for TSG-6 release in vivo. Bikunin and HC3 (i.e., pre-alpha-inhibitor) were also induced by TNF-alpha in primary cultures. Our results suggest that TSG-6 may play an important protective role in bronchial epithelium by increasing the antiprotease screen on the airway lumen.

Endogenous anti-inflammatory substances, inter-alpha-inhibitor and bikunin.

There have been new developments in the elucidation of the biological functions of the inter-alpha-inhibitor (IalphaI) family. The anti-proteolytic activity of the IalphaI family originates from bikunin (also known as urinary trypsin inhibitor). Growing evidence indicates that bikunin is not just an anti-proteolytic agent, but can also be considered an anti-inflammatory agent that suppresses lipopolysaccharide (LPS)-induced cytokine synthesis. Bikunin functions to inhibit calcium influx and extracellular signal-regulated kinase (ERK) signaling via LPS receptors and/or as yet unidentified bikunin signaling receptors. By signaling via the LPS receptor, LPS increases calcium influx and yields phosphorylated ERK, which activates multiple transcription factors, such as nuclear factor kappaB (NF-kappaB) or early growth response-1 (Egr-1), which in turn promote cytokine expression. Deficits in the signaling cascades caused by free or cell-bound bikunin are predicted to down-regulate cytokine expression, render macrophages/neutrophils more inactive, and impair inflammatory processes. This brief review largely focuses on our current understanding of the apparent functions of bikunin, its ligands, the effector molecules with which it interacts, and its regulation.

Identification of bikunin as an endogenous inhibitor of dynorphin convertase in human cerebrospinal fluid.

Dynorphin-converting enzymes constitute a group of peptidases capable of converting dynorphins to enkephalins. Through the action of these enzymes, the dynorphin-related peptides bind to delta-opioid instead of kappa-opioid receptors, leading to a change in the biological function of the neuropeptides. In this article, we describe the identification of the protein bikunin as an endogenous, competitive inhibitor of a dynorphin-converting enzyme in human cerebrospinal fluid. This protein is present together with its target enzyme in the same body fluids. The K(M) value of the convertase was found to be 9 microm, and the K(i) value of the inhibitor was 1.7 nm. The finding indicates that bikunin may play a significant role as a regulatory mechanism of neuropeptides, where one bioactive peptide is converted to a shorter sequence, which in turn, can affect the action of its longer form.

Isolation of a natural inhibitor of human malignant glial cell invasion: inter alpha-trypsin inhibitor heavy chain 2.

Malignant central nervous system (CNS) tumors, such as glioblastoma multiforme, invade the brain and disrupt normal tissue architecture, making complete surgical removal virtually impossible. Here, we have developed and optimized a purification strategy to isolate and identify natural inhibitors of glioma cell invasion in a three-dimensional collagen type I matrix. Inter alpha-trypsin inhibitor heavy chain 2 (ITI H2) was identified from the most inhibitory fractions and its presence was confirmed both as a single protein and in a bikunin-bound form. Stable overexpression in U251 glioma cells validated ITI H2's strong inhibition of human glioma cell invasion together with significant inhibition of cell proliferation and promotion of cell-cell adhesion. Analysis of primary human brain tumors showed significantly higher levels of ITI H2 in normal brain and low-grade tumors compared with high-grade gliomas, indicating an inverse correlation with malignancy. The phosphatidylinositol 3-kinase/Akt signaling cascade seemed to be one of the pathways involved in the effect of ITI H2 on U251 cells. These findings suggest that reduction of ITI H2 expression correlates with brain tumor progression and that targeting factors responsible for its loss or restoring the ITI supply exogenously may serve as potential therapeutic strategies for a variety of CNS tumors.

Redox properties of the lipocalin alpha1-microglobulin: reduction of cytochrome c, hemoglobin, and free iron.

alpha1-Microglobulin (alpha1m) is a 26-kDa plasma and tissue glycoprotein. The protein has a heterogeneous yellow-brown chromophore consisting of small unidentified prosthetic groups localized to a free thiol group (C34) and three lysyl residues (K92, K118, and K130) around the entrance to a hydrophobic pocket. It was recently reported that alpha1m can bind heme and that a C-terminally processed form of alpha1m degrades heme. It is shown here that alpha1m has catalytic reductase and NADH-dehydrogenase-like activities. Cytochrome c, nitroblue tetrazolium (NBT), methemoglobin, and ferricyanide were reduced by alpha1m. Comparison of the reduction rates suggests that methemoglobin is a better substrate than cytochrome c, NBT, and ferricyanide. The reactions with cytochrome c and NBT were mediated by superoxide anions since they were inhibited by superoxide dismutase. The addition of the biological electron donors NADH, NADPH, or ascorbate enhanced the reduction rate of cytochrome c approximately 30-fold. Recombinant alpha1m, which has much less chromophore than plasma and urine alpha1m, was a stronger reductant than the latter alpha1m forms. Site-directed mutagenesis of C34, K92, K118, and K130 and thiol group chemistry showed that the C34 thiol group was involved in the redox reaction but relies upon cooperation with the lysyl residues. The redox properties of alpha1m may provide a physiological protection mechanism against extracellularly exposed heme groups and other oxidants.

Link protein as an enhancer of cumulus cell-oocyte complex expansion.

To investigate the specific components involved in regulating cumulus cell-oocyte complex (COC) expansion in an in vitro mouse experiment, freshly-isolated COC were cultured in the presence of various combinations of FSH (1.0 microg/ml), proteins of the inter-alpha-inhibitor (I alpha I) family (a light chain, also known as bikunin, heavy chains [HC1 + HC2] and I alpha I [0.01-2.0 microg/ml]) and link protein (LP) (0.016-10 microg/ml) for 24 h and were observed for expansion of their cumulus cells (percent of COC with + 3 and + 4 expansion and average projected area). The COC were videotaped in real time at the initiation of culture and after 24 h of culture. FSH alone did not stimulate cumulus expansion under serum-free conditions; however, treatment with I alpha I (0.1-2.0 microg/ml) or heavy chains (10 microg/ml), but not bikunin (10 micro g/ml), in the presence of FSH significantly increased COC expansion, with maximal promotion occurring at 1.0 microg/ ml of I alpha I. Addition of LP (2.0 micro g/ml) to the medium containing I alpha I (1.0 microg/ml) and FSH resulted in significantly higher expansion levels than were observed in response to I alpha I alone, although LP alone (10 microg/ml) had no or very little effect by itself. Anti-I alpha I or anti-LP polyclonal antibody, which inhibits binding of I alpha I and LP, respectively, to hyaluronic acid (HA), markedly reduced expansion of the surrounding cumulus cell extracellular matrices. Therefore, in vitro, LP might serve, in part, to enhance the COC expansion possibly by stabilizing HA-I alpha I (or heavy chains) complex on the surrounding cumulus cell matrices.

Role of alpha(2)-macroglobulin in fever and cytokine responses induced by lipopolysaccharide in mice.

Alpha(2)-macroglobulin (alpha(2)M) is not only a proteinase inhibitor in mammals, but it is also a specific cytokine carrier that binds pro- and anti-inflammatory cytokines implicated in fever, including interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha (TNF-alpha). To define the role of alpha(2)M in regulation of febrile and cytokine responses, wild-type mice and mice deficient in alpha(2)M (alpha(2)M -/-) were injected with lipopolysaccharide (LPS). Changes in body temperature as well as plasma levels of IL-1beta, IL-6, and TNF-alpha and hepatic TNF-alpha mRNA level during fever in alpha(2)M -/- mice were compared with those in wild-type control mice. The alpha(2)M -/- mice developed a short-term markedly attenuated (ANOVA, P < 0.05) fever in response to LPS (2.5 mg/kg ip) compared with the wild-type mice. At 1.5 h after injection of LPS, the plasma concentration of TNF-alpha, but not IL-1beta or IL-6, was significantly lower (by 58%) in the alpha(2)M -/- mice compared with their wild-type controls (ANOVA, P < 0.05). There was no difference in hepatic TNF-alpha mRNA levels between alpha(2)M -/- and wild-type mice 1.5 h after injection of LPS. These data support the hypotheses that 1) alpha(2)M is important for the normal development of LPS-induced fever and 2) a putative mechanism of alpha(2)M involvement in fever is through the inhibition of TNF-alpha clearance. These findings indicate a novel physiological role for alpha(2)M.

Processing of the lipocalin alpha(1)-microglobulin by hemoglobin induces heme-binding and heme-degradation properties.

Alpha(1)-microglobulin is a 26-kd protein, widespread in plasma and tissues and well-conserved among vertebrates. Alpha(1)-microglobulin belongs to the lipocalins, a protein superfamily with highly conserved 3-dimensional structures, forming an internal ligand binding pocket. The protein, isolated from urine, has a heterogeneous yellow-brown chromophore bound covalently to amino acid side groups around the entrance of the lipocalin pocket. Alpha(1)-microglobulin is found in blood both in free form and complex-bound to immunoglobulin A (IgA) via a half-cystine residue at position 34. It is shown here that an alpha(1)-microglobulin species, which we name t-alpha(1)-microglobulin (t = truncated), with a free Cys34 thiol group, lacking its C-terminal tetrapeptide, LIPR, and with a more polar environment around the entrance of the lipocalin pocket, is released from IgA-alpha(1)-microglobulin as well as from free alpha(1)-microglobulin when exposed to the cytosolic side of erythrocyte membranes or to purified oxyhemoglobin. The processed t-alpha(1)-microglobulin binds heme and the alpha(1)-microglobulin-heme complex shows a time-dependent spectral rearrangement, suggestive of degradation of heme concomitantly with formation of a heterogeneous chromophore associated with the protein. The processed t-alpha(1)-microglobulin is found in normal and pathologic human urine, indicating that the cleavage process occurs in vivo. The results suggest that alpha(1)-microglobulin is involved in extracellular heme catabolism.

Defect in SHAP-hyaluronan complex causes severe female infertility. A study by inactivation of the bikunin gene in mice.

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-alpha-trypsin inhibitor (ITI) family, designated as SHAPs (serum-derived hyaluronan-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 fertilization in vivo.