Inter-alpha-inhibitor binding to hyaluronan in the cumulus extracellular matrix is required for optimal ovulation and development of mouse oocytes.

This report characterizes the effects of excess hyaluronan (HA) upon the expansion of the cumulus oocyte complex (COC) within intact follicles and upon ovulation and oocyte viability in mice. Covalent linkage between heavy chains of the inter-alpha-inhibitor (IalphaI) family of serum glycoproteins and HA is necessary for optimal cumulus extracellular matrix (cECM) stabilization and cumulus expansion. Intravenous administration of HA oligosaccharides inhibited the binding of IalphaI to endogenous HA, disrupting the process of expansion and resulting in a reduction in the size of the cumulus mass. Western blot and immunocytochemical analyses of COCs from HA-treated animals demonstrated a reduction of IalphaI heavy chains within the cECM. Additionally, HA-treated immature animals ovulated 56.3% fewer COCs compared to control animals. The developmental potential of COCs in HA-treated animals was also tested. Extended periods of oviductal storage of COCs ovulated by HA-injected adult mice resulted in a reduction of normal embryos and a significant increase in the proportion of fragmented oocytes/embryos. These observations support the view that covalent binding of IalphaI heavy chains to HA is required for optimal cumulus expansion, extrusion of the COCs from the follicle at ovulation, and maintenance of oocyte viability within the oviduct.     

Characterization of complexes formed between TSG-6 and inter-alpha-inhibitor that act as intermediates in the covalent transfer of heavy chains onto hyaluronan

The high molecular mass glycosaminoglycan hyaluronan (HA) can become modified by the covalent attachment of heavy chains (HCs) derived from the serum protein inter-alpha-inhibitor (IalphaI), which is composed of three subunits (HC1, HC2 and bikunin) linked together via a chondroitin sulfate moiety. The formation of HC.HA is likely to play an important role in the stabilization of HA-rich extracellular matrices in the context of inflammatory disease (e.g. arthritis) and ovulation. Here, we have characterized the complexes formed in vitro between purified human IalphaI and recombinant human TSG-6 (an inflammation-associated protein implicated previously in this process) and show that these complexes (i.e. TSG-6 x HC1 and TSG-6 x HC2) act as intermediates in the formation of HC x HA. This is likely to involve two transesterification reactions in which an ester bond linking an HC to chondroitin sulfate in intact IalphaI is transferred first onto TSG-6 and then onto HA. The formation of TSG-6 x HC1 and TSG-6 x C2 complexes was accompanied by the production of bikunin x HC2 and bikunin x HC1 by-products, respectively, which were observed to break down, releasing free bikunin and HCs. Both TSG-6 x HC formation and the subsequent HC transfer are metal ion-dependent processes; these reactions have a requirement for either Mg2+ or Mn2+ and are inhibited by Co2+. TSG-6, which is released upon the transfer of HCs from TSG-6 onto HA, was shown to combine with IalphaI to form new TSG-6 x HC complexes and thus be recycled. The finding that TSG-6 acts as cofactor and catalyst in the production of HC x HA complexes has important implications for our understanding of inflammatory and inflammation-like processes.     

TSG-6 protein binding to glycosaminoglycans: formation of stable complexes with hyaluronan and binding to chondroitin sulfates

TSG-6 protein, up-regulated in inflammatory lesions and in the ovary during ovulation, shows anti-inflammatory activity and plays an essential role in female fertility. Studies in murine models of acute inflammation and experimental arthritis demonstrated that TSG-6 has a strong anti-inflammatory and chondroprotective effect. TSG-6 protein is composed of the N-terminal link module that binds hyaluronan and a C-terminal CUB domain, present in a variety of proteins. Interactions between the isolated link module and hyaluronan have been studied extensively, but little is known about the binding of full-length TSG-6 protein to hyaluronan and other glycosaminoglycans. We show that TSG-6 protein and hyaluronan, in a temperature-dependent fashion, form a stable complex that is resistant to dissociating agents. The formation of such stable complexes may underlie the activities of TSG-6 protein in inflammation and fertility, e.g. the TSG-6-dependent cross-linking of hyaluronan in the cumulus cell-oocyte complex during ovulation. Because adhesion to hyaluronan is involved in cell trafficking in inflammatory processes, we also studied the effect of TSG-6 on cell adhesion. TSG-6 binding to immobilized hyaluronan did not interfere with subsequent adhesion of lymphoid cells. In addition to immobilized hyaluronan, full-length TSG-6 also binds free hyaluronan and all chondroitin sulfate isoforms under physiological conditions. These interactions may contribute to the localization of TSG-6 in cartilage and to its chondroprotective and anti-inflammatory effects in models of arthritis.     

Fusion protein of the hyaluronan binding domain from human TSG-6 with luciferase for assay of hyaluronan

The gene expression plasmid, pET-Lmluc, for the fusion protein of the hyaluronan binding domain from human TSG-6 [product of tumor necrosis factor (TNF)-stimulated gene-6] and luciferase from Renilla reniformis was constructed. The fused gene was expressed in Escherichia coli and the resulted insoluble Lm-luc fusion protein was purified and refolded to recover both the hyaluronan binding capability and the luciferase activity. Hyaluronan as low as 1 ng ml^–1 was detected by using the indirect enzymatic immunological assay with the refolded Lm-luc fusion protein.     

The link module from ovulation- and inflammation-associated protein TSG-6 changes conformation on hyaluronan binding

The solution structure of the Link module from human TSG-6, a hyaladherin with important roles in inflammation and ovulation, has been determined in both its free and hyaluronan-bound conformations. This reveals a well defined hyaluronan-binding groove on one face of the Link module that is closed in the absence of ligand. The groove is lined with amino acids that have been implicated in mediating the interaction with hyaluronan, including two tyrosine residues that appear to form essential intermolecular hydrogen bonds and two basic residues capable of supporting ionic interactions. This is the first structure of a non-enzymic hyaladherin in its active state, and identifies a ligand-induced conformational change that is likely to be conserved across the Link module superfamily. NMR and isothermal titration calorimetry experiments with defined oligosaccharides have allowed us to infer the minimum length of hyaluronan that can be accommodated within the binding site and its polarity in the groove; these data have been used to generate a model of the complex formed between the Link module and a hyaluronan octasaccharide.     

TSG-6 modulates the interaction between hyaluronan and cell surface CD44

Interactions between CD44 and hyaluronan are implicated in the primary adhesion of lymphocytes to endothelium at inflammatory locations. Here we show that preincubation of hyaluronan with full-length recombinant TSG-6 or its Link module domain (Link_TSG6) enhances or induces the binding of hyaluronan to cell surface CD44 on constitutive and inducible cell backgrounds, respectively. These effects are blocked by CD44-specific antibodies and are absent in CD44-negative cells. Enhancement of CD44-mediated interactions of lymphoid cells with hyaluronan by TSG-6 proteins was seen under conditions of flow at shear forces that occur in post-capillary venules. Increases in the number of rolling cells were observed on substrates comprising TSG-6-hyaluronan complexes as compared with a substrate containing hyaluronan alone. In ligand competition experiments, cell surface-bound TSG-6-hyaluronan complexes were more potent than hyaluronan alone in inhibiting cell adhesion to immobilized hyaluronan. Link_TSG6 mutants with impaired hyaluronan binding function had a reduced ability to modulate ligand binding by cell surface CD44. However, some mutants that exhibited close to wild-type hyaluronan binding were found to have either reduced or increased activity, suggesting that some amino acid residues outside of the hyaluronan binding site might be involved in protein self-association, potentially leading to the formation of cross-linked hyaluronan fibers. In turn, cross-linked hyaluronan could increase the binding avidity of CD44 by inducing receptor clustering. The ability of TSG-6 to modulate the interaction of hyaluronan with CD44 has important implications for CD44-mediated cell activity at sites of inflammation, where TSG-6 is expressed.     

Transient invasive migration in mouse cumulus oocyte complexes induced at ovulation by luteinizing hormone

Ovulation, the release of the oocyte from the ovarian follicle, is initiated by the luteinizing hormone surge. It is clear that highly controlled degradation of the follicle and ovarian wall is required for passage of the oocyte and accompanying cumulus cells from the follicle, but the mechanism has not yet been elucidated. Here we show that cumulus oocyte complexes (COCs) adopt transient adhesive, migratory, and matrix-invading capacities at the time of ovulation. We characterized cell adhesion, migration, and invasion in preovulatory and postovulatory mouse COCs collected over a time course post-human chorionic gonadotropin (hCG) administration. Adhesion of dispersed cumulus cells and intact COCs to extracellular matrix proteins present in the ovarian wall (collagens, laminin, and fibronectin) increased significantly after hCG treatment and declined immediately after ovulation. Cumulus cell migration was low in unexpanded, equine chorionic gonadotropin-only treated COCs, but increased 4, 8, and 10 h post-hCG, reaching a peak at 12 h post-hCG that coincided with ovulation. The ability of cumulus cells to migrate was rapidly diminished in COCs isolated from the oviduct within 2 h postovulation. Cell migration was cumulus cell specific and was not observed in granulosa cells. Invasion through three-dimensional collagen I and matrigel barriers by preovulatory expanded COCs was equivalent to that of a known invasive breast cancer cell line (MB-231). Cumulatively, these results demonstrate that cumulus cells in the expanded COC transition to an adhesive, motile, and invasive phenotype in the periovulatory period that may be required for successful release of the oocyte from the ovary at ovulation.     

Influence of hyaluronan accumulation during cumulus expansion on in vitro porcine oocyte maturation

During oocyte maturation, the cumulus-oocyte complexes (COCs) expand dramatically. This phenomenon, which is known as cumulus expansion, is the result of the synthesis and accumulation of hyaluronan in the extracellular space between cumulus cells. The purpose of this study was to investigate the effect of 6-diazo-5-oxo-l-norleucine (DON), an inhibitor of hyaluronan synthesis, on cumulus expansion during in vitro porcine oocyte maturation and hyaluronan accumulation within COCs. Further, this study aimed to examine the influence of hyaluronan accumulation within COCs on the rate of oocyte maturation. Cumulus expansion was observed during in vitro maturation. However, the addition of DON to the maturation medium significantly inhibited cumulus expansion. The total inhibition of hyaluronan accumulation within COCs was observed with the use of confocal microscopy. Moreover, a positive correlation between the area of cumulus expansion and the rate of oocyte maturation was observed. These results demonstrate that the hyaluronan accumulation within the COCs during oocyte maturation affects oocyte maturation. On the basis of these results, we propose that hyaluronan accumulation within the COCs during cumulus expansion is a necessary step in the porcine oocyte maturation process.     

Heterologous cell contacts and metabolic coupling in bovine cumulus oocyte complexes

The integrity of the cumulus cell processes were studied in four categories of bovine cumulus oocyte complexes (COCs) selected on their morphological characteristics. Three different types of cumulus cell process endings (CCPEs) were identified, one penetrating the cortex, another not penetrating the cortex, and a third form was intermediate and more rare in appearance. The process endings that penetrated the cortex frequently made gap junctions with the oolemma. The division of the three types of CCPEs over the four different COC categories was specific for three of the four categories. The first-category COC predominantly possessed the penetrating CCPE, the fourth-category COC possessed predominantly the nonpenetrating CCPE, and the second and third categories had both types of CCPEs. The metabolic coupling of the cumulus-oocyte contacts was assessed by means of incorporation of 3H-choline into the oocyte. The majority of category 4 COCs transferred low levels of choline into the oocyte while the majority of the oocytes of the other three categories transferred high levels of choline into the oocyte. Category 4 includes a smaller proportion of oocytes capable of cleaving after fertilization than the other three categories. This reduced developmental capacity is probably due to the loss of metabolic coupling before the onset of culture.     

Cartilage link protein interacts with neurocan, which shows hyaluronan binding characteristics different from CD44 and TSG-6

The interaction of neurocan with hyaluronan was qualitatively characterized with alkaline phosphatase fusion proteins secreted by mammalian cells. The wild type neurocan hyaluronan binding domain fused to alkaline phosphatase bound to immobilized hyaluronan under physiological as well as moderately hypertonic conditions, whereas its ability to bind to immobilized chondroitin sulfate dropped rapidly with increasing salt concentration. Strong hyaluronan binding ability was still evident when in both link modules within the hyaluronan binding domain a basic amino acid was mutated, which is well conserved among link modules of hyaluronan binding proteins. A strong enhancement of the binding of neurocan to immobilized hyaluronan was observed after preincubation of the immobilized hyaluronan with cartilage link protein. Moreover, this preincubation mediated also the binding of a fusion protein representing only the immunoglobulin module of neurocan linked to alkaline phosphatase, which showed no binding to immobilized hyaluronan alone. The interaction of the neurocan immunoglobulin module with link protein could also be shown by overlay blot analysis. These observations suggest that the hyaluronan binding characteristics of paired link modules are different from those of single link modules, and that the reported temporal co-expression of cartilage link protein and of neurocan in developing brain implicates the possibility of a cooperative function of these molecules.     

Specificity of the tumor necrosis factor-induced protein 6-mediated heavy chain transfer from inter-alpha-trypsin inhibitor to hyaluronan: implications for the assembly of the cumulus extracellular matrix

The formation of the hyaluronan-rich cumulus extracellular matrix is crucial for female fertility and accompanied by a transesterification reaction in which the heavy chains (HCs) of inter-alpha-trypsin inhibitor (IalphaI)-related proteins are covalently transferred to hyaluronan. Tumor necrosis factor-induced protein-6 (TNFIP6) is essential for this transfer reaction. Female mice deficient in TNFIP6 are infertile due to the lack of a correctly formed cumulus matrix. In this report, we characterize the specificity of TNFIP6-mediated HC transfer from IalphaI to hyaluronan. Hyaluronan oligosaccharides with eight or more monosaccharide units are potent acceptors in the HC transfer, with longer oligosaccharides being somewhat more efficient. Epimerization of the N-acetyl-glucosamine residues to N-acetyl-galactosamines (i.e. in chondroitin) still allows the HC transfer although at a significantly lower efficiency. Sulfation of the N-acetyl-galactosamines in dermatan-4-sulfate or chondroitin-6-sulfate prevents the HC transfer. Hyaluronan oligosaccharides disperse cumulus cells from expanding cumulus cell-oocyte complexes with the same size specificity as their HC acceptor specificity. This process is accompanied by the loss of hyaluronan-linked HCs from the cumulus matrix and the appearance of oligosaccharide-linked HCs in the culture medium. Chondroitin interferes with the expansion of cumulus cell-oocyte complexes only when added with exogenous TNFIP6 before endogenous hyaluronan synthesis starts, supporting that chondroitin is a weaker HC acceptor than hyaluronan. Our data indicate that TNFIP6-mediated HC transfer to hyaluronan is a prerequisite for the correct cumulus matrix assembly and hyaluronan oligosaccharides and chondroitin interfere with this assembly by capturing the HCs of the IalphaI-related proteins.     

The effects of plasma-derived extracellular vesicles on cumulus expansion and oocyte maturation in mice

Cumulus cell expansion is required for the ovulation of a fertilizable oocyte. Extracellular vesicles (EVs) are bilayer-lipid membrane vesicles that may be found in a variety of bodily fluids and play an important role in biological processes. This study aimed to examine the effects of plasma-derived EVs on cumulus expansion and in vitro maturation (IVM) of the oocyte. EVswere isolated using ultracentrifugation from the plasma of female mice. The morphology and size of EVs were analyzed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Western blotting allowed us to identify CD63, CD81, CD9, and HSP70 protein markers of EVs; the expression of the genes related to cumulus cell expansion, including hyaluronan synthase 2 (Has2) and prostaglandinendoperoxide synthase 2 (Ptgs2), were assessed using real-time polymerase chain reaction. Plasma-derived EVs labeled with Dil dye were successfully incorporated with cumulus cells during IVM. Plasma-derived EVs significantly induced cumulus expansion and maturation of oocytes. The percentage of oocytes that reached the MII stage was significantly greater in the EVs treatment group compared with other groups. Although treatment with epidermal growth factor (EGF) significantly increased cumulus expansion in cumulus-oocyte complexes (COCs), the impact was less than that seen with plasma-derived EVs. Furthermore, EVs generated from plasma substantially enhanced Has2 and Ptgs2 mRNA expression in the cumulus-oocyte complex. This research indicates that EVs derived from plasma are capable of promoting cumulus expansion and oocyte maturation.     

Determining the molecular basis for the pH-dependent interaction between the link module of human TSG-6 and hyaluronan

TSG-6 is an inflammation-associated hyaluronan (HA)-binding protein that has anti-inflammatory and protective functions in arthritis and asthma as well as a critical role in mammalian ovulation. The interaction between TSG-6 and HA is pH-dependent, with a marked reduction in affinity on increasing the pH from 6.0 to 8.0. Here we have investigated the mechanism underlying this pH dependence using a combined approach of site-directed mutagenesis, NMR, isothermal titration calorimetry and microtiter plate assays. Analysis of single-site mutants of the TSG-6 Link module indicated that the loss in affinity above pH 6.0 is mediated by the change in ionization state of a histidine residue (His(4)) that is not within the HA-binding site. To understand this in molecular terms, the pH-dependent folding profile and the pK(a) values of charged residues within the Link module were determined using NMR. These data indicated that His(4) makes a salt bridge to one side-chain oxygen atom of a buried aspartate residue (Asp(89)), whereas the other oxygen is simultaneously hydrogen-bonded to a key HA-binding residue (Tyr(12)). This molecular network transmits the change in ionization state of His(4) to the HA-binding site, which explains the loss of affinity at high pH. In contrast, simulations of the pH affinity curves indicate that another histidine residue, His(45), is largely responsible for the gain in affinity for HA between pH 3.5 and 6.0. The pH-dependent interaction of TSG-6 with HA (and other ligands) provides a means of differentially regulating the functional activity of this protein in different tissue microenvironments.     

Cyclooxygenase-2 is expressed by cumulus cells during oocyte maturation in cattle.

Prostaglandins could be involved in various aspects of final differentiation of ovarian follicles. Prostaglandins are generated by the cyclooxygenase (cox) pathway. Until now, the expression pattern of isoforms cox-1 and cox-2 of cyclooxygenase in bovine cumulus-oocyte complexes (COCs) was unknown. Using immunodetection procedure, we demonstrated in the present study that cox-2 was expressed by cumulus cells during in vivo and in vitro maturation. Time course induction of cox-2 expression was investigated during in vitro maturation using Western blot analysis. Specific signal of cox-2 was markedly evidenced from 6 hr of culture and increased to reach a maximal level at 24 hr of culture. In vitro, cox-2 expression in COCs was associated with increased concentrations of PGE(2) and PGF(2alpha) in the maturation medium. In addition, the effects of culture conditions on cox-2 expression was considered using RT-PCR and Western-blot analysis. We demonstrated that the addition of 10 ng/ml of EGF to TCM199 clearly increased the expression level of cox-2 mRNA and protein. Higher levels of in vitro cox-2 expression was associated with greater rates of cumulus expansion and oocytes at metaphase II at 24 hr of culture. In conclusion, our present results suggest that cox-2 expression in cumulus cells may be involved in differentiation of COCs that occurs during oocyte maturation.     

16S acetylcholinesterase of the extracellular matrix is assembled within mouse muscle cells in culture.

The present paper examines where the extracellular-matrix (ECM) 16S acetylcholinesterase (AChE, EC 3.1.1.7) is assembled in muscle cells in culture. The existence of an internal pool of 16S AChE was detected by using AChE inhibitors of differing membrane permeability. After irreversible inhibition of all cellular esterase, the newly synthesized 16S form appears in an intracellular compartment and is only later detected on the cell surface. Results show that the ECM 16S AChE is assembled within muscle cells.