Glycosaminoglycan-bound and free inter-alpha-trypsin inhibitor components of follicular fluid.

The proteinase inhibitor inter-alpha trypsin inhibitor (ITI) is a blood-derived protein necessary for normal female fertility. Absence of ITI leads to ovulation of naked oocytes that cannot fertilise. ITI consists of two heavy chains (ITI-HC) and bikunin linked by a chrondroitin sulphate. By binding to hyaluronate, ITI-HC stabilises the extracellular matrix, but ITI-HC also binds to proteoglycans in follicular fluid. In vivo concentrations of ITI components in preovulatory follicular fluid, free as well as bound to hyaluronate or proteoglycan, are unknown. In order to quantify these components, 58 follicular fluids and 13 blood samples were collected in connection with in vitro fertilisation and embryo transfer treatment of 13 women. Quantitation of glycosaminoglycan-bound ITI-HC was performed after separation from free ITI in agarose gel. ITI components were determined by immunoelectrophoresis and hyaluronate by an ELISA method. The follicular fluid concentration of ITI was on average 70% of that in plasma and the concentration of hyaluronate remained low despite follicular production, suggesting that the production of hyaluronate is the rate-limiting step in the formation of the extracellular matrix of the oocyte-cumulus complex. In follicular fluid, the concentration of free ITI-HC was higher than that of glycosaminoglycan-bound ITI-HC. Addition of exogeneous hyaluronate doubled the amount of hyaluronate-bound ITI-HC, further supporting the notion that ITI in follicular fluid is not rate-limiting for cumulus expansion in vivo.     

A novel basal lamina matrix of the stratified epithelium of the ovarian follicle.

Basal laminas are important sheets of specialized extracellular matrix that underlie and surround groups of cells, such as epithelia or endothelia, enabling the cells to orientate their basal/apical polarity and creating a microenvironment for them. Basal laminas can also individually encapsulate whole cells, such as muscle cells, thereby forming a microenvironment but not polarizing the enclosed cells. Other mesenchymal or stromal cells exist with no basal lamina. In the course of studying the bovine follicular basal lamina which underlies the multilayered epithelium of the ovarian follicle, we identified a developmentally regulated novel extracellular matrix (which we call focimatrix for focal intra-epithelial matrix). Focimatrix is composed of basal lamina-like material deposited as plaques or aggregates between the multilayers of the epithelial granulosa cells. The focimatrix does not encapsulate individual or groups of cells and therefore does not form a microenvironment for them. Focimatrix contains collagen type IV subunits alpha1 and alpha2 (but not alpha3-alpha6), and laminin chains alpha1, beta2 and gamma1 (but not alpha2 or beta1), and nidogen-1 and perlecan (but not versican). The amount of focimatrix increases with increasing follicular size, and its appearance precedes the expression by granulosa cells of the enzymes for steroid hormone synthesis, cholesterol side-chain cleavage cytochrome P450 (SCC) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD), in the days preceding ovulation. The expression in granulosa cells of two components examined, nidogen-1 and perlecan, also increases substantially when follicles enlarge to a sufficient size capable of ovulating. Following ovulation the follicular basal lamina is degraded, and presumably focimatrix is too since it is not detected in corpora lutea that develop from the ovulating follicles. During this development the granulosa cells undergo an epithelial-mesenchymal transition (EMT) into luteal cells following ovulation, and substantially increase their expression of steroidogenic enzymes in the process. During EMT epithelial cells lose polarity. Since focimatrix exists on more than one side of the granulosa cells, we propose that it disrupts the polarity induced by the follicular basal lamina in the lead up to ovulation. Hence focimatrix maybe a key part of the follicular/luteal EMT.     

A heparin-binding erythroid cell stimulating factor from fetal bovine serum has the N-terminal sequence of insulin-like growth factor II

An 8 kd heparin-binding peptide which stimulates thymidine incorporation in cultures of fetal calf liver erythroid cells was isolated from fetal bovine serum by affinity chromatography on Heparin-Sepharose, ion exchange chromatography, gel filtration and reversed-phase HPLC. The N-terminal sequence of the isolated peptide was identical to the N-terminal sequence of bovine erythrotropin or insulin-like growth factor II (IGF II). The potential heparin-binding site of IGF II is probably situated in the arginine-rich C-peptide region. The affinities of human recombinant IGF I and II were compared with those of apolipoprotein H (a plasma heparin-binding protein) and bovine insulin in a heparin-affinity column. The retention times were in the order: Apolipoprotein H greater than hrIGF II greater than hrIGF I greater than insulin (no retention). This unusual property of IGF II suggests that it may be captured in the extracellular matrix in a similar way to fibroblast growth factor, interleukin 3 or granulocyte/macrophage colony-stimulating factor.     

Identification of extracellular proteins in the rat cumulus oophorus

We have examined the proteins associated with the mucous matrix of the rat cumulus oophorus and compared them to the composition of rat serum, follicular fluid, ampullary fluid, and oocyte–cumulus cell extract. The cumulus matrix was dispersed using Streptomyces hyaluronidase, and the proteins were analyzed by highresolution two-dimensional polyacrylamide gel electrophoresis and compared with proteins of the serum, proestrous follicular fluid, and postvulatory ampullary fluid and extracts of oocytes and cumulus cells. In addition to albumin and transferrin, which were common to all the fluids analyzed, the cumulus material contained many proteins in common with the follicular fluid and the ampullary fluid. However, the protein extract of the cumulus matrix also contained four major proteins not present in the other fluids analyzed. Two of these proteins were acidic and heterogenous in charge and size (MW ～81,000 and 100,000). The other two proteins were more basic and occurred at MW ～90,000 and 150,000. Our results show that the extracellular matrix of the cumulus contains proteins that are not present in the fluids that surround the oocyte.     

The chemistry of the collagen cross-links. Purification and characterization of cross-linked polymeric peptide material from mature collagen containing unknown amino acids.

A polymeric form of the alpha 1-chain C-terminal peptide alpha 1 CB6 (poly-alpha 1 CB6) was purified from CNBr digests of insoluble bovine tendon type-I-collagen by gel filtration and ion-exchage chromatography. The purified material had a molecular weight of 1.5 x 10(6)-5 x 10(6) on gel filtration and an amino acid content virtually identical with that of monomeric peptide alpha 1 CB6. The material could be adsorbed on affinity gels containing immobilized anti-(alpha 1 CB6-peptide non-helical region) antibodies and was an inhibitor of haemagglutination by the same antibodies of alpha 1 CB6-peptide-coated sheep erythrocytes. Periodate treatment of the material had no effect. Alkali hydrolysates were shown to contain two unknown amino acids, which were purified by gel filtration and ion-exchange chromatography in volatile buffers and are believed to be components of the mature cross-link of collagen.     

Luteinizing hormone-dependent activation of the epidermal growth factor network is essential for ovulation

In the preovulatory ovarian follicle, mammalian oocytes are maintained in prophase meiotic arrest until the luteinizing hormone (LH) surge induces reentry into the first meiotic division. Dramatic changes in the somatic cells surrounding the oocytes and in the follicular wall are also induced by LH and are necessary for ovulation. Here, we provide genetic evidence that LH-dependent transactivation of the epidermal growth factor receptor (EGFR) is indispensable for oocyte reentry into the meiotic cell cycle, for the synthesis of the extracellular matrix surrounding the oocyte that causes cumulus expansion, and for follicle rupture in vivo. Mice deficient in either amphiregulin or epiregulin, two EGFR ligands, display delayed or reduced oocyte maturation and cumulus expansion. In compound-mutant mice in which loss of one EGFR ligand is associated with decreased signaling from a hypomorphic allele of the EGFR, LH no longer signals oocyte meiotic resumption. Moreover, induction of genes involved in cumulus expansion and follicle rupture is compromised in these mice, resulting in impaired ovulation. Thus, these studies demonstrate that LH induction of epidermal growth factor-like growth factors and EGFR transactivation are essential for the regulation of a critical physiological process such as ovulation and provide new strategies for manipulation of fertility.     

Interaction of osteogenin, a heparin binding bone morphogenetic protein, with type IV collagen

Osteogenin, an extracellular matrix component of bone, is a heparin binding differentiation factor that initiates endochondral bone formation in rats when implanted subcutaneously with an insoluble collagenous matrix. We have examined the interaction of osteogenin with various extracellular matrix components including basement membranes. Osteogenin, purified from bovine bone, binds avidly to type IV collagen and to a lesser extent to both type I and IX collagens. Osteogenin binds equally well to both native and denatured type IV collagen. Both alpha 1 and alpha 2 chains of type IV collagen are recognized by osteogenin. Osteogenin binds to a collagen IV affinity column, and is eluted by 6.0 M urea with 1 M NaCl, pH 7.4, and the eluate contained the osteogenic activity as demonstrated in vivo. Binding of osteogenin to collagen IV is not influenced by either laminin or fibronectin. These results imply that osteogenin binding to extracellular matrix components including collagens I and IV and heparin may have physiological relevance, and such interactions may modulate its local action.     

Isolation and characterization of B cell differentiation factor (BCDF) secreted from a human B lymphoblastoid cell line

A subclone of a human B lymphoblastoid cell line, CESS-2, spontaneously secreted a kind of BCDF (B-BCDF) in their culture supernatant without any stimulation. B-BCDF induced IgG and IgM secretions in human B lymphoblastoid cell lines, CESS cells and CL-4 cells, respectively. BCDF-responsive CESS cells expressed IgG on their surface, whereas CESS-2, which were able to secrete B-BCDF, did not express surface IgG. B-BCDF could induce Ig-secretion in SAC-stimulated low density peripheral B cells, but did not induce Ig secretion in nonstimulated B cells. B-BCDF did not show any IL 2, BCGF, or gamma-interferon activities. B-BCDF was highly purified by gel filtration on an AcA-34 column, by chromatofocusing and ion exchange chromatography on an HPLC system, and by gel filtration on an HPLC column. Highly purified preparations showed a single protein band in SDS-PAGE analysis. The m.w. and isoelectric points of the factor were 20,000 and pH 5.1 to 5.2, respectively. The minimum protein amount required for Ig induction in B cell lines was 16 ng/ml.     

Aromatase activity in human adipose tissue stromal cells; the effect of fetal bovine serum fractions on dexamethasone-stimulated aromatization

Aromatization in human adipose stromal cells is stimulated by dexamethasone, but only in the presence of fetal bovine serum (FBS). To determine whether there was a specific fraction of FBS responsible for this stimulation, FBS was fractionated either by a pressure-driven ultrafiltration membrane or by Sephadex gel filtration techniques. The stimulating factor(s) appeared to be in the FBS fraction of 150,000-300,000 Mw by Sephadex filtration. Conversely, FBS fractions with less than 30,000 Mw as separated by the former method inhibited the dexamethasone-stimulated aromatization of cultured adipose stromal cells. Bovine serum albumin, which constituted the major portion of FBS, had no discernible effect on the dexamethasone action on the aromatization of these cells.     

Platelet-derived growth factor in combination with collagen promotes the migration of human skin fibroblasts into a denuded area of a cell monolayer.

Since we have found previously that adult donor skin fibroblasts (TIG-114) migrated more slowly in serum-depleted medium than in medium supplemented with 10% FBS, we tried to identify a factor(s) which promotes fibroblast migration from the edge of a denuded area in a monolayer. In medium supplemented with 10% FBS, the effects of both suramin, a competitor of growth factors at the receptor level, and monensin, an inhibitor of the secretion of extracellular matrix, were examined. Both substances suppressed cell migration, suggesting that growth factors and matrix substances are important for cell migration. Then, we examined the effects of growth factors and extracellular matrix on fibroblast migration in serum-free medium. Platelet-derived growth factor (PDGF), basic fibroblast growth factor, acidic fibroblast growth factor, and transforming growth factor-beta did not stimulate cell migration. Type I collagen, plasma fibronectin, and heparin also did not promote cell migration. However, the combination of PDGF and type I collagen did promote cell migration. Addition of anti-PDGF antibody reduced the stimulatory effect induced by the combination of PDGF and type I collagen. These results suggest that the copresence of growth factors and extracellular matrix regulates fibroblast migration into a denuded area in a monolayer.     

Analysis of the mitogenic effect of fetuin preparations on arterial smooth muscle cells: the role of contaminant platelet-derived growth factor

Fetuin, a major protein of fetal calf serum, partially purified by the method of Pedersen, stimulated growth of aortic smooth muscle cells. More highly purified fetuin preparations stimulated growth less than Pedersen fetuin, as previously described for other cell types, suggesting that this activity is due to a contaminant. Recently bovine alpha 2-macroglobulin or "Embryonin" has been proposed as the mitogenic component of crude fetuin preparations. We found that active fetuin preparations did contain alpha 2-macroglobulin that stimulated smooth muscle cell growth. However, alpha 2-macroglobulin purified directly from platelet-poor bovine plasma or fetuin purified from Pedersen fetuin by gel filtration lacked appreciable mitogenic effect on smooth muscle cells. Since alpha 2-macroglobulin can bind platelet-derived growth factor (PDGF), and since highly acidic fetuin might bind the very basic PDGF molecule non-specifically, we measured the PDGF content of various fetuin preparations and found a good correlation between the PDGF content and mitogenic activity. Gel filtration experiments demonstrated that in Pedersen fetuin PDGF occurred both free, and in association with alpha 2-macroglobulin. We conclude that the principal mitogenic component for smooth muscle cells in crude fetuin preparations is PDGF, since purified bovine alpha 2-macroglobulin or fetuin do not appreciably affect growth of these cells. These results help to resolve a long-standing controversy regarding the nutrition of cultured cells. In addition, we suggest that before alpha 2-macroglobulin or "Embryonin" is accepted as a bona fide growth factor for a given cell type, the role of contamination with PDGF should be assessed.     

Architecture of the hamster oocyte-cumulus complex

The structure of the hamster oocyte-cumulus complex (OCC) has been analyzed to help understand how mammalian sperm penetrate the investing coats of the oocyte. At ovulation, oocytes of most mammalian species are surrounded by a zona pellucida, corona radiata, and cumulus layer. Cells of the cumulus and corona radiata are separated by an extracellular matrix (ECM) that contains hyaluronic acid. In hamsters, the diameter of mature follicular OCCs is 0.61 ± 0.12 mm, whereas in freshly ovulated OCCs in culture medium it is 0.78 ± 0.15 mm. This indicates the OCC expands at or after ovulation. The corona radiata is 1–4 cell layers deep, and corona radiata cells are closely packed (center-to-center distances between adjacent cells in follicular OCCs averaged 14 ± 3 μm). The cumulus layer is 5–8 cell layers deep, and intercellular spaces are much larger (center-to-center distances between adjacent cumulus cells in follicular OCCs averaged 50 ± 20 μm). An ovulated OCC has an approximate volume of 248 × 10⁶ μm³ and was estimated to contain approximately 5,700 cells. Studies with stretched OCCs show that ink particles can readily penetrate the extracellular spaces of the cumulus and corona radiata layers and interact with the ECM, staining it black. At the periphery of the OCC, the ECM appeared discontinuous and formed “cords” containing cumulus cells, whereas closer to the corona radiata the matrix completely filled intercellular spaces. Ink penetrated the corona radiata, but the ECM was difficult to visualize because of the close packing of cells in this layer. Our observations on unfixed OCCs show that cell packing becomes more dense proceeding from the periphery of the OCC to the zona pellucida and that the ECM at the periphery differs from the matrix deeper in the OCC. These data suggest that an incoming sperm would find penetration of the investing coats to increase in difficulty (that is physical resistance would increase) as it got closer to the oocyte. We have also examined the effects of processing for microscopy on the structure of the OCC. Both standard fixation procedures and fixation in the presence of ruthenium red caused condensation of the ECM, especially in the cumulus layer, and thus produced a significant decrease in the diameter of the OCC.     

Cumulus oophorus extracellular matrix: its construction and regulation.

Cumulus oophorus, an investing structure unique to oocytes of higher mammals, is induced to synthesize an extensive extracellular matrix by ovulatory stimulus, leading to the characteristic preovulatory expansion of the cumulus-oocyte complex. The extracellular matrix consists of cumulus cell-secreted hyaluronan, proteoglycans and proteins, as well as extrafollicularly originated SHAPs (serum-derived hyaluronan-associated proteins) that are bound covalently to hyaluronan. The secretion and assembly of matrix molecules by cumulus cells are temporally regulated by factors derived from both mural granulosa cells and oocyte, which synchronize the deposition of the cumulus oophorus matrix with other intrafollicular ovulatory events. The cumulus oophorus matrix is essential for ovulation and subsequent fertilization. Recently, taking advantage of animal models with defined genetic modifications, it has become possible to investigate in vivo the structure of the cumulus oophorus matrix, the regulatory mechanism for matrix deposition and its biological functions. This review focuses on the recent findings on the construction of the cumulus oophorus matrix and the regulation.     

Purification and partial characterization of a plasma inhibitor of tonin

A plasma inhibitor of tonin activity in the rat, was purified by ammonium sulfate precipitation, ion-exchange of chromatography, and gel filtration. Its purity was investigated by analytical electrophoresis on polyacrylamide gel and by ultracentrifugation sedimentation velocity. The molecular weight (360 000) of the purified inhibitor was determined by sodium dodecyl sulfate electrophoresis and its isoelectric point (4.5) by gel isoelectrofocusing. The Stokes radius (640 nm) was evaluated by gel filtration studies and a frictional ratio (f/fo) of 1.95 was calculated from the molecular weight and Stokes radius. Kinetic studies using angiotensin I as substrate showed that the inhibition of tonin by the purified inhibitor was noncompetitive and does not exceed 70%. Electrophoresis showed the same mobility for [125I]tonin bound to plasma proteins and for [125I]tonin bound to the purified inhibitor. The inhibitor may be a protein resembling half of the dimeric protease inhibitor rat alpha 1-macroglobulin or human alpha 2-macroglobulin.     

Acetylcholinesterase from fetal bovine serum. Purification and characterization of soluble G4 enzyme

Acetylcholinesterase (EC 3.1.1.7) from fetal bovine serum (FBS) was purified to electrophoretic homogeneity. The procedure involved procainamide affinity chromatography with native FBS, followed by chromatography on Sepharose 6B and DEAE-Sephadex. The acetylcholinesterase was purified approximately 44,000-fold, and 13 mg was obtained corresponding to an overall yield of about 45%. The purified acetylcholinesterase was stable at 4 degrees C for at least 8 weeks but was labile to freezing; however, in 50% glycerol the enzyme was stable at -20 degrees C for at least 12 weeks. FBS acetylcholinesterase exhibited typical substrate inhibition, had a Km of 120 microM, and a turnover number of 5300 s-1 with the substrate acetylthiocholine. The enzyme was highly sensitive to the specific acetylcholinesterase inhibitor 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one. FBS acetylcholinesterase was characterized as a G4 form of acetylcholinesterase and was distinguished from bovine erythrocyte acetylcholinesterase on the basis of lectin gel binding, [3H] Triton X-100 binding, amino acid composition, number of catalytic subunits/molecule, and hydrodynamic properties. FBS acetylcholinesterase had a Stokes radius of 76 A as judged by gel filtration, and from this a molecular weight of 340,000 daltons was calculated. The enzyme had a subunit weight of approximately 83,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; paraoxon titration indicated a relative active site mass of 75,000 daltons. The amino acid composition of FBS acetylcholinesterase was similar to the human erythrocyte acetylcholinesterase (Rosenberry, T. L., and Scoggin, D. M. (1984) J. Biol. Chem. 259, 5643-5652). A monoclonal antibody directed against human erythrocyte acetylcholinesterase, AE-2, (Fambrough, D. M., Engel, A. G., and Rosenberry, T. L. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1078-1082) cross-reacted with FBS acetylcholinesterase.     

Cell-to-cell communication and ovulation. A study of the cumulus-oocyte complex

Cell-to-cell communication was characterized in cumulus-oocyte complexes from rat ovarian follicles before and after ovulation. Numerous, small gap junctional contacts were present between cumulus cells and oocytes before ovulation. The gap junction are formed on the oocyte surface by cumulus cell processes that transverse the zona pellucida and contact the oolemma. The entire cumulus mass was also connected by gap junctions via cumulus-cumulus interactions. In the hours preceding ovulation, the frequency of gap junctional contacts between cumulus cells and the oocyte was reduced, and the cumulus was disorganized. Electrophysiological measurements indicated that bidirectional ionic coupling was present between the cumulus and oocyte before ovulation. In addition, iontophoretically injected fluorescein dye was tranferred between the oocyte and cumulus cells. Examination of the extent of ionic coupling in cumulus-oocyte specimens before and after ovulation revealed that ionic coupling between the cumulus and oocyte progressively decreased as the time of ovulation approached. In postovulatory specimens, no coupling was detected. Although some proteolytic mechanism may be involved in the disintegration of the cumulus-oocyte complex, neither the cumulus cells nor the oocyte produced detectable levels of plasminogen activator, a protease which is synthesized by membrana granulosa cells. In summary, cell communication is a characterisitc feature of the cumulus-oocyte complex, and this communication is terminated near the time of ovulation. This temporal pattern of the termination of communication between the cumulus and the oocyte may indicate that communication provides a mechanism for regulating the maturation of the oocyte during follicular development before ovulation.     

Characterization of recombinant human granulocyte-colony-stimulating factor produced in mouse cells.

Mouse C127I cells were transformed with a chimeric plasmid consisting of bovine papillomavirus DNA and human granulocyte-colony-stimulating factor (G-CSF) cDNA placed under the control of the SV40 early promoter. The transformed cells secreted constitutively a high level of human G-CSF, 10-20 micrograms/ml in a low-serum medium. The secreted G-CSF has been purified to homogeneity by a two-step procedure including gel filtration and hydrophobic column chromatography. The purified recombinant G-CSF runs as a single band with an apparent Mr of 19,000 on a polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. This value corresponds to that of the native human G-CSF purified from the medium conditioned by human carcinoma CHU-2 cells. The recombinant human G-CSF was as active as native G-CSF in vitro in supporting proliferation of mouse NFS-60 cells and stimulating colony formation from human as well as mouse bone marrow cells. When the recombinant human G-CSF was subcutaneously administrated into mice, a remarkable stimulation of granulopoiesis and splenomegaly was observed.     

Remodeling of extracellular matrix at ovulation of the bovine ovarian follicle

Using immunohistochemistry and RNA analyses we examined the fate of components of a newly identified matrix that develops between granulosa cells (focimatrix, abbreviated from focal intraepithelial matrix) and of the follicular basal lamina in ovulating bovine ovarian follicles. Pre- and postovulatory follicles were generated by treatment with estradiol (Day 1), progesterone (Days 1-10), and prostaglandin analogue (Day 9) with either no further treatment (Group 1, n = 6) and or with 25 mg porcine LH (Day 11, Group 2, n = 8 or Day 10, Group 3, n = 8) and ovariectomy on Day 12 (12-14 hr post LH in Group 2, 38-40.5 hr in Group 3). In the time frame examined no loss of follicular basal lamina laminin chains beta2 and gamma1 or nidogen 1 was observed. In the follicular basal lamina collagen type IV alpha1 and perlecan were present prior to ovulation; after ovulation collagen type IV alpha1 was discontinuously distributed and perlecan was absent. Versican in the theca interna adjacent to the follicular basal lamina in preovulatory follicles was not observed post ovulation, however, the granulosa cells then showed strong cytoplasmic staining for versican. Expression of versican isoforms V0, V1, and V3 was detected at all stages. Focimatrix was observed in preovulatory follicles. It contained collagen type IV alpha1, laminins beta2 and gamma1, nidogen 1 and perlecan and underwent changes in composition similar to that of the follicular basal lamina. In conclusion focimatrix and the follicular basal lamina are degraded at ovulation. Individual components are lost at different times.     

Human transforming growth factor beta.alpha 2-macroglobulin complex is a latent form of transforming growth factor beta

125I-Labeled human platelet-derived transforming growth factor beta (125I-TGF-beta) and human alpha 2-macroglobulin (alpha 2M) formed a complex as demonstrated by 5% native polyacrylamide gel electrophoresis. The 125I-TGF-beta.alpha 2M complex migrated at a position identical to that of the fast migrating form of alpha 2M. Most of the 125I-TGF-beta.alpha 2M complex could be dissociated by acid or urea treatment. When 125I-TGF-beta was incubated with serum, the high molecular weight form of 125I-TGF-beta could be immunoprecipitated by anti-human alpha 2M anti-sera as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. alpha 2M purified from platelet-rich plasma also showed the latent transforming growth factor activity and immunoreactivity of TGF-beta. These results suggest that TGF-beta.alpha 2M complex is a latent form of TGF-beta.