Ultrastructural immunocytochemical localization of fibronectin in the developing rat lung

In a previous study changes in the macrodistribution of fibronectin during rat-lung development were examined. Using the peroxidase-antiperoxidase immunocytochemical technique, we have demonstrated the presence of fibronectin in embryonic, neonatal, and adult rat lung at the ultrastructural level. In the embryo, fibronectin is found both in an intra- and extracellular association with isolated pneumoblasts, and in a periodic distribution along the basal lamina. The neonate displays fibronectin in an intracellular association with early type-I cells and on their basal and luminal surfaces, but not in association with type-II cells. Neonatal basal lamina is diffusely labeled by anti-fibronectin antiserum. Fibronectin in adult tissue is found both intracellularly and on the basal and luminal surfaces of type-I cells but not in type-II cells. The basal lamina and interstitial connective tissue are slightly or non-reactive. These observations confirm and extend our initial suggestion that fibronectin is involved in rat-lung development.     

Ultrastructural and immunofluorescence studies of basal-lamina alterations during mouse-lung morphogenesis

Epithelial differentiation during lung development appears to be influenced by mesenchyme-derived instructions coupled with hormonal regulations. The basal lamina which is associated with progenitor and differentiating epithelia during mouse embryogenesis (Theiler-stages 16-28) was examined by transmission electron microscopy and indirect-immunofluorescence microscopy. During the embryonic phase of lung development, progenitor epithelia for the pulmonary acinus projected microvilli or cytoplasmic "feet" through the basal lamina, which resulted in discontinuities and a close approximation of the adjacent mesenchymal-cell processes. These changes were also associated with the transitory polarization of mesenchymal cells perpendicular to the plane of the basal lamina, which resulted in a sheet of cuboidal mesenchymal cells adjacent to the developing acinar-tubule epithelium. During the embryonic phase of lung development, these specific interstitial or mesenchymal cells stained for heparan-sulfate proteoglycans; no other cell types were immunostained. By Theiler-stage 25, the acinar-tubule epithelia had differentiated into type-II pneumonocytes which contained lamellar bodies and significant amounts of glycogen. Fibronectin, laminin, and heparan-sulfate proteoglycan were localized in the basement membranes during the embryonic, canalicular, and terminal sac phases of lung morphogenesis. A diffuse localization of fibronectin of the interstitial cell surfaces was observed. These observations indicate that major changes in the structure and composition of basal lamina occur during the embryonic and fetal phases of pulmonary-acinus epithelial-cell differentiation and the production of pulmonary surfactant. The major changes in the basal lamina may be partly mediated by mesenchyme-derived instructions for type-II epithelial-cell differentiation.     

Expression and adhesive properties of basement membrane proteins in cerebral capillary endothelial cell cultures

Previous studies have indicated the importance of basement membrane components both for cellular differentiation in general and for the barrier properties of cerebral microvascular endothelial cells in particular. Therefore, we have examined the expression of basement membrane proteins in primary capillary endothelial cell cultures from adult porcine brain. By indirect immunofluorescence, we could detect type IV collagen, fibronectin, and laminin both in vivo (basal lamina of cerebral capillaries) and in vitro (primary culture of cerebral capillary endothelial cells). In culture, these proteins were secreted at the subcellular matrix. Moreover, the interaction between basement membrane constituents and cerebral capillary endothelial cells was studied in adhesion assays. Type IV collagen, fibronectin, and laminin proved to be good adhesive substrata for these cells. Although the number of adherent cells did not differ significantly between the individual proteins, spreading on fibronectin was more pronounced than on type IV collagen or laminin. Our results suggest that type IV collagen, fibronectin, and laminin are not only major components of the cerebral microvascular basal lamina, but also assemble into a protein network, which resembles basement membrane, in cerebral capillary endothelial cell cultures.     

Migratory behavior of cells on embryonic retina basal lamina

In order to study cell translocation in vitro on a physiological substrate a novel cell migration assay was developed using the inner limiting membrane of the avian embryonic retina. The matrix sheet consists of a laminin-rich basal lamina covered by a dense layer of neuroepithelial endfeet. The retina basal lamina does not contain fibronectin. Cells translocating on this substrate displace the neuroepithelial endfeet, leaving behind tracks in the endfeet monolayer. Motility of cells and the relative forward to lateral migration can be quantitated by measuring lengths, widths, and areas of the tracks. Using this assay system, the conditions and patterns of cell migration for a variety of cells have been examined. In the absence of serum all cell types show only minor migratory activity and addition of serum to the culture medium always enhances the rate of cell migration in a saturable, dose-response manner. The serum cannot be replaced by fibronectin or vitronectin (serum spreading factor). For maximum cell migration, serum has to be constantly present in the medium; however, 58% cell migration is obtained in serum-free medium when the matrix is preincubated with serum. According to the area and linearity of the tracks, the migratory behavior of the different cells can be classified into three groups: (i) fibroblasts and the nonpigmented Bowes melanoma cells form straight and long tracks; (ii) glioma, sarcoma, and carcinoma cells from straight but short tracks, and (iii) neuronal tumor cells, epithelial cells, and pigmented B16 melanoma cells form wide and short tracks. Comparative studies with low and high metastatic clones of tumorgenic cell lines show that migratory activity and metastatic potential of cells do not necessarily correlate. Finally, we show that fibroblasts deposit fibronectin fibrils on their paths as they migrate on the basal lamina. Fibronectin trails are also seen when fibroblasts are cultured on plain basal laminae that are pretreated with detergent to remove the endfeet monolayer. Likewise, when fibroblasts are cultured in the presence of antifibronectin antibodies, the fibronectin secreted by cells is detectable. Due to antibody treatment the cellular fibronectin is precipitated and its normal fibril formation is inhibited; however, the translocation of fibroblasts is not impaired.     

Structural features of alveolar wall basement membrane in the adult rat lung

The ultrastructural characteristics of alveolar (ABM) and capillary (CBM) basement membranes in the adult rat lung have been defined using tannic acid fixation, ruthenium red staining, or incubation in guanidine HCl. ABM is dense and amorphous, has 3- to 5-nm filaments in the lamina rara externa (facing the alveolus) that run between the lamina densa and the basal cell surface of the epithelium, has an orderly array of ruthenium red-positive anionic sites that appear predominantly (79%) on the lamina rara externa, and has discontinuities beneath alveolar type II cells but not type I cells that allow penetration of type II cytoplasmic processes into the interstitium of the alveolar wall. The CBM is fibrillar and less compact than ABM, has no lamina rara filaments, and has one fifth the number of ruthenium red- positive anionic sites of ABM that appear predominantly (64%) overlying the lamina densa. Incubation of lung tissue with Flavobacterium heparinum enzyme or with chondroitinase has shown that ABM anionic sites represent heparan sulfate proteoglycans, whereas CBM anionic sites contain this and other sulfated proteoglycans. The CBM fuses in a local fashion with ABM, compartmentalizing the alveolar wall into a thick and thin side and establishing a thin, single, basement-membrane gas-exchange surface between alveolar air, and capillary blood. The potential implications of ABM and CBM ultrastructure for permeability, cell differentiation, and repair and morphogenesis of the lung are discussed.     

Collagen-binding proteins secreted by type II pneumocytes in culture

The alveolar epithelial basement membrane is believed to play important roles in lung development, in maintaining normal alveolar architecture, and in guiding repair following lung injury. However, little is known about the formation of this structure, or of the mechanisms which mediate interactions between the epithelium and specific matrix macromolecules. Since type IV collagen is a major structural component of basement membranes, we investigated the production of type IV collagen-binding proteins by primary cultures of rat lung type II pneumocytes. Cultures were labeled for up to 24 h with 3H-labeled amino acids or [3H]mannose. Soluble collagen-binding proteins which accumulated in the culture medium were isolated by chromatography on collagen-Sepharose and examined by SDS-polyacrylamide gel electrophoresis. The major type IV collagen-binding protein (CBP1) was identified as fibronectin. We also identified a novel disulfide-bonded collagen-binding glycoprotein (CBP2; Mr = 45,000, reduced). This protein was not recognized by polyclonal antibodies to fibronectin, and showed no detectable binding to denatured type I collagen. The protein was resolved from fibronectin and partially purified by sequential chromatography on gelatin and type IV collagen-Sepharose. We suggest that type II pneumocyte-derived collagen-binding proteins contribute to the formation of the epithelial basement membrane and/or mediate the attachment of these cells to collagenous components of the extracellular matrix.     

Effects of Rat Alpha-2 Macroglobulin on Growth in vitro

The patterns of cellular development of primary explants from four tissues from 18-day rat embryos and of Walker 256 carcinosarcoma have been examined in the presence of rat alpha-1 macroglobulin (A1MG), alpha-2 macroglobulin (A2MG), bovine serum albumin (BSA) and of antisera to the foregoing. The following principal results were obtained: 1. A2MG suppresses the formation of cellular carpets around Walker 256 explants whereas, in all other media tested, the neoplastic tissue showed more frequent cellular carpet formation than the embryonic tissues. 2. A1MG increases the frequency of carpet formation by embryonic tissues relative to the other proteins or to control in plain Medium 199. 3. In each of the tested media, embryonic tissues develop peripheral fibroblasts more often than do neoplastic tissues. 4. In cultures of embryo limb explants, and to a lesser degree with other embryonic tissues, A1MG increased and anti-A1MG decreased the extent to which fibroblasts organize into strands and cords.
It is suggested that the suppression of cellular carpet formation by A2MG around Walker 256 explants, but not around embryonic tissue explants, may be related to the peculiar pattern of appearance of this protein in rat sera.     

Development of basal lamina in synaptic and extrasynaptic portions of embryonic rat muscle

Each vertebrate skeletal muscle fiber is ensheathed by a basal lamina (BL) which passes through the synaptic cleft of the neuromuscular junction. In the adult, the synaptic portion of the BL is both functionally and chemically specialized. We have used an immunofluorescence method to compare the development of synaptic and extrasynaptic portions of BL in embryonic rat intercostal muscles. Immunohistochemical staining of adult muscle fibers with monoclonal and serum antibodies defines "synaptic" antigens (including acetylcholinesterase) that are concentrated in synaptic BL, "extrasynaptic" antigens that are concentrated in extrasynaptic regions, and "shared" antigens (including collagen IV, fibronectin, laminin, and a heparan sulfate proteoglycan) that are present in both synaptic and extrasynaptic BL ( Sanes and Chiu , 1983). Synapses appear on newly formed myotubes on embryonic Day 14 (E14; birth is on E22 ). Patches of BL that contain shared and extrasynaptic antigens are present on myotube surfaces by E15, and BL forms a continuous sheath by E17. Shared antigens are present at but not confined to synaptic areas by E15. Two synaptic antigens appear in synaptic areas a day later, and are not detectable extrasynaptically . At least one extrasynaptic antigen is present at immature synapses, and lost or masked by E19 . Thus synaptic BL is not assembled as a unit; rather, components are added, lost, or modified as synaptogenesis proceeds.     

Changes in serum and tissue fibronectin during ontogeny

It has been proved that both in bovine and rat serum fibronectin concentration increases with age. While in foetal bovine the serum fibronectin concentration was 77 +/- 5 micrograms/ml, in adult cows the concentration of this protein reached 150.0 +/- 5.5 micrograms/ml. In newborn rats, the corresponding fibronectin concentration in the serum was 227 +/- 25 micrograms/ml as compared to 455 +/- 47 micrograms/ml in adult individuals. On the contrary, a drop in fibronectin concentration was found in the bovine glomerular basement membrane: fibronectin represents 3.8 +/- 0.6% of total protein in the newborn, while in animals older than two months this protein was not detectable. Accordingly, in newborn rats fibronectin represents 5.1 +/- 0.6% of total protein in the glomerular basement membrane and drops dramatically to 0.4 +/- 0.1% after one year of life with no detectable changes thereafter. In the alveolar basement membrane the changes are similar as in the glomerular basement membrane. While in newborn rats this protein represents 13.2 +/- 0.5% of total basement membrane protein, it drops to 3.2 +/- 0.3% after two years of life.     

Fibronectin synthesis is a marker for peritubular cell contaminants in Sertoli cell-enriched cultures

With indirect immunofluorescent microscopic techniques, we have shown that fibronectin is distributed primarily in or along the basal lamina of the seminiferous tubule boundary tissue in sections of testes from 20-day-old rats. Purified rat Sertoli cell-enriched aggregates, maintained in culture in the presence or absence of serum, exhibit no detectable immunofluorescence with fibronectin antibody, whereas purified peritubular cells in culture do have a positive reaction to fibronectin antibody. Peritubular cells in culture incorporate [35S] methionine into fibronectin which can be immunoprecipitated with a fibronectin antiserum, but Sertoli cells do not. We have used various criteria to estimate the degree of purity of Sertoli cell-enriched preparations. The presence of peritubular myoid cells in conventional Sertoli cell-enriched aggregates, cultured in the presence or absence of serum, can be detected with transmission electron microscopic examination, by the Feulgen staining procedure, and by the immunocytochemical identification of fibronectin. We describe a technique to purify Sertoli cells in conventional Sertoli cell-enriched preparations by treatment with hyaluronidase, resulting in a lesser number of peritubular cells by the above criteria, even in preparations cultured in the presence of serum. Data presented suggest that some of the products previously attributed exclusively to Sertoli cells in Sertoli cell-enriched preparations, particularly those cultured in the presence of serum, may have been contributed by peritubular cells.     

Intraglomerular fibronectin in rat experimental glomerulonephritis.

To clarify the mechanisms of glomerular pericapillary fibronectin deposition in human membranous nephropathy and mesangial proliferative glomerulonephritis, intraglomerular fibronectin distribution was examined by light and electron microscopy using the experimental rat models of Heymann and nephrotoxic serum nephritis. As previously demonstrated by immunofluorescence microscopy (Pettersson and Colvin 1978; Ikeya et al. 1985, 1986), fibronectin was distributed in the mesangial areas and occasionally on percicapillary walls of normal glomeruli, while in nephrotoxic serum nephritis and Heymann nephritis, fibronectin was diffusely located along glomerular capillary walls as well as in the mesangium. By immunoelectron microscopy using the immunogold technique, fibronectin was also noted in the mesangial areas and the lamina densa of the glomerular basement membrane (GBM) in normal glomeruli. In nephrotoxic serum nephritis, fibronectin was seen around mesangial cells situated between endothelial cells and the GBM, suggesting that pericapillary fibronectin in nephrotoxic serum nephritis reflects mesangial extension. However, in Heymann nephritis, it was found uniformly in the lamina rara interna, lamina densa and lamina rara externa of the GBM, indicating no specific relation to glomerular cells. When sections of normal and both experimental nephritis kidneys were incubated with fluorescein isothiocyanate conjugated with rat plasma fibronectin, a linear pattern of fluorescein staining along the glomerular capillary walls was observed in Heymann nephritis but not in normal or nephrotoxic serum nephritic rats. The GBM in Heymann nephritis would thus appear to have an affinity for plasma fibronectin. Based on the above findings, fibronectin in the GBM of rats with Heymann nephritis may reasonably be concluded to originate from the plasma.     

Expression of the integrin subunit alpha8 in murine lung development.

The complex interplay between cells and extracellular matrix (ECM) proteins is critical for lung development. Integrins are key modulators of this interaction. The integrin subunit alpha 8 associates with the beta(1)-subunit to form an RGD-binding integrin. We previously showed that, in adult lung, alpha 8 is expressed in contractile interstitial cells and smooth muscle cells and is upregulated in lung injury. To gain insight into the function of alpha 8 during lung development, we examined the spatiotemporal expression of alpha 8 throughout murine lung development. We compared the distribution of alpha 8 with alpha-smooth muscle actin (alpha SMA), fibronectin (alpha 8 ligand), and cytokeratin. alpha 8 co-localized with alpha SMA and fibronectin in the peribronchial and perivascular regions. In all stages, alpha 8 immunoreactivity was detected diffusely in the mesenchyme except for cells surrounding distal, newly forming airways. alpha 8, alpha SMA, and fibronectin co-localized at tips of secondary septae in the alveolar stage. We conclude that alpha 8 is marker for lung mesenchymal cells starting early in development. alpha 8 is also a marker for smooth muscle cells, expressed as early as alpha SMA. Co-localization of alpha 8 with fibronectin suggests a role in branching morphogenesis. Furthermore, alpha 8 may participate in secondary septation by modulating signals from the extracellular matrix to alveolar myofibroblasts.     

Temporal expression of alpha-smooth muscle actin and drebrin in septal interstitial cells during alveolar maturation.

In rat lung, the definitive alveoli are established during development by the outgrowth of secondary septa from the primary septa present in newborn; however, the mechanism of alveolar formation has not yet been fully clarified. In this study, we characterize the septal interstitial cells in developing alveoli. During the perinatal period, alpha-SMA-containing slender cells were found in the primitive alveolar septa. Alpha-SMA-containing cells were detected at the tips of the septa until postnatal day 21, when the alveolar formation was almost completed, but disappeared in adult. Immunoelectron microscopy demonstrated that alpha-SMA is localized mainly in the cellular protrusions, which are connected with the elastic fibers around the interstitial cells. Developmentally regulated brain protein (drebrin) is also located in the cell extensions containing alpha-SMA in immature alveolar interstitial cells. In adult lung, alpha-SMA-positive cells are located only at the alveolar ducts but are not found in the secondary septa. Desmin is expressed only in alpha-SMA-containing cells at the alveolar ducts but not in those at the tip of alveolar septa. These results suggest that a part of the septal interstitial cells are temporarily alpha-SMA- and drebrin-positive during maturation. Alpha-SMA- and drebrin-containing septal interstitial cells (termed septal myofibroblast-like cells) may play an important role in alveolar formation.     

Evidence for a novel enzymatic mechanism of neural crest cell migration on extracellular glycoconjugate matrices

Migrating embryonic cells have high levels of cell surface galactosyltransferase (GalTase) activity. It has been proposed that GalTase participates during migration by recognizing and binding to terminal N-acetylglucosamine (GlcNAc) residues on glycoconjugates within the extracellular matrix (Shur, B. D., 1982, Dev. Biol. 91:149-162). We tested this hypothesis using migrating neural crest cells as an in vitro model system. Cell surface GalTase activity was perturbed using three independent sets of reagents, and the effects on cell migration were analyzed by time-lapse microphotography. The GalTase modifier protein, alpha-lactalbumin (alpha-LA), was used to inhibit surface GalTase binding to terminal GlcNAc residues in the underlying substrate. alpha-LA inhibited neural crest cell migration on basal lamina-like matrices in a dose-dependent manner, while under identical conditions, alpha-LA had no effect on cell migration on fibronectin. Control proteins, such as lysozyme (structurally homologous to alpha-LA) and bovine serum albumin, did not effect migration on either matrix. Second, the addition of competitive GalTase substrates significantly inhibited neural crest cell migration on basal lamina-like matrices, but as above, had no effect on migration on fibronectin. Comparable concentrations of inappropriate sugars also had no effect on cell migration. Third, addition of the GalTase catalytic substrate, UDPgalactose, produced a dose-dependent increase in the rate of cell migration. Under identical conditions, the inappropriate sugar nucleotide, UDPglucose, had no effect. Quantitative enzyme assays confirmed the presence of GalTase substrates in basal lamina matrices, their absence in fibronectin matrices, and the ability of alpha-LA to inhibit GalTase activity towards basal lamina substrates. Laminin was found to be a principle GalTase substrate in the basal lamina, and when tested in vitro, alpha-LA inhibited cell migration on laminin. Together, these experiments show that neural crest cells have at least two distinct mechanisms for interacting with the substrate during migration, one that is fibronectin-dependent and one that uses GalTase recognition of basal lamina glycoconjugates.     

The alpha-isoform of caveolin-1 is a marker of vasculogenesis in early lung development.

Caveolin-1 is a scaffolding protein component of caveolae, membrane invaginations involved in endocytosis, signal transduction, trans- and intracellular trafficking, and protein sorting. In adult lung, caveolae and caveolin-1 are present in alveolar endothelium and Type I epithelial cells but rarely in Type II cells. We have analyzed patterns of caveolin-1 expression during mouse lung development. Two caveolin-1 mRNAs, full-length and a 5' variant that will translate mainly into caveolin-1alpha and -beta isoforms, are detected by RT-PCR at embryonic day 12 (E12) and afterwards in the developing and adult lung. Immunostaining analysis, starting at E10, shows caveolin-1alpha localized in primitive blood vessels of the forming lung, in an overlapping pattern to the endothelial marker PECAM-1, and later in all blood vessels. Caveolin-1alpha is not detected in fetal or neonatal lung epithelium but is detected in adult epithelial Type I cells. Caveolin-1 was previously shown to be expressed in alveolar Type I cells. These data suggest that expression of caveolin-1 isoforms is differentially regulated in endothelial and epithelial cells during lung development. Caveolin-1alpha is an early marker for lung vasculogenesis, primarily expressed in developing blood vessels. When the lung is fully differentiated postnatally, caveolin-1alpha is also expressed in alveolar Type I cells.     

The apelinergic system in the developing lung: expression and signaling

Apelin and its receptor APJ constitute a signaling pathway best recognized as an important regulator of cardiovascular homeostasis. This multifunctional peptidergic system is currently being described to be involved in embryonic events which extend into vascular, ocular and heart development. Additionally, it is highly expressed in pulmonary tissue. Therefore, the aim of this study was to investigate the role of apelinergic system during fetal lung development. Immunohistochemistry and Western blot analysis were used to characterize apelin and APJ expression levels and cellular localization in normal fetal rat lungs, at five different gestational ages as well as in the adult. Fetal rat lung explants were cultured in vitro with increasing doses of apelin. Treated lung explants were morphometrically analyzed and assessed for MAPK signaling modifications. Both components of the apelinergic system are constitutively expressed in the developing lung, with APJ exhibiting monomeric, dimeric and oligomeric forms in the pulmonary tissue. Pulmonary epithelium also displayed constitutive nuclear localization of the receptor. Fetal apelin expression is higher than adult expression. Apelin supplementation inhibitory effect on branching morphogenesis was associated with a dose dependent decrease in p38 and JNK phosphorylation. The results presented provide the first evidence of the presence of an apelinergic system operating in the developing lung. Our findings also suggest that apelin inhibits fetal lung growth by suppressing p38 and JNK signaling pathways.     

Tissue-dependent regulation of protein tyrosine kinase activity during embryonic development

Protein tyrosine kinase activity was assayed in a variety of chicken tissues during embryonic development and in the adult. In some tissues protein tyrosine kinase activity decreased during embryonic development; however, in other tissues it remained high throughout development, it contrast to the level of protein tyrosine phosphorylation, which decreased during development. The highest levels of tyrosine kinase activity were detected in 17-d embryonic brain although only low levels of protein tyrosine phosphorylation were observed in this tissue. Several alternatives were examined in an effort to determine the mechanism responsible for the low levels of tyrosine phosphorylated proteins in most older embryonic and adult chicken tissues despite the presence of highly active tyrosine kinases. The results show that the regulation of protein tyrosine phosphorylation during embryonic development is complex and varies from tissue to tissue. Furthermore, the results suggest that protein tyrosine phosphatases play an important role in regulating the level of phosphotyrosine in proteins of many older embryonic and adult tissues.     

A Cellular Pathway Involved in Clara Cell to Alveolar Type II Cell Differentiation after Severe Lung Injury

Regeneration of alveolar epithelia following severe pulmonary damage is critical for lung function. We and others have previously shown that Scgb1a1-expressing cells, most likely Clara cells, can give rise to newly generated alveolar type 2 cells (AT2s) in response to severe lung damage induced by either influenza virus infection or bleomycin treatment. In this study, we have investigated cellular pathway underlying the Clara cell to AT2 differentiation. We show that the initial intermediates are bronchiolar epithelial cells that exhibit Clara cell morphology and express Clara cell marker, Scgb1a1, as well as the AT2 cell marker, pro-surfactant protein C (pro-SPC). These cells, referred to as pro-SPC⁺ bronchiolar epithelial cells (or SBECs), gradually lose Scgb1a1 expression and give rise to pro-SPC⁺ cells in the ring structures in the damaged parenchyma, which appear to differentiate into AT2s via a process sharing some features with that observed during alveolar epithelial development in the embryonic lung. These findings suggest that SBECs are intermediates of Clara cell to AT2 differentiation during the repair of alveolar epithelia following severe pulmonary injury.     

Culture of postimplantation rat embryos in rabbit serum for the identification of the growth factor in fractionated rat serum.

The growth factor for postimplantation rat embryos was investigated on the basis of the serum species-specificity in supporting embryonic development in culture. We used rabbit serum as a basal medium for the culture of head-fold stage rat embryos, and examined the effects of various fractions of rat serum on their development. In rabbit serum alone, rat embryos developed poorly. With the rat serum ultrafiltrate of molecular weight (MW) < 300,000, embryonic development improved, but not with the ultrafiltrate of MW < 100,000. With dialyzed rat serum or the globulin fraction of rat serum, embryonic development improved, but the albumin fraction had no effect. It was concluded from these results that some macromolecular growth factor for cultured postimplantation rat embryos was present in the globulin fraction of rat serum. The molecular weight of this growth factor was estimated to be between 65,000 and 300,000. Rabbit serum was considered to be suitable as a medium for the identification of this growth factor.