Basement membrane type IV collagen molecules in the choroid plexus,pia mater and capillaries in the mouse brain

We investigated the differential distribution of basement membrane type IV collagen a chains in the mouse brain by immunohistochemistry using a chain-specific monoclonal antibodies. Subendothelial basement membranes were found to contain alpha1 and alpha2 chains. Basement membranes surrounding smooth muscle cells on blood vascular walls were immunoreactive for alpha1 and alpha2 chains but not for alpha5 and alpha6 chains. Interestingly, the pia mater contained a thin basement membrane which was positive for alpha1, alpha2, alpha5, and alpha6 chains, suggesting that glia limitans superficialis coheres basement membranes containing [alpha1(IV)]2alpha2(IV) and [alpha5(IV)]2alpha6(IV) molecules. In contrast, capillaries always possessed thin basement membranes of [alpha1(IV)]2alpha2(IV) molecules. Cerebrospinal fluid is produced through filtration of blood at the choroid plexus, where two distinct basement membranes were detected by anti-al and anti-alpha2 antibodies. The subendothelial basement membrane appeared to consist of [alpha1(IV)]2alpha2(IV) molecules, whereas the subependymal basement membrane in the choroid plexus was strongly positive for alpha3, alpha4, and alpha5 chains, indicating that the filtering unit was composed of alpha3(IV)alpha4(IV)alpha5(IV) molecules. That the specific localizations of these molecules are shared by renal glomeruli and the choroid plexus leads us to hypothesize that the supramolecular network containing alpha3(IV) alpha4(IV)alpha5(IV) molecules may function as a permeability selective barrier.     

The distributions of type IV collagen alpha chains in basement membranes of human epidermis and skin appendages

Distributions of type IV collagen alpha chains in the basement membrane (BM) of human skin and its appendages were analyzed by immunofluorescent microscopy using chain-specific monoclonal antibodies. The basement membrane beneath the epidermis contained [alpha1(IV)](2)alpha2(IV) and [alpha5(IV)](2)alpha6(IV) but no alpha3(IV)alpha4(IV)alpha5(IV); this held true for at the eccrine sweat glands and glandular ducts, sebaceous glands, hair follicles, and arrector muscles of hair. The secretary portion of the eccrine sweat glands was rich in [alpha1(IV)](2) alpha2(IV) and had less [alpha5(IV)](2)alpha6(IV), while [alpha5(IV)](2) alpha6(IV) was abundant in the ductal portion. In the subepidermal zone, alpha5(IV)/alpha6(IV) chain negative spots (1.9-15.0 microm) were frequently observed. Triple staining samples (Mel.2, alpha2(IV) and alpha5(IV) chains) showed that about 50% of epidermal melanocytes colocalized with such spots. Results suggest that these alpha5(IV)/alpha6(IV) chain negative spots of the subepidermal basement membrane have a particular relationship with melanocytes and are sites for certain interactions between the two.     

The differential distribution of type IV collagen alpha chains in the subepithelial basement membrane of the human alimentary canal

We studied distribution patterns of type IV collagen alpha chains in the subepithelial basement membrane (SBM) of the human gastrointestinal tract - the esophagus through the anal canal - by immunofluorescent microscopy using alpha(IV) chain-specific monoclonal antibodies. The alpha1(IV), alpha2(IV), alpha5(IV), and alpha6(IV) chains were found in the SBM throughout the tract, indicating the localization of [alpha1(IV)](2)alpha2(IV) and [alpha5(IV)](2)alpha6(IV) heterotrimeric molecules. The [alpha1(IV)](2)alpha2(IV) molecule was continuously stained, while the [alpha5(IV)](2)alpha6(IV) molecule was weakly stained in gastric glands and small intestinal crypts. In addition, the SBM at the luminal surface epithelium of the stomach and large intestine contained small amounts of alpha3(IV) and alpha4(IV) chains which combined to form the alpha3(IV)alpha4(IV)alpha5(IV) heterotrimeric molecule with alpha5(IV) chain. The SBM beneath the villous epithelium of the small intestine was also demonstrated to have an alpha3(IV) chain and alpha4(IV) chain. Considering the specific locations of the type IV collagen trimers throughout the gastrointestinal SBM, the supramolecular network containing the alpha3(IV)alpha4(IV)alpha5(IV) molecule appears to function as a selective permeability barrier and /or as a protection against chemical stress from the luminal digestive enzymes.     

Differential distribution of basement membrane type IV collagen alpha1(IV), alpha2(IV), alpha5(IV) and alpha6(IV) chains in colorectal epithelial tumors

In this study, we examined the relationship between the histopathological grade and immunohistochemical localization of six genetically distinct type IV collagen alpha chains, the major component of basement membrane (BM), in normal and neoplastic colorectal tissues. In the normal colorectal mucosa, alpha1/alpha2(IV) and alpha5/alpha6(IV) chains were stained in all epithelial BM. However, alpha3/alpha4(IV) chains were restrictively immunostained in the BM of the apical surface epithelium. Similar immunostaining profiles for alpha1/alpha2(IV) and alpha5/alpha6(IV) chains were observed in tubular adenomas with mild/moderate atypia. However, in intramucosal carcinomas, both alpha1/alpha2(IV) chains were linearly stained in the BM of cancer cell nests, while the assembly of alpha5/alpha6(IV) chains into the BM was inhibited in a discontinuous or negatively stained pattern. The normal colorectal mucosa forms a second network of BM composed of alpha5/alpha6(IV), partly alpha3/alpha4(IV) chains, in addition to the classic network of alpha1/alpha2(IV) chains. The differential immunohistochemical localization of the type IV collagen alpha5/alpha6 chains could be one diagnostic marker for the invasiveness of colorectal cancer.     

Loss of type IV collagen alpha 5 and alpha 6 chains in human invasive prostate carcinomas.

Type IV collagen, a major component of basement membranes, is organized in a network responsible for the mechanical resistance of the basement membranes. It also plays a key role in epithelial cell adhesion to basement membranes. This study was designed to investigate the distribution of type IV collagen alpha-chains in normal, preneoplastic, and malignant prostate basement membranes. For this purpose, immunohistochemistry using specific antibodies raised against the different alpha-chains of type IV collagen was performed in eight normal samples, six prostatic intraepithelial neoplasia, and 20 malignant lesions of the prostate. Our results demonstrate the presence of the "novel" alpha 5 (IV) and alpha 6 (IV) chains along with the "classical" alpha 1 (IV)/alpha 2 (IV) chains in the basement membrane of the normal prostate gland. The alpha 3 (IV) chain was never detected in any prostate specimen. Prostatic intraepithelial neoplasia showed a similar immunostaining pattern to that found in normal glands. In cancer gland basement membranes, we demonstrate for the first time a specific loss of the alpha 5 (IV) and alpha 6 (IV) chains, whereas the classical alpha 1 (IV) and alpha 2 (IV) chains were consistently exhibited. Additionally, type VII collagen colocalized with alpha 5 (IV) collagen chain, and these two proteins, which were always observed in normal and prostatic intraepithelial neoplasia gland basement membranes, were lost in invasive carcinoma basement membranes. This observation raises questions about the possible association or cooperation between alpha 5 (IV)/alpha 6 (IV) chains and anchoring fibrils in prostate glands basement membrane.     

Distribution of α(IV) collagen chains in the ocular anterior segments of adult mice

The distribution of the collagen chains from α1(IV) to α6(IV) could serve as a basis for the characterization of type IV collagen. In this study, immunohistochemistry of the ocular anterior segment of adult mice was performed using specific monoclonal antibodies against each chain in the series from α1(IV) to α6(IV). The results show that the components of type IV collagen in vascular basement membranes are α1(IV) and α2(IV) with or without α5(IV) and α6(IV) chains and those in epithelium and muscle basement membranes are α1(IV), α2(IV), α5(IV), and α6(IV) chains. In corneal endothelium, pigmented epithelium of iris and ciliary body, and trabecular meshwork, α3(IV) and α4(IV) chains are also expressed in addition to α1(IV), α2(IV), α5(IV), and α6(IV) chains. Moreover, we investigated the change in molecular composition in ciliary body during postnatal development. α3(IV) and α4(IV) chains were also expressed in addition to α1(IV), α2(IV), α5(IV), and α6(IV) chains in ciliary pigmented epithelium basement membrane from 7 days after birth. This result suggests that the basement membranes gradually change their biochemical features owing to temporal regulation. Taken together, these findings suggest that the different distribution and the developmental expression of α1(IV) to α6(IV) chains are associated with the tissue-specific function of type IV collagen in basement membranes.     

Remodeling of basement membrane in association with cancer invasion

Type IV collagen the major component of basement membrane (BM), is composed of six genetically distinct alpha chains. In normal breast tissue, benign breast tumors, and in the intraductal components of invasive ductal carcinoma, alpha 1 (IV) and alpha 2 (IV) chains were stained in all BM, whereas alpha 5 (IV) and alpha 6 (IV) chains were restrictively localized in a linear pattern in the epithelial BM. However, in invasive ductal carcinoma, alpha 1 (IV) and alpha 2 (IV) chains were discontinuously or negatively stained in the cancer cell nest, and the assembly of alpha 5 (IV) and alpha 6 (IV) chains into the BM was completely inhibited. The results indicate that the mammary gland forms a second network of BM composed of alpha 5 (IV)/alpha 6 (IV) chains, in addition to the classic network of alpha 1 (IV)/alpha 2 (IV) chains. Remodeling of type IV collagen alpha chains during the development of invasive breast cancer seems to be differentially regulated, and to be associated with modification of histopathological findings.     

A human-mouse chimera of the alpha3alpha4alpha5(IV) collagen protomer rescues the renal phenotype in Col4a3-/- Alport mice

Collagen IV is a major structural component of basement membranes. In the glomerular basement membrane (GBM) of the kidney, the alpha3, alpha4, and alpha5(IV) collagen chains form a distinct network that is essential for the long-term stability of the glomerular filtration barrier, and is absent in most patients affected with Alport syndrome, a progressive inherited nephropathy associated with mutation in COL4A3, COL4A4, or COL4A5 genes. To investigate, in vivo, the regulation of the expression, assembly, and function of the alpha3alpha4alpha5(IV) protomer, we have generated a yeast artificial chromosome transgenic line of mice carrying the human COL4A3-COL4A4 locus. Transgenic mice expressed the human alpha3 and alpha4(IV) chains in a tissue-specific manner. In the kidney, when expressed onto a Col4a3(-/-) background, the human alpha3(IV) chain restored the expression of and co-assembled with the mouse alpha4 and alpha5(IV) chains specifically at sites where the human alpha3(IV) was expressed, demonstrating that the expression of all three chains is required for network assembly. The co-assembly of the human and mouse chains into a hybrid network in the GBM restores a functional GBM and rescues the Alport phenotype, providing further evidence that defective assembly of the alpha3-alpha4-alpha5(IV) protomer, caused by mutations in any of the three chains, is the pathogenic mechanism responsible for the disease. This line of mice, humanized for the alpha3(IV) collagen chain, will also provide a valuable model for studying the pathogenesis of Goodpasture syndrome, an autoimmune disease caused by antibodies against this chain.     

Differential expression of collagen IV alpha1 to alpha6 chains in basement membranes of benign and malignant odontogenic tumors

Type IV collagen, the major component of basement membrane (BM), demonstrates a stage- and position-specific distribution of its isoforms during tooth development. To determine its localization in BM of odontogenic neoplasms, immunohistochemistry using six anti-alpha(IV) chain-specific monoclonal antibodies was performed. Results disclosed that BM demonstrated an irregular alpha(IV) chain profile in malignant odontogenic tumors as compared to benign odontogenic neoplasms. No alpha3(IV) chains were detected. Expression of alpha1(IV)/alpha2(IV) and alpha5(IV)/alpha6(IV) chains was stronger in desmoplastic ( n=3) than in ordinary (n=5) ameloblastomas. The adenomatoid odontogenic tumor ( n=2) distinctly expressed these chains in BM of cribriform areas and hyaline materials (which was also alpha4(IV)-positive), but weakly around epithelial whorls/rosettes/nests and mineralized foci. These five chains also stained BM and tumor cells of ameloblastic fibroma ( n=3) and ameloblastic fibro-odontosarcoma (n=1), but not the inductive hard tissues. Ameloblastic carcinoma ( n=2) showed specific alpha1(IV)/alpha2(IV) chain loss, while primary intraosseous carcinoma ( n=1) demonstrated a discontinuous alpha1(IV)/alpha2(IV) and alpha5(IV)/alpha6(IV) staining pattern. The present results suggest that modification and remodeling of BM collagen IValpha chains occur during odontogenic neoplasms' progression.     

Differential expression of collagen type IV alpha-chains in the tubulointerstitial compartment in experimental chronic serum sickness nephritis.

The expression of collagen type IV chains in the renal tubulointerstitium was investigated during the development of chronic serum sickness (CSS) in rats, a model for immune complex-mediated renal disease. Immunohistochemical studies showed increased expression of alpha4(IV) collagen early during disease development, followed by an increase in alpha1(IV) through alpha3(IV) collagen subchain expression, especially in the tubular basement membrane. Dot-blot and in situ hybridization analysis showed a transient increase in steady-state mRNA levels for all collagen IV subchains during the development of CSS, which was most abundant for alpha1(IV), alpha2(IV), and alpha4(IV). Statistical correlations were found between the mRNA levels of alpha1(IV) and alpha2(IV) collagen and between alpha3(IV) and alpha4(IV), in line with the results of others which showed that these chains are co-distributed as heterotrimer collagen type IV molecules. However, additional correlations were found between the mRNA levels coding for alpha1(IV) and alpha3(IV) collagen, and between alpha1(IV) and alpha4(IV) mRNAs in the course of CSS. These abnormal correlations support the hypothesis that changes occur in the co-expression of the collagen IV subchains during the development of CSS. In addition, a strong correlation was found between the presence in the tubulointerstitium of alpha1(IV) and alpha2(IV) collagen chains, on the one hand, and the tubulointerstitial influx of R73+ and ED1+ cells, on the other, suggesting the involvement of inflammatory cells in the observed alterations in matrix production. Changes in the relative abundance of collagen IV chains in disease states may perturb the collagen IV network in the tubulointerstitial compartment and thereby play a role in the development of renal failure.     

Production and characterization of a monoclonal antibody to human Type IV collagen.

We have produced a monoclonal antibody to human basement membrane Type IV collagen. The antibody reacts with the pepsin-resistant, collagenase-sensitive domain of Type IV collagen isolated from placental membranes, but not with human collagens of Types I, II, III, V, 1alpha, 2alpha, and 3alpha. The antibody precipitates biosynthetically labeled human Type IV procollagen, and the precipitate contains both the alpha1 (IV) and alpha2 (IV) chains, suggesting the occurrence of both of these chains within the same triple-helical molecule. When used in indirect immunofluorescence, the antibody gives brilliant staining of basement membranes from a variety of human tissues but does not stain tissues of bovine, canine, rabbit, rat, or mouse origin. It is suggested that this antibody will be of value in research on the structure of human basement membrane collagen, on the distribution of this collagen in various basement membranes, and particularly for the study of basement membranes in normal human development and pathologic processes.     

Approach to the diagnosis of thin basement membrane nephropathy in females with the use of antibodies to type IV collagen

CONTEXT: Thin basement membrane nephropathy is recognized by a diffusely thin glomerular basement membrane (GBM) ultrastructurally. In contrast to Alport syndrome (AS), there is no GBM thickening, lamellation, or granular inclusions. Morphologically, there is overlap between thin basement membrane nephropathy and AS in female patients in whom there might be only thin GBM and no pathognomonic findings of AS. OBJECTIVE: To determine if the use of antibodies to collagen IV is helpful in making the distinction between thin basement membrane nephropathy and AS in female patients with primarily thin GBMs. DESIGN: We examined renal biopsies from 9 adult female patients with thin GBMs for the presence of alpha1, alpha3, alpha4, and alpha5 chains of type IV collagen by immunofluorescence. RESULTS: In 2 patients with segmental GBM staining, no suggestion for AS was found on physical examination or in their family history. In the remaining 7 patients with normal GBM staining, 4 had family members with end-stage renal disease of unknown etiology, raising the suspicion of X-linked or autosomal-recessive AS. Three patients were presumed to have thin basement membrane nephropathy. CONCLUSION: Segmental GBM staining for alpha3, alpha4, and alpha5 chains of type IV collagen raises the suspicion of AS in the presence of adequate controls and other supporting evidence. Normal GBM staining for alpha3, alpha4, and alpha5 chains of type IV collagen, however, does not exclude AS.     

Loss of expression of type IV collagen alpha5 and alpha6 chains in colorectal cancer associated with the hypermethylation of their promoter region

Type IV collagen, a major component of the basement membrane (BM), is composed of six genetically distinct alpha(IV) chains, alpha1(IV) to alpha6(IV). Their genes are paired on three different chromosomes in a head-to-head arrangement. The alpha5(IV) gene (COL4A5) and the alpha6(IV) gene (COL4A6) are on chromosome Xq22 and are regulated by a bidirectional promoter. Loss of the alpha5(IV)/alpha6(IV) chains in epithelial BM occur in the early stage of cancer invasion. However, the regulatory mechanism of the specific loss of the alpha5(IV)/alpha6(IV) chains during cancer cell invasion is still undetermined. In the present study, we examined the expression of the alpha5(IV)/alpha6(IV) chains and the methylation profiles of the bidirectional promoter region of COL4A5/COL4A6 in colon cancer cell lines and colorectal tumor tissues. The expression of the alpha5(IV)/alpha6(IV) chains was down-regulated in colorectal cancer, and the loss of expression of the alpha5(IV)/alpha6(IV) chains was associated with the hypermethylation of their promoter region. In conclusion, the hypermethylation of the bidirectional promoter region of COL4A5/COL4A6 is one of the events that is responsible for the loss of expression of the alpha5(IV)/alpha6(IV) chains and the remodeling of the epithelial BM during cancer cell invasion.     

Loss of alveolar basement membrane type IV collagen alpha3, alpha4, and alpha5 chains in bronchioloalveolar carcinoma of the lung

Type IV collagen, the major component of basement membrane (BM), is composed of six genetically distinct alpha(IV) chains. This study investigated for the first time the expression of these six alpha(IV) chains immunohistochemically, using alpha(IV) chain-specific monoclonal antibodies, in normal lung and in small (less than 2 cm in diameter) adenocarcinoma of the lung with a bronchioloalveolar growth pattern at the periphery. Small adenocarcinomas were histopathologically classified into three subtypes: bronchioloalveolar carcinoma (BAC) without collapse, BAC with collapse, and adenocarcinoma with bronchioloalveolar features. In normal lung, alveolar BM was composed of alpha1(IV)/alpha2(IV) chains and alpha3(IV)/alpha4(IV)/alpha5(IV) chains. In non-collapsed areas of BAC, alveolar BM was composed of linear alpha1(IV)/alpha2(IV) chains and discontinuous alpha3(IV)/alpha4(IV)/alpha5(IV) chains. In collapsed areas of BAC, alveolar BM was composed of linear and thick alpha1(IV)/alpha2(IV) chains only, because of the complete loss of alpha3(IV)/alpha4(IV)/alpha5(IV) chains. In invasive areas of adenocarcinoma with bronchioloalveolar features, alpha1(IV)/alpha2(IV) chains around the cancer cell nests were disrupted, in addition to the complete loss of alpha3(IV)/alpha4(IV)/alpha5(IV) chains. In conclusion, during the process of stromal invasion of lung adenocarcinoma, type IV collagen of alveolar BM is remodelled from the complete type, composed of alpha1(IV)/alpha2(IV)/alpha3(IV)/alpha4(IV)/alpha5(IV) chains, to the incomplete type, composed of only alpha1(IV)/alpha2(IV) chains, before the disruption of alpha1(IV)/alpha2(IV) chains. These findings may help to clarify the molecular mechanisms of cancer invasion.     

Differential tissular expression and localization of type IV collagen alpha1(IV), alpha2(IV), alpha5(IV), and alpha6(IV) chains and their mRNA in normal breast and in benign and malignant breast tumors

Type IV collagen, the major component of basement membrane (BM), is composed of six genetically distinct alpha chains. We investigated the cellular regulation and origin of these alpha(IV) chains in normal and neoplastic breast tissues by immunohistochemistry by using alpha(IV) chain-specific antibodies and by in situ hybridization. In normal breast, alpha1(IV) and alpha2(IV) chains were stained in all BM, whereas alpha5(IV) and alpha6(IV) chains were restrictively localized in a linear pattern in the BM of the mammary gland. Similar immunostaining profiles were observed in benign breast tumors and in the intraductal components of invasive ductal carcinoma. However, in invasive ductal carcinoma, alpha1(IV) and alpha2(1V) chains were discontinuously or negatively stained in the cancer cell nests, and the assembly of alpha5(IV) and alpha6(IV) chains into the BM was completely inhibited. Coexpression of alpha5(IV) and alpha6(IV) chains was related to the localization of alpha-smooth muscle actin (alpha-SMA)-positive myoepithelial cells. By in situ hybridization, in fibroadenoma and invasive ductal carcinoma, the signals for alpha1(IV) and alpha2(IV) mRNA were abundant in stromal cells. However, the signals for alpha5(IV) and alpha6(IV) mRNA were not seen in any of these cells. In contrast, in intraductal papilloma, coexpression of alpha1 (IV)/alpha2(IV) mRNA and alpha5(IV)/alpha6(IV) mRNA was identified in epithelial cells. The results indicate that the mammary gland forms a second network of BM composed of alpha5(IV)/alpha6(IV) chains, in addition to the classic network of alpha1(IV)/alpha2(IV) chains. The expression of type IV collagen alpha chains seems to be differentially regulated by the epithelial-myoepithelial interaction and to be associated with the invasive potential of breast cancer.     

Comparison of non-collagenous type IV collagen subunits in human glomerular basement membrane, alveolar basement membrane, and placenta

This study examines the similarities and differences in the noncollagenous domain (NC1) of type IV collagen from human glomerular basement membrane (hGBM), alveolar basement membrane (hABM), and placenta (hPBM). Following collagenase digestion, NC1 domain was isolated on Bio-Gel A-0.5m or by cation exchange chromatography on S-Sepharose. NC1 from each source was characterized by SDS PAGE, and two dimension NEPHGE/SDS PAGE. Immunoblotting and ELISA inhibition was performed using antibody probes specific for M28 , M28+, M26 and M24 monomer subunits of human NC1. It was observed that all NC1 subunits were present in hGBM and hABM derived material, however M28 and M28+ monomers were absent in hPBM NC1. These findings indicate that while alpha 1(IV) and alpha 2(IV) collagen chains are present in hGBM, hABM and hPBM, alpha 3(IV) and alpha 4(IV) collagen chains are only found in hGBM and hABM but are absent in hPBM. It can now be appreciated that heterogeneity of alpha (IV) chain composition exists in basement membranes from various organs.     

Partial protein sequence of the globular domain of alpha 4(IV) collagen chain: sites of sequence variability and homology with alpha 2(IV).

The globular domain (NC) of alpha 4(IV) collagen chain was partially sequenced and compared with the NC domain of other collagen IV chains. The alpha 4(IV) NC domain was found to be most closely related to alpha 2(IV) NC domain but distinct from the NC domain of alpha 1(IV), alpha 2(IV), alpha 3(IV) and alpha 5(IV) collagen chains. Partial sequence, representing nearly one half of alpha 4(IV) NC domain, shows 56%, 69%, 51% and 54% identity with the corresponding NC domains of alpha 1(IV), alpha 2(IV), alpha 3(IV) and alpha 5(IV) collagen chains, respectively. A short, highly polar, region of variable sequence is found near the carboxy terminus of alpha 4(IV) NC domain. This sequence corresponds to a non-conserved region among NC domains, suggesting functional specialization at this site. It exhibits high surface probability with predicted structural differences among NC domains. These results confirm uniqueness of alpha 4(IV) NC domain and indicate its structural relatedness to other NC domains of collagen IV.     

Sequential expression of type IV collagen networks: testis as a model and relevance to spermatogenesis

The six alpha chains of type IV collagen are organized into three networks: alpha1/alpha2, alpha3/alpha4/alpha5, and alpha1/alpha2/alpha5/alpha6. A shift from the alpha1/alpha2 to the alpha3/alpha4/alpha5 network occurs in the developing glomerular basement membrane, but how the alpha1/alpha2/alpha5/alpha6 network fits into this sequence is less clear, because the three networks do not colocalize. Here, we studied the seminiferous tubule basement membrane of normal canine testis where all three networks do colocalize: the alpha1/alpha2 network is expressed from birth, the alpha1/alpha2/alpha5/alpha6 network by 5-6 weeks of age, and the alpha3/alpha4/alpha5 network by 2 months of age. A canine model of Alport syndrome allowed study of the absence of alpha3/alpha4/alpha5 and alpha1/alpha2/alpha5/alpha6 networks in testis. In Alport dogs, the seminiferous tubule basement membrane was thinner than in controls. Spermatogenesis began at the same time as with normal dogs; however, the number of mature sperm was significantly reduced in Alport dogs. Thus, it would appear that alpha3/alpha4/alpha5 and alpha1/alpha2/alpha5/alpha6 networks are not essential for onset of spermatogenesis, but long-term function may be compromised by the loss of one or both networks. This situation is analogous to the glomerular basement membrane in Alport syndrome. In conclusion, testis can serve as a model system to study the sequence of type IV collagen network expression.