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

Abstract

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.

Figure 1

Transverse section of seminiferous tubules. A: The outermost cellular layer is composed of Sertoli cells and spermatogonia. Toward the lumen, spermatocytes and spermatozoa are seen at various stages of development. B: The STBM is seen underlying the seminiferous epithelium (SE), in this case, a spermatogonium. Underlying the STBM is an extracellular matrix (ECM) rich in collagen fibrils, and beneath this, one or more layers of peritubular myoid cells (MYO), each bounded by a discontinuous basement membrane (BM). These cells are separated from the interstitium by a second layer of ECM. A, toluidine blue ×200; B, electron micrograph ×50,000.

Figure 2

Distribution of the α1–α6 chains of type IV collagen in normal canine testis. Each chain is shown in a panel of three time points in development: 11 days, 1.5 months, and 3 months of age. The α1 and α2 chains were present in all basement membranes (STBM, interstitial cells, capillaries, and smooth muscle cells) at all time points. The α3 and α4 chains were present only in the STBM and not before 2 months. The α5 and α6 chains appeared in the STBM of normal dogs around 5 weeks of age, in contrast to basement membranes around smooth muscle cells that showed much stronger staining for both the α5 and α6 chains at this time. Interstitial cells were also positive at this time. H&E counterstain, ×400.

Figure 3

Distribution of the α1–α6 chains in the normal canine rete testis. In 11-day-old normal dogs, the basement membranes of the rete testis stain intensely for all six isoforms of type IV collagen, and this pattern continues into adulthood. H&E counterstain, ×200.

Figure 4

Distribution of the α1–α6 chains of type IV collagen in normal human testis. In this biopsy from a normal 14-year-old, all six chains were expressed in the STBM. The pattern of staining was similar to that seen in normal canine testis. In another sample from a normal 12-year-old, only the α1 and α2 chains were present (data not shown). H&E counterstain, ×400.

Figure 5

Distribution of the α1–α6 chains in affected canine testis. The α1 and α2 chains were present in all basement membranes (STBM, capillaries, and interstitial cells) at all times examined (3 months of age is illustrated). The STBM appeared thinner in Alport dogs compared to normal. The α3, α4, α5, and α6 chains were never present in the basement membranes around seminiferous tubules, interstitial cells, or vascular smooth muscle. H&E counterstain, ×400.

Figure 6

Electron microscopy and morphometry of canine STBM. Top: seminiferous tubules of normal and Alport dogs were surrounded by a complete basement membrane. The thickness of the STBM appears decreased in Alport dogs compared to normal age-matched controls (data for 3 months of age are illustrated, magnification ×50,000). At no time point was a multilaminar appearance noted. Bottom: Graph of STBM thickness versus age in normal and Alport dogs. Data points and error bars represent the mean ± SD from 9 normal and 14 affected dogs. In both normal and Alport dogs, the thickness of the STBM decreased to nearly 40% of its original width between 5 weeks and 8 months of age. For any given time point, the width of the STBM in Alport dogs was ∼70% that of age-matched normal dogs. The STBM thickness was significantly less in affected dogs under 4 months of age compared to normal dogs (P = 0.04).

Figure 7

Spermatogenesis is reduced in Alport dogs. The histology of the testis in normal (A and C) and affected (B and D) dogs is presented in representative sections at 8 months (A and B), 4 years (C), and 3 years (D) of age. Spermatogenesis is first observed around 6 months of age in both normal and Alport dogs (data not shown). Spermatogenesis was present at reduced levels in Alport dogs compared to age-matched normal dogs as evident from the decreased number of spermatozoa in the tubular lumen. The dog illustrated in D underwent a renal transplant at 3 months of age and maintained normal renal function thereafter. Bilateral orchidectomy was performed at age 33 months (toluidine blue counterstain, ×200). The reduction of sperm counts in affected dogs was statistically significant in both age groups (P < 0.05).