PMC

Immunostaining of laminin α1 chain in testes of different genotypes. Cross-sections of testes from wild-type, dy^3K/dy^3K, LNα1TG, and dy^3KLNα1TG mice were stained with antibodies against laminin α1 chain. Bar = 80 μm.

Immunostaining of laminin γ3 chain in dy^3KLNα1TG testis. Cross-sections of testes from wild-type and dy^3KLNα1TG mice were stained with antibodies against laminin γ3 chain. Expression of laminin γ3 chain was not restored in dy^3KLNα1TG testis. Bar = 80 μm.

Restoration of the basement membrane in dy^3KLNα1TG testis. Transmission electron microscopy of testis from 5-month-old wild-type and dy^3KLNα1TG mice. The basement membrane is clearly present along the basal surface of the seminiferous tubule of both wild-type and dy^3KLNα1TG mice. Black arrows denote the basement membrane. Bar = 0.25 μm.

ELISA titration of affinity-purified antibodies against laminin γ1 and γ3 chains. A: The laminin γ1 chain antibody was used against γ1 LN/LEa (○) and γ3 LN/LEa (•). B: The laminin γ3 chain antibody was used against γ3 LN/LEa (•) and γ1 LN/LEa (○). Although the laminin γ3 chain antibody showed some binding to the laminin γ1 fragment, no staining was seen in dy^3K/dy^3K testis, which is a rich source of laminin γ1 chain .

Immunostaining of laminin β and γ chains, perlecan, and collagen IV. Cross-sections of testes from wild-type and dy^3K/dy^3K mice were stained with antibodies against laminin β2 and γ1 to γ3 chains, perlecan, and collagen IV. Laminin γ3 chain was absent from dy^3K/dy^3K testis whereas other γ chains, laminin β2 chain, perlecan, and collagen IV appeared normally expressed. Bar = 80 μm.

Immunostaining of laminin α and β chains. Cross-sections of testes from wild-type and dy^3K/dy^3K mice were stained with antibodies against laminin α1 to α5 and β1 chains. Laminin α2 chain was absent from dy^3K/dy^3K testis, whereas other laminin α chains and laminin β1 chain appeared normally expressed. Inset in LMα4 panel shows double staining with laminin α2 chain in red and laminin α4 chain in green. Bar = 80 μm.

Immunoblotting of laminin α1, β1, and γ1 polypeptides. Immunoblotting of protein extracts from testes of wild-type and transgenic mice overexpressing laminin α1 chain (LNα1TG) was performed. The polyclonal antiserum detects all three chains of laminin-111 (laminin α1 chain at 400 kd and laminin β1 and laminin γ1 chains at 200 kd). Both α1 and β1/γ1 chains were up-regulated about twofold. The migration distances of 400- and 200-kd proteins are shown to the left. Coomassie blue-stained loading control is shown in the bottom panel.

Expression pattern of protamine-2, GATA-1, and actin in wild-type and dy^3K/dy^3K testes. A: Cross-sections of testes from wild-type and dy^3K/dy^3K mice were stained with antibodies against protamine-2, GATA-1 (red), and laminin γ1 (green) and with rhodamine-phalloidin. Protamine-2 staining was markedly reduced in dy^3K/dy^3K testis. GATA-1 and laminin γ1 chain double staining demonstrated that the nuclei of Sertoli cells were present near the basement membrane. Actin staining revealed that apical ectoplasmic specializations are missing in dy^3K/dy^3K testis. Bar in top panel = 250 μm and in middle and bottom panels = 80 μm. B: Quantification of protamine-2 and actin staining in wild-type and dy^3K/dy^3K testes. Significantly fewer protamine-2 (P < 0.0008) and actin (P < 0.0004) stainings were observed in dy^3K/dy^3K testis.

Overexpression of laminin α1 chain in testis partially compensates for laminin α2 chain deficiency. Cryosections of testes from 25-day-old (A) and 9-month-old (B) control and dy^3KLNα1TG mice were stained with H&E and antibodies against protamine-2. Histologically, seminiferous tubules of dy^3KLNα1TG mice appeared very similar to those of wild-type mice. C: Quantification of protamine-2 and staining in control and dy^3KLNα1TG testes. Slightly less protamine-2 was detected in 25-day-old (P < 0.2690) but not in 9-month-old (P < 0.8788) dy^3KLNα1TG testis. Bar = 80 μm. D: Quantification of the number of elongated spermatides per seminiferous tubule in 25-day-old wild-type, dy^3K/dy^3K, and dy^3KLNα1TG testes. The number of elongated spermatides in dy^3K/dy^3K testis was significantly smaller than in wild-type testis (P < 0.0001), whereas the number of elongated spermatides in dy^3KLNα1TG testis was not significantly different from wild-type testis (P < 0.6359).

Analyses of testes from wild-type and dy^3K/dy^3K mice. A: Cryosections of testes from wild-type and dy^3K/dy^3K mice were stained with H&E. Seminiferous tubules in wild-type testis had well-developed lumens, whereas lumen formation seemed to be delayed in dy^3K/dy^3K mice. Bar = 80 μm. High-magnification photomicrographs of the seminiferous tubules of wild-type animals showed tubules containing Sertoli cells (blue arrowhead), spermatogonia (black arrowhead), primary spermatocytes (blue arrow), and early and late spermatides (black arrows). The number of spermatides in dy^3K/dy^3K mice were considerably reduced. Bar = 25 μm. B: Transmission electron microscopy of testes from wild-type and dy^3K/dy^3K mice. Asterisks denote the transverse sections of spermatid flagella, which were seen in wild-type but not dy^3K/dy^3K mice. Bar = 1 μm. C: Transmission electron microscopy of testes from wild-type and dy^3K/dy^3K mice. The basement membrane, clearly present along the basal surface of the seminiferous tubule of wild-type mice, was sometimes thinner and disrupted in dy^3K/dy^3K mice. Black arrows denote a normal basement membrane, white arrows denote a thinner basement membrane, and asterisks denote a disrupted basement membrane. Bar = 0.5 μm.