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(American Journal of Pathology. 2005;167:823-833.)
© 2005 American Society for Investigative Pathology

Laminin {alpha}1 Chain Corrects Male Infertility Caused by Absence of Laminin {alpha}2 Chain

Mattias Häger*, Kinga Gawlik*, Alexander Nyström*, Takako Sasaki{dagger} and Madeleine Durbeej*

From the Department of Experimental Medical Science,* Division for Cell and Matrix Biology, University of Lund, Lund, Sweden; and Max-Planck-Institut fur Biochemie,{dagger} Martinsried, Germany


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Laminins are important for basement membrane structure and function. The laminin {alpha}2 chain is a major component of muscle basement membranes, and mutations in the laminin {alpha}2 gene lead to congenital muscular dystrophy in humans and mice. Although the laminin {alpha}2 chain is prominently expressed in testicular basement membranes, its role in testis has remained unclear. Here, we show that laminin {alpha}1, {alpha}2, ß1, ß2, {gamma}1, and {gamma}3 chains are the major laminin chains in basement membranes of seminiferous tubules. In laminin {alpha}2 chain-deficient dy3K/dy3K mice, lack of laminin {alpha}2 chain led to concurrent reduction of laminin {gamma}3 chain and abnormal testicular basement membranes. Seminiferous tubules of laminin {alpha}2 chain-deficient dy3K/dy3K mice displayed a defect in the timing of lumen formation, resulting in production of fewer spermatides. We also demonstrate that overexpression of laminin {alpha}1 chain in testis of dy3K/dy3K mice compensated for laminin {alpha}2 chain deficiency and significantly reversed the appearance of the histopathological features. We thus provide genetic data that laminin {alpha} chains are essential for normal testicular function in vivo.

Basement membranes are thin sheets of extracellular matrix that surround epithelia, endothelia, muscle, fat, and Schwann cells. All basement membranes contain laminins, collagen type IV, nidogens, and heparan sulfate proteoglycans.1 Laminins are heterotrimers of {alpha}-, ß-, and {gamma}-chains. The five {alpha}-, three ß-, and three {gamma}-chains give rise to at least 15 different protein isoforms that differ in their tissue distribution.2 Laminin {alpha}1 and {alpha}5 chains are crucial for early embryonic development and organogenesis, whereas laminin {alpha}2, {alpha}3, and {alpha}4 chains are essential for postnatal development.3 Laminin {alpha}2 chain is deposited in basement membranes of muscle, and laminin {alpha}2 chain deficiency leads to congenital muscular dystrophy MDC1A.4-6 Two knock out models (dyW/dyW and dy3K/dy3K) and three spontaneous mouse strains (dy/dy, dy2J/dy2J, and dyPas/dyPas) representing animal models for congenital muscular dystrophy with laminin {alpha}2 chain deficiency have been reported.7-12 All strains develop severe clinical signs of muscular dystrophy. The laminin {alpha}2 chain is also expressed in other tissues such as the central and peripheral nervous systems, thymus, and testis; and non-muscle defects are associated with laminin {alpha}2 chain deficiency.13-18 Interestingly, dy/dy mice expressing reduced levels of laminin {alpha}2 chain do not reproduce19 (www.jax.org). This may not only be due to muscle weakness, but could also be due to testicular defects. Furthermore, abnormal basement membrane structures are detected in testicular biopsies from men with impaired fertility,20 and in vitro studies point toward a role for collagen IV and laminins in spermatogenesis.21,22 Thus, basement membranes appear significant for spermatogenesis. However, genetic evidence for a role of basement membrane components for spermatogenesis is lacking.

Normal spermatogenesis is an intricate process that takes place in the seminiferous epithelium of the mammalian testis. The seminiferous tubules are lined by small cells called spermatogonia. Alternating with the spermatogonia are the highly polarized epithelial cells, the Sertoli cells, which act as nursery units for the developing sperm. The Sertoli cells are attached to each other, to the spermatogonia, and to the basement membrane of the seminiferous tubule to form the blood-testis barrier. During spermatogenesis, spermatogonia differentiate into spermatocytes that will cross through the blood-testis barrier as they mature and traverse the tubular lumen.23,24

In the present study, we provide genetic evidence that laminin {alpha} chains are vital for spermatogenesis. We first examined the expression pattern of laminin {alpha}, ß, and {gamma} chains in wild-type and laminin {alpha}2 chain-deficient dy3K/dy3K testes. We demonstrate that lack of laminin {alpha}2 chain leads to concomitant loss of the laminin {gamma}3 chain and an abnormal basement membrane. Seminiferous tubules of laminin {alpha}2 chain-deficient dy3K/dy3K mice displayed a defect in the timing of lumen formation. Furthermore, significantly fewer spermatides were produced in testes lacking laminin {alpha}2 chain. Finally, we show that overexpression of laminin {alpha}1 chain in testis of dy3K/dy3K mice compensates for laminin {alpha}2 chain deficiency and partially reverses the appearance of the histopathological features.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Transgenic Mice

Laminin {alpha}2 chain-deficient dy3K/dy3K mice were previously described.7 Transgenic mice deficient in laminin {alpha}2 chain but overexpressing laminin {alpha}1 chain in various tissues (dy3KLN{alpha}1TG mice) were recently described.25

Quantitative Real-Time Polymerase Chain Reaction (PCR)

Total RNA from human testis was obtained from Becton-Dickinson, Franklin Lakes, NJ. Total RNA from mouse testes was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) according to manufacturer’s specifications. First-strand cDNA synthesis from total RNA was performed using Superscript II RT (Invitrogen). Quantitative real-time PCR was performed on a LIGHTCYCLER (Roche Diagnostics, Mannheim, Germany), with quantitation of GAPDH mRNA synthesized by the cells as control.26 Primers and probes were designed by TIB Molbiol (Berlin). Primer pairs were as follows: for human {alpha}1, Fw-ACTTC-CGGCTACCGTGA and Rev-GTTTCGCTGGAAGGCAAT; for human {alpha}2, Fw-GTACCCCAACATCTCCA-CTGTC and Rev-AAGTTGTTTCCAGAAGACGAAGT; for human {alpha}3, Fw-TACCACCTACTGACCACCTCC and Rev-AAGCCGTAGTCCAGAGTTGTC; for human {alpha}4, Fw-AA-GTCCGTCCCAGAAGCA and Rev-CCAAGCGTGGTGTCAGTAG; for human {alpha}5, Fw- CAGCTCCTGAGGACTG-AAGTG and Rev-CCACTGAAGTTGTAAATGGTGC; for human GAPDH, Fw-AACAGCGACACCCACTCCTC and Rev-GGAGGGGAGATTCAGTGTGGT; for mouse {gamma}3, Fw-GCTGGAAGAGCCTTGGAGAA and Rev-GTAGAGGA-GACCTCAGCTTCATT; for mouse {alpha}1, Fw-AGAGATTGTAGATGGCAAGGTC and Rev-TGTTTGATGTGGGCAGGA-TAG; and for mouse GAPDH, Fw-TTGTCAGCAATGCA-TCCTGC and Rev-CCGTTCAGCTCTGGGATGAC. Probes for detection were as follows: for human {alpha}1, fluorescein-CTGGGTTCATACGGCACAAAAGACT and LIGHTCYCLER red (LCR) 640-TTTATCCATCGAGCTGTTTCGTGG; for human {alpha}2, fluorescein-GTGAGCTCCACACTCATGAAAT-CTCTCA and LCR 640-GTCTCGTGTGGCAAGATACATCAGAAGA; for human {alpha}3, fluorescein-GCAGGTCACTC-TGGAAGATGGTTACA and LCR 640-TGAATTGAGCAC-CAGCGATAGC; for human {alpha}4, fluorescein-TACTGAGG-TGGAAAAATCTCTGATGCC and LCR 640-TTATTGAC-TTTCACTATGACCTGTCCATTT; for human {alpha}5, fluorescein-GTCATCGACATACAGCCAGACTCCC and LCR 640-TG-GCATTGCTGTAGAAGGCGAC; for human GAPDH, fluo-rescein-CATGGCCTCCAAGGAGTAAGACCCCT and LCR 640-ACCACCAGCCCCAGCAAGAGCA; for mouse {gamma}3, fluo-rescein-GTCACCTTGGCAGCTTGCACA and LCR 640-CT-GAGGAAGCCCGTGTGAAGGC; for mouse {alpha}1, fluorescein-CTGAAAGCCCCCACACCCATTCCA and LCR 640-TC-GGCAGACACCAACGATCCCATTTA; and for mouse GAPDH, fluorescein-CACCCAGAAGACTGTGGATGGCCCCT and LCR 640-TGGAAAGCTGTGGCGTGATGGCCG. Standard curves were obtained for each set with serial dilutions of RNA. Amplification curves were generated by LIGHTCYCLER software. The number of cycles (n) corresponding to the start of the logarithmic phase of the fluorescence signals was used to calculate the amount of mRNA by the formula 2({Delta}n), where {Delta}n = nGAPDH – nlaminin. Each increase in {Delta}n by 1 equals a twofold increase of specifically amplified mRNA. Statistical significance was examined by using Student’s t-test

Immunofluorescence

Tissues were immersed in Tissue Tek and frozen in liquid nitrogen. Cryosections (8 µm) were either stained with hematoxylin and eosin (H&E) or analyzed by immunofluorescence. Primary antibodies (Abs) were rat monoclonal Abs 200,25 4H8-2 (Alexis Biochemicals, Lausanne, Switzerland), and 1928 (Chemicon, Hampshire, UK) against laminin {alpha}1, laminin {alpha}2, and laminin ß1 chain, respectively, and rat monoclonal Ab N6 against GATA-1 (Santa Cruz, Santa Cruz, CA). The rabbit polyclonal Abs against laminin {alpha}3,27 {alpha}4,28 {alpha}5,29 ß2,30 ß3,27 and {gamma}227 have been described previously. Additional rabbit Abs against laminin {gamma}1 and {gamma}3 chains were raised against their recombinant LN+LEa fragments following the procedure used for laminin {alpha}1 LN+LEa fragment.31,32 Enzyme-linked immunosorbent assay (ELISA) assays followed standard protocols. Rabbit polyclonal Abs against perlecan33 and collagen type IV (Chemicon) and goat polyclonal Ab against protamine-2 (Santa Cruz) were used. Protamine-2 staining was quantified using the Volocity imaging system (Improvision, Inc., Lexington, MA). Statistical significance was examined by using Student’s t-test.

Transmission Electron Microscopy

Transmission electron microscopy was performed as described previously.25

Laminin Extraction and Immunoblotting

Proteins were isolated from 100 mg of wild-type and LN{alpha}1TG testes by brief sonication in 1 mmol/L EDTA in 1x Tris-buffered saline (TBS) with 1:25 dilution of protease inhibitors (Roche Diagnostics). Samples were incubated at 4°C for 1 hour and spun down at 13,000 rpm at 4°C. The supernatants were collected, and the protein concentration was determined using BCA assay (Pierce, Rockford, IL). One hundred micrograms of each sample was boiled for 5 minutes and separated on 5% polyacrylamide-sodium dodecyl sulfate gels for 90 minutes at 100 V. Proteins were transferred to Hybond-P polyvinylidene difluoride membranes (Amersham, Buckinghamshire, UK). Membranes were blocked for 1 hour in 5% nonfat dry milk in 1x TBS with 0.01% Tween-20 and incubated overnight at 4°C with a rabbit polyclonal antibody detecting laminin {alpha}1, ß1, and {gamma}1 chains (Sigma, St. Louis, MO) (1:500). Blots were washed in 1x TBS with 0.01% Tween-20, and subsequently horseradish peroxidase-linked anti-rabbit secondary antibody (Santa Cruz) was applied onto the membranes (1-hour incubation). Detection was performed with ECL kit (Amersham), and the immunoreactive protein bands were visualized on ECL films (Amersham). Equal loading was confirmed by Coomassie blue staining of sodium dodecyl sulfate-polyacrylamide gels.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Expression of Basement Membrane Components in Human and Mouse Testes

To examine the expression of laminin {alpha} chain mRNAs in human testis, we performed quantitative real-time PCR. This demonstrated high levels of laminin {alpha}1 and {alpha}2 chain mRNAs, moderate levels of laminin {alpha}3 and {alpha}5 chain mRNAs, and low levels of laminin {alpha}4 chain mRNA (Table 1) . Thus, the main laminin {alpha} chain mRNAs in testis are laminin {alpha}1 and {alpha}2. The limitations of testis biopsies of patients with known mutations in the gene encoding laminin {alpha}2 chain prompted our use of the dy3K/dy3K mouse, which is deficient in laminin {alpha}2 chain.7 A panel of antibodies was used to study the expression of laminin {alpha}, ß, and {gamma} chains in mouse testis. All antibodies except the laminin {gamma}1 chain antibody have previously been characterized.25,27-30,32 The specificity of the antibodies against laminin {gamma}1 and {gamma}3 chains was verified by ELISA (Figure 1) . As previously reported, laminin {alpha}1 chain was expressed in the basement membrane of wild-type mice.18 In dy3K/dy3K mice, the expression of laminin {alpha}1 chain did not appear to be altered based on immunofluorescence (Figure 2) . As expected, laminin {alpha}2 chain was expressed in the basement membrane of wild-type testis18 and completely absent in dy3K/dy3K mice (Figure 2) . Laminin {alpha}3 chain was confined to blood vessels in testes of both wild-type and dy3K/dy3K mice (Figure 2) . Double staining with laminin {alpha}2 chain revealed that laminin {alpha}4 chain was expressed outside the epithelium, in smooth muscle and in blood vessels (Figure 2) . In human testis, it has also been demonstrated that laminin {alpha}4 chain is expressed in smooth muscle rather than in the basement membrane.34 In testes of both wild-type and dy3K/dy3K mice, weak immunoreactivity for laminin {alpha}5 chain was found in the basement membrane and in blood vessels (Figure 2) .


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  		Table 1. Relative Amounts of Human Laminin {alpha}1, {alpha}2, {alpha}3, {alpha}4, and {alpha}5 mRNA in Testis

 


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  		Figure 1. ELISA titration of affinity-purified antibodies against laminin {gamma}1 and {gamma}3 chains. A: The laminin {gamma}1 chain antibody was used against {gamma}1 LN/LEa ({circ}) and {gamma}3 LN/LEa (•). B: The laminin {gamma}3 chain antibody was used against {gamma}3 LN/LEa (•) and {gamma}1 LN/LEa ({circ}). Although the laminin {gamma}3 chain antibody showed some binding to the laminin {gamma}1 fragment, no staining was seen in dy3K/dy3K testis, which is a rich source of laminin {gamma}1 chain (Figure 3) .

 


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  		Figure 2. Immunostaining of laminin {alpha} and ß chains. Cross-sections of testes from wild-type and dy3K/dy3K mice were stained with antibodies against laminin {alpha}1 to {alpha}5 and ß1 chains. Laminin {alpha}2 chain was absent from dy3K/dy3K testis, whereas other laminin {alpha} chains and laminin ß1 chain appeared normally expressed. Inset in LM{alpha}4 panel shows double staining with laminin {alpha}2 chain in red and laminin {alpha}4 chain in green. Bar = 80 µm.

 
Both laminin ß1 and ß2 chains were expressed in the basement membrane and in blood vessels of testes from wild-type and dy3K/dy3K mice (Figures 2 and 3) , whereas laminin ß3 chain could not be detected (data not shown). Laminin {gamma}1 chain was expressed in the basement membrane and in blood vessels of testes from wild-type and dy3K/dy3K mice (Figure 3) . Similarly, laminin {gamma}2 chain was expressed in the same locations albeit at lower levels (Figure 3) . Previously, it has been shown that laminin {gamma}3 chain immunoreactivity is localized to the apical surface of the seminiferous tubules.35 However, using a similar assay but different antibodies, we found laminin {gamma}3 chain expression in the basement membrane of wild-type testis (Figure 3) . Also, in human testis, laminin {gamma}3 chain expression was confined to the basement membrane region rather than the apical surface of epithelial cells (data not shown). Noticeably, the expression of laminin {gamma}3 chain was absent in dy3K/dy3K mice (Figure 3) . This reduction appeared to be at the translational level because laminin {gamma}3 chain mRNA was expressed at normal levels in testis of dy3K/dy3K mice (Table 2) . Thus, laminin {gamma}3 chain is associated with laminin {alpha}2 chain in the basement membrane of seminiferous tubules and forms a trimer with either laminin ß1 or ß2 chains. Other basement membrane components such as perlecan and collagen type IV appeared normally expressed in basement membranes and blood vessels in testes of both wild-type and dy3K/dy3K mice (Figure 3) .



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  		Figure 3. Immunostaining of laminin ß and {gamma} chains, perlecan, and collagen IV. Cross-sections of testes from wild-type and dy3K/dy3K mice were stained with antibodies against laminin ß2 and {gamma}1 to {gamma}3 chains, perlecan, and collagen IV. Laminin {gamma}3 chain was absent from dy3K/dy3K testis whereas other {gamma} chains, laminin ß2 chain, perlecan, and collagen IV appeared normally expressed. Bar = 80 µm.

 

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  		Table 2. Relative Amounts of Laminin {gamma}3 mRNA in Wild-Type and dy3K/dy3K Testes

 
Histopathological Features of Laminin {alpha}2 Chain-Deficient Testis

Dy3K/dy3K mice die at 4 to 5 weeks of age before reaching the reproductive age of 6 weeks. However, already at the age of 3 weeks, germ cells appear coinciding with the appearance of lumen within seminiferous tubules.36 At 3 weeks of age, dy3K/dy3K mice are significantly smaller than wild-type littermates.7 Commensurate with the overall reduction in body size, there was general reduction in the size of various organs including testis. Organs such as kidney, heart, bladder, and eye showed normal morphology (data not shown), suggesting that development in general is not significantly impaired. However, testis showed abnormal morphology. Examination of sections of testes from 26- to 30-day-old dy3K/dy3K mice revealed that the laminin {alpha}2 chain-deficient males appear to have a defect in the timing of lumen formation. In wild-type testis, lumen had formed in the majority of seminiferous tubules. In contrast, seminiferous tubules of dy3K/dy3K mice contained either small lumens or no lumens (Figure 4A) . Also, a reduced amount of round and elongated spermatides were detected in laminin {alpha}2 chain-deficient testis (Figures 4A and 7D) . The ultrastructure of testes from wild-type and dy3K/dy3K mice at the age of 23 days was examined by electron microscopy. This revealed the presence of spermatid flagella in the wild-type testis but not in dy3K/dy3K testis (Figure 4B) . Previous studies have shown that laminin {alpha}2 chain deficiency results in de-fective muscle and sciatic nerve basement membranes.10,16,37 Also, the basement membrane underlying the Sertoli cells in dy3K/dy3K testis was sometimes thinner and disrupted (Figure 4C) .



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  		Figure 4. Analyses of testes from wild-type and dy3K/dy3K mice. A: Cryosections of testes from wild-type and dy3K/dy3K mice were stained with H&E. Seminiferous tubules in wild-type testis had well-developed lumens, whereas lumen formation seemed to be delayed in dy3K/dy3K 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 dy3K/dy3K mice were considerably reduced. Bar = 25 µm. B: Transmission electron microscopy of testes from wild-type and dy3K/dy3K mice. Asterisks denote the transverse sections of spermatid flagella, which were seen in wild-type but not dy3K/dy3K mice. Bar = 1 µm. C: Transmission electron microscopy of testes from wild-type and dy3K/dy3K mice. The basement membrane, clearly present along the basal surface of the seminiferous tubule of wild-type mice, was sometimes thinner and disrupted in dy3K/dy3K 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.

 
To further show that the number of spermatides is reduced in dy3K/dy3K testis, we stained for the presence of protamines, which are the major DNA-binding proteins in the sperm nucleus.38 Significantly less protamine-2 was expressed in testis of dy3K/dy3K mice (Figure 5, A and B) . Histologically, spermatogonia seemed normal in dy3K/dy3K mice (Figure 4A) . Alternating with the spermatogonia are the Sertoli cells that are identified by their large nuclei near the basement membrane. By histological means, the Sertoli cells also appeared normal in dy3K/dy3K mice (Figure 4A) . In addition, GATA-1 immunostainings confirmed that Sertoli cells were readily detected in dy3K/dy3K testes. In both wild-type and dy3K/dy3K testes, nuclear immunoexpression of GATA-139 was detected close to the basement membrane (Figure 5A) . Ectoplasmic specializations are actin filament-endoplasmic reticulum complexes that occur in Sertoli cells at sites of intercellular attachment. To study the ectoplasmic specializations, we determined the distribution of actin filament using rhodamine-phalloidin in 26-day-old testis. In agreement with previous findings,40,41 actin was concentrated at the basal Sertoli-Sertoli junctions and at the apical Sertoli-spermatid junctions in wild-type testis (Figure 5A) . Although actin staining was normal at the basal part of the seminiferous tubules, significantly reduced staining was observed at the apical side of dy3K/dy3K seminiferous tubules (Figure 5, A and B) . Only focal patches were seen, and these data suggest that apical junctions are not present in the dy3K/dy3K testis.



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  		Figure 5. Expression pattern of protamine-2, GATA-1, and actin in wild-type and dy3K/dy3K testes. A: Cross-sections of testes from wild-type and dy3K/dy3K mice were stained with antibodies against protamine-2, GATA-1 (red), and laminin {gamma}1 (green) and with rhodamine-phalloidin. Protamine-2 staining was markedly reduced in dy3K/dy3K testis. GATA-1 and laminin {gamma}1 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 dy3K/dy3K 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 dy3K/dy3K testes. Significantly fewer protamine-2 (P < 0.0008) and actin (P < 0.0004) stainings were observed in dy3K/dy3K testis.

 
Laminin {alpha}1 Chain Transgene Partially Compensates for Laminin {alpha}2 Chain Deficiency in Testis

Laminin {alpha}1 chain is also prominently expressed in testis, but whether it is important for normal testicular function cannot be easily tested because mice deficient in laminin {alpha}1 chain die during early embryonic development.42,43 Interestingly, laminin {alpha}1 chain mRNA is up-regulated ~2.4-fold in dy3K/dy3K testis, compared with wild-type testis. (Table 3) . However, it seems that this up-regulation is inadequate to compensate for laminin {alpha}2 chain deficiency. We therefore tested whether an additional up-regulation of laminin {alpha}1 chain in testis could compensate for laminin {alpha}2 chain absence. Recently, we generated and characterized mice overexpressing laminin {alpha}1 chain in various tissues including skeletal muscle.25 In testes of these mice (derived from line 12) an approximately fourfold overexpression of laminin {alpha}1 mRNA (Table 3) and a twofold overexpression of the protein was noted, compared with wild-type testis (Figure 6) . The expression of laminin ß1 and laminin {gamma}1 chains was also up-regulated about twofold in transgenic testis (Figure 6) . The transgenic mice were previously mated with dy3K/+ mice to generate dy3KLN{alpha}1TG mice. In these mice, laminin {alpha}1 chain can compensate for laminin {alpha}2 chain deficiency in muscle and reverses the appearance of histopathological features of muscular dystrophy.25 Next, we analyzed testis from 25-day-old dy3KLN{alpha}1TG mice lacking laminin {alpha}2 chain in testis but overexpressing laminin {alpha}1 chain. In testes of these mice, laminin {alpha}1 chain mRNA was up-regulated ~4-fold (Table 3) . To determine whether transgenically expressed laminin {alpha}1 chain is different from endogenous expression, we compared laminin {alpha}1 chain expression in testes of wild-type, dy3K/dy3K, LN{alpha}1TG, and dy3KLN{alpha}1TG mice by immunofluorescence. Laminin {alpha}1 chain was expressed in the basement membrane of seminiferous tubules in wild-type, dy3K/dy3K, LN{alpha}1TG, and dy3KLN{alpha}1TG mice (Figure 7) . This was further verified with double stainings against laminin {alpha}4 chain, which is mainly expressed in smooth muscle outside the epithelium (data not shown). Thus, transgenically expressed laminin {alpha}1 chain appears to be expressed in a similar manner as endogenous laminin {alpha}1 chain.


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  		Table 3. Relative Amounts of Laminin {alpha}1 mRNA in Wild-Type, dy3K/dy3K, LN{alpha}1TG, and dy3KLN{alpha}1TG Testes

 


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  		Figure 6. Immunoblotting of laminin {alpha}1, ß1, and {gamma}1 polypeptides. Immunoblotting of protein extracts from testes of wild-type and transgenic mice overexpressing laminin {alpha}1 chain (LN{alpha}1TG) was performed. The polyclonal antiserum detects all three chains of laminin-111 (laminin {alpha}1 chain at 400 kd and laminin ß1 and laminin {gamma}1 chains at 200 kd). Both {alpha}1 and ß1/{gamma}1 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.

 


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  		Figure 7. Immunostaining of laminin {alpha}1 chain in testes of different genotypes. Cross-sections of testes from wild-type, dy3K/dy3K, LN{alpha}1TG, and dy3KLN{alpha}1TG mice were stained with antibodies against laminin {alpha}1 chain. Bar = 80 µm.

 
Hematoxylin and eosin-stained sections showed that lumen had formed in the majority of seminiferous tubules of dy3KLN{alpha}1TG mice (Figure 8A) . Round and elongated spermatides were readily detected in dy3KLN{alpha}1TG testis (Figure 8, A and D) . Immunofluorescence analysis revealed that there was almost as much protamine-2 in dy3KLN{alpha}1TG testis as in wild-type testis (Figure 8, A and C) . We next analyzed testes from 5- to 9-month-old mice to see whether the effect of laminin {alpha}1 chain overexpression on testicular morphology was sustained. No difference in the amounts of protamine-2 between control and dy3KLN{alpha}1TG testes was seen, suggesting that spermatogenesis proceeds normally in mature dy3KLN{alpha}1TG testis (Figure 8, B and C) . Also, the basement membrane was restored in testis of dy3KLN{alpha}1TG mice (Figure 9) . Moreover, as previously indicated, male dy3KLN{alpha}1TG mice are able to produce offspring.25



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  		Figure 8. Overexpression of laminin {alpha}1 chain in testis partially compensates for laminin {alpha}2 chain deficiency. Cryosections of testes from 25-day-old (A) and 9-month-old (B) control and dy3KLN{alpha}1TG mice were stained with H&E and antibodies against protamine-2. Histologically, seminiferous tubules of dy3KLN{alpha}1TG mice appeared very similar to those of wild-type mice. C: Quantification of protamine-2 and staining in control and dy3KLN{alpha}1TG testes. Slightly less protamine-2 was detected in 25-day-old (P < 0.2690) but not in 9-month-old (P < 0.8788) dy3KLN{alpha}1TG testis. Bar = 80 µm. D: Quantification of the number of elongated spermatides per seminiferous tubule in 25-day-old wild-type, dy3K/dy3K, and dy3KLN{alpha}1TG testes. The number of elongated spermatides in dy3K/dy3K testis was significantly smaller than in wild-type testis (P < 0.0001), whereas the number of elongated spermatides in dy3KLN{alpha}1TG testis was not significantly different from wild-type testis (P < 0.6359).

 


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  		Figure 9. Restoration of the basement membrane in dy3KLN{alpha}1TG testis. Transmission electron microscopy of testis from 5-month-old wild-type and dy3KLN{alpha}1TG mice. The basement membrane is clearly present along the basal surface of the seminiferous tubule of both wild-type and dy3KLN{alpha}1TG mice. Black arrows denote the basement membrane. Bar = 0.25 µm.

 
To test whether rescue of the testis phenotype is due to a laminin isoform containing the {alpha}1 and {gamma}3 chains, we analyzed the expression of laminin {gamma}3 chain in dy3KLN{alpha}1TG testis. Notably, the expression of laminin {gamma}3 was not restored in the basement membrane of the seminiferous tubules (Figure 10) . These data show that loss of laminin {alpha}2 and {gamma}3 chains are compensated for by laminin {alpha}1 chain (which appears not to be associated with laminin {gamma}3 chain) and the ß1/{gamma}1 chains because these chains are up-regulated on overexpression of laminin {alpha}1 chain (Figure 6) .



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  		Figure 10. Immunostaining of laminin {gamma}3 chain in dy3KLN{alpha}1TG testis. Cross-sections of testes from wild-type and dy3KLN{alpha}1TG mice were stained with antibodies against laminin {gamma}3 chain. Expression of laminin {gamma}3 chain was not restored in dy3KLN{alpha}1TG testis. Bar = 80 µm.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Here, we show that the presence of laminin {alpha}2 chain in the basement membrane of seminiferous tubules is important for spermatogenesis. In wild-type mice, the first wave of spermatogenesis begins shortly after birth; the first meiotic divisions occur at about postnatal day 20, and at day 35 the first spermatozoa are produced.44 Although laminin {alpha}2 chain-deficient dy3K/dy3K mice die of unknown causes at about 35 days of age,7 the first wave of spermatogenesis can still be studied. Lack of laminin {alpha}2 chain affected the structure of the basement membrane, which was thinner and disrupted to a variable degree in dy3K/dy3K testis. Seminiferous tubules of laminin {alpha}2 chain-deficient dy3K/dy3K testis displayed a defect in the timing of lumen formation and reduced amounts of round and elongated sperma-tides. These data show that testis maturation is impaired in laminin {alpha}2 chain-deficient mice.

Lack of laminin {alpha}2 chain in the basement membrane of testis resulted in reduced expression of laminin {gamma}3 chain and disruption of the basement membrane as defined by electron microscopy. Other basement membrane components (such as other laminin chains, collagen type IV, and perlecan) were present despite irregular basement membranes. However, the presence of basement membrane components does not necessarily reflect the presence of a basement membrane defined by electron microscopy. Also, it has been shown that the basement membrane in laminin {alpha}2 chain-deficient muscle is disrupted despite the presence of other basement membrane components.25,37 Previously, it has been demonstrated that laminin {gamma}3 chain is the only laminin chain that is not associated with basement membranes.35 In testis, laminin {gamma}3 chain was shown to be expressed on the luminal side of the seminiferous tubule.35 However, using novel antibodies specific for laminin {gamma}3 chain,32 we showed here that laminin {gamma}3 chain is expressed exclusively in the basement membranes of testis and also in other organs (data not shown). Furthermore, because no other laminin chains have been shown to be expressed on apical surfaces of testis epithelium, it is likely that the antibody used by Koch et al35 cross reacts with an unrelated apical antigen. Thus, we suggest that spermatogenesis may be dependent on laminin-213 or laminin-223.45

Besides the {alpha}2 chain, the {alpha}1 chain is the other major laminin {alpha} chain in testis. Dy3K/dy3K testis has elevated levels of laminin {alpha}1 chain. This led us to question whether further increasing laminin {alpha}1 chain levels could rescue the abnormal testicular phenotype. An additional twofold increase in laminin {alpha}1 chain expression did rescue the testicular phenotype. Very likely, overexpression of laminin {alpha}1 chain in testis is the major cause of rescue, but we cannot rule out that the increased expression of laminin {alpha}1 chain in other tissues also is beneficial.

It is noteworthy that overexpression of laminin {alpha}1 chain did not normalize laminin {gamma}3 chain expression in testis, taking into account that laminin {gamma}3 chain mRNA is made in dy3K/dy3K testis. Also in peripheral nerve, loss of laminin {alpha}2 chain leads to a dramatic reduction of laminin {gamma}3 chain expression, and no restoration is seen on forced expression of laminin {alpha}1 chain (K. Gawlik and M. Durbeej, unpublished data). Hence, it appears that laminin {alpha}1 chain does not form a trimer with laminin {gamma}3 chain, at least not in testis or peripheral nerve. Laminin {alpha}1 chain-mediated rescue of the testis defect most likely involves laminin {gamma}1 chain because this chain is up-regulated on overexpression of laminin {alpha}1 chain. Laminin {gamma}2 chain was also shown to be expressed in the basement membrane of the seminiferous tubules, but so far, the laminin {alpha}3 chain is the only {alpha} chain known to associate with laminin {gamma}2 chain.46 Therefore, it is peculiar that laminin {alpha}3 chain expression in testis was confined to blood vessels rather than the epithelial basement membrane. Further studies are needed to identify the {alpha} chains associated with the {gamma}2 chain in the basement membranes of seminiferous tubules.

The detailed mechanisms by which lack of laminin {alpha}2 chain deficiency leads to impaired spermatogenesis remain to be clarified. Basement membranes provide structural stability of organs and send signals to cells through cell surface receptors.47 Laminin {alpha}2 chain receptors include members of the integrin family and dystroglycan.48 Integrins have been identified as laminin binding proteins in Sertoli cells,49 and integrin {alpha}6ß1 and dystroglycan are both confined to the basal compartment of the seminiferous epithelium.18,50,51 Mice deficient in integrin {alpha}6 subunit die at birth,52 and a targeted disruption of dystroglycan results in early embryonic lethality.53 Thus, tissue-specific ablation of the genes could be used to directly study the involvement of these two genes in spermatogenesis. The expression patterns of these two receptors are not altered in dy3K/dy3K testis (data not shown). Nevertheless, signal transduction cascades through laminin {alpha}2 chain and integrins or dystroglycan might be perturbed in dy3K/dy3K testis. In muscle, laminin {alpha}2 chain ap-pears to promote myotube stability by preventing apo-ptosis,54 and in dystrophic muscle, increased apoptotic cell death might be involved in the process of muscle fiber degeneration.7,55 Yet, in laminin {alpha}2 chain-deficient testis, we did not notice increased apoptotic cell death (data not shown).

Finally, to our knowledge, there is no published literature that deals with the issue of MDC1A and fertility, but our data indicate that male patients with laminin {alpha}2 chain-deficient muscular dystrophy may be infertile. Recently, we demonstrated that laminin {alpha}1 chain distinctly reduces muscular dystrophy in laminin {alpha}2 chain-deficient mice.25 Here, we showed that laminin {alpha}1 chain can compensate for laminin {alpha}2 chain absence in testis. Lentiviral vectors have been shown to be efficient in transducing both muscle and Sertoli cells of testis.56,57 Thus, delivery of laminin {alpha}1 chain into muscle and testis using lentiviral vectors will be tested and could constitute promising therapeutic strategies for treatment of muscular dystrophy with associated infertility.


   Acknowledgements
 
We thank Peter Ekblom and Tord Hjalt for critical reading of the manuscript and Shin’ichi Takeda for providing dy3K/dy3K mice.


   Footnotes
 
Address reprint requests to Madeleine Durbeej, Department of Experimental Medical Science, Division for Cell and Matrix Biology, University of Lund, BMC B12, 221 84 Lund, Sweden. E-mail: madeleine.durbeej_hjalt{at}medkem.lu.se

Supported by Vetenskapsrådet and Crafoord, Magnus Bergwall, and Åke Wiberg Foundations.

K.G. and A.N. contributed equally to this work.

Accepted for publication May 19, 2005.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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