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Biology of Reproduction 66, 211-221 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Fyn Tyrosine Kinase in Sertoli Cells Is Involved in Mouse Spermatogenesis1

Mamiko Maekawa2,a, Yoshiro Toyamaa, Masahiro Yasudab, Takeshi Yagib, and Shigeki Yuasaa

a Department of Anatomy and Developmental Biology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan b Laboratory of Neurobiology and Behavioral Genetics, National Institute for Physiological Sciences, Okazaki 444-8585, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fyn is a member of the Src family of non-receptor-type tyrosine kinases and plays an important role in signal transductions regulating cell proliferation and differentiation. Fyn immunoreactivity was localized in the Sertoli cells of mouse testes. Although fyn-deficient adult male mice were fertile, a significant reduction in testis weight and degenerated germ cells were observed at 3 and 4 wk of age. Electron microscopic examination revealed that fyn -/- testis has ultrastructural abnormalities in the specialized junctional structures of the Sertoli cells, the ectoplasmic specializations. Unusual vesicular structures were found in the actin filament layers of the ectoplasmic specializations of mutant mice. Immunohistochemical studies demonstrated that both Fyn and actin filaments were concentrated in the areas of ectoplasmic specializations. At these sites, a high level of phosphotyrosine was also immunostained in wild-type testes, whereas phosphotyrosine immunoreactivity was reduced in fyn -/- testes. Immunoblot analyses revealed that Fyn was mainly distributed within the Triton X-100-insoluble cytoskeletal fraction prepared from wild-type testes, suggesting that Fyn might be associated with cytoskeletal proteins such as actin filaments. These findings suggest that Fyn kinase functions at the ectoplasmic specializations of the Sertoli cells in the testes, regulating the dynamics of cytoskeletal proteins. Fyn-mediated signal transduction in the Sertoli cells may affect the survival and differentiation of germ cells at a specific stage during spermatogenesis.

kinases, Sertoli cells, signal transduction, spermatogenesis, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein tyrosine kinases are involved in various signal transduction pathways regulating cell proliferation and differentiation in a variety of cell types. In the testis, several tyrosine kinases are known to be involved in the spermatogenesis, such as c-Kit, ferT, c-Abl, sp42, and Mak [1–7]. Receptor tyrosine kinase c-Kit is expressed in germ cells, and its ligand, stem cell factor (SCF), is expressed in the Sertoli cells of the testis. The interaction between c-Kit and SCF is essential for the maintenance and/or mitosis of differentiating type A spermatogonia [1] and for meiosis [2]. A testis-specific isoform of the ubiquitously expressed fer tyrosine kinase, termed ferT is expressed in spermatocytes at the pachytene stage of meiotic prophase [3]. Nonreceptor tyrosine kinase c-Abl is expressed predominantly in pachytene spermatocytes and interacts directly with meiotic chromosomes, playing a functional role in meiosis [4]. Tyrosine kinase sp42, originally identified in ejaculated boar spermatozoa, is a male germ cell-specific product in mammalian testes [5]. Mak is a protein kinase that is distantly related to cdc2 kinase and is expressed exclusively in testicular germ cells at and after meiosis [6, 7]. These findings demonstrate that tyrosine kinases control spermatogenesis in cooperation with many regulatory proteins in the testis.

The Src family of nonreceptor tyrosine kinases have been implicated as important regulators of cellular activities, including proliferation, survival, adhesion, and migration. Src protein is localized in the testis. According to Nishio et al. [8], c-Src tyrosine kinase was detected immunohistochemically in various cell types of rat testis, including spermatogonia, spermatocytes, spermatids, Sertoli cells, and Leydig cells. In another study, Tokuchi et al. [9] showed that c-src expression occurred mainly in the Sertoli cells. Wang et al. [10] demonstrated that Src immunoreactivity was predominant in the Sertoli cytoplasm and occasionally found at the Sertoli-germ cell junctions in the testis. Fyn is a member of the Src family of tyrosine kinases, originally identified as a yes-related kinase [11]. Fyn kinase is found in many tissues but is especially abundant in the brain, platelets, and lymphocytes [12]. Its biological functions are diverse, including the regulation of brain function, signaling via T-cell receptors, and adhesion-mediated signaling [13]. However, little is known about the function of Fyn during spermatogenesis. Unlike src-deficient mice, which manifest osteopetrosis [14], fyn-deficient mice do not display an overt phenotype. However, the loss of Fyn function in mice is related to several behavioral defects, such as defective spatial learning [15], abnormal suckling behavior [16], hyperresponsiveness to fear-inducing stimuli [17], enhanced susceptibility to audiogenic seizures [18], and decreased tolerance to ethanol ingestion [19].

In this study, we found that Fyn tyrosine kinase is localized in the Sertoli cells and is especially concentrated at ectoplasmic specializations, unique actin-related junctional structures between Sertoli cells and between spermatids and Sertoli cells. We also examined the testes of fyn -/- mice and determined that Fyn kinase is involved in the survival and differentiation of germ cells by acting on the control of Sertoli cell structure. In addition, we found that the defective spermatogenesis seems to be compensated for when the testis matures.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Experimental Cryptorchidism

Adult male mice (>=2 mo of age) of the C57BL strain were purchased from Charles River Japan (Yokohama, Japan). WBB6F1-W/Wv mice were obtained from Japan SLC (Shizuoka, Japan). The fyn gene-deficient mice [16] were maintained at the National Institute for Physiological Sciences (Okazaki, Japan). Experimental cryptorchidism was induced in adult C57BL mice according to a previously described procedure [20]. The adipose tissue of the caput epididymidis of the testes was sutured to the upper portion of the abdominal wall. Three months after the operation, the animals were killed. WBB6F1-W/Wv mice and cryptorchid mice were used as a model of disturbed spermatogenesis. All animal experiments were performed in accordance with the Guide for Care and Use of Laboratory Animals (1996, National Academy of Science).

Immunohistochemistry and Histology

Animals were anesthetized with pentobarbital and perfused through the heart with 4% paraformaldehyde and 0.5% picric acid in 0.1 M phosphate buffer (pH 7.4). The testes were removed and immersed in the same fixative for 2 h, rinsed with PBS, embedded in OCT compound, and cut at a thickness of 10 µm on a cryostat. The sections were incubated in PBS containing 3% BSA (Sigma Chemical Co., St. Louis, MO) and 5% normal goat serum (NGS; Gibco BRL, Grand Island, NY) for 1 h at room temperature to block the nonspecific binding of the antibody. The sections were then incubated with rat anti-Fyn monoclonal antibody {gamma}C3 [21] at a 1:200 dilution or rabbit anti-phosphotyrosine antibody (Transduction Laboratories, Lexington, KY) at a 1:150 dilution at 4°C for 16 h. After rinsing with PBS, the sections were incubated with biotinylated anti-rat IgG (Vector Laboratories, Burlingame, CA) at a 1:200 dilution or anti-rabbit IgG (Vector Laboratories) at a 1:200 dilution for 1 h at room temperature and then incubated with avidin-biotinylated peroxidase complex (Vectastain ABC Elite kit; Vector Laboratories) for 30 min at room temperature. Immunohistochemical reactions were performed using 3,3-diaminobenzidine (DAB) and H2O2. The sections were then counterstained with Mayer hematoxylin (Wako Pure Chemical Industries, Osaka, Japan). To evaluate the histology, several mice were fixed with Bouin solution, their testes were embedded in paraffin, and sections were prepared and stained with periodic acid-Schiff (PAS) and hematoxylin.

Confocal Laser Microscopy

Fyn and actin filaments were double labeled by the immunofluorescence staining of Fyn and the binding of fluorescein isothiocyanate (FITC)-labeled phalloidin to study the distribution of Fyn at the ectoplasmic specializations. Frozen sections of testes from 3-wk-old C57BL mice that had been prepared as described above and testicular cell preparations that had been mechanically dissociated from C57BL adult mice were used as samples to study the basal and apical ectoplasmic specializations, respectively. Testicular cells were prepared as follows. The testes from adult C57BL mice were decapsulated, minced with a razor blade into small pieces in ice-cold PBS containing 5 mM EDTA, and then triturated by repeated passage for 3 min through a siliconized pipette. The fragmented tissue was centrifuged at a low speed, and the supernatant containing testicular cells and cellular fragments was collected. Cells were fixed in 3% paraformaldehyde for 15 min and allowed to attach to glass slides coated with 3-aminopropyltriethoxysilane (Sigma) [22]. Both samples were incubated in PBS containing 3% BSA and 5% NGS for 1 h at room temperature to block the nonspecific binding of the antibody and then incubated with anti-Fyn antibody (at a 1:50 dilution) at room temperature overnight. After washing with PBS, the slides were incubated with biotinylated anti-rat IgG (Vector Laboratories) at a 1:100 dilution for 1 h at room temperature and subsequently incubated with streptavidin-Texas Red conjugate (Gibco BRL) for 1 h at room temperature. After washing with PBS, the slides were further stained with FITC-labeled phalloidin (Sigma) for 1 h. The samples were mounted with PermaFluor (Immunon, Pittsburgh, PA) and observed using a confocal laser microscope (LSM410; Carl Zeiss, Göttingen, Germany).

Electron Microscopy

Animals were anesthetized with pentobarbital and perfused transcardially with 3% glutaraldehyde in Hepes buffer. The testes were excised and immersed in the same fixative for 2 h. Tissues were postfixed with osmium tetraoxide and embedded in epoxy resin. Ultrathin sections were prepared, stained with uranyl acetate and lead citrate, and used for electron microscopy (JEM 1200EX; JEOL, Tokyo, Japan).

For immunoelectron microscopy, animals were perfused transcardially with a solution of 4% paraformaldehyde, 0.5% picric acid, and 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The testes were removed, cut into small pieces, immersed in the same fixative for 1 h, rinsed with PBS, embedded in 2% gelatin, and sectioned at a thickness of 100 µm using a vibratome. The sections were treated with a solution of 10% NGS in PBS at 4°C for 30 min and then incubated with anti-Fyn antibody at a 1:150 dilution at 4°C for 16 h. After rinsing with PBS, the sections were incubated with horseradish peroxidase-labeled anti-rat IgG antibody (MBL, Nagoya, Japan) at a 1:100 dilution at 4°C for 2 h. To enhance the immunohistochemical reaction, the modified cobalt-glucose oxidase-DAB intensification method [23] was applied. After the reaction, the sections were rinsed with PBS and postfixed with 2.5% glutaraldehyde in PBS for 2 h followed by 1% osmium tetraoxide for 20 min. The sections were then dehydrated and embedded in epoxy resin. Ultrathin sections were prepared and then observed using an electron microscope (JEM 1200EX, JEOL).

Gel Electrophoresis and Immunoblot Analysis

Testicular proteins were fractionated into Triton X-100-soluble and -insoluble fractions. Decapsulated testes from wild-type and fyn -/- adult mice were lysed in a buffer containing 1% Triton X-100, 5 mM EGTA, 1 mM MgCl2, 1 mM PMSF, and 50 mM Tris-HCl, pH 7.5. The lysates were then homogenized in a glass homogenizer on ice. After removing the cellular debris by light centrifugation, the homogenates were centrifuged at 10 000 x g for 10 min at 4°C. The resulting supernatant was then combined with an equal volume of 2x SDS sample buffer (soluble fraction). The Triton-resistant pellet was washed once with the lysis buffer and then solubilized in SDS sample buffer (insoluble cytoskeletal fraction). The ratio of amounts of proteins collected from the soluble and insoluble fractions from testes was about 20:1. Equal amounts of proteins (20 µg/lane) were electrophoresed on 10% SDS-polyacrylamide gels. The gels were then transferred onto polyvinylidene fluoride membranes (Immobilon; Millipore, Bedford, MA). The membrane was blocked for 1 h in Tris-buffered saline (TBS) containing 10% skim milk and then incubated at 4°C overnight in a 1:200 dilution of rat anti-Fyn monoclonal antibody, a 1:40 dilution of mouse anti-Src monoclonal antibody (Calbiochem OP07; Oncogene Research Products, Cambridge, MA) or a 1:500 dilution of mouse anti-phosphotyrosine monoclonal antibody (PY20; Transduction Laboratories). Following incubation, the membranes were washed with TBS containing 0.5% Tween 20 and incubated for 3 h at room temperature in a 1:500 dilution of a phosphatase-labeled goat anti-rat IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD) for the anti-Fyn antibody or a phosphatase-labeled affinity-purified antibody to mouse IgG (Kirkegaard & Perry Laboratories) for the anti-Src and anti-phosphotyrosine antibodies. The resulting bands were visualized using a 5-bromocresyl-3-indolylphosphate/nitroblue tetrazolium alkaline phosphatase substrate kit IV (Vector Laboratories).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunohistochemical Localization of Fyn Tyrosine Kinase in Mouse Testis

Sections of adult mouse testes were examined for Fyn tyrosine kinase localization using a rat monoclonal antibody specific to Fyn. Fyn immunoreactivity in the adult mouse testis showed a different pattern of staining in each tubular section, depending upon the stage of the cycle of seminiferous epithelium (Fig. 1A). Immunoreactivity was not observed in the seminiferous tubules of fyn -/- mice, although nonspecific staining was found in the interstitial cells (Fig. 1B). A higher magnification of the immunostained seminiferous tubules revealed a specific signal in the Sertoli cells (Fig. 1C), which showed the characteristic morphology of Sertoli cells in each stage of the cycle of seminiferous epithelium. An intense accumulation of Fyn immunoreactivity was often found adjacent to the heads of elongate spermatids (Fig. 1C, arrowheads) and at the basal sites of the epithelium (Fig. 1C, arrows), where a unique type of actin-related intercellular junction, the ectoplasmic specializations, was present [24, 25]. Immunoelectron microscopic analyses revealed that Fyn immunoreactivity was localized in the cytoplasm of the Sertoli cells (Fig. 2). In contrast, immunoreactivity was not detected in the germ cells, such as spermatogonia and spermatocytes (Fig. 2).



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  		FIG. 1. Immunohistochemical localization of Fyn tyrosine kinase in wild-type adult mouse testis (A and C). A negative control was prepared from the testis of a fyn -/- adult mouse (B). C) Higher magnification of seminiferous epithelia immunostained with anti-Fyn antibody. Fyn immunoreactivity is localized in the Sertoli cells, which display the appropriate characteristic morphology of Sertoli cells at each stage of the seminiferous epithelium. Concentrated areas of Fyn immunoreativity are often found adjacent to the heads of elongate spermatids (arrowheads) and at the basal sites of the epithelium (arrows), where ectoplasmic specializations are located. Roman numerals indicate the stages of the seminiferus epithelium. Bars = 100 µm (A and B), 20 µm (C)



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  		FIG. 2. Immunoelectron microscopy of adult mouse testis stained with anti-Fyn antibody. Immunoreactivity is found in the cytoplasm of the Sertoli cells but not in the germ cells. Se, Nucleus of a Sertoli cell; Sg, spermatogonium; Sc, spermatocyte. Bar = 2 µm

Cryptorchid mice and W/Wv mutant mice were used to examine the effect of disturbed spermatogenesis on Fyn expression. Unilateral cryptorchidism was experimentally induced in adult mice, and 3 mo later the differentiated germ cells degenerated and disappeared; only type A spermatogonia and Sertoli cells remained in the seminiferous epithelium. Fyn immunoreactivity was observed in the seminiferous epithelium of the cryptorchid testes (Fig. 3A). Nonspecific staining was slightly observed in the basal part of the seminiferous tubules when the primary antibody was omitted (Fig. 3B). Testes from sterile mutant W/Wv mice were also immunostained for Fyn. Only a few germ cells in the most primitive stage of development were found in the testis of the W/Wv mice, and the Fyn immunoreactivity that was detected in the seminiferous tubules was considered localized in the Sertoli cell cytoplasm (Fig. 3C). Slight nonspecific immunoreactivity was also found in the W/Wv testis (Fig. 3D).



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  		FIG. 3. Immunohistochemical localization of Fyn in the testes of a cryptorchid mouse (A and B) and a W/Wv mutant mouse (C and D). A and C) Stained with anti-Fyn monoclonal antibody. B and D) Negative controls prepared by omission of the primary antibody. Bar = 50 µm

Defect in fyn -/- Mouse Testes During Postnatal Development

The morphology of the fyn -/- testes appeared to be normal, and adult fyn -/- male mice were fertile (Fig. 1B). However, a defect during the development of the testes was identified in mutant mice. Testes from fyn -/-, fyn +/-, and wild-type mice were obtained from mice at the ages of 2, 3, 4, and 8 wk and from adults (older than 12 wk) and then weighed. The weight of the fyn -/- testes was significantly reduced at the ages of 3 and 4 wk compared with the fyn +/- and wild-type testes (Table 1). Younger (2 wk old) and older (8 wk old and adult) animals showed no difference in testis weight among the fyn -/-, +/-, and wild-type mice. Furthermore, no significant difference was found between the fyn +/- and wild-type testes. These data indicate that the fyn deficiency causes defective testicular development at 3 and 4 wk after birth but that the deficiency appears to be recovered by the age of 8 wk.


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  		TABLE 1. Comparison of mean (±SD) testis weight (TW) in fyn -/-, fyn +/-, and wild-type (WT) mice during postnatal development

The histology of the fyn -/- and +/- testes at the age of 3 wk was investigated using PAS and hematoxylin staining (Fig. 4). The testes of fyn +/- mice appeared normal when compared with those of wild-type animals (data not shown). In the fyn +/- testis, the lumina of the seminiferous tubules were obvious, and spermatids at step 5 were the most differentiated germ cells (Fig. 4A). In contrast, an obvious defect was observed in the fyn -/- testis at the same age (Fig. 4B). The lumina of the tubules in the fyn -/- testis were not yet well developed, and the diameters of the seminiferous tubules were significantly smaller than those of the fyn +/- testes (113 ± 13 µm for fyn -/-; 123 ± 13 µm for fyn +/-; P < 0.001; 5 animals and 30 tubules/animal examined). Furthermore, a considerable number of germ cells had degenerated in the fyn -/- testes (Fig. 4B; arrows). However, spermatids at step 5 were also found, as observed in the fyn +/- testes.



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  		FIG. 4. Morphology of 3-wk-old mouse testes from fyn +/- (A) and -/- (B) mice. PAS-hematoxylin staining. A) At this age, the lumina (L) are visible in the seminiferous tubules of the fyn +/- testis. B) Many degenerated germ cells are present (arrowheads). Bar = 50 µm

The ultrastructure of testes from fyn -/- mice at the age of 3 wk was examined by electron microscopy. Degenerated germ cells in the seminiferous tubules were identified (Fig. 5), as with light microscopy (Fig. 4B). The cytoplasm had a high electron density, the nuclear envelope was lost, and degenerated mitochondria, multivesicular vacuoles, and the accumulation of membranous structures were observed, suggesting necrosis. Furthermore, the fyn-deficient testes showed unusual structures in the ectoplasmic specializations. A considerable number of vesicular structures (Fig. 6B, arrows) were found in the actin filament layers of the basal ectoplasmic specializations between neighboring Sertoli cells in the fyn -/- testes. Although not all ectoplasmic specializations of fyn -/- testes indicate these unusual vesicular structures, 66% (119/180) of the observed basal ectoplasmic specializations in the testes of 3-wk-old fyn -/- mice had the unusual structures. The percentage of the basal ectoplasmic specializations with these unusual structures was decreased to 33% (48/145) in the adult fyn -/- mice. In addition, the number of unusual vesicular structures per abnormal ectoplasmic specialization appeared to decrease in adult testes (Fig. 6C) compared with 3-wk-old testes (Fig. 6B). None of the wild-type testes exhibited these unusual structures (Fig. 6A). Ectoplasmic specializations are also localized in the regions where the heads of elongate spermatids attached to Sertoli cells at apical sites of the seminiferous epithelium [24, 25]. These apical ectoplasmic specializations also showed similar unusual vesicular structures in fyn -/- testes (Fig. 7, C and D), which were never seen in wild-type animals (Fig. 7, A and B).



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  		FIG. 5. Electron microphotograph of a necrotic germ cell (N) in the seminiferous tubule of a 3-wk-old fyn -/- testis. Normal round spermatids (arrowheads) are adjacent to the necrotic cell. Bar = 1 µm



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  		FIG. 6. Electron microphotographs of wild-type (A) and fyn -/- (B and C) testes obtained at the ages of 3 wk (A and B) and in adults (C). Basal ectoplasmic specializations between adjoining Sertoli cells are visible. A) Actin filaments and endoplasmic reticulum in the ectoplasmic specializations are indicated by arrows and asterisks, respectively. B and C) The fyn -/- testes often show unusual vesicular structures (arrowheads) in the actin filament layers of the ectoplasmic specializations. These structures are not visible in wild-type testis (A). Bar = 0.5 µm



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  		FIG. 7. Electron microphotographs of apical ectoplasmic specializations attached to the head of a spermatid in adult wild-type (A and B) and fyn -/- (C and D) testes. B and D) Higher magnification of the boxed areas in A and C, respectively. Arrowheads in D indicate unusual vesicular structures in the actin filament layer. In contrast, no vesicular structures are seen in B. Bars = 0.5 µm (A and C)

Concentration of Fyn Immunoreactivity in the Ectoplasmic Specializations of Sertoli Cells

An intense accumulation of Fyn immunoreactivity was often found where the basal and apical ectoplasmic specializations were supposed to occur (Fig. 1C, arrows and arrowheads). To confirm the localization of Fyn at the ectoplasmic specializations, the distribution of actin filaments, as examined by fluorescent phallotoxin staining, was used as a marker for basal and apical junction sites. Actin filaments are found in abundant quantities in ectoplasmic specializations [26]. The testes of 3-wk-old mice were double stained with anti-Fyn antibody and FITC-labeled phalloidin to examine the basal ectoplasmic specializations. The results showed that the distribution of actin filaments was largely overlapped by that of Fyn immunoreactivity in the basal part of the seminiferous tubules (Fig. 8A), indicating that Fyn is localized at the basal ectoplasmic specializations. Because apical junctions have not yet formed in 3-wk-old mice, spermatids prepared mechanically from adult testes were used to investigate the apical junctions. Apical ectoplasmic specializations of Sertoli cells are easily detached from the seminiferous epithelium, together with the heads of the spermatids, by the mechanical dissociation of the testes [27, 28]. Thus, cells were mechanically dissociated from the testis and double stained with anti-Fyn antibody and FITC-labeled phalloidin. Fyn immunoreactivity was found around the sickle-shaped heads of the elongate spermatids, and a large portion of the immunoreactivity was overlapped with the actin filaments (Fig. 8B).



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  		FIG. 8. Double staining with anti-Fyn antibody (red) and actin filaments (green). Actin filaments were stained with FITC-labeled phalloidin and used as a marker for the ectoplasmic specializations. A) Frozen section of 3-wk-old wild-type testis. Fyn immunoreactivity overlaps the area containing the actin filaments in the basal ectoplasmic specializations between Sertoli cells (arrowheads). Bar = 50 µm. B) The head of an elongate spermatid that has been mechanically dissociated from a normal adult testis. Bar = 5 µm

Immunohistochemical Localization of Phosphotyrosine in Wild-Type and fyn -/- Testes

Adult testes from wild-type and fyn -/- mice were immunostained using anti-phosphotyrosine polyclonal antibody. Phosphotyrosine immunoreactivity was distributed broadly in the testis and found mainly in the Sertoli cells, spermatogonia, and elongate spermatids (Fig. 9). Strong staining was found in the basal sites of the seminiferous epithelium (Fig. 9B, arrows) and in the sites adjacent to the heads of elongate spermatids (Fig. 9B, arrowheads) where the ectoplasmic specializations were located. In contrast, the fyn -/- testis showed a reduced level of immunoreactivity (Fig. 9, C and D) compared with that of the wild-type testis.



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  		FIG. 9. Immunostaining of adult testes from wild-type (A and B) and fyn -/- (C and D) mice using an anti-phosphotyrosine antibody. The reaction is more intense in the wild-type testis than in the fyn -/- testis. Strong immunostaining is found around the heads of elongate spermatids (arrowheads) and in the basal sites, including the basal junctions (arrows), in the wild-type testis (B). Bars = 100 µm (A and C), 20 µm (B and D)

Distribution of Fyn, Tyrosine-Phosphorylated Proteins, and Src in the Triton X-100-Soluble and -Insoluble Fractions of Mouse Testis Extract

Immunoblot analyses of Fyn (Fig. 10A) and tyrosine-phosphorylated proteins (Fig. 10B) were performed using Triton X-100-soluble and -insoluble cytoskeletal fractions of adult testis extracts. The results revealed that Fyn was abundantly present in the Triton X-100-insoluble cytoskeletal fraction (Fig. 10, lane 3), but only a very faint band of Fyn immunoreactivity was detected in the Triton X-100-soluble fraction (Fig. 10, lane 1). Fyn was not detected in fyn -/- mice (Fig. 10, lanes 2 and 4). Several bands of the tyrosine-phosphorylated proteins were fainter in the fyn -/- testes than in the wild-type testes (Fig. 10B). A band at about 80 000 (p80; Fig. 10B, arrowhead) was prominent in the Triton X-100-insoluble cytoskeletal fraction from the wild-type testis (Fig. 10, lane 7). P80 was very weak in the Triton X-100-soluble fraction (Fig. 10, lane 5). In contrast, the intensity of p80 was significantly reduced in both the soluble and insoluble fractions from the fyn -/- testis (Fig. 10, lanes 6 and 8). Testes from fyn +/- mice were also examined by immunoblot analysis, and Fyn and p80 were detected in the Triton X-100-insoluble fraction as in the wild type. The amount of Fyn in the fyn +/- testis was less than that in the wild-type testis, but no difference in the amount of p80 was seen between the fyn +/- and wild-type testes (data not shown).



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  		FIG. 10. Immunoblot analysis showing the distribution of Fyn (A, lanes 1–4), tyrosine-phosphorylated proteins (B, lanes 5–8), and Src (C, lanes 9–12) in the Triton X-100-soluble (lanes 1, 2, 5, 6, 9, 10) and -insoluble (lanes 3, 4, 7, 8, 11, 12) fractions obtained from wild-type (lanes 1, 3, 5, 7, 9, 11) and fyn -/- (lanes 2, 4, 6, 8, 10, 12) adult mouse testes. A) Fyn is detected in the insoluble cytoskeletal fraction (lane 3), but only a very faint band is found in the soluble fraction (lane 1). B) A prominent band at about Mr 80 000 (p80, arrowhead) is visible in the insoluble fraction from the wild-type testis (lane 7). C) The arrow on the right indicates Src immunoreactivity at Mr 60 000. Bands near Mr 83 000, seen in lanes 9 and 10 (asterisk), represent nonspecific reactivity of the secondary antibody

The distribution of Src protein in the Triton X-100-soluble and -insoluble fractions from adult testes was also determined using an immunoblot analysis (Fig. 10C). Src was detected in both the Triton X-100-soluble and -insoluble fractions, although the band was more intense in the latter fraction. Src protein was also found in both fractions from the fyn -/- testis (Fig. 10, lanes 10 and 12). No difference was observed in the intensity of the band between fyn -/- and wild-type testes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Sertoli cells play an essential role in spermatogenesis; these cells support and nurse the spermatogenic cells so that they may proliferate and differentiate. The interaction between germ cells and Sertoli cells is essential for the regulation of spermatogenesis [29]. Fyn is immunolocalized in Sertoli cells and fyn deficiency causes abnormal Sertoli cell structure and a transient spermatogenic defect during testis development. This study has provided the first evidence that Fyn tyrosine kinase is involved in the control of spermatogenesis.

Fyn at Ectoplasmic Specializations

Fyn protein was predominantly located at the ectoplasmic specializations of the Sertoli cells. Ectoplasmic specializations are adhesive intercellular junctions found at the basal and apical sites of the seminiferous epithelium [24, 25]. At apical sites, the structures are found in regions where the heads of elongate spermatids attach to the Sertoli cells. At basal sites, they are localized in regions of attachment to adjoining Sertoli cells. The general morphology of the structures at the apical Sertoli-spermatid and basal Sertoli-Sertoli sites is very similar, although the ectoplasmic specializations are only on the Sertoli cell side at the apical Sertoli-spermatid sites and on both Sertoli cells sides at the basal Sertoli-Sertoli sites. Ectoplasmic specializations are assembled and disassembled at specific times during the spermatogenic cycle, and the turnover of these junctions is essential for the release of sperm at the apical sites and the movement of spermatocytes from the basal to the adluminal compartments in the basal epithelium.

Several proteins involved in cell adhesion and actin binding have been found at the sites of ectoplasmic specializations. The actin-binding proteins {alpha}-actinin [30] and vinculin [31] have been localized at ectoplasmic specializations by immunofluorescence, and the actin-bundling protein fimbrin has been detected on immunoblots of subcellular fractions containing ectoplasmic specializations [32]. In addition, espin, a novel actin-binding protein [33, 34], and testin, a testosterone-responsive Sertoli cell secretory product [35, 36], are localized at ectoplamic specializations. Adhesion molecule {alpha}6ß1 integrin has been found in ectoplasmic junctions [37]. Mulholland et al. [38] demonstrated the colocalization of ß1 integrin and integrin-linked kinase at these sites, hypothesized that ectoplasmic specializations are structurally maintained and controlled by an integrin-mediated pathway, and suggested the presence of a putative tyrosine kinase at these junctions [38]. Because Fyn kinase is known to be required for integrin signaling [39, 40], the concentration of Fyn kinase in the ectoplasmic specializations indicates that Fyn may be the tyrosine kinase involved in the integrin-mediated signal transduction that occurs at these junctions.

In mammalian Sertoli cells, the ectoplasmic specializations consist of the plasma membrane in the regions of attachment to adjacent cells, a submembrane layer of tightly packed actin filaments, and an attached cistern of the endoplasmic reticulum [24]. The fyn -/- testes exhibited abnormal vesicular structures in the actin filament layers of both apical and basal ectoplasmic specializations, i.e., the sites of contact with spermatids and between Sertoli cells. The origin of these vesicular structures has not been identified. The number and frequency of the abnormal vesicular structures were conspicuous at the age of 3 wk when the transient spermatogenic defect was observed and then decreased in adult fyn-deficient testes, indicating that these abnormal structures may affect germ cell survival. Phosphotyrosine was also abundantly immunostained at these junctions, as previously reported by other researchers [10, 41], suggesting that ectoplasmic specializations are a possible site of tyrosine kinase signaling. These findings support the idea that ectoplasmic specializations serve as junctional devices and as a site of tyrosine kinase (including Fyn)-mediated signal transduction within the Sertoli cell and between Sertoli cells and germ cells.

Fyn and Cytoskeletal Proteins

Some members of the Src family are associated with cytoskeletal proteins and are known to regulate cytoskeletal architecture and cell-cell interactions [42]. Cytoskeleton-related proteins such as paxillin [43], focal adhesion kinase [44], tensin [45], ß-catenin [46], cortactin [47], and actin-filament-associated proteins [48] serve as the substrates of Src kinases. We have considerable data suggesting that Fyn, like other Src kinases, is associated with cytoskeletal molecule(s) in the testis. First, Fyn was abundantly immunolocalized at the sites of ectoplasmic specializations, where actin filaments and other cytoskeletal molecules are located. Second, fyn -/- testes exhibited abnormal vesicular structures in the actin filament layers of the ectoplasmic specializations. Third, Fyn was abundantly present in the Triton X-100-insoluble fraction, which is enriched with cytoskeletal proteins. In contrast, only a very faint band was detected in the Triton X-100-soluble fraction by immunoblot analysis. These findings strongly suggest that Fyn kinase may function in association with cytoskeletal molecules at the ectoplasmic specializations, which undergo dynamic changes during spermatogenesis. Because Fyn and actin filaments were not completely colocalized in the apical ectoplasmic specializations around the heads of the spermatid (Fig. 8B), it is possible that Fyn is not directly associated with actin filaments in the ectoplasmic specializations.

Cell shrinkage by osmotic shock induces Fyn-dependent tyrosine phosphorylation of the cortical actin-binding protein cortactin [49]. The Fyn-mediated signaling pathway has been suggested to contribute to the reorganization of the cytoskeleton following changes in cell size and cell-cell contacts. Fyn may be engaged in a similar function at the ectoplasmic specialization, which also requires the cytoskeleton to be reorganized as it assembles and disassembles during spermatogenesis.

Fyn and Germ Cell Growth

The defect in testis growth observed in fyn -/- mice at 3–4 wk after birth might be due to a massive degeneration of germ cells during this period. At the age of 3 wk, step 5 spermatids were the most differentiated germ cells present in both fyn -/- and wild-type testes. Thus, Fyn kinase in the Sertoli cells may be responsible for the survival of the germ cell and not for the progression of germ cell differentiation. Fyn was immunolocalized even in disorganized testes from cryptorchid and W/Wv mice; Fyn protein exists in Sertoli cells in the absence of germ cells. This result suggests that a part of Fyn function may be independent of germ cells. Because Fyn immunoreactivity was localized throughout the cytoplasm of Sertoli cells in cryptorchid and W/Wv testes as well as normal testes, Fyn kinase may have another function in addition to that at ectoplasmic specializations. Exclusive localization of Fyn in the Sertoli cells suggests that a Fyn-mediated intracellular signal transduction in Sertoli cells may indirectly regulate differentiation and/or proliferation of germ cells in the testis. The molecular mechanism responsible for the effect of the defective Fyn signaling pathway in Sertoli cells on germ cells remains to be elucidated.

Because fyn -/- mice are viable and fertile, Fyn function may not be critical in the adult testis or another tyrosine kinase(s) may compensate for the lack of Fyn. Craig et al. [50] reported that mice devoid of Fer tyrosine kinase activity developed normally and were fertile, suggesting that another kinase may provide a redundant function. Fer is a ubiquitously expressed nonreceptor protein-tyrosine kinase, suggesting a condition similar to that of fyn-deficient testis. Fyn functions upstream of Fer in a cell-volume-regulated pathway [51]. In the case of fyn-deficient mice, compensation by another kinase may contribute to the fertility of these mice. The Src kinase family is a well-known example of functional complementation [52]. Most cells express more than 1 member of the Src family [53], and a loss of an individual Src family member can be compensated for by other Src family members. Because Src is localized in Sertoli cells [9, 10, 41], the function of Src may overlap that of Fyn in the testis. We demonstrated by immunoblot analysis that Src protein was also present in fyn -/- testis, although its amount did not differ from that observed in wild-type testis. The immunoblot analysis showed that Src protein was present in both the Triton X-100-soluble and -insoluble fractions, whereas Fyn was detected predominantly in the insoluble fraction. These data suggest that Src and Fyn kinases may play different roles in the testis, as discussed by Wu et al. [54] in a study on the differential dynamics of Fyn and Src in platelets. Thus, Src or some other Src family member present in Sertoli cells may compensate, at least partially, for the loss of Fyn, enabling male fertility to be maintained in the adult testis. Such compensatory mechanisms may be insufficient during the period of postnatal development, resulting in the transient spermatogenic defects that were observed in 3- and 4-wk-old mice.

Our results indicate that Fyn tyrosine kinase is involved in the regulation of mouse spermatogenesis through the functional control of Sertoli cells, especially at ectoplasmic specializations. We demonstrated that tyrosine phosphorylation of an 80-kDa protein (p80) was significantly decreased in fyn -/- testis compared with wild-type testis and that both Fyn and p80 were abundantly detected in the Triton X-100-insoluble cytoskeletal fraction. Thus, p80 may play a role in a Fyn-mediated signal transduction that supports germ cell growth and differentiation. Further experiments are under way to identify p80 and other molecules that interact with Fyn kinase in the testis to determine the exact mechanism of fyn-mediated signaling in spermatogenesis.


   ACKNOWLEDGMENTS
 
The authors are grateful to Ms. Kyoko Kamimura for her excellent technical assistance.


   FOOTNOTES
 
First decision: 16 July 2001.

1 Supported in part by a Grant-in-Aid (12670007) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to M.M.), a grant from The Inohana Foundation of Chiba University (to M.M.), and a grant from the Ministry of the Environment of Japan (to S.Y.). Back

2 Correspondence. FAX: 81 43 226 2021; maekawa{at}med.m.chiba-u.ac.jp Back

Accepted: September 11, 2001.

Received: June 27, 2001.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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