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BOR - Papers in Press, published online ahead of print December 15, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.035485
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BIOLOGY OF REPRODUCTION 72, 908–915 (2005)
DOI: 10.1095/biolreprod.104.035485
© 2005 by the Society for the Study of Reproduction, Inc.

Expression of CD46 in Developing Rat Spermatozoa: Ultrastructural Localization and Utility as a Marker of the Various Stages of the Seminiferous Tubuli1

Masashi Mizuno3, Claire L. Harris3, Norihiko Suzuki4, Seiichi Matsuo4, and B. Paul Morgan2,3

Complement Biology Group,3 Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Henry Wellcome Building, Heath Park, Cardiff, CF14 4XN, United Kingdom Department of Internal Medicine,4 Clinical Immunology, Nagoya University Graduate School of Medicine,Tsuruma-cho, Showa-ku, Nagoya 466-8550, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Identification of the various stages of the seminal tubule epithelium that are important in spermatogenesis in humans and rodents requires considerable expertise for analysis of ultrastructural appearance under light microscopy. Few good stage-specific markers have been reported to facilitate the process. We recently described characterization of the expression of CD46 (membrane cofactor protein) in the rat using a novel monoclonal antibody. Expression of CD46 was restricted to spermatozoa and their immediate precursors in the testis. In the present study, we used a combination of morphological analyses, known acrosome markers, actin staining, direct nuclear staining, and staining for CD46 to delineate precisely the subcellular location of CD46. Staining of CD46 colocalized with known acrosome markers in late spermatids and mature spermatozoa and was confirmed by electron microscopy to be acrosome-restricted. Expression was first detected in step 7 spermatids, whereas known markers were not expressed until step 9. The CD46 staining pattern differed through spermatid development, and distinct patterns of staining could be identified that, when combined with 4'-6-diamino-2-phenylindole-2HCl nuclear staining, enabled the accurate staging of the seminiferous tubule epithelium in different profiles. This detailed description of the spatiotemporal expression patterns of CD46 provides a valuable tool for analysis of spermatogenesis in the rat. Furthermore, this information will aid ongoing studies regarding the roles of CD46 in acrosome-related spermatozoal functions.

immunology, sperm, spermatid, spermatogenesis, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The seminiferous tubuli of the testis play an essential role in the production and maturation of the male germ cells. The complexity of this task is reflected in the intricate organization of the tubuli, comprising a series of stages of epithelial structure. In humans, six distinct stages have been identified, but in the rat, 14 stages can be distinguished based on the tubular composition and structure. Spermatid development into spermatozoa is equally complex, comprising 19 steps in the rat [1, 2]. When testis sections are examined, different stages of the epithelial cycle will be seen in each section. These usually are differentiated based on multiple microscopic features, including the frequency of meiotic and mitotic figures, spermatid shape, presence and position of elongated spermatids, possession of periodic acid-Schiff (PAS)-positive Golgi apparatus, mitochondrial alignment, acrosome shape, and presence of clusters of elongated spermatids [1, 2]. However, these multiple markers are difficult to evaluate using either light or fluorescence microscopy, provoking a search for more specific markers. A number of markers of the acrosome have been reported in hamsters, humans [3–7], mice [8, 9], and rats [10, 11]. However, the nature of the antigenic marker molecule in most cases, and particularly in the rat, is unclear.

The membrane cofactor protein (MCP), also known as CD46, was first described as a membrane complement regulator that functions as a cofactor with factor I to regulate the activating enzymes of the complement cascade. More recently, human CD46 has been shown to be a multitasking molecule. Among other activities, CD46 is utilized as a receptor for several species of bacteria and viruses [12–16], acts as a costimulatory molecule on T cells [17–20], and is a ligand for DLG4, a cytoskeletal protein that is involved in cell polarization [21]. In humans, CD46 exists as multiple isomeric forms and is broadly distributed [22, 23]. Human spermatozoa express only an isoform comprising the four short consensus repeat (SCR) domains, the short Ser/Thr/Pro-rich domain C, and the Cyt2 variant of the cytoplasmic tail. The N-linked carbohydrate groups in spermatozoal CD46 also are uniquely trimmed to small, simple structures, explaining the low apparent molecular weight of the protein at this site [24]. Spermatozoal CD46 is identical to the previously described acrosome-restricted spermatozoal protein trophoblast-leukocyte common antigen [25– 28], and it has been ascribed roles in fertilization [29–31]. Because of its acrosome-restricted expression pattern, spermatozoal CD46 has been utilized as a specific acrosome marker in humans [32, 33].

In contrast to the widespread distribution in humans, CD46 in rats, mice, and guinea pigs has been shown by mRNA analysis to be expressed predominantly or exclusively in the testis [34–36]. Recent studies of the distribution of CD46 protein in mice and rats confirm the testis-specific localization and suggest that CD46 in each of these species is acrosome-restricted [37, 38]. However, the detailed distribution of CD46 in developing spermatozoa in the various stages of the seminiferous tubuli remains unclear in any species. In the present study, we undertook to analyze in detail the presence and distribution of CD46 in the rat testis at each stage of the seminiferous tubuli to further clarify the roles of CD46 in sperm development and function. Analysis of CD46 expression was found to be a clear and reliable aid to the staging of seminiferous tubules, providing a useful complement to traditional markers.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and Markers for Immunohistochemistry

The monoclonal antibody (mAb) against rat CD46, MM.1, recognizing the SCR2 and SCR3 domains of rat CD46, has been previously described [38]. A second, previously undescribed mAb against rat CD46, MM.2, was generated from a mouse immunized with the SCR1, SCR2, and SCR3 domains of rat CD46 expressed as a fusion protein on human immunoglobulin (Ig) Fc essentially as described previously [38]. This new mAb specifically recognized SCR1 of rat CD46. The mAb 18.6, a kind gift from Prof. H. Moore (University of Sheffield, Sheffield, U.K.), was used as an acrosome marker [4]. This mAb, raised against an undefined epitope on hamster spermatozoa, has been shown to react specifically with the acrosome in rats. All of these mAbs were mouse IgG1. Tetramethylrhodamine isothiocyanate (TRITC)-labeled phalloidin was used to detect F-actin (Sigma-Aldrich, Dorset, U.K.). 4'-6-Diamino-2-phenylindole-2HCl (DAPI; Sigma) was used at a final concentration of 100 ng/ml to stain cell nuclei.

The secondary antibodies used were fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG (Bio-Rad, California) and rhodamine-labeled donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, Philadelphia, PA). The FITC-labeled goat anti-mouse IgG was pretreated with normal rat serum (1:1, v/v) before use. As a further control, we used an in-house isotype-matched IgG1 mAb without any specific binding on rat tissues. The mAb MM.1 was directly FITC-labeled by incubation with N-hydroxysuccinimide-FITC according to the manufacturer's instructions (Pierce, Rockford, IL).

Immunofluorescence and Histological Procedures

Male Wistar rats (age, 12–16 wk) were humanely killed using U.K. Home Office-approved methods, and their testes were removed, cut into blocks, and snap-frozen in isopentane at –40°C. Frozen tissues were sectioned in a cryostat (thickness, 5 µm) and fixed in acetone at room temperature for 5 min.

When double-immunostaining was used, the specificity of staining for each reagent was confirmed before double-staining. To compare the localization of rat CD46 with nuclear shape and F-actin distribution, MM.1 was first incubated on the specimens, followed by FITC-labeled goat-anti mouse IgG with DAPI (final concentration, 100 ng/ml) and TRITC-labeled phalloidin (final concentration, 100 ng/ml). To compare rat CD46 localization and mAb 18.6 staining, specimens were first incubated for 60 min at room temperature with mAb 18.6 (neat tissue-culture supernatant), followed by rhodamine-labeled donkey anti-mouse IgG for 30 min. The specimens were then blocked with the isotype control mouse IgG1 for 15 min and incubated with FITC-labeled mAb MM.1 for 60 min. The specimens were finally embedded with VectaShield (Vector Laboratories, CA) and observed under immunofluorescence (IF) microscopy.

For some analyses, selected sequential sections from the above-mentioned IF study were stained with hematoxylin and eosin (hematoxylin-eosin) to confirm the stage in the seminiferous tubuli according to the protocol described by Hess [1, 2]. As further confirmation of the IF localization, sequential sections also were stained with an avidin-biotin-peroxidase protocol. Briefly, the sections were pretreated with 0.3% H2O2 to block endogenous peroxidase and treated with the avidin-biotin-blocking reagent according to the manufacturer's protocol (Vector Laboratories). The mAb MM.1 was incubated for 30 min on sections, followed by biotin-labeled goat anti-mouse IgG (Sigma). The specimens were processed using the Vectastain Elite ABC kit (Vector Laboratories) according to the manufacturer's instructions. Development was performed using diaminobenzidine tetrahydrochloride (DAB) development reagents (Sigma fast; Sigma). Finally, counterstaining was performed with hematoxylin.

The IF staining pattern for mAb MM.1 in different tubule profiles was characterized with respect to three different aspects. First, intensity of IF staining was graded into five categories: negative (–), very weak (±), weak (+), moderate (++), and strong (+++). Second, positive cells in each profile were classified into one of seven staining patterns as detailed further in the Results and in Chart 1. Third, whether a single staining pattern (representing one step in spermatid development) or two distinct patterns (representing two different stages of spermatid development) were present in the profile was determined.



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  		CHART 1. CD46, DAPI, 18.6, and F-actin staining at different stages of the seminiferous epithelium cycle. The presence of bundle formation (#) of elongated spermatids is shown as –, negative or +, positive. The degree of phalloidin binding (*) to F-actin and mAb 18.6 binding are shown as –, negative; +/–, trace; +, weak; and ++, strong

To confirm specificity of staining, the mAb MM.1 was preincubated with a twofold molar excess of the rat MCP SCR2 and SCR3 Fc-fusion protein overnight at 4°C before testing with immunohistochemistry as described above.

Immunoelectron Microscopic Analysis

To confirm the ultrastructural location of CD46 in rat spermatozoa, we performed immunoelectron microscopic analysis. Harvested testis and epididymis from killed Wistar rats were fixed with periodate-lysine-paraformaldehyde at 4°C for 4 h. Fixed specimens were embedded in OCT compound (Sakura Finetechnical Co., Tokyo, Japan) and snap-frozen in liquid nitrogen. The frozen tissues were cut (thickness, 8 µm), and sections were air-dried. Sections were washed with 10% sucrose in PBS and pretreated in 0.3% H2O2 in methanol for 20 min, followed by 5% goat serum in PBS for 30 min as a block. The treated sections were incubated with mAb MM.1 (7.5 µg/ml in PBS overnight at 4°C), followed by horseradish peroxidase-labeled goat anti-mouse IgG (F(ab'); Histofine Simple Stain MAX-PO(M); Nichirei Corporation, Tokyo, Japan) for 30 min at room temperature. After washing, the sections were fixed in 1% glutaraldehyde for 5 min. The DAB development was performed with the Envision kit/ horseradish peroxidase (Dako Cytomation, Tokyo, Japan). Developed sections were postfixed with 1% OsO4 for 1 h, dehydrated through sequential ethanol dilutions, and embedded in Epon 812 (Nisshin EM Co., Tokyo, Japan). Sections were viewed on a H7100 electron microscope (Hitachi Co. Ltd., Ibaraki, Japan).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunoelectron Microscopy Confirms Acrosome-Restricted Expression of CD46

Using standard immunoelectron microscopic methods, the anti-rat CD46 mAb MM.1 strongly stained the inner membranes of the acrosome in rat epididymis spermatozoa (Fig. 1A). The CD46 staining was clearly visible inside the acrosome and extended to the tip of the spermatozoon (Fig. 1B). The tail portions of all cells were completely negative (Fig. 1, C and D). During spermatogenetic development, CD46 staining was first detected in step 7 spermatids (the earliest step at which CD46 expression was apparent; see below) and found both inside and around the Golgi apparatus as well as in the acrosome precursor (Fig. 1E). In step 8 spermatids, CD46 staining was found inside and around the forming acrosome and inside the Golgi apparatus. At this step, cytoplasmic elongation is apparent, and the Golgi apparatus is migrating to the other side of the nucleus, well separated from the acrosome (Fig. 1F). The CD46 staining was not detected in the outer cell membrane of spermatids at any step.



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  		FIG. 1. Immunoelectron microscopic localization of CD46 in rat epididymal spermatozoa. A) Spermatozoal head showing heavy staining of acrosome (arrows). B) The tip of spermatozoal head showing that CD46 expression is restricted to the inside of acrosome. Arrowheads show the cytoplasm is devoid of CD46 expression. C Spermatozoal tail (longitudinal section). D) Spermatozoal tail (transverse section). E and F) Step 7 (E) and step 8 (F) spermatids. Arrows show CD46 expression in Golgi apparatus, and arrowheads show CD46 expression in the developing acrosome. Original magnification x10 000

Seminiferous Epithelium Staging by DAPI Nuclear Staining and CD46 Staining

Testis sections were double-stained for DAPI and CD46 as described in Materials and Methods. Each pair of plates in Figure 2 comprised the same section, stained for DAPI and CD46 (using mAb MM.1), respectively. The DAPI staining of seminiferous epithelium clearly defined the round nuclei of spermatids at steps 1–9 (Fig. 2, A, C, E, G, I, and K). The CD46 staining was negative on round spermatids in steps 1–6 and was first detected in step 7 spermatids, in which staining was weak and diffuse (Fig. 2H and Chart 1). In step 8 and 9 spermatids, CD46 expression was weak and clearly displaced to one side of the cell (Fig. 2, J and L, and Chart 1). The DAPI staining was observed in characteristic patterns in the elongated nuclei of spermatids at steps 10–19. Intensity of DAPI staining increased through steps 11–13 of spermatid development (Fig. 2, O and Q) and was strongest through steps 14 and 15 (Fig. 2, A and S), likely reflecting nuclear condensation during these steps. After step 16 of rat spermatid development, the intensity of DAPI staining was decreased, suggesting that nuclear condensation was diminished (Fig. 2, G and I). The CD46 was weakly expressed in an elongated distribution to the caudal side in step 10 spermatids (Fig. 2N). Staining was stronger and extended across the tip of the nucleus and to both nuclear edges in spermatids from step 11 to step 14 (Fig. 2, P, R, and T) and was concentrated in the caudal and dorsal sides in spermatids of steps 15 and 16 (Fig. 2, B and D). Staining for CD46 was strong and deviated to the dorsal side of the nucleus, including the tip, in step 17 spermatids (Fig. 2F). In spermatids of steps 18 and 19, staining was strong and clearly sickle-shaped in a typical acrosome distribution (Fig. 2, H and J), resembling the acrosome staining of swim-up spermatozoa as previously reported [38].



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  		FIG. 2. CD46 expression and DAPI nuclear staining at different stages of the seminiferous tubule cycle. Sections were double-stained as detailed in Materials and Methods. The Greek numbers to the left of each set show the relevant stage of the seminiferous tubuli. For each set, DAPI staining is shown on the left and CD46 staining on the right. Each pair of pictures was taken in the same double-stained section. Arrows and italic numbers indicate spermatids at these specific stages in development. An asterisk indicates the lumen side. Original magnification x1000

Chart 1 summarizes the epithelial staging obtained from CD46 and DAPI staining. Other aids for staging of the seminiferous epithelium were provided by the prominent bundling of elongated spermatids from step 10 through step 18 evident from DAPI and CD46 staining (clearly shown in Fig. 2, A–F and M–T), the position of isolated cells and bundles in the walls of the seminiferous tubules, and the presence, in some profiles, of more than one step in spermatid development (Chart 1). With increasing maturity, CD46-stained spermatids were located progressively closer to the lumen of the tubule. Spermatids of step 7 through step 9 were located near the basement membrane and were not bundled (epithelial stages VII–IX), whereas step 10 spermatids were loosely bundled in this same area (stage X). Spermatids of step 11 through step 14 were tightly bundled and predominantly situated in the middle layer of seminiferous tubule profiles (defining seminiferous epithelial stages XI–XIV). Spermatids of step 15 through step 17 were present in bundles in the middle layer (stages I–IV) and also close to Sertoli cells along the basement membrane (stage V). Spermatids of step 18 and step 19 were bundled and predominantly sublumenal (stage VI) or unbundled and lining the lumen (stages VII and VIII) of seminiferous tubuli. Spermatids placed in steps 18 and 19 based on CD46 staining were present by themselves in stage VI profiles (defined as step 18), with step 7 spermatids in stage VII (defining as stage 19) (Fig. 2H), and with step 8 spermatids in stage VIII (Fig. 2J).

Comparison of Seminiferous Epithelium Stagingby Hematoxylin-Eosin Staining and CD46 Staining

Seminiferous epithelial staging is classically performed by staining sections with hematoxylin-eosin and/or PAS. In serial frozen sections of testis, we compared hematoxylin-eosin staining with staining for CD46, with the latter detected either using IF (as in Fig. 2) or a peroxidase method for easier comparison of tissue architecture. Hematoxylin-eosin staining identified ongoing mitosis in spermatogonia, meiosis in spermatocytes, and spermiogenesis in elongated spermatids, with the latter easily identified morphologically in sections (Fig. 3, A1–I1). Staining for CD46 and detection using a peroxidase method gave results essentially identical to those obtained from IF (Fig. 3, compare A2–I2 with sequential sections in A3–I3). Although in many hematoxylin-eosin-stained profiles approximate epithelial staging was possible from morphological analyses and position of stained cells as detailed in the preceding section, CD46 staining provided a clearer distinction between stages as described in Figure 2 and Chart 1.



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  		FIG. 3. Comparison of hematoxylin-eosin staining and CD46 staining in seminiferous epithelium. Each set of plates comprises sequential sections stained with hematoxylin-eosin (1), CD46 with peroxidase detection and hematoxylin counterstaining (2), and CD46 with IF detection (green) and DAPI counterstaining (blue) (3). The Greek numbers to the left of each set show the relevant stage of the seminiferous tubuli. Also shown (J and K) are set incubated with an isotype-matched mAb instead of MM.1 and the relevant detection method applied as a negative control. Original magnification x400

Confirmation of Specificity of CD46 Staining

The mAb MM.1 has been characterized previously as anti-CD46 [38]. To confirm further the specificity of staining with this mAb, we first compared the staining pattern of a novel anti-CD46 mAb, MM.2, generated against a different region of rat CD46 (SCR1). No staining with this mAb was found in a large panel of rat tissues with the sole exception of testis (not shown). The mAb MM.2 gave a staining pattern identical to that of MM.1 in testis sections (Fig. 4, A and B, respectively). As a further test of specificity, we adsorbed the mAb MM.1 with the CD46 SCR2 and SCR3 fusion protein used to generate the mAb. Adsorbed mAb lost all reactivity with testis sections, whereas an irrelevant fusion protein was without effect, confirming the specificity of the mAb (Fig. 4, C and D). Neither MM.1 nor MM.2 stained human spermatozoa or mouse testis in the IF study and Western blot analyses (data not shown).



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  		FIG. 4. Confirmation of specificity of staining for CD46. Serial testis sections were stained with mAb MM.1 raised against SCR1, SCR2, and SCR3 domains of rat CD46 (A) or mAb MM.2 raised against the SCR1 domain of rat CD46 (B). The staining patterns with these two noncompetitive mAb are identical, confirming that the are staining CD46 in testis. The MM.1 mAb was adsorbed with the SCR2 ;pl 3 fusion protein used as immunogen and then applied to testis sections. Staining was completely abolished by this adsorption step (C), whereas incubation with an irrelevant fusion protein had no effect on staining (D). Original magnification x200

Comparison Between Staining for the Acrosome Marker 18.6 and CD46 in Rat Spermatids

Staining with mAb 18.6 has been used as an aid to identifying the acrosome and staging seminiferous epithelium in several species. In the present study, we compared expression in rat testis of CD46 and 18.6 antigen by double-staining. As described above, CD46 expression was first present at step 7 and steadily increased in intensity through spermatid development. Staining with mAb 18.6 was first present at step 9 (Fig. 5A). Staining was weak and patchy through steps 9 and 10 and was much more restricted in distribution compared with CD46 at these stages (Fig. 5, A and B). From step 11 onward, mAb 18.6 staining was stronger and became increasingly coincident with CD46 staining (Fig. 5, C and D), approaching complete overlap by steps 18 and 19 (Figs. 5, E and F, and 6). In step 19 spermatids and in spermatozoa in the lumen of the cauda epididymis, mAb 18.6 and CD46 staining were coincident (Fig. 6).



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  		FIG. 5. Comparison of CD46 with other markers of spermatid maturation. Testis sections were double-stained for CD46 (green) and 18.6 antigen (red) (A–F) or for CD46 (green) and F-actin (red) (G–L). A and G) Step 9 spermatids in stage IX seminiferous epithelium (SE). B and H) Step 10 spermatids in stage X SE. C and I) Step 13 spermatids in stage XIII SE. D and J) Step 17 spermatids in stage V SE. E and K Steps 7 and 19 spermatids in stage VII SE. F and L) Steps 8 and 19 spermatids in stage VIII SE. Arrows show colocalization of CD46 and 18.6 antigen staining in A–F and colocalization of CD46 and F-actin in G–L. An asterisk indicates the lumen side. Inserts in E and F are close-up views of step 19 spermatids. Arrowheads indicate areas where CD46 is expressed in the absence of 18.6 antigen on step 19 spermatids. Original magnification x1000 for all plates and x3 in inserts



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  		FIG. 6. Comparison between CD46 and 18.6 antigen expression in step 19 spermatids and epididymal spermatozoa. To further delineate the distribution patterns of CD46 and 18.6 antigen, sections of testis and epididymis were double-stained as detailed in Materials and Methods. A, C, and E) Step 19 spermatids in stage VII seminiferous epithelium. B, D, and F) Spermatozoa in the lumen of the cauda epididymis. The CD46 staining (green; A and B), 18.6 staining (red; C and D), and combined images (E and F) are shown. Nuclei were counterstained with DAPI (blue). These images confirm that CD46 expression occupies a broader area compared to 18.6 antigen staining in step 19 spermatids (arrows in A and E), whereas in spermatozoa, the expression patterns are identical for the two antigens. Original magnification x1000; each image is magnified x3 from the original pictures

Comparison Between Staining for F-Actin and CD46in Rat Spermatids

The F-actin staining has been used previously in the staging of seminiferous tubule profiles in the rat [39, 40]. In our hands, F-actin expression, detected by phalloidin staining, was first present in spermatids at step 7, which is the step that first expresses CD46, and remained weak through steps 7–10 (Fig. 5, G and H). Through steps 11– 16, the expression of F-actin gradually increased and was strongest at steps 17–19 as previously reported (Fig. 5, I– K) [39, 40]. The association of strong F-actin staining with characteristic CD46 staining patterns in spermatids of steps 17–19 provided independent confirmation of the staging of seminiferous epithelium profiles through stages IV–VII. Stage VIII seminiferous epithelium has been correlated previously with a reduced F-actin staining [39, 40]. In the present study, stage VIII, defined by CD46 staining, with the coexistence of spermatids at step 19 and step 8, was clearly associated with reduced F-actin staining, thus confirming the staging (Fig. 5L).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Accurate staging of spermatogenesis and the seminiferous tubule cycle is necessary for studies of spatiotemporal aspects of protein and gene expression in germ cell development. Current staging of the seminiferous tubuli is dependent on multiple light microscopic findings on hematoxylin-eosin- and PAS-stained sections and requires the skills of a pathology specialist [1, 2]. Most of the markers used require subjective estimations of staining intensity that can be problematic. In particular, few good markers of the developing acrosome are available in humans and experimental animals. During the last few years, CD46 has emerged as a useful acrosome marker in humans, but to our knowledge, it has not been explored in other species. We recently described generation of an mAb against the rat analogue of the complement regulatory molecule CD46 and showed that its expression is limited to the acrosome region in developing and mature spermatozoa [38]. In the present study, we analyzed in detail the staining pattern and distribution of CD46 in developing spermatids and showed that categorization of the different staining patterns, together with position in the seminiferous epithelium profiles, can be accurately related to the various steps of spermatogenesis. The identity of the antigen has been confirmed using mAb against different regions of CD46 and by specific adsorption of the mAb with CD46 fusion proteins. Expression of CD46, first detected on step 7 intermediate spermatids, became stronger and adopted the deviated distribution that is characteristic of the developing acrosome through steps 8–14 of spermatid development. After step 14, CD46 distribution was concentrated into the position of the mature acrosome. We confirmed that rat CD46 was not expressed in early round spermatids or the earlier spermatogenic cells, including spermatogonium and spermatocytes. We used immunoelectron microscopy to delineate further the expression of CD46 on spermatozoa and developing spermatids. In late spermatids and mature spermatozoa, CD46 was acrosome-restricted, with no staining of the residual Golgi apparatus. In the earliest-expressing spermatids (steps 7 and 8), expression was found in the Golgi apparatus and the early acrosome structure. Before step 7, CD46 was not detected either in the Golgi apparatus or in the forming acrosomal vesicle. This sequence of events suggests that CD46 is transferred from the Golgi apparatus to the acrosome during the early stages of acrosome formation. No CD46 expression was detectable on cell membranes at any stage in the development of spermatozoa.

Recognition of the stage of maturation of seminiferous epithelium in tubule profiles is necessary for analyzing the relationship between the different types of spermatogenic cells. Few specific markers are available, and none alone has the power to identify the stage [41–44]. Staining for CD46 provides an acrosome marker that, when combined with DAPI staining and morphological parameters (bundling and position in profile), enables accurate staging. Inclusion of actin filament staining, which is enhanced in late-elongated spermatids and decreased just before completion of sperm transformation and release to the lumen, further supports the staging of spermatids by CD46 expression patterns during these later steps of development [39, 40].

Of note, CD46 expression appeared at earlier stages of spermatid development than the known acrosome marker 18.6 antigen [4]. Expression of CD46 was present from step 7, whereas 18.6 antigen was not detected until step 9. Furthermore, although in the later steps of elongated spermatids and in mature spermatozoa the distribution of CD46 coincided with that of 18.6 antigen and faithfully delineated the acrosome, CD46 distribution before step 12 was broader than that of 18.6 antigen. This early expression of CD46, also apparent in immunoelectron microscopy to include Golgi staining, as noted above, suggests specific roles in spermatogenesis or the maturation of the acrosome.

Human CD46 is now recognized as a multitasking molecule with roles outside complement regulation, including as a receptor for several microorganisms [12–16] and as a costimulatory molecule for T-cell activation [17–20]. The fascinating expression pattern of rat CD46 revealed in the present study is consistent with roles for CD46 in enhancing sperm-egg interaction, as suggested for human CD46 [7, 29–31]. However, in a recent report describing CD46 knockout mice [37], the possibility of a suppressive role for CD46 in fertilization has emerged. The CD46 knockout mice display an accelerated acrosome reaction and enhanced fertility, perhaps suggesting an acrosome-stabilizing role for CD46. In rat, the roles of CD46 remain unclear, but the availability of specific mAb might help with the progress of further analysis concerning the roles of CD46 in the rodent and human reproductive systems. Delineation of the detailed spatiotemporal aspects of CD46 expression in the rat testis will be useful to support these further analyses.


   ACKNOWLEDGMENTS
 
We thank Professor P.M. Johnson (Liverpool University, Liverpool, U.K.) for helpful discussion.


   FOOTNOTES
 
1 Supported by The Wellcome Trust (Programme support no. 068590 to B.P.M.); Visiting Fellow by a Bursary from UWCM (M.M.); and Tawada Clinic, Nagoya (N.S.). Back

2 Correspondence: B.P. Morgan, Complement Biology Group, Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Henry Wellcome Building, Heath Park, Cardiff CF14 4XN, U.K. FAX: 44 29 207 44905; morganbp{at}cardiff.ac.uk Back

Received: 30 August 2004.

First decision: 7 October 2004.

Accepted: 17 November 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hess RA. Spermatogenesis, overview. In: Knobil E, Neil JD (eds.), Encyclopedia of Reproduction, vol. 4. San Diego, CA: Academic Press; 1998:539–545
  2. Hess RA. Quantitative and qualitative characteristics of the stages and transitions in the cycle of the rat seminiferous epithelium: light microscopic observations of perfusion-fixed and plastic-embedded testes. Biol Reprod 1990 43:525-542[Abstract]
  3. Ellis DH, Hartman TD, Moore HD. Maturation and function of the hamster spermatozoon probed with monoclonal antibodies. J Reprod Immunol 1985 7:299-314[CrossRef][Medline]
  4. Moore HD, Smith CA, Hartman TD, Bye AP. Visualization and characterization of the acrosome reaction of human spermatozoa by immunolocalization with monoclonal antibody. Gamete Res 1987 17:245-259[CrossRef][Medline]
  5. Kallajoki M. Suominen J An acrosomal antigen of human spermatozoa and spermatogenic cells characterized with a monoclonal antibody. Int J Androl 1984 7:283-296[Medline]
  6. Homyk M, Anderson DJ, Wolff H, Herr JC. Differential diagnosis of immature germ cells in semen using monoclonal antibody MHS-10 to the intra-acrosomal antigen SP-10. Fertil Steril 1990 53:323-330[Medline]
  7. Hamatani T, Tanabe K, Kamei K, Sakai N, Yamamoto Y, Yoshimura Y. A monoclonal antibody to human SP-10 inhibits in vitro the binding of human sperm to hamster oolemma but not to human zona pellucida. Biol Reprod 2000 62:1201-1208[Abstract/Free Full Text]
  8. Okabe M, Takeda K, Adachi T, Kohama Y, Mimura T, Aonuma S. Studies on sperm capacitation using monoclonal antibody—disappearance of an antigen from the anterior part of mouse sperm head. J Pharmacobiodyn 1986 9:55-60[Medline]
  9. Gerton GL, O'Brien DA, Eddy EM. Antigens recognized by monoclonal antibody to mouse acrosomal components differ in guinea pig spermatogenic cells and sperm. Biol Reprod 1988 39:431-441[Abstract]
  10. Russinova AI, Atanassova NN, Paskaleva ML, Kancheva LS. Acrosomal component of rat round spermatids recognized by a novel monoclonal antibody. Endocr Regul 1998 32:155-159[Medline]
  11. Phillips DM, Jones R, Shalgi R. Alterations in distribution of surface and intracellular antigens during epididymal maturation of rat spermatozoa. Mol Reprod Dev 1991 29:347-356[CrossRef][Medline]
  12. Dorig RE, Marcil A, Chopra A, Richardson CD. The human CD46 molecule is a receptor for measles virus (Edmonston strain). Cell 1993 75:295-305[CrossRef][Medline]
  13. Greenstone HL, Santoro F, Lusso P, Berger EA. Human herpesvirus 6 and measles virus employ distinct CD46 domains for receptor function. J Biol Chem 2002 277:39112-39118[Abstract/Free Full Text]
  14. Naniche D, Varior-Krishnan G, Cervoni F, Wild TF, Rossi B, Rabourdin-Combe C, Gerlier D. Human membrane cofactor protein (CD46) acts as a cellular receptor for measles virus. J Virol 1993 67:6025-6032[Abstract/Free Full Text]
  15. Maisner A, Schneider-Schaulies J, Liszewski MK, Atkinson JP, Herrler G. Binding of measles virus to membrane cofactor protein (CD46): importance of disulfide bonds and N-glycans for the receptor function. J Virol 1994 68:6299-6304[Abstract/Free Full Text]
  16. Okada N, Liszewski MK, Atkinson JP, Caparon M. Membrane cofactor protein (CD46) is a keratinocyte receptor for the M protein of the group A streptococcus. Proc Natl Acad Sci U S A 1995 92:2489-2493[Abstract/Free Full Text]
  17. Astier A, Trescol-Biemont MC, Azocar O, Lamouille B, Rabourdin-Combe C. Cutting edge: CD46, a new costimulatory molecule for T cells, that induces p120CBL and LAT phosphorylation. J Immunol 2000 164:6091-6095[Abstract/Free Full Text]
  18. Zaffran Y, Destaing O, Roux A, Ory S, Nheu T, Jurdic P, Rabourdin-Combe C, Astier AL. CD46/CD3 costimulation induces morphological changes of human T cells and activation of Vav, Rac, and extracellular signal-regulated kinase mitogen-activated protein kinase. J Immunol 2001 167:6780-6785[Abstract/Free Full Text]
  19. Marie JC, Astier AL, Rivailler P, Rabourdin-Combe C, Wild TF, Horvat B. Linking innate and acquired immunity: divergent role of CD46 cytoplasmic domains in T cell-induced inflammation. Nat Immunol 2002 3:659-666[Medline]
  20. Kemper C, Chan AC, Green JM, Brett KA, Murphy KM, Atkinson JP. Activation of human CD4+ cells with CD3 and CD46 induces a T-regulatory cell 1 phenotype. Nature 2003 421:388-392[CrossRef][Medline]
  21. Ludford-Menting MJ, Thomas SJ, Crimeen B, Harris LJ, Loveland BE, Bills M, Ellis S, Russell SM. A functional interaction between CD46 and DLG4: a role for DLG4 in epithelial polarization. J Biol Chem 2002 277:4477-4484[Abstract/Free Full Text]
  22. Post TW, Liszewski MK, Adams EM, Tedja I, Miller EA, Atkinson JP. Membrane cofactor protein of the complement system: alternative splicing of serine/threonine/proline-rich exons and cytoplasmic tails produces multiple isoforms that correlate with protein phenotype. J Exp Med 1991 174:93-102[Abstract/Free Full Text]
  23. Liszewski MK, Tedja I, Atkinson JP. Membrane cofactor protein (CD46) of complement. Processing differences related to alternatively spliced cytoplasmic domains. J Biol Chem 1994 269:10776-10779[Abstract/Free Full Text]
  24. Cervoni F, Oglesby TJ, Adams EM, Milesifluet C, Nickells M, Fenichel P, Atkinson JP, Hsi BL. Identification and characterization of membrane cofactor protein of human spermatozoa. J Immunol 1992 148:1431-1437[Abstract]
  25. Fenichel P, His B, Farahifar D, Donzeau M, Barrier-Delpech D, Yehy C. Evaluation of the human sperm acrosome reaction using a monoclonal antibody, GB24, and fluorescence-activated cell sorter. J Reprod Fertil 1989 87:699-706
  26. Anderson DJ, Michaelson JS, Johnson PM. Trophoblast/leukocyte-common antigen is expressed by human testicular germ cells and appears on the surface of acrosome-reacted sperm. Biol Reprod 1989 41:285-293[Abstract]
  27. Purcell DF, McKenzie IF, Lublin DM, Johnson PM, Atkinson JP, Oglesby TJ, Deacon NJ. The human cell-surface glycoproteins HuLy-m5, membrane co-factor protein (MCP) of the complement system, and trophoblast leukocyte-common (TLX) antigen, are CD46. Immunology 1990 70:155-161[Medline]
  28. Fenichel P, Dohr G, Grivaux C, Cervoni F, Donzeau M, Hsi BL. Localization and characterization of the acrosomal antigen recognized by GB24 on human spermatozoa. Mol Reprod Dev 1990 27:173-178[CrossRef][Medline]
  29. Okabe M, Ying X, Nagira M, Ikawa M, Kohama Y, Mimura T, Tanaka K. Homology of an acrosome-reacted sperm-specific antigen to CD46. J Pharmacobiodyn 1992 15:455-459[Medline]
  30. Taylor CT, Biljan MM, Kingsland CR, Johnson PM. Inhibition of human spermatozoon-oocyte interaction in vitro by monoclonal antibodies to CD46 (membrane cofactor protein). Hum Reprod 1994 9:907-911[Abstract/Free Full Text]
  31. D'Cruz OJ, Lambert H, Haas GG Jr. Expression of CD15 (Lewisx) antigen on human sperm and its role in sperm-egg interaction. Am J Reprod Immunol 1997 37:172-183
  32. Seya T, Hara T, Matsumoto M, Kiyohara H, Nakanishi I, Kinouchi T, Okabe M, Shimizu A, Akedo H. Membrane cofactor protein (MCP, CD46) in seminal plasma and on spermatozoa in normal and "sterile" subjects. Eur J Immunol 1993 23:1322-1327[Medline]
  33. Jaiswal BS, Eisenbach M, Tur-Kaspa I. Detection of partial and complete acrosome reaction in human spermatozoa: which inducers and probes to use?. Mol Hum Reprod 1999 5:214-219[Abstract/Free Full Text]
  34. Hosokawa M, Nonaka M, Okada N, Nonaka M, Okada H. Molecular cloning of guinea pig membrane cofactor protein: preferential expression in testis. J Immunol 1996 157:4946-4952[Abstract]
  35. Tsujimura A, Shida K, Kitamura M, Nomura M, Takeda J, Tanaka H, Matsumoto M, Matsumiya K, Okuyama A, Nishimune Y, Okabe M, Seya T. Molecular cloning of a murine homologue of membrane cofactor protein (CD46): Preferential expression in testicular germ cells. Biochem J 1998 330:163-168
  36. Mead R, Hinchliffe SJ, Morgan BP. Molecular cloning, expression and characterization of the rat analogue of human membrane cofactor protein (MCP/CD46). Immunology 1999 98:137-143[CrossRef][Medline]
  37. Inoue N, Ikawa M, Nakanishi T, Matsumoto M, Nomura M, Seya T, Okabe M. Disruption of mouse CD46 causes an accelerated spontaneous acrosome reaction in sperm. Mol Cell Biol 2003 23:2614-2622[Abstract/Free Full Text]
  38. Mizuno M, Harris CL, Johnson PM, Morgan BP. Rat membrane cofactor protein (MCP; CD46) is expressed only in the acrosome of developing and mature spermatozoa and mediates binding to immobilized activated C3. Biol Reprod 2004 71:1374-1383[Abstract/Free Full Text]
  39. Fouquet JP, Kann ML, Dadoune JP. Immunogold distribution of actin during spermiogenesis in the rat, hamster, monkey, and human. Anat Rec 1989 223:35-42[CrossRef][Medline]
  40. Oko R, Hermo L, Hecht NB. Distribution of actin isoforms within cells of the seminiferous epithelium of the rat testis: evidence for a muscle form of actin in spermatids. Anat Rec 1991 231:63-81[CrossRef][Medline]
  41. Bjørge C, Wiger R, Holme JA, Brunborg G, Andersen R, Dybing E, Søderlund EJ. In vitro toxicity of 1,2-dibromo-3-chloropropane (DBCP) in different testicular cell types from rats. Reprod Toxicol 1995 9:461-473[CrossRef][Medline]
  42. Abou-Haila A, Tulsiani DR. Acid glycohydrolases in rat spermatocytes, spermatids and spermatozoa: enzyme activities, biosynthesis and immunolocalization. Biol Proc Online 2001 3:35-42[CrossRef]
  43. Parvinen M, Vanha-Perttula T. Identification and enzyme quantitation of the stages of the seminiferous epithelial wave in the rat. Anat Rec 1972 174:435-449[CrossRef][Medline]
  44. Shabanowitz PB, DePhilip RM, Crowell JA, Tres LL, Kierszenbaum AL. Temporal appearance and cyclic behavior of Sertoli cell-specific secretory proteins during the development of the rat seminiferous tubule. Biol Reprod 1986 35:745-760[Abstract]



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P. M Johnson, L. E Clift, P. Andrlikova, M. Jursova, B. F Flanagan, J. A Cummerson, P. Stopka, and K. Dvorakova-Hortova
Rapid sperm acrosome reaction in the absence of acrosomal CD46 expression in promiscuous field mice (Apodemus)
Reproduction, December 1, 2007; 134(6): 739 - 747.
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