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Biology of Reproduction 60, 1128-1136 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Calcium-Dependent Actin Filament-Severing Protein Scinderin Levels and Localization in Bovine Testis, Epididymis, and Spermatozoa1

R.-Marc Pelletier2,a, José-María Trifarob, M. Eloísa Carbajala, Yuji Okawaraa, and María Leiza Vitalea

a Department of Pathology and Cell Biology, Faculty of Medicine, Université de Montréal, P. Québec, Canada H3T 1J4 b Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We assessed the levels and localization of the actin filament-severing protein scinderin, in fetal and adult bovine testes, and in spermatozoa during and following the epididymal transit. We performed immunoblots on seminiferous tubules and interstitial cells isolated by enzymatic digestion, and on bovine chromaffin cells, spermatozoa, aorta, and vena cava. Immunoperoxidase labeling was done on Bouin's perfusion-fixed testes and epididymis tissue sections, and on spermatozoa. In addition, immunofluorescence labeling was done on spermatozoa. Immunoblots showed one 80-kDa band in chromaffin cells, fetal and adult tubules, interstitial cells, spermatozoa, aorta, and vena cava. Scinderin levels were higher in fetal than in adult seminiferous tubules but showed no difference between fetal and adult interstitial cells. Scinderin levels were higher in epididymal than in ejaculated spermatozoa. Scinderin was detected in a region corresponding with the subacrosomal space in the round spermatids and with the acrosome in the elongated spermatids. In epididymal spermatozoa, scinderin was localized to the anterior acrosome and the equatorial segment, but in ejaculated spermatozoa, the protein appeared in the acrosome and the post-equatorial segment of the head. In Sertoli cells, scinderin was detected near the cell surface and within the cytoplasm, where it accumulated near the base in a stage-specific manner. In the epididymis, scinderin was localized next to the surface of the cells; in the tail, it collected near the base of the principal cells. In Sertoli cells and epididymal cells, scinderin may contribute to the regulation of tight junctional permeability and to the release of the elongated spermatids by controlling the state of perijunctional actin. In germ cells, scinderin may assist in the shaping of the developing acrosome and influence the fertility of the spermatozoa.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Actin has been identified in both Sertoli cells [1–7] and germ cells, namely, in developing spermatids [5, 8–14] and spermatozoa [15–20].

In the testis, impermeable tight junctions accompanied by microfilaments are situated between Sertoli cells, where they form the anatomical basis of the blood-testis barrier that divides the seminiferous epithelium into a basal and a luminal cellular compartment. At a precise moment of their development, germ cells located in the basal compartment must migrate into the luminal compartment to complete their meiosis and undergo cellular differentiation [1–3, 21, 22]. Exactly how this event takes place remains the subject of numerous scientific investigations. We recently presented data that suggested the control of the monomeric (G-actin) or filamentous (F-actin) state or form of the perijunctional actin may affect the association of the tight junction peripheral protein ZO-1 with other junctional components of the plasma membrane and perhaps in this way play a role in the regulation of the tight junction permeability [7]. Because the state of the perijunctional actin is in part controlled by actin-binding proteins, in the study reported here, we elected to evaluate the variations in the concentration and localization of scinderin, a Ca2+-dependent actin filament-severing protein during testicular development.

Not everybody agrees on the precise location of actin [23–25] in the germ cells, or on whether actin exists predominantly in a monomeric or filamentous state. Yet divergent views may not necessarily need to be contradictory. It is generally accepted that actin exists in equilibrium between G- and F-actin within the cell. The presence of higher amounts of one or the other form of actin in germ cells may reflect short-lived actions of actin-interacting proteins during the germ cell's development and epididymal transit. Individual actions may appear conflicting among themselves, but taken together they show a logical progression of related events designed to meet specific physiological requirements.

It is to try to better understand the role of actin during germ cell development in the testis and maturation in the testicular excretory ducts that we elected to study the changes in the concentration and distribution of the actin filament-severing protein scinderin in the developing bovine testis and in the spermatozoa during and following their epididymal transit.

Scinderin has been reported typically in tissues demonstrating a high secretory activity including adrenal glands, pituitary, brain, testis, kidney, and salivary glands [26, 27]. In chromaffin cells of the adrenal glands, scinderin shows a cytoplasmic distribution as well as a subsurface localization that coincided with cortical actin [28]. Scinderin severs F-actin in a Ca2+-dependent manner and possesses two Ca2+ binding sites [26]. Furthermore, scinderin binds to phosphatidylinositol 4,5-bisphosphate (PIP2), suggesting the protein can interact with the plasma membrane via binding to this particular membranous phospholipid [29].

No studies have yet assessed scinderin expression and localization during testicular development or during the epididymal transit of spermatozoa, two physiological conditions during which modifications of the actin cytoskeleton take place. The present study uses the bull as an animal model and reports the changes in the protein level and in the localization of scinderin in Sertoli cells, germ cells, and epididymal cells during testicular development and following epididymal transit of spermatozoa.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

The bovine testes and epididymides used in this study were obtained immediately after death from the slaughterhouse Les Abattoirs Les Cèdres (Sainte-Anne de Bellevue, PQ, Canada). We used 10 testes from normal 8- to 9-mo-old fetuses and 10 testes from 2-yr-old normal adults. The fetuses were obtained from gestating cows immediately after death. Adrenal glands and part of the ascending aorta and of the superior vena cava were also dissected.

Isolation of Seminiferous Tubules and OtherTissue Preparations

Testes obtained immediately after death were decapsulated, and small pieces of tissue were placed in cold Minimum Essential Medium (MEM) (Gibco BRL, Oakville, ON, Canada). Testicular tissue pieces were incubated 2 x 45 min with 0.25 mg/ml collagenase D (Boehringer-Mannheim, Laval, PQ, Canada) [30] and 0.1 mg/ml soybean trypsin inhibitor (Sigma, St. Louis, MO) at 37°C in a water shaker bath set at 80 cycles/min. The reaction was stopped by addition of an equal volume of MEM. The seminiferous tubules were allowed to settle by gravity while interstitial cells occupied the supernatant. Further separation was achieved by centrifugation at low speed (400 rpm, GS-6R Beckman centrifuge; Beckman, Mississauga, ON, Canada) for 15 min. The two components of the testis bear distinct colors, thus making them easy to identify: the tubules are whitish and the interstitial tissue brownish. The tubules and the interstitial cells were quickly washed in PBS (137 mM NaCl, 3 mM KCl, 8 mM Na2PO4, 1.5 mM KH2PO4, pH 7.4) and homogenized with a glass tissue grinder in PBS containing 1 mM of PMSF (Sigma). Bovine chromaffin cells were isolated as previously described [31]. Tissue fragments from the aorta and the vena cava were homogenized in PBS containing 1 mM PMSF with a Polytron tissue homogenizer (Brinkmann Co., Westbury, NY).

Epididymal Spermatozoa

The epididymal spermatozoa were flushed from the cauda of bovine epididymides with a perfusion of cold PBS through the deferent duct. Spermatozoa were washed twice in PBS, recovered by centrifugation (600–700 rpm, 4 min Beckman gs-6R centrifuge; Beckman), and resuspended in fresh PBS. Typically, most spermatozoa were motile. For immunolabeling they were diluted 1:5 in PBS. For immunoblot analyses, spermatozoa were diluted 1:1 in cold PBS containing 1 mM PMSF and sonicated while on ice using a VWR Sonifier II Cell Disrupter Branson Ultrasonics at maximal setting during three consecutive intervals of 30 sec each.

Ejaculated Spermatozoa

Thanks to the kind help of Dr. Yves Brindle, freshly ejaculated bovine spermatozoa were obtained from the Centre d'Insémination Artificielle du Québec (CIAQ; Sainte Madeleine, PQ, Canada). A total of 10 ejaculates were collected from fertile bulls by means of an artificial vagina. The ejaculates were not pooled; each ejaculate was assessed individually for volume, appearance, and motility. Samples showing less than 70% motile cells were discarded. Samples were diluted 1:1 in a modified [32] Tyrode's medium (TALP) described by Bavister and Yanagimachi [33] (100 mM NaCl, 3.1 mM KCl, 25 mM NaHCO3, 0.29 mM KH2PO4, 21.6 mM lactic acid, 11.5 mM MgCl2, 1 mM pyruvate, 10 mM HEPES, pH 7.4). They were washed twice in TALP medium. Cells were recovered by centrifugation (600–700 rpm, 4 min Beckman gs-6R centrifuge). After the second centrifugation, the spermatozoa were diluted 1:10 in fresh TALP medium and sonicated as described above for the epididymal spermatozoa.

Antibodies against Scinderin

Rabbit polyclonal anti-scinderin #6 was raised against bovine scinderin and has been previously characterized [26–28, 34].

Electrophoresis and Immunoblotting

Twenty-five micrograms of proteins of each tissue and cell homogenate were loaded in a 10% polyacrylamide minigel. Proteins were subjected to electrophoresis and electrotransferred onto nitrocellulose membranes as previously described [35]. Membranes were blocked with 5% skim milk in TBS (140 mM NaCl, 50 mM Tris HCl, pH 7.4) and incubated with a scinderin antiserum #6 (1/300 dilution in 5% skim milk-TBS) for 2 h at 37°C. After extensive washing, membranes were incubated with alkaline phosphatase-conjugated anti-rabbit IgG (1/3000 dilution, in 5% skim milk in TBS) for 1 h at room temperature (RT). The reaction was developed by treatment with a mixture of p-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate toluidine salt.

Preparation of Tissues for Immunolocalization of Scinderin

Testes obtained immediately after death were fixed by perfusion through the testicular artery of 15 ml of PBS pH 7.4, to flush the blood out, followed by 60 ml of Bouin's fixative. Washing the tissue with PBS was indispensable because the acetic acid contained in the Bouin's fixative caused the blood to clot on contact, therefore blocking the vasculature of the testis and preventing the aldehyde mixture to reach the tissue. Perfusion-fixed testicular tissues were further immersed in the same fixative mixture for an additional 36–48 h at RT [7, 36]. For the adrenal glands, the aorta, and the vena cava, tissue fragments were immersion-fixed in Bouin's solution. Tissues were dehydrated in ethanol and cleared in xylene before paraffinization. Five-micrometer-thick sections were mounted on glass slides coated with 3-aminopropyltriethoxysilane (Sigma), deparaffinized, and rehydrated in xylene and ethanol. To inhibit potential endogenous peroxidase activity, tissue sections were exposed to 0.6% hydrogen peroxide (H2O2) in 70% ethanol for 5 min. They were then washed for 5 min in TBS containing 0.1% Tween-20 (TBST). Inactivation of residual picric acid was achieved using a solution of 1% lithium carbonate in 70% ethanol, and free aldehydes were blocked with a 300 mM glycine aqueous solution (pH 7.4) [37]. Spermatozoa were spotted on coated slides, air-dried, and either fixed for 5 min with 3.7% formaldehyde, washed and exposed 5 min to cold (-20°C) acetone, or treated with cold (-20°C) methanol for 5 min followed by cold (-20°C) acetone for 2 min. An exposure of the spermatozoa to methanol followed by acetone gave the best and most consistent results.

Immunolabeling

The sections were first incubated for 30 min at 37°C with 0.5% skim milk in TBST to block the unspecific binding and then incubated overnight at RT with (1:400) scinderin antiserum #6 and then for 40 min with (1:1000) biotinylated anti-rabbit IgG (Amersham Bio/Can Scientific, Mississauga, ON, Canada) followed by (1:200) horseradish-peroxidase (HRP)-conjugated streptavidin (Amersham) [7]. They were washed in TBST and incubated for 10 min at RT in 0.01% H2O2, 0.05% diaminobenzidine tetrachloride (DAB), and 10 mM imidazole [38] in TBS (pH 7.7). The sections were counterstained with methylene blue dye and mounted with Permount (Fisher Scientific Co., Pittsburgh, PA). The recordings of the stage-dependent distribution of scinderin in the adult bovine testis were made using the identification method of the twelve stages of the cycle of the seminiferous epithelium proposed by Berndtson and Desjardins [39].

Fluorescence Microscopy

Spermatozoa were spotted on poly-L-lysine-coated glass coverslips and air-dried. Spermatozoa were fixed-permeabilized by incubating the coverslips in (-20°C) methanol for 10 min. Preparations were washed with PBS and incubated with 3% skim milk in PBS for 60 min at RT to block nonspecific binding. Next, cells were incubated with scinderin antiserum #6 (1/80 dilution in 1% milk in PBS) for 60 min at 37°C. After extensive washing with PBS, cells were incubated for 60 min at 37°C with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG antibody (1/400 dilution in 1% milk in PBS; Sigma) or with biotinylated anti-rabbit IgG (1:1000 dilution; Amersham) followed by streptavidin-Cy3 conjugate (1:400 dilution; Sigma). After being rinsed with PBS, preparations were mounted in PBS:glycerol (1:1) containing 5% 1,4-diazabicyclo[2.2.2]octane (DABCO; Sigma).

Controls

The specificity of scinderin was tested in the bovine adrenals used as a positive control. Moreover, we carried out immunolabeling of bovine testis using another antiserum as an additional positive control, antiserum #7, which is a polyclonal antibody raised against native scinderin and which did not cross-react with gelsolin. For negative controls, we used the primary or the secondary antibody alone. Preimmune serum was also used. In addition, to ascertain further the specificity of scinderin immunolabeling in testicular tissue sections, we performed immunolabeling of paraffin sections of bovine testes with scinderin antiserum #6 preadsorbed with bovine adrenal medulla supernatant.

Protein Measurement

The protein content of the samples was measured by the Bradford dye binding assay (Bio Rad, ON, Canada).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunoblot Analysis

Immunoblot analysis done on bovine chromaffin cells using anti-scinderin showed one band at 80 kDa (Fig. 1). Immunoblots of bovine seminiferous tubules, interstitial tissue, spermatozoa, and blood vessels also showed a unique band bearing the same molecular mass as chromaffin cell scinderin (Fig. 1). The intensity of the band for immunoreactive scinderin was lower in adult than in fetal seminiferous tubules (Fig. 1, lanes aT, fT), but it was similar in fetal interstitial cells and in adult testis (Fig. 1, lanes ait, fit). Scinderin levels were higher in epididymal (Fig. 1, lane pSpz) than in ejaculated spermatozoa (Fig. 1, lane jSpz). Scinderin expression was higher in the vena cava (line aV) than in the aorta (line aA).



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  		FIG. 1. Immunoblot analysis of several bovine tissues with scinderin antiserum. Twenty-five micrograms of proteins of chromaffin cells (CC), seminiferous tubules isolated from adult testes (aT), interstitial tissue cells isolated from adult testes (ait), epididymal spermatozoa (pSpz), ejaculated spermatozoa (jSpz), seminiferous tubules isolated from fetal testes (fT), interstitial tissue cells isolated from fetal testes (fit), aorta from adult bovine (aA), and vena cava from adult cattle (aV) were loaded in a 10% polyacrylamide minigel. Proteins were subjected to electrophoresis and electrotransferred onto nitrocellulose membranes. Membranes were incubated with scinderin antiserum #6 and then with alkaline phosphatase-conjugated secondary antibody. Reactive bands were revealed by treatment with p-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate toluidine salt. The left lane shows the position of molecular weight standards (kaleidoscope; Bio-Rad). The presence of scinderin (SC) is indicated by an arrowhead. The figure is a representative immunoblot of three different experiments using samples from different bulls.

Immunolocalization in the Testis

Positive controls using adrenal glands from adult bulls revealed the presence of scinderin-positive cells in the medulla (not shown). Negative controls using either primary or secondary antibody alone or preimmune serum (Fig. 2, a and b) in fetal (Fig. 2a) and adult (Fig. 2b) testes or preadsorbed scinderin antiserum #6 (not shown) showed no immunoreactivity.



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  		FIG. 2. Immunolocalization of scinderin in the testis. Controls using preimmune serum showed no reaction product in a) fetal and b) adult testes. In fetal testes, scinderin was localized (arrows) in minute dots aligned along the Sertoli cells' plasma membrane regardless of whether the membrane was facing a gonocyte or a neighboring Sertoli cell (c). d–h) Material obtained from adult testes; the roman numerals identify the stage of the cycle of the seminiferous epithelium. In germ cells, scinderin (closed arrows) was associated with the acrosome of the round developing (d–g) as well as of the elongated mature spermatids (arrowheads) (d). d) Scinderin immunoreactivity in a region corresponding with the subacrosomal space in the developing round spermatids. In addition, during all the stages of the seminiferous cycle, scinderin was localized in the Sertoli cells next to the cell surface of the trunk of the cells and of their cytoplasmic processes which surrounds the germ cells (open arrows d, g, h). Within the cytoplasm of Sertoli cells, deposits of scinderin-positive material accumulated near the base of the cell in a stage-specific manner (open arrows near the limiting membrane of the tubule g, h). x780.

In fetal testes, using antiserum #6, scinderin was localized in arrays of minute stained spots aligned along the subsurface of the Sertoli cells' plasma membranes regardless of whether the membranes were facing adjacent Sertoli cells or gonocytes (Fig. 2c). In the adult, scinderin was detected in both germ cells and Sertoli cells (Fig. 2, d–h). In germ cells, the protein was located in a region corresponding with the subacrosomal space in the developing round spermatids (Fig. 2d) and with the remodeling acrosome in the elongated spermatids (Fig. 2, d–g). In Sertoli cells, immunoreactivity was found all along the subsurface of the cells (Fig. 2, d, g, and h). Within the cytoplasm, deposits of scinderin-positive material were more frequent near the base of the Sertoli cells in stages of the cycle of the seminiferous epithelium that preceded the release of elongated spermatids and/or the tight junction disassembly accompanying the translocation of the spermatocytes into the luminal compartment of the seminiferous epithelium (compare Fig. 2g with Fig. 2h). The distribution of scinderin revealed with scinderin antiserum #7 labeling was identical to that we recorded using scinderin antiserum #6 (Figs. 2–4).



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  		FIG. 3. Immunolocalization of scinderin in the fetal epididymides (head [a], body [b], tail [c]) and adult (head [d], body [e], tail [f]). A lumen is clearly evident in epididymides obtained during the 8th month of gestation. During this period (a–c), scinderin was localized near the lateral cell surface of the principal cells near the base (closed arrows) and close to the apical border near the intercellular junctions (open arrows). In tangential sections of adult epididymis (d–f), scinderin immunoreactivity was detected all along the subsurface of the principal cells' lateral plasma membranes (open arrows in d). In the body and particularly in the tail of the epididymis, deposits of scinderin-positive material were detected (closed arrows in e, f) near the basal cells. The curved solid arrows in d–f point to scinderin-positive spermatozoa. x780.



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  		FIG. 4. Immunolocalization of scinderin in epididymal (a, b) and ejaculated (c, d) spermatozoa. Controls using preimmune serum show no reaction product in the head of epididymal spermatozoa; however, staining was detected (open arrowheads in a) in portions of the tails. Immunofluorescence localization of scinderin in epididymal spermatozoa (b) shows small spots of immunoreactivity in the postacrosomal region (arrowhead); the protein is localized principally in the anterior acrosome and in the equatorial segment (arrow). Part of the connecting piece situated caudally to the postacrosomal region is also scinderin-positive. In ejaculated spermatozoa (c), the acrosome (curved arrow) and the postacrosomal region were heavily labeled when immunofluorescence labeling (c) or streptavidin-HRP was used (d). x780 (reproduced at 82%).

Immunolocalization in the Epididymis

Negative controls showed no immunoreactivity in either fetal or adult epididymides (not shown). In the fetal epididymis (Fig. 3, a–c), scinderin was located along the subsurface of the principal cells, chiefly near the cellular contacts. Labeling was particularly heavy in the body (corpus) of the epididymis (Fig. 3b). In the adult epididymis (Fig. 3, d–f), scinderin immunoreactivity was observed in the subsurface region of the principal cells (Fig. 3d) in all three regions (head [caput], body [corpus], and tail [cauda]) of the epididymis. Deposits of scinderin-positive materials were observed near the base of the principal cells (Fig. 3, e and f). The deposits were most frequent and largest in the tail of the epididymis (Fig. 3f). Spermatozoa were positively labeled in all three regions of the epididymis (Fig. 3, d–f).

Immunolocalization in Spermatozoa

Negative controls in epididymal (Fig. 4a) and ejaculated (not shown) spermatozoa showed no immunoreactivity in the head; however, part of the tail, principally the middle piece, appeared slightly stained. In epididymal spermatozoa (Fig. 4b), scinderin labeling was heavy in the anterior acrosome and in the equatorial segment, but it was light in the postacrosomal region. Labeling was also found in part of the connecting piece situated caudally to the postacrosomal region (Fig. 4b). Furthermore, labeling occurred in the middle piece of the tail; however, because controls also showed staining in this part of the tail, some degree of false positive labeling cannot be ruled out. In ejaculated spermatozoa (Fig. 4, c and d), the acrosome and the postacrosomal region were heavily labeled, but the equatorial segment appeared negative. The results obtained were the same whether labeling was achieved using immunofluorescence (Fig. 4c) or streptavidin-HRP conjugate (Fig. 4d). Labeling was also noted in part of the tail with both techniques.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study reports the presence of scinderin in fetal and adult testes, epididymides, spermatozoa, the aorta, and the vena cava. The antiserum #6 we used to generate these data has been characterized in numerous published reports [26–28, 34]. These reports show that the scinderin antiserum we used in the present study recognizes only a protein with a molecular mass of 80 kDa and that the antibody does not cross-react with gelsolin, another actin filament-severing protein of a 90-kDa molecular mass. Thus, it is highly unlikely that the weak band we observed at 90 kDa in some of the testis samples we analyzed would correspond to gelsolin. Moreover, gelsolin has been documented in chromaffin cells [40] and in spermatozoa [20]. However, under our experimental conditions, there was no band at 90 kDa in chromaffin cells and spermatozoa. Therefore, on the basis of these data, we believe that the weaker band at 90 kDa is not gelsolin but probably reflects a testicular protein that may have some cross-reactivity with scinderin antiserum #6. However, we observed no immunolabeling in paraffin sections of bovine testes treated with preadsorbed scinderin antiserum #6. Moreover, when immunolabeling was carried out using scinderin antiserum #7, which was raised against native scinderin and did not cross-react with gelsolin, the scinderin distribution was identical to the one revealed by scinderin antiserum #6 labeling. For all those reasons, we believe that our immunolabeling accurately reflects scinderin presence and distribution in bovine tissues.

Scinderin may not be related exclusively to spermatogenic function. The finding of higher amounts of scinderin in fetal seminiferous tubules and of a cytoplasmic scinderin distribution that changed in a stage-specific manner in adult Sertoli cells suggests that spermatogenesis influences the quantity and localization of the protein in the testis. Moreover, the fluctuation of scinderin levels in spermatozoa following their transit through the epididymis suggests that the protein may participate in the maturation process by ensuring the appearance of sequential changes in the form/state of actin, which may be essential in the fertilizing capacity to the male gametes.

The nucleotide and amino acid sequence analysis revealed that scinderin possesses six domains, each containing three internal sequence motifs and two actin and two PIP2 binding sites, and showed 63% and 53% homology with gelsolin and villin, both being Ca2+-dependent F-actin-severing proteins [41]. Gelsolin, a 90-kDa actin filament-capping and -severing protein that reportedly binds to the barbed ends of actin filament to prevent their growth and to sever the filaments [42–46], under Ca2+ and phosphoinositide control [47], was immunolocalized in capacitated spermatozoa under specific experimental conditions [20]. The present work is the first to document the variations in the concentration and the localization of the Ca2+-dependent actin filament-severing protein scinderin or adseverin, which reportedly shares a similar sequence [48], in testicular and epididymal cells during development, and in spermatozoa during and after their epididymal transit. It is also the first successful attempt to localize the protein in tissue sections.

Scinderin in the Testis

In the germ cells Scinderin immunoreactivity is associated principally with a region corresponding with the subacrosomal space in the round spermatids and with the remodeling acrosome in the elongated spermatids. Because scinderin is a Ca2+-dependent actin filament-severing protein [29, 49], these observations should be viewed in relation to other reports of actin in developing spermatids [5, 8–12, 14] and spermatozoa [15–20, 50].

It is generally believed that within the cell, actin exists in monomeric (G-actin) as well as in filamentous (F-actin) states or forms. Under conditions of intracellular ionic strength, all the cellular actin would be expected to be polymerized; the fact that this has been reported in neither Sertoli cells [1–3, 5–7, 21, 22] nor germ cells [5, 9–12, 14, 51] implies the presence in both cell types of factors that modify the state of actin during the germ cell's development. Actin [8], particularly F-actin [5, 9–12, 14, 51], has been identified within the subacrosomal space of developing spermatids. Collectively, these authors emphasize that F-actin decreases with the completion of the spermatid's development until it becomes virtually undetectable by the time mature spermatids are released from the seminiferous tubules. This suggests that the older the spermatid, the more abundant the G-actin and the less abundant the F-actin. Assuming that the principal role of cortical F-actin is to act as a physical barrier [52, 53] that could rigidify the structure that it accompanies, namely, cell membranes [54], the predominance of one form of actin over the other within the subacrosomal space could influence the shaping of the acrosome to meet specific physiological and structural requirements.

Not surprisingly, scinderin is found in germ cells in which actin has been reported, that is, within a region that corresponds to the subacrosomal space of developing spermatids [5, 8–12, 14]. In vitro, scinderin has been reported to bind to both G-actin and F-actin [26]. Under basal conditions, when intracellular Ca2+ ([Ca2+]i) is low, 90–95% of scinderin is cytoplasmic, while 5–10% is associated with membrane phospholipids, but when [Ca2+]i is high, most of the scinderin becomes associated with actin and the phospholipids [29, 49]. Therefore, one could anticipate that the changes in scinderin distribution reported here in the spermatids and in the spermatozoa during and following their epididymal transit might have been dictated by [Ca2+]i-induced changes in the form of actin.

In Sertoli cells In the testis, Sertoli cells share a morphological feature found in cells of most epithelia: they possess a layer of subsurface or cortical actin that occupies the peripheral cytoplasm and surrounds the perimeter of the cell. In the Sertoli cells, this cortical or peripheral actin is typically sandwiched between cisternae of endoplasmic reticulum (ER) on the intracellular side and the plasma membrane on the extracellular side [1, 21, 55]. Within the Sertoli cell, the monomeric form of actin has been reported in the base, the middle, and the apex [7], while the filamentous form has been shown in the base and the apex of the cell [1–3, 6, 21, 22]. Thus, G-actin was localized in the same sites as F-actin, but, in addition, the monomeric actin was found in sites where F-actin was not detectable. The finding of scinderin in the same sites as cortical G- and F-actin may reflect the role this actin regulatory protein plays on the perijunctional actin filament network that accompanies and possibly influences the function of the Sertoli cell junctions. The use of cytochalasin D and/or of ZO-toxin of cholera, two actin-depolymerizing agents, induces a disruption of perijunctional actin filaments followed by a breakage of the paracellular barrier, suggesting a functional link between actin organization and the tight junction [4, 56]. By analogy with the muscle, the subsurface cisternae of ER that accompany Sertoli cell junctions near the base (joining adjacent Sertoli cells) and the apex (joining Sertoli cells and spermatids) of the epithelium are probably involved in the regulation of local Ca2+ ion concentration in selected regions of the Sertoli cells. The sequestration of [Ca2+]i in the cisternae of ER accompanying the cortical or peripheral actin in the Sertoli cells could induce modifications in affinity of the Ca2+-dependent actin-interacting protein scinderin for either the cytosol, the phospholipids of the membrane, or actin; these modifications, in turn, could prove significant in the physiology of the Sertoli cell junctions that are responsible for the maintenance of the blood-testis barrier [57] or for the release of mature spermatids. In Madin-Darby canine kidney (MDCK) confluent cell cultures, tight junctions failed to develop when extracellular Ca2+ levels were low [58–61]. Elevation of [Ca2+]i concentration during cell injury has been reported to close gap junctions [62, 63], suggesting that both tight and gap junctions are responsive to [Ca2+]i levels. Ca2+-containing precipitates were localized in the cisternae of ER associated with the Sertoli cell junctions at the electron microscope level by ion-capture cytochemistry using combined oxalate and pyroantimonate methods [64]. In smooth- and non-muscle tissues, the Ca2+ storage compartment is believed to be associated with the ER. The appearance of scinderin-positive deposits in the cytoplasm of Sertoli cells during stages of the cycle that precede the release of mature spermatids may reflect transient changes in the availability of [Ca2+]i stored in the cisternae of ER.

Scinderin in Epididymal Cells

Scinderin immunoreactivity in epididymal cells also coincides with the presence of the cortical perijunctional actin filament network reported along the principal cells' lateral plasma membranes [65, 66]. Whether scinderin accompanies the cell junctions in the epididymal cells or in the Sertoli cells, the protein may conceivably have similar effects on cellular contacts.

Scinderin in Spermatozoa

Our immunoblot analyses revealed a decrease in the expression of scinderin in the ejaculated compared to the epididymal spermatozoa. In addition, the protein was located principally in the anterior acrosome and in the equatorial segment in the epididymal spermatozoa, but in the ejaculated spermatozoa, scinderin was no longer detectable in the equatorial segment; it was detected chiefly in the acrosome and in the postacrosomal region. In the head of epididymal spermatozoa, most of the actin detected was nonfilamentous or G-actin [9, 67] except where membrane specialization occurs [68]. F-actin, which was apparently not observed in noncapacitated spermatozoa, was reported to develop in most regions of the gamete during capacitation [69]. The changes reported here in the distribution of the Ca2+-dependent actin filament-severing protein scinderin in the spermatozoa during and following epididymal transit may represent a different response of the gametes to local conditions that altered the [Ca2+]i. Scinderin is a calcium-dependent actin filament-severing protein that has been shown to have a major role in the regulation of exocytosis of secretory vesicles during neurotransmitter release by controlling cortical actin disassembly [26]. Perhaps during their transit through the excretory ducts of the testis, the spermatozoa need to modify the state/form of their actin so as to allow emergence of the acrosome reaction, which has been called "a sperm exocytosis" [70].

The finding of scinderin in the neck and in the tail of the epididymal and ejaculated spermatozoa is somewhat more difficult to justify, perhaps because the participation of actin in the movements of the flagellum is ambiguous. Nevertheless, scinderin immunoreactivity in the epididymal and ejaculated spermatozoa (Fig. 4, b–d) was substantially stronger than the immunofluorescence staining detected in the control (Fig. 4a), suggesting that the protein does exist also in the tail. The presence of actin has been reported in the tail of the spermatozoa in virtually all species studied thus far [14, 20, 23–25, 71–75].

In summary, this study analyses the changes in the concentration and localization of the actin filament-severing protein scinderin, during testicular development, and in spermatozoa during and following the epididymal transit. Immunoblot analyses showed one band at 80 kDa in chromaffin cells, fetal and adult tubules, interstitial cells, aorta and vena cava, and spermatozoa. Scinderin immunoreactivity was associated with a region corresponding with the subacrosomal space and with the acrosome in the spermatids. In epididymal spermatozoa, scinderin was located in the anterior acrosome and in the equatorial segment, but in ejaculated spermatozoa, the protein appeared in the acrosome and the post-equatorial segment of the head. In Sertoli cells, the protein was detected near the cell surface during all the stages of the cycle and within the cytoplasm, where it accumulated near the base in a stage-specific manner. In the epididymis, scinderin was also localized next to the surface of the cells; in the tail (cauda), it collected near the base of the principal cells. The results suggest that scinderin may be involved in actin remodeling in Sertoli cells and epididymal cells, where it may play a role in the control of the state of perijunctional actin and contribute to the regulation of tight junctional permeability, and in germ cells, where it may assist in shaping the developing acrosome and influence the fertility of the spermatozoa.


   FOOTNOTES
 
1 This work was supported in part by Population Council Award B97.071P–9/ICMC to R.M.P.; CORPAQ grant to R.M.P. and M.L.V.; MRC Grant MT-12879 to M.L.V.; MRC grant MT-11169 to J.M.T. M.L.V. is also funded by a scholarship from Fonds de la Recherche en santé du Québec. Back

2 Correspondence: R.-Marc Pelletier, Université de Montréal, Faculty of Medicine, Department of Pathology and Cell Biology, Pavillon Principal, 2900 Edouard-Montpetit blvd. Montréal, P. Québec, Canada H3T 1J4. FAX: 514 485 7932; pellemar{at}ere.umontreal.ca Back

Accepted: December 8, 1998.

Received: August 6, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Dym M, Fawcett DW. The blood-testis barrier in the rat and the physiological compartmentation of the seminiferous epithelium. Biol Reprod 1970; 3:308–326.[Abstract]
  2. Suárez-Quian CA, Dym M. Further observations on the microfilaments bundles of Sertoli cell junctional complexes. Ann N Y Acad Sci 1984; 438:476–480.[Medline]
  3. Vogl AW, Soucy LJ. Arrangement and possible function of actin filament bundles in ectoplasmic specializations of ground squirrel Sertoli cells. J Cell Biol 1985; 100:814–825.[Abstract/Free Full Text]
  4. Madara JL, Barenberg D, Carlson S. Effects of cytochalasin D on occluding junctions of intestinal absorptive cells: further evidence that the cytoskeleton may influence paracellular permeability and junctional charge selectivity. J Cell Biol 1986; 102:2125–2136.[Abstract/Free Full Text]
  5. Vogl AW, Grove BD, Lew GJ. Distribution of actin in Sertoli cell ectoplasmic specializations and associated spermatids in ground squirrel testis. Anat Rec 1986; 215:331–341.[CrossRef][Medline]
  6. 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]
  7. Pelletier R-M, Okawara Y, Vitale ML, Anderson JM. Differential distribution of the tight junction associated protein ZO-1 {alpha} (+) and a (-) isoforms in guinea pig Sertoli cells: a possible association with F-actin and G-actin. Biol Reprod 1997; 57:367–376.[Abstract]
  8. Campanella C, Cabbiani G, Bacetti B, Burrini AG, Pallini V. Avtin and myosin in the vertebrate acrosomal region. J Submicroscop Cytol 1979; 11:53–71.
  9. Welch JE, O'Rand MG. Identification and distribution of actin in spermatogenic cells and spermatozoa of the rabbit. Dev Biol 1985; 109:411–417.[CrossRef][Medline]
  10. Russell LD, Weber JE, Vogl AW. Characterization of filaments within the subacrosomal space of rat spermatids during spermiogenesis. Tissue Cell 1986; 18:887–898.[CrossRef][Medline]
  11. Halenda RM, Primakoff P, Myles DG. Actin filaments, localized to the region of the developing acrosome during early stages, are lost during later stages of guinea pig spermiogenesis. Biol Reprod 1987; 36:491–499.[Abstract]
  12. Fouquet JP, Kann M-L, Dadoune JP. Immunogold distribution of actin during spermiogenesis in the rat, hamster, monkey and human. Anat Rec 1989; 223:35–42.[CrossRef][Medline]
  13. Fouquet JP, Kann ML, Dadoune JP. Immunoelectron microscopic distribution of actin in hamster spermatids and epididymal, capacitated and acrosome-reacted spermatozoa. Tissue Cell 1990; 22:291–300.[CrossRef][Medline]
  14. Fouquet JP, Kann ML. Species-specific localization of actin in mammalian spermatozoa: fact or artefact? Microscop Res Tech 1992; 20:251–258.
  15. Saxena N, Peterson RN, Saxena NK, Russell LD. Microfilaments appear in boar spermatozoa during capacitation in vitro. J Exp Zool 1986; 239:423–427.[CrossRef][Medline]
  16. Castellani-Ceresa L, Brivio MF, Radaelli G. Electron microscopic localization of F-actin in acrosome-reacted boar spermatozoa by means of a phalloidin-FITC complex. J Submicroscop Cytol Pathol 1991; 23:347–349.[Medline]
  17. Castellani-Ceresa L, Brivio MF, Radaelli G. F-Actin in acrosome-reacted boar spermatozoa. Mol Reprod Dev 1992; 33:99–107.[CrossRef][Medline]
  18. Moreno-Fierros L, Hernandez EO, Salgado ZO. F-Actin in guinea pig spermatozoa: its role in calmodulin translocation during acrosome reaction. Mol Reprod Dev 1992; 33:172–181.[CrossRef][Medline]
  19. Vogl AW, Genereux KD, Pfeiffer C. Filamentous actin detected in rat spermatozoa. Tissue Cell 1993; 25:33–48.[CrossRef][Medline]
  20. de las Heras MA, Valcarcel A, Pérez LJ, Moses DF. Actin localization in ram spermatozoa: effect of freezing/thawing, capacitation and calcium ionophore induced acrosomal exocytosis. Tissue Cell 1997; 29:47–53.[CrossRef][Medline]
  21. Pelletier R-M, Friend DS. The Sertoli cell junctional complex: structure and permeability to filipin in the neonatal and adult guinea pig. Am J Anat 1983; 168:213–228.[CrossRef][Medline]
  22. Pelletier R-M. Cyclic modulation of the Sertoli cell junctional complexes in a seasonal breeder: the mink (Mustela vison). Am J Anat 1988; 183:68–102.[CrossRef][Medline]
  23. Lora-Lamia C, Castellani-Ceresa L, Andreetta F, Cotelli F, Brivio MF. Localization and distribution of actin in mammalian sperm heads. J Ultrastruct Mol Struct Res 1986; 96:12–21.[CrossRef][Medline]
  24. Flaherty SP, Winfrey VP, Olson GE. Localization of actin in human, bull, rabbit, and hamster sperm by immunoelectron microscopy. Anat Rec 1988; 221:599–610.[CrossRef][Medline]
  25. Vogl AW. Distribution and function of organized concentrations of actin filaments in mammalian spermatogenic cells and Sertoli cells. Int Rev Cytol 1989; 119:1–56.[Medline]
  26. Rodriguez Del Castillo A, Lemaire S, Tchakarov L. Chromaffin cell scinderin, a novel calcium-dependent actin filament-severing protein. EMBO J 1990; 9:43–52.[Medline]
  27. Tchakarov L, Vitale ML, Jeyapragasan M, Rodriguez Del Castillo A, Trifaró J-M. Expression of scinderin, an actin-severing protein, in different tissues. FEBS Lett 1990; 268:209–212.[CrossRef][Medline]
  28. Vitale ML, Rodriguez Del Castillo A, Tchakarov L, Trifaró J-M. Cortical filamentous actin disassembly and scinderin redistribution during chromaffin cell stimulation precedes exocytosis. A phenomenon not exhibited by gelsolin. J Cell Biol 1991; 113:1057–1067.[Abstract/Free Full Text]
  29. Rodriguez Del Castillo A, Vitale ML, Trifaró J-M. Ca2+ and pH determine the interaction of chromaffin cell scinderin with phosphatidylserine (PS) and phosphatidylinositol 4,5 diphosphate (PIP2) and its cellular distribution during nicotinic stimulation and protein kinase C activation. J Cell Biol 1992; 119:797–810.[Abstract/Free Full Text]
  30. Joshi MS, Anakwe OO, Gerton GL. Preparation and short-term culture of enriched populations of guinea pig spermatocytes and spermatids. J Androl 1990; 11:120–130.[Abstract/Free Full Text]
  31. Trifaró J-M, Lee RWH. Morphological characteristics and stimulation-secretion coupling in bovine adrenal chromaffin cells. Neuroscience 1980; 5:1533–1546.[CrossRef][Medline]
  32. Parrish JJ, Susko-Parrish J, Winer MA, First NL. Capacitation of bovine sperm by heparin. Biol Reprod 1988; 38:1171–1180.[Abstract]
  33. Bavister BD, Yanagimachi R. The effects of sperm extract and energy sources on the motility and acrosome reaction of hamster sperm in vitro. Biol Reprod 1977; 16:228–237.[Abstract]
  34. Rodriguez Del Castillo A, Vitale ML, Tchakarov L, Trifaró J-M. Human platelets contain scinderin, a Ca2+-dependent actin-filament severing protein. Thromb Haemostasis 1992; 67:248–251.[Medline]
  35. Doucet JP, Trifaró J-M. A discontinuous and highly porous sodium dodecyl sulfate-polyacrylamide slab gel system of high resolution. Anal Biochem 1988; 168:265–271.[CrossRef][Medline]
  36. Pelletier R-M. The distribution of connexin 43 is associated with the germ cell differentiation and with the modulation of the Sertoli cell junctional barrier in continual (guinea pig) and seasonal breeder's (mink) testes. J Androl 1995; 16:400–409.[Abstract/Free Full Text]
  37. Oko R, Clermont Y. Light microscopic immunocytochemical study of fibrous sheath and outer dense fiber formation in the rat spermatid. Anat Rec 1989; 225:46–55.[CrossRef][Medline]
  38. Straus W. Imidazole increases the sensitivity of the cytochemical reaction for peroxidase with diaminobenzidine at neutral pH. J Histochem Cytochem 1982; 30:491–493.[Medline]
  39. Berndtson WE, Desjardins C. The cycle of the seminiferous epithelium and spermatogenesis in the bovine testis. Am J Anat 1974; 140:167–178.[CrossRef][Medline]
  40. Bader MF, Trifaró J-M, Langley OK, Thiersé D, Aunis D. Secretory cell actin-binding proteins: identification of a gelsolin-like protein in chromaffin cells. J Cell Biol 1986; 102:636–646.[Abstract/Free Full Text]
  41. Marcu GM, Rodriguez Del Castillo A, Vitale ML, Trifaró J-M. Molecular cloning and functional expression of chromaffin cell scinderin indicates that it belongs to the family of Ca2+-dependent F-actin severing proteins. Mol Cell Biochem 1994; 141:153–165.[CrossRef][Medline]
  42. Yin HL, Zaner KS, Stossel TP. Calcium control of actin gelation. Interaction of gelsolin with actin filaments and regulation of actin gelation. J Biol Chem 1980; 255:9494–9500.[Abstract/Free Full Text]
  43. Yin HL, Albrecht JH, Fattoum A. Identification of gelsolin, a calcium dependent regulatory protein of actin gel-sol transformation, and its intracellular distribution in a variety of cells and tissues. J Cell Biol 1981; 91:901–906.[Abstract/Free Full Text]
  44. Yin HL, Hartwig JH, Maruyama K, Stossel TP. Calcium control of actin filament. Effects of macrophage gelsolin on actin polymerization. J Biol Chem 1981; 256:9693–9697.[Abstract/Free Full Text]
  45. Wang LL, Bryan J. Isolation of calcium-dependent platelet proteins that interact with actin. Cell 1981; 25:637–649.[CrossRef][Medline]
  46. Muallem S, Kwiatkowska K, Xu X, Yin HL. Actin filament disassembly is a sufficient final trigger for exocytosis in nonexcitable cells. J Cell Biol 1995; 124:589–598.[Abstract/Free Full Text]
  47. Yin HL. Gelsolin: calcium- and polyphosphoinositide-regulated actin-modulating protein. Bioessays 1988; 7:176–179.
  48. Nakamura S, Sakurai T, Nonomura Y. Differential expression of bovine adseverin in adrenal gland revealed by in situ hybridization. Cloning of a cDNA for adseverin. J Biol Chem 1994; 269:5890–5896.[Abstract/Free Full Text]
  49. Vitale ML, Rodriguez Del Castillo A, Trifaró J-M. Protein kinase C activation by phorbol esters induces chromaffin cell cortical filamentous actin disassembly and increases the initial rate of exocytosis in response to nicotinic receptor stimulation. Neuroscience 1992; 51:463–474.[CrossRef][Medline]
  50. Peterson RN, Bozzola JJ, Hunt WP, Darabi A. Characterization of membrane-associated actin in boar spermatozoa. J Exp Zool 1990; 253:202–214.[CrossRef][Medline]
  51. Camatini M, Colombo A, Bonfanti P. Cytoskeletal elements in mammalian spermiogenesis and spermatozoa. Microscop Res Tech 1992; 20:232–250.
  52. Vitale ML, Seward EP, Trifaró J-M. Chromaffin cell cortical actin network dynamics control the size of the release-ready vesicle pool and the initial rate of exocytosis. Neuron 1995; 14:353–363.[CrossRef][Medline]
  53. Spungin B, Margalit I, Breitbart H. Sperm exocytosis reconstructed in a cell-free system: evidence of the involvement of phospholipase C and actin filaments in membrane fusion. J Cell Sci 1995; 108:2525–2535.[Abstract]
  54. Pelletier R-M, Vitale ML. Filipin vs enzymatic localization of cholesterol in guinea pig, mink, and mallard duck testicular cells. J Histochem Cytochem 1994; 42:1539–1554.[Abstract]
  55. Gilula NB, Fawcett DW, Aoki A. The Sertoli cell occluding junctions and gap junctions in mature and developing mammalian testis. Dev Biol 1976; 59:142–168.
  56. Fasano AB, Baudry D, Pumplin W. Vibrio cholerae produces a second enterotoxin which affects intestinal tight junctions. Proc Natl Acad Sci USA 1991; 88:5242–5246.[Abstract/Free Full Text]
  57. Pelletier R-M. Blood-tissue barriers in the male reproductive male reproductive system. In: Mártinez-Garciá F, Regadera J (eds.), Male Reproduction A Multidisciplinary Overview. Churchill Communications Europe España ed. Spain: Churchill Communications Europe España; 1998: 183–195.
  58. González-Mariscal L, Contreras RG, Bolívar JJ, Ponse A, Chávez de Ramirea B, Cereijido M. Role of calcium in tight junction formation between epithelial cells. Am J Physiol 1990; 259:C978-C986.
  59. Contreas RG, Miller JH, Zamoro M, González-Mariscal L, Cereijido M. Interaction of calcium with plasma membrane of epithelial (MDCK) cells during junction formation. Am J Physiol 1992; 263:C313-C318.
  60. Nigam SK, Rodriguez-Boulan E, Silver RB. Changes in intracellular calcium during the development of epithelial polarity and junctions. Proc Natl Acad Sci USA 1992; 89:6162–6166.[Abstract/Free Full Text]
  61. Citi S. Protein kinase inhibitors prevent junction dissociation induced by low extracellular calcium in MDCK epithelial cells. J Cell Biol 1992; 117:169–178.[Abstract/Free Full Text]
  62. Oliviera-Castro GM, Loewenstein WR. Junctional membrane permeability: effects of divalents cations. J Membr Biol 1971; 73:69–89.
  63. Spray DC, White RL, Campis de Cavalho AC, Harris AL, Bennett MVL. Gating of gap junctions channels. Biophys J 1984; 45:219–230.[Abstract/Free Full Text]
  64. Ravindranath N, Papadopoulos V, Vornberger W, Zitzmann D, Dym M. Ultrastructural distribution of calcium in the rat testis. Biol Reprod 1994; 51:50–62.[Abstract]
  65. Pelletier R-M. Blood barriers of the epididymis and vas deferens act asynchronously with the blood barrier of the testis in the mink. Microscop Res Tech 1994; 27:333–349.
  66. Pelletier R-M. A freeze-fracture study of cell junctions in the epididymis and vas deferens of a seasonal breeder: the mink (Mustela vison). Microscop Res Tech 1995; 30:37–53.
  67. Ochs D, Wolf DP. Actin in ejaculated human sperm cells. Biol Reprod 1985; 33:1223–1226.[Abstract]
  68. Flaherty SP, Breed WG, Sararis V. Localization of actin in the sperm head of the plains mouse, Pseudomys australis. J Exp Zool 1983; 225:497–500.[CrossRef][Medline]
  69. Rogers BJ, Bastias C, Coulson RL, Russell LD. Cytochalasin D inhibits penetration of hamster eggs by guinea pig and human spermatozoa. J Androl 1989; 10:275–282.[Abstract/Free Full Text]
  70. Yanagimachi R. Mammalian fertilization. In: Knobil E, Neil J (eds.), Physiology of Reproduction. New York: Raven Press; 1994: 189–317.
  71. Flaherty SP, Winfrey VP, Olson GE. Localization of actin in mammalian spermatozoa: a comparison of eight species. Anat Rec 1986; 216:504–515.[CrossRef][Medline]
  72. Camatini M, Casale A, Cifarelli M. Immunocytochemical identification of actin in rabbit spermiogenesis and spermatozoa. Eur J Cell Biol 1987; 45:274–281.
  73. Casale A, Camatini M, Skalli O, Gabbiani G. Characterization of actin isoforms in ejaculated boar spermatozoa. Gamete Res 1988; 20:133–144.[CrossRef][Medline]
  74. Yagi A, Parenko J. Localization of actin, {alpha}-actinin, and tropomyosin in bovine spermatozoa and epididymal epithelium. Anat Rec 1992; 233:61–74.[CrossRef][Medline]
  75. Yagi A, Parenko J. Actin, {alpha}-actinin, and spectrin with specific associations with the proacrosomal and acrosomal domains of bovine spermatozoa. Anat Rec 1995; 241:77–87.[CrossRef][Medline]



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