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BOR - Papers in Press, published online ahead of print October 14, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.004143
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BIOLOGY OF REPRODUCTION 68, 67–76 (2003)
DOI: 10.1095/biolreprod.102.004143
© 2003 by the Society for the Study of Reproduction, Inc.

Male Reproductive Tract

Developmental Association of the Synaptic Activity-Regulated Protein Arc with the Mouse Acrosomal Organelle and the Sperm Tail1

Bernhard Maier3,a, Silvia Medrano3,a, Susan B. Sleightb, Pablo E. Viscontib, and Heidi Scrable2,a

a Department of Neuroscience b Center for Research in Contraceptive and Reproductive Health, Cell Biology Department, University of Virginia, Charlottesville, Virginia 22903


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In neurons, arc (activity regulated, cytoskeleton associated) is an immediate early gene (IEG) that is rapidly and transiently induced by excitatory stimulation. It is believed to mediate rapid strengthening of signaling structures at activated synaptic sites. Unlike most IEGs, arc does not encode nuclear transcription factor, but an effector molecule that associates with the actin cytoskeleton. Cytoskeletal rearrangements of microtubule- and actin-based networks that occur at activated synapses also take place in similar fashion during the formation of the acrosome, the site of regulated exocytosis at fertilization. In this paper, arc is reported to be highly expressed both at the RNA and protein levels in postmeiotic germ cells in the testis of adult mice. Immunofluorescence studies reveal that arc is first present in the perinuclear region of round, elongating, and elongate spermatids, where it colocalizes with the developing acrosome. In isolated mature sperm, arc is present in the acrosomal region of the sperm head, the centriole region of the neck, and the principal piece of the tail. Arc is lost to varying degrees during sperm capacitation and in acrosome-reacted sperm. Phalloidin staining of mature sperm cells reveals an overlapping pattern of filamentous-actin and arc expression. These results support a role for arc and the actin cytoskeleton in the acrosome formation, the sperm acrosome reaction, and in sperm cell motility.

acrosome reaction, gametogenesis, sperm capacitation, sperm motility and transport, spermatogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Arc, also known as Arg3.1, is an immediate early gene (IEG) that is rapidly and transiently induced in neurons in response to physiological and pathological stimuli. These stimuli include long-term potentiation (LTP) [1, 2], a long-lasting increase in synaptic efficacy following high-frequency stimulation of afferent fibers. Unlike other IEGs that are stimulated by neuronal activity, arc is not a transcription factor that regulates the expression of specific downstream genes. Rather, arc is believed to be an effector IEG that affects cellular function by interacting directly with other components of the cell. Arc encodes a 396-amino acid protein that is associated with the actin cytoskeleton and shares a region of homology with the actin-binding protein, {alpha}-spectrin [2]. Arc is unique among activity-regulated genes in that after induction its mRNA is rapidly transported to dendrites where it becomes selectively localized along with its cognate protein to activated postsynaptic sites [3]. Indeed, proteomic studies have identified arc as a component of the multiprotein N-methyl-D-aspartate (NMDA) receptor complex [4] at the synapse, and cellular fractionation studies indicate that arc protein is present at high levels at the postsynaptic density [5]. Based on these observations it has been hypothesized that arc may be synthesized locally in dendrites in close proximity to activated synaptic sites and may mediate activity-dependent cytoskeletal changes at these sites.

It has been reported that arc is found at very low levels in rat peripheral tissues such as kidney, stomach, liver, spleen, lung, muscle, heart [1], and thymus [2], and its expression can be induced by growth factors in both fibroblastic (Rat-6) and neuronal (PC12) cell lines [1, 2]. These observations taken together suggest that the function of arc may not be limited to the brain. Indeed, in mice, arc is expressed ubiquitously early in embryogenesis, and homozygous arc-null mutants exhibit severe growth retardation and improper formation of primary germ layers, and die before gastrulation [6]. Therefore, it appears that arc expression is essential for early embryonic development and patterning in mice. Due to this early embryonic lethality, however, it has not been possible to conduct functional studies of arc during later stages in mammalian development. In particular, there is still no evidence for a developmental function for arc in the brain despite strong predictions of its central role in neurogenesis and synaptogenesis.

Like neurogenesis, spermiogenesis results in the production of a highly specialized and polarized cell, the mature spermatozoon. This complex developmental process continues throughout the lifetime of the animal, which makes it easily accessible for study. Within the seminiferous epithelium of the testis, germ cells go through a series of stereotypical stages that result in the acquisition of the complex morphology of mature sperm cells, including the motility apparatus necessary for fertilization. Following epididymal maturation, sperm cells undergo a complex series of molecular events called capacitation that confer on sperm the ability to fertilize an egg [7, 8]. The interaction of the capacitated sperm head with the zona pellucida of the egg initiates a calcium-dependent signal transduction pathway that results in the exocytosis of components of the acrosome, a large secretory vesicle overlying the sperm nucleus [9, 10], into the cleft between the two gametes. This process of regulated exocytosis in the sperm acrosome reaction uses the actin cytoskeleton, as does regulated exocytosis at the neuronal synapse [11, 12]. During this process, the filamentous cortical network of the cytoskeleton that underlies the plasma membranes of both the neuronal synapse and the sperm head must be partially depolymerized for exocytosis of secretory vesicles to occur [13, 14].

In this study, arc is reported to be activated during spermiogenesis in mice. By combining Northern and Western blot analyses and immunostaining, the time course of arc expression and the distribution of its cognate protein during the entire course of mouse male germ cell development are examined. Arc is shown to be expressed in postmeiotic germ cells and is associated with the formation of the acrosome, where it colocalizes with filamentous-actin (F-actin), suggesting a functional relationship of arc and the actin cytoskeleton in acrosomal exocytosis. Furthermore, arc might play a role in sperm motility because it is found in the centriolar region of the neck and in the principal piece of the tail.


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

All animal experiments were conducted in accordance with the ethical guidelines described in Guide for the Care and Use of Laboratory Animals by the National Research Council. Protocol 2891 was reviewed and approved by the Animal Care and Use Committee of the University of Virginia. ICR mice were originally obtained from Harlan (Indianapolis, IN). Breeding pairs were housed in a 12L:12D room and given food and water ad libitum. First-generation litters were used in these studies.

Materials

All reagents were of reagent grade available from commercial sources.

Probes

Probes were labeled by the method of random priming [15]. The arc probe was derived from a full-length murine cDNA kindly provided by Dr. Paul Worley (Johns Hopkins University, Baltimore, MD). The ß-actin probe was derived from a murine cDNA [16] kindly provided by Margaret Buckingham (Institut Pasteur, Paris, France).

RNA Extraction and Northern Blot Analysis

Tissue from testis or brain was pulverized with a liquid nitrogen-cooled mortar and pestle, and total RNA was isolated using TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH). Approximately 30 µg of total RNA was subjected to electrophoresis in 1% agarose/0.67 M formaldehyde gels and transferred to Biodyne A nylon membrane (Pall Corporation, Ann Arbor, MI). Blots were subsequently UV-crosslinked and prehybridized in 50% formamide, 5x Denhardt solution, 5x SSPE (0.9 M NaCl, 0.05 M NaH2 PO4, 5 mM EDTA, pH 7.4), 200 µg/ml boiled salmon sperm DNA, 1 µg/ml poly(A), and 0.1% SDS at 42°C for 18 h. Hybridizations were performed at 65°C for 36–48 h in a solution containing 50% formamide, 1x Denhardt, 5x SSC, 0.04 M phosphate pH 6.7, 200 µg/ml boiled salmon sperm DNA, 1 µg/ml poly(A) and 0.05% SDS, and 2 x 106 cpm/ml {alpha}-32P-labeled dCTP probes. Unless otherwise indicated, washings were performed as follows: 50% formamide, 2x SSC, and 0.1% SDS for 1 h at room temperature, followed by three washes in 0.1x SSC, 0.1% SDS, one wash for 15 min at room temperature, and two washes for 30 min at 65°C. Blots were autoradiographed at -70°C with Kodak X-Omat Blue XB-1 or Kodak BIOMAX MS film (Kodak, Rochester, NY).

Antibodies

Rabbit anti-arc serum-1 was originally raised against a recombinant His-tagged C-terminal arc fragment (amino acids 132–396) [2] and was a kind gift of Dr. Paul Worley (Baltimore, MD). Rabbit anti-arc 839 was raised in our laboratory against an arc-GST fusion protein containing the same C-terminal fragment (amino acids 132–396). Rabbit anti-arc sc-15325 (Santa Cruz Biotechnology, Santa Cruz, CA) is affinity-purified from a serum raised against the N-terminus of arc (amino acids 1–300). DM1A (Sigma, St. Louis, MO) is a mouse monoclonal immunoglobulin G1 (IgG1) specific for {alpha}-tubulin. All secondary antibodies used in immunofluorescence were Alexa-conjugated antibodies (Molecular Probes, Eugene, OR). Horseradish peroxidase (HRP)-conjugated secondary antibodies were from Pierce (Rockford, IL).

Western Blot Analysis

Protein was isolated from brains and testes using RIPA-buffer containing proteinase and phosphatase inhibitors (Sigma). Equal amounts (50 µg) of protein were electrophoresed on 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Arc was detected using a polyclonal rabbit anti-arc serum (sc-15325, Santa Cruz) at a 1:2000 dilution in PBS containing 5% dry milk and 0.05% Tween-20. After incubation with HRP-conjugated goat anti-rabbit IgG (Pierce) at a 1:10 000 dilution, bands were developed with a chemiluminescent substrate (SuperSignal West Pico, Pierce). For all antibody dilutions and for blocking nonspecific reactions, PBS containing 5% nonfat dry milk, and 0.05% Tween-20 was used.

Immunofluorescence of Testes

Testes were immersion-fixed in Bouin solution (0.9% picric acid, 37% formaldehyde, and 5% glacial acetic acid in 0.9% saline) for 6 h at room temperature, washed in 70% ethanol, embedded in paraffin wax and sectioned at 4 µm. Sections were dewaxed using tissue-clearing solution (Fisher, Newark, DE). For antigen unmasking, rehydrated tissue sections were incubated in 0.1 N HCl at 4°C for 15 min. After washing in PBS, sections were further treated with PBS containing 5 mM MgCl2 and 0.2% Triton X-100 for 5 min. Nonspecific binding was blocked by incubating sections in PBS containing 3% BSA, 2% goat serum, and 0.05% Tween-20. After additional washes in PBS, sections were incubated for 18 h at 4°C with either C-terminus specific polyclonal rabbit anti-arc serum-1, rabbit anti-arc 839, or the N-terminus-specific sc-15325 at 1:500 dilution in PBS + 0.05% Tween-20. Goat anti-rabbit IgG labeled with Alexa 488 or Alexa 568 (Molecular Probes) was used as secondary antibodies at a 1:500 dilution of PBS + 0.05% Tween-20. Sections were counterstained with DAPI staining solution (5 µg/ml in 0.3 M NaCl and 0.3 M sodium citrate) for 3 min. To preserve fluorescent signals, we used Vectashield mounting solution (Vector Laboratories, Burlingame, CA).

Mouse Sperm Isolation, Capacitation, and Induction of Acrosome Reaction

Caudal epididymal sperm were collected from retired breeder males by placing two minced caudae epididymidis into 1 ml of 37°C media containing 100 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5.5 mM glucose, 1 mM pyruvic acid, 4.8 mM L(+)-lactic acid, and hemicalcium salt in 20 mM Hepes pH 7.3. All reagents in media were ultra pure/cell culture-tested (Sigma). Sperm were allowed to swim out for 10 min and the sperm-containing media was removed for the following set of conditions. For noncapacitating conditions, a 500 µl fraction was placed in a round-bottom borosilicate glass tube and then gently overlaid with 1.5 ml of 37°C MW media supplemented with 2 mg/ml Na2CO3. To induce capacitation, 5 mg/ml of fatty acid-free BSA was included in the noncapacitating MW media. Sperm were allowed to swim up into the noncapacitating and capacitating MW media for 1 h in a 37°C 5% CO2/95% O2 incubator. To induce the acrosome reaction, the Ca2+-ionophore A23187 was added for the last 10 min of the 1-h incubation period to a third tube containing capacitation medium. A23187 was dissolved in dimethyl sulfoxide (DMSO). The final concentration of A23187 was 2 µM and the final DMSO content was 0.02%. The upper 1 ml was removed from each tube and the sperm were counted and diluted with the respective MW media to a final concentration of 500 000 sperm cells/ml. Then, 25 µl of diluted sperm was spotted onto a poly-L-lysine coated microscope slide. Sperm were allowed to air-dry at 40°C for approximately 20–30 min and then fixed at room temperature in 4% paraformaldehyde in PBS pH 7.3.

Arc-Adsorption of Rabbit Anti-Arc Antibodies

Eight hundred micrograms of protein of a mouse testis lysate in RIPA-buffer containing a cocktail of protease and phosphatase inhibitors (Sigma) were electrophoresed in 12% SDS-PAGE and transferred to nitrocellulose. The migration positions of arc and tubulin were determined on small strips of the blot using anti-arc and anti-tubulin antibodies and HRP-conjugated secondary antibodies. The migration of the two molecules, arc at 55 kDa and tubulin at 50 kDa, was sufficiently different to separate them on a 12% gel. The 55-kDa band containing arc was excised from the blot, pulverized under liquid nitrogen with a cooled mortar and pestle, and blocked overnight at 4°C with 5% dry milk in PBS + 0.05% Tween-20. Working dilutions of anti-arc (1:300) and anti-tubulin (1:2000) were allowed to react for 3–18 h at 4°C with aliquots of the pulverized nitrocellulose-bound arc in PBS + 0.05% Tween-20. After centrifugation, the supernatants were used in immunofluorescence or Western blot analysis. Alternatively, anti-arc antibodies were adsorbed on purified recombinant arc purified from bacterial extracts.

Immunofluorescence of Sperm Cells

Paraformaldehyde-fixed sperm slides were boiled in 1:100 diluted antigen unmasking solution (Vector Laboratories) for 20 min (four times for 5 min each) in a microwave oven. After slowly cooling to room temperature for approximately 1 h, nonspecific binding was blocked with PBS + 5% fish skin glycerol and 10% normal goat serum (Sigma) for 1 h. Sperm samples were stained for arc with polyclonal rabbit anti-arc serum at a 1:300 dilution in PBS + 0.05% Tween-20. Tubulin in sperm tails was stained with monoclonal antibody DM1A (Sigma) and secondary Alexa 488-labeled goat anti-mouse IgG (Molecular Probes). Nuclei were counterstained with DAPI as described above for testes.

Peanut Agglutinin Staining of Acrosomal Glycoproteins

Peanut agglutinin (PNA) binds preferentially to galactosyl (ß-1,3) N-acetylgalactosamine residues of glycoproteins and glycolipids and is used to evaluate the acrosomal status of mouse sperm cells [17]. In noncapacitated sperm Alexa 488-PNA intensely stains the whole, intact acrosome. In acrosome-reacting or -reacted sperm the PNA staining is shed from the sperm head together with the acrosome.

Testis sections and sperm samples were prepared as described above. Slides were washed in PBS containing calcium and magnesium and incubated with Alexa 488-PNA at 10 µg/ml PBS (containing calcium and magnesium) for 30 min. Slides were washed in PBS and mounted in Vectashield mounting fluid.

Mitochondria Staining

Mitochondria in the sperm midpiece were stained with MitoTracker Green FM or MitoTracker Red CM-H2Xros (Molecular Probes) at a concentration of 100–500 nM.

Phalloidin Staining of Filamentous Actin (F-Actin) in Sperm

Paraformaldehyde-fixed sperm samples were incubated with 1 unit of Alexa 568-phalloidin (Molecular Probes) in 100 µl of PBS for 30 min. Slides were washed in PBS and mounted in Vectashield solution.

Microscopic Imaging

Specifically stained testis and sperm samples were visualized using a Zeiss Axiophot (Zeiss, Thornwood, NY) microscope equipped with appropriate filters for detection of the fluorophores Alexa 488, Alexa 568, and DAPI. We used Zeiss Plan-Neofluar objectives 20x 0.8, 40x 1.3, and 100x 1.3. Digital images were taken with a Sony DSC S-75 camera mounted on the Axiophot. Pictures were imported into Adobe Photoshop 5.0.2 (Adobe Systems, Mountain View, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In Mice, Arc Is Highly Expressed in Both Brain and Testis

The tissue distribution of arc mRNA was studied in adult mice by Northern blot analysis of total RNA extracted from different tissues and hybridized to a full length arc cDNA probe. As expected from previous studies in rats [1, 2], arc mRNA is also abundantly expressed in the brain of mice. However, unlike that reported for rats, we cannot detect any arc mRNA in peripheral tissues of mice such as thymus, lung, liver, kidney, and spleen (Fig. 1A). In contrast to the rat, in which we could not detect arc mRNA in the testis within the limits of Northern blot detection ([1] and data not shown), we found arc mRNA highly expressed in mouse testis (Fig. 1A). Like its brain counterpart, the arc transcript detected in mouse testis has a size of ~3.0 kilobases (kb) (Fig. 1A).



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  		FIG. 1. Arc mRNA and protein expression in the mouse. A) Autoradiogram of arc expression in total RNA isolated from various tissues of adult mice (72-h exposure; Kodak X-Omat Blue XB-1 film). Ethidium-bromide staining of RNA lanes was used as loading control (lower panel). B) Western blot analysis of protein extracts from testis and brain. Equal amounts of protein (50 µg) were analyzed by immunoblot using a polyclonal rabbit anti-arc antibody (sc-15325, Santa Cruz Biotechnology). The three sections of the blot were incubated as indicated in the figure. The arc antibody reveals a major component of 55 kDa in both tissues, which is adsorbed out by preadsorption on nitrocellulose-bound arc.

To determine whether arc is also expressed in testis at the protein level, Western blot analysis was performed on tissue lysates from 5- to 6-wk-old mice using arc-specific antibodies and chemiluminescence. As shown in Figure 1B, a 55 kDa immunoreactive band that comigrated with brain arc appeared as a major component of testis protein extracts. These results confirm that arc is abundantly expressed in the testis of adult mice. Additional, higher molecular weight bands are observed with testis lysate, which could be either translational products of larger transcripts, intermolecular aggregates formed in the lysate, or both. All specifically immunoreactive bands disappeared after the rabbit anti-arc antibody was preadsorbed on nitrocellulose-bound arc or when the first antibody was omitted (Fig. 1B).

Arc Expression Is Developmentally Regulated in Mouse Germ Cells

During postnatal development in mice, extensive differentiation processes take place in brain and testis that result in the production of highly polarized and functionally specialized neurons and sperm cells, respectively. To determine the temporal expression of arc mRNA during sperm cell development, Northern blot analysis was carried out on total RNA extracted from testes of prepubertal and adult mice of different ages. As shown in Figure 2, arc mRNA is expressed at very low levels in testes of 1- and 2-wk-old animals, with a slight increase observed at 2.5 wk. Arc mRNA expression increases dramatically at 3 wk and remains high after that. The significance of this expression pattern is that it overlaps precisely with the first appearance of postmeiotic germ cells in the prepubertal testis. The lower panel in Figure 2 shows the same blot rehybridized to actin. The probe hybridizes to the two size classes of actin mRNAs present in testis: the 2.1-kb actin mRNA, which codes for cytoplasmic ß- and {gamma}-actin isoforms, and the 1.5-kb actin mRNA that codes for smooth muscle {gamma}-actin. Whereas the 1.5-kb actin mRNA is expressed only in postmeiotic germ cells, the 2.1-kb actin mRNA is expressed throughout male germ cell differentiation and constitutes a good loading control [18]. The expression of arc and the 1.5-kb species of {gamma}-actin follow a similar time course, in that both transcripts are developmentally regulated and expressed mainly in postmeiotic germ cells. Figure 2 also shows the developmental pattern of arc mRNA expression in the brain. Arc mRNA expression gradually increases during the first 2 wk postnatally and reaches a maximum between 2.5 and 3 wk after birth. This is similar to the pattern of arc expression during postnatal development in rats [2].



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  		FIG. 2. Developmental expression of arc mRNA in mouse testis and brain. Autoradiogram of arc expression in total RNA from testis and forebrain of prepubertal and mature mice (72-h exposure; Kodak X-Omat Blue XB-1 film). The lower panel shows the same blot rehybridized to ß-actin as a loading control (5-h exposure; Kodak X-Omat Blue XB-1 film). In testis, arc expression is very low in prepubertal animals and dramatically increases 3 wk after birth when most germ cells have completed meiosis and are at the spermatid stage. Arc expression in forebrain of mice gradually increases during the first weeks after birth, reaching a maximum between 2.5 and 3 wk

Arc Resides in the Perinuclear Region of Spermatids

To identify the cellular, spatial, and temporal aspects of arc expression in germ cell development, the developmental expression of arc protein was monitored in testis by immunofluorescence microscopy of Bouin-fixed and paraffin-embedded testes taken successively at 1 to 5 wk after birth. This allowed exploitation of the synchronous nature of germ cell maturation and the ease of inspection of seminiferous tubules of the testis by light microscopy. Perinuclear arc staining is first observed in postmeiotic round spermatids at 3 wk after birth (Fig. 3). As the differentiation of the seminiferous epithelium proceeds, arc can be seen in elongating and elongate spermatids whose nuclei can be easily identified by the pattern of DAPI staining (Fig. 3, lower panel). Arc expression is also observed in Leydig cells of the interstitial islets of 3- and 4-wk-old testes (Figs. 3 and 4). The time course of RNA expression shown in Figure 2, therefore, finds a close correlate in the course of protein expression in postmeiotic cells during spermiogenesis (Fig. 3). The arc-staining by rabbit antibodies raised against the C-terminus as well as N-terminus is highly specific and can be specifically blocked by preadsorption of the antibodies on recombinant arc as shown in the lower panels of Figure 3.



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  		FIG. 3. Developmental expression of arc protein in mouse testis. Sections from testes of 1- to 5-wk-old mice were stained with a polyclonal rabbit anti-arc antiserum and Alexa 488-labeled goat anti-rabbit IgG. Sections were counterstained with DAPI to visualize nuclei as shown in the lower panel. Arc expression starts at 3 wk of postnatal development in postmeiotic round spermatids. At 4 and 5 wk, arc stains the developing acrosome of elongating and elongate spermatids, as well as Leydig cells. Lower panels: preadsorption on recombinant arc abrogates staining by C- and N-terminus specific rabbit anti-arc antibodies. Magnification x400 for 1- to 4-wk-old testes; x200 for testis at 5 wk of age



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  		FIG. 4. Arc and PNA staining patterns in mouse testis. Testis sections were double-stained for arc and PNA using a polyclonal rabbit anti-arc serum, Alexa 568-conjugated goat anti rabbit IgG, and Alexa 488-conjugated PNA (Molecular Probes). Single color images show are staining in red (A) and PNA staining in green (B). In the overlay of red and green images (C), a partial overlap of arc and PNA staining patterns is revealed as areas of yellow staining in postmeiotic spermatids. Images were taken at x400 magnification

Arc Colocalizes with the Glycoproteins of the Acrosomal Organelle

An Alexa 488-labeled PNA and polyclonal rabbit- and Alexa 568-labeled secondary antibodies were employed to study the colocalization of arc and the developing acrosome. PNA is known to stain the acrosome selectively through its exclusive specificity for terminal sugar modifications (ß-galactose and N-acetylgalactosamine) of glycoproteins enriched in this organelle [19]. When testis sections were pretreated with HCl to remove terminal sialic acid residues and expose the glycoprotein sugar side chains, PNA stained all stages of acrosome development: Golgi-phase (steps 1–3), cap phase (steps 4–7), acrosome phase (steps 8–12), and maturation phase (steps 13–16) (Fig. 4A, green). Counterstaining for arc showed a less abundant, but similar staining pattern (Fig. 4B, red). An overlay of the two reactions shows extensive areas of overlapping arc and PNA staining (Fig. 4C, yellow), indicating colocalization of arc protein with glycoproteins of the acrosomal region. Leydig cells in interstitial islands also appear to express arc in cytoplasmic structures, whereas a few islet cells remain unstained and probably are macrophages.

Acrosome Biogenesis and Arc Expression Closely Parallel Each Other

In round spermatids at 3 wk of testis development, arc is found exclusively in a small acrosomal vesicle, in which there is a hole the size of the acrosomal granule that is devoid of staining (Fig. 5A). The perimeter of arc staining then expands to cover approximately one third of the nucleus (Fig. 5B). At 5 wk of development, spermatids of the first cycle undergo elongation in a process that appears to "pull" arc over the elongating nucleus. At the same time, arc appears as a tight, little ring surrounding the presumed acrosomal granule in a new wave of postmeiotic round spermatids that are located just beneath the elongating spermatids in a cross-section of a seminiferous tubule at stages II–III (Fig. 5C). Figure 5D presents a closer view of arc as it is pulled over elongating spermatid nuclei. In Figure 5E (panels 1–8), eight characteristic patterns of arc staining during mouse spermiogenesis are presented in a hypothetical chronological order, paralleling acrosome biogenesis. As shown in Figure 5E (panels 1–4), the angle subtended by the perinuclear area stained for arc extends from approximately 40° to greater than 120°, corresponding to angles described for developing acrosomal areas [20]. Top views of the enlarging umbrella-like acrosome are shown in Figure 5E (panels 5 and 6). The section through the lumen of the acrosomal vesicle leaves a hole-like area devoid of arc staining. Acrosomal structures staining for arc start to expand over the two nuclei in Figure 5E (panel 7) and surround the entire nucleus in elongating spermatids in Figure 5E (panel 8). Arc epitopes are also recognized on maturing spermatozoa during epididymal transit, as shown in Figure 5F.



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  		FIG. 5. Arc localization during acrosome development. Sections from testis and epididymis were stained with a polyclonal rabbit anti-arc antiserum and Alexa 488-labeled goat anti-rabbit IgG. Cell nuclei were counterstained with DAPI, except for E, panel 8, in which arc staining was superimposed on a phase-contrast micrograph. A–D) Arc staining in the developing acrosomes of steps 5–6 (A and B), steps 4 and 15 (C), and step 10 (D) spermatids. E) Panels 1–6 depict single spermatids in which arc staining subtends angles over the nucleus of 40°–150°. This is typical for the developing acrosomal region of steps 2–3 to step 7 spermatids. Top views are given in E, panels 5–6. F) Arc staining pattern in spermatozoa during epididymal transit

Components of Mature Spermatozoa Necessary for Fertilization Are Rich in Arc

Paraformaldehyde-fixed and citric acid-demasked mature spermatozoa were triple-stained for arc (rabbit anti-arc), nuclei (DAPI), and mitochondria (MitoTracker). Arc staining was localized to the acrosomal region of the sperm head and the principal piece of the tail (Fig. 6A, bright green). A slightly fainter, but distinct arc staining in the centriolar area of the neck was observed as highlighted by arrows in Figures 6A and 7a. When the anti-arc antibody was preadsorbed on purified arc protein, it completely failed to stain the sperm head and tail (Fig. 6B). To control for the specificity of arc adsorption, mature spermatozoa were stained for tubulin before and after adsorption of the anti-tubulin antibody on the same arc protein preparation. As shown in Figure 6, C and D, antitubulin stained the axonema of sperm tails with an equally bright green signal before and after adsorption on purified arc, respectively.



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  		FIG. 6. Arc- and tubulin-specific staining of mature spermatozoa. Caudal epididymal sperm stained for arc with polyclonal rabbit anti-arc antiserum and Alexa 488-labeled goat anti-rabbit IgG (A and B) or for tubulin with monoclonal anti-tubulin DM1A antibody and Alexa 488-conjugated goat anti-mouse IgG (C and D). As a control for specificity, anti-arc (B) and anti-tubulin (C) antibodies were preadsorbed on purified arc. Images were taken at x1000 magnification



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  		FIG. 7. Arc and PNA-staining in noncapacitated, capacitated, and acrosome-reacted sperm. Sperm cells were stained for arc, PNA-binding glycoproteins (Alexa 488-PNA), mitochondria (MitoTracker), and nuclei (DAPI). a–c) Arc staining in acrosomes in varying degrees of intactness. An arrowhead in a points to arc staining in the centriolar area of the sperm neck. d–f) PNA-staining of sperm. A white arrowhead in e points to a pair of sperm cells that have lost a significant amount of PNA staining after capacitation. g) An overview of noncapacitated sperm cells. A green left arrow in h shows a sperm tail with arc staining localized to parallel spurs lining the edges. In contrast, a green right arrow points to a patchy tail staining in h. White arrowheads point to the arc staining in the acrosome. All images were taken at x1000 magnification

Capacitation and Acrosome Reaction Alter the Arc Matrix in the Sperm Head

The staining patterns for arc and PNA in noncapacitated, capacitated, and acrosome-reacted sperm were studied by staining for arc and PNA binding glycoproteins in nuclei and mitochondria. Figure 7, a–c depicts sperm with various degrees of intactness of the arc matrix in the sperm head. In noncapacitated sperm preparations, most cells demonstrate a complete and bright staining of the arc matrix (Fig. 7a). In capacitated sperm preparations, about 75% of the sperm cells are intact, as in Figure 7a; the remaining 25% of cells have lost arc staining to various degrees (Fig. 7b). After the acrosome reaction, at least 50% of the sperm cells are motile and 90% are viable as judged by staining sperm cells with the live cell staining dye Calcein-AM and DNA dyes provided in the sperm Live/Dead assay kit (Molecular Probes). Acrosome-reacted sperm preparations showed a high loss of arc staining in about 40% of cells. About 10% of the sperm cells appear to be in the process of shedding the arc matrix, as represented in Figure 7c. The remaining 50% appear to have an intact arc matrix, as represented in Figure 7a. The loss of PNA staining correlated with the loss of arc staining in capacitated and acrosome-reacted sperm (Fig. 7, d–f). The principal piece of the sperm tail typically displays one of two staining patterns for arc. In one pattern, small patches of arc staining are spread over the entire surface of the principal piece (Fig. 7h, green right arrow), whereas in the other, the arc signal is focused into parallel lines along the peripheral edges of the tail (Fig. 7h, green left arrow).

Arc May Be Linked to F-Actin in the Cytoskeleton

Arc has been described as an actin-binding cytoskeleton-associated protein [2]. Therefore, we investigated the presence of F-actin in the acrosomal region of the sperm head, where it might function as a potential binding and interaction partner of arc. Freshly isolated paraformaldehyde-fixed sperm cells were stained with Alexa 568-labeled phalloidin, which specifically detects F-actin [21]. F-actin was found in a rim along the dorsal aspect of the sperm head and in a V-shaped, postacrosomal area (Fig. 8A). PNA staining, which precisely demarcates the acrosome, overlaps with the dorsal, but not the postacrosomal region of phalloidin-stained actin structures in the sperm head (Fig. 8B). The precisely overlapping patterns of arc and actin on the dorsal aspect of the acrosome (Fig. 8A) supports the idea of a possible functional relationship between the two molecules in the sperm head.



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  		FIG. 8. Spatial relationship of F-actin and the acrosome. Paraformaldehyde-fixed sperm were stained with Alexa 488-conjugated PNA and Alexa 568-conjugated Phalloidin (Molecular Probes). Phalloidin-stained F-actin is located in a rim along the dorsal head area and in the postacrosomal V-shaped area (A) as further emphasized by the overlay of PNA and phalloidin staining in B. Images were taken at x1000 magnification


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The immediate early gene arc is induced in the brain by intense neuronal activity and has been implicated in signaling morphological changes at activated synaptic sites [2, 3, 22]. In this paper we show that arc is also highly expressed in mouse testis. A number of other gene products were originally believed to be brain-specific and were later found expressed in testis as well. Some of these include tau [23], the cyclic-nucleotide-gated channel, the cannabinoid receptor agonist anandamide [24], N-cadherin [25, 26], nerve growth factor [27], and molecules involved in vesicle fusion, such as v-snares, t-snares, and the calcium sensor protein synaptotagmin [28]. It has been argued that this apparent indiscriminate expression of neuronal genes during sperm cell differentiation occurs as a simple byproduct of the changes in chromatin structure that take place during repackaging of DNA into the condensed nuclei of mature spermatozoa. It seems more likely, however, that unregulated gene transcription during germ cell development would be incompatible with germ cell survival [29] and that genes expressed in the testis, including arc, are there because they subserve a normal function, such as sperm cell differentiation.

The production of sperm cells is a complex developmental process. During spermiogenesis, postmeiotic germ cells differentiate into mature spermatozoa. This process involves significant structural and biochemical changes, including the shaping of the sperm head and tail, the compaction of nuclear DNA, the formation of the acrosome, and the establishment of plasma membrane domains that are necessary for sperm-egg recognition. Our immunofluorescence studies with sections of mouse testis show that arc is distributed asymmetrically in the cytoplasm of round spermatids coincident with the perinuclear position of the developing acrosome. The time course of arc expression during prepubertal development, the structural dimensions of the subcellular organelles stained with arc, and the observed overlap with the expression of characteristic protein glycosylation patterns support the assumption that arc is an integral part of the developing acrosome.

F-actin was also found in the mature sperm head. This is consistent with the notion that the actin cytoskeleton in mammalian germ cells is involved in the acquisition of the morphology of the mature sperm, for one thing by stabilizing the position and shape of the acrosome with respect to the nucleus [30]. The actin cytoskeleton might also help to establish and maintain plasma membrane domains with specific functions. During ejaculation and transit through the female reproductive tract, significant changes in extracellular ion concentrations and osmolarity are believed to induce capacitation, in which sperm cells gain competence to undergo the acrosome reaction upon binding to the zona pellucida of the egg. Capacitation is accompanied by an efflux of cholesterol from the sperm plasma membrane and a concomitant change in membrane fluidity and the topology of membrane signaling domains [31]. Changes in ion fluxes such as HCO3- and Ca2+ initiate a cascade of adenyl cyclase induction, protein kinase A activation, and protein tyrosine phosphorylation [7]. The actin cytoskeleton plays an important role in regulating ion and proton channels [32, 33], as well as orchestrating membrane signaling pathways [34]. It is therefore feasible that the arc and actin observed in the sperm head are part of a molecular network preparing sperm cells to respond to extracellular stimuli during capacitation and upon binding to the zona pellucida of the egg. During the acrosome reaction, the outer membrane of the acrosome fuses with the plasma membrane, which overlies the sperm head. This forms hybrid membrane vesicles at multiple sites on the anterior sperm head [10, 35], which are subsequently degraded. As a consequence, the inner acrosomal membrane overlying the nucleus is exposed in preparation for subsequent fusion with the egg plasma membrane. Furthermore, the fusion of the outer acrosomal membrane with the sperm head plasma membrane seems to be regulated by actin depolymerization induced by Ca2+ increase and phospholipase A2 activation in human sperm [36]. In marine invertebrates, actin bundling by {alpha}-scruin in the sperm is necessary to facilitate penetration of the zona pellucida at fertilization [37]. A mammalian equivalent of {alpha}-scruin (CPß3) is a major component of the subacrosomal region of human and bovine spermatozoa [38]. The presence of actin in mammalian spermatozoa has been detected with specific antibodies [39–41] and phallotoxins, which bind to F-actin [42, 43]. Furthermore, the spatial relationship between F-actin and arc that was observed in the head region of murine sperm suggests a functional relationship between the two molecules.

The staining pattern of arc in the principal piece of the tail suggests a possible arc association with another cytoskeletal complex, the fibrous sheath surrounding the flagellar axoneme. It can be speculated that arc forms at least two configurations as it interacts with the complex structures of the tail cytoskeleton [44]. In a parallel band configuration, arc could possibly associate with the two longitudinal columns of the fibrous sheath that extend along the length of the sperm tail. This parallel staining is reminiscent of a pattern described recently by Catalano et al. for the intermediate filament-related fibrous sheath protein Fs39 [45]. In the patchy configuration, arc could possibly associate with the transverse ribs that connect the two longitudinal columns of fibrous sheath. This fibrous sheath serves as a scaffold for enzymes involved in cAMP-dependent phosphorylation/dephosphorylation events that propel the sperm forward [46–48]. Because these processes are induced by calcium influx, the recent identification of a testis-specific cation channel in the principal piece of the mouse sperm tail is of particular note [49]. Taken together with the known association of arc with the actin cytoskeleton in the brain [2], our findings support the idea that arc also interacts with the actin skeleton in male germ cells and sperm, where it may play a critical role in the formation of the acrosome, in the acrosome reaction, and in the facilitation of sperm motility.


   ACKNOWLEDGMENTS
 
The authors thank Drs. Jeff Lysiak, Terry Turner, and Jennifer Kirby for their gifts of mammalian RNA and expertise, and for many informative discussions about male reproductive biology. We thank Drs. Paul Worley and Margaret Buckingham for the gifts of arc and cytoplasmic actin cDNAs, respectively, and Dr. Worley for the anti-arc antibodies. We thank the other members of our laboratory, especially Preston Kirby, for technical help and many provocative discussions.


   FOOTNOTES
 
1 This work was supported by National Institutes of Health grants RR11102 (to H.S.) HD38082 (to P.E.V.), and by training grant DK07766 (to B.M.). Back

2 Correspondence. FAX: 804 982 4380; hs2n{at}virginia.edu Back

3 These authors contributed equally to this work Back

Received: 6 February 2002.

First decision: 5 March 2002.

Accepted: 12 July 2002.


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