ARTICLE |
Correspondence to: Peter Redecker, Abt. Anatomie 1, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. E-mail: redecker.peter@mh-hannover.de
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Summary |
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Proteins of the presynaptic exocytic machinery have been found associated with the acrosome of male germ cells, suggesting that the sperm acrosome reaction and neurotransmission at chemical synapses may share some common mechanisms. To substantiate this hypothesis, we studied the expression and ultrastructural localization of prominent pre- and postsynaptic protein components in rat testis. The presynaptic membrane trafficking proteins SV2 and complexin, the vesicular amino acid transporters VGLUT and VIAAT, the postsynaptic scaffolding protein ProSAP/Shank, and the postsynaptic calcium-sensor protein caldendrin, could be identified in germ line cells. Immunogold electron microscopy revealed an association of these proteins with the acrosome. In addition, evidence was obtained for the expression of the plasmalemmal glutamate transporters GLT1 and GLAST in rat sperm. The novel finding that not only presynaptic proteins, which are believed to be involved in membrane fusion processes, but also postsynaptic elements are present at the acrosome sheds new light on its structural organization. Moreover, our data point to a possible role for neuroactive amino acids in reproductive physiology.
(J Histochem Cytochem 51:809819, 2003)
Key Words: chemical synapse, postsynaptic density, shank, vesicular neurotransmitter, transporters, glutamate, GABA, GAD, SV2, complexin, acrosome
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Introduction |
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TESTICULAR GERM CELLS of various mammalian species have recently been shown to contain proteins that serve important functions in synapses of the nervous system. Most of the available data refer to synaptic vesicle trafficking proteins that regulate transport and exocytic membrane fusion of neuronal synaptic vesicles. Members of the SNAP, syntaxin, VAMP, synaptotagmin, and Rab protein families have all been detected in sperm cells, in which they are believed to operate primarily during the acrosome reaction (
Interestingly, other synaptic protein constituents, not implicated in membrane fusion, may also play important roles in male germ cells. For example, testicular germ cells appear to be equipped with distinct neurotransmitter receptor subtypes for the neuroactive amino acids glutamate, GABA, and glycine (
The study of synaptic proteins in the testis is likely to provide novel insights into the molecular architecture and physiology of the male reproductive system. This prompted us to examine the expression and localization of further synaptic proteins in male rat germ cells, using immunohistochemical (IHC) and immunochemical (ICC) techniques. In our study we have included vesicle trafficking proteins of the presynaptic nerve terminal, scaffolding and calcium-binding proteins of the postsynaptic density (PSD) of the central nervous system (CNS), and transporter proteins for amino acid neurotransmitters.
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Materials and Methods |
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Animals and Tissue Preparation for IHC (Light Microscopy)
Adult Wistar rats kept under controlled laboratory conditions were used for the investigation. Principles of laboratory animal care and specific national laws were followed. Rats were sacrificed by decapitation under CO2 anesthesia, and small pieces of the dissected testes were fixed by immersion for 48 hr at 4C in Bouin's solution. In addition, anesthetized animals were transcardially perfused with pre-wash and fixative solution (a mixture of phosphate-buffered 0.5% picric acid and 4% paraformaldehyde) as detailed elsewhere (
Tissue Preparation for Immunoelectron Microscopy
Small testicular specimens were fixed by immersion in solutions containing either phosphate-buffered (0.1 M, pH 7.3) 1% glutaraldehyde and 0.25% picric acid or phosphate-buffered 4% paraformaldehyde and 0.25% picric acid for 4 hr at 4C, followed by embedding of the unosmicated tissue in LR White (London Resin; Hampshire, UK) as described previously (
Immunohistochemistry
Antibodies.
Three different antibodies directed against ProSAP1/Shank2, which have been characterized in detail previously, were used in this study (
IHC Protocol for Light Microscopy
Sections were processed either for the avidinbiotinperoxidase complex (ABC) technique or for immunofluorescence staining as outlined previously (
Method controls consisted of the omission of single steps in the IHC protocol, the application of primary antibodies of unrelated specificities, the use of ascending dilutions of the first antibody or the use of high-molar (0.5 M) PBS as a rinsing solution between the various steps of the IHC protocol.
Immunogold Staining for Electron Microscopy
On-grid labeling of thin sections was performed essentially as outlined elsewhere (
Immunoblotting
Electrophoresis and immunoblotting of total tissue lysates were performed essentially as reported previously (
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Results |
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Light Microscopic IHC
The analysis of testicular immunostaining was focused on the epithelium of the seminiferous tubules. In extension of previous findings on the expression of presynaptic vesicle trafficking proteins in male germ cells, we could identify complexin and SV2 immunoreactivity in these cells. The antibodies directed against complexin1/2 and SV2B labeled the acrosome in round and elongated spermatids (Fig 1A and Fig 1B). Elongated spermatids were only very weakly stained by the SV2A antiserum. In agreement with earlier studies, positive immunoreactions in germ cells were observed with antibodies directed against syntaxin1, synaptotagmin1/2, synaptobrevin1, and synaptobrevin2 (not shown).
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Most interestingly, immunostaining revealed the presence of ProSAP/Shank proteins, core components of the PSD, within seminiferous tubules. Therefore, in addition to weak cytoplasmic immunolabel, which was visible in spermatocytes, immunoreactivity for both ProSAP1/Shank2 (Fig 1C) and ProSAP2/Shank3 was associated with the acrosomes of round and elongated spermatids. We also found discrete ProSAP1/Shank2 immunolabeling in Sertoli cells (Fig 1C) and in the cell cortex of spermatogonia/young spermatocytes (Fig 1C). Acrosomal labeling of spermatids was also detected with the caldendrin antiserum (Fig 1D).
Next, we assessed whether transporter proteins of the neurotransmitters glutamate and GABA are present in rat testis. Both vesicular and plasmalemmal glutamate and GABA transporter proteins appear to be expressed in testicular germ cells. Immunoreactivity for the plasmalemmal glutamate transporter protein GLT1 was detected in the cytoplasm of spermatocytes and was most prominent at the cell surface of round spermatids (Fig 1E). Moreover, the tail of elongated spermatids was clearly labeled by the GLT1 antiserum (not shown). In contrast to strong immunoreactivity for GLAST in Leydig cells, spermatids in the seminiferous epithelium were only moderately immunostained by the GLAST antiserum (not shown). The vesicular glutamate transporter proteins VGLUT1 (Fig 1F) and VGLUT2 and the vesicular GABA transporter VIAAT (Fig 1G) could all be localized in early and mature spermatids, in which the acrosome appeared prominently immunostained. It is noteworthy that immunostainings using an antibody directed against the GABA-synthesizing enzyme GAD67 also led to reproducible staining of acrosomes (Fig 1H).
These results suggest that most of the investigated pre- and postsynaptic proteins may be co-localized in the acrosomal region of spermatids. Immunostaining of serial semithin sections revealed extensive co-localization in the acrosomal region as demonstrated for VGLUT1, VGLUT2, VIAAT, ProSAP1/Shank2, ProSAP2/Shank3, and caldendrin (Fig 2).
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Finally, acrosomal labeling was found in smears of epididymal spermatozoa when they were incubated with antibodies against caldendrin (Fig 3A), ProSAP1/Shank2, ProSAP2/Shank3 (Fig 3C), VGLUT1, VGLUT2, VIAAT (Fig 3E), and GAD67. The acrosomes of epididymal spermatozoa were also weakly immunopositive for SV2B and complexin1/2. Moreover, we noted variable degrees of ProSAP/Shank immunofluorescence in the tails of spermatozoa (Fig 3C), which were also immunopositive for GAD67. Immunoreactivity for GLT1 and GLAST (Fig 3G) was mainly present in the tails of spermatozoa.
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Immunogold Electron Microscopy
To verify the acrosomal immunolabeling observed by light microscopy, we carried out an ultrastructural analysis of LR White-embedded testicular tissue. Immunogold electron microscopy confirmed the tight association of complexin1/2, SV2B, ProSAP1/Shank2, ProSAP2/Shank3, Caldendrin, VGLUT1, VGLUT2, VIAAT, and GAD67 with the acrosome. Gold particles indicating exposed epitopes of the respective synaptic proteins were consistently distributed along the outer and inner acrosomal membrane. This labeling pattern was observed in both round and elongated spermatids. Some examples are depicted in Fig 4.
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Immunoblotting
Western blotting analyses of testis protein homogenates (Fig 5) showed that all antibodies employed in this study detect immunoreactive bands of molecular weights that were also seen in brain homogenates (not shown). Therefore, it is unlikely that a hitherto not characterized crossreactivity of the employed antisera with proteins exclusively expressed in testis has obscured the staining results. Compared to testis homogenates, we noted additional strongly stained bands above 200 kD apparent molecular weight in immunoblotting analyses of ProSAP/Shank proteins in brain homogenates. This observation probably reflects the differential expression of splice variants and/or degradation of these proteins (
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Discussion |
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A steadily growing set of data speak in favor of an important role of presynaptic vesicle trafficking proteins during spermiogenesis and fertilization. Various members of the corresponding protein families have already been implicated in the biogenesis of the acrosome and in the exocytic acrosome reaction (
The most remarkable finding of the present study is that, in the acrosomal region, presynaptic vesicle proteins are co-localized with proteins that are concentrated at the PSD of CNS synapses (-fodrin (
and mGluR5 in cells of the germinal line in rat testis has recently been reported, although no details concerning the ultrastructural localization of the receptors were given (
Interestingly, for the first time we also provide evidence for the concomitant presence of plasmalemmal and vesicular amino acid neurotransmitter transporter proteins in testis. Functional roles for the neuroactive amino acids glutamate, GABA, and glycine in germ line cells are just beginning to emerge. For example, it has been shown that receptors for GABA and glycine are expressed in mammalian sperm (
Remarkably, the results of our study strongly suggest that the acrosome is endowed with the vesicular amino acid transporters VGLUT and VIAAT. VIAAT is a neuronal transporter protein that mediates the uptake of GABA, and probably also of glycine, into synaptic vesicles (
Finally, it must be stressed that the co-localization of VIAAT, VGLUT1, and VGLUT2 in a single vesicular organelle, as shown here for the acrosome, is a unique feature, taking into account that in neural tissue (a) the VGLUT transporters reside on vesicle populations distinct from GABA-containing synaptic vesicles (
In summary, the present study shows that not only pre- but also postsynaptic proteins are assembled in the acrosomal region of male germ cells. This observation should be helpful to provide further new insights into the molecular architecture of sperm cells by addressing the known functional interactions of these proteins that have been found in neurons. It is tempting to speculate that a highly specialized cytoskeleton is attached to acrosomal membranes that closely resembles that of the pre-and postsynaptic cytomatrix. The possibility that the organization of this cytoskeleton is similar to that of the synapse clearly awaits further investigation. The extent of this similarity could provide important insights into basic mechanisms of membrane specialization. Moreover, our results show that neuroactive amino acids may play a more significant role in the physiology of the reproductive system than was previously believed. Finally, the presence of vesicular neurotransmitter transporters in the acrosome supports the view that the acrosome has emerged as a modified secretory granule rather than as a lysosome-like organelle (
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Acknowledgments |
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Supported by grants from the Deutsche Forschungsgemeinschaft DFG BO 1718/2-1 to TMB, and KR 1879/2-1, 2-2 to MRK and EDG, and the Fonds der Chemischen Industrie to EDG.
We gratefully acknowledge the skillful technical assistance of H. Böning and D. von Mayersbach. We thank R. Jahn (Göttingen) for his gifts of antibodies against synaptic vesicle-associated proteins.
Received for publication October 14, 2002; accepted January 22, 2003.
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