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Volume 52 (6): 833-836, 2004
Copyright ©The Histochemical Society, Inc.
BRIEF REPORT |
Electron Microscopic Cytochemistry of Adenylyl Cyclase Activity in Mouse Spermatozoa
Dipartimento di Scienze Biochimiche Biotecnologie Molecolari, Sezione Biochimica Cellulare (LL,IB,AM), e Dipartimento di Medicina Sperimentale (MGR), Sezione Anatomia Umana, Università di Perugia, Perugia, Italia
Correspondence to: Alba Minelli, PhD, MD, Dipartimento Scienze Biochimiche Biotecnologie, Molecolari Sezione Biochimica Cellulare, Via del Giochetto, 06123 Perugia, Italia. E-mail: albaminelli{at}virgilio.it
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Summary |
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 Top Summary Literature Cited |
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We investigated adenylyl cyclase activity of mouse spermatozoa by electron microscopic cytochemistry. Subcellular localization of enzyme activity was determined in the presence and absence of bicarbonate ions. Results confirm the existence in sperm of a bicarbonate-regulated adenylyl cyclase, which suggests microdomain signaling.
(J Histochem Cytochem 52:833–836, 2004)
Key Words: adenylyl cyclase • mouse spermatozoa • localization • electron microscopy
IN MAMMALS, the ubiquitous second messenger cAMP is synthetized by two classes of adenylyl cyclase (AC): the G-protein-responsive transmembrane adenylyl cyclase (mAC) and the bicarbonate-responsive soluble adenylyl cyclase (sAC) (Buck et al. 1999
; Chen et al. 2000).
sAC is molecularly and biochemically distinct from mAC because it is insensitive to G- protein and forskolin (Buck et al. 1999), is soluble and particulate (Zippin et al. 2003), possesses no predicted transmembrane domains, and has catalytic domains that are more closely related to cyanobacterial cyclases than other mammalian cyclases (Chen et al. 2000). cAMP signaling is mediated by multiple effectors, i.e., cAMP-dependent protein kinase (PKA), RAP exchange proteins (EPACs), cAMP-gated ion channels (cNGCs), and A-kinase anchoring proteins (AKAPs). The activation of intracellular targets by cAMP produced only by mAC involves a diffusion process inside the cell. Diffusion lowers specificity and selectivity of signaling and the levels of the cAMP produced must be very high because the cytoplasm contains many cAMP- catabolizing phosphodiesterases (PDEs). Furthermore, it has been established that cAMP diffusion from the plasma membrane is restricted to 
1 µm. Therefore, the existence of a cytosolic form of AC provides new possibilities for cAMP signaling within the cell. Indeed, cytosolic localization of sAC suggests a model whereby cAMP can signal in complexes consisting of both substrate and effector. Such second messenger microdomains provide selective, specific, and efficient activation of individual cAMP effectors.
The fact that PDEs are also complexed with AKAPs indicates that signal terminators can be found in signaling microdomains. It has long been known that a non-conventional bicarbonate-regulated AC is present in maturing male germ cells and spermatozoa, corresponding to the bicarbonate-sensitive cyclase found in testis, where it functions as a bicarbonate sensor (Chen et al. 2000). sAC has also been identified in spermatids and in mature spermatozoa (Jaiswal and Conti 2001), suggesting the involvement of this bicarbonate-regulated enzyme during sperm cell differentiation and sperm capacitation, i.e., the acquisition of fertilizing capacity by ejaculated spermatozoa. Recently, membrane-associated AC (mAC), has been found in mammalian spermatozoa (Baxendale and Fraser 2003). Because of the rising interest in sperm AC and its role in physiological reproductive processes, we present electron microscopic cytochemistry pictures of subcellular localization of AC activity in mouse spermatozoa. Epididymal spermatozoa were extracted from adult mice sacrificed by cervical dislocation. The epididymes removed from the animals were separated from caput regions and cauda regions were suspended in 10 ml of prewarmed human tubal fluid (HTF; Irvine Scientific, Santa Ana, CA), where they were left for a short time to allow the spermatozoa to disperse throughout the medium. Adenylyl cyclase activity distribution was carried out according to Yamamoto et al. (1998).
The assay medium consisted of 80 mM Tris-maleate buffer, pH 7.4, with 8% glucose, 2 mM theophylline, 4 mM MgSO4, 10 mM ouabain, 1 mM levamisole, 2 mM lead nitrate as tracer, and purified 0.5 mM adenylate imidodiphosphate (AMP-PNP) as substrate. After incubation, sperm samples were examined with a PhilipsTEM 400 electron microscope. Experiments performed without AMP-PNP were used as negative controls. To distinguish between sAC and the bicarbonate-insensitive AC form, freshly prepared mouse spermatozoa were divided into three lots of 10 x 106 cells. One lot was used as uncapacitated control and kept in non-capacitating medium (NCM) (132.2 mM NaCl, 2.7 mM KCl, 0.49 mM MgCl2, 0.36 mM Na2HPO4, 5.5 mM glucose, 25 mM Hepes, pH 7.4). The second lot was incubated in HTF and the third lot was incubated in bicarbonate-free HTF. Each lot was incubated for 90 min at 37C in 5% CO2. At this stage, aliquots were withdrawn to assess the capacitative status of samples with Coomassie Blue staining. Samples were then incubated in freshly prepared medium to demonstrate AC activity. AC activity was apparent as a deposit of fine granules of electron-dense reaction product. Fine structures of spermatozoa were well preserved so that a localization of the enzyme could be studied in relation to subcellular structures.
In uncapacitated sperm (Figure 1)
, no reaction products of enzyme activity were observed in the acrosome and head region (Figure 1a), in the mid-piece (Figure 1b), or in the tail (Figure 1c). However, this negative finding does not exclude the possibility of endogenous enzyme activity that is too low to be detected. In sperm capacitated in HTF (Figure 2), enzyme activity was clearly visible. In the head region (Figure 2a), fine granules were localized on the plasma membrane and in the small residual cytoplasmic area, indicating the existence of mAC and sAC activities. In the mid-piece (Figure 2b) and in the tail (Figure 2c), products of reaction were observed as fine granules in the mitochondria and inside the tail. To verify whether the enzyme activity was due to bicarbonate-sensitive AC, sperm were incubated in bicarbonate-free HTF (Figure 3). Under these experimental conditions, the head region still presented an electron-dense deposit attributable to the reaction of bicarbonate-insensitive AC activity, i.e., mAC (Figure 3a), in agreement with recent results (Baxendale and Fraser 2003). Small amounts of reaction products, if any at all, were visible in the mid-piece (Figure 3b) and in the tail (Figure 3c), suggesting that the most abundant form of AC present in these compartments is the bicarbonate-activated form.
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To eliminate the possibility that, under these experimental conditions, we saw sAC localized near carbonic anhydrase, the spermatozoa were incubated for 90 min at 37C in bicarbonate-free HTF under 0% CO2, and forskolin was used as an mAC-specific activator (Figure 3d). AC activity was localized as in Figure 3a, confirming the presence of mAC. It was also absent in the mid-piece and the tail (data not shown). Despite the fact that confocal microscopy and Western blotting results (Zippin et al. 2003
) showed that sAC protein is present in the nuclei of several cell lines and primary fibroblasts, AC activity was not observed in the nuclei of HTF-capacitated spermatozoa (Figure 2a). It should be stressed, however, that spermatozoa are specialized and differentiated cells with highly condensed chromatin until they encounter the oocyte. Therefore, in the activation steps before fertilization these cells do not need genomic events. Nevertheless, the existence of a source of cAMP inside the nucleus cannot be ruled out because it is widely accepted that cAMP functions to regulate gene expression and immunolocalization studies have revealed PKA catalytic and regulatory subunits in the nuclei of several cell lines. Another way of explaining our negative results could derive from the high degree of chromatin condensation that does not allow the cytochemical reaction of AC to occur. Other results of subcellular localization of bicarbonate-sensitive AC agree with sAC compartmentalization findings (Zippin et al. 2003), suggesting that, contrary to its name, sAC, the alternative source for cAMP, is not only a soluble protein but is also specifically targeted to well-defined intracellular compartments, each containing cAMP effectors that are distant from the plasma membrane sources of cAMP. It is becoming clear that molecular anchors, scaffolds, or adaptor proteins allow the signaling events to occur in spatially discrete compartments, in specific regions of most types of cells, as well as in spermatozoa. As already suggested for ATP compartmentalization in spermatozoa, the generation of cAMP also appears to be compartmentalized. In conclusion, specific localization of enzymes such as AC (present results) and PDEs (Lefievre et al. 2002) emphasizes the importance of the local control that these very compartmentalized cells have over enzyme systems composed of cAMP/PKA, AC, and PDEs. |
Acknowledgments |
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We thank Dr M. Kerrigan (Cantab, MA) for valuable linguistic suggestions.
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Footnotes |
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Received for publication August 1, 2003; accepted February 4, 2004
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Literature Cited |
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TopSummaryLiterature Cited |
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Baxendale RW, Fraser LR (2003) Evidence for multiple distinctly localized adenylyl cyclase isoforms in mammalian spermatozoa. Mol Reprod Dev 66:181–189[CrossRef][Medline]
Buck J, Sinclair ML, Schapal L, Cann MJ, Levin LR (1999) Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals. Proc Natl Acad Sci USA 96:79–84
Chen Y, Cann MJ, Litvin TN, Iourgenko V, Sinclair ML, Levin LR, Buck J (2000) Soluble adenylyl cyclase as an evolutionary conserved bicarbonate sensor. Science 289:625–628
Jaiswal BS, Conti M (2001) Identification and functional analysis of splice variants of the germ soluble adenylyl cyclase. J Biol Chem 276:31698–31708
Lefievre L, Jha KI, De Lamirande E, Visconti PE, Gagnon C (2002) Activation of protein kinase A during human sperm capacitation and acrosome reaction. J Androl 23:709–716
Yamamoto S, Kawamura K, James TN (1998) Intracellular distribution of adenylate cyclase in human cardiocytes determined by electron microscopy cytochemistry. Microsc Res Techn 40:479–487[CrossRef][Medline]
Zippin JH, Chen Y, Nahirney P, Kamenetsky M, Wuttke MS, Fischman DA, Levin LR, et al. (2003) Compartimentalization of bicarbonate-sensitive adenylyl cyclase in distinct signaling microdomains. FASEB J 17:82–84
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