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From the Departments of * Pharmacology and
Toxicology and Anatomy and Cell Biology,
Philipps-University Marburg, Germany.
| Correspondence to: Ingrid Boekhoff, Philipps-University Marburg, Department of Pharmacology and Toxicology, Karl-von-Frisch-Straße 1, D-35033 Marburg, Germany (e-mail: boekhoff{at}staff.uni-marburg.de). |
| Received for publication September 12, 2005; accepted for publication December 19, 2005. |
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Abstract |
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Key words: Signal transduction, acrosome reaction, MPDZ
The precision of such complex sequential reactions is usually taken for granted. However, efficiency and specificity of signaling processes are often achieved through temporal and spatial organization of participating molecules (Pawson and Scott, 1997). A common strategy used by cells to ensure specific protein-protein interactions is the organization into functional multiprotein complexes, often realized by PDZ domain proteins (Fanning and Anderson, 1999; Harris and Lim, 2001; Fan and Zhang, 2002; Jelen et al, 2003; van Ham and Hendriks, 2003). The interacting modules of PDZ domain proteins ensure the correct spatial arrangement of transmembrane and cytosolic transduction molecules with respect to each other and/or to specialized regions of a cell, by targeting receptors, ion channels, cytosolic signaling proteins, or other PDZ domains to specific signaling complexes (Fanning and Anderson, 1999). One of the best-studied PDZ-containing proteins is the Drosophila protein INAD, which appears to serve as a scaffold for the G protein–mediated phototransduction cascade in the fly eye: the five PDZ domains in INAD interact with individual proteins of the signal transduction pathway, including phospholipase C ß (PLC-ß), protein kinase C (PKC), and a TRP channel, thus ensuring rapid termination of the photoresponse as well as proper localization of signaling proteins within the rhabdomere (Scott and Zuker, 1998; Montell, 1999; Huber, 2001; Tsunoda et al, 2001; Montell, 2004).
To determine whether the vertebrate INAD-like PDZ domain protein MUPP1, characterized by 13 different PDZ domains (Fan and Zhang, 2002; van Ham and Hendriks, 2003), might be a candidate scaffolding protein mediating sperm signal transduction processes, specific primer pairs matching distinct regions of MUPP1 were used in RT-PCR approaches on cDNA derived from murine testicular tissue as template. Sequence analysis of the amplified PCR fragments demonstrated that MUPP1, previously shown to be highly expressed in distinct areas of the mouse brain (Ullmer et al, 1998; Sitek et al, 2003), also seems be expressed in mouse testicular tissue. To define the testicular cell type(s) in which MUPP1 is detectable at the protein level, a MUPP1-specific antibody was used in immunohistochemical as well as immunocytochemical experiments. The results revealed that MUPP1-specific staining was only detectable in round and elongated spermatids and was strictly confined to the acrosomal region of mature mammalian spermatozoa.
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Experimental Procedures |
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Sperm Preparation
Sperm from adult mice, rats, and bulls were isolated as described
previously (Wennemuth et al,
2000). Briefly, carefully dissected caudae epididymidis were
excised and washed in HS working solution (30 mM HEPES,135 mM NaCl, 5 mM KCl,
2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 10 mM lactic
acid, and 1 mM pyruvic acid), adjusted to pH 7.4 with NaOH. Subsequently,
cleaned tissue was transferred to HS working solution supplemented with 0.5%
BSA and 15 mM NaCO3 (HS/BSA/NaCO3) and incised several
times to allow the sperm to exude to the medium. After a "swim
out" period of 15 mM at 37°C and 5% CO2, the medium was
collected and the sperm was concentrated by centrifugation (5 min, 400 g, RT),
washed 3 times with HS working solution, and used for immunofluorescence and
Western blot analyses.
Capacitation and Acrosome Reaction of Mouse Spermatozoa
For the induction of capacitation, isolated spermatozoa were incubated for
1.5 to 3 hours in HS/BSA/NaCO3 buffer in an atmosphere of 5%
CO2. Each capacitated sample was divided into two aliquots: one was
challenged with the calcium ionophore A23187 (10 to 50 µM) dissolved in HS
(HEPES buffered saline)/BSA/NaCO3, and the other was treated with
diluted DMSO alone to serve as a control. Both aliquots were then incubated at
37°C for 30 minutes and washed 3 times with PBS; subsequently, sperm were
allowed to swim up into the supernatant. An aliquot of the upper phase was
subsequently spotted onto glass microscope slides, and sperm were air-dried
for approximately 20 to 30 minutes at room temperature (RT). Induction of
acrosome reaction was assessed by monitoring the intactness of the sperm head
membranes using FITC-conjugated PNA (Aviles
et al, 1997).
Cell Culture of Germ Cells
The mouse germ cell–derived cell lines GC-1spg and GC-2spd
(Hofmann et al, 1992;
Hofmann et al, 1994), kindly
provided by Prof. G. Aumüller (Department of Anatomy and Cell Biology,
Philipps University Marburg, Germany), were grown in a incubator at 37°C
with 5% CO2 in DMEM supplemented with 1% Pen/Strep and 10% FCS.
cDNA Synthesis and Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Testes and cerebral cortex removed from adult male mice were frozen in
liquid nitrogen and stored at -70°C. For RNA isolation, thawed tissue or
cultured GC-1spg and GC-2spd cells were mixed with Trifast reagent (Peqlab,
Erlangen, Germany), minced on ice, and, subsequently, total RNA was extracted
according to the manufacturer's instructions. After spectrophotometric
determination of total RNA concentration, traces of genomic DNA were removed
by treating 4 µg of RNA with RNase-free DNase I (MBI Fermentas, St
Leon-Rot, Germany), and subsequently reverse-transcribed using the RevertAid
First Strand cDNA Synthesis Kit as described in the manufacturer's
specifications (MBI Fermentas, St. Leon-Rot, Germany).
PCR reactions were performed in a volume of 50 µl containing mouse cDNA from different tissue (20–100 ng), 0.4 mM of each primer, 0.4 mM dNTP, 1.5 mM MgCl2, 2.5 U Taq DNA polymerase (Master-Taq Kit, Eppendorf, Hamburg, Germany), and 5 µl 10 x PCR buffer. The PCR cycling profile was as follows: 5 minutes at 94°C, followed by 33 cycles of denaturation at 94°C for 1 minute, annealing at 60°C for 1 minute, and extension at 72°C for 1 minute.
For amplification of MUPP1 from cDNA derived from mouse tissue, specific primers were used matching a region between PDZ 3 and 4 (Silek et al, 2003). The primers, purchased from MWG Biotech (Ebersberg, Germany), were 5'-ATTGGATCCGCAAGCCAGGAAGCAGAACTTACG-3' (sense, position 1484–1518), and 5'-ATTGAATTCGTTAATTCCCATCC TCTGCC-3' (antisense, position 1684–1715). To exclude an amplification of genomic DNA contamination, only a primer pair spanning exon junctions of the ß-actin gene were used (Ziegler et al, 1992). The ß-actin sense primer was 5'-GGCTACAGCTTCACCACCAC-3' (position 669–688); the antisense primer was 5'-GAGTACTTGCGCTCAGGAGG-3' (position 1074–1093). Based on the primer design, the expected sizes for the PCR products were 250 bp for MUPP1 and 425 bp for ß-actin. Following PCR, 10 µl of the reaction products were analyzed on 1.5% agarose gels, subcloned into pGEM-T Easy (Promega, Mannheim, Germany), and subsequently sequenced (Sequence Laboratories Göttingen, Germany).
Immunocytochemistry and Confocal Microscopy
For immunocytochemical analysis, sperm from different species isolated as
described above were placed on glass slides and allowed to settle for 15
minutes. Adherent cells were subsequently washed with PBS (150 mM NaCl, 1.4 mM
KH2PO4, 8 mM Na2HPO4, pH 7.4), fixed for 2 minutes with
ice-cold (-20°C) methanol, and immediately washed with PBS. All subsequent
steps were performed in a humidified chamber. To reduce nonspecific binding of
antibodies, samples were blocked with PBS supplemented with 10% FCS and
thereafter incubated overnight at 4°C with the anti-MUPP1-antibody diluted
in PBS containing 10% FCS. To check the specificity of antibody binding,
control slides were incubated with PBS/10% FCS only. After removing the
primary antibody by 3 washes for 5 minutes in PBS, cells were incubated with a
dilution of the FITC-conjugated goat anti-rabbit IgG for 1 hour at RT.
Subsequently, slides were washed 3 times with PBS and counterstained with 10
µg/mL propidium iodide. After 3 additional washes in PBS, samples were
coated with fluorescent mounting medium (DAKO) and examined under a Zeiss LSM
510 META laser scanning confocal microscope.
To assess the acrosome reaction, slides were incubated for 30 minutes at RT with a 0.5 µg/mL solution of FITC-coupled PNA diluted in PBS/10% FCS, washed 3 times with PBS, and counterstained with propidium iodide, and subsequently the acrosomal status was evaluated using a confocal laser scanning microscopy.
Immunohistochemistry
Immunohistochemical experiments were performed with paraffin sections of
mouse testis as described recently (Aigner
et al, 2002) as well as Epon-embedded sections of adult mouse
testis. To visualize MUPP1 expression in testicular tissue, semithin sections
of 1 µm were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M
sodium cacodylate buffer (pH 7.3), treated with sodium ethanolate to remove
the embedding medium, and subsequently rehydrated with descending ethanol
solutions (100%, 96%, 80%, and 70%) and distilled water. Antigen retrieval was
performed by an incubation in 10 mM citrate buffer (pH 7.4) at 90°C for 10
minutes. Subsequently, sections were washed in PBS/0.1% Tween 20 and blocked
for 1 hour at RT with 10% normal goat serum in PBS. Control sections were
treated in parallel but were incubated without the primary antibody. The
specific antibody was applied at a dilution of 1:100 in PBS in an overnight
incubation at 4°C in a wet chamber. Sections were washed 3 times with
PBS/0.1% Tween; thereafter, samples were incubated with the Cy3-conjugated
secondary antibody (dilution 1:500). After a 2-hour incubation at room
temperature, excess of secondary antibody was removed by washes as described,
and sections were mounted in fluorescent mounting medium and coverslipped for
microscopic analysis.
SDS/PAGE and Western Blot Analysis
Dissected tissue from mouse cortex, testes and epididymis were homogenized
in buffer containing 10 mM Tris, 2 mM EGTA, 3 mM MgCl2, 0.25%
NP-40, and a protease inhibitor cocktail (Set III, Calbiochem, Schwalbach am
Taunus, Germany). To separate tissue debris, the homogenate was centrifuged
for 10 minutes at 1500 g, and the supernatant was collected. Following
centrifugation at 43 000 rpm for 30 minutes in a Beckman SW 50.1 rotor, the
resulting pellets were resuspended in homogenisation buffer without NP-40, and
the protein concentration was determined according to Bradford
(Bradford, 1976).
For SDS-PAGE, protein samples of mouse tissue and isolated bovine and human sperm, washed 3 times with HS working solution, were mixed with 5 x sample buffer (625 mM Tris/HCl, pH 6.8, 50% glycerol, 5% SDS, 7.5 mM dithiothreitol, 0.05% bromphenol blue) and boiled for 2 minutes; subsequently, 25 µg of protein were applied to each slot of 7% SDS-polyacrylamide gels using the Laemmli buffer system (Laemmli, 1970).
The separated proteins were transferred onto nitrocellulose using a semidry blotting system. The blots were stained with Ponceau S, dried, and stored at 4°C until use. For Western blot analysis, nonspecific binding sites were blocked with 5% nonfat milk powder (Roth, Karlsruhe, Germany) in 10 mM Tris/HCl, pH 8.0, 150 mM NaCl and 0.05% mM Tween 20 (TBST). Subsequently, the blots were incubated overnight at 4°C with specific antibodies against MUPP1 (1:1000 in TBST, containing 3% nonfat milk powder). After 3 washes with TBST, a horseradish peroxidase–conjugated goat anti-rabbit IgG (1:10 000 dilution in TBST with 3% milk powder) was applied. Following 3 washes with TBST, the ECL system (Amersham Biosciences Freiburg, Germany) was used to visualize bound antibodies.
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To define which cell type expresses MUPP1, immunohistochemical experiments were performed by incubating paraffin sections (data not shown) as well as semithin sections of adult mouse testis with the anti-MUPP1 antibody. The superimposed pictures shown in Figure 3C and D demonstrate that immunofluorescence is only detectable in the luminal part of the seminiferous epithelium. A detailed analysis of the spatial MUPP1-expression profile revealed that immunopositive cells within the epithelium are concentrated at distinct stages of spermatogenesis. Whereas no staining was detectable in spermatogonia located in the outer region of the seminiferous tubule, the most prominent immunoreactivity was observed in more differentiated spermatids covering the luminal surface of the seminiferous tubules. To further evaluate the cellular localization of the MUPP1 protein within spermatids, semithin testis sections were examined at a higher magnification (Figure 3F). Most interestingly, immunoreactivity of the MUPP1 antibody was only visible in the acrosomal region of round (Figure 3F, arrowhead) as well as of elongated spermatids (Figure 3F, arrow), whereas no staining was detectable in the tail of elongated spermatids.
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A similar acrosomal protein expression pattern was observed analyzing the subcellular distribution of the anti-MUPP1 antibody staining in isolated rat sperm. Figure 5 shows that antibody staining was not visible in the long tail of rat sperm (Figure 5B), but, in contrast, was strictly confined to the outline of the head where the acrosomal cap is localized (Figure 5C and D).
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To confirm the localization of MUPP1 to the acrosome, the acrosome reaction was induced by incubating capacitated fresh mouse sperm with the calcium ionophore A23187; subsequently, the intactness of sperm head membranes was assessed using FITC-conjugated PNA as a marker (Aviles et al, 1997). Figure 6A shows capacitated spermatozoa stained with FITC-labeled PNA. Lectin binding is detectable over the entire acrosomal cap of spermatozoa, indicating an intact acrosome. In spermatozoa treated with the calcium ionophore A23187, most of the sperm show a reduced patchy fluorescence pattern on the head region, or no staining at all (Figure 6B), signifying that the acrosome reaction has occurred in these spermatozoa. To assess MUPP1 reactivity in acrosome-reacted sperm, immunocytochemical experiments were performed with the anti-MUPP1 antibody. A representative photomicrograph presented in Figure 6C demonstrates that MUPP1 immunofluorescence was lost in the acrosomal region following the acrosome reaction, suggesting that MUPP1 was lost during this process.
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The results described so far strongly suggest that rodent and mammalian sperm express a related, if not identical, form of this PDZ-domain protein. To evaluate whether there are differences in the subcellular distribution pattern of MUPP1 in different mammalian spermatozoa, immunohistochemical experiments were performed with semithin sections of bull testicular tissue, as well as indirect immunofluorescence experiments with isolated bovine and human sperm.
Figure 8A and C present an overview of the immunohistochemical localization of MUPP1 in two different seminiferous tubules of adult bull testis showing various stages of developing spermatozoa. Interestingly, the MUPP1 antibody shows a crescent-shaped labeling pattern within the seminiferous epithelium. At a higher magnification, MUPP1 can be seen to be associated with the acrosome of round developing spermatids (Figure 8B and D).
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To determine the subcellular localization in mature spermatozoa, isolated bovine as well as human sperm were analyzed for MUPP1 antibody reactivity. Figure 9 shows propidium iodide–counterstained bull sperm with their characteristic paddle-shaped heads. It is obvious that no MUPP1-labeling was detectable in the flagellum, but immunoreactivity was exclusively concentrated in the head of the bull spermatozoa (Figure 9C). To evaluate whether bovine sperm also show a confined acrosomal MUPP1 expression pattern, the distinct shape of the acrosome was visualized using an FITC-conjugated PSA as a marker for this organelle (Mendoza et al, 1992) (Figure 9B). The higher magnification shown in Figure 9D clearly demonstrates that staining is only visible in the apical acrosomal region and most notably, in the equatorial segment of the acrosome, the initial site of egg membrane fusion (Flesch and Gadella, 2000).
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Discussion |
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At present, it is generally accepted that PDZ-domain proteins function as structural organizers of signaling molecules in diverse cellular compartments, including neuronal synapses, cell-cell junctions of epithelial cells, and subcompartments of sensory cells (Fanning and Anderson, 1999; Huber, 2001; van Ham and Hendriks, 2003; Kim and Sheng, 2004). The predominant expression of MUPP1 in the anterior acrosomal region as well as in the equatorial segment of bovine and human spermatozoa suggests that MUPP1 may assemble similar, if not identical, signaling molecules mediating primary acrosomal events in fertilizing spermatozoa of different mammalian species. Although these primary processes include extremely different events such as capacitation, egg recognition, Ca2+-triggered exocytotic events, and sperm/egg membrane fusion (Breitbart, 2002; Primakoff and Myles, 2002; Fukami et al, 2003; Lopez-Gonzalez et al, 2003), information about the few described MUPP1-ligands along with the 13 PDZ domains of MUPP1 may provide a framework for testing different hypotheses.
Initially, MUPP1 was identified as a protein that interacts with the C-terminus of the serotonin type 2C receptor (Ullmer et al, 1998; Becamel et al, 2001; Parker et al, 2003). Further studies showed that MUPP1 is also a cytoplasmic ligand for the membrane-spanning NG2 proteoglycan (Barritt et al, 2000), viral transforming oncoproteins (Lee et al, 2000; Massimi et al, 2004), neuronal Rho-GEF (Penzes et al, 2001), the tandem pleckstrin homology domain protein (TAPP1) (Kimber et al, 2002), as well as components of the epithelial cell tight junction (Hamazaki et al, 2002; Jeansonne et al, 2003; Coyne et al, 2004). In addition, recent studies revealed that heterologously-expressed MUPP1 interacts with GST-fusion protein constructs of the receptor tyrosine kinase c-kit (Mancini et al, 2000). C-kit is known to be involved in spermatogenesis (for review see Prasanth et al, 2004), but is also present in the acrosome region of mammalian testicular spermatozoa (Sandlow et al, 1996; Feng et al, 1997; Sandlow et al, 1997). Remarkably, stimulation of sperm with stem cell factor, the endogenous ligand for c-kit, significantly increased the percentage of sperm undergoing an acrosome reaction (Feng et al, 1998; Feng et al, 2005), indicating that c-kit plays a functional role in sperm capacitation and/or the acrosome reaction. Although one might suggest that MUPP1 may function to physically bring together the identified different downstream signaling pathways of c-kit in spermatozoa (for review see Rossi et al, 2003), neither co-immunoprecipitation approaches nor GST pull-down assays with GST–c-kit or MUPP1-fusion proteins and soluble fractions of testicular tissue provide any evidence for an interaction of both proteins in mammalian spermatozoa (data not shown).
In addition to the interaction of MUPP1 with c-kit, recent studies have revealed that MUPP1 is also a component of the NMDA-receptor signaling complex at excitatory synapses of hippocampal neurons (Krapivinsky et al, 2004). Interestingly, MUPP1 interaction with SynGAP, a synaptic GTPase-activating protein, is controlled by an NMDA receptor–induced increase in cytosolic calcium. The observation that calcium allows a controlled PDZ-mediated assembly and disassembly of MUPP1-binding partners, together with the predominant expression profile of MUPP1 within the acrosomal region of spermatozoa and the observed MUPP1 release during acrosomal exocytosis (Figure 6), suggests that MUPP1 might be involved in targeting molecules that elicit calcium-triggered acrosomal exocytosis. Although the acrosome reaction differs from other known exocytotic events in that sperm only contain a single secretory vesicle, acrosomal exocytosis exhibits a striking analogy to the complex process of classical calcium-triggered, regulated exocytosis, known to be mediated by the SNARE protein complex, composed of integral components of the vesicle and plasma membrane as well as soluble bridging factors (for review see Mayer, 2002; Jahn et al, 2003; Sudhof, 2004). Interestingly, proteins homologous to members of the membrane-bridging SNARE protein fusion machinery have recently been shown to be localized to the acrosomal region of spermatozoa and have been demonstrated to be responsible for acrosomal membrane fusion (Michaut et al, 2000; Michaut et al, 2001; Hutt et al, 2002; Ramalho-Santos et al, 2002; Tomes et al, 2002; Yunes et al, 2002; Tomes et al, 2005). Currently, we do not know whether MUPP1 is definitely localized to the sperm cytosol and thus capable of directly interacting with components of the SNARE complex. However, exocytosis is a highly regulated multistage process including targeting, tethering, priming, docking, and fusion. Thus it could be possible that MUPP1 is involved in coordinating and guiding the flow of regulatory information necessary to ensure an unobstructed, sequential acrosomal exocytosis. However, it is also conceivable that MUPP1 may function in physically bringing together components of the SNARE fusion complex with molecules involved in ZP3-induced increase in cytosolic calcium by using calcium as a critical regulator of the MUPP1-protein interaction. In this context, it is of interest that MUPP1 shows striking structural homology to the PDZ-domain protein INAD in photoreceptor cells of Drosophila. Remarkably, INAD is capable of interacting with members of the TRP channel family (Montell, 2004). Thus, it will be interesting to examine whether the calcium-permeable TRPC2 channel, recently described to be involved in producing the acrosome reaction preceding sustained increases in intracellular Ca2+ concentration (Jungnickel et al, 2001), is an interaction partner of MUPP1 in spermatozoa and whether Enkurin, a novel scaffolding protein identified in murine spermatozoa which interacts with TRPC2 (Sutton et al, 2004), is also capable of binding to MUPP1.
Interestingly, recent studies have revealed that PDZ domain proteins also play a crucial role in the establishment of sperm polarity, which is required for spermatid differentiation: polarization of round spermatids requires the assembly of a cell-polarity protein complex, in which the PDZ domain protein PATJ/INADl (Philipp and Flockerzi, 1997) with its 10 PDZ domains binds several partners, thus functioning as an adapter protein linking integral cell adhesion molecules, like JAM-C, to the actin terminal web (Gliki et al, 2004). In addition to their functional role in establishing cell polarity, PDZ domain proteins also appear to be involved in the structural organization of the maintenance of cell polarity. Remarkably, maintenance of polarization in photoreceptor cells is realized by the generation of multiprotein complexes organized by MUPP1 interactions (van de Pavert et al, 2004). The observation that MUPP1 is only detectable in more differentiated round and elongated spermatids (Figures 4 and 8), but not in all generations of spermatogenic cells like JAM-C (Gliki et al, 2004), does not favor a functional role of MUPP1 in establishing cell polarity. However, whether MUPP1 is involved in maintaining sperm polarization, in particular sustaining the sperm acrosome, has to be evaluated in future studies.
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Acknowledgments |
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Footnotes |
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These authors contributed equally to this work. 
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