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From the * Center for Animal Transgenesis and Germ
Cell Research, Department of Clinical Studies, University of Pennsylvania New
Bolton Center, Kennett Square, Pennsylvania;
Department of Biology, University of
Pennsylvania, Philadelphia, Pennsylvania; and
The James A. Baker Institute for Animal
Health, College of Veterinary Medicine, Cornell University, Ithaca, New
York.
| Correspondence to: Regina M Turner, New Bolton Center, University of Pennsylvania School of Veterinary Medicine, 382 West Street Rd, Kennett Square, PA 19348 (e-mail: rmturner{at}vet.upenn.edu). |
| Received for publication October 25, 2006; accepted for publication April 13, 2007. |
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Abstract |
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Key words: Motility, flagellum
There is evidence to suggest that the cAMP-dependent phosphorylation of
flagellar proteins is involved in the initiation and maintenance of sperm
motility (Tash and Means,
1982; Tash and Means,
1983; San Augustin and Witman,
1994). Since PKA is a major downstream target of cAMP in sperm, it
likely that this kinase plays a central role in these phosphorylation events
(Visconti et al, 1997). As
further support of a role for PKA in sperm function, it has been shown that
mice that lack the male germ cell-specific catalytic subunit of PKA
(C
2) are infertile due to several abnormalities, including aberrations
of motility (Nolan et al,
2004).
Extracellular Ca2+ is required for motility in most epididymal sperm samples, and Ca2+ is known to regulate both activated and hyperactivated motility (Suarez et al, 1987; Tash and Means, 1987; Lindemann and Goltz, 1988; White and Aitken, 1989; Yanagimachi, 1994; Ho et al, 2002). One mechanism by which Ca2+ is directly linked to flagellar function is through its regulation of the atypical, "soluble" adenylyl cyclase, sAC, which generates cAMP and is required for sperm motility (Jaiswal and Conti, 2003; Litvin et al, 2003; Esposito et al, 2004).
Calmodulin (CaM) is a ubiquitous, highly conserved, 17-kd protein that serves as a classical intracellular Ca2+ receptor (Means et al, 1982). At least some of the effects of Ca2+ on the flagellum are likely to be achieved through CaM, since inhibition of CaM decreases sperm motility (White and Aitken, 1989; Ahmad et al, 1995; Si and Olds-Clarke, 2000). Interestingly, the effects of Ca2+ on sAC are independent of CaM (Jaiswal and Conti, 2003; Litvin et al, 2003), which suggests that Ca2+ affects motility via multiple pathways, only some of which require CaM.
Since sperm are highly compartmentalized, proteins must be targeted accurately to the appropriate region(s) of the cell. Thus, proteins involved directly in the regulation of motility typically localize to the flagellum. We use indirect immunofluorescence to show that CaM is present in the principal piece of the flagellum. In addition, we show that a portion of sperm CaM is insoluble, consistent with its localization to the cytoskeleton and similar to the extraction profile of a known fibrous sheath (FS) protein (the pro-domain of pro-AKAP4). These findings indicate that a pool of CaM localizes to the flagellum and possibly to the FS, an insoluble accessory structure that is found exclusively in the principal piece of the mammalian flagellum. We also suggest that CaM is involved in the regulation of sperm motility, since a CaM inhibitor decreased motility. This inhibition was largely reversed by stimulation of PKA, but only when lactate and pyruvate were present in the medium. Furthermore, the Ca2+/CaM-dependent protein kinase IIß (CaMKIIß), which is a downstream target of Ca2+/CaM, colocalized with CaM in the principal piece, which suggests that a Ca2+/CaM/CaMKII signaling pathway is present in the sperm principal piece.
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Materials and Methods |
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The secondary antiserum used for most of the immunoblotting experiments was HRP-conjugated donkey anti-rabbit IgG (Amersham Biosciences, Piscataway, NJ). For membranes probed with the anti-CaMKIIß antiserum, an alkaline phosphatase conjugated goat anti-mouse secondary antiserum was used (Jackson Immunoresearch Laboratories, West Grove, Pa). The secondary antiserum used for indirect immunofluorescence was Alexa Fluor 594-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, Ore).
Tissue and Cell Preparation and Protein Isolation
Sperm were collected from the cauda epididymides of retired breeder CD-1
mice (Taconic ICR) (Travis et al,
2001a). Sperm suspensions were washed in PBS that contained a
protease inhibitor cocktail (Complete Tablets; Roche, Indianapolis, Ind).
After the final wash, sperm pellets were mixed with SDS sample buffer that
contained 40 mM DTT and boiled for 5 minutes. Samples were centrifuged and the
supernatants, which contained the extracted proteins, were saved.
Mixed germ cells were prepared from decapsulated adult mouse testes by sequential dissociation with collagenase and trypsin/DNAse I, and individual germ cell types were separated by sedimentation velocity (STAPUT), as previously described (Romrell et al, 1976; Bellve et al, 1977). Protein was extracted from the resulting germ cell pellets as described above for sperm.
The amount of protein in each sample was determined using the Bradford assay with the test sample diluted such that the final DTT and SDS concentrations were within the limits specified by the manufacturer of the assay (Bio-Rad Laboratories, Hercules, Calif).
Immunoblotting
Proteins were separated under reducing conditions by SDS-PAGE on either 9%
(for detection of CaMKII), 4–15% gradient precast (for detection of CaM)
or 12.5% (for detection of the pro domain of pro-AKAP4) gels. After
electrophoresis, proteins were electrophoretically transferred to
polyvinylidene fluoride membranes (Immobilion-P Transfer Membranes, Millipore
Corp, Bedford, Mass; 10 µg protein/lane unless otherwise stated). After
blocking and washing, the membranes were probed for 1 hour at room temperature
or overnight at 4°C with the appropriate primary antibody: anti-CaM
(1:5000 [v/v]), anti-DYSKIPSEN (1:5000 [v/v]) or anti-CaMKIIß (1:500
[v/v]). The membranes were washed and then probed with the appropriate
secondary antibody diluted 1:5000 (v:v), as previously described
(Carrera et al, 1994;
Johnson et al, 1997;
Eshete and Fields, 2001).
After additional washes, blots probed with HRP-conjugated secondary antibodies were overlaid with ECL reagents (Amersham, Piscataway, NJ) and developed before being exposed to Reflection film (New England Nuclear, Boston, Mass). Blots probed with an alkaline phosphatase-conjugated secondary antibody were developed using ECF substrate (Amersham), and fluorescence was detected using the Storm 860 system (Molecular Dynamics, Sunnyvale, Calif).
Immunoblots that were to be probed with the anti-CaM antibody were treated as previously described, to improve the retention of CaM on the membrane (Yang et al, 2001). Briefly, the polyacrylamide gels were presoaked in KP buffer (25 mM KH2/K2HPO4 [pH 7.0]) for 15 minutes prior to electrophoretic transfer to membranes. The membranes were prewet in methanol and washed for 15 minutes in the KP buffer prior to transfer. Transfer of proteins was performed in KP buffer overnight at 4°C. Following transfer, membranes were soaked in 0.2% glutaraldehyde in KP buffer, and then blocked and probed using the techniques described above.
Sperm Fractionation
Fractionation of sperm was carried out as previously described
(Travis et al, 2001b).
Briefly, sperm were lysed by Dounce homogenization and sonication in PBS in
the presence of protease inhibitors (Complete Protease Inhibitor Cocktail;
Boehringer Mannheim, Mannheim, Germany). The mixture then was centrifuged at
10 000 x g for 10 minutes at 4°C and separated into
supernatant (S10) and pellet (P10) fractions. An aliquot of the S10 fraction
was then centrifuged at 100 000 x g for 1 hour, yielding a
pellet of membranes and associated proteins (P100) and a supernatant fraction
(S100). An aliquot of the membrane pellet was suspended in PBS that contained
protease inhibitors and 0.5% Triton X-100, incubated for 15 minutes at
4°C, and centrifuged again at 100 000 x g for 1 hour,
yielding a detergent-soluble supernatant and a detergent-insoluble pellet. The
appearance of a protein in the detergent-insoluble membrane fraction has
sometimes been used to indicate localization to membrane subdomains known as
lipid rafts (regions enriched in sterols and sphingolipids, as opposed to
phospholipids). However, assigning this designation based solely on detergent
insolubility is susceptible to artifact, due to perturbation of the membranes
by the detergent from their physiologically native state
(Munro, 2003). Nonetheless,
the differentiation between detergent-soluble and detergent-insoluble
partitioning is a useful initial biochemical screen, which can provide
information on how a protein might associate with a membrane. We performed 3
separate membrane preparations, and performed immunoblot analysis on each of
the 3 associated blots. Identical results were obtained each time.
Indirect Immunofluorescence of Sperm and Testis Sections
Ten-microliter aliquots of sperm diluted in PBS to 60 x
106 cells/mL were transferred onto microscope slides, smeared, and
air-dried. Cells were then acetone-fixed on the slides for 15 seconds, dried,
and then permeabilized in cold methanol for 15 minutes. Slides were washed
with PBS and blocked for 1 hour at room temperature in 3% non-fat milk, before
being washed again in PBS.
Sperm were incubated in the appropriate secondary antibody diluted to 5 µg/mL in 10% goat serum. The slides were then washed and counterstained with 4',6-diamidino-2-phenylindole (DAPI) before being coverslipped. Images were obtained using a fluorescent microscope (Leica Microsystems, Bannockburn, Ill) equipped with the Open Lab software (Improvision, Lexington, Mass). Paired sample and control images were exposed for equal amounts of time.
Assessment of Sperm Motility Following Addition of CaM Antagonist
N-(6-Aminohexyl)-5-chloro-1-naphthalenesufonamide hydro-chloride (W-7) is a
water soluble and cell-permeable competitive antagonist of CaM (Calbiochem,
San Diego, Calif). The W-7 IC50 values for CaM-dependent enzymes range from 25
µM to 50 µM (Hidaka and Kobayashi,
1992). Concentrations of up to 100 µM have been used to assess
the role of CaM in sperm motility (Si and
Olds-Clarke, 2000; Morita et
al, 2006). A stock solution of W-7 was prepared in DMSO to a final
concentration of 10 mM and stored at –20°C.
Male Swiss Webster mice (Taconic, Germantown, NY) between 10 and 12 weeks of age were killed by cervical dislocation. The cauda epididymides were removed, minced, and placed in a modified Whitten medium (22 mM HEPES [pH 7.4], 1.2 mM MgCl2, 100 mM NaCl, 4.7 mM KCl, 1 mM pyruvic acid, 4.8 mM lactic acid, 3 mM CaCl2, 5.5 mM glucose, 10 mM sodium bicarbonate) (Travis et al, 2001a) for 10 minutes to allow the sperm to swim out. The resulting liquid was filtered through a 70-µm cell strainer (BD Biosciences, San Jose, Calif) to remove tissue debris, and then placed in a water bath at 37°C. The motility of untreated sperm was determined using a computer-assisted sperm motion analysis system (IVOS Analyzer; Hamilton Thorne Research, Beverly, Mass). A progressively motile cell was defined as having Path Velocity (VAP) greater than 79 µm/s and Straightness (STR) greater than 60%. VAP is defined as the total distance a sperm moves over time, while STR is a measure of the straightness of the sperm track (defined as progressive velocity divided by VAP). The sperm solution was loaded into a 20-µm chambered slide (Leja Products, Amsterdam, The Netherlands) and viewed with the 10x objective on the IVOS Analyzer. Distal droplets were excluded from analysis using the sort option with the following parameters: head size greater than 9 pixels and beat frequency of more than 4 µm/s.
To analyze the effects of W-7 on sperm motility, 100-µL aliquots of sperm were placed in different microcentrifuge tubes and incubated with 2.5 µM to 200 µM W-7 in DMSO. Solvent controls were treated with an equivalent amount of DMSO. The resulting mixtures were incubated at 37°C and sperm motility was assessed at 0, 5, 10, 20, and 30 minutes after the addition of DMSO/W-7. The motility results are expressed as both the number of progressively motile cells divided by the number of total cells (% progressive motility) and as the number of motile cells divided by the number of total cells (% total motility).
To test whether components of the sAC/PKA pathway could restore motility to CaM-inhibited cells, sperm suspended in complete medium were treated with 1 mM dibutyryl cAMP (db-cAMP; Sigma Chemical Co, St Louis, Mo) and 100 µM 3-isobutyl-1-methylxanthine (IBMX, Sigma) or 3 mM 8-bromo cAMP (Sigma) and 100 µM IBMX, all diluted in water, 1 minute after treatment with 100 µm W-7. In a parallel assay, the same treatment was given to sperm suspended in medium with lactate and pyruvate omitted, in order to reproduce the conditions used previously (Si and Olds-Clarke, 2000). Sperm motility was recorded prior to and immediately after treatment. Control samples were treated with W-7, followed by water without cAMP/IBMX.
Statistical Analysis
The Wilcoxon signed rank test was used to determine differences between
sample motilities. A P-value of < .05 was considered statistically
significant.
Ethical Guidelines
The care and use of all animals used in this study were within standard
ethical guidelines, as approved by the University of Pennsylvania or Cornell
University Institutional Animal Care and Use Committees.
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Results |
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CaM Is Found in the Soluble and Insoluble Fractions of the Sperm Cell
To determine the solubility of CaM in sperm, we fractionated mouse sperm
and performed immunoblot analysis of each fraction using the anti-CaM
antibody. We identified pools of CaM in the insoluble (P10) sperm fraction,
the soluble S10 sperm fraction, and some of its derivatives, including the
S100, total membrane, and detergent-soluble membrane fractions
(Figure 2). No CaM was found in
the detergent-insoluble membrane pellet.
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Thus, both the pro-domain and CaM are present in an insoluble fraction of cauda epididymal sperm. While the pro-domain was exclusive to this fraction, the CaM fractionation pattern was broader. These findings are consistent with the localization of a pool of CaM to the FS, and suggest that CaM has roles both within and outside the flagellum.
Sperm Motility Is Inhibited by a CaM Antagonist and Partially Recovers After Stimulation of PKA
As CaM is present in the principal piece and since Ca2+ has long
been implicated in the regulation of flagellar motility, we hypothesized that
some of the effects of Ca2+ on sperm motility might be mediated by
CaM. To test this hypothesis, we examined the effects of a CaM antagonist,
W-7, on murine sperm motility. Cauda epididymal sperm were allowed to swim out
into modified Whitten medium, and were then exposed to various concentrations
of W-7. As previously reported, sperm treated with W-7 showed
concentration-dependent declines in total motility and progressive motility,
as compared to controls (data not shown)
(Si and Olds-Clarke, 2000).
The decline in progressive motility was due to declines in both VAP and STR.
The W-7 concentration of 100 µM proved to be the most effective in our
hands (Figure 3). Previous
studies have demonstrated that this reduction in motility is not due to a
toxic effect of W-7 on sperm (Si and
Olds-Clarke, 2000).
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It has been reported that the effects of Ca2+ on sAC are independent of CaM (Jaiswal and Conti, 2003; Litvin et al, 2003). If this is correct, part of the effect of Ca2+/CaM on motility must be achieved through a pathway that is separate from sAC/PKA or from an effect on that pathway either upstream or downstream of sAC. We tested whether components of the sAC/PKA pathway could restore motility to W-7-treated sperm suspended in a complete medium that contained glucose, pyruvate, and lactate. Motility was inhibited by the addition of 100 µM W-7. One minute after exposure to W-7, sperm were treated with either 1 mM db-cAMP and 100 µM of the phosphodiesterase inhibitor IBMX or 3 mM 8-bromo cAMP and 100 µM IBMX. The addition of either db-cAMP/IBMX or 8-bromo cAMP/IBMX resulted in significant increases in both total and progressive sperm motility associated with increases in both VAP and STR (P < .05) (Figure 3). This restored motility approached the motility levels of the untreated controls (P = .06).
The experiment was then repeated in the same medium, except that lactate and pyruvate were omitted (Si and Olds-Clarke, 2000). Under these conditions, there was no increase in either the total or progressive motility of sperm treated with W-7/cAMP/IBMX compared to sperm treated only with W-7 (Figure 3). These data suggest that the stimulation of PKA can partially compensate for the reduction in sperm motility seen following inhibition of CaM by W-7 but that lactate and pyruvate are required for this rescue to occur. In addition, these data confirm that the reduction in sperm motility seen following the addition of W-7 is not due to a toxic effect of W-7 on sperm.
CaMKII Is Present in Sperm and Localizes to the Principal Piece
In somatic cells, many of the actions of Ca2+ are mediated by
its interaction with CaM and the subsequent activation of a variety of
Ca2+/calmodulin-dependent protein kinases (CaMK). Several CaMK
isoforms have been reported in the testis. These include CaMKII, CaMKIV and
Pnck in the mouse, and CamKIIN, CaMK-gr, and CaMKIßI in the rat
(Frangakis et al, 1991;
Jones et al, 1991;
Naito et al, 1997;
Gardner et al, 2000;
Moriya et al, 2000;
Chang et al, 2001). More
recently, CamKIV and CaMKII have been identified in the flagella of
human and bovine sperm, respectively
(Ignotz and Suarez, 2005;
Marin-Briggiler et al, 2005).
CaMKII has also been identified in ascidian sperm, where it plays a role in
sperm motility (Nomura et al,
2004).
Given the reports on the presence of CaMK isoforms in testes and sperm, and
since we had evidence that CaM was involved in the regulation of murine sperm
motility, we searched for a CaMK isoform in murine sperm. Since CamKII had
been reported in murine testes, we performed immunoblot analysis on mouse
sperm protein extracts using an antibody against CaMKIIß and identified a
band of Mr 62 500 (Figure 4A).
This band was identical to a control band identified in hippocampus extract
(Kelly et al, 1984;
Scholz et al, 1988). Note that
the anti-CaMKIIß antibody it not absolutely specific for the
ß-isoform. This antibody also recognizes CaMKII
, particularly if
the isoform is more abundant. Therefore, our results raise the
possibility that the isoform is also present in sperm.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Our findings of an insoluble pool of CaM in sperm and of CaM in the principal piece are consistent with, though not proof of, the existence of a pool of CaM in the FS (Tash and Means, 1987; Tash et al, 1988). It has been suggested that PKA is anchored to the FS through 1 or more AKAPs (Carrera et al, 1994; Mei et al, 1997; Miki and Eddy, 1998; Turner et al, 1998; Mandal et al, 1999; Vijayaraghavan et al, 1999). If CaM also is associated with the FS, then components of both of the major sperm motility signaling pathways (cAMP/PKA and Ca2+ signaling through CaM) find homes in this important accessory structure. Regardless of whether or not CaM is present in the FS, its presence in the flagellar principal piece provides indirect evidence for a role of CaM in the regulation of sperm motility. Inhibition of sperm motility by the addition of the CaM antagonist W-7 provides more direct evidence for this role.
It has previously been reported that 8-br-cAMP and IBMX do not restore motility to W-7-treated sperm when the sperm are incubated in a medium that contains glucose but lacks pyruvate and lactate (Si and Olds-Clarke, 2000). Our results concur with this finding. However, similar to reports on demembranated ascidian sperm (Nomura et al, 2000), we found that if lactate and pyruvate were present in the medium, then IBMX and either 8-br-cAMP or db-cAMP could largely compensate for the loss of function of CaM. Taken together, these findings provide insight into the signaling and metabolic control of mammalian sperm motility. In the mouse, it has been demonstrated that glycolysis produces ATP in the principal piece that is essential for fully normal sperm motility and for the phosphorylation events that are believed to facilitate the regulation of motility (Travis et al, 2001a; Miki et al, 2004; Mukai and Okuno, 2004). Our data show that CaM functions in the principal piece in that, when CaM is inhibited, sperm motility is significantly decreased. In addition, without substrates for oxidative respiration (lactate and pyruvate), and even in the presence of a substrate for glycolysis (glucose), increased intracellular cAMP cannot restore motility to CaM-inhibited sperm. This suggests the possibility that CaM is involved in the regulation of glycolysis or in the utilization of glycolytic ATP. There have been several previous reports linking Ca2+/CaM to the regulation of glycolytic enzymes in somatic cells (Ashkenazy-Shahar et al, 1998; Ashkenazy-Shahar and Beitner, 1999; Singh et al, 2004). Thus, it is possible that W-7, by inhibiting CaM, indirectly inhibits glycolysis.
As they are substrates for oxidative respiration, lactate and pyruvate may be able to compensate partially for the lack of glycolytic ATP by enabling the production of ATP in the midpiece. It has been shown that the production of ATP in the midpiece in the absence of glycolytic ATP can support normal motility for short periods of time (Mukai and Okuno, 2004). It is unclear whether some of this ATP is able to move to the proximal principal piece or whether it remains entirely in the midpiece. Regardless, these data show the partially compensatory abilities of the metabolic pathways that support flagellar motility, and suggest a new potential function for CaM in sperm (ie, the regulation of glycolysis in the principal piece).
Other than the addition of lactate and pyruvate to our medium, there were other more subtle differences between our experiments (in which agonists of the PKA pathway were able to restore motility to CaM-inhibited sperm) and those of Si and Olds-Clarke (in which agonists of the PKA pathway were unable to restore motility to CaM-inhibited sperm). For example, we utilized a different mouse strain in our experiments. Therefore, we can not rule out the possibility that the relationship of the CaM pathway to the PKA pathway may not be identical in all genetic backgrounds.
Although the addition of cAMP/IBMX to W-7-treated sperm significantly increased motility compared to sperm treated with W-7 alone, it should be noted that motility was still slightly but significantly less than the motility seen in the untreated control sample (P = .06 for 8-bromo cAMP/IBMX). This finding still argues strongly for our hypothesis that agonists of the PKA pathway rescue the inhibited CaM pathway. However, another possible explanation for this incomplete restoration of motility is that, at the 100 µM concentration used in our study, W-7 may have been inhibiting other enzymes in addition to CaM. If this was the case, then it is possible that cAMP/IBMX rescues these other pathways, and that the remaining sperm motility deficit that persists in the presence cAMP/IBMX is due to the still-inhibited CaM pathway.
Ca2+ also affects sperm motility through its role as a regulator of the predominant flagellar cyclase, sAC, which catalyzes the synthesis of cAMP to activate the PKA pathway. This cyclase is molecularly and biochemically distinct from the transmembrane ACs (tmACs), in part because sAC is uniquely sensitive to both bicarbonate and Ca2+ (Buck et al, 1999; Chen et al, 2000; Wuttke et al, 2001; Liguori et al, 2004). However, the effects of Ca2+ on sAC are independent of CaM (Jaiswal and Conti, 2003; Litvin et al, 2003). Therefore, although it is known that sAC is required for sperm motility (Esposito et al, 2004) and sAC is regulated by Ca2+, the effect of CaM on motility is not achieved via this cyclase.
These observations support a model in which Ca2+ affects motility at 2 different points within the sAC pathway. Alternatively, separate calcium signaling pathways may exist; one that is independent of CaM (eg, sAC/PKA) and one that is not. Since deletion of the sAC gene results in immotile sperm, it is clear that Ca2+/CaM cannot compensate for the loss of sAC function. However, our data suggest that IBMX/cAMP (ie, agonists of the PKA pathway) can restore motility when CaM is inhibited. Thus, components of the sAC/PKA pathway can compensate for a loss of function in the Ca2+/CaM component of the pathway(s), provided that the metabolic substrates pyruvate and lactate are present. The mechanism for this restoration of motility provides a novel avenue for further investigation. One possible model that is consistent with these data is that Ca2+/CaM functions upstream of sAC/PKA.
In Chlamydomonas, it has been shown that Ca2+ may act
through CaM and CaMKII to control flagellar motility by regulating
dynein-driven microtubule sliding (Smith,
2002). Furthermore, in ascidian sperm, it has recently been
documented that CaMKII mediates sperm-activating and -attracting factor
(SAAF)-induced motility activation (Nomura
et al, 2004). Conflicting data exist regarding a potential role
for CaMK isoforms in mammalian sperm motility. One group has reported that
targeted mutagenesis of the CaMKIV isoform has no effect on male fertility
(Blaeser et al, 2001), while
another group has reported that loss of CaMKIV results in male sterility in
association with decreased sperm motility
(Wu et al, 2000). Inhibitors
of CaMKIV have been reported to decrease human sperm motility
(Marin-Briggiler et al, 2005)
and CaMKII stimulates hyperactivation in bovine sperm
(Ignotz and Suarez, 2005). Our
data indicate that an isoform of CaMKII is present in the principal piece of
murine sperm. This is probably CaMKIIß but it could also be
CaMKII. Taken together, these and additional data
(Weinman et al, 1986;
Bendahmane et al, 2001) suggest
that a Ca2+/CaM/CaMKII pathway in the sperm principal piece is
active in mammalian sperm motility regulation.
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Acknowledgments |
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Footnotes |
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Present address: Laboratorio de Biologia Celular y Molecular, Instituto de
Histologia y Embriologia, CC 56, Facultad de Ciencias Medicas, Universidad
Nacional de Cuyo, 5500 Mendoza, Argentina. 
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