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Biol Reprod. 2008 November; 79(5): 999–1007.
Prepublished online 2008 July 30. doi:  10.1095/biolreprod.107.067058
PMCID: PMC2714999

Cyclic 3′,5′-AMP Causes ADAM1/ADAM2 to Rapidly Diffuse Within the Plasma Membrane of Guinea Pig Sperm1


Because sperm cannot synthesize new proteins as they journey to the egg, they use multiple mechanisms to modify the activity of existing proteins, including changes in the diffusion coefficient of some membrane proteins. Previously, we showed that during capacitation the guinea pig heterodimeric membrane protein ADAM1/ADAM2 (fertilin) transforms from a stationary state to one of rapid diffusion within the lipid bilayer. The cause for this biophysical change, however, was unknown. In this study we examined whether an increase in cAMP, such as occurs during capacitation, could trigger this change. We incubated guinea pig cauda sperm with the membrane-permeable cAMP analog dibutyryl cAMP (db-cAMP) and the phosphodiesterase inhibitor papaverine and first tested for indications of capacitation. We observed hypermotility and acrosome-reaction competence. We then used fluorescence redistribution after photobleaching (FRAP) to measure the lateral mobility of ADAM1/ADAM2 after the db-cAMP treatment. We observed that db-cAMP caused roughly a 12-fold increase in lateral mobility of ADAM1/ADAM2, yielding diffusion similar to that observed for sperm capacitated in vitro. When we repeated the FRAP on testicular sperm incubated in db-cAMP, we found only a modest increase in lateral mobility of ADAM1/ADAM2, which underwent little redistribution. Interestingly, testicular sperm also cannot be induced to undergo capacitation. Together, the data suggest that the release of ADAM1/ADAM2 from its diffusion constraints results from a cAMP-induced signaling pathway that, like others of capacitation, is established during epididymal sperm maturation..

Keywords: ADAM1, ADAM2, cAMP, capacitation, fertilin, fertilization, fluorescent redistribution after photobleaching, FRAP, gamete biology, membrane diffusion, sperm, sperm capacitation, sperm maturation


Sperm must be able to adapt and function not only in the different environments within the male reproductive tract but also through the various milieus of the female reproductive tract. It is noteworthy that sperm accomplish these adaptations and changes in activity without synthesizing new proteins. Once sperm leave the testis, they shed their synthetic machinery to become the extremely streamlined gametes necessary to swim to and fertilize the egg. However, without the ability to produce new proteins, sperm must use other mechanisms to detect and respond to changing environments.

Three mechanisms sperm can use to affect functional changes on preexisting proteins are 1) posttranslational modifications (such as proteolytic cleavage or phosphorylation); 2) the relocalization of proteins from one domain on the sperm to another, thus allowing for new protein-protein and protein-lipid interactions; and 3) changes in a membrane protein's ability to diffuse within the lipid bilayer (such as the alteration of a protein from an immobile to a mobile state within the membrane) [112]. This last type of event can occur by a number of mechanisms, including loss of a binding interaction that restricts protein mobility (e.g., by posttranslational modification of the binding partner), a reduction or increase in cross-linking of a membrane associated network that restricts the free path of the protein, or a change in the surrounding lipid composition such as a change in liquid-ordered lipid domains that could potentially restrict long-range mobility of the protein.

ADAM1/ADAM2 (fertilin) is a heterodimeric, transmembrane protein, the exact function of which remains unclear but which appears important for producing functionally efficient sperm [13]. ADAM1/ADAM2 may require all three of the previously mentioned mechanisms for it to become a fully functional protein. ADAM1 (fertilin α) is proteolytically processed when the sperm is in the testis, while ADAM2 (fertilin β) is proteolytically processed during the sperm's transit through the epididymis [6, 1416]. Previously we showed that the proteolytic cleavage of ADAM2 coincides with the relocalization of ADAM1/ADAM2 from a whole head pattern to consolidation solely into the posterior head domain (PHD) [6]. We demonstrated that the relocalization results from the redistribution of existing ADAM1/ADAM2 from the anterior head domain (AHD) to the PHD and is not due to the removal of ADAM1/ADAM2 from the AHD with concomitant exposure of a previously hidden population of ADAM1/ADAM2 within the PHD [6].

Using fluorescent redistribution after photobleaching (FRAP), we also determined that lateral fluidity of ADAM1/ADAM2 within the membrane exists in two different states depending on the functional stage of the sperm. ADAM1/ADAM2 is highly restricted from diffusing on testicular sperm (when it is found covering the whole sperm head) and in cauda epididymal sperm (when ADAM1/ADAM2 has consolidated into only the PHD). During capacitation, however, ADAM1/ADAM2 becomes highly mobile within the PHD membrane, and its diffusion increases even more after the sperm undergo the acrosome reaction (Fig. 1) [1].

FIG. 1.
Diagram representing the distribution and relative diffusion states of ADAM1/ADAM2 on sperm at various stages of maturation. On testicular sperm, ADAM1/ADAM2 (closed circles) is found distributed over the entire sperm head, and the protein shows little ...

Capacitation is the term given to the preparatory cellular events sperm undergo in the female reproductive tract before they are capable of fertilizing the egg [1719]. Cellular changes in sperm at capacitation include but are not limited to hypermotility, increased protein tyrosine phosphorylation (pY), and membrane lipid reorganization, which culminate in the sperm becoming competent to undergo an acrosome reaction and fertilize the ovum [20]. Capacitation can be induced in vitro for most sperm by incubating them in defined medium supplemented with a combination of calcium, bicarbonate, and BSA or 2-hydroxypropyl-β-cyclodextrin (βCD). BSA or βCD act by removing sterols from the membrane [21, 22], while one function of bicarbonate, enhanced by calcium, is to stimulate soluble adenylyl cyclases (sACs) within the sperm to increase the intracellular concentration of cAMP [2328]. For sperm of many species, the bicarbonate requirement for capacitation can be bypassed if sperm are given a membrane-permeable cAMP analog along with a phosphodiesterase inhibitor (PDEi) to artificially increase their intracellular cAMP concentration [2938]. The cAMP can then be used by various signaling pathways, one of which leads to the stimulation of protein tyrosine kinases (PTK) and results in enhanced pY formation [39, 40].

For this study, we sought to extend the understanding of the role of cAMP in capacitation and the biological events surrounding it that cause sperm to transform into fertilizing gametes. We initially tested whether exogenous cAMP can induce capacitation-associated changes in guinea pig sperm, as it does in other species, leading to hypermotility and competence to undergo an acrosome reaction. Guinea pig sperm, exceptionally, do not require exogenous calcium during in vitro capacitation [41]. When calcium is present in the media, capacitated guinea pig sperm spontaneously undergo the acrosome reaction. We exploited this phenomenon to establish whether sperm incubated in exogenous cAMP achieve capacitation by determining the percentage of sperm in which acrosome reaction was induced on the addition of calcium. Because guinea pig sperm have exceptionally large acrosomes, distinguishing between acrosome-intact and acrosome-reacted sperm was easily done using light microscopy.

We then considered whether cAMP would also trigger the capacitation-associated conversion of ADAM1/ADAM2 from its restricted-diffusion state to its highly mobile state. We tested this directly by incubating cauda epididymal sperm with a cAMP analog and phosphodiesterase inhibitor and used FRAP to measure whether the diffusion properties of ADAM1/ADAM2 altered. Finally, we used testicular sperm, which are unable to undergo cAMP-stimulated capacitation until they have been epididymally matured, to test whether exogenous cAMP would affect the lateral mobility of ADAM1/ADAM2 in these less functionally mature cells.


Antibodies, Reagents, and Animals

All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise noted.

Male Hartley guinea pigs (retired breeders) were purchased from Charles River Laboratories (Wilmington, MA) and housed in the Laboratory Animal Research Center at the Rockefeller University or at the animal facility at the University of Connecticut Health Center. The respective Institutional Animal Care and Usage Committees approved all animal protocols.

The mouse anti-guinea pig ADAM2 monoclonal antibody used has been described previously in Cowan and Myles [42]. For the FRAP experiments, F(ab) fragments of this monoclonal antibody were directly conjugated with rhodamine and used as described in Cowan et al. [1].

Sperm Isolation

Epididymal sperm.

The dissected epididymides were placed into either Hanks balanced salt solution lacking calcium (HBSS-Ca2+) for FRAP studies or Hepes-Mg2+ (1 M NaCl, 1 M KCl, 0.1 M Hepes, 0.1 M dextrose, 1 M MgCl2, pH 7.4) for the remaining studies. Sperm were collected by mincing the epididymides and allowing the sperm to swim free as well as being gently eased out of the lumen by applying slight pressure to the tubule fragments [6]. The sperm were filtered through a 100-μm sieve to remove chunks of epididymal tissue, and the sperm concentration was determined using a hemocytometer.

Testicular sperm.

Testes were removed from euthanized guinea pigs and decapsulated, and the freed seminiferous tubules were minced into fine pieces using razor blades and placed into HBSS-Ca2+ medium containing 0.5 μg/ml DNase I. Cells were pelleted through 7% Percoll in HBSS-Ca2+, resuspended in 60% Percoll in HBSS-Ca2+, and centrifuged at 12 000 × g for 10 min. The band of testicular sperm was removed, diluted 10-fold in Hepes-buffered saline (pH 7.4), pelleted, and resuspended at 108/ml. For photobleaching experiments, sperm are subjected to several additional washes during antibody staining to ensure removal of Percoll, which has been reported to have effects on membrane structure [43]. No significant difference was observed in lateral mobility of ADAM1/ADAM2 between testicular sperm and in elongating spermatids isolated without the use of Percoll [42], indicating that Percoll is not affecting the results.

Dibutyryl-cAMP Treatment of Guinea Pig Sperm

Caudal epididymal or testicular sperm (0.5–1.0 × 107 sperm/ml) were washed into modified Tyrodes medium (mT) (112 mM NaCl, 2.8 mM KCl, 0.5 mM MgCl2, 25 mM NaHCO3, 5.6 mM glucose, 10 mM sodium lactate, 1 mM sodium pyruvate; pH 7.5); 1 mM dibutyryl cAMP (db-cAMP) was added from a 100-mM stock solution made fresh in 10 mM Pipes, pH 7.2. The working concentration of the PDEi papaverine (0.1 mM) was diluted from a 10-mM stock solution freshly made using a newly opened ampoule of DMSO. Sperm were incubated for 30–90 min at 37°C, depending on the experiment. For negative controls, parallel sets of sperm were incubated in mT alone or with vehicle solvents.

Acrosome Reaction Evaluation

After 30 or 90 min of incubation with db-cAMP and papaverine, sperm samples were combined with an equal volume of mT medium supplemented with 4 mM Ca2+ (2 mM Ca2+ final) and incubated an additional 15 min at 37°C to induce the acrosome reaction. Vehicle-treated negative control sperm were similarly sampled and supplemented with calcium and incubated alongside the test samples. At the end of the incubation, samples were fixed in 3% formaldehyde, and the percent of acrosome-reacted sperm was quantified by microscopic examination. At least 200 sperm were counted from each sample.

FRAP Analysis

Following 60 min of incubation with db-cAMP and papaverine, sperm prelabeled with rhodamine-conjugated anti-ADAM2 Fab antibody fragments [1] were prepared for FRAP analysis by mixing with agarose that had been heated and cooled to just above solidification temperature and then quickly spreading in a thin layer on a slide. For the epididymal sperm, only cells with embedded heads but freely moving tails were chosen for FRAP examination to eliminate the risk of examining dead cells [1]. Testicular sperm exhibit little mobility, although in most cases some infrequent twitching spasms were observed. Cells for analysis, therefore, were selected on the basis of exhibiting normal morphology with intact flagellum and acrosome.

Fluorescence redistribution after photobleaching experiments were performed using a Zeiss LSM510 laser scanning confocal microscope [44, 45]. Images were collected using a 63× 1.4 N.A. planapochromat objective with the pinhole fully opened to ensure collection of fluorescence through the entire depth of the specimen. The scan axis was rotated such that the x-axis of the scan was parallel to the long axis of the sperm head, and a region consisting of approximately one-third of the middle section of the sperm head was photobleached using 100% laser power. The first postphotobleach image (t = 0 sec) was collected within 3 msec of the end of the photobleach, and subsequent postphotobleach images were collected immediately thereafter. For epididymal sperm, the time per image ranged from 1.1 to 1.9 sec; for testicular sperm, the time per image ranged from 2.3 to 3.0 sec. Image analysis was performed essentially as described in Cowan et al. [45]. Background values were subtracted from all images, and average intensity values were calculated perpendicular to a line drawn down the long axis of the sperm head.

The analysis of the fluorescence redistribution was performed with the software system MLAB, constructed by Civilized Software, Inc. (Bethesda, MD). The diffusion coefficient (D) and percent mobile fraction (%R) were calculated on the basis of a normal-mode analysis as previously described [45]. Briefly, we treat the data as a normal-mode analysis on a spherical surface of radius r. For an initial condition with bleaching symmetric about the scanning axis and a monodisperse redistribution function, we have

equation image

where c(x, t) is the concentration distribution of the fluorophore at time t, D is the diffusion coefficient of the fluorophore, and P0(x) and P2(x) are the zero- and second-order Legendre polynomials, respectively [46]. For a polydisperse redistribution function, we have [47]

equation image

whereAn external file that holds a picture, illustration, etc.
Object name is bire-79-06-06-ilm1.jpg . For a simple case of a diffusion coefficient and an immobile fraction, we add a constant to μ2(t) [48],

equation image

where R is the so-called mobile fraction.

As was previously found, lateral mobility of ADAM1/ADAM2 in testicular sperm was highly restricted, and because there can be some motion in these cell preparations, we were not able to follow the fluorescence redistribution for a sufficiently long time to obtain accurate values for the fractional recovery. In this case we used a method of cumulants [47] to compute a value for mobility, An external file that holds a picture, illustration, etc.
Object name is bire-79-06-06-ilm2.jpg , equivalent to the average diffusion coefficient of a polydisperse distribution of all labeled molecules of that cell. We use the method of cumulants, where we define the cumulant generating function [47],

equation image

where the mth cumulant of Γ, Km(Γ), is the coefficient of (−3t)m/m! in the MacLaurin expansion of An external file that holds a picture, illustration, etc.
Object name is bire-79-06-06-ilm3.jpg , which thus takes the form of

equation image


equation image
equation image

For a single exponential function, K(−3t; Γ) is linear in t. Deviations from “exponentiality” of μ2(t) thus appear as higher order terms in t.

We fit μ2(t) to an approximate function, that is, A[RX exp(−3Γt) + 1 – R)], where Γ = 2D′/r2 and R is a “mobile” fraction. From there, we then calculate

equation image

which leads to

equation image

Statistical Methods

Statistical significance between groups was determined using the nonparametric Mann-Whitney U-test. Standard deviations are reported for all the FRAP and acrosome reaction data presented.


cAMP Induces Guinea Pig Sperm to Capacitate

We first determined whether cAMP could induce capacitation-associated competence for the acrosome reaction in guinea pig sperm as it does in several other species. Unlike sperm of many other species, guinea pig sperm do not require an exogenous calcium supply during in vitro capacitation. The addition of calcium to the media causes capacitated guinea pig sperm to spontaneously undergo the acrosome reaction and can therefore be used as a bioassay to determine the proportion of sperm that have completed capacitation at any given time point.

For each experiment, cauda epididymal sperm were isolated and incubated with the membrane-permeable cAMP analog, dibutyryl cAMP (db-cAMP), and the PDEi papaverine to determine if this caused the sperm to capacitate. Negative control sperm were incubated with vehicle alone. Based on concentrations of db-cAMP and papaverine used to trigger signaling events of capacitation and/or fertilization in gametes of other species [29, 37, 4954], we incubated 107 cauda sperm/ml in 1 mM db-cAMP and 100 μM papaverine at 37°C. After 30 or 90 min of incubation, calcium was added to the samples to induce the fully capacitated sperm to undergo the acrosome reaction. The sperm were then fixed and the percentage of acrosome-reacted sperm determined. We found on average 60%–67% of sperm to be acrosome-reacted regardless of whether the sperm were treated with db-cAMP for 30 or 90 min (Table 1). To determine if we were working at suboptimal concentrations, we tested the db-cAMP at concentrations of 2, 3, and 10 mM. None of these concentrations improved the level of acrosome reactivity within the population. In fact, the acrosome reactivity averages obtained for the 2- and 3-mM db-cAMP were slightly lower (46% and 53%, respectively, at 90 min). The 10-mM concentration of db-cAMP resulted in lowered sperm motility. Decreasing the concentration of db-cAMP to 0.1 mM resulted in levels of acrosome reaction that were only slightly higher than background. Sperm motility was also assessed by microscopic examination during cAMP incubation. By the 30-min time point, the 1-mM db-cAMP treatment had induced hypermotility, evidenced by vigorous tail thrashing. No hypermotility was observed in the vehicle control samples.

Percentage of acrosome-reacted sperm after cAMP treatment.

cAMP Induces ADAM1/ADAM2 to Exhibit Rapid Lateral Diffusion on Cauda Sperm

Previously we found, using fluorescence redistribution after photobleaching, that the lateral diffusion of ADAM1/ADAM2 within the membrane is highly restricted prior to capacitation, with the majority of ADAM1/ADAM2 showing little or no movement [1]. However, ADAM1/ADAM2 becomes highly diffusible within the membrane during capacitation and remains exceedingly mobile even after the acrosome reaction (Fig. 1) [1]. We thus asked if this increased diffusion for ADAM1/ADAM2 could be induced in cauda sperm by artificially increasing the intracellular levels of cAMP. To test this, we incubated cauda sperm with db-cAMP and papaverine and then determined the D for ADAM1/ADAM2 using FRAP analysis.

Figure 2 (top row) shows four images from a time series of a representative FRAP experiment (the entire times series can be viewed as a movie in Supplemental Movie 1, available online at ). A stripe running anterior-posterior in the posterior head area was photobleached, as indicated in the first postphotobleach image at t = 0 sec. The fluorescence within the photobleached area quickly began to return, and by 15 sec redistribution was essentially complete. An analysis of this experiment is shown in Figure 3 (top row). The leftmost panel (A) shows the fluorescence intensity of ADAM1/ADAM2 averaged along the anterior-posterior axis of the posterior head domain at each point in a line perpendicular to the anterior-posterior axis at each time point. Panel B shows at higher resolution three individual scans from panel A at three selected time points: the initial fluorescence intensity in the PHD prior to bleaching (circles), the loss of fluorescence intensity in the region of the PHD immediately after bleaching (squares), and the final scan after bleaching (triangles) showing the recovery of fluorescence intensity in the bleach region concomitant with a loss of fluorescence intensity in the nonbleached regions. By the 78-sec time point, the fluorescence intensity within the bleached area has equilibrated essentially to its initial distribution, revealing rapid diffusion within the PHD, although the overall level of fluorescence has been reduced because of the photobleaching. Fluorescence redistribution was analyzed on the basis of fitting the data to the change in the second moment of the distribution function over time (panel C) and from which both D as well as the %R (the fraction of the ADAM1/ADAM2 population exhibiting diffusion) were determined [44].

FIG. 2.
Fluorescence redistribution after photobleaching of ADAM1/ADAM2 on sperm treated with db-cAMP and the PDEi papaverine. A representative set of fluorescent redistribution after photobleaching (FRAP) images of db-cAMP-treated cauda-epididymal sperm (top ...
FIG. 3.
Analysis of fluorescence redistribution after photobleaching data. A representative fluorescent redistribution after photobleaching (FRAP) analysis of a db-cAMP-treated epididymal sperm (AC), a db-cAMP-treated testicular sperm (DF), ...

The average lateral diffusion of ADAM1/ADAM2 on cauda sperm treated with db-cAMP and papaverine was (9.8 ± 4.2) × 10−10 cm2/sec and had an averaged %R of 62 ± 16% (n = 22). Thus, the majority of the ADAM1/ADAM2 population (62%) on a sperm was found to be highly diffusible after cAMP treatment, while the remaining 38% of the population was immobile under these conditions. This increase in D was an order of a magnitude larger than our previous measurements of ADAM1/ADAM2 on untreated cauda sperm (0.8 × 10−10 cm2/sec) with a larger portion of the ADAM1/ADAM2 population diffusing than what we had seen previously (%R = 45) as well [1]. Parallel samples of these treated sperm were incubated in calcium to determine the percent acrosome reaction for each experiment and to prove that the drugs added were efficacious. The average acrosome reactivity in these experiments was 64%.

cAMP Does Not Induce Rapid Diffusion of ADAM1/ADAM2 on Testicular Sperm

On testicular sperm, ADAM1/ADAM2 is localized over the entire sperm head and shows little to no lateral diffusion within the membrane (Fig. 1) [1]. Until sperm released from the testis undergo epididymal maturation, they cannot be induced to capacitate or undergo the acrosome reaction [6]. It is hypothesized that this is because the cAMP signaling pathway(s) of capacitation require epididymal maturation before they are fully established. We thus tested whether cAMP would induce the rapid diffusion of ADAM1/ADAM2 in testicular sperm as it did on epididymal sperm. We performed FRAP analysis of ADAM1/ADAM2 on testicular sperm treated with db-cAMP and papaverine as described previously. Although ADAM1/ADAM2 is found over the entire head of testicular sperm, we analyzed its mobility specifically in the posterior head region, where it is located on cauda sperm, although a photobleached stripe was performed along the entire long axis of the sperm head (see Fig. 2 and Supplemental Movie 2, available online at ). In comparison to the findings for cauda sperm, we found that db-cAMP caused only a small increase in the lateral mobility of ADAM1/ADAM2 within the membrane. As shown in Figure 2 (bottom panels), redistribution into the bleach region was very slow and incomplete during the experiment; after more than 100 sec, the bleached region was still somewhat apparent in the fluorescence image. These results are quite similar to redistribution times of control testicular sperm, as the example in panels G and H of Figure 3 show.

The lateral mobility of ADAM1/ADAM2 was at such a low level on the testicular sperm with or without cAMP treatment that redistribution was often not complete during the time scale of the experiment, as indicated in panels F and I of Figure 3, where we see no evidence of a plateau in the distribution function during the time course of the experiment. As a result, we were unable to calculate the %R or D of ADAM1/ADAM2 for these cells directly. To directly compare these cells with the db-cAMP-treated cauda sperm as well as with our previously reported FRAP analyses of ADAM1/ADAM2 on sperm stages [1], we used a previously described method of cumulants [47] to assess lateral mobility by measuring the parameter An external file that holds a picture, illustration, etc.
Object name is bire-79-06-06-ilm4.jpg (see Materials and Methods) for all experiments. These quantified An external file that holds a picture, illustration, etc.
Object name is bire-79-06-06-ilm5.jpg averages showed that ADAM1/ADAM2 on cAMP-treated testicular sperm ([1.1 ± 1.3] × 10−10 cm2/sec) did not undergo the dramatic increase in mobility that was seen on cAMP-treated cauda sperm ([5.9 ± 2.7] × 10−10 cm2/sec) and that this difference was statistically highly significant (P < 0.0001). The addition of cAMP to testicular sperm resulted in modest yet significant (P < 0.03) increase in the An external file that holds a picture, illustration, etc.
Object name is bire-79-06-06-ilm6.jpg of ADAM1/ADAM2 compared to controls ([0.29 ± 0.28] × 10−10 cm2/sec); however, the overall mobility of ADAM1/ADAM2 remained highly restricted, and the An external file that holds a picture, illustration, etc.
Object name is bire-79-06-06-ilm7.jpg was not statistically different (P > 0.3) from ADAM1/ADAM2′s highly restricted An external file that holds a picture, illustration, etc.
Object name is bire-79-06-06-ilm8.jpg state measured on untreated cauda sperm ([0.5 ± 0.41] × 10−10 cm2/sec). Finally, because testicular sperm cannot acrosome react, we verified that the db-cAMP and papaverine added in these experiment were biologically active by incubating parallel samples of cauda sperm, collected from the same animals, with the same drug preparations followed by calcium to induce the acrosome reaction. In all experiments, this bioassay showed that the db-cAMP and papaverine preparations used were biologically effective.

In Figure 4 we compiled all the An external file that holds a picture, illustration, etc.
Object name is bire-79-06-06-ilm9.jpg results for ADAM1/ADAM2 from our current FRAP experiments and plotted them in context with past FRAP experiments after recalculating those data using the method of cumulants. The figure shows that ADAM1/ADAM2 remains highly restricted from diffusing until the sperm undergo capacitation, but if cauda sperm are treated with exogenous cAMP, the restriction of lateral movement is lifted, and the heterodimeric protein increases its rate of diffusion over 10-fold. This dramatic enhancement of diffusion by cAMP, however, is not inducible prior to epididymal maturation, as evidenced by the low mobility of ADAM1/ADAM2 on testicular sperm even after their exposure to cAMP.

FIG. 4.
Scatter plots of the diffusion coefficients for ADAM1/ADAM2 at different stages of sperm maturation and treatments. Mobility for ADAM1/ADAM2 is plotted asAn external file that holds a picture, illustration, etc.
Object name is bire-79-06-06-ilm13.jpg for various stages of sperm maturation and treatments. Each circle represents a fluorescent ...


Previously, we showed that ADAM1/ADAM2 undergoes a change in its diffusion state during capacitation [1]. Because intracellular cAMP rises sharply during the early stages of capacitation, we asked in this study if cAMP signals this increased diffusion rate of ADAM1/ADAM2. Our results show that increasing cAMP levels causes ADAM1/ADAM2 on cauda sperm to switch from its relatively immobile diffusion state to its freely diffusing state. There is precedent for cAMP control of the diffusion state of a membrane protein. Through FRAP analysis, the diffusion coefficient (D) for the membrane protein aquaporin 2 (AQP2) was determined in LLC-PK1 (porcine kidney) cells stably transfected with GFP-AQP2. D was found to be 10-fold fewer cells when treated with the cAMP agonist, forskolin, versus untreated controls [55]. The hypothesized mechanism of action in that case was a cAMP-generated phosphorylation of AQP2, leading to increased AQP2 interaction with cytoskeletal proteins, resulting in the impedance of its diffusion [55]. Although we see the opposite effect on ADAM1/ADAM2 on cauda sperm (cAMP causes an increase in the diffusion coefficient of ADAM1/ADAM2), the compelling aspect of the data is that cAMP in two different systems appears to act as the switching mechanism that alters a protein's membrane diffusion.

The molecular mechanism by which cAMP effects a change in lateral mobility of ADAM1/ADAM2 is unclear. Capacitation is known to entail changes in lipid composition and to affect lipid order in the sperm plasma membrane of a variety of species [9, 50, 53, 5663]. It has been previously reported that during boar sperm capacitation, the plasma membrane architecture is modified through a translocation of phospholipids to the outer leaflet and that cAMP can trigger this lipid scrambling [53, 58, 59, 64]. We do not know if a similar alteration in lipid architecture occurs in guinea pig sperm. However, it is unlikely that such an increase in lipid disorder would be sufficient to account for the changes in the lateral mobility of ADAM1/ADAM2 observed in this report. On testicular sperm and epididymal sperm, ADAM1/ADAM2 is essentially immobile. In the absence of another mechanism, this would mean a complete lack of membrane fluidity. Furthermore, previous studies in guinea pig [2, 65], as well as other species [66], revealed no large increases in global fluidity of the sperm plasma membrane either in different domains or following capacitation. In fact, lipid probes, such as C14diI, have actually shown a moderate, 2- to 3-fold decrease in diffusion coefficient between testicular sperm and cauda epididymal sperm [65] and a further 2-fold decrease in D on acrosome-reacted cells [2].

A more likely explanation for the large (one to two orders of magnitude) increase in lateral mobility of ADAM1/ADAM2 is an alteration in a relatively high-affinity protein-protein or protein-carbohydrate interaction. This hypothesis is strengthened by findings involving the protein PH-20, which is known to occupy the same membrane domains at the same times as ADAM1/ADAM2 [6]. PH-20 is largely immobile on testicular sperm, but on cauda epididymal sperm, it exhibits lateral mobility typical of many membrane proteins (D = 1.8 × 10−10 cm2/sec and %R = 73%), with a further increase in D after the acrosome reaction [2, 67]. Given that two proteins that occupy the same plasma membrane domain exhibit distinctly different temporal patterns for large changes in lateral mobility, we suspect that these are specific effects related to each protein individually rather than a global change in the lipid milieu. The most straightforward hypothesis, therefore, is that ADAM1/ADAM2 complexes with an immobile sperm component and that this binding interaction ceases to occur following cAMP signaling events.

Using the standard bicarbonate and BSA method of in vitro-induced capacitation, we previously observed two subpopulations of sperm (Fig. 4, bracketed data). In one population, ADAM1/ADAM2 was highly mobile, with average D similar to those for ADAM1/ADAM2 on acrosome-reacted sperm. In the second population, ADAM1/ADAM2 showed little or no diffusion, with an average D similar to the constrained mobility of ADAM1/ADAM2 measured for untreated cauda sperm [1]. We hypothesized that these two sperm populations reflected the inefficiency of the in vitro method at inducing capacitation in 100% of the sperm. This was supported by the fact that on average only 40%–50% of these in vitro-capacitated sperm underwent the acrosome reaction when subsequently incubated with calcium chloride [1]. Because FRAP analysis requires that we immobilize swimming sperm in an agarose matrix, we could not use hypermotility as an indicator of capacitation. Therefore, without a means of distinguishing between capacitated and noncapacitated sperm, our results likely contained a mixture of sperm from both states. We speculated that sperm with ADAM1/ADAM2 exhibiting restricted diffusion were sperm that had not capacitated, while the sperm with freely diffusing ADAM1/ADAM2 were those that had capacitated.

Interestingly, we did not observe two such subpopulations in the cAMP-treated cauda sperm; only one population was detected, and it corresponded to the previously mentioned group with freely diffusing ADAM1/ADAM2. The lateral mobility for ADAM1/ADAM2 after cAMP treatment, when calculated as D or as An external file that holds a picture, illustration, etc.
Object name is bire-79-06-06-ilm10.jpg , was 12 times greater than the average mobility measured on untreated cauda sperm. We posit that the release of ADAM1/ADAM2 from some constraint is a cAMP-modulated event and that a threshold of intracellular cAMP must be reached before ADAM1/ADAM2 is released. During capacitation, sAC is stimulated in each sperm to produce cAMP. However, under conventional in vitro-capacitating conditions, each sperm reaches this putative cAMP threshold concentration at a different time because of its individual response to the stimulation of sAC. As a result, at any given sampling, two populations of sperm could be seen: 1) those sperm that have achieved the cAMP threshold and have released ADAM1/ADAM2 to diffuse readily and 2) those sperm whose intracellular levels of cAMP are still below this threshold and so maintain restricted diffusion of ADAM1/ADAM2. When membrane-permeable exogenous cAMP is given to the sperm, all the sperm simultaneously attain the same intracellular level of cAMP. If this concentration of cAMP is above the required threshold in the sperm, then ADAM1/ADAM2 would be converted on all the sperm from the constrained state to the freely diffusing state. This scenario could potentially explain why we detected only one population of sperm with freely diffusing ADAM1/ADAM2. However, even though a threshold may have been reached to convert ADAM1/ADAM2 to a highly mobile state in all the sperm examined, it was not enough to make all the sperm ready to acrosome react. When this population was challenged with calcium chloride, only about 60% of the sperm underwent the acrosome reaction. Therefore, the release of ADAM1/ADAM2 from its restraints may be an event that precedes final acrosome-reactive competence.

We think it is unlikely that cAMP directly releases ADAM1/ADAM2 from its constraints; instead, we postulate that it more likely stimulates a pathway that leads to this event. Cyclic AMP might trigger changes in phosphorylation, as has been suggested for AQP2, or might cause changes in intracellular calcium levels. Treating cauda sperm with the calcium ionophore A23187 and exogenous calcium induces the acrosome reaction in noncapacitated guinea pig cauda sperm [68, 69]. ADAM1/ADAM2 on such sperm is found to be highly mobile within the membrane. It remains unclear precisely how calcium and A23187 enhance ADAM1/ADAM2 diffusion, but collectively the data suggest that both cAMP and calcium have roles in this event.

In an endeavor to elucidate the exact mechanism involved, we examined guinea pig sperm for enhanced protein tyrosine phosphorylation during capacitation but found that guinea pig sperm, unlike mouse and several other species of sperm, showed little or no increase in pY formation during capacitation even though on the same blot, mouse sperm showed an intense increase in pY formation. This does not rule out a role for pY formation during capacitation in guinea pig sperm, but it does indicate yet another difference between these two sperm species. We also tried 2-D gel immunoblotting of acrosome-reacted and acrosome-intact sperm, looking for subtle difference in ADAM2 that might correspond with the change in the diffusion state of ADAM1/ADAM2, but our results were inconclusive. Finally, we have performed yeast two-cell hybrid analysis using the cytoplasmic tail of ADAM2 to try to identify a binding partner that may be anchoring ADAM1/ADAM2 in its constrained state. Although no clear partner has been identified, a few candidates and several unknown proteins warrant further study. Interestingly, among the potential positive and numerous false positives identified, we found an abundance of divalent-cation-binding proteins, often specifically for calcium (unpublished data). This seems to further suggest that ADAM1/ADAM2 may be regulated through an interaction with a calcium-binding protein that may be cAMP regulated.

Regardless of the exact nature of the release event, the cAMP-induced increase in ADAM1/ADAM2 diffusion occurs only on sperm that have completed epididymal maturation. Treatment of testicular sperm (which cannot undergo capacitation or an acrosome reaction—even through calcium ionophore) with db-cAMP and papaverine did not result in significant alteration of the lateral mobility of ADAM1/ADAM2. It is possible that the mechanism that immobilizes ADAM1/ADAM2 within the membrane of testicular sperm is different than the one that keeps ADAM1/ADAM2 immobile on cauda sperm. ADAM1/ADAM2 on testicular sperm is found over the whole head, while on cauda sperm the protein is confined to the PHD [6]. Although two separate mechanisms might be involved in confining ADAM1/ADAM2 in testicular and cauda sperm, the simplest explanation would involve just one tethering mechanism, the release from which is not possible until sperm have completed epididymal maturation. This requirement may be a means to prevent premature release and early activation of the ADAM1/ADAM2, a protein that may have a late-acting function, when sperm are in the female reproductive tract.

The functional consequences of modulating the diffusion of ADAM1/ADAM2 are not clear. The ADAM proteins represent a large family of proteins that are present in sperm but whose roles in fertilization events are not well understood. Mice lacking functional genes for ADAMS 1b, 2, or 3 are infertile, but given that multiple ADAMs appear to interact in ways that stabilize/destabilize each other's surface expression, there remain multiple functional mechanisms that could account for the infertility of the knockout mice. Guinea pig sperm do not appear to express ADAM3, so there are also likely differences in the function of these proteins between mouse and guinea pig. Nevertheless, ADAM1/ADAM2 is a major membrane protein of the sperm surface, and the dramatic change in lateral mobility we have observed is a significant event in capacitation. The magnitude of the change in lateral mobility of ADAM1/ADAM2 is very large. An individual protein that exhibits D in the range of 10−9 cm2/sec would be expected to traverse an area the size of one-half the posterior head domain (~5 μm2) in approximately 40 sec; for D in the range of 10−10 cm2/sec, it would take 7 min, and for D in the range of 10−11 cm2/sec, it would take 70 min. In testicular and cauda epididymal sperm, most of the ADAM1/ADAM2 molecules are even less mobile than 10−11 cm2/sec. Thus, if ADAM1/ADAM2 were involved in a binding event that required recruitment of multiple proteins to the site of interaction, the recruitment event would be unlikely to occur in sperm prior to capacitation but could occur on a physiological time scale after capacitation and/or the acrosome reaction, when D is in the range of 10−9 cm2/sec with a high mobile fraction [1].

These results have positioned us to explore thoroughly the steps in the cAMP-signaling pathway leading to the release and increased diffusion of ADAM1/ADAM2 during capacitation. Further studies may shed light on the operative advantage that this increase in lateral movement provides for ADAM1/ADAM2. Also, more generally, they would provide information regarding previously unknown mechanisms by which synthetically inert sperm can modulate activity of surface proteins and subsequently sperm function.

Supplementary Material

[Supplemental Movies]


We would like to thank Mr. Evan Read for his help with the manuscript illustrations.


1Supported by the National Institute of Health research grant HD038807.


  • Cowan AE, Koppel DE, Vargas LA, Hunnicutt GR. Guinea pig fertilin exhibits restricted lateral mobility in epididymal sperm and becomes freely diffusing during capacitation. Dev Biol 2001; 236: 502–509..509. [PubMed]
  • Cowan AE, Myles DG, Koppel DE. Lateral diffusion of the PH-20 protein on guinea pig sperm: evidence that barriers to diffusion maintain plasma membrane domains in mammalian sperm. J Cell Biol 1987; 104: 917–923..923. [PMC free article] [PubMed]
  • Myles DG, Primakoff P, Koppel DE. A localized surface protein of guinea pig sperm exhibits free diffusion in its domain. J Cell Biol 1984; 98: 1905–1909..1909. [PMC free article] [PubMed]
  • Ladha S, James PS, Clark DC, Howes EA, Jones R. Lateral mobility of plasma membrane lipids in bull spermatozoa: heterogeneity between surface domains and rigidification following cell death. J Cell Sci 1997; 110: 1041–1050..1050. [PubMed]
  • Wolfe CA, James PS, Mackie AR, Ladha S, Jones R. Regionalized lipid diffusion in the plasma membrane of mammalian spermatozoa. Biol Reprod 1998; 59: 1506–1514..1514. [PubMed]
  • Hunnicutt GR, Koppel DE, Myles DG. Analysis of the process of localization of fertilin to the sperm posterior head plasma membrane domain during sperm maturation in the epididymis. Dev Biol 1997; 191: 146–159..159. [PubMed]
  • Jones R. Plasma membrane structure and remodelling during sperm maturation in the epididymis. J Reprod Fertil Suppl 1998; 53: 73–84..84. [PubMed]
  • Cesario MM, Bartles JR. Compartmentalization, processing and redistribution of the plasma membrane protein CE9 on rodent spermatozoa: relationship of the annulus to domain boundaries in the plasma membrane of the tail. J Cell Sci 1994; 107(pt 2):561–570..570. [PubMed]
  • Selvaraj V, Buttke DE, Asano A, McElwee JL, Wolff CA, Nelson JL, Klaus AV, Hunnicutt GR, Travis AJ. GM1 Dynamics as a marker for membrane changes associated with the process of capacitation in murine and bovine spermatozoa. J Androl 2007; 28: 588–599..599. [PubMed]
  • Jones R, Shalgi R, Hoyland J, Phillips DM. Topographical rearrangement of a plasma membrane antigen during capacitation of rat spermatozoa in vitro. Dev Biol 1990; 139: 349–362..362. [PubMed]
  • Bearer EL, Friend DS. Morphology of mammalian sperm membranes during differentiation, maturation, and capacitation. [Review]. J Electron Microsc Tech 1990; 16: 281–297..297. [PubMed]
  • Buffone MG, Calamera JC, Verstraeten SV, Doncel GF. Capacitation-associated protein tyrosine phosphorylation and membrane fluidity changes are impaired in the spermatozoa of asthenozoospermic patients. Reproduction 2005; 129: 697–705..705. [PubMed]
  • Rubinstein E, Ziyyat A, Wolf J-P, Le Naour F, Boucheix C. The molecular players of sperm-egg fusion in mammals. Semin Cell Dev Biol 2006; 17: 254–263..263. [PubMed]
  • Cho C, Ge H, Branciforte D, Primakoff P, Myles DG. Analysis of Mouse fertilin in wild-type and fertilin beta(–/–) sperm: evidence for c-terminal modification, alpha/beta dimerization, and lack of essential role of fertilin alpha in sperm-egg fusion. Dev Biol 2000; 222: 289–295..295. [PubMed]
  • Lum L, Blobel CP. Evidence for distinct serine protease activities with a potential role in processing the sperm protein fertilin. Dev Biol 1997; 191: 131–145..145. [PubMed]
  • Blobel CP, Myles DG, Primakoff P, White JM. Proteolytic processing of a protein involved in sperm-egg fusion correlates with acquisition of fertilization competence. J Cell Biol 1990; 111: 69–78..78. [PMC free article] [PubMed]
  • Shivaji S, Kumar V, Mitra K, Jha KN. Mammalian sperm capacitation: role of phosphotyrosine proteins. Soc Reprod Fertil Suppl 2007; 63: 295–312..312. [PubMed]
  • Salicioni AM, Platt MD, Wertheimer EV, Arcelay E, Allaire A, Sosnik J, Visconti PE. Signalling pathways involved in sperm capacitation. Soc Reprod Fertil Suppl 2007; 65: 245–259..259. [PubMed]
  • Suarez SS, Ho HC. Hyperactivation of mammalian sperm. Cell Mol Biol (Noisy-le-grand) 2003; 49: 351–356..356. [PubMed]
  • Jonge CJD, Barratt CLR. The Sperm Cell: Production, Maturation, Fertilization, Regeneration. Cambridge, UK:: Cambridge University Press;; 2006.
  • Choi YH, Toyoda Y. Cyclodextrin removes cholesterol from mouse sperm and induces capacitation in a protein-free medium. Biol Reprod 1998; 59: 1328–1333..1333. [PubMed]
  • Visconti PE, Galantino-Homer H, Ning X, Moore GD, Valenzuela JP, Jorgez CJ, Alvarez JG, Kopf GS. Cholesterol efflux-mediated signal transduction in mammalian sperm: beta-cyclodextrins initiate transmembrane signaling leading to an increase in protein tyrosine phosphorylation and capacitation. J Biol Chem 1999; 274: 3235–3242..3242. [PubMed]
  • Luconi M, Porazzi I, Ferruzzi P, Marchiani S, Forti G, Baldi E. Tyrosine phosphorylation of the a kinase anchoring protein 3 (AKAP3) and soluble adenylate cyclase are involved in the increase of human sperm motility by bicarbonate. Biol Reprod 2005; 72: 22–32..32. [PubMed]
  • Hess KC, Jones BH, Marquez B, Chen Y, Ord TS, Kamenetsky M, Miyamoto C, Zippin JH, Kopf GS, Suarez SS, Levin LR, Williams CJ, et al. The “soluble” adenylyl cyclase in sperm mediates multiple signaling events required for fertilization. Dev Cell 2005; 9: 249–259..259. [PMC free article] [PubMed]
  • Esposito G, Jaiswal BS, Xie F, Krajnc-Franken MA, Robben TJ, Strik AM, Kuil C, Philipsen RL, van Duin M, Conti M, Gossen JA. Mice deficient for soluble adenylyl cyclase are infertile because of a severe sperm-motility defect. Proc Natl Acad Sci U S A 2004; 101: 2993–2998..2998. [PubMed]
  • Pastor-Soler N, Beaulieu V, Litvin TN, Da Silva N, Chen Y, Brown D, Buck J, Levin LR, Breton S. Bicarbonate-regulated adenylyl cyclase (sAC) is a sensor that regulates pH-dependent V-ATPase recycling. J Biol Chem 2003; 278: 49523–49529..49529. [PubMed]
  • Baxendale RW, Fraser LR. Evidence for multiple distinctly localized adenylyl cyclase isoforms in mammalian spermatozoa. Mol Reprod Dev 2003; 66: 181–189..189. [PubMed]
  • Xie F, Garcia MA, Carlson AE, Schuh SM, Babcock DF, Jaiswal BS, Gossen JA, Esposito G, van Duin M, Conti M. Soluble adenylyl cyclase (sAC) is indispensable for sperm function and fertilization. Dev Biol 2006; 296: 353–362..362. [PubMed]
  • Tardif S, Lefievre L, Gagnon C, Bailey JL. Implication of cAMP during porcine sperm capacitation and protein tyrosine phosphorylation. Mol Reprod Dev 2004; 69: 428–435..435. [PubMed]
  • O'Flaherty C, de Lamirande E, Gagnon C. Phosphorylation of the arginine-X-X-(serine/threonine) motif in human sperm proteins during capacitation: modulation and protein kinase A dependency. Mol Hum Reprod 2004; 10: 355–363..363. [PubMed]
  • Marquez B, Suarez SS. Different signaling pathways in bovine sperm regulate capacitation and hyperactivation. Biol Reprod 2004; 70: 1626–1633..1633. [PubMed]
  • Bennetts L, Lin M, Aitken RJ. Cyclic AMP-dependent tyrosine phosphorylation in tammar wallaby (Macropus eugenii) spermatozoa. J Exp Zool A Comp Exp Biol 2004; 301: 118–130..130. [PubMed]
  • Bajpai M, Doncel GF. Involvement of tyrosine kinase and cAMP-dependent kinase cross-talk in the regulation of human sperm motility. Reproduction 2003; 126: 183–195..195. [PubMed]
  • Harrison RA, Miller NG. cAMP-dependent protein kinase control of plasma membrane lipid architecture in boar sperm. Mol Reprod Dev 2000; 55: 220–228..228. [PubMed]
  • Visconti PE, Stewart-Savage J, Blasco A, Battaglia L, Miranda P, Kopf GS, Tezon JG. Roles of bicarbonate, cAMP, and protein tyrosine phosphorylation on capacitation and the spontaneous acrosome reaction of hamster sperm. Biol Reprod 1999; 61: 76–84..84. [PubMed]
  • Leclerc P, de Lamirande E, Gagnon C. Cyclic adenosine 3′,5′monophosphate-dependent regulation of protein tyrosine phosphorylation in relation to human sperm capacitation and motility. Biol Reprod 1996; 55: 684–692..692. [PubMed]
  • Visconti PE, Moore GD, Bailey JL, Leclerc P, Connors SA, Pan D, Olds-Clarke P, Kopf GS. Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. Development 1995; 121: 1139–1150..1150. [PubMed]
  • Duncan AE, Fraser LR. Cyclic AMP-dependent phosphorylation of epididymal mouse sperm proteins during capacitation in vitro: identification of an M(r) 95,000 phosphotyrosine-containing protein. J Reprod Fertil 1993; 97: 287–299..299. [PubMed]
  • Visconti PE, Johnson LR, Oyaski M, Fornes M, Moss SB, Gerton GL, Kopf GS. Regulation, localization, and anchoring of protein kinase A subunits during mouse sperm capacitation. Dev Biol 1997; 192: 351–363..363. [PubMed]
  • Visconti PE, Ning X, Fornes MW, Alvarez JG, Stein P, Connors SA, Kopf GS. Cholesterol efflux-mediated signal transduction in mammalian sperm: cholesterol release signals an increase in protein tyrosine phosphorylation during mouse sperm capacitation. Dev Biol 1999; 214: 429–443..443. [PubMed]
  • Yanagimachi R, Bhattacharyya A. Acrosome-reacted guinea pig spermatozoa become fusion competent in the presence of extracellular potassium ions. J Exp Zool 1988; 248: 354–360..360. [PubMed]
  • Cowan AE, Myles DG. Biogenesis of surface domains during spermiogenesis in the guinea pig. Dev Biol 1993; 155: 124–133..133. [PubMed]
  • Tanphaichitr N, Zheng YS, Kates M, Abdullah N, Chan A. Cholesterol and phospholipid levels of washed and Percoll gradient centrifuged mouse sperm: presence of lipids possessing inhibitory effects on sperm motility. Mol Reprod Dev 1996; 43: 187–195..195. [PubMed]
  • Cowan AE, Koppel DE, Setlow B, Setlow P. A soluble protein is immobile in dormant spores of Bacillus subtilis but is mobile in germinated spores: implications for spore dormancy. Proc Natl Acad Sci U S A 2003; 100: 4209–4214..4214. [PubMed]
  • Cowan AE, Olivastro EM, Koppel DE, Loshon CA, Setlow B, Setlow P. Lipids in the inner membrane of dormant spores of Bacillus species are largely immobile. Proc Natl Acad Sci U S A 2004; 101: 7733–7738..7738. [PubMed]
  • Koppel DE, Sheetz MP, Schindler M. Lateral diffusion in biological membranes: a normal mode analysis of diffusion on a spherical surface. Biophys J 1980; 30: 187–192..192. [PubMed]
  • Koppel DE. Analysis of macromolecular polydispersity in intensity correlation spectroscopy: the method of cumulants. Journal of Chemical Physics 1972; 57: 4814–4820..4820.
  • Cowan AE, Nakhimovsky L, Myles DG, Koppel DE. Barriers to diffusion of plasma membrane proteins form early during guinea pig spermiogenesis. Biophys J 1997; 73: 507–516..516. [PubMed]
  • Galantino-Homer HL, Visconti PE, Kopf GS. Regulation of protein tyrosine phosphorylation during bovine sperm capacitation by a cyclic adenosine 3′5′-monophosphate-dependent pathway. Biol Reprod 1997; 56: 707–719..719. [PubMed]
  • Osheroff JE, Visconti PE, Valenzuela JP, Travis AJ, Alvarez J, Kopf GS. Regulation of human sperm capacitation by a cholesterol efflux-stimulated signal transduction pathway leading to protein kinase A-mediated up-regulation of protein tyrosine phosphorylation. Mol Hum Reprod 1999; 5: 1017–1026..1026. [PubMed]
  • Baumber J, Meyers SA. Hyperactivated motility in rhesus macaque (Macaca mulatta) spermatozoa. J Androl 2006; 27: 459–468..468. [PubMed]
  • Hoskins DD, Hall ML, Munsterman D. Induction of motility in immature bovine spermatozoa by cyclic AMP phosphodiesterase inhibitors and seminal plasma. Biol Reprod 1975; 13: 168–176..176. [PubMed]
  • Gadella BM, Harrison RA. The capacitating agent bicarbonate induces protein kinase A-dependent changes in phospholipid transbilayer behavior in the sperm plasma membrane. Development 2000; 127: 2407–2420..2420. [PubMed]
  • Hunnicutt GR, Kosfiszer MG, Snell WJ. Cell body and flagellar agglutinins in Chlamydomonas reinhardtii: the cell body plasma membrane is a reservoir for agglutinins whose migration to the flagella is regulated by a functional barrier. J Cell Biol 1990; 111: 1605–1616..1616. [PMC free article] [PubMed]
  • Umenishi F, Verbavatz J-M, Verkman AS. cAMP regulated membrane diffusion of a green fluorescent protein-aquaporin 2 chimera. Biophys J 2000; 78: 1024–1035..1035. [PubMed]
  • van Gestel RA, Brewis IA, Ashton PR, Helms JB, Brouwers JF, Gadella BM. Capacitation-dependent concentration of lipid rafts in the apical ridge head area of porcine sperm cells. Mol Hum Reprod 2005; 11: 583–590..590. [PubMed]
  • Harrison RA, Gadella BM. Bicarbonate-induced membrane processing in sperm capacitation. Theriogenology 2005; 63: 342–351..351. [PubMed]
  • Flesch FM, Brouwers JFHM, Nievelstein PFEM, Verkleij AJ, van Golde LMG, Colenbrander B, Gadella BM. Bicarbonate stimulated phospholipid scrambling induces cholesterol redistribution and enables cholesterol depletion in the sperm plasma membrane. J Cell Sci 2001; 114: 3543–3555..3555. [PubMed]
  • Gadella BM, Miller NG, Colenbrander B, van Golde LM, Harrison RA. Flow cytometric detection of transbilayer movement of fluorescent phospholipid analogues across the boar sperm plasma membrane: elimination of labeling artifacts. Mol Reprod Dev 1999; 53: 108–125..125. [PubMed]
  • Travis AJ, Kopf GS. The role of cholesterol efflux in regulating the fertilization potential of mammalian spermatozoa. J Clin Invest 2002; 110: 731–736..736. [PMC free article] [PubMed]
  • Buttke DE, Nelson JL, Schlegel PN, Hunnicutt GR, Travis AJ. Visualization of GM1 with cholera toxin B in live epididymal versus ejaculated bull, mouse, and human spermatozoa. Biol Reprod 2006; 74: 889–895..895. [PubMed]
  • Shadan S, James PS, Howes EA, Jones R. Cholesterol efflux alters lipid raft stability and distribution during capacitation of boar spermatozoa. Biol Reprod 2004; 71: 253–265..265. [PubMed]
  • Jones R, James PS, Howes L, Bruckbauer A, Klenerman D. Supramolecular organization of the sperm plasma membrane during maturation and capacitation. Asian J Androl 2007; 9: 438–444..444. [PubMed]
  • Gadella BM, Harrison RA. Capacitation induces cyclic adenosine 3′,5′-monophosphate-dependent, but apoptosis-unrelated, exposure of aminophospholipids at the apical head plasma membrane of boar sperm cells. Biol Reprod 2002; 67: 340–350..350. [PubMed]
  • Phelps BM, Primakoff P, Koppel DE, Low MG, Myles DG. Restricted lateral diffusion of PH-20, a PI-anchored sperm membrane protein. Science 1988; 240: 1780–1782..1782. [PubMed]
  • Smith TT, McKinnon-Thompson CA, Wolf DE. Changes in lipid diffusibility in the hamster sperm head plasma membrane during capacitation in vivo and in vitro. Mol Reprod Dev 1998; 50: 86–92..92. [PubMed]
  • Cowan AE, Myles DG, Koppel DE. Migration of the guinea pig sperm membrane protein PH-20 from one localized surface domain to another does not occur by a simple diffusion-trapping mechanism. Dev Biol 1991; 144: 189–198..198. [PubMed]
  • Talbot P, Summers RG, Hylander BL, Keough EM, Franklin LE. The role of calcium in the acrosome reaction: an analysis using ionophore A23187. J Exp Zool 1976; 198: 383–392..392. [PubMed]
  • Summers RG, Talbot P, Keough EM, Hylander BL, Franklin LE. Ionophore A23187 induces acrosome reactions in sea urchin and guinea pig spermatozoa. J Exp Zool 1976; 196: 381–385..385. [PubMed]

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