Modulation of adenylyl cyclase by FPP and adenosine involves stimulatory and inhibitory adenosine receptors and g proteins

FPP and adenosine modulate the adenylyl cyclase (AC)/cAMP signal transduction pathway in mammalian spermatozoa to elicit a biphasic response, initially stimulating capacitation and then inhibiting spontaneous acrosome loss. This study addressed the hypothesis that responses to FPP involve interactions between receptors for FPP and adenosine, the biphasic responses involving stimulatory and inhibitory adenosine receptors. Gln‐FPP, a competitive inhibitor of FPP, significantly inhibited binding of an adenosine analogue and responses to adenosine, especially in capacitated suspensions, consistent with interaction between FPP and adenosine receptors. CGS‐21680 (1 μM), a stimulatory A_2a adenosine receptor agonist, significantly stimulated capacitation and cAMP in uncapacitated cells, while cyclopentyl adenosine (1 μM), an inhibitory A₁ adenosine receptor agonist only affected capacitated cells, inhibiting spontaneous acrosome loss. Responses to FPP and adenosine were inhibited in uncapacitated cells by a selective A_2a antagonist and in capacitated cells by a selective A₁ antagonist; subsequent investigations indicated possible involvement of G proteins. Like FPP, cholera toxin stimulated capacitation and cAMP production in uncapacitated cells, suggesting involvement of a G protein with a Gα_s subunit. In contrast, pertussis toxin prevented FPP's inhibition of both spontaneous acrosome loss and cAMP production, suggesting involvement of a Gα_i/_o subunit. Immunoblotting evidence revealed the presence of proteins of the appropriate molecular weights for Gα_s, Gα_i2, Gα _i3, and Gα_o subunits. This study provides the first direct evidence suggesting the involvement of two different types of adenosine receptors and both Gα_s and Gα_i/_o subunits in the regulation of capacitation, resulting in modulation of AC activity and availability of cAMP. Mol. Reprod. Dev. 53:459–471, 1999. © 1999 Wiley‐Liss, Inc.     

Calcitonin acts as a first messenger to regulate adenylyl cyclase/cAMP and mammalian sperm function

Calcitonin stimulates capacitation in uncapacitated mouse spermatozoa and then inhibits spontaneous acrosome loss in capacitated cells, responses similar to those elicited by fertilization promoting peptide (FPP), a peptide known to regulate the adenylyl cyclase/cAMP pathway. This study investigated the hypothesis that calcitonin also modulates this pathway. Calcitonin significantly stimulated cAMP production in uncapacitated spermatozoa and then inhibited it in capacitated cells; the magnitude of both stimulatory and inhibitory changes was similar to that obtained with FPP but the inhibitory responses to FPP preceded those of calcitonin. This possibly reflects the involvement of two different adenosine receptors in response to FPP compared with one calcitonin receptor. Calcitonin receptors were located on the acrosomal cap and the flagellum, the midpiece having a greater abundance than the principal piece. Although both calcitonin and adenosine receptors are found in the head and flagellum, there was no evidence for cross-talk between them. Chlortetracycline investigations to determine the minimum extracellular Ca(2+) requirement for responses to calcitonin revealed that calcitonin significantly stimulated capacitation in Ca(2+)-deficient medium but FPP did not. Calcitonin also significantly stimulated cAMP production under these conditions, and similarly preincubated suspensions, when diluted into +Ca(2+) medium, were significantly more fertile in vitro than untreated controls. These results indicate that calcitonin, like FPP, acts as a first messenger to regulate the production of cAMP and mammalian sperm function, but the differences in Ca(2+) requirements suggest that calcitonin and FPP may regulate different isoforms of adenylyl cyclase.     

Fertilization promoting peptide and adenosine, acting as first messengers, regulate cAMP production and consequent protein tyrosine phosphorylation in a capacitation-dependent manner

Fertilization promoting peptide (FPP) and adenosine have been shown to act as first messengers, regulating availability of the second messenger cAMP by initially stimulating cAMP production in uncapacitated spermatozoa and then inhibiting it in capacitated cells. This study investigated possible capacitation-related changes in protein tyrosine phosphorylation in response to FPP and adenosine. Time-dependent changes in phosphorylation of proteins of approximately 30-140 kDa were observed in both uncapacitated and capacitated suspensions, the general level of phosphorylation being markedly greater in capacitated cells. In the presence of FPP, phosphorylation was stimulated in uncapacitated but inhibited in capacitated spermatozoa, compared with untreated control samples. Adenosine, cholera toxin, and CGS-21680, a stimulatory A(2a) adenosine receptor agonist, also stimulated phosphorylation in uncapacitated spermatozoa, while Gln-FPP, a competitive inhibitor of FPP, blocked responses to FPP. In capacitated cells, FPP's inhibition of phosphorylation was abolished when cells were treated with FPP in the presence of pertussis toxin. Consistent with the capacitation-dependent effects of FPP and adenosine on cAMP production, these results support the hypothesis that FPP and adenosine modulate sperm function by regulating the AC/cAMP signaling pathway and, consequently, protein tyrosine phosphorylation. Of particular significance is the identification of several phosphoproteins showing FPP-induced alterations in phosphorylation. In uncapacitated spermatozoa, proteins of approximately 116, 95, 82, 75, 66, 56, and 42 kDa showed increased phosphorylation, while in capacitated cells, phosphoproteins of approximately 116, 95, 82, 75, 70, 66, 56, and 50 kDa showed decreased phosphorylation. This suggests that these particular proteins may be involved in stimulation and arrest of capacitation, respectively.     

Adenosine and gpp(NH)p modulate mouse sperm adenylate cyclase

The possible roles of adenosine and the GTP analogue Gpp(NH)p in regulating mouse sperm adenylate cyclase activity were investigated during incubation in vitro under conditions in which after 30 min the spermatozoa are essentially uncapacitated and poorly fertile, whereas after 120 min they are capacitated and highly fertile. Adenylate cyclase activity, assayed in the presence of 1 mM ATP and 2 mM Mn²⁺, was determined by monitoring cAMP production. When adenosine deaminase (1 U/ml) was included in the assay to deplete endogenous adenosine, enzyme activity was decreased in the 30-min suspensions but increased in the 120-min samples (P < 0.02). This suggests that endogenous adenosine has a stimulatory effect on adenylate cyclase in uncapacitated spermatozoa but is inhibitory in capacitated cells. Since the expression of adenosine effects at low nucleoside concentrations usually requires guanine nucleotides, the effect of adding adenosine in the presence of 5 x 10^–5 M Gpp(NH)p was examined. While either endogenous adenosine or adenosine deaminase may have masked low concentration (10^?9?10^?7 M) effects of exogenous adenosine, a marked inhibition (P < 0.001) of adenylate cyclase activity in both uncapacitated and capacitated suspensions was observed with higher concentrations (>10^?5 M) of adenosine. Similar inhibition was also observed in the absence of Gpp(NH)p, suggesting the presence of an inhibitory P site on the enzyme. In further experiments, the effects of Gpp(NH)p in the presence and absence of adenosine deaminase were examined. Activity in 30-min suspensions was stimulated by the guanine nucleotide and in the presence of adenosine deaminase this stimulation was marked, reversing the inhibition seen with adenosine deaminase alone. In capacitated suspensions the opposite profile was observed, with Gpp(NH)p plus adenosine deaminase being inhibitory; again, this was a reversal of the effects obtained in the presence of adenosine deaminase alone, which had stimulated enzyme activity. These results suggest the existence of a stimulatory adenosine receptor site (R_a) on mouse sperm adenylate cyclase that is expressed in uncapacitated spermatozoa and an inhibitory receptor site (R_i) that is expressed in capacitated cells, with guanine nucleotides modifying the final response to adenosine. It is concluded that adenosine and guanine nucleotides may regulate mouse sperm adenylate cyclase activity during capacitation.     

Interactions between a decapacitation factor and mouse spermatozoa appear to involve fucose residues and a GPI-anchored receptor

Epididymal mouse spermatozoa have a surface-associated decapacitation factor (DF) that can be removed precociously by centrifugation, resulting in acceleration of capacitation and increased fertilizing ability. Addition of exogenous DF to capacitated suspensions inhibits fertilizing ability and reverses capacitation in acrosome-intact cells. DF appears to regulate a Ca²⁺-ATPase, located primarily in the postacrosomal region. The present investigations of DF↮spermatozoon interaction indicate that DF can be removed from uncapacitated cells by treatment with phosphatidylinositol-specific phospholipase C (PIC), suggesting the involvement of a glycosylphosphatidylinositol (GPI) moiety. However, exogenous DF cannot reassociate with PIC-treated spermatozoa, suggesting that DF may bind to spermatozoa via a GPI-anchored receptor. DF binding appears to involve fucose residues, since depletion of endogenous DF followed by brief exposure to fucose (0.1–10 mM) prevented DF reassociation with cells. Furthermore, 5 mM fucose could displace DF from uncapacitated cells, accelerating capacitation and resulting in a higher proportion of fertilized oocytes, with increased polyspermy, than obtained with untreated controls. FITC-labelled fucosylated BSA bound specifically to the postacrosomal region, binding being inhibited by both excess fucose and crude DF. UEA I, a lectin with specificity for fucose residues, bound to the postacrosomal region of cells preincubated in fucose but not crude DF, and blocked DF binding to DF-depleted cells. These results are consistent with the DF binding, via fucose residues, to a GPI-anchored receptor. Fucose binding sites are in the same region where Ca²⁺-ATPase, the enzyme regulated by DF, has been localized; these results support the hypothesis that DF modulates capacitation by regulating enzyme activity and hence the intracellular Ca²⁺ concentration. Mol. Reprod. Dev. 51:193–202, 1998. © 1998 Wiley-Liss, Inc.     

Modulation of the function of boar spermatozoa via adenosine and fertilization promoting peptide receptors reduce the incidence of polyspermic penetration into porcine oocytes

Effects of adenosine and pGlu-Glu-ProNH(2) (FPP) on the function and in vitro penetration of boar spermatozoa were examined. First, the effects of dibutyryl cAMP or agonists and antagonists of adenosine receptors (inhibitory adenosine receptors, A1AdR; stimulatory adenosine receptors, A2AdR) on freshly ejaculated spermatozoa were determined by chlortetracycline fluorescence assessment. Capacitation of spermatozoa was stimulated when they were cultured in a medium with dibutyryl cAMP, adenosine, A2AdR agonist, and adenosine plus A1AdR antagonist (CPT). However, acrosome reaction was inhibited only by adenosine. A1AdR agonist did not affect intact spermatozoa. A2AdR antagonist (DMPX) neutralized all of the effects of adenosine. Second, interaction of adenosine and FPP was examined. Gln-FPP, a competitive inhibitor of FPP, and DMPX inhibited the effects of adenosine and FPP, and CPT neutralized the inhibitory effect of FPP on acrosome reaction. Last, the effects of adenosine, FPP, and caffeine on the rate of sperm penetration were examined using frozen-thawed spermatozoa. Adenosine, FPP, and caffeine significantly enhanced the rate of sperm penetration as compared with the case of no additions. Caffeine treatment resulted in a high rate of polyspermic fertilization. In contrast, adenosine and FPP treatments resulted in an increased proportion of normal fertilization in in vitro-matured oocytes. These results suggest that boar spermatozoa can be modulated by the adenylyl cyclase/cAMP pathway via A2AdR in intact cells to induce capacitation and A1AdR in capacitated cells to inhibit spontaneous acrosome loss and that FPP receptors interact with A2AdR in intact cells and with A1AdR in capacitated cells. Furthermore, adenosine and FPP seem to be useful in reducing the incidence of polyspermic penetration.     

A possible mechanism of action for fertilization promoting peptide,a TRH-related tripeptide that promotes capacitation and fertilizing ability in mammalian spermatozoa

Fertilization promoting peptide (FPP), a tripeptide structurally related to thyrotrophin releasing hormone (TRH), has been shown to stimulate capacitation and fertilizing ability in both mouse and human spermatozoa, but the mechanisms of action involved in these responses are currently unknown. In the present study utilizing epididymal mouse spermatozoa, we have compared the ability of FPP, TRH, and pyroglutamylphenylalanineprolineamide (an uncharged structurally related tripeptide found in seminal plasma) to stimulate capacitation. At 50 nM, the mean concentration of FPP found in human seminal plasma, only FPP produced a significant response. This suggests that if a receptor is involved, it is one distinct from the TRH receptor. A significant response to FPP required the presence of extracellular Ca²⁺, with 90 μm Ca²⁺ being sufficient to support a stimulation of capacitation. The addition of FPP to suspensions at later stages of capacitation indicated that the nature of the response changed, such that addition of FPP to capacitated suspensions inhibited spontaneous acrosome reactions; however, FPP-treated, cells were still able to undergo acrosomal exocytosis in response to progesterone, a physiological agonist of acrosomal exocytosis. Because earlier studies had identified a similar capacitation-related change in response to adenosine, being stimulatory early in capacitation and inhibitory later in capacitation, we investigated the possibility that FPP and adenosine might be acting via the same pathway. The combination of FPP plus adenosine, whether used at low, non-stimulatory concentrations or high, maximally-stimulatory concentrations, was more effective in promoting capacitation than either compound used individually. As observed with FPP, addition of adenosine to capacitated cells inhibited spontaneous acrosome loss but did not inhibit exocytosis in response to progesterone. This suggests that the two molecules are affecting a common pathway. Since adenosine, acting via specific cell surface receptors, can stimulate fertilizing ability and adenylate cyclase activity in uncapacitated cells and then inhibit enzyme activity in capacitated cells, we propose that FPP may act by modulating the adenylate cyclase/cyclic AMP signal transduction pathway. In vivo, FPP, which would contact spermatozoa at ejaculation and probably remain bound to cells for some time, could stimulate capacitation as the spermatozoa ascend the female tract; adenosine, present in seminal plasma and the female tract, could either augment FPP's action or replace it if FPP is lost from the cell surface. We therefore suggest that FPP and adenosine, by modulating adenylate cyclase activity to promote capacitation but inhibit spontaneous acrosomal exocytosis, may provide an endogenous mechanism that helps to optimize the fertilizing potential of the few sperm cells that reach the site of fertilization in vivo. © 1996 Wiley-Liss, Inc.     

Adenosine and its analogues, possibly acting at A2 receptors, stimulate mouse sperm fertilizing ability during early stages of capacitation

Earlier studies have provided indirect evidence that the availability of endogenous adenosine can modulate the fertilizing ability of mouse spermatozoa during capacitation. More direct evidence has been sought by evaluating the effect of exogenous adenosine present during the early stages of capacitation. A concentration-dependent stimulation of in-vitro fertilizing ability was observed, with 10 microM- and 100 microM-adenosine significantly increasing the proportion of eggs fertilized compared with drug-free controls. The adenosine-induced stimulation was observed in the presence of 0.01 microM- and 0.1 microM-dipyridamole, an inhibitor of adenosine uptake, suggesting that adenosine is acting at an external site. Comparison of adenosine with its analogues 2'-deoxyadenosine and 2-chloroadenosine indicated that the analogues at 10 microM were able to stimulate fertilization in a manner similar to adenosine. While neither adenosine nor 2'-deoxyadenosine was consistently effective at 1 microM, 2-chloroadenosine significantly stimulated fertilization at both 1 microM and 0.1 microM. In addition, 5'-N-ethylcarboxamidoadenosine (NECA) and (R)-N6-phenylisopropyladenosine (R-PIA), potent analogues in somatic cell systems, proved to be so with mouse sperm suspensions, NECA being stimulatory at greater than or equal to 0.01 microM and R-PIA at greater than or equal to 0.1 microM. Subjective evaluation of motility patterns indicated that more cells exhibited hyperactivated motility in the presence of stimulatory concentrations of adenosine or analogues. Assessment of capacitation state using chlortetracycline fluorescence patterns indicated that incubation in 2'-deoxyadenosine resulted in significantly fewer cells expressing the uncapacitated F pattern and significantly more cells with the capacitated AR (acrosome-reacted) pattern, compared with drug-free counterparts. It is concluded that adenosine promotes capacitation by interacting with externally-directed receptors, possibly on adenylate cyclase to increase the intracellular availability of cyclic adenosine monophosphate (cAMP); cAMP is known to stimulate mouse sperm fertilizing ability. The greater sensitivity to NECA, 2-chloroadenosine and R-PIA, relative to adenosine and 2'-deoxyadenosine, is consistent with interaction at stimulatory A2 adenosine receptors.     

Stimulation by n6-cyclopentyladenosine of A1 adenosine receptors, coupled to galphai2 protein subunit, has a capacitative effect on human spermatozoa

The effects of selective A(1) receptor agonist on human spermatozoa were examined to verify physiological responses and to investigate the signal transduction pathway. N6-Cyclopentyladenosine on uncapacitated spermatozoa did not induce spontaneous acrosome reaction after 5 h capacitation, whereas the number of capacitated spermatozoa, assessed by lysophosphatidylcholine-induced acrosome reaction with Pisum sativum agglutinin staining, was significantly increased. N6-Cyclopentyladenosine was also added to capacitated human spermatozoa to find out whether the agonist could induce the acrosome reaction. Results, although statistically significant, could not be considered biologically significant. A1-Mediated capacitation was followed by the increase of tyrosine phosphorylation of a protein subset ranging between M(r) = 200 000 and 30 000. Stimulation of A1 receptor with the selective agonist elicited an agonist-induced inositol phospholipid hydrolysis leading to a transient rise of inositol triphosphate (IP3). This increase was not induced by A(1) receptor antagonist and was blocked by phospholipase C inhibitor. Coimmunoprecipitation experiments showed that the A(1) receptor is coupled to Galphai2 subunit suggesting that the activation of phospholipase C is mediated by betagamma subunits. In conclusion, the A(1) adenosine receptor in human spermatozoa is coupled to Galphai2, signals via IP3, and affects the capacitative status of ejaculated spermatozoa.     

FPP modulates mammalian sperm function via TCP-11 and the adenylyl cyclase/cAMP pathway

Fertilization promoting peptide (FPP; pGlu-Glu-ProNH₂), which is found in seminal plasma, promotes capacitation but inhibits spontaneous acrosome loss in mammalian spermatozoa in vitro. Adenosine, known to modulate the adenylyl cyclase (AC)/cAMP pathway, elicits these same responses whereas FPP + adenosine produces an enhanced response, leading to the hypothesis that FPP and adenosine modulate the same signal transduction pathway but act via different receptors. TCP-11, the product of a t-complex gene, is the putative receptor for FPP: Fab fragments of anti-TCP-11 antibodies have the same effect as FPP on mouse spermatozoa and Gln-FPP, a competitive inhibitor of FPP, also competitively inhibits responses to the Fab fragments. In the present study, specific binding of ³H-FPP to sperm membranes was significantly inhibited by 200 nM Gln-FPP and anti-TCP-11 Fab fragments (1/25 dilution), thus confirming that FPP, Gln-FPP, and Fab fragments compete for the same binding site. In addition, spermatozoa treated with A23187 to induce the acrosome reaction bound significantly less ³H-FPP than untreated cells, suggesting that a large proportion of the FPP binding sites are associated with the acrosomal cap region; TCP-11 is located in this region. In other experiments, 100 nM FPP significantly stimulated cAMP production in mouse sperm membranes, permeabilized cells and intact cells. Furthermore, Gln-FPP inhibited production of cAMP in response to FPP but not to adenosine (10 μM) or its analogue NECA (100 nM), supporting the involvement of two different receptors. Finally, anti-TCP-11 Fab fragments (1/25 dilution) significantly stimulated cAMP production, whereas low Fab (1/200; nonstimulatory when used alone) plus adenosine (10 μM) significantly enhanced the stimulation of capacitation by adenosine. These results support the hypotheses that TCP-11 is the receptor for FPP and that FPP↔TCP-11 interactions modulate AC/cAMP. Mol. Reprod. Dev. 51:468–476, 1998. © 1998 Wiley-Liss, Inc.     

Inhibition of adenosine-metabolizing enzymes modulates mouse sperm fertilizing ability: A changing role for endogenously generated adenosine during capacitation

The effect of inhibiting adenosine-metabolizing enzymes on sperm fertilizing ability was studied to investigate a possible role for endogenously generated adenosine in the regulation of capacitation. The compounds used have been shown to be effective inhibitors of the relevant enzymes in similarly incubated mouse sperm suspensions. Inhibition of 5′-nucleotidase activity with α,β-methylene adenosine 5′-diphosphate (AMPCP), to reduce available endogenous adenosine, caused a dose-dependent inhibition of the fertilizing ability of partially capacitated spermatozoa, which was significant with 100 and 250 μM AMPCP. Conversely, inhibition of adenosine deaminase with 100 nM coformycin, to increase available endogenous adenosine, promoted the fertilizing ability of partially capacitated spermatozoa when the fertilization rate of control suspensions was low. However, coformycin had no effect on sperm suspensions with moderate fertilizing ability, and it inhibited fertilizing ability when added to capacitated spermatozoa. These data are consistent with a promotion of the early stages of capacitation by endogenously generated adenosine and suggest that sensitivity to adenosine changes as capacitation proceeds. Because the majority of adenosine-metabolizing enzyme activity resides in or is directed toward the extracellular compartment in such suspensions, these effects of adenosine may be mediated at the outer surface of the cell. By interacting with receptors on adenylate cyclase, externally produced adenosine could modulate intracellular levels of cyclic adenosine monophosphate (cAMP), thereby influencing fertilizing ability.     

The role of small molecules in sperm capacitation

Mammalian spermatozoa released into an appropriate environment in vitro can capacitate but then may undergo spontaneous acrosome reactions. Since successful sperm interaction with the zona pellucida of an unfertilized oocyte requires an intact sperm plasma membrane, spontaneous acrosome loss is biologically undesirable because it renders spermatozoa non-fertilizing. Several small molecules (fertilization promoting peptide [FPP], adenosine, calcitonin and adrenaline), found in various body fluids including seminal plasma, have been shown to regulate capacitation in vitro. They initially accelerate capacitation but then inhibit spontaneous acrosome loss, allowing spermatozoa to maintain their fertilizing potential. Specific receptors for all these molecules are present on mammalian spermatozoa and their activation by the appropriate ligands leads to modulation of membrane-associated adenylyl cyclase activity and production of cAMP, stimulating cAMP production in uncapacitated cells and inhibiting it in capacitated cells. Boar spermatozoa have been shown to respond in vitro to adenosine and FPP, suggesting that the addition of these molecules to sperm samples used for artificial insemination could be beneficial in helping spermatozoa maintain fertilizing potential until they reach their target.     

Sperm-oviduct interaction: induction of capacitation and preferential binding of uncapacitated spermatozoa to oviductal epithelial cells in porcine species

After mating, inseminated spermatozoa are transported to the oviduct. They attach to and interact with oviductal epithelial cells (OEC). To investigate sperm-OEC interactions, we used chlortetracycline to study the capacitation status of boar spermatozoa in coculture with homologous OEC and cells of nonreproductive origin (LLC-PK1, porcine kidney epithelial cell line). Boar spermatozoa were cocultured with OEC and LLC-PK1 cells for 15, 60, 120, or 240 min. The proportion of capacitated spermatozoa in coculture with the isthmic and ampullar cells increased significantly (p < 0.05) during incubation. However, most spermatozoa in coculture with LLC-PK1 cells or blank (medium only) remained uncapacitated. In addition, preferential binding of uncapacitated, capacitated, or acrosome-reacted boar spermatozoa to OEC and the other cell type was investigated. Our approach was to vary the proportions of uncapacitated, capacitated, or acrosome-reacted boar spermatozoa in suspension using long preincubation and lysophosphatidylcholine treatment of semen prior to a very short incubation with OEC or LLC-PK1 cells. The results showed that the majority of spermatozoa that were bound to OEC or LLC-PK1 cells were uncapacitated and that a significant relationship existed between the relative proportion of uncapacitated spermatozoa in the control samples and those bound to LLC-PK1 cells (r2 = 0.43, p < 0.005). However, there was no correlation between the proportion of uncapacitated spermatozoa in the control samples and the proportion of those bound to isthmic or ampullar cells. In conclusion, the results clearly demonstrated the specific nature of the sperm-OEC interaction in the porcine species. This interaction is initiated by uncapacitated spermatozoa binding to OEC and is continued by the induction of capacitation in cocultured spermatozoa.     

Characterization of a decapacitation factor associated with epididymal mouse spermatozoa

Earlier studies demonstrated that epididymal mouse spermatozoa have a surface-associated factor which inhibits fertilizing ability in a reversible manner. The factor can be removed from uncapacitated spermatozoa by gentle centrifugation, resulting in immediately highly fertile gametes, and it can be added back to capacitated spermatozoa, resulting in poorly fertile cells in which the acrosome reaction has been blocked. Using such inhibition of in-vitro fertilizing ability as an assay, we have carried out experiments to characterize the factor. It appears to be an anionic polypeptide with Mr of approximately 40,000 (according to its behaviour on gel filtration). It is stable to heating at 100 degrees C for 15 min and is not destroyed by proteases at pH 8.0, yet inhibitory activity decreases during sperm incubation in capacitating conditions and is also destroyed in partially purified preparations by endogenous enzyme action during incubation at pH 5.0. Activity is not adsorbed to either concanavalin A-agarose or wheat-germ agglutinin-agarose, suggesting that terminal mannose and N-acetylglucosamine residues are not abundant. The factor causes rapid changes in the patterns of chlortetracycline fluorescence seen on sperm heads, a parameter used to assess the capacitated state. Removal of the factor from uncapacitated cells results in a shift to a predominance of capacitated patterns, while the addition of crude or partially purified factor to capacitated cells inhibits the acrosome reaction and causes a shift to the uncapacitated pattern in acrosome-intact spermatozoa. The factor therefore behaves as a decapacitation factor. However, it appears to differ from other characterized decapacitation factors in terms both of molecular size and of abundance of mannose and N-acetylglucosamine residues.     

Ganglioside GM1 mediates decapacitation effects of SVS2 on murine spermatozoa

Prior to fertilization, mammalian spermatozoa need to acquire fertilizing ability (capacitation) in the female reproductive tract. On the other hand, capacitated spermatozoa reversibly lose their capacitated state when treated with seminal plasma (decapacitation). Previously, we demonstrated that a mouse seminal plasma protein, SVS2, is a decapacitation factor and regulates sperm fertilizing ability in vivo. Here, we examined the mechanisms of regulation of fertilizing ability by SVS2. Capacitation appears to be mediated by dynamic changes in lipid rafts since release of the cholesterol components of lipid rafts in the sperm plasma membrane is indispensable for capacitation. When the ejaculated spermatozoa were stained with a cholera toxin subunit B (CTB) that preferably interacts with ganglioside GM1, another member of the lipid rafts, the staining pattern of the sperm was the same as the binding pattern of SVS2. Interestingly, SVS2 and CTB competitively bound to the sperm surface with each other, suggesting that the binding targets of both molecules are the same, that is, GM1. Molecular interaction studies by the overlay assay and the quartz crystal microbalance analysis revealed that SVS2 selectively interacts with GM1 rather than with other gangliosides. Furthermore, external addition of GM1 nullified SVS2-induced sperm decapacitation. Thus, ganglioside GM1 is a receptor of SVS2 and plays a crucial role in capacitation in vivo.     

Both fertilization promoting peptide and adenosine stimulate capacitation but inhibit spontaneous acrosome loss in ejaculated boar spermatozoa in vitro

Both fertilization promoting peptide (FPP) and adenosine stimulate capacitation and inhibit spontaneous acrosome loss in epididymal mouse spermatozoa; these responses involve modulation of the adenylyl cyclase (AC)/cAMP signal transduction pathway. However, it was unclear whether these responses were restricted to the mouse or possibly common to many mammalian species. To address this question, the response of boar spermatozoa to FPP and/or adenosine was evaluated. FPP is found in nanomolar concentrations in seminal plasma of several mammals, but not the pig. When cultured in caffeine-containing Medium 199 for 2 hr, chlortetracycline fluorescence evaluation indicated that neither FPP nor adenosine stimulated boar sperm capacitation per se but did inhibit spontaneous acrosome loss. However, in caffeine-free medium, FPP and adenosine both stimulated capacitation and inhibited spontaneous acrosome loss, suggesting that boar spermatozoa have receptors for both FPP and adenosine. Gln-FPP, a competitive inhibitor of FPP in mouse spermatozoa, has recently been shown to inhibit mouse sperm responses to adenosine as well, suggesting that FPP receptors and adenosine receptors interact in some way. Used with boar spermatozoa, Gln-FPP also significantly inhibited responses to both FPP and adenosine. These responses suggest that mechanisms whereby FPP and adenosine can regulate sperm function, via AC/cAMP, are of considerable physiological significance. Mouse, human, and now boar spermatozoa have been shown to respond to FPP, suggesting that these mechanisms may be common to many mammalian species. We also suggest that the effects of FPP and adenosine could also be exploited to maximize monospermic fertilization in porcine in vitro fertilization.     

Surface protein changes in goat spermatozoa during capacitation

Polypeptides of goat sperm surface before and after capacitation were examined by radiolabelling and immunologically using polyclonal antisera. Radioiodination revealed five protein bands having mol wt of 14.8, 72.4, 81, 100 and 128 kDa in uncapacitated ejaculated spermatozoa and only three bands of 23.4, 27 and 72.4 KDa in capacitated spermatozoa. The protein band with mol wt 72.4 kDa was only feebly iodinated in uncapacitated sperm surface but in capacitated spermatozoa it was heavily labelled. Western blot analysis of detergent-extracted proteins using gamma-globulin fraction of antisera raised against purified goat sperm plasma membrane revealed six antigens (17.8, 29.1, 33.4, 45.6, 85.1, 123.2 kDa) in uncapacitated spermatozoa, four (26, 32.1, 40.1, 45.6 kDa) in capacitated spermatozoa and only one (45.6 kDa) in acrosome-reacted spermatozoa. High mol wt proteins were more numerous on the surface of uncapacitated spermatozoa while the capacitated spermatozoa had relatively low mol wt proteins. An apparent effect of capacitation is the metabolism and reorganisation of proteins on goat sperm surface. Polypeptides on capacitated sperm surface revealed through radiolabelling and polyclonal antisera may have a likely receptor(s) role in the recognition and binding to homologous zona pellucida during fertilization.     

Tyrosine phosphoproteome of hamster spermatozoa: Role of glycerol‐3‐phosphate dehydrogenase 2 in sperm capacitation

Capacitation confers on the spermatozoa the competence to fertilize the oocyte. At the molecular level, a cyclic adenosine monophosphate (cAMP) dependent protein tyrosine phosphorylation pathway operates in capacitated spermatozoa, thus resulting in tyrosine phosphorylation of specific proteins. Identification of these tyrosine‐phosphorylated proteins and their function with respect to hyperactivation and acrosome reaction, would unravel the molecular basis of capacitation. With this in view, 21 phosphotyrosine proteins have been identified in capacitated hamster spermatozoa out of which 11 did not identify with any known sperm protein. So, in the present study attempts have been made to ascertain the role of one of these eleven proteins namely glycerol‐3‐phosphate dehydrogenase 2 (GPD2) in hamster sperm capacitation. GPD2 is phosphorylated only in capacitated hamster spermatozoa and is noncanonically localized in the acrosome and principal piece in human, mouse, rat, and hamster spermatozoa, though in somatic cells it is localized in the mitochondria. This noncanonical localization may imply a role of GPD2 in acrosome reaction and hyperactivation. Further, enzymatic activity of GPD2 during capacitation correlates positively with hyperactivation and acrosome reaction thus demonstrating that GPD2 may be required for sperm capacitation.     

The “switching on” of mammalian spermatozoa: Molecular events involved in promotion and regulation of capacitation

Following the discovery of mammalian sperm capacitation and its fundamental importance for the acquisition of fertilizing potential, it has gradually become possible to identify some specific molecules and molecular events that play pivotal roles in the “switching on” of spermatozoa. These are discussed in the context of the promotion and regulation of capacitation, emphasizing differences between commonly used conditions in vitro and the environment in vivo where spermatozoa normally undergo capacitation. Although typical culture media used in vitro do support capacitation, they do not prevent capacitated cells from undergoing spontaneous acrosome reactions and so losing fertilizing potential. This is not a problem in vitro, but could be in vivo where few spermatozoa reach the site of fertilization. Several small molecules, known to be present in vivo, have been shown in vitro to bind to spermatozoa and to regulate capacitation, first accelerating capacitation and then inhibiting spontaneous acrosome reactions, by regulating cAMP production. Since spermatozoa would contact these molecules during and after ejaculation, it is plausible that they serve a similar function in vivo. The mechanisms whereby the presence or absence of decapacitation factors might alter plasma membrane architecture and so alter functionality of a number of membrane‐associated enzymes involved in capacitation are also considered. Finally, several unresolved issues relating to events during capacitation are discussed. Mol. Reprod. Dev. 77: 197–208, 2010. © 2009 Wiley‐Liss, Inc.