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ABSTRACT: Both cyclic AMP (cAMP)/protein kinase A (PKA) and calcium (Ca2+) signaling pathways are known to be involved in the regulation of motility in mammalian sperm. Calmodulin (CaM) is a ubiquitous Ca2+ sensor that has been implicated in the acrosome reaction. In this report, we identify an insoluble pool of CaM in sperm and show that the protein, in addition to its presence in the acrosome, is found in the principal piece of the flagellum. These findings are consistent with, though not proof of, the presence of a pool of CaM in the fibrous sheath. The Ca2+/CaM-dependent protein kinase IIβ (CaMKIIβ), which is a downstream target of Ca2+/CaM, similarly localizes to the principal piece. In addition, we confirm earlier reports that a CaM inhibitor decreases sperm motility. However, we find that this inhibition can be largely reversed by stimulation of PKA if substrates for oxidative respiration are present in the medium. Our results suggest that the Ca2+/CaM/CaMKII signaling pathway in the sperm principal piece is involved in regulating sperm motility, and that this pathway functions either in parallel with or upstream of the cAMP/PKA pathway.
Sperm motility is central to male fertility in mammals. However, we do not have a full understanding of the signaling pathways that regulate flagellar function. Two pathways have emerged as key regulators of normal mammalian activated and hyperactivated motility. These are the cyclic AMP(cAMP)/protein kinase A (PKA) pathway and Ca2+ signaling (Heffner and Storey, 1981; Suarez et al, 1987; Tash and Means, 1987; Lindemann and Goltz, 1988; White and Aitken, 1989; Brokaw, 1991; Yanagimachi, 1994; Ho et al, 2002; Nolan et al, 2004; Marin-Briggiler et al, 2005).
There is evidence to suggest that the cAMP-dependent phosphorylation of flagellar proteins is involved in the initiation and maintenance of sperm motility (Tash and Means, 1982; Tash and Means, 1983; San Augustin and Witman, 1994). Since PKA is a major downstream target of cAMP in sperm, it likely that this kinase plays a central role in these phosphorylation events (Visconti et al, 1997). As further support of a role for PKA in sperm function, it has been shown that mice that lack the male germ cell-specific catalytic subunit of PKA (Cα2) are infertile due to several abnormalities, including aberrations of motility (Nolan et al, 2004).
Extracellular Ca2+ is required for motility in most epididymal sperm samples, and Ca2+ is known to regulate both activated and hyperactivated motility (Suarez et al, 1987; Tash and Means, 1987; Lindemann and Goltz, 1988; White and Aitken, 1989; Yanagimachi, 1994; Ho et al, 2002). One mechanism by which Ca2+ is directly linked to flagellar function is through its regulation of the atypical, “soluble” adenylyl cyclase, sAC, which generates cAMP and is required for sperm motility (Jaiswal and Conti, 2003; Litvin et al, 2003; Esposito et al, 2004).
Calmodulin (CaM) is a ubiquitous, highly conserved, 17-kd protein that serves as a classical intracellular Ca2+ receptor (Means et al, 1982). At least some of the effects of Ca2+ on the flagellum are likely to be achieved through CaM, since inhibition of CaM decreases sperm motility (White and Aitken, 1989; Ahmad et al, 1995; Si and Olds-Clarke, 2000). Interestingly, the effects of Ca2+ on sAC are independent of CaM (Jaiswal and Conti, 2003; Litvin et al, 2003), which suggests that Ca2+ affects motility via multiple pathways, only some of which require CaM.
Since sperm are highly compartmentalized, proteins must be targeted accurately to the appropriate region(s) of the cell. Thus, proteins involved directly in the regulation of motility typically localize to the flagellum. We use indirect immunofluorescence to show that CaM is present in the principal piece of the flagellum. In addition, we show that a portion of sperm CaM is insoluble, consistent with its localization to the cytoskeleton and similar to the extraction profile of a known fibrous sheath (FS) protein (the pro-domain of pro-AKAP4). These findings indicate that a pool of CaM localizes to the flagellum and possibly to the FS, an insoluble accessory structure that is found exclusively in the principal piece of the mammalian flagellum. We also suggest that CaM is involved in the regulation of sperm motility, since a CaM inhibitor decreased motility. This inhibition was largely reversed by stimulation of PKA, but only when lactate and pyruvate were present in the medium. Furthermore, the Ca2+/CaM-dependent protein kinase IIβ (CaMKIIβ), which is a downstream target of Ca2+/CaM, colocalized with CaM in the principal piece, which suggests that a Ca2+/CaM/CaMKII signaling pathway is present in the sperm principal piece.
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Using both indirect immunofluorescence and immunoblotting, we have shown that CaM is present in all germ cell stages, including sperm. The indirect immunofluorescence and fractionation studies reveal a complex distribution pattern of CaM in sperm. Immunofluorescence localized CaM to both the principal piece and the acrosome. The fractionation studies suggest that CaM is found in both insoluble structures (eg, cytoskeletal structures, such as the FS) and soluble structures found in the S10 fraction and its derivatives, the S100, P100, and detergent-soluble membrane fractions. CaM was not found in the detergent-insoluble membrane pellet. Consistent with this broad distribution of CaM in sperm, there is convincing evidence, including the evidence presented here, that Ca2+/CaM is involved in multiple functions in sperm, including motility, capacitation, and the acrosome reaction (Jones et al, 1980; Si and Olds-Clarke, 2000; Bendahmane et al, 2001; Lopez-Gonzalez et al, 2001). Some of the effects of Ca2+/CaM on sperm motility and capacitation may involve calcineurin (Tash et al, 1988; Tash and Bracho, 1994; Carrera et al, 1996). Other studies have identified a link between CaM and the regulation of sperm T-type Ca2+ currents (Lopez-Gonzalez et al, 2001).
Our findings of an insoluble pool of CaM in sperm and of CaM in the principal piece are consistent with, though not proof of, the existence of a pool of CaM in the FS (Tash and Means, 1987; Tash et al, 1988). It has been suggested that PKA is anchored to the FS through 1 or more AKAPs (Carrera et al, 1994; Mei et al, 1997; Miki and Eddy, 1998; Turner et al, 1998; Mandal et al, 1999; Vijayaraghavan et al, 1999). If CaM also is associated with the FS, then components of both of the major sperm motility signaling pathways (cAMP/PKA and Ca2+ signaling through CaM) find homes in this important accessory structure. Regardless of whether or not CaM is present in the FS, its presence in the flagellar principal piece provides indirect evidence for a role of CaM in the regulation of sperm motility. Inhibition of sperm motility by the addition of the CaM antagonist W-7 provides more direct evidence for this role.
It has previously been reported that 8-br-cAMP and IBMX do not restore motility to W-7-treated sperm when the sperm are incubated in a medium that contains glucose but lacks pyruvate and lactate (Si and Olds-Clarke, 2000). Our results concur with this finding. However, similar to reports on demembranated ascidian sperm (Nomura et al, 2000), we found that if lactate and pyruvate were present in the medium, then IBMX and either 8-br-cAMP or db-cAMP could largely compensate for the loss of function of CaM. Taken together, these findings provide insight into the signaling and metabolic control of mammalian sperm motility. In the mouse, it has been demonstrated that glycolysis produces ATP in the principal piece that is essential for fully normal sperm motility and for the phosphorylation events that are believed to facilitate the regulation of motility (Travis et al, 2001a; Miki et al, 2004; Mukai and Okuno, 2004). Our data show that CaM functions in the principal piece in that, when CaM is inhibited, sperm motility is significantly decreased. In addition, without substrates for oxidative respiration (lactate and pyruvate), and even in the presence of a substrate for glycolysis (glucose), increased intracellular cAMP cannot restore motility to CaM-inhibited sperm. This suggests the possibility that CaM is involved in the regulation of glycolysis or in the utilization of glycolytic ATP. There have been several previous reports linking Ca2+/CaM to the regulation of glycolytic enzymes in somatic cells (Ashkenazy-Shahar et al, 1998; Ashkenazy-Shahar and Beitner, 1999; Singh et al, 2004). Thus, it is possible that W-7, by inhibiting CaM, indirectly inhibits glycolysis.
As they are substrates for oxidative respiration, lactate and pyruvate may be able to compensate partially for the lack of glycolytic ATP by enabling the production of ATP in the midpiece. It has been shown that the production of ATP in the midpiece in the absence of glycolytic ATP can support normal motility for short periods of time (Mukai and Okuno, 2004). It is unclear whether some of this ATP is able to move to the proximal principal piece or whether it remains entirely in the midpiece. Regardless, these data show the partially compensatory abilities of the metabolic pathways that support flagellar motility, and suggest a new potential function for CaM in sperm (ie, the regulation of glycolysis in the principal piece).
Other than the addition of lactate and pyruvate to our medium, there were other more subtle differences between our experiments (in which agonists of the PKA pathway were able to restore motility to CaM-inhibited sperm) and those of Si and Olds-Clarke (in which agonists of the PKA pathway were unable to restore motility to CaM-inhibited sperm). For example, we utilized a different mouse strain in our experiments. Therefore, we can not rule out the possibility that the relationship of the CaM pathway to the PKA pathway may not be identical in all genetic backgrounds.
Although the addition of cAMP/IBMX to W-7-treated sperm significantly increased motility compared to sperm treated with W-7 alone, it should be noted that motility was still slightly but significantly less than the motility seen in the untreated control sample (P = .06 for 8-bromo cAMP/IBMX). This finding still argues strongly for our hypothesis that agonists of the PKA pathway rescue the inhibited CaM pathway. However, another possible explanation for this incomplete restoration of motility is that, at the 100 μM concentration used in our study, W-7 may have been inhibiting other enzymes in addition to CaM. If this was the case, then it is possible that cAMP/IBMX rescues these other pathways, and that the remaining sperm motility deficit that persists in the presence cAMP/IBMX is due to the still-inhibited CaM pathway.
Ca2+ also affects sperm motility through its role as a regulator of the predominant flagellar cyclase, sAC, which catalyzes the synthesis of cAMP to activate the PKA pathway. This cyclase is molecularly and biochemically distinct from the transmembrane ACs (tmACs), in part because sAC is uniquely sensitive to both bicarbonate and Ca2+ (Buck et al, 1999; Chen et al, 2000; Wuttke et al, 2001; Liguori et al, 2004). However, the effects of Ca2+ on sAC are independent of CaM (Jaiswal and Conti, 2003; Litvin et al, 2003). Therefore, although it is known that sAC is required for sperm motility (Esposito et al, 2004) and sAC is regulated by Ca2+, the effect of CaM on motility is not achieved via this cyclase.
These observations support a model in which Ca2+ affects motility at 2 different points within the sAC pathway. Alternatively, separate calcium signaling pathways may exist; one that is independent of CaM (eg, sAC/PKA) and one that is not. Since deletion of the sAC gene results in immotile sperm, it is clear that Ca2+/CaM cannot compensate for the loss of sAC function. However, our data suggest that IBMX/cAMP (ie, agonists of the PKA pathway) can restore motility when CaM is inhibited. Thus, components of the sAC/PKA pathway can compensate for a loss of function in the Ca2+/CaM component of the pathway(s), provided that the metabolic substrates pyruvate and lactate are present. The mechanism for this restoration of motility provides a novel avenue for further investigation. One possible model that is consistent with these data is that Ca2+/CaM functions upstream of sAC/PKA.
In Chlamydomonas, it has been shown that Ca2+ may act through CaM and CaMKII to control flagellar motility by regulating dynein-driven microtubule sliding (Smith, 2002). Furthermore, in ascidian sperm, it has recently been documented that CaMKII mediates sperm-activating and -attracting factor (SAAF)-induced motility activation (Nomura et al, 2004). Conflicting data exist regarding a potential role for CaMK isoforms in mammalian sperm motility. One group has reported that targeted mutagenesis of the CaMKIV isoform has no effect on male fertility (Blaeser et al, 2001), while another group has reported that loss of CaMKIV results in male sterility in association with decreased sperm motility (Wu et al, 2000). Inhibitors of CaMKIV have been reported to decrease human sperm motility (Marin-Briggiler et al, 2005) and CaMKII stimulates hyperactivation in bovine sperm (Ignotz and Suarez, 2005). Our data indicate that an isoform of CaMKII is present in the principal piece of murine sperm. This is probably CaMKIIβ but it could also be CaMKIIγ. Taken together, these and additional data (Weinman et al, 1986; Bendahmane et al, 2001) suggest that a Ca2+/CaM/CaMKII pathway in the sperm principal piece is active in mammalian sperm motility regulation.