HCO3 and the cyclic adenosine monophosphate pathway in mammalian sperm

Multiple studies have shown that sperm capacitation is a HCO^-dependent process (Boatman and Robbins, 1991; Gadella and Harrison, 2000; Lee and Storey,

Figure 6.1 Capacitation signaling: proposed sequences of mammalian sperm capacitation. (A) Bicarbonate may enter sperm cells via a Na+/HCC>3 co-transporter or via diffusion as carbon dioxide. Intracellular bicarbonate switches on adenylyl cyclase (AC) and concomitant production of cyclic adenosine monophosphate (cAMP) activates protein kinase A (PKA). The role of cholesterol efflux in the activation of PKA is unclear. Cholesterol efflux may induce increased bicarbonate entry or may directly affect AC. (B) PKA induces tyr (Y) phosphorylation of several substrates (S) most likely via activation of protein tyr kinases (PTK) or inhibition of protein tyr phosphatases (PTP). PKA activation induces plasma membrane changes like in C and/or D. (C) Bilayer redistribution of aminophospholipids. (D) Lateral redistribution of seminolipid and cholesterol. The aminophospholipid scrambling is induced by PKA-dependent activation of a postulated scramblase. Most likely lateral redistribution of cholesterol is required before cholesterol depletion by bovine serum albumin (BSA). (E) The Na+/HCC>3 co-transporter could potentially be a substrate for PKA and may give rise to a sustained increase in HCO3 after its phosphorylation; thus having a positive feedback on cAMP synthesis. (F) Capacitation is also correlated with hyperpolarization of the sperm plasma membrane, which could be due to the opening of a potassium channel that senses alkalinization of the cytosol. (G) The consequent membrane hyperpolarization on turn may open voltage-dependent Ca2+ channels and induce the influx of Ca2+. The increased tyr phosphorylation together with the ionic and lipidic changes in the membrane will induce downstream events involved in the process of capcitation (see Fig. 6.3). PDE: phosphodiesterase. (Adapted from Flesch and Gadella (2000) and Visconti and Kopf (1998).) (see Color plate 7)

1986; Neill and Olds-Clarke, 1987; Shi and Roldan, 1995; Visconti et al., 1995a). Although little is known about the mechanisms of HCO^ transport in sperm, recent evidence strongly suggests that HCO33 transport in these cells is mediated by a member of the Na+/HCO^ co-transporter family, as first described by Romero and Boron (1999). This conclusion is based on findings that HCO^ transport in sperm has the following properties (Demarco et al., 2003):

(1) it is electrogenic,

(3) it increases pHi,

(4) it is blocked by stilbenes, such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS).

The transmembrane movement of HCO33 has been associated with the increase in intracellular pH (pHi) observed during capacitation (Parrish et al., 1989; Zeng et al., 1996). The role of these changes in pHi is not well understood. A second target for HCO33 action is the regulation of cyclic adenosine monophosphate (cAMP) metabolism (Garbers et al., 1982). In 1985, Okamura and collaborators (Okamura and Sugita, 1983) demonstrated that boar semen contained a low molecular weight factor able to induce pig sperm motility; this small factor was also able to stimulate adenylyl cyclase (AC), the enzyme responsible for cAMP synthesis. This factor was later purified to homogeneity and shown to be HCO^ (Okamura et al., 1985). The finding that HCO^ can stimulate sperm AC was later confirmed in bovine and hamster spermatozoa (Garty et al., 1988; Visconti et al., 1990).

The increase in cAMP during capacitation and the stimulation of AC activity in sperm by increased levels of intracellular HCO^ implicate a role for this enzyme and the cAMP signaling pathway in capacitation. Two types of ACs are responsible for cAMP synthesis in eukaryotes; a very well studied family of isoforms, known as transmembrane adenylyl cyclases (tmACs), and a recently isolated soluble adenylyl cyclase (sAC) (Buck et al., 1999). The sAC and tmACs are regulated by different pathways; sAC is insensitive to G-protein or forskolin regulation and is at least 10 times more active in the presence of Mn2+ than in the presence of Mg2+. The identity of the AC activated in capacitating sperm has been the subject of multiple studies; although it is still controversial how many ACs are present in the sperm (Baxendale and Fraser, 2003), multiple evidences demonstrate that one of them is a post-translationally modified form of sAC. This conclusion is supported by the following facts:

(1) Similar to the sperm AC activity, the enzymatic activity of recombinant testicular sAC is stimulated by HCO^ anions (Chen et al., 2000).

(2) Both the sperm AC as well as sAC is 20 times more active in the presence of Mn2+ when compared with the activity in the presence of Mg2+.

(3) Antibodies against the catalytic domain of the testicular sAC recognized two sperm proteins corresponding to the deduced molecular masses of the processed and unprocessed forms of the testicular enzyme (Chen et al., 2000) suggesting that this cyclase remains associated with sperm after spermatogenesis. (4) Antibodies against sAC can immunoprecipitate a fraction containing AC activity from mouse sperm (Chen et al., 2000). Interestingly, the sequence from the catalytic domain of this cyclase has sequence homology to cyanobacterial AC and the cyanobacterial cyclase is also HCO^ dependent (Chen et al., 2000). Although the testis cyclase has been found in the soluble fraction, it is significant that cyclase activity identified in mammalian sperm remains associated with the particulate membrane fraction. Further research should clarify to which extent and how sAC becomes associated to sperm membranes as well as where this process occurs during spermatogenesis or later during epi-didymal maturation.

The HCO^-induced sAC activity results in increasing levels of cAMP in the cell (Harrison and Miller, 2000). One of the main targets for cAMP action is the cAMP-dependent protein kinase A (PKA), which is composed of two regulatory subunits and two catalytic subunits. Both regulatory subunits isoforms (I and II) are present in sperm (Vijayaraghavan et al., 1997; Visconti et al., 1997) but do not share the same sperm localization. PKA catalytic subunit has a unique N-terminal domain in the sperm and it is speculated that this domain is important for the localization of the catalytic subunit within the flagellum after PKA activation (San Agustin et al., 1998). Once activated, PKA phosphorylates various target proteins which are presumed to initiate several signaling pathways (Harrison, 2004; Harrison and Miller, 2000). In pig sperm, the kinetics of the sperm sAC response to bicarbonate is extremely rapid. The cAMP rises to a maximum within 60s (Harrison and Miller, 2000), and the increase in PKA-dependent protein phosphorylation begins within 90s (Harrison, 2004). Functional changes are initiated equally as rapid. It is interesting that cAMP levels fall after their initial rise and then, after some 7 min, begin to rise again (although more slowly); PKA-catalyzed protein phosphorylation follows a similar time course. One possible explanation of this pulse response to bicarbonate with respect to cAMP levels could be the presence of a feedback mechanism in which the stimulated PKA phosphorylates the enzymes responsible for synthesis and degradation of cAMP, thereby modulating their activity (Hanoune and Defer, 2001; Mehats et al., 2002). In this respect the following second rise in cAMP levels appears to surpass this feedback mechanism and has the kinetics of a sustained response to bicarbonate.

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