Molecular Human Reproduction, Vol. 9, No. 12, pp. 739-748, 2003
© European Society of Human Reproduction and Embryology 2003; all rights reserved
Review Article |
First messenger regulation of capacitation via G protein-coupled mechanisms: a tale of serendipity and discovery
Centre for Reproduction, Endocrinology and Diabetes, School of Biomedical Sciences, King’s College London, Guy’s Campus, London Bridge, London SE1 1UL, UK
1 To whom correspondence should be addressed. e-mail: lynn.fraser{at}kcl.ac.uk
Abstract
When placed in a suitable environment, mammalian spermatozoa begin to capacitate and continue until fully capacitated; in vitro, some will ‘over-capacitate’ and undergo spontaneous acrosome loss, undesirable since acrosome-reacted cells are non-fertilizing. Seminal plasma contains several molecules able to bind to specific receptors on spermatozoa, thereby activating/regulating important intracellular signalling pathways. Three such ‘first messengers’ are fertilization promoting peptide (FPP), adenosine and calcitonin, all of which stimulate capacitation and then inhibit spontaneous acrosome reactions by regulating adenylyl cyclase (AC)/cAMP. A recent study has reported the presence in spermatozoa of several membrane-associated AC isoforms, mainly smaller in size than the corresponding ACs in somatic cells, and evidence suggests that more than one of these isoforms may be involved in responses to these first messengers. To regulate AC, FPP receptors appear to interact initially with stimulatory A2A adenosine receptors, which function only in uncapacitated cells, and then with inhibitory A1 receptors, which function only in capacitated cells. In contrast, there appears to be a single population of calcitonin receptors. Responses to cholera and pertussis toxins suggest involvement of G proteins and Gs plus several Gi subunits have been identified in both mouse and human spermatozoa. In particular, G
s and Gi2 are found in the same regions as FPP, adenosine and calcitonin receptors, supporting biochemical evidence for G protein involvement in these responses. In vivo, these first messengers could have a significant effect, helping to maximize the number of capacitated, acrosome-intact (i.e. potentially fertilizing) spermatozoa by regulating what is clearly an important signalling pathway.
Key words: adenosine/adenylyl cyclase/calcitonin/cAMP/FPP/receptors
Introduction
Upon release from the male reproductive tract, mammalian spermatozoa are unable to fertilize, requiring a few to several hours, depending on the species, to be spent in an appropriate environment before they become functional. With time, the spermatozoa undergo post-release maturation events, collectively referred to as ‘capacitation’ (Austin, 1952), that will confer fertilizing ability on them. At least in vitro, once capacitation has begun it will usually continue until the cells are fully capacitated; however, some will ‘over-capacitate’ and undergo a spontaneous acrosome reaction. Biologically, this is undesirable since already acrosome-reacted cells, even if highly motile, are unable to fertilize zona-intact oocytes. This is because important ‘docking’ molecules on the sperm head plasma membrane, necessary for binding to the zona pellucida, are lost as a result of the acrosome reaction (Yanagimachi, 1994).
During the last decade, fertilization promoting peptide (FPP), adenosine and calcitonin, present in seminal plasma and now shown to have specific receptors on mammalian spermatozoa, have been found to elicit responses that regulate capacitation: they initially stimulate capacitation and precocious acquisition of fertilizing ability, then inhibit spontaneous acrosome loss and so preserve fertilizing potential (Fraser and Adeoya-Osiguwa, 2001). Current evidence indicates that all three act as ‘first messengers’ to regulate the production of the ‘second messenger’ cAMP by adenylyl cyclase (AC) in a G protein-regulated manner. This review will trace the investigations undertaken to identify the mechanisms of action of these molecules, the results of which revealed unexpected similarities in responses.
Fertilization promoting peptide
FPP was ‘discovered’ by Cockle and her colleagues (Cockle et al., 1989) who isolated and identified in rabbit prostate tissue the presence of a tripeptide, pGlu–Glu–ProNH2, that was similar to, but distinct from, thyrotrophin releasing hormone (pGlu–His–ProNH2). Subsequent studies identified the same peptide in the prostate glands and/or semen of several other mammals (see Fraser and Adeoya-Osiguwa, 2001). Although also found in the pituitary of several vertebrates (Ashworth et al., 1991; Harvey et al, 1993) and in the thyroid (Cremades et al., 1998), as yet there is little evidence that this tripeptide has a specific biological role in those tissues. In contrast, considerable evidence indicates that the peptide, found in human seminal plasma at quite high concentrations (
50 nmol/l; see Fraser and Adeoya-Osiguwa, 2001), may well play an important role in regulating mammalian sperm function in vivo. Because the peptide was shown to significantly stimulate capacitation and demonstrable fertilizing ability in vitro, it has been named ‘fertilization promoting peptide’ or FPP (Green et al., 1994).
Initial studies evaluated the response of uncapacitated mouse spermatozoa to FPP, using a well characterized in-vitro capacitation and fertilization system (Fraser, 1993). This proved to be advantageous because it utilizes epididymal cells which have had no contact with seminal plasma and hence no contact with FPP. Sperm suspensions were incubated with varying concentrations of FPP for 30 min and then evaluated using chlortetracycline (CTC). CTC is a fluorescent antibiotic that has been shown to bind to mammalian spermatozoa, with the pattern of fluorescence in the sperm head reflecting the functional state of the cell (Ward and Storey, 1984). Its usefulness lies in its ability to allow discrimination between uncapacitated acrosome-intact (non-fertilizing) and capacitated acrosome-intact (potentially fertilizing) spermatozoa, unlike most commonly used microscopical techniques. Three main patterns have been identified: the F pattern, with fluorescence over the entire head, characteristic of uncapacitated acrosome-intact cells; the B pattern, with a fluorescence-free band in the posterior part of the sperm head, characteristic of capacitated acrosome-intact cells; the AR pattern, with dull fluorescence over the whole sperm head, characteristic of capacitated acrosome-reacted cells. These patterns have now been reported for many species, including mouse (Ward and Storey, 1984), human (DasGupta et al., 1993), cattle (Fraser et al., 1995), pig (Wang et al., 1995) and rat (Oberländer et al., 1996).
In the first studies, FPP at 
25 nmol/l was found to significantly stimulate capacitation, with more B pattern cells and correspondingly fewer F pattern cells in the treated suspensions than in the untreated controls; maximal response was obtained with 100 nmol/l and higher concentrations (up to 500 nmol/l) had no additional effect. Interestingly, none of the effective concentrations stimulated the acrosome reaction, a response often observed with treatments that accelerate capacitation. However, these CTC results suggested that treated suspensions would be more fertile and subsequent IVF experiments confirmed this (Green et al., 1994). Successful fertilization requires spermatozoa to express hyperactivated motility and investigations using computer-assisted sperm analysis provided objective evidence that FPP does stimulate hyperactivation (Green et al., 1996c).
It was important to determine whether FPP acts on other mammalian spermatozoa and subsequent studies showed that FPP significantly stimulated human sperm capacitation (CTC analysis) and penetrating ability, using zona-free hamster oocytes to test the latter (Green et al., 1996a). This was interesting since most work on human spermatozoa must utilize ejaculated cells, i.e. cells that have been exposed to seminal plasma and hence to FPP. To evaluate responses to FPP, motile cells were obtained by subjecting ejaculated human semen samples to discontinuous density gradient centrifugation; the fact that cells did respond significantly to exogenous FPP suggests that much of the endogenous peptide was removed by these manipulations. More recently, FPP has also been shown to significantly stimulate capacitation in pig spermatozoa (Funahashi et al., 2000b), another interesting observation since boar seminal plasma contains only low picomolar concentrations of FPP but the spermatozoa appear to still have the appropriate receptors.
Although single compounds usually will have an effect on either uncapacitated or capacitated suspensions, but not both, FPP was different: the addition of FPP to capacitated mouse sperm suspensions resulted in inhibition of the spontaneous acrosome reaction (Green et al., 1996b). Despite this inhibitory action, cells were able to undergo an acrosome reaction in response to either progesterone or unfertilized oocytes, the latter being demonstrated by the increased in-vitro fertility of FPP-treated suspensions. More recent studies have demonstrated a similar effect on capacitated pig (Funahashi et al., 2000a) and human spermatozoa (O.Osiguwa and L.Fraser, unpublished results), with effective concentrations of FPP being similar for all species evaluated. Thus, responses to FPP are physiologically very important, with spermatozoa acquiring fertilizing potential more quickly and then retaining that potential. Such responses in vivo could play an important role in helping to maximize the chances for successful fertilization.
Adenosine
The fact that FPP had an initial stimulatory and a subsequent inhibitory effect on sperm function was reminiscent of earlier studies investigating adenosine’s effects on AC activity and its production of cAMP (e.g. Stein et al., 1986). Stein et al. showed that adenosine stimulated AC activity in uncapacitated mouse spermatozoa but inhibited it in capacitated cells, leading them to suggest that stimulatory adenosine receptors function in uncapacitated cells while inhibitory receptors function in capacitated cells, the latter perhaps providing a mechanism to prevent ‘over-capacitation’. The similarities in the general nature of responses to FPP and adenosine suggested that adenosine should be re-investigated.
Using the same protocols as for FPP, adenosine, which is also found in seminal plasma (Fabiani and Ronquist, 1995), has now been shown to stimulate capacitation, then inhibit spontaneous acrosome loss in both mouse (Green et al., 1996b) and pig spermatozoa (Funahashi et al., 2000b). Insight into a possible mechanism of action for FPP came when combinations of FPP and adenosine were observed to elicit a greater response than with individual treatments. This was especially striking with combinations of low concentrations, which had no significant effect when used individually but together elicited a significant response (Green et al., 1996b). Those results suggested that both FPP and adenosine were working on the same signal transduction pathway and so led to the hypothesis that FPP, like adenosine, acts on AC/cAMP. Numerous studies have now confirmed that hypothesis, with FPP initially stimulating and then inhibiting cAMP production (Adeoya-Osiguwa et al., 1998; Fraser and Adeoya-Osiguwa, 1999; Adeoya-Osiguwa and Fraser, 2002; Figure 5, this review).
|
Receptors for FPP and adenosine
Many different pieces of evidence suggested the presence of separate, specific receptors for FPP and adenosine on spermatozoa, but nothing was known about the former. Serendipitously, a study apparently unrelated to FPP led to the identification of the putative receptor for FPP. Tcp11 is a gene that maps to the t-locus on chromosome 17 in the mouse, a locus known to contain genes that affect male, but not female, fertility (Fraser and Dudley, 1999). The gene appears to be expressed only in the testis, in developing germ cells, and the protein is only synthesized during spermiogenesis (Mazarakis et al., 1991; Hosseini et al., 1994). Structurally, TCP11 has an RGD (arginine–glycine–aspartic acid) sequence and a leucine zipper, but no obvious transmembrane regions. A Tcp11 homologue has been identified on human chromosome 6 (Ragoussis et al., 1992) and recent molecular characterization revealed that the deduced human protein sequence is shorter than, but highly homologous to, the mouse protein (Ma et al., 2002).
The very restricted expression of the protein suggested that it might play a specific role in sperm function. To address this possibility, Fab fragments (needed to avoid sperm agglutination) of a purified IgG fraction of polyclonal anti-TCP11 antiserum were used to localize TCP11 (Figure 1). The protein was detected on the outside of the plasma membrane, in the acrosomal cap and flagellar regions, with variable staining on the midpiece but more consistent and punctuate staining on the principal piece (Fraser et al., 1997b). A similar distribution on human spermatozoa has been observed (M.Tahmasebi, personal communication).
|
In functional tests, uncapacitated mouse sperm suspensions were incubated with the anti-TCP11 antibodies and sampled at two time points. After 30 min, a significant stimulation of capacitation was detected in treated cells (CTC analysis), while after 120 min, a significant inhibition of acrosome loss was observed in the treated suspensions, mimicking results obtained with FPP. Subsequent IVF experiments demonstrated that treated spermatozoa became fertile more quickly than untreated controls and retained fertility during the 2 h preincubation phase, despite the presence of the antibodies and their inhibition of spontaneous acrosome loss (Fraser et al., 1997b). These responses led to the hypothesis that TCP11 is the receptor for FPP; later studies have provided substantial support for this, including demonstrations that antibodies to TCP11 stimulate cAMP synthesis (Adeoya-Osiguwa et al., 1998) and a competitive inhibitor of FPP blocks responses to antibodies (Fraser et al., 1997a).
Adenosine receptors are known to be typical seven transmembrane domain G protein-coupled receptors that often modulate AC activity, and several different isoforms, both inhibitory and stimulatory, have been identified in somatic cells (Palmer and Stiles, 1995). Following the proposal that two populations of adenosine receptors were present on mouse spermatozoa, each functioning in a capacitation-dependent manner (Stein et al., 1986), several studies have provided evidence for the presence and function of stimulatory receptors (mouse: Fraser, 1990; Fraser and Duncan, 1993; human: Fenichel et al., 1996). Using more specific agonists and antagonists, evidence supporting the original proposal of Stein et al. has been obtained: both stimulatory A2A and inhibitory A1 adenosine receptors are present, but A2A receptors function only in uncapacitated spermatozoa while A1 receptors function only in capacitated cells (Fraser and Adeoya-Osiguwa, 1999). Although adenosine receptors fit the profile of receptors able to regulate AC, the apparent lack of transmembrane regions in TCP11 raised an important question: how can TCP11 modulate AC activity? The answer appears to be that FPP receptors somehow interact with adenosine receptors to set up a complete signalling pathway. Evidence for this includes the demonstration that specific antagonists acting only on either A2A or A1 receptors inhibit responses to FPP as well as to adenosine and that a competitive inhibitor of FPP also inhibits responses to adenosine (mouse: Fraser and Adeoya-Osiguwa, 1999; pig: Funahashi et al., 2000b).
This information then led to another question: where are adenosine receptors located? If FPP receptors interact with adenosine receptors, they need to be in the same places. A study evaluating A1 adenosine receptor localization in bull, horse, rat, human and rabbit spermatozoa reported staining, sometimes diffuse in nature, in the acrosomal and postacrosomal regions in most species, along with variable staining in the flagellum (Minelli et al., 2000). More recently, a somewhat different distribution was observed in mouse spermatozoa, with both A2A and A1 receptors being observed on the acrosomal cap (of acrosome-intact cells) and the flagellum, most abundantly on the principal piece (Adeoya-Osiguwa and Fraser, 2002). Because the receptors have a complex transmembrane structure, permeabilization to ensure epitope accessibility was found to give the most consistent results in the latter study. Pilot experiments revealed that different methods used for fixation and permeabilization could have marked effects on the localization and intensity of fluorescence, suggesting that methodological differences may explain the differing results in the two studies.
Interestingly, the intensity of staining was capacitation state-dependent, A2A staining being much stronger on uncapacitated spermatozoa and A1 staining being stronger on capacitated cells, suggesting capacitation-related changes in epitope accessibility (Figure 2). When capacitated suspensions were briefly treated with a purified decapacitation factor (DF) preparation that causes cells to revert to the uncapacitated, non-fertilizing state (Fraser et al., 1990; Rakha et al., 2000), the strong staining for A2A receptors was regenerated. Furthermore, these changes in epitope accessibility appear to reflect changes in accessibility of adenosine binding sites and consequent receptor function: A2A agonists stimulated AC/cAMP only in uncapacitated spermatozoa and A1 agonists inhibited AC/cAMP only in capacitated cells, but A2A agonists were able to stimulate AC/cAMP in capacitated suspensions decapacitated by a brief exposure to DF (Adeoya-Osiguwa and Fraser, 2002). These results thus demonstrate that capacitation is functionally reversible and involves biologically significant changes in the conformation of adenosine receptors and, plausibly, other transmembrane receptors.
|
A recent study, again focusing only on A1 adenosine receptors, proposed that binding of adenosine resulted in activation of the phospholipase C (PLC) signal transduction pathway (Allegrucci et al., 2001) since a transient rise in inositol trisphosphate was observed in uncapacitated human cells treated with the A1-specific agonist cyclopentyladenosine (CPA). However, in several parts of this study, control suspensions were incubated in BSA-containing medium, while experiments were evaluated in BSA-free medium containing CPA. Since BSA plays a general role in maintaining sperm viability and function, while CPA acts specifically on adenosine receptors, it would have been physiologically more informative to evaluate suspensions in BSA-containing medium ± CPA. The suggestion that PLC is involved in capacitation per se is inconsistent with the recent demonstration that PLC plays a role in acrosomal exocytosis in capacitated human spermatozoa rather than stimulating capacitation in uncapacitated cells (O’Toole et al., 1996). Furthermore, two separate studies have shown that CPA has no effect on uncapacitated mouse spermatozoa when assessed using CTC, yet it significantly inhibits acrosome loss and cAMP production in capacitated cells (Fraser and Adeoya-Osiguwa, 1999; Adeoya-Osiguwa and Fraser, 2002).
G proteins
In somatic cells, modulation of AC/cAMP by adenosine receptors involves stimulatory receptors interacting with G proteins containing a stimulatory alpha subunit (Gs) and inhibitory receptors interacting with G proteins containing an inhibitory alpha subunit (Gi/o; Palmer and Stiles, 1995). Given the strong evidence that FPP and adenosine can regulate production of cAMP, involvement of G proteins in these responses was plausible. GTP analogues had been shown to significantly stimulate both cAMP production in permeabilized uncapacitated mouse spermatozoa (Fraser and Duncan, 1993) and capacitation in whole live cells (Fraser and Adeoya-Osiguwa, 1999), consistent with the presence of Gs. Cholera toxin, known to catalyse ADP ribosylation of Gs and so irreversibly activate the protein, stimulated capacitation and production of cAMP in uncapacitated suspensions to the same extent as FPP, but had no detectable effect on capacitated suspensions. In contrast, pertussis toxin, known to inhibit Gi/o, had no effect on uncapacitated cells but abolished FPP’s inhibition of both spontaneous acrosome loss and cAMP production in capacitated cells (Fraser and Adeoya-Osiguwa, 1999). Later investigations of protein tyrosine phosphorylation revealed that FPP, cholera toxin and adenosine all stimulated phosphorylation in uncapacitated suspensions, while FPP inhibited phosphorylation in capacitated suspensions; the inclusion of pertussis toxin in the latter abolished responses to FPP (Adeoya-Osiguwa and Fraser, 2000). Taken together, these results suggest that G proteins are involved in the regulation of AC/cAMP and that unregulated production of cAMP in capacitated spermatozoa can lead to spontaneous acrosome loss.
More direct proof for the presence of several different G subunits was obtained by electrophoresis/Western blotting, using mouse sperm membrane preparations and commercially available antibodies directed against stimulatory and inhibitory G subunits. Consistent with the physiological and biochemical responses to cholera and pertussis toxins, positive evidence for the presence of several isoforms of stimulatory Gs (the most abundant being 48 kDa) as well as inhibitory Gi2, Gi3 and Go, but not Gi1, subunits was obtained (Fraser and Adeoya-Osiguwa, 1999). A number of earlier studies reported finding evidence for inhibitory G subunits but none for stimulatory G subunits in mammalian spermatozoa (e.g. Bentley et al., 1986; Kopf et al., 1986; Glassner et al., 1991; Merlet et al., 1999), leading to the prevailing dogma that spermatozoa do have inhibitory but do not have stimulatory G subunits. Thus, the recent report that mouse spermatozoa have Gs was met with considerable scepticism.
To provide even more convincing evidence for the presence of Gs, mouse and human sperm lysates were analysed using electrophoresis/Western blotting and Gs was detected in both (Baxendale and Fraser, 2003b). The most abundant isoform in both species was Gs-long (Gs-L; 48 kDa) and the next most abundant was Gs-short (Gs-S; 45 kDa, the most common isoform in somatic tissues; Novotny and Svoboda, 1998), results consistent with those obtained using mouse sperm membranes (Fraser and Adeoya-Osiguwa, 1999). The ‘classic’ evidence for the presence of Gs is detection of cholera toxin-catalysed ADP ribosylation of an appropriately sized protein and this has now been achieved. Cholera toxin both enhanced the ADP ribosylation of a protein the correct size for Gs (Figure 3) and stimulated cAMP production in permeabilized mouse spermatozoa, suggesting functional coupling between Gs and AC (Baxendale and Fraser, 2003b). Therefore, a wide array of physiological, biochemical and immunological evidence, obtained in two different species, strongly supports the presence of both stimulatory and inhibitory G subunits in mammalian spermatozoa and makes it plausible that they are involved in regulating AC activity.
|
It is intriguing and puzzling that there has been such apparent difficulty in identifying G
s in spermatozoa and, concomitantly, such determination among many biologists to adhere firmly to a dogma based on negative evidence. An early study, looking for suitable protein substrates that could be ADP ribosylated by cholera and/or pertussis toxins, reported that spermatozoa had neither Gs nor Gi (Hildebrandt et al., 1985), but shortly thereafter, evidence for Gi, but not for Gs, was reported in two new studies using similar methodology (Bentley et al., 1986; Kopf et al., 1986). Other investigators using antibodies to try to detect Gs also failed to find positive evidence (e.g. bovine: Hinsch et al. 1995; human: Hinsch et al., 1992; Merlet et al., 1999); it is difficult to speculate on why those studies obtained negative results, yet the more recent ones have obtained positive results. Only limited details regarding the antibodies used in the former studies are available, but one possibility is that those antibodies may have had relatively low affinity for Gs. Surprisingly, very little attention appears to have been paid to the positive evidence that sea urchin spermatozoa appear to contain Gs (Cuellar-Mata et al., 1995; Ohta et al., 2000), based on Western blotting evidence for the presence of proteins of the correct sizes for Gs (48 and 45 kDa, very similar to results of Fraser and Adeoya-Osiguwa, 1999, and Baxendale and Fraser, 2003b, for mouse and human spermatozoa) and cholera toxin-catalysed ADP ribosylation of a protein of 48 kDa. Even the first study to conclude that spermatozoa (canine) had neither stimulatory nor inhibitory G subunits (Hildebrandt et al., 1985) did actually observe a small amount of an ADP-ribosylated protein of 42 kDa, as well as several smaller ribosylated proteins of 20–25 kDa. These might be full-length and proteolytically cleaved Gs respectively, since Gs is apparently susceptible to proteolysis, particularly when it is ADP ribosylated (Sato-Kusubata et al., 2000).
The next question was: where are the G subunits located? According to the evolving hypothesis, they should be found in the same regions as the FPP and adenosine receptors. Again using both mouse and human spermatozoa, whole cells were prepared for immunolocalization using a range of commercially available antibodies directed against Gs, Golf (related to Gs), Gi1, Gi2, Gi3, Go and Gq/11. Similar patterns of distribution were seen in both species for individual G subunits, with a reasonable signal for all the subunits except for Gi1 (Baxendale and Fraser, 2003b). There was a strong signal for Gs in the acrosomal cap region, the neck and the principal piece of the flagellum, and the inhibitory G subunit with the most similar distribution was Gi2 (Figure 4). On the basis of these results, we have hypothesized that Gs mediates responses involving stimulatory A2A adenosine receptors (and FPP receptors), while Gi2 mediates responses involving inhibitory A1 adenosine receptors (Fraser and Adeoya-Osiguwa, 1999; Adeoya-Osiguwa and Fraser, 2002; Baxendale and Fraser, 2003b). Using human spermatozoa, Allegrucci et al. (Allegrucci et al., 2001) have reported co-immunoprecipitation of A1 receptors and Gi2, results that suggest a direct interaction between the two and so support our hypothesis. The distribution of the other G subunits (Baxendale and Fraser, 2003b) was broadly similar to those reported in other studies (e.g. Merlet et al., 1999); possible functions for them have yet to be identified. Of particular interest in the context of odorant receptors perhaps playing a role in mammalian sperm chemotaxis (Spehr et al., 2003) was the demonstration that Golf, closely related to Gs and known to participate in the olfactory signalling pathway, is present (Figure 4) and found in the same regions as some AC isoforms (see below).
|
Several studies have suggested that a pertussis toxin-sensitive G
subunit is involved in the zona pellucida-induced acrosome reaction, with pertussis toxin blocking acrosomal exocytosis (e.g. Endo et al., 1987; Lee et al., 1992). No specific signalling pathway had been identified until a recent study suggested that the zona-induced acrosome reaction involves stimulation of sperm AC via a pertussis toxin-sensitive inhibitory G protein, the active moiety possibly being the ß
dimer (Leclerc and Kopf, 1999). This is puzzling for two reasons. First, AC stimulation usually involves Gs and secondly, AC inhibition by FPP, adenosine and calcitonin does not interfere with zona-induced acrosome reactions in fertilizing spermatozoa (Green et al., 1994; Funahashi et al., 2000b; Fraser et al., 2001). One possible explanation could be the involvement of different isoforms of AC (see below) in responses to the regulatory molecules and zona glycoproteins. Calcitonin
Calcitonin is a 32 amino acid peptide hormone, usually considered in the context of bone homeostasis, and calcitonin receptors, like adenosine receptors, are seven transmembrane domain G protein-coupled proteins that often modulate AC/cAMP (Pondel, 2000). Calcitonin was found to be present in much higher concentrations in seminal plasma than in blood plasma (Sjöberg et al., 1980) and a subsequent study provided some evidence that calcitonin receptors are present on mammalian spermatozoa (Silvestroni et al., 1987). More recently, calcitonin has been demonstrated to elicit the same capacitation-dependent responses as FPP and adenosine (mouse: Fraser et al., 2001; human: O.Osiguwa and L.Fraser, unpublished data): stimulation of capacitation and fertilizing ability, then inhibition of spontaneous acrosome reactions, the latter response being pertussis toxin-sensitive. Combinations of low, non-stimulatory concentrations of calcitonin and FPP significantly stimulated capacitation in both uncapacitated mouse (Fraser et al., 2001) and human spermatozoa (O.Osiguwa and L.Fraser, unpublished data), leading to the hypothesis that both peptides act on the same signalling pathway, namely AC/cAMP.
In a study testing that hypothesis, calcitonin, like FPP, was shown to significantly stimulate cAMP production in uncapacitated spermatozoa and then inhibit it in capacitated cells (Adeoya-Osiguwa and Fraser, 2003). Interestingly, the slower than usual capacitation occurring in the ‘capacitated’ suspensions made it possible to observe that the inhibitory effects of FPP preceded those of calcitonin but eventually a similar and significant degree of inhibition was observed with both calcitonin and FPP (Figure 5). This may reflect the involvement of two different adenosine receptor populations in response to FPP, allowing a more rapid changeover from stimulation to inhibition, whereas there appears to be only one population of calcitonin receptors. The latter are located on both the acrosomal cap and the flagellum, especially on the midpiece, in similar regions to the other receptors and G protein subunits of interest (Gs, Gi2); there was no evidence for any capacitation-dependent changes in epitope accessibility such as had been detected for adenosine receptors.
AC isoforms
Thus far, nine membrane-bound isoforms of AC (mACs), all of which are G protein-regulated and respond to diverse activating molecules, have been identified in somatic cells. There is also a soluble isoform, sAC, that has been identified in testicular tissue; recent purification and characterization studies have indicated that it is not related to the membrane-bound isoforms (Buck et al., 1999). At 48 kDa, the predominant form of sAC is smaller than mACs found in somatic cells (>100 kDa); it is stimulated by bicarbonate (HCO3–) and Mn2+ but appears to be neither regulated by G proteins nor responsive to known modulators of mAC (Buck et al., 1999; Chen et al., 2000). Many earlier studies reported that mammalian sperm AC is markedly different from somatic cell ACs, being insensitive to G proteins and activators of AC such as fluoride and forskolin (reviewed in Defer et al., 2000), making it likely that much of the AC activity detected in those earlier studies reflected the presence and activity of sAC. However, many of those studies used partially purified subcellular sperm fractions, preparations that might not favour the mACs since their membrane association presumably is required for optimal activity, and so sAC might be the predominant functional isoform. A number of recent studies have either used live or permeabilized whole cells, preparations that may give a better insight into physiological responses because the general cellular architecture that could well be important for signal transduction pathways will be maintained.
Indeed, there have been reports that mammalian sperm have AC activity with properties similar to mAC; it can be stimulated by fluoride (Baxendale and Fraser, 2003a) and forskolin (Leclerc and Kopf, 1995; Baxendale and Fraser, 2003a), the latter response being significantly inhibited by dideoxyadenosine (ddAdo), a specific inhibitor acting on the P site of mAC. Forskolin also stimulated capacitation and acrosome reactions, consistent with a continuous stimulation of AC/cAMP, but inclusion of ddAdo inhibited these responses (Baxendale and Fraser, 2003a). Furthermore, considerable evidence indicates that cAMP production in mammalian spermatozoa is sensitive to bacterial toxins known to act on stimulatory and inhibitory G subunits (see above) and to various GTP analogues such as Gpp(NH)p (Stein et al., 1986; Fraser and Duncan, 1993).
The fact that various first messengers can bind to specific externally directed receptors on intact, live cells and regulate the activity of AC/cAMP is consistent with the involvement of mACs rather than sAC. At present, there is little information in the literature regarding which mACs might be present in mammalian spermatozoa, other than reports that mAC3 (implicated in olfactory signalling; Bakalyar and Reed, 1990) could be detected in immature germ cells (Defer et al., 1998; Gautier-Courteille et al., 1998). However, recent evidence obtained using specific antibodies directed against various somatic cell mAC isoforms indicates the presence of several different mACs (mAC2, 3 and 8 in relative abundance and mAC1 and 4, less so), with distinct localizations in the head and flagellum of mature mouse spermatozoa (Figure 6) (Baxendale and Fraser, 2003a). Using those same antibodies, electrophoresis/Western blotting of both mouse sperm membranes and cell lysates confirmed the presence of a number of proteins although, interestingly, most of the proteins detected are smaller in size (<100 kDa) than the mACs identified in somatic cells. Using a wide array of experimental conditions to minimize proteolytic activity, the same, smaller size, proteins were consistently observed in sperm preparations. However, in testis lysates the antibodies detected both larger (especially >160 kDa) and smaller proteins, and the relevant peptides were able to block the signals in both sperm and testis preparations, indicating that the antibodies were specific for the different mAC isoforms.
|
AC activity, responsive to various known stimulators of mAC activity such as forskolin, GTP analogues and NaF, was also detected in these preparations, but the specific functions of different isoforms remain to be determined. However, new evidence suggests that at least some of the receptors involved in regulating AC/cAMP may interact with different mACs. It is generally accepted that Ca2+ is required for capacitation (de Lamirande et al., 1997) and studies of FPP are consistent with this. Although FPP can bind to mouse spermatozoa in medium lacking CaCl2 (Adeoya-Osiguwa et al., 1998), physiological responses to FPP require the presence of at least 90 µmol/l Ca2+ (Green et al., 1996b). Similar investigations for calcitonin revealed that calcitonin, unlike FPP, can significantly stimulate capacitation (CTC) and cAMP production in sperm suspensions incubated in Ca2+-deficient medium. The inclusion of EGTA to chelate trace amounts of Ca2+ (
13 µmol/l; Fraser, 1982) abolished any response, confirming that very small amounts of extracellular Ca2+ were sufficient for physiological responses. Subsequent IVF experiments using spermatozoa preincubated in Ca2+-deficient medium ± calcitonin, then diluted into medium containing Ca2+ + calcitonin, revealed that suspensions preincubated in the presence of calcitonin were significantly more fertile than the controls preincubated in Ca2+-deficient medium alone (Figure 7) (Adeoya-Osiguwa and Fraser, 2003). The simplest explanation for these differences in responses to calcitonin and FPP is that the respective receptors are regulating different isoforms of mAC, with one requiring less Ca2+ for function than the other. Consistent with that hypothesis, mAC isoforms are known to differ in their requirements for Ca2+ (Defer et al., 2000).
|
Recent evidence suggests that HCO3–, and therefore plausibly sAC, plays an early role in capacitation: within 15 min in the presence of HCO3–, there are marked changes in the sperm membrane lipid architecture, these changes being controlled by cAMP-dependent phosphorylation (Harrison, 2003). It is possible that such membrane modifications might ‘prime’ either the mAC isoforms, or receptors able to regulate mAC activity, by altering their conformation. Even if this is the case, the relative overall contributions made by sAC and mACs to promoting capacitation and acquisition of fertilizing ability, especially in vivo, are not known at present. Given the ubiquitous presence of HCO3– in the female tract, sAC might well be constantly stimulated, resulting in continued production of cAMP; in the absence of regulatory factors, this could ultimately contribute to spontaneous acrosome loss and consequent loss of fertility. However, addition of first messengers such as adenosine and calcitonin (known to be present in vivo) to capacitated cells in vitro results in rapid inhibition of both cAMP production and spontaneous acrosome loss. Since these responses are abolished by pertussis toxin, indicative of G protein and therefore probably mAC rather than sAC involvement, it is plausible that mAC isoforms could play an important role in preserving sperm fertilizing potential. Thus, interplay between mACs and sAC could provide a balanced and biologically important regulation of cAMP levels.
Another possible role for sperm mAC has been identified very recently, although the mechanisms involved are not yet clear. A novel testicular odorant receptor has been shown to be present in mammalian spermatozoa, with evidence that it might function in chemotaxis; responses to bourgeonal, providing a strong chemoattractant signal, were inhibited by inclusion of inhibitors of AC but not inhibitors of PLC (Spehr et al, 2003). Given the similar locations of Golf and AC3 in the head and flagellum of mature spermatozoa (Baxendale and Fraser, 2003a,b), it would appear that all the components of a typical olfactory signalling pathway are present and so plausibly could be involved in responses to chemoattractants.
Conclusions
The initial investigations of pGlu–Glu–ProNH2 began some 10 years ago, asking one apparently simple question: does this peptide, known to be present in nanomolar concentrations in seminal plasma of several mammalian species, have a biological effect on mammalian spermatozoa? That question, once answered in the affirmative, led to many more questions and answers, with subsequent overlap being identified between the mechanisms of action involved in responses to FPP and those relating to adenosine and calcitonin. It has long been known that cAMP is an important second messenger in sperm physiology, but it was somewhat surprising to discover that seminal plasma contains at least three small molecules able to act as first messengers to regulate the activity of AC and its production of cAMP. The recent studies discussed above have confirmed the pivotal importance of cAMP in sperm function and have revealed unexpected complexities in the regulation of AC/cAMP; the proposed interactions are shown in Figure 8. Such regulation is biologically important since unrestrained production of cAMP appears to play a role in spontaneous acrosome loss, an event that abolishes a spermatozoon’s fertilizing potential. Thus, the hypothesis formulated some time ago that the inhibitory responses elicited by adenosine in capacitated spermatozoa might provide a mechanism for preventing ‘over-capacitation’ (Stein et al., 1986) is strongly supported by recent experimental evidence; furthermore, the fact that additional molecules act similarly suggests that these mechanisms play a crucial role in maintaining spermatozoa in a potentially fertilizing state, essential for maximizing the chance of successful fertilization in vivo.
|
Acknowledgements
Recent original research discussed in this review has been supported by grants to L.R.F. from several sources, including The Wellcome Trust, the Kinetique Biomedical Seed Fund and Pfizer Global Research and Development.
References
Adeoya-Osiguwa, S.A. and Fraser, L,R. (2000) Fertilization promoting peptide and adenosine, acting as first messengers, regulate cAMP production and consequent protein tyrosine phosphorylation in a capacitation-dependent manner. Mol. Reprod. Dev., 57, 384–392.[CrossRef][Web of Science][Medline]
Adeoya-Osiguwa, S.A. and Fraser, LR. (2002) Capacitation state-dependent changes in adenosine receptors and their regulation of adenylyl cyclase/cAMP. Mol. Reprod. Dev., 63, 245–255.[CrossRef][Web of Science][Medline]
Adeoya-Osiguwa, S.A. and Fraser, L.R. (2003) Calcitonin acts as a first messenger to regulate adenylyl cyclase/cAMP and mammalian sperm function. Mol. Reprod. Dev., 65, 228–236.[CrossRef][Web of Science][Medline]
Adeoya-Osiguwa, S.A., Dudley, R.K., Hosseini, R. and Fraser, L.R. (1998) FPP modulates mammalian sperm function via TCP-11 and the adenylyl cyclase/cAMP pathway. Mol. Reprod. Dev., 51, 468–476.[CrossRef][Web of Science][Medline]
Allegrucci, C., Liguori, L. and Minelli, A. (2001) Stimulation by N6-cyclopentyladenosine of A1 adenosine receptors, coupled to Gi2 protein subunit, has a capacitative effect on human spermatozoa. Biol. Reprod., 64, 1653–1659.
Ashworth, R.J., Morrell, J.M., Aitken, A., Patel, Y. and Cockle, S.M. (1991) pGlu-Glu-ProNH2 is present in rat anterior and posterior pituitary gland. J. Endocrinol., 129, R1–R4.
Austin, C.R. (1952) The ‘capacitation’ of the mammalian sperm. Nature, 170, 326.[CrossRef][Medline]
Bakalyar, H.A. and Reed, R.R. (1990) Identification of a specialized adenylyl cyclase that may mediate odorant detection. Science, 250, 1403–1406.
Baxendale, R.W. and Fraser, L.R. (2003a) Evidence for multiple distinctly localized adenylyl cyclase isoforms in mammalian spermatozoa. Mol. Reprod. Dev., 66, 181–189.[CrossRef][Web of Science][Medline]
Baxendale, R.W. and Fraser, L.R. (2003b) Immunolocalization of multiple G subunits in mammalian spermatozoa and additional evidence for Gs. Mol. Reprod. Dev., 65, 104–113.[CrossRef][Web of Science][Medline]
Bentley, J.K., Garbers, D.L., Domino, S.E., Noland, T.D. and Van Dop, C. (1986) Spermatozoa contain a guanine nucleotide-binding protein ADP-ribosylated by pertussis toxin. Biochem. Biophys. Res. Commun., 138, 728–734.[CrossRef][Web of Science][Medline]
Buck, J., Sinclair, M.L., Schapal, L., Cann, M.J. and Levin, L.R. (1999) Cytosolic adenylyl cyclase defines a unique signalling molecule in mammals. Proc. Natl Acad. Sci. USA, 96, 79–84.
Chen, Y., Cann, M.J., Litvin, T.N., Iourgenko, V., Sinclair, M.L., Levin, L.R. and Buck, J. (2000) Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor. Science, 289, 625–628.
Cockle, S.M., Aitken, A., Beg, F. and Smyth, D.G. (1989) A novel peptide, pyroglutamylglutamylprolineamide, in the rabbit prostate, structurally related to TRH. J. Biol. Chem., 264, 7788–7791.
Cremades, A., Renafiel, R., Rausell, V., Del Rio-Garcia, J. and Smyth, D.G. (1998) The thyrotrophin-releasing hormone-like peptides pGlu-Phe-Pro amide and pGlu-Glu-Pro amide increase plasma triiodothyronine levels in the mouse; the activity is sensitive to testosterone. Eur. J. Pharmacol., 358, 63–67.[CrossRef][Web of Science][Medline]
Cuellar-Mata, P., Martinez-Cadena, G., Castellano, L.E., Aldana-Veloz, G., Novoa-Martinez, G., Vargas, I., Darszon, A. and Garcia-Soto, J. (1995) Multiple GTP-binding proteins in sea urchin sperm: evidence for Gs and small G-proteins. Dev. Growth Differ., 37, 173–181.
DasGupta, S., Mills, C.L. and Fraser, L.R. (1993) Ca2+-related changes in the capacitation state of human spermatozoa assessed by a chlortetracycline fluorescence assay. J. Reprod. Fertil., 99, 135–143.
Defer, N., Marinx, O., Poyard, M., Lienard, M.O., Jegou, B. and Hanoune, J. (1998) The olfactory adenylyl cyclase type 3 is expressed in male germ cells. FEBS Lett., 424, 216–220.[CrossRef][Web of Science][Medline]
Defer, N., Best-Belpomme, M. and Hanoune, J. (2000) Tissue specificity and physiological relevance of various isoforms of adenylyl cyclase. Am. J. Physiol. Renal Physiol., 279, F400–F416.
de Lamirande, E., Leclerc, P. and Gagnon, C. (1997) Capacitation as a regulatory event that primes spermatozoa for the acrosome reaction and fertilization. Mol. Hum. Reprod., 3, 175–194.
Endo, Y., Lee, M.A. and Kopf, G.S. (1987) Evidence for the role of a guanine nucleotide-binding regulatory protein in the zona pellucida-induced mouse sperm acrosome reaction. Dev. Biol., 119, 210–216.[CrossRef][Web of Science][Medline]
Fabiani, R. and Ronquist, G. (1995) Abundance of guanine, guanosine, inosine and adenosine in human seminal plasma. Int. J. Clin. Lab. Res., 25, 47–51.[Web of Science][Medline]
Fenichel, P., Gharib, A., Emiliozzi, C., Donzeau, M. and Menezo, Y. (1996) Stimulation of human sperm during capacitation in vitro by an adenosine agonist with specificity for A2 receptors. Biol. Reprod., 54, 1405–1411.[Abstract]
Fraser, L. (1982) Ca2+ is required for mouse sperm capacitation and fertilization in vitro. J. Androl., 3, 412–419.[Abstract]
Fraser, L.R. (1990) Adenosine and its analogues, possibly acting at A2 receptors, stimulate mouse sperm fertilizing ability during early stages of capacitation. J. Reprod. Fertil., 89, 463–476.
Fraser, L.R. (1993) In vitro capacitation and fertilization. In Wasserman, P.M. and DePamphilis, M.L. (eds), Guide to Techniques in Mouse Development. Methods Enzymol., 225, 239–253.[Web of Science][Medline]
Fraser, L.R. and Adeoya-Osiguwa, S.A. (1999) Modulation of adenylyl cyclase by FPP and adenosine involves stimulatory and inhibitory adenosine receptors and G proteins. Mol. Reprod. Dev., 53, 459–471.[CrossRef][Web of Science][Medline]
Fraser, L.R. and Adeoya-Osiguwa, S.A. (2001) Fertilization promoting peptide—a possible regulator of sperm function. Vitam. Horm., 63, 1–28.[Web of Science][Medline]
Fraser, L.R. and Dudley, R.K. (1999) New insights into the t-complex and control of sperm function. Bioessays, 21, 304–312.[CrossRef][Web of Science][Medline]
Fraser, L.R and Duncan, A.E. (1993) Adenosine analogues with specificity for A2 receptors bind to mouse spermatozoa and stimulate adenylate cyclase activity in uncapacitated suspensions. J. Reprod. Fertil., 98, 187–194.
Fraser, L.R., Harrison R.A.P. and Herod J.E. (1990) Characterization of a decapacitation factor associated with epididymal mouse spermatozoa. J. Reprod. Fertil., 89, 135–148.
Fraser, L.R., Abeydeera, L.R. and Niwa, K. (1995) Ca2+-regulating mechanisms that modulate bull sperm capacitation and acrosomal exocytosis as determined by chlortetracycline analysis. Mol. Reprod. Dev., 40, 233–241.[CrossRef][Web of Science][Medline]
Fraser, L.R., Hanyaloglu, A. and Cockle, S.M. (1997a) A fertilization promoting peptide (FPP)-related tripeptide competitively inhibits responses to FPP: a cause of male subfertility? Mol. Reprod. Dev., 48, 529–535.[CrossRef][Web of Science][Medline]
Fraser, L.R., Hosseini, R., Hanyaloglu, A., Talmor, A. and Dudley, R.K. (1997b) TCP-11, the product of a mouse t-complex gene, plays a role in stimulation of capacitation and inhibition of the spontaneous acrosome reaction. Mol. Reprod. Dev., 48, 375–382.[CrossRef][Web of Science][Medline]
Fraser, L.R., Pondel, M.C. and Vinson, G.P. (2001) Calcitonin, angiotensin II and FPP significantly modulate mouse sperm function. Mol. Hum. Reprod., 7, 245–253.
Funahashi, H., Fujuwara, T. and Nagai, T. (2000a) Modulation of the function of boar spermatozoa via adenosine and fertilization promoting peptide receptors reduce the incidence of polyspermic penetration into porcine oocytes. Biol. Reprod., 63, 1157–1163.
Funahashi, H., Asano, A., Fujiwara, T., Nagai, T., Niwa, K. and Fraser, L.R. (2000b) Both fertilization promoting peptide and adenosine stimulate capacitation but inhibit spontaneous acrosome loss in boar spermatozoa in vitro. Mol. Reprod. Dev., 55, 117–124.[CrossRef][Web of Science][Medline]
Gautier-Courteille, C., Salanova, M. and Conti, M. (1998) The olfactory adenylyl cyclase III is expressed in rat germ cells during spermiogenesis. Endocrinology, 139, 2588–2599.
Glassner, M., Jones, J., Kligman, I., Woolkalis, M.J., Gerton, G.L. and Kopf, G.S. (1991) Immunocytochemical and biochemical characterization of guanine nucleotide-binding regulatory proteins in mammalian spermatozoa. Dev. Biol., 146, 438–450.[CrossRef][Web of Science][Medline]
Green, C.M., Cockle, S.M., Watson, P.F. and Fraser, L.R. (1994) Stimulating effect of pyroglutamylglutamylprolineamide, a prostatic, TRH-related tripeptide, on mouse sperm capacitation and fertilizing ability in vitro. Mol. Reprod. Dev., 38, 215–221.[CrossRef][Web of Science][Medline]
Green, C.M., Cockle, S.M., Watson, P.F. and Fraser, L.R. (1996a) Fertilization promoting peptide, a tripeptide similar to thyrotrophin-releasing hormone, stimulates the capacitation and fertilizing ability of human spermatozoa in vitro. Hum. Reprod., 11, 830–836.
Green, C.M., Cockle, S.M., Watson, P.F. and Fraser, L.R. (1996b) A possible mechanism of action for fertilization promoting peptide, a TRH-related tripeptide that promotes capacitation and fertilizing ability in mammalian spermatozoa. Mol. Reprod. Dev., 45, 244–252.[CrossRef][Web of Science][Medline]
Green, C.M., Cockle, S.M., Fraser, L.R. and Watson, P.F. (1996c) The effect of FPP on the motility characteristics of mouse spermatozoa. J. Reprod. Fertil. Abstr. Ser., 17, 31.
Harrison, R.A.P. (2003) Cyclic AMP signalling during mammalian sperm capacitation—still largely terra incognita. Reprod. Domest. Anim., 38, 102–110.[CrossRef][Web of Science][Medline]
Harvey, S., Trudeau, V.L., Ashworth, R.J. and Cockle, S.M. (1993) pGlutamylgluamylprolineamide modulation of growth hormone secretion in domestic fowls: Antagonism of thyrotrophin-releasing hormone action? J. Endocrinol., 138, 137–147.
Hildebrandt, J.D., Codina, J., Tash, J.S., Kirchick, H.J., Lipschultz, L., Sekura, R.D. and Birnbaumer, L. (1985) The membrane-bound spermatozoal adenylyl cyclase system does not share coupling characteristics with somatic cell adenylyl cyclases. Endocrinology, 116, 1357–1366.
Hinsch, K.D., Hinsch, E., Aumuller, G., Tychowiecka, I., Schultz, G. and Schill, W.B. (1992) Immunological identification of G protein - and ß-subunits in tail membranes of bovine spermatozoa. Biol. Reprod., 47, 337–346.[Abstract]
Hinsch, K.D., Schwerdel, C., Habermann, B., Schill, W.B., Muller-Schlosser, F. and Hinsch, E. (1995) Identification of heterotrimeric G proteins in human sperm tail membranes. Mol. Reprod. Dev., 40, 345–354.[CrossRef][Web of Science][Medline]
Hosseini, R., Ruddy, S., Bains, S., Hynes, G., Marsh, P., Pizzey, J. and Dudley, K. (1994) The mouse t-complex gene, Tcp-11, is under translational control. Mech. Dev., 47, 73–80.[CrossRef][Web of Science][Medline]
Kopf, G.S., Woolkalis, M.J. and Gerton, G.L. (1986) Evidence for a guanine nucleotide-binding regulatory protein in invertebrate and mammalian sperm. Identification by islet-activating protein-catalyzed ADP-ribosylation and immunochemical methods. J. Biol. Chem., 261, 7327–7331.
Leclerc, P. and Kopf, G.S. (1995) Mouse sperm adenylyl cyclase: general properties and regulation by the zona pellucida. Biol. Reprod., 52, 1227–1233.[Abstract]
Leclerc, P. and Kopf, G.S. (1999) Evidence for the role of heterotrimeric guanine nucleotide-binding regulatory proteins in the regulation of the mouse sperm adenylyl cyclase by the egg’s zona pellucida. J. Androl., 20, 126–134.
Lee, M.A., Check, J.H. and Kopf, G.S. (1992) A guanine nucleotide-binding regulatory protein in human sperm mediates acrosomal exocytosis induced by the human zona pellucida. Mol. Reprod. Dev., 31, 78–86.[CrossRef][Web of Science][Medline]
Ma, Y., Zhang, S., Xia, Q., Zhang, G., Huang, X., Huang, M., Xiao, C., Pan, A., Sun, Y., Lebo, R. and Milunsky, A. (2002) Molecular characterization of the TCP11 gene which is the human homologue of the mouse gene encoding the receptor of fertilization promoting peptide. Mol. Hum. Reprod., 8, 24–31.
Mazarakis, N.D., Nelki, D., Lyon, M.F., Ruddy, S., Evans, E.P., Freemont, P. and Dudley, K. (1991) Isolation and characterisation of a testis-expressed developmentally regulated gene from the distal inversion of the mouse t-complex. Development, 111, 561–571.[Abstract]
Merlet, F., Weinstein, L.S., Goldsmith, P.K., Rarick, T., Hall, J.L., Bisson, J.P. and de Mazancourt, P. (1999) Identification and localization of G protein subunits in human spermatozoa. Mol. Hum. Reprod., 5, 38–45.
Minelli, A., Allegrucci, C., Piomboni, P., Mannucci, R., Lluis, C. and Franco, R. (2000) Immunolocalization of A1 adenosine receptors in mammalian spermatozoa. J. Histochem. Cytochem., 48, 1163–1171.
Novotny, J. and Svoboda, P. (1998) The long (Gs-L) and short (Gs-S) variants of the stimulatory guanine nucleotide-binding protein. Do they behave in an identical way? J. Mol. Endocrinol., 20, 163–173.[Abstract]
Oberländer, G., Yeung, C.H. and Cooper, T.G. (1996) Influence of oral administration of ornidazole on capacitation and the activity of some glycolytic enzymes of rat spermatozoa. J. Reprod. Fertil., 106, 231–239.
Ohta, K., Sato, C., Matsuda, T., Toriyama, M., Vacquier, V.D., Lennarz, W.J. and Kitajima, K. (2000) Co-localization of receptor and transducer proteins in the glycosphingolipid-enriched, low density, detergent-insoluble membrane fraction of sea urchin sperm. Glycoconj. J., 17, 205–214.[CrossRef][Web of Science][Medline]
O’Toole, C.M.B., Roldan, E.R.S. and Fraser, L.R. (1996) A role for diacylglycerol in human sperm acrosomal exocytosis. Mol. Hum. Reprod., 2, 317–326.
Palmer, T.M. and Stiles, G.L. (1995) Neurotransmitter receptors VII: Adenosine receptors. Neuropharmacology, 34, 683–694.[CrossRef][Web of Science][Medline]
Pondel, M. (2000) Calcitonin and calcitonin receptors: bone and beyond. Int. J. Exp. Pathol., 81, 405–422.[CrossRef][Web of Science][Medline]
Ragoussis, J., Senger, G., Mockridge, I., Sanseau, P., Ruddy, S., Dudley, K., Sheer, D. and Trowsdale, J. (1992) A testis expressed zinc finger gene ZNF76 in human 6p21.3 centromeric to the MHC is closely linked to the human homologue of the t-complex gene Tcp-11. Genomics, 14, 673–679.[CrossRef][Web of Science][Medline]
Rakha, A.M., Adeoya-Osiguwa, S.A. and Fraser, L.R. (2000) Purification of a mouse sperm decapacitation factor and its receptor. J. Reprod. Fertil. Abstr. Ser., 26, 25.
Sato-Kusubata, K., Yajima, Y. and Kawashima, S. (2000) Persistent activation of Gs through limited proteolysis by calpain. Biochem. J., 347, 733–740.
Sjöberg, H., Arver, S. and Bucht, E. (1980) High concentration of immunoreactive calcitonin of prostatic origin in human semen. Acta Physiol. Scand., 110, 101–102.[Web of Science][Medline]
Silvestroni, L., Menditto, A., Frajese, G. and Gnessi, G. (1987) Identification of calcitonin receptors in human spermatozoa. J. Clin. Endocrinol. Metabol., 65, 742–746.
Spehr, M., Gisselmann, G., Poplawski, A., Riffell, J.A., Wetzel, C.H., Zimmer, R.K. and Hatt, H. (2003) Identification of a testicular odorant receptor mediating human sperm chemotaxis. Science, 299, 2054–2058.
Stein, D.M., Fraser, L.R. and Monks, N.J. (1986) Adenosine and Gpp(NH)p modulate mouse sperm adenylate cyclase. Gamete Res., 13, 151–158.
Wang, W.H., Abeydeera, L.R., Fraser, L.R. and Niwa, K. (1995) Functional analysis using chlortetracycline fluorescence and in vitro fertilization of frozen-thawed ejaculated boar spermatozoa incubated in a protein-free chemically defined medium. J. Reprod. Fertil., 104, 305–313.
Ward, C.R. and Storey, B.T. (1984) Determination of the time course of capacitation in mouse spermatozoa using a chlortetracycline fluorescence assay. Dev. Biol., 104, 287–296.[CrossRef][Web of Science][Medline]
Yanagimachi, R. (1994) Mammalian fertilization. In Knobil, E. and Neill, J.D. (eds), The Physiology of Reproduction, 2nd Edn. Raven Press, New York, pp. 189–317.
Submitted on May 20, 2003; resubmitted on August 1, 2003. accepted on August 2, 2003
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S.A. Adeoya-Osiguwa and L. R. Fraser Cathine, an amphetamine-related compound, acts on mammalian spermatozoa via beta1- and {alpha}2A-adrenergic receptors in a capacitation state-dependent manner Hum. Reprod., March 1, 2007; 22(3): 756 - 765. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. R. Fraser, E. Beyret, S. R. Milligan, and S. A. Adeoya-Osiguwa Effects of estrogenic xenobiotics on human and mouse spermatozoa Hum. Reprod., May 1, 2006; 21(5): 1184 - 1193. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Gibbons, S. A Adeoya-Osiguwa, and L. R Fraser A mouse sperm decapacitation factor receptor is phosphatidylethanolamine-binding protein 1 Reproduction, October 1, 2005; 130(4): 497 - 508. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Mededovic and L. R Fraser Mechanisms of action of angiotensin II on mammalian sperm function Reproduction, February 1, 2005; 129(2): 211 - 218. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Adeoya-Osiguwa and L. R. Fraser Cathine and norephedrine, both phenylpropanolamines, accelerate capacitation and then inhibit spontaneous acrosome loss Hum. Reprod., January 1, 2005; 20(1): 198 - 207. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Luconi, I. Porazzi, P. Ferruzzi, S. Marchiani, G. Forti, and E. Baldi 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, January 1, 2005; 72(1): 22 - 32. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||