ART: boon or bane?

In 1978, the birth of the first baby through in vitro fertilization resulted in an explosion of new techniques that are now widely used in reproductive medicine and biology around the world. With these advances have also come many concerns for the safety of these techniques. In addition, even newer technologies are on the horizon, such as cloning and the production/use of stem cells. The 18th annual meeting of the European Society of Human Reproduction and Embryology (ESHRE), held in Vienna from 30 June to 3 July 2002, provided a forum for discussion of these and other relevant topics.

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.

Regulation of mammalian sperm capacitation by endogenous molecules.

Capacitation in vitro in mammalian spermatozoa can be regulated by a number of first messengers, including fertilization promoting peptide, adenosine, calcitonin and angiotensin II, all of which are found in seminal plasma. The responses appear to involve several separate signal transduction pathways that have a common end point. These seminal-plasma derived first messengers can bind to specific receptors and directly or indirectly modulate the activity of membrane-associated adenylyl cyclase isoforms and production of the second messenger cAMP. Responses to all of these except angiotensin II involve initial acceleration of cAMP production and capacitation followed by inhibition of both cAMP production and spontaneous acrosome loss, resulting in maintenance of fertilizing potential. Appropriate G proteins and various phosphodiesterase isoforms also appear to be involved. The transition from stimulatory to inhibitory responses involves loss of decapacitation factors (DF) from receptors (DF-R) on the external surface; a DF-R present on both mouse and human spermatozoa has recently been identified as phosphatidylethanolamine-binding protein 1. The presence/absence of DF appears to cause changes in the plasma membrane that then alter the functionality of various membrane-associated proteins, including receptors. Since spermatozoa contact these first messengers at ejaculation, it is plausible that their actions observed in vitro also occur in vivo, allowing these molecules to play a pivotal role in enhancing the chances of successful fertilization.

New insights into possible factors contributing to male subfertility.

Male subfertility contributes significantly to fertility problems in couples. Although semen analysis may identify abnormalities in sperm numbers, morphology and/or motility that might contribute to subfertility, in other instances the semen parameters may appear to be normal, but the spermatozoa might be dysfunctional. A number of endogenous and exogenous factors have now been identified that can significantly affect sperm function in vitro and it is possible that they may have similar effects in vivo. Some endogenous factors maintain the spermatozoa in a non-fertilizing state, to avoid them 'burning out' and losing fertility before they reach an oocyte, while others stimulate spermatozoa to become fertile and then hold them in a state of readiness to fertilize. Exogenous environmental molecules, referred to as xenobiotics, have been shown to continuously stimulate spermatozoa so that they become fertile quickly, but then 'burn out'. Defects relating to the endogenous molecules could result in spermatozoa either never becoming fertile or becoming fertile too quickly and so losing fertilizing potential. By understanding the mechanisms involved in promoting sperm fertilizing ability, it may be possible to develop new therapeutic treatments to overcome such defects. (Reprod Med Biol 2005; 4: 45-53).

Cathine, an amphetamine-related compound, acts on mammalian spermatozoa via beta1- and alpha2A-adrenergic receptors in a capacitation state-dependent manner.

BACKGROUND: Mammalian spermatozoa have been shown to have beta(1,2,3)- and alpha(2A)-adrenergic receptors, the former functioning only in uncapacitated spermatozoa and the latter only in capacitated cells. Cathine, an amphetamine-related metabolite of a compound found in Catha edulis leaves, accelerates capacitation and inhibits spontaneous acrosome loss by regulating cAMP production. This study tested the hypothesis that adrenergic receptors are involved in these responses. METHODS: Uncapacitated and capacitated mouse sperm suspensions were incubated with cathine +/- specific antagonists for alpha(2)- and beta-adrenergic receptors for 35 min, then assessed using chlortetracycline fluorescence. Reversibility of receptor accessibility was assessed by depleting suspensions of endogenous decapacitation factor (DF) and then adding crude DF with/without cathine and antagonists. Effects on tyrosine phosphorylation and calcium requirements for both ligand binding and biological responses were also evaluated. RESULTS: Cathine's acceleration of capacitation was blocked by a beta(1)-antagonist, whereas an alpha(2)-antagonist blocked inhibition of acrosome reactions. Cathine accelerated capacitation in decapacitated cells, a response inhibited by a beta(1)-antagonist; cathine also stimulated tyrosine phosphorylation. Although calcium was not required for binding, it was needed for responses. CONCLUSIONS: Cathine acts at beta(1)-adrenergic receptors in uncapacitated spermatozoa and at alpha(2A)-receptors in capacitated cells; biological activity requires calcium but binding does not. Adrenergic receptor-binding sites can be made reversibly accessible/inaccessible by changing the capacitation state of spermatozoa. These results suggest that amphetamine-related compounds might enhance chances of fertilization in vivo.

The potential impact of novel investigational compounds on human fertility.

There is considerable concern that the incidence of infertility in humans may be increasing, in some instances due to the action of bioactive xenobiotic compounds found in our environment; for example, high concentrations of xenobiotics with estrogenic activity can interfere with normal testicular function and fertility. However, recent studies have shown that very low concentrations of several estrogenic xenobiotics can have subtle, unexpected effects on sperm function. When tested in vitro, these compounds stimulate spermatozoa to become fertile very quickly, but continued stimulation causes them to burn out and lose fertilising ability; similar responses occurring in vivo could reduce fertility. In contrast, several other compounds, structurally related to amfetamine, have been shown to act on spermatozoa in vitro in a positive manner, stimulating cells to 'switch on' quickly and then preventing burnout so that they maintain fertilising potential; similar responses occurring in vivo could enhance fertility. These results could have implications for either reducing or enhancing natural fertility.

Identification of functional alpha2- and beta-adrenergic receptors in mammalian spermatozoa.

BACKGROUND: A recent study of several compounds, structurally related to amphetamine, provided evidence that mammalian spermatozoa might have adrenergic receptors able to regulate cAMP production. The present study investigated this possibility using physiological and immunochemical analyses of mouse and human spermatozoa. METHODS: Antibodies specific for different receptor subtypes were used for Western blotting of mouse and human sperm lysates and for immunocytochemical evaluation of whole mouse and human spermatozoa. Uncapacitated and capacitated mouse spermatozoa were incubated with specific agonists and antagonists for alpha2-, beta1-, beta2- and beta3-adrenergic receptors for approximately 35 min and then assessed using chlortetracycline (CTC) fluorescence. RESULTS: Western blotting revealed proteins of the correct size for all these receptors; immunolocalization indicated their presence on the head, especially acrosomal and neck regions, and flagellum of both mouse and human spermatozoa. CTC results indicated significant responses to agonists for all of the beta-receptors in uncapacitated cells, with agonist effectiveness being beta1 > beta2 > beta3; relevant antagonists blocked responses. In contrast, an agonist and antagonist for alpha2-receptors acted only on capacitated spermatozoa. CONCLUSION: These experiments provide the first good evidence that mammalian spermatozoa have both beta-adrenergic receptors, known to stimulate cAMP production by membrane-associated adenylyl cyclases (mACs), and alpha2-adrenergic receptors, known to inhibit cAMP production by mACs. Responses are capacitation state dependent and provide a mechanism for inhibiting spontaneous acrosome reactions and helping to maintain fertilizing ability. These results suggest that the use of amphetamine-related compounds, either for medical or for social reasons, might have an unexpected positive impact on fertility.

Effects of estrogenic xenobiotics on human and mouse spermatozoa.

OBJECTIVE: To investigate human sperm responsiveness to the estrogenic xenobiotic genistein and seek further information regarding the mechanism of action of estrogenic xenobiotics using mouse spermatozoa. METHODS: Uncapacitated human spermatozoa were incubated with genistein and assessed using chlortetracycline (CTC) fluorescence. CTC was also used to evaluate mouse sperm responses to daidzein and combinations of genistein, 8-prenylnaringenin and nonylphenol. Several steroids were tested to determine structure-function relationships, and possible involvement of cAMP and G proteins in responses was also investigated. RESULTS: Genistein significantly accelerated capacitation and acrosome loss in human spermatozoa, with 1, 10 and 100 nmol/l being equally effective. In mouse spermatozoa, daidzein produced significant responses, and combinations of xenobiotics at low concentrations were more effective than used singly. The compounds appear to act at the cell surface, and responses to three different steroids were nonidentical. A protein kinase-A inhibitor blocked responses to xenobiotics, while genistein and nonylphenol significantly stimulated cAMP production. Pertussis toxin and dideoxyadenosine blocked responses, suggesting involvement of inhibitory G proteins and membrane-associated adenylyl cyclases. CONCLUSION: Human and mouse sperm responses to genistein are very similar, but human gametes appear to be even more sensitive. The mechanism of action may involve unregulated stimulation of cAMP production, leading to significant acrosome loss, undesirable because already acrosome-reacted cells are nonfertilizing. Xenobiotics were even more effective in combination. Since simultaneous exposure to low concentrations of multiple xenobiotics is likely to occur in animals and humans, further investigation is needed to determine whether this could impair fertility.

Mechanisms of action of angiotensin II on mammalian sperm function.

Angiotensin II (AII) stimulates capacitation and fertilizing ability in mammalian spermatozoa, with the binding of AII to its receptors resulting in stimulation of cAMP production in both uncapacitated and capacitated cells. This study investigated possible mechanisms whereby AII affects cAMP availability. The first question was whether extracellular Ca2+ is required for responses in mouse spermatozoa and, using chlortetracycline fluorescence analysis, it was clear that cells responded to AII only when the medium contained CaCl2, with both 90 microM and 1.80 mM supporting a significant acceleration of capacitation. Consistent with those results, AII significantly stimulated cAMP production in both CaCl2-containing media tested, the response being greater in that containing 1.80 mM. Several different agents that might affect the signalling pathway stimulated by AII were then evaluated in uncapacitated suspensions. Chlortetracycline analysis revealed that pertussis toxin abolished responses to AII, suggesting the involvement of an inhibitory Galpha subunit; dideoxyadenosine, a specific membrane-associated adenylyl cyclase (mAC) P-site inhibitor, also blocked responses, suggesting involvement of an mAC. cAMP determinations confirmed that both reagents also abolished AII's stimulation of cAMP. In contrast, nifedipine, a Ca2+ channel blocker, did not inhibit AII's effects on spermatozoa. Finally, in capacitated suspensions, both pertussis toxin and dideoxyadenosine were again shown to block AII's stimulation of cAMP. These results suggest that responses to AII involve an inhibitory G protein and an mAC, but it is likely that AII-receptor coupling does not stimulate directly mAC but rather does so in an indirect manner, perhaps by altering the intracellular Ca2+ concentration.

Mammalian sperm phosphodiesterases and their involvement in receptor-mediated cell signaling important for capacitation.

This study investigated the presence and function of intracellular cyclic nucleotide phosphodiesterases (PDEs) in mature mouse spermatozoa. PCR analysis detected gene transcripts for most of the 11 known PDE families in whole testis, but mainly for PDEs 1, 3, 6, and 8 in spermatozoa. Using specific antibodies, the strongest evidence was obtained for PDE proteins 1, 4, 6, 8, 10, and 11 in both sperm lysates and intact cells. These showed a range of subcellular localizations, with PDE 1A being primarily in the flagellum but PDEs 4D and 10A being in both the acrosomal region and the flagellum, similar to specific G proteins and adenylyl cyclases implicated in cAMP regulation during capacitation. In live spermatozoa, inhibitors selective for PDE 1 (MMPX) and 4 (rolipram) significantly increased cAMP over control levels but only rolipram significantly stimulated capacitation and in-vitro fertilizing ability; this suggests that compartmentalization has functional implications since only PDE 4 was abundant in both head and flagellum. Treatment of spermatozoa with CGS 21680, a stimulatory adenosine receptor agonist, significantly reduced cAMP-PDE activity at the same time-point when it causes increased cAMP. Thus, certain receptor-regulated cAMP processes in spermatozoa may be controlled by changes in both PDE and cyclase activities. In addition to demonstrating for the first time that some of the more recently discovered PDE isoforms, including PDE 6 (usually associated with the retina), are present in mature spermatozoa, this study provides clear evidence that the intracellular location of specific PDEs has important functional significance during capacitation and fertilization.

A mouse sperm decapacitation factor receptor is phosphatidylethanolamine-binding protein 1.

Capacitation is a pivotal event for mammalian spermatozoa, involving the loss of surface proteins known as decapacitation factors (DF) and consequent acquisition of fertilizing ability. Earlier studies showed that a mouse sperm DF binds to a receptor, DF-R, whose attachment to the sperm plasma membrane appears to involve a glycosylphosphatidylinositol (GPI) anchor. In the present study, purification and subsequent sequencing of DF-R has identified this approximately 23 kDa protein as phosphatidylethanolamine-binding protein 1 (PEBP 1). To obtain functional evidence that supports sequence homology data, purified recombinant PEBP 1 and PEBP 2 were evaluated for biological activity. While PEBP 1 was able to remove DF activity in solution at concentrations above approximately 1 nmol/l, PEBP 2 was ineffective, even at 600 nmol/l; this confirmed that DF-R is PEBP 1. Anti-PEBP 1 antiserum recognized recombinant PEBP 1 and a approximately 23 kDa protein in both mouse and human sperm lysates. Immunolocalization studies revealed that DF-R/PEBP 1 is located on the acrosomal cap, the post-acrosomal region and the flagellum of both mouse and human spermatozoa, with epitope accessibility being capacitation state-dependent and reversible. Treatment of cells with a phospholipase able to cleave GPI anchors essentially abolished immunostaining, thus confirming the extracellular location of DF-R/PEBP 1. We suggest that DF-R/PEBP 1 plays its fundamental role in capacitation by causing alterations in the sperm plasma membrane in both head and flagellum, with functional consequences for membrane-associated proteins. Obtaining more detail about DF <--> DF-R interactions could lead to useful applications in both fertility treatments and new contraceptive approaches.

Cathine and norephedrine, both phenylpropanolamines, accelerate capacitation and then inhibit spontaneous acrosome loss.

BACKGROUND: Cathinone, released when Catha edulis leaves (khat) are chewed, has euphoric, stimulatory properties. It is metabolized to the phenylpropanolamines (PPAs) cathine and norephedrine. This study investigated whether PPAs affect mammalian sperm function, using primarily mouse, but also human, spermatozoa. METHODS: Uncapacitated sperm suspensions were treated with cathine, norephedrine, adrenaline and noradrenaline, then assessed using chlortetracycline (CTC) fluorescence. Cathine and adrenaline were also evaluated using in vitro fertilization. Capacitated suspensions were treated with PPAs+/-progesterone and+/-pertussis toxin. Finally, cAMP production was evaluated in uncapacitated and capacitated suspensions. RESULTS: In uncapacitated mouse spermatozoa, cathine, norephedrine, adrenaline and noradrenaline all significantly accelerated capacitation; uncapacitated human spermatozoa responded similarly to cathine. Consistent with these results, cathine- and adrenaline-treated suspensions were significantly more fertile than controls. In capacitated spermatozoa, both PPAs inhibited spontaneous acrosome reactions (ARs) but progesterone could over-ride this inhibition. Pertussis toxin abolished cathine's inhibition of ARs, suggesting G protein involvement. Finally, cathine and adrenaline significantly stimulated cAMP production in uncapacitated suspensions, but significantly inhibited it in capacitated suspensions. CONCLUSIONS: This is the first demonstration that PPAs can directly affect mammalian sperm function, accelerating capacitation and inhibiting spontaneous ARs. These responses correlated with initial stimulation and subsequent inhibition of cAMP production. Adrenaline/noradrenaline elicited similar responses, suggesting the presence of adrenergic receptors. Therefore, regulation of adenylyl cyclase/cAMP in a G protein-mediated fashion by PPAs may possibly involve adrenergic receptors. These results suggest that PPAs, at appropriate doses, might provide a novel approach to enhance natural fertility.

First messenger regulation of mammalian sperm function via adenylyl cyclase/cAMP.

When released into an appropriate environment, mammalian spermatozoa begin to capacitate and then continue until fully capacitated and able to fertilize. During capacitation in vitro, some cells 'over-capacitate' and undergo spontaneous acrosome reactions; this would be highly undesirable in vivo since already acrosome-reacted spermatozoa are non-fertilizing. Recent studies have revealed that seminal plasma contains several small molecules that bind to specific receptors on the sperm plasma membrane and act as 'first messengers', causing biologically important changes in availability of the 'second messenger' cAMP. Fertilization promoting peptide (FPP), calcitonin and adenosine all regulate cAMP production, stimulating it in uncapacitated spermatozoa and then inhibiting it in capacitated cells; in contrast, angiotensin II stimulates cAMP throughout capacitation. The molecules that regulate cAMP appear to do so via G protein-modulated changes in membrane associated adenylyl cyclases (mACs). Both mouse and human spermatozoa have been shown to have Galphas and Galphai2, as well as several isoforms of mAC, located in the same regions as the specific receptors. Thus spermatozoa possess the required elements for several separate signal transduction pathways, many of which regulate mAC/cAMP and so maintain sperm fertilizing ability. In vivo, such responses could increase the chances of successful fertilization.

Angiotensin II stimulates cAMP production and protein tyrosine phosphorylation in mouse spermatozoa.

Angiotensin II (AII), found in seminal plasma, has been shown to stimulate capacitation in uncapacitated mammalian spermatozoa. The present study investigated the location of AII receptors on spermatozoa and AII's mechanism of action. AT1 type receptors for AII are present on the acrosomal cap region and along the whole of the flagellum of both mouse and human spermatozoa. Because combinations of low concentrations of AII and either calcitonin or fertilization-promoting peptide (FPP), both known to regulate the adenylyl cyclase (AC)/cAMP signal transduction pathway, elicited a significant response, this study investigated the hypothesis that these peptides act on the same pathway. AII was shown to significantly stimulate cAMP production in both uncapacitated and capacitated mouse spermatozoa and this was associated with increases in protein tyrosine phosphorylation. Using an anti-phosphotyrosine antibody to visualize the location of tyrosine phosphoproteins within individual cells, AII significantly stimulated phosphorylation within 20 min in both the head, especially in the acrosomal cap region, and the flagellum, especially in the principal piece, of uncapacitated mouse spermatozoa; combined AII + FPP was stimulatory within 5 min. In addition, Western blotting revealed that AII stimulation increased phosphorylation in a number of tyrosine phosphoproteins in both uncapacitated and capacitated mouse spermatozoa, with some being altered only in the latter category of cells. These results support the hypothesis that AII stimulates AC/cAMP in mammalian spermatozoa.

Human sperm responses to calcitonin, angiotensin II and fertilization-promoting peptide in prepared semen samples from normal donors and infertility patients.

BACKGROUND: Fertilization-promoting peptide (FPP), angiotensin II (AII) and calcitonin, present in seminal plasma, have significant effects on mouse sperm function in vitro. This study investigated responses of uncapacitated and capacitated human sperm to these peptides, initially using samples from donors with normal semen parameters and then samples from men attending infertility clinics. METHODS: Prepared suspensions were incubated in the presence/absence of a range of peptide concentrations and assessed using chlortetracycline (CTC) analysis and the hamster oocyte penetration test. RESULTS: In uncapacitated suspensions, maximal stimulatory responses (CTC) were obtained with calcitonin at 0.5-15 nmol/l and AII at 0.3-100 nmol/l; FPP is known to be most effective at 100 nmol/l. All peptides also significantly stimulated sperm penetrating ability. Combinations of peptides at low concentrations, having no detectable effect when used singly, elicited significant responses, suggesting that they work via the same signalling pathway. In suspensions incubated in the presence of fucose to accelerate capacitation and acrosome reactions, both FPP and calcitonin, but not AII, inhibited acrosome loss; however, AII did not interfere with responses to FPP and calcitonin. Unlike samples from 15 donors, some samples from >70 patients had high proportions of capacitated and/or acrosome-reacted cells when assessed immediately following preparation. Even so, the peptides usually elicited responses similar to those obtained with donor samples and combinations of peptides inhibited spontaneous acrosome loss for at least 3 h. CONCLUSIONS: The responses obtained in vitro suggest that these peptides could have significant effects on human sperm function in vivo and could also be used effectively in infertility clinics.

Evidence for multiple distinctly localized adenylyl cyclase isoforms in mammalian spermatozoa.

In addition to a bicarbonate-regulated soluble adenylyl cyclase (sAC), mammalian spermatozoa, like somatic cells, appear to contain receptor/G protein-regulated AC activity that contributes to the modulation of specialized cell processes. This study provides evidence that agents, known to influence somatic membrane-associated AC (mAC) but apparently not germ cell sAC, can modulate cAMP production and functional state in mouse spermatozoa. Specifically, forskolin significantly enhanced cAMP production and capacitation, while inclusion of 2',5'-dideoxyadenosine significantly blocked these responses. Furthermore, GTPgammaS and NaF stimulated cAMP, but GDPbetaS and mastoparan had no apparent effect, consistent with recent evidence that G(s), but not G(i), contributes to AC/cAMP regulation in uncapacitated cells. In addition, intact mouse spermatozoa were screened for all known mAC isoforms by immunolocalization, using commercially available specific antibodies. The most abundant isoforms appeared to be AC2, AC3, and AC8, each with distinct distributions in the acrosomal and flagellar regions; AC1 and AC4 also appeared to be present, although less abundantly, in the midpiece and acrosomal cap regions, respectively. Intriguingly, however, Western blotting revealed that the major immunoreactive proteins in mouse sperm lysates were considerably smaller (approximately 50-60 kDa) than their somatic cell counterparts, suggesting that mature spermatozoa contain multiple mACs which may function in a shortened form. Of particular interest were AC3 and AC8, located in the same regions as, and hence possibly directly associated with, specific cell surface receptors and G proteins that are able to regulate the spermatozoon's acquisition and maintenance of fertilizing ability via changes in AC/cAMP.

Immunolocalization of multiple Galpha subunits in mammalian spermatozoa and additional evidence for Galphas.

Like somatic cells, mammalian spermatozoa appear to contain several different heterotrimeric G protein alpha-subunits that could mediate specialized cell responses. However, the precise Galpha subunits present, their subcellular location and their possible roles are still incompletely defined. In this study, using commercially available specific antibodies, we have shown by immunoblotting that Galpha(s) is present in human and mouse sperm lysates. Immunolocalization using intact spermatozoa from both species revealed this protein to be in the acrosomal cap region and the flagellum, particularly the principal piece. Treatment of permeabilized mouse spermatozoa with cholera toxin led to enhanced ADP-ribosylation of a protein the same size as Galpha(s), as well as an increase in cAMP, providing further proof for Galpha(s). Evidence for the presence and distinct localizations of Galpha(i2), Galpha(i3), Galpha(o), Galpha(q/11), and Galpha(olf) was also obtained. Of particular interest was Galpha(i2) which, like Galpha(s), was present in the acrosomal cap region and flagellum, the same regions where stimulatory and inhibitory adenosine receptors are localized. These observations are consistent with our hypothesis that G proteins mediate adenosine receptor modulation of adenylyl cyclase, with consequent alterations in cAMP production, apparently crucial for the spermatozoon's acquisition and maintenance of fertilizing ability.

Capacitation state-dependent changes in adenosine receptors and their regulation of adenylyl cyclase/cAMP.

This study was designed to localize adenosine receptors and to provide evidence that specific receptors are active only in either uncapacitated or capacitated mouse spermatozoa, where they play a role in regulating cAMP production. Using specific antibodies, stimulatory A(2A) receptors were localized primarily on the acrosomal cap region and the flagellar principal piece. Interestingly, the staining was much more pronounced in uncapacitated than in capacitated spermatozoa, suggesting capacitation-dependent changes in epitope accessibility. A(1) receptors showed a very similar distribution, but the staining was markedly greater in capacitated than in uncapacitated cells. After addition of purified decapacitation factor (DF) to capacitated cells, strong staining for A(2A) was regained, suggesting reversibility in epitope accessibility. Chlortetracycline analysis revealed that an agonist specific for A(2A) receptors had no detectable effect on capacitated cells, but after DF-induced decapacitation, the agonist then stimulated capacitation. That agonist also significantly stimulated cAMP production in uncapacitated cells, had no effect on capacitated cells, but regained the ability to stimulate cAMP in the latter following DF treatment. In contrast, an A(1) agonist inhibited cAMP in capacitated cells. These results indicate that specific adenosine receptors function in a reversible manner in one or other capacitation state, resulting in regulation of cAMP.

First messenger regulation of capacitation via G protein-coupled mechanisms: a tale of serendipity and discovery.

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 A(2A) adenosine receptors, which function only in uncapacitated cells, and then with inhibitory A(1) 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 G(s) plus several G(i) subunits have been identified in both mouse and human spermatozoa. In particular, Galpha(s) and Galpha(i2) 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.