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L R Fraser

Publications and source records attributed to L R Fraser.

At least 19 recordsLinked to original sources
PubMed2001

Calcitonin, angiotensin II and FPP significantly modulate mouse sperm function.

Fertilization-promoting peptide (FPP) regulates the adenylyl cyclase (AC)/cAMP pathway to elicit capacitation-dependent responses, stimulating capacitation in uncapacitated spermatozoa and then arresting it in capacitated cells, thereby inhibiting spontaneous acrosome reactions. Like FPP, calcitonin and angiotensin II are found in seminal plasma and so might affect sperm function; this study investigated responses in uncapacitated and capacitated mouse spermatozoa to these three peptides. Both calcitonin (5 ng/ml) and angiotensin II (1 and 10nmol/l), like FPP (100nmol/l), significantly stimulated capacitation, assessed using chlortetracycline (CTC) fluorescence and fertilization in vitro analyses. Combinations of two or three peptides, at high and low, non-stimulatory concentrations, were more stimulatory than the individual peptides, suggesting that they may act on the same signalling pathway, plausibly AC/cAMP; preliminary data indicate that calcitonin does stimulate cAMP production. In capacitated cells, FPP and calcitonin elicited pertussis toxin-sensitive inhibition of spontaneous acrosome loss, suggesting involvement of inhibitory G proteins; angiotensin II had no detectable effect. When all three peptides were used, angiotensin II did not interfere with inhibitory responses to FPP/calcitonin. These results suggest that angiotensin II, calcitonin and FPP may somehow modulate the AC/cAMP signal transduction pathway, but the precise mechanisms involved have yet to be elucidated.

Angiotensin II↗
PubMed2000

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

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

Acrosome↗
PubMed2000

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

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

Adenosine↗
PubMed1999

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

FPP and adenosine modulate the adenylyl cyclase (AC)/cAMP signal transduction pathway in mammalian spermatozoa to elicit a biphasic response, initially stimulating capacitation and then inhibiting spontaneous acrosome loss. This study addressed the hypothesis that responses to FPP involve interactions between receptors for FPP and adenosine, the biphasic responses involving stimulatory and inhibitory adenosine receptors. Gln-FPP, a competitive inhibitor of FPP, significantly inhibited binding of an adenosine analogue and responses to adenosine, especially in capacitated suspensions, consistent with interaction between FPP and adenosine receptors. CGS-21680 (1 microM), a stimulatory A2a adenosine receptor agonist, significantly stimulated capacitation and cAMP in uncapacitated cells, while cyclopentyl adenosine (1 microM), an inhibitory A1 adenosine receptor agonist only affected capacitated cells, inhibiting spontaneous acrosome loss. Responses to FPP and adenosine were inhibited in uncapacitated cells by a selective A2a antagonist and in capacitated cells by a selective A1 antagonist; subsequent investigations indicated possible involvement of G proteins. Like FPP, cholera toxin stimulated capacitation and cAMP production in uncapacitated cells, suggesting involvement of a G protein with a Galphas subunit. In contrast, pertussis toxin prevented FPP's inhibition of both spontaneous acrosome loss and cAMP production, suggesting involvement of a Galphai/o subunit. Immunoblotting evidence revealed the presence of proteins of the appropriate molecular weights for Galphas, Galphai2, Galpha i3, and Galphao subunits. This study provides the first direct evidence suggesting the involvement of two different types of adenosine receptors and both Galphas and Galphai/o subunits in the regulation of capacitation, resulting in modulation of AC activity and availability of cAMP.

Adenosine↗
PubMed1999

New insights into the t-complex and control of sperm function.

The mouse t-complex, located on chromosome 17, contains genes known to influence male, but not female, fertility. Although some t-complex genes are recessive lethals, t-chromosomes are maintained in the population by transmission ratio distortion. When male mice heterozygous for the t-chromosome mate with wild-type females, most offspring will possess the t-chromosome, indicating a link between t-complex genes and sperm function. Several proteins coded for by t-complex genes have been localised in the sperm flagellum, suggesting roles relating to motility. Another t-complex protein appears able to regulate the adenylyl cyclase/cAMP signal transduction pathway, known to play an important role in capacitation. Defective motility and/or failure to capacitate ("switch on") would result in poorly fertile or infertile spermatozoa. Given the existence of human homologues for many genes in the t-complex and the prevalence of "male factor" infertility, information obtained about the t-complex not only will provide insight into basic biological mechanisms but may be of future clinical relevance as well.

Animals↗
PubMed1999

New insights into possible causes of male infertility.

Male subfertility/infertility is acknowledged to contribute significantly to infertility problems experienced by couples. In some instances, morphological and/or physiological defects known to interfere with normal sperm function can be identified. However, in others, no obvious cause of fertilization failure can be identified. The recent introduction of molecular methods has made it possible to diagnose more subtle defects that could affect the function of spermatozoa produced by some males. For others, though, the problems may result from defects in the physiological mechanisms that need to be activated in spermatozoa so that they 'switch on' functionally following their release from the male reproductive tract. Capacitation, the term applied to this 'switching on', encompasses a number of changes that, collectively, confer fertilizing potential on sperm cells. This article focuses on two extrinsic factors, one a protein and one a very small peptide, that become associated with spermatozoa either in the epididymis or following contact with seminal plasma. These factors modulate capacitation in vitro in ways that could be very relevant to fertilization in vivo, possibly helping to maximize the fertilizing potential of the few cells that reach the site of fertilization. In some men, defects in either of the factors and the systems they modulate could result in defective fertilization. However, by understanding the underlying mechanisms, it may prove possible to develop new diagnostic techniques and new therapeutic treatments to alleviate the infertility.

Calcium-Transporting ATPases↗
PubMed1998

Role of fertilization promoting peptide (FPP) in modulating mammalian sperm function.

Fertilization promoting peptide (FPP), structurally similar to thyrotrophin releasing hormone, is produced by the prostate gland and secreted into seminal plasma. Recent in vitro studies have provided evidence that FPP elicits biologically relevant responses in mouse, human and boar spermatozoa. In the presence of nanomolar concentrations of FPP, spermatozoa become fertilizing more quickly and then are inhibited from undergoing spontaneous acrosome loss, an event that would make them non-fertilizing. In vivo, these responses would be very important in maximizing the availability of potentially fertilizing spermatozoa. Adenosine, which can elicit the same responses as FPP, is known to modulate the adenylyl cyclase(AC)/cAMP signal transduction pathway; current evidence indicates that FPP and adenosine act via separate receptors on the same signal transduction pathway. Mouse spermatozoa are known to have adenosine receptors and a putative receptor for FPP (TCP-11) has been identified. Unlike many surface receptors, TCP-11 has no obvious transmembrane regions whereby modulation of AC could occur. Recent evidence suggests that FPP receptors may dimerize with adenosine receptors to activate the signaling pathway, with stimulatory adenosine receptors involved in the stimulation of capacitation, but inhibitory receptors involved in inhibition of spontaneous acrosome loss. These results indicate that FPP plays an important role in normal sperm function and that it might be used in new therapeutic strategies designed to alleviate some causes of sperm dysfunction.

Animals↗
PubMed1998

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

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

Acrosome↗
PubMed1998

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

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

Adenosine↗
PubMed1998

Sperm capacitation and the acrosome reaction.

To achieve successful fertilization under normal circumstances in vivo, mammalian spermatozoa must first undergo capacitation and then the acrosome reaction, an exocytotic event that allows cells to penetrate the zona pellucida and fuse with the oocyte plasma membrane. These complex events permit spermatozoa to achieve fertilizing ability at the right time in the right place, important considerations since relatively few sperm cells actually reach the site of fertilization in vivo. Several mechanisms that may be involved in regulating the acquisition of fertilizing ability are considered. In vivo, selective pressures placed on the initial population of spermatozoa help ensure that the 'fittest' spermatozoa are able to fertilize. Since intracytoplasmic sperm injection bypasses this selection process, it is best used only in cases where spermatozoa are judged to be incapable of achieving normal fertilization in vitro.

Acrosome↗
PubMed1998

The modulation of sperm function by fertilization promoting peptide.

Fertilization promoting peptide (FPP; pGlu-Glu-ProNH2) is a peptide produced by the prostate gland and then secreted into seminal plasma. Recent studies have shown that the addition of FPP to uncapacitated mouse and human sperm suspensions stimulates capacitation as demonstrated by cytological assessment and increased fertilizing/penetrating ability in vitro, hence its name. Interestingly, the addition of FPP also has an effect on capacitated cells, namely inhibition of spontaneous acrosome loss; these spermatozoa retain high fertilizing ability, however, when tested with unfertilized oocytes. Adenosine, which is known to modulate adenylate cyclase activity, has been shown to elicit responses similar to those obtained with FPP in both uncapacitated and capacitated spermatozoa. Because the use of FPP and adenosine simultaneously is more effective than either used individually, it has been proposed that these two molecules interact with different receptors to modulate the adenylate cyclase/cAMP signal transduction pathway. FPP-related peptides have been found to vary in their biological activity in vitro, the most interesting one being Gln-FPP (pGlu-Gln-ProNH2). This peptide, identified in human seminal plasma and possibly produced by men with prostatic dysfunction, had no intrinsic activity itself but was able to competitively inhibit responses to FPP. Finally, very recent evidence suggests that the protein TCP-11, coded for by a mouse t-complex gene, may be the receptor for FPP. The existence of a human homologue for Tcp-11 suggests that TCP-11 and FPP could well play an important role in human fertility/subfertility. In vitro, FPP's ability to stimulate capacitation might reduce the incidence of delayed fertilization which results in impaired embryonic development and implantation.

Animals↗
PubMed1998

Fertilization promoting peptide: an important regulator of sperm function in vivo?

Fertilization promoting peptide (FPP; pGlu-Glu-ProNH2), a tripeptide structurally related to thyrotrophin releasing hormone, is produced by the prostate gland and released into seminal plasma. Recent studies carried out in vitro have revealed that FPP elicits biologically important responses in both mouse and human spermatozoa. In the presence of physiological concentrations of FPP (50-100 nmol l(-1)), uncapacitated spermatozoa undergo accelerated capacitation and so become potentially fertilizing more quickly, while capacitated spermatozoa are inhibited from undergoing spontaneous acrosomal exocytosis, an event that would make them non-fertilizing. In vivo, these responses would be very important since relatively few spermatozoa reach the site of fertilization; FPP could help to ensure that these were potentially fertilizing cells. A putative receptor (TCP-11) for FPP has been identified in mice. The gene for TCP-11 (which has a human homologue) maps to the t-complex, a region known to contain genes affecting male fertility. Current evidence indicates that FPP and TCP-11 act by modulating the activity of adenylyl cyclase and hence production of cAMP, a signal transduction pathway shown to be important in the acquisition of fertilizing ability. These results suggest that FPP plays an important role in normal fertility and that insufficient FPP could reduce fertility. Prostatic dysfunction can lead to decreased synthesis of FPP and increased synthesis of FPP-related peptides with reduced biological activity, both of which could compromise fertility in vivo. Given that 'male factor' infertility is a common contributor to subfertility in couples, it may prove possible to develop new therapeutic treatments, for at least some males, using FPP. In addition, this ligand-receptor pair could provide a novel target for male contraception.

Animals↗
PubMed1997

TCP-11, the product of a mouse t-complex gene, plays a role in stimulation of capacitation and inhibition of the spontaneous acrosome reaction.

Tcp-11 is a candidate for a distorter gene within the t-complex on mouse chromosome 17; although t-complex genes appear to affect sperm function, relatively little is known about mechanisms whereby these genes might play a specific physiological role. We present evidence that the protein TCP-11 is found on the surface of mature epididymal spermatozoa. Although detected on both the acrosomal cap region of the head and the flagellum of acrosome-intact cells, it is absent from the heads of acrosome-reacted cells. When epididymal spermatozoa were incubated in the presence of anti-TCP-11 IgG Fab fragments for a total of 120 min and assessed using chlortetracycline fluorescence, we observed a stimulation of capacitation and an inhibition of spontaneous acrosome loss, suggestive of enhanced fertility compared with untreated suspensions. In vitro fertilization experiments confirmed that Fab-treated suspensions became fertile more quickly and then maintained high fertility. Because these responses were remarkably similar to those obtained using the TRH-related peptide FPP (fertilization promoting peptide; pGlu-Glu-ProNH2) and adenosine, we investigated responses to Fab fragments, FPP, and adenosine. Results indicated that the Fab fragments appear to work at the same extracellular site as FPP, one that is distinct from the adenosine site of action. Further evidence for this conclusion was obtained using pGlu-Gln-ProNH2, an FPP-related tripeptide known to competitively inhibit responses to FPP; as with FPP, pGlu-Glu-ProNH2 inhibited the stimulatory effect of Fab fragments in a concentration-dependent manner. From these results we suggest that TCP-11 may be the receptor for FPP and that the adenylate clyclase/cyclic AMP pathway may be the signal transduction pathway activated by interactions between extracellular effector molecules (e.g., Fab fragments or FPP acting as an agonist) and TCP-11. A mechanism such as this that promotes capacitation but inhibits spontaneous acrosome loss in vivo would play a very important role by helping to maximize the fertilizing potential of the few spermatozoa that reach the site of fertilization. The fact that there is a human homolog of Tcp-11 suggests that this gene could play an important role in regulation of human, as well as mouse, sperm function.

Acrosome↗
PubMed1997

A fertilization promoting peptide (FPP)-related tripeptide competitively inhibits responses to FPP: a cause of male subfertility?

Fertilization promoting peptide (FPP; pGlu-Glu-ProNH2), a tripeptide structurally related to thyrotrophin releasing hormone (TRH; pGlu-His-ProNH2), is present in the prostate gland and seminal plasma of several mammalian species. FPP has been shown not only to stimulate the capacitation and fertilizing ability of epididymal mouse and ejaculated human spermatozoa, but also to inhibit spontaneous acrosome loss in mouse spermatozoa. These results suggest a possible role in vivo for FPP to maximize the fertilizing potential of the few cells that reach the ampulla. In this study we have investigated the effects of FPP-related peptides on mouse sperm capacitation and the acrosome reaction (using chlortetracycline fluorescence) and in vitro fertilizing ability. Deamidated FPP neither stimulated capacitation when tested at 50-200 nM nor interfered with FPP's stimulation of capacitation. Three neutral peptides (pGlu-Phe-ProNH2, MeO-FPP, pGlu-Gln-ProNH2) were also evaluated. pGlu-Phe-ProNH2, slightly stimulatory when used alone, had no additive effect when used in combination with FPP and the methyl derivative of FPP had no bioactivity itself and did not inhibit responses to FPP. In marked contrast, pGlu-Gln-ProNH2 (Gln-FPP), which had no bioactivity when added to uncapacitated suspensions at 50-100 nM, significantly inhibited FPP's stimulation of capacitation and fertilizing ability in vitro. Furthermore, when Gln-FPP + FPP were added to capacitated suspensions, Gln-FPP prevented FPP's inhibition of spontaneous acrosome loss. Our recent studies have indicated that FPP and adenosine can elicit similar responses but appear to act at different sites. The fact that Gln-FPP inhibited responses to FPP, but not to adenosine, indicates that Gln-FPP is acting at an FPP-specific site. We, therefore, conclude that the specific structure of the FPP molecule is crucial for biological activity. Removal of the terminal amide group abolishes bioactivity and changes to the central amino acid can have significant functional consequences. Since Gln-FPP is a candidate intermediate peptide in the FPP biosynthetic pathway and has been identified in human semen, abnormality in prostate function could lead to release of Gln-FPP along with, or instead of, FPP. Our results suggest that the relative proportions of FPP and related peptides in seminal plasma could have a significant effect on fertility in vivo.

Adenosine↗
PubMed1996

Evidence for Ca(2+)-dependent ATPase activity, stimulated by decapacitation factor and calmodulin, in mouse sperm.

Membrane preparations from mouse sperm heads and tails were used in a gamma 32P-ATP hydrolysis assay to investigate Ca(2+)-dependent ATPase activity. In membranes from sperm heads, but not tails, a Ca2(+)-dependent ATPase that was further stimulated by calmodulin was detected. The addition of partially purified mouse sperm decapacitation factor (DF) to head membrane preparations significantly stimulated Ca(2+)-ATPase activity, this effect being further increased in the presence of DF plus calmodulin; in contrast, no response was observed when the same treatment was applied to tail membranes. Sperm preincubated in the presence of trifluoperazine (TFP), a calmodulin antagonist, were significantly more fertile than cells from the same males incubated in the absence of TFP, indicating that inhibition of calmodulin accelerates capacitation. When sperm cells were preincubated briefly, then gently centrifuged to remove DF and resuspended in medium containing 45Ca2+ +/- DF, their ability to accumulate 45Ca2+ was significantly lower in the early stages after resuspension in the presence of DF than in its absence. These data correlated with chlortetracycline analysis of the sperm functional state. When cells were centrifuged and resuspended in medium only, there was a noticeable shift from the F pattern (characteristic of uncapacitated cells) to the B pattern (characteristic of capacitated cells), but the reintroduction of DF caused a significant reversion to the F pattern. Finally, using a monoclonal antibody to somatic cell Ca2(+)-ATPase, we have obtained evidence that the enzyme is particularly localized to the postacrosomal region of the mouse sperm head; specific binding was observed only in permeabilized cells, indicating that the epitope involved in the binding has an intracellular location. Based on these various pieces of evidence, we propose that when present on mouse sperm, DF stimulates calmodulin-sensitive Ca(2+)-ATPase activity and thus ensures maintenance of a low intracellular Ca2+ concentration. As capacitation proceeds, DF is lost and Ca2(+)-ATPase activity declines, allowing intracellular Ca2+ to rise and promoting capacitation-related changes. The fact that inhibitors of Ca(2+)-ATPase and calmodulin appear to accelerate capacitation in several mammalian species, as determined by chlortetracycline analysis, suggests that Ca(2+)-ATPase activity may play an important role in modulating capacitation in many or even all mammals.

Animals↗
PubMed1996

Role for Ca2+ channels in the signal transduction pathway leading to acrosomal exocytosis in human spermatozoa.

Progesterone interaction with human spermatozoa promotes a rise in intracellular Ca2+ and can trigger acrosomal exocytosis in capacitated cells. We have used nifedipine, a 1,4-dihydropyridine Ca2+ channel antagonist, to investigate the possibility that Ca2+ channels play a role in the progesterone-stimulated exocytotic response. Cells were assessed biochemically for the generation of diacylglycerol (DAG) and microscopically for acrosome loss using chlortetracycline fluorescence. When motile cells were preincubated for 5 hr using culture conditions similar to those used for successful human in vitro fertilization, a short exposure to progesterone significantly stimulated DAG formation and acrosomal exocytosis. The addition of nifedipine (10 and 100 nM), either at time 0 or just prior to progesterone introduction, significantly inhibited both DAG formation and exocytosis, suggesting that Ca2+ channels are involved in the responses observed. Treatment of capacitated cells with a synthetic permeant DAG stimulated exocytosis irrespective of whether nifedipine was present, indicating that Ca2+ channels function prior to DAG generation. The possibility that an influx of Na+, as well as Ca2+, might be involved in the exocytotic pathway was investigated using the monovalent cation ionophores monensin and nigericin. Both significantly stimulated DAG generation and acrosome loss, but the prior inclusion of nifedipine significantly inhibited all responses. These results strongly suggest that the entry of Ca2+ through Ca2+ channels, with characteristics similar to those of L-type, voltage-sensitive Ca2+ channels found in cardiac and skeletal muscle, is a crucial step in the sequence of events leading to exocytosis in progesterone-stimulated human spermatozoa. An influx of Na+ also may play a role, but at a point prior to the opening of Ca2+ channels.

Acrosome↗
PubMed1996

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

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

Animals↗