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.     

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-Gln-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. Mol. Reprod. Dev. 48:375–382, 1997. © 1997 Wiley-Liss, Inc.     

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. Mol. Reprod. Dev. 48:529–535, 1997. © 1997 Wiley-Liss, Inc.     

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.     

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 μM), a stimulatory A_2a adenosine receptor agonist, significantly stimulated capacitation and cAMP in uncapacitated cells, while cyclopentyl adenosine (1 μM), 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 A_2a 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 Gα_s subunit. In contrast, pertussis toxin prevented FPP's inhibition of both spontaneous acrosome loss and cAMP production, suggesting involvement of a Gα_i/_o subunit. Immunoblotting evidence revealed the presence of proteins of the appropriate molecular weights for Gα_s, Gα_i2, Gα _i3, and Gα_o subunits. This study provides the first direct evidence suggesting the involvement of two different types of adenosine receptors and both Gα_s and Gα_i/_o subunits in the regulation of capacitation, resulting in modulation of AC activity and availability of cAMP. Mol. Reprod. Dev. 53:459–471, 1999. © 1999 Wiley‐Liss, Inc.     

Modulation of the function of boar spermatozoa via adenosine and fertilization promoting peptide receptors reduce the incidence of polyspermic penetration into porcine oocytes

Effects of adenosine and pGlu-Glu-ProNH(2) (FPP) on the function and in vitro penetration of boar spermatozoa were examined. First, the effects of dibutyryl cAMP or agonists and antagonists of adenosine receptors (inhibitory adenosine receptors, A1AdR; stimulatory adenosine receptors, A2AdR) on freshly ejaculated spermatozoa were determined by chlortetracycline fluorescence assessment. Capacitation of spermatozoa was stimulated when they were cultured in a medium with dibutyryl cAMP, adenosine, A2AdR agonist, and adenosine plus A1AdR antagonist (CPT). However, acrosome reaction was inhibited only by adenosine. A1AdR agonist did not affect intact spermatozoa. A2AdR antagonist (DMPX) neutralized all of the effects of adenosine. Second, interaction of adenosine and FPP was examined. Gln-FPP, a competitive inhibitor of FPP, and DMPX inhibited the effects of adenosine and FPP, and CPT neutralized the inhibitory effect of FPP on acrosome reaction. Last, the effects of adenosine, FPP, and caffeine on the rate of sperm penetration were examined using frozen-thawed spermatozoa. Adenosine, FPP, and caffeine significantly enhanced the rate of sperm penetration as compared with the case of no additions. Caffeine treatment resulted in a high rate of polyspermic fertilization. In contrast, adenosine and FPP treatments resulted in an increased proportion of normal fertilization in in vitro-matured oocytes. These results suggest that boar spermatozoa can be modulated by the adenylyl cyclase/cAMP pathway via A2AdR in intact cells to induce capacitation and A1AdR in capacitated cells to inhibit spontaneous acrosome loss and that FPP receptors interact with A2AdR in intact cells and with A1AdR in capacitated cells. Furthermore, adenosine and FPP seem to be useful in reducing the incidence of polyspermic penetration.     

The role of small molecules in sperm capacitation

Mammalian spermatozoa released into an appropriate environment in vitro can capacitate but then may undergo spontaneous acrosome reactions. Since successful sperm interaction with the zona pellucida of an unfertilized oocyte requires an intact sperm plasma membrane, spontaneous acrosome loss is biologically undesirable because it renders spermatozoa non-fertilizing. Several small molecules (fertilization promoting peptide [FPP], adenosine, calcitonin and adrenaline), found in various body fluids including seminal plasma, have been shown to regulate capacitation in vitro. They initially accelerate capacitation but then inhibit spontaneous acrosome loss, allowing spermatozoa to maintain their fertilizing potential. Specific receptors for all these molecules are present on mammalian spermatozoa and their activation by the appropriate ligands leads to modulation of membrane-associated adenylyl cyclase activity and production of cAMP, stimulating cAMP production in uncapacitated cells and inhibiting it in capacitated cells. Boar spermatozoa have been shown to respond in vitro to adenosine and FPP, suggesting that the addition of these molecules to sperm samples used for artificial insemination could be beneficial in helping spermatozoa maintain fertilizing potential until they reach their target.     

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 μm 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. © 1996 Wiley-Liss, Inc.     

Calcitonin acts as a first messenger to regulate adenylyl cyclase/cAMP and mammalian sperm function

Calcitonin stimulates capacitation in uncapacitated mouse spermatozoa and then inhibits spontaneous acrosome loss in capacitated cells, responses similar to those elicited by fertilization promoting peptide (FPP), a peptide known to regulate the adenylyl cyclase/cAMP pathway. This study investigated the hypothesis that calcitonin also modulates this pathway. Calcitonin significantly stimulated cAMP production in uncapacitated spermatozoa and then inhibited it in capacitated cells; the magnitude of both stimulatory and inhibitory changes was similar to that obtained with FPP but the inhibitory responses to FPP preceded those of calcitonin. This possibly reflects the involvement of two different adenosine receptors in response to FPP compared with one calcitonin receptor. Calcitonin receptors were located on the acrosomal cap and the flagellum, the midpiece having a greater abundance than the principal piece. Although both calcitonin and adenosine receptors are found in the head and flagellum, there was no evidence for cross-talk between them. Chlortetracycline investigations to determine the minimum extracellular Ca(2+) requirement for responses to calcitonin revealed that calcitonin significantly stimulated capacitation in Ca(2+)-deficient medium but FPP did not. Calcitonin also significantly stimulated cAMP production under these conditions, and similarly preincubated suspensions, when diluted into +Ca(2+) medium, were significantly more fertile in vitro than untreated controls. These results indicate that calcitonin, like FPP, acts as a first messenger to regulate the production of cAMP and mammalian sperm function, but the differences in Ca(2+) requirements suggest that calcitonin and FPP may regulate different isoforms of adenylyl cyclase.     

Tyrosine phosphoproteome of hamster spermatozoa: Role of glycerol‐3‐phosphate dehydrogenase 2 in sperm capacitation

Capacitation confers on the spermatozoa the competence to fertilize the oocyte. At the molecular level, a cyclic adenosine monophosphate (cAMP) dependent protein tyrosine phosphorylation pathway operates in capacitated spermatozoa, thus resulting in tyrosine phosphorylation of specific proteins. Identification of these tyrosine‐phosphorylated proteins and their function with respect to hyperactivation and acrosome reaction, would unravel the molecular basis of capacitation. With this in view, 21 phosphotyrosine proteins have been identified in capacitated hamster spermatozoa out of which 11 did not identify with any known sperm protein. So, in the present study attempts have been made to ascertain the role of one of these eleven proteins namely glycerol‐3‐phosphate dehydrogenase 2 (GPD2) in hamster sperm capacitation. GPD2 is phosphorylated only in capacitated hamster spermatozoa and is noncanonically localized in the acrosome and principal piece in human, mouse, rat, and hamster spermatozoa, though in somatic cells it is localized in the mitochondria. This noncanonical localization may imply a role of GPD2 in acrosome reaction and hyperactivation. Further, enzymatic activity of GPD2 during capacitation correlates positively with hyperactivation and acrosome reaction thus demonstrating that GPD2 may be required for sperm capacitation.     

Cyclic nucleotide metabolism in mouse epididymal spermatozoa during capacitation in vitro

The role of cyclic nucleotides in sperm capacitation is equivocal. Using conditions known to support mouse sperm capacitation after 120 min incubation in vitro, the cAMP and cGMP contents of epididymal spermatozoa were measured and the cGMP/cAMP ratio determined. The initial high cAMP content detected upon release of spermatozoa decreased within 30 min to a lower plateau, which was then maintained throughout incubation. With the cGMP content remaining approximately constant, the cGMP/cAMP ratio increased over 120 min. In the presence of 2 mM caffeine, an increased cAMP content was noted at 0 and 30 min before a fall to the plateau level. To investigate cyclic nucleotide metabolism, adenylate cyclase and phosphodiesterase activities were compared in two sperm populations, one essentially uncapacitated and the other incubated for 120 min. Adenylate cyclase activity, higher in the presence of 2 mM Mn²⁺ compared to Mg²⁺, showed increased activity at 120 min compared to 30 min incubation, while phosphodiesterase activity decreased during this period. The ability of spermatozoa to form adenosine and inosine from cAMP indicated endogenous 5′-nucleotidase and deaminase, as well as phosphodiesterase, activities. Although the endogenous cAMP content appeared to remain constant during the time that acrosome loss, hyperactivated motility and fertilizing ability can be demonstrated, activities of the enzymes responsible for cAMP metabolism indicate an increased potential for cAMP availability and turnover. The increased cGMP/cAMP ratio may also play a role during capacitation.     

Phosphatidylinositol 3-kinase pathway regulates sperm viability but not capacitation on boar spermatozoa

Phosphatidylinositol 3-kinase (PI3-K) plays an important role in cell survival in somatic cells and recent data pointed out a role for this kinase in sperm capacitation and acrosome reaction (AR). This study was undertaken to evaluate the role of PI3-K pathway on porcine spermatozoa capacitation, AR, and viability using two unrelated PI3-K inhibitors, LY294002 and wortmannin. In boar spermatozoa, we have identified the presence of PDK1, PKB/Akt, and PTEN, three of the main key components of the PI3-K pathway. Incubation of boar sperm in a capacitating medium (TCM) caused a significant increase in the percentage of capacitated (25 +/- 2 to 34 +/- 1% P < 0.05, n = 6) and acrosome reacted (1 +/- 1 to 11 +/- 1% P < 0.01, n = 6) spermatozoa compared with sperm in basal medium (TBM). Inhibition of PI3-K did affect neither the capacitation status nor AR nor protein p32 tyrosine phosphorylation of boar spermatozoa incubated in TBM or TCM. Boar sperm viability in TBM was significantly decreased by 40 and 20% after pretreatment with LY294002 or wortmannin, respectively. Similar results were observed after incubation of boar spermatozoa in TCM. Treatment of boar spermatozoa with the analog of cAMP, 8Br-cAMP significantly prevented the reduction on sperm viability. Our results provide evidence for an important role of the PI3-K pathway in the regulation of boar sperm viability and suggests that other signaling pathways different from PI3-K must be activated downstream of cAMP to contribute to regulation of sperm viability. Finally, in our conditions the PI3-K pathway seems not related with boar sperm capacitation or AR.     

In vitro effect of gamma-aminobutyric acid on bovine spermatozoa capacitation

Sperm capacitation is defined as the maturational changes that render a sperm competent for fertilization and occurs in the female reproductive tract. Identification of the factor/s that regulate sperm capacitation would allow the understanding of these phenomena. Among these factors, gamma-aminobutyric acid (GABA) has recently become as a putative modulator of sperm function. The aim of this study was to explore the presence of a GABAergic regulation of bovine sperm capacitation as well as the possible intracellular mechanisms involved. GABA was detected in fresh semen by a sensitive radioreceptor assay (spermatozoa, 0.064 +/- 0.003 nmoles/10(6) cells; seminal plasma, 23.21 +/- 1.16 nmoles/ml). Scatchard analysis of [(3)H]-muscimol binding to sperm membranes yielded a linear plot consistent with a single population of binding sites (K(d) = 3.87 nM, B(max) = 417 fmol/mg prot.). [(3)H]-muscimol specific binding to sperm membranes was significantly inhibited by the GABA A receptor (GABA A-R) antagonist bicuculline and by the agonists muscimol and isoguvacine. Addition of GABA to the incubation medium resulted in a concentration-dependent increase in the percentage of capacitated spermatozoa (chlortetracycline assay). We observed a significant increment on intracellular calcium and cyclic 3',5' adenosine monophosphate (cAMP) concentrations induced by GABA, being the cation influx abolished when the cell suspensions were coincubated with the antagonists bicuculline or picrotoxin. It is concluded that GABA induces sperm capacitation through an intracellular mechanism dependent on calcium influx and cAMP accumulation mediated by a specific GABA A-R.     

Effect of a fertilization-promoting peptide on the fertilizing ability and glycosidase activity in vitro of frozen-thawed spermatozoa in the pig

This study has evaluated the effect of fertilization-promoting peptide (FPP) on the fertilizing ability and glycosidase activity in vitro of frozen-thawed boar spermatozoa. Use of chlortetracycline (CTC) fluorescence analysis, as well as various glycosidase analyses and the oocyte penetration test showed that FPP can promote the fertilizing ability and glycosidase activity of frozen-thawed spermatozoa in vitro. There were significantly (P < 0.05) more acrosome-reacted and penetrated in medium with 100 nM FPP than with 0, 50, 200 or 400 nM. The beta-N-acetylglucosaminidase (beta-GlcNAcase) activity was at least two-fold higher than other glycosidase regardless of FPP concentrations. In the same glycosidase, there were no differences in medium with different concentrations of FPP. The percentages of spermatozoa that reached acrosome reaction were affected by different periods (0, 1, 2, 3 or 4 h) of spermatozoa preincubation and were higher in medium with than without FPP. Penetration rates were decreased with preincubation periods of spermatozoa when oocytes were inseminated with spermatozoa preincubated in medium with and without FPP for the different periods. These rates were higher in spermatozoa preincubated with that than without FPP and had a tendency to increase as time of culture periods when the sperm-oocyte were cultured for 4, 8, 12, 16, 20 or 24 h. The activities of alpha-fucosidase, alpha-mannosidase, beta-galactosidase and beta-GlcNAcase were higher in medium with that than without FPP regardless of periods of sperm preincubation and sperm-oocyte culture. These results suggest that FPP may have a positive role in promoting sperm function and glycosidase activity in the pig.     

Signal transduction pathways that regulate sperm capacitation and the acrosome reaction

Ejaculated spermatozoa must undergo physiological priming as they traverse the female reproductive tract before they can bind to the egg’s extracellular coat, the zona pellucida (ZP), undergo the acrosome reaction, and fertilize the egg. The preparatory changes are the net result of a series of biochemical and functional modifications collectively referred to as capacitation. Accumulated evidence suggests that the event that initiates capacitation is the efflux of cholesterol from the sperm plasma membrane (PM). The efflux increases permeability and fluidity of the sperm PM and causes influx of Ca²⁺ ions that starts a signaling cascade and result in sperm capacitation. The binding of capacitated spermatozoa to ZP further elevates intrasperm Ca²⁺ and starts a new signaling cascade which open up Ca²⁺ channels in the sperm PM and outer acrosomal membrane (OAM) and cause the sperm to undergo acrosomal exocytosis. The hydrolytic action of the acrosomal enzymes released at the site of sperm-egg (zona) binding, along with the hyperactivated beat pattern of the bound spermatozoon, are important factors in directing the sperm to penetrate the ZP and fertilize the egg. The role of Ca²⁺-signaling in sperm capacitation and induction of the acrosome reaction (acrosomal exocytosis) has been of wide interest. However, the precise mechanism(s) of its action remains elusive. In this article, we intend to highlight data from this and other laboratories on Ca²⁺ signaling cascades that regulate sperm functions.     

Implication of cAMP during porcine sperm capacitation and protein tyrosine phosphorylation

Second messengers are involved in sperm fertilizing potential, as both motility and the acrosome reaction are influenced by cAMP. Moreover, the activity of cyclic nucleotides is implicated in the appearance of tyrosine phosphorylated sperm proteins, which is associated with capacitation in the mammalian spermatozoa. Nevertheless, the involvement of the cAMP/protein kinase A (PK-A) pathway during pig sperm capacitation may be different from that observed in other mammals. The objective of the present study was to clarify the cAMP/PK-A pathway during the capacitation of porcine spermatozoa and to evaluate this impact on the p32 sperm tyrosine phosphoprotein appearance. The presence of p32 was assessed after incubating fresh pig sperm with IBMX/db-cAMP, H-89, a PK-A inhibitor or bistyrphostin, a tyrosine kinase inhibitor, in capacitating (CM) or non-capacitating conditions (NCM) by immunoblotting SDS-extracted and separated sperm proteins using an anti-phosphotyrosine antibody. When pig spermatozoa were incubated in CM supplemented with H-89 (50 microM) or bistyrphostin (1.2 microM), capacitation decreased significantly (P < 0.001). The p32 sperm tyrosine phosphoprotein, previously shown to be associated with capacitation of porcine sperm though not necessarily an end point of this phenomenon, was not modulated by IBMX/db-cAMP (100 microM/1 mM), H-89 (50 microM) nor bistyrphostin (1.2 microM). Our results indicate, therefore, that pig sperm are regulated somewhat differently than as described for other mammals, because although the cAMP/PK-A and tyrosine kinase pathways are involved in capacitation, they do not influence the appearance of p32.     

Intracellular signaling cascades induced by relaxin in the stimulation of capacitation and acrosome reaction in fresh and frozen-thawed bovine spermatozoa

Relaxin is one of the 6-kDa peptide hormones, which acts as a pleiotropic endocrine and paracrine factor. Our previous studies revealed that sperm capacitating medium containing relaxin induced capacitation and acrosome reaction (AR) in fresh and frozen-thawed porcine or bovine spermatozoa. However, the intracellular signaling cascades involved with capacitation or AR induced by relaxin was unknown. Therefore, the present study was designed to investigate the intracellular signaling cascades involved with capacitation and AR induced by relaxin in fresh and frozen-thawed bovine spermatozoa. Spermatozoa were incubated in sperm Tyrode's albumin lactate pyruvate (Sp-TALP) medium supplemented with (40 ng ml(-1)) or without relaxin, and subjected to evaluation of chlortetracycline staining pattern, cholesterol efflux, Ca(2+)-influx, intracellular cyclic adenosine monophosphate (cAMP) and protein tyrosine phosphorylation. Capacitation and AR were increased (P<0.05) in both fresh and frozen-thawed spermatozoa incubated with relaxin. Cholesterol effluxes were greater in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa incubated with relaxin than the spermatozoa incubated without relaxin. Ca(2+)-influxes were also significantly stimulated by relaxin in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa. The Sp-TALP medium containing relaxin influenced the generation of intracellular cAMP in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa, and exhibited higher exposure of protein tyrosine phosphorylation in both sperm types than the medium devoid of relaxin. Therefore, the results postulate that relaxin exerts the intracellular signaling cascades involved with capacitation and AR through accelerating the cholesterol efflux, Ca(2+)-influx, intracellular cAMP and protein tyrosine phosphorylation in fresh and frozen-thawed bovine spermatozoa.     

AKAP3 selectively binds PDE4A isoforms in bovine spermatozoa

Cyclic AMP plays an important role in regulating sperm motility and acrosome reaction through activation of cAMP-dependent protein kinase A (PKA). Phosphodiesterases (PDEs) modulate the levels of cyclic nucleotides by catalyzing their degradation. Although PDE inhibitors specific to PDE1 and PDE4 are known to alter sperm motility and capacitation in humans, little is known about the role or subcellular distribution of PDEs in spermatozoa. The localization of PKA is regulated by A-kinase anchoring proteins (AKAPs), which may also control the intracellular distribution of PDE. The present study was undertaken to investigate the role and localization of PDE4 during sperm capacitation. Addition of Rolipram or RS25344, PDE4-specific inhibitors significantly increased the progressive motility of bovine spermatozoa. Immunolocalization techniques detected both PDE4A and AKAP3 (formerly known as AKAP110) in the principal piece of bovine spermatozoa. The PDE4A5 isoform was detected primarily in the Triton X-100-soluble fraction of caudal epididymal spermatozoa. However, in ejaculated spermatozoa it was seen primarily in the SDS-soluble fraction, indicating a shift in PDE4A5 localization into insoluble organelles during sperm capacitation. AKAP3 was detected only in the SDS-soluble fraction of both caudal and ejaculated sperm. Immunoprecipitation experiments using COS cells cotransfected with AKAP3 and either Pde4a5 or Pde4d provide evidence that PDE4A5 but not PDE4D interacts with AKAP3. Pulldown assays using sperm cell lysates confirm this interaction in vitro. These data suggest that AKAP3 binds both PKA and PDE4A and functions as a scaffolding protein in spermatozoa to regulate local cAMP concentrations and modulate sperm functions.     

Vasopressin Effectively Suppresses Male Fertility

Arginine vasopressin (VP) is neurohypophysial hormone has been implicated in stimulating contractile activity of the male reproductive tract in the testis. Higher levels of VP decrease sperm count and motility. However, very little is known about the involvement of VP in controlling mammalian reproductive process. The goal of this study was to confirm that effect of VP receptor (AVPR2) on sperm function in capacitation condition. Deamino [Cys 1, D-ArgS] vasopressin (dDAVP), an AVPR2 agonist that operates only on AVPR2, was used. Also, Mouse spermatozoa were incubated with various concentrations of dDAVP (10⁻¹¹–10⁻⁵ M) and sperm motility, capacitation status, Protein Kinase A activity (PKA), tyrosine phosphorylation, fertilization, and embryo development were assessed using computer-assisted sperm analysis, Combined Hoechst 33258/chlortetracycline fluorescence, Western blotting, and in vitro fertilization, respectively. AVPR2 was placed on the acrosome region and mid-piece in cauda epididymal spermatozoa, but the caput epididymal spermatozoa was mid-piece only. The high dDAVP treatment (10⁻⁸ and 10⁻⁵ M) significantly decreased sperm motility, intracellular pH and PKA substrates (approximately 55 and 22 kDa) and increased Ca²⁺ concentration. The highest concentration treatment significantly decreased PKA substrate (approximately 23 kDa) and tyrosine phosphorylation (approximately 30 kDa). VP detrimentally affected capacitation, acrosome reaction, and embryo development. Treatment with the lowest concentration (10⁻¹¹ M) was not significantly different. Our data have shown that VP stimulates ion transport across sperm membrane through interactions with AVPR2. VP has a detrimental effect in sperm function, fertilization, and embryonic development, suggesting its critical role in the acquisition of fertilizing ability of mouse spermatozoa. These research findings will enable further study to determine molecular mechanism associated with fertility in capacitation and fertilization. It is also an important pivotal precondition to the progress of diagnostic test to identify infertility and to apply male contraception.     

Binding of the anti-human sperm monoclonal antibody HS-11 to bull spermatozoa is correlated with fertility in vitro

The present study aimed to determine if there is bull to bull variation in the binding of the anti-human sperm monoclonal antibody (MAb) HS-11 to bull spermatozoa, and to investigate if there is any correlation between HS-11 binding to spermatozoa and in vitro fertility of the bulls tested. Semen samples of a single collection (split frozen in 0.5-ml straws) from 8 dairy bulls were used. Swim-up separated motile spermatozoa were incubated in 90-microl drops of capacitation medium (TALP+10 microg/ml heparin) at 39 degrees C, 5% CO2, 95% air. At 0, 2, 4 and 6 h of incubation HS-11 was added (1:1000 final concentration), and the MAb binding was assessed by indirect immunofluorescence assay (IIFA). The HS-11 binding was indicated by a bright green fluorescence of the sperm acrosome region. In vitro-matured, good quality bovine oocytes were randomly allocated to spermatozoa of each bull for in vitro fertilization. Sperm samples of 2 to 3 bulls were used in each trial until 4 replicates per bull were attained for IVF (n approximately 100 oocytes/bull) and IIFA experiments. Sperm capacitation status was assessed simultaneously using an egg yolk lysophosphatidylcholine- (LC) induced acrosome reaction assay. The binding of HS-11 to spermatozoa was maximum at 4 h of incubation in most (6/8) of the bull semen samples. Significant (P < 0.01) differences were observed between bulls in the binding of HS-11 to their spermatozoa (range 22 +/- 8 to 52 +/- 5%) at 4 h, but not within replicates. Similarly, variations (P < 0.05) in the cleavage rate were also seen (range 22 +/- 9 to 58 +/- 7%) between bulls. The HS-11 binding and cleavage were significantly correlated (r = 0.43; n = 32; P < 0.05). The highest percentage of spermatozoa underwent acrosome reaction in response to LC treatment at the 4-h incubation period. This and the linear relationship between HS-11 binding and the cleavage rate observed in the present study together strengthen our earlier suggestion that the binding of the monoclonal antibody HS-11 to bull spermatozoa on a time-dependent manner, may indicate capacitation changes. We conclude that 1) between-bull differences exist in HS-11 binding to spermatozoa, and in the cleavage rate, and 2) HS-11 binding to spermatozoa is correlated with fertility, as determined by the cleavage of bovine oocytes matured and fertilized in vitro.