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.     

Regulation of in vitro penetration of frozen-thawed boar spermatozoa by caffeine and adenosine

Effects of caffeine and adenosine on the function and in vitro penetration of frozen-thawed boar spermatozoa were examined. First, the effect on sperm function was determined by the chlortetracycline fluorescence assessment. Both caffeine and adenosine stimulated capacitation of spermatozoa. However, adenosine, but not caffeine, inhibited spontaneous acrosome loss. Second, sperm penetration into in vitro matured oocytes was compared among spermatozoa cultured in the absence or presence of caffeine or adenosine. Both caffeine and adenosine increased the penetration rate (99.1 +/- 0.9% in caffeine, 72.4 +/- 2.0% in adenosine vs. 54.8 +/- 5.1% in controls) but only caffeine decreased drastically the monospermic penetration rate (8.0 +/- 2.3% in caffeine vs. 75.4 +/- 4.8% in adenosine and 78.6 +/- 4.8% in controls). When oocytes were cocultured in various sperm concentrations, the proportion of monospermy changed in inverse proportion to sperm concentration in the presence of caffeine, but did not change in the presence of adenosine. A relatively high number of spermatozoa at the early stage of spontaneous acrosome reaction in the presence of caffeine may be one of the main causes of polyspermic penetration in porcine IVF system. These results indicate that replacement of caffeine with adenosine in fertilization medium improves monospermic penetration by frozen-thawed boar spermatozoa.     

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.     

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.     

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.     

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.     

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 μM) 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 μM) 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. Mol. Reprod. Dev. 51:468–476, 1998. © 1998 Wiley-Liss, Inc.     

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.     

RU486 suppresses progesterone-induced acrosome reaction in boar spermatozoa

The effects of progesterone on the acrosome reaction, as well as the effects of RU486 on the progesterone-induced acrosome reaction in capacitated boar spermatozoa, were investigated. Progesterone, a major steroid that is secreted by the cumulus cells of oocyte, clearly induced the acrosome reaction in a dose-dependent manner in capacitated boar spermatozoa, even though it failed to show similar effects in non-capacitated spermatozoa. RU486, a potent antiprogestin, significantly reduced the effects of progesterone on the progesterone-induced acrosome reaction; however, when treated alone, it showed no inhibitory effects on the acrosome reaction. The inhibitory effects of RU486 were also shown to be dose dependent. These results imply that in addition to the wellknown inducer of the acrosome reaction, zona pellucida, progesterone can also induce the acrosome reaction through its specific receptors on spermatozoa after the spermatozoa undergo capacitation.     

Modulation of the human sperm acrosome reaction by effectors of the adenylate cyclase/cyclic AMP second-messenger pathway.

The acrosome reaction of spermatozoa appears to be analogous to various somatic cell exocytotic events which involve cascade reactions, i.e., transmission of an external signal across the cell membrane resulting in activation of an "amplifier" enzyme and the generation of a second messenger. Using a synchronous acrosome reaction system (De Jonge et al., J. Androl., 10:232-239, '89a), it was found that analogues of the second-messenger cAMP, dibutyryl cAMP (dbcAMP) and 8-bromo cAMP, stimulated the acrosome reaction of capacitated spermatozoa. Additionally, treatment of spermatozoa with either xanthine or non-xanthine phosphodiesterase inhibitors induced a significant (P less than 0.05) increase in the percent acrosome reaction after a period of capacitation in comparison to untreated controls. These results indicate that analogues of cAMP or inhibitors which prevent cAMP hydrolysis can induce the human sperm acrosome reaction. Subsequent experiments were conducted to test whether the amplifier enzyme in the cascade reaction, adenylate cyclase, has a role in the acrosome reaction. Forskolin, an adenylate cyclase stimulator, caused a significant (P less than 0.01) increase in the percent acrosome reaction in comparison to controls. Modulators of adenylate cyclase--adenosine, 2'-0-methyladenosine, and 2',3'-dideoxyadenosine--significantly (P less than 0.01) inhibited the forskolin-induced acrosome reaction. dbcAMP was able to overcome the inhibition by adenosine. Two inhibitors of protein kinase A, the Walsh inhibitor and H-8, caused a significant (P less than 0.01) inhibition of the dbcAMP-induced acrosome reaction. Finally, in the absence of extracellular calcium, dbcAMP induced a significant (P less than 0.01) increase in the acrosome reaction in contrast to A23187. These results suggest that: 1) a molecular mechanism for the human sperm acrosome reaction involves the cAMP second-messenger system; i.e., activation of adenylate cyclase, the amplifier enzyme that produces cAMP, production of cAMP as a second messenger, and activation of cAMP-dependent kinase A; and that 2) activation of adenylate cyclase occurs after calcium influx.     

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.     

Study on the role of a sperm membrane-associated antigen in canine fertilization

The Mab 4B12 produced by us against capacitated boar spermatozoa was found to recognize a protein located in the acrosome portion of capacitated boar spermatozoa which is shared by different animal species, dogs included. It was shown that Mab 4B12 might affect fertilizing ability in vitro of boar spermatozoa. Using indirect immunofluorescence (IIF) test, we provide evidence here that Mab 4B12 stained the acrosome of the capacitated but not of the ejaculated and acrosome-reacted canine spermatozoa. The biological experiments using hemizona assay functional test in this study provide evidence supporting the involvement of Mab 4B12 corresponding antigen in the functional steps required for canine sperm-zona pellucida binding. These results together with the data on cell and tissue specificity of the 4B12 antigen suggest its contraceptive potential for canine fertilization.     

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.     

Sperm capacitation in vitro in the eld's deer

Sperm capacitation was examined in the endangered Eld's deer (Cervus eldi thamin). Sperm motility and viability (percentage of sperm cells with intact membranes) were assessed in vitro over time after attempting to induce capacitation in TALP alone and TALP supplemented with calcium (10 mM CaCl2), dibutyryl cAMP (1 mM dbcAMP), or fetal calf serum (20% FCS). Sperm aliquots were evaluated at 0, 3, 6, 9, and 12 h for motility, viability, and ability to acrosome react after exposure to calcium ionophore (A23187, CI; 10 microM) or lysophosphatidylcholine (LC; 100 microg/mL). Fresh sperm aliquots in TALP + 10 mM CaCl2 exposed to CI had fewer (P < 0.05) intact acrosomes than the TALP control (TALP alone) or dbcAMP and FCS treatments after 9 h. Mean (+/- SEM) percentage of intact acrosomes of spermatozoa incubated in medium with increased CaCl2 declined (P < 0.05) from 80.2 +/- 2.6% (0 h) to 49.7 +/- 7.3% after prolonged incubation (9 h). The proportion of capacitated fresh spermatozoa was not influenced by LC treatment. Capacitation was not induced (P > 0.05) by any of the presumptive sperm capacitators after freeze-thawing. Likewise, neither CI nor LC induced the acrosome reaction (AR) in these spermatozoa, suggesting that the freeze-thawing process may have caused membrane damage. Results revealed that the supplementation of medium with CaCl2 evokes capacitation in some spermatozoa. However, Eld's deer spermatozoa appear remarkably resistant to conventional stimulators of capacitation and the AR.     

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.     

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.     

Evaluation of sperm acrosome reaction in the Asiatic elephant

This study focuses on the effect of chemicals on acrosome reaction in elephant spermatozoa. Semen was collected at the Washington Park Zoo in Portland, Oregon, from an 11-yr-old Asian elephant by artificial vagina (7 ejaculates) and transported to Mahidol University in Bangkok in extender at 4 to 5 degrees C within 24 to 28 h. A total of 500 x 10(6) sperm/mL was used for the control and for each of the 4 treatment groups: 1) cAMP (0.1 mM); 2) caffeine (0.1 mM); 3) Penicillamine hypotaurine and epinephine, PHE (penicillamine 2 mM, hypotaurine 1 mM, epinephine 1 mM); and 4) heparin (10 microg/mL) at 39 degrees C for 2 h. Aliquots were removed and the sperm viability, abnormal morphology, and acrosome status were evaluated by triple stain technique. Transmission electron microscopy (TEM) was used to observe changes of the sperm head membrane in all treatment groups. Trypan blue reliably stained dead spermatozoa, while rose Bengal stained only the spermatozoa with intact acrosomes. The concentration of dead sperm cells was similar in the 4 groups. The percentages of live acrosome-reacted spermatozoa in the control and in groups treated with caffeine, PHE, cAMP and heparin were 19.5 +/- 4.3, 38.1 +/- 4.0, 34.8 +/- 3.7, 29.8 +/- 0.8 and 28.0 +/- 4.2, respectively. The acrosome reaction rate was higher in the treatment groups than in the control (P<0.05). Caffeine and PHE caused significantly higher acrosome reaction of the sperm head than cAMP or heparin (P<0.05). The electron micrographs showed that the acrosome reaction occurred by the presence of apical vesiculation. The results indicated that 1) the triple stain technique allowed for evaluation of both viability and acrosome reaction simultaneously in elephant spermatozoa,2) acrosome reaction occurred at a high rate in all 3 treatment groups. 3) the effects of caffeine and PHE were significantly higher (P<0.05) than of cAMP and heparin, and 4) the data obtained from the triple stain technique corresponded to those from TEM.     

Evaluation of boar spermatozoa penetrating capacity using pig oocytes at the germinal vesicle stage

We evaluated the ability of immature pig oocytes (at germinal vesicle stage) to detect differences in the in vitro penetration rates of boar spermatozoa. In Experiment 1, immature and ovulated oocytes (n=303) were exposed to capacitated boar spermatozoa to determine if the penetrability of immature pig oocytes was comparable to that of ovulated oocytes. The percentages of penetrated oocytes and the mean number of spermatozoa per oocyte were similar for immature (88.82 and 7.42+/-0.41) and ovulated oocytes (90.97 and 7.95+/-0.34, respectively). In Experiment 2, immature oocytes (n=1230) were inseminated with semen from 2 boars (A and B) with satisfactory semen characteristics to establish the variability of in vitro penetrating capacity between the boars. Semen was examined for motility, movement quality, acrosome integrity and plasma membrane integrity at various stages of the in vitro procedure. Although the sperm evaluation results were similar between boars, Boar A exhibited a significantly higher (P<0.001) penetration rate (91.49%) and number of spermatozoa penetrated per oocyte (5.90+/-0.25) than Boar B (52.87% and 2.03+/-0.12, respectively). Increasing the sperm concentration at insemination from 1x10(6) to 10x10(6) cells/ml resulted in an increased penetrating capacity for both boars, and the differences in the number of spermatozoa per oocyte between boars also increased. These results indicate that immature pig oocytes can be used in a homologous in vitro fertilization assay, and that despite similarities in semen characteristics a significant boar effect is evident for parameters of in vitro penetration of oocytes.     

Effects of Adenosine Receptor Subtypes on Hippocampal Extracellular Serotonin Level and Serotonin Reuptake Activity

Abstract: To clarify the effects of adenosine receptor subtypes (A₁, A₂, and A₃) on hippocampal serotoninergic function, hippocampal extracellular serotonin (5-HT) levels were determined by in vivo microdialysis in freely moving rats under various conditions. Both adenosine and an adenosine A₁ receptor agonist, 2-chloro-N⁶-cyclopentyladenosine, decreased extracellular 5-HT levels, whereas an adenosine A₁ receptor antagonist, 8-cyclopentyl-1,3-dimethylxanthine (CPT), and caffeine increased these levels. A selective A_2A receptor agonist (CGS-21680), an adenosine A₂ receptor agonist (PD-125944), an adenosine A₂ receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX), and an adenosine A₃ receptor agonist, N⁶-2-(4-aminophenyl)ethyladenosine (APNEA), did not affect extracellular 5-HT levels. When the adenosine A₁ receptor was blocked by CPT, the hippocampal extracellular 5-HT level was increased by adenosine, CGS-21680, and PD-125944, and decreased by caffeine, DMPX, and APNEA. When both adenosine A₁ and A₂ receptors were blocked by CPT and DMPX, the extracellular 5-HT level was decreased by adenosine, caffeine, and APNEA. The hippocampal extracellular 5-HT level was not affected by administration of APNEA alone, but was decreased by this agent when the adenosine A₁ receptor was blocked, irrespective of whether the adenosine A₂ receptor was functional. These inhibitory effects of adenosine, caffeine, and APNEA on extracellular 5-HT levels, during both adenosine A₁ and A₂ receptor blockade, were inhibited by selective 5-HT reuptake inhibitors. These results indicate that the stimulatory effects of the adenosine A₂ receptor and the inhibitory effects of the A₃ receptor on hippocampal extracellular 5-HT levels are masked by the inhibitory effects of the adenosine A₁ receptor.     

Effect of cooling and seminal plasma on the capacitation status of fresh boar sperm as determined using chlortetracycline assay

Insemination of sows with frozen-thawed spermatozoa results in lower fertility, in part due to spermatozoa having undergone a capacitation-like reaction. The present study employed chlortetracycline (CTC) staining analysis to investigate the effect of adding 20% (v/v) boar seminal plasma (SP) to boar spermatozoa on the temporal progress of capacitation and the acrosome reaction in spermatozoa cooled to 5 degrees C or incubated at 39 degrees C. Based on CTC staining patterns, seminal plasma appeared to reverse capacitation in spermatozoa that had undergone capacitation while incubated at 39 degrees C in a capacitation-supporting medium from 59.7 to 36.6% capacitated (P<0.001). Similarly, the addition of SP to boar spermatozoa cooled to 5 degrees C resulted in both the prevention of the capacitation-like reaction, and the reversal of an established capacitation-like reaction from 63.3 to 34.2% capacitated (P<0.001). These observations indicated that some constituent(s) of boar SP both prevent spermatozoa from undergoing capacitation as well as reverse capacitation in spermatozoa that have already undergone the process.