Showing papers on "Acrosome reaction published in 2000"
TL;DR: Dynamics in adhesive and fusion properties, molecular composition and architecture of the sperm plasma membrane, as well as membrane derived signalling are reviewed.
580 citations
TL;DR: This minireview focuses on the most important aspects of the sperm acrosome, from its formation during sperm development in the testis to its modification in the epididymis and function following sperm-egg interaction.
241 citations
TL;DR: It is shown that ZP3 generates a sustained Ca(2+) influx through a store depletion-operated pathway and that this drives the exocytotic acrosome reaction.
Abstract: Fertilization occurs after the completion of the sperm acrosome reaction, a secretory event that is triggered during gamete adhesion. ZP3, an egg zona pellucida glycoprotein, produces a sustained i...
239 citations
TL;DR: Sperm characteristics, seminal reactive oxygen species, FA of sperm membrane phospholipids, sperm oxidized DNA, 8-OH-dG, and induced acrosome reaction were evaluated including 27 infertile men.
Abstract: We evaluated the effects of combined conventional treatment, oral antioxidants (N-acetyl-cysteine or vitamins A plus E) and essential fatty acids (FA) on sperm biology in an open prospective study including 27 infertile men. The evaluation included sperm characteristics, seminal reactive oxygen species (ROS), FA of sperm membrane phospholipids, sperm oxidized DNA (8-OH-dG), and induced acrosome reaction (AR). Treatment did not improve sperm motility and morphology, nor decrease the concentration of round cells and white blood cells in semen. Sperm concentration increased in oligozoospermic men (7.4+/-1.3 to 12.5+/-1.9 million/ml). Treatment significantly reduced ROS (mean+/-SEM) (775.3+/-372.2 to 150.3+/-105.2 x 10(3)counts/10 second) and 8-OH-dG (45.3+/-10.4 to 16. 8+/-3.3 fmol/microg DNA). Treatment increased the AR (55.1+/-2.2 to 71.6+/-2.2%), the proportion of polyunsaturated FA of the phospholipids, and sperm membrane fluidity. The overall pregnancy rate was 4.5% in 134 months. The per month pregnancy rate tended to be higher in partners of (ex)-smokers (7.15%, n=14,70 months) than in never-smokers (1.6%, n=13,64 months) (OR:4.57, 95% Cl:0.55-38.1).
233 citations
TL;DR: It is concluded that nitric oxide synthase and nitric-oxide-related bioactivity satisfy the primary criteria of an egg activator: they are present in an appropriate place, active at an appropriate time, and are necessary and sufficient for successful fertilization.
Abstract: The early steps that lead to the rise in calcium and egg activation at fertilization are unknown but of great interest--particularly with the advent of in vitro fertilization techniques for treating male infertility and whole-animal cloning by nuclear transfer. This calcium rise is required for egg activation and the subsequent events of development in eggs of all species. Injection of intact sperm or sperm extracts can activate eggs, suggesting that sperm-derived factors may be involved. Here we show that nitric oxide synthase is present at high concentration and active in sperm after activation by the acrosome reaction. An increase in nitrosation within eggs is evident seconds after insemination and precedes the calcium pulse of fertilization. Microinjection of nitric oxide donors or recombinant nitric oxide synthase recapitulates events of egg activation, whereas prior injection of oxyhaemoglobin, a physiological nitric oxide scavenger, prevents egg activation after fertilization. We conclude that nitric oxide synthase and nitric-oxide-related bioactivity satisfy the primary criteria of an egg activator: they are present in an appropriate place, active at an appropriate time, and are necessary and sufficient for successful fertilization.
172 citations
TL;DR: The main signal transduction pathways involved in capacitation and acrosome reaction are summarized and the mechanisms underlying sperm DNA fragmentation are briefly reviewed.
Abstract: Two processes, namely capacitation and acrosome reaction, are of fundamental importance in the fertilization of oocyte by spermatozoon. Physiologically occurring in the female genital tract, capacitation is a complex process, which renders the sperm cell capable for specific interaction with the oocyte. During capacitation, modification of membrane characteristics, enzyme activity and motility properties of spermatozoa render these cells able to penetrate oocyte investments and responsive to stimuli that induce acrosome reaction prior to fertilization. Physiological acrosome reaction occurs upon interaction of the spermatozoon with the zona pellucida protein ZP3. This is followed by liberation of several acrosomal enzymes and other constituents that facilitate penetration of the zona and expose molecules on the sperm equatorial segment that allows fusion of sperm membrane with the oolemma. The molecular mechanisms and the signal transduction pathways mediating the processes of capacitation and acrosome reaction have been partially defined, and appear to involve modifications of intracellular calcium and other ions, lipid transfer and phospholipid remodeling in sperm plasma membrane as well as changes in protein phosphorylation. Some of the kinases and phosphorylated proteins that are involved in the processes of capacitation and acrosome reaction have been now characterized. Characterization of sperm receptors to physiological inducers of acrosome reaction is in progress. This review summarizes the main signal transduction pathways involved in capacitation and acrosome reaction.Furthermore, the mechanisms underlying sperm DNA fragmentation are also briefly reviewed.
125 citations
TL;DR: Sperm SNAREs may be responsible, together with other sperm components, for the asynchronous male DNA decondensation that occurs following intracytoplasmic sperm injection, an assisted reproduction technique that bypasses normal sperm-egg surface interactions.
117 citations
TL;DR: Biophysical analyses have revealed the presence of both high mannose and complex-type N-glycans in murine zona pellucida, and Oligosaccharides terminated with GalNAcβ1–4Gal have been implicated in the secondary binding interaction that occurs following the acrosome reaction.
112 citations
TL;DR: The regression analysis showed that combining the results of sperm membrane integrity assessment post-thawing with those of capacitation status after swim-up provided the best prediction of fertility.
106 citations
TL;DR: Selecting a highly motile sperm population before freezing enhances overall post-thaw spermatozoa quality and reduces the deleterious effects of cryopreservation.
Abstract: The purpose of this study was to examine if selecting a sperm population with improved motion characteristics before freezing reduces the deleterious effects of cryopreservation. Semen specimens from 15 normal donors were divided into two equal aliquots. The first aliquot received no treatment (control), and the second was processed by swim-up from a washed sperm preparation to select a sperm population with better motility and motion characteristics (swim-up). Both aliquots were cryopreserved by the liquid nitrogen vapour method. Percentage motility and motion characteristics were evaluated by computer-assisted semen analysis. Acrosome integrity as well as spontaneous and calcium ionophore-induced acrosome reactions before freezing and after thawing were assessed by fluorescein isothiocyanate conjugated peanut agglutinin combined with a supra vital dye (Hoechst-33258). Swim-up processing enabled selection of a sperm population with better motion characteristics, percentage motility and viability before freezing (P < 0.001), but with no difference in percentage of acrosome-intact spermatozoa (P = 0.63). After thawing, the swim-up specimens exhibited faster velocity and progression than untreated specimens (P < 0.001). They also had higher percentages of spermatozoa with intact acrosomes and spermatozoa able to undergo acrosome reaction in response to calcium ionophore (P < 0.05). Selecting a highly motile sperm population before freezing enhances overall post-thaw spermatozoa quality.
104 citations
TL;DR: It is shown that N-ethylmaleimide-sensitive factor (NSF), a protein essential for most fusion events, is present in the acrosome of several mammalian spermatozoa and that calcium-dependent exocytosis of permeabilized sperm requires active NSF.
Abstract: The acrosome reaction of spermatozoa is a complex, calcium-dependent, regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. However, very little is known about the molecules that mediate and regulate this unique fusion process. Here, we show that N-ethylmaleimide-sensitive factor (NSF), a protein essential for most fusion events, is present in the acrosome of several mammalian spermatozoa. Moreover, we demonstrate that calcium-dependent exocytosis of permeabilized sperm requires active NSF. Previously, we have shown that the addition of the active (GTP-bound) form of the small GTPase Rab3A triggers exocytosis in permeabilized spermatozoa. In the present report we show that Rab3A is necessary for calcium-dependent exocytosis. The activation of Rab3A protects NSF from N-ethylmaleimide inhibition and precludes the exchange of the endogenous protein with recombinant dominant negative mutants of NSF. Furthermore, Rab3A activation of acrosomal exocytosis requires active NSF. Our results suggest that, upon calcium stimulation, Rab3A switches to its active GTP-bound form, triggering the formation of a protein complex in which NSF is protected. This process is suggested to be an essential part of the molecular mechanism of membrane fusion leading to the release of the acrosomal contents.
TL;DR: Results suggest that under experimental conditions, sildenafil triggers human sperm motility and capacitation, probably via its inhibitory action on PDE activity other than type 5 with a resultant rise in cAMP levels.
Abstract: Capacitation is the series of transformations that spermatozoa undergo in the female genital tract in order to bind to the zona pellucida, initiate the acrosome reaction, and fertilize an egg. Cyclic adenosine monophosphate (cAMP) plays an important role in this process and its levels are regulated by 2 key enzymes, adenylyl cyclase and cyclic nucleotide phosphodiesterase (PDE), the latter being involved in cAMP degradation. Evidence was provided for the involvement of PDE in sperm motility and capacitation. Of the 10 gene families of PDE that exist in mammalian tissues, the calcium-calmodulin-dependent (type 1) and the cAMP-specific (type 4) have been found in human spermatozoa. Using sildenafil, we investigated a highly potent cyclic guanosine monophosphate (cGMP)-specific PDE (type 5) inhibitor and whether this PDE is present in human spermatozoa and is involved in sperm functions. Sildenafil inhibited PDE activity of Percoll-washed spermatozoa with an IC 50 of 97 ± 3 and 33 ± 3 μM when cAMP and cGMP, respectively, were used as substrates. Because the IC 50 of sildenafil obtained for PDE type 5 is much lower (2 to 6 nM) than that obtained with sperm PDE, the data suggest that PDE type 5 represents only a small fraction of the whole PDE activity of spermatozoa. Sildenafil causes dose-dependent increases in sperm cAMP levels and capacitation, which are associated with an increase in the levels of tyrosine phosphorylation of 2 fibrous sheath proteins (p105/81). Sperm velocity, amplitude of lateral head displacement, and hyperactivation were increased at 30-180 minutes. Sildenafil did not trigger the acrosome reaction in capacitated spermatozoa. These results suggest that under our experimental conditions, sildenafil triggers human sperm motility and capacitation, probably via its inhibitory action on PDE activity other than type 5 with a resultant rise in cAMP levels.
TL;DR: Evaluation of AR by staining or fluorescent probes can be useful to predict fertilization success and to direct the therapeutic strategy in male infertility.
TL;DR: The results indicate that permeabilized spermatozoa can be used to study the role of macromolecules in the acrosome reaction, Rab3A is present in human spermutozoa, and Rab 3A or another Rab3 isoform is involved in the exocytosis of the Acrosomal granule in human semen.
Abstract: The acrosome reaction is a regulated exocytotic process leading to a massive fusion between the outer acrosomal membrane and the cell membrane. In spite of the great amount of information available related to the acrosome reaction in several species, there is a remarkable paucity about the role of monomeric guanosine triphosphatases (GTPases) of the Rab family—wellestablished participants in exocytosis in other cell types—in the acrosome reaction. Western blot and immunofluorescence analysis indicate that Rab3A is present in human spermatozoa and localizes to the acrosomal region in the sperm head. One difficulty in studying the role of proteins in intact cells is the fact that they are unable to cross the cell membrane. Therefore, we established a working model of streptolysin O-permeabilized human spermatozoa. Permeabilized spermatozoa were able to respond in a regulated way to different stimuli, such as G protein activators and calcium. An acrosomal reaction was also triggered by a Rab3A peptide corresponding to the effector region. More important, recombinant Rab3A protein in the GTP-bound form caused acrosome exocytosis. The same protein loaded with GDP or Rab11 in the GTP-bound form was inactive. Also, recombinant GDI (GDP dissociation inhibitor)—a protein that releases Rab proteins from membrane—inhibited a GTPgS-stimulated acrosome reaction. Our results indicate that 1) permeabilized spermatozoa can be used to study the role of macromolecules in the acrosome reaction, 2) Rab3A is present in human spermatozoa, and 3) Rab3A or another Rab3 isoform is involved in the exocytosis of the acrosomal granule in human spermatozoa.
TL;DR: To test the hypothesis that sperm intracellular calcium level is correlated with in vivo fertility, the fluorescent calcium indicator, indo-1, and flow cytometry were used and there was a significant difference between the 3 most and least fertile bulls over 4 hours of incubation.
Abstract: Cryopreserved bovine semen is less fertile than fresh semen for reasons that have not been fully elucidated. Cryopreservation is known to disrupt the sperm plasma membrane and it induces premature capacitation of a sperm subpopulation, which may be a result of the increased internal calcium levels after thawing. To test the hypothesis that sperm intracellular calcium level is correlated with in vivo fertility, we used the fluorescent calcium indicator, indo-1, and flow cytometry to assess intracellular calcium levels in frozen-thawed sperm from bulls of varying degrees of fertility. We also tested a second hypothesis that the physiological status of sperm, as assessed by the chlortetracycline (CTC) fluorescent assay, is correlated with fertility. As detected by indo-1 fluorescence, the intracellular calcium level is negatively correlated with bull fertility immediately after thawing (P = .0362; n = 3 ejaculates from each of 10 animals). Moreover, there was a significant difference between the 3 most and least fertile bulls over 4 hours of incubation (P < .05; n = 3 ejaculates per bull). Finally, there was a positive correlation between sperm displaying the CTC acrosome reaction pattern and fertility (P = .0014; n = 3 ejaculates from each of 10 bulls).
TL;DR: Progesterone stimulates sperm functions, e.g. hyperactivation, acrosome reaction, binding to oocyte zona pellucida and penetration rate into the hamster oocyte, and modulates sperm function by stimulating a trypsin-like proteolytic activity, the biosynthesis of polyamine, phospholipase A2 activity and protein tyrosine kinase activity in the sperm cell.
Abstract: Progesterone stimulates sperm functions, e.g. hyperactivation, acrosome reaction, binding to oocyte zona pellucida and penetration rate into the hamster oocyte. The physiological relevance of these effects has been shown using female genital tract fluids which modulate sperm function according to their progesterone content. Progesterone interacts with specific sperm binding sites that, unlike the classic nuclear receptors, are located on the plasma membrane of the spermatozoon. Binding studies have revealed the presence of two classes of progesterone receptors in the human spermatozoon, one class has an elevated affinity constant (nanomolar) and is specific for progesterone, whereas the other class has an affinity constant in the micromolar range and binds equally well other hydroxylated progesterone derivatives. Following exposure to progesterone, the main event is a rapid (within seconds) increase of the intracellular free calcium concentration, followed by a sustained rise lasting for several minutes (plateau phase). Both these calcium transients are dependent upon entry of extracellular calcium. The nature of the calcium channel that mediates the effects of progesterone is, currently, unknown. It has been postulated that it may be: (i) part of the progesterone receptor; (ii) voltage-dependent; or (iii) operated by second messengers following activation of the progesterone receptor. Progesterone also modulates sperm function by stimulating a trypsin-like proteolytic activity, the biosynthesis of polyamine (putrescine and spermidine), phospholipase A2 activity and protein tyrosine kinase activity in the sperm cell. Recent studies have shown that chloride ion efflux is vital for progesterone to promote the acrosome reaction. This effect is achieved by interaction with a sperm membrane receptor which resembles the neuronal GABA(A) receptor. Accordingly, GABA(A) receptors have been found in the spermatozoon plasma membrane and GABA stimulates hyperactivation and promotes the acrosome reaction.
TL;DR: The results suggest that CaM participates in the regulation of membrane changes important for mouse sperm capacitation, at a point upstream from cAMP, and that this pathway is at least partially separable from pathways controlling tyrosine phosphorylation and hyperactivation.
Abstract: Although Ca(2+) is of fundamental importance in mammalian sperm capacitation, its downstream targets have not been definitively demonstrated. The purpose of this study was to use the calmodulin (CaM) antagonists W7 and calmidazolium (CZ) to investigate the possible role of CaM, a Ca(2+)-specific binding protein, in capacitation. Sperm membrane changes associated with capacitation were assessed by the B pattern after chlortetracycline staining and by the ability to undergo the acrosome reaction (AR) in response to lysophosphatidylcholine (LPC). The percentage of B pattern sperm was significantly inhibited by W7 or CZ in a concentration-dependent manner. At 100 microM W7 or 10 microM CZ, these inhibitors also significantly reduced the sperm's ability to undergo the LPC-induced AR. Inhibition of the B pattern and the LPC-induced AR was overcome by exogenous cAMP analogues. Treatment of the sperm with 100 microM W7 also resulted in a significant decrease in their ability to fertilize eggs in vitro. At 100 microM, W5, a less potent dechlorinated W7 analogue, had no effect on the B pattern, LPC-induced AR, or fertilization competence. Sperm viability and protein tyrosine phosphorylation were not substantially affected by 100 microM W7 (relative to 100 microM W5) or 10 microM CZ; however, the percentages of motile and hyperactivated sperm were significantly reduced. The antagonist-inhibited sperm motility was restored by dilution in control medium, but not by cAMP analogues. These results suggest that CaM participates in the regulation of membrane changes important for mouse sperm capacitation, at a point upstream from cAMP, and that this pathway is at least partially separable from pathways controlling tyrosine phosphorylation and hyperactivation.
TL;DR: The results from this study show that, according to the variables studied, 20°C is the least harmful of the four temperatures tested for the long-term liquid storage of boar semen.
Abstract: Contents
In this study the effect of long-term storage of liquid boar semen at different temperatures on motility, acrosome integrity and pH was investigated. Additionally, individual variation in sperm tolerance to storage at 10°C were examined. Beltsville Thawing Solution-diluted AI doses from 16 randomly chosen Norwegian Landrace AI boars with proven fertility were split into subsamples and stored at 25, 20, 15 and 10°C, respectively. After 0, 24, 48, 72 and 96 h of storage, sperm motility, acrosome integrity and pH were determined. After 96 h, the initial percentage of motile sperm (77.8%) was significantly reduced to 52.2, 58.8, 50.9 and 42.8% by storage at 25, 20, 15 and 10°C, respectively. After an identical period of time, the percentage of acrosome intact sperm (95.8%) at time 0 became significantly reduced to 91.3, 91.3, 81.5 and 68.3% by storage at 25, 20, 15 and 10°C, respectively. The initial pH (7.21) decreased significantly to 6.96 and 7.06 after 96 h storage at 25 and 20°C, and increased not significantly to 7.25 for storage at 15°C and significantly to 7.29 at 10°C. In conclusion, the results from this study show that, according to the variables studied, 20°C is the least harmful of the four temperatures tested for the long-term liquid storage of boar semen. Furthermore, remarkable differences in the individual resistance of boar semen to long-term storage at 10°C were observed.
TL;DR: Results support the existence of two populations of DE: a major, loosely bound population that is released during capacitation, and a minor strongly Bound population that remains after capacitation and migrates to the ES with the AR, and thus would correspond to the one with a role in gamete fusion.
Abstract: Rat epididymal glycoprotein DE associates with the dorsal region of the sperm head during sperm maturation, migrates to the equatorial segment (ES) with the acrosome reaction (AR), and is involved in gamete membrane fusion. In the present study we examined the association of DE with the sperm surface and the relationship of this interaction with the behavior and function of the protein. Cloning and sequencing of DE revealed a lack of hydrophobic domains and the presence of 16 cysteine residues in the molecule. Experiments in which cauda epididymal sperm were subjected to different extraction procedures indicated that while most of the protein is removable from sperm by mild ionic strength, a low amount of DE, resistant to even 2 M NaCl, can be completely extracted by agents that remove integral proteins. However, the lack of hydrophobic domains in the molecule and the failure of DE to interact with liposomes, does not support a direct insertion of the protein into the lipid bilayer. These results, and the complete extraction of the tightly bound protein by dithiothreitol, suggest that this population would correspond to a peripheral protein bound to a membrane component by strong noncovalent interactions that involve disulfide bonds. While ELISA experiments showed that no protein could be extracted by NaCl from capacitated sperm, indirect immunofluorescence studies revealed the ability of the NaCl-resistant protein to migrate to the ES. Together, these results support the existence of two populations of DE: a major, loosely bound population that is released during capacitation, and a minor strongly bound population that remains after capacitation, migrates to the ES with the AR, and thus would correspond to the one with a role in gamete fusion. Mol. Reprod. Dev. 56:180–188, 2000. © 2000 Wiley-Liss, Inc.
TL;DR: Results strongly suggest an involvement of sperm SGG in direct binding to the ZP in sperm-egg interaction.
Abstract: Sulfogalactosylglycerolipid (SGG) is the major sulfoglycolipid of mammalian male germ cells. Like other sulfoglycolipids, SGG is believed to be involved in cell-cell/extracellular matrix adhesion. Specifically, we investigated whether sperm SGG played a role in sperm-egg interaction. Initially, we produced an affinity-purified, rabbit polyclonal immunoglobulin (Ig) G antibody that specifically recognized SGG (anti-SGG). Indirect immunofluorescence using anti-SGG IgG localized SGG to the convex and concave ridges and the postacrosome of the mouse sperm head. Pretreatment of sperm with anti-SGG IgG/Fab inhibited sperm-zona pellucida (ZP) binding in vitro in a concentration-dependent manner (to a maximum of 62%). This inhibition was observed at the level of primary binding. Sperm treated with anti-SGG IgG underwent the spontaneous and ZP-induced acrosome reaction at the same rate as control sperm treated with preimmune rabbit serum IgG. Fluorescently labeled SGG liposomes were shown to associate specifically with the egg ZP, whereas fluorescently labeled liposomes of galactosylglycerolipid (SGG's parental lipid) and phosphatidylserine (negatively charged like SGG) did not. Furthermore, coincubation of SGG liposomes with sperm and isolated ZP inhibited sperm-ZP binding in a concentration-dependent manner. These results strongly suggest an involvement of sperm SGG in direct binding to the ZP.
TL;DR: Cryopreservation of rhesus sperm as presented here would allow for the cost-effective storage of lineages of nonhuman primates with known genotypes and suggest that either national or international centers could be established as repositories to fill the global needs of sperm for nonhuman primate research.
Abstract: Artificial insemination (AI) and the cryopreservation of sperm with full reproductive capabilities are vital in the armamentarium of infertility clinics and reproductive laboratories. Notwithstanding the fantastic successes with AI and sperm cryopreservation in numerous species, including humans and cattle, these assisted reproductive technologies are less well developed in other species of importance for biomedical research, such as genetically modified mice and nonhuman primates. To that end, AI at high efficiency in the rhesus macaque (Macaca mullata) and the successful cryopreservation of rhesus sperm is presented here, as are the complexities of this primate model due to differences in reproductive tract anatomy and gamete physiology. Cryopreservation had no effect on the ability of sperm to fertilize oocytes in vitro or in vivo. Post-thaw progressive motility was not affected by cryopreservation; however, acrosome integrity was lower for cryopreserved (74.1%) than for fresh sperm (92.7%). Fertilization rates did not differ when fresh (58.1%; n = 32/55) or cryopreserved sperm (63.8%; n = 23/36) were used for in vitro fertilization. Similarly, pregnancy rates did not differ significantly after AI with fresh (57.1%; n = 8/14) or cryopreserved sperm (62.5%; n = 5/8). Seven live rhesus macaques were born following AI with fresh sperm, and three live offspring and two ongoing pregnancies were obtained when cryopreserved sperm were used. Cryopreservation of rhesus sperm as presented here would allow for the cost-effective storage of lineages of nonhuman primates with known genotypes. These results suggest that either national or international centers could be established as repositories to fill the global needs of sperm for nonhuman primate research and to provide the experimental foundation on which to explore and perfect the preservation of sperm from endangered nonhuman primates.
TL;DR: These studies present the first evidence for the participation of PKA in the P4-initiated AR and also suggest that AKAPs are involved in thePKA-mediated events.
Abstract: The signal transduction pathways involved in the progesterone (P(4))-initiated mammalian sperm acrosome reaction (AR) are not fully understood. To investigate the role of the protein kinase A (PKA) pathway in the P(4)-initiated AR, we probed this pathway by pretreating capacitated human sperm with reagents designed to either inhibit PKA activation or disrupt PKA/A kinase anchoring protein (AKAP) interactions. Preincubation with the stearated (membrane permeable) PKA inhibitor, PKI alpha 5-24 (S-PKI alpha 5-24), significantly inhibited the P(4)-initiated AR at 10 microM as compared to stearated control peptide. In contrast, preincubation with 100 microM nonstearated PKI alpha 5-24 did not significantly inhibit versus solvent control. Preincubation with the PKA inhibitor Rp-8-Br-cAMP at 500 microM and 150 microM significantly inhibited the P(4)-initiated AR versus 8-Br-cAMP and versus solvent. Preincubation with the anchoring inhibitory peptide S-Ht-31 significantly stimulated the P(4)-initiated AR at 10, 3, and 1 microM versus inactive control peptide. The stimulation of the P(4)-initiated AR by 3 microM S-Ht31 was significantly inhibited by the addition of 30 microM S-PKI alpha 5-24 prior to the addition of S-Ht31. Preincubation with S-PKI alpha 5-24 (30 microM) partially inhibited the ionomycin (50 microM)-initiated AR. A role for PKA in the P(4)-initiated AR may exist both upstream and downstream of Ca(2+) entry. Our studies present the first evidence for the participation of PKA in the P(4)-initiated AR and also suggest that AKAPs are involved in the PKA-mediated events.
TL;DR: Both the media used to dilute dog sperm and the temperature at which the spermatozoa are handled are important factors to consider when processing spermutozoa for artificial insemination, IVF procedures or preservation.
TL;DR: The finding that higher frequencies of spermatozoa seemed more membrane stable post-thaw, when frozen in Biociphos-Plus®, might indicate that this extender better protects the sperm viability compared with Triladyl®.
Abstract: Contents
Twenty ejaculates from five dairy AI-bulls were used to compare, in a split-sample experiment, the fertility [56 day-non-return-rate (NRR) from more than 14000 AI) and sperm viability post-thaw of semen diluted with an egg yolk- (Triladyl®) or soybean-based (Biociphos-Plus®) commercial extender. The in vitro evaluations were divided in two experiments. Experiment 1 (n = 20) included post-thaw evaluations of motility (subjective and computerized), membrane integrity (CalceinAM/EthD-1, SYBR-14/PI, and osmotic resistance test; ORT), and capacitation status (CTC/EthD-1). Experiment 2 (n = 10) included evaluations of the capacitation-(CTC/EthD-1) and acrosome status (FITC-PSA/EthD-1) during incubation with/without a challenge with solubilized zona pellucida proteins (SZP). No significant difference in the fertility (69.1 ± 0.8 versus 69.2 ± 0.8) results was found between the two extenders. In experiment 1, the computerized motility evaluations post-thaw (CASA) showed higher values for Biociphos-Plus® processed semen for the velocity patterns and lateral sperm head displacement. After 6 h at room temperature (20–22°C) all the CASA motility patterns were significantly higher for Biociphos-Plus®. The proportion of spermatozoa with intact membranes assessed by CalceinAM was significantly higher in Biociphos-Plus® (p < 0.001) compared to Triladyl®, but such difference was not seen when using SYBR-14 or the ORT-assay. When using the CTC/EthD-1 assay, a lower proportion of acrosome reacted (AR) spermatozoa post-thaw (p < 0.01) was found in Biociphos-Plus® processed semen, as well as a tendency (p < 0.07) for a higher number of uncapacitated spermatozoa. In experiment 2, the proportion of uncapacitated spermatozoa was significantly higher for Biociphos-Plus® when semen was incubated (38°C and 5% CO2) without SZP at both 0 (p < 0.001) and 30 min (p < 0.05). Concomitantly, Triladyl® showed a higher percentage of capacitated spermatozoa at 0 (p < 0.01), 30 (p < 0.05) and 120 min (p < 0.05). A higher (p < 0.05) incidence of AR-spermatozoa was seen in Triladyl® at the beginning of the incubation with SZP. No significant difference between extenders was detected for the acrosome status by the FITC-PSA-assay. Incubation with SZP induced acrosome reaction of capacitated spermatozoa in both extenders, which was detected by CTC and FITC-PSA assays. In conclusion, fertility was not affected by Biociphos-Plus® when 15 × 106 of spermatozoa per AI dose were inseminated. The finding that higher frequencies of spermatozoa seemed more membrane stable post-thaw, when frozen in Biociphos-Plus®, might indicate that this extender better protects the sperm viability compared with Triladyl®.
TL;DR: It is suggested that progesterone activates at least two distinct Ca2+ influx pathways, with fast or slow inactivation kinetics, and some sperm show both types of response, and a cyclic nucleotide-mediated process could participate in the progesterOne-induced [Ca2+]i rise.
Abstract: Rises in intracellular Ca2+ concentration ([Ca2+]i) caused by progesterone, an inducer of the acrosome reaction, or by cyclic nucleotides, possible second messengers, were investigated by Ca2+ imaging of the head of individual mouse sperm. Progesterone induced a [Ca2+]i rise in a dose-dependent manner (4–40 μM), primarily in the postacrosomal region. For 20-μM progesterone, Ca2+ responses occurred in 42% of sperm, separated into two types: transient type (60% of responding cells; duration, 1–1.5 min; mean amplitude, 335 nM) and prolonged type (40%; >3 min; 730 nM). Prolonged responses required higher doses of progesterone, and their occurrence was enhanced significantly by preincubation for 2–4 h as compared with transient responses. 8-Bromo-cGMP (0.3–3 mM) induced a [Ca2+]i rise more effectively than did 8-bromo-cAMP. For 1-mM 8-bromo-cGMP, 90% of cells exhibited transient Ca2+ responses (∼1 min; 220 nM), independently of the preincubation time. In Ca2+-free medium, most sperm showed no Ca2+ res...
TL;DR: It can be concluded that Ca2+‐ionophore treatment followed by simultaneous determination PNA‐FITC and EthD‐1 staining can be used to predict the cryopreservability of ejaculates from individual dogs used as donors.
Abstract: The sensitivity of dog sperm cells for extracellular Ca(2+)/Ca(2+)-ionophore challenge was compared to the detrimental effects of an optimized freeze/thawing protocol. Three sperm-rich fractions of ejaculates from 9 dogs were obtained, and one aliquot of each ejaculate was washed in a modified Tyrode's medium (HBT containing 0.1 mM Ca(2+)), without (control sample) and with 2.5 microM Ca(2+)-ionophore (induced sample) and incubated for 60 min at 38 degrees C in humidified atmosphere. Another aliquot from the same semen fractions was diluted, washed in a Tris buffer, and packed into 0.5-ml straws with a Tris buffer containing 7.5 vol % glycerol. The samples were stored for 1 week in liquid nitrogen after a computer-driven three-step freeze protocol and subsequently thawed for 50 sec in a 37 degrees C water bath and reconstituted into HBT. The acrosome integrity was determined using fluorescein-conjugated peanut agglutinin (PNA-FITC) as an acrosomal marker, while the vitality of the sperm cells was simultaneously assessed with the membrane impermeable DNA supravital stain ethidium homodimer 1 (EthD-1) using fluorescence microscopy and flow cytometry. The motility of frozen/thawed sperm samples was evaluated by microscopic as well as computerized motility analyses. Remarkably, the percentage sperm cells that underwent acrosome reactions induced by Ca(2+)-ionophore correlated very positively (r = 0.93) with the amount of acrosome damage observed in cryopreserved sperm samples. Furthermore, the degree of cellular damage induced by Ca(2+)-ionophore treatment correlated very negatively (r = -0.99) with the relative amount of sperm cells that remained motile after cryopreservation. Such clear correlations between Ca(2+)-ionophore induced acrosome reaction and motility parameters for frozen/thawed dog sperm cells were not found, suggesting that the generation of acrosome leakage and sperm immotility are two independent detrimental processes occurring during cryopreservation. From these results it can be concluded that Ca(2+)-ionophore treatment followed by simultaneous determination PNA-FITC and EthD-1 staining can be used to predict the cryopreservability of ejaculates from individual dogs used as donors.
TL;DR: Data suggest that syntaxin 2 may be involved in regulating the acrosomal reaction in rodent spermatozoa, which has some features in common with regulated exocytosis.
Abstract: The acrosome reaction includes a membrane fusion event that is a prerequisite for sperm penetration through the zona pellucida and subsequent fertilization Since SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins have been shown to be key players in membrane fusion during regulated exocytosis in nerve terminals and secretory cells, and since the acrosome reaction has some features in common with regulated exocytosis, we hypothesized that SNARE proteins might also regulate acrosomal exocytosis RT-PCR analysis demonstrated the expression of SNARE proteins, three isoforms of syntaxin 2 (2A, 2B, and 2C) and syntaxin 4A, in rat testes Immunoblot analysis with anti-syntaxin 2 antibody showed that the protein was expressed in rodent spermatozoa, and that it was associated with membrane components of spermatozoa prepared by sucrose density gradient centrifugation Confocal laser scanning microscopy with double immunolabeling revealed that syntaxin 2 was colocalized with acrin 1, a 90 kDa acrosomal protein, over the acrosomal region of spermatozoa but was not associated with the posterior half of head or tail Localization of syntaxin 2 over the acrosomal region was supported by the finding that it was shed from sperm heads during an acrosome reaction induced by calcium ionophore A23187 in vitro In view of the putative role of syntaxin proteins in other membrane fusion systems, these data suggest that syntaxin 2 may be involved in regulating the acrosomal reaction in rodent spermatozoa
TL;DR: Evidence is provided for a novel mechanism of voltage‐gated Ca2+ channel regulation in mammalian spermatogenic cells by two agents that affect sperm capacitation and the acrosome reaction (AR).
TL;DR: It is hypothesized that paternal proteasomes enter the oocyte during fertilization in tight association with the centrioles and may serve a special function during further development which can be associated with the function of a hypothetical proteolysis centre.
Abstract: In the present study we describe the localization of proteasomes in human spermatozoa by means of immunolabelling with different monoclonal and polyclonal antibodies detected by confocal microscopy. Western blotting confirmed the specificity of the antibodies and has shown that proteasomes are present in spermatozoa and in seminal fluid. In spermatozoa proteasomes are concentrated in the neck region where the centrioles are located. Some labelling was also detected at the periphery of the head, but no proteasomal antigens were detected in either the nucleus or associated with the flagellum. Proteasome inhibitors did not affect the motility of the spermatozoa, acrosome reaction nor zona binding. It is hypothesized that paternal proteasomes enter the oocyte during fertilization in tight association with the centrioles and may serve a special function during further development which can be associated with the function of a hypothetical proteolysis centre.
TL;DR: The present study suggests that the AR of human spermatozoa is highly associated with T-type Ca(2+) channels and is mainly mediated by calcium influx through alpha1H T- type Ca( 2+) channels.
Abstract: of nifedipine were required to block AR (IC50 60 µmol/l). Reverse transcription–polymerase chain reaction (RT–PCR) was performed to identify the sub-types of T-type channels present in human testes. Analysis of PCR products showed that only α1H subunits are expressed in testes. The expression of the α1H subunit may be tissue specific since its mRNA was not detected in the human ovary. The present study suggests that the AR of human spermatozoa is highly associated with T-type Ca 2 channels and is mainly mediated by calcium influx through α1H T-type Ca 2 channels.