Is the hook of muroid rodent's sperm related to sperm train formation?

Competition between spermatozoa of rival males to gain fertilizations has led to a wide array of modifications in sperm structure and function. Sperm cells of most muroid rodents have hook-shaped extensions in the apical-ventral tip of the head, but the function of this structure is largely unknown. These 'hooks' may facilitate aggregation of spermatozoa in so-called 'trains', as an adaptation to sperm competition, because sperm in trains may swim faster than free-swimming cells. However, there is controversy regarding the role of the hook in train formation, and in relation to whether it is selected by sperm competition. We examined spermatozoa from muroid rodents with varying levels of sperm competition to assess whether (i) sperm aggregates are common in these taxa, (ii) presence of a hook relates to the formation of sperm aggregations, and (iii) formation of sperm aggregations is explained by sperm competition. Our analyses in 25 muroid species revealed that > 92% of spermatozoa swim individually in all species, with the exception of the wood mouse, Apodemus sylvaticus, which has ~50% spermatozoa swimming freely. Species with hooked spermatozoa had higher sperm competition levels and longer sperm than species whose sperm lack a hook. Neither the presence of hook nor sperm competition levels were related to the percentage of sperm in aggregations. Thus, (i) sperm aggregates in muroid rodents are an exceptional trait found only in a few species, (ii) evolution of the sperm hook is associated to sperm competition levels, but (iii) the hook is unlikely to be related to the formation of sperm aggregates. The evolutionary significance of the sperm head hook thus remains elusive, and future studies should examine potential roles of this pervasive structure in sperm's hydrodynamic efficiency and sperm-female tract interactions.

Sperm head phenotype and male fertility in ram semen.

Although there is ample evidence for the effects of sperm head shape on sperm function, its impact on fertility has not been explored in detail at the intraspecific level in mammals. Here, we assess the relationship between sperm head shape and male fertility in a large-scale study in Manchega sheep (Ovis aries), which have not undergone any selection for fertility. Semen was collected from 83 mature rams, and before insemination, head shapes were measured for five parameters: area, perimeter, length, width, and p2a (perimeter(2)/2×π×area) using a computer-assisted sperm morphometric analysis. In addition, a cluster analysis using sperm head length and p2a factor was performed to determine sperm subpopulations (SPs) structure. Our results show the existence of four sperm SPs, which present different sperm head phenotype: SP1 (large and round), SP2 (short and elongated), SP3 (shortest and round), and SP4 (large and the most elongated). No relationships were found between males' fertility rates and average values of sperm head dimensions. However, differences in fertility rates between rams were strongly associated to the proportion of spermatozoa in an ejaculate SP with short and elongated heads (P < 0.001). These findings show how the heterogeneity in sperm head shape of the ejaculate has an effect on reproductive success, and highlight the important role of modulation of the ejaculate at the intraspecific level.

No evidence of trade-offs in the evolution of sperm numbers and sperm size in mammals.

Post-copulatory sexual selection, in the form sperm competition, has influenced the evolution of several male reproductive traits. However, theory predicts that sperm competition would lead to trade-offs between numbers and size of spermatozoa because increased costs per cell would result in a reduction of sperm number if both traits share the same energetic budget. Theoretical models have proposed that, in large animals, increased sperm size would have minimal fitness advantage compared with increased sperm numbers. Thus, sperm numbers would evolve more rapidly than sperm size under sperm competition pressure. We tested in mammals whether sperm competition maximizes sperm numbers and size, and whether there is a trade-off between these traits. Our results showed that sperm competition maximizes sperm numbers in eutherian and metatherian mammals. There was no evidence of a trade-off between sperm numbers and sperm size in any of the two mammalian clades as we did not observe any significant relationship between sperm numbers and sperm size once the effect of sperm competition was taken into account. Maximization of both numbers and size in mammals may occur because each trait is crucial at different stages in sperm's life; for example size-determined sperm velocity is a key determinant of fertilization success. In addition, numbers and size may also be influenced by diverse energetic budgets required at different stages of sperm formation.

Effects of metabolic rate and sperm competition on the fatty-acid composition of mammalian sperm.

The sperm membrane is a key structure affecting sperm function and thus reproductive success. Spermatozoa are highly specialized and differentiated cells that undergo a long series of processes in the male and female reproductive tracts until they reach the site of fertilization. During this transit, the sperm membrane is prone to damage such as lipid peroxidation. The characteristics and performance of the sperm membrane are strongly determined by the fatty-acid composition of membrane phospholipids. Polyunsaturated fatty-acids (PUFAs) are the most prone to lipid peroxidation. Lipid peroxidation and other types of oxidative damage increase with higher metabolism and with higher levels of sperm competition due to the increased ATP production to fuel higher sperm velocities. Consequently, we hypothesized that, in order to avoid oxidative damage, and the ensuing impairment of sperm function, sperm cells exhibit a negative relationship between PUFA content and mass-specific metabolic rate (MSMR). We also hypothesized that higher sperm competition leads to a reduction in the proportion of sperm PUFAs. We performed a comparative study in mammals and found that high MSMR and high levels of sperm competition both promote a decrease in the proportion of PUFAs that are more prone to lipid peroxidation. The negative relationship between MSMR and these PUFAs in sperm cells is surprising, because a positive relationship is found in all other cell types so far investigated. Our results support the idea that the effects of MSMR and sperm competition on sperm function can operate at very different levels.

Morphometrically-distinct sperm subpopulations defined by a multistep statistical procedure in ram ejaculates: intra- and interindividual variation.

The existence of sperm subpopulations within the mammalian ejaculate has now been widely recognized. However, to the best of our knowledge, no data exist regarding the existence of sperm morphometric subpopulations within the ovine ejaculate. Computer assisted sperm morphometry analysis (ASMA) data and clustering methods were used in this study to identify sperm-head subpopulations in ram semen. Two experiments were carried out. In Experiment 1, ejaculates from 226 mature rams of the Manchega breed belonging to 36 different herds were used. A minimum of 100 sperm heads were analyzed from each male and eight morphometric characteristics for each individual sperm were recorded. Subpopulation analysis was performed in sequential steps: variable group analysis and correlation analysis to select which morphometric characteristics to use in cluster analyses; nonhierarchical clustering analysis using sperm head length and p2a (also known as roundness) shape factor as initial classificatory variables; and hierarchical clustering analysis to obtain the final number of clusters. The clustering analyses, based on 26,306 individual cells, revealed the existence of four sperm subpopulations (SP1, SP2, SP3 and SP4) with different morphometric characteristics. Significant differences in the proportion of spermatozoa in the SP1 and SP3 were found between rams belonging to different herds. In Experiment 2, the intra- and intermale variability on the distribution of sperm subpopulations was assessed. Three ejaculates from each of 21 rams were collected and the same multistep clustering analysis was performed. For all subpopulations defined, the intermale variability resulted in high values, being the intramale variability much lower. This fact would allow the use of sperm head morphometry to characterize a male and might provide valuable information to asses its fertility. In conclusion, our results show that using computer assisted sperm morphometry analysis and multivariate cluster analyses, four sperm subpopulations with different head phenotype were identified in ram ejaculates.

Effect of storage of domestic cat (Felis catus) epididymides at 5 degrees C on sperm quality and cryopreservation.

Postmortem sperm recovery from the epididymides may constitute a powerful tool for the conservation of valuable genetic material. The domestic cat (Felis catus) is a good model for wild felids and, using this model, we have explored the effect of epididymides storage time on sperm motility and percentage of intact acrosomes upon sperm recovery and after cryopreservation. We also examined the effect of time of sperm equilibration with glycerol before freezing on sperm motility and the percentage of intact acrosomes. Motility varied between sperm recovered from epididymides that were stored for different times. Significant differences were seen in the sperm motility index (SMI) before freezing (55.91+/-2.02, 48.21+/-1.47, and 43.03+/-1.32) and after thawing (51.81+/-3.02, 41.90+/-2.14, and 42.35+/-1.95) of sperm recovered from epididymides stored for 0, 48, or 72 h, respectively. The percentage of intact acrosomes did not vary significantly with storage time (average 60.33+/-1.38% before and 52.50+/-1.91% after freezing, respectively). The percentage of normal sperm after different storage times did not differ (average 19.22+/-1.25% normal sperm after recovery). When epididymides were stored for 72 h, time of sperm equilibration with glycerol (30 vs. 120 min) resulted in significant differences in both motility (SMI=39.17+/-2.76 and 45.00+/-2.65, respectively) and the percentage of intact acrosomes (45.76+/-4.91% and 60.67+/-3.64%, respectively) after thawing. In conclusion, best results are achieved when sperm are recovered from epididymides within 24h of cool storage and when they are equilibrated with glycerol during 120 min before freezing. The current results should be useful in the further development of techniques for the rescue and cryostorage of epididymal spermatozoa of endangered felids.

Male reproductive traits, semen cryopreservation, and heterologous in vitro fertilization in the bobcat (Lynx rufus).

There is limited information on bobcat ejaculate traits and sperm cryopreservation and fertilizing ability. Bobcats were electroejaculated under general anesthesia in November (autumn) and April (spring), and endocrine and sperm traits were characterized. Testosterone (mean+/-SEM: 0.90+/-0.15 ng/mL) was not different between sampling times, but cortisol (average: 13.95+/-1.73 microg/dL) was significantly higher in April. Average number of spermatozoa was 10.0+/-3.4 x 10(6) sperm/ejaculate, with values being significantly higher in April. Sperm motility (average 55.7+/-5.8% motile sperm) was not different between sampling times. The proportion of normal spermatozoa in the ejaculate (average: 14.7+/-2.1%) was significantly higher in April, but the percentage of spermatozoa with intact acrosomes (average: 43.7+/-3.8%) was significantly higher in autumn. Spermatozoa were cryopreserved in a Tes-Tris-based diluent (TEST) or Biladyl, both containing 20% egg yolk and 4% glycerol. Diluted sperm were loaded into straws, refrigerated using a programmable thermoblock with a dry chamber, frozen in nitrogen vapors, thawed, and incubated in F-10 medium with 5% fetal bovine serum for up to 3h. After cryopreservation in TEST, there were about 50% motile sperm upon thawing, and survival was high during incubation post-thaw. Cryopreservation in Biladyl led to similar results, but motility decreased substantially during incubation post-thaw. Bobcat spermatozoa fertilized domestic cat oocytes matured in vitro. Fertilization rates were higher for sperm collected in April and cryopreserved in TEST (46%) than for those cryopreserved using Biladyl (<3%). Fertilized oocytes cleaved in culture, and some (27%) reached the morula stage. This study has allowed us to gain further baseline information on bobcat reproduction, explore sperm cryopreservation conditions, and show that fertilizing capacity can be tested using in vitro-matured cat oocytes. These results will be important for future conservation efforts.

What does testosterone do for red deer males?

Testosterone has been proposed to have a dual effect, enhancing sexual traits while depressing parasite resistance in males. Here, we test this hypothesis in red deer, examining males from captive populations during the whole annual cycle and males from natural populations during the breeding season. We first explored the effects of body size, age and sampling date on testosterone to avoid confounding effects. Our results show that in captive populations seasonal changes in testosterone levels were mirrored by changes in testes size, and that during the rut there was a strong correlation between both. In natural populations, males with higher testosterone levels had larger testes, improved sperm quality, smaller burr diameter, stronger antlers, higher haematocrit levels, and increased nematode parasite load. By contrast, no significant relationship was found between testosterone and spleen size or tick parasite load. We conclude that testosterone (i) improves males' reproductive investment and physical stamina, (ii) improves antler strength but reduces burr diameter, and (iii) imposes a cost in terms of depressed parasite resistance.

Use of a neuroleptic in assisted reproduction of the critically endangered Mohor gazelle (Gazella dama mhorr).

Stress is a limiting factor in assisted reproduction in wild animals maintained in captivity and measures to reduce it should improve reproductive success. The effect of the long-acting neuroleptic (LAN) perphenazine enanthate was assessed on ovarian stimulation for the recovery of immature oocytes from Mohor gazelle (Gazella dama mhorr) and their subsequent in vitro maturation, fertilization and embryo culture. The viability of embryos after transfer was also examined. Perphenazine enanthate decreased activity levels and facilitated handling of treated animals when compared to controls. LAN-treated animals showed a more regular pattern of respiratory and heart rates and body temperature than controls; no major differences were found in hematological and biochemical parameters between groups. Perphenazine-treated females had lower plasma cortisol levels during the days of intense handling. No significant differences were found in the number of punctured follicles and recovered oocytes between groups. The percentage of mature oocytes per female was significantly higher in the LAN-group. Fertilization and cleavage rates were not significantly different between groups. Embryos developed in culture but none reached the blastocyst stage, and those transferred to the oviduct of synchronized recipients did not develop to term. In conclusion, treatment of females with perphenazine enanthate during ovarian stimulation did not have negative effects on maturation, fertilization and embryo development in vitro. Moreover, an increase in oocyte maturation rate per female was observed. Thus, the use of LANs could be useful to alleviate the effects of handling-stress during assisted reproductive procedures in wild ungulates.

Superovulation and in vitro oocyte maturation in three species of mice (Mus musculus, Mus spretus and Mus spicilegus).

Mouse oocytes can be obtained via superovulation or using in vitro maturation although several factors, including genetic background, may affect response. Our previous studies have identified various mouse species as models to understand the role of sexual selection on the evolution of sperm traits and function. In order to do comparative studies of sperm-oocyte interaction, we sought reliable methods for oocyte superovulation and in vitro maturation in mature females of three mouse species (genus Mus). When 5 IU pregnant mare's serum gonadotrophin (PMSG) and 5 IU human chorionic gonadotrophin (hCG) were injected 48 h apart, and oocytes collected 14 h post-hCG, good responses were obtained in Mus musculus (18+/-1.3 oocytes/female; mean+/-S.E.M.) and Mus spretus (12+/-0.8), but no ovulation was seen in Mus spicilegus. Changes in PMSG or hCG doses, or longer post-hCG intervals, did not improve results. Use of PMSG/luteinizing hormone (LH) resulted in good responses in M. musculus (19+/-1.2) and M. spretus (12+/-1.1) but not in M. spicilegus (5+/-0.9) with ovulation not increasing with higher LH doses. Follicular puncture 48 h after PMSG followed by in vitro maturation led to a high oocyte yield in the three species (M. musculus, 23+/-0.9; M. spretus, 17+/-1.1; M. spicilegus, 10+/-0.9) with a consistently high maturation rates. In vitro fertilization of both superovulated and in vitro matured oocytes resulted in a high proportion of fertilization (range: 83-87%) in the three species. Thus, in vitro maturation led to high yields in all three species. These results will allow future studies on gamete interaction in these closely related species and the role of sexual selection in gamete compatibility.

Effect of egg yolk, cryoprotectant, and various sugars on semen cryopreservation in endangered Cuvier's gazelle (Gazella cuvieri).

Cryopreservation of spermatozoa from endangered species is a valuable tool for genetic management. Previous studies showed the feasibility of cryopreservation of spermatozoa from various endangered gazelles but have also revealed difficulties with available protocols for semen freezing in Cuvier's gazelle (Gazella cuvieri). Experiments were carried out to investigate the effect of (a) 5% or 20% egg yolk or 4% or 6% glycerol, and (b) addition of sugars (glucose, fructose, lactose and raffinose) on cryopreservation using a Tes-Tris-based diluent (TEST). A diluent containing 13.5% raffinose, 5% or 20% egg yolk, and 6% glycerol (REYG) was also evaluated. Semen was obtained by electroejaculation from 22 G. cuvieri males. Diluted samples were loaded into 0.25 ml straws, cooled to 5 degrees C over 1.5h (-0.16 degrees C/min), equilibrated at that temperature for 2h, frozen in nitrogen vapours for 10 min and plunged into liquid nitrogen. Subsamples were assessed for motility and acrosome integrity upon collection, after refrigeration-equilibration, after freezing and thawing, and 2h after thawing. Use of TEST with 20% egg yolk or with 4% glycerol led to worse motility preservation, whereas TEST with 5% egg yolk and 6% glycerol led to better results. Addition of fructose, lactose or raffinose to TEST resulted in similar or worse preservation of motility than inclusion of glucose. On the other hand, use of a raffinose-based medium with 20% egg yolk and 6% glycerol (REYG) afforded better preservation of motility than use of TEST. With REYG, 20% egg yolk was better than 5% egg yolk for motility preservation. Differences were noted between males in their responses to cryopreservation when using different egg yolk or glycerol concentrations. Moreover, spermatozoa from most males exhibited better cryopreservation with REYG although some were better cryopreserved in TEST. The raffinose-based diluent thus represents an improvement over previous results but more work is needed to better characterize cryopreservation conditions for future routine banking of Cuvier's gazelle spermatozoa.

In vitro oocyte maturation, fertilization and culture after ovum pick-up in an endangered gazelle (Gazella dama mhorr).

The recovery of immature oocytes followed by in vitro maturation, fertilization and culture (IVMFC) allows the rescue of biological material of great genetic value for the establishment of genetic resource banks of endangered species. Studies exist on sperm cryopreservation of endangered Mohor gazelle (Gazella dama mhorr), but no work has been carried out yet on oocyte collection, fertilization and culture in this or related species. The purpose of this study was to develop a protocol for ovarian stimulation for the recovery of oocytes and subsequent IVMFC in the Mohor gazelle using frozen-thawed spermatozoa. Ovum pick-up was performed after ovarian stimulation with a total dose of 5.28 mg of ovine FSH. A total of 35 oocytes were recovered from 56 punctured follicles (62%) (N=6 females). Out of 29 cumulus-oocyte complexes matured in vitro, 3% were found at germinal vesicle stage, 7% at metaphase I, 21% were degenerated, and 69% advanced to metaphase II. Fertilization and cleavage rates of matured oocytes were 40 and 30%, respectively. Embryos cleaved in vitro up to the 6-8 cell stage but none progressed to the blastocyst stage, suggesting the existence of a developmental block and the need to improve culture conditions. Although more studies are needed to improve hormonal stimulation and oocyte harvesting, as well as IVMFC conditions, this study demonstrates for the first time the feasibility of in vitro fertilization with frozen-thawed semen of in vitro matured oocytes collected by ovum pick-up from FSH-stimulated endangered gazelles.

Sperm phospholipases and acrosomal exocytosis.

At the time of fertilization, the sperm cell undergoes regulated exocytosis in response to the oocyte-associated agonists progesterone and zona pellucida. An early response generated by agonist-receptor interaction in spermatozoa is the activation of mechanisms leading to Ca2+ influx, this ion being essential for the activation of phospholipases and for the fusion of the plasma membrane with the outer acrosomal membrane. Both a phosphoinositide-specific, and a phosphatidylcholine-specific phospholipase C are involved in the generation of a variety of diacylglycerol molecular species. Phospholipase D, on the other hand, does not seem to play a significant role in the generation of diacylglycerol. Hydrolysis of phospholipids by phospholipase A2 generates free fatty acids and lysophospholipids; these are important either as substrates for the generation of other metabolites (e.g., eicosanoids) or having a direct, essential action in the final stages of membrane fusion. There is still much work to be done in the future in order to characterize phospholipase isozymes and their regulation during acrosomal exocytosis in spermatozoa.

Inbreeding and reproduction in endangered ungulates: preservation of genetic variation through the Organization of Genetic Resource Banks.

There is a constant increase in the number of species suffering marked reductions in population size. This reduction in size and the lack of genetic flow may lead to a decrease in genetic variability and to matings between close relatives (i.e. inbreeding) with an ensuing reduction in fitness. It is thus important to understand the mechanism underlying the deleterious effects of inbreeding and to develop reproductive biotechnologies that will allow the reduction of inbreeding depression by facilitating gene exchange between populations. The study of three endangered species of gazelles, Cuvier's gazelle (Gazella cuvieri), Mohor gazelle (Gazella dama mhorr) and dorcas gazelle (Gazella dorcas neglecta) has revealed that inbreeding negatively affects several semen parameters (motility, sperm morphology, acrosome integrity). Semen cryopreservation has been achieved in the three species but success varies depending on the diluent employed and the level of inbreeding. Artificial insemination of Mohor gazelles have led to the birth of the first gazelle born using frozen-thawed semen but improvements are needed before this technology can be applied on a routine basis for the genetic management of the populations. Collection of oocytes after ovarian stimulation, followed by in vitro maturation, fertilization and culture has met with some initial success in the Mohor gazelle. These, together with other reproductive technologies, will offer an invaluable help in preserving the maximum of genetic diversity of these and related endangered ungulate species.

The application of reproductive technologies to natural populations of red deer.

Over the past decade, there has been increasing interest in the application of reproductive technology to the conservation and management of natural populations of deer. The application of assisted reproduction technologies within natural population of deer is in its infancy. However, its future potential is enormous, particularly in relation to genetic management or conservation. This paper reviews the present state of such technologies for a wild subspecies of red deer, the Iberian red deer (Cervus elaphus hispanicus), by discussing the major components of oestrous synchronization, semen collection/cryopreservation and insemination techniques. In addition, findings made during the course of studies on natural populations have enormous potential for the understanding of novel reproductive mechanism that may not be uncovered by livestock or human studies. A summary of these results are also reviewed here.

Progesterone primes zona pellucida-induced activation of phospholipase A2 during acrosomal exocytosis in guinea pig spermatozoa.

We investigated, using guinea-pig spermatozoa as a model, whether phospholipase A2 (PLA2) is involved in progesterone or zona pellucida (ZP)-stimulated acrosomal exocytosis, if progesterone enhances ZP-induced activation of PLA2, and mechanisms underlying PLA2 regulation. Spermatozoa were capacitated and labeled in low Ca2+ medium with [14C]choline chloride or [14C]arachidonic acid, washed, and then exposed to millimolar Ca2+ and progesterone and/or ZP. Each agonist stimulated decrease of phosphatidylcholine (PC) and release of arachidonic acid and lysoPC, indicative of PLA2 activation. Aristolochic acid (a PLA2 inhibitor) abrogated lipid changes and exocytosis, indicating that these lipid changes are essential for exocytosis. Exposure of spermatozoa to submaximal concentrations of both progesterone and ZP resulted in a synergistic increase of arachidonic acid and lysoPC releases, and exocytosis, suggesting that, under natural conditions, both agonists interact to bring about acrosomal exocytosis. Progesterone-induced PLA2 activation appears to be mediated by a GABA(A)-like receptor, because bicuculline (a GABA(A) receptor antagonist) blocked arachidonic acid release and exocytosis. In agreement with this, GABA mimicked progesterone actions. ZP-induced activation of PLA2 seemed to be transduced via G(i) proteins because pertussis toxin blocked arachidonic acid release and acrosomal exocytosis. PLA2 may be regulated by PKC because progesterone- or ZP-induced release of arachidonic acid was blocked by the PKC inhibitors staurosporine or chelerythrine chloride. PLA2 could also be regulated by the cAMP-PKA pathway; inclusion of the PKA inhibitor 14-22 amide or H-89 led to a reduction in arachidonic acid release or exocytosis after progesterone or ZP. Taken together, these results suggest that PLA2 plays an essential role in progesterone or ZP-stimulated exocytosis with progesterone priming ZP action.

Sperm cryopreservation in three species of endangered gazelles (Gazella cuvieri, G. dama mhorr, and G. dorcas neglecta).

Long-term storage of semen by cryopreservation, with high recovery rates on thawing, is essential for the establishment of genetic resource banks of endangered species. The purpose of the present study was to evaluate various diluents for the cryopreservation of spermatozoa from three species of gazelles (genus Gazella) in a captive breeding program. The diluents compared were Tes (N-tris(hydroxymethyl)methyl-2 aminoethane sulfonic acid)-Tris with 5% egg yolk and 6% glycerol (TEST) and Triladyl, yolk-citrate, Tris-trehalose, and Tris-lactose-all of them with 20% egg yolk and 6% (Triladyl) or 8% glycerol. Semen was obtained by electroejaculation from 12 G. cuvieri, 12 G. dama, and 13 G. dorcas males. Samples with less than 50% motile sperm, positive endosmosis, or acrosome integrity were not used. Diluted samples were loaded into 0.25-ml straws, cooled slowly to 5 degrees C over 1.5 h (-0.16 degrees C/min), equilibrated at that temperature for 2 h, frozen in nitrogen vapors for 10 min, and plunged into liquid nitrogen. Subsamples were assessed fresh, after refrigeration-equilibration, after freezing and thawing, and 2 h after thawing. Differences were seen between diluents, with best overall recovery rates after freezing and thawing found with Triladyl, TEST, and Tris-trehalose in G. cuvieri, TEST in G. dama, and Triladyl and TEST in G. dorcas. Differences were observed between species in the ability to withstand freezing and thawing, with best results seen in G. dorcas, intermediate results in G. dama, and worst results in G. cuvieri. These differences were inversely related to the average values of inbreeding of these populations. The underlying mechanism responsible for these differences may be a differential resistance to osmotic shock.

Zona pellucida induces activation of phospholipase A2 during acrosomal exocytosis in guinea pig spermatozoa.

Phospholipase A(2) (PLA(2)) is activated in spermatozoa in response to progesterone and Ca(2+) ionophores, but to our knowledge, no study has yet reported zona pellucida (ZP)-induced activation of PLA(2). We investigated whether PLA(2) is involved in ZP-stimulated acrosomal exocytosis, if Ca(2+) is required for activation of PLA(2), and signal transduction pathways modulating PLA(2) using guinea pig sperm as a model. Spermatozoa were capacitated and labeled in low-Ca(2+) medium with [(14)C]choline chloride or [(14)C]arachidonic acid and were then exposed to millimolar Ca(2+) and various reagents and stimulated with ZP. Precapacitated spermatozoa exposed to millimolar Ca(2+) and stimulated with ZP experienced increases in arachidonic acid (AA) and lysophosphatidylcholine (lysoPC) levels and a parallel decrease in phosphatidylcholine level; these changes are indicative of PLA(2) activation. Simulation with ZP also led to acrosomal exocytosis in a high proportion of spermatozoa. Lipid changes and exocytosis were prevented if spermatozoa were exposed to aristolochic acid, a PLA(2) inhibitor, before treatment with ZP. Stimulation with ZP in medium without added Ca(2+) or in medium with millimolar Ca(2+) and EGTA or La(3+) resulted in no lipid changes or exocytosis. Pretreatment with pertussis toxin, a G(i) protein inhibitor, before stimulation with ZP blocked the release of AA and lysoPC as well as acrosomal exocytosis. Exposure of spermatozoa to the diacylglycerol (DAG) kinase inhibitor R59022 before ZP stimulation led to a significant increase in generation of lysoPC and exocytosis. Taken together, these results indicate very strongly that PLA(2) plays an essential role in ZP-induced exocytosis in spermatozoa, that PLA(2) activation requires Ca(2+) internalization, and that PLA(2) activation is regulated by signal transduction pathways involving G proteins and DAG.