Effect of progesterone on bovine sperm capacitation and acrosome reaction

Progesterone (P) appears to stimulate sperm capacitation and/or induce the acrosome reaction (AR) in some species. In bovine, it is now well established that the BSP-A1/-A2 proteins (the major proteins of bovine seminal plasma) promote sperm capacitation. In this study, we investigated the effect of P on bovine sperm cholesterol efflux, capacitation, and the AR. Labeled bovine epididymal sperm were incubated (0-6 h) with different concentrations of P (0.01-10 microg/ml) in the presence or absence of BSP-A1/-A2 proteins (capacitating conditions). At different time intervals, aliquots of sperm were taken to determine the sperm cholesterol efflux, sperm capacitation (AR induced by lysophosphatidylcholine, lyso-PC), and sperm AR. The results show that the presence of P in the media did not affect the membrane cholesterol efflux potential of the BSP-A1/-A2 proteins. P alone did not stimulate the AR with or without lyso-PC unless the epididymal sperm were incubated in capacitating conditions (in the presence of BSP-A1/-A2). When washed ejaculated sperm were continuously incubated with P, the P did not stimulate AR. However, when ejaculated sperm were preincubated (6 h) with heparin (capacitation medium) and then incubated 15 min with P (2 microg/ml), the percentage of AR obtained was similar to that obtained with lyso-PC. The effect of P on sperm AR was concentration dependent with a maximum 2.2-fold increase at 2 microg/ml of P. These results demonstrate a potential role of P in bovine sperm AR but not in capacitation.     

Reactive oxygen species as mediators of sperm capacitation and pathological damage

Oxidative stress plays a major role in the life and death of mammalian spermatozoa. These gametes are professional generators of reactive oxygen species (ROS), which appear to derive from three potential sources: sperm mitochondria, cytosolic L‐amino acid oxidases, and plasma membrane Nicotinamide adenine dinucleotide phosphate oxidases. The oxidative stress created via these sources appears to play a significant role in driving the physiological changes associated with sperm capacitation through the stimulation of a cyclic adenosine monophosphate/Protein kinase A phosphorylation cascade, including the activation of Extracellular signal regulated kinase‐like proteins, massive up‐regulation of tyrosine phosphorylation in the sperm tail, as well as the induction of sterol oxidation. When generated in excess, however, ROS can induce lipid peroxidation that, in turn, disrupts membrane characteristics that are critical for the maintenance of sperm function, including the capacity to fertilize an egg. Furthermore, the lipid aldehydes generated as a consequence of lipid peroxidation bind to proteins in the mitochondrial electron transport chain, triggering yet more ROS generation in a self‐perpetuating cycle. The high levels of oxidative stress created as a result of this process ultimately damage the DNA in the sperm nucleus; indeed, DNA damage in the male germ line appears to be predominantly induced oxidatively, reflecting the vulnerability of these cells to such stress. Extensive evaluation of antioxidants that protect the spermatozoa against oxidative stress while permitting the normal reduction‐oxidation regulation of sperm capacitation is therefore currently being undertaken, and has already proven efficacious in animal models.     

Signaling pathways in sperm capacitation and acrosome reaction.

The binding to the egg's zona pellucida stimulates the spermatozoon to undergo acrosome reaction, a process which enables the sperm to penetrate the egg. Prior to this binding, the spermatozoa underago in the female reproductive tract a series of biochemical transformations, collectively called capacitation. The first event in capacitation is cholesterol efflux leading to the elevation of intracellular calcium and bicarbonate to activate adenylyl cyclase (AC) to produce cyclic-AMP, which activates protein kinase A (PKA) to indirectly phosphorylate certain proteins on tyrosine. During capacitation, there is also an increase in protein tyrosine phosphorylation dependent actin polymerization and in the membrane-bound phospholipase C (PLC). Sperm binding to zona-pellucida causes further activation of cAMP/PKA and protein kinase C (PKC), respectively. PKC opens a calcium channel in the plasma membrane. PKA together with inositol-trisphosphate activate calcium channels in the outer acrosomal membrane, which leads to an increase in cytosolic calcium. The depletion of calcium in the acrosome will activate a store-operated calcium entry mechanism in the plasma membrane, leading to a higher increase in cytosolic calcium, resulting in F-actin dispersion which enable the outer acrosomal and the plasma membrane to come into contact and fuse completing the acrosomal reaction.     

Role of hydrogen peroxide in sperm capacitation and acrosome reaction

The generation of reactive oxygen species (ROS) has been implicated in the regulation of sperm capacitation and acrosome reaction; however, the mechanisms underlying this regulation remain unclear. To examine the cellular processes involved, we studied the effect of different concentrations of hydrogen peroxide (H(2)O(2)) on protein tyrosine phosphorylation under various conditions. Treatment of spermatozoa with H(2)O(2) in medium without heparin caused a time- and dose-dependent increase in protein tyrosine phosphorylation of at least six proteins in which maximal effect was seen after 2 h of incubation with 50 microM H(2)O(2). At much higher concentrations of H(2)O(2) (0.5 mM), there is significant reduction in the phosphorylation level, and no protein tyrosine phosphorylation is observed at 5 mM H(2)O(2) after 4 h of incubation. Exogenous NADPH enhanced protein tyrosine phosphorylation similarly to H(2)O(2). These two agents, but not heparin, induced Ca(2+)-dependent tyrosine phosphorylation of an 80-kDa protein. Treatment with H(2)O(2) (50 microM) caused approximately a twofold increase in cAMP, which is comparable to the effect of bicarbonate, a known activator of soluble adenylyl cyclase in sperm. This report suggests that relatively low concentrations of H(2)O(2) are beneficial for sperm capacitation, but that too high a concentration inhibits this process. We also conclude that H(2)O(2) activates adenylyl cyclase to produce cAMP, leading to protein kinase A-dependent protein tyrosine phosphorylation.     

Reactive oxygen species modulate independent protein phosphorylation pathways during human sperm capacitation

Spermatozoa must undergo capacitation to acquire fertilizing ability. Reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide H2O2, and nitric oxide (NO*), are involved in this process. We investigated the roles and interactions of ROS, the ERK cascade, and the phosphoinositide 3-kinase (PI3K)/Akt axis during human sperm capacitation. Two different agents, fetal cord serum ultrafiltrate and bovine serum albumin, similarly promoted capacitation and the associated phosphorylation of protein tyrosine residues (P-Tyr), threonine-glutamine-tyrosine (P-Thr-Glu-Tyr-P) motif, and MEK-like proteins (P-MEK-like proteins). Components of the ERK pathway modulated these phosphorylation events. ROS increased P-MEK-like proteins and NO* induced P-Thr-Glu-Tyr-P, possibly by acting on or downstream of Ras. The PI3K/Akt axis participated in capacitation and phosphorylation of Tyr and Thr-Glu-Tyr but not MEK-like proteins. H2O2 and NO* induced P-Tyr even in the presence of ERK pathway inhibitors, indicating that ROS also act downstream of this pathway. These new results indicate that ROS act on different transduction elements during sperm capacitation and regulate phosphorylation events that occur in parallel pathways that eventually lead to late phosphorylation of Tyr. These new data reinforce the concept that a complex network of differentially modulated pathways is needed for spermatozoa to become capacitated.     

L-arginine promotes capacitation and acrosome reaction in cryopreserved bovine spermatozoa

Sperm capacitation and acrosome reaction are essential for fertilization and they are considered as part of an oxidative process involving superoxide and hydrogen peroxide. In human spermatozoa, the amino acid L-arginine is a substrate for the nitric oxide synthase (NOS) producing nitric oxide (NO*), a reactive molecule that participates in capacitation as well as in acrosome reaction. L-arginine plays an important role in the physiology of spermatozoa and has been shown to enhance their metabolism and maintain their motility. Moreover, L-arginine has a protective effect on spermatozoa against the sperm plasma membrane lipid peroxidation. In this paper, we have presented, for the first time, the effect of L-arginine on cryopreserved bovine sperm capacitation and acrosome reaction and the possible participation of NOS in both processes. Frozen-thawed bovine spermatozoa have been incubated in TALP medium with different concentrations of L-arginine and the percentages of capacitated and acrosome reacted spermatozoa have been determined. L-arginine induced both capacitation and acrosome reaction. NO* produced by L-arginine has been inhibited or inactivated using NOS inhibitors or NO* scavengers in the incubation medium, respectively. Thus, the effect of NOS inhibitors and NO* scavengers in capacitated and non-capacitated spermatozoa treated with L-arginine has also been monitored. The data presented suggest the participation of NO*, produced by a sperm NOS, in cryopreseved bovine sperm capacitation and acrosome reaction.     

Role of tyrosine phosphorylation in sperm capacitation / acrosome reaction

Capacitation is an important physiological pre-requisite before the sperm cell can acrosome react and fertilize the oocyte. Recent reports from several laboratories have amply documented that the protein phosphorylation especially at tyrosine residues is one of the most important events that occur during capacitation. In this article, we have reviewed the data from our and other laboratories, and have constructed a heuristic model for the mechanisms and molecules involved in capacitation/acrosome reaction.     

Xenoestrogenic compounds promote capacitation and an acrosome reaction in porcine sperm

There is growing evidence that endocrine disruptors bind to hormone receptors; since these receptors are present on the sperm membrane, sperm are potentially a useful model for examining estrogenic activities of endocrine disruptors. The objective of the present study was to compare the effects of two xenoestrogenic compounds (genistein and 4-tert-octylphenol) to those of two steroids (estrogen and progesterone) and heparin on in vitro capacitation and the acrosome reaction in a porcine sperm model. Porcine sperm were incubated with various concentrations (0.001-100 μM) of each chemical for 15 or 30 min, and then capacitation and the acrosome reaction were assessed using chlortetracycline. Estrogen and progesterone were considerably more potent than the other chemicals in stimulating capacitation. Estrogen stimulated sperm capacitation at all tested concentrations after 15 min of incubation (P < 0.05), whereas progesterone stimulated sperm capacitation at all tested concentrations after 15 and 30 min (P < 0.05). The effect of genistein on sperm capacitation was comparable with that of estrogen, and it was the most potent in stimulating the acrosome reaction. Genistein stimulated the acrosome reaction at all tested concentrations after 30 min (P < 0.05). However, 4-tert-octylphenol had the least effect on capacitation and the acrosome reaction. In summary, since all chemicals studied effectively altered capacitation and the acrosome reaction, it was concluded that porcine sperm could be a useful model for in vitro screening of potential endocrine disruptors. It was noteworthy that concurrent comparisons to steroids increased the ability to determine estrogenic characteristics of the tested chemicals.     

Signal transduction pathways that regulate sperm capacitation and the acrosome reaction

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

Reactive oxygen-induced reactive oxygen formation during human sperm capacitation

Physiological processes are often activated by reactive oxygen species (ROS), such as the superoxide anion (O₂^–) and nitric oxide (NO) produced by cells. We studied the interactions between NO and O₂^–, and their generators (NO synthase, NOS, and a still elusive oxidase), in human spermatozoa during capacitation (transformations needed for acquisition of fertility). Albumin, fetal cord serum ultrafiltrate, and L-arginine triggered capacitation and ROS generation (NO and O₂^–) and superoxide dismutase (SOD) and NOS inhibitors prevented all these effects. Surprisingly, capacitation due to exogenous NO (or O₂^–) was also blocked by SOD (or NOS inhibitors). Probes used were proven specific and innocuous on spermatozoa. Whereas O₂^– was needed only for 30 min, the continuous NO generation was essential for hours. Capacitation caused a time-dependent increase in protein tyrosine nitration that was prevented by SOD and NOS inhibitors, suggesting that O₂^– and NO· also act via the formation of ONOO^–. Spermatozoa treated with NO (or O₂^–) initiated a dose-dependent O₂^– (or NO) production, providing, for the first time in cells, a strong evidence for a two-sided ROS-induced ROS generation. Data presented show a close interaction between NO and O₂^– and their generators during sperm capacitation.     

Remodeling of the actin cytoskeleton during mammalian sperm capacitation and acrosome reaction

The sperm acrosome reaction and penetration of the egg follow zona pellucida binding only if the sperm has previously undergone the poorly understood maturation process known as capacitation. We demonstrate here that in vitro capacitation of bull, ram, mouse, and human sperm was accompanied by a time-dependent increase in actin polymerization. Induction of the acrosome reaction in capacitated cells initiated fast F-actin breakdown. Incubation of sperm in media lacking BSA or methyl-beta-cyclodextrin, Ca(2+), or NaHCO(3), components that are all required for capacitation, prevented actin polymerization as well as capacitation, as assessed by the ability of the cells to undergo the acrosome reaction. Inhibition of F-actin formation by cytochalasin D blocked sperm capacitation and reduced the in vitro fertilization rate of metaphase II-arrested mouse eggs. It has been suggested that protein tyrosine phosphorylation may represent an important regulatory pathway that is associated with sperm capacitation. We show here that factors known to stimulate sperm protein tyrosine phosphorylation (i.e., NaHCO(3), cAMP, epidermal growth factor, H(2)O(2), and sodium vanadate) were able to enhance actin polymerization, whereas inhibition of tyrosine kinases prevented F-actin formation. These data suggest that actin polymerization may represent an important regulatory pathway in with sperm capacitation, whereas F-actin breakdown occurs before the acrosome reaction.     

Localisation of low molecular weight sperm antigens during capacitation and acrosome reaction

FITC-labelled sperm-specific antibodies against hamster spermatozoa were utilized as probes in acrosome reaction assays. An indirect immunofluorescence test demonstrated the localisation of two sperm proteins of 19 kDa and 23 kDa on the anterior acrosomal cap region of washed cauda epididymal sperm. These proteins were not detected in reacted acrosome or on immature or immotile sperm. Antisperm agglutinating antibodies specific to these two low molecular weight sperm antigens could be useful probes for evaluating the acrosomal status of mammalian spermatozoa.     

Reactive oxygen species and sperm cells

There is a dynamic interplay between pro- and anti-oxidant substances in human ejaculate. Excessive reactive oxygen species (ROS) generation can overwhelm protective mechanism and initiate changes in lipid and/or protein layers of sperm plasma membranes. Additionally, changes in DNA can be induced. The essential steps of lipid peroxidation have been listed as well as antioxidant substances of semen. A variety of detection techniques of lipid peroxidation have been summarized together with the lipid components of sperm membranes that can be subjected to stress. It is unsolved, a threshold for ROS levels that may induce functional sperm ability or may lead to male infertility.     

Strontium supports human sperm capacitation but not follicular fluid-induced acrosome reaction.

The ability of strontium (Sr(2+)) to replace calcium (Ca(2+)) in maintaining human sperm function has still not been completely characterized. In the present study, acrosome reaction (AR) inducibility in response to human follicular fluid (hFF) was compared in spermatozoa incubated in either Ca(2+)- or Sr(2+)-containing media. Other events related to sperm capacitation, such as protein tyrosine phosphorylation and hyperactivation as well as zona pellucida (ZP) recognition under both conditions, were also analyzed. Spermatozoa incubated overnight in the presence of Sr(2+) were unable to undergo the AR when exposed to hFF. Nevertheless, when spermatozoa were incubated under this condition and then transferred to medium with Ca(2+), sperm response to hFF was similar to that of cells incubated throughout in the presence of Ca(2+). The sperm protein tyrosine phosphorylation patterns and the percentages of sperm motility and hyperactivation were similar after incubation in Ca(2+)- or Sr(2+)-containing media. Under both conditions, the same binding capacity to homologous ZP was observed. Similar results were obtained when EGTA was added in order to chelate traces of Ca(2+) present in Sr(2+) medium. From these results, it can be concluded that Sr(2+) can replace Ca(2+) in supporting capacitation-related events and ZP binding, but not hFF-induced AR of human spermatozoa.     

Reactive oxygen species and protein kinases modulate the level of phospho-MEK-like proteins during human sperm capacitation

Capacitation is an essential process by which spermatozoa acquire fertilizing ability. Reactive oxygen species (ROS), protein kinase A (PKA), protein kinase C (PKC), protein tyrosine kinases (PTKs), and the extracellular signal-regulated protein kinase (ERK or mitogen-activated protein kinase [MAPK]) pathway regulate sperm capacitation. Our aim was to evaluate the phosphorylation of MEK (MAPK kinase or MAP2K) or MEK-like proteins in human sperm capacitation and its modulation by ROS and kinases. Immunoblotting using an anti-phospho-MEK antibody indicated that the phosphorylation of three protein bands (55, 94, and 115 kDa) increased in spermatozoa treated with fetal cord serum ultrafiltrate (FCSu), BSA, or isobutylmethylxanthine plus dibutyryl cAMP as capacitating agents. These phospho-MEK-like proteins are localized along the sperm flagellum. The MEK-inhibitors PD98059 and U126 prevented this phosphorylation, suggesting that these proteins are MEK-like proteins. The ROS scavengers prevented, and the addition of H(2)O(2) or spermine-NONOate (nitric oxide donor) triggered, the increase of phospho-MEK-like proteins. The capacitation-related increases in phospho-MEK-like proteins induced by FCSu, H(2)O(2), and spermine-NONOate were similarly modulated by PKA, PKC, and PTK, suggesting ROS as mediators in this phenomenon. These results indicate that phospho-MEK-like proteins are modulated by ROS and kinases and probably represent an intermediary step between the early events and the late tyrosine phosphorylation associated with capacitation.     

A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa

The abundance of data pertaining to the metabolism of lipids in relation to mammalian fertilization has warranted an effort to assemble a molecular membrane model for the comprehensive visualization of the biochemical events involved in sperm capacitation and the acrosome reaction. Derived both from earlier models as well as from current concepts, our membrane model depicts a lipid bilayer assembly of space-filling molecular models of sterols and phospholipids in dynamic equilibrium with peripheral and integral membrane proteins. A novel feature is the possibility of visualizing individual lipid molecules such as phosphatidylcholine, phosphatidylethanolamine, lysophospholipids, fatty acids, and free or esterified cholesterol. The model illustrates enzymatic reactions which are believed to regulate the permeability and integrity of the plasma membrane overlying the acrosome during interactions between the male gamete and capacitation factors present in fluids of the female genital tract. The use of radioactive lipids as molecular probes for monitoring the metabolism of cholesterol and phosphatidylcholine revealed the presence of (1) steroid sulfatase in hamster cumulus cells, (2) lecithin: cholesterol acyltransferase in human follicular fluid, (3) phospholipase A₂, and (4) lysophospholipase in human spermatozoa. These enzymatic reactions can be integrated into a pathway that provides a link between the concepts of lysophospholipid accumulation in the sperm membranes and alteration of the cholesterol/phospholipid ratio as factors involved in the preparation of the membranes for the acrosome reaction. Capacitation is viewed as a reversible phenomenon which, upon completion, results in a decrease in negative surface charge, an efflux of membrane cholesterol, and an influx of calcium between the plasma and outer acrosomal membranes. Triggered by the entry of calcium, the acrosome reaction involves phospholipase A₂ activation followed by a transient accumulation of unsaturated fatty acids and lysophospholipids implicated in membrane fusion which occurs during the formation of membrane vesicles in spermatozoa undergoing the acrosome reaction.     

Strontium supports capacitation and the acrosome reaction in mouse sperm and rapidly activates mouse eggs

Extracellular Ca²⁺ is required for capacitation and fertilization in the mouse, but very little is known about the ability of other divalent cations to substitute for Ca²⁺. In this study, Sr²⁺, Ba²⁺, and Mg²⁺ were evaluated for their ability to support capacitation, the acrosome reaction, hyperactivated motility, and fertilization. Ba²⁺ proved to be ineffective, but Mg²⁺-containing medium was able to support capacitation to a greater extent than unsupplemented Ca²⁺-deficient media; despite this, Ca²⁺ was required for fertilization. In contrast, Sr²⁺ proved capable of substituting for Ca²⁺ in all events. Furthermore, Sr²⁺-induced responses were indistinguishable from the corresponding Ca²⁺-induced ones: Sperm capacitated at the same rate and underwent the acrosome reaction to the same extent. However, demonstration of sperm:egg fusion in Sr²⁺ required the use of zona-free eggs. This was due not to the inability of the sperm to penetrate the zona but to the very rapid activation and cortical granule release by eggs in response to Sr²⁺. When zona-intact eggs were used, the block to polyspermy had been mounted by the time sperm had penetrated the zona. A 15 min exposure to Sr²⁺ was sufficient to block sperm fusion, but a longer exposure was required to ensure the resumption of meiosis in eggs; such a response was surprising in that the eggs were freshly ovulated and not susceptible to activation by many different treatments. Thus Sr²⁺ can profoundly affect both gametes in the mouse: It substitutes completely for Ca²⁺ in sperm responses and rapidly activates eggs, possibly by displacing Ca²⁺ from intracellular stores into the cytoplasm, where the Ca²⁺ can then trigger the various events of activation.     

The acrosome reaction of Strongylocentrotus purpuratus sperm. Ion requirements and movements

The acrosome reaction of sperm of the sea urchin, Strongylocentrotus purpuratus, is accompanied by ion movements. When the reaction is induced by the addition of egg jelly to sperm suspended in sea water, there is an acid release and an uptake (or exchange) of calcium ions. Verapamil and D600, drugs which block Ca²⁺ channels, inhibit induction of the acrosome reaction, acid release, and ⁴⁵Ca²⁺ uptake; this inhibition is reduced at higher concentrations of external Ca²⁺. Although acid release correlates temporally with extension of the acrosome filament, ⁴⁵Ca²⁺ uptake continues after the acrosome reaction has been completed. Neither the acrosome reaction nor acid release is inhibited by cyanide, azide, dinitrophenol (DNP), or carbonyl cyanide m-chlorophenylhydrazone (CCCP), whereas these metabolic inhibitors partially inhibit Ca²⁺ uptake. Tetraethylammonium (TEA) chloride, an inhibitor of delayed axonal potassium currents, inhibits the acrosome reaction. An increase in ⁸⁶Rb⁺ permeability accompanies the acrosome reaction, suggesting that movement of K⁺ is an important effector of the reaction. In support of this, the acrosome reaction may be triggered with nigericin, an ionophore that catalyzes the electrically neutral exchange of K⁺ and H⁺ across membranes. Induction of the acrosome reaction with nigericin can occur with either Na⁺ or K⁺ as the predominant external monovalent cation, while with jelly it requires external Na⁺. With nigericin, there is a delay in acid release, Ca²⁺ uptake, and filament extension, all of which follow a transient proton uptake. Taken together, these data suggest that triggering of the acrosome reaction involves linked permeability changes for monovalent and divalent ions.