INHIBITION OF RESPIRATION IN SEA URCHIN SPERMATOZOA FOLLOWING INTERACTION WITH FIXED UNFERTILIZED EGGS

The respiration of spermatozoa of the sea urchin, Hemicentrotus pulcherrimus , was found to be sensitive to rotenone, antimycin A, and cyanide. This suggests that sperm respiration results from electron transport which spans the whole mitochondrial respiratory chain. The sperm respiration was inhibited by oligomycin and this inhibition was released by 2, 4-dinitrophenol (DNP). DNP did not stimulate the respiration of spermatozoa in a diluted suspension (2 × 10⁸/ml), where they were swimming vigorously. The ADP level of spermatozoa in the diluted suspension was markedly higher than that in dry sperm. The spermatozoa, which had reacted with unfertilized eggs fixed with glutaraldehyde, were immotile with a quite low respiratory rate. The respiratory rate of the immotile spermatozoa was enhanced by DNP. In the immotile spermatozoa, ADP level was markedly low and the ATP level was as high as that in dry sperm. From these findings, it is concluded that in the swimming spermatozoa respiration coupled with oxidative phosphorylation occurs at the maximum rate. State 3 respiration probably occurs in the swimming spermatozoa. The low respiratory rate of the immotile spermatozoa is assumed to be due to a shortage of ADP and is practically regarded as state 4 respiration.     

Induction of Fertilization Membrane Formation and Cyanide-insensitive Respiration in Sea Urchin Eggs by the Treatment with Dimethylsulfoxide Followed by an Incubation in an Ice Bath

In unfertilized eggs of the sea urchin Hemicentrotus pulcherrimus , fertilization membrane formation was induced by an incubation with dimethylsulfoxide (DMSO) for several min at 20°c followed by another incubation in an ice bath. The number of eggs with fertilization membrane, thus obtained, increased in relation to the concentration of DMSO between 1 and 3% (v/v) and was higher than 75% at concentrations above 3%. Fertilization membrane formation by this treatment occurred in Ca²⁺ free- or Ca²⁺, Mg²⁺ free- artificial sea water containing EGTA (50 mM) and was inhibited by verapamil. In the presence of DMSO, the membrane formation was also induced by 2, 4-dinitrophenol or cyanide in considerable number of eggs at 20°c. Eggs remained fertilizable, even when they were kept with DMSO for 1 hr at 20°c. DMSO slightly enhanced respiratory rate in unfertilized eggs and substantially reduced it in fertilized eggs. DMSO-treated eggs exhibited cyanide-insensitive respiratory burst following chilling in an ice bath or by adding DNP or cyanide, in a similar manner to the burst induced by sperm.     

SCN— Blocks Hardening of the Fertilization Envelope of Sea Urchin Eggs and Adhesion of Blastomeres

Sea urchin eggs kept in artificial sea water (ASW) containing 0.01–0.3 M NaSCN in place of NaCI from within 2 min after insemination formed thin, enlarged fertilization envelopes, which were broken on mild agitation of egg suspensions more easily than those formed in Ca²⁺-free ASW. The blastomeres of almost all embryos derived from eggs treated with 0.2M SCN^— for 1 hr dissociated spontaneously, and did not reassociate with other blastomeres appreciably. Thus SCN^— probably denaturated some compound(s) participating in blastomere binding and hardening of the fertilization envelope. Abnormal arrangements of blastomeres, probably due to incomplete blastomere dissociation, were observed in embryos derived from eggs treated with 0.1 M SCN^— for 1 hr. Treatment of fertilized or unfertilized eggs with 0.05–0.1 M SCN^— for a short period caused concentration-dependent block of morphogenic processes such as formation of the archenteron and pluteus arms in the post-hatching period. The effects of SCN^— on morphogenesis were not inhibited by furosemide or 4,4'-diisothiocyano 2,2'-disulfonic stilbene. Presumably, the denaturation of several compounds in the egg surface by SCN^— causes abnormal morphogenesis of embryos. The inhibitory effects of SCN^— on hardening of the fertilization envelope, blastomere binding and morphogenesis were greater in the absence of Ca²⁺.     

Does the low respiratory rate in unfertilized eggs result mainly from depression of the redox reaction catalyzed by flavoproteins? Analysis of the respiratory system by light-induced release of CO-mediated inhibition

In unfertilized eggs of the sea urchin, the quite low respiratory rate is enhanced by tetramethyl- p -phenylenediamine (TMPD), phenazine methosulfate (PMS) and sperm and this augmentation is completely inhibited by carbon monoxide (CO). Exposure to light releases eggs from this CO-mediated inhibition. The action spectra for photoreactivation of CO-inhibited cytochrome c oxidase in isolated mitochondria and CO-blocked respiration in TMPD-treated eggs were found to be similar to the absorption spectrum of CO-bound cytochrome aa ₃. In PMS-treated eggs and fertilized eggs, the maximum photoreactivation of CO-inhibited respiration occurred at a light fluence rate higher than that for maximum photoreactivation of CO-inhibited respiration in TMPD-treated eggs, with peaks at the same wavelengths as those in the absorption spectrum of reduced cytochrome b. A similar phenomenon was seen for NADH cytochrome c reductase in mitochondria. Thus, cytochrome c oxidase and NADH cytochrome c reductase, whose activities are not altered by fertilization, seem to be functional, even in unfertilized eggs. In unfertilized eggs, difference spectra indicated that PMS and sperm augmented cytochrome b reduction and that TMPD accelerated cytochrome c reduction without cytochrome b reduction. Therefore, it is likely that depression of electron transport to cytochrome b , which is augmented by PMS and sperm, is responsible for the low respiratory rate in unfertilized eggs.     

INHIBITION OF RESPIRATION IN SEA URCHIN SPERMATOZOA FOLLOWIGN INTERACTION WITH FIXED UNFERTILIZED EGGS

The respiratory rate of spermatozoa of the sea urchin, Pseudocentrotus depressus and Hemicentrotus pulcherrimus , became quite low and spermatozoa was immotile, after sperm suspension containing glutaraldehyde-fixed eggs of homologous species was stirred at 20°C for 15 min. The respiratory rate of fresh spermatozoa, introduced to the suspension of immotile spermatozoa thus obtained, was also reduced markedly. The respiration of fresh spermatozoa was not inhibited by adding them to suspension of intact or acrosome reacted spermatozoa. A heat stable and non-dialyzable substance, which inhibited sperm respiration, was removed from the fixed eggs by vigorously stirring the egg suspension for 10 min, when unfertilized eggs were fixed with insufficient amount of glutaraldehyde (10 ml of 1% glutaraldehyde solution to 1 ml egg pellet).     

Stimulation of Unfertilized Eggs of the Echiuroid, Urechis unicinctus by Polyamines

Unfertilized eggs of the echiuroid, Urechis unicinctus , were activated by polyamines, such as putrescine, spermidine and spermine at concentrations above 10 μM. Fertilization membrane elevated and germinal vesicle disappeared in unfertilized eggs kept for several min in sea water containing these polyamines. Following the addition of these polyamines, a decrease of pH value in the egg suspension, occurred in a similar manner as observed following fertilization. Several sec after the addition of polyamines to the egg suspension, the respiratoy rate increased very slightly and the sensitivity of the respiration to 2, 4-dinitrophenol, which was lower in unfertilized eggs than in fertilized eggs, became as high as in fertilized ones. Irregular cleavage occurred in the eggs stimulated by polyamines. The incorporation of [³H]-deoxyadenosine into DNA was initiated by adding polyamines in the unfertilized eggs preloaded with the isotope. The rate of [³H]-leucine incorporation into protein in the preloaded unfertilized eggs was also enhanced by polyamines, in almost the same manner as observed following fertilization.     

Probable participation of phospholipase A2 reaction in the process of fertilization-induced activation of sea urchin eggs

In sea urchin eggs activated by sperm, A23187 or melittin, BPB (4-bromophenacyl bromide, a phospholipase A₂ inhibitor) blocked fertilization envelope formation and transient CN⁻-insensitive respiration in a concentration-dependent manner. BPB had virtually no effect on the increase in [Ca²⁺]_i, (cytosolic Ca²⁺ level), the activity of phosphorylase a and the rate of protein synthesis, as well as acid production and augmentation of CN⁻-sensitive respiration. BPB also inhibited fertilization envelope formation and augmentation of CN⁻-insensitive respiration induced by melittin. Melittin, known to be an activator of phospholipase A₂, induced the envelope formation, acid production, augmentation of CN⁻-insensitive and sensitive respiration, but did not cause any increase in [Ca²⁺]_i, the phosphorylase a activity and the rate of protein synthesis. An activation of phospholipase A₂ induced by Ca²⁺ or melittin seems to result in cortical vesicle discharge and production of fatty acids, which are to be utilized in CN⁻-insensitive lipid peroxidase reactions. Activation of other examined cell functions in eggs activated by sperm or A23187, probably results from Ca²⁺-triggered sequential reactions other than Ca²⁺-caused activation of phospholipase A₂.     

Inhibitory Effects of Diltiazem and Verapamil, Calcium Antagonists, on Fertilization-Induced Increase in the Phosphorylase Reaction in Echiuroid Eggs

The calcium antagonists diltiazem and verapamil at 100 μM caused considerable inhibition of the glycolysis system in recently fertilized eggs of the echiuroid, Urechis unicinctus . The levels of glycolytic intermediates in eggs were found to be higher 5 min after insemination than before fertilization while the levels of adenine nucleotides and inorganic phosphate were almost the same before and after fertilization. Addition of diltiazem or verapamil 30 sec after insemination did not inhibit fertilization, but resulted in maintenance of as low levels of glycolytic intermediates as in unfertilized eggs. The apparent mass action ratio in the phosphorylase step, calculated from the levles of glucose-1-phosphate and inorganic phosphate was normally higher in fertilized eggs than in unfertilized eggs, but was maintained at as low a level as in unfertilized eggs by adding these compounds 30 sec after insemination. Phosphorylase a activity also normally increased after insemination, but was maintained at a low level in fertilized eggs by adding these compounds. These compounds also inhibited the increased ⁴⁵Ca²⁺ uptake normally observed after fertilization. These results suggest that after fertilization, the Ca²⁺ level increases associated with fertilization-induced Ca²⁺ influx and that this stimulates Ca²⁺ dependent protein kinase to phosphorylate phosphorylase b , resulting in an increased rate of the phosphorylase reaction.     

INHIBITION OF RESPIRATION IN SEA URCHIN SPERMATOZOA FOLLOWING INTERACTION WITH FIXED UNFERTILIZED EGGS

The respiratory rate of spermatozoa of the sea urchins, Anthocidaris crassispina, Clypeaster japonicus and Pseudocentrotus depressus , decreases markedly in the presence of homologous unfertilized eggs fixed with glutaraldehyde. No decrease in the rate of respiration occurs in the presence of fixed fertilized eggs. Fixed unfertilized eggs of different sea urchin species do not cause any change in the rate of sperm respiration. Spermatozoa adhere only to the fixed unfertilized eggs of the same species and are removed by a stirring for 5 min on a magnetic stirrer. The spermatozoa thus removed, are immotile and their respiratory rate is quite lower than that of motile spermatozoa in a control suspension stirred for 5 min. Intact spermatozoa adhere to the fixed eggs, from which the attached spermatozoa have been removed, and the respiratory rate of the spermatozoa also becomes quite low.     

MAGNESIUM REQUIREMENT IN FERTILIZATION PROCESS OF SEA URCHINS

The Mg²⁺ requirement in fertilization was investigated in sea urchins. It was found that when sea urchin eggs were inseminated in sea water free of Mg²⁺, little fertilization took place. Even when spermatozoa pre-treated with dissolved egg-jelly to induce the acrosome reaction, which needs Ca²⁺, were used, the fertilization rate remained quite low in the absence of Mg²⁺. In Strongylo-centrotus intermedius , the lowest concentration of Mg²⁺ required for 50% fertilization was 0.05 mM in the presence of 10 mM Ca²⁺, whereas that of calcium was 3 mM in the presence of 49 mM Mg²⁺. These critical concentrations increased when the concentration of the other ion decreased. Removal of Mg²⁺ or Ca²⁺ or both from the suspending medium had little adverse effect on sperm motility. The elevation of the fertilization membrane was also induced by butyric acid independent of the presence or absence of Mg²⁺ and/or Ca²⁺. These results indicate that Mg²⁺ are required at least in some process(es) between acrosome reaction and fertilization membrane elevation, such as sperm penetration or membrane fusion.     

Characteristics of sperm of captive seabass in relation to its fertilization potential

Seabass Dicentrarchus labrax sperm concentration was high (up to 60 × 10⁹ spz ml⁻¹) but decreased significantly at the end of the reproductive season (mid-March) in monthly sampled fish. The spermiation period may be shortened by frequent stripping. Sperm can be prediluted up to 1: 128 in non-activating medium without loss of initial motility and motility duration. Immediately after activation by transfer to sea water, all the spermatozoa were motile for 10 s and then the number of motile cells decreased progressively but sharply to zero, so that the duration of sperm motility was very short (40 s). As a consequence, the fertility of seabass sperm decreased exponentially after 10 s following sperm activation and was zero by 1 min. The sperm requirements for optimal fertilization were c . 66 000 spermatozoa per egg. Scalingup of the experimental insemination procedure yielded better fertilization rates while conserving the individual differences due to the breeder pairs.     

Effects of Sperm-Activating Peptide I on Hemicentrotus pulcherrimus Spermatozoa in High Potassium Sea Water

The effects of sperm-activating peptide I (SAP-I: Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) on Hemicentrotus pulcherrimus spermatozoa in high [K⁺] sea water were examined. In high [K⁺] sea water, the respiration rates and motility of H. pulcherrimus spermatozoa were lower than those in normal sea water. SAP-I did not stimulate the lowered respiration rate or motility, although the peptide bound to the spermatozoa as it does in normal sea water. SAP-I elevated the sperm cGMP level in 100 mM K⁺ sea water (from 0.37 to 4.81 pmol/mg wet weight spermatozoa) more than those in normal sea water (from 0.21 to 0.93 pmol/mg wet weight). A phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) and SAP-I synergistically elevated the cGMP level from 0.35 to 33.08 pmol/mg wet weight in 100 mM K⁺ sea water. However, in high [K⁺] sea water, SAP-I did not increase the cAMP level even in the presence of IBMX. SAP-I caused rapid, transient elevation of the intracellular pH and Ca²⁺ concentration of spermatozoa in normal sea water but not in 100mM K⁺ sea water. SAP-I did not decrease the apparent molecular weight of sperm guanylate cyclase from 131,000 to 128,000 in high [K⁺] sea water. These results suggest that the SAP-I-induced elevation of the cGMP level in sea urchin spermatozoa occurs before or independently of membrane hyperpolarization induced by the opening of K⁺ channels.     

VII. Decrease in the Rate of Respiration in the Spermatozoa of the Sea Urchin, Hemicentrotus Pulcherrimus, Caused by Long Chain Fatty Acyl-CoA-induced Inhibition of the Movement

Spermatozoa of the sea urchin, Hemicentrotus pulcherrimus , showed marked decrease in respiration, and arrested movement after interaction with the fixed eggs. Immotile spermatozoa that had reacted with fixed eggs contained higher levels of long chain fatty acyl-CoAs than normal motile spermatozoa. On treatment with carnitine, the immotile spermatozoa became motile again and their intracellular concentrations of long chain fatty acyl-CoAs decreased. On incubation with anti-mycin A or CN⁻ for 20 min, the motility of normal spermatozoa decreased gradually but their long chain fatty acyl-CoA content changed only slightly. The decrease in sperm motility in the latter case was probably due to decrease in the level of ATP, resulting from inhibition of respiration by antimycin A or CN⁻. The motility of spermatozoa extracted with Triton X-100 was restored by ATP and their movement was inhibited by long chain fatty acyl-CoAs, such as myristoly CoA and palmitoyl-CoA, but was not by short chain fatty acyl-CoAs, such as acetyl-CoA, propionyl CoA and butyryl-CoA. Na-palmitate, Na-myristate and CoA did not inhibit the reactivation of extracted spermatozoa by ATP.     

Activation of Sea Urchin Eggs by Halothane and Its Inhibition by Dantrolene

Eggs of the sea urchin, Hemicentrotus pulcherrimus , were stimulated by halothane, known to induce Ca²⁺ release from sarcosome, to cause fertilization membrane formation in normal and Ca²⁺ free artificial sea water. In the absence of external Ca²⁺, halothane-induced formation of fertilization membrane was inhibited by dantrolene, an inhibitor of Ca²⁺ release from sarcosome, but was not blocked by nifedipine, a Ca²⁺ antagonist specific to Ca²⁺ channels in plasma membrane. Ca²⁺ release from sedimentable fraction isolated from eggs was induced by halothane and was inhibited by dantrolene, but was not blocked by nifedipine. In normal artificial sea water, halothane-caused egg activation was not inhibited either by dantrolene or by nifedipine, but was blocked in the presence of both compounds. ⁴⁵Ca²⁺ influx was substantially stimulated by halothane in eggs exposed to ⁴⁵CaCl₂. Halothane-induced ⁴⁵Ca²⁺ influx into eggs was inhibited by nifedipine but was not blocked by dantrolene. When Ca²⁺ release from intracellular organellae is blocked, Ca²⁺ transport through Ca²⁺ channels in plasma membrane probably acts as a "fail-safe" system to induce an increase in cytosolic Ca²⁺ level, resulting in egg activation.     

Requirement of Extracellular Calcium Ions for the Early Fertilization Events in the Medaka Egg

The requirement for calcium and the change in calcium content in eggs of Oryzias iatipes during the cortical reaction and sperm penetration were examined. Naked eggs failed to exhibit the cortical reaction upon insemination under Ca Mg-free conditions. These eggs exhibited the cortical reaction by reinsemination in the presence of extracellular Ca²⁺. The effect of extracellular Ca²⁺ on sperm penetration could be replaced by one of several divalent cations in the external medium. Unlike the cortical reaction, sperm penetration failed to be induced by microinjection to increase intracellular Ca²⁺. Verapamil significantly reduced the action of extracellular Ca²⁺ or Ba²⁺ of divalent cations examined in fertilization, while TEA and TTX had no effect on fertilization in the presence of these cations. No ⁴⁵Ca uptake into the egg proper was recognized before completion of the cortical reaction. These observations suggest that extracellular divalent cations are indispensable for sperm stimulation of the egg and its penetration into the egg, for which an influx of Ca²⁺ from the external medium is not required.     

The Role of External Potassium Ion in Activation of Sea Urchin Spermatozoa

When sperm of the sea urchin, Hemicentrotus pulcherrimus, are diluted into K⁺-free seawater, the pH of the suspension gradually decreases, whereas a rapid decline in pH is observed following dilution into regular seawater. Sperm motility and respiration are also activated after dilution into K⁺-free seawater, but levels of activity are less than those observed following dilution into regular seawater. Upon addition of 10 mM K⁺ to K⁺-free seawater, rapid acid release occurs and motility and respiratory rate in sperm are reactivated. The effect of K⁺ on respiration was competitive with respect to the external Na⁺ concentrations. Harmaline, a potent inhibitor of Na⁺/K⁺-ATPase, causes a decrease in movement and respiration of the sperm. Harmaline does not inhibit the rapid decline in pH, although it depresses the release of acid from mitochondria. These results suggest that external K⁺ plays an important role in intracellular alkalinization of sea urchin sperm.     

Lack of active K+ uptake in aeroponically grown wheat seedlings

The K⁺ (⁸⁶Rb⁺) uptake and the growth of intact wheat seedlings ( Triticum aestivum L. cv. GK Szeged) grown in 0.5 m M CaCl₂ solution and of seedlings grown on wet filter paper in Petri dishes were compared under different experimental conditions. Aeroponic (AP) and hydroponic (HP) conditions brought about striking differences in the growth of the roots, whereas the shoot growth was not influenced. The dry weight of the roots was higher for the AP plants than for the HP plants. The AP grown seedlings exhibit a low rate of K⁺ uptake, which seems to be a passive process. The effect of 2, 4–dinitrophenol (2, 4–DNP) clearly shows the absence of an active component of the K⁺ uptake in roots grown in air with a high relative humidity. In plants grown under AP conditions the effect of Ca²⁺ on the K⁺ uptake is unfavourable, i.e. there is an inhibition (negative Viets effect). Results relating to the effect of 2,4–DNP suggest that the "negative Viets effect" is a feature of the passive K⁺ uptake. The data suggest that the AP growth conditions play a very important role in the induction and/or development of the ion transport system(s), which becomes impaired under the AP conditions.     

Effects of osmolality and ions on the motility of stripped and testicular sperm of freshwater-and seawater-acclimated tilapia, Oreochromis mossambicus

A significantly higher concentration of testicular spermatozoa was obtained from freshwater Oreochromis mossambicus (9·9×10⁹ spermatozoa ml⁻¹) than seawater O. mossambicus (4·6×10⁹ spermatozoa ml⁻¹). The mean osmolality of the urine of freshwater fish (78·5 mOsmol kg⁻¹) was significantly different from that of seawater fish (304·8 mOsmol kg⁻¹). The mean length of the mid-piece of the spermatozoa together with the tail was more variable in freshwater O. mossambicus (8·80±0·23μm) than in seawater specimens (8·27±0·18 μm). Stripped sperm of freshwater O. mossambicus was highly contaminated by urine which was a good activator of sperm motility in O. mossambicus held in both fresh and sea water. The osmolality for initiation of motility in freshwater O. mossambicus spermatozoa was from 0 to 333 mOsmol kg⁻¹ while for seawater O. mossambicus spermatozoa it was from 0 to 1022 mOsmol kg⁻¹. The optimum osmolality for motility was from 70 to 333 mOsmol kg⁻¹ for freshwater O. mossambicus spermatozoa and from 333 to 645 mOsmol kg⁻¹ for seawater fish. In freshwater O. mossambicus spermatozoa, the presence of 20 mM CaCl₂ increased the permissive osmolality of NaCl from 184 to 645 mOsmol kg⁻¹. For seawater O. mossambicus spermatozoa, solutions of NaCl devoid of CaCl₂ were unable initiate motility, but the addition of 1·5 to 30 mM CaCl₂ to the NaCl solution (0–934 mOsmol kg₁) had a full motility initiating effect.     

Ca2+ release from intracellular stores by thapsigargin in sea urchin eggs: Relationship to larval development and relevance in egg activation

Thapsigargin (Tg), an inhibitor of microsomal Ca²⁺ ATPase, is used as a tool to study the changes in Ca²⁺ sequestration in sea urchin eggs and their relationship to embryonic development. Micromolar amounts of Tg inhibit ATP-dependent Ca²⁺ sequestration in a dose-dependent and non-reversible manner, depending on the bulk of biological material used. IC_5O values are 1 nmol/L and 1–10μmol/L, respectively, in the cortical Ca²⁺ stores (isolated cortices preparation) and in digitonin-permeabilized eggs, a preparation giving access to the deeper reticulum compartment. Micromolar Tg does not induce Ca²⁺ release from ⁴⁵Ca pre-loaded cortices but leads to a loss of 25% of the total Ca²⁺ content from the cortical area. Using microspectrofluorimetry of fura-2-loaded eggs, we found that 10 μmol/L Tg induced a moderate rise in cytosolic Ca²⁺ activity as compared with the fertilization-induced Ca²⁺ transient whether eggs were fertilized or not. Early events related to fertilization as, for example, elevation of the fertilization envelope, proton excretion and sustained increase of amino acid uptake, are triggered by 10μmol/L Tg but with a delayed onset relative to sperm-induced effects. The present findings indicate that although it triggers most fertilization-related events, Tg cannot be considered as a true mitotic agent in sea urchin eggs. When added after fertilization, Tg affects cleavage and the further embryonic development giving rise to abnormalities comparable to the animalized larvae obtained with other compounds responsible for the inhibition of reticular Ca²⁺ sequestration.     

Characterization of sperm motility in sea bass: the effect of heavy metals and physicochemical variables on sperm motility

Computer assisted sperm analysis (CASA) was used to characterize the motility of sea bass Dicentrarchus labrax spermatozoa and to study the effect of several physicochemical variables and heavy metals on sperm swimming performance. Duration of sperm motility in sea bass was very short (<50 s). During the first 20 s all the motility variables measured remained approximately constant, the velocity and linearity of the movement being maximum during this period, while both variables decreased sharply later. While slight variations in pH did not significantly modify sperm swimming performance, changes in osmolality affected all the measured motility variables. Two of the heavy metals tested, Cu²⁺ and Pb²⁺, did not affect sperm motility when the activating media contained up to 100 ppm of the metal salts. In contrast, Hg²⁺ modified the morphology of post-swimming spermatozoa at 0·4–1 ppm (sperm dilution rate 1:39) and completely arrested sperm motility at concentrations as low as 0·1 ppm (sperm dilution rate 1:2500). Assuming a covalent binding to sperm cells, this revealed a finite number of c. 10 million Hg²⁺ binding sites per spermatozoon. Complementary results using demembranated spermatozoa suggested that the main target of HgCl₂ would be located in the plasma membrane and that HgCl₂ would inhibit water channels, hence preventing sperm motility.