Dialysis addition of trehalose/glycerol cryoprotectant allows recovery of cryopreserved mouse spermatozoa with satisfactory fertilizing ability as assessed by yield of live young.

Mouse sperm cryopreservation provides a means for storing the genetic information in genetically modified mice (mutants, transgenics, and "knockouts") in a cost- and space-effective manner. Sperm from this species are highly sensitive to cryodamage, which has impeded their cryopreservation in the past. The cryoprotectant used in this study was 6% glycerol (0.65 M) plus 7.5% trehalose (0.22 M), which was added to a concentrated suspension of sperm from B6SJLF1/J mice in bicarbonate-free buffer by dialysis to minimize osmotic stress on the cells. Sperm suspensions were frozen in 0.25 mL straws and stored in liquid N2. Eggs were obtained from B6SJLF1/J superovulated females. For in vitro fertilization (IVF), 15-25 microL of sperm suspension post-thaw from one straw was added directly to each of three 1.5 mL drops of fertilization medium containing 30 eggs each, for 3 replicates per experiment. The fertilized eggs were scored for blastocyst formation, after which 12 blastocysts from each drop were implanted into pseudopregnant CD-1 females. The number of live pups were then scored at birth. Ten experiments yielded 21.7 +/- 1.4 (SD) blastocysts per 30 eggs inseminated (72%) and 7.3 +/- 0.4 (SD) live pups per 12 blastocysts implanted (61%). The overall yield of live pups was 44 per 100 eggs inseminated (44%). This yield should be satisfactory for maintaining a mouse strain through sperm cryostorage, with restart of the strain through IVF and embryo transfer. The method should also provide improvement in human sperm cryopreservation, as human sperm are less sensitive to cryodamage than are mouse sperm.

Functional relationships between capacitation-dependent cell signaling and compartmentalized metabolic pathways in murine spermatozoa.

Spermatozoa are highly polarized cells with specific metabolic pathways compartmentalized in different regions. Previously, we hypothesized that glycolysis is organized in the fibrous sheath of the flagellum to provide ATP to dynein ATPases that generate motility and to protein kinases that regulate motility. Although a recent report suggested that glucose is not essential for murine sperm capacitation, we demonstrated that glucose (but not lactate or pyruvate) was necessary and sufficient to support the protein tyrosine phosphorylation events associated with capacitation. The effect of glucose on this signaling pathway was downstream of cAMP, and appeared to arise indirectly as a consequence of metabolism as opposed to a direct signaling effect. Moreover, the phosphorylation events were not affected by uncouplers of oxidative respiration, inhibitors of electron transfer, or by a lack of substrates for oxidative respiration in the medium. Further experiments aimed at identifying potential regulators of sperm glycolysis focused on a germ cell-specific isoform of hexokinase, HK1-SC, which localizes to the fibrous sheath. HK1-SC activity and biochemical localization did not change during sperm capacitation, suggesting that glycolysis in sperm is regulated either at the level of substrate availability or by downstream enzymes. These data support the hypothesis that ATP specifically produced by a compartmentalized glycolytic pathway in the principal piece of the flagellum, as opposed to ATP generated by mitochondria in the mid-piece, is strictly required for protein tyrosine phosphorylation events that take place during sperm capacitation. The relationship between these pathways suggests that spermatozoa offer a model system for the study of integration of compartmentalized metabolic and signaling pathways.

Kinetics of onset of mouse sperm acrosome reaction induced by solubilized zona pellucida: fluorimetric determination of loss of pH gradient between acrosomal lumen and medium monitored by dapoxyl (2-aminoethyl) sulfonamide and of intracellular Ca(2+) changes monitored by fluo-3.

The onset of the zona pellucida-induced acrosome reaction in mouse sperm is marked by loss of the pH gradient existing in acrosome-intact sperm between the acidic acrosomal lumen and the suspending medium, due to pore formation between outer acrosomal and plasma membranes. In earlier work, it was shown that this pH gradient loss occurred in single sperm bound to structurally intact zonae pellucidae with a half-time of 2.1 min; the extended kinetics of this loss determined in a sperm population bound to intact zonae was due to a 180-min range of variable lag times. We hypothesized that this lag time range was due to steric constraints imposed by the three-dimensional structure of the structurally intact zona pellucida, and that this constraint should be removed in solubilized zonae. The fluorescent probe, Dapoxyl(TM) (2-aminoethyl)sulfonamide (DAES) allowed a test of this hypothesis in a population of sperm cells. It is a weak base that is non-fluorescent in aqueous solution, but which accumulates in the acidic acrosomal compartment due to the pH gradient with highly enhanced fluorescence; loss of the pH gradient leads to a decrease in fluorescence. The half-time for DAES fluorescence loss in a population of capacitated, acrosome-intact sperm in response to solubilized zona pellucida protein was 2.13 +/- 0.10 min (SEM, n = 9). The agreement between single cell and cell population kinetics validates the hypothesis of steric constraint in the structurally intact zona pellucida. The change in intracellular Ca(2+) concentration in response to solubilized zona pellucida, as monitored with fluo-3, was a rapid increase, followed by a decrease, with a half-time of 0.85 +/- 0.09 min (SEM, n = 6) to a steady state level higher than the initial level, indicating this Ca(2+) transient as the precursor reaction to onset of the zona-induced acrosome reaction.

Determination of the intracellular dissociation constant, K(D), of the fluo-3. Ca(2+) complex in mouse sperm for use in estimating intracellular Ca(2+) concentrations.

In order to calculate the actual, rather than the relative, intracellular Ca(2+) concentration (Ca(2+))(i) in mammalian sperm cells, using fluorescent probes whose fluorescence emission differs between the probe. Ca(2+) complex and free probe, the value of the dissociation constant for the probe. Ca(2+) complex, K(D), is required. Interaction of the probe with cellular components may change the intracellular value of K(D) from that determined in buffered solution. We had previously shown that fluo-3, whose Ca(2+) complex is highly fluorescent whereas free fluo-3 is not, could be used to monitor changes of (Ca(2+))(i) in mouse sperm. In this report, we describe a method for determining K(D) for the fluo-3. Ca(2+) complex in mouse sperm suspended in medium MJB, a medium in which the sperm remain viable, but which contains high Ca(2+). The method involved treating the sperm with ionomycin to provide a plasma membrane Ca(2+) carrier, with nigericin to eliminate pH gradient, and with gramicidin D to eliminate membrane potential, such that (Ca(2+))(i) equilibrates with medium Ca(2+) concentration (Ca(2+))(e), then titrating (Ca(2+))(e) with EGTA in added aliquots to near nil concentration. At EGTA concentrations in excess of total medium Ca(2+), an approximation algorithm was used to calculate (Ca(2+))(e), based on the known K(D) for the EGTA. Ca(2+) complex. The fluorescence of the intracellular fluo-3. Ca(2+) complex, F, decreased with increasing additions of EGTA; (Ca(2+))(i) = (Ca(2+))(e) was plotted as a linear function of F/[F(max) - F]; the slope gives K(D). At 37 degrees C, intracellular K(D) was calculated to be 0.636 +/- 0.018 microM (+/-SEM, n = 8). At 37 degrees C and 20 degrees C, K(D) values in MJB were calculated to be 0.502 +/- 0.022 and 0.578 +/- 0.029 (+/-SEM, n =8 and n = 6), respectively. The higher intracellular K(D) value implies probe interaction with cytosol components, primarily those in the head, as this compartment is the major contributor to sperm fluorescence. Changes in (Ca(2+))(i), monitored with fluo-3 fluorescence, that occur on interaction of capacitated mouse sperm with the zona pellucida and may now be quantified, using 0.636 microM for K(D) of the intracellular fluo-3. Ca(2+) complex.

High PCO does not alter pHi, but raises [Ca2+]i in cultured rat carotid body glomus cells in the absence and presence of CdC12.

We measured the effect of high PCO (500-550 Torr) on the pHi and [Ca2+]i in cultured glomus cells of adult rat carotid body (CB) as a test of the two models currently proposed for the mechanism of CB chemoreception. The metabolic model postulates that the rise in glomus cell [Ca2+]i, the initiating reaction in the signalling pathway leading to chemosensory neural discharge, is due to [Ca2+] release from intracellular Ca2+ stores. The membrane potential model postulates that the rise in [Ca2+]i comes from influx of extracellular Ca2+ through voltage-dependent Ca2+ channels (VDCC) of the L-type. High PCO did not change pHi at PO2 of 120-135 Torr, showing that CO-induced changes in [Ca2+]i are not due to changes in pHi. High PCO caused a highly significant rise in [Ca2+]i from 90+/-12 nM to 675+/-65 nM, both in the absence and in the presence of 200 microM CdCl2, a potent blocker of L-type VDCCs. This result is fully consistent with release of Ca2+ from glomus cell intracellular stores according to metabolic model, but inconsistent with influx of extracellular Ca2+ through VDCCs according to the membrane potential model.

Comparison of glycerol, other polyols, trehalose, and raffinose to provide a defined cryoprotectant medium for mouse sperm cryopreservation.

Most procedures for mouse sperm cryopreservation have utilized raffinose to provide hypertonicity for cell desiccation prior to freezing and glycerol to block intracellular ice formation. Trehalose has been shown in other cell systems to provide positive protection to the plasma membrane and so was examined as a replacement for raffinose. Comparison of 3 and 6% glycerol and 7.5 and 20% sugar showed that 6% glycerol and 7.5% sugar gave maximal protection consistently and so were adopted as standard. Comparison of raffinose and trehalose at this concentration showed trehalose to give significantly better recovery of intact cells: 48 +/- 6% for trehalose, 36 +/- 9% for raffinose (+/- SE, n = 5; arc sine transformed data; P < 0.01). Less hydrophilic polyols should prove more permeant to the membrane than glycerol, enter the cell rapidly, and so possibly inhibit lethal intracellular ice formation effectively. We hypothesized that one of these polyols plus glycerol would be a more effective cryoprotectant than glycerol alone. The polyols tested as supplements to 6% glycerol were propane-1,2-diol, propane-1,3-diol, 1,1,1-tris-(hydroxymethyl)ethane (THME), and 2-ethyl-2-(hydroxymethyl)-propane-1,3-diol (EHMP). With 6% glycerol and 7.5% raffinose or trehalose, the two diols and THME gave less cryoprotection than with glycerol alone, and EHMP reduced postthaw membrane integrity to nil, thus invalidating the hypothesis. Comparison of bicarbonate-containing medium MJB to bicarbonate-free medium NTP, both with 6% glycerol/7.5% trehalose, showed no difference in recovery of membrane-intact cells. For ease of pH maintenance, NTP was chosen for studies of addition prefreeze and removal postthaw of 6% glycerol/7.5% trehalose cryoprotectant with in vitro fertilization as endpoint. Three protocols for cryoprotectant handling were tested: serial addition/dilution; dialysis addition and removal; and dialysis addition and direct insemination without cryoprotectant removal. The last proved significantly superior (P < 0.01), giving 62% fertilized eggs, normalized to controls, compared to 21% for dialysis addition and removal and 32% for serial addition and dilution. The glycerol/trehalose combination thus provides a defined cryoprotectant which, when used with addition by dialysis prefreeze and direct insemination postthaw, yields a satisfactory yield of fertilized eggs in an in vitro fertilization system.

Human sperm glutathione reductase activity in situ reveals limitation in the glutathione antioxidant defense system due to supply of NADPH.

In order to characterize further the antilipoperoxidative enzyme system of human sperm, that part of the system designed to provide reducing equivalents for the reduction of highly reactive and potentially damaging lipid hydroperoxides to relatively inert hydroxylipids was examined. The substrate that provides the reducing equivalents directly to glutathione peroxidase (GPX) is reduced glutathione (GSH), which is in turn oxidized to glutathione disulfide (GSSG). The reducing equivalents needed for regeneration of GSH through the action of glutathione reductase (GRD) are provided by NADPH, produced by the action of glucose-6-phosphate dehydrogenase (G6P-DH) on substrates glucose-6-phosphate and NADP+. The kinetic properties of the enzymes GRD and G6P-DH were determined by standard enzyme activity assay at 24 and 37 degrees C. At 37 degrees C, the Vmax for GRD was found to be 36 nmol/min x 10(8) cells, with Km values for GSSG and NAPH of 150 microM and 16 microM, respectively; the Vmax for G6P-DH was 3.3 nmol/min x 10(8) cells with Km for NADP+ of 8 microM. This suggested that G6P-DH activity was limiting in this reductive pathway. The activity of GRD in situ in intact cells was estimated using the thiol-reactive fluorogenic probe ThioGlo-1, which is cell permeant and reacts rapidly with GSH to give a highly fluorescent adduct. Mixing a suspension of human sperm with the fluorogenic reagent at 37 degrees C gave an initial rapid increase in fluorescence, followed by a slower one. The rapid phase is due to reaction with intracellular GSH already present; the slow phase is due to reaction with GSH generated by the GRD-catalyzed reduction of GSSG. Both rates showed first-order kinetics. Calculation of the maximal rate as NADPH oxidation, attributable to in situ GRD activity, gave the value of 1.0 nmol/min x 10(8) cells, less than the maximum for NADPH production by the dehydrogenase. These results support the suggestion that NADPH production limits the capacity of the pathway leading to hydroperoxide reduction in human sperm. We propose that the antilipoperoxidative defense system of human sperm has just sufficient capacity to allow these cells to fulfill their function but is limited to allow their timely disposal from the female reproductive tract.

Water permeability, Lp, of the mouse sperm plasma membrane and its activation energy are strongly dependent on interaction of the plasma membrane with the sperm cytoskeleton.

Two parameters fundamental to cell cryobiology are the water permeability (hydraulic conductivity), Lp, and its activation energy, EA. The Lp can be calculated from two experimental determinations: the critical osmolality, Osmcrit, at which 50% of the cells lyse, and the time, tcrit, to 50% lysis in a highly hyposmotic medium, based on the assumption that the cells swell to lysis with minimal resistance to swelling. We have reported [Cryobiology 32, 220-238 (1995)] that mouse sperm in hyposmotic medium show minimal swelling and so fail to meet this assumption. The concept that resistance to swelling was due to anchoring of the plasma membrane through cytoskeletal interaction was examined by treating mouse sperm with 5 microM cytochalasin D to depolymerize the cytoskeletal filamentous actin (f-actin), whose presence was established by staining with fluorescently labeled phalloidin. Diminution of fluorescence due to loss of f-actin induced by cytochalasin D was shown by flow cytometry. Mouse sperm treated with cytochalasin D showed tail curling in hyposmotic medium, similar to that observed with bovine and human sperm, indicating that the standard swelling model was applicable to these cells. Two sets of Lp values were calculated from tcrit: one using individual means of Osmcrit and one using the mean of means of Osmcrit between 37 and 4 degrees C, as these individual means were not significantly different. Values (micron.min-1.atm-1), respectively, were 9.95, 7.15 (37 degrees C); 1.51, 0.91 (22 degrees C); 0.54, 0.78 (12 degrees C); 0.47, 0.50 (4 degrees C); 0.33 (0 degree C); and 0.36 (-3 degrees C). Arrhenius plots gave EA = 13.7 and 11.7 kcal/mol, respectively. Values of t1/2 were calculated from the first-order rate constants characterizing the kinetics of cell lysis at the higher four temperatures; Lp values calculated from these, and the two sets of Osmcrit values described were 5.70, 4.09 (37 degrees C); 1.18, 0.71 (22 degrees C); 0.62, 0.90 (12 degrees C); and 0.34, 0.37 (4 degrees C). Arrhenius plots gave EA = 14.2 and 11.0 kcal/mol, respectively. We propose that these EA values are characteristic of the plasma membrane relatively unperturbed by cytoskeletal interactions. In untreated sperm, decrease of Lp with decreasing temperature and presence of cryoprotectant and the cytoskeletal interactions all act to hamper the sperm cells' ability to respond to osmotic stress encountered during freezing and thawing, such that these cells are especially sensitive to cryodamage.

Mechanism of the acceleration of CO2 production from pyruvate in liver mitochondria by HCO3-.

To investigate the mechanism by which HCO3- accelerates pyruvate metabolism in guinea pig liver mitochondria, we measured continuously, at pH 7.4 and 37 degrees C, 13C16O2 production from [1-13C]pyruvate by mass spectrometry and NADH concentration by fluorescence and analyzed total malate, citrate, and beta-hydroxybutyrate produced by standard biochemical methods. When [1-13C]pyruvate is added to the mitochondrial suspension, 13C16O2 concentration rises steeply in the first seconds and then slows to a steady lower rate. Carbonic anhydrase (CA) eliminates this initial phase, which shows that decarboxylation of pyruvate produces CO2, not HCO3-, and it does this more rapidly than it can equilibrate without CA. HCO3- (25 mM) increased 13C16O2 production, O2 consumption and total malate and citrate production and decreased NADH concentration and total beta-hydroxybutyrate production. After obtaining the total amount of 13C16O2, malate, citrate, and beta-hydroxybutyrate produced, we calculated that the addition of 25 mM HCO3- to the suspension medium increased the amount of pyruvate decarboxylated by pyruvate dehydrogenase (PDH) 16% and increased the amount carboxylated by pyruvate carboxylase 300%. This supports our initial proposal that HCO3- accelerates the pyruvate carboxylation, which in turn consumes ATP directly and NADH and acetyl CoA secondarily, all of which increase PDH activity. However, we found no acceleration of pyruvate decarboxylation by 0.5 and 1 microM free Ca2+ concentration, unless the mitochondria were uncoupled and ATP was added.

Biochemistry of the induction and prevention of lipoperoxidative damage in human spermatozoa.

Lipid peroxidation occurs in human sperm cells with damage to the cell plasma membrane, leading to loss of cytosolic components and hence to cell 'death'. The peroxidation may be induced at high rates in the presence of Fe2+ and ascorbate. It occurs at slower rates under physiological conditions as spontaneous lipid peroxidation, which has the following characteristics. The rate is constant over the time required for complete loss of motility in the cells of the sperm sample; one can thus use the time to complete loss of motility (TLM) as a ready measure of the rate. Loss of motility occurs at a characteristic extent of lipid peroxidation, assayed in terms of production of the peroxidative breakdown product, malonaldehyde (MA), that is independent of peroxidation rate. For human sperm, this extent corresponds to 0.1 nmol MA/10(8) cells. Human spermatozoa possess the anti-lipoperoxidative defence enzymes, superoxide dismutase (SOD) and glutathione peroxidase plus glutathione reductase (GPX/GRD). The SOD activity is highly variable between human sperm samples while the activities of GPX and GRD are rather more constant. The rates of production of superoxide anion, O2-, and hydrogen peroxide, H2O2, from human spermatozoa are variable, but their sum calculated in O2- equivalents as O2- + 2H2O2 is quite constant. The variability arises from the variability in SOD activity: all H2O2 produced is from O2- due to the action of SOD. The essential role of SOD as defence enzyme is inferred from the observation that TLM of a given sperm sample is directly proportional to the SOD activity of that sample. The essential role of GPX/GRD is inferred from the observation that inhibition of GPX, either with mercaptosuccinate or with complete oxidation of intracellular reduced glutathione, results in a 20-fold increase in peroxidation rate. The capacity of the GPX/GRD system appears to be limited by the glucose-6-phosphate dehydrogenase-catalysed rate of production of NADPH, the required reductive substrate for GRD. Human spermatozoa appear to have enough anti-lipoperoxidative defensive capacity for lifetimes long enough for fertilization but still short enough for ready removal from the female reproductive tract in good time. Too low a defence capacity could lead to male infertility.

Assessment of the anti-microbial agent C31G as a spermicide: comparison with nonoxynol-9.

The broad-spectrum anti-microbial agent, C31G, containing an equimolar mixture of n-dodecyl-dimethylamine-N-oxide (C12-N-O) and N-(n-dodecyl), N-dimethyl-glycine (C12-betaine), was tested for spermicidal activity in comparison with the currently used spermicide, nonoxynol-9 (N-9). The rate of sperm cell permeabilization by the spermicides, as assayed with the fluorescent probe, TO-PRO-1, increased as the cube of the C31G concentration, while the rate increase was linear with N-9 concentration. At 0.04%, the rate of sperm cell permeabilization with both spermicides is at the limit of rapid measurement. C31G diffuses through cervical mucus at a more rapid rate than does N-9. C31G has long been known to aid wound healing and reduce inflammation, whereas N-9 has been reported to induce vaginal irritation. C31G would, thus, seem to have the spermicidal efficacy, the broad range of anti-microbial activity, and the lack of inflammatory activity that is sought in the ideal vaginal spermicide.

A comparison of three methods for detecting the acrosome reaction in human spermatozoa.

This study was designed to compare three different fluorescent probes to assay the acrosome reaction in human spermatozoa: chlortetracycline (CTC), mannosylated bovine serum albumin (BSA) labelled with fluorescein (MAF), and quinacrine (QN). Normal human sperm ejaculates were washed and allowed to swim up for 30-60 min. Samples were examined under epifluorescence for the percentage of the acrosome reacted spermatozoa, as detected by the three probes. There was no significant differences between samples of fresh, uncapacitated spermatozoa evaluated with CTC, MAF or QN; all gave < 10% reacted. Following capacitation for 3 h, the percentage of spontaneously reacted spermatozoa was higher than in fresh spermatozoa; CTC and MAF gave the same percentage (12%), while QN indicated a higher percentage (18%) of reacted spermatozoa (P < 0.001). Following exposure to ionophore A23187 at 1 h, the percentage of acrosome reactions increased to a mean of 31% as detected with CTC or MAF; the mean percentage (45%) was significantly higher with QN (P < 0.0001). Further incubation up to 2 h with A23187 did not change these percentages. These results suggest that the QN probe detects the onset stage of the acrosome reaction, whereas the CTC and MAF probes detect the later stages in which the acrosomal cap is lost. Use of the two types of probe provides a means for finer resolution of the time course of the acrosome reaction in the human spermatozoa.

Continuous measurement of 13C16O2 production from [13C]pyruvate by intact liver mitochondria: effect of HCO3-.

We have measured continuously the production of mass 45 CO2(13C16O2) from 13C-labeled pyruvate in a guinea pig liver mitochondrial suspension and simultaneously the O2 consumption at 37 degrees C and pH 7.4. The reactions took place in a closed 3-ml volume, stirred, thermoregulated chamber separated from the ion source of a mass spectrometer by a gas-permeable membrane that permitted recording the mass peaks of any gas dissolved in the reaction mixture with a response time as fast as 3 s. If the pyruvate was labeled on C-2, no 13C16O2 was formed, even after 1 h, indicating that C-2 and C-3 were not metabolized in the citric acid cycle. We found that production of 13C16O2 was five times greater in the presence of 25 mM HCO3- than in its absence. A probable mechanism of this CO2/HCO3- effect is carboxylation of pyruvate to oxaloacetate, which would react with acetyl CoA to form citrate and with NADH to form malate, thus removing two major inhibitors of pyruvate dehydrogenase. We conclude that CO2/HCO3- has a potent and hitherto unappreciated regulatory effect on liver pyruvate dehydrogenase.

Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids of human spermatozoa.

Intact human sperm incorporated radiolabelled fatty acids into membrane phospholipids when incubated in medium containing bovine serum albumin as a fatty acid carrier. The polyunsaturated fatty acids were preferentially incorporated into the plasmalogen fraction of phospholipid. Uptake was linear with time over 2 hr; at this time sufficient label was available to determine the loss of fatty acids under conditions of spontaneous lipid peroxidation. Loss of the various phospholipid types, the loss of the various fatty acids from these phospholipids, and the overall loss of fatty acids were all first order. The loss of saturated fatty acids was slow with first order rate constant k1 = 0.003 hr-1; for the polyunsaturated fatty acids, arachidonic and docosahexaenoic acids, k1 = 0.145 and 0.162 hr-1, respectively. The rate of loss of fatty acids from the various phospholipid types was dependent on the type, with loss from phosphatidylethanolamine being the most rapid. Among the phospholipid types, phosphatidylethanolamine was lost at the greatest rate. Analysis of fatty acid loss through oxidation products was determined for radiolabelled arachidonic acid. Under conditions of spontaneous lipid peroxidation at 37 degrees C under air in the absence of albumin, free arachidonic acid was found in the medium, along with minor amounts of hydroxylated derivative. All the hydroperoxy fatty acid remained in the cells. In the presence of albumin, all the hydroperoxy fatty acid was found in the supernatant bound to albumin; none could be detected in the cells. Albumin is known as a very potent inhibitor of lipid peroxidation in sperm; its action may be explained, based on these results, as binding the damaging hydroperoxy fatty acids. These results also indicate that a phospholipase A2 may act in peroxidative defense by excising a hydroperoxy acyl group from phospholipid and providing the hydroperoxy fatty acid product as substrate to glutathione peroxidase. This formulation targets hydroperoxy fatty acid as a key intermediate in peroxidative degradation.

The temperature dependence in the hydraulic conductivity, Lp, of the mouse sperm plasma membrane shows a discontinuity between 4 and 0 degrees C.

Cryopreservation of sperm from transgenic mice would make possible the economical perpetuation of these genetic models. In order to design a protocol for this process, it was first necessary to determine the hydraulic conductivity, Lp, or water permeability, of the plasma membrane as a function of temperature to calculate its activation energy, Ea. This was done with sperm from the caudae epididymides of CD-1 mice, using the hyposmotic cell lysis method, in which the critical osmolality, Osmcrit, defined as the osmolality at which 50% of the cells lyse, was first determined, and then the time, t, to lysis of 50% of the cells in a medium of Osm < Osmcrit was obtained. Values (mean +/- SEM, n = 10) of Osmcrit at 37, 22, 4, and 0 degrees C were 32.1 +/- 2.7, 33.7 +/- 4.1, 30.8 +/- 3.4, and 101.0 +/- 10.8 mOsm, and of t in 17 mOsm medium were 40.6 +/- 2.4, 33.8 +/- 5.7, 25.0 +/- 4.4, and 4.7 +/- 1.2 s, respectively. Values of Lp were calculated from Osmcrit and t by two different algorithms, one based on the high-amplitude swelling model used with sperm from other mammalian species and the other based on a low-amplitude swelling model. Values of Lp in micron.min-1.atm-1 from the high-amplitude swelling model at 37, 22, 4, and 0 degrees C were 1.41 +/- 0.08, 1.88 +/- 0.35, 2.12 +/- 0.19, and 1.13 +/- 0.1, respectively. From the low-amplitude swelling model, the Lp values were 0.025 +/- 0.001, 0.040 +/- 0.008, 0.082 +/- 0.029, and 1.66 +/- 0.23. Examination of mouse sperm in hyposmotic media by microscopy revealed little swelling of the cells, indicating that the low-amplitude swelling model may be the one more applicable to these cells. The temperature dependence, and hence Ea, of Lp shows a marked discontinuity between 4 and 0 degrees C with values calculated from either model. This suggests a membrane phase transition to a more brittle structure in this temperature range, consistent with the observed marked increase in Osmcrit (P < 0.0003) and decrease in t (P < 0.0001) at 0 degrees C compared to the other temperatures. In the presence of 1 M glycerol, there was no discontinuity between 4 and 0 degrees C in the values of Osmcrit, consistent with the ability of glycerol to fluidize the membrane. Low hydraulic conductivity and low-temperature embrittlement of the plasma membrane are proposed as two factors leading to mouse sperm hypersensitivity to cryodamage.

Interactions between gametes leading to fertilization: the sperm's eye view.

Sexual reproduction requires that the gamete carrying the male-derived haploid chromatin join with the gamete carrying the female-derived haploid chromatin during fertilization to produce the diploid zygote. To accomplish this feat, the sperm must not only meet the egg, it must recognize the egg and be recognized in turn by the egg, and in the end must enter and be engulfed by the egg. In this selective overview of gamete interactions that lead to fertilization, encounters of three kinds, followed by the finale of gamete fusion, are considered from the sperm's viewpoint, with particular emphasis on the mammalian species with the mouse as the principal model. The first encounter is with the zona pellucida of the egg, to whose surface the sperm must bind. Mouse sperm appear to have four binding sites for zona ligands. Three interact with sugar moieties of the oligosaccharide chains of the mouse zona glycoprotein ZP3; the fourth binds a peptide backbone arginine. Capacitation is not required for this encounter, but is obligate for the second encounter--induction of the acrosome reaction in the bound sperm. The acrosome reaction is an exocytotic process that makes available the enzymatic machinery needed for sperm penetration the zona which is the end point of a sequence of reactions directed by intracellular signalling systems. In mouse sperm, these systems are presumed to be activated by ligands on ZP3 binding to ligand-specific sperm receptors with consequent aggregation of receptors. No receptor has been identified with certainty, nor have candidates for putative ZP3 ligands been identified. Completion of the acrosome reaction allows the sperm to penetrate the zona and, bind to the egg plasma membrane, thereby completing the third encounter. In the mouse, a 94-kDa protein appears essential for this binding. In the guinea-pig, a sperm plasma membrane protein (formerly PH-30, now fertilin), is a strong candidate for the mediator of the fusion process by which the egg engulfs the sperm. Decondensation of the sperm chromatin reverses the remarkable packing of DNA organized by sperm protamines. Mitochondrial DNA is also engulfed by the egg; the question of whether this DNA makes a small finite, or null, contribution to cytosolic inheritance is still in debate. The puzzles attending these encounters are presented as reminders of the intricacy and fascination, as well as of the vital necessity, of gamete interaction.

Solubilization and partial purification from mouse sperm membranes of the specific binding activity for 3-quinuclidinyl benzilate, a potent inhibitor of the zona pellucida-induced acrosome reaction.

3-Quinuclidinyl benzilate (QNB), a potent antagonist of muscarinic acetylcholine receptors, has been demonstrated to inhibit specifically the zona pellucida (ZP)-induced acrosome reaction (AR) in mouse sperm (Florman and Storey, 1982; Dev Biol 91:121-130). In this study we describe the solubilization and partial purification of the mouse sperm QNB binding activity which may represent a component of the putative receptor complex for ZP on the sperm plasma membrane. Sperm membranes were isolated from cell homogenates of washed, capacitated, epididymal mouse sperm. Scatchard plots of QNB binding to these membranes indicated a single class of binding sites with KD = 7.2 nM and Bmax = 8700 sites/cell. These binding characteristics are similar to those seen with QNB binding to whole cells (Florman and Storey, 1982, J Androl 3:157-164). Sperm membranes were solubilized using 1% digitonin/0.2% cholate, and the resultant detergent-soluble fraction possessed QNB binding activity similar to that of intact membranes. The detergent-soluble fraction maintained intact ZP receptor(s)-G protein coupling in that treatment of this fraction with either ZP or mastoparan resulted in a 35% or 65% increase in specific GTP gamma S binding, respectively. The solubilized membrane preparation was fractionated by gel permeation HPLC. A majority of specific QNB binding activity was confined to one HPLC fraction. Analysis of this fraction by SDS-PAGE revealed a complex of approximately 5 proteins unique to this fraction. The most prominent protein had a M(r) of 72 kDa, which is within the M(r) range for muscarinic receptors. A protein with M(r) = 41 kDa was also present within this fraction. Subsequent pertussis toxin (PTX)-catalyzed ADP-ribosylation of this fraction revealed this protein to be the alpha subunit of the G(i) class of G proteins. Although the QNB binding activity could not be positively identified, we propose that it is contained in one or more of the proteins unique to this fraction and that these proteins, including G(i), may act as part of a sperm receptor complex for the ZP.

Calcium influx into mouse spermatozoa activated by solubilized mouse zona pellucida, monitored with the calcium fluorescent indicator, fluo-3. Inhibition of the influx by three inhibitors of the zona pellucida induced acrosome reaction: tyrphostin A48, pertussis toxin, and 3-quinuclidinyl benzilate.

The fluorescent calcium indicator, fluo-3, was loaded as the membrane permeant tetraacetoxymethyl (AM) ester into cauda epididymal mouse sperm at 25 degrees C for 20 min in the absence of bovine serum albumin (BSA) and presence of the dispersant, Pluronic F-127. Excess indicator was removed by two centrifugation washes at 100g for 10 min, a procedure that did not impair sperm motility. Upon resuspension in medium containing 20 mg/ml BSA to promote capacitation, the sperm cells exhibited readily detectable fluorescence uniformly distributed in the cytoplasm. Cell fluorescence was stable over the time of the experiments and was responsive to changes in intracellular calcium concentration, [Ca2+]i. Initial [Ca2+]i was 231 +/- 58 nM (+/- SE, n = 43). Addition of heat-solubilized mouse zonae pellucidae to capacitated sperm increased [Ca2+]i by 106 +/- 19 nM (+/- SE, n = 18), the higher steady-state concentration being reached after 30 min. Subsequent addition of the non-fluorescent calcium ionophore Br-A23187 resulted in a further increase of 114 +/- 18 nM (+/- SE, n = 18), the higher steady-state concentration being reached after 6 min. The increase in [Ca2+]i induced by solubilized zonae pellucidae was largely blocked by 3-quinuclidinyl benzilate (QNB), an antagonist of muscarinic receptors that was earlier shown to block the zona pellucida induced acrosome reaction in mouse sperm (Florman and Storey, 1982: Dev Biol 91:121-130). This [Ca2+]i increase was completely blocked by the tyrosine kinase inhibitor, tyrphostin A48, and by the inactivator of G1 proteins, pertussis toxin. At the concentrations at which they blocked the zona pellucida-induced increase in [Ca2+]i, all three inhibitors also blocked the zona pellucida-induced acrosome reaction. These results indicate that [Ca2+]i increase in is an early, if not the initial, reaction in the sequence leading to zona pellucida induced acrosomal exocytosis in mouse sperm. The observation that the three inhibitors, each having a different mode of action, all block the zona pellucida induced [Ca2+]i suggests that the sperm plasma membrane receptors mediating the zona pellucida induced acrosome reaction may function as a complex, whose formation is activated by zona pellucida ligand binding.

Mechanism of superoxide dismutase loss from human sperm cells during cryopreservation.

Earlier studies on human sperm cryodamage have shown that plasma membrane stress is the primary process and that phospholipid peroxidation in cryopreserved samples is not inhibited by addition of antioxidants. One consistent effect of cryopreservation is loss of enzymatic activity of the peroxidation defense enzyme, superoxide dismutase (SOD). To clarify this aspect of the freeze-thaw process and to develop a more complete resolution of the reactions leading to cryodamage, we sought to identify which of the two most probable mechanisms, loss of enzyme protein from the cells of denaturation of the protein, operates. If the first operates, cellular enzymatic activity and enzyme protein as identified by immunocytochemistry should give a linear correlation. If the second operates, there should be no correlation. In this study, five individual samples were analyzed before and after cryopreservation for immunoreactive Cu/Zn SOD and cell intactness by flow cytometry, for SOD enzymatic activity by a highly sensitive fluorimetric method, and for motility characteristics by Hamilton-Thorn motility analyzer. Fresh samples were obtained by the "swim-up" method and had > 95% intact cells with > 78% motile cells. After freeze-thaw, about half the cells were intact. SOD enzymatic activity was determined on Triton X-100 cell extracts, a method that removes all enzymatic activity from the cell structure, and compared with immunoreactive SOD in the cells as determined by indirect immunofluorescence mean intensities. Residual immunofluorescence was observed in the cells after Triton X-100 treatment; if this was taken into account, a close linear correlation between SOD enzyme activity and SOD immunoreactivity was obtained (r = 0.90; P = 0.00014). There was no correlation between SOD enzyme activity ratios for cryopreserved and fresh cells and fraction of intact cells after freeze-thaw. We conclude that loss of SOD protein from the subset of cells undergoing acute membrane damage is the most probable primary mechanism of SOD enzymatic activity loss from the sample and that resistance to cryodamage and SOD activity in any given cell are quite independent of one another.

Selective activation of Gi1 and Gi2 in mouse sperm by the zona pellucida, the egg's extracellular matrix.

Mammalian sperm acrosomal exocytosis as induced by the egg's zona pellucida (ZP) appears to be a G protein-mediated event. Previously, we demonstrated that ZP3, the acrosome reaction-inducing component of mouse ZP, activates one or more Gi subtypes in isolated sperm membranes (Ward, C. R., Storey, B. T., and Kopf, G. S. (1992) J. Biol. Chem. 267, 14061-14067). To determine the identity of the Gi subtype(s) activated in the sperm, we examined ligand-dependent decreases in pertussis toxin-catalyzed in vitro [32P]ADP-ribosylation of the different Gi alpha subtypes that were identified by immunoprecipitation using Gi alpha-specific antisera. Membranes treated with solubilized ZP or mastoparan and subsequently [32P]ADP-ribosylated using pertussis toxin displayed 35 and 56% decreases, respectively, in [32P]ADP-ribosylation when compared with controls. These changes were quantitatively similar to the percentage increases in 35S-labeled guanosine 5'-O-(thiotriphosphate) binding in membranes treated with equivalent concentrations of ZP or mastoparan (Ward et al., 1992). Immunoprecipitation with Gi alpha subtype-specific antisera and subsequent autoradiography revealed that mastoparan activated Gi1, Gi2, and Gi3 in the membranes. ZP, in contrast, selectively activated Gi1 and Gi2. These data demonstrate that the egg's extracellular matrix has the ability to activate selectively sperm membrane Gi subtypes.