Roy H. Hammerstedt

Cryopreservation of poultry sperm: the enigma of glycerol.

This review summarizes recent data for cryopreservation of poultry sperm and data establishing the contraceptive effect of glycerol. Successful cryopreservation protocols for bovine sperm are compared to the requirements for rooster sperm, with emphasis on glycerol-induced alterations in avian reproductive systems. It has been shown that molar concentrations of glycerol can affect (a) physical features of the cytoplasm (cytoplasmic organization and viscosity), (b) permeability and stability of the membrane bilayer(s), and (c) noncovalent attachment of proteins to the sperm surface. Perturbing effects of glycerol on sperm metabolism and the essentiality of maintaining bioenergetic balance during the temperature changes associated with any cryopreservation protocol are discussed. Emphasis is placed on the processes in avian reproduction that may be altered by interactions with glycerol. Finally, we discuss the potential value of using available genetic models (lines of roosters differing in the capacity of their sperm to survive a freeze-thaw cycle) to clarify and overcome damage to poultry sperm induced by cryopreservation.

Differential effects of butylated hydroxytoluene analogs on bull sperm subjected to cold-induced membrane stress.

Previous reports established that butylated hydroxy toluene (BHT) minimized cold-induced membrane rupture in sperm from several species. No data regarding the specificity of its effect is available. In this study 25 BHT analogs were tested for their effect on bovine sperm membrane stability. Fourteen were membrane lytic at 25 degrees C and 6 were neither membrane lytic nor membrane stabilizing. The remaining 5 compounds, a family of 2,6-tert-butyl phenols with substitutions at position 4 of hydrogen, methyl (BHT), ethyl, butyl, hexyl, or octyl, afforded effective membrane protection to cold shock. Since membrane protection is a function of both the ability of a compound to partition into the membrane and a molecules effectiveness once there, an analysis of each analogs membrane partitioning, assessed by measuring the cellular analog/cholesterol ratio, showed the following extents of transfer for the analogs: ethyl = butyl greater than methyl = hydrogen greater than hexyl greater than octyl. Thus, an optimum chain length exists for partitioning from micellar donors into cells. A separate experiment established that all analogs, when incorporated in equivalent amounts, protect equally plasma and mitochondrial membranes from cold shock. No effect on acrosomal membrane stability was noted. BHT, but not the other analogs, reduced sperm motility. Addition of egg yolk to extender containing BHT analog protected sperm motility from cold shock but had little effect on membrane stabilization. Analysis of sperm membrane compartments revealed that little to no analog was partitioned into the outer acrosomal membrane or the plasma membrane overlying the acrosome, but rather was localized in other portions of the sperm. We conclude that (a) the effective BHT analogs, if partitioned into the membrane, are indistinguishable with regard to their capacity to eliminate cold-induced membrane lysis; (b) membrane-linked events (e.g., motility) are uniquely disrupted by a subset of this analog family; and (c) when concentrations of egg yolk and BHT analogs are carefully controlled, unique synergistic effects are noted.

Artificial induction of exocytosis in bull sperm

We have investigated an exocytotic event, the acrosome reaction (AR), induced by treatment of bovine sperm with vesicles composed of dilauroyl phosphatidylcholine (PC12). Cell membrane permeability barriers (dye exclusion), acrosomal status (pisum sativum (PSA) lectin binding), and intracellular Ca2+ (Fluo3 fluorescence) were evaluated utilizing flow cytometry and fluorescence microscopy. By these methods the AR is resolved into four kinetically distinct steps: (a) PC12 transfer to the sperm plasma membrane (PM); (b) increased permeability of the PM to extracellular Ca2+; (c) localized leakage of acrosomal contents at the anterior tip of the sperm; and (d) vesiculation of sperm membranes and complete exposure of acrosomal contents. Evidence for PC12 transfer to sperm includes transfer of a fluorescent PC12 analogue from vesicles to cells and the absence of detectable vesicle--cell fusion. The fusion inducing properties of PC12 appear to reside in the lipid head group as neither dilauroylphosphatidylethanolamine nor dilauroylphosphatidylglycerol stimulated the AR. The effect of PC chain length on AR induction closely parallels the aqueous phase solubility of the lipid tested. The rate and extent of the AR depend on the extracellular calcium concentration. Cells treated in the absence of calcium do not undergo the AR, but do so rapidly (less than 1 min) upon subsequent addition of calcium. This role of Ca2+ is partially filled by Sr2+, but not by Ba2+ or Mg2+. The rate of the AR decreases with decreasing temperature and the AR occurs very slowly below 27 degrees C. Simultaneous evaluation of intracellular calcium and acrosomal status reveals the kinetic relationship between Ca2+ influx and the exposure of acrosomal contents. N-Ethylmaleimide preincubation arrests PC12-treated sperm at an intermediate stage in the AR, characterized by punctate PSA binding over the tip of the sperm head. The AR, a developmentally regulated, receptor-mediated fusion event, synchronously induced here in vitro, provides a useful model for mechanistic studies of exocytosis.

Evaluation of sperm quality: Identification of the subfertile male and courses of action

Abstract Use of semen analysis to predict fertility in animal agriculture has yielded a vast literature but no test yielding accurate predictions. Reasons for this disappointing conclusion are complex, but include: (a) unrealistic goals; (b) incomplete inclusion of emerging information on the steps in fertilization into design of an assay; (c) erratic assay technique; and (d) difficulty in explicitly defining success. Each topic is treated in this presentation. Three possible goals for an assay are to: (a) identify subfertile males (can be achieved); (b) estimate current fertility of a male of previously known fertility (may be possible) and (c) identify males who will have superior fertility (impossible). Steps in the fertilization process are analyzed in terms of identifying potential assay methods with maximum predictive power. Sources of assay error are considered, with their effects on assay conclusions listed. If assays are considered in terms of end application, established techniques for developing decision matrices can be used to optimize use of assay output information. Will the information be used as a basis for the culling of otherwise valuable animals or to identify all animals likely to benefit from therapeutic treatments to enhance probability of fertilization? Examples of the latter case are provided.

Changes in ram sperm membranes during epididymal transit.

Abstract The sperm plasma membrane consists of three zones: a lipid bilayer that provides an impermeable barrier to uptake of extracellular, polar molecules; a charged lipid-water interface formed by the phospholipids and membrane-associated proteins; and a charged glycoprotein calyx with absorbed proteins. We quantitatively evaluated all three zones of membranes in testicular, cauda epididymal, and ejaculated ram sperm. Integrity of the lipid bilayer was assessed by measuring the aqueous isolated intracellular volume with water-soluble spin labels and electron spin resonance spectroscopy. Effects of rapid cooling to 0 °C (cold shock), of freezing on solid CO2, and of butylated hydroxytoluene (BHT) on membrane integrity were evaluated. Testicular, cauda epididymal, and ejaculated sperm all had an isolated intracellular volume of 20–24 μm3 per sperm. Isolated intracellular volume of testicular sperm was maintained (>90%) after cold shock, but reduced ~50% by freezing. BHT (1–3 m m ) did not protect testicular sperm against membrane damage. Cauda epididymal and ejaculated sperm retained 20–30% of their isolated intracellular volume after cold shock but not freezing. However, 4 m m BHT prevented freeze damage of ejaculated but not cauda epididymal sperm. The lipid-water interface of the membrane was studied using surface-directed spin labels; the negative charge density was found to increase during epididymal transit. Net charge of the glycoprotein calyx was detected using whole-cell isoelectric focusing. Testicular, cauda epididymal, and ejaculated sperm isofocused at pH 5.2, 4.7, and 4.8 after insertion at pH 6 into the preformed pH gradient. Mixtures of testicular plus cauda epididymal or testicular plus ejaculated sperm were separable. We conclude that each of the three zones of the plasma membrane of sperm changes during epididymal maturation.

An automated method for ATP analysis utilizing the luciferin-luciferase reaction.

Abstract An automated method for the analysis of ATP has been described. This method uses the luciferin-luciferase reaction, with the firefly extract being delivered by means of an automatic pipetter interfaced with an automatic liquid scintillation spectrometer. A range of 10–300 pmoles is obtained with a standard deviation of 5·10%. An analysis rate of approximately 200 samples per hr can be attained. At least 1400 samples can be run from one standard curve when the firefly extract is stabilized with bovine serum albumin and mercaptoethanol.

Effect of incubation temperature on motility and cAMP content of bovine sperm

Abstract We have used a change in temperature to vary sperm motility in order to see if changes in glucose consumption and cellular concentration of ATP, ADP, AMP, and cyclic AMP (cAMP) are correlated with the temperature-dependent control of motility. Effect of temperature on the kinetic parameters of adenylate cyclase and cyclic nucleotide phosphodiesterase also were studied. Glucose consumption rate was independent of adenine nucleotide concentration or energy charge. Furthermore, the percentage of progressively motile sperm and velocity of motile sperm were independent of mean cAMP concentration, in contrast to previously published data presented as evidence for the modulation of progressive motility of sperm via changes in cAMP concentration.

Use of amphiphilic spin labels and whole cell isoelectric focusing to assay charge characteristics of sperm surfaces

Abstract The charge characteristics of the surface of bull and rabbit sperm were analyzed using surface-directed spin labels and whole cell isoelectric focusing. Spin label experiments tested charges believed to be localized at the membrane phospholipid-water interface. Charge properties of the glycoprotein calyx were analyzed with isoelectric focusing. Addition of charged detergents altered spin label spectra without changing the isoelectric focusing pH value. Sperm presumed to differ in the amount of adsorbed protein had different isoelectric focusing pH values, but similar spin label spectra. We conclude that these techniques are capable of monitoring charge domains on the sperm surface: one at the polar surface of the phospholipid membrane and one at the interface between the glycocalyx and the suspending fluid. Furthermore, changes in charge density are induced in unique zones of the cell surface during sperm maturation.

Spin labels for cell surfaces.

We describe the use of spin labels that localize on the outer surfaces of membranes. We use yeast as an example of a simple eucaryotic cell system. These labels should be very useful in probing the surfaces of a variety of vesicle and cell preparations. Most spin labels designed to probe membranes penetrate throughout the membrane system resulting in an average signal of all membranes present. It is necessary to use such probes as the ones described here in order to detect many of the interactions or modifications that appear to be localized on the surfaces of cells. These probes offer the opportunity of detecting physical state changes on or near the membrane surface. In recent years it has been well established that the cell surface is the site of many unique and important events. Virus attachments, antigen-induced antibody formation, hormone binding, lectin recognition, cel lcell recognition, and contact inhibition are all regarded as cell surface events. Spin labels have become widely used as probes of membrane structure anc, function. Most studies of membranes using spin labels measure a signal that reflects an average of both halves of the bilayer and a more general average of all membranes in a given system. Spin labels may have specificity for localizing in a dielectric zone, such as near the membrane interface for some sterol [1] and aliphatic hydrocarbon labels [2], or at differing depths in bilayers depending on the nitroxide position on spin label fatty acids [3]. The spin labels we describe here localize only on the outer surface of the plasma membrane. 2. Materials and methods

Alterations in motility and metabolism associated with sperm interaction with accessory sex gland fluids

Sperm passing through the male tract interact with accessory sex gland fluids during ejaculation. Cellular metabolism is stimulated by this interaction for unknown reasons. These experiments involved calorimetric measurements [P.B. Inskeep and R.H. Hammerstedt (1983) J. Biochem. Biophys. Methods 7, 199-210] on ejaculated sperm (EJS) and cauda epididymal sperm (CES) from bulls to establish the contribution of individual pathways to total cellular ATP synthesis. Parallel incubations outside the calorimeter yielded samples for oxygen consumption measurements and for motility analysis, the major ATP-consuming reaction of sperm. Energy charge values were identical for incubations of EJS and CES with glucose, thereby establishing that the ratios of rates of ATP synthesis and degradation were equivalent for these cells under this incubation condition. The total rate of ATP synthesis was greater for EJS than for CES (5 vs 13 mumol ATP h-1/10(8) cells) with less than 2 mumol ATP h-1 for each cell type coming from degradation of endogenous reserves. Thus, ejaculation is associated with a large increase in catabolic rate that is satisfied by degradation of extracellular glucose. No difference in percentage of motile sperm was noted, but mean velocity was lower for CES (58 micron s-1) than for EJS (85 micron s-1). A difference in forward motility pattern was observed (wig-wag to flipping). We conclude from these data that interaction with accessory sex gland fluids alters ATP requiring activities of sperm, with one obvious alteration being their motility pattern. The increase in ATP requirement is satisfied by increased degradation of extracellular substrates, but not intracellular reserves, to provide sufficient ATP to satisfy cellular needs.