Effects of cold shock and phospholipase A2 on intact boar spermatozoa and sperm head plasma membranes

Head plasma membranes (HPM) isolated from cryopreserved boar spermatozoa show an excessive fluidization, which might be involved in the loss of fertility. The current study assessed the ability of cold shock (5 degrees C) and phospholipase A2 (PA2) to duplicate these effects on membrane structure and to affect 45Ca2+ uptake and gross morphological characteristics of whole, fresh boar-sperm. The HPM from cold-shocked sperm showed a significantly greater rate of fluidization over time than did HPM from control sperm. Addition of PA2 (bee or snake venom, 0.1 or 10.0 ng/ml) to HPM from control sperm caused fluidization similar to cold shocking, but to a lesser degree (P less than 0.05). Cold-shocked intact sperm exhibited severe acrosomal disruption, loss of motility, and increased 45Ca2+ uptake relative to control sperm. Addition of PA2 (bee or snake venom, 0.1, 1.0., 10.0, and 1,000 ng/ml) to control sperm had no effect on gross morphology or motility while maintaining or increasing sperm extrusion of 45Ca2+. Therefore, although PA2 can, to some extent, duplicate the effects of cold shock on HPM molecular organization, its lipid hydrolytic action is insufficient to cause all the gross disruptions of severe thermal shock. Both PA2 and cold shock disrupted HPM structure, but only cold shock increased 45Ca2+ uptake, suggesting that cold shock may be increasing 45Ca2+ uptake in areas other than the head. Cold shock disrupts sperm on three levels; membrane molecular organization, intracellular Ca2+ regulation, and gross morphology/motility.     

PZ-peptidase activity from ejaculated boar spermatozoa

Protein constituents of the boar spermatozoon were fractionated in three components, the hypotonic soluble fraction, the detergent-soluble fraction, and the detergent-insoluble fraction. When all these fractions were assayed spectrophotometrically using the PZ-peptide as substrate, a high value of PZ-peptidase specific activity was observed in the first fraction. Electrophoretic analysis at pH 8.3 of the protein content from the hypotonic soluble fraction revealed the existence of multiple molecular forms capable of hydrolysing the PZ-peptide. The major form was characterized by a surprisingly high value of electrophoretic mobility, index of the presence of numerous negatively charged residues. Biochemical and ultrastructural analyses showed that the hypotonic soluble fraction did not contain intrinsic, and specifically acrosomal, sperm enzymes.     

Effects of 2-hydroxypropyl-beta-cyclodextrin and cholesterol on porcine sperm viability and capacitation status following cold shock or incubation

Porcine sperm are extremely sensitive to the damaging effects of cold shock. It has been shown that cholesterol-binding molecules, such as 2-hydroxypropyl-beta-cyclodextrin (HBCD), improve post-cooling porcine sperm viability when added to an egg yolk-based extender, but also enhance sperm capacitation in other species. The objective of this study was to determine the effects of HBCD and cholesterol 3-sulfate (ChS) on porcine sperm viability and capacitation following cold shock or incubation under conditions that support capacitation using a defined medium. We report here that porcine sperm incubated in medium containing both HBCD and ChS have significantly improved viability following cold shock (10 min at 10 degrees C) when compared to sperm incubated without HBCD or ChS, or with either component alone. Treatment with HBCD plus ChS also completely inhibited the increase in protein tyrosine phosphorylation induced by the cold shock treatment or by incubation for 3 hr under conditions that support capacitation. Two assays of sperm capacitation, the rate of calcium ionophore-induced acrosome reactions and chlortetracycline (CTC) staining, were not significantly altered by HBCD and ChS following cold shock. However, 3-hr incubation with HBCD plus ChS or with 1 mM ChS alone decreased the percentage of sperm undergoing the induced acrosome reaction without significantly affecting viability when compared to the control. These results indicate that the manipulation of sperm plasma membrane cholesterol content affects porcine sperm viability and capacitation status and could therefore be useful to protect sperm from cold shock during cryopreservation by improving viability without promoting premature capacitation.     

Metabolism of glycerol by mature boar spermatozoa.

Mature boar spermatozoa oxidized glycerol to carbon dioxide in the absence of any detectable activity of glycerol kinase. With triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase inhibited by the presence of 3-chloro-1-hydroxypropanone (CHOP), dihydroxyacetone phosphate accumulated in incubates when glycerol-3-phosphate was the substrate, but not when it was glycerol. Both dihydroxyacetone and glyceraldehyde could be used as substrates; in the presence of CHOP, dihydroxyacetone phosphate and fructose-1,6-bisphosphate accumulated when dihydroxyacetone was the substrate, but not when it was glyceraldehyde. The metabolic pathways glycerol----glyceraldehyde----glyceraldehyde 3-phosphate and dihydroxyacetone----dihydroxyacetone phosphate have been shown to operate in these cells.     

Adsorption of seminal plasma proteins by boar spermatozoa

Seminal plasma protein adsorption by boar spermatozoa was examined using ejaculated sperm from vesiculectomized boars and seminal plasma from vasectomized boars. Sperm adsorbed 14 pg protein/sperm in 10 min. When seminal plasma proteins were radiolabeled, most of the adsorbed radiolabel was present in low M(r) proteins, particularly a 12700 M(r) protein. A 349300 M(r) seminal plasma protein was also readily adsorbed. Three radiolabeled seminal plasma proteins (307600, 165400 and 7400 M(r)) were not detected on the sperm; either they are not adsorbed by the sperm or the sperm were previously exposed to these proteins in other accessory sex gland fluids and had already adsorbed them. A 29100 M(r) sperm protein was also radiolabeled (4.9% of the adsorbed radiolabel), although there was no corresponding seminal plasma protein. Large quantities of seminal plasma protein (particularly low M(r) proteins) are adsorbed by sperm not previously exposed to seminal vesicle secretion. The functions of these proteins are yet to be determined.     

Fine structure of boar spermatozoa

Summary The fine structure of epididymal spermatozoa of boars was studied, with special regard to the head cytoplasm, the neck, and the axial filament. Epon embedding and staining with heavy metals was used.The acrosome consists of a moderately opaque, homogeneous substance bounded by a single membrane. Within the distinct equatorial segment, the acrosome is very thin and separated from the nuclear membrane by a narrow rim of moderately dense material, which may be related to the perforatorium of rat spermatozoa.The postnuclear cap consists of a dense, homogenous substance inside the cell membrane and is stainable with phosphotungstic acid.The fibre structures of the neck are surrounded by folded extensions of the nuclear membrane. Two short, dark rods appear in the centre of the neck. The light segments of the coarse, peripheral fibres are merely deep notches in the fibre substance. The coarse peripheral fibres reach their maximal thickness at the anterior end of the middle piece. They taper rapidly anteriorly from this point and more gradually posteriorly. Irregular bridges connect them with each other in the anterior middle piece.The central 9+2 fibrils of the axial filament have distinct arms and spokes in the middle piece and main piece. The subfibrils connected with the arms and spokes appear to be solid, except in the neck and end pieces. The two central fibrils run through the neck to the wall of the proximal centriole. Acknowledgements. We wish to express our sincere gratitude to Dr. B. Afzelius, the Wenner-Gren Institute, Stockholm, for helpful discussions regarding tail fine structure, and to Dr. J. Luft, Department of Anatomy, University of Washington, Seattle, Wash., U.S.A., for the generous supply of ingredients for the Epon embedding procedure.     

Isolation and characteristics of aspartate aminotransferase from boar spermatozoa

1. Gel electrophoresis of aspartate aminotransferase released from boar spermatozoa after cold shock showed one band migrating towards anode. 2. Physico-chemical and kinetic properties of isolated enzyme were similar to cytoplasmic isoenzyme of AAT from somatic tissues.     

Ultrastructural abnormalities of boar spermatozoa

Described here are the main ultrastructural malformations observed in spermatozoa of ejaculates collected from healthy, adult Landrace boars following 2 days of sexual abstinence. Previously semen had been collected 3 times per week. Sperm concentration in the cell-rich fraction of ejaculates was approximately 700,000 sperm/mm(3). The aberrant gamete forms did not exceed 2% of the total number of spermatozoa. Ultrastructural anomalies of spermatozoa were classified into 2 groups: head malformations and tail malformations. These consisted of: 1) spermatozoa with expanded and vacuolated acrosomes, 2) spermatozoa with myelin figures within the perinuclear space, 3) macrocephalic spermatozoa with 2 nuclei and a deformed acrosomal vesicle, 4) spermatozoa with an expanded acrosomal apex, 5) spermatozoa with nuclear vacuoles, 6) macrocephalic spermatozoa with a roundish head, 7) spermatozoa with swollen mitochondria, 8) spermatozoa with additional mitochondria over the mitochondrial sheath, 9) spermatozoa without the central microtubular pair, 10) spermatozoa without some peripheral doublets, 11) spermatozoa with 1 or 2 coiled tails, 12) spermatozoa with a folded tail and a disorganized connecting piece, 13) spermatozoa with a vesiculated tail, and 14) spermatozoa with 2 tails fused by their respective mitochondrial sheaths.     

Substrates for endogenous metabolism by mature boar spermatozoa

Washed boar spermatozoa incubated in the absence of exogenous substrates maintained a high energy charge potential (ECP) for at least 10 h. Addition of bromopyruvate, an inhibitor of stage 2 of the glycolytic pathway, at any time during the incubation caused an immediate decrease in the ECP, indicating that the mobilization of endogenous compounds requires this section of the pathway for the production of lactate, the major mitochondrial substrate for ATP production. Some of the sources of the metabolic substrates have been identified, by NMR and metabolic studies, as di- or triglycerides, to produce glycerol, and membrane phospholipids for the production of glycerol 3-phosphate. Acetylcarnitine contributes acetyl groups early in the incubation; glycerylphosphorylcholine is degraded to glycerol 3-phosphate and choline after about 5 h, and acetate also accumulates after about 5 h. The presence of phosphorylcholine and phosphorylethanolamine later in the incubation indicates that phospholipids are also degraded to glycerol.     

In vitro phagocytosis of boar spermatozoa by neutrophils from peripheral blood of sows

A considerable number of spermatozoa are used in each sow in routine artificial insemination. However, within a few hours after insemination, many spermatozoa are phagocytosed by polymorphonuclear leucocytes. Some aspects of sperm transport in the female genital tract in the sow have been thoroughly investigated, whereas little is known about the mechanisms involved in the phagocytosis of spermatozoa, or about which spermatozoa (fresh, capacitated or dead) are the most susceptible to ingestion by polymorphonuclear leucocytes. In this study, phagocytosis was investigated by use of an in vitro phagocytosis assay. Polymorphonuclear leucocytes were challenged with either untreated, cold-shocked or frozen-thawed spermatozoa, or with spermatozoa that had been treated to induce capacitation in vitro. The influence of serum on phagocytosis was also investigated. Treatment of the semen to induce capacitation in vitro considerably reduced the phagocytosis of spermatozoa, whereas crude treatments like cold-shock or freezing and thawing reduced phagocytosis only in the first 15-30 min of incubation with polymorphonuclear leucocytes. Viable spermatozoa were phagocytosed mainly through a pathway that was independent of complement or other serum components (for example, antibodies). Complement had little effect on phagocytosis of spermatozoa, but did cause acrosomal exocytosis and cell death.     

Loss of acrosin from bovine spermatozoa following cold shock: Protective effects of seminal plasma

Loss of the alkaline proteinase acrosin and other proteins from the acrosome of bovine spermatozoa was investigated following cold shock and/or incubation of the spermatozoa at either 5, 21, or 37 °C for 4 hr. As detected by electrophoretic analyses of the acrosomal material two bands of acrosin activity and 10 proteins were lost from the acrosome after cold shock and incubation for 4 hr at 5 or 21 °C, whereas one acrosin band and 10 protein bands were lost after cold shock and incubation at 37 °C. Only 45% of the total acrosin activity remained in the acrosome after both cold shock and 4-hr incubation at 37 °C. Egg yolk, present at levels above 15%, and seminal plasma prevented much of the loss of acrosin from the cells.     

Genotype-independent transmission of transgenic fluorophore protein by boar spermatozoa

Recently, we generated transposon-transgenic boars (Sus scrofa), which carry three monomeric copies of a fluorophore marker gene. Amazingly, a ubiquitous fluorophore expression in somatic, as well as in germ cells was found. Here, we characterized the prominent fluorophore load in mature spermatozoa of these animals. Sperm samples were analyzed for general fertility parameters, sorted according to X and Y chromosome-bearing sperm fractions, assessed for potential detrimental effects of the reporter, and used for inseminations into estrous sows. Independent of their genotype, all spermatozoa were uniformly fluorescent with a subcellular compartmentalization of the fluorophore protein in postacrosomal sheath, mid piece and tail. Transmission of the fluorophore protein to fertilized oocytes was shown by confocal microscopic analysis of zygotes. The monomeric copies of the transgene segregated during meiosis, rendering a certain fraction of the spermatozoa non-transgenic (about 10% based on analysis of 74 F1 offspring). The genotype-independent transmission of the fluorophore protein by spermatozoa to oocytes represents a non-genetic contribution to the mammalian embryo.     

Identification of capacitation in boar spermatozoa by chlortetracycline staining

The functional status of boar spermatozoa undergoing capacitation in vitro was investigated. Two fluorescent stains were used: chlortetracycline (CTC) and a FITC-conjugated lectin (FITC-PSA). The first has been used for the direct identification of the capacitated boar spermatozoa, while the second, based on the identification of capacitated spermatozoa by their ability to undergo zona-induced acrosome reaction (AR), was used to confirm and validate the CTC assay in this species. Spermatozoa obtained from 5 different boars was washed and incubated under capacitating conditions. Aliquots of spermatozoa were collected at 0, 90 and 180 min of incubation and then stained with CTC or FITC-PSA. After CTC staining, 3 different fluorescent patterns were observed: Pattern A with the fluorescence uniformly distributed on the sperm head, Pattern B with the fluorescence concentrated in the post-acrosomial region, and Pattern C with the fluorescence concentrated in the acrosomial region. The percentage of spermatozoa displaying fluorescent Pattern A decreased throughout the incubation while that of spermatozoa with Pattern C showed a concomitant progressive increase. Pattern B fluorescence remained unchanged throughout the maturation period. Exposure to zonae pellucidae (ZP) brought back the levels of Pattern C fluorescence to basal values. Since only the capacitated spermatozoa are believed to react to ZP, this observation together with the rising incidence of Pattern C throughout maturation suggests that fluorescence in the acrosomial region identifies capacitated spermatozoa. The analysis of acrosome integrity carried out with FITC-PSA showed that the proportion of zona-induced AR was nearly the same as that of spermatozoa displaying Pattern C, thus confirming that CTC staining is suitable for the detection of boar sperm capacitation. In the second part of this study, CTC was used to investigate the effects of sperm origin and storage on the capacitation process. Our finding demonstrates that capacitation kinetics show wide variations in sperm samples derived from different boars; moreover, capacitation is also affected by sperm storage. While fresh semen showed a progressive increase in capacitated spermatozoa, ranging from low levels at the beginning of the culture to 46% at the end of incubation, the refrigerated semen had a relatively high percentage of capacitated spermatozoa at the beginning of culture, but this proportion increased only slightly during the following 90 to 180 min of treatment. These data indicate that CTC can be used to identify capacitated boar spermatozoa, and, because of its rapid and easy execution, it can be used routinely to identify the optimal capacitation time for different sperm samples.     

Detrimental effects of non-functional spermatozoa on the freezability of functional spermatozoa from boar ejaculate

In the present study, the impact of non-functional spermatozoa on the cryopreservation success of functional boar spermatozoa was evaluated. Fifteen sperm-rich ejaculate fractions collected from five fertile boars were frozen with different proportions of induced non-functional sperm (0--native semen sample-, 25, 50 and 75% non-functional spermatozoa). After thawing, the recovery of motile and viable spermatozoa was assessed, and the functional of the spermatozoa was evaluated from plasma membrane fluidity and intracellular reactive oxygen species (ROS) generation upon exposure to capacitation conditions. In addition, the lipid peroxidation of the plasma membrane was assessed by the indirect measurement of malondialdehyde (MDA) generation. The normalized (with respect to a native semen sample) sperm motility (assessed by CASA) and viability (cytometrically assessed after staining with Hoechst 33342, propidium iodide and fluorescein-conjugated peanut agglutinin) decreased (p<0.01) as the proportion of functional spermatozoa in the semen samples before freezing decreased, irrespective of the semen donor. However, the magnitude of the effect differed (p<0.01) among boars. Moreover, semen samples with the largest non-functional sperm subpopulation before freezing showed the highest (p<0.01) levels of MDA after thawing. The thawed viable spermatozoa of semen samples with a high proportion of non-functional spermatozoa before freezing were also functionally different from those of samples with a low proportion of non-functional spermatozoa. These differences consisted of higher (p<0.01) levels of intracellular ROS generation (assessed with 5-(and-6) chloromethyl-20,70-dichlorodihydrofluorescein diacetate acetyl ester; CM-H(2)DCFDA) and increased (p<0.01) membrane fluidity (assessed with Merocyanine 540). These findings indicate that non-functional spermatozoa in the semen samples before freezing negatively influence the freezability of functional spermatozoa.     

Identification of a zona-binding protein from boar spermatozoa as proacrosin

The initial stages of fertilization in vertebrates and invertebrates are thought to involve complementary recognition molecules on spermatozoa and eggs. In a previous work (C. R. Brown and R. Jones, 1987, Development) we described one such putative molecule (a protein of approximate molecular weight 53 kDa) in detergent extracts of boar spermatozoa that has affinity for glycoproteins from the zona pellucida of pig eggs. This molecule has now been identified as proacrosin, the zymogen form of the acrosomal protease acrosin, on the basis of its electrophoretic behavior, the ability of zona glycoproteins to recognize and bind to proacrosin on Western blots, and the cross-reactivity of specific antisera to the 53-kDa molecule and proacrosin. A role is proposed for this enzyme in binding the sperm head to the zona pellucida during the initial stages of sperm-egg interaction.     

Isoelectric focusing of boar spermatozoa.

The isoelectric points of washed spermatozoa from intact boars and from boars after removal of the seminal vesicles were determined using isoelectric focusing on natural pH gradients. Normal boar spermatozoa focused at a higher pH than spermatozoa from boars without seminal vesicles. The isoelectric point of the latter was increased to a value approaching normal by preincubation in normal seminal plasma. This indicates that seminal plasma alters the membrane surface charge of boar spermatozoa on ejaculation.