Boar Sperm Plasma Membranes Research Articles

Effects of riboflavin on boar sperm motility, sperm quality, enzyme activity and antioxidant status during cryopreservation.

ObjectivesThis study was conducted to evaluate the effect of adding riboflavin to boar sperm freezing extender on the challenge of cryopreservation.MethodsDifferent concentrations (0, 5, 10, 15, 20 or 25 μM) of riboflavin were added to the freezing extender. Spermatozoa motility, membrane integrity, acrosomal integrity, mitochondrial membrane potential and enzyme activities were analysed once 10 min after thawing. Q‐PCR was used to detect the mRNA expression of Caspase3, Bcl‐2 and Bax.ResultsThe results showed that the addition of 10 μM riboflavin to boar sperm freezing extender significantly increased the frozen‐thawed sperm progressive motility compared with the control group (p < 0.05). Activities of superoxide dismutase, glutathione peroxidase and catalase improved after adding riboflavin to the extender (p < 0.05). During freezing‐thawing, the boar sperm mitochondrial membrane potential, acrosomal integrity, plasma membrane and DNA at 10 μM in the riboflavin group increased by 6.6%, 9.6%, 5.49% and 5.62% (p < 0.05), respectively, compared with the control group. The addition of 10 μM riboflavin to the extender significantly decreased the malondialdehyde (p < 0.05) content, whereas it increased the ATP content (p < 0.05) of boar sperm during freezing‐thawing. Furthermore, the expression of Caspase‐3 and Bax (p < 0.05) were significantly lower, whereas the expression of BCL‐2 (p < 0.05) was greater than the control group when adding 10 μM riboflavin to the extender.ConclusionsRiboflavin showed cryoprotective capacity to the freezing extender used for boar sperm during the process of freezing‐thawing, and the optimal concentration of riboflavin for the frozen extender was 10 μM.

Relationship between sperm plasma membrane integrity and morphology and fertility following artificial insemination

Sperm quality plays an important role in determining fertility. The aim of the study was to examine the relationship between sperm plasma membrane integrity and morphology, and fertility following artificial insemination (AI). A total of 16 ejaculates were collected from three Large White boars using the gloved hand technique. The semen was extended with a commercial extender. The AI dose contained 80 mL semen sample (3 × 10 9 sperm/mL). Aliquots of diluted semen were assessed for sperm plasma membrane integrity (synthetic binding CD-14 (SYBR+)/propidium iodide (PI-) and sperm morphology (eosin nigrosin). A total of 73 Duroc-type, Large White and nondescript multiparous sows from smallholder farms were inseminated with extended semen samples. Boar sperm plasma membrane integrity and morphology were subjected to one-way analysis of variance (ANOVA). The average boar sperm plasma membrane integrity and normal sperm morphology were 78.6% and 77.2%, respectively. The average conception and farrowing rates following artificial insemination (AI) were 78.1 and 57.5%, respectively. A negative correlation was observed between sperm plasma membrane integrity and fertility. There was a weak positive correlation between normal sperm morphology and conception rate (r = 0.11). Additionally, a relationship was observed between normal sperm morphology and litter size (r = 0.37) and total number born alive (r = 0.03), although relatively low. In conclusion, a negative relationship was found between sperm plasma membrane integrity and fertility. Moreover, there was a relationship between morphologically normal sperm and litter size, as well as number of piglets born alive, although relatively low. Keywords : Boar, Eosin Nigrosin, Semen quality, SYBR14/PI

The mu (μ) and delta (δ) opioid receptors modulate boar sperm motility.

Endogenous and exogenous opioids modulate reproductive functions in target cells via opioid receptors (μ, δ, and κ). Sperm motility is a metric of gamete functionality, and serves as a suitable parameter for in vitro drug-induced toxicity assays. This study identifies the presence and location of opioid receptors in pig spermatozoa as well as their functional response after in vitro challenge with known agonists (morphine [μ]; [D-Pen 2,5]-enkephanile [δ]; and U 50488 [κ]) and antagonists (naloxone [μ]; naltrindole [δ]; and nor-binaltrorphimine [κ]). Only the μ- and δ-opioid receptors were present in the boar sperm plasma membrane, overlying the acrosome, neck, and principal piece. Challenge experiments with agonists and antagonists identified both μ- and δ-opioid receptors as regulators of sperm kinematics, wherein μ maintains or increases sperm movement whereas δ decreases sperm motility over time. Mol. Reprod. Dev. 83: 724-734, 2016 © 2016 Wiley Periodicals, Inc.

Multiple receptors for sulfated Lewis X trisaccharide and sialylated N‐acetyllactosamine are present on the boar sperm plasma membrane contributing to oviduct reservoir formation

After insemination, a sperm reservoir is established in the oviduct by sperm binding to oviductal glycans containing sulfated Lewis X (sLeX) and biantennary sialylated N‐acetyllactosamine (SiLN) motifs. Based on a binding assay to fluoresceinated sLeX, SiLN or N‐acetyllactosamine (LN) glycans, we localized glycan receptors on the sperm plasma membrane. Fluorescent staining revealed two binding regions: the anterior head region (AR) or both the anterior and posterior region (APR). sLeX bound preferentially to the AR (71.0% of sperm) and LN to the APR (98.9% of sperm) whereas SiLN bound to both regions, suggesting the involvement of two distinct receptors. Sperm preferentially bound to sLeX and SiLN compared to LN (61.5, 61.7 and 25.2% of sperm bound respectively, P=0.001), indicating the necessity of sialic acid and multivalency for optimal glycan recognition. Glycan blot analysis identified several proteins that bind specifically to sLeX with apparent molecular weights of 250, 130, 80, 55 and 47 kD. Bands of 70 and 37 kD recognized sLeX and SiLN, whereas a 15kD band recognized all three motifs. Based on these results, we determined that sperm recognition of the sLeX motif is mediated by multiple receptors that may also recognize the SiLN motif. We propose a binding model in which Lewis X and SiLN receptors are components of sperm adhesion to the oviduct. Supported by USDA Grant no. 2011–67015‐20099.

The protective effect of a 17 °C holding time on boar sperm plasma membrane fluidity after exposure to 5 °C

The holding time (HT) is the period during which an ejaculate, either in a raw or diluted state, is held at 17°C before further processing for cold-storage. In boars, the HT positively influences select sperm quality parameters of semen cooled from 15 to 5°C, a range in temperature during which plasma membrane remodeling occurs. Objective insight into the effect of HT on plasma membrane organization remains unknown. Therefore, the present work sought to elucidate if HT contributes to minimizing alterations in boar sperm plasma membrane fluidity at the initial step of the cooling process in a cryopreservation practice (holding at 5°C) and in relation with select sperm quality parameters. Nineteen ejaculates from five boars were collected and processed according to different treatments: T1) Fresh diluted semen, 0h at 17°C; T2) Fresh diluted semen, 24h at 17°C (HT); T3) Sperm from T1 in a lactose-egg yolk (LEY) extender, 3h at 5°C; T4) Sperm from T2 in LEY, 3h at 5°C; T5) Sperm from T1 in LEY, 24h at 5°C; T6) Sperm from T2 in LEY, 24h at 5°C. Sperm motility was assessed using CASA, and sperm plasma membrane integrity and fluidity were evaluated by flow cytometry with dual labeling (M540/YO-PRO®-1). Results demonstrated that the lack of exposure to a HT (T5) results in reduced sample motility compared to those having a HT (T6), with sperm exposed to HT exhibiting less plasma membrane fluidity. Collectively, these results provide empirical evidence that incorporation of a HT in semen processing protects boar sperm against cold injury through maintenance of lipid architecture of the plasma membrane.

The Effects of Dietary Flaxseed on Fresh and Stored Boar Sperm Parameters.

Storage of fresh liquid boar semen for artificial insemination is associated with decreased sperm fertility, viability and motility over time. Boar sperm plasma membranes contain approximately 60% long chain poly-unsaturated fatty acids (PUFA) and the omega-3 (n-3) and omega-6 (-6) fatty acids are essential PUFA which cannot be synthesized by the animal. It has been proposed that mammals evolved on a dietary n-3 to n-6 ratio of 1:1, but the ratio in typical boar diets is 1:6, risking sub-optimal supply of long chain n-3 fatty acids for spermatogenesis. This study evaluated dietary flaxseed as a source of n-3 fatty acids on fresh and stored boar sperm. Three boars were fed for 3wk according to NRC (1998) recommendations for breeding boars, followed by an iso-caloric and iso-nitrogenous diet containing 15% flaxseed for 9 weeks. Semen was collected 2× per week and analyzed for concentration, volume, and function (motility, viability, morphology, membrane fluidity and capacitation status) during weeks 1-3 (W1/3), weeks 6 and 7 (W6/7), and weeks 10 and 11 (W10/11). Sperm function was also assessed after 5 and 7 days of storage (BTS, 17°C) at weeks 3, 7 and 11. Ejaculate volume increased through the 12 wks for each boar while concentration increased for only one boar; total sperm per ejaculate differed among periods. Total motility increased from W6/7 to W10/11 for one boar, but not the others. The percent motile sperm declined from day 0 to 5 of storage at all times, but there was significantly less loss of motility at W10/11 (p < 0.05). Mean number of viable sperm was greater at W6/7 and W10/11 than W1/3, and viability after 5 and 7 days of storage also was significantly better at W6/7 and W10/11 than W1/3 (p < 0.06 and p < 0.05 respectively). Dietary flaxseed increased the percent of morphologically normal sperm, and decreased of percent of sperm with tail abnormalities. Fluidity of the sperm plasma membrane as assessed by flow cytometry increased with the flax diet. Diet did not affect the acrosome reaction in fresh or stored sperm. These results indicate that the magnitude of the loss in sperm motility and viability following storage are reduced following dietary flax supplementation and the fluidity of the sperm plasma membrane is increased. (poster)

Biochemical characterization and membrane fluidity of membranous vesicles isolated from boar seminal plasma

Mammalian seminal plasma contains membranous vesicles (MV), which differ in composition and origin. Among these particles, human prostasomes and equine prostasome-like MV have been the most studied. The aim of the present work is to characterize the biochemical composition and membrane fluidity of MV isolated from boar seminal plasma. The MV from boar seminal plasma were isolated by ultracentrifugation and further purification by gel filtration on Sephadex G-200. The MV were examined by electron microscopy (EM), amount of cholesterol, total phospholipid, protein content, and phospholipid composition were analyzed. Membrane fluidity of MV and spermatozoa were estimated from the electron spin resonance (ESR) spectra of the 5-doxilstearic acid incorporated into the vesicle membranes by the order parameter ( S). The S parameter gives a measure of degree of structural order in the membrane and is defined as the ratio of the spectral anisotropy in the membranes to the maximum anisotropy obtained in a rigidly oriented system. The S parameter takes into consideration that S = 1 for a rapid spin-label motion of about only one axis and S = 0 for a rapid isotropic motion. Intermediate S values between S = 0 and S = 1 represents the consequence of decreased membrane fluidity. The EM revealed the presence of bilaminar and multilaminar electron-dense vesicles. Cholesterol to phospholipid molar ratio from the isolated MV was 1.8. Phospholipid composition showed a predominance of sphingomyelin. The S parameter for porcine MV and for boar spermatozoa was 0.73 ± 0.02 and 0.644 ± 0.008, respectively, with the S for MV being greater ( p < 0.001) than the S for spermatozoa. The high order for S found for boar MV was in agreement with the greater cholesterol/phospholipids ratio and the lesser ratio for phosphatidylcholine/sphingomyelin. Results obtained in the present work indicate that MV isolated from boar semen share many biochemical and morphological characteristics with equine prostasome-like MV and human prostasomes. The characteristics of the porcine MV of the seminal plasma, however, differed from those of boar sperm plasma membranes.

Bicarbonate-induced membrane processing in sperm capacitation

During capacitation, major changes take place in the sperm plasma membrane so as to render it fusogenic and responsive to zona pellucida glycoproteins. However, the mechanisms involved have not been defined. As bicarbonate is known to be the key component that induces capacitation, we have investigated the bicarbonate-dependent changes in the boar sperm's plasma membrane architecture. We have discovered that bicarbonate induces a rapid collapse of phospholipid transverse asymmetry, exposing phosphatidylethanolamine and phosphatidylserine at the outer surface of the lipid bilayer. The collapse, which is reversible, is brought about as a result of activation of the phospholipid scramblase that exchanges phospholipids in a non-specific fashion between the two leaflets of the lipid bilayer. The activation takes place via a cyclic AMP–protein kinase A-dependent pathway and is initiated via stimulation of the so-called ‘soluble’ adenylyl cyclase in the sperm cell by bicarbonate. As a result of the collapse and the concurrent increase in phospholipid exchange, removal of cholesterol by albumin is facilitated (perhaps due to increased lipid packing disorder). This finding is in conflict with earlier surmises that cholesterol loss precedes activation of the cyclic AMP–protein kinase A axis. We have noted that not all cells in a given sperm population show rapid changes in response to bicarbonate stimulation; samples from individual boars also differ in their response. Maturation differences between cells have been found to play an important role in such functional heterogeneity.

CAMP-dependent protein kinase control of plasma membrane lipid architecture in boar sperm.

Bicarbonate/CO(2), a physiological effector of sperm capacitation, has been shown to induce a rapid and reversible change in the lipid architecture of the plasma membrane of live boar sperm: the change is detectable as an increase in the cells' ability to bind the fluorescent dye merocyanine, a characteristic which implied an increase in lipid packing disorder (Harrison et al. 1996. Mol Reprod Dev 45:378-391). Evidence suggested that cAMP may act as a second messenger in the system, and we have therefore investigated this cAMP-dependency in more detail. Bicarbonate stimulates cAMP levels within 1 min in a dose-dependent fashion, prior to parallel increases in merocyanine binding. Although the potent somatic cell adenylyl cyclase activator forskolin is unable to induce significant increases in cAMP or merocyanine binding, increases in merocyanine binding are inducible in a dose-dependent fashion by 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole 3',5'-cyclic monophosphothioate, a cAMP analogue highly specific in its ability to stimulate protein kinase A; moreover, the bicarbonate-induced membrane change is inhibited by H89, a specific protein kinase A inhibitor. Neither bisindolylmaleimide I (protein kinase C inhibitor) nor lavendustin A (protein tyrosine kinase inhibitor) are inhibitory. In the presence of low levels of the potent phosphodiesterase inhibitor papaverine, increases in merocyanine binding are enhanced by okadaic acid and (more effectively) by calyculin (both protein phosphatase inhibitors). We conclude that boar sperm plasma membrane lipid architecture is controlled via a target protein that is dynamically phosphorylated by cAMP-dependent protein kinase and dephosphorylated by protein phosphatase type 1. Mol. Reprod. Dev. 55:220-228, 2000.

Flow cytometric detection of transbilayer movement of fluorescent phospholipid analogues across the boar sperm plasma membrane: Elimination of labeling artifacts

Flow cytometric detection of transbilayer movement of fluorescent phospholipid analogues across the boar sperm plasma membrane: Elimination of labeling artifacts

Flow cytometric detection of transbilayer movement of fluorescent phospholipid analogues across the boar sperm plasma membrane: elimination of labeling artifacts.

Reliable protocols were established for investigating asymmetric distributions of 6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino-caproyl (C6NBD) phospholipids in the plasma membrane of boar sperm cells under physiological conditions. A method based on fluorescence resonance energy transfer was used to ensure that incorporation of the fluorescent phospholipids into the sperm proceeded via monomeric transfer. The total amount of incorporated phospholipid fluorescence and the proportion of translocated phospholipid fluorescence were determined by flow cytometric analysis before, and after, dithionite destruction of outer leaflet fluorescence. Catabolism of incorporated fluorescent phospholipids was blocked with phenylmethylsulfonyl fluoride. Membrane-damaged cells were detected with impermeant DNA stains, thereby enabling their exclusion from subsequent analyses of the flow cytometric data, whence it could be demonstrated that the labeled phospholipids were incorporated only via the outer plasma membrane leaflet in living sperm cells. Phospholipid uptake and internalization was followed at 38 degrees C. After 1 hr of labeling, about 96% of the incorporated C6NBD-phosphatidylserine, 80% of C6NBD-phosphatidylethanolamine, 18% of C6NBD-phosphatidylcholine, and 4% of C6NBD-sphingomyelin were found to have moved across the plasma membrane bilayer to the interior of the spermatozoa. These inward movements of fluorescent phospholipids were ATP-dependent and could be blocked with sulfhydryl reagents. Movements from the inner to the outer leaflet of the sperm plasma membrane were minimal for intact fluorescent phospholipids, but were rapid and ATP-independent for fluorescent lipid metabolites. The described method enables, for the first time, assessment of changes in lipid asymmetry under fertilizing conditions.

Bicarbonate/CO2, an effector of capacitation, induces a rapid and reversible change in the lipid architecture of boar sperm plasma membranes.

Bicarbonate/CO2 is believed to be the key in vitro effector of sperm capacitation, a process which induces major changes in the sperm plasma membrane in preparation for fertilization. In a flow cytometric study, we examined the effect of bicarbonate on boar spermatozoa using merocyanine, an impermeant lipophilic probe which binds to plasma membranes with increasing affinity as their lipid components become more disordered. We found that bicarbonate causes a rapid increase in the ability of live boar spermatozoa to bind merocyanine. First detected about 100 sec after exposure to bicarbonate and largely complete by 300 sec, this increase appears to result from individual cells within the sperm population switching from a low merocyanine-binding state to a high binding state. The majority of live spermatozoa are capable of responding in this way, and do so in proportion to bicarbonate concentration, half-maximal response being induced by about 3 mM bicarbonate; however, overall population response varies greatly between ejaculates. Increased merocyanine stainability is observed over the whole surface area of the cell, and is reversible both with respect to temperature (it is only manifested above 30 degrees C) and with respect to presence of bicarbonate. A similar effect can be induced by phosphodiesterase inhibitors such as isobutylmethylxanthine, and enhanced by a permeant cyclic nucleotide analogue. We conclude that bicarbonate causes a major alteration in sperm plasma membrane lipid architecture, apparently by perturbing enzymic control processes. This novel action of bicarbonate may represent an initial permissive event in the capacitation sequence.

Bicarbonate/CO2, an effector of capacitation, induces a rapid and reversible change in the lipid architecture of boar sperm plasma membranes

Bicarbonate/CO2, an effector of capacitation, induces a rapid and reversible change in the lipid architecture of boar sperm plasma membranes

Topographical distribution of phospholipids in boar sperm plasma and intracellular membranes as revealed by freeze-fracture cytochemistry.

We used fracture-label and label-fracture cytochemistry in conjunction with the phospholipase A2-colloidal gold (PLA2-CG) technique to study the distribution of phospholipids in ejaculated boar spermatozoa. These techniques provide visualization of the topographical distribution of phospholipids in freeze-fractured sperm membranes in a three-dimensional view. In various freeze-fractured boar sperm membranes and crossfractured cytoplasmic structures, quantitative analysis revealed that the nuclear envelope membranes and the nuclear content possessed the highest labeling density of PLA2-CG. Moderate labeling was detected over acrosomal membranes, especially the inner acrosomal membrane. Replicas of both protoplasmic and exoplasmic fracture faces of the plasma membrane of boar sperm head showed a relatively low density of PLA2-CG labeling. Moreover, a differential distribution of phospholipids was seen over the protoplasmic face of the plasma membrane domains of the sperm head, which showed the highest concentration of gold particles in the postacrosomal region, followed by the equatorial segment and the anterior acrosome region. The PLA2-CG labeling densities over the post-acrosomal region and the equatorial segment were significantly higher than that over the anterior acrosome region. In the flagellum, an intense labeling was also seen over crossfractured mitochondria, dense fibers, and fibrous sheath. The protoplasmic fracture face of the plasma membrane over the middle piece, the annulus, and the principal piece was moderately labeled by PLA2-CG. No significant difference in mean labeling density of PLA2-CG was detected among the three membrane domains. In label-fracture preparations, exoplasmic halves of the plasma membrane of the head and the middle piece of the tail were uniformly labeled with PLA2-CG. However, the annulus and principal piece were devoid of PLA2-CG binding sites. These results indicate that differential distribution of phospholipids associated with the boar sperm membranes may reflect phospholipid composition of membrane domains characteristic of special physiological functions.

Boar sperm plasma membrane Ca(2+)-selective channels in planar lipid bilayers.

Entry of Ca2+ through Ca2+ channels is thought to trigger the acrosome reaction of spermatozoa during fertilization. Antagonists of the L-type Ca2+ channel are known to prevent the intracellular Ca2+ (Ca2+) increase and inhibit acrosomal exocytosis in mammalian sperm. Planar bilayer recordings were used to study Ca2- channels incorporated from partially purified boar sperm plasma membranes. With symmetrical 50 mM NaCl and 100 mM BaCl2 on the cis side, single-channel events consistent with Ba2+ flux from cis to trans were observed. These channels were activated by the dihydropyridine agonist (+/-)BAY K 8644 and blocked by the antagonist nitrendipine. Sperm Ca2- channels did not require depolarization for activation and did not inactivate. The (+/-)BAY K 8644 and (S-)BAY K 8644 enantiomers increased apparent open time in a dose-dependent [half-maximal activity constant (K0.5) = 0.9 and 0.3 microM, respectively] manner. Dihydropyridine antagonists nitrendipine (K0.5 = 0.5 microM) and (R+)BAY K 8644 (K0.5 = 2.8 microM) decreased apparent open times. The channels described in this report share some properties with brain, cardiac, and skeletal muscle t tubule Ca2+ channels and may be involved in increasing Cai2+ before the acrosome reaction.

Interaction of non-aggregated boar AWN-1 and AQN-3 with phospholipid matrices. A model for coating of spermadhesins to the sperm surface.

Boar spermadhesins are 12-14 kDa lectins which coat the entire acrosomal cap sperm head surface. The large amount of spermadhesins AQN-1, AQN-2, AQN-3, and AWN-1 present on in vitro capacitated spermatozoa (approximately 7 x 10(6) molecules of each spermadhesin per cell) suggested that they may bind to major component(s) of the sperm surface. We have investigated both the aggregation state of spermadhesins in seminal plasma using gel filtration chromatography, and their ability to bind to the major phospholipids of the boar sperm plasma membrane, i.e. phosphorylcholine and phosphorylethanolamine. The bulk (90%) of spermadhesins AQN-3 and AWN-1 were eluted as aggregated proteins (Mr > 50,000) with the void volume of a Sephadex G-50 column; the remaining 10% of the total amount of seminal plasma AWN-1 and AQN-3 were recovered, together with the whole amount of AQN-1 and AQN-2, in a fraction containing low-molecular-mass proteins (Mr 16,000-30,000). None spermadhesin of either gel-filtration fraction bound to a phosphorylcholine affinity matrix. On the other hand, low-molecular-mass (monomeric or dimeric) AQN-3 and AWN-1 were the only spermadhesins retained in a phosphorylethanolamine affinity column. Both AQN-3 and AWN-1 purified from seminal plasma by reverse-phase HPLC retained their lipid-binding capability. In addition, immobilization of AQN-3 and AWN-1 onto a phosphorylethanolamine matrix did not interfere with the ability of the proteins to bind bovine glycoprotein PDC-109, indicating that the structural determinants for the binding lipid and carbohydrates lay on different structural domains.(ABSTRACT TRUNCATED AT 250 WORDS)

Ion channels in boar sperm plasma membranes: Characterization of a cation selective channel

Plasma membranes isolated from cauda epididymal and ejaculated boar sperm were inserted into planar lipid bilayers and examined for the presence of ion channels. Channel fusion was frequently observed; the most prominent was a nonselective cation channel which conducted K, Na, Cs, Ca, and Ba. Channel opening did not show a strict dependence on voltage but was partially blocked by verapamil, nitrendipine, and ruthenium red. A channel with these characteristics was observed when plasma membranes were isolated by high-pressure nitrogen cavitation (650 psi, 78% sperm head plasma membranes) or at very low nitrogen pressures (50 psi, 90% sperm head plasma membranes), suggesting that this channel may be present in the plasma membrane overlying the sperm head.

Cold-induced ultrastructural changes in bull and boar sperm plasma membranes

The effect of low temperatures on the ultrastructure of the plasma membrane of bull and boar spermatozoa was investigated. Cold-induced changes in the organization of sperm plasma membrane components were demonstrated by the use of fast-freezing combined with freeze-fracture electron microscopy. This preparation technique ensures fixation without artifacts. At 38 °C bull and boar spermatozoa exhibited a random distribution of intramembranous particles over the plasma membrane of both head and tail. Exposure to 0 °C resulted in redistribution of the intramembranous particles: on the head and principal piece of bull spermatozoa and on the principal piece of boar spermatozoa, particle-free areas were observed, whereas on the boar sperm head, particle aggregates were present. The original particle distribution was restored upon rewarming of bull and boar spermatozoa to 38 °C, as well as after freezing and thawing of bull spermatozoa. Dilution of bull and boar semen into Tris-dilution buffer and Beltsville Thaw Solution-dilution buffer, respectively, could not prevent cold-induced redistribution of intramembranous particles. The observed particle reorganization upon cooling was interpreted as the result of lateral phase separation in the plasma membrane. Species-dependent differences in cold-induced ultrastructural changes were considered to be determined by lipid composition and asymmetry of the plasma membrane, and might be related to differences in cold resistance between species.

Identification, isolation, and properties of a plasma membrane protein involved in the adhesion of boar sperm to the porcine zona pellucida.

Boar sperm plasma membranes contain an integral protein (Mr 55 kDa) that apparently functions in the adhesion of sperm to the zona pellucida (Peterson and Hunt: J Cell Biol 105:170a, 1987.) In experiments described in this report, the protein is identified after additional steps of purification involving lectin affinity chromatography and preparative PAGE. An active form of the adhesion protein (APz) develops or becomes first exposed in the corpus epididymis and is fully active in the cauda epididymis; a significant portion of this conformationally labile protein, while integral to the plasma membrane, cannot be solubilized by nonionic detergents and may be associated with the membrane skeleton. APz does not exhibit enzymatic properties thought possibly to be involved in sperm-zona interaction in this and other species. Galactosyltransferase substrates and inhibitors and antiproteases including soybean trypsin inhibitor, pepstatin, leupeptin, and p-aminobenzamidine failed to block sperm from binding to porcine eggs. Boar sperm proacrosin and antiproacrosin antibody failed to inhibit sperm-egg binding. When plasma membranes or fractions containing APz that bind to dextran sulfate agarose were chromatographed on L-fucose agarose, a sugar which binds proacrosin, plasma membrane proteins that bound to the column failed to absorb anti-APz antibody. Anti-APz was absorbed by fractions that did not contain proacrosin. These data indicate that APz is not proacrosin. Since anti-APz monovalent antibody raised from whole cauda or corpus sperm plasma membranes or from chromatographic fractions containing APz completely block capacitated sperm from binding to eggs, and since the ability of this antibody to be absorbed develops as sperm become capable of binding to eggs, we view APz to be the major and perhaps only plasma membrane protein involved in the adhesion of capacitated boar sperm to eggs prior to the acrosome reaction.

Identification of calcium conducting channels in isolated boar sperm plasma membranes

Ion channel recordings were obtained from liposomes containing purified boar sperm plasma membrane proteins using a tip-dip method. Liposomes prepared in HEPES-TRIS and clamped by electrodes containing Ba-HEPES displayed channel activity that was partially inhibited by verapamil or nitrendipine and completely inhibited by La 3 + . Reversal of current at pipette negative voltages was observed only when Ba 2 + ions were also present in the bath solution. These data indicate that channels capable of carrying calcium currents are prominent components of the plasma membrane of mammalian sperm.