A short hypoosmotic swelling test for the prediction of boar sperm fertility

This study was designed to explore the relationship between the ejaculate response to a hypoosmotic swelling test (HOST) and in vivo fertility in a group of 38 boars The hypoosmotic test used was a modification of the HOST that involves a shorter incubation time (5 vs 120 min) and lower osmotic pressure (75 vs 150 mOsm/kg). Ejaculates containing less than 20% abnormal spermatozoa were selected and checked for percentage of motility, percentage of normal acrosomes, percentage of short ORT and percentage of cells showing positive short HOST (sHOST) results Two hundred eightyeight sows were inseminated to obtain in vivo fertility and prolificacy data. No differences were shown between technicians in the sHOST results obtained. Significant differences were recorded between boars in sHOST results (p < 0.002). Only the sHOST result presented a significant correlation with in vivo fertility (r = 0.43, p < 0.01). Short HOST data significantly improved fertility prediction of routine semen analysis tests. Unlike motility and acrosomal status, sHOST values showed a significant decrease when fresh ejaculates (37 degrees C) were stored for 24 h at 15 degrees C, indicating possible damage due to cold shock.     

Detection of early changes in sperm membrane integrity pre-freezing can estimate post-thaw quality of boar spermatozoa

A recently developed triple staining (SNARF-1/YO-PRO-1/ethidium homodimer) was used to assess early changes in boar sperm membrane integrity (MI) with the results of cryopreservation procedures and to seek for correlations among MI-spermatozoa in pre-freeze semen and its freezeability. Ejaculates from five boars were evaluated in the fresh and frozen-thawed (FT) state, and its freezeability defined as % of membrane intactness, MI% (MI%=% of FT-spermatozoa with intact membranes x 100 divided by the % of pre-freeze spermatozoa with intact membranes) estimated. Significant differences were found among boars for freezeability (MI%) and motility post-thaw (%). Interestingly, significant correlations were found between the percentage of YO-PRO-1-positive spermatozoa and freezeability (R=0.440, p<0.01), indicating this new triple staining can be used to safely disclose among ejaculates prior to freezing.     

Immunological approach to the characterization of the outer acrosomal membrane of boar spermatozoa

Antiserum to purified boar spermatozoan outer acrosomal membrane (OAM) was raised in rabbits and adsorbed with boar liver and serum glutaraldehyde cross-linked immunoadsorbents. The IgG fraction of the antiserum was purified by (NH₄)₂SO₄ precipitation followed by ion-exchange chromatography. Indirect immunofluorescence showed bright fluorescent staining of the acrosomal cap of boar spermatozoa and to a lesser extent of the acrosomes of bull and goat spermatozoa after incubation with anti-OAM-IgG. Immuno-electron microscopy further confirmed the specificity of the antibody for the OAM. Preincubation of the anti-OAM-IgG with isolated OAM, completely abolished its reactivity. When tested by ELISA, anti-OAM-IgG reacted with boar, bull, goat, and human spermatozoa; however, its binding activity to boar spermatozoa was significantly greater as compared to spermatozoa from the other species tested. In an effort to identify OAM antigens recognized by this antiserum, the isolated boar OAM was labeled either with ³H or with ¹²⁵I and solubilized by mild detergent treatment. The extracted components were immunoprecipitated with anti-OAM-IgG and protein A-bearing S. aureus and the thus isolated antigens were analysed on SDS-PAGE. The results suggest that anti-OAM-IgG recognized one high molecular ³H-labeled glycoprotein (270 kd), and four ¹²⁵I-labeled polypeptides of lower molecular weight of the boar OAM.     

Solubilization and enrichment of boar sperm membrane proteins

Boar sperm membranes are rather resistent to the solubilizing effect of some detergents. Deoxycholate, an ionic detergent, was efficient in solubilizing sperm proteins but some nonionic detergents like Triton X-100 displayed relatively poor capacity in rendering membrane proteins soluble. This may be due to sperm proteins being attached to submembraneous structures through bonds involving divalent cations, since mixtures of Triton X-100 and ethylenediamine tetraacetic acid (EDTA) were almost as efficient as deoxycholate in solubilizing membrane proteins. Since intact spermatozoa were directly treated with detergents the solubilized proteins comprised a mixture of intracellular and membrane components. To enrich for membrane proteins, affinity chromatography on columns containing different lectins was carried out. SDS polyacryiamide gel electrophoresis of sperm glycoproteins desorbed from the various lectin columns demonstrated that each lectin bound a unique set of components although most glycoproteins were recovered from two or more columns. Columns containing Lens culinaris hemagglutinin yielded more sperm glycoproteins than any of the other lectin columns examined. The predominant amount of the sperm proteins recovered from the Lens culinaris lectin column was membrane derived, as the majority of the proteins were integrated into liposomes. It is concluded that sperm membrane proteins are efficiently solubilized by detergent in the presence of a chelator and that most of the membrane glycoproteins can easily be enriched by affinity chromatography on a lectin column. Proteins obtained in this way should serve as excellent starting material for the isolation of individual sperm membrane proteins.     

Antioxidant supplementation of boar spermatozoa from different fractions of the ejaculate improves cryopreservation: changes in sperm membrane lipid architecture

Previous studies have shown sperm quality after cryopreservation differs depending on the fraction of seminal plasma the boar spermatozoa are contained in. Thus, spermatozoa contained in the first 10 ml of the sperm-rich fraction (portion I) withstand handling procedures (extension, handling and freezing/thawing) better than those contained in the latter part of a fractionated ejaculate (second portion of the sperm-rich fraction and the post-spermatic fraction; portion II). The present study evaluated whether an exogenous antioxidant, the water-soluble vitamin E analogue Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), could, when added to the freezing extender in a split-sample design trial, improve the post-thaw viability and membrane quality of this particular portion of the ejaculate, with particular attention to the status of the plasma membrane. Using a split-sample design, the initial changes in the fluidity status of the sperm plasmalemma after thawing were measured by flow cytometry (FC) after loading with Merocyanine-540 and YO-PRO-1. The FC-derived data revealed a clear ejaculate portion-dependent effect of the antioxidant supplementation. While no beneficial effect of the antioxidant supplementation was visible in spermatozoa from portion I, more spermatozoa with intact membranes were observed in the supplemented samples of portion II, suggesting the protective effect of vitamin E is dependent of the portion of the boar ejaculate considered.     

Partial purification of a boar sperm membrane protein inducing sperm agglutinating antibody

For the development of immunological contraception, attention is being concentrated on the possibility of using a sperm membrane antigen. Boar sperm membrane was extracted with triton-X 100 and fractionated by Sephadex G-150 column chromatography. The glycosylated and nonglycosylated portions of protein peaks from the gel filtration were obtained by fractionating on concanavalin A-Sepharose and eluting the bound protein with 0.3 M methyl mannoside. A glycosylated fraction was found to induce sperm agglutinating antibodies in rabbit. The partially purified protein has a molecular weight of 30 kilodaltons, as determined by sodium dodecyl polyaccyrlamide gel electrophoresis. Further work is planned on the histochemical determination of the origin of this protein and species cross-activity of the antibody.     

Detection of cooling-induced membrane changes in the response of boar sperm to capacitating conditions

There is a need for methods of rapid and sensitive sperm function assessment. As spermatozoa are not able to fertilize an oocyte before having undergone a series of complex physiological changes collectively called capacitation, it is logical to assess sperm function under fertilizing conditions in vitro. In this study, the responsiveness of sperm to capacitating conditions in vitro was monitored by changes in sperm response to ionophore and by changes in the amount of intracellular calcium ions in stored boar semen. Boar semen was diluted at 32 and 20 degrees C and stored for 24 and 72 h at 16 and 10 degrees C. Ionophore-induced changes and increased intracellular calcium ion content in boar spermatozoa were recorded by flow cytometry and found to progress as a function of time during incubation under capacitating conditions. All responsiveness parameters (increases in proportions of membrane-defective spermatozoa, acrosome-reacted spermatozoa, and cells with high intracellular calcium levels) were shown to be sensitive to subtle physiological changes occurring at low storage temperatures. The initial levels of sperm with a high calcium content were higher in semen stored at 10 degrees C, but the accumulation of internal calcium was lower than in semen stored at 16 degrees C. The loss of membrane integrity and increase in the proportion of acrosome-reacted cells were higher in semen stored at 10 degrees C. Dilution at 20 degrees C had no negative effect on membrane integrity or responsiveness to capacitating conditions. There was no significant difference between semen stored for 24 and 72 h in terms of membrane integrity, acrosome reaction, and intracellular calcium after capacitation treatment. However, dynamics of cell death and acrosome reaction in response to capacitating conditions were somewhat accelerated after 72 h storage, especially in semen stored at 10 degrees C. It can be concluded that the simultaneous use of the sperm membrane responsiveness and kinetic parameters is a sensitive tool for the detection of storage-related membrane changes in boar semen.     

Quality of boar spermatozoa from the sperm-peak portion of the ejaculate after simplified freezing in MiniFlatpacks compared to the remaining spermatozoa of the sperm-rich fraction

Boar sperm viability post-thaw differs depending on the ejaculate fraction used, with spermatozoa present in the first 10 mL of the sperm-rich fraction (SRF) (portion 1, P1, sperm-peak portion) displaying the best cryosurvival in vitro compared with that of spermatozoa from the rest of the ejaculate (portion 2 of the SRF plus the post-spermatic fraction), even when using simplified freezing routines. This viability apparently relates to the specific profile of seminal plasma in P1 (i.e., glycoprotein and bicarbonate concentrations, and pH). However, spermatozoa from P1 have not been compared with spermatozoa from the rest of the SRF (SRF-P1, usually 30-40 mL of the SRF), which is routinely used for freezing. We compared P1 with SRF-P1 in terms of sperm kinematics (using the QualiSperm™ system), while membrane integrity (SYBR-14/PI), acrosome integrity (FITC PNA/PI), and sperm membrane stability (Annexin-V) were explored using flow cytometry. As well, total protein concentration and the proteomics of the seminal plasma (SP) of both portions of the SRF were studied using two-dimensional electrophoresis (2DE), mass fingerprinting (MALDI-TOF), and collision-induced dissociation tandem mass spectrometry (CID-MS/MS) on selected peptides. The SRF portions were collected weekly from four mature boars (4-5 replicates per boar, sperm concentration: P1, 1.86 ± 0.20; SRF-P1, 1.25 ± 0.14 × 10⁹ spz/mL) and processed using a quick freezing method in MiniFlatPacks. Post-thaw sperm motility reached 50%, without differences between SRF portions, but with clear inter-boar variation. Neither plasma membrane nor acrosome integrity differed (ns) between fractions. These results indicate that there are no differences in cryosurvival after quick freezing of boar spermatozoa derived from either of the two SRF portions. While P1 and SRF-P1 clearly differed in relative total protein contents, as expected, they displayed very similar protein profiles as assessed using 2DE and mass spectrometry (tryptic peptide mass fingerprint analysis and CID-MS/MS), indicating a similar emission of epididymal protein content.     

The acrosomal membrane of boar sperm: a Golgi-derived membrane poor in glycoconjugates

The acrosome is a large secretory vesicle of the sperm head that carries enzymes responsible for the digestion of the oocyte's investments. The event leads to sperm penetration and allows fertilization to occur. Release of the acrosomal enzymes is mediated by the interaction between sperm acrosomal and plasma membranes (acrosome reaction). Biochemical characterization of the acrosomal membrane has been restrained by a lack of methods to isolate uncontaminated fractions of the membrane. Here, we use new methods to expose the membrane to in situ cytochemical labeling by lectin-gold complexes. We study the topology and relative density of glycoconjugates both across and along the plane of the acrosomal membrane of boar sperm. Detachment of the plasma membrane from glutaraldehyde-fixed cells exposed the cytoplasmic surface of the acrosome to the lectin markers; freeze-fractured halves of the acrosomal membrane were marked by "fracture-label" (Aguas, A. P., and P. Pinto da Silva, 1983, J. Cell Biol. 97:1356-1364). We show that the cytoplasmic surface of the intact acrosome is devoid of binding sites for both concanavalin A (Con A) and wheat germ agglutinin (WGA). By contrast, it contains a high density of neuraminidase-resistant anionic sites detected by cationic ferritin. On freeze-fractured sperm, the receptors for Con A partitioned with the exoplasmic membrane half of the acrosomal membrane. The Con A-binding glycoconjugates were accumulated on the equatorial segment of the membrane. A low density of WGA receptors, as well as of intramembrane particles, was found on the freeze-fracture halves of the acrosomal membrane. The plasma membrane displayed, in the same preparations, a high density of receptors for both Con A and WGA. We conclude that the acrosome is limited by a membrane poor in glycoconjugates, which are exclusively exposed on the exoplasmic side of the bilayer. Regionalization of Con A receptors on the acrosome shows that sperm intracellular membranes, like the sperm surface, express domain distribution of glycocomponents.     

The interaction of living boar sperm and sperm plasma membrane vesicles with the porcine zona pellucida

Enzymological, morphological, and immunological methods were used to characterize further the interaction of noncapacitated boar spermatozoa with the porcine zona pellucida. Transmission electron microscopy showed that sperm usually bind to the zona over the head region of the cell. Only the plasma membrane is involved in this binding. Bound sperm will undergo the acrosome reaction when treated with calcium and the ionophore A23187. The ability of intact sperm to bind to porcine eggs in vitro and the ability of sperm plasma membrane vesicles to absorb univalent antibody to the sperm binding site for the zona were used to determine the effects of various physical, chemical, and enzymological treatments on the sperm binding sites. These sites were resistant to a number of enzymes including proteases and polysaccharidases, but were inactivated by heat and trichloroacetic acid. Binding sites on the zona were inactivated by extracts from small quantities of sperm. Binding was also blocked by Fab antibody to whole zonae absorbed to other swine tissue and by similarly absorbed Fab antibody to sperm plasma membranes. These data provide further support for the presence of zona recognition sites on the plasma membrane of noncapacitated boar sperm. The binding sites on the sperm plasma membrane do not appear to be peripheral membrane proteins nor major constituents of a surface glycocalyx.     

Effect of egg yolk and other reagents upon the zinc of cold-shocked boar spermatozoa

A marked reduction (80.8%) in the zinc uptake by boar spermatozoa cooled to 4 degrees C occurs when the seminal plasma is pretreated with egg yolk-glucose at this temperature. Crude lecithin is less effective (59.8%). Similar pretreatment of the seminal plasma by the polycationic drug Antrypol, which totally removes the zinc-precipitable basic haemagglutinin, does not result in a significant reduction of the sperm zinc uptake at 4 degrees C.     

Use of a fluorescent dye to measure drug-induced changes in the membrane potential of boar spermatozoa.

The fluorescence emission intensity of the dye 3,3′ dipentyloxo-carbocyanine iodide equilibrated with washed boar spermatozoa and valinomycin or gramicidin varies with external potassium or sodium concentration in a manner indicating that dye fluorescence is related to the plasma membrane potential. The membrane potential in turn is shown to be dependent on energy metabolism. The drugs propranolol, lidocaine and diphendydramine, which possess local anesthetic-like properties, induce a rapid depolarization which can be reversed by valinomycin at low drug concentrations and a slower apparently energy dependent depolarization at higher drug concentrations that is not reversible. Low concentrations of these drugs decrease forward progression of sperm but have little effect on the percentage motile cells. Theophylline increases the frequency of contraction of drug-treated cells but not their forward progression, these findings are discussed in terms of the role of the sperm membrane in the control of motility.     

Further characterization of boar sperm plasma membrane proteins with affinity for the porcine zona pellucida

Boar sperm plasma membrane proteins (PMPs) with affinity for the zona pellucida were partially purified from columns of dextran sulfate using a linear salt gradient and a buffered detergent that retained their ability to block directly the binding of uncapacitated and capacitated sperm to isolated porcine oocytes. PMPs that bound most strongly to dextran sulfate (fraction IV) were also most effective in blocking sperm binding to porcine oocytes. These tightly bound proteins also bound to isolated zonae to a greater extent than other fractions. Monovalent antibodies to fraction IV PMPs completely blocked sperm binding to isolated eggs. Fraction IV PMPs lost the ability to inhibit directly the binding to eggs when treated with chaotropic agents and trypsin; the fraction also displayed a tendency to aggregate in the absence of high salt. This property and the affinity of proteins in this fraction for sulfated polysaccharides indicate that specific hydrophilic interactions may play a significant role in sperm-zona attachments.     

Capacitation induces tyrosine phosphorylation of proteins in the boar sperm plasma membrane.

Capacitation (activation) of mammalian spermatozoa is accompanied by protein phosphorylation, elevation of the intracellular calcium concentration and an increased plasma membrane fluidity. The subcellular localization of tyrosine phosphorylation during capacitation have not yet been elucidated. The aim of this study was to investigate whether boar sperm capacitation induces tyrosine phosphorylation of plasma membrane proteins. Capacitation induced tyrosine phosphorylation of 3 proteins (27, 37, and 40 kDa), which coincided with an increase in the plasma membrane fluidity. The importance of the induced tyrosine phosphorylation in sperm binding to the zona pellucida and the induction of the acrosome reaction is discussed.     

Impact of storage prior to cryopreservation on plasma membrane function and fertility of boar sperm

Occasionally, boar semen must be shipped to another location for cryopreservation. We increased the initial holding time for the cooling of extended semen at 15 degrees C from 3 to 24 h to determine the effects on sperm characteristics and fertility. Thirty-one gilts and sows were inseminated once with subsequently cryopreserved and thawed semen. Increasing the holding time from 3 to 24 h had no significant effect on pregnancy rate 23 days after AI with frozen-thawed semen (64.5%) but decreased (P<0.05) embryo number from 15 to 9 and recovered embryos as fraction of CL from 73 to 47%. While the longer holding time at 15 degrees C did decrease potential litter size, the loss incurred was not too great to preclude the incorporation of a longer holding time into the cryopreservation protocol. An experiment was conducted to test the hypothesis that processing and freeze-thawing of boar semen would induce phospholipid scrambling in the plasma membrane similar to that evoked by incubation in bicarbonate-containing media. Merocyanine staining after incubation in the presence and absence of bicarbonate indicated that changes in plasma membrane phospholipid scrambling of processed and cryopreserved sperm differed from those in fresh semen undergoing bicarbonate-induced capacitation. The level of Annexin-V binding in boar spermatozoa increased from 1.6% in live spermatozoa in fresh semen to 18.7% in cryopreserved sperm. Apoptosis is unlikely to operate in mature spermatozoa. Apoptotic morphology in ejaculated spermatozoa is probably a result of incomplete deletion of apoptotic spermatocytes during spermatogenesis. Increased Annexin-V binding in thawed spermatozoa probably results from plasma membrane damage incurred during freezing and thawing.