Acrosomal integrity and capacitation are not influenced by sperm cryopreservation in the giant panda

Sperm cryopreservation and artificial insemination are important management tools for giant panda breeding and the preservation of extant genetic diversity. This study examined the influence of freeze-thawing on sperm function, specifically capacitation. Sperm from nine giant pandas were assessed before and after rapid (- 40 and - 100 degrees C/min) cryopreservation by incubation in HEPES-buffered Ham's F10 medium with and without the capacitation accelerators, 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP). At 0, 3 and 6 h of exposure, aliquots were assessed for sperm motility traits and capacitation, defined as the proportion of sperm with intact acrosomes following exposure to solubilised zonae pellucidae (ursid or felid) or calcium ionophore subtracted from the proportion of sperm with intact acrosomes before exposure. Although mean+/-S.E.M. sperm motility post-thaw (56.1 +/- 3.9% at 0 h) was less (P < 0.05) than pre-freeze (71.7 +/- 6.0%), there was no difference (P > 0.05) in the proportion of acrosome-intact sperm (fresh, 93.0 +/- 1.7% versus cryopreserved-thawed, 81.7 +/- 4.7% at 0 h). Incidence of capacitation was greater (P < 0.05) in fresh sperm incubated with capacitation accelerators IBMX and dbcAMP (9 h: 50.9 +/- 1.1) compared with fresh sperm incubated without accelerators (9 h: 41.2 +/- 1.1%). Frozen-thawed sperm preincubated without accelerators underwent capacitation (49.6 +/- 1.1%) to a greater extent (P < 0.05) compared with these fresh counterparts. Thawed samples with (9 h: 45.9 +/- 1.4%) and without accelerators (9 h: 41.2 +/- 1.1%) did not differ (P > 0.05) during the 9-h incubation. We conclude that giant panda spermatozoa (1) undergo capacitation in vitro with or without chemical accelerators and (2) withstand a rapid cryopreservation protocol, including retaining normal acrosomal integrity and functional capacitation ability.     

Effect of medium on the kinematics of frozen-thawed ram spermatozoa

Cervically inseminated cryopreserved ram spermatozoa have reduced fertility due to poor mucus-penetrating ability. This effect is ameliorated by the addition of 20% (v/v) seminal plasma (SP) to the phosphate-buffered saline (PBS) thawing medium. The aims of this study were to determine whether the impaired mucus penetration was due to alterations in the sperm motility and, if so, whether these alterations were due to the SP or its viscosity, or to the medium components. To this end, artificial SP medium (ASP), a medium which supports motility but not capacitation, was compared with PBS and SP. Thawed, pooled semen from seven mature rams was layered under 1 ml each of PBS, SP and ASP and motile spermatozoa allowed to swim up (37 degrees C, 30 min). Upper regions of the overlays were harvested, and the capacitation status of the spermatozoa in each suspension determined by chlortetracycline (CTC) analysis. Sperm movement was videotaped in 300 microm chambers for both computer-aided sperm analysis assessment and manual flagellar curvature analysis. There was no effect of the culture medium on the concentration of spermatozoa recovered by swim up, nor on the proportion of motile spermatozoa. However, the spermatozoa resuspended in PBS did show changes associated with capacitation in both the CTC-binding patterns and in their movement patterns. These changes were significantly greater than those observed in spermatozoa resuspended in SP or ASP. These results indicated that the differences in sperm movement and function observed in SP medium were not due to changes in viscosity, but rather to components of the medium.     

Fate of lactadherin P47 during post-testicular maturation and capacitation of boar spermatozoa

Polyclonal avian antibody was used partially to characterize the pig sperm lactadherin P47. P47 is a mosaic protein, composed of two epidermal growth factor (EGF)-like domains and two C1/C2 domains. P47 is homologous to the bovine mammary gland protein MGP 53/57 and mouse milk fat globule protein. Expression of P47 along the male genital tract and its localization on spermatozoa during post-testicular maturation and capacitation were studied. P47 was detected in the testis and in all parts of the epididymis by immunohistochemistry and by western blots of tissue extracts. By indirect immunocytochemistry, P47 was localized at the apical ridge of the sperm head in testicular, epididymal and ejaculated spermatozoa. The fluorescence intensity progressed during sperm transit from caput to cauda epididymis, probably caused by the ongoing expression and subsequent accumulation of P47 on the sperm surface. During the time course of capacitation, P47 appears to be unmasked by the release of coating proteins and appears to migrate from the apical ridge onto the entire acrosomal region, showing an intensive fluorescence pattern after 3 h capacitation in vitro. The kinetics of signal changes during in vitro capacitation were different in epididymal and ejaculated spermatozoa, indicating accelerated capacitational plasma membrane destabilization in epididymal spermatozoa.     

Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa

This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment after swim-up for 20 min in sperm-Tyrode's albumen lactate pyruvate. Frozen-thawed spermatozoa from the same stallion were treated in a similar way. Spermatozoa were immobilized and injected into the oocytes using a Piezo drill. Presumptive zygotes were cultured in G1.2 medium for 20 or 96 h after the injection was administered, or were transferred to the oviducts of recipient mares and recovered 96 h later. In addition, bovine oocytes with first polar bodies were injected with the two types of stallion spermatozoa and fixed 20 h after injection to examine pronuclear formation. Fertilization rate (pronucleus formation and cleavage) at 20 h after injection of spermatozoa was not significantly different between fresh and frozen-thawed sperm groups in either equine or bovine oocytes. Pronucleus formation after injection of spermatozoa into bovine oocytes was significantly higher than that for equine oocytes (P < 0.05). There were no significant differences in cleavage rate or average number of nuclei at 96 h between equine oocytes injected with fresh or frozen-thawed spermatozoa. However, embryos developed in vivo for 96 h had a significantly higher number of nuclei in both sperm treatments compared with those cultured in vitro. These results indicate that good activation rates may be obtained after injection of either fresh or frozen-thawed equine spermatozoa without additional activation treatment. Injection of frozen-thawed equine spermatozoa results in similar embryo development to that obtained with fresh equine spermatozoa. In vitro culture of equine zygotes in G1.2 medium results in a similar cleavage rate but reduced number of cells compared with in vivo culture within the oviduct. Bovine oocytes may be useful as models for assessing sperm function in horses.     

Measured effect of collection and cooling conditions on the motility and the water transport parameters at subzero temperatures of equine spermatozoa

The effects of extracellular ice and cryoprotective agents on the measured volumetric shrinkage response and the membrane permeability parameters of equine spermatozoa have been reported previously. The volumetric shrinkage data were obtained using a differential scanning calorimeter technique that was independent of cell shape. The aim of this study was to examine the effects of collection and cooling conditions on the motility and the water transport parameters at subzero temperatures of equine spermatozoa. Stallion semen samples were collected using either a commercial lubricating agent, which caused osmotic stress to the spermatozoa, or water-insoluble Vaseline( trade mark ) as the artificial vagina lubricant. In some experiments, spermatozoa were cooled at 1 degrees C min(-1) from 20 degrees C to 4 degrees C to induce cold shock. An Equitainer was used to achieve control cooling rates (< or = 0.3 degrees C min(-1)) at temperatures > 0 degrees C. The water transport response of spermatozoa that were cold-shocked and osmotically shocked was significantly different from that of control spermatozoa (P < 0.01). Osmotic stress appeared to have an effect on the water transport response, although this effect was not significant. These results indicate that cold shock alters the behaviour of equine spermatozoa in cryopreservation protocols as a result of changes in the water transport properties of the plasma membrane. Although osmotic stress did not significantly affect water transport in equine spermatozoa, it did significantly decrease sperm motility in the extended semen samples (P < 0.01), which would, in turn, lower the quality of cold-stored or cryopreserved spermatozoa.     

Effect of platelet-activating factor (PAF) on stallion sperm motility, capacitation and the acrosome reaction

Phospholipids are an essential component of all mammalian cells; platelet activating factor (PAF=1-O-alkyl-acetyl-sn-glycero-3-phosphocholine) is a signalling phospholipid that has many biological properties in addition to platelet activation. PAF receptors have been detected on stallion spermatozoa; therefore, the aim of this study was to evaluate the effect of synthetic PAF on the motility, capacitation and the acrosome reaction of stallion spermatozoa. Treatment of ten stallion semen samples with 10(-4)-10(-13) mol PAF l(-1) resulted in significant differences in motility and capacitation (r(2)=0.81 and 0.83, respectively). Statistical analysis indicated that PAF also has an effect on acrosome reaction (r(2)=0.20). PAF concentrations, incubation time and their interaction had a highly significant (P<0.01) effect on motility. After capacitation in vitro with PAF, and induction of the acrosome reaction by progesterone, transmission electron microscopy was conducted on the spermatozoa of three stallions to detect the true acrosome reaction. Differences in PAF concentrations were highly significant (r(2) for intact: 97.2; reacted: 89.8; and vesiculated: 98.1). The results indicate that a lower concentration of PAF enhances motility and induces capacitation of stallion spermatozoa, whereas a higher concentration of PAF induces the acrosome reaction.     

Use of computer-assisted sperm motility assessment and multivariate pattern analysis to characterize ejaculate quality in Mohor gazelles (Gazella dama mhorr): effects of body weight, electroejaculation technique and short-term semen storage

Subjective and objective semen assessments were performed on 18 male Mohor gazelles (Gazella dama mhorr). Sperm motility assessments combined with sperm plasma membrane and acrosomal integrity evaluations were undertaken as part of a captive breeding programme. The primary objective was to test methodology for short-term preservation of gazelle semen for artificial insemination (storage in N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulphonic acid-Tris diluent (TEST) for up to 96 h at 17 degrees C). However, the secondary objective was to investigate phenotypic and genotypic influences on semen quality within this small population, which was established in 1971 with only 12 genetic founders. Sperm motility was measured by computer-assisted semen assessment and the data were analysed using a pattern analysis technique to detect and quantify naturally occurring sperm subpopulations within the semen samples. Four sperm subpopulations distinguishable by their motion characteristics were detected. The relative frequencies of two subpopulations (population 2: highly motile, non-linear; and population 4: poorly motile, non-linear) in fresh semen were correlated with the maximum voltage used during electroejaculation. The frequency of subpopulation 2 was negatively correlated with maximum voltage (r = -0.875, P < 0.0001) and the frequency of subpopulation 4 was positively correlated (r = 0.727, P < 0.005). The frequencies of all subpopulations varied significantly among the animals sampled (chi-squared = 2577.6, degrees of freedom = 54, P < 0.0001) and subpopulation 4 was also correlated with body weight (r = -0.59, P < 0.005). Semen stored at 17 degrees C retained motility, plasma membrane and acrosomal integrity for 48 h, but these measures decreased thereafter. The frequency of a sperm subpopulation showing uncoordinated but active motility increased significantly over the first 48 h and then decreased.     

Effect of serum sources and colostral whey on bovine semen quality and spermatozoa immunoglobulin G immunofluorescence

Heifer, bull, fetal calf sera, and colostral whey were used to evaluate the influence of protein concentrations on percent progressive motility, head-to-head agglutination, acrosomal integrity, and immunoglobulin G immunofluorescence of bovine spermatozoa using ejaculates from 10 bulls. In the first experiment, 10% (vol/vol) addition of undiluted colostral whey resulted in the highest head-to-head agglutination, acrosomal integrity, and immunoglobulin G immunofluorescence. Ten percent (vol/vol) addition of whey diluted to a protein concentration equivalent to fetal calf serum produced significantly lower agglutination, acrosomal integrity, and immunoglobulin G immunofluorescence. Fetal calf serum was unable to produce agglutination and immunoglobulin G immunofluorescence of bovine spermatozoa. Heifer and bull sera produced similar responses for all seminal measurements. In Experiment 2, unheated whey and heifer serum resulted in higher response for all variables than heat inactivated whey and heifer serum. Whey treatment produced greater spermatozoal motility, agglutination, acrosomal integrity; and immunoglobulin G immunofluorescence than treatment with heifer serum. Spermatozoal immunofluorescence indicated antibodies in normal whey, bull, and heifer serum bound to spermatozoal membranes at the acrosomal region. Colostral whey was an effective source of agglutinin factor. Normal unheated whey and heifer serum did not cause sperm damage or immobilization.     

Effects of scrotal insulation on viability characteristics of cryopreserved bovine semen.

The effect of a 48-h scrotal insulation on spermatozoal viability (motility and acrosomal integrity), before and after semen cryopreservation, was studied in six young Holstein bulls whose semen was collected twice in succession at 3-d intervals. Motility and acrosomal integrity were measured before and after incubation of semen at 37 degrees C for 3 h. For assessment of results, collection days were grouped: period 1 (control) = d -6, -3, and 0, where d 0 = initiation of scrotal insulation after semen collection; period 2 = d 3, 6, and 9 (sperm presumed in the epididymis or rete testis during scrotal insulation); period 3 = d 12, 15, ... 39 (sperm presumed in spermatogenesis during scrotal insulation). Semen was cryopreserved each collection day until morphologically abnormal cells exceeded 50% of the ejaculate (d 12 to 21). Semen viability before and after freezing was lower in period 3 than in period 1 (P less than .05). These differences coincided with the appearance in period 3 of abnormal sperm morphology and depressed undiluted semen motility, which began on d 12 (P less than .01). Semen collected during period 2 that was extended but unfrozen did not differ from that collected during period 1 in morphology or viability. However, for frozen semen, period 2 was significantly poorer than period 1 for both viability measurements, but only after incubation for 3 h at 37 degrees C postthaw (P less than .05). We conclude that epididymal sperm are adversely affected by elevated testicular temperatures, as noted by their decreased ability to maintain motility and acrosomal integrity following cryopreservation.     

Involvement of tyrosine kinase and cAMP-dependent kinase cross-talk in the regulation of human sperm motility

Tyrosine phosphorylation and its upregulation by cAMP have been associated with capacitation and motility changes of spermatozoa. In the present study, washed spermatozoa were incubated for 6 h in protein-supplemented complete medium with or without kinase inhibitors to verify whether upstream activation of protein kinase A is indispensable for tyrosine phosphorylation and motility changes to occur in capacitating human spermatozoa. H89, a specific protein kinase A inhibitor, significantly inhibited the activity of sperm protein kinase A. However, this inhibition did not alter capacitation-related tyrosine kinase activation. Tyrosine phosphorylated proteins, motion parameters and the incidence of phosphotyrosine-immunoreactive spermatozoa were decreased only slightly. Conversely, genistein, a tyrosine kinase inhibitor which inhibited sperm tyrosine kinase but not protein kinase A, significantly reduced all the parameters studied. Spermatozoa incubated with cAMP and pentoxifylline showed a rapid enhancement of tyrosine phosphorylation and some of the sperm motion parameters, particularly hyperactivation. Inclusion of H89 reduced cAMP stimulation of tyrosine kinase, and tyrosine phosphorylation and motion parameters were reduced almost to basal values. Treatment with genistein reduced tyrosine kinase activity, especially in the soluble fraction of sperm extracts. A decrease in tyrosine phosphorylation of soluble proteins, 105, 81, 55 and 48 kDa, correlated with a significant reduction in sperm motion parameters. Hyperactivation was reduced by tenfold. Tyrosine phosphorylated proteins in the insoluble fraction and the incidence of tyrosine phosphorylated-positive spermatozoa were not reduced markedly. Upstream protein kinase A activation may be a facilitatory rather than an indispensable step in the capacitation-induced tyrosine phosphorylation mediating motility changes in human spermatozoa. Triton-x100 soluble tyrosine phosphorylated proteins, more than their insoluble counterparts, appear to be involved in the modulation of human sperm motion characteristics.     

Role of actin cytoskeleton in mammalian sperm capacitation and the acrosome reaction

In order to fertilize, the mammalian spermatozoa should reside in the female reproductive tract for several hours, during which they undergo a series of biochemical modifications collectively called capacitation. Only capacitated sperm can undergo the acrosome reaction after binding to the egg zona pellucida, a process which enables sperm to penetrate into the egg and fertilize it. Polymerization of globular (G)-actin to filamentous (F)-actin occurs during capacitation, depending on protein kinase A activation, protein tyrosine phosphorylation, and phospholipase D activation. F-actin formation is important for the translocation of phospholipase C from the cytosol to the sperm plasma membrane during capacitation. Prior to the occurrence of the acrosome reaction, the F-actin should undergo depolymerization, a necessary process which enables the outer acrosomal membrane and the overlying plasma membrane to come into close proximity and fuse. The binding of the capacitated sperm to the zona pellucida induces a fast increase in sperm intracellular calcium, activation of actin severing proteins which break down the actin fibers, and allows the acrosome reaction to take place.     

Signal transduction mechanisms involved in in vitro ram sperm capacitation

We validate the chlortetracycline (CTC) technique for the evaluation of capacitation and acrosome reaction-like changes in ram sperm, carrying out a double estimation of the acrosome status after treatment with lysophosphatidylcholine, using fluorescein isocyanate (FITC)-RCA/ethidium homodimer 1 (EthD-1) and CTC/EthD-1. Highly consistent results and a positive correlation between the results of acrosome-reacted sperm evaluated with both techniques were obtained. In this study, we evaluate the effects of ram sperm capacitation of BSA, Ca(2+), NaHCO(3) and cAMP agonists and their influence on the associated protein tyrosine phosphorylation. We found a time-dependent increase in capacitation related to protein tyrosine phosphorylation, either in the absence or the presence of BSA. The addition of an increasing concentration of cholesterol to samples containing BSA did not influence results. The effect of bicarbonate was concentration-dependent, with a significantly lowered value of non-capacitated sperm in the presence 18 and 25 mM. The addition of extracellular calcium did not significantly increase either the proportion of capacitated sperm or the protein tyrosine phosphorylation signalling, although a significantly higher value of acrosome-reacted sperm was found in samples containing 4 mM Ca(2+). cAMP agonists increased capacitated sperm and protein tyrosine phosphorylation signalling. The inhibition of protein kinase A by H-89 caused a decrease in sperm capacitation. Addition of a calcium-entry blocker (Verapamil; Sigma) did not influence results, which suggests that the calcium entry blocker was unable to inhibit the calcium influx associated with capacitation in ram sperm. Our findings might benefit our understanding of the biochemical mechanisms involved in mammalian sperm capacitation and ultimately, fertility.     

A mouse sperm decapacitation factor receptor is phosphatidylethanolamine-binding protein 1

Capacitation is a pivotal event for mammalian spermatozoa, involving the loss of surface proteins known as decapacitation factors (DF) and consequent acquisition of fertilizing ability. Earlier studies showed that a mouse sperm DF binds to a receptor, DF-R, whose attachment to the sperm plasma membrane appears to involve a glycosylphosphatidylinositol (GPI) anchor. In the present study, purification and subsequent sequencing of DF-R has identified this approximately 23 kDa protein as phosphatidylethanolamine-binding protein 1 (PEBP 1). To obtain functional evidence that supports sequence homology data, purified recombinant PEBP 1 and PEBP 2 were evaluated for biological activity. While PEBP 1 was able to remove DF activity in solution at concentrations above approximately 1 nmol/l, PEBP 2 was ineffective, even at 600 nmol/l; this confirmed that DF-R is PEBP 1. Anti-PEBP 1 antiserum recognized recombinant PEBP 1 and a approximately 23 kDa protein in both mouse and human sperm lysates. Immunolocalization studies revealed that DF-R/PEBP 1 is located on the acrosomal cap, the post-acrosomal region and the flagellum of both mouse and human spermatozoa, with epitope accessibility being capacitation state-dependent and reversible. Treatment of cells with a phospholipase able to cleave GPI anchors essentially abolished immunostaining, thus confirming the extracellular location of DF-R/PEBP 1. We suggest that DF-R/PEBP 1 plays its fundamental role in capacitation by causing alterations in the sperm plasma membrane in both head and flagellum, with functional consequences for membrane-associated proteins. Obtaining more detail about DF <--> DF-R interactions could lead to useful applications in both fertility treatments and new contraceptive approaches.     

Capacitation-associated protein tyrosine phosphorylation and membrane fluidity changes are impaired in the spermatozoa of asthenozoospermic patients

Sperm protein tyrosine phosphorylation has been associated with capacitation, motility changes, zona binding, and fertilizing ability. We previously demonstrated that gradient-isolated human sperm subpopulations differ in their plasma membrane composition, their ability to phosphorylate proteins in tyrosine residues, and their capacity to undergo hyperactivation. In this study, we have characterized capacitation-associated changes in protein tyrosine phosphorylation and membrane fluidity in spermatozoa of asthenozoospermic and normozoospermic patients consulting for infertility. Semen samples were studied at baseline and after a capacitating incubation with or without the addition of a permeable cAMP analog and a phosphodiesterase inhibitor. Basic sperm and computer-assisted motion parameters, hyperactivation, protein tyrosine phosphorylation (immunofluorescence and Western blot), and membrane fluidity (fluorescent Laurdan probe) were the main study parameters. In comparison with normozoospermic and proven-fertile donor semen, asthenozoospermic samples showed lower motility, velocity, and amplitude of lateral head displacement, both originally and after a 6-h capacitating incubation. Unlike those in normal samples, asthenozoospermic spermatozoa were unable to increase protein tyrosine phosphorylation during capacitation. Such impairment, however, was overcome when they were incubated with a membrane-permeable cAMP analog and a phosphodiesterase inhibitor, indicating a possible membrane defect. Confirming this hypothesis, plasma membranes of asthenozoospermic sperm showed decreased fluidity (increased Laurdan polarization), even after a capacitating incubation. In conclusion, spermatozoa from functional asthenozoospermic samples may owe their poor motility, and their inability to properly capacitate and develop hyperactivation, to an impairment in the tyrosine phosphorylation of critical proteins caused by decreased membrane fluidity. These findings suggest a molecular pathogenetic mechanism for a common seminal pathology associated with male infertility.     

Selective sperm binding to pig oviductal epithelium in vitro

The sperm reservoir in the caudal isthmus of the oviduct of a number of species is created by binding of spermatozoa to oviductal epithelium. The sperm reservoir fulfills a number of functions such as control of sperm transport, maintenance of sperm viability and modulation of capacitation. The initial capacities of ejaculated and epididymal boar spermatozoa to bind to oviductal epithelium were investigated using a modified pig oviductal explant assay. The number of spermatozoa that bound to 0.01 mm(2) of explant surface was used as the parameter of binding capacity. Binding of spermatozoa to oviductal epithelial explants was dependent in a linear manner on the number of spermatozoa added (P < or = 0.05). No difference was found in initial sperm binding between isthmic and ampullar explants. There was no effect of the stage of the oestrous cycle or the reproductive status of the female donor. There was a significant effect (P < or = 0.05) of the individual boar on the binding index. The binding index correlated negatively with the percentage of spermatozoa with cytoplasmic droplets and the percentage of morphologically abnormal spermatozoa (P < or = 0.05). Epididymal spermatozoa showed significantly lower initial binding capability than did ejaculated spermatozoa from the same boars (P < or = 0.05); therefore, components of seminal plasma may play a role in the binding process. The individual differences revealed by this study and their relation to morphology and contact of spermatozoa with seminal fluid indicate a selective function of sperm-oviduct binding.     

A flow cytometric assay for global estimation of tyrosine phosphorylation associated with capacitation of spermatozoa from two marsupial species, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula)

The phosphorylation of tyrosine residues in cellular proteins is a major signal transduction event during sperm capacitation. In this study protein phosphorylation was monitored using a fluorescein isothiocyanate (FITC)-labeled antiphosphotyrosine monoclonal antibody and a flow cytometric procedure optimized for sperm. Using this technique, the correlation between tyrosine phosphorylation and sperm capacitation was examined in two marsupial species, the brushtail possum and the tammar wallaby and compared with that of ram spermatozoa. The levels of tyrosine phosphorylation in sperm from all three species were increased by the addition of cyclic AMP (cAMP) and vandate, a phosphotyrosine phosphatase inhibitor and were decreased by the addition of the phosphotyrosine kinase inhibitor, staurosporine. Oviductal conditioned media (CM) induced a progressive increase in tyrosine phosphorylation in both marsupial species and also induced morphological transition from a streamlined to a 'T'-shape configuration in brushtail possum spermatozoa but not in tammar wallaby spermatozoa. Transition to the 'T'-shape orientation associated with capacitation in marsupial spermatozoa was observed by 2 h of incubation in both species when tyrosine phosphorylation was increased by higher levels of cAMP i.e. 5 mM dibutyryl cAMP plus 3 mM pentoxyphylline. Thus the tyrosine phosphorylation trigger with CM may differ in these two marsupial species. Ram sperm tyrosine phosphorylation could be increased by addition of lower levels of cAMP (1 mM). These results support the finding that tyrosine phosphorylation is associated with sperm capacitation in marsupials. Similar results were obtained by using SDS PAGE/Western blot analysis of tyrosine phosphorylation in the brushtail possum spermatozoa. The specificity, efficiency and sensitivity of the procedure described here make it applicable for routine assessment of capacitation in large numbers of samples and in other species.     

Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose

This study investigates the ability of sucrose to protect spermatozoa against mitochondrial damage, artificial cryoinduction of capacitation, and acrosome reaction. Spermatozoa were isolated using the swim-up procedure performed using three different media: (a) human tubal fluid (HTF, control) medium; (b) HTF with 1% human serum albumin (HSA); and (c) HTF with 1% HSA and 0.25 M sucrose. From each group, 30 mul suspensions of cells were dropped directly into liquid nitrogen and stored for at least 24 h. Cells were thawed by quickly submerging the spheres in HTF with 1% HSA at 37 degrees C with gentle agitation. Sperm motility, viability, mitochondrial membrane potential integrity, spontaneous capacitation, and acrosome reaction were investigated. Sperm viability, acrosome reaction, and capacitation were detected using the double fluorescence chlortetracycline-Hoechst 33258 staining technique. Mitochondrial function was evaluated using a unique fluorescent cationic dye, 5,5',6,6'-tetrachloro-1-1',3,3'-tetraethyl-benzamidazolocarbocyanin iodide, commonly known as JC-1. The number of progressively motile spermatozoa was significantly higher in the sucrose-supplemented medium group (57.1+/-3.2%, P<0.05) when compared with controls (19.4+/-1.9%). The combination of HSA and sucrose (65.2+/-2.6%) has a stronger cryoprotective effect on the integrity of mitochondrial membrane potential (P<0.05) compared with HSA alone (32.6+/-4.7%). In conclusion, vitrification of human spermatozoa with non-permeable cryoprotectants such as HSA and sucrose can effectively cryopreserve the cells without significant loss of important physiological parameters.     

Binder of sperm 1 and epididymal sperm binding protein 1 are associated with different bull sperm subpopulations

Previously, we showed that binder of sperm 1 (BSP1) and epididymal sperm binding protein 1 (ELSPBP1) proteins are more abundant in the immotile bovine sperm subpopulation following cryopreservation. In this study, we investigated the association of BSP1 and ELSPBP1 with sperm in relation to their ability to survive the cryopreservation process. Fresh and cryopreserved semen samples from the same ejaculate collected from nine Holstein bulls were incubated with a fixable viability probe, fixed and permeabilised and then immunolabelled with rabbit anti-BSP1, rabbit anti-ELSPBP1 or rabbit IgG as negative control. Spermatozoa were then incubated with Alexa 488-conjugated secondary antibody and Hoechst 33342. For each sample, 10?000 'Hoechst positive' events were analysed by flow cytometry. Alternatively, sperm populations were obtained by fluorescence-activated cell sorting. In freshly ejaculated live sperm, two distinct BSP1 detection patterns were revealed: a first population where BSP1 is present along the flagellar region (P1 subpopulation) and a second population where BSP1 is localised on both the flagellar and the acrosomal regions (P3 subpopulation). The dead population presented a BSP1 distribution similar to P3 but with a more intense fluorescence signal (P4 subpopulation). In the corresponding cryopreserved samples, all sperm in the P3 subpopulation were dead while only a small proportion of the P1 subpopulation was dead (P2 subpopulation). ELSPBP1 was detected only in dead spermatozoa and in comparable proportions in both freshly ejaculated and cryopreserved semen. These results show that the presence of BSP1 over the acrosomal region characterises spermatozoa sensitive to cryopreservation and that ELSPBP1 characterises spermatozoa that are already dead at ejaculation.     

Selection of highly fertilization-competent bovine spermatozoa through adhesion to the Fallopian tube epithelium in vitro

Mammalian spermatozoa undergo a marked reduction in number during their journey through the female reproductive tract. One of the checkpoints in the selection of fertilizing spermatozoa may be the transient adhesion to the Fallopian tube epithelium, an event previously shown to play a key role in sperm storage. Bovine spermatozoa adhering to the Fallopian tube epithelium in vitro may be synchronously released by sulphated glycoconjugates. In the present study, experiments were designed to quantify the number of spermatozoa selected through adhesion, and to compare the zona pellucida (ZP) binding and fertilization competence of the initial sperm suspension versus the bound and unbound sperm subpopulations. Results showed that: (1) a fraction accounting for about 30% of the initial sperm suspension was selected by in vitro adhesion to oviductal epithelial cell monolayers; (2) selected spermatozoa, collected after heparin-induced release, had a significantly superior ZP binding and fertilization competence (mean +/- SD: 110 +/- 28 bound spermatozoa per oocyte; % cleavage, mean +/- SEM: 89 +/- 4) compared with both the initial sperm suspension (45 +/- 10 bound spermatozoa per oocyte, P < 0.001; % cleavage: 69 +/- 3, P < 0.05) and the unselected subpopulation (30 +/- 4 bound spermatozoa per oocyte, P < 0.001; % cleavage: 58 +/- 3, P < 0.01). These findings support the hypothesis that binding to oviductal cells is not only beneficial for sperm survival but also represents a crucial step for the selection of spermatozoa endowed with superior fertilization competence.