Biomedical subjects

A M Petrunkina

Publications and source records attributed to A M Petrunkina.

18 recordsLinked to original sources

PubMed2006

Enhanced binding of sperm with superior volume regulation to oviductal epithelium.

The plasma membrane is a key organelle with respect to sperm fertilizing ability. A sensitive way of testing plasma membrane functionality is to examine the sperm ability to moderate its swelling in response to hypo-osmotic stress (volume regulatory ability) using an electronic cell counter to assess cell volume changes. In this study of frozen-thawed bull sperm, we examined the relationship among sperm-oviductal epithelium binding capacity, osmotically induced swelling response, volume regulatory ability, and standard spermatologic parameters. Sperm cell volume distributions were measured under iso-osmotic conditions and after hypo-osmotic stress. The relative volume shift was calculated by comparing modal values of the cell volume distributions during transition from iso-osmotic to hypo-osmotic conditions. Significant correlations were found between volumetric parameters and sperm-oviduct binding capacity. Both the relative volume shift and regulative volume decrease correlated positively and significantly with the sperm-oviduct binding capacity. No significant correlations were found between sperm volumetric parameters and any of the standard sperm parameters with the exception of forward motility of Percoll-washed sperm. However, the use of multiple regression models improved the prediction level for binding capacity when motility parameters were combined with membrane integrity and volumetric parameters (R2 = .84). Spermatozoa of bulls with high nonreturn rates responded to hypotonicity as "perfect osmometers." Subfertile bulls had lower binding indices and deficiencies in volume recovery after hypotonic challenge, indicating that intact volume regulatory ability is a necessary prerequisite for binding to oviductal epithelium and is related to fertility. Volumetric parameters therefore could be used as tools in semen evaluation programs.

Animals↗

PubMed2006

Binding of boar spermatozoa to oviductal epithelium in vitro in relation to sperm morphology and storage time.

In vitro short-term storage of boar semen for up to 72 h before insemination negatively affects fertility, but this often remains undetected during semen quality assessment. One important sperm function is the ability to form the functional sperm reservoir in the oviduct. In the present study, we used the modified oviductal explant assay to study sperm binding to oviductal epithelium in vitro in diluted boar semen stored for 24 or 72 h. First, we determined the kinetics of in vitro sperm binding to oviductal epithelium in relation to co-incubation time of sperm and oviductal tissue pieces. Then, we studied how the binding of sperm to oviductal epithelium was affected by in vitro semen storage and by differences among individual boars. Sperm binding after different incubation times was significantly higher when semen was stored 24 h than after 72-h storage (P < 0.05), and peaked at 30-90 min of incubation. Sperm binding differed between boars (n = 44), and was negatively correlated to the percentage of sperm with cytoplasmic droplets (R = -0.51, P < 0.001). There were no significant changes in motility, acrosome integrity and propidium iodide stainability during the 72-h storage period. However, sperm-binding indices were significantly lower after 72 h in vitro storage than after 24-h storage in sperm from boars with normal semen quality (P < 0.05); in contrast, the binding capacity of sperm from boars with higher percentages of morphologically altered sperm remained at a low level. The sperm-binding capacity of sperm from four of the five boars with known subfertility was lower than the mean binding index minus one standard deviation of the boar population studied here. It is concluded that changes in the plasma membrane associated with in vitro ageing reduce the ability of stored boar sperm to bind to the oviductal epithelium. This study shows the potential of sperm-oviduct binding as a tool to assess both male fertility and changes in sperm function associated with in vitro ageing.

Acrosome↗

PubMed2005

Volume regulatory function and sperm membrane dynamics as parameters for evaluating cryoprotective efficiency of a freezing extender.

In the past years a series of functional assays has been developed to determine the structural, morphological and functional integrity of the plasma membrane and sperm acrosomal membrane. Cell volume regulation is an important physiological function crucial for the success of cryopreservation. In this study, the effects induced by freezing-thawing were judged by evaluating the functional characteristics of frozen-thawed semen samples submitted to secondary stress such as osmotic challenge or incubation under capacitating conditions, following cryopreservation. Prior to freezing, dog semen samples were diluted in the presence or absence of Equex STM Paste, which contains sodium dodecyl sulphate (SDS) as the active ingredient. Cell volume regulation and capacitation and calcium ionophore-induced membrane dynamics were assessed in freshly diluted and frozen-thawed semen samples by electronic volume measurement and flow cytometry. Cryopreservation led to a disturbance of the volume regulatory function and to a rapid decrease in the proportion of acrosome-reacted live spermaotozoa. Extender containing Equex STM Paste had a protective effect on isotonic cell volume, on regulatory function under hypertonic conditions, and on the proportion of live acrosome-reacted cells. The evaluation of the functional state of sperm submitted to secondary stress after freezing-thawing leads to a more subtle characterization of sperm function and helps improve the cryoprotective efficiency of the extender.

Acrosome↗

PubMed2005

Importance of sperm-binding assays for fertility prognosis of porcine spermatozoa.

There has been a considerable effort to establish correlations between the outcome of in vitro sperm-binding assays and the fertility achieved by individual males under conditions of commercial AI. During passage through the oviduct, a fertilizing spermatozoon has to bind to and interact with several targets. Generally, it is assumed that these interactions can be mimicked by in vitro binding assays. However, there is little evidence that assays based on zona binding, zona penetration, or IVF: (a) have been adequately validated; (b) provide data with a high degree of correlation to a boar of average fertility; (c) provide accurate predictions as to pregnancy rate and litter size from a given boar when used for commercial AI. This is due partly to the variability in measurements of pregnancy rate and litter size in a commercial setting and partly to the fact that sperm fertility is multifactorial. A recently developed in vitro test is based on the fact that spermatozoa bind in vivo to oviduct epithelium, creating a functional sperm reservoir, and that fertilization-competent spermatozoa are released in a time-dependent manner from these cells. Mating or insemination occurs usually hours before ovulation thus rendering such temporary sperm binding to the epithelial cells, a prerequisite for successful sperm-oocyte interaction. In vitro binding of porcine spermatozoa to explants derived from fresh oviduct epithelium may provide a useful test system to predict fertility, although detailed validation has not been published. The sperm-oviduct-binding assay tests for multifunctional characteristics of the plasma membrane and may be a valuable in vitro test to identify subfertile boars. We believe that boar subfertility might be indicated in vitro by reduced capacity of his spermatozoa to bind to oviductal cells and that this may provide information as to whether an adequate sperm reservoir will presumably be established in vivo from the sperm population that successfully has passed the barriers of the utero-tubal junction.

Animals↗

PubMed2005

Regulatory and necrotic volume increase in boar spermatozoa.

Spermatozoa of many species initially respond to hypotonicity as perfect osmometers. Thereafter they undergo a regulatory process resulting in a decrease in cell volume, similar to that reported for somatic cells. Regulatory volume increase (RVI), a complementary process which is assumed to occur following initial shrinkage of sperm volume after exposure to a hypertonic medium, has not yet been described in detail for spermatozoa. In this study, we investigated whether spermatozoa are able to regulate their volume after hypertonic stress and whether this ability is maintained in preserved sperm. Cell volume changes were recorded using electronic cell sizing. Sperm response to the ion channels blockers quinidine, tamoxifen, and dydeoxyforskolin, and to protein kinase/phosphatase inhibitors lavendustin, staurosporine, and vanadate was studied to investigate possible mechanisms of RVI. Annexin V staining was used in combination with propidium iodide to determine whether hypertonic stress may induce apoptosis. Overall protein tyrosine phosphorylation under hypertonic conditions was measured via flow cytometry using antiphosphotyrosine antibody. Spermatozoa exposed to hypertonic stress initially responded with an abundant subpopulation according to the perfect osmometer model and recovered their volume from this shrinkage after 20 min. RVI was inhibited by quinidine and tamoxifen, which indicates the involvement of the important cellular ions sodium and chloride in this process. Volume regulatory ability was essentially maintained during storage of liquid semen. However, the response of the sperm population was heterogeneous. A second population raised, containing spermatozoa with larger volumes, which demonstrated irregularities in the volume response with respect to osmotic challenge, ion channel blockers, and storage. Under hypertonic conditions, both protein kinase inhibitors (PKI) led to increased isotonic volumes and to elevated initial relative volumes and subsequent volume decrease. RVI was inhibited by the vanadate. Hypertonic stress did not result in an increase in early apoptotic cells, but produced a shift toward late necrotic cells. Substitution of sodium and chloride by choline and sulfate resulted in decreased isotonic volume of sperm treated with lavendustin. Tyrosine phosphorylation levels were reduced after 20 min under hypertonic conditions. It was concluded that RVI is regulated via a protein tyrosine kinase-dependent pathway, and that dephosphorylation occurs when volume regulation is required. The necrotic volume increase (NVI) is associated with the accumulation of sodium and chloride following uncontrolled opening of the channels. The ability to regulate volume after exposure to hypertonic conditions is important for sperm functionality and can have practical applications in spermatological diagnostics and cryopreservation.

Animals↗

PubMed2004

Role of volume-stimulated osmolyte and anion channels in volume regulation by mammalian sperm.

The ability to maintain cellular volume is an important general physiological function. Swelling induced by hypotonic stress results in the opening of channels, through which ions exit with accompanying water loss (regulatory volume decrease, RVD). RVD has been shown to occur in mammalian sperm, primarily through the opening of quinine-sensitive potassium channels. However, as yet, direct evidence for the participation of anion channels in sperm RVD has been lacking. The chloride channel type ClC-3 is believed to be involved in RVD in other cell types. Using electronic cell sizing for cell volume measurement, the following results were obtained. (i) The anion channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), tamoxifen and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) increased hypotonic swelling in concentration-dependent fashion, whereas verapamil (P-glycoprotein inhibitor) had little effect. The most potent, NPPB and DIDS, blocked RVD without affecting cell membrane integrity at effective concentrations. (ii) When gramicidin was included to dissipate Na+/K+ gradients, major secondary swelling was observed under hypotonic conditions. This secondary swelling could be reduced by NPPB, and suppressed completely by replacing chloride in the medium with sulphate, an ion which does not pass through chloride channels. It was deduced that the initial hypotonic swelling activated an anion channel through which chloride ions could then enter freely down a concentration gradient, owing to the lack of a counter-gradient of potassium. (iii) Taurine, an osmolyte often involved in RVD, does not appear to play a role in sperm RVD because lengthy preincubation with taurine did not alter sperm RVD response. Our observations provide direct evidence that a chloride channel (possibly ClC-3) is involved in the process of volume regulation in mammalian sperm.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2004

Role of potassium channels, the sodium-potassium pump and the cytoskeleton in the control of dog sperm volume.

Response to osmotic shock is an important aspect of mammalian sperm physiology. In this study we recorded volume changes of dog spermatozoa at 39, 33, and 25 degrees C under isotonic conditions and following hypotonic shock. Cell volume measurements were performed electronically in saline solutions of 300 and 150 mOsmol kg(-1), and Percoll-washed preparations were compared with unwashed samples. The involvement of potassium channels in volume control was tested by treatment with quinine, while the involvement of the plasma membrane Na(+)-K+ pump was tested by treatment with ouabain. The role of the cytoskeleton was investigated by treatment with colchicine and cytochalasin D. The number of cell populations observed varied with temperature and tonicity. In both types of sperm preparations, between two and three populations were present under isotonic conditions at 25 degrees C whereas at 39 and 33 degrees C only one population was detected. Hypotonic stress at the higher temperatures caused the single population to swell, whereas at 25 degrees C it resulted in a population of cells whose modal volume was similar to that of the middle isotonic sub-population. Both quinine and the cytoskeletal inhibitors markedly increased swelling both under hypotonic conditions at 39 degrees C and under isotonic conditions at 25 degrees C. However, little or no effect of ouabain was observed. We conclude that in dog spermatozoa swelling in response to hypotonic conditions is minimised through the activity of potassium channels and the presence of an intact cytoskeletal network. Under isotonic conditions at 25 degrees C, a considerable proportion of the sperm population is already swollen; this swelling varies between individual males and appears to be due to lowered cytoskeletal and potassium channel activity.

Animals↗

PubMed2004

Kinetics of protein tyrosine phosphorylation in sperm selected by binding to homologous and heterologous oviductal explants: how specific is the regulation by the oviduct?

Essential steps of the capacitation process take place in the oviductal isthmus. A crucial step in the process of capacitation is the phosphorylation of membrane proteins. The aims of this work were (1) to study the effect of dog sperm binding to oviductal epithelium on tyrosine phosphorylation and (2) to investigate the specificity of regulation of molecular changes by the oviduct of different species by comparing the numbers of canine sperm bound to heterologous (porcine) and homologous epithelium, and the kinetics of tyrosine phosphorylation. Semen was collected from four healthy dogs and washed through a Percoll gradient. Explants, small pieces of epithelium, were cut from porcine and estrous bitch oviducts. During 6 h of coincubation in Tyrode medium, the numbers of bound sperm were counted by microvideographic observation, and the state of tyrosine phosphorylation was determined immunocytochemically after 3, 30, 90, 180 and 360 min. Canine sperm bound in similar numbers to homologous and heterologous explants. Increasing tyrosine phosphorylation of tail proteins and subsequent phosphorylation of sperm head proteins were observed. Binding occurred mainly in sperm with non-phosphorylated heads (approximately 2% phosphorylated), while higher proportions of head-phosphorylated cells were found in unbound populations (approximately 40-60%;P<0.05). The head phosphorylation progressed significantly during incubation in unbound spermatozoa (P<0.05), while it was suppressed in bound suspensions. The rate of tyrosine phosphorylation of sperm tail proteins was higher in cells bound to explants than in unbound cells or in those incubated in control medium. There were no significant differences with respect to the kinetics of tyrosine phosphorylation between the two coincubation systems. These observations support the hypothesis that spermatozoa with non-phosphorylated heads preferentially attach to epithelial cells. Tyrosine phosphorylation of sperm head proteins and capacitation are delayed in spermatozoa in close contact with oviductal epithelium. This mechanism appears to be species-independent, as sperm bound similarly to pig and dog oviduct explants, and similar phosphorylation kinetics were observed in both types of tissue.

Animals↗

PubMed2004

Requirement for an intact cytoskeleton for volume regulation in boar spermatozoa.

Osmotically induced cell swelling triggers a chain of events leading to a net loss of major cell ions and water, resulting in cell volume recovery, a process known as regulatory volume decrease (RVD). In many cell types, there is an evidence that the cytoskeleton may play a role in the initial sensing and transduction of the signal of volume change. In this study, we tested the hypothesis that an intact microfilament and microtubule network is required for volume response and RVD in boar sperm before and after capacitation treatment and whether addition of cytochalasin D and colchicine to the capacitation medium would affect volumetric behaviour. Capacitation is a series of cellular and molecular alterations that enable the spermatozoon to fertilize an oocyte. Cell volume measurements of washed sperm suspensions were performed electronically in Hepes-buffered saline solutions of 300 and 180 mosmol/kg. After exposure to hypoosmotic conditions, boar sperm showed initial swelling (up to 150% of initial volume within 5 min), which was subsequently partially reversed (to about 120-130% after 20 min). Treatment with cytochalasin D led to reduced initial swelling (1 micromol/l) and loss of RVD in washed sperm (1-10 micromol/l) and at the beginning of incubation under capacitating conditions (5 micromol/l). Short treatment with 500 micromol/l colchicine affected the volume regulatory ability in sperm under capacitating conditions but not in washed sperm. No significant differences in cell volume response were observed after subsequent addition of cytochalasin D and colchicine to the suspensions of sperm incubated for 3 h under capacitating conditions. However, the incubation under capacitating conditions in the presence of cytochalasin D led to improved volume regulation at the end of the incubation period (23%). The microfilament network appears to be important for volume regulation in washed boar spermatozoa while intact microtubules do not seem to be necessary for osmotically induced RVD. The changes in cytoskeleton microfilament organization during capacitation, possibly affecting the osmotically induced volume response, appear to occur at the later stages of capacitation, whereas changes in microtubules, related to volume regulatory ability, may be programmed within the first stages of capacitation.

Actin Cytoskeleton↗

PubMed2003

Regulation of capacitation of canine spermatozoa during co-culture with heterologous oviductal epithelial cells.

Progress of essential steps of the capacitation is coordinated in the oviductal isthmus, where sperm are stored in close contact with the epithelium. A crucial capacitational event is the phosphorylation of sperm membrane proteins. Regulation of the tyrosine phosphorylation by the oviduct has not been examined in dog sperm yet. The aim of this work was to study the effect of dog sperm binding to porcine oviductal epithelium on capacitation-induced cellular and molecular changes. Epithelial cells were stripped from the oviducts of post-puberal sows and cultured for 5-7 days at 39 degrees C and 5% CO2 on Biomatrix-covered Chamber slides. Sperm washed through Percoll was co-incubated with the oviductal epithelium cell cultures in a bicarbonate Tyrode's medium. During co-incubation, sperm membrane changes, the state of tyrosine phosphorylation and motility were determined after 3, 30, 90, 180, 240 and 360 min. Significant increases in the percentage of capacitated and dead cells were observed in unbound sperm, while bound sperm remained uncapacitated, live and motile. An increasing tyrosine phosphorylation of tail proteins in bound, unbound and control sperm suspensions and a subsequent phosphorylation of head proteins in unbound and control sperm suspensions were observed. A significant difference regarding head phosphorylation (p < 0.05) was found between sperm bound to oviductal epithelium and unbound sperm. Binding occurred mainly in sperm with non- phosphorylated heads, while higher proportions of phosphorylated cells were found in unbound populations. The head phosphorylation progressed significantly during incubation in unbound spermatozoa (p < 0.05); however, it was suppressed in population of sperm attached to oviductal epithelium. Significant correlations between motility parameters related to hyperactivation and tail phosphorylation were found in unbound sperm. These observations support the hypothesis that spermatozoa with non-phosphorylated heads preferentially attach to epithelial cells. It can be concluded that tyrosine phosphorylation of head membrane proteins and capacitation are delayed in canine spermatozoa being in closed contact with oviductal epithelium.

Animals↗

PubMed2003

Seeking mathematical strategies in sperm function analysis: between scylla and charybdis?

During the last decades, essential progresses in reproductive biotechnology were achieved, implying development of special spermatological techniques. The major problem was to set up simple, rapid, precise and adequate evaluation methods. The key aspect to be considered in all assays of sperm fertilizing function is capacitation. As not all spermatozoa respond to fertilizing conditions in a similar manner, it seems to be logical to assess samples via their response to these specific conditions. For the spermatological practice, the sensitivity of methodology for assessment and analysis of data with respect to differences in individual response, in heterogeneity of the population, and proper temporal characterization of the response is crucial for the improvement of evaluation procedures. Currently, most used statistical analytical tools in spermatology do not always fulfil these essential sensitivity requirements. We structured our paper concerning different fields of mathematical science (distribution analysis, fractal geometry, functional approximation and differentiation) related to the modern insights in sperm function analysis. The spectrum of methods we are going to review in this paper is restricted to basic ideas to illustrate how the accuracy and sensitivity of sperm evaluation assays may be improved by applying adequate elementary tools of the mathematical analysis.

Animals↗

PubMed2003

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.

Animals↗

PubMed2001

Sperm-cell volumetric measurements as parameters in bull semen function evaluation: correlation with nonreturn rate.

Sperm-cell volume, measured electronically by cell counter, is a parameter providing information about the state and integrity of the plasma membrane by determining cell osmotic reactivity (swelling level). Electronic volume measurement is a modification of the hypo-osmotic swelling test, based on the increase in sperm volume in response to hypo-osmotic stress. In this study the volumetric method was applied to bull ejaculates, and the relationships of volumetric parameters, osmolality of seminal plasma, and concentration of sodium and potassium ions in seminal plasma, with the nonreturn rate (NRR) were examined. Significant correlations were found between volumetric parameters, conventional spermatological parameters, and NRR. The relative volume shift of the mean volume correlated significantly with motility before and after thawing (P < 0.05). NRR correlated significantly with iso-osmotic cell volume (- 0.49; P < 0.05) and with the relative volume shift (0.51, P < 0.05). The prediction level of regression models was improved when volumetric parameters (iso-osmotic cell volume) were included in the multiple regression model. Therefore, using electronic volume measurement as a component for fast, correct and valid (up to 50,000 cells), recording sperm-cell population may help to evaluate ejaculate quality more precisely.

Animals↗

PubMed2001

Role of quinine-sensitive ion channels in volume regulation in boar and bull spermatozoa.

The ability to reverse swelling caused by hypo-osmotic stress is an important cell function; in spermatozoa, it is likely to be of consequence during ejaculation and also during the thawing process that terminates cryopreservation. In this study, the time course of boar and bull sperm volume changes after exposure to hypo-osmotic conditions at 39 degrees C was recorded. Cell volume measurements of washed sperm suspensions were performed electronically in Hepes-buffered saline solutions of 300 and 180 mosmol kg(-1) containing 2.5 mmol K(+) l(-1). Treatment with quinine in the presence or absence of the potassium ionophore valinomycin was used to determine whether potassium channels were involved in the reversal of swelling. After exposure to hypo-osmotic conditions, both bull and boar spermatozoa showed initial swelling (up to 200% and 140% of initial volume, respectively, within 5 min), which was subsequently partially reversed (to about 150% and 120%, respectively, after 20 min). Incubation with quinine led to an increase in swelling in both species. However, bull sperm volume was already maximal (up to 294%) after 30 s and declined thereafter, whereas boar sperm volume increased slowly to a maximum of about 220% after 20 min. Valinomycin treatment caused quinine-induced swelling in bull spermatozoa to decrease rapidly to control (no quinine, no valinomycin) values, whereas in quinine-treated boar spermatozoa it had an opposite, enhancing effect. Interpreting these results in the light of data from studies by others on a variety of cell types, it is proposed that swelling-activated potassium channels are involved in regulatory volume decrease in both species of spermatozoa, but that boar spermatozoa may contain fewer swelling-activated chloride channels than do bull spermatozoa.

Animals↗

PubMed2001

Kinetic characterization of the changes in protein tyrosine phosphorylation of membranes, cytosolic Ca2+ concentration and viability in boar sperm populations selected by binding to oviductal epithelial cells.

On reaching the oviduct, spermatozoa are retained in the isthmic region of the oviduct until ovulation occurs. The essential steps of capacitation are co-ordinated in this region. In this study, a primary cell culture system of oviductal epithelial cells was established to investigate sperm binding to oviductal epithelium and modulation of sperm function during incubation under capacitating conditions in co-culture with oviductal epithelial cells. Epithelial cells were stripped from the oviducts of sows and cultivated for 5-7 days on Lab-Tek Chamber slides on Matrigel. The preparations on chamber slides and suspensions of control spermatozoa were incubated for 3 h in Tyrode's albumin lactate pyruvate (TALP) medium. At 3, 30, 60, 90 and 180 min the free-swimming spermatozoa were collected by washing, and membrane integrity, tyrosine phosphorylation patterns and [Ca(2+)](i) of bound, unbound and control spermatozoa were assessed with fluorescent probes (propidium iodide, Cy-3 and fluo-3-AM). The cells bound to oviductal epithelial cells showed reduced cytosolic Ca(2+) concentration, reduced and almost absent tyrosine phosphorylation of membrane proteins and higher viability at the time of the first sampling. Increases in Ca(2+) concentration and cell death occurred much more slowly during incubation in cells bound to oviductal epithelial cells compared with free-swimming spermatozoa, and no changes in tyrosine phosphorylation were observed. The preferential binding of viable, low-Ca(2+) cells with suppressed tyrosine phosphorylation and slower functional modulation of boar spermatozoa attached to oviductal epithelial cells might represent a mechanism for selecting functionally competent spermatozoa and prolonging their lifespan by delaying capacitation in the oviductal reservoir.

Animals↗

PubMed2001

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.

Animals↗

PubMed2000

Heterogeneous osmotic behaviour in boar sperm populations and its relevance for detection of changes in plasma membrane.

The spermatozoa of most mammals behave as 'perfect osmometers'. The volume response to osmolality obeys the Boyle-Van't Hoff relationship (i.e. volume changes are determined by the osmotically active fraction of the cell volume (solids and water)). Most evaluations of osmotic sperm cell behaviour have been based on the mean volume of the cell population. In the present study, both mean and modal volumes of samples of sperm were evaluated. Both mean and modal volumes responded to environmental osmolality via the Boyle-Van't Hoff relationship; however, the modal volume showed a more sensitive response than the mean volume. This was confirmed for both ejaculated and epididymal spermatozoa. After incubation under capacitating conditions, the difference in modal and mean volume response of ejaculated sperm was considerably diminished and, in epididymal sperm, completely abolished. The sperm osmotic behaviour was still consistent with the Boyle-Van't Hoff equation, but the apparent osmotically inactive modal cell volume decreased after exposure to capacitating conditions in both ejaculated and epididymal sperm samples. The changes in epididymal sperm were more intensive. Due to its enhanced sensitivity to environmental osmolality and incubation under capacitating conditions, the modal volume could be used as a parameter for evaluating sperm population response, such as for detecting environmentally or cryopreservation-induced membrane changes.

Animals↗

PubMed2000

Only low levels of spermadhesin AWN are detectable on the surface of live ejaculated boar spermatozoa.

The zona-binding protein family of spermadhesins are constituents of boar seminal plasma that are generally believed to attach to the acrosomal region of spermatozoa and thereby assist sperm interaction with the zona pellucida at fertilization. However, previous studies have yielded conflicting results with respect to amounts of adhesin bound to ejaculated cells, to the distribution of bound adhesin within the sperm population, and the regionalization of binding on the sperm surface. In the present study, spermadhesin AWN in unfixed living suspensions of boar spermatozoa was assessed by means of flow cytometry and immunocytochemistry, using a polyclonal antibody raised in chicken. Direct probing with an Oregon Green conjugate of the antibody was compared with indirect probing using Alexa Fluor-conjugated goat anti-chicken IgG as second antibody. Regardless of staining procedure, the live sub-population showed homogeneously low levels of staining, whereas the dead sub-population showed high (more than 5-fold greater) levels of staining. The live cells were stained about 2-fold more intensely by anti-AWN than by preimmune immunoglobulin, indicating the presence of small amounts of AWN. Immunocytochemistry showed the live cells to be faintly stained all over their surface, whereas staining of the dead cells was largely localized to the acrosomal region. This latter staining was non-specific, preimmune immmunoglobulin resulting in as much bound fluorescence as anti-AWN. Attempts to block non-specific staining with appropriate pretreatment with chicken or goat serum (as compared with routine use of BSA) met with variable and incomplete success, and did not increase staining by anti-AWN relative to preimmune serum in either live or dead cells. It is concluded that limited amounts of spermadhesin AWN bind tightly over the whole surface of live ejaculated boar sperm. However, the acrosomal region of disrupted sperm has an alarming tendency to bind fluoro-conjugates of immunoglobulins non-specifically.

Animals↗