A mouse monoclonal antibody against boar acrosin and antiserum prepared to highly purified acrosin in female rabbits were used to detect the antigen in various fluids and tissues of boars using an indirect immunofluorescence technique. A strong reaction was found in fluid and epithelial tissue of the seminal vesicles as well as in the germinal cells in the testis. No immunoreactivity was detected in tissues of the epididymides and other organs of the boar. The antigens present in seminal vesicle fluid of boars were partially purified by column chromatography. It was demonstrated that two antigens differing in molecular mass were present and both possessed protease and amidase activity. The higher molecular mass antigen eluted from a gel filtration column in a volume identical to that of proacrosin. The same result was obtained in polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE). The low molecular mass antigen was eluted from Sephadex G-75 column together with natural protease inhibitors corresponding in molecular mass to less than 20 kDa. The mobility of the antigen in SDS-PAGE was greater than that of chymotrypsin. It is assumed that the protease from seminal vesicle epithelia resembled acrosin in structure and function. Acrosin may therefore not be specific for spermatozoa.
D. Čechová, V. Jonáková, L. Veselský and E. Töpfer-Petersen
L. Veselský, V. Jonáková, M. L. Sanz, E. Töpfer-Petersen and D. Čechová
Summary. A highly purified 15 kDa glycoprotein isolated from ejaculated spermatozoa was used to raise antisera in female rabbits. An indirect immunofluorescence technique was used to detect the antigen in the seminal vesicle tissue and on the acrosomes of ejaculated, native and capacitated, boar spermatozoa. No immunoreactivity was detected on cells of the seminiferous tubules (spermatogonia, spermatocytes, and spermatids), on spermatozoa in the ductus epididymis and in cells of the epididymal and testicular tissues. These observations support the view that the 15 kDa protein is produced in the seminal vesicle secretory epithelium, and is attached to the sperm plasma membrane during the exposure of spermatozoa to seminal vesicle compounds. The observations that the antigen remained on the acrosome of ejaculated spermatozoa after capacitation and blocked sperm–oocyte binding in vitro suggest that the antigen plays a role in sperm–egg interactions. The strong immunoreactivity exhibited by cumulus cells after incubation of antisera with the porcine egg surrounded by cumulus cells shows the possible importance of the 15 kDa glycoprotein for contact of spermatozoa with cells of the cumulus oophorus surrounding the egg.
Keywords: zona pellucida; binding proteins; sperm–egg interaction; gamete recognition; boar; indirect immunofluorescence
AM Petrunkina, R Gehlhaar, W Drommer, D Waberski and E Topfer-Petersen
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.
D Waberski, F Magnus, F Ardón, A M Petrunkina, K F Weitze and E Töpfer-Petersen
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.
A M Petrunkina, M Hebel, D Waberski, K F Weitze and E Töpfer-Petersen
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 μmol/l) and loss of RVD in washed sperm (1–10 μmol/l) and at the beginning of incubation under capacitating conditions (5 μmol/l). Short treatment with 500 μmol/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.
AM Petrounkina, RA Harrison, R Petzoldt, KF Weitze and E Topfer-Petersen
The osmotic reactivity of boar spermatozoa during incubation in vitro was studied using a hypo-osmotic swelling test in conjunction with electronic measurement of cell volume. Sperm populations showed fluctuations in both iso-osmotic cell volume and hypo-osmotic volume response that fitted mathematical models for periodicity. Significant differences of frequency and amplitude were observed during sperm incubation under capacitating conditions as compared with those under non-capacitating conditions. In addition, different boars showed specific differences in their fluctuation characteristics under capacitating conditions. During incubation under capacitating conditions, a decrease in osmotic reactivity was observed that correlated with a decrease in motility, while the absolute value of the earliest maximum of the osmotic-induced response correlated with an increase in the proportion of discharged acrosomes. The time course of the cyclical behaviour of osmotic reactivity may be a useful parameter for assessing boar sperm response to capacitating conditions.
AM Petrunkina, RA Harrison, M Hebel, KF Weitze and E Topfer-Petersen
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.
AM Petrunkina, J Friedrich, W Drommer, G Bicker, D Waberski and E Topfer-Petersen
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.
AM Petrunkina, A Lakamp, M Gentzel, M Ekhlasi-Hundrieser and E Topfer-Petersen
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.
A M Petrunkina, D Waberski, A R Günzel-Apel and E Töpfer-Petersen
Fertilization success cannot be attributed solely to the absolute number of vital, motile, morphologically normal spermatozoa inseminated into the female but more especially to their functional competence. A range of in vitro tests has therefore been developed to monitor crucial aspects of sperm function: their ability to adapt to changing osmotic conditions, to bind to the oviductal epithelium, and to undergo capacitation in an appropriate and timely manner. The tests employ flow cytometry in conjunction with fluorescent techniques, electronic cell counting, and computer-assisted image area analysis. The highly quantitative analysis provided by electronic sizing and flow cytometry enables assessment of representative cell numbers in a very short time with high reproducibility. More importantly, it allows the detection of physiological heterogeneity within an ejaculate in terms of the development of cell subpopulations and enables the kinetic analysis of changes in living cell suspensions. The tests offer a promising strategy for evaluating fertility in domestic animals. The capability for volume regulation ensures that sperm recover from the tonic shocks experienced at ejaculation and during cryopreservation. Assessment of capacitation in vitro provides valuable information on both the sperm’s ability to respond to fertilizing conditions and the sequence and rates of ongoing capacitation/destabilization processes. The monitoring of response to capacitating conditions in kinetic terms allows the sensitive and adequate detection of sperm populations expressing fertilization attributes and their ability to respond to external stimuli in a timely manner. However, subfertility is likely to be associated with a suboptimal response (i.e. too high or too low) rather than a minimal response.