Loss of acrosin from bovine spermatozoa following cold shock: Protective effects of seminal plasma

Loss of the alkaline proteinase acrosin and other proteins from the acrosome of bovine spermatozoa was investigated following cold shock and/or incubation of the spermatozoa at either 5, 21, or 37 °C for 4 hr. As detected by electrophoretic analyses of the acrosomal material two bands of acrosin activity and 10 proteins were lost from the acrosome after cold shock and incubation for 4 hr at 5 or 21 °C, whereas one acrosin band and 10 protein bands were lost after cold shock and incubation at 37 °C. Only 45% of the total acrosin activity remained in the acrosome after both cold shock and 4-hr incubation at 37 °C. Egg yolk, present at levels above 15%, and seminal plasma prevented much of the loss of acrosin from the cells.     

Proacrosin activation and acrosin release during the guinea pig acrosome reaction

The kinetics of proacrosin activation and release from guinea pig spermatozoa during the nonsynchronous acrosome reaction were studied. Epididymal spermatozoa were incubated at 37 degrees C in a defined medium (pH 7.8) containing 1.7 mM Ca2+. After 195 min, 78% of the motile spermatozoa had undergone the acrosome reaction as determined by light microscopy. Acrosin and proacrosin levels in the spermatozoa and medium were measured at the beginning of the incubation period. Most of the total acrosin activity (78%) was associated with the spermatozoa, of which greater than 90% was in the form of proacrosin. Proacrosin represented a small, stable fraction (23%) of the total acrosin in the medium; it did not activate to acrosin while in the medium. After 195 min, a decrease in sperm-associated total acrosin (42%; p less than 0.05) was accompanied by an increase in the total acrosin level in the medium (115%; P less than 0.05). No change in the relative proacrosin content (percent of total acrosin) was evident in either medium or spermatozoa. Additional experiments quantified acrosin and proacrosin during the progression of the acrosome reaction. Both the loss of sperm-associated total acrosin and the increase in total acrosin levels in the medium were highly correlated with the fraction of acrosome-reacted spermatozoa (r = 0.954 and 0.922, respectively; P less than 0.001). However, the rate of acrosin appearance in the medium was only 60% (P less than 0.001) of the rate of acrosin loss from the spermatozoa. The fractional proacrosin content of spermatozoa (94%) and medium (31%) remained unchanged during the acrosome reaction (r = 0.15 and 0.30, respectively; P greater than 0.1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Acrosomal enzymes of human spermatozoa before and after in vitro capacitation

The effect of in vitro capacitation (events that occur before the acrosome reaction) on the acrosomal enzymes of human spermatozoa was determined. Capacitation of human spermatozoa was assessed by their ability to penetrate denuded hamster oocytes. The activities of a number of enzymes commonly associated with the sperm acrosome, including nonzymogen acrosin, proacrosin, inhibitor-bound acrosin, hyaluronidase, acid phosphatase, beta-glucuronidase, beta-glucosidase, beta-N-acetylglucosaminidase, beta-galactosidase and beta-N-acetylgalactosaminidase were assessed. With the exception of acid phosphatase, no alteration in enzyme activity occurred after 4 h of incubating the spermatozoa under capacitation conditions although gamete fusion took place. The acid phosphatase levels decreased twofold, presumably due to the loss of seminal (prostatic acid phosphatase that loosely adheres to spermatozoa. After 8 h of capacitation, a large decrease in sperm enzyme levels took place only in the case of hyaluronidase, although small decreases were also noted in total acrosin, proacrosin and inhibited acrosin. No new electrophoretically migrating forms of acrosin were observed. Decreases in total acrosin and proacrosin, but not in inhibited acrosin, also occurred when spermatozoa were incubated under noncapacitating conditions for 8 h, indicating that capacitation may specifically cause the release of some acrosin inhibitor from human spermatozoa. It is concluded that, with the possible exception of hyaluronidase, the in vitro capacitation of human spermatozoa does not cause a major change in its acrosomal enzyme content so that these hydrolases are fully present before the acrosome reaction takes place during gamete fusion. Serum albumin appears to protect against the loss of some of these enzymes since the activity of several glycosidases was significantly reduced when the spermatozoa were incubated for 8 h in human serum albumin-free medium.     

Wheat germ agglutinin treatment of follicle cell-free mouse oocytes inhibits fertilization

Controversy exists whether treatment of follicle cell-free oocytes with wheat germ agglutinin (WGA) prevents fertilization. Lack of inhibition in one case has led to the suggestion that acrosin may not be a zona lysin. To re-examine the effect of the WGA, the zona pellucida of follicle cell-free mouse oocytes was made more resistant to proteinase digestion by treatment with 10 or 50 μg/ml WGA. Such WGA-treated oocytes showed decreased fertilizability when washed to remove excess WGA and incubated with capacitated spermatozoa. Oocyte cleavage was used as an end point, because a large number of spermatozoa adhered to the eggs after WGA treatment, making observation of sperm penetration and pronucleus formation unreliable. Resistance to proteinase digestion increased, and the fertilizability decreased with the higher amount of WGA. The action of WGA was most likely not mediated by a direct effect on sperm motility, sperm acrosin activity, sperm binding to the zona pellucida, or oocyte cleavage. WGA did not affect the acrosome reaction of guinea pig spermatozoa. These data show that WGA treatment of follicle cell-free mouse oocytes results in decreased fertilizability, possibly by rendering the zona pellucida more resistant to sperm proteinase digestion.     

Immunolocalization of proacrosin/acrosin in rabbit sperm during acrosome reaction and in spermatozoa recovered from the perivitelline space

The participation of acrosin in mammalian sperm penetration through the zona pellucida has been amply debated. In this paper we report the immunolocalization—by silver enhanced immunogold technique using ACRO-8C10 monoclonal antibody to human acrosin—of proacrosin/acrosin on ejaculated rabbit spermatozoa incubated in vitro in a capacitating medium and on spermatozoa recovered from the perivitelline space. After incubation in a capacitating medium, four different patterns were observed: (1) no labeling on acrosome intact spermatozoa; (2) labeling on the rim of the head; (3) labeling on the whole acrosome area; and (4) no labeling on acrosome reacted spermatozoa. At the start of incubation, spermatozoa with pattern 1 were the most abundant, whereas at the end of the 32 h incubation period, patterns 2 and 3 were the most frequent. On the other hand, 625 perivitelline spermatozoa were recovered from 17 fertilized rabbit eggs, of which 26% were labeled with the anti-acrosin monoclonal antibody ACRO-8C10 in two different areas: (1) only on the equatorial region; and (2) only on the postacrosomal area. These results are consistent with the idea that proacrosin/acrosin remains associated to the acrosome reacted spermatozoa for long periods of time, and that proacrosin/acrosin associated to perivitelline spermatozoa could be responsible for the second penetration of fresh rabbit eggs by perivitelline spermatozoa. © 1994 Wiley-Liss, Inc.     

Immunolocalization of proacrosin/acrosin in bovine sperm and sperm penetration through the zona pellucida

The aim of the present work was to immunolocalize acrosin in bull spermatozoa incubated for up to 6 h in capacitating culture medium (TALP-heparin), in order to study the kinetics of its release during the acrosome reaction and in vitro sperm penetration. Six replicates from semen of one bull were used. Acrosin was localized by the silver-enhanced immunogold technique using anti-bovine acrosin monoclonal antibody ACRO-C2E5. Spermatozoa thus showed the presence of acrosin only at the acrosomal region. Four different patterns were seen: (1) no labeling: (2) intense labeling on the rim of the portion of the acrosome; (3) diffuse label over the entire acrosomal region; and (4) intense label over the entire acrosomal region. Spermatozoa incubated in capacitating medium for 4 h showed that unlabeled (pattern 1) spermatozoa decreased from 72% to 28% difference that was found to be significant (p<0.05). Patterns 3 and 4 increased from about 10% to 20-29%, (p<0.05). With further incubation (4-6 h), pattern 1 increased while patterns 3 and 4 decreased differences were not significant (p0.05). The incidence of pattern 2 did not change through the whole incubation period. Sperm penetration through the zona pellucida of in vitro matured bovine oocytes (57%) or empty zonae pellucida (70.5%) increased (p<0.05) as a function of sperm incubation time in capacitating medium. The presence of acrosin, as determined by the silver-enhanced immunogold technique, was highly correlated with sperm penetration of in vitro mature bovine oocyte (r=0.98) and cryopreserved zonae pellucidae (r=0.93) (p<0.01).     

Determination of acrosin activity of boar spermatozoa by the clinical method: optimization of the assay and changes during short-term storage of semen

A clinical assay to evaluate total acrosin activity developed for human semen has been optimized for use in boar spermatozoa. The main modifications included a decrease of sperm number per assay from 1.0 to 10.0 x 10(6) to 12.5 to 75.0 x 10(3) spermatozoa, and the time of incubation from 180 to 60 min. Linearity of response for differing quantities of spermatozoa was maintained. Extensive washing of spermatozoa was necessary to eliminate seminal plasma, the source of acrosin inhibitors. Seminal plasma that was diluted 1000 times inhibited acrosin activity by about 50%. To abolish the inhibitory effect of seminal plasma it was necessary to use 25,000-fold dilution. Total acrosin activity of boar spermatozoa was about 100 times higher than that of human spermatozoa. Acrosin activity of boar spermatozoa in extended semen decreased during 7 d of storage. These results indicate that the clinical assay of acrosin activity can be used for boar spermatozoa to evaluate the quality of boar semen.     

Characterization of proacrosin/acrosin system after liquid storage and cryopreservation of turkey semen (Meleagris gallopavo)

This study was designed to identify the effect of liquid storage at 4 °C for 48 h and cryopreservation on the proacrosin/acrosin system of turkey spermatozoa. Anti-acrosin I antibodies were produced and used to demonstrate Western blot analysis profile of the proacrosin/acrosin system of sperm and seminal plasma and possible changes in the proacrosin/acrosin system of turkey sperm stored for 2.5, 24, and 48 h or cryopreserved. At the same time acrosin-like activity was examined by the measurement of amidase activity of sperm extracts, sperm suspension, and seminal plasma of turkey semen. A computer-assisted sperm analysis system was used to monitor the sperm motility characteristics of turkey sperm stored for 48 h or cryopreserved. Different profiles of the sperm proacrosin/acrosin system were observed regarding the presence or absence of inhibitors (p-nitrophenyl-p'-guanidine benzoate [NPGB] and Kazal family inhibitor) during the extraction process. When NPGB was present three main bands were observed with the molecular weight ranging from 66 to 35 kDa. Bands corresponding to acrosin I and II were not observed. In sperm extract without NPGB, three or four bands were observed with the molecular weight ranging from 41 to 30 kDa. The bands corresponding to acrosin I and II were observed. During liquid storage a decrease in sperm motility and an increase in sperm-extracted amidase activity were observed. After 24 and 48 h of storage, extracted amidase activity was higher than at 2.5 h by 24% and 31%, respectively. However, no changes in the Western blot analysis profiles of sperm extract and seminal plasma were visible during liquid storage. After cryopreservation a decrease in sperm motility and all sperm motility parameters were observed. In contrast to liquid storage, cryopreservation did not increase extracted amidase activity. However, changes in Western blot analysis profiles were visible in sperm extract and seminal plasma after cryopreservation. After freezing-thawing, additional bands appeared in sperm extract and seminal plasma. These bands were of different molecular weight regarding the presence or absence of NPGB. These data suggest that the mechanism of damage to the proacrosin/acrosin system is different for liquid storage and cryopreservation. Liquid storage seems to increase in the susceptibility of the proacrosin/acrosin system to be activated during extraction. Kazal inhibitors of turkey seminal plasma are involved in the control of proacrosin activation. The disturbances of the proacrosin/acrosin system of turkey spermatozoa can be related to a disturbance in the induction of the acrosome reaction. Our results may be important for a better understanding of the proacrosin/acrosin system of turkey spermatozoa and disturbance to this system during liquid storage and cryopreservation.     

Proteolytic activity of rabbit perivitelline spermatozoa.

Acrosin, an acrosomal serine protease, has been associated with binding of spermatozoa and their penetration through the zona pellucida. This study was aimed at determining whether the remaining proacrosin/acrosin system on rabbit perivitelline spermatozoa still has proteolytic activity and whether this activity is involved in further penetration of unfertilised rabbit eggs. Eight hundred and sixty-five rabbit perivitelline spermatozoa were evaluated by the gelatin-substrate film technique for the detection of acrosin on individual spermatozoan. Fifteen per cent of the studied spermatozoa showed small digestion halos on the gelatin film. The proteolytic activity of rabbit perivitelline spermatozoa was inhibited in the presence of 1 mg/ml of soybean trypsin inhibitor (SBTI) or with 20 micrograms/ml of a mixture of the monoclonal anti-proacrosin/acrosin antibody. In vitro fertilisation occurred in 21.8% of rabbit oocytes co-incubated with perivitelline spermatozoa and was completely inhibited when oocytes were incubated with 600 micrograms/ml of a mixture of three anti-acrosin monoclonal antibodies (ACRO-A8C10, ACRO-C2B10 and ACRO-C5F10). Inseminations in the presence of anti-cholera monoclonal antibody (irrelevant to spermatozoa) resulted in 17.6% fertilisation. These results support the idea that the residual proacrosin/acrosin system in perivitelline spermatozoa might be involved in spermatozoal binding and/or second penetration through the zona pellucida.     

Production,characterization, and use of ionophore-induced,calcium-dependent acrosome reaction in ram spermatozoa

A system has been developed for inducing a calcium-dependent acrosome reaction in ram spermatozoa in vitro using the calcium ionophore A23187. The resultant reaction is accompanied by release of the acrosomal enzymes hyaluronidase and acrosin, but there is no release of the cytoplasmic enzyme glucose 6-phosphate isomerase. In any given cell, the visible acrosome reaction apparently takes place rapidly, but there is a variable delay before the reaction occurs. Under optimum conditions, about 90% of treated spermatozoa show an acrosome reaction within one hour. Preincubation of the spermatozoa with the proteinase inhibitors p-amino-benzamidine or p-nitrophenylguanidinobenzoate allows two stages of the reaction to be distinguished ultrastructurally, a membrane fusion stage followed by a dispersal of the acrosomal matrix. In the presence of the inhibitors, the first stage is delayed but is completed within 1 hour, whereas the second remains largely incomplete. In the presence of calcium, ionophore concentrations which induce an acrosome reaction abolish sperm motility rapidly and completely. However, by adding serum albumin shortly after addition of ionophore, motility can be preserved while the acrosome reaction occurs as usual; the motility pattern observed under these conditions is of the “whip-lash” or “activated” type. Although the motile ionophore-treated spermatozoa were unsuccessful at penetrating normal mature sheep oocytes in vitro, they were able to penetrate zona-free oocytes, after which swelling and decondensation of the sperm head took place.     

Acquisition of zona binding by ram spermatozoa during epididymal passage,as revealed by interaction with rat oocytes

To assess the ability of ram spermatozoa to bind to oocytes, spermatozoa (2.5–200 × 10⁶/500μ1) taken from the rete testis, or from various regions of the epididymis (head, body, and tail) were mixed with cumulus-free heterologous oocytes obtained from immature superovulated rats. After incubation for 30–45 min in Parker 199 Hepes medium at 35°C, testicular spermatozoa were unable to bind to the zona at any of the concentrations used. However, spermatozoa from the middle body of the epididymus were able to bind to the zona and this binding reached a maximum in the distal body and in the tail of the epididymis. The spermatozoa were bound by their heads. Electron microscopy showed that the plasma membrane and the acrosome of the bound sperm remained intact, without any sign of an acrosome reaction.     

The lysis effect of bull spermatozoa on gelatin substrate film methodical investigations.

Proteolytic activity in the acrosomes of ejaculated bull spermatozoa was demonstrated using an autoradiographic film as a gelatin substrate. Incubation of the spermgelatin adducts at +37 degrees C and 94% humidity, which was kept constant by ventilating an incubator with water-saturated compressed air, yielded reproducible results. Gelatin depolymerisation started adjacent to the posterior segment of the acrosome within 30 to 60 s after application of individual spermatozoa to the substrate membrane and, finally, increased to a white circular digestion area enveloping the entire sperm head. The observed gelatinolysis seems to be mainly caused by acrosin, the trypsin-like acrosomal proteinase. This conclusion is supported by the positive correlation (r = +0.83, P is less than or equal to 0.01) found between the mean values of the lysis areas of individual spermatozoa on gelatin films and the acrosin activity of the sperm population measured with Bz-Arg-OEt as substrate after acidic extraction of the spermatozoa. In addition, prior saturation of the substrate layers with acrosin inhibitor (SSPI-I, II) from boar seminal plasma prevented the lysis reaction. Extraction of acrosin from the spermatozoa before application to the gelatin membranes resulted in a complete loss of any proteolytic activity. If spermatozoa were stored for 4 to 6 days at +4 degrees C or -20 degrees C in Tris buffer and afterwards applied to the substrate layer, lysis areas of individual spermatozoa differed markedly. Spermatozoa from undiluted ejaculated frozen at -20 degrees C showed no proteolytic effect on gelatin films. In general, there was a high correlation (r = +0.83, P is less than or equal 0.01) between the number of "living cells" characterized by live-dead staining and the percentage of spermatozoa active on the substrate membranes.     

Inhibition of the human sperm acrosome reaction by proteinase inhibitors

The analogue of the second messenger cAMP, dibutyryl cAMP (dbcAMP), was shown to induce the human sperm acrosome reaction to the same extent as calcium ionophore A23187, providing preliminary evidence for the involvement of the adenylate cydase system in the acrosome reaction (AR) of human spermatozoa. Using the human synchronous acrosome reaction system, proteinase inhibitors were tested for their effect on the dbcAMP-induced human sperm acrosome reaction. The proteinase inhibitor 4′-acctamidophenyl4-guanidinoben-zoate (AGB), an inhibitor of proacrosin activation and of acrosin, when added at either the onset of incubation or to capacitated spermatozoa, 5 min prior to stimulation by dbcAMP, significantly (P < 0.01) inhibited the acrosome reaction at final concentrations of 1 × 10^?4 M to 1 × 10^?6 M in comparison to dbcAMP treatment alone. At concentrations less than 1 × 10^?6 M, no significant inhibitory effect was seen. Similarly, para-aminobenzamidine (pAB), also an inhibitor of proacrosin activation and of acrosin, significantly (P < 0.01) inhibited the dbcAMP-induced acrosome reaction at final concentrations of 1 × 10-4 M to I × 10-6 M when added at either the onset of incubation or to capacitated spermatozoa, 5 min prior to stimulation by dbcAMP, in comparison to stimulation by dbcAMP alone. However, at concentrations less than 1 × 10^?6 M, no significant (P > 0.05) inhibitory effect was seen. These results indicate that a serine proteinase, most likely acrosin, has a role in the human sperm acrosome reaction and suggest that the enzyme functions after the involvement of the adenylate cyclase system.     

Acrosin activity is a good predictor of boar sperm freezability

The main aim of this study was to determine whether acrosin activity could predict boar sperm freezability. For this purpose, we characterized the changes in sperm quality and acrosin activity throughout the cryopreservation procedure of sperm samples from 30 Pietrain boars by analyzing four critical steps: step 1 (extended sperm at 15 °C), step 2 (cooled sperm at 5 °C), step 3 (30 minutes postthaw), and step 4 (240 minutes postthaw). Freezability ejaculate groups were set on the basis of sperm motility and membrane integrity after freeze–thawing. Results obtained highlighted the low predictive value in terms of freezability of sperm motility and kinematics and sperm membrane integrity, as no differences between good and poor freezability ejaculates were seen before cryopreservation. Significant differences (P < 0.05) between ejaculate groups were observed in the cooling step at 5 °C for sperm kinetic parameters, and after thawing for sperm motility and membrane integrity. In contrast, acrosin activity appeared as an indicator of boar sperm freezability because the differences (P < 0.05) between good and poor freezability ejaculates manifested yet in extended samples at 15 °C. On the other hand, we also found that variations in sperm kinematics, membrane lipid disorder, intracellular calcium content, acrosome integrity, and acrosin activity throughout the cryopreservation procedure were indicative of a significant damage in spermatozoa during the cooling step in both ejaculate groups. In conclusion, the main finding of our study is that acrosin activity can be used as a reliable predictor of boar sperm freezability because it differs significantly between good and poor freezability ejaculates yet before freeze–thawing procedures took place, i.e., in the refrigeration step at 15 °C.     

Human sperm acrosomal status,acrosomal responsiveness,and acrosin are predictive of the outcomes of in vitro fertilization: A prospective cohort study

The sperm acrosome reaction (AR) is a physiological secretory course of membrane fusion and hydrolytic enzymes, as well as matrix protein release, enabling spermatozoa to penetrate the egg surroundings. An instable acrosomal status before a specific stimulus, insufficient acrosomal responsiveness, or inadequate enzymatic activity of acrosomal content can be detrimental to male fertility. This prospective cohort study was designed to determine whether three human sperm acrosome evaluation parameters—including spontaneous AR rate, AR after calcium ionophore A23187 challenge (ARIC) rate, and modified Kennedy acrosin activity—can predict fertilization outcomes in vitro and are correlated with male characteristics. A total of 485 eligible couples undergoing in vitro fertilization (IVF) therapy were included in two phases of this study. In a ‘construction phase’, three acrosome evaluation parameters were determined simultaneously in 132 cases, whereas in a ‘validation phase’, the spontaneous AR rate was determined in 353 cases. The results of the ‘construction phase’ revealed that the spontaneous AR rate was the only significant predictor of fertilization outcome (unadjusted odds ratio [OR]?=?0.68, 95% confidence interval [CI]: 0.53–0.88, P?=? 0.003; adjusted OR = 0.64, 95% CI: 0.43–0.95, P?=? 0.03), and the cut-off value for total fertilization failure (TFF) prediction, determined by ROC curve analysis, was 9.91%; higher acrosin activity was shown to predict a higher fertilization rate only when patients were divided into groups (≥25 μIU/10⁶ spermatozoa, 14–25 μIU/10⁶ spermatozoa, <14 μIU/10⁶ spermatozoa). The spontaneous AR rate was negatively correlated with sperm motility, forward progression motility, and normal morphology; modified Kennedy acrosin activity was positively correlated with normal morphology; and the ARIC rate was not correlated with any of the male characteristics. A similar result was obtained for the spontaneous AR rate in the ‘validation phase’, and the cut-off value in predicting TFF was calibrated for 9.52%. Clinically, patients can voluntarily choose spontaneous AR rate alone or in combination with modified Kennedy acrosin activity to predict TFF, and early rescue intracytoplasmic sperm injection (ICSI), half ICSI, or full ICSI should be considered in advance for men with spontaneous AR rates ≥9.52% or spontaneous AR rates ≥9.52% and AE activities <25 μIU/10⁶ spermatozoa.     

Evidences for the presence of chymotrypsin-like activity in human spermatozoa with a role in the acrosome reaction

The effect of chymotrypsin inhibitors and substrates on the human sperm acrosome reaction stimulated by the human zonae pellucidae or follicular fluid were evaluated. Motile spermatozoa, selected by a Percoll gradient, were incubated at 1 × 10⁷ cells/ml, 37°C, and 5% CO₂, After 4.5 hr, the chymotrypsin inhibitor TPCK (N-Tosyl-L-Phenylalanine-Chloromethyl Ketone) or the substrate ATEE (N-Acetyl-L-Tyrosine Ethyl Ester) were added for 30 min. Then, four oocytes were added and the percentage of acrosome-reacted spermatozoa on the zona was determined. TPCK and ATEE inhibited the zona pellucida-induced acrosome reaction. The chymotrypsin inhibitors TPCK and chymostatin and the chymotrypsin substrates ATEE, BTEE (N-Benzoyl-L-Tyrosine Ethyl Ester), Succinyl-Ala-Ala-Phe-7-Amido-4-Methyl-Coumarin (Suc-Ala-Ala-Phe-AMC), and Succinyl-Leu-Leu-Val-Tyr-7-Amido-4-Methyl-Coumarin (Suc-Leu-Leu-Val-Tyr-AMC) inhibited the human follicular fluid-induced acrosome reaction. Sperm extracts exhibited hydrolytic activity toward Suc-Ala-Ala-Phe-AMC and Suc-Leu-Leu-Val-Tyr-AMC. This enzyme activity was abolished by TPCK and chymostatin, was independent of Ca²⁺, and was not modified by 1,10 phenanthroline. In addition, the activity was present in the supernatant after the acrosome reaction was induced with calcium ionophore and in epididymal spermatozoa recovered from the cauda region. Electron microscopic observations indicated that the inhibitors prevented the membrane events of the acrosome reaction. These data suggest an association between human spermatozoa and chymotrypsin-like activity with a possible role in the acrosome reaction. © 1994 Wiley-Liss, Inc.     

Production of mouse fetuses using spermatozoa exposed temporarily to high temperature or continuously to room temperature after freeze-drying in Na+-free/K+-rich EGTA buffer

Present study aimed to determine to what extent freeze-dried spermatozoa were able to withstand high-temperature conditions: transient increase in storage temperature and long-term exposure to room temperature. Mouse spermatozoa were freeze-dried in EGTA/Tris-HCl buffered solution alkalinized using KOH (K-ETBS, pH 7.7), and then stored for up to 7 months at 4 °C or 25 °C. After 2 months’ storage, some of the 4°C-stored spermatozoa were exposed to 40 °C for 1 week or 1 month, then again stored at 4 °C for the remaining storage period. Following storage, rehydrated spermatozoa were injected into mouse oocytes. The resulting zygotes were assessed for chromosome damage, in vitro development up to the blastocyst stage, and post-implantation development to normal fetuses on day 18 of gestation. In storage at 4 °C, one-week exposure to 40 °C had no adverse effect on the chromosome integrity and developmental competence compared to non-exposure to 40 °C (continuous storage at 4 °C). In contrast, one-month exposure to 40 °C caused an increasing level of chromosome damage (36%, P < 0.05) and reduced frequencies of blastocysts (54%, P < 0.05) and normal fetuses (36%, P < 0.05) compared to the frequencies obtained by continuous storage at 4 °C (15%, 82% and 52%, respectively). Storage at 25 °C resulted in accumulation of chromosome damage (27%, P < 0.05), leading to decreased blastocyst formation (63%, P < 0.05). But, the frequency of normal fetus (44%) was not significantly different from that obtained by continuous storage at 4 °C. Consequently, mouse spermatozoa freeze-dried in K-ETBS withstood temporary exposure to 40 °C for 1 week. Chromosome damage accumulated in spermatozoa during storage at 25 °C.     

Immunodetection of acrosin during the acrosome reaction of hamster, guinea-pig and human spermatozoa.

Mammalian sperm acrosomes contain a trypsin-like protease called acrosin which causes limited and specific hydrolysis of the extracellular matrix of the mammalian egg, the zona pellucida. Acrosin was localized on hamster, guinea-pig and human sperm using monoclonal and polyclonal antibodies to human acrosin labelled with colloidal gold. This was visualized directly with transmission electron microscopy, and with light and scanning microscopy after silver enhancement of the colloidal gold probe. Four distinct labelling patterns were found during capacitation and the acrosome reaction in hamster and guinea-pig spermatozoa, and three patterns were found in human spermatozoa. In the hamster, acrosin was not detected on the inner acrosomal surface after the completion of the acrosome reaction, thus correlating with the observation that hamster spermatozoa lose the ability to penetrate the zona after the acrosome reaction. With guinea-pig and human spermatozoa, acrosin was still detected after the completion of the acrosome reaction, thus correlating with the observation that acrosome reacted guinea-pig spermatozoa bind to and penetrate the zona pellucida.     

Evidence for two forms of phospholipase A2 in human semen

The molecular weight of the active unit of phospholipase A₂ (PA₂) in human seminal plasma and spermatozoa was determined using the radiation inactivation technique. Fresh spermatozoa possess more than one form of PA₂ activity as judged by the biphasic nature of the curve obtained during enzyme inactivation. However, when stored frozen for several months followed by a period of heating for 60 min at 60 °C prior to irradiation, the sperm exhibited PA₂ activity, which corresponded to a single low molecular mass form of 12,000 d when radioactive phosphatidylcholine (PC) was used as substrate and 8,000 d when radioactive phosphatidylethanolamine (PE) was used as substrate. In fresh seminal fluid, only one active form of PA₂ was detected as judged by the linear nature of the curve obtained during enzyme inactivation by irradiation. Using PC as substrate, the active unit was again estimated to be 12,000 d, whereas it corresponded to 18,000 d when PE was used. The PA₂ activity associated with normal spermatozoa exhibited a 60% decrease in activity after storage at ?20 °C for 48 hr followed by a heating period of 10 min at 60 °C. Long-term storage of spermatozoa at ?20 °C also resulted in a similar decrease in the deacylation of PC. No further loss of activity was observed during subsequent heat treatment at 60 °C. Seminal plasma, however, showed no loss of activity following short (48 hr at 4 °C or ?20 °C) or long-term storage and subsequent heat treatment. Thus, the behavior of PA₂ when the effect of temperature was studied and in radiation inactivation experiments indicates that the low molecular weight component in the seminal plasma as well as in spermatozoa is temperature resistant. However, in fresh spermatozoa, a second form of PA₂ was found and was sensitive to changes in temperature.     

Acrosin activation follows its surface exposure and precedes membrane fusion in human sperm acrosome reaction

Evidence has accumulated suggesting multiple roles of acrosin in fertilization, including its participation in early steps of gamete recognition and binding. However, the implication of acrosin in many of these processes is not compatible with its presumptive sequestration within the sperm acrosome until a late phase of the acrosome reaction. In an earlier study (J. Tesarik, J. Drahorad, J. Peknicova, 1988, Fertil. Steril. 50, 133-141), we reported the binding of an anti-acrosin monoclonal antibody (MO-AKR.1) to the plasma membrane overlying the acrosome of human spermatozoa starting the acrosome reaction. In this study, we characterized further this antibody with regard to its reactivity with different forms of acrosin and found that it recognizes specifically an active form of this enzyme and does not react with its proenzyme form. MO-AKR.1 was thus used as a probe for in situ analysis of acrosin activation during the acrosome reaction. When suspensions of living spermatozoa were incubated with MO-AKR.1 and with another monoclonal antibody (T6) directed to an intra-acrosomal cytoskeletal protein, significantly more spermatozoa reacted with the former antibody than with the latter; this indicated that some of the spermatozoa showing acrosin immunoreactivity carried activated acrosin on the cell surface, while their acrosome was still impermeable to intra-acrosomal-directed probes. The size of this particular sperm subpopulation was increased by the action of follicular fluid (a natural acrosome reaction inducer), but not ionophore A23187 (an artificial acrosome reaction inducer); it corresponded to the proportion of spermatozoa showing acrosin immunoreactivity on the plasma membrane but neither intra-acrosomal staining nor perceptible membrane perturbations when examined by immunoelectron microscopy. When spermatozoa were pre-incubated with protease inhibitors before the addition of acrosome reaction-inducing agents, the percentage of cells binding MO-AKR.1 was markedly reduced. These data suggest that limited acrosin activation on the sperm plasma membrane is an early event in the physiological acrosome reaction.