Purification and partial characterization of the 17 kDa sperm coating protein from boar seminal plasma.

Sperm coating proteins of 16, 17, and 19 kDa have been purified from boar seminal plasma. The 17 kDa protein has been identified as an antigen recognized by monoclonal antibody ACR.3 and is thus identical to low molecular mass zona pellucida binding protein from boar spermatozoa (Moos et al., 1990). The 17 and 19 kDa proteins are glycosylated and tend to form hetero-complexes. The 17 kDa ACR.3 antigen is sequentially released from the sperm cell surface during capacitation and, after induction of the acrosome reaction, the 16 kDa form was also observed. Immunocytochemical studies on boar reproductive tissues have suggested that the seminal vesicle epithelium may be the source of these proteins.     

The zymogen form of acrosin in testicular,epididymal, and ejaculated spermatozoa from ram

The sperm-specific proteinase acrosin (EC 3.4.21.10) is found in spermatozoa as a zymogen. We have looked for different forms of this zymogen in testicular, epididymal, and ejaculated spermatozoa from ram and have compared total sperm extracts made immediately after cell disruption with extracts made later from isolated sperm heads. We have concluded that the autoactivatable zymogen form, known generally as proacrosin, is the only form of acrosin within intact mature ram spermatozoa; no other zymogen form was detected, although lower levels of proacrosin were found in some samples of testicular spermatozoa. From studies of the activation process, it appears that ram proacrosin is truly autoactivatable; no evidence could be found for the involvement of any auxiliary enzyme. Estimations of the molecular weight of proacrosin using gel chromatography (60,000) and SDS-polyacrylamide gel electrophoresis (51,300) indicated that the zymogen is monomeric. Comparison with the molecular weight of ram acrosin (44,000 or 40,000, using the two respective methods) indicated that a single acrosin molecule is derived from each zymogen molecule. The sperm acrosin inhibitor (molecular weight 11,000 or 8,000) was present in testicular spermatozoa as well as in ejaculated spermatozoa; there was no evidence that it was produced as a result of zymogen activation.     

Rabbit testis proacrosin: Immunological similarities between testis,sperm proacrosins and the initially formed testis acrosin

Anti-rabbit proacrosin IgG was prepared from goat serum following immunization with a homogeneous preparation of rabbit testis proacrosin. The “auto-activation” products of purified testis proacrosin were separated into 68,000 and 34,000 molecular weight (mol wt) acrosins by Sephadex G-100 column chromatography. Immunodiffusion analysis of testis and epididymal sperm proacrosins and acrosins on agarose gel against goat anti-rabbit testis proacrosin showed immunological identity between rabbit testis and sperm proacrosins and the initial testis acrosin (mol wt 68,000). However, the 34,000 mol wt form of testis acrosin showed weaker reaction with the antibody and only partial identity with the proacrosin and the 68,000 mol wt form of acrosin. These results suggest that there is no major structural difference between testis and sperm proacrosins and between proacrosin and the 68,000 mol wt acrosin, but such a structual change occurs when the 34,000 mol wt acrosin is formed.     

Low temperature-induced dimerization of the bovine sperm serine protease,BSp66

BSp120 and BSp66 are trypsin-like serine proteases from bovine spermatozoa. The former is active in cryopreserved sperm samples while the latter shows proteolytic activity in recently obtained fresh sperm. Both proteases are immunologically related and co-localize in the apical portion of the sperm head. In Western blots with specific antibodies, sperm samples incubated with reducing agents showed a decrease in the amount of BSp120, while BSp66 was detected with both anti-BSp120 and anti-BSp66 antibodies. BSp120 was evident in frozen intact spermatozoa after 60 days of semen cryopreservation and the kinetic of appearance of this protein was coincident with the decrease in the amount of BSp66. Identical results were obtained by freezing sperm extracts from fresh semen at -20 degrees C. Our results suggest that BSp120 results from disulfide bond-dimerization of BSp66 and that this process may be induced by temperatures below zero in both intact spermatozoa and in sperm extracts.     

Difference of acrosomal serine protease system between mouse and other rodent sperm

A fraction of acrosomal proteins dispersed during calcium ionophore A23187‐induced acrosome reaction was prepared from cauda epididymal sperm of wild‐type and acrosin‐deficient mice, rat, and hamster. The acrosome‐reacted sperm were further extracted by Nonidet P‐40 to obtain the detergent‐soluble protein fraction. Activities of serine proteases in the two protein fractions were examined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in the presence of gelatin. A mixture of 42‐ and 41‐kDa gelatin‐hydrolyzing proteases was found in both fractions of the wild‐type mouse sperm, whereas the acrosin‐deficient mouse sperm contained the active 42‐kDa protease and apparently lacked the activity of the 41‐kDa protease. However, exogenous bovine pancreatic trypsin compensated for the absence of acrosin in the protein fractions of the mutant mouse sperm; the gelatin‐hydrolyzing activity of the 41‐kDa protease appeared when the sperm proteins of the mutant mice were treated with pancreatic trypsin. Two‐dimensional polyacrylamide gel electrophoresis revealed that the 42‐ and 41‐kDa proteases were distinguished from acrosin by the isoelectric point and immunoreactivity with affinity‐purified antibody against an oligopeptide corresponding to the N‐terminal amino acid sequence of mouse proacrosin. Moreover, the gelatin‐hydrolyzing proteins corresponding to these two proteases were not detected in rat and hamster sperm, in spite of the treatment of the sperm extracts with pancreatic trypsin, and the total amount of gelatin‐hydrolyzing activities in mouse was much smaller than those in rat and hamster. These results may reflect the difference of the serine protease system for the sperm penetration through the egg zona pellucida between mouse and other rodent animals, possibly explaining why the acrosin‐deficient mouse sperm are capable of penetrating the zona pellucida. Dev. Genet. 25:115–122, 1999. © 1999 Wiley‐Liss, Inc.     

Ultrastructural localization of proacrosin and acrosin in ram spermatozoa

Proacrosin and acrosin were localized immunocytochemically at the electron microscope level in ram spermatozoa undergoing an ionophore-induced acrosome reaction. Antigenicity was preserved after fixation with 0.5% w/v ethyl-(dimethylaminopropyl)-carbodimide, and an antibody preparation was used that reacted with all major forms of ram acrosin. All stages of the acrosome reaction could be observed in a single preparation. At the earliest stage, labeling was observed throughout the acrosomal contents, which were just beginning to disperse. As dispersal proceeded, labeling diminished, being associated only with visible remnants of the acrosomal matrix. By the time the acrosome had emptied, almost no labeling could be detected on the inner acrosomal membrane. The relationship between matrix dispersal and proacrosin activation was studied in isolated ram sperm heads. While proacrosin was prevented from activating, the acrosomal matrix remained compact; but as activation proceeded, the matrix decondensed and dispersed in close parallel. By the time proacrosin activation was complete, the acrosomal contents had almost entirely disappeared. We conclude that proacrosin is distributed throughout the acrosomal contents as an intrinsic constituent of the acrosomal matrix. During the acrosome reaction, proacrosin activation occurs, resulting directly in decondensation of the matrix. All the contents of the acrosome including acrosin disperse and, by the time the acrosome is empty and the acrosomal cap is lost, only occasional traces of acrosin remain on the inner acrosomal membrane. Since the acrosomal cap is normally lost during the earliest stages of zona penetration, acrosin's role in fertilization is unclear: it does not appear to be a zona lysin bound to the inner acrosomal membrane.     

Purification and partial peptide sequence analysis of the boar proacrosin binding protein

Boar proacrosin binding protein has been purified and the partial peptide sequence of the CNBr‐digested proacrosin binding protein has been determined. Proacrosin binding protein was purified as a proacrosin and proacrosin binding protein complex from the acid extracts of boar spermatozoa through gel filtration. After the proacrosin binding protein was dissociated from proacrosin by freeze‐thaw method, the proacrosin binding protein was purified through gel filtration. Fractions containing the proacrosin binding protein were pooled and were concentrated by lyophilization and then subjected to CNBr digestion. Four major CNBr‐digested peptides were subjected to N‐terminal peptide sequencing. All four showed the same N‐terminus sequence. Mol. Reprod. Dev. 54:76–80, 1999. © 1999 Wiley‐Liss, Inc.     

Characterization of the functional domains of boar acrosin involved in nonenzymatic binding to homologous zona pellucida glycoproteins

During the first steps of the gamete interaction, the proacrosin/acrosin system seems to play a crucial role in the secondary binding, holding acrosome-reacted spermatozoa during their passage through the zona pellucida. To analyze the functional domains of acrosin, we decided to express recombinant boar acrosin proteins in bacteria and to study their binding capacities to zona pellucida glycoproteins (ZPGPs). The expressed proteins were immunodetected by Western blot with a polyclonal antiacrosin antibody. The recombinant truncated β-acrosin has a typical hyperbolic curve of a zymogen enzymatic activation. Three of the five recombinant forms (truncated β-acrosin, Ser/Ala²²²-truncated β-acrosin, and truncated β-acrosin “heavy chain”) had the ability to bind ZPGPs. The two shorter forms (the amino and carboxy termini of truncated β-acrosin) failed to bind. The catalytic site mutant (Ser/Ala²²²) of truncated β-acrosin does not differ from the recombinant truncated β-acrosin in its mechanism of interaction to ZPGPs, indicating that this secondary binding is done by a nonenzymatic process. Our results show that binding between acrosin and ZPGPs depends on the secondary and tertiary structures of acrosin and does not depend on an active catalytic site. Mol. Reprod. Dev. 49:426–434, 1998. © 1998 Wiley-Liss, Inc.     

Purification and characterization of a sperm motility-dynein ATPase inhibitor from boar seminal plasma.

A sperm motility inhibitor from boar seminal plasma was purified. The purification procedure included dialysis against 0.1 M Tris-HCl containing 0.1 mM DTT and chromatographies on SP-Sephadex C-25 and Phenyl-Sepharose CL-4B. With this procedure, the seminal plasma motility inhibitor (SPMI) preparation was highly purified with a 18% recovery of inhibitory activity. The molecular weight of SPMI in native conditions has been estimated at 50,000 by molecular sieving, but 3 polypeptides with molecular weights of 14,000, 16,000 and 18,000 were observed following polyacrylamide gel electrophoresis in denaturing conditions. SPMI is a thermolabile basic protein that is stable between pH 6 and pH 11. The observations that SPMI effects on motility of demembranated spermatozoa are reversed by Mg.ATP and that SPMI inhibited bull dynein ATPase in a concentration-dependent manner suggest that this protein blocks the motility of demembranated spermatozoa by interfering with dynein arm function.     

Expanded cumuli induce acrosome reaction in boar sperm

The authors investigated acrosomal changes occurring in boar sperm that interact with the expanded cumulus matrix surrounding ovulated pig oocytes. Samples of washed boar sperm obtained from six donors were incubated for 4 hr under capacitating conditions and exposed either to solubilized zonae pellucidae (ZP) or solubilized expanded pig cumuli (SEC) obtained from IVM oocytes. Alternatively, hyaluronic acid, laminin, or fibronectin, components of the extracellular matrix (ECM) were added to capacitated sperm. Acrosomal integrity was evaluated 1hr later by using FITC-PSA staining. Solubilized cumuli induced acrosome reaction (AR) in a dose-dependent manner with a saturating effect exerted at 2.5 SEC/50 μl. Both 500 nM fibronectin and 500 nM laminin stimulated acrosomal exocytosis, the latter being more effective and inducing saturating levels of AR. By contrast, hyaluronic acid did not affect acrosomal status. Preincubation with anti-laminin antibodies completely prevented the inducing activity of SEC without affecting the activity of solubilized ZP. Consistent with these data, the integrin VLA-6, a receptor with high affinity for laminin, was detected by immunoblotting on the plasma membrane of capacitated boar spermatozoa. In addition, its immunoneutralization, obtained with the preincubation of capacitated sperm with the antibody raised against the α chain of VLA-6 integrin, prevented AR upon exposure to laminin or SEC (10.7 ± 3.2 and 10.2 ± 1.0% respectively), while the samples retained their responsiveness to ZP (29.6 ± 1.2%). The results demonstrate that the interaction between laminin, entrapped in the expanded cumuli, and specific integrins present on the sperm membrane can initiate AR, thus taking part in the process of sperm-egg recognition. Mol. Reprod. Dev. 51:445–453, 1998. © 1998 Wiley-Liss, Inc.     

Partial characterization of a 29 kDa cysteine protease purified from Taenia solium metacestodes

A 29 kDa cysteine protease of Taenia solium metacestodes was purified by Mono Q anion-exchanger and Superose 6 HR gel filtration chromatography. The enzyme was effectively inhibited by cysteine protease inhibitors, such as iodoacetic acid (IAA) and trans-epoxy-succinyl-L-leucyl-amido (4-guanidino) butane (E-64) while inhibitors acting on serine- or metallo-proteases did not affect the enzyme activity. The purified enzyme degraded human immunoglobulin G (IgG), collagen and bovine serum albumin (BSA), but human IgG was more susceptible for proteolysis by the enzyme. To define the precise biological roles of the enzyme, more detailed biochemical and functional studies would be required.     

Participation of sperm proteasome in fertilization of the phlebobranch ascidian Ciona intestinalis

Sperm proteasomes are thought to be involved in sperm binding to and in sperm penetration through the vitelline coat of the eggs of the stolidobranch ascidian Halocynthia roretzi. However, it is not known whether they are involved in the fertilization of eggs of other ascidians. Therefore, we investigated whether sperm proteasomes are also involved in the fertilization of the eggs of the primitive phlebobranch ascidian Ciona intestinalis. Fertilization of the eggs of C. intestinalis was potently inhibited by the proteasome inhibitors MG115 and MG132 but not by the cysteine protease inhibitor E-64-d. On the other hand, neither fertilization of the vitelline coat-free eggs nor sperm binding to the vitelline coat was inhibited by the two proteasome inhibitors at a concentration sufficient to inhibit fertilization of intact eggs. These results indicate that the proteasome plays an essential role in sperm penetration through the vitelline coat rather than in sperm binding to the coat or in sperm-egg membrane fusion. The proteasome activity, which was detected in the sperm extract using Suc-Leu-Leu-Val-Tyr-MCA as a substrate, was strongly inhibited by both MG115 and MG132, and was weakly inhibited by chymostatin, whereas neither leupeptin nor E-64-d inhibited the activity. The molecular mass of the enzyme was estimated to be 600-kDa by Superose 12 gel filtration, and the activity in sperm extract was immunoprecipitated with an anti-proteasome antibody. These results indicate that the proteasome present in sperm of C. intestinalis is involved in fertilization, especially in the process of sperm penetration through the vitelline coat, probably functioning as a lysin. Mol. Reprod. Dev. 50:493–498, 1998. © 1998 Wiley-Liss, Inc.     

Purification and partial peptide sequence analysis of the boar 32 kDa sperminogen

Boar 32 kDa sperminogen was purified from acid extracts of washed epididymal spermatozoa, and partial peptide sequence was determined. Boar sperminogen was purified from the acid extracts of boar spermatozoa by gel filtration through Sephadex G-75 column, followed by preparative SDS-PAGE. Gelatin zymographic analysis of the gel-filtered fractions showed that sperminogen was composed of three separate proteolytic bands. Among the three proteolytic bands, the 32 kDa sperminogen band which showed the strongest proteolytic activities upon activation was sliced out and eluted from the gel fragments. The eluted 32 kDa sperminogen was then subjected to peptide sequencing. Since the N-terminus of the 32 kDa sperminogen was blocked for peptide sequencing by Edman degradation method, the internal amino acid sequence of the sperminogen was obtained from the CNBr-digested peptides of sperminogen. The amino acid sequence of the analyzed peptide of the 32 kDa sperminogen showed 100% identity with that of proacrosin.     

Origin of a sperm motility inhibitor from boar seminal plasma

We have investigated the origin of the sperm motility inhibitor (SPMI) from boar seminal plasma. SPMI was measured by its capacity to inhibit the motility of demembranated spermatozoa and by an enzyme-linked immunosorbant assay (ELISA). Among the various reproductive and now reproductive tissues and fluids tested, only the seminal vesicle fluid and seminal plasma contained significant amounts of SPMI biological activity and SPMI antigen. Like other seminal vesicle fluid proteins, SPMI is diluted 6- to 8-fold upon ejaculation. By immunohistochemical detection at the light microscope with antibodies obtained from rabbits immunized with SPMI purified from boar seminal plasma, SPMI was found in the cytosol and/or on the plasma membrane bordering the lumen of the seminal vesicles. At the electron microscope level, SPMI appeared to be present only on the surface of the secretory cells. The data indicate that SPMI originates from a single tissue, the seminal vesicle, and suggest that only the mature form present on the luminal surface of the gland can react with the antibody generated from rabbits immunized with the secreted form of SPMI. © 1993 Wiley-Liss, Inc.     

Valosine containing protein is a substrate of cAMP-activated boar sperm tyrosine kinase

Previously we reported that treatment of boar sperm with cAMP-elevating drugs induces tyrosine phosphorylation of a triton-insoluble 93 kDa protein (p93). We have isolated p93 by preparative SDS electrophoresis and blotting from urea-extracted boar sperm and identified it as a valosine containing protein (VCP) by mass spectrometry and microsequencing. With the use of antibodies to VCP and phosphotyrosine (pY) we found that both p93 and VCP are poorly extractable with triton and are solubilized in > 6 M urea. Furthermore, VCP and p93 overlap on one and two dimensional (1 and 2D) electrophoretic gels, supporting the identity of p93 as a tyrosine-phosphorylated population of VCP. According to immunofluorescence, VCP is localized along the entire sperm tail, in the posterior ring, distal equatorial segment, and postacrosome. In addition, 9-12% sperm contained VCP in the acrosome. The cAMP-elevating treatment did not alter VCP localization but induced tail tyrosine phosphorylation in 15% sperm cells. In those sperm, VCP and pY colocalized in connecting piece and posterior ring.     

Binding of boar spermatozoa to porcine oocytes: Effect of low molecular weight 17 kDa protein

The effect of seminal plasma and low molecular weight ACR.3(17 kDa) protein on boar spermatozoa-porcine oocyte binding was examined. Boar seminal plasma that contains the sperm-adhesive ACR.3 protein was added to spermatozoa prior to their coincubation with oocytes, and the binding capacity of the spermatozoa so treated was compared to that of untreated cells. Similarly, purified ACR.3 protein, that binds to the egg zona pellucida, was added to noncapacitated spermatozoa, and the binding capacity of treated and untreated cells was again evaluated. In the two cases, the treatment of spermatozoa reduced their capacity to bind to the zona pellucida. We propose that the reduction in binding is due to competition for the ACR.3 binding sites on the zona pellucida between the soluble ACR.3 protein and the ACR.3 protein attached to the sperm surface. Furthermore, sperm-ZP binding was examined in the presence of ACR.3 monoclonal antibody, which specifically reacts with ACR.3 protein. Preliminary results show that addition of ACR.3 monoclonal antibody to a suspension of boar spermatozoa prior to their coincubation with oocytes did not markedly change sperm-zona binding in comparison with the control untreated spermatozoa. Our results suggest that ACR.3 protein may mediate the primary sperm-egg zona pellucida binding, and that it is one of the likely candidates for the primary sperm-ZP binding protein. Mol Reprod Dev 46:168–175, 1997. © 1997 Wiley-Liss, Inc.     

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

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

Substrate specificity of ascidian sperm trypsin-like proteases,spermosin and acrosin

In order to investigate systematically the substrate or subsite specificity of two sperm proteases; acrosin and spermosin (a novel trypsin-like protease) of the ascidian, Halocynthia roretzi, the effects of peptidyl-argininals on the purified enzymes as well as on fertilization were examined. Among four benzyloxycarbonyl (Z)-Leu-X-argininals (X = Pro, Leu, Ser, and Gly), Z-Leu-Pro-argininal showed the strongest inhibition toward the spermosin activity. On the P3 site specificity, Val-Pro-argininal derivatives showed a stronger inhibition than a Leu-Pro-argininal derivative, suggesting the preference of Val rather than Leu residue at the P3 position. Similar results were obtained by analyzing the hydrolyzing activity of the fluorogenic peptide substrates: it hydrolyzed Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide (MCA) most efficiently, and Boc-Asp(O-benzyl)-Pro-Arg-MCA was the next best substrate, but Gly-Pro-Arg (or Lys)-MCAs were hardly hydrolyzed. On the other hand, acrosin was found to prefer Leu or Pro residue rather than Gly or Ser residue at the P2 position as revealed by comparing the Ki values of peptidyl-argininals. Detailed kinetic analysis on the inhibitory abilities of peptidyl-argininals toward the purified enzymes and the ascidian fertilization suggested that both acrosin and spermosin are involved in ascidian fertilization. © 1996 Wiley Liss, Inc.     

Paraoxonase 1 activity in the sperm-rich portion of boar ejaculates is positively associated with sperm quality

Associations of the activity of the paraoxonase 1 (PON1) enzyme with boar sperm quality still needs to be characterized, since boar ejaculates present distinct portions with differences in sperm concentration and quality. This study evaluated PON1 activity in the serum, in the distinct portions of boar ejaculates and estimated correlations with sperm quality parameters. Ejaculates and blood samples were collected from six boars for three weeks (two per week per boar; n = 36). Serum and post-spermatic portion PON1 activities were positively correlated (P = 0.01) but were both uncorrelated with the PON1 activity in the sperm-rich portion and in the whole ejaculate (P > 0.05). Differences in PON1 activity among boars were only observed in the sperm-rich portion of the ejaculate (P < 0.05). The PON1 activity in the serum and in the post-spermatic portion was generally negatively correlated with parameters of spermatozoa kinetics (P < 0.05). In the sperm-rich portion, PON1 activity was positively correlated with sperm concentration (P < 0.0001), curvilinear distance and velocity (both P < 0.05) and DNA integrity (P < 0.05), but negatively correlated with straightness and linearity (P < 0.05). Thus, boar ejaculates with increased PON1 activity in the sperm-rich portion may present increased concentration and spermatozoa with acceptable curvilinear velocity and distance and DNA integrity, which suggests that PON1 activity may be a biomarker for potential fertility.     

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.