Dipeptidyl-amino-peptidase IV activity in stored buffy coat smears from human blood

The stability of dipeptidyl-amino-peptidase IV (DAP IV) activity in lymphoid cells of buffy coat smears from human blood was studied during storage for 40 days. Fixed or unfixed smears may be stored at 20 C for up to 24 hr before a decrease in activity occurs. Storage of either fixed or unfixed smears at 4 C, -10 C and -80 C results in a significant loss of activity within 24 hr. However, the cells retain more than 85% of their DAP IV activity for up to 10 days when stored fixed at -80 C. These data underscore the importance of proper processing of slides for DAP IV staining to avoid misinterpretation of results.     

Dipeptidyl-Amino-Peptidase Iv Activity in Stored Buffy Coat Smears from Human Blood

The stability of dipeptidyl-amino-peptidase IV (DAP IV) activity in lymphoid cells of buffy coat smears from human blood was studied during storage for 40 days. Fixed or unfixed smears may be stored at 20 C for up to 24 hr before a decrease in activity occurs. Storage of either fixed or unfixed smears at 4 C, -10 C and -80 C results in a significant loss of activity within 24 hr. However. the cells retain more than 85% of their DAP IV activity for up to 10 days when stored fixed at -80 C. These data underscore the importance of proper processing of slides for DAP IV staining to avoid misinterpretation of results.     

Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid

Human ejaculated spermatozoa were washed through a Percoll gradient, preincubated for 10 hr in a defined medium containing serum albumin, and then induced to undergo rapid acrosome reactions by addition of human follicular fluid or a Sephadex G-75 column fraction of the fluid. Induction by follicular fluid did not occur when the spermatozoa were preincubated for only 0 or 5 hr. The reactions were detected by indirect immunofluorescence using a monoclonal antibody directed against the human sperm acrosomal region. The percentage of acrosomal loss counted by transmission electron microscopy agreed with that counted by immunofluorescence. The apparent molecular weight of the Sephadex G-75 fraction containing the peak of acrosome reaction-inducing activity was 45,000 ± 4,200 (SD). The occurrence of physiological acrosome reactions was supported by: assessing motility (no significant loss of motility occurred during the treatment period when sperm were preincubated with bovine serum albumin), transmission electron microscopy (the ultrastructural criteria for the acrosome reaction were met), and zona-free hamster oocyte binding and penetration (spermatozoa pretreated with the active fraction of follicular fluid, then washed and incubated with oocytes, showed significantly greater binding to and penetration of oocytes). The stimulation of the acrosome reaction by follicular fluid is apparently not due to blood serum contamination; treatment of preincubated spermatozoa with sera from the follicular fluid donors had no effect on the spermatozoa. The nature of the active component(s) in that fraction is currently being investigated.     

Direct contact is required between serum albumin and hamster spermatozoa for capacitation in vitro

A dialysis unit was used to test whether direct physical contact between serum albumin and hamster spermatozoa is required for capacitation and/or the acrosome reaction. Sperm and bovine serum albumin (BSA) were incubated cither together (direct incubation) or separated by a dialysis membrane (indirect incubation). Sperm viability was supported with “sperm motility factors” (hypotaurine and epinephrine) and polyvinylalcohol (PVA). Spermatozoa became capacitated and underwent acrosome reactions when directly incubated in medium containing BSA (TALP-PVA), but did not undergo acrosome reactions when indirectly incubated with BSA (medium TLP-PVA). When sperm were first incubated for 4 hr indirectly with BSA, followed by 4 hr direct incubation with BSA, capacitation did not occur during indirect incubation. These findings indicate that an “intimate association” is necessary between serum albumin and spermatozoa to support capacitation under in vitro incubation conditions. The data are consistent with the concept of direct transfer of compounds from sperm to albumin and/or vice versa during sperm capacitation.     

The use of bright-field microscopy in evaluating bovine acrosome reaction

A novel stain for evaluating the acrosomes of bovine spermatozoa was investigated. Acrosome reactions were induced by incubating spermatozoa enriched by swim-up with 1 mumol/l calcium ionophore A23187 for 1 or 1.5 h, while control samples were incubated in modified Tyrode's medium alone. After fixing in formaldehyde, spermatozoa were stained either with naphthol yellow S plus erythrosin B (NE) or with the novel stain, naphthol yellow S plus aniline blue (NA). The number of spermatozoa that had undergone acrosome reaction was counted and compared with results obtained using differential interference contrast microscopy (DIC). The correlation between the 2 staining methods was high (r=0.99), as was the correlation between NA staining and DIC (r=0.97). Slides stained with NA showed little background staining and the preparations were permanent. The results indicate that NA may be a useful stain for the bright-field evaluation of the bovine acrosome.     

Early nuclear events of in vitro fertilization in the domestic fowl (Gallus domesticus)

In vitro fertilization in the domestic fowl (Gallus domesticus) was investigated by observation of the early nuclear events. Ova retrieved from the fimbria following ovulation were inseminated in vitro with 10(6)-10(7) spermatozoa in Dulbecco's modified Eagle's medium (DMEM) for 10 min and then further incubated in DMEM + albumen for 1, 2, 3, or 4 hr. These eggs were histologically examined by epifluorescent microscopy after staining with 4',6'-diamidino-2-phenylindole (DAPI). Nuclei of spermatozoa at various stages of transformation were observed in the ova incubated for 1-3 hr. Close pairing of two pronuclei, presumed to be male and female juxtaposition, was detected in ova incubated for 4 hr. These data provide direct evidence for the in vitro fertilization of fowl eggs and suggested that the early process of in vitro fertilization is comparable to that of in vivo fertilization.     

Nonspecific esterase activity of human alveolar macrophages in routine cytology

Alpha-naphthyl acetate esterase (ANAE) activity in alveolar macrophages was demonstrated in air-dried smears from samples of 82 sputa, 47 bronchial secretions and 14 bronchial lavages from 113 patients. Enzyme activity was estimated by a semiquantitative scoring method. There was a 7.4% loss of activity after 24 hours of storing the unfixed material, increasing to 14.4% after three days, while storing the air-dried smears for up to four weeks did not change the ANAE activity. The mean cellular esterase activity was correlated to the clinical and cytologic findings. A stringent correlation could be found for patients smoking more than 30 cigarettes a day; they had a 17% increase in activity as compared to nonsmokers. In patients with bronchial asthma, the activity was 18% higher than the total mean. In three patients with pulmonary embolisms, the ANAE activity was also increased. Treatment with a combination of cytostatics and a corticoid caused a severe decrease. No correlation could be found to age, sex, inflammation or malignant or cardiac diseases. These findings indicate that the application of the ANAE reaction to routine cytologic specimens can contribute to the functional characterization of human alveolar macrophages.     

Intracellular signaling cascades induced by relaxin in the stimulation of capacitation and acrosome reaction in fresh and frozen-thawed bovine spermatozoa

Relaxin is one of the 6-kDa peptide hormones, which acts as a pleiotropic endocrine and paracrine factor. Our previous studies revealed that sperm capacitating medium containing relaxin induced capacitation and acrosome reaction (AR) in fresh and frozen-thawed porcine or bovine spermatozoa. However, the intracellular signaling cascades involved with capacitation or AR induced by relaxin was unknown. Therefore, the present study was designed to investigate the intracellular signaling cascades involved with capacitation and AR induced by relaxin in fresh and frozen-thawed bovine spermatozoa. Spermatozoa were incubated in sperm Tyrode's albumin lactate pyruvate (Sp-TALP) medium supplemented with (40 ng ml(-1)) or without relaxin, and subjected to evaluation of chlortetracycline staining pattern, cholesterol efflux, Ca(2+)-influx, intracellular cyclic adenosine monophosphate (cAMP) and protein tyrosine phosphorylation. Capacitation and AR were increased (P<0.05) in both fresh and frozen-thawed spermatozoa incubated with relaxin. Cholesterol effluxes were greater in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa incubated with relaxin than the spermatozoa incubated without relaxin. Ca(2+)-influxes were also significantly stimulated by relaxin in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa. The Sp-TALP medium containing relaxin influenced the generation of intracellular cAMP in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa, and exhibited higher exposure of protein tyrosine phosphorylation in both sperm types than the medium devoid of relaxin. Therefore, the results postulate that relaxin exerts the intracellular signaling cascades involved with capacitation and AR through accelerating the cholesterol efflux, Ca(2+)-influx, intracellular cAMP and protein tyrosine phosphorylation in fresh and frozen-thawed bovine spermatozoa.     

Capacitation and the acrosome reaction in squirrel monkey (Saimiri sciureus) spermatozoa evaluated by the chlortetracycline fluorescence assay

Capacitation and the acrosome reaction in squirrel monkey seminal spermatozoa diluted in Tyrode's medium (TALP) and TC-199 were monitored by a chlortetracycline (CTC) fluorescence assay. Four CTC patterns, similar to those found in human sperm, were readily characterized by fluorescent staining on the heads of the spermatozoa. The appearance of the capacitated (CP) pattern was dependent on the concentration of the bovine serum albumin. Acrosomal loss was observed in a maximum of 15% of the sperm in the populations studied here. Calcium ionophore A23187 (5 μM to 20μM) induced acrosomal loss in 60–70% of capacitated spermatozoa. However in freshly ejaculated sperm incubated under capacitating conditions or in spermatozoa incubated in Ca^{+ +}-free medium, A23187 failed to induce acrosomal loss. Furthermore, spermatozoa incubated in the presence of seminal plasma or spermatozoa obtained following a 1-hour “swim-up” procedure showed an identical timecourse of appearance of the CP pattern, indicating the lack of effect of seminal plasma on capacitation in the squirrel monkey.     

Effect of the cumulus on in vitro fertilization of bovine matured oocytes

The effect of the cumulus on in vitro fertilization in bovines was examined. Follicular oocytes were cultured in medium 199 plus OCS and extra granulosa cells. Frozen-thawed bovine spermatozoa was separated by the swim-up technique, suspended in Talp medium and capacitated with heparin. Fresh sheep and goat semen was incubated for 4 h at room temperature, washed and spermatozoa were then suspended in Talp medium and capacitated by incubation at 38.5 °C and 5% CO₂ in air and heparin.

In experiment 1, cumulus-enclosed oocytes, denuded oocytes and denuded oocytes plus additional cumulus cells were incubated with a reduced concentration of bovine spermatozoa for 8 or 18 h. In Experiment 2, cumulus enclosed and denuded oocytes were incubated with bovine spermatozoa for 4, 6, 8 and 18 h using a sperm concentration adjusted to secure high fertilization rates. In Experiment 3, cumulus-enclosed and denuded bovine oocytes were incubated with either sheep or goat spermatozoa for 18 h. Fertilization rates were then calculated and compared statistically. The results showed that 1) the cumulus improved the fertilization rate only when cumulus cells were associated with the oocytes 2) the timing of sperm penetration was not modified by the cumulus and started at 4 h after sperm incubation and 3) the presence of the cumulus improved the heterologous fertilization rate only when sheep spermatozoa were used. The results suggest that the cumulus improves fertilization rate by providing a capacitation-inducing mechanism and by facilitating the interaction between capacitated spermatozoa and the zona pellucida surface.     

Microtubule organization of lymphocytes and its modulation by patch and cap formation.

Microtubule organization of mouse splenic lymphocytes was investigated by immunofluorescence microscopy using specific tubulin antibody. Each resting lymphocyte was shown to contain well organized microtubules which are associated with a microtubule organization center at one end. Microtubule organization of lymphocytes was readily modulated during patch and cap formation of surface immunoglobulins. These modulations induced by antibodies directed against immunoglobulins were prevented when the cells were incubated with 100 μg ml^?1 of concanavalin A before the addition of antibodies. A modulation of microtubule organization was also detected when incubated in hypertonic medium where cap formation of various receptors was nonspecifically induced in the absence of ligand. The degree to which microtubule organization was modulated, however, was larger in cap formation induced with antibodies than in ligand-independent cap formation, probably because the latter does not require specific patch formation. On the cells forming cap in hypertonic medium, strong staining with tubulin antibody was observed beneath the cap, but the region of cap itself was not stained with tubulin antibody. A mitogenic dose of ConA did not readily induce any significant alteration of staining patterns with tubulin antibody but caused extreme changes in microtubule organization of lymphocytes after incubation for 48 hr. Growing lymphoma cells were shown to have poorly ordered microtubules and related structures. These results seem to suggest that the modulations of microtubule organization in lymphocytes are associated with cell surface events and cell growth.     

Capacitation of fresh bovine spermatozoa on bovine epithelial oviduct cell monolayers

Capacitation of fresh bovine spermatozoa on bovine epithelial oviduct cells was assessed1) by the ability of spermatozoa to fertilize bovine oocytes in vitro and2) by exposure to lysophosphatidylcholine (LC) to induce acrosome reaction in the capacitated spermatozoa. When spermatozoa were incubated on bovine epithelial oviduct cells in B2 medium supplemented with 10% estrous cow serum (ECS) and then exposed to 100 mug/ml LC for 15 minutes, the percentage of acrosome reaction induced increased in a time-dependent course, reaching a plateau after 6 hours. Inversely, when spermatozoa were incubated in B2 + 10% ECS alone, the percentage of acrosome reaction induced by LC didn't fluctuate. The in vitro fertilization rate obtained after incubation of spermatozoa during 6 hours on bovine epithelial oviduct cells in B2 + 10% ECS medium was on average 75% for both the preovulated and ovulated oocytes. The developmental stages observed 18 hours after male and female gamete co-culture were similar to those obtained after in vivo fertilization. This study suggests that incubation of fresh bovine spermatozoa on bovine oviduct epithelial cell monolayers during 6 hours is an efficient method, and one that is close to in vivo capacitation.     

Evaluation of the acrosomal membrane in bovine spermatozoa: effects of proteinase inhibitors

Thawed bovine spermatozoa are characterized by a lack of homogeneity in the acrosomal membrane. Therefore, it is difficult to visualize the acrosome to assess morphology. Synthetic proteinase inhibitors were tested on thawed bovine semen for their effect on the integrity of acrosomal membranes. The proteinase inhibitors 4-nitrophenyl-4-guanidinobenzoate (NPGB) and N-L-p-tosyl-L-lysine-chloromethylketone (TLCK) were added to a medium containing spermatozoa separated on a percoll gradient. After incubation for 30 min at 38 degrees C in 5% CO(2), 95% air (final concentration 1 mM), the action of these inhibitors was controlled by measuring the activity of acrosome proteinases. The acrosomal membrane was evaluated by means of a dual stain procedure (trypan blue, Giemsa). In contrast to spermatozoa that had been incubated with proteinase inhibitor-free solution, samples that had been incubated with TLCK showed homogeneity in 90% of the acrosomal membranes and excellent visualization of the acrosome itself; in the NPGB-treated samples, homogeneous staining was observed in 83% of spermatozoa (P < 0.0005). It is concluded that alteration of the acrosomal membrane in thawed semen is not directly caused by freezing-thawing, but may be due to activation of acrosomal proteinases, which is increased during staining procedures. The addition of proteinase inhibitors before staining offers a new possibility for improved assessment of the acrosome in bovine spermatozoa.     

Characterization of phenoloxidase activity in venom from the ectoparasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

Crude venom isolated from the ectoparasitic wasp Nasonia vitripennis was found to possess phenoloxidase (PO) activity. Enzyme activity was detected by using a modified dot blot analysis approach in which venom samples were applied to nylon membranes and incubated with either L-DOPA or dopamine. Dot formation was most intense with dopamine as the substrate and no activators appeared to be necessary to evoke a melanization reaction. No melanization occurred when venom was incubated in Schneider's insect medium containing 10% fetal bovine serum or when using tyrosine as a substrate, but melanization did occur when larval or pupal plasma from the fly host, Sarcophaga bullata, was exposed to tyrosine. Only fly larval plasma induced an enzyme reaction with the Schneider's insect medium. The PO inhibitor phenylthiourea (PTU) and serine protease inhibitor phenylmethylsulfonylfluoride (PMSF) abolished PO activity in venom and host plasma samples, but glutathione (reduced) only inhibited venom PO. Elicitors of PO activity (sodium dodecyl sulfate and trypsin) had no or a modest effect (increase) on the ability of venom, or larval and pupal plasma to trigger melanization reactions. SDS-PAGE separation of crude venom followed by in-gel staining using L-DOPA as a substrate revealed two venom proteins with PO activity with estimated molecular weights of 68 and 160 kDa. In vitro assays using BTI-TN-5B1-4 cells were performed to determine the importance of venom PO in triggering cellular changes and evoking cell death. When cell monolayers were pre-treated with 10 mM PTU or PMSF prior to venom exposure, the cells were protected from the effects of venom intoxication as evidenced by no observable cellular morphological changes and over 90% cell viability by 24 h after venom treatment. Simultaneous addition of inhibitors with venom or lower concentrations of PMSF were less effective in affording protection. These observations collectively argue that wasp venom PO is unique from that of the fly hosts, and that the venom enzyme is critical in the intoxication pathway leading to cell death.     

Effect of albumin on fertilization of mouse ova in vitro

Serum albumin is an obligatory component of the incubation medium for the fertilization of mouse ova. Normal, untreated bovine serum albumin supports high rates of fertilization of cumulus-free ova both with and without their zonae pellucidae. Heat-treated or trichloroacetic acid-extracted bovine serum albumin is unable to support the fertilization of a majority of zona-intact ova but fertilization of zona-free ova is unimpaired. Spermatozoa incubated in medium containing heated bovine serum albumin fertilize zona-intact ova when 2 mM caffeine is present but the progress of sperm head decondensation is delayed when compared to normal controls. Trichloroacetic acid extracted BSA preferentially and irreversibly inhibits zona penetration by spermatozoa, but this effect is not mediated by an inhibition of spermatozoal motility or zona-binding ability. This effect occurs after only a 10-min preincubation of the spermatozoa in the extracted BSA or when the medium contains only a 10% (v/v) proportion of this albumin. It is estimated that mouse spermatozoa under the conditions used take 2 hr to penetrate the zonae pellucidae of 50% of ova and effect fertilization.     

Effects of platelet activating factor on the motility and acrosome reaction of bovine spermatozoa

The effects of platelet activating factor (PAF) on motility and the acrosome reaction of ejaculated bull spermatozoa were evaluated. Washed spermatozoa (30 x 10(6)/ml) were incubated (39 degrees C) for up to 2 h with 10 to 200 muM PAF in a modified Tyrode's solution (pH 7.4) containing 3 mg/ml bovine serum albumin. Sperm motility was evaluated subjectively and by computer-assisted semen analysis. Percent acrosome-reacted spermatozoa was quantified microscopically from fixed smears following Giemsa staining. Percent fertilization by PAF-treated spermatozoa was determined using in vitro-matured bovine ova. Percent sperm motility decreased with >/= 50 muM PAF, while the rate of motility loss increased with PAF concentration (P<0.001). Percent acrosome reactions increased with PAF concentration during incubation (P<0.001). Acrosomal loss was rapid and complete with 200 muM PAF. At concentrations between 80 to 120 muM PAF, bull spermatozoa underwent acrosome reactions without a rapid loss of motility and penetrated in vitro-matured bovine ova at a rate comparable to that of heparin-capacitated spermatozoa (68 versus 54%, respectively). Incubation of bull spermatozoa with 10 to 50 muM PAF for 45 min had no effect on percent progressive motility, sperm velocity or other motility parameters. These results indicate that PAF can be used to induce acrosome reactions in bull spermatozoa and to promote in vitro fertilization of bovine ova. Under the conditions used in this study, PAF did not stimulate bovine sperm motility.     

A Simple Staining Procedure for Detecting the true Acrosome Reaction in Buffalo (Bubalus bubalis) Spermatozoa

A simple dual staining procedure for detecting the true acrosome reaction in dried smears of buffalo spermatozoa is described. Trypan blue is used first to differentiate live from dead spermatozoa and the dried smears which have been prepared are stained with Giemsa for acrosome evaluation. Four categories of spermatozoa were recognized: A) live, intact acrosome (acrosome pink, postnuclear cap clear); B) dead, intact acrosome (acrosome pink, postnuclear cap blue); C) live, detached acrosome (acrosome clear, postnuclear cap clear); and D) dead, detached acrosome (acrosome clear, postnuclear cap blue). The procedure is simple, rapid and convenient for assessing true acrosome reaction in buffalo spermatozoa. Simultaneous assessment of sperm viability and its acrosomal status in dried smears makes this procedure attractive because the true acrosome reaction can be studied thoroughly at a later state after the incubation period.     

Failure of human spermatozoa to penetrate zona free mouse and rat ova in vitro

When incubated for 8 to 26 hours with zona-free mouse or rat ova, human spermatozoa failed to attach to or penetrate any of the ova. The ova were capable of being fertilized since both intra- and inter-species penetration of spermatozoa and formation of pronuclei occurred between rat and mouse gametes. When mouse spermatozoa were incubated for three to eight hours with rat ova, a high proportion of the ova were penetrated, formation of pronuclei occurred and in 9 out of 36 ova incubated for 40 hours after insemination, regular cleavage and formation of morphologically normal 2-cell embryos occurred. Human spermatozoa retained their morphological integrity and motility only when the culture medium contained purified bovine serum albumin (3 mg/ml) or human serum (5% v/v) and not when unpurified BSA from several different commercial sources was used as a protein source. In this latter medium, the ova of both rats and mice degenerated after 8-hour incubation in the presence of human spermatozoa but not when human spermatozoa were absent or in the presence of either rat or mouse spermatozoa. Electron microscopy indicated that the human spermatozoa incubated for eight hours in medium containing purified BSA had undergone an acrosome reaction. These spermatozoa also attached to and penetrated human oocytes which had been matured in vitro.     

Localization of xanthine oxidoreductase activity using the tissue protectant polyvinyl alcohol and final electron acceptor Tetranitro BT

We have detected xanthine oxidoreductase activity in unfixed cryostat sections of rat and chicken liver, rat duodenum, and bovine mammary gland using the tissue protectant polyvinyl alcohol, the electron carrier 1-methoxyphenazine methosulfate, the final electron acceptor Tetranitro BT, and hypoxanthine as a substrate. Enzyme activity was localized in rat duodenum at lateral membranes and brush borders of enterocytes and in goblet cells and mucus. Hepatocytes in pericentral areas and especially sinusoidal cells showed high activity in rat liver. Xanthine oxidoreductase was also detected in epithelial cells and milk lipid globules of lactating bovine mammary gland, which is known to contain large quantities of the oxidase form of the enzyme. Chicken liver, which contains an inconvertible dehydrogenase form, also showed high activity in sinusoidal cells. Therefore, we conclude that the tetrazolium reaction demonstrates both the dehydrogenase and the oxidase form of xanthine oxidoreductase. Control activity, in the absence of hypoxanthine or in the presence of the competitive inhibitor allopurinol, was low in all tissues studied. Addition of O2 or NAD to the incubation medium did not change the specific reaction in bovine mammary gland or chicken liver, implying that the dehydrogenase and the oxidase form are not dependent on their natural electron acceptors in this tetrazolium salt reaction. We conclude that the present light microscopic method gives specific and precise localization of xanthine oxidoreductase activity in situ.     

Characterization of PH-20 in canine spermatozoa and testis

The purpose of this study was to characterize the sperm membrane protein PH-20 in the dog. Canine spermatozoa were extracted with Triton X- 100 and the presence of PH-20 was determined by immunoblot with an antibody against recombinant macaque PH-20. The hyaluronidase activity of canine PH-20 was determined with substrate gel electrophoresis based upon digestion of hyaluronic acid (HA) incorporated into the separating gels. Hyaluronidase activity was also quantified using a microplate assay. Sperm extracts were incubated at pH 4 or 7 in wells containing agarose and HA. For immunolabeling of PH-20 on canine sperm membranes, canine sperm were fixed and incubated with R-10 primary antibody, and an anti-rabbit IgG-FITC secondary antibody. Samples were visualized by fluorescence microscopy. Non-reducing SDS-PAGE and Western blot of detergent-extracted canine sperm revealed a major band at 50 kDa, and three other bands at 42, 124, and >209 kDa. Substrate PAGE revealed translucent bands of hyaluronidase activity of similar size to bovine testicular hyaluronidase. These bands were markedly more pronounced at pH 4 than at pH 7. The microplate assay also demonstrated that hyaluronidase activity was over four times greater at the acidic pH. Immunolabeling of canine spermatozoa demonstrated that PH-20 is localized to the anterior head region and appeared in the Golgi area of round spermatids as detected by the immunohistochemical staining of the testis. This study provides evidence that PH-20 is present on the membrane of canine spermatozoa and in round spermatids. Canine PH-20 exhibits hyaluronidase activity that is markedly more pronounced at acidic pH.