Biomagnification of cadmium selenide quantum dots in a simple experimental microbial food chain.

Previous studies have shown that engineered nanomaterials can be transferred from prey to predator, but the ecological impacts of this are mostly unknown. In particular, it is not known if these materials can be biomagnified-a process in which higher concentrations of materials accumulate in organisms higher up in the food chain. Here, we show that bare CdSe quantum dots that have accumulated in Pseudomonas aeruginosa bacteria can be transferred to and biomagnified in the Tetrahymena thermophila protozoa that prey on the bacteria. Cadmium concentrations in the protozoa predator were approximately five times higher than their bacterial prey. Quantum-dot-treated bacteria were differentially toxic to the protozoa, in that they inhibited their own digestion in the protozoan food vacuoles. Because the protozoa did not lyse, largely intact quantum dots remain available to higher trophic levels. The observed biomagnification from bacterial prey is significant because bacteria are at the base of environmental food webs. Our findings illustrate the potential for biomagnification as an ecological impact of nanomaterials.

Thoracic outlet syndrome in children and young adults.

OBJECTIVES: Thoracic outlet syndrome has been well described in the population between 25 and 40 years of age, and is less frequently reported in those in the first two decades of life. The objective of this study was to review results with onset of TOS in the first two decades of life to determine type of presentation and outcomes from surgical intervention. METHODS AND MATERIALS: Charts of all patients in the first two decades of life, operated on for TOS between 1994 and 2006 were reviewed with follow-up by clinic visit and phone survey to assess the patients' current level of activity and relief from symptoms. RESULTS: Twelve patients were identified (13 operations), with a mean age of 16.8 years. Acute ischemic symptoms were the initial presentation for 38%, venous TOS in 24%, and neurogenic symptoms in 38%. All patients had symptom relief with surgery with a mean time to resolution of 10.9 weeks. All patients remained symptom free or improved at follow-up. CONCLUSIONS: Vascular TOS is much more common in TOS presenting in the first two decades of life. Surgical intervention for TOS in this population results in long-lasting symptom relief and should be considered for all subtypes of patients.

Differentiated structure and function of primary cultures of monkey oviductal epithelium.

We have established well-differentiated, polarized cultures of monkey oviductal epithelium. Oviductal epithelial cells were isolated by protease digestion and plated on collagen-coated, porous cell culture inserts. About 5 d after plating, cells developed detectable transepithelial electrical resistance of up to 2000 Omega.cm(2) (an index of tight junction formation) and transepithelial voltages of up to 20 mV (an index of vectorial transepithelial ion transport). Measurements of short-circuit current in Ussing chambers indicated that active secretion of Cl was the major transepithelial active ion transport process, and that this was stimulated by elevation of either cAMP or Ca(i). Furthermore, estimates of the volume of mucosal liquid were consistent with Cl secretion mediating fluid secretion. Various microscopical methods showed that the cultures were densely ciliated and contained mature secretory cells. Transport across the oviductal epithelium determines the composition of the oviductal fluid, and the study of the relevant transport processes will be greatly enhanced by well-differentiated cultures of oviductal epithelium of the kind established here.

Pre-arteriotomy guidewire access (PAGA): a crucial maneuver for securing inflow and/or outflow in patients with bulky iliofemoral occlusive disease undergoing combined (open/endovascular) procedures.

PURPOSE: To present our technique of pre-arteriotomy guidewire access (PAGA). SUMMARY: Placement of a guidewire across inflow lesions before performing the arteriotomy during combined endovascular/open procedures while treating patients with complex iliofemoral occlusive disease is a crucial maneouvre. We routinely utilize this approach in patients in whom endarterectomy and/or patch angioplasty is planned as the central part of the revascularization procedure, or when we find an unsuspected severely diseased EIA in a patient undergoing leg revascularization.

The carbohydrate structure of DEFB126, the major component of the cynomolgus Macaque sperm plasma membrane glycocalyx.

Based on the amino-acid sequence of the macaque epididymal secretory protein, ESP 13.2 (Q9BEE3/AJ236909), it has now been classified as beta-defensin DEFB126. DEFB126 is one of the five beta-defensins with genes that are clustered along chromosome 20pl3, and all five proteins have an extended carboxy terminus that continues beyond the 6-cysteine beta-defensin core region. This 60-amino acid carboxyl tail extension of the DEFB126 molecule is extraordinarily rich in threonine and serine (40%), many of which appear to be likely candidates for having O-glycosylation. DEFB126 has been shown to coat the entire surface of cynomolgus macaque sperm as they move through the corpus/caudal region of the epididymis. It is a major glycocalyx barrier to the external environment and is retained until the completion of capacitation. Sperm exposed to fluorescein-conjugated poly-L-lysine or Alexa488-histone showed a very uniform fluorescent labeling pattern over the entire sperm surface, almost identical to that observed with anti-DEFB126 Ig label. Sperm surface components that were released following treatment with caffeine/cAMP (in vitro capacitation) were blotted and probed with three different lectins which are known to recognize terminal sialic acid residues, and all three recognized the 35 kDa DEFB126 band. Neuraminidase treatment of sperm shifted the molecular weight of DEFB126 from 34-36 kDa to approximately 38-40 kDa and removed or greatly inhibited sialic acid-specific lectin recognition. O-Glycanase treatment alone was ineffective at removal of the oligosaccharides, but prior treatment with neuraminidase was sufficient to enable the O-glycanase treatment to effectively change the apparent molecular weight to 10 kDa, confirming that a major portion of the molecular mass is associated with the carbohydrate portion. Western blots of neuraminidase-treated DEFB126 showed strong recognition with a number of lectins that identify beta-galactose and also lectins that recognize the N-acetylgalactosamine-serine/threonine, the proposed connection site of O-glycosylation. All of the lectins that recognized DEFB126 on Western blots were used to fluorescently probe sperm. The fluorescent patterns that were observed with poly-L-lysine, Alexa488-histone, sialic acid-specific lectins, and galactose-specific lectins showed even distributions over the entire sperm surface and the patterns were identical to sperm labeled with anti-DEFB126 Ig, and all but the antibody did not recognize neuraminidase-treated sperm.

Renal complications with aortic surgery.

Because of the limited ability to alter the course of acute renal failure, the vascular surgeon's best strategy is prevention of renal dysfunction. Preoperative screening can identify patients at high risk for acute renal failure after aortic reconstruction. Although the mainstay of preventative therapy is maintenance of adequate renal perfusion, other adjunctive measures are available before, during, and after aortic surgery, which may reduce the incidence of acute renal failure.

Infection is an unusual but serious complication of a femoral artery catheterization site closure device.

Percutaneous devices have been developed to close the femoral artery puncture site after catheterization. Because direct compression is not needed, the devices save time for the treating health-care provider, reduce patient discomfort, and obviate the need for post-catheterization bed rest. Reported complications with use of these devices are similar in nature and frequency to those accompanying direct compression. Complications of infection requiring surgical treatment are exceedingly rare with use of these devices. We describe a series of five catheterization site infections occurring among 1807 patients (0.3%) whose femoral artery puncture was closed with a percutaneous suture closure device. All patients required operative intervention and there was one late death. Physicians should be aware of this uncommon but serious complication to expedite evaluation and treatment of patients with suspected infections from these devices.

Rectourethral fistula and massive rectal bleeding from iodine-125 prostate brachytherapy: a case report.

Iodine-125 brachytherapy is an effective well-tolerated treatment for localized prostate cancer. Gastrointestinal complications of brachytherapy (minor rectal bleeding or tenesmus) are uncommon. Rectal ulceration or rectourethral fistulas after prostate brachytherapy are rare. We present a case of massive refractory rectal bleeding and rectourethral fistula, a complication of prostate brachytherapy never before reported. As a result of the patient's life-threatening symptoms aggressive surgical therapy was necessary.

Impact of UV radiation on the early development of the giant kelp (Macrocystis pyrifera) gametophytes.

The mechanisms and dose-response of UV action on the early development of Macrocystis pyrifera (L.) C. Agardh gametophytes were investigated. Post-release, zoospores undergo germination, germ tube elongation, DNA synthesis, nuclear division and translocation, which were followed for 41 h under laboratory conditions. The spores were exposed to UV radiation before germination (3 h post-release) or before nuclear division (20 h post-release). Biologically effective UV-B doses (BEDDNA300 nm) higher than those used in the experiments are needed for a 50% inhibition in germination (BED50 > 1600 J m-2). Nuclear division/translocation was more sensitive to UV radiation. When the spores were cultured in the dark, UV exposure at both 3 and 20 h post-release resulted in a dose-responsive inhibition of nuclear division/translocation (BED50 64 and 86 J m-2). Culturing in the light indicated recovery in the spores that were irradiated at 3 h post-release (BED50 356 J m-2), whereas no light-dependent recovery occurred within 41 h of culture when irradiated at 20 h post-release (BED50 80 J m-2). The results present a possible mechanism of UV inhibition in early life stages of the giant kelp, suggesting that environmentally relevant UV-B levels can perturb or delay the development and recruitment of the gametophytes by inhibiting nuclear events.

Renal duplex sonography: main renal artery versus hilar analysis.

OBJECTIVE: The purpose of this study was to compare the accuracy of main renal artery Doppler scanning interrogation and hilar analysis to diagnose hemodynamically significant renal artery disease. METHODS: From January 1998 to August 1999, 41 patients had renal duplex sonography with both main renal artery interrogation and hilar analysis followed by angiography. They form the basis of this review. The sample consisted of 24 men and 17 women, with a mean age of 68.9 +/- 10.2 years, who provided 80 kidneys for comparative analysis. Significant renal artery disease revealed through angiography was defined as >/= 60% diameter-reducing stenosis or occlusion. Peak systolic velocity (PSV) (in meters per second) and the presence of poststenotic turbulence (PST) were determined from main renal artery interrogation. Acceleration time (AT) (in milliseconds) was measured by means of hilar analysis. Significant renal artery stenosis was defined by a PSV of 2.0 m/s or more and a PST or an AT more than 100 ms. Sensitivity analyses of both PSV and AT were examined, and 95% CIs were computed. Receiver operating characteristic curves were used to estimate optimal values for PSV and AT. RESULTS: Angiography revealed hemodynamically significant fibromuscular dysplasia in 5 kidneys (4 patients), atherosclerotic stenosis >/= 60% in 48 kidneys (30 patients), and renal artery occlusion in 4 kidneys (4 patients). Kidneys with significant renal artery stenosis had a higher PSV (2.54 +/- 0.11 vs 1.28 +/- 0.08, P <.001) and AT (82.43 +/- 7.2 vs 30.0 +/- 2.8, P <.001) compared with those without stenosis. Compared with angiography, a PSV of 2.0 m/s or more and PST demonstrated a sensitivity of 91%, specificity of 96%, and overall accuracy of 92% for detection of significant renal artery stenosis. Two of five studies with false-negative results reflected diseased polar vessels. By contrast, AT of more than 100 ms had a sensitivity of 32%, specificity of 100%, and overall accuracy of 54%. Receiver operating characteristic curve analysis revealed a PSV of more than 1.8 m/s and an AT of 58 ms or greater as optimal values. With an AT of 58 ms or more, the sensitivity was 58%, and specificity was 96%, with an overall accuracy of 70%. There were no apparent associations between PSV or AT and type or location of renal artery lesion, serum creatinine level, or end-diastolic ratio. CONCLUSION: Main renal artery interrogation is an accurate screening test to detect significant stenosis or occlusion of the main renal artery. Hilar analysis alone does not provide sufficient sensitivity to be used as a sole screening study. Neither method detects the presence of renovascular disease associated with polar vessels.

Management of ischemic nephropathy: dialysis-free survival after surgical repair.

PURPOSE: This retrospective review describes the surgical management of consecutive patients with severe hypertension and ischemic nephropathy due to atherosclerotic renovascular disease. METHODS: From January 1987 through December 1998, a total of 590 patients underwent operative renal artery repair at our center. A subgroup of 232 hypertensive patients (97 women, 135 men; mean age, 66 +/- 8 years) with atherosclerotic renovascular disease and preoperative serum creatinine levels of 1.8 mg/dL or more forms the basis of this report. Change in renal function was determined from glomerular filtration rates estimated from preoperative and postoperative serum creatinine. The influence of selected preoperative parameters and renal function response on time to dialysis and dialysis-free survival was determined by a proportional hazards regression model. RESULTS: In all, 83 patients underwent unilateral renal artery repair and 149 patients underwent bilateral repair, including repair to a solitary kidney in 17 cases. A total of 332 renal arteries were reconstructed, and 32 nephrectomies were performed in these patients. After surgery, there were 17 deaths (7.3%) in the hospital or within 30 days of surgery. Advanced patient age (P =.001; hazard ratio, 1.1; 95% CI [1.1, 1.2]) and congestive heart failure (P =.04; hazard ratio, 2.9 CI [1.0, 8.6]) demonstrated significant and independent associations with perioperative mortality. With a change of 20% or more in EGFR being considered significant, 58% of patients had improved renal function, including 27 patients removed from dialysis dependence; function was unchanged in 35% and worsened in 7%. Follow-up death from all causes or progression to dialysis dependence demonstrated a significant and independent association with early renal function response. Both patients whose function was unchanged (P =.005; hazard ratio, 6.0; CI [2.2, 16.6]) and patients whose function was worsened (P =.03; hazard ratio, 2.2; CI [1.1, 4. 5]) remained at increased risk of death or dialysis dependence. For patients with unchanged renal function after operation, risk of death or dialysis demonstrated a significant interaction with preoperative renal function. In addition to severe preoperative renal dysfunction, diabetes mellitus demonstrated a significant and independent association with follow-up death or dialysis. CONCLUSION: Surgical correction of atherosclerotic renovascular disease can retrieve excretory renal function in selected hypertensive patients with ischemic nephropathy. Patients with improved renal function had a significant and independent increase in dialysis-free survival in comparison with patients whose function was unchanged and patients whose function was worsened after operation. These results add further evidence in support of a prospective, randomized trial designed to define the value of renal artery intervention in patients with ischemic nephropathy.

Soybean trypsin inhibitor as a probe for the acrosome reaction in motile cynomolgus macaque sperm.

Soybean trypsin inhibitor (SBTI) inhibits the catalytic activity of serine proteases, and has been shown to bind to acrosin, an acrosomal hydrolase which is not exposed on the surface of macaque sperm until after the acrosome reaction. Following activation with caffeine and dibutyryl cAMP, cynomolgus macaque sperm were induced to acrosome react with calcium ionophore A23187 in the presence of SBTI and were fixed for ultrastructural observation. Transmission electron microscopy (TEM) revealed secondary labelling of anti-SBTI-IgG with colloidal gold in association with the acrosomal matrix and fused membranes of sperm undergoing the acrosome reaction, but gold labelling was not observed on acrosome-intact sperm. When SBTI was conjugated with the fluorochrome Alexa 488, labelled (acrosome-reacted) sperm showed bright fluorescence that ranged from a patchy or punctate appearance to solid labelling over the region of the acrosomal cap. Following treatment with ionophore, the percentages of total acrosome-reacted sperm (motile and non-motile) as assessed with Alexa-SBTI, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), and TEM were 54.6%, 51.6% and 61.5%, respectively. Measures of acrosomal status with FITC-PSA and Alexa-SBTI were highly correlated (r = 0.94; n = 3). Macaque zonae pellucidae were co-incubated with activated sperm for 1 min and then rinsed in medium containing Alexa-SBTI and immediately observed with epifluorescence microscopy. The mean percentage of Alexa-SBTI-labelled (acrosome-reacted) motile sperm bound to the zona was 45.7 +/- 14 (range: 22-80.4%; n = 4). Fewer than 1% of the motile sperm in suspension surrounding the zonae were acrosome-reacted. Alexa-SBTI had no effect on sperm motility, survival, or zona binding capability.

Identification of bladder tumor-derived hyaluronidase: its similarity to HYAL1.

The glycosaminoglycan hyaluronic acid (HA) and its degrading enzyme, hyaluronidase, are intricately associated with tumor metastasis and angiogenesis. HA promotes tumor cell adhesion and migration, whereas its small fragments stimulate angiogenesis. Such small HA fragments are generated from the degradation of HA by hyaluronidase. We have previously shown (V. B. Lokeshwar et al., Cancer Res., 57: 773-777, 1997) that the HA levels are elevated in the urine and tumor tissues of bladder cancer patients regardless of the tumor grade (G). The hyaluronidase levels were found to be elevated in the urine and tumor tissues of G2 and G3 bladder cancer patients. Furthermore, angiogenic HA fragments were isolated from the urine of G2/G3 bladder cancer patients, which stimulated endothelial cell proliferation, a key event in angiogenesis. In this study, we characterized the bladder tumor-derived hyaluronidase. Analysis of hyaluronidase activity in the culture-conditioned media (CM) of 11 bladder cancer cell lines, using an ELISA-like assay and a substrate (HA)-gel technique, showed that the invasive bladder cancer cell lines secrete elevated levels of a Mr approximately 60,000 hyaluronidase. Reverse transcription-polymerase chain reaction, cloning, and sequence analyses revealed the expression of an HYAL1 transcript in bladder cancer lines. HYAL1 encodes for a hyaluronidase that is present in serum. Immunoblot analysis using an anti-HYAL1 peptide IgG confirmed the presence of a Mr approximately 60,000 HYAL1-related protein in the CM of bladder cancer cell lines, in the urine specimens from G2 and G3 bladder cancer patients, and in the partially purified preparations of bladder tumor-derived hyaluronidase. No HYAL1-related protein was detected in urine specimens from normal individuals, G1 bladder cancer patients, and patients with a history of bladder cancer but no disease at the time of testing. The bladder tumor-derived hyaluronidase present in CM and partially purified preparations was found to have maximum activity at a pH range of 4.1-4.3. The identification of bladder tumor-derived hyaluronidase should help in elucidating its role in bladder tumor progression.

Hyaluronic acid enhances induction of the acrosome reaction of human sperm through interaction with the PH-20 protein.

When capacitated human sperm were treated with hyaluronic acid (HA) for 30 min prior to the addition of progesterone or solubilised human zonae pellucidae, there was a significant increase in the percentage of acrosome reactions. Progesterone treatment alone increased acrosome reactions from 10.5% to 21.8% and pretreatment with 100 micrograms/ml HA resulted in 33.0% acrosome reactions. With zonae pellucidae treatment alone the increase was from 9.0% to 23.5% and with HA pretreatment it was 48.8%. HA treatment alone had no direct effect on acrosome reactions, and the enhancing effect of HA was not removed when sperm were washed prior to the addition of either acrosome reaction agonist. Experiments with sperm 5 min after HA treatment demonstrated that enhancement of acrosome reactions was apparent as early as 1 min after addition of zonae and within 5 min after addition of progesterone. When sperm were pretreated with Fab fragments of anti-PH-20 IgG, then with HA and then with progesterone or zonae pellucidae, there was no enhancement of the acrosome reaction. Fab treatment did not induce acrosome reactions and did not interfere with the action of either agonist in the absence of HA. Sperm that were treated with HA had significantly higher intracellular calcium levels, and pretreatment with Fab reduced this increase to 42.7%. Addition of progesterone to HA-treated sperm was followed by another large increase in intracellular calcium, which was lower when sperm were pretreated with Fab. These results suggest that HA interacts with the PH-20 protein to increase basal levels of intracellular calcium and thereby potentiates the acrosome reaction. The data support the hypothesis that HA in the cumulus matrix may act to prime the fertilising sperm for induction of the acrosome reaction by constituents of the cumulus and/or zona pellucida.

Preliminary observations on responses of embryonic and larval Pacific herring, Clupea pallasi, to neutral fraction biodegradation products of weathered Alaska North Slope oil.

Weathered Alaska North Slope crude oil (ANS 521) was subjected to biodegradation in vigorously stirred incubations for 14 days at 15 +/- 1 degrees C in 20/1000 salinity sterilized seawater, amended with nutrients and inoculated with a hydrocarbon-degrading microorganism (EI2V) isolated from an oil-contaminated beach in Prince William Sound, Alaska. A total of 13.7 mg/L water-soluble neutral fraction (WSF) was recovered from the incubation of weathered ANS 521. Toxicity/ teratogenicity tests were conducted with WSF recovered from the biodegradation system using embryonic and larval Pacific herring, Clupea pallasi. Exposures were begun at 4, 48, and 96 h postfertilization of herring eggs. Exposure concentrations were 1, 10, and 100% of the original concentration of WSF recovered from incubations (redissolved in 20/1000 salinity sterile seawater at 15 +/- 1 degrees C). Sterile 20/1000 salinity seawater without the addition of redissolved neutral fraction was used as a control. Significant (p < or = 0.05) embryo mortality or teratogenic responses were observed at WSF concentrations of 10 and 100%. On days 5 through 8 of embryogenesis, counts of heart contraction rates were significantly lower (p < or = 0.05) at the 100% WSF concentration for embryos exposed beginning at 4 and 48 h postfertilization. Grow-out of larvae from selected exposures was conducted. High mortality was noted in larvae exposed to the 10% WSF concentration beginning at 4 and 48 h postfertilization. Most of these larvae died 5 to 8 days after hatching when they elicited vertebral displacements at a time concurrent with the onset of feeding behavior.

Hyaluronic acid enhances the zona pellucida-induced acrosome reaction of macaque sperm.

Hyaluronic acid (HA) not only surrounds the zona pellucida as part of the cumulus matrix but also is present throughout the zona pellucida and the perivitelline space of many mammalian oocytes. However, most in vitro techniques to study sperm-oocyte interaction eliminate HA from the oocyte through enzymatic digestion and/or do not expose sperm to HA prior to zona pellucida binding. This study explores the effect of preincubation of sperm or oocytes with HA on sperm-zona pellucida binding and subsequent acrosome reaction of bound sperm. Cynomolgus macaque semen was washed. Incubated, chemically capacitated with dibutryl cyclic adenosine monophosphate (dbcAMP) and caffeine, and sperm-zona pellucida binding assays were performed. In one experiment, sperm were pretreated with HA (100 micrograms/ml) during the last 10 minutes of the 30-minute period for chemical capacitation. In another experiment, only the oocytes were preincubated in the media containing HA. In the third experiment, gametes were exposed to HA for 10 minutes after sperm had been allowed to bind to zonae pellucida. The preincubation of either sperm or zonae pellucida with HA enhanced the percentage of bound sperm that were acrosome reacted. However, HA did not affect the number of sperm bound to zonae pellucida. When sperm already bound to the zonae pellucida were exposed to HA, there was no increase in the percentage of bound acrosome-reacted sperm. The HA enhancement of the acrosome reaction of sperm bound to the zona pellucida shown in this study required less than 1 minute of sperm exposure to HA-treated zonae during the zona binding process. However, this enhancement was observed only if the HA exposure preceded sperm-zona binding. This result suggests that HA is interacting with the sperm surface, possibly via a receptor, at the time of initiation of the acrosome reaction. It is unlikely that the effects noted in the current experiments were the result of motility retention or improvement because the full enhancement of the acrosome reaction was observed when only the oocytes were pretreated with HA.

The PH-20 protein in cynomolgus macaque spermatozoa: identification of two different forms exhibiting hyaluronidase activity.

In these experiments, we have characterized the bifunctional sperm protein PH-20 in macaque sperm and studied its hyaluronidase activity. Intact sperm were evaluated before the acrosome reaction (AR), and a soluble form of PH-20 released during acrosomal exocytosis was also investigated. Western blots of SDS-PAGE of acrosome-intact sperm extracts revealed a 64-kDa form of PH-20 was recognized by a polyclonal antibody (R-10) raised in rabbits against purified, recombinant cynomolgus macaque sperm PH-20. The soluble components released during the AR which were recognized by the R-10 antibody included both the 64-kDa form and a 53-kDa form of PH-20. An ELISA-like procedure for determining PH-20 hyaluronidase activity indicated that acrosome-intact sperm exhibited two peaks of hyaluronidase activity near pH 4 and > or = pH 7. The majority of enzyme activity in acrosome-intact sperm extracts occurred at neutral pH, while the soluble hyaluronidase activity released at the AR was predominantly acid-active. Hyaluronidase activity of PH-20 at different pH optima was investigated using hyaluronic acid substrate gel electrophoresis, and results indicated that the 64-kDa polypeptide had a broad range, with the majority of activity at neutral pH (pH 7). The 53-kDa polypeptide in sperm extracts only exhibited activity at acid pH (pH 4). The hyaluronidase activities of both enzymes could be inhibited by apigenin. The soluble PH-20 hyaluronidase activity released during the AR was primarily of the acid-active 53-kDa form. Fine structural localization of PH-20 using Fab fragments of R-10 IgG demonstrated that PH-20 was associated not only with sperm membranes, but also with the dispersing acrosomal contents. These data suggest that the more neutral-active form of PH-20 (64 kDa) is present on the plasma and inner acrosomal membranes and gives rise to the soluble acid-active form at the time of the AR. The generation of the soluble form of PH-20 may result from the action of acrosomal enzymes, which could include proteases, glycosidases, and phospholipases.