In Vivo Degradation of Crosslinked Hyaluronic Acid Fillers by Exogenous Hyaluronidases.

BACKGROUND: An advantage of hyaluronic acid (HA)-based fillers is reversibility. OBJECTIVE: To evaluate the ability of 2 hyaluronidases to degrade 3 HA-based fillers using a novel in vivo model. MATERIALS AND METHODS: Rats were injected with 3 HA fillers (HYC-24L+, VYC-20L, and RES-L) to create a projecting bolus. After 4 days, recombinant human hyaluronidase (HX) or ovine hyaluronidase (VIT) was administered at (1) varying doses (5 U, 10 U, or 30 U per 0.1 mL filler) or (2) different dilutions (10 U diluted 3-fold). The impact of tissue integration was assessed by administering 10 U/0.1 mL filler 4 weeks after filler injection. Three-dimensional images quantified projection loss over 72 hours. RESULTS: Complete loss of projection was achieved for all fillers with the highest HX and VIT doses; lower doses achieved less degradation. No difference in degradation was observed between HYC-24L+ and VYC-20L using HX or VIT. RES-L was slightly more degraded with 10 U VIT but not with 10 U HX. Enzyme dilution resulted in less degradation. Tissue integration did not impact the degree of degradation. CONCLUSION: This model incorporates the biological system while controlling variables including filler depth and volume and location of hyaluronidase delivery. Hyaluronic acid filler degradation by exogenous hyaluronidase was not hindered by differences among fillers.

Histochemical structure and immunolocalisation of the hyaluronan system in the dromedary oviduct.

We investigated the local modulation of some histochemical properties of oviducts of the dromedary (Camelus dromedarius), focusing on the immnolocalisation of hyaluronic acid (HA) synthases (HAS2 and HAS3), hyaluronidases (HYAL2 and HYAL1) and the HA receptor CD44 in the ampulla and isthmus. Abundant acidic mucopolysaccharides (glycosaminoglycans) were detected by Alcian blue staining along the luminal surface of both ciliated and non-ciliated epithelial cells (LE). Staining for HAS2 was higher in the primary epithelial folds of the ampulla compared with the isthmus, especially in secretory cells, adluminal epithelial surface and supranuclear cell domain. HAS3 staining was stronger in the LE of the isthmus than ampulla. HYAL2 was detected in the LE in the ampulla and isthmus and was more intense in the adluminal projections of secretory cells. HYAL1 was weakly detected in the LE with no difference between the ampulla and isthmus. Strong CD44 immunostaining was present in the LE of the ampulla and isthmus. CD44 staining was higher in secretory cells than in ciliated epithelial cells and was higher in the supranuclear region than the basal region of the cytoplasm. In conclusion, we provide evidence that HA synthesis and turnover occur in the camel oviduct. Differences in HAS2 and HAS3 expression suggest regional differences in the molecular size of HA secreted in oviductal fluid that may influence oviduct-gamete interaction in the camel.

Two novel functions of hyaluronidase from Streptococcus agalactiae are enhanced intracellular survival and inhibition of proinflammatory cytokine expression.

Streptococcus agalactiae is the causative agent of septicemia and meningitis in fish. Previous studies have shown that hyaluronidase (Hyl) is an important virulence factor in many Gram-positive bacteria. To investigate the role of S. agalactiae Hyl during interaction with macrophages, we inactivated the gene encoding extracellular hyaluronidase, hylB, in a clinical Hyl(+) isolate. The isogenic hylb mutant (Δhylb) displayed reduced survival in macrophages compared to the wild type and stimulated a significantly higher release of proinflammatory cytokines, such as interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor alpha (TNF-α), than the wild type in macrophages as well as in mice. Furthermore, only Hyl(+) strains could grow utilizing hyaluronic acid (HA) as the sole carbon source, suggesting that Hyl permits the organism to utilize host HA as an energy source. Fifty percent lethal dose (LD50) determinations in zebrafish demonstrated that the hylb mutant was highly attenuated relative to the wild-type strain. Experimental infection of BALB/c mice revealed that bacterial loads in the blood, spleen, and brain at 16 h postinfection were significantly reduced in the ΔhylB mutant compared to those in wild-type-infected mice. In conclusion, hyaluronidase has a strong influence on the intracellular survival of S. agalactiae and proinflammatory cytokine expression, suggesting that it plays a key role in S. agalactiae pathogenicity.

Hyaluronidase 2: a novel germ cell hyaluronidase with epididymal expression and functional roles in mammalian sperm.

To initiate the crucial cell adhesion events necessary for fertilization, sperm must penetrate extracellular matrix barriers containing hyaluronic acid (HA), a task thought to be accomplished by neutral-active hyaluronidases. Here we report that the ~57 kDa hyaluronidase 2 (HYAL2) that in somatic tissues has been highly characterized to be acid-active is present in mouse and human sperm, as detected by Western blot, flow cytometric, and immunoprecipitation assays. Immunofluorescence revealed its presence on the plasma membrane over the acrosome, the midpiece, and proximal principal piece in mice where protein fractionation demonstrated a differential distribution in subcellular compartments. It is significantly more abundant in the acrosome-reacted (P = 0.04) and soluble acrosomal fractions (P = 0.006) (microenvironments where acid-active hyaluronidases function) compared to that of the plasma membrane where neutral hyaluronidases mediate cumulus penetration. Using HA substrate gel electrophoresis, immunoprecipitated HYAL 2 was shown to have catalytic activity at pH 4.0. Colocalization and coimmunoprecipitation assays reveal that HYAL2 is associated with its cofactor, CD44, consistent with CD44-dependent HYAL2 activity. HYAL2 is also present throughout the epididymis, where Hyal2 transcripts were detected, and in the epididymal luminal fluids. In vitro assays demonstrated that HYAL2 can be acquired on the sperm membrane from epididymal luminal fluids, suggesting that it plays a role in epididymal maturation. Because similar biphasic kinetics are seen for HYAL2 and SPAM1 (Sperm adhesion molecule 1), it is likely that HYAL2 plays a redundant role in the catalysis of megadalton HA to its 20 kDa intermediate during fertilization.

Germ-cell hyaluronidases: their roles in sperm function.

Hyaluronidases (hyases) are a family of enzymes that catalyse the breakdown of hyaluronic acid (HA), which is abundant in the extracellular matrix. Two unlinked gene clusters encode these six proteins: three each in the somatic (or ubiquitous) acid-active subgroup and the neutral-active germ-cell subgroup. This review analyses the data on the expression and role of hyases in gamete biology and fertilization, using electronic databases until October 2010. Evidence indicates that hyases are membrane proteins with multifunctional essential, enzymatic and non-enzymatic, roles (cumulus penetration, zona binding and HA receptor) in fertilization. While sperm adhesion molecule-1 (SPAM1), which has neutral and acidic (bimodal) activity, is the widely conserved mammalian sperm hyase, it co-exists with an acidic hyase in murine and human spermatozoa. Thus, sperm function depends on the concerted activity of both germ cell and 'somatic' hyases. Some hyases are in low abundance in the ovary, somatic testicular cells, the male accessory organs and the male and female genital tracts where they are secreted and acquired by spermatozoa. The latter opens up the possibility of treating hyase-deficient spermatozoa via assisted reproductive technology. The findings challenge the existing classification of hyases, and support the notion that hyase activities are polygenic traits controlled by as many as five hyase genes in mice. Multiple sperm hyases may function cooperatively in a quantitative system and/or serve redundant roles. Unsolved problems include functional redundancy, which can be addressed by double gene-knockouts, and identifying the murine hyase(s) involved in zona binding or whether this role shows species specificity.

HYAL4-V1/Chondroitinase (Chase) Drives Gemcitabine Resistance and Predicts Chemotherapy Failure in Patients with Bladder Cancer.

PURPOSE: Gemcitabine-based chemotherapy regimens are first-line for several advanced cancers. Because of better tolerability, gemcitabine + cisplatin is a preferred neoadjuvant, adjuvant, and/or palliative chemotherapy regimen for advanced bladder cancer. Nevertheless, predicting treatment failure and overcoming resistance remain unmet clinical needs. We discovered that splice variant (V1) of HYAL-4 is a first-in-class eukaryotic chondroitinase (Chase), and CD44 is its major substrate. V1 is upregulated in bladder cancer and drives a malignant phenotype. In this study, we investigated whether V1 drives chemotherapy resistance. EXPERIMENTAL DESIGN: V1 expression was measured in muscle-invasive bladder cancer (MIBC) specimens by qRT-PCR and IHC. HYAL-4 wild-type (Wt) and V1 were stably expressed or silenced in normal urothelial and three bladder cancer cell lines. Transfectants were analyzed for chemoresistance and associated mechanism in preclinical models. RESULTS: V1 levels in MIBC specimens of patients who developed metastasis, predicted response to gemcitabine + cisplatin adjuvant/salvage treatment and disease-specific mortality. V1-expressing bladder cells were resistant to gemcitabine but not to cisplatin. V1 expression neither affected gemcitabine influx nor the drug-efflux transporters. Instead, V1 increased gemcitabine metabolism and subsequent efflux of difluorodeoxyuridine, by upregulating cytidine deaminase (CDA) expression through increased CD44-JAK2/STAT3 signaling. CDA inhibitor tetrahydrouridine resensitized V1-expressing cells to gemcitabine. While gemcitabine (25-50 mg/kg) inhibited bladder cancer xenograft growth, V1-expressing tumors were resistant. Low-dose combination of gemcitabine and tetrahydrouridine abrogated the growth of V1 tumors with minimal toxicity. CONCLUSIONS: V1/Chase drives gemcitabine resistance and potentially predicts gemcitabine + cisplatin failure. CDA inhibition resensitizes V1-expressing tumors to gemcitabine. Because several chemotherapy regimens include gemcitabine, our study could have broad significance.

SPAM1 isoforms from two tissue origins are differentially localized within ejaculated bull sperm membranes and have different roles during fertilization.

The deduced amino acid sequence of bull sperm, SPAM1, suggests that it possesses a transmembrane domain between the hyaluronidase and the putative zona pellucida (ZP) binding domains. The objective of this study was to determine the orientation and localization of SPAM1 in order to understand how it could fulfill these two roles. We report that two isoforms of SPAM1 are present on ejaculated bull spermatozoa: one localized on the anterior portion of the sperm head, and the other on the postacrosomal portion of the head. The first isoform is expressed intracellularly, while the second one is detected at the sperm surface with its hyaluronidase domain facing the extracellular environment. Two-dimensional electrophoresis revealed that the two isoforms have different masses (80 and 70 kDa, respectively), and LC/MS/MS analyses confirmed our previously published deduced amino acid sequence of bovine SPAM1. In addition, this approach showed that the 70-kDa isoform differs from the 80-kDa isoform in its C terminus. Our results suggest that the shorter SPAM1 form originates from the epididymis, while the longer one is produced during spermatogenesis. These results clearly demonstrate that ejaculated bull sperm possess two forms of SPAM1: one (epididymal) expressed at the surface, and one (testicular) that interacts with the ZP after the acrosome exocytosis.

Acidic hyaluronidase activity is present in mouse sperm and is reduced in the absence of SPAM1: evidence for a role for hyaluronidase 3 in mouse and human sperm.

The molecular mechanisms underlying sperm penetration of the physical barriers surrounding the oocyte have not been completely delineated. Although neutral-active or "reproductive" hyaluronidases (hyases), exemplified by Sperm Adhesion Molecule 1 (SPAM1), are thought to be responsible for hyaluronan digestion in the egg vestments and for sperm-zona binding, their roles in mouse sperm have been recently questioned. Here we report that acidic "somatic" Hyaluronidase 3 (HYAL3), a homolog of SPAM1 with 74.6% structural similarity, exists in two isoforms in human ( approximately 47 and approximately 55 kDa) and mouse ( approximately 44 and approximately 47 kDa) sperm, where it resides on the plasma membrane over the head and midpiece. Mouse isoforms are differentially distributed in the soluble (SAP), membrane (MBP), and acrosome-reacted (AR) fraction where they are most abundant. Comparisons of zymography of Hyal3 null and wild-type (WT) AR and MBP fractions show significant HYAL3 activity at pH 3 and 4, and less at pH 7. At pH 4, a second acid-active hyase band at approximately 57 kDa is present in the AR fraction. HYAL3 activity was confirmed using immunoprecipitated HYAL3 and spectrophotometry. In total proteins, hyase activity was higher at pH 6 than at 4, where Spam1 nulls had significantly (P < 0.01) diminished activity implicating an acidic optima for murine SPAM1. Although fully fertile, Hyal3 null sperm showed delayed cumulus penetration and reduced acrosomal exocytosis. HYAL3 is expressed in epididymal tissue/fluid, from where it is acquired by caudal mouse sperm in vitro. Our results reveal concerted activity of both neutral- and acid-active hyaluronidases in sperm.

Hyaluronidase 2 and its intriguing role as a cell-entry receptor for oncogenic sheep retroviruses.

Jaagsiekte sheep retrovirus (JSRV) causes lung adenocarcinoma in sheep and goats, while the closely related enzootic nasal tumor virus (ENTV) causes nasal tumors in the same species. The envelope (Env) protein from either virus can transform fibroblasts and epithelial cells in culture, indicating that the Env proteins are responsible for tumorigenesis. However, the primary function of retroviral Env proteins is to mediate virus entry into cells by interacting with specific cell-surface receptors, suggesting that the virus receptor might be a key player in transformation as well. Thus, identification of Hyaluronidase-2 (Hyal2) as the cell-entry receptor for both JSRV and ENTV suggested a role for Hyal2 in oncogenesis. Furthermore, Hyal2 is located in a key lung cancer tumor suppressor locus on chromosome 3p21.3, suggesting that Hyal2 might have a tumor suppressor activity that was disrupted by Env thereby leading to tumorigenesis. However, recent experiments showing that expression of the JSRV or ENTV Env protein in mouse lung can induce lung tumors, even though the viral Env proteins cannot bind to or utilize mouse Hyal2 as a receptor for virus entry into cells, indicate that Hyal2 plays no role in cancer induction by these retroviruses. Hyal2 remains an enigmatic member of the hyaluronidase family given its very low hyaluronidase activity in purified form or when expressed in cultured cells, suggesting that it may have evolved to perform some other as yet unknown function.

Hyalurondiase: both a tumor promoter and suppressor.

Originally termed as the "spreading factor", hyaluronidases (HAases) are present in a variety of toxins and venoms. For example, HAase is the virulent factor of beta-hemolytic Streptococci and it is also present in the venoms of snake, bee, wasp, scorpion, etc, where it aids in the spread of these venoms in the body. In mammals, testicular HAase present in the sperm acrosome is necessary for the fertilization of the ovum. Despite a lot of work on bacterial, invertebrate and testicular HAases, a connection between HAase and cancer was unequivocally established just over a decade ago and the functional significance of HAases in cancer was demonstrated just about a year ago. In this part of the review, we will focus on the recent advances in our understanding of the role of HAases in cancer.

Hyaluronidases in cancer biology.

Hyaluronan is a megadalton glycosaminoglycan polymer critical for maintaining the integrity of the extracellular matrix. It can exist in a protein-bound state with aggregating proteoglycans, where it expands the extracellular matrix and modulates cell-cell interactions. It also exists in lower molecular weight forms that participate in a myriad of biological functions. It is unique in that much of it is degraded within hours of its synthesis. High molecular weight hyaluronan, a reflection of intact healthy tissues, is normally produced by hyaluronan synthases at the plasma membrane. It is catabolized by the action of an extracellular plasma membrane-tethered hyaluronidase that is coordinated with intracellular lysosomal hyaluronidases and exoglycosidases. This occurs in local tissues and lymph, with the remainder being cleared by the sinusoidal liver endothelium upon entering the vascular compartment. Elevated extracellular levels of hyaluronan and its partially catabolized oligomers are found in certain malignancies, potentially due to decoupled synthesis and degradation. Furthermore, partially depolymerized hyaluronan in the extracellular environment may have properties not found in the multivalent high molecular weight polymer in malignancies. Functional perturbations of hyaluronan synthesis and degradation have revealed active roles of the synthases and hyaluronidases in epithelial mesenchymal conversion, stroma and vascular formation, interstitial fluid pressure and chemosensitivity. While at least three confirmed hyaluronidases exist in the human genome (HYAL1, HYALl2 and PH20), functional perturbation of these genes in mice have failed to identify a simple linear catabolic circuit. The family of enzymes responsible for the synthesis and degradation of hyaluronan are being characterized. The fragmented forms of hyaluronan, largely a sign of cellular distress, occur in abundance in many malignancies. These small hyaluronan oligomers are assumed to be largely a result of hyaluronidase activity. Precisely how particular-sized fragments are generated and maintained is not known. Presumably, hyaluronan-binding proteins, in addition to the proteoglycans, participate in this process. Hyaluronidase inhibitors are now recognized, as well as growth factors that enhance the synthetic enzymes. A complete understanding of the anabolic and catabolic systems for hyaluronan may provide new dimensions into our understanding of cancer progression, as well as new opportunities for therapeutic intervention.

Hyaluronidase inhibitors: a biological and therapeutic perspective.

The hyaluronidases (HAases) are a group of less extensively studied glycosidases distributed throughout the animal kingdom and are popularly known as 'spreading factors'. In recent years, HAases received much attention due to their ability to abruptly alter the hyaluronic acid (HA) homeostasis. HAases preferentially degrade HA, which is a megadalton acidic structural polysaccharide found exclusively in the extracellular matrix (ECM) of animals. The HA-HAase system has been suggested to participate in many pathophysiological conditions. The HA degradation in ECM, crack down the structural integrity with an eventual increased tissue permeability that is attributed for the spreading property. The spreading property has been widely accepted in functions including envenomation, acrosomal reaction/ovum fertilization, cancer progression, microbial pathogenesis such as wound infections, pneumonia, and other sepses like, bacteremia and meningitis. HA fragmentation has dual effects; generation of a wide molecular range bioactive oligosaccharides of angiogenic, pro-inflammatory, and immunostimulatory properties; and impairment in the reservoir capacity of ECM that holds metal ions, growth factors, cytokines and various enzymes for signal transduction. Hence, inhibition of HA degradation appears critical and imperative in HAase mediated pathological conditions. HAase inhibitors are thus potent regulators that maintain HA homeostasis and they might serve as anti-inflammatory, anti-aging, anti-microbial, anticancer and anti-venom/toxin and contraceptive agents. In addition, HAase inhibitors may serve as tools to understand several unexplained and complex functions of HAases in HA metabolism. Therefore, this review is expected to provide an integrated update as of 2008 on the HAase inhibitors and their possible role as therapeutics in the management of a wide range of pathological conditions.

Decreased hyaluronan in airway smooth muscle cells from patients with asthma and COPD.

Glycosaminoglycans (GAG) are essential extracellular matrix molecules which regulate tissue flexibility, a parameter that is reduced in airways of patients with asthma and chronic obstructive pulmonary disease (COPD). We investigated the expression of GAG and their metabolising enzymes in primary human airway smooth muscle cells (ASMC) obtained from healthy donors (controls) and patients with asthma or COPD. Total GAG synthesis was assessed by [(3)H]-glucosamine incorporation. GAG were isolated, purified, fractionated by electrophoresis and characterised using specific GAG-degrading enzymes. Secretion of hyaluronic acid (HA) by ASMC from patients with asthma or COPD was significantly decreased compared with controls. RT-PCR analysis and western blotting revealed that this decrease was associated with a significant reduction in the expression of HA synthase-1 and -2 and a significant increase of hyaluronidase-1. Furthermore, the expression of the HA receptor CD44 was significantly decreased, whereas the receptor for HA-mediated motility was not expressed in asthma or COPD. Our results indicate that there is a decreased expression of HA in asthma and COPD associated with a synergistic regulation of HA metabolising enzymes that may regulate the pathological airway remodelling in these lung diseases.

Mouse Hyal3 encodes a 45- to 56-kDa glycoprotein whose overexpression increases hyaluronidase 1 activity in cultured cells.

Hyaluronidases are enzymes that mediate the breakdown of hyaluronan (HA), a large polysaccharide abundant in the extracellular matrix of vertebrate tissues. Six genes have been predicted to encode hyaluronidases in humans, but the protein products of only SPAM1, HYAL1, and HYAL2 have been characterized. We have now expressed the mouse Hyal3 gene product, hyaluronidase 3 (Hyal3), in Baby Hamster Kidney (BHK) cells and demonstrated the presence of multiple forms of Hyal3 ranging from approximately 45 to 56 kDa in expression lysates. Complete and partial digestions of the expressed protein with PNGase F showed three N-linked oligosaccharides accounted for all forms of Hyal3 detected in expression lysates. Most of these oligosaccharides were Endo H sensitive, indicating that they were high mannose or hybrid N-linked oligosaccharides. Subcellular fractionation of Hyal3-expressing BHK cells by density gradient centrifugation revealed most Hyal3 in a low-density vesicular population. Low levels of Hyal3 were detected in higher density vesicles, but no colocalization with the late endosomal/lysosomal marker Lamp1 was found by immunofluorescence microscopy. BHK cells stably expressing Hyal3 had increased acid-active hyaluronidase activity, but no such activity was detected when Hyal3 was transfected into Hyaluronidase 1 (Hyal1)-deficient fibroblasts. Overexpression of Hyal3 in BHK cells increased the Hyal1 protein and mRNA levels, suggesting that the increased hyaluronidase activity in these cells was due to Hyal1 rather than Hyal3. The results indicate that Hyal3 overexpressed in cultured cells lacks intrinsic hyaluronidase activity and that Hyal3 may contribute to HA metabolism by augmenting the activity of Hyal1.

Mammalian hyaluronidase induces ovarian granulosa cell apoptosis and is involved in follicular atresia.

During ovarian folliculogenesis, the vast majority of follicles will undergo atresia by apoptosis, allowing a few dominant follicles to mature. Mammalian hyaluronidases comprise a family of six to seven enzymes sharing the same catalytic domain responsible for hyaluronan hydrolysis. Interestingly, some of these enzymes have been shown to induce apoptosis. In the ovary, expression of three hyaluronidases (Hyal-1, Hyal-2, and Hyal-3) has been documented. However, their precise cellular localization and role in ovarian regulation have not yet been defined. We herein investigated the possible involvement of these enzymes in ovarian atresia. First, we established a mouse model for ovarian atresia (gonadotropin withdrawal by anti-equine chorionic gonadotropin treatment) and showed that the mRNA levels of Hyal-1, Hyal-2, and Hyal-3 were significantly increased in apoptotic granulosa cells as well as in atretic follicles. Second, using ovaries of normally cycling mice, we demonstrated the correlation of Hyal-1 mRNA and protein expression with cleavage of caspase-3. In addition, we showed that expression of all three hyaluronidases induced apoptosis in transfected granulosa cells. Significantly, the induction of apoptosis by hyaluronidases was independent of catalytic activity, because enzymatically inactive Hyal-1 mutant (D157A/E159A) was as efficient as the wild-type enzyme in apoptosis induction. The activation of the extrinsic apoptotic signaling pathway was involved in this induction, because increased levels of cleaved caspase-8, caspase-3, and poly-ADP-ribose polymerase (PARP) were observed upon hyaluronidase ectopic expression. Our present findings provide a better understanding of the role of hyaluronidases in ovarian functions, showing for the first time their involvement in follicular atresia.

[Changes of the neuropil ultrastructure in the CA1 hippocampal area of rat pups after hyaluronidase treatment].

Electron microscopical and electrophysiological studies were carried out in cross-sections of the hippocampus of 4-weeks-old male rat pups (n = 35) to detect the ultrastructural changes in CA1 hippocampal area and the peculiarities of excitatory postsynaptic potential formation in this brain area after the incubation of the sections in the solution of hyaluronidase (10 U/ml), the enzyme which specifically degrades the extracellular matrix glycosaminoglycan--the hyaluronic acid. The reduction of the width of synaptic cleft by 15-25% in the axo-dendritic contacts of the stratum radiatum of CA1 hippocampal area was detected 1.5 min following the application of enzyme, coinciding with the increase of the excitatory postsynaptic potential amplitude. The width of synaptic cleft was further reduced by 45-55% after 4.5 min of incubation; during this period the blockade of signal transmission along the Schaffer's collaterals to CA1 hippocampal area was observed. Thus, the structural and functional state of glycosaminoglycans is one of the factors controlling the efficiency of synaptic transmission in the brain.

Hyalp1 in murine sperm function: evidence for unique and overlapping functions with other reproductive hyaluronidases.

While Sperm adhesion molecule 1 (SPAM1) is the highly conserved mammalian sperm hyaluronidase (hyase), multiple hyases are present in the mouse testis. In this study we show that one of the murine hyases, Hyalp1, which is predominantly expressed in the testis in a 24-kd isoform has neutral enzymatic activity. On sperm, Hyalp1 is localized on the plasma membrane of the anterior head and was shown to have neutral hyase activity for an isoform of approximately 55-56 kd, contributing modestly to the overall neutral hyase activity. This activity is associated with in vitro cumulus penetration, since antibody inhibition of Hyalp1 significantly (P = .034) retarded the rate of penetration of wild-type (WT) sperm. Antibody-inhibited Spam1 null sperm were more severely retarded (P = 4.2 x 10(-19)), suggesting an up-regulation of Hyalp1 in these mice. A functionality test of the hyaluronic acid (HA) receptor domain identified in the N-terminus by in silico analysis revealed that sperm Hyalp1 is significantly (P = .006) involved in the progesterone-induced HA-enhanced acrosome reaction. Finally, developmental reverse transcription polymerase chain reaction (RT-PCR) shows that testicular transcripts of Hyalp1 are detected as early as 6 days postparturition, similar to transcripts for Spam1, suggesting that the gene might also play a role in the developing testes prior to spermiogenesis. Taken together, the findings reveal that Hyalp1 likely has a unique function in the adult testis, and redundant overlapping ones with Spam1 and may compensate for it in Spam1 null mice.

The magic glue hyaluronan and its eraser hyaluronidase: a biological overview.

Hyaluronan (HA) is a multifunctional high molecular weight polysaccharide found throughout the animal kingdom, especially in the extracellular matrix (ECM) of soft connective tissues. HA is thought to participate in many biological processes, and its level is markedly elevated during embryogenesis, cell migration, wound healing, malignant transformation, and tissue turnover. The enzymes that degrade HA, hyaluronidases (HAases) are expressed both in prokaryotes and eukaryotes. These enzymes are known to be involved in physiological and pathological processes ranging from fertilization to aging. Hyaluronidase-mediated degradation of HA increases the permeability of connective tissues and decreases the viscosity of body fluids and is also involved in bacterial pathogenesis, the spread of toxins and venoms, acrosomal reaction/ovum fertilization, and cancer progression. Furthermore, these enzymes may promote direct contact between pathogens and the host cell surfaces. Depolymerization of HA also adversely affects the role of ECM and impairs its activity as a reservoir of growth factors, cytokines and various enzymes involved in signal transduction. Inhibition of HA degradation therefore may be crucial in reducing disease progression and spread of venom/toxins and bacterial pathogens. Hyaluronidase inhibitors are potent, ubiquitous regulating agents that are involved in maintaining the balance between the anabolism and catabolism of HA. Hyaluronidase inhibitors could also serve as contraceptives and anti-tumor agents and possibly have antibacterial and anti-venom/toxin activities. Additionally, these molecules can be used as pharmacological tools to study the physiological and pathophysiological role of HA and hyaluronidases.

Lung regions differently modulate bronchial branching development and extracellular matrix plays a role in regulating the development of chick embryo whole lung.

Normal branching development is dependent on the correlation between cells and extracellular matrix. In this interaction glycosaminoglycans, cytokines and growth factors play a fundamental role. In order to verify the distribution and influence of extracellular matrix and related enzymes on chick embryo lung development, 6 day-old whole lungs were maintained in vitro with testicular hyaluronidase, beta-N-acetyl-D-glucosaminidase and chondrotinase ABC or in linkage with apical, medial and caudal lung regions of 6-day development before and after enzyme treatment. In a separate lung region beta-N-acetyl-D-glucosaminidase and hyaluronidase were determined. Our data show that the whole lung cultures increase bronchial branching development when the medial region is admixed separately, while the separate apical or caudal regions or apical combined with caudal region do not affect bronchial branching development. The enzyme treatment of medial region prevents the branching development in associated whole lung. The bronchial branching development of whole lung cultured in medium containing the enzymes related to glycosaminoglycans turnover is significantly altered. In conclusion, these data show that the different influence of separate apical, medial, caudal lung regions on bronchial branching development is related to the extracellular matrix composition.