Multiple forms of ram and bull sperm hyaluronidase revealed by using monoclonal antibodies

Harrison, Robert A.; Gaunt, Stephen J.

doi:10.1530/jrf.0.0820777

Cited by 17 publications

(12 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This result is also in agreement with the association of the protein with the membrane, because membrane-bound protein PH-20 shows an optimal hyaluronidase activity at neutral pH [15]. Harrison and Gaunt [32] have detected a single 80-kDa bull sperm protein by Western blot analysis using an anti-ram sperm hyaluronidase monoclonal antibody in protein samples solubilized under reducing conditions. As for the p80 described in the present study (Fig.…”

Section: Discussionsupporting

confidence: 80%

Characterization of an 80-Kilodalton Bull Sperm Protein Identified as PH-201

Lalancette

Dorval

Leblanc

et al. 2001

Biology of Reproduction

View full text Add to dashboard Cite

This paper presents the partial characterization and the identification of an 80-kDa protein detected in bull spermatozoa using a monoclonal antibody directed against a 16-amino acid long peptide from the N-terminal domain of the protooncogene p60(src) from the Rous Sarcoma Virus When subjected to two-dimensional electrophoresis, this 80-kDa protein migrated as several isoforms, with an isoelectric point ranging from 7.4 to 8.2. Amino acid sequence analysis of a peptide obtained following trypsin digestion of the bull sperm protein showed homology to the PH-20/hyaluronidase precursor sperm protein. As for PH-20, this bull sperm 80-kDa protein is located at the plasma membrane surface in the postacrosomal region of the head. An increased immunolabeling in the anterior head region of fixed/permeabilized spermatozoa was observed when these cells were incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react lost their labeling almost completely. As for the PH-20 protein, the 80-kDa bull sperm protein possesses a hyaluronidase activity that is higher at pH 7.0 than at pH 4.0 in an in-gel assay. Unlike what has been observed in the guinea pig, mouse, and human PH-20, this 80-kDa protein was not released from the surface of bull spermatozoa by treatment with phosphatidylinositol-specific phospholipase C or with trypsin. However, this protein was not sedimented by a 100,000 x g centrifugation after nitrogen cavitation, which suggests that the 80-kDa protein is loosely attached to the sperm membrane by a yet-unknown mechanism. These results suggest that the 80-kDa bull sperm protein shares many homologies with the sperm PH-20 protein reported in the literature and, most likely, is the bull sperm homologue of the PH-20.

show abstract

Section: Discussionsupporting

confidence: 80%

Characterization of an 80-Kilodalton Bull Sperm Protein Identified as PH-201

Lalancette

Dorval

Leblanc

et al. 2001

Biology of Reproduction

View full text Add to dashboard Cite

show abstract

“…(1982), except that the crude product was not precipitated in diethyl ether but was recrystallized directly from the syrup left after rotary evaporation of the filtered reaction mixture. Monoclonal mouse anti-(ram hyaluronidase) antibody was obtained as ascites fluid from tumours of the 1D6 monoclonal mouse cell line isolated by Harrison & Gaunt (1988). The ascites fluid was used as antibody without further purification.…”

Section: Methods Reagentsmentioning

confidence: 99%

Hyaluronidase in ram semen. Quantitative determination, and isolation of multiple forms

Harrison

1988

Biochemical Journal

View full text Add to dashboard Cite

A study was made of hyaluronidase in ram semen. The end-group assay conditions used to determine activity quantitatively were chosen to ensure reliability as well as sensitivity [Gacesa, Savitsky, Dodgson & Olavesen (1981) Anal. Biochem. 118, 76-84]; they led to 1 W.H.O. Standard International Hyaluronidase Unit displaying 0.1263 EC munit (1 EC unit of activity releases 1 mumol equivalent of N-acetylglucosamine end groups/min at 37 degrees C). All the activity in the semen was shown to be sperm-derived, and intact spermatozoa were estimated to contain 1.23 EC units per 10(9) cells. In a low-ionic-strength medium, only some 20% of the hyaluronidase was extractable, although up to 80% of the activity could be extracted as the ionic strength was increased; further addition of detergent extracted the remainder. During purification of the enzyme, it was found that inclusion of poly(vinyl alcohol) in the media stabilized the activity; detergent inclusion also improved the yield, especially during early stages. As a consequence both of reliable quantitative determination and of stabilization, a number of forms of hyaluronidase could be isolated in high yield, by using anion-exchange chromatography, cation-exchange chromatography, affinity chromatography and gel filtration. The existence of all these forms was confirmed by electrophoresis and immunoblotting with the use of a monoclonal anti-(ram hyaluronidase) antibody, and their presence in very freshly prepared sperm extracts was demonstrated. The specific activity of the isolated major hyaluronidase form was 15.0 EC units/mg; this was equivalent to 119,000 W.H.O. units/mg, higher than any other previously reported values.

show abstract

“…Multiple oligomeric forms of hyaluronidase are present in bull and ram spermatozoa (Harrison and Gaunt, 1988). Hyaluronidases in semen are only of sperm origin.…”

Section: Proteases and Protease Inhibitors Of Spermatozoamentioning

confidence: 99%

“…Hyaluronidases are glycosidase abundant in acrosome especially in principal segment in bull (Mancini et al, 1964) and ram sperm (Morton et al, 1975). In ram sperm outer acrosomal membrane also have half of the hyaluronidase of the sperm with intact acrosome (Harrison et al, 1988).…”

Section: Proteases and Protease Inhibitors Of Spermatozoamentioning

confidence: 99%

Proteases and Proteases Inhibitors of Semen - A Review

Gurupriya¹

2014

Adv. Anim. Vet. Sci.

View full text Add to dashboard Cite

| The ejaculate composition is extremely complicated and variable among livestock species. The seminal proteins and enzymes are vital for ejaculate metabolism, spermatozoa performance, survival, and transport within the feminine reproductive tract. Proteases and proteinase inhibitors are secreted by the accessory sex glands of the male reproductive tract and find mixed with the spermatozoa throughout ejaculation. But an entire understanding of those enzymes and their performance in numerous class species is not obtainable. The performance of the assorted proteinases and protease inhibitors of ejaculate stay a mystery, and only a few studies are conducted concerning the characterization of those enzymes. Throughout this review, we tend to target the current understanding of the key proteases and their inhibitors of ejaculate from various mammalian species.

show abstract

Multiple forms of ram and bull sperm hyaluronidase revealed by using monoclonal antibodies

Cited by 17 publications

References 23 publications

Characterization of an 80-Kilodalton Bull Sperm Protein Identified as PH-201

Characterization of an 80-Kilodalton Bull Sperm Protein Identified as PH-201

Hyaluronidase in ram semen. Quantitative determination, and isolation of multiple forms

Proteases and Proteases Inhibitors of Semen - A Review

Contact Info

Product

Resources

About