Histo-Immunological Localization of Hyaluronidase in the Bull Testis

Mancini, R. E.; Alonso, A.; Barquet, J.; Alvarez, Bryce; Nemirovsky, M.

doi:10.1530/jrf.0.0080325

Cited by 53 publications

(17 citation statements)

References 20 publications

(13 reference statements)

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“…It is clear that a number of proteins unique to spermatozoa cannot be detected until after meiosis: in addition to PLH and PGK-2, the sperm-specific hyaluronidase (30) and certain sperm autoantigens (31) do not appear until after meiosis. We are not aware of previous work that demonstrates increases in mRNA for such proteins after meiosis.…”

Section: Discussionmentioning

confidence: 99%

Quantitation of mRNAs during mouse spermatogenesis: protamine-like histone and phosphoglycerate kinase-2 mRNAs increase after meiosis.

Erickson¹,

Kramer²,

Rittenhouse³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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ABSIRACT Total RNA, prepared from immature or mature mouse testes or from spermatogenic cells separated on the basis of sedimentation velocity, was translated in vitro. Mouse protamine-like histone could be identified as an in vitro translational product when [3]arginine was used as the label. The mRNA for protamine-like histone was detected only after meiosis; the appearance of a peak of radioactivity comigrating with protamine-like histone occurred only when RNA from mature testes or late spermatid cell fractions was translated. Phosphoglycerate kinase-2 (ATP:3-phospho Dglycerate 1-phosphotransferase, EC 2.7.2.3) was identified as an in vitro translational product by affinity chromatography followed by two-dimensional gel electrophoresis or by specific immunoprecipitation when [35Sjmethionine was the labeL The mRNA for phosphoglycerate kinase-2 was detected only in mature testes or late spermatid cell fractions. These translational assays for protamine-like histone and phosphoglycerate kinase-2 mRNAs suggest that these messages are transcribed after meiosis.it is usually considered that sperm function depends little or not at all on the genotype of the sperm but mainly or only on the diploid genotype of the male, and that all four products of meiosis form functional sperm (1). Thus, it has been generally assumed that postmeiotic (i.e., haploid) gene transcription does not occur. There are two lines of argument for the alternative view in mammals First, although Mendelian transmission ratios require gamete equivalence, this could be accomplished in other ways than eliminating postmeiotic gene transcription. The large intercellular bridges connecting all spermatids allow a sharing of gene products and would contribute greatly to gametic equivalence (2). Second, the appearance of sperm specific proteins after meiosis requires new transcription or the use of stored mRNA. There is little evidence for the use of stored mRNA in mammalian embryogenesis, in contrast to the situation in lower vertebrates (3, 4). The situation remains to be explored for mammalian gametogenesis.Evidence for postmeiotic RNA synthesis was first provided by Monesi (5), who demonstrated that early mouse spermatids exposed to short pulses of [3H]uridine showed a small peak of incorporation when examined by autoradiography. Quantitative autoradiographic studies showed that the rate of RNA synthesis per cell decreased only 75% during meiosis in the ram testis; i.e., the RNA synthesis/DNA ratio was essentially unchanged (6). The newly synthesized RNA from separated, postmeiotic spermatogenic cells has been characterized by size and by the percentage of the RNA transcripts that contain poly(A) tracts (7,8). By these criteria, synthesis of putative mRNA remains at about the same level in early postmeiotic as compared to late premeiotic cells.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact...

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Section: Discussionmentioning

confidence: 99%

Quantitation of mRNAs during mouse spermatogenesis: protamine-like histone and phosphoglycerate kinase-2 mRNAs increase after meiosis.

Erickson¹,

Kramer²,

Rittenhouse³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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“…However, low levels of excess Fab antihyaluronidase antibody were apparently carried into the culture media with the inseminating spermatozoa, and it is possible but unlikely that the antibody acted directly on the eggs. (Mancini, Alonso, Barquet, Alverez & Neminirovsky, 1964;Morton, 1975). Some of this hyaluronidase is attached loosely to the sperm surface (O'Rand & Metz, 1974), probably in equilibrium with free hyaluronidase (Swyer, 1947), some is strongly bound to sperm membranes (unpublished), and most is presumably stored in the acrosome body itself.…”

Section: Methodsmentioning

confidence: 96%

Inhibition of fertilization in vitro by treatment of rabbit spermatozoa with univalent isoantibodies to rabbit sperm hyaluronidase

Dunbar¹,

Muñoz²,

Cordle³

et al. 1976

Reproduction

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Isoantibodies to whole semen or epididymal spermatozoa of rabbits inhibit fertilization in vitro (Russo & Metz, 1974a). This inhibition is attributed to a primary blocking of one or more sperm antigens essential for the sperm-egg interaction, and not to a secondary action (e.g. agglutination, complement-dependent immobilization), because univalent (Fab) antibody fragments, which do not agglutinate or immobilize spermatozoa, are also effective. Hyaluronidase, the sperm enzyme required for penetration of the cumulus surrounding the egg, could be one such sperm antigen. This possibility is supported by the fact that cumulus dispersion was inhibited in the experiments of Russo & Metz (1974a) and that hyaluronidase-inhibiting antibodies are present in isoantisera to rabbit semen (Metz, Seiguer & Castro, 1972). It is necessary to employ antibody preparations specific for rabbit sperm hyaluronidase to examine critically the inhibition of fertilization by antihyaluronidase antibodies. We report here such inhibition of rabbit fertilization in vitro by Fab isoantibodies to purified rabbit sperm hyaluronidase. MethodsHyaluronidase was purified from rabbit epididymides (Pel-Freez, Rogers, Arkansas). These were homogenized in batches of approximately 200-300 in 500 ml triton-X buffer (0-1 m-P04, 0-15 M-NaCl, 01 % Triton-X, pH 5-3) using a Waring-type blender. After further homogenization in a glass homogenizer the material was frozen and thawed (10 cycles) and centrifuged (10,000 g, 30 min, 4°C). The hyaluronidase in the supernatant was purified by a modification of the procedure of Borders & Raftery (1968); an initial precipitation with 50% saturated (NH4)2S04 was followed by DEAE chromatography. Hyaluronidase activity was determined by the turbidimetric (Tolksdorf, McCready, McCullough & Schwenk, 1949) or the N-acetylglucosamine (Aronson & Davidson, 1967) assay procedure. The purity of the hyaluronidase obtained was tested for some preparations by acryla¬ mide gel electrophoresis and immunodiffusion against isoantisera to whole semen of rabbits. The least pure hyaluronidase preparations (sp. act. 3600 turbidity reducing units/mg protein) used as immunizing antigens and so tested produced up to 6 protein-staining bands in acrylamide gel elec¬ trophoresis but only one precipitin band, which was identical to the band containing hyaluronidase activity in the acrylamide gel. It was therefore concluded that the hyaluronidase preparations were isoantigenically pure. Isoantisera were prepared by injecting virgin female rabbits of the New Zealand White breed once a week for 3-4 weeks with this isoantigenically pure hyaluronidase (0-2-0-46 mg in 0-5 ml phosphate-buffered saline emulsified in 0-5 ml complete Freund's adjuvant per injection). The isoantisera utilized from these rabbits strongly inhibited rabbit sperm hyaluronidase activity, and did not produce more than one precipitin band during immunodiffusion against concentrated sperm extract, or the immunizing antigen which was sometimes tested. These results are consistent with ...

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“…Morton (1977), who made great efforts to purify his antigen and to characterize the specificity of his resulting antibody preparation, concluded that sites were sensitive to fixation (he used an optimal formaldehyde fixation protocol very similar to our own). On the other hand, Mancini et al (1964), Fléchon & Dubois (1975), Gould & Bernstein (1975), and Sakai et al (1979) all observed labelling of fixed material. However, the antisera used by these authors were raised to antigens whose purity was not established, and proof of specificity of the resulting antisera was not rigorous; the possibility therefore exists that an acrosomal component other than hyaluronidase was being labelled which resisted fixation.…”

Section: Immunocytochemical Reactivitymentioning

confidence: 94%

“…sperm) hyaluronidase have been used both for localization studies (Mancini et al, 1964;Fléchon & Dubois, 1975;Gould & Bernstein, 1975;Morton, 1975Morton, , 1977Sakai et ai, 1979;Gupta & Goldberg, 1981;De Vries et al, 1985) and for inhibition studies (Metz et al, 1972;Dunbar et ai, 1976;Sokolovskaya et al, 1976), to try to define its physiological role. Unfortunately, the enzyme is difficult to purify, and totally specific antibody preparations have rarely been obtained with conventional antisera.…”

Section: Introductionmentioning

confidence: 99%

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

Harrison

Gaunt²

1988

Reproduction

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Histo-Immunological Localization of Hyaluronidase in the Bull Testis

Cited by 53 publications

References 20 publications

Quantitation of mRNAs during mouse spermatogenesis: protamine-like histone and phosphoglycerate kinase-2 mRNAs increase after meiosis.

Quantitation of mRNAs during mouse spermatogenesis: protamine-like histone and phosphoglycerate kinase-2 mRNAs increase after meiosis.

Inhibition of fertilization in vitro by treatment of rabbit spermatozoa with univalent isoantibodies to rabbit sperm hyaluronidase

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

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