Detection of 25-kDa Anti-Agglutinin in Epididymal Plasma and Spermatozoa Collected from Various Regions of Boar Epididymis.

Harayama, Hiroshi; Miyano, Takashi; Masuda, Hiroshi; Miyake, Masakazu; Kato, Seishiro

doi:10.1262/jrd.41.113

Cited by 7 publications

(6 citation statements)

References 26 publications

(34 reference statements)

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“…Regional division of boar epididymis was shown in our previous report [11]. Epididymal fluid was obtained from the central caput (region 2) and distal corpus (region 5) by microperfusion [13] with phosphate-buffered saline (PBS; 136.9 mM NaCl, 2.7 mM KCl, 8.1 mM Na 2 HPO 4 ·12H 2 O and 1.5 mM KH 2 PO 4 ) and from the distal cauda (region 7) by retrograde displacement [11] with PBS.…”

Section: Sample Preparationsupporting

confidence: 55%

“…Epididymal fluid was obtained from the central caput (region 2) and distal corpus (region 5) by microperfusion [13] with phosphate-buffered saline (PBS; 136.9 mM NaCl, 2.7 mM KCl, 8.1 mM Na 2 HPO 4 ·12H 2 O and 1.5 mM KH 2 PO 4 ) and from the distal cauda (region 7) by retrograde displacement [11] with PBS. Epididymal spermatozoa were recovered from the fluid by centrifugation at 400 g for 5 min at room temperature and then washed three times in PBS containing 0.2 mM phenylmethylsulfonyl fluoride (Sigma Chem.…”

Section: Sample Preparationmentioning

confidence: 99%

“…The extract from 2 × 10 6 spermatozoa or epididymal plasma (2.5 µg protein) was loaded into each slot of the gel. SDS-PAGE and electrotransfer to nitrocellulose membrane (Hybond-C super, Amersham Life Science, Buckinghamshire, UK) were performed as described previously [11].…”

Section: Sds-page and Electrotransfermentioning

confidence: 99%

“…tabolism [7], location of a cytoplasmic droplet [6], capacity to undergo the acrosome reaction [8], ability to bind tightly to the zona pellucida [9] and to the egg plasma membrane [5,8], head-to-head agglutinability [5,10,11], polypeptide components [12][13][14] and so on. However, few attempts have been made so far to reveal maturation-dependent changes in the lectin affinity for spermatozoa in boars, although the detailed lectin-binding characteristics of sperm components as well as of sperm oar spermatozoa gradually become mature during epididymal transit and then are stored in B the cauda epididymidis until ejaculation [1,2].…”

mentioning

confidence: 99%

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Lectin-Binding Characteristics of Extracts from Epididymal Boar Spermatozoa.

Harayama

Watanabe²,

Masuda³

et al. 1998

Journal of Reproduction and Development

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Abstract. Glycosylated components of boar spermatozoa and epididymal plasma from the central caput, distal corpus and distal cauda epididymides were compared by electrophoretic and lectin blotting techniques. The four lectin reagents used in this study were Concanavalin A (Con A), Lens Culinaris Agglutinin (LCA), Ricinus Communis Agglutinin I (RCA I) and Peanut Agglutinin (PNA), which were labeled with peroxidase. The affinity of Con A and of LCA increased for three bands and one smear (A: >86.8 kDa, B: 80 kDa, C: 65-75 kDa and D: 45 kDa) and for two bands and one smear (A, B and C) of extracts from spermatozoa, respectively, as they passed through the epididymis. RCA I apparently recognized one band and one smear (G: >86.8 kDa and H: 46-58 kDa) of extracts from the distal cauda spermatozoa, though it hardly or faintly reacted with those from the central caput and distal corpus spermatozoa. PNA showed a similar affinity for band "G" to RCA I. Moreover, bands and smears with similar mobility (A, B, C, D, G and H) were also detected in epididymal plasma by lectin blotting techniques. On the other hand, several sperm components were detected more visibly with Con A (E: 37-41 kDa and F: 32-37 kDa), RCA I (I: 41 kDa) and PNA (I: 41 kDa and J: 47-51 kDa) in sperm extracts from the distal cauda than in those from the central caput, though no corresponding components were observed in epididymal plasma. These epididymal maturation-dependent modifications related to glycosylation in boar spermatozoa are discussed.

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Section: Sample Preparationsupporting

confidence: 55%

Section: Sample Preparationmentioning

confidence: 99%

Section: Sds-page and Electrotransfermentioning

confidence: 99%

mentioning

confidence: 99%

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Lectin-Binding Characteristics of Extracts from Epididymal Boar Spermatozoa.

Harayama

Watanabe²,

Masuda³

et al. 1998

Journal of Reproduction and Development

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“…We recently reported (Harayama et al, 1994(Harayama et al, , 1995(Harayama et al, , 1996(Harayama et al, , 1999 an ''anti-agglutinin'' molecule(s) from boar reproductive tract fluids, apparently distinct from the factors described above, with these features: (a) detection, via a specific polyclonal antibody, in epididymal plasma from the distal epididymis and seminal plasma; (b) capacity to bind strongly to sperm acrosome; (c) molecular mass of about 25 kDa, as assessed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE); (d) ability to effectively reduce head-tohead agglutination of epididymal spermatozoa; (e) possible modification during incubations promoting in vitro capacitation such that the molecule is released from spermatozoa.…”

Section: Introductionmentioning

confidence: 99%

Biochemical characterization of sialoprotein ?anti-agglutinin? purified from boar epididymal and seminal plasma

et al. 2000

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Sialoprotein “anti‐agglutinin,” previously shown to inhibit sperm head‐to‐head agglutination, is found in both boar epididymal and seminal plasma. The present report characterizes anti‐agglutinin by mass spectrometry, by N‐terminal amino acid sequence analysis, and by sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis (PAGE) and Western blotting techniques to assess phosphate content of the molecule. Anti‐agglutinin had the SDS‐PAGE mobility of approximately 25 kDa. By electrospray ionization‐mass spectrometry, however, mass spectra of anti‐agglutinin were characterized by two major peaks (19,379–19,382 Da and 19,395–19,397 Da) and several minor peaks. Mass spectrometry of tryptic peptide fragments of deglycosylated anti‐agglutinin and amino acid sequence analysis revealed that the protein has a unique peptide‐mass fingerprinting of fragments (12,668 Da, 5,209 Da, 1,226 Da, and 1,168 Da) and a novel N‐terminal amino acid sequence (KTDDY AISGA KEEEF YDYME ELYAV), respectively. Additionally Western blot techniques, using commercially available monoclonal antibodies, were used to detect presence of phosphothreonine and phosphoserine substituents, but two different monoclonal antibodies did not detect phosphotyrosine. Moreover, treatment with two different alkaline phosphotases converted the molecule, as assessed by SDS‐PAGE and detection by silver stain, from the parent form of about 25 kDa to forms of approximately 19 kDa (similar to that assigned by mass spectrometry) and/or 15 kDa. Original antiserum generated toward, and reacting with native anti‐agglutinin, reacted only with 19 kDa form. These results are consistent with the conclusion that the native anti‐agglutinin may be a novel protein that is phosphorylated at serine and/or threonine residues. Mol. Reprod. Dev. 55:96–103, 2000. © 2000 Wiley‐Liss, Inc.

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