Binding of mammalian spermatozoa to the zona pellucida of homologous eggs is mediated by specific molecules on their surface membranes. In the present investigation we describe the biogenesis, epididymal processing and cellular distribution of a plasma membrane antigen (2B1) on rat spermatozoa that has a potential role in mediating zona binding. 2B1 is expressed postmeiotically in the testis as a precursor glycoprotein (approximately 60 kDa) that first appears on the plasma membrane of stage 6 to 8 round spermatids. Northern and western blot analyses show that there is a close correlation between the timing of transcription and expression of the glycoprotein on the cell surface. During spermatid elongation 2B1 is excluded from the head domain and is sequestered onto the sperm tail. As spermatozoa pass through the caput epididymidis 2B1 is endoproteolytically cleaved at a specific arginine residue (Arg 312) to produce a heterodimeric glycoprotein (approximately 40 kDa and approximately 19 kDa) containing intramolecular disulphide bridges. Endoproteolysis at Arg 312 also takes place during culture of washed testicular or caput spermatozoa in vitro and can be prevented by serine proteinase inhibitors or enhanced by trypsinisation. However, neither processing in vivo or in vitro has any effect on the domain organisation of 2B1 antigen i.e. it remains localised to the tail. These results support the hypothesis that sperm antigens that are important for fertilization are synthesized as precursor molecules in the testis and are then “activated' during epididymal maturation and capacitation, thereby ensuring that they only become fully functional at the site of fertilization.

REFERENCES

Blobel
C. P.
,
Myles
D. G.
,
Primakoff
P.
,
White
J. M.
(
1990
).
Proteolytic processing of a protein involved in sperm-egg fusion correlates with acquisition of fertilization competence.
J. Cell Biol
111
,
69
78
Braun
R. E.
,
Peschon
J. J.
,
Behringer
R. R.
,
Brinster
R. L.
,
Palmiter
R. D.
(
1989
).
Protamine 3-untranslated sequences regulate temporal translational control and subcellular localisation of growth hormone in spermatids of transgenic mice.
Genes Dev
3
,
793
802
Carroll
D. J.
,
Dikegoros
E.
,
Koppel
D. E.
,
Cowen
A. E.
(
1995
).
Surface expression of the pre-subunit of fertilin is regulated at a post-translational level in guinea pig spermatids.
Dev. Biol
168
,
429
437
Cesario
M. M.
,
Bartles
J. R.
(
1994
).
Compartmentalization, processing and redistribution of the plasma membrane protein CE9 on rodent spermatozoa.
J. Cell Sci
107
,
561
570
Cesario
M. M.
,
Enstrud
K.
,
Hamilton
D. W.
,
Bartles
J. R.
(
1995
).
Biogenesis of the posterior-tail plasma membrane domain of the mammalian spermatozoon: targeting and lateral redistribution of the posterior-tail domain specific transmembrane protein CE9 during spermiogenesis.
Dev. Biol
169
,
473
486
Cowen
A. E.
,
Primakoff
P.
,
Myles
D. G.
(
1986
).
Sperm exocytosis increases the amount of PH-20 antigen on the surface of guinea pig sperm.
J. Cell Biol
103
,
1289
1297
Cowen
A. E.
,
Myles
D. G.
(
1993
).
Biogenesis of surface domains during spermiogenesis in the guinea pig.
Dev. Biol
155
,
124
133
Dubray
G.
,
Bezard
G.
(
1982
).
A highly sensitive periodic acid-silver stain for 1,2-diol groups of glycoproteins and polysaccharides in polyacrylamide gels.
Anal. Biochem
119
,
325
329
Gaunt
S. J.
,
Brown
C. R.
,
Jones
R.
(
1983
).
Identification of mobile and fixed antigens on the plasma membrane of rat spermatozoa using monoclonal antibodies.
Exp. Cell Res
144
,
275
284
Giulian
G. G.
,
Moss
R. L.
,
Greaser
M.
(
1983
).
Improved methodology for analysis and quantitation of proteins on one-dimensional silver-stained slab gels.
Anal. Biochem
129
,
277
287
Hecht
N. B.
(
1986
).
Regulation of haploid expressed genes in male germ cells.
J. Reprod. Fert
88
,
679
693
Huarte
J.
,
Belin
D.
,
Bosco
D.
,
Sappino
A. P.
,
Vassalli
J. D.
(
1987
).
Plasminogen activator and mouse spermatozoa: urokinase synthesis in the male genital tract and binding of the enzyme to the sperm surface.
J. Cell Biol
104
,
1381
1289
Humphries
S. E.
,
Whittall
R.
,
Minty
A.
,
Buckingham
M.
,
Williamson
R.
(
1981
).
There are approximately 20 actin genes in the human gernome.
Nucl. Acids Res
9
,
4895
4908
Jones
R.
(
1978
).
Comparative biochemistry of mammalian epididymal plasma.
Comp. Biochem. Phys
61
,
365
370
Jones
R.
,
Brown
C. R.
,
von Glos
K. I.
,
Gaunt
S. J.
(
1985
).
Development of a maturation antigen on the plasma membrane of rat spermatozoa in the epididymis.
Exp. Cell Res
156
,
31
44
Jones
R.
(
1986
).
Preparation of a plasma membrane-rich fraction from rat spermatozoa.
J. Reprod. Fert
77
,
435
449
Jones
R.
,
Shalgi
R.
,
Hoyland
J.
,
Phillips
D. M.
(
1990
).
Topographical rearrangement of a plasma membrane antigen during capacitation of rat spermatozoa in vitro.
Dev. Biol
139
,
349
369
Koehler
J. K.
(
1978
).
The mammalian sperm surface: studies with specific labelling techniques.
Int. Rev. Cytol
54
,
73
108
Laemmli
U. K.
(
1970
).
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227
,
680
685
Lathrop
W. F.
,
Carmichael
E. P.
,
Myles
D. G.
,
Primakoff
P.
(
1990
).
cDNA cloning reveals the molecular structure of a sperm surface protein, PH-20, involved in sperm-egg adhesion and the wide distribution of its gene among mammals.
J. Cell Biol
111
,
939
2949
Lin
Y.
,
Mahan
K.
,
Lathrop
W. F.
,
Myles
D. G.
,
Primakoff
P.
(
1994
).
A hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus cell layer surrounding the egg.
J. Cell Biol
125
,
1157
1163
Lisanti
M. P.
,
Rodriguez-Boulan
E.
(
1989
).
Glycophospholipid membrane anchoring provides clues to the mechanism of protein sorting in polarised epithelial cells.
Trends Biochem. Sci
15
,
113
118
Myles
D. G.
,
Primakoff
P.
(
1984
).
Localized surface antigens of guinea pig sperm migrate to new regions prior to fertilization.
J. Cell Biol
99
,
1634
1641
Myles
D. G.
,
Kimmel
L. H.
,
Blobel
C. P.
,
White
J. M.
,
Primakoff
P.
(
1994
).
Identification of a binding site in the disintegrin domain of fertilin required for sperm-egg fusion.
Proc. Nat. Acad. Sci. USA
91
,
4195
4198
Nehme
C. L.
,
Cesario
M. M.
,
Myles
D. G.
,
Koppel
D. E.
,
Bartles
J. D.
(
1993
).
Breaching the diffusion barrier that compartmentalizes the transmembrane glycoprotein CE9 to the posterior-tail plasma membrane domain of the rat spermatozoon.
J. Cell Biol
120
,
687
694
O'Farrell
P. H.
(
1974
).
High resolution two-dimensional electrophoresis of proteins.
J. Biol. Chem
250
,
4007
4021
Perry
A. C. F.
,
Jones
R.
,
Hall
L.
(
1993
).
Sequence analysis of monkey acrosin-trypsin inhibitor transcripts and their abundant expression in the epididymis.
Biochim. Biophys. Acta
1172
,
159
160
Perry
A. C. F.
,
Gichuhi
P. M.
,
Jones
R.
,
Hall
L.
(
1995
).
Cloning and analysis of monkey fertilin reveals novel subunit isoforms.
Biochem. J
307
,
843
850
Petruszak
J. A. M.
,
Nehme
C. L.
,
Bartles
J. R.
(
1991
).
Endoproteolytic cleavage in the extracellular domain of the integral plasma membrane protein CE9 precedes its redistribution from the posterior to the anterior tail of the rat spermatozoon during epididymal maturation.
J. Cell Biol
115
,
917
927
Phelps
B. M.
,
Koppel
D. E.
,
Primakoff
P.
,
Myles
D. G.
(
1990
).
Evidence that proteolysis of the surface is an initial step in the mechanism of formation of sperm cell surface domains.
J. Cell Biol
111
,
1839
1847
Primakoff
P.
,
Myles
D. G.
(
1983
).
A map of the guinea pig sperm surface constructed with monoclonal antibodies.
Dev. Biol
98
,
417
428
Primakoff
P.
,
Hyatt
H.
,
Tredick-Kline
J.
(
1987
).
Identification and purification of a sperm surface protein with a potential role in sperm-egg membrane fusion.
J. Cell Biol
104
,
141
149
Scully
N. F.
,
Shafer
J. H.
,
Shur
B. D.
(
1987
).
Spatial and temporal expression of cell surface galactosyltransferase during mouse spermatogenesis and epididymal maturation.
Dev. Biol
124
,
111
124
Shalgi
R.
,
Matityahu
A.
,
Gaunt
S. J.
,
Jones
R.
(
1990
).
Identification of antigens on rat spermatozoa with a potential role in fertilization.
Mol. Reprod. Dev
25
,
286
296
Vanha-Perttula
T.
,
Mather
J. P.
,
Bardin
C. W.
,
Moss
S. B.
,
Bellve
A. R.
(
1985
).
Localization of the angiotensin-converting enzyme activity in testis and epididymis.
Biol. Reprod
33
,
870
877
Yang
Z.
,
Wreford
N. G.
,
de Kretser
D. M.
(
1990
).
A quantitative study of spermatogenesis in the developing rat testis.
Biol. Reprod
43
,
629
635
Yang
B.
,
Yang
B. L.
,
Savani
R. C.
,
Turley
E. A.
(
1994
).
Identification of a common hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein.
EMBO J
13
,
286
296
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