Fertilization in the mouse is initiated by sperm beta 1,4-galactosyltransferase (GalTase) binding to terminal N-acetylglucosamine residues on the zona pellucida glycoprotein ZP3. Binding of ZP3 induces exocytosis of the sperm acrosome, whose contents are believed to digest a penetration slit in the zona matrix through which sperm reach the egg. As a consequence of acrosomal exocytosis, GalTase is redistributed to the lateral aspect of the sperm head, where its function remains unknown. In this location, GalTase could conceivably impede zona penetration by binding to N-acetylglucosamine residues exposed on zona pellucida glycoproteins. Therefore, in this study we investigated the presence and function of acrosomal glycosidases capable of removing the GalTase-binding site from zona pellucida glycoproteins. beta-N-acetylglucosaminidase was found at very high levels in sperm, being more than 20-fold higher than other glycosidases assayed. The specific isozymic variant was identified as beta-hexosaminidase B. beta-N-acetylglucosaminidase was localized to sperm acrosomes by biochemical and indirect immunofluorescence studies and was released during the acrosome reaction, as expected for an enzyme involved in zona penetration. To determine if, in fact, acrosomal beta-N-acetylglucosaminidase facilitated penetration through the zona, an assay was developed using eggs that were rendered incapable of triggering the block to polyspermy. A specific competitive inhibitor of beta-N-acetylglucosaminidase activity, PUGNAC, inhibited sperm penetration of the zona in a dose-dependent manner, whereas a closely related beta-glucosidase inhibitor, PUGLU, had no effect on zona penetration or on beta-N-acetylglucosaminidase activity. Neither glycosidase inhibitor affected sperm motility or induction of the acrosome reaction. These results demonstrate that beta-N-acetylglucosaminidase is found in sperm acrosomes and is released during the acrosome reaction, at which time it facilitates sperm penetration through the zona. These results also imply that sperm have developed mechanisms to prevent the formation of stable interactions between surface receptors and their zona pellucida ligands during penetration.

Reference

Anakwe
O. O.
,
Sharma
S.
,
Hardy
D. M.
,
Gerton
G. L.
(
1991
)
Guinea pig proacrosin is synthesized principally by round spermatids and contains O -linked as well as N -linked oligosaccharide side chains.
Mol. Reprod. Dev
29
,
172
179
Bapat
B.
,
Ethier
M.
,
Neote
K.
,
Mahuran
D.
,
Gravel
R. A.
(
1988
)
Cloning and sequence analysis of a cDNA encoding the-subunit of mouse -hexosaminidase.
FEBS Lett
237
,
191
195
Beccari
T.
,
Hoade
J.
,
Orlacchio
A.
,
Stirling
J. L.
(
1992
)
Cloning and sequence analysis of a cDNA encoding the-subunit of mouse - N -acetylhexosaminidase and comparison with the human enzyme.
Biochem. J
285
,
593
596
Beer
D.
,
Vasella
A.
(
1985
)
Aldonhydroximo-lactones. Preparation and determination of configuration.
Helv. Chim. Acta
68
,
2254
2274
Ben-Yoseph
Y.
,
Reid
J. E.
,
Shapiro
B.
,
Nadler
H. L.
(
1985
)
Diagnosis and carrier detection of Tay-Sachs disease: direct determination of hexosaminidase A using 4-methylumbelliferyl derivatives of-N-acetylglucosamine-6-sulfate and -N-acetylgalactosamine-6-sulfate.
Am. J. Hum.Genet
37
,
733
748
Bleil
J. D.
,
Greve
J. M.
,
Wassarman
P. M.
(
1988
)
Identification of a secondary sperm receptor in the mouse egg zona pellucida, role in maintenance of binding of acrosome-reacted sperm to eggs.
Dev. Biol
128
,
376
385
Bleil
J. D.
,
Wassarman
P. M.
(
1980
)
Mammalian sperm-egg interaction: Identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm.
Cell
20
,
873
882
Bleil
J. D.
,
Wassarman
P. M.
(
1986
)
Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm.
J. Cell Biol
102
,
1363
1371
Bleil
J. D.
,
Wassarman
P. M.
(
1990
)
Identification of a ZP3-binding protein on acrosome-intact mouse sperm by photoaffinity crosslinking.
Proc. Natl. Acad. Sci. USA
87
,
5563
5567
Boatman
D. E.
,
Andrews
J. C.
,
Bavister
B. D.
(
1988
)
A quantitative assay for capacitation, evaluation of multiple sperm penetration through the zona pellucida of salt-stored hamster eggs.
Gamete Res
19
,
19
29
de Vries
J. W. A.
,
Willemson
R.
,
Geuze
H. J.
(
1985
)
Immunocytochemical localization of acrosin and hyaluronidase in epididymal and ejaculated porcine spermatozoa.
Eur. J. Cell Biol
37
,
81
88
Endo
Y.
,
Lee
M. A.
,
Kopf
G. S.
(
1988
)
Characterization of an islet-activating protein-sensitive site in mouse sperm that is involved in the zona pellucida-induced acrosome reaction.
Dev. Biol
129
,
12
24
Farooqui
A.
,
Srivastava
P. N.
(
1980
)
Isolation of- N -acetylhexosaminidase from rabbit semen and its role in fertilization.
Biochem. J
191
,
827
834
Florman
H. M.
,
Bechtol
K. B.
,
Wassarman
P. M.
(
1984
)
Enzymatic dissection of the functions of the mouse egg's receptor function for sperm.
Dev. Biol
106
,
243
255
Godknecht
A.
,
Honegger
T. G.
(
1991
)
Isolation, characterization, and localization of a sperm-bound N -acetylglucosaminidase that is indispensable for fertilization in the ascidian, Phallusia mammillata.
Dev. Biol
143
,
398
407
Goldstein
I. J.
,
Hughes
R. C.
,
Monsigny
M.
,
Osawa
T.
,
Sharon
N.
(
1980
)
What should be called a lectin.
Nature
285
,
66
–.
Jones
R.
,
Brown
C. R.
,
Lancaster
R. T.
(
1988
)
Carbohydrate-binding properties of boar sperm proacrosin and assessment of its role in sperm-egg recognition and adhesion during fertilization.
Development
102
,
781
792
Kaback
M. M.
(
1972
)
Thermal fractionation of serum hexosaminidases: applications to heterozygote detection and diagnosis of Tay Sach's disease.
Meth. Enzymol
28
,
862
867
Kopecny
V.
,
Flechon
J.-E.
(
1981
)
Fate of acrosomal glycoproteins during the acrosome reaction and fertilization, a light and electron microscope autoradiographic study.
Biol. Reprod
24
,
201
216
Kytzia
H.-J.
,
Sandhoff
K.
(
1985
)
Evidence for two different active sites on human-hexosaminidase A: interaction of G m2activator protein with -hexosamindase A.
J. Biol. Chem
260
,
7568
7572
Lambert
C. C.
(
1989
)
Ascidian eggs release glycosidase activity which aids in the block against polyspermy.
Development
105
,
415
420
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
,
2939
2949
Leyton
L.
,
Saling
P.
(
1989
)
95 kd sperm proteins bind ZP3 and serve as tyrosine kinase substrates in response to zona binding.
Cell
57
,
1123
1130
Leyton
L.
,
Saling
P.
(
1989
)
Evidence that aggregation of mouse sperm receptors by ZP3 triggers the acrosome reaction.
J. Cell Biol
108
,
2163
2168
Liotta
L. A.
,
Rao
C. N.
,
Wewer
U. M.
(
1986
)
Biochemical interactions of tumor cells with the basement membrane.
Annu. Rev. Biochem
55
,
1037
1057
Liu
D. Y.
,
Baker
H. W. G.
(
1993
)
Inhibition of acrosin activity with a trypsin inhibitor blocks human sperm penetration of the zona pellucida.
Biol. Reprod
48
,
340
348
Llanos
M.
,
Vigil
P.
,
Salgado
A. M.
,
Morales
P.
(
1993
)
Inhibition of the acrosome reaction by trypsin inhibitors and prevention of penetration of spermatozoa through the human zona pellucida.
J. Reprod. Fert
97
,
173
178
Lopez
L. C.
,
Shur
B. D.
(
1987
)
Redistribution of mouse sperm galactosyltransferase after the acrosome reaction.
J. Cell Biol
105
,
1663
1670
Macek
M. B.
,
Lopez
L. C.
,
Shur
B. D.
(
1991
)
Aggregation of-1,4-galactosyltransferase on mouse sperm induces the acrosome reaction.
Dev. Biol
147
,
440
444
Mahuran
D. J.
(
1991
)
The biochemistry of HEXA and HEXB gene mutations causing Gm2 gangliosidosis.
Biochim. Biophys. Acta
1096
,
87
94
Majumber
G. C.
,
Turkington
R. W.
(
1974
)
Acrosomal and lysosomal isoenzymes of-galactosidase and N -acetyl--glucosaminidase in rat testis.
Biochemistry
13
,
2857
2864
Miller
D. J.
,
Macek
M. B.
,
Shur
B. D.
(
1992
)
Complementarity between sperm surface-1,4-galactosyltransferase and egg-coat ZP3 mediates sperm-egg binding.
Nature
357
,
589
593
Moller
C. C.
,
Bleil
J. D.
,
Kinloch
R. A.
,
Wassarman
P. M.
(
1990
)
Structural and functional relationships between mouse and hamster zona pellucida glycoproteins.
Dev. Biol
137
,
276
286
Mori
E.
,
Takasaki
S.
,
Hedrick
J. L.
,
Wardrip
N. J.
,
Mori
T.
,
Kobata
A.
(
1991
)
Neutral oligosaccharide structures linked to asparagines of porcine zona pellucida glycoproteins.
Biochemistry
30
,
2078
2087
Neill
J. M.
,
Olds-Clarke
P.
(
1988
)
Incubation of mouse sperm with lactate delays capacitation and hyperactivation and lowers fertilization levels in vitro.
Gamete Res
20
,
459
473
Neote
K.
,
Brown
C. A.
,
Mahuran
D. J.
,
Gravel
R. A.
(
1990
)
Translation initiation in the HEXB gene encoding the-subunit of human -hexosaminidase.
J. Biol. Chem
265
,
20799
20806
Proia
R. L.
,
d'Azzo
A.
,
Neufeld
E. F.
(
1984
)
Association of-and -subunits during the biosynthesis of-hexosaminidase in cultured human fibroblasts.
J. Biol. Chem
259
,
3350
3354
Prody
G. A.
,
Greve
L. C.
,
Hedrick
J. L.
(
1985
)
Purification and characterization of an N -acetyl--D-glucosaminidase from cortical granules of Xenopus laevis eggs.
J. Exp. Zool
235
,
335
340
Shur
B. D.
(
1991
)
Cell surface1,4galactosyltransferase: twenty years later.
Glycobiology
1
,
563
575
Shur
B. D.
,
Hall
N. G.
(
1982
)
A role for mouse sperm surface galactosyltransferase in sperm binding to the egg zona pellucida.
J. Cell Biol
95
,
574
579
Shur
B. D.
,
Neely
C. A.
(
1988
)
Plasma membrane association, purification, and partial characterization of mouse sperm1,4-galactosyltransferase.
J. Biol. Chem
263
,
17706
17714
Urch
U. A.
,
Wardrip
N. J.
,
Hedrick
J. L.
(
1985
)
Limited and specific proteolysis of the zona pellucida by acrosin.
J. Exp. Zool
233
,
479
483
Wassarman
P. M.
(
1988
)
Zona pellucida glycoproteins.
Ann. Rev. Biochem
57
,
415
442
This content is only available via PDF.