Mammalian oocytes synthesize and secrete a zona pellucida that surrounds the growing oocytes, ovulated eggs and preimplantation embryos. The extracellular zona matrix is composed of three glycoproteins (ZP1, ZP2, ZP3) that are involved in folliculogenesis, species-specific fertilization, and passage of the early embryo down the oviduct. We have established a mouse line in which Zp3 has been inactivated by homologous recombination with an insertional mutation. Neither Zp3 transcripts nor ZP3 protein was detected in female mice homozygous for the mutation (Zp3−/−), whereas both ZP1 and ZP2 were present in mutant oocytes. Homozygous mutant Zp3−/− mice had follicles with germinal-vesicle-intact oocytes but that lacked a zona pellucida matrix and had a disorganized corona radiata. Although mutant oocytes underwent germinal vesicle breakdown (GVBD) prior to ovulation, the cumulus-oocyte complex was markedly disrupted and the oocytes were often separate from the cumulus cells. After hormone-induced ovulation, cumulus masses were present in the oviducts of homozygous mutant mice, but zona-free eggs were observed in only half of the females and, in these, less than 10% of the normal number [correction of mumber] of eggs were detected. No zona-free 2-cell embryos were recovered from homozygous mutant Zp3−/− female mice after mating with males proven to be fertile, and none became visibly pregnant or produced offspring. These results demonstrate that a genetic defect in a zona pellucida gene causes infertility and, given the conserved nature of the zona pellucida, a similar phenotype is expected in other mammals.

Reference

Accili
D.
,
Drago
J.
,
Lee
E. J.
,
Johnson
M. D.
,
Cool
M. H.
,
Salvatore
P.
,
Asico
L. D.
,
Jose
P. A.
,
Taylor
S. I.
,
Westphal
H.
(
1996
)
Early neonatal death in mice homozygous for a null allele of the insulin receptor gene.
Nat. Genet
12
,
106
109
Anderson
E.
,
Albertini
D. F.
(
1976
)
Gap junctions between the oocyte and companion follicle cells in the mammalian ovary.
J. Cell Biol
71
,
680
686
Bleil
J. D.
,
Beall
C. F.
,
Wassarman
P. M.
(
1981
)
Mammalian sperm-egg interaction: fertilization of mouse eggs triggers modification of the major zona pellucida glycoprotein, ZP2.
Dev. Biol
86
,
189
197
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.
(
1980
)
Structure and function of the zona pellucida: identification and characterization of the proteins of the mouse oocyte's zona pellucida.
Dev. Biol
76
,
185
202
Bleil
J. D.
,
Wassarman
P. M.
(
1980
)
Synthesis of zona pellucida proteins by denuded and follicle-enclosed mouse oocytes during culture in vitro.
Proc. Natl. Acad. Sci. USA
77
,
10291033
–.
Braden
A. W. H.
,
Austin
C. R.
(
1954
)
The number of sperms about the eggs in mammals and its significance for normal fertilization.
Aust. J. Biol. Sci
7
,
543
551
Brambell
F. W. R.
(
1928
)
The development and morphology of the gonads of the mouse. Part III. The growth of the follicles.
Proc. R. Soc. Lond. [Biol.]
103
,
258
272
Bronson
R. A.
,
McLaren
A.
(
1970
)
Transfer to the mouse oviduct of eggs with and without the zona pellucida.
J. Reprod. Fertil
22
,
129
137
Chamberlin
M. E.
,
Dean
J.
(
1989
)
Genomic organization of a sex specific gene: The primary sperm receptor of the mouse zona pellucida.
Dev. Biol
131
,
207
214
Chamberlin
M. E.
,
Dean
J.
(
1990
)
Human homolog of the mouse sperm receptor.
Proc. Natl. Acad. Sci. USA
87
,
6014
6018
East
I. J.
,
Gulyas
B. J.
,
Dean
J.
(
1985
)
Monoclonal antibodies to the murine zona pellucida protein with sperm receptor activity: Effects on fertilization and early development.
Dev. Biol
109
,
268
273
East
I. J.
,
Dean
J.
(
1984
)
Monoclonal antibodies as probes of the distribution of ZP-2, the major sulfated glycoprotein of the murine zona pellucida.
J. Cell Biol
98
,
795
800
Epifano
O.
,
Liang
L.-F.
,
Dean
J.
(
1995
)
Mouse Zp1 encodes a zona pellucida protein homologous to egg envelope proteins in mammals and fish.
J. Biol. Chem
270
,
27254
27258
Epifano
O.
,
Liang
L.-F.
,
Familari
M.
,
Moos
M. C.
Jr.
,
Dean
J.
(
1995
)
Coordinate expression of the three zona pellucida genes during mouse oogenesis.
Development
121
,
1947
1956
Eppig
J. J.
(
1979
)
A comparison between oocyte growth in coculture with granulosa cells and oocytes with granulosa cell-oocyte junctional contact maintained in vitro.
J. Exp. Zool
209
,
345
353
Eppig
J. J.
(
1991
)
Intercommunication between mammalian oocytes and companion somatic cells.
BioEssays
13
,
569
574
Florman
H. M.
,
Storey
B. T.
(
1982
)
Mouse gamete interactions: The zona pellucida is the site of the acrosome reaction leading to fertilization in vitro.
Dev. Biol
91
,
121
130
Gilula
N. B.
,
Epstein
M. L.
,
Beers
W. H.
(
1978
)
Cell-to-cell communication and ovulation. A study of the cumulus-oocyte complex.
J. Cell Biol
78
,
58
75
Harris
J. D.
,
Hibler
D. W.
,
Fontenot
G. K.
,
Hsu
K. T.
,
Yurewicz
E. C.
,
Sacco
A. G.
(
1994
)
Cloning and characterization of zona pellucida genes and cDNAs from a variety of mammalian species: ZPA, ZPB, and ZPC gene families.
DNA Seq
4
,
361
393
Heller
D. T.
,
Cahill
D. M.
,
Schultz
R. M.
(
1981
)
Biochemical studies of mammalian oogenesis: Metabolic cooperativity between granulosa cells and growing mouse oocytes.
Dev. Biol
84
,
455
464
Johnson
G. D.
,
Nogueira Araujo
G. M.
(
1981
)
A simple method of reducing the fading of immunofluorescence during microscopy.
J Immunol. Methods
43
,
349
350
Kinloch
R. A.
,
Roller
R. J.
,
Fimiani
C. M.
,
Wassarman
D. A.
,
Wassarman
P. M.
(
1988
)
Primary structure of the mouse sperm receptor polypeptide determined by genomic cloning.
Proc. Natl. Acad. Sci. USA
85
,
6409
6413
Li
E.
,
Beard
C.
,
Jaenisch
R.
(
1993
)
Role for DNA methylation in genomic imprinting.
Nature
366
,
362
365
Liang
L.-F.
,
Chamow
S. M.
,
Dean
J.
(
1990
)
Oocyte-specific expression of mouse Zp-2: Developmental regulation of the zona pellucida genes.
Mol. Cell. Biol
10
,
1507
1515
Liang
L.-F.
,
Dean
J.
(
1993
)
Conservation of the mammalian secondary sperm receptor genes results in promoter function of the human homologue in heterologous mouse oocytes.
Dev. Biol
156
,
399
408
Lunsford
R. D.
,
Jenkins
N. A.
,
Kozak
C. A.
,
Liang
L.-F.
,
Silan
C. M.
,
Copeland
N. G.
,
Dean
J.
(
1990
)
Genomic mapping of murine Zp-2 and Zp-3, two oocyte-specific loci encoding zona pellucida proteins.
Genomics
6
,
184
187
Mansour
S. L.
,
Thomas
K. R.
,
Capecchi
M. R.
(
1988
)
Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: A general strategy for targeting mutations to non-selectable genes.
Nature
336
,
348
352
McBurney
M. W.
,
Sutherland
L. C.
,
Adra
C. N.
,
Leclair
B.
,
Rudnicki
M. A.
,
Jardine
K.
(
1991
)
The mouse Pgk-1 gene promoter contains an upstream activator sequence.
Nucl. Acids Res
19
,
5755
5761
Modlinski
J. A.
(
1970
)
The role of the zona pellucida in the development of mouse eggs in vivo.
J. Embryol. Exp. Morphol
23
,
539
547
Naito
K.
,
Toyoda
Y.
,
Yanagimachi
R.
(
1992
)
Production of normal mice from oocytes fertilized and developed without zonae pellucidae.
Hum. Reprod
7
,
281
285
Pavlok
A.
,
McLaren
A.
(
1972
)
The role of cumulus cells and the zona pellucida in fertilization of mouse eggs in vitro.
J. Reprod. Fertil
29
,
91
97
Ringuette
M. J.
,
Chamberlin
M. E.
,
Baur
A. W.
,
Sobieski
D. A.
,
Dean
J.
(
1988
)
Molecular analysis of cDNA coding for ZP3, a sperm binding protein of the mouse zona pellucida.
Dev. Biol
127
,
287
295
Saling
P. M.
,
Sowinski
J.
,
Storey
B. T.
(
1979
)
An ultrastructural study of epididymal mouse spermatozoa binding to zonae pellucidae in vitro: Sequential relationship to the acrosome reaction.
J. Exp. Zool
209
,
229
238
Sato
K.
(
1979
)
Polyspermy-preventing mechanisms in mouse eggs fertilized in vitro.
J. Exp. Zool
210
,
353
359
Schultz
R. M.
,
Wassarman
P. M.
(
1977
)
Biochemical studies of mammalian oogenesis: Protein synthesis during oocyte growth and meiotic maturation in the mouse.
J. Cell Sci
24
,
167
194
Shimizu
S.
,
Tsuji
M.
,
Dean
J.
(
1983
)
In vitro biosynthesis of three sulfated glycoproteins of murine zonae pellucidae by oocytes grown in follicle culture.
J. Biol. Chem
258
,
5858
5863
Steptoe
P. C.
,
Edwards
R. C.
(
1978
)
Birth after replantation of a human embryo.
Lancet
2
,
366
–.
Tong
Z.-B.
,
Nelson
L. M.
,
Dean
J.
(
1995
)
Inhibition of zona pellucidagene expression by antisense oligonucleotides injected into mouse oocytes.
J. Biol. Chem
270
,
849
853
Yurewicz
E. C.
,
Hibler
D.
,
Fontenot
G. K.
,
Sacco
A. G.
,
Harris
J.
(
1993
)
Nucleotide sequence of cDNA encoding ZP3 alpha, a sperm-binding glycoprotein from zona pellucida of pig oocyte.
Biochim. Biophys. Acta
1174
,
211
214
This content is only available via PDF.