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(Received for publication, September 15, 1994; and in revised form, November 1, 1994) From the
During murine oogenesis, the zona pellucida proteins (ZP1, ZP2,
and ZP3) are synthesized and secreted to form an extracellular matrix
that surrounds the oocyte and mediates specific biological functions
essential to mammalian fertilization and early development. To
investigate the relationship among the zona proteins during zona matrix
assembly, we have undertaken to inhibit de novo biosynthesis
of specific zona proteins with antisense oligonucleotides complementary
to the 5`-ends of ZP2 (nucleotide position 19-42) and ZP3
(nucleotide 21-44) mRNAs. When injected into the cytoplasm of
growing mouse oocytes, the antisense oligonucleotides targeted specific
zona mRNAs for degradation, as confirmed by a RNase protection assay.
Individual zona pellucida protein synthesis was followed by
immunoprecipitation with ZP2- and ZP3-specific monoclonal antibodies.
New zona protein synthesis from the targeted mRNA was abolished, but
nontargeted zona protein continued to be synthesized. Interestingly,
abolishment of either ZP2 or ZP3 protein synthesis prevented the
incorporation of the other protein into the extracellular zona matrix.
These results suggest that ZP2 and ZP3 proteins are independent of each
other in their biosynthesis but are dependent upon each other for their
incorporation into the zona pellucida matrix. This study provides an
experimental system in which destruction of a targeted mRNA generates a
transient loss-of-expression phenotype during mouse oocyte growth. The zona pellucida is an extracellular matrix that surrounds
mammalian oocytes and mediates initial sperm-egg interactions at
fertilization. The mouse zona is composed of three sulfated
glycoproteins, ZP1 ( The three zona proteins
are assembled into a matrix that first appears in the early stages of
oocyte growth and eventually forms a 7-8-µm-thick coat
surrounding fully grown oocytes, which is distinct from the plasma
membrane. Electron microscopic observations indicate that the zona is a
relatively homogeneous meshwork(5) , the pore size of which
allows the passage of viral particles(6) . It has been proposed
that this mesh is composed of long filaments (1:1 dimers of ZP2 and
ZP3) cross-linked by ZP1, a disulfide-bonded dimeric protein (7) . However, little is known about the molecular mechanism of
zona assembly and the structural relationships between the zona
proteins during this process. There are no reported null mutations
in the mouse zona pellucida genes that would allow an analysis of the
biosynthesis of one zona protein in the absence of another. Therefore,
to examine the influence of each of the zona proteins on the de
novo formation of the zona matrix, we have used antisense
oligonucleotides to target either ZP2 or ZP3 transcripts in growing
mouse oocytes. The specific degradation of either mRNA effectively
abolishes the biosynthesis of the corresponding protein. We find that
the inhibition of either ZP2 or ZP3 protein synthesis prevents the
incorporation of the other protein into the extracellular zona matrix
of growing mouse oocytes.
Alternatively, after the TAB-solubilized
zona proteins were separated by SDS-PAGE, the proteins were transferred
onto a nitrocellulose membrane by electrophoresis at 100 V for 1
h(14) . The blots were rinsed in Tris-buffered saline buffer
(10 mM Tris-HCl, pH 7.4, 140 mM NaCl) supplemented
with 3% bovine serum albumin and incubated with anti-mouse ZP2 and ZP3
monoclonal antibodies (1:1,000) at 4 °C for 2 h. The filter was
washed with Tris-buffered saline buffer containing 0.2% Tween-20 (3
Vertebrate oocytes, as well as most somatic cells, have
highly active endogenous RNase H
activity(8, 15, 16) . The enzyme recognizes
and destroys RNA in a RNA
Figure 1:
ZP2 and ZP3 antisense oligonucleotides
and RNA. The antisense oligonucleotides (above each
transcript) are complementary to the 5`-ends of mouse ZP2 (Nt
19-42) and ZP3 (Nt 21-44) mRNAs. Each oligonucleotide spans
the translation start site of its complementary transcripts. Synthetic
antisense RNA probes complementary to ZP2 (Nt 34-481) and ZP3 (Nt
28-233) shown below each transcript were used to detect
the zona transcripts in RNase protection assays. Coding and noncoding
regions are indicated by hatched and openrectangles, respectively.
Figure 2:
RNase protection assay for detection of
mouse ZP2 and ZP3 mRNAs.
For a further validation, the two probes
were mixed (Fig. 2, lane7) to demonstrate
their ability to target synthetic ZP2 and ZP3 transcripts in a single
sample and their ability to distinguish zona transcripts from
nonspecific RNA (Fig. 2, lane8). Using this
assay, oocyte lysates were then directly hybridized to a mixture of the
two antisense RNA probes, and both ZP2 and ZP3 mRNA could be detected
in as few as 50 oocytes. The specificity of the assay was confirmed by
a comparison of the protected fragments in total RNA isolated from
oocyte with those obtained with synthetic ZP2 and ZP3 mRNAs (Fig. 2, lanes9 and 10).
The RNase protection assay was used to
monitor the degradation of ZP2 and ZP3 mRNA in oocytes injected with
antisense oligonucleotides. Two hours after injection, the targeted ZP2
and ZP3 mRNAs were reduced in size as compared with the protected
fragments of endogenous ZP2 and ZP3 mRNAs in the uninjected oocytes (Fig. 3, lanes2-4). This decrease in size
may represent truncation of the targeted mRNA after 2 h by endogenous
RNase H or the formation of a DNA:RNA duplex (antisense
oligonucleotides/targeted mRNAs) that prevents hybridization of the
3`-end of the RNA probes to the complementary zona transcript (see Fig. 1). In either event, the mRNA would not be available for
translation into protein. The levels of the targeted zona mRNAs
decreased progressively at 4, 6, and 10 h after injection (data not
shown). Although residual amounts of shortened ZP2 were detected even
after 16 h (Fig. 3, lane6), neither full-length
ZP2 nor ZP3 transcripts were detected in oocytes injected with
complementary antisense oligonucleotides (Fig. 3, lanes5-7). Of note is the relative integrity of the
nontargeted zona mRNA under these culture conditions.
Figure 3:
Degradation of zona mRNAs in oocytes
injected with antisense oligonucleotides. Whole oocyte lysates were
hybridized with
In the present study, 50
oocytes were injected with antisense oligonucleotides, allowed to
recover for 2-3 h, and then placed in medium containing
[
Figure 4:
Degradation of zona proteins after
injecting oocytes with antisense oligonucleotides. PanelA, de novo synthesis of zona proteins in oocytes after
16-h culture with
Sixteen hours after injection of 100 oocytes
with antisense oligonucleotides complementary to either ZP2 or ZP3,
zonae pellucidae were isolated with TAB buffer. Half of each sample was
immunoprecipitated with anti-ZP2 and -ZP3 monoclonal antibodies to
detect the newly synthesized zona proteins; the other half was
immunoblotted to confirm the integrity of the zona preparation during
the experimental procedures. Newly synthesized ZP2 and ZP3 proteins
were present in the zona pellucidae of the uninjected and rabbit
Figure 5:
Incorporation of zona proteins into the
zona matrix. PanelA, incorporation of de novo synthesized zona proteins into the zona matrix was detected by
immmunoprecipitation with monoclonal antibodies. Each lane contains immunoprecipitates of solubilized zonae from 50 oocytes
after 16-h culture with
Antisense oligonucleotides microinjected into growing mouse
oocytes can specifically ``knock-out'' ZP2 or ZP3 mRNA, and
the degradation of each can be monitored by a sensitive RNase assay.
The absence of either zona transcript precludes de novo synthesis of the cognate protein and prevents incorporation of
both proteins into the extracellular zona pellucida matrix. These data
indicate that while ZP2 and ZP3 proteins are independently synthesized,
new zona matrix formation is dependent on the coordinate biosynthesis
of both proteins. The role of the recently cloned mouse ZP1 ( At birth, mouse oocytes are normally enclosed in a layer
of flattened granulosa cells surrounded by a basement membrane, forming
units called primordial follicles. At the beginning of follicular
development, granulosa cells become cuboidal and proliferate to form a
stratified epithelium. Concomitant with the onset of granulosa cell
proliferation, the oocyte initiates its own growth, and the zona
pellucida is first observed as extracellular patches that later
coalesce into a uniform matrix surrounding the oocyte. Cytoplasmic
processes from both the oocyte and granulosa cells traverse the zona
matrix providing the basis of oocyte-granulosa cell interactions during
folliculogenesis. Whether the zona pellucida matrix is necessary for
normal follicular development is not known. Oocytes growing in early
follicles are remarkably active both transcriptionally and
translationally. Some mRNAs, including those for histones(23) ,
The
biosynthesis of the zona pellucida involves a series of coordinate
events initiated by the expression of the zona genes and culminating
with the stable formation of an extracellular matrix. Although zona
transcripts are present in low amounts in resting mouse oocytes
(10-15 µm), the abundance of ZP2 and ZP3 mRNA increases
dramatically as oocytes enter their growth phase. In oocytes that are
50 µm in diameter, ZP2 and ZP3 mRNAs represent Morphologic and biochemical evidence
suggests that ZP2:ZP3 dimers participate in an insoluble zona
matrix(7) , although mammalian cell lines, expressing either
recombinant mouse ZP2 (
Volume 270,
Number 2,
Issue of January 13, 1995 pp. 849-853
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)(180-200 kDa), ZP2 (120-140
kDa), and ZP3 (83 kDa)(1, 2) . Each mouse zona
contains 3-5 ng of protein, and approximately 1 µg of zona
can be isolated from a mouse ovary. This paucity of biological material
effectively precludes detailed biochemical analysis of native mouse
zona proteins. However, the primary structure of two mouse zona
proteins has been deduced from full-length cDNAs of the cognate
genes(3, 4) . ZP2 contains 713 amino acids (80,219
Da), and ZP3 contains 424 amino acids (46,307 Da). Each protein has a
signal peptide to direct it into a secretory pathway where it undergoes
posttranslational glycosylation; each has a 20-30-amino acid
hydrophobic domain near its carboxyl terminus capable of forming a
transmembrane domain. Their primary structures suggest little, if any,
other similarity between the two proteins.
Isolation and Culture of Growing Mouse
Oocytes
Growing mouse oocytes were dissected from ovaries of
14-day-old NIH Swiss mice in HEPES-modified Brinster's media
containing bovine serum albumin (0.1 mg/ml) and sodium pyruvate (30
µg/ml). 45-50-µm diameter oocytes, collected by
micropipette, were washed with M199 media and transferred into complete
M199-M media (Earle's modified medium 199, 2 mg/ml bovine serum
albumin, 30 µg/ml sodium pyruvate (Life Technologies, Inc.)) for
further processing. Approximately 100 oocytes were incubated in 100
µl of complete M199-M media under paraffin oil (37 °C, 5%
CO
). For radiolabeling of protein, oocytes were cultured
for 16 h in methionine- and cysteine-free M199-M media (Special Media
Inc.) supplemented with [S]methionine (517
Ci/mmol, ICN) and [
S]cysteine (1084 Ci/mmol,
ICN) each at a final concentration of 0.5 mCi/ml.
Microinjection of Antisense Oligonucleotides into
Oocytes
Antisense oligonucleotides were synthesized (DNA
Synthesizer, Applied Biosystems, model 380B), purified on 6% denaturing
polyacrylamide gels, and dissolved in phosphate-buffered saline (1
µg/µl). Three antisense oligonucleotides (24 Nt) were utilized
for microinjection: 5`-CACCAGCAGGCAGTGGGACAGGAG-3`, complementary to
rabbit 
-globin mRNA (Nt 330-354)(8) ;
5`-CCACCTCGCCATGTTGGAAGGTAC-3`, complementary to mouse ZP2 mRNA (Nt
19-42)(3) ; and 5`-ATAGCTTGACGCCATGGTCCCGCC-3`,
complementary to mouse ZP3 (Nt 21-44)(4) . Approximately
10 pg of each oligonucleotide (10 pl) were injected into the cytoplasm
of individual oocytes with a microinjector (Eppendorf, model 5242).
After injection, the oocytes were washed twice with the culture medium
and transferred into fresh complete M199-M medium for further
incubation as described above.Transcription in Vitro
In vitro transcription was performed following the experimental protocol
provided in a RNA transcription kit (Stratagene). Full-length mouse ZP2
and ZP3 cDNAs subcloned into plasmid pBluescript KS II (3, 4) were used as templates. After linearization of
the plasmid, ZP2 and ZP3 mRNAs were synthesized using T
and
T
RNA polymerase, respectively. Synthetic mRNAs were intact
as judged by electrophoresis in denaturing formamide agarose gels.
Templates for antisense RNA were obtained by subcloning ZP2
(34-481 base pairs) and ZP3 (28-233 base pairs) into
pBluescript KS II. After linearization, antisense ZP2 and ZP3 RNA
probes were prepared by incorporation of
[-P]UTP (3000 Ci/mmol, ICN) into the
transcripts using T
RNA polymerase. Unincorporated
[-P]UTP was removed by precipitation of RNA
probes with 0.5 M sodium acetate and 70% ethanol. The ZP2 (503
Nt) and ZP3 (257 Nt) probes contain 447 and 205 nucleotides
complementary to their respective transcripts; additional sequences at
the 5`-end of each probe were derived from plasmid sequences.
RNase Protection Assay
A RNase protection assay II
kit (Ambion Inc.) was used according to the manufacturer's
instructions to detect ZP2 and ZP3 transcripts. Fifty oocytes were
dissolved in 10 µl of 5 M guanidine thiocyanate (9) and analyzed immediately or stored at -70 °C.
Antisense RNA probes for ZP2 and ZP3 were diluted (2
10
cpm) with hybridization buffer (80% deionized formamide,
100 mM sodium citrate, pH 6.4, 300 mM sodium acetate,
pH 6.4, 1 mM EDTA) and incubated for 24 h at 42 °C with
synthetic ZP2 and ZP3 mRNAs or oocyte lysates in a final volume of 20
µl. Following digestion with RNase A/RNase T1 (0.5 µg/ml RNase
A and 10 units/ml RNase T1) for 1 h at 37 °C, the samples were
precipitated with RNase inactivation/precipitation mixture. The
protected fragments were separated on a 4.5% denaturing polyacrylamide
gel and detected by autoradiography. Fragments from pUC19 digested with Sau3AI and labeled with [-
P]ATP
(6000 Ci/mmol, ICN) using DNA 5`-end labeling system (Promega) were
used as molecular weight markers.
Preparation of Zona Pellucida Proteins
Zonae
pellucidae were selectively solubilized from the S-labeled
oocytes using Tyrode's acidic buffer (TAB) at pH 2.5 as described
previously (10) . Briefly, oocytes were washed with
phosphate-buffered saline containing 0.4% polyvinylpyrrolidone and
transferred to 10 µl of TAB for microscopic visualization. After 20
s of gentle agitation, the fluid containing solubilized zonae
pellucidae was collected. Zona-free oocytes were dissolved by the
addition of 10 µl of 1% SDS, 0.125 M Tris-HCl, pH 6.8.
After heating (1 h, 60 °C), each sample was diluted with either SDS
electrophoresis buffer or immunoprecipitation buffer as described
below.
Immunoprecipitation and Immunoblotting
Samples of
solubilized zonae pellucidae or zona-free oocytes were diluted 100-fold
in phosphate-buffered saline containing 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and bovine serum albumin (1 mg/ml).
After incubation (4 °C, 2 h) with 10 µl of rat monoclonal
antibodies (2 mg/ml) specific to mouse ZP2 (11) and ZP3 (12) in a total volume of 500 µl, the zona proteins were
precipitated with goat anti-rat IgG conjugated to Sepharose 4B beads
according to the manufacturer's instructions (Zymed Laboratories
Inc.) and analyzed by SDS-PAGE (10% acrylamide, 3.5% bisacrylamide) and
fluorography(13) . 20-min incubations). Using alkaline phosphatase-conjugated
sheep anti-rat IgG antibody as a second antibody (diluted 1:1,000),
mouse ZP2 and ZP3 proteins were visualized with
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium premix
solution according to the manufacturer's instruction (Zymed
Laboratories Inc.).
DNA complex. This property has been
exploited experimentally for targeted destruction of specific mRNAs
hybridized with antisense oligonucleotides. Although antisense
oligonucleotides can inhibit gene expression through multiple
mechanisms, mRNA degradation and prevention of protein translation play
major roles(17, 18) . In this study, we monitored the
integrity of ZP2 and ZP3 mRNAs and the synthesis of zona proteins in
mouse oocytes after injection of antisense oligonucleotides (Fig. 1).
RNase Protection Assay for Detection of ZP2 and ZP3
Transcripts
Individual growing mouse oocytes, 50 µm in
diameter, contain approximately 1 pg of ZP2 and 0.4 pg of ZP3
transcripts(3) . A simple and sensitive RNase protection assay
was developed for detection of both zona mRNAs in a single sample.
Synthetic ZP2 and ZP3 transcripts were used to validate the assay (Fig. 2). A P-labeled antisense RNA probe specific
to ZP2 (Nt 34-481, Fig. 1) was hybridized to full-length
ZP2 or ZP3 RNA, digested by RNase A/T1, separated by PAGE, and detected
by autoradiography. As expected, a 447-Nt portion of the 503-Nt ZP2
probe was protected from digestion in the presence of ZP2 transcripts (Fig. 2, lane2) but not in the presence of
ZP3 transcripts (Fig. 2, lane3). Similarly, a
205-Nt portion of the 257-Nt
P-labeled, antisense ZP3 RNA
probe (Nt 28-233, Fig. 1), was protected from RNase
digestion after hybridization with ZP3 transcripts (Fig. 2, lane6) but not ZP2 transcripts (Fig. 2, lane5).
P-labeled ZP2 (503 Nt) and/or ZP3
probe (257 Nt) were hybridized to oocyte RNA and digested with RNase
A/T1. Protected ZP2 (447 Nt) and ZP3 (205 Nt) fragments were detected
after electrophoresis and radiography. Shown is hybridization with ZP2
probe, lanes 1-3; with ZP3 probe, lanes
4-6; and with both ZP2 and ZP3 probes, lanes
7-10; probes prior to treatment with RNase, lanes 1, 4, and 7; probes hybridized with synthetic ZP2 mRNA (20 ng)
alone, lanes 2 and 5; probes hybridized with
synthetic ZP3 mRNA (20 ng) alone, lanes 3 and 6. Both
probes hybridized with yeast tRNA (5 ng), lane 8; with
synthetic ZP2 and ZP3 mRNAs (20 ng each), lane 9; and with
total RNA from 50 oocytes, lane 10. Fragments of pUC19,
digested with Sau3AI and labeled with
[
-
P]ATP, were used as molecular weight
markers, lane M.
Degradation of Zona Pellucida Transcripts by Antisense
Oligonucleotides in Mouse Oocytes
Antisense oligonucleotides (24
Nt) complementary to the 5`-region of mouse ZP2 mRNA (Nt 19-42)
and ZP3 mRNA (Nt 21-44) were designed to include noncoding and
coding regions (Fig. 1). To maintain effective concentrations of
antisense oligonucleotides and ensure the complete destruction of
targeted endogenous zona transcripts, 10 pg of antisense
oligonucleotide(s) were injected into the cytoplasm of each oocyte.
This represents at least a 1000-fold molar excess of oligonucleotide to
targeted ZP2 and ZP3 mRNA.
P-labeled ZP2 and ZP3 antisense RNA probes
and digested with RNase A/T1 at 2 h (lanes 2-4) and 16 h (lanes 5-7) after injection. Probes alone, lane
1; 50 uninjected oocytes, lanes 2 and 5; 50
oocytes injected with ZP2 antisense oligonucleotides, lanes 3 and 6; 50 oocytes injected with ZP3 antisense
oligonucleotides, lanes 4 and 7. LaneM, molecular weight markers.
Inhibition of Targeted Zona Protein Biosynthesis in
Growing Mouse Oocytes
Mouse oocytes cultured in vitro synthesize and secrete ZP2 (120 kDa) and ZP3 (
83 kDa)
proteins containing complex-type
oligosaccharides(10, 19) . ZP2 is synthesized as an
81-kDa polypeptide chain to which six high mannose-type
oligosaccharides are added giving rise to a 91-kDa intermediate. ZP3 is
synthesized as a 44-kDa polypeptide chain to which three or four high
mannose-type oligosaccharides are added to form 53- and 56-kDa
intermediates, respectively. Most or all of the N-linked
oligosaccharides are then processed to complex-type glycans prior to
secretion of the mature glycoproteins into the zona
pellucida(20, 21) .
S]methionine/cysteine until 16 h after the
initial injection. The overall SDS-PAGE profiles of
S-labeled proteins from uninjected oocytes or oocytes
injected with antisense specific to ZP2 or ZP3 mRNA appear to be the
same (Fig. 4A). To examine newly synthesized,
intracellular zona proteins, the zona pellucida was removed with TAB,
and whole cell lysates were immunoprecipitated with monoclonal
antibodies specific to ZP2 and ZP3 (Fig. 4B). Both ZP2
(average, 80 kDa) and ZP3 (average, 55 kDa) precursors were present in
the cytoplasm of the uninjected and rabbit
-globin antisense
oligonucleotide-injected oocytes (Fig. 4B, lanes2 and 3). The biosynthesis of either ZP2 or ZP3
could be specifically inhibited in oocytes injected with antisense
oligonucleotides complementary to ZP2 or ZP3 (Fig. 4B, lanes4 and 5). In each experiment, the
nontargeted zona protein continued to be synthesized. Thus, each zona
protein appears to be synthesized independent of the other in the
oocyte.
S-labeled methionine and cysteine. Whole
cell lysates from 25 oocytes were solubilized in SDS-PAGE sample
buffer: uninjected oocytes, lane 1; oocytes injected with
antisense oligonucleotide specific to ZP2, lane 2; oocytes
injected with antisense oligonucleotide specific to ZP3 mRNA, lane
3. Molecular mass markers (kDa) are indicated on the left. PanelB, immunoprecipitation of newly
synthesized ZP2 and ZP3 proteins in oocytes. Each lane contains
immunoprecipitates of 50 zona-free oocytes after 16-h culture with
S-labeled methionine and cysteine: uninjected oocytes with
no monoclonal antibody added to immunoprecipitation reaction, lane
1; uninjected oocytes with monoclonal antibodies specific to ZP2
and ZP3, lane 2; same as lane2 but using
oocytes injected with antisense oligonucleotide specific rabbit
-globin mRNA, lane 3; same as lane2 but using oocytes injected with antisense oligonucleotide specific
to ZP2, lane 4; same as lane2 but using
oocytes injected with antisense oligonucleotide specific to ZP3, lane 5. Molecular mass markers (kDa) are indicated on the left.
Co-dependence of ZP2 and ZP3 for Incorporation into the
Zona Matrix
Once synthesized, zona proteins are normally
secreted and assembled into an acellular matrix that surrounds growing
oocytes(2, 10) . To determine whether the assembly of
the zona matrix was affected by preventing de novo synthesis
of either ZP2 or ZP3, the zona pellucida surrounding the cultured
oocytes was analyzed.-globin oligonucleotide-injected oocytes (Fig. 5A, lanes3 and 6). However, little, if any, ZP2
or ZP3 protein was detected in zonae pellucidae isolated from the
oocytes injected with either ZP2 or ZP3 antisense oligonucleotides (Fig. 5A, lanes4 and 5).
Thus, both proteins must be efficiently synthesized to add additional
zona proteins to a preexisting zona matrix. To confirm the integrity of
the zona pellucida, solubilized zonae from uninjected oocytes or
oocytes injected with either ZP2 or ZP3 or globin antisense
oligonucleotides were immunoblotted. Intact ZP2 and ZP3 proteins were
detected in all samples. Even though the signal obtained with ZP3 was
less than ZP2 (reflecting both lower amounts of ZP3 and the lower
affinity of the antibody), the degree of ZP2 and ZP3 immunostaining was
constant among the four samples (Fig. 5B, lanes1-4).
S-labeled methionine and cysteine:
uninjected oocytes, no monoclonal antibodies added, lane 1;
uninjected oocytes, no second antibody added, lane 2;
uninjected oocytes with monoclonal antibodies specific to ZP2 and ZP3, lane 3; same as lane3 but using oocytes
injected with antisense oligonucleotide specific to ZP2, lane
4; same as lane3 but using oocytes injected
with antisense oligonucleotide specific to ZP3, lane 5; same
as lane3 but using oocytes injected with antisense
oligonucleotide specific to rabbit
-globin mRNA, lane 6.
Molecular mass markers (kDa) are indicated on the left. PanelB, immunoblot probed with monoclonal antibodies
specific to ZP2 and ZP3. Each lane contains solubilized zonae from 50
oocytes after 16-h culture with S-labeled methionine and
cysteine: uninjected oocytes, lane 1; oocytes injected with
antisense oligonucleotide specific to ZP2, lane 2; oocytes
injected with antisense oligonucleotide specific to ZP3, lane
3; oocytes injected with antisense oligonucleotide specific to
rabbit
-globin mRNA, lane 4. Molecular mass markers (kDa)
are indicated on the left.
)in assembling the zona pellucida remains to be determined.
However, if we assume that ZP1 is synthesized normally in cultured
oocytes, our data suggests that its synthesis is not sufficient for the
incorporation of either ZP2 or ZP3 into the zona matrix. Although these
results may be particular to the accretion of zona proteins in a
preexisting zona matrix (e.g. after the commencement of oocyte
growth), they raise the possibility that the absence of either ZP2 or
ZP3 in early oocytes would preclude in vivo zona pellucida
formation.-tubulin(24) , 
-actin(25) , lactate
dehydrogenase (26) , heat-shock protein 68(27) , and
zona pellucida proteins(3, 4) , are directly
translated into proteins during oocyte growth. Other mRNAs,
hypoxanthine phosphoribosyltransferase(24) , proto-oncogene mos(28) , tissue plasminogen activator(29) ,
and OM-1 and OM-2(30) , are stored in a stable untranslated
form. In general, these dormant mRNAs have a short poly(A) tail of
approximately 15-90 residues. When translational activation
occurs (after meiotic maturation), the tail is elongated by cytoplasmic
polyadenylation (29, 30, 31) . Both antisense
RNA and deoxyoligonucleotides have been injected into growing oocytes
to cause degradation of specific maternal transcripts including those
encoding tissue plasminogen activator(32) , OM-1 (30) and c-mos(16, 33) .1.4% of total
poly(A)
RNA. As the oocyte reaches its full size
(75-80 µm), the amount of ZP2 and ZP3 transcripts declines,
and, in ovulated eggs, the abundance of these two transcripts is less
than 5% of their peak levels(3, 34) . This profile is
very similar to the pattern of de novo biosynthesis of the
zona proteins, which is coordinately regulated during the initial
growth phase and then declines in the latter stages of oocyte growth;
no zona protein synthesis is detected in ovulated
eggs(2, 10) .
)or ZP3 (22, 35) cDNA, secrete soluble zona protein. Precursor
ZP2 and ZP3 proteins contain a signal peptide that directs them into a
secretory pathway, and both undergo posttranslational modifications.
While it is not known if the two proteins complex with one another in
the secretory pathway, at the cell surface, or in the extracellular
space, our data suggest that only the ZP2:ZP3 complex (by itself or
with the addition of ZP1) can participate in zona formation.
)))
These investigations were undertaken in the Laboratory
of Cellular and Developmental Biology, NIDDK, National Institutes of
Health. We thank our colleagues in both laboratories for constructive
suggestions and discussion during this study, and we appreciate the
critical reading of the manuscript by Drs. Robert McIsaac, Ann
Ginsberg, and Robert Simpson.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 P. Wassarman, C Liu, and E. Litscher Constructing the mammalian egg zona pellucida: some new pieces of an old puzzle J. Cell Sci., January 8, 1996; 109(8): 2001 - 2004. [PDF]
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 H. Qi, Z. Williams, and P. M. Wassarman Secretion and Assembly of Zona Pellucida Glycoproteins by Growing Mouse Oocytes Microinjected with Epitope-tagged cDNAs for mZP2 and mZP3 Mol. Biol. Cell, February 1, 2002; 13(2): 530 - 541. [Abstract] [Full Text] [PDF]
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