(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 ()(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.
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
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.).
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 RNADNA 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).
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. 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.
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 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.
In the present study, 50
oocytes were injected with antisense oligonucleotides, allowed to
recover for 2-3 h, and then placed in medium containing
[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.
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 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.
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
-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).
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 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.
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 ()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.
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) ,
-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) .
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 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) .
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 ()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.