©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of Zona Pellucida Gene Expression by Antisense Oligonucleotides Injected Into Mouse Oocytes (*)

(Received for publication, September 15, 1994; and in revised form, November 1, 1994)

Zhi-Bin Tong (1)(§) Lawrence M. Nelson (1) Jurrien Dean (2)

From the  (1)Developmental Endocrinology Branch, NICHD and the (2)Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 (^1)(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.


MATERIALS AND METHODS

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(2)). 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 alpha-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(7) and T(3) 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 [alpha-P]UTP (3000 Ci/mmol, ICN) into the transcripts using T(7) RNA polymerase. Unincorporated [alpha-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 times 10^4 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) .

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 times 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.).


RESULTS

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 RNAbulletDNA 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.



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).


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).

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.

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.



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) .

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 alpha-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 alpha-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.

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 alpha-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 alpha-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 alpha-globin mRNA, lane 4. Molecular mass markers (kDa) are indicated on the left.




DISCUSSION

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 (^2)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) , alpha-tubulin(24) , beta-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 (^3)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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Laboratory of Cellular and Developmental Biology, NIDDK, NIH, Bldg. 6, Rm. B1-26, 6 Center Dr., MSC 2715, Bethesda, MD 20892-2715. Tel.: 301-496-2738; Fax: 301-496-5239.

(^1)
The abbreviations used are: ZP, zona pellucida; TAB, Tyrode's acidic buffer; PAGE, polyacrylamide gel electrophoresis; Nt, nucleotide(s).

(^2)
O. Epifano, unpublished observations.

(^3)
A. Ginsberg, unpublished observations.


ACKNOWLEDGEMENTS

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.


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