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Biology of Reproduction 62, 979-987 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Relationship Between p62 and p56, Two Proteins of the Mammalian Cortical Granule Envelope, and Hyalin, the Major Component of the Echinoderm Hyaline Layer, in Hamsters1

Tanya Hoodbhoya, Edward J. Carroll Jr.3,a, and P. Talbot2,b

a Department of Biology and b Department of Neuroscience, University of California, Riverside, California 92521


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian cortical granules contain two polypeptides (p62 and p56) that are incorporated into the cortical granule envelope after fertilization and function in cleavage of the zygote and the preimplantation blastomeres. Since the echinoderm hyaline layer and mammalian cortical granule envelope are analogous, and since the hyaline layer protein, hyalin, functions in early echinoderm embryogenesis, this study was done to determine whether p62 and p56 and/or other components of the mammalian cortical granule envelope are related to hyalin. A polyclonal antibody (IL2) against purified S. purpuratus hyalin was shown by confocal scanning laser microscopy to bind to hamster cortical granules and to the cortical granule envelope of fertilized hamster oocytes and preimplantation embryos up to the blastocyst stage. In immunoblots, IL2 bound only to 62- and 56-kDa cortical granule proteins that were incorporated into the cortical granule envelope after fertilization. IL2 binding antigens appeared to be resynthesized by preimplantation embryos starting at the 2-cell stage of development. In vivo treatment of 2-cell-stage hamster embryos with IL2 inhibited blastomere cleavage, but treatment of morulae did not inhibit blastocyst implantation. These results support the idea that the mammalian cortical granule envelope proteins, p62/p56, share a common antigenic epitope(s) with echinoderm hyalin, and that p62/p56, like hyalin, play a role in early embryogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cortex of most vertebrate and invertebrate oocytes contains cortical granules that are exocytosed upon fertilization (for review, see [1–3]). Some secreted cortical granule components help establish blocks to polyspermy by modifying oocyte extracellular matrices that bind sperm, e.g., the mammalian zona pellucida, and the echinoderm vitelline and amphibian fertilization layers [4–18]. Other cortical granule components are incorporated into new extracellular matrices that form in the perivitelline space of oocytes after fertilization including the hyaline layer in echinoderms [19] and the cortical granule envelope in mammals and marsupials [20–23]. The hyaline layer has been well characterized and consists of 10–12 polypeptides including a very large glycoprotein known as hyalin [24, 25]. Hyalin is secreted from the cortical granules at fertilization, is later resynthesized and secreted by the echinoderm embryo, and is essential for early echinoderm embryogenesis [19, 26–28]. Hyalin's role in early embryogenesis is mediated by its multiple cell binding domains that allow hyalin to serve as an adhesive substrate for the embryo [26–31]. The interaction of hyalin with the embryonic surface is necessary for morphogenetic movements that occur during early embryogenesis [28]. It is also probable that hyalin participates in other functions ascribed to the hyaline layer itself, including maintenance of the structural integrity of the embryo and the interaction of the blastomeres at the 2-cell stage [32–36].

Constituents of the mammalian cortical granule envelope have recently been characterized [37, 38]. After fertilization, hamster cortical granules secrete at least 12 heavily glycosylated polypeptides that contain the carbohydrates {alpha}-D-mannose, {alpha}-D-methyl-mannopyranoside, galactosyl ß(1,3) N-acetylgalactosamine, N-acetylglucosamine, N-acetylgalactosamine, and/or D-galactose. Nine to ten of these polypeptides become incorporated into the cortical granule envelope and/or zona pellucida after fertilization, and seven to eight of them are still present at the 8-cell stage of preimplantation embryogenesis. Moreover, the binding of a mannose-specific lectin (concanavalin A [Con A]) to the cortical granule envelope of 2-cell-stage hamster embryos inhibits blastomere cleavage, suggesting that one or more mannosylated envelope polypeptide functions in early mammalian embryonic development. A subsequent study showed that two of the hamster cortical granule proteins, p62 and p56, are present in the cortical granules of mouse, rat, pig, and bovine oocytes and are incorporated into the cortical granule envelope after fertilization [37, 38]. Like hyalin, p62 and p56 are also synthesized and secreted by early embryos [39, 40], and these proteins appear to play a role in blastomere cleavage since treatment of fertilized oocytes and cleavage-stage embryos with a polyclonal antibody against p62/p56 inhibits cell division [38–40].

Since p62/p56 and hyalin both become part of postfertilization envelopes in the perivitelline space and both have been implicated in early embryogenesis, the current study was carried out to determine whether these proteins are related. Unfertilized and fertilized hamster oocytes and preimplantation embryos were probed with a polyclonal antibody (IL2) made against purified sea urchin hyalin to determine whether the antibody binds to hamster cortical granules and the cortical granule envelope. Oocyte and preimplantation embryonic proteins were subsequently immunoblotted to establish whether IL2 recognizes p62/p56 and/or other hamster cortical granule components. Finally, 2-cell and late-morula- to blastocyst-stage hamster embryos were treated in vivo with IL2 to determine whether the IL2 binding cortical granule envelope antigens function in mammalian preimplantation embryogenesis and/or in blastocyst hatching and implantation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

Chemicals used to make all culture media, hCG, BSA (fraction V), normal rabbit serum IgG, hyaluronidase, trypsin, paraformaldehyde, Triton X-100, Tween-20, ammonium persulfate, N,N,N',N'-tetramethylethelenediamine (TEMED), polyacrylamide, and 4',6'-diamidino-2-phenylindole (DAPI) were obtained from Sigma Chemical Company (St. Louis, MO). eCG was purchased from Calbiochem (La Jolla, CA). Vectashield mounting medium was obtained from Vector Laboratories (Burlingame, CA). Fluorescein conjugated to goat anti-rabbit IgG was obtained from Miles Laboratory (Elkhart, IN). Square capillary tubes were purchased from In Vitro Dynamics (Rockaway, NJ). Nitrocellulose paper, molecular weight standards, and protein assay kit were obtained from Bio-Rad (Hercules, CA). The enhanced chemiluminescence (ECL) kit was obtained from Amersham Pharmacia Biotech (Piscataway, NJ), and autoradiography film from Du Pont (Boston, MA). Carnation (Los Angeles, CA) nonfat dried milk was purchased from a local supermarket.

Solutions

For dissection and for oocyte and embryo collection for confocal microscopy and SDS-PAGE, Earle's balanced salt solution (EBSS) was made as previously described [41]. For the in vivo studies, Con A conjugated to rhodamine was diluted using Dulbecco's PBS (DPBS), pH 7.4, and the preimplantation embryos and uterine horns were evaluated in 0.1 M PBS, pH 7.3, containing 0.1% BSA. A single-strength working solution of DPBS, pH 7.4, was prepared by dissolving the following salts per liter of deionized water: 0.1 g CaCl2, 0.2 g KCl, 0.2 g KH2PO4, 0.1 g MgCl2·6H2O, 8.0 g NaCl, 2.16 g Na2HPO4. A 0.2 M PBS solution was prepared by mixing 230 ml of solution A (27.6 g of NaH2PO4·H2O in 1 liter of water) with 770 ml of solution B (28.4 g of Na2HPO4 in 1 liter of water). 0.1 M PBS was prepared by dilution, and the pH was adjusted to 7.3. To process oocytes and preimplantation embryos for confocal microscopy, 3.7% paraformaldehyde was made immediately before use in EBSS, pH 7.4. Blocking solution was made fresh by supplementing DPBS, pH 7.4, with 100 mM glycine and 1 mg/ml BSA.

Animals

Golden hamsters (Mesocricetus auratus) were purchased from Harlan Sprague Dawley (San Diego, CA), maintained on a 14L:10D cycle, and fed water and Purina rodent chow (Ralston-Purina, St. Louis, MO) ad libitum. Female hamsters have a 4-day estrous cycle with Day 1 being the day of the vaginal discharge (an external indication of ovulation). Sea urchins (Strongylocentrotus purpuratus) were obtained through the courtesy of Jon Allen at the University of California at Riverside.

Oocyte Collection

Unfertilized follicular oocytes were collected from female hamsters that had received i.p. injections of 25 IU hCG on the evening of Day 3 of the estrous cycle. In some cases, hamsters were superovulated by administering 25 IU of eCG at 1000 h on Day 1 of the estrous cycle, then injecting hCG on Day 3. Unfertilized oviductal oocytes were collected in EBSS/0.5% BSA for confocal laser scanning microscopy (CLSM) or in EBSS/0.1% polyvinylpyrrolidone (PVP) for SDS-PAGE by flushing oviducts with EBSS 14–16 h after hCG administration. To collect in vivo-fertilized oocytes and preimplantation embryos, female hamsters in Day 4 of the estrous cycle were placed in cages containing 1–2 male hamsters. At various times after mating, fertilized oocytes containing two pronuclei and preimplantation embryos up to and including hatched blastocysts were collected in EBSS/0.5% BSA (for CLSM) or in EBSS/0.1% PVP (for SDS-PAGE) by flushing the oviducts with EBSS. Unfertilized and fertilized oocytes were denuded of cumulus cells by incubation in EBSS/0.5% BSA (CLSM) or in EBSS/0.1% PVP (SDS-PAGE) containing 100 IU hyaluronidase for 5 min at room temperature and washed thoroughly with EBSS/0.5% BSA or EBSS/0.1% PVP. Zonae were removed if required by incubating cumulus-free oocytes or preimplantation embryos in EBSS/0.5% BSA or in EBSS/0.1% PVP containing 900 BAEE (N{alpha}-benzoyl-L-arginine ethyl ester) units of bovine pancreatic trypsin for 5–10 min at room temperature.

Sea urchin eggs were obtained by injecting 0.5 M KCl into S. purpuratus and collecting the spawned eggs in a beaker of seawater. The eggs were filtered through a wire mesh to remove debris from the spines. Removal of the jelly layer was accomplished by placing the oocytes in a beaker of sea water containing a pH electrode, and dropping the pH of the water to 5.3 using 1 N HCl. During the dejellying process, which took roughly 2 min, the oocytes were gently stirred with the wide end of a Pasteur pipette; then the pH was raised to 8.0 with 2 N Tris-base. After the eggs settled, the supernatant was gently decanted to remove solubilized jelly, and the eggs were resuspended in an equal volume of fresh seawater. The vitelline envelope was removed by incubating the egg suspension for 10 min, with occasional stirring, with an equal volume of a mixture of 0.1 M Tris-base (unneutralized) and 0.02 M dithiothreitol made up in sea water, pH 9.1. Finally, the eggs were centrifuged briefly at 2000 rpm in a Sorvall (Newtown, CT) centrifuge at 4°C, the supernatant was decanted, and the eggs were resuspended in fresh seawater, pH 8.0.

Confocal Laser Scanning Microscopy

Unfertilized and fertilized oocytes and preimplantation embryos were fixed for 1 h in 3.7% paraformaldehyde in EBSS, pH 7.4, while sea urchin eggs were fixed in 3.7% paraformaldehyde in seawater. After being washed with blocking solution, some oocytes and preimplantation embryos were permeabilized to allow the antibodies to penetrate the oocyte and embryonic plasma membranes by placing them in blocking solution containing 0.1% Triton X-100 for 5 min, and then washed again. Oocytes and preimplantation embryos were incubated for 30 min at room temperature with IL2 antibody diluted either 1:10 or 1:100 with blocking solution. The IL2 antibody, which was made previously in Dr. Ed Carroll's laboratory, recognizes the 11.6s sea urchin hyalin molecule [42]. After being washed thoroughly with blocking solution, oocytes and preimplantation embryos were incubated for 30 min at room temperature with goat anti-rabbit IgG conjugated to fluorescein isothiocyanate (FITC) diluted 1:100 with blocking solution. Control samples were incubated with a 1:100 dilution of preimmune IgG for 30 min at room temperature, followed by anti-rabbit IgG conjugated to FITC for 30 min, or incubated with anti-rabbit IgG conjugated to FITC alone for 30 min. Fixation and antibody labeling steps were done under oil to maintain buffer pH and osmolarity. Oocytes and preimplantation embryos were washed in blocking solution overnight at 4°C.

Oocytes and preimplantation embryos were placed in Vectashield, then transferred into square glass capillary tubes with a wall thickness of 0.1 mm, an inner diameter of 0.2 mm, and a length of 50 mm. The tubes were sealed at the ends with plasticine and secured to glass slides by taping the ends. Optical sections of the oocytes and preimplantation embryos were examined through a Bio-Rad MRC-600 confocal laser scanning microscope to evaluate the intensity and distribution of fluorescence.

Gel Electrophoresis

Mammalian oocytes and preimplantation embryos were solubilized in reducing and denaturing sample buffer at a concentration of approximately four oocytes or preimplantation embryos per microliter. Jelly and vitelline envelope-free sea urchin eggs were solubilized 1:1 (v:v) in double-strength denaturing but nonreducing Laemmli sample buffer. Insoluble sea urchin egg material was removed by centrifugation, and soluble proteins were diluted 1:20 with single-strength Laemmli sample buffer [43]. Oocyte and preimplantation embryonic proteins were separated by one-dimensional SDS-PAGE on a 4% stacking and a 7.5% separating gel according to methods previously described [38, 44]. The electrophoresed proteins were blotted onto nitrocellulose at 100 V for 15 min as previously described [45]. Blots were blocked at room temperature for 1 h, at which time IL2 was added to the blocking solution at a final concentration of 3–15 mg/ml (1:2000–1:10 000 dilution). Control blots were either incubated overnight at 4°C with preimmune IgG or maintained in blocking solution. The immunoblots were detected by ECL using a 1:5000 dilution of horseradish peroxidase (HRP)-conjugated anti-rabbit IgG for 1 h at room temperature according to the manufacturer's instructions. To detect the biotinylated SDS-PAGE standards, the standard lane was incubated in 0.025 mg/ml of HRP-streptavidin for 30 min. The ECL film was developed using a Kodak X-omat automated developer (Eastman Kodak, Rochester, NY).

In Vivo Functional Studies

Female hamsters were mated, and two days after mating, hamsters were anesthetized by i.p. injection of 0.3–0.4 ml of Nembutal (50 mg/ml solution of sodium pentobarbital; Abbott, N. Chicago, IL) delivered gradually. Surgical procedures and injection into the right oviduct were performed using a sterile technique as described previously [46]. A total of 125 or 250 µg of IL2 IgG, 250 µg of preimmune IgG, or 250 µg of the IgG fraction of an antibody against the zona pellucida glycoprotein ZP1 (obtained by courtesy of Dr. Jurrien Dean at National Institutes of Health, Bethesda, MD) in 50 µl of DPBS was injected into the oviduct via the infundibulum using a 1-ml tuberculin syringe fitted with a 30-gauge/1.27-cm needle. The ovary and oviduct were returned to the peritoneal cavity, the incision was sutured and covered with Neosporin, and the animal recovered under a heat-lamp.

The effect(s) of IL2 on blastomere cleavage were evaluated by examining the development of 2-cell preimplantation embryos treated with 250 µg of preimmune IgG, 250 mg of anti-ZP1 IgG, 125 µg of IL2, or 250 µg of IL2 on Day 2 of pregnancy to the 5- to 8-cell stage on Day 3 of pregnancy. The rate of blastomere cleavage in preimplantation embryos developing in the contralateral oviducts served as an internal control for the treated groups. To ensure that IL2 was not having any cytotoxic effects on the preimplantation embryos, the preimplantation embryos were subjected to a trypan blue exclusion assay (0.1% trypan blue in PBS/0.1% BSA for 5 min) to test for embryo viability. In addition, the binding of IL2 to the preimplantation embryos was confirmed by incubating live IL2-treated embryos in a 1:100 dilution of anti-goat IgG conjugated to FITC for 1 h at room temperature, and examining them with a Zeiss epifluorescent microscope (Carl Zeiss, Thornwood, NJ). Digital images were captured with a Spot camera (Diagnostic Instruments, Sterling Heights, MI).

Lastly, the effect of IL2 on implantation was determined by treating morula- to blastocyst-stage preimplantation embryos on the evening of Day 3 of pregnancy with 125 µg of IL2 and flushing the oviducts and uterine horns thoroughly on Day 4 of pregnancy to recover any nonimplanted blastocysts. A failure to find embryos was used as a criterion for implantation since blastocysts normally implant on Day 4 of pregnancy. For both the right and left side of the reproductive tract, the number of corpora lutea was counted to obtain the probable number of preimplantation embryos per uterine horn, and this number was compared to the number of recovered embryos to obtain the percentage of unimplanted embryos. This percentage was subtracted from 100 to obtain the percentage of implanted embryos.

Digital Images

All digital images were processed using PhotoImpact (Ulead, Torrance, CA) or Adobe Photoshop (Adobe Systems Inc., San Jose, CA), and printed with a Tektronix Phaser 440 dye sublimation printer (Tektronix, Inc., Wilsonville, OR).

Statistical Analyses

The percentage of 2-cell preimplantation embryos developing to the 5- to 8-cell stage in preimmune IgG-treated versus IL2-treated preimplantation embryos was analyzed statistically using a one-way ANOVA. When the ANOVA showed significant differences among means, Dunnett's post hoc test was used to compare the individual means of the IL2-treated groups to the preimmune IgG control group. P values of less than 0.05 and 0.01 were considered significant and highly significant, respectively.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Binding of IL2 to the Cortical Granules of Sea Urchin and Hamster Oocytes

The polyclonal antibody IL2 was made against purified hyalin from the cortical granules of S. purpuratus eggs [42]. IL2 bound specifically to the cortical granules of unfertilized S. purpuratus eggs labeled with a 1:100 dilution of the antibody (Fig. 1A). Control S. purpuratus eggs were not labeled by preimmune IgG followed by goat anti-rabbit IgG conjugated to FITC or by secondary antibody alone (data not shown). Hamster cortical granules were not labeled by a 1:100 dilution of IL2 (data not shown); however, a 1:10 dilution of IL2 labeled the cortical granules of permeabilized (Fig. 1B) but not nonpermeabilized (Fig. 1C) unfertilized hamster oviductal oocytes. The area of the oocyte cortex that was not labeled corresponded to a cortical granule-free domain, and the metaphase II spindle lies directly under this domain (Fig. 1B). Control permeabilized hamster oocytes were not labeled by preimmune IgG followed by goat anti-rabbit IgG conjugated to FITC (Fig. 1D) or by secondary antibody alone (data not shown).



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  		FIG. 1. Confocal laser scanning micrographs of unfertilized sea urchin eggs and hamster oviductal oocytes labeled with 1:100 (sea urchin) or 1:10 (hamster) IL2 for 30 min followed by 1:100 goat anti-rabbit IgG conjugated to FITC for 30 min. A) IL2 labeled the cortical granules of permeabilized sea urchin eggs. B, C) IL2 labeled the cortical granules of hamster oocytes if the oocytes were permeabilized (B) but not if they were nonpermeabilized (C). D) Control permeabilized hamster oocytes were not labeled by preimmune IgG followed by secondary antibody. The zona pellucida, which lies about 5 µm from the oolemma and is about 7 µm thick, was not labeled by IL2.

Distribution of the IL2 Binding Antigens in Fertilized Hamster Oocytes and in 2-Cell to Blastocyst-Stage Preimplantation Embryos

To determine whether IL2 labeled the mammalian cortical granule envelope, since this structure is analogous to the echinoderm hyaline layer, fertilized hamster oocytes and preimplantation embryos were incubated with a 1:10 or a 1:100 dilution of IL2 and examined by CLSM. The cortical granule envelope of fertilized hamster oocytes and preimplantation embryos was labeled by both concentrations of IL2 (Fig. 2). IL2 bound solely to the cortical granule envelope of fertilized oocytes (Fig. 2A). No label was observed in the oocyte cytoplasm or in the zona pellucida, even when the neutral density filter was removed and the gain was increased on the confocal microscope to make the zona visible (data not shown). At the 2-cell (Fig. 2B) and 8-cell (Figs. 2, C and D) stages, IL2 labeled the cortical granule envelope as well as the blastomere cortices (Fig. 2, B–D). Furthermore, some of the labeled components appeared to be on the surface of the blastomeres (Fig. 2D). At the blastocyst stage, no label was detected in the perivitelline space; however, the trophoblast cells, but not the inner cell mass, contained punctate granules that were labeled by IL2 (Fig. 2E). This trophoblast-specific labeling pattern was established during compaction of the 8-cell preimplantation embryo, at which time IL2-labeled vesicles were present only in the five outer blastomeres that differentiate into trophoblast cells (data not shown). Control preimplantation embryos were not labeled by preimmune IgG followed by goat anti-rabbit IgG conjugated to FITC, as demonstrated in a 4-cell preimplantation embryo (Fig. 2F), or by secondary antibody alone (data not shown).



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  		FIG. 2. Confocal laser scanning micrographs of permeabilized fertilized hamster oocytes and preimplantation embryos labeled with 1:100 (A–C, E) or 1:10 (D) IL2 for 30 min followed by 1:100 goat anti-rabbit IgG conjugated to FITC for 30 min. A) IL2 labeled the cortical granule envelope but not the cortex or zona of fertilized oocytes. B–D) IL2 labeled the cortical granule envelope (bright spots outside oocyte) and blastomere cortices of 2 (B)- and 8 (C, D)-cell preimplantation embryos. Some labeled components appeared to be on the surface of the blastomeres (D). E) IL2 labeled the cortex of the trophoblast cells but not the inner cell mass, blastocyst cavity, zona, or perivitelline space of zona-intact blastocysts. F) Control 4-cell preimplantation embryos were not labeled by preimmune IgG followed by secondary antibody. A zona pellucida is present in all figures, but is not visible because it was not labeled with the IL2 antibody

Molecular Weight of the IL2 Binding Antigen(s) in Hamster Oocytes and Preimplantation Embryos

IL2 specifically recognized the echinoderm cortical granule glycoprotein, hyalin (350 kDa), as demonstrated on immunoblots of unfertilized sea urchin eggs (Fig. 3A). To determine the molecular weight of the hamster cortical granule antigen(s) that cross-reacts with IL2 and to establish whether the molecular weight of this antigen(s) changes after its incorporation into the cortical granule envelope, hamster oocyte and embryonic proteins were immunoblotted with IL2 (Fig. 3B). IL2 recognized two bands with molecular masses of 62 and 56 kDa (p62/p56) in unfertilized hamster oocytes (Fig. 3B, lane 1); these bands were less abundant in the cytoplasm of fertilized oocytes (Fig. 3B, lane 2). The bands were detected at the 2-cell (Fig. 3B, lane 3) and 8-cell (Fig. 3B, lanes 4 and 5) stages of embryonic development with no apparent change in their molecular size. In addition, the abundance of p62/p56 appeared to increase during preimplantation embryonic development (Fig. 3B, lanes 3–5), with the greatest amount of antigen associated with 8-cell embryos (Fig. 3B, lanes 4 and 5). A significant amount of p62/p56 was present in the cytoplasm of the embryonic blastomeres (Fig. 3B, lane 4), in addition to the perivitelline space and/or blastomere plasma membranes (Fig. 3B, lane 5). No bands were detected on blots of unfertilized oocytes or preimplantation embryos probed with preimmune IgG followed by secondary antibody (data not shown) or by secondary antibody alone (Fig. 3B, lane 6).



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  		FIG. 3. Immunoblots of solubilized proteins from A) unfertilized sea urchin eggs and B) unfertilized and fertilized hamster oocytes and preimplantation embryos. A) Five microliters of an S. purpuratus egg preparation was electrophoresed, and the proteins were immunoblotted with a 1:10 000 dilution of IL2 followed by a 1:5000 dilution of goat anti-rabbit IgG conjugated to HRP. IL2 reacted with a major 350-kDa band in sea urchin eggs. B) The electrophoresed proteins of 100 zona-free unfertilized hamster oviductal oocytes (lanes 1 and 6), 100 zona-free fertilized oocytes (lane 2), 100 zona-intact 2-cell embryos (lane 3), 50 zona-free 8-cell embryos (lane 4), and 50 zona-intact 8-cell preimplantation embryos (lane 5) were immunoblotted with a 1:2000 dilution of IL2 (lanes 1–5) or left in blocking solution (lane 6), followed by a 1:5000 dilution of goat anti-rabbit IgG conjugated to HRP. IL2 reacted with two bands in unfertilized oocytes with molecular masses of 62 and 56 kDa (lane 1), and these bands were less abundant in the cytoplasm of fertilized oocytes (lane 2). The 62- and 56-kDa bands were also observed at the 2 (lane 3)- and 8 (lanes 4 and 5)-cell stages of embryonic development, and these bands were more abundant in these stages than in unfertilized oocytes (lane 1). Moreover, at the 8-cell stage, a significant amount of the 62- and 56-kDa bands was in the cytoplasm itself (lane 4), in addition to the perivitelline space and/or zona (lane 5). No bands were detected on blots of unfertilized oocytes probed with secondary antibody alone (lane 6) or with preimmune IgG followed by the secondary antibody (data not shown). ECL exposure times were 5 min (lanes 1–3) and 10 min (lanes 4–6).

Role of p62/56 in the Second and Third Cleavage Divisions of Hamster Blastomeres

To determine whether p62/56 function in blastomere cleavage in vivo, we examined the development of IL2-treated 2-cell preimplantation embryos to the 5- to 8-cell stage. Treatment of live embryos from IL2-injected oviducts with an anti-rabbit IgG-FITC conjugate revealed that IL2 was associated with these embryos, as shown in the epifluorescent image of a zona-free 2-cell embryo (Fig. 4A). The antibodies presumably reached the surface of the live embryos by passively diffusing through the zona while the embryos were in the oviduct. In contrast, preimplantation embryos recovered from the contralateral oviduct of the same female had bound little IL2, and they had reached the 8-cell stage of development (Fig. 4B). Only antigens associated with the blastomeres were detected in live embryos since the cortical granule envelope and zona pellucida were not preserved in the absence of fixation (Fig. 4, A and B). Since A and B are epifluorescent images of compressed embryos, they do not distinguish between label on the surface and interior of blastomeres. Seventy-four percent of 2-cell preimplantation embryos that were treated with preimmune IgG developed to the 5- to 8-cell stage, as opposed to 30% (P < 0.05) or 22% (P < 0.01) of embryos treated with 125 or 250 µg of IL2, respectively (Fig. 4C). No significant differences (P > 0.05) were observed between the percentages of 2-cell preimplantation embryos developing to the 5- to 8-cell stage in the contralateral oviducts of females treated with preimmune IgG or IL2 (Fig. 4D). The effects of IL2 were specific and not a consequence of a high local concentration of IgG since treatment of 2-cell embryos with 250 µg of the IgG fraction of an antibody against the zona pellucida glycoprotein ZP1 did not inhibit blastomere cleavage. Seventy-five percent of the anti-ZP1-treated 2-cell embryos from two females reached the 5- to 8-cell stage (data not shown).



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  		FIG. 4. The effects of IL2 treatment on the development of 2-cell preimplantation embryos to the 5- to 8-cell stage. Two-cell preimplantation embryos were treated on Day 2 of pregnancy with 250 µg of preimmune IgG, 125 µg of IL2, or 250 µg of IL2; and blastomere cleavage was evaluated on Day 3 of pregnancy. A, B) Live zona-free preimplantation embryos from an oviduct injected with 250 µg of IL2 (A) and from the contralateral oviduct (B) of the same female were incubated with 1:100 goat anti-rabbit IgG conjugated to FITC for 1 h and imaged with a Zeiss epifluorescent microscope. The embryo treated with IL2 was arrested at the 2-cell stage and had more fluorescence than the 8-cell embryo from the contralateral oviduct. C, D) Bar graphs showing the percentage of 2-cell preimplantation embryos that developed to the 5- to 8-cell stage in treated (C) and in contralateral (D) oviducts. C) Seventy-three percent of 2-cell preimplantation embryos treated with 250 µg of preimmune IgG developed to the 5- to 8-cell stage. However, both 125 µg and 250 µg of IL2 significantly inhibited development to the 5- to 8-cell stage. D) No significant differences (P > 0.05) were observed between the percentages of 5- to 8-cell preimplantation embryos in the contralateral oviducts for any treatment. Data are plotted as means ± SD of five experiments. *P < 0.05, **P < 0.01

Role of p62/56 in Hamster Blastocyst Implantation

To determine whether the IL2 on the surface of trophoblast cells or in trophoblast vesicles functions in implantation, morula- to blastocyst-stage preimplantation embryos were treated with IL2 on the evening of Day 3 of pregnancy, and blastocyst implantation was evaluated the following day (Fig. 5). IL2 reached the embryos and was observed in epifluorescent images of recovered morulae that were incubated in vitro with goat anti-rabbit IgG-FITC (not shown). IL2 treatment did not have an effect on implantation since there was no significant difference (P > 0.05) between the percentage of implantation in the IL2-treated and contralateral uterine horns (Fig. 5). However, implantation was not 100% in either horn since 17% and 6% of the preimplantation embryos were still at the morula stage in the treated and contralateral uterine horns, respectively.



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  		FIG. 5. The effects of IL2 treatment on implantation of blastocysts in the uterus. Morula- to blastocyst-stage preimplantation embryos were treated with 125 µg of IL2 on the evening of Day 3 of pregnancy, and their implantation in the uterus was evaluated on Day 4 of pregnancy. No blastocysts were recovered from either injected or contralateral oviducts or uterine horns. The bar graph shows that 94% of the blastocysts implanted in the uterus on the contralateral side, and the percentage of implantation on the treated side (83%) was not significantly decreased (P > 0.05). Data are plotted as means ± SD of three experiments


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The apparent dual origin of p62/p56 from both cortical granules of unfertilized oocytes and the cortex of preimplantation embryos, and their role in mammalian embryonic development parallels the biogenesis and function of a very well-characterized component of the echinoderm hyaline layer known as hyalin. Since both the hyaline layer and the cortical granule envelope contain cortical granule proteins that have been implicated in early embryogenesis, the current study addressed the question of whether hyalin and p62/p56 share similar functional epitopes. A polyclonal antibody (IL2) against hyalin from S. purpuratus cortical granules cross-reacted with 62- and 56-kDa hamster cortical granule proteins that are incorporated into the cortical granule envelope after fertilization. It is most likely that the IL2 binding antigens are the same as the previously characterized p62/p56 [38–40]. However, it is possible that p62 and p56 each consists of several 62- and 56-kDa polypeptides and that IL2 recognizes only two of them. The granular distribution of the IL2 binding antigens within the cortical granule envelope may be due to an aggregation of these antigens by IL2, which is a multivalent polyclonal antibody, or to localization of the IL2 binding antigens in specific "nodes" throughout the envelope. At the ultrastructural level, the envelope is composed of numerous small granules [21–23, 47], but the electron microscopic and CLSM samples were fixed differently and cannot be directly compared. The abundance of the IL2 binding antigens in the cortical granule envelope appeared at the confocal microscope level to increase during preimplantation embryogenesis, and this increase occurred concomitantly with an appearance of antigenic label in the blastomere cortices. This apparent increase in the quantity of the IL2 binding antigens was confirmed at the protein level on immunoblots that showed far more p62/p56 in preimplantation embryos, especially 8-cell-stage embryos, than in unfertilized oocytes. These data are consistent with earlier studies showing that mouse and hamster preimplantation embryos synthesize and secrete p62/p56 [38–40] and further support the conclusion that A-BL2 and IL2 bind to the same antigens.

As mentioned earlier, p62/p56 and hyalin function in early mammalian and echinoderm embryogenesis, respectively [26–28, 34–36, 38, 40, 48]. Previous in vivo studies showed an antibody (A-BL2) against mouse p62/p56 blocked zygote and blastomere cleavage in hamsters but did not interfere with blastocyst implantation in the uterus [38]. In the current study, the polyclonal anti-hyalin antibody IL2 that bound to p62/p56 also inhibited blastomere cleavage in 2-cell hamster embryos, but it did not affect blastocyst implantation. These data show that p62/p56 and hyalin contain immunologically related epitopes, and these epitopes function in early hamster embryonic development.

Although IL2 treatment inhibited mammalian preimplantation embryogenesis, p62/p56 and hyalin may not function in exactly the same way in early mammalian and echinoderm embryonic development, respectively. While antibody inhibition studies have shown that p62/p56 play a role in regulating cell division in mammalian zygotes and preimplantation blastomeres, it is not known if hyalin performs the same function in echinoderm embryos. The hyaline layer, of which hyalin is the major component, is thought to allow the blastomeres of the early echinoderm embryo to interact at the 2-cell stage, since they are not held together by adhesive cell junctions until the 32-cell stage [32–36, 49]. If the hyaline layer is removed, the embryonic blastomeres separate and do not divide until a new hyaline layer is regenerated from vesicles containing hyalin that were not released at fertilization, or from de novo synthesized hyalin [19, 32, 33, 35]. While a lack of blastomere-blastomere interaction in 2-cell echinoderm embryos is thought to be responsible for the inhibition of embryogenesis in the absence of a hyaline layer, it is also possible that blastomere cleavage is halted because hyalin and other component(s) of the hyaline layer are no longer present to signal the blastomeres to divide. In fact, it could be concluded from the sea urchin studies that hyalin does regulate cell division, which does not occur when hyalin is absent or inhibited by antibodies, and that dissociation of blastomeres is a secondary feature of these experiments, unrelated to blastomere cleavage.

The mechanism by which p62/p56 control blastomere cleavage will need to be elucidated. Like other extracellular matrix molecules, p62/p56 may control blastomere cleavage by directly triggering a signal transduction cascade or indirectly by trapping or presenting soluble mitogens that in turn promote signaling and lead to cell divisions, as shown for other systems [50–53]. Some of the IL2 binding antigens do, in fact, appear in close contact with the surface of the hamster preimplantation embryo, in agreement with ultrastructural studies showing that components of the cortical granule envelope interact directly with the plasma membranes of the mammalian embryonic blastomeres [20–22]. In sea urchins, the direct interaction of hyalin with the embryonic surface is essential for embryogenesis [28]. Each molecule of hyalin contains three or more copies of a cell binding domain that anchor the hyaline layer to the surface of the echinoderm embryo [29, 31]. This anchoring probably physically holds the blastomeres together until intercellular adhesions are established and allows hyalin to serve as an adhesive substrate during morphogenesis of the early embryo [26–28, 31, 35].

In summary, we have shown that a polyclonal antibody (IL2) against hyalin, the major component of echinoderm cortical granules, binds to two hamster cortical granule components that have molecular masses of 62 and 56 kDa. The hamster cortical granule antigens appear to be the same as two previously characterized cortical granule components, p62 and p56 [38–40]. These data suggest that p62/p56 and hyalin share common protein and/or carbohydrate epitopes. After fertilization, the IL2 binding antigens are incorporated into the cortical granule envelope, a newly characterized extracellular matrix that is assembled from secreted cortical granule components [21, 23, 37, 54]. Moreover, these antigens are resynthesized during preimplantation embryogenesis, and they function in blastomere cleavage. These data, when taken as a whole, suggest that p62/p56 were derived from hyalin during deuterosome evolution and their IL2 binding epitope(s) was conserved during this process because of its importance in embryogenesis.


   ACKNOWLEDGMENTS
 
We are grateful to Sohail Wasif and Ben Han for their help in preparing the figures for publication. We would like to thank Ms. Min Liu, Dr. Zongmin Zhou, Dr. Manuela Martins-Green, and Dr. Bradley Hyman for their invaluable technical assistance and advice; Dr. Manuela Martins-Green and Dr. Ameae Walker for their suggestions on this manuscript; and Dr. Zongmin Zhou for his help in preparing Figure 3.


   FOOTNOTES
 
First decision: 5 October 1999.

1 This work was supported by NIH grant HD35204, as well as the Academic Senate, the Graduate Student Association, the Irwin P. Newell Award Foundation, and the Graduate Division at the University of California at Riverside. Back

2 Correspondence. FAX: 909 787 4286; talbot{at}citrus.ucr.edu Back

3 Current address: California State University, Northridge College of Science and Mathematics, 18111 Nordhoff Street, Northridge, CA 91330–8238. Back

Accepted: November 18, 1999.

Received: September 9, 1999.


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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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