Growing Mouse Oocytes Research Articles

Centrosome phosphorylation and the developmental expression of meiotic competence in mouse oocytes

Previous studies suggested that the transition from an incompetent to a competent meiotic state during the course of oogenesis in the mouse involved a G 2 M - like cell cycle transition (Wickramasinghe et al., 1991. Dev. Biol. 143, 162). The present studies tested the hypothesis that centrosome phosphorylation, an event normally induced by MPF, is required for this developmental transition and the expression of meiotic competence in cultured growing mouse oocytes. Multiple fluorescence labeling techniques were used to evaluate centrosome number, phosphorylation status, and microtubule nucleating capacity in competent and incompetent oocytes. Experimental conditions were established for reversibly altering the phosphorylation status of the centrosomes and the effects of these treatments on meiotic resumption were examined. Phosphorylated centrosomes nucleating short microtubules were observed in competent oocytes, whereas nonphosphorylated centrosomes and interphase microtubule arrays were found in incompetent oocytes. Upon recovery from nocodazole-induced microtubule depolymerization, short microtubules formed from centrosomes in competent oocytes, whereas long microtubules reappear in the cytoplasm of incompetent oocytes. Perturbation of the phosphorylation state of oocytes with activators of protein kinase A or protein kinase C resulted in the formation of long interphase microtubules in competent oocytes while centrosome phosphorylation was maintained. Treatment of competent oocytes with the phosphorylation inhibitor 6-dimethylaminopurine also led to formation of long microtubules, although under these conditions centrosomes were dephosphorylated. When competent oocytes were treated simultaneously with puromycin and the phosphodiesterase inhibitor isobutyl methylxanthine (IBMX) for 6 hr, centrosomes became dephosphorylated; centrosomes were rephosphorylated when competent oocytes were further cultured in IBMX without puromycin. Conditions that induced centrosome dephosphorylation in competent oocytes resulted in the loss of the ability to express meiotic competence in culture, whereas maintenance of centrosome phosophorylation in these oocytes was correlated with the ability to resume meiosis. These results suggest that the G 2 M transition that occurs when mouse oocytes progress from an incompetent to a competent state in vivo involves the phosphorylation of centrosomes and that the maintenance of centrosome phosphorylation is required for the in vitro expression of meiotic competence.

Meiotic competence acquisition is associated with the appearance of M-phase characteristics in growing mouse oocytes

To determine whether the acquisition of meiotic competence during the growth phase of oogenesis is associated with the appearance of M-phase characteristics, oocytes obtained from 13- to 30-day-old mice were evaluated by fluorescence microscopy with respect to chromatin and microtubule organization, in vitro maturation ability, and the distribution of M-phase phosphoproteins. Meiotically incompetent oocytes were distinguished from their competent counterparts in displaying elaborate interphase-like arrays of cytoplasmic microtubules and dispersed germinal vesicle chromatin. Meiotically competent oocytes were larger in size, exhibited condensation of chromatin around the nucleolus, and displayed a progressive diminution of cytoplasmic microtubules in conjunction with the appearance of multiple microtubule organizing centers. After 24 hr in culture, medium- to large-sized oocytes exhibiting perinucleolar chromatin condensation resume meiosis whereas smaller meiotically incompetent oocytes retain GVs with diffuse chromatin. Moreover, indirect immunofluorescence studies using the M-phase phosphoprotein specific monoclonal antibody MPM-2 indicate that the appearance of reactive cytoplasmic foci is directly correlated with nuclear changes characteristic of meiotically competent oocytes. Thus, the earliest transition to a meiotically competent state during oocyte growth in the immature mouse ovary is characterized by stage-specific and coordinated modifications of nuclear and cytoplasmic components.

Oocyte-specific expression of mouse Zp-2: developmental regulation of the zona pellucida genes.

The zona pellucida surrounds all mammalian oocytes and plays a vital role at fertilization and in early development. The genes that code for two of the mouse zona proteins (ZP2 and ZP3) represent a developmentally regulated set of genes whose expression serves as markers of mouse oocyte growth and differentiation. We previously characterized the single-copy Zp-3 gene and showed that its expression is oocyte specific and restricted to a narrow window of oocyte development. We now define the Zp-2 gene transcript and show that it is coordinately expressed with Zp-3 only during the 2-week growth phase of oogenesis that occurs prior to ovulation. Like Zp-3, the expression of Zp-2 is restricted to oocytes, and, although not detectable in resting oocytes, both ZP2 and ZP3 transcripts accumulate to become very abundant messengers in 50-microns-diameter oocytes. Ovulated eggs contain ZP2 and ZP3 transcripts which are 200 nucleotides shorter than those found in growing oocytes and have an abundance of less than 5% of the peak levels. In an attempt to understand the molecular details associated with the developmentally regulated, tissue-specific gene expression of the zona genes, the Zp-2 genetic locus has been characterized and its 5' flanking sequences have been compared with those of Zp-3. Both genes contain three short (8- to 12-base-pair) DNA sequences of 80 to 88% identity located within 250 base pairs of their transcription start sites.

Oocyte-Specific Expression of Mouse Zp-2: Developmental Regulation of the Zona Pellucida Genes

The zona pellucida surrounds all mammalian oocytes and plays a vital role at fertilization and in early development. The genes that code for two of the mouse zona proteins (ZP2 and ZP3) represent a developmentally regulated set of genes whose expression serves as markers of mouse oocyte growth and differentiation. We previously characterized the single-copy Zp-3 gene and showed that its expression is oocyte specific and restricted to a narrow window of oocyte development. We now define the Zp-2 gene transcript and show that it is coordinately expressed with Zp-3 only during the 2-week growth phase of oogenesis that occurs prior to ovulation. Like Zp-3, the expression of Zp-2 is restricted to oocytes, and, although not detectable in resting oocytes, both ZP2 and ZP3 transcripts accumulate to become very abundant messengers in 50-microns-diameter oocytes. Ovulated eggs contain ZP2 and ZP3 transcripts which are 200 nucleotides shorter than those found in growing oocytes and have an abundance of less than 5% of the peak levels. In an attempt to understand the molecular details associated with the developmentally regulated, tissue-specific gene expression of the zona genes, the Zp-2 genetic locus has been characterized and its 5' flanking sequences have been compared with those of Zp-3. Both genes contain three short (8- to 12-base-pair) DNA sequences of 80 to 88% identity located within 250 base pairs of their transcription start sites.

Developmental capacity of mouse oocytes that grow and mature in culture: The effect of modification of the protocol

Recent progress in the technology for growth and maturation of mouse oocytes in culture is described. The beneficial effect of modified systems for oocyte maturation and for preimplantation development on the developmental capacity of oocytes matured in vitro is demonstrated. The two most important changes in the culture systems are the use of Waymouth medium MB752/1 for oocyte maturation and CZB medium for preimplantation development. The overall result of using the new protocols is that the percentage of oocytes from unprimed and gonadotropin-primed immature mice that develop to blastocysts is approximately doubled. A system for the growth and development of oocytes isolated from preantral follicles that results in oocytes which have a full capacity for maturation, fertilization, and development to live young was developed. Use of Waymouth medium increases oocyte size during growth in vitro compared to other media tested. Continued improvements in the culture systems for oocyte and embryo development in vitro should provide excellent experimental systems for basic studies on the regulation of oocyte and embryo development as well as functional systems having practical applications.

Fertilization of in vitro grown mouse oocytes

Oocyte-granulosa cell complexes, isolated from 10-d-old mice, were cultured for 10 d in hypoxanthine-supplemented medium. The oocytes increased in size, but the resumption of meiosis was completely suppressed over the culture period. The oocytes were cultured for a further 20 h in an hypoxanthine-free medium, after which germinal vesicle breakdown was observed in 10% of oocytes larger than 60 μ, 53% of oocytes larger than 65 μ and 86% of oocytes larger than 70 μ m in diameter. The oocytes grown and matured in vitro were inseminated with epididymal sperm. When the oocytes were examined 6 h after insemination, 72% of them were undergoing fertilization. Most of the fertilized oocytes had a normal male pronucleus, and the percentage of monospermic oocytes was as high as that obtained for oocytes matured in vivo (87% vs 80%). However, the incidence of abnormality in the female pronucleus of in vitro grown and matured oocytes was much higher than that of oocytes matured in vivo (21 vs 1%).

Gene expression during mammalian oogenesis and early embryogenesis: quantification of three messenger RNAs abundant in fully grown mouse oocytes.

Ribonuclease protection assays have been used to quantitatively assess changes in steady-state levels of specific mRNAs during oogenesis and early embryogenesis in mice. The mRNAs encode ZP3 (a glycoprotein that serves as a sperm receptor), LDH-B (heart-type lactate dehydrogenase), and MOM-1 (a protein of unknown function). MOM-1 and LDH-B are expressed in a variety of adult mouse tissues and midgestation embryos, whereas ZP3 expression is restricted completely to oocytes. All three mRNAs are expressed by growing mouse oocytes and accumulate to unusually high levels in fully grown oocytes as compared to somatic cells; 240,000, 200,000 and 74,000 copies mRNA per fully grown oocyte for ZP3, LDH-B and MOM-1, respectively. Steady-state levels of LDH-B and MOM-1 mRNA undergo a modest decline (approximately 20-40%) during ovulation when fully grown oocytes become unfertilized eggs and, in general, mirror the reported change in poly(A)+RNA levels during this period of development. On the other hand, the level of ZP3 mRNA declines dramatically (approximately 98%) during ovulation, from approximately 240,000 copies per oocyte to approximately 5000 copies per unfertilized egg, and ZP3 mRNA is undetectable in fertilized eggs (less than 1000 copies per fertilized egg). MOM-1 mRNA is expressed at relatively low levels in morulae (approximately 2000 copies per embryo) and blastocysts (approximately 5000 copies per embryo), whereas ZP3 mRNA remains undetectable (less than 1000 copies per embryo) at these stages of preimplantation development. These findings are discussed in the context of overall gene expression during oocyte growth, meiotic maturation and early embryogenesis in mice.

Somatic cell-oocyte interactions in mouse oogenesis: Stage-specific regulation of mouse oocyte protein phosphorylation by granulosa cells

The relative rate of synthesis of a number of proteins and the protein phosphorylation pattern of growing and fully grown oocytes were influenced by the presence of granulosa cells. In particular, a 74-kDa phosphorylated protein was detected only in granulosa cell-enclosed growing mouse oocytes. When reaggregated with granulosa cells, the growing oocyte displayed the phosphorylated form of the 74-kDa protein but when oocytes were cultured on Sertoli cell monolayers or in granulosa cell-conditioned medium the 74-kDa protein was not phosphorylated. We propose that (1) granulosa cells regulate protein phosphorylation in mouse oocytes; (2) a 74-kDa protein is phosphorylated only in growing oocytes when surrounded by granulosa cells; and (3) granulosa cells, but not Sertoli cells, are competent to send the appropriate “signal” to the growing oocyte.

Growth and development of mouse oocytes in vitro.

Annals of the New York Academy of SciencesVolume 541, Issue 1 p. 205-210 Growth and Development of Mouse Oocytes in Vitroa JOHN J. EPPIG, JOHN J. EPPIG The Jackson Laboratory Bar Harbor, Maine 04609Search for more papers by this authorSUSAN A. J. DANIEL, SUSAN A. J. DANIEL The Jackson Laboratory Bar Harbor, Maine 04609 IVF Laboratory, University Hospital, 339 Windermere Road, London, Ontario, Canada N6A 5A5.Search for more papers by this authorALLEN C. SCHROEDER, ALLEN C. SCHROEDER Biology Department Gettysburg College Gettysburg, Pennsylvania 17325 eThe Jackson Laboratory, Bar Harbor, Maine 04609.Search for more papers by this author JOHN J. EPPIG, JOHN J. EPPIG The Jackson Laboratory Bar Harbor, Maine 04609Search for more papers by this authorSUSAN A. J. DANIEL, SUSAN A. J. DANIEL The Jackson Laboratory Bar Harbor, Maine 04609 IVF Laboratory, University Hospital, 339 Windermere Road, London, Ontario, Canada N6A 5A5.Search for more papers by this authorALLEN C. SCHROEDER, ALLEN C. SCHROEDER Biology Department Gettysburg College Gettysburg, Pennsylvania 17325 eThe Jackson Laboratory, Bar Harbor, Maine 04609.Search for more papers by this author First published: October 1988 https://doi.org/10.1111/j.1749-6632.1988.tb22257.xCitations: 3 a These research projects were supported by National Institutes of Health Grant HD-20575 to JJE and a postdoctoral fellowship from the Medical Research Council of Canada to SAJD. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume541, Issue1In Vitro Fertilization and Other Assisted ReproductionOctober 1988Pages 205-210 RelatedInformation

Involvement of thiol-disulfide groups in the sensitivity of fully grown mouse oocytes to calcium-free medium.

The lethality caused by calcium-free medium (CFM) to fully grown mouse oocytes significantly decreases if a disulfide reducing agent (dithiothreitol, reduced glutathione, or L-cysteine) is added to the medium. In this condition, most of the surviving oocytes do not spontaneously resume meiosis. We also show that the sulfhydryl content of fully grown oocytes, estimated by monobromobimane labeling, rapidly decreases during culture in CFM. The hypothesis is discussed that lethality of oocytes cultured in CFM may be a consequence of an alteration of thiol-disulfide balance.

The effect of hypoxanthine on mouse oocyte growth and development in vitro: Maintenance of meiotic arrest and gonadotropin-induced oocyte maturation

The concentration of hypoxanthine in mouse follicular fluid has been estimated to be 2–4 m M, and although this concentration maintains meiotic arrest in fully grown mouse oocytes in vitro, oocyte maturation in vivo is not induced by a decrease in the concentration of this purine in follicular fluid (J. J. Eppig, P. F. Ward-Bailey, and D. L. Coleman, Biol. Reprod., 33, 1041–1049, 1985). In the present study, the effect of 2 m M hypoxanthine on oocyte growth and development in vitro was assessed and the ability of gonadotropins to stimulate oocyte maturation in the continued presence of hypoxanthine was determined. Oocyte-granulosa cell complexes were isolated from 10- to 11-day-old mice and cultured in the presence or absence of 2 m M hypoxanthine. Oocytes from 10- to 11-day-old mice are in mid-growth phase and, without further development, are incompetent of undergoing meiotic maturation. During a 12-day culture period the granulosa cell-enclosed oocytes approximately doubled in size and, regardless of the presence or absence of hypoxanthine, 50–70% developed competence to undergo germinal vesicle breakdown (GVBD). Hypoxanthine promoted the continued association of oocytes with their companion granulosa cells during the 12-day culture period, and therefore had a beneficial effect on oocyte development. Most of the oocytes that acquired GVBD competence in the absence of hypoxanthine underwent spontaneous GVBD. In contrast, 95% of the GVBD-competent oocytes were maintained in meiotic arrest by hypoxanthine. Following withdrawal of the hypoxanthine after the 12-day culture, 75% of the GVBD-competent oocytes underwent GVBD. These results show that hypoxanthine, and/or its metabolites, maintains meiotic arrest in oocytes that grow and acquire GVBD competence in vitro. Follicle-stimulating hormone (FSH), but not luteinizing hormone or human chorionic gonadotropin, induced oocyte GVBD in the continued presence of hypoxanthine. FSH stimulated oocyte maturation at a significantly ( P < 0.01) higher frequency than coculture of the granulosa cell-denuded oocytes with granulosa cells in the continued presence of hypoxanthine. FSH did not induce the maturation of denuded oocytes cocultured with granulosa cells. Similar results were obtained using oocyte-cumulus cell complexes isolated from 22-day-old mice wherein ovarian follicular development was promoted by injection of exogenous gonadotropin. Based on these results, is it hypothesized that GVBD was promoted in these culture systems by an FSH-induced positive signal from granulosa/cumulus cells, and not simply by deprivation from the meiosis-arresting action of hypoxanthine.

Electron microscopic study on the bismuth staining of nucleoli in growing mouse oocytes.

The electron microscopic bismuth staining method of Locke and Huie (16) was applied to the nucleoli of dictyate-stage growing mouse oocytes. After bismuth staining following glutaraldehyde fixation (GA-Bi staining), bismuth was largely localized in the fibrillar centers and the adjoining zones of dense fibrillar components not only in the nucleoli of growing oocytes during the unilaminar, bilaminar and plurilaminar follicle stages, but also in the nucleoli of mature antral-follicular oocytes. These results suggest that the materials stained with the GA-Bi method in the nucleoli of mouse oocytes may have no direct correlation with the transcriptional activity of ribosomal DNA.In the nucleoli of antral follicular oocytes, the large spherical bodies, consisting of condensed fibrillar components, were unstained with GA-Bi staining but densely stained with bismuth following formaldehyde fixation (FA-Bi staining). This fact suggested the presence of nuclear basic proteins such as histones and protamines in these nucleolar bodies.

Regulation of development in the fully grown mouse oocyte: chromosome-mediated temporal and spatial differentiation of the cytoplasm and plasma membrane

The relationship between nuclear maturation and the differentiation of the cytoplasm and plasma membrane during resumption of arrested meiosis was investigated by culture of GV-and MII-stage mouse oocytes in the presence and absence of nocodazole. Culture in the presence of nocodazole was associated with dispersal of MI and MII chromosomes throughout the subplasmalemmal cytoplasm. A progression of cortical (thickening of actin filaments) and plasma membrane changes (denudation of microvilli, reduction in cell surface glycoproteins, formation of chromosome-containing evaginations) that normally occurs in proximity to chromosomes associated with intact MI or MII spindles took place only in those regions of the cortical cytoplasm containing the dispersed subplasmalemmal chromosomes. The dispersion and migration of the chromosomes occurred in an apparently random fashion. Fluorescent probe analysis of normal and treated oocytes indicated a stage-specific association between the spatial distribution of chromosomes and mitochondria. Transfer of individual bivalent chromosomes to untreated oocytes at different stages of maturation and to cytoplasts derived from oocytes anucleated prior to GVB demonstrated the necessity of chromosomes for cytoplasmic and plasma membrane differentiation, and that the capacity of the cytoplasm and plasma membrane to differentiate in response to the presence of a chromosome is acquired prior to GVB.

Ethanol-Phosphotungstic Acid Staining of Basic Proteins in the Nucleolar Condensation Occurring in Growing Mouse Oocytes

Ethanol-Phosphotungstic Acid Staining of Basic Proteins in the Nucleolar Condensation Occurring in Growing Mouse Oocytes

Maturation-inhibiting activity in growing mouse oocytes

Growing mouse oocytes incompetent to resume meiosis were fused with fully grown immature mouse oocytes and cultured for 20–24 h. In giant cells that developed, two intact germinal vesicles remained well conserved in all cases. We propose that the cytoplasm of growing oocytes possesses a maturation-inhibiting activity which is able to arrest, after fusion, nuclear maturation in fully grown oocytes competent to mature spontaneously.

Relationship between rates of synthesis and intracellular distribution of ribosomal proteins during oogenesis in the mouse

Ribosomal proteins are synthesized continuously in nonequimolar amounts during oogenesis in the mouse ( M. J. LaMarca and P. M. Wassarman, 1979, Develop. Biol. 73, 103), even though ribosomal proteins are found in equimolar amounts in ribosomes. In this report, the distribution of newly synthesized ribosomal proteins between the cytoplasm and germinal vesicle (nucleus) of fully grown mouse oocytes has been examined. As compared to total newly synthesized protein, ribosomal proteins were found to be highly concentrated in the oocyte's germinal vesicle. Furthermore, an inverse relationship was found between rates of synthesis of individual ribosomal proteins and percentages of newly synthesized ribosomal proteins associated with germinal vesicles. As a result of this relationship, the amounts of newly synthesized ribosomal proteins associated with germinal vesicles approximated an equimolar situation. Even in the presence of actinomycin D, oocytes continued to synthesize ribosomal proteins which were found associated with germinal vesicles in amounts similar to those observed in the absence of the drug. These results suggest that, although synthesis of ribosomal proteins by mouse oocytes is not coordinately regulated, a post-translational mechanism exists for adjusting the stoichiometry of these proteins within the oocyte's germinal vesicle; this mechanism apparently is not dependent upon concomitant ribosomal-RNA synthesis.

Early programming of maturation competence in mouse oogenesis

Growing mouse oocytes incompetent to mature were freed of attached granulosa cells at different stages of growth, and cultured in vitro in the presence of fibroblast monolayers and/or their products. In these culture conditions, although growth was arrested, isolated oocytes survived in vitro for several days, and finally resumed meiosis spontaneously, progressing up to metaphase I. The culture time length needed for in vitro acquisition of the capacity to mature was inversely related to the initial oocyte size at the time of isolation from granulosa cells, and closely corresponded to developmental timing of acquisition of such ability in vivo. We conclude that the acquisition of mouse oocyte competence to mature follows a definite time program, which is independent of the presence of granulosa cells and of heterologous cell contacts, at least within the developmental stages studied.

Regulation of mouse oocyte growth: probable nutritional role for intercellular communication between follicle cells and oocytes in oocyte growth.

Intercellular communication, as determined by two different assay procedures, was established in vitro between mouse oocytes free of adhering follicle cells and monolayers of either follicle or 3T3 cells. Both of these cell types are known to be able to form homologous gap junctions, and follicle cells naturally form heterologous gap junctions with oocytes in vivo. Monolayers of L cells that are communication deficient did not establish intercellular communication with oocytes as determined by the two different assays for intercellular communication. The diameter of oocytes cultured for 4 days in medium or on monolayers of L cells decreased markedly, 9.7 and 13.1 micron, respectively. In contrast, oocytes cultured for 4 days on follicle cell monolayers increased on the average about 4.7 micron in diameter. Oocytes cultured for 4 days on monolayers of 3T3 cells decreased slightly in diameter, i.e., 2.1 micron. Results from these experiments support a nutritional role for intercellular communication between follicle cells and oocytes in oocyte growth.

Amino acid transport systems in growing mouse oocytes

Amino acid transport systems have been studied in isolated mouse oocytes throughout oogenesis. While these cells were lacking the A-transport system of Ehrlich cell ( Christensen, 1975), they were found to be provided with the L- and ASC-systems of Ehrlich cell ( Christensen, 1975). Since, in contrast to the A-system, the L- and ASC-systems mainly work by exchanging internal and external amino acids, we propose that their main function is to actively maintain the appropriate internal balance between different amino acids whithin the growing oocyto.

Role of asparagine-linked oligosaccharides in secretion of glycoproteins of the mouse egg's extracellular coat.

Tunicamycin, an inhibitor of asparagine-linked glycosylation of glycoprotein, has been used here to examine the role of N-linked oligosaccharides in secretion of ZP2 and ZP3, two of the three glycoproteins that constitute the mouse egg's extracellular coat (zona pellucida). In the absence of tunicamycin, growing mouse oocytes cultured in vitro synthesize a 91,000-Mr ZP2 precursor and 53,000 and 56,000 Mr ZP3 precursors. All of these precursors contain high mannose-type oligosaccharides that are processed to complex-type prior to secretion of mature ZP2 (120,000 Mr) and ZP3 (80,000 Mr) (Greve, J. M., Salzmann, G. S., Roller, R. J., and Wassarman, P. M. (1982) Cell 31, 749-759; Salzmann, G. S., Greve, J. M., Roller, R. J., and Wassarman, P. M. (1983) Eur. Mol. Biol. Org. J. 2, 1451-1456). In the presence of 5 micrograms/ml of tunicamycin, growing oocytes cultured in vitro are unable to carry out "core" glycosylation of nascent ZP2 and ZP3. Consequently, under these conditions, ZP2 and ZP3 appear as 81,000 and 44,000 Mr polypeptide chains, respectively. The apparent rates of synthesis of core-glycosylated ZP2 and ZP3 precursors synthesized in the absence of tunicamycin and of precursors synthesized in the presence of the drug are virtually identical. On the other hand, in the presence of tunicamycin, nascent ZP3 is incorporated into the zona pellucida as an extremely heterogeneous species (approximately equal to 51,000 Mr) at about three times the rate observed for mature ZP3 in the absence of tunicamycin. In the presence of tunicamycin, ZP2 is incorporated into the zona pellucida as 81,000 and 76,000 Mr species at about one-sixth the rate observed for mature ZP2 in the absence of the drug. Results of pulse-chase experiments indicate that the low degree of incorporation of ZP2 lacking N-linked oligosaccharides into the zona pellucida is due to a greatly decreased rate of secretion as compared to the core-glycosylated precursor. ZP2 synthesized in the presence of tunicamycin is relatively stable and accumulates intracellularly. These results suggest that N-linked oligosaccharides are necessary for normal secretion of ZP2, but are probably not necessary for ZP3 secretion.