Early Stages Of Oogenesis Research Articles

Transport of axl2p depends on erv14p, an ER-vesicle protein related to the Drosophila cornichon gene product.

COPII-coated ER-derived transport vesicles from Saccharomyces cerevisiae contain a distinct set of membrane-bound polypeptides. One of these polypeptides, termed Erv14p (ER-vesicle protein of 14 kD), corresponds to an open reading frame on yeast chromosome VII that is predicted to encode an integral membrane protein and shares sequence identity with the Drosophila cornichon gene product. Experiments with an epitope-tagged version of Erv14p indicate that this protein localizes to the ER and is selectively packaged into COPII-coated vesicles. Haploid cells that lack Erv14p are viable but display a modest defect in bud site selection because a transmembrane secretory protein, Axl2p, is not efficiently delivered to the cell surface. Axl2p is required for selection of axial growth sites and normally localizes to nascent bud tips or the mother bud neck. In erv14Delta strains, Axl2p accumulates in the ER while other secretory proteins are transported at wild-type rates. We propose that Erv14p is required for the export of specific secretory cargo from the ER. The polarity defect of erv14Delta yeast cells is reminiscent of cornichon mutants, in which egg chambers fail to establish proper asymmetry during early stages of oogenesis. These results suggest an unforeseen conservation in mechanisms producing cell polarity shared between yeast and Drosophila.

Essential role for gamma -tubulin in the acentriolar female meiotic spindle of Drosophila

Microtubule nucleation in vivo requires gamma-tubulin, a highly conserved component of microtubule-organizing centers. In Drosophila melanogaster there are two gamma-tubulin genes, gammaTUB23C and gammaTUB37C. Here we report the cytological and molecular characterization of the 37C isoform. By Western blotting, this protein can only be detected in ovaries and embryos. Antibodies against this isoform predominantly label the centrosomes in embryos from early cleavage divisions until cycle 15, but fail to reveal any particular localization of gamma-tubulin in the developing egg chambers. The loss of function of this gene results in female sterility and has no effect on viability or male fertility. Early stages of oogenesis are unaffected by mutations in this gene, as judged both by morphological criteria and by localization of reporter genes, but the female meiotic spindle is extremely disrupted. Nuclear proliferation within the eggs laid by mutant females is also impaired. We conclude that the expression of the 37C gamma-tubulin isoform of D. melanogaster is under strict developmental regulation and that the organization of the female meiotic spindle requires gamma-tubulin.

A Conserved Element in the Protein-Coding Sequence Is Required for Normal Expression of Replication-Dependent Histone Genes in Developing XenopusEmbryos

Replication-dependent histone genes in the mouse and Xenopusshare a common regulatory element within the protein-encoding sequence called the CRASα element (coding region activating sequence α) which has been shown to mediate normal expression in vivoand to interact with nuclear factors in vitroin a cell cycle-dependent manner. Thus far, the α element has only been studied in rodent cells in culture, and its effect on histone gene expression during development has not been determined. Here we examine the role of the α element in histone gene expression during Xenopusdevelopment which features a switch in histone gene expression from a replication-independent mode in oocytes to a replication-dependent mode in embryos after midblastula stage. In vivoexpression experiments involving wild-type or α-mutant mouse H3.2 genes show that mutation of the CRASα element results in a fourfold decline of expression in embryos, but does not affect expression in oocytes. Two distinct α sequence-specific binding activities were detected in both oocyte and embryonic extracts. A slowly migrating DNA-binding complex was present at relatively constant levels throughout development from the earliest stages of oogenesis through larval stages. In contrast, levels of a rapidly migrating complex were high in stage I and II oocytes, declined in stage II–VI oocytes, remained low in unfertilized eggs and cleavage stage embryos, and rose dramatically after the midblastula transition. The molecular masses of the factors forming the slow and rapidly migrating complexes were estimated to be approximately 110 and 85 kDa, respectively. DNA-binding activity of the 85 kDa α-binding factor was affected by phosphorylation, binding with higher affinity in the dephosphorylated state. The abrupt increase in DNA-binding activity of the 85-kDa α-binding factor at late blastula coincides with the switch to the replication-dependent mode of histone gene expression. We propose that the conserved α element present in the coding sequence of mouse and Xenopuscore histone genes is required for normal replication-dependent histone expression in the developing Xenopusembryo.

Cloning ofXenopus presenilin-α and -β cDNAs and Their Differential Expression in Oogenesis and Embryogenesis

Humanpresenilin(ps)-1and-2genes have recently been shown to be involved in genesis of early-onset familial Alzheimer's disease. By probing with human (H-)ps-1cDNA, we isolated two types of cDNA clones, namedX-ps-α and -β, from aXenopusbrain cDNA library. The encoded proteins, X-PS-α and -β, may correspond to H-PS-1 and -2 with 89.4 and 85.9% similarity, respectively. The strongest expression of these genes was observed in ovaries and in the early stages of oogenesis, although weak or moderate expression was detected ubiquitously for bothX-ps-α and -β genes in multiple tissues. Upon oocyte maturation, theX-ps-α transcript was rapidly degraded, whereas theX-ps-β mRNA level was constant even after fertilization until the midblastula transition. Zygotic expression of these genes became evident only at the tailbud stage. We propose that presenilins may function in preventing cells from undergoing apoptotic degeneration particularly prior to embryonic development and in developmentally matured tissues.

Distinct Stress-Inducible and Developmentally Regulated Heat Shock Transcription Factors inXenopusOocytes

The presence of a maternal pool of heat shock factor (HSF) inXenopusoocytes has been suggested by two lines of evidence from previous studies. First, heat shock response element (HSE)-binding activity is induced in heat-shocked eggs and embryos prior to expression of zygotic HSF. Second, expression from microinjected heat shock protein promoters in oocytes is induced upon heat shock. To date, however, endogenous oocyte HSF molecules have not been detected, nor has induction of HSE-binding activity been directly demonstrated. Here we report the detection of distinct stress-inducible and developmentally regulated HSE-binding activities of endogenous oocyte factors. Exposure of defolliculated oocytes to heat, cadmium, and arsenite resulted in the formation of an HSE-specific complex detectable by gel mobility shift assay. Induction of HSE-binding activity by each of these stressors corresponded to increased expression from a microinjected hsp70 promoter. The stress-inducible HSE-binding complex was recognized by antiserum against mammalian HSF1, but not by HSF2 antiserum, suggesting that aXenopushomologue of HSF1 is the major component of this activity. The HSE-binding activity of HSF1 was induced by stress treatments of stage I through VI oocytes, an indication that it is responsive to stress throughout oogenesis. During recovery from heat shock, the HSF1–HSE complex rapidly declined to control levels, but was induced for prolonged periods in oocytes exposed to continuous stress, a pattern unlike the transient activation previously observed in fertilized eggs or embryos. The kinetics of HSF1 activation in oocytes suggests that a key protein(s) regulating attenuation of the stress response is present at exceedingly low levels or is somehow modified during preembryonic development. We also detected an unusual constitutive HSE-binding complex in unstressed stage I and II oocytes, but not in later stage oocytes, eggs, developing embryos, or A6 cells. This constitutive complex was unaffected by heat or chemical treatments and was not recognized by either HSF1 or HSF2 antiserum. Appearance of the constitutive HSE-binding activity during oogenesis corresponded closely with peak levels of hsp70 mRNA detected by Northern blot analysis of RNA from staged oocytes. We suggest that the constitutive HSE-binding activity in early oocytes is formed by a unique developmentally regulated heat shock factor that may play a role in the expression of heat shock proteins during early stages of oogenesis.

Antral follicles confer developmental competence on oocytes.

This paper addresses the proposition, first advanced by Wilson (1925), that successful embryogenesis depends on an ordered series of events in oogenesis. It is at the completion of this varied set of intracellular changes that the oocyte finally acquires its full capacity to support fertilisation and development. Amongst the earliest nuclear events are those associated with chromosome pairing and meiotic recombination. During the growth phase cell volume increases 300-fold and the cytoplasm becomes the storage site for RNA and protein which will be mobilised during early development. Finally, a short phase of intracellular reprogramming, or maturation, completes the series of events during oogenesis that confer developmental competence upon the oocyte. Follicle cell support is an indispensable requirement for ordered oocyte development and provides the early germline cell with many of the essential nutrients and growth regulators required to ensure progression through the protracted growth phase (see contributions by Cecconi & Rosella and De Feliciet al.this issue). Although different, the interactions between the full-grown oocyte and the antral follicle are no less crucial to the acquisition of competence than those involved in the earlier stages of oogenesis.

Gratuitous mRNA localization in the Drosophila oocyte

Many of the genes that control pattern formation in Drosophila encode mRNAs that are localized to discrete regions of the oocyte during oogenesis. While such localization is generally assumed to be important for the pattern-forming activities of these genes, this has been rigorously demonstrated in only a few cases. Here we address the role of mRNA localization for the dorsoventral patterning gene K10. K10 mRNA is localized to the oocyte's anterior cortex following its transport into the cell during early stages of oogenesis. We show that mutations in cappuccino and spire, which permit K10 mRNA transport, but prevent subsequent anterior localization, do not disrupt the synthesis or localization of K10 protein. We also show that modified K10 transgenes that produce transcripts which are uniformly distributed throughout the oocyte, or which are mislocalized to the oocyte's posterior pole, produce localized and functional K10 protein. We conclude that the anterior localization of K10 mRNA is not important for K10 protein targeting or gene function. We propose that the anterior localization of K10, and probably other mRNAs, is a by-product of mRNA transport and does not necessarily reflect a requirement for localization per se.

Chromosome painting analysis of early oogenesis in human trisomy 18

We have used chromosome 18-specific painting probes to analyze early stages of oogenesis in two human trisomy 18 fetuses. At leptotene, a diffuse, nonlinear chromosomal fluorescence was detected as one (27%), two (42%), or three (31%) signals in 534 cells. The variation in size of these signals implies the possibility of associations between homologs prior to zygotene. At pachytene, about 75% (339/453) of the cells had a trivalent configuration, and almost half of these cells exhibited almost complete triple synapses. Approximately 24% of the pachytene cells demonstrated a bivalent:univalent configuration, and 1% exhibited complete asynapsis. Our data imply that triple synapses may be a regular feature of meiosis involving multivalents.

XLPOU-60, a Xenopus POU-Domain mRNA, Is Oocyte-Specific from Very Early Stages of Oogenesis, and Localised to Presumptive Mesoderm and Ectoderm in the Blastula

POU-domain proteins are a large family of transcriptional regulatory proteins, related to the homeodomain proteins, many of which are implicated in the control of gene expression during early development. We describe here the isolation of a cDNA encoding a Xenopus POU-domain protein, XLPOU-60. The predicted protein sequence of this cDNA is most closely related to the mouse germ line-specific transcription factor Oct-3/4. The XLPOU-60 gene is specifically expressed in oocytes of newly metamorphosed frogs, from the earliest stages at which transcription is known to occur. The mRNA is concentrated in the animal half of fully grown oocytes and is inherited maternally by the embryo, where it remains localised to animal cap and marginal zone cells of the blastula. Transcripts decline abruptly to a low level during gastrulation, but remain detectable throughout larval stages. However, unlike Oct-3/4, the transcript is not detectable in primordial germ cells, and XLPOU-60 is therefore probably not the functional homologue of the murine gene. We suggest that XLPOU-60 is one of the earliest genes to be transcribed in oocyte development, and that the XLPOU-60 protein may therefore be involved in initiating oocyte-specific patterns of transcription. Localisation of the transcript in the embryo may indicate that XLPOU-60 is also required for the initiation of mesoderm- and ectoderm-specific patterns of transcription in the embryo.

Progressive redistribution of alcohol dehydrogenase during vitellogenesis in Drosophila melanogaster: characterization of ADH-positive bodies in mature oocytes.

The use of monoclonal antibodies against Drosophila alcohol dehydrogenase (ADH) provides a powerful tool in the analysis of the tissue and temporal patterns of Adh gene expression. Immunocytochemical techniques at the light- and electron-microscopic levels have been used to determine the distribution of ADH in the ovarian follicles of D. melanogaster during oogenesis. In the early stages of oogenesis, small amounts of ADH are detectable in the cystocytes. At the beginning of vitellogenesis (S7), ADH appears to be located mainly in the nurse cells. From stage S9 onwards, the ADH protein is evenly distributed in the ooplasm until the later stages of oogenesis (S13-14), when multiple ADH-positive bodies of varying size appear in the ooplasm. This change in distribution is a result of the compartmentalization of the ADH protein within the glycogen yolk or beta-spheres. Yolk becomes enclosed within the lumen of the primitive gut during embryonic development, and thus our results suggest a mechanism for the transfer of maternally-inherited enzymes to the gut lumen via yolk spheres.

On the synthesis and destruction of A- and B-type cyclins during oogenesis and meiotic maturation in Xenopus laevis.

We have measured the levels of cyclin mRNAs and polypeptides during oogenesis, progesterone-induced oocyte maturation, and immediately after egg activation in the frog, Xenopus laevis. The mRNA for each cyclin is present at a constant level of approximately 5 x 10(7) molecules per oocyte from the earliest stages of oogenesis until after fertilization. The levels of polypeptides show more complex patterns of accumulation. The B-type cyclins are first detectable in stage IV and V oocytes. Cyclin B2 polypeptide is present at approximately 2 x 10(9) molecules (150 pg) per oocyte by stage VI. The amount increases after progesterone treatment, but returns to its previous level after GVBD and undergoes no further change until it is destroyed at fertilization. Cyclin B1 is present at 4 x 10(8) molecules per oocyte in stage VI oocytes, and rises steadily during maturation, ultimately reaching similar levels to cyclin B2 in unfertilized eggs. Unlike the B-type cyclins, cyclin A is barely detectable in stage VI oocytes, and only starts to be made in significant amounts after oocytes are exposed to progesterone. A portion of all the cyclins are destroyed after germinal vesicle breakdown (GVBD), and cyclins B1 and B2 also experience posttranslational modifications during oocyte maturation. Progesterone strongly stimulates both cyclin and p34cdc2 synthesis in these oocytes, but whereas cyclin synthesis continues in eggs and after fertilization, synthesis of p34cdc2 declines strongly after GVBD. The significance of these results is discussed in terms of the activation and inactivation of maturation-promoting factor.

Expression of two mRNAs encoding EGF-related proteins identifies subregions of sea urchin embryonic ectoderm

Many proteins containing domains related to epidermal growth factor (EGF) function in intercellular interactions that mediate specification of cell fate. We have used in situ hybridization to show that the expression of two EGF-related genes (SpEGF I and SpEGF II) is restricted to the same subset of ectodermal cells in sea urchin pluteus larvae. However, the concentration of EGF I mRNA in different epithelial cells of aboral ectoderm and postoral facial epithelium is constant while that of EGF II mRNA is highly modulated. RNase protection assays show that both genes are activated during the period when ectoderm founder cells are established, i.e., between fourth and fifth and between fifth and sixth cleavages for EGF I and EGF II, respectively. By mesenchyme blastula stage EGF I mRNA reaches maximum abundance (800–1000 copies/expressing cell) as a result of a high transcription rate, while EGF II mRNA peaks at about half that concentration by gastrula stage. EGF I expression begins at early stages of oogenesis while EGF II expression appears to be confined to embryogenesis.

Autonomous fluorescence of eggs of the ascidian Ciona intestinalis

AbstractUnder ultraviolet illumination, eggs of the ascidians Ciona intestinalis and C. savignyi were found to emit specific autonomous fluorescence in the myoplasmic region of egg cytoplasm, the vacuoles of test cells, and the follicular vesicles of follicle cells. The fluroescent egg cytoplasm formed a crescent after ooplasmic segregation and was distributed mainly to presumptive muscle cells. The fluorescence of the test cells appeared at early stages of oogenesis and remained constant during oogenesis and embryogenesis. The number of test cells within the perivitelline space of Ciona unfertilized eggs was estimated to be 1,154 ± 182 (S.D.); at the tailbud stage the number was 1,089 ± 149 (S.D.). These counts suggest that there is no proliferation of test cells from fertilization to at least the tailbud stage. The fluorescence of follicle cells also appeared at early stages of oogenesis and did not change during embryogenesis. The number of follicle cells attached to the chorions of eggs was estimated to be 63 ± 8 (S.D.).

Effect of ouabain on the meiotic maturation of stage IV-V Xenopus laevis oocytes.

Full-grown stage VI Xenopus laevis oocytes (1,200 to 1,300 micron) respond to progesterone stimulation by undergoing a series of physiological and morphological changes that are referred to as meiotic maturation. Oocytes in earlier stages of oogenesis (I through V) do not undergo these changes and remain in prophase arrest when exposed to this steroid. We have found that oocytes ranging from 850 micron (stage IV) to 1,000 micron (stage V) are capable of responding to progesterone under the appropriate conditions. Oocytes greater than or equal to 850 micron in diameter underwent germinal vesicle breakdown (GVBD) after 10-12 hr of exposure to progesterone when ouabain was added to the medium at a concentration greater than 2.5 X 10(-6) M. Under this culture condition, progesterone was now able to induce a 0.3- to 0.4-unit increase in the intracellular pH of stage IV-V oocytes, a 4- to 5-fold increase in 40s ribosomal protein S-6 phosphorylation, and a 2.3-fold increase in their rate of protein synthesis. All of these physiological changes are characteristic of full-grown stage VI oocytes undergoing meiotic maturation. In addition, we have found that oocytes greater than or equal to 750 micron are capable of amplifying maturation promoting factor (MPF) in their cytoplasm leading to GVBD. Therefore, stage IV-V Xenopus oocytes have the potential for undergoing meiotic maturation, but they are blocked at a point in prophase that appears to be alleviated by the combination of progesterone and ouabain.

Fine Structures of Oocytes and Accessory Cells of the Ovary in the Starfish Patiria (Asterina) pectinifera at Different Stages of Oogenesis and After 1-Methyladenine-Induced Maturation: (starfish oogenesis/oocyte maturation/electron microscopy/cytoplasm).

Morphological changes in the growing and maturing oocytes of Patiria (Asterina) pectinifero were studied by electron microscopy. Oogenesis is of the solitary type. An extensive system of rough endoplasmic reticulum (ER) and Golgi complex (GC) develops in the ooplasm forming the cortical, yolk and secretory granules in its peripheral regions. The contents of the latter granules are released from the oocyte and form the vitelline membrane. At early stages of oogenesis, extensive multiplication of mitochondria results in formation of a large aggregate of these organelles in the perinuclear cytoplasm ("yolk nucleus"). After maturation of full grown oocytes has been induced by 1-methyladenine, the membranous cell structures are rapidly rearranged: vast aggregates of ER cisternae in the surface cytoplasm layer and single ER cisternae among yolk granules are disintegrated to small vesicles; the GC is reduced. These processes are suggested to be somehow related to changes in hydration of the cytoplasm and in rigidity of its surface layer. In maturing oocytes, the yolk granules form characteristic linear rows, trabeculae, traversing the cytoplasm and their boundary membranes fuse in zones of contact. Some granules are converted to multivesicular bodies, thus suggesting the activation of hydrolytic enzymes that form part of the yolk in echinoderms.

The cytoskeleton of Xenopus oocytes and its role in development

ABSTRACT Much is known about determinative events in early amphibian embryos, perhaps more than any other animal group. However, as yet, little attention has been focused on the cytoarchitecture of the oocyte, and the way in which this could regulate asymmetries in the egg, which in turn could lead to developmentally important interactions. The changing cytoarchitecture of the Xenopus oocyte is described with the emphasis on the following: - firstly the polarity; the oocyte is not radially symmetrical at early stages of oogenesis, but shows marked polarity. Secondly, several cytoskeletal elements change their distribution during oogenesis, and again during maturation to form a fertilizable egg. Thirdly, monoclonal antibody methods show that the oocyte develops several asymmetries which are retained in the egg and early embryo, and may be lineage related.

Distribution and utilization of 5 S-RNA-binding proteins during the development of Xenopus oocytes.

At early stages of oogenesis in Xenopus laevis most of the ribosomal 5S RNA is complexed with three proteins to form two types of cytoplasmic RNP storage particle. A particle sedimenting at 42S contains 5S RNA and tRNA together with two proteins of Mr 48000 (P48) and Mr 43000 (P43) and a second particle sedimenting at 7S contains 5S RNA plus a protein of Mr 40000 (P40, also known as the transcription factor, TFIIIA). In this report we use antibodies monospecific for each protein to follow the movement of 5S RNA from nucleus to cytoplasm to nucleolus to cytoplasm and to determine the fate of each of the proteins that associate with 5S RNA during these transitions. Both P48 and P43 have roles additional to the formation of the 42S RNP storage particle; P48 is detected in the nucleus during early oogenesis and is cleaved to yield an Mr-33000 fragment that remains associated with 5S RNA that is excess to ribosome requirement during late oogenesis; P43 appears to be cleaved to yield fragments of Mr 28000 and 17000, the latter being present in ribosomal fractions. Apparently, there is no function for P40 in addition to those already described in transcription of 5S RNA genes and in storage of 5S RNA as a 7S RNP particle.

Effects of a (n−3) polyunsaturated fatty acid‐deficient diet on profiles of serum vitellogenin and lipoprotein in vitellogenic trout (Salmo gairdneri)

During the 6 months of vitellogenesis, 3-year-old female trout (Salmo gairdneri) were fed either an enriched (E) or an (n-3) polyunsaturated fatty acid (PUFA)-deficient (D) diet; serum vitellogenin (VG) and lipoproteins (d<1.21 g/ml) were analyzed at the third month of vitellogenesis (September) and at ovulation (December). The serum content of high density lipoproteins (HDL), the major protein class, maintained a mean value of 1500 mg/dl at both stages and with both diets. On the contrary, very low density lipoproteins (VLDL) were 90% higher during vitellogenesis than at spawning time, whereas excess vitellogenin circulated at this period (6580 mg/dl serum with diet E). The diet deficient in (n-3) lowered serum vitellogenin content by 16% in September and by 26% in December. The degree of (n-3) PUFA incorporation moderately decreased in low density lipoproteins (LDL) and in HDL with the (n-3)-deficient diet. The effect was more pronounced for 20∶5. On the other hand, essential 22∶6 was incorporated into vitellogenin at the same rate in September as in December with diet E (23% and 25%, respectively), whereas after a 3-month deficiency, the percentage fell to 12%; this percentage rose again to 19% at spawning time. These findings show that, although stored (n-3) PUFA were not exhausted after a 6-month dietary deficiency, the incorporation of essential fatty acids (EFA) into vitellogenin during the early stages of oogenesis was low, suggesting changes in egg composition that may influence hatching.

Binding of the cortical granule lectin to the jelly envelope in mature perch ova.

Endogenous lectins are present within cortical vesicles of previtellogenic and early vitellogenic oocytes in several species of fish (Krajhanzl et aI., 1984a, b; Nosek et al., 1983). Since the cortical vesicles are a precursor form of cortical granules (Nosek, 1984) and their content is released to the perivitelline space upon activation of the egg, it seemed interesting to follow the fate of lectin during this event. Therefore, indirect immunofluorescence staining and a fluorescent cytochemical marker have been employed for the localization of the specific lectin in mature perch ova before and after activation. Perch lectin (binding specificity towards D-glucose) was isolated from ovaries of the perch (Perca fluviatilis L.) as described previously (Krajhanzl et al., 1978). In extracts prepared from mature ovulated roe, both the agglutinating activity and the content of soluble lectin are very low and the physico-chemical properties of isolated lectin are different from similar preparations obtained at earlier stages of oogenesis (Krajhanzl & Kocourek, 1982). We have evidence that the lectin strongly interacts with some component in the homogenate of mature ova which is not present in similar material prepared from ovaries containing only previtellogenic oocytes, and that this interaction is responsible for the low solubility of tectin. The fate of the lectin in mature perch oocytes was studied in cryostat sections prepared as follows. One part of the freshly stripped ripe roe was rapidly frozen by immersion in liquid nitrogen, sectioned and fixed in Bouin-d'Hollande solution. The other part of the roe was either fertilized in vitro by perch sperm and allowed to swell in water or it was activated by water without prior fertilization. After swelling of the jelly envelope (30 rain) the ova were frozen in liquid nitrogen and cut in a cryostat. Sections were treated with the antibody as described previously (Nosek et aI., 1983) or they were stained with the fluorescent glycosylated marker (Krajhanzl et al., 1984b).

Comparative cytochemistry of the developing ovarian follicles of the dog, rabbit, and mouse: Origin of the zona pellucida

AbstractThe reactivity of the glycoproteins of ovarian follicles of the dog, rabbit, and mouse were compared, using the Alcian blue (pH 0.5) and Alcian yellow (pH 2.5) technique at the light‐microscope level and the periodic acid‐chromic acid‐silver methenamine technique at the electron‐microscope level.In paraffin sections, the rabbit and mouse show the appearance of both zona pellucida and follicular fluid in the earliest growing follicles. In the dog, there is a sequence of development with the follicular fluid appearing late, after much of the zona has been secreted. The zona and follicular fluid are highly sulfated in all animals. Zonae pellucidae of atresia appear to lose all traces of sulfation and become highly acidic. At the electronmicroscope level, oocytes contain little if any reactive glycoprotein material which can be related to zona pellucida formation. The initial appearance of the zona material occurs between follicle cell membranes extending outwards and away from the oocyte. Follicle cells of all species consistently contain a variety of reactive Golgi bodies and granules, with exocytotic vesicles, suggesting active synthesis and secretion of zona material.Our observations suggest that in the early stages of oogenesis, the follicular epithelium is responsible for at least part of the synthesis of the zona pellucida. It is possible therefore that both the oocyte and its follicle cells participate, probably on the sequential basis, in the synthesis of the mammalian zona pellucida.