Quantitative and qualitative cytochemical analysis of changes in polysaccharides-proteins in rat ovulable and atretic ovarian follicles during the estrous cycle

Summary The glycosaminoglycan (periodic acid — Schiff, PAS) and hyaluronic acid (alcian blue) content of the membrana granulosa, zona pellucida and antrum of rat ovarian follicles was analyzed qualitatively and quantitatively during the estrous cycle in three types of follicles: ovulable, early atretic and late atretic. The qualitative analysis consisted of the conjunctive localization of PAS-reactive, fluorescent granules within the membrana granulosa. The quantitative analysis consisted of microdensitometric measurements of PAS and alcian blue staining within the zona pellucida and antrum of the ovulable and atretic follicles. For the localization of PAS granules within the granulosa cells, ovaries were removed on the day of proestrus, fixed in 6% paraformaldehyde, embedded in methacrylate and sectioned. Following the examination of the cells for fluorescence, the same section was stained with PAS and lead-hematoxylin. In ovulable follicles there was no fluorescence in the membrana granulosa while PAS granules occurred exclusively within the cells of the cumulus and corona radiata. In late atretic follicles, fluorescent-PAS reactive granules were located in the granulosa cells at the periphery of the follicle. During early atresia no fluorescence and very few PAS granules were observed in the granulosa cells. Since fluorescence is a marker for some lysosomes, these observations suggest that the PAS granules in the ovulable follicles may not be a type of lysosome. The amount of stain in the zona pellucida and antrum of the three follicular types was quantified using a scanning and integrating microdensitometer. On all days of the estrous cycle, PAS intensity was higher in the zona pellucida than in the antrum of the three follicular types. PAS staining in the respective antra was the same on all days of the estrous cycle. Intrafollicular PAS staining in the zonae pellucidae differed during the cycle. With respect to the zonae pellucidae, staining intensity in the three follicles was identical on estrus. On diestrus-1, staining intensity was the same in the ovulable and early atretic follicles and less in the late atretic follicle. By diestrus-2 and on proestrus, PAS intensity was highest in the zona pellucida of the ovulable follicle and less in the zona pellucida of both types of atretic follicle. In contrast to this pattern of staining, alcian blue staining intensity was identical in the zona pellucida of all follicles throughout the cycle. There was no difference in intra-antral alcian blue staining intensity on estrus and diestrus-2. On diestrus-1 and proestrus, staining intensity was greater in the antrum of the late atretic follicle than in the antra of the other follicular types. These studies indicate that glycosaminoglycan content is greater in the zona pellucida of the ovulable follicle of the rat on the last two days preceding ovulation than in the zona pellucida of either the early or late atretic follicles. In contrast, hyaluronic acid content remains constant in the zona pellucida of the three follicular types throughout the estrous cycle. These studies also give the first indication that, in the rat, the localization of PAS granules exclusively in the cumulus oophorus and corona radiata may be used to identify ovulable follicles.This work was supported by a research grant from the National Institute of Child Health and Human Development, HD-12684     

Defective zonae pellucidae in Zp2-null mice disrupt folliculogenesis, fertility and development

All vertebrate eggs are surrounded by an extracellular matrix. This matrix is known as the zona pellucida in mammals and is critically important for the survival of growing oocytes, successful fertilization and the passage of early embryos through the oviduct. The mouse zona pellucida is composed of three glycoproteins (ZP1, ZP2 and ZP3), each encoded by a single copy gene. Using targeted mutagenesis in embryonic stem cells, Zp2-null mouse lines have been established. ZP1 and ZP3 proteins continue to be synthesized and form a thin zona matrix in early follicles that is not sustained in pre-ovulatory follicles. The abnormal zona matrix does not affect initial folliculogenesis, but there is a significant decrease in the number of antral stage follicles in ovaries isolated from mice lacking a zona pellucida. Few eggs are detected in the oviduct after stimulation with gonadotropins, and no two-cell embryos are recovered after mating Zp2-null females with normal male mice. The structural defect is more severe than that observed in Zp1-null mice, which have decreased fecundity, but not quite as severe as that observed in Zp3-null mice, which never form a visible zona pellucida and are sterile. Although zona-free oocytes matured and fertilized in vitro can progress to the blastocyst stage, the developmental potential of blastocysts derived from either Zp2- or Zp3-null eggs appears compromised and, after transfer to foster mothers, live births have not been observed. Thus, in addition to its role in fertilization and protection of early embryos, these data are consistent with the zona pellucida maintaining interactions between granulosa cells and oocytes during folliculogenesis that are critical to maximize developmental competence of oocytes.     

Immunohistochemical localization of zona pellucida proteins ZPA, ZPB and ZPC in human, cynomolgus monkey and mouse ovaries

The zona pellucida of mammalian oocytes plays an important role in binding and activation of sperm cells during the molecular events leading to fertilization. The genes coding for the three zona pellucida glycoproteins ZPA, ZPB, and ZPC of various species including mouse, dog, and human have been cloned and sequenced by several groups. However, it has remained a matter of debate as to whether the oocytes alone or in conjunction with the surrounding granulosa cells express and deposit these proteins to form the zona pellucida matrix. Addressing this unresolved issue, we assessed the expression and localization of all three zona pellucida proteins in ovaries of human, cynomolgus monkey and mice using immunohistochemical methods. In addition, oocyte-specific expression of ZPC from the primordial stage onward was confirmed by in situ hybridization. In sections of human ovaries, ZPA, ZPB, and ZPC proteins were immunohistochemically detected in the cytoplasms of primordial oocytes and during later stages of folliculogenesis in the zona pellucida matrices of oocytes. In sections fixed with formalin, a clear homogeneous ring was visible around the oocyte and no staining of granulosa cells was observed. In contrast, staining of ZP proteins was also observed between granulosa cells when Bouin's reagent had been used for tissue fixation. Thus, the original zona pellucida architecture was better preserved by formalin fixation. We further demonstrated that dissolution of the zona pellucida of isolated bovine oocytes occurred after they were exposed to Bouin's reagent. In summary, these results demonstrate that in mice, monkeys and humans, zona proteins are expressed and assembled exclusively by the oocyte and not by the granulosa cells. Previously observed results of ZP expression by an involvement of granulosa cells might therefore be the result of an improper fixation of the tissues leading to the disruption of the zona pellucida. Additionally this study highlights the importance of choosing the correct fixative for immunohistochemistry, not only for the usual reason of retaining antigenicity, but rather to retain the entire architectural structure.     

Involvement of cell proliferation in the process of follicular atresia in the guinea pig

Cell morphology and proliferation was investigated in the atretic follicles during estrous cycles in the guinea pig. Ovarian samples on days 1, 4, 8, 12 and 16 of the estrous cycle in the guinea pig were taken in the morning for histologic staining with hematoxylin and eosin (HE), and immunohistochemical staining of the protein proliferating cell nuclear antigen (PCNA). The results indicated that the granulosa cells degenerated and eliminated first in atretic follicles, while the fibroblast-like cells appeared in the innermost layer of theca interna cells. When the fibroblast-like cells migrated to the antrum, they proliferated and formed a new tissue in peripheral to the zona pellucida of the oocyte. Our results also revealed that the orientation of the theca interna cell arrangement changed twice during the process of atresia, and the loose connective tissue in the antrum was critical for follicular atresia. Therefore, follicular atresia was not a simple process of cell death and elimination, but coexisted with cell proliferation. To our knowledge, we have for the first time confirmed cell proliferation and the presence of new tissue in atretic follicles in guinea pigs.     

Topographical localisation of glucidic residues and their variations in the canine zona pellucida during folliculogenesis

SummaryIn the present ultrastructural study, horseradish peroxidase-labelled lectins, in conjunction with antiperoxidase antibody and protein A-gold, were used to characterise and localise the oligosaccharide sequences of zona pellucida glycoproteins at different stages of follicular development in the canine ovary. Deacetylation and sialidase digestion were also performed before lectin cytochemistry. The zona pellucida of oocytes present in unilaminar primary follicles reacts with WGA- and RCA-I-lectins. The zona pellucida of oocytes present in bilaminar and trilaminar secondary follicles displays positivity to WGA, RCA-I, Con-A, UEA-I, and sialidase/SBA. This labelling pattern persists in the zona pellucida of oocytes present in antral tertiary follicles with the exception of WGA and RCA-I reactive sites which are differently distributed throughout the zona pellucida. The topographical distribution of these carbohydrates is not uniform throughout the zona pellucida, indicating the regionalization of oligosaccharide chains within three concentric bands of the zona matrix: an inner surface close to the oocyte plasmamembrane, an intermediate portion and an outer layer in contact with the follicular cells. Our results demonstrated variations in the presence and distribution of the carbohydrate residues in the canine zona pellucida during different stages of follicular growth. We also observed the presence of vesicles in both the ooplasm and granulosa cells, showing a similar lectin binding pattern to that of the zona pellucida.     

The ovarian follicle as a model for the cell-mediated control of tissue growth

Summary Thy-1⁺ cells, producing Thy-1⁺ material, have been demonstrated by the indirect immunoperoxidase technique in the theca of growing ovarian follicles of the rat. OX-2 antigen, known as the minor glycoprotein of rat thymocytes, was detected in granulosa cells of non-growing follicles. Ia⁺ cells of dendritic type and/or activated macrophages were identified in the granulosa of advanced degenerating follicles, and remnants of the zona pellucida exhibited immunoglobulins. In some ovaries immunoglobulins were also bound to the zona pellucida of oocytes of early degenerating antral follicles. Medium-sized antral follicles with degenerating granulosa were occasionally invaded by cells carrying antigens of cytotoxic T lymphocytes or other T lymphocyte subsets, while degenerating large antral follicles were sometimes invaded by cells exhibiting antigen of cells with natural killer function (but not antigens of T lymphocytes). Granulosa cells of some degenerating antral follicles exhibited class-I antigens derived from the major histocompatibility complex. We suggest that cell-mediated control mechanisms of antigen expression and metabolism of tissue cells during their differentiation and degeneration should be considered in addition to the well-documented hormonal dependence of some tissues.     

Light microscopic observations of alterations in staining of the zona pellucida of porcine follicular oocytes: Possible early indication of atresia

Serial sections of porcine ovaries were examined in an attempt to detect early signs of oocyte degeneration/atresia using special staining. Porcine ovaries were fixed in Bouin's fixative and embedded in paraffin using routine techniques. Serial sections (8 μm) were mounted on glass slides and stained with Shorr's S3 and hematoxylin stain. Several criteria were used for examining general histology of the antral follicles: condition of the granulosa layer, antral cavity, the oocyte and its surrounding zona pellucida, and the cumulus layers. A change in the staining characteristic of the zona pellucida was the single most striking observation in all ovaries examined. In presumably healthy follicles, the zona pellucida was uniformly stained green, the granulosa layer was intact with fewer than three pyknotic cells per section, and a uniform basement membrane (stained green) separated the intact theca layers from the remainder of the follicle. In those follicles showing some degree of degenerative changes in the follicular wall, the zona pellucida was stained a bright orange. In the last stages of degeneration, follicles exhibited many pyknotic nuclei throughout the granulosa layers, the layer of granulosa cells was in many cases separated from the basement membrane, and the antrum was infiltrated with lymphocytes. In these follicles, the zona pellucida was always stained orange. Frequently, the zona pellucida was partially stained orange before any detectable changes could be seen in other elements of the follicular wall. Additionally, many non-antral (primary) follicles exhibited oocytes with orange-stained zonae pellucidae. In terminal stages of follicular degeneration, collapsed follicles were infiltrated by connective tissue elements stained bright orange and green. These structures very often contained dying oocytes always with bright orange-stained zonae pellucidae. Scattered throughout the ovarian stroma were many orange-stained remnants of zonae pellucidae. It is thought that perhaps the characteristic staining of the zona pellucida with Shorr's S3 stain may give an early, previously undetectable indication of follicular atresia.     

Abnormal zonae pellucidae in mice lacking ZP1 result in early embryonic loss.

All vertebrates have an egg shell that surrounds ovulated eggs and plays critical roles in gamete recognition. This extracellular matrix is known as the zona pellucida in eutherian mammals and consists of three glycoproteins, ZP1, ZP2 and ZP3 in the mouse. To investigate the role of ZP1 in fertilization and early development, we have used targeted mutagenesis in embryonic stem cells to create mouse lines (Zp1(tm/tm)) lacking ZP1. Although a zona pellucida composed of ZP2 and ZP3 was formed around growing Zp1(tm/tm) oocytes, the matrix was more loosely organized than zonae around normal oocytes. In some Zp1 null follicles, this structural abnormality resulted in ectopic clusters of granulosa cells, lodged between the zona matrix and the oolemma, that perturbed normal folliculogenesis. Comparable numbers of eggs were ovulated from Zp1 null females and normal females following hormonal stimulation. However, after mating with males, fewer two-cell embryos were recovered from Zp1 null females, and their litters were significantly smaller than those produced by normal mice. Therefore, although mouse ZP1 is not essential for sperm binding or fertilization, it is required for the structural integrity of the zona pellucida to minimize precocious hatching and reduced fecundity.     

Radioautographic study of glycoprotein biosynthesis and renewal in the ovarian follicles of mice and the origin of the zona pellucida

Summary L-fucose-³H was injected intravenously into mice which were killed at several time intervals after injection and semi-thin sections of their ovaries were processed for radioautography and analysed quantitatively. At the same time the specific activity of serum glycoproteins was determined. Glycoprotein biosynthesis was demonstrated in the oocytes, granulosa and stromal cells. The silver grain density of the follicular fluid in large follicles reached a peak at 4 h, remained high at 8 h after injection and decreased steadily at the subsequent intervals. It was demonstrated that the labeling pattern of the follicular fluid depends on the secretory activity of the granulosa cells and also on the specific activity of serum glycoproteins. The collapsed zonae pellucidae which represent the highest degree of follicular atresia are able to take up glycoprotein macromolecules. Based on this finding and also on the labeling pattern of the large follicles it was shown that there is very little synthesis of specific glycoproteins for the zona pellucida in large follicles. A more specific labeling of the zona pellucida occurred in the medium follicles. Following the growth of these follicles having a previously labeled zona pellucida, it was demonstrated that this extracellular structure is secreted by the oocyte.     

Expression of rabbit zona pellucida-1 messenger ribonucleic acid during early follicular development

Progress in research on initiation of folliculogenesis has progressed slowly because of a lack of markers for early folliculogenesis. The rabbit zona pellucida protein (ZP1) is synthesized in follicles during early stages of folliculogenesis. In order to establish ZP1 as a marker for initiation of folliculogenesis, in situ hybridization was used to localize ZP1 mRNA in immature follicles. ZP1 mRNA was first detected in oocytes of some but not all primordial follicles. The primordial follicles expressing ZP1 mRNA were located at the cortico-medullary junction, indicating that they were newly activated follicles. ZP1 mRNA accumulated in oocytes of intermediate, primary, and secondary follicles. In contrast, ZP1 mRNA was first detectable in granulosa cells of intermediate follicles and is present in cuboidal granulosa cells of primary and early secondary follicles, but was undetectable in granulosa cells of more mature follicles. These data demonstrate that 1) ZP1 mRNA is expressed in both oocytes and granulosa cells, 2) ZP1 mRNA is initially expressed in oocytes of activated follicles, and 3) ZP1 mRNA is transiently expressed in granulosa cells during early stages of folliculogenesis. Therefore, rabbit ZP1 is a molecular marker that can be used in future studies to measure initiation of folliculogenesis.     

Granulosa Cell Deficient Follicles

The diameters of oocytes in follicles having a single layer of granulosa cells were measured hi four week old mice of various strains. There is a unique population of these follicles hi strains LT/Sv and C58/J in which the oocytes are significantly larger than the oocytes in single granulosa cell layered follicles of other common strains (C57BL/6J, BALB/cJ, and DBA/2J). These unique follicles are referred to as granulosa cell deficient (GCD) follicles since oocytes of these sizes are usually found in follicles with more than a single layer of granulosa cells. The parthenogenetic embryos that give rise to ovarian teratomas in strain LT/Sv are usually found in GCD-follicles. Some of the ova of strains LT/Sv and LTXBP, but not the ova of the other strains, are capable of spontaneous parthenogenetic activation after meiotic maturation. Although the ovulated ova of strain LTXBP are capable of spontaneous parthenogenetic development, the frequency of GCD-follicles and teratocarcinogenesis is low. Therefore, the frequency of ovarian teratocarcinogenesis is correlated with the simultaneous occurrence of two atypical conditions: first, the capability of the matured ova to undergo spontaneous parthenogenetic activation and, second, the high frequency of GCD-follicles.
GCD-follicles containing oocytes with a diameter greater than 65 μm were studied by electron microscopy. The follicles are usually enclosed within a layer of flattened theca-like cells. A basal lamina separates these cells from a single layer of cuboidal granulosa cells. Granulosa cell processes traverse the zona pellucida to contact the oocyte which shows ultrastructural characteristics typical of oocytes in the final growth stages. It is proposed that the GCD-follicles are competent to participate in the normal functions of follicular cells relating to oocyte growth and meiotic maturation.     

Targeted expression of Bcl-2 in mouse oocytes inhibits ovarian follicle atresia and prevents spontaneous and chemotherapy-induced oocyte apoptosis in vitro.

Members of the Bcl-2 family serve as central checkpoints for cell death regulation, and overexpression of Bcl-2 is known to inhibit apoptosis in many cell types. To determine whether targeted expression of Bcl-2 could be used to protect female germ cells from apoptosis, we generated transgenic mice expressing fully functional human Bcl-2 protein only in oocytes. Transgenic mice were produced using a previously characterized 480-bp fragment of the mouse zona pellucida protein-3 (ZP3) gene 5'-flanking region to direct oocyte-specific expression of a human bcl-2 complementary DNA. Immunohistochemical analyses using a human Bcl-2-specific antibody showed that transgene expression was restricted to growing oocytes and was not observed in the surrounding ovarian somatic cells or in any other nonovarian tissues. Histomorphometric analyses revealed that ovaries collected from transgenic female mice possessed significantly fewer atretic small preantral follicles compared with wild-type sisters, resulting in a larger population of healthy maturing follicles per ovary. However, the number of oocytes ovulated in response to exogenous gonadotropin priming and the number of pups per litter were not significantly different among wild-type vs. transgenic female mice. Nonetheless, oocytes obtained from transgenic mice and cultured in vitro were found to be resistant to spontaneous and anticancer drug-induced apoptosis. We conclude that targeted expression of Bcl-2 only in oocytes can be achieved as a means to convey resistance of the female germ line to naturally occurring and chemotherapy-induced apoptosis.     

Morphological investigations on follicular atresia in canine ovaries

Summary Morphologically, canine ovaries show two types of regression pattern in secondary follicles. In type A necrotic changes of the oocytes and the zona pellucida dominate, whereas in type B degeneration, necrobiosis and necrosis of the granulosa prevail. The atretic course of type B regression results in a pseudoantrum and leads, by pseudogrowth, to a structure imitating tertiary follicles. In both type A and type B regression, four consecutive stages of atresia are distinguished by light- and electron microscopy.True tertiary follicles display only one regression pattern which resembles type B of secondary follicles. Early, advanced, late, and terminal stages of atresia are again described.     

A quantitative and qualitative cytochemical analysis of glycosaminoglycan content in the zona pellucida of hamster ovarian follicles

In this study the amount of neutral and acidic glycosaminoglycans in the zona pellucida and antrum of primary, preovulatory, and atretic follicles was analyzed. Serial sections of preovulatory hamster follicles were stained with PAS or alcian blue to estimate the content of neutral and acidic glycosaminoglycans, respectively. The amount of hyaluronic acid, chondroitin sulfate and sialic acid was determined using enzyme digestion procedures followed by alcian blue staining. Microdensitometric analyses showed that the strongest PAS staining was in the zona pellucida of atretic follicles, less staining in preovulatory follicles but more than in the antrum of preovulatory follicles. No hyaluronic acid was found in the zona pellucida of any follicular type, but there was a measurable amount in the antrum of preovulatory follicles. Chondroitin sulfate was present in the zona pellucida of primary and atretic follicles, as well as in the antrum of preovulatory follicles. Sialic acid was present in the antrum and zona pellucida of all follicular types. Sialic acid plays a role in receptor recognition and its presence may reflect the role of the zona pellucida in sperm recognition and fertilization.     

Ultrastructural evaluation of oocytes during atresia in rat ovarian follicles

Mammalian females are born with a finite number of ovarian oocytes, the vast majority of which ultimately undergo degeneration by atresia. The overall process of ovarian follicular atresia has been morphologically well described only in large antral follicles. Additionally, little attention has been focused on ultrastructural changes in the oocyte. Furthermore, most such morphological studies were performed prior to identification of apoptosis as a mechanism of physiological cell death. Therefore, the purpose of this study was to use electron microscopy to compare the process of atretic oocyte degradation in ovarian follicles of female Fischer 344 rats (38 days old) with ultrastructural characteristics of apoptosis. Examination of ovarian follicles revealed that nucleolar segregation, cytoplasmic or nuclear condensation, apoptotic body formation, and chromatin margination along the nuclear membrane are never observed in atretic oocytes during the degenerative process. Instead, early morphological changes in atretic oocytes include retraction of granulosa cell- and oocyte-derived microvilli and condensation of mitochondria and loss of cristae. These occurrences coincide with initiation of granulosa cell apoptosis. After most granulosa cells are lost, more severe changes occur, including segmentation of the oocyte and cytoplasmic vacuolization as atresia progresses. Thus, these results suggest that, during atresia, oocytes are removed by physiological oocyte cell death, a method that does not involve classically described apoptosis.     

A quantitative and qualitative cytochemical analysis of glycosaminoglycan content in the zona pellucida of hamster ovarian follicles

Summary In this study the amount of neutral and acidic glycosaminoglycans in the zona pellucida and antrum of primary, preovulatory, and atretic follicles was analyzed. Serial sections of preovulatory hamster follicles were stained with PAS or alcian blue to estimate the content of neutral and acidic glycosaminoglycans, respectively. The amount of hyaluronic acid, chondroitin sulfate and sialic acid was determined using enzyme digestion procedures followed by alcian blue staining. Microdensitometric analyses showed that the strongest PAS staining was in the zona pellucida of atretic follicles, less staining in preovulatory follicles but more than in the antrum of preovulatory follicles. No hyaluronic acid was found in the zona pellucida of any follicular type, but there was a measurable amount in the antrum of preovulatory follicles. Chondroitin sulfate was present in the zona pellucida of primary and atretic follicles, as well as in the antrum of prevulatory follicles. Sialic acid was present in the antrum and zona pellucida of all follicular types. Sialic acid plays a role in receptor recognition and its presence may reflect the role of the zona pellucida in sperm recognition and fertilization.     

Distribution of the intermediate filament proteins vimentin,keratin, and desmin in the bovine ovary

The distribution of the intermediate filament (IF) proteins desmin, keratin, and vimentin was studied immunohistochemically in bovine ovaries. Special attention was paid to granulosa cells to examine possible marked changes of IF distribution in relation to folliculogenesis during ovarian development. Therefore, ovaries were used from fetuses from 3 months of gestation onward, calves, heifers, and cows. In all ovaries, desmin immunoreactivity was restricted to smooth muscle cells in blood vessel walls. Keratin appeared a characteristic of the ovarian surface epithelium. Co-localization of keratin and vimentin was observed in the epithelium of rete ovarii tubules in fetuses and calves, and in cortical cord epithelium and pregranulosa cells of primordial follicles in fetuses at 3–7 months of gestation. Vimentin was demonstrated in endothelium and in fibroblasts. In addition, vimentin immunoreactivity was present in granulosa cells of primary, secondary, and antral follicles. In antral follicles, these granulosa cells mainly had an elongated appearance and either contained an oblong or a round nucleus. Those with an oblong nucleus were characteristic for atretic antral follicles. In nonatretic follicles, numerous vimentin immunore-active, elongated granulosa cells with a round nucleus were observed, especially in the peripheral granulosa layer and in small (<3 mm in diameter) antral follicles. Additionally, in antral follicles, protrusions of vimentin-positive corona radiata cells were observed, that penetrated the zona pellucida to contact the oocyte. The data show that the distribution of vimentin containing IFs is associated with various aspects of granulosa cell activity, as mitosis, atresia, and intercellular transport. © 1995 Wiley-Liss, Inc.     

In vivo changes in oocyte germinal vesicle related to follicular quality and size at mid-follicular phase during stimulated cycles in the cynomolgus monkey

Histological examination of gonadotrophin stimulated Macaca fascicularis ovaries removed at mid-follicular phase showed that germinal vesicles (GV) could exhibit different configurations in follicles greater than 1000 microns in diameter. We describe 3 types of nuclear organization called GV1 (dispersed and filamentous chromatin), GV2 (clumped and filamentous chromatin) and GV3 (perinucleolar chromatin condensation). Gonadotrophin stimulation and follicular atresia induced modifications in GV chromatin dispersion. Such modifications were of a higher degree in the case of atresia which could even induce in vivo germinal vesicle breakdown (GVBD). Our findings were as follows. The frequency of GV1 oocytes was always low, but was higher in healthy than in atretic follicles, whereas GV3 oocytes were more frequent in atretic compared to healthy follicles; the oocytes which resumed meiosis in vitro were most probably those which were at the GV3 stage at the time of recovery; GV nuclear changes were related to follicle size and quality, but not to oocyte size. The mean follicular size increased from GV1 to GV3 oocyte stages whatever the follicle quality; the nucleus was often observed in a peripheral position even in GV1 oocytes; zona pellucida appearance was related to GV stage and follicle quality and was more often observed to be abnormal or absent in case of GV3 oocytes included in atretic follicles. Oocyte nuclear modifications therefore appear to be a prerequisite to resumption of meiosis.