Aspects of cellular proliferation during follicular atresia in the dog ovary

Summary Autoradiography after pulse labelling with [³H] thymidine was applied to investigate the proliferation processes in the granulosa and theca related to follicular atresia of the dog ovary during metestrus.The number of proliferating cells depends on the follicle type and its atretic stage. There is less proliferation in smaller secondary follicles than either in larger ones or tertiary follicles. While in early atresia tertiary follicles show the highest labelling indices, in advanced atresia the larger secondary follicles are those with the highest values. For each follicle type a decline in the labelling indices can be observed from early to terminal atresia. Tertiary follicles show a precipitous decrease in the labelling index between early and advanced atresia. There is a continuous gradient of proliferation from the center of the follicle over the peripheral granulosa to the theca. In tertiary follicles, an inverse correlation between labelling and necrosis of granulosa cells can be observed.     

Histological and histochemical studies of post-ovulatory and pre-ovultory atretic follicles in Mustelus canis

The atresia of post-ovulatory and pre-ovulatory follicles of the viviparous smooth dogfish, Mustelus canis, is compared for approximately the first fourth of an 11 month gestation. A thick collagenous sheath and numerous tubules in the theca identify the large, folded stage A post-ovulatory follicle. In stage B the tubules have been filled by cells to form “islands.” In stage C the entire structure is greatly diminished, adjacent islands tend to fuse, the collagenous sheath is virtually gone and the granulosa is degenerating. Preovulatory follicles from large, yolky oocytes pass through four stages beginning with yolk phagocytosis by granulosa cells of the villi (stage I), which are long and granular in stage II; villi fuse, theca cells increase greatly, fill with granules (stage III), encroach on the granulosa and disperse it into small groups of cells which finally disappear (stage IV) leaving a mass of thecal cells. A special type of pre-ovulatory follicle from small non-yolky oocyte atresia exhibits prominent thecal tubules and an unusual arrangement of granulosa cells. This follicle appearrs to enlarge during the summer, becoming multilobed; few granules are present. The distribution of lipid in frozen sections, stained by Oil red O, is described for all types of follicles. Schultz and Lewis and Lobban tests for steroids were made on frozen sections with corresponding results. Positive green tests indicating the presence of steroids or possible steroidogenesis were limited to: (1) one post-ovulatory follicle, in the islands; (2) four stage III and seven late stage IV pre-ovulatory yolky atretic follicles; (3) two special atretic follicles. The special atretic follicle appears to be a unique feature of M. canis and it is suggested tentatively that it may be related to viviparity.     

Histological study of follicular atresia in the ovary of the domestic hen (Gallus domesticus)

Semi-serial (1 in 20) sections of ovaries were studied and only two types of atresia were identified--non-bursting and bursting. Smaller, non-yolky follicles (less than 1 mm diameter) showed non-bursting atresia. Atresia in follicles greater than 1 mm diameter was invariably of the bursting type which involved the rupture of the follicular wall, and the extrusion of yolk and cellular debris through the rupture site into the stroma. However, this rupture site was small and consequently was not visible in every section but it could always be seen when the follicle was followed in semi-serial sections. The mitotic index of granulosa cells in bursting atretic follicles was much lower than that for normal follicles. The most common criteria for distinguishing non-bursting atretic follicles were the extremely shrunken, irregularly shaped oocytes and the separation of the granulosa from the theca. In bursting atretic follicles, reliable indications were the presence in the ooplasm of some cells or cellular debris, and disorganization of the yolk and granulosa tissue. The presence of pycnotic nuclei in the granulosa cells was not a consistent feature of all atretic follicles of the hen.     

Theca interna: the other side of bovine follicular atresia

Currently, histological classifications of ovarian follicular atresia are almost exclusively based on the morphology of the membrana granulosa without reference to the theca interna. Atresia in the bovine small antral ovarian follicle has been redefined into antral or basal atresia where cell death commences initially within antral or basal regions of the membrana granulosa, respectively. To examine cell death in the theca interna in the two types of atretic follicles, bovine ovaries were collected and processed for immunohistochemistry and light microscopy. Follicles were classified as healthy, antral atretic, or basal atretic. Follicle diameter was recorded and sections stained with lectin from Bandeiraea simplicifolia to identify endothelial cells or with an antibody to cytochrome P450 cholesterol side-chain cleavage to identify steroidogenic cells and combined with TUNEL labeling to identify dead cells. The numerical density of steroidogenic cells within the theca interna was significantly reduced (P < 0.001) in basal atretic follicles in comparison with other follicles. Cell death was greater in both endothelial cells (P < 0.05) and steroidogenic cells (P < 0.01) of the theca interna of basal atretic follicles compared with healthy and antral atretic follicles. Thus, we conclude that the theca interna is susceptible to cell death early in atresia, particularly in basal atretic follicles.     

DNA degradation in mural granulosa cells of non- and slightly atretic follicles of fresh and cold-stored domestic cat ovaries

Apoptosis of granulosa cells is associated with follicular atresia and may occur before atresia becomes morphologically evident. Detection of DNA fragmentation by in situ end-labeling (ISEL) with terminal transferase allows the histological assessment of apoptotic cells on conventional histological sections. Degradation of DNA also may occur after prolonged cold storage of ovaries caused by the release of lysosomal enzymes. The objectives of this study were to assess follicle atresia and the impact of cold storage for 8, 12, 24, and 48 hr after ovarian excision by assessing DNA degradation in mural granulosa cells of cat ovaries. Follicles were distinguished by morphological criteria as nonatretic (NA), slightly atretic (SA), or atretic, and the mean number (±SEM) of granulosa cells labeled by ISEL was determined. About 50% of follicles showed some sign of atresia independent from the stage of the reproductive cycle of the ovarian donor. Number of ISEL-stained granulosa cells for NA and SA, freshly collected follicles was 7.5 ± 0.6 and 9.3 ± 0.8 cells/field, respectively, compared to 16.2 ± 0.8 cells/field in the wall of atretic follicles (P < 0.001). Fresh NA follicles from luteal phase ovaries had more (P < 0.05) labeled granulosa cells (9.2 ± 0.7 cells/field) than measured in follicles of cats in a follicular phase (5.7 ± 0.7). During cold storage, DNA degradation began within 12 hr (NA, 12.2 ± 0.7 cells/field; SA, 13.3 ± 0.5), both values being different (P < 0.05) from fresh controls. By 24 hr, DNA degradation was at the level of a positive control subjected to DNAse treatment. In summary, results reveal that granulosa cell DNA degeneration precedes the loss of developmental capacity of cat oocytes during atresia and postexcision storage. Finding irreversible changes in granulosa cell DNA after storage of cat ovaries for >12 hr may be important for developing oocyte rescue protocols for rare felids in cases where prolonged storage and transport may be required. Mol. Reprod. Dev. 48:350–355, 1997. © 1997 Wiley-Liss, Inc.     

Expression and Preliminary Functional Profiling of the let-7 Family during Porcine Ovary Follicle Atresia

Most follicles in the mammalian ovary undergo atresia. Granulosa cell apoptosis is a hallmark of follicle atresia. Our previous study using a microRNA (miRNA) microarray showed that the let-7 microRNA family was differentially expressed during follicular atresia. However, whether the let-7 miRNA family members are related to porcine (Sus scrofa) ovary follicular apoptosis is unclear. In the current study, real-time quantitative polymerase chain reaction showed that the expression levels of let-7 family members in follicles and granulosa cells were similar to our microarray data, in which miRNAs let-7a, let-7b, let-7c, and let-7i were significantly decreased in early atretic and progressively atretic porcine ovary follicles compared with healthy follicles, while let-7g was highly expressed during follicle atresia. Furthermore, flow cytometric analysis and Hoechst33342 staining demonstrated that let-7g increased the apoptotic rate of cultured granulosa cells. In addition, let-7 target genes were predicted and annotated by TargetScan, PicTar, gene ontology and Kyoto encyclopedia of genes and genomes pathways. Our data provide new insight into the association between the let-7 miRNA family in granulosa cell programmed death.     

Light microscopic, enzyme biochemical and steroid analytical investigations of follicular atresia in the ovary of domestic goose

Follicular atresia in the ovary of the domestic goose was investigated by light microscopic, steroid RIA and lysosomal enzyme activity measuring methods during the spring reproduction cycle. Degenerative processes are associated with the transformation and proliferation of granulosa, internal and external thecal cells in the follicle. Seven types of atretic follicles were identified on the basis of the presence, absence or dominance of cells containing lipids and synthesizing steroids. Conclusive evidence for the relation between cell type and hormone content was found only in one type: in type 6, stromal glandular cells show an extremely intensive PROG synthesizing activity. In the other types it was shown that glandular type of cells which become proliferative during atresia possess a relatively uniform steroid synthesizing ability. This uniformity is also seen in the high activity of lysosomal enzymes regardless of the size and type of atretic follicles.     

Expression of tPA, LH receptor and inhibin a,βA subunits during follicular atresia in rats

By using DNA 3′-end labeling, immunocytochemistry and mRNA insitu hybridization detection techniques, the expression of inhibin subunits and LH receptor in the granulosa cells and tissue-type plasminogen activator (tPA) in the oocytes has been studied in relation to follicular development and atresia. The results demonstrated that: (i) tPA activity in the oocytes of normal developing follicles is undetectable, and increases significantly in the follicle undergoing atresia; (ii) the production of inhibin subunits in granulosa cells is negatively correlated with the expression of oocyte tPA activity, indicating that they may be an important regulator of oocyte tPA production and follicular development; (iii) in atretic follicles, granulosa cells do not express LH receptor and inhibin subunits. It is therefore suggested that tPA may play a role in oocyte self-destruction and clearance in some of atretic follicles, and inhibin of granulosa-origin might be an inhibitory factor for the translation of tPA in the oocyte. Project supported by the National Natural Science Foundation of China (Grant Nos. 39770099 and 39770290), Chinese Academy of Sciences and Rockefeller Foundation/WHO HPR research project.     

Follicular atresia in the infant human ovary.

The pattern of follicular atresia was studied in nine ovaries from children between the ages 3 months and 8 years. Atretic follicles were found among follicles at all stages of development. The percentage of follicles with signs of atresia became larger as the size of the follicles increased. Only 2% of small follicles (Type 3b) showed signs of atresia, while all follicles greater than 1 mm in diameter (Type 8) were atretic. In follicles of Type 5 and larger, four stages of atresia, which represent consecutive stages of a single atretic process, were defined. The beginning of atresia was characterized by the presence of pyknotic granulosa cells. As atresia progressed, the granulosa layer disappeared, the oocyte became necrotic, the follicle collapsed and the theca cells became hypertrophied. The oocyte can degenerate in several ways: it can be penetrated by cells, the nucleus can become pyknotic or it may complete meiotic prophase. It is suggested that the last event is only possible after the oocyte has reached its full size and has completed RNA synthesis.     

Sphingosine-1-phosphate and ceramide are associated with health and atresia of bovine ovarian antral follicles

The follicle destiny towards ovulation or atresia is multi-factorial in nature and involves outcries, paracrine and endocrine factors that promote cell proliferation and survival (development) or unchain apoptosis as part of the atresia process. In several types of cells, sphingosine-1-phospate (S1P) promotes cellular proliferation and survival, whereas ceramide (CER) triggers cell death, and the S1P/CER ratio may determine the fate of the cell. The aim of present study was to quantify S1P and CER concentrations and their ratio in bovine antral follicles of 8 to 17 mm classified as healthy and atretic antral follicles. Follicles were dissected from cow ovaries collected from a local abattoir. The theca cell layer, the granulosa cells and follicular fluid were separated, and 17β-estradiol (E2) and progesterone (P4) concentrations were measured in the follicular fluid by radioimmunoassay. Based on the E2/P4 ratio, the follicles were classified as healthy (2.2±0.3) or atretic (0.2±0.3). In both follicular compartments (granulosa and theca cell layer), sphingolipids were extracted and S1P and CER concentrations were quantified by HPLC (XTerra RP18; 5 µm, 3.0×150 mm column). Results showed that in both follicular compartments, S1P concentrations were higher in healthy antral follicles than in atretic antral follicles (P<0.05). The concentration of CER in the granulosa cells was higher in atretic antral follicles than in healthy antral follicles, but no differences were observed in the theca cell layer. The S1P/CER ratio in both follicular compartments was also higher in healthy antral follicles. Interestingly, in these follicles, there was a 45-fold greater concentration of S1P than CER in the granulosa cells (P<0.05), whereas in the theca cell layer, S1P had only a 14-fold greater concentration than CER when compared with atretic antral follicles. These results suggest that S1P plays a role in follicle health, increasing cellular proliferation and survival. In contrast, reduction of S1P and the S1P/CER in the antral follicle could trigger cellular death and atresia.     

Expression of tPA, LH receptor and inhibin α, βA subunits during follicular atresia in rats

By using DNA 3′-end labeling, immunocytochemistry and mRNA insitu hybridization detection techniques, the expression of inhibin subunits and LH receptor in the granulosa cells and tissue-type plasminogen activator (tPA) in the oocytes has been studied in relation to follicular development and atresia. The results demonstrated that: (i) tPA activity in the oocytes of normal developing follicles is undetectable, and increases significantly in the follicle undergoing atresia; (ii) the production of inhibin subunits in granulosa cells is negatively correlated with the expression of oocyte tPA activity, indicating that they may be an important regulator of oocyte tPA production and follicular development; (iii) in atretic follicles, granulosa cells do not express LH receptor and inhibin subunits. It is therefore suggested that tPA may play a role in oocyte self-destruction and clearance in some of atretic follicles, and inhibin of granulosa-origin might be an inhibitory factor for the translation of tPA in the oocyte.     

Size-frequency analysis of atresia in cycling rats

The purpose of this study was to delineate when, during follicular growth, the alternative developmental pathways leading to ovulation or atresia diverge. By using computerized image analysis techniques, random samples of healthy and atretic follicles in ovaries of cycling rats were subjected to size-frequency analysis. The vast preponderance of atretic follicles were of the early antral size class (approximately 300-350 micron diameter, 800-1000 granulosa cells in the largest cross-section); atretic small follicles (less than 250 granulosa cells in the largest cross-section) were rare. Follicles in early stages of atresia were uncommon in ovaries of animals killed at estrus, but were found with great frequency in ovaries of animals killed the following day (metestrus). These results suggest that, under normal cyclic conditions, there may be only one major branching point during follicular development when growing follicles become susceptible to atresia. The alternative developmental pathways leading to ovulation and atresia may not diverge until the penultimate stage of growth, immediately preceding the final transformation into a preovulatory follicle.     

Steroidogenic activity of atretic follicles in the cycling hamster ovary and relation to ultrastructural observations

To evaluate the relation between the steroidogenic activity and cell proliferation of individual follicles in mature hamster ovaries during the estrous cycle, the localization of enzymes involved in estrogen biosynthesis and bromodeoxyuridine (BrdU) incorporation were examined immunohistochemically. Moreover, granulosa cells from the early atretic follicle were examined by scanning and transmission electron microscopy. Immunoreactivity for aromatase was localized in the granulosa cells of healthy developing follicles and Graafian follicles, as well as in newly formed granulosa lutein cells. In the healthy follicles of an ovulation cycle, intensity of aromatase immunoreactivity was suddenly decreased on day 3. The theca interna cells of healthy developing follicles were immunopositive for 17-hydroxylase/C17–C20 lyase (17-lyase) from day 2 to the morning of day 4, but on the evening of day 4 most theca interna cells were immunonegative except for only a few cells of the large Graafian follicles. BrdU incorporation was observed in the granulosa cells of healthy developing follicles, in the endothelial cells of capillaries around the developing follicles, and of newly formed corpora lutea. Very early morphological signs of atresia was the pyknotic change of a few granulosa cells lining the antral cavity. In that follicle, the number of BrdU-incorporating granulosa cells was suddenly decreased whilst immunoreactivity of aromatase and 17-lyase were gradually decreased. These data suggest that the mechanism of the loss of aromatase activity from the granulosa cells of atretic follicles appears to differ from that in cycling follicles. Even in the early stage of atresia, the granulosa cells showed remarkable morphological change characteristic of apoptosis, as visualized by scanning and transmission electron microscopy. Cessation of granulosa cell proliferation may occur earlier than apoptotic change and the degeneration of the granulosa cells becomes rapid once atresia starts.     

Expression of tPA, LH receptor and inhibin α, β_A subunits during follicular atresia in rats

By using DNA 3'-end labeling, immunocytochemistry and mRNA in situ hybridization detection techniques, the expression of inhibin subunits and LH receptor in the granulosa cells and tissue-type plasminogen activator (tPA) in the oocytes has been studied in relation to follicular development and atresia. The results demonstrated that: (ⅰ) tPA activity in the oocytes of normal developing follicles is undetectable, and increases significantly in the follicle undergoing atresia; (ⅱ) the production of inhibin subunits in granulosa cells is negatively correlated with the expression of oocyte tPA activity, indicating that they may be an important regulator of oocyte tPA production and follicular development; (ⅲ) in atretic follicles, granulosa cells do not express LH receptor and inhibin subunits. It is therefore suggested that tPA may play a role in oocyte self-destruction and clearance in some of atretic follicles, and inhibin of granulosa-origin might be an inhibitory factor for the translation of tPA in     

Cholesterol side-chain cleavage cytochrome P450 and 3beta-hydroxysteroid dehydrogenase expression and the concentrations of steroid hormones in the follicular fluids of different phenotypes of healthy and atretic bovine ovarian follicles

Bovine ovarian antral follicles exhibit either one or the other of two patterns of granulosa cell death in atresia. Death can commence either from the antrum and progress toward the basal lamina (antral atresia) or the converse (basal atresia). In basal atresia, the remaining live antrally situated cells appeared to continue maturing. Beyond that, little is known about these distinct patterns of atresia. Healthy (nonatretic) follicles also exhibit either one or the other of two patterns of granulosa cell shape, follicular basal lamina ultrastructure or location of younger cells within the membrana granulosa. To examine these different phenotypes, the expression of the steroidogenic enzymes cholesterol side-chain cleavage cytochrome P450 (SCC) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in granulosa cells and concentrations of steroid hormones in follicular fluid were measured in individual histologically classified bovine antral follicles. Healthy follicles first expressed SCC and 3beta-HSD in granulosa cells only when the follicles reached an approximate threshold of 10 mm in diameter. The pattern of expression in antral atretic follicles was the same as healthy follicles. Basal atretic follicles were all <5 mm. In these, the surviving antral granulosa cells expressed SCC and 3beta-HSD. In examining follicles of 3-5 mm, basal atretic follicles were found to have substantially elevated progesterone (P < 0.001) and decreased androstenedione and testosterone compared to healthy and antral atretic follicles. Estradiol was highest in the large healthy follicles, lower in the small healthy follicles, lower still in the antral atretic follicles, and lowest in the basal atretic follicles. Our findings have two major implications. First, the traditional method of identifying atretic follicles by measurement of steroid hormone concentrations may be less valid with small bovine follicles. Second, features of the two forms of follicular atresia are so different as to imply different mechanisms of initiation and regulation.     

Immunocytochemical study of cell proliferation in the cystic ovarian follicles in cows

We examined the frequency of proliferating cells in cystic, atretic and healthy antral follicles to determine whether a disorder of cell proliferation was responsible for the occurrence of bovine cystic follicles. Paraffin sections of healthy follicles and various stages of atretic and cystic follicles were immunostained with mouse monoclonal antibody to proliferating cell nuclear antigen (PCNA). The PCNA-positive cells were counted in 4 different regions of a follicle from the apical to the basal side. In the granulosa layer, a significantly higher frequency of PCNA-positive cells was observed in the healthy follicle in the basal region as compared with the apical region. A similar pattern of PCNA-positive cells population was observed in the granulosa layer of atretic follicles, although the frequency in the basal region was significantly lower in the atretic than the healthy follicle. The rate of cell proliferation in the granulosa layer of cystic follicles was markedly lower at the basal region than that of atretic follicles. In the theca interna, the frequency of PCNA-positive cells in atretic follicles at the early stages was higher than that in cystic follicles at the early stages. These results suggest that in the healthy follicle the proliferative activity of granulosa cells is higher in the basal than the apical region, and that the cell proliferation activity in the granulosa and theca interna may decrease in association with the induction of a follicular cyst.     

Regression in the ovary of Myxine glutinosa L. (Cyclostomata) II. Electron microscope studies of atretic follicles

In the atretic follicle of the open involutionary type an opening in the wall of the follicle is formed through which granulosa cells and yolk platelets are emitted. Migrating cells of the theca layer invade the follicular lumen and absorb phagocytotically residues of granulosa cells. On the other hand, atretic follicles of the closed involutionary type show yolk platelets which remain in the follicular lumen and are dissolved there. The granulated residue of the yolk platelets and the residue of the granulosa cells are absorbed phagocytotically by migrating cells. The follicular atresia of both degenerating types can be regarded as a process exclusively devoted to the purpose of resorbing atretic oocytes. No indications for the production of steroid hormones were found.     

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     

Syndecan-4 expression is associated with follicular atresia in mouse ovary

Ovarian granulosa cells synthesize heparan sulfate proteoglycans (HSPGs), that have anticoagulant properties. Moreover, HSPGs greatly increase in the granulosa cells during follicular atresia. However, the species of ovarian HSPGs have not yet been identified. Syndecan-4 (ryudocan, amphiglycan) is a membrane-spanning HSPG and a member of the syndecan family. Herein, we demonstrate that syndecan-4 is expressed in the granulosa cells of type 4-5b follicles and, most intensely, in those of the atretic follicles in the mouse ovary, as revealed by in situ hybridization. There is no relationship between syndecan-4 expression and age or sexual cycle stage. Compared with syndecan-4 expression, syndecan-1 and -3 are expressed more abundantly in postovulatory follicles and the corpora lutea, but less in the type 4-5b follicles and much less in the atretic follicles. Immunohistochemistry also demonstrates syndecan-4 expression in atretic follicles with apoptosis. The present study has revealed the distinct modes of expression of the syndecan family members, and the association of syndecan-4 expression and apoptosis in ovarian atretic follicles.