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BOR - Papers in Press, published online ahead of print August 20, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.017442
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BIOLOGY OF REPRODUCTION 69, 2022–2028 (2003)
DOI: 10.1095/biolreprod.103.017442
© 2003 by the Society for the Study of Reproduction, Inc.

Ovary

Cholesterol Side-Chain Cleavage Cytochrome P450 and 3ß-Hydroxysteroid Dehydrogenase Expression and the Concentrations of Steroid Hormones in the Follicular Fluids of Different Phenotypes of Healthy and Atretic Bovine Ovarian Follicles1

Helen F. Irving-Rodgers, Malgorzata Krupa, and Raymond J. Rodgers2

Reproductive Medicine Unit, Department of Obstetrics and Gynaecology, University of Adelaide, Adelaide, South Australia 5005, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 3ß-hydroxysteroid dehydrogenase (3ß-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 3ß-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 3ß-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.

follicle, granulosa cells, ovary, steroid hormones


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The developing ovarian follicle undergoes one of two fates—continued growth to ovulation or atresia. In the former situation, on reaching a large ovulatory size (>10 mm in bovine ovaries), the epithelial granulosa cells express the steroidogenic enzymes, cholesterol side-chain cleavage cytochrome P450 (SCC), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), and cytochrome P450 aromatase [1–4]. The former two enzymes catalyze the conversion of cholesterol into pregnenelone and then progesterone, while the latter converts androgens, derived from the theca interna, into estrogens. As the follicle matures, receptors for luteinizing hormone (LH) are expressed on the granulosa cells in these large follicles, accompanied by a decline in expression of follicle-stimulating hormone receptors [5]. Only follicles that have undergone these later maturation events ovulate their oocytes in response to the surge release of LH. Nonovulating follicles, which are the majority, undergo atresia and regression.

Heterogeneity exists within the large pool of healthy follicles <5 mm in diameter [6], from which emerges a wave of follicles on a growth trajectory. Within this heterogeneous population of follicles are two basic types, occurring in equal proportions. One type of healthy follicle has either rounded basal granulosa cells with a conventional single-layered follicular basal lamina and the other has columnar cells with a multilayered follicular basal lamina [7]. Localization of telomerase RNA within granulosa cells has also revealed different populations of healthy antral follicles [8]. In one population of follicles, the youngest cells (expressing telomerase) were located on the antral side of the membrana, whereas in other follicles, they were on the basal side [8]. It is considered from theoretical calculations that the two types of healthy follicles arise by differential rates of antrum expansion relative to the rate of granulosa cell replication [9]. The differential location of older cells within the membrana granulosa may have implications for atresia of follicles.

In atretic follicles, all the original histological subclassifications of atretic bovine follicles [10, 11] have now been resolved into two basic phenotypes—antral (AA) and basal atresia (BA) [12]. The original microscopic descriptions of bovine follicular atresia did not accurately identify BA [12]. In AA follicles, the wave of cell death begins at the antrum and progresses toward the basal lamina; in BA follicles, the direction is reversed. Macrophages breach the follicular basal lamina of BA but not AA follicles [12]. The remaining live cells, now no longer in contact with the follicular basal lamina, have the appearance of maturing granulosa cells. Lussier et al. [13] estimated that approximately 30% of bovine follicles 1.5–3.8 mm in diameter are atretic, and we estimate that about half of these are BA [12], so this form of atresia is not insignificant.

Numerous studies have measured steroid hormones in follicular fluid as a method for distinguishing between healthy and atretic follicles [14–17]. Several studies have shown that healthy follicles have higher concentrations of estradiol than either progesterone or androgen, while atretic follicles have elevated concentrations of progesterone or androgens [14, 15, 17]. Subsequently, researchers have used the ratio of the concentration of estradiol to progesterone in follicular fluid as an indicator of atresia, especially for large follicles. However, these methods of assessing atresia are unsuitable when applied to antral follicles <5 mm in diameter because aromatase is not expressed until later in follicular development [4].

For two reasons, we examined the expression of two steroid-biosynthetic enzymes, SCC and 3ß-HSD, in both small (<5 mm) healthy and atretic follicles and large (>=10 mm) healthy follicles, and measured the levels of steroid hormones in the follicular fluid from individually classified follicles. First, in previous studies of steroid hormones in follicular fluid, healthy and atretic follicles had not been segregated into their different phenotypes. Second, based on our preliminary histological observations suggesting that maturation of granulosa cells occurs in BA follicles, one would predict that steroid hormone synthesis and content are vastly different in this type of follicle, not only relative to healthy follicles but also to other atretic follicles. If this were true, the current biochemical method of classification of atresia would require re-evaluation.


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

Antibodies used have been reported previously [18]. The anti-SCC antibody used was rabbit anti-bovine adrenal mitochondrial SCC [19], obtained from OXYgene (Dallas, TX). The 3ß-HSD antibody used was mouse monoclonal antibody FDO66Q initially raised against JEG choriocarcinoma cells [20] and obtained from Flinders Technologies (Bedford Park, SA, 5042, Australia). FD066Q recognizes a very similar target to human 3ß-HSD type 1 [21, 22]. 3ß-HSD Type II is expressed in ovaries and is 94% homologous with type I in the human. FDO66Q binds to human ovarian follicles and Leydig cells (Bill Kalionis, personal communication), indicating that it cross-reacts with 3ß-HSD type II. FDO66Q also colocalizes to the same cells in bovine ovary (unpublished observation) as a rabbit polyclonal antibody raised against human placental 3-ßHSD and used to immunolocalize 3-ßHSD in human ovary [23].

Immunohistochemistry

For immunohistochemical studies, antral follicles were harvested from the ovaries of young, nonpregnant Bos taurus cows slaughtered at the local abattoir in South Australia and transported to the laboratory in Earle balanced-salt solution (D-2906; Sigma Bio Science, St. Louis, MO) on ice. For small follicles (2–5 mm), two were collected per ovary. For large follicles (6–17 mm), the number of ovaries in which there was one, two, or three follicles collected per ovary were 37, 2, and 1, respectively. Follicles were dissected from each ovary with the aid of a dissecting microscope fitted with an ocular micrometer and were snap frozen in Tissue Tek OCT Tissue Tek compound (Miles Inc., Elkhart, IN). Small (n = 87) or large (n = 29) frozen follicles were bisected and one half of each immersed in 2.5% glutaraldehyde, postfixed in osmium tetroxide, and embedded in epoxy resin, as previously described [24]. Thirteen large follicles were frozen in OCT only.

Methods for immunohistochemistry have been reported previously [18]. Tissue sections were cut from OCT-embedded bovine follicles using a CM 1800 Leica cryostat (Leica Microsystems Pty. Ltd., Victoria, Australia), collected on glass slides treated with 0.01% poly-L-ornithine hydrobromide (P-4638, Sigma Bio Science), and stored at -20°C until use. Unfixed sections were dried under vacuum for 5 min and then incubated in 10% normal donkey serum (D-9663; Sigma Bio Science) in antibody diluent containing 0.55 M sodium chloride and 10 mM sodium phosphate (pH 7.1) for 20 min. Sections were then incubated overnight with primary antibodies (mouse anti-human 3ß-HSD [1:1000]) or rabbit anti-bovine SCC (1 µg/ml IgG). The secondary antibodies used were biotin-SP-conjugated AffiniPure donkey-anti-mouse IgG (Cat # 715-0660151, 1:100) or biotin-SP-conjugated AffiniPure donkey-rabbit IgG (711-066-152, 1:200), followed by Cy3-conjugated-streptavidin (016-160-084, 1:100), from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA), in antibody diluent as above. All incubations were carried out at room temperature in a humidified chamber and, following incubation with primary or secondary antibodies or streptavidin-conjugated reagents, sections were washed (3 x 5 min) in hypertonic phosphate-buffered saline containing 0.274 M sodium chloride, 5.4 mM potassium chloride, 10 mM sodium phosphate, pH 7.2.

Histology

Sections were cut at a thickness of 1 µm with glass knives using a Richert-Jung Ultracut E ultramicrotome (Leica Microsystems Pty. Ltd.), stained with aqueous 1% methylene blue in 1% sodium tetraborate and examined by light microscopy.

Classification of Follicles by Light Microscopy

Follicles were classified on the basis of their morphology as healthy or atretic, using the epoxy resin-embedded half, where available, or frozen sections. Atretic follicles were assessed as undergoing BA or AA [12], and further divided into early, mid, or late stages based on the degree of cellular degeneration during the atresia process. Descriptions of the two forms of atresia are in the Results.

Microscopic Observations and Photography

Sections of bovine ovary stained with methylene blue were examined using an Olympus BX50 microscope and images captured with an Olympus DP12 digital camera system (Olympus Australia Pty. Ltd.). Sections processed for immunofluorescence staining were observed and photographed with an Olympus Vanox AHBT3 epifluorescence microscope with Olympus C35AD-4 camera attachment and photographed with Kodak T-Max 400 black and white film for light microscopy.

Follicular Fluid Collection

For measurement of the concentrations of steroid hormones in follicular fluids, additional ovaries were collected as above and 1–3 small antral follicles were dissected from each ovary (Table 1), the diameters were measured as above, and then the follicles were snap frozen on dry ice. A section of the follicle wall was cut off and immersed in 2.5% glutaraldehyde and processed as above. The remaining frozen follicle was stored at -70°C for subsequent collection of follicular fluid, at which time follicles were thawed on ice, follicular fluid aspirated, and centrifuged at 4°C at 14 000 rpm for 10 min to remove particulate matter.


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  		TABLE 1. Data on the follicles used for assay of the follicular fluid concentrations of progesterone (P), androstenedione (A), testosterone (T), and estradiol (E2).*

Steroid Hormone Assays

Steroids in follicular fluid were all measured by radioimmunoassays (Diagnostic System Laboratories, Webster, TX); progesterone (DSL-3400), androstenedione (DSL-4200), testosterone (DSL-4100), and estradiol (DSL-4400). Follicular fluid samples were diluted as required to be measured on the logarithmic part of the standard curve in 0.1 M phosphate-buffered saline (pH 7.2) containing 0.01% bovine serum albumin (Miles, Kankakee, IL) and 0.1% sodium azide. For the estradiol assay, it was necessary to replace the buffer supplied with our dilution buffer. Sensitivities and intra- and interassay coefficients of variation for each assay are shown in Table 2.


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  		TABLE 2. Parameters of steroid hormone assays

Statistical Analyses

Follicle diameter measurements and steroid concentrations in follicular fluid are presented as means ± SEM. Data were analyzed by ANOVA and Student-Newman-Keuls tests for comparison of the means. Estradiol and estradiol to progesterone ratios were log transformed to normalize data before analysis. P-values of < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Follicle freezing and thawing in fixative caused no detectable damage to cell walls, as detected by light microscopy (Fig. 1). Healthy follicles <5 mm in diameter (Fig. 1, a and b) had a membrana granulosa consisting of compact layers of granulosa cells with round or ovoid nuclei and occasional mitotic figures (Fig. 1a). Basal granulosa cells were in close apposition with the follicular basal lamina and either rounded (Fig. 1a) or columnar (Fig. 1b) in shape, as previously reported [7].



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  		FIG. 1. Light microscopy of healthy follicles (a, b) and atretic follicles (c, d). Healthy follicles have either rounded basal granulosa cells (a) or columnar basal granulosa cells (b). Mitotic figures are occasionally seen [arrow in (a)]. Antral atretic follicles (c) show loss of granulosa cells closest to the antrum and reduced layers of granulosa cells in comparison with healthy follicles. Pyknotic nuclei in antrum (open arrows), pyknotic nuclei in granulosa cells (solid arrows). Basal atretic follicles (d) show death of the basal granulosa cells and hypertrophy of the remaining granulosa cells separated from the basal lamina by a zone containing follicular fluid and granulosa cell debris (asterisk). (a) = 3 mm follicle; (b) = 2.5 mm follicle; (c) = 4 mm follicle; (d) = 2 mm follicle. G, Membrana granulosa; T, theca interna. Scale bar = 20 µm

AA follicles were characterized by numerous pyknotic nuclei present in either the layer(s) of the membrana granulosa closest to the antrum or in the antrum itself and adjacent to the membrana granulosa (Fig. 1c). The layers of granulosa cells closest to the basal lamina were tightly packed at all stages except advanced atresia, when all the layers had degenerated. In contrast, BA follicles exhibited initial destruction of the most basal layer of granulosa cells while the most antral granulosa cells remained healthy and closely opposed to each other (Fig. 1d). The cells in the most basal layer of the membrana granulosa were separated from each other and often from the basal lamina by large intercellular spaces (Fig. 1d). Capillaries and macrophages had breached the basal lamina and were present in the basal area [12]. As previously reported [12], BA was observed only in small (<5 mm) follicles.

Atretic follicles were further classified as early, mid, or late atretic. Early AA follicles had pyknotic nuclei in the antra, mid atretic follicles had pyknotic nuclei within the antral layers, and late atretic follicles had advanced pyknosis such that only 1–2 layers of basal healthy cells remained. Early-stage BA was difficult to differentiate from healthy follicles by light microscopy and was not attempted; mid-BA follicles had dead cells in the basal layer with partial expansion of matrix in this region. In late-BA follicles, extensive basal areas of the former membrana granulosa were occupied by fluid, cell debris, and cells infiltrating from the theca interna.

In healthy follicles, SCC or 3ß-HSD were not detected in granulosa cells by immunohistochemistry until follicles had reached at least 10 mm (Fig. 2 and Table 3). The pattern of expression in the granulosa cells of small AA follicles was similar to healthy follicles of the same size. The granulosa cells of BA follicles expressed both SCC and 3ß-HSD. These follicles only occur at sizes <5 mm, and hence their expression of the steroidogenic enzymes is in contrast with the absence of these enzymes in both healthy and AA follicles of the same sizes.



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  		FIG. 2. Immunofluorescent localization of SCC (a, c, e) and 3ß-HSD (b, d, f, g) in healthy (a, b) and basal (c, d) and antral (e, f, g) atretic follicles. a and b is dual localization on the same section. a, b) = 10-mm follicle; (c) = 2.5-mm follicle; (d) = 4-mm follicle; (e) = 3-mm follicle, (f) = 4.5-mm follicle, (g) = 10-mm follicle. G, Membrana granulosa; T, theca interna. Scale bar = 20 µm for (a–d), 40 µm (e–g).


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  		TABLE 3. Proportion of follicles (per total number of follicles examined) classified by size, health, and type of atresia, expressing cholesterol side-chain cleavage cytochrome P450 (SCC) or 3ß-hydroxysteroid dehydrogenase (3ß-HSD) within the membrana granulosa

The follicular fluid concentrations of progesterone, androstenedione, testosterone, estradiol, and the estradiol to progesterone ratio of healthy and atretic follicles are shown in Figures 3 and 4. Sizes and numbers of follicles used for measurements of follicular fluid steroids are shown in Table 1. Small healthy and atretic follicles averaged 3.6 mm in diameter and large healthy follicles were 12.6 ± 0.6 mm in diameter. BA follicles had a significantly elevated concentration of progesterone compared with either type of small or large healthy follicles or AA follicles (P < 0.001) (Fig. 3). Androstenedione concentration was greater in AA follicles than large healthy or BA follicles, but small healthy follicles did not differ significantly from other follicles (P > 0.05) (Fig. 3). Testosterone concentration was also elevated in small healthy follicles in comparison with large healthy follicles and small follicles undergoing BA, but not AA (P < 0.01) (Fig. 3). In contrast, estradiol was significantly elevated in large healthy follicles (P < 0.001) and significantly elevated in small healthy follicles relative to atretic follicles of the same sizes (P < 0.001) (Fig. 4). All healthy follicles had a significantly elevated ratio of estradiol to progesterone in comparison with BA follicles, with large healthy follicles having the highest estradiol to progesterone ratio and BA the lowest (Fig. 4). AA follicles had a significantly higher estradiol to progesterone ratio than BA follicles (P < 0.001) and a significantly lower ratio than either small healthy follicles (P < 0.05).



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  		FIG. 3. Concentration of progesterone, androstenedione, and testosterone in follicular fluid of different classes of healthy and atretic follicles (mean ± SEM). Comparisons in steroid hormone concentrations were made only within each steroid hormone. Thus, bars within each steroid hormone with different alphabetical superscripts are significantly different from one another. (a, b) P < 0.001, (d, e) P < 0.05; (f, g) P < 0.01



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  		FIG. 4. Concentration of estradiol in follicular fluid and the ratio of the concentrations of estradiol to progesterone in follicular fluid of different classes of healthy and atretic follicles (mean ± SEM). Comparisons in steroid hormone concentrations were made only within each measurement. Thus, bars within each steroid hormone or ratio with different alphabetical superscripts are significantly different from one another, P < 0.001. The number of follicles used in each measurement is shown in brackets


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study of small bovine antral follicles, the AA follicles were similar to healthy follicles in their levels of progesterone, androstenedione, and testosterone, whereas BA follicles had elevated levels of progesterone and reduced levels of androstenedione and testosterone. Estradiol differed between all follicles types and was highest in the healthy follicles, intermediated in the AA follicles, and lowest in the BA follicles. Consistent with these findings, the remaining live granulosa cells of BA follicles expressed the requisite biosynthetic enzymes, SCC and 3ß-HSD. In larger healthy follicles, estradiol was >10-fold greater than that of the smaller follicles, and the granulosa cells expressed SCC and 3ß-HSD. At the large sizes of follicles, the only type of atresia seen was AA, and these follicles also expressed SCC and 3ß-HSD like the healthy follicles.

As healthy follicles enlarge to >5 mm, only follicles with rounded basal granulosa cells were observed. Granulosa cell expression of the steroidogenic enzymes SCC and 3ß-HSD occurred when healthy bovine follicles reached 10 mm in diameter. This is consistent with the previous findings of no detectable SCC in granulosa cells in 2–5-mm follicles [25] and of increased 3ß-HSD mRNA expression in granulosa cells from follicles of increasing size [26]. However, previous studies have detected mRNA for SCC in granulosa cells of follicles [27, 28] far smaller in diameter than when the SCC protein was first detected here. This suggests that the levels of SCC mRNA and protein could be regulated independently or that the immunodetection was less sensitive than the in situ hybridization method used previously. Follicular fluid concentrations of steroids were similar to those reported previously for bovine antral follicles [15, 16, 29]. Follicular fluid of healthy, large antral follicles (>10 mm) in current experiments also had significantly greater estradiol concentrations and a higher ratio of estradiol to progesterone than follicles <5 mm in diameter. Thus, the subclassification of healthy follicles undertaken here did not reveal any new information about these different phenotypes of healthy follicles, suggesting that they do not have functional differences in terms of their steroid biosynthetic capacity.

AA follicles occurred at all sizes examined. Expression of SCC and 3ß-HSD protein by granulosa cells of AA follicles corresponded with that for healthy follicles of the same size. At small sizes, the levels of steroid hormones were similar to healthy follicles. In contrast, in BA follicles, the granulosa cells expressed SCC and 3ß-HSD, as reported previously [18], and the progesterone levels were elevated. Thus, clearly the process of BA is very different from AA and involves the maturation of granulosa cells into progesterone-producing cells. These findings can explain results of two other studies. Bao et al. [26] noted 3ß-HSD mRNA expression in remnant hypertrophied granulosa cells from some 6-mm atretic follicles and Grimes et al. [30] found that 13 out of 177 (7%) of follicles of 5–15 mm had substantially elevated progesterone levels. They designated the follicles as luteinizing atretic. In both studies, the follicles being observed were likely to be BA follicles.

Many other studies have been undertaken to determine the steroid hormone profile of healthy and atretic follicles. McNatty et al. [15] classified follicles according to the macroscopic appearance of the thecal tissue, presence of debris in follicular fluid, the status of the oocyte, and total number of granulosa cells recovered. They found more progesterone and less estradiol in atretic follicles than healthy follicles of a comparable size. Other investigators have also observed an elevated concentration of progesterone [17, 30] in atretic follicles. With respect to the current study, it should be noted that larger sized follicles were examined in the previous studies; nevertheless, Grimes et al. [30] concluded that nonatretic and atretic follicles could not be accurately identified by the ratio of estradiol to progesterone in follicular fluid. Where the ratio of estradiol to progesterone does appear to be useful is in the identification of the potentially dominant follicle [31, 33], and estradiol concentration alone has been proposed as sufficient to assess the health status of large antral follicles [34].

Khandoker et al. [35] individually characterized 2- to 6-mm follicles as healthy or atretic based on the light microscopic appearance of the cumulus-oocyte complex. In atretic follicles, they found increased levels of progesterone, reduced levels of testosterone, and a reduced ratio of estradiol to progesterone, consistent with the present findings. Our results confirm that atretic follicles do have increased capacity to synthesise progesterone, but this is confined to BA follicles. BA follicles also had significantly reduced androstenedione as well as testosterone in comparison with AA follicles, indicating a qualitative change in theca interna of BA follicles. We found significantly lower concentrations of estradiol in both types of atretic follicles <5 mm in diameter relative to healthy follicles. Not inconsistent with these findings, Smith et al. [36] found reduced levels of estradiol in follicles 2–7 mm following emergence of a dominant follicle, a time when larger follicles at least are known to undergo atresia.

Why are there two types of healthy follicles and two types of atretic follicles? We have hypothesized, but not yet shown, that the two phenotypes of healthy follicle could arise merely by different rates of antrum expansion relative to granulosa cell replication [9]. It is well known that follicles expand at different rates. Lussier et al. [13] estimated that small bovine antral follicles (0.14–0.29 mm) grow at a slower rate than larger antral follicles (0.68–3.67 mm). In any event, it has been observed that, in some follicles, the younger cells are nearest the antrum and others are nearest the basal lamina, which could come about by different rates in antrum expansion relative to the rate of granulosa cell replication [9]. One corollary of the different locations of older cells is that, if on atresia the oldest cells die first, one would expect death to be initiated either from the antral side or the basal side, thus producing the two patterns of atresia observed.

During the course of BA, some granulosa cells die as others mature into steroidogenic cells. A most interesting question is why SCC and 3ß-HSD are upregulated and the level of progesterone synthesis increased, especially when this does not occur in AA. One clear difference is that the membrana granulosa becomes detached from the follicular basal lamina in BA but not in AA follicles. Basal laminas are important sheets of specialized extracellular matrix that underlie and surround groups of cells such as epithelia or endothelia (see [37]). Epithelial cells interact with the basal lamina, enabling them to orientate their basal/apical polarity and preventing their death by anoikis [38]. In BA, the remaining live granulosa cells have no anchorage and hence control over their polarity. Epithelial cells lose apical-basal polarity, cellular adhesion molecules, and cell-cell junctions during the process of epithelial-mesenchymal transition (EMT) [39]. Epithelial granulosa cells readily undergo EMT and develop into mesenchymal luteal cells [37], or luteinization as it is colloquially termed, at ovulation when there is degradation of some components of the follicular basal lamina. In undergoing an EMT, the granulosa cells substantially upregulate their capacity for progesterone synthesis. Hence, as an explanation of the behavior of granulosa cells in BA, the remaining live cells commence the process of EMT following their detachment from the basal lamina and consequently upregulate their capacity to secrete progesterone. However, they eventually die, possibly by anoikis, as they appear unable to complete the process of EMT or luteinization.

To accurately assess and classify follicles into different phenotypes of atresia and health, follicles were snap frozen. Frozen follicle were divided into two, part for histological processing and part for collection of follicular fluid. While this has advantages for observing the architecture of the follicle for histological examination, there is the potential for contamination of the follicular fluid by intracellular debris. We acknowledge this but doubt that this contributes to or diminishes the value of the current data because steroid hormone diffuses freely across cell membranes.

In summary, it has been shown that two types of atresia occur in small bovine follicles. The behavior of the cells is very different, suggesting that very different mechanisms exist to bring about the two types of atresia. The results will also help clarify considerably the dichotomy of previous methods used for classifying atresia.


   ACKNOWLEDGMENTS
 
We would like to thank Jenny Hayes for ovary collection, Stephanie Morris for assistance with follicle dissection and histological classification, and Alan Gilmore for assistance with the assay of the steroid hormones.


   FOOTNOTES
 
1 Supported by the National Health and Medical Research Council of Australia, Adelaide University, and the Clive and Vera Ramaciotti Foundation. Back

2 Correspondence: FAX: 61 8 8303 4099; ray.Rodgers{at}Adelaide.edu.au Back

Received: 17 April 2003.

First decision: 5 May 2003.

Accepted: 12 August 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Rodgers RJ, Rodgers HF, Hall PF, Waterman MR, Simpson ER. Immunolocalisation of cholesterol side-chain-cleavage cytochrome P-450 and 17{alpha}-hydroxylase cytochrome P-450 in bovine ovarian follicles. J Reprod Fertil 1986 78:627-638[Abstract/Free Full Text]
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  8. Lavranos TC, Mathis JM, Latham SE, Kalionis B, Shay JW, Rodgers RJ. Evidence for ovarian granulosa stem cells: telomerase activity and localisation of the telomerase RNA component in bovine ovarian follicles. Biol Reprod 1999 61:358-366[Abstract/Free Full Text]
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