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a Department of Animal Science, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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4 mm), medium (5–8 mm), and large (> 8 mm) follicles during follicular atresia occurs by apoptosis. The second objective was to establish an in vitro model system to elucidate the developmental (GC from follicles of different sizes) and hormonal (FSH and insulin-like growth factor-I [IGF-I]) regulation of bovine GC apoptosis during follicular atresia. Bovine ovaries were obtained from a nearby slaughterhouse. Follicles were classified by morphometric criteria as healthy or atretic. Apoptosis in GC from follicles of different sizes was analyzed by both morphological and biochemical methods. Bovine GC were cultured for 48 h at a density of 5 x 106 cells/ml in serum-free media at 39°C to determine the effects of FSH and IGF-I on apoptosis. The results showed that apoptosis occurred in GC from all sizes of follicles. Apoptosis in GC was also detected in some healthy follicles. Degenerate GC displayed the morphological characteristics of apoptosis, including nuclei with marginated chromatin, a single condensed nucleus, multiple nuclear fragments, and/or membrane-bound structures containing variable amounts of chromatin and/or cytoplasm (apoptotic bodies). All GC classified as apoptotic on the basis of their morphology contained fragmented DNA measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique. Cells that had undergone apoptosis were observed mainly in GC and in scattered theca cells. Throughout the GC layer, apoptotic cell death was more prevalent among antral GC than among mural GC. Interestingly, morphological results showed that no apoptosis occurred in cumulus cells. A time-dependent, spontaneous onset of apoptosis occurred in GC from small, medium, and large follicles during in vitro serum-free culture. The rate of DNA fragmentation in the culture of GC from small follicles was higher than that from medium and large follicles. FSH attenuated apoptotic cell death in GC from medium follicles more effectively than in those from small follicles. IGF-I also suppressed apoptosis in cultured GC from small follicles. In conclusion, this study showed that 1) GC death during bovine follicular development and atresia occurs by apoptosis; 2) apoptosis occurs in GC and theca cells; however, apoptosis does not occur in cumulus cells even in atretic antral follicles; 3) GC from all small, medium, and large follicles undergo spontaneous onset of apoptosis when cultured under serum-free conditions; and 4) FSH and IGF-I can attenuate apoptosis in cultured bovine GC.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies have suggested that degenerative changes associated with atresia appear initially in the granulosa cell (GC) layer. The death of GC leads to almost total destruction of the GC layer lining the inner follicular wall [3] and triggers the atresia of the follicles [4]. Recent studies have demonstrated that the death of GC during follicular atresia in ewe, pig, chicken, cow, and rodent ovaries occurs by apoptosis—a physiological, active, and genetically governed process whereby death of cells occurs in a controlled fashion triggered by changes in the levels of specific physiological stimuli [4, 5]. A unique biochemical event in apoptosis is the activation of a Ca2+/Mg2+-dependent endogenous endonuclease. The enzyme cleaves genomic DNA at internucleosomal regions, resulting in DNA fragments the size of 180–200 base pairs (bp). When separated by agarose gel electrophoresis, the DNA fragments can be visualized as a distinctive ladder pattern. The presence of this DNA pattern in cells is considered a hallmark indicator of apoptosis [5–7]. Cells undergo apoptosis under various physiological and experimental conditions and show distinctive morphological features, including condensed cytoplasm and nuclear chromatin coalesced into one or several large masses. As apoptosis continues, the nucleus breaks into several fragments; then the cell breaks up into several membrane-bound smooth-surfaced apoptotic bodies that contain a variety of intact cytoplasmic organelles and some nuclear fragments. Apoptotic bodies are typically phagocytosed by nearby cells or macrophages, or are extruded into body cavities [5–7].
In cows, apoptosis has been demonstrated in GC from follicles (> 4 mm only) and corpora lutea by the DNA ladder pattern [8–10]. It is known that although atresia occurs at all stages of follicle development [1], the majority of follicles undergo degeneration at the early antral stage rather than the preantral and preovulatory stages [11]. Considering that atresia is a stage-dependent process, the study of apoptosis in GC from follicles at different developmental stages is important. To our knowledge, no such study has been reported.
In addition to the detection of oligonucleosomes in isolated DNA, the occurrence of apoptosis may be inferred from the characteristic morphological appearance of degenerating cells, together with the detection of fragmented DNA in single cells in situ through the use of the 3' end-labeling technique (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling [TUNEL]) [12, 13]. TUNEL is a direct and specific method for the in situ visualization of apoptosis at the single-cell level, while preserving tissue architecture [12]. Previous histological studies have identified morphological changes of atretic follicles, including the degeneration and detachment of the GC layer from the basement membrane and the presence of pyknotic nuclei [4, 14, 15]. These morphological features are similar to, but not necessarily specific for, apoptotic cell death. In cows, there is no report on whether the changes in the morphological features of follicular cells during atresia conform to the distinctive morphological features of apoptotic cell death. Also, it is not clear whether cells or cell debris classified as apoptotic on the basis of morphological appearance contains fragmented DNA. In addition, it is not known what cell type(s) are involved in bovine follicle apoptosis.
During follicular development, FSH and insulin-like growth factor-I (IGF-I) have been considered follicle survival factors capable of stimulating estrogen production in vivo and in vitro [16, 17]. Considerable evidence has indicated that the occurrence of apoptosis in individual atretic follicles is correlated with decreased levels of intrafollicular estrogen [14, 15] and aromatase mRNA [14]. A recent study has found that both FSH and IGF-I attenuated apoptosis in cultured porcine GC [18]. In rats, on the other hand, FSH and IGF-I did not attenuate apoptosis in isolated GC in culture. Apoptosis in rat GC was attenuated by FSH and IGF-I only when they were added to cultures of intact preovulatory follicles [19]. In the bovine species, the roles of FSH and IGF-I in apoptosis in follicles are unknown.
The aims of the present study were 1) to determine whether the death of bovine GC from follicles (small [ 4 mm], medium [5–8 mm], and large [> 8 mm]) during follicular atresia occurs by apoptosis in the bovine species by showing a) that DNA fragments extracted from GC form a distinctive ladder pattern when separated electrophoretically, b) that morphological changes in GC during atresia conform with general criteria for apoptotic cell death tested using tissue sections stained with hematoxylin and eosin (H & E), and c) that cells classified as apoptotic on the basis of their morphology contain fragmented DNA as shown by the TUNEL technique; and 2) to establish an in vitro model system to analyze the role of FSH and IGF-I in modulating apoptosis in GC at different follicle developmental stages.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovaries were collected, and follicles were classified as previously described [20]. Briefly, ovaries were collected at a local slaughterhouse and transported to the laboratory (within 2 h of slaughter) on ice in chilled collection medium composed of Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 (1:1) containing 0.1% (w:v) BSA, L-glutamine, and 50 µg/ml gentamicin (Sigma Chemical Co., St. Louis, MO).
The follicles were classified into three groups based on surface diameters: small ( 4 mm), medium (5–8 mm), and large (> 8-mm). Follicle-size categories were selected on the basis of reported gonadotropin dependence and changes in the expression of steroidogenic enzyme and LH receptor mRNAs [21]. Studies have shown that preantral follicles can grow to the antral stage without gonadotropin support. At 4 mm, follicle growth will be halted if FSH is suppressed. From around 8 mm, follicles express LH receptors on GC and require pulsatile LH stimulation to continue growing [21]. Small, medium, and large follicles were then classified as healthy or atretic according to previously established morphological criteria [22]. Healthy follicles had vascularized (pink or red) theca interna and clear amber follicular fluid (FF) with no debris, and they contained > 25% of the maximum number of GC that could be present for a given follicle size (> 25% Gmax). Follicles that did not satisfy any one of these criteria were classified as atretic.
Morphological Analysis
Ovaries (n = 26) containing the follicles of interest (healthy or atretic) were excised with a scalpel and fixed in 4% neutral buffered formalin for 48 h. Fixed tissues were washed in PBS solution, dehydrated through a graded series of ethanol (70–100%), cleared in xylene, embedded in paraffin, and sectioned (5 µm). The sections (n = 50 from 26 ovaries) were deparaffinized in xylene, rehydrated through a graded series of ethanol (100–50%), and then stained with H & E to identify apoptotic cell death.
Histological Assessment of Different Types of Apoptotic Cell Death
Morphological criteria for apoptotic cells and bodies described previously were used [12, 23]. Briefly, apoptotic cells were defined as cells with nuclei containing condensed chromatin that had either aggregated in large compact granular masses that abut on the nuclear membrane (marginated chromatin), had shrunken into a single regularly shaped, dense, homogeneously stained mass (pyknotic appearance), or had fragmented into multiple densely stained masses (multiple fragments). The masses, which appeared to originate from a single cell, clustered together and were situated among, and apparently not internalized by, neighboring viable cells. Apoptotic bodies are remnants of apoptotic cell death. They are defined as discrete membrane-bound structures with roughly spherical or ovoid shape containing variable amounts of condensed chromatin and/or cytoplasm dispersed in the intercellular spaces, and are either extruded into an adjacent lumen or, commonly, phagocytosed by resident tissue cells. An oil-immersion objective (x100) was used to observe cell structure.
In Situ 3' End-Labeling: TUNEL
The internucleosomal DNA fragmentation in follicles was detected using nonradioactive labeling of DNA 3' ends (TUNEL) described by Gavrieli et al. [12] using a FragELTM kit (Oncogene, Cambridge, MA). Briefly, tissue sections of interest (n = 25) were deparaffinized and rehydrated. Sections were then washed in Tris-buffered saline and treated with 20 µg/ml proteinase K for 20 min at room temperature. Tissues were treated with 3% H2O2 for 5 min to inactivate endogenous peroxidase, and their DNA was labeled at 3' ends with biotin-dUTP by incubation with the reaction buffer containing terminal deoxynucleotidyl transferase enzyme for 1.5 h at 37°C. The sections were further incubated with streptavidin-horse radish peroxidase conjugate to detect biotinylated nucleotides for 30 min at room temperature. Diaminobenzidine reacted with the labeled samples to generate an insoluble colored substrate at the site of DNA fragmentation. Finally, sections were counterstained with methyl green to aid in the morphological evaluation and characterization of normal and apoptotic cells as described previously. Negative control sections were processed identically except that the labeling enzyme (terminal deoxynucleotidyl transferase enzyme) was omitted. An oil-immersion objective (x100) was used to observe cell structure.
Recovery of GC
For each follicle size category (healthy and atretic small [n = 150], medium [n = 50], and large [n = 40]), follicles were punctured with an 18-gauge needle, and FF was aspirated. GC collection medium—Ca2+/Mg2+-free buffer (20 mM Tris, 140 mM NaCl, 2 mM EDTA, pH 7.4)—was flushed in and out of the follicles repeatedly. The follicles were then cut into hemispheres, and the interior walls were gently scraped with an inoculating loop to remove GC, leaving the basement membrane and theca cells intact. Media obtained from different categories of follicles were placed in 15-ml centrifuge tubes. The GC were harvested by centrifuging the media at 400 x g for 10 min. Cells were washed three times with collection medium, and cell number and viability were determined using a hemocytometer and the trypan blue dye exclusion method. GC from both healthy and atretic small, medium, and large follicles were then suspended in 1 or 2 ml collection medium and snap-frozen to -70°C for subsequent DNA extraction [24].
DNA Extraction
The DNA isolation procedure was adapted from Tilly and Hsueh [24]. Cells were snap-frozen and stored at -70°C to prevent nonspecific activation of deoxyribonuclease. Cells were first disrupted by addition of homogenization buffer and repeated passage through a pipette. Homogenates were then lysed. The DNA was extracted by the phenol/chloroform/isoamyl alcohol (25:24:1, v:v:v) method and quantitated by absorbance at 260 nm.
Agarose Gel Electrophoresis
The DNA was separated (10–20 µg/lane) according to size in a 2% agarose gel by electrophoresis. Gels were stained with ethidium bromide and washed in double-distilled H2O. The DNA fluorescence was viewed with a UV transilluminator, and the gels were photographed [24]. DNA extraction and agarose gel electrophoresis were carried out 10 times.
GC Culture Conditions
To establish an in vitro model system, 5 x 106 viable GC from healthy small (n = 90), medium (n = 45), and large (n = 35) follicles were cultured in 6-well plates (n = 3 culture wells for each follicle size) with 2 ml DMEM/Ham's F-12 supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin sulfate. The cells were incubated in a humidified 5% CO2 atmosphere at 39°C. After 16-h culture, dead cells were washed off. Only viable GC were left and were attached tightly to the culture plates. These GC were cultured for another 48 h in serum-free DMEM/Ham's F-12 medium. GC from follicles of different sizes were collected from the wells and snap-frozen at 0, 24, and 48 h (i.e., 16, 40, and 64 h from the beginning of culture) for DNA extraction. Three trials were conducted.
Treatment of Cultured GC
GC from healthy small (n = 90) and medium (n = 45) follicles were cultured in the absence or presence of bovine (b) FSH (1 ng/ml; n = 3 culture wells per treatment) for 48 h in a humidified 5% CO2 atmosphere at 39°C. Bovine FSH was obtained from the USDA National Hormone and Pituitary Program (Bethesda, MD). Three trials were conducted. To study the effects of IGF-I on apoptosis, GC from healthy small follicles (n = 90) were cultured with either 10 or 100 ng/ml IGF-I (Sigma Chemical Co.) with and without FSH (1 ng/ml) (n = 3 culture wells per treatment) for 48 h in a humidified 5% CO2 atmosphere at 39°C. After culture, GC were harvested, snap-frozen, and stored at -70°C until processed for DNA extraction. Three trials were conducted. The doses of FSH and IGF-I used in the culture system were determined on the basis of their effects on GC steroidogenesis in vitro [16, 17, 20].
DNA 3' End-Labeling and Quantification
DNA 3' end-labeling is a simple and sensitive autoradiographic method for qualitative and quantitative analysis of apoptosis in minute quantities of tissues and cultured cells [24]. Five hundred nanograms of DNA from samples were labeled at the 3' end with [
32P]dideoxy (dd) ATP (3000 Ci/nmol; Amersham, Arlington Heights, IL) using 25 U terminal transferase enzyme (Boehringer Mannheim, Laval, PQ, Canada) as previously described [24]. After separation of the labeled DNA samples through 2% agarose gels, gels were dried for 2.5 h without heat in a slab-gel dryer and exposed to Kodak X-OMAT films (Eastman Kodak Co., Rochester, NY) at -70°C for 2 h. The amount of radiolabeled ddATP incorporated into low-molecular weight DNA fractions (< 1 kilobase [kb]) for each sample was quantitated by cutting the individual lane containing these fractions from dried gels with a scalpel and measuring the radioactivity of these when immersed in a liquid scintillation cocktail (SCINT-A XF; Packard Instrument Company, Inc., Meriden, CT) in a beta counter (LS 6500 scintillation system; Beckman, Fullerton, CA). The extent of radiolabeled incorporation into low-molecular weight fractions was used to provide a quantitative estimate of the degree of internucleosomal DNA fragmentation among samples.
Data Analysis
Experiments were repeated a minimum of three times. Representative photomicrographs and autoradiograms are presented. Quantitative data represent the mean ± SEM of three cultures expressed as percentage changes compared to the molecular weight standard. The effects of cell types (GC from different size follicles), time/treatment, and cell type-by-time (or treatment) interactions on GC apoptotic death were analyzed using two-way ANOVA. One-way ANOVA was performed on data from IGF-I treatment followed by Student's t-test. A P value of < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Follicles classified as healthy had an intact and well-organized GC layer, with few pyknotic cells observed under higher magnification (Fig. 1a). In early atretic follicles, the GC layer had thinned considerably and in some cases had become either partially or completely detached from the basement membrane (Fig. 1b), with a moderate number of degenerate cells and/or atretic bodies observed. With the progression of atresia, the GC layer was totally disorganized, and numerous degenerate cells and/or atretic bodies were widespread in the follicle (Fig. 1c). Finally, with advanced atresia, the GC layer was virtually absent (Fig. 1d).
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Apoptotic cells and apoptotic bodies were evident in the GC of both morphologically healthy and atretic follicles (Fig. 2). These included cells with nuclei containing marginated chromatin (Fig. 2a), cells with a single small densely stained nucleus (Fig. 2b), cells with multiple densely stained nuclear fragments (Fig. 2c), and membrane-bound structures containing condensed chromatin and/or cytoplasm (apoptotic bodies; Fig. 2d). Cells and subcellular structures with the above-mentioned apoptotic morphological features were found, through the TUNEL method, to contain fragmented DNA (Fig. 3, a–d). Dead cells with morphological features of necrosis (swollen, irregular shape, and/or karyolysis), caused during staining, also were observed (Fig. 3, a–e). In the negative control (Fig. 3e), no labeling was observed, indicating that the labeling procedure was specific.
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Within the membrana granulosa, apoptotic cells and/or bodies were more prevalent among antral GC than among mural GC (Fig. 4a). Many more apoptotic cells and/or apoptotic bodies within and/or along the antral border of the membrane granulosa were found in the atretic follicles than in the healthy follicles (not shown). Apoptotic cells and apoptotic bodies were mainly found in GC as compared with theca cells (Fig. 4b). Apoptosis of cumulus cells collected from morphologically healthy and atretic follicles was also examined. No apoptotic cells or bodies were detected in the oocyte-cumulus cell complex as opposed to the apoptotic cells and/or bodies observed within the GC layer.
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Internucleosomal DNA Fragmentation in Pooled GC from Healthy and Atretic Small, Medium, and Large Follicles
Regardless of follicle size, distinct ladder-like patterns of internucleosomal DNA fragmentation, characteristic of apoptosis, were apparent in pooled GC collected from atretic small, medium, and large follicles (Fig. 5a). Evidence of internucleosomal DNA fragmentation was also detected in some pooled GC collected from healthy follicles (Fig. 5b).
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Spontaneous Onset of Apoptosis in Cultured GC from Follicles at Different Developmental Stages
GC extracted at 0 h contained predominantly intact high-molecular-weight DNA. However, the spontaneous onset of apoptosis, as evidenced by internucleosomal fragmentation of DNA into 185-bp multiples (Fig. 6a), was clearly visible by 24 and 48 h of culture, and the extent of DNA cleavage into low-molecular-weight fragments increased in a time-dependent manner throughout the 48 h of culture of GC from all small, medium, and large follicles (vs. 0 h, P < 0.05). When the increasing rate of apoptosis of GC from follicles of different sizes in serum-free culture was compared, it was found that GC from small follicles had the highest rate (48 h vs. 0 h, P < 0.05, Fig. 6b).
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Suppression of Spontaneous Onset of Apoptosis by FSH and IGF-I in Cultured GC
FSH (1 ng/ml) inhibited spontaneous apoptotic DNA fragmentation in cultured GC from both small and medium follicles (P < 0.05). The inhibitory effect was more apparent on GC from medium follicles than from small follicles (Fig. 7). Treatment with 10 ng/ml of IGF-I suppressed apoptosis in cultured GC from small follicles (P < 0.05), while higher concentrations of IGF-I (100 ng/ml) stimulated it. However, FSH (1 ng/ml) prevented the stimulatory effect of the high dose of IGF-I (100 ng/ml; P < 0.05; Fig. 8).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Internucleosomal DNA fragmentation has been considered to be characteristic of apoptosis and is one of the earliest events [6]. In the present study, oligonucleosome formation was first detected in GC isolated from all small, medium, and large follicles classified as atretic. These results provided biochemical evidence that GC death during ovarian follicular atresia in cows occurs by apoptosis, consistent with the findings in other studies to date in cows [8, 9], ewes [23], and rats [7]. Internucleosomal DNA fragmentation was also detected in GC isolated from healthy follicles, indicating that apoptosis in the bovine species may occur very early in the atretic process before other morphological and biochemical signs of degeneration or dysfunction are evident. Therefore, the morphological criteria used previously [22] for classifying healthy and atretic follicles are not very accurate. Our findings are consistent with one study in cows in which follicles were collected during the luteal phase [9] and a study in ewes [24]. However, in another study on cows [7], oligonucleosomes were not detected in dominant preovulatory follicles recovered during a prostaglandin-induced follicular phase. Also, in all previous reports on rats, pigs and chickens, the presence of oligonucleosomes has been confined solely to DNA isolated from atretic follicles.
In addition to the detection of oligonucleosomes in extracted DNA, the occurrence of apoptosis may also be inferred from the characteristic morphological appearance of degenerating cells, together with the detection of fragmented DNA in single cells in situ using TUNEL [12, 13]. Most previous studies [8, 9, 26] were done using pooled DNA extracted from ovaries or GC homogenates; therefore, morphological changes in GC undergoing apoptosis were unknown. Although the descriptions of nuclear pyknosis and the subsequent formation of atretic bodies are similar to the descriptions of cellular and nuclear changes that occur during apoptotic cell death [27–29], the relationship between the prevalence of pyknosis (conventional histological staining) and apoptosis in GC is not clear in cows. Using classic histological techniques and in situ 3' end-labeling (TUNEL), which can detect apoptosis precisely at the single-cell level without disruption of the tissue morphology [12, 13], the present study related specific morphological features of GC death in follicular atresia (nuclear pyknosis, karyorrhexis, and formation of atretic bodies) to the physiological process of apoptosis. The relationship was supported by a combination of biochemical evidence, classic histological evidence, and in situ histochemical evidence of DNA fragmentation (a hallmark feature of apoptosis). Different cell appearances were observed in atretic and healthy follicles classified by morphological criteria, including cells with a single shrunken and dense nucleus (pyknotic appearance) and cells with marginated chromatin and/or nuclear fragmentation. In agreement with our histological study described above, TUNEL confirmed the presence of fragmented DNA in cells with those morphological appearances in atretic and healthy follicles. These results are consistent with the results of studies in ewes [24] but are in contrast to studies in rats, pigs, and chickens [2, 6, 7, 11], which suggests that apoptosis occurs only in atretic follicles. The reason may be the different criteria used in different studies to classify follicles as healthy or atretic. According to Lussier et al. [30], nonatretic follicles should have intact and normal granulosa layers with the mean pyknotic index per class varying from 0.13% to 0.67%. However, in another study in cows [31], pyknotic cells were observed in the GC layer in 30–60% of estrogen-active large follicles. Thus, the mere presence of pyknotic cells in the GC layer does not imply that they are atretic. The actual relationship between the prevalence of pyknotic cells in the GC layer and their ability to maintain GC function is not known. Together, however, the morphological and biochemical results in the present study strongly indicate that apoptosis may occur to a certain level during normal follicle growth and development, and that apoptotic death of GC may be detectable before other morphological and biochemical signs of degeneration appear in cows.
The present study provides evidence that cells involved in apoptosis during bovine follicular atresia were mainly GC, although occasional theca interna cells also underwent apoptosis. This is consistent with the study in ewes [24] and the concept that during the atretic process GC become pyknotic and die, whereas most of the theca cells are reincorporated into the ovarian interstitium [13]. Apoptotic cells appeared to be disseminated throughout the GC layer. However, a higher prevalence of apoptotic cells and bodies was observed in the antral GC than in mural GC. This may be due to different steroidogenic activity between bovine antral and mural GC [32]. In addition, a recent study suggests that the follicular basement membrane plays an important role in transmitting survival signals and in prevention of apoptosis [33]. In our study, no apoptotic cumulus cells in the oocyte-cumulus cell complex in atretic follicles were observed, even though scattered GC with condensed nuclei and DNA fragmentation were observed in the same follicle. To our knowledge, similar data have not previously been reported for cows. Cumulus cells are a subpopulation of GC that are extruded normally from the follicle along with the oocyte. Dirnfeld et al. [34] reported that in humans, cumulus cells have less steroidogenic capability compared with GC. The process of apoptosis in follicles is associated with decreased levels of aromatase mRNA and FSH- and LH-receptor mRNAs, and these decreased levels are consistent with the decreased response of GC to gonadotropins and decreased estrogen concentrations in FF [14]. Therefore, extra-follicular factors probably affect mural GC, antral GC, and cumulus cells differently. In addition, biochemical studies have provided evidence that cells undergoing apoptosis exhibit distinct morphological changes and DNA fragmentation that may be regulated by an endogenous neutral Ca2+/Mg2+-dependent endonuclease [35]. A recent study has shown that high activity of neutral Ca2+/Mg2+-dependent endonuclease was noted only in GC and not in cumulus cells in porcine atretic follicles [36].
Identification of endocrine or paracrine factors that modulate apoptosis in ovarian cells can provide a basis for elucidating the hormonal regulation of follicle atresia. To study the hormonal regulation of follicle atresia further, an in vitro model system based on serum-free culture of GC, developed in the previous study [20], was established to examine stage-dependent differences in the hormonal regulation of follicle apoptosis. GC isolated from small, medium, and large follicles as classified by their gonadotropin dependence and changes in the expression of steroidogenic enzymes were used to represent different follicular developmental stages [21]. A spontaneous onset of apoptotic cell death was observed from cultured GC in all classes. The reason for the spontaneous onset of apoptotic cell death during the culture may be that increases in nuclear cation (Ca2+/Mg2+) levels in cultured GC activated the existing endonuclease, resulting in DNA fragmentation [19]. These results are similar to those in rats and pigs [18, 19] and may indicate that survival factors are required by GC to overcome apoptotic cell death. With time of culture, the level of spontaneous onset of apoptotic cell death increased. The increased rate of spontaneous apoptosis in the culture of GC from small follicles was highest. This suggested that follicular apoptosis may be stage-specifically regulated and explains why the antral transition is the "bottle neck" for developing follicles [11].
Preantral follicles, although responsive to gonadotropins, can grow until the antral stage without gonadotropin support. Once antral (4-mm onwards), follicular growth will be halted if FSH is suppressed [21]. In the present study, FSH treatment significantly suppressed apoptosis in the culture of GC from medium follicles compared to the culture of GC from small follicles. This suggested that FSH is a follicle survival factor and also indicated again that follicular apoptosis is stage-specifically regulated. Likewise, Chun et al. [37] reported that LH/hCG had a significant suppressive effect on apoptosis of preovulatory follicle but only a marginal effect on apoptosis in early antral follicles. LH/hCG receptors are predominantly in only theca cells of small antral follicles and in both GC and theca cells of preovulatory follicles [16]. In addition to gonadotropins, the development of ovarian follicles is also controlled by locally produced intraovarian regulators such as growth factors and cytokines, which act in a paracrine/autocrine manner. IGF-I can amplify the actions of gonadotropins [16, 38]. In our study, IGF-I (10 ng/ml) attenuated apoptosis while IGF-I (100 ng/ml) increased apoptosis in cultured GC, and a combination treatment of FSH (1 ng/ml) and IGF-I (100 ng/ml) inhibited apoptosis. Our findings were similar to that in pigs in that both FSH and IGF-I (0–250 ng/ml) suppressed apoptosis in cultured GC [18]. However, in rats, FSH and IGF-I attenuated apoptosis in GC only when they were added to the culture of intact preovulatory follicles [19]. The pathways of FSH, IGF-I, IGF binding proteins, and their interactions on regulation of apoptosis in cultured GC are controversial and remain to be elucidated [39]. Species differences may also exist. In our study, the mechanism for the stimulation of apoptosis in cultured GC by the high concentration of IGF-I (100 ng/ml) is not clear. However, a previous study has shown that a high concentration of IGF-I (evident at 
30 ng/ml) inhibits estrogen production by GC from small bovine follicles (1–5 mm) [40]. In vivo, negative correlations were also found in some studies in which follicles 8 mm were collected [41]. In most mammals, follicular atresia is correlated with a decline in estrogen synthesis concomitant with increased progesterone production [7]. Estrogens have been shown to inhibit ovarian GC apoptosis in rats [42].
In summary, our data indicate that 1) apoptosis may occur to a certain level during normal follicular growth and development, and apoptotic death of GC may be detectable before other morphological and biochemical signs of degeneration appear in cows; 2) apoptosis occurs in GC and theca cells; however, apoptosis does not occur in cumulus cells even in atretic antral follicles in bovine ovaries; and 3) FSH and a low concentration of IGF-I are follicle survival factors, and follicle apoptosis is regulated differentially depending upon the stage of follicular development in cows.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Funded by NSERC (Research Grant No. OGP0036609). The results of this study have previously been published as abstracts in the proceedings of the 1997 and 1998 Annual Meeting of the Canadian Society of Animal Science. 
2 Correspondence: R. Rajamahendran, Faculty of Agricultural Sciences, The University of British Columbia, 208-2357 Main Mall, Vancouver, BC, Canada V6T 1Z4. FAX: 604 822 4400; raja{at}unixg.ubc.ca
Accepted: December 14, 1999.
Received: April 5, 1999.
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J. Greenaway, P. A. Gentry, J.-J. Feige, J. LaMarre, and J. J. Petrik Thrombospondin and Vascular Endothelial Growth Factor Are Cyclically Expressed in an Inverse Pattern During Bovine Ovarian Follicle Development Biol Reprod, May 1, 2005; 72(5): 1071 - 1078. [Abstract] [Full Text] [PDF]
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L. J. Clark, H. F. Irving-Rodgers, A. M. Dharmarajan, and R. J. Rodgers Theca Interna: The Other Side of Bovine Follicular Atresia Biol Reprod, October 1, 2004; 71(4): 1071 - 1078. [Abstract] [Full Text] [PDF]
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F. J. Rubio Pomar, B. A.J. Roelen, K. A. Slot, H. T.A. van Tol, B. Colenbrander, and K. J. Teerds Role of Fas-Mediated Apoptosis and Follicle-Stimulating Hormone on the Developmental Capacity of Bovine Cumulus Oocyte Complexes In Vitro Biol Reprod, September 1, 2004; 71(3): 790 - 796. [Abstract] [Full Text] [PDF]
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 J. Greenaway, K. Connor, H. G. Pedersen, B. L. Coomber, J. LaMarre, and J. Petrik Vascular Endothelial Growth Factor and Its Receptor, Flk-1/KDR, Are Cytoprotective in the Extravascular Compartment of the Ovarian Follicle Endocrinology, June 1, 2004; 145(6): 2896 - 2905. [Abstract] [Full Text] [PDF]
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 S. M. Quirk, R. G. Cowan, R. M. Harman, C.-L. Hu, and D. A. Porter Ovarian follicular growth and atresia: The relationship between cell proliferation and survival J Anim Sci, January 1, 2004; 82(13_suppl): E40 - 52. [Abstract] [Full Text] [PDF]
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M. E. Dell'Aquila, M. Albrizio, F. Maritato, P. Minoia, and K. Hinrichs Meiotic Competence of Equine Oocytes and Pronucleus Formation after Intracytoplasmic Sperm Injection (ICSI) as Related to Granulosa Cell Apoptosis Biol Reprod, June 1, 2003; 68(6): 2065 - 2072. [Abstract] [Full Text] [PDF]
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S. Kolle, M. Stojkovic, G. Boie, E. Wolf, and F. Sinowatz Growth Hormone-Related Effects on Apoptosis, Mitosis, and Expression of Connexin 43 in Bovine In Vitro Maturation Cumulus-Oocyte Complexes Biol Reprod, May 1, 2003; 68(5): 1584 - 1589. [Abstract] [Full Text] [PDF]
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K. Tajima, M. Orisaka, K. Hosokawa, A. Amsterdam, and F. Kotsuji Effects of Ovarian Theca Cells on Apoptosis and Proliferation of Granulosa Cells: Changes During Bovine Follicular Maturation Biol Reprod, June 1, 2002; 66(6): 1635 - 1639. [Abstract] [Full Text] |
