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BOR - Papers in Press, published online ahead of print January 21, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.025114
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BIOLOGY OF REPRODUCTION 70, 1475–1484 (2004)
DOI: 10.1095/biolreprod.103.025114
© 2004 by the Society for the Study of Reproduction, Inc.

Ovary

Identification of Genes Involved in Apoptosis and Dominant Follicle Development During Follicular Waves in Cattle1

A.C.O. Evans2,3, J.L.H. Ireland4, M.E. Winn4, P. Lonergan3, G.W. Smith4, P.M. Coussens4, and J.J. Ireland4

Department of Animal Science and Production and the Centre for Integrative Biology,3 Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland Department of Animal Science and Center for Animal Functional Genomics,4 Michigan State University, East Lansing, Michigan 48824


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We hypothesize that granulosa and theca cells from growing dominant follicles, with relatively high intrafollicular concentrations of estradiol, have a greater expression of genes involved in inhibiting apoptosis pathways and lower expression of genes involved in apoptosis pathways than growing subordinate follicles with lower estradiol concentrations. Using the well-characterized bovine dominant follicle model, we collected granulosa and theca cells from individual dominant and the largest subordinate follicle 3 days after initiation of a follicular wave in four animals. Based on ultrasound analysis, both follicle types were in the growth phase at the time of ovariectomy. However, dominant follicles were larger (9.8 ± 1.0 versus 7.6 ± 0.6 mm in diameter, P < 0.05) and had greater intrafollicular concentrations of estradiol (132.2 ±3 8.5 versus 24.1 ± 12.1 ng/ml, P < 0.05), compared with the largest subordinate follicles. We used bovine cDNA microarrays, which contained a total of 1400 genes, including a subset of 53 genes known to be involved in apoptosis pathways, to determine which apoptosis and marker genes from each of the four dominant versus subordinate follicles were potentially differentially expressed. Using a low stringency-screening criterion, 22 genes were identified. Quantitative real-time polymerase chain reaction confirmed that 16 of these genes were differentially expressed. Our novel results demonstrate that the high intrafollicular concentrations of estradiol in growing dominant follicles were positively associated with enhanced expression of mRNAs in granulosa cells for aromatase, LH receptor, estradiol receptor ß, DICE-1, and MCL-1, compared with granulosa cells from subordinate follicles (all survival-associated genes). In contrast, the relatively low intrafollicular concentrations of estradiol in growing subordinate follicles were positively associated with enhanced expression of mRNAs in granulosa cells for ß glycan, cyclo-oxygenase-1, tumor necrosis factor {alpha}, caspase-activated DNase, and DRAK-2, and in theca cells for ß glycan, caspase 13, P58(IPK), Apaf-1, BTG-3, and TS-BCLL, compared with granulosa or theca cells from dominant follicles (genes that are all associated with cell death and/or apoptosis). We suggest that that these genes may be candidate estradiol target genes and that they may be early markers for the final stages of follicle differentiation or initiation of apoptosis and thus selection of dominant follicles during follicular waves.

apoptosis, estradiol, follicle, follicular development


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intraovarian mechanisms regulating growth of a species- specific number of ovulatory follicles, despite rampant follicular atresia, are poorly understood. Although humans and cattle are single-ovulating species, growth of several dozen antral follicles occurs in a wave-like pattern every 7–14 days during each menstrual [1, 2] or estrous cycle [3, 4]. A follicular wave that coincides with the follicular phase of a menstrual or estrous cycle results in development of a dominant ovulatory follicle, whereas dominant follicles that develop in waves asynchronous to the follicular phase undergo atresia. Each follicular wave is preceded by a transient rise in serum FSH concentrations. As FSH declines, a poorly understood selection process occurs whereby a single dominant follicle from the original cohort of growing follicles continues to develop while all other subordinate follicles in the wave undergo atresia [4, 5]. Atresia of ovarian follicles in vertebrates is mediated via apoptosis [6]. Tumor-associated, death, and survival genes regulate the balance between pro- and antiapoptotic factors present within cells [7], and these genes have been described in vitro and to a lesser extent in vivo primarily in various vertebrate laboratory species [6, 8, 9]. Consequently, selection involves prevention or activation of apoptotic pathways that lead to growth of a dominant follicle and atresia of all other individual antral follicles in a follicular wave.

We have taken advantage of the dominant follicle model in cattle to identify intrafollicular factors that regulate selection and survival of dominant follicles during follicular waves. At the early stages of a follicular wave, before ultrasound can identify the dominant follicle based on its size (largest diameter), the follicle destined to become dominant usually has higher intrafollicular concentrations of estradiol, compared with similar-sized follicles destined to become atretic later in the follicular wave [10]. In addition, estradiol is sustained throughout growth of dominant follicles, and estradiol production by follicles in a wave declines before DNA fragmentation (marker of apoptosis) occurs in granulosa cells [11]. Taken together, these findings imply that estradiol has a key role in selection and sustained growth of dominant follicles. In support of this idea, estradiol receptors are expressed in granulosa and theca cells [12]. In addition, numerous in vitro studies demonstrate positive roles for estradiol in mitosis of granulosa cells and gonadotropin action, and that estradiol is required for gonadotropin-induced follicular growth and differentiation [13, 14]. Nevertheless, potential estradiol target genes involved in survival or apoptosis of granulosa and theca cells, which results in follicular growth or atresia, have not been identified. Moreover, the anti- or proapoptotic genes involved in survival or atresia of dominant or subordinate follicles during follicular waves have not been identified. The exception is the Fas antigen and its ligand, which are suppressed in dominant compared with highly atretic subordinate follicles [15, 16],

Based on previous studies of apoptosis during folliculogenesis in laboratory species, coupled with the well-defined patterns of dominant and subordinate follicle growth during follicular waves in cattle and humans, we hypothesized that granulosa and theca cells of growing dominant follicles, with relatively high intrafollicular concentrations of estradiol, would have a greater expression of genes involved in the inhibition of apoptosis and lower expression of genes involved in apoptosis than growing subordinate follicles with lower estradiol concentrations. To test this hypothesis, we utilized bovine cDNA microarrays that contained a platform of 1400 known and unknown genes as a qualitative screen to identify potential estradiol-responsive target genes that were differentially expressed in individual dominant versus subordinate follicles. Here we report results on expression of 53 genes known to be involved in apoptosis pathways in reproductive and nonreproductive tissues. The cDNA microarrays and quantitative real-time polymerase chain reaction (PCR) revealed that 11 genes known to be involved in apoptosis pathways were differentially expressed in granulosa or theca cells of dominant versus subordinate follicles, many of which have not been ascribed a role in the regulation of follicular atresia or apoptosis.


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

Four cross-bred (Hereford x Friesian) beef cows (30–36 months old; 500–550 kg body weight; maintained under standard husbandry conditions) with regular estrous cycles received a luteolytic dose of prostaglandin (Prosolvin; Intervet, Dublin, Ireland) during the luteal phase. Observations for estrus began 24 h after prostaglandin treatment and were performed every 4–8 h. The time of first detection of estrous behavior was designated as Day 0 of the cycle. Starting the day before prostaglandin, growth of ovarian follicles 4 mm in diameter was monitored daily using transrectal ultrasonography [17]. All experimental procedures were licensed by the Department of Health and Children, Ireland, in accordance with the cruelty to animals act (Ireland 1897) and European Community Directive 86/609/EC.

Ovariectomy, Tissue Collection, and RNA Extraction

Ovariectomies were performed by colpotomy [18] between Days 2.5 and 3.5 of the estrous cycle when the dominant follicle of the first follicular wave was first detected. Ovaries were immediately placed in ice-cold PBS on ice and follicles 4 mm in diameter were dissected from both ovaries and placed individually in ice-cold RNAlater (a tissue storage reagent that stabilizes and protects cellular RNA in intact, unfrozen tissue samples; Ambion, Huntingdon, UK). Follicles were measured using a dissecting microscope and 1 mm grid [19], follicular fluid was aspirated, and the follicle cut open and completely immersed in RNAlater. The theca- basement membrane-granulosa layers were collectively peeled from the stroma with forceps. Granulosa cells were then gently scraped from the basement membrane into RNAlater using a fine glass scraper [19]. The theca enriched tissue layer was removed from RNAlater and slightly minced using a scalpel blade, placed in a microcentrifuge tube with 1 ml Trizol (Invitrogen Life Technologies Corp., Carlsbad, CA), and snap frozen in liquid nitrogen. Dispersed granulosa cells were transferred to a microcentrifuge tube in RNAlater and pelleted (10 000 x g for 1 min), the supernatant containing RNAlater was removed, and 1 ml of Trizol was added to the pellet. The pelleted cells were resuspended by gentle vortexing and then snap frozen in liquid nitrogen and stored at –80°C. Total time from ovariectomy to freezing was 25–45 min.

Total RNA was extracted from theca (after homogenization) and granulosa samples in Trizol after aspirating through a 20-gauge needle following the manufacturer's recommendations. RNA was then subjected to DNase treatment (RQ1 RNase-free DNase; Promega, Madison, WI) following the manufacturer's protocol, and the quantity and quality of extracted total RNA was estimated by ultraviolet spectrophotometry and electrophoresis on a 1% denaturing agarose gel.

Hormone Assay

Estradiol concentrations were measured in follicular fluid diluted between 1:100 and 1:10 000 in PBS using a validated RIA [20]. Mean intra- and interassay (n = 2) coefficients of variation were 4.7%, 6.5%, and 5.2% and 7.8%, 7.8%, and 7.0%, respectively, for reference samples with concentrations of 1.78, 3.23, and 7.48 ng/ml (assayed at a dilution of 1:100).

Progesterone concentrations in unextracted follicular fluid were measured in a single RIA as previously described [21]. Sensitivity of the assay was 0.1 ng/ml and the intra-assay coefficient of variation was 4.0% for a reference sample with a concentration of 8.74 ng/ml.

BOTL-4 cDNA Microarray, Preparation of Labeled cDNA, and Microarray Analysis

The cDNA microarrays (BOTL-4) used in these experiments were an expanded version of those described previously [22–25]. Each microarray contained 4800 total spots consisting of 709 bovine EST clone inserts developed from a normalized bovine total leukocyte (BOTL) cDNA library [24] and an additional 627 amplicons representing additional genes including cytokines, receptors, signal transduction molecules, transcription and growth factors, enzymes, cell cycle regulators and cellular components, and 53 genes coding for factors involved in apoptosis [26]. A list of known genes represented on the BOTL-4 microarrays and their sequences can be found at http://www.nbfgc.msu.edu under the Links section. Amplicons were suspended in 50% dimethylsulfoxide (DMSO) and spotted in triplicate on glass slides in a 4-by-12 pattern of 48 patches with 100 spots per patch. In addition to the above genes of interest, various control genes were also included on the microarray. These were 144 spots representing glyceraldehyde-3-phosphate dehydrogenase (three spots in each of 48 patches), 75 spots representing ß-actin, 78 spots representing RPL-19 (ribosomal protein L-19) genes, 48 synthetic Lambda Q gene spots (one per patch), 303 negative control spots (DMSO only spotted), and 144 blank spots (nothing spotted).

Microarray analysis involved synthesis of cDNA from total RNA, labeling of cDNA, hybridization of cDNA to BOTL-4 microarray slides, and imaging of the final hybridized slides [25]. Total RNA (8 µg) from the dominant and largest subordinate follicle of each animal were used as templates in reverse transcription reactions (BD Atlas Powerscript fluorescent labeling system, BD Biosciences Inc., Alameda, CA) using Oligo (dT)15–18 as primer and incorporating amino-tagged deoxyuridine 5-triphosphates (dUTPs). To provide a control for cDNA synthesis and labeling efficiency, as well as for subsequent cDNA microarray hybridization, 650 pg of synthetic lambda Q gene RNA containing an engineered poly A tail were spiked into each cDNA synthesis reaction.

Following first-strand cDNA synthesis, cDNAs from dominant and the largest subordinate follicle of each animal were differentially labeled using N-hydroxysuccinimide (NHS)-activated fluorescent Cy3 or Cy5 dyes (Amersham Pharmacia Ltd., Piscataway, NJ). The dye couples to the cDNA by reacting with the amino functional groups on the incorporated amino-tagged dUTPs. The NHS-Cy3 and NHS-Cy5 dyes were used to label cDNA from dominant and subordinate follicles, respectively, for two animals and to label subordinate and dominant follicles, respectively, for the remaining two animals (flipped-fluor design). Labeled cDNAs were purified to remove unincorporated dyes using a QIAquick purification kit (Qiagen, Valencia, CA). Differentially labeled cDNA from dominant and subordinate follicles were combined within animal and concentrated to less than 10 µl using Microcon 30 spin concentrators (Millipore Corp., Bedford, MA) and added to 100 µl of SlideHyb-3 (Ambion). The hybridization mixture was added to the BOTL-4 microarray slide, and hybridizations were conducted for 18 h in a commercial microarray hybridization station (GeneTAC, Genomics Solutions, Inc., Ann Arbor, MI) using a step- down hybridization protocol (65°C for 3 h, 55°C for 3 h, 50°C for 12 h). Following hybridizations, cDNA microarrays were washed within the hybridization station (42–50°C), rinsed once in 2x saline sodium citrate and once in ddH2O and finally dried by centrifugation in a cushioned 50 ml conical centrifuge tube. Finally, microarrays were scanned using a GeneTAC LS IV microarray scanner and GeneTAC LS software (Genomic Solutions). GeneTAC analyzer software was then used to process microarray images, find spots, integrate robot-spotting files with the microarray image, and finally to create reports of raw spot fluorescence intensities.

Real-Time PCR

The findings from the microarray analyses were used as a qualitative screen to identify genes involved in apoptosis of granulosa and theca cells in dominant versus largest subordinate follicle of four animals. These genes were further compared between follicle types using quantitative real- time reverse transcriptase PCR (Q-RT-PCR) with an Applied Biosystems 7000 DNA sequence detection system (Perkin Elmer Corp., Foster City, CA), as previously described [25]. Our criteria for selecting genes after microarray to validate by Q-RT-PCR were based solely on a differential expression ratio that was significantly (P < 0.05) different from 1. We expected this approach to result in fewer false negatives being discarded. However, the false-positive rate was expected to be higher using this approach, compared with selection of genes after microarray based on level of significance and a high relative expression ratio. False positives following DNA microarray analysis were based on results of Q-RT-PCR analysis. In addition to the apoptosis genes, genes known to be important for follicle development (aromatase, LH receptor and FSH receptor, and ß glycan) were also examined using Q-RT-PCR. The estrogen receptors {alpha} and ß were not present on the microarray but were examined using Q-RT-PCR. ß-Actin was measured in all assays and served as the control gene for normalization of results.

Total RNA (2 µg) from individual follicles was converted into first- strand cDNA [25]. Using 20 ng of template cDNA, Q-RT-PCR was performed in duplicate using SYBR Green PCR Master Mix (Perkin Elmer) with gene-specific primers. All primers were designed using Primer Express software (Perkin Elmer) and were synthesized by Operon Technologies (Alameda, CA). Primer sequences, expected melting temperatures (Tm) (predicted using Primer Express software), and appropriate primer concentrations are shown in Table 1. Forty cycles of PCR were programmed to ensure the threshold crossing point (cycle number) was attained. Fluorescence emission was monitored continuously during cycling. At the completion of cycling, melting curve analysis was carried out to establish the specificity of the amplicons produced and to confirm melting at the predicted Tm. The optimum amount of template and optimum primer concentration were determined by trying a range of values between 20 and 50 ng per reaction and a range of concentrations between 50 and 900 nM, respectively.


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  		TABLE 1. Primer sequences, optimal concentrations, and expected melting point (Tm) using 20 ng cDNA template except for CD40 ligand where 50 ng was optimal

Data Analyses

In general, most cDNA microarray data display bias at both high and low ends of the intensity spectrum, even in the absence of cDNA loading differences [24, 25]. To correct for potential sample and dye intensity biases, data in each microarray were normalized using a robust local regression technique [27] using the LOESS procedure of SAS (version 8, SAS Institute Inc., Cary, NC). Normalized data were then back transformed and the intensity for the dominant follicle divided by the intensity for the subordinate follicle (i.e., without background subtraction) to give an expression ratio value. The likelihood that the four values from the four microarrays (between individual dominant and subordinate follicles within animals) differed from one was determined using Student t-test. All genes that had expression ratios that were significantly different from 1 (irrespective of mean expression ratio) were then subjected to Q-RT-PCR for confirmation of the DNA microarray results.

Q-RT-PCR data were analyzed using the 2-{Delta}{Delta}CT method [28]. Briefly, the relative level of expression of each mRNA in each sample was determined and a ratio of each mRNA to ß-actin (control gene) was calculated (to correct for any differences in efficiency of cDNA production). The relative abundance of the mRNAs was calculated by dividing the dominant by the subordinate (baseline) value (after ß-actin correction) to give a fold change value. Samples were measured in duplicate for each gene of interest, and ß-actin was measured in all samples in every 96-well plate that was subjected to Q-RT-PCR.

Values for dominant and largest subordinate follicles were compared using Student t-test. For the presentation of data in which expression was lower in the dominant, compared with the subordinate follicle, the ratio (<1) was divided into –1 to give a negative relative expression value (–1/[subordinate value/dominant value]). Values are presented as the mean (±SEM).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The growth dynamics and morphological and hormonal characteristics for each individual follicle that was analyzed using DNA microarrays and Q-RT-PCR are shown in Figure 1. Ultrasound analysis demonstrated that diameters of both dominant and subordinate follicles were increasing daily until ovariectomy (Fig. 1a); thus, both follicle types were in the growth rather than static phase of follicular development. Diameters of dominant follicles measured either before ovariectomy by ultrasonography (Fig. 1a) or after follicle dissection were greater (P < 0.01) than the largest subordinate follicles (9.8 ± 1.0 versus 7.6 ± 0.6 mm; Fig. 1b). Each dominant follicle also had a higher concentration of estradiol in follicular fluid, compared with the largest subordinate follicle (132.2 ± 38.5 versus 24.1 ± 12.1 ng/ml, P < 0.05, Fig. 1c). There were no consistent differences in follicular fluid progesterone concentrations between dominant and subordinate follicles (combined means 41.4 ± 7.2 versus 28.6 ± 7.8 ng/ml, P > 0.05). Each dominant follicle and one subordinate follicle (animal D) were classified hormonally as estrogen active (follicular fluid estradiol greater than progesterone concentration, Fig. 1), whereas three subordinate follicles (animals A, B, C) were classified as estrogen inactive (progesterone greater than estradiol) [29]. We have previously shown in cattle that estrogen-active follicles are healthy growing follicles, whereas estrogen-inactive follicles are in the early stages of atresia [30]. Although ultrasound analysis easily distinguished a dominant from a subordinate follicle (based on its larger diameter) in all four animals, the dominant and subordinate follicles for animal A were at an earlier stage of growth and differentiation (based on diameters and intrafollicular estradiol and progesterone concentrations) than animals B, C, and D (Fig. 1c).



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  		FIG. 1. Characteristics of individual dominant and largest subordinate follicles in four individual cows (A, B, C, D) depicting: a) follicle growth (measured by daily transrectal ultrasonography), b) follicle diameter at ovariectomy (measured after dissection), and c) follicular fluid concentrations of estradiol (E2) and progesterone (P4). Arrows indicate the time of ovariectomy

DNA Microarray (Marker Genes)

Associated with the higher intrafollicular concentrations of estradiol in dominant follicles (Fig. 1c) was a greater (P < 0.05) expression of mRNAs for aromatase and FSH and LH receptors in granulosa cells of dominant compared with subordinate follicles (Table 2). Expression of ß-glycan was greater (P < 0.05) in theca cells of subordinate compared with dominant follicles. However, expression of LH receptor mRNA was similar for theca cells and ß-glycan mRNA was similar for granulosa cells of dominant and subordinate follicles (Table 2).


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  		TABLE 2. Relative levels of expression of marker and apoptosis genes that were significantly different between the dominant and largest subordinate follicle in beef cows (n = 4) determined using DNA microarrays

DNA Microarray (Apoptotic Genes)

Of the 53 apoptotic genes on the DNA microarrays, mRNA expression for 18 of these genes (theca = 8; granulosa = 10) was significantly (P < 0.05) different when granulosa or theca cells from dominant versus subordinate follicles were compared (Table 2). Apoptotic genes similar (P > 0.05) in granulosa or theca cells of dominant and subordinate follicles are listed in Table 3. DNA microarrays were used as a qualitative screen for potential estradiol target genes, and the gene selection method was predicted to include false positives, as explained in Materials and Methods. Thus, mRNA expression of each of the genes listed in Table 2 was subjected to Q-RT-PCR.


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  		TABLE 3. Apoptosis genes on the DNA microarray for which the ex pression ratio was not significantly different between the dominant and largest subordinate follicle in beef cows (n = 4)

Quantitative Real-Time PCR (Marker Genes)

Expression of mRNA for estradiol receptor ß, aromatase, and the LH receptor was 1.6 ± 0.2-, 11.6 ± 8.8-, and 2.8 ± 0.5-fold greater (P < 0.02–0.05), respectively in granulosa cells of dominant compared with subordinate follicles (Fig. 2). In contrast, expression of ß-glycan mRNA was 6.9 ± 1.2- and 1.6 ± 0.4-fold greater (P < 0.02–0.05) in granulosa and theca cells, respectively, of subordinate compared with dominant follicles. The mRNA for the estradiol receptors-{alpha} and -ß was detected in both cell types but did not differ among follicle types (P > 0.35), with the exception of estradiol receptor-ß as indicated above. In agreement with the DNA microarray analysis, expression of the LH receptor was similar (P = 0.40) for theca cells of dominant and subordinate follicles using the Q-RT-PCR analysis.



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  		FIG. 2. Relative levels of expression of marker and apoptosis genes that were significantly different between the dominant and largest subordinate follicle in beef cows determined using Q-RT-PCR. The ratio is of the dominant compared with the subordinate follicle. Positive values indicate greater expression in the dominant compared with the subordinate follicle and negative values indicate greater expression in the subordinate compared with the dominant follicle (*P < 0.05; **P < 0.02). The expression ratio of the dominant to the subordinate follicles for three genes in granulosa cells (ß-glycan, CAD, DRAK-2) was only different from 1 (P < 0.05) when animals B, C, and D were considered without animal A. The follicles in animal A were at an earlier stage of growth and differentiation than in animals B, C, and D (see Results), indicating that these three genes may be temporally of later importance than other genes identified

Quantitative Real-Time PCR (Apoptotic Genes)

Of the 18 apoptosis genes identified by DNA microarray (Table 2), 11 were confirmed to be differentially expressed in granulosa or theca cells of dominant compared with subordinate follicles by Q-RT-PCR (Fig. 2). Specifically, mRNA expression for DICE-1 and MCL-1 was greater (P < 0.05) in granulosa cells of dominant compared with subordinate follicles (Fig. 2). In contrast, expression of mRNAs for cyclo-oxygenase-1 (COX1), tumor necrosis factor-{alpha} (TNF{alpha}), caspase-activated DNase (CAD), and DRAK-2 was greater (P < 0.02–0.05) in granulosa cells of subordinate compared with dominant follicles (Fig. 2). Expression of mRNAs for caspase 13, P58(IPK), Apaf-1, BTG-3, and TS-BCLL was greater (P < 0.02–0.05) in theca cells of subordinate compared with dominant follicles (Fig 2).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The combination of the bovine dominant follicle model, qualitative cDNA microarray screening, and Q-RT-PCR has successfully identified 11 genes in granulosa or theca cells of growing dominant and subordinate follicles known to be involved in the regulation of apoptosis in a variety of tissues. This novel set of genes may be early markers for the final stages of follicle differentiation and thus selection of dominant follicles during follicular waves.

The advantages of using the bovine dominant follicle model to identify genes involved in follicle development are that growth can be monitored in vivo and that follicles yield sufficient tissues and fluids for individual analysis. This enables replication in individual animals, rather than pools of tissue, greatly improving reliability of results. Our approach to identify differentially expressed genes relied on statistical determination of ratios of gene expression that were significantly (P < 0.05) different from 1 (no difference in gene expression), rather than use of a predetermined expression ratio. Consequently, we were more likely to identify false-positive genes but less likely to omit potential differentially expressed genes (false negatives). Using this criterion, the DNA microarray identified 22 differentially expressed genes in granulosa or theca cells of dominant versus subordinate follicles. Q-RT-PCR confirmed that expression of 16 of these genes was significantly (P < 0.05) different. The reason for the relatively high false-positive rate may be the qualitative nature of cDNA microarray analysis, coupled with our relaxed standard for selection of differentially expressed genes, as explained earlier.

As depicted in the model for dominant and subordinate follicle growth during a follicular wave (Fig. 3), follicles in our study were collected relatively soon after completion of selection and identification of which follicle in the wave was dominant based on its greater growth rate, larger size, and higher intrafollicular concentrations of estradiol, compared with the largest subordinate follicle. The high intrafollicular concentrations of estradiol in dominant follicles were positively associated with relatively high expression of mRNAs for the estrogen receptor-ß, LH receptor, aromatase, DICE 1, and MCL-1 in granulosa cells of dominant compared with subordinate follicles (Fig. 3). The finding that mRNA for aromatase and the LH receptor were higher in the dominant compared with subordinate follicles and mRNA for LH receptors in the theca and FSH receptors in the granulosa cells were not different supports previous findings that dominant follicles obtained during their early stages of development after selection, whereas FSH secretion is relatively low, are probably dependent on LH for their continued development and enhanced estradiol production [31]. The mechanism that results in LH dependency of a single dominant follicle from a growing cohort is unknown. However, it is well established in laboratory species that estradiol has a positive role in regulation of gonadotropin action, including increased synthesis of LH receptors [13].



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  		FIG. 3. Model depicting differential expression of genes in granulosa (gray) or theca (black) cells of a dominant compared with the largest subordinate follicle during the first wave of follicular development in cattle (oocytes are depicted by small black circles). Associated with growth and high intrafollicular concentrations of estradiol ({uparrow}E2) in the dominant follicle is enhanced expression of mRNAs for estrogen receptor, LH-R, aromatase, MCL-1, and DICE-1 in granulosa cells, compared with granulosa cells of subordinate follicles. Associated with growth, but eventual atresia, and relatively low intrafollicular concentrations of estradiol ({downarrow}E2) in the subordinate follicle is enhanced expression of ß-glycan, DRAK2, TNF, CAD, and COX1 by granulosa cells and enhanced expression of ß-glycan, Apaf-1, BTG-3, caspase 13, TS-BCLL, and P58 (IPK) by theca cells, compared with granulosa or theca cells of dominant follicles. Note that Day 0 is the first day 3- to 4-mm follicles are observed by ultrasound, and this marks the first day of a follicular wave

In cattle, estradiol concentrations are usually highest in follicles destined to become dominant compared with other similar-sized follicles at the beginning of a follicular wave [10], and loss of estradiol production precedes onset of apoptosis in granulosa cells in follicles destined to undergo atresia [11]. In laboratory species, estradiol inhibits, whereas androgens promote, apoptosis in granulosa cells [32, 33]. Moreover, follicle atresia in rats is associated with a decrease in estrogen receptors on granulosa cells [34], and estrogen receptor-ß is the predominant form of the estrogen receptor in the ovary [35]. Although estradiol receptor-ß mRNA and protein are present in granulosa cells of small, medium, and large bovine antral follicles [36], our study shows that expression of estrogen receptor-ß is greater in granulosa cells of dominant compared with subordinate follicles. Growth of the dominant follicle was also associated with enhanced expression of the tumor-associated genes, DICE-1 and MCL-1. DICE-1 has not been detected or a function ascribed to it in ovarian tissues. However, DICE- 1 may be involved in nuclear processes such as DNA repair, transcription, or RNA splicing [37], and expression of this gene is severely downregulated in cell lung carcinomas [38]. MCL-1 is a member of the Bcl-2 family of antiapoptotic proteins and is an early induced gene during myeloblastic leukemia cell differentiation [39]. The antiapoptotic activity of MCL-1, which is positively regulated by gonadotropins in rat follicles [40], may be mediated via its capacity to inhibit members of the proapoptotic family of proteins (including Bok, BOD, BAD, and Bax) or by interactions with Apaf-1, which prevent activation of caspases [41].

Compared with dominant follicles, the relatively low intrafollicular estradiol microenvironment of subordinate follicles in our study was positively associated with enhanced expression of mRNAs for the inhibin coreceptor (ß-glycan, transforming growth factor-ß type III receptor), TNF{alpha}, DRAK2, CAD (also known as DNA fragmentation factor 40), and COX1 in granulosa cells and with mRNAs for ß- glycan, apoptotic protease-activating factor 1 (Apaf-1), B- cell translocation gene member 3 (BTG-3), caspase 13, tumor suppressor for B-cell chronic lymphocytic leukemia (TS-BCLL), and P58 (IPK) in theca cells (Fig. 3). Our novel finding that the inhibin coreceptor was the only gene more highly expressed in both the granulosa and theca cells of subordinate compared with dominant follicles further supports an important role for inhibin in regulation of intrafollicular estradiol production. Inhibin's mechanism of action is currently explained by its high-affinity interaction with ß-glycan, which in turn inhibits activin signaling [42]. Because many activin actions are positive for folliculogenesis, including estradiol production [43], inhibin may act as a physiologically important antagonist of activin action and thus inhibit follicular growth and differentiation. We have previously shown a strong inverse relationship between intrafollicular concentrations of inhibin and estradiol throughout dominant follicle development in cattle, whereas activin-A concentrations remain relatively unchanged [10, 30]. Also, inhibin has a pronounced negative role in regulation of estradiol production by granulosa cells from bovine dominant follicles [44]. Others report that inhibin stimulates androgen production by bovine theca cells [43]. Because androgens promote follicular atresia [45, 46] and estradiol enhances gonadotropin action and follicular growth as mentioned earlier [12], the interaction of inhibin with the inhibin coreceptors during selection may have an important role in regulation of intrafollicular estradiol and androgen production, which in turn may be important for follicular survival and regulation of the genes identified in the present study (Fig. 3).

In the relatively low estradiol milieu of subordinate follicles, numerous genes directly involved in apoptosis were more highly expressed in granulosa and thecal cells, compared with dominant follicles in our study (Fig. 3). In granulosa cells, CAD, which has been detected in human ovaries [47] but has not previously been examined in individual growing follicles, functions downstream of caspases and is the endonuclease directly responsible for DNA fragmentation during apoptosis [48]. TNF{alpha} has a number of roles within ovaries and is most often a proapoptotic factor [49] that suppresses responsiveness of developing follicles to gonadotropins [49], inhibits FSH-stimulated estradiol production by bovine and human granulosa cells [50, 51], and stimulates apoptosis of bovine granulosa cells [52]. In rat and human granulosa cells, TNF{alpha} reduces intracellular levels of Bcl-2 [53], leading to an increase in caspase activity and apoptosis. DRAK1 and DRAK2 are novel serine/threonine kinases exclusively localized to the nucleus in which they phosphorylate unknown downstream targets in apoptotic pathways in NIH 3T3 cells [54]. Although the upstream regulators of DRAKs have not been identified, their kinase domains share high homology with that of death- associated protein kinase, which is involved in apoptotic signaling induced by a number of factors including TNF{alpha} and Fas [55]. Caspase-13 and Apaf-1 were highly expressed in theca cells of subordinate versus dominant follicles. Caspases are a family of proteases that are essential for the initiation and execution of apoptosis. To date, 14 caspases have been identified, many of which exist as orthologs in different species [56]. However, caspase-13 has not been reported in ovaries but has been identified in bovine white blood cells [57]. Although its specific role in the apoptosis cascade is not clear, it may be involved in cytokine (including TNF{alpha})-induced and mitochondria-mediated apoptosis [56]. Apaf-1 is also proapoptotic and is crucial for the recruitment and activation of caspases [41]. Studies in ovarian follicles show that Apaf-1 expression and granulosa cell apoptosis are inhibited after gonadotropin treatment [58]. Also, some ovarian cancer cell lines lack Apaf-1 activity and thus are resistant to apoptosis [59].

The expression of two tumor suppressor genes not previously identified in ovaries was also greater in thecal cells of subordinate compared with dominant follicles. B-cell translocation gene member 3 (BTG-3) is an inhibitor of cell proliferation [60]. Although the mechanism of action is unclear, the BTGs are part of a wider collection of similar molecules that represent a new family of antiproliferative molecules [61]. A tumor suppressor gene that is commonly missing in patients with B-cell chronic lymphocytic leukemia (TS-BCLL) was also elevated in theca cells of subordinate compared with dominant follicles in our study. Deregulated proliferation and absence of cell death is associated with loss of TS-BCLL and leukemia [62]. Hence, the gene functions to promote cell death, a function that is consistent with its possible role in cell death in the theca cells of subordinate follicles in the present experiment.

In addition to the above proapoptotic factors in theca and granulosa cells of the subordinate follicles, we identified mRNA for two factors, COX-1 and P58(IPK), which have greater expression in the largest subordinate than dominant follicle and whose function is unclear in follicles. There are two cyclooxygenase enzyme isoforms (COX-1 and COX-2) involved in the synthesis of prostaglandins and thromboxanes. COX-1 is the "housekeeping" isoform of cyclooxygenase, whereas COX-2 is rapidly induced in response to numerous intracellular and extracellular stimuli and acts in a proinflammatory fashion [63]. COX-2 is involved in ovulation in many species [64] but not COX-1 [65]. COX-2, but not COX-1, is involved in the activation of apoptosis in bovine luteal cells in vitro [66]. The role of COX-1 in tissue development is likely antiprolific but is not clear, and a role for COX-1 in follicle development has not been described. The double-stranded RNA-dependent protein kinase (PKR) induces apoptosis by activation of caspases [67]. P58(IPK) is an inhibitor of PKR and is considered to be a suppressor of apoptosis [68]. Why levels of mRNA for P58(IPK) are greater in the theca of subordinate compared with dominant follicles is unclear. However, activation of PKR leads to suppression of mRNA translation [69]. Thus, P58(IPK)-induced inhibition of PKR suppression may maintain translation of new proteins involved in apoptosis and follicle atresia.

To date, the only apoptotic factors examined in individual developing and regressing ovarian follicles during a follicle wave are Fas antigen and its ligand, which are suppressed in theca and granulosa cells of dominant compared with highly atretic subordinate follicles collected on Day 5 of the estrous cycle [15]. These differences were not observed in the present study, probably because follicles were collected earlier in the estrous cycle (Day 3), which is relatively soon after dominant follicle selection and before widespread DNA fragmentation [11] and probable activation of genes involved in the terminal stages of apoptosis (e.g., Bcl and caspase family proteins). This observation suggests that the 11 genes identified in our study may be early markers for the final stages of follicle differentiation and initiation of apoptosis. Because of the well-established important role of estradiol in follicular growth and differentiation in laboratory species [12], coupled with the enhanced production of estradiol and its receptor in dominant compared with subordinate follicles beginning during the very early stages and continuing throughout a follicular wave in cattle [30], we hypothesize that the genes identified as differentially expressed in granulosa or theca cells isolated from dominant versus largest subordinate may not only be potential estradiol target genes but also be involved in selection of a dominant follicle during follicular waves. Despite this working hypothesis, we recognize that some of the aforementioned genes may be regulated independently of estradiol by factors also known to differ in dominant versus subordinate follicles, including gonadotropins, inhibin, and/or insulin-like growth factor-I [70, 71].

In summary, the dual combination of cDNA microarray screening analysis and Q-RT-PCR is a rapid and reliable approach to assess differential expression of a variety of genes in multiple tissues from individual antral follicles. We examined the expression of 53 genes known to be involved in apoptosis pathways in theca and granulosa cells from follicles with high versus low intrafollicular concentrations of estradiol that may be important for differentiation of dominant and subordinate follicles. On the basis of our findings, we propose the following model. LH and/or FSH binds its receptor on granulosa cells of the dominant follicle, which enhances aromatase and estradiol production and increases estradiol receptor. This promotes the local synthesis of estradiol-target survival genes, DICE and MCL-1. In subordinate follicles, the negative actions of inhibin are mediated via its receptor, ß glycan, to enhance androgen production by theca cells and suppress estradiol production by granulosa cells. This results in a decrease in intrafollicular estradiol concentrations leading to a decrease in the expression of estradiol-target survival genes (above) and an increase in the expression of genes that may otherwise be inhibited by estradiol but that are involved in apoptosis and cell death (COX-1, TNF{alpha}, CAD, DRAK-2 in granulosa cells and caspase 13, P58(IPK), Apaf-1, BTG- 3, and TS-BCLL in theca cells; Fig. 3). Although we hypothesize that estradiol regulates these genes involved in apoptosis pathways, we recognize that they may be regulated by other endocrine and/or local factors within the ovaries. We conclude that of the 53 genes involved in apoptosis that were studied, 11 are differentially expressed in dominant compared with subordinate follicles and that these genes may be early markers of the final stages of follicle differentiation and initiation of apoptosis and thus contribute to the selection of dominant follicles during follicular waves.


   ACKNOWLEDGMENTS
 
We thank P. Duffy, C. O'Meara, and K. Ryan for assistance with tissue collection; S. Sipkovsky, C. Colvin, W. Nobis, and S. Suchyta for assistance with microarrays and PCR; and F. Jimenez-Krassel and N. Hynes for doing the steroid assays.


   FOOTNOTES
 
1 This work was supported by Science Foundation Ireland under Grant No. 02/IN1/B78 to A.C.O.E. and P.L. (the opinions, findings and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the Science Foundation Ireland) and by a grant from the United States Department of Agriculture (USDA 2000-02391) to J.J.I. Back

2 Correspondence: A.C.O. Evans, Department of Animal Science, Faculty of Agriculture, University College Dublin, Belfield, Dublin 4, Ireland. FAX: 353 1 716 1103; alex.evans{at}ucd.ie Back

Received: 3 November 2003.

First decision: 13 December 2003.

Accepted: 15 January 2004.


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 MATERIALS AND METHODS
 RESULTS
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