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BOR - Papers in Press, published online ahead of print February 15, 2006.
Biol Reprod 2006, 10.1095/biolreprod.105.045799
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BIOLOGY OF REPRODUCTION 74, 1051–1059 (2006)
DOI: 10.1095/biolreprod.105.045799
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Molecular Evidence That Growth of Dominant Follicles Involves a Reduction in Follicle-Stimulating Hormone Dependence and an Increase in Luteinizing Hormone Dependence in Cattle1

M. Mihm 2 3, P.J. Baker 3, J.L.H. Ireland 4, G.W. Smith 4, P.M. Coussens 4, A.C.O. Evans 5, and J.J. Ireland 4

Division of Cell Sciences,3 Faculty of Veterinary Medicine, University of Glasgow, Glasgow G61 1QH, United Kingdom Department of Animal Science and Center for Animal Functional Genomics,4 Michigan State University, East Lansing, Michigan 48824 School of Agriculture,5 Food and Veterinary Science and the Conway Institute, University College Dublin, Dublin 4, Ireland

ABSTRACT

The bovine dominant follicle (DF) model was used to identify molecular mechanisms potentially involved in initial growth of DF during the low FSH milieu of ovarian follicular waves. Follicular fluid and RNA from granulosa and theca cells were harvested from 10 individual DF obtained between 2 and 5.5 days after emergence of the first follicular wave of the estrous cycle. Follicular fluid was subjected to RIA to determine estradiol (E) and progesterone (P) concentrations and RNA to cDNA microarray analysis and (or) quantitative real-time PCR. Results showed that DF growth was associated with a decrease in intrafollicular E:P ratio and in mRNA for the FSH receptor, estrogen receptor 2 (ER beta), inhibin alpha, activin A receptor type I, and a proliferation (cyclin D2) and two proapoptotic factors (apoptosis regulatory protein Siva, Fas [TNFRSF6]-associated via death domain) in granulosa cells. In contrast, mRNAs for the LH receptor in granulosa cells and for two antiapoptotic factors (TGFB1-induced antiapoptotic factor 1, LAG1 longevity assurance homolog 4 [Saccharomyces cerevisiae]) and one proapoptotic factor (tumor necrosis factor [ligand] superfamily, member 8) were increased in theca cells. We conclude that the bovine DF provides a unique model to identify novel genes potentially involved in survival and apoptosis of follicular cells and, importantly, to determine the FSH-, estradiol-, and LH-target genes regulating its growth and function. Results provide new molecular evidence for the hypothesis that DF experience a reduction in FSH dependence but acquire increased LH dependence as they grow during the low FSH milieu of follicular waves.

apoptosis, estradiol receptor, follicle-stimulating hormone receptor, follicular development, gene regulation

INTRODUCTION

The gonadotropin-dependent molecular mechanisms underlying growth of dominant follicles during the low serum FSH milieu of ovarian follicular waves [reviewed in 1, 2] are poorly understood in single-ovulating species. This hinders resolution of complex ovarian disorders preventing ovulation, such as polycystic ovarian disease, and inhibits progress toward therapies to precisely regulate dominant follicle development in women and other monovulatory species with agricultural importance, such as cattle.

The synergistic roles of FSH, estradiol, and LH in growth and differentiation of antral follicles in rodent models [3] and the importance of the timing of the alterations in secretion of FSH and LH for development of dominant follicles during follicular waves in cattle [4–6] are well established. However, gonadotropin and estradiol target genes involved in proliferation, survival, and differentiation of granulosa and theca cells during development of dominant follicles are unknown. Because FSH secretion declines [5] while episodic LH secretion increases when the dominant follicle becomes selected [6], growth of dominant follicles during the low FSH milieu of ovarian follicular waves is hypothesized to be associated with decreased expression of the FSH receptor gene but enhanced expression of estradiol receptor ß and LH receptor genes in granulosa cells and of genes involved in proliferation and survival of theca and (or) granulosa cells. To test this hypothesis, the present study used a combination of ultrasound analysis, RIA, microarray and quantitative real-time PCR (qRT-PCR) to 1) identify new genes that potentially regulate proliferation and survival in granulosa and theca cells and 2) firmly establish the association of alterations in expression of newly identified proliferation and survival genes in granulosa and theca cells with alterations both in intrafollicular estradiol and progesterone concentrations and in expression of receptor mRNA for the key hormones that regulate antral follicle growth, FSH, LH, and estradiol. Results from this well-defined "window" of dominant follicle development are expected to provide the foundation for future mechanistic studies designed to determine which genes or their proteins and signaling systems in granulosa or theca cells are regulated by gonadotropins or estradiol.

MATERIALS AND METHODS

Animal Model and Recovery of Ovaries

Ten nonlactating Holstein or Holstein x Hereford cows, 3–11 yr old and in the luteal phase of the estrous cycle, were treated with an intravaginal progesterone device (Eazi-Breed CIDR; Animal Reproductive Technologies Ltd) and injected with the prostaglandin F2{alpha} analogue luprostiol (Prosolvin; Intervet; http://www.intervet.co.uk) before device withdrawal or with luprostiol and the gonadotropin-releasing hormone analogue buserelin (Receptal, Intervet) 48 h later (one cow) to induce precisely timed luteolysis, estrus, and ovulation [7]. From the time of CIDR removal or buserelin treatment, ovarian follicular development was monitored daily by transrectal ultrasound scanning using a 7.5-MHz transducer to determine the time of ovulation. Subsequent ultrasound examinations were carried out at 12-h intervals to detect emergence of the first follicle wave of the cycle. The time of wave emergence was defined as the first day a follicle >3 mm in diameter belonging to the new wave was detected during ultrasound scanning [8]. Dominant follicles were obtained between Days 2 and 5.5 after wave emergence (Fig. 1) following slaughter of cows at the postmortem facility of the Faculty of Veterinary Medicine, University of Glasgow. This time interval coincides with the early phase of rapid growth and enhanced estradiol production of a dominant follicle [4]. Thus, all dominant follicles were recovered on Days 3.5–6.5 of the estrous cycle (Day 0 = onset of estrus). Cows were bred and raised at Cochno Farm, University of Glasgow. Transrectal ultrasound scanning was carried out under license awarded by the Home Office, United Kingdom, according to the Animals and Procedures Act 1986 and in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching.


Figure 1
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  		FIG. 1. a) Growth of the dominant and largest subordinate follicle (plotted to illustrate the size deviation between the two largest follicles in the wave) in 10 individual cows determined by ovarian ultrasound scanning every 12 h from wave emergence to recovery of dominant follicles. b) Schematic illustration of the timing of DF recovery and the reference design for the cDNA microarray study.

Follicle Dissection and Recovery of Follicular Fluid and Granulosa and Theca Cells

Following slaughter, ovaries were removed and placed in ice-cold 0.05 M PBS for transport to the laboratory. Dominant and subordinate follicles belonging to the first follicle wave of the estrous cycle were identified using ovarian diagrams, ultrasound records, and caliper measurements and dissected free from stroma. Follicular fluid was aspirated using a 20- or 25-gauge needle attached to a 1-ml syringe, stored on ice until all dissections were completed, and frozen at –20°C until assay for estradiol (E) and progesterone (P) concentrations. Care was taken for each cow to ensure that dominant follicles were always the largest follicle with highest intrafollicular E compared with other follicles from the same wave [5].

Granulosa and theca interna cells were collected and snap-frozen in liquid nitrogen in 1 ml of TRIzol Reagent (Invitrogen; http://www.invitrogen.com) as described recently [9] using a plastic inoculation loop to scrape granulosa cells from the theca layer. Total time from ovary recovery to freezing of cells was 30–45 min. Granulosa and theca samples in TRIzol were subsequently stored at –80°C until total RNA extraction.

Follicular Fluid Steroid Analysis

Estradiol concentrations in nonextracted follicular fluid samples were determined using a validated RIA [5]. Sensitivity of the assay is 0.04 pg/tube, and the mean intra- (n = 3–4) and interassay (n = 3) coefficients of variation (CV) for a 1.33 pg/tube quality control sample were 12.6% and 2.6%, respectively.

Progesterone concentrations in nonextracted follicular fluid samples were estimated using a validated RIA [10, 11]. Sensitivity of the assay is 0.01 ng/tube and mean intra- (n = 3–4) and interassay (n = 2) CV for a 0.35 ng/tube quality control sample were 8.8% and 7.8%, respectively.

Extraction and Estimation of Quantity and Quality of Total RNA from Individual Granulosa and Theca Samples and Generation of a Pooled Reference Sample

Total RNA extraction and DNAse treatment were carried out as previously described [9]. In addition, a reference sample ("reference") was generated by pooling total RNA extracted from granulosa and theca cell samples of the 10 dominant follicles, with each cell type contributing approximately equally to the reference. Quantity and quality of reference and individual granulosa and theca RNA samples were determined using the Agilent 2100 Bioanalyzer nanochip (Agilent Technologies Inc.; http://www.chem.agilent.com, and 8- and 2-µg aliquots of total RNA were prepared for the cDNA microarray and the qRT-PCR study, respectively.

Prescreening of Granulosa and Theca Cell Gene Expression Using cDNA Microarray

Reference design. A reference design [12] was chosen for cDNA microarray analysis to profile gene expression of individual granulosa or theca cell samples relative to the same reference sample. A schematic of the reference design utilized for the cDNA microarray study is presented in Figure 1b. The reference was generated from granulosa and theca samples of all follicles to minimize the likelihood of extremely low or high cy3/cy5 hybridization ratios due to very different RNA concentrations between reference and individual samples, which may compromise subsequent normalization procedures [12].

Complementary DNA Microarray Gene Expression Profiling. Gene expression profiling of granulosa and theca cells from individual dominant follicles was carried out using the BOTL-5 cDNA microarray as explained previously [9]. In brief, the BOTL-5 was developed from a bovine total leukocyte library (http://www.nbfgc.msu.edu under Links), and the array contains a total of 3889 spots representing 1395 individual genes involved in steroidogenesis, signal transduction, proliferation, cellular life span, and apoptosis. Reverse transcription of total RNA, cDNA purification, dye labeling, and purification of labeled cDNA were carried out using previously described protocols [9, 13]. All individual granulosa and theca cell cDNA samples were labeled with Cy3 and matched with one aliquot of the reference, which was labeled with Cy5 (Fig. 1b). One granulosa and one theca cell sample did not render sufficient total RNA of adequate quality for the microarray study. Hybridizations of individual granulosa and theca samples against reference, followed by image acquisition and analyses, were conducted as recently reported [9].

Quantile normalization of cDNA microarray data before analysis. The log2 transformed Cy3/Cy5 ratios underwent a quantile normalization procedure to ensure equal distribution of ratios across all arrays from the same tissue type. This procedure was considered highly suitable for the reference design utilized in our study, as single relative expression levels were calculated for each gene in each sample, and the technique compares favorably with others in relation to ease of use, speed, and evaluation of variance and bias [14].

Quantitative RT-PCR Study

Abundance of mRNA encoding the gonadotropin receptors and estrogen receptor 2 (ER beta) (ESR2) was examined in granulosa cells using qRT-PCR according to our previously published procedures [9, 13]. Abundance of 17 further transcripts that were identified during cDNA microarray prescreening was also examined in granulosa and theca cells using qRT-PCR, an approach recommended for microarray studies [15] and used in our previous study of follicle mRNA expression [9]. Estimation of cDNA concentrations was carried out using the NanoDrop ND 1000 Spectrophotometer (NanoDrop Technologies; http://www.nanodrop.com), and 5–20 ng of cDNA were added to each reaction. All primers were designed using Primer Express Software v2.0 (Applied Biosystems; http://www.appliedbiosystems.com), synthesized by Operon Technologies (http://www.operon.com), and used at 100–600-nM concentrations. A list of genes, accession numbers, and primers used for qRT-PCR is in Table 1. One of the 10 thecal samples did not render sufficient total RNA of adequate quality for the qRT-PCR study.


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  		TABLE 1. Sequences of primers (5' to 3') used for qRT-PCR.

The amount of transcripts in each sample was normalized using actin, beta (ACTB) as the internal control gene to correct for differences in cDNA loading. Subsequently, relative transcript levels to the reference were determined in individual granulosa or theca samples using the 2–{Delta}{Delta}CT method [16]; thus, the reference was used as the calibrator sample for all genes. The internal control gene ACTB was chosen for the following reasons: it had previously been used in porcine and bovine studies of follicular cell gene expression with no indication of regulation within large antral follicles [10, 17], cDNA microarray analysis demonstrated that mRNA expression was unaltered in dominant follicles during Days 2–5.5 after wave emergence, and very little variation in CT values existed between individual granulosa and theca samples and the reference.

Statistical Analysis

Linear regression analysis was carried out to determine whether follicle parameters or mRNA expression are significantly altered in first-wave dominant follicles recovered between Days 2 and 5.5 after wave emergence, representing the time of predicted rapid growth. Therefore, the slope of the regression line indicates a positive (increase in follicle parameter or mRNA abundance) or negative (decrease in follicle parameter or mRNA abundance) relationship between the follicle parameter or mRNA abundance and dominant follicle "age." Linear regression analysis of microarray data was carried out in SAS/STAT (Statistical Analysis Systems, Inc.; http://www.sas.com), and linear regression analysis in MINITAB v13.1 (http://www.minitab.com) was used to determine if dominant follicle diameter, intrafollicular steroid concentrations, ratio of E:P in follicular fluid, and relative mRNA abundance estimated in granulosa or theca cells using qRT-PCR were altered in growing dominant follicles during Days 2–5.5 after wave emergence. In Figure 2, individual data points in addition to regression results (regression lines, P values, and R2 as a measure of model fit) are shown to allow comparison with previous studies using the bovine dominant follicle model, while regression results are presented in Tables 2 and 3 and in Figures 3 and 4. P values <0.05 are considered significant, and P values from 0.05 to 0.06 are reported as tendencies. Abundance of mRNA relative to the reference (estimated for cDNA microarray data as the cy3/cy5 ratio and for qRT-PCR data using 2–{Delta}{Delta}CT) was log2 transformed before analyses because untransformed data show an abnormally skewed distribution (overexpression is represented by values from 1 to infinite, while underexpression is represented by values only between 0 and 1).


Figure 2
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  		FIG. 2. Changes in diameter following dissection (a), intrafollicular E (b), and P (c) concentrations and estrogenic activity (d) determined in DF during Days 2 to 5.5 after wave emergence. Results from the regression analyses (regression lines, R2, and P values) are presented in addition to individual data points. FF, Follicular fluid; E, estradiol; P, progesterone; E:P ratio, estrogenic activity.


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  		TABLE 2. Results from regression analysis of cDNA microarray data for a subset of selected granulosa and theca cell genes whose expression was determined to be altered in dominant follicles during Days 2 to 5.5 after wave emergence.


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  		TABLE 3. Results from regression analysis of qRT-PCR data for selected granulosa and theca cell genes whose expression was not altered (P > 0.06) in dominant follicles during Days 2 to 5.5 days after wave emergence.


Figure 3
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  		FIG. 3. Changes in the relative mRNA expression of granulosa cell genes regulating hormone response (a), growth factor signaling (b), and proliferation/apoptosis (c) determined by qRT-PCR in 10 DF during Days 2–5.5 after wave emergence. Results from the regression analyses (regression lines, R2, and P values) are presented for each gene. Note that relative gene expression values are log2 transformed; therefore, 0 = no difference to reference, +1 = 2-fold increase relative to reference, and –1 = one-half of reference gene expression.


Figure 4
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  		FIG. 4. Changes in the relative expression of theca cell genes regulating proliferation (a), cellular life span, or apoptosis (b) determined by qRT-PCR in nine DF during Days 2–5.5 after wave emergence. Results from the regression analyses (regression lines, R2, and P values) are presented for each gene. Note that relative gene expression values are log2 transformed; therefore, 0 = no difference to reference, +1 = 2-fold increase relative to reference, and –1 = one-half of reference gene expression.

RESULTS

Growth of Dominant Follicles and Intrafollicular Steroid Concentrations

At the last ultrasound examination, dominant follicles ranged in diameter from 7.7 mm (follicle recovered 2 days after wave emergence) to 16.4 mm (follicle recovered 5 days after wave emergence), and follicles grew 1.8 ± 0.2 mm (mean ± SEM) during the last 24 h before recovery. Measurements taken following dissection revealed that while dominant follicles almost doubled in size (P = 0.01; Fig. 2a), intrafollicular concentrations of E and P were maintained (P > 0.2; Fig. 2, b and c). Although all dominant follicles had higher E than P in follicular fluid and were, therefore, hormonally classified as healthy (= estrogen active [18]), the ratio of E:P in follicular fluid declined 64% during Days 2–5.5 after wave emergence (P < 0.05; Fig. 2d).

Complementary DNA Microarray Prescreening and qRT-PCR Analyses

Preliminary screening with cDNA microarrays revealed alterations (P ≤ 0.05) in the mRNA abundance for 60 granulosa and 53 theca cell genes in growing dominant follicles during Days 2–5.5 after wave emergence (see data in the Supplemental Table 1 available online at http://www.biolreprod.org). From these, only genes involved in the induction or prevention of apoptosis (five genes in granulosa and eight genes in theca cells) and genes known to be regulated by the gonadotropins and estradiol and involved in proliferation, steroidogenesis, and growth factor signaling (five genes) were selected for further study using qRT-PCR (Table 2).

Quantitative real-time PCR analyses showed no changes (P > 0.06) in the mRNA expression of 10 of 18 selected genes prescreened using microarray (Table 3). However, in addition to alterations seen in the mRNA expression for the gonadotropin receptors and ESR2, mRNA expression for the remaining eight selected genes was determined to be altered in growing dominant follicles during Days 2–5.5 after wave emergence. Specifically, in granulosa cells recovered from growing dominant follicles, mRNA expression for the LH receptor (LHCGR) was highly upregulated, while mRNA expression for the FSH receptor (FSHR) and ESR2 was highly downregulated during Days 2–5.5 after wave emergence (P ≤ 0.01; Fig. 3a). Similarly, expression of mRNAs for the activin A receptor type I (ACVR1; Fig. 3b), cyclin D2 (CCND2; Fig. 3c), and apoptosis regulatory protein Siva (SIVA; Fig. 3c) was (P < 0.01) downregulated, while the expression of mRNAs for inhibin alpha (INHA; P = 0.05; Fig. 3b) and Fas (TNFRSF6)-associated via death domain (FADD, P = 0.06; Fig. 3c) tended to be downregulated in dominant follicles during Days 2–5.5 after wave emergence.

In contrast, mRNA expression for TGFB1-induced antiapoptotic factor 1 was highly upregulated (TIAF1; P < 0.01; Fig. 4a), while mRNA expression for LAG1 longevity assurance homolog 4 (Saccharomyces cerevisiae) (LASS4; P = 0.06; Fig. 4b) and tumor necrosis factor (ligand) superfamily, member 8 (TNFSF8; P = 0.05; Fig. 4b), tended to be upregulated in theca cells recovered from growing dominant follicles during Days 2–5.5 after wave emergence.

DISCUSSION

The results of the present study are depicted in our model of dominant follicle growth (Fig. 5) and demonstrate, for the first time during the early growth phase of first-wave dominant follicles in cattle (Fig. 5B), that the alterations in serum concentrations of FSH, LH, and estradiol (Fig. 5A) are associated with 1) a steep decline in intrafollicular ratio of estradiol:progesterone (Fig. 5B); 2) decreased mRNA expression for genes known to be induced by FSH in granulosa cells, including the FSHR, ESR2, INHA, ACVR1, and CCND2 (Fig. 5C, part 1); 3) increased mRNA expression for LHCGR in granulosa cells (Fig. 5C, part 2); and 4) alterations in mRNA expression of numerous genes potentially involved in survival and apoptosis of granulosa and theca cells, including SIVA, FADD, TIAF1, LASS4, and TNFSF8 (Fig. 5C, parts 3 and 4). The present study not only identified five novel genes in granulosa (SIVA, FADD) and theca cells (TIAF1, LASS4, TNFSF8) and five known genes in granulosa cells (FSHR, ESR2, INHA, ACVR1, CCND2) potentially important for the regulation of cellular proliferation, survival, and apoptosis but also provides new molecular evidence for the hypothesis that granulosa cells experience a reduction in FSH dependence but acquire increased LH dependence as dominant follicles grow during follicular waves (Fig. 5D). This shift in gonadotropin dependence at the level of granulosa cells needs to be further addressed in the future.


Figure 5
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  		FIG. 5. Model summarizing the association between systemic hormone concentrations and alterations in follicle function and mRNA expression as first-wave dominant follicles undergo rapid growth from 2 to 5.5 days after wave emergence. A) Serum concentrations of FSH, LH, and E drawn schematically based on [4–6, 22, 24]. B) Diameter increase of DF and relative changes in the ratio of follicular fluid E to progesterone (E:P). C) Alterations in abundance of mRNA for 1) FSH receptor (FSHR) and other key genes known to be induced by FSH in granulosa cells, including estrogen receptor 2 (ESR2), activin A receptor type I (ACVR1), inhibin alpha (INHA), and cyclin D2 (CCND2); 2) LH receptor (LHCGR) in granulosa cells; 3) proapoptotic genes (FADD, SIVA) in granulosa cells; and 4) proliferation (TIAF1), survival (LASS4), and apoptosis (TNFSF8) genes in theca cells. D) Changes in follicle parameters may result from a reduction in FSH dependence and concomitant increase in LH dependence of granulosa cell functions as dominant follicles grow during the low FSH milieu of follicular waves. Note that Day 0 is the first day new follicles larger than 3 mm are detected by ultrasound, and this is the designated day of emergence of the follicular wave.

During their growth from 2 to 5.5 days after wave emergence, sizes achieved by individual dominant follicles were comparable to those of ovulatory follicles [19], although first-wave dominant follicles are generally nonovulatory and undergo atresia because of a lack of LH support [20]. Cessation of growth and reductions in estrogenic activity in first-wave dominant follicles occur simultaneously with the initial decline in LH pulse frequency on Day 8 of the bovine estrous cycle [21–23], which is usually 6 days after emergence of the first follicular wave (Fig. 5A). In the present study, first-wave dominant follicles were recovered before this time point, between Days 3.5 and 6.5 after onset of estrus. Although intrafollicular estradiol and progesterone concentrations showed no significant change during the rapid growth of these dominant follicles because of large variation (exemplified by the two "oldest" dominant follicles, which had 36 and 151 ng/ml E in follicular fluid, respectively), the ratio of E:P, which is a well-characterized hormonal marker for follicle health (i.e., the in vivo capacity of follicles or in vitro capacity of granulosa cells to produce estradiol [18, 24]), declined by more than 60%. Therefore, in contrast to dominant ovulatory follicles, dominant nonovulatory follicles undergo a loss in such estradiol-producing capacity despite their rapid growth. In support of this, two studies reported that systemic estradiol concentrations in heifers decline before Day 8 of the estrous cycle [23, 25], approximately 3 days after emergence of the first follicular wave (Fig. 5A). While the exact cause of this early decrease in intrafollicular E:P ratio during growth of dominant nonovulatory follicles is unknown, it is unlikely to be the result of atresia for several reasons. First, all dominant follicles in the present study were classified as estrogen active because their intrafollicular ratio of E:P was >1. Our previous studies show that estrogen-active follicles are healthy, growing antral follicles with minimal amounts of pyknotic or apoptotic granulosa cells compared with estrogen-inactive (P > E in FF) follicles [18, 26]. Second, all dominant follicles were in a growth phase during Days 3.5–6.5 of the estrous cycle as explained earlier and have the ability to ovulate following prostaglandin-induced luteolysis on Day 7 [27]. Third, mRNA expression for the LH receptor, a marker for differentiation of granulosa cells [3], was enhanced during dominant follicle growth (Fig. 5C, part 2), as was mRNA abundance for genes involved in prevention of apoptosis (Fig. 5C, part 4), while mRNA abundance for proapoptotic genes declined (Fig. 5C, part 3). Nevertheless, previous studies in cattle demonstrate that a decline in intrafollicular estradiol production as follicles grow during waves precedes apoptosis of granulosa cells and onset of follicular atresia [26]. Consequently, this indication of an early decline in the estradiol producing capacity during growth of dominant nonovulatory follicles may have important consequences for the regulation of genes involved in survival and apoptosis of granulosa and theca cells, the balance of which ultimately controls the physiological fate (growth or atresia) of dominant follicles.

Findings from the present study suggest that at the time when systemic FSH concentrations are low (Fig. 5A), the decline in the intrafollicular ratio of E:P (Fig. 5B) is also associated with a diminished capacity of granulosa cells to be stimulated by FSH. First, abundance of mRNAs in granulosa cells for the FSH receptor decreased coincident with dominant follicle growth (Fig. 5C, part 1). This likely results in reductions in FSH binding as dominant follicles grow, especially as previous radioligand binding studies showed a reduced number of FSH receptors in granulosa cells during growth of dominant nonovulatory and ovulatory follicles in cattle [18, 28]. Second, the abundance of mRNAs for several key genes known to be induced by FSH in granulosa cells of growing follicles, including ESR2 [29], ACVR1 [30], INHA [31], and CCND2 [32], also decreased during dominant follicle growth (Fig. 5C, part 1) in the present study. Although the protein products corresponding to each gene were not measured, experiments using a variety of in vivo and in vitro rodent and (or) bovine models usually show positive effects of estradiol, activin, inhibin, and cyclin D2 (stimulates granulosa cell proliferation [32]) on follicular growth, differentiation, and function [reviewed in 30, 33, 34]. In addition, secretion of estradiol and activin is not only stimulated by FSH, but both hormones are, in turn, important mediators of FSH action [35–37]. Thus, the potential diminished synthesis of inhibin alpha and cyclin D2 and receptors for estradiol and activin as a result of reduced gene expression could markedly reduce FSH actions on granulosa cells during dominant follicle development. Taken together, the in vivo observations generated in the present study strongly suggest that FSH-mediated molecular events decline in granulosa cells (Fig. 5D) coincident with dominant follicle growth during the first follicle wave. It is not known whether the same applies to ovulatory dominant follicles growing during the follicular phase. However, this may at least partially explain why FSH treatments administered during growth of the first-wave dominant follicle do not prevent its decline in estrogen activity or delay its atresia [38] or why the increase in serum FSH concentrations at the end of the first wave, which is responsible for the next follicle wave in cattle, does not affect growth or estradiol production of the original first-wave dominant follicle [4, 21]. In fact, a reduction in FSH-mediated stimulation of granulosa cell function may be crucial for growing dominant follicles to prevent abnormal growth and cyst formation and may also be important for atresia of nonovulatory dominant follicles by ensuring loss of dominance and occurrence of new follicular waves. Subordinate follicles, which are usually <8 mm in size, also show reduced ability to synthesize FSHR and diminished capacity to produce estradiol prior to their atresia during follicular waves in cattle [26, 39]. In contrast, granulosa cells obtained from 5–7-mm antral follicles of women with polycystic ovarian disease retain their in vitro capacity to respond to FSH and produce high levels of estradiol [40]. These observations emphasize the potential importance of reductions in FSH-dependent granulosa cell function during the normal sequential development of dominant follicles in single-ovulating species like cattle and humans [41, 42].

The decline in mRNA expression for FSHR and other genes known to be induced by FSH observed in the present study provides molecular evidence that growth and function of dominant follicles do not completely depend on FSH. Indeed, several lines of evidence support a critical role for LH rather than FSH in growth, differentiation, and estradiol production by dominant follicles. First, a rise in serum concentrations of LH occurs just before the dominant follicle becomes selected in cattle [6]. Second, continued growth of dominant follicles during the low FSH milieu of follicular waves in cattle is associated with increased frequency of episodic secretion of LH, while atresia or ovulation of the dominant follicle at the end of a wave is associated with reduced frequency (Fig. 5A) or enhanced episodic LH secretion, respectively [23, 43]. Thus, alterations in episodic secretion of LH have an important role in regulation of dominant follicle growth and function during follicular waves [44]. Third, abundance of mRNA for the LH receptor in granulosa cells increased markedly during dominant follicle growth concomitant with the reduced expression of FSHR and other FSH-induced genes in the present study (Fig. 5C, parts 1 and 2). In support of an increased ability of granulosa cells to respond to LH during dominant follicle growth, in situ hybridization [45] and receptor binding assays [18] also demonstrate an increase in amounts of both LHR mRNA and LH receptor numbers in granulosa cells during growth of dominant follicles in cattle. In addition, it has been shown in the rodent that induction of LHR gene expression causes an increase in LH responsiveness of immature granulosa cells [46]. Increased mRNA expression of the LH receptor also distinguishes dominant from subordinate follicles during follicular waves [9, 45, 47].

Alterations in mRNA abundance during dominant follicle development, which were determined for 10 of 18 selected transcripts using microarray prescreening, were not confirmed at P ≤ 0.06 using qRT-PCR (Table 3). While a further validation of microarray gene expression data using other molecular techniques is recommended, complete agreement between the two molecular approaches is not expected, mostly because of differences in the sequence of clones and PCR products, recognition of sequence variants, or data analysis (normalization) [15]. The present combined microarray/qRT-PCR approach confirmed alterations in transcript levels of eight genes, a result that is concurrent with our previous study of follicle gene expression [9]. Specifically, alterations in the expression of five genes possibly involved in survival or apoptosis in granulosa and theca cells were associated with increased mRNA expression for LHCGR in granulosa cells during dominant follicle growth. Thus, abundance of mRNAs in granulosa cells for the proapoptotic genes SIVA and FADD was decreased during dominant follicle growth (Fig. 5C, part 4), while abundance of mRNAs in theca cells for the antiapoptotic and survival genes TIAF1 and LASS4 was increased (Fig. 5C, part 3). Proapoptotic effects of variants of the SIVA protein have so far been described only in blood and cancer cells [48, 49]. However, the adaptor protein FADD in granulosa cells conveys caspase-mediated death signals following activation of death receptors by tumor necrosis factor alpha (TNF) or fas ligand [50], and both TNF and fas ligand are preferentially expressed in bovine subordinate follicles undergoing atresia [9, 51] and can induce apoptosis in bovine granulosa cells [52, 53]. TIAF1 is antiapoptotic, preventing cytotoxic effects of TNF and FADD [54], whereas longevity assurance gene 1 homologs (such as LASS4) increase cellular lifespan of yeast cells and prohibit uncontrolled cellular proliferation in tumor cells [55, 56]. In addition, mRNA abundance in theca cells for the proapoptotic TNFSF8, a member of the tumor necrosis factor superfamily usually expressed by T and B lymphocytes and embryonic carcinoma cells [57, 58], was increased during dominant follicle growth (Fig. 5C, part 4). These genes have an important role in survival and apoptosis primarily in nonreproductive tissues or cell lines but have not been studied in detail in reproductive cells. Thus, the bovine dominant nonovulatory follicle provides a well-characterized and unique model not only to examine the roles of these novel genes and their mechanism of action in granulosa and theca cells but also to determine if these genes are regulated by FSH, LH, or estradiol during dominant follicle growth. In addition, it remains to be determined whether the regulation of such genes differs between ovulatory and nonovulatory dominant follicles. Taken together, we suggest that the diminished mRNA expression of FSHR and other multiple key genes known to be induced by FSH in granulosa cells of growing follicles, coupled with the enhanced mRNA expression of LHCGR and alterations in numerous genes possibly involved in survival and prevention of apoptosis in granulosa and theca cells, provides new molecular evidence for the hypothesis that granulosa cells increasingly depend on LH and less on FSH (Fig. 5D) as dominant follicles grow during the low FSH milieu of follicular waves.

ACKNOWLEDGMENTS

The authors would like to acknowledge staff at Cochno Farm, University of Glasgow, for animal care and maintenance; S. Sipkovski, S. Suchyta, and C. Colvin for valuable guidance during the microarray and qRT-PCR studies; and R. Tempelman and G. Rosa for advice regarding the reference design and quantile normalization of microarray data.

FOOTNOTES

1 Supported by a fellowship from the Faculty of Veterinary Medicine, University of Glasgow, BBSRC 17/S17355 and BBSRC 36727/1 to M.M. and by National Research Initiative Competitive grants 2000-02255 and 2004-01697 from the USDA Cooperative State Research, Education and Extension Service and the Agricultural Experiment Station at Michigan State University to J.J.I. Back

2 Correspondence: M. Mihm, Division of Cell Sciences, Faculty of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow G61 1QH, U.K. FAX: 44 141 330 5797; Back

101 m.mihm{at}vet.gla.ac.uk Back

Received: 20 July 2005.

First decision: 18 August 2005.

Accepted: 10 February 2006.

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