Differential genome-wide gene expression profiling of bovine largest and second-largest follicles: identification of genes associated with growth of dominant follicles

The final stage of bovine follicular development occurs in a wave-like fashion [1, 2]. During a wave, increase of follicle-stimulating hormone (FSH) induces recruitment of a cohort of follicles beyond 4 mm in diameter and usually a single follicle is selected as a dominant follicle (DF) [3, 4]. Although the DF continues to grow by transition of gonadotropin dependency from FSH to luteinizing hormone (LH) and secretes large quantities of estradiol (E₂), the remaining subordinate follicles (SFs) cease to grow, then undergo atresia [5]. It is well documented that increased expression of LH receptor (LHR) in granulosa cells (GC) and specific changes of intrafollicular factors such as the insulin-like growth factor (IGF) and inhibin-activin-follistatin systems play a critical role in E₂ production in the DF [6, 7]. Therefore, regulatory mechanisms of follicular development are closely associated with complex interactions between follicular local paracrine/autocrine factors and endocrine hormones.

Increasing evidence using global gene expression analysis such as a DNA microarray, suppression subtractive hybridization and serial analysis of gene expression have identified numerous genes in various aspects of bovine follicular development [8‐18]. Some studies compared the gene expression profiles between DF and SF around the time of follicular selection. They showed that DF up-regulates genes regulating E₂ synthesis, anti-apoptosis, cell proliferation and gene transcription. Conversely, SF enhanced the expression of genes associated with pro-apoptosis and cell death compared with the DF [8, 9, 13, 14]. Recent studies found that 93 mostly novel genes were differently expressed in the GC of newly selected DF compared with SF and/or growing cohort follicles whereas most of these genes were down-regulated in the GC of preovulatory follicles during final maturation before the LH surge [15, 17]. Growth of a DF during 2-5.5 days following follicular wave emergence was associated with a decrease in genes encoding proliferation and pro-apoptotic factors and an increase in genes regulating anti-apoptotic factors [12]. An increase in follicular diameter during follicular growth was accompanied by alteration of gene expression regulating some growth factors and cytokines [16, 18]. Ndiaye et al. identified a subset of novel genes down-regulated in preovulatory follicles after human chorionic gonadotropin (hCG) stimulation compared with DF, which may contribute to ovulation and luteinization [11].

In this study, as expected, hierarchical cluster analysis of the microarray data classified F1 and F2 according to differences in gene expression profiles. In each follicular group, characteristic genes determining their developmental status were expressed. The F1 showed greater expression of genes responsible for enhancement of follicular E₂ production than the F2. These genes were gonadotropin receptor (FSHR), steroidogenic enzymes (CYP17, CYP19 and HSD17B1) and inhibin-activin-follistatin system (INHA, INHBA and FST). It is well demonstrated that mRNA expression for FSHR, CYP17, CYP19, INHA and INHBA increases with the progress of bovine follicular development and is greater in DF than SF [8, 12, 29]. On the other hand, the F2 had greater expression of IGFBP5 mRNA than the F1. IGFBP5 mRNA expression dramatically increased in bovine atretic follicles compared with the DF [26]. Intrafollicular levels of IGFBP proteolytic activity and IGFBPs gene expression are important for bioavailability of free IGF within the follicle and play a crucial role for determining follicular dominance and fate [7, 30]. Therefore, we evaluated the F1 were selected DF and the F2 were unselected SF.

Our evaluation of follicular status was confirmed to investigate CYP19 and IGFBP5 mRNA localization in healthy and atretic follicles in experiment 2 using in situ hybridization. CYP19 mRNA was abundantly expressed in healthy follicles but it was also expressed in atretic follicles while IGFBP5 mRNA was detected only in atretic follicles. Both CYP19 and IGFBP5 mRNA is hormonally regulated in bovine follicular cells [31‐34], in addition, in situ hybridization is not quantitative and not be as sensitive as QPCR. Thus, small amounts of CYP19 and IGFBP5 mRNA may be detected or regulated in atretic and healthy follicles, respectively. Since we did not perform sample collection at a specific phase of follicular wave in experiment 1, detailed growth profiles of the follicles we used were unclear. However, our results demonstrate that randomly collected follicles can be divided into several groups by similarities of gene expression profiles among the follicles and suggest that gene expression profiles of examined follicles are closely associated with their development status.

Confirmation of microarray data by QPCR analysis successfully identified a set of genes differentially expressed between the F1 and F2. In addition, possible involvement of these genes in follicular development and/or atresia was further demonstrated to investigate mRNA localization in healthy and atretic follicles. The F1 showed greater expression of genes involved in follicular growth and survivability (AMH, PLA2G1B, SCD2 and TRB2) than the F2. High expression of these genes may be closely associated with the establishment and maintenance of follicular dominance. Although the functional role of AMH in antral follicle development is poorly understood, recent studies showed that both intrafollicular AMH concentration and AMH mRNA expression were highest in small antral follicles and then decreased with follicular growth, suggesting the involvement of AMH in bovine follicular recruitment and/or selection [16, 35, 36]. A recent study showed a significant decrease of AMH mRNA expression in late atretic follicles compared with healthy follicles [36], which is consistent with our present result. Furthermore, in agreement with previous studies [16, 37], our in situ hybridization study showed that AMH mRNA was localized in only GC. High expression and clear localization of AMH mRNA in the GC of healthy large follicles implies that this growth factor has a plausible effect on the development of DF after follicular selection as well as recruitment.

PLA2 enzymes including PLA2G1B hydrolyze fatty acids from the sn-2 position of phospholipids with concomitant formation of lysophospholipids, which serve as precursor for lipid mediators such as lysophosphatidic acid (LPA) [38, 39]. Released LPA has diverse biological activities including cell proliferation and differentiation, suppression of apoptosis and cytoskeleton modulation in reproductive tissues [39]. Because Diouf et al. reported that PLA2G1B mRNA expression in the GC of bovine preovulatory follicle decreased after hCG injection [40], PLA2G1B may mainly contribute to generation of LPA during DF growth before the LH surge.

SCD is a rate-limiting enzyme that catalyzes the synthesis of monounsaturated fatty acids, mainly palmitic and oleic acid [41]. Consistent with our result, SCD mRNA expression in bovine follicles was found to be highest in GC of DF than in cohort follicles before selection or SF [11, 17]. Expression of SCD2 is hormonally regulated during follicular development because both SCD2 mRNA and protein expression in rat large follicles were stimulated by gonadotropin and IGF-I treatment [42]. Increase of monounsaturated acids synthesize by SCD2 activation during DF growth may be required to maintain membrane fluidity [43] and a major lipid reserve of oocytes [44].

Members of the TRB family including TRB2 interact and modulate the activity of mitogen-activated protein kinase (MAPK) which regulates cell proliferation, differentiation, apoptosis and survival [45]. These MAPK cascade protein levels were greater in DF than in SF [46]. In addition, it has been reported that TRB2 mRNA was constantly expressed between bovine small follicles and DF [11]. These studies and our present result suggest the potential role of TRB2 in the regulation of MAPK cascades in the growing DF.

The F2 are characterized by high expression of the genes involved in immune reaction (CCL2, SELP and SPP1). In bovine follicles, expression of CCL2 and SPP1 mRNAs and SELP protein was up-regulated in association with follicular development and ovulation [16, 18, 47]. Our results raise the possibility that these immune-related genes may be involved in bovine follicular atresia as well as follicular development and ovulation. Both CCL2 and SELP mediate induction of leukocyte emigration into extravascular inflammatory sites [48]. Although SPP1, also known as osteopontin, has diverse physiological functions, one of its potent actions is recruitment and retention of macrophages and T cells to inflamed sites [49]. Since number of leukocytes, lymphocytes and activated macrophages are increased in atretic follicles [50], CCL2, SELP and SPP1 participate in the regulation of inflammatory processes during follicular atresia to attract white blood cells.

The F2 are also characterized by high expression of genes regulating tissue remodeling (TIMP1 and PLAUR). Both plasminogen activator (PA)-plasmin and matrix metalloproteinase (MMP) systems play a crucial role in the degradation and remodeling of extracellular matrix associated with follicular development, ovulation and atresia [51]. Urokinase PA (uPA) receptor is a specific cell surface receptor for uPA and its principal role is to localize pericellular plasmin activity to induce extracellular matrix degradation [52]. A previous study showed that there was no difference in mRNA expression levels of uPA between non-atretic and atretic bovine follicles, whereas atretic follicles had lower FF protein level and mRNA expression of a PA inhibitor and higher FF plasmin activity than non-atretic follicles [53]. Therefore, the follicular PA-plasmin system may be primarily regulated by changes in their receptors and inhibitors' expressions. TIMP-1 is an intrinsic inhibitor of MMPs and preferentially binds to MMP-9 [54]. A previous study demonstrated that MMP-9 proenzyme (proMMP-9) protein in FF was detected only in atretic follicles but not in healthy follicles in cattle [55]. Atretic follicles may balance MMPs and TIMP-1 in response to an increase in proMMP-9 to control extracellular matrix degradation by MMP-9.

In the present study, we identified differential expression of two anti-apoptosis factors (GADD45A and GPX3) between the groups. Expression of GADD45A mRNA was greater in the F2 than in the F1. GADD45A controls cell cycle arrest, apoptosis induction and DNA damage repair in response to DNA damaging agents and growth arrest signals of genotoxic stress [56]. Our result implies that the atretic follicles suffered more severe DNA damage than healthy follicles. Indeed, we found in experiment 2 that the atretic follicles expressed GADD45A mRNA in both GC and TL whereas the healthy follicles expressed it only in GC. This result supports our microarray result and suggests an increase in the requirement of GADD45A activity for progression of apoptotic cell death in GC and TC during follicular atresia. On the other hand, GPX3 mRNA was found to be more greatly expressed in the F1 than in the F2. Glutathione peroxidase protects cells against oxidative damage to catalyze the reduction of free hydrogen peroxide and other hydroperoxides [57]. High oxidative stress can trigger apoptosis of follicular cells and induce atresia [58]. In cultured swine GC, GPX3 mRNA expression was upregulated by FSH treatment [59]. Thus GPX3 could prevent cell apoptosis from oxidative stress during growth of the healthy follicles. It is likely that follicular oxidative stress-response enzymes are expressed in a stage-dependent manner since mRNA expression of other anti-oxidative stress enzymes in bovine GC was increased in atretic DF than in healthy DF [60].

Providing a sufficient blood supply is essential for follicular growth [61, 62]. A morphological study has demonstrated that bovine healthy DF has a high density and well developed capillaries in TL whereas atretic follicles has sparse and poorly developed capillaries [63]. In the present study, two genes regulating angiogenesis, PlGF and TSP2, were differentially expressed between the groups. PlGF was expressed most in F1 than in F2 and localized in both GC and TL of healthy follicles but not detected in atretic follicles. PlGF is a member of the vascular endothelial growth factor family and stimulates the proliferation of endothelial cells and supports angiogenesis [64, 65]. Therefore, PlGF may contribute to follicular thecal angiogenesis via paracrine/autocrine action in healthy follicles as well as other angiogenic factors. In contrast to PlGF, TSP-2, a member of the TSP family, acts as a potent inhibitor of angiogenesis and induces endothelial cell apoptosis [66]. In experiment 1, TSP2 mRNA expression was greater in the F2 than in the F1. TSP2 mRNA level in the bovine follicles decreased in accordance with an increase in follicular diameter [67]. The same authors also showed that TSP protein was localized in both GC and TC of small follicles but in only in the GC of large follicles [67]. We demonstrated in experiment 2 that TSP2 mRNA was localized in both GC and TL of atretic follicles while it was expressed in only GC of healthy follicles. Recent studies have demonstrated that mRNA and protein expression of TSP-1, another antiangiogenic TSP, is upregulated in primate GC during progression of follicular atresia [68] and TSP1 mRNA abundance is decreased by IGF-I treatment in cultured porcine GC [69]. Thus, we speculate that TSP2 mRNA expression is maintained at high levels in follicular cells of atretic follicles whereas it decreases in healthy follicles. Highly expressed TSP2 mRNA in the follicles could negatively influence their angiogenesis. It may cause an insufficient supply of substrates essential for follicular growth, thereby affecting follicular hormone production and cell proliferation, and, as a result, inducing atresia.

Microarray and QPCR analysis enabled us to classify uncharacterized bovine follicles and to evaluate their representative follicular status according to differences in global gene expression profiles. Our present study demonstrates that the expression of stage-specific genes in F1 and F2 may be closely associated with follicular growth and atresia. Several genes identified in this study will provide information on the genomic actions of intriguing candidates for the determinant of bovine follicular development.