Do the Theca Layer and Fibroblast Growth Factors Have a Role in Follicular Atresia?1

Ovarian follicular development in the bovine estrous cycle is characterized by a wave-like pattern of successive periods of follicle growth and regression, with the majority of estrous cycles in cows comprising either two or three waves per cycle. Each wave involves recruitment of a cohort of antral follicles, followed by selection of a single dominant follicle that progresses to the pre-ovulatory stage, while the remaining follicles of the wave, the subordinate follicles, undergo atresia. The pituitary gonadotropic hormones, follicle-stimulating hormone (FSH) and luteinizing hormone, are the primary drivers of follicle recruitment and growth, with molecules produced locally within the follicle having secondary but essential roles in dominant follicle selection, while promoting atresia of subordinate follicles [1]. Bidirectional communication between the oocyte and adjacent cumulus granulosa cells results in expansion and mucification of the cumulus-oocyte complex necessary for ovum release at ovulation, mediated by oocyte-secreted peptides (growth differentiation factor 9, bone morphogenic protein [BMP] 15, fibroblast growth factor [FGF] 8) [2, 3]. In turn, molecules from cumulus cells (epidermal growth factor–like peptides, cAMP, and pyruvate) trigger meiotic and cytoplasmic maturation of the oocyte essential for fertilization and embryo development [4]. Similarly, paracrine signals and substrates passing between mural granulosa cells and theca cells of the follicle wall contribute to both granulosa and theca cell proliferation and differentiation essential for continued follicle growth and maturation leading to ovulation. Theca cells supply androgens as precursors for conversion to estrogens by aromatase located exclusively in granulosa cells, and growth factors (insulin-like growth factors [IGFs] and FGFs) that act synergistically with the estrogens to further enhance follicle growth. In turn, estrogens and other growth factors produced by granulosa cells exert regulatory actions back on the theca cells. These closely coordinated activities of the three follicle cell types—oocyte, granulosa cells, theca cells—comprise a regulatory network that results in an intrafollicular milieu necessary for follicle growth and ovulation of a developmentally competent ovum as culmination of the final follicular wave of the cycle [5, 6]. The importance of steroids as mediators of paracrine communication within the follicle has been well recognized since the 1970s and 1980s. Androgens secreted by theca cells not only serve as precursors for conversion to estrogen by aromatase located in granulosa cells [7], but also enhance the stimulatory action of FSH in inducing granulosa cell aromatase [8, 9]. Estrogen also acts in autocrine fashion on granulosa cells, in concert with IGF1, also of granulosa cell origin, in stimulating granulosa cell proliferation. In fact, estradiol concentration in follicular fluid is the most reliable marker of follicle dominance available. Steroids are well suited to function as paracrine regulatory molecules, their small size and lipophilic nature being conducive to their free diffusion between cells and through cell membranes, where binding with intracellular receptors enables direct interaction with DNA to promote transcription of specific genes involved in their characteristic biological responses. FGFs are a large family containing 18 molecules and, additionally, FGF11 to FGF14 are FGF-homologous FGF factors [10]. FGF18 belongs to a subfamily (FGF8) based upon homology and phylogeny and containing FGF8, 17, and 18. There are four receptor genes (single-pass transmembrane domain with intracellular tyrosine kinase activity) and, with alternative splicing, there are seven main isoforms [11]. FGFs can interact with heparan sulfate (HS) glycosaminoglycans, some less well than others, giving these FGFs more of an endocrine function [10]. HS-containing proteoglycans, such as perlecan, collagen XVIII, or agrin, can be found in extracellular matrix. The interaction with matrix reduces the diffusion of FGFs in tissue or acts as a reservoir of FGFs. Thus, even if one cell releases FGF and another nearby cell expresses the receptor, FGF may not reach its target cell if HS matrix proteins are located between the cells. For receptor activation, HS-containing syndecans, of which there are four, are important for coupling FGF to its receptor and, in their dimerization, form an activated receptor complex. The activated receptor complex contains FGF, FGF receptor, and HS glycosaminoglycan in a 2:2:2 ratio [12], and this complex must form first to allow phosphorylation and intracellular signal transmission [13]. The article by Portela et al. in this issue of Biology of Reproduction [14] focuses on expression of FGF18 in bovine follicles, particularly the theca and its role in follicular atresia. They show that FGF18 is expressed by the theca but also localizes to the granulosa cells and in follicular fluid. It is elevated in subordinate relative to dominant follicles, and elevated in atretic follicles as defined by the ratio of estradiol to progesterone, but when granulosa cells were treated with FGF18, steroidogenesis and expression of steroidogenic enzymes declined, cell cycle progression and expression of cell cycle checkpoint regulator declined, and DNA was more fragmented. Based on these findings, the authors suggest that FGF18 can signal from the theca to the granulosa cells and influence the process of atresia. In the bovine follicle, the theca interna cells express at least six FGF family members: FGF1, FGF2, FGF7, FGF8, FGF17, and FGF18, with levels of expression varying with different stages of follicle development and regression. Since first shown as potent granulosa cell mitogens [15], a variety of biological effects within the follicle have been attributed to FGFs, including stimulation of granulosa cell proliferation and extracellular matrix production, angiogenesis, oocyte growth, and follicular atresia [16–24]. Granulosa cells express FGF receptors (FGFr), where their level of expression is regulated by FSH, usually in the presence of IGF1, thus providing compelling evidence for roles of FGFs in paracrine communication from theca to granulosa cells. Several FGFs are also expressed in granulosa cells, making them well placed to serve as autocrine mediators of granulosa cell regulation. In addition to their expression in somatic cells, FGF2, FGF8, and FGF17 also are expressed in bovine oocytes [20, 22, 25]. In rodent follicles, where its expression is restricted to the oocyte, FGF8 has been reported to act synergistically with another oocyte-specific peptide, BMP15, in stimulating glycolysis in cumulus cells, and thus may contribute to the complex changes in the cumulus cell mass that occur in the preovulatory follicle and are essential for ovulation [26]. Follicular atresia in antral follicles largely involves death of granulosa cells. Many recent studies have examined the process of apoptosis, but granulosa cells also die by necrosis, autophagy, and a process similar to cornification (reviewed in [27]). Since death of the granulosa cells in atresia is a coordinated event, as nearly all of the cells die within a short period of time, it probably involves cell-to-cell signaling. This could be due to loss of growth factor support such as transforming growth factor alpha [28] or expression of fas or fas ligand [29, 30] or, as article by Portela et al. [14] considers, FGF18 derived from the theca layer. The concept that the theca layer is involved in atresia has rarely been considered. There have been reports of death of steroidogenic and endothelial cells in the theca interna early in some forms of atresia of bovine follicles as well as loss of expression of insulin-like growth factor 3 in the theca interna (reviewed in [27]), but the suggestion of FGF signaling from the theca to initiate cell death in the granulosa is novel. However, since most of the study by Portela et al. [14] is in vitro, complete proof is still warranted and some conceptual issues need to be addressed to fully confirm their novel hypotheses. For instance, the follicular basal lamina and specialized aggregates of basal lamina matrix called focimatrix [31] contain perlecan and collagen XVIII at different stages of folliculogenesis [31, 32]. Since these molecules contain HS and are located in the membrana granulosa, they potentially pose as barriers for the translocation and action of FGF18 from the theca cells to the granulosa cells. Additionally, syndecans are important for FGF signaling and there have been few studies of these in ovaries in general [33, 34]. Testing a role that FGF18 may play in bovines might pose some logistical issues, but with Merck Serono targeting FGF18 for treatment of osteoarthritis, there may be an opportunity to directly test the role of FGF18 in vivo.

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