1. REVIEW OF LITERATURE
1.4. Folliculogenesis
Folliculogenesis is a process of maturation of the ovarian follicle. Folliculo-genesis involves the recruitment of the follicle into the growing phase, which is controlled by paracrine and autocrine signals produced in the ovary itself, followed by the proliferation and differentiation of the surrounding granulosa and theca cells, which are regulated in addition to the internal signalling by endocrine signals from outside the ovary. This process is under the primary control of two pituitary hormones, gonadotrophins FSH and luteinizing hormone (LH). During the maturation, the follicle grows and goes through the primordial, primary, secondary and preantral stages before it reaches the antral stage (Figure 2).
Figure 2. Light micrographs of the different follicular stages. A – primordial follicles (magnification 400X), B – a transitional follicle from the primordial to the primary stage (magnification 400X), C – primary follicle (upper) (magnification 400X), D – secondary follicle (magnification 400X), E – antral follicle (magnification 100X), F – atretic follicle (magnification 400X). Photos with permission, by Inger Britt Carlsson.
A resting primordial follicle is surrounded by a single layer of flattened granulosa cells (GCs) (Gosden et al. 2002). During the initial stages of folliculogenesis certain resting primordial follicles start to grow due to the expression of kit-ligand (Packer et al. 1994) and retinoblastoma protein (Bukovsky et al. 1995). This step of the folliculogenesis is considered to be gonadotrophin independent, as primordial follicles do not possess FSH receptors (Speroff and Fritz 2005). The development of primary follicle is the first sign of activation and initial follicular recruitment. At this stage the expression of FSH receptors begins. In human ovaries, it has been demonstrated that FSH receptors are expressed in 1/3 of the primary and two-layer secondary follicles and in all multi-layer follicles (Oktay et al. 1997). During the early growth phase the proliferating GCs grow larger and become cuboidal, providing nutrients and different molecular signals to the oocyte (Wandji et al. 1997). The GCs communicate with each other and with the oocyte via gap junctions, composed mainly by connexins (Eppig 1991). Primary GCs start to secrete mucopolysaccharides forming the zona pellucida, a thick layer of glycoproteins and acid proteoglycans around the oocyte. Further proliferation of GCs and follicular enlargement result in formation of secondary follicle.
Two or more layers of GCs surround secondary follicle. GCs associated with secondary follicle possess FSH, estrogen, and androgen receptors (Speroff and Fritz 2005). FSH binds to FSH receptors on the GC surface, stimulating the proliferation of the GCs, while increasing the number of FSH receptors
expressed on their surface (thereby magnifying its own effects), and stimulating the aromatase enzyme. According to the generally accepted “two cell, two gonadotrophin theory” (Hillier et al. 1994), FSH binding to FSH receptor in the GCs activates cyclic adenosine monophosphate (cAMP) to induce p450 aromatase that converts androgens to estrogens. At the same time as GCs proliferate, theca cells respond to LH. The interaction of LH with its receptor on the surface of the theca cells also activates cAMP that in turn initiates the production of androgens from cholesterol. Androgens are subsequently aromatised in the GCs to estrogens, mainly estradiol-17β (E2) (Erickson and Shimasaki 2001). Estrogens are then released into the follicular fluid and circulation to participate in the further regulation of follicular maturation. In the ovary, estrogens influence the GC proliferation, increase the number of FSH receptors and their sensitivity, and stimulate aromatase activity and thus further estrogen biosynthesis. Androstenedione, at low concentrations, promotes aromatase action and estrogen biosynthesis, but high levels of androgens cause follicular atresia (Speroff and Fritz 2005).
Under the influence of gonadotrophins and growth factors the follicle grows and the surrounding stroma stratifies and differentiates, forming theca interna and theca externa with vessels between the two layers. This enables the follicle to gain a blood supply, resulting a direct exposure to factors circulating in the blood (Reynolds et al. 1992). In the preantral stage, a fluid filled antrum starts to develop. From this point, the GCs proliferate and differentiate to mural GCs (MGCs) in the periphery of the follicle and cumulus GCs (CGCs) closest to the oocyte. The follicular fluid accumulation is limited by the level of FSH present (Eppig 1991), reflecting the steroidogenesis of surrounding GCs and theca cells, containing plasma proteins, proteoglycans, prolactin, inhibin and etc (Speroff and Fritz 2005).
A large fluid-filled antrum is a characteristic feature of the mature follicle, named also Graafian follicle or antral follicle (Figure 3). At the antral stage, under the competition of available FSH, most of the follicles go through atresia, and only a few of them reach the pre-ovulatory phase. The dominant follicle has the advantage of higher rate of GC proliferation, therefore for increased number of FSH receptors, higher aromatisation ability, and increase in estrogen production.
The preovulatory gonadotrophin surge is resulted from the positive feedback action of elevated estrogen on the pituitary gland. It is suggested that also progesterone is involved in the stimulation of ovulation (Zalanyi 2001).
Progesterone production starts in the GCs prior to gonadotrophin surge and increases in the corpus luteum after the ovulation. Further, the terminal maturation of the follicle is believed to be stimulated mainly by LH (Sullivan et al. 1999). In the immature follicles LH receptors are expressed in the theca cells, but in antral follicles they are also expressed in GCs (Camp et al. 1991). It has been hypothesised that the maturing follicle continues to develop in the presence of increasing FSH levels because of the capacity to respond to LH
(Sullivan et al. 1999). Following the pre-ovulatory gonadotrophin surge, the dominant follicle releases a mature oocyte that is ready for fertilization (Gougeon 1996).
Figure 3. Mature Graafian follicle in the ovary. Different granulosa cells surrounding the maturing oocyte together with antrum and thecal layers are indicated.
Besides gonadotrophins and estrogens, androgens, other hormones including progesterone and different growth factors are involved in the intraovarian regulation of folliculogenesis. Many proteins belonging to the transforming growth factor β (TGFβ) family are important as local regulators of follicular development and oocyte maturation, such as bone morphogenetic factor 15 (BMP15), growth differentiation factor-9 (GDF-9), anti-Mullerian hormone (AMH), activins and inhibins (Knight and Glister 2006). Other factors include growth hormone (GH) (Sharara and Nieman 1994), insulin and insulin-like growth factors (Erickson and Shimasaki 2001) and members of the epidermal growth factor (EGF)-family (Shimada et al. 2006). Many factors, however, are still unknown.