Morphology of follicular development

Folliculogenesis begins with the formation of primordial follicles in the fetal ovary. The primary oocytes, which are arrested in the meiotic prophase, become surrounded by a squamous layer of pre-granulosa cells around day 90 of gestation to form the primordial follicles of the ovary (RÜSSE 1983). The factors that regulate primordial follicle formation are poorly understood. It has been demonstrated in mice that the c-kit receptor and its ligand play a central role in directing migration and survival of germ cell during embryonic life (DRIANCOURT et al. 2000). Expression of Figlα mRNA (also known as Figα), a gene that encodes a helix–loop–helix transcription factor which co-ordinates expression of structural genes for components of the zona pellucida, has been identified as a important factor during the formation of the primordial follicles (EPPIG 2001).

Primordial follicles constitute a population of resting, non-growing follicles which are progressively depleted during the reproductive life span. The depletion of the primordial follicle pool occurs as the result of two processes: Atresia mainly through apoptosis or entry into the growth phase (KAIPIA and HSUEH 1997; VAN VOORHIS 1998). Primordial follicles contain 4-8 flattened pre-granulosa cells and have a diameter of ~35 µm with an oocyte of approximately 30 µm in diameter [Fig. 2]

(BRAW-TAL and YOSSEFI 1997; FAIR et al. 1997b). Initiation of follicular growth involves the transition of primordial follicles from the quiescent to the growth phase and is characterized by three main events: change in shape of the granulosa cells from squamous to cuboidal, proliferation of granulosa cells and enlargement of the oocytes (BRAW-TAL and YOSSEFI 1997). The mechanism by which the primordial follicles are activated to start growth is still unclear. However, factors such as bone morphogenetic protein-7 (BMP-7), IGF-I, EGF, anti-mullerian inhibiting hormone (AMH), oocyte specific growth and differentiation factor-9 (GDF-9), BMP-2 receptor, BMP-15 and kit ligand are involved in this process, (for review see FAIR 2003).

Entry of the primordial follicle into the growth phase is characterized by conversion of the flattened of pre-granulosa cells surrounding the oocyte into a single layer of cuboidal granulosa cells (FAIR et al. 1997b). This follicle is now termed a primary

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follicle (VAN VOORHIS 1998). Primary follicles contain 8-20 cuboidal granulosa cells forming a single layer around the oocytes. The diameter is increased to ~55 µm, while size of the oocyte is not changed [Fig. 2] (BRAW-TAL and YOSSEFI 1997). In cattle, the first growing follicles appear in the fetal ovary around day 180 of gestation consisting mainly of primary and secondary follicles, the first antral follicles are observed approximately at day 220 of gestation (WANDJI et al. 1992).

Secondary follicles are characterized by a partial or complete double layer of cuboidal granulosa cells. Formation of the zona pellucida is associated with the formation of granulosa cells processes into the oolemma, forming gap junctions between the granulosa cells and oocyte (FAIR et al. 1997b). Theca interna cells can be recognized occasionally as elongated cells attached to the basement membrane (BRAW-TAL and YOSSEFI 1997). In primordial follicles the communication between the oocyte and the granulosa cells is apparently mediated through an endocytotic pathway as indicated by abundant coated pits and vesicles within the oocyte. In secondary follicles, communication is via gap junctions that are formed between the oocyte and granulosa cells. Tertiary follicles are characterized by antrum formation, a multilayer of granulosa cells surrounding the oocyte, formation of the basal lamina and the cumulus cells (FAIR et al. 1997b). These follicles have a clearly defined theca interna and externa and the oocyte is surrounded by a thick zona pellucida (BRAW-TAL and YOSSEFI 1997; FAIR et al. 1997b). Antrum formation starts in follicles ranging 0.14-0.28 mm. Growth of antral follicles can be divided in two phases. Firstly, early growth of follicles can be attributed to an increase in the number of granulosa cells and therefore an increase in the surface of granulosa layer. Secondly, in follicles larger than 2.5 mm, follicular growth results from antrum development rather than an increase of the number of granulosa cells (LUSSIER et al. 1987). The granulosa cells intimately surrounding the oocyte, constitute the cumulus oophorus, whereas the other granulosa cells are referred to as either antral granulosa cells (cells close to the antral cavity) or mural granulosa cells [cells close to the basement membrane of the follicle] (VAN VOORHIS 1998). The oocyte reaches a diameter of 92 µm in small antral follicles (BRAW-TAL and YOSSEFI 1997). In cattle the preovulatory or Graafian follicle is characterized by a diameter of 15 to 20

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mm, an oocyte with a diameter >120 µm, and an expanded cumulus oophorus due to the secretion of hyaluronic acid by the cumulus cells (EPPIG 2001). The extensions of the corona radiata penetrating the zona and reaching the oocyte are retracted to a more superficial position and are ultimately lost (HYTTEL et al. 1997). In this phase meiosis is resumed and the oocyte progresses through the final stages of meiosis I to be arrested at metaphase of meiosis II [Fig. 2] (HYTTEL et al. 1986b; LUSSIER et al.

1987). During the final stage of follicular development, transcription of specific genes, which are expressed transiently prior to follicle rupture, and after biochemical events cause an inflammatory-like reaction resulting in an increase in vasodilatation and hyperemia. The preovulatory follicle becomes a highly vascularized structure with exudation, edema, collagenolysis, cell proliferation, and tissue remodeling before rupture of the so-called follicular stigma is induced (ESPEY 1994; VAN VOORHIS 1998; RICHARDS et al. 2002). Ovulation occurs 25-30 hr after the LH surge (KAIM et al. 2003).

Fig: 2. Relationship between follicle development and oocyte growth in cattle.

Growth of oocytes (surrounded by granulosa cells) is shown schematically in relation to follicle diameter, oocyte diameter and estimated duration of stage of folliculogenesis from the primordial to preovulatory stage. Reproduced from FAIR 2003. Based on data from LUSSIER

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