# Technologies for the Production of Fertilizable Mammalian Oocytes
## 2 The Production of Fertilizable Oocytes  
### Folliculogenesis  
Folliculogenesis is defined as the sequence of processes that allow the formation of a mature ovarian follicle, containing a fertilizable oocyte (Figure 1). It is noteworthy that folliculogenesis fails to produce a large number of mature follicles due to the occurrence of an apoptotic related process (atresia) that naturally reduces the original number of follicles of more than 99%. It has been calculated that in a woman half of the about 7 million of oocytes that are likely produced during embryo and fetal development are eliminated before birth. At the onset of puberty, the about 2 million healthy follicles present in the human ovary at birth are reduced to about 300,000. As this loss continues up to menopause, women throughout their fertile life can produce about 400 healthy mature oocytes. Conversely, sperm production usually keeps up throughout the entire life, and sperm treatments, such as those aimed to enhance motility, do not affect embryo quality [33; 34]. Furthermore, the presence of spermatogonal stem cells allows recovery of fertility also after chemotherapy [35]. The precursors of oocytes are Primordial Germ cells (PGC). PGC actively proliferate during their migration towards the genital ridges under the control of several transcription factors [36]. In the gonads, proliferating PGC have incomplete cytokinesis, evidenced by intracellular bridges [37]. After leaving mitosis and entering meiosis, PGC remain arrested at diplotene of the first meiotic division, commonly known as GV stage. This transition occurs during the first days after birth in mice and during fetal life in domestic animals and primates. Now, a single layer of flattened pre-granulosa cells (pre-GC)  
Figure 1: A mammalian ovary. Panoramic view of a mouse ovary by light microscopy. Follicles at various stages of development are shown. Follicles become responsive to FSH from the secondary stage onward. In these follicles, estrogen production increases. Mid-cycle Luteinizing Hormone (LH) surge induces oocyte meiotic resumption and the ovulated oocyte is arrested at MII. The CL originates from cells of the follicle wall during ovulation, although its precise origin remains controversial. Abbreviations: PF1 (primordial follicle); PF2 (primary follicle); AF (antral follicle); O (oocyte); G (granulosa cells); T (theca cells); OSE (ovarian surface epithelium); CL (corpus luteum). Bar = 1 mm. surrounds the oocyte to form the primordial follicle. In rodents, follicle formation occurs during the first few days after birth, while in primates during fetal life. The primary oocytes are arrested at diplotene of prophase I for a long period of time (resting stage), which lasts months in rodents and years in humans [38]. Despite the presence of mitotically active germ cells inside the mammalian ovary [39; 40], the number of oocytes is fixed early in life, and neo-oogenesis is excluded [41]. Periodically, a cohort of primordial follicles is recruited into growth phase (activation), evidenced by the transition from flattened to proliferating cuboidal GC (Figure 1) [42; 43]. Little is still known about the molecules driving the exit from resting to growth phase. Follicular growth is controlled by local factors and not by gonadotropins [42; 44]. The accurate balancing of activatory (e.g., Anti-Mullerian hormone, AMH [45]) and inhibitory (e.g., Spermat- and oogenesis-specific basic helix-loop-helix-containing protein 2, Sohlh2; and Phosphatase and tensin homolog, PTEN [46]) factors avoids premature exhaustion of primordial follicle pool, with consequent reproductive senescence in animals or menopause in humans. As soon as follicles enter the growth phase and become bilaminar (secondary), GC undergo a differentiation process by which they acquire responsiveness to FSH [43; 47; 48]. Although FSH receptors are present on the GC, FSH is not required up to preentral-to-antral transition [49; 50].