# Technologies for the Production of Fertilizable Mammalian Oocytes
## 3 Methods for Obtaining Fertilizable Oocytes from In Vitro Grown (IVG) Mammalian Follicles
### 2D and 3D Culture Systems  
2D (non-spherical) and 3D (spherical) culture systems have been established to culture secondary and primary/primordial follicles. The latter are usually preferred because able to better survive to chemotherapy and cryopreservation, although they must be cultured inside ovarian cortical strips to avoid massive apoptosis. In the 2D approach, the surface of a dish or membrane is coated with one or more components of extracellular matrix (ECM), which provide structural support for the maintenance of efficient intercellular communications, proper cell differentiation and survival. The mouse was the first selected  
Figure 2: Fully grown mouse oocytes. (**a**) GV stage. To show the GV, surrounding cumulus cells have been mechanically removed. (**b**) MII stage. Expanded cumulus cells have been removed to evidence PB. Abbreviations: GV (germinal vesicle); ZIP (zona pellucida); PB (polar body). Bar = 20 \(\upmu\)m.  
model, because of shorter folliculogenesis and more knowledge about the molecular mechanisms controlling the production of developmentally competent oocytes. The first IVG oocytes able to produce some blastocysts were obtained by [PERSON] and [PERSON], in 1989 [PERSON] and [PERSON] (1989). A few years later, a two-step method based on the combination of ovarian tissue and 2D culture onto collagen gels allowed obtaining the first viable offspring from IVG activated primordial/primary mouse follicles [PERSON] and [PERSON] (1989). Since the development of multistep culture systems better supports each specific developmental stage [PERSON] and [PERSON] (1989), after several technical improvements, mouse primordial follicles were completely differentiated in vitro. The derived embryos were able to produce a healthy colony formed by 35 females and 37 males that survived to adulthood [PERSON] and [PERSON] (1989). Similar results were obtained by others, either by culturing cryopreserved follicles [PERSON] and [PERSON] (1989) or by simply modifying the culture protocols [PERSON] and [PERSON] (1989). However, gene expression, reactive oxygen species (ROS) production, steroid secretion and mitochondria activity of IVG follicles appear altered in comparison with in vivo follicles [PERSON] and [PERSON] (1989). Despite these limits, mouse systems provided useful information for the culture of preantral follicles from large mammals and humans [PERSON] and [PERSON] (1989). The first IVG ovine follicles were obtained by a 2D system, although the enclosed oocytes were far from full developmental competence [PERSON] and [PERSON] (1989); [PERSON] (1989). Beside the disruption of intercellular communications, the altered expression of several key genes, such as those regulating steroid synthesis and apoptosis, can account for reduced in vitro development [PERSON] and [PERSON] (1989). Surprisingly, the methylation profile of three imprinted genes and developmental competence were comparable to that of oocytes obtained in vivo from the early antral follicles [PERSON] and [PERSON] (1989). The 3D culture systems more closely mimic the physiological environment of the ovary. The encapsulation of follicles in various substrates as alginate gels, collagen or Matrigel(r) determines a better preservation of follicle 3D architecture and of functional communications between somatic and germ cells. Matrigel(r) is a basement membrane rich in ECM components and growth factors; alginate is a natural biopolymer obtained from brown algae and characterized by high biocompatibility, structural similarity to ECM and capable to form a gel with well-defined features [PERSON] and [PERSON] (1990). At present, many researchers consider alginate as the best support capable of assuring the production of live offspring [PERSON] and [PERSON] (1989). Despite the technical difficulties experienced in isolation and encapsulation of follicles, alginate system has allowed the successful culture of follicles from dog, goat, baboon and macaque [PERSON] and [PERSON] (1989) (Figure 3).