A. Parque nacional, monumento nacional y reserva nacional

Choosing a specific medium for IVM of immature oocytes was derived by adapting methods developed from culturing other cell types and mimicking the composition of ovarian microenvironment of the enclosed oocyte in the follicle. Complex culture media such as tissue culture medium 199 (TCM-199), Ham’s-F10 and Whitten’s medium buffered with bicarbonate or 4-(2-Hydroxyethyl)-1-piperazine-ethanesulphonic acid (HEPES) among others has been used in rodent IVM culture. Numerous supplements can be added (Bevers et al., 1997; Trounson et al., 1998) to provide the necessary nutrients for the process of oocytes maturation.

The biophysical parameters must be controlled in the culture media as pH (potential hydrogen), carbon dioxide (CO2) and osmolarity. Most in vitro culture media develops a neutral or slightly alkaline pH, being the best results between 7.2 and 7.6. For the CO2, the gas phase used consists of 5% CO2 in air to mimick the follicle and oviduct of mammalian females or 20% of CO2 as in air. Osmolarity ranges between 275 and 290 mOsm.

Despite many supplements can add to the maturation media, normally energy substrates and protein source, as well as different hormones and growth factors are added. Different energy substrates are provided due to the influence on oocyte meiotic and cytoplasmic maturation (Rose-Hellekant et al., 1998; Chung et al., 2007; Garner and Lane, 2013).

This energy source used in most culture media is generally lactate, pyruvate and glucose because they are the main substrates for energy metabolism of oocytes (Gardner and Lane, 2013). Oocyte utilization of pyruvate is dependent upon CCs that can convert glucose or lactate into pyruvate to be used by oocytes (Pool, 2004). Furthermore, glutamine can serve as an energy substrate to improve in vitro nuclear maturation of oocytes. On the other hand, protein source are essential and non-essential amino acids that would be used by protein synthesis, but also can be utilized as an energy source and intracellular buffer. Essential and non-essential amino acids are added to culture media serum-supplemented or serum-free culture media (Gardner and Lane, 1998;

2014; Rezaei et al., 2003). Also Fetal calf serum (FCS) and bovine serum albumin (BSA) are the most used protein sources for in vitro culture (Carolan et al., 1995; Lazzari et al., 2000). FCS contains a complex array of protein components that are required by many cells to grow; it provides a wide variety of macromolecular proteins, low molecular weight nutrients, carrier proteins for water-insoluble components and other compounds necessary for in vitro growth of cells, such as hormones, growth factors, vitamins and attachment factors (Chung et al., 2007; Chian et al., 2004). However, large offspring

Introduction

39 syndrome (LOS) has been described mainly in ruminants with the adition of serum (Farin et al., 2001). Efforts to avoid FCS and BSA as indeterminant compounds in the medium are done in the last decade. Synthetic compounds as polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) can be substitute as well as BSA.

As gonadotrophins (FSH and LH) play an important role in the physiological development and function of follicles in vivo and oocyte maturation as we explained before, some medium includes these supplements. LH probably acts to induce GVBD by an indirect action mediated by CCs because it is believed that there are no LH receptors on oocytes (Yadav et al., 2010; Sha et al., 2010). Besides, E2 may be important in regulating oocyte maturation (Lonergan, 2011) and also is used in some laboratories.

Finally, growth factors are usually present in the culture media. The most used are EGF, IGF-1, TGFα and TGF-β.

EGF has been shown to stimulate in vitro oocyte maturation thus resulting in an increased developmental competence of oocytes in different species (mouse: Das et al., 1991; Smitz et al., 1998; Merriman et al., 1998; De La Fuente et al., 1999; sheep: Guler et al., 2000; cattle: Lonergan et al., 1996; Sakaguchi et al., 2002; pig: Reed et al., 1993;

Coskun and Lin, 1995; Singh et al., 1997; Prochazka et al., 2000, 2003; rat: Dekel and Sherizly, 1985; rabbit: Lorenzo et al., 1996; buffalo: Kumar and Purohit, 2004 and human: Gómez et al., 1993; Goud et al., 1998). It was reported that EGF stimulates granulosa cells proliferation in vitro (pig: May et al., 1992) and estrogen production through aromatase activation (human: Misajon et al., 1999; goat: Behl and Pandey, 2001); modulates steroidogenesis and estrogen production (Hsueh et al.,1981; human:

Misajon et al., 1999). Also EGF is particularly important in controlling oestrogen receptor function (Ignar-Trowbridge et al., 1996) and alone or associated with gonadotropins induces cumulus expansion and promotes nuclear and cytoplasmic maturation of immature oocytes during culture in vitro (Lorenzo et al., 1994; Wang and Niwa, 1995; De La Fuente et al., 1999).

In vitro studies have also shown that EGF maintains follicular survival (Wandji et al., 1996), promotes follicular growth (bovine: Wandji et al., 1996; caprine: Kajarajan et al., 2006; Silva et al., 2013; hamster: Roy, 1993; mouse: Boland and Gosden, 1994 and human: Roy and Kole, 1998), proliferation of granulosa cells obtained from preantral follicles Morbeck et al., 1993) and stimulates antrum formation (Silva et al., 2013).

Introduction

40

IGF-1 promoted nuclear maturation of oocytes, stimulated proliferation and inhibited apoptosis in the surrounding CCs, increased developmental competence of oocytes (Lorenzo et al., 1996; Patiño et al.,2001; Sirotkin et al., 2003; Hunter et al., 2004;

Velazquez et al., 2009). Likewise, IGF-1 is known to stimulate protein synthesis when added to medium for mouse embryos in vitro (Simmen et al., 1993) and known to increase E² production by the theca granulosa cells in serum free culture (Shores et al., 2000).

In some studies, it have been shown that EGF and IGF-1 in combination act synergistically and accelerate the cumulus expansion and the progression of meiosis (Lorenzo et al., 1996; Sakaguchi et al., 2000; Purohit, 2001; Sakaguchi et al., 2002; Isobe and Terada, 2001). In bovine oocyte activation is known to be associated with Ca²⁺

dependent electrical events (Tosti et al., 2002). A combination of EGF and IGF-1 under conditions in vitro stimulates a cascade of events including protein synthesis which generate positive signals for mitotic resumption (Spicer and Chamberlain, 2000). On the other hand, the addiction the protein sources how FCS or BSA could neutralize the accelerating effect of EGF and IGF-1 of the meiosis of in vitro (Sakaguchi et al., 2000) stimulating cytoplasmic oocyte maturation.