Alongside the development of assisted reproduction with its clinical implications, scientists have been working for more than 40 years on techniques to mature follicles and oocytes in vitro, with the aim to improve and widen the applications of reproductive biology. In 1977, John Eppig showed for the first time, that mouse oocytes were able to grow and mature in vitro, if cultured in the presence of granulosa cells, thereby proving the crucial interactions between both cells (Eppig, 1977). Since then, in vitro culture of ovarian follicles and oocytes has provided novel knowledge on follicle biology and the mechanisms involved in oocyte maturation. An important clinical application of in vitro follicle growth and maturation is under development for women desiring to spare their fertility before undergoing gonadotoxic oncology treatments. Oncofertility has emerged as a new discipline, linking reproductive medicine and oncology, and researches treatment alternative treatment options for ovarian tissue transplantation in female cancer patients of reproductive age (Jeruss and Woodruff, 2009, Woodruff, 2010).
Follicle culture has been achieved in vitro, employing 2-dimensional (2D) or adhesive systems, or more recently, in spherical 3-dimensional (3D) systems.
1.3.4.1 Pioneering methods of follicle culture
In 2D culture systems, different types of substrates are used, such as culture dishes (Eppig, 1977, Cortvrindt et al., 1996) or collagen trans-wells (Eppig and Schroeder, 1989), to which the granulosa cells adhere, allowing the follicle to expand on a flat surface.
The follicle structure is remodeled, and although gap-junctions and basal membrane continuity might be disrupted (Desai et al., 2010), this method have been proven successful in producing live offspring in rodents following in vitro follicle culture, oocyte maturation and IVF (Eppig
and Schroeder, 1989). Its application in larger mammals, including human (Abir et al., 1997), is hampered by the abnormal follicle expansion and aberrant paracrine signaling (Ksiazkiewicz, 2006).
1.3.4.2 Encapsulated in vitro follicle growth
To overcome the challenges faced while culturing follicles in 2D, novel non-adherent techniques, allowing for 3D follicle proliferation, have recently been developed, including ‘floating’ and ‘encapsulated’ models (Brito et al., 2014) (Figure 1-15). For the purpose of this thesis, the focus is on the alginate encapsulated 3D follicle culture technique, developed by Dr Teresa Woodruff, in collaboration with Dr Lonnie Shea at Northwestern University, a decade ago.
Figure 1-15 (a) Secondary mouse follicle encapsulated in an alginate bead (edge of bead indicated by arrows). (b) Follicles stained for viability 1 day after encapsulation are healthy. (c,d) Follicles cultured in alginate beads (c) maintain their morphology at day 4 of culture, while follicles cultured on 2D substrates (d) have a disrupted follicular architecture. Scale bar = 30 µm. Reprinted from Kreeger et al, with kind permission of Pergamon, Copyright 2006.
Alginate is a polysaccharide of repeating b-D-mannuronic and a-L-guluronic units, isolated from cell walls of brown algae. It is liquid in its natural form, but cross-links in the presence of calcium, and forms a solid-like hydrogel used to surround the follicle (Figure 1- 16).
Figure 1-16 Alginate is a polysaccharide, derived from seaweed, that crosslinkes in the presence of calcium to form a solid gel, used to encapsulate ovarian follicles. Courtesy to the Woodruff Lab.
Having achieved success with live birth of healthy offspring in mice (Xu et al., 2006a) (Figure 1-17), these 3D model equally support spherical expansion of follicles isolated from larger mammals, including murine, bovine, goat, canine, non-human primate, and human- derived follicles (Xu et al., 2006a, Araujo et al., 2014, Silva et al., 2015, Songsasen et al., 2011, Min Xu, 2009, Xu et al., 2009). Studies across species have enabled comparative assessments
of biology unique to each species, for example, the physical rigidity of the biomaterial, while providing important new insights into the conserved mechanisms governing the follicle development (Woodruff and Shea, 2011).
Figure 1-17 (A) After 8 days of culture, immature follicle reached the pre-ovulatory stage (B) an outer theca cell layer indicated by 3b-hydroxysteroid dehydrogenase staining. (C) Meiotically arrested cultured oocytes (D) Resumption of meiosis after exogenous HCG stimulation. (E) Metaphase II oocytes fertilized in vitro, and (F) resulting in live offspring after transfer into the oviduct of pseudopregnant mouse. Bar 1⁄4 100 mm (A, B), 50 mm (C–E). Reprinted from Xu et al, with kind permission of Mary Ann Liebert INC Publishers, Copyright 2006.
Alginate is non-degradable and non-adhesive, and adaptable, making it a unique tissue- engineered system to support in vitro follicle growth (Kreeger et al., 2005). Through chemical changes, extra-cellular matrix proteins, such as collagen, fibronectin or laminin, can be incorporated in the alginate scaffold, which has shown to improve the rate of meiotically competent oocytes (Kreeger et al., 2006). Additionally, alginate concentrations can be modified, which leads to changes in the rigidity of the alginate bead and consequently variation in the forces exerted on the enclosed follicle. Decreased alginate concentrations have been found to improve follicle growth and oocyte meiotic competence (Xu et al., 2006b, West et al., 2007).
1.3.4.3 Follicle culture medium and additives
In vitro follicle culture is dependent on various additives within the culture medium, such as nutrients, gonadotrophins, energy substrates, antioxidants and vitamins, involved in in vivo endocrine and paracrine regulation of follicle growth and maturation (Brito et al., 2014). Follicle culture protocols may include serum, containing many different substrates and growth factors, or include purified proteins such as albumin and fetuin (Demeestere et al., 2005). The addition of FSH and insulin, transferrin and selenium in follicle culture medium has shown to improve follicle growth and morphology, oocyte maturation and steroidogenesis (Silva et al., 2004, Demeestere et al., 2005). The optimized culture protocol for murine alginate-based follicle culture consists of a-minimal essential medium (MEM), substituted with insulin- selenium-transferrin, FSH, bovine serum albumin (BSA) and fetuin (Xu et al., 2006a).