Towards a 3D culture of mouse ovarian follicles MARTINA BELLI 1,# , GIULIA VIGONE 1,# , VALERIA MERICO 1 , CARLO ALBERTO REDI 1 , MAURIZIO ZUCCOTTI 2* and SILVIA GARAGNA 1,3,4,* 1 Laboratorio di Biologia dello Sviluppo, Dipartimento di Biologia e Biotecnologie 'Lazzaro Spallanzani', Università degli Studi di Pavia, Pavia, 2 Dipartimento di Scienze Biomediche, Biotecnologiche e Traslazionali, Università degli Studi di Parma, 3 Centro di Ingegneria Tissutale, Universita’ degli Studi di Pavia, Pavia and 4 Centro di Eccellenza in Biologia Applicata, Università degli Studi di Pavia, Pavia, Italy. ABSTRACT The ovarian follicle has a three-dimensional (3D) structure in which the oocyte is sur- rounded by tightly connected follicle cells that mediate the action of external signals and nourish the gamete during its maturation. Thus, the maintenance of follicle organization during the whole growth process is crucial for the correct acquisition of developmental competence. In recent years, much attention has been given to in vitro culture systems capable of maintaining follicle architecture. With the aim of providing a quick reference guide, in this review we will summarize the techniques developed for the 3D culture of mouse follicles. KEY WORDS: 3D culture, ovarian follicle, follicle organization, follicle architecture, matrix Introduction In the past few years important improvements have been achieved in the in vitro culture of mammalian ovarian follicles. Some laboratories have even been able to culture follicles from the primordial/primary stage up to their complete maturation, ac- quisition of fertilizability and developmental competence (Eppig and O’Brien 1996). As thoroughly described earlier (Desai et al., 2010), despite the many upgrades suggested throughout the years, a single standard protocol has not yet been agreed, not even with a model species like the mouse. The dificulties and challenges lay in the peculiar features of the follicle: a small “organ” within the ovary that possesses a unique vascular system built up around a 3D structure in which the oocyte is surrounded by companion, tightly connected, follicle cells. The oocyte growth is strictly dependent on autocrine and paracrine bidirectional signaling, the latter through gap junctions and transzonal projections, between the germinal and the somatic components of the follicle (Matzuk et al., 2002; Luciano et al., 2011). Speciically, follicle cells mediate the action of external signals and nourish the oocyte during its maturation. Due to the key role of this cross talk, the maintenance of the follicle three-dimensionality during the whole growth process is crucial for the correct acquisition of the developmental competence. Follicles have been cultured under 2D or 3D systems, both trying to reproduce the complex bidirectional stimuli (e.g., exchange of Int. J. Dev. Biol. 56: 931-937 (2012) doi: 10.1387/ijdb.120175mz www.intjdevbiol.com *Address correspondence to: Maurizio Zuccotti. Dipartimento di Scienze Biomediche, Biotecnologiche eTraslazionali, Università degli Studi di Parma, Via Volturno 39, I-43125, Parma, Italy. e-mail: maurizio.zuccotti@unipr.it or Silvia Garagna. Laboratorio di Biologia dello Sviluppo, Dipartimento di Biologia e Biotecnologie ‘Lazzaro Spallanzani’, Università degli Studi di Pavia, Via Ferrata 9, I-27100, Pavia, Italy. e-mail: silvia.garagna@unipv.it Final, author-corrected PDF published online: 5 February 2013. ISSN: Online 1696-3547, Print 0214-6282 © 2013 UBC Press Printed in Spain Abbreviations used in this paper: 3D, three dimensional; ECM, extracellular matrix; HA, hyaluronic acid; PEG, polyethylene glycol; PVA, polyvinyl alcohol. nutrients, soluble and insoluble signals and hormones) and both with advantages and drawbacks. Historically, 2D systems were the irst to be developed with remarkable results, including the growth of primary follicles to com- plete maturation, fertilizability and full developmental competence (Eppig and Schroeder 1989; Cortvrindt et al., 1996; Eppig and O’Brien 1996). These methods comprise the culture in multi-well plates, microdrops, gel-coated dishes or membranes coated with extracellular matrix (ECM) proteins. Follicles are cultured on a 2D surface that only partly maintains the spatial coniguration of the follicle, letting the follicle cells expand at the bottom of the dish, with the consequent partial loss of the oocyte-follicle cells interactions. For this reason, in recent years, much attention has moved to the use of matrices and culture systems capable of maintaining the follicle 3D architecture. Although this approach is still in its infancy and requires much improvement, some important goals have been achieved. The purpose of this review is to give state-of-the-art of 3D methods that have been developed for the culture of mouse follicles, with the aim of providing an essential reference guide to those who are approaching these techniques with this species.