DOI: 10.21451/1984-3143-AR2019-0051 Proceedings of the 35th Annual Meeting of the European Embryo Transfer Association (AETE); Murcia, Spain, September 12th and 14th, 2019. Intrafollicular barriers and cellular interactions during ovarian follicle development Gabriella Mamede Andrade, Maite del Collado, Flávio Vieira Meirelles, Juliano Coelho da Silveira, Felipe Perecin* 1Faculty of Animal Sciences and Food Engineering, Department of Veterinary Medicine, University of São Paulo, Pirassununga, São Paulo, Brazil. Abstract ridge, colonize, and proliferate. After this highly proliferative period, a human female fetus has th Follicles are composed of different approximately 6-7 million germ cells around the 20 interdependent cell types including oocytes, cumulus, week of gestation, however a vast majority of these granulosa, and theca cells. Follicular cells and oocytes germ cells are lost and approximately 1 to 2 million exchange signaling molecules from the beginning of the oocytes remain viable at birth (Motta et al., 1997; Sun development of the primordial follicles until the et al., 2017). In bovines, the maximum number of germ th moment of ovulation. The follicular structure transforms cells is around 2.5 million at about the 15 week of during folliculogenesis; barriers form between the germ gestation (Erickson, 1966) and thirteen days after birth and the somatic follicular cells, and between the somatic bovine germ cells number decrease approximately to 68 follicular cells. As such, communication systems need thousand. This dramatic loss of germ cells close after to adapt to maintain the exchange of signaling birth occurs in most female mammals (Paulini et al., molecules. Two critical barriers are established at 2014). different stages of development: the zona pellucida, Once mitotic proliferation stops, these germ separating the oocyte and the cumulus cells limiting the cells arrest at meiotic prophase I to form the germ cell communication through specific connections, and the nests (Buehr, 1997; Tilly, 2001; Sun et al., 2017). Close antrum, separating subpopulations of follicular cells. In to birth, breakdown of the germ cell nests occurs with both situations, communication is maintained either by the formation of the primordial follicle. Two cell types the development of specialized connections as characterize this primordial follicle: a primary oocyte transzonal projections or by paracrine signaling and surrounded by a single layer of pre-granulosa cells trafficking of extracellular vesicles through the (Fortune, 1994; BrawTal and Yossefi, 1997; Eppig, follicular fluid. The bidirectional communication 2001). The primordial follicle population in the ovary between the oocytes and the follicle cells is vital for serves as a reservoir for developing follicles and oocytes driving folliculogenesis and oogenesis. These throughout the female reproductive life (Zuckerman, communication systems are associated with essential 1951; Kerr et al., 2013). After puberty, groups of functions related to follicular development, oocyte primordial follicles are periodically recruited to initiate competence, and embryonic quality. Here, we discuss folliculogenesis. the formation of the zona pellucida and antrum during Although the precise mechanisms that regulate folliculogenesis, and their importance in follicle and germline nest breakdown and primordial follicle oocyte development. Moreover, this review discusses formation are mostly unknown (Wang et al., 2017), the current knowledge on the cellular mechanisms such several growth factors and hormones play essential roles as the movement of molecules via transzonal projections, in primordial follicle formation (Pepling, 2012), for and the exchange of extracellular vesicles by follicular example estradiol-17β (E2) and members of the β cells to overcome these barriers to support female gamete transforming growth factor beta (TGF- ) superfamily development. Finally, we highlight the undiscovered (Knight and Glister, 2006; Wang and Roy, 2007; β aspects related to intrafollicular communication among Chakraborty and Roy, 2017). The TGF- family the germ and somatic cells, and between the somatic members are secreted by the oocyte and include bone follicular cells and give our perspective on manipulating morphogenetic protein 15 (BMP15) and growth the above-mentioned cellular communication to improve differentiation factor 9 (GDF9), which act via autocrine reproductive technologies. and paracrine mechanisms, regulating follicle growth and differentiation, as well as granulosa and thecal cell function during follicular development (Dong et al., : cellular communication, extracellular Keywords 1996; Eppig et al., 1997; Gilchrist et al., 2004; Sanfins vesicles, granulosa cells, oocyte, ovarian follicle, et al., 2018). By secreting these members of TGF-β transzonal projections family the oocyte is the main responsible for activating primordial follicles (Eppig, 2001). Introduction: Follicle development Ovarian follicle development is a continuous process that has two different phases: the preantral and The ovarian follicle development starts long antral. The first phase, preantral, is gonadotropin- before birth during the intra-uterine period (Russe, independent and relies on local growth factors. As 1983). The primordial germ cells migrate to the genital folliculogenesis progresses, the follicle becomes _________________________________________ *Corresponding author: [email protected] orcid.org/0000-0003-2009-5863 Received: July 30, 2019 Copyright © The Author(s). Published by CBRA. Accepted: May3, 2019 This is an Open Access article under the Creative Commons Attribution License (CC BY 4.0 license) Andrade et al. Communication through intrafollicular barriers. gonadotropin-responsive and develops until secondary layers (Fair et al., 1997; Hyttel et al., 1997; Guo et al., follicles. The second phase, antral, is gonadotropin- 2016). dependent and is characterized by the presence of the In bovine, from the beginning of antral phase tertiary follicles, which has the presence the antrum, a until a diameter of approximately 8 mm, follicle growth cavity filled with follicular fluid (Dvořák and Tesařík, is stimulated by FSH secreted by the pituitary gland. 1980; Erickson and Shumichi, 2001). This entire Follicles develops by the rapid proliferation of process of growth and differentiation of the follicle is granulosa and theca cells that contribute to the further accompanied by the oocyte growth and acquisition of enlargement of the antrum and the follicle itself. From a competence (El-Hayek and Clarke, 2015; Monniaux, diameter of 8 mm onwards, the follicle develops mainly 2016). by the trophic stimulation of LH, and eventually, after Stimulation by the locally secreted factors the LH surge, will be termed preovulatory follicles activates the primordial follicles initiating the preantral (Eppig et al., 1997). growth phase for development into a primary follicle. At the end of its growth, the dominant follicle Factors responsible for primary follicle development are reaches a plateau phase of non-exponential growth with not fully known; however, it is known that granulosa fewer cell divisions and slower diameter increase cell-derived anti-Mullerian hormone and activins (Girard et al., 2015). Following the preovulatory participate in the regulation of this process (reviewed by gonadotropin surge, follicular cells initiate Matzuk et al., 2002). The primary follicles are morphological, endocrine, and biochemical changes characterized by the presence of an oocyte covered with associated with luteinization process (Smith et al., 1994; a single layer of cuboidal granulosa cells. As the oocyte Revelli et al., 2009). In monovulatory species, only one grows, the granulosa cells proliferate to envelop the follicle continues its growth to become an ovulatory surface of the expanding oocyte (vandenHurk et al., follicle, while the remaining antral follicles regress and 1997). undergo atresia (Hennet and Combelles, 2012). Continuous granulosa cell proliferation results For the follicle formation and its steady growth in multiple layers of cells surrounding the oocyte and during the whole folliculogenesis process, the the follicles are referred to as secondary follicles. At this bidirectional communication within the follicle stage, the formation of the theca cell layer starts, environment is essential for the complete development separated from the granulosa by a basement membrane of the follicle as well as the oocyte. The crosstalk (BrawTal and Yossefi, 1997). At the same time, oocytes between the oocyte and somatic follicular cells and undergo alterations as the formation of cortical granules between the somatic follicular cells occurs through the in the cytoplasm (Fair et al., 1997) and the beginning of interactions mediated by paracrine signaling factors, by mRNA synthesis (McLaughlin et al., 2010). At this gap junctions and, as recently described, by stage, the formation of the zona pellucida (ZP) around extracellular vesicles. The paracrine signaling occurs the oocyte starts, to form the first significant barrier through the secretion of factors from the oocyte or from between the oocyte and the somatic granulosa cells the somatic cells. The gap junctions are structures (BrawTal and Yossefi, 1997; Clarke, 2018) (Fig. 1A). formed by connexins that allow the transport of As the secondary follicle develops, more layers molecules of low molecular weight (<1 kDa) as ions, of granulosa cells form, and an antral cavity