Article Cell polarity during folliculogenesis and oogenesis

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1 RBMOnline - Vol 10. No Reproductive BioMedicine Online; on web 21 February 2005 Article Cell polarity during folliculogenesis and oogenesis Dr Carlos E Plancha Carlos E Plancha received his Medical Degree in 1985 at the Lisbon Medical School, Portugal, and completed his internship in 1987 at the Hospital de Santa Maria in Lisbon. He submitted his PhD thesis on female gamete biology at the Lisbon Medical School in 1996, where he is now Assistant Professor. In 2001 he began collaborating with a Reproductive Medicine Clinic in Lisbon CEMEARE where he now works as an embryologist coordinating the IVF laboratory. Since 2002 he is also the Director of the Biology of Reproduction Unit in the Institute of Molecular Medicine, Lisbon. His main scientific areas of research are oogenesis and folliculogenesis. He teaches and supervises numerous pre- and post-graduate activities and has coordinated nationally and internationally funded research projects in his areas of expertise. Dr Plancha has also been involved in the organization of national and international meetings. He has published several articles in peer-reviewed scientific journals and book chapters. He maintains long-term collaborations with David F Albertini, with whom he is graduate supervisor of Alexandra Sanfins and Patrícia Rodrigues. Carlos E Plancha 1,3, Alexandra Sanfins 1, Patrícia Rodrigues 1,2, David Albertini 2 1 Unidade de Biologia da Reprodução, Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Av. Prof. Egas Moniz, Lisboa, Portugal; 2 Department of Molecular and Integrative Physiology, University of Kansas Medical Centre, 3901 Rainbow Boulevard, Kansas City, KS , USA 3 Correspondence: carlos.plancha@mail.telepac.pt Abstract Polarity is an important aspect of oogenesis and early development for many animal groups, but only recently it has become relevant to the study of mammals. Mammalian oocyte development occurs through tight coordination and interaction between all ovarian structures. In fact, bi-directional communication between the oocyte and its companion granulosa cells (GC) in the ovarian follicle seems essential for GC proliferation, differentiation, and production of a functional female gamete. The transzonal projections (TZP), which are specialized extensions from granulosa cells that terminate on the oolema after crossing the zona pellucida, are major structural components necessary for oocyte GC interaction. Granulosa cell polarity seems to be a necessary requisite for appropriate function of TZP, and the role of FSH as modulator of a polarized phenotype on GC is discussed. This article also discusses oocyte polarity with special reference to the partial loss of polarity that occurs during invitro oocyte maturation and possible implications in the modulation of oocyte competencies. Cytoskeletal markers that may account for oocyte quality were defined and found to be distinct in in-vivo and in-vitro matured oocytes. Implications of partial loss of oocyte polarity during in-vitro maturation, reflected by distinct distribution of these markers, are further discussed. It is also proposed that expression of both somatic and germ cell polarity in the ovarian follicle will ultimately determine acquisition of meiotic, fertilization and developmental competences by the oocyte. Keywords: in-vitro maturation, in-vivo maturation, oocyte, polarity 478 Cell polarity Polarity is an essential feature of development in all organisms, from the 1-cell embryo to the establishment of tissue polarity through the whole organism. It is a characteristic of most cells in metazoan organisms and is associated with asymmetries in cell shape, protein content, organelle distribution and, ultimately, cell function (for review, see Nelson, 2003). Epithelial cells from simple transporting epithelia, as well as neurons, have been frequently used as models for somatic cell polarity (Le Gall et al., 1995; Nelson, 2003). Epithelial cells show polarized organization of their actin and microtubule cytoskeleton to mediate vectorial membrane trafficking and direct specific proteins to their basolateral or apical surfaces (Figure 1). Another example is the polarized way in which neurons are organized to communicate and accomplish vectorial movement of specific neurotransmitters across long distances in the body, or conduct nerve impulses through reversible signals of membrane polarity (Jockusch et al., 2004). Overall, establishment and maintenance of cellular polarity is a conserved feature of vertebrate and invertebrate organisms (Wodarz, 2002; for review see Muller and Bossinger, 2003; Nelson, 2003). Evolutionarily conserved mechanisms were adopted by different cell types to facilitate specific cytoskeleton reorganization and oriented localization of cortical proteins responsible for spatial allocation of resources. Identification of the mammalian homologues of the invertebrate polarity genes

2 Figure 1. Schematic representation of epithelial cells showing polarized cytoskeleton organization and bi-directional membrane trafficking. On the left, organization of the actin and microtubule cytoskeleton with the centrosome as the main microtubule organizing centre; on the right, membrane trafficking to/from the apical and basal membranes, allowing transport of molecules to/from the basal or apical surfaces. PAR = protease-activated receptor. has indeed reinforced the concept that important protein domains involved in maintenance of cellular polarity are evolutionarily conserved (Muller and Bossinger, 2003). Recently, it was shown that a set of genes responsible for establishment of spatial cues is conserved throughout the animal kingdom: the par genes are responsible for polarized organization in the Caenorhabditis elegans zygote, in the female germline cyst of Drosophila, and in mammalian epithelial cells (Wodarz, 2002). Although the determinants of egg polarity have been extensively discussed in Drosophila, C. elegans, and Xenopus (Edwards, 2001; Pellettieri and Seydoux, 2002; Wodarz, 2002), no attempt has been made to search systematically for mammalian homologues in female germ cells in order to draw comparison with mammals regarding germ cells (Edwards, 2001). Thus, the subject of egg polarity in mammals remains as a theme of interesting discussion (Gardner, 1999; Plusa et al., 2002). The model system that has interested the authors for several years is the developing mammalian ovarian follicle. This review will specifically focus on the somatic granulosa cell and the germ cell compartments of the follicle, as further examples of how cell polarity determines appropriate cell function. Granulosa cell polarity This section will discuss the polarity of the main somatic cell constituents of the ovarian follicle, the granulosa cells, as opposed to discussion on the more controversial issue of ovarian follicle polarity. The ovarian follicle consists of an association between a germ cell, the oocyte, and at least two somatic cell types, granulosa and theca cells. Four distinct extracellular matrix (ECM) territories, zona pellucida, antrum, basal lamina and thecal matrix, are also present at specific spatial and time points, modulating follicle cellular interactions and establishing territorial frontiers. These provide the coordinates for the different polarized phenotypes found in ovarian follicles. However, the role of such components in the establishment, maintenance and modulation of granulosa cell (GC) polarity is still poorly understood. Recently, it has been suggested by Irving-Rodgers and coworkers (2004) that GC polarity is induced by contact with the follicular basal lamina, enabling directional secretion and uptake of molecules, and other polarized functions. Previously, it was proposed that the follicular epithelium is similar to other known epithelia (for review, see Rodgers et al., 1999), although being particularly complex, as it expands from a single to a multi-layered epithelium as the follicle grows. In addition, there is the transition from pre- to antral follicle, and the increase in the epithelium compartment through fluid filling. Irving-Rodgers and co-workers (2004) proposed the existence of a novel basal lamina matrix, the focal intraepithelial matrix, which is developmentally regulated and may be involved in the modulation of GC polarity. During follicular development, two sets of specialized GC within the ovarian follicle become apparent: the mural granulosa cells, which overlay the basal lamina; and the cumulus oophorus cells, which locate around the zona pellucida with some cells 479

3 480 making contact with the oolema, the corona radiata cells. The mural cells exhibit a morpho-functional organization with cell cell and cell basal lamina junctions, thus closer to the classical examples of epithelial cells with a clear apical basal axis differentiation (Figure 1). In contrast, besides homologous gap junctions and other cell interactions between themselves, the cumulus cells that constitute the corona radiata also display heterologous gap junctions and other bi-directional interactions with the germ cell (Albertini and Anderson, 1974), through a complex morphological and functional circuitry at the zona pellucida level (Motta et al., 1994; Albertini et al., 2001). Cytoplasmic projections from cumulus cells that transverse the zona pellucida matrix and terminate at the oocyte surface are responsible for the complex bi-directional signalling at the oocyte cumulus cell interface (Albertini et al., 2001). These specialized structures, referred as trans-zonal projections (TZP) (Motta et al., 1994; Albertini et al., 2001), exist in at least two forms: microtubule-containing TZP (MT-TZP), which appear to be involved in paracrine communication, and actin-containing TZP (ACT-TZP), which appear to mediate gap junctional communication and cumulus cell oocyte adhesion (Albertini et al., 2001; Navarro-Costa et al., 2005). The cellular and molecular characterization of these processes has uncovered their dynamic nature throughout folliculogenesis (Albertini et al., 2001; Albertini and Barrett, 2003; Combelles et al., 2004). These are present and very numerous in pre-antral follicles, and there seems to be some degree of retraction after antrum formation in human and mouse oocytes (for review, see Motta et al., 1994; Albertini et al., 2001). At peri-ovulatory stages in the hamster, most ACT-TZP seem to retract (Plancha and Albertini, 1994). Recently, it has been demonstrated that FSH is an important modulator of MT- TZP organization. FSH-like priming reduces cumulus MT- TZP density in FSH-β / mice, which otherwise exhibit enhanced density of TZP with associated decreases in oocyte development (Combelles et al., 2004). Important features of oocyte development that include chromatin remodelling and meiotic competence acquisition have been shown to be induced under conditions where FSH directly modifies oocyte cumulus interaction via TZP (Plancha and Albertini, 1994; Combelles et al., 2004). In short, evidence that FSH modulates the polarized phenotype of GCs, particularly through mediation of TZP dynamics, is gaining further importance. Cellular interactions structurally dependent on TZP may be a specialized mean by which paracrine factors are exchanged between the germ line and the soma during follicular development. It remains to be demonstrated if gonadotrophins are the main modulators for this signalling circuit, and how local regulators come into place. A better understanding of the complex hormonal and paracrine environment that directs the interplay between GC and the oocyte would surely be crucial for answering this question in the near future. Oocyte polarity Establishment and maintenance of polarity is of central importance in the process of oogenesis, particularly during oocyte maturation. Mitochondria distribution in the oocyte is frequently taken as evidence for the existence of polarity in this cell, or at least of an asymmetric localization that could have functional significance (Calarco et al., 1972; Van Blerkom and Runner, 1984; Calarco, 1995). In fact, the localization of the Balbiani body, a concentration of material that contains RNA and basic proteins as well as numerous mitochondria, cisternae of endoplasmic reticulum and Golgi, is first localized at one side of the germinal vesicle (GV) during oocyte growth, and although its role in the mammalian oocyte is not yet clear, it raises questions about the putative functional role of organelle polarization (Zamboni, 1970; Smedt et al., 2000). It has been shown that there is a polarization of mitochondria during oocyte maturation. Van Blerkom and Runner (1984) have shown that mitochondria translocate to the perinuclear region during first metaphase spindle formation and disperse during first polar body extrusion. This translocation is mediated by microtubules in pig oocytes (Sun et al., 2001). Interestingly, in-vitro matured (IVM) pig oocytes exhibit incomplete movement of mitochondria to the inner cytoplasm, hence affecting cytoplasmic maturation (Sun et al., 2001). During meiosis, the oocyte undergoes asymmetric cytokinesis necessary for proper accumulation of maternal factors in the ooplasm and restriction of the material lost during polar body extrusion. The cortical localization of the meiotic spindle with formation of microvillus-free areas and thickening of the actin layer associated with the oocyte cortex supports the existence of a polarized phenotype (Longo and Chen, 1985). Therefore, during oocyte maturation, some link between the spatial regulation of cell cycle cues and the first asymmetric cytokinesis, may serve to preserve a polarized distribution of factors within the oocyte. This polarization may extend after fertilization to ensure proper second asymmetric polar body extrusion. Considering that nuclear positioning may define asymmetrically localized functions within a given cell, it is interesting to note the polarized position of the GV in mammalian oocytes (Raven, 1961). In most mammalian species, the GV is positioned eccentrically (for review, see Lefèvre, 2003); specifically, this has been characterized in the human (Combelles et al., 2002), mouse (Mattson and Albertini, 1990), and hamster (Plancha and Albertini, 1992). However, exceptions to this tendency have been reported in the mouse, where the GV is apparently located centrally and the meiotic spindle, according to some, seems to assemble from a central position and only later migrates to the oocyte cortex (Maro and Verlhac, 2002). Although the mechanisms established to maintain the anchoring of the GV to the oocyte cortex remain unknown, it was recently postulated that central GV positioning reported in mouse or rat oocytes may be due to an artefact of culture (Albertini and Barrett, 2004). Overall, proper coordination between karyokinesis and cytokinesis is essential for fertilization and early embryogenesis. Coordination between the cortical actin cytoskeleton and the meiotic spindle is essential in maintaining proper asymmetric divisions during the final stages of oogenesis. For example, formin-2 is a maternaleffect gene, expressed in oocytes, necessary for the maintenance of the correct positioning of the meiotic spindle to the oocyte cortex (Leader et al., 2002). Fmn-2 / females

4 are sub-fertile and fertilization of Fmn-2 / oocytes results in failure of polar body extrusion, polyploid embryo formation and recurrent pregnancy loss. It is tempting to speculate that proteins that interact with the cortical cytoskeleton may be additionally involved in maintenance of proper spindle position necessary for correct asymmetric divisions. On the other hand, single chromosomes were also found to be able to induce cortical and plasma membrane modifications, normally seen in proximity of intact MI or MII spindle (Van Blerkom and Bell, 1986). Therefore, polarity of cortical cytoplasm could be determined by the presence of a tight set of chromosomes beneath the oolema (Van Blerkom and Bell, 1986). Whether GV and spindle position are due to asymmetric surface signalling coming from the granulosa cells through TZP is still argued (Albertini and Barrett, 2004) and should be considered with caution, since no experimental evidence has been found to suggest that disruption or maintenance of TZP relates to GV and spindle positioning. Partial loss of polarity may account for competence decrease in IVM oocytes Since the seminal experiments of Pincus and Enzmann (1935) and Edwards (1965), it has been known that oocytes released from their follicular environment spontaneously resume meiosis in vitro. However, in-vitro maturation still represents a challenge in assisted reproductive technologies (Hardy et al., 2000), as IVM oocytes exhibit decreased developmental competence relative to their in-vivo matured (IVO) counterparts (Eppig and O Brien, 1998; Schroeder et al., 1988; Mermillod et al., 1999; Moor and Dai, 2001; Trounson et al., 2001). Besides a variety of arguments that might explain the low quality of IVM oocytes, and since cytoskeletal orientation is a key factor for polarity determination, new cellular markers of oocyte quality have recently been proposed, after careful comparative analyses of cytoskeleton organization between IVM and IVO mouse oocytes (Sanfins et al., 2003, 2004). These experiments have allowed the uncovering of previously undetected morphological differences between IVM and IVO mouse oocytes that seem to be of functional significance. However, great care in extrapolating this interpretation to other species, namely to the human, must be exercised, particularly taking into account the known differences in terms of centrosome dynamics and inheritance (Schatten et al., 1986). Centrosome positioning has been considered a critical factor in the establishment of cell polarity, since it directs microtubule organization (for review, see Bornens, 2002). Even though the relevance of distribution and quantity of microtubule organizing centres (MTOC) in mammalian oocytes is still unclear, it has been reported that in Drosophila, maternally derived MTOC are involved in reorganizing oocyte microtubules used to reorient molecular gradients for both anterior/posterior and dorsal/ventral axes (Pellettieri and Seydoux, 2002; Wodarz, 2002). Furthermore, centrosome migration to the mid-body vicinity is required for proper cytokinesis in human fibroblasts (Piel et al., 2001). Regarding the distribution of microtubules and centrosomes, IVM mouse oocytes exhibit barrel shape and larger meiotic spindles with scattered γ-tubulin foci distributed throughout the spindle proper. These oocytes also exhibit a lower number of cytoplasmic MTOC and present increased polar body size. In contrast, IVO mouse oocytes display pointed shape and more compact spindles; with γ-tubulin distribution restricted to the spindle poles. These oocytes show increased cytoplasmic MTOC and reduced polar body size (Figure 2). Furthermore, the distinct organization of these cytoskeleton markers has been shown to be related to distinct cell cycle progression during spindle morphogenesis. While IVM mouse oocytes exhibit protracted and asynchronous meiotic progression, IVO mouse oocytes display synchronous and efficient meiotic progression relying on proper location of cell cycle factors during maturation (Sanfins et al., 2003). Interestingly, in contrast to IVO oocytes, in IVM oocytes the spindle anchoring seems to be partially lost. Considering that the spindles produced in IVM do not underlay the oolema as do IVO spindles, this could reflect a partial loss of polarity of IVM oocytes as described above, namely by perturbing spindle positioning and orientation at the oocyte cortex. Consequently, this abnormal positioning of the spindle compromises successful cytokinesis, resulting in mouse oocytes with increased size of polar bodies. Therefore, oocyte volume, and more importantly MTOC distribution, is partially lost for the first polar body during IVM. This partial loss of polarity of IVM oocytes may be aggravated by an additional observation: IVM oocytes recruit massive amounts of centrosomal material (either γ-tubulin or pericentrin) for M-I and M-II spindle morphogenesis, originating larger spindles and depleting this material from the oocyte cytoplasm (Sanfins et al., 2003, 2004). Consequently, not only is there an initial loss of MTOC during first polar body formation, but due to the increment in the amount of MTOC material that forms the M-II spindle, it is reasonable to suspect that even more will be lost to the second polar body after fertilization. Since cytoplasmic MTOC are believed to support early cleavages (Maro et al., 1985), the loss of MTOC material from IVM mouse oocytes may indicate a restriction in the amount of maternal factors necessary to support early embryogenesis. In clear contrast, retention of cortical MTOC in IVO mouse oocytes allows formation of compact spindles, small polar bodies and proper preservation of accumulated maternal factors in the cytoplasm that will be used during embryogenesis. A working model that illustrates all these features is shown in Figure 3. In conclusion, the defective alignment of the microtubule centrosome complex in murine IVM oocytes results in partial loss of spatial asymmetries, which ultimately may contribute to lower oocyte quality. The implications of these findings in terms of fertilization and developmental competence are currently being tested in mouse oocytes. Extension of these studies to oocytes from other mammalian species such as humans will allow a better understanding of the competencies in both nuclear and cytoplasmic compartments during the process of oocyte maturation. This may ultimately lead to improved quality in assisted reproductive technologies. 481

5 a b Figure 2. In-vivo matured (IVO) (A) and in-vitro matured (IVM) (B) mouse oocytes denoting microtubules (tubulin, green), centrosomes (pericentrin, red) and DNA (Hoechst, blue). Note the pointed-shape spindle of IVO oocytes, contrasting with the barrel-shape spindle of IVM oocytes. In addition, IVO oocytes display a small polar body that does not contain microtubule organizing centres (MTOC); in contrast, IVM oocytes exhibit a large polar body containing MTOC. Figure 3. Working model showing the fundamental differences between in-vivo matured (IVO) and in-vitro matured (IVM) oocytes. Note that IVO oocytes present a small polar body and retain increased amounts of cortical microtubule organizing centres (MTOC) that keep them spatially segregated from the cortically positioned compact spindle. 482

6 Acknowledgements This work was supported by Fundação para a Ciência e a Tecnologia POCTI/ESP/43628/2000 (CEP), #SFRH/BD/2757/2000 (AS), #SFRH/BD/6439/2001 (PR), March of Dimes Birth Defects Foundation (DA) References Albertini DF, Anderson E 1974 The appearance and structure of intercellular connections during the ontogeny of the rabbit ovarian follicle with particular reference to gap junctions. Journal of Cell Science 63, Albertini DF, Barrett SB 2004 The developmental origins of mammalian oocyte polarity. Seminars in Cell and Developmental Biology 15, Albertini DF, Barrett SL 2003 Oocyte somatic cell communication. Reproduction Supplement 61, Albertini DF, Combelles CMH, Benecchi E et al Cellular basis for paracrine regulation of ovarian follicle development. Reproduction 121, Bornens M 2002 Centrosome composition and microtubule anchoring mechanisms. Current Opinion in Cell Biology 14, Calarco PG 1995 Polarization of mitochondria in the unfertilized mouse oocyte. 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