Mouse Oocytes Within Germ Cell Cysts and Primordial Follicles Contain a Balbiani Body
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Mouse oocytes within germ cell cysts and primordial follicles contain a Balbiani body Melissa E. Pepling*†, James E. Wilhelm‡, Ashley L. O’Hara*, Grant W. Gephardt*, and Allan C. Spradling†‡ *Department of Biology, Syracuse University, Syracuse, NY 13244; and ‡Department of Embryology/Howard Hughes Medical Institute, Carnegie Institution of Washington, Baltimore, MD 21210 Contributed by Allan C. Spradling, November 8, 2006 (sent for review October 7, 2006) The Balbiani body or mitochondrial cloud is a large distinctive arises in spermatocytes also in conjunction with mitochondria organelle aggregate found in developing oocytes of many species, (4) and Golgi (17). The mouse chromatoid body contains the but its presence in the mouse has been controversial. Using me31B homolog, Ddx25, which is essential for male fertility and confocal and electron microscopy, we report that a Balbiani body has been implicated in translational regulation (18). Other does arise in mouse neonatal germline cysts and oocytes of components of the chromatoid body include Vasa and Tudor primordial follicles but disperses as follicles begin to grow. The proteins (19, 20) mouse Balbiani body contains a core of Golgi elements surrounded The Balbiani body or mitochondrial cloud is a collection of by mitochondria and associated endoplasmic reticulum. Because of organelles asymmetrically located adjacent to the nucleus in very their stage specificity and perinuclear rather than spherical distri- young oocytes of diverse species (21–23). Balbiani bodies con- bution, these clustered Balbiani body mitochondria may have been tain mitochondria, ER, and granulofibrillar material (GFM) missed previously. The Balbiani body also contains Trailer hitch, a organized in a characteristic manner. Although Balbiani body widely conserved member of a protein complex that associates mitochondria often aggregate in a cloud around the Golgi, in with endoplasmic reticulum/Golgi-like vesicles and transports spe- some species, they extend throughout the perinuclear cytoplasm. cific RNAs during Drosophila oogenesis. Our results provide evi- The Balbiani body GFM morphologically resembles germinal dence that mouse oocytes develop using molecular and develop- granule precursors. A connection to germinal granules has been mental mechanisms widely conserved throughout the animal proven in Xenopus, where GFM has been shown to contain kingdom. germinal granule proteins and RNAs that are incorporated into Drosophila germ plasm ͉ gamete biology ͉ oocyte development ͉ ovary ͉ oogenesis germ cells (15, 16, 24, 25). Likewise in , a key component of the germinal granules, oskar RNA, associates with the newly formed Balbiani body (26). hether eggs and embryos of diverse species initiate de- Mouse oocytes have been thought to be exceptional in lacking Wvelopment by highly divergent or basically similar path- a Balbiani body (1, 2). Early researchers reported that fetal and ways remains a key unresolved issue. One of the strongest BIOLOGY neonatal mouse oocytes contain a perinuclear Golgi body con- arguments for divergent pathways has been the differing origins sisting of small vesicles similar to a Balbiani body (27, 28). DEVELOPMENTAL of germ cells in organisms such as flies, nematodes, and frogs However, subsequent studies failed to confirm these observa- that contain a definitive germ plasm and organisms such as mice and newts that do not (1). In Xenopus and Drosophila, germinal tions, and it is now widely accepted that mouse oocytes lack such granule components are produced early in oogenesis, associate mitochondrial aggregates (1, 2). This situation is surprising, with a distinctive structure known as the Balbiani body, and are because Balbiani bodies have been found in marsupials (29) and subsequently transported and localized within the egg (2). many mammalian species including goat (21), rat (30, 31), Shortly after fertilization, germinal granules are taken up by hamster (32), and humans (33–35), although the function of primordial germ cells where they persist (at least in part) as mammalian Balbiani bodies is unknown. electron-dense particles called nuage. In contrast, none of these The lack of a Balbiani body in mouse oocytes is also hard to processes are thought to take place in mouse oocytes or germ understand in light of similarities in the cellular events that cells. However, several nuage-containing structures have been precede Balbiani body formation in Drosophila, Xenopus, and described in male mouse germ cells, including the chromatoid mouse oogenesis. In both Xenopus and Drosophila, the Balbiani body, chromatoid satellites, RNF17 granules, sponge bodies, and body arises within interconnected germline cysts by the con- spherical particles (3–7). The function of these nuage-containing trolled assembly of organelles and germinal granule components structures in male germ cells is unclear, in part because of the at the time of follicle formation (26, 36). Female mouse germ lack of cytological markers to distinguish one nuage body from cells also generate cysts after reaching the genital ridge (37). As another. the cysts break down around the time of birth, approximately Molecular studies of germinal granules strongly implicate one-third become surrounded by somatic pregranulosa cells to them in the transport, storage, localization, stability, and regu- form primordial follicles (38). At this time, mitochondria orga- lated utilization of mRNA (8). The Vasa DEAD box RNA helicase is found in germ cells and nuage of diverse organisms (9). Drosophila nuage contains Vasa, as well as Maelstrom, Author contributions: J.E.W. and A.C.S. designed research; M.E.P., J.E.W., A.L.O., and Aubergine, and SpindleE, proteins implicated in RNAi (10). G.W.G. performed research; J.E.W. contributed new reagents/analytic tools; M.E.P., A.L.O., G.W.G., and A.C.S. analyzed data; and M.E.P. and A.C.S. wrote the paper. Recently, transport particles used to assemble germinal granule The authors declare no conflict of interest. components have been characterized in Drosophila and shown to Abbreviations: ER, endoplasmic reticulum; GFM, granulofibrillar material; PNDn, postnatal contain conserved protein components, including me31B/ day n; RNP, ribonucleoprotein; P body, processing body; 4E-T, 4E-Transporter; dpc, days Deadsouth (a DEAD box RNA helicase and translational re- postcoitum. pressor), Cup/4E-Transporter (4E-T, an eIF4E-binding pro- †To whom correspondence may be addressed. E-mail: [email protected] or spradling@ tein), and Trailer hitch (11–13). These components also ciw.edu. associate with the endoplasmic reticulum (ER) (14). Before This article contains supporting information online at www.pnas.org/cgi/content/full/ localization within the egg, germinal granules associate with 0609923104/DC1. mitochondria in the Balbiani body (15, 16). The chromatoid body © 2006 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0609923104 PNAS ͉ January 2, 2007 ͉ vol. 104 ͉ no. 1 ͉ 187–192 Downloaded by guest on September 27, 2021 forming primordial follicles, the stages where Balbiani bodies can first be observed in Drosophila and Xenopus ovaries (23). Using electron microscopy, we observed that Golgi stacks are arranged in a distinctive circular manner in young mouse oocytes (Fig. 1 A–D). Masses of mitochondria and closely associated ER either surround these Golgi stacks (Fig. 1A) or extend from it around the perinuclear region (Fig. 1B). Despite the limited amount of cytoplasm present in these oocytes, mitochondrial organization was nonrandom, with a strong preference for a perinuclear vs. a periplasma membrane position. Moreover, these mitochondria showed additional evidence of organization as many associate distinctively with thin sheets of ER during this time (38). Thus, a Balbiani body similar in general structure to those described in other species is present in young mouse oocytes. Oocyte organelles first become organized into a discrete Balbi- ani body on about postnatal day (PND)1, both in oocytes that still remain in cysts and in oocytes that have already assembled into primordial follicles (Fig. 1 B and C). Darkly staining material is sometimes observed associated with mitochondria and resembles descriptions of nuage (Fig. 1E, arrow). This classic arrangement does not persist indefinitely, however. As follicles begin to grow, the mitochondria and ER disperse and become evenly distributed throughout the cytoplasm (Fig. 1F). Golgi and Mitochondria Associate with the Balbiani Body. We used antibodies specific for its component organelles to further characterize the mouse Balbiani body. Because Golgi cisternae lie near its center, we used an antibody against the Golgi localized protein, GM130, to analyze the distribution of Golgi both within the Balbiani body and throughout the oocyte cytoplasm at the level of confocal microscopy. These experi- Fig. 1. Electron micrographs of oocytes in neonatal ovaries. (A) Micrograph ments strongly supported our conclusions based on electron of an oocyte within a germline cyst from PND1 showing a well defined Balbiani microscopy (Fig. 2). GM130 staining was largely confined to a body (arrow) with Golgi surrounded by mitochondria. (B) Micrograph of two semicircular perinuclear structure in the oocytes of newly oocytes from PND3 (top oocyte in a newly formed primordial follicle and bottom oocyte still within a germline cyst) with Balbiani bodies (arrows). (C) formed follicles (Fig. 2A) whose size, distinctive shape, and Micrograph of an oocyte