Abstract
The presence of oogonia in the ovaries of adult females is typical in species with a broadcast spawning reproductive strategy, including invertebrates and lower vertebrates. In sea urchins, difficulties in the study of oogonia arise from the small number of these cells and the lack of specific markers for their identification. Therefore, more reliable methods are needed for identifying and manipulating oogonial cells in quantities sufficient for experimentation. Homologs of the DEAD-box RNA helicase vasa expressed in germline cells have been proposed for use as markers to detect germline cells in diverse species. We have developed a method for the isolation of sea urchin oogonia by using immunocytochemistry with vasa antibodies, together with reverse transcription and the polymerase chain reaction to detect the expression of Sp-vasa and Sp-nanos2 homologs and a morphological approach to identify germline cells in sea urchin ovaries and cell fractions isolated from the ovarian germinal epithelium. This method has allowed us to obtain 15%-18% of small oogonia with 70%-75% purity from the total amount of isolated germ cells. Our findings represent the first methodological basis for obtaining cell populations containing sea urchin oogonia; this method might be useful as a tool for further investigations of the early stages of sea urchin oogenesis.
Similar content being viewed by others
References
Aoki Y, Nakamura S, Ishikawa Y, Tanaka M (2009) Expression and syntenic analyses of four nanos genes in medaka. Zool Sci 26:112–118
Bachvarova RF, Masi T, Drum M, Parker N, Mason K, Patient R, Johnson AD (2004) Gene expression in the axolotl germ line: Axdazl, Axvh, Axoct-4, and Axkit. Dev Dyn 231:871–880
Bhat KM (1999) The posterior determinant gene nanos is required for the maintenance of the adult germline stem cells during Drosophila oogenesis. Genetics 151:1479–1492
Byrne M, Villinski JT, Cisternas P, Siegel RK, Popodi E, Raff RA (1999) Maternal factors and the evolution of developmental mode: evolution of oogenesis in Heliocidaris erythrogramma. Dev Genes Evol 209:275–283
Chatlynne LG (1969) A histochemical study of oogenesis in the sea urchin, Strongylocentrotus purpuratus. Biol Bull 136:167–184
De Felici M (2000) Regulation of primordial germ cell development in the mouse. Int J Dev Biol 44:575–580
DeFalco T, Capel B (2009) Gonad morphogenesis in vertebrates: divergent means to a convergent end. Annu Rev Cell Dev Biol 25:457–482
Di Carlo M, Romancino DP, Montana G, Ghersi G (1994) Spatial distribution of two maternal messengers in Paracentrotus lividus during oogenesis and embryogenesis. Proc Natl Acad Sci USA 91:5622–5626
Draper BW (2010) Using transgenics to study the mechanism of oocyte production in zebrafish. Transgenic Res 19:138–139
Draper BW, McCallum CM, Moens CB (2007) nanos1 is required to maintain oocyte production in adult zebrafish. Dev Biol 305:589–598
Forbes A, Lehmann R (1998) Nanos and Pumilio have critical roles in the development and function of Drosophila germline stem cells. Development 125:679–690
Fuji A (1960a) Studies on the biology of the sea urchin. I. Superficial and histological gonadal changes of two sea urchins, Strongylocentrotus nudus and S. intermedius. Bull Fac Fish Hokkaido Univ 11:1–14
Fuji A (1960b) Studies on the biology of the sea urchin. III. Reproductive cycles of two sea urchins, Strongylocentrotus nudus and S. intermedius in Southern Hokkaido. Bull Fac Fish Hokkaido Univ 11:49–57
Fujii T, Mitsunaga-Nakatsubo K, Saito I, Iida H, Sakamoto N, Akasaka K, Yamamoto T (2006) Developmental expression of HpNanos, the Hemicentrotus pulcherrimus homologue of nanos. Gene Expr Patterns 6:572–577
Gassei K, Ehmcke J, Schlatt S (2009) Efficient enrichment of undifferentiated GFR alpha 1+ spermatogonia from immature rat testis by magnetic activated cell sorting. Cell Tissue Res 337:177–183
Harrington LH, Walker CW, Lesser MP (2007) Stereological analysis of nutritive phagocytes and gametogenic cells during the annual reproductive cycle of the green sea urchin, Strongylocentrotus droebachiensis. Invertebr Biol 126:202–209
Houston DW, King ML (2000) Germ plasm and molecular determinants of germ cell fate. Curr Top Dev Biol 50:155–181
Juliano CE, Voronina E, Stack C, Aldrich M, Cameron AR, Wessel GM (2006) Germ line determinants are not localized early in sea urchin development, but do accumulate in the small micromere lineage. Dev Biol 300:406–415
Juliano CE, Yajima M, Wessel GM (2010) Nanos functions to maintain the fate of the small micromere lineage in the sea urchin embryo. Dev Biol 337:220–232
Kai T, Williams D, Spradling AC (2005) The expression profile of purified Drosophila germline stem cells. Dev Biol 283:486–502
Kobayashi S, Yamada M, Asaoka M, Kitamura T (1996) Essential role of the posterior morphogen nanos for germline development in Drosophila. Nature 380:708–711
Lehmann R, Nüsslein-Volhard C (1991) The maternal gene nanos has a central role in posterior pattern formation of the Drosophila embryo. Development 112:679–691
Liu Y, Wu C, Lyu Q, Yang D, Albertini DF, Keefe DL, Liu L (2007) Germline stem cells and neo-oogenesis in the adult human ovary. Dev Biol 306:112–120
Lobascio AM, Klinger FG, Scaldaferri ML, Farini D, De Felici M (2007) Analysis of programmed cell death in mouse fetal oocytes. Reproduction 134:241–252
Matova N, Cooley L (2001) Comparative aspects of animal oogenesis. Dev Biol 231:291–320
Matsumoto L, Kasamatsu H, Pikó L, Vinograd J (1974) Mitochondrial DNA replication in sea urchin oocytes. J Cell Biol 63:146–159
Millonig G, Bosco M, Giambertone L (1968) Fine structure analysis of oogenesis in sea urchins. J Exp Zool 169:293–313
Nakamura S, Kobayashi K, Nishimura T, Higashijima S, Tanaka M (2010) Identification of germline stem cells in the ovary of the teleost medaka. Science 328:1561–1563
Raz E (2000) The function and regulation of vasa-like genes in germ-cell development. Genome Biol 1:1017
Saiti D, Lacham-Kaplan O (2007) Mouse germ cell development in-vivo and in-vitro. Biomark Insights 2:241–252
Schupbach T, Wieschaus E (1986) Germline autonomy of maternal-effect mutations altering the embryonic body pattern of Drosophila. Dev Biol 113:443–448
Song JL, Wessel GM (2007) Genes involved in the RNA interference pathway are differentially expressed during sea urchin development. Dev Dyn 236:3180–3190
Song JL, Wong JL, Wessel GM (2006) Oogenesis: single cell development and differentiation. Dev Biol 300:385–405
Voronina E, Lopez M, Juliano CE, Gustafson E, Song JL, Extavour C, George S, Oliveri P, McClay D, Wessel G (2008) Vasa protein expression is restricted to the small micromeres of the sea urchin, but is inducible in other lineages early in development. Dev Biol 314:276–286
Walker CW, Lesser MP (1998) Manipulation of food and photoperiod promotes out-of-season gametogenesis in the green sea urchin, Strongylocentrotus droebachiensis: implications for aquaculture. Mar Biol 132:663–676
Walker CW, Unuma T, McGinn NA, Harrington LM, Lesser MP (2001) Reproduction of sea urchins. In: Lawrence JM (ed) Edible sea urchins: biology and ecology, vol 32. Developments in aquaculture and fisheries science. Elsevier, Amsterdam, pp 5–26
Walker CW, Harrington LM, Lesser MP, Fagerberg WR (2005) Nutritive phagocyte incubation chambers provide a structural and nutritive microenvironment for germ cells of Strongylocentrotus droebachiensis, the green sea urchin. Biol Bull 209:31–48
Walker CW, Unuma T, Lesser MP (2007) Gametogenesis and reproduction of sea urchins. In: Lawrence JM (ed) Edible sea urchins: biology and ecology, vol 37. Developments in aquaculture and fisheries science. Elsevier, Amsterdam, pp 11–33
Wang Z, Lin H (2004) Nanos maintains germline stem cell self-renewal by preventing differentiation. Science 303:2016–2019
Wessel GM, Juliano CE, Wong JL, Gustafson EA, Song JL (2010) Molecular markers of oocyte and primordial germ cell development in the sea urchin. In: Harris LG, Boetger A, Walker CW, Lesser MP (eds) Echinoderms: Durham. Taylor and Francis Group, London, pp 517–528
Xu H, Gui J, Hong Y (2005) Differential expression of vasa RNA and protein during spermatogenesis and oogenesis in the gibel carp (Carassius auratus gibelio), a bisexually and gynogenetically reproducing vertebrate. Dev Dyn 233:872–882
Acknowledgements
The authors are grateful to Mrs. Irina Barsegova for her help in editing the manuscript.
Author information
Authors and Affiliations
Corresponding author
Additional information
This work was supported by grants from the Far Eastern Branch of Russian Academy of Sciences (09-II-SB-06-001 and 09-I-P22-04) and Siberian Branch of the Russian Academy of Sciences (N 48).
Rights and permissions
About this article
Cite this article
Yakovlev, K.V., Battulin, N.R., Serov, O.L. et al. Isolation of oogonia from ovaries of the sea urchin Strongylocentrotus nudus . Cell Tissue Res 342, 479–490 (2010). https://doi.org/10.1007/s00441-010-1074-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00441-010-1074-5