Ultrastructure of Differentiating Oocytes and Vitellogenesis in the Giant Freshwater Prawn, Macrobrachium rosenbergii (de Man) NANTAWAN SOONKLANG, 1 CHAITIP WANICHANON, 1 * MICHAEL J. STEWART, 2 PRAPHAPORN STEWART, 3 PRASERT MEERATANA, 4 PETER J. HANNA, 1,5 AND PRASERT SOBHON 1 1 Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand 2 Genecology Research Group, Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, Maroochydore DC, Queensland 4558, Australia 3 Department of Preclinical Science, Faculty of Medicine, Thammasat University, Pathumthani 12120, Thailand 4 Deparment of Biomedical Sciences, Faculty of Allied Health Sciences, Burapha University, Chonburi 20131, Thailand 5 Faculty of Science and Technology, Deakin University, Geelong, Victoria 3220, Australia KEY WORDS Macrobrachium rosenbergii; ultrastructure; ovary; oocyte; vitellogenesis ABSTRACT The ultrastructure of oogenesis in Macrobrachium rosenbergii, with reference to vitellogenesis, has not been reported. We used light and electron microscopy, as well as vitellin (Vn) purification and antibody production, to study the temporal and spatial production of Vn in the ovary by immunofluorescence. Histologically, the ovary is subdivided into cone-shaped ovarian pouches with a central core containing layers of oogonia. These divide to produce oocytes that migrate outwardly and differentiate into mature oocytes. During the course of differentiation, oocytes undergo modifications, including the rearrangement of nuclear chromatin, the accumula- tion of ribosomes, rough endoplasmic reticulum (RER), and lipid, and the formation of secretory and yolk granules, resulting in four stages. Ultrastructurally, early previtellogenic oocytes (Oc 1 ) are characterized by the accumulation of new ribosomal aggregates, translocated from the nucleus. Late previtellogenic oocytes (Oc 2 ) show nuclear heterochromatin with a ‘‘clock face’’ pattern, the presence of RER, and three types of secretory granules. Follicular cells occupy the intercellular spaces and surround the Oc 2 . Early vitellogenic oocytes (Oc 3 ) are larger, with nuclei containing pre- dominantly decondensed euchromatin, and cytoplasm with yolk and secretory granules, and few lipid droplets. Late vitellogenic oocytes (Oc 4 ) are characterized by completely euchromatic nuclei, an indistinct plasma membrane, yolk platelets and secretory granules, and abundant lipid. Vitello- genin (Vg) in ovaries of M. rosenbergii consist of two main bands at MW 90 and 102 kDa. Our data indicates that Vn is present, and probably synthesized in Oc 3 and Oc 4 , but there may be some undetected exogenous Vg production. Microsc. Res. Tech. 75:1402–1415, 2012. V V C 2012 Wiley Periodicals, Inc. INTRODUCTION In decapod crustaceans, which include the giant fresh water prawn, oogenesis begins with mitotic divi- sions of oogonia (Og) at a centralized germinative zone of the ovary (Damrongphol et al., 2001). As secondary Og develop, they increase in size, enter the first stage of meiotic division, and migrate away from the zone of proliferation toward the periphery of the ovarian fol- licles (Kruevaisayawan et al., 2010; Meeratana and Sobhon, 2007; Okumura et al., 2004). At the same time, daughter cells differentiate through four or five stages of primary oocytes, during which they exhibit unique cytological characteristics. It is during this pro- liferative phase (previtellogenesis) that the oocytes produce ribosomes (Ri), rough endoplasmic reticulum (RER), Golgi complexes, and organelles responsible for the synthesis of glycoproteins and lipids in the cyto- plasm (Hinsch and Cone, 1969; Komm and Hinsch, 1987; Kruevaisayawan et al., 2010). Cessation of this stage occurs, when the new oocytes develop into large vitellogenic oocytes that are arrested at prophase I or metaphase I of meiosis. The next phase of oogenesis is characterized by the breakdown of the germinal vesicle (nuclear membrane) and subsequent oocyte matura- tion just before spawning and fertilization (which in some species such as the prawn, Palaemon serratus, occurs at the same time). Once oocytes enter the vitellogenic stage, the hepato- pancreas (Tseng et al., 2001) and follicular cells sur- rounding growing oocytes within the ovary (Meeratana and Sobhon, 2007; Kruevaisayawan et al., 2010) pro- duce yolk protein, vitellogenin (Vg). Vg is a high-molec- ular weight precursor protein, which is associated with lipidic, glycocidic, and carotenoid groups (Zagalsky et al., 1967). The Vg from the heptopancreas is released *Correspondence to: C. Wanichanon, Department of Anatomy, Faculty of Sci- ence, Mahidol University, Bangkok 10400, Thailand. E-mail: chaitip.wan@mahidol.ac.th Received 3 April 2012; accepted in revised form 27 April 2012 Contract grant sponsor: The Commission on Higher Education (CHE), Minis- try of Education (National Research University Project to P.S. and Ph.D. Schol- arship to N. S.); Contract grant sponsor: a TRF-CHE-Mahidol Distinguished Research Professor Grant (P. S.). DOI 10.1002/jemt.22081 Published online 31 May 2012 in Wiley Online Library (wileyonlinelibrary.com). V V C 2012 WILEY PERIODICALS, INC. MICROSCOPY RESEARCH AND TECHNIQUE 75:1402–1415 (2012)