Ultrastructural Aspects of Previtellogenic Oocyte Growth in Hermaphrodite Sharpsnout Seabream, Diplodus Puntazzo (Teleostei, Sparidae)

Acta Biologica Hungarica 60 (2), pp. 159–166 (2009) DOI: 10.1556/ABiol.60.2009.2.3

ULTRASTRUCTURAL ASPECTS OF PREVITELLOGENIC OOCYTE GROWTH IN HERMAPHRODITE SHARPSNOUT SEABREAM, DIPLODUS PUNTAZZO (TELEOSTEI, SPARIDAE)

NAGIHAN GÜLSOY1,2* and SIBEL ÇOLAK3

1 Department of Biology, Faculty of Science, Marmara University, Göztepe 34722 Istanbul, Turkey 2 Department of Basic Science, College of Veterinary Medicine, Mississippi State University, MS 39762 USA 3 Department of Agriculture, Faculty Fisheries, Istanbul University, Laleli, Turkey

(Received: May 6, 2008; accepted: June 2, 2008)

This paper describes various aspects of previtellogenic oocyte growth in sharpsnout seabream, Diplodus puntazzo, is an important marine culture fish species in the Mediterranean. The ultrastructural characteristics of nuclear morphology, nuclear-cytoplasmic ratio and the starting of the follicle envelope formation were described in detail. These cells do not significantly differ from those of the other teleost species. The ultrastructural aspects provide new information on the reproductive biology of Sparidae.

Keywords: Ultrastructure – oocyte – previtellogenic growth – Sparidae – Diplodus puntazzo

INTRODUCTION

The family Sparidae of the fish order Perciformes comprises 106 species worldwide from which 24 species are described from Mediterranean Sea, North-Eastern Atlantic Ocean and Canary Islands [3, 12]. Seabreams are of great importance for the commercial fisheries and fish culture. Within the family Sparidae, sexuality presents a variety of natural modalities, from gonochorism to hermaphrodism [5, 20]. Sharpsnout seabream, Diplodus puntazzo (Cetti, 1777), is one of the species in the family Sparidae. Reproductive cycle of this species has been investigated in captivity during the third year of their life. All examined specimens have both ovarian and testicular tissue in same time and therefore they are named as ambisexual species [5]. Until recently, protandry was considered the most common reproductive style found in seabreams, but protogyny has now been reported in more species [11, 20].

* Corresponding author; e-mail:[email protected]; [email protected]

However, despite the availability of information on hermaphroditism, reproductive characteristics and spawning cycle in sharpsnout seabream [10, 12], detailed study about its oocyte structures failed up to now. The process of oogenesis in the female teleost fishes is divided into two main phases, as previtellogenic and vitellogenic oocyte growth [8, 17]. During the previtellogenic oocyte growth, the oogonia transform into the previtellogenic oocytes; membranous organelles proliferate, various structures occur within the cytoplasm and the nucleus-cytoplasm ratio decreases dramatically. With the formation of cortical alveoli vitellogenesis starts and lasts until ovulation [15, 17]. Ultrastructural aspects of oogenesis have been documented in several teleost species [15], including some perciforms [7] and hermaphrodite fish [2, 6, 14, 16], but not in seabreams. The present study is the first carried out on the ultrastructure of the previtellogenic oocytes, differentiated from oogonia to late previtellogenic stage of oocyte during oogenesis in the hermaphrodite Diplodus puntazzo.

MATERIALS AND METHODS Fish

Adult female specimens of D. puntazzo were caught from wild in the Eastern Aegean Sea and kept in floating nets cages at a commercial fish farm, located 4 km offshore from Izmir Buy, Turkey. The sample specimens were collected monthly from the farm during the period of May 2006 and April 2007. Total length and total weight were recorded and sexes were determined macroscopically.

Transmission electron microscopy

The females were anesthetized (Benzocaine, 1 mg/ml) and their gonads removed, measured, and fixed overnight in 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2 (4 °C). After two washes in buffer, the ovaries were post-fixed in 1% osmium-tetroxide in the same buffer for 1 h in the dark and cool (4 °C), contrasted in block using 0.5% uranyl in aqueous solution for 1 h, dehydrat- ed in acetone series (30% to 100%), embedded in Epon 812. Semithin sections (1 μm) were examined and photographed using digital camera attached to BH-2 microscope after staining with methylene blue. Ultrathin sections (0.6 μm) postcon- trasted in saturated solution of uranyl acetate in 50% alcohol and lead citrate. These prepared ovaries were then examined and photographed by JEOL 1011 transmission electron microscope.

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RESULTS

In D. puntazzo, oogonia are small cells with a high nucleus to cytoplasm ratio. They occur in nests (Inset I) and have a scarce and finely granular cytoplasm which shows round and elongated mitochondria, abundant Golgi complex, poorly developed endoplasmic reticulum and nuage. The nucleus is large, and displays a single nucleolus and a nuclear envelope perforated by a great number of pores (Fig. 1). Oogonia are encapsulated by two or more squamous follicle cells. Early previtellogenic oocytes are also located on the periphery of the ovarian lamellae, either isolated or forming cysts in near of these nests and are still small, with scarce and thickly granular cytoplasm, which shows round mitochondria, Golgi complexes, rough endoplasmic reticulum and nuage bodies (Fig. 1). In their nucleus, chromosomes arrested in the diplotene of meiosis I as in other fishes. When the oocytes start to develop, they usually have a single central nucleolus (Inset II) but immediately afterwards several micronucleoli appear on the internal surface of the nuclear envelope (Fig. 2). During the previtellogenic growth the oocytes are larger than the early previtellogenic ones and grow progressively (Inset II). There are a lot of ribosomes homogeneously distributed, well-developed Golgi complex, endoplasmic reticulum and spherically many mitochondria in their cytoplasm. The basophilic nuclear material moves to the cytoplasm in the oocyte during this stage. It extrudes through the nuclear envelope pores and accumulates in the per-

Fig. 1. Electron micrograph of oogonia (OG) and previtellogenic oocyte (PO) of D. puntazzo. The oogonia have nucleus (n), several mitochondria (*) and endoplasmic reticulum (arrow) and surrounded by squamous follicle cells (fc). The early previtellogenic oocyte has nucleus (n) and nucleoli (nu), Golgi complex (GC) and nuage (na). Inset I: A nest showing oogonia

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Fig. 2. In the previtellogenic oocytes have several micronucleoli (nu) appeared on the internal surface of the nuclear envelope (ne) and many nuage (na) and mitochondria (m) in ooplasm. Inset II: During the previtellogenic growth the previtellogenic oocytes (arrow) are larger than early primary oocytes (arrowhead)

Fig. 3. The cytoplasmic region of previtellogenic oocyte. Nucleus (n), nuclear envelope (ne) and basophilic nuclear material, transfer from the nucleus to the ooplasm (op) are seen. Basophilic aggregates enclose to mitochondria (m) with lamellar cristae and nuage (na), associated with mitochondria. Golgi complexes (GC) are near the cluster

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Fig. 4. The general aspects of the perinuclear and peripheral cytoplasmic region in the previtellogenic oocyte (PO). A. Oocyte shows multiple round nucleoli (nu). B. The electron-lucent vesicles (arrowheads) are seen in the periphery ooplasma (op). Oocyte surrounded by follicle envelope is consisted of squamous follicle cells (fe) with nucleus (n), basal lamina and theca. Inset III: Light microscopy of larger previtellogenic oocyte

Acta Biologica Hungarica 60, 2009 164 NAGIHAN GÜLSOY and SIBEL ÇOLAK inuclear cytoplasm (Fig. 3). Organelles come to enclose to basophilic aggregates as a cluster. Most mitochondrial clusters are near to the nucleus and a smaller number of them are located at the cytoplasm periphery and may, or may not, be associated with nuage (Fig. 3). The nucleus of previtellogenic oocytes is large (~18 – 34 μm in diameter) and shows multiple round nucleoli (Fig. 4A and Inset III). The perinuclear cytoplasm, close to the nuclear envelope, has many electrondense granules (Fig. 4A). In some previtellogenic oocytes, electron-lucent vesicles of different sizes may be seen at the cytoplasm periphery (Fig. 4B). Ultrastructural observations showed that follicular envelope is formed by squamous follicle cells during the oocyte primary growth (Fig. 4B). It consists of two layers. The first one, adjacent to the oocyte is composed of follicular epithelium and is covered by the second layer termed the theca. The basal membrane is present between these two layers. In the plasma membrane and at the apex of follicle cells, short microvilli appear. The zona radiata starts to arise between follicle cells and oocyte (Fig. 5).

Fig. 5. The structure of follicular envelope in two previtellogenic oocytes (PO1 and PO2). In PO1 oolemma start to arise towards the follicles cells, microvilli (mv) are seen. Also electron lucent vesicles (arrowheads) are found in peripheral ooplasm (op). In PO2 (late previtellogenic oocyte) zona radiata starts to accumulate between oolemma and follicle cells. Microvilli are crossed through the zona radiata. Two basal lamina and collagen fiberils are seen in oocyte envelope and stroma, respectively

DISCUSSION

The terminology of oogenesis shows variation according to authors and species. In this study the previtellogenic oocyte development in D. puntazzo was divided into

Acta Biologica Hungarica 60, 2009 Ultrastructure of oocyte growth in seabream 165 early and late previtellogenic oocytes on the basis of histological and ultrastructural observations. These stagings are compatible with the other classifications common- ly used in teleost species [1, 8, 15, 17] as morphological, biochemical and physio- logical events. The present study demonstrated that previtellogenic growth begins with oogonia and lasts until cortical alveolar stage in the oocytes. Ultrastructure of the previtellogenic oocytes of sharpsnout seabream does not differ significantly from those reported previously for other teleost [5, 8, 10–11, 15, 17, 20]. Oogonia are small but have large nuclei with a single nucleolus. Their morphological characteristics are very common in the teleost, reported before [1–2, 6–8, 14–17, 19]. During the initial stage of oogenesis the accumulation of RNA starts from the nucleus towards the ooplasma in oogonia and continues during the previtellogenic growth [9]. Firstly early previtellogenic cells are formed from oogonia having many mitochondria and a basophilic cytoplasm full with free polyribosomes. In the nucleus of early previtellogenic oocytes there are many synaptonemical complexes that these structures are the first prominent morphological signs of the meiosis [8]. The nucleus has multiple nucleoli in this stage indicating the intensive transcription activity of the heteroge- neous nuclear RNA [1, 8, 17, 19]. During the previtellogenic growth the characteristic structures are the electrondense nuages close to the nuclear envelope. It is found in the cytoplasm of previtellogenic oocytes during oogenesis in teleost and various animal species [4, 18]. In the peripheral cytoplasm of D. puntazzo oocytes have some electron lucent vesicles, con- taining exogenous substances derived from the follicle cells and theca via the short microvilli [1, 8, 19]. They get through the channels of the zona radiata to contact the oolemma. In the late previtellogenic oocytes were found to surround a thin follicular envelope. This is derived from prefollicular somatic cells associated with germ cell clusters or nests in the ovigerous lamellae as in other teleost species [8, 9, 13, 18]. The ultrastructural characteristic of the previtellogenic oocytes of the D. puntazzo, as reported in present paper, provide insight into their cellular and subcellular organization during primary growth.

ACKNOWLEDGEMENTS

This study was founded within the framework of a research program financed by Marmara University, Scientific Research Foundation (project number FEN-BGS-27036-0046).

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