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1* E-mail: moghaddam7@yahoo.com

Study of the Zona Radiata Structure in Oocytes of the Persian Sturgeon (Acipenser persicus) before and after Fertilization

Moghaddam, Ali1*; Oryan, Shahrbanoo1; Shabanipour, Nader2

1-Faculty of Biological Science, Kharazmi University, Tehran, IR Iran. 2-Department of Biology, Faculty of Sciences, Guilan University, Rasht, IR Iran.

Received: April 2013 Accepted: August 2013

© 2013 Journal of the Persian Gulf. All rights reserved.

Abstract Egg envelope or otherwise called zona radiata (ZR) is an acellular area that originates next to oolemma externally. It grows and thickens gradually from oolemma and follicular epithelium both. Microscopic studies on the tissue sections hava shown that during developmental stages of oocytes, ZR could not be observed at stage I (Primary growth stage) in Acipenser persicus. It appeared in stage II (Cortical alveolar stage) and during stage III (vitellogenesis), ZR thickness was greatest and had highest complexity. The striated appearance of ZR at this stage, were identified to be pore-canals involved in yolk material transportation. In stage IV (Maturation stage), a decline was observed in ZR thickness and complexity, a process which continued after fertilization. In mature egg, an uneven layer of chorion over ZRe (ZR, external) and a jelatinous coat extrachorion were developed. Immediately after fertilization, ZRi (ZR, internal) turned to a homogenous layer (fertilization envelope), probably because of cortical reaction. 30 minutes after fertilization, two layers recognized in ZRi and chorion and extrachorion were disappeared.

Keywords: Zona radiata, Oocyte, Persian sturgeon, Acipenser persicus

1. Introduction

Fishes are the most successful and diverse and account for more than half of the live vertebrate species (Kaviani et al., 2013). Accurate recognition of the egg coverings of fishes, is a fundamental principal that could illuminate the knowledge of their propagation and breeding. Egg envelope or ZR is an eggshell made between oocyte plasma membrane (oolemma) and follicular epithelium by growing of microvilli of oocyte and follicular cells toward each

other and secretion and deposition of substances between microvilli. The thickness and complexity of ZR changes gradually during developmental stages of ovary under management of both oocyte and follicular epithelium. ZR internal and external architecture differs among many fishes and does not seem to be similar in even all species of one genus (McMillan, 2007).The main function of egg envelope include transport of yolk material in early developmental stages and fixation of a deposited egg to the substratum, sperm attraction, prevention of polyspermy and antibacterial and mechanical protection during spawning, fertilization and post

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fertilization periods. For the developing embryo, the egg envelope enables gas exchange, excretion and transport of nutrients from the external environment (Zelazowska, 2010). For the present study, the Persian sturgeon (Acipenser persicus) was selected as one of the most valuable endemic and vulnerable caviar fishes in southern part of Caspian Sea (Hosseinzadeh et al., 2012). In this research, the growing ZR during ovarian developmental stages and its post-fertilization modifications were evaluated. Such researches are able to provide basic knowledge about successful management, caviar production, artificial propagation and breeding of this valuable species.

2. Materials and Methods

All samples were obtained from Shahid Beheshti sturgeon propagation center (Rasht, Iran). Based on laparoscopy, sonography and morphological properties, mature females were recognized. Some ripe oocytes were taken from hand evacuated ovary. Some of the mature oocytes were copulated with semen to get fertilized eggs. Some fertilized oocytes were massaged with fine clay particle suspension (30 min), to countervail sticky characteristic of egg coverings. Remnants of empty ovary was also collected to identify earlier stages of oocytes. All samples were fixed in bouin’s solution for 24 hrs. and then transferred to 70% methanol. Samples were dehydrated in series of graded methanol, turned transparent by xylene, embedded in paraffin wax and sectioned at a 5 µm thickness by a rotary microtome. Sections were stained by hematoxylin and eosin (H-E universal procedure of staining), mounted permanently and studied under a light microscope. Micrographs provided with a camera, evaluated with TSView software.

2. Results

Primary growth stage (stage I): Oocytes of stage I (average diameter, 183.22 µm) showed normal features of fish oocytes (Fig. 1)

Fig. 1: Oocyte in stage I (Primary growth stage), N: nucleus, n: nucleoli, Cr: chromatin, O: ooplasm.

Cortical alveolar stage (stage II): The average diameter of oocytes at this stage reached 300 µm. Large nucleus tended to animal pole and occupied a major part of the oocyte. The dispersed nucleoli found at stage I, now aggregated showing as larger ones. At initial stage II, the ZR was appeared as a thin layer (2 µm) between oolemma and follicular epithelium, the process which is called zonagenesis. Some clear vesicles called cortical alveoli also appeared in ooplasm.These vesicles originated in ooplasm, migrated toward peripheral ooplasm and lodged next to inner surface of oolemma (Fig. 2a). In continue, with projection of processes or microvilli of oocyte and follicular cells and secretion of substances between them, average diameter of ZR increased and reached 30 µm (Fig. 2b). Follicular cells had different sizes and shapes (spherical, oval and pyramidal). Two stages that mentioned above are previtellogenic stages.

Vitellogenesis (stage III): At vitellogenic stage, average diameter of oocytes was1765.89 µm. The ZR with maximal thickness (average, 201.53 µm) and complexity exhibited two distinct layers; zona radiata interna (ZRi) and zona radiata externa (ZRe). Fence-like striations were obviously seen in each layer and it seems that stria of two layers are in the length of each other (Fig. 3 and Fig. 4b). The canals were pathways of microvilli that originated from oocyte and follicular cells which grow toward each

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other. Canals with pores on the internal and external surfaces of the ZR are named pore-canal system. Yolk granules (material) occupied whole ooplasm. A narrow perivitelline space was recognizable between oolemma and vitelline envelope (ZR). Peripheral ooplasm under oolemma was pigmented.

Fig. 2: Oocyte in stage II (Cortical alveolar stage); a- Initial stage II, b-Late stage II. N: nucleus, n: nucleoli, O: ooplasm, FC: follicular cell (with different shapes and sizes), CA: cortical alveoli, ZR: zona radiata, Cr: chromatin, M: microvilli, PO: peripheral pigmented ooplasm.

Maturation or post-vitellogenic stage (stage IV): Oocytes at this stage reached them, were maximum size and average diameter of them were 2112 µm. Yolk material were united and ooplasm showed a homogenous state. The continuation of canals at the junction site of two layers of the ZR was recognized and some microvilli were observed in the canals (Fig. 4a). The ZR found quite simple architecture with less thickness (average, 161.82 µm) and

complexity. Outside the ZRe, a more thin and chromatic layer named chorion was observed. Surface of chorion were covered with a jelatinous coat or layer called extrachorion (Fig. 4a,b). The extrachorion had a brush-like feature.

Fig. 3: Vegetal pole of oocyte in stage III (vitellogenesis); ZRi: zona radiata interna, ZRe: zona radiata externa, C: canals (Pore-canal system), YM: yolk material, PVS: perivitelline space, PO: peripheral pigmented ooplasm, O: ooplasm.

Fig. 4: a and b- Fine structure of egg envelope in mature oocyte; ZRi: zona radiata interna, ZRe: zona radiata externa, E: extrachorion, Ch: chorion, M: microvilli in the canals.

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Post-fertilization stage: The reduction in thickness (average, 141.02 µm) and pores-canals of the ZR continued. The change of ZRi in animal pole was more apparent than the vegetal pole (Fig. 5a). The striations reduced or disappeared and ZRi elevated and turned to a monolithic state (Fig. 5a). In this stage, ZR is called "fertilization envelope". After at least 30 min. massaging of zygotes in the fine clay particle suspension, the reduction in thickness (average, 63.43 µm) and striation of the ZR was continued yet and ZR turned into a thin and simple area (Fig.5b). Microvilli and canals disappeared but some remnants of them were observed. In ZRi, two simple and distinct layers separated from each other. Chorion and extrachorion also disappeared (Fig. 5b).

Fig. 5: a- Animal pole of fertilized oocyte (zygote), immediately after fertilization. b- zygote, 30 minutes after fertilization and massaging with fine clay particle suspension. ZRi: zona radiata interna, ZRe: zona radiata externa, O: ooplasm, PVS: perivitelline space, Ch: chorion, E: extrachorion, R: remnants of microvilli and ramification of canals.

3. Discussion

In the Persian sturgeon, the ZR wasn’t observed at stage I (primary growth stage) (Fig. 1), though it is suggested to be initiated at this stage in Pseudosciaena crocea (Kaviani et al., 2013). The ZR was first observed at stage II (cortical alveolar stage) as a thin layer around the oocyte (Fig. 2a), which increased gradually in thickness and structural complexity during further development of oocyte passing from stage II to III (Fig. 2b and Fig. 3). Other researchers (Shabanipour and Heidari, 2004, Bahmani et al., 2005 and Shabanipour and Hossayni, 2010) have also reported the originating of the ZR at stage II. It has also been reported in Bryconops affinis at previtellogenic stage and in Akarotaxis nudiceps and Bathyraco marri at mid-stage II (Kaviani et al., 2013). At stage III (vitellogenic stage), the sickness and complexity reached their maximum grade and ZR has two distinct and striated layers; zona radiata interna and zona radiata externa (Fig. 3). These two layers are together named vitelline envelope, which is separated from oolemma with a narrow space called perivitelline space. The ZR made by oocyte, is named primary envelope (Zelazowska, 2010). In Hippocampus erectus and Syngnathus fuscus, three layers of primary envelope have been named Z1, Z2 and Z3 ( Anderson, 1967). The ZR of a number of fish species consists of 2 to 4 protein monomers. These proteins are mainly filamentous (Celius et al., 2000). Stripes or stria of the ZR are canals or pathways that continue from the external surface of the ZR to its internal surface. These canals are passageways of microvilli or processes (Fig. 2b), originating from oolemma and follicular cells toward each other. The external and internal aperture of canals on the ZR surfaces are called pores. The canals have different sizes and pathways but how this inequality in pores-canals and also direction of pathway of canals act in transport of materials, is yet unknown. These properties may demonstrate a type of selective transport. The gradual increment and overall

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better defined organization, exhibits the importance of the ZR for transport of essential materials for yolk synthesis. Vitellogenesis, egg maturation and hatching are very complex processes that need active synthesis and transport of row materials to oocytes (Kaviani et al., 2013). Yolk material (vitellogenin), is a high molecular weight glyco-phospho-lipoprotein. It is synthesized by the liver cells of mature females and secreted into the blood stream from where it is taken up by developing oocytes and cleaved into yolk proteins (Keyvanshokooh and Vaziri, 2008). In mature oocyte, the ZR found quite simple architecture and it,s thickness and complexity (fence-like striations or canals) reduced obviously (Fig. 4.a,b). These changes may exhibit the reduction of nutritional and increment of protective role of the ZR. Outside the ZRe, two layers called chorion and extrachorion are made. The jelatinous coat (extrachorion) has a brush-like feature (Figs. 4.a,b). It is appeared that jelly layer could act for adhesion of benthic oocytes of the Persian sturgeon to the spawning substrates (stones or plants).The studies of Esmaeli and Johal (2005) on the silver carp (Hypophthalmichthyes molitrix), Huysentruyt and Adriaens (2005) on the catfish (Corydoras aeneus) and Heidari et al. (2009) on the Kutum (Rutilus frisii kutum) revealed that generally in teleosts, small protuberances, attaching-filaments or microvilli-like adhesive structures do this role. Chorion and extrachorion that were observed outer to the ZR and synthesized by follicular cells, are named secondary envelopes (Zelazowska, 2010). The changes observed in oocytes and particularly in ZR, in fish and other animals even mammals are physiologically induced by estrogens (Arokwe et al., 1997, Celius et al., 2000 and Hosseinzadeh et al., 2013) and ZR in fish is equivalent of zona pellucida in mammals (Green, 1997, Litscher and wassarman, 2007). Our findings in this study on the prefertilized oocytes, showed that the structure of the ZR in the Persian sturgeon was similar to the Russian sturgeon (Acipenser gueldenstaedtii) (Zelazowska, 2010) and differences were minor. This is to be

expected, because these two species belong to one genus, as comparative ultrastructural studies of the ZR of marine fish eggs in three genera in Perciformes revealed that the studied characters did not differ significantly for the fishes in the same genus, but was significantly different for different genera, even when the genera were in the same family (Li et al., 2000). After fertilization, the reduction in thickness and structural complexity of the ZR continued (Fig. 5). In animal pole, the striated feature of the ZR was lost and the ZR changed to a homogenated and elevated region (Fig. 5a). It is appeared that these changes are the result of cortical reaction, i.e., at the time of fertilization, cortical alveoli attach to the oolemma and exit their contents into perivitelline space by exocytosis and change the structure of the ZR (Iwamatsu and Ohta, 1976, Guraya, 1986). Thus, this layer hardens, elevates and transforms to the fertilization envelope.

Transformation of the ZR by the alveolin and transglutaminase with reduction in micropyle diameter, closes it and blocks polyspermy (Murata et al., 1991). Iwamatsu (1983) showed that when oocytes were dechorionated (i.e. the envelope removed or separated) and massaged with sperms, the polyspermy occured. Our findings showed that in Persian sturgeon, the ZR appears at previtellogenic stage, culminates at vitellogenic stage and reduces in thickness and structural complexity at post-vitellogenic and post-fertilization stages, that could point the change in its role from transportation to protection.

Acknowledgement

The authors are grateful to Shahid Beheshti Sturgeon Propagation Center and F. Nazar Haghighi for her assistance.

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