Journal of the Persian Gulf (Marine Science)/Vol. 6/No. 19/March 2015/06/53-58 Gonad Histology and Gonadosomatic Index Variations during Gonadal Development of Wild Female Tenualosa ilisha Moallem, Zohre 1 ; Abdi, Rahim 1* ; Movahedinia, Abdolali 1 ; Shirali, Solmaz 1 ; Salati, Amir Parviz 2 1- Department of Marine Biology, Faculty of Marine Sciences, Khorramshahr University of Marine Science and Technology, Khorramshahr, IR Iran 2- Department of Fisheries, Faculty of Marine Natural Resources, Khorramshahr University of Marine Science and Technology, Khorramshahr, IR Iran Received: October 2014 Accepted: March 2015 2015 Journal of the Persian Gulf. All rights reserved. Abstract Hilsa shad (Tenualosa ilisha) is an anadromous species which has reproductive migration from the Persian Gulf to the river. Morphological properties and histological changes in oocyte of Hilsa shad in two different environments (salt water and fresh water in Khuzestan province) were studied with respect to its reproductive cycle. Twenty fish samples with average weight of 830.75±3.25 gr, were collected from Bahmanshir (freshwater) and Musa creek (estuarine water). Following biometry (length and weight measurements), pieces of ovary of each fish were fixed in Bouin's fixative solution. Microscopic sections (5µm thickness) from paraffin embedded tissues were prepared and stained with Hematoxylin & Eosin, and then slides were studied under light microscopy. Results showed six phases as follows: chromatin nucleolus, perinucleolus, cortical alveolus, primary vitellogenesis, secondary vitellogenesis and third vitellogenesis were recognized in ovaries of T. ilisha. Three follicular layers such as theca, granulosa and zona radiata were clearly observed at the end of the vitellogenesis. The most oocyte diameters were also measured at the same phase. Ovarian follicles were at the same phase as in the ripe ovaries, indicating Hilsa shad is synchronous fish species in ovarian development. The changes observed in the pattern of oocyte development in T. ilisha was typical of the patterns described in most synchronous spawners studied to date. Keywords: Tenualosa ilisha, Ovary, Oogenesis, Anadromous 1. Introduction Five species of tropical shads (Clupeidae: Tenualosa spp.) live in the estuaries and coastal waters of tropical Asia (Ahmed et al., 2008). The most widespread and well studied species is Tenualosa ilisha. T. Ilisha, belonging to the sub-family alosinea of the family * E-mail: abdir@kmsu.ac.ir clupeidae (Clupeiformes, Pisces), occurs in foreshore areas, estuaries, brackish water lakes and fresh water rivers of the western division of the Indo-Pacific region (Karimi et al., 2013). Its marine distribution extends from Iran and Iraq in the Persian Gulf to the west coast of India in the Arabian Sea and the Bay of Bengal (Gomes et al., 2004). In the Persian Gulf. T. ilisha migrates to Arvand river and Bahmanshir river for 53
Moallem et al. / Gonad Histology and Gonadosomatic Index Variations during Gonadal spawning (Cardenas et al., 2008). Reproduction is a continuous developmental process throughout ontogeny, requiring energetic, ecological, anatomical, biochemical and endocrinological adaptations (Caputo et al., 2000). Information on fish reproduction is also important in aquaculture. The availability of quality seeds and the ability to control fish reproduction are limiting factors in the farming of any commercial species (Merson et al., 2000). This became an important factor to fulfill the demand of continuous supply of table fish and fish seeds throughout the year (Cakici and Ucuncu, 2007). Adequate information about reproductive parameters and developmental biology can meet this requirements (Kunz, 2004). Study on the gonadal histological changes of fish is an important tool for identification of reproductive period which includes developmental phases (Unver and Saraydın, 2012). So, the present study was designed to provide a better understanding of gonadal changes in the female T. ilisha during the natural reproductive cycle. 2. Material and Methods fixed in Bouin s solution, dehydrated through ethanol series and embedded in paraffin, sectioned at 5 μm and stained with hematoxylin and eosin (H&E) for histological examination under light microscopy (Model CX2, Olympus Japan) (Van et al., 2004). The developmental stage and the diameter of the 20 largest oocytes were recorded (Unver and Saraydın, 2012). 2.3. Statistical Analysis All data are presented as (means ± S.D.). Data were checked for normality by Kolmogorov Smirnoff test and analyzed for statistical differences within months by one- way ANOVA. Differences between and within groups were tested using oneway variance analysis followed by the Tukey s comparison test (An, H., Kang, H., Han, H., 2014). Differences were considered significant, when P value was lower than 0.05. The statistical analysis was performed using the SPSS (version 18, Chicago, USA). 3. Results 2.1. Fish and Sample Collection 3.1. Female Gonadal Development Freshwater fish samples were captured by boat fishing from Bahmanshir river and marine fish were captured from Sajjafi port near Hendijan south of Iran. Twenty fish speciemen, with average weight of 830.75±3.25 g in both stations were collected by ear net with mesh size of 80 mm;weight and length were recorded and a piece of fish ovary tissue was removed and placed in Bouin's solution after euthanasia with high doses of ketamine, samples were kept in Bouin's solution for 48 h and then stored in 70% ethanol. 2.2. Histology Mid portion of the right lobe of the gonad was Female's gonad developmental stages were as follows: chromatin nucleolus, perinucleolus, cortical alveoli formation, primary vitellogenesis, secondary vitellogenesis and tertiary vitellogenesis. At first stage, oocytes were spherical, oval and multi-faceted, large nucleus occupies most of the cell. Large nucleoli were found sporadically in nucleoplasm. Cytoplasm was strongly basophilic and stained with hematoxylin (Fig. 1). The average diameter of oocytes in this stage was 7.8 ± 96/80µ. Number of oocytes in the ovaries blades increased in perinucleolus stage and cytoplasm was stained with hematoxylin lightly compared to previous stage (Fig. 2). 54
Journal of the Persian Gulf (Marine Science)/Vol. S 6/No. 19/March 2015/06/53-58 These alveolii occupied the whole cytoplasm. Zona radiata was seen in this stage (Figs 3 to t 4). Fig 1: Section of immature ovary of T. ilisha: oogonia (og),, Stage of chromatin nucleus (green arrow head), Stage of pronucleus (red arrow head), Nucleus (N), Nucleolus (n), Yolk nucleolus (ny) (H&Ex10). Fig 3: Oocytes in i cortical alveolus stage. Cortical alveolus stage (red arrow head) ), Pronucleus (black arrow head), Oogonia (og), Nucleus (N), Nucleolus (n), Cortical alveolus (ca) (H&Ex40). Fig 2: Oocyte in pronucleus stage. Nucleus (N), Nucleolus (n), Yolk nucleolus (ny) (H&Ex40). Most oocytes were spherical in i shape. Nuclei increased in size and also were distinguished with nucleus membrane. Nucleoli, which were arranged around the nucleus increased in number. The average diameter of oocytes in this stage was 49. 15 ± 70.118µ. Characteristic of cortical alveoli stagee was vacuolization of oocytes and subsequent cell enlargement. Small alveoli were formed around the nucleus which increased in numberr and size during this stage as at the end of cortical alveoli stage. 55 Fig 4: Coverage of oocyte in cortical alveoluss stage. Theca ( Th), Zona radiata (Zr)) (H&Ex40). In Primary vitellogenesis (Fig. 5), vitellin granules around the nucleus appeared. Fat vacuoles were seen in large numbers around the core. Att this stage, the oocyte diameter increased to 239.96 ± 20.34µ µm. Number of granules increased and moved to center of cell in secondary vitellogenesis (Figs 6 to 7).
Moallem et e al. / Gonad d Histology and Gonadosomatic Index Variations during Gonadal Cell layers were thickened and zona radiata was stained with eosin heavily. Ooocytes were seen with unaided eye in tertiary vitellogenesis (Fig. 8). Fig 5: Oocytes in primary vitellogenesis stage. Ann oocyte in primary vitellogenesis (blue arrow head with germinal vesicle v in around), Chromatin nucleus (black arrow head), Nucleus (N), Cortical alveolus (ca), Yolk globule (g) (H&Ex40). Fig 8: Oocytes in tertiary vitellogenesis (H&Ex40). Ovarian volume was increased as it occupied most of lumen. Parallell to oocyte development lipid granules increased in size and number and some off them fused. Changes in oocyte diameter and developmental stage in ovaries of T. ilisha females are shown in Fig.9. Oocyte diameter increased significantly during gonadal development as highest value, 399.03±92.01 was recorded in tertiary vitellogenesis (P<0.05). 500 400 Fig 6: An oocyte in secondary vitellogenesis (red arrow head) (H&Ex40). oocyte diameter( µm) 300 200 100 0 A B C D E1 E2 developmental stage E3 Fig 9: Changes in oocyte o diameterr in developmental stage. A: oogonia, B: chromatin nucleus, C: pronucleus, D: cortical alveolus, E1: Primary vitellogenesis, E2: secondary vitellogenesis, E3: tertiary vitellogenesis 4. Discussion Fig 7: Covering of oocyte in secondary vitellogenesis. Theca (Th), Granulosa (Gr), Zona radiata (Zr) (H&Ex40). 56 In the teleost fishes, the process of oogenesis may be divided into five to eightt stages (Andrade et al., 2001). Stages off gonadal development in T. ilisha weree identified according a to the scales described by
Journal of the Persian Gulf (Marine Science)/Vol. 6/No. 19/March 2015/06/53-58 Shinkafi et al. (2011) and divided into 6 periods (Shinkafi et al., 2011). Nucleus occupies the greater part of the cell during the chromatin nucleolus in T. ilisha. Cytoplasm was basophilic that caused by accumulation of abundant free ribosome (Bariche et al., 2013). As cell transformed to perinucleolus stage, nucleoli increased in number, decreased in size and arranged around the inner lining of the nucleus. Similar findings had been reported in Dicentrarchus labrax during chromatin nucleolus stage. Cakici and Ucuncu (2007) in Danio rerio found that each nucleolus split to several smaller nucleoli (Cakici and Ucuncu, 2007). Changes in the nucleus and nucleolus during oocyte growth stages attributed to synthesis process in nucleus. Cortical alveolus is marker of oocyte development at the beginning of the spawning season (Green et al., 2011). The cortical alveoli and follicular layer surrounding the oocytes is the prominent feature of this stage in T. ilisha. Three types of yolk in oocytes of teleosts including lipid droplets, yolk vesicles (alveoli of cortical) and the yolk vesicles had been reported in teleosts (Grier et al., 2009). In most teleosts, intracellular vacuolization in oocyte begins on the sidelines and then proceeds to the center of the oocyte (Timothy et al., 2015). In Paralichthys dentates, Lutjanus synagris and Katsuwonus pelamis alveolus first appeared near the core and then spread around the ooplasm in the vicinity of the cell membrane (Kang et al., 2009). In the genus Phoxinus, alveoli appear simultaneously all around the oocyte. Histological studies showed that the cortical alveoli first appeared around the nucleus and then gradually occupied all of cytoplasm in T. ilisha and zona radiata was first seen in cortical alveoli stage that is common in teleosts, but in Sciadeichthys luniscutis and Genidens genidens zona radiata was seen first during vitellogenesis (Kayaba et al., 2001). At vitellogenesis, oocyte reaches its maximum size. Accumulation of exogenous yolk protein precursors caused the increase in cell size, but in some species accumulation of endogenous species had been reported (McBride et al., 2012). In the initial stage of vitellogenesis, small acidophilic yolk particles begin to stack, in margins of the cytoplasm. Similar findings were reported in Merluccius merluccus and Katsuwonus pelamis (Merson et al., 2000). In mature oocyte of T. Ilisha, heterogeneous yolk vacuoles were seen. At the end of vitellogenesis, due to the accumulation of yolk in the cytoplasm of cells, nucleus starts to migrate to the animal pole. Group synchronous ovaries are those in which at least two size groups of oocytes could be seen simultaneously; Therefore, T. ilisha is considered a group synchronous species as two or three stages of oocytes were seen in each sampling time. Karimi et al. (2013) also reported a similar situation for Acanthopagrus latus in Persian Gulf (Karimi et al., 2013). In conclusion the combination of fish oocytes in Tenualosa ilisha was as such that a high percentage of oocytes grew concurrently at maturity but only small percentage at lower levels were observed. This feature indicates that spawning in this fish occurs at the same time as well. References Ahmed, MS., Sharif, AS., Latifa, GA.,2008. Age, growth and mortality of Hilsa shad, Tenualosa ilisha in the River Meghna, Bangladesh. Asian Journal of Biological Science 1: 69-76. An, H., Kang, H., Han, H., 2014. Isolation and characterization of 26 novel poly-nucleotide microsatellites from short barbeled grunter (Hapalogenys nitens) for genetic analysis. Conservation Genet Resours 6: 669 672. Andrade, RF., Bazzoli, E., Rizzo, E., 2001. Continuous gametogenesis in the neotropical freshwater teleost, Bryconops affinis (Pisces: Characidae). Tissue Cell 33: 524-532. Bariche, M., Torres, M., Azzurro, E., 2013. The presence of the in vasivelion- fish (Pteroismiles) in the Mediterranean Sea. Mediterrane Marine Science 14: 292 294. 57
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