Changes in plasma steroid hormones and gonadal histology associated with sexual maturation in wild southern hake (Merluccius australis)/Cambios en las hormonas esteroidales plasmaticas y en la histologia gonadal asociados a la maduracion sexual a la merluza austral (Merluccius australis).
Southern hake (Merluccius australis) is a demersal gadiform fish species found in the southern hemisphere between Argentina in the Atlantic Ocean (Tingley et al., 1995) and New Zealand in the Pacific Ocean (Aguayo-Hernandez, 1995; Colman, 1995). This species supports important industrial and artisanal fisheries in Chile, Argentina, and New Zealand, which supply local and mainly overseas market of Japan, USA, Spain and Portugal (Sylvia, 1995)
Global southern hake landings had historical peaks of about 65,000 ton between 1987 and 1989 (Sylvia, 1995), but there were dramatically declining catches in later years due to smaller fishing quotas to protect this resource from overexploitation. Nowadays, global southern hake landings are steady at just over 30,000 ton per year with prices around US$10 per kilo. Nevertheless, the global demand for southern hake is growing and the wild capture is declining, creating an undersupplied market for this fish. Indeed, the extent of this dependence has prompted the development of southern hake farming in Chile.
Even though there is an advanced understanding of the biology of the southern hake (Aguayo-Hernandez, 1995; Colman, 1995; Tingley et al., 1995; Bustos et al., 2007; Effer et al., 2013), there are some relevant questions concerning their reproductive biology, particularly the sexual maturation cycle and reproductive endocrinology that still remains unrevealed. To date, it is known that fish reproduction is regulated by a wide variety of abiotic and biotic environmental factors that trigger internal physiological mechanisms responsible for causing sexual maturation of fish (Arcand-Hoy & Benson, 1998).
Wild broodstock fish can spawn naturally in the tank when the environmental conditions are favorable, nevertheless, several fish species exhibit reproductive dysfunctions when they are raised in captivity (Mylonas et al., 2010). Reproductive dysfunctions are usually more seriously in female broodstock and can be associated with final oocyte maturation, ovulation and spawning (Zohar et al., 1988; Peter et al., 1993). These dysfunctions most likely result from the combination of the stress induced by captivity, and the lack of a suitable environment for natural spawning (Schreck et al., 2001; Pankhurst, 2011). Therefore, in the case of the absence or scarcity of natural spawning, several studies have shown that hormone induced spawning is a reliable method of inducing reproduction in these fishes (Zohar & Mylonas, 2001). However, this method has been reported to exhibit negative effects on the quality of gametes and survival rate at later stages of Salmo salar (Crim & Glebe, 1984; Crim et al., 1986), S. trutta (Mylonas et al., 1992), Oncorhynchus nerka (Slater et al., 1995), and O. mykiss (Arabaci et al., 2004). In southern hake farming, one of the most critical aspects is to achieve the spawning of wild broodstock under captive conditions. Wild southern hake broodstock are spawned mainly by the use of hormones, causing a low survival rate of their larvae during weaning from Artemia to dry feed in culture conditions.
Considering the above-mentioned problem, the analysis of blood steroid levels has been used to clarify the optimum time to hormone induce spawning in fish, which can help to obtain higher quality gametes and therefore more suitable larvae, and also prevents the occurrence of over-maturation and follicular atresia of the gametes (Donaldson, 1996). Strictly, the use of quantitative analysis of blood steroid hormones, as a method for predicting the maturation stage of southern hake, involves less handling of broodstock compared to current method of gonadal biopsies, which is an invasive method and requires large samples. Moreover, the knowledge of the reproductive management concepts such as maturation cycle, reproductive endocrinology and gonadal development are scarce in southern hake (Bustos et al., 2007; Effer et al., 2013) and therefore, in order to scale up the commercial farming of southern hake is relevant to research in this important area. Hence, the aim of this study was to identify the gamete developmental stages and characterise plasma sex steroid hormones during the maturation cycle of southern hake.
MATERIALS AND METHODS
The specimens were captured by longline gear at 250-300 m depth in the inland waters of the Reloncavi Sound, Interior Sea of Chiloe in the Lagos Region, Chile (41[degrees]31'S, 72[degrees]44'W). Fishing activities were carried out from September 2011 to January 2012. During this period, fishes were collected every two or three weeks depending on weather conditions. Immediately upon reaching the surface, fishes were sacrificed and the samples of blood and gonads were collected and stored for later analysis.
Seventy six samples of gonads in different maturation stages from 40 females and 36 males of M. australis were collected for histological analysis. The dissected tissue was fixed in 5% formalin for 24 h and stored in 70% ethanol. The fixed tissue was subsequently dehydrated and embedded in the paraffin wax. The waxed tissue were cut in transverse sections of 6-7 [micro]m thickness (Microtome, Leica Microsystems, model RM2125, Bannockburn, IL, USA) and then stained with hematoxylineosin. Sample sections were examined under a light microscope (Leica Microsystems model DM750, Leica, Bannockburn, IL, USA) and classified according to their maturation status as immature, proliferation, growth, maturation and spawning. After the maturation stage was determined, the samples were correlated with the levels of serological steroid.
Analysis of hormonal steroids
Blood samples were extracted from each fish by caudal venipuncture and immediately placed on ice, where they were allowed to clot for 3-6 h. Blood samples were later centrifuged for 15 min at 1500 g (MiniSpin Centrifuge, AG 22331, Merck, Hamburg) and serum stored at -80[degrees]C for later sex steroid hormones analysis.
Quantification of sex steroid hormones was performed by enzyme-immunoassay technique (ELISA) using commercial kits protocols; 17,20[beta]-dihydroxy-4-pregnen-3-one (17,20[beta]P) (Cayman Chemicals Company, MI, USA) and 11-ketotestosterone (11-TK) and 17 [beta]-estradiol (E2) (Mybiosource, Beijing, China).
The results were analyzed using the programme SPSS Statistics 8.0 for Windows (SPSS Inc. Chicago, IL, USA). Normality and homoscedasticity were assessed using the Kolmogorov-Smirnov and Bartlett's test respectively. An analysis of variance (ANOVA) was performed to determine the existence of significant differences among sex hormones levels of each stage of gonadal maturation. Differences in mean values were determined by Tukey's test. The probability level for all statistical tests was set at 0.05.
Gonad morphology and histology Male
The testicles of M. australis are paired organs of similar size and are white. They are composed of several lobes with similar morphology, which are joined to form a U-shaped structure. These organs are located ventral to the swim bladder of the fish.
Histological analysis of the testicles reported the presence of individuals in all maturation states (Table 1). Five individuals were found in an immature stage, which showed absolute dominance of germ cells (Fig. 1a). Eight individuals were found in the stage of proliferation (spermatogenesis), which had a high presence of spermatogonia and spermatocytes and fewer spermatids (Fig. 1b). Ten individuals were found in the growth stage (spermiogenesis), which showed a dominance of spermatids and a considerable amount of sperm, spermatocytes and spermatogonia (Fig. 1c). Nine individuals were found in the stage of maturation (spermiation), which had the exclusive presence of free sperm in the testis lobular lumen (Fig. 1d). Finally, ten individuals were found in the stage of spawning, which showed the presence of a few free spermatozoa and occasionally spermatogonia in the testicles lumen.
The ovaries of M. australis are paired organs in the shape of elongated and bilobed sacs, which are located ventral to the swim bladder. The ovarian wall is transparent and thin, allowing oocytes in advanced stages of maturity to be visible to the naked eye. In the posterior region (caudal) is observed the fusion of the ovaries that extend into a short oviduct, which opens in the urogenital pore.
In the early developing stages, the ovaries showed a light orange colour which becomes more intense with advancing sexual maturity. Histological analysis of the ovaries reported the presence of individuals in all maturation stages except the immature stage (Table 1).
Fifteen individuals were found in the proliferation stage (primary growth), showing the presence of chromatin-nucleolar and perinuclear oocytes (Fig. 2a). Nine individuals were found in the growth stage (vitellogenesis), which had got oocytes in cortical alveoli and early vitellogenic stage. Similarly, the presence of oocytes in earlier stages of oogenesis (Fig. 2b) was also observed.
Eleven individuals were found in the maturation stage, which showed the presence of oocytes with nucleus migration and a noticeable size increase due to hydration (Fig. 2c).
Finally, five individuals were found in the spawning stage, which showed the presence of large amount of post-ovulatory follicles, atretic oocytes and also oocytes in earlier stages of oogenesis (Fig. 2d).
Plasma sex steroid hormonal profile 11-ketotestosterone
Plasma concentrations of 11-KT showed significant differences (P < 0.05) between the males found in the stage of proliferation and those found in the others maturity stages.
In the immature stage, the individuals reached an average plasma 11-KT concentration of 0.23 [+ or -] 0.03 ng [mL.sup.-1]. However, during the proliferation stage, the levels of this hormones in the individuals increased significantly (P < 0.05), reaching the highest levels at maturational with an average of 1.04 [+ or -] 0.45 ng [mL.sup.-1]. As maturation progressed, the concentrations of 11-KT present in individuals began to decrease, progressively reaching averages of 0.32 [+ or -] 0.24, 0.18 [+ or -] 0.07 and 0.15 [+ or -] 0.04 ng [mL.sup.-1] in the stage of growth, maturation and spawning respectively.
Plasma levels of 11-KT in each of stages of testicular maturity of male southern hake described by the histological analysis are shown in (Fig. 3).
Plasma concentrations of E2 in M. australis females exhibited significant differences among the different stages of gonadal development (P < 0.05). These differences were showed among individuals in the growth phase (vitellogenesis) and those in the late stages of development.
In the proliferation stage (primary growth), the females reached an average plasma E2 concentration of 0.29 [+ or -] 0.06 ng [mL.sup.-1]. Afterwards, the concentration of E2 increased significantly (P < 0.05) during the growth stage (vitellogenesis), achieving a mean maximum concentration of 0.62 [+ or -] 0.14 ng [mL.sup.-1].
Subsequent to vitellogenesis, the E2 levels decreased significantly (P < 0.05) as the gonadal development progresses. Females in maturation and spawning stages showed average concentrations of 0.32 [+ or -] 0.24 and 0.13 [+ or -] 0.03 ng [mL.sup.-1] respectively. Plasma levels of E2 related to each of stage of ovarian maturity, described by the histological analysis, are shown in (Fig. 4).
The levels of 17[alpha],20[beta]-DP in the plasma of M. australis males showed no significant differences (P > 0.06) among different stages of gonadal development. However, the concentration of this hormone reached the maximum average of 0.3 [+ or -] 0.1 ng [mL.sup.-1] during the maturation stage (espermiation).
The remaining gonadal development stages had lower levels of 17[alpha],20[beta]-DP, with average values of 0.07 [+ or -] 0.03, 0.11 [+ or -] 0.03, 0.09 [+ or -] 0.03 and 0.04 [+ or -] 0.01 ng [mL.sup.-1] in the immature, proliferation, growth and spawning stages respectively.
Similar to the results reported in males, levels of 17[alpha],20[beta]-DP in females plasma showed no significant differences (P > 0.06) among different stages of gonadal development averaging 17[alpha],20[beta]-DP plasma values of 0.07 [+ or -] 0.02, 0.06 [+ or -] 0.01, 0.06 [+ or -] 0.01, 0.06 [+ or -] 0.01 and 0.06 [+ or -] 0.02 ng [mL.sup.-1] in immature, proliferation, growth, maturation and spawning stages respectively. Plasma levels of 17[alpha],20[beta]-DP in each of stages of ovarian and testicular maturity of southern hake described by the histological analysis are shown in Figures 3 and 4.
Studies concerning the anatomy and physiology of the reproductive system are important in order to understand the biology of fish reproduction. This study represents the first attempt at a detailed histological identification of gamete developmental stages and characterization of plasma sex steroid hormones during the maturation cycle of southern hake (Merluccius australis).
Histological evaluation of the gonad development stages in male and female specimens of M. australis found cells that exhibit all stages of maturation, showing clearly the type of asynchronous ovarian and testicular development of this species.
Testicular histology showed spermatogonia, spermatocytes and spermatids in the lobular wall, whereas spermatozoa were observed free in the lumen. At the stage of spermiation, however, the individuals showed almost exclusively spermatozoa, probably due to the extensive spawning period.
According to the observations in this study, ovarian histology is very similar to that reported in studies with Merluccius hubbsi (Cornejo, 1998; Honji et al., 2006), and M. merluccius (Recasens et al., 2008), exhibiting the same characteristics in each oocyte stage.
Sex steroids concentrations in M. australis were at very low levels compared to studies on Chalcalburnus tarichi (Unal et al., 2005), Perca fluviatilis (Migaud et al., 2003), Coregonus clupeamorfis (Rinchard et al., 2001) and Pleuronectes americanus (Harmin et al., 1995). However, studies on fish with asynchronous gonadal development such as Gobio gudgeon and Verasper variegatus have also reported low steroid concentrations (Rinchard et al., 1993; Koya et al., 2003).
The low concentration of sex steroids present in M. australis could be attributed to the fact that this marine fish is a partial spawner and therefore the levels of circulating sex steroids are diluted as consequence of extensive spawning periods.
The androgen 11-ketostestosterone has been identified as the most important steroid hormone in teleost testes (Borg, 1994). In the present study, fluctuations of serological 11-KT levels were found during testicular maturation. The levels of 11-KT were higher in the stage of spermatogenesis compared to other maturational stages, demonstrating the importance of this hormone in the process of spermatogenesis. The observed levels of 11-KT in M. australis, are consistent with findings reported by studies with Hucho perryi (Amer et al., 2001); Clupeapallasii (Koya et al., 2002); Verasper variegatus (Koya et al., 2003) and Solea senegalensis (Garcia-Lopez et al., 2006), where 11-KT was the most influential androgen for the spermatogenesis stage.
In female teleosts, the level of the E2 has been reported to increase gradually during cortical alveoli phase, peaking in the vitellogenesis phase and then declining prior to the ovulation phase (Mayer et al., 1990; Schulz et al., 2010). Overall, these previous findings are consistent with the present results, where the levels of E2 were higher in the stage of vitellogenesis compared to other maturational stages, indicating that this sex hormone is essential to induce the process of vitellogenesis in female M. australis. Similar results were reported in Engraulis ringens (Cisneros, 2007), Sardinops melanostictus (Murayama et al., 1994), Salvelinus leucomaenis (Kagawa et al., 1981), Mugil cephalus (Tamaru et al., 1991), Acheilognathus rhombea (Shimizu et al., 1985), and Oreochromis mossambicus (Cornish, 1998), where oocytes development was mediated by increasing 17[beta]-estradiol during vitellogenesis stage.
The 17[alpha],20[beta]-DP has been identified as a maturation-inducing steroid (MIS) in several fish species during final oocyte maturation (Yamauchi et al., 1984; Tamaru et al., 1991; Petrino et al., 1993; Murayama et al., 1994), however, this hormone did not show noticeable fluctuation during any maturation stages in M. australis females. Similar findings regarding the consistency in the levels of 17[alpha],20[beta]-DP were reported in Engraulis ringens (Cisneros, 2007) and Dicentrarchus labrax (Prat et al., 1990). Shortrange variations of 17[alpha],20[beta]-DP levels during the process of oocyte final maturation and ovulation in M. australis females could be explained by a lack of blood samples at the precise moment of increase in this hormone. A variety of experiments have shown that the increase in this steroid occurs for a short period of time, just prior to ovulation when the germinal vesicle membrane breaks down (Tamaru et al., 1991; Murayama et al., 1994; Mylonas et al., 1997). Furthermore, in vitro studies carried out by Migaud et al. (2003) showed that 17[alpha],20[beta]-DP is detectable up to two hours after being synthesized, which could also explain the low concentration of this hormone in the analyzed samples. Another explanation for this phenomenon could be that 17[alpha],20[beta]-DP did not act as MIS in M. australis females. Studies carried out with Micropogonias undulatus (Trant & Thomas, 1989), Cynoscion nebulosus (Thomas & Trant, 1989) and Halobatrachus didactylus (Modesto & Canario, 2002) have shown that 17,20[beta],21-trihydroxy-4-pregnen-3-one (17,20[beta],21P) act as MIS instead of 17[alpha],20[beta]-DP. However, the role of 17,20[beta],21P as MIS in M. australis females is still unclear and further investigations are needed.
In southern hake males, levels of 17[alpha],20[beta]-DP showed a slight fluctuation during gonadal development. Conversely, studies with other fish species have shown an increase in the levels of this steroid during the espermiation stage (Vermeirssen et al., 1998, 2000; Koya et al., 2002). The difference between our findings and those reported in the literature could be due to the low number of individuals sampled, the continuous process of spermatogenesis, or the short duration of 17[alpha],20[beta]-DP in the bloodstream.
In conclusion, this study reported that there were serological fluctuations of E2 and 11-KT during gonadal maturation of M. australis, identifying these hormones as the main hormones responsible for vitelogenesis and spermatogenesis respectively. On the other hand, the levels of 17[alpha],20[beta]P-DP did not show fluctuations so, apparently, this hormone is no involved in gonadal maturation of this species. Future research should include the entire maturation cycle of wild and captive M. australis, in order to evaluate the physiological effect of captivity conditions on broodstock of this species. Similarly, additional studies regarding the quantification of gonadotropins (FSH and LH), 17,20[beta],21P and vitellogenin are required for a complete understanding of the reproductive physiology of M. australis.
The authors would like to thank Dr. Karl D. Shearer and Dr. Ivan Valdebenito for their critical review of this manuscript. This research was supported by funding from Chilean National Commission for Scientific and Technological Research (CONICYT) in the frame of the project FONDEF DA09I 1001.
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Received: 12 February 2014; Accepted: 10 March 2015
Manuel Alvarado (1), Edison Serrano (1), Juan Carlos Sanchez (1) & Luis Valladares (2)
(1) Estacion Experimental Quillaipe, Unidad de Gestion Tecnologica, Area de Alimentos y Biotecnologia Fundacion Chile, P.O. Box 27-D, Puerto Montt, Chile
(2) Instituto de Nutricion y Tecnologia de los Alimentos, Universidad de Chile P.O. Box 138-11, Santiago, Chile
Corresponding author: Edison Serrano (firstname.lastname@example.org)
Corresponding editor: Guido Plaza
Caption : Figure 1. Cross sections of M. australis testes showing different maturity stages. a) Testis in immature stage (10x), b) testis in proliferation stage (40x), b) testis in the growth stage (40x), d) testis in the maturation stage (40x). The blue arrows indicate cells in spermatogenesis.
Caption: Figure 2. Cross sections of M. australis ovaries showing different maturity stages. a) Ovary in proliferation stage (4x), b) ovary in growth stage (4x), b) ovary in the maturation stage (4x), d) ovary in the spawning stage (4x). The blue arrows indicate oocyte cells in oogenesis.
Caption: Figure 3. Plasma steroidal profiles of M. australis males during maturation cycle (Mean [+ or -] Standard Error) (P < 0.05).
Caption: Figure 4. Plasma steroidal profiles of M. australis females during maturation cycle (Mean [+ or -] Standard Error) (P < 0.05).
Table 1. Histological classification of gonadal maturity stage of wild southern hake (M. australis). Gonadal maturity Males Females stage (n = 36) (n = 40) Immature 5 0 Proliferation 8 15 Growth 10 9 Maturation 9 11 Spawning 4 5
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|Title Annotation:||Research Article|
|Author:||Alvarado, Manuel; Serrano, Edison; Sanchez, Juan Carlos; Valladares, Luis|
|Publication:||Latin American Journal of Aquatic Research|
|Date:||Sep 1, 2015|
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