Influence of Water Temperature on Induced Reproduction By

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General and Comparative Endocrinology 172 (2011) 400–408

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General and Comparative Endocrinology

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Inﬂuence of water temperature on induced reproduction by hypophysation, sex steroids concentrations and ﬁnal oocyte maturation of the ‘‘curimatã-pacu’’ Prochilodus argenteus (Pisces: Prochilodontidae) ⇑ Fábio P. Arantes a,c, Hélio B. Santos a, Elizete Rizzo a, Yoshimi Sato b, Nilo Bazzoli c,

a Departamento de Morfologia Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, P.O. Box 1686, CEP 30161-970 Belo Horizonte, Minas Gerais, Brazil b Estação de Hidrobiologia e Piscicultura de Três Marias, CODEVASF, P.O. Box 11, CEP 39205-000 Três Marias, Minas Gerais, Brazil c Programa de Pós-Graduação em Zoologia de Vertebrados, Pontifícia Universidade Católica de Minas Gerais, Av. Dom José Gaspar 500, CEP 30535-610 Belo Horizonte, Minas Gerais, Brazil

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Article history: Most fishes with commercial importance from the São Francisco basin are migratory and do not complete Received 25 October 2010 the reproductive cycle in lentic environments, such as hydroelectric plant reservoirs, hence natural stocks Revised 18 March 2011 are declining and there is an urgent need to reduce the pressure of fishing on those wild populations. Accepted 4 April 2011 Therefore, studies on reproductive biology and its relationship with endocrine and environmental factors Available online 8 April 2011 are key to improving the cultivation techniques of Brazilian fish species. This study examined the influence of water temperature on sex steroid concentrations (testosterone, 17b-estradiol and 17a-hydroxy- Keywords: progesterone), spawning efficiency, fecundity, fertilisation rate, larval abnormality rates and involvement Freshwater fish of the cytoskeleton during the final oocyte maturation of Prochilodus argenteus under experimental con- Artificial reproduction Testosterone ditions. The results of our study showed that in captivity, sex steroid plasma concentrations and spawn- 17b-Estradiol ing performance of P. argenteus were clearly different for fish kept in water with different temperature 17a-Hydroxyprogesterone regimes. In lower water temperature (23 °C), it was observed that: 33% of females did not ovulate, fecundity was lower and vitellogenic oocytes after the spawning induction procedure exhibited a smaller diameter. Moreover, concentrations of 17b-estradiol and 17a-hydroxyprogesterone were lower and there was a delay in the final oocyte maturation and, consequently, ovulation and spawning. Our experiments showed direct influence of water temperature in the process of induced spawning of P. argenteus. Changes in water temperature also suggest the tubulin involvement in the nuclear dislocation process and the possible action of actin filaments in the release of polar bodies during final oocyte maturation of P. argenteus. Ó 2011 Elsevier Inc. Open access under the Elsevier OA license.

1. Introduction inducing the resumption of meiosis in teleost oocytes has also been reported [31]. The fish pituitary produces two types of gonadotropin (GTH), Fish final oocyte maturation completes the process of oogenesis follicle stimulating hormone (FSH) and luteinizing hormone (LH) and results in the fertilisable female gamete or egg [14,16,31]. Final [31]. The temporal pattern of secretion in several fishes suggests oocyte maturation is a remarkable transformation of the prophase I that FSH has a dominant role regulating vitellogenic growth of fol- oocyte; a cell specialised for transcription, material uptake, nonex- licles, in part through stimulation of 17b-estradiol (E2) synthesis citable, and unable to osmoregulate in fresh water, to the meta- by ovarian follicles, and E2 regulates ovarian development through phase II egg, a cell different from the oocyte in many ways. In its control of vitellogenin synthesis. LH induced cascade of matura- many teleosts, oocyte maturation is triggered by 17a,20b-DP act- tion-inducing hormone (MIH) synthesis followed by maturation or ing on a steroid–receptor in the cytoplasmic membrane [17,53,61]. metaphase-promoting factor (MPF) production is well studied in This unconventional steroid–receptor interaction leads to the var- fish [30,47]. The 17a,20b-dihydroxy-4-pregnen-3-one (17a,20b- ious non-genomic cellular changes that are the markers for oocyte DP) which has 17a-hydroxyprogesteone as a precursor, has been maturation [31]. identified as MIH in several fish and the action of this MIH in The fully-grown zebrafish oocyte residing in the ovary is cov- ered by three cell layers, the ovarian epithelium, the theca and ⇑ Corresponding author. Fax: +55 3133194269. the follicle cell layer [24,48]. The oocyte and follicle cells have E-mail address: [email protected] (N. Bazzoli). numerous gap junctions in common and, thus, are in chemical

0016-6480 Ó 2011 Elsevier Inc. Open access under the Elsevier OA license. doi:10.1016/j.ygcen.2011.04.007 F.P. Arantes et al. / General and Comparative Endocrinology 172 (2011) 400–408 401 and electrical communication [20]. The oocyte has a high degree of sex steroid concentrations, spawning, fecundity and the involve- endocytic activity and uses receptor-mediated endocytosis to take ment of the cytoskeleton during induced spawning by hypophysa- up vitellogenin transported from the liver. The fully-grown oocyte tion in females of P. argenteus utilising two regimes of water is in prophase I of meiosis with a central, large nucleus or germinal temperature: 23 and 26 °C. As P. argenteus females do not complete vesicle containing many nucleoli and a very high transcriptional their reproductive cycle in the stretch of the river in the São Fran- activity [23]. cisco near the Três Marias hydroelectric dam [3], and the water Fecundity is an important variable for aquaculture, species temperature in this environment has mean values near 23 °C dur- management and conservation [54,57], and it estimates the repro- ing the reproductive period of this species, in our study, the fishes ductive potential of a fish species [4]. Fecundity depends on body of first experimental group (group A) was kept in the tank with size, oocyte diameter, spawning type and may vary as a result of water temperature of 23 °C. According Sato et al. [42] in a water different adaptations to environmental habitats and it provides temperature of 26 °C, P. argenteus showed good results for induced important information for aquaculture, fisheries management, spawning procedures, thus, in our work the second group (group B) and conservation [52,56,58]. was kept in water of 26 °C. The males were kept in a tank with During the final oocyte maturation of the fish, the cytoskeleton water of 26 °C. participates in the ooplasm segregation [19]. Variations in water temperature can cause conformational changes as well as in the dynamics of the cytoskeleton [35,55], as aggregation of microtu- 2. Materials and methods bules occurs when the temperature is high and its breakdown occurs when the temperature decreases [35], thus, interfering in the 2.1. Sampling migration of the nucleus during final oocyte maturation [22]. Actin microfilaments, especially those present in the cortical region, also In order to perform the experiment, 150 specimens of P. argen- participate in the major events of final oocyte maturation in fish, teus were captured in the São Francisco River and kept for 2 years such as cytoplasmic polarisation, formation of the first polar body, in a 200 m3 tank (20 Â 10 Â 1 m) in the Hydrobiology and Fishcul- fertilisation and embryo morphogenesis [5,21,27]. During fertilisa- ture Station of Três Marias, CODEVASF. The fish were feed with pel- tion, the cortical actin participates in the exocytosis of cortical leted feed containing 36% of crude protein, at a level equivalent to granules, formation of the fertilisation cone, assisting the migra- 2% of their biomass per day during the storage period. The experi- tion of the pronucleus and the release of the second polar body [5]. ments were performed in duplicate, first in December 2005 and Efforts to control the reproduction of neotropical migratory fish again in December 2006. In each experiment was used 30 females began many years ago, but its success has been limited by intrinsic and one male, totalling 62 specimens of P. argenteus. factors such as obtaining good breeders, and by extrinsic factors such as temperature, dissolved oxygen and other water properties which have prevented producers from achieving satisfactory re- 2.2. Induced spawning sults of artificial reproduction. The water temperature has great relevance in the reproductive process of many fish species, acting For the experiments, only fish in advanced gonadal maturation on gamete maturation, ovulation and spawning [2]. In culture con- stages were used. Females were selected by external morphologi- ditions, neotropical migratory fish, when kept at temperatures be- cal characteristics indicating that they were ready for spawning low 25 °C, did not respond satisfactory to artificial reproduction induction procedures, such as red urogenital papilla and bulging procedures [40]. coelomic cavity. Males that released sperm with light pressure Prochilodus argenteus, popularly known as curimatã-pacu, is an on the coelomic cavity were selected for the fertilisation endemic species in the São Francisco basin and is the most abun- procedures. dant migratory species in the Três Marias region, representing al- For each repetition, the specimens were divided into two most 50% of the total catch. It has an illiophagous feeding habit groups of 15 females, which were transferred to 2.4 m3 breeding and is the largest member of the Prochilodontidae family, and is tanks (3 Â 1 Â 0.8 m) with constant water circulation and the fol- intensively used in hatcheries for restoring fishery stocks [42,43]. lowing characteristics: a dissolved oxygen concentration ranging P. argenteus performs long-distance migrations upstream for from 5.5 to 6.5 mg LÀ1, pH from 6.3 to 7.5 and an electrical conduc- spawning, and has high fecundity. It exhibits total spawning and tivity of 58 to 87 lScmÀ1. The fish in group A were kept in a breed- its reproductive period extends from November to January in the ing tank with a water temperature of 23 °C, whereas fish in group B rainy season, coinciding with the time of flooding, higher temper- were kept in a tank with water of 26 °C. atures, and long photoperiods. In captivity, the curimatã-pacu pre- The fishes of groups A and B were submitted to induced spawn- pares to reproduce and completing vitellogenesis, however, ing by hypophysation according methodology established by [44]. ovulation and spawning only occur after hormonal induction Crude common carp pituitary extract (CCPE) was injected into the [40,42]. coelomic cavity. Males received a single dose of 5 mg CCPE/kg of The application of hormonal therapies to induce spawning can body weight and females received two doses: a first dose of 1 mg be based on the administration of the gonadotropin-releasing hor- of CCPE/kg and a second one of 5 mg of CCPE/kg, with an interval mone (GnRH), [9,29], by treatment with Ovaprim, a preparation of 14 h in-between doses. Fertilisation was carried out by the dry containing a synthetic GnRH analogue with domperidone [51] or method, using semen from a single male for each year of the using the heteroplastic hypophysation method where commercial experiment. crude carp pituitary extract (CCPE) is injected into the coelomic After the induced spawning procedures, the total length (TL), cavity or intramuscularly [38,45,46,47,59]. However, in commer- body weight (BW) and gonad weight (GW), ie weight of the cial aquaculture of Brazilian freshwater fish, spawning induction spawned oocytes (the weight of free oocytes released plus the free is usually performed with hypophysation, since this methodology, oocytes retained in the ovaries was considered), were taken. We besides being economically beneficial, has high efficiency and pro- calculated the gonadosomatic index (GSI = GW Â 100/BW) and Ful- duces eggs with high rates of fertilisation [44]. ton condition factor (K =BWÂ 100/TL3). The fish were killed by Considering the scarcity of studies on the influence of water transversal section of the cervical medulla following the ethical temperature on final oocyte maturation and spawning of neotrop- principles established by the Brazilian College of Animal Experi- ical migratory teleosts, the objective of our study was to evaluate mentation (COBEA, http://www.cobea.org.br). 402 F.P. Arantes et al. / General and Comparative Endocrinology 172 (2011) 400–408

2.3. Hormonal concentrations dilution (MOL. PROBES) for actin, and mouse anti a-tubulin primary antibody at 1:100 dilution (MOL. PROBES) and goat’s second- For the experimental groups A and B, blood samples were ob- ary antibody Alexa FluorÒ 568 at a 1:150 dilution (MOL. PROBES) tained by tail vein puncture at the following times: 0 h (collected for the tubulin marking. For positive control, we used sections of before the first dose of CCPE and values taken as reference), 14, mice intestine, and for negative control, some sections did not re- 23, 25 and 26 h after the first dose of CCPE. Then the samples were ceive the primary antibodies during treatment. The analysis of the centrifuged for 15 min at 800 rpm. Serum aliquots were kept under slides used for the fluorescence immunohistochemistry procedures refrigeration (À20 °C) until concentrations of sexual steroids were was performed using a confocal microscope and a conventional determined. Testosterone concentrations were determined by fluorescence microscope. chemiluminescence with the Bayer Corporation’s Testosterone ADVIA Centaur test. In order to determine 17 b-estradiol concen- 2.6. Fecundity trations, immunofluorometry with Perkin Elmer’s Estradiol Auto- Ò DELFIA test was used. For 17a-hydroxyprogesterone solid-phase For the purpose of estimating the fecundity in each experimen- radioimmunoassay was used, via the Coat-a-Count 17a-OH pro- tal group, samples of spawned oocytes were collected and gesterone – DPC kit. weighed. Absolute fecundity (AF) was determined by the formula: AF = NOG Â OW, where NOG = number of oocytes per gram of the 2.4. Histology and oocyte diameter sample and OW = total weight of spawned oocytes. Relative fecundity (RF) was also calculated in order to eliminate the interference In order to microscopically monitor the oocyte’s final matura- of fish size on fecundity, and was estimated by the formula: tion events in the two temperature regimes, fragments of the ova- RF = AF/BW, where AF = absolute fecundity, BW = body weight. ries were collected at the same times mentioned above (0 h [reference group], 14, 23, 25 and 26 h after the first doses of CCPE). 2.7. Fertilisation rates and larval abnormalities The fragments were fixed in Bouin’s fluid (24 h) and submitted to routine histological techniques: embedded in glycol methacrylate, After fertilisation, incubation of the eggs of both groups was sectioned with a 5 lm thickness and stained with toluidine blue- performed in funnel-shaped incubators (20 L), and with continually sodium borate. circulating water circulation at 24 °C. The water flow ranged from The vitellogenic oocytes’ diameters (n = 6 females/time/group) 0.5 to 1 L minÀ1 and each incubator received 50 g of eggs. To examine were determined from histological sections of the ovaries before the rates of fertilisation and larval abnormalities, samples of eggs (8– the first hormonal injection, just before spawning and post- 12 h after fertilisation) and newly-hatched larvae were fixed in 4% spawning for each experimental group (A and B). A micrometer neutral formalin. At least 100 eggs and 50 larvae of each sample were attached to the eyepiece of a light microscope was used to take used to estimate the fertilisation rates and larval abnormalities, the measurements. respectively. Analyses were performed using a stereomicroscope.

2.5. Immunohistochemistry 2.8. Statistical analysis During the final oocyte maturation, samples of ovaries were For statistical tests, GraphPad InStat and Prism software were fixed in methanol and dimethyl sulfoxide (DMSO) in a 8:2 ratio used. In order to compare the averages of total length, body weight, for 5 days at À20 °C. Samples were also fixed in 4% paraformalde- oocyte diameter, fecundity between the experimental groups, data hyde in 0.1 M pH 7.3 phosphate buffer for 8–12 h at 4 °C. The sam- normality was tested and Student T test with or without Welch’s ples obtained before the first hormone dose, before the second correction [60] was used. In order to compare hormone concentra- hormone dose, prior to spawning and recently spawned oocytes tions amongst the groups and between times, the Tukey-Kramer were embedded in Paraplast (HistosecÒ MERCK). multiple comparison test was used. Differences were considered The immunoperoxidase technique was used with anti actin significant at a = 0.05 (p < 0.05). monoclonal/mouse IgG (C-2 clone, Santa Cruz) and anti a-tubulin polyclonal/rabbit IgG (clone H-300, Santa Cruz) both at a 1:100 dilution. The visualization was achieved using the LSAB 2 System 3. Results HRP kit by Dako Cytomation (K 0675) containing the goat anti- rabbit or mouse IgG secondary antibody conjugated with biotin 3.1. Spawning and fecundity (1:200) and streptavidin conjugated with peroxidase. The histological sections were deparaffinised, hydrated and kept in PBS, then For the experiments conducted in 2005 and 2006 and for both submitted to blocking endogenous peroxidase with 3% H2O2 in groups of fish the moment of oocyte release was signalled by circu- PBS for 30 min at room temperature. lar movements of the female. For the specimens kept in water at The sections were submitted to the blocking of unspecific bonds 23 °C (group A), spawning was delayed by 2 h compared to those with bovine albumin 2% in PBS buffer for 30 min at room temper- kept at 26 °C (group B). All females spawned in group B, while ature, incubated with primary antibodies in an overnight moist 33% of group A did not ovulate and, therefore, did not spawn. chamber at 4 °C, then with secondary antibody conjugated to bio- The absolute and relative fecundity showed higher levels in group tin for 45 min and finally with streptavidin peroxidase conjugate in B fishes compared to those in group A (Table 1). The females of a moist chamber for 45 min. The peroxidase reaction was revealed group A only started the spawning 2 h after the fish of group B. with DAB in PBS for 8 min at room temperature. The sections were Among the fish that spawned in group A, it was observed that counterstained with hematoxylin. Sections of mice intestine were 25.7% of their vitellogenic oocytes have not started or completed used for positive control, and, for negative control, some sections the process of final oocyte maturation. did not receive the primary antibodies during treatment. The analysis of the peroxidase stained slides was performed under a light 3.2. Histology and oocyte diameter microscope. Actin and tubulin detection was also performed by immunoflu- The morphological events of final oocyte maturation (nuclear orescence using the Alexa FluorÒ 488 Phalloidin kit at a 1:150 dislocation, disruption of the nuclear envelope and finally the F.P. Arantes et al. / General and Comparative Endocrinology 172 (2011) 400–408 403

Table 1 Total length (TL), body weight (BW), absolute fecundity (AF), relative fecundity (RF), fertilisation rate (F), rate of abnormal larvae (AL) and diameter of vitellogenic oocytes immediately prior to spawning (OD) in ﬁsh subjected to induced spawning by hypophysation and kept in two temperature regimes.

Groups n A (23 °C) B (26 °C) Mean ± DP Range Mean ± DP Range TL (cm) 60 30.1 ± 4.0a 23.0–37.0 29.2 ± 4.0a 22.5–37.0 BW (g) 60 351.8 ± 136.7a 140.0–650.0 329.0 ± 141.1a 140.0–650.0 AF 10 32733.0 ± 17743.2a 19285.0–58870.0 44051.0 ± 38.547.3b 14210–10454.5 RF 10 1163.9 ± 580.2a 803.8–2030.0 1457.0 ± 1085.9b 568.4–3168.0 F (%) 10 88.4 ± 13.4a 68.4–96.8 83.08 ± 17.85a 52.1–98.9 AL (%) 10 9.3 ± 9.0a 0.2–17.0 9.5 ± 10.8a 0.2–24.3 OD (lm) 10 887.8 ± 49.3 714.1–965.0 966.61 ± 43.68 887.8–1080.8

Different letters (a,b) means statistical differences between the temperatures. Data expressed in means ± standard deviation. ovulation into the ovarian cavity) were similar in both groups, migration and germinal vesicle breakdown varied from 25.5 h for however, the process was slower in group A. Amongst the females group A to 23.5 h for group B. of the group A which spawned (67%), were observed numerous oo- The oocyte diameter showed increasing values for fish from the cytes who have not started or not completed the process of final reference group (835.41 lm ± 32.74), group A (887.80 lm ± 49.35), oocyte maturation and therefore not ovulated. group B (966.61 lm ± 43.68) and spawned oocytes of group B Before the first CCPE dose, the vitellogenic oocytes presented a (967.97 lm ± 48.42), respectively, and differences were highly sig- central nucleus (Fig. 1A). The main events of the final oocyte mat- nificant, except between group B and spawned oocytes, showing uration began with the first dose of CCPE, with dislocation of the an increase in egg diameter after hormonal induction and a larger nucleus toward the micropylar region in the animal pole diameter for fish kept in water at 26 °C. (Fig. 1B). After the full migration of the nucleus (Fig. 1C and D), there was disruption of the nuclear envelope and finally the 3.3. Hormonal concentration ovulation into the ovarian cavity occurred (Fig. 1E). At this stage, chromosomes in metaphase II, close to the micropylar region, were Plasma concentrations of testosterone increased after the first observed (Fig. 1F). The period of time for germinal vesicle CCPE dose, especially in group A and a quick decreased after the

Fig. 1. Vitellogenic oocytes histological sections of P. argenteus submitted to induced spawning by hypophysation. Oocyte with central nucleus (A), oocyte with nucleus moving toward the micropylar region (B), complete nuclear migration (C and D) nuclear envelope breakdown and ovulation (E); details of chromosomes near the micropylar region in metaphase II (F). OV, vitellogenic oocytes; N, nucleus; Arrow head, micropyle; Asterisk, nuclear material; Arrow, chromosomes in metaphase II. Bars: 100 lm (A–C and E) and 50 lm (D–F). 404 F.P. Arantes et al. / General and Comparative Endocrinology 172 (2011) 400–408

3.4. Immunohistochemistry

Results obtained by immunoperoxidase and immunofluorescence showed actin and tubulin reactivity in the ooplasm and follicular cells of vitellogenic oocytes. During the final oocyte maturation, a continuous collar of actin in the cortical region (Fig. 3A–D) was observed, which became thicker in the animal pole (Fig. 3C). Distribution of tubulin in the entire cortical region (Fig. 4A–D), with intensification in the vegetative (Fig. 4A) and animal (Fig. 4B and D) poles was also observed. Intense reactivity was observed for tubulin surrounding the migrating nucleus (Fig. 4C). Spot markings for tubulin (Fig. 4D) and for DNA (Fig. 4E) were observed in the meiotic spindle parked in metaphase II, near the micropylar region of ovulated oocytes.

3.5. Fertilisation rates and larval abnormalities

High fertilisation rates and low rates of larvae abnormalities were observed for ﬁsh of both experimental groups (Table 1). There were no statistical differences in the fertilisation and larval abnormalities rates between ﬁsh from groups A and B. The most frequent larval abnormality was spine malformation.

4. Discussion

Since the majority of the Brazilian neotropical teleosts with commercial importance are migratory and do not complete their reproductive cycle in captivity or in lentic environments, experimental studies using migratory fish in captivity have been conducted in an attempt to complete their life cycle [1,7,33,39,41,42, 45–47,49]. In artificial reproduction procedures, the control of spawning is one of the most important stages for successful artificial reproduction of fish [44,62], and water parameters such as temperature, dissolved oxygen and pH can limit the success of final oocyte maturation, ovulation and thus spawning. However, studies on the influence of water parameters on the concentrations of sex steroids, oocyte morphology and actin and tubulin involvement in the final oocyte maturation events during artificial reproduction procedures of neotropical fish are scarce. In our study, the period of gonadal maturation of males and females of P. argenteus kept in captivity coincided with the reproductive period of this species in their natural environment, which occurs from November to January, that is, during the hot and rainy season [3]. This has also been recorded for other migratory species, such as Brycon orthotaenia [15], Pseudoplatystoma corruscans [18], Fig. 2. Profiles of sex steroids in P. argenteus female subjected to hypophysation- induced spawning. 0 h = reference values; 14, 23, 25 and 26 h after first dose of Leporinus elongatus [47] and Prochilodus affinis [41]. CCPE. Values are expressed as means ± SD. Different capital letters indicate In this study, fish from both experimental groups showed signs statistical differences between the two experimental groups at the same time. of the spawning moment, usually through repeated circular move- Lowercase letters indicate statistical differences between subsequent times within ments, which has also been observed by Sato et al. [42]. In proce- each experimental group (p < 0.05). dures of induced spawning by hypophysation, changes in fish behaviour are commonly used as indicators for the moment of spawning [10,42,47]. second dose. The variations in the 17 b-estradiol concentrations In our study, by manipulating water temperature alone, it was were not very prominent during the experiment, an increase observed that the spawning performance of P. argenteus was signif- after the first dose followed by a gradual reduction until the icantly different for the two temperature regimes, such that post-spawning was observed in group B, but there was no spawning of the fish kept at 23 °C (group A) was delayed by 2 h increase of 17 b-estradiol after the first dose in group A fish. in relation to those kept at 26 °C (group B), showing that low tem- The concentrations of 17a-hydroxyprogesterone (17a-P) showed peratures may negatively interfere with the spawning process of P. a rapid increase at the moment of spawning, and quick decrease argenteus. Similar interference was also reported for other species, in post-spawning for both experimental groups. Although both such as carp [17], Rhamdia sapo [11], Leporinus elongatus [47], and groups exhibited a similar tendency, group A showed lower con- Anguilla japonica [10]. Besides the delay in spawning, it was also centrations of 17a-P and peak levels with a 2-h delay as com- noted that 33% of fish in group A did not ovulate and consequently pared to fish from group B, what probably caused the delay in did not spawn, whereas in group B, spawning occurred for all fe- spawning (Fig. 2). males. According Dou and colleague [10] fish usually ovulate and F.P. Arantes et al. / General and Comparative Endocrinology 172 (2011) 400–408 405

Fig. 3. Immunohistochemistry for actin detection during final oocyte maturation in P. argenteus subjected to induced spawning. Actin detected by immunoperoxidase (A, B, C – brownish) and immunofluorescence (D – green). Arrows, yolk globules, M, micropylar region; N, nucleus; OV, vitellogenic oocyte, ZP, zona pellucida. Sections with immunoperoxidase were counter-stained with hematoxylin. Bars: 400 lm (A), 20 lm (B–D). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

Fig. 4. Immunohistochemistry for tubulin detection during final oocyte maturation in P. argenteus subjected to induced spawning. Tubulin detected by immunoperoxidase (A and B) and immunofluorescence (C and D – red). Chromosomes in metaphase and follicle cells’ nuclei, labelled with DAPI (E – blue) Arrows, yolk globules; Stars, follicular cells; M, micropylar region; N, nucleus; ZP, zona pellucida; arrowhead, Chromosomes in metaphase II. Sections with immunoperoxidase were counter-stained with hematoxylin. Bars: 20 lm (A, B, D, E); 110 lm (C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.) spawn within a limited range of water temperatures, and our undergoing induced spawning by hypophysation in artificial results indicate that a 26 °C water temperature is more suitable reproduction experiments. Dou et al. [10] also noted lower ovula- for triggering ovulation and spawning in P. argenteus when tion and spawning rates in A. japonica kept in low-temperature 406 F.P. Arantes et al. / General and Comparative Endocrinology 172 (2011) 400–408 waters, as well as delayed spawning. In natural environments, Sato at 26 °C was probably responsible for the larger oocyte diameter of et al. [40] and Arantes et al. [3] also observed the negative influ- the females from this group. ence of low water temperature on the reproductive activity of Investigations involving spawning induction made it possible to migratory fish. Despite the fact that, in this study, ovulation and control the reproductive process of fish in captivity, contributing to spawning performance of P. argenteus females kept in water at the expansion of aquaculture [25]. The use of crude carp pituitary 23 °C were impaired, the mechanism by which temperature alters extract (CCPE) yields positive results for induced ovulation and the performance of spawning of this species is not yet known. presents good fertilisation rates in tropical fish, as observed in 25 In our study, the concentrations of 17a-hydroxyprogesterone species from the São Francisco River [44]. Although this work (17a-P) increased sharply after the application of crude carp pitu- shows the efficiency of injecting CCPE in the artificial reproduction itary extract (CCPE) and decreased abruptly in post-spawning for of P. argenteus, giving fertilisation rates above 80% in both experi- both temperature regimes. Nevertheless, fish kept in water at mental groups, fish maintained at 26 °C showed better rates of 23 °C showed lower 17a-P concentrations and this hormone’s peak spawning and fecundity. happened with a 2-h delay in relation to fish kept in water at 26 °C, We observe that prior to the first dose of CCPE, P. argenteus fe- which probably caused the late spawning and lower spawning per- males had vitellogenic oocytes with a central nucleus. After the centage. Thus, 23 °C was not the ideal water temperature in which first dose of CCPE, dislocation was observed of the nucleus toward CCPE and 17a-P could efficiently work on final oocyte maturation, the micropylar region, followed by disruption of the nuclear enve- ovulation and spawning of mature P. argenteus females. Delay in lope, resumption of meiosis until metaphase II, and culminating in hormonal action and spawning in fish kept in lower temperatures ovulation into the ovarian cavity and spawning. In most teleosts, was also recorded by Dou et al. [10]. The gonadotropins FSH and LH nuclear and cytoplasmic events in vitellogenic oocytes are known require at least two steroidal mediators, 17b-estradiol (E2) and as final oocyte maturation (FOM) [12,32]. Disruption of the nuclear 17a,20b-dihydroxy-4-pregnen-3-one (17a,20b-DP) to perform envelope after its migration to the oocyte cortex has also been ob- oocyte growth and maturation, respectively. The theca cells pro- served in zebrafish [23]. However, Prodon et al. [37] observed that vide the precursor steroids, and the granulosa cells produce the in ascidia the nuclear envelope disruption occurs before the migra- two steroidal mediators under the direct influence of FSH and LH tion of the nuclear structures. [50]. Moreover, in many teleosts, temperature has effects on hor- In this study, the FOM process and ovulation lasted for 23 h in mone function, interfering in final oocyte maturation and ovula- group B fish (26 °C) and 25 h in group A (23 °C). According to Naga- tion [10]. Although measurements of 17a,20b-DP (MIH) have hama et al. [32], final oocyte maturation in teleosts occurs rapidly, not been made in this study due to technical limitations, measure- being regulated by LH and by the maturation-inducing hormone. ments of its precursor, 17a-P, were carried out, and together with The delay in FOM and ovulation events observed in this study, the results of spawning performance, histology and ICC obtained in was probably caused by the late peak (2 h) of 17a-P, which for this study suggests the influence of water temperature on the pro- most teleosts is the precursor of the maturation-inducing hor- cess of final oocyte maturation and ovolation of P. argenteus sub- mone, the 17a,20b-dihydroxy-4-pregnen-3-one [31]. jected to induced spawning hypophysation. In our study, we observed intense reactivity to tubulin in three The concentrations of 17b-estradiol seems not to had direct regions: around the nucleus in migration; in the oocyte’s periph- involvement in the mains events of final oocyte maturation, since ery, especially in the animal pole, and in the ooplasm in the form from middle of the process, concentrations of this steroid showed of bundles between the nucleus and the cortex. This positioning decreasing values, including the spawning moment. There was a of tubulin during nuclear migration in the final oocyte maturation significant increase of testosterone concentrations detected at in P. argenteus, suggests involvement of microtubules in this pro- 14 h time for fish kept in colder water. However, this steroid’s con- cess, as observed in Holothuroideas [28] and in bivalves [36]. After centrations for fish kept in both temperature regimes, at the completion of the migration and disruption of the nuclear enve- spawning moment, showed low levels. This suggests that in P. lope, the reactivity to tubulin decreased in the ooplasm and was argenteus, testosterone probably does not act directly upon final observed near the micropyle, where the meiotic spindle was lo- oocyte maturation. According [31] testosterone induces final oo- cated. The actin collar in the peripheral ooplasm, thicker in the ani- cyte maturation only experimentally and in high concentrations. mal pole, which was detected in vitellogenic oocytes of P. Direct effects of water temperature on the quantity and quality argenteus, is similar to the arrangement observed in other animal of gametes have been reported for fish in captivity and in natural groups [28,36], and may be involved in the release of polar bodies. stocks [6,8]. A recent study of wild P. argenteus in two stretches The exact mechanism of nuclear migration toward the cortex in of the São Francisco River also registered lower fecundity rates in oocytes of teleosts is not completely known, but it involves compo- fish living in lower water-temperature environments [3]. Differ- nents of the cytoskeleton [23]. Studies using Holothuroidea’s oo- ences in fecundity rates may also be related to the age of breeders, cytes [28] showed that in this animal, only the microtubules stocking density and the feeding of the breeders [13,26]. However, seem to be involved in nuclear migration, whereas in ascidian oo- in the present study, these factors were probably not relevant, cytes the actin filaments seems to be responsible for the migration since fish used in the experiment were of the same age, were kept of the meiotic spindle toward the oocyte cortex, besides this, the under the same stocking density and were given the same amount nuclear envelope breaks down in the centre of the oocyte [37]. of food. In summary, this study shows the direct influence of water tem- In this study, the diameters of vitellogenic oocytes of P. argen- perature on plasma concentrations of sex steroids and spawning teus females kept in captivity for 2 years and which did not receive performance of P. argenteus submitted to induced spawning. With CCPE injections were similar to oocyte diameters reported for wild the lower water temperature (23 °C) it was observed that 33% of fish of the same species [3], indicating that confinement does not females did not ovulate, fecundity was lower and the vitellogenic prevent the process of vitellogenesis in migratory fish such as cur- oocytes after the spawning induction procedure showed a smaller imatãs. The same was reported for murray cod, Maccullochella peelii diameter. Moreover, concentrations of 17b-estradiol and 17a- peelii, a freshwater Perciformes [34]. We observed in this study, hydroxyprogesterone were lower and there was a delay in the final however, that at the time of spawning induction, the number of oocyte maturation and consequently on ovulation and spawning, vitellogenic oocytes in fish kept in water at 23 °C was significantly showing the direct influence of water temperature in the spawning lower. Since 17b-estradiol is responsible for vitellogenesis, the ele- process of P. argenteus. Our experiments also suggest the involve- vation of this steroid detected after the first CCPE dose in fish kept ment of tubulin in the nuclear dislocation process and possible F.P. Arantes et al. / General and Comparative Endocrinology 172 (2011) 400–408 407 action of actin filaments in the release of polar bodies during final [29] C.C. Mylonas, Y. Zohar, Use of GnRHa-delivery systems for the control of oocyte maturation of P. argenteus. Our next investigation will focus reproduction in fish, Rev. Fish Biol. Fish. 10 (2001) 463–491. [30] Y. Nagahama, Endocrine regulation of gametogenesis in fish, Int. J. Dev. Biol. 38 on the involvement of tubulin and actin on FOM events. (1994) 217–229. [31] Y. Nagahama, M. Yamashita, Regulation of oocyte maturation in fish, Dev. Growth Differ. 50 (2008) 195–219. References [32] Y. Nagahama, M. Yamashita, T. Tokumoto, Regulation of oocyte growth and maturation in fish, Curr. Top. Dev. Biol. 30 (1994) 103–145. [33] M.Y. Narahara, A.F. Andrade-Talmelli, E.T. Kavamoto, H.M. Godinho, [1] E.F. Andrade-Talmelli, E.T. Kavamoto, M.Y. Narahara, N. Fenerich-Verani, Reprodução Induzida da Pirapitinga-do-Sul, Brycon opalinus (Cuvier, 1819), Reprodução induzida da piabanha, Brycon insignis (Steindachner, 1876), Mantida em Condições de Confinamento, Rev. Bras. Zootec. 31 (2002) 1070– mantida em cativeiro, Rev. Bras. Zootec. 31 (2002) 803–811. 1075. [2] M.V. Anguís, J.P. Cañavate, Spawning of captive Senegal sole (Solea [34] D.M. Newman, P.L. Jones, B.A. Ingram, Temporal dynamics of oocyte senegalensis) under a naturally fluctuating temperature regime, Aquaculture development, plasma sex steroids and somatic energy reserves during 243 (2005) 133–145. seasonal ovarian maturation in captive Murray cod Maccullochella peelii [3] F.P. Arantes, H.P. Santos, E. Rizzo, Y. Sato, N. Bazzoli, Profiles of sex steroids, peelii, Comp. Biochem. Physiol. 148 (2007) 876–887. fecundity, and spawning of the curimatã-pacu Prochilodus argenteus in the São [35] E. Nogales, F.J. Medrano, G.P. Diakun, R.G. Mant, E. Towns-Andrews, J. Bordas, Francisco River, downstream from the Três Marias Dam, Southeastern Brazil, The effect of temperature on the structure of vimblastine-induced polymers of Anim. Reprod. Sci. 118 (2010) 330–336. purified tubulin: detection of a reversible conformational change, J. Mol. Biol. [4] T.B. Bagenal, E. Braum, Eggs and early life history, in: T. Bagenal (Ed.), Methods 254 (1995) 416–430. of Assessment of Fish Production in Fresh Waters. IBP Handbook 3, Blackwell [36] R.M. Pielak, V.A. Gaysinskaya, W.D. Cohen, Cytoskeletal events preceding polar Scientific, Oxford, 1978, pp. 165–201. body formation in activated Spisula eggs, Biol. Bull. 205 (2003) 192–193. [5] K.A. Becker, N.H. Hart, The cortical actin cytoskeleton of unactivated zebrafish [37] F. Prodon, J. Chenevert, C. Sardet, Establishment of animal–vegetal polarity eggs: spatial organization and distribution of filamentous actin, during maturation in ascidian oocytes, Dev. Biol. 290 (2006) 297–311. nonfilamentous actin and myosin-II, Mol. Reprod. Dev. 43 (1996) 536–547. [38] L.A. Sampaio, R.B. Robaldo, A. Bianchini, Hormone-induced ovulation, natural [6] R. Billard, C. Gillet, Vieillissement des ovules et potentialisation par la spawning and larviculture of Brazilian flounder Paralichthys orbignyanus température des effets des micropollutants du milieu aqueux sur les Valenciennes, 1839), Aquac. Res. 39 (2008) 712–717. gamètes chez la truite, Cah. Lab. Hydrob. Mont. 12 (1981) 35–42. [39] Y. Sato, H.P. Godinho, Migratory fishes of the São Francisco River, in: J. [7] M.F.G. Brito, N. Bazzoli, Reproduction of the surubim catfish (Pisces, Carolsfeld, B. Harvey, C. Ross, A. Baer (Eds.), Migratory Fishes of South Pimelodidae) in the São Francisco River, Pirapora Region, Minas Gerais, America: Biology, Fisheries and Conservation Status, World Fisheries Trust/The Brazil, Arq. Bras. Med. Vet. Zootec. 55 (2003) 624–633. World Bank/International Development Research Centre, Victoria, 2003, pp. [8] N.P. Brown, R.J. Shields, N.R. Bromage, The influence of water temperature on 195–222. spawning patterns and egg quality in the Atlantic halibut (Hippoglossus [40] Y. Sato, N. Bazzoli, E. Rizzo, M.B. Boschi, M.O.T. Miranda, Influence of Abaeté hippoglossus L.), Aquaculture 261 (2006) 993–1002. River on the reproduction success of the neotropical migratory teleost [9] L.W. Crim, S. Bettles, Use of GnRHa analogues in fish culture, in: M. Fingerman, Prochilodus argenteus in the São Francisco River, downstream of the Três R. Nagabhushanam, M.F. Thompson (Eds.), Recent Advances in Marine Marias dam, Southeastern Brazil, River Res. Appl. 21 (2005) 939–950. Biotechnology, Vol.: Endocrinology and Reproduction, Oxford and IBH [41] Y. Sato, E.L. Cardoso, A.L. Godinho, H.P. Godinho, Hypophysation of the fish Publishing Co., New Delhi, 1997, pp. 369–382. Prochilodus affinis from the São Francisco basin, Brazil, Arc. Bras. Med. Vet. [10] S.Z. Dou, Y. Yamada, A. Okamura, A. Shinoda, S. Tanaka, K. Tsukamoto, Zootec. 48 (1996) 55–62. Temperature influence on the spawning performance of artificially-matured [42] Y. Sato, E.L. Cardoso, A.L. Godinho, H.P. Godinho, Hypophysation parameters of Japanese eel, Anguilla japonica, in captivity, Environ. Biol. Fish. 82 (2008) 151– the fish Prochilodus marggravii obtained in routine hatchery station conditions, 164. Rev. Bras. Biol. 56 (1996) 59–64. [11] R.A. Espinach, V.G. Amutio, J.P. Arceredtllo, G. Orti, A. Nani, Induced breeding of [43] Y. Sato, N. Fenerich-Verani, A.P.O. Nuner, H.P. Godinho, J.R. Verani, Padrões the South American catfish, Rhamdia sapo (C. & V), Aquaculture 37 (1984) 141– reprodutivos de peixes da bacia do rio São Francisco, in: H.P. Godinho, A.L. 146. Godinho (Eds.), Águas e peixes no São Francisco das Minas Gerais, PUC Minas, [12] J. García-Alonso, A. Nappa, G. Somoza, A. Rey, D. Vizziano, Steroid metabolism Belo Horizonte, MG, 2003, pp. 229–274. in vitro during final oocyte maturation in white croaker Micropogonias furnieri [44] Y. Sato, N. Fenerich-Verani, H.P. Godinho, Reprodução Induzida de Peixes da (Pisces: Scianidae), Braz. J. Biol. 64 (2004) 211–220. Bacia do São Francisco, in: H.P. Godinho, A.L. Godinho (Eds.), Águas e peixes no [13] O. Gennari-Filho, F.M.S. Braga, Fecundidade e desova de Astyanax bimaculatus e São Francisco das Minas Gerais, PUC Minas, Belo Horizonte, MG, 2003, pp. A. schubarti (Characidae, Tetragonopterinae) na represa de Barra Bonita, rio 275–290. Piracicaba, SP. Rev. UNIMAR. 18 (1996) 241–254. [45] Y. Sato, N. Fenerich-Verani, J.R. Verani, H.P. Godinho, E.V. Sampaio, [14] F.W. Goetz, Hormonal control of oocyte final maturation and ovulation in Reproductive traits oh the yellow-mandi catfish Pimelodus maculatus fishes, in: W.S. Hoar, D.J. Randall, E.M. Donaldson (Eds.), Fish Physiology: Lacépède (Osteichthyes, Siluriformes) in captive breeding, Rev. Bras. Zool. 16 Reproduction, Academic Press, New York, 1983, pp. 117–161. (1990) 981–986. [15] T.L. Gonçalves, N. Bazzoli, M.F.G. Brito, Gametogenesis and reproduction of the [46] Y. Sato, N. Fenerich-Verani, J.R. Verani, H.P. Godinho, L.J.S. Vieira, Reprodução matrinxã Brycon orthotaenia (Günther, 1864) (Pisces: Characidae) in the São artificial do dourado Salminus brasiliensis (Pisces: Characidae) da bacia do rio Francisco River, Minas Gerais, Brazil, Braz. J. Biol. 66 (2006) 513–522. São Francisco [Artificial reproduction of the ‘‘dourado’’ Salminus brasiliensis [16] S.S. Guraya, The cell and molecular biology of fish oogenesis, Basel (1986). (Pisces: Characidae) from the São Francisco River Basin], Rev. Bras. Reprod. [17] R. Hanna, Y. Pang, P. Thomas, Y. Zhu, Cell-surface expression, progestin binding, Anim. 21 (1997) 113–116. and rapid nongenomic signaling of zebrafish membrane progestin receptors [47] Y. Sato, N.F. Verani, J.R. Verani, L.J.S. Vieira, H.P. Godinho, Induced reproductive alpha and beta in transfected cells, J. Endocrinol. 190 (2006) 247–260. responses of the neotropical anostomid fish Leporinus elongatus Val. under [18] L. Horvath, Relation between ovulation and water temperature by farmed captive breeding, Aquac. Res. 31 (2000) 189–193. cyprinids, Aquacult. Hung. 1 (1978) 58–65. [48] K. Selman, R.A. Wallace, A. Sarka, X. Qi, Stages of oocyte development in the [19] V.V. Ivanenkov, A.A. Minin, V.N. Meshcheryakov, L.E. Martynova, The effect of zebrafish Brachydanio rerio, J. Morphol. 218 (1993) 203–224. local cortical microfilament disorganization on ooplasmic segregation in the [49] J.A. Senhorine, G.M. Figueiredo, N.A. Fontes, Larvicultura e alevinagem do loach (Misgurnus fossilis) egg, Cell Differ. 22 (1987) 19–28. pacu, Piaractus mesopotamicus (Holmberg, 1887), tambaqui Colossoma [20] R.G. Kessel, H.N. Tung, R. Roberts, H.W. Beams, The presence and distribution macropomum (Cuvier, 1818) e seus respectivos híbridos, Bol. Tec. Cepta. 1 of gap junctions in the oocyte–follicle cell complex of the zebrafish, (1988) 19–30. Brachydanio rerio, J. Submicrosc. Cytol. 17 (1985) 239–253. [50] B. Senthilkumaran, M. Yoshikuni, Y. Nagahama, A shift in steroidogenesis [21] M.W. Klymkowsky, A. Karnovsky, Morphogenesis and the cytoskeleton: occurring in ovarian follicles prior to oocyte maturation, Mol. Cell. Endocrinol. studies of the Xenopus embryo, Dev. Biol. 165 (1994) 372–384. 215 (2004) 11–18. [22] C.A. Lessman, Germinal vesicle migration and dissolution in Rana pipiens [51] V. Sugumar, N. Munuswamy, Induction of population growth, mictic female oocytes: effect of steroids and microtubule poisons, Cell Differ. 20 (1987) 239– production and body size by treatment of a synthetic GnRH analogue in the 251. freshwater rotifer, Brachionus calyciflorus Pallas, Aquaculture 258 (2006) 529– [23] C.A. Lessman, Oocyte maturation: converting the zebrafish oocyte to the 534. fertilizable egg, Gen. Comp. Endocrinol. 161 (2009) 53–57. [52] H.I. Suzuki, A.A. Agostinho, K.O. Winemiller, Relationship between oocyte [24] C.A. Lessman, Oogenesis, nonmammalian vertebrates, in: E. Knobil, J.D. Neill morphology and reproductive strategy in loricariid catfishes of the Paraná (Eds.), Encyclopedia of Reproduction, Academic Press, NY, 1999, pp. 498–508. River, Brazil, J. Fish Biol. 57 (2000) 791–807. [25] B. Levavi-Sivana, R. Vaimana, O. Sachsb, I. Tzchori, Spawning induction and [53] P. Thomas, Y. Pang, Y. Zhu, C. Detweiler, K. Doughty, Multiple rapid progestin hormonal levels during final oocyte maturation in the silver perch (Bidyanus actions and progestin membrane receptor subtypes in fish, Steroids 69 (2004) bidyanus), Aquaculture 229 (2004) 419–431. 567–573. [26] R. Lowe, The fecundity of tilapia species, East Afr. Agric. J. 21 (1955) 45–52. [54] A.E.A. Vazzoler, Biologia da reprodução de peixes teleósteos: teoria e prática, [27] B. Maro, M.H. Verlhac, Polar body formation: new rules for asymmetric SBI, Maringá: EDUEM, São Paulo, 1996. divisions, Nat. Cell Biol. 4 (2002) 81–83. [55] P. Vernier, A.C. Maggs, M.F. Carlier, D. Pantaloni, Analysis of microtubules [28] A. Miyazakia, H.K. Kato, S. Nemoto, Role of microtubules and centrosomes in rigidity using hydrodynamic flow and thermal fluctuations, J. Biol. Chem. 269 the eccentric relocation of the germinal vesicle upon meiosis reinitiation in (1994) 13353–13360. sea-cucumber oocytes, Dev. Biol. 280 (2005) 237–247. 408 F.P. Arantes et al. / General and Comparative Endocrinology 172 (2011) 400–408

[56] R.L. Welcomme, The relationship between fecundity and fertility in the mouth- [60] J.H. Zar, Biostatistical Analysis, fifth ed., Prentice Hall, Upper Saddle River, NJ, brooding cichlid fish Tilapia leucosticta, J. Zool. Lond. 151 (1967) 453–468. 2009. [57] R.L. Welcomme, Fisheries Ecology of Floodplain Rivers, Longman, New York, [61] Y. Zhu, C.D. Rice, Y. Pang, M. Pace, P. Thomas, Cloning, expression, and 1979. characterization of a membrane progestin receptor and evidence it is an [58] P.R. Witthames, M. Greer Walker, M.T. Dinis, C.L. Whiting, The geographical intermediary in meiotic maturation of fish oocytes, Proc. Natl. Acad. Sci. USA variation in the potential annual fecundity of Dover sole Solea solea (L.) from 100 (2003) 2231–2236. European shelf waters during 1991, Neth. J. Sea Res. 34 (1995) 45–58. [62] Y. Zohar, Fish reproduction: its physiology and artificial manipulation, in: M. [59] E. Woynarovich, L. Horváth, The artificial propagation of warm-water finfishes Shilo, S. Sarig (Eds.), Fish Culture in Warm Water Systems: Problems and – a manual for extension. FAO Fisheries Technical Paper, 201 (1980) 183p. Trends, CRC Press, Boca Raton, 1989, pp. 65–119.