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, Brazilb Estação de Hidrobiologia e Piscicultura de Três Marias, CODEVASF, P.O. Box 11, CEP 39205-000 Três Marias, Minas Gerais, Brazilc 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

a r t i c l e i n f o

Article history:Received 25 October 2010Revised 18 March 2011Accepted 4 April 2011Available online 8 April 2011

Keywords:Freshwater fishArtificial reproductionTestosterone17b-Estradiol17a-Hydroxyprogesterone

0016-6480/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.ygcen.2011.04.007

⇑ Corresponding author. Fax: +55 3133194269.E-mail address: bazzoli@pucminas.br (N. Bazzoli).

a b s t r a c t

Most fishes with commercial importance from the São Francisco basin are migratory and do not completethe reproductive cycle in lentic environments, such as hydroelectric plant reservoirs, hence natural stocksare declining and there is an urgent need to reduce the pressure of fishing on those wild populations.Therefore, studies on reproductive biology and its relationship with endocrine and environmental factorsare key to improving the cultivation techniques of Brazilian fish species. This study examined the influ-ence of water temperature on sex steroid concentrations (testosterone, 17b-estradiol and 17a-hydroxy-progesterone), spawning efficiency, fecundity, fertilisation rate, larval abnormality rates and involvementof the cytoskeleton during the final oocyte maturation of Prochilodus argenteus under experimental con-ditions. The results of our study showed that in captivity, sex steroid plasma concentrations and spawn-ing performance of P. argenteus were clearly different for fish kept in water with different temperatureregimes. In lower water temperature (23 �C), it was observed that: 33% of females did not ovulate, fecun-dity was lower and vitellogenic oocytes after the spawning induction procedure exhibited a smallerdiameter. Moreover, concentrations of 17b-estradiol and 17a-hydroxyprogesterone were lower and therewas a delay in the final oocyte maturation and, consequently, ovulation and spawning. Our experimentsshowed 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 processand the possible action of actin filaments in the release of polar bodies during final oocyte maturationof P. argenteus.

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1. Introduction

The fish pituitary produces two types of gonadotropin (GTH),follicle stimulating hormone (FSH) and luteinizing hormone (LH)[31]. The temporal pattern of secretion in several fishes suggeststhat FSH has a dominant role regulating vitellogenic growth of fol-licles, in part through stimulation of 17b-estradiol (E2) synthesisby ovarian follicles, and E2 regulates ovarian development throughits control of vitellogenin synthesis. LH induced cascade of matura-tion-inducing hormone (MIH) synthesis followed by maturation ormetaphase-promoting factor (MPF) production is well studied infish [30,47]. The 17a, 20b-dihydroxy-4-pregnen-3-one (17a, 20b-DP) which has 17a-hydroxyprogesteone as a precursor, has beenidentified as MIH in several fish and the action of this MIH in

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inducing the resumption of meiosis in teleost oocytes has also beenreported [31].

Fish final oocyte maturation completes the process of oogenesisand results in the fertilisable female gamete or egg [14,16,31]. Finaloocyte maturation is a remarkable transformation of the prophase Ioocyte; a cell specialised for transcription, material uptake, nonex-citable, and unable to osmoregulate in fresh water, to the meta-phase II egg, a cell different from the oocyte in many ways. Inmany teleosts, oocyte maturation is triggered by 17a, 20b-DP act-ing on a steroid–receptor in the cytoplasmic membrane [17,53,61].This unconventional steroid–receptor interaction leads to the var-ious non-genomic cellular changes that are the markers for oocytematuration [31].

The fully-grown zebrafish oocyte residing in the ovary is cov-ered by three cell layers, the ovarian epithelium, the theca andthe follicle cell layer [24,48]. The oocyte and follicle cells havenumerous gap junctions in common and, thus, are in chemical

F.P. Arantes et al. / General and Comparative Endocrinology 172 (2011) 400–408 401

and electrical communication [20]. The oocyte has a high degree ofendocytic activity and uses receptor-mediated endocytosis to takeup vitellogenin transported from the liver. The fully-grown oocyteis in prophase I of meiosis with a central, large nucleus or germinalvesicle containing many nucleoli and a very high transcriptionalactivity [23].

Fecundity is an important variable for aquaculture, speciesmanagement and conservation [54,57], and it estimates the repro-ductive potential of a fish species [4]. Fecundity depends on bodysize, oocyte diameter, spawning type and may vary as a result ofdifferent adaptations to environmental habitats and it providesimportant information for aquaculture, fisheries management,and conservation [52,56,58].

During the final oocyte maturation of the fish, the cytoskeletonparticipates in the ooplasm segregation [19]. Variations in watertemperature can cause conformational changes as well as in thedynamics of the cytoskeleton [35,55], as aggregation of microtu-bules occurs when the temperature is high and its breakdown oc-curs when the temperature decreases [35], thus, interfering in themigration of the nucleus during final oocyte maturation [22]. Actinmicrofilaments, especially those present in the cortical region, alsoparticipate in the major events of final oocyte maturation in fish,such as cytoplasmic polarisation, formation of the first polar body,fertilisation and embryo morphogenesis [5,21,27]. During fertilisa-tion, the cortical actin participates in the exocytosis of corticalgranules, formation of the fertilisation cone, assisting the migra-tion of the pronucleus and the release of the second polar body [5].

Efforts to control the reproduction of neotropical migratory fishbegan many years ago, but its success has been limited by intrinsicfactors such as obtaining good breeders, and by extrinsic factorssuch as temperature, dissolved oxygen and other water propertieswhich have prevented producers from achieving satisfactory re-sults of artificial reproduction. The water temperature has greatrelevance in the reproductive process of many fish species, actingon gamete maturation, ovulation and spawning [2]. In culture con-ditions, neotropical migratory fish, when kept at temperatures be-low 25 �C, did not respond satisfactory to artificial reproductionprocedures [40].

Prochilodus argenteus, popularly known as curimatã-pacu, is anendemic species in the São Francisco basin and is the most abun-dant migratory species in the Três Marias region, representing al-most 50% of the total catch. It has an illiophagous feeding habitand is the largest member of the Prochilodontidae family, and isintensively used in hatcheries for restoring fishery stocks [42,43].P. argenteus performs long-distance migrations upstream forspawning, and has high fecundity. It exhibits total spawning andits reproductive period extends from November to January in therainy season, coinciding with the time of flooding, higher temper-atures, and long photoperiods. In captivity, the curimatã-pacu pre-pares to reproduce and completing vitellogenesis, however,ovulation and spawning only occur after hormonal induction[40,42].

The application of hormonal therapies to induce spawning canbe based on the administration of the gonadotropin-releasing hor-mone (GnRH), [9,29], by treatment with Ovaprim, a preparationcontaining a synthetic GnRH analogue with domperidone [51] orusing the heteroplastic hypophysation method where commercialcrude carp pituitary extract (CCPE) is injected into the coelomiccavity or intramuscularly [38,45,46,47,59]. However, in commer-cial aquaculture of Brazilian freshwater fish, spawning inductionis usually performed with hypophysation, since this methodology,besides being economically beneficial, has high efficiency and pro-duces eggs with high rates of fertilisation [44].

Considering the scarcity of studies on the influence of watertemperature on final oocyte maturation and spawning of neotrop-ical migratory teleosts, the objective of our study was to evaluate

sex steroid concentrations, spawning, fecundity and the involve-ment of the cytoskeleton during induced spawning by hypophysa-tion in females of P. argenteus utilising two regimes of watertemperature: 23 and 26 �C. As P. argenteus females do not completetheir reproductive cycle in the stretch of the river in the São Fran-cisco near the Três Marias hydroelectric dam [3], and the watertemperature in this environment has mean values near 23 �C dur-ing the reproductive period of this species, in our study, the fishesof first experimental group (group A) was kept in the tank withwater temperature of 23 �C. According Sato et al. [42] in a watertemperature of 26 �C, P. argenteus showed good results for inducedspawning procedures, thus, in our work the second group (group B)was kept in water of 26 �C. The males were kept in a tank withwater of 26 �C.

2. Materials and methods

2.1. Sampling

In order to perform the experiment, 150 specimens of P. argen-teus were captured in the São Francisco River and kept for 2 yearsin a 200 m3 tank (20 � 10 � 1 m) in the Hydrobiology and Fishcul-ture Station of Três Marias, CODEVASF. The fish were feed with pel-leted feed containing 36% of crude protein, at a level equivalent to2% of their biomass per day during the storage period. The experi-ments were performed in duplicate, first in December 2005 andagain in December 2006. In each experiment was used 30 femalesand one male, totalling 62 specimens of P. argenteus.

2.2. Induced spawning

For the experiments, only fish in advanced gonadal maturationstages were used. Females were selected by external morphologi-cal characteristics indicating that they were ready for spawninginduction procedures, such as red urogenital papilla and bulgingcoelomic cavity. Males that released sperm with light pressureon the coelomic cavity were selected for the fertilisationprocedures.

For each repetition, the specimens were divided into twogroups of 15 females, which were transferred to 2.4 m3 breedingtanks (3 � 1 � 0.8 m) with constant water circulation and the fol-lowing characteristics: a dissolved oxygen concentration rangingfrom 5.5 to 6.5 mg L�1, pH from 6.3 to 7.5 and an electrical conduc-tivity of 58 to 87 lS cm�1. The fish in group A were kept in a breed-ing tank with a water temperature of 23 �C, whereas fish in group Bwere kept in a tank with water of 26 �C.

The fishes of groups A and B were submitted to induced spawn-ing by hypophysation according methodology established by [44].Crude common carp pituitary extract (CCPE) was injected into thecoelomic cavity. Males received a single dose of 5 mg CCPE/kg ofbody weight and females received two doses: a first dose of 1 mgof CCPE/kg and a second one of 5 mg of CCPE/kg, with an intervalof 14 h in-between doses. Fertilisation was carried out by the drymethod, using semen from a single male for each year of theexperiment.

After the induced spawning procedures, the total length (TL),body weight (BW) and gonad weight (GW), ie weight of thespawned oocytes (the weight of free oocytes released plus the freeoocytes retained in the ovaries was considered), were taken. Wecalculated the gonadosomatic index (GSI = GW � 100/BW) and Ful-ton condition factor (K = BW � 100/TL3). The fish were killed bytransversal section of the cervical medulla following the ethicalprinciples established by the Brazilian College of Animal Experi-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

For the experimental groups A and B, blood samples were ob-tained by tail vein puncture at the following times: 0 h (collectedbefore the first dose of CCPE and values taken as reference), 14,23, 25 and 26 h after the first dose of CCPE. Then the samples werecentrifuged for 15 min at 800 rpm. Serum aliquots were kept underrefrigeration (�20 �C) until concentrations of sexual steroids weredetermined. Testosterone concentrations were determined bychemiluminescence with the Bayer Corporation’s TestosteroneADVIA Centaur test. In order to determine 17 b-estradiol concen-trations, immunofluorometry with Perkin Elmer’s Estradiol Auto-DELFIA� test was used. For 17a-hydroxyprogesterone solid-phaseradioimmunoassay was used, via the Coat-a-Count 17a-OH pro-gesterone – DPC kit.

2.4. Histology and oocyte diameter

In order to microscopically monitor the oocyte’s final matura-tion events in the two temperature regimes, fragments of the ova-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).The fragments were fixed in Bouin’s fluid (24 h) and submitted toroutine histological techniques: embedded in glycol methacrylate,sectioned with a 5 lm thickness and stained with toluidine blue-sodium borate.

The vitellogenic oocytes’ diameters (n = 6 females/time/group)were determined from histological sections of the ovaries beforethe first hormonal injection, just before spawning and post-spawning for each experimental group (A and B). A micrometerattached to the eyepiece of a light microscope was used to takethe measurements.

2.5. Immunohistochemistry

During the final oocyte maturation, samples of ovaries werefixed in methanol and dimethyl sulfoxide (DMSO) in a 8:2 ratiofor 5 days at �20 �C. Samples were also fixed in 4% paraformalde-hyde in 0.1 M pH 7.3 phosphate buffer for 8–12 h at 4 �C. The sam-ples obtained before the first hormone dose, before the secondhormone dose, prior to spawning and recently spawned oocyteswere embedded in Paraplast (Histosec� MERCK).

The immunoperoxidase technique was used with anti actinmonoclonal/mouse IgG (C-2 clone, Santa Cruz) and anti a-tubulinpolyclonal/rabbit IgG (clone H-300, Santa Cruz) both at a 1:100dilution. The visualization was achieved using the LSAB 2 SystemHRP kit by Dako Cytomation (K 0675) containing the goat anti-rabbit or mouse IgG secondary antibody conjugated with biotin(1:200) and streptavidin conjugated with peroxidase. The histolog-ical sections were deparaffinised, hydrated and kept in PBS, thensubmitted to blocking endogenous peroxidase with 3% H2O2 inPBS for 30 min at room temperature.

The sections were submitted to the blocking of unspecific bondswith bovine albumin 2% in PBS buffer for 30 min at room temper-ature, incubated with primary antibodies in an overnight moistchamber at 4 �C, then with secondary antibody conjugated to bio-tin for 45 min and finally with streptavidin peroxidase conjugate ina moist chamber for 45 min. The peroxidase reaction was revealedwith DAB in PBS for 8 min at room temperature. The sections werecounterstained with hematoxylin. Sections of mice intestine wereused for positive control, and, for negative control, some sectionsdid not receive the primary antibodies during treatment. The anal-ysis of the peroxidase stained slides was performed under a lightmicroscope.

Actin and tubulin detection was also performed by immunoflu-orescence using the Alexa Fluor� 488 Phalloidin kit at a 1:150

dilution (MOL. PROBES) for actin, and mouse anti a-tubulin pri-mary antibody at 1:100 dilution (MOL. PROBES) and goat’s second-ary antibody Alexa Fluor� 568 at a 1:150 dilution (MOL. PROBES)for the tubulin marking. For positive control, we used sections ofmice intestine, and for negative control, some sections did not re-ceive the primary antibodies during treatment. The analysis of theslides used for the fluorescence immunohistochemistry procedureswas performed using a confocal microscope and a conventionalfluorescence microscope.

2.6. Fecundity

For the purpose of estimating the fecundity in each experimen-tal group, samples of spawned oocytes were collected andweighed. Absolute fecundity (AF) was determined by the formula:AF = NOG � OW, where NOG = number of oocytes per gram of thesample and OW = total weight of spawned oocytes. Relative fecun-dity (RF) was also calculated in order to eliminate the interferenceof fish size on fecundity, and was estimated by the formula:RF = AF/BW, where AF = absolute fecundity, BW = body weight.

2.7. Fertilisation rates and larval abnormalities

After fertilisation, incubation of the eggs of both groups wasperformed in funnel-shaped incubators (20 L), and with continuallycirculating water circulation at 24 �C. The water flow ranged from0.5 to 1 L min�1 and each incubator received 50 g of eggs. To examinethe rates of fertilisation and larval abnormalities, samples of eggs (8–12 h after fertilisation) and newly-hatched larvae were fixed in 4%neutral formalin. At least 100 eggs and 50 larvae of each sample wereused to estimate the fertilisation rates and larval abnormalities,respectively. Analyses were performed using a stereomicroscope.

2.8. Statistical analysis

For statistical tests, GraphPad InStat and Prism software wereused. In order to compare the averages of total length, body weight,oocyte diameter, fecundity between the experimental groups, datanormality was tested and Student T test with or without Welch’scorrection [60] was used. In order to compare hormone concentra-tions amongst the groups and between times, the Tukey-Kramermultiple comparison test was used. Differences were consideredsignificant at a = 0.05 (p < 0.05).

3. Results

3.1. Spawning and fecundity

For the experiments conducted in 2005 and 2006 and for bothgroups of fish the moment of oocyte release was signalled by circu-lar movements of the female. For the specimens kept in water at23 �C (group A), spawning was delayed by 2 h compared to thosekept at 26 �C (group B). All females spawned in group B, while33% of group A did not ovulate and, therefore, did not spawn.The absolute and relative fecundity showed higher levels in groupB fishes compared to those in group A (Table 1). The females ofgroup A only started the spawning 2 h after the fish of group B.Among the fish that spawned in group A, it was observed that25.7% of their vitellogenic oocytes have not started or completedthe process of final oocyte maturation.

3.2. Histology and oocyte diameter

The morphological events of final oocyte maturation (nucleardislocation, disruption of the nuclear envelope and finally the

Table 1Total length (TL), body weight (BW), absolute fecundity (AF), relative fecundity (RF), fertilisation rate (F), rate of abnormal larvae (AL) and diameter ofvitellogenic 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 22.5–37.0BW (g) 60 351.8 ± 136.7a 140.0–650.0 329.0 ± 141.1a 140.0–650.0AF 10 32733.0 ± 17743.2a 19285.0–58870.0 44051.0 ± 38.547.3b 14210–10454.5RF 10 1163.9 ± 580.2a 803.8–2030.0 1457.0 ± 1085.9b 568.4–3168.0F (%) 10 88.4 ± 13.4a 68.4–96.8 83.08 ± 17.85a 52.1–98.9AL (%) 10 9.3 ± 9.0a 0.2–17.0 9.5 ± 10.8a 0.2–24.3OD (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.

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ovulation into the ovarian cavity) were similar in both groups,however, the process was slower in group A. Amongst the femalesof the group A which spawned (67%), were observed numerous oo-cytes who have not started or not completed the process of finaloocyte maturation and therefore not ovulated.

Before the first CCPE dose, the vitellogenic oocytes presented acentral nucleus (Fig. 1A). The main events of the final oocyte mat-uration began with the first dose of CCPE, with dislocation of thenucleus toward the micropylar region in the animal pole(Fig. 1B). After the full migration of the nucleus (Fig. 1C and D),there was disruption of the nuclear envelope and finally theovulation into the ovarian cavity occurred (Fig. 1E). At this stage,chromosomes in metaphase II, close to the micropylar region, wereobserved (Fig. 1F). The period of time for germinal vesicle

Fig. 1. Vitellogenic oocytes histological sections of P. argenteus submitted to induced smoving toward the micropylar region (B), complete nuclear migration (C and D) nuclearregion in metaphase II (F). OV, vitellogenic oocytes; N, nucleus; Arrow head, micropyle; Aand E) and 50 lm (D–F).

migration and germinal vesicle breakdown varied from 25.5 h forgroup A to 23.5 h for group B.

The oocyte diameter showed increasing values for fish from thereference group (835.41 lm ± 32.74), group A (887.80 lm ± 49.35),group B (966.61 lm ± 43.68) and spawned oocytes of group B(967.97 lm ± 48.42), respectively, and differences were highly sig-nificant, except between group B and spawned oocytes, showingan increase in egg diameter after hormonal induction and a largerdiameter for fish kept in water at 26 �C.

3.3. Hormonal concentration

Plasma concentrations of testosterone increased after the firstCCPE dose, especially in group A and a quick decreased after the

pawning by hypophysation. Oocyte with central nucleus (A), oocyte with nucleusenvelope breakdown and ovulation (E); details of chromosomes near the micropylar

sterisk, nuclear material; Arrow, chromosomes in metaphase II. Bars: 100 lm (A–C

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 ofCCPE. Values are expressed as means ± SD. Different capital letters indicatestatistical differences between the two experimental groups at the same time.Lowercase letters indicate statistical differences between subsequent times withineach experimental group (p < 0.05).

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second dose. The variations in the 17 b-estradiol concentrationswere not very prominent during the experiment, an increaseafter the first dose followed by a gradual reduction until thepost-spawning was observed in group B, but there was noincrease of 17 b-estradiol after the first dose in group A fish.The concentrations of 17a-hydroxyprogesterone (17a-P) showeda rapid increase at the moment of spawning, and quick decreasein post-spawning for both experimental groups. Although bothgroups exhibited a similar tendency, group A showed lower con-centrations of 17a-P and peak levels with a 2-h delay as com-pared to fish from group B, what probably caused the delay inspawning (Fig. 2).

3.4. Immunohistochemistry

Results obtained by immunoperoxidase and immunofluores-cence showed actin and tubulin reactivity in the ooplasm and fol-licular cells of vitellogenic oocytes. During the final oocytematuration, 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. 4Band D) poles was also observed. Intense reactivity was observed fortubulin surrounding the migrating nucleus (Fig. 4C). Spot markingsfor tubulin (Fig. 4D) and for DNA (Fig. 4E) were observed in themeiotic spindle parked in metaphase II, near the micropylar regionof ovulated oocytes.

3.5. Fertilisation rates and larval abnormalities

High fertilisation rates and low rates of larvae abnormalitieswere observed for fish of both experimental groups (Table 1). Therewere no statistical differences in the fertilisation and larval abnor-malities rates between fish from groups A and B. The most frequentlarval abnormality was spine malformation.

4. Discussion

Since the majority of the Brazilian neotropical teleosts withcommercial importance are migratory and do not complete theirreproductive cycle in captivity or in lentic environments, experi-mental studies using migratory fish in captivity have been con-ducted in an attempt to complete their life cycle [1,7,33,39,41,42,45–47,49]. In artificial reproduction procedures, the control ofspawning is one of the most important stages for successful artifi-cial reproduction of fish [44,62], and water parameters such astemperature, dissolved oxygen and pH can limit the success of finaloocyte maturation, ovulation and thus spawning. However, studieson the influence of water parameters on the concentrations of sexsteroids, oocyte morphology and actin and tubulin involvement inthe final oocyte maturation events during artificial reproductionprocedures of neotropical fish are scarce.

In our study, the period of gonadal maturation of males and fe-males of P. argenteus kept in captivity coincided with the reproduc-tive period of this species in their natural environment, whichoccurs from November to January, that is, during the hot and rainyseason [3]. This has also been recorded for other migratory species,such as Brycon orthotaenia [15], Pseudoplatystoma corruscans [18],Leporinus elongatus [47] and Prochilodus affinis [41].

In this study, fish from both experimental groups showed signsof the spawning moment, usually through repeated circular move-ments, which has also been observed by Sato et al. [42]. In proce-dures of induced spawning by hypophysation, changes in fishbehaviour are commonly used as indicators for the moment ofspawning [10,42,47].

In our study, by manipulating water temperature alone, it wasobserved that the spawning performance of P. argenteus was signif-icantly different for the two temperature regimes, such thatspawning of the fish kept at 23 �C (group A) was delayed by 2 hin relation to those kept at 26 �C (group B), showing that low tem-peratures may negatively interfere with the spawning process of P.argenteus. Similar interference was also reported for other species,such as carp [17], Rhamdia sapo [11], Leporinus elongatus [47], andAnguilla japonica [10]. Besides the delay in spawning, it was alsonoted that 33% of fish in group A did not ovulate and consequentlydid not spawn, whereas in group B, spawning occurred for all fe-males. According Dou and colleague [10] fish usually ovulate and

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 withimmunoperoxidase 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 isreferred 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 (Aand B) and immunofluorescence (C and D – red). Chromosomes in metaphase and follicle cells’ nuclei, labelled with DAPI (E – blue) Arrows, yolk globules; Stars, follicularcells; M, micropylar region; N, nucleus; ZP, zona pellucida; arrowhead, Chromosomes in metaphase II. Sections with immunoperoxidase were counter-stained withhematoxylin. 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.)

F.P. Arantes et al. / General and Comparative Endocrinology 172 (2011) 400–408 405

spawn within a limited range of water temperatures, and ourresults indicate that a 26 �C water temperature is more suitablefor triggering ovulation and spawning in P. argenteus when

undergoing induced spawning by hypophysation in artificialreproduction experiments. Dou et al. [10] also noted lower ovula-tion and spawning rates in A. japonica kept in low-temperature

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waters, as well as delayed spawning. In natural environments, Satoet al. [40] and Arantes et al. [3] also observed the negative influ-ence of low water temperature on the reproductive activity ofmigratory fish. Despite the fact that, in this study, ovulation andspawning performance of P. argenteus females kept in water at23 �C were impaired, the mechanism by which temperature altersthe performance of spawning of this species is not yet known.

In our study, the concentrations of 17a-hydroxyprogesterone(17a-P) increased sharply after the application of crude carp pitu-itary extract (CCPE) and decreased abruptly in post-spawning forboth temperature regimes. Nevertheless, fish kept in water at23 �C showed lower 17a-P concentrations and this hormone’s peakhappened with a 2-h delay in relation to fish kept in water at 26 �C,which probably caused the late spawning and lower spawning per-centage. Thus, 23 �C was not the ideal water temperature in whichCCPE and 17a-P could efficiently work on final oocyte maturation,ovulation and spawning of mature P. argenteus females. Delay inhormonal action and spawning in fish kept in lower temperatureswas also recorded by Dou et al. [10]. The gonadotropins FSH and LHrequire at least two steroidal mediators, 17b-estradiol (E2) and17a, 20b-dihydroxy-4-pregnen-3-one (17a, 20b-DP) to performoocyte growth and maturation, respectively. The theca cells pro-vide the precursor steroids, and the granulosa cells produce thetwo steroidal mediators under the direct influence of FSH and LH[50]. Moreover, in many teleosts, temperature has effects on hor-mone function, interfering in final oocyte maturation and ovula-tion [10]. Although measurements of 17a, 20b-DP (MIH) havenot been made in this study due to technical limitations, measure-ments of its precursor, 17a-P, were carried out, and together withthe results of spawning performance, histology and ICC obtained inthis study suggests the influence of water temperature on the pro-cess of final oocyte maturation and ovolation of P. argenteus sub-jected to induced spawning hypophysation.

The concentrations of 17b-estradiol seems not to had directinvolvement in the mains events of final oocyte maturation, sincefrom middle of the process, concentrations of this steroid showeddecreasing values, including the spawning moment. There was asignificant increase of testosterone concentrations detected at14 h time for fish kept in colder water. However, this steroid’s con-centrations for fish kept in both temperature regimes, at thespawning moment, showed low levels. This suggests that in P.argenteus, testosterone probably does not act directly upon finaloocyte maturation. According [31] testosterone induces final oo-cyte maturation only experimentally and in high concentrations.

Direct effects of water temperature on the quantity and qualityof gametes have been reported for fish in captivity and in naturalstocks [6,8]. A recent study of wild P. argenteus in two stretchesof the São Francisco River also registered lower fecundity rates infish living in lower water-temperature environments [3]. Differ-ences in fecundity rates may also be related to the age of breeders,stocking density and the feeding of the breeders [13,26]. However,in the present study, these factors were probably not relevant,since fish used in the experiment were of the same age, were keptunder the same stocking density and were given the same amountof food.

In this study, the diameters of vitellogenic oocytes of P. argen-teus females kept in captivity for 2 years and which did not receiveCCPE injections were similar to oocyte diameters reported for wildfish of the same species [3], indicating that confinement does notprevent the process of vitellogenesis in migratory fish such as cur-imatãs. The same was reported for murray cod, Maccullochella peeliipeelii, a freshwater Perciformes [34]. We observed in this study,however, that at the time of spawning induction, the number ofvitellogenic oocytes in fish kept in water at 23 �C was significantlylower. Since 17b-estradiol is responsible for vitellogenesis, the ele-vation of this steroid detected after the first CCPE dose in fish kept

at 26 �C was probably responsible for the larger oocyte diameter ofthe females from this group.

Investigations involving spawning induction made it possible tocontrol the reproductive process of fish in captivity, contributing tothe expansion of aquaculture [25]. The use of crude carp pituitaryextract (CCPE) yields positive results for induced ovulation andpresents good fertilisation rates in tropical fish, as observed in 25species from the São Francisco River [44]. Although this workshows the efficiency of injecting CCPE in the artificial reproductionof P. argenteus, giving fertilisation rates above 80% in both experi-mental groups, fish maintained at 26 �C showed better rates ofspawning and fecundity.

We observe that prior to the first dose of CCPE, P. argenteus fe-males had vitellogenic oocytes with a central nucleus. After thefirst dose of CCPE, dislocation was observed of the nucleus towardthe micropylar region, followed by disruption of the nuclear enve-lope, resumption of meiosis until metaphase II, and culminating inovulation into the ovarian cavity and spawning. In most teleosts,nuclear and cytoplasmic events in vitellogenic oocytes are knownas final oocyte maturation (FOM) [12,32]. Disruption of the nuclearenvelope after its migration to the oocyte cortex has also been ob-served in zebrafish [23]. However, Prodon et al. [37] observed thatin ascidia the nuclear envelope disruption occurs before the migra-tion of the nuclear structures.

In this study, the FOM process and ovulation lasted for 23 h ingroup B fish (26 �C) and 25 h in group A (23 �C). According to Naga-hama et al. [32], final oocyte maturation in teleosts occurs rapidly,being regulated by LH and by the maturation-inducing hormone.The delay in FOM and ovulation events observed in this study,was probably caused by the late peak (2 h) of 17a-P, which formost teleosts is the precursor of the maturation-inducing hor-mone, the 17a, 20b-dihydroxy-4-pregnen-3-one [31].

In our study, we observed intense reactivity to tubulin in threeregions: around the nucleus in migration; in the oocyte’s periph-ery, especially in the animal pole, and in the ooplasm in the formof bundles between the nucleus and the cortex. This positioningof tubulin during nuclear migration in the final oocyte maturationin P. argenteus, suggests involvement of microtubules in this pro-cess, as observed in Holothuroideas [28] and in bivalves [36]. Aftercompletion of the migration and disruption of the nuclear enve-lope, the reactivity to tubulin decreased in the ooplasm and wasobserved near the micropyle, where the meiotic spindle was lo-cated. The actin collar in the peripheral ooplasm, thicker in the ani-mal pole, which was detected in vitellogenic oocytes of P.argenteus, is similar to the arrangement observed in other animalgroups [28,36], and may be involved in the release of polar bodies.The exact mechanism of nuclear migration toward the cortex inoocytes of teleosts is not completely known, but it involves compo-nents of the cytoskeleton [23]. Studies using Holothuroidea’s oo-cytes [28] showed that in this animal, only the microtubulesseem to be involved in nuclear migration, whereas in ascidian oo-cytes the actin filaments seems to be responsible for the migrationof the meiotic spindle toward the oocyte cortex, besides this, thenuclear envelope breaks down in the centre of the oocyte [37].

In summary, this study shows the direct influence of water tem-perature on plasma concentrations of sex steroids and spawningperformance of P. argenteus submitted to induced spawning. Withthe lower water temperature (23 �C) it was observed that 33% offemales did not ovulate, fecundity was lower and the vitellogenicoocytes after the spawning induction procedure showed a smallerdiameter. Moreover, concentrations of 17b-estradiol and 17a-hydroxyprogesterone were lower and there was a delay in the finaloocyte maturation and consequently on ovulation and spawning,showing the direct influence of water temperature in the spawningprocess of P. argenteus. Our experiments also suggest the involve-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 finaloocyte maturation of P. argenteus. Our next investigation will focuson the involvement of tubulin and actin on FOM events.

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