Tributyltin impaired reproductive success in female zebrafish through disrupting oogenesis, reproductive behaviors and serotonin synthesis

Accepted Manuscript Title: Tributyltin impaired reproductive success in female zebrafish through disrupting oogenesis, reproductive behaviors and serotonin synthesis Authors: Wei-Yang Xiao, Ying-Wen Li, Qi-Liang Chen, Zhi-Hao Liu PII: DOI: Reference:

S0166-445X(18)30384-9 https://doi.org/10.1016/j.aquatox.2018.05.009 AQTOX 4938

To appear in:

Aquatic Toxicology

Received date: Revised date: Accepted date:

10-1-2018 8-5-2018 10-5-2018

Please cite this article as: Xiao W-Yang, Li Y-Wen, Chen Q-Liang, Liu Z-Hao, Tributyltin impaired reproductive success in female zebrafish through disrupting oogenesis, reproductive behaviors and serotonin synthesis, Aquatic Toxicology (2010), https://doi.org/10.1016/j.aquatox.2018.05.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Tributyltin impaired reproductive success in female zebrafish through disrupting oogenesis, reproductive behaviors and serotonin

Wei–Yang Xiao, Ying–Wen Li, Qi–Liang Chen, Zhi–Hao Liu*

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synthesis

Chongqing Key Laboratory of Animal Biology, College of Life Sciences, Chongqing

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Normal University, Chongqing 401331, China

Running title: Mechanisms involved in tributyltin disrupted oogenesis and disturbed

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reproductive behaviors in female zebrafish.

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*Corresponding Author at: Chongqing Key Laboratory of Animal Biology,

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College of Life Sciences, Chongqing Normal University, Chongqing 401331, China. Tel.: +86 23 6591 0315; Fax: +86 23 6591 0315.

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In the highlights,

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E-mail Address: [email protected] (Z. Liu).

-TBT impaired reproductive success in zebrafish female

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-TBT altered plasma level of E2 and disrupted oogenesis of the female

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-TBT disturbed reproductive behaviors of the female

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-TBT altered the expressions of genes in oogenesis, reproductive behavior

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

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and serotonin synthesis

Abstract Tributyltin (TBT), an organotin acting as aromatase (Cyp19a1) inhibitor, has been found to disrupt gametogenesis and reproductive behaviors in several fish species.

However, few studies addressing the mechanisms underlying the impaired gametogenesis and reproduction have been reported. In this study, female adults of zebrafish (Danio rerio) were continuously exposed to two nominal concentrations of TBT (100 and 500 ng/L, actual concentrations: 90.8 ± 1.3 ng/L and 470.3 ± 2.7 ng/L, respectively) for 28 days. After exposures, TBT decreased the total egg number, reduced the hatchability and elevated the mortality of the larvae. Decreased

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gonadosomatic index (GSI) and altered percentages of follicles in different

developmental stages (increased early-stage follicles and reduced mid/late-stage

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follicles) were also observed in the ovary of TBT-treated fish. TBT also lowered the

plasma level of 17β-estradiol and suppressed the expressions of cyp19a1a in the ovary. In treated fish, up-regulated expressions of aldhla2, sycp3 and dmc1 were

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present in the ovary, indicating an enhanced level of meiosis. The mRNA level of

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vtg1 was dramatically suppressed in the liver of TBT-treated fish, suggesting an

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insufficient synthesis of Vtg protein, consistent with the decreased percentage of mid/late-stage follicles in the ovaries. Moreover, TBT significantly suppressed the

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reproductive behaviors of the female fish (duration of both sexes simultaneously in

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spawning area, the frequency of meeting and the visit in spawning area) and down-regulated the mRNA levels of genes involved in the regulation of reproductive behaviors (cyp19a1b, gnrh-3 and kiss 2) in the brain. In addition, TBT significantly

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suppressed the expressions of serotonin-related genes, such as tph2 (encoding serotonin synthase), pet1 (marker of serotonin neuron) and kiss 1 (the modulator of

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serotonin synthesis), suggesting that TBT might disrupt the non-reproductive behaviors of zebrafish. The present study demonstrated that TBT may impair the reproductive success of zebrafish females probably through disrupting oogenesis,

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disturbing reproductive behaviors and altering serotonin synthesis. The present study greatly extends our understanding on the reproductive toxicity of TBT on fish.

Keywords: Tributyltin; Plasma E2 level; Oogenesis; Reproductive behavior; Serotonin synthesis; Gene expression

1. Introduction Tributyltin (TBT), an organotin compound, has been used as a biocide in numerous industrial applications, such as antifouling paints for ships and fish nets (Gipperth, 2009). However, the use of TBT was globally banned by the International Marine Organization (IMO) starting in 2003 and totally prohibited by 2008 (IMO, 2001) due to its acute toxicity on the growth and reproduction of aquatic animals (Hu

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et al., 2014). Since then, the concentrations of TBT in aquatic environments have generally dropped below 1 ng/L (Gao et al., 2006). However, continuing high

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concentrations of TBT are still detected in some water samples due to its environmental persistence and its widespread illegal use (Sousa et al., 2009; Garg et al., 2011; Cao et al., 2009). It has been reported that TBT concentrations in bottom

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sediments did not decline in Hungary (Üveges et al., 2007) and Japan (Harino et al.,

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2007) after the regulation or restriction of the use of the compound. The TBT levels

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range from 200 to 400 ng/L in the surface waters of Europe and India (Sousa et al., 2009; Garg et al., 2011). In mainland China, published TBT concentrations from

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water samples range from below 0.5 to 977 ng/L as Sn (Gao et al., 2006; Cao et al.,

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2009).

TBT has been shown to exert anti-Cyp19a1 (aromatase, responsible for estrogen production) activity in animals (Oberdörster and McClellan-Green, 2002; McAllister

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and Kime, 2003). Thus, it has been well documented that TBT decreases Cyp19a1 activity in ovarian cells, changes the estrous cycle, impedes the development of

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ovarian follicles, affects the balance of ovarian hormones, reduces the number of germ cells and affects sexual development (Nakanishi et al., 2002; Grote et al., 2004). Induced masculinization and the altered sex ratios in fish by TBT administration have

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been also observed. In zebrafish, exposure to TBT (0.1-100 ng/L) induced masculinization and biased sex ratio (McAllister and Kime, 2003). The enhanced occurrence of sex-reversed males with typical testes were observed in genetic females of Japanese flounder (Paralichthys olivaceus) treated with TBT (0.1 and 1.0 μg/g diet) (Shimasaki et al., 2003). Disruptions in fish reproduction by TBT exposure have been also observed. In zebrafish males, exposure to TBT during early developmental stages

induced irreversible damages in sperm, such as sperm lacking flagella, decreased motility of sperm, and increased milt volume in adults (McAllister and Kime, 2003). In Japanese whiting (Sillago japonica) exposed to TBT, decreased floating egg percentage, viable hatchability and total number of viable larvae, as well as increased larval deformity were observed (Shimasaki et al., 2006). A 50-day exposure to TBT was also found to decrease the ratio of late-stage follicles to early-stage follicles in

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female cuvier (Sebastiscus marmoratus), indicating the disruption of fish oogenesis

(Zhang et al., 2007). Although many studies have demonstrated the reproductive

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toxicity of TBT on fish gametogenesis, the detailed mechanisms underlying the disruption remain unclear.

In fish females, the quantity and the ratio of follicles in each developmental stage

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are largely determined by the proliferation of oocytes and vitellogenin (Vtg)

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accumulation (Wang et al., 2005; Wang et al., 2017). Arrested oogenesis and

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insufficient production of Vtg are therefore both considered to result in disrupted reproductive success in fish. Meiotic entry and meiotic maintenance are both essential

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for oogenesis. A recent study has confirmed the involvement of retinoic acid (RA) in

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meiotic entry (Rodríguez-Marí et al., 2013). RA is mainly synthesized by Aldh1a2 (RA synthase) in the somatic cells surrounding pre-meiotic oogonia, triggering the meiotic initiation of germ cells (Rodríguez-Marí et al., 2013). Thereafter, Sycp3 (an

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essential component of the synaptonemal complex) is synthesized in these oogonia, indicating their entry into meiosis (Ozaki et al., 2011). In these oocytes, RA is

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subsequently degraded by Cyp26a1 (RA catalase), resulting in reduced level of RA and suppressed expression of sycp3, suggesting the termination of meiotic initiation (Li et al., 2016; Rodríguez-Marí et al., 2013). In addition, Dmc1, which acts

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exclusively during homologous recombination (Sansam and Pezza, 2015), is also a marker of meiosis. The altered expressions of these genes may lead to disrupted oogenesis. Moreover, the growth of developing oocytes is under the action of numerous growth factors that stimulate the production of yolk (e.g., Vtg) in the liver, which is necessary for oocyte growth (Tyler and Sumpter, 1996). Consequently, vtg expression and Vtg production levels are the instructors of oocyte growth and

maturation (Wang et al., 2005). Increasing evidence also showed the impaired reproductive behaviors in animals in response to TBT exposure, which might also lead to disrupted reproduction (Matthiessen et al., 1998; Tian et al., 2015). Reproductive behaviors, like other social behaviors, are mainly controlled by the central nervous system (CNS) and the neuroendocrine systems. It has been suggested that the reproductive behaviors, such

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as courtship, mating, and territorial marking, are possibly affected by gonadal hormones (such as estrogen, androgen and progesterone) through a feedback action on

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the brain (Wu and Shah, 2011). Many studies have thus attributed the disruptions in sexual behavior after exposure to endocrine disruptors to the altered gonadal hormone levels (Nakayama et al., 2004; Tian et al., 2015). In medaka (Oryzias latipes), males

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exposed to TBT were less prone to perform “following” and “dancing” behaviors

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(Nakayama et al., 2004). In male guppies (Poecilia reticulata), TBT exposure

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significantly suppressed the production of 17β-estradiol (E2), resulting in altered sexual characteristics and reproductive behavior (Tian et al., 2015). However, most of

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these studies in TBT-disrupted reproductive behaviors have mainly focused on male

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fish; whether and how the reproductive behavior in female fish may be affected by TBT remains poorly known. A recent study showed that Gnrh systems were not only essential for the regulation of reproduction but were also indispensable for the control

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of reproductive behaviors in animals (Kauffman and Rissman, 2004). To date, two forms of Gnrhs (Gnrh-2 and Gnrh-3) have been identified in zebrafish (Steven et al.,

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2003). Study on the function of Gnrh-2 is extremely scarce and has focused on its possible involvement in reproduction and food intake (Matsuda et al., 2008). In zebrafish, Gnrh-3 is recognized as the actual hypophysiotropic form of Gnrh which is

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involved in the regulation of many aspects of reproduction (Steven et al., 2003). Recent studies further reported that Kisspeptins (particularly Kiss 2) are capable of stimulating the Gnrh neurons in zebrafish (de Roux et al., 2003; Smith et al., 2006; Servili et al., 2011), indicating their involvement in reproductive events (Kitahashi et al., 2009; Servili et al., 2011; Gopurappilly et al., 2013). Disruption in the expressions of gnrh-3 and kiss 2 may therefore give rise to altered productions of these proteins

and subsequently disturbed reproductive behaviors. On the other hand, increased non-reproductive behaviors may also lead to altered reproductive behaviors. The Serotonin (5-HT) neurotransmitter system is an important modulator of neural circuitry and controls a wide range of behavioral and physiological processes, including cognition, circadian rhythms, and mood (Jacobs and Azmitia, 1992). A recent study in zebrafish showed that Kiss 1, which is located in the ventral habenula

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(vHb), takes part in the modulation of 5-HT neurons and the fear responses (Ogawa et

al., 2014; Nathan et al., 2015). In fish, both tryptophan hydroxylase 2 (Tph2) and the

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Pet1 EST factor are essential for 5-HT biosynthesis (McGeer et al., 1973). Disruption in the expressions of these genes may lead to altered productions of these proteins, which probably give rise to decreased 5-HT synthesis, resulting in altered

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non-reproductive behavior (such as fear, anxiety and aggressiveness) and disrupted

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reproductive behaviors (Hendricks et al., 2003; Nathan et al., 2015).

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To better understand the reproductive toxicity of TBT on fish and the underlying mechanisms involved, adult female zebrafish were exposed to two concentrations of

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TBT (100 and 500 ng/L) for 28 days. After exposures, reproductive behaviors

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(duration of both sexes simultaneously in spawning area, frequency of meeting and visits in spawning area) were investigated and the total egg number, hatchability and mortality of the larvae were measured. The reproductive toxicity of TBT on ovarian

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development and oogenesis, evaluated by phenotypic factors (body weight and gonadosomatic index, GSI), plasma level of E2, histology, total oocyte number and

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the percentage of follicles in different developmental stages were further assessed. The mRNA levels of the selected genes mediating E2 production in the ovary (cyp19a1a), vitellogenin production (vtg1) in the liver, meiotic entry (aldh1a2 and

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cyp26a1), meiosis (sycp3 and dmc1), neural regulation of reproductive behavior (cyp19a1b, gnrh-3 and kiss 2) and 5-HT synthesis (kiss 1, tph2 and pet1) were analyzed by quantitative RT-PCR (qRT-PCR). 2. Material and methods 2.1 Chemicals and fish Tributyltin chloride (TBT-Cl, CAT No. 45713-250MG, CAS No. 1461-22-9,

purity > 96%) was obtained from Sigma-Aldrich (USA). Sexually mature female zebrafish (AB strain, 4-months old) were obtained from the China Zebrafish Resource Center and maintained in 200 L glass aquaria under flow-through conditions using dechlorinated and aerated water. The photoperiod was set to 14 h light/10 h dark cycle and water temperature was set at 28 ± 0.5 °C. The fish were fed twice a day with flake food (Shengsuo, Shandong, China) and Artemia

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nauplii (Artemia International LCC, USA). All animal experiments were approved by the Committee of Laboratory Animal Experimentation at Chongqing Normal

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University. 2.2 Experimental designs and sampling

Previously, TBT concentrations in the range of 200-400 ng/L were detected in

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the surface waters of Europe and India (Sousa et al., 2009; Garg et al., 2011).

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Published concentrations of TBT in mainland China reached 977 ng/L as Sn (Cao et

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al., 2009). Recent experiments on TBT have also used a wide range of concentrations (1–100 and 1–500 ng/L TBT) (Tian et al., 2015; Zhang et al., 2007; 2009; 2013).

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According to our pilot study, a 28-day exposure to TBT with 500 ng/L concentration

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was sufficient to decrease total egg number without inducing mortality in female zebrafish. Therefore, two concentrations of TBT (100 and 500 ng/L) were chosen for the present study.

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Following 14 days of acclimation, 108 uniform-sized adult female fish (0.52 ± 0.04 g) were randomly separated into three equal groups (triplicate tanks per group)

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and exposed to TBT with nominal concentrations of 0 (0.05% DMSO), 100, and 500 ng/L in charcoal-dechlorinated tap water for 28 days. During exposures, the water in each tank was replaced daily with fresh dechlorinated tap water containing the

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corresponding chemical concentrations. The actual TBT concentrations in the test tanks were measured once every week in the morning just before water replacement, using automated in-tube solid-phase micro-extraction (SPME) and high-performance liquid chromatography (HPLC) coupled to a quadrupole mass spectrometer (MS) with electrospray (ES) as an ionization source (Wu et al., 2001; Lima et al., 2015). Briefly, 5 mL water samples spiked with different concentrations of TBT were

micro-extracted by SPME according to a published method (Wu et al., 2001). The extracted samples were then transported to the LC column and detector (Agilent Technologies, Palo Alto, CA, USA). A standard curve was established with samples spiked with 0, 20, 50, 100, 200, 500 and 1000 ng/L of TBT. To determine the quality of measurement, a non-spiked water sample and the pure water samples spiked with the same amounts of TBT (100 and 500 ng/L) were assayed. The detection limit of the

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measurements is 50 ng/L. The measured values for the two spiked treatments were 90.8 ± 1.3 ng/L (TBT-low) and 470.3 ± 2.7 ng/L (TBT-high) (mean ± SD, N = 3). The

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fish were fed twice daily with commercial flake diet (Shengsuo, Shandong, China)

and Artemia nauplii (Artemia International LCC, USA). Water temperature was maintained at 28 ± 0.5 °C with a 14:10 h (light: dark) cycle. Water quality was

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monitored once a week in the morning and the parameters were as follows: water

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temperature, 27.5–28.5 °C; pH, 7.2–7.9; dissolved oxygen, 6.2–7.5 mg/L; hardness,

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131.1–140.7 mg/L as CaCO3.

At the endpoint of exposures, the fish were anesthetized with 0.1% MS-222 and

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body weight of the fish in each tank was measured. Mean value of the body weight in

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each tank was set as one biological sample (N = 1) and three samples were obtained in each group (N = 3). The ovaries of three fish in each tank were removed, weighed and used to calculate the GSI (GSI = ovary weight/body weight). The mean value of the

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GSI in each tank was set as one biological sample (N = 1) and three samples were obtained in each group (N = 3). Blood samples of 6 fish were collected for the

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estimation of E2. These fish were also used for the following histology and RNA extraction. The ovaries of 3 fish in each tank were fixed in Bouin’s solution for histological observation and ovaries of the other 3 fish per tank were immediately

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frozen in liquid nitrogen and stored at –80 °C for RNA extraction. The livers and the entire brain were also collected and frozen in liquid nitrogen and then stored at –80 °C for total RNA extraction. Still, we recognized that the whole brain is not an ideal sample and we recommend therefore that additional studies should be performed to examine the region-specific expression of the transcripts. 2.3 Reproductive behavior measurements

After sampling, the reproductive behavior was quantified using one exposed female paired with one non-exposed adult male following the method described previously (Baatrup and Henriksen, 2015). The behavior test tanks (15 cm×20 cm×25 cm) contained 10 cm header tank water (3.0 L) at 28 ± 0.5 °C. The fish pair were then placed in the test tank in darkness approximately 16 h before the test (9 fish in each group, N = 9). During this acclimation period, the two fish were separated by a

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perforated partition wall. To enhance the probability that these non-exposed males

were receptive to reproductive behavior, they were isolated from females for five days

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prior to the behavioral measurement (Hisaoka and Firlit, 1962; Van den Hurk et al., 1987). During the reproduction of zebrafish, the male tries to attract the female and

lead her to the spawning area. He then entices her to spawn with rapid tail oscillations

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against her side (Darrow and Harris, 2004). To ensure a suitable spawning area, a

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plastic tray (7 cm × 7 cm) with transparent 5 mm diameter glass beads was placed in

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one corner of the test tank. After removing the partition wall in the morning, the ensuing scenario was recorded for 20 min following a simulated 5-min light from

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above. Viewed from the tank above, this design resulted in clear silhouettes of the two

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fish, where the female was distinguishably bigger than the male. A SONY DSC-RX100 M4 digital camera (SONY Corporation, Tokyo, Japan) was mounted approximately 50 cm above the test tank. Previous studies showed that TBT affects

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fish ATPase activity and normal behaviors, such as total swimming distance, velocity and activity (Schmidt et al., 2004, 2005; Li and Li, 2015; Li et al., 2016a, b; Liang et

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al., 2017). Therefore, it is possible that altered normal behavior induced by TBT exposure may affect the parameters related to reproductive behaviors. Consequently, the parameters (duration of both sexes simultaneously in spawning area, frequency of

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meeting and visits in spawning area) chosen to evaluate reproductive behaviors were selected because they are unlikely to be affected by normal behaviors. These parameters have also been applied in the other study (Baatrup and Henriksen, 2015). 2.4 Determination of the total egg number, hatchbility and mortality of the larvae After the behavioral test, eggs from 9 pairs of fish in each group (N = 9) were harvested and thoroughly rinsed three times using embryo medium (1 liter each time)

(Westerfield, 2000). The embryos were further raised in embryo medium at 28 ± 0.5 °C with a 14:10 h (light: dark) cycle. The total egg number was counted and hatchability (hatched larva / total egg number) was then calculated. Three days post hatching, the number of survived larvae were counted and were used to calculate the mortality rate ((hatched larvae – survived larvae) / hatched larvae). 2.5 Histological observation and follicle count

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Histological observations were carried out following the method described previously (Yin et al., 2017). Briefly, the ovaries were fixed in Bouin’s solution for 24

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h at room temperature and then dehydrated, embedded in paraffin and sectioned at 4

μm thickness. Sections at the middle part of the ovaries were prepared for hematoxylin and eosin counterstaining. The sections were then imaged using a Nikon

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microscope (Eclipse 90i, Nikon, Japan). Follicles in each developmental stage (PG,

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primary growth, PV, pre-vitellogenic; EV, early-vitellogenic; MV, middle-vitellogenic

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and FG, full-grown) were scored by adapting staging criteria from previously published protocols (Li et al., 2011). The total number of follicles and the number of

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follicles in each developmental stage were determined microscopically and graphed as

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percentage to total follicles as previously described (Zhang et al., 2007). The mean values of these data in each tank was set as one biological sample (N = 1) and three samples were obtained in each group (N = 3).

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2.6 Estimation of plasma E2 level

Blood samples were collected from the zebrafish using a heparinized capillary

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tube (5 μl of 10 μg/mL heparin was used) as previously described (Wang et al., 2017; Yin et al., 2017). The blood of two fish in each tank was pooled for the measurement of E2 level. The collected blood samples were then centrifuged at 5, 000 × g for 20

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min at 4 °C, and the supernatants were separated according to the manufacturer's extraction protocol (Cayman Chemical, Ann Arbor, USA). The plasma level of E2 was measured by competitive enzyme-linked immunosorbent assay kits (Cayman Chemical) following the manufacturer’s instructions. The mean value of the E2 levels in each tank was set as one biological sample (N = 1) and three biological samples in each group were obtained (N = 3). The inter- and intra-assay coefficients of variation

were 4.2 - 9.4% and 5.6 - 6.7%, respectively. 2.7 RNA extraction and qRT-PCR Analyses on the gene expression levels were conducted by the qRT-PCR method described in Wang et al., 2017. Target tissues (ovary, brain and liver) of one fish in each tank were homogenized in 1.0 mL Trizol reagent (Invitrogen, NY, USA). Total RNA extracted according to the manufacturer’s protocol was then quantified using a

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Nano Drop ND-2000 spectrophotometer (Thermo Electron Corporation, Waltham,

USA). The quality of total RNA was verified by RNA electrophoresis. Viewed under

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a Gel Image System (GelDoc XRTM, Bio-Rad, USA), only 28S and 18S bands (28S

band is 2-fold brighter than that of 18S band) were present on the gel. Genomic DNA was removed using DNase I and cDNA was obtained from 500 ng total RNA using

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the TakaRa Reverse Transcription Kit (TaKaRa, Dalian, China). The mRNA levels of

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the target genes were analyzed by qRT-PCR using Bio-Rad CFX96™ Real time PCR

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Detection System (Bio-Rad, USA). Oligonucleotide primers for the target genes and reference genes were selected based on other reference reports (Table 1). The primer

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sets were tested for specificity using RT-PCR and zebrafish wild type adult female cDNA as template to verify production of a single band with the predicted size. Prior

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to the analysis, a qRT-PCR trial of the expression levels of housekeeping genes ef1α and rpl13a in each group was carried out (Xu et al., 2016; Yin et al., 2017). The Sybr

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mix qRT-PCR reactions contained: 0.3 μM forward and reverse primers, 1 × Sybr Green master mix (TaKaRa), and 2 μL diluted cDNA template (1:9 diluted) in a final

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volume of 20 μL. All samples were run in triplicates using 96-well PCR plates (Axygen Biosciences, USA). The mean value of all the fish in each tank was set as one biological sample (N = 1) and three samples were obtained in each group (N = 3).

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The qRT-PCR parameters consisted of an initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s and 72 °C for 10 s. A series of no reverse transcriptase controls were also included for qRT-PCR to avoid trace contamination of genomic DNA. Dissociation curve analysis and a non-template reaction were performed to ensure that only one PCR product was amplified and the stock solutions were not contaminated. The amplification efficiencies of all genes

were approximately equal and ranged from 96.3% to 105.1%. The mRNA levels of genes were quantified relative to the mean values of ef1α and rpl13a using the 2-ΔΔCT method (Livak and Schmittgen, 2001). 2.8 Statistical analyses Quantitative data are presented as the mean ± SEM. Statistical differences were evaluated by one-way ANOVA followed by Tukey’s range test. Analysis was

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performed using the SPSS 20.0 package (SPSS, Chicago, IL, USA), and the mean differences were considered significant P < 0.05.

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3. Results

3.1 Total egg number, hatchability and mortality of the larvae

Both concentrations of TBT significantly decreased the total egg number of

16.119, P = 0.004, Fig. 1A). Compared to the control, TBT also decreased the

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(2,6) =

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zebrafish females (by 50% in the TBT-low group and 80% in the TBT-high group) (F

(F

(2,6) =

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hatchability of the embryos, showing a significant change only in the TBT-high group 7.506, P = 0.022, Fig. 1B). Additionally, the mortality of the larvae in both

(2,6) =

212.397, P < 0.001, Fig. 1C). No larvae survived in the TBT-high group 3

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(F

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TBT-treated groups were significantly increased in a concentration-dependent manner

days post hatching (Fig. 1C).

3.2 Influences of TBT on the body weight and GSI of zebrafish female

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The body weight of the treated fish showed no significant change compared with the control (F in

1.217 P = 0.36, Fig. 2A). However, both concentrations of TBT

significant

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resulted

(2,6) =

reductions

of

the

GSI

in

treated

fish

in

a

concentration-dependent manner (F (2,6) = 30.709, P < 0.001, Fig. 2B).

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3.3 Histology observations and follicle count Histological examination of the ovary in control group revealed the presence of

all development phases of germ cells and a large amount of oocytes (Fig 3.A & D). However, increased number of early-stage follicles and decreased number of late-stage follicles were observed in ovaries of the treated fish (Fig 3.B, C, E & F). Counting microscopically, the total follicle number of the TBT-treated fish decreased significantly in comparison to the control (F

(2,6)

= 193.216, P < 0.001, Fig. 3G).

Moreover, the percentages of early- (PG) and mid/late-stage follicles (EV, MV and FG) increased and decreased significantly, respectively, after TBT exposures (F PG (2,6) = 27.136,P

PG

= 0.001, F

EV (2,6)

= 22.974, P

EV

= 0.002, F

MV (2,6)

= 11.671, P

MV

=

0.009, F FG (2,6) = 22.085, P FG = 0.002 , Fig. 3H). 3.4 Influences of TBT on E2 level in plasma and the mRNA levels of vtg1 in liver The plasma E2 levels of the TBT-treated fish decreased significantly in a

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concentration-dependent manner when compared with the control (F (2,6) = 36.639 P < 0.001) (Fig. 4A). The mRNA level of vtg1 in the liver also showed a significant

(2,6) =

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decline (lower than 50%) in both TBT-treated groups in comparison to the control (F 12.503, P = 0.007) (Fig. 4B).

3.5 Effects of TBT on mRNA levels of genes in E2 production and meiotic

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regulation

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Both concentrations of TBT suppressed the mRNA levels of cyp19a1a in the

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ovaries in a concentration-dependent manner (F (2,6) = 304.968, P < 0.001). However, the mRNA levels of cyp17a1, aldhla2 and dmc1 were significantly elevated (by

= 0.008, F aldhla2 (2,6) = 33.508, P aldhla2 = 0.001, F dmc1 (2,6) = 16.524, P dmc1 =

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cyp17a1

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2-fold) in the fish treated with both concentrations of TBT (F cyp17a1 (2,6) = 11.961, P

0.004). In treated fish, the mRNA level of sycp3 was sharply elevated by 3- to 4-fold, compared to the control (F (2,6) = 64.907, P < 0.001). In contrast, a slight but not

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significant increase in the mRNA level of cyp26a1 in TBT-exposed fish was observed (F (2,6) = 4.878, P = 0.055, Fig. 5).

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3.6 Effects of TBT on the reproductive behaviors of zebrafish Compared to the control (7.5 min staying in spawning area), both concentrations

of TBT sharply decreased the duration that both sexes spent simultaneously in the

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spawning area (approximately 2 min in the TBT-low group and less than 1 min in the TBT-high group) (F

(2,9) =

33.932, P < 0.001, Fig. 6A). Moreover, the frequency of

meeting in both treated groups were sharply decreased (100 and 50 meetings in the TBT–low and –high groups, respectively), compared to the control (more than 150 meetings) (F

(2,9) =

32.654, P < 0.001, Fig. 6B). After exposure, although the visit of

the non-exposed males in spawning area remained unchanged (P>0.05), the visit of

the TBT-exposed female to spawning area declined significantly in comparison to the control (F (2,9) = 10.093, P = 0.009, Fig. 6C). 3.7 Effects of TBT on mRNA levels of genes in behavioral regulation In the brain, the mRNA levels of cyp19a1b and gnrh-3 were dramatically suppressed in both TBT-exposed groups in a concentration-dependent manner (F cyp19a1b (2,6)

= 176.202, P

cyp19a1b

< 0.001, F

gnrh-3 (2,6)

= 116.29, P gnrh-3 < 0.001). Both

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concentrations of TBT also significantly down-regulated the mRNA levels of pet1

and tph2 (F pet1 (2,6) = 21.74, P pet1 = 0.002, F tph2 (2,6) = 19.891, P tph2 = 0.002). Moreover, (2,6) =

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the expression of kiss 1 was significantly suppressed in the TBT-high group (F

14.990, P < 0.018), although it remained unchanged in the TBT-low group (P = 0.991). In addition, both concentrations of TBT down-regulated the mRNA level of (2,6) =

11.769, P =

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kiss 2, showing significant change only in the TBT-high group (F

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0.008, Fig. 7).

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4. Discussion

Previous studies have reported the impaired fertilization success, diminished

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spawned egg number and decreased egg quality of the medaka females exposed to

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organotins (Nakayama et al., 2004; Zhang et al., 2008). In agreement with these studies, our data showed that TBT not only decreased the total egg number and hatchability but also elevated the mortality of the larvae, indicating the toxicity of

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TBT on the quantity and quality of the fish eggs. In fish, normal ovarian maturation is indispensable for successful reproduction and egg production (Nakayama et al., 2004).

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Therefore, disordered ovarian maturation may result in diminished egg production (Nakayama et al., 2004; Tian et al., 2015). In this study, the GSI decreased in a concentration-dependent manner, in agreement with the decreased total egg number

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and total follicle number in treated fish, suggesting an inhibitory role of TBT on fish ovarian maturation (Zhang et al., 2007). In cuvier, TBT exposures resulted in increased percentage of early-stage oocytes in fish (Zhang et al., 2007). Our data also indicated that the percentage of early-stage follicles increased significantly, while that of vitellogenic (EV, MV and FG) follicles decreased in ovaries of the exposed fish, suggesting the disruption of zebrafish oogenesis due to TBT. However, in contrast to

our data, no vitellogenic follicles were observed in cuvier exposed to TBT (1-100 ng/L) (Zhang et al., 2007). We propose that the difference might result from the exposure duration (50 days in cuvier, but only 28 days in our study) or the species-specific effects. In fish, oogenesis is under the direct modulation/control of estrogens (such as E2), which are synthesized by Cyp19a1 (Wang et al., 2017). TBT, which acts as a

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Cyp19a1 suppressor (Oberdörster and McClellan-Green, 2002; McAllister and Kime,

2003), may therefore disrupt oogenesis by altering the plasma E2 level. In cuvier

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treated with TBT, a lower E2 level has been detected (Zhang et al., 2007). The present

study also showed a significantly declined plasma level of E2 in TBT-treated fish in a concentration-dependent manner, consistent with the disrupted oogenesis reported in

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our study and the data in cuvier (Zhang et al., 2007). To date, two forms of Cyp19a1

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(Cyp19a1a and Cyp19a1b) have been identified in teleost fish (Chiang et al., 2001).

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Cyp19a1a is mainly located in the ovary; while Cyp19a1b is predominantly located in the brain (Vizziano-Cantonnet et al., 2011). In fish, both suppressed mRNA levels of

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cyp19a1 gene and lower Cyp19a1 activity result in decreased E2 level (Tian et al.,

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2015; Lima et al., 2015). In male guppies, TBT exposure sharply suppressed the expressions of both cyp19a1a and cyp19a1b in the ovary and brain, respectively, and significantly declined the E2 production (Tian et al., 2015). In zebrafish, azocyclotin

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(another type of organotin) exposure led to significantly down-regulated expression of cyp19a1a in the ovary (Ma et al., 2016). Consistent with these investigations, our data

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also revealed significantly suppressed mRNA levels of cyp19a1a in the ovary and cyp19a1b in the brain in response to TBT exposure. Decreased expressions of these genes may lead to diminished productions of Cyp19a1 proteins and subsequently the

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declined activity of these enzymes. Considering the direct inhibition of TBT on Cyp19a1 activity, our data strongly suggest that TBT may suppress E2 production by inhibiting Cyp19a1 productions (by down-regulating the cyp19a1 expressions) and their activities (both directly and indirectly). In fish, E2 plays pivotal roles in early oogenesis and oocyte growth (Colli-Dula et al., 2014). Altered E2 production may thus lead to disordered oogenesis in fish

(Hua et al., 2016). In accordance to declined E2 level obtained in this study, histological observation further revealed the increased percentage of PG follicles and decreased percentage of vitellogenic follicles in treated fish, suggesting an inhibitory role of the altered E2 level on fish oogenesis. Fish oogenesis consists of various cellular processes, such as the proliferation of germline stem cell, meiotic entry, meiotic maintenance and oocyte growth (Elkouby and Mullins, 2017). The increased

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percentage of PG follicles in TBT-treated fish were thus supposed to be closely related to elevated germ cell proliferation and/or meiosis (Wang et al., 2017).

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Previous reports have shown that Aldh1a2, Cyp26a1 and Sycp3 are involved in the

RA production and meiotic entry in fish (Li et al., 2016; Rodríguez-Marí et al., 2013; Wang et al., 2017). The present study revealed that although the mRNA level of

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cyp26a1 remained unchanged, the mRNA level of aldh1a2 was significantly

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up-regulated in TBT-treated fish, suggesting a possible increase in the production of

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Aldh1a2 protein. In most cases, transcription is the major regulator of gene product formation, and thus the activity of protein changes in the same direction as

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transcription. Our data suggested a probable enhanced activity of Aldh1a2 and

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increased production of RA. Moreover, the expressions of sycp3 and dmc1 (the marker of meiotic maintenance) were also dramatically elevated in TBT-treated fish, indicating the increased productions of these proteins. These data corresponded well

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with the increased percentage of PG follicles, indicating an enhanced level of meiotic entry and maintenance in zebrafish treated with TBT (Sansam and Pezza, 2015; Wang

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et al., 2017).

Oocyte growth is also under the influence of numerous growth factors that

stimulate the production and accumulation of Vtg, which is synthesized in liver (Tyler

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and Sumpter, 1996). The Vtg level and vtg expression in the liver are thus essential for oocyte growth (Wang et al., 2005). In fish, vtg expression is directly regulated by endogenous E2 (Knoebl et al., 2006). Previous reports revealed that higher E2 level (or enhanced estrogenic effect) led to elevated vtg mRNA level and Vtg production (Bowman et al., 2000), while lower E2 levels resulted in decreased expression of vtg (Villeneuve et al., 2009). In Chinese loach (Misgurnus anguillicaudatus), TBT

suppressed the E2-enhanced Vtg synthesis in vivo (Lv et al., 2009). In zebrafish, azocyclotin exposure down-regulated the vtg mRNA levels in the liver (Ma et al., 2016). Consistent with these observations, our data also showed a lower plasma level of E2 and a sharp suppression of vtg1 expression in treated fish. The suppressed vtg1 expression in the liver strongly suggests an insufficient Vtg production for oocyte growth, thus resulting in fewer vitellogenic oocytes in TBT-exposed fish (Fig. 8).

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On the other hand, coordinated reproductive behaviors between sexes are also

indispensable for reproductive success in fish (Weinberger and Klaper, 2014). TBT

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has been reported to disrupt reproductive behaviors in a few fish species. In medaka,

exposure to TBT (1μg/g body weight, dietary) for 3 weeks significantly decreased the sexual behaviors such as “following” and “dancing” (Nakayama et al., 2004). Male

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guppies exposed to environmentally relevant concentrations of TBT (5, 50 and 500

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ng/L) also showed sharply altered reproductive behaviors (Tian et al., 2015). However,

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most of the previous studies have focused on males; the impact of TBT on female reproductive behaviors is little known. The present study revealed that the

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TBT-treated females showed fewer visits to the spawning area; in contrast, the visits

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of the untreated males remained unchanged. Consistent with these results, the duration that both sexes spent simultaneously in the spawning area and the frequency of meeting also decreased significantly in a concentration-dependent manner,

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indicating the disrupted reproductive behavior of the females after TBT exposures. Reproductive behavior of fish is controlled by the central nervous system (Li et

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al., 2017). In various fish species, the Gnrh systems, which play a central role in neuroendocrine regulation, also control reproductive behaviors, such as courtship, mating, nest building and other behaviors (Li et al., 2017). To date, three forms of

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Gnrh (namely, Gnrh-1, Gnrh-2 and Gnrh-3) have been identified in vertebrates (Kim et al., 2011; Parhar et al., 2016); while two forms (Gnrh-2 and Gnrh-3) have been characterized in zebrafish (Steven et al., 2003). Gnrh-3, which is ubiquitously located, is recognized as the actual hypophysiotropic Gnrh form in zebrafish (Steven et al., 2003). Except for its role in reproduction, Gnrh-3 is also localized to extrahypothalamic area (Steven et al., 2003). Gnrh-3 neurons also project their neural

fibers throughout the brain, indicating its multiple roles in regulating social behaviors as a neurotransmitter or neuromodulator (Steven et al., 2003). In goldfish (Carassius auratus) and cichlid fish, Gnrh-3 has been found to stimulate female spawning behavior (Volkoff and Peter, 1999) and regulate nest building and territorial behaviors in male (Ogawa et al., 2006). A recent study in Japanese medaka revealed a novel function of the terminal nerve Gnrh-3 neurons as a gate for activating mating

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preferences based on familiarity (Okuyama et al,. 2014). In agreement to these

findings, the mRNA level of gnrh-3 in TBT-treated fish was dramatically suppressed,

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suggesting an inhibited production of Gnrh-3. The suppressed gnrh-3 expression also agrees well with arrested oogenesis and disrupted reproductive behaviors in TBT-treated fish. Our data suggest therefore that TBT may interfere with the

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reproductive behaviors of female zebrafish through inhibiting gnrh-3 expression and

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production of Gnrh-3 protein.

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It is worth noting that Cyp19a1b, which is responsible for E2 synthesis, is also located in the brain of zebrafish (Kishida and Callard, 2001). Intriguingly, in several

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fish species, E2 has been found to regulate gnrh expression in the brain. In the

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Chinese sturgeon (Acipenser sinensis), a significant increase in gnrh mRNA level was detected in fish receiving E2 implantation (Yue et al., 2013). In zebrafish and yellow catfish (Pelteobagrus fulvidraco), lowered plasma levels of E2 also resulted in sharply

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suppressed gnrh expression (Yin et al., 2017; Wang et al., 2017). These findings indicate that E2 exerts positive feedback effects on the transcription of the gnrh.

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Consistent with the previous data, the present study showed a suppressed mRNA level of cyp19a1b, which might lead to decreased Cyp19a1b production and subsequently lowered activity. These data were in agreement with the declined E2 level and the

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lower level of gnrh-3 expression. Therefore, lower E2 levels probably resulted from the decreased protein production of Cyp19a1b and inhibited activity of Cyp19a1b might be responsible for the suppression of gnrh-3 expression in the brain and the subsequent disturbance in reproductive behaviors. Kisspeptins have been recently reported to be involved in the stimulatory regulation on Gnrh neurons in fish (de Roux et al., 2003; Smith et al., 2006). In most

teleost species, two kisspeptins have been identified (Kiss 1 and Kiss 2) (Kitahashi et al., 2009). In zebrafish, Kiss 2-positive neurons and fibers are in close apposition with Gnrh-3 neurons (Servili et al., 2011), indicating an involvement of Kiss 2 systems in reproductive events by regulating Gnrh-3 (Kitahashi et al., 2009; Servili et al., 2011; Gopurappilly et al., 2013). Moreover, previous reports have demonstrated the stimulatory role of E2 on kiss 2 expression in zebrafish (Servili et al., 2011),

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indicating a positive regulation of E2 on kiss 2 (Mitani et al., 2010). In the present

study, the expression of kiss 2 was significantly suppressed after TBT exposure,

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consistent with the lower plasma level of E2 and the suppressed gnrh-3 expression. The disruption in the expressions of these genes and the alteration in E2 production are thus supposed to be closely related to the disrupted reproductive behaviors in

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TBT-treated fish.

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On the other hand, Kiss 1-expressing cells project only to the interpeduncular

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and raphe nuclei (Servili et al., 2011), indicating the possible involvement of Kiss 1 in non-reproductive functions (Roa et al., 2008). Recent studies showed that alarm

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substance (AS)-evoked fear experience significantly reduced kiss 1 expression in

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zebrafish, while Kiss 1 administration suppressed the AS-evoked fear response through modulating 5-HT synthesis (Ogawa et al., 2014; Nathan et al., 2015). The 5-HT neurotransmitter system is an important modulator of neural circuitry that

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controls a wide range of behavioral and physiological processes (Jacobs and Azmitia, 1992). In fish, tryptophan hydroxylase 2 (Tph2) and the Pet1 EST factor are both

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indispensable for 5-HT biosynthesis (McGeer et al., 1973). In this study, the expression of tph2 in TBT-treated fish was significantly down-regulated, indicating the probable reduced production of Tph2 protein and subsequent decreased enzyme

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activity. Moreover, the expression of pet1, a precise marker of the 5-HT neuron, was also significantly suppressed in both treated groups. These data suggest that TBT may play an inhibitory role on 5-HT synthesis, uptake, and storage (Hendricks et al., 2003). Considering the pivotal role of 5-HT in the mode control, disrupted 5-HT production after TBT exposure may therefore result in altered non-reproductive behavior (such as increased fear, anxiety and aggressiveness) and thereby impairing the reproductive

behavior of the exposed female (Hendricks et al., 2003; Nathan et al., 2015). Consequently, TBT might have altered both reproductive and non-reproductive behaviors of the exposed females and decreased the response of zebrafish females to the attractions of the males during mating (Fig. 8). 5. Conclusion The present study showed that TBT exposure suppressed the expressions of

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cyp19a1a/b and lowered the plasma level of E2. TBT exposure also enhanced the

meiotic entry and meiotic maintenance (reflected by elevated expressions of aldh1a2,

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sycp3 and dmc1) in the ovary, and decreased the vtg1 expression in the liver, thereby altering the percentages of follicles in different developmental stages. Moreover, TBT

exposure disturbed the reproductive behaviors of zebrafish females, probably by

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suppressing E2 production (down-regulation of cyp19a1b expression) in the brain and

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inhibiting the expressions of genes involved in reproductive behavior regulation

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(gnrh-3 and kiss 2). In addition, TBT might also increase the non-reproductive behaviors by suppressing the biosynthesis of 5-HT (inhibiting kiss 1, tph2 and pet1

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expressions). In conclusion, the results of exposure to TBT in zebrafish females

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demonstrated that disrupted oogenesis, disturbed reproductive behaviors and altered 5-HT synthesis induced by TBT may be responsible for the impaired reproductive success of zebrafish females. The present study greatly extends our understanding of

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the underlying mechanisms of reproductive toxicity of TBT on fish. Acknowledgements

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We would like to thank Y. Wang and P. Yin for their assistance with chemical

exposure and excellent sample preparation. This work was funded by the Chongqing Research Program of Basic Research and Frontier Technology (cstc2016jcyjA0133),

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the Scientific and Technological Research Program of Chongqing Municipal Education Commission (KJ130622, KJ1600308), the key project of Chongqing Normal University (13XLZ08) and the Open project fund of ‘Key laboratory of Freshwater Fish Reproduction and development (Ministry of Education, China) (FFRD-2015-02)’, China.

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Table 1 Primers and their sequences of the genes used in the present study

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Gene name (Protein name)

Primer sequences (5' to 3') F: CTCCCGAGTTCATTCAGA

vtg1 (Vitellogenin 1)

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cyp19a1a (Cytochrome P450 19a1a)

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R: ATGACAACTTCACGCAGA

cyp17a1 (cytochrome P450 17a1)

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aldh1a2 (Retinaldehyde dehydrogenase 1a2) cyp26 a1 (Cytochrome P450 26a1)

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sycp3 (Synaptonemal complex protein 3) dmc1 (DNA meiotic recombinase 1)

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cyp19a1b (Cytochrome P450 19a1b) kiss 1 (kisspeptin 1)

A

kiss 2 (kisspeptin 2)

F: TCATCGAGGGCTACAACGTG R: GATCCGAACGGCTGGAAGAA F: TCTGATGAGCCTGGTGAG R: ATGAGCAGTTTGTGGGAG F: TCCAGGAAGCCGACAAGG R: CCAGGGTAGCAAGGTAAGCA F: TGTCAGGAGGATACAGAATAGC R: AGCCGAGGTGTCATGGGT F: GCGTTTGTTTGTTGGATT R: CACCGTCAGTATGTTTGG F: TCGGGATACCAAGATGAT R: ATCTGGATGCCTTTCACC F: ACTAAGCAAGTCCTCCGCTGTGTACC R: TTTAAACATACCGATGCATTGCAGACC F: CAGGGGAACAGACACTCGTC R: CTCTCTTGCCATAGCGGAGG F: TCAATGGAGCGAAGGCAGTT R: CTGTCAGAGTCGCTGGTTGT

gnrh-3 (Gonadotropin releasing hormone subtype

F: TTGGAGGTCAGTCTTTGCCAG

3)

R: CCTCCATTTCACCAACGCTTC

pet1 (plasmacytoma expressed transcript 1) tph2 (tryptophan hydroxylase 2) rpl13a (ribosomal protein L13a) ef1α (Elongation factor 1α)

F: CCATTCAGTTTTCAGGTATTTCC R: GGCTGTGGTAGAGGGTTGGAG F: CCAGGAGTGCCTCATTACCA R: GCTCTGCGTGTAAGGGTTGT F: CCCGCGTGTCTTTCTTTTCC R: CTTGCTTGGCCACAATAGCG F: GATCACTGGTACTTCTCAGGCTGA

Accession NO.

Size (bp)

References

XM_009296387

133

Yin et al., 2017

NM_131154

151

Zhang et al., 2016

XM_005156809

108

Yin et al., 2017

NM_131850

165

Yin et al., 2017

NM212666

110

Yin et al., 2017

NM_001040350

126

Yin et al., 2017

NM_001020782

128

Yin et al., 2017

NM_131642

100

Yin et al., 2017

EF641126

185

Ogawa et al., 2014

NM_00114258

191

Ogawa et al., 2014

NM_182887

76

Yin et al., 2017

EF370169

105

Ogawa et al., 2014

NM_214795

139

Ogawa et al., 2014

NM_212784

111

Xu et al., 2016

NM_131263

121

Yin et al., 2017

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R: GGTGAAAGCCAGGAGGGC

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Fig. 8

Figure legends Fig. 1 Influences of TBT on the total egg number (A), hatchability rate (B) and mortality of the larva (C). TBT-low, 100 ng/L; TBT-high, 500 ng/L. Different letters on bars in each figure

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indicated statistical difference (P<0.05), N=9. Fig. 2 Influences of TBT on the body weight (A) and GSI (B) of zebrafish female. TBT-low, 100 ng/L; TBT-high, 500 ng/L. Different letters on bars in each figure indicated statistical difference

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(P<0.05), N=3.

Fig. 3 Influences of TBT on the ovarian structure (A-F) of zebrafish, total follicle number (G) and the ratio of developing follicles (H). TBT-low, 100 ng/L; TBT-high, 500 ng/L. D, E, F are the

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magnification of the boxed area in A, B, C, respectively. PG, primary growth; PV, pre-vitellogenic;

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EV, early vitellogenic; MV, mid-vitellogenic; FG, full grown. Scale bar, 100μm. Different letters

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on bars in each figure indicated statistical difference (P<0.05), N=3.

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Fig. 4 Influences of TBT on the plasma level of E2 (A) and the mRNA level of vtg1 in liver (B). TBT-low, 100 ng/L; TBT-high, 500 ng/L. Different letters on bars in each figure indicated

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statistical difference (P<0.05), N=3.

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Fig. 5 Influences of TBT exposures on mRNA levels of the genes involved in E2 production and meiosis. TBT-low, 100 ng/L; TBT-high, 500 ng/L. Different letters on bars in each figure indicated

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statistical difference (P<0.05), N=3. Fig. 6 Influences of TBT on the reproductive behaviors of zebrafish. TBT-low, 100 ng/L;

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TBT-high, 500 ng/L. A, duration of both sexes simultaneously in spawning area; B, frequency of meeting; C, visit in spawning area. Different letters on bars in each figure indicated statistical

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difference (P<0.05), N=9. Fig. 7 Influences of TBT on the mRNA levels of genes in reproductive behaviors and serotonin synthesis. TBT-low, 100 ng/L; TBT-high, 500 ng/L. Different letters on bars in each figure indicated statistical difference (P<0.05), N=3. Fig. 8 Schematic diagram of the Tributyltin impaired reproductive success. Red arrow up, elevated expression of genes; green arrow down, suppressed expression of genes, plasma levels of E2.