Tributyltin impaired spermatogenesis and reproductive behavior in male zebrafish
Introduction
Tributyltin (TBT), a widely used organotin compound, has been applied to many industrial usages, for instance the antifouling paint and agricultural pesticides due to its antiseptic, bactericidal and anti-mildew effects (Gipperth, 2009). At the end of last century, studies reported the sexual abnormality in gastropods induced by TBT (Smith, 1981a, b) and International Marine Organization (IMO) banned the use of TBT therefore as active ingredients in antifouling systems of ships (IMO, 2001). Despite the restriction, TBT is still widely and persistently distributed in the environment such as freshwater, estuarine, and coastal ecosystems (Antizar-Ladislao, 2008). TBT was also detected recently in marine sediments and economic animals (Abraham et al., 2017; Santos et al., 2009; Oh, 2009). In Indian and European surface waters, TBT concentrations ranging from 200 to 400 ng/L has been reported (Garg et al., 2011; Sousa et al., 2009). In mainland China, between 1988 and 2003, the concentrations of TBT ranged from undetectable to 976.9 ng Sn/L in waters and from undetectable to 1160 ng/L in sediments (Cao et al., 2009).
TBT has been reported to cause masculinization in female mollusks and biased sex ratio of zebrafish favoring males (Morcillo and Porte, 2000; Santos et al., 2006). Reduced fertility in rats, impeded growth in juvenile Japanese medaka (Oryzias latipes) and impaired immunological abilities in zebrafish (Danio rerio) have also been documented (Harazono et al., 1996; Horie et al., 2018; Zhang et al., 2017).
As an aromatase inhibitor, TBT has been shown to suppress estrogen production in fish through down-regulating aromatase gene expression and/or inhibiting the aromatase activity (Lyssimachou et al., 2006; McAllister and Kime, 2003). Consequently, the reproductive toxicity of TBT has been well documented, such as inhibiting survival and reproduction in Japanese medaka (Horie et al., 2018) and decreasing reproductive success in zebrafish and medaka (McAllister and Kime, 2003; Nakayama et al., 2004). Recent studies also demonstrated the TBT-disturbed testicular development and spermatogenesis in several aquatic organisms (Mochida et al., 2007; Revathi et al., 2014; Zhang et al., 2009). Although disrupted testicular development and spermatogenesis has been reported, the mechanisms involved in reproductive toxicity of TBT in fish remained largely unknown.
A great number of studies have demonstrated the inhibitory role of TBT on the production of estrogen from androgen, which is catalyzed by aromatase, leading to elevation of androgen levels in females (Nakanishi et al., 2005). A 28-day exposure of TBT on zebrafish male also induced elevation in 11-KT/E2 ratio (Liu et al., 2020). Androgens, particularly 11-ketotestosterone (11-KT), play key roles in testicular development and spermatogenesis of fish (Miura and Miura, 2003). Elevated androgen levels were thus supposed to enhance spermatogenesis in fish exposed to TBT. However, previous studies in TBT-treated cuvier (Sebastiscus marmoratus) and xenoestrogen-treated zebrafish showed a suppressed spermatogenesis which strongly suggest that altered androgen level might not be responsible for disturbed spermatogenesis in these species (Wang et al., 2019; Zhang et al., 2009). In contrast, changes of other key factors might be involved in TBT-induced spermatogenic disorders in these animals.
Although the spermatogenesis of fish is directly controlled by hypothalamic-pituitary-gonadal (HPG) axis, a series of local factors in testis play key roles in the modulation of spermatogenesis (Wang et al., 2019). Several cellular processes were indispensible for fish spermatogenesis, for example germ stem cell proliferation, meiosis and apoptosis (Schulz et al., 2010). Many cellular factors are indispensable for each of these cellular processes. It has been well demonstrated that Nanog homeobox is necessary for the S-phase transition and cell proliferation in embryonic development of medaka (Camp et al., 2009) and Cyclind1 protein is required in the regulation of cell transition from G1 phase to G2 phase (Stacey, 2003). Proliferating cell nuclear antigen (PCNA), which acts as a cofactor of DNA polymerases, also plays critical role in germ cell proliferation (Moldovan et al., 2007). All of these factors are all essential for the proliferation of germ stem cells in fish testis (Khillan, 2014; Wang et al., 2019). Moreover, Retinoic acid (RA), the amount of which depends predominantly on its synthase (Aldh1a2) and metabolic enzyme (Cyp26a1), acts as an initiator of meiotic initiation in fish (Feng et al., 2015). Affected entry of spermatogonia into meiosis was present when the expressions of aldh1a2 and cyp26a1 and/or abnormal RA production were observed (Feng et al., 2015). In addition, synaptonemal complex protein 3 (Sycp3) and DNA meiotic recombinase 1 (Dmc1) are indispensable for the formation of synaptonemal complex and recombination respectively in meiotic maintenance (Sansam and Pezza, 2015; Syrjänen et al., 2014). Altered expressions of these genes have been correlated with disturbed meiosis and spermatogenesis in fish (Chen et al., 2016; Li et al., 2016).
Apoptosis has also been recently observed to be involved in regulating the balance of quality and quantity of germ cells in spermatogenesis (Aitken et al., 2011). Two apoptotic (i.e. intrinsic and extrinsic) pathways have been identified so far to participate in apoptosis. The intrinsic pathway is regulated by the interaction of pro-inflammatory factors (such as Bax) and anti-inflammatory factors (such as Bcl-2) and Bax/Bcl-2 imbalances are thus responsible for cell death (Youle and Strasser, 2008). The extrinsic pathway of apoptosis depends mainly on the FAS or tumor necrosis factor receptor (TNFR), which is known as death receptors and induces apoptosis through triggering the activation of procaspases (Espín et al., 2013). In zebrafish, two TNFR subtypes (Tnfrsf1a and Tnfrsf1b) have been identified (Shalaby et al., 1990). Previous studies showed that Tnfrsf1a acts as a pro-inflammatory factor triggering apoptosis or inflammation whereas Tnfrsf1b is an anti-inflammatory factor promoting tissue repair and regeneration (Aggarwal, 2003). Elevated expressions of these genes may thus result in enhanced apoptosis thereby disrupting spermatogenesis in fish.
In addition to spermatogenesis, appropriate reproductive behaviors are also indispensable for successful reproduction (Weinberger and Klaper, 2014). Disordered sexual behaviors have been correlated to reduction in reproductive success of several fish species treated with TBT (Nakayama et al., 2004; Xiao et al., 2018). Reproductive behaviors are under the direct control of neuronal factors and endocrinal factors, such as GnRH and Kiss1/2, in the neuroendocrine system (Zohar et al., 2010). In zebrafish, two forms of GnRHs (GnRH2 and GnRH3) are present and GnRH3 stimulates synthesis and release of the gonadotropins as neuropeptides to play a role in spawning behavior (Ogawa et al., 2006; Steven et al., 2003). Kiss2 has also been reported recently to regulate the reproductive behavior by activating the GnRH system in zebrafish (Shahjahan et al., 2013). E2 in the brain has been recently shown to act on the regulation of Kiss2 and GnRH in fish (Yin et al., 2017; Servili et al., 2011). Moreover in zebrafish, Cyp19a1b which is responsible for E2 production in the brainis located in the brain (Simpson et al., 1994), also indicating a possible involvement of E2 on the regulation of Kiss2 and GnRH In addition, olfactory nervous system cues are also able to stimulate courtship in male adult fish when perceiving of female sex pheromones (Friedrich and Korsching, 1998). When male adult fish perceive the female sex pheromone, the olfactory marker protein b (Ompb) is activated and androgens level in the plasma is elevated, thus promoting their reproductive behavior (Ghosal and Sorensen, 2016). Alteration in the expressions of these genes may impair reproductive behaviors and disrupt reproduction in fish.
In order to further demonstrate the reproductive toxicity of TBT on fish, a 28-day exposure with two nominal concentrations (100 and 500 ng/L) of TBT was conducted using adult male zebrafish. After exposure, reproductive behaviors were investigated and the parameters, such as frequency of meeting; visits of both sexes in spawning area; duration in spawning area; visits of each sex to spawning area, were analyzed. The phenotypic factors, such as testis weight, gonadosomatic index (GSI) and sperm count, were subsequently measured. The reproductive toxicity of TBT on spermatogenesis was also assessed by histology, PCNA-immunostaining (IHC) and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assays. To better understand the underlying mechanisms, the transcriptional expression of genes involved in germ cell proliferation (nanog, cyclind1 and pcna), meiotic initiation (aldh1a2 and cyp26a1), meiotic maintenance (sycp3 and dmc1) and apoptosis (fas and the ratios of bax/bcl-2 and tnfrsf1a/tnfrsf1b) in the testis and reproductive behaviors (cyp19a1b, gnrh3, kiss2 and ompb) in the brain were investigated by quantitative RT-PCR (qRT-PCR).
Section snippets
Chemicals
Tributyltin chloride (CAS No. 1461-22-9, CAT No. 45713-250MG) was obtained from Sigma Aldrich (USA) and the purity of TBT is higher than 96 %.
Experimental fish
Male zebrafish (AB strain, 4-months) were provided by China Zebrafish Resource Center and acclimated for 14-day in 200 L glass aquaria containing aerated-dechlorinated water with a photoperiod of 14 h light/ 10 h darkness at 28 ± 0.5 °C before use. The fish were fed with Artemia nauplii (Artemia International LCC, USA) and flake food daily during
Testis weight, GSI and sperm number
Testis weight and GSI of the treated fish didn’t show significant change in comparison to the control (Fig. 1A & B, P > 0.05). However, the sperm count declined significantly in a concentration-dependent manner in TBT treated groups (Fig. 1C, P < 0.05).
Histology, IHC and TUNEL assays
Spermatocysts containing large amount of germ cells in different developmental stage (such as spermatogonia, spermatocytes, spermatids or spermatozoa) were observed in the testis of control fish (Fig. 2A). In comparison, increased number of
Discussion
Our data showed that TBT exposures didn’t affect the testis weight and GSI of the fish. However, TBT significantly decreased the sperm count in treated fish. In accordance with decreased sperm count in testis, reduced number of spermatozoa and several lacunas probably resulting from sperm loss were observed in treated fish. These findings strongly suggest a reproductive toxicity of TBT on adult male zebrafish.
Spermatogenesis in fish is controlled by HPG axis, particularly by a direct modulation
Conclusion
In the present study, a 28-day exposure of TBT disrupted spermatogenesis in adult male zebrafish, evidenced by decreased number of spermatogonia, increased number of spermatocytes and reduced sperm count. PCNA-immunostaining and TUNEL also demonstrated enhanced proliferation in spermatogonia and apoptosis in spermatocytes. Accordingly, increased mRNA levels of genes involved in meiotic entry and maintenance (aldh1a2, sycp3 and dmc1) and down-regulation of genes responsible for terminus of
Declaration of Competing Interest
The authors have declared that no conflict of interest exists.
CRediT authorship contribution statement
Xue-Rong Lan: Investigation. Ying-Wen Li: Writing - original draft. Qi-Liang Chen: Writing - review & editing. Yan-Jun Shen: Data curation. Zhi-Hao Liu: Funding acquisition, Supervision.
Acknowledgements
We would like to thank WY Xiao and YQ Wang for their assistance with sampling and reproductive behaviors analysis. This work was funded by the Chongqing Research Program of Basic Research and Frontier Technology (cstc2016jcyjA0133); the Scientific and Technological Research Program of Chongqing Municipal Education Commission (KJ1600308); the Open project fund of ‘Key laboratory of Freshwater Fish Reproduction and development (Ministry of Education, China) (FFRD-2015-02)’, China and the Open
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