Accepted Manuscript Title: Semicarbazide disturbs the reproductive system of male zebrafish (Danio rerio) through the GABAergic system Authors: Miao Yu, Yongliang Feng, Xiaona Zhang, Jun Wang, Hua Tian, Wei Wang, Shaoguo Ru PII: DOI: Reference:
S0890-6238(17)30163-6 http://dx.doi.org/10.1016/j.reprotox.2017.08.007 RTX 7562
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Please cite this article as: Yu Miao, Feng Yongliang, Zhang Xiaona, Wang Jun, Tian Hua, Wang Wei, Ru Shaoguo.Semicarbazide disturbs the reproductive system of male zebrafish (Danio rerio) through the GABAergic system.Reproductive Toxicology http://dx.doi.org/10.1016/j.reprotox.2017.08.007 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.
Semicarbazide disturbs the reproductive system of male zebrafish (Danio rerio) through the GABAergic system Miao Yu, Yongliang Feng, Xiaona Zhang, Jun Wang, Hua Tian, Wei Wang, Shaoguo Ru*
College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China
This manuscript has 28 pages, 5 Figures
*Corresponding author College of Marine Life Sciences, Ocean University of China, 5 Yushan Road, Qingdao 266003, Shandong Province, P.R. China Phone: +86–532–82031962 Fax: +86–532–82031962 E-mail: [email protected]
BSI, brain-somatic index; CYP, cytochrome P450; E2, 17β-estradiol; ELISA, enzyme-linked immunosorbent; FSH, follicle-stimulating hormone; fshr, FSH receptor; GABA, γ-aminobutyric acid; GAD, glutamate decarboxylase; gabaa, GABA A receptor; gabab, GABA B receptor; GnRHs, gonadotropin-releasing hormones; gnrhr, GnRH receptor; GtHs, gonadotropic hormones; hmgcr, 3-hydroxy-3-methylglutaryl-CoA reductase; HPG, hypothalamic-pituitary-gonadal; hsd, hydroxy steroid dehydrogenase; LH, luteinizing hormone; lhr, LH receptor; NMDAR, N-methyl-D-aspartate receptor; PC, principal component; PCA, principal component analysis; RT-qPCR, real-time quantitative polymerase chain reaction; SEM, standard error of the mean; sgnrh, salmon GnRH; SMC, semicarbazide; star, steroidogenic acute regulatory protein; T, testosterone; TSI, testicular somatic index.
Male zebrafish were exposed to semicarbazide for 28 d.
Testis histology was affected after semicarbazide exposure.
Semicarbazide exposure reduced plasma testosterone and 17β-estradiol levels.
Semicarbazide exposure altered gene transcription along the GABAergic system.
Semicarbazide disturbs the reproductive system of male zebrafish.
Abstract Semicarbazide (SMC), an emerging water contaminant, exerts anti-estrogenic effects in female zebrafish. However, the exact influence of SMC on male reproduction remains unclear. In this study, adult male zebrafish were exposed to 1-1000 μg/L SMC in a semi-static system for 28 d prior to examining the testicular somatic index (TSI), testis histology, plasma sex hormone levels, and the transcription of genes involved in reproduction. The results showed that testicular morphology was altered and TSI was down-regulated by high concentrations of SMC (≥ 100 μg/L and 1000 μg/L, respectively). Plasma testosterone and 17β-estradiol concentrations were significantly decreased by all of the SMC treatments, along with down-regulation of the corresponding steroidogenic gene transcripts. These changes were associated with the inhibition of gamma-aminobutyric acid synthesis
and function, in addition to the decreased expression of reproductive regulators. Our results contribute to elucidating the mechanisms underlying the adverse reproductive effects of SMC in male zebrafish. Keywords: Semicarbazide; reproductive endocrine disruption; mechanism; gamma-aminobutyric acid; male zebrafish
1. Introduction Semicarbazide (SMC) is a hydrazine derivative that occurs in the environment and food due to the metabolism of nitrofurazone, the partial degradation of azodicarbonamide, and a reaction between hypochlorite and food additives [1-4]. SMC has been detected at levels of up to 53.9 ± 3.1 µg/kg in European jarred baby foods , 260 μg/kg in commercial bread products , and 46.41 ± 21.22 µg/L and 6.46 ± 0.03 µg/kg in sea water and shellfish, respectively, measured in a survey of coastal waters adjacent to the Chaohe river estuary . Published experimental data have indicated that SMC acts as an inhibitor of many enzymes in vertebrates, including glutamate decarboxylase (GAD), lysyloxidase, and semicarbazide-sensitive amine oxidase; reduced activities of these enzymes result in toxic effects such as convulsions , injury to the cardiovascular and skeletal systems [9,10], and suppression of food consumption and body weight gain [11,12] in SMC-treated animals. Moreover, SMC was shown to act as a reproductive endocrine disruptor with anti-estrogenic activity [13-15]. Other well-characterized anti-estrogens such as ICI 182,780, tamoxifen, and some aromatase inhibitors (for example, anastrozole and letrozole) show negative effects on the male reproductive system, although their exact toxic effects differ. Oliveira et al. observed dilated testicular efferent ductules and seminiferous tubules in the absence of any change in the plasma testosterone (T) and luteinizing hormone (LH) concentrations in ICI 182,780-treated male rats , while male rats injected with tamoxifen showed significant decreases in plasma T and LH levels . In another study, oral administration of anastrozole elevated plasma T and follicle-stimulating hormone (FSH) concentrations, while testicular structure and spermatogenesis appeared normal . It is likely that the distinct modes of action of these anti-estrogens lead to the discrepant effects observed in males.
A previous review summarized the different mechanisms underlying alterations in puberty that are driven by endocrine disrupters in rodents, and proposed that anti-estrogenic SMC showed unconventional modes of action . In contrast to many classic anti-estrogens, which act directly on estrogen receptors or aromatase (cytochrome P450 (CYP) 19), the authors of the review suggested that the effects of SMC on neurotransmitter systems (antagonism of the N-methyl-D-aspartate receptor [NMDAR]) might be the key reason for its endocrine-disrupting activities. In our previous study, we also found that SMC interfered with neurotransmission in female zebrafish (Danio rerio) by decreasing GAD transcription and consequently inhibiting the biosynthesis of γ-aminobutyric acid (GABA) . GABA plays an important role in regulating the hypothalamic-pituitary-gonadal (HPG) axis via stimulation of the secretion of gonadotropin-releasing hormones (GnRHs) and gonadotropic hormones (GtHs) , and we demonstrated that this disturbance of the GABAergic system could at least partially explain the altered levels of mRNAs encoding HPG axis proteins, and the concomitant decrease in plasma 17β-estradiol (E2) . These findings indicated that the unconventional modes of SMC action also operated in a teleost species. However, zebrafish literature has reported sex differences in the responses to these neurotransmitter system-disturbing chemicals. For example, exposure to a dopamine antagonist (chlorpromazine) for 2, 4, 14, or 28 d increased the number of differentially expressed genes in females over time, but decreased those observed in males, and these differentially expressed genes were classified into diverse functional classes . For this reason, it is important to clarify whether the unconventional mechanisms of SMC also operate in males, how these differ from those observed in females, and what consequences they have for the male reproductive systems. However, the information available in these fields is fairly limited, especially for male aquatic organisms.
The available genomic resources and other advantages of zebrafish, including their high physiological and genetic homology to humans, cost- and space-effectiveness, ease of genetic and other experimental manipulations, and high fecundity, has ensured their popularity as a vertebrate model for determining the effects and molecular targets of endocrine-disrupting compounds [22,23]. Therefore, we used this attractive species to investigate the effects of SMC on male zebrafish reproduction by examining the testicular somatic index (TSI), brain-somatic index (BSI), testicular histology, and plasma sex hormone levels (T and E2). In addition, the transcriptional levels of genes involved in neurotransmitter systems, the HPG axis, and leptin signaling (which regulates the HPG axis), were quantified to elucidate the potential mechanisms of action of SMC.
2. Materials and methods 2.1. Fish maintenance Wild-type sexually mature (5 months-old) male zebrafish with an average length of 2.87 ± 0.18 cm (mean ± the standard error of the mean [SEM]) and an average weight of 0.36 ± 0.06 g (mean ± SEM) were purchased from a local distributor (Nanshan Aquaria, Qingdao, China). They were acclimated in 50-L glass tanks containing 40 L dechlorinated tap water (~70 fish/tank) for 4 weeks in our laboratory with a light: dark cycle of 14:10 h prior to SMC exposure. The fish were maintained at a temperature of 27 ± 1 °C and a pH of 6.5 ± 0.1, with a dissolved oxygen concentration of 7.0 ± 0.1 mg/L; these basic water quality parameters were monitored twice weekly. Commercial dried cysts of Artemia were purchased from a local supplier (Haifa Company, Shandong, China) and hatched for approximately 24 h under the following optimal conditions, reported by Sorgeloos et al. : constant temperature of 28 °C, 30 ppt salinity, pH of 8.0, dissolved oxygen concentration of 5 mg/L, cyst densities of 2 g/l, and
strong illumination of 2000 lux. Zebrafish were fed twice daily with an equal amount of newly hatched Artemia nauplii. 2.2. Exposure experiment and sampling SMC hydrochloride (CAS No: 563-41-7, purity ≥ 98%) was supplied by Sigma (Shanghai, China). Because of its high hydrophilicity, no cosolvent was added to any of the SMC solutions. The nominal concentrations for SMC were 1, 10, 100, and 1000 μg/L, based on the environmental concentration (46.41 ± 21.22 µg/L)  and the 96-h median lethal concentration (26.29 mg/L) of SMC . Stock solutions were prepared at a concentration of 10 mg/mL in deionized water and used for no longer than 2 weeks. The stock solutions were considered to be stable because a previous study indicated that a 1-mg/mL stock solution of SMC was stable for at least 10 months . Daily stock aliquots were prepared by diluting this stock to produce final concentrations of 100 and 1000 μg/mL. All stock solutions were stored in amber glass vials wrapped with aluminum foil at 4 °C until use. The exposure experiments were conducted in 5-L glass tanks filled with 4 L well-aerated control water or exposure medium. Three replicate tanks, each containing seven male zebrafish, were allocated for water control or SMC treatments. Fish were exposed to 1, 10, 100, or 1000 μg/L SMC for 28 d and a 24-h semi-static renewal procedure was used. The actual concentrations of SMC at the beginning (0 h) and prior to full water renewal (24 h) were measured in a previous study in our laboratory . Since the actual exposure concentrations were close to the nominal ones, the nominal concentrations were used for the results presented throughout this study. The water quality parameters, photoperiod, and feeding were kept consistent with those described in section 2.1. Mortality was recorded daily. After the 28-d exposure, fish were anesthetized in 0.03% MS-222 and weighed. The total weight measurement (Table S1) and blood collection were conducted, based on
the method described previously . The testes and brains were dissected and weighed (Table S1) in order to calculate the TSI and BSI: TSI = 100 × [testes weight (g)/body weight (g)]; BSI = 100 × [brain weight (g)/body weight (g)]. Testes and brains were frozen in liquid nitrogen after dissection and then stored at -80 °C until further analysis. All studies were conducted in accordance with the guidelines for the care and use of laboratory animals of the National Institute for Food and Drug Control of China. 2.3. Histological examination The testes (n = 3; one from each replicate tank) were fixed in Bouin’s solution for 24 h. The fixed testes were then dehydrated in increasing concentrations of ethanol (50–99.9%), cleared in xylene, and embedded in paraffin wax. Samples were step-cut into 5-μm sections, mounted on glass slides, and stained using hematoxylin and eosin (Sigma-Aldrich, Shanghai, China). Sections from the control and SMC groups were examined and photographed under a light microscope (Eclipse E200, Nikon, Tokyo, Japan). 2.4. Quantification of sex hormone concentrations After euthanasia, blood samples from 6 individual fish from the same exposure tank were pooled as one replicate (n = 3; one from each replicate tank) and then immediately centrifuged at 3,000 × g for 10 min. The resulting supernatants were divided equally into two parts for T and E2 analysis. Hormones were extracted from the supernatant (10 μL) 3 times using diethyl ether, as previously described . Plasma sex hormone levels were quantified using commercial ELISA kits (T: Cat No. 582701 and E2: Cat No. 582251) obtained from Cayman Chemical (Ann Arbor, MI, USA), in accordance with the manufacturer’s protocol. The detection limits were 6 pg/mL for T and 19 pg/mL for
E2, and the intra- and inter-assay coefficients of variation were within 10%. The control and SMC exposure samples were analyzed in triplicate, as were the standards. 2.5. Analysis of gene transcription Three pairs of testes or three brains were pooled as one sample to provide sufficient material for gene expression analysis (n = 6; two from each replicate tank). RNA isolation, cDNA synthesis, and real-time quantitative polymerase chain reaction (RT-qPCR) assays were performed as described in our previous work . Briefly, total RNA was extracted from the testes and brains using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). After measuring the RNA concentration, equal amounts of RNA (1 μg) were reverse-transcribed to produce cDNA, using a commercial reverse transcription system (TaKaRa, Dalian, China). All procedures were conducted in accordance with the manufacturer’s instructions. The specific primers for RT-qPCR were obtained from the previous study , with the exception of those employed for nmdar, which were designed using Primer Premier 5.0 (Premierbiosoft, Palo Alto, CA, USA) using the published sequence. Before starting the mRNA expression assays, the amplification efficiencies of the primers were calculated from standard curves constructed using serial dilutions of pooled cDNA. These values ranged from 90-110%. The optimal combination of reference genes was determined by geNorm analyses (https://genorm.cmgg.be/), which showed that β-actin and the elongation factor 1α provided the best reference combination in both testis and brain under SMC treatments. More detailed information about the primers used is presented in Table S2. The amplification procedure was as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min (MasterCycler ep RealPlex4 Real-Time System, Eppendorf, Wesseling-Berzdorf, Germany). Selected gene expression was measured in triplicate, and the analysis was repeated 3 times. The relative mRNA expression levels of the target
genes were normalized to the geometric mean of the two internal control genes  using the 2-ΔΔCt method . The normalized values in the SMC treatment groups were expressed as fold changes, relative to the normalized control values. 2.6. Statistical analysis All statistical analyses were performed using IBM SPSS Statistic 22 (IBM Corp., New York, USA). The Kolmogorov-Smirnov test and Levene’s test were used to check data normality and homoscedasticity, respectively. Data were log-transformed to approximate normality when necessary. Differences between the control and SMC treatment groups were analyzed using one-way analysis of variance, followed by Tukey’s multiple range test. The statistical significance threshold was set at p < 0.05. To investigate the relationship between plasma sex hormone levels and gene transcription, we conducted the following analyses. First, the bivariate correlations between 25 gene transcripts were assessed using Spearman correlation analysis. Principal component analysis (PCA) was then conducted to reduce the dimensionality of the data by transforming the correlated gene variables into relatively fewer numbers of uncorrelated variables, called the “principal component” (PC). For the first two PCs, the correlations between plasma hormone levels and gene transcription were analyzed using regression analysis. Furthermore, the differences between the treatment groups were visualized using the PC1 and PC2 scores for each fish.
3. Results 3.1. Body conditions
The survival rates of both control and SMC treatment groups were 100% during the exposure period. For TSI, the only significant difference from the control group was a decrease in the TSI of fish exposed to the highest SMC concentration (1000 μg/L). The BSI did not vary detectably between any of the study groups (Fig. 1). No tank effects on these somatic indices were observed between the three replicates. 3.2. Testis histology The normal zebrafish testis consists of seminiferous tubules lined with asynchronously developed spermatogenetic cell clusters, called spermatocysts. The spermatogenetic cells can be classified into three types, depending on the developmental stage: spermatogonia; primary or secondary spermatocytes; and spermatids or mature sperm. In a single cyst, all of the cells are at the same developmental stage. The spermatocysts exist next to each other and form a cavity in the center of the tubule, where mature sperm are stored. The testes of the control fish showed a regular arrangement of spermatocysts, with normal successive stages of spermatogenesis, and the cavity was filled with sperm (Fig. 2A). In fish exposed to ≤ 10 μg/L SMC, no gross differences were detected from the controls (Fig. 2B, 2C). In the 100-μg/L treatment group, we observed densely packed sperm and there were many spaces in the seminiferous tubules (Fig. 2D). Fish exposed to 1000 μg/L SMC showed a severely altered testis structure, with a disordered arrangement of spermatocysts, a broken tunica propria, and fewer sperm (Fig. 2E, 2F).
3.3. Plasma sex hormones
Plasma T and E2 levels were significantly reduced in zebrafish exposed to all of the SMC treatments for 28 d, as compared with the controls, but there was no direct linear concentration–response relationship (Fig. 3).
3.4. Gene transcription profiles Exposure to SMC caused significant changes in gene transcription in the brains and testes of male zebrafish. After exposure to SMC for 28 d, brain levels of gad67 mRNA were strongly decreased in fish exposed to 1000 μg/L SMC, and the mRNA expression levels of gad65, GABA B receptor (gabab), GnRH receptor (gnrhr) 1, gnrhr2, and gnrhr4 were significantly down-regulated in the 100- and 1000-μg/L SMC treatment groups. SMC exposure at 10, 100, and 1000 μg/L resulted in significant reductions in the transcription levels of the GABA A receptor (gabaa) and fshβ, while lhβ mRNA levels were significantly decreased in response to all of the tested SMC concentrations. However, chicken GnRH-II showed the opposite direction of change, where its mRNA expression was greatly stimulated by SMC. The transcription of GABA transaminase, nmdar, and salmon GnRH (sgnrh) remained unchanged (Fig. 4A). In the testes, exposure to 100 and 1000 μg/L SMC produced significant down-regulation of 3-hydroxy-3-methylglutaryl-CoA reductase (hmgcr) B, steroidogenic acute regulatory protein (star), 3β hydroxy steroid dehydrogenase (3βhsd), and 17βhsd mRNA levels, and the transcription of the FSH receptor (fshr) and leptinb was significantly decreased in the 10-, 100-, and 1000-μg/L SMC treatment groups. All of the tested SMC concentrations significantly reduced the mRNA levels of LH receptor (lhr), hmgcra, cyp11a, cyp17, cyp19a, and leptina (Fig. 4B).
We assessed the relationship between the mRNA expression levels of the selected genes and the plasma sex hormone levels. Since a Spearman correlation analysis showed that the mRNA levels of the majority of these 25 genes were highly correlated with each other (r > 0.5, p < 0.01; Figure S1), PCA was employed to reduce the number of variables. This analysis of the selected genes showed that PC1 and PC2 could explain 56.31% and 9.58% of the total variance, respectively (Fig. 5, Table S3). PC1 was highly influenced by variables such as gad65, gabaa, gabab, gnrhr1, gnrhr4, leptina, leptinb, hmgcra, star, cyp11a, and cyp17 (loading factor > 0.80, Table S3), and was significantly correlated with plasma levels of T (β = 0.727, p < 0.0001) and E2 (β = 0.780, p < 0.0001). 4. Discussion In our present study we noted a correlation of altered mRNA levels of the HPG axis and decreased plasma T and E2 concentrations by SMC in male zebrafish, which coincided with testicular morphological changes. This suggests an unconventional mode of action for endocrine disruption by SMC in male zebrafish, as has been reported in rats before . However, in contrast with the view that the glutamatergic NMDAR was a potential site of SMC action , we did not detect significant changes in the brain mRNA expression of the nmdar in zebrafish exposed to SMC, as compared with the control group. Instead, we found that SMC induced significant decreases in the expression of the rate-limiting enzyme for GABA synthesis (GAD) and GABA receptors. This indicated that the GABAergic system was the key target for SMC in male zebrafish. In vertebrates, GAD exists as two major isoforms that are known as GAD65 and GAD67, reflecting their approximate molecular masses . GABA receptors are divided into ionotropic (GABAA/C) and metabotropic (GABAB) types . After the 28-d SMC exposure, the mRNA levels of gad65, gad67, gabaa, and gabab were significantly down-regulated in the brains of male zebrafish, indicating that SMC inhibited both the synthesis and
normal biological activity of GABA; this would subsequently influence the production of GtHs. In teleosts, GABA stimulates the secretion of GtHs by enhancing the release and activity of GnRH [32,33], as well as inhibiting the dopaminergic system [34,35]. In the present study, transcription of the major hypophysiotropic form of GnRH (sgnrh) in zebrafish  was not significantly affected by SMC, although the mRNA expression levels of GnRH receptors (gnrhr1, gnrhr2, gnrhr4) and gonadotropins (fshβ, lhβ) were significantly reduced by exposure to SMC for 28 d. This suggested that SMC may reduce the responsiveness of the pituitary to GnRH stimulation by inhibiting the GABAergic system, suppressing the production of GtHs. We also detected concomitant down-regulation of fshr and lhr expression. However, our previous study of female zebrafish showed that 28-d SMC exposure significantly decreased the brain transcription of sgnrh . These findings indicate that there are sex differences in the response of the neuroendocrine system to SMC; these might be related to the neuroprotective effects of estrogens on this system in female animals [37-40]. Therefore, the significant reduction in the level of circulating E2 caused by SMC would abrogate its neuroprotective effects, leading to a decrease in the expression of the hypothalamic hormone, sgnrh. However, E2 may provide less protection to the male neural system because of the relatively low basal E2 concentration. Thus, the SMC-induced decrease in E2 levels in male fish was not associated with a significant change in sgnrh expression, as compared with the control group. Generally, LH is considered to be the main regulator of androgen production in male mammals. LH interacts with its receptor on the testicular Leydig cell membrane and stimulates a cyclic adenosine monophosphate-mediated second messenger pathway. This results in alterations in the mRNA expression or activities of steroidogenic enzymes/proteins, which subsequently affect the de novo production of T from cholesterol . FSH binds to its receptor on Sertoli cells and thus promotes
spermatogonia proliferation and the activation of various factors required for successful spermatid production [42,43]. However, there are species-related differences in these systems. In teleosts, FSH receptor expression was identified in Leydig cells, and this hormone stimulated androgen production both in vitro and in vivo [44,45]. In the present study, the observed reductions in the mRNA levels of lhβ, fshβ, lhr, and fshr would be predicted to inhibit the expression of genes or enzyme activities involved in testicular steroidogenesis, and thus reduce the plasma sex steroid hormone concentrations. Plasma sex hormone levels, which have been established as some of the most integrative and functional reproductive parameters, have been measured in many toxicological studies using zebrafish [46-48]. Our present results showed that 28-d SMC exposure markedly down-regulated plasma T and E2 concentrations in male zebrafish, and that this was accompanied by the reduced expression of genes encoding proteins involved in T and E2 biosynthesis (star, cyp11a, 3βhsd, cyp17, and cyp19a). A previous study in male rats that were treated with SMC for 28 d also reported a significant decrease in plasma androgen levels . The key steps in T synthesis include the first and rate-limiting step, where cholesterol is converted into pregnenolone by CYP11A , and the reactions catalyzed by CYP17, which directs the biosynthesis of steroids toward the sex hormones . If the expression level of CYP11A or CYP17 is modulated, the production of T will be affected [51-53]. Thus, the SMC-induced reduction in plasma T concentrations observed in all exposure groups in the present study was possibly due to the down-regulated transcription of cyp11 and cyp17. In addition, the reduced mRNA expression of star, 3βhsd, and 17βhsd in the presence of 100 or 1000 μg/L SMC made an important contribution to the decreased T concentrations. CYP19 plays a key role in converting T to E2, so the significant down-regulation in cyp19a expression might account for the
reduced E2 levels present in all treatment groups. It is notable that the concentration-response relationships were non-monotonic for plasma T and E2, with a lower T concentration in male zebrafish exposed 10 μg/L SMC than in those exposed to 100 or 1000 μg/L SMC, and a slightly higher E2 concentration in zebrafish exposed to 1000 μg/L SMC than in those exposed to 100 μg/L SMC. Vandenberg et al.  stated that endocrine-disrupting chemicals often show non-monotonic dose-response curves. For example, treatment with 1, 3, 10, 300, or 1000 μg/L prochloraz down-regulated plasma E2 levels, while exposure to 100 μg/L prochloraz slightly up-regulated the plasma E2 levels . Changes in sex hormone levels can result from altered expression of genes encoding proteins involved in T and E2 biosynthesis and in the present study, cyp19a mRNA and E2 levels showed similar SMC concentration-response relationships. However, fish exposed to 10 μg/L SMC had a lower plasma T concentration, and higher mRNA levels of several steroidogenic genes including hmgcrb, cyp17, and 17βhsd, as compared with other SMC exposure groups. This suggested that other factors besides hormone biosynthesis, such as hormone transportation and metabolism, may also regulate plasma sex hormone concentrations, and this will require further investigation. In addition to its regulation by the HPG axis, sex hormone production is under the control of many other direct or indirect regulators, such as leptin. Leptin is a 16-kDa protein that is mainly produced by adipose tissue in mammals , while the gonads express the highest levels of leptin in zebrafish . In addition to regulating appetite, metabolic rate, and energy stores, one of leptin’s most established roles is in modulating reproduction by acting on multiple targets along the HPG axis. In general, leptin enhances the release of GnRHs and GtHs from the hypothalamus and pituitary, respectively, thereby stimulating the reproductive axis [58-60]. Nevertheless, elevated concentrations of leptin can inhibit androgen production by directly affecting Leydig cell function in males [61-63]. In the present study,
significant reductions in leptina and leptinb mRNA expression levels were detected in the male zebrafish testis after SMC exposure for 28 d. This result was consistent with a previous observation of reduced leptin transcription in SMC-treated male rats . A decline in the level of leptin would prevent its stimulation of the secretion of GnRHs and GtHs; this might be one of the mechanisms underlying the observed down-regulation of lhβ and fshβ expression. On the other hand, this might somewhat relieve leptin-mediated inhibition of T production, but this effect was not powerful enough to normalize the T level. Comparison with SMC-exposed female zebrafish revealed sex-related differences in leptin expression, which was significantly elevated in females and reduced in males. This was probably due to sex-specific differences in the expression and activity of leptin [64,65]. T is a well-characterized and highly important androgen in males, playing vital roles in the morphological development of reproductive organs and the expression of conspicuous sexual behaviors, as well as promoting spermatogenesis and secondary sexual characteristics . Nevertheless, many studies have proven that the ‘female’ hormone, E2, also has a role in the regulation of the male reproductive system; this includes promoting the proliferation of spermatogonia, maintaining the normal testis structure, and enhancing the reabsorption of luminal fluid in the efferent ductules [67-69]. Thus, alterations in the plasma concentrations of these sex hormones may lead to histologic changes in the gonads. The present study found that decreases in plasma T and E 2 levels were accompanied by severe alterations in testis structure; the significant reduction of TSI in male zebrafish exposed to 1000 μg/L SMC also corroborates this hypothesis. In addition, high concentrations of SMC can produce reactive oxygen species , which may contribute to the testicular abnormalities observed in the present study. A high concentration of SMC was also reported
to induce marked changes in rat testicular morphology, with shrunken seminiferous tubules and depleted germ cells . We also analyzed the relationships between sex hormone concentrations and gene transcription using PCA. These results indicated that PC1, which was mainly influenced by the mRNA levels of gad65, gabaa, gabab, gnrhr1, gnrhr4, leptina, leptinb, hmgcra, star, cyp11a, and cyp17, was highly correlated with both T and E2 levels. This statistical analysis of the genetic factors that were most closely associated with the changes in plasma sex hormone levels produced findings that were in agreement with the biological conclusions drawn above. In summary, the present study showed that SMC causes male reproductive effects by an unconventional mode of action. The identified SMC-mediated inhibition of GABA synthesis and function would modulate gene transcription along the HPG axis and consequently reduce sex hormone levels and impair testis structure. In addition, SMC-induced changes in the reproductive axis were associated with significant down-regulation of leptina and leptinb. The results of our study highlight the adverse effects of SMC on male zebrafish reproduction, likely through the neurotoxicity of this compound. The effective concentrations of SMC that were identified to alter sex hormones and HPG axis gene expression in this study were lower than the reported environmentally relevant concentrations . Therefore, more attention should be paid to SMC contamination in the aquatic environment. Furthermore, our findings will open new avenues for the investigation of the mechanisms underlying the reproductive endocrine disrupting effects of other exogenous chemicals, especially those that negatively affect neurotransmitter systems. Acknowledgements This work was supported by the National Natural Science Foundation of China [grant number
41676100] and Shandong Provincial Natural Science Foundation, China
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Figure legends Fig. 1 Effects of exposure to the indicated concentrations of semicarbazide (SMC) for 28 d on the testicular somatic index (TSI) and the brain-somatic index (BSI) in male zebrafish. The data represent the mean ± standard error of 14 individual fish; *p < 0.05, as compared with the relevant control group. Fig. 2 Histopathological analyses of male zebrafish testes from control and semicarbazide (SMC)-treated groups (n = 3). No marked differences were observed between the control group (A), 1 μg/L SMC group (B), and 10 μg/L SMC group (C). In the groups exposed to 100 μg/L SMC (D) or 1000 μg/L SMC (E and F), sperm and
indicates the space in seminiferous tubules because of the concentrated
indicates the broken tunica propria (TP). SG, spermatogonium; SC, spermatocyte; ST,
spermatid; SP, sperm; LC, Leydig cells. Fig. 3 Effects of exposure to the indicated concentrations of semicarbazide (SMC) for 28 d on plasma (A) testosterone (T) and (B) 17β-estradiol (E2) levels in male zebrafish. The data represent the mean ± standard deviation of three replicate samples; *p < 0.05, as compared with the relevant control group. Fig. 4 Gene expression profiles in male zebrafish after exposure to semicarbazide (SMC; 0, 1, 10, 100, and 1000 μg/L) for 28 d. Responses in (A) brain and (B) testis are summarized. The results are shown as the mean ± standard deviation (n = 6). Gene expression levels were expressed as fold changes, relative to control; *p < 0.05, as compared with the control group. Fig. 5 Plot of the first two factors in the principal component (PC) analysis of gene transcription. Each dot represents the PC1 and PC2 scores for each fish. The cluster shown as orange represents the control group, while the red, green, purple, and blue ovals represent the fish exposed to semicarbazide (SMC) at 1 μg/L, 10 μg/L, 100 μg/L, and 1000 μg/L, respectively.
Control 1 μg/L 10 μg/L 100 μg/L 1000 μg/L
1800 1600 1400 1200
1000 800 600 400
P l a s m a 2 El e v e l ( p g / m L )
Plasma T level (pg/mL)
0 C o n t r o l 1 μ g / L 1 0 μ g / L1 0 0 μ g /1L0 0 0 μ g / L
C o n t r o l 1 μ g / L 1 0 μ g / L1 0 0 μ g /1L0 0 0 μ g / L
Control 1 μg/L 10 μg/L 100 μg/L 1000 μg/L
* * *
** * *
gnrhr1 gnrhr2 gnrhr4
Control 1 μg/L 10 μg/L 100 μg/L 1000 μg/L
* * *
leptinb hmgcra hmgcrb
cyp11a 3β hsd
cyp17 17β hsd cyp19a
1.5 Control 0.5
10 μg/L -0.5
100 μg/L 1000 μg/L
-1.5 -2.5 -2