Effects of Letrozole on Gonad Differentiation of Carp (Cyprinus carpio).
Letrozole (LET) is a triazole-containing drug that can inhibit the activity of cytochrome P450 aromatase, so as to affect the production of estrogen. In the present study, to detect the effects of letrozole on gonad differentiation, 5dph fry of carp were exposed to different concentrations of LET. At 130 dph, sex ratio was recorded. The results showed that continuous exposure to LET induced the changes of sex ratio. High concentration of LET can promote male-biased sex ratio. In order to reveal the molecular mechanism by which LET caused sex reversal, juvenile carp (120dph) were exposed to 0, 5, 25, 125, and 625ug/L LET for 15 days, and the expression profiles of seven gonad differentiation related genes (Cyp19A, Cyp19B, Ar[alpha], ER[alpha], Dax, Foxl2, Dmrt1) and gonad histological changes were examined.
The results showed that LET could inhibit oocyte growth in female and promote the development of testes in males. For the gonad differentiation related genes, the expression profiles of Cyp19A and Cyp19B show a pattern of increase in short time and low concentration, but decline in long time and high concentration. Ar[alpha] has a trend of increases, ER[alpha] shows downwards trend. Dax and Foxl2 are female-biased genes, their expression profiles show similar trends to Cyp19A in ovary, but downwards trend in testis. Dmrt1 is male-biased genes, it shows a trend of increases in male and female individuals. These findings suggest that continuous exposure to LET may perturb sex differentiation in carp, the molecular mechanism not only involve inhibition of cytochrome P450 aromatase activity, but also the changes of upstream genes in sex differentiation.
So, LET can be used as a reagent to control the sex differentiation of carp, but also can be used to disturb gonad differentiation, and then to reveal the molecular mechanism.
Aromatase inhibitor, Carp (Cyprinus carpio), Letrozole, Endocrine disruption, Gonadal differentiation-related genes.
Sex determination in fish is known to be significantly influenced by environmental factors, such as temperature, pH, exogenous hormones, and pesticides (Devlin and Nagahama, 2002; Adkins-Regan, 1987). Many of these factors are potential endocrine disruptors, which at minute levels are sufficient to cause developmental and reproductive dysfunction in numerous species (Corcoran et al., 2010; Colborn, 1993). In recent years, numerous research efforts have been focused on man-made chemical compounds which can disrupt the endocrine system in vertebrates, including teleosts (Colborn, 1993; Sumpter, 1998; Nakamura et al., 2003). Conazoles are a class of imidazole- or triazole-containing drugs, which have been commonly used as fungicides in agriculture and medicine (Sheehan et al., 1999).
However, several azoles have adverse health effects such as carcinogenesis via CYP (e.g., CYP1A, 2B, 3A)-mediated metabolism in mammals and other non-targeted organisms, and these chemicals are also known as aromatase inhibitors (AIs) which block cytochrome P450 aromatase (P450arom) activity, resulting in a decrease in estrogen production (Allen et al., 2006; Zarn et al., 2003; Taxvig, 2008). P450arom is the steroidogenic enzyme responsible for the conversion of androgen to estrogen. It is widely recognized that AIs play important roles in sexual differentiation and reproduction in all vertebrates studied to date (Hamilton and Piccart, 1999; Requena et al., 2008), and the P450arom gene has been shown to be related to sex differentiation as well as gonadal development in teleost fish (Fukada et al., 1996; Gen et al., 2001; Ijiri et al., 2003; Deng et al., 2009, 2015).
AIs have been widely used in studies of sex differentiation, reproduction and sex change in fish. Letrozole (LET) is a triazole-containing drug, is known to be a potent AI and acts indirectly (EPA, 2002; Smith, 1999; Seralini and Moslemi, 2001). LET can inhibit the activity of P450arom by competitively binding to the heme of the cytochrome P450 subunit, resulting in a reduction in estrogen biosynthesis in tissues (Scott and Keam, 2006). For this reason, LET has been widely used in the therapy of breast and ovarian cancer in postmenopausal women (Haynes et al., 2003; Howell et al., 2005). It is considered a standard AI in sh (Das, 2007; Periasamy, 2007). Some studies have shown that LET can disrupt the endocrine system of fish and cause alterations in both reproduction and development (Sonnenschein and Soto, 1998; Harries et al., 2000; Ana et al., 2016).
LET was also found to inhibit oocyte growth and reduce plasma vitellogenin (VTG) levels in adult female Medaka (Sun et al., 2007). To date, very few studies have been carried out to determine the differences in the expression of genes related to gonadal development and gonadal sex differentiation, which could serve as biomarkers of damage in reproductive organs caused by LET. It is vital to identify the mechanism involved in the effects of LET exposure during several development stages. Genes regulating gonadal differentiation and maintenance of reproductive functions are good candidates for this purpose.
Carp is one of the most important aquaculture species in China. As females grow faster than males, the output of these fish can be increased and the economic benefit improved by controlling the sex. In practice, hormones are used to increase the number of females. In this study, fry and juvenile carp were exposed to LET at environmentally relevant concentrations (0, 5, 25, 125, 625 ug/L) for 130 or 15 days to investigate the expression of the upstream genes associated with sex development in the brain and gonads as well as their histological changes. The results of the present study will improve our understanding of the endocrine-disrupting effects of this chemical in the environment.
MATERIALS AND METHODS
LET (purity>98%) was purchased from Beijing Dezhong-Venture Pharmaceutical Technology Development Co., Ltd. (Beijing, China). As LET has low solubility in water, LET stock solutions were prepared in acetone and diluted with acetone (Fisher Scientific, USA).
Fish and care of fish
Adult and juvenile carp Yellow River carp (Cyprinus carpio) were obtained from the aquaculture sites and maintained at the genetic laboratory (Henan normal university, Xinxiang Henan province, China) in flow-through water tanks with a constant temperature of 25 +- 1AdegC, three tanks per exposure concentration. Embryos were obtained by natural spawning and larvae were cultured in embryo medium following standard procedures. Fry were fed two times daily with commercial flake food.
Effect of LET on sexual differentiation of carp
On day 5 post-hatch (dph), fry were divided into several groups, held in the water containing LET at a concentration of 0, 5, 25, 125, or 625ug/L. To avoid metabolic and microbial breakdown of LET, half of the water was removed and replaced every 3 days with fresh LET-contaminated water. At 130 dph, 100 fish from each group were randomly selected for histological examination of the gonads, each concentration group was set with three replicates.
Water samples (100 mL) were randomly collected from each treatment tank every 3-5 days during the exposure period to confirm the concentration of LET. LET concentrations in the water samples were quantified by an LC system (Dionex Ultimate 3000, Thermo Scientific, San Jose, CA, USA) coupled to a triple quadruple mass spectrometer (TSQ Vantage, Thermo Scientific).
120dph juvenile carp were used to detect the expression profile of genes related to sexual differentiation. Groups of 100 fish were exposed to 0, 5, 25, 125, or 625 ug/L LET under static renewal conditions for 15 days respectively, and 50% of the test solution was renewed every three days. Two control groups were included, one is acetone and the other was kept in water without chemicals. No physical signs of negative health effects were observed, and all sh survived during the 15-day treatment period.
During the treatment period, 20 fish from each group were sacrificed at day 5, 10 and 15, respectively. Brain and gonadal tissues were dissected, the tissues were immediately stored at -80AdegC for gene expression analysis and stored in formaldehyde for subsequent histological observation.
The gonads and brains were fixed in Bouin's solution, embedded in paraffin, and 6 um sections were cut. The sections were then stained with hematoxylin and eosin, observed and photographs obtained under a microscope.
Quantitative real-time PCR (qPCR)
The gonads and brains of five male and female fish were homogenized and prepared at each time point. RNA isolation was performed using TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol. Total RNA concentration was estimated by the absorbance ratio of 260/280 nm, and the quality of RNA was evaluated by 1% agarose gel electrophoresis. cDNA was obtained using a Quantitec Reverse Transcription Kit (Qiagen, Germany), according to the manufacturer's instructions.
Real-time PCR with SYBR green detection was performed using a Light CycleA(r) Real-Time PCR System (Roche Applied Science), according to the operator's guide. The transcription of functionally relevant genes (Cyp19A, Cyp19B, DMRT1, FOXL2, DAX, ER[alpha] and AR[alpha]) was analyzed.
Table I.- Primer sequences used for gene transcription analysis.
(Cytochrome P450 aromatase 1a)###3'-ACTTACTGCGGTGTATTGT-5'
(Cytochrome P450 aromatase 1b)###3'-CGGGTCTCCAGAAATCGGTAGA-5'
(Forkhead transcriptional factor 2 Gene)###3'-CTCATGCCGTTGTAAGTGTTCA-5'
(Dosage-sensitive sex reversal-adrenal hypoplasia gene on the X chromosome)###3'-CCATTCACTGCAAACTGCC-5'
(Estrogen receptor alpha)###3'-TGGACTGGAGCAGAATGA-5'
(Doublesex and mab-3 related transcription factor 1)###3'-GCTCTGAGTGATGGTAACG-5'
All oligonucleotide primers were designed with Primer Premier 5.0. The efficiency and optimal concentration of each primer was tested using adult wild-type male and female carp cDNA (Table I). The expression profile of the target gene was normalized to that of the housekeeping gene [beta]-actin, as the expression of [beta]-actin had almost no influence on the effects of LET exposure according to our preliminary experiment.
Each sample was run in triplicate. Quantification of the expression of each gene was based on the 2-IICt method (Livak and Schmittgen, 2001). Statistical differences between experimental samples were assessed using SPSS 13.0 software. Analysis of variance (ANOVA) followed by Dunnett's post-hoc test were used to determine statistical differences between experimental samples. All data are expressed as means +- standard error of the mean and p<0.05 was considered statistically significant as follows: *p< 0.05, **p25 ug/L groups and 5 ug/L groups at >10d. In female brains, LET exposure had no obvious effect on the expression of Foxl2 at all concentrations at 5d. Foxl2 expression was significantly down-regulated at day 10 and 15 at all concentrations. In the testes, the expression of Foxl2 was decreased with the exception of no obvious change in 5 ug/L group at 5d. In the ovary, Foxl2 was slightly up-regulated at 5d, and was significantly down-regulated at 10d and 15d (Fig. 4).
In male and female brains, exposure to LET caused a decrease to Dax expression, with the exception of less change at day 5 in the 5 ug/L and 25 ug/L treatment groups. In the testes, LET exposure down-regulated the expression of Dax in all treatment group. No effects were observed in the ovaries at 5d, however, Dax expression was inhibited by 125 ug/L and 625 ug/L group at 10d, as well as all concentration groups at 15d (Fig. 5).
In brain and gonad of female, a slightly increase in Er[alpha] expression was observed after exposure to LET for 5d, but it decreased rapidly at all groups for 10d or 15d. Acts of Er[alpha]s in male is similar to female, except that it had shown to decline at 5d. With increased treatment time, the decline trend gets slow (Fig. 6).
In males, increased levels of Ar[alpha] were seen both in the brain and testes compared with the control group. In the testes of LET-exposed males, an increase in Ar[alpha] mRNA was observed in the 5 ug/L and 25 ug/L groups at 10d. In the 625 ug/L group, a significant increase was observed at day 5 (Fig. 7).
The same change was observed in the brain of females. Exposure to LET caused a significant increase in Ar[alpha] mRNA in female brains at all concentrations on day 5. In ovary, the expression of Ar[alpha] was enhanced in a concentration-dependent manner with increased treatment time, markedly increasing to 20% (p25 ug/L at 5d. The down-regulation of Foxl2 and Dax also explained the phenomenon of Oocyte growth slowing.
LET has direct and indirect effects on sex differentiation of carp. Continuous exposure to LET induced the changes of sex ratio in fry of carp. High concentration of LET can promote male-biased sex ratio. For juvenile carp, continuous exposure to LET could to inhibit oocyte growth in female and promote the development of testes and spermatogenesis in males. Our data show that LET exposure altered gene expression in a complex manner. The expressions of genes related to ovarian differentiation, such as Cyp19A, Cyp19B, Dax, Foxl2 and Er[alpha], decreased rapidly, but downstream gene such as Cyp19A, Cyp19B and Er[alpha] presented compensatory responses at short exposure time in low concentration. For the genes related to testes differentiation, their expressions showed increasing trend. So, LET can be used as a reagent to control the sex differentiation of carp, but also can be used to disturb gonad differentiation, and then to reveal the molecular mechanism.
Conflict of interest statement
We declare that we have no conflict of interest.
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|Date:||Dec 31, 2017|
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