Environmental Progestins Progesterone and Drospirenone Alter the

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Environmental Science & Technology

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Environmental progestins progesterone and drospirenone alter the circadian rhythm

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network in zebrafish (Danio rerio)

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Yanbin Zhaoa, Sara Castiglionib, and Karl Fenta,c*

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a

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Gründenstrasse 40, CH–4132 Muttenz, Switzerland

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b

University of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences,

IRCCS – Istituto di Ricerche Farmacologiche “Mario Negri”, Environmental Biomarkers

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Unit, Department of Environmental Health Sciences, Via La Masa 19, I-20156, Milan, Italy

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c

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Pollution Dynamics, Department of Environmental System Sciences, CH–8092 Zürich,

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Switzerland

Swiss Federal Institute of Technology (ETH Zürich), Institute of Biogeochemistry and

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* Corresponding author:

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Karl Fent

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Tel.: +41 61 467 4571; Fax: +41 61 467 47 84

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E-mail: [email protected]; [email protected]

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Abstract

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Progestins alter hormone homeostasis and may result in reproductive effects in humans and

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animals. Thus far, studies in fish have focused on the hypothalamic–pituitary–gonadal

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(HPG)-axis and reproduction, but other effects have little been investigated. Here we report

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that progesterone (P4) and drospirenone (DRS) interfere with regulation of the circadian

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rhythm in fish. Breeding pairs of adult zebrafish were exposed to P4 and DRS at

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concentrations between 7 and 13´650 ng/L for 21 days. Transcriptional analysis revealed

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significant and dose-dependent alterations of the circadian rhythm network in the brain with

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little effects in the gonads. Significant alterations of many target transcripts occurred even at

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environmental relevant concentrations of 7 ng/L P4 and at 99 ng/L DRS. They were fully

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consistent with the well-described circadian rhythm negative/positive feedback loops.

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Transcriptional alterations of the circadian rhythm network were correlated with those in the

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HPG-Liver-axis. Fecundity was decreased at 742 (P4) and 2´763 (DRS) ng/L.

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Dose-dependent alterations in the circadian rhythm network were also observed in F1

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eleuthero-embryos. Our results suggest a potential target of environmental progestins, the

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circadian rhythm network, in addition to the adverse reproductive effects. Forthcoming

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studies should show whether the transcriptional alterations translate into physiological effects.

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TOC


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Introduction

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Naturally excreted progesterone (P4) and its metabolites, as well as synthetic progestins

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that are widely used in human medicine and as growth promoters in livestock enter the

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aquatic environment by wastewater and agricultural run-offs. Consequently, P4 and synthetic

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progestins occur in surface and ground waters in the low ng/L range.1-3 Of those, several were

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most widely detected in surface water, such as P4, levonorgestrel, norgestrel and

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medroxyprogesterone acetate (MPA) at concentrations in the low ng/L to up to over 100

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ng/L.4-7 In animal farm run-offs, concentrations were much higher.1 Besides aquatic wildlife,

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humans may also be exposed to natural and synthetic progestins via contaminated drinking

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water and seafood (in addition to the use of contraceptives).8,9

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Environmental progestins are endocrine-disrupters that mediate their activities mainly

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through nuclear and membrane progesterone receptors. Endogenous P4 is involved in many

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processes including meiotic oocyte maturation, ovulation, spermatogenesis and sperm

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motility.10,11 Therefore, the widespread occurrence of P4 and synthetic progestins in aquatic

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ecosystems raises concerns about their potential endocrine disruptive effects in aquatic

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animals. In fish, progestin exposures resulted in a decrease of fecundity and adverse

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reproductive effects, steroid hormone imbalances, masculinization of females, altered sexual

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behavior, changes in gonadal histology and alteration of sex-development.12-16 Transcriptional

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analysis revealed that pathways involved in the hypothalamic–pituitary–gonadal (HPG)-axis

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were disrupted by several progestins even at environmental relevant concentrations.17,18

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Thus far, activity assessments of progestins in adult vertebrates are limited to the

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HPG-axis and associated specific endocrine targets, including fecundity, hormone levels and 3 ACS Paragon Plus Environment


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reproduction. However, additional endpoints that may result in health impacts are rarely

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investigated. In our previous studies, we demonstrated that P4, drospirenone (DRS),

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dydrogesterone (DDG) and MPA led to transcriptional alterations of genes in the HPG-axis,

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as well as of genes associated with an unexpected target for progestins, such as the circadian

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rhythm network, among others.12,19 Especially for the circadian rhythm network,

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transcriptional alterations of key genes, such as nr1d1, nr1d2b, per1b and cry5, were

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significant. Compared to other pathways, including the HPG-axis, alteration of the circadian

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rhythm network was most prominent, both in adult zebrafish and eleuthero-embryos. The

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transcriptional responses even occurred at environmental relevant concentrations of 3.5 ng/L

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P4 and 4.8 ng/L DDG. Therefore, the question arises, whether the circadian rhythm is a

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significant but unexplored target of progestins.

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Circadian rhythm is an essential timing system in the body driving daily oscillations of a

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variety of crucial cellular and physiological processes, such as cell cycle and its regulations,

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energy metabolism, cardiovascular function, sleep-wake rhythm, insulin secretion, hormone

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secretion and even reproduction.20,21 The core of the circadian rhythm network is conserved in

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vertebrates. It mainly consists of six groups of genes, including CLOCKs, ARNTLs, PERs,

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CRYs, NR1Ds and RORCs, which combine to several key negative and positive feedback

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loops.22 Recently, we showed that two environmental compounds, diazepam and the

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cyanobacterial toxin cyanopeptolin, interferred with the circadian rhythm by altering

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transcripts of key genes.23,24 Whether the circadian rhythm network is a target of

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environmental steroid hormones needs more detailed investigations.

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To test the hypothesis that progestins interfere with the regulation of the circadian 4 ACS Paragon Plus Environment

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rhythm network, here we focused on two important environmental progestins, P4 and DRS,

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which are extensively used in medical treatments and contraception, and which were

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previously shown to adversely affect the HPG-axis in fish.12,19 We aimed at a comprehensive

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analysis of the circadian rhythm network and downstream pathways involved in the HPG-axis,

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apoptosis and cell cycle and its regulation, in addition to reproductive and histological effects.

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Our systematic transcriptional analyses in the brain and gonads of adult zebrafish and in F1

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generation eleuthero-embryos suggest that progestins may interfere with the circadian rhythm

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network, a target of progestins not yet fully recognized.

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Materials and Methods

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Chemicals and Maintenance of Zebrafish. Detailed information can be found in the

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Supporting Information (SI).

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Experimental Design. Adult zebrafish (10 months old) were selected from the culture tank

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and randomly placed into 10 L stainless steel tanks in well-aerated water at 27± 1oC. The

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experimental setup consisted of the following exposure treatments: solvent control (0.01%

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DMSO), increasing concentrations of P4 (nominal 100, 1000 and 10000 ng/L) and increasing

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concentrations of DRS (nominal 500, 5000 and 20000 ng/L). Each treatment consisted of four

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replicates and each replicate (n=4) contained 6 females and 6 males as breeding pairs. The

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concentrations were selected on the basis of previous studies for their transcriptional and

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reproductive effects on zebrafish and fathead minnows.15,19,25 The lowest actual concentration

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was supposed to be environmentally realistic or slightly higher, especially in case of P4,

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where a remarkable decrease of the nominal concentrations in the experimental system was 5 ACS Paragon Plus Environment


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expected.

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The experiment was conducted according to the procedure described previously.12,19 In

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brief, after a three days acclimatization, the experiment started with a pre-exposure period of

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14 days to establish the baseline rate of fecundity for each tank (and spawning group),

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followed by one day of equilibration when chemical-dosing started, and finally, 21 days of the

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progestin exposure period. The whole experiment was performed by use of a flow-through

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system, which ensured a complete change of reconstituted water every 12 h. To ensure the

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water quality, pH value (6.7–7.2) and dissolved oxygen concentration (>70%) were

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continuously measured. The photoperiod was 14:10 h light/dark. During the whole exposure

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period, mortality and any abnormalities in appearance of fish were recorded, but no

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compound related effects occurred. Fish were fed twice daily. Eggs were collected and

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counted during the whole experimental period. Each tank was equipped with a spawning tray,

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which was consisted of a stainless steel frame covered by a stainless steel mesh with a mesh

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size 2.5 mm for eggs to fall through. Every morning about 1.5 hour after the beginning of the

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light period, eggs were collected and transferred to Petri dishes for counting. The study was

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conducted based on OECD Guideline 229/230.

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After termination of the exposure, fish were anesthetized by KoiMed Sleep (1.5 mL/L

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water). Before dissection, two female and two male fish from each replicate (n=8 for each

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gender of each treatment) were randomly selected and measured for wet weight (mg) and

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length (cm), which was used to calculate the condition factor. Four female and four male fish

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from each of the four replicates were then dissected immediately. Brain, liver and gonads of

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two to four fish were pooled (depending on the tissue), transferred to RNAlater and stored at 6 ACS Paragon Plus Environment

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−80 °C for subsequent RNA extraction. Ovaries were collected from individual females and

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weighed in order to assess the gonadosomatic index (GSI = gonad weight (g)/body weight (g)

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× 100). For RNA analysis, ovaries of two fish were pooled in each replicate, whereas four fish

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of each replicate were pooled in case of brain, liver and testis. Pooling was necessary due to

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the small tissue sizes, varying extraction efficiencies and to control for inter-individual

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variability. One female and one male fish per replicate (total of 4 fish per gender per treatment)

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were fixed in Bouin’s after opening of the abdominal area for histological examination. Due

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to practical constraints, time differences between sampling of controls and exposed fish

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spanned up to several hours.

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Chemical Analysis. The analytical methods as described previously15,19 were employed to

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determine P4 and DRS concentrations in exposure waters. The analysis was performed by

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solid phase extraction and liquid chromatography-tandem mass spectrometry (HPLC-MS-

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MS). Recoveries in the investigated matrix were 89.5±4.2% and 85.7 ±1.5% for DRS and P4,

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respectively. The limits of quantification at a signal to noise ratio of 10 were 2 and 0.27 ng/L

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for DRS and P4, respectively. Detailed information about analytical procedures used for

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chemical analysis and the delivered chemical concentrations is provided in the Supporting

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Information (SI Text and Tables S1-S3).

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Histology. Histological analysis of zebrafish gonads was performed as described

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previously.12,15 In brief, one male and one female zebrafish per replicate tank (total of n = 4

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per treatment) were randomly taken after anesthesia, opened at the abdominal site and fixed in 7 ACS Paragon Plus Environment


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Bouin’s solution for about 24 h. After fixation, fish were kept in 70% ethanol for 4-6 weeks,

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and further procedures, including dehydration, embedded in paraffin and stained by standard

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hematoxylin−eosin, were performed. To obtain a comprehensive evaluation of the

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characteristics of each gonad, three cross-sections were taken from different parts along the

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gonad-axis (front, middle, rear) from each individual fish. Subsequently, two sections (out of

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three) with good qualities were examined as previously.12

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RNA Isolation and qRT-PCR Analysis. Total RNA was extracted from the different adult

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zebrafish tissues by use of the RNeasy Mini Kit (Qiagen, Basel, Switzerland). The samples

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were then treated with RNase-free DNase (Qiagen, Basel, Switzerland) to purify the RNA

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from DNA contamination. RNA concentrations and qualities were analyzed using a

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NanoDrop 1000 spectrophotometer (Nanodrop Technologies Inc. Wilmington DE, U.S.); the

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purity of each sample was between 1.8 and 2.0 (260 nm/280 nm ratio). RNA samples were

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then stored at −80 °C for subsequent RT-qPCR analysis.

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The first-strand cDNA synthesis and the relative quantitation in real time RT-PCR were

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performed according to methods described previously.12 In brief, 1 µg RNA was reverse-

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transcribed by MMLV (Promega, Switzerland) in the presence of random hexamers (Roche,

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Switzerland) and dNTP (Sigma–Aldrich, Switzerland). The complete reaction mixture was

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incubated at 37°C for 50 min, following at 95°C 5 min to stop the reaction. RT-PCR was

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conducted on BIO-RAD CFX96 Real-Time PCR Detection System (BIO-RAD, Switzerland)

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using SYBR Green Fluorescence (Roche Diagnostics, Basel, Switzerland) as recommended

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by the manufacture’s guidelines. Two-step real-time PCR profile was used: enzyme activation 8 ACS Paragon Plus Environment

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step at 95°C (10 min) and 40 cycles of 95°C (30 s), 58–62°C (60 s) depending on the target

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transcript, followed by a melting curve analysis post run (65–95°C), which confirmed the

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specificity of chosen primers as well as absence of primer dimers.

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For primers design, Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/)

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was employed. The intron/exon boundary spanning primers were preference to minimize

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DNA contamination. Melting curves were analyzed to ensure that only a single product was

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amplified. Primer details are presented in the Supporting Information (Table S4), and the

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efficiencies were calculated to ensure no significant change between the primer efficiencies of

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target genes and the reference gene, ribosomal protein L13a (RpL13a). RpL13a was selected

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as housekeeping gene for normalization, because it showed high gene expression stability in

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adult and embryonic zebrafish in different progestin treatments and different zebrafish

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tissues.12,15 In the present study, the stability of RpL13a expression was also demonstrated;

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the transcripts displayed very little variation in different treatments, tissues and time during 24

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h (Figure S1). Threshold cycle (CT) values were recorded in the linear phase of amplification

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and the data were analyzed using the delta−delta CT method of relative quantification.

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Transcript indices. Transcript indices for the circadian rhythm network (CRN) and

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HPG-Liver axis (HPG-L) were developed based on their gene expression levels. Since

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dose-dependent alterations in abundance of transcripts were observed for most of the

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circadian and HPG-Liver axis genes under progestin exposures, an average value was used to

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represent the overall expression levels for each treatment for these two clusters to reduce the

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dimension of gene expression data, and to simplify their relationships. In total, 34 circadian 9 ACS Paragon Plus Environment


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rhythm genes in zebrafish brain were used for establishment of CRN index; 11 genes (3 genes

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in brain, 5 genes in gonad and 3 genes in liver) were used for establishment of HPG-L index.

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Detailed information about the approaches for evaluation of transcript indices is provided in

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the Supporting Information.

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Data Analysis and Statistics. The hierarchical clustering (HAC) map was constructed with

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MultiExperimental Viewer v4.9 (Dana-Farber Cancer Institute, Boston, MA, USA). Data

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from the gene expressions were graphically illustrated and statistically analyzed by GraphPad

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Prism 5 (GraphPad Software, San Diego, CA, USA). The significance of differences between

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the solvent control and P4 and DRS exposed adult fish in transcript levels, egg production,

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condition factors and GSI were analyzed by one-way analysis of variance (ANOVA) followed

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by Tukey post-hoc test (95% confidence interval). Results are given as mean ± standard

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deviation (S.D.). Differences were considered as significant at p ≤ 0.05.

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Results and Discussion

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Chemical Analysis. P4 and DRS concentrations were measured during the whole exposure

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period at day 1, 7, 14 and 21. Mean delivered P4 concentrations were 7, 116 and 742 ng/L,

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respectively, which were 88%-93% lower than nominal. Mean delivered DRS concentrations

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were 99, 2´763 and 13´650 ng/L, respectively, which were 32%-80% lower than nominal

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(Table S2). A similar phenomenon was also found in our previous studies after zebrafish

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exposed to the progestins P4, DRS, MPA and DDG.12,19 Especially for P4, the delivered

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concentrations were always observed to be significantly lower than nominal; for instance, a

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similar range of concentration decreases (about 80%) was observed for 14 day exposures of

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adult zebrafish females.12 It should be noted, however, that the delivered low concentrations

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of P4 and DRS were quite stable during the experiment (Table S2), which indicates that the

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exposure was almost constant during the exposure period. As previously observed, a series of

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factors could be responsible for this decrease as for example, adsorption to the flow-through

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system (tubes), fish, particles and debris in the exposure experiment.

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To determine the reasons for these decreases, we further conducted a 24 h static exposure

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experiment for P4 and DRS at one concentration without and with different numbers of fish in

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tanks. The aim was to analyze for the sorption to tanks (and fish) and to determine the

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influence of fish (bioaccumulation) and degradation by light and micro-organisms. As shown

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in Table S3 (supplementary material), rapid adsorptions to stainless steel tanks and spawning

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trays were negligible for both P4 and DRS, as there were almost no alterations after the first

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hour of exposure (concentration at 1 hour). In case of P4, the presence of fish significantly

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reduced the concentration. After 24 h exposure, there were remarkable decreases of the 11 ACS Paragon Plus Environment


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measured concentrations for P4, while almost no alterations occurred for DRS. In case of P4,

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uptake by fish, metabolism and degradation (by photolysis and biodegradation) during the

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exposure were important factors responsible for this decrease. This contributed to about 43%

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and 23% of the decrease, respectively. For DRS, the adsorption to particles and debris, fish

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metabolism and degradation displayed negligible effects in this static experiment.

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As related to our present study, we used a flow-through system equipped with several

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plastic tubes used for chemical delivery with a flow-through rate two times per day. Sorption

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to plastic tubes and relative low flow-through rate would be also reasonable factors

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responsible for both, DRS and P4 decrease, in the 21-days exposure experiment. In summary,

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the results demonstrated that for P4, but not for DRS, adsorption and incorporation into fish,

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as well as degradation during the exposure were important for the decrease. The results also

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indicated the flow-through system characteristics (adsorption by plastic tubes and relative low

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flow-through rate) were further factors responsible for the P4 and DRS decrease. In future

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experiments, the characteristics of compounds will be pre-checked for their physical and

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chemical properties, biotransformation and metabolisms. In addition the delivery system will

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be also carefully controlled for sorption characteristics.

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Transcriptional Alterations of Circadian Rhythm Networks in Adult Fish. The basic

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molecular model of the core circadian rhythm network in zebrafish consists of six groups of

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genes, including several ones of the family of CLOCK, ARNTL, PER, CRY, NR1D and RORC

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genes that regulate the circadian rhythm.26 These genes comprise the core four feedback loops,

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including three negative feedback loops and one positive feedback loop. CLOCK/ARNTL 12 ACS Paragon Plus Environment

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heterodimer states the core position and forms the positive limb of the feedback loops. It

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activates the transcriptions of core circadian genes, period (PERs) and cryptochrome (CRYs),

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which are transcriptional repressors from the negative limb of the feedback loops and interact

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with the CLOCK/ARNTL heterodimer to inhibit its activity, and thereby, negatively regulating

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their own expressions. It also activates transcriptions of nuclear receptor genes, NR1Ds and

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RORs, which form the second group of feedback loops and which activate and repress

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CLOCK/ARNTL transcriptions, respectively.26

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Besides the core circadian gene families, novel circadian genes were also discovered in

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vertebrates, such as NFIL3, DECs, TEFs and DBPs.27-31 Recent studies have demonstrated the

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basic functions of these genes involved in circadian rhythm regulation. For instance, NFIL3,

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also known as E4BP4, which is a basic leucine zipper transcription factor, contains a

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DNA-binding domain closely related to the PAR proteins like dbp, hlf and tef.27 It plays an

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important role in the regulation of the core clock gene per2 and light-entrainment of the

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circadian clock.28 DECs were demonstrated to be the basic helix–loop–helix transcription

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factors and functionally resemble negative feedback components of the mammalian circadian

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clock.29 Similarly, ciart, tefa, tefb and dbpa, dbpb were also supposed to participate in the

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suppressions of CLOCK/ARNTL heterodimers directly or indirectly.27,28,30, 31

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Despite the detailed functions for these novel genes remain to be described, we covered

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all of these circadian rhythm genes for a comprehensive study. Considering that multiple gene

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copies of most of these genes exist in zebrafish due to genome duplication as compared to

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mammals, we performed homologous alignments in the zebrafish genome based on Ensembl

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database. In total, 41 genes were identified and subdivided into 13 groups. The whole picture 13 ACS Paragon Plus Environment


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of the circadian rhythm network is provided in the Supporting Information (Figure S2).

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P4 significantly altered the circadian network in the brain of both female and male

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zebrafish after the 21-days exposure. Hierarchical clustering maps revealed two different

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subdivisions of genes with up-regulations and down-regulations, respectively (Figure 1A). Of

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these 41 circadian rhythm genes, seven were significantly up-regulated and 15 were

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significantly down-regulated at different P4 concentrations in females (Figure 1B). Similarly,

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in males, four genes were significant up-regulated and 20 were significant down-regulated

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(Figure S3, S5). Transcriptional alterations even occurred at environmental relevant

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concentrations of 6.6 ng/L P4 and were consistent with the well-described circadian rhythm

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negative and positive feedback loops.22,26

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Compared to P4, the effects of 99-13´650 ng/L DRS were more pronounced. Yet, the

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pattern of transcriptional responses was quite similar and also fully consistent with the

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circadian rhythm feedback loops (Figure 2, Figure S4). Usually, the fold-changes were higher

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than two times but even reached about 20-times (ciart and per1a) (Figure S5). Strong

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transcriptional alterations even occurred at 99 ng/L DRS in both females and males (Figure

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2B, Figure S4). As shown in Figures 1B, 2B, S3 and S4, the decreased transcripts of core

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circadian genes of the per, cry and nr1d family and the increased clock, arntl and rorc

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transcripts fitted well to these feedback loops, which suggests an alteration of the circadian

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rhythm networks. Whether these alterations translate to physiological endpoints, including

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altered locomotor activity, as well as additional circadian related endpoints (e.g. metabolism)

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should be investigated in forthcoming studies.

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It should be noted that, the transcripts of cry2 and cry4 were significantly up-regulated, 14 ACS Paragon Plus Environment

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which did not fit with other CRYs and the negative feedback loop. The probable reason is that

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there are two groups of CRY genes in vertebrates, of which one group did not response to the

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suppressions on CLOCK/ARNTL heterodimers.32 Though the molecular mechanisms for these

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transcriptional alterations on the circadian rhythm networks by progestins are unknown,

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recent studies revealed progesterone receptor binding sites in several key clock genes, such as

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CLOCKs, PERs and CRYs.33

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Of the additional circadian genes, dec1 and dec2 displayed significant negative feedback

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loops in both female and male zebrafish brain. Similarly, ciart, dbpa, dbpb and tefa, tefb were

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also significantly down-regulated by P4 and DRS (Figure 1B, 2B, S3 and S4). Though there

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exist fewer studies focusing on these transcription factors, they were supposed to participate

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in the suppressions of CLOCK/ARNTL heterodimers directly or indirectly.27,28,30,31 In addition,

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of the six NFIL3s genes, three groups were observed based on their transcriptional alterations.

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Nfil3, nfil3-2 and nfil3-5 were significantly up-regulated, nfil3-6 was significantly

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down-regulated and nfil3-3 and nfil3-4 showed no responses to P4 and DRS. NFIL3 as a basic

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leucine zipper transcription factor contains a DNA-binding domain closely related to the PAR

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proteins dbp, hlf and tef. The nfil3 gene plays a role in the regulation of the core clock gene

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per2 and light-entrainment of the circadian clock.27,28 The results of our present study suggest

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divergent functions of each of the six nfil3 paralogs in zebrafish, which is also consistent with

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their differential expression patterns in zebrafish embryos.34

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Compared to the significant transcriptional alterations in the brain, only slight alterations

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were observed in the gonads. P4 did not induce significant alterations in transcript levels

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compared to the solvent controls even at 742 ng/L (Figure 1A, S6). Exposure to DRS resulted 15 ACS Paragon Plus Environment


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in significant alterations of five genes in the ovary and of only two genes in the testis (Figure

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2A, Figure S6). This fits well to the natural oscillations of clock gene in zebrafish. Peripheral

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oscillations of clock were observed for several organs of zebrafish, such as heart and kidney,

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but not in gonads.35 Whether or not nr1d1 and nfil3-2 are involved in circadian rhythm

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regulations in the gonads is not clear due to their multi-functions. Nr1d1 is a member of the

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Rev-ErbA family of nuclear receptors. Besides circadian rhythm it regulates several important

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physiological processes, such as metabolic homeostasis and inflammation.36 Nfil3-2

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participates in immune responses.28 Consequently, whether progestin related alteration of

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these genes are involved in the circadian rhythm networks or other physiological processes in

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the gonads of zebrafish needs further investigations.

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In our previous study on MPA and DDG, we found that several key circadian rhythm

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genes, such as per1b and cry5, also displayed significant decreases in the zebrafish brain.12

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Here we investigated the circadian rhythm network in more detail, by use of RNA left from

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our previous study.12 In total, six additional genes were measured. Similar as P4 and DRS, the

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core clock/arntl heterodimers and the related positive feedback loop gene, rorcb, were

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significantly induced in response to different doses of MPA and DDG and their binary

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mixtures. Other transcripts, including the three key negative feedback loop genes, displayed

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significant and dose-dependent decreases (Figure S7).

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Our data suggest that progestins alter the regulation of the circadian rhythm network on

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the transcriptional level. The magnitude of transcriptional alterations may differ between the

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progestins. It is also influenced by the sampling time. In our experiments, time differences

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between sampling of controls and exposed fish spanned up to several hours, which may have 16 ACS Paragon Plus Environment

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influenced the magnitude of transcriptional changes. Thus, the fold-changes of the different

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transcripts may have been lower if all sampling would have taken place at the same time

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(which was not feasible for practical terms). To further substantiate our hypothesis that

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progestins alter the circadian network, additional studies focusing on physiological outcomes

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need to be done, as transcriptional effects do not necessarily translate to physiological

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outcomes. Thus, both transcriptional and physiological data are needed to fully understand the

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implications of transcriptional circadian rhythm changes and associated physiological

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consequences,37 including alteration of locomotor activity and metabolism.

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Transcriptional effects on HPG-axis, cell cycle and apoptosis. Circadian rhythm regulates

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a variety of cellular and physiological processes, such as the cell cycle and its regulation,

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energy metabolism, and hormone secretion.20,21 In our previous study, we found that

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alterations of circadian rhythm genes were related to transcriptional alterations of genes

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involved in cell cycle regulation after exposure of zebrafish to MPA and DDG.12 Consequently,

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we aimed at investigating additional effects of P4 and DRS on these pathways. A total of 26

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transcripts of three downstream pathways involved in the HPG-axis, apoptosis and cell cycle

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were measured in the zebrafish brain and gonads. Of ten genes associated with the HPG-axis,

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four genes in females and in males, respectively, displayed significant alterations (Figures 1C,

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2C, Figure S8). Most of the transcripts displayed dose-dependent alterations but the

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fold-changes were usually lower than three times compared with the control. Of these genes,

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significant down-regulations were observed for cyp19b in the brain and had11b2 and cyp17 in

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the gonads, while significant up-regulations were observed for cyp19a in the ovaries. These 17 ACS Paragon Plus Environment


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results were quite consistent with our previous study on MPA and DDG,12 indicating that these

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progestins have similar molecular mechanism of actions. Of eight genes involved in apoptosis,

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only ddb2 displayed a significant and dose-dependent down-regulation in the brain of both

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females and males (Figures 1C, 2C, Figure S8). There were almost no significant alterations

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occurred in gonads (Figure S8). In addition, of eight genes involved in cell cycle, only cdk2,

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cyca1 and cycd1 were significantly down-regulated in males and females in response to high

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doses of P4 and/or DRS (Figure S8).

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Effects on Fecundity and Gonad Histology. In addition to transcriptional effects, we studied

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the effects of P4 and DRS on reproductive physiological outcomes e.g. egg production,

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parameters of growth and gonad histology. In the 14 days pre-exposure period, the egg

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production was consistent and similar across all dose groups and control. When breeding pairs

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were subsequently exposed to 13´650 ng/L DRS, there was an immediate stop of egg

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production. In the 21 days exposure period, both 742 ng/L P4 and 2´763 ng/L DRS led to a

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significant decrease in egg production (Figures 3A, 3B, Figure S9). This is consistent with

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previous data, where a significant decrease in egg production was observed for adult fathead

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minnows exposed to similar doses of P4 and DRS.25,38 Exposure to 742 ng/L P4 and 13´650

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ng/L DRS caused a significant increase in body weight and length in females and males, and

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the condition factor in females (Figure S10). At these concentrations, the ovary weight and

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gonadosomatic index were also significantly induced (Figure S11). The organizational

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architecture of gonads were also altered by P4 and DRS, similar to MPA and DDG.12 An

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increased frequency of oocyte degeneration, manifested as atretic follicles and post-ovulatory 18 ACS Paragon Plus Environment

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follicles, was observed at the high P4 and DRS doses in females (Figure S12). In males, a

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lower percentage of immature spermatocytes and a higher percentage of mature

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spermatocytes were observed (Figure S13). The effects were similar but not identical to the

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effects found in our previous studies with P4 and DRS.15,19

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Correlations among Circadian Rhythm Network, HPG-Liver Axis and Fish

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Reproduction. Circadian rhythm times a variety of cellular, physiological and metabolic

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processes, and suggested to regulate the HPG-axis related activities, hormone secretion and

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even reproduction. In rodents, endogenous circadian clocks in hypothalamic–pituitary plays a

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crucial role in generating timed signals to GnRH neurons to increase neuronal activity, and

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thus stimulate LH release from pituitary gonadotrope cells. Mice, in which the core clock

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gene Bmal1 was knocked out, are infertile, which can be traced to effects on steroid hormone

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production, gametogenesis, and others.39,40 A similar phenomenon was also observed for mice,

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in which the Clock gene was knocked out.40 The extent to which the circadian timing system

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affects teleost reproductive performance is not known, in part, because many of the

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appropriate studies have not been done in fish species, such as zebrafish. The circadian

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rhythm network and HPG-axis are complex molecular networks that are difficult for

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quantification. However, preliminary evidence for their relationships can be addressed based

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on their transcriptional levels, as shown in a similar study with focus on HPG-Liver axis and

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reproduction in Japanese medaka.41

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To get this preliminary concept about the relationships among transcripts of circadian

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rhythm network (CRN), HPG-Liver axis (HPG-L) and fish reproduction, in the present study 19 ACS Paragon Plus Environment


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we reduced the amount of gene expression data to key genes and simplified their relationships

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by considering data on gene expression in the brain, gonads and liver and the fecundity (egg

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production) in zebrafish. To this end, we further measured three key genes, era, er2b and vtg1,

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in the liver as they may be related to reproductive outcomes (Figure S14). On this basis we

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developed the transcript indices for both the circadian rhythm network and the HPG-Liver

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axis, similar to a previous study on prochloraz and ketoconazole, in which a hepatic transcript

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index (HTI) was developed to quantitatively assess the correlation between fecundity and

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hepatic gene expression profiles.41 The detailed method used in the present study is described

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in the supporting information (Figure S15).

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We first analyzed the relationships between transcript indices of the circadian rhythm

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network and the HPG-Liver axis. As shown in Figure 3C, significant correlations between

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these networks occurred for both females and males, with R2=0.84 and p =0.006 and R2=0.95

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and p

Environmental Progestins Progesterone and Drospirenone Alter the

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