Environmental Progestins Progesterone and Drospirenone Alter the

Jul 10, 2015 - Progestins alter hormone homeostasis and may result in reproductive effects in humans and animals. ... and medroxyprogesterone acetate ...
9 downloads 12 Views 1MB Size
Page 1 of 36

Environmental Science & Technology

1

Environmental progestins progesterone and drospirenone alter the circadian rhythm

2

network in zebrafish (Danio rerio)

3 4

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

5 6 7

a

8

Gründenstrasse 40, CH–4132 Muttenz, Switzerland

9

b

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

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

10

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

11

c

12

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

13

Switzerland

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

14 15

* Corresponding author:

16

Karl Fent

17

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

18

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

1 ACS Paragon Plus Environment

Environmental Science & Technology

19

Abstract

20

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

21

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

22

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

23

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

24

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

25

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

26

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

27

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

28

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

29

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

30

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

31

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

32

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

33

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

34

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

35

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

36 37

TOC

2 ACS Paragon Plus Environment

Page 2 of 36

Page 3 of 36

38

Environmental Science & Technology

Introduction

39

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

40

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

41

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

42

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

43

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

44

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

45

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

46

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

47

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

48

Environmental progestins are endocrine-disrupters that mediate their activities mainly

49

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

50

processes including meiotic oocyte maturation, ovulation, spermatogenesis and sperm

51

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

52

ecosystems raises concerns about their potential endocrine disruptive effects in aquatic

53

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

54

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

55

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

56

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

57

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

58

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

59

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

Environmental Science & Technology

60

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

61

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

62

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

63

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

64

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

65

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

66

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

67

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

68

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

69

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

70

significant but unexplored target of progestins.

71

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

72

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

73

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

74

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

75

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

76

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

77

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

78

cyanobacterial toxin cyanopeptolin, interferred with the circadian rhythm by altering

79

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

80

environmental steroid hormones needs more detailed investigations.

81

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

Page 4 of 36

Page 5 of 36

Environmental Science & Technology

82

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

83

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

84

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

85

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

86

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

87

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

88

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

89

network, a target of progestins not yet fully recognized.

90 91

Materials and Methods

92

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

93

Supporting Information (SI).

94

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

95

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

96

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

97

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

98

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

99

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

100

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

101

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

102

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

103

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

Environmental Science & Technology

104

expected.

105

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

106

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

107

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

108

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

109

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

110

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

111

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

112

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

113

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

114

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

115

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

116

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

117

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

118

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

119

conducted based on OECD Guideline 229/230.

120

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

121

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

122

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

123

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

124

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

125

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

Page 6 of 36

Page 7 of 36

Environmental Science & Technology

126

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

127

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

128

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

129

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

130

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

131

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

132

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

133

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

134

spanned up to several hours.

135 136

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

137

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

138

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

139

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

140

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

141

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

142

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

143

Information (SI Text and Tables S1-S3).

144 145

Histology. Histological analysis of zebrafish gonads was performed as described

146

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

147

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

Environmental Science & Technology

148

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

149

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

150

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

151

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

152

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

153

three) with good qualities were examined as previously.12

154 155

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

156

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

157

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

158

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

159

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

160

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

161

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

162

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

163

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

164

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

165

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

166

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

167

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

168

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

169

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

Page 8 of 36

Page 9 of 36

Environmental Science & Technology

170

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

171

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

172

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

173

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

174

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

175

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

176

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

177

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

178

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

179

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

180

adult and embryonic zebrafish in different progestin treatments and different zebrafish

181

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

182

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

183

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

184

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

185 186

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

187

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

188

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

189

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

190

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

191

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

Environmental Science & Technology

192

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

193

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

194

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

195

the Supporting Information.

196 197

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

198

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

199

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

200

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

201

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

202

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

203

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

204

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

205

10 ACS Paragon Plus Environment

Page 10 of 36

Page 11 of 36

Environmental Science & Technology

206

Results and Discussion

207

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

208

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

209

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

210

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

211

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

212

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

213

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

214

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

215

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

216

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

217

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

218

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

219

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

220

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

221

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

222

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

223

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

224

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

225

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

226

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

227

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

Environmental Science & Technology

228

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

229

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

230

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

231

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

232

metabolism and degradation displayed negligible effects in this static experiment.

233

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

234

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

235

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

236

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

237

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

238

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

239

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

240

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

241

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

242

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

243

be also carefully controlled for sorption characteristics.

244 245

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

246

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

247

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

248

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

249

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

Page 12 of 36

Page 13 of 36

Environmental Science & Technology

250

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

251

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

252

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

253

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

254

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

255

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

256

CLOCK/ARNTL transcriptions, respectively.26

257

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

258

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

259

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

260

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

261

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

262

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

263

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

264

factors and functionally resemble negative feedback components of the mammalian circadian

265

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

266

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

267

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

268

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

269

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

270

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

271

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

Environmental Science & Technology

272

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

273

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

274

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

275

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

276

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

277

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

278

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

279

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

280

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

281

negative and positive feedback loops.22,26

282

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

283

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

284

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

285

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

286

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

287

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

288

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

289

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

290

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

291

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

292

should be investigated in forthcoming studies.

293

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

Page 14 of 36

Page 15 of 36

Environmental Science & Technology

294

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

295

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

296

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

297

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

298

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

299

CLOCKs, PERs and CRYs.33

300

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

301

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

302

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

303

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

304

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

305

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

306

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

307

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

308

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

309

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

310

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

311

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

312

their differential expression patterns in zebrafish embryos.34

313

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

314

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

315

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

Environmental Science & Technology

316

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

317

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

318

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

319

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

320

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

321

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

322

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

323

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

324

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

325

the gonads of zebrafish needs further investigations.

326

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

327

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

328

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

329

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

330

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

331

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

332

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

333

significant and dose-dependent decreases (Figure S7).

334

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

335

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

336

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

337

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

Page 16 of 36

Page 17 of 36

Environmental Science & Technology

338

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

339

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

340

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

341

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

342

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

343

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

344

implications of transcriptional circadian rhythm changes and associated physiological

345

consequences,37 including alteration of locomotor activity and metabolism.

346 347

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

348

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

349

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

350

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

351

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

352

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

353

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

354

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

355

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

356

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

357

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

358

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

359

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

Environmental Science & Technology

360

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

361

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

362

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

363

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

364

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

365

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

366

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

367 368

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

369

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

370

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

371

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

372

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

373

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

374

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

375

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

376

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

377

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

378

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

379

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

380

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

381

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

Page 18 of 36

Page 19 of 36

Environmental Science & Technology

382

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

383

lower percentage of immature spermatocytes and a higher percentage of mature

384

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

385

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

386 387

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

388

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

389

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

390

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

391

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

392

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

393

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

394

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

395

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

396

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

397

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

398

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

399

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

400

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

401

reproduction in Japanese medaka.41

402

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

403

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

Environmental Science & Technology

404

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

405

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

406

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

407

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

408

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

409

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

410

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

411

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

412

in the supporting information (Figure S15).

413

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

414

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

415

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

416

and p