Exposure to the Contraceptive Progestin, Gestodene, Alters

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Exposure to the Contraceptive Progestin, Gestodene, Alters Reproductive Behavior, Arrests Egg Deposition, and Masculinizes Development in the Fathead Minnow (Pimephales promelas) Tyler E. Frankel, Michael T. Meyer, Dana Ward Kolpin, Amanda B. Gillis, David A. Alvarez, and Edward F. Orlando Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b00799 • Publication Date (Web): 29 Apr 2016 Downloaded from http://pubs.acs.org on May 5, 2016

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Environmental Science & Technology

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Full Title

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Exposure to the Contraceptive Progestin, Gestodene, Alters Reproductive Behavior,

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Arrests Egg Deposition, and Masculinizes Development in the Fathead Minnow

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(Pimephales promelas)

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Short Title

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Human Contraceptive Progestin, Gestodene, Profoundly Affects Fish Reproduction

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Author List and Affiliations Tyler E. Frankel1, Michael T. Meyer2, Dana W. Kolpin3, Amanda B. Gillis1, David A. Alvarez4, and Edward F. Orlando1*

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University of Maryland, Department of Animal and Avian Sciences, College Park, 20742, USA 2

U.S. Geological Survey, Organic Geochemistry Research Laboratory, 4821 Quail Crest Place, Lawrence, Kansas 66049, USA 3

U.S. Geological Survey, Iowa Water Science Center, 400 S. Clinton Street, Iowa City, IA, 52240, USA 4

U.S. Geological Survey, Columbia Environmental Research Center, 4200 New Haven Road, Columbia, MO, 65201, USA

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*Corresponding author: [email protected], (301) 405-6386, no fax number

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1 ACS Paragon Plus Environment


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Abstract Endogenous progestogens and pharmaceutical progestins enter the environment

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through wastewater treatment plant effluent and agricultural field runoff. Lab studies

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demonstrate strong, negative exposure effects of these chemicals on aquatic vertebrate

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reproduction. Behavior can be a sensitive, early indicator of exposure to environmental

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contaminants associated with altered reproduction, yet is rarely examined in

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ecotoxicology studies. Gestodene is a human contraceptive progestin and a potent

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activator of fish androgen receptors. Our objective was to test the effects of gestodene

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on reproductive behavior and associated egg deposition in the fathead minnow. After

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only one day, males exposed to ng/L of gestodene were more aggressive and less

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interested in courtship and mating, and exposed females displayed less female

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courtship behavior. Interestingly, 25% of the gestodene tanks contained a female that

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drove the male out of the breeding tile and displayed male-typical courtship behaviors

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toward the other female. Gestodene decreased or arrested egg deposition with no

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observed gonadal histopathology. Together, these results suggest that effects on egg

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deposition are primarily due to altered reproductive behavior. The mechanisms by which

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gestodene disrupts behavior are unknown. Nonetheless, the rapid and profound

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alterations of the reproductive biology of gestodene-exposed fish suggest that wild

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populations could be similarly affected.

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Introduction


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There is a continuing concern regarding the effects of endocrine disrupting

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chemicals (EDC) that enter aquatic ecosystems from a variety of sources such as

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wastewater treatment plants, agricultural runoff, and paper mill plants and potentially

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effect the reproductive health of aquatic organisms 1, 2. One of the most studied EDCs is

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17α-ethinylestradiol (EE2), an estrogen that is combined with a progestin in human

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contraceptives and some hormone replacement therapeutics 3. Gestagens include

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endogenous progestogens (e.g. progesterone and 17α,20β-dihydroxy-4-pregnen-3-one)

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and synthetic progestins (e.g. gestodene and levonorgestrel), and are classified as

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compounds that mediate physiological processes and behavior through progesterone

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receptors. In vertebrates, progestogens play critical roles in the control of reproduction,

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including regulation of gamete maturation, gestation, and species-specific behaviors 4-7.

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Progestins are not only used as human pharmaceuticals, but are also commonly used

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as veterinary pharmaceuticals for growth promotion and to synchronize reproduction.

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Thus, they have been frequently detected in water impacted by human and/or livestock

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waste 8-10. Moreover, paper mill effluent has been documented to contain phytosterols

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that are microbially converted to steroid hormones including progesterone 11. As a

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result, gestagens can enter the aquatic environment via a myriad of sources such as

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wastewater treatment plant effluent, runoff from agricultural fields and livestock farms,

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and paper mill effluent. While relatively few in number, studies examining

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environmental concentrations of gestagens have found progestin concentrations up to

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188 ng/L in wastewater treatment plant effluent, 34 ng/L in surface waters, and

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progestogen concentrations in excess of 10,000 ng/L in animal agricultural lagoons 12,

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13

.



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Progestins have been specifically designed as ligands for human nuclear

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progesterone receptors (PR) and have been categorized into generations (I – IV).

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Progestins in each generation share a common parent molecule, with progestins in

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each successive generation exhibiting progressively greater specificity for the human

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PR 14, 15. Several generations have cross-reactivity with the human androgen and other

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steroid hormone receptors, resulting in unwanted side effects in women including

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hirsutism and unwanted weight gain. Previous research using in vitro receptor

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transactivation luciferase assays has shown that some commonly used progestins (e.g.

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gestodene, levonorgestrel, and norethindrone) express high affinity for the fathead

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minnow (Pimephales promelas) nuclear androgen receptor (nAR) and little to no affinity

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for the fathead minnow nPR, indicating that some progestins likely induce biological

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effects through mostly AR mediated pathways 16, 17. In addition, corroborating results

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have recently been published on a distantly related fish, the Murray-Darling rainbowfish

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(Melanotaenia fluviatilis) 18.

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Thus far, research on the exposure effects of gestagens on aquatic wildlife has

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mainly focused on the reproduction of aquatic vertebrates 12, 13, 19. While exposure to

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gestagens has been shown to decrease egg deposition, increase ovarian apoptosis,

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and alter development of secondary sexual characteristics in fishes 17, 20-24, our

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understanding of the cause of these changes remains incomplete. Although behavior

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has been recognized as a sensitive endpoint that is critical for successful reproduction,

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comparatively few studies have examined EDC exposure effects on behavior in fish 2, 25.

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In this study, we investigated the exposure effects of gestodene (GES) on the number of eggs deposited, secondary sex characteristics, and reproductive behaviors of


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fathead minnows. We intentionally selected a relatively short, 8 d exposure period due

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to our interest in examining the potential short-term effects of GES exposure on both

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behavior and egg deposition. The fathead minnow is an established model for aquatic

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toxicology and is part of Tier 1 assays that comprise the USEPA Endocrine Disrupter

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Screen Program, EDSP 26. In addition to the attributes that make fathead minnows

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suitable for the EDSP, they also exhibit relatively sophisticated and easily identifiable

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reproductive behaviors related to territorial defense, nest tending, courtship, and mating

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27

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selected for the exposure because of its documented effects on fathead minnow

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reproduction 17, as well as its strong binding affinity for the fathead minnow AR 16, 17. We

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hypothesized that following GES exposure female fathead minnow morphology would

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be masculinized, egg deposition decreased, and that the reproductive behavior of both

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sexes would be altered.

making them well suited for examining exposure effects on behavior. GES was

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Materials and Methods

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Chemical Stock Preparation

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Superstock solutions were prepared by dissolving GES (Steraloids Inc., CAS

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60282-87-3) in 95% EtOH (Fisher Scientific), aliquoted, and stored at -20 °C. Each day,

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working stock solutions were made by a 1:2000 dilution of the superstock solution with

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95% EtOH in an aluminum foil covered glass container. Fish were exposed using a

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flow-through system to four treatments consisting of water control, EtOH control, 10

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ng/L GES, and 100 ng/L GES. EtOH concentrations for all treatments did not exceed



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0.0000095%. For more information, see Supplemental Information, SI Exposure

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System Design.

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Fathead Minnow Exposure Four-month old adult fathead minnows were obtained (Aquatic Biosystems, CO,

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USA) and quarantined for one month in our lab. After quarantine, one male and two

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female fathead minnows (breeding trio per tank) were transferred from the breeding

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colony to the exposure system and acclimated for two weeks in dechlorinated tap water.

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Fish were fed twice with flake food and once daily with frozen adult brine shrimp. Water

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quality, including water temperature, pH, and dissolved oxygen, was monitored daily,

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and photoperiod was maintained using an automatic timer with lights on at 0800 h. To

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ensure that all trios were reproductively active, all breeding tiles were checked for the

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presence of eggs daily at 1500 h during the acclimation period. Any breeding tiles that

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had been used for spawning were removed from the tank and replaced with a fresh tile.

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Trios that had not deposited eggs by day 5 were replaced with fish from the breeding

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colony. All trios used for this study (n = 8 for H20 control, 10 ng/L GES, and 100 ng/L

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GES, with n = 7 for EtOH control due to tank damage) had laid eggs at least once by

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the conclusion of the acclimation period. All experiments were conducted under an

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approved University of Maryland IACUC protocol (R-13-60). For more information about

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fathead minnows, see SI Research Organisms.

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After the acclimation period, trios were continuously exposed to one of four

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treatments (water control, EtOH control, 10 ng/L GES, and 100 ng/L GES) using a flow

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through system that provided one turnover every 4 h (55 mL / min). The exposure


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consisted of a 24 h transition period, when dosing of GES was initiated, and a

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subsequent 8 d exposure period. For each treatment, water samples were collected

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daily in amber glass bottles at 1300 h dipped into a randomly selected tank and

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immediately frozen at -20 °C. Water samples were frozen upon collection and shipped

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overnight on ice to the USGS Organic Geochemistry Research Laboratory, Lawrence

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KA.

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To determine potential effects on egg deposition, all breeding tiles and surfaces

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of the tank were examined daily at 1500 h, and the number of eggs deposited by each

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trio quantified. To examine reproductive behaviors, a 1 h video was recorded from each

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tank starting at 0800 h on days 1, 2, and 8. To prevent observer bias, video files were

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automatically labelled with random alphanumeric titles by the recording software before

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being analyzed. Post-analysis, results were matched to appropriate tanks and

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treatments using a key generated by the recording software. To analyze the videos, an

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independent observer examined one 30 s segment every 5 min, and specific

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reproductive behaviors displayed by both males and females were quantified in terms of

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frequency or time as they occurred inside the breeding tile (as described in 27). For the

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purpose of this study, we further classified these behaviors into categories involving

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courtship, mating, aggression, and nest tending as described in Table S1.

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After the 8 d exposure period, all fish were euthanized using excess MS-222

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(300 mg/L, pH 7.4) and examined for the presence of secondary sexual characteristics,

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including the presence of nuptial tubercles, dorsal fin spot, and fatpad. One gonad lobe

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was processed for histology and the other snap-frozen in liquid nitrogen for future

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studies. All chemicals were purchased either through Fisher Scientific (Waltham, MA,



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USA) or VWR (Radnor, PA, USA), unless otherwise noted. For each treatment, gonads

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from one-half of randomly chosen fish (8 females and 4 males) were preserved in

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Davidson’s fixative for 24 h and stored in neutral buffered formalin for further histological

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processing and analysis following standard USEPA protocols 26. Gonads from females

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that exhibited masculinized behaviors, and were not randomly selected initially, were

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purposely added to the histology samples to be processed and examined. Fixed tissues

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were embedded in paraffin wax, sectioned on a rotary microtome (5-6 µm), and

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alternating ribbons from half the gonad thickness were mounted on glass slides, stained

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with hematoxylin and eosin, and mounted with glass coverslips. Seven to eleven slides

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were prepared for each gonad and three to five slides (approximately every other one)

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was stained and coverslipped. The middle section on each slide was examined by two

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researchers independently using a Zeiss Axioplan microscope to (1) confirm the sex of

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each fish and (2) check for the presence of three stages of gametogenesis:

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spermatozoa, spermatids, and spermatocytes in testes and vitellogenic, cortical

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alveolar, and pre-follicular oocytes in ovaries.

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Water Chemistry Analysis Upon reception at the USGS Organic Geochemistry Research Laboratory, water

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samples were logged in, thawed, and aliquots pipetted into a 2 mL glass

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chromatography vial to which 5 µL of a 5% tetrasodium EDTA solution was added. The

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bulk sample and the vialed samples were then refrozen until analysis. Samples were

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analyzed using a Waters Corp. (Milford, MA) Acquity H-Class Bio ultrapressure liquid

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chromatograph (UPLC) and AB/Sciex (Ontario, Canada) API 5500 tandem mass


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spectrometer (MS/MS). Samples were analyzed for GES and progesterone-d10 as an

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internal standard. To compensate for potential matrix effects, each sample was

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analyzed unspiked and then spiked using stacked direct aqueous injection of 95 µL of

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sample and 5 µL of a solution (A mix) containing 1.9 ng/mL of internal standard followed

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by 5 µL of a solution (SA mix) containing 1.9 ng/ml of the internal standard and of GES,

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respectively. Addition of 5 µL of the 1.9 ng/µL A and SA solutions provided the

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equivalent mass of the injection of 95 µL of a sample with an analyte concentration of

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100 ng/L. The compounds were separated using a 20 min gradient separation. The

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samples were analyzed by MS/MS using scheduled multiple reaction monitoring

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(SMRM) in positive-ion mode. The SMRM progestin method was set with a 1 min

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acquisition window centered on the retention time for each analyte. The declustering

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potential (DP), entrance potential (EP), collision energy (CE), and collision exit potential

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(CXP) was optimized for each MRM transition pair. Specific UPLC and MS/MS

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conditions are presented in Table S2.

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An 8-point standard curve of solutions ranging from 0.5 to 1000 ng/L was

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analyzed with the samples using stacked injections to draw up 95 µL of standard

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solution and 5 µL of A mix. The experimental samples then were analyzed as A-SA

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pairs as previously stated. A laboratory water blank sample was analyzed after the

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standard curve and after the analysis of each A-SA paired sample. Two MRM

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(protonated molecule-daughter ion) transitions were monitored for each analyte.

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Analytes were identified based on their analyte retention time, defined as the average

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ratio of the two daughter ions of the protonated analyte calculated from the standard

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curve ± 25% and their retention time to each other (± 1 s). Sample runs were



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processed using AB/Sciex Multiquant 3.0 and samples were initially quantitated using

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the 8-point standard curve. The resulting processed file was exported to Excel

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(Microsoft, 15.0) and quantitation was performed using the method of standard addition.

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Statistical Analyses

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Statistical analyses were performed using SAS Studio 3.3 (Cary, NC). All data

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sets were checked to assure normality and homoscedasticity. For the total number of

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eggs deposited, secondary sexual characteristic development (number of tubercle

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number only, as fin spot and fatpad were documented as present or not present), and

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male reproductive behaviors, treatments groups were compared with using a one-way

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ANOVA followed by a pairwise post-hoc analysis using Tukey’s HSD test with

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significance set at p < 0.05. To test for main (treatment, time) and interaction effects on

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daily egg production, a two-way ANOVA was also performed followed by a pairwise

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post-hoc analysis using Tukey’s HSD test with significance set at p < 0.05. We

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considered any display of male behaviors by females to be significant. Male behavior in

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female fish were only present in gestodene treated females and completely absent in

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control female fish, thus we presented them without significance testing. Unless

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otherwise noted, all data are shown as mean ± SEM.

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Results

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Water Chemistry

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Daily measured concentrations of GES found in each treatment are shown in Table S3. No GES was detected in either the water or ethanol (EtOH) control samples.


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Inexplicably, no GES was detected in the 10 ng/L tank on day 3. Following the

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recommendations in the USEPA test guidelines 26, calculations including ½ of the limit

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of quantification (1 ng/L ), thus 0.5 ng/L, and calculations excluding day 3 from the 10

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ng/L treatment are both shown in Table S3. With day 3 excluded, concentrations for the

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10 ng/L treatment ranged from 8 - 12 ng/L, with an overall average of 9.7 ± 0.5 ng/L.

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Concentrations for the 100 ng/L ranged from 40 - 140 ng/L, with an average of 91.2 ±

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11.9 ng/L.

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Gonadal Morphology No treatment effects were observed on the gonadosomatic index, i.e., gonad

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mass as percentage of the total body mass (p = 0.787). Histological examination of the

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gonad confirmed that the initial determination of sex at dissection was accurate, and

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that no intersex or gross pathological differences were detected. Slides were also

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examined for differences in the presence of three stages of gametogenesis

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(spermatozoa, spermatids, spermatocytes in testes; vitellogenic, cortical alveolar, and

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pre-follicular oocytes in ovaries). All stages of gametogenesis were observed in all

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gonads and no treatment-associated differences were observed.

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Egg Deposition For each treatment level, the average daily number of eggs and the total number

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of eggs deposited were calculated (Figure 1). We found that both treatment (p =

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0.0001) and time (p = 0.0072) affected egg deposition and there was no interaction

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between the two variables (p = 0.9904). There was a strong decrease in daily egg



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deposition in the 10 ng/L treatment and a complete cessation of deposition by day 3 for

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the 100 ng/L treatment. No differences were found in the total number of eggs

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deposited between the two controls, and significant decreases were seen for both the

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10 ng/L and 100 ng/L treatments (Figure 1). However, no differences in daily egg or

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total eggs deposited by the fish between the 10 and 100 ng/L treatments were found

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(Figure 1). When the number of eggs deposited each day was compared, significantly

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lower numbers of eggs were deposited on days 1 and 6, and significantly higher

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numbers of eggs deposited on day 2 and there was no difference in egg deposition in

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days 3-5 and 7-8.

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Secondary Sex Characteristics Males from all treatments exhibited nuptial tubercles, fatpad, and dorsal fin spot

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with no visual effects from exposure to GES (Figures 2 and 3). Females from both

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water and EtOH controls did not display tubercles or fatpads. With the exception of a

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single EtOH female, dorsal fin spots were absent from control females. Conversely,

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females showed apparent dose dependent masculinization in both the low and high

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GES treatments, with 40% of the 10 ng/L treated females and 90% of the 100 ng/L

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treated females having nuptial tubercles (Figures 2 and 3). Females exposed to 100

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ng/L of GES developed greater numbers of tubercles than both the controls and 10 ng/L

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treatment (p = 0.045). Regarding the other secondary sex characteristics examined,

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60% of females in the 10 ng/L and 100% of females in the 100 ng/L treatment

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developed dorsal fin spots (Figures 2 and 3). Fatpads were only present in females in


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the 100 ng/L treatment, and at relatively lower frequencies (30%) compared to the other

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male-typical secondary sex characteristics (Figures 2 and 3).

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Male Behavior GES altered various male fathead minnow behaviors, beginning as early as the

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first day of exposure (Video S1). Males from both the 10 ng/L and 100 ng/L treatments

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had fewer approaches to females on days 1, 2 and 8 (p < 0.0001), with no differences

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found between the 10 ng/L and 100 ng/L treatments (Figure 4, p = 0.621). There were

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fewer successful lead attempts by GES treated males compared to control males on

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days 1, 2, and 8, with no differences between the 10 and 100 ng/L treatments (Figure 4,

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p = 0.421). Spawning arch events remained consistent among treatments for days 1

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and 2, but spawning arches in males were completely absent on day 8 within both the

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10 and 100 ng/L treatments. While not significant (p = 0.4213), GES exposed males

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trended toward a greater number of nipping behaviors on day 1. Both 10 ng/L and 100

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ng/L treated males displayed a significant increase in the frequency of nipping

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behaviors on days 2 (p = 0.0088) and 8 (p = 0.0003) (Video S2). No differences were

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found among all treatments on days 1, 2, and 8 for the number of observed ceiling rubs,

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lateral quivers, or total time spent in the breeding tile displayed by males (p > 0.05).

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Female Behavior In addition to the alteration of male behaviors, we also observed the general

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masculinization of behaviors in female fish in the 10 and 100 ng/L treatments. These

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females showed increased aggression towards males in the form of nipping behavior,



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and were observed guarding and cleaning breeding tiles (Video S3) after just one day of

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exposure (Figure 5). Interestingly, in 25% of GES treatment tanks (2 of 8 tanks for both

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10 and 100 ng/L treatments), females exhibited male-typical behaviors such as

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approaching other females (Video S4) and successfully leading them back to the

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spawning tile where an attempt was made to perform lateral quivers (Video S5).

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Females in both the water and EtOH controls did not exhibit any male typical behaviors.

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Discussion While progestins have been widely utilized by humans as a component of birth

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control pharmaceuticals since the 1960s, few studies have examined the exposure

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effects of such chemicals on the reproduction of fish and other aquatic wildlife. Those

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that have been performed have tested their effects over a relatively long 21 - 28 d

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periods of exposure and have measured effects on egg deposition, hormone levels, and

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secondary sex characteristics and did not include a behavioral component. Behavior is

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known to be a sensitive endpoint for fish exposed to endocrine disrupting chemicals and

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other toxicants 1, 2. Given our specific interest in potential effects on behavior, we chose

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a shorter exposure period of 8 d that enabled us to document reproductive behaviors at

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days 1, 2, and 8, yet sufficiently long to measure effects on egg deposition as shown by

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other researchers using progestins similar to GES. Development of secondary sex

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characteristics, gonadosomatic index or GSI, and histopathology were also examined at

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the end of the study.

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We chose GES for the exposure because it was the most potent activator of the fathead minnow nAR in vitro 16 and because of the exposure effects in vivo 17. Although


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there are no known environmental measurements for gestodene yet, concentrations of

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all other progestins have been reported in the single to low hundreds of ng/L 12; thus, we

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felt comfortable using the 0, 10, and 100 ng/L concentrations selected for our study.

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Using liquid chromatography tandem mass spectrometry, we measured the

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concentrations of exposure system samples taken daily, and these measured

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concentrations were in reasonable agreement to the nominal GES concentrations.

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Water quality measurements, including dissolved O2, pH, and temperature were also

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measured daily, and they remained within the recommended range 26.

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While GES exposure did not appear to affect development of secondary sex

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characteristics in male fathead minnows, a strong masculinizing effect was documented

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in females. The masculinization of female secondary sex characteristics was similar in

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scope, but differed in apparent sensitivity of secondary sex characteristics to GES

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exposure to that noted previously 17. Based on our results, the development of dorsal

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fin spots in GES exposed females appears to be the most sensitive of the secondary

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sex characteristics examined, followed by nuptial tubercle formation and fatpad

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formation (the latter of which was only seen in the 100 ng/L treatment). These results

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differ somewhat from a previous study 17 which reported that dorsal fin spot and fatpad

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formation were the most sensitive secondary sex characteristics, and that nuptial

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tubercles were present, but only in the highest, 100 ng/L, exposure concentration.

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Other studies using other 19-nortestosterone derived progestins, norethindrone or

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levonorgestrel, have reported masculinized skin pigmentation or fin spot, as well as

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nuptial tubercles, but neither measured the presence of fatpad formation 21, 22.

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Furthermore, female fathead minnows also developed secondary sex characteristics



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similar to reproductive males when exposed to the environmental androgen, 17β–

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trenbolone, including darkened skin pigmentation, nuptial tubercles, and fatpad 28.

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These studies together with in vitro receptor transactivation research 16, 18 indicate that

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GES and other early generation progestins are producing their effects in fish at least in

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part through AR mediated pathways.

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GES exposure negatively affected the number of eggs deposited by fathead

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minnow trios on breeding tiles, compared to egg deposition in water (1,079 eggs) or

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EtOH (803 eggs) control trios. Importantly, the EtOH control numbers are based on 7

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tanks, whereas the water control (and GES treatment) levels have 8 tanks. If EtOH

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control eggs are adjusted to account for the tank that was damaged (918), and the

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water and adjusted EtOH control number of egg deposited averaged (998), there is a

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63% decline in the number for eggs in the 10 ng/L treatment group (369). Using a

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similar approach, the number of eggs deposited by the 100 ng/L treatment group

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declined by more than 95% from 998 to 46 total eggs deposited in 8 d. Our finding that

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GES exposure precipitously decreases egg deposition by fathead minnow trios agrees

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with previous research17, which found similar decreases in egg deposition, but in a 21 d

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exposure experiment using paired fathead minnows. In combination, these results

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confirm that GES has rapid, strong, and adverse effects on reproduction.

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The deleterious effect of GES on fathead minnow egg deposition is certainly

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important, but how does GES exposure lead to such decreased egg deposition? In the

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following paragraphs, we propose three hypotheses for how GES exposure dramatically

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decreased egg deposition through: (1) dysfunction of the brain-pituitary-gonadal axis

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regulation of reproduction; (2) gonadal toxicity resulting from histopathological


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alterations; and (3) altered reproductive behavior of males, females, or both sexes.

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These hypotheses are not necessarily all-inclusive, and more than one may be involved

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simultaneously in the strong decline and arrest of egg deposition in this study and

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others.

371 372 373

Dysfunction of the Brain-Pituitary-Gonadal Axis Previous studies where progestin exposure causes decreases in egg deposition

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are all 21 - 28 d exposures, which is far longer than our study at 8 d. Some of these

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studies examine histology of the gonad and report effects on gametogenesis in both

376

sexes and hypothesize that normal brain-pituitary-gonadal regulation of gametogenesis

377

has been altered 12, 19. Studies have measured circulating concentration of sex steroid

378

hormones and expression of gonadotropin genes. In female fathead minnows exposed

379

to GES or levonorgestrel, concentration of steroid hormones 17β-estradiol and

380

testosterone were decreased and, in males, testosterone and 11-ketotestosterone were

381

increased 17. Female fathead minnows exposed to norethindrone had similarly

382

decreased circulating concentrations of 17β-estradiol, but testosterone was decreased

383

in males exposed to the highest concentration of this progestin.

384

In similar studies, the frog (Xenopus laevis) was exposed to levonorgestrel for

385

approximately 28 d from Nieuwkoop Faber (NF) stage 46 – 55 (period of sexual

386

differentiation), transferred to water only, and followed by sampling at NF 58 and NF 66,

387

when the expression of luteinizing hormone (LHβ) and follicle stimulating hormone

388

(FSHβ) genes were quantified 29. The expression of LHβ and FSHβ in the brain and

389

pituitary (combined) in both males and females decreased at 10 -9 and 10 -8 M at NF 58



390

and at just the 10 -8 M concentration of levonorgestrel at the older stage, NF 66. While

391

not significant, the authors reported a trend in lower mean value of LHβ in adult female

392

fathead minnows exposed to another progestin, norethindrone 30. Presently, there are

393

no published studies examining the exposure effects on brain neuropeptides

394

(gonadotropin releasing hormone, kisspeptin, gonadotropin-inhibitory hormone etc.) that

395

regulate pituitary gonadotropins. As such, further research is suggested to test this

396

hypothesis.

397 398 399

Gonadal Toxicity Resulting in Histopathology In addition to dysregulation of the brain-pituitary-gonad axis, GES and other

400

progestins could be acting directly upon the gonad and affect gametogenesis directly or

401

affect steroid hormone synthesis. GES is a potent agonist of the fathead minnow AR

402

and as such may produce similar effects as have been reported in previous research

403

where fish have been exposed to 17β-trenbolone, a known environmental androgen.

404

There was no difference in gonadosomatic index (percentage gonad mass relative to

405

body mass), but there were histopathologies documented in fathead minnow males and

406

females exposed to trenbolone for 28 d 28. As characterized by the authors,

407

histopathologies of the testis included hyperproduction of sperm (greatly expanded

408

seminiferous tubule lumens filled with spermatozoa). In the ovaries of trenbolone

409

treated females, there was no evidence of recent ovulation, and there was increased

410

number of pre-ovulatory atretic follicles 28.

411 412

In our study, there were no observable differences in ovaries or testes across treatments. There was no difference in gonadosomatic index, which is a measure of the


Page 18 of 35

Page 19 of 35


413

reproductive state of the fish 31. We supplemented the gonadosomatic index with

414

histological analysis of gonadal sex and three stages of gametogenesis. We confirmed

415

that the initial determination of gonadal sex from gross examination during dissection of

416

the fish and extraction of the gonad was accurate and agreed with the histology. In

417

addition, all ovaries contained vitellogenic, cortical alveolar, and prefollicular oocytes,

418

and all testes contained spermatozoa, spermatids, and spermatocytes. While not

419

quantitative, these histological results enable us to confidently conclude that any effect

420

of GES on the sexual differentiation of the gonad or stages of gametogenesis was not

421

observed. That said, future studies could quantitatively examine at least these three

422

stages of gametogenesis, particularly if the exposure period is longer, to confirm and

423

extend the results of our study. The only other known study with GES did not report

424

histology 17. Importantly, our study differs from other progestin exposure studies in that

425

we did not find treatment effects on the gonads of either sex. These differences indicate

426

that GES may affect egg deposition via different mechanism(s) compared to

427

levonorgestrel and other progestins. It is also possible that effects on the gonad require

428

longer exposure periods to develop and are not observable from an 8 d exposure to

429

GES as was conducted here.

430 431 432

Altered Reproductive Behavior Reproductive behaviors were examined on day 8 to both ascertain the

433

consistency of any exposure effects on behavior observed during days 1 and 2 and to

434

determine if effects were altered by the duration of exposure. To our knowledge, our

435

recently published study on the exposure effects of another progestin, levonorgestrel,



436

on the eastern mosquitofish is the first study to expressly research effects on

437

reproductive behavior 32. Males paired with females exposed to levonorgestrel for 8 d

438

exhibited reduced interest in courtship and mating. One study by another lab provides

439

anecdotal evidence that male fathead minnows exposed to the progestins

440

levonorgestrel and drospirenone for 28 d displayed increased aggression and a lack of

441

interest in the breeding tiles 21.

442

Most fish use excreted amino acids, prostaglandins, and steroid hormones as

443

pheromones 33. Among other behaviors, pheromones enable chemical communication

444

of the state of reproductive readiness and help coordinate reproductive behaviors in

445

fishes. Of the reproductive pheromones used by cyprinid fishes, such as goldfish and

446

carp, females release androgens (androstenedione), progestogens (free and

447

conjugated 4-pregnen-17,20 –diol-3-one, DHP and DHP-S), and prostaglandins

448

(PGF2α), which males use to identify individuals nearing ovulation. Receptors for some

449

of these pheromones have been reported at the mRNA transcript level in the olfactory

450

epithelium of other cyprinid species, goldfish and in zebrafish 34. Environmental

451

measurement of steroid hormones downstream of animal agriculture facilities and

452

human waste water treatment plants have been suggested by others to be potential

453

pheromone signaling disrupters 35, 36.

454

How pheromones work through olfactory receptors to contribute to the brain-

455

pituitary-gonadal regulation of gametogenesis is poorly understood. However, given the

456

presence of membrane progesterone receptors in the olfactory epithelium 37 and that

457

androgenic and progestogenic pheromones are known to orchestrate final maturation of


Page 20 of 35

Page 21 of 35


458

gametes and spawning behavior 38, it is reasonable to hypothesize that GES and other

459

environmental gestagens could alter normal reproductive behavior in exposed fishes.

460

In our study, we found that aqueous exposures to GES caused rapid and

461

profound effects on the normal reproductive behavior (Video S1) of both male and

462

female fathead minnows, and these effects on behavior were maintained through the

463

end of the experiment. In contrast to the effects of GES on secondary sex

464

characteristics, both male and female fathead minnows appear to exhibit altered

465

behaviors after just one day of GES exposure. Males showed less interest in courtship

466

and mating (as evidenced by changes in approach and lead attempts), and showed

467

increased aggression towards females (Video S2). Increased male aggressive behavior

468

and nest acquisition have been noted in other studies examining exposure effects to

469

androgens such as 17α-methyltestosterone and 11-ketotestosterone 39, 40, our results

470

provide further evidence that GES action is mediated through the AR and that GES and

471

likely other 19-nortestosterone derived progestins function as environmental androgens.

472

Females also became more aggressive during GES exposure, accompanied by

473

decreases in their willingness to be led by males and related courtship and spawning

474

behaviors. While low in frequency, females from two of eight tanks in both the 10 ng/L

475

and 100 ng/L treatments were even observed competing with males for territory

476

acquisition and performing male-typical reproductive behaviors such as nest tending

477

(Video S3), approaches towards other females (Video S4), and lateral quiver attempts

478

with other females (Video S5).

479 480

Our research documents that relatively short-term exposure of adult fathead minnows to the human contraceptive progestin, gestodene, at environmentally



481

reasonable concentrations induces rapid and profound negative effects on reproductive

482

behavior, egg deposition, and sexual development. Because of the rapidity of the

483

changes in behavior and the lack of deleterious effects on gonads of gestodene-

484

exposed fish, we conclude that the effect on egg deposition in our study is primarily the

485

result of altered reproductive behavior. We hypothesize that the altered reproductive

486

behavior is due to disrupted pheromonal signaling and or direct effects on the

487

neuroendocrine regulation of reproduction. The alteration of fathead minnow

488

reproductive behaviors may serve as either the initial or main driving force of the

489

observed decline in egg deposition, providing an explanation for the disparity between

490

the rapid shutdown of egg production observed in this and other studies, along with

491

potential subsequent changes in the gonad during longer exposures. Regardless of

492

mechanism, rapid and strong responses from gestodene exposure suggest that

493

exposed populations of wild fish would be similarly affected. Taken together, results of

494

our study and previous research strongly suggest that certain progestins working

495

through fish androgen receptors may be affecting population recruitment and, by

496

extension, aquatic ecosystem health.


Page 22 of 35

Page 23 of 35


497

Supporting Information: This material is available free of charge via the Internet at

498

http://pubs.acs.org.

499

Acknowledgments

500

We thank C. Woods and D. Theisen, for their guidance with the exposure system

501

construction, and B. Bequette, for his assistance with the chemical delivery system.

502

Thanks to Z. Bailey, I. Chambers, J. Held, M. Levitas, C. Matz, and L. Moffatt for their

503

help with dissections. We graciously acknowledge the support of the Department of

504

Interior, U.S. Geological Survey Toxic Substances Hydrology Program, National

505

Institutes of Water Resources Grant (2014MD321G, to EFO), the Morris Animal

506

Foundation Grant (D14ZO-010 to EFO), and the University of Maryland. We sincerely

507

appreciate the editorial suggestions provided by the USGS reviewer. Any use of trade,

508

firm, or product names is for descriptive purposes only and does not imply endorsement

509

by the U.S. Government.



510

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636

Figure Legends

637 638

Figure 1. Mean daily number of eggs deposited (left Y axis) and the total eggs

639

deposited (right Y axis) for the H2O control, ethanol (EtOH) control, 10 ng/L gestodene

640

(GES), and 100 ng/L GES treatments. Sample size was 8 for all groups except EtOH

641

control, which had 7. Letters indicate significant differences for total eggs deposited (p

Exposure to the Contraceptive Progestin, Gestodene, Alters

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