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BIOLOGY OF REPRODUCTION (2012) 86(4):126, 1–7 Published online before print 21 December 2011. DOI 10.1095/biolreprod.111.097378

Disrupted Oogenesis in the Frog Xenopus tropicalis after Exposure to Environmental Progestin Concentrations1 Moa Sa¨fholm,2,3 Anna Norder,3 Jerker Fick,4 and Cecilia Berg3 3

Department of Environmental Toxicology, Uppsala University, Centre for Reproductive Biology (CRU), Uppsala, Sweden 4 Department of Chemistry, Umea˚ University, Umea˚, Sweden

Levonorgestrel is a synthetic progesterone commonly used in pharmaceuticals (e.g., in contraceptives). It is found in sewage treatment plant effluents at concentrations up to 30 ng/L and was recently shown to pose a threat to egg laying in fish. Information on the susceptibility of adult amphibians to progestin toxicity is lacking. The present study aimed to 1) characterize progestogenic effects on the full cycle of oogenesis (egg development) in frogs and 2) determine female amphibians’ susceptibility to reproductive impacts from progestogenic compounds in the environment. Sexually mature female Xenopus tropicalis were exposed to levonorgestrel via the surrounding water for 7 days (0, 51, or 307 ng/L) or 28 days (0, 1.3, 18, 160, or 1240 ng/L). Their ovaries were analyzed histologically with respect to frequencies of immature (in early meiotic prophase I), previtellogenic, vitellogenic, mature, and atretic oocytes. The 28-day exposure caused reduced proportions of oocytes at immature, vitellogenic, and mature stages, and increased proportions of previtellogenic oocytes compared with the control. The lowest tested concentration, 1.3 ng/L, increased the proportions of previtellogenic oocytes and reduced the proportions of vitellogenic oocytes, indicating inhibited vitellogenesis. The present study shows that progestin concentrations found in the aquatic environment impaired oogenesis in adult frogs. Our results indicate that progestogenic effects on oocyte development include interrupted germ cell progression into meiosis and inhibited vitellogenesis. Considering the crucial role of oogenesis in female fertility, our results indicate that progestogenic pollutants may pose a threat to reproduction in wild amphibian populations. amphibians, endocrine disruption, levonorgestrel, meiosis, oocyte development, pharmaceuticals, reproductive toxicology, vitellogenesis

INTRODUCTION Reproductive disorders, including increased incidences of intersex gonads and impaired oocyte development, have been reported in wild aquatic organisms at contaminated sites throughout the world [1–8]. The research into potential causes 1

Supported by the Research Council Formas, the Carl Trygger Foundation, and MistraPharma, a research program supported by the Swedish Foundation for Strategic Environmental Research (Mistra). 2 Correspondence: Moa Sa¨fholm, Department of Environmental Toxicology, Uppsala University, Norbyva¨gen 18A, 75236 Uppsala, Sweden. E-mail: [email protected] Received: 31 October 2011. First decision: 17 November 2011. Accepted: 9 December 2011. Ó 2012 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363

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for these reproductive disorders has mainly focused on environmental chemicals interfering with estrogen or androgen signaling. Progesterone signaling has received very little attention as a potential target for disruption, even though it is a key regulatory pathway in the development and function of the female reproductive organs. Progestins (synthetic progesterone) are pharmaceuticals commonly used in human and veterinary medicine, contraceptives, and other hormonal therapies. These compounds are released into the aquatic environment from sewage treatment plants, pharmaceutical industries, and agricultural areas (farm animal waste). The progestin levonorgestrel (LNG) has been detected in sewage treatment plant effluents and surface waters at concentrations up to 30 and 7 ng/L, respectively [9, 10]. It has also been demonstrated to strongly bioconcentrate in fish caged downstream from a sewage treatment plant [11]. In laboratory experiments, environmentally relevant concentrations of the progestins LNG and norethindrone inhibited egg laying in adult female fish [12, 13]. These findings indicate that progestins may pose a risk to reproduction in wild fish. Further information on the effects of progestins on aquatic wildlife is needed to assess the risk of environmental effects of this type of compounds. Can progestins in the environment act as contraceptives in amphibians? In women, the contraceptive actions of progestins generally include suppression of the gonadotropin surges and luteal serum progesterone concentration, inhibition of progesterone receptor synthesis in the endometrium, and inhibition of final preovulatory oocyte maturation, leading to suppressed ovulation [14–16]. Isoforms of the progesterone receptor have been identified in the brain and reproductive tissues, including ovaries, testes, and oviducts, in amphibians as well as in humans and other mammals [17–27]. In light of the global decline of amphibians and their proven sensitivity to endocrine disruption [28–31] it is important to increase our understanding of amphibians’ susceptibility to adverse effects of progestogenic environmental pollutants. We have previously reported that LNG is a potent developmental toxicant in female frogs [32]. To the best of our knowledge, the effects of adult exposure to progestins on oogenesis and fertility in frogs are not known. Oogenesis is the process by which female germ cells undergo meiosis and differentiation into mature oocytes. The germ cells form oogonia, which proliferate before they enter meiosis. They are referred to as immature or primary oocytes as they enter the prophase of the first meiosis. The oocytes are then arrested in meiotic prophase during the whole process of folliculogenesis until gonadotropin-induced signals trigger them to resume meiosis prior to ovulation [33, 34]. In most mammals the early germ cell differentiation into primary oocytes occurs in fetal life. In contrast, amphibian germ cells enter differentiation into oocytes continuously, throughout life [35]. When the immature oocyte progresses beyond the early diplotene stage of meiotic prophase and is surrounded by

ABSTRACT

¨ FHOLM ET AL. SA TABLE 1. Categorization of oocyte stages in Xenopus tropicalis using the criteria described by Hausen and Riebesell [55]. Stage Stage in vitellogenesis Oocyte stagea Stage in meiotic prophase I a

Immature oocytes

Follicular oocytes

– – Early stages: leptotene, pachytene, early diplotene

Previtellogenic I–II Diplotene

Vitellogenic III–V Mid diplotene

Postvitellogenic VI Late diplotene

According to Hausen and Riebesell [55].

Dissection and Morphometry of Reproductive Organs and Secondary Sex Characteristics The frogs were anesthetized in 0.7% benzocaine solution (Sigma-Aldrich) and killed by decapitation. The body weight and the ovary and oviduct weights were then determined. Cloacal length (enlarged cloaca is a female secondary sex characteristic) and occurrence of nuptial pads on the forearms (morphological characteristics of reproductive active males) were recorded. The ovaries were assessed with respect to the occurrence of darkly pigmented oocytes. The oocytes become darkly pigmented during vitellogenesis, before which they are transparent or white [53]. Gonadosomatic index (GSI) was calculated as: 100 3 weight of both ovaries/body weight. Oviducal-somatic index (OviSI) was calculated as: 100 3 right oviduct weight/body weight. The ovaries were fixed in formaldehyde (4% in phosphate buffer) and processed for histological evaluation.

Ovarian Histology Following dehydration in increasing concentrations of ethanol, part of the ovarian tissue was embedded in hydroxyethyl methacrylate (Leica Histroesin). Transverse sections were cut (2 lm) at two levels of the ovary and stained with hematoxylin-eosin. In one section per individual, the oocytes were scored as immature (i.e., in the early stages of meiotic prophase), previtellogenic, vitellogenic, mature postvitellogenic, or atretic oocytes using the criteria described by Hausen and Riebesell [55] (Table 1). The percentages of immature and follicular oocytes (i.e., those that have progressed beyond the early diplotene stage of meiotic prophase and are surrounded by follicle cells) were estimated by two analysts. The proportions of various stages of follicular oocytes were calculated as percentages of the total number of follicular oocytes. All histological evaluations were made using coded slides.

Chemical Analysis Water samples were extracted and analyzed using an inline solid-phase extraction column coupled to liquid chromatography-tandem mass spectrometry. Details of the chemical analysis and the inline extraction, including chemicals used, chromatographic details, and selected reaction monitoring transitions, are described in Kvarnryd et al. [34].

MATERIALS AND METHODS Animals and Exposure

Statistics

Sexually mature female X. tropicalis (Xenopus1, Inc.) were exposed to LNG (purity 99%; CAS: 797-63-7; Sigma-Aldrich) in separate plastic aquariums (15 L; Ferplast). They were exposed for 7 days to the nominal LNG concentrations of 0, 31.2, or 312 ng/L (0, 0.1, or 1 nM), or for 28 days to 0, 3.12, 31.2, 312, or

The statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software). Data from the 28-day exposure were compared using a KruskalWallis test with Dunn multiple-comparison test. The frequencies of frogs

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3120 ng/L (0, 0.01, 0.1, 1, or 10 nM). Water samples were collected for chemical analysis after water exchange on Day 3 in the short-term experiment and on Day 14 in the long-term experiment. This was considered adequate sampling based on previous measurements showing that the measured LNG concentrations were stable over a period of 2 mo using the same exposure regimen and materials (Sa¨fholm et al., unpublished data). The LNG was dissolved in acetone, with an acetone concentration in the aquaria, including controls, of 0.0002% during the 7-day exposure and 0.0006% during the 28-day exposure. To ensure that all females were in the same reproductive state, human chorionic gonadotropin (Sigma-Aldrich) was injected into the dorsal lymph sac, causing all females to ovulate before exposure started, as described in Gyllenhammar et al. [31]. The frogs were maintained in a 12L:12D cycle in water prepared from seven parts deionized water and three parts copper-free, dechlorinated tap water, keeping a conductivity of 130–150 S/m, a temperature of 278C 6 0.58C, and a pH at 7.5 6 0.3. The exposure was carried out under semistatic conditions, with half of the test solution and water being renewed daily. Three times a week the frogs were fed tropical fish food (Excel; Aquatic Nature). All procedures described in the present study were reviewed and approved by the Uppsala Ethics Committee for Animal Care and Use, and were performed in accordance with guiding principles for the care of laboratory animals.

follicle cells it is referred to as a follicular oocyte. During folliculogenesis, the amphibian oocyte increases in size because of incorporation of the yolk protein vitellogenin. Only a portion of the developing oocytes reach final maturity; the remaining ones undergo atresia (i.e., degeneration and resorption). The balance between the numbers of mature and atretic oocytes is important for controlling the fecundity in amphibians and fish [36, 37]. Progesterone is implicated at several stages of oogenesis, although its mechanisms of action remain to be clarified. Progestogens have been suggested to be involved in 1) the earliest stages of oogenesis (i.e., meiotic entry in fish oocytes [38] and follicular assembly in mammals [39–41]); 2) vitellogenesis in amphibians, reptiles, and fish [42–47]; and 3) promoting the final preovulatory oocyte maturation (i.e., resumption of meiosis I in vitro [23, 48]). However, the actual physiological trigger of resumption of meiosis in vivo seems to be androgens [33]. Because progesterone is readily converted to androgen by the enzyme CYP17 in the oocyte, the promoting role of progesterone in final oocyte maturation seen in vitro may be as a substrate for androgen production [49]. The concentration of the progesterone receptor increases dramatically in the postvitellogenic amphibian oocyte [50]. Xenopus tropicalis is an excellent model organism for studies on reproductive and endocrine toxicity for several reasons [51]. First, X. tropicalis is very sensitive to endocrine-disrupting compounds [30, 31, 52]. Second, because of its relatively short generation time (6 mo), X. tropicalis provides unique possibilities compared with other frog species. Third, its close relative Xenopus laevis is a well-established model for studies on oocyte maturation, so the oocyte stages are well defined [53]. Still another reason is that the organization and components of the hypothalamus-pituitary-gonadal axis are similar to those in mammals [54]. Last, being a water-dwelling species throughout life, X. tropicalis is a suitable model in experimental aquatic toxicology. Most amphibians, also terrestrial species, breed in water and may thereby be exposed to water-borne chemicals during their egg maturation period. Hence, the exposure scenario in the present study is ecologically relevant. The main objectives of the present study were to characterize progestogenic effects on the full cycle of oogenesis in frogs and to determine amphibians’ sensitivity to environmental progestogenic compounds with regard to reproduction-related responses in females. Female X. tropicalis were exposed to LNG via the surrounding water for 7 or 28 days, after which the full cycle of oogenesis (including the earliest stages of meiosis and vitellogenesis) as well as the morphology of reproductive organs and secondary sex characteristics were analyzed.

LOW PROGESTIN CONCENTRATIONS DISRUPT OOGENESIS TABLE 2. Nominal and mean measured concentrations of LNG (SD) in the test aquariums (n ¼ number of aquariums per concentration).

increased the proportions of previtellogenic oocytes and reduced the proportions of vitellogenic and mature oocytes compared with the controls, although the differences were not always statistically significant (Fig. 3). Twenty-eight days of exposure to the three lowest LNG concentrations reduced the proportion of immature oocytes compared with controls. Exposure to 307 ng/L for 7 days significantly increased the percentages of previtellogenic and immature oocytes compared with the controls.

Measured concentrations, ng/La

Nominal concentrations, ng/L 28-Day exposure 3.12 (n ¼ 4) 31.2 (n ¼ 4) 312 (n ¼ 3) 3120 (n ¼ 4) 7-Day exposure 31.2 (n ¼ 6) 312 (n ¼ 4)

1.3 18 160 1240

(0.19) (2.02) (14.76) (214)

51 (12) 307 (45)

DISCUSSION

a

Water samples were collected from all aquariums after water exchange at one occasion in the middle of the exposure period.

displaying nuptial pads were compared using a Fisher exact test. Data from the 7-day exposure were compared using a Kruskal-Wallis test. Oocyte frequencies in the controls and the females exposed to LNG were compared using a MannWhitney test.

RESULTS Chemical Analysis The measured LNG concentrations are shown in Table 2. Levonorgestrel was not detected in the control aquaria. Health Status There was no mortality in any exposure group; neither was there any weight loss or other signs of general toxicity in the experimental animals. Morphometry of Reproductive Organs and Secondary Sex Characteristics The females exposed to the highest LNG concentration (1240 ng/L) for 28 days had significantly decreased GSIs compared with the control females (Table 3), and their ovaries had transparent regions devoid of mature oocytes, visible to the naked eye (Fig. 1). They also had reduced cloacal length compared with the controls (Table 3), and they all displayed nuptial pads on their forelimbs (Fig. 2). Exposure to the lower LNG concentrations did not have any significant effects on GSI, OviSI, or cloacal length (Table 3). Oocyte Development The results from the histological evaluation of oogenesis are shown in Table 4. Levonorgestrel exposure for 28 days

TABLE 3. Reproductive anatomy in female Xenopus tropicalis after adult exposure to LNG.a LNG treatment 28-Day exposure Control (n ¼ 7) 1.3 ng/L (n ¼ 4) 18 ng/L (n ¼ 4) 160 ng/L (n ¼ 3) 1240 ng/L (n ¼ 4) 7-Day exposure Control (n ¼ 4) 51 ng/L (n ¼ 6) 307 ng/L (n ¼ 4)

Body weight, g 14.50 12.52 15.01 14.03 14.03

(2.05) (1.48) (1.40) (2.60) (1.29)

14.66 (0.37) 15.35 (1.97) 13.42 (1.57)

GSI, %b

Cloacal length, mm 2.47 2.18 2.20 2.30 1.93

(0.22) (0.39) (0.39) (0.17) (0.10)*

9.33 11.26 12.61 11.16 3.29

2.41 (0.53) 2.21 (0.63) 1.90 (0.79)

(2.07) (1.22) (1.95) (3.21) (0.48)*

12.10 (2.97) 8.02 (2.82) 9.41 (2.37)

OviSI, %c 1.26 1.56 1.58 1.95 0.87

(0.44) (0.39) (0.13) (0.68) (0.08)

2.03 (0.36) 1.33 (0.49) 1.68 (0.31)

Nuptial pad display, % 0 0 0 0 100** 0 0 0

a

Data are presented as mean (SD). GSI ¼ weight of both ovaries/body weight 3 100. c OviSI ¼ right oviduct weight/body weight 3 100. * Significantly different from control (P , 0.05), Kruskal-Wallis test with Dunn multiple-comparison test. ** Significantly different from control (P , 0.05), Fisher exact test. b

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The study shows that exposure of adult frogs to environmental concentrations of a common progestin, LNG, impaired oogenesis. The impact of progesterone on the final stages of oogenesis (i.e., the resumption of meiosis) has been reported in several studies [23, 48, 56], whereas information on progestogenic effects on early oogenesis is scant. Our results indicate that progestins act on the earliest stages of oogenesis as well as on vitellogenesis in frogs. To our knowledge the present study is the first to report reproductive effects of a progestin in amphibians after adult exposure. Our findings indicate that progestogenic pollutants may pose a threat to oocyte development and, consequently, female fertility in wild amphibian populations at contaminated sites. Levonorgestrel exposure for 28 days (but not for 7 days) dramatically reduced the proportion of mature postvitellogenic oocytes, implying impaired fertility. This finding is in accordance with the contraceptive effects of progestins in humans/mammals (i.e., inhibition of final preovulatory oocyte maturation, which leads to suppressed ovulation [14–16]). Although the frequency of atretic oocytes was not increased in the LNG-exposed females, degeneration of mature oocytes had obviously occurred. Normally the ovaries of sexually mature X. tropicalis consist of about 15% mature oocytes [53]. The loss of mature oocytes following exposure to environmentally relevant progestin concentrations in the present study is an important finding because it indicates that oogenesis and fertility in wild frogs may be at risk from this type of compound. Impaired oocyte development, including increased proportions of immature and previtellogenic oocytes as well as reduced proportions of vitellogenic and mature oocytes, has been reported in wild fish at contaminated sites [5, 6, 8, 57]. Hence, the ovarian abnormalities observed in the present study are similar to those observed in wild fish. To our knowledge, there are no comparable data on oocyte development in wild frogs.

¨ FHOLM ET AL. SA

FIG. 1. Photograph of reproductive organs in adult female Xenopus tropicalis. a) Control frog with ovaries filled with mature, pigmented oocytes. b) Frog exposed to 1240 ng/L LNG for 28 days, with ovaries displaying mainly previtellogenic oocytes and only a few mature eggs. O, ovaries; OD, oviducts.

vitellogenic oocytes is a sensitive endpoint also for progestogenic chemicals. The proportion of immature oocytes in early meiotic prophase I was slightly increased after 7 days’ LNG exposure, whereas after 28 days it was heavily reduced, except in the highest concentration. These findings indicate that chronic exposure of adult frogs to low progestin concentrations inhibits the progression of germ cells into meiosis. We have previously reported that transient LNG exposure during the tadpole period caused a chronic inhibition of oogenesis as well as an inhibition of Mu¨llerian duct (embryonic precursor of the reproductive tract) development, which subsequently resulted in sterile females [32]. In these developmentally exposed females the oocytes were arrested in early meiotic prophase stages (i.e., leptotene, pachytene, and early diplotene). The present results from frogs exposed to LNG as adults suggest that oogenesis was interrupted at even earlier stages (i.e., before or at meiotic entry). Hence, both larval and adult exposure to LNG inhibited early oogenesis in frogs, but at different oocyte stages. The mechanisms regulating the early events in oogenesis (including oogonial proliferation, meiotic entry, and follicular assembly) as well as the impact on these processes by environmental chemicals, are poorly understood [68]. Recently, the estrogenic environmental contaminant bisphenol A was shown to down-regulate a family of genes thought to be important in meiotic entry in the fetal mouse ovary [69]. Our present and previous findings show that the earliest stages of oocyte development are a sensitive target for progestogenic chemicals [32]. The display of nuptial pads on the forelimbs of the females exposed to the highest LNG concentration indicates masculinization. Nuptial pads are androgen-dependent secondary sex characteristics in male frogs and are normally absent in females [70]. This finding is not unexpected, because LNG is derived from testosterone and has a binding affinity of 58% to the human androgen receptor compared with that of testosterone [71]. This suggests that LNG has androgenic effects only at concentrations higher than those that have an impact on oogenesis. Our results are in accordance with findings in fish showing that male secondary sex characteristics (nuptial tubercles) were developed at higher LNG concentrations than those that inhibited egg laying in the females [13]. The present results together with those in fish imply that the observed effects of LNG on oogenesis are in fact progestogenic and not androgenic. A decreased cloacal length was seen in the females exposed

FIG. 2. Photograph of the right forelimb of a control female Xenopus tropicalis (a) and a female exposed to 1240 ng/L LNG (b) for 28 days displaying darkly pigmented nuptial pads (black arrow), a male secondary sex character.

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The observed increased proportion of previtellogenic oocytes and the decrease of vitellogenic oocytes in the LNGexposed females suggest that vitellogenesis was inhibited. This is in accordance with earlier findings showing that progesterone can inhibit both the synthesis and the uptake of vitellogenin into the oocyte [42–45]. Vitellogenin synthesis in the liver has been shown to be modulated by several steroids, most typically estrogens but also progesterone, testosterone, gonadotropins, and growth hormones, suggesting a multihormonal regulation [45, 46, 53, 58–64]. The uptake of vitellogenin into the oocyte is proposed to be estrogen independent and has been shown to be induced by gonadotropins [53]. Several kinds of environmental contaminants, including flame retardants, as well as estrogenic and androgenic compounds, have been shown to impair vitellogenesis as measured as reductions in the number of vitellogenic oocytes in zebrafish [65–67]. Our results suggest that reduction of

LOW PROGESTIN CONCENTRATIONS DISRUPT OOGENESIS TABLE 4. Frequencies of oocyte stages (mean [SD]) in ovaries of female Xenopus tropicalis after exposure to LNG. Follicular oocyte stages, %b LNG treatment 28-Day exposure Control (n ¼ 7) 1.3 ng/L (n ¼ 4) 18 ng/L (n ¼ 4) 160 ng/L (n ¼ 3) 1240 ng/L (n ¼ 4) 7-Day exposure Control (n ¼ 4) 51 ng/L (n ¼ 6) 307 ng/L (n ¼ 4)

Immature oocytes, %a 27 6 2 5 30

(12) (3)** (3)** (5)* (6)

18 (3) 30 (10) 27 (3)*

Previtellogenic oocytes 51 76 67 66 92

Vitellogenic oocytes

(10) (7)* (13) (12) (3)**

37 14 26 28 8

45 (10) 62 (16) 64 (9)*

(5) (3)** (9)* (10) (4)**

39 (9) 23 (10) 24 (9)

Postvitellogenic mature oocytes 13 9 3 6 1

(5) (6) (1)** (4) (1)**

16 (6) 15 (9) 12 (4)

Atretic oocytes 1.25 1.06 1.84 0.72 0.54

(0.79) (0.73) (2.06) (2.06) (0.39)

1.59 (2.02) 2.20 (2.42) 2.59 (2.33)

a

Percentage of oocytes in early meiotic prophase of the estimated total number of oocytes in a histological section of the ovary. Percentage of oocytes in various follicular stages of the total number of follicular oocytes in a histological section of the ovary. * Significantly different from control (P , 0.05), Mann-Whitney test. ** Significantly different from control (P , 0.01), Mann-Whitney test. b

FIG. 3. Photomicrograph from adult female Xenopus tropicalis showing a control ovary (a) with the majority of oocytes in the postvitellogenic mature stage (V); immature oocytes (b) in early meiotic prophase I (arrows) in a control ovary; an ovary of a female exposed to 1.3 ng/L LNG (c) containing mainly previtellogenic oocytes; and an ovary of a female exposed to 1240 ng/L LNG (d and e) containing mainly previtellogenic oocytes and very few mature oocytes. I and II, previtellogenic oocyte stages; III, vitellogenic oocyte. Bars ¼ 1 mm (a, c, and d) and 50 lm (b and e).

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be induced experimentally in female Xenopus by injecting gonadotropins [53]. As far as we know, this is the first study to report on the effects of steroid exposure on the cloacal size in frogs.

to the highest LNG concentration and indicated in those exposed to lower concentrations. Enlargement of the cloaca is a female secondary sex characteristic because it becomes swollen and red at sexual maturity [72]. Enlargement of the cloaca can

¨ FHOLM ET AL. SA

ACKNOWLEDGMENT The authors are grateful to Margareta Mattsson for excellent technical assistance and to Annelie Eriksson, an MSc student who helped us with the exposure and dissections of the frogs.

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In conclusion, the present study shows that environmentally relevant concentrations of a common progestin disrupt oogenesis in the adult frog. To our knowledge, this is the first study to report reproductive effects of adult progestin exposure in amphibians. Androgenic effects of LNG were observed only at the highest tested concentration. Our results indicate that progestogenic effects on oocyte development include interrupted germ cell progression into meiosis and inhibited vitellogenesis. The reduced formation of mature oocytes in the progestin-exposed females implies impaired fertility. Our findings therefore suggest that progestogenic compounds in contaminated aquatic environments pose a risk to reproduction in wild amphibian populations.

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