Search

The combined effects of the novel antifouling biocide tralopyril (TP) nitrile and ocean acidification (OA) on marine organisms are still not well understood, despite the increasing attention given to the toxic effects of emerging pollutants and OA on marine organisms in recent years. In this study, Crassostrea gigas (C. gigas) was exposed to TP, OA, and a combination of TP and OA for 21 days with a 14-day depuration. This study investigated the inter-tissue variability in energy metabolism responses and the impacts on gonadal development in C. gigas under both single and combined exposures to TP and OA. The results indicate that TP exposure and OA resulted in up-regulation of energy metabolism genes in the C. gigas, with tissues exhibiting enhanced aerobic metabolism. Furthermore, OA influences the sex determination of C. gigas, promoting the development of female individuals. Moreover, following depuration, C. gigas is able to restore normal energy metabolism and sexual development through the accumulation of suitable energy reserves. This study provides a valuable reference for the environmental and ecological risk assessment of TP, addressing the research gap in understanding the combined toxicity of TP and OA on aquatic organisms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Studying and discovering the molecular mechanism of Plasmodium sexual development is crucial for the development of transmission blocking drugs and malaria eradication. The aim of this study was to investigate the feasibility of using phosphatase inhibitors as a tool for screening proteins essential for Plasmodium sexual development and to discover proteins affecting the sexual development of malaria parasites., Methods: Differences in protein phosphorylation among Plasmodium gametocytes incubated with BVT-948 under in vitro ookinete culture conditions were evaluated using phosphoproteomic methods. Gene Ontology (GO) analysis was performed to predict the mechanism by which BVT-948 affected gametocyte-ookinete conversion. The functions of 8 putative proteins involved in Plasmodium berghei sexual development were evaluated. Bioinformatic analysis was used to evaluate the possible mechanism of PBANKA_0100800 in gametogenesis and subsequent sexual development., Results: The phosphorylation levels of 265 proteins decreased while those of 67 increased after treatment with BVT-948. Seven of the 8 genes selected for phenotype screening play roles in P. berghei sexual development, and 4 of these were associated with gametocytogenesis. PBANKA_0100800 plays essential roles in gametocyte-ookinete conversion and transmission to mosquitoes., Conclusions: Seven proteins identified by screening affect P. berghei sexual development, suggesting that phosphatase inhibitors can be used for functional protein screening., Competing Interests: Declaration of competing interest Declarations of interest: none., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes developmental and reproductive disorders in pups due to the attenuated luteinizing hormone (LH) production during the perinatal stage; however, the administration of α-lipoic acid (LA) to TCDD-exposed pregnant rats reversed the attenuated LH production. Therefore, reproductive disorders in pups are expected to be ameliorated with LA supplementation. To address this issue, pregnant rats orally received low dose TCDD at gestational day 15 (GD15) and proceeded to parturition. The control received a corn oil vehicle. To examine the preventive effects of LA, supplementation with LA was provided until postnatal day 21. In this study, we demonstrated that maternal administration of LA restored the sexually dimorphic behavior of male and female offspring. TCDD-induced LA insufficiency is likely a direct cause of TCDD reproductive toxicity. In the analysis to clarify the mechanism of the decrease in LA, we found evidence suggesting that TCDD inhibits the synthesis and increases the utilization of S-adenosylmethionine (SAM), a cofactor for LA synthesis, resulting in a decrease in the SAM level. Furthermore, folate metabolism, which is involved in SAM synthesis, is disrupted by TCDD, which may adversely affect infant growth. Maternal supplementation of LA restored SAM to its original level in the fetal hypothalamus; in turn, SAM ameliorated abnormal folate consumption and suppressed aryl hydrocarbon receptor activation induced by TCDD. The study demonstrates that the application of LA could prevent and recover next-generation dioxin reproductive toxicity, which provides the potential to establish effective protective measures against dioxin toxicity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The expression of apelin system has been shown in the adult testis of rat and mice. It has also been emphasized that regulation of testicular activity in early stages is important to sustain normal testicular activity in adulthood. Since the expression of apelin receptor (APJ) has been shown in the adult testis, moreover, developmental expression of APJ and its role has not been explored yet. Thus, we have examined the testicular expression of APJ during postnatal stages with special reference to proliferation, apoptosis and hormone secretion in early postnatal stage. Postnatal analysis showed that circulating apelin was lowest at PND1 and maximum at PND42. Among testosterone, estrogen and androstenedione, only circulating testosterone showed a gradual increase from PND1 to PND42. Testicular expression of APJ was also developmenatly regulated from PND1 to PND42, revealing a positive correlation with circulating apelin, testosterone, and androstenedione. Immunohistochemical study showed that APJ was mainly confined to Leydig cells of early postnatal stages, whereas, seminiferous tubules at PND42 showed immunostaining in the round spermatids. APJ inhibition from PND14-PND20 by ML221 suppressed the testicular proliferation, increased apoptosis and increased estrogen secretion. However, expression of AR was down-regulated by ML221 treatment. Furthermore, ML221 decreased the abundance of p-Akt. In vitro study also showed that APJ antagonist, ML221 decreased AR expression. These results suggests that apelin signaling during early developmental stages might be required to stimulate the germ cell proliferation, and inhibition of apoptosis. Both in vivo and in vitro study have shown that expression of AR was regulated by apelin signaling. Since the first wave spermatogenesis involves proliferation and apoptosis, therefore, further study would be required to unravel the exact mechanism of apelin mediated regulation of testicular activity during early postnatal stages. In conclusion, the present results are an indicative of apelin mediated signaling during early postnatal stage for regulation of germ cell proliferation, apoptosis and AR expression., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

To explore in mice if a 15% food restriction protocol during pregnancy programs the offspring postnatal development, with emphasis on reproductive function, and to assess if ghrelin (Ghrl) administration to mouse dams exerts effects that mimic those obtained under mild caloric restriction. Mice were 15% food-restricted, injected with 4 nmol/animal/day of Ghrl, or injected with the vehicle (control) thorough pregnancy. After birth, the pups did not receive further treatment. Pups born from food-restricted dams (FR pups) were lighter than Ghrl pups at birth, but reached normal weight at adulthood. Ghrl pups were heavier at birth and gained more weight than control pups (C pups). This effect was not associated with plasma IGF-1. FR pups showed a delay in pinna detachment and eye opening, while an advance was observed in Ghrl pups. FR pups showed also impairment in the surface-righting reflex. In both female FR and Ghrl pups, there was an advance in vaginal opening and, in adulthood, FR pups showed a significant decrease in their own litter size and plasma progesterone, and an increase in embryo loss. A delay in testicular descent was evident in male Ghrl pups. Changes in puberty onset were not associated with differences in the expression of Kiss1 in hypothalamic nuclei. Finally, in adulthood, FR pups showed a significant decrease in sperm quality. In conclusion, a mild food restriction thorough gestation exerted programming effects on the offspring, affecting also their reproductive function in adulthood. These effects were not similar to those of intragestational Ghrl administration., (© 2021. Society for Reproductive Investigation.)

Context: Gender incongruence is defined as disharmony between assigned gender and gender identity. Several interventions are liable in this case including genital affirming surgery among other surgical interventions such as harmonization, and also the use of gonadotropin-releasing hormone agonists (GnRHa) for gonadal shielding. This aids in preventing the development of secondary sexual characteristics related to the genetic sex., Objective: Systematically review the treatment of gender incongruity with GnRHa analogues., Data Sources: The data source of this research is from Pubmed-Medline and Embase., Study Selection: Articles published between 2009 and 2019 which studied transgender adolescents treated with GnRHa were carefully selected., Data Extraction: Were extracted: design, sample size, study context, targeted subjects of intervention, outcome measures, and results., Results: Eleven studies were included. The use of GnRHa seems to be well tolerated by the studied population. When started in pubertal transition, it was associated with a more distinct resemblance to body shape than to the affirmed sex. In addition to preventing the irreversible phenotypic changes that occur in cross-hormonal therapy, the use of GnRHa can equally contribute to the mental health of these adolescents., Limitation: There are few consistent studies on the use of GnRHa for gender incongruence., Conclusion: As the population of transgender children and adolescents grows, they acquire knowledge and greater access to the various forms and stages of treatment for sex reassignment. The medical community needs to be adequately prepared to better serve this population and offer the safest resources available.

Overexposure to glucocorticoids during fetal development alters fetal organ growth and maturation patterns, which can result in adverse programming outcomes in adulthood. The aim of this study was to determine whether exposure to dexamethasone (Dx) during the fetal period programmed ovary development and function in infant (16-day-old) and peripubertal (38-day-old) female offspring. Pregnant Wistar rats were separated into control and Dx-treated (0.5mg kg-1) groups and were injected with Dx or an equivalent volume of vehicle on Days 16, 17 and 18 of gestation. Ovaries from 16- and 38-day-old female offspring were prepared for histological and stereological examination. The volume of the ovary and the number of primordial and primary follicles were significantly reduced in prenatally Dx-exposed infant and peripubertal female offspring compared with control offspring. The number of multilaminar follicles was decreased in infant female offspring. In peripubertal females, prenatal exposure to Dx increased the number of multilaminar and large follicles of all classes. Because vaginal opening did not occur up to Day 38 postpartum in the Dx-exposed offspring, the absence of ovulation and corpora lutea is confirmation that the onset of puberty had been delayed. We can conclude that overexposure to glucocorticoids early in life programs ovary development, which may affect fertility in adulthood.

Polychlorinated biphenyls (PCBs), as persistent organic pollutants, are environmental endocrine-disrupting chemicals (EDCs). We aim to investigate the effects of prepubertal exposure to PCBs on the reproductive development and expression and regulation of related genes in rats. Female rats were treated with Aroclor-1221 (A-1221) (4 mg/kg/day, 0.4 mg/kg/day) or castor oil daily from postnatal day (PND) 28 for 2 weeks by gavage. Morphological, histological, hormonal, and biochemical parameters were studied. Lower weight and relative weight of hypothalamus, earlier puberty onset, a longer length of the estrous cycle, lower serum estradiol and progesterone levels, accelerated ovarian folliculogenesis, and higher apoptotic index in the ovary were found. The in vitro fertilization study showed a lower fertilization rate and cleavage rate. The genetic study revealed higher expression of Kiss-1 mRNA and lower expression of GnRH mRNA in the hypothalamus and higher expression of AMH mRNA and lower expression of C-myc mRNA in the ovary. These confirmed the reproductive damage of A-1221 in rats.

Prenatal tobacco-smoke exposure negatively affects the reproductive functions of female offspring and oxidative stress plays a major role at this point. Alpha-lipoic acid (ALA), well known as a biological antioxidant, has been used as a nutritional supplement and as a therapeutic agent in the treatment of certain complications during pregnancy. We aimed to investigate the effects of maternal tobacco-smoke exposure and/or ALA administration on puberty onset, sexual behavior, gonadotrophin levels, apoptosis-related genes, apoptotic cell numbers and oxidative stress markers in the adult female rat offspring. Sprague-Dawley rats were divided into four groups; control, tobacco smoke (TS), TS+ALA and ALA groups. Animals were exposed to TS and/or ALA for 8 weeks before pregnancy and throughout pregnancy. All treatments ended with birth and later newborn female rats were selected for each experimental group. The experiment ended at postnatal day 74-77. Maternal tobacco smoke advanced the onset of puberty in the female offspring of the TS group ( p < 0.05). In all treatment groups; the mean number of anogenital investigations and lordosis quality scores showed a decline, serum luteinizing hormone levels significantly increased ( p < 0.05) and several histopathological changes in ovaries were observed compared to the control group. In addition, an increase in apoptotic marker levels and apoptotic cell numbers was detected in the ovaries of all treatment groups. Decreased TAS and increased TOS levels were detected in all treatment groups compared to control. These findings suggested that maternal tobacco smoke and/or ALA administration may be leading to the impaired reproductive health of female offspring. Abbreviations : ALA: alpha-lipoic acid; LH: luteinizing hormone; FSH: follicle-stimulating hormone; TAS: total antioxidant status; TOS: total oxidant status; Apaf1: apoptotic protease-activating factor 1; Casp3: caspase 3; Casp9: caspase 9; CF: cyst follicles; 4-HNE: 4-Hidroxynonenal; 8-OHdG: 8-hydroxydeoxyguanosine; TUNEL: terminal deoxynucleotidyl transferase-mediated deoxyuridine-biotin nick end labeling; ROS: reactive oxygen species; GnRHR: gonadotropin-releasing hormone receptor; HPG: hypothalamic-pituitary-gonadal; AMPK: AMP-activated protein kinase; ELISA: enzyme-linked immunosorbent assay; cDNA: complementary DNA; qPCR: quantitative real-time PCR; FC: follicular cysts; PF: primary follicle; SF: secondary follicle; GF: graafian follicle; CL: corpus luteum; DF: degenerated follicle; AF: atretic follicle.

Differences in Sex Development (DSD) encompasses many diagnoses, where the development of chromosomal make-up, gonadal development or anatomical development is atypical. XY, DSD is a classification under the recent international consensus statement, and XY females commonly encapsulate disorders of androgen synthesis and androgen action. Complete Androgen Insensitivity Syndrome (CAIS) is the most common XY, DSD diagnosis, which results in an individual having XY chromosomes, but the person is phenotypically female. This article explores the care and management of children and young people with a DSD and focuses on the diagnosis of CAIS in adolescence. Medical and surgical management is discussed, alongside sexual function, gender identity and the psychological impact of the diagnosis. The involvement of the multidisciplinary team is stressed, together with an emphasis on the investment that is needed in psychological and nursing support for girls with CAIS, and their families.

Concern over global amphibian declines and possible links to agrochemical use has led to research on the endocrine disrupting actions of agrochemicals, such as fertilizers, fungicides, insecticides, acaricides, herbicides, metals, and mixtures. Amphibians, like other species, have to partition resources for body maintenance, growth, and reproduction. Recent studies suggest that metabolic impairments induced by endocrine disrupting chemicals, and more particularly agrichemicals, may disrupt physiological constraints associated with these limited resources and could cause deleterious effects on growth and reproduction. Metabolic disruption has hardly been considered for amphibian species following agrichemical exposure. As for metamorphosis, the key thyroid hormone-dependent developmental phase for amphibians, it can either be advanced or delayed by agrichemicals with consequences for juvenile and adult health and survival. While numerous agrichemicals affect anuran sexual development, including sex reversal and intersex in several species, little is known about the mechanisms involved in dysregulation of the sex differentiation processes. Adult anurans display stereotypical male mating calls and female phonotaxis responses leading to successful amplexus and spawning. These are hormone-dependent behaviours at the foundation of reproductive success. Therefore, male vocalizations are highly ecologically-relevant and may be a non-invasive low-cost method for the assessment of endocrine disruption at the population level. While it is clear that agrochemicals disrupt multiple endocrine systems in frogs, very little has been uncovered regarding the molecular and cellular mechanisms at the basis of these actions. This is surprising, given the importance of the frog models to our deep understanding of developmental biology and thyroid hormone action to understand human health. Several agrochemicals were found to have multiple endocrine effects at once (e.g., targeting both the thyroid and gonadal axes); therefore, the assessment of agrochemicals that alter cross-talk between hormonal systems must be further addressed. Given the diversity of life-history traits in Anura, Caudata, and the Gymnophiona, it is essential that studies on endocrine disruption expand to include the lesser known taxa. Research under ecologically-relevant conditions will also be paramount. Closer collaboration between molecular and cellular endocrinologists and ecotoxicologists and ecologists is thus recommended., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Advancements in measurement and modeling capabilities are providing unprecedented access to estimates of chemical exposure and bioactivity. With this influx of new data, there is a need for frameworks that help organize and disseminate information on chemical hazard and exposure in a manner that is accessible and transparent. A case study approach was used to demonstrate integration of the Adverse Outcome Pathway (AOP) and Aggregate Exposure Pathway (AEP) frameworks to support cumulative risk assessment of co-exposure to two phthalate esters that are ubiquitous in the environment and that are associated with disruption of male sexual development in the rat: di(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DnBP). A putative AOP was developed to guide selection of an in vitro assay for derivation of bioactivity values for DEHP and DnBP and their metabolites. AEPs for DEHP and DnBP were used to extract key exposure data as inputs for a physiologically based pharmacokinetic (PBPK) model to predict internal metabolite concentrations. These metabolite concentrations were then combined using in vitro-based relative potency factors for comparison with an internal dose metric, resulting in an estimated margin of safety of ~13,000. This case study provides an adaptable workflow for integrating exposure and toxicity data by coupling AEP and AOP frameworks and using in vitro and in silico methodologies for cumulative risk assessment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Zinc plays an important role in the regulation of insulin-like growth factor-I (IGF-I). IGF system, in turn, has a key role in the development and functions of the reproductive organs. This research was performed to investigate the effects of different sources of zinc on IGF-I gene expression and testicular development in pre-pubertal male Japanese quail. A total of 512 unsexed day-old Japanese quail chicks were randomly divided into 16 groups (4 dietary treatments × 4 replicates) and kept for 35 days. The control group diet was not supplemented with zinc whereas the diets of three groups were supplemented with 25 mg kg -1 zinc oxide (ZnO), zinc oxide nanoparticle (ZnON), and zinc-methionine (Zn-Met). On days 28 and 35, one birds from each subgroup were weighed, bled, and euthanized to evaluate gonado-somatic index (GSI), testicular histology, serum testosterone concentration, cloacal gland index (CGI), and the testicular IGF family gene expression. The results showed that GSI was higher in ZnON (2.307) than control (1.619) on day 35 (P < .05). Germinal epithelium thickness was higher in ZnON (78.88 μm) and Zn-Met (79.73 μm) than control (67.73 μm) on day 35 (P < .05). On day 35, the testosterone concentration was lowest in the control (5.830 ng/ml, P < .05). The CGI of 35-day-old birds was higher in Zn-Met (411.28) than the control (307.59, P < .05). IGF-IR mRNA expression was highest in Zn-Met group on day 28. Therefore, supplementation of diet with Zn-methionine is superior to other sources of zinc for diet supplementation in immature Japanese quail., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The increase in human infertility prevalence due to male reproductive disorders has been associated with extensive endocrine-disrupting chemical (EDC) exposure. Acrylamide (AA) is a compound formed spontaneously during heat processing of some foods that are mainly consumed by children and adolescents. In this study, we evaluated the prepubertal AA exposure effects on male adult reproductive physiology using a prepubertal experimental model to analyze the pubertal development, spermatogenesis hormones levels and genes expression involved in male reproductive function. This study is the first one to use the validated protocol to correlate the AA exposure with puberty development, as well as the AA-induced endocrine disrupting effects on reproductive axis. AA did not affect the age at puberty, the reproductive organ's weight and serum hormonal levels. AA reduces spermatogenesis, induces morphological and functional defects on sperm and alters transcript expression of sexual hormone receptors (Ar and Esr2), the transcript expression of Tnf, Egr2, Rhcg and Lrrc34. These findings suggest that excessive AA consumption may impair their reproductive capacity at adulthood, despite no changes in hormonal profile being observed., Competing Interests: Declaration of Competing Interest All authors declare that they have no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Di-(2-ethylhexyl) phthalate (DEHP) is ubiquitous in the environment and has been proposed to lead to reproductive disruption. In this study, we systematically investigated the effects of different doses of DEHP exposure on female hypothalamic-pituitary-gonadal axis development. Female Sprague-Dawley rats were gavaged with vehicle (corn oil) or DEHP (5 or 500mgkg-1 day-1) during postnatal Days (PNDs) 22-28 or PNDs 22-70. Results demonstrated that the low and high doses of DEHP exerted opposite effects on puberty onset, circulating luteinising hormone, serum oestradiol and progesterone levels, with the low dose (5mgkg-1) promoting and the high dose (500mgkg-1) inhibiting these parameters. Significant dose-related differences were also found in the D500 group with longer oestrous cycle duration, lower ovarian/bodyweight ratio, fewer corpus lutea and more abnormal ovarian stromal tissue in comparison with the oil or D5 groups. Molecular data showed that the hypothalamic Kiss1 mRNA expression in the anteroventral periventricular but not in the arcuate nucleus significantly decreased in the D500 rats and increased in the D5 rats relative to the rats in the oil group. These findings suggested that the kisspeptin system is a potential target for DEHP to disrupt reproductive development and function.

Progesterone (P4) are aquatic contaminants that can impair fish reproduction even in low concentrations. The aim of this study was to investigate the effects of P4 on the sex differentiation, by quantitative determination of transcriptional changes of a candidate target gene ( dax1 , has a function in the sex determination and gonadal differentiation of several vertebrate species) in Misgurnus anguillicaudatus . We first cloned and characterized the full-length cDNAs for the dax1 in M. anguillicaudatus (designated as Ma-dax1 ). Sequence analysis reveals that Ma-dax1 shares high homology with dax1 in other species. Quantitative real-time PCR (qRT-PCR) and in situ hybridization showed that Ma-dax1 gene was highly conserved during vertebrate evolution and involved in a wide range of developmental processes including embryogenesis, central nervous system development and gonad development. For the P4 administration assay, groups of mature fish were exposed for 1, 7, 14, 21 and 28 days to nominal concentrations of 10, 100, and 1000 ng/L P4 in a flow-through system. Quantification of Ma-dax1 transcripts revealed the expression of Ma-dax1 mRNA is altered after P4 treatment in mature gonads. Those showed that P4 could influence the sexual development and sex differentiation in M. anguillicaudatus by disturbing sex differentiation-associated gene expression, and dax1 can be used as a sensitive molecular biomarker for early warning to monitor the environmental progestins chemicals in fresh water environment.

Benzyl butyl phthalate (BBP) is a widely used plasticizer and has raised public health concerns. Here, we report the effects of BBP on the testis development during rat puberty. BBP (0, 10, 100 or 1000 mg/kg) was gavaged to 35-day-old male Sprague Dawley rats for 21 days. The serum testosterone levels, Leydig cell number, the expressions of Leydig and Sertoli cell genes and proteins were measured. The in vitro effects on steroidogenesis and gene expression in immature Leydig cells were observed. BBP significantly increased serum testosterone level at 10 mg/kg but lowered its level at 1000 mg/kg without affecting serum luteinizing hormone and follicle-stimulating hormone levels. BBP increased Leydig cell number at all doses but inhibited steroidogenic capacity per Leydig cell at 1000 mg/kg. BBP significantly increased the ratio of phosphos-AKT2 (pAKT2)/AKT2, and phosphos-ERK1/2 (pERK1/2)/ERK1/2 in the testis. Mono-benzyl phthalate (the metabolite of BBP) inhibited steroidogenesis but BBP did not affect androgen production in immature Leydig cells in vitro. In conclusion, BBP non-linearly regulates Leydig cell development by increasing Leydig cell number but inhibiting steroidogenesis., (Copyright © 2019. Published by Elsevier B.V.)

Study Question: Can mice serve as a translational model to investigate the reproductive effects of testosterone (T) therapy commonly used by transgender men?, Summary Answer: T enanthate subcutaneous injections at 0.45 mg twice weekly can be used in the postpubertal C57BL/6N female mouse to investigate the reproductive effects of T therapy given to transgender men., What Is Known Already: Most models of T treatment in female mice involve prenatal or prepubertal administration, which are not applicable to transgender men who often begin T therapy after puberty. Studies that have looked at the impact of postpubertal T treatment in female mice have generally not investigated reproductive outcomes., Study Design, Size, Duration: A total of 20 C57BL/6N female mice were used for this study. Study groups (n = 5 mice per group) included sesame oil vehicle controls and three doses of T enanthate (0.225, 0.45 and 0.90 mg). Mice were injected subcutaneously twice weekly for 6 weeks., Participants/materials, Setting, Methods: Daily vaginal cytology was performed prior to initiation of treatment to confirm that all mice were cycling. At 8-9 weeks of age, therapy with subcutaneous T enanthate (0.225, 0.45 or 0.90 mg) or the vehicle control was begun. T therapy continued for 6 weeks, at which point mice were sacrificed and compared to control mice sacrificed during diestrus/metestrus. Data collected included daily vaginal cytology, weekly and terminal reproductive hormone levels, terminal body/organ weights/measurements, ovarian follicular distribution/morphology and corpora lutea counts., Main Results and the Role of Chance: Of the mice treated with 0.90 mg T enanthate, two of five mice experienced vaginal prolapse, so this group was excluded from further analysis. T enanthate administration twice weekly at 0.225 or 0.45 mg resulted in cessation of cyclicity and persistent diestrus. One of five mice at the 0.225-mg dose resumed cycling after 2.5 weeks of T therapy. As compared to controls, T-treated mice had sustained elevated T levels and luteinizing hormone (LH) suppression in the terminal blood sample. T-treated mice demonstrated increases in clitoral area and atretic cyst-like late antral follicles (0.45 mg only) as compared to controls. No reduction in primordial, primary, secondary or total antral follicle counts was detected in T-treated mice as compared to controls, and T-treated mice demonstrated an absence of corpora lutea., Limitations, Reasons for Caution: Mouse models can provide us with relevant key findings for further exploration but may not perfectly mirror human reproductive physiology., Wider Implications of the Findings: To our knowledge, this report describes the first mouse model mimicking T therapy given to transgender men that facilitates analysis of reproductive changes. This model allows for future studies comparing duration and reversibility of T-induced changes, on the reproductive and other systems. It supports a role for T therapy in suppressing the hypothalamic-pituitary-gonadal axis in adult female mice as evidenced by LH suppression, persistent diestrus and absence of corpora lutea. The increase in atretic cyst-like late antral follicles aligns with the increased prevalence of polycystic ovary morphology seen in case series of transgender men treated with T therapy. The results also suggest that T therapy does not deplete the ovarian reserve., Study Funding/competing Interest(s): This work was supported by the American Society for Reproductive Medicine/Society of Reproductive Endocrinology and Infertility Grant and NIH R01-HD098233 to M.B.M. and University of Michigan Office of Research funding (U058227). H.M.K. was supported by the Career Training in Reproductive Biology and Medical Scientist Training Program T32 NIH Training Grants (T32-HD079342, T32-GM07863) as well as the Cellular and Molecular Biology Program. The University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core is supported by the Eunice Kennedy Shriver NICHD/NIH (NCTRI) Grant P50-HD28934. E.E.M. consults for Allergan. No other authors have competing interests., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Context: In 46,XY disorders of sexual development (DSD) patients, several factors may affect psychosexual development, leading to gender identity discrepancy and gender change later in life. Prenatal sexual steroid exposure and external genital virilization are considered to influence human psychosexual development, but their roles not completely understood yet., Design: A total of 144 individuals (18 to 60 years of age) with a clinical/molecular diagnosis of 46,XY DSD from a single tertiary center were enrolled. Psychosexual outcomes (gender role, gender identity, and sexual orientation) were assessed using questionnaires and psychological test. The Sinnecker score was used for genital virilization measurement. Prenatal androgen exposure was estimated according to 46,XY DSD etiology., Results: We found a positive association between prenatal androgen exposure and male psychosexual outcomes. Alternatively, prenatal estrogen exposure, age of gonadectomy, and the degree of external genital virilization did not influence any psychosexual outcome. There were 19% (n = 27) with gender change, which was associated with prenatal androgen exposure (P < 0.001) but not with the external genital virilization. The median age of gender change was 15 years, but most of the patients reported the desire for gender change earlier., Conclusions: Prenatal androgen exposure influenced psychosexual development in 46,XY DSD favoring male psychosexuality in all psychosexual outcomes, whereas the degree of external genital virilization did not influence these outcomes. The organizational effect of sexual steroids on psychosexuality at puberty appears to be weak in comparison with the prenatal effects. Prenatal androgen exposure also influenced female-to-male gender change frequency. All 46,XY DSD conditions with prenatal androgen exposure must be followed for gender issues in their management., (Copyright © 2019 Endocrine Society.)

We showed previously that in utero exposure to the cholesterol-lowering drug simvastatin (SMV) during sex differentiation lowers fetal lipids and testicular testosterone production (T Prod) in Hsd:SD rats. Here, the effects of SMV on fetal lipids and T Prod in Crl:CD(SD) rats were correlated with postnatal alterations in F1 males. The current study was conducted in two parts: 1) a prenatal assessment to confirm and further characterize the dose response relationship among previously reported alterations of SMV on fetal T Prod and the fetal lipid profile and 2) a postnatal assessment to determine the effects of SMV exposure during the periods of major organogenesis and/or sexual differentiation on F1 offspring growth and development. We hypothesized that SMV would have adverse effects on postnatal development and sexual differentiation as a consequence of the disruptions of fetal lipid levels and testicular T Prod since fetal cholesterol is essential for normal intrauterine growth and development and steroid synthesis. In the prenatal assessment, SMV was administered orally at 0, 15.6, 31.25, 62.5, 80, 90, 100, and 110 mg SMV/kg/d from GD 14-18, the period that cover the critical window of sex differentiation in the male rat fetus. T Prod was maximally reduced by ~40% at 62.5 mg/kg/d, and higher doses induced overt maternal and toxicity. In the postnatal assessment, SMV was administered at 0, 15.6, 31.25, and 62.5 mg/kg/d from GD 8-18 to determine if it altered postnatal development. We found that exposure during this time frame to 62.5 mg SMV/kg/d reduced pup viability by 92%, decreased neonatal anogenital distance, and altered testis histology and morphology in 17% of the F1 males. In another group, SMV was administered only during the masculinizing window (GD14-18) at 62.5 mg/kg/d to determine if male rat sexual differentiation and postnatal reproductive development were altered. SMV-exposed F1 males displayed female-like areolae/nipples, delayed puberty, and reduced seminal vesicle and levator ani-bulbocavernosus weights. Together, these results demonstrate that in utero exposure to SMV reduces offspring viability and permanently disrupts reproductive tract development in the male offspring. While the effects of high dose, short term in utero exposure to SMV in the adult male are likely androgen-dependent and consistent with the 40% reduction in T Prod in the fetal testes, long-term, lower dose administration induced some effects that were likely not mediated by decreased T Prod., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Over the past two decades, public health has focused on the identification of environmental chemical factors that are able to adversely affect hormonal function, known as endocrine disruptors (EDs). EDs mimic naturally occurring hormones like estrogens and androgens which can in turn interfere with the endocrine system. As a consequence, EDs affect human reproduction as well as post and pre-natal development. In fact, infants can be affected already at prenatal level due to maternal exposure to EDs. In particular, great attention has been given to those chemicals, or their metabolites, that have estrogenic properties or antagonistic effects on the activity of androgen or even inhibiting their production. These compounds have therefore the potential of interfering with important physiological processes, such as masculinization, morphological development of the urogenital system and secondary sexual traits. Animal and in vitro studies have supported the conclusion that endocrine-disrupting chemicals affect the hormone-dependent pathways responsible for male gonadal development, either through direct interaction with hormone receptors or via epigenetic and cell-cycle regulatory modes of action. In human populations, epidemiological studies have reported an overall decline of male fertility and an increased incidence of diseases or congenital malformations of the male reproductive system. The majority of studies point towards an association between exposure to EDs and male and/or female reproductive system disorders, such as infertility, endometriosis, breast cancer, testicular cancer, poor sperm quality and/or function. Despite promising discoveries, a causal relationship between the reproductive disorders and exposure to specific toxicants has yet to be established, due to the complexity of the clinical protocols used, the degree of occupational or environmental exposure, the determination of the variables measured and the sample size of the subjects examined. Despite the lack of consistency in the results of so many studies investigating endocrine-disrupting properties of many different classes of chemicals, the overall conclusion points toward a positive association between exposure to EDs and reproductive system. Future studies should focus on a uniform systems to examine human populations with regard to the exposure to specific EDs and the direct effect on the reproductive system.

The aim of this study was to characterise the molecular structure of the oestrogen receptor ERα and to evaluate the effect of bisphenol A (BPA) on ERα expression during sexual development of the Chinese giant salamander (Andrias davidianus). The ERα cDNA of A. davidianus includes an open reading frame of 1755bp (encoding 584 amino acids), a 219-bp 5' untranslated region (UTR) and a 611-bp 3'UTR. A polyadenylation signal was not found in the 3'UTR. Amino acid sequence analysis showed high homology between ERα of A. davidianus and that of other amphibians, such as Andrias japonicas (99.66% identity) and Rana rugose (81.06% identity). In 3-year-old A. davidianus, highest ERα expression was observed in the liver and gonads. During different developmental stages in A. davidianus (from 1 to 3 years of age), ERα expression in the testes increased gradually. ERα was localised in the epithelial cells of seminiferous lobules and in interstitial cells. ERα-positive cells were more abundant in the interstitial tissue during testicular development. ERα was located in the nucleus of oocytes during ovary development. We found that the sex of 6-month-old A. davidianus larvae could not be distinguished anatomically. The sex ratio did not change after larvae were treated with 10μM BPA for 1 month. However, BPA treatment reduced bodyweight and ERα expression in the gonads in male larvae.

Chemotherapy treatment is a mainstay of anticancer regimens, significantly contributing to the recent increase in childhood cancer survival rates. Conventional cancer therapy targets not only malignant but also healthy cells resulting in side effects including infertility. For prepubertal boys, there are currently no fertility preservation strategies in use, although several potential methods are under investigation. Most of the current knowledge in relation to prepubertal gonadotoxicity has been deduced from adult studies; however, the prepubertal testis is relatively quiescent in comparison to the adult. This review provides an overview of research to date in humans and animals describing chemotherapy-induced prepubertal gonadotoxicity, focusing on direct gonadal damage. Testicular damage is dependent upon the agent, dosage, administration schedule and age/pubertal status at time of treatment. The chemotherapy agents investigated so far target the germ cell population activating apoptotic pathways and may also impair Sertoli cell function. Due to use of combined chemotherapy agents for patients, the impact of individual drugs is hard to define, however, use of in vivo and in vitro animal models can overcome this problem. Furthering our understanding of how chemotherapy agents target the prepubertal testis will provide clarity to patients on the gonadotoxicity of different drugs and aid in the development of cytoprotective agents.

Background: Epiphyseal fusion (EF) marks the completion of longitudinal bone growth, a critical milestone monitored during treatment of skeletal growth and/or developmental disorders. Recently, a trend toward accelerated skeletal maturation in children has been documented. Because current methods for assessing skeletal maturation include children in their reference populations born as early as the 1930s, the timing of EF events in contemporary patients may differ substantially from those standards., Questions/purposes: (1) Do children today initiate the process of EF in the hand and wrist earlier than past generations on which maturity standards are based? (2) Do children today complete EF in the hand and wrist earlier than past generations on which maturity standards are based?, Methods: A total of 1292 children (665 males, 627 females) participating in the Fels Longitudinal Study, born between 1915 and 2006, were included in this retrospective, observational study. Each participant had between one and 39 serial left hand-wrist radiographs during childhood obtained specifically for research purposes. Main outcomes were the chronological age at the first sign of EF initiation (EF-I) and the first chronological age when EF was complete (EF-C) in the radius and ulna, and metacarpals and phalanges of the first, third, and fifth rays according to criteria of the Fels method. EF is a reliable metric with an average κ agreement statistic of 0.91. Penalized B-splines were used to model the changes in EF-I and EF-C ages and to identify changes across continuous birth years with major comparisons between children born in 1935 and 1995., Results: Approximately half of the epiphyses of the hand and wrist examined exhibited earlier EF-I and/or earlier EF-C in children born in 1995 compared with those born in 1935. The age at each milestone (EF-I and EF-C) decreased by as much as 6.7 and 6.8 months in males and 9.8 and 9.7 months in females, respectively. This change occurred gradually over the past century. The more proximal traits (EF of the distal radius, distal ulna, and metacarpals) were more likely to experience a shift in timing, whereas timing of EF in the phalanges remained relatively stable across birth years., Conclusions: A trend has occurred over the past century in the timing of EF, in both initiation and completion of the process, for many of the bones of the hand and wrist. Earlier EF reflects modern population advances in both skeletal and sexual maturation. Shifts in the timing of EF have the potential to influence treatment strategies for skeletal growth and/or developmental disorders such as scoliosis or leg length inequality, moving treatment windows to earlier ages. Earlier EF-I and EF-C identified in this study signals a need to reevaluate the timing of maturational milestones and current standards for skeletal assessment., Level of Evidence: Level II, prognostic study.

Nitrate has the potential to affect steroid production. Nitrate concentrations in streams in agricultural areas may exceed concentrations showing effects in laboratory studies. The effects of nitrate and/or nitrite on endocrine relevant endpoints were tested in zebrafish and brown trout. Zebrafish were exposed in two experiments to nitrate (8.8 to 89 mg NO 3 - /L) and nitrite (3.6 to 19 mg NO 2 - /L) during the period of sexual differentiation and sex ratios were determined. Vitellogenin concentrations were determined in the second experiment. The sex ratio was unaffected by the exposure to nitrate and nitrite. Vitellogenin concentrations were slightly elevated in males (but not females) in all of the groups exposed to nitrate. Juvenile brown trout were exposed to 5.7, 14, and 31 mg NO 3 - /L for 8 days and vitellogenin levels in liver were determined. Vitellogenin concentrations in the females were not affected by exposure, but in the males, there was an overall statistically significant effect of exposure to nitrate with the group exposed to 5.7 mg NO 3 - /L showing a trend of higher vitellogenin concentrations than the control group; levels in the males of the groups exposed to 14 and 31 mg NO 3 - /L were not statistically different from those of the control group. In conclusion, some marginal effect of nitrate in male fish on endocrine activity was observed but the present results for zebrafish, using environmentally relevant concentrations, do not define nitrate and nitrite as endocrine disrupting chemicals according to the generally accepted WHO/IPCS definition because no adverse effects (altered sex ratios) were demonstrated., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The increase of obesity, bad eating habits and the lack of physical exercises are highly related to dyslipidemias. Rosuvastatin is a lipid-lowering drug and has been indicated to prevent cardiovascular diseases and to treat dyslipidemias due to its higher efficiency to reduce serum cholesterol concentrations. This study aimed to evaluate the reproductive adverse effects on sexual maturity due to rosuvastatin exposure in juvenile male rats during prepuberty. Three groups were randomly formed with newly weaned rats: control, whose rats received saline solution 0.9% and rosuvastatin at doses of 3 or 10 mg kg -1 day -1 , administered orally by gavage, from postnatal day 21 until preputial separation (average of 45 days for controls and 49 days for statin-treated animals), indicative of puberty onset. Male rats were maintained until sexual maturity and were killed on postnatal day 110. In the rosuvastatin-treated groups, the results showed diminished follicle-stimulating hormone, luteinizing hormone and testosterone concentrations, increased estradiol and prolactin concentrations, histopathologic alterations on testis and epididymis and decreased sperm quality. Moreover, statin-exposed groups showed decreased expression of androgen receptor on testis and epididymis and lower expression of aquaporin-9 on epididymal epithelium. In conclusion, administration of rosuvastatin to prepubertal male rats provoked long-term hormonal deregulation and impaired reproduction at adulthood., (Copyright © 2018 John Wiley & Sons, Ltd.)

Corn oil, sesame oil, and 10% ethanol in corn oil are commonly used as dosing vehicles in toxicology studies. Since these vegetable oils contain bioactive compounds, it is important for toxicology studies to characterize the toxicities of the dosing vehicles themselves. It has been recently proposed that the width of the genital tubercle (GT), the dorsal-ventral length (D-V length) of the GT, and urethral tube closure in mouse fetuses can be used as novel markers for monitoring sexual development in mice. However, how these parameters are influenced by the dosing vehicles themselves remains unclear. Therefore, we evaluated the effects of corn oil, sesame oil, and 10% ethanol in corn oil on GT width, D-V length, and GT morphology in ICR mice. Our results showed that all three vehicles influenced GT width and D-V length, but not GT morphology, suggesting that the effects of dosing vehicles themselves might need to be considered when GT width or D-V length is used as a parameter to evaluate the effects of chemicals on GT development.

Critical windows of development are often more sensitive to endocrine disruption. The murine pituitary gland has two critical windows of development: embryonic gland establishment and neonatal hormone cell expansion. During embryonic development, one environmentally ubiquitous endocrine-disrupting chemical, bisphenol A (BPA), has been shown to alter pituitary development by increasing proliferation and gonadotrope number in females but not males. However, the effects of exposure during the neonatal period have not been examined. Therefore, we dosed pups from postnatal day (PND)0 to PND7 with 0.05, 0.5, and 50 μg/kg/d BPA, environmentally relevant doses, or 50 μg/kg/d estradiol (E2). Mice were collected after dosing at PND7 and at 5 weeks. Dosing mice neonatally with BPA caused sex-specific gene expression changes distinct from those observed with embryonic exposure. At PND7, pituitary Pit1 messenger RNA (mRNA) expression was decreased with BPA 0.05 and 0.5 μg/kg/d in males only. Expression of Pomc mRNA was decreased at 0.5 μg/kg/d BPA in males and at 0.5 and 50 μg/kg/d BPA in females. Similarly, E2 decreased Pomc mRNA in both males and females. However, no noticeable corresponding changes were found in protein expression. Both E2 and BPA suppressed Pomc mRNA in pituitary organ cultures; this repression appeared to be mediated by estrogen receptor-α and estrogen receptor-β in females and G protein-coupled estrogen receptor in males, as determined by estrogen receptor subtype-selective agonists. These data demonstrated that BPA exposure during neonatal pituitary development has unique sex-specific effects on gene expression and that Pomc repression in males and females can occur through different mechanisms., (Copyright © 2018 Endocrine Society.)

Ziram [zinc, bis (dimethyldithiocarbamate)] is an agricultural dithiocarbamate fungicide. By virtual screening, we have identified that ziram is a potential endocrine disruptor. To investigate its effects on pubertal development of Leydig cells, 35-day-old male Sprague Dawley rats orally received ziram (2 or 4 mg/kg/d) for 4 weeks and immature Leydig cells isolated from 35-day-old rat testes were treated with ziram (0.5-50 μM in vitro). Serum hormones, Leydig cell number and specific gene or protein expression levels after in vivo treatment were determined and medium androgen levels were measured as well as apoptosis of Leydig cells after in vitro treatment were determined. In vivo exposure to ziram lowered testosterone and follicle-stimulating hormone levels, and reduced Leydig cell number, and downregulated Leydig cell specific gene or protein expression levels. Ziram exposure in vitro inhibited androgen production and steroidogenic enzyme activities in Leydig cells by downregulating expression levels of P450 cholesterol side cleavage enzyme (Cyp11a1), 3β-hydroxysteroid dehydrogenase 1 (Hsd3b1), 17α-hydroxylase/17,20-lyase (Cyp17a1), and 17β-hydroxysteroid dehydrogenase 3 (Hsd17b3) via downregulating the steroidogenic factor 1 (Nr5a1) at a concentration as low as 5 μM. In conclusion, ziram exposure disrupts Leydig cell development during puberty possibly via downregulating Nr5a1., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology.All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The United States Consumer Product Safety Commission (Commission or CPSC) issues this final rule prohibiting children's toys and child care articles that contain concentrations of more than 0.1 percent of diisononyl phthalate (DINP), diisobutyl phthalate (DIBP), di-n-pentyl phthalate (DPENP), di-n-hexyl phthalate (DHEXP), and dicyclohexyl phthalate (DCHP). Section 108 of the Consumer Product Safety Improvement Act of 2008 (CPSIA) established permanent and interim prohibitions on the sale of certain consumer products containing specific phthalates. That provision also directed the CPSC to convene a Chronic Hazard Advisory Panel (CHAP) to study the effects on children's health of all phthalates and phthalate alternatives as used in children's toys and child care articles and to provide recommendations to the Commission regarding whether any phthalates or phthalate alternatives, other than those already permanently prohibited, should be prohibited. The CPSIA requires the Commission to promulgate a final rule after receiving the final CHAP report. This rule fulfills that requirement.

Controlled ovarian hyperstimulation (COH) is widely used for the treatment of infertility, while the long-term effects of COH on the reproductive function in female offspring are currently unknown. Based on the fact that COH could cause high E2 levels in women throughout pregnancy and excess estrogenic exposure during fetal development is harmful to subsequent adult ovarian function, we assumed the hypothesis that COH disrupts reproductive function in female offspring. To test this hypothesis, COH was induced in mice to obtain female offspring by pregnant mare serum gonadotropin (PMSG) and HCG, and then we evaluated pubertal transition, serum levels of E2, anti-Mullerian hormone (AMH), FSH and LH, mRNA expressions of Esr1 , Amhr2 , Fshr and Lhcgr in ovaries, number of follicles and ovarian histology. We also investigated the apoptosis of follicles by TUNEL; the mRNA expressions of Fas , FasL , Bax , Bcl2 , and caspase 3, 8 and 9 by quantitative real-time PCR; and the protein expressions of cleaved-caspase (CASP) 3, 8 and 9 by Western blot. Moreover, we further observed estrous cyclicity in young adult offspring, performed follicle counting and measured the level of AMH in both serum and ovary. COH could induce detrimental pregnancy outcomes, as well as delayed pubertal transition and irregular estrous cycle due to the aberrant growth and maturation of follicles in female offspring. Our novel findings add new evidence to better understand the potential risks of COH on the reproductive function in female offspring, raising the awareness that COH could exert adverse effects on female offspring, rather than just obtain more oocytes for fertilization., (© 2017 Society for Reproduction and Fertility.)

Purpose of Review: Male reproductive disorders are common and increasing in incidence in many countries. Environmental factors (including pharmaceuticals) have been implicated in the development of these disorders. This review aims to summarize the emerging epidemiological and experimental evidence for a potential role of in-utero exposure to analgesics in the development of male reproductive disorders., Recent Findings: A number of epidemiological studies have demonstrated an association between in utero exposure to analgesics and the development of cryptorchidism, although these findings are not consistent across all studies. Where present, these associations primarily relate to exposure during the second trimester of pregnancy. In vivo and in vitro experimental studies have demonstrated variable effects of exposure to analgesics on Leydig cell function in the fetal testis of rodents, particularly in terms of testosterone production. These effects frequently involve exposures that are in excess of those to which humans are exposed. Investigation of the effects of analgesics on human fetal testis have also demonstrated effects on Leydig cell function. Variation in species, model system, dosage and timing of exposure is likely to contribute to differences in the findings between studies., Summary: There is increasing evidence for analgesic effects on the developing testis that have the potential to impair male reproductive function. However, the importance of these findings in relation to human-relevant exposures and the risk of male reproductive disorders remain unclear.

Maternal treatment with dexamethasone (Dex) in threatening preterm delivery alters activities at the hypothalamic-pituitary-adrenal axis in the offspring. This alteration may interfere with reproductive function. The impact of gestational Dex exposure on male reproductive function of the offspring was investigated. A total of 25 pregnant rats randomly assigned to five groups (n=5) were treated with normal saline (control), Dex (100 μg/kg/day sc) during gestation days (GD) 1-7, 8-14, 15-21 and 1-21, respectively. Birth weight, anogenital distance (AGD), pubertal age, sperm parameters, hormonal profile and histopathology of testis and epididymis were determined in the F1 and F2 offspring. Results showed a significant increase (P<0.05) in pubertal age, serum corticosterone and gonadotropin-releasing hormone (GnRH) levels in the male offspring of DexGD 15-21 and 1-21 groups and a significant decrease (P<0.05) in serum testosterone, luteinizing hormone, birth weight and AGD at birth in the male F1 offspring. In the F2 offspring, there was a significant reduction (P<0.05) in serum corticosterone, testosterone, follicle-stimulating hormone and GnRH when compared with the control. Dex treatment at GD 15-21 and 1-21 significantly reduced (P<0.05) sperm motility and normal morphology in the F1 and F2 offspring. Maternal Dex treatment in rats during late gestation may disrupt sexual development markers in the F1 and F2 male offspring.

N-Nitrosodiethylamine (DEN), a well-known hepatocarcinogen, is found in certain food products as such or as a metabolic byproduct. This study investigated the effects of DEN on sexual development, gametogenesis, and oocyte maturation in Japanese medaka (Oryzias latipes). DEN reduced the germ cell number dose-dependently during early stages of sexual differentiation in XX larvae, resulting in underdeveloped ovaries in adulthood at low doses. This effect was sex-specific as no such changes were seen in XY larvae. Furthermore, XX and XY larvae that were exposed at a low dose during early life showed a significant reduction in body weight in adulthood. Gonads in sexually immature adult medaka males and females exposed to DEN were in advanced stages in comparison to that of the controls. Gonado-somatic indices were significantly high in treated males and females. DEN induced oocyte maturation in vitro, which was inhibited by cordycepin, demonstrating that it stimulated oocyte maturation through polyadenylation of cyclin B mRNA as in the case of the endogenous maturation-inducing hormone. Altogether, our results have proven that DEN could disrupt or mimic the signaling pathways involved in germ cell development, proliferation, and maturation., (© 2017 S. Karger AG, Basel.)

Methylphenidate (MPH), a psychoactive agent that acts mainly by blocking the uptake of dopamine, is the main drug used to treat Attention Deficit Hyperactivity Disorder in children and adolescents. During development, important changes in brain architecture and plasticity occur, these changes, sensitive to exposure to stimulant drugs, are important in the control of GnRH secretion, influencing the release of sex hormones throughout the ovarian cycle. This study investigated the effects of repeated treatment with MPH during development on reproductive parameters of adult female rats. Wistar rats received MPH 2.5mg/kg, MPH 5.0mg/kg, or tap water (gavage) from postnatal day (PND) 21 to PND 60. From PND 75, one subgroup of females was selected for evaluation of estrous cycle, estradiol levels, weight of sexual organs, and histomorphological analysis of ovary follicles and uterus. In another subgroup, the sexual and maternal behaviors were evaluated at PND 90 and on lactational day 5, respectively. No significant alterations were observed in the MPH groups. This study demonstrated that repeated administration of MPH during the period corresponding to childhood to early adulthood does not interfere in the reproductive function of female rats in adulthood., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Due to their terrestrial habitats and aquatic reproduction, many amphibians are both very vulnerable and highly suitable bioindicators. The plasticizer bisphenol A (BPA) is one of the most produced chemical substances worldwide, and knowledge on its impacts on humans and animals is mounting. BPA is used for the industrial production of polycarbonate plastics and epoxy resins and found in a multitude of consumer products. Studies on BPA have involved mammals, fish and the fully aquatic anuran model Xenopus laevis. However, our knowledge about the sexual development of non-model, often semi-terrestrial anuran amphibians remains poor. Using a recently developed experimental design, we simultaneously applied BPA to two non-model species (Hyla arborea, Hylidae; Bufo viridis, Bufonidae) and the model X. laevis (Pipidae), compared their genetic and phenotypic sex for detection of sex reversals, and studied sexual development, focusing on anatomical and histological features of gonads. We compared three concentrations of BPA (0.023, 2.28 and 228 μg/L) to control groups in a high-standard flow-through-system, and tested whether conclusions, drawn from the model species, can be extrapolated to non-model anurans. In contrast to previous studies on fish and Xenopus, often involving dosages much higher than most environmental pollution data, we show that BPA causes neither the development of mixed sex nor of sex-reversed individuals (few, seemingly BPA-independent sex reversals) in all focal species. However, environmentally relevant concentrations, as low as 0.023 μg/L, were sufficient to provoke species-specific anatomically and histologically detectable impairments of gonads, and affected morphological traits of metamorphs. As the intensity of these effects differed between the three species, our data imply that BPA diversely affects amphibians with different evolutionary history, sex determination systems and larval ecologies. These results highlight the role of amphibians as a sensitive group that is responsive to environmental pollution., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Antenatal betamethasone is used for accelerating fetal lung maturation for women at risk of preterm birth. Altered sperm parameters were reported in adult rats after intrauterine exposure to betamethasone. In this study, male rat offspring were assessed for reproductive development after dam exposure to betamethasone (0.1mg/kg) or vehicle on Days 12, 13, 18 and 19 of pregnancy. The treatment resulted in reduction in the offspring body weight, delay in preputial separation, decreased seminal vesicle weight, testosterone levels and fertility, and increased testicular weight. In the testis, morphologically abnormal seminiferous tubules were observed, characterized by an irregular cell distribution with Sertoli cell that were displaced towards the tubular lumen. These cells expressed both Connexin 43 (Cx43) and Proliferative Nuclear Cell Antigen (PCNA). In conclusion, intrauterine betamethasone treatment appears to promote reproductive programming and impairment of rat sexual development and fertility due to, at least in part, unusual testicular disorders., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Rapamycin inhibits the nutrient-sensing TOR pathway and extends life span in a wide range of organisms. Although life-span extension usually differs between the sexes, the reason for this is poorly understood. Because TOR influences growth, rapamycin likely affects life-history traits such as growth and reproduction. Sexes have different life-history strategies, and theory predicts that they will resolve the tradeoffs between growth, reproduction, and life span differently. Specifically, in taxa with female-biased sexual size dimorphism, reduced growth may have smaller effects on male fitness. We investigated the effects of juvenile, adult, or life-long rapamycin treatment on growth, reproduction, life span, and individual fitness in the outcrossing nematode Caenorhabditis remanei Life-long exposure to rapamycin always resulted in the strongest response, whereas postreproductive exposure did not affect life span. Although rapamycin resulted in longer life span and smaller size in males, male individual fitness was not affected. In contrast, size and fitness were negatively affected in females, whereas life span was only extended under high rapamycin concentrations. Our results support the hypothesis that rapamycin affects key life-history traits in a sex-specific manner. We argue that the fitness cost of life-span extension will be sex specific and propose that the smaller sex generally pay less while enjoying stronger life-span increase., (© The Author 2015. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The Fish Sexual Development Test (FSDT) is a non-reproductive test to assess adverse effects of endocrine disrupting chemicals. With the present study it was intended to evaluate whether gene expression endpoints would serve as predictive markers of endocrine disruption in a FSDT. For proof-of-concept, a FSDT according to the OECD TG 234 was conducted with the non-steroidal aromatase inhibitor fadrozole (test concentrations: 10μg/L, 32μg/L, 100μg/L) using zebrafish (Danio rerio). Gene expression analyses using quantitative RT-PCR were included at 48h, 96h, 28days and 63days post fertilization (hpf, dpf). The selection of genes aimed at finding molecular endpoints which could be directly linked to the adverse apical effects of aromatase inhibition. The most prominent effects of fadrozole exposure on the sexual development of zebrafish were a complete sex ratio shift towards males and an acceleration of gonad maturation already at low fadrozole concentrations (10μg/L). Due to the specific inhibition of the aromatase enzyme (Cyp19) by fadrozole and thus, the conversion of C19-androgens to C18-estrogens, the steroid hormone balance controlling the sex ratio of zebrafish was altered. The resulting key event is the regulation of directly estrogen-responsive genes. Subsequently, gene expression of vitellogenin 1 (vtg1) and of the aromatase cyp19a1b isoform (cyp19a1b), were down-regulated upon fadrozole treatment compared to controls. For example, mRNA levels of vtg1 were down-regulated compared to the controls as early as 48 hpf and 96 hpf. Further regulated genes cumulated in pathways suggested to be controlled by endocrine mechanisms, like the steroid and terpenoid synthesis pathway (e.g. mevalonate (diphospho) decarboxylase (mvd), lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase; lss), methylsterol monooxygenase 1 (sc4mol)) and in lipid transport/metabolic processes (steroidogenic acute regulatory protein (star), apolipoprotein Eb (apoEb)). Taken together, this study demonstrated that the existing Adverse Outcome Pathway (AOP) for aromatase inhibition in fish can be translated to the life-stage of sexual differentiation. We were further able to identify MoA-specific marker gene expression which can be instrumental in defining new measurable key events (KE) of existing or new AOPs related to endocrine disruption., (Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.)

Exposure to endocrine disrupting chemicals (EDCs) during development can have negative consequences later in life. In this study we investigated the effect of perinatal exposure to mixtures of human relevant EDCs on the female reproductive system. Rat dams were exposed to a mixture of phthalates, pesticides, UV-filters, bisphenol A, butylparaben, as well as paracetamol. The compounds were tested together (Totalmix) or in subgroups with anti-androgenic (AAmix) or estrogenic (Emix) potentials. Paracetamol was tested separately. In pre-pubertal rats, a significant reduction in primordial follicle numbers was seen in AAmix and PM groups, and reduced plasma levels of prolactin was seen in AAmix. In one-year-old animals, the incidence of irregular estrous cycles was higher after Totalmix-exposure and reduced ovary weights were seen in Totalmix, AAmix, and PM groups. These findings resemble premature ovarian insufficiency in humans, and raises concern regarding potential effects of mixtures of EDCs on female reproductive function., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Exposure of wildlife to endocrine disrupting chemicals (EDCs) is not necessarily continuous. Due to seasonal changes and variable industrial and agricultural activities it often occurs intermittently. Thus, it is possible that aquatic organisms may be more affected by periodic peak exposure than by chronic exposure. Therefore, an experimental scenario including an exposure from 2h to 90 days post-fertilization (dpf) and a subsequent recovery period until 150 dpf was chosen to assess the potential reversibility of the effects of sex steroids on sexual and gonad development of zebrafish (Danio rerio). The aim of this study was to investigate the persistence of the endocrine effects of an estrogen (EE2-17α-ethinylestradiol, 4ng/L), an inhibitor of estrogen synthesis (Fad-fadrozole, 50μg/L) or their binary mixture (Mix-EE2+ Fad, 4ng/L+50μg/L). Afterwards, a semi-quantitative histological assessment was used to investigate histopathological changes on gonad differentiation and development. The data showed that fadrozole, alone or in combination with EE2, permanently disrupts the sexual development, inducing masculinization and causing severe pathological alterations in testis, such as intersex associated to the enlargement of sperm ducts, interstitial changes, asynchronous development and detachment of basal membrane. After exposures to both EDCs and their mixture, the gonad histopathology revealed interstitial proteinaceous fluid deposits and, in ovaries, there were atretic oocytes, and presumably degenerative mineralization. On the other hand, the gonadal changes induced by EE2 alone seem to be partially reversible when the exposure regime changed to a recovery period. In addition, EE2 enhanced zebrafish growth in both genders, with male fish presenting signs of early obesity such as the presence of adipocytes in testis. Moreover, sex ratio was slightly skewed toward females, at 90 and 105 dpf, in zebrafish exposed to EE2. The data further indicate that long-term studies on impacts of single EDCs and their mixtures with recovery periods are crucial to reveal the possibility of sex reversal and pathological changes of gonads that can adversely affect breeding., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The aim of the present study was to investigate if the effects of the androgen, dihydrotestosterone (DHT) on the sexual development in juvenile Murray rainbowfish (Melanotaenia fluviatilis) are canceled out by the anti-androgen, flutamide. Fish (60 days post hatch) were exposed to 250ng/L of DHT, 25μg/L of flutamide (Flu-low), 250μg/L of flutamide (Flu-high), DHT+Flu low and DHT+Flu high. After 35 days of exposure, lengths and weights of the fish were measured and the condition factor (CF) calculated; vitellogenin (VTG) concentrations were measured in tail tissue; sex steroid hormones (17β-estradiol [E2] and 11-keto testosterone [11-KT]) were measured in the head tissue and abdominal regions were used in histological investigation of the gonads. Treatment with DHT reduced the body-length of both male and female fish, an effect which was canceled out by low and high concentrations of flutamide. However, flutamide (low or high) could not nullify the DHT-induced reduction in the CF in either sex. The E2 levels were reduced only in female fish after exposure to DHT but returned to normal after treatment with Flu-high. DHT increased the levels of 11-KT and decreased the E2/11-KT ratio in both sexes. Flu-high, but not Flu-low, could nullify these effects. Both DHT and flutamide (low or high) induced VTG production and this effect persisted when both chemicals were co-administered. Treatment with DHT did not affect gonadal cell development in the testes. However, the female fish treated with DHT contained ovaries in early-vitellogenic stage in comparison to the pre-vitellogenic ovaries in control fish. Co-treatment with flutamide (low or high) resulted in oocyte atresia. The results from the present study suggest that treatment with Flu-high could cancel out DHT-induced effects only on the hormonal profile and body-length in both male and female fish. Juvenile fish co-treated with DHT and flutamide (low or high) had high VTG levels and low CF. In addition, the ovaries in female fish were atretic. These data represent potential adverse effects on the ability of the fish to reproduce successfully., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Fipronil, a phenylpyrazole insecticide, is used in agriculture, veterinary medicine, and public health. Because this insecticide is considered a potential endocrine disruptor, the aim of this study was to examine the influence of perinatal exposure to fipronil on neonatal female reproductive system development. Pregnant rats were exposed (via gavage) daily to fipronil (0.03, 0.3, or 3 mg/kg) from gestational day 15 to day 7 after birth, and effects on the reproductive functions assessed on postnatal day (PND) 22. No signs of maternal toxicity were observed during daily treatment with fipronil. Perinatal exposure to the highest dose of fipronil (3 mg/kg) delayed the age of vaginal opening (VO) and first estrus without markedly affecting the anogenital distance (AGD). Further, exposure to 0.3 mg/kg fipronil produced a significantly shorter estrus cycle and reduced number of cycles during the period of evaluation. However, the other reproductive parameters analyzed, including fertility, hormone levels, sexual behavior, and histology of ovaries and uterus, displayed no marked alterations. In this experimental model, fipronil interfered with development of neonatal female reproductive system as evidenced by delay in VO and estrus cycle alterations without apparent significant effects on fertility. Further studies are needed to identify the mechanisms of action associated with the observed female reproductive system changes.

The chemical ultraviolet (UV) filter benzophenone-3 (BP-3) is suspected to be an endocrine disruptor based on results from in vitro and in vivo testing. However, studies including endpoints of endocrine adversity are lacking. The present study investigated the potential endocrine-disrupting effects of BP-3 in zebrafish (Danio rerio) in the Fish Sexual Development Test (Organisation for Economic Co-operation and Development TG 234) and a 12-d adult male zebrafish study. In TG 234, exposure from 0 d to 60 d posthatch caused a monotone dose-dependent skewing of the phenotypic sex ratio toward fewer males and more female zebrafish (no observed effect concentration [NOEC]: 191 μg/L, lowest observed effect concentration [LOEC]: 388 μg/L). Besides, gonad maturation was affected in both female fish (NOEC 191 μg/L, LOEC 388 μg/L) and male fish (NOEC 388 μg/L, LOEC 470 μg/L). Exposure to BP-3 did not affect the vitellogenin concentration in TG 234. After 12 d exposure of adult male zebrafish, a slight yet significant increase in the vitellogenin concentration was observed at 268 μg/L but not at 63 μg/L and 437 μg/L BP-3. Skewing of the sex ratio is a marker of an endocrine-mediated mechanism as well as a marker of adversity, and therefore the conclusion of the present study is that BP-3 is an endocrine-disrupting chemical in accordance with the World Health Organization's definition., (© 2015 SETAC.)

Exposure to environmental estrogens in wastewater treatment works (WwTW) effluents induces feminized responses in male fish, including the development of eggs in male testes. However, the impacts on the offspring of exposed fish are not well understood. In this study, we examined whether roach (Rutilus rutilus) from mothers that had been exposed to an undiluted WwTW effluent from early life to sexual maturity had altered susceptibility to gonadal feminization and an impaired capacity to reproduce. For males from both WwTW effluent exposed mothers and dilution water exposed mothers, effluent exposure for up to 3 years and 9 months induced feminized male gonads, although the intersex condition was relatively mild. There was no difference in the severity of gonadal feminization in roach derived from either WwTW effluent exposed or dilution water exposed mothers. Furthermore, a breeding study revealed that roach with effluent-exposed mothers reproduced with an equal success as roach with mothers exposed to clean water. Roach exposed to the effluent for 3 years in this study were able to reproduce successfully. Our findings provide no evidence for impacts of WwTW effluent exposure on reproduction or gonadal disruption in roach down the female germ line and add to existing evidence that male roach with a mild intersex condition are able to breed competitively.

Industrial use of aniline is increasing worldwide with production estimated to surpass 5.6 million metric tons in 2016. Exposure to aniline occurs via air, diet, and water augmenting the risk of exposing a large number of individuals. Early observations suggest that aniline is metabolized to paracetamol/acetaminophen, likely explaining the omnipresence of low concentrations of paracetamol in European populations. This is of concern as recent studies implicate paracetamol as a disrupter of reproduction. Here, we show through steroidogenic profiling that exposure to aniline led to increased levels of the Δ4 steroids, suggesting that the activity of CYP21 was decreased. By contrast, paracetamol decreased levels of androgens likely through inhibition of CYP17A1 activity. We confirm that aniline in vivo is rapidly converted to paracetamol by the liver. Intrauterine exposure to aniline and paracetamol in environmental and pharmaceutical relevant doses resulted in shortening of the anogenital distance in mice, a sensitive marker of fetal androgen levels that in humans is associated with reproductive malformations and later life reproductive disorders. In conclusion, our results provide evidence for a scenario where aniline, through its conversion into antiandrogenic paracetamol, impairs male reproductive development., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The aim of the present study was to investigate the persistence of endocrine effects by prochloraz, a fungicide known to have multiple effects on the endocrine system of vertebrates. Since discontinuous exposure is particularly relevant in aquatic ecosystems, an exposure scenario with an exposure phase and a subsequent recovery period was chosen to assess the potential for reversibility of effects by prochloraz on the sexual development of zebrafish (Danio rerio). Zebrafish were exposed to different concentrations of prochloraz (10-300 μg/L) until 60 days post hatch (dph), which includes the period of sexual differentiation. For the subsequent 40 days, fish were either held in clean water for depuration or under further continuous exposure. Histological investigations of the gonads revealed persistent effects on sexual differentiation. The sex ratio was skewed towards males and significantly more intersex individuals were found after exposure to prochloraz at 60 dph. No intersex fish, but masculinized sex ratios were still present after the depuration period, documenting that prochloraz irreversibly affects the sexual development of zebrafish.

Current knowledge on zebrafish (Danio rerio) sex determination suggests that this trait has a polygenic genetic basis, although environmental factors, such as endocrine disrupting chemicals (EDC), may also be involved in modeling or disturbing the species sex differentiation and development. This study aimed to assess how sex steroids imbalance triggers impact on sex differentiation and gonad development in zebrafish. Fish where exposed to an estrogen (EE2, i.e. 17α-ethinylestradiol, 4ng/L), to an inhibitor of estrogen synthesis (Fad, i.e. fadrozole, 50μg/L) or to their binary mixture (Mix-EE2+ Fad, 4ng/L+50μg/L), from 2h to 60 days post-fertilization (dpf). Afterwards, a quantitative (stereological) analysis using light microscopy, based on systematic sampling, was made at 35 and 60dpf, to identify alterations on gonad differentiation and development. During the sex differentiation period, our histological data showed that not all zebrafish males develop a "juvenile ovary", contrarily to what is currently taken for granted. Furthermore, the stereological analysis suggests that EE2 alone enhanced both zebrafish growth and gonad development. On the other hand, exposure to Fad affected the sexual development in zebrafish, inducing masculinization of the specimens, with some degree of intersex observed in males. In addition, the binary mixture allowed identifying sex-dependent roles of steroid hormones in the general growth and gonad development of zebrafish, with estrogens acting as growth promoters in females and being essential for ovary development. Data further support that sex-specific and single EDC impact studies are important, but clearly not sufficient to understand what may occur in the environment., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The objective of this study was to determine if artificially reared male lambs differ from those reared by their mothers in their reproductive development and sexual behaviour during the first breeding season and in their serum testosterone to a GnRH challenge at the end of the first breeding season. Lambs were assigned to two experimental groups: 1) artificially reared lambs, separated from their dams 24-36h after birth (Week 0) and fed sheep milk until 10weeks of age (group AR, n=14); and 2) lambs reared by their dams until 10weeks of age (group DR, n=13). Reproductive parameters and sexual behaviour were recorded from Weeks 9 to 39. The GnRH challenge was performed on Week 40. Body weight, scrotal circumference, gonado-somatic index, testosterone concentration and sperm parameters were unaffected by group, but increased with age (P<0.0001). Lambs reared by their mothers had greater values of gonado-somatic index on Weeks 9, 16 and 19 (P<0.05), and tended to reach puberty earlier than AR (22.9±0.7 vs. 25.1±1.1weeks, respectively, P=0.087). Lambs reared by their mothers presented more lateral approaches and mount attempts than AR (P<0.05), and DR lambs presented more mounts on Weeks 32 and 39 than AR (P<0.05). Blood testosterone concentrations 3.5 and 4h after the GnRH challenge were higher in AR than in DR lambs (P<0.05). In conclusion mother rearing promoted sexual behaviour and reproductive performance of male lambs., (Copyright © 2015. Published by Elsevier Inc.)