Intersex Occurrence in Rainbow Trout (Oncorhynchus mykiss) Male Fry Chronically Exposed to Ethynylestradiol

This study aimed to investigate the male-to-female morphological and physiological transdifferentiation process in rainbow trout (Oncorhynchus mykiss) exposed to exogenous estrogens. The first objective was to elucidate whether trout develop intersex gonads under exposure to low levels of estrogen. To this end, the gonads of an all-male population of fry exposed chronically (from 60 to 136 days post fertilization – dpf) to several doses (from environmentally relevant 0.01 µg/L to supra-environmental levels: 0.1, 1 and 10 µg/L) of the potent synthetic estrogen ethynylestradiol (EE2) were examined histologically. The morphological evaluations were underpinned by the analysis of gonad steroid (testosterone, estradiol and 11-ketotestosterone) levels and of brain and gonad gene expression, including estrogen-responsive genes and genes involved in sex differentiation in (gonads: cyp19a1a, ER isoforms, vtg, dmrt1, sox9a2; sdY; cyp11b; brain: cyp19a1b, ER isoforms). Intersex gonads were observed from the first concentration used (0.01 µg EE2/L) and sexual inversion could be detected from 0.1 µg EE2/L. This was accompanied by a linear decrease in 11-KT levels, whereas no effect on E2 and T levels was observed. Q-PCR results from the gonads showed downregulation of testicular markers (dmrt1, sox9a2; sdY; cyp11b) with increasing EE2 exposure concentrations, and upregulation of the female vtg gene. No evidence was found for a direct involvement of aromatase in the sex conversion process. The results from this study provide evidence that gonads of male trout respond to estrogen exposure by intersex formation and, with increasing concentration, by morphological and physiological conversion to phenotypic ovaries. However, supra-environmental estrogen concentrations are needed to induce these changes.     

Estrogènes et spermatozoïdes

Aromatase is the terminal enzyme responsible for estrogen biosynthesis; it is present in the endoplasmic reticulum membrane of steroidogenic cells in vertebrates. This enzyme functions with the ubiquitous reductase as the electron donor. The aromatase gene is unique and its expression is regulated in a tissue and more precisely in a cell-specific fashion via the alternative use of several promoters located in the first exons. This enzymatic complex is generally involved in development, reproduction, sexual differentiation and behaviour, but also in bone and lipid metabolism, brain functions and diseases such as breast and testicular tumors. The aromatase gene expression and its transduction in a fully active protein in testicular somatic cells and germ cells together with the widespread distribution of estrogen receptors (ERα & β) in the testis and the genital tract of the male, are clearly in favor of a physiological role for estrogens in the spermatogenesis processings especialy in sperm maturation. Therefore, we begin to understand the physiopathological roles of the estrogens in males indeed, the aromatase deficiency is associated, with severe bone maturation problems and sterility in man. Conversely, it is also obvious that estrogens in excess are responsible of the impaired spermatogenesis. These female hormones (or the ratio androgens/estrogens) do play a physiological role in the development and maintenance of male gonadal functions and obviously, several steps are concerned especially the sperm production and maturation.     

Antiapoptotic Factor Humanin Is Expressed in Normal and Tumoral Pituitary Cells and Protects Them from TNF-α-Induced Apoptosis

Humanin (HN) is a 24-amino acid peptide with cytoprotective action in several cell types such as neurons and testicular germ cells. Rattin (HNr), a homologous peptide of HN expressed in several adult rat tissues, also has antiapoptotic action. In the present work, we demonstrated by immunocytochemical analysis and flow cytometry the expression of HNr in the anterior pituitary of female and male adult rats as well as in pituitary tumor GH3 cells. HNr was localized in lactotropes and somatotropes. The expression of HNr was lower in females than in males, and was inhibited by estrogens in pituitary cells from both ovariectomized female and orquidectomized male rats. However, the expression of HNr in pituitary tumor cells was not regulated by estrogens. We also evaluated HN action on the proapoptotic effect of TNF-α in anterior pituitary cells assessed by the TUNEL method. HN (5 µM) per se did not modify basal apoptosis of anterior pituitary cells but completely blocked the proapoptotic effect of TNF-α in total anterior pituitary cells, lactotropes and somatotropes from both female and male rats. Also, HN inhibited the apoptotic effect of TNF-α on pituitary tumor cells. In summary, our results demonstrate that HNr is present in the anterior pituitary gland, its expression showing sexual dimorphism, which suggests that gonadal steroids may be involved in the regulation of HNr expression in this gland. Antiapoptotic action of HN in anterior pituitary cells suggests that this peptide could be involved in the homeostasis of this gland. HNr is present and functional in GH3 cells, but it lacks regulation by estrogens, suggesting that HN could participate in the pathogenesis of pituitary tumors.     

Impaired reproduction in three-spined sticklebacks exposed to ethinyl estradiol as juveniles

To investigate the population-level effects of exposure to environmental endocrine disrupters, a mesocosm-scale study was carried out in which the reproductive performance of groups of free-spawning three-spined sticklebacks, Gasterosteus aculeatus, exposed as juveniles to a model estrogen, was assessed. Juvenile sticklebacks were exposed to ethinyl estradiol (EE(2)) at measured concentrations of (mean +/- SEM) 1.75 +/- 0.37 ng L(-1) and 27.7 +/- 1.08 ng L(-1) for 4 wk posthatch and then reared thereafter in pristine lake water until they reached adulthood. Exposure to the higher EE(2) concentration resulted in the occurrence of ovotestis among males, whereas no gonadal abnormalities were evident among males exposed to the lower concentration of EE(2). In addition, when spawning was allowed in the mesocosm environment, fewer nests were built per male, and fewer eggs were deposited per nest, in the group exposed to 27.7 ng L(-1). Males from this group also exhibited a less intense nuptial coloration than control males. In the group exposed to 1.75 ng L(-1) EE(2) posthatch, significantly fewer nests were built than in the control group. These results demonstrate that the timing of exposure to estrogenic contaminants, in developmental terms, is critically important. Short-term exposure to estrogens as juveniles can clearly influence reproductive performance as adults, despite all growth and development subsequent to the exposure period taking place in an estrogen-free environment. In addition, these results suggest that reproductive dysfunction can occur even in fish with no gross abnormalities in gonadal structure. This suggests that the absence of gonadal intersex is not a reliable indicator of the reproductive potential, or estrogen-exposure history, of fish populations or the only important factor involved in compromising the reproduction of estrogen-exposed fish.     

A Single Neonatal Injection of Ethinyl Estradiol Impairs Passive Avoidance Learning and Reduces Expression of Estrogen Receptor α in the Hippocampus and Cortex of Adult Female Rats

Although perinatal exposure of female rats to estrogenic compounds produces irreversible changes in brain function, it is still unclear how the amount and timing of exposure to those substances affect learning function, or if exposure alters estrogen receptor α (ERα) expression in the hippocampus and cortex. In adult female rats, we investigated the effects of neonatal exposure to a model estrogenic compound, ethinyl estradiol (EE), on passive avoidance learning and ERα expression. Female Wistar-Imamichi rats were subcutaneously injected with oil, 0.02 mg/kg EE, 2 mg/kg EE, or 20 mg/kg 17β-estradiol within 24 h after birth. All females were tested for passive avoidance learning at the age of 6 weeks. Neonatal 0.02 mg/kg EE administration significantly disrupted passive avoidance compared with oil treatment in gonadally intact females. In a second experiment, another set of experimental females, treated as described above, was ovariectomized under pentobarbital anesthesia at 10 weeks of age. At 15–17 weeks of age, half of each group received a subcutaneous injection of 5 μg estradiol benzoate a day before the passive avoidance learning test. Passive avoidance learning behavior was impaired by the 0.02 mg/kg EE dose, but notably only in the estradiol benzoate-injected group. At 17–19 weeks of age, hippocampal and cortical samples were collected from rats with or without the 5 μg estradiol benzoate injection, and western blots used to determine ERα expression. A significant decrease in ERα expression was observed in the hippocampus of the estradiol-injected, neonatal EE-treated females. The results demonstrated that exposure to EE immediately after birth decreased learning ability in adult female rats, and that this may be at least partly mediated by the decreased expression of ERα in the hippocampus.     

Effect of Regular Circus Physical Exercises on Lymphocytes in Overweight Children

Obesity associated with a sedentary lifestyle can lead to changes in the immune system balance resulting in the development of inflammatory diseases. The aim of this study was to compare lymphocyte activation mechanisms between overweight children practicing regular circus physical exercises with non-exercised children. The study comprised 60 pubescent children randomly divided into 4 groups: Overweight Children (OWC) (10.67 ± 0.22 years old), Overweight Exercised Children (OWE) (10.00 ± 0.41 years old), Eutrophic Children (EC) (11.00 ± 0.29 years old) and Eutrophic Exercised Children (EE) (10.60 ± 0.29 years old). OWE and EE groups practiced circus activities twice a week, for 4.3 ± 0.5 and 4.4 ± 0.5 months, respectively. Percentage of T regulatory cells (Treg) and the expression of CD95 and CD25 in CD4⁺ lymphocytes were evaluated by flow cytometry. Lymphocyte proliferation capacity was measured by [¹⁴C]-thymidine incorporation and mRNA expression of IL-35, TGF-beta, IL-2 and IL-10 by real-time PCR. Lymphocyte proliferation was higher in OWC and OWE groups compared with the EC (3509 ± 887; 2694 ± 560, and 1768 ± 208 cpm, respectively) and EE (2313 ± 111 cpm) groups. CD95 expression on lymphocytes was augmented in the EC (953.9 ± 101.2) and EE groups (736.7 ± 194.6) compared with the OWC (522.1 ± 125.2) and OWE groups (551.6 ± 144.5). CTLA-4 expression was also lower in the OWC and OWE groups compared with the EC and EE groups. Percentage of Treg, IL-35, and IL-10 mRNA expression were lower in the OWC and OWE groups compared with the EC and EE groups. In conclusion, overweight children present altered immune system balance characterized by elevated lymphocyte proliferation due to a decrease in T regulatory cell percentage. These effects were partially reverted by moderate physical exercise, as demonstrated by decreased lymphocyte proliferation.     

Do microplastics impair male dominance interactions in fish? A test of the vector hypothesis

Microplastics (MPs) are widespread in aquatic environments and have become a critical environmental issue in recent years due to their adverse impacts on the physiology, reproduction, and survival of aquatic animals. Exposure to MPs also has the potential to induce sub‐lethal behavioral changes that can affect individual fitness, but these effects are understudied. Many plastic additives introduced during the manufacture of MPs are known endocrine‐disrupting chemicals (EDCs) that mimic the action of natural hormones, alter sexual and competitive behavior, and impair reproductive success in fish. In addition, EDCs and other aquatic contaminants may adhere to MPs in the environment, the latter of which may serve as transport vectors for these compounds (i.e., the vector hypothesis). In this study, we staged territorial contests between control males, and males exposed to virgin MP particles or to MPs previously immersed in one of two environmentally relevant concentrations of 17‐alpha ethinyl estradiol (EE2; 5 ng/L and 25 ng/L) to evaluate the independent and synergistic effects of exposure to MPs and a common environmental estrogen on male–male aggression and competitive territory acquisition in a freshwater fish, Pimephales promelas. Short‐term (30 days) dietary exposure to MPs did not impair the ability of males to successfully compete for and obtain a breeding territory. Overall levels of aggression in control and exposed males were also similar across trial series. These results help to fill a critical knowledge gap regarding the direct and indirect (vector‐borne) effects of MPs on the reproductive behavior of aquatic vertebrates in freshwater systems.     

Gadd45g Is Essential for Primary Sex Determination,Male Fertility and Testis Development

In humans and most mammals, differentiation of the embryonic gonad into ovaries or testes is controlled by the Y-linked gene SRY. Here we show a role for the Gadd45g protein in this primary sex differentiation. We characterized mice deficient in Gadd45a, Gadd45b and Gadd45g, as well as double-knockout mice for Gadd45ab, Gadd45ag and Gadd45bg, and found a specific role for Gadd45g in male fertility and testis development. Gadd45g-deficient XY mice on a mixed 129/C57BL/6 background showed varying degrees of disorders of sexual development (DSD), ranging from male infertility to an intersex phenotype or complete gonadal dysgenesis (CGD). On a pure C57BL/6 (B6) background, all Gadd45g^−/− XY mice were born as completely sex-reversed XY-females, whereas lack of Gadd45a and/or Gadd45b did not affect primary sex determination or testis development. Gadd45g expression was similar in female and male embryonic gonads, and peaked around the time of sex differentiation at 11.5 days post-coitum (dpc). The molecular cause of the sex reversal was the failure of Gadd45g^−/− XY gonads to achieve the SRY expression threshold necessary for testes differentiation, resulting in ovary and Müllerian duct development. These results identify Gadd45g as a candidate gene for male infertility and 46,XY sex reversal in humans.     

Testicular development and serum levels of gonadal steroids during the annual reproductive cycle of captive Japanese sardine

Gonad and blood samples were taken throughout the year from captive males of the Japanese sardine,Sardinops melanostictus, and changes in serum levels of gonadal steroids were examined in relation to the annual gonadal cycle. On the basis of testicular histology, the annual gonadal cycle was divisible into four periods: immature (July–September), spermatogenesis (October–December), spermiation (January–April), and post-spawning (May–June). The pattern of seasonal changes in the gonadosomatic index (GSI) was inversely correlated with that of water temperature, and reflected the degree of testicular maturity. The serum testosterone level was relatively low during spermatogenesis (2.2–2.5 ng/ml), rose markedly around the time of spermiation (7.7–24.6 ng/ml), and became low after spawning and during immature periods (0.6–0.7 ng/ml). The serum 17α,20β-dihydroxy-4-pregnen-3-one level was high in males with spermatogenic or spermiating testes (0.6–1.0 ng/ml), but became low (0.2 ng/ ml during the post-spawning period and was undetectable in immature fish. Although 11-ketotestosterone was detectable in some fish, the values obtained were thought to reflect cross-reactivity of the antiserum employed with testosterone. These findings are discussed in relation to male reproduction of the Japanese sardine and steroidal regulation of spermatogenesis and spermiation in other teleosts.     

The Tyrosine Kinase Inhibitor Sunitinib Affects Ovulation but Not Ovarian Reserve in Mouse: A Preclinical Study

The aim of the study was to evaluate ovarian toxicity of tyrosine kinase inhibitor (TKI) sunitinib, since only scarce data are available on gonadal function after this treatment. Six-week-old female mice received orally, once daily, vehicle or sunitinib (50 mg/kg/d) during 5 weeks. Fertility parameters were analyzed from ovulation to litter assessment. Sunitinib exposure significantly reduced (i) corpora lutea number per ovary (1.1 ± 0.38 in sunitinib group versus 4 ± 0.79 in control group, p<0.01) and (ii) serum Anti Müllerian hormone (AMH) levels in sunitinib treated mice (12.01 ± 1.16) compared to control mice (14.33 ± 0.87 ng/ml, p< 0.05). However, primordial and growing follicles numbers per ovary were not different in both groups. After treatment withdrawal, female mice in both groups were able to obtain litters. These data could be helpful to counsel clinicians and patients, when fertility preservation methods are discussed, before TKI treatment in girls and young women.     

The Influence of Shc Proteins and Aging on Whole Body Energy Expenditure and Substrate Utilization in Mice

While it has been proposed that Shc family of adaptor proteins may influence aging by regulating insulin signaling and energy metabolism, the overall impact of Shc proteins on whole body energy metabolism has yet to be elucidated. Thus, the purpose of this study was to determine the influence of Shc proteins and aging on whole body energy metabolism in a mouse model under ambient conditions (22°C) and acute cold exposure (12°C for 24 hours). Using indirect respiration calorimetry, we investigated the impact of Shc proteins and aging on EE and substrate utilization (RQ) in p66 Shc−/− (ShcKO) and wild-type (WT) mice. Calorimetry measurements were completed in 3, 15, and 27 mo mice at 22°C and 12°C. At both temperatures and when analyzed across all age groups, ShcKO mice demonstrated lower 24 h total EE values than that of WT mice when EE data was expressed as either kJ per mouse, or adjusted by body weight or crude organ mass (ORGAN) (P≤0.01 for all). The ShcKO mice also had higher (P<0.05) fed state RQ values than WT animals at 22°C, consistent with an increase in glucose utilization. However, Shc proteins did not influence age-related changes in energy expenditure or RQ. Age had a significant impact on EE at 22°C, regardless of how EE data was expressed (P<0.05), demonstrating a pattern of increase in EE from age 3 to 15 mo, followed by a decrease in EE at 27 mo. These results indicate a decline in whole body EE with advanced age in mice, independent of changes in body weight (BW) or fat free mass (FFM). The results of this study indicate that both Shc proteins and aging should be considered as factors that influence energy expenditure in mice.     

Pubertal exposure to estrogenic chemicals affects behavior in juvenile and adult male rats

In this paper, we tested the hypothesis that exposure to estrogens of different source and estrogenic potency at early puberty could affect the development of socio-sexual behavior in the male rat. Puberty is regarded as a second stage of the ontogenetic period, in the sexual maturation of mammals, particularly sensitive to gonadal hormone milieu. We treated animals orally, from postnatal day 23 to 30, with an environmentally compatible dose of bisphenol A (BPA, 40 microg/kg/day) and with a dosage of ethinylestradiol (EE, 0.4 microg/kg/day) comparable to the human oral contraceptives. Exposure to EE altered the temporal pattern of male sexual activity, reducing performance, in the adult animals; slight modifications, in the same direction, were observed with BPA. Short-term behavioral effects were observed in the treated animals, both with BPA and EE: the exploratory drive, directed to a stimulus object and to the environment, as well as to conspecifics, was reduced in the juveniles. Modifications in the circulating T levels were observed after treatments: T was reduced in the juveniles, both with BPA and EE. The decrement persisted in the adult animals but reached significance only in the BPA group. On the whole, effects of pubertal exposure on behavior are more marked with EE than BPA. This can be due to the much higher estrogenic potency of EE; the direction of the behavioral effects of BPA, compared with EE, is however indicative of an estrogenic mechanism.     

Life-stage-dependent sensitivity of zebrafish (Danio rerio) to estrogen exposure

The aim of this study was to identify periods in zebrafish (Danio rerio) development when estrogen exposure has long-term consequences on reproductive capabilities at the adult stage. To this end, zebrafish were exposed to 10 ng/L ethynylestradiol (EE(2)) during three stages of gonadal differentiation: (i) the juvenile hermaphroditic stage when gonads display the morphology of an immature ovary (in our zebrafish colony this lasted from 15 to 42 days post-fertilization [dpf]), (ii) the gonad transition stage when the hermaphroditic gonad differentiates into either testes or ovary (from 43 to day 71 dpf), and (iii) the premature stage of testicular and ovarian development (from 72 to 99 dpf). The consequences of stage-specific exposure to EE(2) were assessed by determining time to first spawning, fecundity (number of eggs per female per day), fertilization success (percentage of fertilized eggs) and sex ratio of the adults. Exposure during the gonad transition period induced a delay in the onset of spawning and a significant reduction of fecundity and fertilization success, whereas exposure during the hermaphroditic stage or during the premature stage had no significant impact on the reproductive parameters of adult fish. The results from this experiment pointed to the gonad transition stage as being most susceptible to persistent effects of developmental estrogen exposure. In a second experiment, the concentration dependency of the EE(2)effects was evaluated by exposing zebrafish during the gonad transition stage (43-71 dpf) to 1.67, 3 or 10 ng EE(2)/L. Significant effects of EE(2) on adult reproduction were found with 3 and 10 ng EE(2)/L, but not with 1.67 ng/L. Histological examination of the gonads revealed that at termination of EE(2) exposure (71 dpf), all individuals in the 3 and 10 ng EE(2)/L treatment possessed ovaries. However, this feminising effect appeared to be reversible since at the adult stage (190 dpf), both fish with ovaries and with testes were found. Thus, EE(2) exposure during the gonad transition stage seems to have no persistent effect on gonad histology but on reproductive capabilities.     

Mise au point Estrogènes et reproduction chez le mâle

Aromatase is the terminal enzyme responsible for estrogen biosynthesis. Besides somatic cells, the aromatase gene expression and its transduction in a fully active protein in germ cells of rodent testes in one hand, the widespread distribution of estrogen receptors (ERα and ERβ) in the genital tract of the male in an other hand, are clearly in favour of a physiological role for estrogens in the regulation of mammalian testicular functions. In mouse and man, the aromatase deficiency is associated with severe bone maturation problems and sterility. Therefore, together with gonadotrophins and androgens, estrogens (or the balance androgens/estrogens) likely play a physiological role (either directly or via testicular somatic cells) in maintenance of male gonadal functions and obviously, several steps are concerned especially the spermatid production (both in terms of quality and number) and epididymal sperm maturation.     

Mutation of cyp19a1b results in sterile males due to efferent duct obstruction in Nile tilapia

The involvement of estrogen in male fertility has been well established in mammals. However, less is known about the role of estrogen in fish male reproduction. Our recent study revealed that Cyp19a1a deficiency had no effect on fertility in male fish. In this study, expression of Cyp19a1b, but not Cyp19a1a, was detected by immunohistochemistry in Leydig cells of tilapia testes. cyp19a1b mutation resulted in a significant decrease in the concentration of 17β‐estradiol in serum and sterility in XY fish, as no offspring were obtained when crossed with control XX fish at 240 days after hatching (dah). No sperm was obtained from the mature mutants by in vitro extrusion. Further examination of the mutant gonads revealed excessive semen accumulation and testicular hypertrophy. Semen collected from the mutant testes during autopsy contained sperm with a normal morphology that showed no significant differences in motility, VCL, BCF, STR, or fertility compared with control sperm. Efferent ducts from the mutant testes, which had low‐convolution levels, fewer branches, and no blood vessels observed inside the walls, were significantly smaller in size. qRT‐PCR analyses showed downregulated expression of ion exchange genes. There was increased apoptosis in the epithelial cells of the efferent ducts and other somatic cells of the testes as revealed by TUNEL staining, as well as upregulation of apoptosis gene expression in the mutants. At 360 dah, mutant fish showed testicular atrophy and efferent duct fibrosis. These results demonstrated that estrogen deficiency caused by Cyp19a1b mutation resulted in male sterility due to efferent duct obstruction.     

Profiling lipid metabolites yields unique information on sex- and time-dependent responses of fathead minnows (Pimephales promelas) exposed to 17α-ethynylestradiol

Alterations in hepatic lipid profiles of fathead minnows (FHM) exposed to the synthetic estrogen 17α-ethynylestradiol (EE2) were determined using ¹H-NMR spectroscopy-based metabolite profiling. The exposures were conducted using either 10 ng/l or 100 ng/l EE2 via a continuous flow water delivery system. Livers were collected at 1, 4, and 8 days of the exposure and 8 days after the chemical was removed from the water (i.e. an 8 day depuration). The exposure resulted in a number of sex-specific changes in lipid profiles that were also highly time dependent. Those metabolites most affected by exposure included phosphatidylcholine, diglycerides, triglycerides and cholesterol. In addition, changes in the length and degree of unsaturation of hepatic fatty acids were observed. Lipid profiles in plasma for fish collected on the 4th day of exposure were also analyzed in order to provide further insights into changes observed in hepatic metabolite changes. Using validated partial least-squares discriminant analysis (PLS-DA), the response trajectories of the male liver lipid profiles at both exposure concentrations were compared. This analysis indicated that the males exposed to the low concentration of EE2 (10 ng/l) were largely able to recover from the exposure once the chemical was removed from the water. Conversely, the males exposed to the high concentration (100 ng/l) did not appear to recover from the exposure despite the 8 day depuration.