Maternal exposure to mixtures of dienestrol, linuron and flutamide. Part I: Feminization effects on male rat offspring.

Exposure to endocrine-disrupting compounds (EDCs) during pregnancy can result in negative health effects in later generations, including sex changes and feminization. The present study assessed the feminization effects on male offspring rats of three EDCs: Dienestrol (DIES), Linuron (LIN), and Flutamide (FLU). Sexually mature female rats were exposed from gestation day (GD) 6 until postnatal day (PND) 21 to: 0.37, 0.75, 1.5, 3.12 or 6.25 µg/kg/day of DIES, 1.5, 3, 6, 12.5, 25 or 50 mg/kg/day of LIN, 3.5, 6.7, 12.5, 25 or 50 mg/kg/day of FLU, and the following mixtures: FLU + DIES (mg/kg/day+µg/kg/day), 3.5 + 0.37, or 3.5 + 3, 25 + 0.37, or 25 + 3; FLU + LIN (mg/kg/day + mg/kg/day), 3.5 + 12.5, or 25 + 12.5; and DIES + LIN (µg/kg/day + mg/kg/day), 0.37 + 12.5, or 3 + 12.5. Anogenital distance (AGD), nipple retention (NR) and cryptorchidism were evaluated. FLU produced a decrease of AGD, an increase of NR, and an increase of cryptorchidism at the highest dose. None of these three endpoints were significantly affected by LIN or DIES treatments alone. Combinations of FLU + LIN and FLU + DIES increased NR, and decreased AGD, while DIES + LIN did not produce any effects in male pups. Results show that FLU is able to induce feminization in male pups, while binary combinations of LIN and DIES did not modify the effects produced by FLU.

Breast Development in Transwomen After 1 Year of Cross-Sex Hormone Therapy: Results of a Prospective Multicenter Study.

Context: Breast development is a key feature of feminization and therefore important to transwomen (male-to-female transgender persons). It is not exactly known when breast development starts after initiating cross-sex hormone therapy (CHT) and how much growth may be expected. Objective: To investigate breast development in transwomen during their first year of CHT and whether clinical or laboratory parameters predict breast development. Design: This study was performed as part of the European Network for the Investigation of Gender Incongruence, which is a prospective multicenter cohort study. Setting: Gender clinics in Amsterdam, Ghent, and Florence. Participants: Transwomen who completed the first year of CHT (n = 229). Intervention: CHT. Main Outcome Measures: Breast development in centimeter and cup size. Results: The median age of the included transwomen was 28 years (range, 18 to 69). Mean breast-chest difference increased to 7.9 ± 3.1 cm after 1 year of CHT, mainly resulting in less than an AAA cup size (48.7%). Main breast development occurred in the first 6 months of therapy. Serum estradiol levels did not predict breast development after 1 year of CHT (first quartile, 3.6 cm [95% confidence interval (CI), 2.7 to 4.5], second quartile, 3.2 cm [95% CI, 2.3 to 4.2], third quartile, 4.4 cm [95% CI, 3.5 to 5.3], and fourth quartile, 3.6 cm [95% CI, 2.7 to 4.5]). Conclusion: This study shows that, after 1 year of CHT, breast development is modest and occurs primarily in the first 6 months. No clinical or laboratory parameters were found that predict breast development.

Voice feminization in male-to-female transgendered clients after Wendler's glottoplasty with vs. without voice therapy support.

The objective of this study was to evaluate the medium-term results of Wendler's glottoplasty surgery (WG) and the effects of post-operative voice therapy in a group of male-to-female transsexuals. This is a retrospective study of 18 transsexuals who voluntarily underwent WG between 2010 and 2014 at a single hospital. Ten of the subjects underwent an additional voice therapy training. The group was assessed pre- vs. post-treatments with a limited battery of measures consisting of fundamental frequency (Fo), maximum phonation time, the TSEQ transgender self-assessment questionnaire, and perceptual assessment of the voice (Visual Analog Scale and a simplified version of the classical Hirano-GRBAS scale) by inter-rater agreement. The surgical procedure consisted of a de-epithelialization of the anterior third of both vocal folds; this area was sutured, and the surface of both vocal folds was vaporized with a laser diode. The results showed a significant increase in vocal tone and feminization of voice in all participants, including a significant increase in Fo 12 months after treatment. Significant improvements were also shown in other evaluated measures, such as self-reported satisfaction and the degree of feminization of the voice. However, no improvements in maximum phonation time were observed. The use of voice therapy appears decisive for optimal improvement of this class of patients. WG applied appropriately by well-trained hands is thus a very effective and less traumatic procedure than other techniques that aim for an acceptable feminization of the voice in MtoF transgendered clients.

Neonatal Masculinization Blocks Increased Excitatory Synaptic Input in Female Rat Nucleus Accumbens Core.

Steroid sex hormones and genetic sex regulate the phenotypes of motivated behaviors and relevant disorders. Most studies seeking to elucidate the underlying neuroendocrine mechanisms have focused on how 17ß-estradiol modulates the role of dopamine in striatal brain regions, which express membrane-associated estrogen receptors. Dopamine action is an important component of striatal function, but excitatory synaptic neurotransmission has also emerged as a key striatal substrate and target of estradiol action. Here, we focus on excitatory synaptic input onto medium spiny neurons (MSNs) in the striatal region nucleus accumbens core (AcbC). In adult AcbC, miniature excitatory postsynaptic current (mEPSC) frequency is increased in female compared with male MSNs. We tested whether increased mEPSC frequency in female MSNs exists before puberty, whether this increased excitability is due to the absence of estradiol or testosterone during the early developmental critical period, and whether it is accompanied by stable neuron intrinsic membrane properties. We found that mEPSC frequency is increased in female compared with male MSNs before puberty. Increased mEPSC frequency in female MSNs is abolished after neonatal estradiol or testosterone exposure. MSN intrinsic membrane properties did not differ by sex. These data indicate that neonatal masculinization via estradiol and/or testosterone action is sufficient for down-regulating excitatory synaptic input onto MSNs. We conclude that excitatory synaptic input onto AcbC MSNs is organized long before adulthood via steroid sex hormone action, providing new insight into a mechanism by which sex differences in motivated behavior and other AbcC functions may be generated or compromised.

Transgenic Chickens Overexpressing Aromatase Have High Estrogen Levels but Maintain a Predominantly Male Phenotype.

Estrogens play a key role in sexual differentiation of both the gonads and external traits in birds. The production of estrogen occurs via a well-characterized steroidogenic pathway, which is a multistep process involving several enzymes, including cytochrome P450 aromatase. In chicken embryos, the aromatase gene (CYP19A1) is expressed female-specifically from the time of gonadal sex differentiation. Ectopic overexpression of aromatase in male chicken embryos induces gonadal sex reversal, and male embryos treated with estradiol become feminized; however, this is not permanent. To test whether a continuous supply of estrogen in adult chickens could induce stable male to female sex reversal, 2 transgenic male chickens overexpressing aromatase were generated using the Tol2/transposase system. These birds had robust ectopic aromatase expression, which resulted in the production of high serum levels of estradiol. Transgenic males had female-like wattle and comb growth and feathering, but they retained male weights, displayed leg spurs, and developed testes. Despite the small sample size, this data strongly suggests that high levels of circulating estrogen are insufficient to maintain a female gonadal phenotype in adult birds. Previous observations of gynandromorph birds and embryos with mixed sex chimeric gonads have highlighted the role of cell autonomous sex identity in chickens. This might imply that in the study described here, direct genetic effects of the male chromosomes largely prevailed over the hormonal profile of the aromatase transgenic birds. This data therefore support the emerging view of at least partial cell autonomous sex development in birds. However, a larger study will confirm this intriguing observation.

[CHANGES IN THE BRAIN TESTOSTERONE METABOLISM AND SEXUAL BEHAVIOR IN MALE RATS PRENATALLY EXPOSED TO METHYLDOPA AND STRESS].

The changes of aromatase and 5α-reductase activities were studied in preoptic area (POA) and medial basal hypothalamus of 10-days-old and sexual behavior in 3-month-old male offsprings of rats exposed daily to noradrenaline antagonist methyldopa (400 mg/kg per os) 30 minutes prior to 1-hour immobilization during the last week of pregnancy (from 15th to 21st day). Prenatal stress caused aromatase activity lowering in the POA of developing brain and feminization (appearance of lordosis) and demasculinization of sexual behavior (prolongation of latent periods to the first mounting and first intromission as well as of the first ejaculation and postejaculation refractory period) in young male offspring. Oral methyldopa used prior to pregnant females stressing prevented early effect of prenatal stress on aromatase activity in the POA and normalized the male sexual behavior in young male rats by shortening both latent period to the first ejaculation and postejaculation refractory period, and an increase of numbers of ejaculation. The data obtained indicate that brain noradrenergic system plays significant role in the mechanisms of metabolic- and behavioral disturbances developing in male rats exposed to prenatal stress.

Intersex gonad differentiation in cultured Russian (Acipenser gueldenstaedtii) and Siberian (Acipenser baerii) sturgeon.

Among sturgeons, the occurrence of individuals with gonads containing both testis and ovary components is considered pathological, and such fish are described as intersex individuals or intersexes. Intersexes are observed in both wild and cultured populations of sturgeon, usually at low frequencies. In the present study, intersex Russian (Acipenser gueldenstaedtii) and Siberian (Acipenser baerii) sturgeons constituted 30% of the studied populations. Macroscopically, intersex gonads were recognizable from 500 days posthatching (dph). Initially, gonads with predominantly male characteristics (testis-ova) were observed, but in older fish gonads with predominantly female traits (ova-testis) were more frequent. Using microscopic analysis, intersex gonads were discernible by 130-200 dph. Observations of intersex germinal epithelium development and analysis of sex distribution in the study populations indicated that feminization was occurring. Histological analysis revealed that differentiation of the germinal epithelium in such gonads was accompanied by various morphological alterations (transformations) that were described using quantitative and localization criteria. The most common type of transformations, massive subepithelial transformations, was manifested by the presence of abundant female germinal tissue located under the gonad surface epithelium in the developing testis. These transformations were identified in the early development stage (100-200 dph). In this type of transformation, differentiation of female germinal tissue at the gonad surface and male tissue at the mesorchium/mesovarium resulted in complete formation of both male and female germinal epithelia within the same gonad.

Effects of feminizing microsporidia on the masculinizing function of the androgenic gland in Gammarus duebeni.

Feminizing parasites enhance their vertical transmission to the host offspring by converting genetic male hosts into phenotypic females. Crustacea are the only invertebrates where sexual differentiation is controlled by a specialised endocrine organ, the androgenic gland, rather than by the gonads. We showed that a feminizing microsporidian Microsporidium sp. inhibits androgenic gland differentiation. We investigated the effect of Microsporidium sp. and a second feminizing microsporidium, Nosema granulosis, on the masculinizing function of the androgenic gland in Gammarus duebeni. Androgenic gland implants had a masculinizing effect on the sexual characteristics and sexual behaviour of recipient female hosts, reflecting the masculinizing function of the androgenic gland. Individuals that had received androgenic glands showed changed morphology in comparison with controls; they were bigger overall, they lost their oostegite marginal setae, developed calceoli and acquired a male-like behaviour. This effect was observed in uninfected females, as well as in females infected with the Microsporidium sp. The masculinizing effect of androgenic gland implants was smaller in N. granulosis infected individuals. N. granulosis and Microsporidium sp. fall into distinct clades of the Microspora. It appears that these divergent parasites both act by inhibiting the development of the androgenic gland. However, they differ in their ability to inhibit the host's response to the hormone that controls male sexual differentiation.

Atrazine induces complete feminization and chemical castration in male African clawed frogs (Xenopus laevis).

The herbicide atrazine is one of the most commonly applied pesticides in the world. As a result, atrazine is the most commonly detected pesticide contaminant of ground, surface, and drinking water. Atrazine is also a potent endocrine disruptor that is active at low, ecologically relevant concentrations. Previous studies showed that atrazine adversely affects amphibian larval development. The present study demonstrates the reproductive consequences of atrazine exposure in adult amphibians. Atrazine-exposed males were both demasculinized (chemically castrated) and completely feminized as adults. Ten percent of the exposed genetic males developed into functional females that copulated with unexposed males and produced viable eggs. Atrazine-exposed males suffered from depressed testosterone, decreased breeding gland size, demasculinized/feminized laryngeal development, suppressed mating behavior, reduced spermatogenesis, and decreased fertility. These data are consistent with effects of atrazine observed in other vertebrate classes. The present findings exemplify the role that atrazine and other endocrine-disrupting pesticides likely play in global amphibian declines.

Prevalence of low bone mass in relation to estrogen treatment and body composition in male-to-female transsexual persons.

Bone health is a parameter of interest in the daily follow-up of male-to-female (M --> F) transsexual persons both before and after sex reassignment surgery (SRS) due to an intensely changing hormonal milieu. We have studied body composition, areal, geometric, and volumetric bone parameters, using DXA and peripheral quantitative computed tomography at different sites in 50 M --> F transsexual persons, at least 3 yr after the start of the hormonal treatment and 1 yr after SRS. In this cross-sectional study, hormone levels and markers of bone metabolism were assessed using immunoassays. Prevalence of low bone mass as defined by a Z-score < or = -2.0 according to DXA criteria was 26% at lumbar spine and 2% at the total hip. We found no major differences in hormonal parameters between participants with a Z-score < or = or > -2.0. Markers of bone turnover were comparable between subjects with or without low bone mass, indicating a stable bone turnover at the time of investigation. No significant differences in bone size or density were observed between patients on transdermal vs. oral estrogens. Low bone mass is not uncommon in M --> F transsexual persons. Smaller bone size, and a strikingly lower muscle mass compared with men appear to underlie these findings.

Stress experienced in utero reduces sexual dichotomies in neurogenesis, microenvironment, and cell death in the adult rat hippocampus.

Hippocampal function and plasticity differ with gender, but the regulatory mechanisms underlying sex differences remain elusive and may be established early in life. The present study sought to elucidate sex differences in hippocampal plasticity under normal developmental conditions and in response to repetitive, predictable versus varied, unpredictable prenatal stress (PS). Adult male and diestrous female offspring of pregnant rats exposed to no stress (control), repetitive stress (PS-restraint), or a randomized sequence of varied stressors (PS-random) during the last week of pregnancy were examined for hippocampal proliferation, neurogenesis, cell death, and local microenvironment using endogenous markers. Regional volume was also estimated by stereology. Control animals had comparable proliferation and regional volume regardless of sex, but females had lower neurogenesis compared to males. Increased cell death and differential hippocampal precursor kinetics both appear to contribute to reduced neurogenesis in females. Reduced local interleukin-1beta (IL-1beta) immunoreactivity (IR) in females argues for a mechanistic role for the anti-apoptotic cytokine in driving sex differences in cell death. Prenatal stress significantly impacted the hippocampus, with both stress paradigms causing robust decreases in actively proliferating cells in males and females. Several other hippocampal measures were feminized in males such as precursor kinetics, IL-1beta-IR density, and cell death, reducing or abolishing some sex differences. The findings expand our understanding of the mechanisms underlying sex differences and highlight the critical role early stress can play on the balance between proliferation, neurogenesis, cell death, and hippocampal microenvironment in adulthood.

Effects of androgens and estradiol on spine synapse formation in the prefrontal cortex of normal and testicular feminization mutant male rats.

Recent studies suggest that, in female monkeys and rats, estrogens elicit dendritic spine synapse formation in the prefrontal cortex, an area that, similar to the hippocampus, plays a critical role in cognition. However, whether gonadal hormones induce synaptic remodeling in the male prefrontal cortex remains unknown. Here we report that gonadectomy reduced, whereas administration of 5alpha-dihydrotestosterone or estradiol-benzoate to castrated male rats increased, the number of medial prefrontal cortical (mPFC) spine synapses, with estradiol-benzoate being less effective than 5alpha-dihydrotestosterone. To investigate whether the androgen receptor contributes to the mediation of these changes, we compared the response of testicular feminization mutant (Tfm) male rats to that of wild-type animals. The number of mPFC spine synapses in gonadally intact Tfm rats and 5alpha-dihydrotestosterone-treated castrated Tfm males was considerably reduced compared to intact wild-type animals, whereas the synaptogenic effect of estradiol-benzoate was surprisingly enhanced in Tfm rats. These data are consistent with the hypothesis that remodeling of spine synapses in the prefrontal cortex may contribute to the cognitive effect of gonadal steroids. Our findings in Tfm animals indicate that androgen receptors may mediate a large part of the synaptogenic action of androgens in the mPFC of adult males. However, because this effect of 5alpha-dihydrotestosterone is not completely lost in Tfm rats, additional mechanisms may also be involved.

Role of androgens in fetal testis development and dysgenesis.

This study sought to establish whether reduced androgen levels/action in the fetal rat testis induced by di(n-butyl) phthalate (DBP) contributes to dysgenetic features, namely reduced Sertoli cell number, occurrence of multinucleated gonocytes (MNG), and Leydig cell aggregation. Pregnant rats were administered treatments or cotreatments designed to manipulate testosterone levels [DBP, testosterone propionate (TP)] or action [flutamide, 7,12-dimethyl-benz[a]anthracene (DMBA)]. The aforementioned end points were analyzed and related to intratesticular testosterone (ITT) levels and peripheral androgen action (anogenital distance). Dysgenetic features were also evaluated in mice with inactivation of the androgen receptor (testicular feminized or ARKO mice). Exposure to DBP alone, or combined with flutamide, DMBA, or TP, resulted in reduced Sertoli cell number and ITT levels, as did exposure to TP alone; coadministration of DBP + TP caused the most severe reduction in both parameters. A positive correlation between ITT levels and Sertoli cell number was found (r = 0.791; P = 0.019). Similarly, exposure to DBP alone, or as a cotreatment, significantly increased occurrence of MNG and Leydig cell aggregation, and these were negatively correlated with ITT levels. Exposure to flutamide or DMBA alone had no significant effect on these dysgenetic end points. These findings suggest that reduced ITT decreases fetal Sertoli cell numbers and might be involved in Leydig cell aggregation and MNG. However, of these three end points, only Sertoli cell number was affected significantly in ARKO/testicular feminized mice with absent androgen action. Therefore, induction of MNG and Leydig cell aggregation might result from DBP-induced effects other than suppression of ITT levels.

Spatial memory performance in androgen insensitive male rats.

Masculinization of the developing rodent brain critically depends on the process of aromatization of circulating testosterone (T) to its estrogenic metabolite 17beta-estradiol, which subsequently interacts with estrogen receptors to permanently masculinize the brain. However, it remains unclear what role other androgenic mechanisms may play in the process of masculinization. A novel way of examining this is through the study of male rats that express the tfm mutation of the androgen receptor (AR) gene; such males are fully androgen insensitive and manifest a female phenotype due to a failure of AR-mediated masculinization of peripheral structures. Because tfm-affected males develop secretory testes and have near-normal T titers during development, aromatization would be expected to proceed normally, and brain mechanisms may be developmentally masculinized despite the feminized periphery. We compared tfm-affected males (X(tfm)Y) with normal males and females in the Morris Water Maze, a task in which males typically perform better than females. Performance of tfm-affected males was intermediate between that of normal males and females. While an overall male superiority was found in the task, the X(tfm)Y group reached male-typical escape latencies faster than females. Furthermore, in the X(tfm)Y group, the granule cell layer of the dentate gyrus was significantly larger than in females. These results support the suggestion that that AR mediated mechanisms contribute to the masculinization of spatial behaviours and hippocampal morphology, and this may be independent of estrogenic processes.

Opioids are responsible for neurochemical feminization of the brain in prenatally stressed male rats.

OBJECTIVES: To study the role of opioids in early postnatal changes of the hypothalamic testosterone metabolism and catecholamine content underlying feminizing effect of prenatal stress on male sexual behavior in rats. MATERIAL AND METHODS: 10 day-old male and female offspring from mothers given naltrexone prior to daily 1-hour restraint during the last gestational week were used in the study. Aromatase and 5 alpha-reductase activities, noradrenaline and dopamine contents in the brain preoptic area and medial basal hypothalamus were studied by thin layer chromatography, radiometric and spectrofluorimetric techniques. RESULTS: Sexual dimorphism of testosterone metabolism enzymes activity and catecholamine content in discrete brain regions of 10 day-old rat pups was found. Prenatal stress attenuated these gender-related differences. Naltrexone pre-treatment of stressed dams prevented modifying effect of prenatal stress on aromatase activity and noradrenaline content in the male preoptic area. CONCLUSIONS: The results of the study demonstrate preventive effect of naltrexone on stress-induced alterations of testosterone aromatization and noradrenaline concentration in the developing brain preoptic area associated with neuroendocrine control of the male sexual behavior in adult rats. These findings indicate that endogenic opioids mediate detrimental effect of prenatal stress on neurochemical determinants of the brain sexual differentiation that may underlie feminization of the male sexual behavior.

Intersex and gender assignment; the third way?

The birth of a new baby is one of the greatest wonders of nature and one of the most exciting events known to man. The first question that is usually posed by the mother or father is "is it a boy or a girl?"; without this information the new parents cannot even formulate the second question which is usually "is he/she alright?". It is no wonder that the birth of a child with complex genital anomalies where the sex of rearing is uncertain at birth, presents difficult clinical and ethical issues.

Sexual and olfactory preference in noncopulating male rats.

In some species including rats, mice, gerbils, and rams, apparently normal males fail to copulate when repeatedly tested with receptive females. These animals are called "noncopulators (NC)," and the cause of this behavioral deficit is unknown. It has been shown that NC rats do not have hormonal alterations or deficits in the mechanisms that control penile function. The present study was designed to examine (Experiment 1) whether NC male rats prefer receptive females to sexually active males. In addition, the olfactory preference for bedding soiled from estrous or for anestrous bedding was investigated. These tests were performed in NC and copulating (C) male rats when the subjects were intact, gonadectomized (GDX), or GDX and treated with high doses of testosterone propionate (TP). Our results demonstrate that NC rats do not display sexual behavior even after high TP treatment. While C male rats have a clear preference for receptive females as opposed to a sexually active male, NC rats do not. In all hormonal conditions, the preference shown by NC rats for estrous bedding was significantly reduced in comparison to that seen in C rats. TP treatment in NC rats did not modify either partner or odor preference. In Experiment 2, we evaluated if NC rats are feminized and if it could be easier to induce feminine-like behavior by hormone treatment with estradiol benzoate (EB) or with EB plus progesterone (P) (EB+P). Odor preference for estrous or male bedding under these hormonal conditions was also compared. No differences between NC and C rats were found in feminine sexual behavior. In the olfactory test, we found that NC rats prefer odors from receptive females as opposed to male odors, but this preference is reduced compared to that of C rats. Males treated with EB or EB+P show no preference for female odors. These results demonstrate that treatment with EB or EB+P does not increase feminine sexual behavior in NC rats.

Ovarian hormones can organize the rat corpus callosum in adulthood.

The rat corpus callosum (CC) is larger in males than females, and is responsive to hormone manipulations during development. Previous data suggest that CC sensitivity to testosterone ends by postnatal day 8 (P8). In contrast, responsivity to ovarian hormones extends as late as P25. The current series of experiments investigates whether ovarian hormone effects on the callosum are permanent and whether CC sensitivity to ovarian hormones extends beyond P25. We found that P70 ovariectomy (Ovx) did not affect callosal size, suggesting that ovarian hormone exposure sometime prior to P70 is sufficient to feminize the CC, and that once the callosum is feminized, the effects can not be reversed. We also found that P25 ovariectomy enlarged, or defeminized, adult female CC, whereas ovary transfer starting on P55 or P70 counteracted this enlarging effect, resulting in feminized adult CC. Thus, although a previously feminized callosum is not affected by P70 ovarian hormone removal, a not-yet feminized callosum can still be feminized after P70. These findings indicate that there is flexibility in the developmental window within which the female brain is responsive to the active feminization process initiated by ovarian hormones.