Active feminization of the preoptic area occurs independently of the gonads in Amphiprion ocellaris.

Sex differences in the anatomy and physiology of the vertebrate preoptic area (POA) arise during development, and influence sex-specific reproductive functions later in life. Relative to masculinization, mechanisms for feminization of the POA are not well understood. The purpose of this study was to induce sex change from male to female in the anemonefish Amphiprion ocellaris, and track the timing of changes in POA cytoarchitecture, composition of the gonads and circulating sex steroid levels. Reproductive males were paired together and then sampled after 3 weeks, 6 months, 1 year and 3 years. Results show that as males change sex into females, number of medium cells in the anterior POA (parvocellular region) approximately double to female levels over the course of several months to 1 year. Feminization of gonads, and plasma sex steroids occur independently, on a variable timescale, up to years after POA sex change has completed. Findings suggest the process of POA feminization is orchestrated by factors originating from within the brain as opposed to being cued from the gonads, consistent with the dominant hypothesis in mammals. Anemonefish provide an opportunity to explore active mechanisms responsible for female brain development in an individual with male gonads and circulating sex steroid levels.

Feminization imprinted by developmental growth hormone.

Previously, we identified early developmental exposure to growth hormone (GH) as the requisite organizer responsible for programming the masculinization of the hepatic cytochromes P450 (CYP)-dependent drug metabolizing enzymes (Das et al., 2014, 2017). In spite of the generally held dogma that mammalian feminization requires no hormonal imprinting, numerous reports that the sex-dependent regulation and expression of hepatic CYPs in females are permanent and irreversible would suggest otherwise. Consequently, we selectively blocked GH secretion in a cohort of newborn female rats, some of whom received concurrent GH replacement or GH releasing factor. As adults, the feminine circulating GH profile was restored in the treated animals. Two categories of CYPs were measured. The principal and basically female specific CYP2C12 and CYP2C7; both completely and solely dependent on the adult feminine continuous GH profile for expression, and the female predominant CYP2C6 and CYP2E1 whose expression is maximum in the absence of plasma GH, suppressed by the feminine GH profile but more so by the masculine episodic GH profile. Our findings indicate that early developmental exposure to GH imprints the inchoate CYP2C12 and CYP2C7 in the differentiating liver to be solely dependent on the feminine GH profile for expression in the adult female. In contrast, adult expression of CYP2C6 and CYP2E1 in the female rat appears to require no GH imprinting.

Soybean isoflavone exposure does not have feminizing effects on men: a critical examination of the clinical evidence.

OBJECTIVE: To critically evaluate the clinical evidence, and when not available, the animal data, most relevant to concerns that isoflavone exposure in the form of supplements or soy foods has feminizing effects on men. DESIGN: Medline literature review and cross-reference of published data. RESULT(S): In contrast to the results of some rodent studies, findings from a recently published metaanalysis and subsequently published studies show that neither isoflavone supplements nor isoflavone-rich soy affect total or free testosterone (T) levels. Similarly, there is essentially no evidence from the nine identified clinical studies that isoflavone exposure affects circulating estrogen levels in men. Clinical evidence also indicates that isoflavones have no effect on sperm or semen parameters, although only three intervention studies were identified and none were longer than 3 months in duration. Finally, findings from animal studies suggesting that isoflavones increase the risk of erectile dysfunction are not applicable to men, because of differences in isoflavone metabolism between rodents and humans and the excessively high amount of isoflavones to which the animals were exposed. CONCLUSION(S): The intervention data indicate that isoflavones do not exert feminizing effects on men at intake levels equal to and even considerably higher than are typical for Asian males.

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.

Absence of gonadotropin-releasing hormone 1 and Kiss1 activation in alpha-fetoprotein knockout mice: prenatal estrogens defeminize the potential to show preovulatory luteinizing hormone surges.

Sex differences in gonadal function are driven by either cyclical (females) or tonic (males) hypothalamic GnRH1 release and, subsequently, gonadotrophin (LH and FSH) secretion from the pituitary. This sex difference seems to depend on the perinatal actions of gonadal hormones on the hypothalamus. We used alpha-fetoprotein (AFP) knockout mice (Afp(-/-)) to study the mechanisms by which estrogens affect the sexual differentiation of the GnRH1 system. Afp(-/-) mice lack the protective actions of AFP against estrogens circulating during embryonic development, leading to infertility probably due to a hypothalamic dysfunction. Therefore, we first determined whether Afp(-/-) females are capable of showing a steroid-induced preovulatory LH surge by FOS/GnRH1 immunohistochemistry and RIA of plasma LH levels. Because the KISS1/GPR54 system is a key upstream regulator of the GnRH1 system as well as being sexually dimorphic, we also analyzed whether Kisspeptin-10 neurons were activated in Afp(-/-) mice after treatment with estradiol and progesterone. We found that the GnRH1 and Kisspeptin-10 neuronal systems are defeminized in Afp(-/-) females because they did not show either steroid-induced LH surges or significant FOS/GnRH1 double labeling. Furthermore, Kisspeptin-10 immunoreactivity and neural activation, measured by the number of double-labeled FOS/Kisspeptin-10 cells, were lower in Afp(-/-) females, suggesting a down-regulation of GnRH1 function. Thus, the sex difference in the ability to show preovulatory LH surges depends on the prenatal actions of estrogens in the male hypothalamus and, thus, is lost in Afp(-/-) females because they lack AFP to protect them against the defeminizing effects of estrogens during prenatal development.

Feminizing/demasculinizing effects of polychlorinated biphenyls on the secondary sexual development of Xenopus laevis.

We have previously demonstrated that polychlorinated biphenyls (PCBs) have caused phenotypic feminization/demasculinization of gonadal development in Xenopus laevis. Whether PCBs affect secondary sexual development has remained unknown. In this study, X. laevis tadpoles were exposed to Aroclor1254 and PCB(3) from stage 46/47 (system of Nieuwkoop and Faber) for up to 1 month postmetamorphosis. After 24 months postmetamorphosis, the degree of secondary sexual development was examined. Male oviducts were observed in some of the PCB-exposed male frogs, but not in control males. These male oviducts had not completely developed in histological structure when compared with mature female oviducts. Larynx weight and width of PCB-exposed males were significantly less than those of control males. Laryngeal histology showed that PCBs inhibited cartilaginous and muscular development of male frogs, i.e. elastic cartilages had not completely developed and laryngeal muscle fibers were smaller. In a further study on adult male frogs, a decrease in serum testosterone level was found in PCB-exposed frogs compared with controls, but serum estradiol level was not significantly affected. Our study suggests that PCBs can cause phenotypic feminization/demasculinization of male genital ducts and larynges, and these effects may, in part, result from the decrease in serum testosterone level in X. laevis.

Feminization of wild carp, Cyprinus carpio, in a polluted environment: plasma steroid hormones, gonadal morphology and xenobiotic metabolizing system.

Wild carp, Cyprinus carpio, were sampled in January and March 2000 in a section of the Anoia River (NE Spain) known to be polluted by estrogenic compounds. At each sampling time, three groups were distinguished: (1) apparently normal males; (2) apparently normal females; and (3) affected fish. The latter were characterized by the simultaneous development of male and female tissue in their gonads at a macroscopical level (six out of 31 fish sampled at this particular point), or testicular atrophy (three out of 31). Plasmatic and hepatic vitellogenin (VTG) levels and plasma testosterone (T) and estradiol (E2) were measured to observe the particular estrogenic response of the affected fish. Moreover, the response in the xenobiotic metabolizing capacity in liver was tested. This involved the analysis of mixed function oxygenase (MFO) system such as: total cytochrome P450 content, NAD(P)H cytochrome c reductases and the associated CYP1A1, EROD activity. Also, glutathione S-transferase (GST) and UDP-glucuronosyltransferase (UDPGT) as detoxifying enzymes were measured. Our results showed: (1) a highly variable VTG content in all fish groups; (2) an increase in sex hormones content in March for the female group; and (3) an enhanced xenobiotics metabolism in the affected fish group, measured as total cytochrome P450, EROD activity in the January survey and cytosolic GST in March. The observed increase in VTG, sex hormones and in most of the enzymatic activities from January to March that could also be attributed to higher water temperature.

Clinical feminisation and sex hormone concentrations in Nigerian men with cirrhosis with and without hepatocellular carcinoma.

The study explored the incidence of clinical feminisation and the sex hormone levels of 18 Nigerian patients with liver cirrhosis (LC) alone and 18 patients with LC and hepatocellular carcinoma (HCC). The incidence (11%) of clinical feminisation in Nigerian patients was lower than values reported from other countries and there was no association between feminising signs and the sex hormone levels of the patients. Plasma oestradiol and sex hormone-binding globulin (SHBG) levels were significantly higher and testosterone lower in patients with liver diseases than in 18 age-matched normal controls. Serum concentrations of oestradiol were also found to be significantly higher in patients with LC alone than in those with LC and HCC. A possible promotive role for oestrogens in the development of HCC from the cirrhotic liver is discussed.

Endocrine investigations in two cases of feminizing Leydig cell tumour.

Two patients, aged 32 and 35 years, presented with gynaecomastia and a unilateral testicular tumour which proved to be a Leydig cell tumour. Pre-operative samples taken at 08.00 h on different days showed marked elevation of plasma oestradiol in the first patient, and very slight irregular oestradiol elevation in the second, plasma oestrone within the normal range in both patients, reduced plasma testosterone in the first patient and reduced or normal testosterone in the second, and low or low-normal serum LH and FSH in both patients. One of the patients received an oral dose of 100 mg of clomiphene citrate for 3 consecutive days which induced a rise in LH and FSH and a decrease in the 17-hydroxyprogesterone/androstenedione ratio. These data suggest the inhibiting effect of endogenous hyperoestrogenism on testicular steroidogenesis owing to both the reduction of gonadotropin secretion and a direct local negative effect on C 17,20-lyase. After human chorionic gonadotropin stimulation, oestradiol response was increased and abnormally prolonged, a finding which may be helpful when diagnosing a feminizing Leydig cell tumour; testosterone reached normal values. After removal of the tumoural testis, gynaecomastia regressed within a few days, gonadotropins increased, oestrogens dropped, testosterone and 5 alpha-dihydrotestosterone normalized in one patient but remained low in the other at day 30. The Leydig cells outside the tumour appeared morphologically normal, but the count gave evidence of juxtatumoural Leydig cell hyperplasia in areas where the tumour was well encapsulated while showing a significant reduction at a distance from the tumour and in the contralateral testis by comparison with control testes.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgen-oestrogen imbalance in men with chronic alcoholism and fatty liver.

The plasma concentrations of oestrone (E1), oestradiol (E2), oestrone sulphate (E1S), testosterone (T), sex hormone binding globulin (SHBG) and apparent free testosterone (AFTC) were measured in 20 normal-weight men with chronic alcoholism and fatty liver. Twenty normal-weight male blood donors matched for age acted as controls. In the alcoholic subjects (patients) the plasma levels of E1, E2 and SHBG were significantly elevated, whereas the concentrations of E1S and AFTC were significantly decreased. No difference in plasma T was seen between the two groups. Both patients and controls showed a significant correlation between SHBG and E2, whereas a significant correlation between SHBG and T, and between E2 and T, was only found in the controls. Eight of the patients showed signs and/or symptoms of hypogonadism or feminization. Only patients with hypogonadism and/or feminization showed significantly higher plasma levels for E2 and decreased T/E2 ratio than matched controls. The ratio E1S/E1 was in every case lower than in the control and tended to be even lower in the patients with hypogonadism and/or feminization.

Testicular feminization syndrome. An unusual case with high estrogen levels.

In an unusual case of testicular feminization, the patient had a high estrogen and low testosterone level. The source of this female hormone excess may have been functioning gonadal stroma of either female or male derivation. All other aspects of the case were consistent with classic examples of the syndrome.

Massive extranglandular aromatization of plasma androstenedione resulting in feminization of a prepubertal boy.

This report describes the mechanism of origin and the quantity of estrogen produced in a prepubertal boy who developed severe feminization at 8 yr of age as the result of a heretofore undescribed metabolic abnormality. The clinical findings were gynecomastia and accelerated linear growth and bone maturation. At the time feminization developed, there were no signs of growth or development of the otherwise normal prepubertal male external genitalia or any increase of muscle mass that normally accompanies male puberty. The hyperestrogenism was found to be the consequence of massive extraglandular conversion of plasma androstenedione to estrone. During a 6-mo period of study, the plasma production rate of androstenedione ranged from 1.2 to 1.6 mg/day. More than 55% of plasma androstenedione was metabolized by aromatization to estrone which, in turn, was extensively sulfurylated in the tissue sites of aromatization before its entry into the blood. Thus, estrone sulfate was the final product in the aromatizing sites, and the plasma production rate of estrone sulfate derived from plasma androstenedione was 782 mug/24 h. The extent of extraglandular conversion of plasma androstenedione to estrone measured in this boy was 50 times that observed in two normal prepubertal boys. Moreover, 94% of the extraglandular aromatization occurred in extrahepatic sites. The metabolic clearance rate of plasma androstenedione, 2,380 liters/day per m(2), was markedly increased in this boy. Approximately 1,500 liters of plasma androstenedione clearance was accounted for by extrahepatic, extraglandular aromatization. The fractional conversion of testosterone to estradiol, 0.16, was 50 times greater in this boy than that observed in normal young adult men. The total extent of aromatization of plasma prehormones was even greater in this boy inasmuch as evidence was obtained that aromatization of 16-hydroxysteroids, e.g. 16alpha-hydroxy androstenedione and 16alpha-hydroxy dehydroisoandrosterone (sulfate), resulted in estriol formation independent of estrone formation. Thus, extensive extrahepatic, extraglandular aromatization resulted in advanced feminization in this prepubertal boy by a previously undescribed metabolic abnormality.

Measurement of human luteinizing hormone in plasma by radioimmunoassay.

The recent isolation of highly purified human pituitary luteinizing hormone (LH) has permitted the development of a sensitive and specific radioimmunoassay for this hormone in plasma. Results of this immunoassay system employing anti-LH serum agree closely with previous reports for the measurement of plasma LH in which immunoassays employing cross-reactive antisera to human chorionic gonadotropin were used. The immunoassay and bioassay of LH in several crude and partially purified pituitary and urinary extracts show acceptable agreement. The sensitivity of the LH immunoassay (0.2 mmug/ml) is adequate to measure LH levels in almost half of all prepuberal children and in all but a few normal adults. A small, but significant, rise in plasma LH level occurs at pubescence in both boys and girls. In women, plasma LH level varies with both age and the phase of the menstrual cycle. The mean LH concentration in nine normal women during the follicular phase (1.2 mmug/ml was found to be significantly higher than during the luteal phase (1.0 mmug/ml). At midcycle, the mean peak LH level was 10.2 mmug/ml. In a large group of normal women, the mean plasma LH concentration rose significantly at menopause to a level of 5.8 mmug/ml during the fifth decade and 10.5 mmug/ml during the seventh decade. A small, but significant, rise in plasma LH concentration also occurred in men from the third and fourth decades (0.7 mmug/ml to the seventh and eighth decades (1.7 mmug/ml). Both estrogen and testosterone suppress plasma LH levels, but marked variation in response exists. The immunoassay serves as a useful diagnostic tool in evaluating men with gonadal failure, amenorrheic women of reproductive age, and postmenopausal women suspected of hypopituitarism. From the half-time disappearance of LH-(131)I in plasma (mean 69 min) and the calculated volume of distribution (2.5-2.8 liters) it has been determined that approximately 30 mug of LH is secreted per day in men, and in women except at midcycle, at which time the release of LH is estimated to be 10-15 times this basal rate.