Combinations of Physiologic Estrogens with Xenoestrogens Alter ERK Phosphorylation Profiles in Rat Pituitary Cells

Background

Estrogens are potent nongenomic phospho-activators of extracellular-signal–regulated kinases (ERKs). A major concern about the toxicity of xenoestrogens (XEs) is potential alteration of responses to physiologic estrogens when XEs are present simultaneously.

Objectives

We examined estrogen-induced ERK activation, comparing the abilities of structurally related XEs (alkylphenols and bisphenol A) to alter ERK responses induced by physiologic concentrations (1 nM) of estradiol (E₂), estrone (E₁), and estriol (E₃).

Methods

We quantified hormone/mimetic-induced ERK phosphorylations in the GH₃/B6/F10 rat pituitary cell line using a plate immunoassay, comparing effects with those on cell proliferation and by estrogen receptor subtype-selective ligands.

Results

Alone, these structurally related XEs activate ERKs in an oscillating temporal pattern similar (but not identical) to that with physiologic estrogens. The potency of all estrogens was similar (active between femtomolar and nanomolar concentrations). XEs potently disrupted physiologic estrogen signaling at low, environmentally relevant concentrations. Generally, XEs potentiated (at the lowest, subpicomolar concentrations) and attenuated (at the highest, picomolar to 100 nM concentrations) the actions of the physiologic estrogens. Some XEs showed pronounced nonmonotonic responses/inhibitions. The phosphorylated ERK and proliferative responses to receptor-selective ligands were only partially correlated.

Conclusions

XEs are both imperfect potent estrogens and endocrine disruptors; the more efficacious an XE, the more it disrupts actions of physiologic estrogens. This ability to disrupt physiologic estrogen signaling suggests that XEs may disturb normal functioning at life stages where actions of particular estrogens are important (e.g., development, reproductive cycling, pregnancy, menopause).     

Xenoestrogen-induced ERK-1 and ERK-2 activation via multiple membrane-initiated signaling pathways

Xenoestrogens can mimic or antagonize the activity of physiological estrogens, and the suggested mechanism of xenoestrogen action involves binding to estrogen receptors (ERs). However, the failure of various in vitro or in vivo assays to show strong genomic activity of xenoestrogens compared with estradiol (E2) makes it difficult to explain their ability to cause abnormalities in animal (and perhaps human) reproductive functions via this pathway of steroid action. E2 has also been shown to initiate rapid intracellular signaling, such as changes in levels of intracellular calcium, cAMP, and nitric oxide, and activations of a variety of kinases, via action at the membrane. In this study, we demonstrate that several xenoestrogens can rapidly activate extracellular-regulated kinases (ERKs) in the pituitary tumor cell line GH3/B6/F10, which expresses high levels of the membrane receptor for ER-alpha (mER). We tested a phytoestrogen (coumestrol), organochlorine pesticides or their metabolites (endosulfan, dieldrin, and DDE), and detergent by-products of plastics manufacturing (p-nonylphenol and bisphenol A). These xenoestrogens (except bisphenolA) produced rapid (3-30 min after application), concentration (10(-14)-10(-8) M)-dependent ERK-1/2 phosphorylation but with distinctly different activation patterns. To identify signaling pathways involved in ERK activation, we used specific inhibitors of ERs, epidermal growth factor receptors, Ca2+ signaling, Src and phosphoinositide-3 kinases, and a membrane structure disruption agent. Multiple inhibitors blocked ERK activation, suggesting simultaneous use of multiple pathways and complex signaling web interactions. However, inhibitors differentially affected each xenoestrogen response examined. These actions may help to explain the distinct abilities of xenoestrogens to disrupt reproductive functions at low concentrations.     

HYALURONIC ACID INCREASES MOTILITY/INTRACELLULAR CA 2+ CONCENTRATION IN HUMAN SPERM IN VITRO

This study investigated the mechanisms of the stimulatory effect of hyaluronic acid on motility in human sperm in vitro. A method, involving the measurement of forward progression through an agarose gel, was used to measure sperm motility quantitatively. Changes in intracellular Ca 2+ concentrations in sperm were detected using the fluorescent dye Fluo-3. The effects of hyaluronic acid (6.5, 65, 650 ng/mL) and nifedipine (32 nM) on sperm motility were investigated. The effects of hyaluronic acid, nifedipine (32 nM), A23187 (32 µM), and a monoclonal antibody to human CD44 (1 µg/mL) on changes in intracellular CA 2+ concentrations were investigated. Hyaluronic acid significantly ( p <. 008) stimulated sperm motility and this was partially inhibited by nifedipine.A23187 significantly ( p <. 005) increased intracellular CA 2+ concentrations. Hyaluronic acid significantly ( p <. 04) increased intracellular Ca 2+ concentrations and this was inhibited by nifedipine and a monoclonal antibody to human CD44. Hyaluronic acid stimulated human sperm motility by increasing intracellular Ca 2+ concentration, partially via an influx of extracellular Ca 2+.     

Alkylphenol Xenoestrogens with Varying Carbon Chain Lengths Differentially and Potently Activate Signaling and Functional Responses in GH3/B6/F10 Somatomammotropes

Background

Alkylphenols varying in their side-chain lengths [ethyl-, propyl-, octyl-, and nonylphenol (EP, PP, OP, and NP, respectively)] and bisphenol A (BPA) represent a large group of structurally related xenoestrogens that have endocrine-disruptive effects. Their rapid nongenomic effects that depend on structure for cell signaling and resulting functions are unknown.

Objectives

We compared nongenomic estrogenic activities of alkylphenols with BPA and 17β-estradiol (E₂) in membrane estrogen receptor-α–enriched GH₃/B₆/F10 pituitary tumor cells. These actions included calcium (Ca) signaling, prolactin (PRL) release, extracellular-regulated kinase (ERK) phosphorylation, and cell proliferation.

Methods

We imaged Ca using fura-2, measured PRL release via radioimmunoassay, detected ERK phosphorylation by fixed cell immunoassay, and estimated cell number using the crystal violet assay.

Results

All compounds caused increases in Ca oscillation frequency and intracellular Ca volume at 100 fM to 1 nM concentrations, although long-chain alkylphenols were most effective. All estrogens caused rapid PRL release at concentrations as low as 1 fM to 10 pM; the potency of EP, PP, and NP exceeded that of E₂. All compounds at 1 nM produced similar increases in ERK phosphorylation, causing rapid peaks at 2.5–5 min, followed by inactivation and additional 60-min peaks (except for BPA). Dose–response patterns of ERK activation at 5 min were similar for E₂, BPA, and PP, whereas EP caused larger effects. Only E₂ and NP increased cell number. Some rapid estrogenic responses showed correlations with the hydrophobicity of estrogenic molecules; the more hydrophobic OP and NP were superior at Ca and cell proliferation responses, whereas the less hydrophobic EP and PP were better at ERK activations.

Conclusions

Alkylphenols are potent estrogens in evoking these nongenomic responses contributing to complex functions; their hydrophobicity can largely predict these behaviors.     

大豆异黄酮对妇女围绝经期症状和性激素的影响

目的了解大豆异黄酮对妇女围绝经期症状的改善作用和对雌激素的影响。方法将已经出现围绝经期症状的45～55岁女性50例,随机单盲分为大豆异黄酮组和安慰剂组,观察8周后的围绝经期症状和雌激素水平。结果大豆异黄酮组试验后BMI增加明显(P0.05);潮热及出汗、Kupperman评分等多项指标有明显改善;雌二醇、促卵泡生成素、睾酮和孕酮有显著性差异(P0.01或P0.05)。与安慰剂组比较,潮热及出汗、性生活状况和Kupperman评分等多项指标差异有显著性(P0.01);除孕酮外其它雌激素指标差异均有显著性(P0.01或P0.05)。安慰剂组试验前后比较虽然检测指标有变化,但无统计学意义。结论大豆异黄酮作为植物雌激素对改善围绝经期症状和提高雌激素水平有广泛的应用前景。     

地卓西平对甲基汞致大鼠脑皮质细胞内钙稳态失调的影响

目的研究地卓西平对甲基汞致大鼠脑皮质细胞内钙稳态失调的影响。方法 Wistar大鼠32只,按体重随机分成4组,每组8只。第1组为对照组,腹腔注射0.9%氯化钠;第2组为低剂量甲基汞染毒组,腹腔注射4μmol/kg的甲基汞溶液;第3组为高剂量甲基汞染毒组,腹腔注射12μmol/kg的甲基汞溶液;第4组为地卓西平预处理组,隔日皮下注射0.3μmol/kg地卓西平,2 h后腹腔注射12μmol/kg的甲基汞溶液;注射容量均为5 ml/kg,染毒4周,每周5次。观察神经细胞内Ca2＋浓度及其凋亡情况,同时测定脑皮质Hg含量和与维持钙稳态相关的酶Na＋-K＋-ATPase和Ca2＋-ATPase活性。结果染毒4周后,随着染毒剂量的增加,脑皮质Hg含量和细胞内Ca2＋浓度均明显升高（P〈0.01）;细胞凋亡率明显高于对照组（P〈0.01）;脑皮质Na＋-K＋-ATPase和Ca2＋-ATPase活性均不同程度的受到抑制。地卓西平预处理可以明显缓解神经细胞凋亡和细胞内Ca2＋超载,对Na＋-K＋-ATPase和Ca2＋-ATPase活性也有一定程度的恢复作用。结论甲基汞通过抑制与维持钙稳态相关的酶Na＋-K＋-ATPase和Ca2＋-ATPase活性,干扰神经细胞内钙稳态,进而造成神经细胞凋亡。地卓西平可以阻断Ca2＋通道对钙稳态失调引起的细胞损伤有拮抗作用。     

Effect of estrogens on prolactin secretion in transsexual subjects

The effect of estrogens on the secretion of prolactin in 8 different groups of transsexual subjects was studied. Two different types of estrogens, estradiol or its conjugate and ethinyl estradiol, were used. Different doses and durations of exposure were employed. Plasma levels of prolactin and SHBG after estrogen exposure were compared with corresponding levels before treatment. Results showed that for estrogens to exert an enhancing effect on the secretion of prolactin, three factors needed to be considered: (i) the absolute concentration of estrogen, (ii) the duration of exposure, and (iii) whether levels of SHBG are sufficiently altered to change the concentration of free estrogen. It appears that there exist both time and dose thresholds for effective enhancement of prolactin secretion by estrogen. Estradiol and its conjugate are more likely to induce hyperprolactinemia than ethinyl estradiol. The reduced effect of the latter is probably related to its ability to induce large and rapid increases in SHBG binding which probably results in unaltered free estrogen concentration. In the light of this study, treatment of male transsexuals with high doses of estradiol in its conjugate forms must be viewed with caution. Without the financial support from the National University at Singapore, this project would not be feasible.     

含铅、无铅汽油尾气排出颗粒提取物的细胞膜毒性比较

[目的]通过实验室细胞染毒模型来揭示无铅汽油的使用能否减低汽车尾气颗粒有机提取物的细胞膜毒性。[方法]在发动机台架试验上，分别使用市售90#含铅及无铅两种汽油来制造尾气。收集尾气的颗粒物及其吸附有机物，并检测尾气中HC与C0浓度。将体外培养中国仓鼠肺细胞(CHL)暴露于尾气的提取物中，通过检测细胞培养液中乳酸脱氢酶（LDH)及细胞内K^ 及Ca^2 浓度来分析细胞内外物质交换的变化，从而说明不同汽油排出尾气颗粒有机提取物的细胞膜毒性。[结果]随着对细胞染毒剂量的增加，培养液中的LDH浓度以及细胞内的Ca^2 浓度有增加的趋势，而细胞内K^ 浓度有降低的趋势，这种变化呈剂量—反应关系。统计学检验表明，无铅汽油尾气颗粒提取物的细胞膜毒性与对照组及其他实验组有显著性的差别。[结论]无铅汽油的使用能减少尾气中C0、HC及颗粒物的排放。与含铅汽油相比较，无铅汽油的颗粒吸附有机物的细胞膜毒性有所降低。     

Effects of Single and Mixed Estrogens on Single and Combined Cultures of D. subspicatus and P. subcapitata

This paper investigates the effect of estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) individually and mixed at equal proportions (1:1:1) on Desmodesmus subspicatus and Pseudokirchneriella subcapitata in single and combined cultures (S+) at different exposure times based on algal growth (in vivo chlorophyll fluorescence and cell counting) and coenobium formation. EE2 and E2 were more toxic to individual and combined (S+) cultures than was E1. The frequency of coenobium formation by D. subspicatus increased significantly for all estrogens and all concentrations. After 96 h, D. subspicatus prevailed in S+. The results of the exposure to E+ suggested a less-than-additive effect on D. subspicatus and S+ and additive effect on P. subcapitata. Toxic effects occurred for both species exposed to E+ with individual estrogen concentrations below the NOEC of each species. Assays must include changes in response due to the exposure of more than one species to more than one estrogen.     

Bisphenol S Disrupts Estradiol-Induced Nongenomic Signaling in a Rat Pituitary Cell Line: Effects on Cell Functions

Background: Bisphenol A (BPA) is a well-known endocrine disruptor that imperfectly mimics the effects of physiologic estrogens via membrane-bound estrogen receptors (mERα, mERβ, and GPER/GPR30), thereby initiating nongenomic signaling. Bisphenol S (BPS) is an alternative to BPA in plastic consumer products and thermal paper.Objective: To characterize the nongenomic activities of BPS, we examined signaling pathways it evoked in GH₃/B₆/F₁₀ rat pituitary cells alone and together with the physiologic estrogen estradiol (E₂). Extracellular signal-regulated kinase (ERK)– and c-Jun-N-terminal kinase (JNK)–specific phosphorylations were examined for their correlation to three functional responses: proliferation, caspase activation, and prolactin (PRL) release.Methods: We detected ERK and JNK phosphorylations by fixed-cell immunoassays, identified the predominant mER initiating the signaling with selective inhibitors, estimated cell numbers by crystal violet assays, measured caspase activity by cleavage of fluorescent caspase substrates, and measured PRL release by radioimmunoassay.Results: BPS phosphoactivated ERK within 2.5 min in a nonmonotonic dose-dependent manner (10^–15 to 10^–7 M). When combined with 10^–9 M E₂, the physiologic estrogen’s ERK response was attenuated. BPS could not activate JNK, but it greatly enhanced E₂-induced JNK activity. BPS induced cell proliferation at low concentrations (femtomolar to nanomolar), similar to E_2. Combinations of both estrogens reduced cell numbers below those of the vehicle control and also activated caspases. Earlier activation of caspase 8 versus caspase 9 demonstrated that BPS initiates apoptosis via the extrinsic pathway, consistent with activation via a membrane receptor. BPS also inhibited rapid (≤ 1 min) E₂-induced PRL release.Conclusion: BPS, once considered a safe substitute for BPA, disrupts membrane-initiated E₂-induced cell signaling, leading to altered cell proliferation, cell death, and PRL release.     

Angiotensin II increases intracellular calcium concentration in pig endometrial stromal cells through type 1 angiotensin receptors,but does not stimulate phospholipase C activity or prostaglandin F2alpha secretion

Although the presence of endometrial receptors for angiotensin (Ang) II has been demonstrated, a specific function for AngII in the uterus has not been identified. Cytosolic free Ca2+ concentration [Ca2+]i, phospholipase C (PLC) activity and prostaglandin (PG) F2alpha secretion in response to AngII and oxytocin (OT) were measured in pig endometrial stromal cells collected 16 days after oestrus. Treatment with 100 nM OT or AngII increased (P<0.001) [Ca2+]i in stromal cells similarly (720 +/- 34 v. 690 +/- 33 pM, respectively). Subsequent administration of OT or AngII to the same cells induced smaller [Ca2+]i increases (25% or 35% of the initial responses, respectively) that occurred only if the second exposure to the same agent took place at least 5 min after the first. When administered sequentially, OT and AngII each induced a full response within 1 min of the previous treatment, regardless of which peptide was applied first. Whereas OT increased PLC activity and PGF2alpha secretion in stromal cells (P<0.01), AngII did not increase either PLC activity or PGF2alpha secretion. Type I AngII (AT1) receptors were present on stromal cells, whereas AT2 receptors were absent. Therefore, the effect of AngII in stromal cells was mediated via AT1 receptors. That AngII increased [Ca2+]i in stromal cells, but did not increase PLC or PGF2alpha secretion, indicates that either AngII releases a pool of Ca2+ through a mechanism that is not mediated by PLC and is not involved in PGF2alpha secretion or that a mechanism for PGF2alpha production other than one involving Ca2+ may exist.     

Predicted exposures to steroid estrogens in U.K. rivers correlate with widespread sexual disruption in wild fish populations

Steroidal estrogens, originating principally from human excretion, are likely to play a major role in causing widespread endocrine disruption in wild populations of the roach (Rutilus rutilus), a common cyprinid fish, in rivers contaminated by treated sewage effluents. Given the extent of this problem, risk assessment models are needed to predict the location and severity of endocrine disruption in river catchments and to identify areas where regulation of sewage discharges to remove these contaminants is necessary. In this study we attempted to correlate the extent of endocrine disruption in roach in British rivers, with their predicted exposure to steroid estrogens derived from the human population. The predictions of steroid estrogen exposure at each river site were determined by combining the modeled concentrations of the individual steroid estrogens [17beta-estradiol (E2), estrone (E1), and 17alpha-ethinylestradiol (EE2)] in each sewage effluent with their predicted dilution in the immediate receiving water. This model was applied to 45 sites on 39 rivers throughout the United Kingdom. Each site studied was then categorized as either high, medium, or low "risk" on the basis of the assumed additive potency of the three steroid estrogens calculated from data derived from published studies in various cyprinid fish species. We sampled 1,438 wild roach from the predicted high-, medium-, and low-risk river sites and examined them for evidence and severity of endocrine disruption. Both the incidence and the severity of intersex in wild roach were significantly correlated with the predicted concentrations of the natural estrogens (E1 and E2) and the synthetic contraceptive pill estrogen (EE2) present. Predicted steroid estrogen exposure was, however, less well correlated with the plasma vitellogenin concentration measured in the same fish. Moreover, we found no correlation between any of the end points measured in the roach and the proportion of industrial effluents entering the rivers we studied. Overall, our results provide further and substantive evidence to support the hypothesis that steroidal estrogens play a major role in causing intersex in wild freshwater fish in rivers in the United Kingdom and clearly show that the location and severity of these endocrine-disrupting effects can be predicted.     

妊高征眼底视网膜血管病变与雌激素 孕激素水平的相关性研究

目的 :探讨血清 6种雌激素、孕激素在妊高征眼底视网膜血管病变发病中的作用以及与临床症状的相关性 ,研究妊高征患者眼底视网膜病变的分子机制。方法 :随机选择符合诊断标准的 75例住院妊高征病人作为研究组 ,正常足月妊娠待产孕妇 2 0例为对照组。专人检查妊高征病人产前、产后眼底视网膜病变情况 ,采用放射免疫分析法检测轻、中、重度妊高征产前和产后血清 6种激素水平。结果 :重度妊高征组血清 FSH水平显著高于正常妊娠组 ,而血清 PRL、 E2水平明显低于正常妊娠组 ;轻度、中度妊高征组血清 PRL水平显著低于正常妊娠组 ;中度妊高征组血清 E2水平显著低于正常妊娠组 ;妊高征患者产后血清 FSH水平明显低于正常妊娠组 ,而产后血清 PRL 水平显著高于正常妊娠组。血清孕酮、雌二醇水平与妊高征眼底病变高度相关。结论 :血清雌激素、孕激素水平与妊高征病情严重程度有关 ,可能在妊高征眼底视网膜病变发生发展过程中发挥重要作用。     

Soy protein isolate increases urinary estrogens and the ratio of 2:16alpha-hydroxyestrone in men at high risk of prostate cancer

Specific estrogen metabolites may initiate and promote hormone-related cancers. In epidemiological studies, significantly lower excretion of urinary estradiol (E2) and lower ratio of urinary 2-hydroxy estrogens to 16alpha-hydroxyestrone (2:16 OH-E1) have been reported in prostate cancer cases compared to controls. Although soy supplementation has been shown to increase the ratio 2:16 OH-E1 in women, no studies to our knowledge have investigated the effects of soy supplementation on estrogen metabolism in men. The objective of this randomized controlled trial was to determine the effects of soy protein isolate consumption on estrogen metabolism in men at high risk for developing advanced prostate cancer. Fifty-eight men supplemented their habitual diets with 1 of 3 protein isolates: 1) isoflavone-rich soy protein isolate (SPI+) (107 mg isoflavones/d); 2) alcohol-washed soy protein isolate (SPI-) (<6 mg isoflavones/d); or 3) milk protein isolate (MPI), each providing 40 g protein/d. At 0, 3, and 6 mo of supplementation, the urinary estrogen metabolite profile was measured by GC-MS. Both soy groups had higher E2 excretion than the MPI group at 3 and 6 mo. After 6 mo of supplementation, the SPI+ group had a significantly higher urinary 2:16 OH-E1 ratio than the MPI group. Increased urinary E2 excretion and 2:16 OH-E1 ratio in men consuming soy protein isolate are consistent with studies in postmenopausal women and suggest that soy consumption may be beneficial in men at high risk of progressing to advanced prostate cancer as a result of effects on endogenous estrogen metabolism.     

Chemical and biological analysis of endocrine-disrupting hormones and estrogenic activity in an advanced sewage treatment plant

The steroid hormones estrone (E(1)), 17beta-estradiol (E(2)), estriol (E(3)), 17alpha-ethinylestradiol (EE(2)), and their conjugated forms were surveyed throughout an advanced sewage treatment plant (STP). The estrogen concentrations in water and sludge samples, collected in October 2004 and April 2005, were determined by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. Simultaneously, the estrogenic activity was quantified using estrogen-responsive reporter cell lines (MELN) to investigate the behavior of overall estrogenic compounds. The estrogen concentrations in the inlet ranged from 200 to 500 ng/L, with the contribution of conjugated forms being higher than 50%. The major estrogens in influent were E(1) and E(3). The estrogenic activity was between 25 and 130 ng/L of E(2) equivalents (EEQs). Estrogen concentrations and estrogenicity measured in the inlet and in primary treated sewage were similar, showing a weak impact of primary treatment on hormone removal. In contrast, both estrogen concentration and estrogenicity decreased during biological treatment, with high removal efficiencies (>90%). Estrone, E(2), and EE(2) persisted in the treated water below 10 ng/L, whereas the estrogenicity was lower than 5 ng/L of EEQs. Estrogen mass flux in the effluent and sludge represented less than 2 and 4%, respectively, of the inlet. Consequently, the fraction of estrogens sorbed into the sludge was very small, and biodegradation was the main vehicle for estrogen elimination. This dual approach, comparing chemical and biological analysis, allowed us to confirm that most of the estrogenic activity occurring in this STP, which receives mainly domestic sewage, resulted from sex hormones.     

激素补充治疗中不同雌激素的特点

雌激素是性激素补充治疗的核心。目前临床应用的雌激素种类繁多。本文以用药途径为主线,详细介绍不同雌激素的特点。临床上需个体化选择适宜药物。对于缓解潮热出汗症状、预防骨质疏松症而言,全身用药,包括各种口服雌激素和经皮雌激素,均可达到满意的疗效。对于有肝胆功能障碍或血栓形成高危因素者,经皮雌激素更有优势。对于仅存在阴道局部症状者,建议局部用药。建议使用最低有效剂量。     

Effects of oral contraceptive steroids on pituitary prolactin secretion.

Serum prolactin (PRL) levels in 16 premenopausal subjects on synthetic estrogen and progestin were measured during 19 treatment cycles. The studies were performed during the estrogen segments of the treatment cycle (days 7–16) in 10 subjects receiving sequential contraceptives, and between days 9–20 in 6 subjects receiving combination type contraceptives. Because of the episodic nature of PRL release, the transverse mean of the frequent samples obtained over a period of four hours was used for quantitative comparison of subjects receiving the contraceptive steroids with normal subjects. Significant increases in prolactin concentrations were noted in both sequential (P < 0.001) and combination (P < 0.05) pill users as compared to normals. The mean serum PRL level of subjects on sequential type pill was significantly greater (P < 0.01) than that of subjects on combination type pill. This data suggests that the estrogen component of the pill exerts a positive effect on pituitary prolactin secretion.     

Neuroendocrine response to estrogen and brain differentiation in heterosexuals,homosexuals, and transsexuals

Since 1964, we have found positive estrogen feedback to be a relatively sex-specific reaction of the hypothalamo-hypophyseal system in rats as well as in human beings. It is dependent on the estrogen-convertible androgen level during sexual brain differentiation and also on estrogen priming in adulthood. The lower the estrogen-convertible androgen or primary estrogen level during brain differentiation, the higher the evocability of a positive estrogen action on LH secretion in later life. In clinical studies, we induced a positive estrogen feedback luteinizing hormone secretion in most intact homosexual men, in clear-cut contrast to intact heterosexual or bisexual men. In addition, the evocability of a positive estrogen feedback was also demonstrable in most homosexual male-to-female transsexuals in significant contrast to hetero-or bisexual male-to-female transsexuals. The following relations have been found between sex hormone levels during brain differentiation and sex-specific responses in adulthood: (i) Estrogens, which are mostly converted from androgens, are responsible for the sex-specific organization of gonadotropin secretion and hence the evocability of a positive estrogen feedback in later life; (ii) both estrogens and androgens, occurring during brain differentiation, predetermine sexual orientation, and (iii) androgens, without conversion to estrogens, are responsible for the sex-specific organization of gender role behavior. Furthermore, the organization periods for sex-specific gonadotropin secretion, sexual orientation, and gender role behavior are not identical but overlapping. Thus, combinations as well as dissociations between deviation of the neuroendocrine organization of sex-specific gonadotropin secretion, sexual orientation, and gender role behavior may occur. A revised version of a paper presented at the annual meeting of the International Academy of Sex Research, Amsterdam, September 16–20, 1986.