Local Estradiol Synthesis Research Articles

Unkeito promotes follicle development by restoring reduced follicle-stimulating hormone responsiveness in rats with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common disorder resulting in irregular menstruation and infertility due to improper follicular development and ovulation. PCOS pathogenesis is mediated by downregulated follicle-stimulating hormone receptor (FSHR) expression in granulosa cells (GCs); however, the underlying mechanism remains elusive. Unkeito (UKT) is a traditional Japanese medicine used to treat irregular menstruation in patients with PCOS. In this study, we aimed to confirm the effectiveness of UKT in PCOS by focusing on follicle-stimulating hormone (FSH) responsiveness. A rat model of PCOS was generated by prenatal treatment with 5α-dihydrotestosterone. Female offspring (3-week-old) rats were fed a UKT mixed diet or a normal diet daily. To compare the PCOS phenotype in rats, the estrous cycle, hormone profiles, and ovarian morphology were evaluated. To further examine the role of FSH, molecular, genetic, and immunohistological analyses were performed using ovarian tissues and primary cultured GCs from normal and PCOS model rats. UKT increased the number of antral and preovulatory follicles and restored the irregular estrous cycle in PCOS rats. The gene expression levels of FSHR and bone morphogenetic protein (BMP)-2 and BMP-6 were significantly decreased in the ovarian GCs of PCOS rats compared to those in normal rats. UKT treatment increased FSHR staining in the small antral follicles and upregulated Fshr and Bmps expression in the ovary and GCs of PCOS rats. There was no change in serum gonadotropin levels. In primary cultured GCs stimulated by FSH, UKT enhanced estradiol production, accompanied by increased intracellular cyclic adenosine monophosphate levels, and upregulated the expression of genes encoding the enzymes involved in local estradiol synthesis, namely Cyp19a1 and Hsd17b. Furthermore, UKT elevated the expression of Star and Cyp11a1, involved in progesterone production in cultured GCs in the presence of FSH. UKT stimulates ovarian follicle development by potentiating FSH responsiveness by upregulating BMP-2 and BMP-6 expression, resulting in the recovery of estrous cycle abnormalities in PCOS rats. Restoring the FSHR dysfunction in the small antral follicles may alleviate the PCOS phenotype.

Lipoxin A4 inhibits the development of endometriosis in a mouse model by suppressing local estradiol synthesis

We investigated whether lipoxin A4 (LXA4) inhibits the development of endometriosis by suppressing local estradiol synthesis. An endometriosis mouse model was constructed by surgical transplantation to subcutanous tissue sites. The treatment group received daily injections of LXA4 (10 μg/Kg) for 21days after which lesions were recovered. We measured 17β-HSD1, 17β-HSD2, CYP11A1, CYP19A1, CYP17A1, and estrogen receptor mRNA expression levels using real-time RT-PCR. In addition, immunohistochemistry was performed to determine protein expression and localization. After LXA4 administration, the volume of endometrial lesions was significantly reduced. Administration of LXA4 resulted in a more rudimentary architecture with a reduced number of developed glands surrounded by a small amount of stroma. LXA4 downregulated the mRNA and protein expression levels of 17β-HSD1, CYP11A1, CYP19A1, CYP17A1, ERα, and ERβ. Furthermore, LXA4 downregulated the expression of ERβ, aromatase expression, and 17β-HSD1 enzyme activity, which affected local estradiol production, resulting in reduced endometriosis. Results from our endometriosis mouse model showed that treatment with LXA4 reduced expression of enzymes and receptors associated or implicated with estrogen-dependent regulation of extra-uterine tissue. We believe that LXA4 has a potential therapeutic value for the treatment of endometriosis.

Sex‐dependency of oestrogen‐induced structural synaptic plasticity: Inhibition of aromatase versus application of estradiol in rodents

Sex-dependent differences in learning and memory formation in humans have been frequently shown. The mechanisms underlying the formation and retention of memories are assumed to involve synaptic plasticity in the hippocampus. Estradiol was shown to effect synaptic plasticity in the hippocampus of rodents. The effects after exogenous application of estradiol to animals frequently produce inconsistent results, in particular, if sex is not considered in the studies. Recently we provided evidence that locally synthesized estradiol plays an essential role on synaptic connectivity in the hippocampus of females but not of male mice. In females, inhibition of local estradiol synthesis leads to synapse loss, which results from impairment of long-term potentiation and dephosphorylation of cofilin, and thereby the destabilization of postsynaptic dendritic spines. This sex-dependency was also seen in the classical aromatase knock-out mouse. Intriguingly, no differences between sexes have been found in a conditional forebrain-specific aromatase knock-out mouse. Altogether, the findings underscore the necessity of including 'Sex as a Biological Variable' in studies of sex steroid-induced synaptic plasticity.

Evidence that inflammation promotes estradiol synthesis in human cerebellum during early childhood

Discovering and characterizing critical and sensitive periods in brain development is essential for unraveling the myriad variables that impact disease risk. In previous work, we identified a critical period in cerebellar development in the rat that depends upon an intrinsic gene expression program and links increased prostaglandin production to local estradiol synthesis by stimulating Cyp19a, the estradiol synthetic enzyme, aromatase. This intrinsic critical period is sensitive to disruption by either inflammation or administration of cyclooxygenase (COX) inhibitors, ultimately impacting Purkinje cell dendritic growth. In a first step towards determining if a similar sensitive period exists in humans, the same gene expression profile was characterized in post-mortem cerebellar tissue of 58 children aged 0 to 9 years. Subjects were categorized as experiencing inflammation or not at the time of death. In individuals experiencing inflammation and over 1 year of age, there was a significant increase in the messenger RNA (mRNA) of the COX-1 and COX-2 enzymes and this strongly correlated with mRNA levels of aromatase. A step-wise linear model accounted for 94% of the variance in aromatase mRNA levels by co-variance with the COX enzymes, prostaglandin E2 synthase and other inflammatory mediators (Toll-like receptor 4), and Purkinje cell markers (calbindin, estrogen receptor 2). The influence of inflammation on these measures was not seen in subjects younger than 1 year. These data suggest a sensitive period to inflammation in the human cerebellum begins at about 1 year of age and may provide insight into sources of vulnerability of very young children to either inflammation or drugs designed to treat it.

Hippocampal Aromatization Modulates Spatial Memory and Characteristics of the Synaptic Membrane in the Male Zebra Finch.

The estrogen-synthesizing enzyme aromatase is abundant at the synapse in the zebra finch hippocampus (HP), and its inhibition impairs spatial memory function. To more fully test the role of local estradiol (E2) synthesis in memory, the HP of adult male zebra finches was exposed to either control pellets or those containing the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD), ATD and E2, ATD and the G protein-coupled estrogen receptor (GPER) agonist G1, or the antagonist G15 alone. Birds were tested for spatial memory acquisition and performance, and HP levels of the postsynaptic protein PSD95 were measured. ATD-treated birds took longer to reach criterion than control birds, whereas acquisition in ATD+E2 and ATD+G1 birds was indistinguishable from control and ATD treatments. Interestingly, all G15 birds failed to acquire the task. Following a retention interval, ATD birds took the longest to reach the (formerly) baited cup and made the most mistakes. ATD+E2 animals displayed the lowest retention latencies and made fewer mistakes than ATD-treated birds, and ATD+G1 birds did not significantly differ from controls in retention latencies. The amount of PSD95 in the HP was lowest in ATD-treated animals compared with birds with silicone-only-implanted craniotomies, ATD+E2, and ATD+G1 birds, who did not differ in this expression. Thus, spatial memory acquisition and performance appear aromatase and E2 dependent, an effect more reliably revealed after consolidation and/or recall compared to acquisition. E2 may exert this effect via GPERs, resulting in an increase in PSD95 levels that may modify receptor activity or intracellular signaling pathways to increase synaptic strength.

Increased levels of enzymes involved in local estradiol synthesis in chronic obstructive pulmonary disease

IntroductionSteroid hormones are involved in lung development, pulmonary inflammation, and lung cancer. Estrogen signaling and exposure may play a role in pulmonary disorders, including COPD. In both genders, estrogens can be generated locally in the lungs and this contributes importantly to the tissue exposure to these steroids. ObjectiveTo characterize and assess differences in localization of estrogen receptors and enzymes involved in the local generation of estrogens in COPD. MethodsEstrogen Receptor alpha (ERα/ESR1), Estrogen Receptor beta (ERβ/ESR2) and G-protein-coupled estrogen receptor 1 (GPER) were explored by real-time (RT)-PCR analysis (mRNA expression), immunohistochemistry and western blotting in controls and COPD patients.mRNA expression of the enzymes involved in the local estrogen generation – i.e. aromatase (CYP19A1), 17beta-hydroxysteroid dehydrogenases (17β-HSDs) 1, 2, 4, 5, 7 and 12, steroid sulfatase (STS) and sulfotransferase (SULT1E1) - were analyzed by RT-PCR. ResultsERα, ERβ and GPER were expressed in lung tissue, but no differences were observed between patients and controls. The main enzymes involved in local estrogen generation were also present in both normal and COPD lung tissue. In lungs of COPD patients compared with controls, we observed increased expression of the enzymes 17β-HSD type 1 and aromatase (positive association), both involved in the local synthesis of active estrogens. ConclusionAll ER subtypes are present in the lung. The shift in local mRNA level of estrogen metabolic enzymes suggests that exposure to estrogens is involved in the pathogenesis of COPD.

A Critical Period in Purkinje Cell Development Is Mediated by Local Estradiol Synthesis, Disrupted by Inflammation, and Has Enduring Consequences Only for Males.

Identifying and understanding critical periods in brain development is essential to decoding the long-term impact of widespread, poorly defined, and frequently occurring insults such as inflammation. Using the laboratory rat Rattus norvegicus, we have discovered a narrowly constrained critical period in Purkinje neuron development subject to dysregulation by inflammation. The onset and offset of heightened vulnerability are attributed to a tightly orchestrated gene expression profile present only during the second postnatal week and not the first or third weeks. Genes expressed during this time code for enzymes and receptors which are critical not only for prostaglandin production and activity but also for estradiol production via the aromatase enzyme and estradiol action via the α isoform of the estrogen receptor. The two synthetic pathways are connected by prostaglandin E2 (PGE2) activation of the aromatase enzyme, as we reported previously (Dean et al., 2012b) and confirm here. Dysregulation of the PGE2-estradiol pathway during the second week by treatment with PGE2 or lipopolysaccharides produces enduring consequences as a result of reduced growth of Purkinje dendritic trees and impaired juvenile social play behavior, but only in males. The deleterious consequences of inflammation locally in the cerebellum are prevented by peripheral treatment with the cyclooxygenase inhibitor nimesulide or the aromatase inhibitor formestane. These findings highlight a novel regulatory pathway that creates a critical period in brain development vulnerable to dysregulation by inflammation. The cerebellum is increasingly appreciated for its role in social, emotional, and cognitive behaviors. It is consistently and severely affected in neuropsychiatric disorders originating during development, such as autism spectrum disorder and schizophrenia. We have identified a critical period in rat development during the second week of life that is dysregulated by inflammatory insults. An intrinsic program of gene expression determines the critical period. The enduring consequences of inflammation during the second postnatal week are stunted dendrites of the cerebellum's principal neurons, Purkinje cells, and impairments in later social behavior. These changes are not evident if inflammation occurs during the first or third week, highlighting the importance of fine-grained analyses of developmental processes and the factors that influence them.

Pituitary Aromatase P450 May Be Involved in Maintenance of the Population of Luteinizing Hormone-Positive Pituitary Cells in Mice

As aromatase P450 is located in several pituitary cells, testosterone can be transformed into 17β-estradiol in the gland by the enzyme. The possible role of this transformation in pituitary function remains to be elucidated, but some evidence suggests a physiological and pathophysiological role for pituitary aromatase. To determine its relevance in the modulation of pituitary function, mainly associated with reproduction, luteinizing hormone (LH)-positive cells in the hypophysis of female and male aromatase knockout (ArKO) mice were studied. In all LH-positive cells, significant increases in the cellular (p < 0.01) and nuclear (p < 0.05) areas were found in the ArKO mice compared to the wild-type mice. In the ArKO mice, LH-positive cells were more abundant (p < 0.01); they were characterized by a stronger cytoplasmic reaction and the cells were more polygonal and exhibited more short, thick cytoplasmic prolongations than those in the wild-type mice. Moreover, LH-positive cells showed a greater proliferation rate in the ArKO mice compared to the wild-type mice (p < 0.01). These findings suggest that the local production of estradiol mediated by pituitary aromatase is necessary for the regulation of LH-positive gonadotropic cells, exerting an autoparacrine inhibitory regulation. These results could underlie the higher pituitary aromatase expression observed in male versus female mice. Similar effects were found in ArKO male and female mice, suggesting that in both sexes the effects of estrogens on maintenance of the LH-positive pituitary cell population could be related to the local aromatization of testosterone to estradiol inside the hypophysis. Therefore, aromatase could modulate pituitary LH-positive cells in males through local estradiol synthesis.

Ovariectomy and Subsequent Treatment with Estrogen Receptor Agonists Tune the Innate Immune System of the Hippocampus in Middle-Aged Female Rats

The innate immune system including microglia has a major contribution to maintenance of the physiological functions of the hippocampus by permanent monitoring of the neural milieu and elimination of tissue-damaging threats. The hippocampus is vulnerable to age-related changes ranging from gene expression to network connectivity. The risk of hippocampal deterioration increases with the decline of gonadal hormone supply. To explore the impact of hormone milieu on the function of the innate immune system in middle-aged female rats, we compared mRNA expression in the hippocampus after gonadal hormone withdrawal, with or without subsequent estrogen replacement using estradiol and isotype-selective estrogen receptor (ER) agonists. Targeted profiling assessed the status of the innate immune system (macrophage-associated receptors, complement, inhibitory neuronal ligands), local estradiol synthesis (P450 aromatase) and estrogen reception (ER). Results established upregulation of macrophage-associated (Cd45, Iba1, Cd68, Cd11b, Cd18, Fcgr1a, Fcgr2b) and complement (C3, factor B, properdin) genes in response to ovariectomy. Ovariectomy upregulated Cd22 and downregulated semaphorin3A (Sema3a) expression, indicating altered neuronal regulation of microglia. Ovariectomy also led to downregulation of aromatase and upregulation of ERα gene. Of note, analogous changes were observed in the hippocampus of postmenopausal women. In ovariectomized rats, estradiol replacement attenuated Iba1, Cd11b, Fcgr1a, C3, increased mannose receptor Mrc1, Cd163 and reversed Sema3a expression. In contrast, reduced expression of aromatase was not reversed by estradiol. While the effects of ERα agonist closely resembled those of estradiol, ERβ agonist was also capable of attenuating the expression of several macrophage-associated and complement genes. These data together indicate that the innate immune system of the aging hippocampus is highly responsive to the gonadal hormone milieu. In ovariectomized female rats, estradiol replacement exerts potent immunomodulatory effects including attenuation of microglia sensitization, initiation of M2-like activation and modulation of complement expression by targeting hippocampal neurons and glial cells through ERα and ERβ.

Estradiol responsiveness of synaptopodin in hippocampal neurons is mediated by estrogen receptor β

In the hippocampus, synaptic proteins, as studied so far, have been shown to be upregulated by 17β-estradiol, while inhibition of local estradiol synthesis consistently downregulates them. As an exception to this rule, we have previously shown that synaptopodin, an actin-associated postsynaptic protein, is downregulated in response to estradiol in dissociated cultured hippocampal neurons. In this study, we show, unexpectedly, that synaptopodin is downregulated in the hippocampus of aromatase knock-out mice and that inhibition of neuronal estradiol synthesis using the aromatase inhibitor letrozole also downregulates synaptopodin in these cultures. Moreover, the effects of estradiol and letrozole are additive, suggesting a subtle balance between available ligand and receptor. Using selective estrogen receptor agonists and antagonists, we consequently studied the effects of estrogen receptor subtypes on synaptopodin expression in our hippocampal cultures. We found that estradiol-induced downregulation of synaptopodin is mediated by estrogen receptor β. Estrogen receptor β in turn, is upregulated in response to intracellular estradiol ablation following inhibition of estradiol synthesis by letrozole in dissociated hippocampal cultures, as well as in the hippocampus of the aromatase knock-out mouse. Thus, it appears that both the application of estradiol, via binding to estrogen receptor β, and letrozole, via upregulation of estrogen receptor β, eventually result in a downregulation of synaptopodin. Our data show that the synaptic plasticity caused by estradiol is subject to a subtle balance of the levels of estrogen receptor subtypes regulated by the available ligands. In addition, both seem to be part of a homeostatic feedback mechanism.

Sexual dimorphism in estrogen-induced synaptogenesis in the adult hippocampus

It has long been known that estradiol influences synaptic plasticity in the female hippocampus. The density of dendritic spines varies during the estrous cycle and correlates positively with varying levels of estradiol in serum. In accordance, ovariectomy results in a loss of spines that can be rescued by estradiol treatment in animals, suggesting that estradiol originating from the ovaries induces spine formation in the hippocampus. More recent studies point to a role of hippocampus-derived estradiol in synaptogenesis in the female hippocampus, rather than of estradiol of ovarian origin. In our studies, we have shown that inhibition of hippocampal estrogen synthesis results in spine synapse loss in female animals and, more importantly, also in ovariectomized animals. Surprisingly, inhibition of local estradiol synthesis had no effect on synapse formation in males, in spite of a similar capacity to synthesize estradiol in male and female hippocampal neurons. In females, neuro-sexual steroid production is promoted by hypothalamic, cyclic GnRH release and likely underlies the estrus cyclicity of spine synapse density in the hippocampus. As a result, peripheral serum concentrations of estradiol determine the amount of estradiol synthesis in the hippocampus. This paradigm may also be true in males. In support of this hypothesis, we found that the content of estradiol in hippocampal tissue is higher in female compared to that in male animals, with low levels of estradiol in serum and tonic and acyclical GnRH release. In summary, our data point to important sex-specific differences in sexual steroid-induced synaptogenesis.

The Stimulation of HSD17B7 Expression by Estradiol Provides a Powerful Feed-Forward Mechanism for Estradiol Biosynthesis in Breast Cancer Cells

Our laboratory has previously cloned and purified an ovarian protein found to be a novel 17β-hydroxysteroid dehydrogenase type 7 enzyme (HSD17B7) (formerly prolactin receptor-associated protein) that converts the weak estrogen, estrone, to the highly potent estradiol. The regulation of this enzyme has not yet been explored. In this report, we show high expression of HSD17B7 in human ductal carcinoma and breast cancer cell lines and present evidence for a strong up-regulation of this enzyme by estradiol at the level of mRNA, protein expression, and promoter activity in MCF-7 cells. The effect of estradiol is mediated by estrogen receptor (ER)α, whereas ERβ prevents this stimulation. ER antagonists, ICI 182,780 and 4-hydroxytamoxifen, prevent estradiol-induced stimulation of the endogenously expressed HSD17B7, suggesting that these inhibitors not only block estradiol action but also its production. We have identified a -185-bp region of the hsd17b7 promoter that is highly conserved among rat, mouse, and human and confers regulation by estradiol in MCF-7 cells. This region is devoid of a classical estradiol-response element but contains a nuclear factor 1 (NF1) site that is essential for estradiol action. We found that estradiol stimulates the recruitment and DNA binding of NF1 to this region of the hsd17b7 promoter. Furthermore, knockdown of NF1 family members, NF1B, NF1A, and NF1X, completely prevents induction of this gene by estradiol. In summary, our findings demonstrate that estradiol stimulates HSD17B7 transcriptional activity in breast cancer cells through a novel mechanism requiring NF1 and strongly suggest a positive feedback mechanism to increase local estradiol synthesis causing growth of estrogen-dependent breast cancers.

17β‐Hydroxysteroid Dehydrogenases in Human Breast Cancer

Estrogen plays a pivotal role in development and progression of human breast carcinoma. Before menopause the main source of estrogen in women is circulating estrogen secreted from the ovary, but following menopause the source changes to the hormone that is converted from circulating adrenal androgens in peripheral tissues. Therefore, adrenal androgens have to be converted to estrogen to stimulate breast carcinoma cells. In these steps, several enzymes such as aromatase, steroid sulfatase, and 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) are involved in the production of estrogens. The reaction related to 17beta-HSDs activity is one of the last steps of estradiol biosynthesis, and 14 isozymes of 17beta-HSD have been identified at this juncture. The balance of the relative expression levels of 17beta-HSD isozymes in human breast carcinomas is thought to play a pivotal role in supply of estradiol to estrogen receptor positive carcinoma cells. Understanding the character of 17beta-HSD isozymes in human breast carcinoma thus provides important information on the mechanisms of biosynthesis of estradiol in breast carcinoma and for development of a therapeutic agent targeted for inhibition of local estradiol synthesis in breast carcinoma cells. In the present review we summarize the roles played by 17beta-HSDs in human breast carcinoma to obtain a better understanding of the properties of 17beta-HSDs in human breast carcinoma.

Cholesterol‐promoted synaptogenesis requires the conversion of cholesterol to estradiol in the hippocampus

Cholesterol of glial origin promotes synaptogenesis (Mauch et al., (2001) Science 294:1354-1357). Because in the hippocampus local estradiol synthesis is essential for synaptogenesis, we addressed the question of whether cholesterol-promoted synapse formation results from the function of cholesterol as a precursor of estradiol synthesis in this brain area. To this end, we treated hippocampal cultures with cholesterol, estradiol, or with letrozole, a potent aromatase inhibitor. Cholesterol increased neuronal estradiol release into the medium, the number of spine synapses in hippocampal slice cultures, and immunoreactivity of synaptic proteins in dispersed cultures. Simultaneous application of cholesterol and letrozole or blockade of estrogen receptors by ICI 182 780 abolished cholesterol-induced synapse formation. As a further approach, we inhibited the access of cholesterol to the first enzyme of steroidogenesis by knock-down of steroidogenic acute regulatory protein, the rate-limiting step in steroidogenesis. A rescue of reduced synaptic protein expression in transfected cells was achieved by estradiol but not by cholesterol. Our data indicate that in the hippocampus cholesterol-promoted synapse formation requires the conversion of cholesterol to estradiol.

Resting intracellular calcium concentration, depolarizing Gamma-Aminobutyric Acid and possible role of local estradiol synthesis in the developing male and female hippocampus

The maturation of the hippocampus is impacted by a multitude of factors, including the regulation of intracellular calcium levels. Depolarizing actions of Gamma-Aminobutyric Acid (GABA) can profoundly alter intracellular calcium in immature hippocampal neurons via influx through voltage-gated calcium channels. We here report fundamental sex differences in properties of depolarizing GABA responses and in resting intracellular calcium in neonatal cultured hippocampal neurons. The effects of the estrogen receptor antagonist, ICI 182,780, and the estradiol-synthesis inhibitor, formestane, indicate the sex differences in depolarizing GABA responses are at least in part due to de novo estradiol synthesis by female neurons, whereas a sex difference in resting calcium is independent of steroids. We postulate that local estradiol synthesis in cultured female hippocampal neurons affects the kinetics of either the GABAA receptor or voltage sensitive calcium channels. These data highlight the fact that immature hippocampal neurons exhibit fundamentally different physiological properties in males versus females. Elucidating how and where immature male and female neurons differ is essential for a complete understanding of normal rodent brain development.

In search of neuroprotective therapies based on the mechanisms of estrogens

Although estradiol is a neuroprotective factor, estrogen therapy in older women increases the risk of adverse cognitive outcomes and poses additional peripheral risks, requiring careful use of estrogenic compounds as treatments for neurodegenerative conditions or neural injury. Potential alternatives to estrogen therapy to promote neuroprotection might include treatment with molecules that are able to interact with estrogen receptors, with alternative mechanisms of action, or with molecules that induce local estradiol synthesis in the brain, or a combination of all. However, before considering the broad clinical applications, more basic research is required to clarify the mechanisms of action and potential risks of some of these estrogen-based treatments.

Castration reduces male testosterone, estradiol, and territorial aggression, but not paternal behavior in biparental dwarf hamsters ( Phodopus campbelli)

Biparental male hamsters, Phodopus campbelli, act as midwives during the birth of their litter and are highly responsive to an experimentally displaced pup. They also have high peripheral concentrations of estradiol, a hormone with known roles in maternal behavior. Surgical castration during the gestation of their first litter was used to investigate the source of that estradiol and the functional role of testicular sex steroids in paternal responsiveness. In Experiment I, castration reduced both testosterone and estradiol concentrations, confirming that the testes were the primary source of estradiol. However, neither paternal responsiveness nor multiple measures of reproductive success were altered by the castration. Aggression directed towards an intruder, however, was reduced by castration. In Experiment II, removal of prior experience with birth or pups also failed to alter paternal responsiveness in castrated males. Although the present results do not preclude a role for local estradiol synthesis in the brain, results do not support an association between high circulating estradiol in males and their paternal behavior.