Neonatal Estrogen Research Articles

FRI282 Sexually Dimorphic Responses Of Kisspeptin Expression To Neonatal Exposure To Estrogen In Rats

Abstract Disclosure: B. Marzbanabbasabadi: None. P. Lin: None. C. Park: None. C. Ko: None. Kisspeptin neurons (Kiss1 neurons) are located mainly in anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) of hypothalamus and play a key role as regulators of GnRH secretion in all mammalian species. In both females and males, ARC contains more Kiss1 neurons than AVPV. Importantly, unlike ARC, AVPV displays sexual dimorphism; female AVPV contains significantly more Kiss1 neurons than males’. Recent studies showed that neonatal exposure to estrogen decreased Kiss1 expression in the hypothalamus, impacting fertility, in both sexes. However, whether such impact was driven by decreased number of Kiss1 neurons or expression of Kiss1 gene is not known. In this study, we tested our hypothesis that neonatal estrogen exposure decreases both the number of Kiss1 neurons and the Kiss1 gene expression in AVPV, in a gender-dependent manner. This hypothesis was tested in rats by treating them with estradiol benzoate (EB, a prodrug that is converted to estradiol) and measuring the effect by counting the number of Kiss1 neurons (Kiss1 mRNA expressing cells) and comparing Kiss1 mRNA expression levels between EB-exposed and control hypothalami. Male and female rat pups were injected, on postnatal day 1, subcutaneously with 0.3 mg EB via microsphere that was designed to release EB for 2 weeks; unexposed age matching pups were used as controls (n=6-7/group). Male rats were euthanized at 2.5 months of age and their entire brains were collected. Female brains were also sampled at 2.5 months of age, but after synchronizing their estrous cycle by injecting them with PMSG and hCG following an established superovulation protocol. The brains were serially sectioned and the sections from AVPV and caudal region of ARC where Kiss1 neurons most highly populated were identified and used for localizing Kiss1 neurons and quantitating Kiss1 mRNA expression level, using RNAscope. Results showed that in the AVPV, EB-exposed rats had less Kiss1 neurons both in males (8.7±1.9 vs. 42.2±7.9 Kiss1 neurons/section in control, p=0.02) and females (26.0±5.65 vs. 141.6±15.8 Kiss1 neurons/section in control, p=0.03) and showed lower Kiss1 mRNA expression in males (0.33 folds, p=0.03) and in females (0.44 folds, p=0.04) as well. In the ARC, EB-exposed rats had less Kiss1 neurons in females (27.5±4.3 vs. 56.0±2.4/section in control, p=0.00), but had same number in males (31.7±2.3 vs. 42.8±7.6 kiss1 neurons/section in control, p=0.22). EB exposure increased Kiss1 mRNA expression in both males (2.39 folds, p=0.00) and females (1.55 folds, p=0.05). In conclusion, this study showed that neonatal exposure to estrogen alters the number of Kiss1 neurons and Kiss1 mRNA expression in AVPV in a gender-dependent manner. In addition, neonatal estrogen exposure also alters Kiss1 neuron numbers and Kiss1 gene expression in the ARC as well. Presentation: Friday, June 16, 2023

Neonatal estrogen induces male-like expression of astroglial markers of maturation and plasticity in the neocortex of female mice

Astroglia play a crucial role in various aspects of neurodevelopment including building, maintaining, and modulating neuronal circuits that underly complex behaviours in the neocortex. Telencephalic regions exhibit sex differences in neuronal networks that arise early in development. Astroglia express receptors for gonadal hormones responsible for the organization of sex differences, such as estrogen, placing them in a key position to modulate sex differences in the development of neuronal networks. Astroglial cells express specific proteins related to their morphology, function, and maturation. We have previously shown that P7-P14 is a key transition period for neocortical astroglial maturation and that males reach a mature phenotype earlier than females, at P7. In this study, we investigated whether administration of perinatal estradiol to female mice is sufficient to masculinize astroglial protein and gene expression related to maturation that we previously observed at P7. We found that canonical astroglial markers like glial fibrillary acidic protein and glutamine synthetase are not affected by perinatal estrogen, but markers of astroglial maturation, Vimentin, Aldh1a1, Dio2, and the number of actively dividing astroglia are masculinized by perinatal estradiol administration. These findings suggest that sex differences in neocortical astroglial maturation are at least in-part due to the role of perinatal estrogen. Given the higher prevalence of neurodevelopmental disorders in males compared to females and the involvement of astroglia in virtually all neurodevelopmental disorders, further research is needed to determine other contributions to sex differences in neocortical astroglial cells.

Single neonatal estrogen implant sterilizes female animals by decreasing hypothalamic KISS1 expression

Reproductive sterilization by surgical gonadectomy is strongly advocated to help manage animal populations, especially domesticated pets, and to prevent reproductive behaviors and diseases. This study explored the use of a single-injection method to induce sterility in female animals as an alternative to surgical ovariohysterectomy. The idea was based on our recent finding that repetitive daily injection of estrogen into neonatal rats disrupted hypothalamic expression of Kisspeptin (KISS1), the neuropeptide that triggers and regulates pulsatile secretion of GnRH. Neonatal female rats were dosed with estradiol benzoate (EB) either by daily injections for 11 days or by subcutaneous implantation of an EB-containing silicone capsule designed to release EB over 2–3 weeks. Rats treated by either method did not exhibit estrous cyclicity, were anovulatory, and became infertile. The EB-treated rats had fewer hypothalamic Kisspeptin neurons, but the GnRH-LH axis remained responsive to Kisspeptin stimulation. Because it would be desirable to use a biodegradable carrier that is also easier to handle, an injectable EB carrier was developed from PLGA microspheres to provide pharmacokinetics comparable to the EB-containing silicone capsule. A single neonatal injection of EB-microspheres at an equivalent dosage resulted in sterility in the female rat. In neonatal female Beagle dogs, implantation of an EB-containing silicone capsule also reduced ovarian follicle development and significantly inhibited KISS1 expression in the hypothalamus. None of the treatments produced any concerning health effects, other than infertility. Therefore, further development of this technology for sterilization in domestic female animals, such as dogs and cats is worthy of investigation.

OP-12 - Three-dimensional and immunohistochemical analysis of sex steroid receptors in the developing murine mesonephros

Sex steroids are essential for the development of the reproductive tracts, and their function is mediated by activating their receptors. The expression patterns of the receptors have been investigated in previous studies, but the results differ. One of the reasons is a lack of a method to distinguish each region of the reproductive tracts. To distinguish the cranial and caudal Wolffian duct (WD), which contribute to the common efferent duct and epididymal duct, respectively, it is necessary to check the connection with the mesonephric tubules (MTs) that differentiate into the efferent ductules. In this study, we examined the expression patterns of sex steroid receptors in detail to combine the three-dimensional analysis. Serial paraffin sections of the developing murine mesonephros were prepared, and immunohistochemistry for sex steroid receptors, such as estrogen receptor (ESR1), androgen receptor (AR), and progesterone receptor (PR), was performed. Additionally, the sections in which Collagen Type IV was detected were used to reconstruct the MTs and WD. ESR1 was found in the mesenchymal cells around the WD, and the MTs and cranial WD also became positive from the middle period of the development, but the caudal WD was negative for ESR1. AR was first found at the distal end (gonadal side) of the MTs and weakly in the mesenchymal cells. The epithelial expression became strong in the whole length of the MTs and cranial portion of the WD in the middle period of the development. Finally, the caudal part of the WD also showed strong positive just before birth. PR was weakly positive in the MTs and cranial WD, but the distal end of the MTs where the rete cells were adherent was negative for PR. Androgen secreted from the gonad probably acts on the distal end of the MTs to maintain and develop the efferent tubules. ESR1 and PR may also contribute to the development of the efferent duct. Additionally, estrogen treatment in the neonatal male mice induces epididymal inflammation after puberty, but its mechanisms are still unclear. This study found the ESR1 expression in not epithelial cells but mesenchymal cells around the caudal WD. These results suggest that neonatal estrogen treatment probably affects the mesenchymal cells around the epididymal duct, resulting in the inability to suppress the autoimmune response to spermatozoa after puberty.

Role of nuclear and membrane estrogen signaling pathways in the male and female reproductive tract

Estrogen signaling through the main estrogen receptor, estrogen receptor 1 (ESR1; also known as ERα), is essential for normal female and male reproductive function. Historically, studies of estrogen action have focused on the classical genomic pathway. Although this is clearly the major pathway for steroid hormone actions, these hormones also signal through rapid non-classical effects involving cell membrane actions. Reports of rapid effects of estrogens extend for more than half a century, but recent results have expanded understanding of the identity, structure, function and overall importance of membrane receptors in estrogen responses. Key findings in this field were the immunohistochemical detection of ESR1 in cell membranes and demonstration that a portion of newly synthesized ESR1 is routed to the membrane by palmitoylation. These receptors in the membrane can then signal through protein kinases and other mechanisms following ligand binding to alter cell function. Another crucial advance in the field was development of transgenic mice expressing normal amounts of functional nuclear ESR1 (nESR1) but lacking membrane ESR1 (mESR1). Both male and female transgenic mice lacking mESR1 were infertile as adults, and both sexes had extensive reproductive abnormalities. Transgenic mice lacking mESR1 were highly protected from deleterious effects of neonatal estrogen administration, and estrogen effects on the histone methyltransferase Enhancer of Zeste homolog 2 that are mediated through mESR1 could have significant effects on epigenetic imprinting. In summary, signaling through mESR1 is essential for normal male and female reproductive function and fertility, and is a critical enabler of normal estrogen responses in vivo. Although the precise role of mESR1 in estrogen responses remains to be established, future research in this area should clarify its mechanism of action and lead to a better understanding of how mESR1 signaling works with classical genomic signaling through nESR1 to promote full estrogenic responses.

Hypothalamic miR-30 regulates puberty onset via repression of the puberty-suppressing factor, Mkrn3.

Mkrn3, the maternally imprinted gene encoding the makorin RING-finger protein-3, has recently emerged as putative pubertal repressor, as evidenced by central precocity caused by MKRN3 mutations in humans; yet, the molecular underpinnings of this key regulatory action remain largely unexplored. We report herein that the microRNA, miR-30, with three binding sites in a highly conserved region of its 3′ UTR, operates as repressor of Mkrn3 to control pubertal onset. Hypothalamic miR-30b expression increased, while Mkrn3 mRNA and protein content decreased, during rat postnatal maturation. Neonatal estrogen exposure, causing pubertal alterations, enhanced hypothalamic Mkrn3 and suppressed miR-30b expression in female rats. Functional in vitro analyses demonstrated a strong repressive action of miR-30b on Mkrn3 3′ UTR. Moreover, central infusion during the juvenile period of target site blockers, tailored to prevent miR-30 binding to Mkrn3 3′ UTR, reversed the prepubertal down-regulation of hypothalamic Mkrn3 protein and delayed female puberty. Collectively, our data unveil a novel hypothalamic miRNA pathway, involving miR-30, with a prominent role in the control of puberty via Mkrn3 repression. These findings expand our current understanding of the molecular basis of puberty and its disease states.

Neonatal Estrogen Causes Irreversible Male Infertility via Specific Suppressive Action on Hypothalamic Kiss1 Neurons.

Aberrant exposure to estrogen-like compounds during the critical developmental period may cause improper hypothalamic programming, thus resulting in reproductive dysfunction in adulthood in male mammals. Kisspeptin-neurokinin B-dynorphin A (KNDy) neurons in the arcuate nucleus (ARC) have been suggested to govern tonic GnRH/gonadotropin release to control reproduction in male mammals. In this study, we report that chronic exposure to supraphysiological levels of estrogen during the neonatal period caused an irreversible suppression of KNDy genes in the ARC, resulting in reproductive dysfunction in male rats. Daily estradiol benzoate (EB) administration from days 0 to 10 postpartum caused smaller seminiferous tubules, abnormal spermatogenesis, and a decrease in plasma testosterone in adult male rats. The neonatal EB treatment profoundly suppressed LH pulse and ARC KNDy gene expression at adulthood, but it failed to affect the number of GnRH gene-expressing cells in male rats. The EB treatment failed to affect gene expression of other neuropeptides, such as GHRH, proopiomelanocortin, and agouti-related protein in the ARC, suggesting that ARC KNDy neurons would be a specific target of neonatal estrogen to cause male reproductive dysfunction. Because LH secretory responses to kisspeptin challenge and GnRH expression were spared in male rats with the EB treatment, LH pulse suppression is most probably due to ARC KNDy deficiency. Taken together, the current study indicates that chronic exposure to estrogenic chemicals in the developing brain causes a defect of ARC KNDy neurons, resulting in an inhibition of pulsatile GnRH/LH release and the failure of spermatogenesis and steroidogenesis.

Differentiation Patterns of Uterine Carcinomas and Precursor Lesions Induced by Neonatal Estrogen Exposure in Mice.

Developmental exposure to estrogenic chemicals is an established risk factor for cancer of the female reproductive tract. This increase in risk has been associated with disruption of normal patterns of cellular differentiation during critical stages of morphogenesis. The goal of this study was to document uterine epithelial phenotypes over time following neonatal treatment with the synthetic estrogen diethylstilbestrol (DES) or the soy phytoestrogen genistein (GEN) in female CD-1 mice. Both DES and GEN induced three distinct populations of abnormal endometrial epithelial cells: luminal (SIX1+/P63-/CK14-/CK18+), basal (SIX1+/P63+/CK14+/CK18-), and mixed/bipotential (SIX1+/P63-/CK14+/CK18+), which were all established by early adulthood. In older animals, DES and GEN resulted in uterine carcinomas with mixed glandular, basal, and squamous cell elements. All carcinomas were composed largely of the three abnormal cell types. These findings identify novel epithelial differentiation patterns in the uterus and support the idea that disruption of cellular programming in early development can influence cancer risk later in life.

Proteomic changes during adult stage in pre-optic, hypothalamus, hippocampus and pituitary regions of female rat brain following neonatal exposure to estradiol-17β

Although neonatal exposure to estrogen or estrogenic compounds results in irreversible changes in the brain function and reproductive abnormalities during adulthood but the underlying mechanisms are still largely unknown. The present study has attempted to compare the protein profiles of sexually dimorphic brain regions of adult female rats which were exposed to estradiol- 17β during neonatal period. The total proteins extracted from pre-optic area (POA), hypothalamus, hippocampus and pituitary of control and neonatally E2 treated female rats was subjected to 2D-SDS-PAGE and differentially expressed proteins were identified by MALDI TOF/TOF-MS. Our results revealed that a total of 21 protein spots which were identified as differentially expressed in all the four regions analyzed; the differential expression was further validated by RT-PCR and western blotting. The differentially expressed proteins such as 14-3-3 zeta/delta (POA), LMNA (hippocampus), Axin2 (hypothalamus), Syntaxin-7 (hippocampus), prolactin and somatotropin (pituitary) which have very important functions in the process of neuronal differentiation, migration, axon outgrowth, formation of dendritic spine density and synaptic plasticity and memory have not been previously reported in association with neonatal estrogen exposure. The affected brain functions are very important for the establishment of sex specific brain morphology and behavior. Our results suggest that the differentially expressed proteins may play an important role in irreversible changes in the brain function as well as reproductive abnormalities observed in the female rats during adulthood.

Long-Term Neonatal Estrogen Exposure Causes Irreversible Inhibition of LH Pulses by Suppressing Arcuate Kisspeptin Expression via Estrogen Receptors α and β in Female Rodents.

Exposure to estrogen during the developmental period causes reproductive dysfunction in mammals, because the developing brain is highly sensitive to estrogens. In the present study, we report that long-term exposure to supraphysiological doses of estrogen during the neonatal critical period causes irreversible suppression of Kiss1/kisspeptin expression in the arcuate nucleus (ARC) via estrogen receptor-alpha (ERα) and ERβ, resulting in reproductive dysfunction in female rats. Daily estradiol-benzoate (EB) administration from days 0 to 10 postpartum caused persistent vaginal diestrus in female rats. The female rats showed profound suppression of pulsatile luteinizing hormone (LH) release and ARC Kiss1/kisspeptin expression even after ovariectomy at adulthood. In contrast, female rats treated with a single EB injection at day 5 postpartum exhibited persistent vaginal estrus and showed comparable LH pulses and numbers of ARC Kiss1-expressing cells to vehicle-treated controls after ovariectomy at adulthood. Because the LH secretory response to exogenous kisspeptin was spared in female rats with neonatal long-term estrogen exposure, the LH pulse suppression was most probably due to ARC kisspeptin deficiency. Furthermore, neonatal estrogen might act through both ERα and ERβ, because EB exposure significantly reduced the number of ARC Kiss1-expressing cells in wild-type mice but not in ERα or ERβ knockout mice. Taken together, long-term exposure to supraphysiological doses of estrogen in the developing brain might cause defects in ARC kisspeptin neurons via ERα and ERβ, resulting in inhibition of pulsatile LH release and lack of estrous cyclicity.

新生仔期のエストロジェン暴露による弓状核kisspeptin抑制を介したパルス状黄体形成ホルモン（LH）分泌不全メカニズム

新生仔期のエストロジェン暴露による弓状核kisspeptin抑制を介したパルス状黄体形成ホルモン（LH）分泌不全メカニズム

Testicular expression of the Lin28/let-7 system: Hormonal regulation and changes during postnatal maturation and after manipulations of puberty.

The Lin28/let-7 system, which includes the RNA-binding proteins, Lin28a/Lin28b, and let-7 miRNAs, has emerged as putative regulator of puberty and male gametogenesis; yet, its expression pattern and regulation in postnatal testis remain ill defined. We report herein expression profiles of Lin28 and let-7 members, and related mir-145 and mir-132, in rat testis during postnatal maturation and in models of altered puberty and hormonal deregulation. Neonatal expression of Lin28a and Lin28b was low and rose markedly during the infantile period; yet, expression patterns diverged thereafter, with persistently elevated levels only for Lin28b, which peaked at puberty. Let-7a, let-7b, mir-132 and mir-145 showed profiles opposite to Lin28b. In fact, let-7b and mir-145 were abundant in pachytene spermatocytes, but absent in elongating spermatids, where high expression of Lin28b was previously reported. Perturbation of puberty by neonatal estrogenization reverted the Lin28/let-7 expression ratio; expression changes were also detected in other models of delayed puberty, due to early photoperiod or nutritional manipulations. In addition, hypophysectomy or growth hormone (GH) deficiency revealed regulation of this system by gonadotropins and GH. Our data document the expression profiles of the Lin28/let-7 system in rat testis along postnatal/pubertal maturation, and their perturbation in models of pubertal and hormonal manipulation.

Developmental exposure to estrogen alters differentiation and epigenetic programming in a human fetal prostate xenograft model.

Prostate cancer is the most frequent non-cutaneous malignancy in men. There is strong evidence in rodents that neonatal estrogen exposure plays a role in the development of this disease. However, there is little information regarding the effects of estrogen in human fetal prostate tissue. This study explored early life estrogen exposure, with and without a secondary estrogen and testosterone treatment in a human fetal prostate xenograft model. Histopathological lesions, proliferation, and serum hormone levels were evaluated at 7, 30, 90, and 200-day time-points after xenografting. The expression of 40 key genes involved in prostatic glandular and stromal growth, cell-cycle progression, apoptosis, hormone receptors and tumor suppressors was evaluated using a custom PCR array. Epigenome-wide analysis of DNA methylation was performed on whole tissue, and laser capture-microdissection (LCM) isolated epithelial and stromal compartments of 200-day prostate xenografts. Combined initial plus secondary estrogenic exposures had the most severe tissue changes as revealed by the presence of hyperplastic glands at day 200. Gene expression changes corresponded with the cellular events in the KEGG prostate cancer pathway, indicating that initial plus secondary exposure to estrogen altered the PI3K-Akt signaling pathway, ultimately resulting in apoptosis inhibition and an increase in cell cycle progression. DNA methylation revealed that differentially methylated CpG sites significantly predominate in the stromal compartment as a result of estrogen-treatment, thereby providing new targets for future investigation. By using human fetal prostate tissue and eliminating the need for species extrapolation, this study provides novel insights into the gene expression and epigenetic effects related to prostate carcinogenesis following early life estrogen exposure.

Neonatal estrogen exposure results in biphasic age-dependent effects on the skeletal development of male mice.

Peak bone mass, one of the most important predictors for fracture risk later in life, is attained during puberty and adolescence and influenced by neonatal and pubertal sex-specific gonadal hormones and GH-IGF-I secretion patterns. This study examined the effects of brief neonatal estrogen (NE) exposure on growth and skeletal development in C57BL/6J mice. A single injection of 100-μg estradiol or vehicle was administered on the first day of life. Growth parameters were monitored and skeletal phenotyping performed at 16 weeks in female mice and at 4 and 16 weeks in male mice. NE exposure negatively impacted adult femoral length in both sexes, but adult body weight, areal bone density, and bone strength in female mice were unaffected. In contrast, somatic growth was attenuated in estrogen-exposed male mice throughout the study period. At the prepubertal time point, the estrogen-exposed males exhibited higher bone mineral density, cortical volume, and cortical thickness compared with controls. However, by the time of peak bone mass acquisition, the early skeletal findings had reversed; estrogen-exposed mice had lower bone density with reduced cross-sectional area, cortical volume, and cortical thickness, resulting in cortical bones that were less resistant to fracture. NE exposure also resulted in reduced testicular volume and lower circulating IGF-I. Male mice exposed to estrogen on the first day of life experience age-dependent changes in skeletal development. Prepubertal animals experience greater endocortical bone acquisition as a result of estrogen exposure. However, by adulthood, continued developmental changes result in overall reduced skeletal integrity.

Activated vitamin D3 and pro-activated vitamin D3 attenuate induction of permanent changes caused by neonatal estrogen exposure in the mouse vagina.

Exposure of mice to a high dose of estrogens including diethylstilbestrol (DES) during the neonatal period modifies the developmental plan of the genital tract, which leads to various permanent changes in physiology, morphology and gene expression. These changes include development of an abnormal vaginal epithelium lined with hyperplastic mucinous cells accompanied by Tff1 gene expression in mice. Here, the influence of vitamin D on the direct effect of estrogen on the developing mouse vagina was examined. The mid-vagina of neonatal mice was cultured in a serum-free medium containing estradiol-17β (E2) and various concentrations of 1,25-dihydroxyvitamin D3 (1,25(OH)2D) ex vivo and then was transplanted under the renal capsule of ovariectomized host mice for 35 days. Exposure to E2 alone caused the vaginal tissue to develop estrogen-independent epithelial hyperplasia and to express TFF1 mRNA, while addition of a low nanomolar amount of 1,25(OH)2D added at the same time as E2 to the culture medium attenuated the effects of estrogen. Expression of vitamin D receptor was also evident in the neonatal mouse vagina. Interestingly, addition of 25-hydroxyvitamin D3, a pro-activated form of vitamin D, at the micromolar level was found to be potent in disrupting the developmental effects of E2, while cholecalciferol was not at least at the dose examined. Correspondingly, expression of Cyp27B1, a kidney-specific 25-hydroxyvitamin D hydroxylase, was evident in the neonatal mouse vagina when examined by RT-PCR. In addition, simultaneous administration of 1,25(OH)2D successfully attenuated DES-induced ovary-independent hyperplasia in the vagina in neonatal mice in vivo. Thus, manipulation of vitamin D influenced the harmful effects of estrogens on mouse vaginal development.

Persistently Altered Epigenetic Marks in the Mouse Uterus After Neonatal Estrogen Exposure

Neonatal exposure to diethylstilbestrol (DES) causes permanent alterations in female reproductive tract gene expression, infertility, and uterine cancer in mice. To determine whether epigenetic mechanisms could explain these phenotypes, we first tested whether DES altered uterine expression of chromatin-modifying proteins. DES treatment significantly reduced expression of methylcytosine dioxygenase TET oncogene family, member 1 (TET1) on postnatal day 5; this decrease was correlated with a subtle decrease in DNA 5-hydroxymethylcytosine in adults. There were also significant reductions in histone methyltransferase enhancer of zeste homolog 2 (EZH2), histone lysine acetyltransferase 2A (KAT2A), and histone deacetylases HDAC1, HDAC2, and HDAC3. Uterine chromatin immunoprecipitation was used to analyze the locus-specific association of modified histones with 2 genes, lactoferrin (Ltf) and sine oculis homeobox 1 (Six1), which are permanently upregulated in adults after neonatal DES treatment. Three histone modifications associated with active transcription, histone H3 lysine 9 acetylation (H3K9ac), H3 lysine 4 trimethylation (H3K4me3), and H4 lysine 5 acetylation (H4K5ac) were enriched at specific Ltf promoter regions after DES treatment, but this enrichment was not maintained in adults. H3K9ac, H4K5ac, and H3K4me3 were enriched at Six1 exon 1 immediately after neonatal DES treatment. As adults, DES-treated mice had greater differences in H4K5ac and H3K4me3 occupancy at Six1 exon 1 and new differences in these histone marks at an upstream region. These findings indicate that neonatal DES exposure temporarily alters expression of multiple chromatin-modifying proteins and persistently alters epigenetic marks in the adult uterus at the Six1 locus, suggesting a mechanism for developmental exposures leading to altered reproductive function and increased cancer risk.

Neonatal estrogen treatment with β-estradiol 17-cypionate induces in post-pubertal mice inflammation in the ductuli efferentes, epididymis, and vas deferens, but not in the testis, provoking obstructive azoospermia

Neonatal estrogen treatment (NET) induces morphological changes in male reproductive organs. NET with β-estradiol 17-cypionate is reported to induce inflammation with stromal-epithelial abnormalities in the prostate and seminal vesicles in post-pubertal mice. The aim of this study was to investigate the histopathology of the testis, ductuli efferentes, epididymis, and vas deferens in mice after NET with β-estradiol 17-cypionate. No morphological changes in these organs were observed until 4weeks after NET. However, some inflammatory cells were found in epididymis and vas deferens 6weeks after NET. Eight weeks after NET, inflammatory cells spread to the ductuli efferentes and inflammation was severe from 6 to 12weeks after NET. Inflammatory cells were never seen in the whole testis, but cystic dilatation of the rete testes with spermatogenic disturbance was found around the mediastinum testis. Many inflammatory cells emigrated into the lumen of the epididymis, resulting in complete absence of spermatozoa in the vas deferens. Most of the inflammatory cells penetrating into the epithelial layers of epididymal ducts were neutrophils. These results indicate that in post-pubertal mice, NET with β-estradiol 17-cypionate induces inflammation in the ductuli efferentes, epididymis, and vas deferens, but not in the testis, provoking obstructive azoospermia.

The effects of gonadal steroid manipulation on the expression of Kiss1 mRNA in rat arcuate nucleus during postnatal development

Kisspeptins, encoded by Kiss1 gene, play pivotal roles in the regulation of reproduction. Recently, several studies reported a sex difference in Kiss1 expression in the arcuate nucleus (ARC) during the neonatal period. In this study, we investigated the effect of gonadal steroid manipulation on the sex difference in Kiss1 expression in ARC of rats. At neonatal and prepubertal stages, females had a greater number of Kiss1 neurons than the males. Gonadectomy at those stages resulted in significant increases in the Kiss1 neuron number and the sex differences disappeared. We also confirmed the expression of estrogen receptor α in kisspeptin neurons in neonates. Altogether, our results indicate that ARC Kiss1 expression is negatively regulated by gonadal steroids from early postnatal stages, and that the sex difference in ARC Kiss1 expression is attributed to the difference in circulating gonadal steroid levels. We also found that neonatal estrogenization inhibits Kiss1 expression and impairs negative feedback system.

Neurokinin B and the control of the gonadotropic axis in the rat: developmental changes, sexual dimorphism, and regulation by gonadal steroids.

Neurokinin B (NKB), encoded by Tac2 in rodents, and its receptor, NK3R, have recently emerged as important regulators of reproduction; NKB has been proposed to stimulate kisspeptin output onto GnRH neurons. Accordingly, NKB has been shown to induce gonadotropin release in several species; yet, null or even inhibitory effects of NKB have been also reported. The basis for these discrepant findings, as well as other key aspects of NKB function, remains unknown. We report here that in the rat, LH responses to the NK3R agonist, senktide, display a salient sexual dimorphism, with persistent stimulation in females, regardless of the stage of postnatal development, and lack of LH responses in males from puberty onward. Such dimorphism was independent of the predominant sex steroid after puberty, because testosterone administration to adult females failed to prevent LH responses to senktide, and LH responsiveness was not restored in adult males treated with estradiol or the nonaromatizable androgen, dihydrotestosterone. Yet, removal of sex steroids by gonadectomy switched senktide effects to inhibitory, both in adult male and female rats. Sexual dimorphism was also evident in the numbers of NKB-positive neurons in the arcuate nucleus (ARC), which were higher in adult female rats. This is likely the result of differences in sex steroid milieu during early periods of brain differentiation, because neonatal exposures to high doses of estrogen decreased ARC NKB neurons at later developmental stages. Likewise, neonatal estrogenization resulted in lower serum LH levels that were normalized by senktide administration. Finally, we document that the ability of estrogen to inhibit hypothalamic Tac2 expression seems region specific, because estrogen administration decreased Tac2 levels in the ARC but increased them in the lateral hypothalamus. Altogether, our data provide a deeper insight into relevant aspects of NKB function as major regulator of the gonadotropic axis in the rat, including maturational changes, sexual dimorphism, and differential regulation by sex steroids.

Probing the role of estrogen receptor isoforms in neonatal programming of neuroendocrine and behavioral functions

Sex differences in the activity of the hypothalamus-pituitary-adrenal (HPA) axis in rats are programmed by neonatal estrogens; exposure of female neonates to estradiol (E2) leads to overt defeminization of endocrine and behavioral functions in adulthood. E2 activates both estrogen receptor isoforms (ERa and ERb); these are widely expressed in the brain, and differentially regulate HPA axis activity in adulthood. However, the contributions of each ER isoform to the sex-specific organization of the neural mechanisms governing HPA axis function remain unknown. ERa, ERb agonists (PPT and DPN, respectively) or E2 were administered to female rats on days 1-10 of life. Animals subsequently underwent endocrine (HPA axis and reproductive) and behavioral profiling (anxiety-related and reproductive) in adulthood, and patterns of expression of relevant genes were monitored in limbic structures post mortem. Exposure of neonatal females to PPT or DPN led to distinctly different HPA secretory profiles, neither of which completely recapitulated the effects of E2. Thus, whereas impaired glucocorticoid negative feedback was the most prominent effect of PPT treatment, increased basal corticosterone secretion was the most obvious characteristic of DPN-treated animals. Behavioral analysis revealed higher anxiety levels in PPT-treated animals, similar to those observed in E2-treated female neonates and control males; in contrast, DPN treatment was associated with reduced anxiety-like behavior. Parallel treatment-specific alterations in the expression of the genes encoding mineralocorticoid (MR) and glucocorticoid (GR) receptors in the hippocampus and amygdala and altered expression of ERb mRNA in discrete brain regions, as well as disturbed ovarian activity, were also found; together, they suggest potential mechanisms that could account for the different endocrine and behavioral phenotypes observed. Defeminization of HPA axis activity and associated anxiety-related behavior depends on balanced activation of ERa and ERb during early postnatal life, rather than on the activation of a specific ER isoform. Long-term E2-, PPT- and DPN-induced alterations in the expression levels of GR and MR in the hippocampus and amygdala, as well as disrupted ovarian activity appear to be largely responsible for eliciting and maintaining the aberrant endocrine and behavioral phenotypes induced by estrogenization of neonatal females.