Nonclassical androgen and estrogen signaling is essential for normal spermatogenesis.

Signaling by androgens through androgen receptor (AR) is essential to complete spermatogenesis in the testis. Similarly, loss of the main estrogen receptor, estrogen receptor 1 (ESR1; also known as ERα), results in male infertility, due in part to indirect deleterious effects on the seminiferous epithelium and spermatogenesis. Effects of steroid hormones are induced primarily through genomic changes induced by hormone-mediated activation of their intracellular receptors and subsequent effects on nuclear gene transcription. However, androgens and estrogens also signal through rapid nonclassical pathways involving actions initiated at the cell membrane. Here we review the data that nonclassical androgen and estrogen signaling pathways support processes essential for male fertility in the testis and reproductive tract. The recent development of transgenic mice lacking nonclassical AR or ESR1 signaling but retaining genomic nuclear signaling has provided a powerful tool to elucidate the function of nonclassical signaling in the overall response to androgens and estrogens. Results from these mice have emphasized that nonclassical signaling is essential for full responses to these hormones, and absence of either nonclassical or classical AR or ESR1 pathways produces abnormalities in spermatogenesis and the male reproductive tract. Although additional work is required to fully understand how classical and nonclassical receptor signaling synergize to produce full steroid hormone responses, here we summarize the known physiological functions of the classical and nonclassical androgen and estrogen signaling pathways in the testis and reproductive tract.

Estrogens in Male Physiology.

Estrogens have historically been associated with female reproduction, but work over the last two decades established that estrogens and their main nuclear receptors (ESR1 and ESR2) and G protein-coupled estrogen receptor (GPER) also regulate male reproductive and nonreproductive organs. 17ß-Estradiol (E2) is measureable in blood of men and males of other species, but in rete testis fluids, E2 reaches concentrations normally found only in females and in some species nanomolar concentrations of estrone sulfate are found in semen. Aromatase, which converts androgens to estrogens, is expressed in Leydig cells, seminiferous epithelium, and other male organs. Early studies showed E2 binding in numerous male tissues, and ESR1 and ESR2 each show unique distributions and actions in males. Exogenous estrogen treatment produced male reproductive pathologies in laboratory animals and men, especially during development, and studies with transgenic mice with compromised estrogen signaling demonstrated an E2 role in normal male physiology. Efferent ductules and epididymal functions are dependent on estrogen signaling through ESR1, whose loss impaired ion transport and water reabsorption, resulting in abnormal sperm. Loss of ESR1 or aromatase also produces effects on nonreproductive targets such as brain, adipose, skeletal muscle, bone, cardiovascular, and immune tissues. Expression of GPER is extensive in male tracts, suggesting a possible role for E2 signaling through this receptor in male reproduction. Recent evidence also indicates that membrane ESR1 has critical roles in male reproduction. Thus estrogens are important physiological regulators in males, and future studies may reveal additional roles for estrogen signaling in various target tissues.

Exploring community insights on antimicrobial resistance in Nepal: a formative qualitative study.

BACKGROUND: Antimicrobial resistance (AMR) is the process by which microbes evolve mechanisms to survive the medicines designed to destroy them i.e. antimicrobials (AMs). Despite being a natural process, AMR is being hastened by the abuse of AMs. In context of Nepal, there is limited information on drivers of AMR and barriers in addressing it from a community perspective. This study explores the local language and terminology used around AMs in the community, commonly used AMs and reasons for their usage, how these AMs are sourced, and the perceived barriers to addressing AMR via One Health approach. METHODS: A phenomenological study design was utilized with applied qualitative research theoretically framed as pragmatism. Twelve in-depth interviews and informal discussions with a One Health focus, were purposively conducted with wide range of stakeholders and community resident of Kapilvastu municipality of Nepal during April 2022. The acquired data was analyzed manually via a thematic framework approach. The study obtained ethical approval from ethical review board of Nepal Health Research Council and University of Leeds. RESULTS: Nepali and Awadhi languages does not have specific words for AMs or AMR, which is understandable by the community people. Rather, community use full explanatory sentences. People use AMs but have incomplete knowledge about them and they have their own local words for these medicines. The knowledge and usage of AMs across human and animal health is impacted by socio-structural factors, limited Government regulation, inadequate supply of AMs in local government health facilities and the presence of various unregulated health providers that co-exist within the health system. Novel ideas such as the use of visual and smart technology, for instance mobile phones and social media exposure, can enable access to information about AMs and AMR. CONCLUSION: This study shows that terminology that is understandable by the community referring to AMs and AMR in Nepali and Awadhi languages does not exist, but full explanatory sentences and colloquial names are used. Despite regular utilisation, communities have incomplete knowledge regarding AMs. Since, knowledge alone cannot improve behaviour, behavioural interventions are required to address AMR via community engagement to co-produce their own solutions. TRIAL REGISTRATION: Not applicable.

Practitioner Review: Effectiveness and mechanisms of change in participatory arts-based programmes for promoting youth mental health and well-being - a systematic review.

BACKGROUND: Participatory arts-based (PAB) programmes refer to a diverse range of community programmes involving active engagement in the creation process that appear helpful to several aspects of children's and young people's (CYP) mental health and well-being. This mixed-methods systematic review synthesises evidence relating to the effectiveness and mechanisms of change in PAB programmes for youth. METHOD: Studies were identified following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses approach. Eleven electronic databases were searched for studies of PAB programmes conducted with CYP (aged 4-25 years), which reported mental health and well-being effectiveness outcomes and/or mechanisms of change. A mixed-methods appraisal tool assessed study quality. A narrative synthesis was conducted of effectiveness and challenges in capturing this. Findings relating to reported mechanisms of change were integrated via a metasummary. RESULTS: Twenty-two studies were included. Evidence of effectiveness from quantitative studies was limited by methodological issues. The metasummary identified mechanisms of change resonant with those proposed in talking therapies. Additionally, PAB programmes appear beneficial to CYP by fostering a therapeutic space characterised by subverting restrictive social rules, communitas that is not perceived as coercive, and inviting play and embodied understanding. CONCLUSIONS: There is good evidence that there are therapeutic processes in PAB programmes. There is a need for more transdisciplinary work to increase understanding of context-mechanism-outcome pathways, including the role played by different art stimuli and practices. Going forward, transdisciplinary teams are needed to quantify short- and long-term mental health and well-being outcomes and to investigate optimal programme durations in relation to population and need. Such teams would also be best placed to work on resolving inter-disciplinary methodological tensions.

Total Ankle Replacement Versus Arthrodesis for End-Stage Ankle Osteoarthritis: A Randomized Controlled Trial.

BACKGROUND: End-stage ankle osteoarthritis causes severe pain and disability. There are no randomized trials comparing the 2 main surgical treatments: total ankle replacement (TAR) and ankle fusion (AF). OBJECTIVE: To determine which treatment is superior in terms of clinical scores and adverse events. DESIGN: A multicenter, parallel-group, open-label randomized trial. (ISRCTN registry number: 60672307). SETTING: 17 National Health Service trusts across the United Kingdom. PATIENTS: Patients with end-stage ankle osteoarthritis, aged 50 to 85 years, and suitable for either procedure. INTERVENTION: Patients were randomly assigned to TAR or AF surgical treatment. MEASUREMENTS: The primary outcome was change in Manchester-Oxford Foot Questionnaire walking/standing (MOXFQ-W/S) domain scores between baseline and 52 weeks after surgery. No blinding was possible. RESULTS: Between 6 March 2015 and 10 January 2019, a total of 303 patients were randomly assigned; mean age was 68 years, and 71% were men. Twenty-one patients withdrew before surgery, and 281 clinical scores were analyzed. At 52 weeks, the mean MOXFQ-W/S scores improved for both groups. The adjusted difference in the change in MOXFQ-W/S scores from baseline was -5.6 (95% CI, -12.5 to 1.4), showing that TAR improved more than AF, but the difference was not considered clinically or statistically significant. The number of adverse events was similar between groups (109 vs. 104), but there were more wound healing issues in the TAR group and more thromboembolic events and nonunion in the AF group. The symptomatic nonunion rate for AF was 7%. A post hoc analysis suggested superiority of fixed-bearing TAR over AF (-11.1 [CI, -19.3 to -2.9]). LIMITATION: Only 52-week data; pragmatic design creates heterogeneity of implants and surgical techniques. CONCLUSION: Both TAR and AF improve MOXFQ-W/S and had similar clinical scores and number of harms. Total ankle replacement had greater wound healing complications and nerve injuries, whereas AF had greater thromboembolism and nonunion, with a symptomatic nonunion rate of 7%. PRIMARY FUNDING SOURCE: National Institute for Health and Care Research Heath Technology Assessment Programme.

Functions of Steroid Hormones in the Male Reproductive Tract as Revealed by Mouse Models.

Steroid hormones are capable of diffusing through cell membranes to bind with intracellular receptors to regulate numerous physiological processes. Three classes of steroid hormones, namely androgens, estrogens and glucocorticoids, contribute to the development of the reproductive system and the maintenance of fertility. During the past 30 years, mouse models have been produced in which the expression of genes encoding steroid hormone receptors has been enhanced, partially compromised or eliminated. These mouse models have revealed many of the physiological processes regulated by androgens, estrogens and to a more limited extent glucocorticoids in the testis and male accessory organs. In this review, advances provided by mouse models that have facilitated a better understanding of the molecular regulation of testis and reproductive tract processes by steroid hormones are discussed.

Multiple Lesions Contribute to Infertility in Males Lacking Autoimmune Regulator.

Male factors, including those of autoimmune origin, contribute to approximately 50% of infertility cases in humans. However, the mechanisms underlying autoimmune male infertility are poorly understood. Deficiency in autoimmune regulator (AIRE) impairs central immune tolerance because of diminished expression of self-antigens in the thymus. Humans with AIRE mutations and mice with engineered ablation of Aire develop multiorgan autoimmunity and infertility. To determine the immune targets contributing to infertility in male Aire-deficient (-/-) mice, Aire-/- or wild-type (WT) males were paired with WT females. Aire-/- males exhibited dramatically reduced mating frequency and fertility, hypogonadism, and reduced serum testosterone. Approximately 15% of mice exhibited lymphocytic infiltration into the testis, accompanied by atrophy, azoospermia, and reduced numbers of mitotically active germ cells; the remaining mice showed normal testicular morphology, sperm counts, and motility. However, spermatozoa from all Aire-/- mice were defective in their ability to fertilize WT oocytes in vitro. Lymphocytic infiltration into the epididymis, seminal vesicle, and prostate gland was evident. Aire-/- male mice generated autoreactive antibodies in an age-dependent manner against sperm, testis, epididymis, prostate gland, and seminal vesicle. Finally, expression of Aire was evident in the seminiferous epithelium in an age-dependent manner, as well as in the prostate gland. These findings suggest that Aire-dependent central tolerance plays a critical role in maintaining male fertility by stemming autoimmunity against multiple reproductive targets.

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.

Cornification and classical versus nonclassical androgen receptor signaling in mouse penile/preputial development.

Mouse penile development is androgen-dependent. During development of male and female external genitalia, an internal ectodermal epithelial structure forms called the preputial lamina. At puberty the male preputial lamina canalizes to create the preputial space, effectively splitting into two layers: (a) the epithelial lining of the prepuce and (b) the surface epithelium of the penis. The female preputial lamina does not canalize, and instead remodels into the inverted U-shaped clitoral lamina of the adult female mouse. Androgen-dependent penile development was studied in transgenic mice with pathway-selective AR mutant transgenes through which AR signaling was activated either via the classical (AR-C) or the nonclassical pathway (AR-NC). Penile development and canalization of the preputial lamina was observed in AR-C and wild-type male mice naturally having both AR-C and AR-NC pathways. Conversely, clitoral development occurred in AR null (lacking both AR-C and AR-NC pathways) and AR-NC mice. The process of canalization of the preputial lamina seen in wild-type, AR-C and AR-C/AR-NC male mice involved cornification of the preputial lamina which involved up-regulation of keratin 10 and loricrin. Such up-regulation of these epidermal proteins was absent in the developing and adult clitoral lamina seen in wild-type female mice and AR-NC and AR null male (XY) mice. Thus, signaling through AR-C is sufficient to initiate and promote penile development and canalization of the preputial lamina, a process involving epithelial cornification.

Spatial transcriptomics analysis of uterine gene expression in enhancer of zeste homolog 2 conditional knockout mice†.

Histone proteins undergo various modifications that alter chromatin structure, including addition of methyl groups. Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that methylates lysine residue 27, and thereby suppresses gene expression. EZH2 plays integral roles in the uterus and other reproductive organs. We have previously shown that conditional deletion of uterine EZH2 results in increased proliferation of luminal and glandular epithelial cells, and RNA-seq analyses reveal several uterine transcriptomic changes in Ezh2 conditional (c) knockout (KO) mice that can affect estrogen signaling pathways. To pinpoint the origin of such gene expression changes, we used the recently developed spatial transcriptomics (ST) method with the hypotheses that Ezh2cKO mice would predominantly demonstrate changes in epithelial cells and/or ablation of this gene would disrupt normal epithelial/stromal gene expression patterns. Uteri were collected from ovariectomized adult WT and Ezh2cKO mice and analyzed by ST. Asb4, Cxcl14, Dio2, and Igfbp5 were increased, Sult1d1, Mt3, and Lcn2 were reduced in Ezh2cKO uterine epithelium vs. WT epithelium. For Ezh2cKO uterine stroma, differentially expressed key hub genes included Cald1, Fbln1, Myh11, Acta2, and Tagln. Conditional loss of uterine Ezh2 also appears to shift the balance of gene expression profiles in epithelial vs. stromal tissue toward uterine epithelial cell and gland development and proliferation, consistent with uterine gland hyperplasia in these mice. Current findings provide further insight into how EZH2 may selectively affect uterine epithelial and stromal compartments. Additionally, these transcriptome data might provide mechanistic understanding and valuable biomarkers for human endometrial disorders with epigenetic underpinnings.

Androgen-independent events in penile development in humans and animals.

The common view on penile development is that it is androgen-dependent, based first and foremost on the fact that the genital tubercle forms a penis in males and a clitoris in females. However, critical examination of the complex processes involved in human penile development reveals that many individual steps in development of the genital tubercle are common to both males and females, and thus can be interpreted as androgen-independent. For certain developmental events this conclusion is bolstered by observations in androgen-insensitive patients and androgen receptor mutant mice. Events in genital tubercle development that are common to human males and females include: formation of (a) the genital tubercle, (b) the urethral plate, (c) the urethral groove, (d) the glans, (e) the prepuce and (f) the corporal body. For humans 6 of 13 individual developmental steps in penile development were interpreted as androgen-independent. For mice 5 of 11 individual developmental steps were found to be androgen-independent, which were verified through analysis of androgen-insensitive mutants. Observations from development of external genitalia of other species (moles and spotted hyena) provide further examples of androgen-independent events in penile development. These observations support the counter-intuitive idea that penile development involves both androgen-independent and androgen-dependent processes.

Mice lacking uterine enhancer of zeste homolog 2 have transcriptomic changes associated with uterine epithelial proliferation.

Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that suppresses gene expression. Previously, we developed a conditional null model where EZH2 is knocked out in uterus. Deletion of uterine EZH2 increased proliferation of luminal and glandular epithelial cells. Herein, we used RNA-Seq in wild-type (WT) and EZH2 conditional knockout (Ezh2cKO) uteri to obtain mechanistic insights into the gene expression changes that underpin the pathogenesis observed in these mice. Ovariectomized adult Ezh2cKO mice were treated with vehicle (V) or 17ß-estradiol (E2; 1 ng/g). Uteri were collected at postnatal day (PND) 75 for RNA-Seq or immunostaining for epithelial proliferation. Weighted gene coexpression network analysis was used to link uterine gene expression patterns and epithelial proliferation. In V-treated mice, 88 transcripts were differentially expressed (DEG) in Ezh2cKO mice, and Bmp5, Crabp2, Lgr5, and Sprr2f were upregulated. E2 treatment resulted in 40 DEG with Krt5, Krt15, Olig3, Crabp1, and Serpinb7 upregulated in Ezh2cKO compared with control mice. Transcript analysis relative to proliferation rates revealed two module eigengenes correlated with epithelial proliferation in WT V vs. Ezh2cKO V and WT E2 vs. Ezh2cKO E2 mice, with a positive relationship in the former and inverse in the latter. Notably, the ESR1, Wnt, and Hippo signaling pathways were among those functionally enriched in Ezh2cKO females. Current results reveal unique gene expression patterns in Ezh2cKO uterus and provide insight into how loss of this critical epigenetic regulator assumingly contributes to uterine abnormalities.

Mice lacking membrane estrogen receptor 1 are protected from reproductive pathologies resulting from developmental estrogen exposure†.

Both membrane and nuclear fractions of estrogen receptor 1 (ESR1) mediate 17ß-estradiol (E2) actions. Mice expressing nuclear (n)ESR1 but lacking membrane (m)ESR1 (nuclear-only estrogen receptor 1 [NOER] mice) show reduced E2 responsivity and reproductive abnormalities culminating in adult male and female infertility. Using this model, we investigated whether reproductive pathologies caused by the synthetic estrogen diethylstilbestrol (DES) are mitigated by mESR1 ablation. Homozygous and heterozygous wild-type (WT and HET, respectively) and NOER male and female mice were subcutaneously injected with DES (1 mg/kg body weight [BW]) or vehicle daily from postnatal day (PND) 1-5. Uterine histology was assessed in select DES-treated females at PND 5, whereas others were ovariectomized at PND 60 and treated with E2 (10 µg/kg BW) or vehicle 2 weeks later. Neonatal DES exposure resulted in ovary-independent epithelial proliferation in the vagina and uterus of WT but not NOER females. Neonatal DES treatment also induced ovary-independent adult expression of classical E2-induced transcripts (e.g., lactoferrin [Ltf] and enhancer of zeste homolog 2 [Ezh2]) in WT but not NOER mice. At PND 90, DES-treated WT and HET males showed smaller testes and a high incidence of bacterial pyogranulomatous inflammation encompassing the testes, epididymis and occasionally the ductus deferens with spread to lumbar lymph nodes; such changes were largely absent in NOER males. Results indicate that male and female NOER mice are protected from deleterious effects of neonatal DES, and thus mESR1 signaling is required for adult manifestation of DES-induced reproductive pathologies in both sexes.

The histone methyltransferase EZH2 is required for normal uterine development and function in mice†.

Enhancer of zeste homolog 2 (EZH2) is a rate-limiting catalytic subunit of a histone methyltransferase, polycomb repressive complex, which silences gene activity through the repressive histone mark H3K27me3. EZH2 is critical for epigenetic effects of early estrogen treatment, and may be involved in uterine development and pathologies. We investigated EZH2 expression, regulation, and its role in uterine development/function. Uterine epithelial EZH2 expression was associated with proliferation and was high neonatally then declined by weaning. Pre-weaning uterine EZH2 expression was comparable in wild-type and estrogen receptor 1 knockout mice, showing neonatal EZH2 expression is ESR1 independent. Epithelial EZH2 was upregulated by 17ß-estradiol (E2) and inhibited by progesterone in adult uteri from ovariectomized mice. To investigate the uterine role of EZH2, we developed a EZH2 conditional knockout (Ezh2cKO) mouse using a cre recombinase driven by the progesterone receptor (Pgr) promoter that produced Ezh2cKO mice lacking EZH2 in Pgr-expressing tissues (e.g. uterus, mammary glands). In Ezh2cKO uteri, EZH2 was deleted neonatally. These uteri had reduced H3K27me3, were larger than WT, and showed adult cystic endometrial hyperplasia. Ovary-independent uterine epithelial proliferation and increased numbers of highly proliferative uterine glands were seen in adult Ezh2cKO mice. Female Ezh2cKO mice were initially subfertile, and then became infertile by 9 months. Mammary gland development in Ezh2cKO mice was inhibited. In summary, uterine EZH2 expression is developmentally and hormonally regulated, and its loss causes aberrant uterine epithelial proliferation, uterine hypertrophy, and cystic endometrial hyperplasia, indicating a critical role in uterine development and function.

Tissue interactions and estrogenic response during human female fetal reproductive tract development.

The role of tissue interactions was explored to determine whether epithelial differentiation within the developing human reproductive tract is induced and specified by mesenchyme in tissue recombinants composed of mouse vaginal mesenchyme + human uterine tubal epithelium (mVgM+hTubE). The tissue recombinants were grown in DES-treated ovariectomized athymic mice. After 2-4 weeks of in vivo growth, several vaginal specific features were expressed in the human tubal epithelium. The mesenchyme-induced effects included morphological change as well as expression of several immunohistochemical markers. Although the mesenchyme-induced shift in vaginal differentiation in the human tubal epithelium was not complete, the partial induction of vaginal markers in human tubal epithelium verifies the importance of mesenchymal-epithelial interactions in development of the human female reproductive tract. In a separate experiment, DES-induction of uterine epithelial progesterone receptor (PGR) and estrogen receptor 1 (ESR1) was explored in tissue recombinants composed of wild-type or Esr1KO mouse uterine mesenchyme + human fetal uterine epithelium (wt UtM+hUtE and Esr1KO UtM+hUtE). The rationale of this experiment was to determine whether DES-induction of PGR and ESR1 is mediated directly via epithelial ESR1 or indirectly (paracrine mechanism) via mesenchymal ESR1. DES-induction of uterine epithelial ESR1 and PGR in Esr1KO UtM+hUtE tissue recombinants (devoid of mesenchymal ESR1) formally eliminates the paracrine mechanism and demonstrates that DES induction of human uterine epithelial ESR1 and PGR is directly mediated via epithelial ESR1.

Estrogen in the male: a historical perspective.

Estrogens have traditionally been considered female hormones. Nevertheless, the presence of estrogen in males has been known for over 90 years. Initial studies suggested that estrogen was deleterious to male reproduction because exogenous treatments induced developmental abnormalities. However, demonstrations of estrogen synthesis in the testis and high concentrations of 17ß-estradiol in rete testis fluid suggested that the female hormone might have a function in normal male reproduction. Identification of estrogen receptors and development of biological radioisotope methods to assess estradiol binding revealed that the male reproductive tract expresses estrogen receptor extensively from the neonatal period to adulthood. This indicated a role for estrogens in normal development, especially in efferent ductules, whose epithelium is the first in the male reproductive tract to express estrogen receptor during development and a site of exceedingly high expression. In the 1990s, a paradigm shift occurred in our understanding of estrogen function in the male, ushered in by knockout mouse models where estrogen production or expression of its receptors was not present. These knockout animals revealed that estrogen's main receptor (estrogen receptor 1 [ESR1]) is essential for male fertility and development of efferent ductules, epididymis, and prostate, and that loss of only the membrane fraction of ESR1 was sufficient to induce extensive male reproductive abnormalities and infertility. This review provides perspectives on the major discoveries and developments that led to our current knowledge of estrogen's importance in the male reproductive tract and shaped our evolving concept of estrogen's physiological role in the male.

Mouse hypospadias: A critical examination and definition.

Hypospadias is a common malformation whose etiology is based upon perturbation of normal penile development. The mouse has been previously used as a model of hypospadias, despite an unacceptably wide range of definitions for this malformation. The current paper presents objective criteria and a definition of mouse hypospadias. Accordingly, diethylstilbestrol (DES) induced penile malformations were examined at 60 days postnatal (P60) in mice treated with DES over the age range of 12 days embryonic to 20 days postnatal (E12-P20). DES-induced hypospadias involves malformation of the urethral meatus, which is most severe in DES E12-P10, DES P0-P10 and DES P5-P15 groups, and less so or absent in the other treatment groups. A frenulum-like ventral tether between the penis and the prepuce was seen in the most severely affected DES-treated mice. Internal penile morphology was also altered in the DES E12-P10, DES P0-P10 and DES P5-P15 groups (with little effect in the other DES treatment groups). Thus, adverse effects of DES are a function of the period of DES treatment and most severe in the P0-P10 period. In "estrogen mutant mice" (NERKI, ßERKO, αERKO and AROM+) hypospadias was only seen in AROM+ male mice having genetically-engineered elevation is serum estrogen. Significantly, mouse hypospadias was only seen distally at and near the urethral meatus where epithelial fusion events are known to take place and never in the penile midshaft, where urethral formation occurs via an entirely different morphogenetic process.

Neonatal uterine and vaginal cell proliferation and adenogenesis are independent of estrogen receptor 1 (ESR1) in the mouse.

Neonatal uterus and vagina express estrogen receptor 1 (ESR1) and respond mitogenically to exogenous estrogens. However, neonatal ovariectomy does not inhibit preweaning uterine cell proliferation, indicating that this process is estrogen independent. Extensive literature suggests that ESR1 can be activated by growth factors in a ligand-independent manner and drive uterine cell proliferation. Alternatively, neonatal uterine cell proliferation could be ESR1 independent despite its obligatory role in adult luminal epithelial proliferation. To determine ESR1's role in uterine and vaginal development, we analyzed cell proliferation, apoptosis, and uterine gland development (adenogenesis) in wild-type (WT) and Esr1 knockout (Esr1KO) mice from Postnatal Day 2 to Postnatal Day 60. Uterine and vaginal cell proliferation, apoptosis, and uterine adenogenesis were comparable in WT and Esr1KO mice before weaning. By Days 29-60, glands had regressed, and uterine cell proliferation was reduced in Esr1KO mice in contrast to continued adenogenesis and proliferation in WT. Apoptosis in Esr1KO uterine epithelium was not increased compared to WT at any age, indicating that differences in cell proliferation, rather than apoptosis, cause divergence of uterine size in these two groups at puberty. Similarly, vaginal epithelial proliferation was reduced, and the epithelium became atrophic in Esr1KO mice by 29 days of age and later in Esr1KO mice. These results indicate that preweaning uterine and vaginal development is ESR1 independent but becomes dependent on ESR1 by Day 29 on. It is not yet clear what mechanisms drive preweaning vaginal and uterine development, but ligand-independent activation of ESR1 is not involved.

Maximal Dexamethasone Inhibition of Luminal Epithelial Proliferation Involves Progesterone Receptor (PR)- and Non-PR-Mediated Mechanisms in Neonatal Mouse Uterus.

Progesterone (P4) and the synthetic glucocorticoid dexamethasone (Dex) inhibit luminal epithelial (LE) proliferation in neonatal mouse uteri. This study determined the roles of progesterone receptor and estrogen receptor 1 (PR and ESR1, respectively) in P4- and Dex-induced inhibition of LE proliferation using PR knockout (PRKO) and Esr1 knockout (Esr1KO) mice. Wild-type (WT), heterozygous, and homozygous PRKO female pups were injected with vehicle, P4 (40 µg/g body weight), or Dex (4 or 40 µg/g body weight) on Postnatal Day 5, then 24 h later immunostained for markers of cell proliferation. In WT and heterozygous mice, P4 sharply reduced LE proliferation, and Dex produced dose-responsive decreases equaling those of P4 at the higher dose. Critically, although both doses of Dex similarly decreased proliferation compared to vehicle-treated PRKOs, treatment of PRKO pups with the high Dex dose (40 µg/g) did not inhibit LE as much as treatments of WT mice with this Dex dose or with P4. Stromal proliferation was stimulated by P4 in WT but not PRKO mice, and Dex did not alter stromal proliferation. Uteri of all genotypes strongly expressed glucocorticoid receptor (GR), demonstrating that impaired Dex effects in PRKOs did not reflect GR deficiency. Furthermore, inhibition of LE proliferation by Dex (40 µg/g body weight) in Esr1KO mice was normal, so this process does not involve ESR1. In summary, inhibitory Dex effects on LE proliferation occur partially through non-PR-mediated mechanisms, presumably GR, as indicated by Dex inhibition of LE proliferation in PRKOs. However, maximal inhibitory Dex effects on uterine LE proliferation are not seen in PRKO mice with even high Dex, indicating that maximal Dex effects in WT mice also involve PR.