Endocrinology

Female infertility remains a major women's reproductive health challenge, with impaired endometrial receptivity contributing to recurrent implantation failure, early pregnancy loss, and pregnancy complications. Mechanistic studies of human early pregnancy are limited by the lack of physiologically relevant models, and existing in vitro endometrial models rely on exogenous hormone treatment and do not recapitulate the dynamic ovarian control of endometrial growth and differentiation. Here, as a proof of concept, we developed a 3D in vitro ovarian follicle-endometrial organoid co-culture model. Immature murine follicles were cultured in vitro to obtain preovulatory follicles, followed by human chorionic gonadotropin (hCG) stimulation to induce ovulation and luteinization. Luteinized follicles were co-cultured with human endometrial stroma organoids for 6 days. The results showed that luteinized follicles generated a dynamic endocrine environment characterized by progressively increasing progesterone levels and declining estradiol levels. Follicle co-culture induced morphological differentiation and expression of decidualization markers in human endo-organoids. Transcriptomic analysis revealed strong genome-wide concordance between follicle co-culture and conventional decidualization induction using estradiol/progesterone/cAMP (EPC), with significant enrichment of decidualization-associated genes and pathways under both conditions. Nevertheless, the two conditions also exhibited distinct transcriptional states, with EPC preferentially enriched for more mature decidualization-associated programs, whereas luteinized follicle co-culture retained features of the early stage of decidualization. Functional perturbation testing revealed that the progesterone receptor (PR) antagonist RU486 attenuated co-culture-induced endometrial decidualization. Together, these results establish a new in vitro new approach methodology (NAM) that recapitulates ovarian control of endometrial decidualization. This platform provides a human-relevant NAM model to investigate the physiology and pathophysiology of endometrial decidualization, and to evaluate how drugs and environmental chemicals perturb uterine receptivity and early pregnancy.

Objectives: The bile acid nuclear receptor Farnesoid X Receptor (FXR, NR1H4) is a major regulator of metabolism and energy homeostasis in peripheral organs. It modulates bile acid, glucose, and lipid metabolism, as well as fat mass and body weight. However, FXR is also expressed in the brain, particularly in the hypothalamus, a key center for the regulation of energy homeostasis. Although one study has demonstrated a role for brain FXR activation in energy balance, its specific hypothalamic role is still unknown. Here, we examined the role of FXR in the mediobasal hypothalamus in the regulation of energy balance. Methods: We used a genetic approach combined with metabolic phenotyping to determine the effect of FXR invalidation in the mediobasal hypothalamus on metabolic parameters involved in the central regulation of energy homeostasis. Results: Our results demonstrate that hypothalamic FXR deficiency induces a positive energy balance, resulting in a reduction in energy expenditure due to alterations in glucose metabolism accompanied by structural changes in white adipose tissues. Conclusion: This study uncovers a previously unrecognized role for hypothalamic FXR in the central homeostatic control of energy balance, providing new insights into its contribution to peripheral glucose metabolism and adipose tissue structural remodeling.

Non-functioning gonadotroph (NFG) tumors are the most common type of non-functioning pituitary adenomas and can cause significant symptoms due to mass effect. However, the molecular programs underlying NFG tumor growth and their relationship to the normal anterior pituitary gland (APG) are poorly understood. To gain a deeper understanding of NFG tumor biology in the context of the normal APG, we performed single-cell/nucleus RNA-sequencing on 21 NFG tumors and 8 APG samples in human, generating the largest transcriptomic dataset of its kind. Single-cell/nucleus sequencing yielded 77,342, cells from APG samples and 152,649 cells from NFG tumor samples. Differential expression analysis within the APG identified novel marker genes of each neuroendocrine cell type and defined distinct transcriptional signatures of anterior and posterior pituitary stem cell populations. Comparison with tumor transcriptomes revealed that NFG tumor cells most closely resemble gonadotrophs, while also showing significant enrichment of thyrotroph and somatotroph markers. Notably, there was no significant overlap with stem cell markers, suggesting that NFG tumors most likely originate from differentiated gonadotrophs but can take on transcriptional characteristics of other neuroendocrine cell types. Pseudobulk profiling of NFG tumor cells demonstrated that tumor volume is significantly positively correlated with 83 genes including known oncogenes CAD, BRF2, and SOX12, along with 16 zinc finger transcription factors. Lastly, analysis of the tumor microenvironment revealed proportional increases in myeloid, endothelial, and mural cell populations in tumor samples compared to APG samples and highlighted cross talk between tumor and endothelial, mesenchymal, and immune populations via VEGF, PDGF, and MIF signaling pathways respectively. This study identifies novel transcriptomic signatures determining cell type identity in both APG neuroendocrine and NFG tumor cells. Our findings elucidate molecular programs driving NFG lineage infidelity and tumor growth, highlighting candidate predictors of patient outcomes and potential targets for therapeutic intervention.

This was a cohort study of 1,663 subjects (1196 women, 467 men; median age 48 years, IQR 37-57) with thyroid nodules recruited from 2020 to 2025 in five tertiary medical centers. Cytological/histopathological diagnoses were established by an independent review committee, and preoperative serum thyroid hormone metabolites (THMs) were tested using liquid chromatography tandem mass spectrometry. A diagnostic model on this serum THM test to distinguish between differentiated thyroid cancer (DTC) and benign thyroid nodule (BTN) was trained on 872 retrospective subjects (454 DTC and 418 BTN), followed, prospectively, by model validation on 392 subjects (195 DTC and 197 BTN) and a second validation on 399 subjects (300 DTC and 99 BTN). Following successful diagnostic model training, the model validation demonstrated a diagnostic specificity of 99.5% (196/197), sensitivity of 92.3% (180/195), positive predictive value (PPV) of 99.4%, negative predictive value (NPV) of 92.9%, and diagnostic accuracy of 95.9% (95% CI, 90.8 to 100.0%) for DTC. Combination of the two validation cohorts showed a diagnostic specificity of 99.7% (295/296), sensitivity of 92.1% (456/495), PPV of 99.8%, NPV of 88.3%, and diagnostic accuracy of 94.9% (95% CI, 91.5 to 98.0%) for DTC. The test on indeterminate Bethesda categories III-IV (139 DTC, 87 BTN) showed a diagnostic specificity of 100.0% (87/87), sensitivity of 93.5% (130/139), PPV of 100.0%, NPV of 90.6%, and diagnostic accuracy of 96.0% (95% CI, 89.2 to 100.0%) for DTC. This study develops a novel molecular blood test of serum THMs for preoperative diagnostic evaluation of thyroid nodules, which displays a high PPV and decent NPV with an excellent overall diagnostic accuracy for DTC.

Several different mechanisms of regeneration have been proposed to explain repair of the endometrial luminal epithelium (LE) following menstrual/estrous cyclicity, parturition or other damaging insults to the uterine mucosa. These include activity of an as-yet unidentified epithelial stem cell population, mesenchymal transdifferentiation, or integration of bone marrow-derived cells. Here, we used a novel Cag-Flox-STOP-Flox-Pgrmc2-3XFlag (Pgrmc2-3XFlag) reporter mouse coupled with a recently developed GE-specific cre recombinase mouse (Prss29-Cre) to determine if and to what extent the GE contributes to regeneration of the LE following parturition and during aging in nulliparous mice. To initiate regeneration in the endometrium, control (Pgrmc2-3XFlag) and reporter (Prss29-Cre;Pgrmc2-3XFlag) mice were placed with males for one pregnancy. Postpartum (pp) uterine tissues were then collected 5 days (1x5dpp), 3-weeks (1x3wpp) and 7-weeks (1x7wpp) later. A fourth set of uterine tissues was collected 3-weeks after three pregnancies (3x3wpp). The number of glands did not change at any time point. Indelibly marked FLAG+ GE cells were detected by immunohistochemistry and immunofluorescence in GE as expected. As demonstrated in serial uterine sections of uteri from all four treatment groups, GE cells migrated toward the LE and formed junctional points (JP) where GE cells transitioned from cuboidal GE cells to pseudostratified columnar LE cells. Transitioning cells at junctional points expressed markers of both GE (i.e., FOXA2) and LE (i.e., CALB1) tissues. Following transition, GE-derived LE cells expressed PGR and ESR1 consistent with surrounding developmentally-derived LE cells. About 17 FLAG+ LE cell islands were also observed in 1x5dpp 4 mm longitudinal sections at sites distant from GE-LE junctional points. Interestingly, the number of islands did not change from 1x5dpp to 1x7wpp. However, the total number of GE-derived FLAG+ LE cells observed in each island decreased from 1x5dpp to 1x3wpp, but then tended (p=0.07) to increase by 1x7wpp. There was no difference in the number of GE-LE junctional points or GE-derived FLAG+ LE islands between 1x3wpp and 3x3wpp tissues. However, a significant increase in the total (p=0.02) and average (p=0.02) number of FLAG+ LE cells was observed in 3x3wpp LE islands highlighting the proliferative expansion of these cells over the intervening ~80 days. GE-LE junctional points were also observed in 5-month old nulliparous Prss29-Cre;Pgrmc2-3XFlag mice at diestrus, but not in younger 6-week old mice. Collectively, these findings establish the GE as a quantifiable cellular source for LE regeneration during postpartum involution and as part of normal LE cell turnover during the estrous cycle in nulliparous mice. We propose a mechanism whereby GE cells within glandular structures are not terminally differentiated, but rather are stably differentiated to perform glandular function. However, when needed and based on the level of endometrial damage, GE cells can dedifferentiate, become integrated in to LE tissue and then reprogram to take on an LE phenotype as part of the regenerative process following parturition and during estrous cyclicity over time.

Conserved RNA binding proteins (RBPs) regulate key steps of gene expression including mRNA processing, export, localization, stability and translation. Human ZC3H14 is a conserved RBP that regulates pre-mRNA processing in neurons and loss of ZC3H14 leads to neurological defects. Studies of Nab2, the Drosophila orthologue of ZC3H14, have identified potential target RNAs involved in metabolism, suggesting Nab2 may influence neurometabolic circuitry. Here, we show a female-specific increase in dilp2 and dilp5 mRNA levels. The dilps encode insulin-like peptides that signal from the brain insulin producing cells (IPCs) to peripheral tissues. Nab2null females have enlarged lipid droplets in the fat body, a tissue analogous to human adipose tissue and liver. Notably, neuronal depletion of Nab2 increases lipid droplet size while neuronal expression of Nab2 in Nab2null female rescues this phenotype supporting a role for Nab2 in a neuronal circuit that regulates dilp levels. Furthermore, depletion of dilp2 or dilp5 from IPCs rescues the enlarged lipid droplet phenotype in Nab2null females indicating that elevated dilp2/dilp5 contributes to enlarged lipid droplets. Together, these data support a female-specific role for Nab2 in brain neurons to support insulin signaling and fat storage, expanding the known functions of RBPs linking neuronal function and metabolic homeostasis.

Prostate cancer (PC) remains a significant health concern as the second leading cause of cancer-related death in American men. Androgen deprivation therapies represent the standard of care for patients with PC, regardless of disease stage. Gonadotropin-releasing hormone (GnRH) antagonists, such as Degarelix, are commonly used hormone therapies for PC and work by suppressing testosterone production. Previous studies indicate a prostate involution-regeneration cycle following surgical castration and testosterone addback. However, whether a similar response occurs following pharmacologic androgen deprivation with agents such as Degarelix remains poorly understood. In particular, the temporal dynamics of prostate involution and subsequent regeneration after chemical castration have not been systematically defined. Our study utilizes immunocompetent mouse models to evaluate the systemic and temporal effects of Degarelix treatment and determine its efficacy in suppressing PC tumor growth. We show that continuous Degarelix treatment significantly suppressed systemic testosterone levels in mice, whereas testosterone levels returned to physiological baseline following Degarelix withdrawal. Gross anatomical, H&E, and Trichrome analyses demonstrated that Degarelix induced castration resulted in prostate involution, characterized by reduced gland size and increased stromal remodeling. This phenotype was reversed after Degarelix withdrawal, highlighting the reversibility of pharmacological androgen deprivation. Immunofluorescence staining revealed changes in basal and luminal epithelial cell populations within the prostatic gland, indicating changes in prostate glandular architecture following Degarelix induced castration and subsequent regeneration. Next, we evaluated the impact of chemical castration on PC tumor growth. Although Degarelix initially suppressed tumor growth, this response was not sustained through the study endpoint, suggesting the emergence of acquired resistance to androgen deprivation. These findings establish this immunocompetent model as a physiologically relevant platform for dissecting the mechanisms that drive therapeutic resistance and tumor progression following androgen deprivation. Our findings highlight the effectiveness of chemical castration with Degarelix and establish a robust model to study both therapeutic responses and the emergence of castration resistance in PC.

Functional ovarian plasticity depends on the granulosa cell (GC) fate determination, differentiation, oocyte support and steroid production. After birth, GC identity must be maintained in differentiating follicles, while from puberty, ovarian plasticity is cyclically challenged by gonadotropic hormones. TRIM28-FOXL2 interactions form a key chromatin component maintaining ovarian identity programs and repressing testis-specific genes. This TRIM28 role depends on its E3-SUMO-ligase activity, suggesting SUMOylation in GC can be regulated by transient hormonal cues, although hormonal involvement in modulating GC identity remains to be established. Here, we demonstrated the in vivo interplay between gonadotropic-PKA-signaling and hypo-SUMOylation waves underlying ovarian GC plasticity. In vitro TRIM28 mutagenesis and phospho-proteomics demonstrated direct PKA inhibitory control on TRIM28. This prompted us to highlight SUMOylation alterations in Carney Complex ovarian lesions resulting from chronic PKA activity from rare genetic mutations. Using mutant models, transcriptomics and TRIM28/FOXL2-ChIP-Seq analyses, we specified the in vivo PKA inhibitory mechanisms on TRIM28/FOXL2 chromatin binding and its consequences on adult ovarian somatic cell identity. Together, our data support an integrative model where gonadotrophin pulses create transient windows of PKA-mediated TRIM28 phosphorylation, hypo-SUMOylation, and FOXL2-TRIM28 destabilization. This positions SUMOylation dynamics as central integrator of hormonal signaling and epigenetic identity maintenance in the ovary.

Prenatal alcohol exposure (PAE) can produce persistent alterations in mammary development and increase susceptibility to mammary tumorigenesis, but the early molecular events underlying these effects remain poorly understood. Here, we examined whether PAE alters the molecular program of the mammary gland at birth. Pregnant MMTV-ErbB2 mice received control or ethanol-containing liquid diets during gestation, and mammary tissues were collected from newborn female offspring. RNA sequencing revealed exposure-level-dependent transcriptional reprogramming, with substantially broader alterations following moderate compared with lower ethanol exposure. Pathway analyses identified prominent changes in metabolic and growth-regulatory networks, including insulin signaling, with coordinated alterations in components of the insulin/IGF-IRS-PI3K/AKT pathway. Biochemical analyses further demonstrated exposure-dependent changes in IGFBP expression and IRS1 abundance and phosphorylation, supporting remodeling of insulin/IGF-associated signaling. Transcriptomic analyses also revealed enrichment of RNA metabolic and translational processes and, particularly following moderate exposure, enhanced DNA-replication, cell-cycle, and mitotic programs. These molecular signatures were accompanied by increased proliferative features in newborn mammary tissue. Together, these findings demonstrate that PAE establishes substantial molecular reprogramming of the mammary gland by birth and identify altered insulin/metabolic signaling and proliferative programming as prominent features of this early developmental response.

Progesterone governs the establishment and maintenance of pregnancy in ruminants, acting through the nuclear progesterone receptor (PGR) and, potentially, through membrane progesterone receptors (mPRs) of the PAQR family. The cellular distribution of mPRs in ruminant reproductive tissues, however, remains undefined. We generated and validated rabbit polyclonal antibodies against the intracellular C-terminal peptides of bovine PAQR6 (mPR{delta}) and PAQR8 (mPR{beta}), and used them, with quantitative PCR of the uterus and conceptus, to map these receptors in the ovine uterus and conceptus and in the bovine ovary. Both antisera showed high ELISA titer against their immunizing peptides, negligible preimmune reactivity, and complete loss of tissue signal after peptide pre-absorption. By qPCR, PAQR6, PAQR8 and PGR transcript levels did not change significantly across the peri-implantation period in either the conceptus or the endometrium; in contrast, the progesterone-responsive gene ZBTB16 increased markedly in the conceptus (up to ~45-fold by day 21, p < 0.05 at days 17, 19 and 21), whereas FKBP5 showed a numerical increase without reaching statistical significance. Both genes remained relatively stable in the endometrium, indicating conceptus-specific changes in progesterone-responsive gene expression. At the protein level, PAQR6 and PAQR8 localized to the luminal and glandular epithelium of the endometrium across the cycle and early pregnancy, and to follicular (granulosa) and luteal cells of the ovary. Dual immunofluorescence in the ovine endometrium showed that both PAQR6 and PAQR8 are enriched at the apical surface of the luminal epithelium and are spatially distinct from the basolateral adherens-junction marker E-cadherin. These results provide a protein-level map of PAQR6 and PAQR8 in the ruminant reproductive tract, identify the apical epithelial surface as a site of receptor enrichment, and show that the endometrial epithelium retains membrane progesterone receptors while progesterone-responsive gene expression changes dynamically in the conceptus during the peri-implantation period. The validated ruminant-reactive antibodies provide a resource for future functional studies.

Aims/hypothesis Fasting plasma uridine concentrations are elevated in obese mice and in individuals with type 2 diabetes (T2D), suggesting that uridine may function as a metabolic signal that adjusts insulin secretion to changes in metabolic demand. Here, we tested this hypothesis by determining whether uridine potentiates glucose-stimulated insulin secretion (GSIS) in mouse and human islets and by identifying uridine phosphorylase 1 (UPP1), the principal enzyme responsible for systemic uridine catabolism, as a key mediator of this effect. Methods Whole-body Upp1 knockout (KO) and {beta}-cell-specific Upp1 knockout (KO{beta}) mice were used to determine the requirement for UPP1 in uridine-potentiated glucose-stimulated insulin secretion (GSIS) and glucose-stimulated MAPK activation. Leptin-deficient and diet-induced obese mouse models were used to assess the impact of obesity on uridine responsiveness. UPP1 protein abundance and subcellular localization were examined in islets from obese mice and human donors spanning a range of glycemic control. Integrated single-nucleus RNA sequencing and ATAC sequencing were performed on wild-type and Upp1 KO mouse islets to characterize {beta}-cell molecular remodeling associated with UPP1 deficiency and altered responses to uridine. Results Uridine potentiated GSIS in islets from chow-fed and high-fat diet-fed mice, but not in leptin-deficient obese mice. Uridine also potentiated GSIS in human islets, although this effect progressively declined with worsening glycemic control. In mouse islets, uridine potentiation was accompanied by transient activation of the MAPK pathway. Both uridine-potentiated GSIS and uridine-enhanced glucose-stimulated MAPK activation were abolished in islets from Upp1 KO and KO{beta} mice. The requirement for a transient MAPK signaling was further supported by pharmacological inhibition of MEK and constitutive activation of MEK1. Leptin-deficient islets exhibited impaired glucose-induced MAPK activation together with increased UPP1 protein expression. In human islets, UPP1 protein abundance positively correlated with donor HbA1c and displayed heterogeneous subcellular localization. Integrated single-nucleus multiomic analysis identified UPP1 as an important regulator of {beta}-cell identity, function, and transcriptional responses to uridine. Conclusions These findings establish UPP1 as a key regulator linking pyrimidine metabolism to {beta}-cell function and identify impaired uridine responsiveness as a potential contributor to and/or consequence of {beta}-cell dysfunction during the progression of T2D.

Uterine leiomyomas, commonly known as fibroids, are benign tumors of the female reproductive tract. They are the most common tumor of the female reproductive system and the leading cause of hysterectomy in the United States. Progesterone, estrogen, and their receptors are important drivers for the development and growth of leiomyomas. How estrogen drives leiomyoma growth remains unclear. We have previously shown that simvastatin can inhibit leiomyoma growth by modulating intracellular calcium. This suggests that calcium signaling is important for leiomyoma growth. We therefore hypothesized that estrogen may drive leiomyoma growth by stimulating calcium release through the membrane estrogen receptor GPR30 (also known as GPER). We found that estrogen dose-dependently induced calcium release in leiomyoma cells. The GPR30-specific agonist G-1 also induced calcium release, indicating that GPR30 mediates estrogen-mediated calcium release. The GPR30 antagonist G-15 blocked estrogen-induced calcium release and significantly reduced leiomyoma proliferation. Our results demonstrate that GPR30 is a major driver of estrogen-dependent growth in leiomyoma cells by modulating intracellular calcium. These findings may have high therapeutic relevance to leiomyomas and other estrogen-dependent tumors.

The success of modern antiretroviral therapy (ART) has increased the life expectancy of people living with HIV to levels approaching that of uninfected individuals. For women living with HIV (WLWH), this means that more will survive to undergo menopause and experience the consequences of decreased ovarian hormone levels, particularly estrogen (E2). The recent change in federal guidance for use of postmenopausal hormone therapy is increasing demand for both E2-alone and E2+progestogen formulations to control adverse symptoms of menopause. The consequences and efficacy of hormone therapy in WLWH are thus an important issue for WLWH and their healthcare providers. The role of E2 replacement in postmenopausal WLWH is a significant issue because of its potential effects on control the viral reservoir and its demonstrated beneficial metabolic effects in uninfected postmenopausal women. To address these questions, we employed a novel nonhuman primate model of postmenopausal WLWH undergoing E2 replacement. Reproductively competent female rhesus macaques were infected with simian immunodeficiency virus (SIV) and then subjected to a daily ART regimen. After complete suppression of plasma viremia, all animals were ovariectomized (OVX) and then implanted with Silastic capsules containing either cholesterol vehicle or sufficient E2 to restore pre-OVX plasma levels. Plasma and cell-associated viral dynamics, immune responses, body composition, systemic and tissue-specific metabolic parameters, cytokine profiles, and parameters of bone health were followed longitudinally from baseline through 34 weeks of E2 deficiency or replacement. We found that E2 status did not significantly affect plasma or tissue viral dynamics or overall metabolic homeostasis. However, E2 replacement exerted beneficial effects on several aspects of bone health in spite of a chronic inflammatory state that persisted following effective ART suppression of the SIV reservoir. Our findings suggest that hormone therapy, specifically E2 replacement, offers benefit to WLWH, particularly with respect to bone loss.

Progesterone is essential for the establishment and maintenance of pregnancy, primarily through the classical nuclear progesterone receptor (PGR). However, progesterone also exerts rapid, non-genomic effects through membrane progesterone receptors, including members of the progestin and adipoQ receptor (PAQR) family, whose cellular distribution and potential roles at the human maternal-fetal interface remain poorly understood. We therefore sought to define the cell-type-specific expression landscape of classical and membrane progesterone receptors during early human pregnancy. We analyzed publicly available single-cell RNA-sequencing datasets of the first-trimester human maternal-fetal interface to characterize the expression of PGR and PAQR family members across trophoblast, immune, stromal, and vascular cell populations. Major expression patterns were examined across independent datasets and further assessed using spatial transcriptomic data. PGR expression was predominantly localized to decidual stromal and perivascular populations, whereas individual PAQR family members showed distinct cellular distributions. PAQR6 was enriched in decidual natural killer cells, PAQR7 was broadly expressed in trophoblast populations, PAQR8 was prominently expressed in fetal stromal and macrophage populations, including Hofbauer cells, and PAQR9 showed prominent enrichment in syncytiotrophoblasts. Selected cell-type-specific patterns were further supported by spatial transcriptomic analysis. These findings provide a cell-type-resolved framework for investigating how distinct progesterone receptor systems mediate progesterone signaling across maternal and fetal cellular compartments during early human placentation.

In utero alcohol exposure (IUE) can alter mammary development and increase mammary tumor susceptibility in experimental models. However, most previous studies have relied on carcinogen-induced or genetically tumor-prone models, leaving it unresolved whether prenatal alcohol exposure produces mammary developmental and molecular changes before the introduction of an oncogenic challenge. Here, we addressed this question using non-tumorigenic C57BL/6 mice. Pregnant dams received a Lieber-DeCarli liquid diet containing 1.7% (v/v) ethanol or an isocaloric control diet from gestational day 11 to 19, and mammary glands from female offspring were examined during puberty (postnatal day 40, PND40) and young adulthood (PND70). At PND40, IUE enhanced mammary ductal morphogenesis, increased terminal end bud numbers, and elevated epithelial proliferation. These developmental alterations persisted at PND70, with increased lateral branching and proliferative activity. IUE also altered mammary epithelial composition, expanding the basal/myoepithelial compartment and the CD24highCD49fhigh mammary repopulating unit/stem cell-enriched population. Mammary epithelial cells from IUE-exposed offspring exhibited increased colony-forming cells (CFCs), mammosphere formation, and 3D Matrigel growth, indicating enhanced stem/progenitor-associated activity. At the molecular level, IUE increased ER; expression and phosphorylation together with Cyclin D1, c-Myc, E2F1, and Bcl-2, and enhanced AKT, ERK, and STAT3 phosphorylation, IL-6 expression, and SOX2 at PND40. Importantly, major components of this signaling phenotype remained altered at PND70, including ER;-associated signaling, AKT/ERK activation, and IL-6/STAT3 signaling, whereas the pubertal increase in SOX2 was not sustained. Together, these findings demonstrate that prenatal alcohol exposure induces profound and persistent histomorphological, cellular, and signaling alterations in the mammary gland in the absence of a carcinogenic challenge or genetically introduced oncogenic driver. These results identify intrinsic developmental reprogramming of normal mammary tissue as a potential basis through which prenatal alcohol exposure may modify subsequent responses to tumor-promoting or other environmental challenges.

Alcohol consumption is an established risk factor for breast cancer, with a particularly strong association with estrogen receptor-positive (ER+) disease. Although the estrogenic activity of alcohol is well recognized, how alcohol-induced ER signaling is coupled to growth factor receptor pathways that promote tumor cell growth and progression remains incompletely understood. Here, we identify neuregulin-1 (NRG1) as a functional mediator, linking alcohol-induced ER activity to ErbB3 receptor tyrosine kinase signaling in ER+ breast cancer cells. Under estrogen-depleted conditions, alcohol induced proliferation, clonogenic growth, and S-phase progression in MCF-7 and T47D cells. Alcohol concurrently increased ER phosphorylation and transcriptional activity, including enhanced ER occupancy at the endogenous TFF1/pS2 regulatory region, and activated ErbB3 and downstream Akt, ERK, and p38 signaling. Notably, alcohol induced NRG1 expression in both cell lines. Pharmacological inhibition of ER signaling with ICI 182,780 (fulvestrant) substantially attenuated NRG1 induction, ErbB3/RTK activation, and alcohol-promoted growth, indicating that NRG1 induction and engagement of RTK signaling are strongly dependent on functional ER signaling. Conversely, shRNA-mediated NRG1 depletion suppressed alcohol-induced proliferation, clonogenic growth, and cell-cycle progression and markedly reduced alcohol-induced migratory and invasive phenotypes. NRG1 depletion also attenuated ErbB3/Akt/ERK signaling while reducing ER activation and ERE-dependent transcription, demonstrating a functional contribution of NRG1 to both arms of the signaling response. Together, these findings support a reciprocal ER-NRG1-ErbB3 signaling circuit in which alcohol-induced ER activity promotes NRG1 expression, while NRG1-dependent ErbB3 signaling reinforces ER activity and tumor-promoting phenotypes. Our study identifies NRG1 as a previously unrecognized molecular link between the estrogenic activity of alcohol and growth factor receptor signaling and provides a mechanistic framework for understanding alcohol-associated promotion of ER+ breast cancer.

During embryogenesis, the cortical and medullary compartments of the adrenal gland emerge from two distinct sources: intermediate mesoderm-derived steroidogenic progenitors that comprise the cortex, and migratory neural crest-derived catecholaminergic progenitors that give rise to the medulla. The distinct cellular origins and paracrine signaling environments that foster induction, differentiation, and/or migration of cortical and medullary layers present challenges to recapitulating their co-emergence and incorporation in a human pluripotent stem cell (hPSC) based system. To address this, we developed an approach for modeling the adreno-gonadal primordium (AGP) in compound organoids that are organized along a gradient of NR5A1 expression and which unexpectedly incorporate SOX10-expressing Schwann cell precursors that ultimately generate chromaffin cells. Deconstruction of paracrine signaling within AGP-like organoids (AGPLOs) identified a fibroblast growth factor 9 (FGF9)-driven mechanism that expands bridge cell-like chromaffin progenitors and correlates to paracrine signaling cues encountered by physiological correlates along their migration corridor to the cortex in vivo. Combination of NR5A1-expressing cells with chromaffin cell clusters generated corticomedullar-like assembloids (CMLAs) that generated steroids and catecholamines and were responsive to adrenocorticotropic hormone. This work establishes a platform for modeling the reciprocal signaling relationships that drive adrenal gland development and function in health and disease and provides an approach for generating hPSC-derived adrenal cells/tissues that can be applied therapeutically.

The association of cytoplasmic localization of VDR in TNBC tumors and disease aggressivity prompted investigation of its regulation by XPO1. In TNBC cell lines, the XPO1 inhibitors selinexor and eltanexor increased nuclear VDR localization. Their effects were particularly strong when combined with vitamin D treatment. Similarly, XPO1 knockdown increased nuclear VDR levels, both alone and in combination with vitamin D. These results identify XPO1 as an important regulator of VDR subcellular distribution. XPO1 inhibition also enhanced vitamin D-dependent VDR transcriptional activity. Indeed, selinexor, eltanexor, and XPO1 silencing increased vitamin D-dependent CYP24A1 reporter activity and the expression of genes such as CYP24A1 or CDH1. RNA sequencing showed that XPO1 modulation markedly reshaped the vitamin D transcriptional response. The affected genes were mainly involved in metabolism, cell-cycle regulation, protein localization, and cellular organization. Functional analyses demonstrated that cytoplasmic VDR promoted TNBC cell proliferation. Conversely, nuclear VDR was required for the antiproliferative effects of vitamin D. Accordingly, XPO1 inhibition or depletion potentiated vitamin D-mediated growth suppression. This finding suggested functional cooperation between XPO1-dependent export and VDR activity. Finally, high VDR expression was associated with poor disease-free or overall survival only in tumors with high XPO1 expression. Overall, XPO1 negatively regulates VDR nuclear localization and vitamin D responsiveness in TNBC. Targeting XPO1 may therefore enhance the therapeutic potential of vitamin D-based strategies in TNBC.

The ovary is one of the first organs to age, with declines in oocyte quantity and quality leading to infertility and loss of endocrine function. Why the ovary ages earlier than somatic tissues has remained enigmatic but may relate to its highly dynamic physiology and substantial demand for cellular debris clearance. Each ovulation forms a corpus luteum (CL), a transient endocrine structure which supports pregnancy but regresses in its absence. Because CLs have a short functional lifespan but prolonged structural regression, they represent a substantial source of ovarian debris. We hypothesized that post-ovulatory debris clearance becomes impaired with age and contributes to ovarian fibroinflammation. Using a CL-labeling system, we demonstrated that CLs from reproductively old mice show dysregulated regression, resulting in an accumulation of persistent CLs (pCLs) which appeared concurrently with ovarian multinucleated giant cells (MNGCs). Laser capture microdissection, RNA sequencing, and histological evaluation of CLs, pCLs, and MNGCs demonstrated that pCLs were focal hotspots of proinflammatory and debris-associated signatures enriched in lipids, extracellular matrix, and immune cells. Although pCLs retained luteal features, they were transcriptionally more similar to MNGCs than CLs. Consistent with this, an ex vivo CL model acquired MNGC-associated molecular features during extended culture, suggesting that pCLs may represent sites of MNGC formation. Culture of isolated follicles or ovarian stromal organoids with factors from MNGC-enriched explants induced compartment-specific fibroinflammatory responses. These findings provide novel mechanistic insight into ovarian aging, whereby post-ovulatory debris clearance is compromised with age, leading to pCL accumulation, MNGC formation, and a fibroinflammatory milieu.

Synchrony between time-of-day and food intake is critical for maintaining homeostasis while desynchrony between these cues caused by repeated bouts of mistimed eating (ME) is profoundly disruptive to organismal health. However, it is unknown whether the same is true for a single bout of ME. While it is generally accepted that mammals recover quickly from acute alterations in feeding schedule and that chrono-disruption requires chronic, repeated changes, there have been no dedicated investigations of acute ME to suggest otherwise. Therefore, we exposed female and male C57Bl6/J mice to a single bout of ME or timed eating (TE) and continuously measured metabolic and behavioral responses for five days. A single bout of ME was sufficient to disrupt the daily rhythms of metabolic and behavioral outcomes for several days. Energy expenditure and wheel running activity were disrupted and light:dark variation in food intake and macronutrient utilization were altered for several days. Finally, ME changed the amount of time spent resting in for several days post-ME. The TE paradigm disrupted macronutrient utilization for two days but otherwise had no effect on metabolism or behavior. Our results demonstrate that one instance of ME alone has lasting impacts on daily rhythmicity and is an understudied source of circadian disruption.