Endocrinology

Prolactinomas, the most common secretory pituitary tumour subtype, frequently occur in patients with Multiple Endocrine Neoplasia type 1, caused by germline MEN1 mutations encoding menin. While menin loss is well established in MEN1-associated prolactinomas, its role in sporadic tumours remains unclear. We investigated menin expression, subcellular localization, and downstream signalling in two murine models of non-MEN1 prolactinomas, the dopamine D2-receptor knockout and the hCG{beta}-subunit-overexpressing mice, in which only females develop prolactinoma. Pituitary Men1 expression, analysed by qPCR, remained unchanged despite the genotype, in both sexes. However, in prolactinomas, lactotrophs exhibited a marked loss of nuclear MEN1 immunostained, with protein restricted to the cytoplasm. Male mice pituitaries retained nuclear MEN1 localization regardless their genotype. Loss of nuclear menin in prolactinomas was associated with reduced p27 and Pten expression, increased Ccnd1 expression, and enhanced pAKT. Moreover, by using in vivo pharmacological and surgical approaches we demonstrated that dopamine-agonist treatment preserved nuclear menin in lactotrophs, whereas dopamine blockade or estradiol induced its nuclear loss. Importantly, analysis of human pituitary biopsies confirmed nuclear and cytoplasmic menin localization in lactotrophs from normal pituitaries, and in prolactinomas from both genders following dopamine agonist therapy. However, in a prolactinoma from an untreated female, nuclear menin was partially lost. Therefore, our findings identify a state of functional MEN1-deficiency in sporadic prolactinomas (characterized by preserved MEN1 expression), but its exclusion from the nucleus (linked to activation of proliferative pathways, impaired tumour suppressor signalling, and tumour development) highlights the restoration of nuclear MEN1 localization as a potential therapeutic strategy.

In mammals, ovarian follicle development is a highly coordinated process that underlies female fertility. Granulosa cells expressing anti-Mullerian hormone (AMH) are widely used as a marker of growing follicles. However, the in vivo roles of granulosa cells in follicular development and female fertility remain unclear. Here, we analyzed AMH-toxin receptor-mediated cell knockout (AMH-TRECK) transgenic (Tg) mice on a NOG background, in which AMH-expressing granulosa cells are specifically depleted by diphtheria toxin (DT). We first found that, after a single DT injection into postnatal AMH-TRECK Tg females, AMH-expressing granulosa cells in primary and secondary follicles exhibited cleaved caspase-3 signals 1 day later and were depleted 4 days later. Second, after repeated DT injections weekly from 1 to 7 weeks of age in AMH-TRECK Tg females, antral follicles and corpora lutea were rarely observed, and the numbers of primordial, primary, and secondary follicles were decreased. Following PMSG and hCG stimulation, repeated DT-injected Tg females exhibited a reduced number of ovulated oocytes with a low proportion of mature oocytes, resulting in reduced IVF rates and fertility. Further, after a cessation of repeated DT treatment, ovarian weight and follicular development recovered: the numbers of primary, secondary, and antral follicles were recovered, whereas the primordial follicle pool remains reduced. We conclude that selective depletion of AMH-expressing granulosa cells in vivo impairs follicular development and fertility. Our model enables assessment of the in vivo effects of granulosa cell depletion and may provide a useful platform for future transplantation-based studies to understand complex follicular dynamics.

Pregnancy requires maternal pancreatic {beta}-cells adaptations to increased insulin demand, yet the embryo-derived signals contributing to this response remain poorly defined. Here we identify preimplantation factor (PIF), an embryo-derived peptide present in maternal circulation from early gestation, as a regulator of {beta}-cell adaptation. In a murine model of gestational diabetes mellitus (GDM), circulating PIF levels were reduced during mid-gestation, indicating that this endogenous signal is altered under gestational metabolic dysfunction. Conversely, chronic exposure of non-pregnant female mice to synthetic PIF (sPIF) recapitulated key temporal features of gestational {beta}-cell adaptation, including early {beta}-cell proliferation, increased glucose-stimulated circulating C-peptide, expansion of {beta}-cell mass and sustained enhancement of ex vivo glucose-stimulated insulin secretion (GSIS). Mechanistically, sPIF activated rapid ERK-, AKT- and PKA-dependent signaling that converged on estrogen receptor alpha (ER) phosphorylation and nuclear translocation. Pharmacological inhibition and genetic silencing demonstrated that ER is required for full propagation of the functional and kinase responses. This kinase-ER axis was conserved in human islets, where sPIF enhanced insulin secretion in an ER-dependent manner. These findings identify PIF as an embryo-derived metabolic signal supporting maternal {beta}-cell compensation and suggest that reduced PIF availability may contribute to inadequate {beta}-cell adaptation in GDM.

Targeted mass spectrometry (multiple-reaction monitoring, MRM) enables reproducible, multiplexed quantification of plasma proteins, but whether a fixed targeted panel can resolve endocrine disorders with overlapping systemic features is unknown. We analyzed a 256-protein targeted panel (1,894 peptides; 3,790 transitions) quantified in 57 participants spanning autoimmune thyroid disease (Hashimotos thyroiditis, n=7; Graves disease, n=5) and growth-hormone deficiency (GHD; partial, n=26; complete, n=19). Protein abundances were obtained by transition summation, log2 transformation, and per-sample median normalization. We applied unsupervised analysis (PCA, PERMANOVA), differential expression (limma), an ordered severity-trend test, and leave-one-out cross-validated classification with feature selection performed strictly inside each fold. All 256 proteins were quantified in every sample (median inter-sample r=0.875). PC1 (36% variance) separated autoimmune thyroid disease from GHD (p=0.016), whereas the global four-group structure was not significant (PERMANOVA p=0.17). No protein reached FDR<0.05, but the autoimmune-versus-GHD contrast was strongly enriched for low p-values (26 proteins at p<0.05; binomial p=5.4x10-4). The signal was biologically coherent: immunoglobulin/B-cell-receptor proteins, including CD79A, were lower, whereas proteasome subunits (PSMC5, PSMC3) and the NF-{kappa}B subunit RELA were higher in autoimmune disease. A cross-validated classifier separated the two classes (AUC 0.72; permutation p=0.05; eight proteins selected in all folds), whereas GHD severity was not predictable (AUC 0.31). A fixed 256-protein targeted panel reproducibly captures an immunoglobulin/B-cell-receptor and proteasome/NF-{kappa}B axis that distinguishes autoimmune thyroid disease from GHD but cannot resolve within-class severity.

Inhibin A and B (InhA and InhB) are potent negative regulators of follicle-stimulating hormone (FSH), which is essential for female fertility. InhA and InhB are heterodimeric transforming growth factor {beta} (TGF-{beta}) family ligands consisting of an inhibin (inh) subunit disulfide linked to an inhibin {beta}A or {beta}B (inh{beta}A, inh{beta}B) subunit, respectively. During purification, inhibins must be separated from activins (inh{beta}A or inh{beta}B homodimers), which always form when producing inhibins, and have the opposite effect on FSH synthesis. Current inhibin purification methods are technically challenging, require reagents that are not generally available, result in low purity or contamination with activins, or employ substitutions within the growth factor domain to favor inhibin formation over activin. We developed a simple and high-yielding expression and purification system for inhibins by transfection of suspension-cultured mammalian cells and tandem affinity chromatography, followed by reverse-phase purification of the mature ligands. We purified 1.4 mg (n = 3, SD = 0.47) and 0.093 mg (n = 3, SD = 0.043) of wild-type InhA and InhB per liter of culture medium with little to no contaminating activin A or B, at equal to higher purity and bioactivity than commercially available inhibins. Thus, we present a simple, robust, and versatile system for producing inhibins to interrogate their functions and mechanisms of action in vitro and in vivo.

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.

Background and AimsPregnancy requires dynamic, stage-specific adaptations in maternal liver metabolism and growth to sustain fetal development while preserving systemic homeostasis. Estrogen signaling, which significantly increases during pregnancy, is primarily mediated in hepatocytes by estrogen receptor (ER). Although hepatic ER regulates female liver metabolism under non-pregnant conditions, its role in pregnancy-induced hepatic remodeling remains unclear. MethodsWe studied non-pregnant and pregnant control and liver-specific ER knockout (LERKO) mice across gestational stages using longitudinal physiological measurements, liver transcriptomics, targeted metabolomics, histological assessment of cell proliferation, and metabolic phenotyping. ResultsIn control mice, pregnancy elicited sequential hepatic remodeling characterized by early induction of cell-cycle programs, a mid-gestational peak in hepatocyte proliferation with transient suppression of selected metabolic pathways, and late reactivation of specific metabolic programs. Chronic hepatic ER deficiency alters this temporal pattern. LERKO livers showed premature activation of proliferative and anabolic transcriptional programs, changes in amino acid- and fatty acid-related metabolic pathways, and altered temporal regulation of AKT-mTORC1-related signaling. At mid-gestation, LERKO mice displayed reduced hepatocyte proliferation, altered expression of metabolic and insulin-related genes, blunted gestational glucose adaptation without overt evidence of systemic insulin resistance, and changes in the light/dark-phase metabolic patterns. ConclusionsThese findings suggest that hepatic ER is required for the appropriate stage-specific coupling of liver growth, metabolic remodeling, and insulin-responsive signaling during pregnancy. Its loss is associated with gestational hepatic maladaptation and systemic metabolic phenotypes, providing a framework for investigating estrogen-dependent mechanisms underlying pregnancy-associated metabolic and liver disorders. HighlightsHepatic ER is required for stage-specific liver remodeling during pregnancy. Loss of hepatic ER alters temporal coupling of liver growth and metabolism. LERKO mice show early changes in amino acid- and fatty acid-related pathways. Hepatic ER loss reduces proliferation and alters gestational glucose adaptation. Hepatic ER loss is associated with altered light/dark-phase metabolic organization. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/743939v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@d52bborg.highwire.dtl.DTLVardef@b27511org.highwire.dtl.DTLVardef@23b286org.highwire.dtl.DTLVardef@19d9314_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

Objectives- Cell death and inflammatory pathways play important roles in adaptations to nutrient overload and metabolic dysfunction. This study investigates the metabolic consequences that arise from the dual disruption of both caspase 1 (Casp1) and receptor interacting protein kinase 3 (Ripk3) in mice fed a control or obesity-inducing diet. Methods- Male and female wild-type (WT), Casp1/11 knockout (KO), Ripk3 KO and Casp1/11/Ripk3 double knockout (DKO) mice were fed a matched low-fat or a 60% kcal high fat diet, followed by metabolic phenotyping. Islets were isolated from WT and DKO mice for measures of dynamic glucose-stimulated insulin and somatostatin (Sst) secretion. Islet architecture and cellular composition were assessed in WT and DKO mice by immunofluorescent staining of intact pancreatic sections. Pharmacological inhibition of Casp1 (Ac-YVAD-cmk) and Ripk3 (GSK872) was performed in WT and DKO mice using isolated islets and in vivo administration. Exogenous hormones were administered prior to glucose injection to test in vivo responses. Results- High-fat feeding resulted in increased adiposity in male, but not female mice, with single or double deletion of Casp1/11 and Ripk3. These mice also exhibited markers of impaired glucose tolerance and insulin sensitivity. Interestingly, when both Casp1 and Ripk3 were deleted or inhibited in mice fed a low-fat diet, mice experienced reductions in glucose excursion following administration of glucose due to increased plasma insulin levels. This increase in insulin secretion was recapitulated in isolated islets ex vivo and was independent of changes in the proportions of -, {beta}-, and {delta}-cells within the islet. There were significant reductions in the percentage of urocortin-3 (Ucn3)-positive {beta}-cells in DKO mice compared to control, suggesting altered Ucn3-Sst signaling; however, only exogenous Sst (Octreotide) and not Ucn3 was able to correct the decreased glucose excursion. Conclusions- Loss or inhibition of both Casp1 and Ripk3 fundamentally alter islet responses to glucose. Our findings highlight that endogenous Casp1 and Ripk3 act independently of inflammatory or cell death signals to coordinate normal glucose-stimulated insulin release.

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.

Background: Biological sex and endocrine signaling influence cancer biology, immune response, and therapeutic outcomes. Recent evidence suggests that testosterone signaling may exert brain context dependent protective effects in glioblastoma through the hypothalamic-pituitary-adrenal axis, reduced glucocorticoid-mediated immune suppression, and altered tumor-immune interactions. We assessed whether testosterone replacement therapy (TRT) exposure was associated with survival in solid tumor central nervous system (CNS) metastases and glioblastoma (GBM, IDHwildtype, WHO grade 4), settings in which post-diagnosis survival and TRT timing can be clinically defined. Methods: We performed a retrospective Mayo Clinic cohort study of adult patients with molecularly confirmed glioblastoma and solid tumor CNS metastases confirmed from neuroimaging reports using large language model-assisted adjudication. TRT exposure was defined by testosterone-specific prescription evidence within prespecified peri-diagnostic windows. Overall survival was evaluated using propensity score-matched Cox models, 24 month administratively censored Cox models, time-dependent Cox sensitivity analyses, and 24 month restricted mean survival time. Results: In the pooled solid tumor CNS metastasis cohort, TRT exposure was associated with improved overall survival after propensity score matching (HR 0.80, 95% CI 0.65 to 0.98, p=0.029) and a 3.22-month improvement in 24 month restricted mean survival time. In glioblastoma, TRT exposure was similarly associated with improved overall survival after propensity score matching (HR 0.56, 95% CI 0.38 to 0.82, p=0.003) and a 5.81-month improvement in 24 month restricted mean survival time. Conclusions: TRT exposure was associated with improved survival in CNS metastases and glioblastoma. These hypothesis-generating findings support prospective studies incorporating TRT timing, hormone levels, corticosteroid exposure, immune correlates, and tumor-specific stratification.

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.

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.

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.

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.

AbstractAdrenocortical carcinoma (ACC) is a rare and highly aggressive endocrine malignancy originating from the adrenal cortex with limited effective treatment options. The underlying pathophysiology of ACC is uniquely characterized by abnormal steroid production and increased metabolic activity, highlighting the critical role of mitochondria in adrenal steroid hormone biosynthesis and tumor metabolism. In this study, we investigated the therapeutic potential of TR-107, a novel and highly selective small-molecule agonist targeting the mitochondrial protease ClpP. Pharmacologic hyperactivation of ClpP disrupts mitochondrial proteostasis and bioenergetics and has shown promising antitumor activity in various preclinical models. Our results demonstrated that TR-107 induces potent dose-dependent cytotoxic effects at nanomolar concentrations in ACC cell lines NCI-H295R and mACC3 as well as short-term ACC patient-derived organoid (PDO) models, markedly reducing cell viability and confluency in vitro. Metabolic analyses revealed that TR-107 significantly impaired oxygen consumption, indicating a disruption of oxidative phosphorylation and substantial attenuation of basal cellular respiration. Mechanistic studies showed dose-dependent increases in reactive oxygen species (ROS) levels and upregulation of proteins involved in mediating the ferroptotic rheostat. Pharmacokinetic assessment uncovered that TR-107 was not a substrate of the ABCB1 (MDR1/P-glycoprotein) efflux transporter, suggesting potential to overcome common multidrug resistance mechanisms. Given the importance of IGF-2 signaling in ACC, we further explored the combinatorial effects of TR-107 with IGF-1 receptor (IGF-1R) inhibitors and discovered that co-treatment produced synergistic reductions in cell viability across NCI-H295R, mACC3, and ACC PDOs. Collectively, these findings support the potential of mitochondrial ClpP hyperactivation as a promising therapeutic strategy for ACC and demonstrate that TR-107 exhibits significant antitumor activity as a monotherapy or in combination with IGF-1R inhibitors. These findings provide a strong rationale for advancing ClpP agonists into clinical development for the management of ACC.

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.