Endocrinology

Polycystic Ovarian Syndrome (PCOS) is a complex endocrine disorder characterised by hyperandrogenism, oligo- or anovulation, and polycystic ovaries. Endocrine dysfunction in PCOS disrupts both hormonal and neurotransmitter balance, contributing to the psychological distress frequently reported by affected individuals. Although hormonal imbalances have been associated with memory impairments, their specific contribution to cognitive dysfunction in PCOS remains incompletely understood. In this study, we investigated the impact of PCOS on the hippocampus, a brain region critical for memory formation and highly sensitive to sex steroid modulation. A dehydroepiandrosterone (DHEA)-induced PCOS mouse model was employed to assess anxiety-like behaviour, locomotion, and memory. In the open field test (OFT), DHEA-treated mice spent significantly less time in the central zones and travelled a shorter total distance compared with controls, indicating increased anxiety-like behaviour. DHEA treatment also resulted in significantly impaired performance in both the object location test (OLT) and novel object recognition test (NORT), as reflected by a reduced discrimination index. Analysis of hippocampal immediate early gene expression using qRT-PCR revealed altered transcription of memory-related markers, including downregulation of Npas4 and Grin2a, and upregulation of Grin1, Arc, Egr1, and Egr2. Collectively, these findings suggest that elevated androgen levels induce anxiety- and depression-like behaviours and impair cognitive function, including spatial, recognition, and motor learning abilities, in PCOS. Our results further indicate that disrupted cortex-hippocampus communication may underlie these cognitive deficits, underscoring the importance of evaluating memory and cognitive health in women with PCOS to support brain health and overall well-being.

Polycystic ovary syndrome (PCOS) is characterized by reproductive and metabolic disturbances and is associated with increased symptoms of anxiety and depression. Circulating adiponectin, an insulin-sensitizing adipokine, is reduced in women with PCOS, and low adiponectin has been linked to impaired mental health, particularly in females. We investigated whether low serum adiponectin is associated with impaired mental health in women with PCOS and whether adiponectin deficiency exacerbates anxiety-like behaviour in a PCOS-like mouse model. Serum adiponectin was measured in women with (n=179) and without PCOS (n=228), stratified by body mass index (BMI). Health-related quality of life was assessed using the SF-36, generating physical and mental component scores. In parallel, the prenatal androgenization (PNA) PCOS-like mouse model was combined with adiponectin-deficient mice (APNhet) to assess the impact of reduced adiponectin on anxiety-like behaviour with and without prenatal androgen exposure. Women with PCOS had lower total and high molecular weight adiponectin levels compared with controls. Adiponectin positively correlated with mental component scores in women with BMI <30, but not in those with obesity. Free testosterone was inversely correlated with adiponectin. In mice, PNA induced anxiety-like behaviour, however, reduced adiponectin did not exacerbate this phenotype. Although APNhet PNA mice showed 65% lower serum adiponectin levels and reproductive dysfunction, they displayed improved metabolic function. Unlike women with PCOS, adult PNA mice were not hyperandrogenic. These findings suggest that adiponectin is associated with mental health in non-obese women, but reduced adiponectin alone does not induce anxiety-like behaviour in the absence of hyperandrogenism. The differing patterns observed across BMI categories, as well as between the human cohort and experimental data, underscore the complexity of the mechanisms underlying mental health disturbances in PCOS.

Pregnancy leads to many adaptations in the maternal body, most of which are reversible. However, reproductive experience can also result in permanent effects. Here, we investigated how pregnancy influences the somatotrophic system and the lasting effects of reproductive experience on the maternal organism. Reproductive experience induced a pronounced increase in lean body mass and longitudinal growth in both wild-type and growth hormone (GH)-deficient mice compared with age-matched virgins. Body growth was primarily observed during the first pregnancy, whereas a second gestation was mostly associated with increased adiposity. Data from a cohort of women with isolated GH deficiency (IGHD) caused by a loss-of-function mutation in the GHRHR gene revealed that nulliparous women were 7 cm shorter than those with one or more pregnancies. Increased GH secretion was observed in pregnant wild-type mice but not in pregnant GHRHR-deficient mice. Pregnancy-induced body growth is preserved despite disruption of GH-, ghrelin-, and estrogen-related signaling pathways. In conclusion, reproductive experience induces permanent changes in the maternal organism, promoting body growth in models that allow this response. Pregnancy-induced body growth appears to be independent of GH action. These findings underscore the need for further studies to investigate the long-lasting consequences of reproductive experience in females.

Psychosocial stressors are key contributors to ovarian functional decline. Chronic unpredictable mild stress (CUMS) is widely used to model stress-induced premature ovarian insufficiency (POI) in mice; however, current animal models do not adequately reflect middle-aged women, who represent a key population exposed to chronic psychosocial stress, nor do they capture the dynamic progression toward POI. Here, female C57BL/6 mice aged 2 or 6 months were subjected to CUMS for 8 or 12 weeks. Estrous cyclicity, endocrine profiles, ovarian histology, and transcriptomic changes in HPO axis-related tissues were systematically analyzed. After 8 weeks of exposure, 2-month-old mice exhibited impaired pituitary responsiveness to estradiol negative feedback, as evidenced by dysregulated FSH secretion, indicating reduced stress tolerance compared with 6-month-old mice. Following 12 weeks of CUMS exposure, both age groups showed significant reductions in ovarian size and follicle numbers across all developmental stages. These findings demonstrate that CUMS induces an age-dependent progression toward POI, with short-term exposure eliciting compensatory phases preceding overt ovarian insufficiency, accompanied by distinct endocrine and reproductive alterations and differential responsiveness of the HPO axis. Transcriptomic analyses revealed age-dependent stress responses: ovaries of 2-month-old mice displayed marked activation of inflammatory and immune-related pathways, whereas 6-month-old mice showed sustained upregulation of protein kinase-related signaling networks. Notably, the 6-month-old CUMS model more closely recapitulates stress-associated reproductive aging in adult women. In briefCUMS has been widely used to establish mouse models of psychosocial stress-induced POI. However, current animal models do not adequately reflect middle-aged women, who represent a key population exposed to chronic psychosocial stress, nor do they capture the dynamic progression toward premature ovarian insufficiency (POI). In this study, we demonstrate that different durations of CUMS exposure induce distinct stages of ovarian dysfunction in both young and middle-aged mice, with short-term exposure driving age-dependent compensatory phases and prolonged exposure leading to overt POI, both accompanied by divergent endocrine and reproductive alterations, alongside age-dependent changes in HPO axis responsiveness to CUMS. Notably, the 6-month-old CUMS model shows greater clinical relevance in recapitulating chronic psychosocial stress and stress-related reproductive aging in adult women.

Early detection of ovulation and pregnancy in the common marmoset is crucial for reproductive studies, yet hCG kits lack cross-reactivity with marmoset CG, and current methods remain labor-intensive. Here, we developed monoclonal antibodies against marmoset CG and CG{beta}, and established a non-invasive immunochromatographic CG assay. By eliminating invasive blood sampling, this assay supports 3Rs principles and enables practical endocrine monitoring. The assay detected urinary CG surges preceding ovulation, enabling efficient embryo recovery through artificial insemination (75%). Early pregnancy was detected at approximately 17 days post-ovulation. In addition, pregnancy detection in squirrel monkeys suggests conservation of CG features among certain New World primates. Overall, this simple, non-invasive assay provides a practical tool for marmoset research and establishes a foundation for future conservation-oriented reproductive monitoring following appropriate species-specific validation.

The human endometrium undergoes cyclic, hormone-driven remodeling that establishes a transient window of receptivity required for embryo implantation, placentation, and maintenance of pregnancy. Decidualization of endometrial stromal cells is a central component of this process and can be induced in vitro using cAMP alone or in combination with ovarian steroid hormones (EPC: estradiol, progesterone, and cAMP). Although cAMP activates the core decidual transcriptional program, whether hormone supplementation induces a more physiologically relevant response remains unclear, particularly in 3D endometrial organoid (Endo-organoid) models which have emerged as a new alternative methodology (NAM). Here, we compared morphological and transcriptomic responses of human endometrial stromal cell-derived Endo-organoids undergoing decidualization induced by cAMP or EPC stimulation. EPC-treated Endo-organoids exhibited enhanced structural remodeling and more advanced morphological transformation compared with cAMP-treated organoids. RNA-seq analysis revealed substantial overlap in canonical decidual gene expression between the two conditions, but EPC induced broader transcriptional and pathway-level changes, including enrichment of metabolic, stress-response, and differentiation-related processes. Together, these findings demonstrate that while cAMP activates the core decidual program, EPC elicits a broader and more physiologically relevant decidualization response in 3D human Endo-organoids, providing guidance for optimizing Endo-organoids to study endometrial receptivity, implantation, and early pregnancy success.

Early human pregnancy is a critical period characterized by rapid growth and extensive maternal-fetal communication that influence maternal and fetal outcomes. Circulating extracellular vesicles (EVs) have the capacity to capture cargo that reflect these processes in real-time; however, signatures of EV subtypes during early pregnancy are poorly defined. Here we quantified mitochondrial DNA (mtDNA) and performed transcriptomic and proteomic profiling of small ([~]100 nm) and large ([~]200 nm) plasma EVs from n=10 normal pregnancies (11-15 weeks) to define subtype-specific molecular signatures. mtDNA and mitochondrial protein content were more abundant in large EVs (lEVs). lEVs also contained a more complex set of long RNAs enriched for placental, immune, and mitochondrial-related transcripts compared with small EVs (sEVs). Proteomic profiling showed enrichment of canonical EV markers and extracellular matrix proteins in sEVs, whereas lEVs were preferentially associated with pregnancy-specific proteins, including proteins related to placental hormone production. MicroRNAs (miRNAs) accounted for [~]25% of small RNAs in both EV subtypes with miR-223 and miR-16 enriched in lEVs and miR-639 enriched in sEVs. These data together, support a model where small and large plasma EVs have distinct, yet complementary signatures reporting systemic adaptations during the critical 11-15 week transition period. This work establishes a foundational framework for future studies linking EV signatures to placental dysfunction and adverse outcomes.

BackgroundSteroid sulfatase (STS) cleaves sulfate groups from steroid hormones. In humans, STS deficiency is associated with X-linked ichthyosis (a dermatological disorder), neurodevelopmental/mood conditions, and cardiac arrhythmias. Until recently, no single-gene knockout mammalian model existed to investigate these associations; previous work in such a model has been limited to skin phenotypes. MethodsWe generated a novel C57BL/6J mouse model with a deletion in critical exon 2 of Sts. We then examined gene expression and enzyme activity in liver and brain samples of homozygous mice, and assessed the breeding performance and health of male and female deletion-carriers. Subsequently, we compared performance across a range of behavioural paradigms in wildtype and homozygous male and female mice: elevated plus maze, open field, rotarod, spontaneous alternation, and acoustic startle/prepulse inhibition. We also investigated serum steroid hormone levels by liquid chromatography-mass spectrometry and measured heart weights and two morphological indices (bodyweight/tibia length) post mortem. ResultsHomozygous mice almost completely lacked STS expression/activity. Genetically-altered mice exhibited grossly-normal breeding performance, health, and endocrinology. Homozygous mice were more active, and had higher normalised heart weights, than wildtype mice. We also found significant genotype x sex interactions on bodyweight, and on two behavioural measures (potentially reflecting lower anxiety in homozygous males and heightened anxiety in homozygous females). ConclusionsThe Sts-deletion mouse represents an experimentally-tractable model in which to identify and characterise phenotypes associated with STS deficiency. The mechanistic basis of the genotype-phenotype associations described here requires further investigation, and whether such associations translate to humans remains to be tested.

The population of kisspeptin neurons located in the rostral periventricular area of the third ventricle (RP3V) is thought to have a key role in generating the GnRH surge that triggers ovulation. Using a modified GCaMP fibre photometry procedure, we have been able to record the in vivo population activity of RP3VKISS neurons across the estrous cycle of female mice. A marked increase in GCaMP activity was detected beginning on the afternoon of proestrus that lasted in total for 13{+/-}1 hours. This was comprised of slow baseline oscillations with a period of 91{+/-}4 min and associated with high frequency rapid transients. Very little oscillating baseline or transient activity was detected at other stages of the estrous cycle. Concurrent blood sampling showed that the peak of the LH surge occurred 3.5{+/-}1.1 h after the first baseline RP3VKISS neuron baseline oscillation on the afternoon of proestrus. The time of onset of RP3VKISS neuron oscillations varied between mice and across subsequent proestrous stages in the same mice. To assess the impact of estradiol on RP3VKISS neuron activity, mice were ovariectomized and given an incremental estradiol replacement regimen. Minimal patterned GCaMP activity was found in OVX mice, and this was not changed acutely by any of the estradiol treatments. However, on the afternoon of the expected LH surge, the same oscillating baseline activity with associated transients occurred for 7.1{+/-}0.5 h. These observations reveal an unexpected prolonged oscillatory pattern of RP3VKISS neuron activity that is dependent on estrogen and underlies the preovulatory LH surge as well as potentially other facets of reproductive behavior.

Maternal pancreatic {beta}-cells undergo functional and structural changes to adapt to increased metabolic demands during pregnancy. Lactogen signaling via the prolactin receptor (PRLR) contributes to these adaptations by increasing {beta}-cell mass, insulin transcription and glucose-stimulated insulin secretion[1-4]. In other lactogen-responsive tissues such as the mammary glands and specific hypothalamic nuclei, gestation induces epigenetic changes, some of which persist long after birth[5, 6]. We have previously found that prolactin treatment in islets regulates the expression of epigenetic modifiers[7, 8]. However, whether lactogen signaling in {beta}-cells mediates epigenetic changes to regulate chromatin accessibility has not been examined. Therefore, our objective was to determine whether PRLR signaling alters chromatin accessibility of {beta}-cells to facilitate transcriptional regulation. Using single-cell ATAC-sequencing, we identified differentially accessible regions (DARs) in {beta}-cells which had 718 overrepresented motifs following prolactin treatment of murine islets. Validating this approach, these included motifs bound by established PRLR signaling effectors such as the STAT family of transcription factors (TFs). Using RNA-sequencing we identified transcriptional changes in 41 TFs whose motifs were overrepresented in DARs, including several previously linked to PRLR signaling within {beta}-cells, including Myc, Mafb and Esr1. Importantly, we also identified TFs not previously associated with PRLR signaling, including OVOL2 an established regulator of epigenetic landscape within cells. OVOL2 is a transcription factor involved in EMT inhibition and energy homeostasis with unknown roles in pancreatic {beta}-cells. Here, we establish that OVOL2 acts as a negative regulator of lactogen-dependent effects on {beta}-cell proliferation, establishing a novel regulator of PRLR signaling.

Pregnancy is a period of extensive metabolic rewiring. Insulin secreting {beta}-cells respond to the metabolic challenges of pregnancy by increasing their mass and size and by altering secretory patterns to maintain glucose homeostasis. If glucose metabolism is not tightly controlled, gestational diabetes may develop. Most studies on {beta}-cell adaptation during pregnancy are derived from rodent models, making translation to the vastly different human gestational setting challenging. In this work, we performed an extensive characterization of pancreatic adaptations throughout porcine pregnancy. Pigs have a long gestational period (114 days) and share a similar size and metabolism to humans, making them an ideal model to bridge the knowledge gap between rodents and humans. By analyzing pancreatic samples from early and late gestational ages, we captured the full trajectory of endocrine remodeling. We observed pregnancy-driven remodeling of endocrine cell types, marked by preferential expansion of pancreatic polypeptide-secreting cells. Proteomic characterization of the pancreas from early and late gestation showed a downregulation of SLC20A2 and ZCCHC7, identifying new protein targets involved in physiological endocrine cell adaptation. Overall, our comprehensive characterization of pancreatic adaptations in the pig model helps bridge the translational gap between rodents and humans and highlights previously unrecognized proteins with therapeutic potential for gestational diabetes.

Human embryo implantation, occurring approximately one week after fertilization, remains poorly understood due to ethical and technical limitations of in vivo investigation. To overcome these barriers, and model this critical developmental event, encompassing peri- and early post-implantation stages, we used an in vitro embryo attachment model composed of donor-derived endometrial epithelial cells forming an open-faced endometrial layer (OFEL) and human stem cell-derived blastoids recapitulating human day 5 blastocysts in peri-implantation model. Following attachment, developmental progression was further investigated on laminin-coated substrates to capture early post-implantation dynamics. Despite its central role as the primary endocrine signal of early pregnancy, human chorionic gonadotropin (hCG) remains largely uncharacterized in this context. Here, we describe the transcriptomic profile of blastoid-endometrial co-cultures relative to OFEL alone, identifying CGA and CGB3/5/8 as among the most strongly upregulated genes following blastoid attachment to hormonally stimulated OFEL. Consistent with these findings, immunoassays and luteinizing hormone/choriogonadotropin receptor (LHCGR) activation assays of conditioned media confirmed the secretion of heterodimeric, biologically active hCG and its free subunits in co-cultures, but not in endometrial layers alone. Notably, the hyperglycosylated hCG heterodimer was the predominant isoform detected. Co-culture with the endometrial component significantly increased hCG secretion compared with blastoids cultured alone, an effect further enhanced by hormonal priming in the peri-implantation model. Collectively, these findings indicate that a hormonally primed endometrial environment not only promotes blastoid attachment but also amplifies embryonic hCG production and bioactivity, underscoring the importance of maternal endocrine cues in early embryo-endometrium communication. Furthermore, our peri- and early post-implantation models recapitulate key aspects of reciprocal endocrine signaling between embryonic and endometrial tissues, providing a tractable experimental framework to investigate embryo-endometrium crosstalk.

The secretion of glucagon from the pancreatic alpha () cell within the islets of Langerhans is physiologically regulated by nutrients (glucose, amino acids, fatty acids), neurotransmitters, and paracrine hormones. Insulin and somatostatin form an intra-islet paracrine network to control glucagon secretion through direct inhibitory effects on cell secretory granule exocytosis. In a potential new cellular pathway for the regulation of glucagon secretion, we have previously identified the neuronal trafficking protein Stathmin-2 (Stmn2) as a negative regulator of glucagon trafficking and secretion by directing glucagon to degradative lysosomes. In this study, we examined if insulin and somatostatin direct glucagon to lysosomes in a Stmn2-dependent manner as part of their paracrine mechanisms. Using the TC1-6 glucagon-secreting cell line and confocal microscopy of both fixed and live cells, we show that insulin and somatostatin direct glucagon, glucagon+LAMP1+ vesicles, and LAMP1-RFP to the intracellular region, away from sites of exocytosis. As visualized in live cells, insulin treatment resulted in the rapid retrograde transport of lysosomes from the cell periphery, and this effect was lost under siRNA-mediated silencing of Stmn2. Somatostatin appeared to enhance the intracellular retention of lysosomes, also in a Stmn2-dependent manner. We determined a possible mechanism for Stmn2 in the regulation of lysosome transport in TC1-6 cells through the Arf-like small GTPase Arl8, indicating that Stmn2 may function in lysosomal positioning along microtubules. We propose that Stmn2-mediated lysosomal transport may be a potential new pathway, in addition to inhibition of secretory granule exocytosis, through which insulin and somatostatin regulate glucagon secretion.

ObjectiveG protein-coupled receptor 180 (GPR180) has been implicated in systemic energy metabolism, primarily in adipose tissue and the liver. Given impaired whole-body glucose tolerance following GPR180 dysfunction, we aimed to determine whether GPR180 regulates pancreatic {beta}-cell function. We investigated whether GPR180 contributes to {beta}-cell insulin secretion by modulating metabolic processes that couple glucose sensing to mitochondrial energy production. MethodsPhenotyping of whole-body (Gpr180 -/-) and {beta} cell-specific Gpr180 (bGpr180-KO) knockout mice was combined with gain- and loss-of-function studies in MIN6 cells. Glucose-stimulated insulin secretion, pancreatic endocrine architecture and identity, transcriptomic and metabolic profiles, as well as mitochondrial function were assessed using in vivo and in vitro approaches, including metabolic challenge tests, histology, RNA sequencing, targeted metabolomics, respirometry, and transmission electron microscopy. ResultsLoss of GPR180 impaired first-phase insulin secretion and glucose tolerance without affecting insulin sensitivity. These defects were {beta}-cell-autonomous, as confirmed in the bGpr180-KO mice and in MIN6 cells. Functional studies revealed that GPR180 regulates mitochondrial substrate utilization, anaplerotic support of the TCA cycle, and ATP generation without affecting glucose uptake or mitochondrial biogenesis. In particular, Gpr180-deficient {beta} cells showed mitochondrial membrane depolarization, reduced oxygen consumption, and endoplasmic reticulum remodeling, altering the local mitochondrial microenvironment. In vivo, Gpr180 deletion in {beta} cells led to downregulation of mitochondrial gene programs in islets, along with altered endocrine cell identity. ConclusionsGPR180 is a previously unrecognized regulator of pancreatic {beta}-cell metabolic competence and identity, linking defects in insulin secretion with alterations in mitochondrial function and endocrine cell identity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/720098v1_ufig1.gif" ALT="Figure 1"> View larger version (87K): org.highwire.dtl.DTLVardef@1a441ecorg.highwire.dtl.DTLVardef@e41e02org.highwire.dtl.DTLVardef@6e2212org.highwire.dtl.DTLVardef@7ee07a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Mitochondrial homeostasis, governed by the balance between biogenesis and mitophagy, is essential for steroidogenesis in adrenocortical cells. While the requirement of active mitochondria for steroid synthesis is well-established, the hormonal regulation of genes governing mitochondrial function remains poorly understood. This study investigated whether angiotensin II (Ang II) and the cAMP/PKA pathway modulate the expression of key regulatory factors involved in mitochondrial biogenesis and redox status in the human adrenocortical H295R cell line. Using real-time qPCR and Western blot, we show that Ang II and 8Br-cAMP --a permeant analogue of cAMP-- modulate NRF-1, Nrf2, UCP2, and ANT1 impacting on mitochondrial biogenesis, antioxidant defense, and respiratory activity. These molecular changes correlated with increased mitochondrial membrane polarization, as confirmed by MitoTracker red staining. Interestingly, Ang II stimulation promoted a time-dependent increase in TFAM levels, a key transcription factor in mitochondria, which correlates with the increase in mitochondrial DNA (mtDNA) content. The rate of oxygen consumption (OCR) and mitochondrial parameters were determined, with results showing that Ang II led to a significant increase in basal and maximum respiration, ATP production, and proton leak. These findings suggest that hormone stimulation favors mitochondrial activity, thereby enhancing the bioenergetic capacity of adrenocortical cells. Furthermore, treatment with the uncoupler CCCP triggered a retrograde signaling response, upregulating nuclear-encoded mitochondrial genes to counteract mitochondrial membrane depolarization. Our findings demonstrate for the first time that hormonal signals directly modulate the mitochondrial genetic program in H295R human adrenocortical cells, optimizing the bioenergetic platform required for efficient steroidogenic function.

The oviduct provides the dynamic microenvironment that supports fertilization and early embryo development yet replicating its hormonally regulated secretory activity in vitro remains a major challenge. Here, we established bovine oviductal epithelial organoids that reproduce the structural polarity and endocrine responsiveness of the native oviduct. Exposure to either estradiol or progesterone resulted in distinct transcriptomic and proteomic landscapes that were characteristic of the follicular and luteal phases, respectively. This included the upregulation of canonical phase-specific markers, such as OVGP1, NTS, HP and TGM2. Proteomic profiling of organoid-derived secretions (ODS) revealed extensive overlap with in vivo oviductal fluid. Integration of transcriptomic and proteomic datasets by multi-omics factor analysis identified coherent biological signatures defining each hormonal state. Functionally, ODS obtained from estradiol-treated organoids enhanced sperm capacitation and acrosome reaction, recapitulating the activity of follicular-phase oviductal fluid. These findings demonstrate that hormonally responsive oviductal organoids generate bioactive secretions that emulate the molecular and functional features of the native oviductal environment, providing a sustainable and physiologically relevant platform for studying gamete-maternal communication and improving assisted reproduction technologies.

Background and AimsAdult pancreas-derived islet progenitor cells (IPCs) have recently been shown to expand in culture and differentiate into endocrine-like organoids. However, translation of this approach to a clinically compatible workflow requires cell enrichment strategies and validation using tissue obtained during real-world clinical procedures. Here, we adapted our previously described IPC platform to non-endocrine pancreatic tissue fractions generated during clinical islet isolation procedures and evaluated their capacity to generate functional islet organoids. MethodsNon-endocrine pancreatic tissue fractions obtained during clinical islet isolation were expanded ex vivo and enriched using fluorescence-activated cell sorting (FACS) for CD81 and CD9, surface markers previously identified in IPC populations. Sorted cells were expanded, induced to form IPC clusters, and differentiated with ISX9 to generate islet organoids. Differentiation was assessed by gene expression analysis, flow cytometry, immunofluorescence, calcium flux assays, glucose-stimulated insulin and glucagon secretion, and single-cell RNA sequencing. ResultsClinically derived non-endocrine cell fractions yielded expandable IPC populations expressing progenitor-associated markers. FACS-purified and expanded CD81+/CD9+ IPCs were enriched with BMPR1A and P2RY1. Sorted cells generated three-dimensional BMPR1A+ and RGS16+ IPC clusters. IPC clusters differentiated into islet organoids with upregulated expression of canonical beta-and alpha-cell transcription factors. Single-cell transcriptomic profiling revealed activation of coordinated endocrine gene programs and alignment with reference human islet endocrine signatures, while the undifferentiated IPC compartment was marked by enrichment of PTX3, FST, CEMIP, and GREM1. Terminally differentiated cells exhibited depolarization-induced calcium influx and glucose-regulated insulin and glucagon secretion. ConclusionsThese findings establish an adaptable workflow for expansion and production of functional islet organoids recovered from clinically derived pancreatic tissue. This strategy may provide an unlimited autologous source of adult progenitor-derived islets for future islet cell replacement therapies in diabetes.

Pancreatic beta cells produce and secrete insulin to maintain glucose homeostasis. Due to their high secretory activity, beta cells rely heavily on endoplasmic reticulum (ER) function and are particularly susceptible to ER stress, which contributes to beta cell dysfunction in diabetes. However, the transcriptional mechanisms linking ER stress to beta cell failure remain poorly understood. In this study, we investigated the role of the transcription factor Mef2a in ER stress-mediated beta cell dysfunction using primary mouse islet cells. ER stress induced by thapsigargin increased Mef2a expression and activated canonical unfolded protein response (UPR) pathways. Overexpression of Mef2a reduced beta cell proliferation, suppressed expression of key beta cell transcription factors including Pdx1, MafA, NeuroD1, and Nkx6.1, and impaired glucose-stimulated insulin secretion. Mef2a overexpression also altered mitochondrial respiration, characterized by reduced glucose-coupled respiration and increased maximal respiratory capacity. In contrast, Mef2a knockdown attenuated ER stress induced activation of ATF6 and IRE1/XBP1 dependent UPR genes. Importantly, reducing Mef2a expression preserved beta cell identity gene expression and improved insulin secretion during ER stress induced by thapsigargin or tunicamycin. Together, these findings identify Mef2a as a stress-responsive regulator that contributes to ER stress-mediated beta cell dysfunction and suggest that modulating Mef2a activity may help preserve beta cell function during metabolic stress.

(1) Aims and hypothesisLoss-of-function mutations in SLC30A8, encoding the zinc ion (Zn2+) transporter ZnT8 in pancreatic beta cells, lower type 2 diabetes risk dose-dependently, but the underlying mechanisms remain unclear. Here, we combine proteomic, transcriptomic and functional approaches in human stem cell-derived islet-like clusters bearing common alleles or the inactivating variant R138X. We hypothesized that this variant protects against the deleterious effect of Zn2+ depletion on cell survival and function. (2) MethodsHuman embryonic stem cells INS(GFP/w) (MEL1), and CRISPR/Cas9-derived heterozygous or homozygous R138X lines were differentiated into stem cell-derived islet-like clusters. Intracellular Zn2+ levels were reduced using the chelator N,N,N',N'-tetrakis(2-pyridylmethyl)-1,2-ethanediamine (TPEN). Apoptosis was assessed by TUNEL staining and protein expression by immunofluorescence. Glucose-stimulated calcium (Ca2+) dynamics were measured using the intracellular probe (Cal590) and insulin secretion by homogenous time-resolved fluorescence. Transcriptomic profiling was performed by bulk mRNA sequencing and proteomics by liquid chromatography-tandem mass spectrometry. (3) ResultsIntracellular Zn2+ depletion increased apoptosis in wild-type islet-like clusters, whereas R138X clusters were protected. R138X heterozygous clusters showed a mild increase in GCG+ cells and R138X homozygous clusters exhibited increased NKX6.1+ cells, without affecting polyhormonal populations. These changes were reversed under Zn2+ depletion. Transcriptomic and proteomic analyses, assessing genotype effects while accounting for Zn2+ depletion, showed that R138X clusters (versus wild-type) exhibited upregulation of genes and proteins involved in vesicle trafficking, secretion, Ca{superscript 2} signaling and mitochondrial metabolism, consistent with enhanced glucose-stimulated insulin secretion in homozygous clusters. Conversely, genes and proteins associated with extracellular matrix remodeling, metal-ion handling, apoptosis and cellular stress were downregulated. R138X clusters displayed altered Ca2+ signaling, with decreased area under the curve and oscillation amplitude, but increased frequency. These differences were reversed by TPEN, while Zn2+ depletion impaired Ca2+ response in wild-type clusters. Despite lowered overall activity, R138X homozygous clusters showed enhanced overall cell-cell connectivity, reversed by TPEN treatment. The opposite effects were observed in R138X heterozygous clusters, showing improved connectivity and activity under Zn2+ depletion. (4) Conclusion and interpretationIntracellular Zn2+ depletion compromises islet-like cluster identity and function, while the R138X variant confers protection against these effects. Under Zn2+-depleted conditions, ZnT8 deficiency promotes a more mature and metabolically active state of the R138X clusters, with enhanced Ca2+ signaling and insulin secretion, supported by a structural remodeling and the downregulation of apoptosis and cellular stress. These findings highlight the therapeutic potential of targeting ZnT8 in type 2 diabetes and support its relevance for further improving cell-based therapies. Research in ContextO_ST_ABSWhat is already know about this subject?C_ST_ABSO_LIRare inactivating mutations in the insulin granule-associated zinc transporter gene, SLC30A8/ZnT8, drive lowered type 2 diabetes risk. C_LIO_LIPrevious studies have indicated that apoptosis is lowered, and glucose-stimulated insulin secretion enhanced, after ZnT8 inactivation. C_LIO_LIThe molecular mechanisms underlying these changes are unclear. C_LI What is the key question?O_LIHow do inactivating mutations in SL30A8/ZnT8 lead to lowered apoptosis and enhanced insulin secretion from stem cell-derived islet-like clusters, and is altered susceptibility to intracellular zinc depletion involved? C_LI What are the new findings?O_LIThe rare inactivating R138X mutation in SLC30A8 leads to gene dose-dependent changes in the transcriptome and proteome of islet-like clusters. C_LIO_LIChanges include upregulation of maturity and downregulation of immaturity genes. C_LIO_LIDepletion of intracellular Zn2+ exaggerates the protective effects of the inactivating mutation on apoptosis and insulin secretion C_LI How might this impact on clinical practice in the foreseeable future?O_LIOur findings suggest that careful monitoring of both dietary zinc intake and of circulating levels of zinc ions, whose effects are mitigated in SLC30A8 mutation carriers, may be helpful in some populations to lower diabetes risk. C_LI

Primary ovarian insufficiency (POI) and related infertility, early menopause, and endocrine disorders due to hormonal deficiency are major side effects in young female cancer patients undergoing cancer therapy. Current strategies preserving the fertility and hormonal functions of the ovary remain imperfect due to concerns of feasibility, efficacy, or safety. Herein, we identified c-Jun N-terminal kinase (JNK) as a pivotal regulator of the DNA damage response (DDR) signaling in oocytes of primordial follicles in response to DNA-damaging cancer therapy. Using pharmacological JNK inhibition and a genetically modified mouse model with oocyte-specific JNK deletion, together with histological, bioinformatic, and molecular approaches, we demonstrated that JNK inhibition prevented chemotherapy-induced oocyte apoptosis and POI, and preserved long-term reproductive cycles and fertility. Mechanistically, JNK was activated in response to chemotherapy-induced DNA damage in oocytes of primordial follicles, causing activation of transcription factor TAp63 and subsequent oocyte apoptosis, ultimately resulting in diminished ovarian reserve and POI. A more clinically relevant breast cancer-bearing mouse model revealed that JNK inhibition preserved the ovarian reserve without compromising anti-cancer efficacy of chemotherapy. Together, our study identifies oocyte-intrinsic JNK as a promising target for developing ovarian protectants and safeguarding reproductive health and fertility in young female cancer survivors.