The Journal of Steroid Biochemistry and Molecular Biology

Menopausal hormone therapy (MHT) is prescribed for climacteric symptoms including hot flushes and weight gain and contains estrogens such as 17 beta-estradiol (17{beta}E2). However, estrogen receptor activation by MHT may increase reproductive cancers and cardiovascular event risk in some people. As the protective metabolic effects of 17{beta}E2 are partly mediated through the arcuate nucleus of the hypothalamus, restricting 17{beta}E2 actions to the brain could serve as a safer mechanism of MHT. 10{beta},17{beta}-Dihydroxyestra-1,4-dien-3-one (DHED) is a prodrug of 17{beta}E2 which is enzymatically converted to the parent hormone exclusively within the brain. DHED has demonstrated positive benefit in rodent models of centrally-mediated maladies including hot flushes, depression and cognitive decline, without peripheral hormonal burden. Therefore, we hypothesized that DHED treatment in obese female mice would act within the hypothalamus to provide the same beneficial metabolic effects as 17{beta}E2. Female mice were ovariectomized, placed on a high fat diet and split into either control, DHED, or 17{beta}E2 treatment groups. Body weight, uterus weight and glucose tolerance were recorded along with gonadal hormone receptor expression in the brain. Delivery of DHED at a similar dose as 17{beta}E2 failed to improve metabolic parameters or recapitulate the hypothalamic responses induced by 17{beta}E2. Delivery of DHED at higher doses, which elicited estrogen-like actions within the brain, still failed to improve metabolic health. Our findings suggest that peripheral actions, in addition to hypothalamic targets, may be required to mediate 17{beta}E2s protective effects on metabolism and that brain-targeted MHT may be unsuitable for improving metabolic health during menopause.

PurposeMaternal high-fat diet (mHFD) induces metabolic disturbances that lead to inflammatory responses in the offsprings brain. The retina, as part of the central nervous system, may be similarly affected. This study aimed to determine how mHFD affects microglial and Muller cell activity in the retinas of offspring and assess how these effects depend on sex and the female estrous cycle. MethodsFemale C57Bl/6J mice were fed a control diet (CD, 10% fat) or a high-fat diet (HFD, 60% fat) from weaning to lactation. The offspring were weaned to a normal rodent diet. Retinal structure and glial cells were assessed using immunohistochemical labeling of retinal ganglion cells, Muller glia, astrocytes, phagocytic and inflammatory markers. Observed retinal changes in female offspring were correlated with the estrous cycle stages. ResultsmHFD induced subtle retinal structural changes and sex-specific alterations in glial cells of offspring peripheral retina. Male offspring exhibited a reduced microglial area, accompanied by increased phagocytic capacity, whereas females showed the opposite pattern. Under mCD, the microglial area and its phagocytic and metabolic activity fluctuated with the female estrous cycle, while mHFD diminished the differences between phases. Additionally, mHFD reduced Muller glial reactivity in females, indicating disrupted glial communication. ConclusionsOur findings demonstrate that mHFD has a sex-specific effect on the offsprings peripheral retina, affecting the response of retinal microglia to female reproductive hormones.

BackgroundThe effects of dexamethasone during in vitro human osteogenesis present a complex picture. On one side, dexamethasone promotes the osteogenic differentiation of human bone marrow mesenchymal stromal cells (BMSCs) by downregulating SOX9. On the other side, it simultaneously promotes adipogenesis through the upregulation of PPARG. The regulation of SOX9 and PPARG levels appears to be mediated by the transactivation function of the glucocorticoid receptor (GR), suggesting an indirect effect of dexamethasone on SOX9 downregulation. This study aims to determine whether PPAR-{gamma} affects the expression levels of SOX9, as suggested by several studies. MethodsHuman BMSCs were isolated from bone marrow and cultured in different osteogenic induction media containing 10 or 100 nM dexamethasone. Undifferentiated cells were used as control. Cells were treated either with a pharmacological PPAR-{gamma} inhibitor (T0070907) or with a PPARG-targeting siRNA. Differentiation markers or PPAR-{gamma} target genes were analysed by RT-qPCR. Mineral deposition was assessed by Alizarin Red staining. Two-way ANOVA followed by a Sidak multiple comparison test was used to compare the effects of treatments. ResultsPharmacological inhibition of PPAR-{gamma} had a mild effect on the expression of PPAR-{gamma} target genes but hindered adipocyte formation. Neither RUNX2 nor SOX9 expression were affected by T0070907. siRNA treatment successfully downregulated PPARG expression, as well as that of PPAR-{gamma} target genes LPL, LPAR1, and ADIPOQ. Contrary to expectations, RUNX2 was significantly downregulated by the PPARG-siRNA treatment during osteogenic differentiation both in the absence and presence of dexamethasone, while SOX9 levels were downregulated in undifferentiated cells. Overall, Alizarin Red staining analysis showed no change in mineralization levels when PPARG expression or activity was inhibited. ConclusionsUnderstanding how dexamethasone regulates human BMSC differentiation is crucial to refine current in vitro models. These results suggest that PPAR-{gamma} is not involved in SOX9 or RUNX2 repression during in vitro osteogenic differentiation of human cells.

Purpose: To evaluate the efficacy and safety of oral L-ergothioneine (EGT) in improving ovarian reserve and clinical symptoms in women with diminished ovarian reserve (DOR). As a proof-ofconcept study, we explored correlations between hormonal shifts and symptom amelioration. Methods: This single-center, open-label trial enrolled 40 women (aged 35-45 years) with DOR (baseline AMH: 1.0-3.0 ng/mL) and menstrual disorders. Participants received oral EGT (120 mg/day) for three consecutive menstrual cycles. The primary outcome was the change in serum AMH. Secondary outcomes included sex hormones (FSH, E2), antral follicle count, and validated clinical questionnaires (modified Kupperman Index [KI], PSQI, SF-36, and Menstrual Symptom Score). Results: Thirty-six participants completed the intervention without product-related adverse events. EGT significantly improved core ovarian markers: mean AMH increased from 1.79 {+/-} 0.71 to 2.47 {+/-} 1.52 ng/mL (p = 0.029). Concurrently, basal FSH decreased (8.22 {+/-} 2.93 to 7.05 {+/-} 2.47 mIU/mL, p = 0.032) and E2 increased (46.00 {+/-} 22.70 to 63.46 {+/-} 50.10 pg/mL, p = 0.030). Clinical assessments showed progressive reductions in KI (5.42 {+/-} 3.66 to 1.90 {+/-} 2.16, p < 0.0001) and PSQI scores (6.89 {+/-} 1.82 to 5.50 {+/-} 1.40, p < 0.0001), alongside improved menstrual and SF-36 scores (p < 0.001). Subgroup analysis revealed upward AMH trends across both the 35-39 and 40-45 age cohorts. Crucially, endocrine restoration ({Delta}FSH) significantly correlated with improvements in sleep quality ({Delta}PSQI, r = 0.43, p < 0.05) and E2 increases (r = -0.46, p < 0.05), linking hormonal stabilization directly to systemic relief. Conclusion: Oral EGT safely enhances serum AMH and optimizes the FSH/E2 balance in women with DOR, yielding substantial relief from peri-menopausal and sleep disturbances. This pilot proofof- concept study provides the first clinical evidence supporting EGT's systemic benefits in reproductive aging, laying the groundwork for future placebo-controlled trials. Trial Registration: ChiCTR2500104484; Prospectively registered on 2025-06-18. Keywords: L-Ergothioneine, diminished ovarian reserve, anti-Mullerian hormone (AMH), oxidative stress, clinical trial

Perinatal development of the mammary gland is regulated by hormonal signals that influence cell proliferation, extracellular matrix remodeling, immune cell recruitment, and intracellular signaling. While the role of estrogen in mammary gland development is well established, the impact of androgens remains less understood. To address this gap, we inhibited androgen signaling in utero using the anti-androgen flutamide (FLU) and investigated the effects on mammary gland development in rats. Using an integrative strategy combining histology, transcriptomics, lipidomics, cytokine profiling, and high-resolution imaging, mammary tissue were analyzed at pre-puberty (postnatal days (PND) 21), peri-puberty (PND46), and adulthood (PND9O). FLU exposure induced subtle, yet significant, alterations in mammary morphology and molecular signatures. At PND2l, the FLU exposed group exhibited an increased number of adipocytes with reduced size. Transcriptomic analysis revealed differentially expressed genes at PND2l and enrichment in pathways related to androgen response and immune signaling, but minimal changes at later developmental stages. Lipidomic profiling showed transient disruption in long-chain fatty acid composition at early developmental stages. Cytokine profiling revealed a reduced adaptive immune response at PND46 and PND9O, and second harmonic generation imaging demonstrated changes in collagen fiber orientation and density across all developmental stages. These data indicate that prenatal androgen signaling is essential for proper stromal development and the establishment of early transcriptional networks in the mammary gland, with only minor long-term effects on glandular architecture in adult nulliparous females.

BackgroundAdrenal steroid hormone production is essential for systemic stress adaptation and metabolic homeostasis, and it is tightly regulated by oxygen availability. Previously, we demonstrated that acute hypoxia suppresses adrenal steroidogenesis through HIF-1-dependent induction of microRNAs (miRNAs) that target key steroidogenic enzymes. However, the mechanisms by which HIF-1 controls miRNA expression and activity in this context remain unclear. MethodsTo address this issue, we mapped the genome-wide HIF-1 binding landscape in murine adrenocortical cells using Cleavage Under Targets & Tagmentation (CUT&Tag). We integrated this data with gene expression analyses following pharmacological HIF-1 stabilization, physiological hypoxia, and genetic HIF-1 depletion to distinguish HIF-1-dependent effects from broader hypoxia-driven responses. ResultsWe detected HIF-1 binding at loci encoding steroidogenic enzymes and steroidogenesis-associated miRNAs. Unexpectedly, we also detected binding at genes involved in miRNA biogenesis and function, including components of the nuclear microprocessor complex and the cytoplasmic RNA-induced silencing complex (RISC). Functional analyses revealed that hypoxia broadly represses the expression of miRNA-processing genes through both HIF-1-dependent and -independent mechanisms. Notably, HIF-1 selectively modulated or counteracted this repression in a gene-specific manner, indicating a regulatory role beyond direct transcriptional activation. ConclusionsThese findings reveal an unrecognized layer of hypoxia-driven cell communication, wherein HIF-1 coordinates the transcriptional and post-transcriptional regulation of adrenal steroidogenesis by shaping the miRNA-processing landscape. This work extends our understanding of how oxygen-sensitive signaling pathways integrate gene expression and RNA-based regulatory mechanisms to control endocrine function.

The placenta is an important producer of hormones essential for fetal development. Insulin-like growth factor 1 (IGF1) is a hormone primarily produced in the placenta in utero and is an important regulator of various developmental pathways including those in heart and liver. Embryonic disruptions in these developmental pathways can lead to lifelong changes and are often associated with chronic disease. Further, the placenta has sex-specific impacts on offspring development in response to hormonal changes. Previous work has shown that altered expression of Igf1 in the placenta results in sexually dimorphic changes to placental and fetal developmental outcomes. Here, mice underwent placental-targeted CRISPR manipulation for overexpression or insufficiency of Igf1. At the time of euthanasia, heart and liver tissues were collected and weighed. This dataset presents the heart and liver mass of these postnatal mice. There was a significant increase in proportional heart mass in placental Igf1 overexpression adult female mice and a trending increase in proportional liver mass in placental Igf1 overexpression adult male mice. No significant changes in heart or liver mass were seen in placental Igf1 insufficiency mice. These data provide insight into the impact of placental IGF1 on long-term heart and liver development. VALUE OF THE DATAO_LIThere is significant evidence for the role of early genetic changes in influencing long-term health outcomes, as laid out by the Developmental Origins of Health and Disease (DOHaD) hypothesis [1]. According to this hypothesis, genetic factors may be critical in determining the timing and severity of chronic disease, with varying effects based on sex. Genetics of the placenta, which makes up the maternal-fetal interface, plays an important role in modulating exposures associated with the DOHaD hypothesis [2]. C_LIO_LIThe placenta provides essential hormones to the fetus during pregnancy [3]. Placental changes are associated with the development of chronic disease and metabolic changes [4,5]. Disruptions in placental functions have been linked to defects including congenital heart disease which affects approximately 40,000 babies each year in the United States [6,7]. The placenta is also linked to metabolic diseases later in life such as nonalcoholic fatty liver disease, a chronic liver disease which has increased in prevalence by over 50% from 1990 to 2019 [5,8,9]. C_LIO_LIInsulin-like growth factor 1 (IGF1) is a placentally produced factor that regulates pathways involved in fetal growth and development and has been shown to be critical in growth of the heart and liver [10-13]. Despite the importance of the placenta and IGF1 in heart and liver growth, specific links between placental Igf1 expression and developmental outcomes remain understudied. C_LIO_LIPlacental function is known to have sex-specific impacts on fetal growth [14]. Further, Igf1 expression in the placenta is linked to differences in offspring developmental outcomes by sex [15]. Placental Igf1 overexpression and insufficiency affect offspring in a sexually dimorphic manner. IGF1 is a hormone and interacts with sex hormones, likely contributing to sex differences in response to changes in Igf1 expression [16]. Further research, including the work done to produce this dataset, may help clarify the role of placenta Igf1 expression in fetal outcomes, specifically regarding sex differences. C_LIO_LIThe data presented in this paper provide insight into the effects of placental Insulin-like growth factor 1 overexpression and insufficiency on adult heart and liver mass. More research is needed to understand specific functional impacts on these organs. Further, understanding the effects of placental genetic changes may support the development of future treatments and therapies for placental insufficiencies. C_LI

BackgroundKidney stones, a prevalent urological disorder, are increasingly associated with potential skeletal health issues, including reduced bone mineral density (BMD) and an elevated risk of osteoporosis. However, the underlying mechanisms and subgroup-specific associations have not yet been adequately explored. MethodsThis study used data from a nationally representative survey with a weighted complex sampling design. A total of 6,464 participants were enrolled in the study. We performed weighted and unweighted comparative analyses, multivariate linear regression, mediation analysis, and subgroup evaluations to examine the association between kidney stones and BMD of the femoral neck and lumbar spine. Potential mediators, including the systemic immune-inflammation index (SII), estimated glomerular filtration rate (eGFR), and calcium-to-phosphorus (CaP) ratio, were investigated. ResultsThe presence of kidney stones was significantly associated with lower femoral neck BMD ({beta} =-0.015, p = 0.046) after adjusting for confounding factors. The CaP ratio was identified as a significant mediator (average causal mediation effect [ACME] = 0.00077, p = 0.028), whereas the SII and eGFR did not show significant mediating effects. Stratified analyses revealed stronger associations in participants aged < 50 years and in those without chronic kidney disease (CKD). No significant interactions according to gender were detected. ConclusionKidney stones are independently associated with reduced BMD, which is partially mediated by altered calcium-phosphorus homeostasis. These findings highlight the importance of monitoring bone health in patients with kidney stones, particularly in younger and non-CKD populations, and suggest that dietary mineral balance may play a critical role in bone-stone interaction.

Salt-sensitive hypertension, a condition in which the blood pressure (BP) increases with an increase in salt intake, is influenced by behavioral, genetic, and environmental factors. Salt sensitivity is associated with variants of the G protein-coupled receptor kinase 4{gamma} (GRK4{gamma}) and the renal sodium bicarbonate cotransporter 2 (NBCe2), encoded by the solute carrier family 4 member 5 (SLC4A5). The R>65L variant (rs2960306) of human GRK4 (hGRK4{gamma} 65L) contributes to salt sensitivity through a signaling pathway and gene-gene interaction with SLC4A5. Global expression of GRK4{gamma} 65L in transgenic mice results in salt-sensitive hypertension, due in part to an increase in endogenous GRK4 and angiotensin type 1 receptor (AT1R) expression. Grk4 knockout (Grk4-/-) mice have decreased blood pressure and are salt-resistant. The expression of hGRK4{gamma} 65L only in the kidney of Grk4-/- mice increases BP in response to a high salt diet. The renal expression of SLC4A5 is increased in hGRK4{gamma} 65L transgenic mice, relative to mice expressing wild-type (WT) human GRK4 (hGRK4 65L), without endogenous mGrk4. Human renal proximal tubule cells (hRPTCs) endogenously expressing GRK4 WT and SLC4A5 WT, SLC4A5 variants, GRK4 65L, and both GRK4 65L and SLC4A5 variants were studied. SLC4A5 expression is increased in hRPTCs expressing GRK4 65L and in cells expressing both GRK4 65L and SLC4A5 variants compared with GRK4 WT and SLC4A5 WT. Luminal and basolateral sodium transport in hRPTCs is increased in the presence of both hGRK4 65L and SLC4A5 variants. GRK4 interacts with nuclear histone deacetylases (HDACs). Mice expressing hGRK4 65L only in the kidney have decreased expression but increased phosphorylation of HDAC1. HDAC1 expression is decreased and HDAC1 but not HDAC2 phosphorylation is increased in hRPTCs expressing both hGRK4 65L and SLC4A5 variants. The presence of hGRK4{gamma} 65L decreased HDAC1 expression but increased AT1R expression in the kidneys of mice on high salt diet. Our results show that GRK4{gamma} 65L causes salt-sensitive hypertension by increasing renal SLC4A5 and AT1R expressions by inhibiting the HDAC1 pathway.

Kidneys play an essential role in balancing fluid and electrolyte levels. Two mouse strains, C57Bl/6 and 129S2/SV, are routinely used to study renal physiology in laboratory settings, and prior observations suggest that significant differences in salt and water handling exist between them. This study aims to further establish the sources of these observed differences at both expressional and functional levels, in male and female mice. At baseline, male 129S2/SV mice displayed decreased Na+ and increased K+ plasma concentrations compared to C57Bl/6 males, while no statistical differences were observed between female mice. Interestingly, 129S2/SV male mice had lower glomerular density than C57Bl/6 males. Immunoblotting shows that 129S2/SV mice of both sexes had increased expression of NHE3 and NKCC2 compared to their C57Bl/6 counterparts. Both total and phosphorylated NCC were more abundant in female mice as compared to males, indicating sexual dimorphism. Furthermore, 129S2/SV females had higher expression of total and phosphorylated NCC compared to C57Bl/6 females. In contrast, the expression of SGLT2, ENaC subunits, and Na+/K+-ATPase were comparable between C57Bl/6 and 129S2/SV mice of both sexes. When challenged with diuretics intended to block NKCC2, NCC or ENaC, 129S2/SV male mice responded with a smaller diuresis and natriuresis than their C57Bl/6 counterparts. Taken together, our data suggest that differential expression of key Na+ transporters along the nephron contributes to differences in Na+/K+ homeostasis between these two mouse strains. NEW & NOTEWORTHYWe assessed the influence of genetic background on the expression of key Na+ transporters along the nephron in two commonly used inbred mouse strains, C57Bl/6 and 129S2/SV. We found that the kidney expression of NHE3, NKCC2, and NCC are strain dependent. Additionally, murine strain significantly contributes to the diuretic responses induced by hydrochlorothiazide, amiloride, and furosemide.

AimsThe Mediterranean diet is associated with reduced cardiometabolic risk, yet its physiological effects during pregnancy and its impact on placental metabolism remain incompletely understood. This study aimed to determine whether maternal adherence to a Mediterranean diet during pregnancy influences placental lipid metabolism and signalling pathways involved in nutrient handling, tissue remodelling, and inflammation, and to assess their relationship with pregnancy outcomes. MethodsPlacental samples and clinical outcome data were analysed from pregnant women participating in an unblinded randomized clinical trial of a Mediterranean diet intervention. Placental lipid composition was quantified and the expression of genes and signalling pathways involved in lipid metabolism, nutrient transport, inflammation, and tissue remodelling was evaluated. ResultsMaternal adherence to a Mediterranean diet during pregnancy was associated with significant alterations in placental lipid composition, including reduced C18:0 and C24:0 and increased C18:1n9c, C20:3n6, and C22:0, with lower total short-chain fatty acids and higher monounsaturated fatty acids. Placental expression of lipid metabolism regulators ALOX15 and PPAR{gamma} was reduced, alongside downregulation of AKT and p38 MAPK signalling pathways. Placentas from mothers adhering to the Mediterranean diet also showed lower expression of amino acid and glucose transporters SLC3A2 and SLC2A1, as well as altered inflammatory and extracellular matrix remodelling markers, including decreased SOCS3 and GHR and increased PAI1 and MMP3. ConclusionsMaternal adherence to a Mediterranean diet during pregnancy modifies placental lipid composition and regulates pathways involved in lipid handling, nutrient transport, inflammation, and tissue remodelling, providing insight into mechanisms linking maternal diet with placental metabolic function.

Tissue-nonspecific alkaline phosphatase (TNAP) is a ubiquitous enzyme whose substrates are various phosphorylated extracellular molecules including pyridoxal phosphate (vitamin B6) and adenine nucleotides. Dysfunctions of TNAP result in hypophosphatasia, a rare disease characterized by defective bone mineralization and impaired brain functions. In the brain, TNAP expression peaks during development and is associated with various steps of neurogenesis. However, the influence of TNAP activity on neurogenesis remains poorly understood in its cellular and molecular aspects. Here we used the SK-N-SH D human neuroblastoma cell line as a cell culture model to further investigate the involvement of TNAP in neuronal precursor proliferation and neuronal differentiation. We also used 1H-NMR-based metabolomics to investigate the molecular correlates of TNAP action on SK-N-SH D cell proliferation and differentiation. We first observed an increase in alkaline phosphatase (AP) activity when the cells were placed in differentiation medium. We next found that inhibiting TNAP with a specific inhibitor (MLS-0038949) impeded neuroblastoma cell proliferation. TNAP inhibition also hindered neuronal differentiation, as evidenced by a decrease in the number of neurite-bearing cells. In contrast, neurite length was not affected by TNAP inhibition, suggesting that TNAP controls neurite sprouting, but not neurite outgrowth per se. The metabolomic results indicate that proliferation and differentiation are associated with a decrease in the amounts of proteinogenic amino acids as well as that of compounds potentially involved in lipid production. This analysis also revealed that proliferation and differentiation are associated with increased glutathione levels and decreased amounts of hypotaurine and taurine, supporting proposals that organosulfur compounds play an important role in these processes. Since pyridoxine was present in the culture media, these results suggest that TNAP is involved in neurogenesis through mechanisms in addition to its role in vitamin B6 metabolism and may instead involve the ectonucleotidase activity (or an unidentified activity) of TNAP.

BackgroundDexamethasone (DEX) is used in vitro to promote osteogenic differentiation of human bone marrow mesenchymal stromal cells (hBMSCs). In clinical use, however, glucocorticoids induce osteoblast and osteocyte apoptosis while increasing osteoclast survival, leading overall to osteoporosis and high fracture risk. The overall impact of DEX on the differentiation of human progenitor cells remains contradictory and not fully understood, highlighting the need for further investigation using sequencing approaches as in vitro results will naturally influence further translational research. MethodshBMSCs were induced to osteogenic differentiation for 7 days using different concentrations of either DEX or the nonsteroidal glucocorticoid receptor agonist (+)-ZK216348. cDNA library preparation and RNA sequencing (RNAseq) were performed using Oxford Nanopore Technologies. Differentially expressed genes and pathways associated to the transactivation or transrepression activity of DEX were identified. Sequencing results were validated by qPCR, protein analysis, and with a functional assay on peripheral blood mononuclear cells to determine the overall effect of the BMSC supernatant. ResultsHierarchical clustering of RNAseq data identified eight subclusters with shared regulatory patterns. Enrichment analysis revealed that both upregulated and downregulated genes are involved in ossification and extracellular matrix organization pathways. Several pro- and anti-inflammatory genes were differentially regulated. qPCR analysis validated the upregulation of CXCL1, CXCL8, IL18, and COL8A1, while MMP1 and CXCL12 expression decreased in response to DEX. Comparing DEX results with those obtained using (+)-ZK216348 helped distinguish the potential mechanisms regulating the expression of specific genes. Notably, CXCL8 upregulation occurred through transactivation, whereas COL8A1 upregulation is downstream of a transrepressed gene. Further in vitro experiments confirmed that DEX significantly increased CXCL8 expression and IL-8 secretion. However, hPBMC responses indicated no significant pro- or anti-inflammatory effects from hBMSC conditioned medium. ConclusionsIn conclusion, the effects of DEX on the transcriptome of hBMSCs in a pro-osteogenic environment do not fully replicate the acquisition of an osteogenic phenotype. Several genes associated with ossification, extracellular matrix organization, and inflammation were dysregulated. The unique expression patterns of pro-inflammatory cytokines and collagen types warrant further investigation to elucidate their roles in osteogenic differentiation and bone homeostasis.

Bisphenol A (BPA) and Bisphenol S (BPS) exposure disrupt ovarian function and granulosa cell (GC) steroidogenesis. Extracellular vesicles (EVs) and their miRNA cargo, as mediators of cellular response to environmental stimuli, might be involved in fertility and folliculogenesis. This study explored modulation of microRNA expression after 48h BPA or BPS exposure (10 {micro}M) in ovine primary GC and EVs from corresponding conditioned medium (CM EVs). Small RNA sequencing of control (0h) and 48h treated GC, CM EVs as well as follicular fluid EVs allowed identification of 533 ovine miRNAs, including 129 new sequences. BPA did not alter miRNA expression in GC, while BPS decreased cellular oar-24b miR. In contrast, BPA modified expression of 4 miRNAs in CM-EVs, including 3 new sequences, and two miRNAs were modified by BPS. Both compounds reduced expression of sequence homologous to miR-1306. Further studies are required to decipher their roles in bisphenol toxicity in GC.

BackgroundDiabetes mellitus leads to skeletal muscle dysfunction associated with loss of strength, impaired blood perfusion, lipid accumulation, and inflammation. The opening of large-pore channels has been linked to increased membrane permeability and inflammatory signaling in several pathologies. Boldine, an alkaloid from Peumus boldus, blocks large-pore channel activity and exhibits antioxidant and anti-inflammatory properties. This study evaluated whether boldine prevents skeletal muscle alterations induced by diabetes and explored potential underlying mechanisms. MethodsDiabetes was induced in male C57BL/6J mice using streptozotocin (STZ, 40 mg/kg/day for 5 days). Diabetic mice were treated with boldine (50 mg/kg/day) for four weeks. Muscle strength and resting membrane potential were analyzed in vivo. Also, right gastrocnemius muscle blood perfusion at basal and after acetylcholine (10 M) stimulation were analyzed in vivo. Lipid accumulation was assessed using Oil Red O staining, and CD31 immunodetection was used to evaluate capillary density. mRNA levels of NLRP3 were evaluated in muscle by qPCR. In human myoblasts (AB1167) cultured under low (8 mM) or high glucose (25 mM) conditions, with or without boldine, membrane permeability (ethidium uptake), intracellular Ca{superscript 2} (Fura-2), nitric oxide (DAF-FM), and levels of NLRP3 and Casp1 (qPCR) and reactivity PPAR{gamma} (Immunofluorescence) were determined. ResultsSTZ mice showed reduced muscle strength and depolarized resting membrane potential, both prevented by boldine. Basal muscle perfusion was [~]20% lower in diabetic mice (160.1 {+/-} 17.2 vs. 199.1 {+/-} 13.8 units), whereas boldine preserved perfusion (184.6 {+/-} 14.3 units). Oil Red O-positive fibers increased to 52.4 {+/-} 3.6% in diabetic mice and decreased to 15.2 {+/-} 4.1% with boldine (control: 3.1 {+/-} 1.3%; p<0.05). NLRP3 mRNA increased 17.7 {+/-} 2.8-fold in diabetic muscle and was reduced by [~]50% with boldine. In myoblasts, high glucose increased ethidium uptake, nitric oxide production, NLRP3 and caspase-1 expression, and nuclear PPAR{gamma} ([~]45% positive nuclei); all effects were prevented by boldine. ConclusionsBoldine preserves skeletal muscle function and vascular reactivity in diabetes and prevents lipid accumulation and inflammasome activation both in vivo and in vitro. These effects are associated with inhibition of large-pore channel activity and attenuation of downstream calcium-dependent, inflammatory, and adipogenic pathways, supporting boldine as a promising therapeutic candidate for diabetes-associated skeletal muscle dysfunction. Graphical abstractIn myoblasts, high glucose activates large-pore channels, elevating cytoplasmic Ca{superscript 2} concentration and nitric oxide generation, which increases the activity of Cx-formed hemichannels, raises the levels of inflammasome components, and promotes lipid accumulation. In STZ-diabetic mice, de novo expression of large-pore channels in skeletal muscles contributes to reduced blood perfusion, accumulation of intramuscular fat, muscle weakness, and reduced resting membrane potential of myofibers. Boldine inhibits large-pore channel activity, preventing these alterations and preserving muscle physiology in vivo. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/707704v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@19179b4org.highwire.dtl.DTLVardef@1cd3d21org.highwire.dtl.DTLVardef@16851d6org.highwire.dtl.DTLVardef@1d4e77c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Steroidal alkaloids may be responsible for some of the health benefits of a tomato rich diet, but little is known about their metabolic fate after consumption. The objective of this study was to elucidate the pharmacokinetic parameters of plasma steroidal alkaloids and to define their bioavailability and metabolism following a single tomato containing meal. Healthy subjects (n = 11, 6M/5F) consumed 505 g of tomato juice following a two-week tomato washout and blood plasma were collected post-prandially at 11 time points over 12-hours. Plasma steroidal alkaloids were analyzed using UHPLC-MS. The fractional absorption of steroidal alkaloids was 11.8 {+/-} 7% and over 99% of the absorbed dose were present as metabolized products. The maximum concentration of total plasma steroidal alkaloids in subjects was 406.5 {+/-} 377.0 nmol/L occurring at 6 hours after consumption, with an AUC0-12hr of 2529.0 {+/-} 1644.8 nmol*h/L. Liver S9 enzymatic synthesis of steroidal alkaloid metabolites including trihydroxy-tomatidine and sulfonated dihydroxy-tomatidine improved confidence in compound identification. This study reports the first pharmacokinetic data for tomato steroidal alkaloids, demonstrating moderate absorption and extensive metabolism after tomato juice consumption. These data provide context for future studies investigating the potential role that these compounds may play in human health.

Cannabis (marijuana) is the most widely used recreational drug in the USA accounting for about 62 million users in 2024. Among cannabis users, 26% are of prime reproductive age (18-25 years). Delta-9 tetrahydrocannabinol (THC) is the principal psychoactive component of cannabis and has been detected in human seminal fluids. Although abundant evidence indicates adverse effects of THC exposure on spermatogenesis in different species, acute effects of THC on postejaculatory sperm including fertilization potential and subsequent carryover effects on embryo development are largely unknown. The present study was designed to provide missing information on structural and mechanistic effects of THC exposure to postejaculatory sperm function by evaluating sperm indices often overlooked or masked during clinical evaluation. A bovine embryo continuum model was employed to determine effects of THC on sperm structure, kinematics, bioenergetics, and binding mechanisms. Effects of THC on the sperm genomic and epigenomic landscape were determined, complemented by paternal carry over effects on embryo development as a human translational model to elucidate paternal effects on future development, and to mirror sperm exposure during transport within the female reproductive tract. Cryopreserved bovine sperm from three bulls were independently exposed to physiologically relevant concentrations of THC (0 and 32nM, n = 2 individual replicates/bull) for 24 h under non-capacitating conditions at 25{degrees}C followed by quantification of sperm kinematics at 37{degrees}C. Samples of THC-exposed sperm and vehicle-control (0.1% DMSO) were collected in replicate following immediate addition of THC (0 h) and again at 24 h. DNA damage, acrosome integrity, bioenergetics, changes to DNA methylation and embryo development were quantified. Data were analyzed by logistic regression with a generalized linear mixed effect model. Computer-assisted sperm assessment revealed a reduction in progressive motility of THC-exposed sperm after 24 h while other parameters were not affected. Acrosome integrity as determined by flowcytometric analysis with FITC-PSA was severely compromised in THC-exposed sperm (P [&le;] 0.05), despite no detectable difference in capacitation status using merocyanine staining. Similarly, DNA integrity as determined by TUNEL assay was significantly impaired after 24 h of THC exposure (P [&le;] 0.05). Mechanistic effects of THC were explored through characterization of the transmembrane G-protein coupled cannabinoid 1 receptor (CB1). CB1 is expressed in the post-acrosomal region and its abundance decreased as compared to unexposed sperm. Alterations to the methylation landscape of sperm were then determined after 24 h of THC exposure through whole-genome Enzymatic Methyl Sequencing. PCA analysis indicated that sperm from different males formed distinct clusters, implying individual differences among bulls, while the effects of THC exposure produced tighter clusters. Paternal carryover effects on embryos derived by in vitro fertilization from THC exposed sperm had reduced 2-cell cleavage, 8-16 cell morula development, and reduced blastocyst development compared to unexposed sperm (46% vs. 33%). In conclusion, post-ejaculatory mammalian sperm exposure to THC compromises acrosome integrity, induces DNA damage, changes the sperm methylome, and reduces developmental potential. Collectively, these data implicate new considerations for recreational and clinical use of cannabis that impact cellular and molecular mechanisms important for sperm function with detrimental consequences for gamete interaction and embryo development.

BackgroundDeoxycorticosterone acetate (DOCA)-salt induces greater increases in blood pressure (BP) and a more pro-inflammatory T cell profile in males compared to females. T cells contribute to DOCA-salt hypertension, however, the mechanisms driving T cell activation remain unclear. The NLRP3 inflammasome has been implicated in DOCA hypertension in male mice. Little is known regarding NLRP3 in females. The goal of the current study was to test the hypothesis that NLRP3 contributes to greater increases in BP and renal inflammation with DOCA in males vs. females. MethodsRenal NLRP3 protein levels were measured in normotensive and hypertensive male and female subjects and in male and female Sprague Dawley uni-nephrectomized (UNX) control and DOCA-salt rats. Additional 11-wk-old Sprague Dawley rats were UNX and randomized to: 1) DOCA + vehicle or 2) DOCA + the NLRP3 inhibitor MCC950 (10 mg/kg/day in saline) from 11-14 wks of age. At 14-wks-of-age rats were euthanized, terminal plasma samples and remaining kidneys were collected for flow cytometric analysis of T cells. ResultsRenal NLRP3 levels were significantly greater in hypertensive males and females vs. normotensive controls. DOCA increased BP in both sexes, with greater elevations in males. MCC950 attenuated DOCA-induced increases in BP in male, but not female rats. MCC950 decreased circulating and renal CD4 and Th17 cells in both sexes, although the effect was greater in males. ConclusionDespite both males and females exhibiting an increase in NLRP3 in hypertension, NLRP3 contributes to BP elevations only in DOCA-salt males.

Background: A previous meta-analysis by Singh-Ospina et al. (2017) suggested that Gender affirming hormone treatment (GAHT) does not change transgender mens bone mineral density (BMD) at any clinically relevant site; emerging studies and advances in synthesis methods necessitate an updated evaluation. The primary aim was to update the bone measures of Singh-Ospina et al. (2017), with the secondary aim to expand measures to how GAHT affects musculoskeletal health. Methods: A systematic review with meta-analysis was conducted using studies published in English up to 31 July 2024, identified through three electronic databases (PubMed, Embase, SportDiscus), and final cross-referencing in summer 2025. Primary outcomes were longitudinal changes in femoral neck (FN), lumbar spine (LS), and total hip (TH) bone mineral density (BMD). Secondary outcomes included body composition and muscle strength. Standardised effect sizes (Hedges g) were pooled using the inverse heterogeneity (IVhet) model. Results: GAHT (4 years) was not associated with significant longitudinal changes in FN, LS, or TH BMD. In contrast, substantial anabolic effects were observed, including increases in BMI (g = 0.13), body mass (g = 0.18), fat-free mass (g = 0.59), and muscle strength (g = 0.86). Heterogeneity was high for muscle strength, FN and TH BMD, limiting confidence in pooled estimates. Conversely, changes in LS BMD, BMI, body mass and fat-free mass demonstrated low heterogeneity and greater consistency across studies. Conclusion: Masculinising GAHT does not negatively affect clinically relevant BMD sites while reliably increasing lean mass and muscle strength; however, the evidence base remains methodologically weak and highly variable, particularly for FN and TH. The need for continued clinical monitoring of bone health and muscle function, alongside high-quality longitudinal research incorporating advanced imaging modalities such as HR pQCT is emphasised. Strengthening the evidence base will be essential for clarifying long-term skeletal trajectories as transgender men age. PROSPERO registration: CRD42024573102

Polycystic ovary syndrome (PCOS) is a heterogenous reproductive disorder that is often associated with metabolic dysfunction, as well as comorbidities such as pregnancy complications. Although metabolic traits like hyperinsulinemia (i.e., elevated insulin without hypoglycemia) likely exacerbate the reproductive and metabolic features of PCOS, the precise impacts of specific metabolic traits on PCOS pathogenesis, symptom severity, and comorbidity incidence are not known. The aim of our study was to investigate the relationships between insulin levels, PCOS-like traits, and pregnancy complications by limiting endogenous insulin production in a mouse model of PCOS. Using Ins1-null mice with modulated Ins2 gene dosage (Ins1-/-:Ins2+/- versus Ins1-/-:Ins2+/+ littermates), we longitudinally assessed metabolic and reproductive phenotypes in PCOS-like mice generated via prenatal anti-Mullerian hormone (PAMH) exposure. We observed mild reproductive characteristics of PCOS in PAMH mice of both genotypes, including increased anogenital distances, delayed puberty, and disrupted estrous cycling, but did not detect robust PAMH-induced metabolic changes across six months. In the absence of PAMH-aggravated metabolic dysfunction or hyperinsulinemia--even in mice fed a high-fat, high-sucrose diet--reducing Ins2 gene dosage did not notably change most measured traits. However, high-fat, high-sucrose-fed PAMH pregnant dams exhibited a diminished pregnancy-induced insulinogenic response and a trend for reduced {beta}-cell mass compared to control mice, together with superior blood glucose homeostasis despite the physiological challenges of pregnancy. Therefore, while Ins1-null PAMH mice did not manifest pronounced PCOS-like metabolic features, prenatal AMH exposure can cause shifts in metabolic homeostasis during pregnancy.