This study aimed to investigate the role of STOX1 in trophoblast pyroptosis during preeclampsia (PE) and elucidate its underlying molecular mechanisms. A trophoblast hypoxia cell model was established by exposing HTR-8/SVneo cells to hypoxic conditions. The results showed that STOX1 messenger RNA expression was significantly elevated in placental tissues from PE patients as well as in hypoxia-treated HTR-8/SVneo cells. Hypoxia markedly induced pyroptosis in HTR-8/SVneo cells, whereas STOX1 silencing enhanced cell proliferation and migration while suppressing programmed cell death. Moreover, STOX1 knockdown reduced the levels of reactive oxygen species and malondialdehyde, increased superoxide dismutase (SOD) activity, and decreased the expression of pyroptosis-associated inflammatory cytokines, including interleukin-1β (IL-1β) and interleukin-18 (IL-18). In addition, silencing STOX1 downregulated the expression of inflammasome-related proteins, including NLRP3, ASC, cleaved caspase-1, and GSDMD-N. Mechanistically, STOX1 knockdown activated the Nrf2/HO-1 signaling pathway, as evidenced by increased expression of Nrf2 and HO-1 and decreased expression of Keap1. Rescue experiments further demonstrated that co-silencing Nrf2 partially reversed the inhibitory effects of STOX1 knockdown on pyroptosis in hypoxia-treated HTR-8/SVneo cells. Collectively, these findings indicate that STOX1 is upregulated in PE and that STOX1 silencing alleviates oxidative stress and inhibits caspase-1-mediated trophoblast pyroptosis through activation of the Nrf2 signaling pathway. Consequently, STOX1 may serve as a promising therapeutic target for the prevention and treatment of preeclampsia.
Diminished ovarian reserve (DOR) is associated with poor reproductive outcomes; however, the underlying molecular mechanisms-particularly those involving the regulation of granulosa cell (GC) function by exosomal microRNAs (miRNAs) derived from follicular fluid-remain unclear. This study integrated clinical analysis with molecular profiling to investigate the regulatory effects of DOR patient-derived follicular fluid exosomes on GC function. Clinical data confirmed significantly poorer embryological and clinical outcomes in DOR patients. Follicular fluid was collected from patients with normal ovarian reserve (NOR) and those with DOR. Exosomes and GCs were subsequently isolated from the respective follicular fluid samples. Molecular profiling of follicular fluid exosomes and GCs from both groups identified differentially expressed miRNAs and mRNAs via next-generation sequencing. Integrated bioinformatics analysis and dual‑luciferase reporter assays confirmed that exosomal hsa‑miR‑135a‑5p directly targets and suppresses BIRC5 expression in GCs. Functional experiments demonstrated that incubating NOR GCs with DOR exosomes not only significantly downregulated BIRC5 but also induced marked cellular autophagy and mitochondrial dysfunction. These findings reveal a novel exosome‑mediated regulatory pathway in which DOR follicular fluid exosomes deliver miR‑135a‑5p to recipient GCs, repress the anti‑apoptotic gene BIRC5, and disrupt cellular homeostasis, thereby providing a potential mechanistic explanation for the compromised follicular microenvironment and adverse clinical outcomes in DOR.
Successful reproduction in eutherian mammals requires intimate connections between the embryo and uterus, involving adhesion, attachment and placentation, with or without invasion of the endometrium. The complex dialogue between embryo and uterus involves numerous molecules and pathways. The study of the mechanisms driving early embryo-uterus interactions has historically been challenging, particularly discovering the embryo-expressed molecules. Some components of the interactions have been known for decades, but many have been revealed in recent years through innovative transcriptome studies, studies of the contents and functions of extracellular particles, use of in vitro three-dimensional organoids, genetic studies, approaches for dissecting the regulation of maternal immune system cells, and many other approaches. This review focuses on embryo-expressed molecules that support implantation and embryo attachment, implantation and pregnancy establishment. Both classical and recent discoveries are examined, encompassing results from human, murine, livestock and comparative studies. Opportunities for further mechanistic insights are considered. Additionally, the review considers the potential for agricultural and clinical applications of the more recently identified molecular players and mechanisms for enhancing reproductive success or for providing new approaches to contraception.
Post-ovulatory aging (POA) reduces oocyte quality, yet effective interventions remain limited. Here we demonstrate that early intervention with vitamin K2 (menaquinone-4 isoform, MK-4) alleviates POA by inhibiting iron-disorder-induced oxidative stress. Western blot revealed that the expressions of ferroptosis suppressor protein 1 (FSP1) and vitamin K epoxide reductase-like protein (VKORC1L1) were significantly decreased in mouse POA oocytes; however, MK-4 attenuated these changes. Further, MK-4 inhibited ferritinophagy by downregulating NCOA4 and LC3II, markedly restored ferritin level, reduced intracellular Fe2+ amount, thus attenuating oxidative stress. These results were characterized by reducing lipid peroxidation marker malondialdehyde (MDA) and acyl-CoA synthetase long chain family member 4 (ACSL4) expression, decreasing γ-H2AX and 8-Hydroxy-2'-deoxyguanosine (8-OHdG), and enhancing expression of the mitochondrial antioxidant enzyme superoxide dismutase 1 (SOD1). Additionally, MK-4 suppressed mitophagy by downregulating PINK1 and Parkin, and upregulating Rab7 improving mitochondrial membrane potential. Consequently, abnormalities in chromosomes, spindles, and the cytoskeleton were significantly ameliorated in POA oocytes receiving early MK-4 intervention. Notably, in vitro-aging oocytes expressed higher FSP1 and VKORC1L1 but lower ACSL4 and MDA versus the in vivo counterparts, suggesting a milder lipid peroxidation in vitro. Collectively, our findings uncover a novel anti-aging mechanism triggered by MK-4, highlighting its potential to establish a preventive strategy for female reproductive aging.
ABSTRACT Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy‐specific liver disorder characterized by elevated maternal bile acids and an increased risk of adverse fetal outcomes. Although placental dysfunction is a key contributor to ICP pathogenesis, the underlying mechanisms remain incompletely understood. Here, we combined a Sprague–Dawley rat model of ICP with spatial metabolomics to delineate region‐specific metabolic alterations within the placenta. Multivariate analysis revealed distinct metabolic signatures between ICP and control placentas, with pronounced reprogramming in trophoblast‐enriched regions. Differential metabolite and pathway analyses identified significant perturbations in glycerophospholipid metabolism, pyruvate metabolism, phospholipase D signaling, and the tricarboxylic acid cycle. Notably, phosphatidic acid (PA) was broadly elevated, whereas its downstream product phosphatidylinositol 4,5‐bisphosphate (PIP 2 ) exhibited spatially restricted accumulation in trophoblasts. Mechanistically, taurocholic acid exposure in HTR‐8/SVneo cells induced upregulation of PIP5K1, leading to PIP 2 accumulation and increased trophoblast apoptosis via disruption of the Bcl‐2/Bax balance. Silencing of PIP5K1 restored PIP 2 homeostasis and attenuated apoptosis. Importantly, these molecular alterations were recapitulated in placental tissues from ICP patients, confirming activation of the PIP5K1–PIP2 axis in vivo. Collectively, our findings identify a spatially resolved PA–PIP5K1–PIP 2 lipid signaling cascade that links bile acid–induced metabolic stress to trophoblast apoptosis and placental dysfunction in ICP.
Seminal plasma proteins are vital for sperm metabolism, protection against oxidative stress, fertility, and early embryo development. A substantial body of data evidence that seminal plasma proteomes are highly species-specific. However, this information remains dispersed across the literature, hindering comparative analyses and limiting the identification of potential, conserved fertility-related biomarkers. To address this gap, we developed SemProtDB (https://semprotdb.org/), the first centralized database of seminal plasma from domestic mammals. It consolidates data from 28 studies, featuring nearly 18,000 protein entries from 8 major species: Bos taurus, Bubalus bubalis, Capra hircus, Ovis aries, Equus caballus, Equus asinus, Canis lupus familiaris, and Sus scrofa. With more than 1500 proteins manually curated, the database provides a user-friendly interface linked to UniProt, NCBI, STRING, and source publications. SemProtDB represents an unprecedented resource for reproductive biology, facilitating comparative and translational research to better understand the molecular mechanisms underlying fertility across species.
Animal cloning, while promising for livestock breeding and genetic improvement, is hampered by low efficiency largely due to placental abnormalities. This study investigated this issue in cloned pigs at the 35th day of gestation, where a 69.8% developmental abnormality rate was observed. Morphological analyses revealed structural defects in placentas from failed pregnancies, including delayed fold development and abnormal areolae formation, coupled with reduced endometrial glands and maternal-side blood vessels. Immunohistochemistry demonstrated impaired placental function, evidenced by aberrant E-cadherin expression compromising trophoblast adhesion, and significantly reduced glycogen/polysaccharide secretion. Furthermore, Plet1 expression was disrupted. Transcriptomic profiling indicated that upregulated genes were enriched in cell-cell tight junctions, while downregulated genes were associated with oxygen binding, oxidative stress, stromal differentiation, and vascular development. Cross-species comparison with cloned mice suggested conserved genetic mechanisms underlying placental defects. These findings provide a crucial theoretical foundation for elucidating the mechanisms of placental failure in animal cloning and for developing strategies to enhance its efficiency.
Understanding the molecular mechanisms that guided early animal evolution is a central challenge of evolutionary developmental biology. Sponges (Porifera), as one of the earliest-branching metazoan lineages, are crucial for reconstructing the ancestral genetic toolkit for development. Here, we present the first transcriptomic analysis of embryogenesis in the demosponge Halisarca dujardinii, characterizing gene expression across three key stages: cleavage, blastula, and prelarva. We identified significant transcriptional shifts, including upregulation of cadherins during blastula formation, suggesting the establishment of adherens junctions, and a wave of kinase expression in the prelarva. Furthermore, we phylogenetically classified and analyzed the developmental expression of 186 transcription factors from the zinc finger, homeobox, Forkhead, and T-box families. Strikingly, spatial expression analysis revealed that Wnt ligands and T-box genes are expressed in complementary domains along the embryonic anterior-posterior axis, with HduWntL specifically marking the future posterior pole from the blastula stage. Our results demonstrate that despite its morphological simplicity, H. dujardinii employs a complex repertoire of developmental transcription factors and signaling molecules, and provides evidence for the early specification of the primary body axis, offering new insights into the evolution of developmental mechanisms in metazoans.
Flow cytometric sorting can be used to select sperm subpopulations with defined physiological or molecular traits; however, the impact of sorting on sperm function and subsequent embryo development in the porcine model remains unresolved. The objective of this study was to evaluate the effect of fluorescence-activated cell sorting (FACS) on boar sperm motility and developmental competence following in vitro fertilization (IVF). Pooled semen from boars was divided into sorted and unsorted (control) treatments and analyzed for motility and kinematic parameters using computer-aided sperm analysis (CASA) with both population-level (conventional, population-averaged) metrics and single-cell track data collected before and after sorting. Oocytes were fertilized with either treatment, and cleavage (≥ 2-cell stage) and blastocyst development were evaluated. Cleavage and blastocyst percentages were reduced in the sorted (48.8% ± 9.9% and 8.5% ± 4.2%) compared with control (92.2% ± 1.5% and 28.4% ± 2.8%, respectively; p < 0.001) group. Population-level CASA revealed statistically significant but biologically modest changes in motility and kinematic parameters following sperm sorting. In contrast, single-cell CASA coupled with k-means clustering identified four distinct motility subpopulations and demonstrated that sorting redistributed existing motility states, characterized by depletion of fast, progressive sperm and enrichment of less energetically demanding populations. These findings suggest that FACS alters the composition of sperm motility subpopulations, alongside a marked reduction in post-fertilization developmental competence in porcine IVF.
Sex determination in Porifera remains one of the least understood aspects of early metazoan biology despite the group's key phylogenetic position. Sponges display exceptional diversity in sexual systems-ranging from stable gonochorism to sequential hermaphroditism and sex reversal-yet lack morphological dimorphism and any discrete gonadal structures, blurring the boundary between sex determination and gametogenesis. Here we synthesize current knowledge on sexual systems, environmental and genetic influences on sexual fate, and the evolutionary origins of sexual systems in sponges. Evidence for environmental modulation is substantial, particularly temperature-driven shifts in sex ratios and reproductive timing, but environmental sex determination remains unsupported by causal evidence yet. Conversely, conserved molecular components of metazoan sex-determining pathways, such as fem-1 and Dmrt genes, are present in several sponge lineages, though their functional roles remain unresolved. We also evaluate the hypothesis that microbial symbionts contribute to sexual allocation by buffering the energetic costs of gametogenesis. Overall, sponges exhibit an evolutionarily labile system in which sexual fate may emerge from the interaction of conserved genetic modules, environmental cues, and physiological constraints. Clarifying these mechanisms will be essential for understanding the origins of sex determination in Metazoa and for predicting reproductive resilience under environmental change.
In vitro production (IVP) of bovine embryos often results in lower developmental competence compared to in vivo-derived embryos. This disparity is further exacerbated by oocyte cryopreservation (vitrification). This study aimed to investigate the molecular basis of reduced embryo quality by profiling the transcriptomic impact of in vitro maturation (IVM) and vitrification on bovine blastocysts.Day 7 blastocysts from three groups were analyzed: (1) in vivo: control embryos produced entirely in vivo, (2) IVF: embryos from fresh oocytes subjected to IVM and in vitro fertilization, and (3) V_IVF: embryos from vitrified-thawed oocytes subjected to IVM and fertilization. Whole-blastocyst transcriptome profiling was performed using single-embryo RNA sequencing (SUPeR-seq; n = 3 blastocysts per group) and analyzed without cellular dissociation. Developmental competence was significantly reduced in the V_IVF group (~ 11.8% blastocyst rate) compared to the IVF group (~ 41.5%) and in vivo controls. Transcriptome analysis revealed 362 differentially expressed mRNAs in V_IVF embryos and 78 in IVF embryos compared to in vivo controls. Genes such as ATP1A1, GOT1L1, and QSOX1 were significantly altered, influencing pathways related to oxidative phosphorylation, lysosome activity, and chromatin organization. In addition, 694 DE-lncRNAs in the V_IVF group and 187 in the IVF group were identified. Exonic circRNAs (608) were detected with differential expression and enrichment in signaling and epigenetic pathways, including Hedgehog signaling and stem cell pluripotency regulation. Integrated network analysis revealed coordinated dysregulation across mRNA, lncRNA, and circRNA layers, suggesting disruption of multi-layered regulatory circuits. Overall, this study presents a detailed transcriptomic landscape of bovine blastocysts affected by IVM and vitrification.
Premature ovarian insufficiency (POI) patients usually have a few residual dormant primordial follicles that are difficult to activate. We investigated the effects of branched-chain amino acids (BCAAs) on mouse primordial follicle activation. Leucine, but not isoleucine and valine, promoted primordial follicle activation in the cultured neonatal mouse ovaries. Furthermore, leucine significantly elevated the levels of phosphorylated protein kinase B (p-Akt) and phosphorylated forkhead Box O3a (p-FOXO3a), and increased the number of oocytes exhibiting nuclear export of FOXO3a in mouse ovaries. LY294002 and PI3K-IN-1, the inhibitors of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, reversed the promoting effects of leucine on mouse primordial follicle activation. Importantly, intraperitoneal injection of leucine in 3-day-old neonatal mice and oral administration of leucine in 21-day-old mice with or without cyclophosphamide treatment significantly increased the number of growing follicles. Thus, leucine activates mouse primordial follicles through the PI3K/Akt pathway. Leucine may be a potential oral supplement for treating infertility in POI patients with residual dormant primordial follicles.
Tris-2,3-dibromopropyl isocyanurate (TBC), a brominated flame retardant widely used in industrial applications, raises increasing concerns due to its potential reproductive toxicity. While the role of apoptosis in TBC-induced cytotoxicity is partially understood, its effects on autophagy in reproductive cells remain underexplored. Therefore, the present study aimed to determine the impact of TBC on autophagy-related processes in GC-1 (spg) spermatogenic cell line as an in vitro model of testis-derived cells. Our findings demonstrate that TBC significantly reduces cellular metabolic activity in a time-dependent manner. Molecular analysis revealed an upregulation of PPARγ gene expression, accompanied by a decrease in phosphorylated mTOR (p-mTOR) levels and a concurrent increase in phosphorylated IRE1 (p-IRE1), indicating activation of endoplasmic reticulum (ER) stress pathways. Furthermore, TBC exposure was associated with modulation of autophagy-related markers, suggesting a potential adaptive or compensatory cellular response to stress conditions. Cell cycle analysis showed arrest in the G0/G1 phase, while ROS quantification indicated elevated reactive oxygen species production upon treatment. Collectively, our data suggest that TBC disrupts cell homeostasis through ER stress-associated signaling, oxidative stress, and cell cycle regulation, which may contribute to its potential reproductive toxicity.
Sex differentiation, the process by which an organism develops as a male or female, can be influenced by the environment in many species. High temperature, or pollutant exposure, can lead to skewed sex-ratios and genotype-phenotype mismatches that can seriously threaten populations and biodiversity. Understanding the mechanisms involved in sex differentiation is thus of crucial importance. In the present study, we analyzed the DNA methylation and transcriptional changes that occur during sex differentiation in zebrafish. Our results support the fact that DNA methylation is not only involved in sex differentiation but also in sex maintenance. Initiation of sex differentiation was associated with a DNA demethylation event. Subsequently, a second DNA methylation remodeling event, which coincided with the loss of bi-potentiality, was observed in males. Among the genes under study, the methylation level of the aromatase gene was found to be the most robust sex marker. However, genes encoding for transcription factors of the ZBTB family offered a more precocious marker since sex-specific changes in their transcription levels preceded changes in DNA methylation. In addition, their transcription levels were found be sensitive to a known masculinizing factor, i.e. temperature. Our results thus support an unexpected role of ZBTB proteins in sex differentiation.
Ovarian aging is characterized by the reduction of the follicular pool and a progressive loss in oocyte quality. Although these changes have initially been attributed to oocyte alterations, increasing evidence indicates that stromal deterioration plays a central role in this process. Stromal remodeling involves complex interactions among structural, molecular, and cell communication disruptions. This review discusses the main ovarian stroma shifts related to aging, emphasizing ECM remodeling, biomechanical changes, and cell signaling dysfunction. In light of the multifactorial nature of ovarian aging, no single therapeutic strategy is likely to fully address stromal dysfunction. In this context, Mesenchymal Stromal Cells (MSCs) and their secreted Extracellular Vesicles (EVs) have emerged as promising approaches, given their ability to deliver a diverse and functionally complementary set of bioactive molecules that collectively modulate inflammation, support extracellular matrix remodeling, and restore cell communication. Notably, EVs retain key paracrine effects of MSCs while offering advantages in safety and manufacturability, supporting the transition from cell-based to cell-free therapies. Accordingly, this review also examines this therapeutic shift, highlighting the role of MSCs and EVs in stromal regeneration. By integrating ovarian biology with biotechnological bioprocesses, we discuss key translational challenges-including scalability, quality control, and regulatory considerations-that are critical for the development of effective stroma-targeted interventions aimed at preserving or restoring ovarian function.
Epimedium brevicornu Maxim (Yinyanghuo) is widely used to treat reproductive disorders. Icariin (ICA), the main active ingredient of Epimedium brevicornu Maxim, can alleviate age-related testicular dysfunction and Sertoli cell injury. However, whether ICA can regulate the autophagic activity of Sertoli cells and thus improve cell function remains unclear. The present study aimed to evaluate the protective effects of ICA on mitochondrial biogenesis, lysosome biogenesis, and autophagic activity in D-Galactose (D-gal)-induced Sertoli cell. TM4 cells were pretreated with ICA and then exposed to D-gal. After treatment, Sertoli cell viability was detected. Oxidative stress, apoptosis, mitochondrial biogenesis, lysosomal biogenesis, and autophagy-related protein and mRNA levels were analyzed. Sertoli cells were stained with Lyso-Tracker and Mito-Tracker to visualize lysosomes and mitochondria. ICA treatment significantly increased the Sertoli cell viability and protected against D-gal-induced oxidative stress. Meanwhile, ICA treatment significantly increased the expression levels of TFEB and LAMP2 proteins and promoted lysosomal biogenesis. Moreover, ICA treatment increased the expression of PINK1, Parkin, ATG5, ULK1, and LC3-II proteins, activated autophagic activity, and thus improved Sertoli cell function and increased GDNF and Claudin5 protein levels. ICA treatment could alleviate oxidative stress, increase mitochondrial biogenesis, and improve Sertoli cell function by activating autophagy and promoting lysosomal biogenesis.
Diminished ovarian reserve (DOR) is a major cause of female infertility. In recent years, traditional Chinese medicine has displayed unique ameliorating effects on DOR. This study aims to investigate the therapeutic efficacy and molecular mechanisms of Bushen Zhuluan Decoction (BSZLD) in DOR. A rat model of DOR was established and treated with either BSZLD or Progynova (positive control). Estrous cycle monitoring, ovarian index calculation, hematoxylin-eosin staining, and enzyme-linked immunosorbent assay were performed to assess ovarian reserve function. TUNEL staining, immunohistochemistry, and Western blotting were conducted to evaluate ovarian cell apoptosis. To elucidate the pharmacological basis of BSZLD, ultra-high-performance liquid chromatography-tandem mass spectrometry was applied. Network pharmacology analysis, machine learning algorithms, and molecular docking were integrated to predict key active compounds and pathways. The role of the sirtuin 1 (SIRT1) and its downstream target hypoxia-inducible factor 1alpha (HIF-1α) signaling in BSZLD-mediated effects was verified using short hairpin RNA (shRNA) lentiviral-mediated interference. BSZLD significantly restored estrous cycle disturbances, increased ovarian index and follicle counts, and reduced atretic follicles and ovarian cell apoptosis in DOR rats. Serum anti-Müllerian hormone and estradiol levels were elevated, while follicle-stimulating hormone and luteinizing hormone levels were reduced. The SIRT1/HIF-1α signaling pathway was a core regulatory axis for BSZLD action. BSZLD markedly upregulated SIRT1 expression while suppressing HIF-1α expression. Silencing SIRT1 using shRNA reduced SIRT1 levels, elevated HIF-1α expression, and significantly attenuated the therapeutic effects of BSZLD. BSZLD attenuates DOR in rats by activating the SIRT1/HIF-1α signaling, thereby suppressing ovarian cell apoptosis and restoring ovarian reserve function.
Endometriosis is a common gynecological disease with high recurrence rates after surgery and the lesions keep unlimited proliferative capacity. The effect of garcinol on cell proliferation has not been investigated in endometriosis. Herein, we studied the effect of garcinol on endometriosis by using the immortalized human endometriotic epithelial cell line 12Z and stromal cell line iheESCs. RTCA and EdU assays were used to assess cell proliferation. RNA-Seq was used to discover the differential expression gene (DEG) profile and GSEA, KEGG, protein-protein interaction network and the transcription factor interaction network were analyzed. We found that garcinol inhibited the cell proliferation and S phase DNA synthesis of 12Z and iheESCs cells. There were 548 DEGs in the transcription profiles of the two cell lines. The GSEA results showed that garcinol could inhibit the DNA replication and cell cycle pathways. Among the cell cycle related genes, p21 was increased, while cyclin B1, cyclin E1, CDK2, CDK4, Myc, p27 and E2F1 were significantly decreased after garcinol treatment. The level of cell cycle M phase marker, pH3 Ser10, was also reduced by garcinol. Garcinol could regulate the Akt/c-Jun/ERK1/2 signaling pathways. Garcinol could regulate the protein-protein interaction network and the transcription factor interaction network, in which garcinol increased the transcription factor ATF3 and FOSB expression. In conclusion, garcinol could inhibit the proliferation of endometriosis cells in vitro.
Delivery of large molecular complexes into spermatozoa remains a challenge in reproductive biotechnology. Teleost sperm possess highly compacted chromatin and minimal cytoplasmic volume, limiting intracellular access of ribonucleoprotein assemblies. Here, we evaluated whether CRISPR-Cas9 ribonucleoprotein (RNP) complexes can be introduced into Nile tilapia spermatozoa using capillary electroporation while preserving cellular integrity. RNP uptake was quantified via EGFP fluorescence, and sperm performance was assessed through motility, membrane integrity, mitochondrial activity, and DNA fragmentation analyses. Approximately 10%-13% of spermatozoa exhibited detectable EGFP signal following electroporation. Increased pulse numbers reduced motility and mitochondrial activity, whereas membrane integrity was preserved and no increase in DNA fragmentation was detected. A single pulse (1250 V, 40 ms) provided the best balance between RNP uptake and functional preservation. These findings support capillary electroporation as a minimally disruptive strategy for CRISPR-RNP delivery in teleost reproductive biology.
