Specificity protein 1 (SP1) is the most active member of the specificity protein and Krüppel-like factor (Sp/KLF) family and is widely expressed across all mammalian cell types. However, more detailed studies on the role of SP1 in preimplantation embryonic development are needed. Here, we analyzed the role of SP1 in the development of preimplantation mouse embryos through supplementation with a small molecular inhibitor (plicamycin) and microinjection of Sp1 siRNA. We found that SP1 was indispensable for zygotic genome activation (ZGA) and the morula-to-blastocyst transition. Plicamycin supplementation arrested embryo development at the 2-cell stage and resulted in aberrant RNA polymerase II preconfiguration. Sp1 expression knockdown by Sp1 siRNA microinjection caused most embryos to arrest at the morula stage, and the expression of NANOG, POU5F1 and CDX2 significantly decreased. Both plicamycin supplementation and Sp1 siRNA microinjection decreased embryonic H3K4me3 levels and increased H3K9me3 levels. Moreover, when Sp1 was overexpressed, the embryos were arrested at the 2-cell stage, the H3K4me3 level increased, and the H3K9me3 level decreased. In conclusion, our findings demonstrate that SP1 is crucial for mouse preimplantation embryonic development through the regulation of gene expression and histone modifications.

The exRNA transcriptome of the preimplantation embryo culture medium constitutes a comprehensive profile of embryonic RNAs, offering a non-invasive resource for elucidating developmental status. To delineate its relationship with the maternal-to-zygotic (MZT) transition and evaluate its predictive potential for developmental outcomes, we performed systematic exRNA transcriptome profiling of individually cultured cleavage-stage embryos. This study included 34 spent culture medium (SECM) samples for day 3 in vitro preimplantation cleavage embryos from 30 patients undergoing IVF/ICSI. 24 morphologically high-grade samples (Grade I, n = 12; Grade II, n = 12) and 10 low grade samples (Grade III, n = 6; abnormal PNs, n = 2; arrested embryos, n = 2) were included. Embryo transfer after the SECM collection was traced for subsequent clinical pregnancy outcome. ExRNA transcriptome from cleavage SECM showed 81.78% concordance with embryonic gene expression profiles. 1,058 differentially expressed exRNA markers were identified across embryo morphological grading groups, revealing MZT-related molecular dynamics prior to blastocyst formation. A 300-gene signature significantly correlated with embryonic developmental potential was established. The accuracy and robustness of the models were validated using clinical samples. The LogitBoost model built on exRNA profile yielded an AUC value above 0.95, indicating encouraging performance for embryo assessment. This study establishes SECM exRNA profiling as a non-invasive method for capturing key molecular events during early embryo development, particularly MZT activation. The identified exRNA biomarkers and machine learning models provide a promising framework for the objective assessment of embryo developmental potential, warranting further validation to overcome the limitations of conventional morphology-based selection in IVF.

Antenatal corticosteroid (ANS) therapy and chorioamnionitis are common antecedents of preterm birth, yet their effects on the developing fetal ovary remain poorly understood. Long-term follow-up of 45% of participants from the Auckland Steroid Trial reported that high-dose antenatal betamethasone exposure was associated with a modest delay in female reproductive maturation and a non-significant two-year reduction in menopause age (Lord et al., 2025), raising concerns regarding potential effects on ovarian lifespan. Using a sheep model of pregnancy, we investigated the impact of antenatal betamethasone exposure and lipopolysaccharide (LPS)-induced chorioamnionitis on fetal ovarian development. Date-mated Merino ewes were randomized to saline control, ANS, or chorioamnionitis treatment groups administered 2 or 8 days before delivery. Female lambs were delivered preterm at 124±1 days' gestation, underwent standardized 30 minutes ventilation, and ovarian tissues were collected for transcriptomic and histological analyses. RNA sequencing demonstrated marked transcriptional alterations following both ANS and chorioamnionitis, affecting pathways involved in cell cycle regulation, ovarian development, and cellular function. Histological assessment identified changes in follicle populations. ANS exposure was associated with increased markers of apoptosis, including significantly elevated TUNEL and caspase-3 staining. Chorioamnionitis exposure resulted in significant reduction of primary follicle numbers and trends toward increased tertiary and atretic follicles. Markers of ovarian and stromal cell populations were unchanged. Although limited by sample size and assessment of individual rather than combined exposures, these findings demonstrate that both ANS and chorioamnionitis induce acute molecular and structural changes in the developing fetal ovary, with potential implications for ovarian reserve, reproductive lifespan, and long-term female reproductive health.

Ovulation is the process by which an egg is released from an ovarian follicle, ready for fertilisation. It is a multi-scale transformation that converts endocrine cues into coordinated tissue and extracellular matrix remodelling, culminating in physical rupture of the follicle wall. Live imaging has reshaped understanding by replacing static snapshots with direct observation of sequence and timing. Here, we outline the architecture of the preovulatory follicle and the LH-driven signalling programmes that coordinate cumulus expansion, oocyte maturation, tissue remodelling and rupture. We then chart key imaging milestones-from early time-lapse of exteriorised ovaries, to clinical endoscopy in humans, perfused ovary preparations, intravital multiphoton microscopy, and ex vivo live imaging of isolated follicles. Finally, we summarise emerging approaches to probe physical regulation, including intrafollicular pressure measurements and complementary methods to quantify tissue and matrix mechanics. Together, these advances show that ovulation is a staged programme that demands quantitative, time-resolved measurements.

Ovarian endometrioma (OMA), an endometriosis subtype, involves hemorrhagic cysts formed by ectopic endometrial-like tissue, causing pelvic pain, inflammation, and infertility by disrupting ovarian function. Despite its impact, the single-cell dynamics in OMA follicular fluid are poorly understood. We performed scRNA-seq (10x Genomics) on follicular fluid from a patient's affected (2793 cells) and unaffected (4699 cells; control) ovaries. Analysis revealed four cell types, including a novel TCR+ macrophage subtype. Affected-side granulosa cells showed abnormal developmental trajectories linked to dysregulated steroid pathways. Immune cells exhibited heightened inflammation and reduced macrophage phagocytosis. Cell-cell communication highlighted prominent MIF and MK signaling from granulosa cells. This study provides the first single-cell transcriptomic profile of OMA follicular fluid, elucidating aberrant granulosa cell development, immune dysregulation, and distinctive cellular interactions, enhancing understanding of OMA's impact on follicular development and suggesting potential diagnostic/therapeutic targets.

Progesterone resistance in endometriosis reduces the efficacy of progestin. The non-classical progesterone receptor, progesterone receptor membrane component 1 (PGRMC1) may regulate progesterone signaling in the normal endometrium, but its expression and role in endometriosis remain unknown. We aimed to investigate PGRMC1 expression in endometriosis and its role in progesterone resistance. PGRMC1 expression in normal endometrium (NE), eutopic endometrium in patients with endometriosis (EE), and ovarian endometrioma (OE) was examined using immunohistochemistry. Cultured OE stromal cells (SCs) were subjected to PGRMC1 knockdown using siRNA and treated with AG205 (a PGRMC1 inhibitor) and RU486 (a PR inhibitor) under progesterone stimulation. Progesterone responsiveness was assessed by measuring IGFBP1 and PRL expression through real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). PGRMC1 expression in NESCs and EESCs was significantly lower in the secretory phase than in the proliferative phase (P < 0.01), with no similar changes observed in OE. Following siRNA-mediated PGRMC1 knockdown in OESCs, IGFBP1 and PRL expression was significantly upregulated (P < 0.05). Notably, treatment with siRNA and progestin induced significantly higher IGFBP1 and PRL expression than treatment with progestin alone (P < 0.05). AG205 induced similar effects on IGFBP1and PRL expression (P < 0.05), suggesting that suppression of PGRMC1 expression enhances progestin responsiveness in OESCs. However, AG205-induced IGFBP1 expression was abolished following treatment with RU486, suggesting that PGRMC1 acts through PR. In conclusion, suppressing PGRMC1, which is consistently expressed throughout the menstrual cycle in endometriotic lesions, may restore progesterone sensitivity and improve the efficacy of progestin-based therapies for endometriosis.

In brief: Redox imbalance compromises granulosa cell survival and follicle fate. This study identifies the mitochondrial-derived peptide Humanin (HN) as a cytoprotective factor that protects granulosa cells under oxidative condItions. Abstract: Granulosa cell function is essential for proper ovarian physiology. Redox imbalance compromises granulosa cell survival, thereby impacting follicle fate within the ovary. Humanin (HN), a small mitochondrial-derived peptide, exerts cytoprotective effects in several tissues under pro-oxidant conditions. The present study aimed to evaluate the cytoprotective effects of HN under oxidative conditions in granulosa cells, using a human granulosa cell line (KGN) and an in-vitro rat ovary culture to assess its action within the ovarian microenvironment. KGN cells showed a significant increase in endogenous HN mRNA expression in response to oxidative conditions induced by H2O2. Upon oxidative insult, exogenous HN enhanced cellular antioxidant capacity by significantly increasing catalase activity levels, without modifying superoxide dismutase expression or activity, or redox status in KGN cells. Importantly, HN significantly decreased H2O2-induced granulosa cell apoptosis in KGN cells, as determined by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. This protective effect was associated with modulation of key apoptosis-related genes, including reduced expression of BAX and the caspase-3 precursor (CASP3), while BCL2 expression remained unchanged. Consistently, in rat ovaries, HN protected granulosa cells of antral follicles under oxidative conditions. In conclusion, our findings support a cytoprotective role of HN in granulosa cells under oxidative conditions, which may contribute to the maintenance of antral follicle survival within the ovarian microenvironment.

The ovary is a structurally complex organ whose function depends on precisely coordinated interactions among multiple cell types. Spatially resolved transcriptomics (ST) has emerged as a powerful complement to single-cell RNA sequencing (scRNA-seq), enabling gene expression profiling within intact tissue and preserving the spatial context that dissociation-based methods inherently lack. This review provides a comprehensive overview of the major ST platforms, including sequencing-based technologies (Visium, Visium HD, Stereo-seq, and GeoMx) and imaging-based technologies (Xenium, MERSCOPE, and CosMx), with a focus on their distinct technical features, resolution trade-offs, and suitability for ovarian research. We survey 40 published studies applying ST to ovarian biology, spanning atlases, ovarian aging, follicle development and ovulation, and ovarian cancer. We also discuss typical computational analyses as well as their challenges specific to ovary, including cell segmentation of morphologically diverse cell populations, deconvolution of mixed-cell capture spots in sequencing-based platforms, quality control, batch correction, and spatially aware downstream analyses encompassing trajectory inference, cell-cell interaction modeling, gene regulatory network reconstruction and more. Across these biological contexts, multi-modal integration, pairing ST with scRNA-seq, spatial proteomics, or chromatin accessibility profiling, has proven increasingly valuable for resolving the full molecular complexity of ovarian biology. Nevertheless, some challenges persist, and no single platform is universally optimal for all research questions. Thoughtful alignment between biological objectives, tissue scale, and platform capability will be critical for advancing ST from descriptive mapping toward mechanistic and clinically translatable discovery.

Polyendocrine metabolic ovarian syndrome (PMOS), formerly termed polycystic ovary syndrome (PCOS), is a reproductive disorder with heterogeneous symptoms and severity. Despite extensive research documenting chronic immune dysfunction as a hallmark of PMOS, the specific mechanisms of immune activation remain poorly understood. Emerging evidence suggests that gut-derived bacterial endotoxins, particularly lipopolysaccharide (LPS), can breach intestinal barriers and trigger systemic inflammation via Toll-like receptor 4 (TLR4). This study examined the role of TLR4 in PMOS-like pathology using a letrozole (LET)-induced mouse model. In LET-treated wild-type female mice, serum LPS and its carrier protein LBP were elevated compared to LET-treated TLR4-/- mice. Additionally, TLR4 deficiency attenuated multiple PMOS-like features, including elevated luteinizing hormone, anovulation, and metabolic dysfunction. LET-treated TLR4-/- mice also preserved estrous cycling and fertility, maintained gut barrier integrity, and reduced inflammatory markers. These findings support TLR4 as an important contributor to multiple features of PMOS-like pathology. This novel work highlights TLR4-mediated inflammation as a potential target for anti-inflammatory treatments in women with PMOS.

Oviductal fluid (OF) proteins, such as HSPA5, play essential roles in reproductive physiology, including zona pellucida (ZP)-sperm interactions. Hypothesizing that HSPA5 interacts with oocytes, we evaluated its effects during the last 4 h of bovine in vitro cumulus-oocyte complex (COC) maturation and subsequent embryo development. COCs were aspirated from slaughterhouse-derived cow ovaries and matured for 20 h in base medium (BM). The medium was then partially replaced with BM (control) or BM supplemented with recombinant HSPA5 (100 ng/ml) for the final 4 h of maturation. After maturation, COCs were either denuded for analysis or used for in vitro fertilization (IVF) and culture (IVC) to assess embryo development to the blastocyst stage. To investigate HSPA5's role, we assessed the mitochondrial membrane potential (MMP), reactive oxygen species (ROS), glutathione (GSH) content in COCs and maturation medium, zona pellucida (ZP) solubility, oocyte penetration, blastocyst rates, and gene expression. Recombinant HSPA5 (rHSPA5) enhanced oocyte mitochondrial activity (p = 0.0006) and significantly increased the blastocyst yield (p = 0.04). Blastocysts from rHSPA5-treated oocytes showed upregulation of 19 genes associated with embryo quality, DNA methylation, cell growth, development and oxidative stress. Although rHSPA5 did not alter the monospermic fertilization rate or oxidative stress in oocytes, it improved mitochondrial function, supported blastocyst development, and modulated embryonic transcriptional profiles. These findings suggest that rHSPA5 plays a beneficial role during the final phase of oocyte maturation, contributing to improved embryo quality and developmental competence in vitro.

In brief: The most effective way of activating mammalian eggs involves causing multiple cytosolic Ca2+ transients-difficult to artificially induce in most species in a standard laboratory setting. We show that the halogenated anaesthetic isoflurane can be used to cause multiple large Ca2+ transients and egg activation in mouse eggs in a simple, reliable, and adjustable way. Abstract: Mammalian eggs are activated at fertilization by a series of sperm-induced increases in cytosolic Ca2+. The Ca2+ ionophores A23187 and ionomycin are used to stimulate a single large Ca2+ transient to induce mammalian egg activation after intracytoplasmic sperm injection but this response is of unclear effectiveness. Some protocols involve two or more incubations in Ca2+ ionophore, but we now report that repeated application of either A23187 or ionomycin in mouse eggs leads to Ca2+ transients of decreasing amplitude, or to egg lysis. In contrast we show that the halogenated anaesthetic isoflurane causes a large Ca2+ increase in mouse eggs, but when washed out and reapplied to eggs, it repeatedly generates another large Ca2+ transient. We also found that, after fertilization or PLCz1 injection, brief exposure to Ca2+ ionophores caused one Ca2+ rise but then inhibited further Ca2+ oscillations in eggs. In contrast, brief application of isoflurane to fertilizing or PLCz1-injected eggs enhanced ongoing induced Ca2+ oscillations. The triple application of isoflurane triggered mouse egg activation more effectively than A23187 or ionomycin. Isoflurane is volatile and rapidly evaporates from culture medium, but we demonstrate that isoflurane can be reliably used to cause Ca2+ increases and egg activation when it is added to the mineral oil that covers the drops of medium into which eggs are placed. Our data suggest that isoflurane can be used to induce multiple large amplitude Ca2+ transients and promote efficient egg activation with a simple and adjustable protocol.

Fibroblast Growth Factor 9 (FGF9), as one of the fibroblast growth factor family, is vital for testis formation. Additionally, histone modifications, especially H3K4me3, have been reported to be important in steroidogenesis. However, the regulation of SLC differentiation by FGF9 and the role of histone modifications in this process remain unclear. Adult male Sprague-Dawley rats were treated with ethane dimethane sulfonate to deplete LCs, followed by intratesticular injection of FGF9 from post EDS day 14 to 28 at 0, 10, and 100 ng/testis. qPCR and Western blot analyses were used to detect the gene expression and protein levels of testosterone(T) synthesis. ChIP-seq was employed to identify the H3K4me3-binding regions and binding levels. The results showed that FGF9 treatment led to an increase in serum T levels, LC number and the expression of LC-specific genes (Lhr, Scarb1, Stard1, Cyp11a1, Cyp17a1, Hsd17b3, and Hsd11b1). FGF9 markedly increased H3K4me3 protein levels and decreased H3K9me3 protein level. FGF9 promoted H3K4me3 on the promoter regions of Scarb1, Stard1 and Srd5a1 in vivo. In vitro studies demonstrated that FGF9 increased medium T levels and stimulated the incorporation of EdU, a marker of cell proliferation, into stem LCs after tubule culture. Moreover, Fgfr1 siRNA and WDR5-0103, the histone methyltransferases, could reverse the effect of FGF9 on promoting T production. This study supports previous ones demonstrating that FGF9 stimulates the proliferation and differentiation of stem LCs in an EDS-treated model and in vitro tubule culture model through H3K4me3 histone modifications.

In brief: The RNA-binding protein Pumilio-2 (PUM2) is expressed by human granulosa cells and granulosa cell tumor cells, including KGN cells. Fundamental roles in the regulation of the innate immune abilities, proliferation, and steroidogenesis of KGN granulosa cells became apparent upon downregulation. Abstract: The RNA-binding protein Pumilio-2 (PUM2), encoded by PUM2, is known as a translational repressor with important roles -during embryonic development, cell differentiation, and synapse function. Information on its roles in the ovary were sparse, yet databank mining followed by immunohistochemistry revealed expression in nonhuman primate ovarian granulosa cells (GCs). Expression was also found in human in vitro fertilization-derived GCs, samples of human GC tumors (GCTs), and KGN cells, a well-established GCT-derived cell line. KGN cells were used to explore the roles of PUM2. A small interfering RNA (siRNA) targeting PUM2 reduced PUM2 levels in KGN cells, as seen in a proteomic analysis, which also revealed that a total of 51 -proteins were present at lower levels, while the abundance of 66 proteins was increased. Gene Ontology term analyses showed that the pathways mainly decreased were "response to virus," "defence response to virus," and "innate immune response." PUM2 may thus be a regulator of the innate immune abilities of GCs. The most increased pathway was "cell division," in line with increased proliferation of siRNA-transfected KGN cells. This underlies the increased ability to close the gap in scratch assays, as individual cell velocity or directionality of migration were comparable to controls. Downregulation of PUM2 also impaired the steroidogenic potential of KGN cells (lower HSD3B2 levels, lower progesterone in supernatant). Taken together, the results indicate fundamental roles of PUM2 not only in in the regulation of proliferation and steroidogenesis of human granulosa cells, but also in regulation of their innate immune abilities.

Over the last decade, mammalian sperm metabolism has moved from a binary view centered on glycolysis versus oxidative phosphorylation to an integrated model in which ATP production, redox homeostasis, metabolic plasticity, and cellular heterogeneity are functionally linked. This conceptual shift has relevance in the stallion, whose spermatozoa display strong mitochondrial engagement, marked redox sensitivity, and substantial responsiveness to media composition and preservation conditions. Here, we review advances from 2016 to 2026 that reshaped the field, with emphasis on extracellular flux analysis, multiparametric and label-free flow cytometry, proteomics, phosphoproteomics, metabolomics, and emerging stable-isotope approaches. We discuss evidence indicating that stallion spermatozoa operate within an oxidative framework in which mitochondrial respiration is supported by glycolytic input, whereas auxiliary pathways involving lactate-pyruvate cycling, and possibly neutral lipid mobilization and glycerol-glycerol-3-phosphate interconversion, may contribute primarily to redox stabilization and metabolic flexibility rather than to bulk ATP production. We further examine how these concepts inform diagnostics, semen preservation, cryopreservation, and intracytoplasmic sperm injection, emphasizing that single parameters are weak predictors of fertility and that integrated metabolic phenotypes are more informative. Finally, we identify major unresolved issues, including the need to standardize media composition and bioenergetic assays, distinguish pathway capacity from flux and coupling efficiency, and link metabolic phenotypes to fertility endpoints using rigorous experimental and statistical designs. A systems view of sperm metabolism offers a rational framework to improve equine fertility technologies and to explain inter-stallion variation in reproductive performance.