Maestro (MRO) is a transcription factor that has a role in regulating gene expression. The MRO expression pattern was examined in a unique set of human granulosa cells (GCs) and follicles collected across the ovulatory period of a natural menstrual cycle. MRO mRNA was elevated (70-fold) in women during the early ovulatory phase (12h to ≤18h post hCG administration; LH analog) and remained elevated (35-fold) in the late ovulatory phase (>18h to ≤ 34h) when compared to the preovulatory state (before the LH surge). Immunohistochemistry of whole follicles from women demonstrated positive MRO signal in GCs during the early and late ovulatory phases. Employing a cultured human granulosa luteal cell (hGLCs) model, we further examined MRO regulation in human granulosa cells from IVF patients. Treating hGLCs in vitro with hCG elevated MRO expression by 22-fold at 6h, 48-fold at 12h, and then expression slightly decreased to 13-fold at 24h. Similarly, the strongest MRO protein expression was detected at 12h. Next, LH/hCG signaling pathways regulating MRO expression were investigated using inhibitors specific to each signaling pathway. Our data revealed that hCG regulated MRO expression through LH-dependent classical signaling pathways such as PKA, PKC, and PI3K, with MAPK partially regulating its expression. Moreover, the EGF and progesterone pathways, important in the periovulatory period, were found to be involved in MRO regulation. These data, taken together, demonstrate that MRO is poised to play a role in the coordination of periovulatory events through the EGF and progesterone signaling pathways.

Hybridization is common among animals and plants, but often leads to sterility due to improper chromosome pairing. Some hybrids overcome sterility through modified gametogenesis, enabling clonal or hemiclonal reproduction. In water frog hybrids from the Pelophylax esculentus complex, hybridogenesis typically ensures hemiclonal reproduction. These hybrids inherit haploid chromosomal sets (n = 13) from both parental species, Pelophylax lessonae and Pelophylax ridibundus. During hybrid gametogenesis, one genome is eliminated while the other is clonally transmitted. We analyzed hybrid males and females from Central Europe, where hybrids coexist with P. lessonae and with both parental species, to assess gametogenic stability and whether the P. lessonae genome is consistently eliminated. Most hybrids followed canonical gametogenesis with premeiotic elimination of the P. lessonae genome and endoreplication of the P. ridibundus genome, yielding haploid P. ridibundus gametes. Nevertheless, gametogenesis was frequently altered in males but not in females. Some hybrid males had spermatocytes with 13 or 26 univalents, 26 bivalents, or tetravalents, indicating variability in genome elimination and endoreplication. Some males produced diploid or haploid spermatids with P. ridibundus and P. lessonae genomes. Comparative genome hybridization revealed that some males with altered gametogenesis carried unusual chromosomes with mixed R and L signals. Furthermore, rare triploid males showed substitutions of P. lessonae chromosomes to P. ridibundus chromosomes. Despite altered gametogenesis, they produced spermatocytes and haploid spermatids with P. lessonae chromosomes and two P. ridibundus chromosomes that escaped elimination. Overall, despite a generally consistent pattern of genome transmission, hybrid males exhibit frequent deviations from canonical hybridogenesis across populations.

Fish oogonial stem cells (OSCs) are vital for fish reproduction research and germplasm conservation, yet stable long-term in vitro culture of fish OSCs remains a major technical bottleneck. Herein, we established an efficient in vitro culture system (termed L15SP) for OSCs derived from Paralichthys olivaceus, consisting of L-15 medium supplemented with15% fetal bovine serum (FBS), 2 μg/L basic fibroblast growth factor (bFGF), 2 μg/L leukemia inhibitory factor (LIF), 50 μmol/L β-mercaptoethanol (β-ME), 1% fish serum, and 18 g/L embryo extract protein, cultured at 23 °C. This system enabled stable long-term passage of P. olivaceus OSCs. Further investigations revealed that interferon-induced transmembrane protein 3 (Ifitm3) was highly expressed on the membrane of P. olivaceus OSCs. Functional assays demonstrated that Ifitm3 significantly enhanced proliferation, migration, and stemness maintenance of long-term cultured OSCs. Ifitm3 positively modulated the expression of key PI3K-AKT signaling components, including pik3cb, pik3r1, and akt2, to regulate downstream OSC functional genes. Upon pathway activation, Ifitm3 facilitated PIP3 enrichment at the OSC plasma membrane, thereby amplifying PI3K-AKT signaling. In conclusion, Ifitm3 governed OSC biological functions by potentiating PI3K-AKT signal transduction. This study establishes a reliable long-term culture system for fish OSCs and identifies a novel functional marker for OSC identification, providing valuable support for fish germplasm preservation and genetic breeding.

Stress adversely affects various aspects of the female reproductive process, including follicle development, maturation, ovulation, luteal activity, and embryo implantation. Restraint stress reduces the number of implantated embryos. The precise mechanisms by which stress affects embryo implantation are not fully understood. In this study, a mouse model of restraint stress was used to investigate the effects of stress on uterine receptivity and the underlying mechanisms. Mice were randomly divided into three groups. The groups were the Control group, Stress group, and Ab-IL-6 treatment group. Mice in the Stress and Ab-IL-6 treatment groups underwent restraint stress in perforated 50 ml centrifuge tubes for 4 hours daily over 7 consecutive days. Results indicated that the number of implanted blastocysts and total litter size in the stress group were significantly lower than those in the control group. These findings indicate that restraint stress leads to high IL-6 expression in the endometrium. This increased expression may affect the endometrium's receptivity, resulting in a decreased number of implanted blastocysts. Furthermore, we demonstrated that IL-6 regulated autophagy through the IL-6/JAK2/BECN1 pathway in Ishikawa cells and mouse endometrial epithelial cells, and treatment with an IL-6 antibody significantly mitigated the adverse effects of stress-induced blastocyst implantation. Our findings demonstrated that restraint stress had a negative influence on endometrial receptivity via the IL-6/JAK2/BECN1 pathway. This effect led to a reduction in the number of implanted embryos. Consequently, inhibiting IL-6 expression may represent a potential therapeutic strategy for preventing stress-induced implantation failure.

Adropin, a peptide involved in energy homeostasis, acts via G protein-coupled receptor 19 (GPR19). This study examined the expression of adropin/GPR19 in porcine ovarian follicles and assessed the effects of adropin on granulosa cells (Gc) function and oocyte maturation in vitro. Levels of adropin/GPR19 were analyzed in porcine ovarian follicles and follicular fluid during the estrous cycle. Then, Gc isolated from medium-sized follicles were cultured with gonadotropins, steroids, or adropin (0.1-100 nM) to evaluate adropin/GPR19 expression, Gc function, and signaling pathways using pharmacological inhibitors. Additionally, the effect of adropin on oocyte maturation was assessed after in vitro maturation of cumulus-oocyte complexes from prepubertal pigs. We noted that adropin expression increased in the whole porcine ovarian follicle, while GPR19 decreased during the estrous cycle. Adropin protein expression and secretion to culture medium and GPR19 protein increased with gonadotropin and steroid treatment. Both proteins are localized to Gc and theca cells. Functionally, adropin reduced Gc viability via AKT signaling, inhibited proliferation and cell cycle progression via ERK1/2, suppressed estradiol secretion and CYP19A1 expression via PKA, increased progesterone secretion, cleaved caspase-3 expression and its activity, and inhibited autophagy-related markers. Adropinsimultaneously enhancing oocyte maturation, progesterone secretion, and lipid utilization in cumulus-oocyte complexes. These findings indicate that adropin may regulate porcine follicular development through its actions on Gc and oocytes.

Preeclampsia (PE) is a severe pregnancy-specific disorder featured by insufficient extravillous trophoblast (EVT) invasion and impaired spiral artery remodeling. Pregnancy-specific beta-1-glycoprotein 2 (PSG2), a placenta-derived protein, is significantly elevated in the serum of PE patients, yet its exact role and underlying mechanism in trophoblast function remain largely unknown. We demonstrated that PSG2 was remarkably upregulated in PE placentas and its expression correlated positively with blood pressure and proteinuria, and negatively with gestational age and birth weight. In HTR-8/SVneo trophoblast cells, PSG2 overexpression suppressed proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT), downregulated MMP2 and MMP9 expression, and induced S-phase cell cycle arrest and late apoptosis. Conversely, PSG2 knockdown exerted opposing effects without influencing cell cycle distribution or apoptosis. Immunofluorescence staining verified the co-localization of PSG2 and TGF-β1 in trophoblasts and placental tissues. Mechanistically, PSG2 activated the TGF-β/Smad3 signaling pathway, and the TGFBR1 inhibitor SB431542 effectively reversed PSG2-mediated inhibition of EMT, MMP2/MMP9 expression, and trophoblast functional impairment. In conclusion, highly expressed PSG2 in PE placentas contributes to PE pathogenesis by activating the TGF-β/Smad3 pathway, thereby inhibiting EMT and MMPs expression and attenuating trophoblast proliferation, migration, and invasion. These findings provide a novel molecular basis for understanding PE pathogenesis.

Maternal obesity compromises placental development and fetal growth, yet the mechanisms remain unknown. Here, we investigated the impact of high-fat diet (HFD) on placental energy homeostasis in mice. Female C57BL/6 mice were fed a control diet or an HFD, with a subset receiving FerroTerminator-1 (FOT1) to modulate metabolic dysfunction. At embryonic day 18.5, maternal metabolic status, fetal growth, placental morphology, energy metabolism, and oxidative stress were assessed. HFD-fed dams developed dyslipidemia and insulin resistance, and exhibited placental lipid accumulation and impaired labyrinth zone development, resulting in reduced fetal weight and altered fetal liver lipid and glycogen contents. The placenta showed mitochondrial damage, accompanied by reduced ATP content, mitochondrial membrane potential, mitochondrial DNA copy number, and suppressed respiratory chain complex activities. Placental metabolomic profiling showed that HFD was associated with altered levels of metabolites in glycolysis and oxidative phosphorylation pathways. These alterations coincided with placental iron-associated oxidative stress, evidenced by elevated non-heme iron, lipid peroxidation products, upregulation of transferrin receptor (TFRC) and lysophosphatidylcholine acyltransferase 3 (LPCAT3), downregulation of ferritin heavy chain 1 (FTH1) and solute carrier family 40 member 1 (SLC40A1), a reduced glutathione to oxidized glutathione ratio, and impaired glutathione peroxidase 4 (GPX4) expression and activity. Meanwhile, FOT1 administration attenuated placental oxidative stress, partially restored ATP production and mitochondrial function, and improved fetal growth in HFD-fed mice. Collectively, maternal obesity was associated with placental iron-associated oxidative stress and bioenergetic impairment, which may contribute to reduced placental efficiency and FGR.

Flame retardants are a diverse class of environmental contaminants widely used to reduce flammability in consumer products, leading to pervasive human exposure. Among these, polybrominated flame retardants (PBFRs), including polybrominated diphenyl ethers (PBDEs), and organophosphate ester flame retardants (OPEs) have raised significant concern due to their potential to disrupt reproductive health. Increasing experimental, epidemiological, and mechanistic evidence demonstrates that both PBDEs and OPEs adversely affect both the male and female reproductive systems. In males, exposure to these compounds is associated with impaired spermatogenesis, altered sperm quality, disrupted steroidogenesis, and structural and functional damage to the testis. Mechanistic studies reveal direct toxicity to Leydig, Sertoli, and germ cells, driven by mitochondrial dysfunction, oxidative stress, endocrine disruption, and cytoskeletal disorganization that may compromise both fertility and offspring health. In females, PBDEs and OPEs disrupt ovarian folliculogenesis, steroid hormone synthesis, and oocyte quality, while also altering uterine receptivity. These effects are mediated through perturbations of the hypothalamic-pituitary-gonadal axis, interference with nuclear and membrane hormone receptors, mitochondrial injury, oxidative stress, and dysregulation of signaling pathways essential for follicle maturation and ovulation. Both classes of flame retardants can bioaccumulate and cross critical biological barriers, including the placenta, raising concerns about developmental and transgenerational effects. Collectively, the evidence underscores that PBFRs and OPEs pose significant risks to reproductive function in both sexes, acting through molecular mechanisms that disrupt gonadal development, endocrine signaling, and cellular homeostasis. Understanding these shared and sex-specific pathways is essential for improving human health risk assessment and guiding regulatory strategies.

Endocrine-disrupting chemicals (EDCs) constitute a broad class of toxicants capable of altering hormone synthesis, transport, and receptor activity. Phthalate esters, a major subgroup of EDCs, are pervasive in consumer, industrial, and medical products and readily leach from these materials, resulting in human exposure through ingestion, inhalation, dermal absorption, and medical treatments. After absorption, phthalates undergo extensive biotransformation and distribute systemically, including to ovarian tissue and follicular fluid. Epidemiological studies consistently associate phthalate burden with diminished ovarian reserve, altered reproductive hormones, early menopause, pregnancy loss, and reproductive disorders. Notably, phthalate metabolites detected in follicular fluid correlate with reduced antral follicle count, impaired oocyte maturation, decreased fertilization rates, and poor medically assisted reproduction outcomes. Experimental evidence from mouse and bovine models supports these associations, demonstrating that phthalate exposure reduces antral follicle counts, disrupts steroidogenesis, causes oxidative stress, and promotes apoptosis. In vitro and mechanistic studies show that phthalates impair follicular function and hormone biosynthesis by disrupting lipid metabolism, mitochondrial function, microRNA expression, and pathways governing redox balance, inflammation, cell cycle and apoptosis. These disruptions contribute to impaired folliculogenesis, increased follicular atresia, ovulation deficits, and compromised oocyte competence. Although substantial descriptive evidence exists, significant mechanistic gaps still remain, highlighting the need for research using biologically relevant phthalate doses and approaches capable of distinguishing effects of individual congeners from mixtures.

The golden hamster, renowned for its representative PIWI-piRNA pathway profile among mammals, has emerged as a promising animal model for investigating female mammalian fertility. Prior research uncovered that the disruption of Piwil1 leads to complete infertility of female golden hamsters, with embryos derived from homozygous knockout (Piwil1-/-) females mated with wild-type males arresting at the two-cell stage. However, the underlying deep mechanisms remain unexplored. In the current study, we utilized highly sensitive single-cell mass spectrometry to generate proteome data of five developmental stages from oocytes to embryos in both wild-type (WT) and Piwil1-deficient golden hamsters, thus profiling the dynamic proteome landscape during oocyte-to-embryo transition (OET). Integrative analyses highlighted the temporal dynamics and complexity of hamster oocytes and embryos. Notably, we observed that classical maternal proteins in hamsters exhibit a more intricate pattern compared to mice, while PIWIL1 deficiency led to aberrant expression of proteins with diverse functions in oocytes and early embryos. Analysis of Multi-omics data indicated that PIWIL1 primarily regulates differentially expressed proteins at the post-translational level during OET. Specifically, the stability of TDRD1, essential for embryogenesis and gametogenesis, is modulated by PIWIL1, and could be its downstream target. Our study provides an extensive database that offers valuable insights into mammalian oocyte and early embryo development, and represents an invaluable resource for further mechanistic studies to deepen the understanding of developmental regulation.

Endometriosis (EMS) is characterised by a disrupted peritoneal immune microenvironment where M2 macrophage polarization and impaired phagocytosis promote lesion survival. Single-cell RNA-seq (ScRNAseq) of the peritoneal macrophages from EMS patient revealed elevated Triggering Receptor Expressed on Myeloid cells 2 (TREM2) and signal regulatory protein α (SIRPα). The expression of TREM2 and SIRPa is positively correlated, and each are positively correlated with estrogen response. Mouse EMS model was established by intraperitoneal injection of estrogen primed mouse endometrial fragments into recipient female mice. In vitro estrogen treatment of RAW 264.7 cells indicated an increasing TREM2/SIRPα expression and enhancing phagocytosis of lesion cells derived from WT EMS ectopic lesions. Peritoneal macrophage from mice with EMS were analyzed using flow cytometry for the expression of TREM2 and SIRPα. The assessment of F4/80, CD206, and SIRPα expressing cells within the lesions were performed through the implementation of multi-color immunohistochemistry (mIHC). Small peritoneal macrophages (SpMs, F4/80low CD11blow) were markedly increased in the EMS model. Trem2 knockout mice, as the recipient, showed smaller ectopic lesion size and less lesion formation. TREM2 deficiency inhibited SpM polarization into M2 and downregulated their SIRPα expression, while enhancing phagocytic activity. Mechanistically, estrogen pretreatment upregulated TREM2, which correlated with SIRPα upregulation and impaired phagocytosis. Under estrogen exposure, TREM2 knockdown RAW 264.7 cells potentiated phagocytosis. Thus, TREM2 is an important contributing pathogenic factor in promoting EMS by enhancing M2 polarization and suppressing phagocytosis via SIRPα upregulation.

Normal neurodevelopment relies on effective placental oxygen transfer and the specialized circulatory shunts that direct oxygen-rich blood to key fetal organs. The tone of these shunts, particularly the ductus venosus, is critical for routing oxygen and nutrient-rich blood towards the fetal brain. However, these shunts are sensitive to both oxygenation status and sympathetic innervation. Notably, fetal hypoxia increases shunting through the ductus venosus via enhanced hepatic sympathetic activation, a mechanism that redistributes oxygen rich blood toward vital organs. Thus, we hypothesized that hyperoxygenation may decrease shunting through the ductus venosus via hepatic sympathetic regulation but may be reversed if combined with an α-adrenergic agonist. Pregnant ewes (n = 8) underwent fetal catheterization surgery at 116-117 days gestation. At 120-124 days gestation, ewes were anaesthetized, and MRI scans were performed during normoxia, hyperoxia, and hyperoxia + phenylephrine infusion to measure blood flow and oxygenation within the major fetal vessels using phase-contrast and T2 oximetry MRI techniques, respectively. Hyperoxia had minimal effects on fetal haemodynamics. However, with the addition of phenylephrine, there was increased flow through the ductus venosus and ascending aorta but decreased flow toward the brain via the carotid arteries. Hyperoxia + phenylephrine increased overall fetal oxygen delivery but decreased cerebral oxygen delivery, despite no change in ductus venosus shunting (DV/UV flow ratio). There was no change in fetal or cerebral oxygen consumption. In conclusion, hyperoxia + phenylephrine reduced cerebral oxygenation, indicating that increased ductus venosus flow does not translate to improved cerebral oxygen delivery when carotid vascular resistance is elevated.

Intrafollicular transfer of immature oocytes (IFIOT) is a promising embryo production strategy, but outcomes remain poor and inconsistent. We investigated the effects of the injection procedure and the number of injected cumulus-oocyte complexes (COCs) on the biochemical and molecular environment of follicles. First, dominant follicles from hormonally synchronized heifers were either not injected (NIFIOT), injected with medium only (IFIOTM), or injected with medium containing COCs (IFIOT-COCs). Follicular fluid (FF) was analyzed for cell-free DNA (cfDNA), oxidative stress markers (GPx activity and FRAP), steroid hormones, and metabolomic profiles using UPLC-HRMS. Injection procedure increased cfDNA levels and altered steroidogenesis, with higher progesterone levels, lower estradiol levels, and lower E2/P4 ratios in the injected follicles. Metabolomic analysis revealed changes in amino acid and anti-inflammatory pathways on the injected follicle compared to the non injected. In addition, follicles were injected with 25 or 50 COCs (IFIOT25 and IFIOT50) to assess the impact of oocyte number on the follicular environment. IFIOT25 follicles showed higher GPx activity and estradiol concentrations compared to IFIOT50, whereas IFIOT50 follicles exhibited the downregulation of redox- and stress-related genes (SOD1, GPX4, NFE2L2, HSP70, and CASP3) and lower embryo yield compared to IFIOT25 (42 vs 52%, respectively). Metabolomic analysis indicated alterations in lipid and energy metabolism, with key metabolites (creatine, l-acetylcarnitine, dodecanoic acid, and panthenol) reduced in the IFIOT50 group, suggesting impaired metabolic homeostasis at higher oocyte density. Overall, both the injection process and oocyte number influenced follicular physiology, potentially affecting oocyte quality, highlighting the need to refine IFIOT protocols.

For most animals, fertilization of an egg by more than one sperm is embryonically lethal, and eggs therefore deploy multiple blocks to prevent additional sperm from entering an already fertilized egg. While Zn2+ release has emerged as a conserved feature of the slow block, its precise role in external fertilizers remains unclear. Here, we demonstrate that Xenopus laevis fertilization triggers a robust efflux of Zn2+ and that insemination of X. laevis eggs in physiologically relevant concentrations of Zn2+ potently and reversibly suppresses early development. Our findings are consistent with the hypothesis that extracellular Zn2+ inhibits fertilization through multiple mechanisms. Extracellular Zn2+ prevents the fertilization-evoked depolarization, also called the fast block to polyspermy, by acting upstream of the Ca2+-activated Cl- channel TMEM16A. Zn2+ also directly reduces sperm fertilizing capacity, although eggs are significantly more sensitive than sperm. Furthermore, Zn2+ coordinates with the egg's extracellular matrix to stabilize the glycoprotein-rich jelly coat, rendering it resistant to reductive removal and suggesting a structural mechanism by which Zn2+ limits sperm entry. The inhibitory effects of Zn2+ are fully reversed by the extracellular chelator ZX1, reinforcing the conclusion that Zn2+ functions as a dynamic extracellular regulator rather than an intracellular messenger. Our findings establish Zn2+ as a multi-level signal that reinforces fertilization barriers by targeting both gametes and modifying the egg's extracellular environment.

Endometrial decidualization is essential for successful embryo implantation and pregnancy establishment. This study investigates the role of high mobility group box 1 (HMGB1) in endometrial stromal cells (hESCs) during decidualization. We found that HMGB1 expression was upregulated during decidualization in vitro and in normal decidual tissues but was reduced in decidua from patients with recurrent spontaneous abortion (RSA). HMGB1 Knockdown in hESCs impaired decidualization by disrupting stromal cell differentiation, reducing the expression of prolactin (PRL) and insulin like growth factor binding protein 1 (IGFBP1), and promoting aberrant cell proliferation. HMGB1 deficiency resulted in mitochondrial dysfunction, characterized by impaired oxidative phosphorylation, reduced ATP production, and altered mitochondrial morphology and membrane potential. Mechanistically, HMGB1 regulated the expression of PPARG coactivator 1 alpha (PPARGC1A), a key regulator of mitochondrial biogenesis and oxidative metabolism, suggesting that HMGB1 contributes to decidualization through PPARGC1A mediated metabolic regulation. Furthermore, reduced PPARGC1A expression in RSA decidua further supports the involvement of the HMGB1-PPARGC1A axis in decidual dysfunction. In addition, HMGB1 exhibited dynamic expression patterns in the uterus of early pregnant mice, with enriched expression in decidual regions at implantation sites. Collectively, these findings identify HMGB1 as an important regulator of decidualization by coordinating mitochondrial function and cellular energy metabolism, providing new insights into the molecular mechanisms underlying decidual dysfunction and pregnancy complications.