During oocyte growth, substantial epigenetic programming occurs to establish a distinctive epigenome including appropriately patterned DNA methylation and histone modifications. Oocyte epigenetic programming must be tightly spatiotemporally regulated to ensure that a wide variety of epigenetic modifiers correctly establish their respective modifications to mediate precise control of gene expression. Furthermore, epigenetic modifications in oocytes include canonical and non-canonical genomic imprints, which are transmitted through meiosis to offspring. Significantly, disruptions in oocyte epigenetic programming can cause aberrant developmental outcomes in the next generation mediated by altered imprinting. Polycomb repressive complex 2 is an important epigenetic modifier that establishes histone 3 lysine 27 trimethylation and non-canonical imprints during mouse oogenesis, which are important for normal offspring development. While it is widely recognised that altered oocyte epigenetic programming can disrupt offspring development, mechanisms controlling maternal epigenetic inheritance remain poorly understood. The possibility remains that non-canonical imprinting exists in humans, although this requires confirmation. This review discusses mouse and human oocyte epigenetic programming including interactions between various epigenetic modifiers and modifications that form the unique oocyte epigenome. Understanding how oocyte epigenetic programming is regulated will be crucial in discerning how changes to the oocyte epigenome can disrupt epigenetic memory and alter developmental outcomes in offspring.
CONTEXT:Inhibiting phosphodiesterase (PDE) activity to increase cyclic adenosine monophosphate (cAMP) concentrations is a core strategy in artificial sperm activation. PDE3, a key member of the PDE family, comprises two subtypes, PDE3A and PDE3B. However, their specific expression patterns and functions in human sperm remain incompletely understood. AIMS:To analyze the expression characteristics of PDE3A and PDE3B in human sperm and investigate the regulatory effects of PDE3 inhibitor milrinone on asthenozoospermic sperm function and the related mechanisms. METHODS:The expression of PDE3A and PDE3B was detected using reverse transcription polymerase chain reaction (RT-PCR), western blot and immunofluorescence. Changes in asthenozoospermic sperm functions after milrinone treatment were detected using computer-assisted sperm analysis (CASA), methylcellulose viscous penetration assay, and fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) staining. Moreover, intracellular factors closely correlated with sperm function regulation were measured using enzyme-linked immunosorbent assay (ELISA), flow cytometry, and metabolome analysis. KEY RESULTS:PDE3B expression level was greater than was the expression level of PDE3A in human sperm. Sperm function analysis showed that milrinone significantly enhanced asthenozoospermic sperm motility, mucus penetration ability, and the acrosome reaction. With respect to intracellular signals, milrinone activated the cAMP‒protein kinase A (PKA) signaling pathway, and metabolomic analysis showed that milrinone-induced differentially abundant metabolites (DAMs) were associated with the citrate cycle and fatty acid degradation pathways, suggesting that it regulates energy metabolism. CONCLUSIONS:These results demonstrated that milrinone primarily targets PDE3B to improve asthenozoospermic sperm function by regulating cAMP‒PKA signaling pathway and energy metabolism. IMPLICATIONS:The present study has gained new insight into the molecular basis of cAMP signaling regulation in human sperm and provides theoretical and experimental support for the potential application of milrinone for asthenozoospermic sperm activation.
CONTEXT:Trophectoderm (TE) biopsy combined with embryo vitrification is increasingly being used; however, concerns remain regarding its effects on embryo survival and physiological stability after warming. AIMS:We aimed to evaluate whether TE biopsy performed prior to vitrification compromises early post-warming recovery and physiological responses of bovine embryos. METHODS:Expanded blastocysts were allocated into two groups, namely, Control and Biopsy. After warming, embryos were evaluated at 0, 4, 24, and 48 h for re-expansion and hatching rates. Mitochondrial activity, oxidative stress, antioxidant activity estimated by intracellular thiol content, and total cell number were assessed after 48 h. KEY RESULTS:Re-expansion differed between groups at 0 h (Control 13.40% vs Biopsy 33.90%; P = 0.005) and 4 h (Control 93.81% vs Biopsy 84.75%; P = 0.038), with no differences at 24 or 48 h (P ≥ 0.346). Hatching rates were similar at 4 h (P = 0.447), but higher in the Biopsy group at 24 h (77.56% vs 53.35%; P = 0.005) and 48 h (90.79% vs 77.39%; P = 0.027). The Biopsy group showed higher mitochondrial activity (P < 0.001), reactive oxygen species (P = 0.009), intracellular thiol content (P < 0.001), and total cell number (216.68 ± 9.75 vs 186.82 ± 10.22; P = 0.038). CONCLUSIONS:TE biopsy prior to vitrification induced oxidative stress, increased mitochondrial activity and increased thiol content, but did not compromise early post-warming recovery of bovine embryos. IMPLICATIONS:TE biopsy before vitrification is safe under short-term in vitro conditions.
CONTEXT:Pre-eclampsia (PE) involves placental dysfunction and immune dysregulation, but the cell-type localization of tryptophan-kynurenine-nicotinamide adenine dinucleotide (Trp-Kyn-NAD+) axis-related genes in PE placentas remains unclear. AIMS:This study aimed to localize Trp-Kyn-NAD+ axis-related expression across placental cell types and prioritize candidate axis-related markers using public transcriptomic datasets. METHODS:The placental microarray dataset GSE75010 was used for discovery, GSE24129 for supportive external evaluation, and the single-cell RNA sequencing dataset GSE173193 for descriptive cell-type localization. Single-cell annotation, combined axis-gene localization, differential-expression and co-expression analyses, fixed four-gene model evaluation, immune-signature analysis, exploratory ligand-receptor expression scoring and transcription factor-gene co-expression analysis were performed. KEY RESULTS:Combined single-cell analysis showed non-uniform localization of Trp-Kyn-NAD+ axis-related expression across placental cell types. Microarray-based screening prioritized AFMID, ENPP1, IDO1, and NT5E as candidate axis-related genes. The fixed four-gene model showed moderate internal discrimination, and results from the small external cohort were interpreted as supportive. Candidate genes were also associated with immune-signature variation, and ligand-receptor expression scoring identified exploratory communication-related transcriptional patterns. CONCLUSIONS:By prioritizing AFMID, ENPP1, IDO1, and NT5E and mapping their cell-type localization, this study placed established IDO1-related evidence within a broader Trp-Kyn-NAD+ axis-related transcriptomic context. AFMID, ENPP1, and NT5E represent additional candidates for evaluation in larger cohorts and experimental systems. IMPLICATIONS:These findings provide a cell-type-resolved transcriptomic context for prioritizing Trp-Kyn-NAD+ axis-related candidates for further validation in PE.
Aims A comparative study was conducted on the sperm motility kinematics of six species within the subfamily Leuciscinae, aiming to elucidate the potential relationship between variations in sperm motility parameters and both the water currents at their spawning grounds and their mating behaviour. Methods Computer-assisted sperm analysis (CASA) was performed to evaluate nine motility parameters. Water currents at the spawning grounds and mating behaviours were compiled based on literature data. Key results This study identified both similarities and differences in sperm motility characteristics. Differences in sperm motility kinematics primarily concerned velocity at activation and the total duration of movement. Conclusions In rheophilic Leuciscinae spawners, such as the vimba bream and chub, a prolonged motility duration or lower sperm velocities were observed compared to still-water spawners. Still-water spawners, such as the territorial freshwater bream and the non-territorial white bream, which exhibits reported courtship tactics, showed intermediate sperm velocities. The roach and rudd, which display lek-like mating behaviour, presented the highest initial velocities. Implications Further research is required to confirm that sperm traits in Leuciscinae are strongly shaped by environmental conditions and mating systems, as well as to provide detailed descriptions of the multi-faceted biological factors that underlie breeding success across different species.
Somatic cell nuclear transfer (SCNT, or cloning) in wild animal species is considered a potential tool in biodiversity conservation to restore genetic diversity in animal populations and overcome the detrimental effects of inbreeding, climate change, or diseases in rare and endangered animal populations. In January 2026, the Committee for Companion Animals, Non-Domestic and Endangered Species (CANDES) of the International Embryo Technology Society (IETS) organized a round table discussion on future directions in wildlife cloning. This paper reflects the content of discussions among experts in assisted reproductive technologies (ARTs) and specialists in wild animal conservation. After reviewing the advantages and limitations of SCNT, several research priorities were identified. Importantly, strengthening open communication and data exchange among experts was highly recommended to ensure that progress in the field is effective. It was also emphasized that regulatory and ethical frameworks are still required to better integrate this technology into animal conservation efforts. Overall, SCNT remains a highly specialized technology with a distinct, yet currently limited, role in biodiversity conservation. Its true value does not lie in immediate population recovery, but in its capacity to preserve genetic material, generate critical scientific insight, and inspire new collaborations across different disciplines.
CONTEXT:Artificial insemination in farmed crocodiles is limited by poor sperm survival after cryopreservation, requiring alternative preservation strategies. AIMS:This study evaluated the effects of three extenders on saltwater crocodile (Crocodylus porosus) semen during chilled storage for up to 14 days and assessed post freeze-drying (FD) sperm functional integrity. METHODS:Semen was collected from six adult males. Samples were then diluted in a commercial milk-based extender, Hanks' balanced salt solution (HBSS), or HBSS supplemented with 5% bovine serum albumin (BSA) and stored at 5°C, with sperm assessed at 24 h, 48 h, 72 h, 7 days, and 14 days. Freeze-drying was performed using standard protocols. KEY RESULTS:Ejaculate characteristics (mean ± standard error of the mean; s.e.m.) included volume 1.3 ± 0.3 mL, sperm concentration 3.32 ± 0.2 × 109 sperm/mL, total motility 65.0 ± 3.9%, viability 97.8 ± 0.7%, and normal morphology 66.8 ± 6.6%. After 72 h of storage, motility, viability, and normal morphology were maintained across all extenders, with the milk-based extender outperforming the HBSS-based extenders. Cytoplasmic droplets, loose heads, and absent midpieces were the predominant abnormalities observed. Following rehydration, freeze-dried spermatozoa showed viability of 23.0 ± 1.0%, normal morphology of 15.0 ± 3.4%, mitochondrial mass in 55.8 ± 2.7%, and DNA fragmentation of 6.2 ± 0.7%, with no evidence of motility recovered. CONCLUSIONS:Our results suggest that commercial milk-based extenders support chilled storage of crocodile semen for up to 3 days, while representing the first report of spermatozoa FD in a reptile species. IMPLICATIONS:Chilled semen storage supports semen transport for crocodile artificial insemination or cryopreservation programs, whereas freeze-drying expands future options for reptilian genetic preservation.
CONTEXT:Intracytoplasmic sperm injection (ICSI) is the primary method for equine in vitro embryo production, yet outcomes may be influenced by sperm selection techniques. AIMS:This study compared four isolation methods: EquiPure™, Vetmotl™, Felix™, and Samson™, to assess effects on sperm quality and embryo development. METHODS:Semen was collected from four fertile stallions, processed and evaluated for sperm motility, viability, DNA integrity, and yield of high-quality cells relative to input. Isolated samples were compared to non-isolated (centrifuged) samples. Spermatozoa were used for ICSI with in vitro-matured oocytes, and cleavage and blastocyst rates were recorded. KEY RESULTS:Compared with unfractionated samples, sperm quality (motility and viability) was improved in samples recovered with VetMotl, Felix and Samson while DNA integrity was improved in Vetmotl-isolated samples. When concentration of recovered cells was considered, Samson, EquiPure, and Felix yielded significantly more motile, viable, and DNA-intact spermatozoa than Vetmotl, showing the lowest recovery. Samson achieved the greatest recovery of progressively motile cells. Isolation with all methods supported in vitro embryo production; cleavage (51.2-69.4%) and blastocyst rates (20.5-24.5%) did not differ significantly between methods. CONCLUSIONS:Several established and emerging methods for sperm isolation are effective in isolating high-quality spermatozoa suitable for use in embryo production by ICSI. VetMotl, Felix, and Samson performed comparably to conventional methods, supporting their clinical utility. IMPLICATIONS:Isolation methods vary in efficiency, labour intensiveness and sperm selection stringency, with potential implications for sperm storage, low dose insemination and IVF. Further studies incorporating multiple stallions and embryo transfer outcomes are needed to confirm reproductive relevance.
CONTEXT:Hypoxia could have an impact on spermatogenesis. However, the mechanism by which hypoxia affects spermatogonial stem cells (SSC) is still unknown. AIMS:This study aims to elucidate the impact of hypoxia on the proliferation of mouse SSC and explore the underlying mechanism. METHODS:We established the hypoxia cultured C18-4 cell model using CoCl2. Cell counting kit-8 and 5-ethynyl-2'-deoxyuridine assays were used to assess C18-4 cells proliferation. RNA sequencing was performed to explore the differentially expressed genes. Western blotting and real-time quantitative polymerase chain reaction were used to detect the expression of hypoxia-inducible factor-1α and iron metabolism-related proteins. The small interfering RNA assay was used to knock down the expression of transferrin receptor complex (TFRC) in hypoxia cultured C18-4 cells. KEY RESULTS:We found that CoCl2 decreased the proliferation of C18-4 cells. Based on the RNA sequencing, we found that iron metabolism imbalance and ferroptosis might play crucial roles in the hypoxia-induced injury to C18-4 cells. Western blotting and real-time quantitative polymerase chain reaction analyses detected the upregulation of TFRC, ferritin heavy chain, ferritin light chain and ACSL4, as well as the downregulation of ferroportin and glutathione peroxidase 4 in hypoxia cultured C18-4 cells. Notably, knock down of Tfrc restored C18-4 cell proliferation, which had been inhibited by CoCl2. CONCLUSIONS:This study demonstrates that hypoxia inhibits C18-4 cell proliferation through the effects of TFRC on iron metabolism and ferroptosis. IMPLICATIONS:The hypoxia could induce iron metabolism imbalance and ferroptosis in SSC. Regulating the expression of TFRC could protect SSC from hypoxic injury.
CONTEXT:Tissue fibrosis is described as the excessive accumulation of extracellular matrix (ECM) components. This process is associated with inflammation, and the interaction between fibroblasts and immune cells plays a key role, driving the progression of fibrosis. Results of recent study suggest that interleukin (IL)-12 may play a role in the processes associated with the development of fibrosis. This proinflammatory cytokine is synthesized and released by macrophages, dendritic cells, and B cells. AIMS:The objective of this study was to determine: (i) the expression of IL-12 and its receptor in different mare endometrial categories; (ii) the effects of IL-12 on the expression of ECM-related factors; and (iii) endometrial fibroblast functional characteristics. METHODS:The mRNA expression of IL-12 subunits and IL-12 receptor was determined using quantiative polymerase chain reaction (qPCR). The expression of ECM-related factors and functional characteristics in endometrial fibroblasts were determined using qPCR, Western blotting, zymography with bromodeoxyuridine and scratch assays. KEY RESULTS:IL-12Rβ2 mRNA expression was upregulated in category IIB endometrium during the mid-luteal phase compared with the follicular phase of the estrous cycle. The IL-12 treatment of endometrial fibroblasts increased mRNA expression of COL1A1, COL3A1, ACTA2, and LOXL2, as well as matrix metalloproteinase (MMP)-9 and MMP3, with elevated pro-MMP2 and MMP9 gelatinolytic activity. Moreover, IL-12 reduced fibroblast proliferation after 96 h, without effect on migration. CONCLUSIONS:The findings indicate that IL-12 has a direct impact on the mRNA expression of fibrotic markers, MMP-2 and MMP-9 gelatinolytic activity and fibroblast proliferation. IMPLICATIONS:This suggests a potential role for IL-12 in the processes associated with ECM remodeling.
