Search Results (166)

Porcine cumulus–oocyte complexes (COCs) undergo dynamic morphological and molecular changes during maturation; however, cell type- and stage-dependent transcript patterns of Ras-related factors in cumulus cells and oocytes remain unclear. This study investigated changes in Ras-related factors during porcine in vitro maturation (IVM). Porcine COCs were cultured under IVM conditions supplemented with follicle-stimulating hormone (FSH), human chorionic gonadotropin (hCG), and epidermal growth factor (EGF). Cumulus cells and oocytes were collected at 0, 22, and 44 h of IVM, and cumulus expansion was monitored using time-lapse imaging. Hormone receptor-related factors, CX43, Ras family members, and their RasGAP and RasGEF regulators were analyzed using RT-PCR and qPCR with band-intensity analysis. Progressive cumulus expansion was observed during IVM. In cumulus cells, R-Ras transcript abundance increased at 22 and 44 h, whereas RASA1 transcript abundance was lower at 44 h. In oocytes, R-Ras and H-Ras transcript abundance decreased during maturation. These findings indicate that selected Ras family members and their regulators exhibit distinct cell type- and stage-dependent transcript patterns during porcine COC maturation. These transcript-level findings provide a basis for future protein-level and functional analyses. Full article

Improvement in the in vitro maturation (IVM) of oocyte quality is a gateway to enhancing the efficiency of in vitro embryo production. The anti-Müllerian hormone (AMH) is a crucial hormone secreted by granulosa cells that effectively suppresses primordial follicle recruitment and regulates follicular growth and development. This study was designed to investigate the role of AMH on the IVM of sheep oocytes. In this current study, oocytes in vitro were cultured in media supplemented with AMH. We comprehensively analyzed the impact of AMH on various developmental parameters of sheep oocytes, such as cellular activity, cortical granules (CGs) migration, cytoskeleton and mitochondrial function of oocytes. Furthermore, Smart-seq2 single-cell RNA sequencing (scRNA-seq) was employed to elucidate the oocytes’ development. The results showed that treatment with 100 ng/mL improved the maturation rate of the oocytes, the normal distribution rate of cortical granules and mitochondrial function, while reducing the rate of spindle abnormalities in oocytes. A total of 741 differentially expressed genes (DEGs) were observed between the FSH_12 h and AMH_12 h groups, and 746 DEGs were observed between the FSH_24 h and A+F groups. KEGG pathway analysis revealed that the FSH_12 h and AMH_12 h groups significant enrichment in DEGs were associated with p53, MAPK, PI3K-Akt and TGF-beta signaling pathways, and the FSH_12 h and AMH_24 h groups significant enrichment in DEGs were associated with cAMP, AMPK, Hedgehog and estrogen signaling pathways. These findings suggest that AMH may regulates oocytes IVM via several candidate signaling pathways. Our results provide preliminary clues for exploring the regulatory mechanism of sheep oocyte maturation and optimizing relevant culture systems. Full article

Granulosa cells (GCs), an element of the ovarian follicle, are crucial for oocyte maturation, folliculogenesis, and steroidogenesis. Granulosa cells play a crucial role in fertilization by providing metabolic and hormonal support to the oocyte, maintaining its quality and regulating its meiotic arrest. Oocyte quality and fertilization efficiency depend on the proper activity of GCs, especially their mutual communication, providing metabolic support and protecting against oxidative stress. When interrupted, they may take part in the pathogenesis of polycystic ovary syndrome, premature ovarian failure, primary ovarian insufficiency, and diminished ovarian reserve. GCs are enclosed in the antrum where they communicate with surrounding cells, create a dynamic microenvironment, and regulate hormone biosynthesis. To analyze molecular mechanisms regulating endogenous signaling, it is important to consider the dynamic transcriptomic response of porcine GCs during in vitro culturing over 48, 96, and 144 h. Transcriptomic analysis revealed a variable and dynamic transcriptional upregulation of genes associated with cellular response to endogenous and external stimuli, chemical compound metabolism, vascular development, and GCs migration. Also, proven by Gene Ontology (GO) enrichment analysis, the following terms were highlighted: “cellular response to chemical stimulus” and “cellular response to organic substance”. Specific genes, such as HSD3B1, POSTN, LOX, SERPINB2, ITGB3, ANKRD1, SLC1A1, and SFRP2, exhibited significant expression changes, suggesting extensive GCs self-regulation and metabolism changes. Further analysis indicates improvements in cellular response to a cytokine stimulus, growth factor response, hormone response, enzyme-linked receptor protein signaling, and positive regulation of cell migration. These findings suggest interweaving of regulatory mechanisms underlying intercellular communication in GCs during in vitro culturing, despite the lack of signals from the native ovarian environment. Further investigating interplays of detecting pathways will provide a more comprehensive understanding and even insights into the potential clinical use of the knowledge about the role of GCs in folliculogenesis, oocyte maturation and ovulation. Full article

Advanced maternal age is a significant social and clinical issue associated with the natural decline in a woman’s ovarian reserve. This prospective, single-center study included women with primary infertility who presented to the Gyncentrum Clinic in Katowice and analyzed 77 ovarian follicles. The study group consisted of patients of advanced reproductive age with diminished ovarian reserve, who underwent hormonal stimulation in preparation for oocyte retrieval. Each metaphase II (MII) oocyte was fertilized in vitro and cultured individually in a time-lapse incubator. Follicular fluid obtained during oocyte retrieval was collected separately from each follicle and used for non-invasive biochemical analysis of prognostic factors using a Multiplex assay. The concentrations of interleukin 10 (IL-10), granulocyte colony-stimulating factor (G-CSF), granulocyte–macrophage colony-stimulating factor (GM-CSF), and C-type natriuretic peptide (CNP) were evaluated. The analysis showed that lower concentrations of GM-CSF and CNP were associated with an increased probability of oocyte fertilization, whereas higher levels of IL-10 and G-CSF had the greatest impact on blastocyst formation. This model was supported by continuous embryo monitoring. An eightfold increase in the likelihood of blastocyst formation was observed when early embryo cleavage occurred between 25 and 27 h after insemination. Furthermore, prolonged duration of the first cytokinesis reduced the probability of blastocyst development, while an extended cell cycle at the two-blastomere stage significantly affected further embryo development. These findings may support non-invasive embryo selection strategies. Full article

Hair follicle (HF) stem cells are multipotent adult stem cells that play a key role in the hair follicle cycle. However, it remains poorly understood how the outer root sheath (ORS)—specifically, the stem cells in the bulge region of the hair follicle—promotes skin repair. This study aims to investigate the role of bulge stem cells in tissue growth and repair and to determine whether the ORS of transplanted hair follicles can facilitate skin repair. We further seek to elucidate the mechanisms by which bulge stem cells contribute to hair follicle development, regeneration, and skin wound healing. In this study, hair follicle samples were obtained from neonatal mice using microdissection. Hair follicle morphology was assessed by Sirius red staining, H&E staining, and transmission electron microscopy. Immunofluorescence staining was used to detect changes in CD34 and SOX9 protein expression. Additionally, microdissection-based hair follicle transplantation and Western blotting were employed to investigate protein activation and inhibition in the Wnt/β-catenin signaling pathway. The results show that the hair follicle bulge, inner root sheath, and dermal papilla all increase in size as hair follicles grow, with each structure growing relatively rapidly on day 7. Treatment with Teplinovivint effectively inhibits the expression of Wnt/β-catenin signaling pathway-related proteins and hair follicle stem cell markers. Damaged hair follicle tissues cultured in vitro are capable of self-repair. At the transplantation site, the skin gradually closes as the outer root sheath wound heals. In contrast, the central region of the outer root sheath becomes progressively filled with numerous dividing cells and extracellular matrix. The inner portion of the outer root sheath is densely populated with cells, and the markers CD34 and SOX9 are also widely distributed. This indicates that activation of the Wnt/β-catenin signaling pathway enhances the proliferation and differentiation of hair follicle stem cells, thereby promoting hair follicle growth, repair of damaged follicles, and healing of skin wounds. Furthermore, this study demonstrates the feasibility of using transplanted outer root sheath (ORS) to repair skin wounds—specifically, the potential to achieve large-scale hair regeneration from a limited number of hair follicle stem cells—providing a new approach for the clinical treatment of skin injury disorders. Nevertheless, achieving long-term, stable, and scalable clinical translation of ORS stem cells for hair follicle regeneration remains a major challenge. Full article

The present study investigated the effects of propionic acid (PA) supplementation during the in vitro growth of oocytes derived from bovine early antral follicles (EAFs) on oocyte quality and granulosa cell (GC) characteristics. Oocyte–GC complexes (OGCs) were collected from bovine ovaries and cultured for 14 days in medium supplemented with or without 0.01 mM PA. Oocytes and GCs obtained from large antral follicles were used as in vivo controls. Oocytes cultured with PA exhibited higher nuclear maturation and fertilization rates than those cultured without PA. PA supplementation increased lipid content and ATP levels in both oocytes and GCs and enhanced glucose consumption in OGCs. However, compared with in vivo-grown oocytes, in vitro-grown oocytes showed lower ATP levels, lipid content, mitochondrial membrane potential, and fertilization ability. RNA-seq followed by K-medoids clustering analysis revealed that PA-induced genes in GCs showing expression patterns similar to those in vivo were mainly associated with oxidative phosphorylation, whereas genes showing expression patterns distinct from the in vivo profile were associated with the PI3K–Akt signaling pathway. In conclusion, PA improves the quality of in vitro-grown oocytes and is associated with enhanced energy status in both oocytes and GCs. However, some PA-induced gene expression patterns in GCs differed from those observed in vivo. Full article

Hair follicles (HFs) are highly accessible mini-organs that house diverse somatic and stem cell populations with broad therapeutic potential. In this study, we investigate the untapped utility of plucked HFs as a non-invasive tissue source for regenerative medicine. We demonstrate the successful isolation and expansion of keratinocytes and mesenchymal stem cells (MSCs) from plucked follicles using an enzyme-free explant culture method. HF-derived keratinocytes retained their epithelial identity and were efficiently reprogrammed into induced pluripotent stem cells (iPSCs). These iPSCs were further directed toward definitive endoderm and pancreatic progenitor fates, confirming their robust autologous regenerative capacity. Flow cytometric analysis of HF-MSCs validated a characteristic mesenchymal profile, and these cells exhibited classical trilineage plasticity alongside the ability to differentiate into dopaminergic neural progenitors. Furthermore, proteomic and vesicular characterization of the autologous HF secretome (aHFS) revealed a rich enrichment of regenerative cytokines and exosomes. The aHFS demonstrated potent wound-healing bioactivity in vitro. Collectively, these findings establish the plucked hair follicle as a highly practical, scalable source for both cell-based and cell-free therapies, highlighting the clinical value of early-stage follicular biobanking for personalized medicine. Full article

Increased heat levels can affect follicle development, oocyte maturation, and bovine fertility by disrupting amino acid (AA) metabolism and oocyte competence. This study aimed to explore the effect of heat shock on AA metabolism in bovine cumulus cells (CCs) or denuded oocytes (DOs). CCs and DOs were separately cultured for 24 h in vitro at 38.5 °C (control group), 39.5 °C (moderate heat shock-CC, MHS-CC group), or 40.5 °C (high heat shock-CC, HHS-CC group). AA levels were analyzed in 24-h in vitro maturation media using high-performance liquid chromatography. The findings indicated that the HHS-CC group consumed more AAs than the control (p = 0.04) or MHS-CC group (p = 0.03). Compared with the control and HHS-CC groups, the MHS-CC group exhibited elevated alanine levels (p = 0.02). The MHS-CC (p = 0.03) and HHS-CC (p = 0.03) groups exhibited significantly greater glutamine depletion than the control group. The HHS-DO group exhibited significant lysine depletion (p < 0.01) but produced more tryptophan than the control and MHS-DO groups (p = 0.02). In contrast to the control and MHS-DO groups, the HHS-DO group displayed a notably elevated level of appearance (p = 0.005) and net balance (p = 0.005) for all AAs. The findings imply that heat shock may alter the metabolism of certain AAs in CCs and DOs, thereby affecting the developmental competence of bovine oocytes. Full article

Ovarian tissue cryopreservation is the primary fertility preservation strategy for prepubertal girls and patients requiring urgent gonadotoxic therapy. However, the risk of reintroducing malignant cells has prompted the development of safer alternatives, including follicle isolation followed by three-dimensional scaffold encapsulation for transplantation. Fibrin is a promising biomaterial for bioengineered ovary construction, although its ability to support early human follicle maintenance remains unclear. Follicles isolated from cryopreserved ovarian tissues of six patients were encapsulated within fibrin scaffolds of graded concentrations (high, medium, low). After 7 days of in vitro culture, follicle survival and diameter change were quantified. A total of 282 follicles (45.4 ± 10.1 µm) were embedded into fibrin scaffolds. After culture, 237 viable follicles were detected, yielding an overall survival of 84%. Follicle diameter increased to 58.8 ± 12.0 µm. Follicle survival rates were comparable across groups, while mean follicle diameter was 56.3 ± 12.5 µm (high), 61.9 ± 13.4 µm (medium), and 57.4 ± 9.3 µm (low). Follicles cultured in medium-concentration fibrin demonstrated significantly larger diameters compared with both high and low groups (p < 0.05), with no difference between high and low groups. Fibrin-based bioprosthetic ovary scaffolds support short-term in vitro maintenance of isolated human follicles, preserving spherical morphology and granulosa cell layer integrity. Medium-concentration fibrin was associated with greater follicle diameter expansion compared with higher and lower concentrations, indicating that scaffold composition influences early morphometric changes during in vitro follicle culture. Full article

Scalp microbes are recognized as contributors to hair loss by influencing scalp homeostasis and hair growth. However, the in vitro anti-hair loss activity of microbial culture media derived from healthy scalps remains unclear. In this study, resident microbes from 20 Korean participants with healthy scalps and hair were isolated, and Staphylococcus capitis was used to produce S. capitis ferment filtrate (SCFF). SCFF anti-hair loss activity was evaluated in human follicle dermal papilla cells (HFDPCs) and human adult low-Calcium High-Temperature (HaCaT) keratinocytes via proliferation assays, qPCR, immunocytochemistry, and SA-β-gal staining at 250–1000 μg/mL. SCFF increased cell density after 48 h in a concentration-dependent manner. In HFDPCs, SCFF controlled growth (KGF, IGF-1, and HGF) and androgen (AR and TGF-β2) factors, regulating key mRNAs for hair growth. SCFF mitigated scalp and hair aging by promoting sirtuins 1 and 7 and collagen type 13, while suppressing p21 and X-Gal staining. In HaCaT cells, SCFF exhibited a scalp barrier-strengthening effect by significantly increasing filaggrin and involucrin levels. It suppressed reactive oxidative stress and exhibited DPPH and ABTS radical scavenging activity. These results suggest that SCFF may modulate key pathways associated with hair loss by promoting scalp and hair anti-aging, barrier strengthening, enhancing antioxidant activity, and supporting hair growth. Full article

Androgenetic alopecia (AGA) and telogen effluvium (TE) are common hair loss disorders characterized by dysregulated hair follicle cycling and impaired dermal papilla cell function. Emerging evidence indicates that exosomes are key mediators of intercellular communication, largely through their microRNA (miRNA) cargo. Milk-derived exosomes (Mi-Exos) represent an accessible and biologically active source of regulatory miRNAs with potential relevance for hair disorders. This study evaluated the in vitro effects of bovine milk-derived exosomes (MEV-miRNAs) on human hair follicles. MEV-miRNAs were enriched in miRNA families (Let-7, miR-21, miR-30, miR-200, and miR-148/152) previously implicated in hair follicle regulation. Viability/metabolic activity of hair follicle dermal papilla (HFDP) cells was assessed, and human hair follicles were cultured ex vivo to measure shaft elongation and modulation of the WNT signaling pathway by qRT-PCR. MEV-miRNAs significantly increased HFDP cell viability after 24 h compared with controls. Human hair follicles showed a non-significant trend toward increased elongation following treatment. Gene expression analysis revealed significant up-regulation of key WNT pathway components, including WNT2, WNT5B, WNT10A, WNT11, MMP7, WISP1, and NKD1, indicating modulation of WNT-associated pathways implicated in hair follicle growth and cycling. Overall, MEV-miRNAs exhibit positive modulatory effects on signaling pathways, supporting their potential as a novel therapeutic strategy for AGA and TE. Full article

Cryopreservation of preantral follicles (PAFs) is a promising tool for gene conservation and fertility preservation. However, standardized protocols for the cryopreservation and in vitro culture of isolated follicles—particularly in pigs—are still lacking. This study aimed to analyze the survival and developmental potential of porcine PAFs vitrified using two different methods: open pulled straw (OPS) and cryotube (CT). Ovaries of Hungarian Large White sows were collected from a local slaughterhouse and enzymatically digested to isolate preantral follicles. Morphologically normal follicles were assigned to three groups: fresh control, OPS-vitrified, and CT-vitrified. All follicles were cultured for 10 days in FSH-supplemented medium, with growth, survival, and estradiol (E2) production monitored. Survival rate was lower in the CT group (83.3%) than that of the control and OPS (97.4% and 94.4%, respectively). The follicular area was consistently larger in control than in CT and OPS, with no difference between vitrified groups. E2 production varied among treatments: OPS follicles showed lower E2 levels on Day 2, no differences were detected on Day 7, and CT follicles produced less E2 on Day 10. These results indicate that OPS is the more suitable vitrification method for porcine PAFs and that the culture system supports hormone production; however, it may require refinement to provide long-term follicle maintenance. Full article

The increasing biomedical and conservation interest in porcine species has driven the development of advanced in vitro follicle culture systems designed to preserve genetic diversity and accurately model key stages of folliculogenesis. This study assessed a three-dimensional (3D) alginate-based system for the in vitro culture of porcine preantral follicles, aiming to overcome the structural limitations of conventional two-dimensional (2D) methods. A total of six experimental groups were established, consisting of group-cultured (four follicles/well) or individually cultured (one follicle/well) follicles maintained either without alginate (0%) or encapsulated in 0.5% or 1% alginate for 14 days in media supplemented with FSH, EGF, and IGF-I, with LH added from day 9. Follicular development was assessed by morphometric evaluation, image-based and histological analyses, and quantification of steroid hormones in media collected every 48 h. Group-cultured follicles encapsulated in 0.5% alginate most effectively maintained their 3D architecture, reached the largest diameters, and progressed more uniformly compared with other groups. In contrast, follicles cultured without alginate rapidly lost structural integrity, showed granulosa cell migration, and decreased in size, whereas those encapsulated in 1% alginate exhibited restricted growth. Estradiol and testosterone concentrations increased over time in the 0.5% alginate group, were lowest without alginate, and intermediate in 1% alginate. Individually cultured follicles exhibited reduced growth and lower total hormone production compared with group-cultured follicles; however, when normalized per-follicle, steroid secretion, particularly in the 0.5% alginate group, was enhanced, indicating increased steroidogenic efficiency on a per-follicle basis. These findings indicate that 0.5% alginate provides an optimal balance between structural support and physiological steroidogenesis during preantral follicle culture. This 3D system improves the biological relevance of porcine follicle culture and may support future applications in reproductive biology, conservation, and genetic resource preservation. Full article

Oocyte-granulosa cell complexes (OGCs) cultivation is crucial for advancing reproductive biotechnology but remains incomplete and needs further optimization. Insulin-like growth factor-I (IGF-I) regulates granulosa cell proliferation and apoptosis, and numerous studies have confirmed its role in promoting ovarian follicle development. Porcine follicular fluid (PFF) contains factors beneficial for oocyte growth, which may enhance oocyte development. To investigate whether IGF-I and PFF improve the in vitro culture efficiency of porcine OGCs, we cultured OGCs with IGF-I (0, 10, 50, 100 ng/mL) and PFF (from 3 to 6 mm follicles) at concentrations of 0, 2.5%, 5%, 10%, respectively. The results revealed that 50 and 100 ng/mL IGF-I significantly increased the antrum formation rate of OGCs (from 61.11 ± 7.35% to 88.89 ± 7.35%) and diameter growth of oocytes (from 108.77 ± 0.27 µm to 114.94 ± 0.58 and 113.29 ± 0.50 µm, respectively). However, only the 50 ng/mL group, but not the 100 ng/mL group, significantly improved the maturation rate (38.13 ± 3.77% vs. 25.00 ± 3.27%, p < 0.05) of oocytes. Additionally, 50 ng/mL IGF-I downregulated BAX (a pro-apoptotic gene) and upregulated BCL-2 (an anti-apoptotic factor) in granulosa cells, ultimately reducing apoptosis. In contrast, none of the PFF doses used in this study induced the formation of enclosed antrum-like structures in OGCs, nor did they significantly enhance their in vitro development. Our findings demonstrate that 50 ng/mL IGF-I effectively promotes the in vitro growth of porcine early antral follicle-derived OGCs by reducing apoptosis, whereas tested PFF concentrations had no beneficial effects and induced abnormal granulosa cell growth. How PFF modulates the adherent and spreading growth of granulosa cells has not been fully elucidated and requires further clarification. Full article

Sodium butyrate (NaBu), a common feed additive, has been shown to enhance reproductive performance in livestock and poultry. However, whether NaBu exerts this effect by directly regulating follicular development remains unclear. In this study, a three-dimensional (3D) in vitro culture system of mouse preantral follicles was used to investigate the effects of NaBu on follicular growth, hormone secretion, maturation of oocytes, and subsequent embryonic development. Preantral follicles were treated with different doses of NaBu on the fourth day of culture. Subsequently, the mature oocytes (MII stage) were released from the follicles on the ninth day and subjected to parthenogenetic activation for developmental assessment. The results showed that 0.10 mM NaBu treatment could significantly promote follicular growth, antral formation, and oocyte maturation. Furthermore, NaBu also significantly increased estradiol (E₂) secretion, improved follicular structure, and maintained cellular viability. qPCR analysis revealed that NaBu significantly increased the mRNA levels of STAR, CYP11A1, and CYP1B1. In addition, it significantly enhanced the distribution and organization of F-actin, with increases in the mRNA levels of GDF9, BMP15, and CX37. NaBu treatment significantly reduced intracellular ROS levels and increased the mRNA levels of NRF2 and SOD1, while SOD2 and GSR showed increasing trends without significant differences. NaBu significantly improved oocyte cytoskeletal organization and the morphology of the spindle, but it did not lead to a significant increase in the rates of cleavage and blastocyst formation after parthenogenetic activation. Collectively, these findings indicate that NaBu promotes follicular development and improves oocyte quality, at least partly, by enhancing steroidogenesis, alleviating oxidative stress, and maintaining cytoskeletal integrity, providing insight into its potential application for improving reproductive performance in livestock and poultry. Full article