Search Results (66)

This study investigates the effect of tryptophan treatment on aged pig oocytes, focusing on its potential to reduce oxidative stress and improve oocyte quality. An oxidative stress model was induced using hydrogen peroxide (H₂O₂) to mimic aging effects on oocytes. Fresh ovaries from young sows were collected, and oocytes were aspirated and cultured for in vitro maturation. Oocytes in the H₂O₂ and the H₂O₂+Trp groups were exposed to 100 µM H₂O₂ for 30 min, with the H₂O₂+Trp group receiving an additional 50 µM tryptophan supplementation. RNA-sequencing was performed to study the underlying mechanism through which tryptophan mitigated the H₂O₂-induced oxidative stress in oocytes. The results demonstrated that tryptophan supplementation significantly reduced oxidative stress markers such as H₂O₂ and malonaldehyde (MDA) while restoring key antioxidant enzymes such as superoxide dismutase (SOD), and catalase (CAT) confirming its antioxidant role. Furthermore, tryptophan improved cumulus cell expansion, and oocyte quality, which were compromised by oxidative stress. Transcriptomics study revealed the enrichment of several KEGG pathways, such as P13K-Akt signaling pathways as a critical regulator of cell survival and function, emphasizing the protective effects of tryptophan on oocyte integrity. Moreover, the protein–protein interaction (PPI) network identified several hub genes in the tryptophan-treated group compared with H₂O₂, including TIMP1, CCN2, and MMP12 as key players in ECM remodeling and cellular adhesion, which are critical for restoring oocyte quality. These findings suggest that tryptophan supplementation not only mitigated oxidative stress but also modulated gene expression related to cellular functions and stress response. These results propose that tryptophan could be a valuable therapeutic strategy for improving reproductive outcomes in aging sows and other mammals facing age-related oocyte dysfunction. Full article

Although prior studies have identified cumulus cells (CCs)-expressed genes and miRNAs that regulate cumulus expansion and/or CC apoptosis and may serve as markers for selecting competent oocytes and embryos, there remains an urgent need to identify CCs-expressed genes and miRNAs whose expression levels are directly correlated with oocyte developmental potential (DP). In this study, we first established CC models from mouse cumulus-oocyte complexes (COCs) that exhibited significantly different DP following in vitro or in vivo maturation. Subsequently, we performed mRNA/miRNA sequencing and functional analyses using these in vitro and in vivo CC models. We identified and validated Spp1, Fn1, Sdc1, and Ngf as DP-beneficial genes; Fos and Jun as DP-detrimental genes; and miR-7686-5p, miR-133a-3p, novel-miR-239, novel-miR-193, and miR-339-5p as DP-detrimental miRNAs. Finally, by employing a well-in-well activation/embryo culture system that enables tracking the COC origin of CCs and embryos, we further validated Spp1 and Ngf as DP-beneficial genes, Jun as the DP-detrimental gene, and miR-7686-5p, novel-miR-239, and miR-339-5p as DP-detrimental miRNAs. In conclusion, we identified and validated new sets of CCs-expressed genes and miRNAs whose expression levels were directly correlated with oocyte DP. These newly identified genes and miRNAs may serve as potential biomarkers for selecting competent oocytes and embryos. Full article

Both the livestock and biomedical fields require a large supply of high-quality mature oocytes. However, the in vitro maturation (IVM) process often leads to an accumulation of reactive oxygen species (ROS), which can cause defects in oocyte meiosis and embryo development, ultimately compromising oocyte quality. Urolithin A (UA), known for its antioxidant properties, has not been thoroughly investigated for its potential to mitigate the negative effects of oxidative stress during the in vitro culturing of oocytes, and its underlying mechanism is not well understood. In this study, an in vitro oxidative stress model was established using porcine oocytes treated with H₂O₂, followed by exposure to varying concentrations of UA. The results revealed that 30 μM UA significantly improved both the quality of oocyte culture and the developmental potential of the resulting embryos. UA was found to enhance oocyte autophagy, reduce oxidative stress-induced mitochondrial damage, and restore mitochondrial function. Additionally, it lowered ROS and DNA damage levels in the oocytes, maintained proper spindle/chromosome alignment and actin cytoskeleton structure, promoted nuclear maturation, prevented abnormal cortical granule distribution, and supported oocyte cytoplasmic maturation. As a result, UA alleviated oxidative stress-induced defects in oocyte maturation and cumulus cell expansion, thereby improving the developmental potential and quality of parthenogenetic embryos. After supplementation with UA, pig parthenogenetic embryo pluripotency-related genes (Nanog and Sox2) and antiapoptotic genes (Bcl2) were upregulated, while proapoptotic genes (Bax) were downregulated. In conclusion, this study suggests that adding UA during IVM can effectively mitigate the adverse effects of oxidative stress on porcine oocytes, presenting a promising strategy for enhancing their developmental potential in vitro. Full article

This study evaluated the effects of serum (egpS) and follicular fluid (egpFF) from estrus guinea pigs as in vitro maturation (IVM) supplements for natural cycle guinea pig oocytes. Two experiments were conducted: the first assessed three IVM media, a commercial medium (CMOM) and two homemade media (HMOM-P and HMOM-S), for oocyte in vitro maturation using 615 bovine oocytes, while the second evaluated the incorporation of 5%, 10%, and 20% egpFF or egpS into IVM media for 1744 guinea pig oocytes. Initially, we optimized the IVM base medium using bovine oocytes to determine the most suitable culture conditions. The results obtained from these experiments served as a critical foundation for subsequent supplementation trials conducted with guinea pig oocytes. The oocytes were cultured in 70 μL drops under controlled atmospheric conditions, and maturation rates were assessed based on cumulus cell expansion and nuclear progression. The HMOM-S medium significantly enhanced cumulus cell expansion (72.5 ± 2.88%) compared with the CMOM medium (57.7 ± 5.33%; p < 0.05). Supplementation with egpFF at 5% (68.8 ± 6.22%), 10% (76.3 ± 5.39%), and 20% (80.9 ± 6.22%) significantly improved both cumulus cell expansion and nuclear progression in high-quality oocytes (types A and B), compared to the control group (43.3 ± 4.82%). Conversely, supplementation with egpS did not have a significant effect on cumulus cell expansion (p > 0.05); however, it notably improved nuclear maturation in low-quality oocytes (type C) at concentrations of 10% and 20% (p < 0.05). This resulted in an overall improvement in maturation outcomes, particularly for oocytes with compromised initial quality. These findings demonstrated that the IVM of guinea pig oocytes using HMOM-S medium was significantly enhanced by the presence of egpFF, whereas egpS supplementation exhibited a less pronounced effect on IVM outcomes. Full article

In bovine embryos, the microRNA (miRNA) expression has been profiled at each stage of early development in vitro. The miRNAomic analysis of spent media has the potential to reveal characteristics of embryo health; however, applications are limited without categorizing miRNA profiles by embryo quality. Time-lapse imaging has shown the timing of embryo development in vitro may be indicative of their developmental potential. The study aimed to characterize miRNAs in the spent media of bovine embryos with different growth rates during the pre-implantation phase. Bovine cumulus–oocyte complexes were aspirated from ovaries, fertilized, and cultured to blastocyst stage of development. At the 2-cell, 8-cell, and blastocyst stage, each microdrop of 30 presumptive zygotes were classified as slow- or fast-growing based on the percentage of embryos that had reached the desired morphological stage. A comparative analysis was performed on the spent media of slow- and fast-growing embryos using the results of a GeneChip miRNA 4.0 array hybridization. In total, 34 differentially expressed miRNAs were identified between the comparison groups: 14 miRNAs were found in the 2-cell samples, 7 in the 8-cell samples, and 12 in the blastocyst samples. The results demonstrate distinct miRNAs populations can be identified between slow- and fast-growing embryos, highlighting the novel biomarkers of developmental potential at each stage of pre-implantation development. Full article

Differences in structural and functional properties between oocytes and cumulus cells (CCs) may cause low vitrification efficiency for cumulus–oocyte complexes (COCs). We have suggested that the disconnection of CCs and oocytes in order to further cryopreservation in various ways will positively affect the viability after thawing, while further co-culture in vitro will contribute to the restoration of lost intercellular gap junctions. This study aimed to determine the optimal method of cryopreservation of the suspension of CCs to mature GV oocytes in vitro and to determine the level of mRNA expression of the genes (GJA1, GJA4; BCL2, BAX) and gene-specific epigenetic marks (DNMT3A) after cryopreservation and in vitro maturation (IVM) in various culture systems. We have shown that the slow freezing of CCs in microstraws preserved the largest number of viable cells with intact DNA compared with the methods of vitrification and slow freezing in microdroplets. Cryopreservation caused the upregulation of the genes Cx37 and Cx43 in the oocytes to restore gap junctions between cells. In conclusion, the presence of CCs in the co-culture system during IVM of oocytes played an important role in the regulation of the expression of the intercellular proteins Cx37 and Cx43, apoptotic changes, and oocyte methylation. Slow freezing in microstraws was considered to be an optimal method for cryopreservation of CCs. Full article

(1) Background: In this paper we aim to study the relationship between the expression levels of molecules involved in apoptotic/survival pathways, considered as molecular markers of oocyte competence (i.e., AKT, p-AKT, ERK1/2, and p-ERK1/2) in mural granulosa cells (MGCs) and the administration of r-FSH alone or combined with exogenous r-LH, in ovarian stimulation protocol. Moreover, we aim to evaluate oocyte competence by comparing normally cleaved embryos that were transferred in the uterus, with embryos that were arrested during in vitro culture. (2) Methods: The study included 34 normo-responder women undergoing ICSI procedures. All subjects were divided into two groups. Group A consisted of 18 women stimulated with r-FSH and used as a control group; Group B consisted of 14 women stimulated with r-FSH combined with r-LH. The MGCs were obtained from individual follicles. Immunoblot analyses were carried out to analyze the AKT, p-AKT, ERK1/2, and p-ERK1/2 levels in MGCs and to correlate them with the ovarian stimulation protocol. Furthermore, the oocyte competence was evaluated, for each follicle, according to the development of the embryo during in vitro culture and the pregnancy outcome. (3) Results: We found no significant difference in the levels of molecules in isolated MGCs between groups A and B. These results, in light of our previous research, suggest for the first time, to our knowledge, that cumulus cells and mural granulosa cells in the same follicle show different expression levels of molecules involved in the apoptotic mechanism. (4) Conclusions: Our results could clarify some controversial data in the literature where cumulative cell pools of cumulus and granulosa were analyzed, described as ovarian follicle cells, and used as markers of oocyte competence. In this paper, we found evidence that cumulus and granulosa cells need to be analyzed separately. Full article

Gap junctional connection (GJC) in the cumulus–oocyte complex (COC) provides necessary support for message communication and nutrient transmission required for mammalian oocyte maturation. Cyclic adenosine monophosphate (cAMP) is not only a prerequisite for regulating oocyte meiosis, but also the key intercellular factor for affecting GJC function in COCs. However, there are no reports on whether cAMP regulates connexin 37 (Cx37) expression, one of the main connexin proteins, in sheep COCs. In this study, the expression of Cx37 protein and gene in immature sheep COC was detected using immunohistochemistry and PCR. Subsequently, the effect of cAMP on Cx37 expression in sheep COCs cultured in a gonadotropin-free culture system for 10 min or 60 min was evaluated using competitive ELISA, real-time fluorescent quantitative PCR (RT-qPCR), and Western blot. The results showed that the Cx37 protein was present in sheep oocytes and cumulus cells; the same results were found with respect to GJA4 gene expression. In the gonadotropin-free culture system, compared to the control, significantly higher levels of cAMP as well as Cx37 gene and protein expression were found in sheep COCs following treatment in vitro with Forskolin and IBMX (100 μM and 500 μM)) for 10 min (p < 0.05). Compared to the controls (at 10 or 60 min), cAMP levels in sheep COCs were significantly elevated as a result of Forskolin and IBMX treatment (p < 0.05). Following culturing in vitro for 10 min or 60 min, Forskolin and IBMX treatment can significantly promote Cx37 expression in sheep COCs (p < 0.05), a phenomenon which can be counteracted when the culture media is supplemented with RP-cAMP, a cAMP-specific competitive inhibitor operating through suppression of the protein kinase A (PKA). In summary, this study reports the preliminary regulatory mechanism of cAMP involved in Cx37 expression for the first time, and provides a novel explanation for the interaction between cAMP and GJC communication during sheep COC culturing in vitro. Full article

Recent studies have established that exosomes (EXs) derived from follicular fluid (FF) can promote oocyte development. However, the specific sources of these EXs and their regulatory mechanisms remain elusive. It is universally acknowledged that oocyte development requires signal communication between granulosa cells (GCs) and oocytes. However, the role of GC-secreted EXs and their functions are poorly understood. This study aimed to investigate the role of porcine granulosa-cell-derived exosomes (GC-EXs) in oocyte development. In this study, we constructed an in vitro model of porcine GCs and collected and identified GC-EXs. We confirmed that porcine GCs can secrete EXs and investigated the role of GC-EXs in regulating oocyte development by supplementing them to cumulus–oocyte complexes (COCs) cultured in vitro. Specifically, GC-EXs increase the cumulus expansion index (CEI), promote the expansion of the cumulus, alleviate reactive oxygen species (ROS), and increase mitochondrial membrane potential (MMP), resulting in improved oocyte development. Additionally, we conducted small RNA sequencing of GC-EXs and hypothesized that miR-148a-3p, the highest-expressed microRNA (miRNA), may be the key miRNA. Our study determined that transfection of miR-148a-3p mimics exerts effects comparable to the addition of EXs. Meanwhile, bioinformatics prediction, dual luciferase reporter gene assay, and RT-qPCR identified DOCK6 as the target gene of miR-148a-3p. In summary, our results demonstrated that GC-EXs may improve oocyte antioxidant capacity and promote oocyte development through miR-148a-3p by targeting DOCK6. Full article

Our previous study established that chrysoeriol (CHE) can reduce reactive oxygen species (ROS) accumulation, apoptosis, and autophagy in vitro culture (IVC) of porcine embryos. However, the role of CHE in oocyte maturation and lipid homeostasis is unclear. Herein, we aimed to elucidate the effect of CHE on porcine oocyte competence in vitro maturation (IVM) and subsequent embryo development. The study chooses parthenogenetic activated porcine oocytes as the research model. The study revealed that the cumulus expansion index and related gene expressions are significantly elevated after supplementing 1 μM CHE. Although there were no significant differences in nuclear maturation and cleavage rates, the blastocyst formation rate and total cell numbers were significantly increased in the 1 μM CHE group. In addition, CHE improved the expression of genes related to oocyte and embryo development. ROS was significantly downregulated in all CHE treatment groups, and intracellular GSH (glutathione) was significantly upregulated in 0.01, 0.1, and 1 μM CHE groups. The immunofluorescence results indicated that mitochondrial membrane potential (MMP) and lipid droplet (LD), fatty acid (FA), ATP, and functional mitochondria contents significantly increased with 1 μM CHE compared to the control. Furthermore, CHE increased the expression of genes related to lipid metabolism, mitochondrial biogenesis, and β-oxidation. Full article

Zearalenone (ZEN), a non-steroidal estrogenic fungal toxin widely present in forage, food, and their ingredients, poses a serious threat to animal and human reproductive health. ZEN also threatens ovine, a major source of human food and breeding stock. However, the mechanisms underlying the impact of ZEN on the in vitro maturation (IVM) of ovine oocytes remain unclear. This study aimed to elucidate these mechanisms using the Smart-seq2 technology. A total of 146 differentially expressed genes were obtained, using Smart-seq2, from sheep oocytes cultured in vitro after ZEN treatment. ZEN treatment inhibited RUNX2 and SPP1 expression in the PI3K signaling pathway, leading to the downregulation of THBS1 and ultimately the downregulation of TNFAIP6; ZEN can also decrease TNFAIP6 by reducing PTPRC and ITGAM. Both inhibit in vitro maturation of ovine oocytes and proliferation of cumulus cells by downregulating TNFAIP6. These findings provide data and a theoretical basis for elucidating ZEN’s toxicity mechanisms, screening therapeutic drugs, and reducing ZEN-related losses in the ovine industry. Full article

Oocyte developmental competence is the ability of a mature oocyte to be fertilized and subsequently support embryonic development. Such competence is gained during folliculogenesis and is facilitated by the bidirectional communication into a compacted cumulus–oocyte complex (COC). Human tissue inhibitor of metalloproteinases-1 (TIMP1) participates in biological processes, including cell growth, differentiation, and apoptosis. This study aimed to evaluate the influence of TIMP1 as a growth factor on the in vitro maturation (IVM) culture of bovine COCs to improve oocyte developmental competence. All TIMP1 treatments (50, 100, and 150 ng/mL) favored the COCs’ compaction structure (p < 0.05). TIMP1 at 150 ng/mL produced more oocytes in metaphase II compared to the other treatments (p < 0.05). The 150 ng/mL TIMP1 generated oocytes with the most (p < 0.05) cortical granules below the plasma membrane (pattern I). In a parthenogenesis assay, oocyte IVM in 50 ng/mL of TIMP1 produced the most blastocyst compared to the other treatments (p < 0.05). The Principal Component Analysis (PCA) showed that 50 ng/mL of TIMP1 was the best condition to develop oocyte competence because it was associated with the COC compact and cortical granule pattern I. TIMP1 influences the development of oocyte competence when added to the IVM culture medium of COCs. Full article

Maturation is a critical step in the development of an oocyte, and it is during this time that the oocyte advances to metaphase II (MII) of the meiotic cycle and acquires developmental competence to be fertilized and become an embryo. However, in vitro maturation (IVM) remains one of the limiting steps in the in vitro production of embryos (IVP), with a variable percentage of oocytes reaching the MII stage and unpredictable levels of developmental competence. Understanding the dynamics of oocyte maturation is essential for the optimization of IVM culture conditions and subsequent IVP outcomes. Thus, the aim of this study was to elucidate the transcriptome dynamics of oocyte maturation by comparing transcriptomic changes during in vitro maturation in both oocytes and their surrounding cumulus cells. Cumulus–oocyte complexes were obtained from antral follicles and divided into two groups: immature and in vitro-matured (MII). RNA was extracted separately from oocytes (OC) and cumulus cells (CC), followed by library preparation and RNA sequencing. A total of 13,918 gene transcripts were identified in OC, with 538 differentially expressed genes (DEG) between immature OC and in vitro-matured OC. In CC, 13,104 genes were expressed with 871 DEG. Gene ontology (GO) analysis showed an association between the DEGs and pathways relating to nuclear maturation in OC and GTPase activity, extracellular matrix organization, and collagen trimers in CC. Additionally, the follicle-stimulating hormone receptor gene (FSHR) and luteinizing hormone/choriogonadotropin receptor gene (LHCGR) showed differential expressions between CC-MII and immature CC samples. Overall, these results serve as a foundation to further investigate the biological pathways relevant to oocyte maturation in horses and pave the road to improve the IVP outcomes and the overall clinical management of equine assisted reproductive technologies (ART). Full article

Salidroside (Sal) possesses several pharmacological activities, such as antiaging, and anti-inflammatory, antioxidant, anticancer activities, and proliferation-promoting activities, but the effects of Sal on oocytes have rarely been reported. In the present study, we evaluated the beneficial effects of Sal, which is mainly found in the roots of Rhodiola. Porcine cumulus oocyte complexes were cultured in IVM medium supplemented (with 250 μmol/L) with Sal or not supplemented with Sal. The maturation rate in the Sal group increased from 88.34 ± 4.32% to 94.12 ± 2.29%, and the blastocyst rate in the Sal group increased from 30.35 ± 3.20% to 52.14 ± 7.32% compared with that in the control group. The experimental groups showed significant improvements in the cumulus expansion area. Sal reduced oocyte levels of reactive oxygen species (ROS) and enhanced intracellular GSH levels. Sal supplementation enhanced the mitochondrial membrane potential (MMP), ATP level, and mtDNA copy number, which shows that Sal enhances the cytoplasmic maturation of oocytes. Oocytes in the Sal group exhibited slowed apoptosis and reduced DNA breakage. Cell cycle signals and oocyte meiosis play important roles in oocyte maturation. The mRNA expressions of the MAPK pathway and MAPK phosphorylation increased significantly in the Sal group. The mRNA expression of the oocyte meiosis gene also increased significantly. These results show that Sal enhances the nuclear maturation of oocytes. Moreover, Sal increased the number of blastocyst cells, the proliferation of blastocysts, and the expressions of pluripotency genes. Sal down-regulated apoptosis-related genes and the apoptotic cell rate of blastocysts. In summary, our results demonstrate that Sal is helpful to improving the quality of porcine oocytes in vitro, and their subsequent embryonic development. Full article

In vivo-matured oocytes exhibit higher developmental competence than those matured in vitro but mimicking the in vivo environment by in vitro conditions has been challenging. Until now, conventional two-dimensional (2D) systems have been used for in vitro maturation of bovine cumulus-oocytes-complexes (COCs). However, using such systems present certain limitations. Therefore, alternative low-cost methodologies may help to optimize oocyte in vitro maturation. Here, we used two different systems to culture COCs and evaluate their potential influence on embryo development and quality. In the first system, we used treated fumed silica particles to create a 3D microenvironment (liquid marbles; LM) to mature COCs. In the second system, we cultured COCs in 96-well plates with different dimensions (flat, ultra-low attachment round-bottom, and v-shaped 96-well plates). In both systems, the nuclear maturation rate remained similar to the control in 2D, showing that most oocytes reached metaphase II. However, the subsequent blastocyst rate remained lower in the liquid marble system compared with the 96-well plates and control 2D systems. Interestingly, a lower total cell number was found in the resulting embryos from both systems (LM and 96-well plates) compared with the control. In conclusion, oocytes matured in liquid marbles or 96-well plates showed no remarkable change in terms of meiotic resumption. None of the surface geometries influenced embryo development while oocyte maturation in liquid marbles led to reduced embryo development. These findings show that different geometry during maturation did not have a large impact on oocyte and embryo development. Lower embryo production after in vitro maturation in liquid marbles was probably detected because in vitro maturation was performed in serum-free medium, which makes oocytes more sensitive to possible toxic effects from the environment. Full article