Cortical Granule Distribution Research Articles

Establishing a 3D-cultured system based on alginate-hydrogel embedding benefits the in vitro maturation of porcine Oocytes

The in vitro maturation (IVM) quality of oocytes is directly related to the subsequent developmental potential of embryos and a fundamental of in vitro embryo production. However, conventional IVM methods fail to maintain the gap-junction intercellular communication (GJIC) between cumulus–oocyte complexes (COCs), which leads to insufficient oocyte maturation. Herein, we investigated the effects of three different three-dimensional (3D) culture methods on oocyte development in vitro, optimized of the alginate-hydrogel embedding method, and assessed the effects of the alginate-hydrogel embedding method on subsequent embryonic developmental potential of oocytes after IVM and parthenogenetic activation (PA). The results showed that Matrigel embedding and alginate-hydrogel embedding benefited the embryonic developmental potential of oocytes after IVM and PA. With the further optimization of alginate-hydrogel embedding, including crosslinking and decrosslinking of parameters, we established a 3D culture system that can significantly increase oocyte maturation and the blastocyst rate of embryos after PA (27.2 ± 1.5 vs 36.7 ± 2.8, P < 0.05). This 3D culture system produced oocytes with markedly increased mitochondrial intensity and membrane potential, which reduced the abnormalities of spindle formation and cortical granule distribution. The alginate-hydrogel embedding system can also remarkably enhance the GJIC between COCs. In summary, based on alginate-hydrogel embedding, we established a 3D culture system that can improve the IVM quality of porcine oocytes, possibly by enhancing GJIC.

Follicular fluid exosome-derived miR-339-5p enhances in vitro maturation of porcine oocytes via targeting SFPQ, a regulator of the ERK1/2 pathway

In this study, we aimed to investigate cytoplasmic maturation and miRNA expression of mature oocytes cultured in porcine follicular fluid exosomes. We also examined the effect of miR-339-5p on oocyte maturation. Twenty eight differentially expressed miRNAs were detected using miRNA-seq. We then transfected cumulus oocyte complexes with miR-339-5p mimics and inhibitor during culture. The results showed that exosomes increased endoplasmic reticulum levels and the amount of lipid droplets, and decreased ROS levels, lipid droplet size, and percentage of oocytes with abnormal cortical granule distribution. Overexpressing miR-339-5p significantly decreased cumulus expansion genes, oocyte maturation-related genes, target gene proline/glutamine-rich splicing factor (SFPQ), ERK1/2 phosphorylation levels, oocyte maturation rate, blastocyst rate, and lipid droplet number, but increased lipid droplet size and the ratio of oocytes with abnormal cortical granule distribution. Inhibiting miR-339-5p reversed the decrease observed during overexpression. Mitochondrial membrane potential and ROS levels did not differ significantly between groups. In summary, exosomes promote oocyte cytoplasmic maturation and miR-339-5p regulating ERK1/2 activity through SFPQ expression, thereby elevating oocyte maturation and blastocyst formation rate in vitro.

Cyanidin-3-O-glucoside protects Zearalenone-induced in vitro maturation disorders of porcine oocytes by alleviating NOX4-dependent oxidative stress and endoplasmic reticulum stress in cumulus cells

Zearalenone (ZEN) is widely found in foodstuffs and has serious harmful effects on female fertility, especially in pigs. Cyanidin-3-O-glucoside (C3G), a type of anthocyanin, exists in most dark fruits and vegetables; it has many positive dietary effects including as an antioxidant, anti-inflammatory, or anti-apoptotic agent. However, the beneficial effects of C3G alongside ZEN-induced damage in porcine oocytes and the underlying molecular mechanism have not been investigated. In this work, porcine cumulus-oocyte complexes (COCs) were divided into Control (Ctrl), ZEN, ZEN + C3G (Z + C), and C3G, and treated for 44–46 h in vitro. The results showed that C3G could alleviate ZEN-induced disorders of first polar body (PBI) extrusion, abnormalities of spindle assembly, cortical granule distribution, and mitochondrial distribution; these results were produced via restoring transzonal projections (TZPs), and inhibiting nicotinamide adenine dinucleotide phosphate oxidase (NOX4)-dependent oxidative stress and ‘glucose regulatory protein 78/protein kinase-like endoplasmic reticulum kinase/α subunit of eukaryotic initiation factor 2α/activating transcription factor 4/C/EBP-homologous protein’ (GRP78/PERK/eIF2α/ATF4/CHOP)-mediated endoplasmic reticulum stress (ERS) during oocyte maturation. Moreover, the over-expression of NOX4 in cumulus cells could result in a significant increase in ROS levels and ER fluorescence intensity in oocytes. In conclusion, C3G promoted in vitro maturation of porcine oocytes exposed to ZEN via mitigating NOX4-dependent oxidative stress and ERS in cumulus cells. These results contribute to our comprehension of the molecular mechanisms underlying the protective effects of C3G against ZEN toxicity in porcine oocytes, and they provide a novel theoretical foundation and strategy for future applications of C3G in the improvement of female reproduction.

Ursolic acid alleviates meiotic abnormalities induced by 3-nitropropionic acid in mouse oocytes

3-nitropropionic acid (3-NPA), a toxic metabolite produced by mold, is mainly found in moldy sugarcane. 3-NPA inhibits the activity of succinate dehydrogenase that can induce oxidative stress injury in cells, reduce ATP production and induce oxidative stress in mouse ovaries to cause reproductive disorders. Ursolic acid (UA) has a variety of biological activities and is a pentacyclic triterpene compound found in many plants. This experiment aimed to investigate the cytotoxicity of 3-NPA during mouse oocyte in vitro maturation and the protective effects of UA on oocytes challenged with 3-NPA. The results showed that UA could alleviate 3-NPA-induced oocyte meiotic maturation failure. Specifically, 3-NPA induced a decrease in the first polar body extrusion rate of oocytes, abnormal distribution of cortical granules, and an increase in the proportion of spindle abnormalities. In addition, 3-NPA caused mitochondrial dysfunction and induced oxidative stress, including decreases in the GSH, mitochondrial membrane potential and ATP levels, and increases in the ROS levels, and these effects led to apoptosis and autophagy. The addition of UA could significantly improve the adverse effects caused by 3-NPA. In general, our data show that 3-NPA affects the normal development of oocytes during the in vitro culture, and the addition of UA can effectively repair the damage caused by 3-NPA to oocytes.

Mogroside V alleviates the heat stress-induced disruption of the porcine oocyte in vitro maturation

Heat stress (HS) is a stressor that negatively affect female reproduction. Specially, oocytes are very sensitive to HS. It has been demonstrated that some active compounds can protect oocyte from HS. We previously found that Mogroside V (MV), extracted from Siraitia grosvenorii (Luo Han Guo), can protect oocyte from many kinds of stresses. However, how MV alleviates HS-induced disruption of oocyte maturation remains unknown. In this study, we treated the HS-induced porcine oocytes with MV to examine their maturation and quality. Our findings demonstrate that MV can effectively alleviate HS-induced porcine oocyte abnormal cumulus cell expansion, decrease of first polar body extrusion rate, spindle assembly and chromosome separation abnormalities, indicating MV attenuates oocyte mature defects. We further observed that MV can effectively alleviate HS-induced cortical granule distribution abnormality and decrease of blastocyst formation rate after parthenogenesis activation. In addition, MV treatment reversed mitochondrial dysfunction and lipid droplet content decrease, reduced reactive oxygen species levels, early apoptosis and DNA damage in porcine oocytes after HS. Collectively, this study suggests that MV can effectively protect porcine oocytes from HS.

Alpha-lipoic acid supplementation reverses the declining quality of oocytes exposed to cyclophosphamide

Cyclophosphamide (CY) is a chemotherapeutic drug that is commonly used to treat malignancies of the ovary, breast, and hematology, as well as autoimmune disorders. As a cofactor of mitochondrial multienzyme complexes, alpha lipoic acid (ALA) is well known for its antioxidant characteristics, which operate directly on the scavenging of reactive oxygen species (ROS) and indirectly on the intracellular recycling of other antioxidants. However, the underlying mechanisms through which CY exerts its toxic effects on meiosis and oocyte quality, as well as a viable approach for protecting oocyte quality and preserving fertility, remain unknown. In present study, immunostaining and fluorescence intensity quantification were applied to assess the effects of CY and ALA supplementation on the key processes during the oocyte meiotic maturation. Our results show that supplementing oocytes with ALA, a well-known antioxidant and free radical scavenger, can reverse CY-induced oocyte meiotic maturation failure. Specifically, we found that CY exposure caused oocyte meiotic failure by disrupting meiotic organelle dynamics and arrangement, as well as a prominently impaired cytoskeleton assembly. In addition, CY caused an abnormal distribution of mitochondrion and cortical granules, two indicators of oocyte cytoplasmic maturation. More importantly, we show that ALA supplementation effectively reverses CY-induced meiotic failure and oocyte quality decline by suppressing oxidative stress-induced DNA damage and apoptosis in oocytes. Collectively, our data reveal that ALA supplementation is a feasible approach to protect oocytes from CY-exposed deterioration, providing a better understanding of the mechanisms involved in chemotherapy-induced meiotic failure.

Effects of Alginic Acid on the Porcine Granulosa Cells and Maturation of Porcine Oocytes.

Alginic acid (AA) from brown algae is a marine organic compound. There is extensive use of AA in the food industry and healthcare, suggesting a high probability of AA exposure. The present study investigates the effects of AA on porcine ovarian granulosa cells (GCs) and oocytes to explore its mechanism in female reproduction because of its adverse effects on reproduction. The study adds 20 µM AA to the porcine primary ovarian GCs medium and porcine oocyte in vitro maturation (IVM) medium. Estrogen and progesterone levels are downregulated in GCs. Reactive oxygen species are excessive, and the antioxidant capacity declines. Then mitochondria-mediated apoptosis pathway is involved in GCs apoptosis. In addition, scores of autophagosomes are found in the experimental cells. Furthermore, AA significantly inhibits the proliferation of GCs around cumulus-oocyte complexes (COCs) accompanied by abnormal spindle assembly, chromosome arrangement disorder, and aberrant cortical granules distribution in oocytes, leading to a decreased oocyte maturation rate. These findings suggest that 20 µM AA is toxic to sow reproduction by interfering with estrogen production, oxidative stress, mitochondria-mediated apoptosis, autophagy in GCs of sows, and oocyte maturation.

CAPA-IVM improves the cytoplasmic quality of in vitro-matured oocytes from unstimulated mice

Ovarian tissue oocyte (OTO) in vitro maturation (IVM) is a strategy to improve fertility preservation efficiency. Here, the effects of capacitation IVM (CAPA-IVM) on OTO function were investigated. Immature cumulus–oocyte complexes (COCs) from unstimulated 28-day-old mouse ovaries (mimicking OTOs) underwent CAPA-IVM, standard IVM (S-IVM) or in vivo maturation following ovarian stimulation (OS; positive control), and oocyte meiotic maturation and cytoplasmic quality were assessed. CAPA-IVM resulted in improved oocyte meiotic maturation (P < 0.05) and cumulus expansion (P < 0.0001) compared to S-IVM, with expansion comparable to the OS group. MII OTO ROS was lower after CAPA-IVM than S-IVM (P < 0.0001) but not as low as in the OS group (P = 0.036). CAPA-IVM resulted in a better oocyte mitochondrial distribution than S-IVM (P < 0.05) and was similar to the OS group (P > 0.05). Mitochondrial membrane potential in MII OTOs was higher after CAPA-IVM than S-IVM and OS (P < 0.0001). Compared with S-IVM, CAPA-IVM resulted in lower rates of spindle/chromosome configuration and cortical granule distribution abnormalities (P < 0.05), which were similar to OS levels (P > 0.05). MII OTO intracellular Ca2+ levels were similar in the CAPA-IVM and OS groups (P > 0.05), while S-IVM decreased intracellular Ca2+ (P < 0.05). CAPA-IVM and S-IVM decreased mitochondrial Ca2+ levels (P < 0.05). CAPA-IVM increased expression of antioxidant genes (Sod2 and Sirt1) and Egfr (P < 0.05) but not apoptotic genes (Bcl2, Bax and Bcl2/Bax; P > 0.05). CAPA-IVM increased the OTO maturation rate and quality of oocytes from unstimulated mice to the extent that many features of oocyte cytoplasmic quality were comparable to superovulated in vivo matured oocytes.

Emamectin benzoate exposure impaired porcine oocyte maturation

Emamectin benzoate (EB) is a widely used insecticide that can damage the central nervous and immune systems. EB exposure significantly reduced the number of eggs laid, hatching rate, and developmental rate of lower organisms such as nematodes. However, effects of EB exposure on the maturation of higher animals such as porcine oocytes remains unknown. Here we reported that EB exposure severely impaired porcine oocyte maturation. EB exposure with 200 μM prevented cumulus expansion and reduced the rates of first polar body (pb1) extrusion, cleavage and blastocyst after parthenogenetic activation. Moreover, EB exposure disrupted spindle organization, chromosome alignment, and polymerization of microfilaments, but also apparently decreased the levels of acetylated α-tubulin (Ac-Tub) in oocytes. In addition, EB exposure perturbed mitochondria distribution and increased levels of reactive oxygen species (ROS), but did not affect the distribution of cortical granules (CGs) in oocytes. Excessive ROS caused DNA damage accumulation and induced early apoptosis of oocytes. EB exposure led to the abnormal expression of cumulus expansion and apoptosis-associated genes. Altogether, these results demonstrate that EB exposure impaired nuclear and cytoplasmic maturation of porcine oocytes probably through oxidative stress and early apoptosis.

Oviduct epithelial cell‑derived extracellular vesicles promote the developmental competence of IVF porcine embryos.

Assisted reproductive technology has increased the efficiency of animal reproduction. However, polyspermy is a significant limitation of porcine invitro fertilization (IVF). Therefore, reducing the polyspermy rate and improving monospermic embryos is crucial. Recent studies have reported that oviductal fluid, along with its contents of extracellular vesicles (EVs), enhanced the fertilization process and supported embryo development. Consequently, the present study investigated the effects of porcine oviduct epithelial cells (OEC‑EVs) on sperm‑oocyte interactions during porcine IVF and evaluated invitro embryo developmental competence outcomes. During IVF embryo development, the cleavage rate was significantly higher in the group treated with 50ng/ml OEC‑EVs compared with the control group (67.6±2.5 vs. 57.3±1.9; P<0.05). Furthermore, the OEC‑EV group had significantly more embryos (16.4±1.2 vs. 10.2±0.8; P<0.05), and the polyspermy rate significantly decreased (32.9±2.5 vs. 43.8±3.1; P<0.05) compared with that of the control group. Additionally, the fluorescence intensities of cortical granules (3.56±0.47 vs. 2.15±0.24; P<0.05) and active mitochondria (8.14±0.34 vs. 5.96±0.38; P<0.05) were significantly higher in the OEC‑EV group compared with those in the control group. In conclusion, OEC‑EV adsorption and penetration crosstalk between sperm and oocytes was observed. OEC‑EV treatment was demonstrated to significantly improve the concentration and distribution of cortical granules in oocytes. Furthermore, OEC‑EVs also increased oocyte mitochondrial activity, reduced polyspermy and increased the IVF success rate.

Velvet antler water extract protects porcine oocytes from lipopolysaccharide-induced meiotic defects.

Previous studies have demonstrated that lipopolysaccharide (LPS), as a central toxic factor of gram-negative bacteria, can induce oxidative stress and cellular inflammation to result in the impairment of female fertility in different organisms. Particularly, it has harmful effects on the oocyte quality and subsequent embryonic development. However, the approach concerning how to prevent oocytes from LPS-induced deterioration still remains largely unexplored. We assessed the effective influences of velvet antler water extract (VAWE) by immunostaining and fluorescence intensity quantification on the meiotic maturation, mitochondrial function and sperm binding ability of oocytes under oxidative stress. Here, we report that VAWE treatment restores the quality of porcine oocytes exposed to LPS. Specifically, LPS exposure contributed to the failed oocyte maturation, reduced sperm binding ability and fertilization capability by disturbing the dynamics and arrangement of meiotic apparatuses and organelles, including spindle assembly, chromosome alignment, actin polymerization, mitochondrial dynamics and cortical granule distribution, the indicators of oocyte nuclear and cytoplasmic maturation. Notably, VAWE treatment recovered these meiotic defects by removing the LPS-induced excessive ROS and thus inhibiting the apoptosis. Collectively, our study illustrates that VAWE treatment is a feasible strategy to improve the oocyte quality deteriorated by the LPS-induced oxidative stress.

Coculture with porcine luteal cells during in vitro porcine oocyte maturation affects lipid content, cortical reaction and zona pellucida ultrastructure.

In pigs, in vitro fertilisation (IVF) is associated with high polyspermy rates, and for this reason, in vitro embryo production (IVP) is still an inefficient biotechnology. Coculture with somatic cells is an alternative to improve suboptimal in vitro maturation (IVM) conditions. This study was conducted to test a coculture system of porcine luteal cells (PLC) and cumulus-oocyte complexes (COC) to improve oocyte metabolism. COC were matured in vitro with PLC. Oocyte lipid content, mitochondrial activity, zona pellucida (ZP) digestibility and pore size, cortical reaction and in vitro embryo development were assessed. Coculture reduced cytoplasmic lipid content in the oocyte cytoplasm without increasing mitochondrial activity. Although ZP digestibility and ZP pore number were not different between culture systems, ZP pores were smaller in the coculture. Coculture impacted the distribution of cortical granules as they were found immediately under the oolemma, and more of them had released their content in the ZP. Coculture with porcine luteal cells during IVM increased monospermic penetration and embryo development after IVF. The coculture of COC with PLC affects the metabolism of the oocyte and benefits monospermic penetration and embryo development. The coculture system with PLC could be an alternative for the conventional maturation medium in pigs.

Resveratrol ameliorates the defects of meiotic maturation in lipopolysaccharide exposed porcine oocytes

Lipopolysaccharide (LPS), a significant virulence factor of gram-negative bacteria, adversely affects female reproduction, especially the maturation and early embryonic development of oocytes, through inducing of inflammatory and oxidative stress-associated toxic responses. Resveratrol (Res), a potent antioxidant, exhibits many beneficial effects on the maturation and developmental competence of oocytes. However, it is unclear whether Res can restore LPS-induced defects in the maturation of oocytes during meiosis. In this study, we used porcine oocytes to explore the protective effects of Res and its underlying mechanism against the toxic impacts of LPS exposure on meiotic maturation and developmental competence of oocytes during meiosis. The oocytes were randomly assigned to a control, LPS-exposed or Res-supplemented group. Nuclear and cytoplasmic maturation was assessed after 26 h (MI) or 44 h (MII) of in vitro maturation (IVM). Our results showed that 10 µM Res significantly improved the rates of oocyte maturation and blastocyst formation after exposure to 15 µg/mL LPS. In addition, Res preserved the normal spindle/chromosome structure and maintained acetylated tubulin levels, actin polymerization and cortical granules (CGs) distribution. Additionally, Res protected mitochondrial content and function, scavenges reactive oxygen species (ROS), and reduced DNA damage and apoptosis in LPS-exposed oocytes. Furthermore, inhibition of SIRT1 by its specific inhibitor EX527 suppressed the recovery of ROS levels, mitochondrial content, and spindle/chromosome structure by Res supplementation. In summary, this study shows that Res can alleviate the impacts of LPS-induced toxicity on meiosis in porcine oocytes by upregulating SIRT1, which ameliorates oxidative stress and increasing mitochondrial content.

Metformin promotes in vitro maturation of oocytes from aged mice by attenuating mitochondrial oxidative stress via SIRT3-dependent SOD2ac.

Human female fecundity decreases irreversibly as chronological age rises, adversely affecting oocyte quality, consequently worsening pregnancy outcomes and increasing the extent of birth defects. The first-line type 2 diabetes treatment metformin has been associated with delayed aging and reduction of oxidative stress; yet it remains unclear if metformin confers any benefits for oocytes from aged mice, particularly in the context of the assisted human reproductive technology (ART) known as in vitro maturation (IVM). Here, we found that adding metformin into the M16 culture medium of oocytes from aged mice significantly improved both oocyte maturation and early embryonic development. This study showed that metformin reduced the extent of meiotic defects and maintained a normal distribution of cortical granules (CGs). RNA-seq analysis of metformin-treated oocytes revealed genes apparently involved in the reduction of mitochondrial ROS. Further, the results supported that the metformin improved mitochondrial function, reduced apoptosis, increased the extent of autophagy, and reduced mitochondrial ROS via SIRT3-mediated acetylation status of SOD2K68 in oocytes from aged mice. Thus, this finding demonstrated a protective effect for metformin against the decreased quality of oocytes from aged mice to potentially improve ART success rates and illustrated a potential strategy to prevent or delay reproductive aging.

Alpha-lipoic acid supplementation restores the meiotic competency and fertilization capacity of porcine oocytes induced by arsenite.

Arsenite is known as a well-known endocrine disrupting chemicals, and reported to be associated with an increased incidence of negative health effects, including reproductive disorders and dysfunction of the endocrine system. However, it still lacks of the research regarding the beneficial effects of ALA on arsenite exposed oocytes, and the underlying mechanisms have not been determined. Here, we report that supplementation of alpha-lipoic acid (ALA), a strong antioxidant naturally present in all cells of the humans, is able to restore the declined meiotic competency and fertilization capacity of porcine oocytes induced by arsenite. Notably, ALA recovers the defective nuclear and cytoplasmic maturation of porcine oocytes caused by arsenite exposure, including the impaired spindle formation and actin polymerization, the defective mitochondrion integrity and cortical granules distribution. Also, ALA recovers the compromised sperm binding ability to maintain the fertilization potential of arsenite-exposed oocytes. Importantly, ALA suppresses the oxidative stress by reducing the levels of ROS and inhibits the occurrence of DNA damage along with apoptosis. Above all, we provide a new perspective for the application of ALA in effectively preventing the declined oocyte quality induced by environmental EDCs.

Octocrylene exposure impairs mouse oocyte quality by inducing spindle defects and mitochondria dysfunction

One of organic ultraviolet (UV) filters, Octocrylene (OCL), is mainly used in various cosmetic products, which is being frequently detected in soil, sediment, aquatic systems and food chain. There is evidence confirmed the reproductive toxicity of OCL in Japanese medaka. However, less was known about the effects of OCL exposure on oocyte quality. Here, we investigated the impacts of OCL on mouse oocyte maturation and quality by exposing oocytes to OCL in vitro at concentrations of 8, 22, 30, 40 and 50 nM. The results showed that OCL markedly reduced mouse oocyte germinal vesicle breakdown (GVBD) at 50 nM and polar body extrusion (PBE) rates at 40 and 50 nM. OCL exposure further disrupted spindle assembly and chromosome alignment, finally inducing aneuploid. Mitochondrial function was also damaged by OCL exposure, leading to ROS overproduction and apoptosis in oocytes. Moreover, OCL treatment impaired the distribution of cortical granules and sperm binding ability of oocytes. In summary, these data demonstrated that OCL could disturb the oocyte meiotic maturation and reduce oocyte quality.

Protective effect of resveratrol against cadmium-induced toxicity on ovine oocyte in vitro maturation and fertilization

BackgroundHeavy metal cadmium (Cd) is a widespread environmental contaminant with a potential toxicity that might negatively affect female reproduction and fertility. It has been reported that Cd exposure impaired the quality of oocytes and led to a defective maturation and fertilization, through oxidative stress induction. Resveratrol (Res) is a natural polyphenol with strong antioxidant properties that exhibited protective role in preventing oocyte redox homeostasis disruption and quality decline. Here, we explored whether the addition of Res to in vitro maturation (IVM) medium might act as a protection against Cd-induced toxicity on ovine oocyte maturation and fertilization. Firstly, we evaluated the effect of supplementing IVM medium with two different Res concentrations (1 and 2 μmol/L) on nuclear maturation and fertilization of oocytes matured under CdCl2 (2 μmol/L) exposure. Therefore, the concentration of 1 μmol/L Res was selected to analyse the effects of this compound on intracellular ROS levels, mitochondrial (mt) distribution and activity, chromatin configuration, cytoskeleton morphology, cortical granules (CGs) distribution and mRNA expression of genes associated with cellular response to oxidative stress (i.e. SIRT1, SOD 1, GPX1, GSR, CAT) in Cd-exposed in vitro matured oocytes.ResultsWe found that 1 μmol/L Res restored the reduced oocyte meiotic competence induced by Cd exposure as well as, Res sustained oocyte ability to be normally fertilized and decreased polyspermic fertilization at both tested concentrations. Moreover, we demonstrated that 1 μmol/L Res mitigated Cd-induced alterations of oocyte cytoplasmic maturation by reducing reactive oxygen species (ROS) accumulation, preventing mt dysfunction, maintaining the correct meiotic spindle and cortical F-actin assembly and the normal cortical granule distribution as well as up-regulating SIRT1, SOD1 and GPX1 genes.ConclusionsTaken together, our findings highlighted the beneficial influence exerted by Res in preventing Cd-induced disturbance of nuclear and cytoplasmic maturation and subsequent fertilization in ovine oocytes. Res treatment may help to establish defence strategies counteracting Cd-induced toxicity on the female gamete.

Granulosa Cells Improved Mare Oocyte Cytoplasmic Maturation by Providing Collagens.

Assisted reproductive technology has important clinical applications and commercial values in the horse industry. However, this approach is limited largely by the low efficiency of oocyte in vitro maturation (IVM), especially cytoplasmic maturation. To improve the efficiency of mare oocyte IVM, we evaluated the effects of co-culture with cumulus–oocyte complexes (COCs) and granulosa cells (GCs) from follicles with small (<15 mm) and large diameters (>35 mm). Our results showed that oocyte nucleus maturation was not significantly improved by co-culturing with GCs. Interestingly, the cytoplasmic maturation of oocytes, defined by the distribution of cortical granules and mitochondria, as well as reactive oxygen species (ROS) levels, improved dramatically by co-culture with GCs, especially those derived from small follicles. Moreover, GCs promoted cumulus cell expansion by upregulating the expression of BMP15 in oocytes. To determine the mechanism underlying the effects of GCs, the transcriptomes of GCs from large and small follicles were compared. Expression levels of COL1A2, COL6A1, and COL6A2 were significantly higher in GCs from small follicles than in those from large follicles. These three genes were enriched in the extracellular matrix proteins-receptor interaction pathway and were involved in the regulation of collagens. Taken together, our results suggest that co-culture with GCs is beneficial to oocyte cytoplasmic maturation, and the increased expression of COL1A2, COL6A1, and COL6A2 improve the mare oocyte IVM system via the regulation of collagen.

Nicotinamide mononucleotide supplementation improves the quality of porcine oocytes under heat stress

BackgroundElevated ambient temperature-caused heat stress is a major concern for livestock production due to its negative impact on animal feed intake, growth, reproduction, and health. Particularly, the germ cells are extremely sensitive to the heat stress. However, the effective approach and strategy regarding how to protect mammalian oocytes from heat stress-induced defects have not been determined.MethodsGerminal vesicle (GV) porcine oocytes were cultured at 41.5 °C for 24 h to induce heat stress, and then cultured at 38.5 °C to the specific developmental stage for subsequent analysis. Nicotinamide mononucleotide (NMN) was dissolved in water to 1 mol/L for a stock solution and further diluted with the maturation medium to the final concentrations of 10 μmol/L, 20 μmol/L, 50 μmol/L or 100 μmol/L, respectively, during heat stress. Immunostaining and fluorescence intensity quantification were applied to assess the effects of heat stress and NMN supplementation on the key processes during the oocyte meiotic maturation.ResultsHere, we report that NMN supplementation improves the quality of porcine oocytes under heat stress. Specifically, we found that heat stress resulted in oocyte maturation failure by disturbing the dynamics of meiotic organelles, including the cytoskeleton assembly, cortical granule distribution and mitochondrial function. In addition, heat stress induced the production of excessive reactive oxygen species (ROS) and DNA damage, leading to the occurrence of apoptosis in oocytes and subsequent embryonic development arrest. More importantly, we validated that supplementation of NMN during heat stress restored the meiotic defects during porcine oocyte maturation.ConclusionsTaken together, our study documents that NMN supplementation is an effective approach to improve the quality of oocytes under heat stress by promoting both nuclear and cytoplasmic maturation.

Effect of Passage Number of Conditioned Medium Collected from Equine Amniotic Fluid Mesenchymal Stem Cells: Porcine Oocyte Maturation and Embryo Development.

Oocyte in vitro maturation (IVM) is the most important first step in in vitro embryo production. One prerequisite for the success of IVM in oocytes is to provide a rich culture microenvironment that meets the nutritional needs of developing oocytes. We applied different equine amniotic fluid mesenchymal stem cell conditioned medium (eAFMSC-CM) from passages 7, 18, and 27 to porcine oocytes during IVM to determine its effects on oocyte development and subsequent embryo development, specifically. The eAFMSC-CM from passage 7 (eAFMSC-CMp7) has a considerable impact on 9 genes: BAX, BCL2, SOD2, NRF2, TNFAIP6, PTGS2, HAS2, Cx37, and Cx43, which are associated with cumulus cell mediated oocyte maturation. GSH levels and distribution of mitochondrial and cortical granules were significantly increased in oocytes incubated with eAFMSC-CMp7. In addition, catalase and superoxide dismutase activities were high after IVM 44 h with eAFMSC-CMp7. After in vitro fertilization, blastocyst quality was significantly increased in the eAFMSC-CMp7 group compared to control. Lastly, the antioxidant effect of eAFMSC-CMp7 substantially regulated the expression of apoptosis, pluripotency related genes and decreased autophagy activity in blastocysts. Taken together, this study demonstrated that the eAFMSC-CMp7 enhanced the cytoplasmic maturation of oocytes and subsequent embryonic development by generating high antioxidant activity.