Distribution Of Mitochondria In Oocytes Research Articles

Effects of vitrification on mitochondrial ultrastructure and membrane potential and its distribution in mouse oocytes

BACKGROUND: Vitrification is commonly used for in vitro fertilization and has significant impact on gametes. OBJECTIVE: To investigate changes in ultrastructure, membrane potential (ΔΨm) and distribution of mitochondria in mouse oocytes after vitrification.MATERIALS AND METHODS: Mouse oocytes were divided into three groups: one group as fresh control, one group for the toxicity test (treated with cryoprotectant but without vitrification), and the other for vitrification. RESULTS: Most mitochondria in oocytes were damaged after cooling and warming, being rough and fuzzy in appearance, even swollen and broken. The ΔΨm of the toxicity test group and the vitrification group was 0.320±0.030 and 0.244±0.038, respectively, in comparison to the fresh group (0.398±0.043). The ΔΨm of the vitrified oocytes was significantly lower than fresh oocytes and the toxicity test oocytes (P<0.05), but there was no significant difference between fresh oocytes and the toxicity test oocytes (P>0.05). Mitochondria in fresh oocytes were denser and strained stronger, with 59.5% distributed homogeneously and 36.4% polarized. The majority of mitochondria in the toxicity-tested oocytes were clustered (69.3%) and only a small portion were distributed homogeneously (19.6%), while mitochondria in vitrified oocytes were clustered (56.3%) and deficient (24.4%), and their fluorescent staining was weak and blurred. There was a significant disruption in mitochondrial function after vitrification. CONCLUSION: Vitrification alters the ultrastructure, membrane potential and distribution of mitochondria in oocytes, most likely caused by toxicity and mechanical injury.

How to beat the bull: Lycopene as a tool to improve in vitro fertilization efficiency in bulls with high polyspermy

High levels of reactive oxygen species (ROS) derived from in vitro conditions compromise oocyte quality and subsequent polyspermy prevention by the zona and membrane block. Antioxidant supplementation, like lycopene, during in vitro maturation (IVM) mitigates ROS effects, yet, its efficacy in blocking polyspermy remains uncertain. This study aims to evaluate the effect of lycopene supplementation during IVM on oocyte maturation, fertilization, and developmental parameters. To this end, bovine oocytes were supplemented with 0.2 µM lycopene and fertilized with semen from three bulls. The three bulls showed different fertilization potential in vitro, with bull 1 showing the highest penetration and polyspermy rates and the lowest in vitro fertilization (IVF) efficiency. Interestingly, in bull 1, the treatment with lycopene improved IVF efficiency (p = 0.043) and reduced the polyspermy rate (p = 0.028). However, none of these effects were observed in bulls 2 and 3. Bulls with higher penetration rates exhibited better blastocyst rates although those rates did not seem to be associated with polyspermy or IVF efficiency. Oocyte mitochondrial distribution and activity and cortical granule migration and distribution were not influenced by lycopene. In conclusion, we demonstrated that lycopene addition during oocyte maturation had a positive impact on IVF efficiency by reducing polyspermy rates in a bull-dependent manner. The reduction in polyspermy rates was not caused by changes in cortical granule migration or oocyte mitochondrial distribution. Lycopene must therefore induce other changes in the oocyte that lower the in vitro penetration rates of specific bulls prone to polyspermy.

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.

Effects of Yu Linzhu on ovarian function and oocyte mitochondria in natural aging mice

Objective: To study the effect of Yu Linzhu on ovarian function and mitochondria in natural aging mice.Methods: Female BALB/c mice were selected as normal group at 7–8 weeks and natural aging group at 9 months. The natural aging group was divided into Yu Linzhu intervention group and non-intervention group by intragastric administration once a day for 6 weeks. The morphology and blood flow of ovary were observed by ultrasound. Ovarian morphology and follicle were observed by HE staining. Hormone levels were analyzed by ELISA. Serum oxidative stress were detected by radioimmunoassay. The distribution of mitochondria in oocytes was observed by fluorescence staining. The ultrastructure of oocytes and the morphology of mitochondria were observed under electron microscope. The mitochondrial membrane potential was detected by JC-1.Results: Two groups of aging mice had serious disturbance of estrus cycle. The ovarian area of the mice in the aging non-intervention group was smaller than that in the normal group, and the ovarian area of the mice in the aging intervention group recovered. The ovarian blood flow was weak or even disappear in the aging non-intervention group, and the blood flow in the intervention group was improved. The ovarian volume of mice in the non-intervention group was smaller than that in the normal group. Some ovarian tissues were adhered to the surrounding tissues. While in the intervention group, the ovarian volume increased, the degree of adhesion decreased, the infiltration of ovarian interstitial lymphocytes decreased, and the zona pellucida recovered. Granular cell arrangement returned neatly, egg cell shape recover regular and the number also increased. In the non-intervention group, E2 (Estrogen), AMH (Anti-Mullerian hormone) decreased (P = 0.0092 and P = 0.0334, respectively), FSH (Follicle stimulating hormone) increased (P < 0.0001). In the intervention group, FSH decreased (P = 0.0002), LH (luteinizing hormone) decreased and E2, AMH increased. In the non-intervention group, GSH-Px (Glutathione peroxidase) decreased (P = 0.0129), SOD (Superoxide dismutase) decreased, ROS (reactive oxidative species), MDA (Malondialdehyde) increased. In the aging intervention group, ROS, MDA decreased and GSH-Px increased. In the non-intervention group, mitochondrial expression was scattered at the concentrated distribution point, the length of mitochondria was mostly long and the average volume increased, the density decreased, the number decreased and some mitochondria fused, and lesions such as swelling, vacuolar degeneration and inclusion body formation, membrane potential decreased (P = 0.0002). In the aging intervention group, mitochondria were evenly distributed, the mitochondria were basically round, the distribution density was moderate, the inner ridge was clear, and the membrane potential of the aging intervention group increased.Conclusion: Yu Linzhu can improve the ovarian function of natural aging mice by improving the mitochondrial function of oocytes.

Obesity-induced oocyte mitochondrial defects are partially prevented and rescued by supplementation with co-enzyme Q10 in a mouse model.

Does supplementation with co-enzyme Q10 (CoQ10) improve the oocyte mitochondrial abnormalities associated with obesity in mice? In an obese mouse model, CoQ10 improves the mitochondrial function of oocytes. Obesity impairs oocyte quality. Oocytes from mice fed a high-fat/high-sugar (HF/HS) diet have abnormalities in mitochondrial distribution and function and in meiotic progression. Mice were randomly assigned to a normal, chow diet or an isocaloric HF/HS diet for 12 weeks. After 6 weeks on the diet, half of the mice receiving a normal diet and half of the mice receiving a HF/HS diet were randomly assigned to receive CoQ10 supplementation injections for the remaining 6 weeks. Dietary intervention was initiated on C57Bl6 female mice at 4 weeks of age, CoQ10 versus vehicle injections were assigned at 10 weeks, and assays were conducted at 16 weeks of age. Mice were super-ovulated, and oocytes were collected and stained to assess mitochondrial distribution, quantify reactive oxygen species (ROS), assess meiotic spindle formation, and measure metabolites. In vitro fertilization was performed, and blastocyst embryos were transferred into control mice. Oocyte number, fertilization rate, blastulation rate and implantation rate were compared between the four cohorts. Bivariate statistics were performed appropriately. HF/HS mice weighed significantly more than normal diet mice (29 versus 22 g, P< 0.001). CoQ10 supplementation did not influence weight. Levels of ATP, citrate, and phosphocreatine were lower and ROS levels were higher in HF/HS mice than in controls (P< 0.001). CoQ10 supplementation significantly increased the levels of metabolites and decreased ROS levels in oocytes from normal diet mice but not in oocytes from HF/HS mice. However, CoQ10 completely prevented the mitochondrial distribution abnormalities observed in the HF/HS mice. Overall, CoQ10 supplementation significantly increased the percentage of normal spindle and chromosome alignment (92.3 versus 80.2%, P= 0.039). In the sub-analysis by diet, the difference did not reach statistical significance. When undergoing IVF, there were no statistically significant differences in the number of mature oocytes, the fertilization rate, blastocyst formation rates, implantation rates, resorption rates or litter size between HF/HS mice receiving CoQ10 or vehicle injections. Experiments were limited to one species and strain of mice. The majority of experiments were performed after ovulation induction, which may not represent natural cycle fertility. Improvement in oocyte mitochondrial distribution and function of normal, chow-fed mice and HF/HS-fed mice demonstrates the importance of CoQ10 and the efficiency of the mitochondrial respiratory chain in oocyte competence. Clinical studies are now needed to evaluate the therapeutic potential of CoQ10 in women's reproductive health. C.E.B. received support from the National Research Training Program in Reproductive Medicine sponsored by the National Institute of Health (T32 HD040135-13) and the Scientific Advisory Board of Vivere Health. K.H.M received support from the American Diabetes Association and the National Institute of Health (R01 HD083895). There are no conflicts of interest to declare. This study is not a clinical trial.

Changes in the distribution of mitochondria before and after in vitro maturation of human oocytes and the effect of in vitro maturation on mitochondria distribution

To clarify the relationship between oocyte maturation and mitochondria distribution and assess the effects of in vitro maturation (IVM) on the distribution of mitochondria in human oocytes. Prospective randomized trial. Hospital-based IVF center. One hundred fifty-eight patients undergoing intracytoplasmic sperm injection (ICSI) treatment for male factors or combined with oviduct infertility and fifteen patients undergoing controlled ovarian hyperstimulation followed by coitus or IUI. Of all the 284 immature oocytes, 140 were fixed directly. The others were prepared for IVM before they were fixed. All the 21 oocytes matured in vivo were fixed directly and stained for mitochondria. Both immature and mature oocytes were stained by Mito Tracker Green FM. The distribution of mitochondria was observed using a confocal laser scanning microscope. Mitochondrial distribution. Three mitochondria distribution patterns were identified: peripheral, semiperipheral, and evenly diffused. A peripheral distribution of mitochondria was presented by 64.1% (50/78) of the germinal vesicle (GV) oocytes; 45.2% (28/62) of the meiosis I oocytes maintained the peripheral distribution; and 38.7% (24/62) presented a diffused status. After IVM, 75.5% (80/106) of the oocytes displayed an evenly diffused type of distribution. The mitochondria were more abundant in the inner cytoplasm than in the peripheral region in most of the oocytes matured in vivo. There are obvious changes in the distribution of mitochondria in human oocytes before and after maturation. Distribution of mitochondria in oocytes matured in vitro is slightly different from that of oocytes matured in vivo. The results may partially explain the reduced developmental potential of oocytes matured in vitro compared with those matured in vivo.

Comparative study of cytology of cynomolgus monkey oocytes matured in vivo and in vitro

This study was conducted to compare 1) the capacity for maturation of oocytes from cynomolgus monkeys cultured in vitro before or after injection of r-hCG, 2) spindle integrity, and 3) the distribution of mitochondria in oocytes. In total, 31 stimulation cycles from 14 animals were performed. The oocytes were analyzed before or after hCG treatment and maturation in vitro. Beginning within 3 days after initiation of menstruation, adult monkeys were given 60 IU r-hFSH for 6 days, followed by 60 IU r-hFSH and 60 IU r-hLH for 2 days to stimulate multiple follicle growth. The GnRH antagonist, Antide, was administered daily (0.5 mg/kg body weight) to prevent an endogenous LH surge. Follicle development was monitored by serum estradiol concentrations and ultrasonography. Follicle aspiration was performed on anesthesized monkeys without hCG or 12, 24, or 36 h after receiving 1000 IU r-hCG. The oocytes were cultured in M199 medium containing 10% FBS and 75 μIU/ml FSH and LH for a total (in vivo and in vitro) of 40–44 h post-hCG. The maturity of oocytes was indicated by cumulus expansion and the emission of polar bodies. After removing the cumulus cells, oocytes were fixed in 3.7% formaldehyde. The spindles were visualized by using an α-tubulin antibody. Oocytes possessing a bipolar-shaped spindle with well-aligned chromosomes at the equator were considered to be normal. Finally the distribution of mitochondria was studied using MitoTracker® Red 580 and confocal microscopy. Oocytes cultured without in vivo exposure to hCG had a maturation rate of 79.6% (82/111 GV). In comparison, 82.0% (41/51 GV) and 93.8% (60/64 GVBD) of oocytes retrieved at 12h or 24 h after hCG had matured, respectively. In total, 84.5% of oocytes (82/97) retrieved at 36h after hCG had reached MII at the time of aspiration. There was no significant difference in maturation of oocytes that had been matured longer in vivo (p<0.05). The majority of oocytes in all four groups possessed abnormal spindles with asymmetric, tripolar and multipolar spindles, as well as irregular bipolar formations and unaligned chromosomes (Table 1). Spindle abnormalities were generally associated with chromosome displacement (97.9%: 142 with chromosome displacement /145 with abnormal spindles). Mitochondria were distributed mainly around the cortical region in MII oocytes, whereas in GV and GVBD oocytes they were throughout the ooplasm. The duration of culture did not significantly affect the proportion of oocytes undergoing maturation, nor did it affect the incidence of cytological anomalies. The high incidence of defects of spindle formation and chromosome alignment indicates a poor prognosis for post-fertilization development, which is comparable to human oocytes. This non-human primate appears to be a good model for studying oogenesis and embryogenesis in humans.