Oocytes In Medium Research Articles

Folic acids promote in vitro maturation of bovine oocytes by inhibition of ferroptosis via upregulated glutathione and downregulated Fe2+ accumulation

Bovine embryos by in vitro fertilization have become the primary source of commercial embryo transfers globally. However, the developmental capacity of in vitro maturation (IVM) oocytes is considerably lower than that of in vivo maturation (IVO) oocytes, owing to the production of reactive oxygen species (ROS) via mitochondrial metabolism, which was higher in IVM oocytes than in IVO oocytes. To avoid the negative effects of ROS on embryo quality, folic acid (FA) was supplemented directly into the IVM medium to antagonize ROS production, however, the mechanisms remain unknown. In the present study, five levels of FA (0, 25, 50, 100, and 200 µM) were supplemented into the bovine oocyte culture medium. The maturation, cleavage, and blastocyst formation rates increased by 8.95 %, 6.94 %, and 4.36 %, respectively, in the 50 µM group compared to the 0 µM group. Moreover, 7904 differential genes were identified between 0 µM and 50 µM groups by transcriptome sequencing, and they were mainly enriched in 8 pathways. The glutathione, ROS, and Fe2+ levels in oocytes were found to be associated with ferroptosis. Our results revealed that 50 µM FA promoted the IVM of bovine oocytes and affected the expression of genes involved in the ferroptosis pathway. The downregulation of TFR1 and STEAP3 led to a decrease in intracellular Fe2+ accumulation, and the upregulation of GCL increased oocyte GSH levels, thereby reducing the production of ROS in the ferroptosis pathway. Our study provides a new insight into the molecular mechanisms by which FA promotes bovine oocyte development in vitro.

Exploring the impact of heat stress on oocyte maturation and embryo development in dairy cattle using a culture medium supplemented with vitamins E, C, and coenzyme Q10

Heat stress is a significant factor affecting the fertility of dairy cattle due to the generation of free radicals. In assisted reproductive techniques, the inclusion of protective antioxidants becomes crucial to mitigate potential cellular damage. This study aimed to explore the impact of supplementing vitamins E, C, and coenzyme Q10 into the oocyte culture medium, with the goal of ameliorating the adverse effects of heat stress on oocyte maturation and embryo development in dairy cattle. A group of fifty Holstein dairy cows were synchronized, and their oocytes were harvested using the ovum pick-up method. High-quality oocytes were subjected to in vitro maturation (IVM) and in vitro fertilization (IVF) procedures, utilizing a culture medium containing, no supplements (Group 1), 100 μM of vitamins E (Group 2) and C (Group 3), along with 50 μM of coenzyme Q10 (Group 4). The ensuing zygotes were cultured, and the ensuing embryos were evaluated for blastocyst formation by the seventh day. An analysis of the blastocysts' inner cell mass (ICM) and trophectoderm (TE) cells was also conducted. The findings revealed that the group receiving supplementation of vitamin E and coenzyme Q10 exhibited significantly higher maturation and cleavage rates in comparison to both the control and the vitamin C groups. Furthermore, the count of ICM, TE, and blastocyst cells was notably elevated in the vitamin E supplemented group when compared to the control group. In summary, the effectiveness of vitamin E in enhancing IVM, IVF, and embryo development under conditions of heat stress surpassed that of vitamin C and coenzyme Q10.

Chrysoeriol Improves In Vitro Porcine Embryo Development by Reducing Oxidative Stress and Autophagy

Chrysoeriol (CHE) is a flavonoid substance that exists in many plants. It has various physiological and pharmacological effects, including anti-inflammatory, antioxidant, anti-tumor, and protective activity, especially for the cardiovascular system and liver. Among common livestock embryos, porcine embryos are often considered high-quality objects for studying the antioxidant mechanisms of oocytes. Because porcine embryos contain high levels of lipids, they are more vulnerable to external stimuli, which affect development. Our study explored the influence of CHE supplementation on oxidative stress in porcine oocytes and its possible mechanisms. Different concentrations of CHE (0, 0.1, 1, and 3 µM) were supplemented in the in vitro culture medium of the porcine oocytes. The results showed that supplementation with 1 µM CHE significantly increased the blastocyst rate and total cell number of embryos in vitro. After finding the beneficial effects of CHE, we measured reactive oxygen species (ROS), glutathione (GSH), and mitochondrial membrane potential (MMP) when the oocytes reached the 4-cell stage of development and determined the levels of apoptosis, cell proliferation, and autophagy at the blastocyst stage of development. The expression levels of some related genes were preliminarily detected by qRT-PCR. The results showed that the apoptosis of blastocysts in the CHE-treated culture also decreased compared with the untreated culture. Furthermore, CHE downregulated intracellular ROS and increased GSH in the embryos. CHE was also shown to improve the activity of mitochondria and inhibit the occurrence of autophagy. In addition, antioxidant-related genes (SOD1, SOD2, and CAT) and cell pluripotency-related genes (SOX2, OCT4, and NANOG) were upregulated. At the same time, apoptosis-related (Caspase 3) and autophagy-related (LC3B) genes showed a downward trend after supplementation with CHE. These results indicate that CHE improved the development of porcine embryos in vitro by reducing oxidative stress and autophagy levels.

The Effects of an In Vitro Oocyte Maturation System and Chlorogenic Acid Supplementation during Embryo Culture on the Development of Porcine Cloned Embryos Derived from Native Vietnamese Ban Pigs

The aim of this study was to improve the production efficiency of Vietnamese native Ban pig embryos using somatic cell nuclear transfer (SCNT). Fibroblast cells from Ban pigs were injected into the enucleated cytoplasts of crossbred gilts, and the reconstructed embryos were subsequently cultured. In the first experiment, cytoplasts were isolated from oocytes matured in either a defined porcine oocyte medium (POM) or in TCM199 medium supplemented with porcine follicular fluid. Both media were supplemented with gonadotropic hormones, either for the first 22 h of in vitro maturation (IVM) or for the entire 44 h of IVM. In the second experiment, the reconstructed SCNT embryos were cultured with or without 50 μM chlorogenic acid (CGA). Furthermore, this study examined parthenogenetic embryos. The IVM medium and duration of hormone treatment did not affect embryo development. CGA supplementation to the culture medium significantly increased blastocyst formation rates in parthenogenetic embryos but not in SCNT embryos. However, CGA supplementation significantly reduced the apoptotic index in blastocysts regardless of embryo source. In conclusion, the IVM method did not affect SCNT embryo production, while CGA supplementation during embryo culture improved the quality of SCNT embryos in indigenous pig breeds.

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.

Oocyte Culture Medium (OCM)

Oocyte Culture Medium (OCM)

The effect of curcumin on embryonic in vitro development in experimental polycystic ovary syndrome: An experimental study.

BackgroundPolycystic ovary syndrome (PCOS) is a common disease in women. Some plant compounds which have antioxidant properties, such as curcumin, may be useful for these patients when delivered orally or in vitro.Objective The aim of this study was to evaluate the impact of PCOS on oocyte quality and the effect of curcumin on in vitro fertilization of oocytes.Materials and Methods In this experimental study, Naval Medical Research Institute mice aged six to eight wk were used. Mice were divided into five experimental groups (control, experimental PCOS, curcumin 6, 12 and 24 μM). To induce experimental PCOS, estradiol valerate (100 mg/kg, IP) was injected. The total antioxidant capacity and production of malondialdehyde in ovarian tissue and blood serum were evaluated in all groups. Finally, 6, 12 and 24 μM of curcumin were added to the culture medium of the PCOS group oocytes and development in the different groups was evaluated.ResultsA high percentage of oocytes for fertilization were not in good condition in terms of number and quality in the group of PCOS. The addition of curcumin to the embryo culture medium was associated with a higher percentage of fertilized oocytes, two-cells and blastocysts. This increase was significant at a concentration of 24 μM (p 0.01).ConclusionGiven that adding curcumin seemed to improve fetal growth and prevent the harmful effects of oxygen free radicals on the culture medium, it is recommended to add a certain concentration of curcumin under normal conditions without oxidative stress.

A primary effect of palmitic acid on mouse oocytes is the disruption of the structure of the endoplasmic reticulum.

Exposure of mouse oocytes to saturated fatty acids (FAs) such as palmitic acid (PA) has been shown to increase lipid content and cause an endoplasmic reticulum (ER) stress response and changes in the mitochondrial redox state. PA can also disrupt Ca2+ stores in other cell types. The links between these intracellular changes, or whether they are prevented by mono-unsaturated FAs such as oleic acid (OA), is unclear. Here, we have investigated the effects of FAs on mouse oocytes, that are maturated in vitro, using coherent anti-Stokes Raman scattering and two-photon fluorescence microscopy. When oocytes were matured in the presence of PA, there were changes in the aggregation pattern and size of lipid droplets that were mitigated by co-incubation in OA. Maturation in PA alone also caused a distinctive disruption of the ER structure. This effect was prevented by incubation of OA with PA. In contrast, maturation of mouse oocytes in medium containing PA was not associated with any significant change in the redox state of mitochondria or the Ca2+ content of intracellular stores. These data suggest that a primary effect of saturated FAs such as PA on oocytes is to disrupt the structure of the ER and this is not due to an effect on the mitochondria or Ca2+ stores.

Supplementation of the In Vitro Maturation Culture Medium of Mouse Oocytes with Growth Hormone Improves Pregnancy Outcomes.

This study aimed to examine the effects of adding growth hormone (GH) into the in vitro maturation (IVM) culture medium of mouse oocytes on pregnancy outcomes. Cumulus-oocyte complexes (COCs) were cultured in a medium with (GH group, 100 ng/mL) or without (Con group) GH. Thereafter, chromosome morphology, spindle morphology, and mitochondrial function were examined. Embryo development and blastocyst quality after in vitro fertilization were evaluated. After the embryo transfer, the implantation sites and pregnancy outcomes were evaluated. The oocyte maturation rate of the GH group (81.8 ± 9.6%) was compared to that of the Con group (81.3 ± 6.9%, P = 0.928). The proportion of morphologically abnormal spindles in GH-treated oocytes (7.1 ± 0.9%) was significantly lower than control oocytes (13.7 ± 1.3%, P = 0.032), whereas the proportion of morphologically abnormal chromosomes and mitochondrial distribution was similar between the groups. The mitochondrial membrane potential (P < 0.001) and ATP concentration (P < 0.001) in GH-exposed oocytes were higher than those in control oocytes. After fertilization, the blastocyst rate in the GH group (33.8 ± 13.2%) was significantly higher than the Con group (16.2 ± 2.0%, P = 0.003). In addition, inner cell mass (ICM) number (13.91 ± 3.48 vs. 7.00 ± 1.91, P < 0.001), total cell number (47.45 ± 8.39 vs. 37.71 ± 4.15, P = 0.007), and the ratio of ICM/total cell number (29.9 ± 8.2% vs. 18.6 ± 5.0%, P = 0.002) of blastocyst were all higher in GH group. The implantation rate (71.2 ± 1.9% vs. 39.4 ± 16.4%, P < 0.001) and litter size (8.50 ± 3.99 vs. 3.00 ± 1.22, P = 0.018) were significantly higher in the GH group. Although addition of GH into IVM culture medium does not improve oocyte maturation rate, it improves oocyte and embryo quality, which leads to better embryo development and pregnancy outcomes.

Optimization of donor cell cycle synchrony, maturation media and embryo culture system for somatic cell nuclear transfer in the critically endangered Vietnamese Ỉ pig

Our aim was to establish an efficient culture system to produce embryos by SCNT of the endangered Vietnamese Ỉ pig. Reducing the serum concentration from 10.0% to 0.2% during culture efficiently synchronized Ỉ pig fibroblasts used as donor cells at the G0/G1 stage. Oocyte maturation in a defined porcine oocyte medium (POM) supplemented with EGF and gonadotrophins resulted in higher cleavage and blastocyst rates compared with a non-defined POM containing pig follicular fluid (but without EGF) and both the defined and non-defined variants of NCSU-37. For embryo culture PZM3 and PZM5 media were superior to NCSU-37, in terms of the percentage of cleaved embryos. Addition of serum to PZM3 medium on Day 5 of culture (Day 0 = SCNT) improved blastocyst development. When SCNT embryos were transferred at the blastocyst stage, 7 of 11 recipients became pregnant. However, live offspring were not obtained. In conclusion, we established a system for the production of Ỉ pig embryos by SCNT and achieved blastocyst production rate at 26.4% by improving culture systems for donor cells, oocytes and embryos culture. Transfer of embryos resulted in pregnancies; however, live offspring were not obtained.

Design and Microfabrication of An On-Chip Oocyte Maturation System for Reduction of Apoptosis.

Objective:In customary assisted reproductive technology (ART), oocyte culture occurs in static micro drops of Petri dishes with vast media volume; while, the in vivo condition is dynamic. In this study, we aimed to improve the maturation efficiency of mammalian oocytes by designing an optimal microchamber array to obtain the integration of oocyte trapping and maturation within a microfluidic device and evaluate the role of microfluidic culture condition in lipid peroxidation level of the culture medium, in vitro matured oocytes apoptosis, and its comparison with the conventional static system.Materials and Methods:In this experimental research, immature oocytes were collected from ovaries of the Naval Medical Research Institute (NMRI) mice. Oocytes were randomly laid in static and dynamic (passive < active)in vitro maturation culture medium for 24 hours. The lipid peroxidation level in oocyte culture media was assessed by measuring the concentration of malondialdehyde (MDA), and the rate of apoptosis in in vitro matured oocytes was assessed by the TUNEL assay after a-24 hour maturation period.Results:The MDA concentration in both dynamic oocyte maturation media were significantly lower than the static medium (0.003 and 0.002 vs. 0.13 µmol/L, P<0.01). Moreover, the rate of apoptosis in matured oocytes after a-24 hour maturation period was significantly lower in passive dynamic and active dynamic groups compared with the static group (16%, 15% vs. 35%, P<0.01).Conclusion:The dynamic culture for in vitro oocyte maturation (IVM) improves the viability of IVM oocytes in comparison with the static culture condition.

30 The importance of cumulus cells for the survival and timing of meiotic resumption of porcine oocytes vitrified at the immature stage

Previous research revealed that vitrification at the immature (the germinal vesicle, GV) stage triggers premature meiotic resumption in cumulus-enclosed porcine oocytes and causes a damage in gap junctions (Appeltant et al. 2017 Reprod. Fertil. Dev. 29, 2419-2429). However, the correlation between the two phenomena was not investigated yet. The present research was conducted to clarify whether premature meiotic resumption is caused by gap junction disruption and to assess the importance of cumulus cells for the survival of porcine oocytes vitrified at the GV stage. Cumulus–oocyte complexes (COCs) were collected from 3- to 6-mm antral follicles of slaughtered gilts. Immediately after collection, approximately half of them were denuded mechanically (DOs). In each replicate, groups of COCs and DOs were processed without vitrification (control groups). Treatment groups of COCs and DOs were vitrified on Cryotop sheets in a combination of 17.5% propylene glycol and 17.5% ethylene glycol and warmed in 0.4M sucrose. The oocytes were then cultured for 22h in a chemically defined porcine oocyte medium (POM) supplemented with 10ngmL−1 epidermal growth factor, 10IUmL−1 equine chorionic gonadotrophin, 10IUmL−1 human chorionic gonadotrophin, and 1mM dibutyryl cAMP. After culture, COCs were denuded and oocyte survival was assessed by morphological evaluation of membrane integrity under a stereo microscope. Then, live oocytes were fixed and stained with 1% orcein and nuclear status was evaluated under a phase-contrast microscope. The experiment was replicated 5 times. Data were analysed by ANOVA followed by Tukey’s multiple comparisons test. After vitrification and culture, the survival rate in the COC group was higher (P&amp;lt;0.05) than that of the DO group (160/191=84.7±3.4% vs. 153/237=65.0±6.2%, respectively) but reduced (P&amp;lt;0.05) compared with those in the control COC and DO groups (138/143=96.6±1.0% and 152/153=99.3±0.6%, respectively). The majority of the control COCs and DOs were at the GV stage with similar percentages (95.6±2.2% and 94.0±2.2%, respectively). In contrast, the percentages of oocytes at the GV stage in the vitrified COC and DO groups were reduced (71.6±9.4% and 45.7±10.5%, respectively; P&amp;lt;0.05) compared with the control groups, which were associated with increased frequencies of diakinesis and MI stages. Percentages of oocytes at the GV stage in the vitrified COC and DO groups were not significantly different (P=0.23). In conclusion, cumulus cells can prevent vitrification-related membrane damage of oocytes. Furthermore, vitrification induced premature meiosis both in the cumulus-enclosed and denuded oocytes even in the presence of the meiotic inhibitor, dibutyryl cAMP. Nevertheless, cumulus removal without vitrification did not induce premature meiosis in the oocytes. Therefore, disruption in communication with cumulus cells might not be the primary reason for premature meiosis in vitrified oocytes.

Bovine in vitro embryo production using media prepared with Milli-Q® Water or nanowater

The aim of this study was to compare the efficiency of in vitro embryo production (IVP) following the collection of bovine ovaries and 22-h in vitro maturation (IVM) of oocytes in media prepared with Milli-Q® Water (n = 509 oocytes) or nanowater (NW; n = 304 oocytes). The mean cleavage (63.8 ± 4.6 % vs. 63.6 ± 6.1 %, respectively; mean ± SEM) and blastocyst formation rate (16.3 ± 3.4 % vs. 16.7 ± 6.7 % of presumptive zygotes, respectively) did not vary (P > 0.05; Student t-test) between the two types of media diluents. NW is a safe substitute for Milli-Q® Water for IVM of bovine oocytes.

Effectiveness of in vitro maturation and in vitro fertilization techniques in pigs

In vitro maturation and in vitro fertilization techniques in pigs have progressed considerably in recent years. Many reports focus on the factors affecting in vitro maturation that lead to normal male pronuclear formation or monospermy after fertilization in vitro. It is suggested that pig follicular fluid (pFF), follicle somatic cells and various hormones are important factors for the maintenance of cytoplasmic maturation of oocytes in vitro, but that fetal calf serum (FCS), which is generally added to maturation medium, is detrimental. A series of experiments clearly indicate that the glutathione (GSH) content of matured oocytes increases greatly when maturation medium is supplemented with cysteine, a precursor of GSH, and the rates of male pronuclear formation increase in parallel with the increasing GSH content. To prevent polyspermy, conditions of maturation and of fertilization in vitro are important. Culture of oocytes in medium with FCS for the first 24 h and with BSA for the second 24 h decreases the incidence of polyspermy, without a significant effect on nuclear maturation. However, it has been shown that secretory macromolecules of the oviduct may reduce the incidence of polyspermy by interacting with fertilizing spermatozoa rather than with oocytes. A reduction of polyspermy by treating spermatozoa with pFF is also reported. In addition to the many improvements in the methodology of in vitro fertilization using unfrozen spermatozoa in pigs, techniques for fertilizing oocytes in vitro with frozen epididymal and ejaculated spermatozoa have also recently been developed.

Prediction of major microRNAs in follicular fluid regulating porcine oocyte development

The aim of the present study was to identify key microRNAs (miRNAs) in porcine follicular fluid (FF) that regulate oocyte growth. miRNAs contained in FF were determined by small RNA-seq of exosome RNA. Upstream regulator miRNA was determined by ingenuity pathway analysis using differentially expressed genes in granulosa cells (GCs) between small follicles (1-2 mm in diameter) and large follicles (3-5 mm), and between follicles containing oocytes of high developmental ability and follicles containing oocytes of low developmental ability. The candidate miRNAs overlapping among the three miRNAs group were determined. Lastly, the effect of supplementation with FF, exosome-depleted FFs, or each miRNA on in vitro oocyte growth was examined. The miRNAs determined were miR-17, -27, -92a, and -145. These miRNAs were found in the spent culture medium of oocytes and granulosa cells complexes and serum by small RNA sequencing. Culturing of oocytes and granulosa cells complexes collected from porcine early antral follicles (0.5-0.7 mm in diameter) with FF for 14 days improved oocyte growth; depletion of exosomes from the FFs neutralized the beneficial effect observed. miR-92a mimic increased the antrum formation and diameter, together with acetylated levels of H4K12 in oocytes. In addition, supplementation of miRNA mimics miR-17b, -92a, and -145b improved the rate of chromatin configuration, and miR-17b and -92a mimics improved the developmental ability of oocytes to the blastocyst stage. miR-17, -92a, and -145 are major miRNA candidates in follicular fluids regulating oocyte growth.

The Effect of Melatonin on Mitochondrial Function and Autophagy in In Vitro Matured Oocytes of Aged Mice.

Objective This study examined the in vitro effect of melatonin on the protein synthesis of mitochondria, as well as autophagy in matured oocytes of aged mice. Materials and Methods In this experimental study, germinal vesicles (GV) oocytes were collected from aged (with the age of six-months-old) and young mice (with age range of 6-8 weeks old) and then cultured in the in vitro culture medium (IVM) for 24 hours to each metaphase II (MII) oocytes and then supplemented with melatonin at a concentration of 10 μM. The culture medium of MII oocytes was devoid of melatonin. Afterward, the expression of the SIRT-1 and LC3 was assessed by immunocytochemistry. ATP-dependent luciferin-luciferase bioluminescence assay was employed for the measurement of the ATP contents. Intracellular reactive oxygen specious (ROS) was detected by DCFH-DA, and the total antioxidant capacity (TAC) level was determined by TAC assay. Results The expression of SIRT-1 and LC3, as well as the measurement of the ATP content, was significantly increased in oocytes treated with melatonin compared with the oocytes receiving no treatment. Moreover, TAC was considerably higher in melatonin-treated oocytes than oocytes receiving no treatment. On the other hand, the level of ROS was significantly decreased in oocytes treated with melatonin in comparison with the untreated oocytes. The results indicated that melatonin considerably improved the development of oocytes as well. ConclusionAccording to the data, melatonin increased mitochondrial function and autophagy via an increase in the expression of SIRT1 and LC3, as well as the ATP contents while it decreased the levels of ROS and increased TAC in oocytes derived from aged mice.

36 The effects of E-64 on the developmental competence of porcine oocytes vitrified at the germinal vesicle stage

Previous studies reported the activation of the apoptotic cascade by vitrification in mature porcine oocytes (Vallorani et al. 2012 Anim. Reprod. Sci. 135, 68-74) and that the cathepsin B inhibitor E-64 improved developmental competence of bovine oocytes via an antiapoptotic effect (Balboula et al. 2013 Reproduction 146, 407-417). The present study was carried out to test whether E-64 affected the developmental competency of porcine oocytes vitrified at the germinal vesicle stage. Cumulus-enclosed porcine oocytes were vitrified in microdrops and warmed by our method (Somfai et al. 2015 J. Reprod. Dev. 61, 571-579). Then, the oocytes were subjected to in vitro maturation (IVM) for 46h in a chemically defined porcine oocyte medium supplemented with 10ng mL−1 of epidermal growth factor, 10IU mL−1 of eCG, and 10IU mL−1 of hCG and during the first 22h of IVM with 1mM dibutyryl cyclic adenosine monophosphate. Then, cumulus-oocyte complexes were fertilized in vitro and presumptive zygotes were cultured in 50-µL drops of porcine zygote medium-3 for 7 days in 6-well dishes covered by paraffin oil in an atmosphere of 5% CO2, 5% O2, and 90% N2 at 39°C. On Day 5 (Day 0=IVF), the porcine zygote medium-3 was supplemented with 10% (vol/vol) FCS. The effects of 1.0μM of E-64 supplementation during IVM of non-vitrified and vitrified cumulus-oocyte complexes were investigated in a 2×2 factorial design. Survival rates after IVM, cleavage rates on Day 2, blastocyst rates, and total cell numbers in blastocysts on Day 7 were compared among groups. The experiment was replicated 5 times. Results were analysed by ANOVA and Tukey’s multiple comparison test. The percentages of live oocytes were statistically similar when oocytes were matured in the absence or presence of E-64 both in non-vitrified (99.2% v. 99.6%, respectively) and vitrified (94.3% v. 90.8%, respectively) groups. Similarly, IVM without or with E-64 supplementation had no effect on subsequent cleavage and blastocyst development rates in non-vitrified (67.4% v. 71.2% and 38.7% v. 43.2%, respectively) and vitrified (46.8% v. 48.8% and 14.6% v. 22.8%, respectively) oocytes. Irrespective of E-64 treatment, all survival and developmental rates in the vitrified groups were significantly lower (P&amp;lt;0.05) compared with those of their non-vitrified counterparts except for the blastocyst development rate in the E-64-treated vitrified group, which did not differ significantly from those of the non-vitrified groups with or without E-64 treatment. There was no statistical difference in mean blastocyst cell numbers among the groups, ranging between 86.5±15.8 and 118±10.6. In conclusion, E-64 treatment had no effect on embryo production rates, which suggests that in our system, cathepsin-mediated apoptosis during IVM might not be the factor to limit embryo production using either fresh oocytes or those vitrified at the immature stage. This work was supported by JST/JICA SATREPS.

L-15 Oocyte Culture Medium (OCM; 70%)

L-15 Oocyte Culture Medium (OCM; 70%)

PH: the silent variable significantly impacting meiotic spindle assembly in mouse oocytes.

Temperature fluctuation negatively impacts the assembly and function of the meiotic spindle, but does pH have a similar effect? Polarized light microscopy was used to study the spindle in living mouse oocytes under different pH conditions. Female mice (n = 53) were superovulated, and oocytes collected, denuded and allocated to treatment groups. All experiments were performed at 37°C, and standard bicarbonate-buffered medium was used either pre-equilibrated in 6% CO2 or unequilibrated (in ambient CO2). Mean oocyte spindle retardance was measured over time in response to changing pH. Spindles were also assessed to understand whether this effect was reversible, by using a fixed pH in a zwitterionic buffer. The data show the spindle is impacted by pH fluctuation, with mean retardance significantly higher at pH 7.4-7.5 than at the point of media equilibration (P<0.001). This effect was found to be reversible; retardance significantly decreased after transition of the oocytes from pH 7.43 or pH 7.53 back to the original pre-equilibration pH of 7.32 (P<0.05). This study has shown that the meiotic spindle in mouse oocytes is highly sensitive to changes in oocyte culture media pH. If comparable in humans, this has significance as to the pH level of culture media currently used in assisted reproductive technology clinics worldwide, and reinforces the requirement for stringent control over extrinsic variables in the IVF laboratory.

Melatonin attenuates postovulatory oocyte dysfunction by regulating SIRT1 expression.

The quality of postovulatory metaphase II oocytes undergoes a time-dependent deterioration as a result of the aging process. Melatonin is considered to be an anti-aging agent. However, the underlying mechanisms of how melatonin improves the quality of postovulatory aged oocytes remain largely unclear. In this study, by using mouse model, we found that there were elevated reactive oxygen species levels and impaired mitochondrial function demonstrated by reduced mitochondrial membrane potential and increased mitochondrial aggregation in oocytes aged 24 h, accompanied by an increased number of meiotic errors, unregulated autophagy-related proteins and early apoptosis, which led to decreased oocyte quality and disrupted developmental competence. However, all of these events can be largely prevented by supplementing the oocyte culture medium with 10-3 M melatonin. Additionally, we found that the expression of sirtuin family members (SIRT1, 2 and 3) was dramatically reduced in aged oocytes. In addition, in vitro supplementation with melatonin significantly upregulated the expression of SIRT1 and antioxidant enzyme MnSOD, but this action was not observed for SIRT2 and SIRT3. Furthermore, the protective effect of melatonin on the delay of oocyte aging vanished when the SIRT1 inhibitor EX527 was used to simultaneously treat the oocytes with melatonin. Consistent with this finding, we found that the postovulatory oocyte aging process was markedly attenuated when the oocytes were treated with the SIRT1 activator SRT1720. In conclusion, our data strongly indicate that melatonin delays postovulatory mouse oocyte aging via a SIRT1-MnSOD-dependent pathway, which may provide a molecular mechanism support for the further application of melatonin in the assisted reproductive technology field.