Where in the tissues of Danio rerio is more H2O2 produced during acute hypoxia?

The lack of oxygen (O2) causes changes in the cell functioning. Modeling hypoxic conditions in vitro is challenging given that different cell types exhibit different sensitivities to tissue O2 levels. We present an effective in vivo platform for assessing various tissue and organ parameters in Danio rerio larvae under acute hypoxic conditions. Our system allows simultaneous positioning of multiple individuals within a chamber where O2 level in the water can be precisely and promptly regulated, all while conducting microscopy. We applied this approach in combination with a genetically encoded pH-biosensor SypHer3s and a highly H2O2-sensitive Hyper7 biosensor. Hypoxia causes H2O2 production in areas of brain, heart and skeletal muscles, exclusively in the mitochondrial matrix; it is noteworthy that H2O2 does not penetrate into the cytosol and is neutralized in the matrix upon reoxygenation. Hypoxia causes pronounced tissue acidosis, expressed by a decrease in pH by 0.4-0.6 units everywhere. Using imaging photoplethysmography, we measured in D.rerio fry real-time heart rate decrease under conditions of hypoxia and subsequent reoxygenation. Our observations in this experimental system lead to the hypothesis that mitochondria are the only source of H2O2 in cells of D.rerio under hypoxia.

Redox differences between rat neonatal and adult cardiomyocytes under hypoxia.

It is generally accepted that oxidative stress plays a key role in the development of ischemia-reperfusion injury in ischemic heart disease. However, the mechanisms how reactive oxygen species trigger cellular damage are not fully understood. Our study investigates redox state and highly reactive substances within neonatal and adult cardiomyocytes under hypoxia conditions. We have found that hypoxia induced an increase in H2O2 production in adult cardiomyocytes, while neonatal cardiomyocytes experienced a decrease in H2O2 levels. This finding correlates with our observation of the difference between the electron transport chain (ETC) properties and mitochondria amount in adult and neonatal cells. We demonstrated that in adult cardiomyocytes hypoxia caused the significant increase in the ETC loading with electrons compared to normoxia. On the contrary, in neonatal cardiomyocytes ETC loading with electrons was similar under both normoxic and hypoxic conditions that could be due to ETC non-functional state and the absence of the electrons transfer to O2 under normoxia. In addition to the variations in H2O2 production, we also noted consistent pH dynamics under hypoxic conditions. Notably, the pH levels exhibited a similar decrease in both cell types, thus, acidosis is a more universal cellular response to hypoxia. We also demonstrated that the amount of mitochondria and the levels of cardiac isoforms of troponin I, troponin T, myoglobin and GAPDH were significantly higher in adult cardiomyocytes compared to neonatal ones. Remarkably, we found out that under hypoxia, the levels of cardiac isoforms of troponin T, myoglobin, and GAPDH were elevated in adult cardiomyocytes, while their level in neonatal cells remained unchanged. Obtained data contribute to the understanding of the mechanisms of neonatal cardiomyocytes' resistance to hypoxia and the ability to maintain the metabolic homeostasis in contrast to adult ones.

Hyperglycemia exacerbates ischemic stroke not through increased generation of hydrogen peroxide.

Diabetes is one of the significant risk factors for ischemic stroke. Hyperglycemia exacerbates the pathogenesis of stroke, leading to more extensive cerebral damage and, as a result, to more severe consequences. However, the mechanism whereby the hyperglycemic status in diabetes affects biochemical processes during the development of ischemic injury is still not fully understood. In the present work, we record for the first time the real-time dynamics of H2O2 in the matrix of neuronal mitochondria in vitro in culture and in vivo in the brain tissues of rats during development of ischemic stroke under conditions of hyperglycemia and normal glucose levels. To accomplish this, we used a highly sensitive HyPer7 biosensor and a fiber-optic interface technology. We demonstrated that a high glycemic status does not affect the generation of H2O2 in the tissues of the ischemic core, while significantly exacerbating the consequences of pathogenesis. For the first time using Raman microspectroscopy approach, we have shown how a sharp increase in the blood glucose level increases the relative amount of reduced cytochromes in the mitochondrial electron transport chain in neurons under normal conditions in awake mice.

Controlled hatching at the prescribed site using femtosecond laser for zona pellucida drilling at the early blastocyst stage.

PURPOSE: To study whether the application of femtosecond laser pulses for zona pellucida (ZP) drilling of blastocysts at the embryonic or abembryonic poles can promote hatching to start immediately through the hole formed and ensure high hatching rates and embryo viability. METHODS: Mouse blastocyst (E3.5) ZP were microdissected with femtosecond laser pulses (514-nm wavelength, 280-fs pulse duration, 2.5-kHz repetition rate) close to the trophoblast or inner cell mass (ICM). The sizes of the holes formed were in the range of 4.5-8.5 µm. Additional longitudinal incisions (5-7-µm long) on either side of the hole were created to determine whether hatching had started at the correct position. Embryos post-laser-assisted ZP drilling and intact embryos were cultured under standard conditions for 2 days; embryo quality was assessed twice daily. The hatching rates and in vitro and in vivo implantation rates (only for embryos with ZP dissected close to the ICM) were estimated. RESULTS: Femtosecond laser-assisted ZP drilling at the early blastocyst stage facilitated embryo hatching to start at the artificial opening with probability approaching 100%. Despite the artificial opening's small size, no embryo trapping during hatching was observed. Both experimental groups had higher hatching rates than the control groups (93.3-94.7% vs. 83.3-85.7%, respectively). The in vitro implantation rate was comparable with that of the control group (92.3% vs. 95.4%). No statistically significant differences were obtained in the in vivo implantation rates between the experimental and control groups. CONCLUSIONS: Blastocyst-stage femtosecond laser microsurgery of ZP is fast and delicate and enables the hatching process to be initiated in a controlled manner through a relatively small opening, with no embryo trapping.

The in vitro Analysis of Quality of Ovarian Follicle Culture Systems Using Time-Lapse Microscopy and Quantitative Real-Time PCR.

BACKGROUND: The aim of ovarian follicle in vitro culture is to obtain mature oocytes. To evaluate the efficiency of in vitro culture system, the status of the cultured oocyte can be analyzed. METHODS: The preantral ovarian follicles retrieved from 14-day-old C57Bl/6J mice were cultured in 3D alginate hydrogel. The status of oocytes obtained from mature (3 months old, group A) and immature (3 weeks old, group B) mice was compared to the status of oocytes retrieved from ovarian follicles cultured in vitro (Group C) using qRT-PCR analysis and time-lapse microscopy. In the qRT-PCR analysis, 8 samples for group A (80 oocytes), 8 samples for group B (80 oocytes), and 6 samples for group C (60 oocytes) were included. Time-lapse analysis was performed in group A (oocytes n=31), group B (n=45), and group C (n=21). Statistical analysis was done by Kruskal-Wallis and chi-square tests and differences were considered statistically significant if p<0,05. RESULTS: The diameter of group C oocytes is lower in comparison to group A oocytes (67 µm vs. 75 µm, correspondingly). Groups B and C oocytes exhibited delayed meiosis in comparison to group A oocytes. Expression levels of six oocyte maturation genes (Ccnb, CDK1, Ccnh, Wee2, Mos and Epab) were evaluated using qRT-PCR analysis. Expression levels of Ccnh and Epab are lowered in group C oocytes compared to the expression levels of these genes in groups A and B oocytes (p< 0.05). CONCLUSION: Oocytes obtained after ovarian follicles in vitro culture have reduced development competence, future fundamental changes of in vitro culture systems can be expected.

Application of femtosecond laser microsurgery in assisted reproductive technologies for preimplantation embryo tagging.

Femtosecond laser pulses were applied for precise alphanumeric code engraving on the zona pellucida (ZP) of mouse zygotes for individual embryo marking and their identification. The optimal range of laser pulse energies required for safe ZP microsurgery has been determined. ZP was marked with codes in three different planes to simplify the process of embryo identification. No decrease in developmental rates and no morphological changes of embryos post laser-assisted engraving have been observed. ZP thickness of embryos post laser-assisted code engraving has been shown to differ significantly from that of control group embryos at the hatching stage. Due to moderate ZP thinning as compared to its initial width at 0.5 dpc (days post coitum), readability of the code degrades slightly and it still remains recognizable even at hatching stage. Our results demonstrate that application of femtosecond laser radiation could be an effective approach for noninvasive direct embryo tagging, enabling embryo identification for the whole period of preimplantation development.

Femtosecond laser is effective tool for zona pellucida engraving and tagging of preimplantation mammalian embryos.

PURPOSE: Our purpose was to study whether application of femtosecond laser pulses for alphanumeric code marking in the volume of zona pellucida (ZP) could be effective and reliable approach for direct tagging of preimplantation embryos. METHODS: Femtosecond laser pulses (wavelength of 514 nm, pulse duration of 280 fs, repetition rate of 2.5 kHz, pulse energy of 20 nJ) were applied for precise alphanumeric code engraving on the ZP of mouse embryos at the zygote stage for individual embryo marking and their accurate identification. Embryo quality assessment every 24 h post laser-assisted marking as well as immunofluorescence staining (for ICM/TE cell number ratio calculation) were performed. RESULTS: Initial experiments have started with embryo marking in a single equatorial plane. The codes engraved could be clearly recognized until the thinning of the ZP prior to hatching. Since embryo may change its orientation during the ART cycle, multi-plane code engraving seems to be more practical for simplifying the process of code searching and embryo identification. We have marked the ZP in three planes, and no decrease in developmental rates as well as no morphological changes of embryos post laser-assisted engraving have been observed as compared to control group embryos. CONCLUSIONS: Our results demonstrate the suitability of femtosecond laser as a novel tool for noninvasive embryo tagging, enabling embryo identification from day 0.5 post coitum to at least early blastocyst stage. Thus, the versatility and the potential use of femtosecond lasers in the field of developmental biology and assisted reproduction have been shown.

Molecular Mechanisms of Prophase I Meiotic Arrest Maintenance and Meiotic Resumption in Mammalian Oocytes.

Mechanisms of meiotic prophase I arrest maintenance (germinal vesicle [GV] stage) and meiotic resumption (germinal vesicle breakdown [GVBD] stage) in mammalian oocytes seem to be very complicated. These processes are regulated via multiple molecular cascades at transcriptional, translational, and posttranslational levels, and many of them are interrelated. There are many molecular cascades of meiosis maintaining and meiotic resumption in oocyte which are orchestrated by multiple molecules produced by pituitary gland and follicular cells. Furthermore, many of these molecular cascades are duplicated, thus ensuring the stability of the entire system. Understanding mechanisms of oocyte maturation is essential to assess the oocyte status, develop effective protocols of oocyte in vitro maturation, and design novel contraceptive drugs. Mechanisms of meiotic arrest maintenance at prophase I and meiotic resumption in mammalian oocytes are covered in the present article.

Influence of gonadotropins on ovarian follicle growth and development in vivo and in vitro.

Gonadotropins are the key regulators of ovarian follicles development. They are applied in therapeutic practice in assisted reproductive technology clinics. In the present review we discuss the basic gonadotropic hormones - recombinant human follicle-stimulating hormone, its derivatives, luteinizing hormone and gonadotropin serum of pregnant mares, their origin, and application in ovarian follicle systems in in vitro culture systems.