Methotrexate impaired in-vivo matured mouse oocyte quality and the possible mechanisms.

BACKGROUND: Methotrexate (MTX) is an antifolate agent which is widely used in clinic for treating malignancies, rheumatoid arthritis and ectopic pregnancy. As reported, MTX has side effects on gastrointestinal system, nervous system and reproductive system, while its potential damages on oocyte quality are still unclear. It is known that oocyte quality is essential for healthy conception and the forthcoming embryo development. Thus, this work studied the effects of MTX on the oocyte quality. RESULTS: We established MTX model mice by single treatment with 5 mg/Kg MTX. Both morphological and molecular biology studies were performed to assess the in-vivo matured oocytes quality and to analyze the related mechanisms. The in-vivo matured oocytes from MTX-treated mice had poor in-vitro fertilization ability, and the resulting embryo formation rates and blastocyst quality were lower than the control group. We found that the in-vivo matured MTX-treated mouse oocytes displayed abnormal transcript expressions for genes of key enzymes in the folate cycles. MTX increased the rate of abnormal chromosome alignment and affected the regulation of chromosome separation via disrupting the spindle morphology and reducing the mRNA expressions of MAD2 and Sgo1. MTX reduced the DNA methylation levels in the in-vivo matured oocytes, and further studies showed that MTX altered the expressions of DNMT1 and DNMT 3b, and may also affect the levels of the methyl donor and its metabolite. CONCLUSIONS: MTX impaired the in-vivo matured mouse oocyte quality by disturbing folate metabolism and affecting chromosome stability and methylation modification.

[Intensity loss of two-photon excitation fluorescence microscopy images of mouse oocyte chromosomes].

As an optical microscope with high resolution, two-photon excitation (TPE) fluorescence microscope is widely used in noninvasive 3D optical imaging of biological samples. Compared with confocal laser scanning microscope, TPE fluorescence microscope provides a deeper detecting depth. In spite of that, the image quality of sample always declines as the detecting depth increases when a noninvasive 3D optical imaging of thicker samples is performed. Mouse oocytes with a large diameter, which play an important role in clinical and biological fields, have obvious absorption and scattering effects. In the present paper, we performed compensation for two-photon fluorescence images of mouse oocyte chromosomes. Using volume as a parameter, the attenuation degree of these chromosomes was also studied. The result of our data suggested that there exists a severe axial intensity loss in two-photon microscopic images of mouse oocytes due to the absorption and scattering effects. It is necessary to make compensation for these images of mouse oocyte chromosomes obtained from two-photon microscopic system. It will be specially needed in studying the quantitative three-dimensional information of mouse oocytes.

[A method for real-time observation of intracellular free Ca2+ concentration in single bovine retinal neurons].

In order to observe the neurotrophin's influence on intracellular free calcium concentration, the test neuron was labeled by fluorescence indicator Fluo-3, and imaged by self-built real-time fluorescence microscopy system. The authors observed the changes in intracellular free calcium concentration ([Ca2+]i) in the bovine retinal neurons under the influence of four kinds of neurotrophins such as brain derived neurotrophic factor BDNF. On account of the fact that fluorescence indicator's intensity decays over time, it is necessary to apply a "decay removal correction" to the fluorescence intensity in order to show the fluorescence intensity that solely represents [Ca2+]i. The corrected data shows an increase after adding neurotrophins to neurons, which is consistent with similar results published by other groups. Thus, our method of imaging living cells is feasible, and "decay removal correction" is reliable.

[Visualization of the age-related changes in expressions of DNA methyltransferases in mouse oocytes using two-photon imaging system].

Quantum dot (QD) is widely used as fluorescent labeling dye for its strong anti-fluorescence quenching, high quantum yield, wide absorption spectra, and narrow emission spectra. In the present paper, QD 585-labeled DNA methyltransferases (Dnmts) and Hoechst 33342-labeled chromosomes were imaged simultaneously using two-photon imaging system. The authors' results showed that aging mouse oocytes may be not suitable for in-vitro maturation: both the localizations and expression levels of Dnmts in in-vitro matured oocytes of aging mice were changed, and these changes may be related to the abnormal DNA methylation modification.

Age-related changes in the localization of DNA methyltransferases during meiotic maturation in mouse oocytes.

The effects of maternal aging on the localization of DNA methyltransferases were evaluated during mouse oocyte maturation using fluorescence staining. And we conclude that maternal aging affects the cytoplasmic-to-nuclear trafficking of DNA methyltransferases in mouse oocytes during the time from germinal vesicle breakdown to metaphase I.

[Observation on cytoskeleton and root tip growth of Arabidopsis thaliana by two-photon laser scanning microscopy].

Cytoskeleton plays an important role in plant cells and their movements, developments, and so on. With MS culture medium, the present paper cultured GFP-fused Arabidopsis thaliana in asepsis condition, which completed its whole lifecycle in the lab, starting from germination, going through plant growth, blossom, and ending at fructification. During this process, the authors used two-photon laser scanning microscopy (TPLSM) which was suitable for 4D observation on large, thick, live samples to observe cytoskeleton in seed, root tip, vessel and root hair of the Arabidopsis thaliana. Dynamic root tip growth as well as the growing speed was also observed. Applying IAA to the Arabidopsis thaliana, the authors found that the growing speed of root tip was enhanced by about 3.8 times.

[Observation of double-stained African green monkey kidney COS-7 cells using total internal reflection double-channel fluorescence microscopy].

Using a Dual-View wavelength splitter, double-stained African green monkey kidney COS-7 cells, transfected with pEGFP-Myosin 15a and costained with Rhodamine-filopodia were observed based on an ICCD(intensified charge couple device) fluorescence micro-imaging systems. Total internal reflection fluorescence microscopy was used to observe the overexpression of Myosin 15a to the tips of the elongation filopodia. An approach to collecting fluorescence in two channels and avoiding spectra crosstalk was employed to observe Myosin 15a and filopodia distribution in African green monkey kidney COS-7 cells. High sensitivity TIRF technology and two channels imaging method provided a wide application in bio-medical studies.

[Study on real-time imaging of single stretched DNA molecules by total internal reflection fluorescence microscopy].

Total internal reflection fluorescence microscopy (TIRF) is a powerful tool for single molecule study, since only a thin layer of about 200 nanometers is excited by the evanescent wave, resulting in high sensitivity of detection and high signal-to-noise ratio of images. Molecular combing is a convenient and efficient way to stretch DNA molecules with the help of the binding force between DNA molecule and solid surface, as well as the lateral force introduced by ambient fluid flow. In the present paper, real-time fluorescence imaging of single DNA molecules was carried out with these two techniques. Clear images of single stretched DNA were obtained, while photocleavage of DNA-YOYO-1 complex was found to be naturally avoided under TIRF imaging conditions. Photobleaching of the complexes was investigated in real-time, and was greatly reduced by synchronizing the excitation of light (laser) and the exposure of detector (ICCD). The method optimized the experimental conditions for long-lasting real-time observation and imaging of single stretched DNA molecules, so as to lay a foundation for visually studying the kinetic processes of interactions between DNA and proteins.

[DNA nanosensor fluorescence imaging microscopy].

Quantum dots have many excellent optical properties such as high quantum yield, long fluorescence lifetime, wide excitation spectrum and narrow emission spectrum, tunable emission wavelength and so on, thus have become a newpopular type of fluorescence probes in these years. Quantum-dot-based DNA nanosensor comprising streptavidin-conjugated quantum dots, capture probes with biotin and reporter probes with Cy5 was designed to detect DNA or RNA segments. Capture probes and reporter probes were connected by the target DNA or RNA segments so that quantum dots and Cy5s could be together and FRET (fluorescence resonance energy transfer) could be detected. In the present work, quantum-dot-based DNA nanosensor was combined with ICCD fluorescence microscopy imaging system through the authors' experiments. Using the total internal reflection fluorescence (TIRF), FRET between quantum dots and Cy5s was recorded by ICCD showing that segments of singlestranded target DNA with 30-base length were detected in solution using DNA nanosensor. When Cy5-ssDNA-Biotins were added into streptavi din-conjugated quantum dots in solution, by real time recording, the FRET efficiency was found to increase with time, which indicated the process of streptavidin-conjugated quantum dots capturing Cy5-ssDNA-Biotins. It was also observed that streptavidin-conjugated quantum dots and Cy5-ssDNA-Biotins could both enter living Chinese hamster ovary cells and have FRET. The process of streptavidin-conjugated quantum dots capturing Cy5-ssDNA-Biotins was detected in the cells as well and Cy5s were photobleached after a long time of irradiation. It has been proved that detecting DNA or RNA segments in living cells with DNA nanosensor is possible.

[Study of the relationship between apoptosis and intracellular pH in single living cells using a three-channel real-time fluorescence imaging method].

Using a Dual-View wavelength splitter, a custom-made filter block and only a single intensified charge-coupled device (ICCD), a three-channel real-time fluorescence imaging method for single living cell research was established based on an ICCD fast fluorescence micro-imaging system. The relevant calibrating method for images was also developed to eliminate the influence introduced by spectral crosstalk. Double-labeled with two fluorescent indicators, Annexin V-FITC and SNARF-1, mouse thymocyte was treated with S-nitrosoglutathione (GSNO) and the relationship between their apoptosis and intracellular pH changes was studied in real-time at the single living cell level. The results not only showed the different regularities of intracellular pH changes between apoptosis induced by GSNO and spontaneous apoptosis, but also provided evidence for the application of the three-channel real-time fluorescence imaging method to bio-medical studies.

[ICCD-based fast fluorescence micro-imaging technique and preliminary application in living cells].

A fast fluorescence micro-imaging system using mainly intensified charge couple device (ICCD), argon-ion laser, and xenon lamp was set up, and its preliminary application in living cells was presented. Real-time observation and imaging of fast concentration and distribution changing of intracellular Ca2+ labeled by Fluo-3, a fluorescent Ca2+ indicator, in the proliferation process of rat cerebral micro-vessels endothelial cells (rCMECs) were carried out, and curves of artificial intensity versus imaging sequence of four typical points were obtained. It is shown that the ICCD-based fast fluorescence micro-imaging system is a powerful tool for recording the real-time fast processes in living cells.

[Application of two-photon excitation fluorescence imaging to real-time investigation of mouse preimplantation embryo].

The blue shift of a two-photon excitation absorption peak allows the application of a single wavelength to the simultaneous excitation of several fluorochromes with disparate emission characteristics. An output at 730 nm of a mode-locked femtosecond Ti-sapphire laser was used to excite four different commonly used fluorochromes, namely Hoechst 33342, Fluo-4, PI and Indo-1, and characteristic fluorescence images were obtained using (455 +/- 15) nm, (540 +/- 15)nm, (580 +/- 16) nm and (500 +/- 15) nm filters respectively. An approach to exciting with a single wavelength, staining with two different fluorochromes, and collecting fluorescence in two separate channels was employed to study mouse preimplantation embryos by 3D and 4D real-time imaging. Combined with the merits of two-photon excitation fluorescence imaging, such as better penetration, less photon-damage, and higher signal-to-noise ratio, this approach was supposed to be a novel multi-parameter investigating tool for mouse preimplantation embryos development study.

Real-time imaging of viable-apoptotic switch in GSNO-induced mouse thymocyte apoptosis.

Many scientists are focusing on the apoptotic-necrotic or the necrotic-apoptotic switch and its mechanism, but little attention has been paid to the viable-apoptotic switch. Most of the techniques and methods used for detecting apoptosis are performed on fixed samples, yielding static information of specific time points. We have studied the viable-apoptotic switch in S-nitrosoglutathione (GSNO)-induced mouse thymocyte apoptosis in real-time by means of a novel technique, intensified charge coupled device (ICCD)-based real-time fluorescence micro-imaging, coupled with Annexin V-FITC labeling for phosphatidylserine (PS) translocation in cell membrane. We have successfully recorded the initiating time points (mostly at 2 h) of the viable-apoptotic switch in GSNO-initiated apoptosis, as well as shown the real-time differences between living and apoptotic thymocytes. These findings suggest that NO is also a switch molecule for the conversion from viable to apoptotic cell. Thymocytes cotreated by N-G-monomethyl-L-arginine acetate salt (L-NMMA) provide further evidence for this suggestion, as well as for the suggestion that L-NMMA prolongs the early stage of thymocyte apoptosis rather than strongly blocks it.

[Study on multi-photon excited fluorescence combined with capillary electrophoresis].

This paper describes a new method, multiphoton excitation fluorescence detection combined with capillary electrophoresis separation. The excitation source was a self mode-locked femtosecond titanium-sapphire laser (Spectra-physics Inc.), producing a stream of pulses with a pulse duration of about 100 fs at 82 MHz repetition rate. Its average power was about 200 mW at 750 nm. The laser beam was focused into a thin wall flow cell of capillary electrophoresis. A high numerical aperture objective (100 x NA 1.25) was chosen to focus the laser beam and to collect the fluorescence emitted by detecting molecules. Then the fluorescence was detected by a fast response PMT. All data were acquired and processed by a microcomputer. For three biological molecules, 5HT, FAD and NADH, it was demonstrated that they can be separated and detected efficiently by this method with three-photon and two-photon excitation respectively using only one 750 nm laser beam. The detection limits were 1.0 x 10(-6) mol x L(-1) for 5HT, 7.4 x 10(-7) mol x L(-1) for FAD and 9.8 x 10(-7) mol x L(-1) for NADH using the criterion of three standard deviations above background. The results are much better than those with UV absorption detection by one or two magnitudes.

[Measurement of methane 13C/12C isotope ratio of slurry gas from oil wells by laser frequency-modulation absorption spectroscopy].

In this paper, we determined the ratio of C13/C12 of methane in slurry gas from oil wells by the method of laser frequency modulation absorption spectroscopy that has many advantages such as high precision, high spectroscopic resolution and rapidly and simply dealing with samples. This technique provided us with a subsidiary method for oil-gas source exploration. The method of laser frequency modulation absorption spectroscopy is based on the method of laser frequency absorption spectroscopy and is more accurate. And this method can be expanded to apply to others aspects easily. We only need change other appropriate laser and select right absorption peak, then we can determined the ration and concentration of diverse gases.