collection of germinal vesicle oocytes from the ovaries of cd-1 mice and induction of dna double-strand breaks

Identificación del daño en el ADN en ovocitos de ratones con profase detenida

The process of meiosis in mammalian female germ cells is initiated in the ovaries before birth. The total number of oocytes is established in the ovaries primarily during embryogenesis. Oocytes enter meiosis and remain arrested at prophase I1. After the onset of puberty and the production and endocrine action of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), oocytes may reinitiate and complete meiosis2. In humans, prophase arrest can last for up to 50 years3. The cell divisions following the entry into meiosis I are asymmetric, resulting in the production of a small polar body and an oocyte that retains its size. Thus, most cytoplasmic components are stored in the ooplasm during early embryogenesis4. Then, the oocytes enter meiosis II, without reforming their nucleus or decondensing their chromosomes, and remain arrested at metaphase II until fertilisation5.
A unique characteristic that distinguishes oocytes from somatic cells is the state of arrest in prophase I, when the oocyte possesses an intact nucleus (germinal vesicle [GV] arrest), referred to as the GV stage6. Based on the chromatin organization, GV-stage oocytes are classified into two categories: non-surrounded nucleolus (NSN) and surrounded nucleolus (SN)7,8. In NSN GV-stage oocytes, the chromatin spreads throughout the whole nuclear region, and transcription is active, while in SN oocytes, the chromatin forms a compact ring that surrounds the nucleolus, and transcription is silent9. Both types of GV-stage oocytes show meiotic competence; they enter meiosis at the same rate, but the NSN oocytes present low developmental capacity and cannot develop beyond the two-cell stage embryo10.
The protracted state of prophase I arrest increases the incidence of DNA damage accumulation11. Therefore, DNA damage response mechanisms in oocytes are essential for allowing the production of gametes with genetic integrity and for ensuring that the resulting embryo has a physiological chromosomal content.
A central aspect of the DNA damage response is DNA repair. The main pathways for DSB repair in eukaryotic cells include non-homologous end joining (NHEJ), homologous recombination (HR), and alternative NHEJ12,13,14,15. NHEJ is a faster but more error-prone mechanism, while HR requires more time to be completed but has high fidelity16.
There is not enough knowledge about the mechanisms that oocytes use for DNA damage repair. Studies have shown that DNA damage induced in fully grown mammalian oocytes by the use of genotoxic agents, such as etoposide, doxorubicin, or UVB or ionizing radiation, does not affect the timing and rates of exit from prophase I arrest17. Oocytes can undergo GV breakdown (GVBD) even in the presence of elevated levels of damage. This damage can be determined by the observation of &#947;H2AX. This phosphorylated form of H2AX (&#947;&#919;2&#913;&#935;) is a DSB marker, which is located at the site of breaks and functions as a scaffold to help repair factors and proteins to accumulate at broken ends18.
The absence of cell cycle arrest following DNA damage is due to an insufficient DNA damage checkpoint that allows oocytes with unrepaired DNA to re-enter meiosis. Following high levels of DNA damage, a checkpoint can maintain prophase arrest through the activation of an ATM/ Chk1-dependent pathway. The limited checkpoint response to DSBs is due to the limited activation of ATM17,19. In the M-phase of meiosis I, research has shown that DNA damage may activate a spindle assembly checkpoint (SAC)-induced meiosis I checkpoint, which prevents the activation of the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) and, therefore, M-phase exit. Moreover, the ablation of SAC proteins overcomes the state of M-phase arrest, thus underlining the importance of the SAC in the establishment of the meiosis I chekpoint20.
As previous research clearly shows, DSBs cannot induce a robust prophase checkpoint in mouse oocytes. If such damage is left unrepaired, it could lead to embryos carrying chromosomal abnormalities. Therefore, it is important to study the DNA damage response at different stages of female gametogenesis to better understand the unique pathways that oocytes use to cope with potential genetic insults.

Autor destacado: Comprensión de la respuesta al daño del ADN en ovocitos de mamíferos y embriones preimplantacionales 

Detección de roturas de ADN bicatenario en ovocitos de ratón

Our research aims to understand how the cell cycle is regulated in mammalian oocytes and in preimplantation embryos. A key aspect of our research involves examination of repair capacity and DNA damage response in different maturation states is in oocytes and also in preimplantation embryos. We are particularly interested in understanding how the checkpoints respond and which repair mechanisms establish after potential DNA damage.Our lab was the first to mechanistically examine the DNA damage response in mammalian oocytes. We have shown the absence of the G2 prophase DNA damage checkpoint in mammalian oocytes. And we observed the importance of the spindle assembly checkpoint in establishing metaphase arrest after damage.By using this protocol, it is possible to detect DNA double strand breaks with accuracy and to determine whether oocytes have DNA damage and whether DNA damage is being repaired. This protocol involves the use of immunofluorescence which allows us to identify the exact sites of damage to DNA.

Watch this Scientific Journal Video about Collection of Germinal Vesicle Oocytes from the Ovaries of CD-1 Mice and Induction of DNA Double-Strand Breaks at JoVE.com

Collection of Germinal Vesicle Oocytes from the Ovaries of CD-1 Mice and Induction of DNA Double-Strand Breaks  

Recolección de ovocitos de vesículas germinales de los ovarios de ratones CD-1 e inducción de roturas de doble cadena de ADN

Para comenzar, prepare gotas de M2 IBMX Medium en una placa plástica de cultivo de tejidos y coloque la placa en un bloque caliente a 37 grados centígrados durante al menos 30 minutos antes del aislamiento de ovocitos. Diseccionar los ovarios de los ratones sacrificados y colocarlos en un tubo inferior redondo de cinco mililitros con M2 IBMX. Transfiera los ovarios a una tapa de plástico que contenga 1.5 mililitros de M2 IBMX.Extirpar cualquier tejido adiposo periovárico o segmentos de trompas de Falopio, y liberar los complejos de ovocitos del cúmulo mediante la perforación mecánica de los ovarios con una aguja de calibre 27. Transferir los complejos ovocitarios cúmulos a una placa de cultivo con gotas de M2 IBMX. Y elimine las células del cúmulo mediante pipeteo repetido con una pipeta de vidrio de orificio estrecho.Seleccionar el núcleo rodeado de la vesícula germinal o los ovocitos en estadio GV, y transferirlos en una gota de medio M2 IBMX sobre bloque caliente a 37 grados centígrados protegidos de la luz. Después de inducir roturas de doble cadena con etopósido, coloque los ovocitos en estadio GV en gotas del agente genotóxico durante una hora en el bloque caliente en la oscuridad. Añadir gotas de medio de cultivo M16 suplementado con IBMX de 400 micromolares a los ovocitos para mantenerlos detenidos durante un periodo prolongado.Colocar los ovocitos en una incubadora a 37 grados centígrados y 5% de dióxido de carbono.

collection-germinal-vesicle-oocytes-from-ovaries-cd-1-mice-induction

Research

JoVE Journal

Developmental Biology

Collection of Germinal Vesicle Oocytes from the Ovaries of CD-1 Mice and Induction of DNA Double-Strand Breaks

546 vistas.  University of Ioannina. Para comenzar, prepare gotas de M2 IBMX Medium en una placa plástica de cultivo de tejidos y coloque la placa en un bloque caliente a 37 grados centígrados durante al menos 30 minutos antes del aislamiento de ovocitos. Diseccionar los ovarios de los ratones sacrificados y colocarlos en un tubo inferior redondo de cinco mililitros con M2 IBMX. Transfiera los ovarios a una tapa de plástico que contenga 1.5 mililitros de M2 IBMX.Extirpar cualquier tejido adiposo periovárico o segmento...

Video: Collection of Germinal Vesicle Oocytes from the Ovaries of CD-1 Mice and Induction of DNA Double-Strand Breaks  

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis. This protracted state of arrest makes them extremely susceptible to accumulating DNA-damaging insults, which affect the genetic integrity of the female gametes and, therefore, the genetic integrity of the future embryo. 
Consequently, the development of an accurate method to detect DNA damage, which is the first step for the establishment of DNA damage response mechanisms, is of vital importance. This paper describes a common protocol to test the presence and progress of DNA damage in prophase-arrested oocytes during a period of 20 h. Specifically, we dissect mouse ovaries, retrieve the cumulus-oocyte complexes (COCs), remove the cumulus cells from the COCs, and culture the oocytes in &#924;2 medium containing 3-isobutyl-1-methylxanthine to maintain the state of arrest. Thereafter, the oocytes are treated with the cytotoxic, antineoplasmic drug, etoposide, to engender double-strand breaks (DSBs). 
By using immunofluorescence and confocal microscopy, we detect and quantify the levels of the core protein &#947;H2AX, which is the phosphorylated form of the histone H2AX. H2AX becomes phosphorylated at the sites of DSBs after DNA damage. The inability to restore DNA integrity following DNA damage in oocytes can lead to infertility, birth defects, and increased rates of spontaneous abortions. Therefore, the understanding of DNA damage response mechanisms and, at the same time, the establishment of an intact method for studying these mechanisms are essential for reproductive biology research.

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain ...

Investigación

Biología del Desarrollo

To begin, prepare drops of M2 IBMX Medium in a plastic tissue culture dish, and place the dish on a hot block at 37 degrees Celsius for at least 30 minutes before oocyte isolation. Dissect the ovaries of the euthanized mice and place them in a five milliliter round bottom tube with M2 IBMX. Transfer the ovaries to a plastic lid containing 1.5 milliliters of M2 IBMX.Remove any peri-ovarian adipose tissue, or fallopian tube segments, and release the cumulus oocyte complexes by mechanical perforation of the ovaries with a 27-gauge needle. Transfer the cumulus oocyte complexes to a culture dish with drops of M2 IBMX. And remove the cumulus cells by repeated pipetting using a narrow bore glass pasture pipette.Select the surrounded nucleus germinal vesicle or GV stage oocytes, and transfer them in a drop of M2 IBMX medium on hot block at 37 degrees Celsius protected from light. After inducing double strand breaks using etoposide, place the GV stage oocytes in drops of the genotoxic agent for one hour on the hot block in the dark. Add drops of M16 culture medium supplemented with 400 micromolar IBMX to the oocytes to keep them arrested for a prolonged period.Place the oocytes in an incubator at 37 degrees Celsius and 5%carbon dioxide.

Fixation and Confocal Imaging of Germinal Vesicle-Oocyte Collected from CD-1 Mice

Place the control and etoposide-treated germinal vesicle, or GV, oocytes in different plastic tissue culture dishes with PFA-Tx 100 buffer for 40 minutes at room temperature. Rinse the oocytes in three distinct 50-microliter washing buffers at room temperature and place them in 25 microliters of blocking buffer on a hot block for one hour. Next, prepare the primary antibody that recognizes gamma H2AX.Dilute the primary antibody with the blocking buffer at a one to 200 ratio and immerse the oocytes in 15-microliter drops at four degrees Celsius overnight. Wash the oocytes in three different 50-microliter washing buffers. Prepare the Alexa Fluor 488 conjugated growth anti-rabbit secondary antibody.After diluting it with the blocking buffer, immerse the oocytes in 15-microliter drops of the antibody for one hour on a 37 degrees Celsius hot block protected from light. Take a far-red fluorescent DNA dye DRAQ7 and transfer the oocytes to it for 10 minutes at room temperature in the dark before washing them with three different washing buffers. Add small drops of washing buffer to the oocytes in a 35-millimeter glass-bottom Petri dish for confocal microscopy.Switch on the laser controller and the lasers in the confocal system. After turning on the microscope controller and the lamps, open the confocal software and choose the 40X oil lens. Place the dish with the oocytes into the specimen holder and try to focus on the oocytes by moving the stage on X, Y, and Z axes using the joystick.Set the laser power, the gain, and the pinhole size independently for each experiment to minimize any saturation. For each oocyte, set the area of interest specifically in the nucleus at the DNA area. Define the borders of the DNA area and adjust the Z-step size to three micrometers.Then start the scanning. Save the images for each cell in the selected folder. Open the ImageJ software and click on image, followed by color, and split channels.To split all the channels. Click on lookup table and choose the preferred colors for each channel Click on image, color, and merge channels to merge the channels for gamma H2AX and DNA. In the surrounded nucleolus oocytes with high levels of DNA damage, click on image, stacks, Z-project, and with the freehand selections command, select the entire DNA area.Click on analyze, followed by measure to measure the gamma H2AX fluorescence and copy the measurements into an Excel file. The gamma H2AX fluorescence in the surrounded nucleolus GV stage oocytes zero hours after the etoposide treatment is shown in this figure. The gamma H2AX increases immediately after the exposure at all the etoposide concentrations, and the increase is concentration-dependent.The gamma H2AX fluorescence in the surrounded nucleolus GV stage oocytes 20 hours after the etoposide treatment is shown here. Gamma H2AX reduces 20 hours after the exposure at all the etoposide concentrations. After protracted prophase arrest, the GV stage oocytes showed the capacity to reduce the gamma H2AX foci number and intensity, implying the presence of active repair processes in the GV stage arrested oocytes.