This work was supported by NIGMS (R01GM11755) to P.W.J and by a training grant fellowship from the National Cancer Institute (NIH) (CA009110) to G.H. and J.H.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Johns Hopkins University. Experiments were performed on wild-type C57BL/6J mice.
1. Harvesting Fetal or Neonatal Ovaries and Preparation of Prophase Chromatin Spreads
<ol>
	<li>To extract embryos at 14.5&#8211;19.5 days post-coitum (dpc) sacrifice the pregnant females via cervical dislocation or CO2 asphyxiation according to IACUC guidelines. 
	NOTE: For postnatal day 1&#8211;5 ovaries skip to step 1.3. Figure 1 summarizes the meiotic prophase stages enriched at different embryonic and post-natal ages.</li>
	<li>Open the abdominopelvic cavity using sterile scissors, making a V-shaped opening. Dissect out the maternal uterine horn, separate the embryos from the placenta, and transfer embryos into 35 mm petri dishes containing 3 mL of pre-warmed 1x phosphate buffer saline (PBS) at 37 &#176;C. 
	NOTE: A fliptop incubator set at 37 &#176;C can be used to maintain temperature. In addition, a temperature regulated glass stage can also be used to maintain temperature during ovary manipulation.</li>
	<li>With 3.5 inch surgical scissors, sacrifice embryos or pups via decapitation according to IACUC guidelines. Place decapitated embryos or pups in pre-warmed PBS prior to further dissection.</li>
	<li>Dissect one embryo or pup at a time in a separate 35 mm petri dish containing 3 mL of pre-warmed PBS at 37 &#176;C. Proceed by cutting along the ventral midline of the posterior half of the embryo, along the anterior half below the forelimbs and directly above the hind-limbs and tail as outlined in Figure 2A&#8211;B.</li>
	<li>Open the abdomen using 3.5 inch surgical scissors. Using fine-tipped forceps displace or remove the liver and loops of bowel, exposing the ovaries in a new 35 mm petri dish containing 3 mL of pre-warmed PBS at 37 &#176;C (see guide in Figure 2B). The ovaries are located immediately below and behind the kidney towards the posterior wall of the peritoneal cavity (Figure 2B&#8211;C). A guide for differentiating between male and female gonads is provided in Figure 2D. 
	NOTE: For optimal conditions, rapidly dissect out the ovaries after pregnant female is sacrificed.</li>
	<li>Remove both ovaries from each female fetus using a pair of fine-tipped forceps under a dissection scope and place in a watch glasses or separate wells of a multi-well plate containing 0.5 to 1.0 mL of pre-warmed PBS and maintain at 37 &#176;C.</li>
	<li>Place each pair of ovaries in 0.5 mL hypo-extraction buffer (17 mM trisodium citrate dihydrate, 50 mM sucrose, 5 mM EDTA, 0.5 mM DTT, 30 mM Tris-HCl, protease inhibitor, pH 8.2) in a clean watch glass or a small well of a 9-well plate, making sure to immerse the ovaries completely. Incubate for at least 15 min, but no more than 30 min. 
	NOTE: Make hypo-extraction buffer fresh and use within 2 h of DTT addition.</li>
	<li>During incubation, using a hydrophobic barrier PAP pen, draw two 22 x 22 mm2 squares on a clean glass 25 x 75 mm2, 1 mm thick microscope slide as shown in Figure 3A. 
	NOTE: Slides can be prepared ahead of time.</li>
	<li>Pipette 45&#8211;50 &#181;L of 100 mM sucrose onto a clean slide and transfer one pair of ovaries to the drop.</li>
	<li>Using the sharp ends of two 27 G needles, tease the ovaries apart to release cells into the sucrose solution. With forceps, remove large pieces of ovary and carefully pipette sucrose solution to disperse cells.</li>
	<li>Place 40 &#181;L of fixative solution (1% paraformaldehyde (PFA), 0.2% detergent (see Table of Materials), pH 9.2) into each square of the prepared slide. With a 200 &#181;l pipette tip, spread the fixative solution evenly over the slide surface.</li>
	<li>Pipette 20 &#181;L of the sucrose mix onto the each square of the slide containing the fixative. 
	NOTE: This equates to using one pair of ovaries per slide.</li>
	<li>Incubate the slides in a closed humid chamber overnight at room temperature. 
	NOTE: The overnight incubation can range around (12-15 hrs) at room temperature.</li>
	<li>Open the chamber lid and allow the slides to air-dry completely.</li>
	<li>Wash slides in a Coplin jar containing 50 mL of 0.4% wetting agent-PBS solution (see Table of Materials) for 2 min, air dry, and proceed to immunolabelling protocol (step 6). 
	NOTE: It may be possible to store the slides at -80 &#176;C for later use, but this varies depending on the protein of interest. For optimal results proceed immediately to the immunolabelling protocol (step 6).</li>
</ol>
2. Metaphase I Oocyte Collection
<ol>
	<li>To maximize the number of antral follicles isolated from each mouse, intraperitoneally inject sexually mature virgin female mice with 5 IU of pregnant mare&#39;s serum gonadotropin (PMSG, also known as equine chorionic gonadotropin (eCG)). 
	NOTE: For optimal oocyte yield, mice should be 1 to 3 months of age, as the number of oocytes harvested will decrease with age. To prepare PMSG, dissolve bottle of 5,000 IU in 100 mL of sterile PBS (5 U/0.1 mL) Store in single-use aliquots of 600 &#181;L at -20 &#176;C.</li>
	<li>After 44&#8211;48 h, prepare collection medium, minimum essential medium alpha (MEM&#945;) medium supplemented with 5% fetal bovine serum (FBS) and 3 mg/mL bovine serum albumin (BSA; MEM&#945;/BSA/FBS). Sterilize media through a 0.2 &#181;m pore filter. Decant 2.5 mL of MEM&#945;/BSA/FBS medium into one glass dish per mouse and warm to 37 &#176;C in a 5% CO2 incubator. 
	NOTE: Other collection and culture media that are commercially available (M2 and M16) are also commonly used. 
	Caution: We recommend removing culture dishes from the 5% CO2 incubator one at a time for limited duration to minimize ambient air exposure during manipulation steps.</li>
	<li>Sacrifice the mice via cervical dislocation or CO2 asphyxiation according to IACUC guidelines.</li>
	<li>Open the abdominopelvic cavity using sterile scissors, making a large V-shaped opening. Using forceps, displace intestines towards the head of the mouse. Locate each uterine horn; the ovaries are present proximal to the rib cage. Hold the oviduct with fine forceps and cut the fat superior to the ovary using dissection scissors (Figure 4). Continue to hold the oviduct, and with another set of fine forceps release the ovary from the bursa and place into collection dish containing MEM&#945;/BSA/FBS medium. 
	NOTE: As in step 1, it is imperative to keep the ovaries at 37 &#176;C and maintain at 5% CO2. A fliptop incubator hooked up to 5% CO2 mix can be used to allow for optimal maintenance of temperature and CO2 levels. In addition, a temperature regulated glass stage should also be used to maintain temperature during oocyte manipulation.</li>
	<li>Using a 1 mL syringe with a 27-gauge needle (or similar size), release cumulus oocyte complexes by manually puncturing the large antral follicles.</li>
	<li>While observing through a dissection microscope, collect GV-staged oocytes using a mouth-operated glass pipette or capillary, or a hand-operated micrometer-syringe (Figure 5). Only collect oocytes that are released from antral follicles. GV oocytes have a diameter of approximately 90 &#181;m. GV oocytes may be surrounded by 2-3 layers of granulosa cells, and have a total diameter of around 200 &#181;m.</li>
	<li>Culture oocytes in a clean watch glass or a 9-well plate containing MEM&#945;/BSA/FBS medium for 6 h to reach metaphase I at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Proceed to step 4.</li>
</ol>
3. Metaphase II (MII) Oocyte Collection
<ol>
	<li>To maximize oocytes isolated from each mouse, intraperitoneally inject sexually mature virgin female mice with 5 IU of PMSG. After 44&#8211;48 h, intraperitoneally inject with 5 IU of human chorionic gonadotropin (hCG). 
	NOTE: For optimal oocyte yield, mice should be 1 to 3 months of age, as the number of oocytes harvested will decrease with age. To prepare hCG, dissolve a bottle of 10,000 U in 200 mL PBS (5 U/0.1 mL). Store in single-use aliquots of 600 &#181;L at -20 &#176;C.</li>
	<li>After 12-14 hrs, prepare MEM&#945;/BSA/FBS collection medium, as described in step 2.2, above.</li>
	<li>Sacrifice the mice via cervical dislocation or CO2 asphyxiation according to IACUC guidelines.</li>
	<li>Open the abdominopelvic cavity using sterile scissors, making a large V-shaped opening. Using forceps, displace intestines towards the head of the mouse. Locate each uterine horn, the ovaries are present proximal to the rib cage. Hold the oviduct with fine forceps and cut the fat superior to the ovary using dissection scissors (Figure 4). Then remove the ovary and oviduct and place into collection dish containing MEM&#945;/BSA/FBS medium. 
	NOTE: As in steps 1 and 2, it is imperative to keep the ovaries at 37 &#176;C and maintain at 5% CO2. A fliptop incubator hooked up to 5% CO2 mix can be used to allow for optimal maintenance of temperature and CO2 levels. In addition, a temperature regulated glass stage should also be used to maintain temperature during oocyte manipulation.</li>
	<li>Using a 1 mL syringe with a 27 G (or similar size) or sharp forceps, tear a hole in the ampulla of the oviduct to release the MII oocytes. 
	NOTE: Be careful not to damage the oocytes in the ampulla. The ampulla will appear swollen and translucent such that the oocytes are visible (Figure 4).</li>
	<li>Harvest MII oocytes from the ampulla of the oviduct into a new dish with collection medium using a mouth-operated glass pipette or capillary, or a hand-operated micrometer-syringe (Figure 5).</li>
	<li>Proceed to step 4.</li>
</ol>
4. Oocyte Denuding and Zona Pellucida Removal
<ol>
	<li>Prepare 300 IU/mL of hyaluronidase in MEM&#945; medium supplemented with 3 mg/mL BSA to denude oocytes of surrounding cumulus cells (2.5 mL/mouse) in a watch glass, and keep at 37 &#176;C, 5% CO2. 
	NOTE: Hyaluronidase efficacy sharply decreases after 1 h of preparation. For MII oocytes, hyaluronidase treatment is not required.</li>
	<li>Expose oocyte-cumulus cell complexes to 300 IU/mL of hyaluronidase in MEM&#945;/BSA for 3 min to denude oocytes of surrounding cumulus cells. 
	Caution: Do not exceed 3 min exposure to hyaluronidase, as it will damage the oocytes.</li>
	<li>To wash the oocytes, transfer oocytes to a fresh warmed MEM&#945;/BSA medium dish. Using a small mouth-operated glass pipette or capillary (slightly larger than the diameter of the oocyte, which is approximately 90 &#181;m), pipette the complexes up and down to completely detach the cumulus cells. Allow the oocytes to recover in the incubator while preparing for the next step.</li>
	<li>Warm acidic Tyrode&#39;s solution, MEM&#945;/BSA, and Waymouth&#39;s media to 37 &#176;C (500 &#181;L/mouse). Place 300 &#181;L in each well of small 9-well glass plates.</li>
	<li>To remove the zona pellucida (ZP) transfer 5&#8211;10 oocytes into Tyrode&#39;s solution. Expose the oocytes for only 30-45 s to the solution. 
	NOTE: Too little exposure to the solution will not remove the ZP completely, which will prevent the oocytes from bursting and chromosomes from spreading. Too much exposure will damage and kill the oocyte. Watching the ZP dissolve under the dissection microscope can aid in optimizing exposure time. 
	Caution: Using fresh Tyrode&#39;s solution is critical, as its function declines with age. Use a fresh well of Tyrode&#39;s solution for each group of oocytes treated to ensure uniform digestion of the ZP.</li>
	<li>Immediately after ZP removal, wash the oocytes by transferring into warmed MEM&#945;/BSA/FBS medium. Repeat this wash step. 
	NOTE: Be careful when transferring, as oocytes will easily stick together and to the glass pipette or capillary following removal of the ZP. Pre-wetting the glass pipette or capillary in Waymouth&#39;s medium will minimize oocytes sticking together.</li>
	<li>Transfer and allow the oocytes to recover for 30 minutes in Waymouth&#39;s medium at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Proceed to step 5.</li>
</ol>
5. MI and MII Oocyte Chromatin Spreads
<ol>
	<li>Using a PAP pen, draw a 11 x 44 mm2 rectangle on the glass slide as shown in Figure 6A.</li>
	<li>Coat the slide with a thin layer of fixative (1% paraformaldehyde, 0.2% Triton-X 100 in H2O, pH 9.2) by pipetting 100 &#181;L of fixative onto the slide and rocking the slide back and forth. Tap the slide to get rid of excess fixative.</li>
	<li>Pick up between 5-10 oocytes with a small mouth pipette needle with as little media as possible and drag the needle in a line across the fixative-coated slide while depositing the oocytes from 1 cm above the slide into the fixative solution. Perform this step using a dissection microscope to ensure that the oocytes have been deposited and that they have burst. 
	NOTE: The oocytes should burst immediately on the slide. The height at which the oocytes are deposited impact the degree of chromatin spreading. See representative results in Figure 6 for further details.</li>
	<li>Incubate slides at room temperature in a closed humidified chamber overnight to allow the chromatin to adhere.</li>
	<li>Allow the slides to air-dry completely and rinse slides in a Coplin jar containing 50 mL of 0.4% wetting agent-H2O, pH 8.0.</li>
	<li>Proceed to step 6.</li>
</ol>
6. Immunolabelling
<ol>
	<li>Prepare the antibody dilution buffer (ADB) described in Table 1.</li>
	<li>Prepare two Coplin jars containing 50 mL of wash buffer (WB) solution (10% ADB diluted in PBS), and one Coplin jar containing 50 mL WB and 0.05% detergent solution (e.g., add 250 &#181;L of 10% detergent to 50 mL WB).</li>
	<li>Wash the slides that were prepared in section 1 and 5 of this protocol for 10 min in one Coplin jar containing 50 mL of WB. 
	Caution: Do not let the slides dry at any point during immunolabelling.</li>
	<li>Wash the slides for 10 min in the Coplin jar containing 50 mL WB and 0.05% detergent solution. Then, wash the slides in the remaining Coplin jar containing 50 mL of WB solution for 10 min.</li>
	<li>Tap off excess liquid and cover slide with 100 &#181;L of selected primary antibodies diluted in ADB. Incubate at 4 &#176;C overnight in a closed humid chamber. 
	NOTE: Incubation can be shortened to 2 to 3 hrs at 37 &#176;C. Use smaller volumes of antibody (e.g., 50 &#181;L) by covering the slide with a coverslip or parafilm.</li>
	<li>Rinse slides in a Coplin jar containing 50 mL of 0.2% wetting agent-PBS solution, pH 8.0.</li>
	<li>Repeat steps 6.2 to 6.5.</li>
	<li>Tap off excess liquid and cover slide with 100 &#181;L of selected secondary antibodies diluted in ADB. Incubate slides 1 to 2 hrs at 37 &#176;C in a closed humid chamber.</li>
	<li>Wash slides 2 times for 10 min in Coplin jars containing 50 mL of 0.2% wetting agent-PBS solution, pH 8.0.</li>
	<li>Wash slides 2 times for 5 min in Coplin jars containing 50 mL of 0.2% wetting agent-H2O solution, pH 8.0.</li>
</ol>
7. Mounting slides
<ol>
	<li>For prophase chromatin spreads prepared in step 2, add 100 &#181;L of mounting medium containing 4&#39;, 6-diamidino-2-phenylindole (DAPI, 1.5&#181;g/mL). For MI and MII chromatin spreads prepared in step 5, add 50 &#181;L of mounting medium containing DAPI (1.5&#181;g/mL). Gently blot away excess liquid.</li>
	<li>Place a 22 mm x 60 mm coverslip on top and seal with clear nail polish. Store in a slide box at 4 &#176;C or -20 &#176;C until assessment via fluorescence microscopy. 
	NOTE: Tap lightly on cover slip to rid of excess mounting medium before sealing with nail polish.</li>
</ol>

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The authors have nothing to disclose.

Chromatin spread preparations allow researchers to chronologically study female mammalian meiosis and the dynamic localization of proteins involved. The embryonic and neonatal chromatin spreads allow for close analysis of events throughout meiotic prophase. Metaphase I and metaphase II chromatin spreads can be used to distinguish single sister chromatids from paired sisters and paired homologous chromosomes, as well as assess ploidy. By comparison, the protocol described here can be advantageous compared to whole oocyte immunolabelling, which frequently produces high levels of background signal that decreases the resolution of chromatin-bound proteins. Furthermore, chromatin spread techniques represent an attractive alternative to live-cell imaging, which requires a large investment of time and specialized equipment.
Embryonic and neonatal ovaries can be difficult to locate and extract. We recommend carefully extracting all other organs and material besides the kidneys in a separate dish containing PBS before searching for the ovaries, and using a fresh dish of PBS for each embryo/pup (Figure 2B). If the embryo/pup is male, the testis will be distinguishable by the tubule formation as well as the location of the gonads (Figure 2D). As male embryos develop, the testes will descend towards the penis, becoming larger and oval-shaped.
The oocyte chromatin spread technique requires mastery of oocyte collection and manipulation using mouth-operated glass pipette or capillary. We recommend practicing controlling the mouth pipette before performing this protocol. Pulling the proper size glass pipette/capillary for collection and denuding oocytes will increase chances of success and efficiency by minimizing time oocytes are outside of the incubator as well as in the Tyrode&#39;s solution. Correct timing for ZP removal is extremely important for the success of this method. If the oocytes are incubated in Tyrode&#39;s solution for an insufficient amount of time, the ZP will not be completely removed. This prevents the oocytes from bursting efficiently on the slide, as can be seen in Figure 6E. However, if the oocytes are incubated in Tyrode&#39;s solution for too long, oocyte quality and downstream immunofluorescence staining will be severely compromised. We recommend watching the oocytes under a microscope to determine the exact time the ZP dissolves in Tyrode&#39;s solution. Adding only 5&#8211;10 oocytes at a time to Tyrode&#39;s solution will minimize excessive exposure. If the oocytes are released too high above the PFA-coated slide, the chromosomes can be spread too far apart (Figure 6F). Optimal results are found when oocytes are released 1 cm above the slide. Other factors that can hinder results include prolonged exposure to decreased CO2 levels in ambient air during oocyte manipulation steps. This causes pH fluctuations in the media that are detrimental to oocyte quality, and is apparent by progressive color change of the media from orange-red to pink. The buffering capacity of the media also decreases over time; therefore, we do not recommend using media that has been stored (4 &#176;C) for longer than 1 week. M2 medium, which does not require CO2 to be buffered, is commonly used as an alternative.
A limitation to chromatin spread preparations is the lack of visualization of the chromatin within the native context of the cell. Cellular material outside of the nucleus cannot be visualized and spindle formation cannot be assessed. Therefore, other methods can be used to assess oogenesis in addition to chromatin spreads, such as whole oocyte immunolabelling after monastrol treatment, which collapses the bi-polar spindle into a mono-polar spindle22. This allows sister chromatids to be assessed within the context of the cell. Collectively, the chromatin spread methods described here can easily be applied to assess chromatin morphology and chromosome ploidy throughout oogenesis in novel transgenic and mutant mouse models.

During spermatogenesis, large semi-synchronous waves of meiotic germ cells are rapidly and continuously replenished in the testis at the onset of puberty and throughout adulthood1. In contrast to males, meiosis in females is initiated solely during fetal development. Following birth, oocytes remain arrested in a prolonged dictyate stage of prophase I with an intact germinal vesicle (GV; nuclear envelope) until puberty. At the onset of puberty, a subset of oocytes are cyclically selected to undergo growth and maturation, marking the initiation of meiotic resumption. Meiotic resumption in fully-grown oocytes is manifested by the disappearance of the GV in a process known as germinal vesicle breakdown (GVBD). The oocyte then undergoes chromosome condensation and segregation, followed by polar body extrusion. Oocytes become arrested upon progression to MII and are stimulated to complete the second and final meiotic division only after fertilization.
Female fertility is highly dependent on the success of meiotic prophase I progression. Key to this is formation of a physical linkage between homologous chromosomes known as the chiasmata, which is mediated by repair of induced DNA double strand breaks (DSBs) via crossover recombination2. This process occurs within the context of a dynamic protein-rich scaffold known as the synaptonemal complex (SC) that forms between homologous chromosomes to facilitate their synapsis3. The SC is a zipper-like tripartite structure that consists of two parallel lateral elements connected by central region proteins that holds homologs together throughout ongoing DNA repair. Prior to synapsis, precursors of the lateral elements, called axial elements, form between sister chromatids. Synaptonemal complex proteins such as SYCP2 and SYCP3 form axial elements that colocalize to the sister-chromatid cohesion axes during early prophase. These later serve as binding sites for the transverse filament protein, SYCP1, which facilitates central element assembly and synapsis between aligned homologs4. In mouse oocytes, complete synapsis is indicated by the presence of 20 completely overlapping SYCP3 and SYCP1 stretches, which can be visualized by using chromatin spread preparations. Synapsis is completed upon entry into the pachytene substage, whereby mature crossovers that are destined to form chiasmata between homologs are decorated with mutL homolog (MLH1/3) dimers to promote their accurate processing5,6,7. The structural maintenance of chromosomes (SMC) complexes, including cohesin, condensin, and the SMC5/6 complex, are important for the regulation of chromosome dynamics and structure throughout meiosis8,9,10,11,12. Collectively, these events ensure proper bi-orientation of homologous chromosomes to opposing spindle poles following disassembly of the SC.
The meiotic cell cycle is a powerful model to examine the roles of various proteins in genome maintenance due to the programmed induction and subsequent repair of DNA DSBs. Furthermore, mammalian meiosis is also a relevant model for the study of epigenetic modifications and imprinting13. However, it is technically difficult to assess these events during female meiosis, which takes place in fetal and neonatal ovaries in mammals (Figure 1). Prophase I can be divided into 5 substages: leptonema, zygonema, pachynema, diplonema and dictyate. Herein, we describe how to isolate and distinguish between fetal and neonatal ovaries and testes (Figure 2). Adapted from previously described methods, this manuscript also outlines a protocol (step 1) with video demonstration for preparation of female meiotic prophase I chromatin spreads14,15,16,17. When coupled with immunolabelling, as described in steps 6-7, this protocol enables detailed microscopic analysis of prophase I events in oocytes.
Oogenesis is error-prone, and chromosome missegregation events during the first meiotic division represent the most common source of genetic disease in progeny18,19. In this manuscript (protocol step 2), we describe a protocol in which mature GV-staged oocytes are extracted from primed ovaries of adult female mice. Under supportive conditions, fully-grown oocytes undergo luteinizing hormone-independent resumption of meiosis following isolation and culture20. Following meiotic resumption, oocytes progress through meiosis I, then arrest at metaphase II. Oocytes remain arrested at metaphase II, unless fertilized. In steps 2&#8211;5, we adapt previously reported protocols with video demonstration to describe how to collect, culture and prepare MI and MII oocytes for chromatin spread preparations21. This chromatin spreading technique allows for clear immunolabelling of proteins associated with chromosomes. Furthermore, this protocol can also be used to distinguish between bivalent and univalent chromosomes, and can further resolve single sister chromatids to facilitate assessment of oocyte ploidy. Therefore, in addition to revealing localization patterns of meiotic proteins, this protocol can also serve as an invaluable tool for elucidating potential causes of chromosome missegregation during MI and MII.

<table><tbody><tr><td>10X Phosphate buffered saline (PBS) pH 7.4</td><td>Quality Biological</td><td>119-069-161</td><td></td></tr><tr><td>60 mm x 15 mm petri dishes</td><td>Denville</td><td>T1106</td><td></td></tr><tr><td>35mm x 12mm petri dishes</td><td>Denville</td><td>T1103</td><td></td></tr><tr><td>Fine forceps</td><td>VWR</td><td>300-050</td><td></td></tr><tr><td>Micro dissection scissors</td><td>Ted Pella</td><td>1340</td><td></td></tr><tr><td>Dissecting&amp;nbsp;scissors, sharp tip, 41/2&quot;</td><td>VWR</td><td>82027-578</td><td></td></tr><tr><td>Watch glass square 1 5/8</td><td>Carolina Biological Supply Company</td><td>742300</td><td>Autoclave before each use</td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S8501</td><td></td></tr><tr><td>Trisodium Citrate Dihydrate</td><td>Sigma</td><td>S1804</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E6758</td><td></td></tr><tr><td>Trizma Base</td><td>Sigma</td><td>T1503</td><td></td></tr><tr><td>1,4-Dithiothreitol (DTT)</td><td>Sigma</td><td>646563</td><td>Add to hypotonic buffer immediately before use</td></tr><tr><td>50x Protease Inhibitor</td><td>Roche</td><td>11873580001</td><td>Add to hypotonic buffer immediately before use</td></tr><tr><td>Turberculin syringe with needle (1cc, 27 G x 1/2 in)</td><td>Becton, Dickinson (BD)</td><td>309623</td><td></td></tr><tr><td>Gold seal ultra frost glass slides (25X75mm, 1mm thick)</td><td>Thermo</td><td>3063-002</td><td></td></tr><tr><td>Super PAP pen, large (liquid blocker)</td><td>Electron Microscopy Sciences (EMS)</td><td>71310</td><td></td></tr><tr><td>16% Paraformaldehyde aqueous</td><td>Electron Microscopy Sciences (EMS)</td><td>15710</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td>Detergent in manuscript</td></tr><tr><td>Immuno stain moisture chamber</td><td>Evergreen</td><td>240-9020-Z10</td><td></td></tr><tr><td>Coplin jars</td><td>Fisher</td><td>08-815</td><td></td></tr><tr><td>Photo-flo 200</td><td>Electron Microscopy Sciences (EMS)</td><td>74257</td><td>Wetting agent in manuscript</td></tr><tr><td>Pregnant mare serum gonadotropin (PMSG)</td><td>Sigma</td><td>G4877</td><td>Store aliquots at -20C</td></tr><tr><td>Human chorionic gonadotropin (HCG)</td><td>Sigma</td><td>C0434</td><td>Store aliquots at -20C</td></tr><tr><td>PYREX Spot Plates with nine concave cavities</td><td>Fisher</td><td>13-748B</td><td>Autoclave before each use</td></tr><tr><td>Glass capillary</td><td>Fisher</td><td>22260943</td><td>For making oocyte collection needles</td></tr><tr><td>Brand Parafilm M</td><td>Sigma</td><td>BR701501</td><td>For making oocyte collection needles</td></tr><tr><td>Latex rubber tubing (3.2 mm inner diameter x 6.4 mm outer diameter)</td><td>Fisher</td><td>14-178-5B</td><td>For making oocyte collection needles</td></tr><tr><td>Latex rubber tubing (6.4 mm inner diameter x 11.1 mm outer diameter)</td><td>Fisher</td><td>14-178-5D</td><td>For making oocyte collection needles</td></tr><tr><td>Flexible silicone rubber nosepiece, hard plastic mouthpiece and 15 inches of latex tubing</td><td>Sigma</td><td>A5177-5EA</td><td>For making oocyte collection needles</td></tr><tr><td>Mitutoyo micrometer head</td><td>Mituoyo</td><td>150-208</td><td>For making oocyte collection needles</td></tr><tr><td>Syringe 5 mL</td><td>BD Biosciences</td><td>309646</td><td>For making oocyte collection needles</td></tr><tr><td>Syringe filter 0.45 &amp;mu;m filter</td><td>Corning</td><td>431220</td><td>For making oocyte collection needles</td></tr><tr><td>Glass Pasteur Pipette 5 3/4&quot;</td><td>Fisher</td><td>13-678-6A</td><td>For making oocyte collection needles</td></tr><tr><td>83 x 0.5 mm pipette tip</td><td>Denville</td><td>P3080</td><td>For making oocyte collection needles</td></tr><tr><td>&amp;alpha;-MEM</td><td>Invitrogen</td><td>11415049</td><td></td></tr><tr><td>Waymouth&#039;s media</td><td>Life Technologies</td><td>11220035</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Fisher</td><td>A3160602</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma</td><td>A3311</td><td>For oocyte culture media</td></tr><tr><td>500ml filter units 0.22um</td><td>Denville</td><td>F5227</td><td></td></tr><tr><td>Hyaluronidase</td><td>Sigma</td><td>H3506</td><td></td></tr><tr><td>Tyrode&amp;rsquo;s solution</td><td>Sigma</td><td>T1788</td><td>Store aliquots at -20C</td></tr><tr><td>Horse serum</td><td>Sigma</td><td>H-1270</td><td>For ADB/wash buffer</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma</td><td>A1470</td><td>For ADB/wash buffer</td></tr><tr><td>VECTASHIELD antifade mounting medium with DAPI</td><td>Vector Labs</td><td>H-1200</td><td></td></tr><tr><td>Microscope cover slides (22mmx60mm)</td><td>Fisher</td><td>12-544-G</td><td></td></tr><tr><td>Clear nail polish</td><td>Amazon</td><td>N/A</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Microscopes&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>SteREO Discovery.V8</td><td>Zeiss</td><td>495015-0001-000</td><td></td></tr><tr><td>Observer Z1</td><td>Zeiss</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Primary Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mouse anti-MLH1</td><td>Life Technologies</td><td>MA5-15431</td><td>Dilution factor- 1:100</td></tr><tr><td>Rabbit anti-SYCP1</td><td>Life Technologies</td><td>PA1-16763</td><td>Dilution factor- 1:1000</td></tr><tr><td>Goat anti-SCP3</td><td>Santa Cruz</td><td>sc-20845</td><td>Dilution factor- 1:50</td></tr><tr><td>Human anti-centromere protein</td><td>Antibodies Incorporated</td><td>15-235</td><td>Dilution factor- 1:100</td></tr><tr><td>Rabbit anti- TOPOII</td><td>Abcam</td><td>ab109524</td><td>Dilution factor- 1:100</td></tr><tr><td>Mouse anti-Histone H4 (di methyl K20, tri methyl K20)</td><td>Abcam</td><td>ab78517</td><td>Dilution factor- 1:500</td></tr><tr><td>Rabbit anti-Rec8</td><td>Courtesy of Dr. Karen Schindler</td><td>N/A</td><td>Dilution factor- 1:10,000</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Secondary Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Donkey anti-goat IgG (H+L) Alexa Fluor 568 conjugate</td><td>Thermo-Fisher Scientific</td><td>A-11057</td><td>Dilution factor- 1:500</td></tr><tr><td>Donkey anti-mouse IgG (H+L) Alexa Fluor 488 conjugate</td><td>Thermo-Fisher Scientific</td><td>A-21202</td><td>Dilution factor- 1:500</td></tr><tr><td>Donkey anti-rabbit IgG (H+L) Alexa Fluor 647 conjugate</td><td>Thermo-Fisher Scientific</td><td>A-31573</td><td>Dilution factor- 1:500</td></tr><tr><td>Goat anti-mouse IgG (H+L) Alexa Fluor 568 conjugate</td><td>Thermo-Fisher Scientific</td><td>A-11004</td><td>Dilution factor- 1:500</td></tr><tr><td>Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td><td>Thermo-Fisher Scientific</td><td>A-11013</td><td>Dilution factor- 1:500</td></tr><tr><td>Goat anti-rabbit IgG (H+L) Alexa Fluor 568 conjugate</td><td>Thermo-Fisher Scientific</td><td>A-11011</td><td>Dilution factor- 1:500</td></tr></tbody></table>

chromatin spread preparations for the analysis of mouse oocyte progression from prophase to metaphase ii

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

We have described two techniques for visualizing and assessing meiotic chromosomes in oocytes. The first technique is catered toward assessing prophase progression in embryonic and neonatal ovaries. Prophase chromatin spread preparations are incredibly valuable for visualizing numerous dynamic processes during meiosis, including chromosome pairing, synapsis and desynapsis, homologous recombination, and epigenetic chromosome remodeling. Here, we have demonstrated the utility of this method for robust visualization and quantitative analysis of crossover formation in oocytes harvested from C57BL/6J embryos (Figure 3B and 3C). To enrich for cells in the pachytene substage, mouse embryos were retrieved at 18.5 dpc (Figure 1). Two major hallmarks of the pachytene substage of prophase I are the completion of synapsis between homologs, and the formation of at least one MLH1/3-positive crossover per homolog pair. Complete synapsis between homologs is manifested by the presence of 20 overlapping SYCP3 and SYCP1 stretches. To characterize crossover formation in wild-type oocytes we immunolabeled prophase chromatin spread preparations using antibodies that detect SYCP3, SYCP1 and MLH1, and stained DNA using DAPI. Figure 3B depicts a representative image of a pachytene stage oocyte, which is evidenced by the presence of 27 MLH1 foci distributed along 20 fully assembled SC structures. The average number of MLH1-positive crossovers detected in wild-type oocytes was 24 &#177;3 (Figure 3C, N = 15 oocytes). Figure 3D shows SMC6 enriched at the pericentromeric heterochromatin (PCH) region and along the chromosome arms, and MLH1 foci distributed along the SC at the pachytene stage. Figure 3E depicts a pachytene staged oocyte where the chromosome spread preparation was sub-optimal and individual chromosomes are indistinguishable, preventing accurate assessment of SYCP3, SMC6 and MLH1. Sub-optimal chromosome spreads can be observed when incubation of ovaries in hypo-extraction buffer is for too long or not for long enough.
The second technique described can be used to assess chromatin morphology in oocytes following meiotic resumption (Figure 6B-D). Protein localization, as well as ploidy, can easily be assessed, exposing potential causes of chromosome segregation errors. Figure 6B depicts a MI oocyte showing 20 chromosomes and clear centromere and pericentromeric heterochromatin staining. Figure 6C is a zoomed image where topoisomerase II&#945; (TOPOII) can be seen along the chromosome arms and the PCH. Figure 6D depicts a MII oocyte where paired sister chromatids can be distinguished by chromosome and centromere morphology. Common errors seen when attempting this technique include oocytes not bursting when released onto the PFA-coated slide, as well as chromosomes spreading too far apart (Figure 6E&#8211;F). If the ZP is not removed completely, the chromosomes will remain associated to the spindle, as shown in Figure 6E. If the oocytes are dropped too high from the PFA-coated slide, the chromosomes can be spread too far apart as depicted in Figure 6F.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56736/56736fig1.jpg" /> 
Figure 1: Meiotic prophase timeline during female embryonic and neonatal development. Blue contours indicate populations of specific prophase sub-stage germ cells (leptotene, zygotene, pachytene, and diplotene/dictyate stages) observed during embryonic and neonatal development. Increasing dark blue color indicates the timing at which the specific prophase sub-stage becomes more abundant. This figure was adapted from reference2. <a href="https://cloudflare.jove.com/files/ftp_upload/56736/56736fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56736/56736fig2.jpg" /> 
Figure 2: Ovary extraction from embryos and neonatal female pups. (A, B) The first cut (1) is made above the forelimbs to decapitate the embryo at the head/neck junction immediately upon retrieval from the maternal uterine horn. The second cut (2) is made along the ventral midline of the posterior half of the embryo, followed by incisions along the anterior half below the forelimbs (3). A final cut is made for removal the hind limbs and tail (4). (A) Frontal view schematic of dissection cuts for isolation of ovaries from female pups. (B) Side view schematic of dissection cuts for isolation of ovaries with relative positions of internal organs. Regions shown in light red include the liver and intestines, which are removed during dissection. The dorsal wall and associated organs are shown in light blue. This region includes the ovaries, which are attached to the inferior poles of the kidneys at the top of the uterine horn. (C) Frontal schematic view of dorsal wall of embryo following removal of the liver and intestines. (D) Schematic representation of morphological differences between male and female gonads at approximately 15&#8211;18 dpc. <a href="https://cloudflare.jove.com/files/ftp_upload/56736/56736fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56736/56736fig3.jpg" /> 
Figure 3: Representative prophase chromatin spread preparations. (A) Glass slide schematic for prophase chromatin spread preparations. Black square outlines represent liquid blocker pen outlines. (B, C) Representative images and quantification of crossover formation in wild-type pachytene stage oocytes using prophase chromatin spread preparation methods described in step 1. (B) Chromatin spreads were performed using ovaries fromC57BL/6J mouse embryos isolated at 18.5 dpc. Chromatin spreads were immunolabeled with antibodies against the SC lateral element protein SYCP3 (red), and the SC central element protein SYCP1 (magenta), MLH1 (green, crossover event marker), and DAPI (blue, DNA). Scale bar = 10 &#181;m. (C) Dot plot of MLH1 foci counts obtained from 15 pachytene stage chromatin spread preparations. Error bars represent standard deviation. (D, E) Comparison of optimal (D) and sub-optimal (E) prophase spread preparations, respectively. Chromatin spreads were immunolabeled with antibodies against SMC6 (red), MLH1 (green), and SYCP3 (blue). Scale bar = 10 &#181;m. <a href="https://cloudflare.jove.com/files/ftp_upload/56736/56736fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56736/56736fig4.jpg" /> 
Figure 4: Schematic diagram of adult ovary. This diagram depicts the anatomy of the adult mouse ovary, oviduct, ampulla and uterus. Mouse ovaries should be removed by careful incision of ligaments connecting the ovaries to the inferior poles of the kidneys, and the posterior wall of the abdomen. <a href="https://cloudflare.jove.com/files/ftp_upload/56736/56736fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56736/56736fig5.jpg" /> 
Figure 5: Images of mouth-operated glass pipette, glass capillary and hand-operated micrometer-syringe used for oocyte manipulation. (A) The glass pipette oocyte manipulator is composed of the following components in sequence: mouth piece, latex tubing (3.2 mm inner diameter (ID) x 6.4 mm outer diameter (OD)), 1 mL pipette tip, latex tubing (6.4 mm ID x 11.1 mm OD) and glass Pasteur pipette. The end of the glass Pasteur pipette has been heated over a flame and the end pulled to create a fine-tipped end (Figure 5A1). (B) The glass capillary oocyte manipulator is composed of the following components in sequence: mouth piece, 0.45 &#181;m filter (optional), latex tubing (3.2 mm ID x 6.4 mm OD), 1 mL pipette tip, latex tubing (6.4 mm ID x 11.1 mm OD) and 70 &#181;l glass capillary. The glass capillary tubes are heated over a flame at the center and pulled in opposite directions to create two capillaries with fine tipped ends (Figure 5B1). (C) The hand-operated micrometer-syringe is composed of the following components in sequence: Mitutoyo 150-208 micrometer head (middle size, 0-1&#34; range, 0.001&#34; graduation), 5 mL syringe, latex tubing (3.2 mm ID x 6.4 mm OD), 1 mL pipette tip, latex tubing (6.4 mm ID x 11.1 mm OD), 1 mL pipette tip and a 83 x 0.5 mm2 gel loading tip. The Mitutoyo 150-208 micrometer head is firmly inserted into the 5 mL syringe (Figure 5C1). To ensure the gel loading tip is fastened securely, the connecting 1 mL pipette tip is cut (Figure 5C2). <a href="https://cloudflare.jove.com/files/ftp_upload/56736/56736fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56736/56736fig6.jpg" /> 
Figure 6: Representative metaphase I and II oocyte chromatin spread preparations. (A) Slide schematic for metaphase I and II oocyte chromatin spread preparations. Oocytes (circles) released via mouth-operated glass pipette or capillary in a straight line (following the arrow). Black rectangle outline represents liquid blocker pen outline. (B, C) Optimal metaphase I chromatin spread preparation. Metaphase I chromosomes were stained with DAPI (blue, DNA), and immunolabeled for CEN (green, kinetochore/centromere marker). Scale bars = 10 &#181;m. (B) Antibodies against Histone H4 (di methyl K20, tri methyl K20) were used to label pericentromeric heterochromatin (PCH). (C) Antibodies against Topoisomerase II&#945; (TOPOII) were used to label the PCH and chromosome axes. (D) Optimal metaphase II chromatin spread preparation. Metaphase II chromosomes were stained with DAPI (blue, DNA), and immunolabeled for CEN (green), and Histone H4 (di-methyl K20, tri-methyl K20) to label the PCH. Scale bar: 10&#181;m. (E, F) Poor metaphase I chromatin spread preparations. Chromosomes were stained with DAPI (blue, DNA), and immunolabeled for CEN (green) and the meiotic cohesin component, REC8. Scale bars = 10 &#181;m (E) An oocyte that did not burst upon release onto PFA-coated slide. (F) Chromosomes that were spread too far apart. <a href="https://cloudflare.jove.com/files/ftp_upload/56736/56736fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Item</td>
			<td>Amount</td>
			<td>Final Concentration</td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>50 mL</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>1.5g</td>
			<td>3% (w/v)</td>
		</tr>
		<tr>
			<td>&#160;Horse Serum</td>
			<td>5 mL</td>
			<td>10% (v/v)</td>
		</tr>
		<tr>
			<td>10% detergent (see 
			&#160;Table of Materials) in PBS</td>
			<td>250 &#956;L</td>
			<td>0.05% (v/v)&#160;</td>
		</tr>
	</tbody>
</table>
Table 1: Antibody dilution buffer (ADB) recipe. Store ADB at 4 &#176;C or freeze stocks at -20 &#176;C if making larger quantities. ADB can become contaminated, so make sure good aseptic techniques are used and assess the solution for contamination prior to each use. Smaller aliquots of ADB can also be prepared to minimize contamination.

Watch this Scientific Journal Video about Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II at JoVE.com

Preparaty do rozprzestrzeniania się chromatyny do analizy progresji oocytów myszy od profazy do metafazy II | Genetyka | Wideo

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for ...

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Unit 2

Meiosis

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15.3K Views.  Johns Hopkins University Bloomberg School of Public Health. Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

Video: Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded Nucleolus) or unable (NSN, Not Surrounded Nucleolus) to develop to the blastocyst stage after in vitro maturation (IVM) and in vitro fertilization (IVF). It makes use of Hoeschst33342 (or any other DNA intercalating dye) able to bind to the heterochromatin of the nucleolus showing a ring in the SN oocytes or not, like in the NSN oocytes. This represents the easiest and quickest way to sort both antral oocytes that can be eventually used for IVM or IVF procedures. 
Briefly, the protocol consists of the following steps: hormone injection to stimulate follicular growth; isolation of the oocytes at the GV stage from the antral compartment by puncturing the ovary with a sterile needle; preparation of thin glass pipettes for mouth pipetting of the oocytes; sorting of the oocytes with Hoechst33342 prepared at a supravital concentration; IVM, IVF or any other molecular/cellular analysis.
Unfortunately there are still few evidences to sort SN and NSN oocytes using less invasive techniques. If and once they will be identified, they could be potentially applied to human assisted reproductive technologies, although with several aspects that should be modified. To date, this technique has potential implications to dramatically increase IVM and IVF successful procedures in both endangered and species with economic interest.

Here we present a protocol for the characterization of mouse antral oocytes based on nucleolar organization.

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

Developmental Biology

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected.
Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted ...

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding the chromosome events that take place during meiotic prophase, including the pairing, synapsis, and recombination events that ensure proper chromosome segregation. While the mouse has been an important model for understanding the molecular mechanisms underlying these processes, not all meiotic events in this system are analogous to human meiosis. We recently demonstrated the exciting potential of the zebrafish as a model of human spermatogenesis. Here we describe, in detail, our methods to visualize meiotic chromosomes and associated proteins in chromosome spread preparations. These preparations have the advantage of allowing high resolution analysis of chromosome structures. First, we describe the procedure for dissecting testes from adult zebrafish, followed by cell dissociation, lysis, and spreading of the chromosomes. Next, we describe the procedure for detecting the localization of meiotic chromosome proteins, by immunofluorescence detection, and nucleic acid sequences, by fluorescence in situ hybridization (FISH). These techniques comprise a useful set of tools for the cytological analysis of meiotic chromatin architecture in the zebrafish system. Researchers in the zebrafish community should be able to quickly master these techniques and incorporate them into their standard analyses of reproductive function.

Nuclear surface spreads are an indispensable tool for studying chromosome events during meiosis. Here we demonstrate a method to prepare and visualize meiotic chromosomes during prophase I from zebrafish spermatocytes.

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding ...

Human Egg Maturity Assessment and Its Clinical Application

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the egg&#39;s developmental competence. Presence of the first polar body (PB) together with the meiotic spindle indicates completion of the oocyte maturation and the egg&#39;s readiness for fertilization. In clinical practice, it is customary to assume that all oocytes displaying a PB are mature metaphase (MII) oocytes. However, PB extrusion precedes the formation of the bipolar MII spindle. This asynchrony makes the mere presence of PB an unreliable marker of oocyte maturity. Noninvasive spindle imaging using polarized light microscopy (PLM) allows quick and easy inspection of whether the PB-displaying oocyte actually reassembled a meiotic spindle prior to ICSI. Here, we present a standard protocol to perform human egg maturity assessment in the clinical laboratory. We also show how to optimize the time of ICSI with respect to the oocyte&#39;s developmental stage in order to prevent premature sperm injection of late-maturing oocytes. Using this approach, even immature oocytes extruding PB in vitro can be clinically utilized. Affirmation that MII spindle is present prior to sperm injection and individual adjustment of the time of ICSI is particularly important in poor prognosis in-vitro fertilization (IVF) cycles with a low number of oocytes available for fertilization.

We provide an outline of the clinical protocol for non-invasive assessment of human egg maturity using polarized light microscopy.

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the ...

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give rise to aneuploidy occur during meiosis in oocytes, but why oocyte meiosis is error-prone is still not fully understood. During cell division, cells prevent errors in chromosome segregation by activating the spindle assembly checkpoint (SAC). This control mechanism relies on detecting kinetochore (KT)-microtubule (MT) attachments and sensing tension generated by spindle fibers. When KTs are unattached, the SAC is activated and prevents cell-cycle progression. The SAC is activated first by MPS1 kinase, which triggers the recruitment and formation of the mitotic checkpoint complex (MCC), composed of MAD1, MAD2, BUB3, and BUBR1. Then, the MCC diffuses into the cytoplasm and sequesters CDC20, an anaphase-promoting complex/cyclosome (APC/C) activator. Once KTs become attached to microtubules and chromosomes are aligned at the metaphase plate, the SAC is silenced, CDC20 is released, and the APC/C is activated, triggering the degradation of Cyclin B and Securin, thereby allowing anaphase onset. Compared to somatic cells, the SAC in oocytes is not as effective because cells can undergo anaphase despite having unattached KTs. Understanding why the SAC is more permissive and if this permissiveness is one of the causes of chromosome segregation errors in oocytes still needs further investigation. The present protocol describes the three techniques to comprehensively evaluate SAC integrity in mouse oocytes. These techniques include using nocodazole to depolymerize MTs to evaluate the SAC response, tracking SAC silencing by following the kinetics of Securin destruction, and evaluating the recruitment of MAD2 to KTs by immunofluorescence. Together these techniques probe mechanisms needed to produce healthy eggs by providing a complete evaluation of SAC integrity.

Error in chromosome segregation is a common feature in oocytes. Therefore, studying the spindle assembly checkpoint gives important clues about the mechanisms needed to produce healthy eggs. The present protocol describes three complementary assays to evaluate spindle assembly checkpoint integrity in mouse oocytes.

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The study of chromosome behavior in oocytes from model organisms holds much promise to uncover the molecular basis of the susceptibility of human oocytes to aneuploidy. Drosophila melanogaster is amenable to genetic manipulation, with over 100 years of research, community, and technique development. Visualizing chromosome behavior and spindle assembly in Drosophila oocytes has particular challenges, however, due primarily to the presence of membranes surrounding the oocyte that are impenetrable to antibodies. We describe here protocols for the collection, preparation, and imaging of meiosis I spindle assembly and chromosome behavior in Drosophila oocytes, which allow the molecular dissection of chromosome segregation in this important model organism.

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

We present protocols for the collection, preparation, and imaging of mature Drosophila oocytes. These methods allow the visualization of chromosome behavior and spindle assembly and function during meiosis.

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

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Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Human Egg Maturity Assessment and Its Clinical Application

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes