Zygotic Fluorescence Recovery After Photo-bleaching Analysis for Chromatin Looseness That Allows Full-term Development

Podsumowanie

Chromatin looseness appears to be involved in the developmental potential of blastomeres. However, it is not known whether chromatin looseness can be used as a reliable index for the developmental potential for embryos. Here, an experimental system in which chromatin looseness-evaluated zygotes can develop to full term has been described.

Streszczenie

Live imaging is a powerful tool that allows for the analysis of molecular events during ontogenesis. Recently, chromatin looseness or openness has been shown to be involved in the cellular differentiation potential of pluripotent embryonic stem cells. It was previously reported that compared with embryonic stem cells, zygotes harbor an extremely loosened chromatin structure, suggesting its association with their totipotency. However, until now, it has not been addressed whether this extremely loosened/open chromatin structure is important for embryonic developmental potential. In the present study, to examine this hypothesis, an experimental system in which zygotes that were analyzed by fluorescence recovery after photo-bleaching can develop to term without any significant damage was developed. Importantly, this experimental system needs only a thermos-plate heater in addition to a confocal laser scanning microscope. The findings of this study suggest that fluorescence recovery after photo-bleaching analysis (FRAP) analysis can be used to investigate whether the molecular events in zygotic chromatin are important for full-term development.

Wprowadzenie

After fertilization, the chromatin structure is altered dynamically, and zygotic chromatin structure is then eventually established^1,2. During this period, in paternal pronuclei, the dominant chromatin protein is changed from protamine into histone. The resulting chromatin is extremely different from that of sperms and female oocytes in several points (e.g., histone variant composition, histone modification). Thus, formed zygotic chromatin is thought to be important for subsequent embryonic development. However, despite efforts to reveal the details of zygotic chromatin structure over long periods, methods to evaluate the quality of zygotes or to predict their full-term development at the one-cell stage by analyzing their chromatin structure have never been established.

In the previous study, it is discovered that zygotes have an extremely loosened chromatin structure³. Currently, chromatin looseness or openness is believed to be an important factor for cellular differentiation potential in embryonic stem (ES) cells⁴. ES cells do not exhibit homogeneity in nature, but are rather heterogeneous; in ES cell colonies, some transiently acquire a higher differentiation potential comparable to blastomeres of two-cell stage embryos. During this transition into the two-cell like state, chromatin looseness in ES cells changes into what is comparable with two-cell stage embryos⁵. Thus, chromatin looseness seems to be important for cellular differentiation potential and it is possible that extensively open chromatin in zygotes is useful for the evaluation of zygotic developmental potential.

Live imaging is a powerful tool that allows for the analysis of molecular events during ontogenesis since this method allows for subsequent development and even full-term development⁶. As one of the live imaging methods, FRAP analysis has been used to examine chromatin looseness in preimplantation embryos and ES cells^3,4,5. If zygotic chromatin looseness can be analyzed without a detrimental effect on full-term development by FRAP analysis, it may be a valuable tool for the evaluation of the quality of embryos at the one-cell stage. However, the effects on full-term development by this experimental method have not been examined. Recently, an experimental system using FRAP to evaluate zygotic chromatin looseness was developed. Because this was a new observation system for zygotes, it was termed as zygotic FRAP (zFRAP). zFRAP did not critically affect full-term development and has been reported elsewhere⁷. In this report, the protocol of this experimental method is described.

Protokół

This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the University of Yamanashi. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Yamanashi (Permit Number: A24-50). All surgeries were performed under tribromoethanol anesthesia, and all efforts were made to minimize suffering. An illustrated overview of the procedures and time-table are shown in Figure 1 and Table 1, respectively.

1. Preparation of Messenger RNA (In Vitro Transcription of eGFP-H2B mRNA)

1. Linearize 5 µg of template DNA pTOPO-eGFP-H2B^3,7 with 30 U of Not I in 50 µL at 37 °C overnight.
2. Collect 1.5 µL of digested DNA for confirmation whether completely digested by electrophoresis.
   1. Apply 1.5 µL and 3 - 5 µL of undigested DNA with loading dye to the well of 2% agarose gel, respectively, and subject to electrophoresis.
      NOTE: If completely digested, a single band (approx. 5 kb) is observed. Undigested DNA is used as a control, which appears at a different position.
3. Add 151.5 µL of ddH₂O and 200 µL of phenol/chloroform/isoamyl alcohol (25:24:1) to the remaining digested DNA.
   1. Mix vigorously by vortex.
   2. Centrifuge at the maximum speed for 15 min.
4. Recover the supernatant and then add 200 µL of chloroform.
   1. Mix vigorously by vortex.
   2. Centrifuge at maximum speed for 15 min.
5. Recover the supernatant and then add 20 µL of 3M sodium acetate and 1.5 µL of ethachinmate.
   1. Mix vigorously by vortex.
   2. Add 550 µL of 100% ethanol and then mix vigorously by vortex.
   3. Centrifuge at maximum speed for 15 min.
   4. Discard the supernatant and then wash the pellet with 70% ethanol.
   5. Dry the pellet by evaporation for 5 - 10 min.
6. Dissolve precipitated plasmid DNA in 8 µL of nuclease-free water.
7. Assess plasmid concentration (expected concentration is about 300 - 500 ng/µL) with nanodrop by following manufacturer's instruction.
8. Perform in vitro transcription for mRNA encoding eGFP-H2B with 1 µg of purified DNA as a template in 20 µL of total reaction volume by following the manufacturer's instructions.
   1. After in vitro transcription, add 36 μL of water then recover 1.5 µL from 56 µL of synthesized mRNA for analysis by electrophoresis (as without poly A).
9. Perform poly A tailing for synthesized mRNA in a 100 µL reaction volume by following the manufacturer's instructions.
10. Add 60 µL of lithium chloride precipitation solution to the poly A tailed mRNA of eGFP-H2B and then mix vigorously.
    1. Cool at -30 °C for 30 min.
    2. Centrifuge at maximum speed for 15 min.
    3. Discard the supernatant and then wash the pellet with 70% ethanol.
    4. Dry the pellet by evaporation for 5 - 10 min.
11. Dissolve the pellet with 20 µL of nuclease-free water by heating at 55 °C for 30 min.
12. Assess the concentration of the precipitated mRNA with nanodrop. Dilute to 500 ng/µL with nuclease-free water and store at -80 °C until use.
13. Confirm preferred mRNA production; subject 1 µL of mRNA with/without (obtained in 1.8.1 and 1.12 steps, respectively) poly A tail mixed with 1.5 µL of 0.1 mg/mL ethidium bromide and 3 µL of sample loading dye to the electrophoresis analysis. Applied volume was 5.5 µL.
    NOTE: If Poly A tailing was successful, the shifted band compared to the mRNA without poly A tailing should be observed (Figure 2A).

2. Preparation of Vasectomized Male Mice

1. Weigh the ICR male mice at 8 - 12 months using a weight scale.
2. Intraperitoneally inject male mice with 0.7 - 0.9 mL 1.2% tribromoethanol anesthesia.
   NOTE: The amount of anesthesia depends on the size of the mouse. For example, a mouse weighing 40 g is injected with 0.8 mL of tribromoethanol anesthesia.
3. Wet the berry with 70% ethanol.
4. Make a small cut (3 - 5 mm) on the berry and then make an incision on the muscle wall with the help of scissors.
   NOTE: Scissors and forceps should be cleaned with 70% ethanol before starting the surgery.
5. Grip a fat pad with forceps and gently pull it out. Then, the testis and vas deferens appear.
   NOTE: Vas deferens is a U-shaped tube and with a red blood vessel.
6. Tie off the vas deferens at two points with surgical sutures and then cut out the middle part between the tied points.
7. Gently push back the fat pad and testis into the berry.
   1. Repeat the surgery with the remaining testis.
8. Sew the muscle wall with two stitches with surgical suture.

3. IVF

1. Inject 8 - 10-week old female B6D2F1 (BDF1) mice intraperitoneally with 7.5 IU equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) at 46 - 50 h interval.
2. Prepare drops of 300 µL human tubal fluid (HTF)⁸ media for capacitation and insemination 1 day before IVF in a 30-mm dish, cover these drops with the mineral oil on the laboratory bench and then preincubate in an incubator overnight.
3. Sacrifice matured male ICR mice by cervical dislocation. Dissect cauda epididymis with a 27-G needle and collect spermatozoon. Culture them for capacitation in 300 µL of HTF media for 1 - 2 h at 38 °C.
4. 16 h after hCG injection, sacrifice super ovulated female mice by cervical dislocation. Transfer the oviduct ampulla into mineral oil next to HTF media and then draw out cumulus cells and oocytes complex from this oviduct ampulla by a 30-G needle into the preincubated insemination-HTF medium.
   NOTE: Some female mice do not often show ovulation despite super ovulation treatment. Enlarged oviduct ampulla is a sign of ovulation.
5. After capacitation, transfer 2 - 3 µL of the capacitated spermatozoon into insemination-HTF media in which the ovulated oocytes were collected.
6. 1 h after insemination, collect the fertilized zygotes and treat them with hyaluronidase at 100 µg/mL for 10 min in CZB⁹ media.
   NOTE: When insemination is performed properly, 1 h after insemination, the fertilized zygotes with the 2^nd polar body are observed. If less or low-quality sperm is used, only little zygotes with the 2^nd polar body are observed. Also, if many sperms are used for insemination, polyspermy occurs. Proper insemination leads to zygotes with male and female pronuclei. By using these criteria, it is possible to find whether insemination is done well or not.
   1. After washing in CZB media, subject the zygotes with a second polar body to microinjection with eGFP-H2B mRNA.

4. Preparation of Chamber and Microinjection Pipettes

1. Dilute RNA encoding eGFP-H2B using nuclease-free water to obtain a concentration of 250 ng/µL.
2. Prepare 2 - 3 drops of PVP-HTF and eGFP-H2B drops mRNA, and 5 - 6 drops of Hepes buffered CZB (H-CZB) drops on the 60-mm dish. Cover these drops with mineral oil.
   NOTE: These preparations can be performed on the laboratory bench. There is no requirement for using laminar airflow cabinet.
3. Pull borosilicate glass with a micropipette puller (e.g., P = 500, heat = 825, pull = 30, vel = 120, and time = 200)
4. After breaking the tip of the pipette, bend the pulled glass microinjection pipette close to the tip (−300 µm back) at around 30° using a microforge.

5. Microinjection

1. Turn off the plate heater on the stage of micromanipulator to prevent the killing of the zygotes with a piezo during mRNA injection.
2. Wash and coat the inside of injection needles in HEPES-buffered-HTF containing 10% PVP (PVP-HTF).
3. Collect the fertilized zygotes with a 2^nd polar body (Figure 2B) and transfer 20 - 25 zygotes onto the chamber of the microscope stage attached with a micromanipulator.
4. Fill the diluted eGFP-H2B mRNA into borosilicate glass capillaries from the tips.
5. Hold the zygote with a holding pipette and then break the zona pellucida and the cytosolic membrane with a piezo drive.
6. Inject about 10 pL of mRNA into the cytoplasm of the zygotes. Finish mRNA-injection within 10 min to prevent damage caused by longer culturing the zygotes outside the incubator.
   NOTE: The injection volume should be as large as the 2^nd polar body. If the amount of mRNA injected is too large, it is possible to cause the free eGFP-H2B fraction, which may affect the mobile fraction.
7. 10 min after microinjection, wash in at least 3 drops and then culture the injected zygotes in CZB at 38 °C until zFRAP analysis.

6. zFRAP Analysis

1. 1 h prior to zFRAP analysis, turn on the laser and hot plate attached to the stage of the confocal microscope. Set the temperature at 38 °C.
2. Put 6 - 10 µL of HEPES-buffered CZB media covered with mineral oil on the 60-mm glass bottom dish and warm on the heater until zygote collection. 
3. 8 - 12 h after insemination, collect 5-10 eGFP-H2B-expressing zygotes and transfer into 6 - 10 µL of pre-warmed HEPES-buffered CZB medium drop.
   NOTE: To avoid longer culturing outside incubator, 5 - 10 eGFP-H2B-expressing zygotes were used at each analysis. 5 to 10 zygotes can be analyzed within 1 h.
4. Set the bleaching and imaging conditions according to the manufacturer's instructions. The case a confocal laser scanning microscope (Table of Materials) is shown below:
   1. Use 60X oil lens (60X 60X/1.35 oil).
      NOTE: Lenses with higher magnification (higher numerical aperture) show higher sensitivity.
   2. Set scanning speed as 4.0 µs/Pixel and size as "512" on "Acquisition Setting" (Supplemental Figure 1).
   3. Increase Digital zoom to "5" (Supplemental Figure 1).
      NOTE: A higher zoom is required to recognize whether focus drift occurs or not.
   4. Open dye list (Supplemental Figure 1) and then chose "EGFP". Set observation time as 1.6 s (interval is set as "free run" setting) (Supplemental Figure 1)
      NOTE: More observation points may cause more detailed data, but it may also cause phototoxicity. In the zFRAP analysis, a 1.6 s observation time seems to be enough to detect the parental asymmetry of chromatin looseness.
   5. Set the number of pictures as 12 in total (Supplemental Figure 1).
   6. Start "Stimulus setting" panel and then click "Main" on UseScanner. Set scanning speed as 10.0 µs/Pixel. Click "Activation in Series" and then set PreActivation as "3 frames" and Activation Time as "5 sec" (Supplemental Figure 1).
   7. Click "Focus x 2" and set the focus and then "Stop".
   8. Set bleaching and imaging laser power (477 nm) at 110 and 15 µW, respectively, by adjusting "laser gage power" (Supplemental Figure 1).
      NOTE: Very strong bleaching may cause a larger bleached area, which causes difficulties for the quantitative analysis. For measurement of the laser power, use a power meter. It is important to confirm the laser power to avoid the effect of the time-related deterioration of the laser power.
   9. Click "Clip Rect" (Supplemental Figure 1) in Stimulus Setting panel. Set the region of interest (ROI; red; ROI #1B) as 40 x 40 pixels (7.6 µm²). Prepare a reference region (REF; green; ROI #2), and a background (BG; blue; ROI #3) using "copy ROI" and "paste ROI" (right-click button on the mouse).
      NOTE: Pixel size can be set through "ROI manager" which can be called by clicking the right button of the mouse when the cursor is on the ROI. Larger ROIs are thought to be better since they can standardize the dispersion of the zFRAP score. However, too large of an ROI cannot be used for this analysis because it makes it difficult to avoid the nucleolus precursor body (NPB). eGFP-H2B in this compartment was thought to be free from chromatin and showed remarkable higher mobility³. ROI and REF areas should be separated as much as possible. If these two regions are close, bleaching may also affect the region of REF.
   10. Click "Live" on the tool bar and then start "Live Plot" (Supplemental Figure 1).
       NOTE: Live plot indicates fluorescence signal intensity within each ROI and is used for adjusting laser power using HV. Note that if ROI is not selected, no intensity would be detected on Live plot panel.
   11. Determining the ROI position
       NOTE: ROI can be moved by dragging into the desired position in pronuclei. (1) Be sure to avoid the nucleolus, and (2) fit the entire ROI into the nucleoplasm. Do not contain the region outside the area of the nuclear membrane (cytoplasm) in ROI. The localization of eGFP-H2B is highly restricted into the nucleoplasm so that it is easy to find whether ROI contains cytoplasm or not by the fluorescence of eGFP-H2B. Male pronuclei tend to be larger than those of females, which are often localized near the 2^nd polar body. While using these criteria, judge the male or female pronuclei.
   12. Click "Focus x 2" (Supplemental Figure 1) and then adjust the HV power gage of the channel for EGFP into fluorescence intensity to around 2000 (fluorescence intensity can be seen in the "live plot" window).
5. Click "Time" and then "XY" (Supplemental Figure 1) to start the zFRAP analysis.
   NOTE: If "Time" button is suitably selected, "t" appears on the "XY" button.
   1. Click "Series Done" (Supplemental Figure 1), which appears after FRAP imaging near the "XY button" and then obtain FRAP-analyzed data.
   2. Save the file "2D View-Image XXXXX" as a preferred named file (oib. file format) by "save as" (Ctrl + Shift + S can be also used).
   3. Select ROI, REF, and BG and then click (Ctrl+A can be also used) "Series Analysis" in "2D View Image" window.
   4. Obtain row FRAP data and then click "save" button in the live plot window.
      NOTE: Row FRAP quantitative data is saved as a csv file.
6. For no zFRAP control, put some of the zygotes injected with mRNA on the drop, which is near to the rim of the glass bottom dishes to avoid the effect from fluorescence observation.

7. Data Calculation

1. While using the obtained quantitative data, make the graph of the "recovery curve" and "mobile fraction" by Excel as shown in the references^3,7 with some modifications (see below and supplemental excel file).
2. Calculate the relative intensity by subtracting the value of BG from those of ROI and REF in 12 pictures for each time point.
3. Divide the value of ROI by that of the REF. As a result, 12 standardized relative intensity scores at each time point can be obtained.
4. Calculate the average of the relative score for ROI in 3 images taken before photo bleaching.
5. Calculate the recovery rate. Divide the 12 obtained relative scores for ROI in the images taken at each time point (obtained at 7.1.2.) by the average of the relative score before photo bleaching (obtained at 7.1.3.). Draw recovery curve with using these scores (Figure 2E).
6. Calculate the bleaching rate.
   NOTE: Bleaching rate (%) = 100 - recovery rate of 4^th image.
7. Calculate the mobile fraction (MF) by using the formula as follows^10,11,12
   MF (%) = 12^th recovery rate (at the end point) - 4^th recovery rate/ bleaching rate × 100.

8. Embryo Transfer

1. Mate matured ICR females at 8 - 12 months with vasectomized male ICR mice the night before the embryo transfer by letting them be in the same cage overnight.
2. Collect the embryos that were zFRAP-analyzed and determined to develop to the two-cell stage.
3. Intraperitoneally inject female mice with 0.7 - 0.9 mL 1.2% tribromoethanol anesthesia or with 0.35 - 0.45 mL of 0.75/4/5mg/kg medetomidine/midazolam/butorphanol mixed agents prior to embryo transfer.
   NOTE: The volume of anesthesia is determined by the body weight of mice. Tribromoethanol: 0.2 mL/10 g of body weight; medetomidine/midazolam/butorphanol mixed agents: 0.1 mL/ 10 g of body weight.
   1. Transfer these two-cell stage embryos into the oviducts of pseudopregnant female ICR mice with a vaginal plug at 0.5 days post coitum (dpc).
   2. After the embryo transfer, put the female mice on the heater set at 37 °C until they wake up.
4. At 18.5 dpc, sacrifice the surrogate mother by cervical dislocation and recover the pups derived from zFRAP-analyzed zygotes by cesarean section. Some of the recovered pups were delivered to the foster mother and their body weights were assessed each week.

Wyniki

zFRAP analysis with eGFP-H2B

Properly produced mRNA encoding eGFP-H2B, which is seen as a shifted band caused by poly A tailing (Figure 2A), was injected into the cytoplasm of zygotes with a 2nd polar body at 1 - 3 h after insemination (Figure 2B). Eight to 12 h after insemination, zygotes with 2 pronuclei showing the expression of eGFP-H2B were collected and subjected to ...

Dyskusje

As revealed in this study, the zFRAP analysis does not cause critical damage to full-term development, suggesting this method is a very useful tool to reveal the association between molecular events and the embryonic developmental potential. During reprogramming, into the two-cell like state of ES cells, by which the differential potential of those cells becomes as high as that of two-cell stage embryos, chromatin looseness changes into that comparable with two-cell stage embryos⁵. Accordingly, it...

Materiały

Name	Company	Catalog Number	Comments
Confocal laser scaning microscope	Olympus	FV1200	FV1000 can be also used for FRAP analysis (reference #7)
Thermo plate	Tokai Hit	TP-110RH26
Inverted microscope	Olympus	IX71
Micro manupilator	Narishige	MMO-202ND
Pieze Micro Micromanipulator	Prime tech	PMAS-CT150
35 mm culture dish	Falcom	351008	35 x 100 mm style; for IVF
60 mm culture dish	Falcom	351007	60 x 15 mm style; for embryo culture
50 mm culture dish	Falcom	351006	50 x 9 mm style; for manipulation on the stage
50 mm glass bottom dish	Matsunami	D910400	50 mm dish, 27 mm φ hole size; for FRAP analysis
Mineral oil	Organic spceiality chemicals	625071	For FRAP analysis on the glass bottom dishes
Mineral oil	Sigma	M8410-1L	For IVF and embryo culture
glass capillary	Drummond	1-000-1000	For handling mouse zygotes
Borosilicate glass	Prime tech	B100-75-10-PT	For microinjection of mRNA
Micropipett puller	Sutter instrument	P-97/IVF	For preparation of the injection or holding neadle (out side diameter: 80 µm, inner diameter: 10 µm)
Microforge	Narishige	MF-900
mMESSAGE MACHINE SP6 Transcription kit	Thermo Fisher scientific	AM1340
Poly (A) tailing kit	Thermo Fisher scientific	AM1350
PVP solution 10% (PVP-HTF)	IrvineSceientific	99311	HEPES buffered HTF containing 10% PVP
Sodium HEPES	Sigma	H3784
pTOPO eGFP-H2B			Template plasmid for eGFP-H2B mRNA (reference #6)
NorthernMax Gly Sample loading Dye	Thermo Fisher scientific	AM8551	For electrophoresis of in vitro transcribed mRNA
Phenol chloroform isamyl alcohol	nacalai tesque	25970-14
Chloroform	nacalai tesque	08401-65
Not I	TOYOBO	NOT-111X
Ethachinmate	WAKO	318-01793
3664 Otical power meter	Hioki	3664	Power meter for laser power
Stereomicmicroscope	Olympus	SZX16