Poslední úprava: RNDr. Nataša Šebková, Ph.D. (25.02.2022)
I. PART - Permanent sections
Developmental stages of Xenopus laevis
oocytes
stage 2 cells
stage 4 cells
stage 8 cells
stage 16 cells
early blastula
Tailbud stage
tadpole
Cross-sections
testis - Xenopus laevis
ovarium - Xenopus laevis
testis - mouse
ovarium – mouse
fertilized oocytes Xenopus laevis – pronucleus
sperm cells:
mouse
newt
earthworm
hamster
Xenopus laevis
II. PART - Maturation of porcine oocytes and in vitro fertilization
Background
Oogenesis in mammals
Female gametes undergo three developmental stages: 1. division, 2. growth and 3. maturation.
1. Division – amplification of oogonia by mitosis (total number is established before the birth)
2. Growth – synthesis of proteins, RNAs and other components importing for the early embryonic development. Oocyte growth is accompanied with the origin and growth of follicle. Its fluid is strong inhibitor of oocyte maturation. In the follicle the oocyte is arrested in prometaphase of the first meiotic division (1st meiotic arrest). The nucleus is called Germinal Vesicle (GV).
3. Maturation – fully grown Graf´s follicle bursts (LH peak) and the oocyte is ejected in to the oviductal ampula. Oocyte is not further inhibited by the follicular fluid and the resumption of meiosis has occurred. Germinal vesicle starts to break down (GVBD) and the chromosomes condense to the metaphase figure (metaphase I). After the short anaphase I – telophase I stage, chromosomes again condense to the form of metaphase II, where in the most mammals the oocyte waits for the fertilization (second meiotic arrest). The first polar body should be observable under zona pellucida. Oocyte maturation is driven by p34cdc2 and cyclin B. Both factors interact and create MPF complex (Maturation Promoting Factor). Level of its active form is the highest in metaphase I and metaphase II. In contrary the lowest level is observable during anaphase I – telophase I.
Fertilization in mammals
Sperm cells undergo process of capacitation in the female reproductive tract. Then they are capable to fertilize the oocyte. Contact of both gametes is usually observable in the upper third of the oviduct. Sperm cells have to go through several layers of cumulus cells. Shortly after the contact with zona pellucida, acrosomal reaction has occurred (exocytosis of proteolytic enzymes). Inhibition of polyspermy is achieved by the 1st. fast block (change of the membrane polarity). Then cortical granula placed under cytoplasmic membrane release their content. As a result, sperm cells outside the oocyte are not further capable to bind to zona pellucida (2nd slow block). Sperm penetration activates the oocyte by releasing of Ca2+ ions. The meiosis is accomplished and the second polar body is seen under zona pellucida. Sperm head and oocyte chromatin decondense to form male and female pronuclei. Both pronuclei undergo S phase (DNA replication). In mammals rather than fusion separate condensation to chromosomes occurs in the frame of mitotic prophase. Then chromosomes from both parents are intermixed and the true somatic nuclei arise in the two-cell embryo.
Practical part
Maturation of porcine oocytes
Six hours after ovulation the oocyte undergo breakdown of the germinal vesicle (GVBD). Metaphase I is formed between 20 – 24 hours and metaphase II after 42 – 48 hours
Aim for the 1st day
Isolation of porcine oocytes from follicles by aspiration
Stab the follicles of all ovaries by needle coupled with 10 ml syringe and aspirate the follicular fluid. Then transfer the fluid to the 3 cm Petri dish. During this work take also the photo of ovary under stereomicroscope (Fig. I)
Observe the follicular fluid under stereomicroscope and aspirate the individual oocytes enveloped by layers of cumulus cells by thin glass capillary. Transfer oocytes to the µl 50 drop of M2 medium covered by mineral oil (4 drops x 50 µl in 3 cm Petri dish). Observe isolated oocytes under stereomicroscope. Porcine oocytes are dark (lipid granula). Count oocytes take a photo of oocytes with cumulus cells (Fig. A) and separate them into two groups. In the first one there will be one third of total number of oocytes and you will fix them after 24 hours of cultivation (metaphase I stage). The second group, should be formed by the remaining two thirds. This group is intended for full maturation (42-48 hours, metaphase II stage) with subsequent in vitro fertilization by boar sperm cells.
Transfer the first group of oocytes into the 4-well dish filled with 500 µl of M199 medium with 4 mg of GPBoS/ml (Growth Proteins of Bovine Serum) covered by 500 µl of paraffin oil by glass capillary. The second group will be transferred in the same manner to another 4-well dish. Both dishes will be then cultivated in the incubator at 38.5 °C and 5% of CO2 atmosphere.
Aim for the 2nd day
Fixation of the first group of oocytes after 24 h maturation
Take out the 4-well dish with oocytes intended to fixation after 24 h maturation from thermostat, capture a photo quickly under stereomicroscope (Fig. B), add á 25 µl of 0,1% Hyaluridonase in each well and mix it up by pipetting. The enzyme disrupts the contacts between cumulus cells and oocytes. After 3-5 min transfer oocytes into the drop of M2 medium placed in 3 cm Petri dish (prepared similar like day before) by glass capillary. Remove the rest of cumulus cells by aspirating of oocytes with narrow glass capillary with diameter corresponding to the size of entire oocyte. After this step observe oocytes and place emphasis on clearly visible zona pellucida. Transfer oocytes without any cumulus cells into 500 µl of 4% formaldehyde solution in a 4-well dish and leave in the fridge for next day staining.
Aims for the 3rd day
1. In vitro fertilization (IVF)
Sperm cells intended for IVF will be stained by vital days (Hoechst 33258 – sperm heads + Mitotracker Red CMX Ros – mitochondrions in the neck and part of mid-piece) as follows:
Transfer 3 ml of sperm suspension into 10 ml tube with the blue cap by Pasteur pipette. Centrifuge the tube at 700 g (2.650 RPM – centrifuge MPW-340) for 5 min. Remove the supernatant by Pasteur pipette and add 7 ml of sterile PBS + 0,01% PVA (polyvinylalcohol). Mix well by aspirating and centrifuge in the manner as previous. Repeat this step once again. Remove the supernatant and resuspend the pellet in 2 ml of sterile PBS + 0.01% PVA. From this suspension transfer 500 µl into Eppendorf tube and add 5 µl of Mitotracker (1 mM solution in DMSO) and 2 µl of Hoechst 33258 (1 mg/ml). Mix well by aspirating. Apply 990 µl of distillate water into 1,5 ml Eppendorf tube. Take 10 µl of sperm suspension and transfer it to the tube with water. Mix well by shaking (sperm cells are diluted 100 x and are immobile). This suspension is intended for the sperm counting under Burker´s chamber.
Count the sperm cells as follows:
Take 10 µl from the 100 x diluted sperm suspension and fill the Burker´s chamber. Place the chamber under microscope and count sperm cells in 16 large squares (framed by triple line). Don´t count the sperms laying between large squares. Repeat this step twice in another place of Burker´s chamber. Count the concentration of sperm cells in one ml as follows:
X = average number of sperm cells in the 16 large squares x 15.625 x 100
Explanation:
Area of one large square = 0.04 mm2, depth = 0.1 mm
Volume of liquid above one large square = 0.04 x 0.1 = 0,004 mm3 (µl)
Volume of liquid above 16 large squares = 0.004 x 16 = 0.064 mm3 (µl)
Recounting for 1 cm3 (ml) = 1000 / 0.064 = 15.625 (factor in the above mentioned formula)
Sperm cells were diluted 100 x. So we have to multiply the result by 100.
Meanwhile the part of working group count the sperm cells, others can take a photo of oocytes with cumulus cells in 4-well dish (Fig. C) and denude these oocytes (second group of oocytes). Cumulus cells are removed in the same manner as in oocytes matured up to metaphase I. Observe denuded oocytes under stereomicroscope, place emphasis on finding the first polar body (it should be clearly visible under zona pellucida as a small sphere object) and take a photo (Fig. F). Presence of this polar body is the evidence that oocyte is fully matured (metaphase II). Divide the oocytes into two half groups. The first group will be fixed for 1-2 h in 500 µl of 4% formaldehyde at room temperature and second group will be used for IVF.
Meanwhile the sperm cells incubated with vital dyes are labelled (30-45 min). Take such volume from the sperm suspension so as after dilution with 500 µl of fertilization medium (mTBM with 2 mg of BSA/ml in the 4-well dish) you obtain the final concentration 500.000/ml. Mix the suspension in the well and add the denuded oocytes. Incubate the dish for 1-2 h in the incubator at 38.5°C and 5% of CO2 atmosphere. Then you can observe the contact of both gametes under stereomicroscope and under fluorescence microscopy. Sperm heads are labeled in blue, the neck and part of midpiece (mitochondrion) are stained in red (Fig. H).
For observation of stained sperms, prepare cleaned glass slide and coverslip. The sperm incubated in Ependorf tube at 17 ° C is will dilute 1: 1 with distilled water. This is done by adding 250 μl of H2O to the new Ependorf tube and adding 250 μl of stained sperms. Apply 10 μl of the resulting sperm suspension to the glass slide and cover with coverslip. Frame the coverslip with nail polish and after thorough drying observe sperms under a fluorescence microscope. The sperm head is dyed blue (Hoechst 33258), the neck, and the mid-piece is marked in red (Mitotracker). By combining photos from individual fluorescence channels and photos from the phase contrast, you get the final photo (Figure G).
2. Staining of specimens with oocytes fixed after 24 h (MI) and 42-46 h (MII) maturation.
Prepare two 3 cm Petri dish with 2 drops PBS + PVA covered by mineral oil. The dishes are marked with MI and MII. Transfer the oocytes from fixation to 4-well plates (one from yesterday-oocytes in MI, the other from morning-oocytes in MII) to the first drop on both plates and incubate for 5 minutes. Then transfer the oocytes to the second drop and incubate again for 5 minutes.
We prepare two glass slides from ethanol bath (dry and polish) and mark them MI and MII. Depending on the size of the coverslip, we put some vaseline from the prepared syringe into the "future corners". Using pipette transfer 10 μl of DAPI mounting media into the center of the field bounded with vaseline. Transfer the washed oocytes to the DAPI medium drop and leave them for 2 minutes. Carefully apply a cleaned coverslip and gently press the corners by tweezers. BE CAREFULL, OOCYTES ARE FRAGILE AND CAN BE DAMAGED BY THE STRONG PRESSURE.
After this apply the nail polish around the whole coverslip to prevent evaporation and coverslip movement. Observe the specimen under fluorescence microscopy and try to find metaphase I (Fig. D) and metaphase II (Fig. E).
III. PART - Caenorhabditis elegans
Caenorhabditis elegans is a small free-living bacteriovorous nematode found in soil environments. It has two sexes: predominating hermaphrodites (self-fertilizing, sex chromosomes XX) and rare males (0.05% of the total population, sex chromosomes X0).
In laboratory, C. elegans is an important model organism. On one hand, it is a multicellular eukaryotic organism; on the other hand, it is still a simple transparent animal, allowing analyses of many processes in the intact organism. Furthermore, it has a short generation time – approx. 3 to 4 days. The embryonic development of the worm is deterministic with largely invariant cell lineages. The developmental fate of every single somatic cell (959 in the adult hermaphrodite; 1031 in the adult male) has been mapped out.
Homeobox (Hox) genes encode for transcription factors characterized by highly conserved homeodomain. In general, Hox genes play a central role in specifying anterior–posterior axis and segment identity. C. elegans has a reduced and dispersed set of Hox genes. Despite obvious evolutionary re-arrangement of Hox genes, they are important for cell fates specification along the body axis. However, unlike in many other animals, Hox gene expression in C. elegans is dependent on cell lineage rather than position.
Today, you will visualize expression of three different Hox genes in C. elegans: mab-5 (ftz, fushi tarazu), lin-39 (Scr, sex combs reduced or Hox5) and egl-5 (Abd-B, abdominal B). You will work with animals carrying lacZ reporter driven by promoter belonging to one of above mentioned Hox genes.
Pool worms from the plate into 800 μl M9 buffer
Spin down 1000g/30 sec ~ 3000 rpm / 30 sec., discard supernatant carefully
Wash with 800 μl dH2O, spin down, discard supernatant carefully
Speedvac 20 min.
Incubate with 300 μl acetone (on ice) 2 min. 2x, spin down, discard supernatant
Let the pelleted animals dry out
Add 250 μl staining solution, develop in dark
Rinse with 800 μl M9 buffer
Mount and observe under the microscope
Staining solution for 1ml
1M NaH2PO4 33 μl
0.5M Na2HPO4 332 μl
0.1M MgCl2 1 μl
0.3M K3Fe(CN)6 16.5 μl
0.3M K4Fe(CN)6 16.5 μl
1%SDS 4 μl
2% X-gal 12.5 μl
100% formamide 3 μl
water 581.5 μl
DAPI staining
DAPI (4',6-diamidino-2-phenylindole) is a fluorescent stain that binds strongly to dsDNA, thus enabling visualization of the chromosomes / nuclei.
We will make use of DAPI staining to observe the overall morphology of C. elegans in different developmental stages.
Pool worms from the plate into M9 buffer
Spin down 1000g/30 sec ~ 3000 rpm / 30 sec.
Fix animals with 300 μl methanol (20 min., on ice)
Spin down, discard supernatant carefully
Incubate in 300 μl acetone (10 min., on ice)
Wash with M9 buffer twice, spin down, discard supernatant carefully
Incubate 30-40min. with 100 μl DAPI (1 μg/ml), protect from light!!!
Wash with M9 buffer, spin down, discard supernatant carefully
Mount and observe under the fluorescence microscope
