Poslední úprava: doc. RNDr. Ing. Vladimír Krylov, Ph.D. (15.01.2019)
Practical course is focused on the broadering of theoretical knowledge, acquired during semestral lecture from developmental biology. The major emphasis is on sexual propagation and embryonic development in selected model organisms. Students have an opportunity to observe histological fixed specimens. They can try the micromanipulation techniques for isolation of porcine oocytes from ovaries in practice. Half of oocytes is fixed in MI stage and half is fully matured up to metaphase II. Then students perform in vitro fertilization (IVF) using boar sperm. In addition visualization of Hox genes expression domains in C. elegans is another aim for students attended this practical course.
Literatura - angličtina
Poslední úprava: doc. RNDr. Ing. Vladimír Krylov, Ph.D. (22.04.2015)
Gilbert S.F: Developmental Biology. 10th edition and previous, Sunderland (MA): Sinauer Associates, parts of chapters from 10th edition are available on website: http://10e.devbio.com/contents.php?sub=1&art=1&full=1
Wolpert, L.: Principles of Development (2. vydání), Oxford University Press, 2002.
Sylabus - angličtina
Poslední úprava: doc. RNDr. Ing. Vladimír Krylov, Ph.D. (19.05.2020)
I. PART - PERMANENT SECTIONS
Developmental stages of Xenopus laevis
Developmental stages:
1. oocytes
2. stage 2 cells
3. stage 4 cells
4. stage 8 cells
5. stage 32 cells
6. blastula
7. early gastrula stage
8. middle and late gastrula stage
9. neurula stage
10. tail bud stage 1
11. tail bud stage 2
12. tadpole 1
13. tadpole 2
14. tadpole 3
Cross-sections
testis - Xenopus laevis
ovarium - Xenopus laevis
testis - mouse
ovarium - mouse
fertilized oocytes Xenopus laevis - pronucleus
fertilized oocytes Xenopus laevis - male and female pronucleus - fusion
sperm:
mouse
newt
earthworm
rooster
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 folicle. Its fluid is strong inhibitor of oocyte maturation. In the folicle 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 folicle bursts (LH peak) and the oocyte is ejected in to the oviductal ampula. Oocyte is not further inhibited by the folicular fluid and the resumtion of meiosis has occured. 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 cyklin 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 occured (exocytosis of proteolytical enzymes). Inhibition of polyspermy is achieved by the 1st. fast block (change of the membrane polarity). Then cortical granula placed under cytoplasmic membrane relase their content. As a result sperm cells outside the oocyte are not further capapble 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 oocytary chromatin decondense to form male and female pronuclei. Both pronuclei undergo S phase (DNA replication). In mammals rather then fusion separate condensation to chromosomes occures 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 folices by aspiration
Stab the folicles of all ovaries by needle coupled with 10 ml syringe and aspirate the folicular fluid. Then transfer the fluid to the 6 cm Petri dish.
Observe the folicular fluid under stereobinolupe and aspirate the individual oocytes enveloped by layers of cumulus cells by thin glass capilar. Transfer the oocytes in to the 3 cm Petri dish filled with 2 ml of manipulating medium (HTF with 2 mg BSA/ml. Observe isolated oocytes under stereobinolupe. Porcine oocytes are dark (lipid granula). Count oocytes and separate them into two groups. In the first one there will be lower quality oocytes and you will fix them after 24 hours of cultivation (metaphase I stage). The second group, should be formed by high quality oocytes (uniform dark cytoplasm and rich cumulus envelope). 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 parafine oil by glass capilar. The second group will be transferd in the same manner in to the another 4-well dish. Both dishes will be then cultivated in the incubater 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
Add á 25 µl of 0,1% Hyaluridonase in each well of 4-well dish with oocytes intended to fixation after 24 h maturation and mix up and down by pipeting. The enzyme disrupts the contacts between cumulus cells and oocytes. After 3-5 min transfer oocytes into the drop of HTF medium placed in 6 cm Petri dish by glass capilar. Remove the rest of cumulus cells by aspirating of oocytes with narrow glass capilar with diameter corresponding to the size of entire oocyte. After this step observe oocytes and place emphasis on clearly visible zona pellucida. Fix oocytes as follows:
Dry the microscopic slide placed in the ethanol bath by paper towel. Immerse the cover slip into the same bath and dry it out in the same manner. Apply small piece of vaseline from injection syringe into future corners with respect to the size of the cover slip (it is very important that the cover slip will be placed in the bottom of microscopic slide after transfering of oocytes). Transfer denuded oocytes into the center of field framed by the vaseline spots Put carefully the coverslip on the vaseline and push the corners by tweezers. BE CAREFULL, OOCYTES ARE FRAGILE AND CAN BE DAMAGED BY THE STRONG PRESSURE. Put the prepared specimen into the cuvet with 12 ml of fixative (methanol:acetic acid, 3:1).
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 1.000 g (2.650 RPM - centrifuge MPW-340) for 5 min. Remove the supernatant by Pasteur pipette and add 7 ml of strille 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. Add 5 µl of Mitotracker (1 mM roztok v DMSO) and 2 µl of Hoechst 33258 (1 mg/ml). Mix well by aspirating. Apply 990 µl of destilate water into 1,5 ml Eppendorff 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. Incubate the rest of beginning suspension at 17°C for 30-45 min.
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 multiplicate the result by 100.
Meanwhile the part of working group count the sperm cells, others can denude fully maturated 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 stereobinolupe and place emphasis on finding the first polar body (it should be clearly visible under zona pellucida as a small sphere object). Presence of this polar body is the evidence that oocyte is fully matured (metaphase II)
Meanwhile the sperm cells incubated with vital dyes are labelled (30-45 min). Take the such volume from the sperm suspension so as afer dilution with 500 µl of fertilization medium (mTBM with 2 mg of BSA/ml in the 4-well dish) you obtain the final concentration of sperm cells 500.000/ml. Mix the suspension in the well and add the denuded oocytes. Incubate the dish for 1-2 h in the incubater at 38,5°C and 5% of CO2 atmosphere. Then you can observe the contact of both gametes under stereobinolupe and under inverted fluorescence microscopy. Sperm heads are labeled in blue, the neck and part of midpiece (mitochondrions) are stained in red.
2. Staining of specimens with oocytes fixed after 24 h maturation.
Take out the specimens from the cuvet with fixative and dry out the bottom. Apply the small volume of acetorcein solution on the upper edge of coverslip by 1 ml Pasteur pipete. Put the small piece of paper towel to the bottom edge of coverslip and let the acetoorcein solution fill the whole area under coverslip. After 5 min apply the small volume of acetoglycerol on the upper edge of the coverslip and in the same manner let the solution fill the area with oocytes. During application of acetoglycerol check the oocytes under stereobinolupe (oocytes can migrate. If so, carefully push the vaseline corners by tweezers) After this apply the nail polish around the vhole coverslip to prevent evaporation. Observe the specimen under microscopy with phase contrast. Oocyte cytoplasm is stained in pink. Chromosomes in metaphase I figure are in dark red.
III PART. EMBRYONIC DEVELOPMENT OF C. ELEGANS
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.
