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The overall goal of this protocol is to carry out the in vitro maturation of zebra fish oocytes and, if desired, to functionally manipulate maternal gene products, prior to fertilization. This method can help answer key questions in the field of developmental biology, pertaining to the importance of maternal contribution to the embryo. The main advantage of this technique is to culture oocytes in-vitro, followed by fertilization and production of viable embryos.

And, if desired, allows for functional manipulation of maternal gene products that act in the early embryo immediately after fertilization. To begin, eight to 10 days prior to the in vitro culture manipulation, use a fishnet to transfer multiple sets of a single male and a single female fish of the desired strain to a mating tank. Leave them in the mating tank overnight.

After allowing the fish to mate through the following afternoon, use a fishnet to separate the females that yield eggs during mating and place them into a separate tank. Feed these females twice daily with a food mixture containing approximately an equal amount of brine shrimp and fish food flakes. Under sterile conditions, add 20 milliliters of Leibovitz's L-15 Medium with l-glutamine, PH 7.0, to a 50 milliliter conical tube.

Then, use 10 Normal sodium hydroxide to bring it to PH 9.0. Add 9 milliliters of the L-15 Medium to two separate 50 milliliter conical tubes. Then, label one tube DHP and the other DHP.

In the DHP tube, add 10 microliters of DHP, 490 microliters of DH2O, and 500 microliters of 10%BSA. In the DHP tube, add 100 microliters of 10 milligrams per milliliter gentamicin, 400 microliters of DH2O, and 500 microliters of 10%BSA. To carry out dissection of oocytes, begin by preparing a 0.2%tricaine stock solution in DH2O, buffered to PH 7.0 with one molar tris PH 9.0.

Keep this solution at 4 degrees Celsius. Zero to four hours prior to the end of the daily light cycle in the facility, in a 250 milliliter beaker, add 20 milliliters of 0.2%tricaine stock solution, PH 7.0, to 80 milliliters of fish water and mix. It is critical to initiate the procedure within four hours of the end of the day light cycle to which the fish are accustomed.

Otherwise, the oocytes will not mature properly. Using a spoon, collect the euthanized fish from the tricaine solution, and rinse them briefly in fish water. Then, place the fish onto a paper towel to absorb excess water.

Use a clean razor blade to decapitate the euthanized fish at the level of the pectoral fin. Then, with dissecting scissors, make a longitudinal incision on the ventral side of the fish, extending from the anterior end to the anal area. Place the fish on a Petri dish under a dissecting microscope with incident light.

Then, using a pair of dissecting forceps, isolate ovary portions and transfer the tissue to a 35 by 10 millimeter culture dish, containing four milliliters of Leibovitz's L-15 Meidum, DHP. Now, with dissecting forceps, gently dissociate the oocytes from the follicular mass. Sort the early stage four oocytes, which are at near maximal size, and characterized by a dark opaque cytoplasm, and a readily-apparent germinal vesicle, or GV, located asymmetrically within the oocyte.

Discard oocytes at earlier stages, and translucent mature stage 5 eggs. Use a glass pasture pipette to transfer the early stage four oocytes to a second 35 by 10 millimeter plastic culture dish, containing four milliliters of Leibovitz's L-15 Medium, plus DHP. Transfer minimal amounts of minus DHP medium to the dish, containing the plus DHP solution.

If injecting mRNA, immediately prior to the injection, use RNA grade reverse osmosis water and 0.2 molar potassium chloride to dilute the MRNA to a final solution of 0.1 molar potassium chloride. If injecting Morpholinos, or MOs, immediately prior to the injection, dilute the MOs to the desired concentration using RNA grade reverse osmosis water, and 0.2 molar potassium chloride, to achieve a final concentration of 0.1 molar potassium chloride. Then, to carry out injection, manually hold oocytes with fine forceps, and, using a needle made with a pulled glass capillary pipette, inject approximately one nanoliter into wild-type stage four oocytes.

Continue incubating the immature and injected oocytes in DHP medium at 26.5 degrees Celsius. Checking approximately every 30 minutes to ensure that the oocytes remain intact, and are undergoing proper maturation, by becoming progressively translucent. Use a pasture pipette to remove any lysing oocytes.

And discard them in a lab waste beaker, to maintain the quality of the medium. With approximately half of the volume of fresh DHP medium, exchange the culture medium to maintain a clear solution, depending on the amount of oocyte lysis. When the majority of the oocytes have become translucent, and have a GV that is no longer apparent, use extra-fine forceps to make a tear in the follicular membrane, in a region with increased space between the oocyte and the membrane.

Peel off a portion of the membrane, and while holding down the peeled portion, roll the oocyte out of the membrane. Another critical step is the removal of the follicular membrane. If this layer is not fully removed, fertilization and the chorion expansion will be impeded.

Transfer the defolliculated oocytes in a minimal volume of medium, to a Petri dish with a few drops of culture DHP medium, and proceed to fertilization. Near the end of the oocyte maturation step, and prior to defolliculation, prepare a sperm solution using testes from five males in 500 microliters of Hank's Solution. Add 10 to 50 microliters of sperm solution to the defolliculated oocytes in DHP culture medium.

Then, wait 10 seconds. Using a pipette, add a few drops of E3 medium to the oocytes. Wait one minute, and then use E3 medium to flood the plate.

After allowing the fertilized embryos to develop, use a dissecting microscope with transmitted light optics to observe the progression through the cleavage stages to ensure successful fertilization, as previously described for fertilization and staging. As seen by the expression of GFP, oocytes from in vitro culture are able to produce protein from exogenous mRNA throughout oocyte development in as little as two hours. However, only oocytes that initiate culture conditions at stage four of oogenesis can develop into mature stage five oocytes.

In this experiment, injected wild-type aurora B mRNA rescues the phenotypic effects of a mutation in its corresponding gene, cellular island. The embryos from a control group are also allowed to develop to show the corresponding mutant phenotype. The injection of a translation-blocking Morpholino can successfully phenocopy the known mutant phenotype, as shown by the injection of lrmp-MO into oocytes, which mimics the mutant phenotype of the corresponding mutation futile cycle.

mRNA coding can also be injected into either wild-type or mutant oocytes to visualize the corresponding products within the early embryo. For example, as seen here, Sass6-mCherry protein localizes to foci labeled by the centrosome marker Gamma tubulin. Once mastered, this technique can be done in approximately 5 to 6 hours, if it is performed properly.

While attempting this procedure, it is important to remember not to rush the procedure, and allow for proper maturation of oocytes before defolliculation and fertilization. Following this procedure, other methods like fixation of embryos, can be performed in order to answer additional questions. Such as rescue of your gene of interest, or localization of RNA or protein.

After its development, this technique paved the way for researchers in the field of early embryo genesis to explore the contribution of maternal gene products in zebrafish. After watching this video, you should have a good understanding of how to functionally manipulate maternal gene products via in vitro maturation of zebra fish oocytes.