Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

All zebrafish were handled in strict accordance with good animal practice, as defined by the relevant national and/or local animal welfare bodies, and all animal work was approved by the appropriate committee (University of Wisconsin-Madison assurance number A3368-01). Animals were maintained under standard conditions at 26.5 °C.

1. Pre-selection of Females

NOTE: Oocytes in adult females typically span a range of developmental stages, from stage I to V (Figure 2). Purging the females of preexisting oocytes through a successful natural mating increases the synchronization of oocyte staging, as newly developing oocytes appear as a cohort^3,14. Within about 8 days post-purge, most oocytes are in early stage IV, which is optimal for the initiation of in vitro maturation. Such synchronization increases the yield of oocytes that can go through the full process of in vitro maturation, facilitating experimental manipulation. See previous descriptions¹³ for details on setting up fish in paired matings and on fish water composition (here, 14 g of ocean salt and 150 g of NaHCO₃ per 1,000 L of reverse-osmosis water, pH 6.5-8.5 (preferred range: 6.8-7.5) and a conductivity of 180-360 μS). See Brand et al.¹³ for additional recipes.

1. Eight to ten days prior to the in vitro culture manipulation, use a fish net to transfer a single male and a single female fish of the desired strain to a mating tank. Pair multiple sets. Leave them in the mating tank overnight. Allow them to mate during the evening and through the afternoon of the following day.
2. Use a fish net 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. The amount of food should be enough to provide approximately 20 min, but not more, of feeding time.

2. Preparation of Maturation Medium

NOTE: Prepare maturation medium on the day of the in vitro maturation experiment, within 1 h of removing the oocytes from the female (see section 3).

1. Add 20 mL of Leibovitz's L-15 medium with L-glutamine, pH 7.0, to a 50 mL conical tube under sterile conditions. Bring it to pH 9.0 with 10 N NaOH.
2. Add 9 mL of Leibovitz's L-15 medium, pH 9.0, to 2 separate 50 mL conical tubes. Label 1 tube "+DHP" and the other "-DHP."
3. In the +DHP tube, add 10 µL of 17α-20β-dihydroxy-4 pregnen-3-one (DHP), 490 µL of dH₂O, and 500 µL of 10% bovine serum albumin (BSA).
4. In the -DHP tube, add 100 µL of 10 mg/mL gentamycin, 400 µL of dH₂O, and 500 µL of 10% BSA.

3. Dissection of the Oocytes and Initiation of the In Vitro Culture

NOTE: The in vitro culture experiment is carried out with pre-selected females 8-10 days after they release eggs through natural mating. Use maturation medium made the same day (see section 2). Oocytes mature appropriately if the dissection is begun near the end of the fish diurnal cycle (determined in the laboratory by pre-set artificial lighting in the fish facility)¹³, possibly mimicking processes occurring through natural mating. This implies that most steps in the protocol must be carried out in the evening if fish are housed in a facility with a standard light cycle (e.g., beginning step 3.2 at 6 pm in a facility with an 8 am to 10 pm light period), although other work time-periods are possible with an appropriately time-shifted light cycle.

1. Prepare a 0.2% tricaine stock solution in dH₂O, buffered to pH 7.0 with 1 M Tris, pH 9.0, and keep this solution at 4 °C. This can be prepared ahead of time.
2. Initiate the experiment 0-4 h prior to the end of the daily light cycle in the facility. In a 250-mL beaker, add 20 mL of 0.2% tricaine stock solution, pH 7.0, to 80 mL of fish water and mix.
3. Transfer the pre-selected females to tricaine solution and euthanize them by overexposure. Leave the females in the tricaine solution for 15 min after the cessation of gill movement.
4. Using a spoon, collect the euthanized fish from the tricaine solution and rinse them briefly in fish water. Place the fish onto a paper towel to absorb excess water.
5. Use a clean razor blade to decapitate the euthanized fish at the level of the pectoral fin. Using dissecting scissors, make a longitudinal incision on the ventral side of the fish, extending from the anterior end to the anal area.
6. Place the fish on a petri dish under a dissecting microscope with incident light. Using a pair of dissecting forceps, transfer ovary portions to a 35 x 10 mm culture dish containing 4 mL of Leibovitz's L-15 medium +DHP.
   NOTE: The ovaries, which contain developing oocytes, will appear as opaque and clumpy structures within the internal body cavity.
7. Using dissecting forceps, gently dissociate the oocytes from the follicular masses. Sort the early stage IV oocytes (Figure 2B; prior to GV breakdown, at near-maximal size and characterized by a dark, opaque cytoplasm and a readily apparent GV located asymmetrically within the oocyte). Discard oocytes at earlier stages (Figure 2A) and translucent, mature stage V eggs (similar to those in Figure 2C but present in the ovaries prior to DHP treatment).
   NOTE: Oocytes are selected for maturation in early stage IV, the earliest stage that results in mature, stage V oocytes after in vitro culture conditions (Figure 2C and D)³. Because stage IV oocytes are actively producing products, manipulation at this stage allows for the expression of exogenous products through RNA injection or for the reduction of gene function via introduced MOs.
8. Use a glass Pasteur pipet to transfer the early stage IV oocytes to a second 35 x 10 mm plastic culture dish containing 4 mL of Leibovitz's L-15 medium +DHP. Transfer minimal amounts of -DHP medium to the dish containing the +DHP solution.

4. Oocyte Microinjections

NOTE: Isolated stage IV oocytes undergoing in vitro maturation can be microinjected to introduce reagents for functional manipulation or tagged protein expression. The microinjection of reagents, such as mRNA and MOs (see section 4), is typically carried out when the oocytes are undergoing in vitro maturation in +DHP medium, prior to defolliculation. If desired, in order to allow more time to conduct manipulations prior to oocyte maturation, the injections can also be carried out in -DHP medium, prior to maturation, for at least 2 h. They can subsequently be transferred to +DHP medium.

1. Prepare mRNAs using a standard expression kit. Keep them at -80 °C as a 100-500 pg/µL stock for injection at a final concentration of 50-500 pg/µL (200 pg/µL is recommended) in RNA-grade water. Prepare MOs at a concentration of 4 ng/µL in water according to the manufacturer's instructions. Inject them at final concentrations of 2 ng/µL (or as empirically determined).
   NOTE: Injections are typically carried out in +DHP medium prior to defolliculation (see the note in section 5).
2. If injecting mRNA, immediately prior to the injection, dilute the mRNA using RNA-grade reverse-osmosis water and 0.2 M KCl to achieve a final solution of 0.1 M KCl.
3. If injecting MOs, immediately prior to the injection, dilute the MOs to the desired concentration using reverse-osmosis water and 0.2 M KCl to achieve a final solution of 0.1 M KCl.
4. Manually hold oocytes with fine forceps and inject approximately 1 nL into wild-type stage IV oocytes using a needle made with a pulled-glass capillary pipette.
   1. Prepare the glass needles by pulling heated glass capillary tubes, loading the solution to be injected, and breaking the needle tip with forceps, as previously described in protocols for standard injection into zebrafish early embryos¹⁵.
      NOTE: It is helpful if the needle has a gradual taper rather than an abrupt one; this allows more flexibility in terms of the location of the break in the needle tip and also helps to prevent damage to the injected embryo. Using the same needle and solution as for the embryo injections, adjust the injected volume by pre-determining the microinjector pressure settings that produce the desired volume. These settings can be determined by mock-injecting into a drop of mineral oil on a microscope stage calibration slide (0.01 mm) and adjusting the microinjector settings to obtain the desired diameter in the bolus of injected solution, as previously described¹⁵.

5. Oocyte Maturation and Defolliculation

NOTE: During oocyte maturation, the oocytes will become progressively translucent, which allows for the assessment of successful culture conditions.

1. Continue incubating the immature (and, if appropriate, injected) oocytes in +DHP medium at 26.5 °C, checking periodically (every 30 min) to ensure that the oocytes remain intact and are undergoing proper maturation by becoming progressively translucent (see Figure 2C).
   1. Remove any lysing oocytes with a Pasteur pipette and discard them in a lab waste beaker to maintain the quality of the medium. Exchange the culture medium with approximately a half-volume of fresh +DHP medium to maintain a clear solution, depending on the amount of oocyte lysis.
   2. Allow the in vitro maturation to proceed until a majority of the oocytes become translucent and have a GV that is no longer apparent (approximately 2 h in DHP treatment, see Figure 2C as compared to D). View them under a dissecting microscope with transmitted light optics.
2. Remove the outermost follicular membrane from each matured oocyte. 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 roll the oocyte out of the membrane while holding down the peeled part.
   NOTE: The underlying chorionic membrane will typically remain in close association with the egg during this process; after egg activation, it expands to form a protective layer for the embryo.
3. Transfer the defolliculated oocytes in a minimal volume of medium (typically, transfer about 5-10 mature oocytes in less than 20 µL) to a petri dish with a few drops of culture (+DHP) medium and proceed to fertilization.

6. Fertilization of In Vitro Cultured Oocytes

NOTE: The sperm solution on ice, prepared below, will maintain its potency for about 2 h.

1. Prepare a sperm solution near the end of the oocyte maturation step and prior to defolliculation using testes from five males in 500 µL of Hanks' solution, as described previously^16,17.
2. Add 10-50 µL of sperm solution to the defolliculated oocytes in +DHP culture medium. Wait 10 s.
3. Using a pipette, add a few drops of embryonic (E3) medium to the oocytes. Wait 1 min and then flood the plate with E3 medium.
   NOTE: The composition of E3 medium is as follows: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM MgSO₄, and 1-5% Methylene Blue¹³.
4. Repeat steps 5.3, 6.2, and 6.3 to obtain greater numbers of fertilized embryos.
5. Allow 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 fertilization¹⁷ and staging¹⁸.