Mouse Oocyte In Vitro Maturation, Fertilization, and Culture of Preimplantation Embryos

Our research focuses on controlled in vitro maturation and fertilization of mouse oocyte, an early embryonic development of pre-implantation embryos. Existing methods often separate in vitro maturation from in vitro fertilization in embryo culture. Our protocol integrated these steps and support efficient developmental rates.

To begin, place the euthanized female C57BL/6 mouse aged 8 to 10 weeks in a supine position 48 hours after pregnant mare serum gonadotropin injection. Spray the abdomen with 70%ethanol to wet the fur and prevent hair from entering the abdominal cavity during ovary isolation. Use fine dissecting scissors to make a small lower abdominal skin incision and gently open the skin with gloved hands to expose the peritoneal membrane.

After opening the peritoneal membrane and gently moving the intestines aside, locate the uterine horn and follow it toward the ovary. Identify the ovary near the kidney, typically enclosed by a fat pad. Use fine dissecting scissors to separate the ovary from the uterine horn and trim away excess connective tissue and fat.

Immediately transfer the ovaries to pre-warmed M2 medium supplemented with Isobutyl methylxanthine or IBMX. Then collect the contralateral ovary and transfer it to the same dish. Place the dish under a stereo microscope and visualize the ovary prior to follicle puncture and cumulus-oocyte complexes or COCs collection.

For COCs collection, gently puncture visible antral follicles with a sterile syringe needle to release COCs in the M2 medium. Use a finely pulled glass Pasteur pipette to collect the released COCs. Transfer them to a clean 35 millimeter dish containing two milliliters of pre-warmed M2 medium supplemented with 0.2 millimolar IBMX.

Wash the COCs by serially moving them to clean areas of the dish, leaving detached granulosa cells and tissue debris behind. After debris removal, transfer the COCs to the in vitro maturation or IVM wash dish and wash them by sequential transfer through three 100 microliter drops of IVM wash medium. Transfer the washed COCs into 100 microliter drops of IVM medium with 20 to 30 COCs per drop.

Culture them at 37 degrees Celsius in 5%carbon dioxide for 14 hours. Remove the cauda epididymis from a 10 to 16-week-old euthanized DBA2 male mouse avoiding fat, blood, and tissue fluid. Transfer both cauda epididymis into a 300 microliter drop of pre-equilibrated G-IVF medium for sperm release and capacitation.

Immobilize the cauda epididymis using a pair of forceps. Make five to seven cuts in the cauda epididymis with a sterile syringe needle and shred the tissue to allow the release of concentrated sperm into the media. Incubate the dish at 37 degrees Celsius in 5%carbon dioxide for one hour to allow sperm capacitation.

After 14 hours of in vitro maturation, wash the COCs by sequential transfer through three 100 microliter drops of in vitro fertilization or IVF wash medium. Transfer the washed COCs into the IVF insemination drops with 20 to 30 COCs per 100 microliter drop. Pre-incubate the fertilization dish containing COCs at 37 degrees Celsius in 5%carbon dioxide for 30 minutes before insemination.

After capacitation, determine the sperm concentration using a hemocytometer. Based on the sperm concentration, calculate the required inoculum volume. Add capacitated sperm to each IVF drop to achieve a final concentration of four to 10 times 10 to the power of five sperm cells per milliliter.

Incubate the IVF dishes containing COCs and sperm at 37 degrees Celsius in a 5%carbon dioxide incubator for four to six hours. After incubation, aspirate the presumptive zygotes with a finely pulled Pasteur pipette in minimal volume. Transfer them into the IVF wash drops.

Gently pipette within each drop to remove excess sperm and debris. Wash the presumptive zygotes by sequential transfer through three IVF wash drops. Move the washed presumptive zygotes to the in vitro culture or IVC wash dish and sequentially transfer them through three pre-warmed embryo culture medium drops, minimizing IVF medium carryover.

Place the presumptive zygotes into embryo culture medium with 20 to 30 embryos per 100 microliter drop. Culture them at 37 degrees Celsius in 5%carbon dioxide. After six to eight hours, assess fertilization by scoring the presence of male and female pronuclei, and discard oocytes that retain only one polar body and show no visible pronuclei.

After 24 hours, check and count the number of two-cell embryos and calculate the two-cell rate as the number of two-cell embryos divided by the number of inseminated oocytes. Next, remove and discard uncleaved embryos. Continue to culture the embryos under the same incubation conditions until 96 hours post-insemination.

Assess blastocyst formation at 96 hours post-insemination based on morphological criteria. After 14 hours of in vitro maturation, COCs typically exhibit cumulus expansion and intact morphology, allowing quantification of myotic maturation by denuding a designated cohort and scoring first polar body or PB1 extrusion. The average maturation rate based on PB1 extrusion rates across three independent replicates was 81.3%Following insemination, fertilization was assessed after six to eight hours by scoring the formation of male and female pronuclei.

Cleavage was evaluated 24 hours after insemination using the two-cell rate. The average two-cell rates across three independent replicates were 87.6%The developmental competence was evaluated by further culturing embryos in vitro, and by assessing blastocyst formation at 96 hours post-insemination. The average blastocyst formation rate across three independent replicates was 52.6%Poor quality cleavage outcomes include embryos that fail to cleave, fragmented embryos, or embryos with unevenly sized blastomeres.

Suboptimal developmental outcomes during extended culture included arrest at the two-cell stage, poorly expanded blastocysts, or embryos with obvious fragmentation and abnormal morphology. Stable temperature, optimal kits, and careful handing are critical for successful embryonic development under controlling visual conditions. Embryos generated using this protocol can be utilized for imaging, gene expression analysis, vitrification, or embryo transfer.

Researchers can use this protocol to evaluate the genetic, pharmacological, or counter-condition effects on embryonic development.