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

Summary

This protocol describes procedures for in vitro maturation of mouse oocytes, in vitro fertilization, and subsequent embryo culture to the blastocyst stage.

Abstract

Mouse oocyte in vitro maturation, combined with in vitro fertilization and subsequent embryo culture, enables the generation of preimplantation embryos under defined conditions. These procedures facilitate the investigation of the biological functions and molecular mechanisms underlying oocyte maturation, fertilization, and early embryo development. However, the successful application of this approach depends on careful handling of oocytes and sperm, appropriate culture conditions, and accurate assessment of developmental progression at each stage. Here, we describe methods for isolating immature cumulus–oocyte complexes (COCs) from mouse ovaries, in vitro maturation of oocytes, in vitro fertilization, and in vitro culture of embryos to the blastocyst stage. These procedures include preparing and equilibrating media and culture dishes, isolating and selecting COCs after ovarian dissection, collecting sperm and capacitating them, performing insemination under standardized conditions, and subsequently culturing embryos for developmental assessment. Developmental progression is evaluated using morphological criteria, including the formation of male and female pronuclei, cleavage to the two-cell stage, and blastocyst formation. This protocol can be integrated into studies of oocyte maturation and maternal factor function, providing a valuable tool for basic research in mammalian reproductive biology.

Introduction

Understanding the oocyte quality and developmental competence is essential for successful fertilization and subsequent embryonic development^1,2. Oocyte maturation requires not only accurate chromosome segregation during nuclear maturation, but also cytoplasmic maturation, including organelle redistribution and the accumulation of maternal mRNAs and proteins³. Early embryonic development relies on maternally provided mRNAs and proteins, and the maternal stores in the oocyte sustain preimplantation development. However, the identities and functions of many maternal regulators of mammalian embryogenesis remain incompletely understood⁴. Therefore, further systematic studies are needed to elucidate the mechanisms underlying oocyte maturation, fertilization, and embryo development.

The mouse is a widely used model system for investigating preimplantation embryo development. In vitro maturation (IVM) refers to the collection of immature oocytes from the ovary and their maturation under controlled laboratory conditions⁵. Subsequently, in vitro fertilization (IVF) enables mature oocytes to be co-incubated with capacitated sperm to generate zygotes for early embryonic development studies. However, relative to conventional IVF using in vivo–matured oocytes, IVM-derived oocytes often exhibit lower maturation and fertilization rates and reduced developmental competence⁶. These limitations are largely attributed to asynchrony between nuclear and cytoplasmic maturation under in vitro culture conditions. Moreover, meiotic spindle stability in mouse oocytes is sensitive to changes in pH and temperature during IVM, and spindle disturbance may contribute to maturation arrest⁷. Consequently, the reduced competence of IVM-derived oocytes remains a major barrier, limiting efficient embryo production for developmental and mechanistic studies.

Mouse preimplantation embryos can also be obtained by collecting in vivo–derived zygotes and culturing them in vitro (IVC). In vivo–derived zygotes collected from the oviducts provide a relatively synchronized starting point for experiments focused on post-fertilization events⁸. However, oocyte maturation and the accumulation of maternal factors occur prior to fertilization in vivo; thus, in vivo–derived zygotes are less amenable to experimental manipulation of maturation conditions or maternal stores to assess their effects on subsequent embryo development⁴. Therefore, optimizing IVM and IVF protocols and adopting standardized operating procedures with rigorous quality control (e.g., strict management of temperature, pH, and handling practices) are essential to minimize procedure-related variability and protect oocyte and embryo developmental competence.

Here, we describe an IVM–IVF–IVC protocol detailing media preparation, COCs isolation and selection, IVM and IVF procedures, and in vitro embryo culture (Figure 1). The protocol incorporates practical, stage-specific checkpoints for routine quality control, including first polar body (PB1) extrusion, formation of male and female pronuclei (2PN), two-cell cleavage, and blastocyst formation. This protocol enables reproducible and efficient generation of preimplantation embryos under defined conditions, supporting mechanistic studies, culture optimization, and downstream applications in developmental and reproductive biology.

Protocol

All animal experiments were approved by the Institutional Animal Care and Use Committee of Northwest A&F University, China (permit number: 20230503).

1. Animal maintenance

1. Procure wild-type female C57BL/6 and male DBA/2 mice and keep them in specific pathogen-free (SPF) conditions, with a 12 h light-dark cycle at a temperature of 20–22 °C and 55% ± 10% humidity. Provide a breeding diet and water ad libitum. House male mice singly (one male per cage).

2. Preparation of media

1. To prepare a 50 IU/mL PMSG working solution, dissolve 5,000 IU pregnant mare serum gonadotropin (PMSG) in 100 mL of sterile phosphate-buffered saline (PBS).
   NOTE: Aliquot the solution into 1 mL single-use portions and store them at –20 °C. Thaw each aliquot at room temperature immediately before use.
2. To prepare IVM culture medium, add the ingredients listed in Table 1 to 100 mL of Medium 199 (M199) base medium. Adjust the pH to 7.3–7.4, then sterile-filter the medium through a 0.22 µm filter into sterile medium bottles. Label the bottle “IVM” with initials, date made, and expiration date, and store at 4 °C for up to 1 month.
3. To prepare a 0.1% (w/v) hyaluronidase solution, dissolve 10 mg hyaluronidase in 10 mL of M2 medium.
   NOTE: Store in single-use aliquots of 1 mL at –20 °C and warm to 37 °C before use.

3. Preparation of dishes (Figure 2)

NOTE: Adjust the number of IVM, IVF, and IVC drops according to the expected numbers of COCs and embryos, using 20–30 COCs per 100 µL drop for IVM and IVF and 20–30 embryos per 100 µL drop for embryo culture.

1. Prepare an appropriate number of 35 mm oocyte collection dishes, each containing 2 mL of M2 medium supplemented with 0.2 mM 3-isobutyl-1-methylxanthine (IBMX). Use one dish for ovary puncture and COC collection (one dish per two females), and another for washing COCs. Prewarm the dishes to 37 °C before use.
2. Prepare IVM dishes.
   1. In a 60 mm dish, prepare five 100 µL drops of IVM medium for washing (label the dish as “IVM wash”).
   2. In a separate 60 mm dish, prepare multiple 100 µL drops of IVM medium for maturation (label the dish as “IVM”).
   3. Carefully add sufficient mineral oil to cover the drops, and equilibrate the dishes at 37 °C in a 5% CO₂ incubator for at least 2 h before starting oocyte collection.
3. Prepare IVF dishes.
   1. In a 60 mm dish, prepare one or two 300 µL drops of preequilibrated embryo culture medium for sperm release and capacitation (label the dish as “Sperm capacitation”).
      NOTE: The second capacitation drop is prepared as a reserve for cases in which sperm motility in the first drop is insufficient, a new epididymal sperm sample must be collected and capacitated, or sperm concentration needs to be adjusted before insemination.
   2. Prepare two separate 60 mm dishes, each containing five 100 µL drops of preequilibrated culture medium for washing (label the dish as “IVF wash”).
      NOTE: Use one “IVF wash” dish to wash IVM-matured COCs before insemination, and use the other “IVF wash” dish to wash presumptive zygotes after co-incubation to remove cumulus cells and residual sperm.
   3. In a separate 60 mm dish, prepare multiple 100 µL drops of preequilibrated embryo culture medium for insemination (label the dish as “IVF”).
      NOTE: Preequilibrated embryo culture medium is used for both sperm capacitation and insemination to maintain consistent conditions during sperm preparation and fertilization.
   4. Carefully add sufficient mineral oil to cover the drops, and equilibrate the dishes at 37 °C in a 5% CO₂ incubator for at least 12 h before starting the sperm capacitation procedure.
4. Prepare IVC dishes.
   1. In a 60 mm dish, prepare five 100 µL drops of preequilibrated embryo culture medium for washing embryos (label the dish as “IVC wash”).
   2. In a separate 60 mm dish, prepare multiple 100 µL drops of preequilibrated embryo culture medium for embryo culture (label the dish as “IVC”).
   3. Carefully add sufficient mineral oil to cover the drops, and equilibrate the dishes at 37 °C in a 5% CO₂ incubator for at least 12 h before starting embryo culture.

4. Ovary collection

1. Administer 10 IU PMSG intraperitoneally to 8–10-week-old female C57BL/6 mice.
2. At 48 h after PMSG injection, euthanize the mouse by cervical dislocation and position it supine. Spray the abdomen with 70% ethanol to wet the fur and prevent it from entering the abdominal cavity.
3. Using fine dissecting scissors, make a small lower abdominal skin incision and gently open the skin with gloved hands to expose the peritoneal membrane.
4. Open the peritoneal membrane and gently move 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.
5. Use fine dissecting scissors to separate the ovary from the uterine horn, and trim away excess connective tissue and fat.
6. Immediately transfer the ovaries to pre-warmed M2 medium supplemented with IBMX. Repeat the procedure to collect the contralateral ovary and transfer it to the same dish.
7. Place the dish under a stereomicroscope and proceed with follicle puncture and COCs collection.

5. COCs collection and culture

1. Under a stereomicroscope, gently puncture visible antral follicles with a sterile syringe needle to release COCs in the M2 medium.
   NOTE: When collecting mouse GV-stage oocytes, preferentially puncture visibly large antral follicles on the ovarian surface, which typically appear bulging and semi-translucent with a clear antral cavity.
2. Using a finely pulled glass Pasteur pipette, collect the released COCs and transfer them to a clean 35-mm dish containing pre-warmed M2 medium supplemented with IBMX.
3. Wash the COCs by serial transfer to a clean area of the 35 mm dish as needed to remove detached granulosa cells and tissue debris, leaving debris behind at each transfer (Figure 3).
   NOTE: Use only COCs with three or more compact cumulus-cell layers and uniformly granulated cytoplasm without clear spaces for the subsequent steps^9,10. Denuded GV oocytes and partially cumulus-enclosed GV oocytes are excluded from this protocol. Under our experimental conditions, an average of 26 immature COCs was obtained per female mouse.
4. After debris removal, transfer the COCs to the IVM wash dish and wash them by sequential transfer through five 100 µL drops of IVM wash medium.
   NOTE: During transfers, minimize carryover volume to prevent cross-contamination between drops.
5. Transfer the washed COCs into 100 µL drops of IVM medium (20–30 COCs per drop) and culture at 37 °C in 5% CO₂ for 14 h.
   NOTE: Collect, wash, and transfer COCs to IVM medium with minimal delay to help maintain developmental potential.
6. (Optional) After IVM, briefly incubate the COCs in 1 mg/mL hyaluronidase and gently pipette them repeatedly with a micropipette until the cumulus cells are completely removed.
   NOTE: Limit hyaluronidase exposure to no more than 3 min, as prolonged exposure may damage the oocytes.
7. (Optional) Wash the denuded oocytes in fresh M2 medium on a heated stage, then assess oocyte maturation under a stereomicroscope.
8. (Optional) Oocytes with an extruded first polar body are considered mature and at the metaphase II (MII) stage. Calculate the maturation rate as the number of oocytes with an extruded first polar body divided by the total number of oocytes cultured.
   NOTE: Steps 5.6–5.8 are used to assess oocyte maturation after IVM by denuding a designated assessment cohort before PB1 scoring. These steps are not required for COCs that proceed directly to IVF.

6. Sperm collection and preparation

NOTE: This section describes the collection and capacitation of fresh sperm. Prior to insemination, freshly released sperm should be incubated in preequilibrated embryo culture medium at 37 °C for 1 h to allow capacitation.

1. Euthanize one mature male DBA/2 mouse (10–16 weeks of age) and remove its cauda epididymides, avoiding as much fat, blood, and tissue fluid as possible (Figure 4).
2. For each male, transfer both cauda epididymides into a 300 µL drop of preequilibrated G-IVF medium for sperm release and capacitation.
3. Immobilize the cauda epididymis using a pair of forceps, make five to seven cuts in the cauda epididymis with a sterile syringe needle, and gently squeeze the tissue to release concentrated sperm.
   NOTE: Evaluate sperm motility and proceed only when the majority of sperm show progressive motility (PR), defined as active forward movement, rather than non-progressive motility (NP) or immotility¹¹.
4. Incubate the dish at 37 °C in 5% CO₂ for 1 h to allow sperm capacitation.

7. In vitro fertilization

1. After 14 h of IVM, wash the COCs by sequential transfer through five 100 µL drops of IVF wash medium, and then transfer the washed COCs into the IVF insemination drops (20–30 COCs per 100 µL drop).
2. Preincubate the fertilization dish containing COCs at 37 °C in 5% CO₂ for 30 min before insemination.
3. After capacitation, determine sperm concentration using a hemocytometer.
4. Based on the sperm concentration, calculate the required inoculum volume and add capacitated sperm to each IVF drop to achieve a final concentration of 4 × 10⁵ –1 × 10⁶ sperm/mL.
   NOTE: After sperm addition, confirm that motile sperm are present in each insemination drop.
5. Incubate the IVF dishes containing COCs and sperm at 37 °C in a 5% CO₂ incubator for 4–6 h.

8. Embryo culture

1. After 4–6 h of incubation, use a finely pulled glass Pasteur pipette with an inner diameter slightly larger than the oocyte to aspirate presumptive zygotes in minimal volume and transfer them into the IVF wash drops.
2. Wash the presumptive zygotes by sequential transfer through five IVF wash drops, gently pipetting within each drop to remove excess sperm and debris (Figure 5).
3. Transfer the washed presumptive zygotes to the IVC wash dish and wash them by sequential transfer through five prewarmed embryo culture medium wash drops, minimizing carryover of IVF medium.
4. Place the presumptive zygotes into embryo culture medium culture drops and culture at 37 °C in 5% CO₂ (20–30 embryos per 100 µL drop).
5. At 6–8 h after insemination, assess fertilization under a stereomicroscope by scoring the presence of male and female pronuclei, and discard oocytes that retain only one polar body and show no visible pronuclei.
6. At 24 h after insemination, count the number of two-cell embryos and calculate the two-cell rate as (two-cell embryos / inseminated oocytes).
7. After counting, remove and discard the uncleaved embryos, and continue culturing the remaining cleaved embryos.
   NOTE: At this stage, embryos may be vitrified, transferred to recipient females, or maintained in culture until the blastocyst stage.
8. Continue to culture the embryos under the same incubation conditions until 96 hours post-insemination (hpi). Assess blastocyst formation at 96 hpi based on morphological criteria.
   NOTE: No further medium change is required during this period. A normal blastocyst is identified by the presence of a visible blastocoel cavity, a distinct inner cell mass, and an intact trophectoderm layer.

Results

Using this protocol, mouse COCs were sequentially processed through in vitro maturation, in vitro fertilization, and subsequent in vitro embryo culture, resulting in reproducible generation of preimplantation embryos and development to the blastocyst stage under the conditions described. After 14 h of IVM, COCs typically showed cumulus expansion and intact oocyte morphology. Oocyte meiotic maturation was quantified by denuding a designated assessment cohort after IVM and scoring PB1 extrusion (...

Discussion

This protocol provides a standardized procedure for mouse oocyte in vitro maturation, in vitro fertilization, and in vitro culture using immature COCs, enabling reproducible generation and evaluation of preimplantation embryos through the blastocyst stage under defined in vitro conditions. In many studies, IVM outcomes are sensitive to laboratory-specific differences in media, incubation times, and handling, which can reduce developmental competence after IVM¹²<...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35 mm Petri dish	Thermo Fisher Scientific	150255
60 mm Petri dish	Thermo Fisher Scientific	150270
Bovine Serum Albumin	Sigma-Aldrich	A8806
Breeding diet (rodent breeder chow)	Keao Xieli (Beijing ,China)	N/A
CO2 incubator	Thermo Fisher Scientific	HERAcell 150i
D-glucose	Sigma-Aldrich	G7021
Disposable Pasteur Pipet	Kimble	DWK63A53WT-1000EA
Epidermal growth factor	Gibco	PHG0313
Fine dissecting scissors	Sigma-Aldrich	S3146-1EA
Fine-tip forceps	Rhino	SW14
Follicle-stimulating hormone	Millipore	869001
G-1 PLUS	Vitrolife	10128
G-IVF PLUS	Vitrolife	10136	(preequilibriated) embryo culture medium
Hemocytometer	Millicell	MDH-4N1
Hyaluronidase	Sigma-Aldrich	H4274
IBMX (3-Isobutyl-1-methylxanthine)	Selleck	S5836
KnockOut Serum Replacement	Gibco	A3181501
L-Cysteine	Sigma-Aldrich	C7352
Luteinizing hormone	Millipore	869003
M2 medium	Sigma-Aldrich	M7167
Medium 199	Gibco	11150-059
Micropipette	Gilson	F167350
Mineral oil	Sigma-Aldrich	M8410
Phosphate-buffered saline	Gibco	20012027
Pregnant Mare Serum Gonadotropin	Ningbo Second hormone factory (Ningbo, China)	N/A
Sodium pyruvate	Sigma-Aldrich	P4562
Stereo microscope	Olympus	SZX10
Sterile medium bottles	Nalgene	46600-586
Sterile syringe needle	BD Biosciences	309623	1 mL, 27G1/2
Syringe Filter Unit	Millipore	SLGP033R
Thermo Plate	Tokai Hit	TPi-SZX2X