May 25, 2026

Individual in vitro growth and maturation of oocytes derived from mouse secondary follicles

DOI
https://dx.doi.org/10.17504/protocols.io.14egn5p1qg5d/v1
  • Hidetaka Tasaki1
  • 1Assisted Reproductive Technology Center, Okayama University, Okayama, Japan
Hidetaka Tasaki
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DOI: https://dx.doi.org/10.17504/protocols.io.14egn5p1qg5d/v1
Protocol Citation: Hidetaka Tasaki 2026. Individual in vitro growth and maturation of oocytes derived from mouse secondary follicles. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn5p1qg5d/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 23, 2026
Last Modified: May 25, 2026
Protocol  Integer ID: 317803
Keywords: mouse, oocyte, secondary follicle, in vitro growth, IVG, in vitro maturation, IVM, individual follicle culture, cumulus–oocyte complex, COC, metaphase II oocyte, IVGM, ovary, reproductive biology, Biological techniques, oocyte growth to germinal vesicle, oocyte maturation, mouse oocyte, oocyte growth, germinal vesicle, maturation, Developmental biology, ovarian follicles, in vitro culture, tracing ovarian follicles growth, ovarian follicles growth, development of follicle, mouse follicles from secondary stage, mouse follicle, follicle, 3d culture, maturation of oocyte, maturation competence of each individual follicle, mouse secondary follicles this protocol, isolation of secondary follicle, derived oocyte, secondary follicle, mouse ovary, individual follicle, downstream analyses of oocyte quality, oocyte complex, follicle, oocyte quality, oocyte, individual in vitro growth, vitro growth, maturation, maturation competence, metaphase ii
Disclaimer
This protocol involves the use of mice. All animal procedures must be performed in accordance with institutional and national guidelines and approved by the relevant animal care and use committee or equivalent authority. Users should validate and optimize the procedure under their own laboratory conditions.
Abstract
This protocol describes the isolation of secondary follicles from mouse ovaries and their individual culture (one follicle per well) in low-attachment 96-well plates to support in vitro growth (IVG) over 12 days. Follicles that reach ≥300 μm in diameter are then subjected to in vitro maturation (IVM): the cumulus–oocyte complex (COC) recovered from each follicle is matured individually in a separate 10 μL drop to the metaphase II (MII) stage. Because both growth and maturation are performed one oocyte at a time, the growth trajectory and the maturation competence of each individual follicle-derived oocyte can be traced end to end. The procedure has been applied to ICR, C57BL/6 (B6), and B6D2F1 females across a wide age range (14–50 weeks). The resulting in vitro-grown and matured (IVGM) oocytes can be used for downstream analyses of oocyte quality and developmental competence.
Materials
Reagents
  1. Female mice — ICR, C57BL/6 (B6), or B6D2F1; 14–50 weeks old
  2. Leibovitz's L-15 medium (Gibco / Thermo Fisher Scientific, cat. no. 11415064)
  3. MEM α (αMEM) (Sigma-Aldrich, cat. no. M8042)
  4. M2 medium (Sigma-Aldrich, cat. no. MR-105-D)
  5. Fetal bovine serum (FBS) (Gibco / Thermo Fisher Scientific, cat. no. 10437-028)
  6. Poly(vinyl alcohol) (PVA) (Sigma-Aldrich, cat. no. P8136)
  7. Polyvinylpyrrolidone (PVP360) (Sigma-Aldrich, cat. no. PVP360)
  8. Antibiotic–antimycotic solution (Sigma-Aldrich, cat. no. A5955)
  9. Collagenase type IA (Sigma-Aldrich, cat. no. C9891)
  10. DNase I (FUJIFILM Wako Pure Chemical, cat. no. 043-26773)
  11. L-glutamine (Gibco / Thermo Fisher Scientific, cat. no. 25030081)
  12. 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) (Tokyo Chemical Industry, cat. no. G0394)
  13. Follicle-stimulating hormone (FSH; Gonal-f) (Merck)
  14. Insulin–transferrin–sodium selenite (ITS) (Sigma-Aldrich, cat. no. I3146)
  15. Penicillin–streptomycin (Gibco / Thermo Fisher Scientific, cat. no. 15140122)
  16. Epidermal growth factor (EGF) (Gibco / Thermo Fisher Scientific, cat. no. 53003-018)
  17. Human chorionic gonadotropin (hCG) (ASKA Animal Health)
  18. Hyaluronidase (Sigma-Aldrich, cat. no. H3506)
  19. Mineral oil (for IVM microdrops)


Equipment
  1. Low-attachment 96-well plates (Sumitomo Bakelite, cat. no. MS-9096V)
  2. 26 G needles (for mincing ovaries)
  3. Fine glass (mouth-controlled) capillaries, including one with an L-shaped tip (bent by heating) for seeding follicles into wells (Step 7)
  4. Culture dishes (e.g., 35 mm or 60 mm dishes for isolation; dish for IVM microdrops)
  5. Stereomicroscope with calibrated reticle / eyepiece graticule (for diameter measurement)
  6. CO₂ incubator (37 °C, 5% CO₂, humidified atmosphere)
  7. Micropipettes and sterile tips
  8. Syringe filters, 0.22 μm and 0.45 μm (use 0.45 μm for the PVP-containing IVG medium)
Before start
  • Pre-warm all culture media to 37 °C before use; equilibrate culture media (IVG and IVM) in the CO₂ incubator for at least 2–3 h before adding follicles/COCs.
  • Perform follicle isolation as quickly as possible to maintain follicle viability.
  • Handle follicles and COCs with fine glass (mouth-controlled) capillaries whose bore is matched to the size of the structure being moved, for gentle, low-stress transfer.
  • Maintain sterile technique and filter-sterilize all media after supplementation. IVG medium contains PVP and must be filtered through a 0.45 μm filter — a 0.22 μm filter will clog. Other media are filtered through 0.22 μm.
Procedure: Solutions
Handling medium — Dissolve all components in Leibovitz's L-15: 2.5% (v/v) FBS, 1 mg/mL PVA, and 1% (v/v) antibiotic–antimycotic solution. Filter-sterilize (0.22 μm) and store at 4 °C.
IVG medium
  • Base: Dissolve 1.5 mM AA-2G and 1% (v/v) penicillin–streptomycin in αMEM. Add 2% (w/v) PVP onto the surface of the medium and let it dissolve overnight at 4 °C. The next day, filter-sterilize through a 0.45 μm filter and store at 4 °C.
  • Add fresh on the day of use: L-glutamine, FBS, FSH, ITS.
  • Final composition: αMEM + 2%(w/v) PVP + 2 mM L-glutamine + 1.5 mM AA-2G + 5%(v/v) FBS + 0.1 IU/mL FSH + 1%(v/v) ITS + 1%(v/v) penicillin–streptomycin.
IVM medium
  • Base: Dissolve 1.5 mM AA-2G and 1% (v/v) penicillin–streptomycin in αMEM. Filter-sterilize (0.22 μm) and store at 4 °C.
  • Add fresh on the day of use: L-glutamine, FBS, FSH, EGF, hCG.
  • Final composition: αMEM + 2 mM L-glutamine + 1.5 mM AA-2G + 5%(v/v) FBS + 0.1 IU/mL FSH + 5 ng/mL EGF + 1.2 IU/mL hCG + 1%(v/v) penicillin–streptomycin.
Enzyme solution — Prepared on the day of use from frozen 100× stocks of collagenase type IA and DNase I, diluted in handling medium to 2 mg/mL collagenase type IA and 0.2 mg/mL DNase I. Warm before use.
Hyaluronidase solution — M2 medium supplemented with 1 mg/mL hyaluronidase; store frozen as aliquots and warm before use.
Procedure: Ovary collection and secondary follicle isolation
Euthanize female mice in accordance with your institutional animal care guidelines, and collect both ovaries.
Immediately mince the ovaries with 26 G needles in handling medium.
Transfer the minced ovarian tissue into enzyme solution and incubate at 37 °C for 20 min.
Transfer the ovarian fragments to fresh handling medium and release follicles by gentle pipetting. Use a wide-bore tip — cut the pipette tip to match the size of the tissue — to minimize mechanical damage to the follicles.
Under the stereomicroscope, carefully remove adhering stromal and thecal tissue from the isolated follicles by repeated pipetting with a fine glass capillary.
Select secondary follicles 100–120 μm in diameter with intact, healthy morphology (a centrally located oocyte enclosed by intact granulosa cell layers). Residual thecal/stromal tissue may remain after isolation. Repeated gentle pipetting is used to minimize attached stromal tissue, but complete removal cannot be confirmed reliably under the stereomicroscope. Apply the same cleaning criterion consistently across experiments.
Procedure: Individual IVG culture
Using a fine glass capillary with the tip bent into an L-shape (formed by heating), transfer one follicle per well into a low-attachment 96-well plate containing 100 μL of IVG medium per well. Do not use the outermost ring of wells (the perimeter row and column) for culture; instead, fill these wells with autoclaved sterile water to limit evaporation from the medium-containing wells.
Culture the follicles for 12 days at 37 °C in a humidified atmosphere of 5% CO₂.
Replace half (50 μL) of the medium with fresh IVG medium every other day.
(Optional) Record follicle diameter at defined intervals under the stereomicroscope to trace the growth dynamics of each individual follicle (see Anticipated results).
Procedure: Individual IVM culture
After 12 days of culture, select follicles that have grown to ≥300 μm in diameter and transfer them to IVM medium.
Mechanically isolate the cumulus–oocyte complex (COC) from each follicle using a fine glass capillary.
Place one COC per 10 μL drop of IVM medium under mineral oil and culture at 37 °C for 16-17 h in a humidified atmosphere of 5% CO₂. Maturing each COC individually keeps it linked to its source follicle, so the MII outcome can be traced back to each individual follicle (see Note 2).
Procedure: Cumulus removal and MII oocyte selection
After maturation, completely remove the cumulus cells by gentle pipetting in hyaluronidase solution (M2 + 1 mg/mL hyaluronidase).
Select fully denuded oocytes that have extruded the first polar body as MII oocytes.
Notes / Troubleshooting
Enzymatic digestion. Over-digestion damages follicles and can denude oocytes; under-digestion leaves stromal/thecal tissue attached. Adjust the 20 min incubation if needed for your ovary batch, and complete mechanical cleaning promptly.
Individual traceability. Each follicle is grown in its own well and each COC is matured in its own 10 μL drop, so the entire IVG→IVM trajectory of every oocyte stays linked to its source follicle.
Timing
Ovary collection and follicle isolation: ~1–2 h IVG culture: 12 days IVM culture: 16–17 h Cumulus removal and MII selection: ~30 min Total: ~13 days
Protocol references
Morohaku, K. et al. Complete in vitro generation of fertile oocytes from mouse primordial germ cells. Proc. Natl. Acad. Sci. U. S. A. 113, 9021-9026, doi:10.1073/pnas.1603817113 (2016).