Table 1.
Maturation and competence of oocytes derived from cultured follicles in mice
| Strain | Age | Initial follicle stage and isolation method | Culture system and interval | Basal media, supplement and O2 level | Oocyte assessment and outcomes | References |
|---|---|---|---|---|---|---|
| B6SJLF1 | Postnatal Day 0 |
Ovarian organ culture followed by collagenase/DNase isolation of oocyte-granulosa cell complexes from primordial follicles; |
Original protocol
1. Ovarian tissue culture for 8 days; 8 ovaries per membrane insert for 8 days 2. Granulosa cell-oocyte complexes individual culture on transwell membranes in wells for 8 days; 3. Granulosa cell-oocyte complexes individual culture on membranes in wells for 6 days 4. Granulosa cell–oocyte complexes individual culture on membranes for 15 h–17 h |
Original protocol
1. Waymouth + pyruvic acid, 3 mg/ml BSA, 1 mg/ml fetuin, 5 μg/ml insulin, 0.5 ng/ml rhFSH, 1 ng/ml EGF 2. Waymouth + pyruvic, 3 mg/ml BSA, fetuin, 100 ng/ml rhFSH Revised protocol 3. αMEM, 3 mg/ml BSA, 1 mg/ml fetuin, 5 μg/ml insulin 4. αMEM, 3 mg/ml BSA, 1 mg/ml fetuin, 5 μg/ml insulin,100 ng/ml rhFSH, 1 ng/ml EGF 1–4: 5% O2, 5% CO2, 90% N2 |
Original protocol 17% MII 33% cleaved 2-cell 15% blastocyst; n = 20 Hatching blastocysts rarely seen Revised protocol 44% MII 53% cleaved 2-cell 23% blastocyst; n = 75 Hatching blastocysts observed 59/1160 (5.7%) live pups |
[30] |
| C57BL/6 × CBA/Ca | 32-day old | Primary follicles Mechanical |
1. Individual culture in 96-well V- or U-well plates for 5–6 days 2. Individual culture with or without rhLH on final day of culture 3. COCs removed, IVM with or without EGF |
1. αMEM, 5% serum from 3–4 week old mice, 5 μg/ml insulin, 1 IU/ml rhFSH, 2. 0 or 1 IU rhLH added on Day 5 3. Leibovitz, 5% mouse serum, 15 mg/ml BSA or M16, 5% FCS, 1 IU/ml rhFSH, 10 ng/ml EGF 5% CO2 in air |
0% 2-cell or blastocyst without EGF 9% 2-cell, 100% blastocyst with EGF Day 6 41% 2-cell, 100% blastocyst with EGF Days 5–6 1 live pup from IVM + EGF |
[31] |
| C57BL/6 × CBA/Ca | 14-day old | Preantral – primary/ early secondary Pederson type 3b and 4 (100–130 μm diameter) Mechanical |
Individual culture in droplets under oil for up to 14 days; 12 days optimal 20 follicles/60 mm Petri dish |
1. αMEM glutamax, 5% heat-inactivated FBS, 5 μg/ml insulin; with 100 mIU/ml rhFSH or 100 mIU/m rhFSH +10 mIU/ml rhLH or100 mIU/ml rhFSH +100 mIU rhLH 2. IVM or ovulatory stimulus of 5 ng/ml hCG + 5–50 ng/ml EGF 5% CO2 in air |
23–30% MII on Day 9 without ovulatory stimulus 60% MII with ovulatory stimulus regardless of rhFSH alone or rhFSH + rhLH treatment 50% fertilization in vitro 50% hatched blastocysts Live offspring obtained (efficiency unknown) |
[32, 33] |
| C57BL/6 × CBA/Ca | 16-day old | Multi-layer secondary Pederson Type 5b (150–180 μm diameter) Two-layer secondary Mechanical |
1. 0.25–1.5% (w/v) alginate-embedded individual culture in 96-well plates for 8 days; followed by maturation 2. Alginate lyase to recover follicles for ovulatory maturation |
1. αMEM, 3 mg/ml BSA, 5 μg/ml insulin, fetuin,10 mIU/ml rhFSH 2. αMEM, 10% FCS, 1.5 mIU/ml hCG, 5 ng/ml EGF COC removed, 5% CO2 in air |
Multi-layer secondary in 1.5% alginate: 70/99 (71%) MII 60/86 (68%) fertilized 20 zygotes transferred to recipients 4 normal, fertile offspring (20%) Two-layer secondary in 0.25% alginate: 32/76 (42%) 2-cell embryos 9/32 (29%) blastocysts |
[35, 36] |
| CF1 B6SJLF1 |
12-day old 12-day old |
Secondary; mechanical Early secondary; collagenase digestion |
1. 0.25% (w/v) alginate-embedded individual culture in 96-well plates for 10 or 12 days 2. Alginate lyase to recover follicles for ovulatory maturation 1a. Collagen-impregnated membranes for 10 days 1b. Oocyte maturation of COCs |
1. αMEM, 3 mg/ml BSA, 5 μg/ml insulin, 1 mg/ml fetuin, 10 mIU/ml rhFSH 2. αMEM, 10% FCS, 1.5 IU/ml hCG, 5 ng/ml EGF 1a. αMEM, 3 mg/ml BSA, 1 mg/ml fetuin, 5 μg/ml insulin 1b. αMEM, 3 mg/ml BSA, 1 mg/ml fetuin, 10 ng/ml EGF 5% CO2 in air |
Secondary 3D culture: 100/134 (75%) MII 19/100 (19%) fertilization 1/19 (5%) blastocyst rate Early secondary 2D culture: 64% MII 81% fertilization |
[37] |
| C57BL/6 × CBA/Ca | 8-day old | Ovarian organ culture followed by dissection of individual secondary follicles Mechanical |
1. Ovaries placed on well inserts in 24-well plates, 6 ovaries per well, for 4 days 2. 0.25% (w/v) alginate or fibrin-0.25% (w/v) alginate-embedded culture in 96-well plates for 12 days 3. Cumulus-enclosed oocytes removed from antral follicles for maturation |
1–2. αMEM, 3 mg/ml BSA, 1 mg/ml fetuin, 5 ng/ml insulin, 10 mIU/ml rhFSH, 1. αMEM, 10% FCS, 1.5 mIU/ml rhFSH, 5 ng/ml EGF 5% CO2 |
Alginate 59/96 (61%) MII 19/59 (33%) 2-cell embryos Fibrin-alginate 44/50 (88%) MII 24/44 (54%) 2-cell embryos |
[38] |
| BDF1 from C57BL/6 females × DBA/2 males | 16-day old 6-day old |
Preantral follicles (125–140 μm diameter) Primary/early secondary Pederson type 3a and 3b (<100 μm diameter) Mechanical |
1. Embedded in collagen gel for 9 days 2. Removed from gel with collagenase, transferred to 12-well plates with collagen-coated membranes for 8 days 3. Follicles removed from membrane, induce oocyte maturation |
1. αMEM, 5% FCS, 10 μg/ml insulin, 1 mIU/ml rhFSH, 1 ng/ml EGF 2. αMEM, 5% FCS, 10 μg/ml insulin, 100 mIU/ml rhFSH 3. Ovulatory stimulus of 2.5 mIU/ml hCG, 10 ng/ml EGF 1–2: 5% O2, 5% CO2, 90% N2 |
Preantrals 80/134 (60%) MII 47/80 (59%) fertilized 42/47 (89%) 2-cell 130 two-cell embryos cryopreserved; 53 transferred to 4 recipients; 3 pregnant; 2 live fertile offspring (4%) Early Secondary – no MII, no embryo/ET data |
[39] |
| B6D2F1 (C57BL/6 N × DBA/2) | 10-day old | Secondary follicles Mechanical |
1. 0.1% collagenase after isolation, placed on membrane in well plates for 12 days 2. COC removed from antral follicles for maturation |
1. αMEM, 2% PVP, 5% FBS, 0.1 IU/ml rhFSH; tested optimal MW for PVP and optimal O2 2. αMEM, 5% FBS, 0.1 IU/ml rhFSH, 1.2 IU/ml hCG, 4 ng/ml EGF 5% CO2 in air for PVP experiments 5, 7, 10 and 20% O2 for oxygen experiments |
PVP 360 K 164/230 (71%) MII 49/82 (60%) blastocyst PVP 1300 K 179/238 (75%) MII 34/54 (63%) blastocyst 7% O2 in PVP 360 K 277/366 (76%) MII 58/69 (84%) blastocyst 20% O2 in PVP 360 K 273/294 (93%) MII 70/117 (60%) blastocyst |
[40] |
| CBA × C57BL/6 | 12 days | Secondary (type 4) Mechanical |
1. 0.15% (w/v) alginate-encapsulation 2. COC removed for IVM 12 days |
1. αMEM, 5% FCS, 10 μg/ml insulin, 100 mIU/ml rhFSH, 10 mIU/ml rhLH, 1 mM L-carnitine 2. αMEM, 10% FCS, 1.5 mIU/ml hCG 6% CO2 in 94% air |
~50% MII ~40% 2-cell 2.3-fold increase in fertilization over control 40% blastocyst from surviving follicles 53% blastocyst from antral follicles 3.1-fold increase in blastocyst over control |
[98] |
2D, 2-dimensional; 3D, 3-dimensional; BSA, bovine serum albumin; CO2, carbon dioxide; COC, cumulus–oocyte complex; DNase, deoxyribonuclease; EGF, epidermal growth factor; ET, embryo transfer; FBS, fetal bovine serum; FCS, fetal calf serum; hCG, human chorionic gonadotropin; IVM, in vitro maturation; rhFSH, recombinant human follicle-stimulating hormone; rhLH, recombinant human luteinizing hormone; M16, media 16; αMEM, alpha minimum essential medium; MII, metaphase II; MW, molecular weight; N2, nitrogen; O2, oxygen; PVP, polyvinylpyrrolidone.