Table 2. Chronological advances in in vitro culture of ovine preantral follicles*.
| References/Base medium | Follicle Category/Culture system | Tested compounds/Main findings |
| Arunakumari et al., 2010 - TCM199 Bicarbonate | Secondary - Isolated 2D vs. 3D (Agar-coated plates - 6 days) | Different concentrations and association of ITS, IGF-I, insulin, GH, and TGF-β with 1 µg/ml T4 + 2 µg/ml FSH (Microdrops vs. Agar gel) - Combination of 1µg/ml T4, 2 µg/ml FSH, 10 ng/ml IGF-I, and 1 mIU/ml GH ↑ antrum formation, MII oocytes No differences between culture systems. 25% 2-cell embryo, and 16% morula. |
| Barboni et al., 2011 - α-MEM (2% FCS, 1% ITS ,1 µg/ml FSH) | Secondary - Isolated 2D (14 days) | Oocyte nuclear epigenetic maturation from early antral follicles grown (in vivo vs. in vitro) - Similar methylation profile in in vivo and in vitro. Similar oocyte maturation, fertilization, and embryo production from in vivo and in vitro grown EAFs. 32% Fertilization, 90% <16-cell embryos and 10% >16-cell embryos |
| Luz et al., 2012 - α-MEM(10 µg/ml INS, 5.5 µg/ml TRAN, 5.5 ng/ml SEL, 3 mg/ml BSA, 2 mM GLUT, 2 mM HYPO, 50 µg/ml AA) | Secondary - Isolated 2D (18 days) | 10 and 50 ng/ml LIF alone or in combination with FSH (100 ng/ml D0-D6; 1000 ng/ml D6-D18; 200 ng/ml D13-D18) - 50 ng/ml LIF ↑extrusion. 1 Morula after IVF |
| Luz et al., 2013 - α-MEM(10 µg/ml INS, 5.5 µg/ml TRAN, 5.5 ng/ml SEL, 3 mg/ml BSA, 2 mM GLUT, 2 mM HYPO, 50 µg/ml AA and 50 ng/ml LIF) | Secondary - Isolated 2D (18 days) | 50 ng/ml IGF-I, and 50 ng/ml KL, alone or in combination - KL ↑oocyte meiotic resumption. 58.3% eight-cell stage parthenotes. |
| Bertoldo et al., 2014 - Waymouth MB 752/1 (25 mg/l PYR, 6.25 µg/ml INS, 6.25 µg/ml TRAN, 6.25 µg/ml SEL, 1.25 mg/ml BSA, 5.35 µg/ml LA) | Primordial and primary follicles - In situ (6 days) | 25, 50 or 100 ng/ml BMP4 with or without 50 ng/ml FSH -50 ng/ml BMP4 - ↑ follicle and oocyte diameters. Protected primordial follicles from apoptosis |
| Lakshminarayana et al., 2014 - TCM199 (1µg/ml T4, 2.5 µg/ml FSH, 10 ng/ml IGF-I and 1 mIU/ml GH) | Secondary - Isolated 2D (6 days) | Expression of P450 aromatase gene in cumulus cells and oocytes at different follicular stages (in vivo vs. in vitro). In vitro culture ↓ P450 aromatase expression. |
| Chakravarthi et al., 2015 - TCM199 (1µg/ml T4, 2.5 µg/ml FSH, 10 ng/ml IGF-I and 1 mIU/ml GH) | Secondary - Isolated 2D (6 days) | Quantitative expression of Bcl2 and Bax genes in cumulus cells and oocytes at different follicular stages (in vivo vs. in vitro) - Bcl2 to Bax ratio in oocytes and cumulus cells differed between in vivo and in vitro grown antral follicles |
| Chakravarthi et al., 2016a - TCM199 (1µg/ml T4, 2.5 µg/ml FSH, 10 ng/ml IGF-I and 1 mIU/ml GH) | Secondary - Isolated 2D (6 days) | Quantitative expression of CX32 and CX43 genes in cumulus cells and oocytes at different follicular stages (in vivo vs. in vitro) - In vitro follicle culture ↓ expression of CX32 and CX43. |
| Chakravarthi et al., 2016b - TCM199 (1µg/ml T4, 2.5 µg/ml FSH, 10 ng/ml IGF-I, 1 mIU/ml GH) | Secondary - Isolated 2D (6 days) | Quantitative expression of CCNB1 and CCND1 genes in cumulus cells and oocytes at different follicular stages (in vivo vs. in vitro) - In vitro follicle culture unbalanced the expression pattern of CCNB1 and CCND1 |
| Sadeghnia et al., 2016 - α-MEM (10 µg/ml INS, 5.5 µg/ml TRAN, 5.5 ng/ml SEL, 3 mg/ml BSA, 100 ng/ml FSH, 100 ng/ml GDF-9, 50 µg/ml AA) | Primordial and primary - In situ (8 days) and Isolated 3D (8 days) | 0.5, 1 or 2% alginate (ovarian tissue and isolated follicles) - Alginate encapsulation ↓ number of secondary follicles. Encapsulation ↑follicle growth |
| Kamalamma et al., 2016 - TCM199 (alone or supplemented by 1 µg/ml T4, 2.5 µg/ml FSH, 10 ng/ml IGF-I, 1 mIU/ml GH) | Secondary - Isolated 2D (6 days) | 0-1000 ng/ml leptin and 10 ng/ml human leptin vs. 10 ng/ml ovine leptin - ↑growing follicles, diameter, antrum formation and oocyte maturationcin TCM199 with supplementation and 10 ng/ml human or ovine leptin. |
| Kona et al., 2016 - TCM199 (1µg/ml T4, 2.5 µg/ml FSH, 10 ng/ml IGF-I, 1 mIU/ml GH) | Secondary - Isolated 2D (6 days) | Quantitative expression of GDF9 and BMP15 genes in cumulus cells and oocytes at different follicular stages (in vivo vs. in vitro) -In vitro follicle culture altered the stage-specific changes in the expression of GDF9 and BMP15 |
| Lins et al., 2107 - α-MEM(10 ng/ml INS, 3 mg/ml BSA, 2 mM GLUT, 2 mM HYPO) | Secondary - Isolated 2D (12days) | 0.1, 1 or 10 µg/ml rutin alone or in combination with 5.5 µg/ml TRA, 5 ng/ml SEL, and 50 ng/ml AA - Similar normal follicles (%) and fully-grown oocytes (%) between 0.1 µg/ml rutin alone and the combination of the three antioxidants |
| Nandi et al., 2017 - α-MEM (1% ITS, 3 mg/ml BSA, 2 mM GLUT, 2 mM HYPO, 7 µg/ml FSH and 0.23 mM PYR) | Secondary - Isolated 2D (7 and 14 days) | Ammonia, urea, NEFA, and BHB - Minimum concentrations to impair follicle function. |
| Mbemya et al., 2017 - α-MEM (10 µg/ml INS, 5.5 µg/ml TRAN, 5 ng/ml SEL, 1.25 mg/ml BSA, 2 mM GLUT, 2 mM HYPO) | Primordial and primary - In situ (7 days) | 50 ng/ml FSH vs. 0.3, 1.25, or 5 mg/ml J.insularis. 300 µg/ml Anethole with 0.3 mg/ml J.insularis, or 50 ng/ml FSH - 0.3 ng/ml J.insularis maintained follicle morphology. |
*All the results are compared to control group. Abbreviations: 2D, two-dimensional culture system; 3D, three-dimensional culture system; EAFs, early antral follicles; BSA, bovine serum albumin; GLUT, glutamine; INS, Insulin; TRAN, Transferrin; SEL, Selenium AA, acid ascorbic; Hypo, Hypoxanthine; PYR, Pyruvate; Bcl2, B-cell leukemia/lymphoma-2; Bax, Bcl2-associated X protein; CCNB1, cyclin B1; CCND1, cyclin D1; CX32, connexin 32; CX43, connexin 43; TGF-β, transforming growth factor-β; GDF-9, growth and differentiation factor 9; BMP-15, bone morphogenetic protein 15; BMP4, bone morphogenetic protein 4; FCS, fetal calf serum; ITS, commercial insulin transferrin selenium; FSH, follicle-stimulating hormone; IGF-I, insulin-like growth factor; GH, growth hormone; TNF-α, tumor necrosis factor-alpha; KL, Kit ligand; LIF, leukemia inhibitory factor; LA, linoleic acid; LPF, large preantral follicles; NEFA, non-esterified fatty acids; BHB, β-hydroxybutyric acid.