title: Growth and antrum formation of bovine primary follicles in long-term culture in vitro.
abstract: Successful antral formation in vitro from bovine preantral follicles (145-170 μm) has been described previously, but antrum formation from the primary follicle (50-70 μm) has not yet been achieved in vitro. The aim of the study was to establish an optimal culture system supporting the growth and maturation of bovine primary follicles (50-70 μm) in vitro. Bovine primary follicles were cultured in a three-dimensional culture system for 13 or 21 days in alpha-minimum essential medium. Various treatments including follicle stimulating hormone (FSH), luteinizing hormone (LH), 17β-estradiol (E2), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were tested. The follicular diameter and antrum formation rate were recorded, and follicular maturation markers (P450 aromatase, CYP19A1; anti-Mullerian hormone, AMH; growth differentiation factor-9, GDF9; bone morphogenetic protein-15, BMP15; and type III transforming growth factor β receptor, TGFβR3) were analyzed by real-time RT-PCR. After 21 days of culture under each treatment condition, the follicular diameter was significantly enlarged in the presence of FSH + LH + E2 + bFGF or FSH + LH + E2 + bFGF + EGF (p<0.05). An addition of 50 ng/ml bFGF or bFGF +25 ng/ml EGF initiated antrum formation by day 19 and day 17 of culture, and the antral cavity formation rate was 16.7% and 33.3% by 21 days of culture, respectively. The expression of follicular maturation markers (CYP19A1, AMH, GDF9, BMP15 and TGFβR3) was significantly altered. We conclude that addition of 50 ng/ml bFGF +25 ng/ml EGF to media containing FSH + LH + E2 turned out to be the most effective optimized culture conditions to support the growth and maturation of bovine primary follicles in vitro.
authors.affiliation: State Key Laboratory for Agro-Biotechnology, College of Biological Science, China Agricultural University, Beijing 100193, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Sun
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Li
mesh_terms: D000818:Animals; D054304:Anti-Mullerian Hormone / Q000096:biosynthesis; D001141:Aromatase / Q000096:biosynthesis; D055430:Bone Morphogenetic Protein 15 / Q000096:biosynthesis; D002417:Cattle; D003470:Culture Media; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D055429:Growth Differentiation Factor 9 / Q000096:biosynthesis; D007986:Luteinizing Hormone / Q000494:pharmacology; D006080:Ovarian Follicle / Q000187:drug effects / Q000502:physiology*; D046509:Tissue Culture Techniques / Q000379:methods*
chemical_list: D055430:Bone Morphogenetic Protein 15; D003470:Culture Media; D055429:Growth Differentiation Factor 9; D004958:Estradiol; D054304:Anti-Mullerian Hormone; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone; D001141:Aromatase
keywords: Antrum formation; Bovine; Growth factors; Hormones; Primary follicle