title: Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells in vitro.
abstract: Oocyte control of granulosa and theca cell function may be mediated by several growth factors via a local feedback loop(s) between these cell types. This study examined both the role of oocyte-secreted factors on granulosa and thecal cells, cultured independently and in co-culture, and the effect of stem cell factor (SCF); a granulosa cell derived peptide that appears to have multiple roles in follicle development. Granulosa and theca cells were isolated from 2-6 mm healthy follicles of mature porcine ovaries and cultured under serum-free conditions, supplemented with: 100 ng/ml LR3 IGF-1, 10 ng/ml insulin, 100 ng/ml testosterone, 0-10 ng/ml SCF, 1 ng/ml FSH (granulosa), 0.01 ng/ml LH (theca) or 1 ng/ml FSH and 0.01 ng/ml LH (co-culture) and with/without oocyte conditioned medium (OCM) or 5 oocytes. Cells were cultured in 96 well plates for 144 h, after which viable cell numbers were determined. Medium was replaced every 48 h and spent medium analysed for steroids. Oocyte secreted factors were shown to stimulate both granulosa cell proliferation (P < 0.001) and oestradiol production (P < 0.001) by granulosa cells throughout culture. In contrast, oocyte secreted factors suppressed granulosa cell progesterone production after both 48 and 144 hours (P < 0.001). Thecal cell numbers were increased by oocyte secreted factors (P = 0.02), together with a suppression in progesterone and androstenedione synthesis after 48 hours (P < 0.001) and after 144 hours (P = 0.02), respectively. Oocyte secreted factors also increased viable cell numbers (P < 0.001) in co-cultures together with suppression of progesterone (P < 0.001) and oestradiol (P < 0.001). In granulosa cell only cultures, SCF increased progesterone production in a dose dependent manner (P < 0.001), whereas progesterone synthesis by theca cells was reduced in a dose dependent manner (P = 0.002). Co-cultured cells demonstrated an increase in progesterone production with increasing SCF dose (P < 0.001) and an increase in oestradiol synthesis at the highest dose of SCF (100 ng/ml). In summary, these findings demonstrate the presence of a co-ordinated paracrine interaction between somatic cells and germ cells, whereby oocyte derived signals interact locally to mediate granulosa and theca cell function. SCF has a role in modulating this local interaction. In conclusion, the oocyte is an effective modulator of granulosa-theca interactions, one role being the inhibition of luteinization.
authors.affiliation: School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD, UK. victoria.brankin@nottingham.ac.uk
authors.forename: Victoria
authors.initials: V
authors.lastname: Brankin
authors.forename: Marcus R P
authors.initials: MR
authors.lastname: Mitchell
authors.forename: Bob
authors.initials: B
authors.lastname: Webb
authors.forename: Morag G
authors.initials: MG
authors.lastname: Hunter
mesh_terms: D000735:Androstenedione / Q000096:biosynthesis; D000818:Animals; D001685:Biological Factors / Q000378:metabolism / Q000494:pharmacology*; D002450:Cell Communication; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D018920:Coculture Techniques; D017077:Culture Media, Conditioned / Q000494:pharmacology; D016895:Culture Media, Serum-Free; D003864:Depression, Chemical; D004958:Estradiol / Q000096:biosynthesis; D025461:Feedback, Physiological; D005260:Female; D006107:Granulosa Cells / Q000187:drug effects* / Q000378:metabolism; D034461:Luteinization / Q000187:drug effects; D009865:Oocytes / Q000378:metabolism*; D019899:Paracrine Communication; D011374:Progesterone / Q000096:biosynthesis; D019089:Stem Cell Factor / Q000494:pharmacology*; D013552:Swine; D013799:Theca Cells / Q000187:drug effects* / Q000378:metabolism
chemical_list: D001685:Biological Factors; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D019089:Stem Cell Factor; D000735:Androstenedione; D011374:Progesterone; D004958:Estradiol