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Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells in vitro.

Brankin V, Mitchell MR, Webb B, Hunter MG - Reprod. Biol. Endocrinol. (2003)

Bottom Line: 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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD, UK. victoria.brankin@nottingham.ac.uk

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.

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Mean (± sed) (A) log10 progesterone production by theca cells after 48 hours; (B) log10 androstenedione production by theca cells after 144 hours; (C) log10 oestradiol production by theca cells after 48 and (D) 144 hours in serum free culture supplemented with either 0, 10 or 100 ng/ml hSCF ± 5 oocytes per well. Values are from 3 independent cultures, each having 4 replicates. There was a significant effect of treatment in (A): P = 0.002, (B): P < 0.001, (C): P = 0.031 and (D): P < 0.001. Bars with different superscripts are significantly (P < 0.05) different.
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Figure 6: Mean (± sed) (A) log10 progesterone production by theca cells after 48 hours; (B) log10 androstenedione production by theca cells after 144 hours; (C) log10 oestradiol production by theca cells after 48 and (D) 144 hours in serum free culture supplemented with either 0, 10 or 100 ng/ml hSCF ± 5 oocytes per well. Values are from 3 independent cultures, each having 4 replicates. There was a significant effect of treatment in (A): P = 0.002, (B): P < 0.001, (C): P = 0.031 and (D): P < 0.001. Bars with different superscripts are significantly (P < 0.05) different.

Mentions: Viable theca cell numbers were significantly reduced by SCF (P < 0.001) and these were further reduced with oocyte co-culture (P = 0.05). In contrast to granulosa cells, SCF elicited an overall decrease in progesterone after 48 hours (P = 0.002), which was further reduced in oocyte cultures at 0 and 10 ng/ml SCF (Figure 6A). However, theca cells co-cultured with oocytes and supplemented with 100 ng/ml SCF significantly increased progesterone synthesis (P = 0.05). After 144 hours in culture, there was an overall significant (P < 0.001) effect of SCF dose and oocyte co-culture on androstenedione production (Figure 6B). Androstenedione production was significantly (P < 0.05) increased when theca cells were supplemented with 10 and 100 ng/ml SCF, both with and without oocyte co-culture, compared to controls. Theca cells co-cultured with oocytes secreted less androstenedione at SCF doses of 10 or 100 ng/ml, compared to oocyte free cultures and this difference was significant (P < 0.05) in the presence of 100 ng/ml SCF. Interestingly, this pattern of androstenedione synthesis was observed after 48 hours, but did not reach statistical significance (P = 0.08). In contrast to granulosa cells, thecal cells demonstrated a SCF dose dependent decrease in oestradiol production both with and without oocyte co-cultures after 48 (P = 0.031) and 144 (P < 0.001) hours (Figure 6C and 6D). Similar to granulosa cells, oocyte co-culture increased oestradiol synthesis in the absence of SCF after 48 hours in culture. However, after 144 hours in culture, this effect was reversed and oocyte co-culture in the absence of SCF decreased oestradiol production. Also, 10 ng/ml SCF increased oestradiol synthesis above that of controls (P = 0.05).


Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells in vitro.

Brankin V, Mitchell MR, Webb B, Hunter MG - Reprod. Biol. Endocrinol. (2003)

Mean (± sed) (A) log10 progesterone production by theca cells after 48 hours; (B) log10 androstenedione production by theca cells after 144 hours; (C) log10 oestradiol production by theca cells after 48 and (D) 144 hours in serum free culture supplemented with either 0, 10 or 100 ng/ml hSCF ± 5 oocytes per well. Values are from 3 independent cultures, each having 4 replicates. There was a significant effect of treatment in (A): P = 0.002, (B): P < 0.001, (C): P = 0.031 and (D): P < 0.001. Bars with different superscripts are significantly (P < 0.05) different.
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Related In: Results  -  Collection

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Figure 6: Mean (± sed) (A) log10 progesterone production by theca cells after 48 hours; (B) log10 androstenedione production by theca cells after 144 hours; (C) log10 oestradiol production by theca cells after 48 and (D) 144 hours in serum free culture supplemented with either 0, 10 or 100 ng/ml hSCF ± 5 oocytes per well. Values are from 3 independent cultures, each having 4 replicates. There was a significant effect of treatment in (A): P = 0.002, (B): P < 0.001, (C): P = 0.031 and (D): P < 0.001. Bars with different superscripts are significantly (P < 0.05) different.
Mentions: Viable theca cell numbers were significantly reduced by SCF (P < 0.001) and these were further reduced with oocyte co-culture (P = 0.05). In contrast to granulosa cells, SCF elicited an overall decrease in progesterone after 48 hours (P = 0.002), which was further reduced in oocyte cultures at 0 and 10 ng/ml SCF (Figure 6A). However, theca cells co-cultured with oocytes and supplemented with 100 ng/ml SCF significantly increased progesterone synthesis (P = 0.05). After 144 hours in culture, there was an overall significant (P < 0.001) effect of SCF dose and oocyte co-culture on androstenedione production (Figure 6B). Androstenedione production was significantly (P < 0.05) increased when theca cells were supplemented with 10 and 100 ng/ml SCF, both with and without oocyte co-culture, compared to controls. Theca cells co-cultured with oocytes secreted less androstenedione at SCF doses of 10 or 100 ng/ml, compared to oocyte free cultures and this difference was significant (P < 0.05) in the presence of 100 ng/ml SCF. Interestingly, this pattern of androstenedione synthesis was observed after 48 hours, but did not reach statistical significance (P = 0.08). In contrast to granulosa cells, thecal cells demonstrated a SCF dose dependent decrease in oestradiol production both with and without oocyte co-cultures after 48 (P = 0.031) and 144 (P < 0.001) hours (Figure 6C and 6D). Similar to granulosa cells, oocyte co-culture increased oestradiol synthesis in the absence of SCF after 48 hours in culture. However, after 144 hours in culture, this effect was reversed and oocyte co-culture in the absence of SCF decreased oestradiol production. Also, 10 ng/ml SCF increased oestradiol synthesis above that of controls (P = 0.05).

Bottom Line: 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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD, UK. victoria.brankin@nottingham.ac.uk

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.

Show MeSH
Related in: MedlinePlus