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Effects of recombinant activins on steroidogenesis in human granulosa-lutein cells.

The Journal of clinical endocrinology and metabolism (2014-07-26)
Hsun-Ming Chang, Jung-Chien Cheng, Christian Klausen, Elizabeth L Taylor, Peter C K Leung
ABSTRACT

Exerting a broad range of biological effects in various tissues, activins are homo- or heterodimers of activin/inhibin β-subunits (βA, βB, βC, and βE in humans). Although activins A (βAβA), B (βBβB), AB (βAβB), and AC (βAβC) have been demonstrated in the female reproductive system, little is known about their individual functions in the ovary. To investigate the biological roles and activities of activins in regulating steroidogenesis in human granulosa cells. Human granulosa-lutein cells obtained from 32 patients undergoing in vitro fertilization were used to investigate the effects of activin A, B, AB, and AC on the expression of steroidogenic enzymes and steroid production. An academic research center. mRNA and protein levels were examined by reverse transcription quantitative real-time PCR and Western blot analysis, respectively. The production of estradiol and progesterone was measured by enzyme immunoassay. P450 aromatase, FSH receptor, and estradiol levels were increased, whereas steroidogenic acute regulatory protein (StAR), LH receptor, and progesterone levels were decreased after treatment with activin A, B, and AB, but not activin AC. FSH or LH induced the production of aromatase/estradiol and StAR/progesterone; however, pretreatment with activin A, B, or AB enhanced the effects of gonadotropins on aromatase/estradiol, but suppressed their effects on StAR/progesterone. Treatment with activin A, B, or AB induced the phosphorylation of SMA- and MAD-related proteins (SMAD2 and 3), whereas activin AC had no such effects. Furthermore, co-culture of activin AC (1-100 ng/mL) with activin A (25 ng/mL) did not alter the effects of activin A on P450 aromatase or StAR mRNA levels. Activin A, B, and AB have similar effects on steroidogenesis in human granulosa cells. In contrast, activin AC is not biologically active and does not act as a competitive antagonist.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Streptomycin sulfate salt, powder
Sigma-Aldrich
Streptomycin sulfate salt, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Progesterone, ≥99%
Sigma-Aldrich
Progesterone, powder, BioReagent, suitable for cell culture
Supelco
Progesterone, Pharmaceutical Secondary Standard; Certified Reference Material
Supelco
Streptomycin solution, ~1 mg/mL in 1 mM EDTA, analytical standard
Sigma-Aldrich
Streptomycin sulfate salt, powder, BioXtra, suitable for mouse embryo cell culture
Sigma-Aldrich
Activin A human, ≥95% (SDS-PAGE), recombinant, expressed in baculovirus infected insect cells, lyophilized powder, suitable for cell culture
Streptomycin sulfate, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Progesterone, γ-irradiated, BioXtra, suitable for cell culture
Sigma-Aldrich
Progesterone, meets USP testing specifications
Sigma-Aldrich
Activin A human, recombinant, expressed in E. coli, ≥97% (SDS-PAGE), ≥97% (HPLC), suitable for cell culture
Sigma-Aldrich
Activin A human, recombinant, expressed in HEK 293 cells, HumanKine®, suitable for cell culture
USP
Progesterone, United States Pharmacopeia (USP) Reference Standard
Supelco
Progesterone, VETRANAL®, analytical standard
Progesterone for system suitability, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Activin A active human, Animal-component free, recombinant, expressed in Nicotiana, >97% (SDS-PAGE)
Progesterone for peak identification, European Pharmacopoeia (EP) Reference Standard
Progesterone, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Activin A human, recombinant, expressed in human cells, ≥95% (SDS-PAGE)