Activin Stimulation of Inhibin Secretion and Messenger RNA Levels in Cultured Granulosa Cells

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Abstract
Recent reports suggest that activin (the dimer of inhibin β subunits with FSH-releasing activity) has specific receptors on ovarian granulosa cells. The present study examined the effects of purified porcine activin on inhibin secretion and mRNA levels in granulosa cells obtained from immature, estrogentreated rats. Cells were cultured for 48 h in culture media, or media containing FSH (10 ng/ml) and/or activin (30 ng/ml). Western blot analyses performed with affinity-purified antisera to inhibin α- and βA-subunits revealed that treatment with either FSH or activin increased the secretion of inhibin αβ dimer (Mr 30,000), with a further increase after cotreatment. These results were confirmed by an inhibin α-subunit RIA, which revealed 7-, 14-, and 71-fold increases in the secretion of immunoreactive inhibin-α by activin, FSH, and activin plus FSH, respectively. TGFβ, a structural homolog of activin, also stimulated inhibin release, whereas follistatin was ineffective. Total RNA from cultured cells was hybridized with 32P-labeled inhibin α-subunit cRNA or β-actin cDNA probes, and inhibin-α message levels were normalized with β-actin mRNA levels. Northern blot analysis revealed that treatment with FSH and activin increased hybridization of a 1.5 kilobase (kb) message, corresponding to the inhibin α-subunit mRNA. Slot blot analyses indicated a 6- and 8-fold stimulation of inhibin α-subunit mRNA levels by FSH and activin, respectively. The induction of α-subunit mRNA by activin was augmented by cotreatment with FSH and was time and dose dependent (3–30 ng/ml, apparent ED50: 0.8 nm), consistent with the demonstration of specific 125I-activin binding in granulosa cells with a dissociation constant (Kd) of 0.14 ± 0.04 nm. Although TGFβ also stimulated α-subunit mRNA levels, purified porcine inhibin (αβ dimer) did not modulate inhibin α-subunit mRNA content. In addition, hybridization analysis with a βA-subunit cRNA probe revealed that treatment with FSH and/or activin increased hybridization of a predominant 6.8 kilobase βA-subunit message. While treatment with activin alone had no effect on extracellular cAMP levels, activin potentiated the stimulatory effect of FSH and forskolin on cAMP production. These findings indicate that activin is capable of inducing inhibin mRNA and protein expression as well as augmenting cAMP production in cultured granulosa cells, providing a model to study the induction of inhibin gene expression by activin and related protein.