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Thyroid hormones are fundamental regulators of cellular differentiation, development, and metabolism. Their receptors are expressed in reproductive tissues, including the ovary, and dysregulation of thyroid hormone homeostasis has been associated with menstrual disturbances, infertility, and adverse pregnancy outcomes. Bone morphogenetic protein (BMP) ligands and their receptors are functionally involved in gonadotropin-induced ovarian steroidogenesis in an autocrine or paracrine manner. In this study, we examined the effects of thyroid hormones on steroidogenesis and their interplay with BMP signaling by using human granulosa-like KGN cells and primary rat granulosa cells (GCs). In KGN cells, triiodothyronine (T3) enhanced forskolin-induced expression of key steroidogenic enzymes involved in both estradiol biosynthesis and progesterone synthesis/metabolism, whereas thyroxine (T4) exerted minimal effects. In rat GCs, T3 treatment increased follicle-stimulating hormone (FSH)-stimulated estradiol production without altering progesterone output. T3 pretreatment attenuated BMP-6-induced phosphorylation of Smad1/5/9 in KGN cells, accompanied by upregulation of inhibitory Smad6 and downregulation of the BMP type II receptor. Conversely, BMP-6 stimulation elevated thyroid hormone receptor β expression, indicating reciprocal regulatory interactions between thyroid hormone and BMP pathways. Collectively, these findings suggest that thyroid hormones modulate steroidogenesis, at least in part, through suppression of endogenous BMP-6 signaling in granulosa cells.

Abstract Disclosure: Y. Soejima: None. K. Yamamoto: None. N. Iwata: None. K. Motohashi: None. A. Suyama: None. Y. Nakano: None. F. Otsuka: None. The transforming growth factor (TGF)-β superfamily, including bone morphogenetic protein (BMP), plays a pivotal role in reproductive function. TGF-β and BMP bind to their receptors and induce expression of target genes through Smad-mediated signaling. Smad6 and Smad7, also known as inhibitory Smads, function as negative regulators of TGF-β and BMP signaling. Although it has been reported that Smad6 and Smad7 are expressed in the ovary, their direct effects on follicular steroidogenesis have not been fully elucidated. In the present study, we investigated the influence of inhibitory Smads on ovarian BMP signaling and follicular steroidogenesis by using human granulosa cell tumor-derived KGN cells and primary cultured rat ovarian granulosa cells. In KGN cells, it was confirmed that knockdown (KD) of Smad6 and Smad7 by siRNA suppressed Smad6 and Smad7 mRNA expression, respectively. Under Smad6 KD or Smad7 KD conditions, BMP receptor signaling, detected by Id-1 mRNA levels, was enhanced in the presence of BMP-15. Under Smad6 KD conditions, BMP-15 stimulation suppressed forskolin-induced StAR and P450scc mRNA levels, enhanced 3βHSD expression, but did not affect P450arom expression. On the other hand, under Smad7 KD conditions, BMP-15 stimulation suppressed the expression of StAR and P450scc, while the expression of 3βHSD and P450arom was not affected. The expression of 20α-HSD, a progesterone degradation enzyme, remained unchanged by BMP-15 stimulation under Smad6 KD conditions but was increased under Smad7 KD conditions. Furthermore, BMP-15 stimulation suppressed the expression of clock genes including Clock and Bmal1b under the KD effects of Smad6 but not that of Smad7. Notably, in the primary culture of rat ovarian granulosa cells, simultaneous KD of Smad6 and Smad7 significantly suppressed progesterone synthesis levels, whereas it did not affect estradiol production. Collectively, it was revealed that the coexistence of inhibitory Smad6 and Smad7 regulates BMP receptor signaling in ovarian granulosa cells, which may lead to the integrated modulation of follicular steroidogenesis by BMP-15. Presentation: Monday, July 14, 2025