Multispecies study: low-dose tributyltin impairs ovarian theca cell cholesterol homeostasis through the RXR pathway in five mammalian species including humans

Abstract

Tributyltin (TBT), an organotin chemical used as a catalyst and biocide, can stimulate cholesterol efflux in non-steroidogenic cells. Since cholesterol is the first limiting step for sex hormone production, we hypothesized that TBT disrupts intracellular cholesterol transport and impairs steroidogenesis in ovarian theca cells. We investigated TBT's effect on cholesterol trafficking, luteinization, and steroidogenesis in theca cells of five species (human, sheep, cow, pig, and mice). Primary theca cells were exposed to an environmentally relevant dose of TBT (1 or 10 ng/ml) and/or retinoid X receptor (RXR) antagonist. The expression of RXRα in sheep theca cells was knocked down using shRNA. Steroidogenic enzymes, cholesterol transport factors, and nuclear receptors were measured by RT-qPCR and Western blotting, and intracellular cholesterol, progesterone, and testosterone secretion by ELISA. TBT upregulated StAR and ABCA1 in ovine cells, and SREBF1 mRNA in theca cells. TBT also reduced intracellular cholesterol and upregulated ABCA1 protein expression but did not alter testosterone or progesterone production. RXR antagonist and RXRα knockdown demonstrates that TBT's effect is partially through RXR. TBT's effect on ABCA1 and StAR expression was recapitulated in all five species. TBT, at an environmentally relevant dose, stimulates theca cell cholesterol extracellular efflux via the RXR pathway, triggers a compensatory upregulation of StAR that regulates cholesterol transfer into the mitochondria and SREBF1 for de novo cholesterol synthesis. Similar results were obtained in all five species evaluated (human, sheep, cow, pig, and mice) and are supportive of TBT's conserved mechanism of action across mammalian species.

Keywords: Cholesterol; Luteinization; Retinoid X receptor; Steroidogenesis; Theca cells; Tributyltin.

Figure 1.

Effect of TBT exposure on mRNA expression in pre-luteinized and luteinized primary ovine theca cells. mRNA expression (mean ± SEM) of steroidogenic enzymes HSD3β1, HSD17β1 and CYP11A1 and transport protein (StAR) in pre-luteinized (A) and luteinized (B) ovine primary theca cells exposed to 1 ng/ml TBT (gray bars), 10 ng/ml TBT (closed bars) or vehicle (control group; open bars). Asterisks denote differences among treatments (P < 0.05). N=3 primary cultured cell lines per group.

Figure 2.

Effect of TBT exposure on mRNA expression in pre-luteinized and luteinized ovine primary theca cells. mRNA expression (mean ± SEM) of cholesterol transport factors (TSPO, LDLR, SR-B1, ABCA1) and cholesterol biosynthesis factors (HSL, CREB1, SREBF1) in primary ovine pre-luteinized (A) and luteinized (B) ovine primary theca cells exposed to 1 ng/ml TBT (gray bars), 10 ng/ml TBT (closed bars) or vehicle (control group; open bars). Asterisks denote differences among treatments (P < 0.05). N=3 primary cultured cell lines per group.

Figure 3.

Effect of TBT exposure on intracellular cholesterol content and steroid hormones production in pre-luteinized and luteinized ovine primary theca cells. Top Panels: Intracellular cholesterol content (left panels) and testosterone production (right panels) (mean ± SEM) in pre- luteinized ovine primary theca cells exposed to 1 ng/ml TBT (gray bars), 10 ng/ml TBT (closed bars) or vehicle (control group; open bars). Bottom Panels: Intracellular cholesterol content (left panels) and progesterone production (right panels) (mean ± SEM) in luteinized ovine primary theca cells exposed to 1 ng/ml TBT (gray bars), 10 ng/ml TBT (closed bars) or vehicle (control group; open bars). Asterisks denote differences among treatments (P < 0.05). N=3 primary cultured cell lines per group.

Figure 4.

Effect of TBT exposure on mRNA (ABCA1 and StAR; A) and protein (ABCA1; B) expression (mean ± SEM) in pre-luteinized ovine primary theca cells exposed to 1 ng/ml TBT (gray bars), 10 ng/ml TBT (closed bars) and / or RXR antagonist (UVI3003; 0.05, 2, 4 and 8 μM) or vehicle (control group; open bars). Asterisks denote differences among treatments (P < 0.05). N=3 primary cultured cell lines per group.

Figure 5.

RXRα knockdown efficiency (mean ± SEM) of mRNA (A) and protein (B) in pre- luteinized ovine primary theca cells. Effect of TBT exposure on mRNA (ABCA1; C and StAR; D) and protein (ABCA1; E) expression (mean ± SEM) in RXRα knockdown or scramble controls in pre-luteinized ovine primary theca cells exposed to 1 ng/ml TBT (gray bars), 10 ng/ml TBT (closed bars) and vehicle (control group; open bars). Asterisks denote differences among treatments (P < 0.05). N=3 primary cultured cell lines per group. Values are normalized to scramble shRNA control cells.

Figure 6.

Effect of TBT exposure on mRNA expression (mean ± SEM) of cholesterol transporters (ABCA1 and StAR) in pre-luteinized (left) and luteinized (right) porcine (top), bovine (middle), and mouse (bottom) primary theca cells exposed to 1 ng/ml TBT (gray bars), 10 ng/ml TBT (closed bars) or vehicle (control group; open bars). Asterisks denote differences among treatments (P < 0.05). One primary cultured cell line per species was used.

Figure 7.

Top Panels: Effect of TBT exposure on mRNA expression (mean ± SEM) of cholesterol transporters (ABCA1 and StAR) in pre-luteinized (A) and luteinized (B) human primary ovarian theca cells exposed to 1 ng/ml TBT (gray bars), 10 ng/ml TBT (closed bars) or vehicle (control group; open bars). Bottom Panels: Effect of TBT exposure on ABCA1 and StAR mRNA expression (mean ± SEM) in pre-luteinized human primary theca cells exposed to 10 ng/ml TBT (light gray bars), RXR antagonist (UVI3003; 8 μM; dark gray), the combination of TBT (10 ng/ml) and RXR antagonist (8 μM), or vehicle (control group; open bars). Asterisks denote differences among treatments (P < 0.05). N=3 primary cultured cell lines per group.