Mice lacking Mrp1 have reduced testicular steroid hormone levels and alterations in steroid biosynthetic enzymes

Abstract

The multidrug resistance-associated protein 1 (MRP1/ABCC1) is a member of the ABC active transporter family that can transport several steroid hormone conjugates, including 17beta-estradiol glucuronide, dehydroepiandrosterone sulfate (DHEAS), and estrone 3-sulfate. The present study investigated the role that MRP1 plays in maintaining proper hormone levels in the serum and testes. Serum and testicular steroid hormone levels were examined in both wild-type mice and Mrp1 null mice. Serum testosterone levels were reduced 5-fold in mice lacking Mrp1, while testicular androstenedione, testosterone, estradiol, and dehydroepiandrosterone (DHEA) were significantly reduced by 1.7- to 4.5-fold in Mrp1 knockout mice. Investigating the mechanisms responsible for the reduction in steroid hormones in Mrp1-/- mice revealed no differences in the expression or activity of enzymes that inactivate steroids, the sulfotransferases or glucuronosyltransferases. However, steroid biosynthetic enzyme levels in the testes were altered. Cyp17 protein levels were increased by 1.6-fold, while Cyp17 activity using progesterone as a substrate was also increased by 1.4- to 2.0-fold in mice lacking Mrp1. Additionally, the ratio of 17beta-hydroxysteroid dehydrogenase to 3beta-hydroxysteroid dehydrogenase, and steroidogenic factor 1 to 3beta-hydroxysteroid dehydrogenase were significantly increased in the testes of Mrp1-/- mice. These results indicate that Mrp1-/- mice have lowered steroid hormones levels, and suggests that upregulation of steroid biosynthetic enzymes may be an attempt to maintain proper steroid hormone homeostasis.

Figure 1. Alterations in steroid hormone levels in the serum

All serum samples were run in duplicate or triplicate. Values are the average ± standard deviation of 4-6 mice per group. Statistical differences (*) were determined using Student's t-test (p≤0.05).

Figure 2. Alterations in steroid hormone concentrations in the testes

All samples testicular samples were run in duplicate or triplicate. Values are the average ± standard deviation for 5-6 mice per group. Statistical differences (*) were determined using Student's t-test (p≤0.05).

Figure 3. Sulfotransferase and glucuronosyltransferase expression and activity do not differ in the testes of FVB and FVB/Mrp1-/- mice

Sult1e1 and Ugt2b RNA expression was determined by QPCR (A). The data is expressed in number of molecules/100ng cDNA ± standard deviation. All samples were normalized to 18S rRNA, with each sample run in triplicate (n=5=6). SULT1E1 activity was measured by EST metabolite formation and is expressed as pmol/mg protein (B). All samples were run in triplicate (n=6) and the data is expressed as the average of two separate assays. UGT2B protein levels were determined by immunoblotting (C) and quantified by densitometry, using GAPDH as a loading control. Data is expressed as GAPDH-corrected raw intensity values (n=4) and are the average of two separate blots (D).

Figure 4. The ratio of 17β-HSD3 to 3β-HSD1 and SF-1 to 3β-HSD1 mRNA expression is increased in Mrp1-/- mice

The ratio of 17β-HSD3 to 3β-HSD1 mRNA expression (A) and SF-1 to 3β-HSD1 (B) for each individual mouse was calculated. Statistical differences (*) were determined using Student's t-test (p≤0.05).

Figure 5. Increased testicular Cyp17 protein expression and activity in Mrp1-/- mice

Protein levels of Cyp17 were determined by immunoblotting (A) and quantified by densitometry (B), using actin as a loading control. Data is expressed as actin-corrected raw density values (n=4) and are representative of three separate blots. Cyp17 activity was determined by scintillation counting (C), and the data is presented as the average ± standard deviation (n=6) of 2 different assays. Statistical differences (*) were determined using Student's t-test (p≤0.05).