Sex hormone-binding globulin regulation of androgen bioactivity in vivo: validation of the free hormone hypothesis

Abstract

Sex hormone-binding globulin (SHBG) is the high-affinity binding protein for androgens and estrogens. According to the free hormone hypothesis, SHBG modulates the bioactivity of sex steroids by limiting their diffusion into target tissues. Still, the in vivo physiological role of circulating SHBG remains unclear, especially since mice and rats lack circulating SHBG post-natally. To test the free hormone hypothesis in vivo, we examined total and free sex steroid concentrations and bioactivity on target organs in mice expressing a human SHBG transgene. SHBG increased total androgen and estrogen concentrations via hypothalamic-pituitary feedback regulation and prolonged ligand half-life. Despite markedly raised total sex steroid concentrations, free testosterone was unaffected while sex steroid bioactivity on male and female reproductive organs was attenuated. This occurred via a ligand-dependent, genotype-independent mechanism according to in vitro seminal vesicle organ cultures. These results provide compelling support for the determination of free or bioavailable sex steroid concentrations in medicine, and clarify important comparative differences between translational mouse models and human endocrinology.

Figure 1. Endocrine profile of SHBG-Tg mice.

(A) Human SHBG serum concentrations (shown as mean ± SD) in male and female SHBG-Tg mice of different ages (n = 7–10 per gender and age except 24-week-old females, n = 3). ****P < 0.0001 vs. female SHBG-Tg mice of same age group. ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺⁺P < 0.0001 vs. 3-week-old mice of same gender. (B) Human SHBG expression (relative to mouse 18S) in livers of female and male WT and SHBG-Tg mice (n = 3 for WT and n = 5 for SHBG-Tg mice of each gender). ***P = 0.0002 for difference between SHBG-Tg females and males. (C) Concentrations in serum (left from vertical dotted line) and urine (right from vertical dotted line) of selected steroid hormones in 24-week-old WT and SHBG-Tg male mice (n = 5 per group). *P < 0.05 vs. WT mice. (D) Serum E2 in 12-week-old WT and SHBG-Tg male mice, either sham-operated and given empty placebo implants, or orchidectomized with continuous-release s.c. T replacement. The limit of quantification (LOQ) of the LC-MS/MS method (1.3 pg/mL) is indicated by the horizontal dotted line. *P < 0.05, n as indicated by individual replicates in each group. (E) Serum E2 in 12-week-old orchidectomized male mice given s.c. undiluted E2 implants of different lengths as indicated, or female WT and SHBG-Tg mice. The limit of quantification (LOQ) of the LC-MS/MS method (1.3 pg/mL) is indicated by the horizontal dotted line. ****P < 0.0001, n as indicated by individual replicates in each group.

Figure 2. Evidence of hypogonadism in 24-week-old male SHBG-Tg mice.

(A,B) Serum total and free T. n = 5 biological replicates per group (to obtain sufficient volume, sera of 2 mice were pooled if necessary). **P < 0.01 (C) Serum luteinizing hormone (n = 7–9 per group). *P < 0.05. (D) Free T by equilibrium dialysis in WT and SHBG-Tg mice which were orchidectomized and given s.c. T replacement (ORX+T). n = 4 per group, *P < 0.05. (E,F) Seminal vesicle and levator ani/bulbocavernosus complex (LA/BC) muscle weights. Individual replicates, mean ± SEM are shown. ***P < 0.001, ****P < 0.0001.

Figure 3. Effect of SHBG on androgen-induced branching morphogenesis in seminal vesicle organ cultures.

For each genotype, two representative microphotographs at day 3 of culture are displayed. All pictures scaled identically (1 mm scale bar shown in first photograph only). (A) Unstimulated condition showing lack of epithelial folding in the absence of androgens. (B) Induction of branching morphogenesis (arrowheads in two panels) by 1 nM DHT in both genotypes. (C) Suppression of DHT-induced branching morphogenesis by SHBG in the media. (D) Induction of epithelial folding by 1 nM mibolerone in both genotypes. (E) Lack of suppression of mibolerone-induced branching morphogenesis by SHBG.

Figure 4. SHBG inhibits the stimulatory effects of sex steroids on male and female reproductive organs.

(A,B) Seminal vesicle and LA/BC muscle weight of 12-week-old WT and SHBG-Tg mice following 3 weeks of orchidectomy (ORX), with placebo, testosterone (ORX+T) or DHT replacement (ORX+DHT). n = 5–10 for each genotype and treatment group. ****P < 0.0001 compared to ORX in same genotype. ⁺⁺⁺⁺P < 0.0001 vs. WT in same treatment group. (C) Uterus weight of female WT or SHBG-Tg mice following ovariectomy (OVX) with either placebo, E2 (diluted 1/16 w/w with cholesterol) replacement (OVX+E2) or DHT replacement (OVX+DHT) replacement between 18 and 20 weeks of age, respectively. n = 5–14 per group. (D) Serum E2 concentrations in WT and SHBG-Tg following OVX+E2 replacement (n = 6 randomly selected mice per group). **P  < 0.01 vs. WT by Mann-Whitney U test.

Figure 5. Effects of SHBG on sex steroid pharmacokinetics and biodistribution.

(A) Half-life of tritium-labeled DHT following i.v. injection. n = 4–9 per group. **P < 0.01. (B) Half-life of tritium-labeled T following i.v. injection. n = 3–6 per group. *P < 0.05. (C) Half-life of tritium-labeled mibolerone following i.v. injection. n = 6–10 per group. (D) Testosterone concentrations at 8 or 60 minutes following i.v. T injection. n = 6–10 per group. **P < 0.01 compared to 8 minute timepoint in same genotype. (E) Concentration of E2 in bone homogenates. n = 4 per group. *P < 0.05. (F) Relative mRNA expression of mouse aromatase (Cyp19a1) in the bones (tibia and femur) of 24-week-old WT and SHBG-Tg male mice. n = 9–11 per group.

Figure 6. Summary of differences in sex steroid endocrinology between men and mice.

(A) In men, circulating androgens are derived from the testes and adrenals. Circulating E2 is estimated to be ~85% derived from aromatization in peripheral adipose tissues, with the remainder secreted directly from the testis. SHBG binds androgens as well as estrogens and prevents their entry into target tissues (indicated by striped arrows). Within target tissues, T can also be converted locally into E2. Conjugation and feedback regulation occur within tissues and thus at the level of the free/bioactive sex steroids. Water-soluble conjugation products are removed by hepatic and renal clearance. (B) In contrast, male mice have lower androgen and undetectable E2 concentrations due to (a) less pronounced adrenal secretion of precursor steroids, (b) possibly due to markedly lower peripheral aromatization due to absence in mice of the alternative promoter which drives peripheral CYP19A1 expression in humans, and (c) lack of SHBG, which facilitates rapid entry of both sex steroids into target tissues. Thus, estrogens act mainly as local hormones in target tissues of male mice. Lower gonadotropin concentrations in male mice are at least in part due to lack of SHBG.