Free Hormone Hypothesis Research Articles

Comparison of free and total 25-hydroxyvitamin D in normal human pregnancy

Vitamin D status correct monitoring during pregnancy is critically important for both maternal and fetal health. 25-Hydroxyvitamin D (25(OH)D) – a prohormone of a biologically active 1,25-dihydroxyvitamin D (1,25(OH)2D), despite the lack of biological activity, during the past decades has been routinely used as a main biomarker characterizing vitamin D status. About 85% of 25(OH)D in the bloodstream is bound to its specific carrier – vitamin D-binding protein (DBP), the remaining 15% are loosely bound to albumin, and only less than 0.1% are free in the circulation (“free 25(OH)D”). Total 25(OH)D is the sum of DBP-bound, albumin-bound and free 25(OH)D. According to a “free hormone hypothesis”, only free 25(OH)D is able to induce a biological effect. Normal pregnancy is characterized by elevated serum DBP levels, and due to this fact the diagnostic strength of serum total 25(OH)D has been questioned. Free 25(OH)D might be a better characteristic of vitamin D status in this settings.We aimed to compare the diagnostic strength of a routine total 25(OH)D with directly measured free 25(OH)D in normal pregnancy by comparing the association strength between free and total 25(OH)D with biomarkers of bone health (PTH, calcium, bone-specific alkaline phosphatase (BSAP)), lipid metabolism (adiponectin, LDL, HDL), kidney function (urea), endocrine parameters (T4, T3, TSH), and group B water-soluble vitamins.The study was conducted in 368 healthy white pregnant women – residents of north-east Germany.Free 25(OH)D showed an overall better associations with gestational age, markers of bone metabolism (calcium (rho = 0.141, p = 0.007 with free 25(OH)D; rho = 0.060, p = 0.251 with total 25(OH)D) and BSAP (rho = −0.203, p < 0.001 with free 25(OH)D; rho = −0.108, p = 0.038 with total 25(OH)D), lipid metabolism parameters (adiponectin (rho = 0.142, p = 0.008 with free 25(OH)D; rho = 0.054, p = 0.307 with total 25(OH)D), LDL cholesterol (rho = −0.191, p < 0.001 with free 25(OH)D; rho = 0.033, p = 0.539 with total 25(OH)D)) and a kidney function marker (urea (rho = 0.114, p = 0.032 with free 25(OH)D; rho = 0.008, p = 0.887 with total 25(OH)D)) than total 25(OH)D.In conclusion, the current study revealed that free 25(OH)D is a more precise determinant of the vitamin D status during normal human pregnancy than total 25(OH)D. In the settings of normal pregnancy, free 25(OH)D revealed better associations with markers of bone metabolism (calcium, BSAP), lipid metabolism (adiponectin, LDL cholesterol, LDL/HDL ratio) and kidney function (urea) than total 25(OH)D.

The Role of Vitamin D Binding Protein, Total and Free 25-Hydroxyvitamin D in Diabetes.

Vitamin D is important for bone health, but may also have extra-skeletal effects. Vitamin D and its binding protein DBP have immunological effects and may therefore be important in the development of type 1 diabetes (T1DM), and low serum levels of 25-hydroxyvitamin D (25(OH)D) are associated with later development of type 2 diabetes (T2DM). However, it has so far been difficult to convincingly show an effect of vitamin D supplementation on prevention or treatment of diabetes. The serum level of 25(OH)D has traditionally been used as a marker of a subject's vitamin D status. This measurement includes both 25(OH)D bound to DBP and albumin as well as the free from of 25(OH)D. However, according to the free hormone hypothesis, the free form is the biologically active. Previously the free form of 25(OH)D had to be calculated based on measurements of 25(OH)D, DBP, and albumin, but recently a method for direct measurement of free 25(OH)D has become commercially available. This is important in clinical conditions where the amount of DBP is affected, and has caused a renewed interest in which vitamin D metabolite to measure in clinical situations. In the present review the relations between DBP, total and free 25(OH)D in T1DM and T2DM are described.

Vitamin D deficiency in children: a challenging diagnosis!

The concern about the assessment of vitamin D status is growing. Numerous publications warn about the high prevalence of vitamin D deficiency, as well as the potential role of vitamin D in non-bone health outcomes. The status of vitamin D is usually assessed by measuring serum total 25-hydroxyvitamin D (25OHD) concentration. This is the major circulating form of vitamin D and keeps an inverse correlation with serum parathyroid hormone (PTH) concentration. A value of 25OHD of 20 ng/ml is generally assumed as threshold of vitamin D sufficiency in epidemiologic studies because serum PTH tends to increase when the 25OHD concentration stands below this value. In pediatric population, very few studies have analyzed this issue and the negative relationship between serum 25OHD and serum PTH is not clear, which is the suitable circulating concentration of 25OHD and the threshold of deficiency being matters of controversy. The majority of 25OHD circulates in serum tightly bound to a globulin (DBP). According to the free hormone hypothesis, protein-bound hormones are not biologically available and it is the free form that exerts or facilitates the physiologic actions. If this is true, factors that affect DBP may alter the interpretation of total serum 25OHD measurements.

Abstract A089: Challenging the free hormone hypothesis for vitamin D in the prostate has implications for the prostate cancer disparity in African American men

Abstract African American (AA) men are disproportionately at risk for both prostate cancer (PCa) and vitamin D (vitD) deficiency compared to European American (EA) men. Based on the numerous chemopreventative properties of vitD and its status with PCa aggressiveness and mortality, vitD deficiency has been hypothesized as a biologic contributor to the PCa disparity in AA men. We recently reported that despite having deficient levels of vitD in the serum, AAs have higher levels of vitD in their prostate tissue compared to EAs. This suggests a mechanism of active vitD transport across the membrane and challenges the longstanding free hormone hypothesis, which asserts that the activity of a hormone is due to the bioavailable fraction and its passive diffusion across the membrane. The majority of VitD circulates bound to D binding protein (DBP) and is sequestered in the serum, rendering it unavailable for passive diffusion. A mechanism of active DBP-vitD endocytosis has been well characterized in the kidney by the extracellular receptor megalin. We hypothesized that megalin is functional in prostate epithelium and facilitates adequate prostatic vitD levels in AAs with vitD deficiency. Here, we report megalin protein expression in prostate tissue, LNCaP, and primary human prostate cells. Megalin function was further assessed in primary human-derived organoids treated with 25D in the presence or absence of DBP. Surprisingly, vitD import and metabolism occurred faster in +DBP conditions and suggests that DBP-vitD active transport is more efficient than passive diffusion of vitD alone. VDR activation was diminished in a dose-responsive manner by addition of a megalin antagonist. Additionally, megalin protein levels in PCa were significantly lower compared to benign (p&amp;lt;0.05) on a tissue microarray quantified by immunofluorescent staining. In summary, our data support a model of megalin-mediated active transport for vitD in the prostate and demonstrate ancestry-related and PCa differences in megalin regulation. These findings challenge the free hormone hypothesis and postulate that vitD deficiency is more complex than previously thought. Citation Format: Zachary Richards, Larisa Nonn. Challenging the free hormone hypothesis for vitamin D in the prostate has implications for the prostate cancer disparity in African American men [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A089.

A Reappraisal of Testosterone's Binding in Circulation: Physiological and Clinical Implications.

In the circulation, testosterone and other sex hormones are bound to binding proteins, which play an important role in regulating their transport, distribution, metabolism, and biological activity. According to the free hormone hypothesis, which has been debated extensively, only the unbound or free fraction is biologically active in target tissues. Consequently, accurate determination of the partitioning of testosterone between bound and free fractions is central to our understanding of how its delivery to the target tissues and biological activity are regulated and consequently to the diagnosis and treatment of androgen disorders in men and women. Here, we present a historical perspective on the evolution of our understanding of the binding of testosterone to circulating binding proteins. On the basis of an appraisal of the literature as well as experimental data, we show that the assumptions of stoichiometry, binding dynamics, and the affinity of the prevailing models of testosterone binding to sex hormone-binding globulin and human serum albumin are not supported by published experimental data and are most likely inaccurate. This review offers some guiding principles for the application of free testosterone measurements in the diagnosis and treatment of patients with androgen disorders. The growing number of testosterone prescriptions and widely recognized problems with the direct measurement as well as the computation of free testosterone concentrations render this critical review timely and clinically relevant.

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

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.

Effects of sex hormone-binding globulin (SHBG) on androgen bioactivity in vitro

Biochemical assessments of androgen status (hyper- or hypoandrogenism) are usually based on serum testosterone concentrations. According to the free hormone hypothesis, sex hormone-binding globulin (SHBG) determines free and bioavailable testosterone concentrations. Previous studies have suggested that in vitro androgen bioassay results may also be influenced by SHBG and correlate with free or bioavailable testosterone concentrations. To test this hypothesis, we established a stable HEK293 cell line with high expression of the human androgen receptor (AR) and a luciferase reporter downstream of a classical androgen response element. Importantly, we demonstrate that bioassay results are sensitive to dilution effects which increase apparent bioactivity in an SHBG-dependent manner. We therefore adopted a method using undiluted serum, which reduced cell proliferation but did not significantly affect the luciferase signal, cell viability or cytotoxicity. To correct for serum matrix effects, we applied signal correction based on internal controls with AR agonists or antagonists. Using this method, we provide direct evidence that in vitro androgen bioactivity reflects the inhibitory effects of SHBG, and correlates with free or bioavailable testosterone concentrations in adult hypogonadal men receiving androgen replacement therapy. In men receiving anti-androgens, serum bioactivity decreased tenfold while serum testosterone concentrations decreased only four-fold. Further pilot results in prostate cancer patients showed that androgen synthesis inhibitors result in more complete inhibition of androgen bioactivity than gonadorelin-based androgen deprivation therapy, even in patients whose testosterone concentrations were undetectable by mass spectrometry. We conclude that in vitro androgen reporter bioassays are useful tools to study how androgen bioactivity in serum is determined by androgens, anti-androgens as well as SHBG, provided that dilution and matrix effects are accounted for.

Assessing prenatal and neonatal gonadal steroid exposure for studies of human development: methodological and theoretical challenges.

Assessing prenatal and neonatal gonadal steroid exposure for studies of human development: methodological and theoretical challenges.

Maximal Testosterone Suppression in Prostate Cancer—Free vs Total Testosterone

Testosterone remains a key target in the treatment of advanced prostate cancer. The relationship of free testosterone to prostate cancer treatment and outcomes remains largely unexplored. A consensus of prostate cancer experts was convened in 2013 to review current knowledge surrounding relationship of total and free testosterone to prostate cancer, discuss the free hormone hypothesis, and highlight future avenues for therapeutics. Free testosterone may better reflect prostate cancer tissue androgen levels than serum total testosterone concentration. Free testosterone deserves more research regarding its relation to clinical outcomes.

Vitamin D Binding Protein Impact on 25-Hydroxyvitamin D Levels under Different Physiologic and Pathologic Conditions.

There is a high prevalence of vitamin D deficiency worldwide, but how to define vitamin D deficiency is controversial. Currently, the plasma concentration of total 25-hydroxyvitamin D [25(OH)D] is considered an indicator of vitamin D status. The free hormone hypothesis states that protein-bound hormones are inactive while unbound hormones are free to exert biological activity. The majority of circulating 25(OH)D and 1,25(OH)2D is tightly bound to vitamin D binding protein (DBP), 10–15% is bound to albumin, and less than 1% of circulating vitamin D exists in an unbound form. While DBP is relatively stable in most healthy populations, a recent study showed that there are gene polymorphisms associated with race and ethnicity that could alter DBP levels and binding affinity. Furthermore, in some clinical situations, total vitamin D levels are altered and knowing whether DBP is also altered may have treatment implications. The aim of this review is to assess DBP concentration in different physiological and pathophysiological conditions. We suggest that DBP should be considered in the interpretation of 25(OH)D levels.

Constraints, concerns and considerations about the necessity of estimating free glucocorticoid concentrations for field endocrine studies

Summary We evaluate the utility of measuring corticosteroid‐binding globulins (CBG) and estimating free glucocorticoid (GC) concentrations for field endocrine studies. We assert that for three general reasons, measurement of free GCs might not be more useful than total GCs for many studies. First, estimates of so‐called ‘free’ GCs are likely inaccurate, in part because of the following: (i) other factors in the blood also bind GCs, (ii) CBG binds plasma steroids other than GCs and (iii) CBG binding affinity can vary with local conditions, such as enzymatic activity and tissue temperature. Second, evidence suggests an active role for CBG‐bound GCs, CBG or both, in the vertebrate stress response, calling into question the validity and generality of the free hormone hypothesis. Third, free and total GCs function over different time frames. Free GCs are likely important in the seconds‐to‐minutes time‐scale of interaction with tissues, but total GCs could function at minutes‐to‐hours time‐scales by serving as the reservoir to continue supplying GCs to tissues. As transcription regulators, most GC effects manifest in hours; thus, total GCs would be the most appropriate measure for estimating total biological impact. Our understanding of the biochemistry and the biological actions of both GCs and CBG indicates that total GC concentrations are currently less prone to error and more biologically interpretable than estimates of free hormone. Although further work is necessary, total GC titres currently remain the most accurate and informative estimates of stress hormone levels to address biological questions in nature.

The free hormone hypothesis: Correlation of decreases in PSA with free testosterone rather than total testosterone in men with advanced prostate cancer treated with GTx-758.

e16015 Background: Androgen deprivation therapy (ADT) improves disease-free survival but disease progression is related, in part, to ineffective castration. The free hormone hypothesis states that the biological activity of steroid hormones is affected by its unbound (free) rather than its protein-bound concentration. Serum total testosterone (T) concentrations predominantly reflect the T bound to plasma proteins and do not accurately predict prostatic levels of T. Methods: In a Phase II study (G200705), men with advanced prostate cancer (n=159) were randomized to receive 1000 mg or 2000 mg GTx-758 daily or leuprolide as their initial ADT. Serum total T (mass spectrometry), free T (equilibrium dialysis), SHBG and PSA concentrations were measured. A second Phase II study (G20007) was performed in men (n=9) with CRPC who then received GTx-758 2000 mg daily. Results: Although both treatments reduced serum total T levels to &lt; 50 ng/dL, leuprolide decreased them to a greater extent. However, GTx-758 caused greater reductions in serum PSA, suggesting that total T concentrations did not accurately reflect the suppression of androgen activity. Both dosages of GTx-758 reduced free T levels to a greater extent (mean of 0.7 and 0.4 pg/ml at day 60, and 0.4 and 0.4 pg/ml on day 90, respectively) than leuprolide (mean of 1.4 pg/ml on day 60 and 1.4 pg/ml on day 90; p values &lt;0.03). Similar clinical results were observed in CRPC patients where GTx-758 daily resulted in a 71% decrease in %free T and clinically relevant PSA reductions in men maintained on ADT with LHRH agonists. As a result of adverse events at higher doses of GTx-758, the trial was stopped early. Conclusions: The ERα agonist, GTx-758, reduced the biologically active form of T, free T, to significantly lower levels than leuprolide. Reductions in PSA appeared to be more highly associated with changes in free T. These data provide compelling evidence to support the free hormone hypothesis and suggest that serum free T concentrations would provide a better measure of therapeutic efficacy in ADT than total T. A Phase II clinical trial utilizing lower doses of GTx-758 (G200712) is currently being performed. Clinical trial information: NCT01326312.

Mediating free glucocorticoid levels in the blood of vertebrates: are corticosteroid‐binding proteins always necessary?

Summary Glucocorticoids (GC) are integral to the stress response of vertebrates to environmental challenges. Their impact is immediate and widespread, and prolonged exposure can result in major activational and organizational changes. The vertebrate body has two mechanisms to limit GC impact: first, a rapid negative feedback system to turn off their release, and second, a protein – corticosteroid‐binding globulin (CBG) – to prevent them being free in the blood. Species used in biomedical research have CBG levels that normally bind 90–95% of blood GC. This evidence was the basis for the ‘Free Hormone Hypothesis,’ which posits that only unbound, free hormone is available for use by tissues and is biologically active. High levels of free GC typically occur only under conditions of chronic stress and are associated with major suppression of key body functions. The hypothesis proposes that the primary role of CBG is to render GCs unavailable, thereby preventing tissue exposure. From a field study in southern Ontario on northern (Glaucomys sabrinus) and southern (Glaucomys volans) flying squirrels, we found virtually no CBG binding capacity in their plasma, resulting in only about 10% of cortisol being bound throughout the year. However, neither species showed any evidence of being physiologically compromised. This presents a major challenge to the potential consequences of chronically high levels of free GC. To assess the generality of this finding, we carried out a phylogenetic comparison of 91 vertebrate species in which both GC and CBG levels were measured. In 93%, CBG levels were sufficient to bind about 90% of circulating GCs. This evidence is thus concordant with that from biomedical research. However, both flying squirrel species and four species of New World monkeys were extreme outliers. These two groups had the highest GC concentrations, but relatively little binding capacity (10% or less of their GC was bound). An ancestor state reconstruction of the proportion of GCs bound indicated that the flying squirrels and New World monkeys evolved this character state from ancestors that followed the common 90% bound pattern. It also indicated that the original vertebrates – the earliest fishes – have most of their GC in the free state and little or no steroid‐binding protein. We thus demonstrate a dichotomous pattern with respect to CBG: a dominant branch, where high levels of CBG bind most of the GC, applies to the majority of vertebrates; and a secondary branch, where low levels of CBG bind almost none of the GC, applies to a very small subset. For the latter, the critical unknown is how these species mitigate the impact of the high free GC levels and how such a dramatic trait shift could evolve.

Evaluating stress in natural populations of vertebrates: total CORT is not good enough

Summary Our goal in this review is to discuss how measures beyond simple quantification of total glucocorticoid levels are needed in comparative studies of stress. We need to measure corticosteroid binding globulin – CBG – and further downstream performance metrics to properly evaluate the significance and impact of stress in wild populations. We briefly cover the current literature, discuss methods that may enable detection of chronic stress and point to directions for future research to continue to clarify this field. CBG may regulate access of hormones to tissues, but disagreement remains as to the functional importance of total vs. free vs. bound hormone in the plasma. Here we focus on recent studies providing solid evidence supporting the free hormone hypothesis. These studies unequivocally indicate that the biologically active fraction consists only of the portion that is free (not total levels in the blood). We also present the ‘reservoir hormone hypothesis’, indicating the relevance of CBG‐bound hormone in the plasma. The vast majority of physiological studies on stress in natural populations only measure blood corticosterone or its faecal metabolites. However, downstream metrics (e.g. immune function, oxidative stress and body mass changes) can assess the physiological impact of the changes in corticosterone and CBG and ultimately of the adaptiveness of these changes. Here we will discuss some of the most promising factors that can be measured downstream of the plasma and the rationale for their measurement, how to do it, and an introduction to the evidence. Although we draw on biomedical findings for some of this insight, we recognized that findings from a few mammalian laboratory species (primarily rats and mice) may not apply to the vast diversity of vertebrate species from fish to birds. In large measure we are still in the natural history phase of the accumulation of fundamental knowledge in terms of trying to extract general principles in the stress biology of natural populations. Our review outlines what is needed to lay the foundation for these principles.

Bioavailable vitamin D in chronic kidney disease

Most of the major vitamin D metabolites 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D circulates in a tightly bound state to vitamin D-binding protein (DBP), rendering this fraction unavailable for biological action. A smaller fraction, loosely bound to albumin or circulating freely, is bioavailable, and hence bioactive. This Commentary discusses the free hormone hypothesis and the role of DBP in vitamin D metabolism.

Hyporesponsiveness to Glucocorticoids in Mice Genetically Deficient for the Corticosteroid Binding Globulin

Corticosteroid binding globulin (CBG) is the carrier for glucocorticoids in plasma. The protein is believed to keep the steroids inactive and to regulate the amount of free hormone acting on target tissues (free hormone hypothesis). Here, we generated a mouse model genetically deficient for CBG to test the contribution of the carrier to glucocorticoid action and adrenocortical stress response. The absence of CBG resulted in a lack of corticosterone binding activity in serum and in an approximately 10-fold increase in free corticosterone levels in CBG-null mice, consistent with its role in regulation of circulating free hormone levels. Surprisingly, cbg(-/-) animals did not exhibit features seen in organisms with enhanced glucocorticoid signaling. Rather, the mice exhibited increased activity of the pituitary axis of hormonal control, normal levels of gluconeogenetic enzymes, and fatigue, as well as an aggravated response to septic shock, indicating an inability to appropriately respond to the excess free corticosterone in the absence of CBG. Thus, our data suggest an active role for CBG in bioavailability, local delivery, and/or cellular signal transduction of glucocorticoids that extends beyond a function as a mere cargo transporter.

Does Megalin Mediate Steroid Hormone Uptake?

Androgens and estrogens circulate through the bloodstream bound to sex hormone binding globulin (SHBG), in what has been believed to be an inactive form that prevents these small, lipophilic steroids from diffusing across the plasma membrane to activate intracellular targets (see Adams). A research group that previously showed that the low-density lipoprotein receptor-related protein megalin acts as an endocytic receptor for carrier-bound vitamins A and D has now implicated megalin in the uptake of SHBG-bound sex steroids. Hammes et al. showed that 125 I-labeled SHBG was taken up and degraded by rat choriocarcinoma (BN16) cells, which express megalin, and that this was blocked by receptor-associated protein (RAP), an antagonist of ligand binding to megalin. Most circulating sex steroids are bound to carrier, and the ability of RAP to inhibit [ 3 H]testosterone uptake depended on the ratio of SHBG and testosterone, such that inhibition was maximal when most of the testosterone was bound, whereas RAP was ineffective when most of the testosterone was free. RAP also inhibited the uptake of SHBG-bound dihydrotestosterone (DHT) and 17β-estradiol. Uptake of fluorescein isothiocyanate (FITC)-labeled DHT together with SHBG was confirmed with confocal immunofluorescence microscopy; moreover, DHT internalized together with SHBG activated an androgen-sensitive reporter. The phenotypes of megalin knockout mice were consistent with impaired sex-steroid signaling: Megalin —/— females exhibited vaginal blockade whereas megalin —/— males exhibited impaired descent of the testes. Further, megalin-deficient embryonic tissues were resistant to the effects of exogenous androgens. Thus, the authors conclude that megalin plays a role in the cellular uptake of carrier-bound sex steroids that is critical to the proper development of steroid-responsive tissues. A. Hammes, T. K. Andreassen, R. Spoelgen, J. Raila, N. Hubner, H. Schulz, J. Metzger, F. J. Schweigert, P. B. Luppa, A. Nykjaer, T. E. Willnow, Role of endocytosis in cellular uptake of sex steroids. Cell 122 , 751-762 (2005). [PubMed] J. S. Adams, "Bound" to work: The free hormone hypothesis revisited. Cell 122 , 647-649 (2005). [PubMed]

“Bound” to Work: The Free Hormone Hypothesis Revisited

Megalin is a member of the low-density lipoprotein receptor-related protein (LRP) family. The plasma membrane-anchored LRPs serve as receptors for a wide variety of extracellular ligands, promoting their entry into cells by endocytosis of the receptor-ligand complex. In this issue of Cell, Hammes et al. (2005) show that resistance (insensitivity) to sex steroid hormones is encountered in animals lacking megalin. These data provide important insights into an endocytic mechanism for the uptake of sex steroids by mammalian cells.

Role of Endocytosis in Cellular Uptake of Sex Steroids

Androgens and estrogens are transported bound to the sex hormone binding globulin (SHBG). SHBG is believed to keep sex steroids inactive and to control the amount of free hormones that enter cells by passive diffusion. Contrary to the free hormone hypothesis, we demonstrate that megalin, an endocytic receptor in reproductive tissues, acts as a pathway for cellular uptake of biologically active androgens and estrogens bound to SHBG. In line with this function, lack of receptor expression in megalin knockout mice results in impaired descent of the testes into the scrotum in males and blockade of vagina opening in females. Both processes are critically dependent on sex-steroid signaling, and similar defects are seen in animals treated with androgen- or estrogen-receptor antagonists. Thus, our findings uncover the existence of endocytic pathways for protein bound androgens and estrogens and their crucial role in development of the reproductive organs.

Possible new mechanism of cortisol action in female reproductive organs: physiological implications of the free hormone hypothesis.

The so-called free hormone hypothesis predicts that the biological activity of a given steroid correlates with the free protein-unbound concentration rather than with the total concentration (i.e. free plus protein-bound). Cortisol is a glucocorticoid with many diverse functions and the free hormone hypothesis seems to apply well to the observed effects of cortisol. The ovaries express glucocorticoid receptors and are affected by cortisol, but lack the necessary enzymes for cortisol synthesis. Ovarian follicles modulate the biological activity of cortisol by (1) follicular production of especially progesterone and 17 alpha-hydroxy-progesterone which, within the follicle, reach levels that displace cortisol from its binding proteins, in particular, cortisol-binding protein, making it available for biological action and (2) a developmental regulated expression of two types of 11 beta-hydroxysteroid dehydrogenase (i.e. 11 beta-HSD type 1 and type 2), which oppose the action of one another, the 11 beta-HSD type 2 predominantly inactivating cortisol to cortisone, while 11 beta-HSD type 1 reverses this reaction. As a result, a high concentration of cortisol available for biological action is present in the preovulatory follicle just prior to ovulation and it has been suggested that cortisol may function to reduce the inflammatory-like reactions occurring in connection with ovulation. This paper suggests (1) that the function of the oviduct is also affected by the high levels of free cortisol released in preovulatory follicular fluid at ovulation and (2) that formation and function of the corpus luteum benefits from a high local concentration of free cortisol, whereas the surrounding developing follicles may experience negative effects. If this hypothesis proves correct it may describe a new physiological mechanism by which cortisol interacts with the female reproductive organs, showing that the biologically active concentration of a steroid locally can be regulated to serve specific functions.