Sex hormone-binding globulin: Anatomy and physiology of a new regulatory system

Sex hormone-binding globulin (SHBG) is a plasma glycoprotein that binds a number of circulating steroid hormones (testosterone, dihydrotestosterone and estradiol) with high affinity, thus regulating their free concentration in plasma. In addition to binding steroids, SHBG itself binds to receptor sites on plasma membranes with somewhat unusual kinetics. Both the off and on rates are quite slow. The steroid-binding and membrane-binding functions are interwined in what is clearly an allosteric relationship. Occupation of SHBG's steroid-binding site by a steroid inhibits its ability to bind to its membrane receptor-binding site. This inhibition is not related to a steroid's biological activity. Metabolites of steroids without biological activity, e.g. 2-methoxyestradiol, actively inhibit SHBG's interaction with its membrane receptor. However, if unliganded SHBG is allowed to bind to its receptor on intact cells, and an appropriate steroid hormone then is introduced, adenylate cyclase is activated and intracellular cAMP increases. This function is specific for steroids with biological activity, 2-methoxyestradiol has no activity in this arena. These observations demonstrate a potentially important role for SHBG as a regulator of cell function. They also demonstrate an additional mode of action of steroid hormones, one that does not require that the steroid interact with a steroid receptor.     

Hsd3b2 associated in modulating steroid hormone synthesis pathway regulates the differentiation of chicken embryonic stem cells into spermatogonial stem cells

Steroid hormones regulate differentiation of various types of cell during embryogenesis. Testosterone is one of the androgens that bind to receptors to regulate gene expression and promote spermatogenesis. Our results showed that testosterone, as a product of steroid hormones synthesis pathway, could facilitate the differentiation of embryonic stem cells (ESCs) into spermatogonial stem cells (SSCs). The analysis of the steroid hormones synthesis pathway demonstrated that 3beta‐hydroxysteroid dehydrogenase2 (Hsd3b2) plays a major role in the synthesis of testosterone. In the absence of Hsd3b2, the expression of downstream genes such as Cyp1a1, Ugt1a1, and Hsd17b7 was not maintained. This reduction is probably due to the down‐regulation of the steroid hormones synthesis pathway. Furthermore, qRT‐PCR, immunofluorescence, and flow cytometry analysis confirmed that the steroid hormones synthesis pathway could facilitate the differentiation of ESCs. Altogether, these results lead to a model in which Hsd3b2 regulates ESCs differentiation via modulating the activity of steroid hormones synthesis pathway.     

Uptake, distribution and binding of vertebrate and invertebrate steroid hormones and time-dependence of ponasterone A binding in Calliphora vicina. Comparisons among cholesterol, corticosterone, cortisol, dexamethasone, 5 alpha-dihydrotestosterone, 1,25-dihydroxyvitamin D3, ecdysone, estradiol-17 beta, ponasterone A, progesterone, and testosterone.

The presence of specific binding sites for radiolabelled vertebrate-type and arthropod-type steroid hormones was investigated in several organs including salivary gland, and central nervous system of third instar Calliphora vicina larvae by thaw-mount autoradiography. Ponasterone A, a 20-hydroxyecdysone agonist and 20-hydroxyecdysone are the only steroids which bind to nuclear high affinity binding sites. These binding sites are DNA associated while nucleoli show no tracer binding. Ecdysone, an endogenous 20-hydroxyecdysone precursor, is taken up by target cells but no significant nuclear binding occurs. 1,25-Dihydroxyvitamin D3 concentrates in cytoplasm only and its uptake is highest compared to all other steroids. Progesterone and testosterone show weak accumulation in the cytoplasm, while for cholesterol, corticosterone, cortisol, dexamethasone, dihydrotestosterone and estradiol-17 beta, no noticeable uptake occurs. For ponasterone A, a clear time dependence of uptake and intracellular distribution is visible, suggesting the existence and involvement of specific ecdysteroid uptake and transport mechanisms. These results suggest the presence of binding sites for various mammalian steroids in insects. Whether vertebrate steroid hormones or metabolites of them play a role in insects or whether the uptake and binding is based on chemical similarities alone without biological significance remains to be further investigated.     

Plasma testosterone transport in primates

All primate species, including Old and New World primates and prosimians have a plasma testosterone-estradiol binding globulin (TeBG), which is a glycoprotein and has a similar mobility in polyacrylamide gel electrophoresis. In New World primates the TeBG binding capacity for [3H]testosterone was higher and its affinity lower than in Old World primates. These changes were associated with high unbound plasma testosterone concentrations in these species. Binding parameters of TeBG in prosimian species varied markedly. Thus, in primate evolution TeBG was conserved despite marked differences in binding characteristics. In New World primates changes are associated with high total and unbound testosterone, a finding concordant with alterations of other steroid hormones concentration in these species with "generalized steroid hormone resistance".     

Interaction of testosterone, corticosterone and corticosterone binding globulin in the white-throated sparrow (Zonotrichia albicollis)

Plasma binding globulins bind steroid hormones and are thought to regulate hormone access to tissues. Mammals have both sex steroid binding globulin (SSBG) and corticosteroid binding globulin (CBG). Birds, however, have no detectable SSBG, leading to the early conclusion that birds have no plasma regulation of sex steroids. CBG, however, can bind androgens with relatively high affinity. In birds, therefore, the control of androgenic effects may be tightly regulated by glucocorticoid physiology because glucocorticoids compete with androgens for CBG binding sites. We report levels of total testosterone (T), total corticosterone, CBG, and estimated free T in the males, the more aggressive morph had higher levels of total T; female morphs did not differ. Approximately 96% of T was bound to CBG, but a lack of morph or sex-specific differences in corticosterone titers or CBG capacity caused patterns of free T to mirror those of total T. While CBG has the potential to greatly influence T availability to tissues, in this species interactions between T, CBG and corticosterone do not appear to alter general patterns of T availability to tissues.     

Correlation between plasma steroid hormones and vitellogenin profiles and lunar periodicity in the female golden rabbitfish, Siganus guttatus (Bloch)

Characteristics of the lunar reproductive cycle in the golden rabbitfish, Siganus guttatus, were determined by histological observations of ovarian development, and immunological measurements of plasma steroid hormones, estradiol-17beta (E2), testosterone (T), 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) and 17alpha,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S), and vitellogenin (VTG). Ovarian and plasma samples were collected every week according to the lunar phases from May to July. Weekly change of gonadosomatic index (GSI) showed two peaks at the first lunar quarter in June and July. Yolky oocytes were also observed around this time. Histological observations revealed that the vitellogenic oocytes appeared again 1 week after spawning and developed synchronously. These results suggest that this species is a multiple spawner and the oocyte development is in a group-synchronous manner. Plasma steroid hormones (E2, T, DHP and 20beta-S) and VTG levels changed in parallel with changes in GSI. The peak of plasma VTG level occurred prior to spawning. These cyclic changes of plasma steroid hormones and VTG support the hypothesis that lunar periodicity is the major factor in stimulating reproductive activity of S. guttatus.     

Uptake,distribution and binding of vertebrate and invertebrate steroid hormones and time-dependence of ponasterone A binding in Calliphora vicina

Summary The presence of specific binding sites for radiolabelled vertebrate-type and arthropod-type steroid hormones was investigated in several organs including salivary gland, and central nervous system of third instar Calliphora vicina larvae by thaw-mount autoradiography. Ponasterone A, a 20-hydroxyecdysone agonist and 20-hydroxyecdysone are the only steroids which bind to nuclear high affinity binding sites. These binding sites are DNA associated while nucleoli show no tracer binding. Ecdysone, an endogenous 20-hydroxyecdysone precursor, is taken up by target cells but no significant nuclear binding occurs. 1,25-Dihydroxyvitamin D₃ concentrates in cytoplasm only and its uptake is highest compared to all other steroids. Progesterone and testosterone show weak accumulation in the cytoplasm, while for cholesterol, corticosterone, cortisol, dexamethasone, dihydrotestosterone and estradiol-17, no noticeable uptake occurs. For ponasterone A, a clear time dependence of uptake and intracellular distribution is visible, suggesting the existence and involvement of specific ecdysteroid uptake and transport mechanisms. These results suggest the presence of binding sites for various mammalian steroids in insects. Whether vertebrate steroid hormones or metabolites of them play a role in insects or whether the uptake and binding is based on chemical similarities alone without biological significance remains to be further investigated.     

Fluorescence study of steroid hormone binding activity of Helix pomatia agglutinin

Helix pomatia agglutinin (HPA) is a N-acetylgalactosamine (GalNAc) binding lectin, found in the reproductive gland of a Roman snail. The present study has shown that HPA, in addition to its carbohydrate binding capacity possesses a hydrophobic binding activity. This protein binds with high affinity (k(D)=1.9-2.4 microM) steroid hormones: testosterone and progesterone, identified as putative ligands for the animal lectin HPA. Additionally, we have found that this lectin also interacts with adenine (k(D)=5.4+/-0.5 microM) and arylaminonaphthalene sulfonate TNS (k(D)=12+/-0.3 microM). Binding of HPA to hormones and adenine was accompanied by a significant increase of the intrinsic Trp fluorescence (up to 50%), characterizing the conformational changes in the lectin molecule. The hyperbolic shape of the binding curves indicated one high affinity site for the two steroid hormones and adenine, and more than one hydrophobic site for TNS, showed by the sigmoidal curve fit and Hill coefficient of (n(H)=1.5+/-0.2). Hormones and adenine compete for an identical binding site, suggested to occupy the central hydrophobic cavity of the HPA hexamer. Fluorescence resonance energy transfer (FRET) was applied to calculate the intramolecular distance between TNS and Trp chromophores.     

Studies on steroid binding proteins in normal tissues and tumor cell lines

Steroid binding proteins bind steroid hormones with high affinity and their function is to carry those hormones in the extracellular compartment. Since their discovery more than fifty years ago, many reports concerning their physicochemical structures and functions have contributed to the better understanding of those proteins. Recent advances in recombinant DNA technology have led to the availability of molecular probes for these proteins, and new approaches have been used to analyse their gene structures as well as the regulation of their synthesis. In the present report, we will review the new findings of the last five years which include the cloning and sequencing of the cDNAs and genes for corticosteroid binding globulin, testosterone estradiol binding globulin and androgen binding protein, as well as the tissue distribution and regulation of their mRNAs in normal tissues and cancer cell lines.     

Synthesis and receptor binding of polynuclear organometallic estradiol derivatives

Twelve novel organometallic derivatives of estradiol were synthesized with the aim of utilizing organometallic cold bioprobes as radioisotopic labels substitutes for steroid hormone receptor assays. For this purpose, we envisaged the attachment of several stable cobalt, molybdenum, osmium carbonyl clusters (tetra- and pentanuclear species) at estradiol 17 alpha-, 16 alpha-, 2- or 4-positions. The binding affinity of these new complexes for uterine estradiol receptor has been measured by the competitive binding method. The results show that the 17 alpha-position can tolerate substitution by bulky organometallic groups (especially in the case of cobalt and molybdenum carbonyl clusters). Estradiol derivatives which are functionalized at C-4 and C-16 alpha bind estradiol receptor with reasonable affinity and the RBA values are the same for the complexed and uncomplexed hormones. The 2- position is more sensitive to organometallic substitution and the complexation at the 2- alkyne results in a dramatic decrease of the RBA values. These results show that the attachment of polynuclear moieties in estradiol 17 alpha-, 4- and 16 alpha-, positions gives rise to compounds which are of potential utility in a new non-radioisotopic receptor assay since the metal-carbonyl markers are readily detected by high-sensitivity Fourier-transform infra-red spectroscopy.     

The control of the interaction of sex hormone-binding globulin with its receptor by steroid hormones

Sex hormone-binding globulins (SHBG) is a plasma glycoprotein that binds certain steroids. It, in turn, binds to a specific receptor on cell membranes. This work was undertaken to investigate the role of steroids in the interaction of SHBG with its receptor. Because the probe for the interaction of SHBG with its receptor is 125I-SHBG, we first showed that 125I-SHBG binds [3H]dihydrotestosterone (DHT) at 4 degrees C and 37 degrees C with KD values similar to those published previously for pure radioinert SHBG. 125I-SHBG could be prevented from binding to its receptor by a variety of steroids whose relative inhibitory activity (dihydrotestosterone much greater than 2-methoxyestradiol greater than testosterone greater than estradiol much greater than methyltrienolone greater than cortisol) was almost identical to their relative ability to bind to SHBG. Because significant binding of [3H]DHT to the SHBG receptor could not be demonstrated, steroid inhibition of SHBG binding must be noncompetitive. If steroids bound to SHBG prevent binding to the SHBG receptor, then liganded SHBG should have a higher apparent KD for its receptor than unliganded SHBG. This is the case. The KD was 0.86 +/- 0.25 nM for the high affinity receptor site using liganded SHBG and 0.19 +/- 0.024 nM for unliganded SHBG. Thus, only liganded SHBG assumes a conformation that prohibits interaction with the SHBG receptor. However, when unliganded SHBG was prebound to its receptor, it retained its ability to bind [3H] DHT. The model that emerges from these observations is as follows. Unliganded SHBG can bind either steroids or receptor in a reversible reaction; SHBG bound to a steroid cannot bind to the receptor, but unliganded SHBG that first binds to the receptor can subsequently bind steroids.     

Effect of human chorionic gonadotropin derivatives on leydig cell function.

BACKGROUND: Several human chorionic gonadotropin (hCG) derivatives have been detected in healthy human subjects, indicating that they may play a role in cell function. These hCG derivatives include deglycosylated hCG, proteolytic digestion products of hCG and free alpha and beta subunits of the hormone. It is well documented that testicular Leydig cells are responsive to luteinising hormone (LH) or its analogue hCG. These hormones have high affinity for LH/hCG receptors on the plasma membrane. METHODS: We designed functional and binding studies to compare the effects of native hCG and several hCG derivatives on a rat Leydig cell system. The molecular weight of the hCG derivatives was determined by SDS-PAGE and the binding affinity to LH/hCG receptors was measured by a radioligand assay. In addition, their ability to produce testosterone, cyclic AMP and arachidonic acid release was also studied. RESULTS: These hCG derivatives, with the exception of the free beta subunit, were able to bind to LH/hCG plasma membrane receptors with different affinities than that of native hCG. In addition, hCG derivatives did not increase intracellular cAMP levels or arachidonic acid release. However, they did increase testosterone production. CONCLUSION: Taken together, the results of this study lead us to suggest that these hCG derivatives may regulate the action of the native hormone in Leydig cells and are, thus, molecules of physiological relevance.     

Rat alpha 1-microglobulin. Purification from urine and synthesis by hepatocyte monolayers

Rat alpha 1-microglobulin was isolated from the urine of rats treated with sodium chromate, and was purified by the use of gel chromatography, affinity chromatography on concanavalin-A-Sepharose and ion-exchange chromatography. The protein was heterogeneous in charge, had a tendency to form dimers, and was associated with a brown-coloured chromophore. The size of the protein (25 kDa) was similar to guinea pig alpha 1-microglobulin but smaller than the human protein, when measured with sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Immunological cross-reaction with human and guinea pig alpha 1-microglobulin was demonstrated. The concentration of alpha 1-microglobulin in rat serum was 16.4 mg/l (SD = 8.5 mg/l, n = 13) and rat serum alpha 1-microglobulin was eluted from a gel chromatography column at two different positions corresponding to monomeric alpha 1-microglobulin and IgA. The latter alpha 1-microglobulin activity could be absorbed by anti-IgA serum. Rat alpha 1-microglobulin and albumin were continuously released into the medium of rat hepatocyte monolayers, and alpha 1-microglobulin was isolated from the medium by the use of immunoprecipitation with anti-(alpha 1-microglobulin). Tritiated leucine, added to the medium, was incorporated into the protein, suggesting a de novo synthesis of alpha 1-microglobulin by the hepatocytes. The size of hepatic alpha 1-microglobulin was similar to that of purified urinary rat alpha 1-microglobulin, when determined with sodium dodecyl sulfate/polyacrylamide gel electrophoresis.     

Sex Specific Binding of Steroid Hormones to Microsomal Membranes of Rat Liver

Direct studies of the binding of oestradiol and testosterone to male and female membranes in vitro show that there are sex specific binding sites which have a high affinity for steroid hormones.     

Effect of sex and prior exposure to a cafeteria diet on the distribution of sex hormones between plasma and blood cells

It is generally assumed that steroid hormones are carried in the blood free and/or bound to plasma proteins. We investigated whether blood cells were also able to bind/carry sex-related hormones: estrone, estradiol, DHEA and testosterone. Wistar male and female rats were fed a cafeteria diet for 30 days, which induced overweight. The rats were fed the standard rat diet for 15 additional days to minimize the immediate effects of excess ingested energy. Controls were always kept on standard diet. After the rats were killed, their blood was used for 1) measuring plasma hormone levels, 2) determining the binding of labeled hormones to washed red blood cells (RBC), 3) incubating whole blood with labeled hormones and determining the distribution of label between plasma and packed cells, discounting the trapped plasma volume, 4) determining free plasma hormone using labeled hormones, both through membrane ultrafiltration and dextran-charcoal removal. The results were computed individually for each rat. Cells retained up to 32% estrone, and down to 10% of testosterone, with marked differences due to sex and diet (the latter only for estrogens, not for DHEA and testosterone). Sex and diet also affected the concentrations of all hormones, with no significant diet effects for estradiol and DHEA, but with considerable interaction between both factors. Binding to RBC was non-specific for all hormones. Estrogen distribution in plasma compartments was affected by sex and diet. In conclusion: a) there is a large non-specific RBC-carried compartment for estrone, estradiol, DHEA and testosterone deeply affected by sex; b) Prior exposure to a cafeteria (hyperlipidic) diet induced hormone distribution changes, affected by sex, which hint at sex-related structural differences in RBC membranes; c) We postulate that the RBC compartment may contribute to maintain free (i.e., fully active) sex hormone levels in a way similar to plasma proteins non-specific binding.     

Counter current transfer in the female adnex

The utero-ovarian veins and lymph vessels are intimately connected with the ovarian artery in the human female and in domestic animals, with the exception of the horse and the human female. A direct, local exchange of molecules from veins and lymph vessels to arteries (counter current transfer) has been documented for this anatomic structure. Countercurrent transfer of certain inert gases (133xenon, 85krypton), of prostaglandins (PGF2 alpha), of steroid hormones (e.g. progesterone, estradiol, testosterone), and of small peptide hormones (oxytocin, relaxin) has been shown to occur in laboratory and domestic animals as well as in the human female. The transfer of the inert gases takes place within seconds. The transfer of steroid hormones and peptides is detectable within minutes while the transfer of PGF2 alpha is delayed for 20 minutes. Red blood cells or albumin are not transferred. The existence of the local transfer is postulated to be of importance for: 1) the pregnancy/non-pregnancy signal from the uterus and tube to the ovary. The signal may be a combination of a luteotrophic signal from the embryo and lack of a "non-pregnant" luteolytic signal from the endometrium, the latter probably being PGF2 alpha in some species; 2) the unilateral influence of the ovarian hormones on the function of the ovarian, tubal, and possibly uterine tissues. An active corpus luteum may create in a mono-ovulatory animal a higher progesterone level in arterial blood supplying the ipsilateral tube and ovarian interstitial tissue than on equivalent contralateral organs.     

Gender differences in the vasomotor effects of different steroid hormones in rat pulmonary and coronary arteries.

It is well recognised that oestrogens possess vasodilatory properties, and similar responses to testosterone have been demonstrated. However, vasomotor effects of other steroid hormones have not been well described. Direct comparisons of the relative vasoactivity of different steroid hormones in different vascular beds in male and female genders have not been made. Coronary and pulmonary arteries from adult Wistar rats were mounted in a wire myograph, loaded to 100 and 17 mmHg respectively, maximally pre-contracted with 1 x 10(-4) M prostaglandin-F-2-alpha, and dose response curves to 1 x 10(-6) to 1 x 10(-3) or 3 x 10(-3) M of 17 beta-oestradiol, testosterone, progesterone, and cortisol dissolved in water were constructed. Addition of each steroid hormone caused acute, dose dependent dilatation in coronary and pulmonary vessels. In coronary arteries the order of activity was testosterone > progesterone > 17 beta-oestradiol > cortisol, p < 0.001. In pulmonary arteries, the order of activity was progesterone > testosterone > cortisol > 17 beta-oestradiol, p < 0.001. Pulmonary arteries from male animals were more sensitive to the effects of testosterone than those from female animals, p = 0.003, whereas coronary arteries from female animals were more sensitive to the effects of 17 beta-oestradiol than those from male animals, p < 0.001. We have demonstrated significant differences in the in vitro vasomotor effects of different steroid hormones in two distinct vascular beds. Gender differences in vasomotor responses to steroid hormones may play a role in the aetiology of vasospastic diseases.     

Endotoxin induced changes in serum estrogen in male rats: influence of testicular maturation

The influence of acute endotoxin (Endo) administration on adrenal and testicular serum hormones, corticosterone (B), progesterone (P4), 17 alpha OH progesterone (17 alpha OH P4), androstenedione (delta 4), testosterone (T), estrone (E1) and estradiol (E2) was studied in male rats aged 8, 12 and 15 weeks. The present study confirms that the concentrations of circulating steroid hormones in male rats vary with age, and indicate that the adrenal glands mature before the testes. The steroid response to Endo is age-dependent. B, P4, 17 alpha OH P4 was increased and T decreased in all animals. But, there was a very significant increase in estrogens (E1 and E2) and a decrease in delta 4 only in male rats aged 12 weeks and over. The lack of an estrogen response to Endo injection in 8 week-old rats may indicate that the reduced sensitivity (refractory period) to Endo (which has been reported to last until 21 days of age) continues longer. The reduced sensitivity to Endo which occurs in young rats could be due in part to the absence of adrenal-testicular cooperation as a result of partial testicular immaturity.     

Estrophilic 3 alpha,3 beta,17 beta,20 alpha-hydroxysteroid dehydrogenase from rabbit liver--II. Mechanisms of enzyme-steroid interaction

Binding of [3H]estradiol, [3H]testosterone and [3H]progesterone to purified NADP-dependent estrophilic 3 alpha,3 beta,17 beta,20 alpha-hydroxysteroid dehydrogenase (EHSD) from rabbit liver cytosol has been examined. The three steroids bind to the enzyme with moderate [corrected] affinity (Ka congruent to 10(7) [corrected] M-1 at 4 degrees C) and equal binding capacity. High-rates were shown for both association and dissociation processes. The steroids competitively inhibited the binding of each other to EHSD. At the same time, their relative binding affinities (RBA) were dependent on the nature of [3H]ligand. The results of RBA determinations for 72 steroids and their analogues by inhibition of [3H]progesterone binding to EHSD suggest that androgens and gestagens bind preferentially to the same site on EHSD molecule, while estrogens (at least by their D-ring) bind to another site. The assumption that EHSD molecule has more than one binding site for steroids is corroborated by (i) substrate inhibition revealed for a number of steroids; (ii) the estrogen ability to potentiate 20 alpha-reduction of progesterone; (iii) stimulatory effect of 5 alpha (beta)-androstane-3 alpha (beta), 17 beta-diols on [3H]testosterone and progesterone binding; and (iv) reciprocal effect of NADP on [3H]estradiol and [3H]testosterone binding to EHSD. Significant differences in sensitivity to pH and changes in NaCl concentration upon metabolism and binding of various steroids have been found. At concentrations of 16 mM dithiothreitol potentiated catalytic conversion of some steroids and had no effect on metabolism of others. Both the affinity for steroids and binding capacity of EHSD are found to be cofactor-dependent. It is speculated that EHSD has a complex active center including at least two mutually influencing steroid-binding sites tightly related with cofactor-binding site. The polyfunctionality of EHSD may be due to both the excess of functional protein groups that form individual constellations upon binding of any steroid and also to conformational lability of EHSD molecule implying alternative orientations of steroids at the binding site.