Catherine Grenot

FSH regulates cultured Leydig cell function via Sertoli cell proteins: an in vitro study.

The effects of follicular stimulating hormone (FSH) on testicular steroidogenic activity has been studied by testing the capacity of conditioned medium (CM) by both unstimulated (control) Sertoli cells (C-CM) and FSH stimulated Sertoli cells (FSH-CM) to influence porcine cultured Leydig cell activity. Leydig cells cultured in FSH-CM for 48 hrs, as compared to C-CM, show a significant (P less than 0.05) increase in [125I]-hCG binding (150% +/- 4) and hCG-stimulated testosterone (T) secretion (266% +/- 42). In addition, the stimulating effect of FSH-CM on Leydig cell function as compared to C-CM, is trypsin sensitive, non dialyzable, heat stable, acid resistant and is chromatographed following gel filtration (Sephadex G 100) into two different peaks of activity. These data suggest that FSH regulates Leydig cell function via (at least two types of) Sertoli cell secreted proteins.

Influence of glycosylation on the clearance of recombinant human sex hormone-binding globulin from rabbit blood

Human sex hormone-binding globulin (hSHBG) is a plasma glycoprotein that binds sex steroids with high affinity. Variations in hSHBG glycosylation contribute to its electrophoretic microheterogeneity, but the functional significance of different SHBG glycoforms is unknown. Carbohydrates may influence the biological activities and half-lives of glycoproteins and we have examined how oligosaccharides at specific sites influence the plasma clearance of hSHBG in vivo. To accomplish this, fully-glycosylated hSHBG, or hSHBG mutants lacking specific oligosaccharides chains, were expressed in Chinese hamster ovary (CHO) cells and purified by immunoaffinity chromatography. The purified recombinant proteins were then biotinylated to study their plasma half-lives after intravenous injection into rabbits. When compared to hSHBG isolated from serum, recombinant hSHBG migrates with a slightly larger average molecular size during denaturing polyacrylamide gel electrophoresis. This is due to a greater proportion (33-39% vs. 3%) of more highly branched N-linked oligosaccharides on the recombinant proteins. When injected into rabbits, the disappearance of recombinant hSHBG showed two exponential components, as previously shown for natural hSHBG in the same animal model. The mean +/- S.E.M. plasma half-lives of recombinant hSHBG (t 1/2alpha 0.11+/-0.03 h and t 1/2beta 18.94+/-1.65 h) are shorter than previously measured for natural hSHBG (t 1/2alpha 3.43+/-0.72 h and t 1/2beta 38.18+/-7.22 h) and this is likely due to differences in the composition of their N-linked oligosaccharides. An O-linked chain at Thr7 does not influence the plasma clearance of hSHBG in the presence or absence of N-linked carbohydrates at Asn351 and Asn367. However, a 1.5-1.6 fold (p<0.03) increase in plasma half-life of variants lacking both N-glycosylation sites was observed and this is probably due to the fact these variants are not recognized by the asialoglycoprotein receptor-mediated clearance system. Removal of either N-glycosylation consensus site also increased (p<0.0001) the plasma half-life of hSHBG by 2.3 2.4 fold. Thus, the metabolic clearance of hSHBG appears to be determined by the number of N-linked oligosaccharides rather than their location.

Isolation of antibodies to steroid hormones by affinity chromatography on antigen-linked sepharose: An efficient electrophoretic elution procedure

Abstract An electrophoretic elution procedure of antibodies retained on affinity columns is described. It afforded a 60% recovery of the binding activity of a high affinity (Ka ∼ 10 10 M −1 ) antiserum to 5α-dihydrotestosterone retained on antigen-linked Sepharose 4B affinity columns. These purified unbound antibodies, (Ka ∼ 10 10 M −1 ) when applied again on identical antigen-linked affinity columns, were all retained and totally recovered after a new electrophoretic elution. Comparable results were obtained by elution with 1M NH 4 OH. The residual 40% binding activity remaining on the antigen-linked Sepharose gel after electrophoretic elution was totally recovered by elution with an excess of 5α-dihydrotestosterone. It corresponded to antibodies of higher affinity (Ka ∼ 10 11 M −1 ). On the other hand the residual 40% fraction of antibodies resistant to NH 4 OH elution was denaturated.

Paracrine control of Leydig cell activity by FSH dependent proteins from sertoli cells: An in vitro study

The regulating effect of follicle-stimulating hormone (FSH) on Leydig cell function was studied using a model of immature porcine Leydig and Sertoli cells cultured in a hormone supplemented defined medium. FSH pretreatment for 2 days of Leydig cells cultured alone was with no effect. FSH pretreatment of Leydig cells cocultured with Sertoli cells increases Leydig cell activity in an FSH dose-dependent manner with a maximal effect observed at 50 ng/ml porcine FSH (pFSH). Leydig cells cultured for 2 days in conditioned medium (CM) by FSH stimulated (FSH-CM) Sertoli cells, as compared to CM by unstimulated (control) (C-CM) Sertoli cells show an increase of their activity with a maximal effect observed at 50 ng/ml pFSH. Leydig cells cultured in CM as compared to non CM, show a marked development of organelles (smooth endoplasmic reticulum and mitochondria) involved in the steroidogenic activity. The activity of FSH-CM as compared to C-CM on Leydig cell function was non dialyzable and trypsin sensitive. These data suggest that Sertoli cells exert a regulatory action on Leydig cell steroidogenic activity via FSH dependent secreted proteins.

A low α-linolenic intake during early life increases adiposity in the adult guinea pig

BackgroundThe composition of dietary fatty acids (FA) during early life may impact adult adipose tissue (AT) development. We investigated the effects of α-linolenic acid (ALA) intake during the suckling/weaning period on AT development and metabolic markers in the guinea pig (GP).MethodsNewborn GP were fed a 27%-fat diet (w/w %) with high (10%-ALA group), moderate (2.4%-ALA group) or low (0.8%-ALA group) ALA content (w/w % as total FA) until they were 21 days old (d21). Then all animals were switched to a 15%-fat diet containing 2% ALA (as total FA) until 136 days of age (d136).ResultsALA and docosapentaenoic acid measured in plasma triglycerides (TG) at d21 decreased with decreasing ALA intake. Total body fat mass was not different between groups at d21. Adipose tissue TG synthesis rates and proliferation rate of total adipose cells, as assessed by 2H2O labelling, were unchanged between groups at d21, while hepatic de novo lipogenesis was significantly 2-fold increased in the 0.8%-ALA group. In older GP, the 0.8%-ALA group showed a significant 15-%-increased total fat mass (d79 and d107, p < 0.01) and epididymal AT weight (d136) and tended to show higher insulinemia compared to the 10%-ALA group. In addition, proliferation rate of cells in the subcutaneous AT was higher in the 0.8%-ALA (15.2 ± 1.3% new cells/5d) than in the 10%-ALA group (8.6 ± 1.7% new cells/5d, p = 0.021) at d136. AT eicosanoid profiles were not associated with the increase of AT cell proliferation.ConclusionA low ALA intake during early postnatal life promotes an increased adiposity in the adult GP.

Isolation of antibodies of improved specificity after fractionation on antigen-sepharose affinity columns of antisera to bovine serum albumin conjugates of C-3- and C-7-linked (O-Carboxymethyl)Oximino- and hemisuccinamido derivatives of 5α-dihydrdtestosterone

Abstract Rabbit antisera to bovine serum albumin (BSA) conjugates of 3-(O-carboxymethyl)oximino-, 7-(O-carboxymethyl)oximino- and 7β-hemi-succinamido derivatives of 5α-dihydrotestosterone (DHT) were applied to four affinity columns bearing respectively these three antigens and a fourth 3β-hemisuccinamido-5α-androstan-17β-ol-BSA antigen as ligands. The antibodies retained on the columns were totally desorbed by an excess of DHT, but in DHT-bound form, whereas 1M mh 4 oh and electrophoretic elution allowed a recovery of 60% of the retained antibodies in unbound form. The antibody fractions (40%) remaining on the columns after NH 4 OH or electrophoretic elution were totally recovered by addition of DHT following the electrophoretic elution only. All the DHT-bound fractions were dissociated by dialysis but with a 70% loss of binding activity. The association constants for DHT of most of the antibody fractions were similar to those of the crude antisera (Ka ~ 10 10 M −1 ), with the exception of the antibodies recovered from the antibody fractions resistant to electrophoretic elution which had higher affinities (Ka ~ 2.0 to 30 × 10 10 M −1 ). The specificity charts of the antisera were in some cases considerably modified after fractionation, according to the choice of the ligand employed in the affinity columns as well as of the elution methods. The lowest cross-reactions with testosterone were observed after elution with 1M NH 4 OH (17–20%) or electrophoresis (23–25%) of the anti -7-(O-carboxymethyl)oximino-DHT antisera fractions retained on 3β-hemisuccinamido-5α-androstan-17β-ol-BSA-Sepharose columns.

Intravenous injection of human sex steroid hormone-binding globulin in mouse decreases blood clearance rate and testicular accumulation of orally administered [2-125I]iodobisphenol A

Bisphenol A, an environmental compound with estrogenic activity, has been shown to bind human sex steroid hormone-binding globulin (hSHBG), the main plasma transport protein which regulates the metabolism of androgens and estrogens and limits their access to target organs. The present study was conducted to determine whether physiologically relevant concentrations of hSHBG can influence the blood clearance rate of bisphenol A and its accumulation in the testes. A radioactive [2-125I]iodobisphenol tracer was synthesized with an association constant (Ka) for binding to hSHBG of 0.14 +/- 0.01 x 10(6) M(-1) at 37 degrees C, a value much lower than for [2-125I]iodoestradiol, which was also synthesized. We used i.v. injection of immunopurified hSHBG in adult male mice to maintain hSHBG levels within the physiologically possible range for humans (27-267 nM) before gavage administration of [2-125I]iodobisphenol or [2-125I]iodoestradiol, for measuring the blood clearance rate of radioactive signal in blood samples taken during the following 120 min. Testicular accumulation of radioactivity was measured 24 h and 48 h after gavage of [2-125]iodobisphenol A. In mice receiving immunopurified hSHBG or vehicle, the time-dependent blood clearance of radioactivity exhibited a bi-exponential decrease which indicated alpha-diffusion and beta-elimination phases for both radioactive ligands. The presence of circulating hSHBG significantly and dose-dependently lowered the clearance rate of radioactivity. However, much higher circulating levels of hSHBG were required to retard the blood clearance of [2-125I]iodobisphenol A as compared to those required for [2-125I]iodoestradiol, in keeping with the important difference in their respective Ka value for binding to SHBG. In addition, mice treated with hSHBG exhibited significantly (P = 0.036) reduced testicular accumulation of radioactivity 24 h and 48 h after ingestion of [2-125I]iodobisphenol A. Provided that the binding properties of bisphenol A for hSHBG are not substantially different from those measured for [2-125I]iodobisphenol A, these findings suggest that, although hSHBG binds 2-mono-iodobisphenol A with a relatively low binding affinity, high enough concentrations of circulating hSHBG (range concentrations between 85 and 267 nM) are potentially able to exert a protective effect against exposure to bisphenol A.

Synthesis and characterization by 1H and 13C nuclear magnetic resonance spectroscopy of 17α-cyano, 17α-aminomethyl, and 17α-alkylamidomethyl derivatives of 5α-dihydrotestosterone and testosterone

Abstract 17α-Aminomethyl, 17α-acetamidomethyl, and 17α-hemiglutaramidomethyl derivatives of dihydrotestosterone and testosterone have been prepared by hydrocyanation of 3,3′-(ethylenedioxy)-5α-androstan-17-one and 3,3′-ethylenedioxyandrost-5-en-17-one, reduction of the corresponding acetylated 17α-cyanohydrins with lithium aluminium hydride, and acylation of the resulting 17α-aminomethyl derivatives with either acetic anhydride or the mono acid chloride of glutaric acid mono methyl ester. Saponification of the 17α-hemiglutaramidomethyl methyl esters gave the corresponding hemiglutaramido derivatives, while acid hydrolysis of the 3-ethylene ketal group of 17α-acetamidomethyl and 17α-hemiglutaramidomethyl derivatives regenerated the 3-oxo and 3-oxo-4-ene functions. The 17α-configuration of 17-substituted steroids was determined by 1 H and 13 C NMR and confirmed by comparing with NMR data for 17α- and 17β-cyano-17-hydroxyandrost-4-en-3-one, 17β-cyano-3.3′-(ethylenedioxy)androst-5-en-17-ol, 17α-alkynyl, and 17α-hexanoic derivatives of dihydrotestosterone and testosterone, of known 17-configurations. Several ambiguous assignments of 13 C NMR signals of 17α-substituted steroids and unsubstituted 17β-hydroxy or 17-oxo precursors have been resolved using steroid analogs deuterated at positions C5–7, or C16 for androstane derivatives, and at positions C6–7, or C7 for androstene derivatives. 17α-Aminomethyl and 17α-alkylamidomethyl derivatives of dihydrotestosterone and testosterone are useful intermediates for the access to potential ligands of androgen-binding proteins necessary for affinity chromatography purification or affinity-labeling experiments.

Improvement of specificity of anti-testosterone and anti-5α-dihydrotestosterone rabbit antibodies by immunotolerance techniques

Anti-testosterone (T) and anti-5 alpha-dihydrotestosterone (DHT) antibodies were raised in rabbits after preimmunization with 17 beta-hemisuccinamido-, 7 beta-hemiglutaramido- and 3 beta-hemiglutaramido haptens of DHT and T covalently linked to D-glutamic acid-D-lysine (D-GL) copolymer, followed by immunization with the corresponding haptens covalently linked to bovine serum albumin. Preimmunization with DHT-D-GL in the case of anti-T-antibodies or with T-D-GL, in the case of anti-DHT antibodies significantly lowered the T-DHT cross-reactivity in all cases, the most striking effect being observed with anti-17 beta-hemisuccinamido-T antibodies (CR less than 1%). The evolution with time of the binding characteristics was also studied, showing that in several cases the lowered T-DHT cross-reactivity could be maintained after several booster injections until a useful titer was reached. The better results obtained with 17 beta-hemisuccinamido haptens suggest that the structure of the hapten exerts a strong influence on the induction of immunotolerance.

New approach for measurement of non-SHBG-bound testosterone in human plasma

Testosterone (T) circulates in the blood tightly bound to sex hormone-binding globulin (SHBG) and weakly to albumin. Measuring protein unbound T (free) or non-SHBG-bound T rather than total T has been recommended for the evaluation of androgen disorders in humans. Ammonium sulfate precipitation has been widely used to separate [SHBG-T] complex from free and albumin-bound T. To achieve more specificity in this separation, we used monoclonal anti-SHBG antibody and developed a suitable and convenient immunoassay for measuring non-SHBG-bound T. Magnetic beads were covalently coupled to a monoclonal anti-SHBG antibody to capture [SHBG-T] complex from plasma samples. Magnetic separation was then performed to allow measurement of non-SHBG-bound T in the supernatant by direct radioimmunoassay. When 300 microL of plasma samples were incubated at room temperature with 10 microL of anti-SHBG beads, residual SHBG concentration was undetectable in the supernatant. The specificity of proteins retained on anti-SHBG beads was further demonstrated by peptide mass fingerprint on a MALDI-TOF analyzer. The non-specific adsorption of T on beads was low (5%), and dissociation of T from SHBG-T complex was less than 5% after 180 min of incubation. The plasma concentrations of non-SHBG-bound T using anti-SHBG beads were highly correlated to those obtained using ammonium sulfate precipitation. We conclude that SHBG immunocapture is a highly specific and useful tool for an experimental direct measurement of plasma non-SHBG-bound T. This methodology is also convenient and appropriate for routine and automated assay.