Induction (or stimulation) of prolactin and growth hormone production in a rat pituitary tumor cell line by bromodeoxyuridine.

Under basal conditions, a rat pituitary tumor cell line (C8 11RAP) does not secrete any detectable PrL, FSH, and LH, and secretes only minute amounts of GH (27.1 +/- 0.5 ng/10(6) cells.24 h), as evaluated by RIA. Bromodeoxyuridine (BrdUrd) added to the culture medium induced the accumulation of PRL into cells and medium, increased that of GH, but did not induce that of LH or FSH. The amount of radioimmunoassayable PRL and GH accumulated in the medium increased after a lag period of 15 days and was drug concentration dependent. Maximal accumulation was 232.9 +/- 36.8 and 493.6 +/- 41.5 ng/10(6) cells.24 h for PRL and GH, respectively, at 50 micrograms/ml BrdUrd. In the presence of BrdUrd (greater than or equal to 20 micrograms/ml), the cells grew more slowly and were more strongly attached to the flasks. All of the effects induced by BrdUrd were reversible. PRL and GH were characterized by three methods; 1) radiocompetition with increasing dilution of samples; 2) Sephadex chromatography, followed by RIAs; and 3) sodium dodecyl sulfate-polyacrylamide gel electrophoresis done on the immunoprecipitate of the proteins secreted by cells incubated with [3H]leucine. Chronic treatment with TRH (3 X 10(-6) M) of cells grown without BrdUrd was unable to increase the production of GH or to induce that of PRL. On the other hand, after the same treatment of cells cultured in the presence of BrdUrd, the amounts of PRL accumulated in the culture medium or cells were increased 2- to 7-fold over unstimulated levels; under the same conditions, GH accumulation in the medium was also increased, but this augmentation was less than that of PRL. These results indicate that BrdUrd simultaneously induces or stimulates the production of PRL and GH in C8 11RAP cells, and that TRH increases the production of both hormones only in BrdUrd-treated cells.

Human variant sex hormone-binding globulin (SHBG) with an additional carbohydrate chain has a reduced clearance rate in rabbit.

Sex hormone-binding globulin (SHBG) is the specific plasma transport protein for sex steroid hormones in humans. Considerable variation in SHBG plasma concentration exists between individuals, irrespective of gender, body weight, or thyroid status. In the present work, the influence of carbohydrate chains on the half-life of human SHBG (hSHBG) was investigated using a rabbit model. A variant hSHBG, with a point mutation in exon 8 (GAC --> AAC) encoding an amino acid substitution (Asp327Asn), which introduces an additional consensus site for N-glycosylation, has recently been identified. This mutation suppresses a recognition site for the restriction enzyme Bbs-I, allowing the development of a simple restriction-fragments length polymorphism (RFLP) screening procedure. In a population of patients (272 female and 49 male) consulting in our Endocrinology Clinic, 48 patients (41 female and 7 male) were heterozygous for the variant hSHBG allele and 3 (2 female and 1 male) were homozygous. In this population, the total variant allele frequency was 0.083. The hSHBG genotype, as determined by RFLP, corresponded in all cases to the phenotype as determined by the migration profile of hSHBG by Western blot analysis. The influence of such an additional glycosylation site on the biological half-life of variant hSHBG was investigated. SHBG from serum of patients carrying one of the three hSHBG genotypes was purified and labeled with biotin, then injected into rabbits, as we have recently described for rabbit SHBG. Biotinylated hSHBG was captured from rabbit serum samples on tubes coated with an anti-hSHBG antibody and detected by luminometry with the streptavidine-alkaline phosphatase-dioxetane (AMPPD) system. The results showed that the half-life value was significantly higher (P < 0.05) for SHBG purified from homozygous variant serum (t1/2 beta = 51.43 +/- 1.15 and 63.63 +/- 3.92 h, for male and a female subjects SHBG respectively) than for SHBG purified from heterozygous variant serum (t1/2 beta = 40.19 +/- 0.12 h) or wild-type (t1/2 beta = 38.18 +/- 7.22 h). This study demonstrated that an additional carbohydrate chain on hSHBG decreases the clearance rate of this protein. The low frequency of this variant allele means that further study will be required to determine whether it is associated with higher serum SHBG concentration.

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.

Use of non-radioactive labels for half-life measurement of sex hormone-binding globulin in the rabbit.

The purpose of this study was to investigate two methods for labeling rabbit sex hormone-binding globulin (rSHBG) with non-radioactive material, biotin (B) and europium (Eu3+), in order to obtain stable labeled SHBG and measure in vivo its metabolism and distribution. The obtained half-life values were compared with [125I]rSHBG half-lives. rSHBG was first isolated by immunoaffinity chromatography using an immobilized monoclonal anti-human SHBG (hSHBG) antibody that cross-reacts with rSHBG. This purified rSHBG was labeled by either biotin-X-N-hydroxysuccinimide ester (rSHBG-B), Eu3(+)-diethylenetriaminepentaacetic dianhydride, or Eu(3+)-isothiocyanatobenzyldiethylenetriamine-tetraacetic acid reagents (rSHBG-Eu3+) or by 125I using Bolton and Hunter reagent ([125I]rSHBG). The labeling procedure preserved the main properties of native SHBG: interaction with the lectine concanavaline A-Sepharose, recognition by anti-hSHBG monoclonal antibody, and, although lower than in native SHBG, the binding affinity for 5 alpha-dihydrotestosterone. These characteristics were the prerequisite for reliable measurement of the metabolism of labeled SHBG. Labeled rSHBG was injected into various rabbits with blood sampling at 2 min and at 1, 2, 4, 8, 12, 24, 48, 72, and 96 h after injection. rSHBG-B or desiaylated rSHBG-B and rSHBG-Eu3+ were captured from serum samples by tubes coated with anti-hSHBG antibody prior to the following detection procedure: biotin was detected by luminometry with the [streptavidin-alkaline phosphatase-dioxetane (AMPPD)] system and europium by time-resolved fluorimetry. [125I]rSHBG was detected by measurement of radioactivity either directly on serum or after fixation on concanavaline A-Sepharose.(ABSTRACT TRUNCATED AT 250 WORDS)

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

17 alpha-Aminomethyl, 17 alpha-acetamidomethyl, and 17 alpha-hemiglutaramidomethyl derivatives of dihydrotestosterone and testosterone have been prepared by hydrocyanation of 3,3'-(ethylenedioxy)-5 alpha-androstan-17-one and 3,3'-ethylenedioxyandrost-5-en-17-one, reduction of the corresponding acetylated 17 alpha-cyanohydrins with lithium aluminium hydride, and acylation of the resulting 17 alpha-aminomethyl derivatives with either acetic anhydride or the mono acid chloride of glutaric acid mono methyl ester. Saponification of the 17 alpha-hemiglutaramidomethyl methyl esters gave the corresponding hemiglutaramido derivatives, while acid hydrolysis of the 3-ethylene ketal group of 17 alpha-acetamidomethyl and 17 alpha-hemiglutaramidomethyl derivatives regenerated the 3-oxo and 3-oxo-4-ene functions. The 17 alpha-configuration of 17-substituted steroids was determined by 1H and 13C NMR and confirmed by comparing with NMR data for 17 alpha- and 17 beta-cyano-17-hydroxyandrost-4-en-3-one, 17 beta-cyano-3,3'-(ethylenedioxy)androst-5-en-17-ol, 17 alpha-alkynyl, and 17 alpha-hexanoic derivatives of dihydrotestosterone and testosterone, of known 17-configurations. Several ambiguous assignments of 13C NMR signals of 17 alpha-substituted steroids and unsubstituted 17 beta-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. 17 alpha-Aminomethyl and 17 alpha-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.

Synthesis and characterization by 1H and 13C nuclear magnetic resonance spectroscopy of 17 alpha-hexanoic derivatives of 5 alpha-dihydrotestosterone and testosterone.

The synthesis and characterization of 17 alpha-(6'-hexanoic acid) derivatives of 5 alpha-dihydrotestosterone and testosterone, useful as ligands for affinity chromatography purification or as precursors for affinity-labeling of androgen-binding proteins, is described. Alkynylation of 3-ethylenedioxy-, 3 beta-hydroxy-, and 3 beta,5-dihydroxy-5 alpha-androstan-17-one precursors with the potassium derivative of 5-hexyn-1-ol led to the corresponding 17 alpha-(6'-hydroxyhex-1'-ynyl) derivatives, which were hydrogenated over 10% Pt-C catalyst to give 17 alpha-(6'-hydroxyhexyl) derivatives. Chromic acid oxidation of the primary hydroxy group of the 3-ethylenedioxy-17-hexyl intermediate into carboxylic acid followed by acid cleavage of the 3-ketal group gave 17 alpha-(5'-carboxypentyl)-5 alpha-dihydrotestosterone, which was also obtained directly by chromic acid oxidation of the 3 beta-hydroxy intermediate. Chromic acid oxidation of the primary hydroxy group of the 3 beta,5 alpha-dihydroxy precursor resulted in a 5 alpha-hydroxy-3-oxo intermediate, which was dehydrated to give 17 alpha-(5'-carboxypentyl)testosterone. The 17 alpha configuration of these derivatives and of synthetic precursors was established by comparing their molecular rotations and their 1H and 13C nuclear magnetic resonance (NMR) spectra including solvent effects, with data reported for 17 alpha- or 17 beta-substituted steroid analogs as well as with 1H and 13C NMR reference data recorded in this work for 17 alpha-ethynyltestosterone, 17 alpha-ethynyl-19-nortestosterone, 17 alpha-ethyl-19-nortestosterone, 17 alpha-methyltestosterone, and 17 alpha-methyl-5 alpha-dihydrotestosterone.

Synthesis of (5-azido-2-nitrobenzoyl)amido, (4-azido-2-nitrophenyl)amino, and (5-azido-2-nitro-3,4, 6-trifluorophenyl)amino derivatives of 17alpha-methylamino-, 17alpha-ethylamino-, and 17alpha-propylamino-5alpha-dihydrotestosterone as reagents of different linker lengths for the photoaffinity labeling of sex hormone binding globulins and androgen receptors.

The photoactivable aryl azide reagents, N-(5-azido-2-nitrobenzoyl)oxysuccinimide, 4-azido-1-fluoro-2-nitrobenzene, and 4-azido-1-nitro-2,4,5, 6-tetrafluorobenzene have been condensed at the extremity of three 17alpha-aminomethyl, 17alpha-aminoethyl, and 17alpha-aminopropyl side-chains introduced on (17S)-spiro-(3, 3-dimethoxy)-5alpha-androstan-17beta,2'-oxirane either directly, by ammonolysis, in the first case, or by conversion to nitrile intermediates with cyano or cyanomethyl anions and subsequent reduction to amines with lithium aluminum hydride, in the two other cases. The 3,3-dimethoxy group of these photoreagents was cleaved by acidolysis to a 3-ketone, which was reduced with sodium borohydride to a 3beta-alcohol. All of these compounds were characterized by (1)H- and (13)C-NMR as well as by (1)H, (13)C heteronuclear 2D NMR, which helped to resolve ambiguous assignments. Significant differences of substituent-induced effects on (13)C NMR signals were observed according to the 17alpha-side-chain length, the structure of the terminal aryl azide groups, and the solvent, showing a different behavior of N-5-azido-2-nitrobenzoyl derivatives as compared with 4-azido-2-nitrophenylamino and 5-azido-2-nitro-3,4, 6-trifluorophenylamino derivatives. The N-5-azido-2-nitrobenzoyl conjugates of the three 17alpha-aminomethyl, aminoethyl, and aminopropyl derivatives of 5alpha-dihydrotestosterone were tested as ligands for purified human sex hormone-binding globulin and for the cytosolic androgen receptor of rat ventral prostate by competition experiments with tritiated 5alpha-dihydrotestosterone. The increasing lengths of the aminomethyl, aminoethyl, and aminopropyl spacer arms of N-5-azido-2-nitrobenzoyl conjugates were found to correspond to decreasing relative binding affinities for sex hormone-binding globulin (0.76, 0.47, and 0.10, respectively, versus 1.00 for 5alpha-dihydrotestosterone) while only the longer aminoethyl and aminopropyl conjugates interacted significantly with the androgen receptors (0.05 and 0.10, respectively).

Metabolic adaptations of overwintering European common lizards (Lacerta vivipara).

The European common lizard Lacerta vivipara, a reptile of cold-temperate climates, provides us an interesting model of low-temperature adaptation. Indeed its unique cold-hardiness strategy, which employs both freeze tolerance and freeze avoidance, may be seen as the primary reason for its large distribution, which extends from Spain to beyond the Arctic circle. To study the metabolism supporting this capacity, we used three techniques: two techniques of calorimetry (oxygen consumption and thermogenesis) and nuclear magnetic resonance spectroscopy. These techniques were used to examine the metabolic balance and the different molecular pathways used between three different periods through the year (September, January, and May). The results show a significant 20% augmentation of winter anaerobic metabolism compared to other periods of the year. This is mainly because of an activation of the lactic fermentation pathway leading to an increase of lactate concentration (>34% in winter). Furthermore, glucose, which increases some 245% in winter, is used as antifreeze and metabolic substrate. Furthermore, this study provides evidence that the physiological adaptations of the common lizard differ from those of other ectotherms such as Rana sylvatica. Concentrations of alanine and glycerol, commonly used as antifreeze by many overwintering ectotherms, do not increase during winter.

Supercooling, ice inoculation and freeze tolerance in the European common lizard, Lacerta vivipara.

The European common lizard (Lacerta vivipara) is widely distributed throughout Eurasia and is one of the few Palaearctic reptiles occurring above the Arctic Circle. We investigated the cold-hardiness of L. vivipara from France which routinely encounter sub-zero temperatures within their shallow hibernation burrows. In the laboratory, cold-acclimated lizards exposed to subfreezing temperatures as low as -3.5 degrees C could remain unfrozen (supercooled) for at least 3 weeks so long as their microenvironment was dry. In contrast, specimens cooled in contact with ambient ice crystals began to freeze within several hours. However, such susceptibility to inoculative freezing was not necessarily deleterious since L. vivipara readily tolerated the freezing of its tissues, with body surface temperatures as low as -3.0 degrees C during trials lasting up to 3 days. Freezing survival was promoted by relatively low post-nucleation cooling rates (< or = 0.1 degrees C.h-1) and apparently was associated with an accumulation of the putative cryoprotectant, glucose. The cold-hardiness strategy of L. vivipara may depend on both supercooling and freeze tolerance capacities, since this combination would afford the greatest likelihood of surviving winter in its dynamic thermal and hydric microenvironment.

Water turnover evaluated by the tritiated water method and by the nutritional method in European hedgehog (Erinaceus europaeus L.).

The total body water (TBW), the fractional water turnover rate (WTR), and the water flux rates (WFR) were studied for the first time by the tritiated-water method in 10 hedgehogs under laboratory conditions during the year. To validate this method, we compared the result to the nutritional method. The TBW varied inversely with body mass (BM) during both the active and the hibernation periods. The WTR varied from 16% to 21% during the hibernation period and after the reproduction. The higher values (from 22% to 26%) were recorded during the reproduction period and during the preparation for hibernation. The same variations in WFR were recorded during the active and hibernation period. The net gain or loss of water followed the gain and loss of BM. The tritiated water (THO) method has been validated by the fact that the same variations in WFR during each month have been measured by the THO method and the nutritional method. Moreover, the THO method can be used to estimate the changes in energy metabolism of hedgehogs in relation with the seasons.

Pharmacokinetics of butixocort 21-propionate, budesonide, and beclomethasone dipropionate in the rat after intratracheal, intravenous, and oral treatments.

The disposition of butixocort 21-propionate (JO 1222) in the rat after intratracheal treatment with a pulmonary aerosol was compared to that of budesonide (BUD) and beclomethasone dipropionate (BDP) using tritium-labeled aerosols. Each aerosol canister delivered 50 micrograms of 3H-labeled steroid through an intratracheal catheter directly into the lung. Rats were sacrificed at different times after treatment. Lung and plasma concentrations of unchanged drug and active metabolites were measured by HPLC using a radioactivity detector. Tritium-labeled drugs (1.5 mg/kg) also were administered by the iv and oral routes for the purpose of comparing their systemic availability. The extent of biotransformation of all three steroids in the lung was very limited. JO 1222 was metabolized to the active compounds butixocort (JO 1717) and butixocort 21-methyl (JO 1605). BDP was transformed primarily to beclomethasone monopropionate (BMP); BUD was not metabolized in the lung. In contrast to these results, large differences were observed between plasma concentrations and systemic availability of the three drugs. BUD was rapidly absorbed from the lung, and its plasma elimination half-life was about 3 hr. BDP was hydrolyzed rapidly into its active metabolite BMP after intratracheal and iv treatments; BDP was not observed in plasma after oral treatment. Additionally, plasma concentrations of BMP were higher than those of BUD administered at the same doses. Assuming that BDP was transformed entirely into BMP, the oral bioavailability of BMP at 1.5 mg/kg was around 72%, while that of BUD was 15%. JO 1222 has a distinct pharmacokinetic profile due to the extensive metabolic clearance of both the unchanged drug and its active metabolites JO 1717 and JO 1605.(ABSTRACT TRUNCATED AT 250 WORDS)

[Changes in the internal temperature and the cardiac rhythm of the Saharian lizard, Varanus griseus Daud., using radiotelemetry]. / Etude de l'évolution de la température interne et du rythme cardiaque chez le lézard saharien: Varanus griseus Daud. par radiotélémétrie

The evolution of the internal temperature and of the heart rate, recorded by telemetry, has been studied on Varanus griseus, by using transmitters of reduced size, in the natural conditions of Sahara. A parallelism between these two parameters exists. However, an anticipation of the heart rate increase during sun basking has been established. The hearing and cooling dynamics have been specified; a hypothesis on the mechanism of winter dormancy entrance has been proposed.

Identification of Trp-371 as the main site of specific photoaffinity labeling of corticosteroid binding globulin using delta 6 derivatives of cortisol, corticosterone, and progesterone as unsubstituted photoreagents.

Immunopurified human corticosteroid binding globulin (CBG) was photolabeled with delta 6-[3H]cortisol, delta 6-[4-14C]cortisol, delta 6-[3H]corticosterone, and delta 6-[3H]progesterone. The maximal levels of specific incorporation, as estimated with tritiated photoreagents, were 0.21, 0.14, and 0.08 mol of label/mol of CBG, respectively. Tryptic cleavage of photolabeled CBG gave in all cases a major radioactive peptide that was no longer detectable when a 100-fold molar excess of cortisol was added to the photoreagents. Edman sequencing of tryptic peptides photolabeled with delta 6-[3H]cortisol or delta 6-[3H]corticosterone showed that these peptides correspond to residues 357-378 of the human CBG sequence. The major peak of radioactivity of these peptides was eluted at the 15th cycle (Trp-371). The radioactive tryptic peptides photolabeled with the four steroid photoreagents were subcleaved with alpha-chymotrypsin. The major part of radioactivity was recovered in the T-[*X]-S-S-L-F hexapeptide 370-375 (major peptide) and in the D-H-F-T-[*X]-S-S-L-F nonapeptide 367-375, at the second and fifth Edman cycles, respectively, whereas no PTH derivative could be identified at these cycles, thus suggesting Trp-371 as the main site of photolabeling for all tested photoreagents. Mass spectrometry of tryptic peptides photolabeled with delta 6-[3H]cortisol and delta 6-[3H]corticosterone and of chymotryptic peptides photolabeled with delta 6-[3H]cortisol, delta 6-[3H]corticosterone, and delta 6-[3H]progesterone showed molecular masses corresponding to the addition of delta 6-steroid photoreagents to the peptide.

Variation of plasma sex steroid concentrations in wild and captive populations of Hermann's tortoise (Testudo hermanni hermanni) in Southern France.

Plasma levels of sex steroids in both males and females of the endangered Hermann's tortoise (Testudo hermanni hermanni) were measured throughout their active period in a wild population in the Massif des Maures, France, and in a nearby captive population at Le Village des Tortues in Gonfaron. Both plasma progesterone and testosterone were elevated in males at emergence from winter dormancy, and plasma progesterone levels were significantly higher in wild than in captive males. Plasma testosterone in males then fell to the lowest levels (10 ng ml(-1)) during the nesting season from April to June followed by an elevated plateau during summer, with levels reaching 80 ng ml(-1), presumably concomitant with spermiogenesis. Plasma testosterone increased in all females during autumn, an indication of follicular growth, and remained high on emergence from hibernation, to peak during April, although levels were lower in the captive population. Plasma progesterone also peaked during April and May, presumably related to ovulation, but, again, these changes were less marked in the captive than in the wild females. Measurements of testosterone, progesterone and 17beta-oestradiol in the captive females during their period of oviposition in spring suggested that some females did ovulate and lay eggs, whereas others did not. Differences in sex steroid levels between captive and wild populations of Hermann's tortoise may indicate a problem with ovulation and/or with stress in a proportion of captive females.

Evaluation of active specific immunization against paraquat toxicity in rats.

Four groups of Wistar rats were immunized against a BSA-paraquat derivative antigen, when lethally poisoned with paraquat dichloride via intraperitoneal route. No significant correlation in survival rate was observed between immunized (5/45) and control (0/26) rats, but a significant correlation (p less than 0.05) in the mean survival time was noted in immunized rats as compared to controls (9.1 +/- 16.8 days and 2.0 +/- 0.8 days versus 2.1 +/- 2.4 days and 1.0 +/- 0.1 days respectively).

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.

Detection of testosterone misuse: comparison of two chromatographic sample preparation methods for gas chromatographic-combustion/isotope ratio mass spectrometric analysis.

Two chromatographic methods, reversed-phase liquid chromatography (LC) and immunoaffinity chromatography (IAC), were compared in the preparation of purified testosterone extracts suitable for gas chromatography-combustion/isotope ratio mass spectrometry (GC-C-IRMS) analysis. We have shown previously that GC-C-IRMS is a promising means of detection of testosterone misuse in sport. The two clean-up procedures afford sufficient recovery and adequate purity of testosterone. LC presents several advantages over IAC: access to other urinary steroids, longer column life, no need for special equipment and no antibody preparation. For IAC, the antibodies to testosterone must be selected with care for high affinity and low cross-reactivity. Nevertheless, IAC is of some interest in our experiments, the recovery is slightly better for low concentrations of urinary testosterone and IAC does not induce isotopic discrimination even in overloading experiments. This is the first report on sample preparation by IAC prior to GC-C-IRMS and carbon isotope ratio values for urinary epitestosterone. The carbon isotope ratio test can identify users' urines missed by the testosterone to epitestosterone ratio (T/E > 6) test.