Structural organization and regulation of the gene for the androgen-dependent glutathione peroxidase-like protein specific to the mouse epididymis.

Genomic clones containing the gene for the glutathione peroxidase-like androgen-regulated murine epididymal protein of 24 kilodaltons (arMEP24) were isolated. A 9-kilobase DNA fragment was sequenced and found to contain the entire coding region of the gene, which is divided into five exons. The exact sizes and boundaries of the exon blocks were deduced by comparison with the cDNA sequence. One major and four weak transcription initiation sites in the epididymis were localized by primer extension. The promoter of the gene does not contain a conventional TATA box immediately up-stream of the start site; rather, the sequence TATCA occurs at residue -35. Two CAAT boxes in opposite orientation and two putative binding sites for the transcription factor Sp1 were identified up-stream of the TATA-like box. To localize the cis-acting sequences responsible for androgen regulation of expression, fragments of the arMEP24 gene promoter region were cloned in front of the luciferase (LUC) reporter gene and cotransfected with an androgen receptor expression vector into CV-1 cells in a transient assay. LUC activities of CV-1 cells grown in the presence of various concentrations of 5 alpha-dihydrotestosterone were compared to LUC activities of untreated controls. The DNA fragment containing up to 200 nucleotides up-stream from the major transcription start site was sufficient for the full promoter activity, but not for the responsiveness to androgen induction. Depending on the 5 alpha-dihydrotestosterone concentration, a 2- to 4-fold induction of LUC activity was found if a -1797 to -167 arMEP24 gene fragment was used linked to the reporter gene driven by either the homologous promoter or the heterologous thymidine kinase promoter. Two or three copies of the imperfect palindromic sequence TGTTGAgagAGAACA, found at position -896 to -882 in the gene and resembling the consensus steroid hormone-responsive element, are able to confer androgen regulation to the thymidine kinase promoter independently of their orientation. These findings support evidence that transcriptional regulation of the arMEP24 gene occurs via the sequence TGTTGAgagAGAACA. Homologies found in the sequence up-stream of the promoter with several putative binding sites for erythroid-specific trans-acting regulatory proteins are discussed. Finally, the arMEP24 gene is located by in situ hybridization in the [A2-A4] region of mouse chromosome 13.

Insulin binding to Leydig cells and insulin levels in testicular interstitial fluid at different stages of development in the rat.

Binding of insulin to purified intact Leydig cells (LC) and LC membranes, and levels of insulin in plasma and testicular interstitial fluid (IF) were quantitatively evaluated in rats at three stages of development. Specific insulin binding to intact LC increased significantly with age. Scatchard analysis of the binding data always gave curvilinear plots; the number of high affinity binding sites/LC were 2590 +/- 514, 3977 +/- 701 and 8342 +/- 2039 at 21, 40 and 70 days respectively. When the results were expressed per micrograms membrane protein, the maximal specific insulin binding also increased between 21 and 40 days but did not significantly change thereafter. With ageing, insulin levels in testicular IF decreased (3.05 +/- 0.30, 2.48 +/- 0.22 and 1.66 +/- 0.13 micrograms/l in 21-, 40- and 70-day-old rats) whereas plasma insulin increased. Taken together, these results suggest (1) that the intratesticular environment in this hormone cannot be evaluated by plasma insulin levels--testicular IF insulin concentration is probably a better index and (2) that insulin may play a role in the development of LC function during sexual maturation.

Regulation of the epididymal glutathione peroxidase-like protein in the mouse: dependence upon androgens and testicular factors.

The protein MEP24 was previously described as a glutathione peroxidase-like molecule specifically secreted by the mouse caput epididymidis. Recently, its binding to the head of spermatozoa was demonstrated. Here, the regulation of MEP24 expression was studied by analyzing transcriptional and translational activities in the epididymis (1) of adult mice castrated on day 60 and given various substitutive testosterone (T) treatments from day 90 and (2) of hemicastrated adult animals. In castrated mice, T treatment induced a significant rise in plasma T and 5 alpha-dihydrotestosterone (DHT) concentrations that greatly exceeded the control values. Owing to efficient regulation, however, the epididymal T and DHT levels were never higher than those of the controls. The restoration of MEP24 mRNA accumulation was complete when the epididymal DHT content returned to its normal value. However, when estimated in a cell-free system, the in vitro translatable MEP24 mRNA level never exceeded 70% of control values, even though the DHT and accumulated mRNAs were restored by 100% or more. In hemicastrates, the T content was normal on the castrated side, while the DHT content exhibited a significant decrease (47%). In this case, the MEP24 mRNA accumulation reached 88% of the normal value, but the translation rate, both in vitro and in vivo, was only about 50%. Ultrastructural studies showed that the normal rough endoplasmic reticulum organization in segment I cells is dependent upon the presence of testicular fluid in the epididymal duct lumen. Thus, this report shows that the MEP24 mRNA steady-state level is completely recovered in the presence of a normal epididymal DHT content, while restoration of the regulation of translation is just partial. This could be related to the cell organization but seems mainly dependent upon the presence of specific mRNA-associated factors which are probably under the control of androgens and/or molecules carried by the testicular fluid.

Quantification of endogenous testosterone and dihydrotestosterone in fetal tissues from male guinea-pig.

Testosterone (17 beta -hydroxy-4-androsten-3-one ; T) and dihydrotestosterone (17 beta -hydroxy-5 alpha -androstan-3-one ; DHT) concentrations were determined by radioimmunoassay in different fetal tissues taken from male guinea-pigs. Androgen concentrations were maximal in the components of the Wolffian duct (vas deferens, epididymis, seminal vesicle) and the urogenital sinus (urogenital tubercle, prostate) when these tissues are differentiating between days 28 and 36 (T = 320 to 1450 and DHT = 200 to 860 pg/10 mg of tissue). During the same period circulating testosterone is taken up by the non-specific tissues (intestine, diaphragm) to a lesser degree (150 to 250 pg/10 mg) as well as by hypothalamus and hypophysis (100 to 170 pg/organ). After this uptake phase, T declines in the non-specific tissues to minimal concentrations (less than 10 pg/10 mg). Compared to the situations in the diaphragm and intestine, DHT concentrations were significantly higher in both urogenital sinus and Wolffian duct components, and T concentrations were significantly higher only in the Wolffian ducts components. In the bladder, T and DHT levels were significantly higher than those of the androgen-independent tissues.

Alterations in the androgen metabolism of the female guinea-pig during pregnancy.

The peripheral conversion of testosterone (T) into androstenedione (delta 4) and dihydrotestosterone (DHT) were studied in pregnant (days 32, 48 and 67 post coitum) and non-pregnant anesthetized guinea-pigs by constant intravenous infusion of tritium labeled T. Endogenous levels were measured by radioimmunoassay, and the three labeled androgens (T*, DHT* and delta 4*) counted after celite chromatography. The endogenous T, DHT and delta 4 plasma concentrations were respectively 5, 7 and 20 times higher in pregnant females than they were in cyclic ones. In the non-pregnant guinea-pig, no part of the delta 4 or DHT pools was derived from the peripheral conversion of testosterone. On days 32, 48 and 67 of gestation the fractions of the delta 4 pool originating from T were 9.1, 13.1 and 15.1%, and the fractions of DHT pool coming from T were 5.1, 22.1 and 21.3% respectively.

Changes in the metabolic clearance rate and plasma production rate of testosterone in the pregnant guinea-pig.

The metabolic clearance rate of testosterone (MCRT) in the pregnant and non-pregnant guinea-pig was established by a constant infusion method to steady state levels of 3H testosterone (T). Simultaneously, the concentration of endogenous T was determined by radioimmunoassay and the production rate of T (PRT) was calculated from the product of MCRT and endogenous T concentration. Results indicate important alterations in the three parameters between non-pregnant (MCRT = 64.0 +/- 2.6 1/24 hr; T concentration = 51 +/- 8 pg/ml; PRT = 3.24 +/- 0.13 micrograms/24 h) and the pregnant guinea-pig. In these animals mean values from day 43 were about 33 1/24 h for MCRT, 378 pg/ml for T concentrations and 12.5 micrograms/24 h for PRT. The significance of these results is discussed in relation to the specific binding of testosterone with the progesterone binding globulin during pregnancy in this species.

The androgens in the guinea-pig foetus throughout the embryonic development.

Testosterone (T) and dihydrotestosterone (DHT) content and concentration were measured by radioimmunoassay in plasma, gonads, adrenals, and sexual ducts of 235 male and 214 female guinea-pig foetuses at various stages of gestation after gonadal and adrenal differentiation occurred. In the male foetus, a sharp increase in the content and concentration of T and, to a lesser degree of DHT, is observed in the testis, the plasma and the ducts at the time of sexual differentiation between days 28 and 36. Thereafter androgen concentrations in these tissues decrease and rise again after day 52. In the female foetus, levels of T and DHT in the ovary are low throughout foetal life and do not account for the relatively high concentrations found in the plasma; in sexual ducts, T concentrations decrease at the time of differentiation. DHT levels are low at every stage. In both sexes, T and DHT are present in the adrenal. No sexual dimorphism is observed in the evolution of T and DHT adrenal content and concentration.

[Evolution of testosterone and dihydrotestosterone in plasma, testicle and ovary of the guinea pig throughout fetal life]. / Evolution des teneurs en testostérone et dihydrotestostérone dans le plasma, le testicule et l'ovaire chez le cobaye au cours de la vie faetale.

High testosterone concentrations were found in the plasma of male fetus at the stage of sexual differentiation. Then the fetal testis is the principal source of circulating testosterone; its action on the Wolffian duct would occur by the systemic pathway. In female fetus, androgens were synthesized by the gonad and the relatively high levels found in the plasma have probably another source.

[Levels of testosterone and dihydrotestosterone in guinea pig sex canals during embryonic life]. / Teneurs en testostérone et dihydrotestostérone des canaux sexuels du cobaye au cours de la vie embryonnaire.

At the undifferentiated stage, testosterone and dihydrotestosterone levels were comparable in sexual ducts of both sexes. An hormonal sexual dimorphism appeared at the time of differentiation: while testosterone rapidly decreased in the female ducts, it considerably increased in those of the male. Testosterone rather than DHT seems to be responsible for the wolffian duct stabilization in the male foetus.

[Testosterone and dihydrotestosterone content of the testis during guinea pig]. / Teneurs en testostérone et en dihydrotestostérone du testicule au cours du dévellppement embryonnaire chez le cobaye

The fetal guinea-pig testis synthetizes testosterone and reduces it in 5 alpha-dihydrotestosterone. The embryonic period which goes from the gonadla differentiation (on day 23) up to the beginning of the sex ducts differentiation (on day 29) is characterized in male guinea-pig by a very important testicular load in testosterone and DHT.

Testosterone and dihydrotestosterone in amniotic fluid and foeto-placental membranes from the guinea pig.

Testosterone (T) and dihydrotestosterone (DHT) in the amniotic fluid (AF) and foeto-placental membranes (FPM) (yolk-sac + amnion) from 180 male and female guinea-pig foetuses were determined by radioimmunoassay on days 28, 32, 36, 40, 44, 48, 52, 56, 60, 64 of gestation. In male foetus the evolution of androgens in the fluid is characterized by two sharp rises, the former at the time of sexual differentiation on day 32 (T = 219.1 +/- 39.1 and DHT = 74.7 +/- 10.0 fmol/ml) the latter, which affects only DHT, on day 52 (DHT = 68.5 +/- 10.3 fmol/ml). In female foetus, AF T concentrations (mean = 36.3 fmol/ml) are comparable to the lowest T concentrations observed in male, while DHT concentrations (less than 2.5 fmol/ml), are significantly lower than those observed in the male (mean = 28.9 fmol/ml) and without any overlap in the values. Thus, in guinea pig, DHT, but not testosterone allows to predict accurately the foetal sex at any stage studied. Testosterone and DHT are also present in FPM and their concentrations were comparable for male and female foetuses in most stages, values varying between 400 and 1600 fmol/g of tissue with a light predominance of DHT compared with T; in both sexes, general evolution of androgens is marked by a rapid drop between days 28 and 44 followed by a significant increase between days 44 and 64. Possible origins of androgens in AF and FMP are discussed.

Maternal testosterone and sexual differentiation of the male guinea-pig foetus.

The part of foetal testosterone originating from the mother was explored in the guinea-pig at the time of sexual duct differentiation on day 32 of gestation. Using constant intravenous infusion of tritiated testosterone in the maternal compartment, total radioactivity (RA) and radioactive testosterone ([3H] T) and dihydrotestosterone ([3H] DHT) were measured in the maternal plasma, whole foetus and various foetal tissues, including the genital tract and gonads, brain and liver. After [3H] T was infused to the mother, significant amounts of total RA were found in foetuses of both sexes. The two metabolites isolated from the whole foetus contributed in various amounts to the RA present: 8% for [3H] DHT and 2 to 3% for [3H] T; no significant differences in the amounts of foetal T derived from maternal T (around 5 pg/g) were found between male and female foetuses. The brain, liver and genital tract and gonads concentrated more RA than the whole foetus, but only the male genital tract and gonads concentrated more RA than maternal plasma. Only DHT was isolated from the brain and liver of both sexes (8 to 10% of the RA found), but neither of the two hormones was found in the genital tract and gonads of either male or females foetuses. The results indicated that maternal testosterone and its metabolite, DHT, were not directly involved in the differentiation of sexual ducts in the male guinea-pig foetus.

Aromatase activity in purified Leydig cells from adult rat. Comparative effects of insulin, IGF-I, and hCG.

The comparative effects of insulin and IGF-I on aromatization in adult rat purified Leydig cells were examined to elucidate the mechanism of action of the peptides in testicular steroidogenesis. Aromatase activity was measured in short-time incubations, using the tritiated water release method with [1 beta-3H] and androstenedione as substrate. In the presence of varying concentrations of substrate, the apparent Km for androstenedione was 0.945 mol/l; treatment of cells with insulin, IGF-I and hCG markedly increased the apparent maximal velocity, without modifying Km; peptides were more potent in aromatase stimulation than hCG alone or in combination with either peptides. When related to time (0-4 h) and expressed as percent of control values, aromatase activity in the presence of insulin, IGF-I and/or hCG exhibited a significant and transient increase at 15-30 min. In order to clarify the nature of this early stimulation, the effects of dibutyryl cAMP, various antibiotics, and cytochalasin B on treated Leydig cells were analysed. Results indicated that insulin and IGF-I action on aromatization was not cAMP-dependent; peptides could intervene by increasing RNA and protein, but not DNA, synthesis; they were also effective in glucose transport. These data suggest that insulin and IGF-I are able to modulate aromatization in Leydig cells.

Transfer of maternal testosterone to the guinea pig fetus during the last third of pregnancy.

Testosterone (T) transfer from mother to fetuses was explored in anesthetized guinea pigs on Days 48 and 67 of pregnancy using a constant intravenous infusion of tritium-labeled T ([3H]T). Labeled dihydrotestosterone ([3H] DHT), androstenedione ([3H] delta 4) and [3H] T were estimated after celite chromatography in maternal plasma, umbilical venous plasma, fetal arteriovenous plasma and fetal tissue homogenates including liver, genital tract with gonads, hypothalamic area, hypophysis and remaining brain. Total radioactivity was measured by counting a part of the homogenates. Results indicate that very low concentrations of [3H] T and [3H] delta 4 were found in umbilical and fetal plasma, whereas [3H] DHT levels were relatively high. A minute part of maternal T was transferred to male and female fetuses: 0.7 to 1.9 pg/ml, and that represented 0.4 to 5.6% of fetal T. The difference observed between umbilical and fetal [3H] T showed that 80% of the fetal T coming from the mother was metabolized by the fetuses. This result is discussed in relationship to the high radioactive concentrations present in fetal liver. Concentrations of total radioactivity were high in fetal tissues, and the three nervous tissues exhibited a significant difference in their capacity to fix radioactivity. There was a complete absence of the three labeled androgens in the hypophysis. However, [3H] DHT was present in all other tissues, and gonads + genital tract in the male fetus exhibited [3H] DHT concentrations significantly higher than those detected in the female fetus.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of insulin binding and comparative action of insulin and insulin-like growth factor I on purified Leydig cells from the adult rat.

Insulin binding and insulin action were characterized in adult rat Leydig cells, purified on discontinuous Percoll gradients. Binding of [125I]-porcine insulin was found to be dependent on time, temperature, cell concentration and Leydig cell specific gravity. Competition relative to porcine insulin (100) was as follows: insulin-like growth factor I (IGF-I) : less than 1; proinsulin : 5; guinea-pig insulin : 2; hCG, ovine prolactin and bovine GH : 0. High and low affinity binding sites for insulin were identified on purified Leydig cells with Ka values of 1.2 X 10(9) and 0.3 X 10(8) M-1, with 10,300 and 34,000 binding sites per cells, respectively. Using primary cultures of Leydig cells in serum-free medium, the action of insulin on steroidogenesis was studied and compared with IGF-I action. Insulin and IGF-I used at 1-35 nM enhanced basal testosterone production in a dose-dependent manner; the effect was significant 4 h after administration. Insulin or IGF-I also potentiated the effect of hCG on steroidogenesis during short-term incubation (4 h). Insulin was shown to improve hCG responsiveness without modifying sensitivity to hCG. Moreover, neither cell number nor hCG-binding was altered by insulin, IGF-I or a combination of the two. Concomitant treatment with insulin and IGF-I at half-maximal and maximally effective doses, in the presence or absence of hCG, indicated that the two factors synergized in the stimulation of testosterone production via a common saturable mechanism.

[Androgen-dependent protein secreted by mouse caput epididymis shows high homologies with different glutathione peroxidases]. / Une protéine androgéno-dépendante sécrétée par la tête de l'épididyme de souris présente des homologies élevées avec différentes glutathion peroxydases.

Principal cells of the mouse caput epididymidis synthesize and secrete a 24 kDa protein able to bind to the head of the spermatozoa. Sequencing of several clones selected from cDNA and genomic libraries, combined with the microsequencing of the NH2 terminus of the protein allowed to reconstitute the entire primary structure of the mature 24 kDa protein. It revealed 81% homology with a human plasma glutathione peroxidase and 61% homology with a mouse erythrocyte glutathione peroxidase. This enzyme, once secreted in the epididymal fluid, might protect sperm membrane lipids, particularly those of the acrosomal part, against peroxidation.

Changes in the concentrations of testosterone and androstenedione in the plasma and testis of the guinea-pig from birth to death.

Testosterone and androstenedione concentrations in the plasma and testis of male guinea-pigs were estimated by gas chromatography at intervals (36 stages) from birth to death. Four main periods of androgenic activity were recognized. The neonatal period, from birth to Day 16, is characterized by a precocious but transient peak in plasma testosterone concentration at Days 2 and 3. The pubertal period from Days 16 to 90 can be subdivided into a prepubertal period starting on Day 16 and marked by a sudden linear increase in plasma and testicular testosterone concentration together with an increase in testicular and seminal vesicle weight, and a postpubertal period, from Day 50 (the time of hormonal puberty) to Day 90, characterized by high and stable androgen levels while testicular and genital tract development continues. Adulthood spans the period between Months 3-6 and Month 24; plasma and testicular concentrations of testosterone and androstenedione are stable but lower than those observed during puberty. The period of senescence occurs between Months 24 and 28 and is marked by a fall in testosterone secretion and involution of the seminiferous tubules and accessory sex glands.

In vitro expression of a mouse tissue specific glutathione-peroxidase-like protein lacking the selenocysteine can protect stably transfected mammalian cells against oxidative damage.

The complete sequence of the mouse epididymal protein (MEP24) was cloned. It contains a 663 bp open-reading frame that, after conceptual translation, shows extensive identity with proteins belonging to the glutathione peroxidase (GPX) family. However, a major difference between GPX5 (MEP24) and other known GPXs concerns a protein domain known to be critical for GPX function. To find out what could be the physiological function of such a protein in the mouse epididymis, we have used a mammalian expression system to overexpress the GPX5 protein. Cells constitutively expressing the GPX5 protein were generated and assayed for their ability to metabolize regular substrates of GPX enzymes. Data presented here show that the GPX5-expressing cells can metabolize hydrogen peroxide in a manner that is consistent with a peroxidase activity. However, the substrate preference of the GPX5-expressing cells and their apparent insensitivity to a regular inhibitor of GPX enzymes suggest that the GPX5 protein belongs to a particular class of GPX proteins. Involvement of this protein in the physiology of the mouse epididymis is discussed.