Stimulation of adult rat Leydig cell aromatase activity by a Sertoli cell factor.

The paracrine control of adult rat Leydig cell aromatase activity was investigated in vitro. After a 24-h preculture period of Percoll-purified Leydig cells (2.5-5 X 10(5) cells), 17 beta-estradiol synthesis reached a maximum at 5 h in the presence of exogenous testosterone (200 ng/ml) as substrate, with or without LH (100 ng/ml), and remained stable for a further 24 h. Aromatase activity was stimulated 2.5-fold by LH. The addition of seminiferous tubule culture medium (STM) from normal, neonatally hemicastrated, or prepubertally irradiated rats as well as Sertoli cell culture medium prepared from these animals enhanced both basal and LH-dependent aromatase activities during 5 h; this effect was diminished after 24 h of culture. When seminiferous tubules (200 mm) were cocultured with Leydig cells, a greater stimulation of 17 beta-estradiol production was observed compared to culture with STM. The association of Sertoli and germ cells with purified Leydig cells further enhanced aromatase activity. These results demonstrate that a Sertoli cell factor regulates Leydig cell aromatase activity. This factor is of proteic nature, thermolabile, has a mol wt ranging between 10,000-50,000, and is different from the LHRH-like substance. This compound is tissue and species specific, since it is not present in rat serum, other cell line media, or guinea pig and mouse STM. Its secretion is independent from FSH and testosterone controls. The stimulation of aromatase activity by this factor requires protein synthesis.

Effects of seminiferous tubule secreted factor(s) on Leydig cell cyclic AMP production in mature rat.

The effects of spent media from seminiferous tubules (STM) on Percoll-purified rat Leydig cells were investigated. Intracellular and extracellular cyclic AMP (cAMP) accumulation and testosterone production were measured. After a 5 h incubation period, STM reduces both the basal and LH-dependent cAMP levels (38 and 20%, respectively for intra- and extracellular cAMP) while, simultaneously, a stimulation of testosterone production is observed (47 to 50%, respectively in the absence or presence of LH). The reduction of cAMP levels observed after 5 h is likely to be due to the potentiating effect of the STM factor on the LH-dependent initial rise of the cAMP level which, in turn, induces a desensitization of the Leydig cell adenylate cyclase. This substance is a thermolabile protein (Mr greater than 50 000) produced by the Sertoli cell, independent of FSH and testosterone controls, and different from the LHRH-like substance.

Effect of phorbol ester and phospholipase C on LH-stimulated steroidogenesis in purified rat Leydig cells.

When the phorbol ester, 4 beta-phorbol-12-myristate-13-acetate (PMA) or bacterial phospholipase C (PL-C) is added to a preparation of purified adult rat Leydig cells, containing 2 mM CaCl2, a time- and dose-dependent decreases of LH-stimulated testosterone production is observed. After a 3 h stimulation with oLH (100 ng/ml), PMA (100 ng/ml) and PL-C (1.6 U/ml) do not affect the cell viability or the hCG specific binding, while cAMP accumulation is significantly reduced; cAMP-stimulated steroidogenesis is diminished only in the presence of PL-C. These observations suggest that in vitro: (i) activated Ca2+- and phospholipid-dependent protein kinase is implicated in the regulation of rat Leydig cell steroidogenesis by LH at a step before the adenylate cyclase; (ii) phospholipids play an important role in cAMP-stimulated testosterone synthesis.

Androgen-binding proteins in sheep epididymis: characterization of a cytoplasmic androgen receptor in the ram epididymis.

An androgen receptor (Rc) was demonstrated in caput, corpus and cauda epididymal cytosols of the ram. This receptor had a high affinity for 5 alpha-dihydrotestosterone (Kd = 5.2 X 10(-9) mol/l) and could be distinguished from the androgen-binding protein (ABP) by several characteristics. On polyacrylamide-gel electrophoresis, Rc had a mobility of 0.37 and ABP 0.61; Rc sedimented in the 9S region of a linear sucrose gradient whereas ABP migrated in the 4.3S region; the molecular weights were 192 000 and 90 000 for Rc and ABP; their isoelectric points were 5.7 and 4.8-5.0; they were proteinaceous components since they were destroyed by proteolytic enzymes and heating (50 degrees C for Rc and 60 degrees C for ABP); they exhibited different half-times of dissociation:20 h at 0 degree C for Rc and 6 min for ABP, which is in agreement with their respective physiological roles, intra- and extracellular transport of androgens. The content of Rc-binding sites in caput epididymis was 18, in corpus 4 and in cauda 22 fmol/mg protein.

Androgen-binding proteins in sheep epididymis: age-related effects on androgen-binding protein, cytosolic androgen receptor and testosterone concentrations. Correlations with histological studies.

The androgen-binding protein (ABP) and the cytosol androgen receptor (Rc) were measured in the epididymis of sheep aged 50, 120 and 200 days. Specific binding protein was not detected at 50 days (infantile); near puberty (120 days), ABP was more concentrated in caput and cauda epididymal cytosols (117 and 183 fmol/mg protein respectively) than in corpus (53 fmol/mg) although Rc levels were low (3-5 fmol/mg). In postpubertal rams (200 days), ABP and Rc concentrations were higher in caput and cauda than in corpus epididymis. Testosterone concentrations at 50 days were not statistically different along the epididymis and varied from 0.3 to 0.8 pmol/mg protein. In 120-day-old animals, testosterone was more concentrated in caput and corpus (0.45 and 0.41 pmol/mg) than in cauda (0.17 pmol/mg); at 200 days, the testosterone contents were low (0.10-0.17 pmol/mg) in all parts of the epididymis. A ten-fold increase in plasma testosterone concentrations was observed between 50 and 200 days (1.31 to 11.71 nmol/l). Histological studies of the epididymis in the three groups of animals showed that the cell differentiation started in the cauda where the principal epithelial cells were higher (47-56 micron) than in the caput (31-38 micron) at 50 days. The principal cells of the caput were two- to threefold higher in postpubertal rams than in infantile lambs, a finding which is correlated with the levels of ABP and Rc. This may suggest an important physiological role of this region in the induction of sperm maturation.

Hormonal regulation of androgen-binding protein in lamb testes.

Androgen-binding protein (ABP) was measured in the testes of 50-day-old lambs. The animals were hypophysectomized and treatment lasting for 5 days was begun 15 days after surgery. In hypophysectomized but otherwise untreated lambs (control group), no 5 alpha-dihydrotestosterone binding was detectable in testicular cytosol. One out of four lambs gave a positive response with FSH treatment (25 fmol ABP/mg protein), whereas a restoration of the synthesis of ABP was noted in all LH-treated animals (19 +/- 9 (S.E.M.) fmol ABP/mg, n = 4). No synergism between the two gonadotrophins was observed in lambs treated simultaneously with FSH and LH (19 +/- 4 fmol ABP/mg, n = 5). Testosterone treatment elicited a greater response (37 +/- 9 fmol ABP/mg, n = 5) than FSH or LH alone and the response was not increased by the simultaneous addition of FSH (38 +/- 10 fmol ABP/mg, n = 5). Whatever the treatment, no influence was observed either on the number of supporting cells (undifferentiated Sertoli cells) or the length of the seminiferous tubules (P>0.05); the diameter of tubules was significantly increased in the group treated with FSH and LH. It is postulated that testosterone may have a direct effect on the production of ABP by the supporting cells of the impuberal lamb.

Adult rat Sertoli cells secrete a factor or factors which modulate Leydig cell function.

Production of testosterone and oestradiol-17 beta by Leydig cells from adult rats was stimulated by LH or dibutyryl cyclic AMP (10 and 2.5-fold respectively). The addition of spent medium from normal, hemicastrated or gamma-irradiated rat seminiferous tubule cultures, as well as from Sertoli cell cultures, to purified Leydig cells further enhanced both basal (44 and 53% for testosterone and oestradiol-17 beta respectively) and LH-stimulated (56 and 18%) steroid output. Simultaneously, a decrease (20-30%) in intracellular cyclic AMP levels was observed. This stimulating factor (or factors) secreted by the Sertoli cells is different from LHRH, is of proteinic nature and has a molecular weight ranging between 10,000 and 50,000; its synthesis is not controlled by FSH nor by testosterone. This factor(s) involved in rat Leydig cell steroidogenesis, at a step beyond the adenylate cyclase, does not require protein synthesis for testosterone formation whereas it does for oestradiol-17 beta production. It should be noted that a germ cell-Sertoli cell interaction modulates the synthesis of this factor(s).

Equine cytochrome P450 aromatase exhibits an estrogen 2-hydroxylase activity in vitro.

Aromatase (estrogen synthetase) is a steroidogenic enzyme complex which catalyzes the conversion of androgens to estrogens (termed aromatization). This enzyme was purified from adult equine testis to homogeneity by five chromatographic steps. The ability of purified and reconstituted equine aromatase to exhibit an estrogen 2-hydroxylase activity was tested and compared to testosterone aromatization. Enzymatic activities were assessed by tritiated water release from labelled estradiol and testosterone. Kinetic analysis of estradiol 2-hydroxylation showed an apparent K(m) of 23 microM and a V(max) of 18 nmol/min/mg, whereas the values for testosterone aromatization were a K(m) of 15.7 nM and a V(max) of 34.6 pmol/min/mg. A specific antiserum raised against purified testicular equine P450arom and known to inhibit aromatase activity [1] was also found to inhibit the estrogen hydroxylase activity of equine placental microsomes in a dose-dependent manner with an IC50 value of 15 microl serum: 0.5 ml incubate. The estrogen hydroxylase activity was inhibited in a dose-dependent manner by two classes of aromatase inhibitors, i.e. steroidal-- (4-hydroxyandrostenedione and 7alpha-([4-aminophenyl]thio)-androst-4-ene-3, 17-dione)--and non-steroidal--(fadrozole and miconazole). The IC50 values were approximately 300 and 890 nM for 4-hydroxyandrostenedione and 7alpha-([4-aminophenyl]thio)-androst-4-ene-3, 17-dione, and 92 and 285 nM, for fadrozole and miconazole, respectively. Furthermore, 4-hydroxyandrostenedione caused a time-dependent inactivation of estrogen hydroxylase activity. We conclude that equine aromatase is able to use estradiol as a substrate, and converts it to catechol estradiol in vitro, possibly using the active site of aromatization. This is the first demonstration that equine aromatase functions as an estrogen 2-hydroxylase, in addition to transforming androgens into estrogen.

Germ cell-Sertoli cell interactions and production of testosterone by purified Leydig cells from mature rat.

The addition of seminiferous tubule (ST) culture medium (STM) prepared from testes of either busulfan-treated (Bus) or cryptorchid (Cryp) or genetically sterile (hd) rats, to Percoll purified Leydig cells leads to a further increase of LH-stimulated testosterone (T) output (26, 43 and 14%, respectively). Taking into account that the Sertoli cell number per cm of ST is 2.6, 1.8 and 1.4-fold greater in Bus, Cryp and hd rats than in controls, the above STM effects on T output, expressed per 10(6) Sertoli cells are in fact lower (63, 44 and 43%, respectively) that those of control STM. Similar results have been obtained for the STM transferrin levels which are decreased, 74, 67 and 45%, respectively in Bus, Cryp and hd animals. So, it is likely that the Sertoli cell secretion of both the paracrine factor involved on Leydig cell T production and the transferrin is influenced mainly by spermatids and to a lesser extent by spermatocytes of mature rat testis.

Postprandial lipoprotein metabolism in normotriglyceridemic non-insulin-dependent diabetic patients: influence of apolipoprotein E polymorphism.

Non-insulin-dependent diabetes mellitus (NIDDM) is associated with postprandial lipoprotein clearance defects that are correlated with the fasting hypertriglyceridemia widely observed in NIDDM patients. The aim of this study was to determine if such postprandial disturbances are found in NIDDM patients strictly normotriglyceridemic in the fasting state, and if the apolipoprotein E (apo E) polymorphism influences postprandial metabolism of intestinally derived lipoproteins. The vitamin A-fat loading test was used in 18 normotriglyceridemic NIDDM patients and seven normotriglyceridemic obese controls, and postprandial triglyceride (TG) and retinyl palmitate (RP) concentrations were evaluated in total plasma, and in the chylomicron (Sf > 1,000) and nonchylomicron (Sf < 1,000) fractions isolated by ultracentrifugation. NIDDM patients exhibited an amplified response of both TG and RP as compared with obese controls in the three fractions. Incremental TG response to the oral fat load was strongly correlated with fasting TG level (r = .80, P < .0001) in the whole study population. Postprandial lipoprotein profiles were distinguished in NIDDM patients according to apo E phenotype: despite normal fasting TG levels in E3/3 (n = 6), E2/3 (n = 6), and E3/4 (n = 6), postprandial RP response was twofold to threefold higher in E2/3 and E3/4 patients than in the common E3/3 phenotype. Contrasting lower postprandial TG increment and lower fasting and postprandial high-density lipoprotein (HDL) and HDL3 cholesterol levels were observed in E3/4 versus E3/3 patients, possibly reflecting modifications in lipid content of the postprandial lipoproteins driven by a differential lipid transfer activity depending on apo E isoform. These data indicate an enhanced postprandial lipemia in normotriglyceridemic NIDDM patients, and demonstrate the influence of apo E polymorphism on their lipoprotein clearance. Postprandial alterations of lipoprotein remnants may thus accelerate atherogenesis even in normotriglyceridemic NIDDM patients.

Human Sertoli cells in vitro. Lactate, estradiol-17 beta and transferrin production.

Human Sertoli cell parameters, namely lactate, estradiol-17 beta, and transferrin production, were determined after a 24-hour incubation with either human follicle stimulating hormone (FSH) or dbcAMP in the presence or absence of testosterone plus a phosphodiesterase inhibitor (1-methyl-3-isobutylxanthine; MIX). Testicular tissues were obtained from 10 young patients (mean age, 29 years); using a 3-step enzymatic treatment, Sertoli cell enriched preparations (> 92%) were studied after 4 days as primary cultures. No significant changes in lactate, estradiol-17 beta, and transferrin outputs have been observed according to age in patients ranging in age from 16 years to 47 years. Sertoli cell production of the compounds is controlled by testosterone plus MIX; FSH (or dbcAMP) treatment only slightly improves their synthesis. It is suggested that human Sertoli cell function, as far as the parameters measured in this study are concerned, is likely regulated by cAMP-dependent and independent pathways.

Combined diagnostic value of biochemical markers in acute pancreatitis.

Eighty-three patients suffering from upper abdominal pain were studied to evaluate the contribution of commonly used biochemical markers in the diagnosis of acute pancreatitis. On admission to hospital, serum amylase, lipase, total bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and gamma-glutamyl transferase activities were measured. By stepwise logistic discrimination, only two determinations appeared to be of clinical value: lipase and alkaline phosphatase activities. A classification rule was established including these two measurements and its diagnostic performance evaluated by a jackknifed method amounted .83%. ROC curves were used to assess sensitivity and specificity. Our study clearly shows that serum lipase measurements should be preferred to amylase measurements, and that our two-test classification rule provides an efficient aid in clinical decision-making.

Validation of a new system for androgen binding protein measurement.

A new technique that permits measurement of Androgen-Binding Protein (ABP) is validated by reproducibility, linearity and correlation studies. Using this apparatus allowing Scatchard plot analysis, it is also possible to measure association and dissociation rate constants. In addition, it is a very useful tool for a rapid screening of ABP binding capacity during a chromatographic stepwise purification.

In vitro pregnenolone metabolism by mouse adrenal gland: II-Biosynthesis of androgens.

Adrenal gland homogenates from four different strains of mice were incubated with (4-14 C)-pregnenolone and a NADPH generating system. The most important androgen synthesized was dehydroepiandrosterone; testosterone and progesterone were synthesized to a lesser extent and the production of androstenedione was very low. The highest synthetic activities were found in the high mammary tumor strain of mice (C3H x RIII) Fl; they were increased by ovariectomy, particularly when performed at two months of age. In the other strains, they were lower, specially in the low mammary tumor strain C 57 BL. However, the 3 beta-hydroxysteroid dehydrogenase / delta 5, 4 isomerase activity was not modified by ovariectomy in the high mammary tumor strain whereas it was increased in the low mammary tumor strains. These results indicate that the androgen synthesis in mouse adrenal depends on factors such as age, sex, endocrine status (ovariectomy) but also on susceptibility to mammary tumor development.

Changes in androgen binding protein (ABP) production following continuous low dose gamma irradiation (IR) of adult rat.

Continuous low dose gamma irradiation induces a progressive degeneration of germ cells with a concomittant increase in blood FSH; however, the Sertoli cell function is not too much altered since serum ABP level is normal and it is likely that the decrease of epididymal ABP content is the consequence of a reduction in seminiferous tubule fluid excretion. Obviously, spermatids seems to be involved in the regulation of Sertoli cell ABP synthesis.

[Male infertility in 1984]. / Stérilité masculine en 1984.

The study of male sterility has not benefited as much from progress in fundamental research as that of female sterility, but nevertheless its diagnosis and, to a lesser extent, its treatment have developed considerably over the past few years. With respect to diagnosis, substantial progress has been made in sperm analysis. The techniques of the spermatogram and the spermocytogram, performed under the conditions defined by David et al. (Feuil. Biol., 15, 47, 1974) allow a better classification of the different anomalies of spermatozoa. Ultrastructural studies now being carried out in several laboratories should provide a more exact description of these anomalies. More precise studies of normal sperm have defined the limits of normality (Schwartz et al., J. Reprod. Fert., 57, 391, 1979). The study of the functional capacities of spermatozoa has also advanced considerably. The postcoital test is still routinely used, but it is subject to many causes of error. For this reason it must be complemented by an in vitro analysis of spermatozoon penetration into the zona pellucida, and possibly of spermatozoon penetration into the hamster ovum (Yanagimashi et al., Biol. Reprod., 15, 471, 1976). With respect to seminal plasma, research on antispermatic antibodies both in men and in women should expend with the help of simpler and more reliable techniques. The study of recently discovered constituents of seminal plasma (oligoelements, acetyl carnitine, specific proteins, etc.) should lead to a more accurate determination of the defective organ.(ABSTRACT TRUNCATED AT 250 WORDS)

[Value of the study of total alkaline phosphatases and bone isoenzyme in a population of subjects with osteoporosis]. / Intérêt de l'étude des phosphatases alcalines totales et de l'isoenzyme osseuse dans une population de sujets atteints d'ostéoporose.

The authors have compared the values of total and bone serum alkaline phosphatases in an osteoporotic population and a control group. The total activity of alkaline phosphatases was determined by a kinetic method at 30 degrees C and alkaline phosphatase isoenzymes were separated by agarose gel electrophoresis in presence or absence of wheat germ lectin. The authors have confirmed reported data concerning the physiological variations of alkaline phosphatases in the control group; they have therefore divided each group in several sub-populations according to age and sex in order to obtain accurate comparisons. Mean values of total and bone alkaline phosphatases were greater in the osteoporotic population than in the control group regardless of age or sex. Nevertheless, significant differences were obtained only with 50 to 75 year-old subjects (men or women) for total alkaline phosphatases and with 50 to 75 year-old women for bone alkaline phosphatase.

[Physiological variations of LpA1, HDL2 and principal lipid markers in athletes]. / Variations physiologiques de la LpA1, des HDL2 et des principaux marqueurs lipidiques chez l'homme sportif.

An original approach of the influence of physical activities on lipid metabolism is presented in this work: the authors studied the physiological variations of the lipoparticle LpA1, high-density lipoprotein subfraction HDL2 and the most important lipid markers in serum of a presumably healthy population of 55 high-level sportsmen. They were 18-45 years old and practised endurance sports, whether individual (cycling, long-distance running), or collective activities (soccer, basketball). The authors observed an important increase of LpA1 (+20%, p < 0.001) and C-HDL2 (+23.3%, p < 0.01) after an intense physical activity (CK = 430 +/- 312 U/l); they also found a good correlation between LpA1 and HDL2 (r = 0.85, p < 0.0001) and between LpA1 and CK (r = 0.62, p < 0.001).

Determination of metallothioneins by HPLC: application to zinc metabolism.

Metallothioneins (MT) are well known Zn and Cu tissue proteins. Their level is mainly regulated by Zn. Therefore, they have been suggested as an index of Zn status. The determination of MT has been performed by HPLC (fitted with UV-detector) following a preparation phase (heating, ultracentrifugation and filtration). This method allows direct quantification of MT in tissues and shows a good correlation between MT concentrations and peak heights (r2 = 0.995; a = 774.73; b = -148.38) from 0 to 10 micrograms/100 microL. MT determination by RP-HPLC method allows to assess the tissue zinc status as displayed by the correlation between hepatic zinc and metallothionein concentrations (r2 = 0.82; p < 0.001). Variations of MT in response to Zn intakes are observed within 24 hours and for a physiological range of dietary Zn intakes.

[Sertoli cells. Functional aspects compared in rats, pigs and man]. / La cellule de Sertoli. Aspects fonctionnels comparés chez le rat, le porc et l'homme.

In the mammalian testis, the Sertoli cell plays a key role in the development and maintenance of spermatogenesis. Indeed, within the seminiferous tubule, the structure of the Sertoli cells and the specialized junctions between them and the neighbouring germ cells, contribute to create the sophisticated microenvironment and to bring all the nutriments require for a full development of germ cells. In this review, we have compared the main Sertoli cell functions in the rat and the pig to the available data concerning the human. We have included our recent results obtained from testicular tissues of 15 young men (mean age: 29 years) from which we have prepared the Sertoli cells. In addition to the lactate and estradiol-17 beta syntheses, the human Sertoli cell produces several proteins, namely transferrin, ferritin and inhibin under the control of germ cells.