Development of fibroblast-type-II cell communications in fetal rabbit lung organ culture.

The development of the fetal lung is regulated by fibroblast-type-II cell communications which involve fibroblast pneumonocyte factor (FPF). FPF production is positively regulated by glucocorticoids and negatively regulated by dihydrotestosterone (DHT) and transforming growth-factor beta (TGF-beta). We studied whether DHT or TGF-beta affected other steps in the process of lung maturation, by studying how the developing lung in organ culture would respond to exogenously supplied FPF after DHT or TGF-beta exposure. Fetal rabbit (day 19 of gestation) lung organ cultures were prepared and cultured in the presence of cortisol, DHT or TGF-beta. After seven days, the media were replaced with serum-free medium containing either cortisol or FPF conditioned medium. The incorporation of [14C]glycerol into surfactant lamellar body DSPC was studied over 24 h as the index of surfactant synthesis. Results were compared to simultaneous control cultures. Treatment had no significant effect on tissue protein concentration or on the efficiency of lamellar body recovery. Cortisol stimulated baseline incorporation of glycerol into DSPC. This was inhibited by DHT, such that DHT plus cortisol treatment was no different from untreated controls. FPF stimulated the incorporation of glycerol into DSPC, and did so even after culture treatment with DHT. Cultures treated with TGF-beta exhibited glycerol incorporation similar to untreated controls. After TGF-beta exposure, FPF did not stimulate glycerol incorporation into DSPC. We conclude that DHT interferes with progression of lung development by delaying the appearance of FPF production by the fibroblast. TGF-beta, on the other hand, inhibits other elements of lung maturation besides FPF production. We speculate that TGF-beta interferes with type-II cell development such that the cell cannot respond to FPF.     

Percentage binding of testosterone and dihydrotestosterone and unbound testosterone and dihydrotestosterone in rabbit maternal and fetal plasma during sexual organogenesis.

The percentages of bound testosterone (17 beta-hydroxy-4-androsten-3-one; T) and dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one; DHT) and their unbound concentrations were determined in pregnant rabbits and their fetuses from the 18th day of gestation to birth. T and DHT were also measured in fetal testes. In the testis, the total T/total DHT ratio, very high at 22 days (73.7 +/- 15.2), decreased until birth (6.7 +/- 0.8). In male fetuses the concentrations of total and unbound circulating T and DHT were always low and did not show any peak during sexual organogenesis. The percent binding of T (from 73.0 +/- 0.5 to 77.6 +/- 0.6) and DHT (from 76.5 to 83.7 +/- 1.1) in fetuses were similar in both sexes and significantly lower than those measured in mothers (T: from 87.2 +/- 0.6 to 91.6 +/- 0.9; DHT: from 87.3 +/- 0.9 to 93.8 +/- 0.9).     

Dihydrotestosterone blocks fetal lung fibroblast-pneumonocyte factor at a pretranslational level

Fibroblast pneumonocyte factor (FPF) synthesis by fetal rat lung fibroblasts is augmented during gestation in the presence of cortisol. The control and cortisol-augmented levels of FPF production, as determined by FPF ability to stimulate saturated phosphotidylcholine synthesis by lung epithelial Type II cells, is delayed during development in fibroblasts derived from male fetuses as compared to those derived from female fetuses. The mechanism by which this delay occurs has been addressed. Pregnant rats treated in vivo with dihydrotestosterone (DHT) showed decreased FPF activity from control or cortisol-treated fibroblasts derived from 20-day-old male or female fetuses. In vitro translated proteins of size-fractionated lung RNA from 19-day-old fibroblasts that were pretreated with DHT in vitro showed decreased FPF activity compared to nontreated samples. This decreased FPF activity was present even if the DHT-pretreated cells were stimulated with cortisol prior to RNA preparation. Using a mouse model of testicular feminization that contains no receptors for androgens showed no change in the cortisol augmented FPF activity when the fibroblasts were pretreated with DHT. These data taken together suggest that the delayed FPF production of male-derived lung fibroblasts is a physiologic process which requires androgen receptors, and the mechanism by which androgens inhibit FPF production appears to affect events occurring mainly at a pretranslational level.     

Changes in pulmonary surfactant during bacterial pneumonia

In pneumonia, bacteria induce changes in pulmonary surfactant. These changes are mediated by bacteria directly on secreted surfactant or indirectly through pulmonary type II epithelial cells. The bacterial component most likely responsible is endotoxin since gram-negative bacteria more often induce these changes than gram-positive bacteria. Also, endotoxin and gram-negative bacteria induce similar changes in surfactant. The interaction of bacteria or endotoxin with secreted surfactant results in changes in the physical (i.e. density and surface tension) properties of surfactant. In addition, gram-negative bacteria or endotoxin can injure type II epithelial cells causing them to produce abnormal quantities of surfactant, abnormal concentrations of phospholipids in surfactant, and abnormal compositions (i.e. type and saturation of fatty acids) of PC. The L/S ratio, the concentration of PG, and the amount of palmitic acid in PC are all significantly lower. The changes in surfactant have a deleterious effect on lung function characterized by significant decreases in total lung capacity, static compliance, diffusing capacity, and arterial PO₂ and a significant increase in mean pulmonary arterial pressure. Also decreased concentrations of surfactant or an altered surfactant composition can result in the anatomic changes commonly seen in pneumonia such as pulmonary edema, hemorrhage, and atelectasis.Abbreviations BAL Bronchoalveolar lavage - LPS lipopolysaccharide - PC phosphatidylcholine - PG phosphatidylglycerol - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - LPC lysophosphatidylcholine - SPH sphingomyelin - DPPC dipalmitoylphosphatidylcholine - L/S lecithin/sphingomyelin     

Evaluation of intra-amniotic surfactant administration for lung maturation in preterm sheep

We aimed to evaluate the effects of intra-amniotic surfactant administration on alveolar lecithin/sphingomyelin ratio, density of type II pneumocytes, and fetal lung function in preterm merino sheep. Pregnant ewes at 119 days gestation either received 200 mg intra-amniotic surfactant (n=4) or saline solution (n=4). After 24 h, the lambs were delivered by hysterotomy and mechanically ventilated. Lecithin/sphingomyelin ratios in alveolar fluid, inflating pressure–volume relationships, and type II pneumocyte counts in histological specimens were compared among the groups. All of the lambs completed the protocol. Mean lecithin/sphingomyelin ratio increased significantly in amniotic (p=0.03) and alveolar fluid (p=0.03) samples of surfactant-treated animals. Lung function in terms of pressure–volume curves did not differ between two groups. Type II pneumocyte density tended to be higher (p=0.057) after intra-amniotic surfactant administration. Single-dose treatment with intra-amniotic surfactant seems to improve amniotic and alveolar lecithin/sphingomyelin ratio questionably by increasing alveolar type II cells. Pressure–volume relationships from inflation of the lungs might be unaltered with intra-amniotic surfactant treatment.     

Regional differences in the testosterone to dihydrotestosterone ratio in the epididymis and vas deferens of adult mice

The concentrations of testosterone and dihydrotestosterone (DHT) were measured in the testis and in different segments of the epididymis and vas deferens of adult mice. There were marked regional variations in the concentrations of testosterone and DHT from the testis to the caudal part of the vas deferens. In the testis, testosterone was the predominant androgen (364 +/- 90 ng/g) while DHT was weakly represented (8 +/- 2 ng/g). Qualitative and quantitative changes occurred in epididymis: DHT was the main steroid in the caput (29.3 +/- 2.7 ng/g) and corpus (33.1 +/- 4.4 ng/g) while testosterone and DHT were in similar quantities in the cauda (18.6 +/- 2.6 and 19.0 +/- 2.7 ng/g, respectively). The proximal region of the vas deferens contained higher amounts (71.4 +/- 8.0 ng/g) of androgens (testosterone + DHT) than did the caput epididymidis (39.1 +/- 3.3 ng/g). Testosterone was the predominant androgen in each part of the vas deferens and its concentrations decreased from the proximal (64.5 +/- 7.5 ng/g) to the caudal (26.9 +/- 4.3 ng/g) region. Castration and section of the efferent ducts of the testis showed that the epididymis received testosterone essentially via the blood supply and that epididymal DHT was produced locally from circulating testosterone.     

Glucose effects on lung surfactant kinetics in conscious pigs

The primary goal of this study was to investigate the effects of glucose infusion on surfactant phosphatidylcholine (PC) metabolic kinetics in the lungs. A new stable isotope tracer model was used in which [1,2-(13)C(2)]acetate and uniformly labeled [U-(13)C(16)]palmitate were infused in 12 normal overnight-fasted pigs to quantify lung surfactant kinetics with or without glucose infusion (24 mg. kg(-1). min(-1)). With glucose infusion, the rate of surfactant PC incorporation from de novo synthesized palmitate increased from the control value of 2.1 +/- 0.2 to 15.5 +/- 1.9 nmol PC-bound palmitate. h(-1). g wet lung(-1) (P < 0.05), whereas the incorporation rate from plasma preformed palmitate decreased from the control value of 20.9 +/- 1.9 to 11.6 +/- 1.1 nmol palmitate. h(-1). g wet lung(-1) (P < 0.05). The palmitate composition in lamellar body surfactant PC increased from the control value of 61.7 +/- 2.1% to 75.9 +/- 0.6% (P < 0.05). The surfactant PC secretion rate decreased from the control value of 239.0 +/- 26.1 to 81.9 +/- 5.3 nmol PC-bound palmitate. h(-1). g wet lung(-1) (P < 0.05). We conclude that, whereas surfactant secretion was inhibited by glucose infusion, neither total surfactant PC synthesis nor the surfactant PC pool size was significantly affected due to an increased reliance on de novo synthesized fatty acids.     

Estimation by gas chromatography-mass spectrometry with selected ion monitoring of urinary excretion rates of 3 alpha-androstanediol during/after i.v. administration of 13C-labelled testosterone in man

To study in vivo the conversion of testosterone (T) into its metabolites, dihydro-testosterone (DHT) and 5 alpha-androstane-3 alpha, 17 beta diol (3 alpha-Diol) the urinary excretion rates of these steroids were determined by mass spectrometry in 6 healthy men during/after the i.v. infusion (t = 4 h) of 20 mg [13C]testosterone. In addition, plasma concentrations of T, DHT and 3 alpha-Diol were determined by radioimmunoassay. During steady state conditions at the end of the 4-h infusion of [13C]T the increase in the plasma concentrations of T from, basal, 405 +/- 140 ng/dl to 4205 +/- 804 ng/dl was paralleled by an increase in the plasma concentrations of DHT to 106.4 +/- 62.5 ng/dl) (basal: 30.8 +/- 21.8 ng/dl), and of 3 alpha-Diol to 32.2 +/- 12.5 ng/dl (basal: 12.5 +/- 13.9 ng/dl). Plasma concentrations of T, DHT and 3 alpha-Diol then returned to basal concentrations within 24 hours. Using mass-spectrometry we found a cumulative renal excretion of 13C-labelled T of 15.6 +/- 9.6 micrograms/24 h, equivalent to 0.08 +/- 0.05% of the infused amount (20 mg) of [13C]T. Whereas urinary excretion of [13C]DHT was below the level of detection by mass-spectrometry the cumulative excretion of [13C]3 alpha-Diol was 67.7 +/- 19.9 micrograms/24 hours which is equivalent to 0.3 +/- 0.1% of the infused dose of 13C-labelled testosterone. These data suggest that the determination of urinary 3 alpha-Diol by mass-spectrometry during/after the infusion of stable-labelled testosterone represents an alternative to the use of radioactive label for turnover studies.     

Epidermal growth factor influences the developmental clock regulating maturation of the fetal lung fibroblast

Growth factors may play a significant role in regulating the orderly progression of organ growth and differentiation during fetal development. We hypothesized that epidermal growth factor (EGF) would help regulate the development of surfactant synthesis in the fetal lung by influencing fibroblast-epithelial cell interactions. The effect of EGF (10 ng per ml) on the ability of the fetal lung fibroblast to produce fibroblast pneumonocyte factor (FPF) was studied in sex-specific fibroblasts cultured from day 16, day 17 or day 18 fetal mouse lungs. FPF which is normally not produced by day 16 fibroblasts, is found only in female fibroblasts on day 17, and then in both males and females on day 18. EGF advanced this pattern such that female fibroblasts produced activity on day 16 and fibroblasts from both sexes produced FPF activity on day 17 and day 18. Fibroblasts from an androgen receptor-deficient mouse model confirmed that the effect of EGF was sex-specific and related to the state of development of the fetal lung. We conclude that EGF advances the fetal lung fibroblast through specific stages of development. It appears, therefore, to help control the timing of the clock regulating fetal lung maturation.     

The influence of experimentally produced oligohydramnios on lung growth and pulmonary surfactant content in fetal rabbits.

To study the effect of oligohydramnios on lung growth and biochemical lung development in fetal rabbits, amniotic fluid was drained through a tube inserted into the maternal peritoneal cavity on the 23 day of gestation. Littermate fetuses without an amniotic shunt were used as controls. The fetuses were delivered abdominally on the 28 day of gestation. In a total of 8 pregnant does, 17 fetuses underwent amniotic shunting and 22 fetuses were used as controls. The amniotic shunt produced a significant reduction in the amniotic fluid volume. There were no differences in the wet weights of the fetal body, liver or brain between the two groups. However, the amniotic shunt significantly decreased the wet weight of the fetal lung, fetal lung wet weight/body weight ratio, and protein concentration per lung as compared to the control fetuses. In the fetal liver and brain tissues, no changes were found in the concentrations of total phospholipids, phosphatidylcholine (PC) or disaturated phosphatidylcholine (DSPC, the main component of lung surfactant) per g of wet tissue and per mg of protein. However, the lungs of the fetuses with amniotic shunts contained significantly more PC and DSPC, and the L/S ratio was higher than in the control fetuses. These results suggest that the oligohydramnios produced by an amniotic shunt causes pulmonary hypoplasia, but raises the pulmonary surfactant content of fetal rabbit lung.     

Quantification of surfactant pool sizes in rabbit lung during perinatal development

Methods are presented for the quantitative isolation of surfactants from fetal and newborn rabbit alveolar lavage returns and post-lavaged lung tissue homogenates. The phospholipid content of both fractions progressively increased between 27 days gestation and term (31 days). The tissue-stored fraction increased approximately 16-fold (from 0.48 +/- 0.13 to 7.83 +/- 0.86 mg/g dry lung) and the alveolar fraction more than 30-fold (from 0.08 +/- 0.02 to 2.69 +/- 0.52 mg/g dry lung). Developmental changes in phospholipid composition were also observed. Tissue-stored surfactant was prepared using differential and density gradient centrifugation. Alveolar surfactant was isolated during fetal development as a high-speed pellet following a one-step differential centrifugation. There was little change in the phospholipid content of fetal alveolar lavage supernatant (range 0.12 +/- 0.04 to 0.28 +/- 0.09 mg/g dry lung). By the first postnatal day the phospholipid content of both lavage fractions significantly increased (pellet, 7.51 +/- 1.79; supernatant, 4.01 +/- 1.36 mg/g dry lung) and both were identified as surfactant. This increase in alveolar surfactant was accompanied by an approximately twofold decrease (to 3.81 +/- 1.1 mg/g dry lung) in the tissue-stored fraction. These data provide a quantitative profile of surfactant accumulation and secretion in developing rabbit lung.     

Testosterone processing by pituitary cells in culture: an examination of the role of 5 alpha-reduction in androgen action on the gonadotroph

Dispersed rat pituitary cells were exposed to [1,2,6,7-3H]testosterone ([3H]T, 10(-8) M) to assess the role of 5 alpha-reduction in T regulation of gonadotroph secretion. After 4 to 48 hours of exposure, [3H]T metabolites isolated by thin-layer chromatography were characterized in medium and cell homogenates as well as bound to androgen receptors salt-extracted from purified nuclear pellets. Receptor-bound 5 alpha-[3H]dihydrotestosterone ([3H]DHT)/total [3H]androgens rose progressively from 16% at 4 hours to more than 50% at 48 hours. Coincubation with 4-MA (10- to 1,000-fold molar excess) or testosterone-17 beta-carboxylic acid (TCA; 1,000-fold excess) reduced receptor-bound [3H]DHT/[3H]androgen to less than 10% and 20%, respectively, but elevated [3H]T-receptor levels. Despite inhibiting 5 alpha-reductase activity, TCA and 4-MA had no effect on T suppression of gonadotropin-releasing hormone-stimulated luteinizing hormone secretion or T enhancement of total (cell + secreted) follicle-stimulating hormone levels. The results suggest that 5 alpha-reduction to DHT is not essential for the expression of the direct influences of T on gonadotropin synthesis and secretion in rat gonadotrophs.     

Synthesis of surfactant components by cultured type II cells from human lung

We examined the effect of monolayer culture on surfactant phospholipids and proteins of type II cells isolated from human adult and fetal lung. Type II cells were prepared from cultured explants of fetal lung (16-24 weeks gestation) and from adult surgical specimens. Cells were maintained for up to 6 days on plastic tissue culture dishes. Although incorporation of [methyl-3H]choline into phosphatidylcholine (PC) by fetal cells was similar on day 1 and day 5 of culture, saturation of PC fell from 35 to 26%. In addition, there was decreased distribution of labeled acetate into PC, whereas distribution into other phospholipids increased or did not change. The decrease in saturation of newly synthesized PC was not altered by triiodothyronine (T3) and dexamethasone treatment or by culture as mixed type II cell/fibroblast monolayers. The content of surfactant protein SP-A (28-36 kDa) in fetal cells, as measured by ELISA and immunofluorescence microscopy, rose during the first day and then fell to undetectable levels by the fifth. Synthesis of SP-A, as measured by [35S]methionine labeling and immunoprecipitation, was detectable on day 1 but not thereafter. Levels of mRNAs for SP-A and for the two lipophilic surfactant proteins SP-B (18 kDa) and SP-C (5 kDa) fell with half-times of maximally 24 h. In contrast, total protein synthesis measured by [35S]methionine incorporation increased and then plateaued. In adult cells, the content of SP-A and its mRNA decreased during culture, with time-courses similar to those for fetal cells. We conclude that in monolayer culture on plastic culture dishes, human type II cells lose their ability to synthesize both phospholipids and proteins of surfactant. The control of type II cell differentiation under these conditions appears to be at a pretranslational level.     

The development of surfactant synthesis in fetal rabbit lung organ culture exhibits a sex dimorphism

Sex differences in amniotic fluid and lung lavage surfactant have been found. Although these studies suggest that augmented fetal surfactant synthesis occurs earlier in the female fetus, there is little direct evidence for a sex difference in fetal surfactant synthesis. We studied the synthesis of surfactant by evaluating the appearance of labelled phospholipids in lamellar bodies recovered from sex-specific organ culture of fetal rabbit lungs. Furthermore, we studied the ability of dexamethasone to stimulate surfactant synthesis in male and female fetal lungs. Organ culture was begun on day 21 of gestation. After 5 days the incorporation of [1,3-14C]glycerol into phosphatidylcholine (PC), disaturated phosphatidylcholine, phosphatidylinositol (PI), and phosphatidylglycerol was studied. Female lungs in organ culture synthesized more disaturated PC per milligram protein than male lungs. In the presence of dexamethasone (10(-8) M) and dihydrotestosterone (10(-8) M) an increased synthesis was noted in the female cultures of PC (270%), disaturated PC (234%), PI (281%), and phosphatidylglycerol (754%). No significant increase in the synthesis of PC or disaturated PC was observed in the male cultures. However in the male cultures smaller increases in the synthesis of PI (193%) and of phosphatidylglycerol (360%) were observed. Overall, dexamethasone stimulated synthesis in females but not in males such that significant differences in the synthesis of all phospholipids were found in the presence of 10(-8) M dexamethasone. These studies show that the synthesis of surfactant in the fetal lung is sexually dimorphic, as is the ability of dexamethasone to regulate synthesis. An understanding of the mechanism which causes these differences may provide important insight into the control of the developmental clock which regulates the orderly progression of development.     

Sphingomyelin metabolites inhibit sphingomyelin synthase and CTP:phosphocholine cytidylyltransferase

Tissue injury in inflammation involves the release of several cytokines that activate sphingomyelinases and generate ceramide. In the lung, the impaired metabolism of surfactant phosphatidylcholine (PC) accompanies this acute and chronic injury. These effects are long-lived and extend beyond the time frame over which tumor necrosis factor (TNF)-alpha and interleukin-1beta are elevated. In this paper, we demonstrate that in H441 lung cells these two processes, cytokine-induced metabolism of sphingomyelin and the inhibition of PC metabolism, are directly interrelated. First, metabolites of sphingomyelin hydrolysis themselves inhibit key enzymes necessary for restoring homeostasis between sphingomyelin and its metabolites. Ceramide stimulates sphingomyelinases as effectively as TNF-alpha, thereby amplifying the sphingomyelinase activation, and TNF-alpha, ceramide, and sphingosine all inhibit PC:ceramide phosphocholine transferase (sphingomyelin synthase), the enzyme that restores homeostasis between sphingomyelin and ceramide pools. Second, ceramide inhibits PC synthesis, probably because of its effects on CTP:phosphocholine cytidylyltransferase, the rate-limiting enzymatic step in de novo PC synthesis. The data presented here suggest that TNF-alpha may be an inhibitor of phospholipid metabolism in inflammatory tissue injury. These actions may be amplified because of the ability of metabolites of sphingomyelin to inhibit the pathways that should restore the normal ceramide-sphingomyelin homeostasis.     

Coordination of growth and differentiation in the fetal lung

The male fetal lung begins to synthesize surfactant later in gestation than the female. This delay appears to be caused by androgens. We hypothesized that male fetal lung differentiation is delayed as a consequence of an extended phase of growth which is elicited by androgens. We observed that in vivo fetal lung protein synthesis relative to DNA synthesis peaked earlier in gestation in the female fetal lung and that this event was synchronous with the onset of differentiation. Pregnant rats were treated with dihydrotestosterone (DHT) during pregnancy, and fetal lung growth parameters were measured. Lung wet weight, dry weight, and DNA and protein concentrations were significantly elevated by DHT treatment. Type II cells and fibroblasts were isolated from lungs of DHT-treated fetuses. The number of total cells recovered was increased by 30%; the number of type II cells recovered was increased by 87%; and the number of fibroblasts recovered was increased by 42%. The type II cells which were recovered exhibited increased incorporation of [3H]thymidine into DNA and a reduced ratio of radiolabeled protein to radiolabeled DNA compared to that of cells from control lungs. Further studies were done in vitro with fibroblasts and type II cells isolated from untreated fetal rat lungs. Treatment of the fibroblasts with DHT during culture caused an increase in thymidine incorporation into DNA. This effect was not blocked by simultaneous treatment with cortisol, which normally causes reduced DNA synthesis and induces fibroblast differentiation. Treatment of the type II cells with DHT in culture caused a dose-dependent increase in cell number but a decrease in synthesis of disaturated phosphatidylcholine. These studies provide more direct evidence of the interrelationships between the control of growth and the control of differentiation in the fetal lung. DHT, a signal which delays the onset of expression of differentiation, also induces growth. We conclude that the controls of growth and of differentiation of the fetal lung are reciprocally linked.     

The effects of diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (4-MA) and (4R)-5,10-SECO-19-norpregna-4,5-diene-3,10,20-trione (SECO) on androgen biosynthesis in the rat testis and epididymis

We have investigated the effects of two 4-ene-steroid 5 alpha-reductase inhibitors, diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (4-MA) and (4R)-5,10-seco-19-norpregna-4, 5-diene-3,10,20-trione (SECO), on testicular and epididymal androgen biosynthesis. Kinetic analyses revealed that both compounds inhibited epididymal DHT biosynthesis. 4-MA was a competitive inhibitor of epididymal nuclear and microsomal 4-ene-steroid 5 alpha-reductases (3-oxo-5 alpha-steroid: NADP 4-ene-oxidoreductase EC 1.3.1.22) with Kiapp values of 12.8 and 15.1 nmol/l compared to the respective Kmapp values of 185 and 240 nmol/l. Values for the Vmaxapp were always within 70-130% of the control. SECO at 1.0 mumol/l, also inhibited epididymal nuclear and microsomal 4-ene-steroid-5 alpha-reductases, causing respectively 2.9 and 5.2-fold increases in Kmapp. The Vmaxapp values were unchanged. However, SECO concentrations of 5 and 25 mumol/l abolished 4-ene-steroid 5 alpha-reductase activity at all testosterone concentrations. To examine the specificity of these compounds, we investigated their effects on the enzymes that convert pregnenolone to testosterone. Rat testis microsomes converted pregnenolone to testosterone via the 4-ene-3-oxo pathway, with the major metabolites being progesterone, 17-hydroxyprogesterone, 4-androstenedione and testosterone; some 17-hydroxypregnenolone was also formed. Very small amounts of dehydroepiandrosterone (DHA) and 5-androstenediol were detected. SECO, at a concentration that completely inhibited epididymal 4-ene-steroid 5 alpha-reductase activity, did not alter the metabolic profile of pregnenolone metabolism. However, 4-MA prevented the appearance of 4-ene steroids, and large quantities of 17-hydroxypregnenolone and DHA accumulated, suggesting that inhibition of the 3 beta-hydroxysteroid: NAD(P)+ oxidoreductase (EC 1.1.1.51) and 3-oxosteroid 5-ene-4-ene-isomerase (EC 5.3.3.1) [3 beta-hydroxysteroid dehydrogenase-isomerase] was occurring. Optimal conditions for the microsomal conversion of DHA to 4-androstenedione were determined; kinetic analyses of the 3 beta-hydroxysteroid dehydrogenase-isomerase activity revealed that 4-MA inhibited this reaction non-competitively, reducing Vmaxapp values to 25% of the control. The Kiapp determined from the intercept replot, was 121 nmol/l, and the Kmapp was always between 90 and 130% of the control value. It is concluded that SECO is more specific than 4-MA in its effects on androgen biosynthesis in the testis and epididymis and that both these drugs should provide useful tools in assessments of the relative contributions of 5 alpha-reduced androgens to androgen dependent processes.     

Characterization of plasma membrane lipids and luteinizing hormone receptors of ovine corpora lutea during luteolysis and early pregnancy

Lipid composition of plasma membranes from luteal cells was examined to determine whether changes in this organelle occur during regression and maintenance of the corpus luteum in nonpregnant (NP) and pregnant (P) ewes, respectively. Forty ewes were assigned to be killed on Day 13 or 15 of the estrous cycle (D13-NP and D15-NP) or pregnancy (D13-P and D15-P). Purification of luteal plasma membranes on discontinuous sucrose gradients yielded two fractions, designated F1 and F2, that exhibited the greatest enrichment of 5'-nucleotidase activity (five- and fourfold, respectively) over that of the homogenate. These fractions also yielded the lowest contamination by endoplasmic reticulum as represented by nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome C reductase activity and mitochondrial membranes as indicated by succinate dehydrogenase activity. Predominant phospholipids identified in membranes obtained from all groups were phosphatidylcholine (PC, 48.9 +/- 0.6% of total phospholipid), phosphatidylethanolamine (PE, 33.3 +/- 0.4%), sphingomyelin (SPH, 9.7 +/- 0.3%), phosphatidylserine (PS, 3.5 +/- 0.2%), and phosphatidylinositol (PI, 4.0 +/- 0.5%). No changes in microgram phospholipid/mg membrane protein were observed for any luteal phospholipid on D13 and 15 of the estrous cycle or pregnancy. No significant changes in the relative percentages of major fatty acids present in PC (palmitic [16:0], oleic [18:1]), PE (stearic [18:0], 18:1 and arachidonic [20:4]), or PS (18:0, 18:1, docosatetraenoic [22:4]), nor in the ratios of unsaturated (U) to saturated (S) fatty acids in these phospholipids were observed. Significant differences in unsaturated fatty acids of chain length greater than 20 carbons present in minor quantities in PC, PE, and PS were detected between NP and P ewes as well as between days within reproductive stage. The profile of major fatty acids present in PI revealed decreases in 18:0 and 20:4 in D15-NP and increases in 22:4 and docosapentaenoic acid (22:5) in luteal membranes of both D13- and D15-NP ewes relative to the levels of these fatty acids in PI of corresponding groups of pregnant ewes. There was a general trend for 20:4 levels of PC and PI in membranes of D15-NP ewes to be inversely related to those of D15-P ewes. Collectively, these changes were reflected by an increased U:S fatty acid ratio in luteal membrane PI during the estrous cycle. Specific binding of [125I] iodo-human chorionic gonadotropin to luteal plasma membranes from NP and P ewes on D13 and 15 (6/group) revealed similar affinities and concentrations of unoccupied luteinizing hormone (LH) receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Self-administration of estrogen and dihydrotestosterone in male hamsters

Anabolic-androgenic steroids (AAS) are drugs of abuse. Previous studies have shown that male and female hamsters self-administer testosterone (T) and other AAS, suggesting that androgens are reinforcing in a context where athletic performance is irrelevant. AAS are synthetic derivatives of T, which may be aromatizable to estrogen and/or reducible to dihydrotestosterone (DHT). However, we do not know which metabolites of T are reinforcing. To determine if DHT, estradiol (E(2)), or DHT + E(2) are reinforcing, we tested intracerebroventricular (icv) self-administration in male hamsters. The hypothesis was that androgen reinforcement is sensitive to both androgenic and estrogenic T metabolites. If so, hamsters would self-administer DHT, E(2), and DHT + E(2). Twenty four castrated male hamsters (n = 8/group) received icv cannulas and sc T implants for physiologic androgen replacement. One week later, hamsters self-administered DHT (0.1, 1.0, 2.0 microg/microl), E(2) (0.001, 0.01, 0.02 microg/microl), or DHT + E(2), each for 8 days in increasing concentration (4 h/day). Operant chambers were equipped with an active and inactive nose-poke. At the medium concentration, hamsters self-administered DHT (active nose-poke: 47.9 +/- 13.9 responses/4 h vs. inactive: 18.7 +/- 4.8), E(2) (active: 44.8 +/- 14.9 vs. inactive: 16.6 +/- 2.6), and DHT + E(2) (active: 19.1 +/- 2.4 vs. inactive: 10.4 +/- 2.4, P < 0.05). At the highest concentration, males self-administered DHT (active: 28.3 +/- 7.7 vs. inactive: 15.0 +/- 3.5, P < 0.05) and DHT + E(2) (active: 22.6 +/- 3.8 vs. inactive: 11.6 +/- 2.5, P < 0.05), but not E(2). Hamsters did not self-administer the lowest concentrations of DHT, E(2), or DHT + E(2). These results support our hypothesis that both androgenic and estrogenic T metabolites are reinforcing. Together, they do not exert synergistic effects.     

Lipidomics of cellular and secreted phospholipids from differentiated human fetal type II alveolar epithelial cells

Maturation of fetal alveolar type II epithelial cells in utero is characterized by specific changes to lung surfactant phospholipids. Here, we quantified the effects of hormonal differentiation in vitro on the molecular specificity of cellular and secreted phospholipids from human fetal type II epithelial cells using electrospray ionization mass spectrometry. Differentiation, assessed by morphology and changes in gene expression, was accompanied by restricted and specific modifications to cell phospholipids, principally enrichments of shorter chain species of phosphatidylcholine (PC) and phosphatidylinositol, that were not observed in fetal lung fibroblasts. Treatment of differentiated epithelial cells with secretagogues stimulated the secretion of functional surfactant-containing surfactant proteins B and C (SP-B and SP-C). Secreted material was further enriched in this same set of phospholipid species but was characterized by increased contents of short-chain monounsaturated and disaturated species other than dipalmitoyl PC (PC16:0/16:0), principally palmitoylmyristoyl PC (PC16:0/14:0) and palmitoylpalmitoleoyl PC (PC16:0/16:1). Mixtures of these PC molecular species, phosphatidylglycerol, and SP-B and SP-C were functionally active and rapidly generated low surface tension on compression in a pulsating bubble surfactometer. These results suggest that hormonally differentiated human fetal type II cells do not select the molecular composition of surfactant phospholipid on the basis of saturation but, more likely, on the basis of acyl chain length.