Automated High-Performance Analysis of Lung Morphometry.

Automation of lung morphometric analysis is an asset in the study of lung pathophysiology because it is an assurance of robustness, reproducibility, and rapidity. The novel automated morphometric approach presented here meets these criteria. This new method collects multiple parameters, allowing quantitative elucidation of the pathophysiology of the developing and mature lungs. The automated morphometric analysis is reliable and allows the analysis of a greater proportion of each lung together with a higher number of samples and superior reproducibility than manual analysis. The use of this method revealed that treatment with 80% oxygen and lung development presented an opposite effect on most of the analyzed parameters. In conclusion, this novel approach allowed the collection of new fundamental morphometric data on lung development and a deeper comprehension of the effect of hyperoxia.

Ovine model of congenital chest wall and spine deformity: From birth to 3 months follow-up.

Background: The evolution and treatment of lung alterations related to congenital spine and chest wall deformities (CWD) are poorly understood. Most animal models of CWD created postnatally were not evaluated for respiratory function. The goal of our study was to evaluate the effects of a CWD induced in utero on lung growth and function in an ovine model. Methods: A CWD was induced in utero at 70-75 days of gestation in 14 ovine fetuses by resection of the 7th and 8th left ribs. Each non-operated twin fetus was taken as control. Respiratory mechanics was studied postnatally in the first week and at 1, 2, and 3 months. Post-mortem respiratory mechanics and lung histomorphometry were also assessed at 3 months. Results: Eight out of 14 CWD lambs (57%) and 14 control lambs survived the postnatal period. One severe and five mild deformities were induced. At birth, inspiratory capacity (25 vs. 32 mL/kg in controls), and dynamic (1.4 vs. 1.8 mL/cmH2O/kg), and static (2.0 vs. 2.5 mL/cmH2O/kg) respiratory system compliances were decreased in CWD lambs. Apart from a slight decrease in inspiratory capacity at 1 month of life, no other differences were observed in respiratory mechanics measured in vivo thereafter. Postmortem measurements found a significant decrease in lung compliance-for each lung and for both lungs taken together-in CWD lambs. No differences in lung histology were detected at 3 months in CWD animals compared to controls. Conclusions: Our study is the first to assess the effects of a prenatally induced CWD on lung development and function from birth to 3 months in an ovine model. Our results show no significant differences in lung histomorphometry at 3 months in CWD lambs compared to controls. Resolution at 1 month of the alterations in respiratory mechanics present at birth may be related to the challenge in inducing severe deformities.

Role of gender in morbidity and mortality of extremely premature neonates.

We investigated the effect of gender on survival and short-term outcomes of extremely premature infants (≤27 weeks) born in Canada. The records of infants admitted between 2000 and 2005 to a neonatal intensive care unit participating in the Canadian Neonatal Network were reviewed for infant gender, birth weight, gestational age, outborn status, Score for Neonatal Acute Physiology II, and antenatal corticosteroid exposure. The following outcomes were recorded: survival at final discharge, necrotizing enterocolitis, bronchopulmonary dysplasia (BPD), intraventricular hemorrhage grade ≥3, retinopathy grade ≥3, days on ventilation, and length of hospital stay. Among 2744 extremely premature infants, 1480 (54%) were male and 1264 (46%) were female. Mean birth weight of female neonates was significantly lower at each week of gestational age. Although no significant difference in survival at discharge was found between genders overall, the prevalence of BPD, combined adverse outcomes, and mortality for infants born between 24 and 26 weeks were significantly higher in males. This study suggests that, in the postsurfactant era, males remain at higher risk of respiratory complications and may have higher mortality when born between 24 and 26 weeks of gestation.

Promoting the "3Rs" principle in developmental biology with early and convenient diagnosis of pregnancy in mice.

The 3Rs stand for Replacement of animals in experiments, Reduction in the number of experimental animals, and Refinement of experiments to minimize animal pain and stress. We propose to address Reduction and Refinement in the use of mice as experimental models in developmental research. This study focuses on the maternal percentage of weight increase at gestational day 8 (%WI(GD8)) to diagnose pregnancy early in BALB/c mice. We documented sensitivity, specificity, false positive and negative rates and probability of pregnancy associated with %WI(GD8). This predictive model of pregnancy allows for significant reduction in the number of mice to be sacrificed in developmental research. Reported observations and literature suggest that this model is independent of litter size and should be applicable to other mice strains. This procedure allows mice pregnancy detection before midgestation and proposes an ethically sound approach to experimental animal use by optimizing the number of mice used and refining animal manipulation.

Role of fetal sex in the outcome of antenatal glucocorticoid treatment to prevent respiratory distress syndrome: systematic review and meta-analysis.

BACKGROUND: Antenatal glucocorticoid (AGC) therapy has been associated with a decrease in respiratory distress syndrome (RDS). While preterm males remain at greater risk of RDS than females, the role of fetal sex in AGC response is not well known. OBJECTIVES: To review the available evidence regarding the effect of fetal sex in the prevention of RDS using AGC. METHOD: We conducted a systematic review and meta-analysis of RCTs to compare the effect of AGC in male and female infants with regard to the rates of RDS, intra-ventricular hemorrhage (IVH) grades III and IV, and neonatal mortality. Random effects with 95% confidence intervals were assessed in both groups and relative risks were compared using mixed regression. RESULTS: From 248 potentially eligible articles, we included eight in the analysis for a total of 1109 male and 968 female infants. Both male and female infants had a significant decrease in the risks, but no difference between the sexes was observed in terms of reduction in RDS (RR 0.50; 95% CI 0.33 to 0.77 for males, and RR 0.57; 95% CI 0.43 to 0.75 for females, P = 0.99), reduction in IVH (P = 0.98), and reduction in neonatal mortality (P = 0.43). In a sub-analysis, use of betamethasone was associated with a significant decrease in the rate of RDS in males (RR 0.29; 95% CI 0.15 to 0.57) but dexamethasone was not (RR 0.78; 95% CI 0.57 to 1.07). Conversely, dexamethasone use was significantly helpful in females (RR 0.51; 95% CI 0.32 to 0.81) but betamethasone was not (RR 0.62; 95% CI 0.38 to 1.00). CONCLUSION: The effect of AGC for prevention of RDS is similar in females and males. However, futures studies should investigate the type of AGC according to fetal/neonatal sex.

[Words from yesterday and today's pandemic: what medicine after COVID?] / Mots d'hier et pandémie d'aujourd'hui : quelle médecine après la crise du Covid ?

Words from yesterday and today's pandemic: what medicine after covid? The covid-19 pandemic is a new phenomenon, unknown to our modern society, which has features in common with the plagues of history. We will try to answer two questions. Is the covid pandemic a crisis? How will the pandemic change our approach to health? The pandemic has generated a crisis, that is to say an event en¬dowed with a power of shock that goes beyond its integration into a causal series, an event with health, political, economic, and cultural consequences. To respond to this, our health establishments, our faculties, and all health players will have to work together to review their practices. Ethical choices will be necessary to find equitable solutions. These choices and actions have in common that they are conceived in an interdisciplinary reflection. We address some aspects: science and research, information and communica¬tion, ethics, health governance, patient participation.

Mots d'hier et pandémie d'aujourd'hui : quelle médecine après la crise du covid ? La pandémie de covid-19 est un phénomène nouveau pour notre société moderne mais qui possède des traits communs avec des fléaux de l'histoire. Cette pandémie est-elle une crise ? En quoi devrait-elle modifier notre approche de la santé ? La pandémie a généré une crise, c'est-à-dire un événement doté d'une puissance d'ébranlement qui dépasse son intégration dans une série causale, un événement aux conséquences sanitaires, politiques, économiques et culturelles. Pour la résoudre, nos éta¬blissements de santé, nos facultés, tous les acteurs de santé devront travailler ensemble et revoir leurs pratiques. Des choix éthiques s'imposeront pour trouver des solutions équitables. Ces choix et ces actions ont en commun de se concevoir dans une réflexion interdisciplinaire, dans les domaines tels que : science et recherche, information et communication, éthique, gouvernance sanitaire ou encore participation des patients.

Apolipoprotein C-II and lipoprotein lipase show a temporal and geographic correlation with surfactant lipid synthesis in preparation for birth.

BACKGROUND: Fatty acids are precursors in the synthesis of surfactant phospholipids. Recently, we showed expression of apolipoprotein C-II (apoC-II), the essential cofactor of lipoprotein lipase (LPL), in the fetal mouse lung and found the protein on the day of the surge of surfactant synthesis (gestation day 17.5) in secretory granule-like structures in the distal epithelium. In the present study, we will answer the following questions: Does apoC-II protein localization change according to the stage of lung development, thus according to the need in surfactant? Are LPL molecules translocated to the luminal surface of capillaries? Do the sites of apoC-II and LPL gene expression change according to the stage of lung development and to protein localization? RESULTS: The present study investigated whether the sites of apoC-II and LPL mRNA and protein accumulation are regulated in the mouse lung between gestation day 15 and postnatal day 10. The major sites of apoC-II and LPL gene expression changed over time and were found mainly in the distal epithelium at the end of gestation but not after birth. Accumulation of apoC-II in secretory granule-like structures was not systematically observed, but was found in the distal epithelium only at the end of gestation and soon after birth, mainly in epithelia with no or small lumina. A noticeable increase in surfactant lipid content was measured before the end of gestation day 18, which correlates temporally with the presence of apoC-II in secretory granules in distal epithelium with no or small lumina but not with large lumina. LPL was detected in capillaries at all the developmental times studied. CONCLUSIONS: This study demonstrates that apoC-II and LPL mRNAs correlate temporally and geographically with surfactant lipid synthesis in preparation for birth and suggests that fatty acid recruitment from the circulation by apoC-II-activated LPL is regionally modulated by apoC-II secretion. We propose a model where apoC-II is retained in secretory granules in distal epithelial cells until the lumina reaches a minimum size, and is then secreted when the rate of surfactant production becomes optimal.

Expression of genes related to the hypothalamic-pituitary-adrenal axis in murine fetal lungs in late gestation.

BACKGROUND: Lung maturation is modulated by several factors, including glucocorticoids. Expression of hypothalamic-pituitary-adrenal (HPA) axis-related components, with proposed or described local regulatory systems analogous to the HPA axis, was reported in peripheral tissues. Here, HPA axis-related genes were studied in the mouse developing lung during a period overlapping the surge of surfactant production. METHODS: Expression of genes encoding for corticotropin-releasing hormone (CRH), CRH receptors (CRHR) 1 and 2beta, CRH-binding protein, proopiomelanocortin (POMC), melanocortin receptor 2 (MC2R), and glucocorticoid receptor was quantified by real-time PCR and localized by in situ hydridization in fetal lungs at gestational days (GD) 15.5, 16.5, and 17.5, and was also quantified in primary mesenchymal- and epithelial cell-enriched cultures. In addition, the capability of CRH and adrenocorticotropic hormone (ACTH) to stimulate pulmonary expression of enzymes involved in the adrenal pathway of glucocorticoid synthesis was addressed, as well as the glucocorticoid production by fetal lung explants. RESULTS: We report that all the studied genes are expressed in fetal lungs according to different patterns. On GD 15.5, Mc2r showed peaks in expression in samples that have previously presented high mRNA levels for glucocorticoid synthesizing enzymes, including 11beta-hydroxylase (Cyp11b1). Crhr1 mRNA co-localized with Pomc mRNA in cells surrounding the proximal epithelium on GD 15.5 and 16.5. A transition in expression sites toward distal epithelial cells was observed between GD 15.5 and 17.5 for all the studied genes. CRH or ACTH stimulation of genes involved in the adrenal pathway of glucocorticoid synthesis was not observed in lung explants on GD 15.5, whereas CRH significantly increased expression of 21-hydroxylase (Cyp21a1) on GD 17.5. A deoxycorticosterone production by fetal lung explants was observed. CONCLUSIONS: Temporal and spatial modulations of expression of HPA axis-related genes in late gestation are consistent with roles for these genes in lung development. Our data are likely to lead to valuable insights in relation to lung diseases originating from lung immaturity.

Gene expression profile of androgen modulated genes in the murine fetal developing lung.

BACKGROUND: Accumulating evidences suggest that sex affects lung development. Indeed, a higher incidence of respiratory distress syndrome is observed in male compared to female preterm neonates at comparable developmental stage and experimental studies demonstrated an androgen-related delay in male lung maturation. However, the precise mechanisms underlying these deleterious effects of androgens in lung maturation are only partially understood. METHODS: To build up a better understanding of the effect of androgens on lung development, we analyzed by microarrays the expression of genes showing a sexual difference and those modulated by androgens. Lungs of murine fetuses resulting from a timely mating window of 1 hour were studied at gestational day 17 (GD17) and GD18, corresponding to the period of surge of surfactant production. Using injections of the antiandrogen flutamide to pregnant mice, we hunted for genes in fetal lungs which are transcriptionally modulated by androgens. RESULTS: Results revealed that 1844 genes were expressed with a sexual difference at GD17 and 833 at GD18. Many genes were significantly modulated by flutamide: 1597 at GD17 and 1775 at GD18. Datasets were analyzed by using in silico tools for reconstruction of cellular pathways. Between GD17 and GD18, male lungs showed an intensive transcriptional activity of proliferative pathways along with the onset of lung differentiation. Among the genes showing a sex difference or an antiandrogen modulation of their expression, we specifically identified androgen receptor interacting genes, surfactant related genes in particularly those involved in the pathway leading to phospholipid synthesis, and several genes of lung development regulator pathways. Among these latter, some genes related to Shh, FGF, TGF-beta, BMP, and Wnt signaling are modulated by sex and/or antiandrogen treatment. CONCLUSION: Our results show clearly that there is a real delay in lung maturation between male and female in this period, the latter pursuing already lung maturation while the proper is not yet fully engaged in the differentiation processes at GD17. In addition, this study provides a list of genes which are under the control of androgens within the lung at the moment of surge of surfactant production in murine fetal lung.

Levels of dihydrotestosterone, testosterone, androstenedione, and estradiol in canalicular, saccular, and alveolar mouse lungs.

Androgens and estrogens are known regulators of fetal and postnatal lung development, but their levels in the developing lung have never been determined. We present here, for the first time, a gas chromatography-mass spectrometry (GC/MS) quantification of dihydrotestosterone, testosterone, androstenedione, and estradiol in canalicular, saccular, and alveolar stage lungs of both sexes. Testosterone, androstenedione, and estradiol were observed in all the analyzed lung samples from gestation day (GD) 16.5 to postnatal day (PN) 30, totalizing 383 individual mice. Levels of these three steroids decreased between birth and PN 5. In contrast, dihydrotestosterone was detected only in male samples on GD 19.5, PN 0, and PN 30. A significant sex difference was observed for testosterone and androstenedione but not for estradiol. Steroid levels were also determined in skinned hind legs for comparison. Three-way analysis of variance revealed that tissue (lung or leg) had a significant effect on testosterone levels for both sexes, but not on androstenedione and estradiol levels. Low but significant testosterone and androstenedione levels were observed in all the females and in prepubertal male samples. These levels must be sufficient to induce androgen receptor activation, as suggested by our recent report showing the presence of androgen receptor in the nucleus of several lung cells in corresponding developmental ages and sexes.

Steroid-converting enzymes in human ovarian carcinomas.

Anti-estrogen therapies for treating ovarian carcinoma have had mixed outcomes suggesting some tumors may be estrogen-dependent. We assayed the activity levels of 17beta-hydroxysteroid dehydrogenase (17beta-HSD), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD/3-KSR) and estrone sulfatase in a series of ovarian epithelial carcinomas. 17beta-HSD activity ratios with estradiol (E(2)) and testosterone (T), and inhibition by isoform-specific inhibitors were used to estimate the contributions of 17beta-HSD isoforms. Activity levels were highest for estrone sulfatase, followed, respectively by 17beta-HSD, 3alpha-HSD/3-KSR, and 3beta-HSD. E(2)/T activity ratios varied widely between samples. A 17beta-HSD type 1 inhibition pattern was observed in 23% of the samples and a type 2 pattern in 25%. E(2) formation from estrone sulfate (E(1)S) was detected in 98% (47/48) of the samples. 17beta-HSD type 1, type 2 and type 5 mRNA was detected in matched primary tumor and metastases. Evaluation of 17beta-HSD and sulfatase activity levels, activity ratios and inhibition patterns may help predict tumor response to endocrine therapy.

Role of transforming growth factor-beta1 in gene expression and activity of estradiol and progesterone-generating enzymes in FSH-stimulated bovine granulosa cells.

Survival and inhibitory factors regulate steroidogenesis and determine the fate of developing follicles. The objective of this study was to determine the role of transforming growth factor-beta1 (TGFB1) in the regulation of estradiol-17beta (E(2)) and progesterone (P(4)) secretion in FSH-stimulated bovine granulosa cells. Granulosa cells were obtained from 2 to 5 mm follicles and cultured in serum-free medium. FSH dose (1 and 10 ng/ml for 6 days) and time in culture (2, 4, and 6 days with 1 ng/ml FSH) increased E(2) secretion and mRNA expression of E(2)-related enzymes cytochrome P450 aromatase (CYP19A1) and 17beta-hydroxysteroid dehydrogenase type 1 (HSD17B1), but not HSD17B7. TGFB1 in the presence of FSH (1 ng/ml) inhibited E(2) secretion, and decreased mRNA expression of FSH receptor (FSHR), CYP19A1, and HSD17B1, but not HSD17B7. FSH dose did not affect P(4) secretion and mRNA expression of 3beta-hydroxysteroid dehydrogenase (HSD3B) and alpha-glutathione S-transferase (GSTA), but inhibited the amount of steroidogenic acute regulatory protein (STAR) mRNA. Conversely, P(4) and mRNA expression of STAR, cytochrome P450 side-chain cleavage (CYP11A1), HSD3B, and GSTA increased with time in culture. TGFB1 inhibited P(4) secretion and decreased mRNA expression of STAR, CYP11A1, HSD3B, and GSTA. TGFB1 modified the formation of granulosa cell clumps and reduced total cell protein. Finally, TGFB1 decreased conversion of androgens to E(2), but did not decrease the conversion of estrone (E(1)) to E(2) and pregnenolone to P(4). Overall, these results indicate that TGFB1 counteracts stimulation of E(2) and P(4) synthesis in granulosa cells by inhibiting key enzymes involved in the conversion of androgens to E(2) and cholesterol to P(4) without shutting down HSD17B reducing activity and HSD3B activity.

Essential Intracrine Androgenic Action in Lung Development for Both Sexes.

Albeit their recognized negative effects on lung maturation, androgens have been proposed to play an essential positive role in lung development. This work aimed to evaluate the impact of blocking endogenous androgen and estrogen actions and to study the effect of an excess of androgen and estrogen during the end of saccular stage and the beginning of the alveolar stage on lung development. This was performed with normal oxygen atmosphere and with hyperoxia, a model of alveolar simplification, which is observed in new bronchopulmonary dysplasia. Mouse lung samples were collected on postnatal day 9 after exposure to 21% or 80% oxygen (postnatal days 1 to 4), and after administration (postnatal days 3 to 8) of vehicle, pure antiandrogen (flutamide), dihydrotestosterone, pure antiestrogen (fulvestrant), or 17ß-estradiol. With 21% oxygen, the major effects on morphometric parameters were induced by flutamide. In contrast, with hyperoxia, both flutamide and dihydrotestosterone had similar effects on several morphometric parameters. For instance, a decrease in the relative frequency of closed areas (mainly composed of saccules/alveoli) < 1000 µm2 and an increase for those > 2500 µm2 were observed after flutamide administration. In conclusion, during the junction between the saccular and the alveolar stages, endogenous androgens play an essential intracrine role in lung development for both sexes while an excess of androgens are deleterious when combined with a hyperoxia treatment, but not with normal oxygen levels. Endogenous estrogens have no effects on the lungs during the developmental window studied, while exogenous estrogens had only isolated effects on some morphometric parameters.

Human type 2 17 beta-hydroxysteroid dehydrogenase mRNA and protein distribution in placental villi at mid and term pregnancy.

BACKGROUND: During human pregnancy, the placental villi produces high amounts of estradiol. This steroid is secreted by the syncytium, which is directly in contact with maternal blood. Estradiol has to cross placental foetal vessels to reach foetal circulation. The enzyme 17beta-hydroxysteroid dehydrogenase type 2 (17beta-HSD2) was detected in placental endothelial cells of foetal vessels inside the villi. This enzyme catalyzes the conversion of estradiol to estrone, and of testosterone to androstenedione. It was proposed that estradiol level into foetal circulation could be regulated by 17beta-HSD2. METHODS: We obtained placentas from 10 to 26 6/7 weeks of pregnancy from women undergoing voluntary termination of pregnancy, term placentas were collected after normal spontaneous vaginal deliveries. We quantified 17beta-HSD2 mRNA levels in mid-gestation and term human placenta by RT-QPCR. We produced a new anti-17beta-HSD2 antibody to study its spatio-temporal expression by immunohistochemistry. We also compared steroid levels (testosterone, estrone and estradiol) and 17beta-HSD2 mRNA and protein levels between term placenta and endometrium. RESULTS: High 17beta-HSD2 mRNA and protein levels were found in both mid-gestation and term placentas. However, we showed that 17beta-HSD2 mRNA levels increase by 2.27 fold between mid-gestation and term. This period coincides with a transitional phase in the development of the villous vasculature. In mid-gestation placenta, high levels of 17beta-HSD2 were found in mesenchymal villi and immature intermediate villi, more precisely in endothelial cells of the stromal channel. At term, high levels of 17beta-HSD2 were found in the numerous sinusoidal capillaries of terminal villi. 17beta-HSD2 mRNA and protein levels in term placentas were respectively 25.4 fold and 30 to 60 fold higher than in the endometrium. Steroid levels were also significantly higher in term placenta than in the endometrium. CONCLUSION: The spatial and temporal expression of 17beta-HSD2 in the placenta during pregnancy and the comparison of 17beta-HSD2 expression and steroid levels between placental villi and endometrium are compatible with a role in the modulation of active and inactive forms of estrogens. Our observations strongly support the hypothesis that 17beta-HSD2 acts as a barrier decreasing estradiol secretion rates in the foetal circulation.

Mouse 3alpha-hydroxysteroid dehydrogenase mRNA: a marker of lung maturity.

Lung maturation is delayed in male fetuses compared to females and androgens are responsible of this delay. On the other hand, a normal role was proposed for androgens in the developing lung based on a correlation between expression of type 5 17beta-hydroxysteroid dehydrogenase (HSD), which catalyzes testosterone synthesis, and the emergence of mature type II pneumonocytes, a developmental event associated with the surge of surfactant synthesis. All these observations underline the importance of the metabolism of androgens in the developing lung. Here, we report a study on the expression of genes involved in the metabolism of the most potent androgen, 5alpha-dihydrotestosterone, in the mouse fetal lung between gestation days 15.5 and 18.5. Synthesis and inactivation of 5alpha-dihydrotestosterone occur through 5alpha-reductase and 3alpha-HSD activities, respectively. Type 1 5alpha-reductase was expressed throughout the gestation time window analyzed at fairly constant levels with no gender difference, except that a slight decrease was observed on gestation day 18.5. In contrast, expression of m3alpha-HSD presented a marked increase on gestation day 17.5, when the maturation of type II pneumonocytes occurs, and followed its progression at least until gestation day 18.5. In conclusion, our data show that m3alpha-HSD mRNA is a reliable marker of lung maturity in normal pregnancy.

Dynamic modulation of Cyp21a1 (21-hydroxylase) expression sites in the mouse developing lung.

21-hydroxylase is expressed in the developing lung where it is proposed as a local source of glucocorticoids playing important roles in lung development. We have studied the precise sites of Cyp21a1 expression in the developing mouse lung from the pseudoglandular stage (gestation day (GD) 15.5) to the alveolar stage (postnatal day (PND) 15) by in situ hybridization. Cyp21a1-mRNA was found mainly in epithelial cells from GD 15.5 to PND 5, but the precise site of expression shifted from the distal epithelium during the pseudoglandular and the canalicular stages including the distal epithelium without lumina, to the proximal epithelium and the wall of developing saccules during the perinatal period (GD 19.5 and PND 0), and to the wall of developing saccules and septa, most probably in type I pneumonocytes (PTI), on PND 5. Cyp21a1 expression changed from PTI cells to capillary endothelial cells of the same distal structures during alveolarization. The mesenchyme was generally negative. Endothelial cells forming large vessels were negative. However the tunica adventitia surrounding arteries was Cyp21a1-positive, while several veins were surrounded by a Cyp21a1-positive layer. In conclusion, Cyp21a1 remains expressed in the most distal structure of the developing lung even though these structures are changing, but its expression is not restricted to these areas. Taken together, our data show the highly dynamic modulation of Cyp21a1 expression sites, consistent with the evolving structures of the developing lung.

17Beta-HSD type 5 expression and the emergence of differentiated epithelial Type II cells in fetal lung: a novel role for androgen during the surge of surfactant.

Lung maturation is delayed in male fetuses compared to female fetuses. This has been attributed to higher levels of androgens in the male lung. We previously showed that the genes encoding for the 17beta-hydroxysteroid dehydrogenase (HSD) type 5 (conversion of androstenedione in testosterone) and type 2 (the opposite reaction) are, respectively, expressed in the human epithelial Type II (PTII)-like A549 cells and in human lung fibroblasts. Here, we aim to explain the physiological relevance of androgen synthesis by PTII cells. We showed that 17beta-HSD type 2 and type 5 genes are both up-regulated in correlation with the emergence of mature PTII cells in both male and female developing lungs of the fetal mouse. In contrast, the androgen receptor gene is expressed at similar levels in both sexes with no temporal regulation. In conclusion, the expression profile of the 17beta-HSD type 5 gene does not explain the presence of higher levels of androgens in the male fetal lung but that androgen synthesis must be a normal characteristic of mature PTII cells for both sexes. The production of androgens after the emergence of mature PTII cells should negatively regulate PTII cell maturation and thus, a novel and normal role for androgens in cell reprogramming is proposed.

Sexually dimorphic gene expression that overlaps maturation of type II pneumonocytes in fetal mouse lungs.

BACKGROUND: In human, respiratory distress of the neonates, which occurs in prematurity, is prevalent in male. Late in gestation, maturation of type II pneumonocytes, and consequently the surge of surfactant synthesis are delayed in male fetuses compared with female fetuses. Although the presence of higher levels of androgens in male fetuses is thought to explain this sex difference, the identity of genes involved in lung maturation that are differentially modulated according to fetal sex is unknown. We have studied the sex difference in developing mouse lung by gene profiling during a three-day gestational window preceding and including the emergence of mature PTII cells (the surge of surfactant synthesis in the mouse occurs on GD 17.5). METHODS: Total RNA was extracted from lungs of male and female fetal mice (gestation days 15.5, 16.5, and 17.5), converted to cRNA, labeled with biotin, and hybridized to oligonucleotide microarrays (Affymetrix MOE430A). Analysis of data was performed using MAS5.0, LFCM and Genesis softwares. RESULTS: Many genes involved in lung maturation were expressed with no sex difference. Of the approximative 14,000 transcripts covered by the arrays, only 83 genes presented a sex difference at one or more time points between GDs 15.5 and 17.5. They include genes involved in hormone metabolism and regulation (i.e. steroidogenesis pathways), apoptosis, signal transduction, transcriptional regulation, and lipid metabolism with four apolipoprotein genes. Genes involved in immune functions and other metabolisms also displayed a sex difference. CONCLUSION: Among these sexually dimorphic genes, some may be candidates for a role in lung maturation. Indeed, on GD 17.5, the sex difference in surfactant lipids correlates with the sex difference in pulmonary expression of apolipoprotein genes, which are involved in lipid transport. This suggests a role for these genes in the surge of surfactant synthesis. Our results would help to identify novel genes involved in the physiopathology of the respiratory distress of the neonates.

CYP21A2 expression is localized in the developing distal epithelium of the human perinatal lung and is compatible with in situ production and intracrine actions of active glucocorticoids.

Glucocorticoids play essential roles in lung development. We investigated for expression of CYP21A2 (21-hydroxylase) as well as for the presence of the corresponding protein and identification of CYP21A2-expressing cells in several human developing lungs. Expression of some related genes was also assessed. CYP21A2 and CYP17A1 (P450c17) mRNAs were found in all the 34 lung samples from 17 to 40 weeks' gestation at variable levels. No correlation was found according to sex but a correlation with age was detected for CYP17A1 only. In contrast, CYP11B1 (11ß-hydroxylase)- and CYP11B2 (aldosterone synthase)-mRNAs were not detected. Significant levels of the CYP21A2 protein were detected in all the analyzed samples, while only very low signals were detected for CYP17A1 protein. In situ hybridization revealed that CYP21A2 was almost exclusively expressed in the distal epithelium. It was reported that the lung distal epithelium of human fetuses also express 11ß-hydroxysteroid dehydrogenase type 2, which catalyzes cortisol inactivation into cortisone. Based on this information, intracrine glucocorticoid actions should take place from CYP21A2 products through the glucocorticoid receptor in the absence of cortisol. In contrast, mineralocorticoid receptor activation did not seem to depend on deoxycorticosterone produced from local activity of CYP21A2 because of the reported circulating amounts of aldosterone.