Eric Boucher

Apolipoprotein A-I, A-II, C-II, and H expression in the developing lung and sex difference in surfactant lipids

A sex difference in surfactant lipids is associated with a higher incidence of respiratory distress syndrome for males in cases of preterm birth. In animal models, the sex difference in surfactant lipids was shown to be androgen receptor-dependent. This report examines expression of apolipoprotein (apo)A-I, apoA-II, apoC-II, apoE, apoH, and lipoprotein lipase (LPL) by quantitative real-time PCR in pools of male and female fetal lung tissues from various mouse litters from gestation day (GD) 15.5 to 18.5, and in various adult tissues. Although the expression profiles of ApoA-I, ApoA-II, ApoC-II, and ApoH are complex, these genes are co-regulated and they all present a sex difference (P=0.0896, 0.0896, 0.0195, and 0.0607 respectively) with higher expression for females for several litters. Pulmonary expression of apoA-I, apoA-II, and apoH were specific to the developing lung. ApoE and LPL mRNAs showed a significant increase from GD 17.5 to 18.5. An increase in apoA-I-, apoA-II-, apoC-II-, and apoH-mRNA accumulation was observed from GD 16.5 to 17.5 in correlation with the emergence of mature type II pneumonocytes. These four apolipoprotein genes are co-regulated with type 2 and 5 17beta-hydroxysteroid dehydrogenases, which are respectively involved in inactivation and synthesis of androgens. Finally, apoC-II was detected by immunohistochemistry in epithelial cells of the distal epithelium. Positive signals looking like secretory granules were located near the basal membrane. Our results are compatible with a role for apolipoproteins in lipid metabolism and transport in the developing lung in association with the sex difference in surfactant lipid synthesis.

Androgen receptor and 17β-HSD type 2 regulation in neonatal mouse lung development

A QPCR analysis of androgen receptor and several androgen metabolizing genes was performed during the saccular and alveolar stages of mouse lung development. Androgen receptor expression showed a statistically significant increase during the alveolar stage while levels of 17beta-hydroxysteroid dehydrogenase type 2 (17beta-HSD 2) expression significantly decreased at the end of the saccular stage and remained low throughout the alveolar period. 17beta-HSD 1, 17beta-HSD 5, 5alpha-reductase type 1, and mouse 3alpha-HSD did not present such a regulation. The androgen receptor protein was primarily detected in the nucleus of airway epithelial cells and of a subset of respiratory epithelial cells. 17beta-HSD 2 mRNA co-localized with androgen receptor protein during saccularization, but was absent from airway epithelium during alveolarization. Taken together, our results demonstrate temporal and spatial regulation of androgen receptor and 17beta-HSD 2 during the sacculo-alveolar transition period of mouse lung development suggesting control of androgen action.

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.

Ontogeny of adrenal-like glucocorticoid synthesis pathway and of 20α-hydroxysteroid dehydrogenase in the mouse lung

BackgroundGlucocorticoids exert recognized positive effects on lung development. The genes involved in the classical pathway of glucocorticoid synthesis normally occurring in adrenals were found to be expressed on gestation day (GD) 15.5 in the developing mouse lung. Recently, expression of two of these genes was also detected on GD 17.5 suggesting a more complex temporal regulation than previously expected. Here, we deepen the knowledge on expression of “adrenal” glucocorticoid synthesis genes in the mouse lung during the perinatal period and we also study expression of the gene encoding for the steroid inactivating enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD).ResultsWe performed an ontogenic study of P450scc, 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase 1 (3β-HSD1), 21-hydroxylase, 11β-hydroxylase, 11β-HSD1, and 11β-HSD2 expression up to post natal day (PN) 15. The substrate (progesterone) and the product (deoxycorticosterone) of 21-hydroxylase are substrates of 20α-HSD, thus 20α-HSD (Akr1c18) gene expression was investigated. In lung samples collected between GD 15.5 and PN 15, 11β-hydroxylase was only detected on GD 15.5. In contrast, all the other tested genes were expressed throughout the analyzed period with different temporal expression patterns. P450scc, 21-hydroxylase, 20α-HSD and 11β-HSD2 mRNA levels increased after birth with different patterns including an increase from PN 3 with a possible sex difference for 21-hydroxylase mRNA. Also, the 21-hydroxylase protein was observed by Western blot in perinatal lungs with higher levels after birth.ConclusionProgesterone is present at high levels during gestation and the product of 21-hydroxylase, deoxycorticosterone, can bind the glucocorticoid receptor with an affinity close to that of corticosterone. Detection of 21-hydroxylase at the protein level during antenatal lung development is the first evidence that the adrenal-like glucocorticoid synthesis pathway detected during lung development has the machinery to produce glucocorticoids in the fetal lung. Glucocorticoids from lung 21-hydroxylase appear to modulate lung ontogenesis through paracrine/intracrine actions.

Sex-specific perinatal expression of glutathione peroxidases during mouse lung development.

Reports indicate that antioxidant enzymes like the glutathione peroxidases (GPx) can be regulated by sex steroids. The GPx, a major class of antioxidants involved in H(2)O(2) and lipid hydroperoxides neutralization, showed an age- and sex-specific expression in many adult organs including the lung. High levels of androgens in the male lung are known to delay the surge of surfactant synthesis during gestation in several species. However, the impact of male androgens on antioxidant GPx early in life remains to be determined. The objective was to study the lung sex-specific expression of GPx during BALB/c mouse perinatal development. The mRNA expression of four seleno-dependent Gpx (Gpx1 to 4) in the lung of both sexes was characterized by real-time PCR from gestational day 15 to postnatal day 30, covering the entire canalicular, saccular and alveolar stages. Immunohistochemistry of GPx-1, -3 and -4, and seleno-dependent GPx enzymatic assays were also performed in the lung. We found a transient lower Gpx1 mRNA level in male than in female lungs during the first 5 days after birth, corresponding to the saccular phase. This dimorphic expression was concomitant to a sex difference in GPx enzymatic activity corrected for blood. It is, to our knowledge, the first report of a sex dimorphism for murine lung enzymatic antioxidant defenses during the perinatal period.