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Published ahead of print on April 29, 2004, doi:10.1164/rccm.200312-1680OC
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American Journal of Respiratory and Critical Care Medicine Vol 170. pp. 296-305, (2004)
© 2004 American Thoracic Society

Original Article

A Link between Lung Androgen Metabolism and the Emergence of Mature Epithelial Type II Cells

Pierre R. Provost, Marc Simard and Yves Tremblay

Laboratory of Ontogeny and Reproduction, Department of Obstetrics and Gynecology, and Centre de Recherche en Biologie de la Reproduction, Faculty of Medicine, Laval University, Quebec, Quebec, Canada

Correspondence and reprint requests should be addressed to Dr. Yves Tremblay, Ph.D., Ontogeny and Reproduction, Room T-1-58, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, 2705 Laurier Boul, G1V 4G2, Quebec, Quebec, Canada. E-mail: yves.tremblay{at}crchul.ulaval.ca


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lung maturation is delayed in male fetuses compared with female fetuses, which has been attributed to higher levels of androgens in the male lung. Our previous studies demonstrated that the genes encoding for the 17ß-hydroxysteroid dehydrogenase (17ß-HSD) type 5 (androstenedione -> testosterone) and type 2 (the opposite reaction) are expressed in human epithelial type II (PTII)–like A549 cells and in human lung fibroblasts, respectively. Here, we aim to explain the physiological relevance of androgen synthesis by PTII cells. We showed that both 17ß-HSD type 2 and type 5 genes are upregulated in correlation with the emergence of mature PTII cells in both male and female developing lungs of the mouse. In contrast, the androgen receptor gene is expressed equally in both sexes with no temporal regulation. We conclude that the expression profile of the 17ß-HSD type 5 gene does not explain the presence of higher levels of androgen in the male fetal lung, but that androgen synthesis must be a normal feature 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 role for androgens in cell reprogramming is suggested.

Key Words: androgen receptor, 17ß-hydroxysteroid dehydrogenases • hyaline membrane disease • lung maturation • real-time polymerase chain reaction

A prevalence for male delayed lung maturation has been observed in respiratory distress of the neonate (or hyaline membrane disease) (1–5). The major cause of this pathology is surfactant deficiency, a situation occurring when type II pneumonocyte (PTII) cell maturation is not completed at the time of delivery.

PTII cell maturation occurs late in pregnancy. PTII-fibroblast cell communication includes positive and negative regulators that control PTII cell maturation, which ends by the synthesis of surfactant by these epithelial cells (6). Some cytokines, including epidermal growth factor (EGF) (7, 8), neuregulin-1 (9), and keratinocyte growth factor (10), stimulate the production of surfactant components, whereas others, such as transforming growth factor–ß1 (TGF-ß1) (7, 11–13), downregulate surfactant production (14). Among steroid hormones, glucocorticoids accelerate the maturation process (15), whereas sex steroids have opposing effects with estrogens accelerating and androgens inhibiting lung maturation at comparable developmental time points (16, 17).

Androgens are particularly of interest because we know that PTII cell maturation is delayed in the male compared with the female (18). Androgens have been shown to delay fetal lung maturation both in vitro (19) and in vivo (1, 20). In addition, administration of the antiandrogen, flutamide, eliminated the sex difference in surfactant production by increasing the male surfactant level to that of the females (20). This effect of androgens depends on the presence of the androgen receptor, which has been detected in both human (21) and rabbit (22) fetal lung tissues. Moreover, in the mouse model of testicular feminization (Tfm mouse) in which male carriers of the X-linked Tfm mutation have no functional androgen receptors, the surfactant level of the Tfm male fetuses is the same as the females (1). Therefore, the sex difference in PTII cell maturation should be linked to higher levels of androgens in the male developing lung. In vitro experiments have suggested that the primary target of androgens might be lung fibroblasts (19).

We have studied the expression of genes related to androgen metabolism in human lung cell lines. We have demonstrated that the epithelial-like A549 cells isolated from a human male subject synthesize testosterone via the expression of 17ß-hydroxysteroid dehydrogenase (HSD) type 5 (23) (a general pathway of androgen metabolism in peripheral tissues is shown in Figure 1) . In these A549 cells, 5{alpha}-dihydrotestosterone (5 {alpha}-DHT), the most potent androgen, is formed by 5{alpha}-reductase type 1, but is rapidly converted into 5{alpha}-androstan-3{alpha},17ß-diol by the 3{alpha}-HSD type 3. Therefore, testosterone is the main active androgen secreted by these cells and it can have a paracrine androgenic action on fibroblasts. Next, we demonstrated that human lung fibroblasts inactivate testosterone into androstenedione via the expression of 17ß-HSD type 2 (24). This result is interesting considering that the role of fibroblasts in PTII cell maturation is androgen sensitive.



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  		Figure 1. General pathway of androgen metabolism in peripheral tissues. 17ß-hydroxysteroid dehydrogenase (HSD), 3ß-HSD, 5{alpha}-reductase, and 3{alpha}-HSD activities are encoded by more than one gene, each being tissue- and cell-type specific. The 17ß-HSD activity is not actually in equilibrium. Although both directions are possible, each isoform catalyzes predominantly one reaction.

 
Because androgens are negative regulators of PTII cell maturation, the physiologic reason of androgen synthesis by PTII cells is unclear. Aiming to find the physiologic relevance of androgen synthesis by PTII cells in the developing lung, we performed an ontogeny study of 17ß-HSD type 2, type 3, and type 5, and androgen receptor gene expression in male and female mice in a gestation-time window overlapping the emergence of mature PTII cells (gestation days [GDs] 15.5, 16.5, 17.5, and 18.5 [term, GD 19]). A sex difference in surfactant production was previously reported in the mouse on GD 17.5 (1). The mouse was selected because, like the human, it expresses the 17ß-HSD type 5 gene (25). Other laboratory animal models, including the rat, have no identified 17ß-HSD type 5 gene. Surfactant protein C (SP-C) was included as a control. The expression of this gene is stimulated in mature PTII cells, but with no sex difference (26). We show that 17ß-HSD type 2 and type 5 genes, and the SP-C genes are upregulated from GD 16.5 to 17.5.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Balb/C mice were mated during the night (mating window ± 8 hours), and the day of copulatory plug was considered as GD 0.5. Pregnant females were killed by exposure to a CO2 atmosphere. Protocols were approved by the Animal Care and Use Committee and the Institutional Review Board of the Centre de Recherche du Centre Hospitalier, Universitaire de Québec (protocol no. 2002–2080). Male and female fetuses from five pregnant mice were studied for each time of gestation, except for GD 17.5, in which six pregnant animals were killed. The fetal sex was identified by examination of the genital tract using a dissecting microscope at a magnification of x15. Fetal lungs were collected, and one pool of tissues was prepared for each sex and each pregnant animal (total of 42 pooled samples). Table 1 shows the number of fetuses for each sample, and the relative positions of male and female fetuses in the pregnant uterus.


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  		TABLE 1. Pregnant mice and fetuses

 
RNA Extraction and Complementary DNA Synthesis
Total RNA was extracted using Tri-reagent, a mixture of phenol and guanidine thiocyanate in a monophasic solution (Molecular Research Center, Cincinnati, OH), as described (27). Each RNA sample was then purified on a CsCl gradient, as described (28), using a TLA 120.2 rotor (Beckman, Mississauga, ON, Canada) in an Optima MAX centrifuge (Beckman). After densitometric determination of RNA concentrations, an aliquot of 4 µg of total RNA was DNase treated (DNase I, 0.25 unit/µg of total RNA), and reverse transcribed (Superscript II, Invitrogen, Carlsbad, CA), using hexameric random primer (pd[N]6; Invitrogen) in a final volume of 20 µl.

Real-Time Quantitative Polymerase Chain Reaction
The same complementary DNA (cDNA) preparations were used for the study of all the genes analyzed. For the study of 17ß-HSD type 5, a second series of cDNA samples was prepared and used to confirm the results obtained with the first series of samples. Real-time PCR reactions were performed on a LightCycler Instrument (Roche, Montreal, PQ, Canada) using LightCycler-FastStart DNA Master SYBR Green I Kits (Roche). PCR primers used are described in Table 2 . Reactions were performed according to the protocol of the manufacturer with 0.5 µM of each primer (final concentration), 3 mM MgCl2 (4 mM MgCl2 for 17ß-HSD type 5), and an amount of cDNA sample corresponding to 10 ng (SP-C), 20 ng glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 100 ng (17ß-HSD type 2, androgen receptor, keratinocyte growth factor, neuregulin-1, epidermal growth factor receptor, leukemia inhibitory factor, or insulin-like growth factor I), or 160 ng (17ß-HSD type 5 or type 3) of total RNA input in a final volume of 20 µl. Capillaries were heated for 10 minutes at 95°C for enzyme activation. Subsequently, 35 to 40 polymerase chain reaction (PCR) cycles were applied: step one, 0 seconds at 95°C; step two, 5 seconds at the indicated annealing temperature (Table 2); step three, 20 seconds at 72°C; and step four, 5 seconds at the indicated temperature for acquisition (Table 2). "Acquisition" refers to the reading of fluorescence signals by the instrument. We always program a separate step for acquisition using a temperature just below that at which the amplicon starts to denature. This temperature was determined for each gene from a denaturation curve obtained previously in a separate PCR run. This precaution allows us to subtract the contribution of dimerized primers, if any, from the total fluorescence signal. At the end of each run, samples were heated to 95°C with a temperature transition rate of 0.2°C/second to construct dissociation curves to check that single PCR products were obtained. For each gene analyzed, some PCR reactions were randomly selected, 5 µl were tested by 2% agarose gel electrophoresis, and the remaining 15 µl were purified using a QIAquick PCR purification kit (QIAGEN, Montreal, PQ, Canada), and subjected to DNA sequencing to confirm the specificity of the PCR reactions. The length of each specific amplicon was confirmed to that predicted. For all of the genes, except GAPDH and leukemia inhibitory factor, the selected primers encompass at least one intron, and no amplification from genomic DNA was detected. A standard curve for real-time PCR was prepared for each gene analyzed. Standard DNAs were prepared from amplicons previously obtained by PCR, purified, sequenced, and calibrated by electrophoresis on an agarose gel. One dilution used for the standard curve was included in duplicates or triplicates in each PCR run. The software supplied by the manufacturer (LightCycler Software, Version 3.5, Montreal, PQ, Canada) was used to import the standard curve and calculate the amount of PCR products. All the results presented were normalized by the amount of GAPDH messenger RNA (mRNA) determined by real-time PCR from the same cDNA preparations. It should be noted that there was only one pool of tissues for each sex and each litter (n = 1). When the results are presented for each individual pooled sample, the mean and SD values are calculated from two or three real-time PCR reactions.


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  		TABLE 2. Analyzed genes and real-time polymerase chain reaction conditions

 
Statistical Analysis
A paired t test procedure was used to compare expression levels of several genes in male versus female fetal lungs at each GD. A level of p > 0.05 was not considered statistically significant. Analyses were performed using the JMP statistical software (SAS Institute, Cary, NC).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
SP-C
In the mouse, the surge of surfactant production appears to occur on GD 17.5, as indicated by the appearance of lamellar bodies (29), an increase in surface activity in the mouse lung homogenate (29), and by increases in the activity of some enzymes involved in pulmonary lipid metabolism (30, 31). Moreover, a sex difference in the level of surfactant lipid production was observed in the mouse on GD 17.5 (1). To characterize the Balb/c mouse, SP-C mRNA levels were measured in fetal mouse lung mRNA samples from males and females from GDs 15.5–18.5. SP-C mRNA is a reliable marker of mature PTII cells (32, 33), but in contrast to surfactant lipids, SP-C mRNA levels did not show a sex difference (26). As shown in Figure 2 , a strong increase in SP-C mRNA levels was observed on GD 17.5 with no sex difference. SP-C mRNA levels follow their progression for both sexes from GD 17.5–18.5. A greater variation from subject to subject was observed on GD 18.5, but with no statistical difference between male and female fetuses.



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  		Figure 2. Relative surfactant protein C (SP-C) messenger RNA (mRNA) levels in the developing mouse lung of male and female fetuses. SP-C mRNA levels were determined by real-time polymerase chain reaction (PCR) in the lung of male and female fetuses from the 21 pregnant females presented in Table 1. Each point represents the mean ± SEM at the indicated sex and gestation time. Individual values were normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the mean value obtained for males on gestation day (GD) 15.5 was fixed as onefold. Individual values (± SD) obtained on GD 18.5 and used to calculate the mean are presented in the inset. Numbers on the x-axis of the inset correspond to the number of pregnant females (Table 1). *Paired t test (male vs. female): p > 0.10. When the individual values from pregnant female no. 6 are excluded; p > 0.12.

 
17ß-HSD Type 5
In the human, 17ß-HSD type 5 is highly homologous to 3{alpha}-HSD type 1 (87% identity) and type 3 (90% identity), the later being expressed in the A549 lung epithelial-like cells (23). At the time of the experiment there was no 3{alpha}-HSD cDNA sequence reported for the mouse, and one for the rat. Therefore, primers for mouse 17ß-HSD type 5 were carefully selected by comparison of human and mouse 17ß-HSD type 5, and the human and rat 3{alpha}-HSD cDNA sequences. To test the specificity of these primers in real-time PCR, we have first characterized the steroidogenic pathway of the NIH-3T3 cell line (No. CRL-1658, ATCC, Manassas, VA). There was strong 3{alpha}-HSD activity, but no 17ß-HSD type 5 activity in these mouse cells (data not shown). cDNA was prepared from NIH-3T3 cells and tested by real-time PCR with the selected primers for 17ß-HSD type 5 and GAPDH. A cDNA sample from fetal male lung obtained on GD 17.5 was also included. The results showed a specific amplicon for GAPDH from both samples, no PCR product for 17ß-HSD type 5 from NIH-3T3 cDNA, and a specific amplicon of the predicted molecular weight for 17ß-HSD type 5 from the fetal lung cDNA sample (data not shown). The nucleotide sequence of this amplicon was 100% homologous to that of the mouse 17ß-HSD type 5. We thus considered the primers/PCR parameters as specific to the 17ß-HSD type 5 gene. We recently obtained the sequence of a mouse 3{alpha}-HSD cDNA from Dr. Van Luu-The (personal communication). The study of this nucleotide sequence revealed that the 3'-end of both primers corresponded to 17ß-HSD type 5–specific regions and that some nucleotides within the amplicon are also gene-specific.

Levels of 17ß-HSD type 5 mRNA were measured in male and female fetal lung tissues isolated from 21 pregnant females killed from GDs 15.5–18.5 (one pool per sex per mother). A sex difference was observed on GD 15.5 for four of the five pregnant mothers, with higher levels in the female fetal lung samples compared with those of the male (Figure 3) . Low 17ß-HSD type 5 mRNA levels were observed on GD 16.5, but a marked increase was observed on GD 17.5 for both sexes. Examination of the individual values (Figure 3A) reveals that a sex difference exists on GD 17.5 for all animals, but that higher expression was observed in males for three of the six animals, whereas the opposite situation occurred in the three other pregnant females. These results were reproduced with a second series of cDNA preparations (data not shown). The mean values of 17ß-HSD type 5 expression decrease for both sexes on GD 18.5, as shown in Figure 3C, in which the values from the pregnant female no. 31 were excluded.



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  		Figure 3. Expression of the 17ß-HSD type 5 gene in the developing mouse lung of male and female fetuses. Levels of 17ß-HSD type 5 mRNA were determined by real-time PCR in the lung of male and female fetuses from the 21 pregnant females presented in Table 1. (A and B) Individual values obtained for each pool are indicated (A, ± SD), except the values corresponding to animal no. 31, which are not included in B. For each gestation time in B, male and female values are indicated on the left and on the right, respectively. (C) Means ± SEM of relative 17ß-HSD type 5 mRNA levels versus gestation time (day). The values were calculated from the results presented in A excluding the values from animal no. 31. All individual values were normalized by GAPDH, and the mean value obtained for males on GD 16.5 was fixed as onefold.

 
17ß-HSD Type 2
Similar results were obtained for 17ß-HSD type 2, including a sex difference favoring the females on GD 15.5, a decrease of the mean value of expression for females on GD 16.5 with very low values for both sexes, and a marked increase for both sexes on GD 17.5 followed by a decrease for both sexes on GD 18.5 (excluding the values from the pregnant female no. 31) (Figure 4B) . Again, when the results obtained for individual samples are considered, a sex difference is observed on GD 17.5, which favors male or female fetuses depending on the pregnant mother (Figure 4A). With the exception of pregnant mother no. 20, both 17ß-HSD types 2 and 5 show a sex difference favoring the same gender for all the pregnant mice on GD 17.5 and 18.5. The amplitude of expression also presents similarities for both genes between pregnant mothers with high values for males (animals no. 19 and no. 34), for females (animal no. 44), and for both sexes (animal no. 31) compared with other animals. Therefore, the two genes appear to be coregulated in the developing lung.



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  		Figure 4. Expression of the 17ß-HSD type 2 gene in the developing mouse lung of male and female fetuses. A and B correspond to Figures 3A and 3C, respectively, except that the primers used were specific to 17ß-HSD type 2. The values corresponding to pregnant animals nos. 31 and 72 were excluded to calculate the means presented in B. Individual values were normalized by GAPDH, and the mean value obtained for males on GD 16.5 was fixed as onefold.

 
Androgen Receptor and 17ß-HSD Type 3
Levels of androgen receptor and 17ß-HSD type 3 mRNAs were studied by real-time PCR. Messenger RNA of 17ß-HSD type 3 was barely detectable in all the samples (data not shown and Table 3) . Androgen receptor mRNA was easily detected in all the samples, but means showed no sex difference or temporal regulation (Figure 5B) . Only 3 of the 21 pregnant mothers show a sex difference (twofold or more) when individual values are considered (Figure 5A).


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  		TABLE 3. Expression of genes relative to gapdh mRNA levels

 


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  		Figure 5. Expression of the androgen receptor gene in the developing mouse lung of male and female fetuses. A and B correspond to Figures 3A and 3C, respectively, except that the primers used were specific to androgen receptor gene. In contrast to Figure 4, the values corresponding to pregnant animals nos. 31 and 72 were included to calculate the means presented in B. Individual values were normalized by GAPDH and the mean value obtained for males on GD 15.5 was fixed as onefold.

 
Paired t Test
Statistical analysis of the sex difference was performed from the results presented in Figures 3–5 for the 17ß-HSD type 5, 17ß-HSD type 2, and androgen receptor genes using a paired t test procedure (male versus female) (n = 5 or 6 litters). No significant sex difference was found. However, type 2 statistical errors (no detection of a real difference) are possible because some groups may in fact contain subgroups, as evidenced from individual values.

Relative Expression of the Analyzed Genes
By using a standard curve for each gene, we were able to determine the abundance of specific cDNA template (in ng/100 ng of RNA input) for each cDNA sample and for each gene. Then, the value obtained for each gene was divided by the value obtained for GAPDH for each sample and this ratio was used to compare the level of expression for from gene to gene. These results are presented in Table 3. The higher level of expression was observed for the SP-C gene, which had mRNA levels close to those of GAPDH on GD17.5. Very low levels of mRNA were observed for both 17ß-HSD types 2 and 5 on GD 16.5. Levels of 17ß-HSD type 3 mRNA were at the limit of detection for all samples during all the gestation-time windows studied. The mean of the values obtained on GD 17.5 for 17ß-HSD type 5 is very close to that determined for 17ß-HSD type 2, and is around sevenfold lower than that of androgen receptor.

17ß-HSD Type 5 Catalyzes Testosterone Synthesis from Androstenedione—17ß-HSD Type 2 Catalyzes the Opposite Reaction
Our studies on human lung cell lines have demonstrated that these two genes are not expressed in the same cell type. Comparison of the expression levels of these genes is interesting in that the amplitude of the paracrine action of testosterone can be influenced by the relative abundance of 17ß-HSD types 2 and 5 mRNAs. Quantitative values obtained for 17ß-HSD type 2 were then divided by those of 17ß-HSD type 5 for each pregnant female. This ratio gives a value close to one for the majority of the samples collected on GD 17.5 and 18.5 (Figure 6) . However, this ratio varies from two to nine on GD 15.5 for the majority of the samples, with higher values for the female fetuses in three of the five pregnant females.



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  		Figure 6. Relative abundance of 17ß-HSD types 2 and 5 mRNA in the developing lung of male and female fetuses. The quantitative values (± SD) obtained by real-time PCR for 17ß-HSD type 2 were divided by their corresponding values (± SD) obtained for 17ß-HSD type 5. Values are from the same experiments as those presented in Figures 3 and 4 for 17ß-HSD type 5 and type 2, respectively. The horizontal line corresponds to a ratio of 1.

 
Several Genes Play a Role in Lung Maturation
Knowing that the 17ß-HSD type 2 and type 5 genes present a marked sex difference in expression in fetal lungs for pregnant animals nos. 19 and 44 (with higher expression for males and females, respectively), and no expression for both sexes for fetuses from animal no. 10 (Figures 3A and 4A), the cDNA samples from these animals were used to see whether several genes known to play a role in lung maturation are coregulated with these steroidogenic genes. Results from real-time PCR revealed that the leukemia inhibitory factor (34), epidermal growth factor receptor (35, 36), insulin-like growth factor I (34), neuregulin-1(9), and keratinocyte growth factor (10) genes are all significantly expressed in the developing lung of male and female fetuses for all these three pregnant animals (Figure 7 and Table 3). No strong increase from GD 16.5 (no. 10) to GD 17.5 (no. 19 and 44) was observed for any of these genes (Figure 7). The abundance of mRNAs varied from gene to gene with a difference of 50-fold from the lower (leukemia inhibitory factor) to the higher (insulin-like growth factor I) expressed gene (Table 3). None of these genes were coregulated with the 17ß-HSD type 2 and type 5 genes from sample to sample.



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  		Figure 7. Relative mRNA levels in the developing mouse lung of male and female fetuses for several genes playing a role in lung maturation. Relative mRNA levels were determined by real-time PCR in the male and female fetal lung cDNA samples of pregnant animals no. 10 (GD 16.5), no. 19, and no. 44 (GD 17.5) for (A) leukemia inhibitory factor (LIF), (B) epidermal growth factor receptor (EGF-R), (C) insulin-like growth I (IGF-I), (D) neuregulin-1, and (E) keratinocyte growth factor (KGF) genes. Values (± SD) are normalized by GAPDH, and the values obtained for males from pregnant animal no. 19 were fixed as onefold.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Androgens are known as negative regulators of PTII cell maturation, and the delay of lung maturation observed in male fetuses compared with female fetuses has been attributed to higher levels of androgens in the male developing lung. Our previous data have demonstrated that testosterone is produced and secreted by the human PTII-like A549 cells through expression of the 17ß-HSD type 5 (23). However, the exact physiological role of androgen production by PTII cells remains unclear. Here, for the first time, we demonstrate that the 17ß-HSD type 5 gene is upregulated on GD 17.5 in parallel with the strong increase of SP-C mRNA, the later indicating the emergence of mature PTII cells. Moreover, this increase of 17ß-HSD type 5 mRNA levels was observed in both male and female lungs. The expression profile obtained for the 17ß-HSD type 5 gene does not explain the presence of higher levels of androgens in the male lung before the occurrence of mature PTII cells as it is expected to explain the delay in PTII cell maturation observed for males. Therefore, another source of androgens, or higher expression of the 17ß-HSD type 5 gene in the male lung before GD 15.5 could explain the sex difference in lung maturation. However, our results strongly suggest that PTII cells of both sexes produce androgens when they become mature. This important information suggests that androgens arising from 17ß-HSD type 5 could be produced by the mature PTII cells as physiologic modulators to negatively regulate the maturation process in both male and female fetuses. According to this hypothesis, the presence of androgens from another source in the male lung before the emergence of mature PTII cells is likely to initiate this regulatory event before completion of PTII cell maturation, which would result in a delay in lung maturation for male fetuses.

Our results show that there is no sex difference in 17ß-HSD type 5 expression on GD 17.5 when the average is considered. However, a sex difference in 17ß-HSD type 5 mRNA levels was observed for each pregnant animal, favoring male fetuses for three of the six pregnant females, and favoring female fetuses for the remaining pregnant animals. Visual examination of the data presented in Table 1 did not reveal any evidence that the relative amount or the relative position of male and female fetuses in the uterus could explain the expression profile of the 17ß-HSD type 5 gene on GD 17.5. Considering the time window of animal mating, 17ß-HSD type 5 expression could present a narrow peak on GD 17.5, which may arise at different times for male and female fetuses (Figure 8) . This is compatible with the delay in lung maturation observed for male fetuses. It should be noted, however, that we have assumed that 17ß-HSD type 5 expression in vivo occurs in PTII cells on GD 17.5, and that further investigation is required to confirm this point and to know whether this gene is expressed in the same cell type on GDs 15.5, 17.5, and 18.5.



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  		Figure 8. Hypothesis explaining the expression profile of the 17ß-HSD types 2 and 5 genes in the lung on GD 17. A narrow peak of expression occurring on GD17 at different times in the male and female fetal lungs within a period of 16 hours corresponding to the time of mating could explain why a sex difference in the level of expression is detected for all litters (with higher levels for the males for some litters, and higher levels for the females for the remaining litters), whereas no significant sex difference is observed from the mean (n = 6 litters). Our results do not allow us to determine the sex in which 17ßHSD types 2 and 5 expression are delayed compared with the other sex. We suggest that expression of both genes is delayed for males based on the delay of PTII cell maturation reported for males.

 
A sex difference in 17ß-HSD type 5 mRNA levels was detected on GD 15.5 favoring female fetuses. This would indicate that higher levels of androgens are produced by female fetuses on GD 15.5, whereas higher levels were expected for males. First, as mentioned, the sex difference could be explained by another source of androgens. Second, it is interesting to examine the 17ß-HSD type 2/type 5 mRNA ratio. Expression of 17ß-HSD type 2 and type 5 are likely to occur in different cell types in vivo as we showed in vitro (23, 24). A variation in the 17ß-HSD type 2/type 5 mRNA ratio suggests a variation in the efficiency of testosterone to produce a paracrine androgenic response. This ratio is close to 1 on GDs 17.5 and 18.5, but it is clearly higher on GD 15.5, indicating a more pronounced action of androgens on GD 17.5. We conclude that the sex difference in 17ß-HSD type 5 expression on GD 15.5 does not imply that androgen receptor occupancy is higher for all the androgen receptor-expressing cells of the female lung compared with the male lung.

Our results also clearly indicate that 17ß-HSD types 2 and 5 genes are coregulated on GD 17.5. With only one exception, pregnant animals presenting higher levels of 17ß-HSD type 5 mRNA in fetal male lungs also showed higher levels of 17ß-HSD type 2 mRNA, whereas the other pregnant females had higher levels of expression for both genes in fetal female lungs. Using the same cDNA preparations, the expression of the androgen receptor gene shows no sex difference and no temporal regulation. Once produced in peripheral tissues from a circulating steroid precursor, active steroid hormone can act directly in the cell in which it is produced (intracrinology) or it can be secreted and act on an adjacent cells (paracrinology). We have demonstrated in PTII-like cells (A549) that testosterone is produced and secreted, whereas 5{alpha}-dihydrotesterone is produced but rapidly converted into 3{alpha},17ß-diol (23). In this case, both testosterone and 5{alpha}-dihydrotesterone can exert an intracrine action on PTII cells through the intracellular androgen receptor, whereas testosterone is the main secreted androgen. In the developing lung, 17ß-HSD type 5 can then participate in the production of both intracrine and paracrine factors, whereas 17ß-HSD type 2 can modulate the paracrine action of testosterone. Therefore, coregulation of 17ß-HSD type 2 and type 5 on GD 17 suggests that the paracrine action of testosterone should be finely regulated.

In addition to its major role in androgen synthesis in peripheral tissues, the 17ß-HSD type 5 enzyme has also been reported to have a prostaglandin D2 11-keto reductase activity in many tissues, including the lung (37). Whereas such a role is not ruled out in the developing lung, our results suggest that 17ß-HSD type 5 is expressed mainly for androgen formation in this tissue. Effectively, the 17ß-HSD type 5 and type 2 genes are clearly coregulated in the developing lung both overtime and from sample to sample, whereas the 17ß-HSD type 2 enzyme is known to be an exclusive steroidogenic enzyme.

In an attempt to find another source of androgen in the developing lung, 17ß-HSD type 3 expression has been studied. No significant expression was found. This is not surprising because this gene is considered to be testis specific (38). In fact, very little 17ß-HSD type 3 mRNA was detected (Table 3). This level was at the limit of detection by real-time PCR, and we think that it corresponds only to basal promoter activity. The mean of 17ß-HSD type 5 mRNA levels obtained on GD 17.5 was 10-fold higher than that obtained for 17ß-HSD type 3 mRNA. Even though this level of expression seems to be low compared with the level of androgen receptor mRNA, we have to keep in mind that PTII cells are not abundant in the lung, and that fibroblasts, which express the androgen receptor gene (21) do not express the 17ß-HSD type 5 gene (24). Low steroidogenic gene mRNA levels are also compatible with significant levels of enzymatic activity in cell culture models (24).

We have suggested that 17ß-HSD type 5 expression on GD 17.5 could occur in PTII cells after their maturation to downregulate the maturation process. This action of androgens arising from 17ß-HSD type 5 expression could be exerted through downregulation of a known positive regulator of lung maturation. In an attempt to find a correlation between expression of 17ß-HSD type 5 and one or more positive regulators, mRNA levels of leukemia inhibitory factor, epidermal growth factor receptor, insulin-like growth factor I, neuregulin-1, and keratinocyte growth factor genes were determined in a selection of samples. None of these genes are coregulated with the 17ß-HSD type 5 gene. In fact, similar levels or only discrete differences between the analyzed samples were observed for all of the analyzed genes. This does not mean that these genes are not regulated as a consequence of 17ß-HSD type 5 expression. In fact, a delay could be required to detect such a modulation. Alternatively, androgens could modulate other gene(s) playing a role in lung maturation.

It should be noted that Tfm males survive with no functional androgen receptor (38). If we assume that only one androgen receptor gene is expressed in the developing lung, and that its activity is completely abolished in the Tfm mouse, then the contribution of 17ß-HSD type 5 expression in lung development does not seem to be essential for survival. However, this does not signify that the increase of 17ß-HSD type 5 expression observed on GD 17.5 is not necessary for normal lung development. In fact, the rate of mortality, the incidence of chronic lung disease, and the morphology of the neonatal lung have never been compared between Tfm and normal males. Based on the well-recognized negative effect of androgens on PTII cell maturation, our results suggest that androgens are produced by mature PTII cells to downregulate fibroblast-epithelial cell communication, driving PTII cell maturation. In fact, androgens may accelerate cell reprogramming after the emergence of mature PTII cells. Such a role could be essential to get optimal lung development, which is compatible with low incidence of mortality and chronic lung diseases, and efficient pulmonary functions.

As yet, in lung maturation, androgens were only viewed as a potential cause of pathology for the male premature infant. This report, rather, suggests that this negative regulatory effect of androgens on PTII cell maturation is in fact a physiological event playing a role in cell reprogramming after the emergence of mature PTII cells, and that this developmental event is initiated before completion of PTII cell maturation in the male fetal lung, delaying PTII cell maturation. Although this hypothesis remains to be confirmed, it represents an interesting way to explain why androgens are produced by PTII cells, while they are recognized as negative regulators of the PTII cell maturation process.


   FOOTNOTES
 
Supported by the Natural Sciences and Engineering Research Council of Canada, Association Pulmonaire du Québec, La Chaire Jeanne et Jean-Louis Lévesques, Fond de recherches sur les maladies infantiles du Québec, and the Canadian Institutes of Health Research (Y.T.).

Conflict of Interest Statement: P.R.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; M.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; Y.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this article.

Received in original form December 10, 2003; accepted in final form April 28, 2004


   REFERENCES
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 RESULTS
 DISCUSSION
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