Hyaline membrane disease (respiratory distress of the neonate) occurs primarily in premature infants. A major cause of this disease is surfactant deficiency. Hyaline membrane disease and surfactant synthesis are both affected by fetal sex. Indeed, the surge of surfactant synthesis is normally delayed in the developing male lung when compared with the female, while hyaline membrane disease is prevalent in males 12345.

Surfactant synthesis occurs in type II pneumonocytes (PTII) following maturation of these cells, which is promoted by fibroblast-PTII cell communication 5. This maturation process is stimulated by glucocorticoids 6 and involves some cytokines including epidermal growth factor (EGF) 78, neuregulin-1 9, and keratinocyte growth factor (KGF) 10 as positive regulators and transforming growth factor-β1 (TGF-β1) 11121314 as negative regulator.

Androgens have been shown to both delay fetal lung maturation in vivo 315 and block the stimulatory effect of corticosteroids on surfactant synthesis in vitro 16. These effects are mediated through binding of androgens to androgen receptors 31517, which are present in both male and female lung tissues 1819. There is an active androgen metabolism in the developing lung where androgen synthesis 1820 and inactivation 1821 occur. In mice, many genes involved in androgen metabolism are regulated specifically on gestation day (GD) 17.5 in parallel with the emergence of mature PTII cells (18 and unpublished data).

The surge of surfactant synthesis occurs on GD 17.5 in the mouse 318 with a sex difference in pulmonary surfactant lipid levels 3. Male mice carrying the Tfm gene (male with testicular feminization), which have no functional androgen receptors, have surfactant levels comparable with those of normal females at a comparable developmental time point 3. Therefore, the mouse is a good model to study the effect of fetal sex on the timing of the developmental events related to the surge of surfactant synthesis.

Knowing that the surge of surfactant synthesis is delayed in male fetal mouse lungs compared with females, we were interested to identify genes that are expressed with a sex difference during the gestational period that overlaps the surge of surfactant production. We found, by microarray analysis, genes exhibiting a sex difference in expression in lung development during a three-day window preceding and including the emergence of mature PTII cells. To date, no gene profiling study of sex differences in the embryonic lung tissue exists. Using Affymetrix technology, we have studied about 14,000 transcripts and variants with more than 22,600 probe sets in male and female fetal lungs on GDs 15.5, 16.5, and 17.5.

Pregnant Balb/C mice were sacrificed on GDs 15.5, 16.5, and 17.5. Total RNA prepared from male and female fetal lungs, from each litter, was pooled in order to obtain the following six samples: GD 15.5 males; GD 15.5 females (each from 2 litters); GD 16.5 males; GD 16.5 females (each from 2 litters); GD 17.5 males; and GD 17.5 females (each from 1 litter) (number of fetuses per RNA preparation were respectively of 6, 9, 7, 9, 2, and 4). Previously, the litters corresponding to GDs 15.5 and 17.5 have shown sex differences in expression for several genes 1827. Microarray analysis was performed using six probes prepared from these RNA samples and 12 GeneChip Mouse Genome 430 A array (in duplicate). Values obtained for each gene were compared between sexes for each GD.

This study targeted elucidation of a sex difference in the fetal lung during a three-day gestation period overlapping PTII cell maturation. This sex difference results in more concern for a poorer prognosis for respiratory distress in premature male infants. The genes identified here, with a sexually dimorphic expression during the gestation period examined in the mouse, likely contain key genes involved in PTII cell maturation. Further studies are essential to identify and to characterize these key genes, as our data indicate that PTII cell maturation is not the only aspect of lung development affected by sex during this period of gestation.

Analysis of the data has to be performed in the context of lung development where a transient delay in the surge of surfactant synthesis is observed. Therefore, as in the case of the surge of surfactant synthesis, some genes can be subject to a transient delay in expression for one sex. The case of the Cyp21a1 gene clearly illustrates this occurrence. Recently, we showed that Cyp21a1, a gene involved in corticosteroid synthesis, is expressed specifically on GD 15 in the fetal mouse lung 27. Of the six litters studied on GD 15.5 and obtained using the same mating window of ± 8 hours, two litters presented high expression of Cyp21a1 in females only, whereas one litter had high expression of this gene only in male fetuses. The three remaining litters did not show any elevated levels of expression for this gene (Figure 3). The exact gestation time at which the pregnant females were sacrificed varied from litter to litter according to the time where the females were mated during the mating window of ± 8h. Knowing that Balb/C is an inbred strain, variations from litter to litter should represent the expression pattern of Cyp21a1 gene at different times on GD 15. Consequently, our results strongly suggested that Cyp21a1 should present a narrow peak of expression on GD 15 with a delay for one sex 27. In the present study, expression of Cyp21a1 (Id. #30) was detected by gene profiling only in males on GD 15.5. Real-time PCR analysis of the two litters pooled to prepare probes on GD15.5 revealed that Cyp21a1 was expressed at high levels only in male lungs (one out of two litters) (data not shown). Therefore, our microarray results are compatible with our previous report 27. In this example, the fact that we detected expression only in male fetuses by microarrays depended only on the litters used. Therefore, for the analysis of our microarray results, the identity of the sex where expression is higher compared to the other sex has to be considered with caution. Other animals, mated some hours before or after the pregnant females used in this study within the same mating window, could show elevated expression levels for the other sex, or even present no sex difference at all, as a consequence of a delayed expression in one sex.

Recently, we reported that all the genes involved in corticosteroid synthesis from cholesterol in the mature adrenal gland are transiently expressed in the developing mouse lung on GD 15 27. Hsd3b1 (Id. #28) encodes the 3β-hydroxysteroid dehydrogenase type 1 enzyme that is involved in this cascade of reactions (pregnenolone → progesterone) along with Cyp21a1 (Id. #30) (progesterone → deoxycorticosterone) as mentioned already. Both genes were also found by microarray analysis to be expressed transiently on GD 15 with a sex difference.

Some other genes related to steroid hormone metabolism/regulation are expressed also with a sex difference in fetal mouse lungs. This is the case for Akr1b7 (Id. #29) which showed a sex difference in expression on GD 15.5. This gene encodes an aldose reductase-like protein whose major function is detoxification of isocaproaldehyde generated by conversion of cholesterol to pregnenolone by the enzyme P450scc 34, the latter also being expressed on GD 15.5 in the mouse fetal lung 27. Scgb1a1 gene (Id. #53) is expressed also on GD 15.5 with a sex difference. This gene encodes the secretoglobin family 1 A member 1, also called Clara cell secretory protein (CCSP), which is required for the appearance of Clara cells secretory granules and thus participates to the composition of the epithelial lining fluid 35. Scgb1a1 gene can be induced by glucocorticoids 36 and CCSP can act as an anti-inflammatory protein 37. It was also shown to bind progesterone 38. Some other genes were not detected by microarrays although they are known to be expressed with a sex difference in lung development as evidenced by real-time PCR. These genes include 17β-hydroxysteroid dehydrogenase (HSD) type 2 and type 5 18 which are expressed at low levels in the lung, probably below the minimal threshold of sensitivity of the microarray technology.

Some genes involved in the metabolism of non-steroidal hormones were also found to be expressed differentially between genders. This is the case for Ttr (Id. #32), which codes for transthyretin, a common plasma carrier protein for thyroid hormones and vitamin A metabolites 39. Interestingly, retinoids are important regulators of normal epithelial cell differentiation and proliferation 40 and are involved in lung development 41. Ren1 (Id. #33) encodes for renin 1 and presented a sex difference in expression on GD 15.5. Genes of the renin-angiotensin system are known to be expressed in the fetal lung and, interestingly, angiotensin II can present mitogenic effects on human lung fibroblasts through the activation of the type 1 angiotensin II receptor 42. In our study, Ace (angiotensin II converting enzyme), Agtr1, and Agtr2 (angiotensin II receptor type 1 and type 2) are expressed with no sex difference (data not shown).

Our results demonstrate that genes coding for apolipoprotein (apo) AI (Id. #45), apoAII (Id. #47), apoCII (Id. #46), and apoH (Id. #44) are co-expressed in the developing lung. To our knowledge, none of these genes has been found expressed in the lung. All of these genes present a sex difference in favor of females in the developing lung on GD 15.5 and 17.5, with no detectable expression on GD 16.5. As demonstrated by real-time PCR, 17βHSD types 2 and 5 were also expressed on GDs 15.5 and 17.5 with a sex difference for the majority of litters, but not on GD16.5 18. Our results suggest that the expression of these four apolipoprotein genes in the developing lung is under active modulation. 17β-HSD types 5 and 2 genes are involved in androgen synthesis and inactivation, respectively. Such a similar pattern of expression for these apolipoproteins and 17β-HSD genes may suggest a common regulatory mechanism, or an effect of androgens on expression of these apolipoprotein genes. Apolipoproteins are constituents of circulating lipoproteins. ApoCII is an essential cofactor/activator of lipoprotein lipase (LPL) 4344 while apoH was shown to enhance LPL 45. This enzyme catalyses the hydrolysis of the triacylglycerol component of circulating lipoproteins (chylomicrons and very low density lipoproteins (VLDLs)). It was reported that the presence and activity of LPL in the lung may be important for surfactant production 46. LPL expression was detected with no sex difference. ApoAI 47 and apoAII 48 are major protein components of high density lipoproteins (HDLs). ApoAI is a potent activator of lecithin:cholesterol acyltransferase (LCAT), an HDL-associated enzyme playing a role in reverse cholesterol transport. It is possible that the sex difference in expression of four apolipoprotein genes in the lung could explain in part the sex difference in surfactant lipids observed on GD 17.5; although such a role in the surge of surfactant synthesis has never been suspected for these genes before the present study. It is also noteworthy that many genes involved in lipid metabolism were expressed with no sex difference during each gestational day studied (data not shown). For example, this is the case for genes encoding for LDL receptor, LDL receptor-related protein 1, phospholipid transfer protein, and HMG-CoA-reductase (3-hydroxy-3-methylglutaryl-Coenzyme A reductase), the latter being known to catalyze the rate-limiting step of cholesterol synthesis.

Along with other pathways, fibroblast growth factors (FGFs) and TGFβ signaling pathways have been shown to be involved in many aspects of lung development. We observed expression of many genes that belong to these pathways (Table 3). However, in the present study, these genes exhibit no sexually dimorphic expression. This is in line with our previous study by real-time PCR that showed expression of 5 genes involved in lung maturation on GDs 16.5 and 17.5 with no sex difference, namely, LIF, IGF-I, KGF (FGF-7), EGF-R, and neuregulin-1 18. Although it is possible that these genes could be expressed with a sex difference within a time window not covered by our studies, it seems that the sex difference in PTII cell maturation could rely on other genes.

Tissue remodeling and structure also seem subject to sexual differences in gene expression. Indeed, two genes involved in apoptosis, namely, Rock1 (Rho-associated coiled-coil forming kinase 1) (Id. #1) and Casp8ap2 (caspase 8 associated protein 2) (Id. #2) are differentially expressed on GD 16.5 between genders, while many genes involved in cell adhesion are expressed with a sex difference on GD 15.5. In addition, IGF signaling pathway has been shown to be important in mouse lung development where it is involved in regulation of cell proliferation and differentiation 314950. Igfbp1 (Id. #9) presented a sex difference in expression on GD 15.5, whereas Igfbp1 deficiency was associated with massive hepatocyte apoptosis 51.

In preparation for important immunological challenges related to its function, the developing lung acquires many elements associated with innate and adaptive immunity. Our data suggest that modulation of several genes involved in immune functions of the lung is subject to sex differences. Indeed, the predominating functional category of genes showing sexual dimorphism on GD 16.5 concerns "immune functions". It has been shown that many signaling pathways involved in lung morphogenesis and immune responses are crosslinked 52. These include TTF-1, GATA6 and HNF-3β transcription factors; and FGF- and NF-κ B-dependant signaling pathways. However, we did not detect any sex difference in expression for these genes.

This study revealed that many genes are expressed with a gender difference in the fetal lung. Although we focused on a brief gestational period overlapping the surge of surfactant synthesis, our data demonstrates that PTII cell maturation is not the only aspect of lung development under the influence of fetal sex. We suggest that, among the genes identified here, some are related to the transient delay in lung maturation observed for males. This would help identify novel genes involved in the physiopathology of respiratory distress of the neonate.

The author(s) declare that they have no competing interests.

MS participated in the design of the study, performed the data analysis and wrote the manuscript. PRP participated in the design of the study and helped to draft the manuscript. YT conceived the study, participated in its design and in its coordination. All authors read and approved the final manuscript.