Expression of androgen- and glucocorticoid-related genes during saccular and alveolar stages of mouse lung development

Factors such as gestational age, birth weight and foetal sex are good predictors for the health outcome of extreme premature infants, but individuals tend to respond differently to antenatal and postnatal treatments, presumably because of difference in lung maturation. As such, a cocktail of markers (ex.: high 3aHSD, low 17b-HSD2 and high AR levels) evaluating the level of maturity of different aspect of the lung would be useful in order to “tailor” treatments to each infant, in order to minimize negative sequellea. The stages of developement in my study (saccular to alveolar) are the stages at which extreme premature infants receive treatment. Learning more about androgen and glucocorticoid action and metabolism in the murine lung at those stage, could provide insight on the potentially different timing of the glucocorticoid treatment premature infants according to their sex. The difficulty in the application of mouse data to a clinical setup lies with the availability of the material. With the exception of the L/S ratio and PG content in the amniotic fluid, most of the remaining biological markers of lung maturation developed in animal models (i.e.: rat, mouse, lamb) requires lung tissue, which cannot be obtained from extreme premature infants. As such, most of the information learned in animal models of lung development is fundamental in nature and often cannot be applied directly to the bedside of extreme premature infants, but provide insight.

Hi Heather,
Thanks your questions and your comments. I agree that I have to be very cautious when transferring towards the clinic. Results in mice, rat or rabbit should indeed be seen as preliminary to lager studies or studies done in sheep (preferred in lung developement compared to pork) or in human.

1. Q :What are the characteristics of the Balb/C mouse strain? Which of the characteristics was important in your choice to use the strain as your model animal?
A : The Balb/C strain was chosen for two main reason. First, it is a inbred strain which as the advantage of high genetic homogeneity. This greatly reduces inter-litter and inter-individual variability. Second, it was in this strain that the sex-difference in the surfactant surge was the most extensively studied (papers by Nielsen in the late 80’s to mid 90’s). Since my project is the logical progression of those results, we decided to continue using this strain.

2. Q: You said "The increased number of sample and genetic homogeneity allows us to look for a possible sexual dimorphism of androgen or glucocorticoid regulation by the developing lung". How did you increase the sample number and the genetic homogeneity?
A: Thank you for pointing that out. Re-reading it, I also find the phrasing a bit confusing...
I was talking about an increased number of samples compared to what I would have been able to collect if I was working with human samples. As you surely realize those samples are extremely hard to come by, even with a protocol that has been approved by an ethics committee. The use of a mouse model also allows us to collect lung representing a higher number of days (intermediates between development stages) and to study terminal lung development in its entirety. That would have been impossible in human or other models such as rabbit or sheep. As I have explained in my response to your first question the Balb/C is an inbreed strain with high genetic homogeneity, especially compared to a human population.

3. Q: Why did you choose the five housekeeping genes you did for studying normalization? Do they have any significance in lung maturation?
A: The five housekeeping genes chosen for normalization do not have any significance in lung maturation. In fact we chose them exactly because they are not implicated in lung maturation. They were chosen because they because they have been shown to be stably expressed in a variety of tissues and under many conditions, which makes them good normalization gene candidates. Also, they represent different levels of expression (low copy number to high copy number). Without going into details, in QPCR, genes that are expressed at a low level are more susceptible to biases than genes expressed at high levels. Since some of our studied genes are expressed at low levels (ex.:17b-HSD2), we needed to included housekeeping genes that also are expressed at low levels in the calculation of our normalization factor.

4. Q: Other than lower basal corticosterone levels, what is associated with the stress hyporesponsive period?
A:The principal characteristic of the SHRP is the hyporesponsiveness in response to stress in regard to corticosterone and ACTH production. The SHRP is also characterise by high expression of CRH in the hypothalamus (paraventricular nucleus) and a steady increase of GR in the Expression levels of GR in the hippocampus until the end of the period.

Q:Is there a similar period observed in extreme premature infants?
A: Excellent question! As far as I know, there is no SHRP in human, at least human babies born at term have fully functioning HPA axis. Developement of the HPA axis is still the subject of investigation, but I will go back and check the literature to see if a similar period exists during the same lung development stages. I’ll try to get back at you before Thursday.

Hi Alex,
Thanks for your questions.
1)
Q :Why does it seem to be the case that male fetuses are more at risk than female fetuses? Not being an expert in your field, I have noticed this trend among a few of the STIRRHS presentations to date. Is this vulnerability thought to be linked solely to testosterone levels or to a combination of factors? I find it curious as in my line of work, which focuses more on psychosocial stressors and HIV transmission, we often perceive women to be more at risk. Any thoughts you have would be greatly appreciated.
A : The male disadvantage (at least for lungs and RDS) is pretty much only linked to higher testosterone levels. Androgens have been shown to delay the surge of surfactant (a complex lipid and protein secretion necessary to normal lung function that act by lowering the surface tension of the alveoli, which in turn prevent their collapse). This has been demonstrated in a number of animal models. What remains unclear is how the punctual increase in the levels of testosterone associated with testicular differentiation, which occurs several weeks (days in mouse) before surfactant production, causes the delay in lung maturation. Also, at the same gestational age, some male fetuses fair far better than females (opposite to the statistics). This suggests that there must be other biological factors at work here... My project studies lung maturation, but prematurely born boys are also behind girls for other system as well, that also contribute to their poorer general outcome. As for a psychosocial factor, I’m not aware that extreme premature males have any disadvantages at the onset, other than they usually have a poorer diagnostic. The graduate student responsible for the psychosocial section of the discussion forum did point out that infants born prematurely show more cognitive and social problems at almost all the ages studied, and that boy were more affected by those problems than girls. Whether this male disadvantage is specifically due to the poorer outcome of premature boys, reflects the higher prevalence of those problems in boys in the general population or results from a specific psychosocial male disadvantage is still unclear in the literature and was the subject of debate during the multidisciplinary phase of our forum. What do you think? Another question that was raised during the forum was the existence of a sex-bias (positive or negative) concerning the length or aggressiveness of the treatments that the parents are willing to put their offspring trough. Do you think that this exists? Do you think it could be responsible for a male disadvantage in mortality or morbidity?
I think you figured it out already, but the forum did give me insight into my project. There are A LOT of places where are STILL in the process of formulating a consensus opinion. That’s probably what I find both the most challenging and what I get out the most of.
Eric

Hi Martin,
I really enjoy reading your questions. I hope my answers will shed some light onto them.

Q: With regard to multiple versus single courses of antenatal glucocorticoids in fetuses at risk of preterm labour, are you aware of benefit studies accounting for fetal gender?
A: I’m not aware that there have been any studies showing differential outcome of single vs single courses of antenatal glucocorticoids in males or females. My understanding is that the principal interest for multiple courses comes in the fact that the efficiency of the single dose is limited to about 4 to 7 days after administration. This is a problem when labour is successfully arrested for more than 7 days, after the initial glucocorticoids treatment. The problem with multiple doses is that is increases the chances of neurodevelopmental damages or HPA axis reprogramming, compared to a single dose.

Q: Could it be that due to 'androgen disadvantage' male fetuses in fact would show lower rates of RDS if administered multiple glucocorticoid courses?
A: Male foetuses do show lower rates of RDS, even with single doses. The problem is that they show higher rates than treated females. This suggests that the sex difference is established before glucocorticoids action can take place. As such, even if they have “more catching up to do” then females, I do not think that multiple doses will result in equal rates of rates of RDS in males and females, females will still have lungs that are more mature than males.

Q: Gender differences have been described for chemo- and baroreceptor sensitivity. Since these systems are relevant for RDS, I wonder to what extent they may also contribute to its higher prevalence in males.
A: I’m not aware of any studies that have looked at this directly, but it certainly could be a contributing factor. It might be implicated in the higher rates of ventilation induced BPD in boys compared to girls. Very interesting idea, I’ll certainly look into it in more detail and introduce it to the discussion forum.

It was suggested by Doan et al. 2004, that 'estradiol secreted during late gestation is necessary for respiratory adjustment to pregnancy and is required for adequate development of respiratory and metabolic control in the offspring'.
Joseph et al., 2000, wrote that ‘GENDER-RELATED DIFFERENCES in the physiological responses to hypoxia have been reported by several studies that demonstrated a better capacity of women and female rats to adapt to hypoxia.'
Q: As such, should one consider early postnatal administration of estradiol and/or progesterone to male neonates?
A: Very interesting idea! Estradiol has indeed been shown to accelerate lung development. I’ve found a couple of very promising papers that explored this line of treatment. For exemple, in a baboon model of BPD, McCummin et al 2009 found that post-natal Estradiol treatment upregulated nitric oxide synthase, resulting in “favorable cardiovascular impact, enhanced pulmonary function, and lowered requirements for ventilatory support”. Of course, the benefit of such a treatment should be weighed against the obvious possible detrimental sexual development effects. I’m also sure that females would equally benefit from this avenue.

Again, thanks for the very thought-provoking insights!

Eric