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.
Recently, bone morphogenetic protein (BMP) 4 has been shown to inhibit FSH secretion in ewe. The detection of BMP4 mRNA and BMP receptors in the pituitary suggests that BMP4 can exert paracrine actions on FSH production. This work aimed at determining whether BMP4 and/or BMP receptor mRNA as well as activin/inhibin subunit mRNA fluctuates during the estrous cycle when FSHb mRNA and FSH release changed. The estrous cycles of ewes were synchronized with progestagen sponges. Ewes were killed in late follicular stage (nZ5), before the secondary FSH surge (nZ4), and in luteal phase (nZ4). Using quantitative reverse transcription-PCR, we showed that the levels of mRNA for BMP4, BMP receptor, the inhibitor of differentiation 2 (Id2), a target gene of BMP4, and noggin did not change significantly across the estrous cycle.In contrast, the level of activin bB mRNA and the percentage of immunoreactive cells for activin bB-subunit were higher before the secondary surge of FSH compared to other groups. In ewe pituitary cell cultures, activin, GnRH, or estradiol17b (E 2 ) did not significantly affect the levels of BMP4, BMP receptor, and Id2 mRNA. E 2 , but not GnRH, increased the level of activin bB mRNA. Moreover, the in vitro FSH release was not modified by noggin, a BMP antagonist. In contrast, SB431542, an inhibitor of activin pathway, inhibited FSH release. Collectively, our data showed that pituitary BMP4 would not play a crucial role in the regulation of FSH production during the estrous cycle, whereas local activin B would be a major stimulus of FSH synthesis necessary for the secondary FSH surge.
Bone morphogenetic proteins (BMPs) regulate diverse cellular responses during embryogenesis and in adulthood including cell differentiation, proliferation, and death in various tissues. In the adult pituitary, BMPs participate in the control of hormone secretion and cell proliferation, suggesting a potential endocrine/paracrine role for BMPs, but some of the mechanisms are unclear. Here, using a bioactivity test based on embryonic cells (C3H10T1/2) transfected with a BMP-responsive element, we sought to determine whether pituitary cells secrete BMPs or BMP antagonists. Interestingly, we found that pituitary-conditioned medium contains a factor that inhibits action of BMP-2 and -4. Combining surface plasmon resonance and high-resolution mass spectrometry helped pinpoint this factor as thrombospondin-1 (TSP-1). Surface plasmon resonance and co-immunoprecipitation confirmed that recombinant human TSP-1 can bind BMP-2 and -4 and antagonize their effects on C3H10T1/2 cells. Moreover, TSP-1 inhibited the action of serum BMPs. We also report that the von Willebrand type C domain of TSP-1 is likely responsible for this BMP-2/4-binding activity, an assertion based on sequence similarity that TSP-1 shares with the von Willebrand type C domain of Crossveinless 2 (CV-2), a BMP antagonist and member of the chordin family. In summary, we identified for the first time TSP-1 as a BMP-2/-4 antagonist and presented a structural basis for the physical interaction between TSP-1 and BMP-4. We propose that TSP-1 could regulate bioavailability of BMPs, either produced locally or reaching the pituitary via blood circulation. In conclusion, our findings provide new insights into the involvement of TSP-1 in the BMP-2/-4 mechanisms of action.
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