Prolactin and Brown Adipocytes Figure 2. Alteration of thermogenesis in PRLR KO mice. A) PRLR KO newborn mice are more sensitive to cold exposure. Newborn animals were maintained in a 4uC environment and their survival was measured as a function of time (min). Kaplan-Meier representation of survival rate of KO (n = 6) and WT (n = 8) mice reveals a statistical difference (p,0.022). B) UCP1 mRNA expression in BAT of PRLR KO mice was significantly reduced compared to WT littermates. Quantitative real time PCR analysis of UCP1 mRNA expression was performed as described above. Results, expressed as attomol of UCP1/fmol of 18S, represent mean6SE of 15–16 animals (*** p,0.001). C) Western blot analysis of UCP1 expression in BAT of PRLR KO compared to WT mice. BAT of newborn animals were collected, lysed and 2 mg of proteins from homogenates were processed for immunoblotting with specific antibodies against UCP1 and ERK1/2. Quantification of UCP1 signal ratio was measured by densitometry using the Quantity One software (Biorad) and a significant decreased was detected in PRLR KO mice vs controls (***p,0.001, n = 6 per group). D) b3 adrenoceptor expression in BAT of PRLR KO mice was significantly reduced compared to WT littermates. Results, expressed as attomol of b3AdR /fmol of 18S, represent mean6SE of 15–16 animals (*p,0.05). E) Representative transmission electron-microscopic images of brown adipocytes of interscapular BAT from a WT newborn mouse and PRLR KO littermate. WT brown adipocytes exhibited intracytoplasmic lipid vacuoles and numerous mitochondria. Note that the mitochondria are extremely dense, compacted and characterized by their round shape and packed cristae encompassing the entire organelles (see arrow). Virtually, no cytoplasmic compartment is visible. In contrast, mitochondria in PRLR KO brown adipocytes are smaller, less packed and presented with elongated and fusiform features. The amount of cristae seems to be reduced (inset). Magnification X 15,000. doi:10.1371/journal.pone.0001535.g002 PLoS ONE | www.plosone.org 4 February 2008 | Issue 2 | e1535