and mammary gland tumorigenesis [11,12,13,14,15,16,17]. Akt1 ablation has been shown to delay mammary gland growth during pregnancy and lactation, and accelerates the rate of involution following lactation [11,14]. Conversely, constitutively active Akt1 delays epithelial cell apoptosis and mammary gland involution [18]. Of note, Akt1-deficient dams produce less milk during nursing, and consequently have smaller pup weights regardless of the pup genotype [11,19]. It has been previously shown that Akt1 ablation delays the onset of puberty and results in subfertility in C57/Bl6 mice [20]. Akt1 deficiency also increases the levels of the hormone estradiol and decreases the levels of progesterone at postnatal day 50; however, hormone levels in wild-type and Akt1-deficient mice are comparable by postnatal day 90 [20]. Interestingly, estradiol and progesterone are responsible for ductal growth and alveolar expansion in mammary glands, respectively [3]. These previous studies led us to examine the molecular mechanisms underlying the requirement for Akt1 in mammary gland development and function. Although Akt1 is implicated in lactation and involution as well as mammary carcinogenesis, its functional roles in regulating mam- mary gland development and influencing adult mammary gland structure are not yet fully understood. In this study, we show that Akt1 is essential for proper mammary gland composition during development and for appropriate gene expression patterns in adulthood. We found that Akt1 deficiency delayed mammary gland development, with fewer TEBs during puberty and fewer and smaller alveolar buds in adulthood, potentially due to in- creased apoptosis in the epithelial compartment. Our data further indicate that Akt1 deficiency resulted in gene expression changes and several altered biological processes, which could affect cellular composition and function, leading to impaired lactation. Specif- ically, in both adult virgin Akt12/2 mammary glands and pseudopregnant Akt12/2 glands we found down-regulation of Btn1a1, which has previously been shown to be critical for milk lipid secretion [21]. We also show that suppression of Akt1 in MCF7 human breast epithelial cells decreases expression of Btn1a1, indicating a role for Akt1 in the regulation of Btn1a1. Suppression of Btn1a1 decreased expression of Btn1a1 but not Akt1, suggesting that Btn1a1 functions downstream of Akt1. Taken together, our results indicate that Akt1 regulates mammary gland postnatal development as well as lobuloalveolar development through the regulation of apoptosis and gene expression. Materials and Methods Ethics Statement The Brown University Institutional Animal Care and Use Committee (IACUC) approved all experimental animal protocols in compliance with National Institute of Health guidelines. The IACUC approval number is 0909078 and date of approval was 10/ 7/2010. Animals Akt1 heterozygous mice (Mus musculus) on a C57/BL6 background were obtained from the laboratory of Dr. Morris Birnbaum (University of Pennsylvania, Philadelphia, PA). Hetero- zygous pairs were mated to obtain Akt1 +/+ and Akt1 2/2 females for experimental procedures. Animals were housed in polycarbonate cages and were given access to water and feed ad libidum. Animals were sacrificed by CO2 asphyxiation at PND (postnatal day) 11, PND 25, PND 50 or PND 70. Mammary gland whole mounts Inguinal mammary glands from the fourth and ninth positions were removed, fixed in Carnoy’s solution for 2–4 hours, stained Akt1 in Mammary Development overnight in carmine solution, rinsed in alcohol and cleared in xylene overnight. Whole mounts were examined and images were taken using a Zeiss Discovery V8 stereomicroscope equipped with an Axiocam MRc camera and Axiovert Software (Carl Zeiss Canada Ltd., Canada). The criterion for scoring varied depending upon the age of the mouse group being evaluated. The primary duct length was measured at PND 11 and PND 25 using the curve spline measurement feature. Since the mammary glands are significantly larger at PND 50 and 70, the length of growth past the lymph node was measured instead of the primary duct length by using the length measurement feature. Ductal outgrowth was measured using the outline spline measurement feature as the area occupied by the entire mammary gland for all time points. Terminal end buds and alveolar buds were measured for all time points in the Zone C area of the mammary gland, which for this study is defined as the area comprised of the outer 1 mm of the entire mammary gland [22]. Terminal end buds were defined as having a diameter of $100 mm. The number of total buds was comprised of terminal end buds ($100 mm) and alveolar buds (,100 mm). The percentage of terminal end buds was calculated by dividing the number of terminal end buds by the number of total buds. The scores from the fourth and ninth position mammary glands from each animal were averaged and the mean was used to represent that animal. Histological analysis Mammary gland whole mounts were manually embedded in paraffin and cut into 7-mm sections for histological analysis. Fol- lowing deparaffinization, mammary gland sections were washed in 0.1% lithium carbonate to remove carmine aluminum stain. Mam- mary sections were stained with hematoxylin and eosin (H&E) and martius yellow, soluble blue, brilliant crystal (MSB) stain for fibrin to examine basic pathology. Alveolar bud diameters were measured on the Aperio ImageScope v10.2 (Aperio Technologies Vista, CA). At least 20 measurements per animal were taken and the mean was used to represent that animal. Western blots Thoracic mammary glands were pulverized in liquid nitrogen then lysed in cold RIPA buffer (20 mM Tris 8.0, 150 mM NaCl, 0.5% sodium deoxyocholate, 0.1% SDS, 1% NP-40, 10 mM sodium pyrophosphate, 10 mM sodium fluoride) supplemented with 60 ml/ml HALT protease/phosphatase inhibitors (Thermo Scientific, Waltham, MA) and 1 mM PMSF (Roche, Indianapolis, IN). Lysates were incubated on ice for 20 minutes with frequent vortexing and cleared twice by centrifugation (13,200 rpm, 10 minutes, 4uC). MCF7 cells were lysed in cold lysis buffer (50 mM Tris pH 8.0, 250 mM NaCl, 1% NP-40, 0.1% SDS, 5 mM EDTA, 2 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 10 mM sodium fluoride), incubated on ice for 20 minutes and cleared by centrifugation (13,200 rpm, 10 min- utes, 4uC). Protein concentrations were determined using the DC Protein Assay (Bio-Rad). Fifty micrograms of total protein was subjected to SDS-PAGE and transferred onto Immobilon-P PVDF (Millipore, Billerica, MA). Membranes were blocked for 60 min- utes at room temperature in 5% non-fat milk/Tris-buffered saline/0.1% Tween (TBST). Membranes were incubated for 2 hours at room temperature then overnight at 4uC in one the following antibodies: AKT1 (#2938, 1:1000 in 5% BSA/TBST), AKT2 (#2964, 1:1000 in 5% BSA/TBST), AKT3 (#3788, 1:1000 in 5% BSA/TBST), or GAPDH (AM4300, 1:4000, Applied Biosystems). All antibodies were obtained from Cell Signaling Technology (Danvers, MA) unless indicated otherwise. Membranes were washed 3 times (5 minutes per wash) in TBST, PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e24432