Lactation failure in Src knockout mice is due to impaired secretory activation

<p>Abstract</p> <p>Background</p> <p>Mammary gland development culminates in lactation and is orchestrated by numerous stimuli and signaling pathways. The Src family of nonreceptor tyrosine kinases plays a pivotal role in cell signaling. In order to determine if Src plays a role in mammary gland development we have examined mammary gland development and function during pregnancy and lactation in mice in which expression of Src has been eliminated.</p> <p>Results</p> <p>We have characterized a lactation defect in the Src-/- mice which results in the death of over 80% of the litters nursed by Src-/- dams. Mammary gland development during pregnancy appears normal in these mice; however secretory activation does not seem to occur. Serum prolactin levels are normal in Src-/- mice compared to wildtype controls. Expression of the prolactin receptor at both the RNA and protein level was decreased in Src-/- mice following the transition from pregnancy to lactation, as was phosphorylation of STAT5 and expression of milk protein genes. These results suggest that secretory activation, which occurs following parturition, does not occur completely in Src-/- mice. Failed secretory activation results in precocious involution in the mammary glands of Src-/- even when pups were suckling. Involution was accelerated following pup withdrawal perhaps as a result of incomplete secretory activation. In vitro differentiation of mammary epithelial cells from Src-/- mice resulted in diminished production of milk proteins compared to the amount of milk proteins produced by Src+/+ cells, indicating a direct role for Src in regulating the transcription/translation of milk protein genes in mammary epithelial cells.</p> <p>Conclusion</p> <p>Src is an essential signaling modulator in mammary gland development as Src-/- mice exhibit a block in secretory activation that results in lactation failure and precocious involution. Src appears to be required for increased expression of the prolactin receptor and successful downstream signaling, and alveolar cell organization.</p

An integrated systems biology approach to the study of preterm birth using "-omic" technology - a guideline for research

Preterm birth is the leading cause of neonatal mortality and perinatal morbidity. The etiology of preterm is multi-factorial and still unclear. As evidence increases for a genetic contribution to PTB, so does the need to explore genomics, transcriptomics, proteomics and metabolomics in its study. This review suggests research guidelines for the conduct of high throughput systems biology investigations into preterm birth with the expectation that this will facilitate the sharing of samples and data internationally through consortia, generating the power needed to study preterm birth using integrated "-omics" technologies. The issues to be addressed include: (1) integrated "-omics" approaches, (2) phenotyping, (3) sample collection, (4) data management-integrative databases, (5) international consortia and (6) translational feasibility. This manuscript is the product of discussions initiated by the "-Omics" Working Group at the Preterm Birth International Collaborative Meeting held at the World Health Organization, Geneva, Switzerland in April 2009

U vrtlogu tijela, riječi i prostora – nova propitivanja hrvatske drame i kazališta (Krležini dani u Osijeku 2005., priredio Branko Hećimović)

+/- (B, E, H and K) and Src-/- (C, F, I and L) mice at P6 (A-C), P12 (D-F) and P18 (G-I). Histological sections from mammary glands at P18 are also shown for each genotype (J-L). In panels A to I scale bars represent 5 mm. In panels J to L scale bars represent 100 μm.<p><b>Copyright information:</b></p><p>Taken from "Lactation failure in Src knockout mice is due to impaired secretory activation"</p><p>http://www.biomedcentral.com/1471-213X/8/6</p><p>BMC Developmental Biology 2008;8():6-6.</p><p>Published online 23 Jan 2008</p><p>PMCID:PMC2266720.</p><p></p

Lactation failure in Src knockout mice is due to impaired secretory activation-6

of pregnancy (lanes 1–6), lactation day 2 (lanes 7–12) and lactation day 9 (lanes 13–18). Three separate mice were used per genotype and developmental stage. Protein lysates were prepared as described in the Materials and Methods sections, and immunoblotting conducted to detect phosphorylation of STAT5 (top panel), the amount of total STAT5 (middle panel), and the amount of actin, as a loading control (bottom panel). B) Total RNA was isolated from the number 4 mammary gland of wildtype and knockout mice on day 18 of pregnancy, lactation days 2 and 9 and involution day 2, three mice per genotype and developmental stage were used. cDNA was synthesized from 1 μg of total RNA and quantitative RT-PCR was performed using primers and probe specific for β-casein. β-casein message levels were normalized to GAPDH for each sample and the graph represents the mean (± SEM) relative amount of the triplicate tissue samples.<p><b>Copyright information:</b></p><p>Taken from "Lactation failure in Src knockout mice is due to impaired secretory activation"</p><p>http://www.biomedcentral.com/1471-213X/8/6</p><p>BMC Developmental Biology 2008;8():6-6.</p><p>Published online 23 Jan 2008</p><p>PMCID:PMC2266720.</p><p></p

Lactation failure in Src knockout mice is due to impaired secretory activation-4

C+/- (D-F), and Src-/- (G-I) mice at P18 (A, D, and G) and L2 (B, C, E, F, H and I). The sections shown in A, B, D, E, G and H were stained anti-ADRP antibody to outline the cytoplasmic lipid droplets (red), Oregon-Green 488-conjugated WPA to outline the surface of secretory alveoli (green), and DAPI to stain the nuclei of mammary epithelial cells (blue). Hematoxylin and eosin-stained sections of mammary glands at L2 are shown in C, F, and I. The bar in panel H represents 10 μm and the bars in panels C, F and I represent 100 μm. Luminal space is indicated by the letter "L", the white arrowheads indicate cytoplasmic lipid droplets, and the white arrows indicates regions where staining with WGA represents atypical patterns not observed in wildtype mice. The black arrows in panel I indicate large cytoplasmic lipid droplets not observed in wildtype mice.<p><b>Copyright information:</b></p><p>Taken from "Lactation failure in Src knockout mice is due to impaired secretory activation"</p><p>http://www.biomedcentral.com/1471-213X/8/6</p><p>BMC Developmental Biology 2008;8():6-6.</p><p>Published online 23 Jan 2008</p><p>PMCID:PMC2266720.</p><p></p

Lactation failure in Src knockout mice is due to impaired secretory activation-7

, Src+/- (lanes 4–6 and 13–15) and Src+/+ (lanes 7–9 and 16–18) mice were cultured on Matrigel in growth medium for 3 days then the medium was changed to differentiation medium and the cells were cultured for a further 24 or 72 hours in the presence or absence of prolactin. Cellular proteins were extracted by lysis in NET buffer. A) Immunoblotting was performed to detect phosphorylation of Src (tyrosine) (top panel), total levels of Src protein (middle panel) and the amount of ERK 1&2 (bottom panel) as a loading control. B) Immunoblotting was performed to detect phosphorylation of STAT5 (tyrosine) (top panel), total levels of STAT5 (middle panel) and the amount of ERK 1&2 (bottom panel) as a loading control. C) Immunoblotting was performed to detect β-casein expression (top panel), the levels of Raf and the amount of ERK 1&2 as loading controls (middle and bottom panels).<p><b>Copyright information:</b></p><p>Taken from "Lactation failure in Src knockout mice is due to impaired secretory activation"</p><p>http://www.biomedcentral.com/1471-213X/8/6</p><p>BMC Developmental Biology 2008;8():6-6.</p><p>Published online 23 Jan 2008</p><p>PMCID:PMC2266720.</p><p></p

Lactation failure in Src knockout mice is due to impaired secretory activation-11

+/- (B, E, H and K) and Src-/- (C, F, I and L) mice at P6 (A-C), P12 (D-F) and P18 (G-I). Histological sections from mammary glands at P18 are also shown for each genotype (J-L). In panels A to I scale bars represent 5 mm. In panels J to L scale bars represent 100 μm.<p><b>Copyright information:</b></p><p>Taken from "Lactation failure in Src knockout mice is due to impaired secretory activation"</p><p>http://www.biomedcentral.com/1471-213X/8/6</p><p>BMC Developmental Biology 2008;8():6-6.</p><p>Published online 23 Jan 2008</p><p>PMCID:PMC2266720.</p><p></p

Lactation failure in Src knockout mice is due to impaired secretory activation-1

C+/- (B, E, H and K) and Src-/- (C, F, I and L) mice at P6 (A-C), P12 (D-F) and P18 (G-I). Histological sections from mammary glands at P18 are also shown for each genotype (J-L). In panels A to I scale bars represent 5 mm. In panels J to L scale bars represent 100 μm.<p><b>Copyright information:</b></p><p>Taken from "Lactation failure in Src knockout mice is due to impaired secretory activation"</p><p>http://www.biomedcentral.com/1471-213X/8/6</p><p>BMC Developmental Biology 2008;8():6-6.</p><p>Published online 23 Jan 2008</p><p>PMCID:PMC2266720.</p><p></p

Lactation failure in Src knockout mice is due to impaired secretory activation-8

Mice are shown at I 1 (A, G), I2 (B, H), I4 (C, I), I6 (D, J), I8 (E, K) and I14 (F, L). Scale bars represent 250 μm.<p><b>Copyright information:</b></p><p>Taken from "Lactation failure in Src knockout mice is due to impaired secretory activation"</p><p>http://www.biomedcentral.com/1471-213X/8/6</p><p>BMC Developmental Biology 2008;8():6-6.</p><p>Published online 23 Jan 2008</p><p>PMCID:PMC2266720.</p><p></p