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

Watkin H, Richert MM, Lewis A, Terrell K, McManaman JP, Anderson SM - BMC Dev. Biol. (2008)

Bottom Line: Failed secretory activation results in precocious involution in the mammary glands of Src-/- even when pups were suckling.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.Src appears to be required for increased expression of the prolactin receptor and successful downstream signaling, and alveolar cell organization.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Colorado Health Sciences Center, Research Complex I, South Tower, Mail Stop 8104, 12801 East 17th Avenue, Aurora, CO 80045, USA. harriet.watkin@uchsc.edu

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

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Serum prolactin levels are normal in Src knockout mice however, prolactin receptor expression is reduced. A) Blood was drawn from Src-/-, Src+/- and Src+/+ mice at P17 and L2. Prolactin levels were measured using a radioimmune assay and the amount of PRL plotted as the mean (± SEM) of 5 mice for each genotype at the indicated developmental stages. Welch's t test was used to evaluate the statistical significance (defined as P < 0.05). Src-/- to Src+/+ at P17 P = 0.26, Src+/- to Src+/+ at P17 P = 0.27. Src-/- to Src+/+ at L2 P = 0.89, Src+/- to Src+/+ at L2 P = 0.08. B) Total RNA was isolated from the number 4 mammary gland of wildtype and knockout mice at P18, L2, L9, and I 2; three mice were used per genotype and developmental stage. cDNA was synthesized from 1 μg of total RNA and quantitative RT-PCR was performed using primers and probe specific for the long isoform of the prolactin receptor. PRLR message levels were normalized to GAPDH for each sample and the graph represents the mean (± SEM) relative amount of the triplicate tissue samples. C) The number 4 mammary gland was removed from Src-/- and Src+/+ mice at P18 (lanes 1–6), L 2 (lanes 7–12) and L 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 the total amount of PRLR expression (top panel), and the amount of actin, as a loading control (bottom panel).
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Figure 6: Serum prolactin levels are normal in Src knockout mice however, prolactin receptor expression is reduced. A) Blood was drawn from Src-/-, Src+/- and Src+/+ mice at P17 and L2. Prolactin levels were measured using a radioimmune assay and the amount of PRL plotted as the mean (± SEM) of 5 mice for each genotype at the indicated developmental stages. Welch's t test was used to evaluate the statistical significance (defined as P < 0.05). Src-/- to Src+/+ at P17 P = 0.26, Src+/- to Src+/+ at P17 P = 0.27. Src-/- to Src+/+ at L2 P = 0.89, Src+/- to Src+/+ at L2 P = 0.08. B) Total RNA was isolated from the number 4 mammary gland of wildtype and knockout mice at P18, L2, L9, and I 2; three mice were used per genotype and developmental stage. cDNA was synthesized from 1 μg of total RNA and quantitative RT-PCR was performed using primers and probe specific for the long isoform of the prolactin receptor. PRLR message levels were normalized to GAPDH for each sample and the graph represents the mean (± SEM) relative amount of the triplicate tissue samples. C) The number 4 mammary gland was removed from Src-/- and Src+/+ mice at P18 (lanes 1–6), L 2 (lanes 7–12) and L 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 the total amount of PRLR expression (top panel), and the amount of actin, as a loading control (bottom panel).

Mentions: The hormone PRL was first identified by its ability to stimulate mammary gland development and lactation in rabbits and it has since been demonstrated as an essential modulator of proliferation and differentiation of alveoli during pregnancy and milk protein expression during lactation [39-41]. Due to the critical role of PRL in lactogenesis, the amount of serum PRL in Src+/+, Src+/- and Src-/- mice was quantitated to determine whether a reduction in serum PRL could account for the lactation failure in Src-/- mice. Serum PRL was determined in five Src+/+, Src+/- and Src-/- mice at P17 and L2 and were found to be comparable in both the Src+/+ and Src-/- mice (Figure 6A) thus eliminating diminished serum PRL as the mechanism underlying the observed lactation failure in Src-/- mice. It is interesting to note that serum PRL levels were elevated in the Src+/- mice at both times examined, however the increase is not significant.


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

Watkin H, Richert MM, Lewis A, Terrell K, McManaman JP, Anderson SM - BMC Dev. Biol. (2008)

Serum prolactin levels are normal in Src knockout mice however, prolactin receptor expression is reduced. A) Blood was drawn from Src-/-, Src+/- and Src+/+ mice at P17 and L2. Prolactin levels were measured using a radioimmune assay and the amount of PRL plotted as the mean (± SEM) of 5 mice for each genotype at the indicated developmental stages. Welch's t test was used to evaluate the statistical significance (defined as P < 0.05). Src-/- to Src+/+ at P17 P = 0.26, Src+/- to Src+/+ at P17 P = 0.27. Src-/- to Src+/+ at L2 P = 0.89, Src+/- to Src+/+ at L2 P = 0.08. B) Total RNA was isolated from the number 4 mammary gland of wildtype and knockout mice at P18, L2, L9, and I 2; three mice were used per genotype and developmental stage. cDNA was synthesized from 1 μg of total RNA and quantitative RT-PCR was performed using primers and probe specific for the long isoform of the prolactin receptor. PRLR message levels were normalized to GAPDH for each sample and the graph represents the mean (± SEM) relative amount of the triplicate tissue samples. C) The number 4 mammary gland was removed from Src-/- and Src+/+ mice at P18 (lanes 1–6), L 2 (lanes 7–12) and L 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 the total amount of PRLR expression (top panel), and the amount of actin, as a loading control (bottom panel).
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Figure 6: Serum prolactin levels are normal in Src knockout mice however, prolactin receptor expression is reduced. A) Blood was drawn from Src-/-, Src+/- and Src+/+ mice at P17 and L2. Prolactin levels were measured using a radioimmune assay and the amount of PRL plotted as the mean (± SEM) of 5 mice for each genotype at the indicated developmental stages. Welch's t test was used to evaluate the statistical significance (defined as P < 0.05). Src-/- to Src+/+ at P17 P = 0.26, Src+/- to Src+/+ at P17 P = 0.27. Src-/- to Src+/+ at L2 P = 0.89, Src+/- to Src+/+ at L2 P = 0.08. B) Total RNA was isolated from the number 4 mammary gland of wildtype and knockout mice at P18, L2, L9, and I 2; three mice were used per genotype and developmental stage. cDNA was synthesized from 1 μg of total RNA and quantitative RT-PCR was performed using primers and probe specific for the long isoform of the prolactin receptor. PRLR message levels were normalized to GAPDH for each sample and the graph represents the mean (± SEM) relative amount of the triplicate tissue samples. C) The number 4 mammary gland was removed from Src-/- and Src+/+ mice at P18 (lanes 1–6), L 2 (lanes 7–12) and L 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 the total amount of PRLR expression (top panel), and the amount of actin, as a loading control (bottom panel).
Mentions: The hormone PRL was first identified by its ability to stimulate mammary gland development and lactation in rabbits and it has since been demonstrated as an essential modulator of proliferation and differentiation of alveoli during pregnancy and milk protein expression during lactation [39-41]. Due to the critical role of PRL in lactogenesis, the amount of serum PRL in Src+/+, Src+/- and Src-/- mice was quantitated to determine whether a reduction in serum PRL could account for the lactation failure in Src-/- mice. Serum PRL was determined in five Src+/+, Src+/- and Src-/- mice at P17 and L2 and were found to be comparable in both the Src+/+ and Src-/- mice (Figure 6A) thus eliminating diminished serum PRL as the mechanism underlying the observed lactation failure in Src-/- mice. It is interesting to note that serum PRL levels were elevated in the Src+/- mice at both times examined, however the increase is not significant.

Bottom Line: Failed secretory activation results in precocious involution in the mammary glands of Src-/- even when pups were suckling.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.Src appears to be required for increased expression of the prolactin receptor and successful downstream signaling, and alveolar cell organization.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Colorado Health Sciences Center, Research Complex I, South Tower, Mail Stop 8104, 12801 East 17th Avenue, Aurora, CO 80045, USA. harriet.watkin@uchsc.edu

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus