Differentiation Of Mouse Mammary Gland Research Articles

Involvement of autocrine mechanism of transforming growth factor-beta in the functional differentiation of pregnant mouse mammary gland.

We studied the presence and possible role of the autocrine mechanism of transforming growth factor-beta (TGF-beta) in pregnant mouse mammary gland. Northern blot analysis revealed the expression of the TGF-beta 1 gene transcript at 2.5 kb in mammary epithelial cells isolated from virgin and mid-pregnant mice. The TGF-beta activity was higher in the conditioned medium from mid-pregnant mouse mammary explants than that from virgin explants by a bioassay system using mink lung epithelial cells. A binding study using [125I]TGF-beta 1 as a ligand showed that pregnant mouse mammary epithelial cells possessed a single class of high-affinity TGF-beta 1 binding sites (Kd = 28.0 pmol/l, 1.2 x 10(4) sites per cell). These results suggested the presence of a TGF-beta autocrine mechanism in pregnant mouse mammary epithelial cells. Next, we examined the effect of TGF-beta 1 on the functional differentiation of pregnant mouse mammary gland. Transforming growth factor-beta 1 inhibited alpha-lactalbumin production in cultured mammary explants from mid-pregnant mice in a dose-dependent manner without inhibiting DNA synthesis. All these results suggest that TGF-beta may play a role in regulating the functional differentiation of mouse mammary glands during pregnancy.

1,25-Dihydroxycholecalciferol receptor regulation in hormonally induced differentiation of mouse mammary gland in culture.

Mammary explants from pregnant mouse cultured in the presence of insulin, cortisol, and PRL express their differentiated function by producing milk proteins such as casein. Sucrose density gradient analysis of high salt extract of cultured explants showed the presence of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3 specific binding activity sedimenting at 3.3 S. Specific 1,25-(OH)2D3 binding activity in mammary explants varied as a function of the hormonal milieu in culture. The highest activity was found in explants cultured in the presence of insulin, cortisol, and PRL, whereas only a marginal activity was present in explants cultured with insulin. The comparison of 1,25-(OH)2D3 binding activity in explants cultured with insulin and with the three hormone combination by Scatchard plot analysis indicated that the synergistic actions of insulin, cortisol, and PRL increased the number of 1,25-(OH)2D3 specific binding proteins as much as 6-fold; however, the affinity constant of the binding protein was relatively constant in the two systems. Time course studies showed that the increase in 1,25-(OH)2D3 binding activity was detectable as early as 12 h after the addition of cortisol and PRL to insulin-primed explants. The increase was inhibited by the presence of actinomycin D and cycloheximide, suggesting that the hormonal stimulation of 1,25-(OH)2D3 binding activity involves both transcriptional and translational process. These results indicate that insulin, cortisol, and PRL stimulate the specific 1,25-(OH)2D3 binding activity during the induction of mammary functional differentiation when milk protein synthesis takes place.

Variation of DNA polymerase activities and DNA synthesis in mouse mammary gland during pregnancy and early lactation

The rate of DNA synthesis and the activity of DNA polymerases and thymidine kinase were measured during the endocrine-regulated cellular growth and differentiation of mouse mammary gland. Using specific assays, the activity of the DNA polymerases, α, β and γ, was determined in tissue extracts of mammary glands of mice at various stages of pregnancy and early lactation. In addition, extracts of the mammary tissue of virgin, mid-pregnant and early lactating mice were fractionated on sucrose density gradients, and the activity of DNA polymerase α and β was assayed in the gradient fractions. It was demonstrated that the activity of DNA polymerase α varied considerably during pregnancy and after parturition, showing peaks on day 12 of pregnancy and days 3–4 of lactation. In pregnancy, there was an apparently parallel correlation between the amount of DNA-polymerase-α activity and the rate at which the cells incorporated labelled thymidine into DNA, but the relationship was less clearly expressed during early lactation. The activity of the DNA polymerases, β and γ as well as that of thymidine kinase showed little variation during these periods. Thus, in the developing mammary gland, no correlation was found between DNA synthesis and the activity of the DNA polymerases, β and γ, or thymidine kinase.

Involvement of collagen formation in the hormonally induced functional differentiation of mouse mammary gland in organ culture.

In mammary gland organ culture from midpregnant mice, synergistic actions of insulin, cortisol, and prolactin stimulate differentiation of mammary epithelium and induce the synthesis of the milk proteins casein and alpha-lactalbumin. In the present study we examined the production of collagen and its function in the hormone-dependent development of mammary gland in vitro. The measurement of collagen production by the hydroxyproline assay and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the accumulation of collagen type I and type III in cultured mammary explants increased with the addition of insulin, cortisol, and prolactin to a chemically defined medium. When an analog of proline, L-azetidine-2-carboxylic acid (LACA), was added at a concentration of 80 microgram/ml with insulin, cortisol, and prolactin at the beginning of culture, collagen production in cultured tissue was inhibited by 75% during a 3-day incubation period. This agent also inhibited the synthesis of casein and alpha-lactalbumin by about 77 and 70%, respectively. The inhibitory effect of LACA could be prevented by L-proline; concomitant addition of L-proline (80 microgram/ml) with LACA (80 microgram/ml) resulted in complete restoration of milk protein synthesis to normal levels. Measurement of the amount of milk protein mRNAs in mammary explants by a cell-free translation assay demonstrated that LACA reduced the hormone-stimulated accumulation of casein mRNA and alpha-lactalbumin mRNA by 79 and 76%, respectively. LACA, however, produced little inhibition of DNA synthesis in cultured tissue. These results suggest that collagen production may be involved in the phenotypic expression of milk protein genes during hormonal induction of mammary epithelial differentiation in vitro.

Expression of a Phenotype of Normal Differentiation in Cultured Mammary Glands is Promoted by Epidermal Growth Factor and Blocked by Cyclic Adenine Nucleotide and Prostaglandins

Chemical inducers of development have been found to alter the functional normal differentiation of mouse mammary gland in culture. Normal development, indicated by formation of lobuloalveolar structures, and normal differentiation, evidenced as casein synthesis, were induced in cultured whole mammary glands of virgin Balb/c mice using the hormones insulin, prolactin, aldosterone, and hydrocortisone (IPAH) in serum-free medium. Following removal of all added hormones except insulin, which is needed for general maintenance of mammary epithelial cells, the lobuloalveoli involuted (regressed) as a result of the death of their differentiated cells. A second round of normal development and differentiation was then elicited by the combination of epidermal growth factor (EGF) and IPAH. The cultured glands were found to synthesize seven molecular species of casein in both the first and second normal developmental cycles. Another series of experiments examined the effects of abnormal development and aberrant differentiation on the normal differentiation phenotype. Mammary glands undergoing the first round of normal development and differentiation were simultaneously induced to undergo an abnormal course of development (squamous metaplasia) and differentiation (excessive keratin production) by the additional use of agents known to elevate the level of intracellular cyclic AMP, namely dibutyryl cyclic AMP (dbcAMP), prostaglandins (PG) E1, E2 and B1, and papaverine (pap). The complete mixture of dbcAMP, pap, and the PGs blocked casein synthesis. DbcAMP and pap, in the absence of PG, markedly depressed the production, while the three PGs alone were almost totally inhibitory. Two indications emerge from the present findings: First, EGF appears to play a positive inductive role in the functional normal differentiation of the mammary gland. Second, cyclic adenine nucleotide and specific prostaglandins may mediate the negative de-inductive regulation of that process.

A role of thyroid hormones in differentiation of mouse mammary gland [formula omitted

Addition of thyroxine or triiodothyronine to cultures of mammary gland explants in the presence of insulin, hydrocortisone and prolactin, results in a selective enhancement of the activity of the milk protein, α-lactalbumin. This effect, which is specific for the L-isomer of the thyroid hormones, is not mediated through diffusible activators of the enzyme activity.

DNA synthesis in the absence of cell reproduction during functional differentiation of mouse mammary gland

DNA synthesis, mitotic indices and DNA content of mouse mammary gland at different stages of pregnancy and lactation were determined. 3H-Thymidine specific activity of mid-pregnancy mammary gland DNA was 3 times higher than the lactating gland at its peak period of DNA replication. But, the frequency of mammary cells incorporating 3H-Thymidine was equally high in both the pregnant and lactating tissues. Consistent with its low specific activity of the DNA, the average number of silver grains per nucleus of the 6-day lactating gland was nearly 60% less than that of the pregnant mammary tissue. The nucleolus/chromatin grain ratio in the lactating tissue was significantly greater than in the mammary gland of pregnancy. In spite of the consistent rise of the labeling index between the 3rd and 6th days of lactation, the low mitotic index of the lactating gland virtually remained unaltered during this interval, whereas the high mitotic index of the pregnant tissue was proportionate to its labeling index. The total mammary tissue DNA increased significantly from the 15th day of pregnancy to the 6th day of lactation. A rise of nearly 30% per nuclear DNA content was also evident from 3 to 6 days of lactation. The results indicate that DNA synthesis in a high proportion of cells in the lactating gland may not represent premitotic DNA replication and suggest a partial replication of the cellular genome during early lactogenesis. The significance of these results with respect to “gene amplification” has been discussed.

Steroid structural requirements for mammary gland differentiation in vitro.

The structural characteristics of the steroid molecule required for mammary gland differentiation in vitro have been studied. For moderate activity at 3 ×10-7M, the 4-pregnen nucleus must have the following substituents: 1) a keto group at position 20; and 2) 11β-ol or 21- ol. It is not known whether the 3-keto group is required for activity. For high activity at 3 × 10-7M, in addition to a keto group at position 20, the requirements include an a-hydroxyl group at position 17 or an aldehyde group at 18, and either 11β-ol or 11-one. Introduction of an 11α-ol group into Reichstein’s S, e.g., 11α-hydrocortisone, obliterates the activity of the parent compound. Reduction of the Δ4 double bond of 11β- hydrocortisone to the 5β-dihydro form abolishes all activity, although a small amount of activity is retained in the 5α-dihydro form. The results obtained are consistent with the possibility that both aldosterone and corticosterone are involved in mouse mammary gland differentiation in vivo. (Endocrinology 80: 11...

Hormone-dependent differentiation of mammary gland: sequence of action of hormones in relation to cell cycle.

Differentiation of mouse mammary gland in vitro requires insulin, hydrocortisone, and prolactin. The epithelial cells must first divide in order to synthesize casein in response to these hormones. Insulin is required for the initiation of DNA synthesis and is also necessary during G(1) phase (after mitosis). Prolactin can elicit the overt differentiative responses after mitosis. Activity of hydrocortisone precedes that of prolactin, that is, after mitosis it is not capable of eliciting the differentiative response.