Yale J. Topper

A histological and biochemical study of hormone-dependent differentiation of mammary gland tissue in vitro.

Abstract Mammary gland tissue from pregnant mice undergoes specific changes in structure and function when incubated in vitro on synthetic medium containing insulin, hydrocortisone, and prolactin. This combination of hormones increases the rate of synthesis of one group of phosphoproteins, casein, 300–550%, while the synthesis of other phosphoproteins is only slightly stimulated. This selective biosynthetic response is paralleled by specific histological changes in the alveolar epithelium of the gland. The combination of specific changes in cell and tissue structure and selective stimulation of one group of milk proteins constitutes an example of cellular differentiation under hormonal control.

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 G1 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.

Hormone-dependent development of milk protein synthesis in mammary gland in vitro.

Abstract 1. 1. Insulin, hydrocortisone and prolactin acting synergistically selectively stimulate in vitro the synthesis of milk proteins by the pregnant mouse mammary gland. 2. 2. Synthesis of soluble cytoplasmic non-milk protein derived from epithelial cells is stimulated maximally by insulin alone and is believed to reflect proliferation. 3. 3. There is a coordinate increase in the synthesis of the casein components throughout mammary gland development. 4. 4. The increase of α-lactalbumin and β-lactoglobulin synthesis is not coordinate throughout mammary gland development. Thus as the lactational state is approached the ratio β-lactoglobulin to α-lactalbumin increases. 5. 5. Culture of mid-pregnancy mammary gland demonstrates that the 3 hormone combination effects stimulation of casein and whey protein synthesis of the same magnitude, with the same time course, and that the ratio of whey proteins to casein synthesized corresponds well with the ratio found in mouse milk.

Multiple hormone interactions in the development of mammary gland in vitro.

Publisher Summary Multiple hormone dependencies provide the investigator with opportunities for chemical dissection that facilitate analysis of sequential events occurring during development. This, in turn, aids the delineation of the action of individual hormones. This chapter discusses some of the hormone-mediated processes that occur during development of the mouse mammary gland in vitro. During the first three postnatal weeks, the cells in the epithelial cord undergo little, if any, proliferation. In the course of the next month or two, presumably under the influence of larger amounts of estrogen and progesterone during puberty, the epithelial cells multiply so as to form ducts and primitive alveoli along the connective tissue septa. With the attainment of sexual maturity at the age of 2–3 months, mammary epithelial-cell proliferation virtually ceases. During pregnancy and the early postpartum period, epithelial growth resumes so that the lactating gland contains ducts and alveoli in profusion. Functional and cytological innovations also take place at this time. Within a few weeks after the end of lactation, the gland reassumes the histological and functional profile characteristic of the tissue in the mature nonpregnant animal. The cycle is repeated with the onset of another pregnancy. In all the studies described in the chapter, an organ-culture method was used. Expiants were supported on siliconized lens paper that floated on Medium 199 containing penicillin and various combinations of crystalline beef insulin, hydrocortisone, and ovine prolactin.

The induction of milk protein synthesis in post-mitotic mammary epithelial cells exposed to prolactin

Abstract Prolactin causes the emergence of casein synthesis by mammary epithelial cells formed in vitro in the presence of insulin and hydrocortisone. Prolactin similarly exerts a stimulatory effect on the synthesis of α-lactalbumin and β-lactoglobulin.

Transcriptional and post-transcriptional roles of glucocorticoid in the expression of the rat 25,000 molecular weight casein gene.

In the presence of hydrocortisone, plus insulin and prolactin, rat mammary explants transcribe the 25K casein gene, and the half-life of the transcript is about 20 h. The first detectable effect of the withdrawal of glucocorticoid is reduction of the half-life of the casein mRNA to about 1 h, with full retention of the rate of transcription of the casein gene. A later effect of the withdrawal of glucocorticoid is loss of the ability to transcribe the casein gene. Both aberrations can be rectified by hydrocortisone in vitro. The instability of the 25K casein mRNA in the absence of glucocorticoid appears to be related to degradation in the nucleus. Accumulation of actin mRNA in the nuclear fraction is not dependent on glucocorticoid. The results indicate that glucocorticoid exerts a profound, selective effect on the stabilization of rat 25K casein mRNA, and is essential for the transcription of the casein gene. These effects can be dissociated temporally from one another.

Casein biosynthesis: Evidence for phosphorylation of precursor proteins

Abstract 1. 1. To determine whether casein is phosphorylated after completion of the polypeptide chains, the incorporation of [ 32 P]P i in the presence of puromycyin was studied using mouse mammary gland explants. The rate of incorporation of [ 32 P]P i into casein was much greater than that of 14 C-labeled amino acids for the first 2 h, after which [ 32 P]P i incorporation proceeded in parallel with 14 C-labeled amino acid incorporation. 2. 2. The proteins made in the presence of puremycin and isolated by rennin-Ca 2+ precipitation are authentic casein, as shown by starch-gel electrophoresis. The casein electrophoretic components are phosphorylated at approximately the same relative rates in the presence or absence of puromycin. 3. 3. Chase experiments revealed no exchanged of the 32 P in the biosynthetic casein. 4. 4. It is concluded that a pool of unphosphorylated or incompletely phosphorylated casein exists in mammary gland, and that phosphorylation of the polypeptide chains is a major biosynthetic mechanism for incorporation of phosphate into casein.

PROGESTERONE EFFECTS ON GALACTOSE METABOLISM IN PREPUBERTAL PATIENTS WITH CONGENITAL GALACTOSEMIA AND IN RATS MAINTAINED ON HIGH GALACTOSE DIETS

In a recent publication (1) it was reported that in zitro addition of certain steroid hormones causes a 200 to 300 per cent stimulation of galactose-1-C14 oxidation to C1402 by rabbit liver slices. Progesterone, androsterone and testosterone produced maximal stimulatory effects. Further data have been obtained (2) which demonstrate a) that of many tissues tested, only liver and intestine respond to the steroids; b) the same steroid specific effect can be obtained in a 100,000 x G supernatant fraction of rabbit liver homogenates; and c) the in vitro steroid stimulation of galactose oxidation is mediated by an indirect stimulation of the uridinediphosphogalactose-4-epimerase (UDPGal4-epimerase) reaction. During the course of these investigations some in vivo effects of progesterone on galactose oxidation were investigated. The results of progesterone administration to three prepubertal galactosemic subjects and to young rats maintained on high galactose diets serve as the basis for this report.

A direct effect of prolactin and placental lactogen on mammary epithelial nuclei

Abstract Placental lactogen and prolactin stimulate the rate of RNA synthesis by nuclei isolated from mammary epithelial cells. The effect is tissue specific. It is suggested that these protein hormones may perform some of their functions within the mammary cell.

Retention of glucocorticoid by isolated mammary tissue may complicate interpretation of results from invitro experiments

When mammary gland explants from mid-pregnant rats were incubated with insulin (5 μg/ml) and [3H]cortisol (5 μg/ml) for one day, the tissue accumulated 1.69 μg cortisol/g wet tissue. During a second incubation with insulin and prolactin (5 μg/ml), only 20% of the steroid was lost per day. Such retention of glucocorticoid had an important biological consequence: the tissue exposed for one day to insulin and cortisol showed a transient stimulation of casein synthesis during a subsequent, five-day incubation with insulin and prolactin. No casein synthesis was detected, if the first culture medium contained only insulin. In conclusion, mammary gland explants from mid-pregnant rats require a glucocorticoid for casein synthesis, but this requirement may be obscured if the explants are initially incubated in medium containing cortisol, since they are capable of accumulating and retaining this steroid. Similar interpretative difficulties may arise in studies on other steroid-tissue relationships.