HORMONAL EFFECTS ON RAT MAMMARY GLAND IN VITRO
Authors: Peters J.M. , van Marle J. , Ariëns A.Th. 
Published: October 2009

ABSTRACT
The mammogenic and lactogenic effect of various hormones on rat mammary gland has been studied in vitro using an organ culture technique. The effects have been assessed mainly by light microscopy. The study has been confined mainly to the effects and interactions of the lactogenic protein hormones, corticosteroids and progesterone. It establishes the importance of the presence of insulin and serum and of the type of serum for the hormonal effects in vitro. These effects have been studied mainly on the explants obtained from 13 days pregnant rats. In addition explants from non-pregnant rats and at various stages of pregnancy and lactation were studied.

2.1. Experiments with mammary glands of pregnant rats

Prolactin
The mammogenic effect of prolactin manifested itself as increased alveolar development, and the presence of mitotic activity and thymidine incorporation. A high concentration of virgin rat serum in the medium was necessary for the growth response. Human, rabbit, fetal calf or horse serum could not or only partly replace rat serum. The mammogenic effect was obtained with rat, human, bovine and to a lesser extent with ovine prolactin preparations.
High mitotic activity was present in the pregnant rat mammary gland explants after 24 h of culture especially if insulin was added. Prolactin was not needed for the occurrence of this wave of mitoses. The effect of prolactin on mitotic activity became distinct after 72 h. Prolactin also increased the thymidine incorporation after 24 and 72 h of culture.
Although some effect was observed at concentrations between 10 to 27 ng/ml rat prolactin in the medium, a sharp increase in response occurred in the range of 27 to 115 ng/ml medium. At higher concentrations the response obtained in a 3 days culture did not increase. The prolactin concentration in the medium remained fairly constant during the three days of culture.
A mammogenic response was also obtained with lactating rat serum and it correlated with the prolactin level measured in the sera by radioimmunoassay.
In addition to a proliferative effect prolactin had a weak effect on secretion: it promoted the accumulation of small intracellular fat droplets and promoted some intraluminal secretion and formation of intracellular fat vacuoles locally in the explants. However, the secretory response remained incomplete, even after 6 days of culture.
Progesterone, like prolactin had a proliferative effect on pregnant rat mammary gland in vitro in a medium containing virgin rat serum and insulin. At low dose levels a synergism could be shown for the two hormones. Testosterone had a weak proliferative effect, but a synergism with prolactin was not observed. An interaction between prolactin on the one hand and oestradiol (except for an inhibition at high concentrations) or thyroxin, on the other hand, could not be demonstrated.
A high concentration of insulin was favourable for an optimal effect of prolactin, but a proliferative effect could still be demonstrated in the absence of insulin added to the medium.

Human placental lactogen
Human placental lactogen (hPL) has a proliferative and some lactogenic effect on pregnant rat mammary gland in vitro. For the proliferative effect rat serum must be added to the medium. HPL combined with virgin rat serum can simulate - with some quantitative differences - the effect of 13 days pregnant rat serum in vitro. HPL was approximately four times less active than rat prolactin in a medium containing rat serum. The activity present in one ml of 13 days pregnant rat serum was comparable to 1 to 5 μg rat prolactin added to one ml of virgin rat serum.
Rabbit anti-hPL serum had a toxic effect on the rat mammary gland explants. No conclusive evidence was found for a cross-reactivity between hPL and rat chorionic mammotrophin, present in pregnant rat serum.

Growth hormone, ACTH
Human and porcine growth hormone preparations showed mammogenic activity in vitro, but their activity was much less - on a weight basis - than that of rat prolactin. Evidence for an effect of adrenocorticotropic hormone was equivocal.

Corticosteroids
The corticosteroids induce a secretory response: intraluminal secretion and intracellular fat vacuoles appear. Rat serum was needed for an optimal effect. Heterologous sera could not replace rat serum. A maximum secretory response with cortisol was obtained when the culture medium contained rat serum as well as insulin and a lactogenic protein hormone. When only one of these factors was present the response was virtually absent. When two factors were present a response developed but it remained submaximal.
Prolactin potentiated the corticosteroid effect. The potentiation was dose-related. A similar effect was exerted by growth hormone preparations and human and rat lactogenic hormones of placental origin. The cortisol-potentiating effect of pregnant rat serum appeared on day 8 of pregnancy and disappeared to a large extent shortly after parturition. The cortisol-potentiating effect of lactating rat serum was less predictable.
An effect of adrenocorticotropic hormone, progesterone, testosterone or thyroxin on the cortisol-induced response could not be demonstrated. Oestradiol may slightly stimulate the secretory effect.
The mammary gland of virgin rats or of rats early in pregnancy appeared to be less sensitive to cortisol and aldosterone than the mammary gland of rats late in pregnancy. The secretory response to cortisol developed in 13 days pregnant rat explants only after two days of culture and reached a maximum around day 6. The presence of a lactogenic protein hormone did not accelerate the response. The lag period of the cortisol effect was reduced in explants of rats later in pregnancy. The reaction to the corticosteroids was dose-related. The addition of 0.17 μg cortisol per ml medium gave a response. Both corticosterone and aldosterone produced an effect similar to that of cortisol, but higher concentrations were needed. Some effect could be obtained with deoxycorticosterone but at relatively high concentrations. With the corticosteroids added, the number of mitoses induced by the mammogenic hormones tended to decrease.

Progesterone
Progesterone had a mammogenic effect, which appeared to be less than that of the lactogenic protein hormones. It had no lactogenic effect. High concentrations of oestradiol suppressed the effect of progesterone. An interaction with testosterone was not found. Insulin was needed for a maximum effect.

2.2. Experiments with mammary glands of lactating rats
When mammary explants obtained from lactating rats were cultured in a medium to which only virgin rat serum and insulin had been added, secretion persisted only on the first day of culture. High concentrations of glucose in the medium stimulated the appearance of fat vacuoles. On the second day a wave of mitoses was observed. Thereafter a rapid involution followed.
Prolactin added to a medium containing virgin rat serum had a proliferative effect on lactating rat explants: it increased thymidine incorporation and mitotic activity on day 2 and 3 of culture. It had also some effect on secretion: it seemed to decelerate the breakdown of the accumulated secretion products. Cortisol had a strong effect on secretion: in its presence the secretory aspect of lumina filled with eosinophilic material, and fat vacuoles was maintained for 3 days. This effect was observed with mammary explants obtained at various stages of lactation. A synergism between prolactin and cortisol could not be demonstrated.
After addition of lactating rat serum instead of virgin rat serum the same results were obtained. When prolactin was replaced by the placental lactogen present in pregnant rat serum, an effect on mammary explants from lactating rats could not be demonstrated, while a synergism between the lactogen and cortisol could still be shown in explants from late pregnant rats. An effect of progesterone together with oestradiol was not seen either in the presence or in the absence of cortisol.
A sequence of hormonal control is proposed for the development of the mammary gland and the induction of secretion. In this sequence the lactogenic protein hormones and progesterone regulate proliferation, the lactogenic hormones (with the corticosteroids) regulate the morphological differentiation, while for a full secretory response increased corticosteroid levels are necessary. The lactogenic protein hormones potentiate the corticosteroid effect, but cannot induce secretion at high concentrations. The proposed sequence may explain some of the developments occurring in vivo.
Rat serum and insulin are essential for the in vitro condition. Arguments for the assumption that only "stem cells" are able to divide, were not found.