Induction of mammary prolactin receptors and lactose synthesis after ovariectomy in the pregnant mouse.

The development of prolactin receptors in the mammary gland after ovariectomy was investigated in pregnant KA mice. Mice were ovariectomized on day 13 of pregnancy and used for the determination of the amount of specific binding of 125I-labelled prolactin to the mammary tissue, and the contents of lactose and nucleic acids in the mammary gland 0, 8, 24, and 72 hr after the operation. The specific binding of 125I-labelled prolactin, lactose and RNA contents in the mammary gland remained low until 8 hr, sharply increased 24 hr and decreased 72 hr after ovariectomy. When ovariectomized mice were treated with 0.2 mg progesterone, pregnancy was maintained and an increase (1.5-fold) in the amount of specific binding was observed with an increase of lactose content. Five mg progesterone completely inhibited lactose synthesis. Cortisol administered with progesterone did not show any specific change at the dose used (0.5 to 10 mg). Although the amount of specific binding was also increased after hysterectomy, this increase (2-fold) did not fully cover the increase after ovariectomy (3-fold). These results suggest that the recepter site for prolactin is induced before the initiation of lactose synthesis caused by ovariectomy during pregnancy.     

Suckling-induced prolactin release potentiates mifepristone-induced lactogenesis in pregnant rats

Suckling, starting at 19:00 h on Day 18 of pregnancy, induced a significant increase in serum prolactin concentration at 20:00 h on Day 19 of pregnancy, but no increase in mammary gland casein or lactose content. Mifepristone (2 mg/kg) injection at 08:00 h on Day 19 of pregnancy induced significant increases in casein, but not in lactose, 24 h after administration. Mifepristone alone did not induce prolactin secretion, indicating that lactogenesis was induced by placental lactogen in the absence of progesterone action. When mifepristone was injected into suckling rats, serum prolactin concentrations were higher than in the untreated suckling rats. Casein in these rats increased significantly 12 h after mifepristone administration and lactose at 24 h after. If the suckling mifepristone-treated rats were given two injections of bromocriptine (1.5 mg/kg) at 12:00 h on Days 18 and 19 of pregnancy, serum prolactin concentrations were not increased by suckling, but casein and lactose concentrations in the mammary gland showed values similar to those obtained in the mifepristone-treated non-suckling rats. Mifepristone can therefore potentiate suckling-induced prolactin release in pregnant rats, demonstrating a direct central inhibitory action of progesterone on prolactin secretion. This suckling-induced prolactin secretion, unable to induce casein or lactose synthesis in the presence of progesterone, enhanced significantly synthesis of these milk components in the absence of progesterone action (rats treated with mifepristone). Fatty acid synthase, which is stimulated by the suckling stimulus in lactating rats, was not modified by mifepristone or suckling in pregnant rats.     

Lactogenesis induced by ovariectomy in pregnant rats and its regulation by oestrogen and progesterone

Ovariectomy on day 19 of pregnancy augmented galactosyl transferase activity 24 h after surgery preceding by 6 h the significant alpha-lactalbumin accumulation. Progesterone, injected immediately after ovariectomy showed a clear inhibitory effect on both galactosyl transferase and alpha-lactalbumin concentration, measured 30 h after ovariectomy. However, once the synthesis of lactose has been induced, progesterone is no longer inhibitory. Oestrogen induced a significant increase in lactose synthetase activity but no effect was obtained on galactosyl transferase activity. Progesterone, in a time and dose dependent relationship, was capable of preventing the effect of estrogen on lactogenesis. The lactogenic action of oestrogen in ovariectomized pregnant rats might be due to a direct effect at the mammary gland level facilitating the action of prolactin or through an indirect effect mediated via an increase on prolactin release.     

Progesterone and prolactin are both required for suppression of the induction of rat alpha-lactalbumin activity

Progesterone prevents lactation during pregnancy. This anti-lactogenic effect includes suppression of the advent of alpha-lactalbumin activity, an effect which prevents the formation of lactose. Alpha lactalbumin activity can be induced to some extent in pregnant rat mammary explants by insulin and hydrocortisone alone, and to a greater extent with prolactin in addition, or with EGF in addition. Physiological levels of progesterone markedly inhibit the induction in the presence of prolactin plus insulin and hydrocortisone, only weakly inhibit in the presence of insulin and hydrocortisone alone, and have no inhibitory effect in the presence of EGF plus insulin and hydrocortisone. Prolactin permits some inhibition in the presence of EGF. The results suggest that progesterone does not subvert the essential insulin or glucocorticoid signals. It also appears that transduction of the prolactin signal is required in order that progesterone effectively block induction of alpha-lactalbumin activity.     

Effect of estrogen and placental lactogen on lactogenesis in pregnant rats

The removal of the corpora lutea or ovariectomy on Day 18 of pregnancy induced a rise in serum prolactin 24 h after surgery with a rapid decline to control values 4 h after the surge, only in the ovariectomized group. When hysterectomy was performed in addition to luteectomy or ovariectomy a similar rise in prolactin was obtained. Lactose synthetase activity in mammary tissue was significantly higher in the luteectomized and luteectohysterectomized rats when compared with ovariectomized, ovariohysterectomized rats and the sham-operated group. Estrogen treatment 12 h after ovariectomy increased serum prolactin and lactose synthetase activity to values similar to those measured in luteectomized rats, but this increase was significantly greater when compared with the ovariectomized-nontreated group. Treatment with Tamoxifen did not decrease serum prolactin in the luteectomized rats but lactose synthetase was reduced to values similar to that obtained in ovariectomized rats. Treatment with 2 bromo-alpha-ergocryptine-mesylate (CB-154) prevented the rise in serum prolactin in the ovariectomized, luteectomized and luteectohysterectomized groups, but lactose synthetase activity was lowered to control values (sham-operated rats) only in the luteectohysterectomized rats. According to these findings, rat placental lactogen in the absence of prolactin and progesterone induces lactose synthesis. Estrogen facilitates prolactin but not placental lactogen action on lactose synthetase activity.     

Re-examination of the putative roles of insulin and prolactin in the regulation of lipid deposition and lipogenesis in vivo in mammary gland and white and brown adipose tissue of lactating rats and litter-removed rats.

1. The effects of various treatments to alter either plasma prolactin (bromocryptine administration or removal of litter) or the metabolic activity of the mammary gland (unilateral or complete teat sealing) on the disposal of oral [14C]lipid between 14CO2 production and [14C]lipid accumulation in tissues of lactating rats were studied. In addition, the rates of lipogenesis in vivo were measured in mammary gland, brown and white adipose tissue and liver. 2. Bromocryptine administration lowered plasma prolactin, but did not alter [14C]lipid accumulation in mammary gland or in white and brown adipose tissue. 3. In contrast, complete sealing of teats results in no change in plasma prolactin, but a 90% decrease in [14C]lipid accumulation in mammary gland and a 4-fold increase in white and brown adipose tissue. The rate of lipogenesis in mammary gland was decreased by 95%, but there was no change in the rate in white and brown adipose tissue. Unilateral sealing of teats resulted in a decrease in [14C]lipid accumulation in white adipose tissue. 4. Removal of the litter for 24 h (low prolactin) produced a similar pattern to complete teat sealing, except that there was a 6-fold increase in lipogenesis in white adipose tissue. Re-suckling for 5 h increased plasma prolactin, but did not alter the response seen in litter-removed lactating rats. 5. Changes in lipoprotein lipase activity and in plasma insulin paralleled the reciprocal changes in [14C]lipid accumulation in white and brown adipose tissue and in mammary gland. 6. It is concluded that the plasma insulin is more important than prolactin in regulating lipid deposition in adipose tissue during lactation, and that any effects of prolactin must be indirect.     

Effect of prolactin and glucocorticoids on P-enolpyruvate carboxykinase activity in liver and mammary gland from diabetic and lactating rats

Summary The administration of 2 bromo--ergocryptine, to reduce serum prolactin decreased the activity of cytosolic P-enolpyruvatc carboxykinase (GTP) (EC4.1.1.32) about 50% in both liver and mammary gland of lactating animals. Adrenalectomy had similar effects to those of bromo-a-ergocryptine. In contrast, there was a 50% increase in enzyme activity in the mammary gland of diabetic, lactating rats and a 10-fold increase in liver as compared with normal rats. P-enolpyruvate carboxykinase activity in mammary gland as liver is coordinately regulated by prolactin, glucocorticoids and insulin.     

The effect of hypophysectomy and subsequent replacement therapy with sheep prolactin or bovine growth hormone on the lactose synthetase activity of rabbit mammary gland

1. The effects of hypophysectomy and replacement therapy with sheep prolactin and bovine growth hormone on the lactose synthetase activity of the mammary glands of lactating rabbits were studied. 2. There was an approximately fourfold decline in the lactose synthetase activity of homogenates calculated on a DNA basis within 6-7 days of hypophysectomy. Prolactin reversed this decline but growth hormone had no effect. 3. Changes in the properties of a particulate fraction isolated from the glands indicated that a decline in the effective concentration of alpha-lactalbumin was one factor contributing to the decreased lactose synthetase activity after hypophysectomy. 4. As the changes in lactose output produced by hypophysectomy and prolactin therapy are much greater than the changes in total lactose synthetase activity it is concluded that the activity of this enzyme is not the main factor controlling lactose output under these conditions.     

Effect of indomethacin on the lactogenic action of prolactin

The action of indomethacin on the lactogenic activity of prolactin has been evaluated usind the technique of rabbit mammary gland organ culture. Indomethacin is totally unable to inhibit prolactin action as estimated by lactose synthetase activity and casein synthesis. These data suggest, as opposed to previous works, that prostaglandins are not involved in the mechanism of prolactin action on lactogenesis.     

A possible role for progesterone in the preovulatory gonadotropin surge through modulation of LHRH degrading activity

The preovulatory surge of gonadotropins is triggered by estradiol and enhanced to its full magnitude by progesterone. Progesterone may exert this effect through several mechanisms. One of the mechanisms is through the ability of progesterone to induce an increase in the hypothalamic content and release of LHRH. The purpose of this study was to determine if progesterone might not act through yet another mechanism and facilitate LHRH release of the proestrous gonadotropin surge through modulation of luteinizing hormone releasing hormone (LHRH) degrading activity. Sixty-day-old Sprague-Dawley rats were ovariectomized; 14 days later, the estradiol-progesterone milieu of proestrous was mimicked in these animals through the use of estradiol containing silastic implants and subcutaneous progesterone injections. The LHRH degrading activity of the hypothalamus, pituitary and serum were monitored subsequently at preselected time points. In the hypothalamus, estradiol alone was capable of inducing significant increase in degrading activity; progesterone alone had no effect; however, progesterone subsequent to estradiol priming suppressed the increase induced by estradiol alone. In the pituitary, neither estradiol alone nor progesterone alone nor progesterone subsequent to estradiol priming had any significant effect on degrading activity. In the serum, estradiol induced a rapid and significant increase in activity; progesterone alone suppressed activity; progesterone subsequent to estradiol priming induced a similar but more rapid suppression. Therefore, the overall tendency was for estradiol to stimulate and progesterone to suppress LHRH degrading activity in the tissues studied. The results of this study indicate that progesterone has the capacity to suppress LHRH degrading activity and may be one of the mechanisms capable of increasing the availability of LHRH to the anterior pituitary gland thereby facilitating the preovulatory gonadotropin surges.     

Gamma-glutamyltransferase activity in mammary gland of pregnant rats and its regulation by ovarian hormones, prolactin and placental lactogen.

Ovariectomy and ovariectomy plus hysterectomy on day 18 of pregnancy increased gamma-glutamyltransferase activity in the mammary gland. The withdrawal of progesterone and the subsequent release of prolactin are responsible for the rise in enzyme activity. Rat placental lactogen in the absence of prolactin and progesterone is able to induce gamma-glutamyltransferase activity.     

The control of the form and function of the ribosomes in androgen-dependent tissues by testosterone

1. The ribosome content of the rat ventral prostate gland is controlled by the concentrations of circulating androgens and the polyribosomal complement of the total population of ribosomes is acutely dependent on androgenic stimulation. After the administration of testosterone to castrated rats in vivo, there is a pronounced increase in the amounts of heavy (150-240S) polyribosomes. 2. These results are consistent with a pronounced increase in the mRNA and rRNA content of the prostate gland after the administration of testosterone in vivo. 3. From studies conducted both in vitro, the heavy prostate polyribosomes formed after androgenic stimulation are particularly active in protein synthesis. 4. The androgen-stimulated increase in the formation of prostate polyribosomes has a mandatory requirement for sustained RNA and protein synthesis. 5. Since the androgen-mediated increase in prostate polyribosomes may also be suppressed by the concomitant administration of certain anti-androgenic steroids in vivo, the response in polyribosome formation is probably initiated by the binding of a metabolite of testosterone, 5alpha-dihydrotestosterone, in the prostate gland. 6. The relevance of these findings to the pronounced increase in protein synthesis in androgen-dependent tissues after hormonal stimulation is discussed.     

Stimulation of 17 beta-hydroxy steroid dehydrogenase in the rat anterior pituitary gland by progesterone

Anterior pituitary gland and hypothalamic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity was measured in the immature castrated estradiol primed rat to determine if differences in enzyme activity could explain the progesterone induced reduction of bound estradiol nuclear receptors of the anterior pituitary gland but not the hypothalamus. Higher levels of 17 beta-HSD activity were found in the anterior pituitary gland as compared to the hypothalamus. The enzyme activity in the anterior pituitary gland was stimulated by progesterone administered either in combination with estradiol for 4 days or as a single injection following 4 days of estradiol priming. No progesterone effects were found on hypothalamic 17 beta-HSD. Under the experimental conditions used, progesterone administration did not alter uterine 17 beta-HSD. An increase in anterior pituitary gland and uterine 17 beta-HSD was also induced by estrogen administration.     

The relationship between adenosine diphosphate-ribosylation and mammary gland differentiation

Poly(adenosine diphosphate [ADP]-ribosyl)ation, although associated with differentiation in many systems, exhibited a reciprocal relationship with mammary gland differentiation, and both the synthetic and degradatory pathways complemented each other in this regard. Poly(ADP-ribosyl)synthetase activity declined during pregnancy and lactation, while poly(ADP-ribose) degradatory activity rose late in pregnancy and peaked during lactation. In explant cultures, similar changes occurred and appeared to be under separate hormonal control; prolactin suppressed the synthetase activity, whereas insulin stimulated the poly(ADP-ribosyl)glycohydrolase activity. This latter effect may be mediated by a decline in cAMP levels for the following reasons: the glycohydrolase is known to be inhibited by cAMp, insulin decreased cAMP concentrations in mammary explants by 70%, and cholera toxin blocked the effects of insulin on poly(ADP-ribose) degradation. This reciprocal relationship between poly(ADP-ribosyl)ation and mammary gland differentiation is further supported by pharmacological studies: in the presence of insulin, cortisol, and prolactin, an inhibitor of the synthetase stimulated alpha-lactalbumin three-fold over hormone stimulation alone. However, this inhibitor was unable to induce differentiation in the absence of prolactin. Therefore, although there is a close association between a decline in enzyme activity and mammary differentiation, the data are insufficient to support a causal relationship.     

Regulation of the activity of ornithine decarboxylase and S-adenosylmethionine decarboxylase in mammary gland and liver of lactating rats. Effects of starvation, prolactin and insulin deficiency.

1. Starvation caused a marked decrease in the activity of ornithine decarboxylase in mammary gland, together with a lesser decrease in the activity of S-adenosylmethionine decarboxylase and a marked fall in milk production. Liver ornithine decarboxylase and S-adenosylmethionine decarboxylase activities were unaffected. 2. Refeeding for 2.5 h was without effect on ornithine decarboxylase in mammary gland, but it returned the S-adenosylmethionine decarboxylase activity in mammary gland to control values and elevated both ornithine decarboxylase and S-adenosylmethionine decarboxylase in liver. 3. Refeeding for 5 h returned the activity of ornithine decarboxylase in mammary gland to fed-state values and resulted in further increases in S-adenosylmethionine decarboxylase in mammary gland and liver and in ornithine decarboxylase in liver. 4. Prolactin deficiency in fed rats resulted in decreased milk production and decreased activity of ornithine decarboxylase in mammary gland. The increase in ornithine decarboxylase activity normally seen after refeeding starved rats for 5 h was completely blocked by prolactin deficiency. 5. In fed rats, injection of streptozotocin 2.5 h before death caused a decrease in the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase in mammary gland, which could be reversed by simultaneous injection of insulin. Insulin deficiency also prevented the increase in S-adenosylmethionine decarboxylase in liver and mammary gland normally observed after refeeding starved rats for 2.5 h.     

Prolactin and progesterone receptors in pregnancy-dependent mammary tumors in GR/A mice

In this study, cellular prolactin receptors and cytosolic progesterone receptors were examined and compared in pregnancy-dependent mammary tumors (PDMT) and in normal mammary glands of pregnant GR/A mice. PDMT and normal mammary glands were examined in the same animal, thus assuring an identical hormonal environment. The PDMT cells had a larger capacity to bind prolactin or the synthetic progesterone, R5020, than did the normal mammary gland. While the dissociation constant (Kd) value for prolactin binding to normal mammary epithelial cells was similar to that of PDMT cells, PDMT cells had 2.2 times more prolactin receptors than the normal cells. Progesterone binding activity was detected only in PDMT, but not in the normal mammary cells. The receptor concentration and the Kd value for progesterone binding of PDMT were 606 fmol/mg protein and 3.53 nM, respectively. It appears, therefore, that normal regulation of these receptors may be altered within the PDMT cells. The increased growth responsiveness of PDMT to the hormones of pregnancy, especially prolactin, progesterone, and placental lactogen, may be a function of a sharp increase in the level of cellular receptors for these mammotropic hormones.     

Prolactin as a factor in the uterine response to progesterone in rabbits

The direct effect of prolactin on uteroglobin production and on uterine endometrial oestrogen and progesterone receptor concentrations was tested by using ovariectomized rabbits (at least 12 weeks) treated with prolactin; prolactin + progesterone; prolactin + oestradiol + progesterone; oestradiol + progesterone; or progesterone alone. Prolactin treatment produced a significant (P less than 0.05) increase in the concentration of cytosolic oestrogen and progesterone receptors, restoring the concentrations to values found at oestrus. However, the concentration of nuclear receptors remained low. In the remaining treatment categories there was no significant (P greater than 0.05) increase in the concentration of oestrogen and progesterone receptors compared with those in ovariectomized controls. However, the sequential treatment of ovariectomized animals with prolactin + progesterone stimulated uteroglobin production to a concentration equal to that found in intact rabbits on the 5th day of pregnancy. This was not achieved by prolactin or progesterone alone or with oestradiol. These results suggest that prolactin acts as an essential factor in the rabbit uterine response to progesterone, perhaps by the modulation of progesterone receptor activity.     

Regulation of milk protein synthesis by progesterone in cultured mouse mammary gland

The effect of progesterone on the synthesis of milk proteins, casein and alpha-lactalbumin was investigated by culturing mammary explants from mid-pregnant mice in serum-free medium. The addition of progesterone at concentrations above 10 ng/ml inhibited both the casein and alpha-lactalbumin accumulation that were induced by the synergistic actions of insulin, prolactin and cortisol. The maximal inhibition was attained at a progesterone concentration of 100 ng/ml. The maximal level of inhibition of the alpha-lactalbumin accumulation was about 90% in the presence of insulin and prolactin or insulin, prolactin and 0.01 microgram/ml of cortisol. The inhibition of the casein accumulation by progesterone was about 80% in the presence of insulin and prolactin, and about 40% in the presence of insulin, prolactin and 1 microgram/ml of cortisol, indicating that cortisol partially antagonized the action of progesterone on the casein synthesis. When the inhibitory effect of progesterone on the accumulation of both alpha-lactalbumin and casein was examined in cultured mammary tissues from virgin, early pregnant, mid-pregnant and late pregnant mice, the degree of inhibition was markedly reduced in tissue from late pregnant mice. This indicates that the susceptibility of mammary gland to the inhibitory action of progesterone varies with the developmental stage of the tissue.     

Prolactin induction of casein mRNA in organ culture. A model system for studying peptide hormone regulation of gene expression

The peptide hormone, prolactin, when added to organ explants of rat mammary gland, rapidly (within 1 h) induced the accumulation of casein mRNA. Casein mRNA sequences, as determined by hybridization with a specific cDNA probe, were shown to increase for up to 48 h after prolactin addition. The magnitude of this response was dependent upon the day of pregnancy at which the tissue was placed in culture. Maximal levels of induction (as great as 45-fold) were obtained using tissue from 15-day pregnant rats. Further data indicate that two steroid hormones, hydrocortisone and progesterone, were able to modulate the prolactin-induced accumulation of casein mRNA. The continuous presence of hydrocortisone was not necessary for prolactin induction of casein mRNA. However, the presence of hydrocortisone was required for maximal accumulation of casein mRNA. The induction of casein mRNA by prolactin was inhibited in a dose-dependent manner by the simultaneous addition of progesterone to the organ culture. Thus, hydrocortisone appears to potentiate the prolactin induction of casein mRNA, whereas progesterone is able to prevent casein mRNA accumulation. Since mammary gland organ culture is performed in a serum-free, chemically defined medium, this system allows a detailed examination of the mechanims by which a peptide hormone regulates the rapid accumulation of a specific mRNA.