Role of ovarian secretions in mammary gland development and function in ruminants

The mammary gland is a dynamic organ that undergoes cyclic developmental and regressive changes during the lifetime of a female mammal. Mammogenesis begins during embryonic life with the development of the first mammary gland rudiments and ductal system. After birth, during the pre-pubertal period, the ductal growth of the mammary parenchyma occurs through the fat pad. In most of the ruminant species allometric mammary parenchyma development begins with the onset of cyclic ovarian secretions activity. The two main hormones secreted during an ovarian cycle are estradiol and progesterone. These steroid hormones are derived from cholesterol and are synthesized by theca and granulosa cells in ovaries. During puberty, the mammary parenchyma develops in a compact, highly arborescent parenchymal mass surrounded by a dense connective matrix. Ductal elongation and lobulo-alveolar development are accomplished during growth and pregnancy to prepare for future milk production. At the end of lactation, the mammary gland undergoes involution, which corresponds to a regression of the secretory tissue, a reduction in the alveolar size and a loss of mammary epithelial cells (MECs). Ovarian steroids (estradiol and progesterone) appear to be key regulators of the different stages of mammogenesis and mammary function. Through this review, the role and the importance of ovarian steroids on mammary gland and on MECs is described.     

Glucocorticoid stimulation of choline kinase activity during the development of mouse mammary gland.

Mouse mammary gland contains choline kinase activity that can be stimulated by polyamines. Developmental studies show that the activity of choline kinase in mammary gland is low in both virgin and nonpregnant primiparous animals but increases severalfold during pregnancy and reaches a maximal level during the lactation period. Similar increases in enzyme activity are observed by cultivation of tissue explants in the presence of insulin, cortisol, and prolactin, a combination of hormones which induces the ultrastructural and biochemical changes associated with the development of mammary gland during pregnancy and lactation. The increase in enzyme activity in cultured explants is dependent only on the actions of both insulin and cortisol and parallels the formation of rough endoplasmic reticulum, which is effected by the same combination of hormones. The hormonal stimulation of choline kinase activity appears to involve the action of spermidine, a polyamine which accumulates in the cells under the influence of cortisol and mimicks the effect of cortisol on milk-protein synthesis in cultured explants.     

Effect of mammogenic hormones on the expression of FGF7, FGF10 and their receptor in mouse mammary gland

To investigate the regulation of estrogen, progesterone and prolactin stimulating the development of mammary gland, the Kunming mice were used as experimental animals in this study. Through the experiment in vitro, the effect of mammogenic hormones were systematically investigated on expression of FGF7 and FGF10 and their receptor in different periods. The results are as follows: in mammary glands of mice, 17 beta-estradiol increased the expression of FGF7; progesterone did not affect the expression of FGF7; prolactin up-regulated the expression of FGF7 significantly in pregnancy and lactation. 17 beta-estradiol increased the expression of FGF10; progesterone and prolactin reduced the expression of FGF10 significantly in virgin; prolactin significantly increased the expression of FGF10 in pregnancy. When 17 beta-estradiol in the body was in relatively high proportion, it would lower the expression of KGFR; while 17 beta-estradiol in the body was in relatively low proportion, it would increase the expression of KGFR. Low concentration of progesterone increased the expression of KGFR and high progesterone did not affect the expression of KGFR. Prolactin increased the expression of KGFR significantly in pregnancy and lactation.     

Peroxisome proliferator-activated receptor-binding protein null mutation results in defective mammary gland development

A conditional null mutation of peroxisome proliferator-activated receptor-binding protein (PBP) gene was generated to understand its role in mammary gland development. PBP-deficient mammary glands exhibited retarded ductal elongation during puberty, and decreased alveolar density during pregnancy and lactation. PBP-deficient mammary glands could not produce milk to nurse pups during lactation. Both the mammary ductal elongation in response to estrogen treatment and the mammary lobuloalveolar proliferation stimulated by estrogen plus progesterone were attenuated in PBP-deficient mammary glands. The proliferation index was decreased in PBP-deficient mammary glands. PBP-deficient mammary epithelial cells expressed abundant beta-casein, whey acidic protein, and WDNM1 mRNA, indicating a relatively intact differentiated function. PBP-deficient epithelial cells were unable to form mammospheres, which were considered to be derived from mammary progenitor/stem cells. We conclude that PBP plays a pivotal role in the normal mammary gland development.     

Prolactin binding in rat mammary gland during pregnancy and lactation.

Lactogenic hormones from the placenta and pituitary are primarily responsible for the growth and function of the mammary gland during pregnancy and lactation. In the present study we described the optimal conditions for the measurement of 125I-labeled ovine prolactin binding to mammary gland slices of pregnant and lactating rats. Prolactin binding is saturable (Kd approx. 2.36 - 10(-9) M), hormone specific and destroyed by proteases. The hormonal environments of pregnancy and lactation dramatically influence the availability and measurement of prolactin binding sites. Whereas binding consistently appears to be low in mammary glands removed from rats during pregnancy, binding levels rise 7--8-fold shortly after birth and remain high during the 22 days of lactation. However, the removal of the ovaries and gravid uteri at specific times during pregnancy results in a prompt 3--6-fold increase in prolactin binding. Elevated levels in potential prolactin binding capacity appear in mammary tissue coincident with the reported rise in serum rat placental lactogen between the eighth and eleventh days. We suggest that high levels of this lactogenic hormone promote the appearance of prolactin binding sites during pregnancy and mask the sites such that they are not available for measurement in vitro.     

p19ARF determines the balance between normal cell proliferation rate and apoptosis during mammary gland development

This study demonstrated, for the first time, the following events related to p19(ARF) involvement in mammary gland development: 1) Progesterone appears to regulate p19(ARF) in normal mammary gland during pregnancy. 2) p19(ARF) expression levels increased sixfold during pregnancy, and the protein level plateaus during lactation. 3) During involution, p19(ARF) protein level remained at high levels at 2 and 8 days of involution and then, declined sharply at day 15. Absence of p19(ARF) in mammary epithelial cells leads to two major changes, 1) a delay in the early phase of involution concomitant with downregulation of p21(Cip1) and decrease in apoptosis, and 2) p19(ARF) null cells are immortal in vivo measured by serial transplantion, which is partly attributed to complete absence of p21(Cip1) compared with WT cells. Although, p19(ARF) is dispensable in mammary alveologenesis, as evidenced by normal differentiation in the mammary gland of pregnant p19(ARF) null mice, the upregulation of p19(ARF) by progesterone in the WT cells and the weakness of p21(Cip1) in mammary epithelial cells lacking p19(ARF) strongly suggest that the functional role(s) of p19(ARF) in mammary gland development is critical to sustain normal cell proliferation rate during pregnancy and normal apoptosis in involution possibly through the p53-dependent pathway.     

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.     

Effect of mammogenic hormones on the expression of dFGF7, FGF10 and their receptor in mouse mammary gland

To investigate the regulation of estrogen, progesterone and prolactin stimulating the development of mammary gland, the Kunming mice were used as experimental animals in this study.Through the ex-periment in vitro, the effect of mammogenic hormones were systematically investigated on expression of FGF7 and FGF10 and their receptor in different periods.The results are as follows:in mammary glands of mice, 17 beta-estradiol increased the expression of FGF7;progesterone did not affect the expression of FGF7;prolactin up-regulated the expression of FGF7 significantly in pregnancy and lac-tation.17 beta-estradiol increased the expression of FGF10;progesterone and prolactin reduced the expression of FGF10 significantly in virgin;prolactin significantly increased the expression of FGF10 in pregnancy.When 17 beta-estradiol in the body was in relatively high proportion, it would lower the ex-pression of KGFR;while 17 beta-estradiol in the body was in relatively low proportion, it would increase the expression of KGFR.Low concentration of progesterone increased the expression of KGFR and high progesterone did not affect the expression of KGFR.Prolactin increased the expression of KGFR significantly in pregnancy and lactation.     

Hormone/growth factor interactions mediating epithelial/stromal communication in mammary gland development and carcinogenesis

Epithelial/mesenchymal interactions begin during embryonic development of the mammary gland and continue throughout mammary gland development into adult life. Stromal and epithelial growth factors that may mediate interactions between these compartments of the mammary gland are reviewed. Since mammogenic hormones are the primary regulators of mammary gland development, special consideration is given to hormonal regulation of growth factors in order to explore the integration of hormones and growth factors in the regulation of mammary gland growth and neoplasia. Examination of hormonal regulation of the fibroblast growth factor (FGF)-7/FGFR2-IIIb receptor system in the mammary gland reveals that mammogenic hormones differentially regulate the synthesis of stromal growth factors and their epithelial receptors. These effects serve to optimize the action of estrogen and progesterone on mammary gland development and illustrate that the ratio of these two hormones is critical in regulating this growth factor axis. The role of stromal/epithelial mitogenic microenvironments in modulating the genotype and phenotype of preneoplastic and neoplastic lesions by chemical carcinogens is discussed. Finally, changes in growth factor expression during mammary tumor progression are described to illustrate the relative roles that stromally-derived and epithelial-derived growth factors may play during progression to hormone independent tumor growth.     

Hormonal regulation of retinoic acid-binding proteins in the mammary gland.

A cytosolic retinoic acid-binding (RAB) protein that sediments specifically as a 2S component on sucrose density gradients was detected in the mammary glands of virgin, pregnant and lactating rats. Mammary cytosol from pregnant rats contained significantly higher concentrations of cytosolic RAB protein than did cytosol from either virgin or lactating rats. The glands of pregnant animals exhibited increased concentration of cytosolic RAB protein during the first 5 days of pregnancy, and a steady decline was observed thereafter. The concentration of cytosolic RAB protein dropped to the value observed during lactation on the day 20 of pregnancy. Moreover, throughout lactation, low concentrations of cytosolic RAB protein were maintained. Daily treatment of virgin and lactating animals with 5 micrograms of oestradiol-17 beta for 1 week increased cytosolic RAB protein to concentrations comparable with those seen in pregnant rats. Progesterone, however, did not affect the mammary cytosolic RAB protein content of virgin rats. These results suggest hormonal involvement in the regulation of cytosolic RAB protein concentration of mammary gland during differentiation.     

Estradiol, progesterone and prolactin modulate mammary gland morphogenesis in adult female plains vizcacha (Lagostomus maximus)

We studied for the first time the mammary gland morphogenesis and its hormonal modulation by immunolocalizing estradiol, progesterone and prolactin receptors (ER, PR and PRLR) in adult females of Lagostomus maximus, a caviomorph rodent which shows a pseudo-ovulatory process at mid-gestation. Mammary ductal system of non-pregnant females lacks expression of both ERα and ERβ. Yet throughout pregnancy, ERα and ERβ levels increase as well as the expression of PR. These increments are concomitant with ductal branching and alveolar differentiation. Even though mammary gland morphology is quite similar to that described for other rodents, alveolar proliferation and differentiation are accelerated towards the second half of pregnancy, once pseudo-ovulation had occurred. Moreover, this exponential growth correlates with an increment of both progesterone and estradiol serum-induced pseudo-ovulation. As expected, PR and PRLR are strongly expressed in the alveolar epithelium during pregnancy and lactation. Strikingly, PRLR is also present in ductal epithelia of cycling glands suggesting that prolactin function may not be restricted to its trophic effect on mammary glands of pregnant and lactating females, but it also regulates other physiological processes in mammary glands of non-pregnant animals. In conclusion, this report suggests that pseudo-ovulation at mid-gestation may be associated to L. maximus mammary gland growth and differentiation. The rise in P and E2-induced pseudo-ovulation as well as the increased expression of their receptors, all events that correlate with the development of a more elaborated and differentiated ductal network, pinpoint a possible relation between this peculiar physiological event and mammary gland morphogenesis.     

Developmental role of the SNF1-related kinase Hunk in pregnancy-induced changes in the mammary gland

The steroid hormones 17 beta-estradiol and progesterone play a central role in the pathogenesis of breast cancer and regulate key phases of mammary gland development. This suggests that developmental regulatory molecules whose activity is influenced by ovarian hormones may also contribute to mammary carcinogenesis. In a screen designed to identify protein kinases expressed in the mammary gland, we previously identified a novel SNF1-related serine/threonine kinase, Hunk (hormonally upregulated Neu-associated kinase). During postnatal mammary development, Hunk mRNA expression is restricted to a subset of mammary epithelial cells and is temporally regulated with highest levels of expression occurring during early pregnancy. In addition, treatment of mice with 17 beta-estradiol and progesterone results in the rapid and synergistic upregulation of Hunk expression in a subset of mammary epithelial cells, suggesting that the expression of this kinase may be regulated by ovarian hormones. Consistent with the tightly regulated pattern of Hunk expression during pregnancy, mammary glands from transgenic mice engineered to misexpress Hunk in the mammary epithelium manifest temporally distinct defects in epithelial proliferation and differentiation during pregnancy, and fail to undergo normal lobuloalveolar development. Together, these observations suggest that Hunk may contribute to changes in the mammary gland that occur during pregnancy in response to ovarian hormones.     

Mouse mammary epithelial histamine system.

Histamine is suggested to play a role in mammary gland growth regulation, differentiation and functioning during pregnancy and lactation. Two pools of histamine are thought to be involved in these processes: mastocyte- and epithelial cell related histamine. In the present study we focused on epithelial cells. Immunohistochemistry has shown that the epithelial cells positive for histamine and L-histidine decarboxylase (HDC), the primary enzyme regulating histamine biosynthesis, were mainly found in cells forming alveolar structures in the mammary gland. Cultured primary mouse mammary epithelial cells (MMEC) expressed strong HDC immunoreactivity, especially dividing cells and non-differentiated ones. Histidine decarboxylase activity undergoes significant changes during pregnancy and lactation. Pregnancy associated intensive growth of the mammary gland coincided with an increase and the first days of lactation with a decrease of HDC protein expression. Binding studies with mammary tissue membranes and epithelial cell membranes revealed the presence of H1 and H3 but not H2 receptors. Summarizing, our data have shown that mammary epithelial cells are capable of synthesizing and excreting histamine and they bear histamine receptors. These findings further substantiate the role of histamine in mammary gland physiology.     

Pattern of expression of the KGF receptor and its ligands KGF and FGF-10 during postnatal mouse mammary gland development

The expression of the KGF receptor (KGFR) and its stromal ligands, KGF and FGF-10, was compared during mouse mammary gland development. KGFR expression in mammary parenchyma is maximal in mature virgin mice, declines during pregnancy and lactation, but rises after weaning. The rise in KGFR mRNA in the virgin animal corresponds to parenchymal growth. The fall in KGFR expression in pregnancy is driven by hormone-induced alveolar differentiation since the level of KGFR mRNA is 5-fold higher in isolated ductal cells compared to alveolar cells. KGF and FGF-10 expression patterns differ during ductal development. FGF-10 is also expressed at about a 15-fold higher molar level than KGF. During pregnancy and lactation, expression of KGF and FGF-10 decreases in intact fat pads but is unchanged in parenchyma-free fat pads. Thus, the decrease in KGF and FGF-10 expression observed in intact glands during pregnancy and lactation is not a direct consequence of the changing hormonal milieu but more likely reflects an increase in the ratio of epithelium to stroma. Differences in the level and pattern of expression of mRNA for KGF, FGF-10, and the KGFR during postnatal development of the mouse mammary gland are a result of morphological development, changes in the ratio of stroma to epithelium, and hormonal regulation of cell differentiation. These changes suggest that the biological roles that these growth factors play are regulated by fluctuations in both growth factor and growth factor receptor expression and that KGF and FGF-10 may have different regulatory functions.     

Prolactin binding in rat mammary gland during pregnancy and lactation

Lactogenic hormones from the placenta and pituitary are primarily responsible for the growth and function of the mammary gland during pregnancy and lactation. In the present study we describe the optimal conditions for the measurement of ¹²⁵I-labeled ovine prolactin binding to mammary gland slices of pregnant and lactating rats. Prolactin binding is saturable (K_{d approx. 2.36 · 10^?9 M)}, hormone specific and destroyed by proteases. The hormonal environments of pregnancy and lacation dramatically influence the availability and measurement of prolactin binding sites. Whereas binding consistently appears to be low in mammary glands removed from rats during pregnancy, binding levels rise 7–8-fold shortly after birth and remain high during the 22 days of lactation. However, the removal of the ovaries and gravid uteri at specific times during pregnancy results in prompt 3–6-fold increase in prolactin binding. Elevated levels in potential prolactin binding capacity appear in mammary tissue coincident with the reported rise in serum rat placental lactogen between the eight and eleventh days. We suggest that high levels of this lactogenic hormone promote the appearance of prolactin binding sites during pregnancy and mask the sites such that they are not available for measurement in vitro.     

Hormonal activation of mammary gland peroxidase.

Ultrastructural cytochemistry was used to detect an endogenous peroxidase in the rat mammary gland. The enzyme was identified only during the latter half of pregnancy and during lactation, indicating its possible dependence upon hormones. To test this hypothesis, specific hormones associated with the development and differentiation of the mammary gland were used both in vivo and in vitro in an effort to induce, or unmask, the activity of the enzyme. Estrogen injected into nonpregnant rats induced some peroxidase activity in the mammary glands of a few animals. Two hormone combinations tested in organ cultures of mouse mammary gland were able to activate the enzyme: (1) dexamethasone + insulin and (2) dexamethasone + insulin + prolactin.