Transgene expression in mammary glands of newborn rats

Transgene expression in the mammary glands of newborn rats was studied to establish an early selection system for transgenic animals producing exogenous proteins in their milk during lactation. A fusion gene composed of the bovine alpha S1 casein gene promoter and the human growth hormone gene was microinjected into rat embryos. Transgenic lines that produced human growth hormone in their milk were established and used in this study. Immediately after birth, and without any hormone treatment, human growth hormone was found in the extracts of mammary glands from both male and female rats derived from the line secreting human growth hormone in their milk. The expression of the transgene in mammary glands of newborn rats was also detected by the presence of human growth hormone mRNA. Nontransgenic newborn rats did not express the human growth hormone gene in their mammary glands, while the mRNA for rat alpha casein, an endogenous milk protein, was found in all mammary glands from both transgenic and nontransgenic neonates. These results show that analyzing the expression of transgenes in the mammary glands of neonates is a valuable tool to select the desired transgenic animals and to shorten the selection schedules establishing the transgenic animals. © 1996 Wiley-Liss, Inc.     

Distinction between progestin- and glucocorticoid-binding sites in mammary glands. Apparent lack of cytoplasmic progesterone receptors in lactating mammary glands.

The cytosol fraction of the lactating mammary glands of mice does not appear to contain detectable amounts of progesterone receptors. Mixing experiments indicate that the absence of receptors is not due to interference by other factors in the cytosol. However, in the cytosol of mammary glands, there is specific binding or progestins to certain low-affinity sites which have characteristics of specific glucocorticoid-binding sites.     

Lactose synthetase activities in rat and mouse mammary glands

The activities of lactose synthetase (in the presence and absence of α-lactalbumin), galactosyltransferase and α-lactalbumin levels were determined in rat and mouse mammary glands during pregnancy, lactation and involution. In the rat, essentially none of the above activities were detected prior to parturition. This was followed by a sharp increase with the maximum occurring during late lactation and then by a rapid drop during involution. In the mouse, detectable levels of all the activities occurred at 15 days of pregnancy and these levels increased during pregnancy and reached a maximum level during early lactation and again sharp decreases occurred during involution. The study shows that mammary gland development occurs at an earlier date in the mouse than in the rat.     

Effects of mammary engorgement and feed withdrawal on microvascular function in lactating goat mammary glands

The responses of the mammary microvasculature in lactating goats (n = 8) during feed withdrawal (18-20 h) and mammary engorgement (26-28 h of milk accumulation) were compared using an indicator-dilution technique with FITC-albumin and [(14)C]sucrose as the intravascular and diffusible indicators, respectively. Feed withdrawal and mammary engorgement caused a 50-60% decrease in mammary arterial flow and in the permeability-surface area product (PS) values for sucrose. Only feed withdrawal increased the mean transit time [from 17.3 to 30.0 s, SE of the difference (SED) = 2.16, P < 0.01] of FITC-albumin, whereas only mammary engorgement reduced sucrose extraction (0.63 to 0.51, SED = 0.04, P < 0.05). Mammary engorgement also caused a substantial reduction in the sucrose-accessible extravascular space from 92 to 44 ml (SED = 15.2, P < 0.01). In a separate experiment using five goats, milking after mammary engorgement did not immediately restore arterial flow or sucrose extraction, indicating that the effect of milk accumulation was not mediated simply via increased intramammary pressure. In conclusion, feed withdrawal resulted in slower flow in the capillary bed but apparently no change in capillary recruitment, whereas mammary engorgement caused capillary derecruitment.     

Steroid receptors in mammary glands and tumors of hysterectomised rats

Epidemiological survey suggests that longer exposure of the breast to sex steroids may be one of the factors involved in increased risk for cancer. Using an experimental model of bilaterally hysterectomised rats, the sex steroid receptors in the mammary glands during growth as well as incidence and hormonal characteristics of chemically induced mammary tumors have been studied. Though steroid receptors were detectable in the mammary glands of the model earlier than in intact rats, the incidence or hormone dependency of the mammary tumors in the two groups were not considerably altered.     

Histone phosphorylation in explants of mouse mammary glands and tumors

Rates of histone phosphorylation were measured in explants of mammary glands from mouse strains with high and low tumor incidence. Explants of hormone dependent and independent mouse mammary tumors were also investigated. All mouse strains studied showed predominant phosphorylation of H2A histone at serine and threonine residues. No differences in rates of H2A phosphorylation in glands were found between strains having different mammary tumor susceptibility. Hormone-dependent GR mouse mammary tumors also showed high H2A phosphorylation, but in some tumors also H1 and H3 were phosphorylated. Hormone-dependent GR tumors had 2–5 times higher histone phosphorylation at serine and threonine than hormone-independent tumors.     

Characterization of a progestin-binding component in lactating rat mammary glands

Experiments were carried out to identify progestin-binding receptors in the mammary gland where casein synthesis is known to be inhibited by this hormone. A progestin-binding component with high affinity, low capacity and a sedimentation coefficient of 8.8 S was isolated from the cytosol of lactating rat mammary glands. This component strongly bound [³H]R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) with a dissociation constant of 3.9 · 10^?9 M under low-salt conditions and with that of 8.2 · 10^?10 M in the presence of 0.3 M KCl. Specificity studies showed a higher degree of progestin specificity under high salt conditions. In the absence of KCl, binding of [³H]-R5020 was inhibited by unlabeled glucocorticoid in the same degree as unlabeled progestin, but the inhibition by glucocorticoid was greatly diminished by the presence of 0.3 M KCl. These observations suggest that the [³H]R5020-binding-component is the progestin receptor and that its function may be regulated by the concentration of glucocorticoid and salt.     

Initiation of lipogenic enzyme activities in rat mammary glands.

The activities of acetyl-CoA carboxylase, ATP citrate-lyase and fatty acid synthetase remained low until parturition at 22 days of gestation and increased significantly within 1 day post partum. Administration of progesterone on days 20 and 21 and at parturition abolished the increases for at least 48 h after parturition. Removal of the pups of normal rats prevented the increases in activities of acetyl-CoA carboxylase and ATP citrate-lyase, but not of fatty acid synthetase, and administration of prolactin corticosterone or insulin did not stimulate activity. Tissue from suckled glands in which the ducts had been ligated at parturition showed no increase in the activities of acetyl-CoA carboxylase and ATP citrate-lyase within 24 h, whereas fatty acid synthetase activity was similar to that in the sham-operated contralateral glands. Foetoplacentectomy on day 18 increased the activity of fatty acid synthetase but not of acetyl-CoA carboxylase and ATP citrate-lyase; suckling of these dams by foster pups increased both acetyl-CoA carboxylase and ATP citrate-lyase.     

Candida albicans double-stranded DNA can participate in the host defense against disseminated candidiasis

In the present work, we studied the in vitro immunomodulatory properties of double-stranded Candida albicans DNA and its protective effect in murine disseminated candidiasis. DNA induced the production of TNF-alpha by peritoneal macrophages and splenocytes in vitro through a chloroquine-dependent mechanism. Yeast DNA acted synergistically with IFN-gamma in triggering the secretion of nitric oxide by macrophages and enabled them to stimulate the proliferation of T cells in response to soluble anti-CD3. The effect of DNA on splenocytes is associated with an enhanced synthesis of IFN-gamma, IL-2 and IL-10. In vivo, DNA decreased the mortality and lowered the kidney contamination in mice intraperitoneally inoculated with C. albicans simultaneously with an increase in the specific proliferative response and cytokine production. The present results indicate that C. albicans DNA can provide protection against disseminated infection.