Morphogenetic characteristics of the mammary glands of autoimmune F1 mice (NZB x NZW)

The morphogenesis of mammary glands was studied in the normal and autoimmune F1(NZW X NZB) mice. In the lactation cycle of the autoimmune mice the normal course of structural-functional rearrangements of parenchyma and stroma in the developing and involuting mammary glands was disturbed. A conclusion has been reached that the modification of stromal elements, first of all involved in the autoimmune disease, is the leading link in the abnormal development of mammary glands.     

Quantitative collection of milk and active recombinant proteins from the mammary glands of transgenic mice

Summary

Transgenic mice expressing foreign genes specifically in their mammary glands have been obtained by several groups in the world. The mouse is generally considered as a good reference animal to evaluate the efficiency of gene constructs to be used in larger mammals for the preparation of the corresponding recombinant proteins at an industrial scale. The method described here shows that mammary glands from lactating mice separated from their pups for one day spontaneously released 1.5 ml milk when stored at O'C. The proteins of milk obtained by this method were essentially similar to those obtained after milking. Human growth hormone (hGH) gene under the control of the rabbit whey acidic (WAP) gene promoter was expressed at a high level in the milk of transgenic mice (4 mg/ml milk in the mice examined here). hGH was present in milk obtained after milking or after the incubation of the mammary glands at O'C. In both cases, the hormone was present in essentially similar concentration, undegraded and biologically active (as judged by its prolactin‐like activity). The method depicted here is very simple and can be applied easily to many mice. Its major limitation is that it implies the breeding and the sacrifice of a relatively large number of animals. One gram of crude recombinant protein can be virtually obtained in this way with about 200 lactating mice from their milk containing the proteins at the concentration of 3‐4 mg/ml. The milk of transgenic mice can therefore be considered as a practical source of recombinant proteins for biochemical and pharmaceutical studies.     

Formation of a steroidal allyl alcohol in the mammary glands of mice

After incubation of [ 4-¹⁴C )progesterone with cell-free homogenates of mouse mammary gland in the presence of NADPH, [¹⁴C]-labeled 4-pregnene-3α, 20α-diol was identified as a metabolite, besides 20α-hydroxy-4-pregnen-3-one which was the major metabolite.     

Expression of full-length human pro-urokinase in mammary glands of transgenic mice

Human pro-urokinase expressed in the mammary glands of transgenic animals is quickly activated and converted to urokinase by proteases that are present in the milk. Thus, it is nearly impossible to isolate full-sized pro-urokinase from the milk of transgenic animals. To solve this problem, we constructed transgenic mice that express human pro-urokinase and modified ecotin, which is a potent serine protease inhibitor from E. coli, in their mammary glands. The gene encoding ecotin was modified so as to enhance its specificity for the human urokinase-type plasminogen activator. Co-expression of modified ecotin and human pro-urokinase in the mammary glands allows for purification of full-length human pro-urokinase from these transgenic mice. The results described here suggest a general way of preventing the activation of zymogens that are expressed in the mammary glands of transgenic animals by co-expression of a zymogen along with a protease inhibitor.     

Purification and characterization of mouse alpha-lactalbumin from lactating mammary glands

The whey protein, alpha-lactalbumin, was purified from lactating mammary glands of mice at high yields. It exists as two major charge forms (pI values of 6.2 and 5.8) with similar molecular weights (approx. 14600). Antibodies prepared against these peptides precipitate newly synthesized and secreted alpha-lactalbumin from organ cultures of mid-pregnancy mammary glands. The antibody is specific for mouse alpha-lactalbumin as it does not react with mouse casein, mouse serum or purified bovine alpha-lactalbumin or galactosyl transferase. In addition, it blocks enzymatic activity of alpha-lactalbumin in mouse milk but has no effect on guinea pig or human milk. A very sensitive radioimmunoassay has been developed with this antibody which can detect alpha-lactalbumin levels as low as 0.25 ng.     

Progesterone stimulates DNA synthesis and lobulo-alveolar development in mammary glands in ovariectomized mice

The objective of this study was to determine whether sustained progesterone (P) use in the absence of estrogen could influence mammary development in mice. Three-week-old intact or ovariectomized mice were primed with subcutaneous (s.c.) cholesterol (C), estrogen (E), P, or estrogen and progesterone (E/P) together. Nine days after priming, mammary glands were removed and incubated as a whole organ in media supplemented with various combinations of lactogenic hormones. After 5 days in whole organ culture, glands were removed and end buds, alveolar buds and lobulo-alveoli were quantified. Glands from mice primed with C or E developed significantly less lobulo-alveoli than glands from mice primed with P or E/P. While the development was greater in animals treated with E/P compared to those treated with P, it was clear that P in the absence of E could still induce lobulo-alveolar development. We have shown in this paper that P, in the absence of E, can stimulate cell proliferation during priming. Subsequently, the P primed glands can differentiate in response to lactogenic hormones. J. Cell. Physiol. 180:298–304, 1999. © 1999 Wiley-Liss, Inc.     

Differential glycosylation of rhLf expressed in the mammary gland of transgenic mice

Differential glycosylation of natural hLf and rhLf from hLf-transgenic mice, which harbored a 146 Kb BAC insert that includes the intact hLf gene sequence, was studied in the present report. There were significant differences between the immunoblotting results of rhLf and natural hLf, which were denatured with nonreducing SDS sample buffer. The differences disappeared after rhLf and natural hLf samples were digested with N-glycosidase F, respectively. The results showed that there were significant differences (P<0.01) between the glycosylation of natural hLf and rhLf that were purified, respectively, from milk samples of seven hLf-transgenic mouse lines.     

Immunohistochemical detection of mammary tumor virus antigens in sweat and sebaceous glands of mice

Mammary tumor virus (MTV) antigens in both sexes of GRS/A, SHN and C3H mice were examined in the sweat and sebaceous glands by immunoperoxidase technique using antiserum against gp52, envelope protein, or p27, core protein. Balb/c mice were used for reciprocal foster nursing with these inbreds to discriminate the expression of endogenous MTV from that of exogenous MTV. Both antigens were first detected around the age of 4 months in the sweat glands of mice with endogenous GR- or SHN- MTV. A linear staining of gp52 was seen along the luminal borders of glandular cells, and the reaction products for gp52 were demonstrated on the apical cell membranes, where no virion could be seen ultrastructurally. A diffuse staining of p27 was found in the cytoplasm of some glandular cells, where MTV particles could not be detected. In the sebaceous gland of the same mice, however, only p27 was first detected at the age of 60 days. A dot-like staining of p27 was found in the perinuclear region of some glandular cells, where an aggregation of intracytoplasmic A particles could be seen under an electron microscope. These positive stainings were unrelated to sex. In such skin appendages of all examined C3H mice and Balb/c mice with GR- or SHN- MTV, no antigen expression could be seen up to the age of 500 days. Therefore, some genes might be able to regulate the expression of endogenous MTV antigens in the skin appendages, while their glandular cells would have no receptor for exogenous MTV, namely the so-called "milk factor".     

Immunoelectron microscopic localization of mammary tumor virus (MTV) antigens in normal and cancerous mammary glands of mice

Peroxidase-labeled Fab' fragments of rabbit antisera against gp52 (major envelope protein) and A-particles of mammary tumor virus (MTV) were prepared and used for investigation by immunoelectron microscopy of the replication cycle of MTV-specific envelope and core antigens in normal and malignant mammary gland cells of female mice. The specificity of the antisera was proven by absorption tests and lack of reactivity to MTV-free mammary tissues. Periodate-lysine-paraformaldehyde (PLP) fixation sufficiently preserved the antigenicity of gp52, while Zamboni's fixative was useful to preserve the core antigen. Saponin pretreatment was necessary to reveal the intracellular antigen of A particles but had no influence on gp52. In addition to its presence at the envelope of D particles, gp52 was clearly associated with the biomembrane system, including the nuclear membrane, endoplasmic reticulum, Golgi apparatus and plasma membrane independent of the presence of virus particles. In mammary tumors, a significant level of gp52 antigen was often expressed on the entire cell surface membrane. In contrast, it was localized only to the apical plasma membrane in normal mammary gland cells. A particle antigens were confined to the intracytoplasmic A particles, usually visible as clusters, and to the inner part of B particles. These ultrastructural findings support the available biochemical data on the morphogenesis of MTV particles.     

Purification and properties of cathepsin D from the mammary glands of lactating rabbits

Cathepsin D was purified from the lactating rabbit mammary gland by a rapid procedure, which included fractionation with (NH4)2SO4, acid precipitation, double affinity chromatography on pepstatin-Sepharose 4B and gel filtration on Sephadex G-100, resulting in approximately 360-fold purification of the enzyme over the homogenate and approximately 16% recovery. After isoelectric focusing, the enzyme dissociated into four (pI 5.8, 6.3, 6.5 and 7.2) multiple forms, but appeared homogeneous on polyacrylamide gel electrophoresis. Cathepsin D has a Mr of 45 kDa as determined by Sephadex G-100 column chromatography. On sodium dodecylsulfate/polyacrylamide gel electrophoresis the enzyme gave a single protein band, corresponding to Mr of 45 kDa. The amino acid composition of the enzyme is similar to that of cathepsins D from other tissues. A single N-terminal amino acid was glycine. Cathepsin D contains 6.4% carbohydrates consisting of mannose, galactose, fucose and glucosamine at a ratio of 3:9:2:2. Cathepsin D is inhibited by pepstatin with Ki of 2.5 X 10(-9) M and irreversibly by N-diazoacetyl-N'-2.4-dinitrophenyl-ethylene diamine. The enzyme hydrolyzes bovine hemoglobin with the maximal activity at pH 3.0 with Km = 10(-5) M and HLeu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe with Km = 4 X 10(-5) M and Rcat = 0.95 s-1. The major cleavage sites were Leu15-Tyr16, Phe24-Phe25 and Phe25-Tyr26 during hydrolysis of the oxidized insulin B-chain by cathepsin D.