ARTICLE |
Correspondence to: Takuya Kanazawa, College of Agriculture, Ibaraki University, Ami-machi, Ibaraki 300-0393, Japan. E-mail: takuyak@msv.ipc.ibaraki.ac.jp
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Summary |
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We generated monoclonal antibodies (MAbs) against mouse s1- and ß-casein and used them to survey casein immunochemically in mammary glands of mice at peri-coitous and pregnant stages. Two MAb-producing hybridoma cells, designated MC
1 cell and MCß1 cell, were established. Each antibody, when used in Western blotting, recognized specifically mouse
s1- and ß-casein among a wide spectrum of proteins of both a lactating mammary homogenate and mouse skim milk. Immunohistochemistry revealed
s1- and ß-casein in sections of lactating mammary glands. Staining was found in substances in the lumen and cytoplasm of duct and alveolar cells, particularly in rough endoplasmic reticulum and the Golgi apparatus. Mammary glands at Days 2, 4, 6, 8, and 14 of pregnancy showed positive staining specific to both
s1- and ß-casein in the lumen and cytoplasm of duct cells, whereas the glands at estrus and Day 0 of pregnancy were positive mainly for
s1-casein. Semiquantitative Western blotting analysis of both casein components in epithelial cell fractions from glands during pregnancy confirmed that intra-epithelial
s1- and ß-casein changed during three phases, elevated from trace levels to detectable levels during initial stages of pregnancy (Days 0, 2, and 4), declined to lower levels during mid-pregnancy (Days 6 and 8), and then rose to high levels during late pregnancy (Day 14).
(J Histochem Cytochem 50:257264, 2002)
Key Words:
mouse s1-casein, mouse ß-casein, monoclonal antibody, immunohistochemistry, mouse mammary gland
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Introduction |
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THE FATE OF MAMMARY EPITHELIAL CELLS, including milk-secreting cells, is determined during embryogenesis in the mouse (-casein have confirmed that the combination of the three hormones, i.e., prolactin, insulin and glucocorticoids, stimulates casein secretion by the cells of mice in mid-pregnancy (
s1-casein is different from that for other major casein components (unpublished observations). These observations imply that mammary epithelial cells of virgin mice are as capable of differentiating as are those of pregnant mice and that
s1-casein might be expressed differentially in vivo. However, our studies and those of others concerning casein synthesis have used polyclonal antibodies, and it is therefore difficult to exclude the possibility of detecting crossreactive non-casein proteins and to examine each casein component selectively. In the present study we generated two monoclonal antibodies (MAbs) against mouse
s1- and ß-casein and have surveyed each casein in mammary glands of mice at peri-coitous and pregnant stages using IHC to clarify the timing of expression of each casein component. Furthermore, we have quantified casein components in the mammary epithelium by Western blotting to assess differentiated function during pregnancy.
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Materials and Methods |
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Animals
Inbred KA strain mice, aged 10 to 24 weeks, were used for collecting milk and mammary glands. SpragueDawley strain rats and Balb/c strain nu/nu mice, used for generation of MAbs, were purchased from Charles River Japan (Tokyo, Japan). These animals were maintained with MF chunk diet (Oriental Yeast; Tokyo, Japan) and water ad libitum in a biohazard protection condition, which conformed to the Ethics Review Committee for Animal Experimentation of the University of Tokyo. The estrous cycle of the mice was determined by vaginal smear observation. The days of vaginal plug and parturition were counted as Day 0 of pregnancy and Day 1 of lactation, respectively.
Preparation of MAbs Against Mouse Casein Components
Mouse whole casein was collected and polymerized using glutaraldehyde as described previously (
Western Blotting for Mouse Casein Components
A lactating mammary homogenate was prepared. A lactating mouse at Day 10 of lactation was separated from suckling pups immediately before sacrifice. The mammary tissues were removed and homogenized in 10 vol of ice-cold PBS, pH 7.4, containing 0.25 M sucrose, 1 mM phenylmethylsulfonyl fluoride (Nacalai; Kyoto, Japan) and pepstatin A (2 µg/ml; Sigma), and were centrifuged at 3000 x g for 30 min at 4C. The supernatant was collected (mammary homogenate). Epithelial cells were collected from mammary glands of mice by collagenase digestion, as previously described (
Immunohistochemical Detection of Casein Components
Inguinal mammary glands (fourth glands) were removed from mice at various reproductive stages. Mammary glands at Day 10 of lactation were removed immediately after separation of the mother from suckling pups. The tissues were fixed in Bouin's solution for 1 hr and then embedded in paraffin using standard procedures. Tissue sections of 12 µm were dewaxed in xylene and then treated with 0.3% hydrogen peroxide in methanol for 30 min at room temperature (RT) to inactivate endogenous peroxidase activity. Sections were rehydrated in a descending series of ethanol and blocked in 1% gelatin in PBS (pH 7.4). Sections were then incubated with rat anti-mouse casein MAbs (1 µg/ml in blocking solution) for 1.5 hr, then with goat anti-rat IgG (10 µg/ml in blocking solution; TAGO) for 1.5 hr, and finally with rat PAP (20 µg/ml in blocking solution; Serotech) for 1 hr. The localization of peroxidase was detected by color reaction for 5 min at RT using 4,4'-diaminobenzidine tetrahydrochloride (Dojin; Osaka, Japan) as substrate. As controls, primary antibodies were substituted with normal rat IgG. When a mixture of the two types of anti-mouse casein MAbs was used as primary antibody, peroxidase-conjugated goat anti-rat IgG (10 µg/ml; TAGO) was used as the second antibody and staining was performed as described above.
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Results |
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Production and Characterization of MAbs Against Mouse Casein Components
Antibody-producing hybridoma cells were screened by ELISA and two hybridoma clones, MC1 and MCß1, were established after three sequential clonings by the limiting dilution method. Each hybridoma clone successfully produced ascites antibody when injected into abdominal cavities of nude mice. MC
1 and MCß1 produced rat IgG2a and IgG1, respectively, by typing based on Ouchterlony's gel double-diffusion method (data not shown). These two types of antibodies, purified from ascites by salting-out with ammonium sulfate and anion-exchange column chromatography, ran as single bands on SDS-PAGE under non-reducing conditions (data not shown). SDS-PAGE under reducing conditions revealed that MC
1 and MCß1 are composed of heavy chains of similar molecular weights (52 kD) and distinctive light chains (29 kD and 32 kD, respectively) (data not shown).
Western blotting was performed to identify antigens recognized by these antibodies (Fig 1). As shown in our previous report (s1-, ß-,
-casein, and other minor components, in mouse skim milk and a lactating mouse mammary gland homogenate (Fig 1, Lanes 6 and 7). Rat MAbs specifically recognized a single class of mouse casein component among many classes of proteins: MC
1 and MCß1 stained
s1- and ß-casein, respectively, in a homogenate of a lactating mammary gland as well as in mouse skim milk (Fig 1, Lanes 4, 5, 8, and 9). Two faint bands that ran above and below the major band of
s1-casein are presumably hyperphosphorylated and dephosphorylated products, respectively (
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Immunohistochemical Survey of Casein Components in Mouse Mammary Glands at Various Reproductive Stages
In the tissue sections prepared from lactating mammary glands, positive staining for both s1- and ß-casein was observed when MC
1 and MCß1 were included in the first step of antibody incubation (Fig 2A and Fig 2B). Staining was localized both on the granular substances in the lumen (asterisks) and in most cells surrounding the lumen of the glands, including alveoli and small ducts (small arrowheads) and large ducts (large arrowheads). In contrast, no positive staining was observed when primary antibody was excluded from the immunoreaction or when primary antibody was substituted with normal rat IgG (Fig 2C). The PAP method, in general, produces more sensitive staining but also tends to produce more diffuse staining. Therefore, we next examined localization of casein components in the casein-positive cells by a standard indirect method using a mixture of MC
1 and MCß1 as a primary antibody and peroxidase-conjugated goat anti-rat IgG as the secondary antibody (Fig 2D). The strongest positive staining in the cytoplasm localized predominantly in a particular area between the apical region and nucleus (small arrows), which corresponds to endoplasmic reticulum, the Golgi complex, and/or secretory vesicles (
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It is important to clarify the timing of casein synthesis in vivo to understand the mechanism of differentiation of mammary epithelial cells. We therefore surveyed casein components in sections prepared from mammary glands at estrus of cycling mice and at Days 0, 2, 4, 6, 8, and 14 of first pregnancies. The PAP method, using MC1 and MCß1 as primary antibodies, produced
s1- or ß-casein-specific positive staining at Days 2, 4, 6, 8, and 14 of first pregnancies (Fig 3). Positive staining was obvious both in the apical region of the luminal cytoplasm and in lumens of the glands at Days 2, 4, and 14, but the staining was more restricted to glandular alveoli and was less intense in the glands at Days 6 and 8. Granular substances in the lumens of alveoli and ducts were strongly stained, whereas the cytoplasm of luminal and duct cells was weakly or diffusely stained. In contrast to the glands at Day 2 or later, mammary glands at estrus and at Day 0 of pregnancy exhibited clearly positive staining for
s1-casein but only faintly positive staining for ß-casein. Although casein-specific positive staining was detected, few distended lumens were observed in the glands of pregnant mice, which was in contrast to the lactating glands.
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Western Blotting Survey of Casein Components in Mammary Epithelial Cells from Mice at Various Stages of Pregnancy
To confirm the casein components in mammary epithelial cells of mice at early stages of pregnancy, Western blotting was employed. Epithelial cell fractions were collected from mammary glands of individual mice at days 0, 2, 4, 6, 8, 10, 12, and 14 of first pregnancies and the respective cell extracts prepared from the same number of cells were subjected to SDS-urea-PAGE and were analyzed for casein by Western blotting using the PAP method. As shown in Fig 4, both s1- and ß-casein were detected in cells during pregnancy (data on cells at Days 10 and 12 are not shown). Intensities of both
s1- and ß-casein bands at Days 0, 2, 6, and 8 were very faint, presumably due to lower sensitivity compared to the present IHC, although densitometrical scanning detected each casein band in these samples. Major casein bands exhibited the same mobility as did those from mouse whole casein. Minor bands below the major band were presumably due to degraded products of casein generated during the cell preparation procedure, because these bands were not detected in the homogenate samples as shown in Fig 1, Lanes 4 and 8. Densitometrical quantification of the casein bands revealed that intracellular casein components changed in good correlation with the results from IHC. Although intensities of both
s1- and ß-casein bands at Day 0 were low as trace levels, both of the caseins were elevated from Day 0 to Days 2 and 4, decreased from Day 4 to Days 6 and 8, and then again elevated at Day 14 (Table 1). These three phases of change were statistically significant (Student's t-test, p<0.05). The relative value of
s1-casein tended to be higher than that of ß-casein at Days 0 and 2 of pregnancy. This correlated well with the results from IHC showing that sections at Days 0 and 2 of pregnancy exhibited clearly positive staining for
s1-casein but only faintly positive staining for ß-casein.
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Discussion |
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We have generated anti-mouse s1- and ß-casein MAbs and have clearly demonstrated that both
s1- and ß-casein components are present in mammary epithelium of mice at the estrous stage of cycling virgins and throughout stages of pregnancy.
It has been reported that primary mammary cells dissociated from unprimed mature mice require 3 to 4 weeks of preculture in medium (horse serum, FBS, and insulin) to synthesize casein in response to a lactogenic combination of hormones (insulin, prolactin, cortisol, and aldosterone) (s1- and ß-casein components by Western blotting, which enabled us to examine each casein component rigorously. Furthermore, we purified these antibodies before use as probes for casein. These technical improvements make the present results more convincing.
We have further examined mammary glands around coitous stages, using the PAP method, which is much more sensitive than the standard indirect method used for IHC in earlier studies (
The present IHC study has also revealed that casein components are present in the cytoplasm of almost all luminal epithelial cells of lactating glands, including those lining large ducts. This observation contradicts an earlier observation that casein-specific staining localizes only to cells of alveoli and small ducts (
In conclusion, the present IHC study, using newly generated anti-mouse s1- and ß-casein MAbs, demonstrated that the casein level in mammary epithelium changed in three phases, by elevating from trace to a detectable level during initial stages of pregnancy (Days 0, 2, and 4), declining to a lower level during mid-pregnancy (Days 6, 8, and 10), and then rising to a high level during late pregnancy (Days 14 and later). Our present study also demonstrated both casein components in duct epithelium, which suggested the potentially similar ability of luminal epithelial cells in the mammary glands to synthesize casein.
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Footnotes |
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2 Present address: Department of Animal Science, Nippon Veterinary and Animal Science University, Musashino, Tokyo, Japan.
Received for publication May 29, 2001; accepted October 3, 2001.
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