人Bcl-2乳腺定位表达转基因小鼠的建立

以 p CAT、p WB和 p WC为原始质粒 ,构建 WAP启动子调控的 Bcl- 2乳腺组织特异性表达载体 p WBS。载体用Bgl +Sal +Pvu 酶切 ,回收 3.0 kb的基因片段 WAP- Bcl- 2 - SV40 Poly A,通过显微注射方法导入 C5 7BL / 6 J×DBA/ 2 JF1代小鼠受精卵的雄原核内。共注射 10 0 0枚卵 ,将存活的约 6 0 0枚卵分别移植至 2 9只假孕母鼠输卵管内 ,获得仔鼠 5 5只。PCR检测 ,阳性鼠 12只。Southern blot检测 ,阳性鼠 4只 ,其中公、母鼠各 2只 ,在饲养过程中 ,2只公鼠均意外死亡。Western blot证实 ,1只母鼠 Bcl- 2表达阳性 ,其 F1代的 40只小鼠中 ,有 18只呈 PCR阳性 ,其中母鼠8只 ,在 5个月的反复怀孕过程中 ,未观察到乳房有任何肿瘤或肿块的出现 ,证实 Bcl- 2单独在转基因鼠乳腺中表达不会引起转基因鼠乳腺癌的发生。     

Effect of vitamin A deficiency on mammary gland development and susceptibility to mastitis through intramammary infusion with Staphylococcus aureus in mice

Weanling mice were fed 0 or 150 micrograms retinol equivalent/kg of diet for 5 weeks, were bred, and allowed to complete gestation. On day 3 of lactation, all mice were separated from their litters for 1 hour and were then anesthetized. The 4th right or left mammary gland was inoculated with 0.1 ml of S Aureus (10(10) colony-forming units/0.1 ml). Exactly 24 hours after inoculation, the mice were euthanatized and the mammary glands were removed and fixed for histologic evaluations. Vitamin A-deficient dams had smaller litter size and lower liver stores of vitamin A; however, deficiency was not severe enough to produce external signs of vitamin A deficiency in the dams. Morphologic studies showed large areas of adipose tissue, greatly reduced ductal and lobule-alveolar development, and decreased total secretory activity in mammary glands from vitamin A-deficient females. On the other hand, mammary glands from vitamin A-supplemented mice had extensive lobule-alveolar development and highly distended alveoli. Extensive necrosis of alveolar tissue was observed in staphylococcus-infused mammary glands of all mice. Large numbers of leukocytes and cell debris were present in the lumen of alveoli and ducts. However, mammary glands from vitamin A-deficient females had more extensive pathologic damage compared with corresponding glands from vitamin A-supplemented mice. Results indicted that vitamin A-deficient mice had reduced mammary development and increased pathologic damage to the mammary gland after intramammary challenge with staphylococcus.     

Inhibitory function of whey acidic protein in the cell-cycle progression of mouse mammary epithelial cells (EpH4/K6 cells)

Although the biological role for whey acidic protein (WAP) in milk has been suggested, its true function is not known. This paper describes evidence for WAP function in the cell-cycle progression of EpH4/K6 (EpH4), mammary epithelial cells in vitro. The forced expression of exogenous WAP significantly impaired the proliferation of EpH4 cells, whereas it did not affect that of NIH3T3 cells. Apoptosis was not enhanced in the EpH4 cells with stable expression of WAP (WAP-clonal EpH4 cells). The analyses of BrdU incorporation revealed that forced WAP expression significantly reduced incorporation of BrdU in WAP-clonal EpH4 cells compared with control cells transfected with empty plasmid. Among G1 cyclins, the level expression of cyclins D1 was significantly lower in the WAP-clonal EpH4 cells than in control cells. The inhibitory action of WAP on the proliferation of EpH4 cells was enhanced by the presence of extracellular matrix (ECM), but not by the presence of a single component comprising ECM. The cultured medium of WAP-clonal EpH4 cells inhibited the proliferation of control cells without WAP expression. The present results indicate that WAP plays a negative regulatory role in the cell-cycle progression of mammary epithelial cells through an autocrine/paracrine mechanism.     

Characteristics of Rabbit Transgenic Mammary Gland Expressing Recombinant Human Factor VIII

The objective of this research was to compare (i) the content of milk protein and recombinant human factor VIII (rhFVIII) in the milk of transgenic and non-transgenic rabbit females at three lactations and (ii) histological structure, ultrastructural morphology and occurrence of apoptosis in rabbit transgenic and non-transgenic mammary gland during third lactation and involution. Significant differences (t_0.05) in milk protein content were found between transgenic and non-transgenic at all three lactations. The percentage of apoptotic cells was significantly higher (t_0.01) in non-transgenic ones compared with transgenic mammary gland tissues (6.5% versus 2.4%) taken at the involution stage. Morphometrical analysis of histological preparations at the involution stage detected a significantly higher (t_0.05) relative volume of lumen in transgenic animals compared with non-transgenic ones (60.00 versus 46.51%). Ultrastructural morphology of the transgenic mammary gland epithelium at the involution stage revealed an increased relative volume of protein globules (t_0.05); at the lactation stage, a significantly higher volume of mitochondria (13.8%) compared with the non-transgenic (9.8%) ones was observed. These results, although revealing differences in some parameters of ultrastructure and histology, indicate no harmful effect of the mouse whey acid protein-hFVIII transgene expression on the state of mammary gland of transgenic rabbit females.     

大鼠不同时期乳腺组织中雌激素孕激素及催乳素的变化规律

为探讨雌激素(E2)、孕激素(P)及催乳素(Prl)在大鼠不同时期乳腺组织中的变化规律,选雌性SD大鼠42只,分别为处女鼠、妊娠鼠(6d、12d、18 d)、泌乳鼠(6d、12d、18 d),每个时期6只.在指定的时间点处死大鼠取乳腺组织.用放免(RIA)组织匀浆中E2、P及Prl水平进行定量研究,用统计学方法进行处理,观察这三种激素的变化规律.结果显示E2从处女期到整个妊娠期,其呈现下降趋势.而分娩后E2水平急剧升高,后又有所下降,但维持在较高水平;在乳腺发育不同时期,P在乳腺组织中整体水平一直呈现下降趋势;Prl水平以处女期最高;随着妊娠进行逐渐降低,整个乳腺发育过程中Prl整体上几乎一直呈下降趋势.表明E2、P及Prl在促进乳腺发育及维持和启动泌乳方面发挥了重要的作用.     

人c-myc乳腺定位表达转基因小鼠的建立

以含有 1.6 kb的小鼠乳清酸蛋白 (WAP)上游调序列的 p CAT- WAP和含有人 c- myc c DNA的 p WM为原始质粒 ,构建了 WAP启动子调控下的 c- myc乳腺定位表达载体 p WCS。载体用 Bgl Bam H 酶切 ,回收 3.5 kb的基因片段 WAP- c- myc- SV40 Poly A,通过显微注射方法导入 C5 7BL/ 6 J× DBA/ 2 JF1 代小鼠受精卵的雄原核内。共注射10 0 0枚卵 ,将存活的约 6 0 0枚卵分别移植至 2 9只假孕母鼠输卵管内 ,获得仔鼠 45只。PCR检测 ,阳性鼠 9只 ,South-ern blot检测 ,阳性鼠 3只 ,其中 1只母鼠 ,2只公鼠 ,在饲养过程中 ,1只母鼠意外死亡。对 2只转基因公鼠的 F1代PCR检测表明 ,仅 1只公鼠具有遗传性 ,在所生的 2 7只 F1代小鼠中有 13只为 PCR阳性。     

Production of the mouse whey acidic protein in transgenic pigs during lactation.

The mouse whey acidic protein (WAP) gene was introduced into the genome of pigs and its expression was analyzed in the mammary gland. Mouse WAP was detected in milk of lactating females from five lines at levels between .5 and 1.5 g/liter, thereby representing as much as 2% of the total milk proteins. The corresponding mRNA was expressed in mammary tissue at levels similar to those of pig beta-lactoglobulin and beta-casein. The pattern of WAP secretion in three pigs over a period of 6 wk was quantitatively similar to that of pig beta-lactoglobulin. From the eight transgenic pigs analyzed, three successfully completed one lactational period, but five pigs stopped lactating a few days after parturition. Our results show that it is possible to produce large quantities of a foreign protein in milk of pigs over a full lactational period. However, expression of WAP can compromise the mammary gland and render it nonfunctional.     

Localization of fibroblast growth factor I (acid fibroblast growth factor) and its mRNA in the bovine mammary gland during mammogenesis, lactation and involution

Growth factors are involved in development and function of the mammary gland. The aim of this study was the localization of fibroblast growth factor 1 (FGF-1) and its mRNA in the bovine mammary gland during different developmental and functional stages. Mammary tissue was obtained from German Brown Swiss cows (n = 23) during defined stages of mammogenesis (before and during pregnancy), lactogenesis, peak lactation and involution. The distribution of FGF-1 mRNA was studied using non-radioactive in situ hybridization, the corresponding FGF-protein was analysed using immunohistochemistry [avidin-biotin peroxidase complex (ABC)-method]. A moderate to distinct staining for FGF-mRNA was found in the epithelium of ducts and developing alveoli during mammogenesis. Post-partum at the same cellular locations, a considerable amount of FGF-1 mRNA, was seen that decreased during lactation. Also during early involution clear staining for FGF-mRNA could still be observed. Immunoreactive FGF-1 was found in considerable concentration in the epithelium of the mammary gland in heifers. The staining intensity generally decreased somewhat during mammogenesis and lactation, but could be always clearly demonstrated in the secretory epithelial cells of alveoli and glandular ducts. Also during the first day after the end of milking, the epithelium displayed a moderate to distinct epithelial immunostaining. Notably, After 4 weeks of involution, in many alveoli a shedding of the FGF-1 positive luminal cell layer was found. In our localization studies, no strict correlation between FGF-1 mRNA and its corresponding protein was found. The various reasons for this finding are discussed.     

Effects of coumestrol administration to maternal mice during pregnancy and lactation on immunoblogulin A‐secreting cells in mammary glands

Mortality and morbidity of neonates continue to be major problems in humans and animals, and immunoblogulin A (IgA) provides protection against microbial antigens at mucosal surfaces. The present study was conducted to clarify the effects of coumestrol administration to maternal mice during pregnancy and lactation on IgA antibody‐secreting cells (ASC) in mammary glands in lactating mice. From 6.5 to 16.5 days post coitus and 1 to 13 days post partum (dpp), maternal mice were administered coumestrol at 200 μg/kg body weight/day. Coumestrol administration increased the number of IgA ASC and the messenger RNA expression of IgA C‐region and vascular cell adhesion molecule‐1 in mammary glands of maternal mice at 14 dpp, but coumestrol administration had no effect on the number of IgA ASC in the ileum. Coumestrol administration increased serum IgA concentration in maternal mice at 14 dpp, but IgA concentrations in serum, stomach contents, intestine and feces of neonatal mice were not affected by treatment. These results imply that coumestrol administration to maternal mice during pregnancy and lactation is effective in increasing the numbers of IgA ASC in mammary glands during lactation owing to the activated messenger RNA expressions of IgA C‐region and vascular cell adhesion molecule‐1 in mammary gland.     

Pregnancy and lactation affect the microvasculature of the mammary gland in mice.

Microvascular changes in the mammary gland in mice during pregnancy and lactation were investigated by scanning electron microscopy (SEM) with a corrosion cast method, transmission electron microscopy (TEM) and morphometry. By SEM, duct-associated capillary plexuses were sparsely distributed to branch into adipocytes during virgin period. With advance in pregnancy, both branches from the capillary plexuses and branches from the vessels surrounding adipocytes extended further to form capillary networks. The basket-like architecture was completed by day 18 of pregnancy. These findings may indicate that angiogenesis occurs frequently during this period. During lactation, the basket-like architecture still remained and the capillaries surrounding alveoli meandered. After weaning, the regression of microvasculature followed the degeneration of alveoli. By TEM and morphometry, the density of pinocytotic vesicles (PVs) (number of PVs per microns 2 of endothelium cytoplasm) increased twofold from day 18 of pregnancy to day 5 of lactation, furthermore increased threefold from days 10 to 20 of lactation, and subsequently decreased after weaning. Marginal folds and microvillous processes gradually increased in length with advance in pregnancy, reached the maximum from days 5 to 15 of lactation, and thereafter decreased. In addition, the capillaries with thinner walls were in close contact with alveoli during the late stage of pregnancy and during lactation. Furthermore, the alveolar epithelial cells had well-developed basal infoldings during lactation. These findings suggest that the capillaries play an important role in transporting materials necessary for milk production.     

Pathogenesis of Brucella abortus infection of the mammary gland and supramammary lymph node of the goat

Goats, both in late pregnancy and soon after parturition, were inoculated intravenously with Brucella abortus, and mammary glands and supramammary lymph nodes were examined by light and electron microscopy at 2 to 55 days post-inoculation. After 7 days, lymphoplasmacytic, histiocytic interstitial mastitis with a lobular and periductal distribution were detected microscopically. Brucellae, identified in tissues with immunoperoxidase staining and antibody-coated colloidal gold stain, were first seen in macrophages and neutrophils throughout mammary parenchyma, but most commonly in mammary alveoli. In subsequent samples, infected phagocytes progressively increased in number, especially in ductal and alveolar lumina, and adjacent parenchyma. B. abortus was in phagosomes and phagolysosomes in macrophages and neutrophils; degenerate and necrotic phagocytes were often filled with brucellae. Extracellular brucellae were associated with ruptured necrotic infected phagocytes. Supramammary lymph nodes draining infected mammary glands were enlarged. Lymphofollicular hyperplasia, medullary plasmacytosis, and sinus histiocytosis were seen microscopically. Brucellae were seen exclusively in macrophages, which were most often located in subcapsular and cortical sinuses. This study suggests that phagocytic leukocytes 1) transport brucellae into mammary glands; 2) provide a site for intracellular replication in mammary secretions; and 3) transport brucellae from mammary glands to supramammary lymph nodes.     

Ultrasonographic assessment of the feline mammary gland

The aim of this study is to characterise the feline mammary echotexture using B-mode ultrasonography, which is not routinely used to examine the feline mammary gland. Using a 5-9 MHz linear transducer the ultrasonographic appearance of non-stimulated and stimulated mammary glands was determined in 35 mature intact non-pregnant, pregnant and lactating queens aged from 16 months to 8 years. In intact non-pregnant queens, mammary glands are fairly underdeveloped and on the ultrasonograms they appear with a regular hypoechoic texture and generally show a thickness of less than 2.0mm. The stimulated mammary tissue typically presents a more hyperechoic appearance compared to the non-stimulated gland and a fine granular echotexture. Maximum echogenicity of the mammary gland is reached during lactation. In late pregnancy, the mammary glands reach 6-9 mm in thickness. During lactation, the size of the glands depends on the existence of a suckling stimulus, with the suckled glands reaching about 11 mm in thickness. Ductal structures can only be imaged during late pregnancy and lactation. Ultrasonographic evaluation of the feline mammary gland can become a valuable diagnostic tool to characterise physiological changes and may further contribute to a better characterisation of diseased mammary tissue.     

Whey acidic protein (WAP) depresses the proliferation of mouse (MMT) and human (MCF-7) mammary tumor cells

We previously reported that the enforced expression of exogenous whey acidic protein (WAP) significantly inhibited the proliferation of mouse mammary epithelial cells (HC11 and EpH4/H6 cells). This paper presents the first evidence that WAP also depresses the proliferation of mammary tumor cells from mouse (MMT cells) and human (MCF-7 cells). We established WAP-clonal MMT and MCF-7 cell lines, and confirmed the secretion of WAP from the WAP-clonal cells into culture medium. The enforced expression of WAP significantly inhibited the proliferation of MMT and MCF-7 cells in in vitro culture. FACScan analyses revealed that G0/G1 phase cell-cycle progression was disordered and elongated in the WAP-clonal MMT and MCF-7 cells compared to that of the control cells. The expression of cyclin D1 was significantly decreased in the WAP-clonal MMT and MCF-7 cells, suggesting that progression from the G1 to the S phase was delayed in the WAP-clonal cells. The present results indicate that WAP plays a negative regulatory role in the cell-cycle progression of mammary tumor cells via a paracrine mechanism.     

Involution and redevelopment of the mammary gland in goats. Histological-histochemical studies after a lactation time of 8-10 months

7 chamois-coloured mountain goats were used to investigate histologically and histochemically the processes of involution and redevelopment of the mammary gland after a lactation period of 8 to 10 months. Tissue specimens were obtained by incision biopsy at drying-off two months prepartum and afterwards in intervals of 8-16 days up to parturition. The findings of this investigation were compared with results of two former investigations, in which involution and proliferation of the gland were studied separately, thus precluding an overlapping of the two processes. At drying-off, after an 8 to 10 month-lactation, sections indicative of active lactation occurred concomitantly with sections at various stages of involution, as well as early stages of redeveloping alveoli. At 16 days post drying-off, the sections indicating lactation at first examination were in a stage of maximum involution and transformation. Thus, the time required for involution was reduced by half compared to drying-off at peak lactation. At 32 days post drying-off, stages of proliferation predominated with only a few involuting glands. Specimens obtained thereafter contained only redeveloping glands. The histological and histochemical differentiation between areas of involution and those of proliferation may present difficulties during the mid-portion of the dry period. There appears to be an association between the length of the time interval from drying-off to parturition and the rate of tissue transformation in the caprine mammary gland; the rate is increased when the duration of the dry period is reduced.     

Serum levels of stanniocalcin-1 in Holstein heifers and cows

Stanniocalcin-1 is a hormone that possesses both paracrine and endocrine functions and has recently been identified in mammals. While paracrine functions have been determined for several organs, the role of circulating stanniocalcin-1 in cattle is still unclear but, observations in mice and cows suggest that stanniocalcin-1 plays a role in both gestation and lactation. The changes in serum stanniocalcin-1 content in different physiological states have never been evaluated in ruminants. We measured stanniocalcin-1 levels in sera from cattle ranging in age from post-weaned calves to 17-month-old heifers and in sera from cows during lactation and pregnancy. Our results indicate that the blood concentration of stanniocalcin-1 is increased by pregnancy, but not by lactation. The highest levels of stanniocalcin-1 were found in young calves and during the non-lactating period preceding calving. This suggests that stanniocalcin-1 is important for gestation and the preparation of the mammary gland for lactation.     

Sexual dimorphism of the normal rat mammary gland.

Sexual dimorphism is a routinely encountered but previously unreported feature of the normal rat mammary gland. Forty male and 40 female F344 rats, 19 weeks of age, were examined retrospectively to document histologic differences that are very apparent when mammary glands of males and females of the same age are compared. Development of the mammary gland starts in utero, and there is reported to be little morphologic difference between males and females at birth. By 19 weeks of age, however, the differences are very noticeable. The mammary glands of females, comprised of scattered tubular ducts and alveolar structures that have distinct lumina lined, usually, by a single layer of cuboidal epithelium, are characterized as tubuloalveolar. In young males, mammary tissue is generally more florid than in females of the same age. There are, scattered within the hypodermis, larger, more contiguous, lobular groups of cells that are distinct for their lack of obvious tubular or ductal orientation. The cells, arranged into alveoli, are generally characterized by abundant, foamy, eosinophilic cytoplasm containing distinct, variably-sized vacuoles. Alveolar lumina are mostly indistinct but may contain evidence of secretory activity. To emphasize the importance of this difference, selected results of a routine toxicity study are discussed in which the mammary glands of male rats, exposed for 52 weeks to cis-N-(1-benzyl-2-methylpyrrolidin-3-yl)-5-chloro-2-methoxy-4- methyl-aminobenzamide, a reported dopamine antagonist, underwent distinctive histomorphologic changes. As a result of exposure to this compound, the lobuloalveolar structure described as normal for male rats assumed characteristics of a tubuloalveolar morphology indistinguishable from that seen in the unexposed females.(ABSTRACT TRUNCATED AT 250 WORDS)