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, tvansmission e]eetron microscopy (TEM) and morphometry. By SEM, duct-associated capillary plexuses were sparsety distributed to branch into adipocytes during virgin period. With advance in pregnancy, both branches from the capMary plexuses and branches from the vessels surrounding adipocytes extended further to form capiUary networks. Thc basket-like architecture was completcd by day 18 ofpregnancy. These findings may indicate that angiogenesis occurs frequently during this periud. During ]actation, thc baskct-]ike architecture still remained and the capillaries surrounding alveoli rneandered. After weaning. tbe regression of microvasct]lature followed the degeneration of alveoli. By TEM and morphometry, the density of pinocytotic vesicles (PVs) (number of PVs per stm2 of endothe]ium cytoplasm) increased twofold from day lg of pregnancy to day S of lactation, furthermore increased threefold from days 10 to 20 of tactation. and subscquently decreased ufter weaning. Marginal folds and microvilious processes gradually increased in length with advance in pregnancy, reached the maximum from days 5 te 15 of lactation, and thereafter deereased. In additlon, the capMaries with thinner wa[Is were in close contact with atveoli during the ]ate stage of pregnancy and during lactation. Furthermore, the alveolar epithelial cells had well-developcd basal infoldings during ]actation. These findings suggest that the capillaries play an important role in transporting materials necessary for milk production,-KEy woRDs: corrosion east. tnatnmary gland capil]ary. microvascu]ature, morphometry, mouse. ---------.L Vet. iLfed. Sci. 54(5),937-943,1992 The mammary gland is an accessory gland that develops especially during pregnancy and lactation, There have been a number of reports on the endocrine factors which regulate the development of the gland according to the functional states of milk production and secretion [14, 231. The deve}opment of blood vessels is thought to play an important roie in both mammogenesis and lactogenesis, since a large quantity of blood is required to produce mjlk E8, 251, Thc morphological relationship between the mammary parenchyma and the blood vessels has been investigated in the mouse mammary glands by light microscopy [5, 15, 21, 24]. To our knowledge, however, there is no report on the development of microvascular architecture of the mammary gland except for a report in the rat by Yasugi et al. [26]. Recently, a vascular corrosion casting method for scanning electron microscopy (SEM) l13] has bcen applied to the studies of vascular architectures in a vast variety of organs. In the present study, a vascular corrosion casting method for SEM was employed to clarify the three-dimensional development of microvasculature of the mammary gland in the mouse. UItrastructural changes in capillaries are reported to be involved closely in the capillary permeability l3, 4, 7, 9. 11, 16, 17, 19, 20. 27]. Although Sandborn [18], and Stirling and Chandler [22] described the fine capiLlary structure of the mammary gland during in non-lactating period, ultrastructural changes to be expected to occur in various physiological states have not yet bc¢ n reported. In the present study, morphological changcs in capillaries of the mouse mammary gland from virgin through pregnancy. Iactation and post-weaning stages were examined by transmission electron microscopy (TEM). Moreover, a morphometric analysis was attempted to assess quantitative changes in the morphological features. MATERIALS AND METHODS Eighty five jCL-ICR female mice, bred and maintained as a colony in our laboratory, in stages of virgin (90 day-old), pregnancy (5, 10, 15 and 18 days of pregnancy), lactation (5, 10. 15 and 20 days after partum) and post-weaning (5, 10 and 15 days after weaning) were used in this study. Each stage Japanese Society of Veterinary Science NII-Electronic Library Service apaneseSociety f eterinary cience 938 M. MATSUMOTO, EV'AL. group consisted of 7 or more animals. During lactation, each mother mouse was housed with her 8 to 10 pups. Al} the anirnals were supplied with food and water ad libitum. For SEM, vascular corrosjon casts were prepared according to Murakami's method [131. In brief, the animals were pei'fused with Ringer's solution containing heparin sodium (4 IU/ml) and with Mercox (rL-2R (Duinippon Ink and Chemicals, Inc., Tokyo, Japan), through the thoracic aorta. After polimerization of resin, the first abdomino-inguinal mammary glands werc excised and soaked in 209Z・ KOH so[ution overnlght at 6{)OC to remove the surrounding soft tissues, Dehydrated and ttir-dried vascular casts were exposed to osinic vapor, coated with gold by sputtering in a vacuum eyaporator and exarnined with a jSM-25 scanning electron microscope at 15 kV. For TEM, sma}L pieces wcre cut out from each of the first abdomino-inguinal mammary g]ands. The samplcs were immediately fixed in 2.5% glutaraldehyde in O.1 M phosphate buffer for 2 hr at 40C. They were rinsed in the same buffer and post-fixed in 2% osmium tetroxide in O.1 M phosphate buffer for 2 hr at 40C. Tltey were dehydrated in a gradcd scrics of ethanol and embedded in Epon 812. Thin sections were cut on an ultramicrotome, stained with urany] acetate and lead citrate, and observed with a JEM-leOC transinission ctectron microscope at 80 kV. For morphornetry, 3 or more animals were used for each stage. Ten or rnorc cross sections of mammary capillaries were obtained from different anima}s and tissuc blocks, TEM micrographs of capillarics were taken at a rnagnification of × 10,OOO, The photographs were eniargcd to yield working prints of rnagnification x 25.000, The area of endotheliurn cytoplasm wlthout the nucleus region, the length of margjnal folds and thc length of mjcrovillous processes were measured by an image analyzer (Nikon (]osmozone Is). Pinocytotic vesicles (PVs) were counted on enlarged photographs and tlie densjty of PVs was calculated as the number of PVs per pm2 of endothelial eytoplasm. The data were statistically analyzcd by Studentis t-test.