ABSTRACT

The ultrastructure and morphometry of the endothelial cells of pectoral mammary gland capillaries of female Wistar rats were studied through the reproductive cycle with an electron microscope and image analyser. Subendothelial spaces were absent in the mammary gland capillaries. The density of pinocytotic vesicles (PV) in the capillaries (number of PV per µm² of endothelial cytoplasm) was significantly increased during pregnancy, reaching maximum values during lactation, and which subsequently decreased during the post-wening stage. The capillaries showed the maximum changes of PV during pregnancy and lactation, with two- and four-fold increases during the respective periods. The density of mitochondria and the length of marginal folds and microvillus processes were also increased significantly (P>0.05) during pregnancy and lactation. The length of marginal folds and microvillus processes were increased two-fold during the lactating period. It is assumed that the ultrastructural changes of endothelial cells of mammary gland capillaries are closely related during the reproductive cycle, and certainly with the functional demand of the mammary glands.

Key words: ultrastructure, morphometry, endothelial cells, mammary gland capillaries, Wistar rats

Introduction

The fine structure of mammalian arteries and capillaries has been extensively investigated, with the basic structures of various types of vessel having been well established (PALADE, 1953; MURAD, 1970; NOGUCHI et al., 1987; MATSUMOTO et al., 1992; AWAL et al., 1996).

It is now well established that mammary glands show marked development during pregnancy and lactation. The morphological relationship between the mammary parenchyma and the mammary gland capillaries has been well studied in mouse mammary glands (WAGNER and ROBERTA, 1991; MATSUMOTO et al., 1992). We previously described the role of endothelial cells in the arteries and capillaries of the abdomino-inguinal mammary glands of Wistar rats during the reproductive stage (AWAL et al., 1996). A review of available literature revealed no information regarding ultrastructural studies in the capillaries of pectoral mammary glands during the reproductive cycle.

The present study was therefore conducted to clarify the ultrastructural changes of the endothelial cells of pectoral mammary gland capillaries, as mammary parenchyma shows a cyclic proliferation during the reproductive cycle.

Materials and methods

A total of 12 female Wistar rats at the stages of virgin (90-day-old), pregnancy (15 days of pregnancy), lactation (10 days of lactation) and post-weaning (10 days after weaning) was used during the investigation. They were bred and maintained as a closed colony in a laboratory. Three rats at each of the above stages were studied. The day following an overnight mating was counted as day 1 of gestation. The rats were fed a commercial diet (Oriental East Co., Ltd.) and water ad libitum. During lactation, each mother rat was housed together with her (8-12) pups.

The animals were anaesthetised with Nembutalâ 50 µm/g body mass by intraperitoneal injection and were perfused from the left ventricle with a physiological saline solution, followed by a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer. The mammary tissues from each pectoral mammary gland were carefully extracted in pieces. The tissues were then fixed in the same buffer and post-fixed in 1% osmium tetroxide in 0.1 M phosphate buffer for 2 h at 4 ^oC. After rinsing in the same buffer the specimens were dehydrated in a graded ethanol series and embedded in Epon 812. Thin sections were cut on an ultramicrotome (Dimond knives), coated with copper grids, stained with uranyl acetate and lead citrate and observed with an H-7000 KU transmission electron microscope (TEM) at 75 kV.

For morphometry, more than 30 cross-sections of the endothelial of mammary gland capillaries were obtained from different rats and tissue blocks. Electron micrographs of the endothelium were obtained at a magnification of ×25,000. The area of the endothelial cytoplasm, excluding the nucleus, and the length of the marginal folds (Mf) and microvillus processes were measured by an image analyser (Nikon Cosmozone Is). Pinocytotic vesicles (PV) and mitochondria (Mt) were counted on enlarged photographs. The density of PV and of mitochondria was calculated as the number per µm² of endothelial cytoplasm. The data were statistically analysed by Student's t-test.

Results and discussion

The endothelial cells lining the mammary gland capillaries in the pectoral region formed a continuous sheet, with no intracellular gaps. Subendothelial spaces were absent (Fig. 1a-d). We reported in our previous study (AWAL et al., 1996) that the cytoplasm of the endothelial cells contained well-defined mitochondria (Mt), strands of endoplasmic reticulum, ribosomes, Golgi complex and numerous pinocytotic vesicles (PV). Although the distribution of pinocytotic vesicles was scattered throughout the cytoplasm, the mitochondria (Mt) were frequently concentrated near or around the nucleus of the cells. The marginal folds and microvillus processes (Mf) were projected from the luminal surfaces of cells (Fig. 1b-d). Similar morphological characters were also observed in mice and Wistar rats (MATSUMOTO et al., 1992; AWAL et al., 1996).

Fig. 1a) Virgin stage. Least number of pinocytotic vesicles (PV) observed. ×25,000
Fig. 1b) Pregnant stage. Showing long marginal fold, mitochondria (Mt) and increased number of pinocytotic vesicles compared to that of virgin stage. ×25,000

The least number of PV and mitochondria was observed in the virgin stage (Fig. 1a), with the marginal folds and microvillus processes in the virgin stage being short. The number of PV and mitochondria, as well as the length of marginal folds and microvillus processes, increased during pregnancy (Fig. 1b), and reached maximum values during the lactating period (Fig. 1c), subsequently decreasing following post-weaning (Fig. 1d). AWAL et al. (1996), reported that in the capillary endothelium of abdomino-inguinal mammary glands in Wistar rats the number of PV, mitochondrial and the length of marginal folds and microvillus processes were gradually increased during pregnancy, reaching peak values during lactation, which then decreased following weaning. The present findings are in full accord with their descriptions (AWAL et al., 1996).

Fig. 1c) Lactating stage. Numerous pinocytotic vesicles and long marginal fold (Mf) observed. ×25,000
Fig. 1d) Post-weaning stage. The number of pinocytotic vesicles (PV) and length of marginal folds decline. ×25,000

During morphometrical studies, the density of PV and mitochondria, the length of marginal folds and microvillus processes were significantly (P>0.05) increased during pregnancy, reaching maximum values during the lactating period, subsequently decreasing following post-weaning (Fig. 2a-c). The present findings accord well with the descriptions of MATSUMOTO et al. (1992) and AWAL et al. (1996).

Fig. 2a-c. Graphs depicting morphometrical changes in mammary gland capillaries from the virgin stage, through pregnancy, lactation and post-weaning. Each column shows the mean value, and the vertical bar SEM. P<0.05 is considered statistically significant.
Fig. 2a. Changes in the density of pinocytotic vesicles (PV). Density in the mammary gland capillaries is significantly higher in lactation than at any other stage, significantly higher in the pregnant period than at the post-weaning and virgin stages, and significantly higher in the post-weaning period than at the virgin stage.
Fig. 2b. Density of mitochondria (Mt). Density in mammary gland capillaries at the virgin stage is significantly lower than at any of the other three stages.
Fig. 2c. Changes in length of marginal folds (Mf) and microvillus processes. Length in the mammary gland capillaries is significantly greater in lactation than at the virgin stage.

Although many hypotheses have been presented in the literature regarding the functions of PV at the cellular level (JENNINGS and FLOWEREY, 1967; BRUNS and PALADE, 1968; OGAWA et al., 1993), it is commonly agreed that these vesicles are involved in the active transport of fluid and nutrients across the cell walls. Recent physiological and morphological studies have revealed that the endothelium constitutes a dynamic system which influences the physiology of both the blood vessel wall and the surrounding tissues (CLOUGH and MICHEL, 1981). The number of PV and the length of marginal folds and microvillus processes increased during the period of late pregnancy and the middle of lactation in the endothelial cells of mouse mammary gland capillaries, and suggests possibilities of the participation of these structures with the uptake of fluid and particles, or as a barrier acting to attenuate blood velocity (MATSUMOTO et al., 1992). The increased number of PV and the length of marginal folds and microvillus processes in pregnant and lactating periods observed in the present study is direct evidence supportive of this hypothesis.

In our previous study (AWAL et al., 1995), we noted that both the diameter and thickness of the arterial walls supplying the abdomino-inguinal mammary glands increased significantly during pregnancy, reached their maximum values in lactation and declined at the post-weaning stage. It seems clear that the artery supplying the mammary glands and the capillaries plays an increased functional role in responding to augmented circulatory and physiological demand during that period. The vessels supplying the mammary glands and the capillary are undoubtedly more active during pregnancy and lactation than during the virgin and post-weaning stages. The requirement of the vessel wall for nutrient and energy to work smoothly increases during the former periods. It is assumed that the nutrients are absorbed from the lumen of the vessel by the PV and are subsequently utilised by the vessel wall. The energy utilised by the cells is generated by mitochondria.

Conclusion

It has been suggested that the increased density of PV and mitochondria, and the increased length of marginal folds and microvillus processes during pregnancy and lactation, are exclusively dependent upon the functional circulatory demand of the pectoral mammary gland capillaries. The decrease in the density and length of these structures observed at the post-weaning stage is assumed to be due to decreased blood flow and the comparatively reduced functional role of the vessels, as apparently a lesser amount of blood is utilised by the involuted mammary glands.

Acknowledgment
The authors wish to thank Prof. Dr. T. Nishio, Laboratory of Veterinary Pharmacology, Kagoshima University, for providing experimental animals (Wistar rat). This study was supported by the Ministry of Education, Science, Sports and Culture (Monbusho), Japan.

References

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Received: 12 January 1999
Accepted: 30 March 1999

AWAL, M. A., M. MATSUMOTO, H. NISHINAKAGAWA: Ultrastrukturno i morfolosko istrazivanje endotelnih stanica kapilara grudnih mlijecnih zlijezda Wistar stakora tijekom reprodukcijskog ciklusa. Vet. arhiv 69, 105-113, 1999.