VETERINARSKI ARHIV 69 (2),
105-113, 1999

ISSN 1331-8055 Published in
Croatia

Ultrastructural and morphometrical
studies on the endothelial cells of the pectoral mammary gland capillaries in
Wistar rats during the reproductive cycle

Mohammad Abdul Awal1*,
Mitsuharu Matsumoto2,
and Hayao Nishinakagawa2

1Department of
Anatomy and Histology, Faculty of Veterinary Science,
Bangladesh Agricultural
University, Mymensingh, Bangladesh

2Laboratory of
Veterinary Anatomy, Faculty of Agriculture, Kagoshima University,
Korimoto,
Kagoshima, Japan

* Contact address:
Prof. Dr.
Mohammad Abdul Awal,
Department of Anatomy and Histology, Bangladesh
Agricultural University, Mymensingh-2202, Bangladesh,
Phone: 88 091 556
957/Ext.-2589; Fax: 88 091 55 810

AWAL, M. A., M. MATSUMOTO, H.
NISHINAKAGAWA: Ultrastructural and morphometrical studies on the endothelial
cells of the pectoral mammary gland capillaries in Wistar rats during the
reproductive cycle. Vet. arhiv 69, 105-113, 1999.

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

AWAL, M. A., M. MATSUMOTO, H.
NISHINAKAGAWA (1995): Morphometrical changes of the arterial walls of main
arteries from heart to abdomino-inguinal mammary glands of rat from virgin
through pregnancy, lactation and post-weaning. J. Vet. Med. Sci. 57, 251-256.

AWAL, M. A., M. MATSUMOTO, Y.
TOYOSHIMA, H. NISHINAKAGAWA (1996): Ultrastructural and morphological studies on
the endothelial cells of arteries supplying the abdomino-inguinal mammary gland
of rats during the reproductive cycle. J. Vet. Med. 58, 29-34.

BRUNS, R. R., G. E. PALADE (1968):
Studies on blood arteries. 11. Transport of ferritin molecules across the wall
of muscle capillaries. J. Cell Biol. 37, 277-299.

CLOUGH, G., C. C. MICHEL (1981): The
role of vesicles in the transport of ferritin through frog endothelium. J.
Physiol. 315, 127-142.

JENNINGS, M. S., L. FLOREY (1967): An
investigation of some properties of endothelium related to capillary
permeability. Proc. R. Soc. Lond. 167, 39-63.

MATSUMOTO, M., H. NISHINAKAGAWA, M.
KUROHMARU, Y. HAYASHI, J. OTSUKA (1992): Pregnancy and lactation affect the
microvasculature of the mammary gland in mice. J. Vet. Med. Sci. 54, 937-943.

MURAD, T. M. (1970): Ultrastructural
study of rat mammary gland during pregnancy. Anat. Rec. 167, 17-36.

NOGUCHI, Y., Y. SHIBATA, T. YAMMATO
(1987): Endothelial vesicular system in rapid-frozen muscle capillaries revealed
by serial sectioning and deep etching. Anat. Rec. 217, 355-360.

OGAWA, K., T. WATABE, K. TANIGUCHI
(1993): Transport pathways for macromolecules in the aortic endothelium. 1.
Transendothelial channels revealed by three-dimensional reconstruction using
serial sections. Anat. Rec. 236, 653-663.

PALADE, G. E. (1953): Fine structure of
blood capillaries. J. Appl. Physics. 24, 1424 (abstr).

WAGNER, D. D., B. ROBERTA (1991): Von
Willebrand factor and the endothelium. Mayo Clin. Proc. 66, 621-627.

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.

SAZETAK

Uporabom elektronskog mikroskopa i
analizatora slike istrazivane su ultrastruktura i morfometrija endotelnih
stanica kapilara grudnih mlijecnih zlijezda zenskih Wistar stakora tijekom
reprodukcijskog ciklusa. Gustoca pinocitotskih mjehurica (PV) u kapilarama (broj
PV po µm2 endotelne citoplazme) znacajno je porasla tijekom
gravidnosti i dosizala najvece vrijednosti u laktaciji, te postupno opadala u
razdoblju nakon odbica. Broj PV povecao se dva puta u gravidnosti, a cetiri puta
u laktaciji. Gustoca mitohondrija i duljina rubnih nabora i broj mikrovila
takoder su znacajno (P>0.05) porasli tijekom graviditeta i laktacije. U
laktaciji su duljina rubnih nabora i mikrovila bili dva puta veci.
Pretpostavljamo da su ultrastrukturne promjene endotelnih stanica kapilara
grudnih mlijecnih zlijezda usko povezane sa reprodukcijskim ciklusom, a sigurno
su povezane s funkcionalnim potrebama mlijecnih zlijezda.

Kljucne rijeci: ultrastruktura,
morfometrija, endotelne stanice, kapilare mlijecnih zlijezda, Wistar stakori

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