MICROSCOPIC ANALYSIS OF THE UTERINE ENDOMETRIUM IN POSTPARTURIENT EWES

Krajniãáková M., E. Bekeová, a. Lenhardt, V. Cigánková, I. Valock ̆, I. Maraãek: Microscopic Analysis of the Uterine Endometrium in Postparturient Ewes. Acta Vet. Brno 1999,

In our conditions, uterine involution in Slovak Merino sheep lambed in February was found to be finished by day 34 post partum Krajniãáková et al. (1994).The aim of this study was to obtain data on the course of histological changes in the endometrial structures and entire covering of caruncles with epithelium after autumn lambing of sheep of the same breed kept in our conditions.

Materials and Methods
Twelve Slovak Merino ewes were included in the experiment.The animals, weighing about 40-50 kg, and 3-5year-old, were kept on a commercial farm.We observed a physiological course of delivery in all animals including departing of placenta.Puerperium in all the ewes proceeded without any complications.The animals (n=12) lambed in September.They were on pasture and were housed 3 weeks before parturition.The ewes were fed meadow hay, fodder beet, maize silage, feed mixture BAK and concentrates.Feed mixture BAK is composed of extracted flax meal, wheat flour, wheat bran, malt flour, wheat meal, barley, salt, mineral additive III, and biofactor additive.Water and salt were given ad libitum.The ewes were killed (n=3) on days 7, 17, 25 and 34 post partum.Immediately after lambing, the reproductive apparatus was excised and samples were taken from the caruncular region.Samples were fixed in 10% formalin.The samples were dehydrated and embedded in paraffin, and 7-10 µm thin sections were stained with hematoxylin-eosin.The sections were observed using projection microscope.One mm 3 thin sections for analysis in the electron microscope were fixed for 3 h by immersion in 3% glutaraldehyde and subsequently for 1 h in 2% osmium oxide in phosphate solution (pH 7.4); then they were gradually dehydrated in increasing series of alcohol, rinsed in propylene oxide and embedded into Durcupan ACM (Fluka).Semi-thin sections were stained with toluidine blue and examined in the electron microscope JEM 1200 Ex.The electron micrographs were made by the projection microscope Olympus CH2 with a camera.

Results
Endometrial histological structure in the caruncular region of sheep lambed in September is presented in Figures 1 to 8 (Plates II-V).Fig. 1 shows the uterine sample of a sheep on day 7 post partum; the epithelium above the caruncle was entirely destroyed.Caruncles had large spaces without glands, sharply demarcated from the glandular part of the endometrium.Branches of the uterine artery were slightly dilated and filled with erythrocytes.In the glandular part of the endometrium, the volume of some glands was enlarged and the lumen of secretory parts was dilated.In the endometrial tunica propria, which was rich in cells, fibrocytes, migratory cells and reticular fibres prevailed.In the electron micrograph (Fig. 2), there were microvilli (mv) on the apical surface of cells.Damaged mitochondria (m) and dilated cisterns of the endoplasmic reticulum (er) were in the cytoplasm.
On day 17 (Plate III, Fig. 3) post partum, the epithelium of the endometrium occurred, gradually covering a relatively sizeable caruncle.A multi-layered cylindrical epithelium in some parts changed into an one-layered flat one.The uterine glands were subject to gradual regression and only a small number of them had still apparently dilated lumen of secretory parts.Fig. 4 shows degenerated glandular epithelium, cells were marked with nuclear pyknosis, disintegration of cells and their release into the glandular lumen.Clean-cut subepithelial cumulation of fatty cells (mc), macrophages and sporadic lymphocytes occurred in the endometrium (Plate IV, Fig. 5).
On day 25 (Fig. 6) after lambing, the endometrium in the caruncular region was covered with epithelium.The caruncle was markedly reduced compared to day 17, and from the glandular part of endometrium it was sharply demarcated by a band of reticular fibres.Numerous uterine glands, whose lumen was markedly reduced, occurred in the lamina propria.
On day 34 post partum (Plate V, Fig. 7) the endometrial mucosa was intact.The endometrium was entirely covered with epithelium.Spaces without glands formed a narrow band that was localised close under the basal membrane of the mucosa.The electron micrograph (Fig. 8) shows a part of epithelial surface of the uterine endometrium, where numerous microvilli (mv) occurred on the apical surface.The cellular ultrastructure was undamaged.Numerous secretory granules (sg) occurred in the cytoplasm.

Discussion
Evaluation of the morphological structure of the endometrium on day 7 post partum revealed that the epithelium in the caruncular region was entirely degenerated.Branches of the uterine artery were dilated and filled with erythrocytes.Our results are similar to those reported by V a n W y k (1972) between days 4 and 12 of the postparturient period in sheep.Similar findings were reported by G r e y l i n g and V a n N i e k e r k (1991) in goat post partum.At the observed time interval, damaged mitochondria in the cytoplasm of epithelial cells and dilated cisterns of the endoplasmic reticulum were observed.U h r í n (1992) recorded changes in mitochondria, even their death, by the end of the sexual cycle, when there was also dilation of cisterns of the endoplasmic reticulum.Our findings of the structural changes in these organelles may be connected with gradually progressive proliferation in the postparturient period.
The histological analysis of the results on day 17 post partum indicates progressive process of involution of the uterus.The epithelium of the endometrium gradually covered still large caruncle.The glandular epithelium was considerably degenerated and released cells were present in the lumen.In the endometrium, there were more marked numbers of macrophages and lymphocytes.Regarding the physiological course of involutional and reparatory processes of the uterus, their functionality appears to be inevitable.Results of our observations on the day evaluated are in agreement with the data obtained on day 20 post partum in goat (Greyling and Van Niekerk 1991) and findings of Kudláã (1985) post partum in cows.
Our findings on day 25 post partum indicate more marked proliferation of the superficial epithelium of the endometrium in the caruncular region.In the lamina propria, there are uterine glands with markedly reduced lumen.These findings do not differ from those of the postparturient period of sheep lambed in February ( K r a j n i ã á k o v á et al. 1994).
On day 34, intact endometrial mucosa was detected in our experimental group.The endometrium was covered with epithelium in the whole extent.The involution process of the uterus in sheep according to V a n W y k et al. ( 1972) is finished until day 28 post partum.C r o w d e r et al. (1982) found complete uterine involution by day 32 after lambing.Evaluation of the morphological structure of the epithelium of the endometrium of uterus in the caruncular region in our experiment confirms its reparation up to day 34 post partum.This fact is also confirmed by the undamaged structure of cells and numerous microvilli on the cells of luminal epithelium of the endometrium.The histological picture of similar character is reported by • È a s t n (1993) in cycling heifers.G r a i g and J o l l i e (1984) found that luminal cells of the endometrium in rats react to administration of estrogens by formation of microvilli.Results of K o c h et al. (1985) obtained after administration of ovarian steroids to rats are in agreement with these authors.The fact that formation of microvilli is stimulated by estrogens is confirmed also by our findings of concentrations of ovarian hormones in the animals under study ( K r a j n i ã á k o v á et al. 1996).The presence of numerous secretory granules in the cytoplasm of cells that were found on day 34 post partum, is manifestation of their higher metabolic and secretory activity as reported by • È a s t n (1993) in cycling animals.Our results indicate that in Slovak Merino sheep lambing out of season (September), the involutional process is completed by day 34 after parturition similar to the results found in sheep that lambed in February.Completed covering of the whole endometrial surface, including the protruded margins of caruncles with the epithelium was recorded on day 34 after lambing of our experimental ewes.

Fig. 1 .
Fig. 1.Caruncular region of the endometrium on day 7 post partum.The epithelium above the caruncle is entirely degenerated.The volume of some glands is enlarged.HE.Bar = 40 µm

Fig. 6 .
Fig. 6.Micrograph presents the endometrium in the caruncular region (day 25 post partum).Caruncle (c) is reduced and it is demarcated from the glandular part of the endometrium by a band of reticular fibres (r.f.)In the lamina propria (l.p.) uterine glands (g) are visible.HE.Bar = 40 µm

Fig. 8 .
Fig. 8. Electron micrograph shows the undamaged cellular structure of the endometrial epithelium and in the cytoplasm there are secretory granules (sg).There are microvilli (m.v.) on the cell surface.Toluidine blue.Bar = 0.5 µm