Resident Macrophages and Lymphocytes in the Canine Endometrium

Resident immune cells play a major role in endometrial immunity and in tissue homoeostasis. This study aimed to analyse the distribution of macrophages, B and T lymphocytes (respectively, Mø, B‐Lym and T‐Lym) in the canine endometrium throughout the oestrous cycle and in late involution (at the proestrus stage post‐parturition). An immunohistochemistry technique was used on samples from 50 post‐pubertal healthy female dogs, of which five in late post‐partum. The distribution of resident immune cells was analysed in three endometrial layers (superficial, intermediate and basal areas). Mø, B‐Lym and T‐Lym were demonstrated to reside in the endometrium in all the stages of the canine cycle; their numbers being considerably higher during late involution. T‐Lym were scattered in the stroma or amidst the glandular epithelium, constituting the predominant immune cell population in anestrus and proestrus, but decreased in number at all other stages. Endometrial B‐Lym remained fairly constant during the canine cycle, although its numbers were higher in late involution. Mø counts were higher during anestrus compared to the other stages, the cells being displaced into the superficial endometrial layer. Mø demonstrated the highest level in late involution samples, forming small aggregates below the surface epithelium. The number of immune cells was not normally distributed, suggesting the influence of individual factors, such as age or parity, not explored herein due to limited sample availability. Still, this study provides important information for the interpretation of endometrial biopsies in dogs and for the understanding of the increased susceptibility to uterine infection during dioestrus found in the bitch.     

Immunohistochemical Localization of the Progesterone and Oestrogen α Receptors in the Uterine Horns of the African Giant Rat (Cricetomys gambianus)

The present study investigated the immunolocalization of the progesterone and oestrogen α receptors in the uterine horns of the African giant rat during the oestrous cycle. The progesterone and oestrogen α receptors were demonstrated in various cellular constituents of the endometrium, myometrium and perimetrium. The intensity of progesterone and oestrogen α receptor immunostaining in the endometrial and myometrial layers of the uterine horns varied during the oestrous cycle. The intensity of oestrogen α receptor immunoreactivity in the luminal epithelium was high during pro‐oestrus, oestrus and dioestrus. Progesterone and oestrogen α receptor immunoreactivity in the endometrial epithelia was absent during metoestrus. Moderate to strong immunostaining for the progesterone and oestrogen α receptors was demonstrated in the myometrial smooth muscle cells during pro‐oestrus, oestrus and dioestrus. The intensity of progesterone and oestrogen α receptor immunostaining in the myometrial smooth muscle cells was low during metoestrus. Stromal cells in the perimetrium consistently expressed progesterone and oestrogen α receptor immunoreactivity throughout the oestrous cycle. The findings of the study indicate that in the giant rat the immunolocalization of the progesterone and oestrogen α receptors, in endometrial and myometrial regions of the uterine horns, varies during the oestrous cycle.     

Endometrial Phospholipase A2 Activity During the Oestrous Cycle and Early Pregnancy in Mares

The aim of this study was to determine phospholipase A2 (PLA2) kinetics and activity in the mare’s endometrium during the oestrous cycle and early pregnancy. Phospholipase A2 is responsible for the liberation of arachidonic acid from phospholipids, which is the first limiting step in prostaglandins synthesis. Phospholipase A2 activity was measured using an assay based on the liberation of oleic acid from 1‐palmitoyl‐2‐[¹⁴C] oleoyl phosphatidylcholine. The enzyme was shown to be calcium dependent, to have an optimum pH of 8 and an apparent Michaelis constant of 127 μm . Enzyme activity was low in the endometrium of early luteal phase tissue but increased significantly (p < 0.001) during the late luteal phase (5.39 ± 0.16; 3.48 ± 0.33, 6.85 ± 0.59, and 9.96 thinsp;± thinsp;1.23 thinsp;nmol oleic acid released/mg protein at oestrus, and Days 3, 8 and 14 after ovulation, respectively). The mean PLA2 activity in endometrial tissue from pregnant mares (4.23 ± 0.74) was significantly lower (p < 0.01) than from cyclic animals during late dioestrus (9.96 ± 1.23). The results indicate that PLA2 activity in equine endometrium changes with the stage of the oestrous cycle and thus may be influenced by systemic hormone concentrations. The inhibitory effects of conceptus products on secretion of prostaglandin during early pregnancy were associated with a competitive inhibitor that decreased endometrial PLA2 activity.     

Influence of breed and oestrous cycle on endometrial gland surface density in the mare

REASON FOR PERFORMING STUDY: The diffuse noninvasive epitheliochorial equine placenta develops an intimate and complex interdigitation with the maternal endometrium throughout gestation to maximise surface contact and, consequently, optimise nutritional and gaseous maternofetal exchanges. A significant reduction occurs in the surface density of microcotyledons on the placentae of Welsh Pony vs. Thoroughbred mares that may relate to a difference in either the number or density of endometrial glands between these breeds. OBJECTIVES: To examine this hypothesis and to determine the influence of the oestrous cycle upon the development and surface density of endometrial glands. METHODS: Endometrial biopsies were taken under videoendoscopic visual control from the base of a uterine horn from young, fertile, Welsh Pony and Thoroughbred mares at defined stages of the oestrous cycle. Computer-assisted morphometric analysis then permitted the surface density of endometrial glands within the stratum spongiosum to be assessed. CONCLUSIONS: There was a statistically significant reduction in endometrial gland surface density in the Welsh Pony vs. Thoroughbred mares during both oestrus and dioestrus. POTENTIAL RELEVANCE: A substantial upregulation of epidermal growth factor (EGF) mRNA in the epithelial cells lining the apical portions of endometrial glands has been demonstrated in pregnant mares between Days 35 and 40 after ovulation, coincident with the onset of interdigitation between the allantochorion and endometrium to form the microcotyledonary placenta. The increased surface density of endometrial glands noted in the uteri of Thoroughbred mares might account for the greater surface density of placental microcotyledons in this breed.     

Endometrial cytology of the normal bitch throughout the reproductive cycle

Samples for endometrial cytology were collected both from live bitches using transcervical uterine cannulation (n=48) and postmortem (n=10). The cells identified were endometrial epithelial cells, leucocytes, erythrocytes, spermatozoa, bacteria and cervical or vaginal cells. The endometrial epithelial cells varied morphologically throughout the reproductive cycle and had signs of degeneration during late dioestrus and during early and mid-anoestrus following dioestrus and postpartum. Neutrophils were the most common leucocytes observed during pro-oestrus, oestrus, dioestrus and early pregnancy, and lymphocytes during anoestrus. Macrophages were frequently seen during anoestrus. Erythrocytes were found in variable numbers at all stages of the reproductive cycle. Spermatozoa were detected in samples collected during oestrus and early pregnancy in bitches which had their last mating one to three days previously. Bacteria were commonly observed during pro-oestrus and oestrus. Cornified cervical or vaginal cells were present during pro-oestrus and oestrus. This study demonstrated that the numbers, types, proportions and morphology of ceils in endometrial cytological samples from normal bitches varied throughout the reproductive cycle.     

Immunohistochemical Studies on Oestrogen Receptor Alpha (ERα) and the Proliferative Marker Ki-67 in the Sow Uterus at Different Stages of the Oestrous Cycle

In order to better understand physiological changes during the different stages of the oestrous cycle, immunohistochemistry was used in the present study to investigate the distribution of oestrogen receptor alpha (ERα) as well as the proliferative marker Ki‐67, in the sow uterus during the oestrous cycle. Uterine samples were collected from multiparous sows with normal reproductive performance at selected stages of the oestrous cycle: at late dioestrus (d 17), prooestrus (d 19), oestrous (d 1), early dioestrus (d 4) and dioestrus (d 11–12), respectively. The tissue samples were fixed in 10% formaldehyde, embedded in paraffin and subjected to immunohistochemistry using monoclonal antibodies against ERα (C‐311) and Ki‐67 (MM‐1). In general, the immunostaining of both ERα and Ki‐67 was confined to nuclei of the target cells. Variations were seen, not only at the different stages of the oestrous cycle, but also in the different tissue compartments of the uterus. In the epithelia, the strongest ERα staining and highest amount of positive Ki‐67 cells were found at early dioestrus. In the myometrium, the highest levels of staining of both ERα and Ki‐67 positive cells were found at pro‐oestrus and oestrus. For the proliferative marker, Ki‐67, no positive cells were found at dioestrus and late dioestrus in the epithelium and myometrium. In the connective tissue stroma (subepithelial layer), the highest number of ERα positive cells were found at oestrus, which was significantly different compared with other stages (p≤0.05), whereas the levels of Ki‐67 positive cells were relatively low and did not differ between the stages examined. Significant correlations between the number of ERα positive cells in the stroma and Ki‐67 positive cells in the epithelia were observed. This suggests indirect regulatory mechanisms on epithelial proliferation via ERα in the stroma. In conclusion, these findings in the sow uterus show that the presence of ERα as well as Ki‐67 protein varies not only between different stages of the oestrous cycle but also between different tissue compartments of the uterus. These findings indicate various regulatory mechanisms and stress the importance of localising ERα and proliferating cells in different uterine tissues.     

Expression and Hormonal Regulation of IL-11Rα in Canine Uterus During Early Pregnancy

Embryo implantation is critical for the successful establishment of pregnancy. Interleukin-11 (IL-11) is essential for adequate decidualization in the mouse and human via binding to the specific IL-11 receptor α (IL-11Rα). But the expression and regulation of IL-11 and IL-11Rα in the canine endometrium remain unknown. The aim of this study was to investigate the differential expression of IL-11Rα in canine uterus during early pregnancy and its regulation under different conditions by in situ hybridization. Interleukin-11Rα mRNA was mainly localized in glandular epithelium in canine uterus. There was a low level of IL-11Rα expression in the glandular epithelium on days 6, 12 and 17 of pregnancy. On day 20 of pregnancy when embryo implanted, IL-11Rα mRNA was highly expressed in the glandular epithelium surrounding the embryo, but not in the luminal epithelium and stroma. On day 23 of pregnancy, the expression of IL-11Rα mRNA maintained a constant level compared with the expression of day 20 and increased on day 28 of pregnancy. During the oestrous cycle, a high level of IL-11Rα mRNA expression was seen in the oestrous uterus. Progesterone slightly induced the expression of IL-11Rα mRNA in the ovariectomized canine uterus. These results suggest that IL-11Rα expression is closely related to canine implantation and up-regulated by progesterone.     

Expression and Hormonal Regulation of E‐Cadherin in Canine Uterus During Early Pregnancy

E‐cadherin, a Ca^{2 +} ‐dependent cell adhesion molecule, is necessary for endometrial receptivity to blastocyst implantation. The aim of this study was to investigate the differential expression of E‐cadherin in canine uterus during early pregnancy and its regulation under different conditions by in situ hybridization. E‐cadherin mRNA expression was at a low level in the glandular epithelium on days 6, 12 and 17 of pregnancy. On days 20 and 23 of pregnancy, E‐cadherin mRNA was highly expressed in the glandular epithelium surrounding the embryo, but not in the luminal epithelium and declined in villi and placenta on day 28 of pregnancy. During oestrous cycle, a moderate level of E‐cadherin mRNA expression was found in the luminal and glandular epithelium of canine uteri at oestrus stage. The same expression was also found at anoestrus stage. Progesterone slightly induced the expression of E‐cadherin mRNA in the luminal and glandular epithelium of ovariectomized canine uterus. These results suggest that E‐cadherin expression is closely related to canine implantation and can be up‐regulated by progesterone.     

Tissue concentrations of eosinophilia in the bovine oviduct and uterus of different stages of the oestrous cycle

Numbers of eosinophils in the bovine oviduct and uterus were determined during the oestrous cycle. The eosinophil numbers in the oviduct (ampulla and isthmus) and horn of the uterus during oestrus were significantly higher than during dioestrus. The number of eosinophils in the uterine cervix was lower than in the uterine horn for all stages of the oestrous cycle. In the oviduct, eosinophils accumulated in the lamina propria of the tunica mucosa, in the tunica muscularis and in the connective tissue of the tunica serosa. In the uterus, they were concentrated mainly in the upper parts of the stroma in the endometrium. Degranulation of eosinophils was observed during oestrus when they increased in number in the oviduct and uterus.     

Results of histochemical and biochemical studies of uterus of gilts during the estrous cycle]

Alkaline phosphatase activity as well as lipid, polysaccharide, glycogen, and acid mucopolysaccharide levels in the uteri of 79 gilts were histochemically tested during the first three sexual cycles. Intra-cyclic alkaline phosphatase activity exhibited clearly pronounced variations in the surface epithelium, with maximum values reached in metoestrus, and moderate variations in the endometrial stroma, with maximum values reached in dioestrus. Cycle-dependent variations were recordable also from the glycogen and lipid levels in the surface and glandular epithelia and from the acid mucopolysaccharide levels in the endometrial stroma. The activity of alkaline phosphatase as well as the levels of glycogen and acid mucopolysaccharides during the first cycle still were lower than those in the second and third. Biochemical examinations of the endometrium revealed significant cycle-dependent variations in the activities of alkaline phosphatase, inorganic pyrophosphatase, and succinate-dehydrogenase as well as in the concentrations of soluble protein and glycogen, with maximum values being recordable on the tenth day of cycle. No significant intracyclic variations were recordable from the activities of acid phosphatase as well as of aspartate alanine-amino transferase.     

Detection of the hyaluronan receptor CD44 in the bovine oviductal epithelium

Hyaluronan is involved in fundamental reproductive events such as sperm storage in the female reproductive tract, fertilization, and early embryo development, these functions are presumably mediated by its major cell surface receptor, CD44. The present study was conducted to investigate the presence and localization of CD44 in the bovine oviductal epithelium, using immunohistochemical and Western blot methods on tissue sections and epithelial cell extracts collected from the uterotubal junction (UTJ), isthmus, and ampulla of animals in the oestrus or luteal phase of the oestrous cycle. While positive immunolabelling for CD44 was found on the ad-luminal surface and supra-nuclear region of epithelial cells in all tubal segments investigated, in the UTJ, there were epithelial cells in which the entire cytoplasm positively stained. We found no differences in terms of CD44-positive staining between the different stages of the oestrous cycle. Presence of CD44 was detected by Western blotting in the tubal epithelium as a single band at 200 kDa. Although it appeared in all tubal segments, the expression of CD44 protein was more accentuated in the sperm reservoir (UTJ) than in the other segments. This is the first time CD44 has been detected in the epithelium of the tubal sperm reservoir in cattle, suggesting a pathway for the action of hyaluronan in this segment.     

Expression of oestrogen receptor,androgen receptor and progesterone nuclear receptor in sheep uterus during the oestrous cycle

Oestrogen, androgen and progesterone are involved in the regulation of uterine physiological functions, with the participation of the following proteins: oestrogen receptor (ER), androgen receptor (AR) and progesterone nuclear receptor (PGR). In this study, we used immunohistochemistry to detect the localization of ERα, ERβ, AR and PGR in sheep uterus. Additionally, we used real‐time polymerase chain reaction (RT‐qPCR) and Western blot technique to analyse their expression profiles at different stages of sheep oestrous cycle in the endometrium and myometrium. Immunohistochemical analysis showed that ERα, ERβ, AR and PGR were present in sheep uterus in oestrus, mainly in the uterine luminal epithelium, stroma, gland and myometrium. Real‐time polymerase chain reaction results showed that in the endometrium, ERα expression level was highest in oestrus. ERβ and PGR, instead, were highly expressed in pro‐oestrus. In the myometrium, ERα was highly expressed in both oestrus and pro‐oestrus, and ERβ was highly expressed in oestrus and dioestrus. Progesterone nuclear receptor expression was highest in oestrus, followed by metoestrus. In the endometrium, both receptors ERα and ERβ were abundant in pro‐oestrus, while the maximum AR protein content was found in oestrus. At this stage of the oestrous cycle, PGR protein concentration in the myometrium was significantly lower than those observed in other stages. These results suggest that these receptors are important for sheep reproductive function, as their expression at mRNA and protein levels exhibits particular time‐ and tissue‐specific profiles along the oestrous cycle.     

The Sow Endosalpinx at Different Stages of the Oestrous Cycle and at Anoestrus: Studies on Morphological Changes and Infiltration by Cells of the Immune System

The aim of this study was to investigate the morphological changes of the sow endosalpinx and the distribution of leukocytes throughout the oestrous cycle and at anoestrus. Nineteen crossbred sows (Swedish Landrace x Swedish Yorkshire) at late dioestrus (three), prooestrus (three), oestrus (three), early dioestrus (three), dioestrus (three) and anoestrus (four) were used. Oviductal samples from three different parts (isthmus, ampulla and infundibulum), taken immediately after slaughter, were fixed, embedded in plastic resin and stained with toluidine blue or stored in a freezer at -70 degrees C until analysed by immunohistochemistry (prooestrus and anoestrus) with an avidin-biotin peroxidase method. Quantitative and qualitative examinations of oviductal epithelium and subepithelial connective tissue were performed by light microscopy. During all stages, a lower degree of morphological changes (pseudostratification, mitosis and secretory granules) was found in the isthmus compared with ampulla and infundibulum. In ampulla and infundibulum, pseudostratification, mitotic activity and secretory granules of the epithelium were high at prooestrus/oestrus. Cytoplasmic protrusions of epithelial cells with some extruded nuclei were prominent in ampulla and infundibulum at all stages except for oestrus and early dioestrus. Lymphocytes as well as CD2- and CD3-positive cells were the predominant immune cells in the epithelial layer. The numbers of lymphocytes and CD3-positive cells did not differ among segments and stages. Numbers of CD2-positive cells did not differ between prooestrus and anoestrus while the numbers were significantly higher in the infundibulum than in ampulla and isthmus. Neutrophils were only occasionally found and mainly in the infundibulum. In the subepithelial connective tissue layer, the two most commonly observed immune cell types were lymphocytes and plasma cells. The numbers of lymphocytes as well as CD2- and CD3-positive cells was lower in isthmus than in the other segments (p < or = 0.001). Higher numbers of plasma cells (p < or = 0.001) were found in infundibulum than in ampulla and isthmus. The numbers of lymphocytes and plasma cells were not significantly different between stages of the oestrous cycle. However, the number of neutrophils differed and were highest at prooestrus in ampulla and infundibulum. The numbers of CD2-, CD3- and CD79-positive cells did not differ between prooestrus and anoestrus whereas for CD14- and SWC3-positive cells, the numbers were higher at prooestrus (p < or = 0.05) than at anoestrus. In the oviduct, the morphology differed in ampulla and infundibulum with oestrous cycle stages, which indicates an effect by ovarian steroid hormones. The immune cell infiltration was less influenced by cyclic changes. However, the immune cell infiltration (in the connective tissue) in the upper part, especially infundibulum, differed significantly from the one in the lower part, isthmus, indicating different immune functions within various parts of the oviduct.     

Apoptotic cell death in the porcine endometrium during the oestrous cycle

It has been reported that apoptosis plays an essential role in controlling the physiological cell kinetics in the human and rodent endometrium but this type of death has never been studied in the porcine endometrium. The aim of this study was to investigate the apoptotic cell death in the porcine endometrium during the middle (Days 9-11) and late (Day 13) luteal phase, during the luteolysis (Day 15) and early follicular phase (Days 17-19) of the oestrous cycle. Apoptotic cells were identified by in situ DNA 3'-end labelling method. It was revealed that the greatest number of apoptotic cells in the luminal and glandular epithelium was found on Days 17-19 and on Day 15 of the oestrous cycle, respectively. In the stroma, the greatest number of these cells was found on Days 9-11. Our data have shown that in the porcine endometrium, both epithelial and stromal cells undergo apoptosis and that the number of apoptotic cells varies depending on the phase of the oestrous cycle.     

The oestrous cycle and early pregnancy--a new concept of local endocrine regulation

Facts discovered in recent decades have compelled us to revise long-established views on the physiological regulation of cyclic adjustments to the reproductive system in preparation for pregnancy in females. Evidence has been presented to show that changes in the uterine blood supply induced by the oestrogen/progesterone ratio in the blood and cytokines are important in the regulation of the secretory function of the endometrium. Progressive reduction in uterine blood flow during the luteal phase of the oestrous cycle causes regressive changes in endometrial cells and release of prostaglandin (PG) F(2 alpha), resulting in initiation of luteolysis. Retrograde transfer of PGF(2 alpha) in the area of the mesometrium vasculature is an important element in the mechanism protecting the corpora lutea against luteolysis before day 12 of the porcine oestrous cycle and during early pregnancy and pseudopregnancy. Results of many studies presented in this review indicate that PGF(2 alpha) pulses in uterine venous blood during the follicular phase of the oestrous cycle may not be due to PGF(2 alpha) secretion by endometrial cells, but occur due to remodeling of the endometrium and pulsatile exretion of PGF(2 alpha) in accordance with rhythmic uterine contractions caused by oxytocin.     

Histological Study of Canine Mammary Gland During the Oestrous Cycle

The development of the mammary gland is a puzzling phenomenon and the research on this field has been focussed mostly on the carcinogenesis, with a less goal‐oriented concern in basic histology. In order to determine the histological features of normal mammary gland in the different oestrous phases we used 39 non‐pregnant female dogs of various breeds and ages. The animals were grouped in: pre‐pubertal, pro‐oestrous, oestrous, early and late dioestrous, early and late anoestrous phases. Major changes of the canine mammary histology throughout the oestrous cycle were identified in this study. A rudimentary gland with few ducts in the base of the nipple was observed in pre‐pubertal female individuals and pubertal pro‐oestrous female ones. In the oestrus, small inactive lobules associated with ductal branching and inconspicuous regressive changes were observed, while in early dioestrus, a ductal arborization was present. In late dioestrus, a complete lobuloalveolar differentiation and secretory capacity was achieved. The regressive histological features were abundant on early anoestrus, and markedly generalized on late anoestrus. The regressive process was longer in the more caudal gland pairs. This work provides baseline knowledge of canine mammary gland that may be relevant for interspecies comparative purposes and for pathologists dealing with mammary gland tumours.     

Apoptosis in the porcine uterine endometrium during the estrous cycle, early pregnancy and post partum

The mammalian uterus changes dramatically during the estrous cycle, pregnancy, and involution post partum. Dynamic changes in the uterine endometrium are a type of homeostasis and proceed with proliferation and exclusion of cells. Homeostasis of the uterus is closely related to apoptosis involving various hormones and cytokines. The objective of the present study was to determine the morphological features and occurrence of apoptosis in the porcine endometrium during the estrous cycle, early pregnancy, and post partum. Cyclic changes in the morphology of the surface epithelium were observed during the estrous cycle. The heights of surface epithelia were significantly high on day 4 of the estrous cycle and the early pregnancy. The heights of the surface epithelium remained low from days 1 to 31 post partum. We then used terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end-labeling (TUNEL) of the 3'-terminal of fragmented DNA, which is effective for detection of apoptosis in various tissues. We found that apoptosis in the porcine endometrium contributed to homeostasis of the endometrium during the estrous cycle through control of cell proliferation and exclusion. Conversely, apoptosis on days 4 and 8 of gestation before the implantation window depended on the plasma estrogen and progesterone levels; however, suppressive homeostasis of apoptosis occurred at the time of implantation on days 15, 18 and 21 of gestation. Our study is the first to demonstrate apoptotic cell death in the porcine endometrium directly by TUNEL method. The results strongly suggest that uterine homeostasis is mainly controlled by apoptosis during the estrous cycle and early pregnancy.