Immunolocalization of E-cadherin and ß-catenin in the cyclic and early pregnant canine endometrium.

Putative changes in E-cadherin and ß-catenin during implantation in dogs are of interest to study, as they are relevant proteins for epithelial integrity. E-cadherin and ß-catenin were immunolocalized in the canine endometrium during the estrous cycle and early pregnancy, using monoclonal antibodies. Both proteins were detected in all types of endometrial epithelia (surface epithelium [SE], superficial glandular, and deep glandular epithelia) at all stages of the estrous cycle and in early placental structures. E-cadherin depicted a gradient of intensity apparently being lowest in the SE to progressively increase toward the deepness of the endometrial glands, regardless of the stage of estrous cycle. The overall immunostaining was, however, weaker at diestrus. In pregnant samples, the trophoblast was conspicuously immunolabeled compared with the endometrial surface lining epithelium. In the latter, the cytoplasmic pattern predominated over the membrane-bound, as was also seen in the decidual cells of the placental labyrinth. In the early placenta, only trophoblast cells and lacunae retained membrane signals. ß-Catenin membrane labeling appeared relatively constant throughout the cycle, although a tendency toward a decrease in intensity was detected at the secretory stages. In addition, a dislocation of the immunoreaction from membrane to the cytoplasm was observed in both the SE and the glandular epithelia at particular stages of the cycle. In early pregnancy, a loss of the membranous pattern was observed in the SE and labyrinth, but neither on trophoblast nor in lacunae. The results show the existence of a softening of the adherens junctional complex in progestagen-dominated stages favoring embryo-maternal interactions and endometrial invasion during canine implantation.

Localization of tumor necrosis factor in the canine testis, epididymis and spermatozoa.

Tumor necrosis factor (TNF), formerly known as Tumor necrosis factor alpha is now regarded as a natural component of the mammalian seminal plasma (SP). Although not completely clarified, its functions in the SP have been associated with paradoxal roles, such as sperm survival in the female genital tract, while at high levels negatively affect sperm survival and fertility potential. Recently, it has been discovered that canine inseminated spermatozoa display a strong immunoreaction for TNF when lining the female endometrium. As a continuation of this finding, the present work aimed at documenting TNF localization in the canine testes and epididymis and in freshly ejaculated spermatozoa (SPZ) through immunohisto- or cytochemistry. Immunoreaction for TNF was found in all samples used. In the dog testis, TNF immunoexpression was limited to the seminiferous tubules, where late round spermatids (SPD) showed weak intensity of immunostaining, while elongating and elongated SPD evidenced moderate and the residual bodies a strong intensity. In the epididymis, a gradual progressive increase of TNF immunolabelling was found throughout the epididymal regions, ranging from a weak intensity at the caput epididymis to a moderate intensity at the cauda. TNF immunolabelling was found in mature SPZ during the epididymal transit and also in freshly ejaculated SPZ, which showed a strong midpiece immunolabelling. Data presented here provide important information on expression of TNF in spermatozoa, which is acquired by the SPZ during their formation at the testis. It further provides the basis for subsequent studies on the physiological importance of cytokines in sperm function.

Expression of four canine leukocyte adhesion factors in fresh and stored whole blood samples evaluated using a no-lyse, no-wash method.

Expression of four leukocyte adhesion factors on canine leukocytes was studied by flow cytometry using a no-lyse, no-wash method. The effect of fixation and storage for up to 14 days in 1% paraformaldehyde on labelled samples and within assay variation was evaluated. Monoclonal antibodies directed to monocyte marker CD14, and to adhesion molecules CD11a, CD18, CD32 and CD49d were used. Cell surface marker, cell population, time, and the interactions between time and cell marker significantly affected expression of cell adhesion factors. For CD18, there was a significant difference in mean fluorescence intensity (MFI) between fresh and stored samples (P<0.001), but no significant difference between stored samples. The MFIs of CD11a and CD49d were not significantly affected by fixation and storage. The CVs differed significantly depending on cell marker (P<0.001) and cell population (P=0.005). Fixation and storage did not significantly affect the CV. In conclusion, a no-lyse, no-wash method can be applied to canine leukocytes. The effect of fixation and storage on the resulting MFI differs between monoclonal antibodies, and should be evaluated for each antibody before use. The coefficient of variation was generally acceptable, and high CVs were related to a low MFIs or low numbers of analysed cells.

Differentiation between pyometra and cystic endometrial hyperplasia/mucometra in bitches by prostaglandin F2alpha metabolite analysis.

Bitches with pyometra are potential emergency cases which may be clinically difficult to differentiate from cases of cystic endometrial hyperplasia (CEH) in combination with mucometra. In the present study plasma prostaglandin F(2alpha), as measured by its main metabolite 15-keto-13,14-dihydro-PGF(2alpha) (PG-metabolite) concentrations, blood biochemical and hematological parameters were measured in 59 bitches with pyometra, 10 bitches with CEH and nine controls to determine if PG-metabolite could differentiate between the three uterine conditions. Bitches with pyometra had significantly higher plasma levels of PG-metabolite than bitches with CEH (P=0.002) and the controls (P=0.002). PG-metabolite analysis alone had a high sensitivity (98.3%) and a high specificity (80.0%) for the differentiation of pyometra versus CEH in bitches where fluid in the uterus was diagnosed. When a combination of PG-metabolite and percentage band neutrophils (PBN) was used for differentiation of the two diagnoses, a sensitivity of 100% and specificity of 90.0% was obtained. This means that the combination of PG-metabolite and PBN analysis allows for differentiation between cases of pyometra and CEH. If the PG-metabolite level in a bitch is >or=4,524 pmol l(-1), there is a 99% probability of the diagnosis pyometra versus CEH. Levels of PG-metabolite >or=3,054 pmol l(-1), >or=2,388 pmol l(-1) or>or=1,666 pmol l(-1) indicates a 95%, 90% or 80% probability of pyometra, respectively. At high PG-metabolite levels (above about 3,000 pmol l(-1)), PG-metabolite alone is enough for differentiation of pyometra versus CEH. The results of the present study showed that PG-metabolite analysis is valuable in the diagnosis and prediction of severity of uterine diseases.