Caspase-3-mediated apoptosis and cell proliferation in the equine endometrium during the oestrous cycle.

Cell proliferation and apoptosis are hormone-dependent physiological processes involved in endometrial growth and regression. The aims of the present study were: (1) to evaluate endometrial cell proliferation using proliferating cell nuclear antigen (PCNA) expression; (2) to evaluate the induction of endometrial cell death by the expression of active caspase-3 and the apoptotic phenotype visualised by DNA fragmentation; and (3) to relate these observations to endometrial tissue dynamics in the equine endometrium throughout the oestrous cycle. Endometria were assigned to follicular and luteal phases based on ovarian structures and plasma progesterone. Cell proliferation and active caspase-3-mediated apoptosis were expressed in both phases of the oestrous cycle. In the luteal phase, PCNA expression was higher than in the follicular phase. Highest PCNA activity was noted in the luminal and glandular structures. Active caspase-3 staining was increased in luminal epithelium and deep glandular cells during the luteal phase. However, in the follicular phase, stromal cells showed greater active caspase-3 expression. Only a few apoptotic endometrial cells were detected by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) and these cells were mostly present in luminal and glandular structures. A simultaneous increase in DNA, cell proliferation and protein synthesis was observed in the endometrium during the mid-luteal phase. This suggests that cell hyperplasia occurs at the time the histotroph is needed for eventual embryo nourishment.

Endometrial nitric oxide production and nitric oxide synthases in the equine endometrium: Relationship with microvascular density during the estrous cycle.

Nitric oxide (NO) plays an important role in angiogenesis and in the regulation of the blood flow. This study was carried out to investigate (i) the effects of endogenous estrogens and progestins and exogenous progesterone (P(4)) (5 ng/ml or 1 microg/ml) or estradiol 17beta (E(2)beta) (50 pg/ml or 1 microg/ml) on in vitro endometrial NO synthesis; (ii) the presence of different isoforms of NO synthase; (iii) and their relationship to microvascular density in the equine endometrium during the estrous cycle. NOS expression was also evaluated in the myometrium. Expression of endothelial and inducible forms of NOS in the uterus was assessed by Western blot and immunocytochemistry. Vascular density in endometrial tissue was determined on histologic sections. In the luteal phase, compared to the follicular phase, endometrial NO production increased without exogenous hormones and with exogenous E(2)beta (1 microg/ml). Although immunocytochemistry revealed iNOS and eNOS expression in the endometrium, no positive signal for iNOS was detected by Western blot. Endothelial NOS was observed in endometrial glands, endothelial cells, fibroblasts, blood and lymphatic vessels. Endometrial eNOS expression was the highest in the follicular and mid-luteal phases while it was found to be the lowest in the early luteal phase. In the follicular phase, hyperplasia of endometrial tissue with respect to myometrium was detected. No difference in vascular density was present between phases. All together, NO may play some roles in both proliferative and secretory phases of endometrial development in the mare.

Peripheral blood neutrophil function and lymphocyte subpopulations in cycling mares.

The purpose of this study was to evaluate different parameters of the immune status in the mare, during the follicular and the luteal phases of the oestrous cycle, in two consecutive years. Functional competence of peripheral blood neutrophils, such as chemotaxis, phagocytosis and oxidative burst was assessed under physiological cyclic conditions (Exp. I). In the second year of this study (Exp. II), besides peripheral blood neutrophil phagocytosis and oxidative burst analysis, circulating lymphocyte subsets were also characterized. The reproductive status in a total of 17 adult cycling mares was evaluated by ultrasonography and further confirmed by plasma progesterone levels. Chemotaxis tests were performed using porous membranes inserted in transwell chambers. Lipopolysaccharide (LPS) from Escherichia coli and N-formyl-Met-Leu-Phe (fMLP) were used as chemoattractants. Measurement of phagocytosis and oxidative burst in blood neutrophils were assessed by flow cytometry using commercially available kits. Quantification of T-lymphocyte subsets was assessed by indirect immunofluorescence staining after incubation with monoclonal antibodies specific for CD2, CD3, CD4 and CD8 cell markers by flow cytometry. Natural killer cells and B cells were estimated mathematically. No significant difference was found in migration, phagocytosis and oxidative burst at either phase of the oestrous cycle. Statistical analysis of total white blood cell counts also showed no significant difference between either phase of the oestrous cycle, although there was a tendency for blood neutrophils to increase in number under the progesterone influence (p = 0.09). Lymphocytic subpopulations did not differ throughout the oestrous cycle. Overall, our results suggest that luteal and follicular phases in cycling mares may not influence the immune status of the mare.