Endometrial progesterone and estrogen receptors were studied by immunocytochemistry using monoclonal antibodies during the menstrual cycle in normal women. We initially compared immunocytochemical staining of progesterone and estradiol receptors on endometrial fragments obtained by either aspiration or endometrial biopsy and found that immunocytochemistry could be performed easily on tissue obtained in either way. The immunocytochemical studies showed that the concentration and distribution of receptors changed markedly during the normal menstrual cycle. These changes were distributed in three characteristic phases. During phase I, corresponding to the midfollicular period (days 7-8), a small proportion (25%) of stromal and glandular cells stained positively for the progesterone receptor, whereas estrogen receptor staining was more intense and more frequent (50% of cells). Phase II, which included both the late follicular and early luteal periods (days 9-19), was characterized by a marked staining of progesterone receptors in the majority of glandular cells (75%) and somewhat less abundant and less frequent staining in stromal cells (50%). Estrogen receptor staining was present in about half of the glandular and stromal cells. Phase III, the mid- and late luteal period (days 21-27), was characterized by the disappearance of estrogen and progesterone receptor staining in glandular cells, although faint staining for both receptors was found in stromal cells. These variations in progesterone receptor staining are potentially useful for determining the effect of progesterone on endometrial maturation.

Five monoclonal antibodies were used for the immunocytochemical study of mammalian progesterone receptor (PR). Initial studies were aimed at defining the optimal experimental conditions for the detection of the receptor, with special emphasis on techniques likely to be used in clinical determinations and in immunoelectron microscopic localization. Specific immunoperoxidase staining was observed either in fixed, frozen sections or in sections of paraffin-embedded tissue. The latter method allowed a better preservation of cellular structures. Among the eight fixatives tested, glutaraldehyde, picric-acid formaldehyde, and paraformaldehyde proved satisfactory. Both indirect immunoperoxidase and the indirect antibody peroxidase-antiperoxidase methods could be used. In immature rabbits or castrated guinea pigs primed by estrogen, i.e. in conditions where its ligand was absent (or present in very low concentration), the PR was confined to the nucleus of immunoreactive cells. This was the case for all the cell types of the endometrium and the myometrium, for the immunostained cells of the oviduct, cervix, vagina, pituitary gland, and for the very weakly stained cells in the liver. No staining was observed in nontarget tissues for progesterone, such as diaphragm, spleen, and small intestine. Nuclear staining was also absent when various control antibodies replaced anti-PR antibodies. This result thus generalizes the observations made on the estrogen receptor, showing that there is no translocation of the receptor from cytoplasm to nucleus under the influence of the hormone. Moreover, a marked heterogeneity in immunostaining was observed among cells of the same type in several tissues, suggesting that there could be large differences in the hormonal sensitivity of individual cells. Cellular distribution of PR immunoreactivity was studied in the uterus, cervix, oviduct, and pituitary gland of rabbits and in the uterus and vagina of guinea pigs. A labeling was observed in all the cell types of the uterus (luminal and glandular epithelium, stroma, and muscularis). In the cervix, nuclear immunostaining was observed in the connective tissue of the lamina propria and in some epithelial and muscle cells. In the vagina, PR immunoreactivity was seen in the basal layers of the stratified squamous epithelium, in the connective tissue of the lamina propria, and in the smooth muscle. In the oviduct, the luminal epithelium, the connective tissue, and the muscularis were stained. In the pituitary gland, selective nuclear labeling was observed in a few scattered cells.

Angiogenesis is fundamental for human endometrial development and differentiation necessary for implantation. These vascular changes are thought to be mediated by the vascular endothelial growth factor (VEGF), whose specific receptors have not been examined in detail thus far. We conducted the present study to determine, by immunocytochemistry and computerized image analysis of the functionalis, the expression and modulation of the receptors Flk-1/KDR and Flt-1, which mediate VEGF effects on endothelial mitogenicity, chemotaxis, and capillary permeability. VEGF receptors are expressed mainly in endometrial endothelial cells, with variations of intensity and number of stained capillaries related to the phase of the cycle. The number of capillaries immunostained for Flk-1/KDR was maximal in the proliferative phase (ratio Flk-1/CD34: 1), twice as high as the number of Flt-1-expressing capillaries (ratio Flt-1/CD34: 0.47). The staining intensity for Flk-1 decreased during the late proliferative and early secretory phases, to increase again in the midsecretory period. The number of Flt-1-labeled capillaries was about 2-fold higher in the secretory than in the proliferative phase; however, the proportion of Flt-1-positive cells did not change, owing to the associated increase in vascular density that characterizes progression of the functionalis from the proliferative to the secretory stage. The staining intensity for Flt-1 was higher during the late proliferative and secretory phases (especially in the midsecretory phase) and the premenstrual period. In contrast, the proportion of capillaries expressing Flk-1/KDR decreased in the secretory phase (ratio Flk-1/Von Willebrand factor: 0.55). Enhanced expression of Flk-1/KDR, and of Flt-1, on narrow capillary strands at the beginning of and during the proliferative phase may account for the rapid capillary growth associated with endometrial regeneration following menstrual shedding. The high coexpression of Flk-1/KDR and Flt-1 observed on capillaries during the midsecretory period correlates with an increase of endometrial microvascular density and of permeability characteristic of this phase of the cycle, which is a prerequisite for implantation. Finally, strong expression of Flt-1, but not Flk-1/KDR, was observed on dilated capillaries during the premenstrual period and the late proliferative phase, suggesting preferential association of Flt-1 with nonproliferating capillaries at those times; activation of this receptor by VEGF could be involved in premenstrual vascular hyperpermeability, edema, and extravasation of leukocytes. In addition to the endothelial localization, we found that epithelial cells expressed Flt-1 and Flk-1/KDR. We conclude that Flt-1 and Flk-1/KDR in the functionalis are modulated in parallel or independently according to the phase of the cycle, and that these changes are responsible for VEGF actions on endometrial vascular growth and permeability. The molecular mechanisms concerning these regulations will require further investigation.

Modifications of uterine blood flow are implicated in many important aspects of reproductive physiology and in several of their pathological disorders. These modifications are hormonally regulated but remain poorly understood, and various complex mechanisms have been proposed. The aim of this study was to investigate the presence and some characteristics of estrogen receptors (ER) and progesterone receptors (PR) in uterine blood vessels. Using monoclonal antibodies and immunocytochemistry we observed the presence of ER and PR in muscle cells (tunica media) of uterine arteries of rabbits and women. ER or PR immunoreactivity was not detected in the endothelium of uterine arteries nor in uterine capillaries or veins. Staining for both receptors was also present in arterial walls from the fallopian tube (isthmus and ampulla) and vagina but not in arteries of nonreproductive tissues (intestinal, renal, hepatic, femoral, and pulmonary arteries, aorta). PR immuno-staining was increased by estrogen in all cell types of the rabbit uterus, but the doses necessary to provoke an intense nuclear staining in uterine arteries were higher than those required for observing strong labeling in glandular, stromal, or myometrial cells. These results suggest that, contrary to many hypotheses previously put forward, sex steroid hormones may regulate uterine blood flow through a direct effect on uterine arterial walls.