Immunocytochemical Localization of Progesterone Receptor in the Reproductive Tract of Adult Female Rats

BIOLOGY OF REPRODUCTION 48, 205-213 (1993) Immunocytochemical Localization of Progesterone Receptor in the Reproductive Tract of Adult Female Rats YASUHIKO OHTA, 1' 2 TOMOMI SATO,3 and TAISEN IGUCHI 3 Department of Biology, 3 Faculty of General Education, Tottori University, Tottori 680, Japan Department of Biology, 3 Yokohama City University, 22-2 Seto, Kanazawaku, Yokohama 236, Japan ABSTRACT INTRODUCTION ture- and light-controlled room (22 - 2°C, lights-on from 0500 to 1900 h). All rats were given a standard laboratory pellet diet (Clea Co., Tokyo, Japan) and water ad libitum. At the beginning of the experiment, the rats showed regular 4-day estrous cycles. In the first set of experiments, virgin female rats were killed at approximately 1100 h on each day of the estrous cycle. Some were ovariectomized 1 or 2 days prior to autopsy. To determine the effects of ovarian steroids on PR expression in the uterus and vagina, nine groups of rats were ovariectomized at estrus. Five groups of ovariectomized rats were killed 1, 2, 3, 4, and 20 days after ovariectomy, respectively, while the remaining four groups underwent one of the following four types of treatment commencing 20 days after the operation: three daily s.c. injections of 0.2 pzg estradiol-173 (E2 ) dissolved in 0.1 ml sesame oil; two daily s.c. injections of 3 mg progesterone (P) in 0.1 ml oil following three daily s.c. injections of 0.1 ml oil vehicle only; three daily injections of 0.2 Rg E2 or oil vehicle only followed by 2-day treatment with 3 mg P. These rats were killed on the day following the last injection. In the second set of experiments, mated females were divided into five groups and killed around 1100 h on Days 0 (day of detection of the vaginal plug = Day 0), 1, 2, 3, and 4 of pregnancy, respectively. In addition, four groups of rats were ovariectomized at estrus to examine the relationship between ovarian steroids and PR expression in the uterus and vagina during early pregnancy. Three groups of the ovariectomized rats were given one to three daily s.c. Steroid hormones are bound to cytoplasmic receptor molecules of their target cells with a high affinity and specificity. Varying concentrations of the uterine estrogen and progesterone receptors (ER and PR) during the estrous cycle in laboratory rodents have been reported [1-7]. A number of studies have suggested that the variation in the ovarian production of estrogen and progestin during the estrous cycle is reflected in the uterine levels of their respective cytoplasmic receptor proteins [8-15]. The concentration of the PR in the uterus has also been measured in some rodents during pregnancy [4, 6, 12, 16-18]. On the other hand, ER has been demonstrated in the uterus of laboratory rodents and primates through use of immunocytochemical methods [19-25]. However, the uterine localization of PR has been investigated mainly in primates [26-30] and insufficiently studied in laboratory rodents [31]. The present study, therefore, was undertaken to examine the localization of PR in the reproductive tract of female rats during the estrous cycle and early pregnancy. MATERIALS AND METHODS Adult female rats (60-90 days of age) of the T strain used in the present experiment were maintained in a temperaAccepted September 5, 1992. Received March 4, 1992. 'Correspondence: Dr. Yasuhiko Ohta, Department of Biology, School of General Education, Tottori University, Tottori 680, Japan. FAX: 0857-31-0930. 205 The distribution of the progesterone receptor (PR) was investigated immunocytochemically in female reproductive tracts of rats during the estrous cycle and early pregnancy through use of an anti-PR monoclonal antibody. PR was localized predominantly in the nuclei of epithelial, stromal, and muscle cells in the uterus and vagina during the estrous cycle. In the uterus, the nuclei of epithelial cells were stained intensively at diestrus, while the PR staining of the stromal cells was more intense at proestrus than at any other stage of the cycle. PR expression during the cycle in muscle cells of the myometrium was similar to that in the endometrial stromal cells. In the vagina, however, PR expression during the cycle was approximately the same among epithelial, stromal, and muscle cells, the nuclei of which were stained deeply at proestrus. Ovariectomy at various stages of the cycle altered the PR expression appearing in the uterus and vagina during the cycle. In ovariectomized rats, estrogen increased the PR immunoreaction of various types of cells examined in the uterus and vagina except for the uterine epithelial cells. The reaction of these uterine epithelial cells was decreased by estrogen but was increased by progesterone given after estrogen; however, progesterone given alone reduced the reaction. In the epithelial and stromal cells of the uterus, intensity of the staining was increased after mating, reaching maximum on Day 3 of pregnancy, and then decreased on Day 4 (day of implantation), while in epithelial and stromal cells of the vagina the staining remained weak during early pregnancy. The uterine cells of ovariectomized rats given hormone treatment to promote a deciduogenic reaction showed PR expression similar to that in the pregnant females. These results indicate that PR expression in the uterus and vagina is regulated by ovarian steroids during the estrous cycle and early pregnancy. 206 OHTA ET AL. RESULTS PR Expression in the Uterus and Vagina during the Estrous Cycle PR was localized predominantly in the nuclei of epithelial and stromal cells in both uterus and vagina during the estrous cycle, although PR staining at different stages of the cycle showed marked variation in intensity (Figs. 1 and 2, a-c). In the uterus, the nuclei of the endometrial stromal uterus 4 3 a ' 3 2 0 -E Proestrus 1·I·I·I·I·I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Estrus Metestrus Diestrus Proestrus r vagina 4 3 C 0 C 2 1 0 01 j I I I Proestrus Estrus Metestrus Diestrus Proestrus Days of estrous cycle FIG. 1. PR expression in the uterus and vagina during the estrous cycle. Intensity of PR staining is shown by 0 (negative), 1 (slight), 2 (weak) 3 (moderate), and 4 (strong). 0[, epithelial cells; 0, stromal cells; A, muscle cells; l', 0', and A', epithelial, stromal, and muscle cells in rats ovariec2 2 tomized at estrus; O , 0;2, and A , epithelial, stromal, and muscle cells in 3 3 3 rats ovariectomized at metestrus; 0 , 0 , and A, epithelial, stromal, and muscle cells in rats ovariectomized at diestrus. cells were stained intensely at proestrus as compared with those at any other stage of the cycle, while the staining intensity in the epithelial cells was low at proestrus (Fig. 2a). The uterine epithelial cells stained with maximal intensity at diestrus (Fig. 2c), whereas at estrus and metestrus, the values were almost negative (Fig. 2b). The staining of the stromal cells at metestrus was minimal in intensity. In the muscle cells of the (...truncated)