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Despite the vital physiological role of endometrial regeneration during the menstrual cycle and the various pathological implications of abnormal growth of endometrial epithelial cells, the local factors and regulatory mechanisms involved in endometrial regeneration and growth have not been well characterized. Here, we examine the pattern, hormone dependence, and potential functions of Wnt7a (wingless-type MMTV integration site family member 7a), which is known to play a critical role in the formation of the mouse endometrial epithelium during embryonic development, in both human and artificially cycling rhesus macaque endometrium, and using a potent Wnt-antagonist in a mouse model of endometrial regeneration. Wnt7a transcript levels were examined using quantitative real-time PCR and in situ hybridization, and immunohistochemistry was performed to detect Ki-67 and 3,5-bromodeoxyuridine. Stringent, fully conditional Wnt inhibition was achieved by adenoviral expression of Dickkopf-1 during artificial endometrial regeneration in mice. In macaques, Wnt7a expression was confined to the newly formed luminal epithelium (LE) and upper glands during the postmenstrual repair phase. The signal increased in the LE during the proliferative phase but decreased in the upper glands and was undetectable in the glands by the late proliferative phase. Interestingly, Wnt7a was completely suppressed in the LE and remained undetectable in other cell types after 7 d of progesterone treatment. The pattern of Wnt7a expression in the human endometrium was similar to that in macaques. Blockade of Wnt signaling during endometrial regeneration in mice resulted in a dramatic delay in reepithelialization and degeneration of glands and LE. These results strongly suggest, for the first time, a role for Wnt7a in postmenstrual regeneration and proliferation of endometrial glands and LE in primates, and its dramatic suppression by progesterone is likely essential for secretory transformation of the epithelium.

We have found that microwave (MW) stabilization greatly improves detection of the estrogen receptor (ER) in frozen sections of rhesus monkey oviduct by immunocytochemistry (ICC). Fresh samples of fimbriae were MW-irradiated, frozen, and then cryosectioned. The frozen sections were also MW-treated and then fixed in a paraformaldehyde-based fixative before ICC processing. A parallel set of samples from each monkey were frozen, sectioned and processed for ICC without any MW treatment. MW stabilization clearly increased immunostaining intensity with either of two ER-specific monoclonal antibodies, namely, H222 and 1D5. The greatest increase was noted in tissues collected from spayed or progesterone-treated animals. An antibody dilution series indicated that MW stabilization increased the sensitivity approximately 20- to 40-fold. In addition, we incubated spayed macaque fimbriae at 4 C in the presence of 10 nM [3H]Moxestrol and then either froze the tissues immediately (non-MW) or treated them with MW. Slide-mounted cryosections of non-MW and MW-treated tissue were then incubated with either a Tris-EDTA buffer (low salt) or the same buffer containing 4 M KCl (high salt). The quantity of [3H]Moxestrol-occupied ER extracted from the frozen sections by each buffer was determined by a sucrose gradient shift assay. The low salt buffer extracted significantly more radiolabeled ER from non-MW sections than from MW-treated sections (P < 0.01), whereas the high salt buffer extracted equal amounts of ER from both the MW-treated and non-MW sections. MW-irradiation enhanced ICC detectability of ER in frozen sections by greatly reducing the amount of ER extracted during the various washes used during normal ICC processing.

We previously reported that RU486 can reverse progesterone (P)-induced suppression of the estrogen receptor (ER) in the uterus of pregnant rhesus monkeys, but we did not determine whether estrogen (E) could act through its receptor in the presence of P and RU486. To pursue this question, we treated spayed rhesus monkeys with various hormonal regimens and evaluated the effects of E in the oviduct and endometrium, with and without RU486 treatment, on ER and progestin receptor (PR) levels, morphology, apoptosis, and degree of proliferation. The latter was assessed immunocytochemically with a monoclonal antibody (Ki-67) against a nuclear antigen present only in proliferating cells. Animals were treated for 2 weeks with 17 beta-estradiol (E2) and then for 2 weeks with E2 plus P to produce a regressed oviduct and a secretory endometrium. The animals were then treated for 2 more weeks with four different treatments, as follows: I) E2, P, and vehicle; II) E2 and vehicle; III) E2, P, and RU486; and IV) E2 and RU486 (n = 4 each). In group I, menstruation did not occur, and the endometrium exhibited stromal cell enlargement, extensive development of the spiral arteries, few Ki-67-positive cells, and low levels of ER and PR. Oviducts in group I remained regressed, Ki-67-positive epithelial cells were few, and levels of ER and PR were low. In contrast, in groups II, III, and IV, the oviducts had responded to E2 and were fully ciliated and secretory, with elevated levels of ER and nuclear PR. All animals in these three groups menstruated and then regenerated their endometrium. The regenerated endometria expressed elevations in ER, nuclear PR, and epithelial Ki-67 index. However, the endometria of RU486-treated monkeys in groups III and IV had significantly more epithelial cell death by apoptosis, increased stromal cell compaction, and fewer Ki-67-positive stromal cells than in the E2 controls (group II). In group IV, RU486 caused a significant decrease in endometrial weight. Thus, RU486 blocked P action and allowed E2 to act in a normal fashion in the oviduct and endometrium on several end points, but it also had antiproliferative effects that opposed E2 action, especially in the endometrium.

Previous immunocytochemical studies indicate that receptor regulation varies in different uterine cell types. In primates, progesterone (P) suppresses estrogen receptor (ER) in glandular epithelial cells in the functionalis, but fails to suppress ER in the glandular epithelial (GE) cells of the basalis. P also fails to suppress ER in the perivascular stromal and smooth muscle cells of the spiral arteries in the functionalis. We used nonradioactive in situ hybridization to determine whether similar cell type differences occur at the ER mRNA level. We used digoxigenin-labeled oligodeoxynucleotides (oligo-DNAs; 45-mer) as probes and detected the hybrids immunocytochemically with horseradish peroxidase-labeled antidigoxigenin antibody. This technique can discriminate between positive and negative cells in closely packed histological associations. In spayed monkeys, most of the GE cells as well as endometrial stromal cells were positive for ER mRNA, while all vascular smooth muscle, endothelium, and perivascular stromal cells were negative. Estradiol treatment for 14 days markedly increased ER mRNA staining in the GE cells, most stromal cells, and the vascular smooth muscle and perivascular stromal cells of spiral arteries in the functionalis. However, in the basalis, these components of the spiral arteries were negative as were the small basal arteries of the basalis. In most positive cells, ER mRNA was not homogeneously distributed in the cytoplasm, but, rather, was concentrated in their perinuclear regions. The GE cells in the basalis had especially intense concentrations of perinuclear signal at their apical poles. After sequential estradiol plus P treatment, the signal was greatly reduced in the GE cells of the functionalis, but not in the GE cells of the basalis or in the vascular smooth muscle or perivascular stromal cells of the spiral arteries of the functionalis. In myometrium, ER mRNA was localized to the perinuclear region of smooth muscle cells, but the staining intensity was not dramatically affected by hormonal manipulation. Unexpectedly, we observed clusters of stromal cells characterized by extremely high positive signals for ER mRNA ("hot cells") at the endometrial/myometrial border and deeper in the connective tissue of the myometrium, although such cells did not express high levels of ER protein. In general, however, the cellular distribution of ER mRNA and its hormonal regulation paralleled those of ER protein.

PRL increases during pregnancy in primates with rising levels of placental estradiol (E) and progesterone (P). However, while E will increase PRL secretion in monkey pituitary cell cultures, P has no effect. We recently localized progestin receptors (PR) to gonadotropes, but not lactotropes, with an immunocytochemical technique to double stain monkey pituitary cell cultures. The following studies were performed to confirm the immunocytochemical localization of PR in intact pituitary tissue and to determine the effect of E and P on the levels of estrogen receptors (ER) and PR in the pituitary. ER and PR levels were determined in the endometrium of the same animals for an internal comparison. Thirteen adult cycling female cynomolgus monkeys were ovariectomized and treated for 28 days with 1) an empty Silastic capsule (Spay), 2) a 2-cm E-filled capsule (E), or 3) a 2-cm E-filled capsule for 14 days plus a 6-cm P-filled capsule implanted for an additional 14 days (E + P). Blood samples were drawn daily for assay of serum E, P, and PRL levels. Serum PRL was not significantly affected by E, but the sequential addition of P significantly increased serum PRL levels over those observed in Spray animals. The anterior pituitary and endometrium were removed for measurement of ER and PR levels by a sucrose gradient shift assay incorporating monoclonal antibodies against ER and PR. Pituitary ER levels did not vary significantly with steroid treatment (158.2 +/- 33.6, 135.5 +/- 24.9, 104.3 +/- 13.4 fmol/mg DNA in Spay, E, and E + P animals, respectively). Pituitary PR levels were undetectable in Spay animals, were induced by E (393.3 +/- 53.4 fmol/mg DNA), and were suppressed to undetectable levels by the addition of P. A portion of the pituitary was frozen for immunocytochemical single staining for ER, PR, PRL, and LH and double staining for PRL + PR and LH + PR. ER staining was observed in many parenchymal cells, but there was no apparent change with steroid treatment. PR staining was absent in the Spay animals; many PR-positive cells were observed in E-treated females, and only a small number of faintly staining cells were detected in the E + P animals. Double staining for PRL + PR and LH + PR revealed PR in gonadotropes, but not lactotropes. In conclusion, PR, but not ER, are regulated by E and P in the monkey pituitary. Importantly, PR is regulated within gonadotropes, but not lactotropes. Therefore, P probably increases PRL secretion through a hypothalamic action.

The distribution of estrogen receptors (ER), progesterone receptors (PR), PRL, and gonadotropins in different cell types of the monkey pituitary was examined by immunocytochemical (ICC) labeling of pituitary cell cultures. Dispersed monkey pituitary cells were cultured on extracellular matrix and in serum-free medium for 6-14 days. Individual cultures were singly stained for ER, PR, PRL, LH, and FSH or double labeled for PR and one of the protein hormones. ICC reaction product localizes over the nuclei of cells that are positive for the steroid receptors, whereas reaction product localizes over the cytoplasm of cells that are positive for the protein hormones. Sixty-one percent of the parenchymal cells were positive for PRL, while 1-3% were positive for LH or FSH. Sixty-two percent of the parenchymal cells were ER positive. ER staining was localized over the nuclei of two morphologically distinct cell types. One cell type is smaller and more prevalent than the second cell type. Based on single staining for each of the protein hormones, we propose that the smaller cells are lactotropes, and the larger cells are gonadotropes. PR-positive cells averaged 7.7% of the parenchymal cells. Double ICC staining for PR and the protein hormones demonstrated that PR localize in the nuclei of gonadotropes, but not lactotropes, of monkey pituitary cell cultures. The absence of PR in lactotropes is consistent with our observation that progesterone has no direct effect on PRL secretion in monkey pituitary cell cultures. In contrast, the presence of PR in gonadotropes suggests that progesterone may act directly at the pituitary to modulate gonadotropin secretion in the primate. In conclusion, ER are present in both lactotropes and gonadotropes. PR are present in gonadotropes, but not lactotropes, of the primate pituitary.

Uteri were removed and blood was drawn from hamsters during the estrous cycle, on the eighth day of pregnancy, and after different hormonal treatments. Serum estradiol (E2) and progesterone (P) levels were determined by RIA. Portions of the uteri were prepared for light and electron microscopy. Endogenous uterine nuclear E2 receptor was measured by exchange assay. Large increments of nuclear E2 receptor occurred when the level of serum E2 was rising and that of serum P was falling (diestrus day 2 through proestrus morning); decrements occurred when the serum E2 level was falling and the serum P level was rising (proestrus evening through diestrus day 1). Ovariectomy on proestrus morning led to a decline in the amount of nuclear E2 receptor, and this decline was prevented by administration of E2 at the time of ovariectomy. P depleted nuclear E2 receptor in the presence of high levels of serum E2. The uterine luminal epithelium passed through a phase of mitosis and hypertrophy (diestrus day 2 through proestrus morning), a brief phase suggestive of secretion (proestrus evening), a phase of degeneration (estrus), and a phase of quiescence (diestrus day I). Ovariectomy on proestrus morning led within 24 h to degenerative changes identical to those seen during estrus, and these changes were prevented by treatment with E2 at the time of ovariectomy. P induced these degenerative changes in the presence of high levels of serum E2. After 8 days of pregnancy, the epithelium resembled that seen during diestrus day 1. Elevated uterine nuclear E2 receptor levels were associated with growth and hypertrophy of the luminal epithelium, and severely depressed levels were associated with the degenerative and quiescent phases of the epithelium.

Both estradiol and progesterone may act locally to modulate ovarian function in various species. This study examined the distribution of estradiol and progesterone receptors (ER and PR, respectively) within the primate ovary throughout the menstrual cycle. Ovaries were collected from rhesus or cynomolgus monkeys during the early, mid-, and late (n = 3-6/stage) follicular and luteal phases of the cycle. The tissues were processed for indirect immunocytochemical localization of receptors with specific monoclonal antibodies against ER (H222 and D75) and PR (JZB39). Specific immunocytochemical staining, as determined by comparing adjacent tissue sections incubated with either receptor antibodies or a nonspecific antibody, was exclusively nuclear. Both ER and PR were localized in the germinal epithelium of ovaries at all stages of the cycle. ER was not detected in any other ovarian structure (i.e. stroma, follicles, interstitial tissue, or corpora lutea) regardless of the stage of development. However, ER was detected in other estrogen-responsive tissues, e.g. the oviduct of the monkey and corpora lutea of the pseudopregnant rabbit. In the monkey ovary, PR was detected in stromal and interstitial tissues as well as theca interna and externa of healthy and atretic follicles at all stages of the cycle. The granulosa cells of some primordial and primary follicles demonstrated staining for PR. However, the granulosa layer of follicles that developed beyond the primary stage were consistently negative for PR. Only the granulosa layer of large preovulatory follicles that showed signs of luteinization after the LH surge showed staining for PR equivalent to that in the theca. Monkey corpora lutea exhibited specific nuclear staining for PR. Moreover, the percentage of receptor-positive nuclei in the corpus luteum varied (P less than 0.05) between the early (28 +/- 3%), mid (48 +/- 1%)-, and late (4 +/- 2%) luteal phase of the cycle. Nonfunctional (serum progesterone less than 0.5 ng/ml) regressing corpora lutea did not exhibit for staining for PR. Luteal cells that were PR positive also contained histochemically detectable 3 beta-hydroxysteroid dehydrogenase. These data are consistent with the concept of a receptor-mediated autocrine or paracrine role for progestins, but not estrogens in the gametogenic and endocrine functions of the primate ovary throughout the menstrual cycle.

Monoclonal antibodies to the estrogen receptor (anti-ER) were used to develop an immunocytochemical method to detect ERs in frozen sections of the macaque reproductive tract. Specific nuclear, but not cytoplasmic, staining occurred with two different methods: direct, in which an antiestrophilin -horseradish peroxidase conjugate was used as the first antibody, and indirect, in which a mixture of antiestrophilins was used in the first incubation step. Nuclear staining was absent when various control antibodies replaced the anti-ER. In uteri from spayed monkeys treated with estradiol (E2) for 14 days, nuclear staining was always present. In uteri from similar animals treated for an additional 14 days with E2 and progesterone, nuclear staining was almost completely absent. Mean endometrial nuclear ER levels, measured by an exchange assay, were 5-fold greater in the E2-treated than in the E2-plus progesterone-treated group. In addition, when samples of estrogenized uterus and oviduct were incubated for 60 min in vitro with 100 nM E2, the intensity of nuclear staining increased in parallel with an increase in the concentration of nuclear ER. The nuclei of stromal, smooth muscle, and epithelial cells of the estrogenized oviduct, cervix, and vagina as well as smooth muscle cells of the estrogenized myometrium were also receptor positive. Nontarget tissues, such as duodenum, colon, esophagus, and skeletal muscle, contained no cells that showed specific nuclear staining. Some staining of cytoplasmic and extracellular components occurred in all preparations. These latter reactions were nonspecific, because they were present in many nontarget tissues or when control antibodies replaced the anti-ER. With current methods, only nuclear ERs can be reliably localized in frozen sections of monkey tissues with monoclonal antiestrophilins .

Four weeks or more after being spayed, cats were either left untreated or treated with various regimens of estradiol (E2) and progesterone (P) in silastic implants. Oviducts and uteri were then removed and examined for morphological changes as well as for the amount and compartmentalization of the estrogen receptor. E2 treatment restored a full complement of ciliated and secretory cells to the oviduct, and subsequent P treatment (with or without continuous E2) of an E2-restored oviduct produced an atrophied epithelium equivalent to that resulting from E2 withdrawal. In the uterus, P treatment after E2 priming did not lead to E2-withdrawal symptoms but rather to a new stage of differentiation characterized by altered and increased secretory activity. In both organs E2 caused dramatic elevations in the E2-receptor content in the nuclear as well as in the cytoplasmic compartments. Also (in both organs) P treatment after E2 priming reduced the E2-receptor content in the nuclear and cytoplasmic compartments to the levels present in spayed cats. The degree of suppression caused by P in both organs was similar to that induced by E2 withdrawal. Suppression of the E2-receptor system by P correlated with suppressed function inthe oviduct but with increased growth and secretory activity in the uterus.

Dispersed monkey pituitary cells were cultured in serum-free medium and on extracellular matrix for 30 days. After 10-14 days with only insulin, transferrin, and selenium (ITS), the addition of estradiol (E) significantly increased PRL secretion compared to that in vehicle-treated controls. Simultaneous addition of E plus progesterone (P) increased PRL secretion similarly to E alone. PRL secretion in cultures treated with E plus P after 10 days induction by E was also similar to that in cultures maintained continuously in E. PRL secretion declined in wells switched from E to P and in wells switched from E back to vehicle, relative to that in wells maintained continuously in E. Estrogen receptors (ER) were detected in whole pituitary tissue and in serum-free pituitary cultures with a monoclonal antiestrogen receptor antibody (H222) in a sucrose density gradient shift assay. Immunocytochemical staining for ER with the same antibody also showed positive cell nuclei in serum-free pituitary cultures. In summary, ER are maintained in monkey pituitary cells during tissue culture, and PRL production can be further increased by E treatment after long term serum-free culture. Neither simultaneous nor sequential E plus P treatment alters PRL secretion compared to E alone.

We examined changes in estrogen receptors (ERs) in endometrial stromal and epithelial cells in cynomolgus macaques during artificially induced menstruation and repair. We used Silastic implants filled with either estradiol (E2) or progesterone (P) to treat spayed animals for 14 days with E2 followed by 14 days of E2 plus P. We then withdrew the P (but not the E2) implants and removed uteri 0, 0.5, 1, 2, 3, 4, 5, 7, and 14 days later. Uterine tissues were assayed biochemically for ER content, fixed for histology and frozen for immunocytochemistry of ER with monoclonal antiestrophilins. On day 0, ER levels in endometrium were low [1330 +/- 201 (n = 9) fmol/mg DNA]. An increase in total receptor was evident by 3-4 days of P withdrawal 2762 +/- 190 (n = 6) fmol/mg DNA; P less than 0.001]. Total receptor concentrations increased linearly with time from 0.5-7 days of P withdrawal (r = 0.88). On day 0, staining for nuclear ER in the glandular epithelium and stroma of zones I, II, and III of the endometrium was negative. Beginning at 12-24 h and continuing through 4 days of P withdrawal, nuclear staining became detectable and increased in intensity only in endometrial stromal fibroblasts and myometrial smooth muscle cells. The glandular epithelium of the endometrium did not develop nuclear staining until 5-7 days of P withdrawal, coincident with a 10-fold increase in the mitotic index of the epithelium of the upper zones. Thus, the increase in endometrial ER levels that occurred during the first 5 days of an induced luteal-follicular transition took place almost exclusively in stromal fibroblasts.

We used immunocytochemistry (ICC) with monoclonal antibodies to the estrogen receptor (ER) to localize ER in the oviducts, uteri, and cervix of untreated, estrogen-treated, and estrogen-progestin-treated spayed macaques. We also used binding assays with labeled estrogens to quantify nuclear and cytosolic ER levels in parallel samples of the same tissues. In untreated spayed animals, cytosolic ER levels were much higher than nuclear ER levels, but all specific staining was nuclear. After treatment for 14 days with estradiol (E2), the degree of staining for ER in cell nuclei in the oviduct, cervix, and endometrium had increased, and there were significant increases in both nuclear and cytosolic ER levels. In the myometrium, ER levels and ICC staining of nuclei increased minimally with E2 treatment. In animals treated for 2 weeks with E2 followed by 2 weeks with E2 and progesterone (P; sequential P treatment) the degree of nuclear ER staining in the oviduct, endometrium, and cervix greatly decreased, and cytosolic and nuclear levels of ER declined significantly. In the myometrium of such animals there was a minimal decrease in the degree of staining and a nonsignificant decline in cytosolic and nuclear ER levels. Sequential P treatment reduced the degree of nuclear staining in the oviduct and endometrium below that found in spayed animals; however, such treatment only lowered the amount of cytosolic, not nuclear, ER significantly below spayed levels in those same tissues. Some animals were treated sequentially with P and sampled 1, 3, 12, and 24 h after the onset of P treatment. By 1 h, nuclear ER levels in the endometrium were significantly suppressed, but cytosolic levels were not lowered until 3 h of treatment; ICC staining was also not substantially reduced until 3 h of P treatment. In the oviduct, nuclear ER levels were significantly reduced by 1 h of P treatment, but cytosolic levels were not lowered until after 12-24 h of P treatment; the degree of nuclear staining in the oviduct was also not substantially reduced until 12-24 h of P treatment. In myometrium, there was no significant decline in ER in nuclear or cytosolic fractions or any substantial decrease in the degree of nuclear staining at any time during this treatment. These observations suggest that the ER detected by ICC in the nuclei of target cells in frozen sections represents the total ER detectable by binding assays in cytosolic and nuclear fractions.(ABSTRACT TRUNCATED AT 400 WORDS)

We examined nuclear estrogen receptors (ER) and progestin receptors (PR) in the rhesus monkey prostate. Tissues were obtained from six intact males, five untreated castrates, six castrates treated with testosterone (T) for 6 weeks, and four castrates treated with estradiol (E2) for 6 weeks. Samples of the caudal lobe were either assayed biochemically for ER or stained immunocytochemically (ICC) with monoclonal antibodies against the ER or PR. Prostates from untreated castrates had significantly more ER than tissues from intact or T-treated castrates. In E2-treated castrates, ER number increased compared to that in intact and T-treated castrates. With ICC, ER was found only in the nuclei of the fibroblasts and smooth muscle cells of the stroma, not the glandular, ductal, or urethral epithelial cells. Intact and T-treated castrates had a very small number of positive cells, while untreated and E2-treated castrates had a significantly increased number of positive ER cells in the fibromuscular stroma. With ICC, PR was absent in intact or T-treated animals and barely evident in untreated castrates, but was significantly increased in the fibromuscular stroma of E2-treated castrates. The histological preparations indicated there was no stromal hypertrophy in the E2-treated castrates, but the E2 treatment did cause dilation of the glandular acini. Aromatase activity was measured in prostatic microsomes with a radiometric assay. Levels were low (3-30 fmol/h.mg protein) compared to those in brain and placenta, and no differences in activity were seen between castrates and T-treated castrates. Our data demonstrate that androgens can suppress the level of nuclear ER in the rhesus prostate, and that E2 treatment of castrates can induce PR in the same cells as those that contain ER. Thus, under appropriate conditions, estrogens could affect the rhesus prostate through a receptor-mediated pathway.