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Background: Clinicians routinely prescribe progestins along with estrogens during menopausal hormone therapy (HT) to block estrogen-dependent endometrial proliferation. Breakthrough bleeding (BTB) can negate the utility of this treatment. Because progestin antagonists also inhibit estrogen-dependent endometrial proliferation in women and macaques, we used a menopausal macaque model to determine whether a potent progestin antagonist (ZK 230 211, Schering AG; ZK) combined with estrogen would provide a novel mode of HT., Method: Ovariectomized rhesus macaques were treated for 5 months with either estradiol (E(2)) alone, E(2) + progesterone (two doses) or E(2) + ZK (0.01, 0.05 or 0.25 mg/kg)., Results: In the E(2) + progesterone groups, progesterone suppressed endometrial proliferation and induced a thick decidualized endometrium. In the E(2) + ZK 230 211 groups, all doses of ZK blocked endometrial proliferation and induced endometrial atrophy. In all ZK-treated groups, the atrophied endometrium contained some dilated glands lined by an inactive, flattened, non-mitotic epithelium. BTB was much lower in the E(2) + ZK groups (17 days of spotting, all groups) than in the E(2) and E(2) + progesterone groups (155 bleeding days, all groups). ZK suppressed E(2) effects in the cervix, but not in the vagina, oviduct or mammary glands. All serum chemistry and lipid profiles were normal., Conclusion: The ability of ZK to block estrogen-dependent endometrial proliferation, induce endometrial atrophy and suppress BTB in a menopausal macaque model indicates that progestin antagonists may provide a novel mode of HT.

The human endometrium displays characteristic features, both structural and functional, across the menstrual cycle. It is the sex steroid hormones, oestrogen and progesterone, that drive the endometrium through the different phases of the cycle. Oestrogen and progesterone act sequentially to regulate cellular concentrations of their respective receptors, this interaction initiates gene transcription. Thereafter a cascade of local events prepares the endometrium for implantation, but in the absence of pregnancy, progesterone withdrawal leads to menstruation and cyclic repair. Withdrawal of progesterone from an oestrogen-progesterone primed endometrium is the initiating event for the cascade of molecular and cellular interactions that result in menstruation. Progesterone withdrawal first affects cells with progesterone receptors. Early events in the menstrual process are vasoconstriction and cytokine up-regulation. The activation of lytic mechanisms is a later event and involves cells that may lack progesterone receptors, for example, uterine leucocytes and epithelial cells. Hence progesterone withdrawal results in a local increase of inflammatory mediators and the enzymes responsible for tissue breakdown. The total complex of local factors implicated in normal menstrual and aberrant menstrual bleeding are yet to be fully defined.

Two experiments were conducted to determine how RU486 affected oestradiol action in the non-human primate female reproductive tract. In the first experiment, spayed rhesus monkeys were first treated with a sequential regimen of oestradiol and progesterone to cause regression and deciliation in the oviduct and progestational development in the endometrium. The ability of oestradiol to stimulate oviductal differentiation and endometrial regeneration in the presence of RU486, progesterone, and progesterone plus RU486 was then tested. RU486 was found to block the ability of oestradiol to increase endometrial growth but did not prevent oestradiol-dependent oviductal differentiation. Also, in the endometrium but not the oviduct, RU486 action differed from simple progesterone withdrawal by causing stromal compaction and glandular apoptosis. In the second experiment, spayed monkeys were first treated with oestradiol for 2 weeks to produce a fully differentiated, ciliated-secretory oviduct and an hypertrophied, proliferative endometrium. The ability of oestradiol to maintain these conditions in oviduct and endometrium during treatment with RU486, progesterone, and progesterone plus RU486 was then tested. It was found that RU486 suppressed the ability of oestradiol to maintain the endometrium in a hypertrophied state but not its ability to maintain the oviduct in a fully ciliated-secretory state. As before, RU486 induced extensive glandular apoptosis and stromal compaction in the endometrium, but not the oviduct. These endometrial effects appeared to be due to a combination of a decrease in proliferation, an increase in epithelial cell death by apoptosis, an increase in stromal compaction and a concomitant decrease in interstitial fluid content, all of which led to a decrease in endometrial wet weight and thickness. These effects of RU486 on oestradiol action were similar in the presence and absence of progesterone and were therefore separate and distinct from the classical antiprogestin effects of RU486. Because RU486 did not inhibit the ability of oestradiol to either stimulate or maintain oviductal differentiation or wet weight, these results suggest that the endometrium is much more sensitive to the anti-proliferative effects of RU486 than the oviducts.

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.

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.

Proenkephalin mRNA has previously been shown to be expressed in the rodent uterus with varying levels during the estrous cycle. To examine for the potential regulation of proenkephalin gene expression by steroid hormones in a primate displaying a menstrual cycle and to define the functional tissue within the uterus expressing this transcript, we have used Northern blot analysis of extracted RNA from isolated uterine tissue subtypes from normal adult rhesus macaques obtained during the menstrual cycle and from ovariectomized females under different physiological steroid hormone treatments. A strong band of proenkephalin mRNA of 1.3 kilobases was detected almost exclusively in the proliferative endometrium from monkeys in the follicular phase of the cycle. No proenkephalin mRNA was detected in secretory endometrium obtained from monkeys in the luteal phase. When ovariectomized macaques were implanted with silastic capsules of 17 beta-estradiol, proenkephalin mRNA was detected in the endometrium but not the myometrium of the estradiol-treated animals. No proenkephalin mRNA was detected in ovariectomized control animals. Under these conditions, we were unable to detect proenkephalin mRNA in ovariectomized macaques implanted with separate silastic capsules of 17 beta-estradiol and progesterone or in decidual tissue from early or late pregnancy. These results suggest that in the primate uterus 1) proenkephalin mRNA is expressed primarily in the endometrium of the uterus, 2) expression of the proenkephalin gene is regulated by 17 beta-estradiol in the endometrium, and 3) this effect of estradiol is antagonized by progesterone.

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.

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 compared the morphology of ovaries from fetal rhesus monkeys at two different stages of gestation with the ability of ovarian tissue to synthesize androgens and estrogens in vitro. Ovaries collected between 80 and 104 days of gestation contained single-layered primordial follicles but no multilayered or antral follicles. Within these ovaries we found theca-like interstitial cells which contained abundant smooth endoplasmic reticulum and lipid droplets. These ovaries produced androstenedione and dehydropiandrosterone (but not estradiol) in vitro in the absence of exogenous substrates. Ovaries collected between Days 124 and 153 of gestation, however, contained numerous, well-developed multilayered and antral follicles. The ultrastructural characteristics of the thecal and granulosa cell layers were similar to those of adult ovarian follicles. These ovaries synthesized large amounts of androstenedione, dehydroepiandrosterone, and estradiol in vitro. Identity of the estradiol was confirmed by incubating homogenates of late gestation ovaries with [14C]progesterone, by isolation of estradiol and formation of its derivative, and by recrystallization to constant specific activity. In vitro cyclic AMP synthesis was stimulated by Pergonal (luteinizing hormone [LH] + follicle stimulating hormone [FSH] ) only in ovaries which contained multilayered and antral follicles, an indication that fetal ovaries contain gonadotropin receptors during late stages of development. These data indicate that fetal ovaries of rhesus monkeys attain the capacity for de novo estrogen biosynthesis and to respond to gonadotropins during late gestation, when multilayered and antral follicles have developed.

Basal body replication during estrogen-driven ciliogenesis in the rhesus monkey (Macaca mulatta) oviduct has been studied by stereomicroscopy, rotation photography, and serial section analysis. Two pathways for basal body production are described: acentriolar basal body formation (major pathway) where procentrioles are generated from a spherical aggregate of fibers; and centriolar basal body formation, where procentrioles are generated by the diplosomal centrioles. In both pathways, the first step in procentriole formation is the arrangement of a fibrous granule precursor into an annulus. A cartwheel structure, present within the lumen of the annulus, is composed of a central cylinder with a core, spoke components, and anchor filaments. Tubule formation consists of an initiation and a growth phase. The A tubule of each triplet set first forms within the wall material of the annulus in juxtaposition to a spoke of the cartwheel. After all nine A tubules are initiated, B and C tubules begin to form. The initiation of all three tubules occurs sequentially around the procentriole. Simultaneous with tubule initiation is a nonsequential growth of each tubule. The tubules lengthen and the procentriole is complete when it is about 200 mmicro long. The procentriole increases in length and diameter during its maturation into a basal body. The addition of a basal foot, nine alar sheets, and a rootlet completes the maturation process. Fibrous granules are also closely associated with the formation of these basal body accessory structures.