Sheri Hild‐Petito

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.

Changes in the organization and composition of apical cell surface glycoconjugates accompany the transition of luminal epithelial cells from the prereceptive state of the uterus in many species. In spite of the biological and clinical significance of this process, few molecular markers have arisen as useful predictors of uterine receptivity. Recent studies in mice demonstrate that the transmembrane mucin glycoprotein, Muc-1, is abundantly expressed at the apical surface of luminal epithelia under most conditions and is invariably reduced in receptive uteri. These and other observations have led to the suggestion that mucins serve an antiadhesive role and function to maintain a nonreceptive uterine state. A pan-species Muc-1 specific antibody recognizing a peptide motif conserved in the cytoplasmic domain of Muc-1 was used to examine the temporal and spatial expression of cell-associated Muc-1 in baboon uteri under a variety of conditions, including the pre- and perimplantation periods. Muc-1 expression was not driven by estrogen influences alone, but required progesterone action. In animals exposed to both steroids, Muc-1 was expressed at low moderate levels in epithelia of the basalis and functionalis regions. The highest expression of Muc-1 was detected in surface epithelium of the preimplantation phase, i.e., up to Day 8 (Day 0 = day of ovulation), or in ovariectomized animals receiving a steroid hormone regime that mimicked this phase (14 days of estrogen priming followed by 7 days of estrogen plus progesterone). Continued exposure to both hormones, i.e., as seen at Days 10-12 or in ovariectomized baboons given 14 days of estrogen plus progesterone treatment after estrogen priming, resulted in marked reduction of Muc-1 expression in the surface epithelium; however, staining patterns in the glandular epithelium were unchanged by this treatment. The expression of Muc-1 on the surface epithelium during the prereceptive phase was associated with the presence of both estrogen and progestin receptors in these epithelia. Muc-1 expression was reduced by neither antiestrogen treatment during the prereceptive stage nor antiprogestin treatment through to the receptive phase. Furthermore, persistent Muc-1 expression in the functionalis and basalis epithelium correlated with expression of progestin receptors. Thus, Muc-1 expression appeared to be progesterone-dependent rather than estrogen-dependent. It is concluded that Muc-1 expression in surface epithelium serves as a marker of the prereceptive phase in the baboon and that loss of Muc-1 from surface epithelium correlates with generation of a receptive uterine state.

Ovarian androgens may act locally to modulate follicular and luteal function in various species. This study examined the distribution of androgen receptors within the primate ovary throughout the menstrual cycle. Ovaries were collected from rhesus and cynomolgus monkeys during the early, mid-, and late (n = 3-5 per stage) follicular and luteal phases of the cycle. The tissues were processed for indirect immunocytochemical localization of androgen receptors with a specific monoclonal antibody against human androgen receptor (AN1-15). In addition, ovaries (n = 3) were collected from rhesus monkeys for biochemical detection of androgen receptor using 3H-androgen and AN1-15. Specific immunocytochemical staining, as determined by comparing adjacent tissue sections incubated with either AN1-15 or a nonspecific control antibody, was exclusively nuclear. Androgen receptor was detected in the germinal epithelium and ovarian stroma at all stages of the cycle. The thecal and granulosa cells of growing follicles, and of many but not all atretic follicles, contained androgen receptors. Luteinizing granulosa cells of the periovulatory follicle and luteal cells from the early and midluteal phase stained intensely for androgen receptor. Regressing corpora lutea of the late luteal phase also stained for androgen receptor; however, fully regressed corpora lutea in the early follicular phase of the next cycle did not exhibit receptor staining. Luteal cells that were androgen receptor-positive also stained histochemically for the presence of 3 beta-hydroxysteroid dehydrogenase. Sucrose gradient analysis with radiolabeled androgen demonstrated a shift in the androgen receptor peak in monkey ovarian tissue upon addition of AN1-15, confirming the presence of androgen receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Although estrogen and progesterone are essential for the establishment of pregnancy in primates, localization of their specific receptors in uterine cell types during pregnancy has not been investigated. Therefore, uteri were obtained from baboons during the menstrual cycle, after steroid treatment, or during early pregnancy on days 18, 25 and 32 postovulation. Uterine and placental tissues were also collected from baboons during late pregnancy. Tissues were processed for indirect immunocytochemical localization with specific monoclonal antibodies against estrogen receptor (ER; H222) and progestin receptor (PR; JZB39). Identification of specific cell types was confirmed by iron-hematoxylin/van Gieson and Gimori's stains. Specific staining for steroid receptors was detected only in the nucleus. In the absence of ovarian steroid hormones (ovariectomized baboons), ER were present in glandular epithelium, stroma, and myometrial smooth muscle cells (SMC). In contrast, PR were absent from all uterine cell types. In the estrogen-dominated (follicular and estradiol treatment) uterus, ER and PR were detected in the nuclei of glandular and surface epithelium, stroma, and myometrial SMC. Elevated progesterone levels (luteal or after progesterone treatment) resulted in a loss of nuclear ER in stroma and epithelium, except in the deep glandular epithelium in the basalis. PR was maintained in the stroma throughout the endometrium, but was detected only in the epithelium of the deep glands. The myometrial SMC contained both ER and PR. In early pregnancy, ER was absent from the glands and stroma as early as day 18 postovulation, but was present in the wall of spiral arteries, blood vessels, and myometrial SMC. On day 18 postovulation, staining for PR was absent from all glandular epithelium, but was maintained in the stroma surrounding the glands and spiral arteries, the wall of spiral arteries, blood vessels, and myometrial SMC. Stroma away from glandular epithelium contained few PR-positive cells. This staining pattern persisted throughout early pregnancy. No apparent differences in ER and PR localization were evident between the implantation and nonimplantation sites of the endometrium and myometrium. In late pregnancy, ER were only present in the SMC of the myometrium; however, PR were detected in stroma and myometrial SMC. The maternally derived decidua expressed PR, but not ER, in the majority of cells. In contrast, fetally derived tissues, placenta, and amnio-chorion, did not contain either ER or PR at any stage of pregnancy. Clearly, ER and PR persist in particular uterine cell compartments despite the continual high levels of progesterone in pregnancy and, thus, support a receptor-mediated mechanism for estrogen and progesterone regulation of implantation and pregnancy.

The objective of this study was to determine whether progestin-only contraceptives induce thinning of the vaginal epithelium in nonhuman primates. Eight intact rhesus monkeys (four per group) were treated with either a single intramuscular injection of 30 mg of Depo-Provera or a subcutaneous insertion of Norplant-II (2 x 75 mg rods; day 0). Norplant-II rods were removed 90 days after insertion. Vaginal biopsies were obtained during a pretreatment menstrual cycle and following treatment on days 10, 30, 60, 118, and 146. Formalin-fixed vaginal biopsies were evaluated for epithelial thickness and the degree of keratinization. The circulating levels of estradiol, progesterone, medroxyprogesterone acetate (MPA), or levonorgestrel (LNG) were monitored throughout the study by specific radioimmunoassays. Circulating levels of estradiol and progesterone confirmed the stage of the menstrual cycle in which pretreatment biopsies were obtained. Following treatment with Depo-Provera, serum levels of MPA increased to 2.3 +/- 0.6 ng/ml (x +/- SE, n = 4) within 24 hr. Serum levels of MPA were maximal on day 14 (5.5 +/- 0.9 ng/ml), dropped below 1 ng/ml by day 50, and were nondetectable by day 70. Circulating levels of LNG were elevated 24 hr after insertion of Norplant-II (5.8 +/- 3.0 ng/ml), peaked on day 2 (7.6 +/- 4.2 ng/ml), remained between 1.4 and 6.2 ng/ml from days 14 to 90, and were nondetectable by day 118, the first serum sample after removal of Norplant-II. There were no significant differences (p > 0.05) in the epithelial thickness (microm), number of epithelial cell layers, or type of epithelium present in vaginal biopsies obtained during the follicular or luteal phases of the pretreatment menstrual cycle. Conversely, a pronounced effect of progestin treatment was observed on the vaginal epithelium. There were no significant differences (p > 0.05) between the two progestin treatment groups, but a significant effect (p < 0.05) over time was observed (two-way ANOVA). Compared with pretreatment menstrual cycle controls, the vaginal epithelial thickness was decreased (p < 0.05) by day 30 or 60 following Norplant-II insertion or Depo-Provera injection, respectively. The number of epithelial cell layers was also decreased (p < 0.05) on days 30 and/or 60 in progestin-treated monkeys compared with pretreatment control cycles. Following removal of Norplant-II or metabolic excretion of MPA, the vaginal epithellium regenerated and the thickness was no longer different (p > 0.05) from the pretreatment control cycle. These data demonstrate that progestin-only contraceptives induced thinning of the vaginal epithelium in rhesus monkeys, and this effect was rapidly reversible following physical or metabolic removal of the progestin.