Chronic hyperandrogenemia and western-style diet beginning at puberty reduces fertility and increases metabolic dysfunction during pregnancy in young adult, female macaques.

STUDY QUESTION: What are the impacts of elevated testosterone (T) and an obesogenic western-style diet (WSD), either independently or together, on fertility and metabolic adaptations of pregnancy in primates? SUMMARY ANSWER: Testosterone increases the time to achieve pregnancy, while a WSD reduces overall fertility, and the combination of testosterone and WSD additionally impairs glucose tolerance and causes pregnancy loss. WHAT IS KNOWN ALREADY: Both hyperandrogenemia and obesity are hallmarks of polycystic ovary syndrome, which is a leading cause of infertility among women worldwide. Female macaques receiving T and WSD beginning at puberty show increased metabolic, ovarian and uterine dysfunction in the non-pregnant state by 3 years of treatment. STUDY DESIGN, SIZE, DURATION: The same cohort of female rhesus macaques continued treatments from the time of puberty (2.5 years) to 4 years, including this fertility trial. There were four groups (n = 9-10/group): controls (C), T-treated (T; average total serum level 1.35 ng/ml), WSD-treated, and combined T and WSD-treated (T + WSD) females. PARTICIPANTS/MATERIALS, SETTING, METHODS: Females, which were typically having menstrual cycles, were paired for 4 days with a proven male breeder following the late follicular rise in circulating estradiol (≥100 pg/ml). The presence of sperm in the reproductive tract was used to confirm mating. Animals went through up to three successive rounds of mating until they became pregnant, as confirmed by a rise in circulating mCG during the late luteal phase and ultrasound evidence of a gestational sac at Day 30 post-mating (GD30). Placental vascular parameters were also measured at GD30. Metabolic measurements consisted of fasting levels of blood glucose and insulin at approximately GD30, 60, 90 and 115, as well as an intravenous (iv) glucose tolerance test (GTT) at GD115. MAIN RESULTS AND THE ROLE OF CHANCE: While all animals in the C and T groups eventually became pregnant, T-treated females on average had a greater interval to achieve pregnancy (P < 0.05). However, only ~70% of animals in the WSD and T + WSD groups became pregnant (P < 0.004). One pregnancy in T + WSD group resulted in an anembryonic pregnancy which miscarried around GD60, while another T + WSD female conceived with a rare identical twin pregnancy which required cessation due to impending fetal loss at GD106. Thus, the number of viable fetuses was less in the T + WSD group, compared to C, T or WSD. Placental blood volume at GD30 was reduced in all treatments compared to the C group (P < 0.05). Maternal P4 levels were elevated in the WSD (P < 0.03) group and E2 levels were elevated in T + WSD animals (P < 0.05). An increase in serum A4 levels throughout gestation was observed in all groups (P < 0.03) except WSD (P = 0.3). All groups displayed increased insulin resistance with pregnancy, as measured from the ivGTT during pregnancy. However, only the T + WSD group had a significant increase in fasting glucose levels and glucose clearance during the GTT indicating a worsened glucose tolerance. WSD treatment decreased female fetuses third trimester weights, but there was an interaction between WSD and T to increase female fetal weight when normalized to maternal weight. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: The small number of pregnancies in the WSD and T + WSD groups hampers the ability to make definitive conclusions on effects during gestation. Also, the high fertility rate in the controls indicates the cohort was at their breeding prime age, which may impair the ability to observe subtle fertility defects. The low number of fetuses used for male and female analysis requires additional studies. WIDER IMPLICATIONS OF THE FINDINGS: The current findings strongly suggest that both hyperandrogenemia and obesity have detrimental effects on fertility and gestation in primates, which may be directly relevant to women with polycystic ovary syndrome. STUDY FUNDING/COMPETING INTEREST(S): All ONPRC Cores and Units were supported by NIH Grant P51 OD011092 awarded to ONPRC. Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) of the National Institutes of Health (NIH) under Award Number P50HD071836 (to R.L.S.). The authors have no competing conflict of interests to disclose.

Chronic combined hyperandrogenemia and western-style diet in young female rhesus macaques causes greater metabolic impairments compared to either treatment alone.

STUDY QUESTION: Does developmental exposure to the combination of hyperandrogenemia and western-style diet (WSD) worsen adult metabolic function compared to either treatment alone? SUMMARY ANSWER: Young female rhesus macaques treated for 3 years, beginning at menarche, with combined testosterone (T) and WSD have increased weight gain and insulin resistance compared to controls and animals treated with either T or WSD alone. WHAT IS KNOWN ALREADY: Hyperandrogenemia is a well-established component of polycystic ovary syndrome (PCOS) and can be observed in peripubertal girls, indicating a potential pubertal onset of the disease. Obesity is often associated with hyperandrogenemia in peripubertal girls, and overweight girls appear to be at higher risk for the development of PCOS later in life. STUDY DESIGN, SIZE, DURATION: Juvenile (2.5- year old) female rhesus macaques were divided into four groups (n = 10/group): control animals receiving cholesterol implants and a control diet with 15% of calories derived from fat (C), animals receiving T implants (mean serum levels: 1.35 ± 0.01 ng/ml) and a control diet (T), animals receiving a cholesterol implant and a WSD with 36% of calories derived from fat (WSD) and animals receiving a T implant and a WSD (T + WSD). Animals were maintained on the treatments for 36 months and were 5.5 years old at study completion. PARTICIPANTS/MATERIALS, SETTING, METHODS: Metabolic testing consisted of body measurements including weight, dual-energy X-ray absorptiometry scans, activity monitoring, and glucose tolerance testing at zero months and at least once every 12 months for the remainder of the study. Indirect calorimetry and serum hormone assays were performed following 36 months of treatment. MAIN RESULTS AND THE ROLE OF CHANCE: Body weight and fat mass gain were significantly increased in T + WSD at 24 and 36 months of treatment compared to the other three groups. Log transformed fasting insulin and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) were significantly increased in T + WSD animals at 3 years of treatment compared to all other groups. T-treatment caused a greater rate of decline in activity after 18 months, while food intake and metabolic rate were largely unaffected by treatments. LIMITATIONS REASONS FOR CAUTION: Variability was present in the metabolic parameters measured; however, this is similar to the heterogeneity observed in human populations. WIDER IMPLICATIONS OF THE FINDINGS: Chronic hyperandrogenemia beginning at puberty may exacerbate metabolic dysfunction in women consuming a WSD and account for the increased rates of obesity and insulin resistance observed in PCOS patients. Counseling of female patient populations with elevated androgens about the potential benefit of consuming a lower fat diet could improve long-term metabolic health outcomes. STUDY FUNDING/COMPETING INTEREST(S): Eunice Kennedy Shriver National Institute of Child Health & Human Development P50HD071836 and Oregon National Primate Center Grant P51 OD011092. The authors have no competing conflict of interests to disclose.

Localization and hormonal regulation of endometrial matrix metalloproteinase-26 in the rhesus macaque.

BACKGROUND: The current understanding of hormonal regulation of matrix metalloproteinase-26 (MMP-26) in the primate endometrium is incomplete. The goal of this work was to clarify estrogen and progesterone regulation of MMP-26 in the endometrium of ovariectomized, hormone-treated rhesus macaques. METHODS: Ovariectomized rhesus macaques (n= 66) were treated with estradiol (E(2)), E(2) plus progesterone, E(2) followed by progesterone alone or no hormone. Endometrium was collected from the hormone-treated animals during the early, mid- and late proliferative and secretory phases of the artificial menstrual cycle. MMP-26 expression was quantified by real-time PCR, and MMP-26 transcript and protein were localized by in situ hybridization and immunohistochemistry and correlated with estrogen receptor 1 and progesterone receptor (PGR). RESULTS: MMP-26 was localized to glandular epithelium and was undetectable in the endometrial stroma and vasculature. MMP-26 transcript levels were minimal in the hormone-deprived macaques and treatment with E(2) alone did not affect MMP-26 levels. Treatment with progesterone both in the presence and absence of E(2) stimulated MMP-26 expression in the early and mid-secretory phases (P < 0.001). MMP-26 expression preceded decidualization of endometrial stroma. MMP-26 levels then declined to baseline in the late secretory phase (P < 0.01) despite continued E(2) plus progesterone treatment. Loss of detectable MMP-26 expression in the late secretory phase was correlated with late secretory phase loss of glandular epithelial PGR. CONCLUSIONS: Endometrial MMP-26 expression is dependent on the presence of progesterone in the early secretory phase and then gradually becomes refractory to progesterone stimulation in the late secretory phase. In the macaque, MMP-26 is a marker of the pre-decidual, secretory endometrium. During the second half of the late secretory phase, and during decidualization, MMP-26 loses its response to progesterone concurrent with the loss of epithelial PGR. The decline in MMP-26 levels between the mid- and late secretory phases may play a role in the receptive window for embryo implantation.

Progesterone-dependent expression of keratinocyte growth factor mRNA in stromal cells of the primate endometrium: keratinocyte growth factor as a progestomedin.

In vitro studies have shown that keratinocyte growth factor (KGF, also known as FGF-7) is secreted by fibroblasts and is mitogenic specifically for epithelial cells. Therefore, KGF may be an important paracrine mediator of epithelial cell proliferation in vivo. Because stromal cells are thought to influence glandular proliferation in the primate endometrium, we investigated the hormonal regulation and cellular localization of KGF mRNA expression in the rhesus monkey uterus. Tissues were obtained both from naturally cycling monkeys in the follicular and luteal phases of the cycle, and from spayed monkeys that were either untreated or treated with estradiol (E2) alone, E2 followed by progesterone (P), E2 plus P, or E2 plus P plus an antiprogestin (RU 486). Northern blot analysis of total RNA with 32P-labeled probes revealed that the level of KGF mRNA in the endometrium was 70-100-fold greater in the luteal phase or after P treatment than in untreated, E2-treated, or follicular phase animals. Northern analysis also showed that KGF mRNA was present in the myometrium but was unaffected by hormonal state. RU 486 treatment prevented the P-induced elevation of endometrial KGF mRNA. P-dependent elevation of endometrial KGF expression was confirmed by measurement of KGF protein in tissue extracts using a two-site enzyme-linked immunosorbent assay. In situ hybridization with nonradioactive digoxigenin-labeled cDNA probes revealed that the KGF mRNA signal, which was present only in stromal and smooth muscle cells, was substantially increased by P primarily in the stromal cells located in the basalis region. Smooth muscle cells in the myometrium and the walls of the spiral arteries also expressed KGF mRNA, but the degree of this expression did not differ with hormonal state. P treatment led to increased proliferation in the glandular epithelium of the basalis region and to extensive growth of the spiral arteries. We conclude that the P-dependent increase in endometrial KGF resulted from a dual action of P: (a) a P-dependent induction of KGF expression in stromal cells, especially those in the basalis (zones III and IV), and (b) a P-dependent increase in the number of KGF-positive vascular smooth muscle cells caused by the proliferation of the spiral arteries. KGF is one of the first examples in primates of a P-induced, stromally derived growth factor that might function as a progestomedin.

Intravenous neutralization of vascular endothelial growth factor reduces vascular function/permeability of the ovary and prevents development of OHSS-like symptoms in rhesus monkeys.

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is a disorder associated with elevated serum VEGFA following chorionic gonadotropin (hCG) exposure in controlled ovarian stimulation (COS) cycles in women. In this study, we tested the effect of intravenous VEGFA neutralization on OHSS-like symptoms and vascular function in rhesus macaques during COS cycles. METHODS: Monkeys (n = 8) were treated with 3 COS protocols and assigned randomly to groups as follows: 1) COS alone (Control, n = 5); 2) COS + VEGF mAb Avastin 19 ± 5 h before hCG (Avastin pre-hCG; n = 6); 3) COS + Avastin 3-4 days post-hCG (Avastin post-hCG; n = 4); 4) COS + Simulated Early Pregnancy (SEP n = 3); or 5) COS + SEP + Avastin (SEP + Avastin n = 3). Follicles were aspirated 36 h post-hCG, fluid was collected from one follicle for analysis of steroid and vascular hormone content. Remaining follicles were aspirated, and luteinized granulosa cells (LGCs) cultured for 24 h. Ovarian/uterine vascular flow (VF) and blood volume (BV) were analyzed by contrast enhanced ultrasound (CEUS) before hCG bolus and 6-8 days post-hCG bolus/time of peak SEP response. Ovarian permeability to albumin was analyzed by Dynamic Contrast Enhanced-MRI (DCE-MRI) post-hCG. RESULTS: Abdominal fluid was present in 4/5 Control, 2/6 Avastin pre-hCG, and 3/4 Avastin post-hCG females. Neutralization of VEGFA before hCG reduced ovarian VF, BV, and permeability to albumin (P < 0.05), while only ovarian VF and permeability were reduced in Avastin-post hCG group (P < 0.05). There was no effect of Avastin on ovarian vascular function during COS + SEP. VEGF levels in follicular fluid were reduced 78-fold by Avastin pre-hCG, and LGCs exposed to Avastin in vivo also released 4-fold less VEGF into culture media (P < 0.05). Culture medium of LGCs exposed to VEGFA neutralization in vivo had lower levels of P4 and ANGPT1, and an increased ratio of ANGPT2/1 (P < 0.05). Uterine VF was reduced by SEP + Avastin in the basalis/junctional zone (P < 0.05). CONCLUSIONS: Avastin treatment before hCG prevents the development of symptoms associated with ovarian hyperstimulation syndrome. In vitro data suggest neutralization of VEGFA alters expression of other vascular factors typically induced by hCG in the luteinizing follicle. Neutralization of VEGFA action alters the vascular function of the basalis zone of the uterus during simulated early pregnancy, indicating a potential effect on embryo implantation.

Microwave stabilization enhances immunocytochemical detection of estrogen receptor in frozen sections of macaque oviduct.

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.

Estrogen action in the reproductive tract of rhesus monkeys during antiprogestin treatment.

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.

RU 486 action after estrogen priming in the endometrium and oviducts of rhesus monkeys (Macaca mulatta).

Recently, we reported that in rhesus monkeys, RU 486 treatment could inhibit the ability of estradiol (E2) to stimulate endometrial regeneration without inhibiting E2-dependent oviductal regeneration. In that work, RU 486 had been administered at the end of an artificial luteal phase when the oviducts were regressed, the endometrium was in a late secretory state, and estrogen and progestin receptors (ER and PR, respectively) were at minimal levels in both organs. In the current work, we administered RU 486 after 2 weeks of estrogen priming when the oviducts were fully differentiated, the endometrium was in a proliferative state, and ER and PR levels were maximal. Our goal was to determine whether the degree to which RU 486 inhibited E2 action in either organ varied depending on their initial state. Spayed rhesus monkeys were primed with E2 for 2 weeks and then treated in four different ways for an additional 2 weeks as follows: I) E2; II) E2 plus P; III) E2, P, and RU 486; and IV) E2 plus RU 486. Menstruation was not induced by any of the four treatments. In group I, continuous treatment with E2 maintained a typical proliferative endometrium with abundant Ki-67-positive cells, low levels of apoptosis, and elevated ER and PR; the oviducts were also maintained in a fully ciliated-secretory state. In group II, P induced a typical progestational secretory state in the endometrium, with few proliferating (Ki-67-positive) epithelial cells, undetectable apoptosis, and decreased ER and PR; as expected, the oviducts were fully regressed, with few Ki-67-positive or ciliated epithelial cells and low levels of ER and PR. In the endometria of monkeys treated with RU 486 and E2, either with (group III) or without (group IV) P, the effects of E2 on wet weight, thickness, and epithelial proliferation were more dramatically inhibited than in our previous study. However, the oviducts of the RU 486-treated animals had remained in a hypertrophied, fully ciliated-secretory state as in our previous study, with elevated ER and nuclear PR, although epithelial proliferation was suppressed. The degree to which RU 486 can inhibit estrogen-dependent growth in the endometrium can apparently be affected by the state of differentiation and/or steroid receptor level at the time the antiprogestin treatment is begun, but this effect is not evident in the oviduct, which shows only modest antiproliferative effects of the RU 486 treatment.

Radioligand binding assay of progesterone receptors in the primate corpus luteum after in vivo treatment with the 3 beta-hydroxysteroid dehydrogenase inhibitor, trilostane.

We and others have detected progesterone receptors (PR) in the primate (macaque and human) corpus luteum by immunocytochemistry. However, we have been unable to measure PR in the corpus luteum by conventional ligand binding assay, presumably because high endogenous concentrations of progesterone (P) in luteal tissue prevented specific binding of radiolabeled hormone to PR during the assays. To test this hypothesis, we treated monkeys with the 3 beta-hydroxysteroid dehydrogenase inhibitor, Trilostane, to reduce levels of endogenous P before conducting binding assays for PR in luteal tissue. To obtain adequate tissue for saturation analysis, rhesus monkeys (n = 6) were superovulated by treating them with hFSH beginning at menses (day 1) for 6 days, then with hFSH and hLH (days 6-9), followed by hCG (day 10). Trilostane (600 mg) was given 5 days after hCG treatment (day 15), and binding assays were conducted 18 h later. Trilostane significantly reduced mean (+/- SE) serum levels of P from 97.8 +/- 16 to 2.7 +/- 1.3 nmol/L within 18 h (P < 0.001). Strong nuclear staining of PR was detected by immunocytochemistry in both Trilostane-treated and control (not Trilostane-treated) tissue, but ligand binding was measurable only in Trilostane-treated monkeys. Scatchard transformations of saturation curves revealed high affinity binding of [3H] R5020 in luteal cytosol and nuclear extracts with an approximate dissociation constant (Kd) of 4.8 and 1.37 nmol/L, respectively. Also, the PR-specific monoclonal antibody, JZB-39, shifted a [3H]R5020-bound luteal macromolecule on sucrose gradients. Mean cytosolic and nuclear binding of [3H]R5020 were 0.31 +/- 0.09 and 0.06 +/- 0.02 fmol/micrograms DNA, respectively. Similar binding of [3H]R5020 was demonstrated in corpora lutea obtained during spontaneous menstrual cycles after Trilostane treatment (n = 3). These results show unequivocally that the PR in macaque luteal tissue can bind P with high affinity and suggest a receptor-mediated action of P in the primate corpus luteum.

Effects of keratinocyte growth factor in the endometrium of rhesus macaques during the luteal-follicular transition.

We previously reported that keratinocyte growth factor (KGF) is up-regulated by the action of progesterone (P) in the primate endometrium, and we suggested that this protein is a likely mediator of P-dependent stromal-epithelial paracrine interactions in this tissue. At the end of the menstrual cycle, P levels fall, and the abundance of endometrial KGF transcripts decreases approximately 9-fold. In macaques, withdrawal of P induces the luteal-follicular transition (LFT), marked by menstrual sloughing of the functionalis zone and apoptotic regression of the basalis zone. Because KGF levels fall so dramatically during the LFT, we hypothesized that replacement with exogenous KGF during the LFT would prevent some of the endometrial changes seen after P withdrawal. Here we describe two studies of the effects of exogenously administered KGF during the LFT in rhesus macaques. In one experiment we administered KGF systemically to ovariectomized, juvenile rhesus macaques during an LFT induced by hormonal manipulations. KGF had dramatic proliferative effects on the bladder and salivary glands, known targets of KGF, but did not affect cell proliferation in the endometrium or block menstrual sloughing and bleeding. However, KGF strongly inhibited apoptosis in the basalis zone, increased glandular sacculation and folding in this zone, and had a marked trophic effect on the spiral arteries. In the second experiment we installed oviductal catheters in ovariectomized adult rhesus macaques and infused KGF directly into the uterine lumen during a hormonally induced LFT. Again, arteriotrophic, antiapoptotic, and basalis gland sacculation effects were observed in the absence of any effect on cell proliferation. We concluded that although KGF is mitogenic for many epithelial cell types, it does not play this role in the primate endometrium. Its most important roles may be to stimulate spiral artery growth and inhibit glandular apoptosis during the nonfertile menstrual cycle. Because its expression rises coincident with the time of implantation and because spiral arteries are essential to successful establishment of pregnancy, the role of KGF in the fertile menstrual cycle deserves further study.

Estrogen receptor beta, but not estrogen receptor alpha, is present in the vascular endothelium of the human and nonhuman primate endometrium.

Estrogen action is dependent upon the presence of specific ligand-activated receptors in target tissues. The aim of the present experiments was to compare the spatial and temporal pattern of expression of estrogen receptor beta (ERbeta) with that of ERalpha in full thickness endometrial samples (from the superficial to the basal zone) obtained from both women and rhesus macaques. Immunohistochemical localization with specific antibodies revealed that ERalpha and ERbeta were both expressed in nuclei of the glands and stroma. Consistent with previous studies, expression of ERalpha declined in the glands and stroma of the functionalis during the secretory phase. The luminal epithelium also displayed positive immunoreactivity for ERbeta. Expression of ERbeta declined in glandular cell nuclei, but not stroma, within the functionalis during the late secretory phase. Levels of expression of ERalpha and ERbeta in all cellular compartments remained unchanged in the basalis. Both receptor subtypes were detected on Western blots using proteins extracted from uterine samples obtained throughout the menstrual cycle. There was a striking contrast between the pattern of expression of ERalpha and ERbeta in the vascular endothelium and the perivascular cells surrounding endometrial blood vessels; only ERbeta was present in the endothelial cell population, although both forms of ER were expressed in perivascular cells. We conclude that estrogen action(s) within the vascular endothelium in the endometrium may be mediated via direct binding to the ERbeta isoform and that these cells could therefore be a target for agonists or antagonists that selectively target the beta form of the ER.

Progesterone withdrawal up-regulates vascular endothelial growth factor receptor type 2 in the superficial zone stroma of the human and macaque endometrium: potential relevance to menstruation.

Several reports indicate that vascular endothelial growth factor (VEGF) expression is increased in endometrial glands and stroma during the menstrual phase in the human endometrium. Here we report that VEGF receptor type 2 (KDR), normally expressed only in the vascular endothelium, was dramatically up-regulated in the stromal cells of the superficial endometrial zones during the premenstrual phase in both human and macaque endometrium. This increase was detectable by Northern analysis, in situ hybridization, and immunocytochemistry and was cell specific, zone specific, cycle phase specific, and VEGF receptor type specific. That is, it only occurred during the premenstrual/menstrual phase, did not occur in glandular epithelium, endothelium, or stromal cells of the deepest endometrial zones, and was not observed for VEGF receptor type 1. The upregulation of stromal KDR was induced by progesterone (P) withdrawal in both women and macaques, and adding back P 24 h after P withdrawal in macaques blocked stromal, but not vascular, endothelial KDR expression. Promatrix metalloproteinase-1 (MMP-1) was coordinately up-regulated in the same stromal cell population by P withdrawal. Because of reports that VEGF can enhance MMP expression, we hypothesize that VEGF-KDR interactions may influence MMP expression in the superficial zones of the primate endometrium during the premenstrual phase, and that these interactions play a role in the induction of menstruation.

Progesterone antagonists increase androgen receptor expression in the rhesus macaque and human endometrium.

Antiprogestins (APs) inhibit estradiol (E(2))-stimulated endometrial growth in women and nonhuman primates, but the mechanism of this "antiestrogenic" action is unknown. Here, we report that APs up-regulate endometrial androgen receptor (AR) in both women and macaques, an effect that might play a role in the antiproliferative effects of APs on the primate endometrium. In addition, because there are discrepancies in the literature on the regulation and localization of AR in the primate endometrium, we used both in situ hybridization and immunocytochemistry to evaluate hormonal influences on endometrial AR in women and macaques. In ovariectomized macaques, the following treatments were given for 4 weeks each: E(2) alone, E(2) + progesterone (P), E(2) + mifepristone (RU 486), and E(2) + P + RU 486. In women, samples were obtained during the normal menstrual cycle and after treatment with either RU 486 for 30 days at 2 mg/day, or after a single oral administration of 200 mg RU 486 on cycle day LH + 2. In macaques, E(2) significantly increased AR expression above vehicle controls; E(2) + RU 486 increased binding further; E(2) + P decreased AR binding; and E(2) + P + RU 486 treatment caused an intermediate elevation in AR binding. In macaques treated with E(2) alone, stromal AR staining was predominant, and P treatment suppressed that staining. E(2) + RU 486 or E(2) + P + RU 486 treatment produced a striking up-regulation of glandular epithelial AR staining and enhanced the stromal AR signal. In situ hybridization analyses confirmed the immunocytochemistry data. Similar induction of glandular AR staining and enhanced stromal AR staining were obtained in macaques treated with ZK 137 316 and ZK 230 211. During the natural cycle in women, stromal AR staining predominated and was greater in the proliferative than the late secretory phase. RU 486 treatment of women up-regulated glandular epithelial AR staining after either daily treatment for 30 days with 2 mg/day or after a single oral dose of 200 mg. In summary, endometrial AR was highest in the stroma during the human proliferative phase (or during E(2) treatment in macaques) and lowest during the late secretory phase in women (or after E(2) + P treatment in macaques). In both species, RU 486 induced AR expression in the glands and enhanced AR expression in stromal cells. Because androgens can antagonize E(2) action, enhanced endometrial AR expression induced by APs could play a role in the antiproliferative, "antiestrogenic" effects of APs in primates.

Keratinocyte growth factor and its receptor in the rhesus macaque placenta during the course of gestation.

Keratinocyte growth factor (KGF) is synthesized and secreted exclusively by mesenchymal cells, and acts through its receptor (KGFR) to stimulate epithelial proliferation. In vivo, KGF and KGFR comprise a mesenchymal-epithelial cell paracrine system that can mediate epithelial cell mitosis. In preliminary work, we noted that KGF was expressed in the rhesus monkey placenta, and we now report on the expression of placental KGF and KGFR mRNAs during the course of gestation in this species. In-situ hybridization revealed that during early gestation, KGF mRNA was strongly expressed in placental mesenchymal cells. These cells, which were also immunoreactive for vimentin, were mainly located on the periphery of the mesenchymal cores of both anchoring and floating villi. KGFR mRNA was expressed in the adjacent trophoblastic epithelium, which was immunoreactive for cytokeratin. In-situ hybridization revealed that KGF mRNA expression was very high in the youngest placentae (34-days gestation) and decreased gradually to minimal levels by late gestation (157 days). Northern blot analysis indicated also that the KGF MRNA signal was strongest in early gestation samples and weakest by late gestation. Analysis for KGFR mRNA by a reverse transcriptase-polymerase chain reaction technique showed that KGFR mRNA expression could be detected at all stages. However, in-situ hybridization indicated that KGFR mRNA expression was highest in early gestation placentae and least in the oldest placentae. Autoradiographs of frozen sections of placenta that had been incubated with [125I]KGF to detect receptor binding showed that grain density over the trophoblast was highest in the youngest and least in the oldest placentae. PCNA and Ki-67 expression followed this same temporal trend. We conclude that the KGF/KGFR system may be important in proliferation of the placental trophoblast during early- to mid-pregnancy in rhesus monkeys.

Evidence for a potential role of neuropeptide Y in ovine corpus luteum function.

Neuropeptide Y (NPY) is a neurohormone that is typically associated with food intake, but it has also been reported to affect the production of progesterone from luteal tissue in vitro. However, NPY has not been previously immunolocalized in the ovine ovary or in the corpus luteum (CL) of any species, and the effects of this neurohormone on luteal function in vivo are not known. Thus, we performed fluorescent immunohistochemistry (IHC) to localize NPY in the ovine ovary and used avidin-biotin immunocytochemistry (ICC) to further define the intracellular localization within follicles and the CL. We then infused NPY directly into the arterial supply of the autotransplanted ovaries of sheep to determine the in vivo effect of exogenous NPY on ovarian blood flow and on the luteal secretion rate of progesterone and oxytocin. Immunohistochemistry revealed that the NPY antigen was localized to cells within the follicles and CL, in the nerve fibers of the ovarian stroma, and in the vessels of the ovarian hilus. In the follicle, the NPY antigen was localized to nerves and vessels within the theca interna layer, and strong staining was observed in the granulosal cells of antral follicles. In the CL, NPY was localized in large luteal cells and in the vascular pericytes and/or endothelial cells of blood vessels, found dispersed throughout the gland and within the luteal capsule. In vivo incremental infusions of NPY at 1, 10, 100, and 1,000 ng/min, each for a 30-min period, into the arterial supply of the transplanted ovary of sheep bearing a CL 11 d of age increased (P< or =0.05) ovarian blood flow. The intra-arterial infusions of NPY also increased (P< or =0.05) in a dose-dependent manner the secretion rate of oxytocin, which was positively correlated (P< or =0.05) with the observed increase in ovarian blood flow. The infusions of NPY had a minimal effect on the secretion rate of progesterone, and similar intra-arterial infusions of NPY into sheep with ovarian transplants bearing a CL over 30 d of age had no significant effect on ovarian blood flow or on the secretion rate of progesterone. These results suggest that NPY acts on the luteal vascular system and the large luteal cells to rapidly stimulate blood flow and the secretion of oxytocin, respectively, which collectively implies a putative role for NPY during the process of luteolysis when increasing amounts of oxytocin are secreted from the ovine CL in response to uterine pulses of prostaglandin F2alpha.

Assessment of progestin-only therapy for endometriosis in macaque.

BACKGROUND: Endometriosis is a condition where endometrium-like tissue forms lesions at ectopic sites outside the uterus. In women, oral contraceptive pills and progestins are often prescribed as therapy for early stage endometriosis. In contrast, in macaques the disease is frequently advanced at the time of diagnosis and ovariectomy is the standard therapy. However, surgery is contraindicated in many patients. A review of 15 endometriosis cases over the past 10 years at the Oregon National Primate Research Center (ONPRC) revealed that 5 failed to show improvement after ovariectomy and were subsequently euthanized. Therefore, our goal was to assess the feasibility of treating endometriosis in macaques with chronic progesterone (P) as an alternative therapy for the disease. METHODS: Seven adult rhesus macaques with advanced endometriosis were identified by clinical symptoms and endometriosis was confirmed by abdominal palpation, ultrasound examination, and/or aspiration of menstrual blood from abdominal cysts. The patients were chronically treated with Silastic capsules that released 5-7 ng P /ml in blood for up to 20 months. During treatment the patients were assessed daily and scored numerically for appetite, activity, attitude, abdominal discomfort and menstruation by the Clinical Veterinary staff. The patients were then re-examined by abdominal palpation and ultrasound for the disease at the end of treatment. RESULTS: During the first 2 weeks of treatment, endometriotic symptoms improved significantly in all the patients (P < 0.05). This was associated with a significant increase in body weight and significant reduction in abdominal discomfort and menstrual bleeding. Two of the patients gradually developed increased symptoms of the disease after 5 months of treatment. Post-treatment abdominal examination revealed that 2/5 patients continued to have an abdominal mass even though symptoms were suppressed. CONCLUSIONS: We conclude that continuous P treatment of rhesus monkeys provides therapeutic benefit to reduce symptoms of endometriosis and may provide an option for cases where ovariectomy is contraindicated.

Progesterone withdrawal up-regulates fibronectin and integrins during menstruation and repair in the rhesus macaque endometrium.

BACKGROUND: Fibronectin (FN) is a component of the extracellular matrix that participates in wound healing in various tissues as an adhesive ligand for integrins (Itgs). To determine whether these molecules play similar roles during menstrual repair, we evaluated the expression and localization of FN and specific Itgs in the primate endometrium under hormonally controlled conditions. METHODS: Ovariectomized rhesus macaques were treated for 2 weeks with estradiol (E(2)) followed by E(2) with progesterone for 2 weeks. On day 28, progesterone was withdrawn and uteri were collected during menstruation, postmenstrual repair, and the proliferative and secretory phases. Analysis was by focused microarray, real time PCR, in situ hybridization and immunocytochemistry. RESULTS: Progesterone withdrawal induced significant elevations of FN, Itg alpha5 and Itg beta1 transcripts during menstruation as compared to day 28 (FN: P < 0.01; Itg alpha5: P < 0.05; Itg beta1: P < 0.05; real time PCR). These increases were concentrated in the glandular epithelium (FN) and stroma (Itg alpha5beta1) of the uppermost zones. Cyclic changes in Itg alpha3 occurred in the glandular epithelium. CONCLUSIONS: Spatially and temporally restricted peaks of expression of FN and its Itg receptors are closely correllated with menstruation and postmenstrual repair in the primate endometrium.

Chronic progesterone antagonist-estradiol therapy suppresses breakthrough bleeding and endometrial proliferation in a menopausal macaque model.

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.

Oestrogen action in the endometrium and oviduct of rhesus monkeys during RU486 treatment.

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.