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Continual administration of low doses of the antiprogestin ZK 137 316 was previously reported to permit ovarian/menstrual cyclicity, but disrupt endometrial growth in macaques. The contraceptive efficacy of this regimen was tested in female rhesus monkeys (10 per group) treated daily with vehicle (controls), 0.01 or 0.03 mg ZK 137 316 per kg body weight for 30 days before and during continual co-habitation with males of proven fertility. Treatment continued until confirmation of pregnancy or for 5 months after pair-housing with males. Mating and vaginal sperm were evident in all females. A cumulative pregnancy rate of 90% (9/10) was observed in the controls. Of the 10 animals receiving 0.01 mg/kg, four conceived during the first 2 months of pairing (P = 0.06) with no further conceptions. No pregnancies were observed in the 0.03 mg/kg group (P < 0.01). Timely, overt menses occurred at a higher frequency in the 0.01 mg/kg group than the 0.03 mg/kg group. However, corpora lutea were present in ovaries from both groups during the last treatment cycle, indicating that ovarian cycles occurred. Thus, chronic administration of low-dose ZK 137 316 that permits continued ovarian cyclicity and a high incidence of timely menses, prevents pregnancy in non-human primates. This regimen may provide a novel method of contraception for women. [ABSTRACT FROM AUTHOR]

Large doses of antiprogestin typically disrupt menstrual cyclicity. A chronic low-dose regimen of the potent new antiprogestin ZK 137 316, which permits continued menstrual cyclicity but alters gonadal-reproductive tract activity, was established. Rhesus monkeys received a vehicle (n = 6) or 0.01 (n = 8), 0.03 (n = 8) or 0.1 (n = 5) mg ZK 137 316/kg body weight daily for five menstrual cycles (C-1 to C-5). Oestradiol, progesterone and gonadotrophin profiles were normal during cycles involving vehicle and 0.01 and 0.03 mg ZK 137 316/kg body weight. In the 0.1 mg/kg group, mid-cycle oestradiol and gonadotrophin surges, and subsequent progesterone production, were absent in C-3 and C-5. Ovarian cyclicity was accompanied by timely menstruation in the vehicle and 0.01 mg/kg groups. By C-3, half the animals in the 0.03 mg/kg group and all animals in the 0.1 mg/kg group were amenorrhoeic. A corpus luteum was noted during the mid-luteal phase of C-5 in the vehicle, 0.01 mg/kg and 0.03 mg/kg groups. Large antral and cystic follicles were evident in the 0.1 mg/kg group. Thus, a daily treatment with 0.01 mg/kg ZK 136 317 permitted normal menstrual cyclicity in macaques. While the daily administration of 0.03 mg/kg ZK 136 317 allowed ovarian cyclicity, menstruation was disrupted in some animals. Increasing the dose to 0.1 mg./kg antagonized pituitary function and resulted in anovulation and amenorrhoea. A chronic low-dose regimen of the antiprogestin ZK 137 316, which permits normal ovarian/menstrual cyclicity, has potential as a contraceptive in women. [ABSTRACT FROM PUBLISHER]

The long-term effects of the antiprogestin ZK 137 316 on reproductive tract morphology in rhesus macaques were investigated. The monkeys were injected daily (i.m.) for five menstrual cycles with vehicle or 0.01, 0.03 or 0.1 mg ZK 137 316/kg body weight. Reproductive tracts (n = 3/group) were collected during the mid-luteal phase (day 8) of the fifth cycle in the control, 0.01 and 0.03 mg/kg groups, or 6-7 days after the oestradiol peak in the 0.1 mg/kg group. ZK 137 316 treatment resulted in a dose-dependent atrophy of the endometrium, marked by a reduced mitotic activity in the glands, compaction of the stroma, degradation of spiral arteries and dilation of veins. There was no effect of ZK 137 316 on myometrial or oviductal weight. Treatment with 0.1 and 0.03 mg/kg, but not 0.01 mg/kg resulted in fully ciliated and secretory oviducts, indicating a dose-dependent blockade of progesterone antagonism of oestrogen-dependent oviductal differentiation. In the endometrium, the suppressive action of progesterone on oestrogen and progestin receptors was also blocked by ZK 137 316 in a dose-dependent manner. However, endometrial atrophy appeared due to inhibition of progesterone action together with a blockade of oestrogen-dependent proliferation. The profoundly suppressed endometrium produced by chronic low-dose ZK 137 316 treatment is unlikely to support implantation. Such treatment may therefore provide a novel contraceptive modality. [ABSTRACT FROM PUBLISHER]

AKI after cardiac surgery remains strongly associated with mortality and lacks effective treatment or prevention. Preclinical studies suggest that cell-based interventions may influence functional recovery. We conducted a phase 2, randomized, double-blind, placebo-controlled trial in 27 centers across North America to determine the safety and efficacy of allogeneic human mesenchymal stem cells (MSCs) in reducing the time to recovery from AKI after cardiac surgery. We randomized 156 adult subjects undergoing cardiac surgery with evidence of early AKI to receive intra-aortic MSCs (AC607; n =67) or placebo ( n =68). The primary outcome was the time to recovery of kidney function defined as return of postintervention creatinine level to baseline. The median time to recovery of kidney function was 15 days with AC607 and 12 days with placebo (25th, 75th percentile range, 10-29 versus 6-21, respectively; hazard ratio, 0.81; 95% confidence interval, 0.53 to 1.24; P =0.32). We did not detect a significant difference between groups in 30-day all-cause mortality (16.7% with AC607; 11.8% with placebo) or dialysis (10.6% with AC607; 7.4% with placebo). At follow-up, 12 patients who received AC607 and six patients who received placebo had died. Rates of other adverse events did not differ between groups. In these patients with AKI after cardiac surgery, administration of allogeneic MSCs did not decrease the time to recovery of kidney function. Our results contrast with those in preclinical studies and provide important information regarding the potential effects of MSCs in this setting., (Copyright © 2018 by the American Society of Nephrology.)

Background: Data on differences in toxicity and efficacy of chemotherapy and radiotherapy among different ethnic groups is limited. We evaluated differences in toxicity, tolerability and clinical outcome of Ashkenazi and non-Ashkenazi Jews receiving postoperative chemoradiation for locally advanced gastric cancer (LAGC)., Patients and Methods: Between 6/2000-12/2007, 84 Ashkenazi patients and 60 non-Ashkenazi patients underwent chemoradiation following resection of LAGC (INT-116 trial)., Results: Patients' and tumor characteristics were comparable. Ashkenazi patients experienced significantly higher rates of fatigue, anorexia, and grade 3-4 dysphagia, as well as a trend for a higher rate of diarrhea. The incidence of other toxicities, dose adjustments of chemotherapy and radiotherapy and patient prognosis did not differ., Conclusion: This study shows higher rates of various toxicities among Ashkenazi patients receiving postoperative chemoradiation for LAGC compared to non-Ashkenazi patients. To our knowledge, this is the first study comparing treatment toxicity, tolerability and outcome between these two groups.

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.

Background: In a porcine model, we investigated the impact of sudden stent graft occlusion of thoracic intercostal arteries after open lumbar segmental artery (SA) ligation., Methods: After randomization into two groups, 20 juvenile Yorkshire pigs (27.1±0.6 kg) underwent open lumbar SA sacrifice (T13-L5) followed by endovascular coverage of all thoracic SAs (T4-T12) at 32°C, either in a single operation (group 1) or in two stages separated by seven days (group 2). Collateral network pressure (CNP) was monitored by catheterization of the SA L1, and postoperative hind limb function was assessed using a modified Tarlov score., Results: The CNP in group 1 decreased to 34% of baseline, whereas CNP after lumbar SA ligation in group 2 fell to 55% of baseline (74±2.4 to 25±3.6 mm Hg vs 74±4.5 to 41±5.5 mm Hg; p<0.0001). Subsequent thoracic stenting (group 2) led to another significant but milder drop (p=0.002 versus stage 1) from the restored CNP (71±4.2 to 54±4.9 mm Hg). Five of ten pigs in group 1 suffered paraplegia, in contrast to none in group 2 (median Tarlov score 6, vs 9; p=0.0031). Histopathologic analysis showed more severe ischemic damage to the lower thoracic (p=0.05) and lumbar spinal cord (p=0.002) in group 1., Conclusions: These results underline the potential of the staged approach in hybrid procedures. Furthermore they highlight the need for established adjuncts for preventing paraplegia in hybrid and pure stent-graft protocols in which sudden occlusion of multiple SAs occurs., (Copyright © 2011 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Objective: Prevention of paraplegia after repair of thoracoabdominal aortic aneurysm requires understanding the anatomy and physiology of the spinal cord blood supply. Recent laboratory studies and clinical observations suggest that a robust collateral network must exist to explain preservation of spinal cord perfusion when segmental vessels are interrupted. An anatomic study was undertaken., Methods: Twelve juvenile Yorkshire pigs underwent aortic cannulation and infusion of a low-viscosity acrylic resin at physiologic pressures. After curing of the resin and digestion of all organic tissue, the anatomy of the blood supply to the spinal cord was studied grossly and with light and electron microscopy., Results: All vascular structures at least 8 μm in diameter were preserved. Thoracic and lumbar segmental arteries give rise not only to the anterior spinal artery but to an extensive paraspinous network feeding the erector spinae, iliopsoas, and associated muscles. The anterior spinal artery, mean diameter 134 ± 20 μm, is connected at multiple points to repetitive circular epidural arteries with mean diameters of 150 ± 26 μm. The capacity of the paraspinous muscular network is 25-fold the capacity of the circular epidural arterial network and anterior spinal artery combined. Extensive arterial collateralization is apparent between the intraspinal and paraspinous networks, and within each network. Only 75% of all segmental arteries provide direct anterior spinal artery-supplying branches., Conclusions: The anterior spinal artery is only one component of an extensive paraspinous and intraspinal collateral vascular network. This network provides an anatomic explanation of the physiological resiliency of spinal cord perfusion when segmental arteries are sacrificed during thoracoabdominal aortic aneurysm repair., (Copyright © 2011 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

Objective: A comprehensive strategy to prevent paraplegia after open surgical or endovascular repair of thoracoabdominal aortic aneurysms requires a thorough understanding of the response of the collateral network to extensive segmental artery sacrifice., Methods: Ten Yorkshire pigs underwent perfusion with a low-viscosity acrylic resin. With the use of cardiopulmonary bypass, 2 animals each were perfused in the native state and immediately, 6 hours, 24 hours, and 5 days after sacrifice of all segmental arteries (T4-L5). After digestion of surrounding tissue, the vascular cast of the collateral network underwent analysis of arterial and arteriolar diameters and the density and spatial orientation of the vasculature using light and scanning electron microscopy., Results: Within 24 hours, the diameter of the anterior spinal artery had increased significantly, and within 5 days the anterior spinal artery and the epidural arterial network had enlarged in diameter by 80% to 100% (P < .0001). By 5 days, the density of the intramuscular paraspinous vessels had increased (P < .0001), a shift of size distribution from small to larger arterioles was seen (P = .0002), and a significant realignment of arterioles parallel to the spinal cord had occurred (P = .0005)., Conclusions: Within 5 days after segmental artery occlusion, profound anatomic alterations in the intraspinal and paraspinous arteries and arterioles occurred, providing the anatomic substrate for preservation of spinal cord blood flow via collateral pathways., (Copyright © 2011 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

Objective: We describe the long-term results of aortic arch replacement using a trifurcated graft, including an assessment of survival, neurologic complications, and graft patency., Methods: A retrospective review was conducted on data from 206 consecutive patients (125 male; median age, 67 years; range, 20-87 years) who had a trifurcated graft used for aortic arch replacement between September 1999 and September 2009. Seventy-four patients (35.9%) had chronic dissection, 68 patients (33.0%) had atherosclerotic aneurysms, and 39 patients (18.9%) had degenerative disease. Ninety-one patients (44.2%) had undergone previous cardiac surgery., Results: An elephant trunk was placed in 190 patients (92.2%) and completed in 101 patients (53.1%), with an interval of less than 365 days between stages in 94 of 101 patients. Hospital mortality was 6.8% (14/206). Adverse outcome (death/stroke within the first year postoperatively) occurred in 27.7% of patients (57/206; 50 deaths/7 strokes). Among 152 1-year survivors, the annual rates of transient ischemic attack and stroke were 0.85% and 1.1%, respectively. At 6 years, 75% of patients were still alive, compared with 92% in a matched New York State control population (P < .001). Follow-up computed tomography scans (189 studies in 176/206 patients [85.4%]) revealed 100% patency of the trifurcated graft limbs at a mean of 2.3 years., Conclusions: Aortic arch replacement using a trifurcated graft is highly durable, with excellent patency in the branch grafts, and is associated with a low incidence of cerebral embolization. However, the long-term outcome in these patients is compromised by extensive comorbidities., (Copyright © 2010 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

Background: Endovascular repair of descending thoracic and thoracoabdominal aortic aneurysms is an appealing alternative to the standard surgical approach, but precludes revascularization of segmental arteries (SAs). For safer surgical and endovascular repairs, an accurate prediction of the risk of paraplegia in relation to the extent of SA sacrifice is needed., Methods: From January 1994 to October 2008, 609 patients (mean age, 63 ± 14 years) underwent surgical descending thoracic or thoracoabdominal aortic aneurysm repair without SA reimplantation. Three hundred seventy-six patients (62%) were male; 159 (26%) had urgent or emergent operation; 199 (33%) had previous aortic surgery. Somatosensory- or motor-evoked potential monitoring and cerebrospinal fluid drainage were routinely performed., Results: Hospital mortality was 10.7% (65 patients). Spinal cord injury (SCI) occurred in 3.4% (21 patients). The extent of resection-expressed as the number of SAs sacrificed (p = 0.007)-and the need for visceral artery reimplantation (p = 0.03) were independent risk factors for paraplegia. Further analysis identified four risk groups (p < 0.0001): fewer than 8 SAs sacrificed (group A, SCI = 1.2%); sacrifice of 8 to 12 SAs with proximal origin in the upper thorax (group B, SCI = 3.7%); 8 to 12 SAs sacrificed beginning in the lower thorax (group C, SCI = 15.4%); and 13 or more SAs sacrificed (group D, SCI = 12.5%). This four-group model more accurately predicts SCI risk than the Crawford classification (goodness of fit c statistic: 0.748 versus 0.640)., Conclusions: The extent of SA sacrifice is the most powerful predictor of paraplegia risk. For aneurysms of moderate extent, a more distal location involving the abdominal aorta increases the risk of spinal cord injury. Sacrifice of fewer than 8 SAs is associated with a very low paraplegia risk regardless of location., (Copyright © 2010 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: In a pig model, we compared spinal cord injury after extensive segmental artery (SA) sacrifice in a single stage with recovery after a two-stage procedure: lumbar artery followed by thoracic SA sacrifice., Methods: Twenty juvenile Yorkshire pigs were randomly assigned to undergo extensive SA sacrifice at 32 degrees C in a single operation (group 1, n = 10), or thoracic SA ligation 7 days after lumbar artery sacrifice (group 2, n = 10). Spinal cord perfusion pressure (SCPP) was monitored using a catheter placed in the distal stump of L1. Hind limb function was evaluated intraoperatively using motor-evoked potentials and for 5 days postoperatively using a modified Tarlov score., Results: Motor-evoked potentials were intact in all pigs until 1 hour after surgery. All pigs in group 2 fully recovered hind limb function, whereas 40% in group 1 experienced paraplegia (median Tarlov scores 9 versus 7; p = 0.004). Group 1 SCPP fell to 28 +/- 6 mm Hg after SA sacrifice, compared with 44 +/- 8 mm Hg in group 2 (p < 0.0001). After sacrifice of all residual SAs, SCPP in group 2 remained consistently greater than 85% of baseline, significantly higher than group 1 SCPP from end clamping until 72 hours (p = 0.0002). Histopathologic analysis showed more severe ischemic damage to the lower thoracic (p < 0.001) and lumbar spinal cord (p = 0.01) in group 1., Conclusions: In contrast with the single-stage approach, a two-stage procedure, starting with ligation of six or fewer lumbar SAs, leads to only a mild drop in SCPP and stimulates vascular remodeling, minimizing the impact of subsequent SA sacrifice on spinal cord function. The greater safety of extensive SA sacrifice when undertaken in two stages has important implications for endovascular and hybrid aneurysm repair., (2010 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: The optimal treatment of chronic distal aortic dissection remains controversial, with endovascular stent-graft techniques challenging traditional surgery., Methods: From January 1994 to April 2007, 104 patients (82 male, median age 60.5 years) with chronic distal aortic dissection underwent surgical repair, 0 to 21 years after initial diagnosis of acute type A or B dissection (median 2.1 years). Twenty-three (22%) patients underwent urgent-emergent surgery. Mean aortic diameter was 6.9 +/- 1.4 cm. Indications for surgery, other than aortic expansion, were pain in 6 (6%) patients, malperfusion in 6 (6%), and rupture in 11 (11%). Forty-nine (47%) had previous cardioaortic surgery (29% dissection-related), 21 (20%) had coronary artery disease, 12 (12%) had Marfan syndrome, and 4 (4%) were on chronic dialysis. Twenty-six (25%) had a thrombosed false lumen. Thirty (29%) patients required reimplantation of visceral arteries; 8.3 +/- 2.7 segmental artery pairs were sacrificed., Results: Hospital mortality was 9.6% (10 patients). Paraplegia occurred in 5 (4.8%). Twenty-seven patients (26%) experienced adverse outcome (death within one year, paraplegia, stroke, or dialysis). Adverse outcome was associated with atheroma (p = 0.04, odds ratio = 4.3). Survival was 78% at 1, 68% at 5, and 59% at 10 years (average follow-up, 7.7 +/- 4.1 years). Freedom from distal aortic reoperation was 99% at 1, 93% at 5, and 83% at 10 years. After one year, patients enjoyed longevity equivalent to a normal age-sex matched population (standardized mortality ratio = 1.38, p = 0.23). By multivariate analysis, atheroma (p = 0.0005, relative risk = 9.32) and age (p = 0.0003, relative risk = 1.15/year) were risk factors for long-term survival., Conclusions: The efficacy of open repair for distal chronic dissection is highlighted by normal survival after the first year, and a low reoperation-reintervention rate., (Copyright (c) 2010 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: The levonorgestrel-releasing intrauterine system (LNG-IUS) is a highly effective contraceptive. However, during early months of use unscheduled vaginal bleeding is common, sometimes leading to discontinuation. This study aimed to determine whether intermittent administration of progesterone receptor modulator CDB-2914 would suppress unscheduled bleeding during the first 4 months after insertion of the LNG-IUS., Methods: CDB-2914 150 mg, in divided doses, or placebo tablets, were administered over three consecutive days starting on Days 21, 49 and 77 after LNG-IUS insertion, in a double-blind randomized controlled trial of women aged 19-49 years, newly starting use of LNG-IUS. Daily bleeding diaries were completed for 6 months, and summarized across blocks as percentage days bleeding/spotting (BS%)., Results: Of 69 women randomized to receive CDB-2914, and 67 placebo, 61 and 55, respectively, completed the trial. BS% decreased with time in both arms, but showed a much steeper treatment-phase gradient in the placebo arm (P < 0.0001), so that a benefit of CDB-2914 in the 28 days after first treatment (-11% points, 95% CI -19 to -2), converted to a disadvantage by 64 days after the third treatment (+10% points, 95% CI 1-18)., Conclusions: The effect of CDB-2914 on BS% was initially beneficial but then by third treatment was disadvantageous. Nevertheless, only 3% (4/136) of all women discontinued LNG-IUS. These findings give insight into possible mechanisms and suggest future research directions. ISRCTN Trial no. ISRCTN58283041; EudraCT no. 2006-006511-72.

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.

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.

Macaques are menstruating nonhuman primates that provide important animal models for studies of hormonal regulation in the uterus. In women and macaques the decline of progesterone (P) at the end of the cycle triggers endometrial expression of a variety of matrix metalloproteinase (MMP) enzymes that participate in tissue breakdown and menstrual sloughing. To determine the minimal duration of P withdrawal required to induce menses, we assessed the effects of adding P back at various time points after P withdrawal on both frank bleeding patterns and endometrial MMP expression. Artificial menstrual cycles were induced by treating the animals sequentially with implants releasing estradiol (E2) and progesterone (P). To assess bleeding patterns, P implants were removed at the end of a cycle and then added back at 12, 24, 30, 36, 40, 48, 60, or 72 hours (h) after the initial P withdrawal. Observational analysis of frank bleeding patterns showed that P replacement at 12 and 24 h blocked menses, replacement at 36 h reduced menses but replacement after 36 h failed to block menses. These data indicate that in macaques, a critical period of P withdrawal exists and lasts approximately 36 h. In other similarly cycled animals, we withdrew P and then added P back either during (12-24 h) or after (48 h) the critical period, removed the uterus 24 h after P add back and evaluated endometrial MMP expression. Immunocytochemistry showed that replacement of P during the critical period suppressed MMP-1, -2 and -3 expression along with menses, but replacement of P at 48 h, which failed to suppress mense, suppressed MMP-1 and MMP-3 but did not block MMP-2. We concluded that upregulation of MMPs is essential to menses induction, but that after the critical period, menses will occur even if some MMPs are experimentally blocked.

This review focuses on the complex events that occur in the endometrium after progesterone is withdrawn (or blocked) and menstrual bleeding ensues. A detailed understanding of these local mechanisms will enhance our knowledge of disturbed endometrial/uterine function--including problems with excessively heavy menstrual bleeding, endometriosis and breakthrough bleeding with progestin only contraception. The development of novel strategies to manage these clinically significant problems depends on such new understanding as does the development of new contraceptives which avoid the endometrial side effect of breakthrough bleeding.

Angiopoietin-1 (Ang-1) is an important angiogenic factor that has not been thoroughly studied in the primate endometrium. We evaluated the endometrial expression of Ang-1 and its receptor, Tie2, during induced menstrual cycles in rhesus macaques. Tie2 expression was confined to the vascular endothelium without marked change during the cycle. However, Ang-1 expression varied considerably during the cycle. In the proliferative phase, Ang-1 was only expressed in the basal zone glands, and this expression was estradiol (E2) dependent. In the early- to mid-secretory phase, Ang-1 expression spread to the upper glands, luminal epithelium and the vascular smooth muscle cells (VSMC) of spiral arteries. In the late secretory phase, the signal disappeared from the glands but remained elevated in the VSMC of spiral arteries. Notably, there was a significant correlation between VSMC proliferation and Ang-1 expression in the VSMC of the spiral arteries. Progesterone (P) withdrawal in the early secretory phase induced a decline in Ang-1 expression in the glands and VSMC of spiral arteries along with a complete suppression of VSMC proliferation. These data suggest, for the first time, that Ang-1 may play a key role in the P-dependent growth of the unique spiral arteries in the primate endometrium.

This article reviews the effects of estradiol (E(2)), progesterone (P) and P receptor antagonists (PA) on the rhesus macaque endometrium. Ovariectomized macaques can be treated with implants of estradiol (E(2)) and P to induce precisely controlled, artificial menstrual cycles. During these cycles, treatment with E(2) alone induces an artificial proliferative phase marked by extensive endometrial epithelial cell proliferation and increased expression of stromal and epithelial estrogen receptor (ER) and P receptor (PR). Androgen receptor (AR) is also upregulated by E(2) but is expressed only by the endometrial stroma. Progesterone acts on the E(2) primed endometrium to induce secretory differentiation and causes suppression of epithelial and stromal ER, epithelial PR, and stromal AR in the functionalis zone. However, epithelial ER and PR are retained in the basalis zone during the secretory phase. When potent P antagonists (PA) are administered acutely at the end of an E(2) + P induced cycle, menses typically ensues similar to P withdrawal at the end of the menstrual cycle. When PAs are administered chronically there is significant blockage of all P- dependent effects including upregulation of ER, PR and AR and suppression of glandular secretory function. However, chronic PA administration also inhibits estrogen-dependent endometrial cell proliferation and growth. This endometrial antiproliferative effect is the basis of the clinical use of PA to control various diseases such as endometriosis.

Estradiol (E) and progesterone (P) act on the primate endometrium to induce dramatic changes in the vascular system during the menstrual cycle. These changes include vessel breakdown and bleeding during menses, heightened angiogenesis during the early proliferative phase, and extensive growth of the spiral arteries in the luteal phase of the cycle. Because steroid hormone action is dependent upon the presence of specific nuclear receptors in target tissues, we used immunocytochemistry with receptor-specific monoclonal antibodies to characterize the spatial and temporal expression of estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta), progesterone receptor PR and androgen (A) receptor (AR) in the endometrial vessels of rhesus macaques (Macaca mulatta). The only sex steroid receptor that was present in the endothelium and smooth muscle walls of endometrial vessels was ERbeta. ERalpha, PR, and AR were not detectable in either the endothelium or vascular smooth muscle cells of primate endometrial vessels. However, all of these receptors were strongly expressed by the perivascular stroma, and in these cells, all were modulated by the changes in levels of E and P during the cycle. We concluded that any direct effects of E on endometrial vessels would be mediated by ERbeta, and that the actions of P and A, and possibly some of E, were indirectly mediated through perivascular stromal cells.

Background: The levonorgestrel-releasing intrauterine system (LNG-IUS) is a highly effective contraceptive. However, unscheduled breakthrough bleeding (BTB), leads to discontinuation in a proportion of users. The LNG-IUS down-regulates endometrial progesterone and estrogen receptors and this may play a role in the mechanism responsible for BTB. LNG is an androgenic progestogen and so we examined the regulation of the androgen receptor (AR) in endometrium exposed to intrauterine LNG. Furthermore, as the enzyme 17beta-hydroxysteroid dehydrogenase type 2 (17betaHSD2) regulates intracellular levels of estrogens, progestins and androgens, we evaluated the changes in expression of 17betaHSD2 in the same tissue endometrial samples., Methods: Immunohistochemistry and real time quantitative RT-PCR were used to compare protein and mRNA expression of AR and 17betaHSD2 in endometrial biopsies from women with normal menstrual cycles and those using a LNG-IUS., Results: Immunohistochemistry showed that AR and 17betaHSD2, which were immunolocalized to the stroma and glands of endometrium respectively, were both suppressed by LNG-IUS treatment, though moderate staining of 17betaHSD2 was evident 1 month after insertion of the LNG-IUS. AR mRNA expression was down-regulated in LNG-exposed endometrium when compared with the proliferative phase of the menstrual cycle. 17betaHSD2 mRNA was significantly increased 3 months (but not 6-12 months) after LNG-IUS insertion., Conclusions: Endometrial intracellular estradiol levels would have been suppressed by 17betaHSD2 during the first few, but not the later, months of LNG-IUS action, and the lowered endometrial estradiol level may contribute to the frequent BTB evident in the early months of LNG-IUS use. The subsequent decline in 17betaHSD2 would lead to elevated local intracellular estradiol in the later months, when the BTB tends to subside. The suppression of AR by the LNG-IUS may also play a role in BTB, as elevated AR has been associated with amenorrhoea.

Background: Determination of the mitotic index in sections of endometrium stained with haematoxylin and eosin (H&E) is difficult and time-consuming. We assessed the value of two mitotic markers as immunocytochemical reagents for measuring mitotic rates in endometrium., Methods: Mitotic protein monoclonal antibody 2 (MPM-2) and anti-phosphorylated histone H3 (Phospho H3) were applied to paraffin sections of rhesus macaque and human endometrium., Results: In estrogen-treated macaque endometrium the mean +/- SEM mitotic indices were: H&E 1.5 +/- 0.25%, Phospho H3 antibody 1.02 +/- 0.23% and MPM-2 antibody 0.69 +/- 0.17%; these were not statistically significantly different, but the Phospho H3 antibody gave a stronger and cleaner signal than the MPM-2 antibody. Comparisons were made between a computer-determined Phospho H3 index, the H&E-determined mitotic index and the Ki-67 index in samples of human endometrium across the cycle. All revealed that the highest proliferative rate occurred during the follicular phase, but the Phospho H3 and the mitotic indices were more highly correlated (R(2) = 0.89, P < 0.001) than the Ki-67 and mitotic indices (R(2) = 0.74, P < 0.05)., Conclusions: The exceptionally high contrast staining and the excellent correlation between the Phospho H3 and mitotic indices validates the specificity of the Phospho H3 antibody as a new tool for the assessment of endometrial mitotic activity.

Background: A need exists for a therapy that lowers parathyroid hormone (PTH) without increasing calcium x phosphorus in patients with secondary hyperparathyroidism. The calcimimetic AMG 073 increases the sensitivity of the parathyroid calcium-sensing receptor to extracellular calcium, thereby reducing PTH secretion. Consequently, AMG 073 may provide a novel therapy for secondary hyperparathyroidism., Methods: Seventy-eight hemodialysis patients with secondary hyperparathyroidism were enrolled into this 18-week, double-blind, randomized, placebo-controlled, dose titration study. Daily oral AMG 073 doses were administered to determine the effect on PTH, serum calcium, phosphorus, and calcium x phosphorus., Results: The mean baseline PTH was similar in patients administered AMG 073 or placebo (632 +/- 280.1 pg/mL vs. 637 +/- 455.9 pg/mL, respectively). PTH decreased by 26.0% in the AMG 073-treated group, compared with an increase of 22.0% in the placebo group (P < 0.001). A greater proportion in the AMG 073 group (38%) had a decrease in PTH >or=30%, compared with the placebo group (8%) (P = 0.001). Decreases in PTH were independent of baseline vitamin D usage. Patients receiving AMG 073 had an 11.9% decrease in calcium x phosphorus compared with a 10.9% increase in the placebo group (P < 0.001). Use of vitamin D sterols, as well as both calcium and noncalcium-containing phosphate binders. were similar between treatment groups. Administration of AMG 073 was safe and well tolerated in this 18-week study., Conclusions: The calcimimetic AMG 073 decreases both PTH and calcium x phosphorus levels in hemodialysis patients with secondary hyperparathyroidism.

Hepatocyte growth factor/scatter factor (HGF/SF) induces proliferation, motility and morphogenesis of cells that express the proto-oncogene for the tyrosine kinase receptor, c-Met. Because these cellular events occur in the endometrium during the menstrual cycle and in placenta during development, we have initiated studies of this growth factor in these tissues from macaques. Several HGF/SF alternatively spliced transcripts have been previously reported in other tissues. However, expression of HGF/SF isoforms in the endometrium has not been studied. Here we describe the relative transcript amounts of HGF/SF isoforms in the endometrium and placenta using RNase protection analyses. During these analyses, we discovered two unexpected protected bands that were found through sequence analyses to represent isoforms similar to the previously reported NK1 and NK2 except that they encode a five amino acid deletion in the first kringle domain. We designated these two isoforms as dNK1 and dNK2. Endometrium expressed all of the isoforms; however, dNK2 was consistently expressed at higher levels than NK2 transcripts. In contrast, placenta expressed NK2 and dNK2 mRNA at equal levels, and both NK1 and dNK1 were undetectable in placenta. HGF/SF function in endometrium and placenta may involve complex interactions between the isoforms of HGF/SF and those of c-Met.

Background: A reliable means of menstrual suppression would greatly improve the quality of life for women. Information is lacking on the direct endometrial effects and appropriate dosages of new antiprogestins that may be useful for this purpose., Methods: The current work evaluated three different systems in macaque monkeys. First, the range of doses of two relatively new antiprogestins, ZK 137 316 and ZK 230 211, that would block progesterone action directly on the endometrium in artificially cycled, spayed rhesus macaques; second, the direct endometrial effects of ZK 230 211, a type III antiprogestin; and third, investigation of whether endometrial-suppressive doses administered chronically to intact, cycling monkeys could be used for reversible, menstrual suppression., Results: The results in naturally cycling animals showed that ZK 137 316 blocked menstruation in all animals, but doses of 0.05 mg/kg blocked ovulation in 55.5% of animals and doses of 0.1 mg/kg blocked ovulation in 66.6% of the animals. However, all doses of ZK 230 211 that blocked menstruation also blocked ovulation. All progesterone antagonist (PA)-treated animals, regardless of dose, maintained normal follicular phase concentrations of oestradiol and returned to normal menstrual cyclicity within 15--41 days post-treatment. Therefore ZK 137 316, depending on dose, can allow ovulation but block menstruation, while ZK 230 211, a much more potent PA, blocks both ovulation and menstruation at all effective doses. Both PAs block unopposed oestrogenic action on the endometrium through their antiproliferative effects., Conclusions: Reversible amenorrhoea can be achieved with these two PAs, and they can protect the endometrium from the effects of unopposed oestrogen whether or not ovulation is blocked. Chronic, low dose PA treatment may provide a new option for women who wish to suppress their menstrual periods.

Matrix metalloproteinases (MMPs) are highly expressed in the human endometrium during menstruation, and these enzymes participate in the cyclic destruction and regeneration characteristic of the primate endometrium. To examine hormonal regulation of MMPs in vivo, we evaluated MMP expression and localization in the endometrium of ovariectomized rhesus macaques under various hormonal conditions. Although all MMPs were up-regulated by progesterone (P4) withdrawal, their expression declined spontaneously after menstruation in the absence of P4. Of 7 MMPs examined, only matrilysin and stromelysin-3 were suppressed any further when P4 levels were experimentally re-elevated. MMP expression was confined to the upper functionalis zone during menstruation, but after menstrual breakdown was complete, matrilysin and the tissue inhibitor of MMPs, TIMP-1, shifted expression from the functionalis to the basalis zone in the absence of both estradiol and P4. The spiral arteries in the functionalis, but not the basalis, were intense foci of MMP and TIMP-1 expression. Menstruation and MMP expression after P4 withdrawal were similar in both the presence and absence of estradiol. In sum, endometrial MMPs in vivo are strongly up-regulated by P4 withdrawal, but zone-specific tissue gradients greatly influence the pattern and degree of MMP expression.

The long-term effects of the antiprogestin ZK 137 316 on reproductive tract morphology in rhesus macaques were investigated. The monkeys were injected daily (i.m.) for five menstrual cycles with vehicle or 0.01, 0.03 or 0.1 mg ZK 137 316/kg body weight. Reproductive tracts (n = 3/ group) were collected during the mid-luteal phase (day 8) of the fifth cycle in the control, 0.01 and 0.03 mg/kg groups, or 6-7 days after the oestradiol peak in the 0.1 mg/kg group. ZK 137 316 treatment resulted in a dose-dependent atrophy of the endometrium, marked by reduced mitotic activity in the glands, compaction of the stroma, degradation of spiral arteries and dilation of veins. There was no effect of ZK 137 316 on myometrial or oviductal weight. Treatment with 0.1 and 0.03 mg/kg, but not 0.01 mg/kg resulted in fully ciliated and secretory oviducts, indicating a dose-dependent blockade of progesterone antagonism of oestrogen-dependent oviductal differentiation. In the endometrium, the suppressive action of progesterone on oestrogen and progestin receptors was also blocked by ZK 137 316 in a dose-dependent manner. However, endometrial atrophy appeared due to inhibition of progesterone action together with a blockade of oestrogen-dependent proliferation. The profoundly suppressed endometrium produced by chronic low-dose ZK 137 316 treatment is unlikely to support implantation. Such treatment may therefore provide a novel contraceptive modality.

Large doses of antiprogestin typically disrupt menstrual cyclicity. A chronic low-dose regimen of the potent new antiprogestin ZK 137 316, which permits continued menstrual cyclicity but alters gonadal-reproductive tract activity, was established. Rhesus monkeys received vehicle (n = 6) or 0.01 (n = 8), 0.03 (n = 8) or 0.1 (n = 5) mg ZK 137 316/kg body weight daily for five menstrual cycles (C-1 to C-5). Oestradiol, progesterone and gonadotrophin profiles were normal during cycles involving vehicle and 0.01 and 0.03 mg ZK 137 316/kg body weight. In the 0.1 mg/kg group, mid-cycle oestradiol and gonadotrophin surges, and subsequent progesterone production, were absent in C-3 and C-5. Ovarian cyclicity was accompanied by timely menstruation in the vehicle and 0.01 mg/kg groups. By C-3, half the animals in the 0.03 mg/kg group and all animals in the 0.1 mg/kg group were amenorrhoeic. A corpus luteum was noted during the mid-luteal phase of C-5 in the vehicle, 0.01 mg/kg and 0.03 mg/kg groups. Large antral and cystic follicles were evident in the 0.1 mg/kg group. Thus, a daily treatment with 0.01 mg/kg ZK 136317 permitted normal menstrual cyclicity in macaques. While the daily administration of 0.03 mg/kg ZK 136 317 allowed ovarian cyclicity, menstruation was disrupted in some animals. Increasing the dose to 0.1 mg/kg antagonized pituitary function and resulted in anovulation and amenorrhoea. A chronic low-dose regimen of the antiprogestin ZK 137 316, which permits normal ovarian/menstrual cyclicity, has potential as a contraceptive in women.

The objective of this study was to detect and characterize a secreted oviduct-specific glycoprotein (OGP) in the rhesus macaque (Macaca mulatta) and to compare the characteristics of this OGP to those previously characterized in baboons and women. Oviducts were obtained from untreated ovariectomized rhesus and from ovariectomized rhesus either treated with estradiol (E2) for 14 days or treated sequentially with E2 for 14 days and then with E2 plus progesterone (P4) for an additional 14 days. Segments of oviducts were either fixed for morphological analysis, cultured for OGP synthesis and release, or frozen for RNA analysis. The proteins present in the culture media were separated by one-dimensional SDS-PAGE, and OGP was detected on Western blots using polyclonal antibodies generated against the reduced form of baboon OGP or a 17-amino acid segment of the baboon core protein. Cross-reacting antigens were present in the 120-kDa region, identical to what was observed for baboon and human OGP. Indirect immunogold localization of OGP on thin sections demonstrated specific clustering of gold particles over the apical secretory granules of the secretory cells of the oviductal epithelium. A cDNA was generated using RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE), and sequenced. The total transcript was 2237 nucleotides in length plus a poly(A) tail. The largest open reading frame was 624 amino acids, which would produce a protein of 69.3 kDa. The nucleotide sequence was more than 95% identical to the nucleotide sequences of baboon and human OGP. Northern blots revealed a single message at 2.4 kilobases (kb) in oviduct samples obtained from E2-treated rhesus. This message was absent in oviducts obtained from untreated ovariectomized and from sequential E2 plus P4-treated rhesus macaques. In summary, the rhesus oviduct synthesizes and secretes an OGP in the presence of E2 that is immunologically and structurally similar to the baboon and human OGP. The presence of a highly homologous glycoprotein in several primates suggests a similar function for OGP in the reproductive process.

Rhesus monkeys are useful models in which to examine the hormonal regulation of endometrial matrix metalloproteinases (MMP) and to evaluate the role of MMP in uterine bleeding. Artificial 28 day menstrual cycles can be induced in ovariectomized monkeys by inserting an oestradiol implant for 2 weeks, then inserting a progesterone implant for 2 weeks, and then, with the oestradiol implant remaining in place, removing and reinserting the progesterone implant at 2 week intervals. To examine MMP during menses, we established such cycles and removed uteri by hysterectomy at closely spaced intervals before, during and after menses, as well as at later time points. Some samples were also obtained during menses induced by the withdrawal of both progesterone and oestradiol. We examined mRNA of the following MMP by Northern blotting: matrilysin, stromelysin-1, stromelysin-2, stromelysin-3 and the tissue inhibitor of MMP TIMP-1. The expression of these MMP mRNA increased substantially by 2-3 days after progesterone withdrawal, whether or not oestradiol was maintained. The expression of some of the MMP (stromelysins-1 and -2) returned very rapidly to baseline levels by 5 days after progesterone withdrawal, while the expression of others (matrilysin, stromelysin-3 and TIMP-1) declined more slowly, reaching a baseline level by 10 days after progesterone withdrawal, with little or no further decline after progesterone concentrations rose during the induced luteal phase. Immunocytochemical studies showed that matrilysin was expressed primarily in the glands of the upper functionalis. In other work with the rhesus monkey model, we used a s.c. endometrial autograft technique in which pieces of endometrium were autotransplanted to the abdominal skin. During menses in the grafts, matrilysin was expressed in the glands of the grafts similar to the glands in the eutopic endometrium. Endometrial autografts can serve as a useful model for the study of MMP in uterine bleeding.

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.

We measured androgen receptors (AR) and 5 alpha-reductase activity (5 alpha RA) in the ductuli efferentes and epididymides from adult rhesus macaques. Tissue samples were either assayed biochemically for AR or stained immunocytochemically (ICC) with a monoclonal antibody against AR. To estimate 5 alpha RA, tissue microsomes were incubated with [1 alpha,2 alpha-3H]testosterone, and the [3H]dihydrotestosterone formed was quantified. We found significant regional differences in the levels of both 5 alpha RA and AR in the excurrent ducts. In general, both enzyme activity and AR levels were higher in the caput and corpus epididymis than in ductuli efferentes and cauda epididymis. With ICC, positive nuclear AR staining was detected in all epithelial cell types, whereas variable numbers of stromal cells were positively stained. Our data demonstrate that there are segmental differences in the concentrations of 5 alpha RA and AR in epididymis and suggest that there may be regional differences in the regulation of epididymal functions by androgen.

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)

Specific nuclear staining for progesterone receptor (PR) was detected by immunocytochemistry in human granulosa cells (GCs) obtained from in vitro fertilization protocols. The percent of PR-positive cells (60% to 80%) remained unchanged during 7 days of culture in media containing fetal calf serum, in the absence or presence of human chorionic gonadotropin (hCG) or the progesterone antagonist RU486. Progesterone (P) production by GCs cultured on extracellular matrix from bovine corneal endothelial cells was stimulated by hCG and prostaglandin E2 (PGE2). However, addition of RU486 or human relaxin had no effect on control, hCG-, or PGE2-stimulated P production. Thus, the receptor data are consistent with an autocrine action of P in luteinizing GCs, but initial experiments in cell culture did not define a role for P or relaxin in modulating luteal steroidogenesis.

We obtained the testes, ductuli efferentes, and epididymides from adult rhesus and cynomolgus macaques and examined these tissues for estrogen receptors (ER) with immunocytochemistry (ICC) and a sucrose gradient assay. Both techniques employed monoclonal antibodies prepared against ER, and both showed that high concentrations of ER were present OFFy in the ductuli efferentes. Moreover, all specific staining was confined to the nuclei of the nonciliated, absorptive epithelial cells. The quantity of salt-extractable ER in the ductuli efferentes (834 +/- 161 [SEM] fmol/mg DNA [n = 8]) did not differ significantly from the amounts measured with the identical assay in oviducts and endometrium of estrogenized female macaques. Testes and epididymides of macaques had no specific staining by ICC and barely detectable amounts by biochemical analysis (7 +/- 4 [n = 3], 8 +/- 2 [n = 5], 33 +/- 16 [n = 3], and 6 +/- 3 [n = 8] fmol/mg DNA for testis and caput, corpus, and cauda epididymis, respectively). The functional significance of the high levels of ER in the ductuli efferentes of macaques remains to be determined.

With the aid of monoclonal antibodies specific to the estrogen and progestin receptors, we have examined the cellular localization of these proteins in the reproductive tract of male and female macaques. Two striking findings have resulted from our work with these new reagents. First, these receptors are detectable only in cell nuclei, regardless of hormonal treatment, and second, they are often detectable in stromal, but not epithelial cells when the epithelial cells undergo various estrogen or progestin-dependent events. The latter observation has led us to conclude that stromal cell-epithelial cell interactions may play previously unappreciated roles in the hormonal control of the primate reproductive tract. The lines of evidence that have drawn us to this conclusion will be reviewed.

Sequential steroid administration of estradiol (E2) and progesterone (P) in spayed pigtailed macaques was used to precisely control the time course of sex skin swelling. After removal of the P implant, the sex skin swelled considerably and the water content of the sex skin increased manyfold over that of back skin. During the swelling phase, hyaluronate biosynthesis in sex skin increased dramatically compared with back skin of the same animals. Collagen synthesis also increased but to a lesser extent. Estrogen receptor levels were undetectable in back skin and very low in spayed animals that had been treated with both E2 and P. After removal of the P implant, both the level of estrogen receptor and the rate of hyaluronate biosynthesis increased. Immunocytochemistry with monoclonal antibodies against the estrogen receptor showed that the dermal fibroblast was the only cell type to stain positively for estrogen receptor. We conclude that the sex skin swelling that follows P withdrawal in pigtailed macaques bearing E2 implants is mediated by estrogen receptors in dermal fibroblasts and is a result of increased hyaluronic acid synthesis by these cells.

Immunocytochemical, biochemical, and histologic analysis of endometriotic lesions and endometria from rhesus macaques with endometriosis revealed several distinctions between ectopic and eutopic endometrium. In lesions, unlike endometrium, neither the mean percentages of estrogen receptor positive (ER+) cells nor the total ER content changed significantly during the menstrual cycle. In eutopic endometria, ER staining in both stromal and epithelial cells increased and decreased synchronously during the cycle, but in endometriotic lesions, such synchrony was lacking. Moreover, in lesions, unlike endometria, the percentage of ER+ cells was low in the stroma and highly variable in epithelium throughout the cycle. These data, taken together, indicate a defect in the hormonal regulation of ER in endometriotic lesions of monkeys.

Acute infection with Chlamydia trachomatis serotype E was established in monkey fallopian tube fimbriae by subcutaneous implantation. Depending upon monkey species, from eight to 20 implants could be established in each animal. Animals were given estrogen before percutaneous inoculation of the autografts with Chlamydia. Acute inflammatory changes were found in homografts examined in the first week after infection, with chronic inflammatory changes noted thereafter. Chlamydial inclusions were detected within fimbrial epithelial cells up to seven days postinoculation by fluorescent-antibody staining and immunoperoxidase staining with C. trachomatis-specific monoclonal antibody. Organisms were recovered from autografts up to five days after infection. Analysis of serum antibody by microimmunofluorescence revealed that serotype E-specific antibody of both IgM and IgG classes was produced after infection. We conclude that subcutaneously implanted fallopian tube autografts may provide a useful primate model for kinetic studies of chlamydial infection and immunity.

We sampled oviducts and endometria of 27 cynomolgus macaques during the menstrual cycle and measured the concentration of nuclear and cytoplasmic estrogen receptors in these tissues by exchange assay. We assessed the stage of the cycle by correlating serum estradiol (E2) and progesterone (P), as measured by radioimmunoassay, with the morphological condition of the ovaries, oviducts and endometrium of each animal. We have previously identified a series of oviductal stages that reflected the orderly sequence of cytological changes in the oviduct during the cycle, and we normalized receptor measurements to these stages. The amounts of nuclear and cytoplasmic estrogen receptor in both the oviduct and the endometrium were approximately twofold greater in the follicular phase than in the luteal phase. In the follicular phase, elevated receptor levels were associated with oviductal proliferation and differentiation, as well as with endometrial proliferation. During the luteal phase, lowered levels were correlated with atrophy and dedifferentiation in the oviduct, but with hypertrophy and progestational development in the endometrium. When the luteal phase of one cycle ends and the follicular phase of the next begins, it is a decline in serum P, not a rise in serum E2, that precedes the elevation in estrogen receptor level and the onset of proliferation in the oviduct and endometrium. Proliferation of the reproductive tract and elevations in nuclear estrogen receptor levels during the early follicular phase can therefore be viewed as consequences of the release of the system from antagonism by P.

The mechanisms by which uranyl nitrate (UN) is toxic to the proximal tubule are incompletely understood. To define these further we studied potassium (K+) transport and oxygen consumption (QO2) in rabbit proximal tubule suspensions in vitro immediately after exposure to UN using extracellular O2- and K+-sensitive electrodes. UN caused a cumulative dose-dependent inhibition of proximal tubule QO2, with a threshold concentration of 5 x 10(-5) M. Kinetic analysis suggested two patterns of cell injury: a higher affinity inhibition of QO2 with a Ki of 5 x 10(-4) M, and a lower affinity inhibition of QO2 with a Ki of 10 mM. QO2 was studied in detail in the presence of these Ki concentrations of UN to define the initial cellular events. The results indicated that different cellular processes displayed different sensitivities to UN. At submillimolar concentrations UN caused progressive selective inhibition of ouabain-insensitive QO2 (15% inhibition at 2 minutes). Ouabain-sensitive QO2 and nystatin-stimulated QO2 were not affected, suggesting that Na+,K+-ATPase activity and its coupling to mitochondrial ATP synthesis were intact. Direct measurement of proximal tubule net K+ flux confirmed that Na+,K+-ATPase activity was unchanged. Similarly, UN did not inhibit basal (state 4) or ADP-stimulated (state 3) mitochondrial QO2 in digitonin-permeabilized tubules, confirming that the mitochondria were intact. In contrast, higher concentrations of UN (greater than or equal to 1 mM) caused rapid inhibition of QO2 and net K+ efflux, due to inhibition of Na+,K+-ATPase activity and mitochondrial injury.(ABSTRACT TRUNCATED AT 250 WORDS)