The Nonhuman Primate Models of Menopause Workshop was held on the National Institutes of Health campus in January 2001. The purpose of this workshop, sponsored by the National Institute on Aging, was to review what is known about the female reproductive aging process in various species of monkeys (particularly rhesus, baboons, cynomolgus, and chimpanzees), including hormone profiles during the menopausal transition, occurrence of hot flashes, extent of age-related and menopause-associated changes in hormone levels on metabolism, bone loss, and impaired cardiovascular and cognitive function. Many aspects of the female reproductive aging process appear to be concordant between humans and these monkey species, but several important features may be species-specific. Those features that appear to parallel human menopause and aging include general similarity of hormone profiles across the menopausal transition, progression to cycle termination through irregular cycles, declining fertility with age, age-related gains in weight and percentage body fat content (with tendencies toward insulin resistance and glucose intolerance), increased low-density lipoprotein cholesterol and decreased high-density lipoprotein cholesterol, declines in serum dehydroepiandrosterone, similarities in temperature-regulation systems, protective responses to estrogen replacement following ovariectomy in terms of bone metabolism, lipid profiles, and cognitive changes. Important differences include relatively short postmenopausal life span, timing in menopause-related changes in hormone secretion, and seasonal menstrual cycles. In addition, the question of whether ovariectomy in young adults is an appropriate model for the consequences of natural or surgical menopause in middle-aged and older adults is unresolved, and the numbers of older female animals available for research on menopause are very limited. The use of animal models is seen by workshop participants to be crucial for a mechanistic understanding of the human menopausal process and its connections to postmenopausal health problems; however, extensive in-depth and broad-based research is required to determine if nonhuman primates are appropriate models of human menopause.

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There is an increasing public interest in foods and dietary supplements containing phytoestrogens for the maintenance of health. A workshop was convened to assess evidence for the potential benefits of phytoestrogen-containing foods or supplements on diseases or conditions affecting older populations. Preclinical, clinical, and epidemiologic data on the cardiovascular system, various cancers, bone diseases, and menopausal symptoms were the focus of the discussions. Research on the basis of consumer food choices as well as a presentation from the FDA regarding approval of the use of soy foods to reduce the risk of cardiovascular disease were also presented. Based on the information presented, isoflavone-containing soy foods may have favorable effects on the cardiovascular system, but major knowledge gaps still exist regarding effects ofphytoestrogen supplements on bone diseases, various cancers, menopausal symptoms, and cognitive function.

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Several brominated androgen derivatives were tested for their ability to inactivate microsomal aromatase from term human placenta. In the experimental protocol, the microsomal homogenate was incubated either with androstenedione or a brominated derivative of androstenedione (16alpha-bromo-6-ketoandrostenedione, 16alpha-bromoandrostenedione, 7alpha-(3'-bromoacetoxypropyl)androstenedione, 6alpha-bromoandrostenedione, or 6beta-bromoandrostenedione) and reduced nicotinamide adenine dinucleotide phosphate in a nitrogen saturated buffer composed of glycerol, ethylenediaminetetraacetic acid, and dithiothreitol in tris(hydroxymethyl)aminomethane hydrochloride (pH 7.4) under nitrogen at 4 degrees C with shaking. After the incubation period, the microsomes were recovered by centrifugation and washed once before determining aromatase specific activity. The brominated androgen derivatives which inactivated aromatase were 7alpha-(3'-bromoacetoxypropyl)androstenedione and 6alpha-bromoandrostenedione. The structures of 6alpha- and 6beta-bromoandrostenedione were unequivocally established by single crystal x-ray diffraction techniques. The extent of the enzyme inactivation by 6alpha-bromoandrostenedione was linearly proportional to the logarithm of its concentration. The evidence that this inactivation occurs at the aromatase active site is that androstenedione, when coincubated with 6alpha-bromoandrostenedione, protected aromatase from this inactivation. Progesterone provided much less protection than androstenedione. Furthermore, both 6alpha- and 6beta-bromoandrostenedione are competitive inhibitors of androstenedione aromatization, as determined by a Lineweaver-Burk plot, and 6alpha-bromoandrostenedione gives the same type I cytochrome P-450 binding spectrum with placental microsomes as androstenedione. These data suggest that 6alpha-bromandrostenedione is effective as an active-site-directed inhibitor of placental microsomal aromatase.

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In previous investigations of human placental estrogen synthetase (aromatase), activity was found in the microsomal and mitochondrial fractions after homogenization and subcellular fractionation of placental tissue in isotonic sucrose. We report here that the majority of the placental aromatase sediments in a much heavier particulate fraction than the microsomal or mitochondrial fractions. After low speed centrifugation of the placental homogenate, only about 20% of the homogenate aromatase activity was measured in the supernatant, the precursor of the microsomal and mitochondrial fractions. Even after extensive homogenization in high speed shearing-type blenders and tight-fitting glass homogenizers the majority of the placental aromatase (more than 70%) consistently sedimented at very low centrifugal forces (100 x g ro 10 min) independent of whether the suspending medium contained up to 1.2M sucrose, or IM NaCl, or 50% glycerol. By selectively separating components of the low speed pellet via a sieving procedure or manual dissection of placental tissue, we found that the majority of the enzyme activity is associated with placental chorionic villus fragments. Several extensively washed preparations of villus fragments with aromatase specific activities similar to that found in placental microsomes were synthesis in the villus aromatase fraction was determined to be 1beta,2beta-H removal which is identical with that by placental microsomes and human ovaries. Evidence for the presence of cytochrome P-450 in the villus fraction was obtained by measuring the carbon monoxide-dithionite difference spectrum.