Competitive product inhibition of aromatase by natural estrogens

In order to better understand the function of aromatase, we carried out kinetic analyses to asses the ability of natural estrogens, estrone (E₁), estradiol (E₂), 16-OHE₁, and estriol (E₃), to inhibit aromatization. Human placental microsomes (50 μg protein) were incubated for 5 min at 37°C with [1β-³H]testosterone (1.24 × 10³ dpm ³H/ng, 35–150 nM) or [1β-³H,4-¹⁴C]androstenedione (3.05 × 10³ dpm ³H/ng, ³H/¹⁴C = 19.3, 7–65 nM) as substrate in the presence of NADPH, with and without natural estrogens as putative inhibitors. Aromatase activity was assessed by tritium released to water from the 1β-position of the substrates. Natural estrogens showed competitive product inhibition against androgen aromatization. The K_i of E₁, E₂, 16-OHE₁, and E₃ for testosterone aromatization was 1.5, 2.2, 95, and 162 μM, respectively, where the K_m of aromatase was 61.8 ± 2.0 nM (n = 5) for testosterone. The K_i of E₁, E₂, 16-OHE₁, and E₃ for androstenedione aromatization was 10.6, 5.5, 252, and 1182 μM, respectively, where the K_m of aromatase was 35.4 ± 4.1 nM (n = 4) for androstenedione. These results show that estrogens inhibit the process of andrigen aromatization and indicate that natural estrogens regulate their own synthesis by the product inhibition mechanism in vivo. Since natural estrogens bind to the active site of human placental aromatase P-450 complex as competitive inhibitors, natural estrogens might be further metabolized by aromatase. This suggests that human placental estrogen 2-hydroxylase activity is catalyzed by the active site of aromatase cytochrome P-450 and also agrees with the fact that the level of catecholestrogens in maternal plasma increases during pregnancy. The relative affinities and concentration of androgens and estrogens would control estrogen and catecholestrogen biosynthesis by aromatase.     

Treatment with high-dose estrogen (diethylstilbestrol) significantly decreases plasma estrogen and androgen levels but does not influence in vivo aromatization in postmenopausal breast cancer patients

While growth factors and hormones are known to influence aromatase expression in experimental systems, little is know about potential factors influencing peripheral aromatization in postmenopausal women. The fact that peripheral aromatase activity is higher in old compared to young women and the finding of relatively high tissue estradiol (E₂) concentrations after the menopause suggests peripheral aromatization could be influenced by estrogen concentration. To test this hypothesis, we determined plasma hormone levels (n = 9) and in vivo aromatization (n = 3) in postmenopausal women suffering from advanced breast cancer before and during treatment (4 weeks) with diethylstilbestrol (DES) 5 mg three times daily. Plasma levels of cortisol (C), corticosteroid-binding globulin (CBG), and sex hormone binding globulin (SHBG) were significantly increased in all patients (P < 0.05 for all). While we found no change in total body aromatization and plasma estrone (E₁) levels, estradiol (E₂) and estrone sulfate (E₁S) were suppressed by a mean of 48.8 and 68.2%, respectively (P = 0.043 and 0.008). Surprisingly, plasma levels of androstenedione (A), testosterone (T), dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) were also suppressed by a mean in the range of 32.1 to 52.6% (P < 0.05 for all androgens). In contrast, no change in plasma progesterone or 17-hydroxyprogesterone was found. Thus, one possible explanation to our findings could be that DES administered in high doses reduces 17,20-lyase activity in the adrenal gland.     

Peripheral aromatization: studies on controlling factors

Using constant infusions of 3H-labeled androgens and 14C-labeled estrogens with measurements of radiolabeled estrogens in blood and/or urine we have carried out studies on the peripheral aromatization of androgens in humans, nonhuman primates, sheep, and rabbits. In the human, aromatization is increased in women as they become postmenopausal, although the mechanism remains uncertain. In humans and cynomolgus monkeys the administration of ACTH and/or glucocorticoids does not increase peripheral aromatization, but results in a slight decrease in the aromatization of androstenedione. The administration of l-thyroxine to cynomolgus monkeys increases peripheral aromatization of androstenedione from basal, 1.16 +/- 0.15%, to 1.71 +/- 0.14% probably due to increased tissue blood flow. The aromatization of testosterone is not affected, probably due to an increase in sex hormone-binding globulin. Peripheral aromatization occurs to a similar degree in humans, rhesus and cynomolgus monkeys, and baboons, but is much lower in sheep and rabbits. The compound 10-(2-propynyl)-estr-4-ene-3,17-dione is an effective inhibitor of the peripheral aromatization of both androstenedione and testosterone.     

Multiple functions of aromatase and the active site structure; aromatase is the placental estrogen 2-hydroxylase

Androgen aromatase was found to also be estrogen 2-hydroxylase. The substrate specificity among androgens and estrogens and multiplicity of aromatase reactions were further studied. Through purification of human placental microsomal cytochrome P-450 by monoclonal antibody-based immunoaffinity chromatography and gradient elution on hydroxyapatite, aromatase and estradiol 2-hydroxylase activities were co-purified into a single band cytochrome P-450 with approx. 600-fold increase of both specific activities, while other cytochrome P-450 enzyme activities found in the microsomes were completely eliminated. The purified P-450 showed M_r of 55 kDa, specific heme content of 12.9 ± 2.6 nmol·mg⁻¹ (±SD, N = 4), reconstituted aromatase activity of 111 ± 19 nmol·min⁻¹·mmg⁻¹ and estradiol 2-hydroxylase activity of 5.85 ± 1.23 nmol·min⁻¹·mg⁻¹. We found no evidence for the existence of catechol estrogen synthetase without concomitant aromatase activity. The identity of the P-450 for the two different hormone synthetases was further confirmed by analysis of the two activities in the stable expression system in Chinese hamster ovarian cells transfected with human placental aromatase cDNA, pH β-Aro. Kinetic analysis of estradiol 2-hydroxylation by the purified and reconstituted aromatase P-450 in 0.1 M phosphate buffer (pH 7.6) showed K_m of 1.58 μM and V_max of 8.9 nmol·min⁻¹·mg⁻¹. A significant shift of the optimum pH and V_max, but not the K_m, for placental estrogen 2-hydroxylase was observed between microsomal and purified preparations. Testosterone and androstenedione competitively inhibited estradiol 2-hydroxylation, and estrone and estradiol competitively inhibited aromatization of both testosterone and androstenedione. Estrone and estradiol showed K_i of 4.8 and 7.3 μM, respectively, for testosterone aromatization, and 5.0 and 8.1 μM, respectively, for androstenedione aromatization. Androstenedione and testosterone showed K_i of 0.32 and 0.61 μM, respectively, for estradiol 2-hydroxylation. Our studies showed that aromatase P-450 functions as estrogen 2-hydroxylase as well as androgen 19-, 1β-,and 2β-hydroxylase and aromatase. The results indicate that placental aromatase is responsible for the highly elevated levels of the catechol estrogen and 19-hydroxyandrogen during pregnancy. These results also indicate that the active site structure holds the steroid ssubstrates to face their β-side of the A-ring to the heme, tilted in such a way as to make the 2-position of estrogens and 19-, 1-, and 2-positions of androgens available for monooxygenation.     

Variation in δC values for the seagrass Thalassia testudinum and its relations to mangrove carbon

Carbon isotope ratios (¹³C/¹²C) were measured for the leaves of the seagrass Thalassia testudinum Banks ex König and carbonates of shells collected at the seagrass beds from seven sites along the coast of southern Florida, U.S.A. The δ¹³C values of seagrass leaves ranged from −7.3 to −16.3‰ among different study sites, with a significantly lower mean value for seagrass leaves from those sites near mangrove forests (−12.8 ± 1.1‰) than those far from mangrove forests (−8.3 ± 0.9‰; P < 0.05). Furthermore, seagrass leaves from a shallow water area had significantly lower δ¹³C values than those found in a deep water area (P < 0.01). There was no significant variation in δ¹³C values between young and mature leaves (P = 0.59) or between the tip and base of a leaf blade (P = 0.46). Carbonates of shells also showed a significantly lower mean δ¹³C value in the mangrove areas (−2.3 ± 0.6‰) than in the non-mangrove areas (0.6 ± 0.3‰; P <0.025). In addition, the δ¹³C values of seagrass leaves were significantly correlated with those of shell carbonates (δ¹³C seagrass leaf = −9.1 + 1.3δ¹³C shell carbonate (R² = 0.83, P < 0.01)). These results indicated that the input of carbon dioxide from the mineralization of mangrove detritus caused the variation in carbon isotope ratios of seagrass leaves among different sites in this study.     

Sequential endocrine changes and behaviour during oestrus and metoestrus in repeat breeder and virgin heifers

The aim of the experiment was to study the oestrous behaviour and the peripheral blood plasma profiles of luteinizing hormone (LH), progesterone and the prostaglandin metabolite, 15-keto-13,14-dihydro-PGF₂, during oestrus and metoestrus in repeat breeder (RBH) and virgin heifers (VH). Ten animals of each category were utilized. The RBH had a history of at least three inseminations without conception, and the VH were sexually mature animals not previously inseminated or mated. Oestrous symptoms were recorded and blood collected from the onset of prooestrus to 7 days after oestrus. The animals were inseminated during oestrus and their embryos were collected by a nonsurgical technique 7 days after insemination. The morphology of the embryos was evaluated.

The duration of oestrus was longer (P < 0.05) in the RBH (31.5 ± 3.6 h) than in the VH (23.8 ± 2.0 h). No differences in duration of prooestrus or in the interval from the end of oestrus to postoestrous bleeding were found between the heifer categories. The interval from the onset of oestrus to the preovulatory LH peak was longer (P < 0.05) in the RBH (12.2 ± 2.8 h) than in the VH (4.8 ± 1.5 h). There was a lower LH release in the RBH than in the VH, measured as the magnitude of the preovulatory LH peak (P < 0.05; 28.0 ± 4.0 vs. 40.7 ± 3.6 μg/l) or as the area under the curve of the LH peak (P < 0.01; 1141 ± 164 vs. 1765 ± 144 mm²). The progesterone levels were higher (P < 0.05) in the RBH than in the VH during the interval 5–48 h and lower (P < 0.05) during the interval 121–168 h after the LH peak. Peaks of the prostaglandin metabolite were seen during oestrus in both heifer groups. There were more prostaglandin metabolite peaks in the RBH than in the VH during the interval 13–24 h after the LH peak. Fewer normal embryos (P < 0.05) and more degenerated embryos (P < 0.01) were found in the RBH than in the VH group 7 days after insemination. No apparent relation was found between the morphology of the embryos and the hormonal changes.

The result of the study indicates a hormonal imbalance in the RBH. The hormonal asynchronism starts before or early in oestrus, which presumably leads to a sequence of improper hormonal changes responsible for the elevated embryonic loss in repeat breeder animals.     

Steroid metabolism in the gonads of a patient with testicular feminization. II. Metabolism of c19- and c18-steroids in vitro.

The metabolism of C19- and C18-steroids, in particular, the aromatization of androstenedione and testosterone, the interconversion of androgens to estrogens and the 5alpha-reductase activity of a right abdominal (r) and a left inguinal (l) testis of a patient with testicular feminization, are reported. Aromatization and 5alpha-reductase activity were also evaluated in tissue from the left ductus diferens (ld). The following results were obtained: 1. aromatization of androstenedione to estrone 2.52% (r), 0.02% (l), 0.94% (ld); 2. aromatization of testosterone to estradiol 0.58% (r), 2.88% (l); 3. conversion of androstenedione to testosterone 95.65% (r), 98.07% (l); 4. conversion of testosterone to androstenedione 33.14% (r), 53.65% (l); 5. conversion of estrone to estradiol85.29% (r), 100% (l), 6. conversion of estradiol to estrone 33.12% (r), 32.33% (l); 7.5alpha-reduction of testosterone to 5alpha-dihydrotestosterone 12.01% (r), 13.64% (l) and 4.10% (ld). A lack of 5alpha-reductase activity was not found in the tissues examined as stated in the literature. Estrogen production in these testes was demonstrated by the aromatization of androstenedione and testosterone to estrone and estradiol and is reflected in the difference of the estradiol concentration measured in spermatic and peripheral blood of the same patient (168 versus 33 pg/ml).     

Plasma estrogens,progesterone and chorionic gonadotropin in pregnant rhesus monkeys (Macaca mulatta) after ovariectomy

Maternal peripheral plasma concentrations of estrone (E₁), estradiol-17β (E₂), and progesterone (P) were determined in 4 rhesus monkey ovariectomized in early pregnancy (22–24 days). After ovariectomy, plasma concentrations of E₁ and E₂ were basal for 1 to 2 weeks. In contrast, slightly higher estrogen levels, which may be attributed to the ovaries, were found in intact pregnant monkeys. E₂ levels increased rapidly after this and exceeded those of E₁ until the 5th month of gestation. From that time until parturition, E₁ levels equaled or exceed those of E₂ in most instances. The pattern of P concentrations was similar to that observed in intact monkeys. Urinary chorionic gonadotropin (MCG) levels in ovariectomized monkeys were not significantly differen from those found in normal pregnancies. Thus, the patterns for circulating E_l, E₂ and P, as well as for the excretion of MCG,. after ovari-ectomy were remarkably similar to those found in intact, pregnant rhesus monkeys, indicating minimal ovarian influence.     

Observations on progesterone production and clearance in normal pregnant and fetectomized rhesus monkeys (Macaca mulatta).

The metabolic clearance rate (MCR) and production rate (PR) of progesterone were measured in rhesus monkeys both before and during intravenous infusion of progesterone at rates which approximately doubled or tripled the peripheral plasma levels. The monkeys were normally pregnant or fetectomized and were studied during the second half of pregnancy. Raising the peripheral plasma levels did not significantly after the MCR or the PR of progesterone. We conclude that peripheral progesterone levels are not the factor which controls the PR of progesterone in rhesus monkeys.     

Plasma progesterone profiles and variation in cyclic ovarian activity throughout the year in indigenous goats in Zimbabwe

Eleven non-pregnant indigenous goats were bled three times a week from 1 June 1988 to 31 May 1989. They were fed hay ad libitum supplemented by concentrates, and housed indoors in single pens until November when the goats were divided between two large pens. Plasma progesterone profiles were used to calculate cycle length: 73.3% (99/135) of cycles, mean length 22.0 ± 0.3 days, were classified as normal (N). N cycles had a mean luteal phase of 17.5 ± 0.2 days followed by a peri-ovulatory period of 4.5 ± 0.2 days. Cycles greater than 30 days long were classified as extended (E). These had normal length luteal phases followed by basal progesterone for 15.4 ± 0.5 days (n = 22) or 38.9 ± 4.1 days (n = 10) before the next cycle, giving cycle lengths of 33.6 ± 0.7 days and 56.3 ± 0.9 days, respectively. Four E cycles (mean length 117.0 ± 30 days) had a persistent corpus luteum followed by basal progesterone of 17.8 ± 5.6 days in duration.

The distribution of N versus E cycles varied between animals and was significantly influenced by season (P < 0.005) and housing (P < 0.005), but not body condition. The proportion of N cycles was highest in the cool, dry winter months from June to August, fell during the hot, rainy months from September to February, and rose again between March and May. The proportion of N cycles was higher for goats housed in single pens, however the effect of season and housing were confounded by the move from single to group pens in November.     

Reduction of 8-hydroxyguanine in human leukocyte DNA by physical exercise

We investigated the effect of physical exercise on the level of 8-hydroxyguanine (8-OH-Gua), a form of oxidative DNA damage, and its repair activity in human peripheral leukocytes. Whole blood samples were collected by venipuncture from 21 healthy male volunteers (10 trained athletes and 13 untrained men), aged 19-50 years, both before and after physical exercise. Trained athletes showed a lower level of 8-OH-Gua (2.4 ± 0.5/10⁶ Gua, p = 0.0032) before exercise when compared to that of untrained men (6.2 ± 3.5). The mean levels of 8-OH-Gua of untrained subjects decreased significantly (p = 0.0057) from 6.2 ± 3.5/10⁶ Gua (mean ± SD/10⁶ Gua) to 3.3 ± 1.4/10⁶ Gua after physical exercise. On the other hand, the mean levels of repair activity of untrained subjects significantly increased after exercise (p = 0.0093) from 0.037 ± 0.024 (mean DNA cleavage ratio ± SD) to 0.056 ± 0.036. In the trained athletes 8-OH-Gua level and its repair activity were not changed before and after the exercise. We also observed inter-individual differences in 8-OH-Gua levels and its repair activities. These results suggest that physical exercise causes both rapid and long-range reduction of oxidative DNA damage in human leukocytes, with individually different efficiencies.     

Stereochemistry of 1,2-hydrogen loss during aromatization in the brain

The stereochemistry of hydrogen loss from C-1 and C-2 during aromatization in rat brain was studied using androstenedione containing a known distribution of isotopic label. Comparison of the tritium content of the estrone obtained from the aromatization of androstenedione labeled predominantly in the 1 alpha,2 alpha positions with that in estrone obtained from a parallel incubation using substrate with label in the 1 beta,2 beta orientation gave an estrone alpha/beta ratio of 3.6. This ratio compares with a calculated value of 4.3 for an aromatization mechanism involving loss of the 1 beta,2 beta-hydrogens. The distortion from the predicted value is due to the loss of tritium from the alpha-substrate which is unrelated to aromatization. The ratio determined experimentally is compatible with 2 beta-tritium loss since random or alpha-elimination from C-2 would yield alpha/beta ratios of 2.2 and 1.3 respectively. In an analogous manner the stereochemistry of tritium loss at C-1 was determined using [1 alpha-3H] and [1 beta-3H]androstenedione. The alpha/beta ratio of the isolated estrone was 3.6 which is in good agreement with the calculated value of 3.3 for 1 beta-tritium elimination. Our results therefore show that estrogen formation in the brain occurs with the same stereospecificity of hydrogen loss at C-1 and C-2 as in placental microsomes.     

Androgen and estrogen dynamics: stability over a two year interval in peri-menopausal women

As part of a study on hormones and bone density in peri-menopausal women, metabolic clearance rates (MCR), and interconversions of androgens and estrogens and the peripheral aromatization of androgens were measured twice 2 yr apart. Measurements of clearance rates and interconversions were made from blood samples obtained during constant infusions of [3H]androgens and [14C]estrogens. Measurements of peripheral aromatization were made from the estrogen glucuronides in a pooled 4-day urine collection timed from the start of the infusions. The women were divided into 3 groups: Group A (n = 15) were having menstrual cycles throughout the 2 yr interval; Group B (n = 11) were having menstrual cycles at the time of Study 1 but had been amenorrheic for at least 1 yr at the time of Study 2; Group C (n = 28) were amenorrheic for at least 1 yr at the time of Study 1 and had remained amenorrheic through Study 2. The MCRs for testosterone, androstenedione, estrone and estradiol were not different for Study 1 and Study 2 in any of the groups. The interconversions of the androgens were similar in both studies for all groups. The conversion of estrone to estradiol decreased in Group A, otherwise the interconversions of the estrogens did not vary between the studies for the other groups. The peripheral aromatization of androstenedione, but not of testosterone, was significantly greater at study 2 compared to Study 1 for all groups. We conclude that the MCRs and interconversions of androgens and of estrogens are stable over time, but that the peripheral aromatization of androstenedione increases over a 2 yr interval. This increase may be menopausal and/or age related.     

Flash-induced absorption changes in Photosystem I,Radical pair or triplet state formation?

Absorption changes induced in chlorophyll protein (CP 1) particles by short laser flashes have been analyzed in order to decide whether a state lasting for a few microseconds at 21°C or 800 μs at 10 K corresponds to the biradical P-700⁺ ... A⁻₁ (A₁ being a chlorophyll a) or to a triplet state produced in a submicrosecond recombination of the preceding state. At 21°C the spectrum of the flash-induced ΔA (720–870 nm) presents a flat-topped band from 740 to 820 nm, clearly different from that of P-700⁺. A saturation curve (ΔA vs. laser energy), obtained with a 2 or 10 ns laser pulse, indicates that ΔA saturates at a value 2- or 3-times smaller than that expected on the basis of the chemical oxidation of P-700. At 21°C the size of flash-induced ΔA is slightly decreased (5–15%) when the sample is subjected to a 400 G magnetic field. The kinetics of decay are not affected; they are not affected either by the oxygen concentration. At 10 K the spectrum of the flash-induced ΔA has been measured between 650 and 1700 nm. Between 650 and 720 nm, the spectrum presents only one major negative peak at 702 nm; it is quite different from that due to the chemical oxidation of P-700 (which has additional peaks at 688 and 677 nm). Between 720 and 870 nm, the spectrum is identical to that obtained at 21°C. Above 870 nm, the spectrum includes a broad band around 1250 nm, which is absent in P-700⁺. A saturation curve leads to a maximum ΔA greater than that at 21°C and which is also greater with a 1 μs dye laser flash than with a 10 ns ruby laser flash. An analysis of the spectral data indicates that these do not fit correctly with the hypothesis of a contribution of P-700⁺ and of a chlorophyll a anion radical. They fit more closely with the hypothesis of a triplet state of P-700, a hypothesis which is discussed in relation to other experimental data.     

Lipoprotein-associated paf (LA-paf) was found in washed human platelets and monocyte/macrophage-like U937 cells

Beyond cholesterol, inflammatory ether phospholipids such as platelet-activating factor (paf) may play a role in atherogenesis. (1) We detected a paf-like compound (‘LA-paf’) associated with human serum lipoproteins, mainly in LDL but not with the lipoprotein-poor fraction. (2) LA-paf was also found in washed human platelets, from where it was partially released during platelet aggregation in response to paf (50 nM) or thrombin (1 U). In addition, resident monocyte/macrophage-like U937 cells carried huge amounts of LA-paf (41 ng per 10⁷ cells) and metabolized added [³H]paf to a labelled compound co-eluting with the retention time of LA-paf in standard HPLC. (3) Functionally, LA-paf had a comparable potency to synthetic paf, because LA-paf aggregated washed aspirin-treated platelets in a concentration-dependent manner. The specific paf receptor antagonist WEB2086 inhibited the platelet aggregation induced by three distinct LA-paf preparations as compared with synthetic paf with similar inhibitory concentrations (IC₅₀: 35.6 ± 12.8, 24.0 ± 4.0, 38.0 ± 15.8 nM for LA-paf, and 43.6 ± 6.5 nM for synthetic paf), indicating that LA-paf interacted with paf receptors. (4) However, LA-paf had a distinct retention time using high-pressure liquid chromatography (HPLC) as compared with synthetic paf. LA-paf eluted at 9–15 min and synthetic paf at 21–24 min. In addition, total and non-specific [³H]paf binding to intact washed human platelets was affected differently by the two unlabelled agonists: while LA-paf increased total and non-specific (but not specific) binding in a significant manner (P < 0.002 and P < 0.007) as LDL did (P < 0.006 and P < 0.03), synthetic paf decreased total binding (P < 0.03). Similarly, low-density lipoproteins (LDL) increased significantly the total [³H]paf binding. In contrast, paf did not affect specific [¹²⁵I]LDL binding to human fibroblasts. Our results show the presence of LA-paf in lipoproteins,     

Effects of ovarian tissue reduction on the menstrual cycle: persistent normalcy after near-total oophorectomy

These experiments were designed to evaluate whether removal of approximately 95% visible ovarian tissue would interrupt the short- or long-term regulation of cyclic ovarian function. On cycle Days 2 4 (onset of menses = Day 1), the entire left ovary and approximately 90% of the right ovary were removed from three cycling cynomolgus monkeys. After approximately 95% ovariectomy, there was an acute elevation of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which lasted 11 +/- 2 days. A midcycle-like gonadotropin surge occurred 20 +/- 3 days following approximately 95% ovariectomy; the next menses occurred 19 +/- 1 days later. Follicular phase patterns of estradiol preceded the midcycle gonadotropin surge, and luteal phase progesterone levels indicated subsequent ovulation. Two of three monkeys resumed normal menstrual cyclicity in the following cycle with follicular phase, luteal phase, and menstrual cycle lengths similar to pretreatment levels. Histological examination of the ovarian remnant removed on Day 21 of the next cycle revealed a morphologically normal corpus luteum and many small follicles. A second group of 6 rhesus monkeys also underwent approximately 95% ovariectomy for long-term evaluation of menstrual cyclicity; typical 28-day menstrual cycle patterns were observed in 4 of the 6 monkeys for 5 mo, with 2 of these 3 animals maintaining regular menstrual cycles for 1 yr. In summary, our data suggest that normal ovarian function, i.e. recruitment, selection, and dominance of the ovulatory follicle, ovulation, and subsequent corpus luteum function, is maintained with only approximately 5% of functional ovarian tissue remaining.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of genistein on steroid hormone production in the pregnant rhesus monkey.

Genistein is a phytoestrogen found in soy beans. Phytoestrogens have been reported to cause reproductive problems in sheep and rats. This research was conducted to determine the effects of genistein fed to rhesus monkeys during pregnancy, with specific interest on fetal growth and steroidogenesis in the maternal-fetoplacental unit. Two groups of five monkeys each were selected in early stages of pregnancy. One group was administered genistein in a fruit treat each weekday until Cesarean section 10 days prior to term. The second, control group, received fruit treats without genistein. Maternal blood samples were collected on Tuesday and Friday of each week. At delivery, samples were collected from the maternal peripheral circulation, uterine veins, uterine-ovarian veins, and the fetal heart. Comparisons between control and genistein-treated monkeys revealed no differences in the maternal weight gained during pregnancy, or in fetal weights or placental weights at delivery. Serum was assayed by radioimmunoassay (RIA) for estradiol, progesterone, dehydroepiandrosterone sulfate (DHEA-S), and estrone. No significant differences (P > 0.05) were noted in progesterone or DHEA-S levels at delivery or during the pregnancy; however, estradiol levels were higher (P < 0.05) in the four areas studied at delivery and in the maternal blood with advancing gestation. Additionally, estrone levels tended to increase more rapidly (P = 0. 057) in the maternal blood of monkeys receiving genistein than in untreated controls, suggesting that genistein may stimulate the deconjugation of estrone in the gut, thus allowing its reabsorption into the peripheral circulation and conversion to estradiol.     

Maternal estrogen and progesterone levels after hypophysectomy in early pregnancy and in term fetuses or newborn monkeys

Pregnant rhesus monkeys ($\text{Macaca}$$\text{mulatta}$) were hypophysectomized at 8–10 weeks gestation to determine effects on plasma levels of estrone (E₁), estradiol-17β (E₂) and progesterone (P). The first group of monkeys was subsequently fetectomized At 107–114 days. After hypophysectomy there was an initial decrease in maternal peripheral plasma E₂ followed by a rise to preoperative levels within 4–5 weeks. Plasma levels of e₁ and P were not markedly altered. After fetectomy, peripheral estrogen concentrations, especially E₂, declined markedly. In the second experimental series, we have examined the effects of maternal hypophysectomy on levels of E₁, E₂ and P either (1) in both mother and newborn baby or (2) in mother, term fetus and umbilical vein. Groups of hypophysectomized and intact pregnant monkeys (3 each) were delivered by cesarean section at the expected time of parturition. Other hypophysectomized and intact monkeys (2 each) delivered normally. E₂ levels were elevated significantly in plasma of hypophysectomized monkeys at the time of cesarean delivery and in newborn babies of hypophysectomized mothers shortly after parturition. Except for these differences, the maternal hypophysis apparently is not a major factor in the control of E₁, E₂ and P concentrations in pregnant rhesus monkeys.     

A method to reduce the invasiveness of fetal catheterization in the cow

Surgical intervention in general anesthesia (GA) of the cow in late gestation is a stressful condition for both mother and fetus, potentially leading to premature delivery or fetal death. The present study hypothesized that fetal catheterization at days 246–253 (90% of gestation) is done with less physical and metabolic stress for the mother and fetus, when the surgery is performed on a standing cow and local anesthesia (LA) rather than on a recumbent cow in general anesthesia. Fetal and uterine maternal intra-vascular catheters were implanted during general anesthesia (GA, n=24) or local analgesia (LA, n=7). Blood gases and metabolite levels in the fetal calves and their mothers were measured during surgery and for 5 days post-operatively. During surgery, venous blood pH was higher (7.44±0.01 versus 7.39±0.01, P<0.05) and hemoglobin and oxygen contents lower in LA cows compared with GA cows (9.3±0.3 mg/dl versus 11.8±0.2 mg/dl, P<0.001 and 10.1±0.3 ml/dl versus 12.6±0.6 ml/dl, P<0.05). The differences between the two groups of fetuses reflected those of their dams in that LA fetuses showed lower arterial oxygen pressure (18.3±1.4 mmHg versus 24.8±1.4 mmHg, P<0.05) and hemoglobin (7.81±0.30 mg/dl versus 9.22±0.21 mg/dl P<0.01) and furthermore, they also showed higher blood glucose (2.4±0.2 mM versus 1.4±0.1 mM, P<0.01). During the 5 days post-surgery, 10 GA fetuses (42%) and 1 LA fetus (14%) died in utero. Bacterial contamination was implicated in six of the GA deaths and in the one LA death. In the dams with surviving calves, differences in hemoglobin (9.49±0.21 mg/dl versus 11.17±0.23 mg/dl P<0.001) and O₂ct (10.9±0.3 ml/dl versus 12.5±0.5 ml/dl, P<0.05) were still present, and in addition, blood glucose was higher in LA versus GA cows (4.3±0.2 mM versus 3.8±0.1 mM, P<0.05). The choice of surgical method did not affect post-surgery blood chemistry in the surviving fetuses, except that the higher blood glucose in the LA fetuses at surgery tended to be maintained also post-operatively (2.0±0.2 mM versus 1.5±0.1 mM, P=0.07). The observed differences in blood chemistry parameters between the two methods of surgery and possibly in the fetal death may be explained by differences in catheterization method and the associated differences in physical and metabolic stress during and after surgery. Thus, surgery upon a standing cow in local anesthesia should be considered as an alternative to surgery in universal anesthesia for fetal catheterization in the cow in late gestation.