OBJECTIVE

this analysis was to investigate the effects of dehydroepiandrosterone (DHEA) on cardiovascular risk factors in older women with frailty characteristics.

METHODS

the study was a double-blind, randomised, placebo-controlled trial of 99 women (mean 76.6 +/- 6.0 year) with the low DHEA-S level and frailty.

METHODS

participants received 50 mg/day DHEA or placebo for 6 months; all received calcium (1,000-1,200 mg/day diet) and supplement (combined) and cholecalciferol (1,000 IU/day). Women participated in 90-min twice weekly exercise regimens, either chair aerobics or yoga.

METHODS

assessment of outcome variables included hormone levels (DHEA-S, oestradiol, oestrone, testosterone and sex hormone-binding globulin (SHBG)), lipid profiles (total cholesterol, high density lipoprotein (HDL) cholesterol, low density lipoprotein (LDL) cholesterol and triglycerides), body composition measured by dual energy absorptiometry, glucose levels and blood pressure (BP).

RESULTS

eighty-seven women (88%) completed 6 months of study; 88% were pre-frail demonstrating 1-2 frailty characteristics and 12% were frail with>> or =3 characteristics. There were significant changes in all hormone levels including DHEA-S, oestradiol, oestrone and testosterone and a decline in SHBG levels in those taking DHEA supplements. In spite of changes in hormone levels, there were no significant changes in cardiovascular risk factors including lipid profiles, body or abdominal fat, fasting glucose or BP.

CONCLUSIONS

research to date has not shown consistent effects of DHEA on cardiovascular risk, and this study adds to the literature that short-term therapy with DHEA is safe for older women in relation to cardiovascular risk factors. This study is novel in that we recruited women with evidence of physical frailty.

Oestrogens exert important effects on the reproductive as well as many other organ systems in both men and women. The history of the discovery of oestrogens, the mechanisms of their synthesis, and their therapeutic applications are very important components of the fabric of endocrinology. These aspects provide the rationale for highlighting several key components of this story. Two investigators, Edward Doisy and Alfred Butenandt, purified and crystalized oestrone nearly simultaneously in 1929, and Doisy later discovered oestriol and oestradiol. Butenandt won the Nobel Prize for this work and Doisy's had to await his purification of vitamin K. Early investigators quickly recognized that oestrogens must be synthesized from androgens and later investigators called this process aromatization. The aromatase enzyme was then characterized, its mechanism determined, and its structure identified after successful crystallization. With the development of knock-out methodology, the precise effects of oestrogen in males and females were defined and clinical syndromes of deficiency and excess described. Their discovery ultimately led to the development of oral contraceptives, treatment of menopausal symptoms, therapies for breast cancer, and induction of fertility, among others. The history of the use of oestrogens for postmenopausal women to relieve symptoms has been characterized by cyclic periods of enthusiasm and concern. The individuals involved in these studies, the innovative thinking required, and the detailed understanding made possible by evolving biologic and molecular techniques provide many lessons for current endocrinologists.

Although the majority of pregnancy failures occur during the embryonic period, reports indicate that approximately 5% of detected pregnancies are lost during the fetal period, underlining the fact that fetal death is a substantial cause of economic loss. However, examination for fetal development or death during pregnancy is not performed routinely in domestic animals, and reference curves for normal fetal growth are, therefore, scarce. In this paper, the numerous possible causes of fetal death are reviewed briefly, with emphasis on the role of placental problems in fetal death and impaired fetal viability. In this respect, the role of placental insufficiency as a cause of pregnancy loss in twin pregnancies in monotocous species is well known, whereas the abnormal placental development leading to retarded fetal growth during pregnancies in recipients of in vitro produced (IVP) or nuclear transfer (NT) embryos has been less extensively documented. Fetal viability or death can be evaluated using hormonal, chemical and ultrasonographic parameters. For example, the viability of the feto-placental unit can be examined by measuring maternal plasma concentrations of oestrone sulphate or the placental proteins, including pregnancy-associated glycoprotein (PAG) and pregnancy-specific protein B-60 (PSPB-60). Low concentrations of any of these three indicate either no pregnancy, or if pregnancy was confirmed earlier, fetal death and abnormally high or low levels can indicate fetal abnormality. Ultrasound can be used to examine the fetal heart rate (FHR), the incidence of fetal movements (FM), the appearance of fetal fluids and the development of the fetus and placenta. However, although abnormal FHRs have been correlated to subsequent fetal death, it is important to remember that there is a large physiological variation in FHR at the end of gestation, due to different behavioural states and differences in FM patterns. Although monitoring fetal viability and death using hormonal and ultrasonographic evaluations is possible during pregnancy in domestic animals, there is considerable physiological variations in the 'normal' values. Therefore, suitable combinations of tests need to be identified and more accurate reference values generated before such approaches can be considered reliable for monitoring the status of individual fetuses.

Using radioimmunological methods, levels of cortisol, dehydroepiandrosterone, androstenedione, testosterone, oestrogens (oestradiol + oestrone), progesterone and 17 alpha-hydroxyprogesterone were determined in adipose tissue and peripheral blood obtained during surgical treatment of patients with non-endocrine diseases. The steroid content of human adipose tissue was observed to be extremely high relative to that in the general circulation, giving a tissue/serum ratio of 0.4 to 13.2. The concentration of steroids decreased in the following order: dehydroepiandrosterone greater than cortisol greater than androstenedione greater than progesterone greater than testosterone greater than 17 alpha-hydroxyprogesterone greater than oestradiol + oestrone. This sequence is different from that found in blood. When anthropometric variables were taken into consideration, the adipose tissue mass of severely obese subjects contained a steroid pool far greater than that in the total blood volume.

Oestrone and dihydrotestosterone had no significant action. Other steroids inhibited maturation. The stage of maturation most affected and the median effective concentration (MEC) at this stage varied with different steroids. The predominant effect of pregnenolone (MEC = 6.4 microM), progesterone (MEC = 5.3 microM), androstenedione (MEC = 28.0 microM) and oestradiol-17 beta (MEC = 23.0 microM) was to block maturation after the resumption of meiosis but before completion of the first meiotic division. Testosterone was also effective at this stage (MEC = 23.5 microM) but at higher concentrations it prevented germinal vesicle breakdown (MEC = 40 microM) without causing oocyte degeneration. The inhibitory actions of all steroids were reversible in oocytes exposed for 4 or 18 h.

The shortening of the time interval between the onset of oestrus and ovulation in sows by the transcervical administration of seminal plasma was investigated in 23 German Landrace gilts, using the technique of single horn infusions (Mariensee model) in combination with the transcutaneous sonographic monitoring of ovaries. Preparative surgery comprised the detachment of the left uterine horn from the corpus, leaving the caudal end open to the peritoneal cavity but sealing the corpus wound. The left ovary was loosely tied to the ventral abdominal wall for better sonographic distinction. The animals were used in two to four consecutive cycles. After detection of oestrus by the teaser boar, the patent (right) horns were filled by transcervical infusion of 100 ml of a variety of test solutions. Ovulation was probed by transcutaneous sonography at intervals of 4 h thereafter. Native seminal plasma provoked ovulation in the ipsilateral ovary of the treated horn 10.7 h earlier than in the contralateral ovary. This effect was reduced to 7.3 h after charcoal treatment of seminal plasma; addition of 10 micrograms oestradiol restored the effect in full, while 10 micrograms of oestradiol in PBS shortened the time interval to only 3.3 h versus the control ovary. Little effect was seen with oestrone sulfate, none with prostaglandins in PBS or with PBS alone. The preliminary characterization of the nonsteroidal component of seminal plasma advancing ipsilateral ovulation after transcervical infusion suggests a proteinaceous nature. The activity resides in the 1-10 kDa fraction separated by ultrafiltration and is lost after treatment with pronase.

Aromatase activity and concentrations of cortisol, progesterone and testosterone were measured in samples of breast and abdominal adipose tissue obtained from both pre- and postmenopausal subjects. Enzyme activity was determined by the incorporation of tritium from [1 beta-3H]androstenedione into water and found to be in close agreement to that measured when tritium labelled oestrone (E1) and oestradiol (E2) were isolated. No significant difference in enzyme activity was noted between breast and abdominal adipose tissue. Increased aromatase activity was not observed in adipose tissue taken from a subject with endometrial cancer. Cortisol concentrations were found to be significantly higher (P less than 0.05) in abdominal as compared to breast tissue. Without attaining statistical significance progesterone concentrations were higher in abdominal as compared to breast adipose tissue. Aromatase activity was not related to either cortisol or testosterone tissue concentration, but an inverse relationship between progesterone concentration and aromatase activity was observed (r = 0.542, P less than 0.02). On the basis of results obtained a hypothesis for the increased conversion of androgen to oestrogen as seen after the menopause has been proposed.

1. It was confirmed that bilirubin glucuronyltransferase can be obtained in solubilized form from rat liver microsomes. 2. Michaelis-Menten kinetics were not followed by the enzyme with bilirubin as substrate when the bilirubin/albumin ratio was varied. High concentrations of bilirubin were inhibitory. 3. The K(m) for UDP-glucuronic acid at the optimum bilirubin concentration was 0.46mm. 4. Low concentrations of Ca(2+) were inhibitory in the absence of Mg(2+) but stimulatory in its presence; the converse applied for EDTA. 5. UDP-N-acetylglucosamine and UDP-glucose enhanced conjugation by untreated, but not by solubilized microsomes. 6. The apparent 9.5-fold increase in activity after solubilization was probably due to the absence of UDP-glucuronic acid pyrophosphatase activity in the solubilized preparation. 7. The activation of solubilized enzyme activity by ATP was considered to be a result of chelation of inhibitory metal ions. 8. The solubilized enzyme activity was inhibited by UMP and UDP. The effect of UMP was not competitive with respect to UDP-glucuronic acid. 9. A number of steroids inhibited the solubilized enzyme activity. The competitive effects of stilboestrol, oestrone sulphate and 3beta-hydroxyandrost-5-en-17-one, with respect to UDP-glucuronic acid, may be explained on an allosteric basis.

To explore the possibility that the wide variation in bone loss among oophorectomised women might be due to differences in adrenal androgens or their biosynthetic pathways, 18 women (10 with very fast and 8 with very slow bone loss) were selected. Serum levels of nine adrenal steroids, including the major androgens and cortisol, were measured under basal conditions and after overnight suppression followed by acute corticotropin stimulation. In addition, basal serum oestrone, oestradiol, dehydroepiandrosterone sulphate, sex-hormone-binding-globulin, corticosteroid binding globulin, and urinary free cortisol were measured. The only significant differences found were that women who lost bone rapidly had significantly higher urinary free-cortisol excretion (p less than 0.001) and a paradoxically diminished cortisol response to corticotropin. These data make it unlikely that endogenous adrenal androgens or oestrogens are a major factor in preventing bone loss after cessation of ovarian function; cortisol by its catabolic effect, however, may be a significant factor in causing osteoporosis.

BACKGROUND

It has been suggested that the relative importance of oestrogen-metabolising pathways may affect the risk of oestrogen-dependent tumours including endometrial cancer. One hypothesis is that the 2-hydroxy pathway is protective, whereas the 16α-hydroxy pathway is harmful.

METHODS

We conducted a case-control study nested within three prospective cohorts to assess whether the circulating 2-hydroxyestrone : 16α-hydroxyestrone (2-OHE1 : 16α-OHE1) ratio is inversely associated with endometrial cancer risk in postmenopausal women. A total of 179 cases and 336 controls, matching cases on cohort, age and date of blood donation, were included. Levels of 2-OHE1 and 16α-OHE1 were measured using a monoclonal antibody-based enzyme assay.

RESULTS

Endometrial cancer risk increased with increasing levels of both metabolites, with odds ratios in the top tertiles of 2.4 (95% CI=1.3, 4.6; P(trend)=0.007) for 2-OHE1 and 1.9 (95% CI=1.1, 3.5; P(trend)=0.03) for 16α-OHE1 in analyses adjusting for endometrial cancer risk factors. These associations were attenuated and no longer statistically significant after further adjustment for oestrone or oestradiol levels. No significant association was observed for the 2-OHE1 : 16α-OHE1 ratio.

CONCLUSIONS

Our results do not support the hypothesis that greater metabolism of oestrogen via the 2-OH pathway, relative to the 16α-OH pathway, protects against endometrial cancer.

In order to gain basic understanding in the ecotoxicity of endocrine disrupting chemicals or EDCs (including natural chemicals and some pharmaceuticals), many international research groups are currently testing these chemicals using aquatic invertebrates. This paper discusses relevant examples to address key questions: which aquatic invertebrates are likely to be vulnerable to mammalian and non-mammalian EDCs; and which types of invertebrate chronic tests might be most sensitive and cost-effective to address potential environmental exposures? For a full review of invertebrate endocrine disrupter research see Endocrine Disruption in Invertebrates: Endocrinology, Testing and Assessment (1999). As an example, crustaceans are a particular focus of EDC research, reflecting their abundance in nature, commercial importance and their inclusion in the regulatory assessment schemes for active pharmaceutical ingredients (APIs). There is a diverse literature on the developmental and reproductive effects of mammalian EDCs in Crustacea, although there is growing evidence that such effects are probably not mediated via arthropod hormone systems. For example, recent studies in Europe using a marine copepod (Tisbe battagliai) life-cycle test have evaluated ecdysteroid agonists (e.g. 20-hydroxyecdysone), oestrogen agonists (e.g. diethylstilbestrol (DES), 17beta-oestradiol, oestrone and 17alpha-ethynylestradiol) and the pharmaceutical anti-oestrogen (ZM189, 154). While 20-hydroxyecdysone and DES were highly toxic, the other compounds tested show no significant toxicity to copepods. Furthermore, in vitro studies indicate that these environmental EDCs and several related APIs are not active against the ecdysteroid receptor. Therefore, other undefined modes of action appear to be responsible for crustacean toxicity in vivo and caution should be exercised before ascribing any apical effects to endogenous endocrine mechanisms, or before crustacean "EDC" data are extrapolated to other invertebrate taxa.

OBJECTIVE

To explore the effect of different degrees of oestrogenization on male voiding, by treating adult castrated and 5alpha-dihydrotestosterone (DHT)-maintained male mice with different doses of oestrogens, as exposure of male mice to excessive amounts of oestrogens can cause bladder outlet obstruction (BOO); in addition, male mice lacking oestrogen receptor (ER)alpha (ERKO) or ERbeta (BERKO) were studied to assess the importance of ER subtypes.

METHODS

Castrated, DHT-maintained adult mice were treated with 17beta-oestradiol (E(2); 50 and 250 microg/kg) or oestrone (E(1); 5, 50 and 500 microg/kg) daily for 10 days. Control mice were treated only with the vehicle. BERKO and ERKO mice, and their wild-type littermates used as their controls, remained untreated. Under anaesthesia, the bladder and distal urethra were exposed to record simultaneously the bladder pressure and urinary flow rate from the distal urethra.

RESULTS

E(2)-treated mice showed obstructive voiding, seen as increased bladder pressure, decreased average flow rate and prolonged micturition time. This was also evident when a high dose (500 microg/kg) of E(1) was used. After treatment with a dose of 50 microg/kg, the urodynamic variables were similar to those in the control mice. Surprisingly, after treatment with a low dose (5 microg/kg) all urodynamic variables improved. There was a minor increase in the bladder pressure in BERKO mice; ERKO mice had a significantly lower urinary flow rate.

CONCLUSIONS

High doses of oestrogens caused BOO in castrated, DHT-maintained male mice. A small dose of E(1) had a positive effect on voiding, suggesting that oestrogens are needed for normal male voiding. Reduced urinary flow rates in ERKO mice suggest that oestrogen effects on voiding are mediated at least partly via ERalpha.

Obese subjects have increased bone density relative to non-obese subjects yet this relationship is not fully understood. We examined whether alterations in sex hormones or binding proteins might explain the effect of obesity on osteoporosis in 83 premenopausal women from the San Antonio Heart Study, a population-based study of diabetes. We measured total testosterone, oestradiol, oestrone, sex hormone binding globulin (SHBG), and serum dehydroepiandrosterone sulphate (DHEA-SO4). Bone density was assessed by a Hologic dual photon absorptometer. Lumbar spine and femoral neck density were positively correlated with body mass index (BMI). In addition, femoral neck density was positively correlated with DHEA-SO4. BMI was negatively correlated with SHBG. After adjustment for sex hormones by multiple linear regression a positive association between bone density and obesity still exists suggesting that the association between obesity and bone density is at least partially independent of sex steroids in premenopausal women.

The question of whether melanoma tumours have classical oestrogen receptors (ERs) is unresolved, but epidemiological data clearly show a survival benefit for female patients with metastatic melanoma. The aims of this study were to examine to what extent the presence of ER in melanoma tumours might relate to disease progression and whether disease progression relates to patients' sex steroid status. Additionally, levels of two soluble adhesion molecules [circulating intercellular adhesion molecule-1 (sICAM-1) and circulating vascular cell adhesion molecule-1 (sVCAM-1)] were examined as independent, possibly prognostic indicators of disease progression. ER immunocytochemical assay identified only two lesions (out of 69 investigated) which had any evidence of the receptors, and staining in these lesions was very modest. No significant changes in oestrone or androstenedione levels were noted for male or female patients with disease progression. As expected, oestradiol levels reflected the menopausal status of the female patients but, for all post-menopausal female patients and male patients, there was no significant relationship to tumour stage. However, a significant decrease in sex hormone-binding globulin occurred with disease progression in male but not female patients, and sex differences in the levels of soluble adhesion molecules were also seen in advanced metastatic disease.

43 postmenopausal breast cancer patients were treated orally with the aromatase inhibitor formestane (4-hydroxyandrostenedione) at daily doses of 62.5, 125, 250 or 500 mg for 4 weeks followed by 250 mg daily for a further 4 weeks. For some patients, 62.5 mg did not suppress serum oestradiol levels maximally. The doses of 250 and 500 mg did not differ in their effectiveness. Oestrone levels were suppressed by all doses of formestane but no consistent changes of aldosterone, cortisol or 17-hydroxyprogesterone occurred. Serum levels of sex hormone binding globulin fell by about 15% during treatment with 250 mg formestane reflecting its minor androgenic activity. The maximum concentration and area under the curve of serum formestane levels after the first dose varied in an approximately linear manner with dose. It is concluded that formestane is an effective, specific suppressant of oestradiol levels via the oral route requiring no more than 250 mg to be given daily.

Abnormal steroid secretion may contribute to anovulation in insulin dependent diabetic patients with amenorrhoea. We have measured serum sex hormone-binding globulin (SHBG) and free and bound oestrogen and androgen levels in 17 such patients. As controls we included 17 patients with insulin dependent diabetes mellitus and normal menstrual cycles, 21 regularly menstruating normal women (both sampled during early follicular phase), and 23 non-diabetic patients with amenorrhoea. The diabetic patients with normal cycles had significantly higher serum concentrations of delta 4-androstenedione and testosterone than the normal women (P less than 0.01). The amenorrhoeic diabetics in contrast had significantly lower serum concentrations of SHBG, 5 alpha-dihydrotestosterone and free and total oestradiol-17 beta than either group of menstruating women (P less than 0.05), and significantly lower concentrations of delta 4-androstenedione (P less than 0.01), dehydroepiandrosterone sulphate (P less than 0.01), testosterone (P less than 0.01), and oestrone (P less than 0.05), than the cycling diabetics. The two amenorrhoeic groups had similar free and bound sex hormone concentrations except that delta 4-androstenedione levels were significantly lower in the diabetics (P less than 0.01). We conclude that the low sex hormone levels in diabetic women with amenorrhea may be due to suppression of the hypothalamic-pituitary axis in view of the impaired LH secretion found in these patients and that excess androgen secretion seems not to be of aetiological importance in amenorrhea related to diabetes mellitus. The decreased steroid levels in amenorrheic diabetics is due to their suppressed ovarian function while the increased androgen levels in diabetics with regular cycles are probably of ovarian origin.

Several studies indicate that the beneficial or harmful effects of oestrogens in stroke are dose-dependent. Rats are amongst the most frequently used animals in these studies, which calls for thoroughly validated methods for administering 17β-oestradiol to rats. In an earlier study we characterised three different administration methods for 17β-oestradiol over 42 days. The present study assesses the concentrations in a short time perspective, with the addition of a novel peroral method. Female Sprague-Dawley rats were ovariectomised and administered 17β-oestradiol by subcutaneous injections, silastic capsules, pellets and orally (in the nut-cream Nutella(®)), respectively. One group received 17β-oestradiol by silastic capsules without previous washout time. Blood samples were obtained after 30 minutes, 1, 2, 4, 8, 12, 24, 48 and 168 hours and serum 17β-oestradiol (and oestrone sulphate in some samples) was subsequently analysed. For long-term characterisation, one group treated perorally was blood sampled after 2, 7, 14, 21, 28, 35 and 42 days. At sacrifice, uterine horns were weighed and subcutaneous tissue samples were taken for histological assessment. The pellets, silastic capsule and injection groups produced serum 17β-oestradiol concentrations that were initially several orders of magnitude higher than physiological levels, while the peroral groups had 17β-oestradiol levels that were within the physiological range during the entire experiment. The peroral method is a promising option for administering 17β-oestradiol if physiological levels or similarity to women's oral hormone therapy are desired. Uterine weights were found to be a very crude measure of oestrogen exposure.

BACKGROUND

Oral oestrogen replacement therapy increases levels of C-reactive protein (CRP). CRP is an established strong predictor of cardiovascular events. It is unknown whether endogenous oestrogen levels are associated with CRP. We therefore studied the relationship between endogenous sex hormones and CRP in healthy postmenopausal women emphasizing the role of body composition as peripheral fat is both a main source of oestrogen production after menopause and an endocrine tissue with inflammatory activities.

METHODS

The study population comprised 889 women participating in the PROSPECT study, an ongoing population-based cohort study. Information on risk factors was collected by questionnaires and clinical examination. Endogenous sex hormone levels and CRP were measured with double antibody radio immuno assay (RIA) from fasting plasma samples. In this cross-sectional study, associations between risk factors and lnCRP were studied using linear regression models.

RESULTS

Increases in oestrone and free oestradiol levels and the free androgen index were related to an increase in lnCRP of 1.19, 1.23 and 1.21 mg dL(-1) respectively. Body mass index (BMI), waist circumference and physical activity were strongly related to CRP levels, independent of age and other cardiovascular risk factors. Levels of all sex steroids but dehydroepiandrostenedione decreased with age. In age-adjusted analyses, an increase in waist circumference or BMI by one quartile was associated with a 1.28-fold and 1.26-fold increase in CRP. The relationship between endogenous hormones and CRP was modestly attenuated but remained highly significant after adjustment for body composition, physical activity and other traditional cardiovascular risk factors.

CONCLUSIONS

Our findings show that in postmenopausal women high levels of endogenous oestrogenic and androgenic sex steroids coincide with high CRP levels. This was only explained in part by markers of body composition or intra-abdominal fat.

Twenty-four postmenopausal women with vaginal atrophy due to oestrogen deficiency were treated with 17 beta-oestradiol administered as vaginal tablets containing 10 and 25 micrograms, respectively, in a slow-release system (Vagifem, Novo Nordisk, Denmark). All the women were treated for 2 weeks with each dose in a double-blind, cross-over study. Plasma concentrations of unconjugated oestradiol and unconjugated oestrone were measured at regular intervals for 24 h on days 1 and 14 of each treatment regimen. Cytological and clinical evaluations of the vaginal and urethral epithelium were also carried out. Initially, when the epithelium was still atrophic, dose-dependent absorption of oestradiol was demonstrated. After 14 days of treatment maturation of the vaginal epithelium was seen with both regimens and the absorption of oestradiol then declined significantly on both the 10 and the 25 micrograms dose. Oestrone levels remained unchanged and gonadotrophin levels were unaffected during treatment. Vaginal cytology showed maturation on both the 10 and the 25 micrograms dose, whereas urethral cytology showed a reduction in parabasal cells that was significant only on 25 micrograms. Clinical and subjective improvement was apparent on both doses and acceptance of treatment was good.

A 28-year-old male pseudohermaphrodite with gynaecomastia was raised as a female until the age of 17 years, at which time he developed masculine features (deepening of the voice, development of facial hair, male distribution of body hair and male body habitus) and assumed a male gender role. He had a small phallus with perineal urethra, absence of labioscrotal fusion, presence of vaginal pouch and undescended testes. The testicular biopsy showed hyalinization of the tubular basement membrane, lack of spermatogenesis and hyperplastic Leydig cells. Baseline peripheral plasma studies showed androstenedione concentrations ten times normal, low testosterone, elevated oestrone and elevated gonadotrophins. The in vitro incubation of testicular tissue showed no significant conversion of androstenedione to testosterone. However, two types of peripheral tissues, skin fibroblasts and erythrocytes, had a normal conversion, as did the body overall as measured by the technique of androstenedione constant infusion. These studies demonstrate that the 17-ketosteroid reductase deficiency of the patient was limited to the testes.

Cyclical changes in concentration of plasma progesterone, urinary oestrone-conjugates and urinary luteinizing hormone (LH) were compared in young and older cotton-top tamarins (Saguinus oedipus) and saddle-backed tamarins (S. fuscicollis). A group of six young adult tamarin females (4-5 years of age) was sampled over eight periods of 6-8 weeks and six older (14-20 years of age) females were sampled over thirteen periods. There was hormonal evidence of ovulation in all of the sampling periods for young females; in five of thirteen periods, older females displayed no evidence of ovulation. Of the six older females, two were anovulatory in one sampling period, while one female displayed no evidence of ovulation in any of three sampling periods. Generally, females over 17 years of age either did not ovulate or displayed abnormally long periods of moderate concentrations of progesterone and oestrone conjugates. Basal concentrations of LH differed in individuals, but were not always higher in older females. In contrast to patterns of reproductive senescence in other primates, older, anovulatory tamarins displayed moderate concentrations of urinary oestrone conjugates (5-50 micrograms/mg creatinine) and plasma progesterone (8-19 ng/ml), both of which are hormones of probable luteal origin in these species. This result suggests continued production of steroids by the luteal cells of the prominent interstitial gland in reproductively senescent tamarins. This suggestion was reinforced by histological examination of the ovaries of four older, anovulatory females; few primary follicles were found. Three females had no normal antral follicles, but all females had large luteal masses. The presence of functional luteal cells in the older ovaries, which do not experience regular follicular development, may distinguish ovarian ageing in New World primates from that of Old World primates.

Human oestrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD1) catalyses the final step in the biosynthesis of all active oestrogens. Here we report the steady-state kinetics for 17beta-HSD1 at 37 degrees C and pH 7.5, using a homogeneous enzyme preparation with oestrone, dehydroepiandrosterone (DHEA) or dihydrotestosterone (DHT) as substrate and NADP(H) as the cofactor. Kinetic studies made over a wide range of oestrone concentrations (10 nM-10 microM) revealed a typical substrate-inhibition phenomenon. Data analysis using the substrate-inhibition equation v=V.[s]/[K(m)+[s](1+[s]/K(i))] gave a K(m) of 0.07+/-0.01 microM, a k(cat) (for the dimer) of 1.5+/-0.1 s(-1), a specificity of 21 microM(-1) x s(-1) and a K(i) of 1.3 microM. When NADH was used instead of NADPH, substrate inhibition was no longer observed and the kinetic constants were significantly modified to 0.42+/-0.07 microM for the K(m), 0.8+/-0.04 s(-1) for the k(cat) and 1.9 microM(-1) x s(-1) for the specificity. The modification of an amino acid in the cofactor-binding site (Leu36Asp) eliminated the substrate inhibition observed in the presence of NADPH, confirming the NADPH-dependence of the phenomenon. The possible formation of an enzyme-NADP(+)-oestrone dead-end complex during the substrate-inhibition process is supported by the competitive inhibition of oestradiol oxidation by oestrone. Kinetic studies performed with either DHEA (K(m)=24+/-4 microM; k(cat)=0.47+/-0.06 s(-1); specificity=0.002 microM(-1) x s(-1)) or DHT (K(m)=26+/-6 microM; k(cat)=0.2+/-0.02 s(-1); specificity=0.0008 microM(-1) x s(-1)) in the presence of NADP(H) resulted in low specificities and no substrate inhibition. Taken together, our results demonstrate that the high specificity of 17beta-HSD1 towards oestrone is coupled with an NADPH-dependent substrate inhibition, suggesting that both the specificity and the enzyme control are provided for the cognate substrate.