Effects of clofibrate on the metabolism of progesterone and oestradiol in rat liver microsomal fraction

1. The substrate conversion of [4-(14)C]progesterone and [4-(14)C]oestradiol during incubation with the liver microsomal fraction from both control and clofibrate-treated rats amounted to about 10-15 and 20-25% respectively. 2. The metabolites of progesterone formed by preparations from control rats were hydroxylated in the 16alpha-position (14%), the 6beta-position (12%) and the 2alpha-position (7%). Of the products formed from oestradiol 12% were recovered as a 16alpha-hydroxylated derivative whereas 5% had a 6beta- and 2% a 6alpha-hydroxyl group. 3. Clofibrate affected the microsomal metabolism of both progesterone and oestradiol. It induced 7alpha-hydroxylation of both compounds, metabolic conversions not found in control rats. The 6beta-hydroxylation of progesterone and the 6alpha-hydroxylation of oestradiol were enhanced by a factor of 2 and 3.5 respectively. The 2alpha-hydroxylation, and the 20alpha- and 20beta-hydroxy steroid reduction of progesterone were significantly decreased as were the 16alpha- and the 6beta-hydroxylation of oestradiol.     

The effects of androgens and cortisol on the in vivo metabolism of oestradiol

We have examined the effect of co-administration of dehydroepiandrosterone sulphate, 5-androstenediol or cortisol on the metabolic clearance rate of oestradiol (MCR-E2) and conversion of oestradiol to oestrone (CRE2E1). Previous studies have shown that these androgens influence the metabolism of oestradiol in vitro while cortisol alters the distribution of oestradiol in plasma. The MCR-E2 and CRE2E1 were measured after 2.5 and 5 h of [3H]oestradiol infusion with co-infusion of androgen or cortisol starting after 2.5 h of tracer infusion. For one subject who did not receive co-infusion of another steroid no significant change in MCR-E2 or CRE2E1 occurred over the 5-h period. For other subjects, however, the MCR-E2 decreased by 18 +/- 7% (mean +/- SD) while the CRE2E1 increased by 45 +/- 12%. It is possible that these results are due to: changes in the distribution of oestradiol in plasma; differences in the metabolism of oestradiol bound to albumin or SHBG, or an effect of androgens or cortisol on the uptake of [3H]oestradiol by the liver.     

The use of nonradiolabelled steroid infusions to investigate the origin of oestrone sulphate in postmenopausal women

Infusion of nonradiolabelled dehydroepiandrosterone sulphate (DHA-S) has been used to investigate the possible formation of oestrone sulphate via a sulphated conjugate of androstenedione. The metabolic clearance rate (MCR) of DHA-S also was measured and the mean value (25 1/24h) was similar to values reported using isotopic techniques. Although conversion of DHA-S to 5-androstenediol, a steroid with oestrogenic properties, was detected during infusion of DHA-S, there were no significant increases in plasma levels of conjugated androstenedione or oestrone sulphate. The MCR's oestrone sulphate measured using infusion of nonradiolabelled steroid in two menopausal women were 99 1/24h and 121 1/24h. For one woman, the production rate of oestrone sulphate, calculated from the conversion of oestrone and oestradiol to oestrone sulphate (151 nmol/day) was similar to the measured production rate of oestrone sulphate (144 nmol/day). It is concluded that in menopausal women, oestrone sulphate is derived from conversion of oestrone and oestradiol with no formation occurring via conjugated androstenedione.     

Arrest of somatic embryo development in grapevine: histological characterization and the effect of ABA,BAP and zeatin in stimulating plantlet development

In an effort to understand the causes of arrest of somatic embryo development, generally observed in grapevine (Vitis sp.), histological studies were undertaken, using two cultivars (CH76 and 41B) which differ in their ability to develop into plants. Embryos with a high conversion rate (70%; CH76) formed a well-structured and functional shoot apex between two thread-like cotyledons. In contrast, embryos with a low conversion rate (10%; 41B) formed a normal root apex but lacked a well-structured shoot apex and developed a wide range of aberrant forms in the intercotyledonary area: uncontrolled cellular proliferation, formation of adventitious buds, over-growth of cotyledonary or leaf meristems. ABA increased the conversion rate of 41B embryos from 10% to 20%, but failed to improve embryo morphology. Zeatin and BAP promoted growth of 41B somatic embryos, but generated a high level of abnormalities and failed to improve conversion rate. Applied in combination with ABA, these PGRs increased the frequency of cotyledonary embryos, but decreased the conversion rate.     

A comparative study of the interaction of oestradiol and the steroidal pure antioestrogen, ICI 164,384, with the molybdate-stabilized oestrogen receptor

The kinetics of binding of oestradiol and the steroidal pure antioestrogen ICI 164,384 to the molybdate-stabilized oestrogen receptor, partially purified from pig and human uterine tissue, were determined. ICI 164,384 bound directly to the oestrogen receptor protein and the kinetic parameters of this interaction were, in general, similar to those for the binding of oestradiol, regardless of the source of the receptor protein. However, the rate of association of oestradiol, regardless of the source of the receptor protein. However, the rate of association of the antagonist with the receptor protein was slower when compared to that of oestradiol. Furthermore, the concentration of binding sites for the two ligands was of the same order. The binding of oestradiol resulted in a steroid-receptor complex which could be transformed in vitro, to a form with increased affinity for DNA-cellulose. However, the complex formed between ICI 164,384 and the receptor protein did not show increased affinity for DNA-cellulose when exposed to conditions that transformed agonist-receptor complexes. Therefore, the binding of ICI 164,384 to the oestrogen receptor protein results in a suppression of the transformation process. A similar suppression in vivo may account for the pure antagonist properties of ICI 164,384.     

17 Beta-hydroxysteroid oxidoreductase activity: age-dependent profile in rat liver and kinetic properties of the hepatic microsomal enzyme in relation to cytochrome P450-dependent steroid hydroxylation

The functional relationship between the microsomal cytochrome P450 and 17 beta-hydroxysteroid oxidoreductase (HSOR) enzymes involved in steroid metabolism was investigated in rat liver. In male and female rat hepatic microsomes the NADPH-dependent conversion of androstenedione (AD) to testosterone (T) was approx. 4-fold greater at 6 weeks of age than in 1 week old animals. In hepatic microsomes from 15 week old rats the activity of the HSOR pathway was greater in males than in females (1.51 compared to 0.80 nmol T formed/min/mg protein). However, oestradiol administration to intact adult male rats did not decrease HSOR activity. Thus, androgen is not essential for maintenance of HSOR enzymes. Instead, it is likely that irreversible androgen imprinting of the HSOR enzyme occurs during the prepubertal period. The in vitro characteristics of HSOR activity were also assessed. The Km for NADH-dependent reduction of AD to T was 9.2 microM and the Vmax was 3.0 nmol/min/mg protein but the NAD-mediated formation of AD from T did not follow Michaelis-Menton kinetics. pH markedly influenced HSOR-mediated AD/T interconversion with 17-ketosteroid reduction facilitated at low pH, and 17 beta-hydroxysteroid dehydrogenation about 2-fold more efficient at pH 8.0 than at pH 5.5. Product steroid activation of HSOR activity was noted. 17 beta-Hydroxysteroids, including T and oestradiol, activated the rate of conversion of AD to T and 17-ketosteroids such as oestrone and AD activated the NAD-dependent dehydrogenation of T. Activation was not observed at low steroid substrate concentrations so that it was not possible to analyse this phenomenon by a conventional kinetic approach.     

Morphological and functional characterization of large antral follicles in three breeds of sheep with different ovulation rates

Morphological and functional features of large ovarian follicles from three breeds of sheep, with different ovulation rates (Finnish Landrace N = 12, Finnish Landrace X Scottish Blackface N = 16, Merino X Scottish Blackface N = 16) were compared by integrating three techniques; ink labelling, in-vitro oestradiol production and morphological classification. The follicles were removed at two stages of the follicular phase, 1 (PG + 1) or 2 (PG + 2) days after PGF-2 alpha treatment and compared after monitoring their rates of growth with the use of ink labelling. After ovariectomy all follicles greater than or equal to 1 mm in diameter were dissected, and the 8 largest were incubated individually for 2 h to assess their ability to secrete oestradiol and testosterone. After incubation the follicles were processed for histological examination and checked for atresia. An analysis of the follicle population was based on in-vitro oestradiol secretion rates in all three breeds; an oestrogen-active population producing 500-8100 pg oestradiol/ml/h and an oestrogen-inactive population producing 0-499 pg oestradiol/ml/h. A comparison of the 3 approaches demonstrated agreement on 94.3 +/- 1.2% of occasions. Ink-labelling demonstrated that all follicles identified as oestrogen-active were increasing in size. Within oestrogen-active follicles significant correlations were detected between oestradiol production and testosterone production (r = 0.42), oestradiol production and granulosa cell number (r = 0.45) and between oestradiol production and mitotic index (r = -0.38). A regression model fitting breed, stage of atresia, granulosa cell number, in-vitro testosterone production and mitotic index demonstrated that granulosa cell number is a characteristic which contributes significantly to the variation of in-vitro oestradiol production in oestrogen-active and oestrogen-inactive follicles. There was no significant difference between breeds in the mean number of ink-labelled follicles growing from Day PG - 1 to Day PG + 1. There was a significant difference between the breeds in the number of ink-labelled follicles growing between Days PG + 1 and PG + 2 (Days 1 and 2 of the follicular phase), the number being similar to the ovulation rate for the breed. The majority of the oestrogen-active follicles had been recruited by Day PG - 1, although in the Finnish Landrace genotypes more than 30% were recruited on or after Day PG + 1 compared to less than 10% in Merino x Scottish Blackface ewes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of pituitary grafting on ovarian oestradiol secretion in the hypophysectomized chick embryo.

Chick embryos were hypophysectomized by partial decapitation at the stage of 42 h of incubation and grafted with a hypophysis from a 12-days-old donor embryo on the chorio-allantoic membrane at 9 1/2 days. Two days later, their ovary was removed for organ culture and its oestradiol secretion rate was compared to that of the ovary of hypophysectomized, non grafted control embryos. The oestradiol secretion rate in the grafted embryos was almost twice that in the hypophysioprivic embryos and in the range of that in normal embryos. This result suggests that the hypophysis controls oestradiol secretion of the ovary in the 11 1/2-days-old chick embryo.     

Non-classical inhibition of uricase by cyanide.

The existence of unoccupied nuclear oestradiol-receptor sites in normal human endometrium was investigated. Nuclei were prepared from endometrial samples obtained by curettage and exposed to [3H]oestradiol, which became maximmaly bound at 0 degrees C within 1 h. This result contrasted with the binding kinetics of oestradiol--receptor complexes, since the exchange of hormone took at least 3 h at 30 degrees C and no displacement occurred at 0 degrees C. Before concluding that the nuclear sites were unoccupied, the presence of endogenous low-affinity ligands was excluded, because the association rate of oestradiol was unchanged after nuclei were stripped from their putative ligands, and the displacement of oestrone bound to nuclear receptor by oestradiol was very slow at 0 degrees C. The available sites had high affinity for oestradiol (KD 1.3 nM) and binding-specificity characteristics of oestradiol receptors. Similar results were observed with crude and purified nuclear preparations. It was concluded that a significant proportion of nuclear oestradiol receptors in normal human endometrium is unoccupied by endogenous hormones.     

Effect of A-nor steroids and oestradiol on progesterone production by human luteal cells

Cell suspensions were prepared from human corpora lutea obtained during the mid-luteal phase. Progesterone production was assessed after short-term incubation of luteal cell suspensions. Luteal cells were very sensitive to hCG, the concentration required for 50% maximum response being 0.01 i.u./ml, and the response was 5 times higher than the basal production. Oestradiol (1-100 microM) induced a significant dose-related decrease in both basal and hCG-stimulated progesterone production. The A-nor steroidal compounds anordrin and AF-45 reduced hCG-stimulated progesterone production only at the high concentration of 100 microM. The ED50 values were approximately 3 microM, 75 microM and 100 microM for oestradiol, AF-45 and anordrin respectively. Anordrin showed no significant effects on basal progesterone production. In addition, oestradiol markedly inhibited the activity of 3 beta-hydroxysteroid dehydrogenase in luteal cells, expressed by the conversion of pregnenolone to progesterone, but the inhibitory effects of anordrin and AF-45 were negligible or relatively low. The effects of anordrin and AF-45 were different from those of oestradiol on progesterone production by human luteal cells in vitro, indicating that neither substance is likely to be a useful luteolytic agent in women.     

Metabolic clearance and production rates of oestradiol and progesterone during pubertal and postpubertal development in gilts

The crossbred gilts studied were aged 80 days (prepubertal), 180 days (prepubertal or postpubertal) and 260 days (postpubertal or pregnant). Estimates of metabolic clearance rate (MCR) of oestradiol and progesterone were consistently less (21 and 27%) in plasma than in blood, and these differences were not influenced by age of gilt. The MCR (1/day per kg body weight) for oestradiol and progesterone in plasma was greater (P less than 0.05) for 80-day-old prepubertal gilts than for older gilts. The MCR values of oestradiol and progesterone were similar in 180-day-old and 260-day-old gilts independent of reproductive state. Production rate (PR) of oestradiol and progesterone increased with age (80-180 days), and age and reproductive state differences were much more pronounced for PR of progesterone than of oestradiol. These results support the hypothesis that a reduction in the MCR and an increase in PR of oestradiol and progesterone in the gilt are associated with the process of pubertal development, and changes in gonadal steroid concentrations appear not to alter the MCR of oestradiol and progesterone.     

Evidence that oestrogen exerts an equivalent negative feedback action on LH secretion in male and female ferrets

Gonadally intact male ferrets in breeding condition, which received an aromatase inhibitor, 1,4,6-androstatriene-3,17-dione (ATD) s.c. in Silastic capsules, had significantly more LH pulses and higher mean LH concentrations in plasma than did control males implanted with empty capsules. Aromatase activity in the hypothalamus + preoptic area and temporal lobe was strongly suppressed by ATD treatment whereas circulating concentrations of testosterone and oestradiol were not affected. These results suggest that oestradiol, formed via neural aromatization of circulating testosterone, contributes to the feedback regulation of LH secretion in breeding male ferrets just as oestradiol of ovarian origin controls LH secretion in females. No sex difference was observed in the rate at which mean plasma LH concentrations rose after the removal from gonadectomized ferrets of s.c. Silastic capsules containing oestradiol. Daily s.c. injections of oestradiol in oil caused an equivalent, dose-dependent inhibition of LH pulse frequency and mean LH concentrations in plasma of male and female ferrets. These findings suggest that the negative feedback control of pulsatile LH secretion by oestrogen is not sexually differentiated in this reflexly ovulating species. The ferret appears to differ from spontaneously ovulating mammalian species in which the female is generally more sensitive than the male to the inhibitory feedback action of oestradiol on LH secretion.     

Changes in oestrogen-synthesizing ability of preovulatory bovine follicles relative to the peak of LH

Preovulatory bovine follicles (n = 28) were collected at different times after the onset of standing oestrus until shortly before ovulation. In-vitro conversion of tritiated androstenedione in the presence of NADPH by homogenates of the follicular wall was compared in phases relative to the LH peak. During phase 0 (before the LH surge) conversion into oestradiol-17 beta was high and production of oestrone was about 8-fold lower. During phases 1 (0-6 h after the LH peak) and 2A (6-14 h after the LH peak) the production of oestradiol and oestrone remained constant; the percentage of remaining androstenedione increased. In phase 2B (14-20 h after the LH peak) conversion into oestradiol and oestrone had decreased to about one third correlating with a higher percentage of remaining androstenedione. In phase 3 (20 h after the LH peak until ovulation) conversion into oestradiol and oestrone remained constant. The ratio between the production of oestrone and oestradiol remained constant throughout the phases of preovulatory development (0.13), indicating a concurrent inhibition of aromatase and 17 beta-hydroxysteroid dehydrogenase activities. Conversion into 19-hydroxyandrostenedione showed a pattern similar to that of oestradiol, and testosterone was produced in minute quantities. The results indicate that in preovulatory bovine follicles eventual inhibition of aromatization takes place at about 14 h after the preovulatory LH peak.     

Increase in uterine peroxidase activity in the rat uterus during oestrogen hyperaemia.

The administration of oestrogen results in increased arterial blood flow in all mammalian species studied to date, but its mechanism of action has not been elucidated. Because an interval of 30-60 min is observed between oestrogen injection and uterine hyperaemia, it has been suggested that a vasoactive intermediate is involved and recent evidence suggests that catechol oestrogens are the vasoactive oestrogen intermediates. Uterine peroxidase catalyses the conversion of oestrogens to their catechol forms and thus may play an important role in oestrogen-induced uterine hyperaemia. The present studies evaluated the time course and dose-response effects of oestrogen on uterine peroxidase activity and related these to changes in uterine blood volume, an index of uterine hyperaemia in immature rats. These data demonstrated that the minimal effective hyperaemic dose of oestradiol also increased (P less than 0.05) uterine peroxidase activity. The oestradiol-induced increase in uterine peroxidase activity preceded significant increases in uterine blood volume (1 h versus 2 h, respectively). These data are consistent with a role for peroxidase-mediated conversion of oestradiol to catechol oestradiol in facilitating uterine hyperaemia in rats.     

Comparison of serum oestrogen concentrations in post-menopausal women taking oestrone sulphate and oestradiol.

Mean serum concentrations of oestradiol-17beta, oestrone, and oestrone sulphate in postmenopausal women were the same when measured up to six hours after treatment with either piperazine oestrone sulphate 1.5 mg or oestradiol valerate 2 mg. Maximum concentrations of oestradiol were less than those of oestrone, but oestrone sulphate reached concentrations about 30 times higher than those of oestrone. The rapid conversion of oestradiol valerate to oestrone and oestrone sulphate does not support the suggestion that in menopausal women oestradiol is less likely to be associated with a risk of endometrial carcinoma than oestrone sulphate, since the two preparations appear to become identical after ingestion.     

Validation and use of centrifugal ultrafiltration-dialysis in the measurement of percent free oestradiol in serum

The validity of the centrifugal ultrafiltration-dialysis method in the measurement of the percentage of oestradiol and testosterone which is not bound to protein (free) in human serum was assessed. In an initial validation, the values of % free steroid obtained for oestradiol and testosterone in protein-free ultrafiltrates of human serum were higher than expected. Filter paper discs used in the system were shown to adsorb [3H]oestradiol from protein-free solution. Their replacement with glass-fibre discs, which did not adsorb oestradiol, decreased but did not eliminate the discrepancy between observed and expected results for % free oestradiol in ultrafiltrates. The use of polyethylene rather than polypropylene vials then reduced the discrepancy to insignificant levels. The estimates for % free oestradiol in serum were reduced by these modifications.     

Gonadotrophin induced development of the "special zone" in the adrenal cortex of immature female possums (Trichosurus vulpecula) with concomitant activation of steroid reductases

The effect of gonadotrophin and oestradiol administration on adrenocortical special zone (S.Z.) development and steroidogenesis was studied in immature female possums. Adrenals were examined histologically to determine S.Z. formation, and cell-free homogenates were incubated with 3H progesterone in the presence of an NADPH-generating system. Treatment with PMS + hCG, resulted in the development of S.Z.s. varying in volume from 10 to 60% of the total adrenal gland. This response was independent of ovarian status (i.e. immature or multifollicular). Treatment with porcine FSH (NIH-FSH-P2) also induced development of a S.Z. Oestradiol treatment was ineffective. The appearance of the S.Z. was associated with a change in steroidogenesis. The adrenals of controls produced cortisol and corticosterone in yields of approx. 70%, while these products were less than 22% in the animals with S.Z.s. The major conversion products in the treated animals were 5 beta-reduced pregnane derivatives, in yields ranging from 67 to 93%. The yields of products from the oestradiol treated animals closely resembled those of the controls. It was concluded that FSH is capable of inducing the development of an adrenocortical S.Z. in immature female possums and consequently stimulating adrenal steroid reduction. It appeared that oestradiol was not involved in this process.     

Effects of oestradiol on LH, FSH and prolactin in ovariectomized ewes

This study was conducted to test the hypothesis that the rate (dose/time) at which oestradiol-17 beta (oestradiol) is presented to the hypothalamo-pituitary axis influences secretion of LH, FSH and prolactin. A computer-controlled infusion system was used to produce linearly increasing serum concentrations of oestradiol in ovariectomized ewes over a period of 60 h. Serum samples were collected from ewes every 2 h from 8 h before to 92 h after start of infusion, and assayed for oestradiol, LH, FSH and prolactin. Rates of oestradiol increase were categorized into high (0.61-1.78 pg/h), medium (0.13-0.60 pg/h) and low (0.01-0.12 pg/h). Ewes receiving high rates of oestradiol (N = 11) responded with a surge of LH 12.7 +/- 2.0 h after oestradiol began to increase, whereas ewes receiving medium (N = 15) and low (N = 11) rates of oestradiol responded with a surge of LH at 19.4 +/- 1.7 and 30.9 +/- 2.0 h, respectively. None of the surges of LH was accompanied by a surge of FSH. Serum concentrations of FSH decreased and prolactin increased in ewes receiving high and medium rates of oestradiol, when compared to saline-infused ewes (N = 8; P less than 0.05). We conclude that rate of increase in serum concentrations of oestradiol controls the time of the surge of LH and secretion of prolactin and FSH in ovariectomized ewes. We also suggest that the mechanism by which oestradiol induces a surge of LH may be different from the mechanism by which oestradiol induces a surge of FSH.     

Increased hepatic lipase activity and increased direct removal of very-low-density lipoprotein remnants in Watanabe heritable hyperlipidaemic (WHHL) rabbits treated with ethinyl oestradiol.

We studied the effects of ethinyl oestradiol on the serum concentrations and metabolism of very-low- and low-density lipoproteins (VLDL and LDL) in Watanabe heritable hyperlipidaemic (WHHL) homozygous rabbits, an animal model for familial hypercholesterolaemia. The results were compared with those in untreated homozygotes as well as in heterozygotes treated or not with ethinyl oestradiol. The gain in body weight was similar in all groups. Treatment with ethinyl oestradiol resulted in the homozygotes in an approx. 80% decrease in the concentrations of lipids and apoprotein B in the d less than 1.019 lipoprotein fraction; those in the LDL fraction did not change. In the heterozygotes, basal serum lipids and apoprotein B levels in the d less than 1.019 fraction were low; ethinyl oestradiol treatment especially affected the LDL fraction (cholesterol -84%, apoprotein B -64%). Turnover experiments with 125I-labelled VLDL revealed that, on treatment with ethinyl oestradiol, the fractional catabolic rate in homozygous rabbits increased 2-fold. The secretion rates of lipids and protein in the d less than 1.019 fraction as estimated after injection of Triton WR-1339 was not decreased. In homozygotes and heterozygotes increases in post-heparin hepatic lipase activity of 62 and 80% respectively were observed, with no changes in lipoprotein lipase activity. We conclude that ethinyl oestradiol induces in homozygous WHHL rabbits a direct removal of VLDL and VLDL remnants from the plasma, apparently due to an increase in hepatic lipase activity.     

Characterization and regulation of the 3beta-hydroxysteroid dehydrogenase isomerase enzyme in the rat sciatic nerve

In the peripheral nervous system, progesterone (PROG) has a stimulatory effect on myelination. It could be derived from local synthesis, as Schwann cells in culture express the 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and convert pregnenolone (PREG) to PROG. Although 3beta-HSD mRNA can be detected by RT-PCR in peripheral nerves, the activity of the enzyme has so far not been demonstrated and characterized in nerve tissue. In this study, we show that homogenates prepared from rat sciatic nerves contain a functional 3beta-HSD enzyme and we have analysed its kinetic properties and its regulation by steroids. The activity of 3beta-HSD in homogenates was evaluated using 3H-labelled PREG as a substrate and NAD+ as a cofactor, the levels of steroids formed were calculated either by extrapolating the relationship between tritiated peaks obtained by TLC to the initial amount of PREG, or by gas chromatography/mass spectrometry determination. A rapid increase in PROG formation was found between 0 and 50 min of incubation and no further significant changes were observed between 1 and 4 h. The calculated Km value (1.06 +/- 0.19 microm) was close to the values described for the 3beta-HSD type-I and type-IV isoforms. Trilostane, a competitive inhibitor of the 3beta-HSD caused a potent inhibition of the rate of conversion of PREG to PROG (IC50 = 4.06 +/- 2.58 microm). When the effects of different steroids were tested, both oestradiol and PROG significantly inhibited the conversion of PREG to PROG.