The hypophysis in the regulation of androgen and oestrogen dependent enzyme activities of steroid hormone metabolism in rat liver cytosol.

In order to determine whether the gonadal and hypophyseal modes of regulation recently reported for the microsomal enzymes of hepatic steroid metabolism are also valid for cytoplasmic enzymes, three enzymes whose activities exhibit sex differences (male:female activity ratio shown in brackets), 5beta-reductase(1.7:1), 20alpha-hydroxysteroid dehydrogenase(5 : 1) and 17beta-hydroxysteroid dehydrogenase (4:1), as well as one enzyme whose activity shows no sex difference, 3beta-hydroxy-delta5-steroid dehydrogenase, were investigated after various interferences with the endocrine balance (gonadectomy, hypophysectomy, combination of both operations, administration of testosterone or oestradiol). From the results of this and a previous study the following statements can be made about the endocrine control of hepatic enzyme activities. Those enzymes whose activities show sex differences are either androgen or oestrogen dependent; the sex hormone acts in either an inductive or repressive manner. 1) Criteria for androgen dependency are the feminization of enzyme activity after testectomy or inhibition of testicular function by administration of oestradiol; masculinization of the enzyme activity after administration of testosterone to male or female castrates. Using these criteria the following enzymes investigated in this laboratory fall into this category: all microsomal enzymes which show sex differences in their activity (3alpha-, 3beta-, delta4-3beta, 20-hydroxysteroid dehydrogenase; cortisone alpha-reductase; steroid hydroxylases and 16alpha-hydroxylase) as well as the cytoplasmic 20alpha-hydroxysteroid dehydrogenase. Apart from the single exception of 20alpha-hydroxy-steroid dehydrogenase the presence of the hypophysis is obligatory for the androgen to be effective. The hypophysis does not only work in a permissive manner, but participates in establishing the sex specific activity levels in a manner which is antagonistic to the androgen action. 2) Criteria for oestrogen dependency are that the female animal reacts to gonadectomy, as well as to the inhibition of ovarian function after testosterone administration, by a masculinization of the enzyme activities. After administration of oestradiol, but not gonadectomy, the male animal exhibits typical female activity. Using these criteria the cytoplasmic 5beta-reductase and 17beta-hydroxysteroid dehydrogenase are oestrogen dependent. The repressive oestrogen effect observed on 17beta-hydroxysteroid dehydrogenase is antagonistic to hypophyseal action, whereas in the case of 5beta-reductase it is synergistic. 3) The activities of cytoplasmic 3beta-hydroxy-delta5-steroid dehydrogenase and microsomal 7alpha-hydroxylase show no sex differences and are not influenced by any interference with the endocrine balance.     

Regulatory enzymes of carbohydrate and energy metabolism in the rabbit blastocyst.

The activities of phosphofructokinase, pyruvate kinase, citrate synthase and creatine kinase were determined in blastocysts from rabbits at 144 h post coitum and in similar blastocysts cultured for 24 h with or without oestradiol-17beta (1 microgrm/ml). There was a significant increase in all the enzymes during the 24-h culture period but oestradiol had no effect.     

Molecular epidemiology of breast cancer: genetic variation in steroid hormone metabolism

The age-specific incidence rate of breast cancer in women rises until menopause, levels off and then rises again at a much lower rate indicating a possible hormonal influence on the disease risk. A large amount of evidence has implicated hormones and other compounds with oestrogen activity in the pathogenesis of certain endocrine cancers, particularly breast cancer. Widely dispersed hormone-like chemicals, capable of disrupting the endocrine system and interfering with proliferation, have been described. Compounds such as dioxins, some polychlorinated biphenyls and the plastic ingredient bisphenol-A have been shown to interfere with human reproduction and hormonal regulation. The levels of these foreign compounds as well as the levels of endogenous oestradiol may influence the risk of breast cancer. Endogenous oestradiol is synthesised in the ovarian theca cells of premenopausal women or in the stromal adipose cells of the breast of postmenopausal women and minor quantities in peripheral tissue. These cells, as well as breast cancer tissue, express all the necessary enzymes for this synthesis: CYP17, CYP11a, CYP19, hydroxysteroid hydrogenase, steroid sulphatase as well as enzymes further hydroxylating oestradiol such as CYP1A1, CYP3A4, CYP1B1. Polymorphisms in these enzymes may have a possible role in the link between environmental estrogens and hormone-like substances and the interindividual risk of breast cancer.     

Oestradiol and progesterone: soluble receptor levels and metabolism in the uterus of the ovariectomized ewe

Ovariectomized ewes received injections designed to mimic to some extent oestradiol and progesterone secretion during early pregnancy (maintenance progesterone), during oestrus (oestrous oestradiol) and during the luteal phase of the previous cycle (priming progesterone). The animals were killed at times equivalent to 1, 4 or 7 days after oestrus in those animals which had received oestrous oestradiol. The level of soluble oestradiol and progesterone receptors in whole uterus, and [3H]oestradiol and [3H]progesterone metabolism by uterus minces were measured. Oestradiol receptor level was highest on day 1 in those animals receiving oestrous oestradiol with no significant effect at any stage of the inclusion or omission of priming or maintenance progesterone. Progesterone receptor level was also high on day 1 in those animals receiving oestrous oestradiol with high levels maintained to day 4. Again, inclusion of priming or maintenance progesterone was without effect. In animals not receiving oestrous oestradiol the level of both receptors was uniformly low. Metabolism of [3H]oestradiol was low and not affected by treatment. [3H]Progesterone metabolism, although more variable, was also low and not affected by treatment.     

Immunolocalization of alpha-fetoprotein in the ovary and hypophysis of immature female rats

Summary Alpha-fetoprotein (AFP), an oestrogen-binding protein, has been localized in the ovary and hypophysis of prepuberal rats. In the ovary, AFP was localized in secondary follicles, its distribution being limited to the liquor folliculi and the zona pellucida. In the hypophysis, AFP was only found in blood vessels. The intra-ovarian, localization of the protein is consistent with our previous hypothesis that AFP might act as a regulator of ovarian activity by decreasing the local free oestradiol level.     

Development and characteristics of an oestrogen sulphotransferase in placenta and uterus of the pregnant mouse. Comparison between mouse and rat.

The mouse placenta possesses a soluble oestrogen sulphotransferase activity which increases markedly from at least 12 days of gestation until term. At about 16 days of gestation, a similar activity is found in the uterus. This activity also increases until term and disappears rapidly post partum. The uterine enzyme activity appears to require the presence of the foetal unit for its onset, since unoccupied horns, whether their endometrial stromal cells are differentiated to decidual cells or not, are essentially devoid of it. Uterine cytosols from non-pregnant mice are also inactive in this respect. In late gestation, the uterine sulphotransferase is confined to the decidua basalis, the areas to which the placentas are attached. The sulphotransferase(s) of placenta and uterus has an absolute requirement for 3'-phosphoadenosine 5'-phosphosulphate, and possesses little activity in the absence of exogenous thiol groups. Stimulation is also seen in the presence of Mn2+, Mg2+ or Ca2+. Oestrone and oestradiol, and to a lesser degree oestriol, are substrates for the enzyme(s), whereas testosterone, cortisol and dehydroepiandrosterone are not. Oestrone and oestradiol at higher concentrations (1.0-1.5 microM) completely inhibit the enzyme(s). These enzymes could play a role in altering tissue concentrations of active oestrogens during gestation in the mouse. Oestrogen sulphotransferase activity is low or absent in reproductive tissues of the pregnant rat.     

Enzyme leakage during cryopreservation of ram spermatozoa

Spermatozoal motility and enzyme leakage during cryopreservation of ram spermatozoa was evaluated in the presence and absence of antifreeze proteins (AFP). Loss of spermatozoal motility due to the cryopreservation process was reduced by 10% in the presence of AFP. Samples of the diluted semen at various stages of the cryopreservation process were assayed for aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), acid phosphatase (ACP) and lactate dehydrogenase (LDH). The levels of AST, ALT and ACP were low in ram spermatozoa and were not considered suitable for the objective measurement of spermatozoal damage. ALP leaked during the cooling and freezing process, whereas LDH leakage was prevalent during cooling, freezing and post-thaw incubation of spermatozoa. Changes in ALP and LDH could be used as marker enzymes in the development of semen processing protocols and of semen diluents.     

Influence of 5 alpha-reduced androgens on oestradiol secretion by granulosa cells of pregnant mare serum gonadotrophin treated immature rats

The effect of aromatizable androgens (testosterone and androstenedione) and naturally occurring 5 alpha-androstane, 3 alpha 17 beta-diol and 5 alpha-androstane, -3 beta, 17 beta-diol on oestradiol secretion by granulosa cells isolated from preovulatory follicles of PMSG-primed immature rats was investigated. The amount of oestradiol secreted by granulosa cells in the absence of exogenous aromatizable androgen in a 4h incubation was negligible. However, the addition of testosterone or androstenedione resulted in concentration dependent increases in oestradiol secretion. The 5 alpha-reduced androgens inhibit oestradiol and oFSH-stimulated oestradiol secretion by the granulosa cells in the presence of exogenous testosterone. The least potent of the androgens tested in causing this effect being the 5 alpha-androstane-3 alpha, 17 beta-diol. This result suggests that the naturally occurring 5 alpha-reduced androgens have a direct effect on androgen-aromatizing enzymes. The effect of these androgens may have an important connotation with respect to the control of the onset of puberty and regulation of ovarian oestradiol secretion within the microenvironment of an ovarian follicle.     

The effects of ovariectomy and injected oestradiol monobenzoate on polypeptide metabolism in the hypothalamus

1. The activities of two groups of peptidases separated from a homogenate of rabbit hypothalamus were determined (a) in adult female animals, (b) in ovariectomized animals and (c) in intact female animals after injection of oestradiol monobenzoate (15-120mug.). 2. Ovariectomy decreased the enzyme activity initially; the activity in the particulate group of enzymes subsequently returned to normal whereas the activity of the supernatant fraction was less than normal 8 months after operation. 3. Injection of oestradiol monobenzoate increased the enzyme activity in the supernatant fraction to that observed in pregnant animals and in suckled lactating animals. 4. There is a correlation between changes in enzyme activity of the supernatant fraction and conditions that are known to influence gonadotrophin secretion.     

Sulphation as a metabolic pathway for oestradiol in the sea urchin Strongylocentrotus franciscanus

1. Aerobic incubation of [¹⁴C]oestradiol, in the presence of surviving gut tissue of the sea urchin Strongylocentrotus franciscanus, or a soluble enzyme system prepared therefrom, resulted in rapid formation of a water-soluble metabolite, identified as oestradiol 3-sulphate. 2. No evidence was obtained for the formation of other metabolic derivatives by the urchin-gut enzymes, or for the presence of the sulphating capacity in any other tissues of the organism under the conditions used. 3. The data are consistent with the possibility that oestradiol, previously detected in the gonads, is synthesized therein and excreted in a conjugated, highly water-soluble form via the gut.     

Alpha-fetoprotein can regulate growth in the uterus of the immature and adult ovariectomized mouse

This is a report of development of (1) a 3-day immature mouse bioassay for alpha-fetoprotein (AFP) to increase the working range in uterine wet weights over-coming seasonality, and (2) a bioassay for AFP performed with ovariectomized adult mice to increase sensitivity. Mouse AFP was isolated from amniotic fluid and purified by polyacrylamide gel electrophoresis followed by Blue Sepharose chromatography. The uterine growth evoked by the injection of 1.0 microgram AFP together with excess molar oestradiol (0.5 microgram) over a 23-h period was compared in immature ovariectomized adult Nylar mice. The 3-day assay with immature mice was rendered usable in any season, with sensitivity comparable to the 1-day assay. Increased sensitivity, however, was demonstrated by utilization of AFP in a 1-day assay with the adult ovariectomized mouse.     

In vitro effects of cytosolic inhibitor and opiates on the binding of [3H]oestradiol to nuclear type II binding sites of rat uterus and hypothalamus

The effect of cytosolic ultrafiltrates prepared from intact rat uteri, brain hemispheres and hypothalami and of some opiate analogues on oestradiol binding to nuclear type II sites in rat uterus and hypothalamus was studied. Opiate binding in nuclear fraction of rat uteri was also evaluated. Both uterine and hypothalamic low affinity nuclear oestradiol binding was inhibited by filtrate from uteri, while only hypothalamic nuclear binding was decreased in presence of hypothalamic filtrate. Filtrate from brain was ineffective on nuclear oestradiol binding of the studied tissues. Concentration dependent inhibition of uterine nuclear oestradiol binding could be demonstrated by some opiate analogues in vitro. Specific low affinity nuclear binding of opiate antagonist naloxone and agonist dihydromorphine was observed in rat uteri which could be inhibited by uterine filtrate and oestradiol but not by hypothalamic filtrate or other steroids. Present findings support the probable intracellular interplay of opiates and oestradiol action and suggest that cytosolic inhibitor factor might be involved.     

3H]Oestradiol metabolism by 18-day rat interstitial cells in culture and the effect of FSH: presence of 16 alpha-hydroxylase

The metabolism of [3H]oestradiol by interstitial cells in culture prepared from 18-day old rat testes was investigated. Interstitial cells were able to convert [3H]oestradiol to [3H]oestriol as confirmed by recrystallization of oestriol to constant specific activity from samples containing cells and not from controls. This demonstrated for the first time the presence of 16 alpha-hydroxylase in rat testicular interstitial cells. The effect of in vitro FSH treatment on the cells in culture was also investigated. FSH failed to affect 16 alpha-hydroxylase activity since we could not demonstrate a significant difference between treated and untreated preparations. The 16 alpha-hydroxylation of phenolic steroids is widely regarded as the major pathway of oestrogen metabolism in mammals. This metabolic step significantly reduces the biological activity of oestradiol. The presence of 16 alpha-hydroxylase in interstitial cells suggests that it may play a role in inactivating the oestradiol that is produced in the Leydig cells and thus prevent its intracellular accumulation. Such activity may conceivably play a role in the overall local "fine tuning" of androgen biosynthesis.     

Central metabolism in Acinetobacter sp. grown on ethanol

The ethanol-grown cells of the mutant Acinetobacter sp. strain 1NG, incapable of producing exopolysaccharides, were analyzed for the activity of enzymes of the tricarboxylic acid (TCA) cycle and some biosynthetic pathways. In spite of the presence of both key enzymes (isocitrate lyase and malate synthase) of the glyoxylate cycle, these cells also contained all enzymes of the TCA cycle, which presumably serves biosynthetic functions. This was evident from the high activity of isocitrate dehydrogenase and glutamate dehydrogenase and the low activity of 2-oxoglutarate dehydrogenase. Pyruvate was formed in the reaction catalyzed by oxaloacetate decarboxylase, whereas phosphoenolpyruvate (PEP) was synthesized by the two key enzymes (PEP carboxykinase and PEP synthase) of gluconeogenesis. The proportion between these enzymes was different in the exponential and the stationary growth phases. The addition of the C4-dicarboxylic acid fumarate to the ethanol-containing growth medium led to a 1.5- to 2-fold increase in the activity of enzymes of the glyoxylate cycle, as well as of fumarate hydratase, malate dehydrogenase, PEP synthase, and PEP carboxykinase (the activity of the latter enzyme increased by more than 7.5 times). The data obtained can be used to improve the biotechnology of production of the microbial exopolysaccharide ethapolan on C2-substrates.     

Effect of alpha-fetoprotein on arachidonic acid metabolism in the preadipocyte cell line OB 17

Previous work has shown that alpha-fetoprotein (AFP) is able to bind specifically polyunsaturated fatty acids, one of the major ligands being arachidonic acid (C20:4). In the present study, we demonstrate that AFP is able to reduce the metabolism of exogenous C20:4 by obl7 cells. Both prostaglandins and lipoxygenase products formation were reduced when cells were maintained in the presence of AFP. This decrease was counterbalanced by a higher release of C20:4 into the culture medium by the cells. The amount of C20:4 incorporated into cellular lipids was decreased but the distribution of C20:4 in the different lipid classes remained unchanged. The modification of C20:4 metabolism by AFP might be of primordial importance in developmental biology and may shed a new light on the physiological actions of AFP which have been described in the past years such as ovarian inhibition, cell growth control and immuno-suppressive activity.     

Comparative study of nuclear binding sites for oestradiol in rat testicular and uterine tissue. Determination of low amounts of specific binding site by an [3H] oestradiol-exchange method.

1. An [3H]oestradiol-exchange method was developed for the determination of oestradiol-receptor complexes in the nuclear fraction of immature rat testicular tissue. This method permits the determination of nuclear oestradiol-receptor sites in the presence of a relatively large amount of non-specific oestradiol binding present in testicular nuclei. After incubation of nuclei for 60min at 20 degrees C in the presence of [3H]oestradiol with or without a 1000-fold excess of non-radioactive diethylstilboestrol, specific binding can be determined quantitatively in the KCl-extractabe fraction, which contains 40% of the total receptor population. 2. The amount of receptor-bound steroid present in the 0.4m-KCl extract of testicular neclei remained constant during incubation at 20 degrees C. For uterine nuclei incubated with [3H]oestradiol at 37 degrees C a shift of specifically bound [3H]oestradiol occurred from the KCl-soluble fraction to the KCl-insoluble fraction. 3. In intact rat testis, about 20% of the total receptor concentration was present in its nuclear form. Hypophysectomy 5 days before measurement resulted in a twofold decrease in the amount of receptor, which was present mainly in the cytosol. After injection of choriogonadotropin to intact animals, the total receptor concentration increased threefold. 4. This nuclear exchange method might be useful for determination of occupied specific receptor sites in tissues with relatively low contents of specific receptors.     

Effect of LH on progesterone and oestradiol production in vivo and in vitro by preovulatory rat follicles

Temporal changes in follicular oestradiol production induced in vitro and in vivo by LH were studied. In-vitro changes were measured by incubating preovulatory rat follicles for 12 h, changing the medium every 2 h. Follicles isolated at various intervals after an injection of 10 i.u. hCG were incubated for 2 h to measure changes in oestradiol production in vivo. In both studies there was an increase in oestradiol production lasting 4 h followed by a sharp decline. Progesterone production was also increased by LH in vitro or hCG in vivo, but remained high. A second exposure to LH did not raise oestradiol synthesis, but increased progesterone synthesis in vitro only. The decline in oestradiol production is most probably due to a decrease in C17-20 lyase activity, because addition of testosterone, but not of 17 alpha-hydroxyprogesterone, increased oestradiol production. Incubation of preovulatory follicles in the absence of LH or incubation of follicles derived from animals in which the spontaneous LH surge was blocked by an injection of pentobarbitone sodium also resulted in a decrease of oestradiol and an increase in progesterone production. This oestrogen-progesterone shift was also caused by a decrease in C17-20 lyase activity. The results demonstrate that the changes in steroid production in vivo and in vitro are similar and occur in the presence and absence of LH. It is concluded that the decrease in oestradiol production is dependent on the decrease in the activity of enzymes converting progesterone to aromatizable androgens.