Evidence for the biosynthesis of DHEA from cholesterol by first-trimester human placental tissue: source of androgens.

With a view to establishing whether first-trimester human placentas possess the ability to synthesize DHEA from cholesterol, homogenates of this tissue obtained from two groups of women undergoing elective termination of normally progressing pregnancy between 10 - 12 weeks gestation (n = 5, age 23 - 29 years and n = 5, age 21 - 27 years) were incubated separately with [26-(14)C]cholesterol for the generation of [14C]isocaproic acid + pregnenolone and [7n-3H]pregnenolone for the biosynthesis of [3H]DHEA. Controls consisted of homogenates heated in a boiling water bath for 10 min. Using the reverse-isotope dilution analysis, desmolase efficiency expressed as mean specific activity of [14C]isocaproic acid varied from 282 to 725 dpm/mmol, while that of 17 alpha-hydroxylase and steroid C-17,20-lyase, catalyzed conversion of [7n-3H]pregnenolone to [3H]DHEA varied from 3498 to 26 258 dpm/mmol. The corresponding efficiencies of enzymicconversion varied between 5.8 x 10( -2) and 1.5 x 10( -1) % for [14C]isocaproic acid, but between 5.5 x 10( -2) and 4.1 x 10( -1) % for [3H]DHEA. No such metabolite was evident in the controls of heat-denatured homogenates. These are the first study results to demonstrate that early placentas are capable of converting cholesterol to pregnenolone to DHEA, contrary to the widely held concept of DHEA production by fetal and maternal adrenal glands. This finding has important physiological implications and could provide a new dimension to the concept of fetoplacental steroidogenesis.     

Evidence for de novo cholesterol synthesis by term human fetal amnion and chorion: a comparative study using the reverse-isotope dilution technique

The cholesterol biosynthetic activity was assessed using [2-(14)C]-acetate as substrate in the homogenates of amnion and chorion obtained from women (n = 6, age 26-39 years) after spontaneous labour at term (37-40 weeks of gestation) having uncomplicated pregnancies. Reverse-isotope dilution analysis gave positive identification of [(14)C]-cholesterol acetate in all incubations of viable tissues. This metabolite was not evident in heat-denatured homogenates which served as controls. The extent of enzymic conversion for amnion at 2.6 x 10(-3) to 0.19% was persistently higher than that of the chorion at 1.7 x 10(-3) to 9.0 x 10(-3)%. The results indicate that human term fetal membranes possess the full complement of enzymes to catalyze the transformation of acetate to cholesterol. This study provides evidence that fetal membranes possess the capacity for de novo cholesterol biosynthesis, the sterol being essential for steroidogenesis as well as in embryo viability during pregnancy.     

Steroid production by dispersed cells from fetal membranes and intrauterine tissues of sheep

The hypothesis was examined that the fetal membranes and the endometrium and myometrium of pregnant sheep have the ability to produce oestrogens and progesterone from exogenous precursors, and that this capacity might change during the course of pregnancy, and in relation to the onset of parturition. Cells were dispersed from samples of myometrium, endometrium, allantois, chorion and amnion from sheep at Day 50, Days 130-135 of pregnancy, and at term, in labour, and were incubated in the presence of pregnenolone and 20 alpha-dihydroprogesterone as potential precursors for progesterone production, and oestrone sulphate and androstenedione as potential precursors for oestrogen production. In addition, the metabolism of radioactive progesterone and oestrone sulphate by the dispersed cells was examined. Pregnenolone was converted to progesterone in significant amounts by dispersed cells from chorion and endometrium only. At Day 130 and at term this conversion was blocked by the addition of trilostane, an inhibitor of 3 beta-hydroxysteroid dehydrogenase activity. There was no significant change in the net production of progesterone from exogenous pregnenolone with gestation. 20 alpha-Dihydroprogesterone was converted to progesterone by all tissues, and at each stage of gestation. Formation of progesterone from 20 alpha-dihydroprogesterone was invariably greater than that from pregnenolone, but did not change with pregnancy. Oestrone sulphate was converted to oestrone and oestradiol by all tissues. In the myometrium and chorion this conversion was lower at term than at Day 50 of pregnancy. In contrast, there was very little conversion of androstenedione to unconjugated oestrogen, minimal activity being demonstrable only in dispersed cells from the chorion in some preparations. Radioactive progesterone was converted to radiochemically pure 17 alpha-hydroxyprogesterone by chorion, and to radiochemically pure 20 alpha-dihydroprogesterone by amnion, chorion, allantois and endometrium obtained at term pregnancy. At term [3H]oestrone sulphate was converted to radiochemically pure oestrone by all tissues. We conclude that there is a tissue-specific distribution of different steroid metabolizing enzyme activities in the fetal membranes and intrauterine tissues of pregnant sheep. Of the substrates examined, 20 alpha-dihydroprogesterone and oestrone sulphate were preferred for progesterone and oestrogen production, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

3beta-Hydroxysteroid dehydrogenase activity in human fetal membranes.

A 3beta-hydroxysteroid dehydrogenase (3betaHSD) was demonstrated in term human fetal membranes (chorion and amnion) with both dehydroepiandrosterone (3beta-hydroxy-5-androsten-17-one) and pregnenolone (3beta-hydroxy-5-pregnen-20-one as substrates, and the subcellular distribution substrate and nucleotide specificity of the enzyme was studied. In both membranes the microsomal fraction (particles which sedimented at 105,000 g after 90 min) had the highest specific activity. The chorion was more active than the amnion but the enzyme in both tissues had similar substrate and nucleotide specificity. NAD was the preferred cofactor, and pregnenolone was a better substrate than dehydroepiandrosterone in the presence of NAD. However, with NADP as cofactor both steroids were equally good substrates. When the 3beta-hydroxysteroid dehydrogenase activity of chorion microsomes was compared with that of placental microsomes, the specific activities were found to be of the same order of magnitude, and the substrate, nucleotide specificity and steroid binding properties were almost identical.     

Metabolism of prostaglandins E2 and F2 alpha by human fetal membranes.

Prostaglandin E2 (PGE2) is important in the early stages of human labour, leading particularly to cervical ripening and dilatation. The source of PGE2 is thought to be either the amnion or the decidua, but the chorion interposes between the amnion and the target tissues, namely the myometrium and cervix. In order to investigate the role of the chorion in modulating prostanoid production, [3H]PGE2 was added to the amnion side of fetal membranes, and the production of metabolites on both sides of the fetal membrane followed by HPLC. The major metabolite was 13,14-dihydro-15-oxo-PGE2 with smaller amounts of 13,14-dihydro-15-oxo-PGA2 and PGB2. The production of all metabolites of PGE2 was time dependent. [3H]PGF2 alpha, which is normally produced by the decidua, was also added to fetal membranes and found to be metabolised to 13,14-dihydro-15-oxo-PGF2 alpha and PGE2. These results suggest that the metabolic enzymes in the chorion may determine intra-uterine levels of prostaglandins, and may also determine the identity of the eicosanoids released by intact fetal membranes.     

Estrogen sulfatase and steroid sulfatase activities in intrauterine tissues of the pregnant guinea pig

The possible role of intrauterine estrogen sulfatase and steroid sulfatase around the time of parturition in the guinea pig was investigated. [3H]Estrone sulfate or [3H]pregnenolone sulfate was incubated with intrauterine tissues. Estrogen sulfatase was found in placenta, endometrium, decidua basalis, amnion and chorion. The presence of steroid sulfatase was established in endometrium and decidua basalis but not in placenta or the fetal membranes. Examination of activities in early (days 32-35), mid (days 44-46) and late (within 5 days of parturition) gestation revealed no significant change in estrogen sulfatase specific activity in decidua basalis. However, in chorion and endometrium this activity was seen to increase approx. 12-fold (P less than 0.001) and 2.8-fold (P less than 0.001), respectively, from early to late gestation. In placenta, estrogen sulfatase activity appeared to increase 2.4-fold (P less than 0.001) and in amnion it decreased 2.8-fold (P less than 0.002). Steroid sulfatase activity in decidua basalis did not change during gestation, while activity in endometrium was found to increase by a factor of 5.3 (P less than 0.001), from early to late gestation. The increases, both in estrogen sulfatase activity in chorion, endometrium and placenta and in steroid sulfatase activity in endometrium, occurred primarily within the final 3 weeks of gestation. In contrast, the decrease in estrogen sulfatase activity in amnion occurred principally between the fifth and sixth weeks of gestation. Analysis of radiolabelled metabolites indicated that estradiol and progesterone could be produced via estrogen sulfatase and steroid sulfatase activities in certain tissues. Subcellular fractionation of tissues revealed that the greatest specific activity and total activity, in all cases, was associated with the 105,000 g pellet. Significant activity was also detected in the 750 and 10,000 g pellets but not in the 105,000 g supernatant. Radioimmunoassay of endogenous estradiol-17 beta (estradiol) in chorion extracts revealed a 6.3-fold increase in the hormone from mid to late gestation. Estradiol levels in endometrium and myometrium did not appear to change during this time. It was concluded that increased estrogen sulfatase activity in guinea pig chorion in late gestation occurs along with elevated levels of the hormone estradiol which may be important for parturition in this species.     

Steroid metabolism in corpora lutea of the western spotted skunk (Spilogale putorius latifrons)

The present study reports steroid metabolism by corpora lutea (CL) obtained from skunks with diapausing embryos ('delay' CL) and with activated embryos (activated CL). CL from both reproductive periods were incubated with various radioactive precursors. Control incubations without any tissue or with 50 microliter of packed skunk blood cells were also conducted simultaneously. Incubation of skunk CL with [3H]-pregnenolone for 3 h resulted in 36% of the precursor accumulating as progesterone. Metabolism of [3H]dehydroepiandrosterone (DHEA) to androstenedione proceeded with approximately the same amount of product accumulating (34-46%) as was observed in the conversion of pregnenolone to progesterone. These results suggest that delta 5 isomerase, 3 beta-hydroxysteroid dehydrogenase, is the most prominent enzyme in skunk CL. Metabolism of [3H]pregnenolone to 17 alpha-hydroxypregnenolone and [3H]progesterone to 17 alpha-hydroxyprogesterone occurred at low rates (1-7%), suggesting the presence of C21 steroid 17 alpha-hydroxylase in skunk CL. Aromatase activity, as estimated by measuring accumulation of oestradiol-17 beta from [3H]testosterone, was demonstrated in activated CL. These results suggest that skunk CL appear to metabolize steroids in a manner similar to CL of other mustelids such as the ferret and American badger.     

Local stimulation of prostaglandin production by corticotropin-releasing hormone in human fetal membranes and placenta

Corticotropin-releasing hormone is produced by the human placenta and fetal membranes, but its physiological significance is not established. We examined the possibility that CRH might affect prostaglandin output by these intra-uterine tissues. Primary cultures of amnion, chorion, decidua and placenta were established from tissue obtained from women at term elective cesarean section were maintained in the presence of increasing concentrations of synthetic hCRH. PG output at 48h was measured by radioimmunoassay. hCRH stimulated PGE2 output by amnion, chorion and placenta, but not by decidual tissue. PGF2 alpha output was stimulated in amnion, decidua and placenta but not chorion, whereas output of 13, 14-dihydro-15-keto PGF2 alpha was stimulated in all four tissues. We conclude that hCRH stimulates prostaglandin output by human placenta, decidua and the fetal membranes, raising the possibility of paracrine or autocrine interactions between CRH and prostaglandins in vivo.     

Specific glucocorticoid binding in human uterine tissues, placenta and fetal membranes

The binding of [3H]dexamethasone to cytosol fractions of human myometrium, endometrium, decidua, chorion, amnion and placenta has been studied. All tissues examined contained high affinity, low capacity binding sites with high specificity for glucocorticoids. Maximum specific binding of [3H]dexamethasone was reached after about 10 h at 0-4 degrees C and remained stable for at least the next 12 h. Sucrose density gradient analysis showed that the binding macromolecules sedimented at 7.9 S in hypotonic solutions and at 4.35 in solutions containing 0.4 M KCl. In the presence of sodium molybdate, the sedimentation coefficients shifted both in the absence and presence of 0.4 M KCl to 8.9 and 5.7 S, respectively. The apparent equilibrium dissociation constants (Kd) of the glucocorticoid binding sites were similar in most tissues, ranging between 1 and 6 nM, with the exception of the placenta in which the binding sites showed a higher Kd (13-22 nM). In all tissues studied, the binding affinities were similar in nonpregnant and pregnant patients and in patients at different stages of pregnancy or in labor. The concentration of the binding sites in the different tissues ranged from 11 to 268 fmol/mg protein, higher concentrations being found in myometrium, placenta and amnion and lower concentrations found in endometrium, chorion and decidua. The number of binding sites was higher in the myometrium of nonpregnant than pregnant women, but was similar in the myometrium of women at term pregnancy before or during labor. In the placenta, the number of binding sites increased significantly from early pregnancy to midpregnancy, while in chorion, amnion and decidua the number of binding sites did not change during pregnancy. It is concluded that human uterine tissues, placenta and fetal membranes contain specific binding sites with properties characteristic of glucocorticoid receptors suggesting that these tissues may respond directly to glucocorticoids.     

Identification of angiotensin II receptor and determination of its subtypes in rabbit and guinea pig fetal tissue]

Membrane angiotensin II receptors were measured in trophoblastic tissues using a 2-step procedure. The first step consisted of the relative measurement performed at a fixed 125I[Sar1 Ile8]AII concentration of 0.15 nM in order to determine which tissues had a sufficient number of binding sites for studying the competition curves. The second consisted of determining the maximal binding (Bmax) and the dissociation constant (Kd) for [Sar1 Ile8] AII and the receptor subtypes in these tissues. The relative binding measurement revealed a significant number of occupied sites in rabbit fetal placenta and chorion (159 +/- 17 and 51 +/- 10 fmol/mg proteins) and in guinea pig chorion (132 +/- 12). The mean values of the other trophoblastic tissues were 3-10-fold lower in the 2 species. The competition curves obtained from tissues with high angiotensin II binding receptors showed the predominance of the AT2 subtype in rabbit fetal placenta (AT1/AT2 = 25/75) and of the AT1 receptor in guinea pig chorion (97/3) and in rabbit chorion (90/10). The [SAR1 Ile8] AII affinity (Kd) obtained from Scatchard plot analysis was 1.2 +/- 0.2 nM (n = 5) in fetal placenta and 1.2 (n = 1) in rabbit chorion and 0.5 +/- 0.1 (n = 3) in guinea pig chorion. In these tissues, the respective Bmax values were 1,281 +/- 115 (n = 5), 263 (n = 1) and 1,188 +/- 134 fmol/mg proteins (n = 3). These findings indicate that rabbit fetal placenta and chorion and guinea pig chorion are the most important sites of action for the renin-angiotensin system present in trophoblastic tissues.     

The biosynthesis of some androst-16-enes from C21 and C19 steroids in boar testicular and adrenal tissue

1. The formation of androst-16-enes from [4-(14)C]progesterone has been investigated with long-term incubations and short-term kinetic studies. After 4hr., 1.7 and 10.3% respectively of 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes were formed in boar testis minces, but much smaller yields were obtained in boar adrenal. Both tissues formed small quantities of androsta-4,16-dien-3-one. 2. The amounts of androst-4-ene-3,17-dione and testosterone isolated were small, suggesting that androst-16-ene formation may occur preferentially in the boar testis. 3. In the absence of tissue no radioactive androst-16-enes were formed. 4. Incubation of both [4-(14)C]pregnenolone and [7alpha-(3)H]progesterone resulted in 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes containing (3)H/(14)C ratios of near unity and confirmed that both C(21) steroids were precursors. A similar incubation with 17alpha-hydroxy[4-(14)C]-progesterone and [7alpha-(3)H]progesterone gave the same Delta(16)-alcohols, but they contained only (3)H, indicating that side-chain cleavage of pregnenolone and progesterone occurred before 17alpha-hydroxylation. 5. Dehydroepiandrosterone, testosterone, testosterone acetate and 16-dehydroprogesterone were not found to be precursors of Delta(16)-steroids. 6. A pathway is proposed for the biosynthesis of 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes from pregnenolone and progesterone; this may involve androsta-4,16-dien-3-one as an intermediate, but excludes 17alpha-hydroxyprogesterone, testosterone and dehydroepiandrosterone.     

Direct contribution of inducible nitric oxide synthase expression to apoptosis induction in primary smooth chorion trophoblast cells of human fetal membrane tissues

We have previously demonstrated that apoptosis induction is observed only in smooth chorion laeve trophoblast cells, and not in amnion epithelial cells of human fetal membrane tissues prepared at the term. Apoptosis induction was suppressed by the presence of an inhibitor specific for inducible nitric oxide synthase (iNOS), suggesting that intracellular oxidative stress plays a critical role in this process. In this study, we transfected the iNOS gene into primary cultured chorion and amnion cells to examine the direct contribution of iNOS gene expression to the apoptosis induction in these cells. We identified a significant increase in the levels of iNOS protein expression and nitrite accumulation in both chorion and amnion cells after the iNOS gene transfection. However, the induction of apoptosis was observed in an approximately 70% of chorion cells transfected with iNOS gene. Transfection of the iNOS gene into chorion cells resulted in the activation of p38 mitogen-activated protein kinase (MAPK) and downregulation of hemeoxygenase-1 protein expression, whereas no such events were observed in the transfected amnion cells. These results suggest that apoptosis induced in the chorion trophoblast cells by the iNOS gene expression is closely linked to a physiological consequence, such as the rupture of fetal membranes.     

Phospholipase A activity in human and ovine uterine tissues.

Phospholipase A was assayed in crude lysosomal fractions of human decidua, amnion, chorion and myometrium. Activity was present in all the tissues and was highest in decidua and amnion which contained enzymes with pH optima of 6.5-8.0 and 7.2 respectively. Comparison of the activities in tissues obtained before labor with others obtained during labor showed no differences. In sheep, the fetal membranes contained a greater activity of phospholipase A than placenta and myometrium. Stimulation of the fetal adrenals with corticotrophin caused a marked increase in activity in both amnion and chorioallantois. It is concluded that the human amnion and the ovine amnion and chorioallantois could participate in the biosynthesis of prostaglandins by releasing stored arachidonic acid.     

Cellular permeation pathways in a leaky epithelium: the human amniochorion

The presence of cellular permeation pathways in human fetal membranes at term was evaluated. Electrical parameters (transepithelial potential [TEP], and conductance [Gt], and intracellular potentials [cell PD]), and water and urea diffusional coefficients (Pdw, Pdu), were determined in Ussing-like chambers. In amniochorion, the TEP was practically 0 (0.1 +/- 0.03 mV), and the Gt very high (144 +/- 14 mS/cm2). The Cell PD of amnion cells was -37 +/- 3 mV. Increasing the [K+] of the amniotic perfusate between 5.8 and 125.8 mM depolarized the cells with a slope of 23 mV. The deletion of Na+ hyperpolarized the cells, whereas amiloride and ouabain depolarized them. The Pdw and Pdu were determined in intact amnion and chorion and in their epithelial cell layers. The Pdw/Pdu ratio in amnion was 4.0, and 7.0 in its cell layer; the ratio in chorion was 2.5, and 3.3 in its cell layer. The amniochorion is a leaky structure, but its cellular layers possess definite transcellular permeation pathways. The ionic conductances in amnion cells are complex, with the Cell PD being determined by at least K+ and Na+ conductances, and ouabain- and amiloride-sensitive pathways. The amnion is a more effective diffusional barrier to water and urea than chorion is; its diffusional characteristics are comparable to those of nystatin-treated lecithin: cholesterol bilayers and the membranes of human erythrocytes.     

Estrogen sulfotransferase and 17 beta-hydroxysteroid dehydrogenase activities in guinea-pig chorion through gestation.

Estrogen sulfotransferase (EST) activity measured under optimal in vitro conditions in the 105,000 g cytosols (HSS) of homogenized intrauterine tissues (amnion, chorion, endometrium, decidua basalis and placenta) from guinea-pigs at the 50th day of gestation indicated that the highest specific activity occurred in the chorion. EST activity in the chorion increased from day 34 (early gestation) to peak around day 45 (mid-gestation), before significantly decreasing around day 50 and further declining to barely detectable levels beyond day 60 (late gestation, the onset of parturition). 17 beta-Hydroxysteroid dehydrogenase (17 beta-HSD) activity in the chorion was almost completely membrane associated. The specific activity of the 17 beta-HSD reduction reaction in the 105,000 g pellet was 2.5-fold higher at mid-gestation than at late gestation, while the specific activity of the 17 beta-HSD oxidation reaction was 1.7-fold higher at mid-gestation as compared with late gestation. When intact pieces of chorion tissue from mid- and late gestation were incubated with 5 nM [3H]estradiol (E2), approx. 80% of the recovered free estrogen was E1 (estrone). Only chorion from animals at the onset of parturition were able to produce detectable amounts of E2 from 5 nM [3H]E1. Under the same experimental conditions the ratio of estradiol sulfate (E2S) to estrone sulfate (E1S) isolated from the media and methanol washes of late gestation chorion tissue was 3-4 times greater than for the day 45 tissue.     

Phospholipase A activity in human and ovine uterine tissues

Phospholipase A was assayed in crude lysosomal fractions of human decidua, amnion, chorion and myometrium. Activity was present in all the tissues and was highest in decidua and amnion which contained enzymes with pH optima of 6.5 – 8.0 and 7.2 respectively. Comparison of the activities in tissues obtained before labor with others obtained during labor showed no differences. In sheep, the fetal membranes contained a greater activity of phospholipase A than placenta and myometrium. Stimulation of the fetal adrenals with corticotrophin caused a marked increase in activity in both amnion and chorioallantois. It is concluded that the human amnion and the ovine amnion and chorioallantois could participate in the biosynthesis of prostaglandins by releasing stored arachidonic acid.     

3β-hydroxysteroid dehydrogenase activity in tissues of the human fetus determined with 5α-androstane-3β,17β-diol and dehydroepiandrosterone as substrates

3β-Hydroxysteroid dehydrogenase (3β-HSD)/Δ^5→4-isomerase activity in steroidogenic tissues is required for the synthesis of biologically active steroids. Previously, by use of dehydroepiandrosterone (3β-hydroxy-5-androsten-17-one, DHEA) as substrate, it was established that in addition to steroidogenic tissues 3β-HSD/Δ^5→4-isomerase activity also is expressed in extraglandular tissues of the human fetus. In the present study, we attempted to determine whether the C-5,C-6-double bond of DHEA serves to influence 3β-HSD activity. For this purpose, we compared the efficiencies of a 3β-hydroxy-5-ene steroid (DHEA) and a 3β-hydroxy-5α-reduced steroid (5α-androstane-3β,17β-diol, 5α-A-diol) as substrates for the enzyme. The apparent Michaelis constant (K_m) for 5α-A-diol in midtrimester placenta, fetal liver, and fetal skin tissues was at least one order of magnitude higher than that for DHEA, viz the apparent K_m of placental 3β-HSD for 5α-A-diol was in the range of 18 to 40 μmol/l (n = 3) vs 0.45 to 4 μmol/l for DHEA (n = 3); for the liver enzyme, 17 μmol/l for 5α-A-diol and 0.60 μmol/l for DHEA, and for the skin enzyme 14 and 0.18 μmol/l, respectively. Moreover, in 13 human fetal tissues evaluated the maximal velocities obtained with 5α-A-diol as substrate were higher than those obtained with DHEA. A similar finding in regard to K_ms and rates of product formation was obtained by use of purified placental 3β-HSD with DHEA, pregnenolone, and 3β-hydroxy-5α-androstan-17-one (epiandrosterone) as substrates: the K_m of 3β-HSD for DHEA was 2.8 μmol/l, for pregnenolone 1.9 μmol/l, and for epiandrosterone 25 μmol/l. The specific activity of the purified enzyme with pregnenolone as substrate was 27 nmol/mg protein·min and, with epiandrosterone, 127 nmol/mg protein·min. With placental homogenate as the source of 3β-HSD, DHEA at a constant level of 5 μmol/l behaved as a competitive inhibitor when the radiolabeled substrate, [³H]5α-A-diol, was present in concentrations of 20 to 60 μmol/l, but a lower substrate concentrations the inhibition was of the mixed type; similar results were obtained with [³H]DHEA as the substrate at variable concentrations in the presence of a fixed concentration of 5α-A-diol (40 μmol/l). These findings are indicative that both steroids bind to a common site on the enzyme, however, the binding affinity for these steroids appear to differ markedly as suggested by the respective K_ms. Studies of inactivation of purified placental 3β-HSD/Δ^5→4-isomerase by an irreversible inhibitor, viz 5,10-secoestr-4-yne-3,10,17-trione, were suggestive that the placental protein adopts different conformations depending on whether the steroidal substrate has a 5α-configuration, e.g. epiandrosterone, or a C-5,C-6-double bond e.g. DHEA or pregnenolone. The lower rates of product formation obtained with placenta and fetal tissues by use of 3β-hydroxy-5-ene steroids as substrates when compared with those obtained with 3β-hydroxy-5α-reduced steroids may be explained by a combination of factors, including: (i) inhibition of 3β-HSD activity by end products of metabolism of 3β-hydroxy-5-ene steroids, e.g. 4-androstene-3,17-dione formed with DHEA as substrate; (ii) higher binding affinity of the enzyme for 3β-hydroxy-5-ene steroids—and possibly for their 3-oxo-5-ene metabolites; (iii) lack of a requirement for the isomerization step with 5α-reduced steroids as substrates, and (iv) the possible presence in fetal tissues of an enzyme with 3β-HSD activity only (i.e. no Δ^5→4-isomerase).     

Arachidonic acid metabolism by rabbit fetal membranes of various gestational ages

The ability of rabbit fetal membranes to convert arachidonic acid to both lipoxygenase and cyclooxygenase products was studied by separation and identification of products derived from incubations of 1-14C-arachidonate with subcellular fractions obtained by differential centrifugation of tissue homogenates. Both amnion and splanchnopleure (the chorion-equivalent of the rabbit) produced a mixture of 11-, 12-, and 15-hydroxyeicosatetraenoic acids when stimulated by calcium ions; these products were produced in greater quantity in middle pregnancy (20-22d) than later (28-30d). Cyclooxygenase products included PGD2, PGE2, TxB2 and PGF2 alpha, all of which were made more actively in late pregnancy than the middle in both amnion (which was more active) and chorion-equivalent. These data suggest that arachidonate metabolism by rabbit fetal membranes in middle pregnancy is directed primarily toward production of monohydroxy fatty acids, but that as pregnancy nears term, the PG-producing enzymes are induced, preparing the uterine smooth muscle for parturition.     

Tissue-specific concentration changes of estrone and estradiol during spontaneous and ACTH-induced parturition in sheep

Changes in estrogen production are considered important in the sequence of events leading to parturition. We sought tissue-specific changes in the concentration of unconjugated estrone (E1) and estradiol (E2) in intrauterine fetal (amnion, chorion) and maternal (endometrium, myometrium) tissues during normal pregnancy, labour, and ACTH-induced labour in sheep. The mean concentrations of E1 and E2 in the fetal membranes were higher than in endometrium and myometrium. In amnion there were no consistent changes in estrone concentrations with gestation, although estradiol concentrations increased between day 130 and term. In the endometrium there were increases in both estrone and estradiol between day 100 and term, whereas in the myometrium increases in the concentrations of E1 and E2 occurred between days 130-135 and term. Animals showing a labourlike pattern of uterine contractions after intrafetal ACTH administration did not show significant differences in estrone or estradiol concentrations in amnion, chorion, or endometrium compared with saline-infused controls. However, there was a progressive increase in the concentration of estrone and estradiol in the myometrium during ACTH-induced labour. We conclude that changes in the concentrations of estrone and estradiol in intrauterine tissues vary between the tissues studied and the two estrogens. In general, estrogen concentrations increased towards term, but this trend was more marked in the maternal than fetal tissues. The changes in estrone concentrations in myometrium, but not in the other tissues, were replicated during ACTH-induced labour. Our results would be compatible with the suggestion that tissue-specific changes in estrogen concentrations may contribute to the local intrauterine steroid milieu during pregnancy and at term.     

Steroidogenesis in interstitial cells and microsomal fraction of immature pig testes.

Testicular interstitial cells (greater than 90% viable) obtained from 6-day-old and 3-6-week-old piglets were capable of producing dehydroepiandrosterone (DHEA, 5-10 ng/500,000 cells) and responded to hCG (60 mi.u./ml), dibutyryl-cAMP (1 mmol/l) and cholera toxin (5 ng/ml) with a 2-3-fold increase in DHEA. Aminoglutethimide (100 mumol/l) abolished the response. Testosterone was produced in comparatively minor quantities (less than 0.3 ng/500,000 cells) and was unaffected by stimulation or inhibition. When cells from both age groups were incubated with [14C]- or [3H]-pregnenolone (360 and 3.0 nmol/l), 17-hydroxypregnenolone (15%) and DHEA (5-10%) were the major metabolites on the androgen pathway and 5,16-androstadien-3 beta-ol (andien-beta, 5-10%) and 4,16-androstadien-3-one (dienone, 5-10%) on the 16-androstene pathway. Stimulation and inhibition of endogenous steroidogenesis did not alter the metabolism of exogenous pregnenolone, the same metabolites being found in the same proportions at similar times. Microsomal enzyme activities accurately reflected the metabolic profile of pregnenolone metabolism seen in intact cells, with low activities for 17 beta-HSD, 3 beta-HSD-isomerase, and 16-ene-5 alpha-reductase being observed. Since steroidogenic capacity, enzyme complement and pregnenolone metabolism were the same in testes from both age groups, the differences in Leydig cell activity observed in vivo would not appear to be consequences of changes in steroidogenic enzymes or responsiveness to gonadotrophin stimulation. The lack of effect of stimulation and inhibition of steroidogenesis on the cellular metabolism of exogenous pregnenolone suggests that the endogenous and exogenous supplies of pregnenolone are metabolized by different populations of enzymes. The relative magnitudes of these populations indicate that most of the steroidogenic enzymes in the interstitial cells are not involved in the normal response to trophic stimulation.