Plasma and Urinary Sulfate Determination in a Cohort with Autism

Sulfate is important for mammalian development but is not routinely measured in clinical settings. The renal NaS1 sulfate transporter maintains circulating sulfate levels and is linked to renal sulfate wasting in mice. Some autistic individuals exhibit renal sulfate wasting, but the etiology is yet unknown. We measured plasma and urinary sulfate levels, calculated the fractional excretion index (FEI) of sulfate, and screened for two loss-of-function NaS1 sequence variants (R12X and N174S) in 23 autistic individuals. The FEI sulfate values ranged from 0.13 to 0.50. NaS1 variants were detected in 18 of the 23 individuals (11 heterozygous N174S, four homozygous N174S, two heterozygous R12X, and one individual carried both R12X and N174S). Those individuals with neither sequence variant had FEI sulfate ≤ 0.34, whereas FEI sulfate ≥ 0.35 was found in about 60 % (11 of 18) of individuals that had R12X and/or N174S. This study links renal sulfate wasting with loss-of-function NaS1 sequence variants in humans.     

Aggressive interactions differentially modulate local and systemic levels of corticosterone and DHEA in a wild songbird

During the nonbreeding season, when gonadal androgen synthesis is basal, recent evidence suggests that neurosteroids regulate the aggression of male song sparrows. In particular, dehydroepiandrosterone (DHEA) is rapidly converted in the brain to androgens in response to aggressive interactions. In other species, aggressive encounters increase systemic glucocorticoid levels. However, the relationship between aggression and local steroid levels is not well understood. Here, during the breeding and nonbreeding seasons, we tested the effects of a simulated territorial intrusion (STI) on DHEA and corticosterone levels in the brachial and jugular plasma. Jugular plasma is enriched with neurosteroids and provides an indirect index of brain steroid levels. Further, during the nonbreeding season, we directly measured steroid levels in the brain and peripheral tissues. Both breeding and nonbreeding males displayed robust aggressive responses to STI. During the breeding season, STI increased brachial and jugular corticosterone levels and jugular DHEA levels. During the nonbreeding season, STI did not affect plasma corticosterone levels, but increased jugular DHEA levels. During the nonbreeding season, STI did not affect brain levels of corticosterone or DHEA. However, STI did increase corticosterone and DHEA concentrations in the liver and corticosterone concentrations in the pectoral muscle. These data suggest that 1) aggressive social interactions affect neurosteroid levels in both seasons and 2) local steroid synthesis in peripheral tissues may mobilize energy reserves to fuel aggression in the nonbreeding season. Local steroid synthesis in brain, liver or muscle may serve to avoid the costs of systemic increases in corticosterone and testosterone.     

Influence of oral dehydroepiandrosterone (DHEA) on urinary steroid metabolites in males and females

Oral dehydroepiandrosterone (DHEA) replacement therapy may have a multitude of potential beneficial effects and exerts its action mainly via peripheral bioconversion to androgens (and estrogens). A daily dose of 50-mg DHEA has been shown by us and others to restore low endogenous serum DHEA concentrations to normal youthful levels followed by an increase in circulating androgens and estrogens. As the hepatic first-pass effect may lead to a non physiological metabolism of DHEA after oral ingestion we studied the influence of two single DHEA doses (50 and 100 mg) on the excretion of steroid metabolites in 14 elderly males [age 58.8+/-5.1 years (mean +/- SEM)] with endogenous DHEAS levels <1500 ng/ml and in 9 healthy females (age 23.3+/-4.1 years) with transient suppression of endogenous DHEA secretion induced by dexamethasone (dex) pretreatment (4x0.5 mg/day/4 days). Urinary steroid profiles in the elderly males were compared to the steroid patterns found in 15 healthy young men (age 28.9+/-5.1 years). In the females the results were compared to their individual baseline excretion without dex pretreatment. Urinary steroid determinations were carried out by semiautomatic capillary gas-liquid chromatography. In both genders DHEA administration induced significant increases in urinary DHEA (females: baseline vs. 50 mg vs. 100 mg: 361+/-131 vs. 510+/-264 vs. 1541+/-587 microg/day; males: placebo vs. 50 mg vs. 100 mg: 434+/-154 vs. 1174+/-309 vs. 4751+/-1059 microg/day) as well as in the major DHEA metabolites androsterone (A) and etiocholanolone (Et). Fifty mg DHEA led to an excretion of DHEA and its metabolites only slightly above baseline levels found in young females and in young men, respectively, whereas 100 mg induced clearly supraphysiological values. After 50 mg DHEA the ratios of urinary DHEA metabolites (A/DHEA, Et/DHEA) were not significantly different between elderly males vs. young male volunteers and young healthy females versus their individual baseline levels. In conclusion, an oral dose of 30 to 50 mg DHEA restores a physiological urinary steroid profile in subjects with DHEA deficiency without evidence for a relevant hepatic first-pass effect on urinary metabolites.     

Steroid concentrations in plasma, whole blood and brain: effects of saline perfusion to remove blood contamination from brain

The brain and other organs locally synthesize steroids. Local synthesis is suggested when steroid levels are higher in tissue than in the circulation. However, measurement of both circulating and tissue steroid levels are subject to methodological considerations. For example, plasma samples are commonly used to estimate circulating steroid levels in whole blood, but steroid levels in plasma and whole blood could differ. In addition, tissue steroid measurements might be affected by blood contamination, which can be addressed experimentally by using saline perfusion to remove blood. In Study 1, we measured corticosterone and testosterone (T) levels in zebra finch (Taeniopygia guttata) plasma, whole blood, and red blood cells (RBC). We also compared corticosterone in plasma, whole blood, and RBC at baseline and after 60 min restraint stress. In Study 2, we quantified corticosterone, dehydroepiandrosterone (DHEA), T, and 17β-estradiol (E₂) levels in the brains of sham-perfused or saline-perfused subjects. In Study 1, corticosterone and T concentrations were highest in plasma, significantly lower in whole blood, and lowest in RBC. In Study 2, saline perfusion unexpectedly increased corticosterone levels in the rostral telencephalon but not other regions. In contrast, saline perfusion decreased DHEA levels in caudal telencephalon and diencephalon. Saline perfusion also increased E₂ levels in caudal telencephalon. In summary, when comparing local and systemic steroid levels, the inclusion of whole blood samples should prove useful. Moreover, blood contamination has little or no effect on measurement of brain steroid levels, suggesting that saline perfusion is not necessary prior to brain collection. Indeed, saline perfusion itself may elevate and lower steroid concentrations in a rapid, region-specific manner.     

Regulation of the sodium/sulfate co-transporter by farnesoid X receptor alpha

Fxralpha is known to regulate a variety of metabolic processes, including bile acid, cholesterol, and carbohydrate metabolism. In this study, we show direct evidence that Fxralpha is a key player in maintaining sulfate homeostasis. We identified and characterized the sodium/sulfate co-transporter (NaS-1; Slc13a1) as an Fxralpha target gene expressed in the kidney and intestine. Electromobility shift assays, chromatin immunoprecipitation, and promoter reporter studies identified a single functional Fxralpha response element in the second intron of the mouse Slc13a1 gene. Treatment of wild-type mice with GW4064, a synthetic Fxralpha agonist, induced Slc13a1 mRNA in the intestine and kidney. Slc13a1 mRNA was also induced in the kidney and intestine of wild-type, but not Fxralpha-/- mice, after treatment with the hepatotoxin alpha-naphthylisothiocyanate, which is known to result in elevated blood bile acid levels. Finally, we observed a decrease in Slc13a1 mRNA in the kidney and intestine of Fxralpha-/- mice and a corresponding increase in urinary excretion of free sulfates as compared with wild-type mice. These results demonstrate that mouse Slc13a1 is a novel Fxralpha target gene expressed in the kidney and intestine and that in the absence of Fxralpha, mice waste sulfate into the urine. Thus, Fxralpha is necessary for normal sulfate homeostasis in vivo.     

Antioxidant activity of dioscorea and dehydroepiandrosterone (DHEA) in older humans

Dioscorea is a yam steroid extract used in commercial steroid synthesis and consumed by people. DHEA is a steroid which declines with age, but without known activity. This study was designed to determine whether dioscorea supplementation could increase serum dehydroepiandrosterone sulfate (DHEAS) in humans and modulate lipid levels in older people. The subjects were selected volunteers aged 65–82 years. The serum DHEAS level, lipid peroxidation and lipid profile were assessed. Three weeks of dioscorea supplementation had no affect on serum DHEAS level. However DHEA intake of 85 mg/day increased serum DHEA levels 100.3 %. DHEA and dioscorea significantly reduced serum lipid peroxidation, lowered serum triglycerides, phospholipid and increased HDL levels. Both DHEA and the steroid yam extract, dioscorea, have significant activities as antioxidant to modify serum lipid levels.     

Decrease in serum dehydroepiandrosterone level after fenofibrate treatment in males with hyperlipidemia

The influence of steroid hormones on plasma lipids and lipoproteins was confirmed by many studies. On the other hand, the effect of plasma lipids on metabolism of steroid hormones has so far not been examined. The objective of this research project was to determine (1) the levels of cortisol, testosterone, estradiol, dehydroepiandrosterone (DHEA), its sulfate (DHEAS), 7-hydroxylated DHEA, and SHBG in men suffering from mixed hyperlipidemia (HPL) (n=23, age 46.1+/-7.9 years) in comparison with healthy male volunteers (n=17, age 45.1+/-15.6 years); (2) whether therapy with fenofibrate influences the levels of the above mentioned steroids and SHBG; (3) what are the correlations between lipids and steroids in healthy males and HPL patients before and after therapy. Compared to controls, untreated patients had significantly higher estradiol and free testosterone index (IFT) levels (p<0.0003 and p<0.02, respectively) and significantly lower SHBG (p<0.02). Due to fenofibrate therapy, a significant decrease of TC, TG, and DHEA levels occurred (mean decrease: 14 %, 52 % and 21 %, respectively). Triglycerides correlated negatively with testosterone and SHBG in healthy subjects. HDL-C correlated positively and consequently, atherogenic index correlated negatively with 7-hydroxylated epimers of DHEA in treated patients. This is the first study dealing with the influence of fenofibrate administration on the steroid levels. Taking together, the most important is the finding of decrease DHEA levels after fenofibrate therapy. It could be explained, at least in part, by the effect of the fenofibrateon on the biosynthesis of DHEA and its regulation.     

Physiological roles of renal anion transporters NaS1 and Sat1

This review will briefly summarize current knowledge on the renal anion transporters sodium-sulfate cotransporter-1 (NaS1; Slc13a1) and sulfate-anion transporter-1 (Sat1; Slc26a1). NaS1 and Sat1 mediate renal proximal tubular sulfate reabsorption and thereby regulate blood sulfate levels. Sat1 also mediates renal oxalate transport and controls blood oxalate levels. Targeted disruption of murine NaS1 and Sat1 leads to hyposulfatemia and hypersulfaturia. Sat1 null mice also exhibit hyperoxalemia, hyperoxaluria, and calcium oxalate urolithiasis. NaS1 and Sat1 null mice also have other phenotypes that result due to changes in blood sulfate and oxalate levels. Experimental data indicate that NaS1 is essential for maintaining sulfate homeostasis, whereas Sat1 controls both sulfate and oxalate homeostasis in vivo.     

Dehydroepiandrosterone feeding and protein phosphorylation, phosphatases, and lipogenic enzymes in mouse liver

Dehydroepiandrosterone (DHEA) treatment is effective in preventing or delaying the onset of various genetic and induced disorders of mice and rats. Associated with the beneficial therapeutic effects exerted by action of this steroid is the development of hepatomegaly. To determine whether the changes associated with hepatomegaly also involve alterations in activities of tissue enzymes, we evaluated the effects of DHEA (0.45% in food, w/w) on hepatic protein kinases, phosphatases, and lipogenic enzymes in mice of various strains. The rates of fatty acid and cholesterol syntheses also were evaluated. DHEA administration resulted in profound changes in the sodium dodecylsulfate-polyacrylamide gel electrophoresis patterns of endogenous radiophosphorylated proteins obtained by incubation of liver homogenates with (gamma-32P]ATP. These changes were dependent upon the medium used for homogenization. Thus, when homogenates of liver tissue of DHEA-treated mice were prepared in Tris buffer containing sucrose (0.25 M) there was a marked decrease in phosphorylation of the proteins of relative molecular weight approximately 116,000 (Mr approximately 116,000), approximately 82,000, approximately 80,000, approximately 58,000, approximately 56,000, approximately 48,000, approximately 34,000, and approximately 31,000 compared with controls. With liver homogenates of DHEA-treated mice prepared in Tris buffer alone, there was a marked increase in phosphorylation of the proteins of Mr approximately 70,000, approximately 49,000, approximately 34,000, approximately 31,000, and 28,000 compared with controls. Moreover, the specific activity of kinases for endogenous protein acceptors in liver of control mice was higher than that in liver of DHEA-treated animals. The specific activities of casein kinase, cAMP-dependent protein kinase, and cGMP-dependent protein kinase remained unchanged with DHEA treatment, but the specific activity of histone kinase was increased approximately 30%. Long-term administration of DHEA also was associated with increases in the specific activities of liver AMPase and GTPase (approximately two times), but not of other nucleotidases, alkaline phosphatase, acid phosphatase, glucose-6-phosphatase, or phosphotyrosine phosphatase. The activity of hepatic NADP-linked malic enzyme was increased significantly (two to three times) by DHEA treatment of female mice of three different strains, but was unchanged in male C57BL/6 mice. The specific activities of hepatic glucose-6-phosphate dehydrogenase, NADP-linked isocitrate dehydrogenase, and ATP-citrate lyase were not affected significantly by DHEA treatment of mice. The rate of hepatic lipogenesis, determined by incorporation of tritium from 3H2O into fatty acids, was decreased approximately 70% in DHEA-treated mice, while the rate of cholesterol synthesis was increased approximately 44% compared with controls.     

Skeletal muscle differentiation: role of dehydroepiandrosterone sulfate

Dehydroepiandrosterone (DHEA) and its sulfonated form dehydroepiandrosterone sulfate (DHEAS) are the main circulating steroid hormones and many epidemiological studies show an inverse relationship between DHEA/DHEAS levels and muscle loss for which the primary cause is the accelerated protein breakdown. The aim of this work was to determine whether DHEA/DHEAS supplementation in differentiating C2C12 skeletal muscle cells might influence the expression of the atrophy-related ubiquitin ligase, MuRF-1, and thereby impact key molecules of the differentiation program. DHEA is the prohormone crucial for sex steroid synthesis, and DHEAS is thought to be its reservoir. However, our preliminary experiments showed that DHEAS, but not DHEA, is able to influence MuRF-1 expression. Therefore, we treated differentiating C2C12 cells with various concentrations of DHEAS and analyzed the expression of MuRF-1, Hsp70, myosin heavy chain (MHC), myogenin, and the activity of creatine kinase. We observed that DHEAS at physiological concentrations downregulates MuRF-1 expression and affects muscle differentiation, as shown by the increased levels of MHC, which is a sarcomeric protein that undergoes MuRF-1-dependent degradation, and also by an increase in creatine kinase activity and myogenin expression, which are two other well-known markers of differentiation. Moreover, we found that DHEAS might have a protective effect on differentiating cells as suggested by the augmented levels of Hsp70, a member of heat shock proteins family that, besides its cytoprotective action, seems to have a regulatory role on key atrophy genes such as MuRF-1. In conclusion, our data shed light on the role of DHEAS at physiologic concentrations in maintaining muscle mass.     

Decrease of core body temperature in mice by dehydroepiandrosterone

Dietary dehydroepiandrosterone (DHEA) reduces food intake in mice, and this response is under genetic control. Moreover, both food restriction and DHEA can prevent or ameliorate certain diseases and mediate other biological effects. Mice fed DHEA (0.45% w/w of food) and mice pair-fed to these mice (food restricted) for 8 weeks were tested for changes in body temperature. DHEA was more efficient than food restriction alone in causing hypothermia. DHEA injected intraperitoneally also induced hypothermia that reached a nadir at 1 to 2 hr, and slowly recovered by 20 to 24 hr. This effect was dose dependent (0.5-50 mg). Each mouse strain tested (four) was susceptible to this effect, suggesting that the genetics differ for induction of hypophagia and induction of hypothermia. Because serotonin and dopamine can regulate (decrease) body temperature, we treated mice with haloperidol (dopamine receptor antagonist), 5,7-dihydroxytryptamine (serotonin production inhibitor), or ritanserin (serotonin receptor antagonist) prior to injection of DHEA. All of these agents increased rather than decreased the hypothermic effects of DHEA. DHEA metabolites that are proximate (5-androstene-3beta, 17beta-diol and androstenedione) or further downstream (estradiol-17beta) were much less effective than DHEA in inducing hypothermia. However, the DHEA analog, 16alpha-chloroepiandrosterone, was as active as DHEA. Thus, DHEA administered parentally seems to act directly on temperature-regulating sites in the body. These results suggest that DHEA induces hypothermia independent of its ability to cause food restriction, to affect serotonin or dopamine functions, or to act via its downstream steroid metabolites.     

Pharmacokinetics of oral dehydroepiandrosterone (DHEA) in the ovariectomised cynomolgus monkey

Humans and primates are unique in having adrenals that secrete large amounts of DHEA and DHEA-S in the circulation. These steroids act as precursors of active androgens and estrogen's in a long series of peripheral target intracrine tissues. The marked decline of serum DHEA and DHEA-S concentrations with age in men and women has been incriminated in the development of various pathologies. This study provides detailed information on the effect of a single 50mg oral dose of DHEA on circulating estrogen's as well as androgens and their metabolites over 10h in adult ovariectomised (OVX) Cynomolgus monkeys. Serum DHEA, DHEA-S, testosterone (Testo) and androstenedione (4-dione) concentrations increased rapidly with a maximal value at approximately 1h after DHEA administration followed by a 60-80% decrease during the next 2-6h. An important sulfatation of DHEA occurs through first hepatic pass, thus, leading to a marked increase in serum DHEA-S. Serum androst-5-ene-3beta,17beta-diol and androsterone glucuronide (ADT-G) levels remained elevated on a plateau for 6h. Androstan-3alpha,17beta-diol-glucuronide, estradiol and estrone levels remained unchanged. The present data indicate the predominant transformation of the adrenal precursor DHEA into active androgens in peripheral tissues and support the importance of measurement of circulating glucuronide derivatives as index of peripheral or intracrine androgen formation and action.     

Cholesterol substrate pools and steroid hormone levels are normal in the face of mutational inactivation of NPC1 protein

Mutational inactivation of NPC1 largely blocks the movement of LDL-derived cholesterol from the lysosome to the metabolically active, cytosolic pool of sterol that is the substrate for steroid hormone production. Such a block might, in theory, lead to deficiencies in circulating levels of testosterone, progesterone, and corticosterone. However, there are at least two other sources for cellular cholesterol, de novo synthesis and scavenger receptor class B type I-mediated uptake of HDL cholesteryl ester (CE). In this study, we measured the rates of net cholesterol acquisition by these three pathways in the adrenal, ovary, and testis. In all three organs, the majority (81-98%) of cholesterol acquisition came from the selective uptake of CE from HDL and de novo synthesis. Furthermore, in the npc1(-/-)mouse, the cytosolic storage pool of CE in a tissue such as the adrenal remained constant (approximately 25 mg/g). As a result of these alternative pathways, the plasma concentrations of testosterone (3.5 vs. 2.5 ng/ml), progesterone (8.5 vs. 6.7 ng/ml), and corticosterone (391 vs. 134 ng/ml) were either the same or elevated in the npc1(-/-)mouse, compared with the control animal. Thus, impairment of cholesterol acquisition through the NPC1-dependent, clathrin-coated pit pathway did not limit the availability of cholesterol substrate for steroid hormone synthesis in the steroidogenic cells.     

Urinary excretion of free progestins, androgens, and estradiol after injection of (1-24)ACTH in the Mongolian gerbil

Injection of a "long-acting" synthetic adrenocorticotrophin [(1-24)ACTH, 20 IU/animal] into Mongolian gerbils resulted in a 3.1 fold increase of urinary free testosterone excretion over 2 days. It was accompanied by an elevation of urinary free progesterone (2.1 fold), 17-hydroxyprogesterone (2.5 fold), DHEA (2.8 fold) and androstenedione (3.0 fold) excretion. Similarly, administration of human chorionic gonadotrophin (HCG, 100 IU/animal) increased urinary excretion of free testosterone (2.3 fold), progesterone (4.1 fold), 17-hydroxyprogesterone (2.9 fold), DHEA (4.6 fold), androstenedione (5.4 fold) and of estradiol (2.9 fold). Data presented in this work show that the measurement of urinary free steroid excretion represents a reliable index for the secretory activity of the adrenal-gonadal-axis, and that it may in some aspects be more practicable than the measurement of steroid plasma levels, especially in small laboratory animals, enabling us to monitor the excretion of various steroids over longer time periods without stressing the animals by handling/or blood sampling.     

Interleukin-6 and cachexia in ApcMin/+ mice

The Apc(Min/+) mouse has a mutation in the Apc tumor suppressor gene and develops intestinal polyps, beginning at 4 wk of age. This mouse develops cachexia by 6 mo, characterized by significant loss of muscle and fat tissue. The purpose of the present study was to determine the role of circulating interleukin-6 (IL-6) and the polyp burden for the development of cachexia in Apc(Min/+) mice. At 26 wk of age, mice exhibiting severe cachectic symptoms had a 61% decrease in gastrocnemius muscle weight, complete loss of epididymal fat, a 10-fold increase in circulating IL-6 levels, and an 89% increase in intestinal polyps compared with mildly cachectic animals. Apc(Min/+)/IL-6(-/-) mice did not lose gastrocnemius muscle mass or epididymal fat pad mass while overall polyp number decreased by 32% compared with Apc(Min/+) mice. Plasmid-based IL-6 overexpression in Apc(Min/+)/IL-6(-/-) mice led to a decrease in gastrocnemius muscle mass and epididymal fat pad mass and increased intestinal polyp burden. IL-6 overexpression did not induce cachexia in non-tumor-bearing mice. These data demonstrate that IL-6 is necessary for the onset of adipose and skeletal muscle wasting in the Apc(Min/+) mouse and that circulating IL-6 can regulate Apc(Min/+) mouse tumor burden.     

Therapeutic effect of glycyrrhetinic acid in MRL lpr/lpr mice: implications of alteration of corticosteroid metabolism.

Glycyrrhetinic acid (GA) inhibits 11beta-hydroxysteroid dehydrogenase and increases the levels and thus the action of endogenous glucocorticoid. We considered that GA could be used effectively for treatment of autoimmune diseases that have been treated by synthetic glucocorticoids. In this report, we demonstrated that GA delayed the development of autoimmune disease in spontaneously autoimmune strain MRL lpr/lpr (referred to as lpr) mice. GA was administered via drinking water at approximately 5 mg/kg/day for 170 days. An increase of urine protein levels in the mice treated with GA was delayed as compared to the control mice. After GA treatment began, urinary protein levels in the GA-treated mice were found to be significantly lower than vehicle-treated mice (p<0.05) between days 18 to 50. At 3 weeks of GA treatment serum IgG levels were lowered significantly in comparison with the control mice (p<0.03). In this circumstance, 11beta-HSD activities in liver and kidney were significantly inhibited by GA treatment (p<0.03, p<0.04 respectively). Concentration of corticosterone and dehydrocorticosterone in liver significantly increased after 3 weeks of GA treatment (p<0.02, p<0.01 respectively). In contrast to the local tissue levels of corticosteroids, the serum concentration of dehydrocorticosterone significantly decreased with GA treatment (p<0.02). These data suggest that GA could modify the local and systemic homeostasis of steroid metabolism in lpr mice. We concluded that the continuous treatment of GA is able to retard the development of autoimmune disease by suppressing urinary protein excretion and serum IgG levels in lpr mice. Modulation of local tissue levels as well as serum levels of corticosteroid by GA may thus be implicated in the therapeutic efficacy of GA.     

Deletion of the mouse P450c17 gene causes early embryonic lethality

Dehydroepiandrosterone (DHEA), a 19-carbon precursor of sex steroids, is abundantly produced in the human but not the mouse adrenal. However, mice produce DHEA and DHEA-sulfate (DHEAS) in the fetal brain. DHEA stimulates axonal growth from specific populations of mouse neocortical neurons in vitro, while DHEAS stimulates dendritic growth from those cells. The synthesis of DHEA and sex steroids, but not mouse glucocorticoids and mineralocorticoids, requires P450c17, which catalyzes both 17 alpha-hydroxylase and 17,20-lyase activities. We hypothesized that P450c17-knockout mice would have disordered sex steroid synthesis and disordered brain DHEA production and thus provide phenotypic clues about the functions of DHEA in mouse brain development. We deleted the mouse P450c17 gene in 127/SvJ mice and obtained several lines of mice from two lines of targeted embryonic stem cells. Heterozygotes were phenotypically and reproductively normal, but in all mouse lines, P450c17(-/-) zygotes died by embryonic day 7, prior to gastrulation. The cause of this early lethality is unknown, as there is no known function of fetal steroids at embryonic day 7. Immunocytochemistry identified P450c17 in embryonic endoderm in E7 wild-type and heterozygous embryos, but its function in these cells is unknown. Enzyme assays of wild-type embryos showed a rapid rise in 17-hydroxylase activity between E6 and E7 and the presence of C(17,20)-lyase activity at E7. Treatment of pregnant females with subcutaneous pellets releasing DHEA or 17-OH pregnenolone at a constant rate failed to rescue P450c17(-/-) fetuses. Treatment of normal pregnant females with pellets releasing pregnenolone or progesterone did not cause fetal demise. These data suggest that steroid products of P450c17 have heretofore-unknown essential functions in early embryonic mouse development.