Prolactin receptor-associated protein/17beta-hydroxysteroid dehydrogenase type 7 gene (Hsd17b7) plays a crucial role in embryonic development and fetal survival

Our laboratory has previously cloned and purified a protein named PRAP (prolactin receptor-associated protein) that was shown to be a novel 17beta-hydroxysteroid dehydrogenase (HSD) enzyme with dual activity. This enzyme, renamed HSD17B7 or PRAP/17beta-HSD7, converts estrone to estradiol and is also involved in cholesterol biosynthesis. The major site of its expression is the corpus luteum of a great number of species including rodents and humans. To examine the functional significance of HSD17B7 in pregnancy, we generated a knockout mouse model with targeted deletions of exons 1-4 of this gene. We anticipated a mouse with a severe fertility defect due to its inability to regulate estrogen levels during pregnancy. The heterozygous mutant mice are normal in their development and gross anatomy. The females cycle normally, and both male and female are fertile with normal litter size. To our surprise, the breeding of heterozygous mice yielded no viable HSD17B7 null mice. However, we found HSD17B7 null embryo alive in utero on d 8.5 and d 9.5. By d 10.5, the fetuses grow and suffer from severe brain malformation and heart defect. Because the brain depends on in situ cholesterol biosynthesis for its development beginning at d 10, the major cause of fetal death appears to be due to the cholesterol synthetic activity of this enzyme. By ablating HSD17B7 function, we have uncovered, in vivo, an important requirement for this enzyme during fetal development.     

Regulation of 17-beta hydroxysteroid dehydrogenase type 2 in human placental endothelial cells

17-beta hydroxysteroid dehydrogenase type 2 (HSD17B2) oxidizes estradiol to estrone, testosterone to androstenedione, and 20 alpha-dihydroprogesterone to progesterone. HSD17B2 is highly expressed in human placental tissue where it is localized to placental endothelial cells lining the fetal compartment. The aim of this study was to investigate the effects of potential regulatory factors including progesterone, estradiol, and retinoic acid (RA) onHSD17B2 expression in primary human placental endothelial cells in culture.HSD17B2 mRNA expression was not regulated by progesterone, the progesterone agonist R5020, or estradiol treatment. RA significantly induced HSD17B2 mRNA levels and enzyme activity in a dose- and time-dependent manner. Maximal stimulation occurred at Hour 48 at an RA concentration of 10(-6) M. Both retinoic acid receptor alpha (RARA) and retinoid X receptor alpha (RXRA) were readily detected by immunoblotting in isolated placental endothelial cells. RNA interference directed against RARA or RXRA led to reduced basal levels of HSD17B2 mRNA levels and significantly abolished RA-stimulated HSD17B2 expression. Together, these data indicate that regulation of HSD17B2 mRNA levels and enzymatic activity by RA in the placenta is mediated by RARA and RXRA.     

Steroidogenic enzymes and stem cell markers are upregulated during androgen deprivation in prostate cancer

Considerable levels of testosterone and dihydrotestosterone (DHT) are found in prostate cancer (PCa) tissue after androgen deprivation therapy. Treatment of surviving cancer-initiating cells and the ability to metabolize steroids from precursors may be the keystones for the appearance of recurrent tumors. To study this hypothesis, we assessed the expression of several steroidogenic enzymes and stem cell markers in clinical PCa samples and cell cultures during androgen depletion. Gene expression profiles were determined by microarray or qRT-PCR. In addition, we measured cell viability and analyzed stem cell marker expression in DuCaP cells by immunocytochemistry. Seventy patient samples from different stages of PCa, and the PCa cell line DuCaP were included in this study. The androgen receptor (AR) and enzymes (AKR1C3, HSD17B2, HSD17B3, UGT2B15 and UGT2B17 ) that are involved in the metabolism of adrenal steroids were upregulated in castration resistant prostate cancer (CRPC). In vitro, some DuCaP cells survived androgen depletion, and eventually gave rise to a culture adapted to these conditions. During and after this transition, most of the steroidogenic enzymes were upregulated. These cells also are enriched with stem/progenitor cell markers cytokeratin 5 (CK5) and ATP-binding cassette sub-family G member 2 (ABCG2). Similarly, putative stem/progenitor cell markers CK5, c-Kit, nestin, CD44, c-met, ALDH1A1, α2-integrin, CD133, ABCG2, CXCR4 and POU5F1 were upregulated in clinical CRPC. The upregulation of steroidogenic enzymes and stem cell markers in recurrent tumors suggests that cancer initiating cells can expand by adaptation to their T/DHT deprived environment. Therapies targeting the metabolism of adrenal steroids by the tumor may prove effective in preventing tumor regrowth.     

Investigation of Testosterone,Androstenone, and Estradiol Metabolism in HepG2 Cells and Primary Culture Pig Hepatocytes and Their Effects on 17βHSD7 Gene Expression

Steroid metabolism is important in various species. The accumulation of androgen metabolite, androstenone, in pig adipose tissue is negatively associated with pork flavor, odour and makes the meat unfit for human consumption. The 17β-hydroxysteroid dehydrogenase type 7 (17βHSD7) expressed abundantly in porcine liver, and it was previously suggested to be associated with androstenone levels. Understanding the enzymes and metabolic pathways responsible for androstenone as well as other steroids metabolism is important for improving the meat quality. At the same time, metabolism of steroids is known to be species- and tissue-specific. Therefore it is important to investigate between-species variations in the hepatic steroid metabolism and to elucidate the role of 17βHSD7 in this process. Here we used an effective methodological approach, liquid chromatography coupled with mass spectrometry, to investigate species-specific metabolism of androstenone, testosterone and beta-estradiol in HepG2 cell line, and pig cultured hepatocytes. Species- and concentration-depended effect of steroids on 17βHSD7 gene expression was also investigated. It was demonstrated that the investigated steroids can regulate the 17βHSD7 gene expression in HepG2 and primary cultured porcine hepatocytes in a concentration-dependent and species-dependent pattern. Investigation of steroid metabolites demonstrated that androstenone formed a 3′-hydroxy compound 3β-hydroxy-5α-androst-16-ene. Testosterone was metabolized to 4-androstene-3,17-dione. Estrone was found as the metabolite for β-estradiol. Inhibition study with 17βHSD inhibitor apigenin showed that apigenin didn’t affect androstenone metabolism. Apigenin at high concentration (50 µM) tends to inhibit testosterone metabolism but this inhibition effect was negligible. Beta-estradiol metabolism was notably inhibited with apigenin at high concentration. The study also established that the level of testosterone and β-estradiol metabolites was markedly increased after co-incubation with high concentration of apigenin. This study established that 17βHSD7 is not the key enzyme responsible for androstenone and testosterone metabolism in porcine liver cells.     

Lack of association between common polymorphisms in the 17beta-hydroxysteroid dehydrogenase type V gene (HSD17B5) and precocious pubarche

BACKGROUND: 17beta-Hydroxysteroid dehydrogenase (type V; HSD17B5) is a key enzyme involved in testosterone production in females. A single nucleotide polymorphism (SNP) in the promoter region of its gene was recently found to be associated with polycystic ovary syndrome (PCOS) and its related hyperandrogenaemia. Precocious pubarche (PP) is a clinical entity pointing to adrenal androgen excess from mid-childhood onward and is associated with ovarian androgen excess from puberty onward. It is therefore a strong risk factor for PCOS. METHODS: To investigate associations between this promoter SNP along with three exonic SNPs (one non-synonymous and two synonymous) from the same gene, and PP, a case-control study was performed in 190 girls with PP (84 of which were also tested for functional ovarian hyperandrogenism) from Barcelona, Spain and 71 healthy controls. Clinical features and hormone concentrations relevant to hyperandrogenism were compared by HSD17B5 genotype and haplotype. RESULTS: Neither HSD17B5 genotypes nor haplotype were associated with PP, or subsequent androgen excess in girls from Barcelona (all P>0.05). CONCLUSIONS: HSD17B5 SNPs predicted to have functional effects do not appear to be a risk factor for PP in girls from Barcelona, despite these girls being at high risk of developing androgen excess in adulthood.     

Effect of nomegestrol acetate on estrogen biosynthesis and transformation in MCF-7 and T47-D breast cancer cells

Although ovaries serve as the primary source of estrogen for pre-menopausal women, after menopause estrogen biosynthesis from circulating precursors occurs in peripheral tissues by the action of several enzymes, 17beta-hydroxysteroid dehydrogenase 1 (17beta-HSD1), aromatase and estrogen sulfatase. In the breast, both normal and tumoral tissues have been shown to be capable of synthesizing estrogens, and this local estrogen production can be implicated in the development of breast tumors. In these tissues, estradiol (E(2)) can be synthesized by three pathways: (1) estrone sulfatase transforms estrogen sulfates into bioactive estrogens, (2) 17beta-HSD1 converts estrone (E(1)) into E(2), (3) aromatase which converts androgens into estrogens is also present and contributes to the in situ synthesis of active estrogens but to a far lesser extent than estrone sulfatase. Quantitative assessment of E(2) formation in human breast tumors indicates that metabolism of estrone sulfate (E(1)S) via the sulfatase pathway produces 100-500 times more E(2) than androgen aromatization. Breast tissue also possesses the estrogen sulfotransferase involved in the conversion of estrogens into their sulfates that are biologically inactive. In the present review, we summarized the action of the 19-nor-progestin nomegestrol acetate (NOMAC) on the sulfatase, 17beta-HSD1 and sulfotransferase activities in the hormone-dependent MCF-7 and T47-D human breast cancer cell lines. Using physiological doses of substrates NOMAC blocks very significantly the conversion of E(1)S to E(2). It inhibits the transformation of E(1) to E(2). NOMAC has a stimulatory effect on sulfotransferase activity in both cell lines, with a strong stimulating effect at low doses but only a weak effect at high concentrations. The effects on the three enzymes are always stronger in the progesterone-receptor rich T47-D cell line as compared with the MCF-7 cell line. Besides, no effect is found for NOMAC on the transformation of androstenedione to E(1) in the aromatase-rich choriocarcinoma cell line JEG-3. In conclusion, the inhibitory effect provoked by NOMAC on the enzymes involved in the biosynthesis of E(2) (sulfatase and 17HSD pathways) in estrogen-dependent breast cancer, as well as the stimulatory effect on the formation of the inactive E(1)S, can open attractive perspectives for future clinical trials.     

Age-related changes of plasma steroids in normal adult males

Plasma cortisol, 17-hydroxyprogesterone (17-OH-P), testosterone (T), 5α-dihydrotestosterone (DHT, estrone (E₁) and estradiol (E₂), were measured in 94 normal adult men aged between 20–99. using RIA methods after chromatographic separation of steroids on Sephadex LH-20 columns.All plasma steroids except 17-OH-P, were age dependent: cortisol, testosterone and DHT decreased significantly with age, whereas estrone and estradiol were significantly increased in elderly men. Cortisol, testosterone. T/DHT ratio and estradiol levels were significantly correlated with age.The age related changes of plasma steroids in elderly men, were suggestive of decreased cortisol secretion, and decreased testicular function with increased peripheral conversion of androgens into estrogens. Testosterone was positively correlated with its precursor (17-OH-P) and respectively its peripheral metabolites (DHT and E₂). The negative correlation between estrone and 17-OH-P found in elderly men, suggested that increased estrogen level in aging males may be considered able to inhibit the testicular androgen production.     

Acetylation targets HSD17B4 for degradation via the CMA pathway in response to estrone

Dysregulation of hormone metabolism is implicated in human breast cancer. 17β-hydroxysteroid dehydrogenase type 4 (HSD17B4) catalyzes the conversion of estradiol (E2) to estrone (E1), and is associated with the pathogenesis and development of various cancers. Here we show that E1 upregulates HSD17B4 acetylation at lysine 669 (K669) and thereby promotes HSD17B4 degradation via chaperone-mediated autophagy (CMA), while a single mutation at K669 reverses the degradation and confers migratory and invasive properties to MCF7 cells upon E1 treatment. CREBBP and SIRT3 dynamically control K669 acetylation level of HSD17B4 in response to E1. More importantly, K669 acetylation is inversely correlated with HSD17B4 in human breast cancer tissues. Our study reveals a crosstalk between acetylation and CMA degradation in HSD17B4 regulation, and a critical role of the regulation in the malignant progression of breast cancer.     

Human 17beta-hydroxysteroid dehydrogenases types 1, 2, and 3 catalyze bi-directional equilibrium reactions, rather than unidirectional metabolism, in HEK-293 cells

Human 17beta-hydroxysteroid dehydrogenases (17betaHSDs) catalyze the interconversion of weak and potent androgen and estrogen pairs. Although the reactions using purified enzymes can be driven in either direction, these enzymes appear to function unidirectionally in intact cells: only reductive reactions for 17betaHSD1 and 17beta HSD3 and only oxidative reactions for 17betaHSD2. We show that, after exhaustive incubations with either 17beta-hydroxy- or 17-ketosteroid, the medium for HEK-293 cells expressing 17betaHSD1 or 17betaHSD3 contains a 92:8 ratio of reduced:oxidized steroid. Similarly, 17betaHSD2 yields a >95:5 ratio of oxidized:reduced steroids for both androgens and estrogens. Dual-isotope kinetic measurements show that the rates of the forward and reverse reactions are identical at these functional equilibrium states in intact cells for all three 17betaHSD isoforms, and these rates are much faster than those estimated from single-isotope flux studies. Mutation L36D converts 17betaHSD1 to an oxidative enzyme in intact cells, reversing the equilibrium distribution of estradiol:estrone to 5:95; however, the rates of the forward and reverse reactions at equilibrium are equal and comparable to those of the wild-type enzymes. The co-expression of 17betaHSD2 paradoxically increases the potency of estrone in transactivation assays, demonstrating the physiological relevance of "backwards" metabolism to estradiol. We conclude that 17betaHSD types 1, 2, and 3 catalyze both oxidative and reductive reactions in HEK-293 cells at intrinsic rates that are much faster than those estimated from single-isotope studies. These 17betaHSD isoforms do not drive steroid flux in one direction but rather may achieve functional equilibria in intact cells, reflecting thermodynamically driven steroid distributions.     

Non-steroidal anti-inflammatory drugs interact with testosterone glucuronidation

Testosterone and epitestosterone are secreted mainly as glucuronide metabolites and the urinary ratio of testosterone glucuronide to epitestosterone glucuronide, often called T/E, serves as a marker for possible anabolic steroids abuse by athletes. UDP-glucuronosyltransferase (UGT) 2B17 is the most important catalyst of testosterone glucuronidation. The T/E might be affected by drugs that interact with UGT2B17, or other enzymes that contribute to testosterone glucuronidation. Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used by sportsmen and we have examined the effect of two NSAIDs, diclofenac and ibuprofen, on testosterone and epitestosterone glucuronidation in human liver microsomes. In parallel, we have studied the inhibitory effect of these NSAIDs on recombinant UGT2B17 and UGT2B15, as well as other human hepatic UGTs that revealed low but detectable testosterone glucuronidation activity, namely UGT1A3, UGT1A4, UGT1A9 and UGT2B7. Both diclofenac and ibuprofen inhibited testosterone glucuronidation in microsomes, as well as UGT2B15 and UGT2B17. Interestingly, UGT2B15 was more sensitive than UGT2B17 to the two drugs, particularly to ibuprofen. Human liver microsomes lacking functional UGT2B17 exhibited significantly higher sensitivity to ibuprofen, suggesting that UGT2B15 plays a major role in the residual testosterone glucuronidation activity in UGT2B17-deficient individuals. Nonetheless, a minor contribution of other UGTs, particularly UGT1A9, to testosterone glucuronidation in such individuals cannot be ruled out at this stage. The epitestosterone glucuronidation activity of human liver microsomes was largely insensitive to ibuprofen and diclofenac. Taken together, the results highlight potential interactions between NSAIDs and androgen glucuronidation with possible implications for the validity of doping tests.     

Steroid metabolizing enzymes associated with the microvillar membrane of human placenta

17 beta-Hydroxysteroid oxidoreductase, as well as estrone sulfate and dehydroepiandrosterone sulfate sulfatases, were found in the plasma membrane of microvilli of the fetal syncytiotrophoblast. Because of their location, these enzymes may influence feto-maternal transfer of steroids circulating as sulfates, the utilization of sulfated estrogen precursors and the proportion of estrone and estradiol delivered towards fetal and maternal circulations. Microvillar vesicles isolated from human term placentas were disrupted in hypotonic medium to obtain a membrane preparation. A fraction of the estradiol 17 beta-oxidoreductase (E2DH) activity in the vesicle remained associated to the membrane after disruption and treatment with 2 M NaCl. The membrane-associated activity was resistant to inhibition with trypsin and did not react with a polyclonal antibody which neutralized cytosolic E2DH activity. The membrane-associated enzyme was solubilized with a cholate-glycerol buffer solution and purified on Sephadex G-100. The estimated molecular weight of the solubilized enzyme (137 kDa) appears to correspond to a tetramer since it was found to be about twice the size of the cytosolic enzyme. Both enzymes focused in polyacrylamide gels at pH 5.2. The Km relative to E2 of the membrane-associated E2DH (1.3 microM) differs from those of mitochondrial (0.43 microM), microsomal (0.69 microM) and cytosolic (11 microM) fractions. The cytosolic and the microvillar membrane associated 17 beta-hydroxysteroid oxidoreductases also differ in their specificity for C18 and C19 steroid substrates and in their pH dependence patterns. Sulfatases acting on estrone sulfate and dehydroepiandrosterone sulfate in microvillar membranes were insensitive to trypsin and as resistant to washes with 2 M NaCl as alkaline phosphatase. This data indicated that steroid sulfatases are also microvillar membrane associated enzymes of potential physiologic importance in the hydrolysis of estrogen precursors.     

Characterization of type 12 17beta-hydroxysteroid dehydrogenase, an isoform of type 3 17beta-hydroxysteroid dehydrogenase responsible for estradiol formation in women

A novel 17beta-hydroxysteroid dehydrogenase (17beta-HSD) chronologically named type 12 17beta-HSD (17beta-HSD12), that transforms estrone (E1) into estradiol (E2) was identified by sequence similarity with type 3 17beta-HSD (17beta-HSD3) that catalyzes the formation of testosterone from androstenedione in the testis. Both are encoded by large genes spanning 11 exons, most of them showing identical size. Using human embryonic kidney-293 cells stably expressing 17beta-HSD12, we have found that the enzyme catalyzes selectively and efficiently the transformation of E1 into E2, thus identifying its role in estrogen formation, in contrast with 17beta-HSD3, the enzyme involved in the biosynthesis of the androgen testosterone in the testis. Using real-time PCR to quantify mRNA in a series of human tissues, the expression levels of 17beta-HSD12 as well as two other enzymes that perform the same transformation of E1 into E2, namely type 1 17beta-HSD and type 7 17beta-HSD, it was found that 17beta-HSD12 mRNA is the most highly expressed in the ovary and mammary gland. To obtain a better understanding of the structural basis of the difference in substrate specificity between 17beta-HSD3 and 17beta-HSD12, we have performed tridimensional structure modelization using the coordinates of type 1 17beta-HSD and site-directed mutagenesis. The results show the potential role of bulky amino acid F234 in 17beta-HSD12 that blocks the entrance of androstenedione. Overall, our results strongly suggest that 17beta-HSD12 is the major estrogenic 17beta-HSD responsible for the conversion of E1 to E2 in women, especially in the ovary, the predominant source of estrogens before menopause.     

Two 17beta-hydroxysteroid dehydrogenases (17HSDs) of estradiol biosynthesis: 17HSD type 1 and type 7.

Two 17β-hydroxysteroid dehydrogenases (17HSDs), type 1 and type 7, are enzymes of estradiol biosynthesis, in addition to which rodent type 1 enzymes are also able to catalyze androgens. Both of the 17HSDs are abundantly expressed in ovaries, the type 1 enzyme in granulosa cells and type 7 in luteinized cells. The expression of 17HSD7, which has also been described as a prolactin receptor-associated protein (PRAP), is particularly up-regulated in corpus luteum during the second half of rodent pregnancy. A moderate or slight signal for mouse 17HSD7/PRAP mRNA has also been demonstrated in samples of placenta and mammary gland, for example. Human, but not rodent, 17HSD1 is expressed in placenta, breast epithelium and endometrium in addition to ovaries. A cell-specific enhancer, silencer and promoter in the hHSD17B1 gene participate in the regulation of type 1 enzyme expression. The enhancer consists of several subunits, including a retinoic acid response element, the silencer has a binding motif for GATA factors, and the proximal promoter contains adjacent and competing AP-2 and Sp binding sites.     

Two 17β-hydroxysteroid dehydrogenases (17HSDs) of estradiol biosynthesis: 17HSD type 1 and type 7

Two 17β-hydroxysteroid dehydrogenases (17HSDs), type 1 and type 7, are enzymes of estradiol biosynthesis, in addition to which rodent type 1 enzymes are also able to catalyze androgens. Both of the 17HSDs are abundantly expressed in ovaries, the type 1 enzyme in granulosa cells and type 7 in luteinized cells. The expression of 17HSD7, which has also been described as a prolactin receptor-associated protein (PRAP), is particularly up-regulated in corpus luteum during the second half of rodent pregnancy. A moderate or slight signal for mouse 17HSD7/PRAP mRNA has also been demonstrated in samples of placenta and mammary gland, for example. Human, but not rodent, 17HSD1 is expressed in placenta, breast epithelium and endometrium in addition to ovaries. A cell-specific enhancer, silencer and promoter in the hHSD17B1 gene participate in the regulation of type 1 enzyme expression. The enhancer consists of several subunits, including a retinoic acid response element, the silencer has a binding motif for GATA factors, and the proximal promoter contains adjacent and competing AP-2 and Sp binding sites.     

Multiple steroid metabolic pathways in ZR-75-1 human breast cancer cells

In order to characterize the main enzymatic systems involved in androgen and estrogen formation as well as metabolism in ZR-75-1 human breast cancer cells, incubation of intact cells was performed for 12 or 24 h at 37 degrees C with tritiated estradiol (E2), estrone (E1), androst-5-ene-3 beta, 17 beta-diol (5-ene-diol), dehydroepiandrosterone (DHEA), testosterone (T), androstenedione (4-ene-dione), dihydrotestosterone (DHT) or androsterone (ADT). The extra- and intracellular steroids were extracted, separated into free steroids, sulfates and non-polar derivatives (FAE) and identified by HPLC coupled to a Berthold radioactivity monitor. Following incubation with E2, 5-ene-diol or T, E1, DHEA and 4-ene-dione were the main products, respectively, thus indicating high levels of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD). When 4-ene-dione was used, on the other hand, a high level of transformation into 5 alpha-androstane-3,17-dione (A-dione), Epi-ADT and ADT was found, thus indicating the presence of high levels of 5 alpha-reductase as well as 3 alpha- and 3 beta-hydroxysteroid dehydrogenase. Moreover, some T was formed, due to oxidation by 17 beta-HSD. No estrogen was detected with the androgen precursors T or 4-ene-dione, thus indicating the absence of significant aromatase activity. Moreover, significant amounts of sulfates and non-polar derivatives were found with all the above-mentioned substrates. The present study shows that ZR-75-1 human breast cancer cells possess most of the enzymatic systems involved in androgen and estrogen formation and metabolism, thus offering an excellent model for studies of the control of sex steroid formation and action in breast cancer tissue.     

Increased estradiol-estrone quotient in rat liver by hydroxysteroids: an effect of the specific hydrogen transfer between steroids]

After addition of estrone to rat liver slices, a quotient of estradiol/estrone of ca. 0.1 is reached within 1 - 2 min. By additional application of 17 beta-hydroxysteroids this quotient is changed in the direction of estradiol, although the applied concentrations of both steroids are far below the concentration of the cytoplasmic redox couple NADH/NAD. Of all the steroids tested, testosterone had the strongest influence on the quotient, especially in the liver of female rats. This influence is smaller in the livers of male rats and infantile animals. The changing of the E2/E1 quotient by testosterone can be inhibited by the antiandrogen cyproteron acetate. Steroids with hydroxy groups at C-3 or C-20 or high concentrations of non-steroids, which can be oxidized by NAD, change the E2/E1 quotient only minimally. The experiments demonstrate that in liver, the redox couple estradiol/estrone is not in equilibrium with the main redox couple of the cytoplasmic NADH/NAD. Only on account of this fact it is possible that relatively low concentrations of testosterone change the E2/E1 quotient via the C-17 leads to C-17 hydrogen transfer between steroids. Biological consequences are discussed.     

Estrogen production by fetal rat gonads

Aromatase activity in fetal rat testes and ovaries was demonstrated by the conversion of tritiated testosterone or 19-hydroxyandrostenedione into estrone and estradiol, which were identified and quantified by double isotopic dilution and recrystallization to constant specific activity. Testes formed mostly estradiol, ovaries mostly estrone. Aromatase activity was stimulated by cAMP in both the testes and ovaries as early as 17 days of fetal life. Stimulation by FSH was noted at this same stage in the testis, but not before 3–4 days after birth in the ovary. LH was without effect on aromatase activity in both kinds of gonads. Basal estrogen secretion was non-existent or undetectable in both the testes and ovaries in fetal stages. In the presence of cAMP and as early as 17 days of fetal life, the testes released estradiol, as early as 14 days the ovaries released estrone. Estrogen secretion was stimulated by LH and FSH at fetal stages in the testis and at infantile stages in the ovary. Responsiveness to gonadotrophins closely followed the appearance of the receptors.