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Female sex steroid hormones, including estradiol (E2) and progesterone (P4), serve significant physiological roles in pregnancy. In particular, E2 and P4 influence placenta formation, maintain pregnancy and stimulate milk production. These hormones are produced by ovaries, adrenal glands and the placenta, of which the latter is a major endocrine organ during pregnancy. However, the mechanism of hormone production during pregnancy remains unclear. In the present study, the regulation of steroid hormones and steroidogenic enzymes was examined in human placenta according to gestational age. In human placental tissues, expression levels of steroidogenic enzymes were determined with reverse transcription‑quantitative polymerase chain reaction and western blotting. The mRNA and protein expression of CYP17A1, HSD17B3 and CYP19A1, which are associated with the synthesis of dehydroepiandrosterone (DHEA) and E2, was elevated at different gestational ages in human placenta. In addition, to evaluate the correlation between serum and placental‑produced hormones, steroid hormone levels, including pregnenolone (PG), DHEA, P4, testosterone (T) and E2, were examined in serum and placenta. Serum and placenta expression of DHEA and E2 increased with gestational age, whereas T and P4 were differently regulated in placenta and serum. To confirm the mechanism of steroidogenesis in vitro, placental BeWo cells were treated with E2 and P4, which are the most important hormones during pregnancy. The mRNA and protein expression of steroidogenic enzymes was significantly altered by E2 in vitro. These results demonstrated that concentration of steroid hormones was differently regulated by steroidogenic enzymes in the placenta depending on the type of the hormones, which may be critical to maintain pregnancy. Show less

Mammary epithelial cells (MECs) affect milk production capacity during lactation and are critical for the maintenance of tissue homeostasis. Our previous studies have revealed that the expression of miR-152 was increased significantly in MECs of cows with high milk production. In the present study, bioinformatics analysis identified ACAA2 and HSD17B12 as the potential targets of miR-152, which were further validated by dual-luciferase repoter assay. In addition, the expressions of miR-152 was shown to be negatively correlated with levels of mRNA and protein of ACAA2, HSD17B12 genes by qPCR and western bot analysis. Furthermore, transfection with miR-152 significantly up-regulated triglyceride production, promoted proliferation and inhibited apoptosis in MECs. Furthermore, overexpression of ACAA2 and HSD17B12 could inhibit triglyceride production, cells proliferation and induce apoptosis; but sh234-ACAA2-181/sh234-HSD17B12-474 could reverse the trend. These findings suggested that miR-152 could significantly influence triglyceride production and suppress apoptosis, possibly via the expression of target genes ACAA2 and HSD17B12. Show less

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most abundant and carcinogenic tobacco-specific nitrosamine in tobacco and tobacco smoke. The major metabolic pathway for NNK is carbonyl reduction to form the (R) and (S) enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) which, like NNK, is a potent lung carcinogen. The goal of this study was to characterize NNAL enantiomer formation in human lung and identify the enzymes responsible for this activity. While (S)-NNAL was the major enantiomer of NNAL formed in incubations with NNK in lung cytosolic fractions, (R)-NNAL comprised ~60 and ~95% of the total NNAL formed in lung whole cell lysates and microsomes, respectively. In studies examining the role of individual recombinant cytosolic reductase enzymes in lung NNAL enantiomer formation, AKR1C1, AKR1C2, AKR1C3, AKR1C4 and CBR1 all exhibited (S)-NNAL-formation activity. To identify the microsomal enzymes responsible for (R)-NNAL formation, 28 microsomal reductase enzymes were screened for expression by real-time PCR in normal human lung. HSD17β6, HSD17β12, KDSR, NSDHL, RDH10, RDH11 and SDR16C5 were all expressed at levels ≥HSD11β1, the only previously reported microsomal reductase enzyme with NNK-reducing activity, with HSD17β12 the most highly expressed. Of these lung-expressing enzymes, only HSD17β12 exhibited activity against NNK, forming primarily (>95%) (R)-NNAL, a pattern consistent with that observed in lung microsomes. siRNA knock-down of HSD17β12 resulted in significant decreases in (R)-NNAL-formation activity in HEK293 cells. These data suggest that both cytosolic and microsomal enzymes are active against NNK and that HSD17β12 is the major active microsomal reductase that contributes to (R)-NNAL formation in human lung. Show less

17-beta hydroxysteroid dehydrogenase type 3 (17βHSD-3) enzyme catalyzes the conversion of androstenedione (Δ4) to testosterone (T) in the testes of the developing fetus, thus playing a crucial role in the differentiation of the gonads and in establishing the male sex phenotype. Any mutation in the encoding gene (HSD17B3) can lead to varying degrees of undervirilization of the affected male, ranging from completely undervirilized external female genitalia to predominantly male with micropenis and hypospadias. We present here an infant who was referred to our clinic because of ambiguous genitalia at birth. Gonads were palpable in the inguinal canal bilaterally and no Müllerian structures were identified on pelvic ultrasound. Because of a low T/Δ4 ratio after a human chorionic gonadotropin stimulation test, a tentative diagnosis of 17βHSD-3 deficiency was made which was confirmed after genetic analysis of the HSD17B3 gene of the patient. The molecular analysis identified compound heterozygosity of two previously described mutations and could offer some further validation for the idea of a founder effect for 655-1;G→A mutation in the Greek population. Show less

Essential thrombocytosis (ET) is a chronic myeloproliferative disorder with an unregulated surplus of platelets. Complications of ET include stroke, heart attack, and formation of blood clots. Although platelet-enhancing mutations have been identified in ET cohorts, genetic networks causally implicated in thrombotic risk remain unestablished. In this study, we aim to identify novel ET-related miRNA-mRNA regulatory networks through comparisons of transcriptomes between healthy controls and ET patients. Four network discovery algorithms have been employed, including (a) Pearson correlation network, (b) sparse supervised canonical correlation analysis (sSCCA), (c) sparse partial correlation network analysis (SPACE), and, (d) (sparse) Bayesian network analysis-all through a combined data-driven and knowledge-based analysis. The result predicts a close relationship between an 8-miRNA set (miR-9, miR-490-5p, miR-490-3p, miR-182, miR-34a, miR-196b, miR-34b*, miR-181a-2*) and a 9-mRNA set (CAV2, LAPTM4B, TIMP1, PKIG, WASF1, MMP1, ERVH-4, NME4, HSD17B12). The majority of the identified variables have been linked to hematologic functions by a number of studies. Furthermore, it is observed that the selected mRNAs are highly relevant to ET disease, and provide an initial framework for dissecting both platelet-enhancing and functional consequences of dysregulated platelet production. Show less

The aim of this study was to characterize the expression of hydroxysteroid (17β) dehydrogenase type 12 (HSD17B12), an enzyme involved in the synthesis of arachidonic acid (AA), in ovarian cancer, and to study its coexpression with its upstream and downstream enzymes in the AA pathway, namely elongation of very long chain fatty acids protein 5 (ELOVL5) and cyclooxygenase-2 (COX-2), respectively. Samples from benign and malignant ovarian neoplastic lesions were immunohistochemically stained with HSD17B12, ELOVL5, and COX-2. The staining intensities were quantified with the QuantCenter program, and the results were confirmed with visual inspection. Statistical significances were calculated with the Student t test, the Mann-Whitney test, linear regression, or ANOVA. The expression of the HSD17B12, ELOVL5, and COX-2 enzymes increased according to the grade of the endometrioid ovarian adenocarcinomas. In contrast, in serous adenocarcinomas, staining with ELOVL5 was constantly weak, whereas the expression of HSD17B12 and COX-2 increased with the grade or FIGO stage of the cancer, respectively. The expression of HSD17B12 increased along with the severity of ovarian cancer, and the expression mimicked COX-2 expression and intensity. This further suggests the involvement of HSD17B12 in AA production, and its coexpression with COX-2 indicates a role for the enzyme in the increased prostaglandin production during ovarian cancer progression. Show less

Decabromodiphenyl ether (BDE-209), a congener of polybrominated diphenyl ethers (PBDEs), is used as flame retardant and affects thyroid homeostasis. Thyroid hormones (THs) play crucial role in Leydig cell differentiation and steroidogenesis during early life. Present study examined the effect of maternal BDE-209 exposure during lactation on testicular steroidogenesis and spermatogenesis in relation to thyroid hormone receptor alpha 1 (THRα1) and possible mechanism(s) of its action in prepubertal Parkes mice offspring. Lactating female Parkes mice were orally gavaged with 500, and 700 mg/kg body weight of BDE-209 in corn oil from postnatal day (PND) 1 to PND 28. Lactating mothers and male pups were sacrificed on PND 28. Maternal BDE-209 exposure markedly affected testicular histopathology, steroidogenesis and germ cell dynamics with downregulated expressions of various steroidogenic markers in mice offspring. Serum THs levels were markedly reduced in both pups and lactating mothers compared to controls. Expression of proliferating cell nuclear antigen and THRα1 also deceased in testes of BDE-209-exposed mice offspring. In silico analysis by molecular docking was performed successfully for steroidogenic facor-1 (SF-1) and THRα1 with BDE-209 and T Show less

17β-Hydroxysteroid dehydrogenase type 3 (17β-HSD3) is a major player in human endocrinology, being one of the most important enzymes involved in testosterone production. To capitalize on the discovery of RM-532-105, a steroidal 17β-HSD3 inhibitor, we explored the effect of its backbone configuration on inhibitory activity, androgenic profile, and metabolic stability. Two modifications that greatly alter the natural shape of steroids, i.e. inversion of the methyl on carbon 13 (13α-CH Show less

The presence of splicing sequence variants in genes responsible for sex development in humans may compromise correct biosynthesis of proteins involved in the normal development of gonads and external genitalia. In a cohort of Brazilian patients, we identified mutations in HSD17B3 and SRD5A2 which are both required for human sexual differentiation. A number of these mutations occurred within regions potentially critical for splicing regulation. Minigenes were used to validate the functional effect of mutations in both genes. We evaluated the c.277 + 2 T > G mutation in HSD17B3, and the c.544 G > A, c.548-44 T > G and c.278delG mutations in SRD5A2. We demonstrated that these mutations altered the splicing pattern of these genes. In a genomic era these results illustrate, and remind us, that sequence variants within exon-intron boundaries, which are primarily identified for diagnostic purposes and have unknown pathogenicity, need to be assessed with regards to their impact not only on protein expression, but also on mRNA splicing. Show less

Mutations in the HSD17B3 gene are associated with a 46,XY disorder of sexual development (46,XY DSD) as a result of low testosterone production during embryogenesis. To elucidate the molecular basis of the disorder by chemically analyzing four missense mutations in HSD17B3 (T54A, M164T, L194P, G289S) from Egyptian patients with 46,XY DSD. Expression plasmids for wild-type 17β-hydroxysteroid hydrogenase type 3 (17β-HSD3) and mutant enzymes generated by site-directed mutagenesis were transiently transfected into human HEK-293 cells. Protein expression was verified by western blotting and activity was determined by measuring the conversion of radiolabeled Δ Testosterone formation by wild-type and mutant 17β-HSD3 enzymes was compared. Mutations T54A and L194P, despite normal protein expression, completely abolished 17β-HSD3 activity, explaining their severe 46,XY DSD phenotype. Mutant M164T could still produce testosterone, albeit with significantly lower activity compared with wild-type 17β-HSD3, resulting in ambiguous genitalia or a microphallus at birth. The substitution G289S represented a polymorphism exhibiting comparable activity to wild-type 17β-HSD3. Sequencing of the SRD5A2 gene in three siblings bearing the HSD17B3 G289S polymorphism disclosed the homozygous Y91H mutation in the former gene, thus explaining the 46,XY DSD presentations. Molecular modeling analyses supported the biochemical observations and predicted a disruption of cofactor binding by mutations T54A and M164T and of substrate binding by L196P, resulting in the loss of enzyme activity. In contrast, the G289S substitution was predicted to disturb neither the three-dimensional structure nor enzyme activity. Biochemical analysis of mutant 17β-HSD3 enzymes is necessary to understand genotype-phenotype relationships. Biochemical analysis combined with molecular modeling provides insight into disease mechanism. However, the stability of mutant proteins in vivo cannot be predicted by this approach. The 17β-HSD3 G289S substitution, previously reported in other patients with 46,XY DSD, is a polymorphism that does not cause the disorder; thus, further sequence analysis was required and disclosed a mutation in SRD5A2, explaining the cause of 46,XY DSD in these patients. Engeli RT, Tsachaki M, Hassan HA, et al. Biochemical Analysis of Four Missense Mutations in the HSD17B3 Gene Associated With 46,XY Disorders of Sex Development in Egyptian Patients. J Sex Med 2017;14:1165-1174. Show less

17β-Hydroxysteroid dehydrogenase type 3 (17β-HSD3) converts the inactive Δ4-androstenedione (A) to testosterone (T). Its deficiency is the most common testosterone biosynthesis defect that results in 46,XY Disorders Of Sex Development (DSD). However, the disease is difficult to distinguish from other 46,XY DSD for similar clinical phenotypes. Therefore, genetic testing provides good criteria for the diagnosis of the disease. In this study, HSD17B3 gene was examined in 3 unrelated Chinese patients with 46,XY DSD. Direct sequencing and quantitative PCR of HSD17B3 gene revealed the presence of a compound heterozygous mutation (p.I60T/exon1 deletion) in Patient 1, a homozygous (p.I60T) mutation in Patient 2 and a frameshift mutation (p.V25Efs∗54) and an exon1 deletion in Patient 3. All of the mutations have not been reported previously. These novel mutations may expand the mutation database of HSD17B3 gene and provide us new insights into the molecular mechanism of 17β-HSD3 deficiency. It is noteworthy that when direct sequence analysis showed a rare homozygous mutation in patients with non-consanguineous parents, "apparent homozygosity" should be taken into an account and the intragenic deletion should be screened. In addition, when single mutation was found in patients with disease in recessive heredity mode, the intragenic deletion should also be screened. Show less

17β-Hydroxysteroid dehydrogenase type 3 (17β-HSD3) is expressed almost exclusively in the testes and specifically converts the weak androgenic androstenedione to active testosterone (T) in the presence of NADPH. Additionally, studies have demonstrated that 17β-HSD3 is over-expressed in hormone-dependent prostate cancer. T, which interacts with the androgen receptor (AR), eventually stimulates the growth of prostate cancer cells. Defects in T synthesis or action impair the development of the male phenotype during embryogenesis and cause the autosomal recessive disorder male pseudohermaphroditism. Affected individuals are often born with female-appearing external genitalia and are reared as females. Since 17β-HSD3 plays a central role in T production, it has been recognized as a promising therapeutic target to reduce the circulating level of androgens and to suppress androgen-sensitive tumor proliferation. In recent decades, improvements have been made in the development of 17β-HSD3 inhibitors. Herein, we give an overview of the main structure and function of human 17β-HSD3 and summarize steroidal and non-steroidal inhibitors of 17β-HSD3, which can be a potential target for prostate cancer. Show less

In the fight against androgen-sensitive prostate cancer, the enzyme 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3) is an attractive therapeutic target considering its key role in the formation of androgenic steroids. In this study, we attempted to assess the in vivo efficacy of the compound RM-532-105, an androsterone derivative developed as an inhibitor of 17β-HSD3, in the prostate cancer model of androgen-sensitive LAPC-4 cells xenografted in nude mice. RM-532-105 did not inhibit the tumor growth induced by 4-androstene-3,17-dione (4-dione); rather, the levels of the androgens testosterone (T) and dihydrotestosterone (DHT) increased within the tumors. In plasma, however, DHT levels increased but T levels did not. In troubleshooting experiments, the non-androgenic potential of RM-532-105 was confirmed by two different assays (LAPC-4 proliferation and androgen receptor transcriptional activity assays). The enzyme 5α-reductase was also revealed to be the predominant enzyme metabolizing 4-dione in LAPC-4 cells, yielding 5α-androstane-3,17-dione and not T. Other 17β-HSDs than 17β-HSD3 seem responsible in the androgen synthesis. From experiments with LAPC-4 cells, we fortuitously came across the interesting finding that 17β-HSD3 inhibitor RM-532-105 is concentrated inside tumors. Show less

The purpose of this study was to identify genomic regions associated with carcass traits in an experimental Nelore cattle population. The studied data set contained 2,306 ultrasound records for longissimus muscle area (LMA), 1,832 for backfat thickness (BF), and 1,830 for rump fat thickness (RF). A high-density SNP panel (BovineHD BeadChip assay 700k, Illumina Inc., San Diego, CA) was used for genotyping. After genomic data quality control, 437,197 SNPs from 761 animals were available, of which 721 had phenotypes for LMA, 669 for BF, and 718 for RF. The SNP solutions were estimated using a single-step genomic BLUP approach (ssGWAS), which calculated the variance for windows of 50 consecutive SNPs and the regions that accounted for more than 0.5% of the additive genetic variance were used to search for candidate genes. The results indicated that 12, 18, and 15 different windows were associated to LMA, BF, and RF, respectively. Confirming the polygenic nature of the studied traits, 43, 65, and 53 genes were found in those associated windows, respectively for LMA, BF, and RF. Among the candidate genes, some of them, which already had their functions associated with the expression of energy metabolism, were found associated with fat deposition in this study. In addition, ALKBH3 and HSD17B12 genes, which are related in fibroblast death and metabolism of steroids, were found associated with LMA. The results presented here should help to better understand the genetic and physiologic mechanism regulating the muscle tissue deposition and subcutaneous fat cover expression of Zebu animals. The identification of candidate genes should contribute for Zebu breeding programs in order to consider carcass traits as selection criteria in their genetic evaluation. Show less

17β-hydroxysteroid dehydrogenase (17β-HSD) type 3 deficiency is an autosomal recessive disorder with diminished testosterone synthesis and consequently underandrogenisation. 46,XY patients with 17β-HSD type 3 deficiency are often assigned a female sex at birth but have a high virilisation potential at the time of puberty. We studied four 46,XY patients with 17β-HSD type 3 deficiency at puberty with regard to the underlying mutations, the hormone values, and the clinical findings. Three patients were initially assigned a female sex and 1 was assigned a male sex. All had relevant mutations in the HSD17B3 gene. The 2 patients with deleterious mutations had lower testosterone values at the time of puberty than the patients with possible residual activity of 17β-HSD type 3. One of the latter patients changed to male gender. All 4 patients with 17β-HSD type 3 deficiency synthesized relevant amounts (>0.7 µg/L) of testosterone at puberty, which lead to variable androgenisation. In patients with presumable residual activity of the mutated enzyme, testosterone values in the male reference range can be achieved, thereby inducing male pubertal development. These patients should possibly be assigned a male sex. Any surgical intervention should be avoided until the patients are old enough to consider their options of medical and surgical intervention.
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The aim of this study was to use targeted next-generation sequencing (TNGS) including all known genes associated with 46,XY disorders of sex development (DSD) for a fast molecular genetic diagnosis. Twenty pediatric patients were recruited, and 56 genes related to 46,XY DSD were sequenced using TNGS. The time elapsed between initial appointment and final diagnosis as well as the mean expenditure was determined. A total of 9 (45%) mutations in 4 different genes were identified. Mutations in the HSD17B3 gene were observed in 6 (30%) patients. A heterozygous mutation in WT1 gene and a hemizygous mutation in SRY gene were detected in patients with gonadal dysgenesis. One patient had a homozygous mutation in LHCGR gene. Prior to the molecular diagnosis, the mean number of clinical visits, time elapsed until diagnosis, and expenditure were 27.4 ± 14.6 visits, 5.9 ± 4.1 years per patient, and USD 2,142 ± 1,038, respectively. With TNGS, time elapsed until diagnosis was significantly reduced (3 days), and expenditure per patient was only one third of the conventional approach (USD 761). TNGS is an efficient, rapid, and cost-effective technique for mutation detection in 46,XY DSD. Show less

17β-hydroxysteroid dehydrogenase 3 deficiency consists of a defect in the last phase of steroidogenesis, in which androstenedione is converted into testosterone and estrone into estradiol. External genitalia range from female-like to atypical genitalia and most affected males are raised as females. Virilization in subjects with 17β-HSD3 deficiency occurs at the time of puberty and several of them change to male social sex. In male social sex patients, testes can be safely maintained, as long as they are positioned inside the scrotum The phenotype of 46,XY DSD due to 17β-HSD3 deficiency is extremely variable and clinically indistinguishable from other causes of 46,XY DSD such as partial androgen insensitivity syndrome and 5α-reductase 2 deficiency. Laboratory diagnosis is based on a low testosterone/androstenedione ratio due to high serum levels of androstenedione and low levels of testosterone. The disorder is caused by a homozygous or compound heterozygous mutations in the HSD17B3 gene that encodes the 17β-HSD3 isoenzyme leading to an impairment of the conversion of 17-keto into 17-hydroxysteroids. Molecular genetic testing confirms the diagnosis and provides the orientation for genetic counseling. Our proposal in this article is to review the previously reported cases of 17β-HSD3 deficiency adding our own cases. Show less

17β-Hydroxysteroid dehydrogenase type 3 (17β-HSD3) is expressed almost exclusively in the testis and converts Δ4-androstene-3,17-dione to testosterone. Mutations in the HSD17B3 gene causing 17β-HSD3 deficiency are responsible for a rare recessive form of 46, XY Disorders of Sex Development (46, XY DSD). We report novel cases of Tunisian patients with 17β-HSD3 deficiency due to previously reported mutations, i.e. p.C206X and p.G133R, as well as a case with the novel compound heterozygous mutations p.C206X and p.Q176P. Moreover, the previously reported polymorphism p.G289S was identified in a heterozygous state in combination with a novel non-coding variant c.54G>T, also in a heterozygous state, in a male patient presenting with micropenis and low testosterone levels. The identification of four different mutations in a cohort of eight patients confirms the generally observed genetic heterogeneity of 17β-HSD3 deficiency. Nevertheless, analysis of DNA from 272 randomly selected healthy controls from the same geographic area (region of Sfax) revealed a high carrier frequency for the p.C206X mutation of approximately 1 in 40. Genotype reconstruction of the affected pedigree members revealed that all p.C206X mutation carriers harbored the same haplotype, indicating inheritance of the mutation from a common ancestor. Thus, the identification of a founder effect and the elevated carrier frequency of the p.C206X mutation emphasize the importance to consider this mutation in the diagnosis and genetic counseling of affected 17β-HSD3 deficiency pedigrees in Tunisia. Show less

The steroidogenic enzyme 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3) is a therapeutic target in the management of androgen-sensitive diseases such as prostate cancer and benign prostate hyperplasia. In this Letter, we designed and synthesized the first fluorescent inhibitor of this enzyme by combining a fluorogenic dansyl moiety to the chemical structure of a known inhibitor of 17β-HSD3. The synthesized compound 3 is a potent fluorogenic compound (λex=348 nm and λ em=498 nm). It crosses the cell membrane, keeps its fluorescent properties and is distributed inside the LNCaP cells overexpressing 17β-HSD3, where it inhibits the transformation of 4-androstene-3,17-dione into the androgen testosterone (IC50=262 nM). Show less

17β-hydroxysteroid dehydrogenase type 3 (17βHSD3) deficiency is a rare cause of disorder of sex development (DSD) due to impaired conversion of androstenedione to testosterone. Traditionally, the diagnosis was determined by βHCG-stimulated ratios of testosterone:androstenedione < 0.8. An otherwise phenotypically female infant presented with bilateral inguinal masses and a 46,XY karyotype. βHCG stimulation (1500 IU IM for 2 days) suggested 17βHSD3 deficiency although androstenedione was only minimally stimulated (4.5 nmol/L to 5.4 nmol/L). Expedient genetic testing for the HSD17B3 gene provided the unequivocal diagnosis. We advocate for urgent genetic testing in rare causes of DSD as indeterminate hormone results can delay diagnosis and prolong intervention. Show less

Men with Klinefelter syndrome (KS) show hypergonadotropic hypogonadism, but the pathogenesis of hypotestosteronemia remains unclear. Testicular steroidogenesis in KS men was evaluated over three decades ago after human chorionic gonadotropin (hCG) stimulation, but inconclusive results were obtained. Intriguingly, some recent studies show increased intratesticular testosterone concentrations in men with KS. To analyze serum steroid profile, as a proxy of testicular steroidogenesis, after hCG stimulation in KS compared with control men. A prospective, longitudinal, case-control, clinical trial. Thirteen KS patients (36±9 years) not receiving testosterone (TS) replacement therapy and 12 eugonadic controls (32±8 years) were enrolled. Serum steroids were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) at baseline and for five consecutive days after intramuscular injection of 5000IU hCG. Progesterone (P), 17-hydroxyprogesterone (17OHP), TS, and estradiol (E2) showed a significant increase (P<0.001) after hCG stimulation in both groups. On the contrary, androstenedione (AS) and dehydroepiandrosterone did not increase after hCG stimulation. The 17OHP/P ratio increased in both groups (P<0.001), the TS/AS ratio (17β-hydroxysteroid dehydrogenase type 3 (17βHSD3) activity) did not increase after hCG in any group, and the E2/TS ratio (aromatase activity) increased significantly in both groups (P=0.009 in KS and P<0.001 in controls). Luteinizing hormone decreased after hCG in both groups (P=0.014 in KS and P<0.001 in controls), whereas follicle-stimulating hormone decreased only in control men (P<0.001). This study demonstrates for the first time using LC-MS/MS that Leydig cells of KS men are able to respond to hCG stimulation and that the first steps of steroidogenesis are fully functional. However, the TS production in KS men is impaired, possibly related to reduced hydroxysteroid deydrogenase activity due to an unfavorable intratesticular metabolic state. Show less

We evaluated 69 SNPs in genes previously related to fertility and production traits for their relationship to daughter pregnancy rate (DPR), cow conception rate (CCR) and heifer conception rate (HCR) in a separate population of Holstein cows grouped according to their predicted transmitting ability (PTA) [≤-1 (n = 1287) and ≥1.5 (n = 1036)] for DPR. Genotyping was performed using Sequenom MassARRAY(®) . There were a total of 39 SNPs associated with the three fertility traits. The SNPs that explained the greater proportion of the genetic variation for DPR were COQ9 (3.2%), EPAS1 (1.0%), CAST (1.0%), C7H19orf60 (1.0%) and MRPL48 (1.0%); for CCR were GOLGA4 (2.4%), COQ9 (1.8%), EPAS1 (1.1%) and MRPL48 (0.8%); and for HCR were HSD17B7 (1.0%), AP3B1 (0.8%), HSD17B12 (0.7%) and CACNA1D (0.6%). Inclusion of 39 SNPs previously associated with DPR in the genetic evaluation system increased the reliability of PTA for DPR by 0.20%. Many of the genes represented by SNPs associated with fertility are involved in steroidogenesis or are regulated by steroids. A large proportion of SNPs previously associated with genetic merit for fertility in Holstein bulls maintained their association in a separate population of cows. The inclusion of these genes in genetic evaluation can improve reliabilities of genomic estimates for fertility. Show less

Leukemia inhibitory factor (LIF) has many physiological roles. However, its effects on Leydig cell development are still unclear. Rat immature and adult Leydig cells were cultured with different concentrations of LIF alone or in combination with luteinizing hormone (LH) for 24 h. LIF (1 and 10 ng/ml) significantly increased androgen production in immature Leydig cells, but had no effects on testosterone production in adult Leydig cells. Further studies revealed that LIF dose-dependently increased Star and Hsd17b3 expression levels in immature Leydig cells. Gene microarray revealed that the upregulation of anti-oxidative genes and Star might contribute to LIF-induced androgen production. In conclusion, LIF has stimulatory effects on androgen production in rat immature Leydig cells. Show less

Mutations in the HSD17B3 gene resulting in 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3) deficiency cause 46, XY Disorders of Sex Development (46, XY DSD). Approximately 40 different mutations in HSD17B3 have been reported; only few mutant enzymes have been mechanistically investigated. Here, we report novel compound heterozygous mutations in HSD17B3, composed of the nonsense mutation C206X and the missense mutation G133R, in three Tunisian patients from two non-consanguineous families. Mutants C206X and G133R were constructed by site-directed mutagenesis and expressed in HEK-293 cells. The truncated C206X enzyme, lacking part of the substrate binding pocket, was moderately expressed and completely lost its enzymatic activity. Wild-type 17β-HSD3 and mutant G133R showed comparable expression levels and intracellular localization. The conversion of Δ4-androstene-3,17-dione (androstenedione) to testosterone was almost completely abolished for mutant G133R compared with wild-type 17β-HSD3. To obtain further mechanistic insight, G133 was mutated to alanine, phenylalanine and glutamine. G133Q and G133F were almost completely inactive, whereas G133A displayed about 70% of wild-type activity. Sequence analysis revealed that G133 on 17β-HSD3 is located in a motif highly conserved in 17β-HSDs and other short-chain dehydrogenase/reductase (SDR) enzymes. A homology model of 17β-HSD3 predicted that arginine or any other bulky residue at position 133 causes steric hindrance of cofactor NADPH binding, whereas substrate binding seems to be unaffected. The results indicate an essential role of G133 in the arrangement of the cofactor binding pocket, thus explaining the loss-of-function of 17β-HSD3 mutant G133R in the patients investigated. Show less

The hydroxysteroid (17beta) dehydrogenase (HSD17B)12 gene belongs to the hydroxysteroid (17β) dehydrogenase superfamily, and it has been implicated in the conversion of estrone to estradiol as well as in the synthesis of arachidonic acid (AA). AA is a precursor of prostaglandins, which are involved in the regulation of female reproduction, prompting us to study the role of HSD17B12 enzyme in the ovarian function. We found a broad expression of HSD17B12 enzyme in both human and mouse ovaries. The enzyme was localized in the theca interna, corpus luteum, granulosa cells, oocytes, and surface epithelium. Interestingly, haploinsufficiency of the HSD17B12 gene in female mice resulted in subfertility, indicating an important role for HSD17B12 enzyme in the ovarian function. In line with significantly increased length of the diestrous phase, the HSD17B Show less

To observe the effect of electroacupuncture (EA) intervention on expression of cytochrome P 450 side chain cleavage (P 450 scc) and 17 β-hydroxysteroid dehydrogenase 3 (17 β-HSD3) in the testis in partial androgen deficiency of aging male (PADAM) rats so as to reveal its mechanism underlying improving PADAM. Thirty male SD rats were randomly and equally divided into control, model, and EA groups. The PADAM model was established by intraperitoneal injection of cyclophosphamide (20 mg · kg(-1) · d(-1)), once daily for 5 days. EA (20-30 Hz, 1-3 mA) was applied to bilateral "Shenshu" (BL 23) and "Guanyuan" (CV 4) for 15 min, once daily for 8 weeks. Serum total testosterone (TT) and free testosterone (FT) levels were measured using enzyme-linked immunosorbent assay (ELISA). The expression levels of P 450 scc/17 β-HSD3 proteins and mRNA in the testis tissue were assayed by immunohistochemistry, Western bolt (WB) and RT-polymerase chain reaction (RT-PCR), separately. Compared with the control group, both serum TT, FT levels and expression levels of P450 scc/17 β-HSD3 proteins and mRNA in the testis tissue in the model group were significantly down-regulated (P<0.01). After EA intervention, compared with the model group, the cyclophosphamide-induced decrease of serum TT, FT levels and the expression levels of P 450 scc/17 β-HSD3 proteins and mRNA in the testis was reversed in the EA group (P<0.01). EA intervention is effective in up-regulating serum TT and FT, testicular P 450 scc and 17 β-HSD3 proteins and mRNA levels in PADAM rats, which may be one of its mechanisms underlying improvement of PADAM. Show less