An integrated approach of network pharmacology, molecular docking, and experimental verification uncovers kaempferol as the effective modulator of HSD17B1 for treatment of endometrial cancer.

BACKGROUND: Endometrial cancer (EC) is one of the most common gynecological malignancies globally, and the development of innovative, effective drugs against EC remains a key issue. Phytoestrogen kaempferol exhibits anti-cancer effects, but the action mechanisms are still unclear. METHOD: MTT assays, colony-forming assays, flow cytometry, scratch healing, and transwell assays were used to evaluate the proliferation, apoptosis, cell cycle, migration, and invasion of both ER-subtype EC cells. Xenograft experiments were used to assess the effects of kaempferol inhibition on tumor growth. Next-generation RNA sequencing was used to compare the gene expression levels in vehicle-treated versus kaempferol-treated Ishikawa and HEC-1-A cells. A network pharmacology and molecular docking technique were applied to identify the anti-cancer mechanism of kaempferol, including the building of target-pathway network. GO analysis and KEGG pathway enrichment analysis were used to identify cancer-related targets. Finally, the study validated the mRNA and protein expression using real-time quantitative PCR, western blotting, and immunohistochemical analysis. RESULTS: Kaempferol was found to suppress the proliferation, promote apoptosis, and limit the tumor-forming, scratch healing, invasion, and migration capacities of EC cells. Kaempferol inhibited tumor growth and promotes apoptosis in a human endometrial cancer xenograft mouse model. No significant toxicity of kaempferol was found in human monocytes and normal cell lines at non-cytotoxic concentrations. No adverse effects or significant changes in body weight or organ coefficients were observed in 3-7 weeks' kaempferol-treated animals. The RNA sequencing, network pharmacology, and molecular docking approaches identified the overall survival-related differentially expressed gene HSD17B1. Interestingly, kaempferol upregulated HSD17B1 expression and sensitivity in ER-negative EC cells. Kaempferol differentially regulated PPARG expression in EC cells of different ER subtypes, independent of its effect on ESR1. HSD17B1 and HSD17B1-associated genes, such as ESR1, ESRRA, PPARG, AKT1, and AKR1C1\2\3, were involved in several estrogen metabolism pathways, such as steroid binding, 17-beta-hydroxysteroid dehydrogenase (NADP+) activity, steroid hormone biosynthesis, and regulation of hormone levels. The molecular basis of the effects of kaempferol treatment was evaluated. CONCLUSIONS: Kaempferol is a novel therapeutic candidate for EC via HSD17B1-related estrogen metabolism pathways. These results provide new insights into the efficiency of the medical translation of phytoestrogens.

How experimental competition changes ovarian gene activity in free-living birds: Implications for steroidogenesis, maternal effects, and beyond.

The ovary plays an important role in mediating both a female's response to her social environment and communicating it to her developing offspring via maternal effects. Past work has focused on how ovarian hormones respond to competition, but we know little about how the broader ovarian transcriptomic landscape changes, either during or after competition, giving us a narrow perspective on how socially induced phenotypes arise. Here, we experimentally generated social competition among wild, cavity-nesting female birds (tree swallows, Tachycineta bicolor), a species in which females lack a socially induced rise in circulating testosterone but they nevertheless increase allocation to eggs. After territory settlement, we reduced availability of nesting cavities, generating heightened competition; within 24 h we reversed the manipulation, causing aggressive interactions to subside. We measured ovarian transcriptomic responses at the peak of competition and 48 h later, along with date-matched controls. Network analyses indicated that competing females experienced an immediate and temporary decrease in the expression of genes involved in the early stages of steroidogenesis, and this was moderately correlated with plasma testosterone; however, two days after competition had ended, there was a marked increase in the expression of genes involved in the final stages of steroidogenesis, including HSD17B1. Gene networks related to the cell cycle, muscle performance, and extracellular matrix organization also displayed altered activity. Although the functional consequences of these findings are unclear, they shed light on socially responsive ovarian genomic mechanisms that could potentially exert lasting effects on behavior, reproduction, and maternal effects.

Overexpression of Human Estrogen Biosynthetic Enzyme Hydroxysteroid (17beta) Dehydrogenase Type 1 Induces Adenomyosis-like Phenotype in Transgenic Mice.

Hydroxysteroid (17beta) dehydrogenase type 1 (HSD17B1) is an enzyme that converts estrone to estradiol, while adenomyosis is an estrogen-dependent disease with poorly understood pathophysiology. In the present study, we show that mice universally over-expressing human estrogen biosynthetic enzyme HSD17B1 (HSD17B1TG mice) present with adenomyosis phenotype, characterized by histological and molecular evaluation. The first adenomyotic changes with endometrial glands partially or fully infiltrated into the myometrium appeared at the age of 5.5 months in HSD17B1TG females and became more prominent with increasing age. Preceding the phenotype, increased myometrial smooth muscle actin positivity and increased amount of glandular myofibroblast cells were observed in HSD17B1TG uteri. This was accompanied by transcriptomic upregulation of inflammatory and estrogen signaling pathways. Further, the genes upregulated in the HSD17B1TG uterus were enriched with genes previously observed to be induced in the human adenomyotic uterus, including several genes of the NFKB pathway. A 6-week-long HSD17B1 inhibitor treatment reduced the occurrence of the adenomyotic changes by 5-fold, whereas no effect was observed in the vehicle-treated HSD17B1TG mice, suggesting that estrogen is the main upstream regulator of adenomyosis-induced uterine signaling pathways. HSD17B1 is considered as a promising drug target to inhibit estrogen-dependent growth of endometrial disorders. The present data indicate that HSD17B1 over-expression in TG mice results in adenomyotic changes reversed by HSD17B1 inhibitor treatment and HSD17B1 is, thus, a potential novel drug target for adenomyosis.

A single intronic single nucleotide polymorphism in splicing site of steroidogenic enzyme hsd17b1 is associated with phenotypic sex in oyster pompano, Trachinotus anak.

Teleosts show varied master sex determining (MSD) genes and sex determination (SD) mechanisms, with frequent turnovers of sex chromosomes. Tracing the origins of MSD genes and turnovers of sex chromosomes in a taxonomic group is of particular interest in evolutionary biology. Oyster pompano (Trachinotus anak), a marine fish, belongs to the family Carangidae, in which 17b-hydroxysteroid dehydrogenase 1 (hsd17b1) has repeatedly evolved to an MSD gene. Whole-genome resequencing identified a single nucleotide polymorphism (SNP) at chromosome 24 to be strictly associated with phenotypic sex, with females being the heterozygous sex. This SNP is located in a splicing site at the first exon/intron boundary of hsd17b1. The Z-linked SNP results in malfunction of all spliced isoforms, whereas the W-linked isoforms were predicted to have open reading frames that are conserved among vertebrates, suggesting that hsd17b1 is a female-determining gene. The differential alternative splicing patterns of ZZ and ZW genotypes were consistently observed both in undifferentiated stages and differentiated gonads. We observed elevated recombination around the SD locus and no differentiation between Z and W chromosomes. The extreme diversity of mutational mechanisms that hsd17b1 evolves to an MSD gene highlights frequent in situ turnovers between sex chromosomes in the Carangidae.

FoxA2 and p53 regulate the transcription of HSD17B1 in ovarian granulosa cells of pigs.

The oestrogens have been highly implicated in the fertility of female animals. It is widely known that the oestrogens are primarily synthetized by the ovarian granulosa cells (GCs), and the final and essential step of this process is to catalyse the oestrone to the more active oestradiol by the protein coded by hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1) gene. However, the molecular mechanism regarding the transcription of HSD17B1 remains to be fully elucidated in ovarian GCs. In this study, the 5'-deletion, luciferase assay and chromatin immunoprecipitation (ChIP) were utilized to explore the molecular regulation of transcription of HSD17B1 with the porcine ovarian GCs as the cellular model. After the deletions with -2105 to -1754 bp, -1753 to -1429 bp, -1430 to -1081 bp and -1082 to -730 bp, the relative luciferase activity of HSD17B1 promoter did not change significantly, but the deletion of -731 to -332 bp significantly increased the relative luciferase activity of HSD17B1 promoter, and an insertion (GTTT) that might raise the transcription of HSD17B1 was identified at -401 bp of HSD17B1. These findings suggested the region from -731 to +38 bp was the core promoter of HSD17B1, and the region between -731 to -332 bp might be a silence element for HSD17B1. Furthermore, the forkhead box A2 (FoxA2) directly bound at -412 to -401 bp to negatively but p53 bound at -383 to -374 bp to positively regulate the transcription and translation of HSD17B1 in ovarian GCs. These findings will improve our understanding on HSD17B1-mediated oestrogens and provide useful information for further investigations into fertility of females.

Hydroxysteroid (17ß) dehydrogenase 1 expressed by Sertoli cells contributes to steroid synthesis and is required for male fertility.

The pituitary gonadotrophins and testosterone are the main hormonal regulators of spermatogenesis, but estradiol is also known to play a role in the process. The hormonal responses in the testis are partially mediated by somatic Sertoli cells that provide nutritional and physical support for differentiating male germ cells. Hydroxysteroid (17ß) dehydrogenase 1 (HSD17B1) is a steroidogenic enzyme that especially catalyzes the conversion of low potent 17keto-steroids to highly potent 17ß-hydroxysteroids. In this study, we show that Hsd17b1 is highly expressed in Sertoli cells of fetal and newborn mice, and HSD17B1 knockout males present with disrupted spermatogenesis with major defects, particularly in the head shape of elongating spermatids. The cell-cell junctions between Sertoli cells and germ cells were disrupted in the HSD17B1 knockout mice. This resulted in complications in the orientation of elongating spermatids in the seminiferous epithelium, reduced sperm production, and morphologically abnormal spermatozoa. We also showed that the Sertoli cell-expressed HSD17B1 participates in testicular steroid synthesis, evidenced by a compensatory up-regulation of HSD17B3 in Leydig cells. These results revealed a novel role for HSD17B1 in the control of spermatogenesis and male fertility, and that Sertoli cells significantly contribute to steroid synthesis in the testis.-Hakkarainen, J., Zhang, F.-P., Jokela, H., Mayerhofer, A., Behr, R., Cisneros-Montalvo, S., Nurmio, M., Toppari, J., Ohlsson, C., Kotaja, N., Sipilä, P., Poutanen, M. Hydroxysteroid (17ß) dehydrogenase 1 expressed by Sertoli cells contributes to steroid synthesis and is required for male fertility.

Insights into teleost sex determination from the Seriola dorsalis genome assembly.

BACKGROUND: The assembly and annotation of a genome is a valuable resource for a species, with applications ranging from conservation genomics to gene discovery. Genomic resource development is especially important for species in culture, such as the California Yellowtail (Seriola dorsalis), the likely candidate for the establishment of commercial offshore aquaculture production in southern California. Genomic resource development for this species will improve the understanding of sex and other phenotypic traits, and allow for rapid increases in genetic improvement for and economic gain in culture production. RESULTS: We describe the assembly and annotation of the S. dorsalis genome, and present resequencing data from 45 male and 45 female wild-caught S. dorsalis used to identify a sex-determining region and marker in this species. The genome assembly captured approximately 93% of the total 685 MB genome with an average coverage depth of 180×. Using the assembled genome, resequencing data from the 90 fish were aligned to place boundaries on the sex-determining region. Sex-specific markers were developed based on a female-specific, 61 nucleotide deletion identified in that region. We hypothesize that Estradiol 17-beta-dehydrogenase is the putative sex-determining gene and propose a plausible genetic mechanism for ZW sex determination in S. dorsalis involving a female-specific deletion of a transcription factor binding motif that may be targeted by Sox3. CONCLUSIONS: Understanding the mechanism of sex determination and development of assays to determine sex is critical both for management of wild fisheries and for development of efficient and sustainable aquaculture practices. In addition, this genome assembly for S. dorsalis will be a substantial resource for a variety of future research applications.

Macronutrient intakes and serum oestrogen, and interaction with polymorphisms in CYP19A1 and HSD17B1 genes: a cross-sectional study in postmenopausal Japanese women.

Although higher circulating levels of oestrogen are related to postmenopausal breast cancer risk, limited information is available regarding effects of diet on endogenous oestrogen. Thus, we examined associations between macronutrient intakes and serum oestrogen with consideration of polymorphisms in oestrogen-metabolising genes. In this cross-sectional study, 784 naturally menopaused Japanese women aged 47-69 years were selected from participants of the Japan Multi-Institutional Collaborative Cohort Study. We documented dietary intakes, measured serum concentrations of oestrone (E1) and oestradiol (E2) and genotyped polymorphisms in oestrogen-metabolising CYP19A1 (rs4441215 and rs936306) and HSD17B1 (rs605059) genes. Trends and interactions were examined using linear regression models. In addition, we calculated the ratios of the oestrogen concentrations of the second to the highest quartiles (Q2-Q4) of dietary intake to those of the lowest quartiles (Q1). After adjustment for potential confounders, E2 was significantly associated with intake of carbohydrate and noodles; ratios of Q4 v. Q1 were 1·15 (95 % CI 1·04, 1·28) and 1·15 (95 % CI 1·04, 1·26), respectively. In contrast, E2 levels were inversely associated with intake of total energy, SFA and n-3 highly unsaturated fatty acids (n-3 HUFA); ratios of Q4 v. Q1 were 0·90 (95 % CI 0·82, 0·99), 0·89 (95 % CI 0·81, 0·98) and 0·91 (95 % CI 0·83, 1·00), respectively. In stratified analysis by polymorphisms, the rs605059 genotype of HSD17B1 significantly modified associations of E2 with intake of n-3 HUFA and fish; the associations were limited to those with the CC genotype. Macronutrient intakes were associated with serum E2 level, and these associations may be modified by HSD17B1 polymorphism in postmenopausal women.

Involvement of 17ß-hydroxysteroid dehydrogenase type gene 1 937 A>G polymorphism in infertility in Polish Caucasian women with endometriosis.

PURPOSE: Endometriosis is considered to be an estrogen-related chronic inflammatory disease. The 17ß-hydroxysteroid dehydrogenase 1 (HSD17B1) converts estrone to 17ß estradiol. The role of HSD17B1 937 A>G (rs605059) single nucleotide polymorphism (SNP) in development of endometriosis is still disputable. This study evaluated the association of the HSD17B1 937 A>G (rs605059) SNP with infertile women affected by endometriosis from Polish Caucasian population. METHODS: The genotyping of cases (n = 290) and fertile women (n = 410) was conducted by high-resolution melting curve analysis. RESULTS: Statistical analysis demonstrated that the HSD17B1 937 A>G SNP is associated with endometriosis in stages I and II. The p trend and p allelic values calculated for the HSD17B1 937 A>G polymorphism were statistically significant and were equal to 0.001 and 0.0009, respectively. There was a significant association for the dominant model: (AG + GG vs AA) OR = 1.973 (95% CI = 1.178-3.304), p = 0.009, and for the recessive model: (GG vs AG + AA) OR = 1.806 (95% CI = 1.178-2.770), p = 0.006. However, we did not find statistical association of HSD17B1 937 A>G polymorphism with all infertile women with endometriosis or infertile women with endometriosis in stages III and IV. CONCLUSION: Our genetic study demonstrated HSD17B1 937 G variant as a risk factor for infertility in women with stage I and II endometriosis in Polish Caucasian patients.

Polymorphism of HSD17B1 Ser312Gly with Cancer Risk: Evidence from 66,147 Subjects.

Hydroxysteroid (17-beta)dehydrogenase 1(HSD17B1) plays a central role in sex steroid hormone metabolism. HSD17B1 polymorphic variants may contribute to cancer susceptibility. Numerous investigations have been conducted to assess the association between HSD17B1 Ser312Gly polymorphism and cancer risk in multiple ethnicities, yet these have produced inconsistent results. We therefore performed this comprehensive meta-analysis to attempt to provide a quality assessment of the association of interest. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to evaluate the strength of associations. After a systematic literature search of several major public databases, 20 studies involving 29,460 cases and 36,687 controls were included in this meta-analysis. No significant association was found between HSD17B1 Ser312Gly polymorphism and cancer risk. However, Ser312Gly polymorphism showed a significantly decreased risk for Caucasians (there were 44,284 Caucasians for analysis, comprising 19,889 cases and 24,395 controls) in the subgroup analysis by ethnicity (dominant: OR = 0.958, 95% CI = 0.919-0.998; and allele comparing: OR = 0.973, 95% CI = 0.947-0.999). And there was the same trend towards risk in the population-based (PB) controls (homozygous: OR = 0.951, 95% CI = 0.908-0.997 and allele comparing: OR = 0.976, 95% CI = 0.954-0.999), but not among Asians or hospital-based (HB) controls. In addition, no association was observed in the stratified analysis for breast cancer studies by source of control, ethnicity and quality score. These findings suggested that the HSD17B1 Ser312Gly polymorphism might confer genetic cancer susceptibility in an ethnic-dependent manner, especially among Caucasians. Well-designed, large-scale studies are warranted to validate these findings.

Sex-specific patterns and deregulation of endocrine pathways in the gene expression profiles of Bangladeshi adults exposed to arsenic contaminated drinking water.

Arsenic contamination of drinking water occurs globally and is associated with numerous diseases including skin, lung and bladder cancers, and cardiovascular disease. Recent research indicates that arsenic may be an endocrine disruptor. This study was conducted to evaluate the nature of gene expression changes among males and females exposed to arsenic contaminated water in Bangladesh at high and low doses. Twenty-nine (55% male) Bangladeshi adults with water arsenic exposure ranging from 50 to 1000 µg/L were selected from the Folic Acid Creatinine Trial. RNA was extracted from peripheral blood mononuclear cells for gene expression profiling using Affymetrix 1.0 ST arrays. Differentially expressed genes were assessed between high and low exposure groups for males and females separately and findings were validated using quantitative real-time PCR. There were 534 and 645 differentially expressed genes (p<0.05) in the peripheral blood mononuclear cells of males and females, respectively, when high and low water arsenic exposure groups were compared. Only 43 genes overlapped between the two sexes, with 29 changing in opposite directions. Despite the difference in gene sets both males and females exhibited common biological changes including deregulation of 17ß-hydroxysteroid dehydrogenase enzymes, deregulation of genes downstream of Sp1 (specificity protein 1) transcription factor, and prediction of estrogen receptor alpha as a key hub in cardiovascular networks. Arsenic-exposed adults exhibit sex-specific gene expression profiles that implicate involvement of the endocrine system. Due to arsenic's possible role as an endocrine disruptor, exposure thresholds for arsenic may require different parameters for males and females.

Identification of Hydroxysteroid Dehydrogenase Type 1 As a Potential Bladder Tumor Marker.

Background: The 17beta-hydroxysteroid dehydrogenase type 1 (HSD17B) family has been implicated in the prognosis and treatment prediction of various malignancies; however, its association with bladder cancer (BLCA) remains unclear. This study aimed to evaluate the potential of HSD17B1, as a prognostic biomarker, for the survival of patients with BLCA and to determine its effectiveness as a supplemental biomarker for BLCA. Methods: A series of bioinformatics techniques were applied to investigate the expression of HSD17B1 in different types of cancer and its potential association with the prognosis of BLCA patients using diverse databases. The UALCAN, Human Protein Atlas, cBioPortal, Metascape, GEPIA, MethSurv, and TIMER were employed to analyze expression differences, mutation status, enrichment analysis, overall survival, methylation, and immune-infiltrating cells. The real-time reverse transcription-PCR (qRT-PCR) was implemented to detect the messenger ribonucleic acid (mRNA) expression levels of HSD17B1 in vitro. Results: Elevated mRNA and protein levels of HSD17B1, surpassing normal levels, were observed in BLCA samples. In addition, the BLCA patients with higher mRNA expression level of HSD17B1 significantly reduced the overall survival. Also, several immune infiltrating cells, including mast cell resting CIBERSORT-ABS, have been identified as tumor-associated biomarker genes, with the potential to significantly influence the immunological environment. Finally, qRT-PCR analysis revealed a significant upregulation of HSD17B1 mRNA expression level in the cancer cells compared to the human 293T cells, which was consistent with the bioinformatics data. Conclusion: There is a strong correlation between the elevated HSD17B1 expression and positive prognosis in patients with BLCA. Therefore, HSD17B1 can be used as a prognostic biomarker in these patients.

HSD17B1 Compensates for HSD17B3 Deficiency in Fetal Mouse Testis but Not in Adults.

Hydroxysteroid (17ß) dehydrogenase (HSD17B) enzymes convert 17-ketosteroids to 17beta-hydroxysteroids, an essential step in testosterone biosynthesis. Human XY individuals with inactivating HSD17B3 mutations are born with female-appearing external genitalia due to testosterone deficiency. However, at puberty their testosterone production reactivates, indicating HSD17B3-independent testosterone synthesis. We have recently shown that Hsd17b3 knockout (3-KO) male mice display a similar endocrine imbalance, with high serum androstenedione and testosterone in adulthood, but milder undermasculinization than humans. Here, we studied whether HSD17B1 is responsible for the remaining HSD17B activity in the 3-KO male mice by generating a Ser134Ala point mutation that disrupted the enzymatic activity of HSD17B1 (1-KO) followed by breeding Hsd17b1/Hsd17b3 double-KO (DKO) mice. In contrast to 3-KO, inactivation of both HSD17B3 and HSD17B1 in mice results in a dramatic drop in testosterone synthesis during the fetal period. This resulted in a female-like anogenital distance at birth, and adult DKO males displayed more severe undermasculinization than 3-KO, including more strongly reduced weight of seminal vesicles, levator ani, epididymis, and testis. However, qualitatively normal spermatogenesis was detected in adult DKO males. Furthermore, similar to 3-KO mice, high serum testosterone was still detected in adult DKO mice, accompanied by upregulation of various steroidogenic enzymes. The data show that HSD17B1 compensates for HSD17B3 deficiency in fetal mouse testis but is not the enzyme responsible for testosterone synthesis in adult mice with inactivated HSD17B3. Therefore, other enzymes are able to convert androstenedione to testosterone in the adult mouse testis and presumably also in the human testis.

Transcriptomic profiling of progesterone in the male fathead minnow (Pimephales promelas) testis.

P4 is a hormone with diverse functions that include roles in reproduction, growth, and development. The objectives of this study were to examine the effects of P4 on androgen production in the mature teleost testis and to identify molecular signaling cascades regulated by P4 to improve understanding of its role in male reproduction. Fathead minnow (FHM) testis explants were treated in vitro with two concentrations of P4 (10(-8) and 10(-6) M) for 6 and 12 h. P4 significantly increased testosterone (T) production in the FHM testis but did not affect 11-ketotestosterone. Gene network analysis revealed that insulin growth factor (Igf1) and tumor necrosis factor receptor (Tnfr) signaling was significantly depressed with P4 treatment after 12h. There was also a 20% increase in a gene network for follicle-stimulating hormone secretion and an 18% decrease in genes involved in vasopressin signaling. Genes in steroid metabolism (e.g. star, cyp19a, 11bhsd) were not significantly affected by P4 treatments in this study, and it is hypothesized that pre-existing molecular machinery may be more involved in the increased production of T rather than the de novo expression of steroid-related transcripts and receptors. There was a significant decrease in prostaglandin E synthase 3b (cytosolic) (ptges3b) after treatment with P4, suggesting that there is cross talk between P4 and prostaglandin pathways in the reproductive testis. P4 has a role in regulating steroid production in the male testis and may do so by modulating gene networks related to endocrine pathways, such as Igf1, Tnfr, and vasopressin.

Local production of 17ß-oestradiol in the endometrium during the implantation window: a pilot study.

Abstract: Sex steroids are converted to bioactive metabolites and vice versa by endometrial steroid-metabolising enzymes. Studies indicate that alterations in this metabolism might affect endometrial receptivity. This pilot study determined whether the endometrial formation and inactivation of 17ß-oestradiol differed between the supposedly embryo-receptive endometrium and non-receptive endometrium of women undergoing IVF/intracytoplasmic sperm injection (ICSI). Endometrial biopsies were obtained from IVF/ICSI patients 5-8 days after ovulation in a natural cycle, prior to their second IVF/ICSI cycle with fresh embryo transfer (ET). Endometrial biopsies from patients who achieved clinical pregnancy after fresh ET (n = 15) were compared with endometrial biopsies from patients that did not conceive after fresh ET (n = 15). Formation of 17ß-oestradiol (oxidative 17ß-hydroxysteroid dehydrogenases (HSDs)), oestrone (reductive HSD17Bs) and inhibition of HSD17B1 activity were determined by high-performance liquid chromatography. The endometrial transcriptome was profiled using RNA sequencing followed by principal component analysis and differentially expressed gene analysis. The false discovery rate-adjusted P < 0.05 and log fold change >0.5 were selected as the screening threshold. Formation and inactivation of 17ß-oestradiol resulted similar between groups. Inhibition of HSD17B1 activity was significantly higher in the non-pregnant group when only primary infertile women (n = 12) were considered (27.1%, n = 5 vs 16.2%, n = 7, P = 0.04). Gene expression analysis confirmed the presence of HSD17B1 (encoding HSD17B1), HSD17B2 (encoding HSD17B2) and 33 of 46 analysed steroid metabolising enzymes in the endometrium. In the primary infertile subgroup (n = 10) 12 DEGs were found including LINC02349 which has been linked to implantation. However, the exact relationship between steroid-metabolising enzyme activity, expression and implantation outcome requires further investigation in larger, well-defined patient groups. Lay summary: Sex hormones are produced and broken down by enzymes that can be found in the endometrium (the inner lining of the womb). This enzyme activity might influence the chances of becoming pregnant. We compared (i) enzyme activity in the endometrium of 15 women who did and 15 women who did not become pregnant in their second in vitro fertilisation attempt, (ii) how enzyme activity can be blocked by an inhibitor, and (iii) differences in gene expression (the process by which instructions in our DNA are converted into a product). Enzyme activity was similar between groups. We found that in women who have never been pregnant in the past, inhibition of enzyme activity was higher and found differences in a gene that has been linked to the implantation of the embryo, but future studies should be performed in larger, well-defined patient groups to confirm these findings.

Comparison of Placental HSD17B1 Expression and Its Regulation in Various Mammalian Species.

During mammalian gestation, large amounts of progesterone are produced by the placenta and circulate for the maintenance of pregnancy. In contrast, primary plasma estrogens are different between species. To account for this difference, we compared the expression of ovarian and placental steroidogenic genes in various mammalian species (mouse, guinea pig, porcine, ovine, bovine, and human). Consistent with the ability to synthesize progesterone, CYP11A1/Cyp11a1, and bi-functional HSD3B/Hsd3b genes were expressed in all species. CYP17A1/Cyp17a1 was expressed in the placenta of all species, excluding humans. CYP19A/Cyp19a1 was expressed in all placental estrogen-producing species, whereas estradiol-producing HSD17B1 was only strongly expressed in the human placenta. The promoter region of HSD17B1 in various species possesses a well-conserved SP1 site that was activated in human placental cell line JEG-3 cells. However, DNA methylation analyses in the ovine placenta showed that the SP1-site in the promoter region of HSD17B1 was completely methylated. These results indicate that epigenetic regulation of HSD17B1 expression is important for species-specific placental sex steroid production. Because human HSD17B1 showed strong activity for the conversion of androstenedione into testosterone, similar to HSD17B1/Hsd17b1 in other species, we also discuss the biological significance of human placental HSD17B1 based on the symptoms of aromatase-deficient patients.

17ß-Hydroxysteroid dehydrogenases types 1 and 2: Enzymatic assays based on radiometric and mass-spectrometric detection.

The 17ß-hydroxysteroid dehydrogenase type 1 (HSD17B1) has a key role in estrogen biosynthesis as it catalyzes the reduction of estrone to the most potent estrogen, estradiol. Estradiol has a high affinity for estrogen receptors and thus stimulates their transactivation, which leads to cell proliferation and numerous other effects. HSD17B2 catalyzes the oxidation of estradiol to the less potent estrone, thereby decreasing estrogen receptor activation, which results in reduction of estrogen-associated effects. HSD17B1 and HSD17B2 overexpressing E.coli homogenates or recombinant enzymes can be used for screening and development of drugs against various pathologies such as cancer, endometriosis or osteoporosis. Here we describe the preparation of HSD17B1 and HSD17B2 bacterial homogenates and purified recombinant HSD17B1 protein as enzyme sources as well as enzymatic assays based on radiometric and mass-spectrometric detection for enzyme characterization.

Mixed-Culture Propagation of Uterine-Tissue-Resident Macrophages and Their Expression Properties of Steroidogenic Molecules.

Tissue-resident macrophages (Mø) play tissue/organ-specific roles, and the physiological/pathological implications of uterine Mø in fertility and infertility are not yet fully understood. Herein, we report a simple propagation method for tissue-resident Mø by mixed culture with the respective tissue/organ-residing cells as the niche. We successfully propagated mouse uterine Mø by mixed culture with fibroblastic cells that exhibited properties of endometrial stromal cells. Propagated mouse uterine Mø were CD206- and arginase-1-positive; iNOS- and MHC-II-negative, indicating M2 polarization; and highly phagocytic, similar to endometrial Mø. Furthermore, uterine Mø were observed to express steroidogenic molecules including SRD5A1 and exhibited gap junction formation, likely with endometrial stromal cells. Accordingly, uterine Mø propagated by mixed culture may provide a new tool for studying immune-endocrine interactions related to fertility and infertility, particularly androgen's intracrine actions in preparing the uterine tissue environment to support implantation and pregnancy as well as in the etiology of endometriosis.

Site-Specific Regulation of Sulfatase and Aromatase Pathways for Estrogen Production in Endometriosis.

Endometriosis is a highly prevalent gynecological disease characterized by lesions in different sites. Regulation of specific estrogen pathways may favor the formation of distinct microenvironments and the progression of endometriosis. However, no study has simultaneously evaluated the gene and protein regulation of the main estrogen-synthesizing enzymes in endometriosis. Thus, our goals were to study the relationship between gene and protein expression of aromatase (CYP19A1 or ARO), steroid sulfatase (STS), and hydroxysteroid 17-beta dehydrogenase (HSD17B1) in superficial (SUP), ovarian (OMA), and deep infiltrating (DIE) endometriotic lesion sites as well as in the eutopic endometrium of patients with (EE) and without (control) endometriosis in the same and large cohort of patients. The site-specific expression of these enzymes within different cells (glandular and stromal components) was also explored. The study included 108 patients surgically diagnosed with endometriosis who provided biopsies of EE and endometriotic lesions and 16 disease-free patients who collected normal endometrium tissue. Our results showed that CYP19A1 was detected in all endometriosis tissues and was in higher levels than in control. Unique patterns of the STS and HSD17B1 levels showed that they were most closely regulated in all tissues, with manifestation at greater levels in DIE compared to the other endometriotic lesion sites, OMA and SUP. Gene and protein expression of ARO, STS, and HSD17B1 occurred at different rates in endometriotic sites or EE. The distinctive levels of these estrogen-synthesizing enzymes in each endometriotic site support the hypothesis of a tissue microenvironment that can both influence and be influenced by the expression of different estrogenic pathways, locally affecting the availability of estrogen needed for maintenance and progression of endometriotic lesions.

Activin A Determines Steroid Levels and Composition in the Fetal Testis.

Activin A promotes fetal mouse testis development, including driving Sertoli cell proliferation and cord morphogenesis, but its mechanisms of action are undefined. We performed ribonucleic acid sequencing (RNA-seq) on testicular somatic cells from fetal activin A-deficient mice (Inhba KO) and wildtype littermates at embryonic day (E) E13.5 and E15.5. Analysis of whole gonads provided validation, and cultures with a pathway inhibitor discerned acute from chronic effects of altered activin A bioactivity. Activin A deficiency predominantly affects the Sertoli cell transcriptome. New candidate targets include Minar1, Sel1l3, Vnn1, Sfrp4, Masp1, Nell1, Tthy1 and Prss12. Importantly, the testosterone (T) biosynthetic enzymes present in fetal Sertoli cells, Hsd17b1 and Hsd17b3, were identified as activin-responsive. Activin-deficient testes contained elevated androstenedione (A4), displayed an Inhba gene dose-dependent A4/T ratio, and contained 11-keto androgens. The remarkable accumulation of lipid droplets in both Sertoli and germ cells at E15.5 indicated impaired lipid metabolism in the absence of activin A. This demonstrated for the first time that activin A acts on Sertoli cells to determine local steroid production during fetal testis development. These outcomes reveal how compounds that perturb fetal steroidogenesis can function through cell-specific mechanisms and can indicate how altered activin levels in utero may impact testis development.