Journal: Human Reproduction Open

Article Title: Progressively diminished estrogen signaling concordant with increased fibrosis in ectopic endometrium

doi: 10.1093/hropen/hoaf028

Figure Lengend Snippet: Results of immunohistochemistry analysis of proteins involved in the estrogen signaling pathway . The representative photomicrographs (left panel) of the immunostaining of StAR, HSD3β2, aromatase, HSD17β1, and estrogen receptors ERα, ERβ, and G-protein coupled ER (GPER) in normal endometrium (CT), ovarian endometrioma (OE), adenomyosis (AD), and deep endometriosis (DE) tissue samples, along with data summary (right panel). Sample sizes: CT: n = 20; OE: n = 19; AD: n = 20; DE: n = 20. Magnification: × 400. Scale bar= 50 μm. Symbols for statistical significance levels: NS: P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001 (by Wilcoxon’s rank test, *: compared with the CT group).

Article Snippet: HSD17β1 , ProteinTech Group , 25334-1-AP , 1:1000/1:3000.

Techniques: Immunohistochemistry, Immunostaining

Journal: Human Reproduction Open

Article Title: Progressively diminished estrogen signaling concordant with increased fibrosis in ectopic endometrium

doi: 10.1093/hropen/hoaf028

Figure Lengend Snippet: Correlation between the extent of lesional fibrosis and epithelial staining levels of markers of estrogen signaling . Scatter plots showing the relationship between the extent of lesional fibrosis and the staining levels of StAR ( A ), HSD3β2 ( B ), aromatase ( C ), HSD17β1 ( D ), ERα ( E ), ERβ ( F ), and G-protein coupled ER (GPER) ( G ) in ovarian endometrioma (OE), adenomyosis (AD), and deep endometriosis (DE) tissue samples. In all plots, the dashed line represents the linear regression line, and Pearson’s correlation coefficient, along with its statistical significance level, is shown. Symbols for statistical significance levels: NS: P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: HSD17β1 , ProteinTech Group , 25334-1-AP , 1:1000/1:3000.

Techniques: Staining

Journal: Human Reproduction Open

Article Title: Progressively diminished estrogen signaling concordant with increased fibrosis in ectopic endometrium

doi: 10.1093/hropen/hoaf028

Figure Lengend Snippet: Correlation between the extent of lesional fibrosis and stromal staining levels of markers of estrogen signaling . Scatter plots showing the relationship between the extent of lesional fibrosis and the staining levels of StAR ( A ), HSD3β2 ( B ), aromatase ( C ), HSD17β1 ( D ), ERβ ( E ), and G-protein coupled ER (GPER) ( F ) in ovarian endometrioma (OE), adenomyosis (AD), and deep endometriosis (DE) tissue samples. In all plots, the dashed line represents the linear regression line, and Pearson’s correlation coefficient, along with its statistical significance level, is shown. Symbols for statistical significance levels: NS: P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: HSD17β1 , ProteinTech Group , 25334-1-AP , 1:1000/1:3000.

Techniques: Staining

Journal: Human Reproduction Open

Article Title: Progressively diminished estrogen signaling concordant with increased fibrosis in ectopic endometrium

doi: 10.1093/hropen/hoaf028

Figure Lengend Snippet: Results of protein expression analysis and ELISA results . Relative fold change in protein expression of StAR ( A ), HSD3β2 ( B ), aromatase ( C ), HSD17β1 ( D ), ERα ( E ), ERβ ( F ), and G-protein coupled ER (GPER) ( G ). The GAPDH expression levels were served as a loading control. All data were expressed as fold change in protein expression relative to the CT group. ( H ) Estradiol concentrations measured by ELISA in supernatant of cultured cells derived from different tissues. Symbols of statistical significance levels: NS: P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001 (by Wilcoxon’s rank test, compared with the CT group). Data are represented in means±SDs. CT, control endometrium; OE, ovarian endometriomas; AD, adenomyosis; DE, deep endometriosis.

Article Snippet: HSD17β1 , ProteinTech Group , 25334-1-AP , 1:1000/1:3000.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control, Cell Culture, Derivative Assay

Journal: Endocrinology

Article Title: Female Mice with HSD17B1 Inactivation Show Mild Hyperandrogenism without Notable Impact on Reproductive Function or Bone

doi: 10.1210/endocr/bqaf167

Figure Lengend Snippet: Human and mouse HSD17B1 substrate binding modes. (A) 2D representations of E1 (substrate) and E2 (product). (B) The minimized human E1-HSD17B1 complex indicates that the C3-hydroxyl can form a double H-bond with both Glu283 and His222, and the C17-keto group can also optimally H-bond with Ser143 and Tyr156. (C) After the E1-to-E2 reaction, the C17-hydroxyl positioning of E2 at the catalytic end is compromised due to the proximity of multiple H-bond donors. (D) In mouse HSD17B1, Gly222 is present at the noncatalytic end of the enzyme, instead of the histidine, but the carboxylate group of Glu283 can H-bond directly with the C3-hydroxyl of E1. (E) Mutating Ser143 to alanine compromises or abolishes the C17-keto coordination needed for acquiring the proton from NAD (P)H via the phenol ring of Tyr156. (F) 2D representations of A4 (substrate) and T (product). (G) With the minimized human A4-HSD17B1 complex, the C17-keto group H-bonds with the epsilon protonated His222, preferring a binding mode that is reverse and nonproductive for the reaction. (H) Likewise, in the energy-minimized human T-HSD17B1 complex, the C17-hydroxyl also acquires the double H-bond arrangement with Glu283 and His222. (I) In the modeled mouse A4-HSD17B1 complex, the reverse binding mode is not favored as there are no suitable H-bond donors present at the noncatalytic end. The protein residues are shown as stick models with grey backbones. The ligands and cofactors (NADP+) are shown as ball-and-stick models with magenta/blue or green backbone, respectively. Human and mouse HSD17B1 residue numbering is taken from the UniProt entries DHB1_HUMAN ( P14061 ) and DHB1_MOUSE ( P51656 ). Abbreviations: A4, androstenedione; E1, estrone; E2, estradiol; HSD17B1, 17β-hydroxysteroid dehydrogenase 1; NADP, nicotinamide adenine dinucleotide phosphate; T, testosterone.

Article Snippet: Briefly, the point mutation was introduced to a wild-type (WT) mouse Hsd17b1 expression plasmid (Origene) with a Q5 Site Directed Mutagenesis Kit (NE Biolabs) according to manufacturer instructions, with the following primers: forward 5′-GTGACCGCGGCAGTGGGAGGCTT-3′, reverse 5′-CAGCACACGCCCAGAGTGGC-3′.

Techniques: Binding Assay, Residue

Journal: Endocrinology

Article Title: Female Mice with HSD17B1 Inactivation Show Mild Hyperandrogenism without Notable Impact on Reproductive Function or Bone

doi: 10.1210/endocr/bqaf167

Figure Lengend Snippet: HSD17B1 inactivating mutation affected ovarian steroidogenesis. (A) Conversion of E1 to E2 in MCF-7 cells transfected with Ser143Ala mutant (Mut) HSD17B1 is greatly impaired in comparison with cells transfected with WT HSD17B1, being equal to nontransfected cells (Sham). (B) No change was observed in Hsd17b1 mRNA expression in adult mouse ovaries analyzed by quantitative PCR (n = 4). (C) Intraovarian E1, A4, and DHT concentrations were increased in 6-month-old females, but other steroids were unchanged (n = 13). (D) Serum E1, A4, and T concentrations were also increased in the same animals. (E) Serum LH concentrations were increased in 6- to 7-month-old females (WT n = 11, HSD17B1-KI n = 12). (F) Ovary/serum steroid concentration ratio of A4 was increased, and T decreased in the HSD17B1-KI females. (G) The ratio of T to A4 was decreased in the HSD17B1-KI ovary but unchanged in serum. In (A), data are presented as means and SD; in (B-F), data are presented as individual values, with lines indicating means; and in (C, D, F), data were log 2 -transformed prior to analysis. *= P < .05, **= P < .01, ***= P < .001. Abbreviations: A4, androstenedione; DHT, dihydrotestosterone; E1, estrone; E2, estradiol; HSD17B1, 17β-hydroxysteroid dehydrogenase 1; HSD17B1-KI, 17β-hydroxysteroid dehydrogenase 1 Ser143Ala knock-in; T, testosterone; WT, wild-type.

Article Snippet: Briefly, the point mutation was introduced to a wild-type (WT) mouse Hsd17b1 expression plasmid (Origene) with a Q5 Site Directed Mutagenesis Kit (NE Biolabs) according to manufacturer instructions, with the following primers: forward 5′-GTGACCGCGGCAGTGGGAGGCTT-3′, reverse 5′-CAGCACACGCCCAGAGTGGC-3′.

Techniques: Mutagenesis, Transfection, Comparison, Expressing, Real-time Polymerase Chain Reaction, Concentration Assay, Transformation Assay, Knock-In

Journal: Endocrinology

Article Title: Female Mice with HSD17B1 Inactivation Show Mild Hyperandrogenism without Notable Impact on Reproductive Function or Bone

doi: 10.1210/endocr/bqaf167

Figure Lengend Snippet: Female development and reproduction were not overtly disturbed by HSD17B1 inactivation. (A) Anogenital distance did not differ between WT (n = 13) and HSD17B1-KI (n = 13) females at the ages of 5 and 8 weeks, nor did (B) the timing of the onset of puberty. (C) Although the HSD17B1-KI females cycled mostly normally, a small increase was seen in the number of total days spent in the metestrus stage over the 4 cycles followed. (D) Representative diagrams of estrous cycle progression. (E) Mean time between spontaneous delivery of litters was delayed in HSD17B1-KI females in constant breeding for 2 months (n = 10). (F) Number of pups in litters after spontaneous delivery did not differ between the groups, nor did (G) weights of pups over the first 3 weeks after birth. (H) Ovary weights did not differ between WT and HSD17B1-KI females at 2 months of age (WT n = 10, HSD17B1-KI n = 11) but were decreased in HSD17B1-KI at 6 months of age (n = 10). (I) Representative ovarian histology, showing current and regressing corpora lutea and the prominent interstitial cells in HSD17B1-KI in comparison to WT histology. (J) Weights of ovaries did not differ between groups (n = 7) after stimulation with PMSG and hCG, nor did (K) the number of released oocytes after superovulation. In (A, B, E, F, H, J, K), data are presented as individual values, with lines indicating the means; in (C, G), data are presented as means and SD. In (I), scale bars in wider field images indicate 200 µm and in closer images 100 µm. * P < .05, ** P < .01. Abbreviations: hCG, human chorionic gonadotropin; HSD17B1, 17β-hydroxysteroid dehydrogenase 1; HSD17B1-KI, 17β-hydroxysteroid dehydrogenase 1 Ser143Ala knock-in; PMSG, pregnant mare serum gonadotropin; WT, wild-type.

Article Snippet: Briefly, the point mutation was introduced to a wild-type (WT) mouse Hsd17b1 expression plasmid (Origene) with a Q5 Site Directed Mutagenesis Kit (NE Biolabs) according to manufacturer instructions, with the following primers: forward 5′-GTGACCGCGGCAGTGGGAGGCTT-3′, reverse 5′-CAGCACACGCCCAGAGTGGC-3′.

Techniques: Comparison, Knock-In

Journal: Endocrinology

Article Title: Female Mice with HSD17B1 Inactivation Show Mild Hyperandrogenism without Notable Impact on Reproductive Function or Bone

doi: 10.1210/endocr/bqaf167

Figure Lengend Snippet: Ovarian gene expression was affected by HSD17B1 inactivation. (A) Relative ovarian expression of Star mRNA was increased in 2-month-old HSD17B1-KI females at the estrus stage of the cycle (n = 5) but not at 4 (WT n = 9, HSD17B1/KI n = 11) or 6 months (WT n = 8, HSD17B1-KI n = 7). (B) An increasing trend was visible in HSD17B1-KI ovary Cyp17a1 expression in the same animals and was significant at 6 months. (C) mRNA sequencing revealed an upregulation of Hsd3b6 in 6-month-old HSD17B1-KI, whereas (D) Hsd17b2 was downregulated. (E) Ar , as well as (F) androgen-responsive genes Sult1e1 , Insl3 , Nr5a1 , and Vcam1, were also observed to be upregulated in mRNA sequencing data. (G) STRING interaction analysis and clustering of the differentially expressed genes in 6-month-old females further revealed several clusters of interest upregulated in HSD17B1-KI. In (A-F), data are presented as individual values, with lines indicating the means. * P < .05, ** P < .01, *** P < .001. Abbreviations: HSD17B1, 17β-hydroxysteroid dehydrogenase 1; HSD17B1-KI, 17β-hydroxysteroid dehydrogenase 1 Ser143Ala knock-in; WT, wild-type.

Article Snippet: Briefly, the point mutation was introduced to a wild-type (WT) mouse Hsd17b1 expression plasmid (Origene) with a Q5 Site Directed Mutagenesis Kit (NE Biolabs) according to manufacturer instructions, with the following primers: forward 5′-GTGACCGCGGCAGTGGGAGGCTT-3′, reverse 5′-CAGCACACGCCCAGAGTGGC-3′.

Techniques: Gene Expression, Expressing, Sequencing, Knock-In

Journal: Endocrinology

Article Title: Female Mice with HSD17B1 Inactivation Show Mild Hyperandrogenism without Notable Impact on Reproductive Function or Bone

doi: 10.1210/endocr/bqaf167

Figure Lengend Snippet: Bone parameters were unchanged in females with HSD17B1 inactivation. (A) Representative reconstructed micro-computed tomography images of bone microstructure in WT and HSD17B1-KI femur. Blue represents the highest tissue mineral density. (B) Femur and tibia length did not differ between WT and HSD17B1-KI females at 2 (WT n = 10, HSD17B1-KI n = 11) or 6 months of age (WT n = 10, HSD17B1-KI n = 10). (C) No differences were observed in femur diameter (sagittal or coronal) at 2 months or 6 months either. (D) Representative images of TRAcP staining of the tibia of WT and HSD17B1-KI females at the age of 6 months, with arrows indicating multinucleated TRAcP-positive osteoclasts below the growth plate, with (E) showing the similar number of osteoclasts counted in histological sections at 2 months or 6 months in both WT and HSD17B1-KI females. In (E), scale bars in wider field pictures represent 1000 µm and in closer pictures 200 µm. In (B, C, E), data are presented as individual values, with lines indicating the means. * P < .05, ** P < .01, *** P < .001. Abbreviations: HSD17B1, 17β-hydroxysteroid dehydrogenase 1; HSD17B1-KI, 17β-hydroxysteroid dehydrogenase 1 Ser143Ala knock-in; TRAcP, tartrate-resistant acid phosphatase; WT, wild-type.

Article Snippet: Briefly, the point mutation was introduced to a wild-type (WT) mouse Hsd17b1 expression plasmid (Origene) with a Q5 Site Directed Mutagenesis Kit (NE Biolabs) according to manufacturer instructions, with the following primers: forward 5′-GTGACCGCGGCAGTGGGAGGCTT-3′, reverse 5′-CAGCACACGCCCAGAGTGGC-3′.

Techniques: Micro-CT, Staining, Knock-In

Journal: International Journal of Molecular Sciences

Article Title: Electromagnetic Field (EMF) Radiation Alters Estrogen Release from the Pig Myometrium during the Peri-Implantation Period

doi: 10.3390/ijms22062920

Figure Lengend Snippet: The primary antibodies used for immunofluorescence and Western blotting analyses.

Article Snippet: Anti-17 β HSD , Primary , Orb137855 (Biorbyt) , Rabbit , 1 μg/mL.

Techniques: Immunofluorescence, Western Blot, Concentration Assay

Journal: Toxicology and applied pharmacology

Article Title: Dioxin exposure reduces the steroidogenic capacity of mouse antral follicles mainly at the level of HSD17B1 without altering atresia

doi: 10.1016/j.taap.2012.07.031

Figure Lengend Snippet: qPCR primer abbreviations, accession numbers, and sequences.

Article Snippet: The primary antibodies and concentrations used were as follows: CYP11A1 (C-16) sc-18043, 1:100 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 3β-HSD (P-18) sc-30820, 1:200 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), HSD17B1 NBP1-56295, 0.5 μg/mL (Novus Biologicals, Littleton, CO), CYP19 (C-16) sc-14245, 1:200 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and AHR BML-SA210, 1:5000 (Enzo Life Sciences, Plymouth Meeting, PA).

Techniques: Sequencing

Journal: Toxicology and applied pharmacology

Article Title: Dioxin exposure reduces the steroidogenic capacity of mouse antral follicles mainly at the level of HSD17B1 without altering atresia

doi: 10.1016/j.taap.2012.07.031

Figure Lengend Snippet: Effect of 96 h in vitro TCDD exposure on the expression of steroidogenic enzyme transcripts in antral follicles. At the end of each of the 96 h cultures, 8–16 follicles from each treatment group were pooled and immediately snap frozen in liquid nitrogen and assayed by qPCR for levels of (A) Star (B) Cyp11a1, (C) Cyp17a1, (D) Hsd3b1, (E) Hsd17b1, and (F) Cyp19a1. Levels of these key steroidogeneic enzymes were normalized to β-actin (Actb). Data are expressed as mean relative expression ratios ±SEM calculated from 3–4 separate culture experiments. Columns with asterisks indicate *p≤0.05, **p≤0.01, ***p≤0.001.

Article Snippet: The primary antibodies and concentrations used were as follows: CYP11A1 (C-16) sc-18043, 1:100 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 3β-HSD (P-18) sc-30820, 1:200 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), HSD17B1 NBP1-56295, 0.5 μg/mL (Novus Biologicals, Littleton, CO), CYP19 (C-16) sc-14245, 1:200 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and AHR BML-SA210, 1:5000 (Enzo Life Sciences, Plymouth Meeting, PA).

Techniques: In Vitro, Expressing

Journal: Toxicology and applied pharmacology

Article Title: Dioxin exposure reduces the steroidogenic capacity of mouse antral follicles mainly at the level of HSD17B1 without altering atresia

doi: 10.1016/j.taap.2012.07.031

Figure Lengend Snippet: Effect of 48 and 96 h in vitro TCDD exposure on HSD17B1 and CYP19A1 protein expression in antral follicles. At the end of each of the cultures, 12–16 follicles from each treatment group were pooled and immediately snap frozen in liquid nitrogen and analyzed by western blot for HSD17B1 (A–D) and CYP19A1 (E–H) protein levels. Antral follicles (250–350 μm) (F), ovary (O), heart (H), lung (L), and a biopsy of skeletal muscle (M) were removed from a PND 33 female mouse and analyzed as positive and negative controls. Densitometric units of the HSD17B1 and CYP19A1 protein bands were normalized to the densitometric units of the corresponding ACTB protein bands on each blot for quantification. Data are expressed as mean relative expression ratios±SEM calculated from four separate culture experiments (graphs A and E represent the 48 h cultures and graphs C and G represent the 96 h cultures). B, D, F, and H are representative images of the protein bands from western blots for HSD17B1 and CYP19A1, with an image of the corresponding ACTB protein bands that were used as loading controls below. V = vehicle control, T=1 nM TCDD, and X = blank lane.

Article Snippet: The primary antibodies and concentrations used were as follows: CYP11A1 (C-16) sc-18043, 1:100 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 3β-HSD (P-18) sc-30820, 1:200 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), HSD17B1 NBP1-56295, 0.5 μg/mL (Novus Biologicals, Littleton, CO), CYP19 (C-16) sc-14245, 1:200 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and AHR BML-SA210, 1:5000 (Enzo Life Sciences, Plymouth Meeting, PA).

Techniques: In Vitro, Expressing, Western Blot, Control

Journal: Toxicology and applied pharmacology

Article Title: Dioxin exposure reduces the steroidogenic capacity of mouse antral follicles mainly at the level of HSD17B1 without altering atresia

doi: 10.1016/j.taap.2012.07.031

Figure Lengend Snippet: TCDD-induced transcript and protien level changes in antral at 48 and 96 h.

Article Snippet: The primary antibodies and concentrations used were as follows: CYP11A1 (C-16) sc-18043, 1:100 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 3β-HSD (P-18) sc-30820, 1:200 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), HSD17B1 NBP1-56295, 0.5 μg/mL (Novus Biologicals, Littleton, CO), CYP19 (C-16) sc-14245, 1:200 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and AHR BML-SA210, 1:5000 (Enzo Life Sciences, Plymouth Meeting, PA).

Techniques: