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Introduction: Chronic kidney disease (CKD) presents a critical global health challenge, marked by the progressive decline of renal function. This study explores the role of the 3β-hydroxysteroid dehydrogenase type 2 enzyme (HSD3B2) and the steroid hormone biosynthesis pathway in CKD pathogenesis and progression., Methods: Using an adenine-induced CKD mouse model, we conducted an untargeted metabolomic analysis of plasma samples to identify key metabolite alterations associated with CKD. Immunohistochemistry, Western blotting, and qPCR analyses were performed to confirm HSD3B2 expression in both human and mouse tissues. Additionally, Nephroseq and Human Protein Atlas data were utilized to assess the correlation between HSD3B2 and kidney function. Functional studies were conducted on HK2 cells with HSD3B2 knockdown to evaluate the impact on cell proliferation and apoptosis., Results: Metabolic characteristics revealed significant shifts in CKD, with 61 metabolites increased and 65 metabolites decreased, highlighting the disruption in steroid hormone biosynthesis pathways influenced by HSD3B2. A detailed examination of seven key metabolites underscored the enzyme's central role. HSD3B2 exhibited a strong correlation with kidney function, supported by data from Nephroseq and the Human Protein Atlas. Immunohistochemistry, Western blotting, and qPCR analyses confirmed a drastic reduction in HSD3B2 expression in CKD-affected kidneys. Suppressed proliferation and increased apoptosis rates in HSD3B2 knocked down HK2 cells further demonstrated the enzyme's significance in regulating renal pathophysiology., Discussion: These findings underscore the potential of HSD3B2 as a clinical diagnostic and therapeutic target in CKD. While further studies are warranted to fully elucidate the mechanisms, our results provide valuable insights into the intricate interplay between steroid hormone biosynthesis and CKD. This offers a promising avenue for precision medicine approaches and personalized treatment strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zuo, Zha, Zhang, Yang, Jiang, Wang, Zhang, Chen and He.)

3β-Hydroxysteroid dehydrogenases (3β-HSDs) catalyze the oxidative conversion of delta (5)-ene-3-beta-hydroxy steroids and ketosteroids. Human 3β-HSD type 2 (HSD3B2) is predominantly expressed in gonadal and adrenal steroidogenic cells for producing all classes of active steroid hormones. Mutations in HSD3B2 gene cause a rare form of congenital adrenal hyperplasia with varying degree of salt wasting and incomplete masculinization, resulting from reduced production of corticoids and androgens. Therefore, evaluation of the HSD3B2 enzymatic activity in both pathways for each steroid hormone production is important for accurately understanding and diagnosing this disorder. Using progesterone receptor (PR)- and androgen receptor (AR)-mediated transactivation, we adapted a method that easily evaluates enzymatic activity of HSD3B2 by quantifying the conversion from substrates [pregnenolone (P5) and dehydroepiandrosterone (DHEA)] to (progesterone and androstenedione). HEK293 cells were transduced to express human HSD3B2, and incubated medium containing P5 or DHEA. Depending on the incubation time with HSD3B2-expressing cells, the culture media progressively increased luciferase activities in CV-1 cells, transfected with the PR/AR expression vector and progesterone-/androgen-responsive reporter. Culture media from human and other mammalian HSD3B1-expressing cells also increased the luciferase activities. HEK293 cells expressing various missense mutations in the HSD3B2 gene revealed the potential of this system to evaluate the relationship between the enzymatic activities of mutant proteins and patient phenotype., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Yazawa, Watanabe, Yokohama, Imamichi, Hasegawa, Nakajima, Kitano, Ida, Sato, Islam, Umezawa, Takahashi, Kato, Jahan and Kawabe.)

BACKGROUNDMetastatic hormone-sensitive prostate cancer (mHSPC) is androgen dependent, and its treatment includes androgen deprivation therapy (ADT) with gonadal testosterone suppression. Since 2014, overall survival (OS) has been prolonged with addition of other systemic therapies, such as adrenal androgen synthesis blockers, potent androgen receptor blockers, or docetaxel, to ADT. HSD3B1 encodes the rate-limiting enzyme for nongonadal androgen synthesis, 3β-hydroxysteroid dehydrogenase-1, and has a common adrenal-permissive missense-encoding variant that confers increased synthesis of potent androgens from nongonadal precursor steroids and poorer prostate cancer outcomes.METHODSOur prespecified hypothesis was that poor outcome associated with inheritance of the adrenal-permissive HSD3B1 allele with ADT alone is reversed in patients with low-volume (LV) mHSPC with up-front ADT plus addition of androgen receptor (AR) antagonists to inhibit the effect of adrenal androgens. HSD3B1 genotype was obtained in 287 patients with LV disease treated with ADT + AR antagonist only in the phase III Enzalutamide in First Line Androgen Deprivation Therapy for Metastatic Prostate Cancer (ENZAMET) trial and was associated with clinical outcomes.RESULTSPatients who inherited the adrenal-permissive HSD3B1 allele had more favorable 5-year clinical progression-free survival and OS when treated with ADT plus enzalutamide or ADT plus nonsteroidal antiandrogen compared with their counterparts who did not have adrenal-permissive HSD3B1 inheritance. HSD3B1 was also associated with OS after accounting for known clinical variables. Patients with both genotypes benefited from early enzalutamide.CONCLUSIONThese data demonstrated an inherited physiologic driver of prostate cancer mortality is associated with clinical outcomes and is potentially pharmacologically reversible.FUNDINGNational Cancer Institute, NIH; Department of Defense; Prostate Cancer Foundation, Australian National Health and Medical Research Council.

HSD3B1 encodes 3β-hydroxysteroid dehydrogenase-1, which converts adrenal dehydroepiandrosterone to 5α-dihydrotestosterone and is inherited in adrenal-permissive (AP) or adrenal-restrictive forms. The AP allele is linked to castration resistance, mainly in low-volume tumors. Here, we investigate the association of HSD3B1 alleles with outcomes in ARCHES, a multinational, double-blind, randomized, placebo-controlled phase 3 trial that demonstrated clinical benefit with enzalutamide plus androgen deprivation therapy (ADT) in men with metastatic hormone-sensitive prostate cancer (mHSPC) compared to those treated with placebo plus ADT. There are no significant differences between genotypes for clinical efficacy endpoints. Enzalutamide significantly improves radiographic progression-free survival and overall survival vs. placebo irrespective of HSD3B1 status. Men with the AP genotype have higher post-progression mortality and treatment-emergent adverse events, including hypertension, cardiovascular events, and gynecomastia, but a lower fracture rate. Overall, enzalutamide is beneficial in men with mHSPC independent of the HSD3B1 genotype. Inherited polymorphisms of HSD3B1 may account for differential toxicities., Competing Interests: Declaration of interests N.S. reports consulting for Celgene and Roivant, research funding from Astellas, and a filed patent application by Cleveland Clinic for a method of steroid-dependent disease treatment based on HSD3B1. A.A.A. reports honoraria from Aculeus Therapeutics, Amgen, Astellas, AstraZeneca, Bayer, Bristol Myers Squibb, Ipsen, Janssen, Merck, Merck Serono, Novartis, Noxopharm, Pfizer, Sanofi, Telix, and Tolmar; consulting for Aculeus Therapeutics, Amgen, Astellas, AstraZeneca, Bayer, Bristol Myers Squibb, Ipsen, Janssen, Merck, Merck Serono, Novartis, Noxopharm, Pfizer, Sanofi, Telix, and Tolmar; speakers’ bureau for Amgen, Astellas, Bayer, Bristol Myers Squibb, Ipsen, Janssen, Merck Serono, and Novartis; grants from Aptevo (institutional), Astellas (institutional), Astellas (investigator), AstraZeneca (institutional), AstraZeneca (investigator), Bionomics (institutional), Bristol Myers Squibb (institutional), Exelixis (institutional), Gilead Sciences (institutional), GlaxoSmithKline (institutional), Hinova (institutional), Ipsen (institutional), Janssen (institutional), Lilly (institutional), MedImmune (institutional), Merck Serono (institutional), Merck Serono (investigator), Merck Sharpe & Dohme (institutional), Novartis (institutional), Pfizer (institutional), Sanofi Aventis (institutional), and Synthorx (institutional); travel/accommodation reimbursement from Amgen, Astellas, Bayer, Janssen, Merck Serono, Pfizer, Sanofi, and Tolmar; participation on an advisory board for Amgen, Astellas, AstraZeneca, Bayer, Bristol Myers Squibb, Ipsen, Janssen, Merck, Merck Serono, Novartis, Noxopharm, Pfizer, Sanofi, Telix, and Tolmar; and participation as the chair of the Urologic Oncology Group for the Clinical Oncology Society of Australia and as a chair of the Translational Research Subcommittee and member of the Scientific Advisory Committee for the ANZUP Cancer Trials Group. J.W.D.H. reports consulting for Astellas. M.W. is an employee of Astellas and reports ownership of stock of ADMA Biologics, CASI, Merck, and Seelos. F.Z. is an employee of Pfizer and reports ownership of stock of Pfizer and AlloVir and patents with AlloVir. J.S. is an employee of Astellas and reports ownership of stock of AstraZeneca. G.P.H. is an employee of Astellas. A.S. reports consulting for Alere, Astellas, Bayer, Bristol Myers Squibb, Ferring, Ipsen, Janssen, Roche, Steba Biotech, and Synergo; research funding from Amgen, Astellas, AstraZeneca, Bayer, Cepheid, GenomeDx, Immatics, Janssen, Johnson & Johnson, Karl Storz, Medivation, Novartis, and Roche; patents for A290/99 implantable incontinence device, AT00/0001:C-Trap implantable device to treat urinary incontinence, and 2018/6579 gene-expression signature for subtype and prognostic prediction of renal cell carcinoma; expert testimony for GBA Pharma; and travel support from Amgen, Astellas, AstraZeneca, CureVac, Ferring, Ipsen, Janssen, and Sanofi/Aventis. A.J.A. reports research support (institutional) to Duke from the NIH/NCI, PCF/Movember, DOD, Astellas, Pfizer, Bayer, Janssen, Dendreon, BMS, AstraZeneca, Merck, Forma, Celgene, Amgen, and Novartis and personal compensation from consulting or advising relationships with Astellas, AstraZeneca, Bayer, Bristol Myers Squibb, Celgene, Clovis, Dendreon, Epic Sciences, Exact Sciences, Exelixis, Forma, GoodRx, Janssen, Merck, Myovant, Novartis, Pfizer, and Point., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Androgen receptor signaling is crucial for the development of treatment resistance in prostate cancer. Among steroidogenic enzymes, 3β-hydroxysteroid dehydrogenases (3βHSDs) play critical roles in extragonadal androgen synthesis, especially 3βHSD1. Increased expression of 3βHSDs is observed in castration-resistant prostate cancer tumors compared with primary prostate tumors, indicating their involvement in castration resistance. Recent studies link 3βHSD1 to resistance to androgen receptor signaling inhibitors. The regulation of 3βHSD1 expression involves various factors, including transcription factors, microenvironmental influences, and posttranscriptional modifications. Additionally, the clinical significance of HSD3B1 genotypes, particularly the rs1047303 variant, has been extensively studied. The impact of HSD3B1 genotypes on treatment outcomes varies according to the therapy administered, suggesting the potential of HSD3B1 genotyping for personalized medicine. Targeting 3βHSDs may be a promising strategy for prostate cancer management. Overall, understanding the roles of 3βHSDs and their genetic variations may enable the development and optimization of novel treatments for prostate cancer.

The effects of low-dose radiation (LDR, ≤0.1 Gy) on living organisms have been the hot areas of radiation biology but do not reach a definitive conclusion yet. So far, few studies have adequately accounted for the male reproductive system responses to LDR, particularly the regulation of testosterone content. Hence, this study was designed to evaluate the effects of LDR on Leydig cells and testicular tissue, especially the ability to synthesize testosterone. We found that less than 0.2-Gy 60 Co gamma rays did not cause significant changes in the hemogram index and the body weight; also, pathological examination did not find obvious structural alterations in testis, epididymis, and other radiation-sensitive organs. Consistently, the results from in vitro showed that only more than 0.5-Gy gamma rays could induce remarkable DNA damage, cycle arrest, and apoptosis. Notably, LDR disturbed the contents of testosterone in mice serums and culture supernatants of TM3 cells and dose dependently increased the expression of 3β-HSD. After cotreatment with trilostane (Tril), the inhibitor of 3β-HSD, increased testosterone could be partially reversed. Besides, DNA damage repair-related enzymes, including DNMT1, DNMT3B, and Sirt1, were increased in irradiated TM3 cells, accompanying by evident demethylation in the gene body of 3β-HSD. In conclusion, our results strongly suggest that LDR could induce obvious perturbation in the synthesis of testosterone without causing organic damage, during which DNA demethylation modification of 3β-HSD might play a crucial role and would be a potential target to prevent LDR-induced male reproductive damage., (© 2021 John Wiley & Sons, Ltd.)

BACKGROUNDGenetics of estrogen synthesis and breast cancer risk has been elusive. The 1245A→C missense-encoding polymorphism in HSD3B1, which is common in White populations, is functionally adrenal permissive and increases synthesis of the aromatase substrate androstenedione. We hypothesized that homozygous inheritance of the adrenal-permissive HSD3B1(1245C) is associated with postmenopausal estrogen receptor-positive (ER-positive) breast cancer.METHODSA prospective study of postmenopausal ER-driven breast cancer was done for determination of HSD3B1 and circulating steroids. Validation was performed in 2 other cohorts. Adrenal-permissive genotype frequency was compared between postmenopausal ER-positive breast cancer, the general population, and postmenopausal ER-negative breast cancer.RESULTSProspective and validation studies had 157 and 538 patients, respectively, for the primary analysis of genotype frequency by ER status in White female breast cancer patients who were postmenopausal at diagnosis. The adrenal-permissive genotype frequency in postmenopausal White women with estrogen-driven breast cancer in the prospective cohort was 17.5% (21/120) compared with 5.4% (2/37) for ER-negative breast cancer (P = 0.108) and 9.6% (429/4451) in the general population (P = 0.0077). Adrenal-permissive genotype frequency for estrogen-driven postmenopausal breast cancer was validated using Cambridge and The Cancer Genome Atlas data sets: 14.4% (56/389) compared with 6.0% (9/149) for ER-negative breast cancer (P = 0.007) and the general population (P = 0.005). Circulating androstenedione concentration was higher with the adrenal-permissive genotype (P = 0.03).CONCLUSIONAdrenal-permissive genotype is associated with estrogen-driven postmenopausal breast cancer. These findings link genetic inheritance of endogenous estrogen exposure to estrogen-driven breast cancer.FUNDINGNational Cancer Institute, NIH (R01CA236780, R01CA172382, and P30-CA008748); and Prostate Cancer Foundation Challenge Award.

Endogenous Cushing syndrome (CS) is an endocrine disorder marked by excess cortisol production rendering patients susceptible to visceral obesity, dyslipidemia, hypertension, osteoporosis and diabetes mellitus. Adrenal CS is characterized by autonomous production of cortisol from cortisol-producing adenomas (CPA) via adrenocorticotropic hormone-independent mechanisms. A limited number of studies have quantified the steroid profiles in sera from patients with CS. To understand the intratumoral steroid biosynthesis, we quantified 19 steroids by mass spectrometry in optimal cutting temperature compound (OCT)-embedded 24 CPA tissue from patients with overt CS (OCS, n = 10) and mild autonomous cortisol excess (MACE, n = 14). Where available, normal CPA-adjacent adrenal tissue (AdjN) was also collected and used for comparison (n = 8). Immunohistochemistry (IHC) for CYP17A1 and HSD3B2, two steroidogenic enzymes required for cortisol synthesis, was performed on OCT sections to confirm the presence of tumor tissue and guided subsequent steroid extraction from the tumor. LC-MS/MS was used to quantify steroids extracted from CPA and AdjN. Our data indicated that CPA demonstrated increased concentrations of cortisol, cortisone, 11-deoxycortisol, corticosterone, progesterone, 17OH-progesterone and 16OH-progesterone as compared to AdjN (p < 0.05). Compared to OCS, MACE patient CPA tissue displayed higher concentrations of corticosterone, 18OH-corticosterone, 21-deoxycortisol, progesterone, and 17OH-progesterone (p < 0.05). These findings also demonstrate that OCT-embedded tissue can be used to define intra-tissue steroid profiles, which will have application for steroid-producing and steroid-responsive tumors., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Problem: The miRNAs show placenta-specific expression patterns, which alter during pregnancy-related complications. In present study, the role of miR-27b-5p during forskolin-mediated BeWo cells fusion has been investigated., Method of Study: The fusion of BeWo cells in response to forskolin treatment (25 µM) was studied by desmoplakin I+II staining. Expression profile of miR-27b-5p by qRT-PCR and its targets HSD3β1 and WNT2B by qRT-PCR and in Western blot were studied. The effect of overexpression of miR-27b-5p and silencing of HSD3β1 & WNT2B by siRNA on forskolin-mediated BeWo cells fusion and secretion of hCG and progesterone by ELISA was investigated., Results: Time-dependent down-regulation in the expression of miR-27b-5p in forskolin-treated BeWo cells has been confirmed by qRT-PCR. Overexpression of miR-27b-5p significantly inhibits forskolin-mediated BeWo cells fusion as well as hCG & progesterone secretion. HSD3β1 and WNT2B were identified as targets of miR-27b-5p and are up-regulated in forskolin-treated BeWo cells. Overexpression of miR-27b-5p in BeWo cells downregulates their expression. Further, luciferase reporter assay revealed that miR-27b-5p directly target expression of both HSD3β1 and WNT2B. Silencing of both HSD3β1 and WNT2B leads to a significant reduction in forskolin-mediated BeWo cells fusion with concomitant decrease in the secretion of progesterone or/and hCG. Decrease in forskolin-mediated cells fusion observed in miR-27b-5p mimic transfected BeWo cells could be rescued by the overexpression of both HSD3β1 and WNT2B., Conclusion: These observations suggest that reduced miR-27b-5p in forskolin-treated BeWo cells leads to increased secretion of progesterone and hCG due to loss of repressional control on HSD3β1 and WNT2B., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: While previous studies indicate that the zonae reticularis (ZR) and glomerulosa (ZG) diminish with aging, little is known about age-related transformations of the zona fasciculata (ZF)., Objectives: To investigate the morphological and functional changes of the adrenal cortex across adulthood, with emphasis on (i) the understudied ZF and (ii) sexual dimorphisms., Methods: We used immunohistochemistry to evaluate the expression of aldosterone synthase (CYP11B2), visinin-like protein 1 (VSNL1), 3β-hydroxysteroid dehydrogenase type II (HSD3B2), 11β-hydroxylase (CYP11B1), and cytochrome b5 type A (CYB5A) in adrenal glands from 60 adults (30 men), aged 18 to 86. Additionally, we employed mass spectrometry to quantify the morning serum concentrations of cortisol, 11-deoxycortisol (11dF), 17α-hydroxyprogesterone, 11-deoxycorticosterone, corticosterone, and androstenedione in 149 pairs of age- and body mass index-matched men and women, age 21 to 95 years., Results: The total cortical area was positively correlated with age (r = 0.34, P = 0.008). Both the total (VSNL1-positive) and functional ZG (CYP11B2-positive) areas declined with aging in men (r = -0.57 and -0.67, P < 0.01), but not in women. The CYB5A-positive area declined with age in both sexes (r = -0.76, P < 0.0001). In contrast, the estimated ZF area correlated positively with age in men (r = 0.59, P = 0.0006) and women (r = 0.49, P = 0.007), while CYP11B1-positive area remained unchanged across ages. Serum cortisol, corticosterone, and 11-deoxycorticosterone levels were stable across ages, while 11dF levels increased slightly with age (r = 0.16, P = 0.007)., Conclusion: Unlike the ZG and ZR, the ZF and the total adrenal cortex areas enlarge with aging. An abrupt decline of the ZG occurs with age in men only, possibly contributing to sexual dimorphism in cardiovascular risk., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The testis expresses many long noncoding RNAs (lncRNAs), but their functions and overview of lncRNA variety are not well understood. The mouse Prss/Tessp locus contains six serine protease genes and two lncRNAs that have been suggested to play important roles in spermatogenesis. Here, we found a novel testis-specific lncRNA, Start ( Steroidogenesis activating lncRNA in testis ), in this locus. Start is 1822 nucleotides in length and was found to be localized mostly in the cytosol of germ cells and Leydig cells, although nuclear localization was also observed. Start -knockout (KO) mice generated by the CRISPR/Cas9 system were fertile and showed no morphological abnormality in adults. However, in adult Start -KO testes, RNA-seq and qRT-PCR analyses revealed an increase in the expression of steroidogenic genes such as Star and Hsd3b1 , while ELISA analysis revealed that the testosterone levels in serum and testis were significantly low. Interestingly, at 8 days postpartum, both steroidogenic gene expression and testosterone level were decreased in Start -KO mice. Since overexpression of Start in two Leydig-derived cell lines resulted in elevation of the expression of steroidogenic genes including Star and Hsd3b1 , Start is likely to be involved in their upregulation. The increase in expression of steroidogenic genes in adult Start -KO testes might be caused by a secondary effect via the androgen receptor autocrine pathway or the hypothalamus-pituitary-gonadal axis. Additionally, we observed a reduced number of Leydig cells at 8 days postpartum. Collectively, our results strongly suggest that Start is a regulator of steroidogenesis in Leydig cells. The current study provides an insight into the overall picture of the function of testis lncRNAs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Otsuka, Matsubara, Shiraishi, Takei, Satoh, Terao, Takada, Kotani, Satake and Kimura.)

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in reproductive-age women. Reduced progesterone levels are associated with luteal phase deficiency in women with PCOS. The levels of C-X-C motif chemokine ligand-14 (CXCL14) were previously reported to be decreased in human-luteinized granulosa (hGL) cells derived from PCOS patients. However, the function of CXCL14 in hGL cells and whether CXCL14 affects the synthesis of progesterone in hGL cells remain unclear. In the present study, the levels of CXCL14 were reduced in follicular fluid and hGL cells in PCOS patients, accompanied by decreased progesterone levels in follicular fluid and decreased steroidogenic acute regulatory (STAR) expression in hGL cells. CXCL14 administration partially reversed the low progesterone production and STAR expression in hGL cells obtained from PCOS patients. In primary hGL cells, CXCL14 upregulated STAR expression and progesterone production. CXCL14 activated the phosphorylation of cyclic adenosine monophosphate response element-binding protein (CREB) and CREB inhibitor attenuated the modulation of StAR expression by CXCL14. P38 and Jun N-terminal kinase (JNK) pathways were also activated by CXCL14 and inhibition of p38 and JNK attenuated the increase of phosphorylation of CREB, STAR expression and progesterone production caused by CXCL14. Our findings revealed the novel role of CXCL14 in upregulation of STAR expression and progesterone synthesis through CREB phosphorylation via activation of p38 and JNK pathways in hGL cells. This is likely contributing to the dysfunction in steroidogenesis in granulosa cells from PCOS patients., (Copyright © 2020 Elsevier Inc. All rights reserved.)

An electromagnetic field (EMF) may have effects on female reproduction. This study was conducted to determine whether EMF [50 and 120 Hz, 2 and 4 h of incubation in the presence or absence of progesterone (P 4 , 10 -5 M)] affects androgen synthesis and release from the pig endometrium. Endometrial slices were collected from pigs (n = 5) during the fetal peri-implantation period (i.e., days 15-16 of gestation) and treated in vitro with EMF. The selected endometrial slices were treated with P 4 to determine whether this hormone has effects on protection of the tissue from EMF radiation. The CYP17A1 and HSD3B1 mRNA transcript abundance, steroid 17αhydroxylase/17, 20-lyase (cytochrome P450c17) and hydroxyΔ5steroid dehydrogenase/3β and steroidΔisomerase (3βHSD) protein abundance were examined using Real-Time PCR and Western Blot procedures, respectively. In media collected after incubation, the concentrations of androstenedione (A 4 ) and testosterone (T) were quantified used a RIA. When P 4 was added to the culture medium, EMF radiation had suppressive effects on endometrial T release after 2 and 4 h of incubation when the EMF treatment was occurring and increased A 4 release after 4 h of incubation with EMF at 120 Hz. When there was no inclusion of P 4 , release of A 4 was decreased after 2 h of EMF treatment at 120 Hz and after 4 h of EMF treatment at 50 and 120 Hz. Progesterone did not have functions that protected the pig endometrium against EMF radiation during the fetal peri-implantation period., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The objective was to investigate effects of progesterone (P 4 ) dose on abundance of luteinizing hormone receptor (LHCGR), aromatase (CYP19A1), 3β-hydroxysteroid dehydrogenase (HSD3B1), and other steroidogenic mRNA transcripts in granulosa cells from dominant follicles. Nellore heifers were assigned to one of six groups: new, first-use controlled internal drug release device (CIDR1) inserted for 5 days (Large-P 4 -dose-D5; n = 7) or 6 days (Large-P 4 -dose-D6; n = 8), prostaglandin (PG)F 2α administered on D0 and 1 previously-used CIDR (CIDR3) inserted for 5 days (Small- P 4 -dose-D5; n = 8) or 6 days (Small-P 4 -dose-D6; n = 8), CIDR1 inserted on D0 and removed plus PGF 2α on D5 (Large-P 4 -dose-proestrus (PE); n = 7), and CIDR3 and PGF 2α on D0 and 1, CIDR3 removed plus PGF 2α on D5 (Small-P 4 -dose-PE; n = 7). Duration of P 4 treatment (D5 compared to D6) affected abundances of CYP19A1 mRNA transcripts, with there being greater abundances on D6 than D5 (P ≤ 0.05). Heifers treated with the large dose of P 4 had a smaller dominant follicle, less serum and intra-follicular estradiol (E 2 ) concentrations (P ≤ 0.05) and lesser LHCGR, CYP19A1, and HSD3B1 transcript abundances (P ≤ 0.05). Heifers treated to induce PE had a larger follicle diameter (P = 0.09), greater intra-follicular E 2 concentrations and larger abundances of CYP19A1 mRNA transcript (P ≤ 0.05) than heifers of the D6 group. Overall, treatment with larger doses of P 4 resulted in lesser abundances of LHCGR, HSD3B1, and CYP19A1 mRNA transcripts; thus, potentially leading to development of smaller dominant follicles and lesser E 2 concentrations., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Sheep is usually a monovular animal; superovulation technology is used to increase the number of offspring per individual and shorten generation intervals. To date, mature FSH superstimulatory treatments have been successfully used in sheep breeding, but much remains unknown about genes, pathways, and biological functions involved in follicular development. Therefore, in this study, we performed transcriptome profiling of small follicles (SFs; 2-2.5 mm), medium follicles (MFs; 3.5-4.5 mm), and large follicles (LFs; > 6 mm) in Mongolian ewes after FSH superstimulation. Furthermore, we identified differentially expressed genes and performed Kyoto Encyclopedia of Genes and Genomes pathway and Gene Ontology enrichment analyses in 3 separate pairwise comparisons. We found that ovarian steroidogenesis was significantly enriched in the SFs versus MFs analysis; the associated genes, cytochrome P450 family 19 (CYP19) and Hydroxy-delta-5-steroid dehydrogenase 3 beta- and steroid delta-isomerase 1 (HSD3B1), were significantly upregulated. Moreover, proline metabolism, glutathione metabolism, and PPAR signaling pathways were significantly enriched in the LFs versus SFs analysis; the associated genes, glutamate-cysteine ligase modifier subunit (GCLM) and cystathionine gamma-lyase (CTH), were significantly upregulated, whereas peroxisome proliferator-activated receptor gamma (PPARγ) was significantly downregulated. In summary, our study provides basic data and possible biological direction to further explore the molecular mechanism of sheep follicular development after FSH superstimulation., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Dehydroepiandrosterone (DHEA) and DHEA sulfate together are abundant adrenal steroids whose physiological effects are mediated through their conversion to potent downstream androgens. 3β-Hydroxysteroid dehydrogenase isotype 1 (3βHSD1) facilitates the rate-limiting step of DHEA metabolism and gates the flux of substrate into the distal portion of the androgen synthesis pathway. Notably, a germline, missense-encoding change, HSD3B1(1245C), results in expression of 3βHSD1 protein that is resistant to degradation, yielding greater potent androgen production in the periphery. In contrast, HSD3B1(1245A) encodes 3βHSD1 protein that is easily degraded, limiting peripheral androgen synthesis. These adrenal-permissive (AP) and adrenal-restrictive (AR) alleles have recently been associated with divergent outcomes in androgen-sensitive disease states, underscoring the need to reevaluate DHEA metabolism using HSD3B1 genetics., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: The aberrant expression of genes involved in androgen metabolism and genetic contribution are unclear in hypospadias., Methods: We compared gene expression profiles by RNA sequencing from five non-hypospadiac foreskins, five mild hypospadiac foreskins, and five severe hypospadiac foreskins. In addition, to identify rare coding variants with large effects on hypospadias risk, we carried out whole exome sequencing in three patients in a hypospadias family., Results: The average expression of androgen receptor (AR) and CYP19A1 were significantly decreased in severe hypospadias (p < .01) and mild hypospadias (p < .05), whereas expression of several other androgen metabolism enzymes, including CYP3A4, HSD17B14, HSD3B7, HSD17B7, CYP11A1 were exclusively significantly expressed in severe hypospadias (p < .05). Compound rare damaging mutants of AR gene with HSD3B1 and SLC25A5 genes were identified in the different severe hypospadias., Conclusions: In conclusion, our findings demonstrated that dysregulation of AR and CYP19A1 could play a crucial role in the development of hypospadias. Inconsistent AR expression may be caused by the feedback loop of ESR1 signaling or combined genetic effects with other risk genes. This findings complement the possible role of AR triggered mechanism in the development of hypospadias., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Iridoids are plant-derived terpenoids with a rich array of bioactivities. The key step in iridoid skeleton formation is the reduction of 8-oxogeranial by certain members of the progesterone 5β-reductase/iridoid synthase (PRISE) family of short-chain alcohol dehydrogenases. Other members of the PRISE family have previously been implicated in the biosynthesis of the triterpenoid class of cardenolides, which requires the reduction of progesterone. Here, we explore the occurrence and activity of PRISE across major lineages of plants. We observed trace activities toward either 8-oxogeranial or progesterone in all PRISEs, including those from nonseed plants and green algae. Phylogenetic analysis, coupled with enzymatic assays, show that these activities appear to have become specialized in specific angiosperm lineages. This broad analysis of the PRISE family provides insight into how these enzymes evolved in plants and also suggests that iridoid synthase activity is an ancestral trait in all land plants, which might have contributed to the rise of iridoid metabolites.

An electromagnetic field (EMF) has been found to affect reproductive processes in females. The aim of this study was to determine the effect of low, non-ionizing EMF radiation on the steroidogenic activity of myometrium collected from pigs during the fetal peri-implantation period. Myometrial slices were treated with an EMF (50 and 120 Hz, 2 and 4 h of incubation) and examined for the aromatase cytochrome P450 17α-hydroxylase/C17-20lyase (CYP17A1) and 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (HSD3B1) mRNA transcript abundance, cytochrome P450c17 and 3βHSD protein abundance and the secretion of androstenedione (A 4 ) and testosterone (T). To determine whether progesterone (P 4 ) functions as a protectant from EMF radiation, the selected slices were treated with P 4 . In slices incubated without P 4 , EMF at 50 Hz altered cytochrome P450c17 protein abundance (4 h), HSD3B1 mRNA transcript abundance (4 h) and A 4 release (2 h) as well as T release (2 h) in P 4 -treated slices. The EMF at 120 Hz in non P 4 -treated slices altered A 4 release (2 and 4 h) whereas in P 4 -treated slices altered CYP17A1 mRNA transcript abundance (4 h), 3βHSD protein abundance (4 h), A 4 (4 h) and T release (2 h). In conclusion, EMF radiation in the myometrium collected during the peri-implantation period alters the CYP17A1 and HSD3B1 mRNA transcript and encoded protein abundance, and androgen release due to the time of treatment and P 4 presence or absence. The P 4 did not function directly as an obvious protector against EMF radiation in the myometrium of pigs during the fetal peri-implantation period., Competing Interests: Declaration of Competing Interest The authors hereby confirm that neither the manuscript nor any part of it has been published or is being considered for publication elsewhere. None of the authors of the above manuscript has declared any conflict of interest which may arise from being named as an author of the manuscript., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Acne vulgaris is a chronic inflammatory disease of the skin resulting from androgen-induced increased sebum production and altered keratinization. Nicotinamide (NAM), an amide form of vitamin B3 with a well-established safety profile, has shown good therapeutic potential in treating acne and its complications. NAM has anti-inflammatory effects and reduces sebum but its function in androgen biosynthesis remains unknown. In this study, we used a widely used cell model, starved human adrenal NCI-H295R cells, to examine the effects of NAM in androgen production and its mediated network changes. By treating NCI-H295R cells with 1-25 mmol/L of NAM, we found that cell viability was only slightly inhibited at the highest dose (25 mmol/L). NAM reduced testosterone production in a dose-dependent manner. Transcriptomic analysis demonstrated that key enzymes of androgen biosynthesis were significantly decreased under NAM treatment. In addition, gene set enrichment analysis (GSEA) showed that gene sets of cell cycle, steroid biosynthesis, TGFβ signalling, and targets of IGF1 or IGF2 were enriched in NAM-treated cells. Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway and Gene ontology (GO) analysis of the differentially expressed genes also suggested that steroidogenesis and SMAD signalling were affected by NAM. Overall, these crucial genes and pathways might form a complex network in NAM-treated NCI-H295R cells and result in androgen reduction. These findings help explain the potential molecular actions of NAM in acne vulgaris, and position NAM as a candidate for the treatment of other hyperandrogenic disorders., (© 2020 The Authors. Clinical and Experimental Pharmacology and Physiology published by John Wiley & Sons Australia, Ltd.)

Orexin A (OXA) is primarily known for its involvement in the regulation of feeding behaviour, energy metabolism and sleep/wake cycle. Nevertheless, studies indicate its engagement in the regulation of the porcine reproductive system. Therefore, the aim of this study was to investigate OXA effect (1, 10, 100 nM), in the presence or absence of the selective orexin receptor type 1 antagonist (SB-3348667; 1 μM), on the gene expression of key steroidogenic enzymes: steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage enzyme (CYP11A1) and 3β-hydroxysteroid dehydrogenase (HSD3B1), as well as on progesterone (P 4 ) and androstenedione (A 4 ) secretion. Endometrial and myometrial tissue explants were collected from gilts on days 10 to 11, 12 to 13, 15 to 16 and 27 to 28 of pregnancy, and on days 10 to 11 of the oestrous cycle (n = 5 per studied period of pregnancy or mid-luteal phase of the oestrous cycle). Gene expression was evaluated by real-time PCR. The level of steroid hormones secreted into the culture medium was examined by radioimmunoassay (RIA). In the present study, in the endometrium, OXA significantly stimulated StAR expression on days 12 to 13, CYP11A1 expression on days 27 to 28 and HSD3B1 expression on days 15 to 16 of pregnancy. Further, in this tissue, OXA decreased StAR mRNA level on days 10 to 11, CYP11A1 mRNA level on days 15 to 16, as well as HSD3B1 mRNA level on days 10 to 11 and 12 to 13 of gestation. Regarding the myometrium, OXA stimulated CYP11A1 gene expression on days 15 to 16 of pregnancy. In this tissue, OXA decreased StAR transcript content on days 15 to 16 and CYP11A1 mRNA level on days 27 to 28. We also demonstrated that OXA alone enhanced P 4 secretion in the endometrium on days 10 to 11 and 12 to 13 of gestation. OXA alone has no significant effect on endometrial and myometrial A 4 secretion, whereas OXA in combination with OX1R antagonist increased this hormone secretion during all studied stages of pregnancy. Therefore, we can conclude that OXA may affect de novo synthesis and secretion of P 4 and A 4 in the porcine uterus via participating in the regulation of key steroidogenic enzymes gene expression, as well as modulating steroid hormones secretion during early pregnancy and mid-luteal phase of the oestrous cycle in pigs. However, further research is required to explain the exact role of OXA in the porcine uterus., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Asthma resistance to glucocorticoid treatment is a major health problem with unclear etiology. Glucocorticoids inhibit adrenal androgen production. However, androgens have potential benefits in asthma. HSD3B1 encodes for 3β-hydroxysteroid dehydrogenase-1 (3β-HSD1), which catalyzes peripheral conversion from adrenal dehydroepiandrosterone (DHEA) to potent androgens and has a germline missense-encoding polymorphism. The adrenal restrictive HSD3B1 (1245A) allele limits conversion, whereas the adrenal permissive HSD3B1 (1245C) allele increases DHEA metabolism to potent androgens. In the Severe Asthma Research Program (SARP) III cohort, we determined the association between DHEA-sulfate and percentage predicted forced expiratory volume in 1 s (FEV 1 PP). HSD3B1 (1245) genotypes were assessed, and association between adrenal restrictive and adrenal permissive alleles and FEV 1 PP in patients with (GC) and without (noGC) daily oral glucocorticoid treatment was determined ( n = 318). Validation was performed in a second cohort (SARP I&II; n = 184). DHEA-sulfate is associated with FEV 1 PP and is suppressed with GC treatment. GC patients homozygous for the adrenal restrictive genotype have lower FEV 1 PP compared with noGC patients (54.3% vs. 75.1%; P < 0.001). In patients with the homozygous adrenal permissive genotype, there was no FEV 1 PP difference in GC vs. noGC patients (73.4% vs. 78.9%; P = 0.39). Results were independently confirmed: FEV 1 PP for homozygous adrenal restrictive genotype in GC vs. noGC is 49.8 vs. 63.4 ( P < 0.001), and for homozygous adrenal permissive genotype, it is 66.7 vs. 67.7 ( P = 0.92). The adrenal restrictive HSD3B1 (1245) genotype is associated with GC resistance. This effect appears to be driven by GC suppression of 3β-HSD1 substrate. Our results suggest opportunities for prediction of GC resistance and pharmacologic intervention., Competing Interests: Competing interest statement: Cleveland Clinic has applied for patents on HSD3B1., (Copyright © 2020 the Author(s). Published by PNAS.)

Placental syncytiotrophoblast (ST) is considered as the main placental endocrine tissue secreting progesterone, a steroid essential for maintenance of pregnancy. However, each step of progestins production has been poorly investigated in villous cytotrophoblast (VCT) regarding ST formation. We aimed to characterize progestins production during human differentiation of VCT into ST. VCTs were isolated from term placenta and cultivated, with or without forskolin (FSK), to stimulate trophoblast differentiation. Secreted progestins concentrations were determined by immuno-assay and Gas Chromatography-tandem mass spectrometry. Intracellular expression of cholesterol transporter and enzymes involved in steroidogenesis were studied by immunofluorescence, western-blot, and RT-qPCR. Progesterone and pregnenolone are produced by VCT and their secretion increases with VCT differentiation while 17-hydroxyprogesterone concentration remains undetectable. HSD3B1 enzyme expression increases whereas MLN64, the cholesterol placental mitochondrial transporter and P450SCC expressions do not. FSK induces progestins production. Progestins placental synthesis is effective since VCT and increases with ST formation thanks to mitochondria., (Copyright © 2019 Elsevier B.V. All rights reserved.)

To assess the effects of 4-nitrophenol (PNP) and 3-methyl-4-nitrophenol (PNMC) on steroidogenesis in the chicken ovary, white (WF, 1-4 mm) and yellowish (YF, 4-8 mm) prehierarchical follicles were incubated in a medium supplemented with PNP or PNMC (10 -8 -10 -4  M), ovine LH (oLH; 10 ng/mL), and combinations of oLH with PNP or PNMC (10 -6  M). Testosterone (T) and estradiol (E2) concentrations in media and mRNA expression for steroidogenic proteins (STAR, HSD3B1, and CYP19A1), and LH receptors (LHR), estrogen receptor α (ESR1) and β (ESR2) in follicles were determined by RIA and real-time qPCR, respectively. PNP and PNMC decreased T and E2 secretion by the WF and YF, and oLH-stimulated T secretion from these follicles. PNP decreased basal STAR and HSD3B1 mRNA levels both in the WF and YF, and CYP19A1 mRNAs in the WF. PNP reduced oLH-affected mRNA expression of these genes in the YF. PNMC inhibited basal STAR, HSD3B1, and CYP19A1 mRNA expression in the WF, but not in the YF. PNMC reduced oLH-stimulated STAR and CYP19A1 expression in the YF and WF, respectively. PNP decreased basal mRNA expression of LHR, ESR1, and ESR2 in the WF, but it increased ESR1 and ESR2 mRNA levels in the YF. PNMC reduced both basal and oLH-affected LHR, ESR1, and ESR2 mRNA expression in the WF; however, it did not influence expression of these genes in the YF. We suggest that nitrophenols by influencing sex steroid synthesis and transcription of LH and estrogen receptors in prehierarchical ovarian follicles may impair their development and selection to the preovulatory hierarchy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

CREBZF, including the two isoforms SMILE (long isoform of CREBZF) and Zhangfei (short isoform of CREBZF), has been identified as a novel transcriptional coregulator of a variety of nuclear receptors. Our previous studies found that SMILE is expressed in the mouse uterine luminal and glandular epithelium and is upregulated by estrogen. In the present study, CREBZF was age-dependently and -specifically expressed in mouse interstitial Leydig cells during sexual maturation. The expression pattern of CREBZF exhibited an age-related increase, and SMILE was the dominant isoform in the mouse testis. Although hCG did not affect CREBZF expression, CREBZF silencing significantly inhibited hCG-stimulated testosterone production in primary Leydig cells and MLTC-1 cells. Meanwhile, the serum concentration of testosterone was significantly decreased after microinjection of lentiviral-mediated shRNA-CREBZF into the mature mouse testis. In addition, CREBZF silencing markedly decreased P450c17, 17β-HSD, and 3β-HSD expression following hCG stimulation in primary Leydig cells, and this inhibitory effect was obviously reversed by overexpression of CREBZF. Furthermore, CREBZF significantly upregulated the mRNA levels of Nr4a1 and Nr5a1, which are the essential orphan nuclear receptors for steroidogenic gene expression. Together our data indicate that CREBZF promotes hCG-induced testosterone production in mouse Leydig cells by affecting Nr4a1 and Nr5a1 expression levels and subsequently increasing the expression of steroidogenic genes such as 3β-HSD, 17β-HSD, and P450c17, suggesting a potential important role of CREBZF in testicular testosterone synthesis., (© 2019 Wiley Periodicals, Inc.)

Triptolide (TP), a major active ingredient of Tripterygium wilfordii, exerts potent immunosuppressive effects in the treatment of rheumatoid arthritis but is not widely used in clinical practice due to its multiorgan toxicity, particularly hepatotoxicity, nephrotoxicity, and reproductive toxicity. An LC-MS/MS approach was employed to explore the endocrine-disrupting effects of TP. The endocrine-disrupting effects of various concentrations (0-100 nM) of TP for 48 hour were firstly investigated using an in vitro model (H295R cell line). It was found that TP did not decrease cell viability. The transcriptional levels of steroidogenic enzymes in H295R cells were assessed by quantificational real-time polymerase chain reaction. The possible adrenal and endocrine effects of oral administration of TP (0, 50, and 500 μg/kg) for 28 days on both normal and collagen-induced arthritis (CIA) rats were also explored. The serum and adrenal tissue hormone levels (corticosterone and progesterone) and adrenal histopathology were analyzed, with the results that TP significantly decreased the level of cortisol in H295R cells and the level of plasma corticosterone in both normal and CIA rats. Histological alterations in adrenal cortex were observed at the dose of 500 μg/kg. Exposure to TP for 48 hour had an obvious inhibitory effect on the messenger RNA transcript levels of HSD3B2, CYP21A2, CYP17A1, and CYP11B1, which is essential for the synthesis of corticosteroids. In a word, TP leads to the disorder of corticosteroid synthesis and secretion, and corticosteroid may be a potential biomarker for the treatment of multiorgan toxicity of TP., (© 2019 Wiley Periodicals, Inc.)

Objective: Fetal macrosomia is a major risk factor for shoulder dystocia, which can lead to birth asphyxia, maternal and neonatal traumatic injuries, and perinatal death. If macrosomia is diagnosed in the antenatal period, labour can be induced to decrease shoulder dystocia. But current clinical methods to diagnose fetal macrosomia antenatally perform with poor accuracy. Therefore, improved methods to accurately diagnose fetal macrosomia are required. Blood biomarkers that predict fetal macrosomia could be one such novel diagnostic strategy. We undertook a nested case-control study from a prospective collection of 1000 blood samples collected at 36 weeks' gestation. We analysed plasma samples from 52 women who subsequently delivered a macrosomic (> 95th centile for gestational age) infant and 106 controls. Circulating concentrations of the proteins COBLL1, CSH1, HSD3B1, EGFL6, XAGE3, S100P, PAPPA-1, ERBB2 were assessed for their ability to predict macrosomic infants., Results: We did not identify any significant changes in the plasma concentrations of COBLL1, CSH1, HSD3B1, EGFL6, XAGE3, S100P, PAPPA-1, ERBB2 from women who subsequently delivered macrosomic neonates relative to control samples. Although we have not identified any potential biomarkers of fetal macrosomia, we have ruled out these particular eight protein candidates.

Adiponectin is an adipocyte derived cytokine implicated in energy homeostasis, insulin resistance and is involved in the regulation of reproduction both centrally and peripherally in animals. The present study was conducted to investigate adiponectin (ADIPOQ) and its receptors ADIPOR1 and ADIPOR2 abundance of mRNA transcript and protein in different stages of corpora lutea (CL) development during the estrous cycle of water buffalo and to determine the effect of adiponectin on cultured luteal cells of water buffalo (Bubalus bubalis). The results indicate adiponectin, ADIPOR1, and ADIPOR2 were present in buffalo corpora lutea (CL) throughout the estrous cycle. The abundance of adiponectin and its receptors was greater in the early and regressing and was less in mid- and late-stages of CL functionality. Adiponectin and its receptors were localized in the cytoplasm of small and large luteal cells. Furthermore, luteal cells were cultured in the in-vitro culture system and were treated with 1 and 10 μg/mL dose of adiponectin for 48 h. Adiponectin at both doses decreased (P < 0.05) progesterone (P 4 ) secretion from cultured luteal cells and also suppressed the abundance of factors involved in P 4 productionv [Steroidogenic Acute Regulatory Protein (STAR), cytochrome P45011A1 (CYP11A1) and 3β-hydroxysteroid dehydrogenase (HSD3B1) at the 10 μg/mL dose as compared to adiponectin non-supplemented cells]. In conclusion, results of the present study indicate adiponectin and its receptors are present in bubaline CL and adiponectin inhibits P 4 production in cultured luteal cells. The findings indicate adiponectin affects luteal dynamics and reproductive functions in water buffalo., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Human placental 3β-hydroxysteroid dehydrogenase/steroid Δ5, 4-isomerase 1 (HSD3B1), a high-affinity type I enzyme, uses pregnenolone to make progesterone, which is critical for maintenance of pregnancy. HSD3B1 is located in the mitochondrion and the smooth endoplasmic reticulum of placental cells and is encoded by HSD3B1 gene. HSD3B1 contains GATA and TEF-5 regulatory elements. Many endocrine disruptors, including phthalates, methoxychlor and its metabolite, organotins, and gossypol directly inhibit placental HSD3B1 thus blocking progesterone production. In this review, we discuss the placental HSD3B1, its gene regulation, biochemistry, subcellular location, and inhibitors from the environment., (Copyright © 2019 Elsevier B.V. All rights reserved.)

To investigate whether autophagy and subcellular changes are involved in the corpus luteum after heat exposure, a total of 30 early pregnant mice were divided equally into heat stress (HS) and non-HS (NHS) groups (n = 15). Mice in the HS group were exposed to 40.5 ± 0.2 ℃ for 7 consecutive days. Ovaries were collected for immunohistochemistry (IHC), western blot (WB) analysis and transmission electron microscopy (TEM). Serum was collected to determine progesterone by RIA and uteri were collected to count the implantation sites. Results showed that heat exposure increased rectal temperature, decreased body weight and number of implantation sites. WB analysis revealed that ovarian expression of LC3B and Atg7 was up-regulated, while p62 was down-regulated in the HS group. IHC results demonstrated that ovarian staining intensity of LC3B was more intense in the HS group than that of the NHS group. LC3B was mainly localized in the granulosa cells, oocytes and luteal steroidogenic cells of the HS group. TEM results revealed double-layered separated membranes indicative of autophagosomes in the luteal steroidogenic cells of the HS group. Moreover, TEM showed that the mitochondrial cristae became dearth, structure-less, swollen after HS. Additionally, the nucleus expanded and accumulation of lipid droplets increased after HS. Results also showed that heat exposure decreased serum progesterone level and ovarian P450scc expression. These results indicate that HS enhanced autophagy and altered the subcellular structure of luteal steroidogenic cells, which may contribute to interfering with the maintenance of luteal function in early pregnant mice., (Copyright © 2019 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Objectives: The current study aims to identify markers that would reflect the number of Leydig cells present in the testis, to help determine whether labour-intensive methods such as stereology are necessary. We used our well-characterised Sertoli cell ablation model in which we have empirically established the size of the Leydig cell population, to try to identify transcriptional biomarkers indicative of population size., Results: Following characterisation of the Leydig cell population after Sertoli cell ablation in neonatal life or adulthood, we identified Hsd3b1 transcript levels as a potential indicator of Leydig cell number with utility for informing decision-making on whether to engage in time-consuming stereological cell counting analysis., Competing Interests: The authors have declared that no competing interests exist.

Context: The ovaries and adrenals are sources of androgens in women. Although dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), and testosterone (T) all decline with age, these C19 steroids correlate poorly with parameters of androgen action in postmenopausal women., Objective: To comprehensively compare the androgen profiles of pre- and postmenopausal women., Methods: We quantified 19 steroids-including DHEA; DHEAS; T; androstenedione (A4); and the following adrenal-specific 11-oxygenated C19 steroids (11oxyandrogens): 11β-hydroxytestosterone (11OHT), 11-ketotestosterone (11KT), 11β-hydroxyandrostenedione (11OHA4), and 11-ketoandrostenedione (11KA4)-using liquid chromatography-tandem mass spectrometry in morning serum obtained from 100 premenopausal (age 20 to 40 years) and 100 postmenopausal (age ≥ 60 years) women. Double immunofluorescence of 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2) with cytochrome b5 (CYB5A) or sulfotransferase 2A1 (SULT2A1) was performed in normal adrenal glands obtained from eight premenopausal and eight postmenopausal women., Results: DHEA, DHEAS, A4, and T were significantly higher in pre- than in postmenopausal women (2.9, 2.8, 2.9, and 1.6-fold, respectively; P < 0.0001). In contrast, the 11-oxyandrogens did not decrease with aging, and the 11OHT/T and 11OHA4/A4 ratios showed strong positive correlations with age (r = 0.5 and 0.8, respectively; P < 0.0001). Double immunofluorescence analysis showed that with the involution of the zona reticularis in the old adrenals, the sharp zonal segregation of HSD3B2 and CYB5A becomes less distinct, and areas of HSD3B2 and CYB5A overlap are observed., Conclusions: Unlike DHEA, DHEAS, A4, and T, the 11oxyandrogens do not decline in aging women. Structural changes within the adrenal cortex might explain the evolution of androgen profiles in aging women., (Copyright © 2019 Endocrine Society.)

Steroid hormones play essential roles for living organisms. It has been long and well established that the endoplasmic reticulum (ER) and mitochondria are essential sites for steroid hormone biosynthesis because several steroidogenic enzymes are located in these organelles. The adrenal gland lipid droplet (LD) proteomes from human, macaque monkey, and rodent are analyzed, revealing that steroidogenic enzymes are also present in abundance on LDs. The enzymes found include 3β-hydroxysteroid dehydrogenase (HSD3B) and estradiol 17β-dehydrogenase 11 (HSD17B11). Analyses by Western blot and subcellular localization consistently demonstrate that HSD3B2 is localized on LDs. Furthermore, in vitro experiments confirm that the isolated LDs from HeLa cell stably expressing HSD3B2 or from rat adrenal glands have the capacity to convert pregnenolone to progesterone. Collectively, these data suggest that LDs may be important sites of steroid hormone metabolism. These findings may bring novel insights into the biosynthesis and metabolism of steroid hormones and the development of treatments for adrenal disorders., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Lambda-cyhalothrin (LCT) is a widely used broad-spectrum pyrethroid insecticide and is expected to cause deleterious effects on the male reproductive system. However, the effects of LCT on Leydig cell development during puberty are unclear. The current study addressed these effects. Twenty-eight-day-old male Sprague Dawley rats orally received LCT (0, 0.25, 0.5 or 1 mg/kg body weight/day) for 30 days. The levels of serum testosterone, luteinizing hormone, and follicle-stimulating hormone, Leydig cell number, and its specific gene and protein expression were determined. LCT exposure lowered serum testosterone levels at doses of 0.5 and 1 mg/kg and luteinizing hormone levels at a dose of 1 mg/kg, but increased follicle-stimulating hormone levels at doses of 0.5 and 1 mg/kg. LCT lowered Star and Hsd3b1 mRNA or their protein levels at a dose of 1 mg/kg. Immature Leydig cells were purified from pubertal rats and treated with different concentrations of LCT for 24 h and medium androgen levels, Leydig cell mRNA and protein levels, the mitochondrial membrane potential (△Ψm), and the apoptotic rate of immature Leydig cells were investigated. LCT inhibited androgen production at 5 μM and downregulated Scarb1 at 0.05 μM, Hsd3b1 and Hsd11b1 at 0.5 μM, and Cyp11a1 at 5 μM. LCT also decreased △Ψm at 0.5 and 50 μM. In conclusion, LCT can influence the function of Leydig cells., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Steroid synthesis is required for pregnancy maintenance and for parturition, but comparatively little is known about the major metabolic routes that influence circulating concentrations. Dietary intake changes progesterone and estradiol concentrations in pregnant ewes but whether this reflects placental synthesis is unknown. Progesterone metabolism by 5alpha-reduction is a major metabolic route in other species and can influence the onset of parturition. Therefore, studies were conducted to (1) determine placental enzyme activity, progesterone, and estradiol measured by immunoassay in late gestation ewes on low-, moderate-, and high-nutritional planes, (2) to assess the significance of 5alpha-reduction of progesterone in determining progesterone concentrations in late gestation ewes (gestation day 145) given finasteride to inhibit 5alpha-reductase metabolism. In the second experiment, steroid profiles were examined comprehensively in blood and tissues by liquid chromatography tandem mass spectrometry for the first time in this species. Dietary intake altered progesterone and estradiol serum concentrations but without correlated changes in placental 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase/17,20-lyase cytochrome P450 or aromatase activity. 5alpha-reduced pregnane metabolites were identified in ewes at 145 days of gestation, but concentrations were lower than those of progesterone. Finasteride inhibited 5alpha-reduced progesterone metabolism but did not impact serum progesterone concentrations in these ewes. We conclude that (1) diet-induced changes in serum progesterone and estradiol concentrations are not likely a result of altered placental synthesis of sex steroid but most likely by their metabolism, and (2) metabolism by 5α-reduction is not a major determinant of systemic progesterone concentrations in late gestation ewes.

Iridoid synthases belong to the family of short-chain dehydrogenase/reductase involved in the biosynthesis of iridoids. Despite having high sequence and structural homology with progesterone 5β-reductase, these enzymes exhibit differential substrate specificities. Previously, two loops, L1 and L2 at substrate-binding pocket, were suggested to be involved in generating substrate specificity. However, the structural basis of specificity determinants was elusive. Here, combining sequence and structural analysis, site-directed mutagenesis, and molecular dynamics simulations, we have shown that iridoid synthase contains two channels for substrate entry whose geometries are altered by L1-L2 dynamics, primarily orchestrated by interactions of residues Glu161 and Gly162 of L1 and Asn358 of L2. A complex interplay of these interactions confer the substrate specificity to the enzyme., (© 2018 Federation of European Biochemical Societies.)

Background: A common germline variant in HSD3B1(1245A>C) encodes for a hyperactive 3β-hydroxysteroid dehydrogenase 1 (3βHSD1) missense that increases metabolic flux from extragonadal precursor steroids to DHT synthesis in prostate cancer. Enabling of extragonadal DHT synthesis by HSD3B1(1245C) predicts for more rapid clinical resistance to castration and sensitivity to extragonadal androgen synthesis inhibition. HSD3B1(1245C) thus appears to define a subgroup of patients who benefit from blocking extragonadal androgens. However, abiraterone, which is administered to block extragonadal androgens, is a steroidal drug that is metabolized by 3βHSD1 to multiple steroidal metabolites, including 3-keto-5α-abiraterone, which stimulates the androgen receptor. Our objective was to determine if HSD3B1(1245C) inheritance is associated with increased 3-keto-5α-abiraterone synthesis in patients., Methods: First, we characterized the pharmacokinetics of 7 steroidal abiraterone metabolites in 15 healthy volunteers. Second, we determined the association between serum 3-keto-5α-abiraterone levels and HSD3B1 genotype in 30 patients treated with abiraterone acetate (AA) after correcting for the determined pharmacokinetics., Results: Patients who inherit 0, 1, and 2 copies of HSD3B1(1245C) have a stepwise increase in normalized 3-keto-5α-abiraterone (0.04 ng/ml, 2.60 ng/ml, and 2.70 ng/ml, respectively; P = 0.002)., Conclusion: Increased generation of 3-keto-5α-abiraterone in patients with HSD3B1(1245C) might partially negate abiraterone benefits in these patients who are otherwise more likely to benefit from CYP17A1 inhibition., Funding: Prostate Cancer Foundation Challenge Award, National Cancer Institute.

HSD3B2 plays a crucial role in steroid hormone biosynthesis and is thus of particular interest in hormone dependent tumors such as prostate cancer. To clarify the clinical relevance of HSD3B2 expression in prostate cancer, we analyzed HSD3B2 protein expression by immunohistochemistry on our preexisting tissue microarray with 12.247 annotated cancers. Compared with normal tissue cytoplasmic HSD3B2 staining was stronger in prostate cancers. In 9371 interpretable cancers, HSD3B2 expression was found in 95.5% of cancers and was considered weak in 29.9%, moderate in 40.7% and strong in 24.9%. HSD3B2 up regulation was linked to advanced pathological tumor stage (pT), high Gleason grade, elevated preoperative PSA levels (p < 0.0001 each), lymph node metastasis (p = 0.0019), accelerated cell proliferation (p < 0.0001), androgen receptor (AR) expression (p < 0.0001), and early biochemical recurrence (p < 0.0001). HSD3B2 up regulation was only marginally more frequent in ERG positive (98%) than in ERG negative cancers (94%; p < 0.0001) and was strongly linked to deletions of 5q and 6q (p < 0.0001 each). Multivariate analyses showed that the prognostic impact of HSD3B2 expression was independent of established preoperative, but not of postoperative prognostic parameters. In summary, the results of our study demonstrate that HSD3B2 is strongly up regulated in a fraction of prostate cancers that are characterized by increased AR signaling, adverse tumor phenotype and early biochemical recurrence., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Humans are exposed not only to single endocrine disruptors, but also to chemical mixtures that can adversely affect their reproductive health. Steroidogenesis in reproductive tissues is emerging as the key target of endocrine disruptor action. Here, we analyzed the effect of environmental chemical mixtures with estrogenic activity on steroidogenic processes in immature rat granulosa cells and whether the observed steroidogenic effects were mediated through estrogen receptors. Extracts from untreated wastewater were prepared by solid-phase extraction and silica gel fractionation. ER-CALUX assay showed that the polar fractions of wastewater exerted different levels of estrogenic activity. Exposure of immature granulosa cells to the polar fraction exerting 9 ng of 17β-estradiol equivalents per liter of water of estrogenic activity increased mRNA expression of the key enzymes of progesterone biosynthetic pathway Star and Hsd3b1, but did not alter the level of Cyp19a1 and Lhr. Addition of estrogen receptor inhibitor ICI 182 780 prevented the estrogenic mixture-induced increase in Hsd3b1, but not Star mRNA level in immature granulosa cells. These results indicate that the environmental chemical mixtures with estrogenic activity exert endocrine disrupting effects by augmenting the progesterone biosynthetic pathway in immature rat granulosa cells, which is an effect achieved in part through activation of the estrogen receptors., (Copyright © 2018 John Wiley & Sons, Ltd.)

Intratumoral androgen biosynthesis has been recognized as an essential factor of castration-resistant prostate cancer. The present study investigated the effects of curcumin on the inhibition of intracrine androgen synthesis in prostate cancer. Human prostate cancer cell lines, LNCaP and 22Rv1 cells were incubated with or without curcumin after which cell proliferation was measured at 0, 24, 48 and 72 hours, respectively. Prostate tissues from the transgenic adenocarcinoma of the mouse prostate (TRAMP) model were obtained after 1-month oral administration of 200 mg/kg/d curcumin. Testosterone and dihydrotestosterone concentrations in LNCaP prostate cancer cells were determined through LC-MS/MS assay. Curcumin inhibited cell proliferation and induced apoptosis of prostate cancer cells in a dose-dependent manner. Curcumin decreased the expression of steroidogenic acute regulatory proteins, CYP11A1 and HSD3B2 in prostate cancer cell lines, supporting the decrease of testosterone production. After 1-month oral administration of curcumin, Aldo-Keto reductase 1C2 (AKR1C2) expression was elevated. Simultaneously, decreased testosterone levels in the prostate tissues were observed in the TRAMP mice. Meanwhile, curcumin treatments considerably increased the expression of AKR1C2 in prostate cancer cell lines, supporting the decrease of dihydrotestosterone. Taken together, these results suggest that curcumin's natural bioactive compounds could have potent anticancer properties due to suppression of androgen production, and this could have therapeutic effects on prostate cancer., (© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Patients with advanced prostate cancer who receive androgen deprivation therapy (ADT) almost invariably develop castration-resistant disease. The mechanism of resistance is largely based on synthesis of intratumoral androgens from adrenal precursors, requiring enzymatic action of 3β-hydroxysteroid dehydrogenase/Δ 5→4 isomerase 1 (3β-HSD1), encoded by HSD3B1. A nucleotide polymorphism (1245A>C) in HSD3B1 results in a protein variant with increased steady-state levels and subsequently increased androgen synthesis from extragonadal precursors. Multiple clinical studies have shown that patients with the variant allele have significantly worse outcomes after ADT than those without, indicating that HSD3B1 variant status is a predictive biomarker of shortened ADT response. In addition, inheritance of the HSD3B1 variant is associated with extended responses to 17α-hydroxylase/17,20-lyase (CYP17A1) inhibition with a nonsteroidal agent, adding to evidence of increased tumour dependence on extragonadal androgens in patients who inherited the HSD3B1 variant. However, steroidal drugs with a 3β-hydroxyl, Δ 5 -structure, such as abiraterone, are also metabolized by 3β-HSD1, and 5α-abiraterone, a downstream metabolite, has been shown to activate the androgen receptor, potentially driving cancer progression. These data indicate a potential requirement to modify the treatment framework of patients harbouring variant HSD3B1.

In the present study, we have tested the beneficial effects of forskolin in protecting the mancozeb-induced reproductive toxicity in rats. Adult male Wistar rats were exposed to either mancozeb (500 mg/kg body weight/day) or forskolin (5 mg/kg body weight/day) or both for 65 days and analyzed for spermatogenesis and steroidogenesis and testicular and epididymal oxidative toxicity. A significant decrease in daily sperm production, epididymal sperm count, motile, viable, and hypo-osmotic swelling-tail swelled sperm was observed in mancozeb-treated rats. The activity levels of testicular 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase and circulatory testosterone levels were significantly decreased in mancozeb-treated rats. Exposure to mancozeb resulted in a significant decrease in glutathione levels and superoxide dismutase and catalase activity levels with an increase in lipid peroxidation levels in the testes and epididymis. Coadministration of forskolin mitigated the mancozeb-induced oxidative toxicity and suppressed steroidogenesis and spermatogenesis., (© 2017 Wiley Periodicals, Inc.)

The participation of aberrant receptors and intra-adrenal ACTH in hyperplastic tissue are considered mechanisms that regulate hypercortisolism in PMAH. Additionally, germline ARMC5 mutations have been described as the most frequent genetic abnormality found in patients diagnosed with PMAH. Previous functional studies analyzed ARMC5 role using H295R cells. Therefore, we investigated the role of ARMC5 in cell cultures obtained from PMAH nodules containing steroidogenic cells, aberrant receptors and intra-adrenal ACTH. ARMC5 silencing in non-mutated PMAH cell cultures decreased steroidogenesis-related genes and increased CCNE1 mRNA expression and proliferative capacity without affecting cell viability. Additionally, ARMC5 overexpression induced cell death in PMAH mutated cell cultures, thereby decreasing cell viability. We confirmed the role of ARMC5 as an important pro-apoptotic protein involved in PMAH-related steroidogenesis. We also report for the first time the involvement of ARMC5 in controlling proliferation and regulating cell cycle in PMAH cell cultures; these effects need to be explored further., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Steroid hormones regulate differentiation of various types of cell during embryogenesis. Testosterone is one of the androgens that bind to receptors to regulate gene expression and promote spermatogenesis. Our results showed that testosterone, as a product of steroid hormones synthesis pathway, could facilitate the differentiation of embryonic stem cells (ESCs) into spermatogonial stem cells (SSCs). The analysis of the steroid hormones synthesis pathway demonstrated that 3beta-hydroxysteroid dehydrogenase2 (Hsd3b2) plays a major role in the synthesis of testosterone. In the absence of Hsd3b2, the expression of downstream genes such as Cyp1a1, Ugt1a1, and Hsd17b7 was not maintained. This reduction is probably due to the down-regulation of the steroid hormones synthesis pathway. Furthermore, qRT-PCR, immunofluorescence, and flow cytometry analysis confirmed that the steroid hormones synthesis pathway could facilitate the differentiation of ESCs. Altogether, these results lead to a model in which Hsd3b2 regulates ESCs differentiation via modulating the activity of steroid hormones synthesis pathway., (© 2017 Wiley Periodicals, Inc.)

To clarify the molecular mechanism of ethylene glycol monomethyl ether (EGME)-induced testicular toxicity, the potential for EGME-related changes in transcript levels of genes including spermatocyte-specific genes was evaluated in the testis of rats given single dosing of EGME at 200, 600, or 2000 mg/kg. Furthermore, the contribution of decreased testicular testosterone on EGME-induced spermatocyte toxicity was investigated by comparing to transcriptional profile due to a testosterone synthesis inhibitor, ketoconazole (KET), at 30 or 300 mg/kg. EGME at 600 mg/kg or more dose-dependently caused testicular toxicity characterized by degeneration and necrosis of spermatocytes at stage VII-XIV seminiferous tubules. The spermatocyte injury was well correlated with decreased spermatocyte-specific gene expression. Analysis of upstream regulators by the Ingenuity Pathways Analysis system suggested that up-regulation of oxidative stress, protein kinase activation, and histone acetylation was involved in EGME-induced spermatocyte toxicity. Interestingly, KET decreased testicular testosterone to a similar extent compared to the EGME treatment, but KET at up to 300 mg/kg did not show any histopathological abnormality or change in the expression of spermatocyte-specific genes. These results suggested that the decreased testicular testosterone have little impact on EGME-induced spermatocyte injury. In contrast, KET showed trends toward increases in Hsd3b2 and Hsd17b2 mRNAs, presumably resulting from inhibition of androgen synthesis. Transcriptome analysis clearly demonstrated the differential effects of EGME and KET on androgen synthesis. In conclusion, EGME caused spermatocyte toxicity correlated with decreased expression of spermatocyte-specific genes. Furthermore, oxidative stress, protein kinase activation, and histone acetylation were suggested to be involved in EGME-induced testicular toxicity.

Background: Human 3β-hydroxysteroid dehydrogenase type 1 (HSD3B1) plays a vital role in steroidogenesis in breast tumors and may therefore be a suitable target for treatment of breast cancer. This study investigated the role of HSD3B1 in the pathogenesis of breast cancer in clinical and experimental settings., Methods: Expression of HSD3B1 in primary tumors of 258 breast cancer patients was evaluated by immunohistochemistry. Screening of breast cancer cell lines indicated that triple-negative MDA-MB-231 cells expressed HSD3B1. The effects from genetic and pharmacologic inhibition of HSD3B1 were assessed in vitro and in vivo., Results: The findings showed that 44% of the 258 breast cancers were HSD3B1-positive. The HSD3B1-positivity was associated with advanced-stage disease (p = 0.009) and reduced recurrence-free survival (p = 0.048) but not with tumor subtype or estrogen receptor status. Silencing of HSD3B1 or treatment with an HSD3B1 inhibitor (trilostane) reduced colony formation in breast cancer cells. Knockdown of HSD3B1 inhibited cell proliferation and migration. Analysis of a murine xenograft tumor model indicated that trilostane significantly slowed tumor growth., Conclusions: Expression of HSD3B1 in breast cancer is negatively associated with prognosis. The study found HSD3B1 to be a potential therapeutic target for breast cancer independent of estrogen receptor status.

The human 3β-hydroxysteroid dehydrogenase/isomerase (HSD3B) enzymes catalyze the conversion of 3β-hydroxy Δ5-6 steroids into 3-keto Δ4-5 steroids, which is required for the synthesis of the mature steroid hormones secreted by the adrenal and gonads. The human has 2 isozymes, the HSD3B1 that is traditionally located in placenta and extra-adrenal tissues and the HSD3B2 that is expressed in the adrenal and gonads. Mice with both cryptochrome 1 and 2 genes deletion were recently found to have salt-sensitive hypertension and hyperaldosteronism. These deletions were also associated with overexpression of the Hsd3b6 enzyme, the homolog of the human HSD3B1, in the zona glomerulosa which was believed to explain the hyperaldosteronism. A report using antibodies against human HSD3B1 suggested that it was expressed in the zona glomerulosa of normal human adrenals and in patients with idiopathic hyperaldosteronism and the HSD3B2 expressed in both the zona fasciculata and glomerulosa. We have developed specific monoclonal antibodies against the human HSD3B1 and HSD3B2 isozymes and found that the main enzyme expressed in the zona glomerulosa was the HSD3B2. Faint staining of the adrenal was also obtained using the anti-HSD3B1antibody only at high concentrations of antibody. This study fails to confirm that HSD3B1 expression in the human zona glomerulosa and double immunofluorescence clearly shows that the HSD3B2 is expressed in the zona glomerulosa and fasciculata and in the zona glomerulosa HSD3B2 is co-expressed with aldosterone synthase (CYP11B2)., (Published by Elsevier Inc.)

Equine fetuses have substantial circulating pregnenolone concentrations and thus have been postulated to provide significant substrate for placental 5α-reduced pregnane production, but the fetal site of pregnenolone synthesis remains unclear. The current studies investigated steroid concentrations in blood, adrenal glands, gonads and placenta from fetuses (4, 6, 9 and 10 months of gestational age (GA)), as well as tissue steroidogenic enzyme transcript levels. Pregnenolone and dehydroepiandrosterone (DHEA) were the most abundant steroids in fetal blood, pregnenolone was consistently higher but decreased progressively with GA. Tissue steroid concentrations generally paralleled those in serum with time. Adrenal and gonadal tissue pregnenolone concentrations were similar and 100-fold higher than those in allantochorion. DHEA was far higher in gonads than adrenals and progesterone was higher in adrenals than gonads. Androstenedione decreased with GA in adrenals but not in gonads. Transcript analysis generally supported these data. CYP17A1 was higher in fetal gonads than adrenals or allantochorion, and HSD3B1 was higher in fetal adrenals and allantochorion than gonads. CYP11A1 transcript was also significantly higher in adrenals and gonads than allantochorion and CYP19 and SRD5A1 transcripts were higher in allantochorion than either fetal adrenals or gonads. Given these data, and their much greater size, the fetal gonads are the source of DHEA and likely contribute more than fetal adrenal glands to circulating fetal pregnenolone concentrations. Low CYP11A1 but high HSD3B1 and SRD5A1 transcript abundance in allantochorion, and low tissue pregnenolone, suggests that endogenous placental pregnenolone synthesis is low and likely contributes little to equine placental 5α-reduced pregnane secretion., (© 2017 Society for Reproduction and Fertility.)

Testicular Leydig cells produce androgens essential for proper male reproductive development and fertility. Here, we describe a new Leydig cell ablation model based on Cre/Lox recombination of mouse Gata4 and Gata6 , two genes implicated in the transcriptional regulation of steroidogenesis. The testicular interstitium of adult Gata4 flox/flox ; Gata6 flox/flox mice was injected with adenoviral vectors encoding Cre + GFP (Ad-Cre-IRES-GFP) or GFP alone (Ad-GFP). The vectors efficiently and selectively transduced Leydig cells, as evidenced by GFP reporter expression. Three days after Ad-Cre-IRES-GFP injection, expression of androgen biosynthetic genes ( Hsd3b1 , Cyp17a1 and Hsd17b3 ) was reduced, whereas expression of another Leydig cell marker, Insl3 , was unchanged. Six days after Ad-Cre-IRES-GFP treatment, the testicular interstitium was devoid of Leydig cells, and there was a concomitant loss of all Leydig cell markers. Chromatin condensation, nuclear fragmentation, mitochondrial swelling, and other ultrastructural changes were evident in the degenerating Leydig cells. Liquid chromatography-tandem mass spectrometry demonstrated reduced levels of androstenedione and testosterone in testes from mice injected with Ad-Cre-IRES-GFP. Late effects of treatment included testicular atrophy, infertility and the accumulation of lymphoid cells in the testicular interstitium. We conclude that adenoviral-mediated gene delivery is an expeditious way to probe Leydig cell function in vivo Our findings reinforce the notion that GATA factors are key regulators of steroidogenesis and testicular somatic cell survival.Free Finnish abstract: A Finnish translation of this abstract is freely available at http://www.reproduction-online.org/content/154/4/455/suppl/DC2., (© 2017 Society for Reproduction and Fertility.)