Expression Of 3β-HSD Research Articles

Androgens Modulate Rat Granulosa Cell Steroidogenesis.

Paracrine interactions between ovarian theca-interstitial cells (TICs) and granulosa cells (GCs) play an important role in the regulation of follicular steroidogenesis. Androgens serve as substrates for aromatization as well as affect GC function. This study evaluated the effects of co-culture of GC with TICs and the role of testosterone (T) and 5-alpha-dihydrotestosterone (DHT), and estradiol (E2) in modulation of GC expression of genes involved in the production of progesterone: 3β-hydroxysteroid dehydrogenase/Δ5-4 isomerase (Hsd3b) and cholesterol side-chain cleavage (Cyp11). GCs obtained from immature Sprague-Dawley rats and were cultured in chemically defined media without or with TICs, DHT, or T. Hsd3b and Cyp11 transcripts were analyzed by qt-PCR. Co-culture of GCs with TICs stimulated Hsd3b and CYP11 expression in GCs. DHT and T induced a concentration-dependent upregulation of Hsd3b and CYP11 expression, as well as increased progesterone concentrations in spent media. E2 also increased expression of Hsd3b, and Cyp11. Effects of androgens were abrogated in the presence of an anti-androgen bicalutamide and the antiestrogen ICI 182780 (ICI). In conclusion, present findings demonstrate that androgens upregulate production of progesterone in GCs; these effects are likely due to a combination of direct action on androgen receptors and effects mediated by estrogen receptors.

Relationship between the expression of HSD3B2, SRD5A2 and androgen receptor in hypospadias patients

Testosterone is converted into dihydrotestosterone (DHT) by steroid-5-alpha-reductase. DHT induces the development of the external genitalia, both through the effect on the androgen receptor (AR) steroid-5-alpha-reductase type 2 (SRD5A2) and Hydroxy-delta-5-steroid dehydrogenase,3 beta- and steroid delta-isomerase 2 (HSD3B2) influenced testosterone production. The gene encoding SRD5A2 is particularly interesting because this enzyme is expressed during male genital development around the ventral part of the remodelling urethra and it converts testosterone to the more potent androgen DHT, which induces formation of the external genitalia. The clinical presentation of HSD3B2 deficiency is heterogeneous and may range from salt-losing congenital hyperplasia to premature pubarche, mild hyperandrogenism and the possibility of abnormalities in male genital development, such as hypospadias. From September 2017 – December 2018, we reviewed 80 patients, comprising 70 patients who underwent repair of hypospadias (46 proximal hypospadias (group 1) and 24 distal hypospadias (group 2)) without DSD, and 10 patients with no known anomaly who underwent circumcision (group 3), prospectively. We used periurethral dartos obtained during surgery. The expression of SRD5A2, HSD3B2 and AR was investigated by quantitative real time polymerase chain reaction (qRT-PCR). Median age was 5 years. The median of SRD5A2 was 22,68 (8,57-34,29) and mean of HSD3B2 and AR were 17,14 (2,12 ± 29,85) and 6,96 (1,62±13,92). We found a statistically significant difference between the expression of SRD5A2, HSD3B2 and AR in group 1 vs 3 and group 2 vs 3 but no difference in group 1 vs 2. We found positive correlation between SRD5A2 and HSD3B2 in hypospadias (r=0.38; p=0.001) but no correlation between both of them with androgen receptor. The correlation between SRD5A2 and HSD3B2 might influence the testosterone production but had no effect in androgen receptor.

Adrenomedullin Rescues Testicular Steroidogenesis of Leydig Cells by Suppressing TGF-β1 <i>via</i> PI3K/Akt-Dependent Activation of GSK-3β/β-Catenin Signaling Pathway

Backgroud: Lipopolysaccharide (LPS) exposure can induce Leydig cell dysfunction. This study aims to investigate whether adrenomedullin (ADM) rescues the steroidogenic functions of Leydig cells by suppressing TGF-β1 via PI3K/Akt/ GSK-3β/β-catenin signaling. Methods: Rats were given LPS and injected with an adeno-associated virus vector that expressed ADM (Ad-ADM). Plasma concentrations of testosterone and estradiol were measured. Testes were collected and analyzed by gene expression profiling, western blot and immunofluorescence staining. Primary Leydig cells were treated with various concentrations of LPS and ADM. Cell viability and proliferation and medium concentrations of testosterone and estradiol were detected. Gene expression and protein l evels were detected for steroidogenic enzymes, TGF-β1, PI3K, Akt, GSK-3β and β-catenin. Role of ADM in the regulation of TGF-β1 promoter was confirmed by ChIP and Co-IP. Finding: ADM reversed the decreased number of Leydig cells and plasma concentrations of testosterone and estradiol by rescuing the expression of SF-1, 17β-HSD, LRH1, CYP19, StAR, P450scc, 3β-HSD and CYP17. ADM inhibited the LPS-induced cytotoxicity and reversed the cell proliferation by rescuing the expression of Ki-67. ADM also rescued the expression of SF-1, 17β-HSD, LRH1, CYP19, StAR, P450scc, 3β-HSD and CYP17 and the medium concentrations of testosterone and estradiol in LPS-induced Leydig cells. ADM ameliorated LPS-induced TGF-β1 expression in LPS-exposed Leydig cells. ADM enhanced the phosphorylation of PI3K, Akt and GSK-3β but reduced the activation of β-catenin which interacted with TCF4 and then bound to TGF-β1 gene promoter. Interpretation: ADM may rescue the steroidogenic functions of Leydig cells by suppressing TGF-β1 via the PI3K/Akt/GSK-3β/β-catenin signaling. Funding Statement: This study was supported by the National Science Foundation of China, Beijing, China (Grant Nos.: 81402100, 81401190), Natural Science Foundation of Hunan Province, China (Grant Nos.: 2019JJ40269, 2020JJ4542), Foundation for “Young Medical Talents” of Jiangsu Province, China (Grant No.: QNRC2016840), and Key Guidance Project of University of South China, China (Grant No.: USCKF201902K01). Declaration of Interests: The authors declare no competing financial interests with regard to the publication of this paper. Ethics Approval Statement: Procedures involving rats were approved by the Local Ethics Committee for the Care and Use of Laboratory Animals of University of South China and were conducted in accordance with the national guidelines and protocols of the National Institutes of Health.

Acephate interferes with androgen synthesis in rat immature Leydig cells.

Acephate is an organophosphate pesticide. It is widely used. However, whether it inhibits androgen synthesis and metabolism remains unclear. In the current study, we investigated the effect of acephate on the inhibition of androgen synthetic and metabolic pathways in rat immature Leydig cells after 3-h culture. Acephate inhibited basal androgen output in a dose-dependent manner with the inhibition starting at 0.5μM. It significantly inhibited luteinizing hormone and 8-Br-cAMP stimulated androgen output at 50μM. It significantly inhibited progesterone-mediated androgen output at 50μM. Further study demonstrated that acephate down-regulated the expression of Hsd3b1 and its protein at ≥ 0.5μM, Lhcgr at 5μM and Star at 50μM. Acephate directly blocked rat testicular HSD3B1 activity at 50μM. Acephate did not affect other androgen synthetic and metabolic enzyme activities as well as ROS production, proliferation, and apoptosis of immature Leydig cells. In conclusion, acephate targets LHCGR, STAR, and HSD3B1, thus blocking androgen synthesis in rat immature Leydig cells and HSD3B1 is being the most sensitive target of acephate.

Long-term triphenyltin exposure disrupts adrenal function in adult male rats.

Triphenyltin is an organotin, which is widely used as a fungicide in agriculture. Here, we reported the effects of triphenyltin on adrenal function in adult male rats. Adult male Sprague Dawley rats were daily gavaged with triphenyltin (0, 0.5, 1, and 2 mg/kg body weight) from postnatal day 56-86. Triphenyltin significantly decreased serum corticosterone levels at 1 and 2 mg/kg without affecting serum levels of aldosterone and adrenocorticotropic hormone. Triphenyltin increased thickness of zona glomerulosa without affecting that of zona fasciculata. Triphenyltin did not affect cell number in zona fasciculata and zona glomerulosa. Triphenyltin down-regulated the expression of Scarb1, Star, Cyp11a1, Hsd3b1, Cyp21, Cyp11b1, and Hsd11b1 at 1 and/or 2 mg/kg while it up-regulated the expression of At1, Nr4a2, and Hsd11b2 at 2 mg/kg. Triphenyltin activated the phosphorylation of AMPKα while suppressed the phosphorylation of AKT1 and SIRT1/PGC-1α in rat adrenals invivo and H295R cells invitro. Invitro, triphenyltin also induced ROS production in H295R cells at 100 nM, a concentration at which no apoptosis was induced. In conclusion, triphenyltin disrupts glucocorticoid synthesis in rat adrenal cortex via several mechanisms: 1) lowering AKT1 phosphorylation and SIRT1/PGC-1α levels; 2) activating AMPKα; and 3) possibly inducing ROS production.

Anti-Müllerian hormone inhibits luteinizing hormone-induced androstenedione synthesis in porcine theca cells

AMH (Anti-Müllerian Hormone) is involved in the regulation of follicle growth initiation and inhibits FSH-induced aromatase expression and estrogen production in granulosa cells. However, the function of AMH in steroidogenesis by theca cells remains unclear. The aim of this study is to investigate the role of AMH as a regulator of the basal and stimulated steroid production by pig granulosa cells (pGCs) and theca cells (pTCs). PGCs and pTCs were incubated with hormones AMH, LH (luteinizing hormone), FSH (follicle stimulating hormone), individually or in combination. The expression of CYP19A1, HSD3B1, CYP11A1, LHCGR, and CYP17A1 mRNA were evaluated by quantitative reverse transcriptase PCR. In pGCs, 10 ng/mL AMH significantly decreased the FSH-stimulated effect on FSHR and CYP19A1 expression and estradiol production. In pTCs, LH treatment significantly increased the expression of HSD3B1, CYP11A1, LHCGR, and androstenedione or progesterone production (P < 0.05). Additionally, 10 ng/mL AMH also significantly decreased the LH-stimulated effects on the expression of HSD3B1, CYP11A1, CYP17A1, LHCGR and androstenedione production. Transfection with siAMHR2-I abolished the suppressive effects of AMH on LH-induced HSD3B1 expression and androstenedione production. Taken together, these results demonstrate that AMH is involved in FSH induced estradiol production in pGCs and LH induced androstenedione production in pTCs by regulating the steroidogenesis pathway.

Metabolism of cholesterol and progesterone is differentially regulated in primary trophoblastic subtypes and might be disturbed in recurrent miscarriages

During pregnancy, extravillous trophoblasts (EVTs) invade the maternal decidua and remodel the local vasculature to establish blood supply for the growing fetus. Compromised EVT function has been linked to aberrant pregnancy associated with maternal and fetal morbidity and mortality. However, metabolic features of this invasive trophoblast subtype are largely unknown. Using primary human trophoblasts isolated from first trimester placental tissues, we show that cellular cholesterol homeostasis is differentially regulated in EVTs compared with villous cytotrophoblasts. Utilizing RNA-sequencing, gene set-enrichment analysis, and functional validation, we provide evidence that EVTs display increased levels of free and esterified cholesterol. Accordingly, EVTs are characterized by increased expression of the HDL-receptor, scavenger receptor class B type I, and reduced expression of the LXR and its target genes. We further reveal that EVTs express elevated levels of hydroxy-delta-5-steroid dehydrogenase 3 beta- and steroid delta-isomerase 1 (HSD3B1) (a rate-limiting enzyme in progesterone synthesis) and are capable of secreting progesterone. Increasing cholesterol export by LXR activation reduced progesterone secretion in an ABCA1-dependent manner. Importantly, HSD3B1 expression was decreased in EVTs of idiopathic recurrent spontaneous abortions, pointing toward compromised progesterone metabolism in EVTs of early miscarriages. Here, we provide insights into the regulation of cholesterol and progesterone metabolism in trophoblastic subtypes and its putative relevance in human miscarriage.

The effect of orexin a on the StAR, CYP11A1 and HSD3B1 gene expression, as well as progesterone and androstenedione secretion in the porcine uterus during early pregnancy and the oestrous cycle

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 (P4) and androstenedione (A4) 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 P4 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 A4 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 P4 and A4 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.

Dimethoate blocks pubertal differentiation of Leydig cells in rats.

Dimethoate is an organophosphate pesticide. It is widely used in agriculture. However, whether it blocks pubertal development of Leydig cells remains unknown. In the current study, we exposed male Sprague Dawley rats with 7.5 and 15 mg kg-1 dimethoate from postnatal day 35-56. We also exposed Leydig cells isolated from 35-day-old rats for 3 h. Dimethoate reduced serum testosterone levels at 7.5 and 15 mg kg-1 but increased serum luteinizing hormone and follicle stimulating hormone levels at 15 mg kg-1. Dimethoate did not influence Leydig cell number but reduced Leydig cell size and down-regulated Star, Cyp11a1, and Hsd3b1 in Leydig cells as well as their protein expression. Dimethoate inhibited basal androgen output in a dose-dependent manner with the inhibition starting at 0.05 μM. It significantly inhibited luteinizing hormone and 8Br-cAMP stimulated androgen outputs at 50 μM. It significantly inhibited 22R-hydroxycholesterol and progesterone-mediated androgen outputs at 50 μM. Further study demonstrated that dimethoate also down-regulated the expression of Star, Cyp11a1, and Hsd3b1 at 5 or 50 μM invitro. Dimethoate did not directly inhibit rat testicular steroidogenic enzyme activities at 50 μM. In conclusion, dimethoate targets Star, Cyp11a1, and Hsd3b1 transcription, thus blocking Leydig cell differentiation during puberty.

Gonadal disruption after single dose exposure of prothioconazole and prothioconazole-desthio in male lizards (Eremias argus)

Prothioconazole (PTC) is a widely used triazole fungicide with low toxicity, and its desulfurization metabolite, prothioconazole-desthio (PTC-d), is reported to have higher reproductive toxicity to mammals. However, little is known about the reproductive toxicity, much less endocrine disrupting effect, of these two chemicals on reptiles. In this study, we investigated the effects of single dose of PTC/PTC-d (100 mg kg−1 body weight) exposure on the pathomorphism of testes and epididymides, serum sex steroid hormones (testosterone and 17β-estradiol) and transcription of steroidogenic-related genes (STARD, cyp11A, cyp17, cyp19A, 17β-HSD, 3β-HSD, AR and ER-α) in gonads of male lizards (Eremias argus). Although structural disorder existed in PTC-d exposure group, severe gonadal disruption, especially suppression of spermatogenesis was only observed in testis after PTC treatment, which consequently led to the lack of spermatozoa in epididymal ducts. Consistent with this result, T/E2 value in PTC exposure was elevated to a significant higher level compared with control and continually increased over time, while T/E2 value in the PTC-d exposure group slightly increased only at 12 h. These results demonstrated a more serious disruption of PTC on male lizard gonads than PTC-d. In addition, the expression of cyp17 gene was inhibited at 6 h, however, was induced at 12 h, and exhibited negative correlations with STARD, cyp11A and 3β-HSD after PTC exposure at each timepoint. In PTC-d group, the expression of STARD and 3β-HSD were significantly down-regulated, in contrast, cyp11A and cyp17 were up-regulated, and each gene showed consistent changes over time. For 17β-HSD, no significance was observed in both treated groups. This study was the first to compare the gonadal disruption of PTC and PTC-d in male lizards and elucidated that these two chemicals influenced the physiological function of male gonads through differential transcriptional modulation.

Aconitine inhibits androgen synthesis enzymes by rat immature Leydig cells via down-regulating androgen synthetic enzyme expression in vitro

Aconitine might have reproductive toxicity and the effects of aconitine on androgen synthesis in Leydig cells remain unclear. Here, we explore how aconitine affects androgen synthesis and metabolism in rat immature Leydig cells in vitro. Immature Leydig cells were isolated from 35-day-old male Sprague Dawley rats and cultured with 0–50 μM aconitine for 3 h in combination with LH, 8Br-cAMP, 22R-hydroxycholesterol, pregnenolone, progesterone, androstenedione, testosterone, and dihydrotestosterone, respectively. Medium androgens were measured. The levels of Leydig cell mRNAs, Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14, were measured by qPCR. ROS and apoptosis were determined after 24-h aconitine treatment. Aconitine inhibited basal androgen production in Leydig cells at 0.05 μM and the higher concentrations. Aconitine blocked pregnenolone, progesterone, and androstenedione mediated androgen outputs without affecting 22R-hydroxycholesterol-mediated androgen production at 5 μM. Aconitine also inhibited LH and 8Br-cAMP stimulated androgen outputs at 5 μM. Further investigation showed that aconitine blocked androgen synthesis via down-regulating the expression of Scarb1, Hsd3b1, Cyp17a1, and Hsd17b3. At 50 μM, aconitine also induced ROS generation and increased apoptotic rate of Leydig cells. Aconitine lowered serum testosterone levels at 1.5 mg/kg after 7 days of oral exposure from postnatal day 35. In conclusion, aconitine inhibits androgen synthesis.

Hypothyroidism Alters the Uterine Lipid Levels in Pregnant Rabbits and Affects the Fetal Size.

BackgroundHypothyroidism has been related to low-weight births, abortion and prematurity, which have been associated with changes in the content of glycogen and vascularization of the placenta. Since hypothyroidism can cause dyslipidemia, it may affect the lipid content in the uterus affecting the development of fetuses.ObjectiveTo investigate the effect of hypothyroidism on the lipid levels in serum and uterus during pregnancy and their possible association with the size of fetuses.MethodsAdult female rabbits were grouped in control (n = 6) and hypothyroid (n = 6; treated with methimazole for 29 days before and 19 days after copulation). Food intake and body weight were daily registered. At gestational day 19 (GD19), dams were sacrificed under an overdose of anesthesia. Morphometric measures of fetuses were taken. Total cholesterol (TC), triglyceride (TAG), and glucose concentrations were quantified in blood, uterus and ovaries of dams. The expression of uterine 3β-hydroxysteroid dehydrogenase (3β-HSD) was quantified by Western blot.ResultsHypothyroidism reduced food intake and body weight of dams, as well as promoted low abdominal diameters of fetuses. It did not induce dyslipidemia and hyperglycemia at GD19 and did not modify the content of lipids in the ovary. However, it reduced the content of TAG and TC in the uterus, which was associated with uterine hyperplasia and an increased expression of 3β-HSD in the uterus.ConclusionHypothyroidism alters the lipid content in the uterus that might subsequently affect the energy production and lipid signaling important to fetal development.

Expression and functional role of bone morphogenetic proteins (BMPs) in placenta during different stages of pregnancy in water buffalo (Bubalus bubalis)

The objective of this study was to document the expression and functional role of BMPs in the placental (caruncle; CAR, cotyledon; COT) during different stages of pregnancy in water buffalo. Samples collected from Early pregnancy 1 (EP1); Early pregnancy 2 (EP2), Mid pregnancy (MP), Late pregnancy (LP) while the third stage of oestrus cycle (NP) was taken as control. Also, the synergistic role of BMP4/BMP7 or combination on mRNA expression of vWF, PCNA, StAR, CYP11A1, 3βHSD, and BAX were studied in trophoblast cells cultured (TCC) during an early stage. The qPCR and immunoblotting studies revealed that BMP2, BMPR1A, BMPR1B, and BMPR2 mRNA level was significantly (p < 0.05) upregulated during early pregnancy in COTs while in CARs it was significantly upregulated (p < 0.05) during all the stages of pregnancy.BMP4 mRNA level was significantly upregulated (p < 0.05) during early pregnancy in COTs as well as in CARs. BMP6 expression was significantly upregulated (p < 0.05) during early and late stages of pregnancy. BMP7 mRNA level was upregulated (p < 0.05) during the late stage of pregnancy in COTs. At 100 ng/ml, the BMP4 maximally stimulated the transcripts of StAR, CYP11A1, and 3βHSD while BMP7 maximally stimulated the transcripts of 3βHSD that paralleled with P4 accretion in the media (P < 0.05). BMP4 as well as BMP7 upregulated the transcripts of PCNA, vWF, and downregulated BAX in the TCC (P < 0.05). In conclusion, BMPs are expressed in a regulated manner with stage-specific differences in the placenta and promotes the angiogenesis, proliferation, cell survivability, and steroidogenesis thereby regulating placental function in an autocrine/paracrine manner in water buffalo.

Primary human testicular PDGFRα+ cells are multipotent and can be differentiated into cells with Leydig cell characteristics in vitro.

STUDY QUESTIONIs it possible to differentiate primary human testicular platelet-derived growth factor receptor alpha positive (PDGFRα+) cells into functional Leydig cells?SUMMARY ANSWERAlthough human testicular PDGFRα+ cells are multipotent and are capable of differentiating into steroidogenic cells with Leydig cell characteristics, they are not able to produce testosterone after differentiation.WHAT IS KNOWN ALREADYIn rodents, stem Leydig cells (SLCs) that have been identified and isolated using the marker PDGFRα can give rise to adult testosterone-producing Leydig cells after appropriate differentiation in vitro. Although PDGFRα+ cells have also been identified in human testicular tissue, so far there is no evidence that these cells are true human SLCs that can differentiate into functional Leydig cells in vitro or in vivo.STUDY DESIGN, SIZE, DURATIONWe isolated testicular cells enriched for interstitial cells from frozen–thawed fragments of testicular tissue from four human donors. Depending on the obtained cell number, PDGFRα+-sorted cells of three to four donors were exposed to differentiation conditions in vitro to stimulate development into adipocytes, osteocytes, chondrocytes or into Leydig cells. We compared their cell characteristics with cells directly after sorting and cells in propagation conditions. To investigate their differentiation potential in vivo, PDGFRα+-sorted cells were transplanted in the testis of 12 luteinizing hormone receptor-knockout (LuRKO) mice of which 6 mice received immunosuppression treatment. An additional six mice did not receive cell transplantation and were used as a control.PARTICIPANTS/MATERIALS, SETTING, METHODSHuman testicular interstitial cells were cultured to Passage 3 and FACS sorted for HLA-A,B,C+/CD34−/PDGFRα+. We examined their mesenchymal stromal cell (MSC) membrane protein expression by FACS analyses. Furthermore, we investigated lineage-specific staining and gene expression after MSC trilineage differentiation. For the differentiation into Leydig cells, PDGFRα+-sorted cells were cultured in either proliferation or differentiation medium for 28 days, after which they were stimulated either with or without hCG, forskolin or dbcAMP for 24 h to examine the increase in gene expression of steroidogenic enzymes using qPCR. In addition, testosterone, androstenedione and progesterone levels were measured in the culture medium. We also transplanted human PDGFRα+-sorted testicular interstitial cells into the testis of LuRKO mice. Serum was collected at several time points after transplantation, and testosterone was measured. Twenty weeks after transplantation testes were collected for histological examination.MAIN RESULTS AND THE ROLE OF CHANCEFrom primary cultured human testicular interstitial cells at Passage 3, we could obtain a population of HLA-A,B,C+/CD34−/PDGFRα+ cells by FACS. The sorted cells showed characteristics of MSC and were able to differentiate into adipocytes, chondrocytes and osteocytes. Upon directed differentiation into Leydig cells in vitro, we observed a significant increase in the expression of HSD3B2 and INSL3. After 24 h stimulation with forskolin or dbcAMP, a significantly increased expression of STAR and CYP11A1 was observed. The cells already expressed HSD17B3 and CYP17A1 before differentiation but the expression of these genes were not significantly increased after differentiation and stimulation. Testosterone levels could not be detected in the medium in any of the stimulation conditions, but after stimulation with forskolin or dbcAMP, androstenedione and progesterone were detected in culture medium. After transplantation of the human cells into the testes of LuRKO mice, no significant increase in serum testosterone levels was found compared to the controls. Also, no human cells were identified in the interstitium of mice testes 20 weeks after transplantation.LARGE SCALE DATAN/ALIMITATIONS, REASONS FOR CAUTIONThis study was performed using tissue from only four donors because of limitations in donor material. Because of the need of sufficient cell numbers, we first propagated cells to passage 3 before FACS of the desired cell population was performed. We cannot rule out this propagation of the cells resulted in loss of stem cell properties.WIDER IMPLICATIONS OF THE FINDINGSA lot of information on Leydig cell development is obtained from rodent studies, while the knowledge on human Leydig cell development is very limited. Our study shows that human testicular interstitial PDGFRα+ cells have different characteristics compared to rodent testicular PDGFRα+ cells in gene expression levels of steroidogenic enzymes and potential to differentiate in adult Leydig cells under comparable culture conditions. This emphasizes the need for confirming results from rodent studies in the human situation to be able to translate this knowledge to the human conditions, to eventually contribute to improvements of testosterone replacement therapies or establishing alternative cell therapies in the future, potentially based on SLCs.STUDY FUNDING/COMPETING INTEREST(S)This study was funded by Amsterdam UMC, location AMC, Amsterdam, the Netherlands. All authors declare no competing interests.

Nitrophenols suppress steroidogenesis in prehierarchical chicken ovarian follicles by targeting STAR, HSD3B1, and CYP19A1 and downregulating LH and estrogen receptor expression

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.

Expression of GnRH receptor and 3βHSD during meiosis and foliculogénesis in Columba livia (Aves: Columbiformes): Histological and immunohistochemical analysis

During the ovarian ontogeny in birds, five fundamental events can be recognized: migration and colonization of the primordial germ cells, differentiation and proliferation of oogonies, an organization of germinal nests, beginning of the meiotic process and folliculogenesis. The knowledge of these events is fundamental for the interpretation of the processes involved in the differentiation of female gametes. However, there are only references for some model species such as Gallus gallus domesticus and Coturnix coturnix. In a previous study, the histological structure of embryonic ovaries of Columba livia was revealed. Therefore, the objective of this work is to characterize the processes of meiosis and folliculogenesis C. livia from the analysis of the expression of the GnRH receptor, the 3βHSD enzyme and the cell proliferation protein PCNA in embryonic and postnatal ovaries. Therefore, the expression of GnRHR, 3βHSD, and PCNA was revealed in histological testicular and ovarian preparations in embryos (stages 41–43) and neonates (2, 5, 7, 10 and 75 days post-hatching). The present study demonstrates that the fate of germline cells is dictated by their location during gonadal development. Thus, the germline cells located in the cortex of the left gonad enter meiosis, while those in the right gonad and those in the medulla of the left ovary fail to go into meiosis. This indicates that somatic signals, instead of an autonomous cellular mechanism, regulate the entry of the germline cells into meiosis in the C. livia embryo.Future studies will be focused on the analysis of proteins associated with meiotic events and folliculogenesis in embryonic and neonatal ovaries of C. livia, to evaluate the regulation of meiosis in vitro.

The Footprints of Oxidative Stress and Mitochondrial Impairment in Arsenic Trioxide-Induced Testosterone Release Suppression in Pubertal and Mature F1-Male Balb/c Mice via the Downregulation of 3β-HSD, 17β-HSD, and CYP11a Expression.

Exposure to arsenic (AS) causes abnormalities in the reproductive system; however, the precise cellular pathway of AS toxicity on steroidogenesis in developing F1-male mice has not been clearly defined. In this study, paternal mice were treated with arsenic trioxide (As2O3; 0, 0.2, 2, and 20ppm in drinking water) from 5weeks before mating until weaning and continued for male offspring from weaning until maturity (in vivo). Additionally, Leydig cells (LCs) were isolated from the testes of sacrificed F1-intact mature male mice and incubated with As2O3 (0, 1, 10, and 100μM) for 48h (in vitro). Biomarkers of mitochondrial impairment, oxidative stress, and several steroidogenic genes, including the steroidogenic acute regulatory (StAR) protein, cytochrome P450 side-chain cleaving enzyme (P450scc; Cyp11a), 3β-hydroxysteroid dehydrogenase (3β-HSD), and 17β-hydroxysteroid dehydrogenase (17β-HSD), were evaluated. High doses of As2O3 interrupted testosterone (T) biosynthesis and T-related gene expression in these experimental models. Altogether, overconsumption of As2O3 can cause testicular and LC toxicity through mitochondrial-related pathways and oxidative stress indices as well as downregulation of androgenic-related genes in mice and isolated LCs. These results could lead to the development of preventive/therapeutic procedures against As2O3-induced reproductive toxicity. Graphical Abstract Mohammad Mehdi Ommati and Reza Heidari contributed equally to this study.

MitoQ ameliorates testicular damage induced by gamma irradiation in rats: Modulation of mitochondrial apoptosis and steroidogenesis

AimsThe deleterious effect of gamma radiation on testicular tissue is a challenging problem in nuclear medicine. This study investigated the potential radioprotective effect of mitoquinol (MitoQ), a mitochondria-targeted antioxidant, against testicular damage induced by gamma irradiation in rats. Main methodsRats were allocated into four groups. The first group served as the control, the second group received MitoQ (2 mg / kg / day; i.p.) for seven days, the third group was exposed to gamma radiation (5 Gy as a single dose) and the last group received MitoQ prior to irradiation. Rats were sacrificed. Then, sperm analyses and the serum testosterone were determined. Moreover, evaluation of mitochondrial oxidative stress parameters (SOD, GSH and GPx) as well as apoptosis indicators (cytochrome-c, Bax, Bcl-2 and caspase-3) was performed. Additionally, analysis of steroidogensis biomarkers (StAR, 3β-HSD and 17β-HSD) and histopathological investigations were carried out. Key findingsMitoQ replenished mitochondrial SOD, GPx and GSH indicating its strong antioxidant effect in addition to its energy preservation manifested by the elevated ATP. MitoQ inhibited the intrinsic apoptosis via diminution of Bax, cytochrome-c and caspase-3 and alleviation of Bcl-2. This antioxidant conferred protection to steroidogenesis as verified by the increase in testosterone and the up-regulation of StAR, 3β-HSD and 17β-HSD expression; these effects might be correlated with its antioxidant/anti-apoptotic potential. Histopathological and sperm analyses corroborated the biochemical findings. SignificanceThis study identifies MitoQ as a novel agent for the management of testicular toxicity induced by gamma irradiation.

Features of the fetal gonad in androgen synthesis in the postpubertal testis are preserved in complete androgen insensitivity syndrome due to a novel genetic splice site donor variant in androgen receptor gene intron 1

Mutations in the X-linked androgen receptor (AR) gene cause complete androgen insensitivity syndrome (CAIS). CAIS may cause congenital sexual development disorder, which frequently develops into testicular tumors. Here, we describe a novel splice-site intron 1 mutation in AR leading to improper splicing and AR protein absence in CAIS gonads. We characterized a patient’s postpubertal gonadal steroidogenic enzyme expression profile. Localization of both CYP11A1 and CYP17A1 enzymes was restricted to both Leydig tumor cells and adjacent to tumor gonadal tissues. Sertoli cells of the CAIS gonad showed abundant HSD17B3 protein, which is an adult Leydig cell marker that enables the conversion of androstenedione to testosterone. Such HSD17B3 expression is typical for fetal-type Sertoli cells in rodents. The postpubertal CAIS gonad of our patient was completely devoid of androgen signaling pathway activity. Plausibly, the postpubertal Leydig cells consisted of two distinct cell populations: postpubertal fetal-type Leydig cells that persisted as androgen-independent cells and immature adult Leydig cells that failed to differentiate. Taken together, in this CAIS postpubertal testis, both Leydig and fetal-type Sertoli cells participated in testosterone production. Our results indicate the importance of molecular analysis as well as the characterization of steroidogenic enzyme profiling in the CAIS patient’s gonad.

Bioaccumulation and biochemical effects of ethylhexyl methoxy cinnamate and its main transformation products in zebrafish

The purpose of this study was to investigate the bioaccumulation and biochemical responses exposed to one of the main organic ultraviolet (UV) pollutants in the environment, ethylhexyl methoxy cinnamate (EHMC), and its main transformation product, either alone or in combination in zebrafish (Danio rerio). Four-month-old zebrafish were exposed to EHMC (34.4, 344 nmol/L) solution for 14 days, the species and contents of EHMC transformation products in zebrafish were determined and 3,5-dichloro-2-hydroxyacetophenone (3,5DCl2HAcP) was the one with the highest concentration in transformation products. Then, zebrafish were exposed to EHMC, 3,5DCl2HAcP alone and mixed solution for 21 days. At 7, 14 and 21 d, the related indexes of antioxidant defense system were determined. Results showed that both EHMC and 3,5DCl2HAcP can lead to the increase of malondialdehyde (MDA) and glutathione (GSH) contents, superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) activities in visceral mass compared with the corresponding control group, thus produced oxidative stress effect in organism and 3,5DCl2HAcP even showed stronger oxidative stress than EHMC. The effects of the two lower concentration co-exposure groups were similar and more significant to that of single exposure groups, while excessive oxidative stress occurred at the highest co-exposure group indicated by the decrease of GSH content, SOD, CAT, GR activities and the continued increase of MDA content. At 21 d, estradiol (E2), vitellogenin (Vtg) and testosterone (T) contents, estrogen receptor (Esr), progesterone receptor (Pgr), androgen receptor (Ar), Vtg1, P450 aromatase (Cyp19a1) and 17β-hydroxysteroid dehydrogenase (Hsd17b3) expression were all significantly increased when exposed to 3,5DCl2HAcP alone, showing complex estrogen and androgen effects. When exposed to EHMC alone, E2 and Vtg contents, Esr, Pgr, Vtg1, Cyp19a1 and Hsd17b1 gene expression levels decreased significantly, and T content and Ar and Hsd17b3 expression increased significantly, indicated that EHMC can produce anti-estrogen and androgen effect. Last, the decrease of estrogen effect and increase of androgen effect in co-exposure group suggested that 3,5DCl2HAcP might weaken the estrogen effect and promote the androgen effect of EHMC.