P450 Side-chain Cleavage Enzyme Research Articles

Seasonal expressions of the translocator protein (18 kDa), voltage-dependent anion channel, and steroidogenic acute regulatory protein in the scent glands of muskrats (Ondatra zibethicus)

Steroidogenesis machinery involves the steroidogenic acute regulatory protein (StAR), which regulates cholesterol transfer within the mitochondria, and the transport of cholesterol via a channel composed of 18-kDa translocator protein (TSPO), the voltage-dependent anion channel (VDAC) plus some accessory proteins. In this study, we investigated the immunolocalizations and expressions of StAR, TSPO, VDAC and cytochrome P450 side chain cleavage enzyme (P450scc, CYP11A1) in the scent glands of muskrats (Ondatra zibethicus) during the breeding and non-breeding periods. StAR, TSPO, VDAC and CYP11A1 were immunolocalized in the scent glandular, interstitial and epithelial cells in both breeding and non-breeding seasons with stronger immunostaining in the breeding season. The mRNA expression levels of StAR, TSPO, VDAC and CYP11A1 were higher in the scent glands of the breeding season than those of the non-breeding season. The circulating follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T) as well as scent glandular T and dihydrotestosterone (DHT) concentrations were also significantly higher in the breeding season. Additionally, the transcriptomic study in the scent glands identified that differentially expressed genes might be related to the lipid metabolic process, integral component of membrane, and steroid hormone receptor activity and hormone activity using GO analysis. Further in vitro study verified that StAR, TSPO, VDAC and CYP11A1 expression levels increased significantly after human chorionic gonadotropin, hCG/FSH treatment compared with the control group. The KEGG pathway enriched by differentially expressed genes detected to be involved in endocrine system or amino acid metabolism. These findings suggested that the scent glands of the muskrats have ability to synthesize steroids de novo, and that the steroid hormones may have an important regulatory role in the scent glandular function via an autocrine/paracrine pathway.

Cholesterol side-chain cleavage enzyme deficiency caused by a novel homozygous variant in P450 side-chain cleavage enzyme gene (CYP11A1) in a 46,XX Korean girl

Cholesterol side-chain cleavage enzyme deficiency caused by a novel homozygous variant in P450 side-chain cleavage enzyme gene (<i>CYP11A1</i>) in a 46,XX Korean girl

The Over-Irradiation Metabolite Derivative, 24-Hydroxylumister-ol3, Reduces UV-Induced Damage in Skin.

The hormonal form of vitamin D3, 1,25(OH)2D3, reduces UV-induced DNA damage. UV exposure initiates pre-vitamin D3 production in the skin, and continued UV exposure photoisomerizes pre-vitamin D3 to produce "over-irradiation products" such as lumisterol3 (L3). Cytochrome P450 side-chain cleavage enzyme (CYP11A1) in skin catalyzes the conversion of L3 to produce three main derivatives: 24-hydroxy-L3 [24(OH)L3], 22-hydroxy-L3 [22(OH)L3], and 20,22-dihydroxy-L3 [20,22(OH)L3]. The current study investigated the photoprotective properties of the major over-irradiation metabolite, 24(OH)L3, in human primary keratinocytes and human skin explants. The results indicated that treatment immediately after UV with either 24(OH)L3 or 1,25(OH)2D3 reduced UV-induced cyclobutane pyrimidine dimers and oxidative DNA damage, with similar concentration response curves in keratinocytes, although in skin explants, 1,25(OH)2D3 was more potent. The reductions in DNA damage by both compounds were, at least in part, the result of increased DNA repair through increased energy availability via increased glycolysis, as well as increased DNA damage recognition proteins in the nucleotide excision repair pathway. Reductions in UV-induced DNA photolesions by either compound occurred in the presence of lower reactive oxygen species. The results indicated that under in vitro and ex vivo conditions, 24(OH)L3 provided photoprotection against UV damage similar to that of 1,25(OH)2D3.

Probiotic and prebiotic supplementation ameliorates chronic restraint stress-induced male reproductive dysfunction.

Restraint stress (RS) can induce male reproductive deficits by activating the hypothalamic-pituitary-adrenal (HPA) axis and causing oxidative stress. Previous studies have shown that probiotics can alleviate neurological and metabolic disorders induced by stress. However, the effects of probiotics on RS-induced reproductive deficits have not been fully elucidated. This study aimed to investigate whether Lactobacillus rhamnosus NCDC-610 (Probiotic-1) and Lactobacillus fermentum NCDC-400 (Probiotic-2) with prebiotic (fructooligosaccharides (FOS)) could prevent RS-induced reproductive deficits. C57BL6/J mice were subjected to RS for four hours daily before oral administration of probiotics (4 × 109 CFU per mice) either separately or concurrently with FOS. The results showed that oral administration of Probiotic-1 and Probiotic-2 protected against RS-induced sperm deficits, including sperm count, motility, morphology, and histopathology of testes, and improved intestinal health. Furthermore, Probiotic-1 and Probiotic-2 prevented RS-induced changes in testosterone levels by up-regulating the expressions of steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage enzyme (P450scc), and 17β-hydroxysteroid dehydrogenase (17βHSD) in the testes. Additionally, Probiotic-1 and Probiotic-2 increased the activities of catalase and superoxide dismutase and reduced the fold change of interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-alpha (TNF-α), indicating a protective effect against RS-induced oxidative stress. Oral administration of Probiotic-1 and Probiotic-2, either separately or concurrently with FOS (probiotic dose of 4 × 109 CFU per mice and prebiotic 5% w/v), prevented RS-induced activation of the HPA axis and improved male fertility. These findings suggest that L. rhamnosus NCDC-610 and L. fermentum NCDC-400 are safe and effective probiotics for mitigating stress-induced male reproductive deficits.

HB-EGF upregulates StAR expression and stimulates progesterone production through ERK1/2 signaling in human granulosa-lutein cells

BackgroundHeparin-binding epidermal growth factor-like growth factor (HB-EGF) belongs to the epidermal growth factor (EGF) family of growth factors. HB-EGF and its receptors, epidermal growth factor receptor (EGFR) and HER4, are expressed in the human corpus luteum. HB-EGF has been shown to regulate luteal function by preventing cell apoptosis. Steroidogenesis is the primary function of the human corpus luteum. Steroidogenic acute regulatory protein (StAR) plays a critical role in steroidogenesis. StAR expression and progesterone (P4) production in human granulosa-lutein (hGL) cells have been shown to be upregulated by a ligand of EGFR, amphiregulin. However, whether HB-EGF can achieve the same effects remains unknown.MethodsA steroidogenic human ovarian granulosa-like tumor cell line, KGN, and primary culture of hGL cells obtained from patients undergoing in vitro fertilization treatment were used as experimental models. The underlying molecular mechanisms mediating the effects of HB-EGF on StAR expression and P4 production were explored by a series of in vitro experiments.ResultsWestern blot showed that EGFR, HER2, and HER4 were expressed in both KGN and hGL cells. Treatment with HB-EGF for 24 h induced StAR expression but did not affect the expression of steroidogenesis-related enzymes, P450 side chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase, and aromatase. Using pharmacological inhibitors and a siRNA-mediated knockdown approach, we showed that EGFR, HER4, but not HER2, were required for HB-EGF-stimulated StAR expression and P4 production. In addition, HB-EGF-induced upregulations of StAR expression and P4 production were mediated by the activation of the ERK1/2 signaling pathway.ConclusionThis study increases the understanding of the physiological role of HB-EGF in human luteal functions.7G2SU9-j2App9A69cYCW26Video

Regulation of cortisol production during chronic social stress in rainbow trout

Chronic stress resulting from social interactions impacts the endocrine stress response in many vertebrates, including teleost fishes. Juvenile rainbow trout held in pairs form a dominance hierarchy with the subordinate individual exhibiting chronic elevation of plasma cortisol and an attenuated cortisol response to an additional acute stressor. The current study investigated the mechanisms underlying this apparent dichotomy in cortisol production at the level of the head kidney (adrenal homolog). Following four days of chronic social stress, subordinate rainbow trout exhibited elevated plasma cortisol levels that correlated with basal cortisol production by the head kidney in vitro. Subordinate trout had higher transcript abundances of steroidogenic acute regulatory protein and cytochrome p450 side chain cleavage enzyme, which facilitate key steps in steroidogenesis, as well as two paralogs of steroidogenic factor 1. Despite elevation of basal steroidogenesis, acute cortisol production in response to ACTH (in vivo and in vitro) was lower in subordinate trout. Transcript abundances of the ACTH receptor accessory proteins were elevated in subordinate fish, but head kidney cortisol production in response to a cAMP analogue was lower than in dominant fish. Together, the data suggest that the attenuated acute cortisol response of subordinate trout reflects limitations on cortisol production downstream of cAMP signalling in steroidogenic cells of the head kidney, despite the increased basal abundance of key components of the steroidogenic pathway.

The Intracellular Cholesterol Pool in Steroidogenic Cells Plays a Role in Autophagy, Basal Steroidogenesis and Mitochondrial Dynamics

In steroidogenesis, cholesterol‐ the precursor substrate molecule for all steroid hormones is central and the framework of steroidogenesis across steroidogenic cells is constructed around it— including its cellular uptake, storage in intracellular lipid droplets, mobilization upon steroidogenic stimulation, and finally, its transport to the mitochondria where cholesterol is converted to pregnenolone by the P450 side chain cleavage enzyme. Thus, cholesterol and the mitochondria are highly interconnected in all steroidogenic cells. Moreover, accruing evidence suggests that autophagy and mitochondrial dynamics are important cellular events in the regulation of trophic hormone‐induced cholesterol homeostasis and steroidogenesis. However, a potential role of cholesterol in the regulation of steroidogenic events and factors remain unexplored. We tested the hypothesis that cholesterol plays a role in the regulation of cell‐intrinsic factors and events involving steroidogenesis. We used an intermediary approach to investigate the role of cholesterol in the regulation of steroidogenesis in different steroidogenic cell types such as MA‐10 cells (a murine Leydig cell line), BeWo cells (placental cell line) producing different steroid hormones. Steroidogenic cells were cultured in a lipoprotein‐depleted medium that leads to the depletion of the intracellular cholesterol pool and lipid droplets. Furthermore, cholesterol deprivation induced steroidogenic events. To get a snapshot of cholesterol deprived (CD)‐induced changes in lipid droplets, autophagy, and mitochondrial dynamics at an ultrastructural level we analyzed steroidogenic cells using transmission electron microscopy. As expected, cholesterol deprivation resulted in the depletion or loss of intracellular lipid droplets and the cholesterol pool across steroidogenic cell lines. Moreover, a difference in autophagy‐related structural changes was observed between cells cultured under the normal and CD conditions. In general, the structural characteristics of autophagy were more apparent in the CD group when compared with the normal control group across different steroidogenic cell types. Moreover, a change in mitochondrial shape was observed between the cells from two groups. For instance, the elongated or tubular mitochondria showing signs of mitochondrial dynamics (fusion or fission) were more common in the CD experimental group. Importantly, an increase in lysosome size and numbers were consistently found in cells under CD conditions which inversely correlated with the depletion of lipid droplets and the cholesterol pool, suggesting a compensatory increase in cholesterol mobilization. Taken together, this data suggests that cholesterol indeed plays a role in the regulation of autophagy and mitochondrial dynamics across steroidogenic cells in a context‐dependent manner. Such effects of cholesterol deprivation on autophagy and mitochondrial dynamics were not observed in the non‐steroidogenic cells such as HepG2 (human hepatoma cells) and H4IIE (rat hepatoma cells), indicating that cholesterol insufficiency‐induced changes in steroidogenic cells are specific to steroidogenesis. Thus, we unraveled this previously unknown role of cholesterol in steroidogenesis beyond being a mere substrate for steroid hormones. The implications of our findings are broad and offer new insights into trophic hormone‐dependent and independent steroidogenesis during development, as well as in health and disease.

At the crossroads of fertility and metabolism: the importance of AMPK-dependent signaling in female infertility associated with hyperandrogenism.

What biological processes are linked to the signaling of the energy sensor 5'-AMP-activated protein kinase (AMPK) in mouse and human granulosa cells (GCs)? The lack of α1AMPK in GCs impacted cell cycle, adhesion, lipid metabolism and induced a hyperandrogenic response. AMPK is expressed in the ovarian follicle, and its activation by pharmacological medications, such as metformin, inhibits the production of steroids. Polycystic ovary syndrome (PCOS) is responsible for infertility in approximately 5-20% of women of childbearing age and possible treatments include reducing body weight, improving lifestyle and the administration of a combination of drugs to improve insulin resistance, such as metformin. AMPK signaling was evaluated by analyzing differential gene expression in immortalized human granulosa cells (KGNs) with and without silencing α1AMPK using CRISPR/Cas9. In vivo studies included the use of a α1AMPK knock-out mouse model to evaluate the role of α1AMPK in folliculogenesis and fertility. Expression of α1AMPK was evaluated in primary human granulosa-luteal cells retrieved from women undergoing IVF with and without a lean PCOS phenotype (i.e. BMI: 18-25 kg/m2). α1AMPK was disrupted in KGN cells and a transgenic mouse model. Cell viability, proliferation and metabolism were evaluated. Androgen production was evaluated by analyzing protein levels of relevant enzymes in the steroid pathway by western blots, and steroid levels obtained from in vitro and in vivo models by mass spectrometry. Differential gene expression in human GC was obtained by RNA sequencing. Analysis of in vivo murine folliculogenesis was performed by histology and immunochemistry, including evaluation of the anti-Müllerian hormone (AMH) marker. The α1AMPK gene expression was evaluated by quantitative RT-PCR in primary GCs obtained from women with the lean PCOS phenotype (n = 8) and without PCOS (n = 9). Silencing of α1AMPK in KGN increased cell proliferation (P < 0.05 versus control, n = 4), promoted the use of fatty acids over glucose, and induced a hyperandrogenic response resulting from upregulation of two of the enzymes involved in steroid production, namely 3β-hydroxysteroid dehydrogenase (3βHSD) and P450 side-chain cleavage enzyme (P450scc) (P < 0.05, n = 3). Female mice deficient in α1AMPK had a 30% decrease in their ovulation rate (P < 0.05, n = 7) and litter size, a hyperandrogenic response (P < 0.05, n = 7) with higher levels of 3βHSD and p450scc levels in the ovaries, and an increase in the population of antral follicles (P < 0.01, n = 10) compared to controls. Primary GCs from lean women with PCOS had lower α1AMPK mRNA expression levels than the control group (P < 0.05, n = 8-9). The FastQ files and metadata were submitted to the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB46048. The human KGN is a not fully differentiated, transformed cell line. As such, to confirm the role of AMPK in GC and the PCOS phenotype, this model was compared to two others: an α1AMPK transgenic mouse model and primary differentiated granulosa-lutein cells from non-obese women undergoing IVF (with and without PCOS). A clear limitation is the small number of patients with PCOS utilized in this study and that the collection of human GCs was performed after hormonal stimulation. Our results reveal that AMPK is directly involved in steroid production in human GCs. In addition, AMPK signaling was associated with other processes frequently reported as dysfunctional in PCOS models, such as cell adhesion, lipid metabolism and inflammation. Silencing of α1AMPK in KGN promoted folliculogenesis, with increases in AMH. Evaluating the expression of the α1AMPK subunit could be considered as a marker of interest in infertility cases related to hormonal imbalances and metabolic disorders, including PCOS. This study was financially supported by the Institut National de la Recherche Agronomique (INRA) and the national programme « FERTiNERGY » funded by the French National Research Agency (ANR). The authors report no intellectual or financial conflicts of interest related to this work. R.K. is identified as personnel of the International Agency for Research on Cancer/World Health Organization. R.K. alone is responsible for the views expressed in this article and she does not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer/World Health Organization. N/A.

Growth differentiation factor-11 downregulates steroidogenic acute regulatory protein expression through ALK5-mediated SMAD3 signaling pathway in human granulosa-lutein cells

BackgroundGrowth differentiation factor-11 (GDF-11) belongs to the transforming growth factor-β (TGF-β) superfamily. To date, the expression of GDF-11 in the ovary and its role in regulating ovarian function are completely unknown. Ovarian granulosa cell-mediated steroidogenesis plays a pivotal role in maintaining normal female reproductive function. GDF-11 and GDF-8 share high sequence similarity and exhibit many similar features and functions. Steroidogenic acute regulatory protein (StAR) regulates the rate-limiting step in steroidogenesis and its expression can be downregulated by GDF-8. Polycystic ovary syndrome (PCOS) is the most common cause of female infertility. The expression levels of GDF-8 are upregulated in the human follicular fluid and granulosa-lutein (hGL) cells of PCOS patients. However, whether similar results can be observed for the GDF-11 needs to be determined.MethodsThe effect of GDF-11 on StAR expression and the underlying molecular mechanisms were explored by a series of in vitro experiments in a primary culture of hGL cells obtained from patients undergoing in vitro fertilization (IVF) treatment. Human follicular fluid samples were obtained from 36 non-PCOS patients and 36 PCOS patients. GDF-11 levels in follicular fluid were measured by ELISA.ResultsGDF-11 downregulates StAR expression, whereas the expression levels of the P450 side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD) are not affected by GDF-11 in hGL cells. Using pharmacological inhibitors and a siRNA-mediated approach, we reveal that ALK5 but not ALK4 mediates the suppressive effect of GDF-11 on StAR expression. Although GDF-11 activates both SMAD2 and SMAD3 signaling pathways, only SMAD3 is involved in the GDF-11-induced downregulation of StAR expression. In addition, we show that SMAD1/5/8, ERK1/2, and PI3K/AKT signaling pathways are not activated by GDF-11 in hGL cells. RT-qPCR and ELISA detect GDF-11 mRNA expression in hGL cells and GDF-11 protein expression in human follicular fluid, respectively. Interestingly, unlike GDF-8, the expression levels of GDF-11 are not varied in hGL cells and follicular fluid between non-PCOS and PCOS patients.ConclusionsThis study increases the understanding of the biological function of GDF-11 and provides important insights into the regulation of ovarian steroidogenesis.

Widespread alterations to hypothalamic-pituitary-gonadal (HPG) axis signaling underlie high temperature reproductive inhibition in the eurythermal sheepshead minnow (Cyprinodon variegatus)

Fish experiencing abnormally high or prolonged elevations in temperature can exhibit impaired reproduction, even for species adapted to warm water environments. Such high temperature inhibition of reproduction has been linked to diminished gonadal steroidogenesis, but the mechanisms whereby hypothalamic-pituitary-gonadal (HPG) axis signaling is impacted by high temperature are not fully understood. Here, we characterized differences in HPG status in adult sheepshead minnow (Cyprinodon variegatus), a eurythermal salt marsh and estuarine species of eastern North America, exposed for 14 d to temperatures of 27 °C or 37 °C. Males and females at 37 °C had lower gonadosomatic index (GSI) values compared to fish at 27 °C, and females at 37 °C had fewer spawning capable eggs and lower circulating 17β-estradiol (E2). Gene transcripts encoding gonadotropin-inhibitory hormone (gnih) and gonadotropin-releasing hormone-3 (gnrh3) were higher in relative abundance in the hypothalamus of both sexes at 37 °C. While pituitary mRNAs for the β-subunits of follicle-stimulating hormone (fshβ) and luteinizing hormone (lhβ) were lowered only in males at 37 °C, Fsh and Lh receptor mRNA levels in the gonads were at lower relative levels in both the ovary and testis of fish at 37 °C. Females at 37 °C also showed reduced ovarian mRNA levels for steroid acute regulatory protein (star), P450 side-chain cleavage enzyme (cyp11a1), 3β-hydroxysteroid dehydrogenase (3βhsd), 17β-hydroxysteroid dehydrogenase (hsd17β3), and ovarian aromatase (cyp19a1a). Females at the higher 37 °C temperature also had a lower liver expression of mRNAs encoding estrogen receptor α (esr1) and several vitellogenin and choriogenin genes, but elevated mRNA levels for hepatic sex hormone-binding globulin (shbg). Our results substantiate prior findings that exposure of fish to high temperature can inhibit gonadal steroidogenesis and oogenesis, and point to declines in reproductive performance emerging from alterations at several levels of HPG axis signaling including increased hypothalamic Gnih expression, depressed gonadal steroidogenesis, and reduced egg yolk and egg envelope protein production in the liver.

Epigallocatechin-3-gallate stimulates StAR expression and progesterone production in human granulosa cells through the 67-kDa laminin receptor-mediated CREB signaling pathway.

Epigallocatechin-3-gallate (EGCG) is the most abundant and biologically active catechins extracted from green tea. The health benefits of EGCG have been extendedly studied. Ovarian steroidogenesis plays a pivotal role in maintaining normal reproductive function. Granulosa cells in the ovary are essential for steroid hormone production. To date, the effect of EGCG on steroidogenesis in human granulosa cells remains unclear. In the present study, we examine the physiological concentrations of EGCG on steroidogenesis in a steroidogenic human granulosa-like tumor cell line, KGN. Our results demonstrate that treatment with EGCG upregulates steroidogenic acute regulatory protein (StAR) expression and increases progesterone (P4) production. EGCG does not affect the expression levels of other steroidogenesis-related enzymes, such as P450 side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase, and aromatase. In addition, we identify the expression of 67-kDa laminin receptor (67LR) in KGN cells. Moreover, EGCG-induced StAR expression and P4 production require the 67LR-mediated activation of the PKA-CREB signaling pathway. These results provide a better understanding of the function of EGCG on ovarian steroidogenesis, which may lead to the development of alternative therapeutic approaches for reproductive disorders.

BMP-9 downregulates StAR expression and progesterone production by activating both SMAD1/5/8 and SMAD2/3 signaling pathways in human granulosa-lutein cells obtained from gonadotropins induced ovarian cycles

Bone morphogenetic proteins (BMPs) are expressed in different cell types of the human ovarian follicle and play important roles in the regulation of ovarian function. BMP-9, also known as growth differentiation factor-2 (GDF-2), belongs to the transforming growth factor-beta (TGF-β) superfamily. BMP-9 is mainly synthesized in the liver and secreted into the blood which allows it to regulate various physiological and pathological functions. To date, the expression of BMP-9 in the human ovary and its function in human granulosa cells remains unknown. In the present study, we detect the protein expression of BMP-9 in the human follicular fluid. Using the primary culture of human granulosa-lutein (hGL) cells obtained from patients undergoing in vitro fertilization as a cell model, we show that treatment with BMP-9 downregulates steroidogenic acute regulatory protein (StAR) expression and suppresses progesterone (P4) production. The expression levels of the P450 side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD) are not affected by BMP-9 treatment. Mechanistically, treatment of hGL cells with BMP-9 activates both SMAD1/5/8 and SMAD2/3 signaling pathways. Blocking the activations of SMAD1/5/8 and SMAD2/3 by pharmacological inhibitors or knockdown of SMAD4 attenuates the inhibitory effects of BMP-9 on StAR expression and P4 production. This study reveals a novel function of BMP-9 in the regulation of ovarian steroidogenesis.

Prohibitin-1 Transgenic Mice Revealed an Important Role of Prohibitin in Testicular Steroidogenesis

Testosterone, the male sex hormone, plays an important role in the sexual development and fertility. Consequently, its deficiency causes infertility, obesity, osteoporosis and cardiovascular diseases. Leydig cells (LCs) are the testicular interstitial cells responsible for the biosynthesis of testosterone in response to luteinizing hormone (LH) from the pituitary. Cholesterol is the essential substrate for steroidogenesis which is translocated from the cytosol to the mitochondria where it gets converted to pregnenolone (by P450 side chain cleavage enzyme). Subsequently, pregnenolone translocate to endoplasmic reticulum where action of various enzymes results in the biosynthesis of testosterone. Prohibitin-1 (PHB1) is an evolutionary conserved ubiquitously expressed protein with cell compartment and cell-type specific functions. Mitochondrial function of PHB1 has been widely studied but its role in testicular steroidogenesis is unexplored. Recently, we have reported two transgenic mice models of PHB1, PHB-Tg and mutant-PHB-Tg (mPHB-Tg), expressing PHB1 or Y114F (mutant PHB1) respectively under the control of Fabp-4 gene promoter. During phenotypic characterization of these mice models, we observed a drastic size/weight difference in the testis of PHB-Tg and mPHB-Tg when compared with wild type mice. The mPHB-Tg mice testis was significantly smaller than the PHB-Tg and wild type mice. Further analysis of mPHB-Tg testis revealed wider testicular interstitium with LC hyperplasia and elongated seminiferous tubules. Ultrastructure investigation revealed that LCs of mPHB-Tg mice have prominent nucleus with increased number of mitochondria and lipid droplets. In addition, electron microscopic images of mPHB-Tg mice LCs revealed a sign of lipophagy and mitophagy. This prompted us to measure testosterone levels in these mice; surprisingly mPHB-Tg mice showed significantly higher testosterone levels as compared to PHB-Tg and wild type mice. Furthermore, testicular lysates and primary LCs cell lysates from transgenic mice models revealed that overexpression of PHB/mPHB in LCs inversely effect expression levels of steroidogenic acute regulatory protein (StAR). Moreover, co-immunoprecipitation of PHB1 displayed an interaction with StAR, P450scc and LC3 further revealing a key role of PHB1 in cholesterol translocation, testicular steroidogenesis and autophagy. Taken together, this finding suggests that PHB1 plays a multifaceted role in testicular steroidogenesis from determining testis size to the translocation of cholesterol into the mitochondria, in maintaining lipid homeostasis and biosynthesis of testosterone. Implications of our findings are broad because cholesterol translocation to the mitochondria and its subsequent utilization for steroidogenesis is conserved in all steroidogenic tissues.

Amodiaquine promotes testosterone production and de novo synthesis of cholesterol and triglycerides in Leydig cells

Testosterone is a hormone essential for male reproductive function. It is produced primarily by Leydig cells in the testicle through activation of steroidogenic acute regulatory protein and a series of steroidogenic enzymes, including a cytochrome P450 side-chain cleavage enzyme (cytochome P450 family 11 subfamily A member 1), 17α-hydroxylase (cytochrome P450 family 17 subfamily A member 1), and 3β-hydroxysteroid dehydrogenase. These steroidogenic enzymes are mainly regulated at the transcriptional level, and their expression is increased by the nuclear receptor 4A1. However, the effect on Leydig cell function of a small molecule-activating ligand, amodiaquine (AQ), is unknown. We found that AQ effectively and significantly increased testosterone production in TM3 and primary Leydig cells through enhanced expression of steroidogenic acute regulatory protein, cytochome P450 family 11 subfamily A member 1, cytochrome P450 family 17 subfamily A member 1, and 3β-hydroxysteroid dehydrogenase. Concurrently, AQ dose-dependently increased the expression of 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme in the cholesterol synthesis pathway, through induction of the transcriptional and DNA-binding activities of nuclear receptor 4A1, contributing to increased cholesterol synthesis in Leydig cells. Furthermore, AQ increased the expression of fatty acid synthase and diacylglycerol acyltransferase and potentiated de novo synthesis of fatty acids and triglycerides (TGs). Lipidomics profiling further confirmed a significant elevation of intracellular lipid and TG levels by AQ in Leydig cells. These results demonstrated that AQ effectively promotes testosterone production and de novo synthesis of cholesterol and TG in Leydig cells, indicating that AQ may be beneficial for treating patients with Leydig cell dysfunction and subsequent testosterone deficiency.

Characterization of ovarian morphology and reproductive hormones in Zhedong white geese (Anser cygnoides domesticus) during the reproductive cycle.

Zhedong white goose (Anser cygnoides domesticus) is a native Chinese breed with strong broodiness and low egg production, which is related to the physiology of reproduction. However, thus far, the physiology of goose reproduction has not been well elucidated. In the present study, the ovarian morphology and reproductive hormones of Zhedong white geese were investigated during the reproductive cycle (the laying and brooding periods). The results showed that the surface of the ovary was atrophied and follicular atresia appeared to some extent in the brooding period compared with the laying period. The concentrations of follicle-stimulating hormone, progesterone and luteinizing hormone were significantly higher than those in the brooding period (p<0.05). In contrast, the concentrations of prolactin (PRL) and anti-Müllerian hormone (AMH) in the laying period were significantly lower than those in the brooding period (p<0.05). In addition, the mRNA expression levels of PRL, AMH, dopamine-β-hydroxylase (DβH) and cytochrome P450 side-chain cleavage enzyme (P450scc) were detected in the hypothalamus, pituitary and ovaries by using real-time polymerase chain reaction. The results showed that AMH mRNA was expressed specifically in ovary tissue. The expression levels of DβH and PRL in the brooding period was significantly higher than those in the laying period in the three tissues, especially in the early and middle stages of the brooding period. Moreover, AMH mRNA expression in the ovaries presented the same trend. In addition, P450scc mRNA was highly expressed in both the ovary and pituitary in the laying period. These results revealed the remarkable features of ovarian morphology and characterized the hormonal pattern and expression profile during the reproductive cycle, all of which contribute to understanding the differences in reproductive physiology between the laying and brooding periods in Zhedong white geese.

Newcastle disease virus induces testicular damage and disrupts steroidogenesis in specific pathogen free roosters

Newcastle disease (ND), which is caused by Newcastle disease virus (NDV), can cause heavy economic losses to the poultry industry worldwide. It is characterised by extensive pathologies of the digestive, respiratory, and nervous systems and can cause severe damage to the reproductive system of egg-laying hens. However, it is unknown whether NDV replicates in the male reproductive system of chickens and induces any pathologies. In this study, we selected a representative strain (i.e. ZJ1) of the most common genotype (i.e. VII) of NDV to investigate whether NDV can induce histological, hormonal, and inflammatory responses in the testes of specific pathogen free (SPF) roosters. NDV infection increased the expression of toll like receptor TLR3, TLR7, MDA5, IFN-α, IFN-β, IFN-γ, IL-8, and CXCLi1 in the testes of NDV-infected roosters at 5 days post-infection (dpi). Severe histological changes, including decrease in the number of Sertoli cells and individualized, shrunken spermatogonia with pyknotic nuclei, were observed at 3 dpi. At 5 dpi, the spermatogenic columns were disorganized, and there were fewer cells, which were replaced by necrotic cells, lipid vacuoles, and proteinaceous homogenous material. A significant decrease in the plasma concentrations of testosterone and luteinizing hormone (LH) and the mRNA expression of their receptors in the testes, steroidogenic acute regulatory protein, cytochrome P450 side-chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase in the NDV-infected group was observed relative to those in the control group (P < 0.05). Collectively, these results indicate that NDV infection induces a severe inflammatory response and histological changes, which decrease the steroidogenesis.

Prohibitin‐1 plays a multifaceted role in mediating luteinizing hormone‐induced steroidogenesis in Leydig cells.

Leydig cells (LCs) present in the interstitium between seminiferous tubules in the testis are responsible for the biosynthesis of testosterone from cholesterol in response to luteinizing hormone (LH) from the pituitary. The principal pathways of testosterone biosynthesis, the identities of the main steroidogenic enzymes, and genetic disorders of most of these enzymes have been elucidated. However, many important questions related to LH signaling and cholesterol transport remain unknown. For example, the mechanisms of StAR‐mediated cholesterol transport from the outer mitochondrial membrane to cytochrome P450 side chain cleavage (P450scc or Cyp11a1) enzyme in the inner mitochondrial membrane, which is a critical step in steroidogenesis, remain elusive. It is conceivable that we are missing some important players involved in this process.Prohibitin‐1 (PHB1) is a pleiotropic protein with cell compartment‐specific functions, including the phosphorylation‐dependent membrane signaling and mitochondrial chaperone, which appears to require heterodimerization with its homologous protein prohibitin‐2 (PHB2) in the inner mitochondrial membrane. Previously, we have reported transgenic mouse models expressing PHB1 (PHB‐Tg) or Y114F‐PHB1 (mPHB‐Tg) from the fatty acid binding protein‐4 (Fabp4) gene promoter. During their phenotypic characterization, unexpectedly, we found that male mPHB‐Tg mice, but not PHB‐Tg mice, have high testosterone levels. Subsequent analysis revealed that it was due to leaky overexpression of m/PHB1 in LCs. This prompted us to investigate whether LH regulates PHB1 in LCs. A dose and time‐dependent effect of human chorionic gonadotropin, hCG (pituitary analog of LH) was found on PHB1 expression levels in LCs. To define the cell compartment‐specific role of PHB1 in LCs, we investigated the functional status of LH signaling in testes/LCs and ultrastructure of LCs from transgenic mice. A difference in phospho‐ERK levels was found between PHB‐Tg and mPHB‐Tg, which was higher in mPHB1 expressing cells. A similar effect was found in PHB1 manipulated MA‐10 cells (a model LC line). A parallel change in ultrastructural features of LCs (mitochondrial structure and lipid droplets) was found in PHB and mPHB overexpressing cells. Moreover, co‐immunoprecipitation experiments showed that PHB1 and PHB2 interact with P450scc and/or StAR. Analysis of PHBs protein sequences revealed the presence of multiple CRAC and/or CARC motifs (cholesterol recognition motifs) in both proteins. In aggregate, this finding suggests that PHB1 is a LH regulated protein in LCs and plays a multifaceted role in LH‐induced steroidogenesis, including ERK activation and potentially in the functional coupling of StAR and P450scc across mitochondrial membrane for steroidogenesis. The scope of our findings is broad because the basics of cholesterol transport across mitochondrial membranes are similar in different steroidogenic tissues.

The P450 side-chain cleavage enzyme Cyp11a2 facilitates steroidogenesis in zebrafish.

Cytochrome P450 side-chain cleavage enzyme, encoded by the CYP11A1 gene, catalyzes the first and rate-limiting step of steroid hormone biosynthesis. Previous morpholino-knockdown studies in zebrafish suggested cyp11a2 is a functional equivalent of human CYP11A1 and is essential for interrenal steroidogenesis in zebrafish larvae. The role of Cyp11a2 in adult zebrafish, particularly in gonadal steroidogenesis, remains elusive. To explore the role of Cyp11a2 in adults, we developed zebrafish mutant lines by creating deletions in cyp11a2 using the CRISPR/Cas9 genomic engineering approach. Homozygous cyp11a2 mutant zebrafish larvae showed an upregulation of the hypothalamic-pituitary-interrenal axis. Furthermore, these Cyp11a2-deficient zebrafish demonstrated profound glucocorticoid and androgen deficiencies. Cyp11a2 homozygotes only developed into males with feminized secondary sex characteristics. Adult cyp11a2 -/- mutant fish showed a lack of natural breeding behaviors. Histological characterization revealed disorganized testicular structure and significantly decreased numbers of mature spermatozoa. These findings are further supported by the downregulation of the expression of several pro-male genes in the testes of cyp11a2 homozygous zebrafish, including sox9a, dmrt1 and amh. Moreover, the spermatogonia markers nanos2 and piwil1 were upregulated, while the spermatocytes marker sycp3 and spermatids marker odf3b were downregulated in the testes of cyp11a2 homozygous mutants. Our expression analysis is consistent with our histological studies, suggesting that spermatogonia are the predominant cell types in the testes of cyp11a2 homozygous mutants. Our work thus demonstrates the crucial role of Cyp11a2 in interrenal and gonadal steroidogenesis in zebrafish larvae and adults.

Long-term outcome of partial P450 side-chain cleavage enzyme deficiency in three brothers: the importance of early diagnosis

ObjectiveCYP11A1 mutations cause P450 side-chain cleavage (scc) deficiency, a rare form of congenital adrenal hyperplasia with a wide clinical spectrum. We detail the phenotype and evolution in a male sibship identified by HaloPlex targeted capture array.Family studyThe youngest of three brothers from a non-consanguineous Scottish family presented with hyperpigmentation at 3.7 years. Investigation showed grossly impaired glucocorticoid function with ACTH elevation, moderately impaired mineralocorticoid function, and normal external genitalia. The older brothers were found to be pigmented also, with glucocorticoid impairment but normal electrolytes. Linkage studies in 2002 showed that all three brothers had inherited the same critical regions of the maternal X chromosome suggesting an X-linked disorder, but analysis of NR0B1 (DAX-1, adrenal hypoplasia) and ABCD1 (adrenoleukodystrophy) were negative.In 2016, next-generation sequencing revealed compound heterozygosity for the rs6161 variant in CYP11A1 (c.940G>A, p.Glu314Lys), together with a severely disruptive frameshift mutation (c.790_802del, K264Lfs*5). The brothers were stable on hydrocortisone and fludrocortisone replacement, testicular volumes (15–20 mL), and serum testosterone levels (24.7, 33.3, and 27.2 nmol/L) were normal, but FSH (41.2 µ/L) was elevated in the proband. The latter had undergone left orchidectomy for suspected malignancy at the age of 25 years and was attending a fertility clinic for oligospermia. Initial histology was reported as showing nodular Leydig cell hyperplasia. However, histological review using CD56 staining confirmed testicular adrenal rest cell tumour (TART).ConclusionThis kinship with partial P450scc deficiency demonstrates the importance of precise diagnosis in primary adrenal insufficiency to ensure appropriate counselling and management, particularly of TART.

Inhibition of Cytochrome P450 Side-Chain Cleavage Attenuates the Development of Mechanical Allodynia by Reducing Spinal D-Serine Production in a Murine Model of Neuropathic Pain.

Research indicates that neurosteroids are locally synthesized in the central nervous system and play an important modulatory role in nociception. While the neurosteroidogenic enzyme, cytochrome P450 side-chain cleavage enzyme (P450scc), is the initiating enzyme of steroidogenesis, P450scc has not been examined under the pathophysiological conditions associated with peripheral neuropathy. Thus, we investigated whether chronic constriction injury (CCI) of the sciatic nerve increases the expression of P450scc in the spinal cord and whether this increase modulates serine racemase (Srr) expression and D-serine production contributing to the development of neuropathic pain. CCI increased the immunoreactivity of P450scc in astrocytes of the ipsilateral lumbar spinal cord dorsal horn. Intrathecal administration of the P450scc inhibitor, aminoglutethimide, during the induction phase of neuropathic pain (days 0 to 3 post-surgery) significantly suppressed the CCI-induced development of mechanical allodynia and thermal hyperalgesia, the increased expression of astrocyte Srr in both the total and cytosol levels, and the increases in D-serine immunoreactivity at day 3 post-surgery. By contrast, intrathecal administration of aminoglutethimide during the maintenance phase of pain (days 14 to 17 post-surgery) had no effect on the developed neuropathic pain nor the expression of spinal Srr and D-serine immunoreactivity at day 17 post-surgery. Intrathecal administration of exogenous D-serine during the induction phase of neuropathic pain (days 0 to 3 post-surgery) restored the development of mechanical allodynia, but not the thermal hyperalgesia, that were suppressed by aminoglutethimide administration. Collectively, these results demonstrate that spinal P450scc increases the expression of astrocyte Srr and D-serine production, ultimately contributing to the development of mechanical allodynia induced by peripheral nerve injury.