Expression and functional analysis of Fushi Tarazu transcription factor 1 (FTZ-F1) in the regulation of steroid hormones during the gonad development of Fujian Oyster, Crassostrea angulata.

Crassostrea angulata, a major shellfish cultivated in Southern China, has experienced a notable surge in commercial value in recent years. Understanding the molecular mechanisms governing their reproductive processes holds significant implications for advancing aquaculture practices. In this study, we cloned the orphan nuclear receptor gene, Fushi Tarazu transcription factor 1 (FTZ-F1), of C. angulata and investigated its functional role in the gonadal development. The full-length cDNA of FTZ-F1 spans 2357 bp and encodes a protein sequence of 530 amino acids. Notably, the amino acid sequence of FTZ-F1 in C. angulata shares remarkable similarity with its homologues in other species, particularly in the DNA-binding region (>90%) and ligand-binding region (>44%). In C. angulata, the highest expression level of FTZ-F1 was observed in the ovary, exhibiting more than a 200-fold increase during the maturation stage compared to the initiation stage (P < 0.001). Specifically, FTZ-F1 was mainly expressed in the follicular cells surrounding the oocytes of C. angulata. Upon inhibiting FTZ-F1 gene expression in C. angulata through RNA interference (RNAi), a substantial reduction in the expression of genes involved in the synthesis of sex steroids in the gonads, including 3ß-HSD, Cyp17, and follistatin, was observed. In addition, estradiol (E2) and testosterone (T) levels also showed a decrease upon FTZ-F1 silencing, resulting in a delayed gonadal development. These results indicate that FTZ-F1 acts as a steroidogenic factor, participating in the synthesis and regulation of steroid hormones and thus playing an important role in the reproductive and endocrine systems within oysters.

An intracellular VHH targeting the Luteinizing Hormone receptor modulates G protein-dependent signaling and steroidogenesis.

Luteinizing hormone (LH) is essential for reproduction, controlling ovulation and steroidogenesis. Its receptor (LHR) recruits various transducers leading to the activation of a complex signaling network. We recently identified iPRC1, the first variable fragment from heavy-chain-only antibody (VHH) interacting with intracellular loop 3 (ICL3) of the follicle-stimulating hormone receptor (FSHR). Because of the high sequence similarity of the human FSHR and LHR (LHCGR), here we examined the ability of the iPRC1 intra-VHH to modulate LHCGR activity. In this study, we demonstrated that iPRC1 binds LHCGR, to a greater extent when the receptor was stimulated by the hormone. In addition, it decreased LH-induced cAMP production, cAMP-responsive element-dependent transcription, progesterone and testosterone production. These impairments are not due to Gs nor ß-arrestin recruitment to the LHCGR. Consequently, iPRC1 is the first intra-VHH to bind and modulate LHCGR biological activity, including steroidogenesis. It should help further understand signaling mechanisms elicited at this receptor and their outcomes on reproduction.

Steroids-producing nodules: a two-layered adrenocortical nodular structure as a precursor lesion of cortisol-producing adenoma.

BACKGROUND: The human adrenal cortex consists of three functionally and structurally distinct layers; zona glomerulosa, zona fasciculata (zF), and zona reticularis (zR), and produces adrenal steroid hormones in a layer-specific manner; aldosterone, cortisol, and adrenal androgens, respectively. Cortisol-producing adenomas (CPAs) occur mostly as a result of somatic mutations associated with the protein kinase A pathway. However, how CPAs develop after adrenocortical cells acquire genetic mutations, remains poorly understood. METHODS: We conducted integrated approaches combining the detailed histopathologic studies with genetic, RNA-sequencing, and spatially resolved transcriptome (SRT) analyses for the adrenal cortices adjacent to human adrenocortical tumours. FINDINGS: Histopathological analysis revealed an adrenocortical nodular structure that exhibits the two-layered zF- and zR-like structure. The nodular structures harbour GNAS somatic mutations, known as a driver mutation of CPAs, and confer cell proliferative and autonomous steroidogenic capacities, which we termed steroids-producing nodules (SPNs). RNA-sequencing coupled with SRT analysis suggests that the expansion of the zF-like structure contributes to the formation of CPAs, whereas the zR-like structure is characterised by a macrophage-mediated immune response. INTERPRETATION: We postulate that CPAs arise from a precursor lesion, SPNs, where two distinct cell populations might contribute differently to adrenocortical tumorigenesis. Our data also provide clues to the molecular mechanisms underlying the layered structures of human adrenocortical tissues. FUNDING: KAKENHI, The Uehara Memorial Foundation, Daiwa Securities Health Foundation, Kaibara Morikazu Medical Science Promotion Foundation, Secom Science and Technology Foundation, ONO Medical Research Foundation, and Japan Foundation for Applied Enzymology.

Research progress in steroidal saponins from the genus Polygonatum: Chemical components, biosynthetic pathways and pharmacological effects.

The genus Polygonatum Mill. belongs to the Liliaceae family, which is widely distributed all over the world. Modern studies have found that Polygonatum plants are very rich in chemical compounds such as saponins, polysaccharides and flavonoids. Steroidal saponins are the most commonly studied saponins in the genus Polygonatum and a total of 156 compounds have been isolated from 10 species of the genus. These molecules possess antitumor, immunoregulatory, anti-inflammatory, antibacterial, antiviral, hypoglycemic, lipid-lowering and anti-osteoporotic activities. In this review, we summarize recent advances in studies of the chemical constituents of steroidal saponins from Polygonatum, including their structural characteristics, possible biosynthetic pathways and pharmacological effects. Then, the relationship between the structure and some physiological activities is considered. This review aims to provide reference for further exploitation and utilization of the genus Polygonatum.

Alterations in gonadotropin, prolactin, androgen and estrogen receptor and steroidogenesis-associated gene expression in gander testes in relation to the annual period.

Physiological mechanisms of seasonal changes in testicular function in birds are not fully elucidated. The balance between androgens and estrogens and testis sensitivity for gonadotropin and gonadal steroids are still unclear. The aim of the study was to examine: (1) the changes in circulating and intra-testicular steroid hormone levels and their relationship; (2) the mRNA expression of testicular gonadotropin, prolactin (PRL), progesterone (P4), androgen, and estrogen receptors, and (3) key steroidogenesis processes-related genes with immunofluorescent localization of aromatase in gander testes during the annual period. Testes from ganders (n = 25) in the first reproduction season were obtained at five breeding stages, i.e., prebreeding (PrB), peak of reproduction (PR), postbreeding (PoB), nonbreeding (NB), and onset of reproduction (OR). Males were kept under breeding conditions. It was found that plasma P4 levels decreased at the PoB and NB stages, whereas intra-testicular P4 was the highest in the NB stage. Intra-testicular estradiol (E2) levels were higher at the PoB and NB stages than the other stages, whereas testosterone (T) levels showed a nearly opposite pattern. The plasma estradiol-to-testosterone ratios were higher at the PrB, PoB and NB stages compared to other stages. The transcript abundances for luteinizing hormone receptor (LHR), PRL receptor (PRLR), estrogen receptor alpha (ERα), and estrogen receptor beta (ERß) also change in testicular tissue during the annual period. Moreover, StAR mRNA expression was upregulated at the PoB and NB stages, and CYP11A1 transcript level was the highest at the PoB stage. Stage-dependent changes in the CYP19A1 mRNA and aromatase protein levels with higher abundances of transcript at PoB and NB stages and protein at the NB stage were observed. Localization and immunofluorescent signal intensity for aromatase also differed in relation to the examined stages. It may be suggested that differential E2 levels, as well as aromatase expression and localization across annual stages are responsible for the seasonal activation/inactivation stages of testis spermatogenesis in domestic ganders. These data strongly suggest a role of aromatase in the control of gander steroidogenesis as changes in this enzyme level are associated with alternation in gonadal steroid hormones. In addition, joint action with others hormones, like PRL and LH, seems to be important in the final effect of seasonal reproduction potential.

Expression and localization of apelin and apelin receptor (APJ) in buffalo ovarian follicles and corpus luteum and the in-vitro effect of apelin on steroidogenesis and survival of granulosa cells.

Apelin is an adipose tissue-derived hormone with many physiological functions, including the regulation of female reproduction. It acts through an orphan G protein-coupled receptor APJ/APLNR. The present study aimed to investigate the expression of apelin and its receptor APJ in the ovarian follicles and corpus luteum (CL) and the role of apelin on steroidogenesis and cell survival. Ovarian follicles were classified into four groups based on size and estradiol (E2) level in the follicular fluid as follows: (i) F1 (4-6 mm; <0.5 ng/mL) (ii) F2 (7-9 mm; 0.5-5 ng/mL) (iii) F3 (10-13 mm; 5-40 ng/mL) and (iv) F4 (dominant/pre-ovulatory follicle) (>13 mm; >180 ng/mL). The corpora lutea (CL) were categorized into early (CL1), mid (CL2), late luteal (CL3), and regressing (CL4) CL stages. Expression of apelin increased with follicle size, with significantly greatest in the dominant or pre-ovulatory follicle (P < 0.05). Expression of APJ was greater in large and dominant follicles than in small and medium follicles (P < 0.05). In CL, the mRNA and protein abundance of apelin and apelin receptor was greater during mid (CL2) and late luteal (CL3) stages as compared to early (CL1) and regressing (CL4) stages (P < 0.05). Both the factors were localized in granulosa and theca cells of follicles and small and large luteal cells of CL. The pattern of the intensity of immunofluorescence was similar to mRNA and protein expression. Granulosa cells were cultured in vitro and treated at 1, 10, and 10 ng/mL apelin-13 either alone or in the presence of the follicle-stimulating hormone (FSH) (30 ng/mL) or insulin-like growth factor-I (IGF-I) (10 ng/mL) for 48 h. The luteal cells were treated with apelin-13 at 1, 10, and 100 ng/mL doses for 48 h. Apelin treatment at 10 and 100 ng/ml significantly (P < 0.05) increased E2 secretion, cytochrome P450 aromatase or CYP19A1 expression in GC. In luteal cells, apelin at 10 ng/mL and 100 ng/mL significantly (P < 0.05) increased progesterone (P4) secretion and HSD3B1 expression. In GCs, apelin, either alone or in combination, increased PCNA expression and inhibited CASPASE3 expression suggesting its role in cell survival. In conclusion, this study provides novel evidence for the presence of apelin and receptor APJ in ovarian follicles and corpora lutea and the stimulatory effect on E2 and P4 production and promotes GC survival in buffalo, suggesting the role of apelin in follicular and luteal functions in buffalo.

Molecular characterization of TRIB1 gene and its role in regulation of steroidogenesis in bos grunniens granulosa cells.

To explore the expression pattern of the TRIB1 gene in yak follicles and its effect on the steroidogenesis of granulosa cells (GCs). Here, 4-5 years old female yaks were treated as the subjects. Immunohistochemically assay found that TRIB1 protein was expressed in different developmental follicles. Among different cell types of follicles, including cumulus cells (CCs), granulosa cells (GCs) and theca cells (TCs), the TRIB1 protein was most abundant in GCs (P < 0.0001). In addition, we cloned the coding sequence (CDS) of the yak TRIB1 gene, which is 1119 bp, encoding 372 amino acids (AA). The amino acid sequence homology of TRIB1 is >80% to those of other species, except for zebrafish. To further explore the function of TRIB1 in steroidogenesis, the pcDNA3.1(+)-TRIB1 eukaryotic expression vector was constructed and then transfected into GCs. The data showed that overexpression of TRIB1 significantly reduced the progesterone (P4) secretion of granulosa cells measured by ELISA assay (P < 0.05), but not Estradiol (E2) secretion. Consistently, TRIB1 gain-of-function downregulated the mRNA levels of steroidogenesis related genes steroidogenic acute regulatory protein (StAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) (P < 0.01), while cytochrome P450 family 17 subfamily A member 1 (CYP17A1) and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) had no significant difference (P > 0.05). Interestingly, mito-tracker staining showed that mitochondrial number significantly decreased in TRIB1 overexpressed GCs (P < 0.01). Further, overexpression of TRIB1 inhibited mRNA levels of mitochondrial biogenesis related genes, including Mitochondrial transcription factor (TFAM) and Peroxisome proliferator-activated receptor alpha co-activator (PPARGC1A) (P < 0.05). Conclusively, this work indicates that TRIB1 inhibited progesterone synthesis of GCs might be involved in the reduction of the mitochondria number.

Identification of novel lipid metabolic biomarkers associated with poor adrenocortical carcinoma prognosis using integrated bioinformatics.

BACKGROUND: Adrenocortical carcinoma while rare, often presents with advanced metastatic disease carrying a 5-year survival of <15%. Despite adrenocortical carcinoma tumors having high avidity for cholesterol, the role of lipids in adrenocortical carcinoma has not been well described. Therefore, we performed an integrated bioinformatic analysis to identify novel lipid biomarkers correlating with poor survival that may help identify adrenocortical carcinoma tumor progression or therapy resistance. METHODS: A meta-analysis of collated adrenocortical carcinoma studies from the correlation engine identified lipid metabolism genes differentially expressed between adrenocortical carcinoma and the normal adrenal, which were then selected for enrichment analysis by the Database for Annotation, Visualization and Integrated Discovery database. A protein-protein interaction network of genes was constructed using Search Tool for the Retrieval of Interacting Genes/Proteins and Cytoscape. Top hub genes identified were validated using the Xena database. Survival analysis of hub genes was performed in the R2 genomic analysis platform using The Cancer Genome Atlas program data set. RESULTS: Examination of pathways by correlation engine identified a unique subset of lipid metabolism-related genes that are differentially regulated in adrenocortical carcinoma tumors versus normal tissues (P < .01). Enrichment pathway analysis in Database for Annotation, Visualization and Integrated Discovery indicated that genes involved in sphingolipid, steroid, and peroxisome proliferator-activated receptor-α metabolism is upregulated in adrenocortical carcinoma, whereas glycerol phospholipid, fatty acid, and phosphatidylinositol metabolism are downregulated. Survival analysis of differentially regulated genes indicated that upregulation of SGPL1, FDFT1, SQLE and downregulation of PIK3C2B, PIK3CD, SYNJ2, DGAT1, PLA2G16, PLD1, GPD1 are all significantly associated with poor overall survival (P < .05) in adrenocortical carcinoma patients. CONCLUSION: Upregulation of sphingolipid and steroid synthesis genes and downregulation of phosphatidylinositol and glycerol phospholipid metabolism are associated with worse survival in patients with adrenocortical carcinoma.

Insulin induces steroidogenesis in canine luteal cells via PI3K-MEK-MAPK.

Glucose uptake increases in canine luteal cells under insulin treatment. We hypothesize that insulin also increases luteal cell steroidogenesis. Dogs underwent elective ovariohysterectomy from days 10-60 post ovulation and their corpora lutea (CL) and blood samples were collected. Deep RNA sequencing determined differentially expressed genes in CL; those related to insulin signaling and steroidogenesis were validated in vivo by qPCR and their respective proteins by Western blotting and immunofluorescence. Next, luteal cell cultures were stimulated with insulin with or without inhibition of MAPK14, MAP2K1 and PI3K. Studied proteins except P450 aromatase showed the same expression pattern of coding genes in vivo. The expression of HSD3B and CYP19A1 was higher in insulin-treated cells (P < 0.005). Following respective pathway blockades, the culture medium had decreased concentrations of progesterone (P4) and 17b-estradiol (E2) (P < 0.01). Our results indicate that insulin increases HSD3B and CYP19A1 expression via MAPK and PI3K, and contributes to the regulation of P4 and E2 production in canine luteal cells.

Long-term in vitro exposure of human granulosa cells to the mixture of endocrine disrupting chemicals found in human follicular fluid disrupts steroidogenesis.

Most in vitro studies examine the effects of a single ED or a mixture of EDs on granulosa cells using short-term exposure; however, this approach is unlikely to reflect long-term, real-life exposures that are common in humans. We established an in vitro model that mimics long-term exposure of granulosa cells to real-life ED mixture. Human granulosa cells, HGrC1, were exposed to the mixture consisting of bisphenol A, polychlorinated biphenyl 153, benzo[a]pyrene, and perfluorooctanesulfonate in concentrations found in human follicular fluid (MIX) for 48 h and 4 weeks. Only long-term exposure to MIX decreased estradiol production after 2 and 3 weeks, and CYP19A1 protein after 2 weeks of exposure. By week 4, the cells restored estradiol production and CYP19A1 protein level. MIX increased basal progesterone production after 3 and 4 weeks of exposure but did not affect STAR and CYP11A1 mRNA. Cells that had been exposed to MIX for 4 weeks showed augmentation of forskolin-stimulated progesterone production. These results demonstrate that only long-term exposure to MIX alters steroidogenesis in HGrC1. This study also revealed that adverse effects of MIX on steroidogenesis in HGrC1 occurred a few weeks into MIX exposure and that this effect can be transient.

Mechanisms of MEHP Inhibitory Action and Analysis of Potential Replacement Plasticizers on Leydig Cell Steroidogenesis.

Steroid production in Leydig cells is stimulated mainly by the pituitary luteinizing hormone, which leads to increased expression of genes involved in steroidogenesis, including the gene encoding the steroidogenic acute regulatory (STAR) protein. Mono(2-ethylhexyl)phthalate (MEHP), the active metabolite of the widely used plasticizer DEHP, is known to disrupt Leydig steroidogenesis but its mechanisms of action remain poorly understood. We found that MEHP caused a significant reduction in hormone-induced steroid hormone production in two Leydig cell lines, MA-10 and MLTC-1. Consistent with disrupted cholesterol transport, we found that MEHP represses cAMP-induced Star promoter activity. MEHP responsiveness was mapped to the proximal Star promoter, which contains multiple binding sites for several transcription factors. In addition to STAR, we found that MEHP also reduced the levels of ferredoxin reductase, a protein essential for electron transport during steroidogenesis. Finally, we tested new plasticizers as alternatives to phthalates. Two plasticizers, dioctyl succinate and 1,6-hexanediol dibenzoate, had no significant effect on hormone-induced steroidogenesis. Our current findings reveal that MEHP represses steroidogenesis by affecting cholesterol transport and its conversion into pregnenolone. We also found that two novel molecules with desirable plasticizer properties have no impact on Leydig cell steroidogenesis and could be suitable phthalate replacements.

Management and Medical Therapy of Mild Hypercortisolism.

Mild hypercortisolism (mHC) is defined as an excessive cortisol secretion, without the classical manifestations of clinically overt Cushing's syndrome. This condition increases the risk of bone fragility, neuropsychological alterations, hypertension, diabetes, cardiovascular events and mortality. At variance with Cushing's syndrome, mHC is not rare, with it estimated to be present in up to 2% of individuals older than 60 years, with higher prevalence (up to 10%) in individuals with uncontrolled hypertension and/or diabetes or with unexplainable bone fragility. Measuring cortisol after a 1 mg overnight dexamethasone suppression test is the first-line test for searching for mHC, and the degree of cortisol suppression is associated with the presence of cortisol-related consequences and mortality. Among the additional tests used for diagnosing mHC in doubtful cases, the basal morning plasma adrenocorticotroph hormone, 24-h urinary free cortisol and/or late-night salivary cortisol could be measured, particularly in patients with possible cortisol-related complications, such as hypertension and diabetes. Surgery is considered as a possible therapeutic option in patients with munilateral adrenal incidentalomas and mHC since it improves diabetes and hypertension and reduces the fracture risk. In patients with mHC and bilateral adrenal adenomas, in whom surgery would lead to persistent hypocortisolism, and in patients refusing surgery or in whom surgery is not feasible, medical therapy is needed. Currently, promising though scarce data have been provided on the possible use of pituitary-directed agents, such as the multi-ligand somatostatin analog pasireotide or the dopamine agonist cabergoline for the-nowadays-rare patients with pituitary mHC. In the more frequently adrenal mHC, encouraging data are available for metyrapone, a steroidogenesis inhibitor acting mainly against the adrenal 11-ßhydroxylase, while data on osilodrostat and levoketoconazole, other new steroidogenesis inhibitors, are still needed in patients with mHC. Finally, on the basis of promising data with mifepristone, a non-selective glucocorticoid receptor antagonist, in patients with mild cortisol hypersecretion, a randomized placebo-controlled study is ongoing for assessing the efficacy and safety of relacorilant, a selective glucocorticoid receptor antagonist, for patients with mild adrenal hypercortisolism and diabetes mellitus/impaired glucose tolerance and/or uncontrolled systolic hypertension.

Mechanism of the Clinically Relevant E305G Mutation in Human P450 CYP17A1.

Steroid metabolism in humans originates from cholesterol and involves several enzyme reactions including dehydrogenation, hydroxylation, and carbon-carbon bond cleavage that occur at regio- and stereo-specific points in the four-membered ring structure. Cytochrome P450s occur at critical junctions that control the production of the male sex hormones (androgens), the female hormones (estrogens) as well as the mineralocorticoids and glucocorticoids. An important branch point in human androgen production is catalyzed by cytochrome P450 CYP17A1 and involves an initial Compound I-mediated hydroxylation at the 17-position of either progesterone (PROG) or pregnenolone (PREG) to form 17-hydroxy derivatives, 17OH-PROG and 17OH-PREG, with approximately similar efficiencies. Subsequent processing of the 17-hydroxy substrates involves a C17-C20 bond scission (lyase) activity that is heavily favored for 17OH-PREG in humans. The mechanism for this lyase reaction has been debated for several decades, some workers favoring a Compound I-mediated process, with others arguing that a ferric peroxo- is the active oxidant. Mutations in CYP17A1 can have profound clinical manifestations. For example, the replacement of the glutamic acid side with a glycine chain at position 305 in the CYP17A1 structure causes a clinically relevant steroidopathy; E305G CYP17A1 displays a dramatic decrease in the production of dehydroepiandrosterone from pregnenolone but surprisingly increases the activity of the enzyme toward the formation of androstenedione from progesterone. To better understand the functional consequences of this mutation, we self-assembled wild-type and the E305G mutant of CYP17A1 into nanodiscs and examined the detailed catalytic mechanism. We measured substrate binding, spin state conversion, and solvent isotope effects in the hydroxylation and lyase pathways for these substrates. Given that, following electron transfer, the ferric peroxo- species is the common intermediate for both mechanisms, we used resonance Raman spectroscopy to monitor the positioning of important hydrogen-bonding interactions of the 17-OH group with the heme-bound peroxide. We discovered that the E305G mutation changes the orientation of the lyase substrate in the active site, which alters a critical hydrogen bonding of the 17-alcohol to the iron-bound peroxide. The observed switch in substrate specificity of the enzyme is consistent with this result if the hydrogen bonding to the proximal peroxo oxygen is necessary for a proposed nucleophilic peroxoanion-mediated mechanism for CYP17A1 in carbon-carbon bond scission.

In Vitro Analysis of Deoxynivalenol Influence on Steroidogenesis in Prostate.

Deoxynivalenol (DON) is a type-B trichothecene mycotoxin produced by Fusarium species, reported to be the most common mycotoxin present in food and feed products. DON is known to affect the production of testosterone, follicle stimulating hormone (FSH) and luteinizing hormone (LH) in male rats, consequently affecting reproductive endpoints. Our previous study showed that DON induces oxidative stress in prostate cancer (PCa) cells, however the effect of DON on the intratumor steroidogenesis in PCa and normal prostate cells was not investigated. In this study human normal (PNT1A) and prostate cancer cell lines with different hormonal sensitivity (PC-3, DU-145, LNCaP) were exposed to DON treatment alone or in combination with dehydroepiandrosterone (DHEA) for 48 h. The results of the study demonstrated that exposure to DON alone or in combination with DHEA had a stimulatory effect on the release of estradiol and testosterone and also affected progesterone secretion. Moreover, significant changes were observed in the expression of genes related to steroidogenesis. Taken together, these results indicate that DON might affect the process of steroidogenesis in the prostate, demonstrating potential reproductive effects in humans.

MEHP induces alteration of mitochondrial function and inhibition of steroid biosynthesis in MA-10 mouse tumor Leydig cells.

Di-(2-ethylhexyl) phthalate (DEHP) is a plasticizer that is widely used in manufacturing. Previous studies have shown that mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of DEHP, has inhibitory effects on luteinizing hormone (LH)-stimulated steroid biosynthesis by Leydig cells. The molecular mechanisms underlying its effects, however, remain unclear. In the present study, we examined the effects of MEHP on changes in mitochondrial function in relationship to reduced progesterone formation by MA-10 mouse tumor Leydig cells. Treatment of MA-10 cells with MEHP (0-300 µM for 24 h) resulted in dose-dependent inhibition of LH-stimulated progesterone biosynthesis. Biochemical analysis data revealed that the levels of the mature steroidogenic acute regulatory protein (STAR), a protein that works at the outer mitochondrial membrane to facilitate the translocation of cholesterol for steroid formation, was significantly reduced in response to MEHP exposures. MEHP also caused reductions in MA-10 cell mitochondrial membrane potential (ΔΨm) and mitochondrial respiration as evidenced by decreases in the ability of the mitochondria to consume molecular oxygen. Additionally, significant increases in the generation of mitochondrial superoxide were observed. Taken together, these results indicate that MEHP inhibits steroid formation in MA-10 cells at least in part by its effects on mitochondrial function.

Contraceptive potential of Andrographis paniculata is via androgen suppression and not induction of oxidative stress in male Wistar rats.

Andrographis paniculata has been shown to be associated with male reproductive dysfunction, although the available data are scarce and inconsistent, and the associated mechanisms are elusive. Hormonal mechanism via hypothalamic-pituitary-testicular axis, and non-hormonal mechanism primarily through oxidative stress, are involved in the modulation of male reproductive function. We therefore, hypothesized that suppression of hypothalamic-pituitary-testicular axis and/or oxidative stress is involved in Andrographis paniculata-induced reproductive dysfunction. Male Wistar rats received either vehicle or Andrographis paniculata in varying doses of 250, 500, and 1000 mg/kg body weight daily for 8 weeks. Treatment with Andrographis paniculata led to reduced sperm count, motility, and viability. Andrographis paniculata treatment also resulted in distorted spermatogenesis and reduced serum testosterone. On the other hand, Andrographis paniculata led to reduction in the testicular content of malondialdehyde, nitric oxide, TNF-α, and IL-6, and testicular activities of xanthine oxidase and myeloperoxidase, but raised testicular levels of reduced glutathione content and enhanced activity of super oxide dismutase. However, body weight gain, and absolute and relative reproductive organ weights were similar across all the groups. These findings demonstrate that Andrographis paniculata induces reproductive toxicity via suppression of testosterone and not induction of oxidative stress. Therefore, Andrographis paniculata could be a potential and safe male contraceptive.

Increase in Akkermansiaceae in Gut Microbiota of Prostate Cancer-Bearing Mice.

Gut microbiota are reported to be associated with many diseases, including cancers. Several bacterial taxa have been shown to be associated with cancer development or response to treatment. However, longitudinal microbiota alterations during the development of cancers are relatively unexplored. To better understand how microbiota changes, we profiled the gut microbiota composition from prostate cancer-bearing mice and control mice at five different time points. Distinct gut microbiota differences were found between cancer-bearing mice and control mice. Akkermansiaceae was found to be significantly higher in the first three weeks in cancer-bearing mice, which implies its role in the early stage of cancer colonization. We also found that Bifidobacteriaceae and Enterococcaceae were more abundant in the second and last sampling week, respectively. The increments of Akkermansiaceae, Bifidobacteriaceae and Enterococcaceae were previously found to be associated with responses to immunotherapy, which suggests links between these bacteria families and cancers. Additionally, our function analysis showed that the bacterial taxa carrying steroid biosynthesis and butirosin and neomycin biosynthesis were increased, whereas those carrying naphthalene degradation decreased in cancer-bearing mice. Our work identified the bacteria taxa altered during prostate cancer progression and provided a resource of longitudinal microbiota profiles during cancer development in a mouse model.

Mangiferin mitigates di-(2-ethylhexyl) phthalate-induced testicular injury in rats by modulating oxidative stress-mediated signals, inflammatory cascades, apoptotic pathways, and steroidogenesis.

Di-(2-ethylhexyl) phthalate (DEHP) is an endocrine disruptor that causes reproductive defects in male animal models. This study was conducted to explore the plausible modulatory effects of mangiferin (MF) against DEHP-induced testicular injury in rats. Thirty-two adult male albino rats were allocated into four groups. Two groups were given DEHP (2 g/kg/day, p.o) for 14 days. One of these groups was treated with MF (20 mg/kg/day, i.p) for 7 days before and 14 days after DEHP administration. A vehicle-treated control was included, and another group of rats was given MF only. Results revealed that MF treatment suppressed oxidative testicular injury by amplifying the mRNA expression of nuclear factor-erythroid 2 related factor-2 (Nrf2) and increasing hemoxygenase-1 (HO-1), glutathione, and total antioxidant capacity (TAC) levels. This treatment also enhanced superoxide dismutase activity, but it decreased malondialdehyde and nitric oxide levels. MF had an anti-inflammatory characteristic, as demonstrated by the downregulation of the mRNA of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). The content of tumor necrosis factor-alpha also decreased. MF modulated the apoptotic pathway by suppressing the mRNA of cytochrome c (Cyt c), Fas ligand content, Bax IHC expression, caspase-3 activity and cleaved caspase-3 IHC expression. It also upregulated the expression levels of heat-shock protein 70 (HSP70) and B-cell lymphoma 2. Moreover, MF upregulated the mRNA expression levels of HSP70 and c-kit and enriched the content of steroidogenic acute regulatory (StAR) protein, which were reflected in serum testosterone levels. This result indicated that MF played crucial roles in steroidogenesis and spermatogenesis. Besides, the activities of testicular marker enzymes, namely, acid and alkaline phosphatases, and lactate dehydrogenase, significantly increased. Histopathological observations provided evidence supporting the biochemical and molecular measurements. In conclusion, MF provided protective mechanisms against the DEHP-mediated deterioration of testicular functions partially through its antioxidant, anti-inflammatory, and anti-apoptotic properties. It also involved the restoration of steroidogenesis and spermatogenesis through the modulation of Nrf2/HO-1, NF-κB/Cyt c/HSP70, and c-Kit signaling cascades.

Comparative transcriptome analysis reveals potential testosterone function-related regulatory genes/pathways of Leydig cells in immature and mature buffalo (Bubalus bubalis) testes.

Leydig cells (LCs) are testosterone-generating endocrine cells that are located outside the seminiferous tubules in the testis, and testosterone is fundamental for retaining spermatogenesis and male fertility. In buffalo, adult Leydig cells (ALCs) are developed by immature Leydig cells (ILCs) in the postnatal testes. However, the genes/pathways associated to the regulation of testosterone secretion function during the development of postnatal LCs remains comprehensively unidentified. The present study comparatively analyzed the transcriptome profiles of ILC and ALC in buffalo with significant differences in testosterone secretion. Differentially expressed genes (DEGs) analysis identified 972 and 1,091 annotated genes that were significantly up- and down-regulated in buffalo ALC. Functional enrichment analysis showed that cAMP signaling being the most significantly enriched pathway, and testosterone synthesis and lipid transport-related genes/pathways were upregulated in ALC. Furthermore, gene set enrichment analysis (GSEA) shows that cAMP signaling and steroid hormone biosynthesis were activated in ALC, demonstrating that cAMP signaling may serve as a positive regulatory pathway in the maintenance of testosterone function during postnatal development of LCs. Protein-protein interaction (PPI) networks analysis highlighted that ADCY8, ADCY2, POMC, CHRM2, SST, PTGER3, SSTR2, SSTR1, NPY1R, and HTR1D as hub genes in the cAMP signaling pathway. In conclusion, this study identified key genes and pathways associated in the regulation of testosterone secretion function during the ILC-ALC transition in buffalo based on bioinformatics analysis, and these key genes might be deeply involved in cAMP generation to influencing testosterone levels in LCs. The results suggest that ALCs might increase testosterone levels by enhancing cAMP production than ILCs. Our data will enhance the understanding of developmental mechanism studies related to testosterone function and provide preliminary evidence for molecular mechanisms of LCs regulating spermatogenesis.

Flutamide treatment reveals a relationship between steroidogenic activity of Leydig cells and ultrastructure of their mitochondria.

Our present knowledge on interrelation between morphology/ultrastructure of mitochondria of the Leydig cell and its steroidogenic function is far from satisfactory and needs additional studies. Here, we analyzed the effects of blockade of androgen receptor, triggered by exposure to flutamide, on the expression of steroidogenic proteins (1) and ultrastructure of Leydig cells' constituents (2). We demonstrated that increase in the expression level of steroidogenic (StAR, CYP11A1, 3ß-HSD, and CYP19A1) proteins (and respective mRNAs) in rat testicular tissue as well as elevation of intratesticular sex steroid hormone (testosterone and estradiol) levels observed in treated animals correspond well to morphological alterations of the Leydig cell ultrastructure. Most importantly, up-regulation of steroidogenic proteins' expression apparently correlates with considerable multiplication of Leydig cell mitochondria and subsequent formation of local mitochondrial networks. Interestingly, we showed also that the above-mentioned processes were associated with elevated transcription of Drp1 and Mfn2 genes, encoding proteins implicated in mitochondrial dynamics. Collectively, our studies emphasize the importance of mitochondrial homeostasis to the steroidogenic function of Leydig cells.