[Uncovering the molecular mechanisms behind steroidal saponin accumulation in Liriope muscari (Decne.) Baily through transcriptome sequencing and bioinformatics analysis].

The leaves and roots of Liriope muscari (Decne.) Baily were subjected to high-throughput Illumina transcriptome sequencing. Bioinformatics analysis was used to investigate the enzyme genes and key transcription factors involved in regulating the accumulation of steroidal saponins, which are the main active ingredient in L. muscari. These analyses aimed to reveal the molecular mechanism behind steroidal saponin accumulation. The sequencing results of L. muscari revealed 31 enzymes, including AACT, CAS, DXS and DXR, that are involved in the synthesis of steroidal saponins. Among these enzymes, 16 were in the synthesis of terpenoid skeleton, 3 were involved in the synthesis of sesquiterpene and triterpene, and 12 were involved in the synthesis of steroidal compound. Differential gene expression identified 15 metabolic enzymes coded by 34 differentially expressed genes (DEGs) in the leaves and roots, which were associated with steroidal saponin synthesis. Further analysis using gene co-expression patterns showed that 14 metabolic enzymes coded by 31 DEGs were co-expressed. In addition, analysis using gene co-expression analysis and PlantTFDB's transcription factor analysis tool predicted the involvement of 8 transcription factors, including GAI, PIF4, PIL6, ERF8, SVP, LHCA4, NF-YB3 and DOF2.4, in regulating 6 metabolic enzymes such as DXS, DXR, HMGR, DHCR7, DHCR24, and CAS. These eight transcription factors were predicted to play important roles in regulating steroidal saponin accumulation in L. muscari. Promoter analysis of these transcription factors indicated that their main regulatory mechanisms involve processes such as abscisic acid response, drought-induction stress response and light response, especially abscisic acid responsive elements (ABRE) response and MYB binding site involved in drought-inducibility (MBS) response pathway. Furthermore, qRT-PCR analysis of these eight key transcription factors demonstrated their specific differences in the leaves and roots.

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.

The Key Role of Peroxisomes in Follicular Growth, Oocyte Maturation, Ovulation, and Steroid Biosynthesis.

The absence of peroxisomes can cause disease in the human reproductive system, including the ovaries. The available peroxisomal gene-knockout female mouse models, which exhibit pathological changes in the ovary and reduced fertility, are listed in this review. Our review article provides the first systematic presentation of peroxisomal regulation and its possible functions in the ovary. Our immunofluorescence results reveal that peroxisomes are present in all cell types in the ovary; however, peroxisomes exhibit different numerical abundances and strong heterogeneity in their protein composition among distinct ovarian cell types. The peroxisomal compartment is strongly altered during follicular development and during oocyte maturation, which suggests that peroxisomes play protective roles in oocytes against oxidative stress and lipotoxicity during ovulation and in the survival of oocytes before conception. In addition, the peroxisomal compartment is involved in steroid synthesis, and peroxisomal dysfunction leads to disorder in the sexual hormone production process. However, an understanding of the cellular and molecular mechanisms underlying these physiological and pathological processes is lacking. To date, no effective treatment for peroxisome-related disease has been developed, and only supportive methods are available. Thus, further investigation is needed to resolve peroxisome deficiency in the ovary and eventually promote female fertility.

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.

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.

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.

Is there impact of the SARS-CoV-2 pandemic on steroidogenesis and fertility?

In December of 2019, several cases of unknown atypical respiratory diseases emerged in Wuhan, Hubei Province in China. After preliminary research, it was stated that the disease is transmittable between humans and was named COVID-19. Over the course of next months, it spread all over the world by air and sea transport and caused a global pandemic which affects life of everyone now-a-days. A large number of countries, have since been forced to take precautions such as curfews, lockdowns, wearing facemasks etc. Even with vaccines being produced in mass numbers, lack of targeted therapy continues to be a major problem. According to studies so far it seems that elderly people are more vulnerable to severe symptoms while children tend to by asymptomatic or have milder form the disease. In our review, we focused on gathering data about the virus itself, its characteristics, paths of transmission, and its effect on hormone production and secretion. In such, there is insufficient information in the literature worldwide, especially the ones that focus on the effect of COVID-19 on individual organs systems within the human body. Hence, the present evidence-based study focused on the possible effects of COVID-19 on adrenal gland and gonads i.e. on the process of steroidogenesis and fertility.

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.

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.

UPLC-TOF-MS Method for Simultaneous Quantification of Steroid Hormones in Tissue Homogenates of Zebrafish with Solid-Phase Extraction.

The quantification of steroid hormones of individual zebrafish (Danio rerio) provides perspective to understand endogenous hormone function. A UPLC-TOF-MS method was developed to provide a reproducible, sensitive, and efficient assay to determine the concentration of steroid hormones, including cortisol, testosterone, androstenedione, 11-deoxycortisol, 11-deoxycorticosterone, and 17-hydroxyprogesterone in whole-body homogenates of each zebrafish. Solid-phase extraction was used to sample matrix clean-up and acquired a recovery from 89.7% to 107.9%. The analytes were separated on an Aquity BEH C18 column using gradient elution. Mass spectrometric analysis was performed by single reaction monitoring (SRM) using positive electrospray ionization mode. The total running time was 6 min, which was greatly shortened compared with a previously reported method. The developed method exhibited excellent linearity for all the analytes, with regression coefficients higher than 0.99. The limit of detection varied between 0.1 and 0.5 ng/L and the limit of quantification was 0.5-1.7 ng/L for all analytes. The precision of the method was assessed on replicate measurements and was found to be in the ranges of 1.9 % to 6.6% and 4.3% to 8.6%, for intra- and inter-day analysis, respectively. This method was validated according to FDA guidance and applied to determine steroid hormone levels in the tissue homogenate of zebrafish acutely treated with caffeine and ethanol.

Embryonic Steroids Control Developmental Programming of Energy Balance.

Glucose is a major energy source for growth. At birth, neonates must change their energy source from maternal supply to its own glucose production. The mechanism of this transition has not been clearly elucidated. To evaluate the possible roles of steroids in this transition, here we examine the defects associated with energy production of a mouse line that cannot synthesize steroids de novo due to the disruption of its Cyp11a1 (cytochrome P450 family 11 subfamily A member 1) gene. The Cyp11a1 null embryos had insufficient blood insulin and failed to store glycogen in the liver since embryonic day 16.5. Their blood glucose dropped soon after maternal deprivation, and the expression of hepatic gluconeogenic and glycogenic genes were reduced. Insulin was synthesized in the mutant fetal pancreas but failed to be secreted. Maternal glucocorticoid supply rescued the amounts of blood glucose, insulin, and liver glycogen in the fetus but did not restore expression of genes for glycogen synthesis, indicating the requirement of de novo glucocorticoid synthesis for glycogen storage. Thus, our investigation of Cyp11a1 null embryos reveals that the energy homeostasis is established before birth, and fetal steroids are required for the regulation of glycogen synthesis, hepatic gluconeogenesis, and insulin secretion at the fetal stage.

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.

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.

Upregulation of Low-Density Lipoprotein Receptor of the Steroidogenesis Pathway in the Cumulus Cells Is Associated with the Maturation of Oocytes and Achievement of Pregnancy.

The maturation of the oocyte is influenced by cumulus cells (CCs) and associated with pregnancy rate, whereas the influencing factors have not been completely elucidated in the CCs. In this study, we identified new regulators of CCs for high-quality oocytes and successful pregnancies during assisted reproductive techniques. CCs were collected from cumulus-oocyte complexes (COCs) in young (≤33 years old) and old (≥40 years old) women undergoing intracytoplasmic sperm injection (ICSI) procedures. We screened for factors differentially expressed between young vs. old CCs and pregnancy vs. non-pregnancy using whole mRNA-seq-next-generation sequencing (NGS). We characterized the transcriptome of the CCs to identify factors critical for achieving pregnancy in IVF cycles. Women in the young and old pregnancy groups exhibited the up- and downregulation of multiple genes compared with the non-pregnancy groups, revealing the differential regulation of several specific genes involved in ovarian steroidogenesis in CCs. It was shown that the low-density lipoprotein (LDL) receptor to the steroidogenesis pathway was upregulated in CCs with higher maturity rates of oocytes in the pregnancy group. In conclusion, a higher pregnancy rate is related to the signaling pathway of steroidogenesis by the LDL receptor in infertile women undergoing IVF procedures.

Early Embryo Exposure to Assisted Reproductive Manipulation Induced Subtle Changes in Liver Epigenetics with No Apparent Negative Health Consequences in Rabbit.

Embryo manipulation is a requisite step in assisted reproductive technology (ART). Therefore, it is of great necessity to appraise the safety of ART and investigate the long-term effect, including lipid metabolism, on ART-conceived offspring. Augmenting our ART rabbit model to investigate lipid metabolic outcomes in offspring longitudinally, we detected variations in hepatic DNA methylation ART offspring in the F3 generation for embryonic exposure (multiple ovulation, vitrification and embryo transfer). Through adult liver metabolomics and proteomics, we identified changes mainly related to lipid metabolism (e.g., polyunsaturated fatty acids, steroids, steroid hormone). We also found that DNA methylation analysis was linked to changes in lipid metabolism and apoptosis genes. Nevertheless, these differences did not apparently alter the general health status. Thus, our findings suggest that ART is likely to be a player in embryo epigenetic events related to hepatic homeostasis alteration in adulthood.

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.