Steroid Synthesis Research Articles

The proteome and metabolome changes distinguish the effect of dietary energy levels on the development of ovary in chicken during sexual maturity

To deeply understanding the impact of peripheral energy level on the development of ovaries during the sexual maturation of chicken, in this study, the ovaries and serum of sexually mature and immature chickens at the same age from different energy level groups were collected, and the proteome and metabolome were detected. The results of ovarian and serum metabolomics revealed that dietary energy levels affected the energy metabolism and fatty acid oxidation of ovary in chicken, including the up-regulated expression of dihydroacetone phosphate and α-linolenic acid in high energy level groups. The results of proteomics showed that peripheral energy levels affected the catecholamine biosynthesis and metabolism in ovary before sexual maturation. The integrating analysis revealed that increased energy flux may influence ovarian development by regulating cholesterol reserves and steroid hormone synthesis in the ovaries. In vitro, the cultivation of chicken primary granulosa cells showed that sterol carrier protein 2 played a role in fatty acid synthesis and metabolism but did not significantly affect progesterone synthesis. Overall, dietary energy levels may be involved in the development of the ovaries during sexual maturation by influencing energy metabolism, biosynthesis of unsaturated fatty acids and steroid hormone within the ovaries.

Comparative transcriptomic and metabolomic analyses reveal key regulatory gene for methyl jasmonate-induced steroidal saponins synthesis in Dioscorea composita

Dioscorea composita (D. composita) is a perennial herb with abundant steroidal saponins that have gained worldwide attention for their remarkable efficacy in cardiovascular diseases. However, few studies have been worked on the regulatory network of steroidal saponins biosynthesis under phytohormone induced. In this study, we combined the transcriptome and metabolome analysis to reveal the variation of diosgenin and steroidal saponins in transcriptional and metabolism levels under methyl-jasmonate (MeJA) treatment. Although the application of MeJA indeed significantly increased the accumulation of diosgenin of D. composita, different types of steroidal saponins exhibited different accumulation patterns. Consistently, the expression levels of UDP-glycosyltransferases and Cytochrome P450 monooxygenases genes that highly related to the accumulation of steroidal saponins were either up- or down-regulated. Correlation analyses of transcription factors (TFs)-steroidal saponins and structural genes-TFs were further to identified the TFs potentially involved in the regulation of steroidal saponins biosynthesis. Silencing of DcWRKY11 in Dioscorea composita decreases the accumulation of steroidal saponins by regulating the expression steroidal saponins synthesis genes, suggesting that DcWRKY11 is a positive regulator in the regulation of steroidal saponins biosynthesis. Our findings take a deeper understanding of the regulatory network of MeJA-mediated steroidal saponins biosynthesis in D. composita.

Synergistic cooperation between the β-catenin and SF1 regulates progesterone synthesis in laying hen ovarian granulosa cells

The development of ovarian follicles in poultry is a key factor affecting the performance of egg production. Ovarian follicle development is regulated via the Wnt/β-catenin signaling pathway, and β-catenin, encoded by CTNNB1, is a core component of this pathway. In this study, using ovary GCs from laying hens, we investigated the regulatory role of CTNNB1 in steroid synthesis. We found that CTNNB1 significantly regulates the expression of StAR and CYP11A1 (key genes related to progesterone synthesis) and the secretion of progesterone (P4). Furthermore, simultaneous overexpression of CTNNB1 and SF1 resulted in significantly higher levels of CYP11A1 and secretion of P4 than in cells overexpressing CTNNB1 or SF1 alone. We also found that in GCs overexpressing SF1, levels of CYP11A1 and secreted P4 were significantly greater than in controls. Silencing of CYP11A1 resulted in the inhibition of P4 secretion while overexpression of SF1 in CYP11A1-silenced cells restored P4 secretion to normal levels. Together, these results indicate that synergistic cooperation between the β-catenin and SF1 regulates progesterone synthesis in laying hen ovarian hierarchical granulosa cells to promote CYP11A1 expression.

Elevated RBP4 in Subclinical Ketosis Cows Inhibits Follicular Granulosa Cell Proliferation and Steroid Hormone Synthesis.

The mechanism by which subclinical ketosis (SCK) causes postpartum reproductive disorders in dairy cows remains unclear. In this study, cows within the day 14 to 21 postpartum period were categorized into the SCK group or the control group. Subsequently, they were monitored until 45 d to 60 d postpartum and divided into the SCK anestrus group (SCK-AE, n = 12) and the control estrus group (C-E, n = 12). In comparison to the C-E group, the RBP4 and p-AKT of the SCK-AE group exhibited increased levels in serum, liver, and ovaries. In the in vitro experimental cultivation of granulosa cells (GCs), after adding RBP4, cell proliferation, steroid hormone secretion and synthesis, and GLUT4 secretion were inhibited, and cell apoptosis was exacerbated. After silencing STRA6 (RBP4 receptor), cell proliferation and steroid hormone secretion and synthesis, as well as the inhibition of GLUT4, were alleviated, and the situation of cell apoptosis also improved. The SC79 activator could promote the phosphorylation of AKT, thus alleviating the increased cell proliferation, steroid hormone secretion and synthesis, GLUT4 inhibition, and apoptosis rate in cow GCs induced by RBP4 stimulation. Our research indicates that elevated RBP4 levels in SCK cows inhibit the proliferation, apoptosis, and steroid hormone synthesis of GCs through the STRA6 receptor and the PI3K/AKT pathway.

ALKBH5 Reduces BMP15 mRNA Stability and Regulates Bovine Puberty Initiation Through an m6A-Dependent Pathway.

The timing of puberty significantly influences subsequent reproductive performance in cattle. N6-methyladenosine (m6A) is a key epigenetic modification involved in the regulation of pubertal onset. However, limited research has investigated alterations in m6A methylation within the hypothalamic-pituitary-ovarian (HPO) axis during the onset of puberty. In this study, combined analysis of methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and RNA sequencing (RNA-seq) is used to describe the overall modification pattern of m6A in the HPO axis, while GSEA, KEGG, and GO analyses are used to describe the enrichment pathways of differentially expressed genes and differentially methylated genes. The m6A modifications of the differential genes KL, IGSF10, PAPPA2, and BMP15 and the pathways of cell adhesion molecules (CAMs), TGF-β, cell cycle, and steroid hormone synthesis may play roles in regulating the function of the HPO axis tissue during pubertal transition. Notably, BMP15's m6A modification depends on the action of the demethylase ALKBH5, which is recognized by the reader protein YTHDF2, promoting bovine granulosa cell proliferation, steroid production, and estrogen secretion. This study reveals for the first time the modification mechanism of BMP15 m6A during the initiation of bovine puberty, which will provide useful information for improving the reproductive efficiency of Chinese beef cattle.

Neonatal reference intervals for serum steroid hormone concentrations measured by LC-MS/MS.

Congenital adrenal hyperplasia (CAH) is a rare, inherited disorder of adrenal steroid synthesis. In many countries it is part of the neonatal screening program enabling early diagnosis and treatment. In case of an abnormal neonatal screening result or when other differences of sexual development (DSD) are suspected, measurement of serum steroid hormones using liquid chromatography coupled to mass spectrometry (LC-MS/MS) is needed for further diagnosis. However, reliable age- and sex-specific reference intervals (RIs) for serum steroid hormones during the neonatal period are missing. We therefore aimed to establish LC-MS/MS based RIs for serum steroid hormones in neonates. Serum was obtained from healthy term neonates at two time points: 130 samples at day 3-8 (T1, time of the neonatal screening) and 126 samples at day 13-15 (T2,two weeks old). Concentrations of cortisol, cortisone, corticosterone, 11-deoxycortisol, 21-deoxycortisol, 11-deoxycorticosterone, testosterone, androstenedione, and 17-hydroxyprogesterone (17-OHP) were measured using LC-MS/MS. RIs (in nmol/L) were established for T1 and T2: cortisone (19.3-215;18.0-212), cortisol (10.0-407;8.4-446), corticosterone (<31;<50), 11-deoxycortisol (0.73-4.6;0.70-3.6), 17-OHP (<4.9;<5.1), androstenedione (0.3-1.8;0.3-2.7), 11-deoxycorticosterone (<0.2;<0.2), and 21-deoxycortisol (<1;<1), respectively. Testosterone differed between boys and girls: RIs at T1 and T2 for boys were 0.27-4.3 and 0.63-13.9, and for girls<0.30 and <0.47, respectively. We established LC-MS/MS based RIs for cortisol, cortisone, corticosterone, 11-deoxycortisol, 21-deoxycortisol, 11-deoxycorticosterone, testosterone, androstenedione, and 17-OHP in neonates in the first and second week of life.

Environmental Heat Stress Decreases Sperm Motility by Disrupting the Diurnal Rhythms of Rumen Microbes and Metabolites in Hu Rams.

Heat stress (HS) has become a common stressor, owing to the increasing frequency of extreme high-temperature weather triggered by global warming, which has seriously affected the reproductive capacity of important livestock such as sheep. However, little is known about whether HS reduces sperm motility by inducing circadian rhythm disorders in rumen microorganisms and metabolites in sheep. In this study, the year-round reproduction of two-year-old Hu rams was selected, and the samples were collected in May and July 2022 at average environmental temperatures between 18.71 °C and 33.58 °C, respectively. The experiment revealed that the mean temperature-humidity index was 86.34 in July, indicating that Hu rams suffered from HS. Our research revealed that HS significantly decreased sperm motility in Hu rams. Microbiome analysis further revealed that HS reshaped the composition and circadian rhythm of rumen microorganisms, leading to the circadian disruption of microorganisms that drive cortisol and testosterone synthesis. Serum indicators further confirmed that HS significantly increased the concentrations of cortisol during the daytime and decreased the testosterone concentration at the highest body temperature. Untargeted metabolomics analysis revealed that the circadian rhythm of rumen fluid metabolites in the HS group was enriched by the cortisol and steroid synthesis pathways. Moreover, HS downregulated metabolites, such as kaempferol and L-tryptophan in rumen fluid and seminal plasma, which are associated with promotion of spermatogenesis and sperm motility; furthermore, these metabolites were found to be strongly positively correlated with Veillonellaceae_UCG_001. Overall, this study revealed the relationship between the HS-induced circadian rhythm disruption of rumen microorganisms and metabolites and sperm motility decline. Our findings provide a new perspective for further interventions in enhancing sheep sperm motility with regard to the circadian time scale.

Perfluorooctane sulfonate (PFOS) and benzo[a]pyrene (BaP) synergistically induce neurotoxicity in C6 rat glioma cells via the activation of neurotransmitter and Cyp1a1-mediated steroid hormone synthesis pathways

Humans are often exposed to complex mixtures of multiple pollutants rather than a single pollutant. However, the combined toxic effects and the molecular mechanism of PFOS and BaP remain poorly understood. In this study, two typical environmental pollutants, perfluorooctane sulfonate acid (PFOS) and benzo[a]pyrene (BaP), were selected to investigate their combined neurotoxic effects on rat C6 glioma cells at environmentally relevant concentrations. The results showed that coexposure to low-dose PFOS and BaP induced greater toxicity (synergistic effect) than did single exposure. PFOS-BaP coexposure had stronger toxic effects on inducing oxidative stress and promoting early apoptosis. Targeted metabolomics confirmed that increased levels of the neurotransmitters 5-hydroxytryptophan, dopamine, tryptophan and serotonin disturb the phenylalanine, tyrosine and tryptophan biosynthesis pathways. Mechanistically, exposure to a low-dose PFOS-BaP binary mixture induces steroid hormone synthesis disorder through the activation of Cyp1a1 and Hsd17b8 (steroid hormone synthesis genes) and Dhcr24 and Dhcr7 (cholesterol synthesis genes). These findings are useful for comprehensively and systematically elucidating the biological safety of PFOS-BaP and its potential threats to human health.

Transcriptome and Metabolome Reveal Accumulation of Key Metabolites with Medicinal Properties of Phylloporia pulla.

Phylloporia pulla, a macrofungal species in the Hymenochaetales, Basidiomycota, is known to enhance the nutritional and bioactive properties of rice through co-fermentation; however, its own secondary metabolites are not well understood. In this study, an integrative analysis of transcriptome and metabolome data revealed that the accumulation of steroids, steroid derivatives, and triterpenoids in P. pulla peaks during the mid-growth stage, while the genes associated with these metabolites show higher expression levels from the early to mid-growth stages. Weighted gene co-expression network analysis identified several modules containing candidate genes involved in the synthesis of steroids, steroid derivatives, and triterpenoids. Specifically, six key hub genes were identified, along with their connectivity to other related genes, as potential catalysts in converting the precursor lanosterol to celastrol. This study enhances our understanding of the secondary metabolites of P. pulla and is essential for the selective utilization of these bioactive compounds.

Functional diversity of oxidosqualene cyclases in genus Oryza.

Triterpene skeletons, catalyzing by 2,3-oxidosqualene cyclases (OSCs), are essential for synthesis of steroids and triterpenoids. In japonica rice cultivars Zhonghua11, a total of 12 OsOSCs have been found. While the catalytic functions of OsOSC1, 3, 4, 9, and 10 remain unclear, the functions of the other OsOSCs have been well studied. In this study, we conducted a comprehensive analysis of 12 OSC genes within genus Oryza with the aid of 63 genomes from cultivated and wild rice. We found that OSC genes are relatively conserved within genus Oryza with a few exceptions. Collinearity analysis further suggested that, throughout the evolutionary history of genus Oryza, the OSC genes have not undergone significant rearrangements or losses. Further functional analysis of 5 uncharacterized OSCs revealed that OsOSC10 was a friedelin synthase, which affected the development of rice grains. Additionally, the reconstructed ancestral sequences of Oryza OSC3 and Oryza OSC9 had lupeol synthase and poaceatapetol synthase activity, respectively. The discovery of friedelin synthase in rice unlocks a new catalytic path and biological function of OsOSC10. The pan-genome analysis of OSCs within genus Oryza gives insights into the evolutionary trajectory and products diversity of Oryza OSCs.

Assessing Alcohol Genes Targets in Mouse Liver

Background: Alcohol is a risk factor for liver diseases. There is a correlation between alcohol consumption and fatty liver disease. Experiences have shown gene expression alteration in the liver following alcohol consumption. Since the molecular mechanism of connection between alcohol consumption and fatty liver disease needs a clear perspective, this study aims to explore the significant genes that are targeted by alcohol in the liver of mice.Methods: Gene expression profiles of mice livers fed with alcohol were retrieved from the Gene Expression Omnibus (GEO) database and compared with the control samples. Data are pre-evaluated with the GEO2R program, and the significant differentially expressed genes (DEGs) are analyzed via gene expression evaluations and regulatory network assessment. Results: Among the 25619 dysregulated genes, 78 significant DEGs were pointed out. Gene expression analysis showed that most extremely dysregulated genes are up-regulated and belong to the cytochrome P450 genes family. Finally, cytochrome P450 and glutathione S-transferase genes family, as well as hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 4, were introduced as the critical targeted genes.Conclusion: In conclusion, detoxification of xenobiotics, cellular metabolism and homeostasis, the pathogenesis of some liver diseases, synthesis of several prostaglandins and steroid hormones, and inflammation fibrosis in the liver are possibly associated with alcohol consumption.

Mating behaviour and cholesterol nutritional strategies promoted ovarian development of female swimming crab (Portunus trituberculatus).

Female crabs enter a stage of rapid ovarian development after mating, and cholesterol is a substrate for steroid hormone synthesis. Therefore, in this experiment, an 8-week feeding trial was conducted to investigate the effects of mating treatments (mated crab and unmated crab) and three dietary cholesterol levels (0·09 %, 0·79 % and 1·40 %) on ovarian development, cholesterol metabolism and steroid hormones metabolism of adult female swimming crab (Portunus trituberculatus). The results indicated that crabs fed the diet with 0·79 % cholesterol significantly increased gonadosomatic index (GSI) and vitellogenin (VTG) content than other treatments in the same mating status. Moreover, mated crabs had markedly increased GSI and VTG content in the ovary and hepatopancreas than unmated crabs. The histological observation found that exogenous vitellogenic oocytes appeared in the mated crabs, while previtellogenic oocytes and endogenous vitellogenic oocytes were the primary oocytes in unmated crabs. The transmission electron microscopy analysis showed that when fed diet with 0·79 % cholesterol, the unmated crabs contained more rough endoplasmic reticulum and mated crabs had higher yolk content than other treatments. Furthermore, mating treatment and dietary 0·79 % cholesterol level both promoted cholesterol deposition by up-regulation of the mRNA and protein expression levels of class B scavenger receptors 1 (Srb1), while stimulating the secretion of steroid hormones by up-regulation of the mRNA and protein expression of steroidogenic acute regulatory protein (Star). Overall, the present results indicated that mating behaviour plays a leading role in promoting ovarian development, and dietary 0·79 % cholesterol level can further promote ovarian development after mating.

Integrated Transcriptomics and Metabolomics Studies Reveal Steroid Biosynthesis Pathway and BCL2 Inhibitory Diazo-Progesterone of Drimia indica for Conservation and Sustainable Utilization.

This study is the first report on the sequence of the transcriptome of Drimia indica, a non-model plant with medicinal properties found in a forest tribal belt, using the Illumina NovaSeq platform. The primary objectives of this study were to elucidate the gene expression profiles in different tissues, identify key regulatory genes and pathways involved in secondary metabolite biosynthesis, and explore the plant's potential pharmacological properties. The study generated 670087 unigenes from both leaves and roots and identified putative homologs of annotated sequences against UniProt/Swiss-Prot and KEGG databases. The functional annotation of the identified unigenes revealed the secondary metabolite biosynthetic process as the most prominent pathway, with gene enrichment analysis predominantly accounting for secondary metabolite pathways, such as terpenoid, steroid, flavonoid, alkaloid, selenocompound, and cortisol synthesis. The study also identified regulatory genes NAC, Bhlh, WRKY, and C2H2 on the transcriptome dataset. The functionally annotated unigenes suggested phytocompounds in Drimia indica to have multi-potent properties, such as anti-cancer, anti-inflammatory, and anti-diabetic activities, which has been further validated by GC-MS-based metabolite profiling. Notably, we have identified two novel molecules, di-azo progesterone and 4H-pyran-4-one 2,3-dihydro-3,5-dihydroxy- 6-methyl, with potential BCL2 inhibitory anticancer properties, supported by stable binding interactions observed in molecular docking and dynamics simulations. Additionally, an abundance of mono-nucleotide SSR markers has been identified, useful for genetic diversity studies. This study provides a foundational understanding of the molecular mechanisms in Drimia indica, highlighting its potential as a source for novel therapeutic agents and contributing valuable insights for future pharmacological and agricultural applications. However, further in vivo studies are warranted to confirm these findings and validate their pharmacological efficacy and therapeutic potential. The SSR markers identified also offer valuable tools for molecular genetics, plant breeding, and sustainable drug development.

12547 Spatiotemporal Analysis Of Steroidogenesis Pathway Genes Expression In Mouse Fetal Leydig Cells

Abstract Disclosure: K. Jiang: Other; Self; NIH ORIP P51OD011106&amp;S10OD028626. A. Kothandapani: None. Z. Fu: None. T.W. Kearse: None. J. Jorgensen: Grant Recipient; Self; NIH R01HD090660. Male mouse embryos experience a masculinization programming window (MPW, embryonic day, E13-16), during which a critical threshold of androgens is produced to ensure male-pattern development. Insufficient fetal androgens during the MPW leads to variations of masculinization, such as cryptorchidism and hypospadias. In rodents, fetal Leydig cells (FLC) are the primary source of androgens; however, our knowledge about their maturation and steroid synthesis regulation is limited. Hence, the objective for this research is to reveal spatiotemporal patterns of steroidogenic pathway genes in mouse fetal testes using high-resolution technologies. To understand the temporal aspect of steroidogenesis, we quantified transcript numbers of steroidogenic pathway genes via copy number qPCR from E11 to postnatal day 6 (P6). Results showed a gradual increase in transcripts as FLCs differentiate until E14, which then surge during the MPW until E16, followed by a rapid drop in expression to pre-FLC differentiation levels by the time of birth (P0). The profound changes persisted after data were normalized to FLC numbers, suggesting active stimulation and downregulation phases of transcription regulation. Next, to assess androgen output in relation to gene transcript levels, we collected testes at 24 hour intervals from E12 to P3 to measure progesterone, androstenedione, and testosterone via LC/MS/MS; results are pending. Guided by the temporal expression profile, we examined the spatial patterns of Star (steroidogenic acute regulatory protein) at the onset (E13), peak (E16), and end (P0) of steroidogenesis using single-molecule fluorescent in situ hybridization (smFISH). We were surprised to observe three different stages for Star transcription based on the subcellular localization of it: Stage I-only at gene loci in the nucleus, newly differentiated; Stage II-in both the nucleus and cytoplasm, peak activity; and Stage III-in cytoplasm only, repressed transcription. The proportion of Stage I FLC decreased over time (15% at E13, 2% at E16, and 0.4% at P0). Most (55%) FLCs at E16 were at Stage II, when testes exhibit maximum steroidogenic gene transcript numbers. Stage III FLCs dominated in P0 testes with 68% of the total FLC number, reflecting diminishing steroidogenesis. Of note, we also discovered that the global testis pattern for Star transcripts exhibited a unique pattern. At the onset of differentiation (E13), FLCs accumulated in the middle of the dorsal-ventral axis and at either pole of the anterior-posterior axis. By E16 and at P0, FLCs were evenly distributed throughout the testis. In conclusion, we observed that FLC steroidogenic pathway gene transcription is not synchronized but individual FLCs contribute to global testis androgen output as they differentiate in a coordinated pattern towards maximal output to coordinate masculinization. Presentation: 6/1/2024

12547 Spatiotemporal Analysis Of Steroidogenesis Pathway Genes Expression In Mouse Fetal Leydig Cells

Abstract Disclosure: K. Jiang: Other; Self; NIH ORIP P51OD011106&amp;S10OD028626. A. Kothandapani: None. Z. Fu: None. T.W. Kearse: None. J. Jorgensen: Grant Recipient; Self; NIH R01HD090660. Male mouse embryos experience a masculinization programming window (MPW, embryonic day, E13-16), during which a critical threshold of androgens is produced to ensure male-pattern development. Insufficient fetal androgens during the MPW leads to variations of masculinization, such as cryptorchidism and hypospadias. In rodents, fetal Leydig cells (FLC) are the primary source of androgens; however, our knowledge about their maturation and steroid synthesis regulation is limited. Hence, the objective for this research is to reveal spatiotemporal patterns of steroidogenic pathway genes in mouse fetal testes using high-resolution technologies. To understand the temporal aspect of steroidogenesis, we quantified transcript numbers of steroidogenic pathway genes via copy number qPCR from E11 to postnatal day 6 (P6). Results showed a gradual increase in transcripts as FLCs differentiate until E14, which then surge during the MPW until E16, followed by a rapid drop in expression to pre-FLC differentiation levels by the time of birth (P0). The profound changes persisted after data were normalized to FLC numbers, suggesting active stimulation and downregulation phases of transcription regulation. Next, to assess androgen output in relation to gene transcript levels, we collected testes at 24 hour intervals from E12 to P3 to measure progesterone, androstenedione, and testosterone via LC/MS/MS; results are pending. Guided by the temporal expression profile, we examined the spatial patterns of Star (steroidogenic acute regulatory protein) at the onset (E13), peak (E16), and end (P0) of steroidogenesis using single-molecule fluorescent in situ hybridization (smFISH). We were surprised to observe three different stages for Star transcription based on the subcellular localization of it: Stage I-only at gene loci in the nucleus, newly differentiated; Stage II-in both the nucleus and cytoplasm, peak activity; and Stage III-in cytoplasm only, repressed transcription. The proportion of Stage I FLC decreased over time (15% at E13, 2% at E16, and 0.4% at P0). Most (55%) FLCs at E16 were at Stage II, when testes exhibit maximum steroidogenic gene transcript numbers. Stage III FLCs dominated in P0 testes with 68% of the total FLC number, reflecting diminishing steroidogenesis. Of note, we also discovered that the global testis pattern for Star transcripts exhibited a unique pattern. At the onset of differentiation (E13), FLCs accumulated in the middle of the dorsal-ventral axis and at either pole of the anterior-posterior axis. By E16 and at P0, FLCs were evenly distributed throughout the testis. In conclusion, we observed that FLC steroidogenic pathway gene transcription is not synchronized but individual FLCs contribute to global testis androgen output as they differentiate in a coordinated pattern towards maximal output to coordinate masculinization. Presentation: 6/1/2024

12674 Spatiotemporal Analysis Of Steroidogenesis Pathway Genes Expression In Mouse Fetal Leydig Cells

Abstract Disclosure: K. Jiang: Other; Self; NIH ORIP P51OD011106&amp;S10OD028626. A. Kothandapani: None. Z. Fu: None. T.W. Kearse: None. J. Jorgensen: Grant Recipient; Self; NIH R01HD090660. Male mouse embryos experience a masculinization programming window (MPW, embryonic day, E13-16), during which a critical threshold of androgens is produced to ensure male-pattern development. Insufficient fetal androgens during the MPW leads to variations of masculinization, such as cryptorchidism and hypospadias. In rodents, fetal Leydig cells (FLC) are the primary source of androgens; however, our knowledge about their maturation and steroid synthesis regulation is limited. Hence, the objective for this research is to reveal spatiotemporal patterns of steroidogenic pathway genes in mouse fetal testes using high-resolution technologies. To understand the temporal aspect of steroidogenesis, we quantified transcript numbers of steroidogenic pathway genes via copy number qPCR from E11 to postnatal day 6 (P6). Results showed a gradual increase in transcripts as FLCs differentiate until E14, which then surge during the MPW until E16, followed by a rapid drop in expression to pre-FLC differentiation levels by the time of birth (P0). The profound changes persisted after data were normalized to FLC numbers, suggesting active stimulation and downregulation phases of transcription regulation. Next, to assess androgen output in relation to gene transcript levels, we collected testes at 24 hour intervals from E12 to P3 to measure progesterone, androstenedione, and testosterone via LC/MS/MS; results are pending. Guided by the temporal expression profile, we examined the spatial patterns of Star (steroidogenic acute regulatory protein) at the onset (E13), peak (E16), and end (P0) of steroidogenesis using single-molecule fluorescent in situ hybridization (smFISH). We were surprised to observe three different stages for Star transcription based on the subcellular localization of it: Stage I-only at gene loci in the nucleus, newly differentiated; Stage II-in both the nucleus and cytoplasm, peak activity; and Stage III-in cytoplasm only, repressed transcription. The proportion of Stage I FLC decreased over time (15% at E13, 2% at E16, and 0.4% at P0). Most (55%) FLCs at E16 were at Stage II, when testes exhibit maximum steroidogenic gene transcript numbers. Stage III FLCs dominated in P0 testes with 68% of the total FLC number, reflecting diminishing steroidogenesis. Of note, we also discovered that the global testis pattern for Star transcripts exhibited a unique pattern. At the onset of differentiation (E13), FLCs accumulated in the middle of the dorsal-ventral axis and at either pole of the anterior-posterior axis. By E16 and at P0, FLCs were evenly distributed throughout the testis. In conclusion, we observed that FLC steroidogenic pathway gene transcription is not synchronized but individual FLCs contribute to global testis androgen output as they differentiate in a coordinated pattern towards maximal output to coordinate masculinization. Presentation: 6/1/2024

12674 Spatiotemporal Analysis Of Steroidogenesis Pathway Genes Expression In Mouse Fetal Leydig Cells

Abstract Disclosure: K. Jiang: Other; Self; NIH ORIP P51OD011106&amp;S10OD028626. A. Kothandapani: None. Z. Fu: None. T.W. Kearse: None. J. Jorgensen: Grant Recipient; Self; NIH R01HD090660. Male mouse embryos experience a masculinization programming window (MPW, embryonic day, E13-16), during which a critical threshold of androgens is produced to ensure male-pattern development. Insufficient fetal androgens during the MPW leads to variations of masculinization, such as cryptorchidism and hypospadias. In rodents, fetal Leydig cells (FLC) are the primary source of androgens; however, our knowledge about their maturation and steroid synthesis regulation is limited. Hence, the objective for this research is to reveal spatiotemporal patterns of steroidogenic pathway genes in mouse fetal testes using high-resolution technologies. To understand the temporal aspect of steroidogenesis, we quantified transcript numbers of steroidogenic pathway genes via copy number qPCR from E11 to postnatal day 6 (P6). Results showed a gradual increase in transcripts as FLCs differentiate until E14, which then surge during the MPW until E16, followed by a rapid drop in expression to pre-FLC differentiation levels by the time of birth (P0). The profound changes persisted after data were normalized to FLC numbers, suggesting active stimulation and downregulation phases of transcription regulation. Next, to assess androgen output in relation to gene transcript levels, we collected testes at 24 hour intervals from E12 to P3 to measure progesterone, androstenedione, and testosterone via LC/MS/MS; results are pending. Guided by the temporal expression profile, we examined the spatial patterns of Star (steroidogenic acute regulatory protein) at the onset (E13), peak (E16), and end (P0) of steroidogenesis using single-molecule fluorescent in situ hybridization (smFISH). We were surprised to observe three different stages for Star transcription based on the subcellular localization of it: Stage I-only at gene loci in the nucleus, newly differentiated; Stage II-in both the nucleus and cytoplasm, peak activity; and Stage III-in cytoplasm only, repressed transcription. The proportion of Stage I FLC decreased over time (15% at E13, 2% at E16, and 0.4% at P0). Most (55%) FLCs at E16 were at Stage II, when testes exhibit maximum steroidogenic gene transcript numbers. Stage III FLCs dominated in P0 testes with 68% of the total FLC number, reflecting diminishing steroidogenesis. Of note, we also discovered that the global testis pattern for Star transcripts exhibited a unique pattern. At the onset of differentiation (E13), FLCs accumulated in the middle of the dorsal-ventral axis and at either pole of the anterior-posterior axis. By E16 and at P0, FLCs were evenly distributed throughout the testis. In conclusion, we observed that FLC steroidogenic pathway gene transcription is not synchronized but individual FLCs contribute to global testis androgen output as they differentiate in a coordinated pattern towards maximal output to coordinate masculinization. Presentation: 6/1/2024

Positive effects of a mixture of fish oil and soybean oil as a dietary lipid source on the ovarian development and health of female giant river prawn, Macrobrachium rosenbergii broodstock

Lipids are key nutrients that affect the development of the ovary in aquatic animals. This study aimed to investigate the effects of different lipid sources on the ovarian development and health of Macrobrachium rosenbergii broodstock. The M. rosenbergii broodstock, with an initial body weight of 8.07±0.74 g, were fed diets containing 5 % fish oil (FO), soya bean oil (SO), rapeseed oil (RO), coconut oil (CO), and a 1:1 mixture of fish oil and soybean oil (MO) as lipid sources for a period of 56 days. The results indicated that there were no significant differences in survival rate (SR) and hepatopancreatic index (HSI) among the different groups (P > 0.05). However, the specific growth rate (SGR) and gonadosomatic index (GSI) in the MO group were significantly higher than other groups (P < 0.05). Additionally, fatty acid levels in diets directly influenced the fatty acid composition of prawn hepatopancreas, showing a similar trend to that observed in the diet itself. Furthermore, compared to the other trial groups, haemolymph levels of 17-β estradiol (E2) and progesterone (PROG) were significantly increased in the MO group (P < 0.05). The ovarian section also exhibited better histomorphology and larger oocyte diameter in this group. Moreover, total antioxidant capacity was significantly increased in the MO group (P < 0.05). Saturated fatty acids in RO and CO groups could promote the expression of lipid synthesis-related genes fatty acid synthase (fas) and acetyl-CoA carboxylase (acc), concurrently suppressing the expression of lipid catabolism-related genes acyl-CoA oxidase-1 (acox-1) and AMP-activated protein kinase alpha (ampkα) (P < 0.05). Conversely, polyunsaturated fatty acids in the MO group significantly up-regulated the expression levels of fatty acid binding protein-3 (fabp-3) and peroxisome proliferator-activated receptor gamma (pparγ) (P < 0.05). Based on the findings of hepatopancreatic tissue structure, it was determined that the use of a single vegetable oil resulted in some damage to the hepatopancreatic tissue structure of M. rosenbergii broodstock, leading to increased oxidative stress. In conclusion, it is recommended that a 1:1 mixture of fish oil and soybean oil be used as a dietary lipid source to effectively meet the nutritional requirements during the ovarian development period of M. rosenbergii broodstock. This combination also enhances antioxidant properties, promotes the synthesis of haemolymph steroid hormones and lipid metabolism in female prawns, thereby facilitating ovarian development and overall health of M. rosenbergii broodstock.

Multi-omics and bioinformatics for the investigation of therapeutic mechanism of roucongrong pill against postmenopausal osteoporosis

Ethnopharmacological relevanceThe Roucongrong Pill (RCRP), originating from the historical General Medical Collection of Royal Benevolence, is frequently used to treat postmenopausal osteoporosis (PMOP). Despite its prevalent application, the specific anti-osteoporotic mechanisms of RCRP remain to be elucidated. Aim of the studyThis study aims to elucidate the therapeutic mechanism of RCRP in the context of ovariectomy (OVX)-induced PMOP in rats. By employing an integrative approach, the research combines medicinal chemistry, gut microbiota (GM) profiling, metabolomics, MetOrigin traceability, network pharmacology, molecular docking, and molecular dynamics simulations to deliver a comprehensive analysis. Materials and methodsSprague-Dawley (SD) rats underwent bilateral OVX to establish a PMOP model. The therapeutic efficacy of RCRP was evaluated through bone metrics (BMD, bone strength, BV/TV, Tb.Sp), hematoxylin and eosin (H&E) histological assessment, and bone metabolism markers (OPG, BALP, TRACP-5b, β-CTX, RANKL). Fecal metabolomics and 16S rDNA sequencing were employed to assess the influence of RCRP on GM and metabolite profiles. Furthermore, MetOrigin facilitated the traceability analysis of relevant metabolites. Molecular docking identified potential RCRP compounds with anti-PMOP activity, while their stability and protein interactions were assessed through molecular dynamics simulations. Network pharmacology further confirms the targets of action. ResultsRCRP alleviated PMOP in rats, enhancing bone strength, cortical and trabecular BMD, BV/TV, and serum OPG levels, while reducing Tb.Sp, serum BALP, TRACP-5b, β-CTX, and RANKL concentrations. A total of twenty-six distinct metabolites were identified, of which ten—tribufos, sulfoacetic acid, betamethasone dipropionate, 9-oxooctadeca-10,12,15-trienoic acid, menatetrenone, piperlongumine, maltopentaose, enol-phenylpyruvate, catechol, pentaacetate, and (+)-2-methylpropanoic acid—exhibited correlations with six GM species: Turicibacter, Roseburia, Colidextribacter, Helicobacter, Odoribacter, and Lachnoclostridium, as determined by Spearman's correlation analysis. Notably, MetOrigin revealed the microbial metabolism of taurine and hypotaurine, along with host-specific steroid hormone synthesis. Computational docking studies demonstrated robust interactions between five RCRP-derived steroids (hydroxyecdysone, corticosterone, trilostane, 5α-androstan-3,6,17-trione, and cortisol) and key enzymes (estradiol 17α-dehydrogenase and UDP-glucuronosyltransferase), suggesting a potential enhancement of therapeutic efficacy against PMOP. Furthermore, molecular dynamics simulations indicated stable interactions between hydroxyecdysone and two proteins, with binding free energies of −67.427 kJ/mol and −156.948 kJ/mol, respectively. Through network pharmacology and molecular docking approaches, potential targets of these metabolites were identified, including estrogen receptors ESR1 and ESR2, dual specificity phosphatase 6 (DUSP6), sex hormone-binding globulin (SHBG), prostaglandin E receptor 4 (PTGER4), cannabinoid receptor 2 (CNR2), cathepsin K (CTSK), and androgen receptor (AR). ConclusionsRCRP effectively mitigates OVX-induced bone loss in PMOP rats by modulating GM and associated metabolites, along with their potential targets and key metabolic pathways, including taurine and hypotaurine metabolism, as well as steroid hormone biosynthesis. These findings offer new insights into the therapeutic mechanisms by which RCRP may alleviate PMOP.

Shatavarin-IV rescues the Di (2-ethylhexyl) phthalate (DEHP) induced oxidative stress in rat granulosa cells in vitro

Studies provide notable evidence that oxidative stress (OS) mediated reactive oxygen species (ROS) disturb reproductive health. We have shown in our previous publication that exposure of Di-(2-ethylhexyl) phthalate (DEHP), induces OS mediated ROS generation which inhibits steroid synthesis. In the present study, we demonstrated the ameliorative/protective effects of one of the steroidal saponins, i.e., Shatavarin-IV, isolated from the roots of Asparagus racemosus against DEHP induced OS in rat granulosa cells. Granulosa cells were exposed with DEHP alone (400μM), Shatavarin-IV alone (8μg/ml), and a combination of DEHP + Shatavarin-IV (400μM + 8μg/ml) in vitro for 24 hrs. Intracellular ROS, OS/hypoxia, mitochondrial membrane potential, steroid-responsive genes expression were analyzed. The results revealed that the effective dose of DEHP (400µg) significantly increased OS compared to the control by increasing ROS levels, mitochondrial membrane potential, and β-galactosidase activity with a higher level of apoptotic genes (Bax, Caspase-3) expression at mRNA level. Further, DEHP significantly (p<0.05) reduced mRNA expression of steroidogenic responsive genes (StAR, CYP17A1 and CYP19A1) in granulosa cells treated with above combination compared to control. Interestingly, co-treatment of DEHP + Shatavarin-IV significantly suppressed the DEHP induced OS, ROS, β-galactosidase levels and enhanced steroidogeneic and apoptotic gene expression activities, which suggests that Shatavarin-IV rescued DEHP-induced changes that may useful for the prevention of DEHP- induced reproductive toxicity.