Stard 3 (Steroidogenic Acute Regulatory-Related Lipid Transfer Domain-Containing Protein 3) Play Important Role in Preadipocyte Differentiation

Aim: To evaluate Stard 3’s effects and relative mechanisms in preadipocyto differentiation by vitro study. Materials and Methods: The 3T3-L1 cell were divided into 5 groups as NC, si-Stard 3, ROS agonist, ROS inhibitor and si-Stard 3+ROS agonist groups. The cell of different groups were evaluated by Oil red O staining and Triglyceride. Evaluating ROS production by DHE and NBT assay. Using RT-qPCR and WB methods to evaluate gene and protein expressions. Results: Compared with NC group, Triglyceride, DHE fluorescence intensity and NBT positive rate were significantly down-regulation in si-Stard 3 and ROS inhibitor groups (P < 0.001, respectively), and were significantly up-regulation in ROS agonist group (P < 0.001, respectively); However, with si-Stard 3 transfection and ROS agonist treatment, compared with si-Stard 3 group, Triglyceride, DHE fluorescence intensity and NBT positive rate were significantly increased in si-Stard 3+ROS agonist group (P < 0.001, respectively). With RT-qPCR and WB assay, Compared with NC group, Stard 3 gene and protein expressions of si-Stard 3 and si-Stard 3+ROS agonist group were significantly depressed (P < 0.001, respectively), AMPK, PPARγ, CEBPα and FABP4 gene expressions were significantly differences in si-Stard 3, ROS agonist and ROS inhibitor groups (P < 0.001, respectively) and p-AMPK, PPARγ, CEBPα and FABP4 protein expressions were significantly differences in si-Stard 3, ROS agonist and ROS inhibitor groups (P < 0.001, respectively), with si-Stard 3 transfection and ROS agonist the relative gene and protein expressions were significantly resumed compared with si-Stard 3 group (P < 0.001, respectively). Conclusion: Stard 3 knockdown had effects to suppress 3T3-L1 cells transformation into adipocytes in vitro study.

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.

STARD3: A Prospective Target for Cancer Therapy

Cancer is one of the major causes of death in developed countries and current therapies are based on surgery, chemotherapeutic agents, and radiation. To overcome side effects induced by chemo- and radiotherapy, in recent decades, targeted therapies have been proposed in second and even first lines. Targeted drugs act on the essential pathways involved in tumor induction, progression, and metastasis, basically all the hallmark of cancers. Among emerging pathways, the cholesterol metabolic pathway is a strong candidate for this purpose. Cancer cells have an accelerated metabolic rate and require a continuous supply of cholesterol for cell division and membrane renewal. Steroidogenic acute regulatory related lipid transfer (START) proteins are a family of proteins involved in the transfer of lipids and some of them are important in non-vesicular cholesterol transportation within the cell. The alteration of their expression levels is implicated in several diseases, including cancers. In this review, we report the latest discoveries on StAR-related lipid transfer protein domain 3 (STARD3), a member of the START family, which has a potential role in cancer, focusing on the structural and biochemical characteristics and mechanisms that regulate its activity. The role of the STARD3 protein as a molecular target for the development of cancer therapies is also discussed. As STARD3 is a key protein in the cholesterol movement in cancer cells, it is of interest to identify inhibitors able to block its activity.

DAPL1 is a novel regulator of testosterone production in Leydig cells of mouse testis

Leydig cells in the testes produce testosterone in the presence of gonadotropins. Therefore, male testosterone levels must oscillate within a healthy spectrum, given that elevated testosterone levels augment the risk of cardiovascular disorders. We observed that the expression of death-associated protein-like 1 (DAPL1), which is involved in the early stages of epithelial differentiation and apoptosis, is considerably higher in the testes of sexually mature mice than in other tissues. Accordingly, Dapl1-null mice were constructed to evaluate this variation. Notably, in these mice, the testicular levels of steroidogenic acute regulatory protein (StAR) and serum testosterone levels were significantly elevated on postnatal day 49. The findings were confirmed in vitro using I-10 mouse testis-derived tumor cells. The in vivo and in vitro data revealed the DAPL1-regulated the expression of StAR involving altered transcription of critical proteins in the protein kinase A and CREB/CREM pathways in Leydig cells. The collective findings implicate DAPL1 as an important factor for steroidogenesis regulation, and DAPL1 deregulation may be related to high endogenous levels of testosterone.

Variations in the Initial Presentation of a Rare Congenital Adrenal Hyperplasia: Steroidogenic Acute Regulatory Deficiency

Background: Steroidogenic Acute Regulatory (StAR) deficiency is a rare form of congenital adrenal hyperplasia characterized by dysregulated cholesterol transport mediated by StAR enzyme across mitochondrial membranes. Adrenal dysfunction is due to the two-hit hypothesis: 1) defective StAR protein and 2) cholesterol accumulation in the adrenals and gonads. With variable cellular damage, adrenal crisis can occur early or late. Clinical cases: We present two cases of StAR deficiency with contrasting presentations. Case 1: A 9-day old ex full term female from a nonconsanguineous union presented to a rural hospital with hypothermia, lethargy, and poor feeding. She had hypoglycemia 41 mg/dL (60–105), hyponatremia 120 mEq/L (135–145), hyperkalemia 7.7 mEq/L (3.5–5.5) and cortisol &lt; 0.4 ug/dL (4.5–23). Baby was started on hydrocortisone (HCT) 100 mg/m2 and one-time fludrocortisone (FCT). She decompensated requiring chest compressions, intubation and pressors. She was transferred to our institution. Newborn screen was normal; she had typical female external genitalia. US demonstrated a uterus; ovaries and adrenals were not identified. Upon extubation and clinical improvement, her HCT was weaned to physiologic doses. She became hyponatremic requiring FCT and salt supplements. Post-HCT wean, ACTH level was 304 pg/mL (7–63) with aldosterone &lt; 4.0 ng/dL (6.5–86). Karyotype was 46,XX. Genetic analysis revealed a novel heterozygous likely pathogenic variant in the STAR gene, (STAR c.65-12_68del variant) without defect in the other STAR gene. Case 2: A 9-month-old ex full-term female of Iraqi descent from a nonconsanguineous union presented with fatigue, poor oral intake and weight loss from 50%-ile to 3%-ile. She had hyponatremia 122 mEq/L, hyperkalemia 8.0 mEq/L, but was normoglycemic. She was normotensive; EKG was normal. Parents noted progressive hyperpigmentation including her gums, palmar and plantar creases. She had typical external female genitalia with a hypoplastic clitoris (2 mm x 2 mm). ACTH stimulation test showed low cortisol (0.5 ug/dL) at 60 minutes. She was treated with HCT 100 mg/m2 for 5 days, then tapered to maintenance dosing, with FCT and salt supplements. Her ACTH level returned &gt; 5000 pg/ml. Aldosterone, 17-OH-Progesterone, 17-OH-Pregnenolone, 11-Deoxycortisol and androstenedione were undetectable. Pelvic US did not identify uterus or ovaries. Pelvic MRI identified bilateral inguinal testes with enlarged adrenal glands. Karyotype was 46, XY. We suspected StAR deficiency with sex-reversal. Genetic analysis revealed a known homozygous mutation in STAR (c.545G&gt;A). Conclusion: StAR deficiency is clinically indistinguishable from P450scc deficiency and genetic testing is needed. Both entities can present with early or delayed adrenal crisis. While classic for StAR deficiency, adrenal enlargement is inconsistent. Karyotype is vital to identify sex reversal.

Oocyte-Secreted Factors Strongly Stimulate sFRP4 Expression in Human Cumulus Cells

Secreted frizzled-related protein-4 (SFRP4) belongs to a family of soluble ovarian-expressed proteins that participate in female reproduction, particularly in rodents. In humans, SFRP4 is highly expressed in cumulus cells. However, the mechanisms that stimulate SFRP4 in cumulus cells have not been examined. We hypothesise that oocyte-secreted factors such as growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are involved in the regulation of SFRP4. Human cumulus cells were collected from patients undergoing fertility treatments and treated with GDF9 or BMP15 or their combination in the presence of follicle-stimulating hormone (FSH) or vehicle. FSH treatment significantly decreased SFRP4 mRNA levels when compared with nontreated cells. However, SFRP4 mRNA levels were increased significantly by GDF9 plus BMP15 in a concentration-dependent manner in the presence or absence of FSH. The combination of GDF9 plus BMP15 also increased SFRP4 protein levels and decreased the activity of the β-catenin/TCF-responsive promoter significantly. GDF9 plus BMP15 inhibited steroidogenic acute regulatory protein and luteinising hormone/choriogonadotrophin (LH/hCG) receptor stimulation by FSH, while treatment with SFRP4 blocked the stimulatory effect of FSH on these genes. The evidence demonstrates that GDF9 and BMP15 act in coordination to stimulate SFRP4 expression and suggests that SFRP4 mediates the anti-luteinising effects of the oocyte in human cumulus cells.

Effects of Pre-ovulatory Follicular Fluid of Repeat Breeder Dairy Cows on Bovine Fertility Transcriptomic Markers and Oocytes Maturation and Fertilization Capacity

The current study aimed to determine the effects of the preovulatory follicular fluid (FF) of normal heifer (NH) and repeat breeder cows with subclinical endometritis (SCE) or without (nSCE) on oocyte maturation (Experiment 1) and fertilization rates (Experiment 2). Moreover, the pattern of gene expression of cumulus oocyte-complexes was evaluated in Experiment 1. In Experiment 1, nuclear maturation in the nSCE group was higher, compared to that in the SCE group (P = 0.05). In addition, the oocyte nuclear maturation in the normal heifer was significantly higher, in comparison to that of SCE groups (P &lt; 0.05). Furthermore, the mean percentage of normal oocyte fertilization was higher in the nSCE group, compared to that in the SCE group (P &lt; 0.05). The expressions of growth differentiation factor, GDF9; steroidogenic acute regulatory, StAR and follicle-stimulating hormone receptor, FSHr in the NH group were significantly higher, compared to those in SCE and nSCE groups (P &lt; 0.05). Moreover, the expressions of all genes in the nSCE group were not significant, in comparison to those in the SCE group (P &gt; 0.05). The supplementation of oocyte maturation medium with FF from pre-ovulatory follicles of repeat breeder cows resulted in less oocyte maturation and cumulus cell expansion. In conclusion, the lower fertility in RB cows could be ascribed to the lower oocyte maturation rate and less expression of GDF9, StAR, and FSHr in the cumulus-oocyte complexes.

Evolutionary Analysis of the Steroidogenic Acute Regulatory Protein-Related Lipid Transfer Domain and Its Response to Salt Stress In Vitis Vinifera

This study aimed to enhance the understanding of the steroidogenic acute regulatory protein-related lipid transfer (START) domain in Vitis vinifera. A total of 23 members of the VvSTARD gene family were found, which could be divided into five groups. The analyses of the gene codon preference, selective pressure, and tandem replication events of the VvSTARD, AtSTARD, and OsSTARD genomes indicated that tandem replication events occured in grapes, Arabidopsis, and rice genomes. Eight lipid transporter proteins were found in the tertiary structure of the STARD gene family in grapes. The analysis of the expression profiles of the three species microarrays showed that the expression sites of the STARD gene and the response to abiotic stress in the same subgroup had similar characteristics. In addition, quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze the expression of the STARD gene family in grape leaves in response to different hormones and abiotic stresses, and the obtained results were the same as those predicted by the cis-elements and the expression profiles. Furthermore, 35S:STARD5:EGFP was successfully constructed to verify the subcellular prediction results, and the results showed that STARD5 was located in the nucleus. Through the identification of salt tolerance of transgenic tomato, STARD5 was found to regulate the salt stress of plants. Collectively, these data indicated that the VvSTARD gene family plays an important role in response to salt stress.

Dose-Dependent Effect of Polystyrene Microplastics on the Testicular Tissues of the Male Sprague Dawley Rats

Due to the continuous increase in polystyrene microplastics (PS MPs) incorporation in the environment, growing number of adverse effects on living organisms and ecosystem have become a global concern. Therefore, current study was planned to elucidate the impacts of 5 different concentrations control, 2, 20, 200, and 2000 μgL-1 of PS MPs on testicular tissues of rats. PS MPs significantly reduced the activities of antioxidant enzymes (catalase, superoxide dismutase and peroxidase) as well as total protein contents, while elevated the level of lipid peroxidation and reactive oxygen species. Moreover, expressions of steroidogenic enzymes (3β-hydroxysteroid dehydrogenase, 17β-hydroxysteroid dehydrogenase and steroidogenic acute regulatory protein) as well as the levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) in plasma, intra-testicular testosterone and plasma testosterone were reduced and a significant ( P < 0.05) reduction was noticed in the sperm count, motility and viability. Furthermore, PS MPs significantly up-regulated the expressions of Bax and caspase-3, while down-regulated the Bcl-2 expression. The histomorphological assessment revealed significant damages in the testicles as well as decrease in the number of germ cells (spermatogenic, spermatocytes and spermatids). Collectively, PS MPs generated oxidative stress (OS) and caused potential damage to the testicles of rats in a dose-dependent manner.