Prohormone Research Articles

SUN-002 Hormonal Profile of Ovarian Sertoli-Leydig Cell Tumor

Background: Sertoli-Leydig cell tumors account for less than 1% of ovarian tumors, and information about their biochemical markers has been lacking. Objective: The objective was to characterize the hormonal profile of Sertoli-Leydig cell tumor, which should be helpful in recognizing this rare condition in the future. Methods: We reviewed test results including serum total and free testosterone, steroid hormone precursors, and inhibin B levels in a 17-year-old adolescent girl with ovarian Sertoli-Leydig cell tumor who developed secondary amenorrhea for 6 months, deepening of the voice, acne, and severe hirsutism. Results: Our patient had serum testosterone 641 ng/dL (expected 20 - 38), dihydrotestosterone 42.5 ng/dL (expected 3 - 18), 17-OH progesterone 659 ng/dL (expected 20 - 265), androstenedione 869 ng/dL (expected 50 - 224), 17-OH pregnenolone 760 ng/dL (expected 53 - 357), DHEA 1250 ng/dL (expected 4 - 491), and DHEA-S of 366 mcg/dL (expected 44 - 248). Inhibin B level was 321 pg/mL (expected <136); inhibin A was normal. Anti-mullerian hormone, a-fetoprotein, carcinoembryonic antigen, and CA-125 tumor markers were not elevated. Karyotype was female 46,XX. Dexamethasone 0.5 mg QID PO for 4 days resulted in plasma ACTH <5.0 pg/mL and serum cortisol <1.0 mcg/dL, total testosterone 611 ng/dL, free testosterone 25.1 ng/dL (expected <0.04 - 1.09 ng/dL), and 17-OH progesterone 887 ng/dL. Abdomen and pelvis MRI demonstrated a right ovarian mass primarily solid with high cellularity, measured 4.4 x 3.9 cm; there was at least moderate diffuse enhancement of the mass after contrast administration; adrenal glands were normal. Surgical pathology of the resected right ovary revealed moderately to poorly differentiated Sertoli-Leydig cell tumor. Single antibody immunostain procedures with appropriate controls showed a staining pattern supportive of this rare diagnosis: WT-1 showed moderate nuclear staining, calretinin showed a strong positive stain, and CK showed a patchy moderate staining pattern; immunostains for myogenin, desmin, and EMA were negative. Genetic testing revealed a germline heterozygous mutation in DICER1 gene, c3737del, p.Asn1246Metfs*12, establishing the diagnosis of DICER1 syndrome, an autosomal dominant disorder predisposing to cancer. Menses resumed one month after tumor resection. Conclusions: High serum 17-OH progesterone, androstenedione, 17-OH pregnenolone, and DHEA levels used as indicators of adrenocortical function could be markers of an ovarian tumor. If serum 17-OH progesterone and testosterone remain high when cortisol and plasma ACTH are suppressed on Dexamethasone test, a source of 17-OH progesterone and testosterone is other than ACTH-dependent adrenal one. High serum inhibin B level may be sign of an ovarian tumor. Patients with Sertoli-Leydig cell tumor should be screened for DICER1 gene syndrome to assess risk for other rare neoplasms.

SUN-291 Presence of Aberrant Adrenocorticotropic Hormone Precursors in Two Cases of McCune- Albright Syndrome

Background: McCune-Albright syndrome (MAS) is a rare disorder. MAS is caused by an activating postzygotic somatic mutation in the GNAS, and, is classically defined by the occurrence of fibrous dysplasia (FD), café-au-lait skin macules, and precocious puberty. Autonomous GH and/or PRL production in MAS has been reported. However, there have been no reports of ACTH excess in MAS. Method: Plasma ACTH and serum cortisol (F) levels were assessed using electrochemiluminescence immunoassays (Eclusys ACTHTM and Eclusys Cortisol IITM, respectively; Roche Diagnostics K.K., Tokyo, Japan).Clinical Cases: Case1; 42-year-old man showed craniofacial deformities and suffered from multiple bone fractures. He was diagnosed with FD at the age of 23 years. Café-au-lait macules were found on his back. He had slightly acromegaloid features. He showed no cushingoid features. Pituitary adenoma or hyperplasia was not detected by MRI. The diagnosis of GH excess was confirmed by no suppression of serum GH levels by a 75-g oral glucose tolerance test (nadir GH: 2.34 ng/mL) and an elevated serum IGF-I level (307 ng/mL; normal range: 92-257 ng/mL). The patient was treated with monthly subcutaneous lanreotide injection and then GH excess was well controlled. Basal ACTH and F levels in blood were 40.6-63.4 pg/mL and 8.0-10.5 μg/dL, respectively. The urinary free cortisol (UFC) level was 53 μg/day. Autonomous F excess was excluded by the level of midnight F (1.2 μg/dL) and the level of F (0.2 μg/dL) after a low-dose (1 mg) dexamethasone suppression test (DST). Case2; A 32-year-old man was diagnosed with MAS and gigantism at the Pediatrics Department at the age of 5 years. Treatment of GH excess was well controlled by monthly octreotide depot. He had no acromegaloid features and no cushingoid features. Café-au-lait macules were observed from the left flank to the back. Pituitary adenoma or hyperplasia was not detected by MRI. Basal ACTH and F levels in blood were 35.5-73.1 pg/mL and 7.0-11.7 μg/dL, respectively. The UFC level was 61 μg/day. Autonomous F excess was excluded by the level of F (<0.2 μg/dL) after a low-dose (0.5 mg) DST.Possibility of primary adrenal insufficiency was excluded by ACTH stimulation test and/or insulin tolerance test in both cases. The involvement of 11β-HSD1 by GH excess and PC1/3 deficiency were also excluded. Gel exclusion chromatography was then performed. POMC and pro-ACTH were detected and the aberrant ACTH/normal ACTH ratio was 42% in both cases. Conclusion: This is the first report of the presence of aberrant ACTH precursors, particularly POMC, in MAS. A high ratio of circulating ACTH to F may suggest secretion of inactive ACTH precursors in MAS. Further investigations are required to determine whether GNAS mutations or other mechanisms are involved in the presence of aberrant ACTH precursors in MAS.

SAT-734 HSD3B1 Expression in the Human Immune System

3β-hydroxysteroid dehydrogenase-1 (3βHSD1), catalyzing conversion of dehydroepiandrosterone (DHEA) to Δ 4-androstenedione, is an essential enzyme in the pathway toward production of biologically active androgens such as dihydrotestosterone from the adrenally produced precursor DHEA sulfate, the most predominant steroid hormone in circulation. We previously identified, in the gene (HSD3B1) that encodes 3βHSD1, a germline gain-of-function missense-encoding variant that has now been validated in several studies as predicting more rapid progression in prostate cancer patients treated with gonadal testosterone deprivation. Production of androgens from adrenal precursors is important not just in the context of prostate cancer but in other physiologic and pathophysiologic processes, which could include asthma. Androgens are associated with better lung function in both asthma and healthy cohorts, and increasing circulating androgen levels in males help explain the switchover in asthma being more common in boys than girls but then more common in women than men. A main treatment for asthma, as well as other inflammatory processes, is administration of glucocorticoids, yet unresponsiveness to glucocorticoids in a subset of patients remains a major problem. Systemic glucocorticoid administration suppresses adrenally produced DHEA and DHEA-S, suggesting a depleted pool for biologically active androgen production as a mechanism for glucocorticoid resistance. Our surprising preliminary data support a link between glucocorticoid responsiveness and the more active HSD3B1 allele: patients homozygous for the adrenal-permissive HSD3B1(1245C) allele exhibit better response to oral glucocorticoids than those homozygous for the adrenal-restrictive HSD3B1(1245A), with heterozygous patients falling in the middle. This suggests a model in which patients with the more active (adrenal-permissive) form of 3βHSD1 produce sufficient androgens despite the depleted pool of precursor hormones whereas patients with the less active (adrenal-restrictive) form cannot. To further elucidate the link between 3βHSD1 activity and immune response, we assayed HSD3B1 expression in different types of white blood cells. Leukocyte subsets from asthma patients and healthy controls were purified using fluorescence-activated cell sorting, and HSD3B1 expression was analyzed using qPCR. White blood cells of several types expressed HSD3B1 at levels comparable to or greater than both prostate cancer and placental choriocarcinoma cell lines with very robust 3βHSD1 activity. Further determining the cell type specific expression and activity of this key enzyme is an important step in unraveling the link between the HSD3B1 polymorphism and asthma along with potentially many other immune processes.

Chloroplast-associated molecular patterns as concept for fine-tuned operational retrograde signalling.

Chloroplasts compose about one-quarter of the mesophyll cell volume and contain about 60% of the cell protein. Photosynthetic carbon assimilation is the dominating metabolism in illuminated leaves. To optimize the resource expenditure in these costly organelles and to control and adjust chloroplast metabolism, an intensive transfer of information between nucleus-cytoplasm and chloroplasts occurs in both directions as anterograde and retrograde signalling. Recent research identified multiple retrograde pathways that use metabolite transfer and include reaction products of lipids and carotenoids with reactive oxygen species (ROS). Other pathways use metabolites of carbon, sulfur and nitrogen metabolism, low molecular weight antioxidants and hormone precursors to carry information between the cell compartments. This review focuses on redox- and ROS-related retrograde signalling pathways. In analogy to the microbe-associated molecular pattern, we propose the term 'chloroplast-associated molecular pattern' which connects chloroplast performance to extrachloroplast processes such as nuclear gene transcription, posttranscriptional processing, including translation, and RNA and protein fate. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

The interaction between group 2 innate lymphoid cells and lipid metabolism

Abstract Innate lymphoid cells (ILCs) have been found in many tissue sites but are enriched in barrier surfaces such as intestine, lung and skin. They lack antigen-specific receptors yet respond to alarmin signals that are released by tissue, to resemble the cytokine production and effector function of T cells. ILCs play important roles in host defense, tissue repair, autoimmune disorders, inflammation, metabolic homeostasis and more. Recent progress in immunometabolism has highlighted the diverse roles of lipid metabolism in the response and function of immune cells including lymphocytes. But the interaction between ILC2s and lipid metabolism is poorly understood. Our preliminary studies show that, upon activation, intestinal ILC2s significantly upregulate the expression of several genes that are important for cholesterol metabolism. Cholesterol is an essential structural component of mammalian cell membranes and is the precursor for bile acids, vitamin D and steroid hormones. Cholesterol metabolism disorder leads to severe diseases such as hypercholesterolemia, atherosclerosis and diabetes. By using various mouse models and multi-disciplinary experimental approaches, we will study if and how cholesterol regulates the homeostasis, activation and cytokine production of ILC2s and if cholesterol regulation on ILC2s contributes to host defense against helminth infection. We will also investigate if ILC2s in turn modulate lipid, especially cholesterol, metabolism and if ILC2s play any role in hypercholesterolemia or atherosclerosis.

Progesterone and breast

Progesterone (Pg) is a pregnancy-related hormone that prepares the endometrium for the implantation of the fertilized zygote and suppresses myometrial contractility for the maintenance of pregnancy. At high concentrations, it acts as a natural immunosuppressant avoiding the rejection of a half allogeneic foetus. It is the precursor of many other related steroid hormones, but what is its role in the human breast? In this chapter, we will discuss some aspects related to Pg and its role in breast development and in the neoplastic disease. Understanding the mechanisms related to Pg-induced effects in the normal and neoplastic mammary gland will light the way to exploit this hormone signalling pathway therapeutically. We will introduce some aspects of the effects of progestins in normal breast development, breast cancer risk and in neoplastic growth, and we will describe ongoing clinical trials in breast cancer using progestins or antiprogestins.

Elevated Circulating Cardiac Troponins in Chronically Catheterized Pre‐and Post‐Natal Sheep

BACKGROUNDCirculating markers of cardiac damage have been reported to be elevated in cord blood of healthy infants. This elevation is reported to be independent of the stress of labor, as elevations are also reported in the cord blood of healthy infants delivered by Caesarian section prior to labor. We hypothesized that circulating markers of cardiac damage are elevated in the late‐term fetus prior to delivery.METHODSThree fetal sheep were surgically instrumented with indwelling catheters in the carotid artery and coronary sinus. Sheep were recovered from surgery, and daily blood samples were taken to determine circulating levels of cardiac troponin I (cTnI), c‐reactive protein (CRP), and N‐terminal pro hormone BNP (nt‐ProBNP) using the Siemens DIMENSION VISTA, and cardiac troponin T (cTnT) using the Roche cobas e601 STAT. Data were visualized to determine trends.RESULTSWe found that circulating cTnI and cTnT levels tend to decline in the week preceding birth. Troponins also were elevated following natural vaginal delivery. CRP and nt‐ProBNP levels were not detectable.CONCLUSIONSThese data support our hypothesis that a cardiac process is releasing cardiac troponins to circulate 1–2 weeks prior to birth. Further investigation is required to determine if this is a transient event, or if levels are high throughout fetal development. We also found that levels decline as term approaches, but that birth re‐stimulates release of these markers of cardiac damage.Support or Funding InformationOHSU Medical Research FoundationCirculating markers of cardiac damage in chronically catheterized perinatal sheep. The x‐intercept represents day of surgery, while the dashed line represents the day of birth. Solid symbols = blood samples from the carotid artery. Open symbols = blood samples from the coronary sinus.Figure 1

The effect of RAS blockers on the clinical characteristics of COVID-19 patients with hypertension.

BackgroundCoronavirus disease 2019 (COVID-19), caused by a novel coronavirus (designated as SARS-CoV-2) has become a pandemic worldwide. Based on the current reports, hypertension may be associated with increased risk of sever condition in hospitalized COVID-19 patients. Angiotensin-converting enzyme 2 (ACE2) was recently identified to functional receptor of SARS-CoV-2. Previous experimental data revealed ACE2 level was increased following treatment with ACE inhibitors (ACEIs) and angiotensin receptor blockers (ARBs). Currently doctors concern whether these commonly used renin-angiotensin system (RAS) blockers-ACEIs/ARBs may increase the severity of COVID-19.MethodsWe extracted data regarding 50 hospitalized hypertension patients with laboratory confirmed COVID-19 in the Renmin Hospital of Wuhan University from Feb 7 to Mar 03, 2020. These patients were grouped into RAS blockers group (Group A, n=20) and non-RAS blockers group (Group B, n=30) according to the basic blood pressure medications. All patients continued to use pre-admission antihypertensive drugs. Clinical severity (symptoms, laboratory and chest CT findings, etc.), clinical course, and short time outcome were analyzed after hospital admission.ResultsTen (50%) and seventeen (56.7%) of the Group A and Group B participants were males (P=0.643), and the average age was 52.65±13.12 and 67.77±12.84 years (P=0.000), respectively. The blood pressure of both groups was under effective control. There was no significant difference in clinical severity, clinical course and in-hospital mortality between Group A and Group B. Serum cardiac troponin I (cTnI) (P=0.03), and N-terminal (NT)-pro hormone BNP (NT-proBNP) (P=0.04) showed significant lower level in Group A than in Group B. But the patients with more than 0.04ng/mL or elevated NT-proBNP level had no statistical significance between the two groups. In patients over 65 years or under 65 years, cTnI or NT-proBNP level showed no difference between the two groups.ConclusionsWe observed there was no obvious difference in clinical characteristics between RAS blockers and non-RAS blockers groups. These data suggest ACEIs/ARBs may have few effects on increasing the clinical severe conditions of COVID-19.

Allopregnanolone: From molecular pathophysiology to therapeutics. A historical perspective

Allopregnanolone is synthesized in the central nervous system either de novo from cholesterol or from steroid hormone precursors like progesterone and pregnenolone. Over the past 30 years, direct and rapid, non-genomic actions of allopregnanolone and its derivatives via GABAA receptors have been demonstrated. Changes in brain levels of allopregnanolone during pregnancy and in the postpartum period, or during exposure to protracted stress appear to play a crucial role in the pathophysiology of mood disorders. The discovery that allopregnanolone at low (nanomolar) concentrations elicits marked anxiolytic, anti-stress and antidepressant effects by facilitating allosterically the action of GABA at extrasynaptic GABAA receptors has provided new perspectives for the discovery of novel drugs useful for the treatment of mood disorders. These findings have led to the seminal clinical studies that recently demonstrated that treatment with allopregnanolone (i.e., brexanolone) can dramatically and rapidly improve the symptoms of postpartum depression in many patients.

Feed and animal nutrition: insect as animal feed

In the last ten years, insects have been widely recognized for food and feed. Many studies using insects (Black soldier fly, cricket and silkworm pupae) as feed to explore their nutritional value and apply it in some animal rations as an alternative source of protein and fat. Data showed that Black soldier fly (BSF) has high protein and fat, especially lauric acid which potential as an antibacterial pathogen. In vitro study showed that BSF extracted could phagocyte 99% of pathogen bacteria such as E coli and S aureus. Meanwhile, cricket has also high fat and protein, including glutamic acid for brain development, and mineral Fe has to support hemoglobin synthesis. Silkworm pupae contain 67.48% of unsaturated fatty acid with linoleic acid (omega 6) and linolenic acid (omega 3) which very good as a steroid hormone precursor. Application insects to substitute imported ingredients (Casein, fish, meat bone, and soybean meal) in poultry diets resulted in a good performance and the animal still healthy with low mortality. The BSF and cricket meal as part of milk replacer could produce ADG of pre-weaning goat/sheep around 100-120 g/h/d, meanwhile if those ingredients are used as part of creep feed, the daily gain of post-weaning goat/sheep could be more than 150 g/h/d. Application of silkworm meal as a substitute for the fish meal could improve egg quality and quantity of laying quail. It was concluded that insects have the potential to be used as a protein source in the poultry and pre-weaning small ruminant rations without any negative effect on the animal.

The Effect of Ethanol Extract Karamunting (Melastoma Malabathricum) Leaf of Ovarian Maturation of Mud Crab (Scylla sp) in Traditional Ponds

Karamunting is a plant that contains cholesterol. Cholesterol is needed by mud crabs as precursors of reproductive hormones in ovarian development. This study aims to determine the effect of karamunting leaf extract which is injected with mangrove crabs (Scylla sp) on the level of ovarian maturity. The stages of the research included extraction of karamunting leaves using ethanol 70%, experiments by injecting the parent crabs consisting of 3 treatments A (control), B treatment (0.25 mg / g body weight), C treatment (0.5 mg / g body weight) maintained for 20 days by feeding trash fish and measuring parameters including morphological ovarian development, somatic gonad index (GSI), and hepatosomatic index (HSI). The results showed that by giving karamunting extract at the end of the GSI study on control (1.70%), treatment A (5.61%), treatment B (4.24%) while on ovarian development with morphological observation at the end of the study on treatment ovarian control develops in the TKG II phase, A treatment in the TKG V phase while treatment B in TKG IV. This shows that by giving karamunting extract can provide the development of mud crab ovaries.

Acute Statin Administration Reduces Levels of Steroid Hormone Precursors.

Cholesterol-lowering statin drugs are used by approximately 25% of US adults 45 years of age and older and frequency of use is even higher among the elderly. Cholesterol provides the substrate for steroid hormone synthesis and its intracellular concentrations are tightly regulated. Our aim was to evaluate whether statin use acutely changes the circulating levels of cortisol, other glucocorticoid precursor molecules and their metabolites. Fourteen subjects not taking statins were administered a single oral dose (2 mg) of pitavastatin. Blood samples collected at baseline and 24 h post-treatment were analyzed for plasma cholesterol and steroid hormone profile. A parallel study in mice entailed the administration of atorvastatin (10 mg/kg) via orogastric delivery for three consecutive days. Cholesterol and corticosterone levels were quantified at baseline and at 1-day and 1-week post-treatment. Several precursor molecules in the steroidogenic pathway (corticosterone, cortisone, and 11-deoxycortisol) were significantly decreased 24 h after administration of a single dose of pitavastatin in human study subjects. Their circulating cholesterol concentrations were unchanged. In mice, there were no significant differences in serum cholesterol or corticosterone at 1-day or 1-week post-treatment compared to both pre-treatment baseline levels and control group levels. We conclude that acute dysregulation of the production of certain glucocorticoid precursor molecules was observed after a single treatment with a lipophilic statin drug. This may be of clinical relevance for individuals with underlying or subclinical adrenal insufficiency.

The structure of human thyroglobulin.

SummaryThyroglobulin is the protein precursor of thyroid hormones, which are essential for growth, development and control of metabolism in vertebrates1,2. Hormone synthesis from thyroglobulin (TG) occurs in the thyroid gland via the iodination and coupling of pairs of tyrosines and is completed by TG proteolysis3. Tyrosine proximity within TG is thought to enable the coupling reaction but hormonogenic tyrosines have not been clearly identified and the lack of a three-dimensional structure of TG has prevented mechanistic understanding4. Here we present the structure of full-length human thyroglobulin at ~3.5 Å resolution determined by electron cryomicroscopy (cryo-EM). We identified all hormonogenic tyrosine pairs in the structure and verified them via site-directed mutagenesis and in vitro hormone production assays using human TG expressed in HEK cells. Analysis revealed that proximity, flexibility and solvent exposure of the tyrosines are the key characteristics of hormonogenic sites. Transferring the reaction sites from TG to an engineered tyrosine donor-acceptor pair in the unrelated bacterial maltose binding protein (MBP) yielded hormone production with efficiency comparable to TG. Our study provides a framework to further understand the production and regulation of thyroid hormones.

Acute responses of comprehensive gonadosteroids and corticosteroids to resistance exercise before and after 10weeks of supervised strength training.

What is the central question of this study? Although acute responses of the principal gonadosteroid and corticosteroid hormones to resistance exercise are well documented, there is no information regarding how the key lower-concentration intermediary hormones respond and potentially influence these hormonal pathways. What is the main finding and its importance? This study provides evidence for cascading conversions of some gonadosteroids, and the data suggest that the testosterone concentration increases independently of these hormones. These findings challenge future studies to determine the exact physiological roles of the lower-concentration gonadosteroids and corticosteroids during and immediately after resistance exercise. Resistance training is a potent stimulus for muscle growth, and steroid hormones are known to play a role in this adaptation. However, very little is known about the acute exercise-induced gonadosteroid and corticosteroid hormone responses, including those of key lower-concentration intermediate hormones. The present study determined the acute responses of these steroid hormone families using quantitative ultra-high performance liquid chromatography tandem mass spectrometry after resistance exercise in strength-trained men. Venous and fingertip blood samples were obtained pre-, mid-, 5min post- and 15min post-resistance exercise, both before and after 10weeks of supervised resistance training. The experimental resistance exercise sessions consisted of three sets of 10 repetitions of bilateral leg-press exercise and three sets of 10 repetitions of unilateral knee-extension exercise, with 2 and 1min recovery between sets, respectively. Statistically significant (P<0.05) increases in the concentration of hormones in the gonadosteroid [including dehydroepiandrosterone (DHEA), androstenedione, testosterone and estrone] and the corticosteroid (including cortisol, corticosterone and cortisone) families were demonstrated after both experimental resistance exercise sessions, irrespective of training status. Correlation analyses revealed relationships between the following hormones: (i) DHEA and androstenedione; (ii) DHEA and cortisol; (iii) androstenedione and estrone; and (iv) 11-deoxycortisol and cortisol. Testosterone appears to increase acutely and independently of other intermediary hormones after resistance exercise. In conclusion, lower-concentration intermediary gonadosteroids (e.g. estrone) and corticosteroids (e.g. corticosterone) respond robustly to resistance exercise in strength-trained men, although it seems that testosterone concentrations are regulated by factors other than the availability of precursor hormones and changes in plasma volume.

Novel Insights into Dietary Phytosterol Utilization and Its Fate in Honey Bees (Apis mellifera L.).

Poor nutrition is an important factor in global bee population declines. A significant gap in knowledge persists regarding the role of various nutrients (especially micronutrients) in honey bees. Sterols are essential micronutrients in insect diets and play a physiologically vital role as precursors of important molting hormones and building blocks of cellular membranes. Sterol requirements and metabolism in honey bees are poorly understood. Among all pollen sterols, 24-methylenecholesterol is considered the key phytosterol required by honey bees. Nurse bees assimilate this sterol from dietary sources and store it in their tissues as endogenous sterol, to be transferred to the growing larvae through brood food. This study examined the duration of replacement of such endogenous sterols in honey bees. The dietary 13C-labeled isotopomer of 24-methylenecholesterol added to artificial bee diet showed differential, progressive in vivo assimilation across various honey bee tissues. Significantly higher survival, diet consumption, head protein content and abdominal lipid content were observed in the dietary sterol-supplemented group than in the control group. These findings provide novel insights into phytosterol utilization and temporal pattern of endogenous 24-methylenecholesterol replacement in honey bees.

Role of Akt and mammalian target of rapamycin signalling in insulin-like growth factor 1-mediated cell proliferation in porcine Sertoli cells.

The critical role of insulin-like growth factor (IGF) 1 in promoting Sertoli cell proliferation invivo and invitro has been established, but its downstream signalling mechanisms remain unknown. In addition to mitogenic effects, a role for IGF1 in mediating cholesterol biosynthesis within testes has been implied. The aims of this study were to investigate the roles of: (1) phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (mTOR) signalling in IGF1-mediated Sertoli cell proliferation; and (2) IGF1 in mediating cholesterol biosynthesis in Sertoli cells. Primary cultures of Sertoli cells were prepared from 1-week-old porcine testes. On Day 3 of culture, Sertoli cells were treated with 300ng mL-1 IGF1, alone or in combination with inhibitors of IGF1 receptor (2μM picropodophyllotoxin), Akt (1μM wortmannin) or mTOR (200nM rapamycin). Cells were cultured for 30min and phosphorylation levels of Akt, mTOR and p70 ribosomal protein S6 kinase (p70S6K) were determined by immunoblotting. Cell proliferation and quantitative polymerase chain reaction assays were conducted using cells cultured for 24h. IGF1 increased phosphorylation of Akt, mTOR and p70S6K and cell proliferation, and these effects were inhibited by inhibitors of IGF1R, Akt and mTOR. Furthermore, IGF1 upregulated the expression of cholesterol biosynthetic genes (3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS1) and cytochrome P450, family 5, subfamily A, polypeptide 1 (CYP5A1)), but not sterol regulatory element-binding transcription factor 1 (SREBF1). Increased phosphorylation of p70S6K, a major downstream target of mTOR, and upregulated expression of genes involved in cholesterol biosynthesis are indicative of the key role played by IGF1 in regulating the synthesis of cholesterol, the precursor for steroid hormones.

In silico prediction of neuropeptides from the neural ganglia of Pacific abalone Haliotis discus hannai (Mollusca: Gastropoda)

Neuropeptides play a central role in the regulation of reproduction, growth, development, and various other physiological processes. In our study, 26 neuropeptide precursors were identified from the pleuropedal and cerebral ganglia of Pacific abalone using transcriptome analysis. Of them, two (neuromacin and neuroglian) were potentially novel and two (myomodulin and FMRFamide) were involved in the reproductive regulation of mollusks. A BLAST search indicated that most of these neuropeptides exhibited the highest homologies with neuropeptides of other protostomian species. Myomodulin, FMRFamide, and molluscan insulin-related peptides shared 87%, 72%, and 88% sequence identities with homologous peptides of the tropical gastropod mollusk, Haliotis asinina. In silico analysis revealed that these identified neuropeptide precursors were likely to be extracellular secreted proteins. Alignment of multiple cerebrin, FFamide, and insulin-related peptide sequences illustrated that most of their biologically active residues were highly conserved with other invertebrate homologous neuropeptide residues. Three-dimensional (3D) structures of adipokinetic hormone, allatostain (A, B, and C), allatotropin, cerebrin, clionin, conopressin, elevenin, and neuromycin precursors in H. discus hannai exhibited a helix-loop-helix structure. Based on phylogenetic analysis, buccalin A1 and A2, allatostain B, and FCAP neuropeptide precursors of Pacific abalone formed a clade with other gastropod and bivalve mollusks neuropeptide precursors. This study provides a novel insight for further functional studies of abalone and other gastropod mollusks.

Steroid sulfatase inhibiting lanostane triterpenes – Structure activity relationship and in silico insights

Steroid sulfatase (STS) transforms hormone precursors into active steroids. Thus, it represents a target of intense research regarding hormone-dependent cancers. In this study, three ligand-based pharmacophore models were developed to identify STS inhibitors from natural sources. In a pharmacophore-based virtual screening of a curated molecular TCM database, lanostane-type triterpenes (LTTs) were predicted as STS ligands. Three traditionally used polypores rich in LTTs, i.e., Ganoderma lucidum Karst., Gloeophyllum odoratum Imazeki, and Fomitopsis pinicola Karst., were selected as starting materials. Based on eighteen thereof isolated LTTs a structure activity relationship for this compound class was established with piptolinic acid D (1), pinicolic acid B (2), and ganoderol A (3) being the most pronounced and first natural product STS inhibitors with IC50 values between 10 and 16 µM. Molecular docking studies proposed crucial ligand target interactions and a prediction tool for these natural compounds correlating with experimental findings.

Evaluating Effects of a Critical Micronutrient (24-Methylenecholesterol) on Honey Bee Physiology.

Although poor nutrition is cited as one of the crucial factors in global pollinator decline, the requirements and role of several important nutrients (especially micronutrients) in honey bees are not well understood. Micronutrients, viz. phytosterols, play a physiologically vital role in insects as precursors of important molting hormones and building blocks of cellular membranes. There is a gap in comprehensive understanding of the impacts of dietary sterols on honey bee physiology. In the present study, we investigated the role of 24-methylenecholesterol—a key phytosterol—in honey bee nutritional physiology. Artificial diets with varying concentrations of 24-methylenecholesterol (0%, 0.1%. 0.25%, 0.5%, 0.75%, and 1% dry diet weight) were formulated and fed to honey bees in a laboratory cage experiment. Survival, diet consumption, head protein content, and abdominal lipid contents were significantly higher in dietary sterol-supplemented bees. Our findings provide additional insights regarding the role of this important sterol in honey bee nutritional physiology. The insights gleaned from this study could also advance the understanding of sterol metabolism and regulation in other bee species that are dependent on pollen for sterols, and assist in formulation of a more complete artificial diet for honey bees (Apis mellifera Linnaeus, 1758) (Hymenoptera: Apidae).

Proteomic and Genomic Endocrine Responses in Oxytocin Knockout Mice

Gene knockout animal models have proven extremely useful for studying the effects of a single gene product in systems where human studies are not an option. The nine amino acid peptide, oxytocin, has been shown to play dual roles as a peptide hormone in parturition and milk production; and as a neuropeptide in social and maternal behavior. In this study, we investigated the effect of the absence or presence of a functioning oxytocin allele on other endocrine genes. Taking advantage of an oxytocin-null murine model (OTKO-/-), gene expression levels were assayed for the prohormones; pro-oxytocin, pro-vasopressin, and proopiomelanocortin; processing enzymes, prohormone convertase 1, prohormone convertase 2 and carboxypeptidase E; and transcription factors, c-Fos, and c-Jun in OT+/+ and OTKO-/- animals. Significant changes in gene expression levels were found to occur in cortex and hypothalamic tissues of OTKO-/- mice. Specifically, there was an up-regulation of POMC, PC2, c-Fos and c-Jun in hypothalamus. PC2 was also found to be up-regulated in the cortex. Western blot analysis of the affected tissues showed similar results. We conclude that the absence of the peptide hormone oxytocin can affect other peptide hormone systems, including processing enzymes and transcription factors.