Steroidogenic Gene Expression Research Articles

Genome-Wide Identification and Characterization of the BRD Family in Nile Tilapia (Oreochromis niloticus).

Simple SummaryNile tilapia is a good model for genome-wide identification and examination of the expression and role of gene families. In this study, we identified 54 bromodomain genes (BRDs) divided into eight subfamilies in Nile tilapia. Phylogenetic analysis revealed a high conservation of the BRDs family in vertebrates, with BRDs expansion due to fish-specific duplications. Most of the BRDs displayed sexually dimorphic expression in the gonads at 90 and 180 dah (days after hatching), including 21 testis-dominated genes (brdt, brd4a and brd2b, etc.), and 9 ovary-dominated genes (brd3b, brd2a and kat2a, etc.). Male fish treated with JQ1 (BET subfamily inhibitor) displayed abnormal spermatogenesis. The numbers of germ cells were reduced and the expression of steroidogenic enzyme genes was downregulated, while the expression of apoptosis-promoting genes was elevated in the testes of treated fish.The bromodomain (BRD) proteins specifically recognize the N-acetyllysine motifs, which is a key event in the reading process of epigenetic marks. BRDs are evolutionarily highly conserved. Over recent years, BRDs attracted great interest because of their important roles in biological processes. However, the genome-wide identification of this family was not carried out in many animal groups, in particular, in teleosts. Moreover, the expression patterns were not reported for any of the members in this family, and the role of the BRD family was not extensively studied in fish reproduction. In this study, we identified 16 to 120 BRD genes in 24 representative species. BRDs expanded significantly in vertebrates. Phylogenetic analysis showed that the BRD family was divided into eight subfamilies (I–VIII). Transcriptome analysis showed that BRDs in Nile tilapia (Oreochromis niloticus) exhibited different expression patterns in different tissues, suggesting that these genes may play different roles in growth and development. Gonadal transcriptome analysis showed that most of the BRDs display sexually dimorphic expression in the gonads at 90 and 180 dah (days after hatching), including 21 testis-dominated genes (brdt, brd4a and brd2b, etc.), and nine ovary-dominated genes (brd3b, brd2a and kat2a, etc.). Consistent with transcriptomic data, the results of qRT-PCR and fluorescence in situ hybridization showed that brdt expression was higher in the testis than in the ovary, suggesting its critical role in the spermatogenesis of the tilapia. Male fish treated with JQ1 (BET subfamily inhibitor) displayed abnormal spermatogenesis. The numbers of germ cells were reduced, and the expression of steroidogenic enzyme genes was downregulated, while the expression of apoptosis-promoting genes was elevated in the testis tissue of treated fish. Our data provide insights into the evolution and expression of BRD genes, which is helpful for understanding their critical roles in sex differentiation and gonadal development in teleosts.

Exposure of carbon nanotubes affects testis and brain of common carp

Carbon nanotubes production has been rapidly increasing for many potential applications, however, the environmental impact of this nanomaterial needs to be comprehended. The present work focused on unraveling the effects of single-walled carbon nanotubes (SWCNT) in the common carp, Cyprinus carpio. The physicochemical properties of SWCNT were analyzed with X-ray diffraction, Fourier transforms infra-red, UV-Vis absorption, transmission electron microscopy (TEM), and Raman spectroscopy before testing for exposure impact. The effects of SWCNT, were investigated by exposing to two doses viz., 10 and 50 μg/L, for 7 days in adult common carp, in vivo. Expression of key steroidogenic and transcription factor genes related to testis and brain were downregulated after the treatment. The concomitant decreases in serum testosterone and 11-ketotestosterone levels revealed the impact of SWCNT after exposure. Further, SWCNT exposure induced antioxidant enzymes namely glutathione-S-transferases, superoxide dismutase, and catalase in both testis and brain. Concurrently, histological and TEM analysis of testis revealed structural disarray. In addition, SWCNT treatment, in testicular and brain primary cell cultures decreased cell viability with an increase of reactive oxygen species levels, leading to a significant elevation of apoptotic cells. In line with this, low mitochondrial membrane potential and DNA damage were also observed during post SWCNT treatment. Taken together, transient exposure of SWCNT causes toxic effects and alters testicular and brain function in the common carp. Thus, the discharge of carbon nanotubes poses a greater risk to the aquatic environment warranting regulatory measures.

Brown adipose tissue activation with ginsenoside compound K ameliorates polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common metabolic and endocrine disease affecting women of reproductive age. Due to its complex aetiology, there is no currently effective cure for PCOS. Brown adipose tissue (BAT) activity is significantly decreased in PCOS patients, and BAT activation has beneficial effects in animal models of PCOS. Here, we investigated the effect of ginsenoside compound K (CK) in an animal model of PCOS and its mechanism of BAT activation. Primary brown adipocytes, Db/Db mice and dehydroepiandrosterone (DHEA)-induced PCOS rats were used. The core body temperature, oxygen consumption, energy metabolism related gene and protein expression were assessed to identify the effect of CK on overall energy metabolism. Oestrous cycle, serum sex hormone, ovarian steroidogenic enzyme gene expression and ovarian morphology were also evaluated following CK treatment. Our results indicated that CK treatment could significantly protect against body weight gain in Db/Db mice via BAT activation. Furthermore, we found that CK treatment could normalize hyperandrogenism, oestrous cyclicity, normalize steroidogenic enzyme expression and decrease the number of cystic follicles in PCOS rats. Interestingly, as a potential endocrine intermediate, C-X-C motif chemokine ligand-14 protein (CXCL14) was significantly up-regulated following CK administration. In addition, exogenous CXC14 supplementation was found to reverse DHEA-induced PCOS in a phenotypically similar manner to CK treatment. In summary, CK treatment significantly activates BAT, increases CXCL14 expression and ameliorates PCOS. These findings suggest that CK might be a potential drug candidate for PCOS treatment.

Cross-talk between down-regulation of steroidogenic genes expression and oxidative and apoptotic biomarkers in testes induced by administration of tramadol and boldenone and their combination in male albino rats.

Objective(s):The testis is the male reproductive gland or gonad having two vital functions: to produce both sperm and androgens, primarily testosterone. The study aimed to investigate the effect of tramadol and boldenone injected alone or in combination for 2 months in rats on testicular function.Materials and Methods:Group 1, normal control; Group 2, tramadol HCl (TRAM) (20 mg/kg bwt.) (IP); Group 3, boldenone undecylenate (BOLD) (5 mg/kg bwt) (i.m); Group 4, combination of TRAM (20 mg/kg bwt.) and BOLD (5 mg/kg); respectively for 2 months. Results:TRAM and BOLD alone and in combination showed deteriorated testicular functions, lowered serum steroid levels (FSH, LH, and testosterone), elevation in oxidative biomarkers (MDA & NO) and reduction in GSH and SOD, down-regulation of StaR and HSD17B3 as well as histopathological testicular assessment using H&E staining revealing massive degenerative changes in the seminiferous epithelium and vacuolar changes of most of the spermatogenic stages in both TRAM and BOLD groups. PAS stain showed an intensive reaction in the interstitial tissue between the tubules in the TRAM group. Masson trichrome stain showed abundant collagen fiber deposits in the tunica albuginea with congested BV in the TRAM group. Conclusion:The study illuminated the hazard of administration of these drugs for a long period as well as the prominent deleterious effects reported on concurrent use of both drugs.

Polystyrene microplastics induced male reproductive toxicity and transgenerational effects in freshwater prawn

Microplastics have become pervasive environmental pollutants, especially in freshwater rivers and lakes. However, how freshwater prawns' reproductive system is affected by polystyrene microplastics (PS-MPs) remains incompletely understood. Thus, the present study aimed to determine the effect of PS-MPs on the male reproductive system and offspring larval immunity in oriental river prawn. Acute exposure to PS-MPs decreased the survival rate and heart rate of prawn larvae. After chronic exposure to PS-MPs (2 and 20 mg/L) for four weeks, the oxidative stress generation in testis tissue indicated a negative impact on male prawn testicular function. PS-MPs disrupted testicular germ cell quality and caused sex hormone imbalance, leading to reduced hatching success and survival of F1 larvae, despite not being exposed to PS-MPs. Steroidogenic gene expression was altered and apoptosis-related genes had higher expression in the gonads after parental exposure to PS-MPs. Decreased immunity-related enzyme activities were observed in F1 larvae with/without continued PS-MPs exposure, compared with those in untreated prawns. A concentration-dependent increase in bioaccumulation of PS-MPs in different tissues of larval offspring was observed. Thus, PS-MPs had multiple effects on male reproductive dysfunction and transgenerational toxicity in prawns. Our findings provide a novel insight into the reproductive toxicity mechanism of microplastics in freshwater crustaceans.

Age-related endoplasmic reticulum stress represses testosterone synthesis via attenuation of the circadian clock in Leydig cells

Senile animals exhibit a high risk of elevated endoplasmic reticulum (ER) stress, attenuated circadian clock, and impaired steroidogenesis in testes. However, how these three processes are intertwined in mouse Leydig cells remains unclear. In this study, a mouse model of aging and hydrogen peroxide (H2O2)-induced senescent TM3 Leydig cells were used to dissect the connections among ER stress, circadian oscillators, and steroidogenesis in Leydig cells. Additionally, thapsigargin (Tg, 60 nM)/tunicamycin (Tm, 60 ng/mL)-induced ER stress were established to investigate the underlying mechanisms by which ER stress regulated testosterone synthesis via circadian clock-related signaling pathways in TM3 cells and primary Leydig cells. Elevated ER stress, attenuated circadian clock, and diminished steroidogenesis were detected in the testes of aged mice (24-month-old) and H2O2-induced (200 μM) senescent TM3 cells in comparison with their control groups. Tg/Tm-induced ER stress reduced the transcription of the circadian clock and steroidogenic genes in TM3 cells and LH-treated (100 ng/mL) primary Leydig cells. Furthermore, 4-phenylbutyric acid (4-PBA, 1 μM), an inhibitor of ER stress, alleviated the inhibitory effect of Tg-mediated ER stress on Per2:Luc oscillations in primary Leydig cells isolated from mPer2Luc knock-in mice, and attenuated the repressive effect of H2O2-induced or Tg-mediated ER stress on the transcription of circadian clock and steroidogenic genes expression and testosterone synthesis in TM3 cells. Collectively, these data indicate that age-related ER stress represses testosterone synthesis via attenuation of the circadian clock in Leydig cells.

Deletion of Wt1 during early gonadogenesis leads to differences of sex development in male and female adult mice.

Assessing the role of the WT1 transcription factor (WT1) during early gonad differentiation and its impact on adult sex development has been difficult due to the complete gonadal agenesis and embryonic lethality exhibited by Wt1KO mouse models. Here, we generated Wt1LoxP/GFP;Wt1Cre mice, the first Wt1KO mouse model that reaches adulthood with a dramatically reduced Wt1 expression during early gonadogenesis. Wt1LoxP/GFP;Wt1Cre mice lacked mature gonads and displayed genital tracts containing both male and female genital structures and ambiguous genitalia. We found that WT1 is necessary for the activation of both male and female sex-determining pathways, as embryonic mutant gonads failed to upregulate the expression of the genes specific for each genetic programme. The gonads of Wt1LoxP/GFP;Wt1Cre mice showed a lack of production of Sertoli and pre-granulosa cells and a reduced number of germ cells. NR5A1 and the steroidogenic genes expression was modulated differently in XY and XX Wt1LoxP/GFP;Wt1Cre gonads, explaining the mutant phenotypes. Further studies of the XX Wt1LoxP/GFP;Wt1Cre gonads revealed that deletion of WT1 at an early stage impaired the differentiation of several cell types including somatic cells and the ovarian epithelium. Through the characterisation of this Wt1KO mouse model, we show that the deletion of Wt1 during early gonadogenesis produces dramatic defects in adult sex development.

Effects of sugar cane extract on steroidogenesis in testicular interstitial cells of male Japanese quail (Coturnix japonica)

Sugar cane extract (SCE) is the end product of glucose, fructose, and sucrose elimination in molasses. SCE has various biological effects, such as anti-inflammation and antioxidation, and it is commonly found in animal feed. The present research is aimed at investigating the reproductive endocrine influence of SCE in male Japanese quails (Coturnix japonica) by feeding SCE containing food. In addition, in vitro Leydig cell culture was conducted to clarify the mechanism of SCE's influence. Our results showed that SCE feed extended the latency to the first neck grab, decreased male quail testis and epididymis weights, cloaca gland size, and reduced serum testosterone concentrations. Steroidogenic enzymes 3βHSD, 17βHSD, P450c17, and P450scc gene expression in the testis were decreased in the SCE groups. Western blot analysis showed decreased 3βHSD in the testis after feeding SCE. Isolated testicular interstitial cells cultured with SCE and ovine-LH suppressed testosterone secretion and 3βHSD gene expression. In conclusion, SCE as a feed additive has an impact on the sexual behavior and reproductive function of male Japanese quail, with the suppression of steroidogenesis in the Leydig cell. Our results may provide beneficial information to the livestock management and the poultry industry.

Effect of retained placenta and clinical mastitis on reproduction parameters, immune response, and steroidogenic receptors gene expression in postpartum crossbred dairy cows.

The purpose of this study was to evaluate the effect of retained placenta (RP) and clinical mastitis (CM) on the reproductive efficiency of crossbred dairy cows during the postpartum period and the effect in some innate immune system indicators. For this, two experiments were carried out. In the first, a total of 232 cows were evaluated and divided as: healthy control (n = 184), RP (n = 22), and CM (n = 26) groups. The RP and CM was evaluated until 30 days postpartum (DPP) and reproductive rates were measured. In experiment 2, cows were divided in control (n = 10), RP (n = 10), and CM (n = 30) groups. Between 40 and 50 DPP, clinical, gynecological examination and endometrial cytobrush were performed to evaluate subclinical endometritis (SE) and gene expression of interleukins 1β (IL-1β) and 6 (IL-6), chemokine ligand 5 (CCL5), estrogen α (ESR1), and progesterone (PGR) receptors by qRT-PCR analysis. In experiment 1, the conception rate at 1st artificial insemination (AI) was lower in RP and CM groups and pregnancy rate at 150 days decreased in CM group. Calving-to-1st AI interval and days open were shorter in healthy cows. In experiment 2, the occurrence of SE was 26.7% and higher in RP and CM groups. The expression of IL-1β increased in RP and CM groups, while IL-6 was less expressed in RP group. The CCL5, ESR1, and PGR were similar between groups. In conclusion, cows with RP and CM had their reproductive efficiency negatively affected and had they initial pro-inflammatory response improved by the increase of IL-β.

Phospholipase D2 Loss Impairs Low Salt‐Induced Increases in Steroidogenic Gene Expression with No Effect on Serum Aldosterone Levels

Primary aldosteronism (PA), in which plasma aldosterone levels are normal or elevated relative to suppressed plasma renin levels, is the most frequent cause of secondary hypertension. PA accounts for 5‐10% of hypertension cases and up to 20% in those with resistant hypertension. Aldosterone is a mineralocorticoid hormone responsible for maintaining fluid volume, electrolyte balance, and blood pressure homeostasis; it is tightly regulated by the renin‐angiotensin‐aldosterone system. Our laboratory has previously shown that phospholipase D2 (PLD2) mediates angiotensin II‐induced aldosterone synthesis and secretion in vitro. Therefore, we hypothesized that PLD2 loss would result in decreased aldosterone production in vivo. To test this hypothesis, 11‐week‐old male and female wild‐type and PLD2‐/‐ mice were fed a high salt (1% NaCl) or a low salt (0.02% NaCl; to raise endogenous serum angiotensin II levels) diet for 24 hours. Adrenal steroidogenic gene expression and serum aldosterone, corticosterone, and electrolytes were measured by qRT‐PCR, radioimmunoassay, ELISA, and atomic absorption spectrometry, respectively. The PLD2‐/‐ male mice exhibited increased StAR, Dab2, and CYP11B1 expression; further, PLD2 loss impaired low salt‐induced increases in StAR, Dab2, and CYP11B1 expression; however, PLD2 loss had no effect on CYP11B2 expression or serum aldosterone, Na+, K+, and corticosterone levels in these male mice. These data suggest that increased steroidogenic enzyme expression may compensate for PLD2 loss in order to maintain aldosterone secretion in the males. On the other hand, the PLD2‐/‐ female mice exhibited decreased StAR and CYP11B1 expression and serum Na+ levels as well as impaired low salt‐induced increases in StAR, CYP11B2, Dab2, and CYP11B1 expression. PLD2 loss had no effect on serum aldosterone, K+, or corticosterone levels in these female mice. It is possible that other compensatory mechanisms (not involving steroidogenic enzymes) might be involved in maintaining aldosterone secretion in the females. Together, our findings suggest a likely importance of PLD2 in mediating aldosterone production and steroidogenic enzyme gene expression, with differential effects in male and female mice.

Very Low‐Density Lipoprotein (VLDL) Signals Through Sphingosine‐1‐Phosphate Receptor 1, Upon Binding to VLDL Receptor, to Induce Aldosterone Synthesis and Secretion in Human Adrenocortical Cells

Hypertension is prevalent in 46% of the US adult population with 40% of cases being attributable to obesity. Obese individuals have high serum levels of very low‐density lipoprotein (VLDL) and sphingosine‐1‐phosphate (S1P). VLDL and S1P have been shown to stimulate aldosterone production in multiple zona glomerulosa cell models, with aldosterone thought to be the link between hypertension and obesity. S1P is transported in blood bound to lipoproteins such as VLDL, low‐density lipoprotein (LDL), and high‐density lipoprotein (HDL); the VLDL particle contains the highest S1P levels. S1P in HDL has been shown to promote interactions between the scavenger receptor class B, type I (SR‐BI) and S1P receptor 1 (S1PR1). We hypothesized that like HDL, VLDL will signal through S1PRs, upon binding to SR‐BI; therefore, VLDL‐induced aldosterone secretion will be inhibited by S1PR and SR‐BI antagonists. Human adrenocortical (HAC15) cells were treated with VLDL and/or an S1PR1 antagonist (Ex26) or an anti‐SR‐BI blocking antibody for 24 h. Steroidogenic gene expression and aldosterone secretion were monitored by qRT‐PCR and radioimmunoassay, respectively. Ex26 significantly inhibited VLDL‐induced increases in CYP11B2 (22‐fold) and StAR (1.5‐fold) expression and aldosterone secretion (5‐fold) by 43%, 10%, and 36%, respectively. Unexpectedly, the anti‐SR‐BI antibody enhanced VLDL‐induced increases in CYP11B2 (29‐fold) and NR4A1 (2.2‐fold) expression by 17% and 33%, respectively; no effect was observed on VLDL‐induced increases in StAR and NR4A2 expression. However, when the VLDL receptor was blocked with recombinant human LDL receptor related protein associated protein 1, VLDL‐induced increases in CYP11B2 (2.8‐fold) and NR4A2 (5.3‐fold) expression were significantly reduced by 42% and 67%, respectively. Our results, therefore, indicate that VLDL signals through S1PR1, upon binding to the VLDL receptor and not SR‐BI, to induce aldosterone synthesis and secretion. Our study warrants further investigation into VLDL‐induced steroidogenic signaling pathways which may lead to the identification of novel therapeutic targets like S1PR1 to potentially treat obesity‐associated hypertension.

Transcriptome Reveals Granulosa Cells Coping through Redox, Inflammatory and Metabolic Mechanisms under Acute Heat Stress.

Heat stress affects granulosa cells (GCs) and the ovarian follicular microenvironment, causing poor oocyte developmental competence and fertility. This study aimed to investigate the physical responses and global transcriptomic changes in bovine GCs to acute heat stress (43 °C for 2 h) in vitro. Heat-stressed GCs exhibited transient proliferation senescence and resumed proliferation at 48 h post-stress, while post-stress immediate culture-media change had a relatively positive effect on proliferation resumption. Increased accumulation of reactive oxygen species and apoptosis was observed in the heat-stress group. In spite of the upregulation of inflammatory (CYCS, TLR2, TLR4, IL6, etc.), pro-apoptotic (BAD, BAX, TNFSF9, MAP3K7, TNFRSF6B, FADD, TRADD, RIPK3, etc.) and caspase executioner genes (CASP3, CASP8, CASP9), antioxidants and anti-apoptotic genes (HMOX1, NOS2, CAT, SOD, BCL2L1, GPX4, etc.) were also upregulated in heat-stressed GCs. Progesterone and estrogen hormones, along with steroidogenic gene expression, declined significantly, in spite of the upregulation of genes involved in cholesterol synthesis. Out of 12,385 differentially expressed genes (DEGs), 330 significant DEGs (75 upregulated, 225 downregulated) were subjected to KEGG functional pathway annotation, gene ontology enrichment, STRING network analyses and manual querying of DEGs for meaningful molecular mechanisms. High inflammatory response was found to be responsible for oxidative-stress-mediated apoptosis of GCs and nodes towards the involvement of the NF-κB pathway and repression of the Nrf2 pathway. Downregulation of MDM4, TP53, PIDD1, PARP3, MAPK14 and MYC, and upregulation of STK26, STK33, TGFB2, CDKN1A and CDKN2A, at the interface of the MAPK and p53 signaling pathway, can be attributed to transient cellular senescence and apoptosis in GCs. The background working of the AMPK pathway through upregulation of AKT1, AMPK, SIRT1, PYGM, SLC2A4 and SERBP1 genes, and downregulation of PPARGCIA, IGF2, PPARA, SLC27A3, SLC16A3, TSC1/2, KCNJ2, KCNJ16, etc., evidence the repression of cellular transcriptional activity and energetic homeostasis modifications in response to heat stress. This study presents detailed responses of acute-heat-stressed GCs at physical, transcriptional and pathway levels and presents interesting insights into future studies regarding GC adaptation and their interaction with oocytes and the reproductive system at the ovarian level.

Placental structure, function, and mitochondrial phenotype relate to fetal size in each fetal sex in mice†.

Fetal growth depends on placental function, which requires energy from mitochondria. Here we investigated whether mitochondrial function in the placenta relates to the growth of the lightest and heaviest fetuses of each sex within the litter of mice. Placentas from the lightest and heaviest fetuses were taken to evaluate placenta morphology (stereology), mitochondrial energetics (high-resolution respirometry), mitochondrial regulators, nutrient transporters, hormone handling, and signaling pathways (qPCR and Western blotting). We found that mitochondrial complex I and II oxygen consumption rate was greater for placentas supporting the lightest female fetuses, although placental complex I abundance of the lightest females and complexes III and V of the lightest males were decreased compared to their heaviest counterparts. Expression of mitochondrial biogenesis (Nrf1) and fission (Drp1 and Fis1) genes was lower in the placenta from the lightest females, whilst biogenesis-related gene Tfam was greater in the placenta of the lightest male fetuses. In addition, placental morphology and steroidogenic gene (Cyp17a1 and Cyp11a1) expression were aberrant for the lightest females, but glucose transporter (Slc2a1) expression was lower in only the lightest males versus their heaviest counterparts. Differences in intra-litter placental phenotype were related to changes in the expression of hormone-responsive (androgen receptor) and metabolic signaling (AMPK, AKT, and PPARγ) pathways. Thus, in normal mouse pregnancy, placental structure, function, and mitochondrial phenotype are differentially responsive to the growth of the female and male fetus. This study may inform the design of sex-specific therapies for placental insufficiency and fetal growth abnormalities with life-long benefits for the offspring.

Vitamin E rescues valproic acid-induced testicular injury in rats: Role of autophagy

AimsValproic acid (VPA), a commonly used antiepileptic drug, can induce testicular oxidative stress and injury. Altered autophagic response usually follows testicular injury. The study aims to evaluate the role of autophagy in the protective effect of the antioxidant vitamin E (Vit E) against VPA-induced testicular injury. Materials and methodsVPA (100, 300, and 500 mg/kg/day) was administered for 8 days. The protective group received both Vit E (50 mg/kg) and VPA (500 mg/kg). The testicular weight, sperm analysis, and serum testosterone concentration, as well as testicular histopathology, steroidogenic gene expression, and oxidative stress markers were evaluated. The mRNA or protein expression of autophagy-related proteins [adenosine monophosphate-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), microtubule-associated protein light chain 3 (LC3), Beclin1, and p62] were measured using RT-PCR or immunohistochemistry. Key findingsVPA resulted in lower testes weight and sperm quality with aberrant morphology. VPA dose-dependently induced testicular oxidative stress, which was associated with decreased steroidogenic gene expression and serum testosterone levels, as well as deteriorated histopathology. These biochemical and histological changes were also associated with autophagy induction (higher LC3 and Beclin1, and lower p62) that was lost with the highest toxic dose (500 mg/kg). The attenuated autophagy with the highest dose was accompanied by AMPK downregulation and mTOR upregulation. Vit E protected against VPA-mediated oxidative stress and toxicity while also restoring autophagic response and AMPK/mTOR levels. SignificanceThe study highlights vitamin E as a valuable protective asset against VPA-induced testicular injury, possibly through AMPK-mTOR-dependent autophagy induction.

Perfluorooctanoic Acid Disrupts Ovarian Steroidogenesis and Folliculogenesis in Adult Mice.

Perfluorooctanoic acid (PFOA) is a synthetic fluorosurfactant used in the manufacturing of fluorotelomers. Although PFOA is no longer produced in the United States, it is environmentally persistent and found in imported food packaging, cookware, and textiles. Previous studies have identified developmental toxicity of PFOA, but little is known about the effects of PFOA on the adult ovary. Thus, this study examined the effects of PFOA on hormone levels, ovarian steroidogenic gene expression, and folliculogenesis in mice in vitro and in vivo. For the in vitro studies, antral follicles from adult female mice were cultured with vehicle control or 1, 10, or 100 μg/ml PFOA for 96 h. For the in vivo studies, adult CD-1 female mice were orally dosed with vehicle control or 1, 5, 10, or 20 mg/kg/day PFOA for 10 days. Gene expression of steroidogenic enzymes, levels of sex steroid hormones, and follicle counts were analyzed. In vitro, PFOA (100 μg/ml) significantly decreased follicle growth, estradiol and estrone levels, and gene expression of StaR, Cyp11a1, and Hsd3b1 compared with controls. In vivo, exposure to PFOA significantly decreased progesterone and pregnenolone levels (5 mg/kg), increased testosterone levels (1 mg/kg), and increased gene expression of Cyp19a1 (1 mg/kg) compared with controls. Exposure to PFOA also significantly altered follicle counts by decreasing primordial follicles and increasing preantral and antral follicles (5 and 10 mg/kg) compared with controls. Collectively, these data show that PFOA disrupts adult ovarian function in a nonmonotonic matter and may pose a risk for premature ovarian failure.

Ovarian Toxicity Induced by Aluminum Chloride: Alteration of Cyp19a1, Pcna, Puma, and Map1lc3b genes Expression

Aluminum (Al) is an abundant metal with wide application in our daily lives including medicine, industry, cosmetics, and packaging. After entrance to the body, aluminum binds to transferrin and reaches different tissues. Al is a metalloestrogen that can lead to oxidative stress (OxS) and endocrine disruption. No detailed study can be found addressing the effect of Al on the ovary and granulosa cells (GCs). In this study, the focus is on the treated ovaries and GCs of NMRI mice exposed to low, middle, and high doses of aluminum chloride (AlCl3) via in vitro and in vivo assays. The steroidogenic, proliferative, apoptotic, and autophagic-related genes were examined. Up-regulated expression of steroidogenic and proliferative genes was detected. The observed apoptotic and autophagic genes had variable expression. Interrupted ovarian structure, disrupted folliculogenesis, presence of Call-Exner bodies, overexpression of steroidogenic gene, and unbalanced apoptosis/autophagy and proliferation resembled features of granulosa cell tumor (GCT).

A Chronic-Low-Dose Exposing of DEHP with OECD TG 443 Altered the Histological Characteristics and Steroidogeic Gene Expression of Adrenal Gland in Female Mice.

Phthalates and their metabolites are well-known endocrine disrupting chemicals. Di-(2-ethylhexyl) phthalate (DEHP) has been widely used in industry and the exposing possibility to adult is high. In this study, DEHP was treated (133 μg/L and 1,330 μg/L in drinking water) according to the OECD test guideline 443 to mature female mice and their adrenal gland were examined for histological characteristics and steroidogenic gene expression. The wet weight of the adrenal gland was increased in all administrated groups compared to control. The diameter of zona fasciculata (ZF) was increased by DEHP in both outer ZF and inner ZF but there was no difference in morphology of the cells and arrangements into zona between groups. In addition, the arrangement of extracellular matrix was not different between control and DEHP groups. CYP11B1 was mainly localized at ZF and the intensity was not different between groups. DAX1 was localized in zona glomerulosa (ZG) and ZF, and its expression levels were decreased by DEHP administration. Its level was lower in DEHP133 group than DEHP1330 group. On the other hand, CYP17A1 was localized in ZG of DEHP1330 group. These results suggest that chronic low-dose DEHP exposing may modify the microstructure and function of the adrenal cortical cortex.

Cyp17a1 is Required for Female Sex Determination and Male Fertility by Regulating Sex Steroid Biosynthesis in Fish.

In teleost fish, sex steroids are involved in sex determination, sex differentiation, and fertility. Cyp17a1 (Cytochrome P450 family 17 subfamily A member 1) is thought to play essential roles in fish steroidogenesis. Therefore, to further understand its roles in steroidogenesis, sex determination, and fertility in fish, we constructed a cyp17a1 gene mutant in Nile tilapia (Oreochromis niloticus). In XX fish, mutation of the cyp17a1 gene led to a female-to-male sex reversal with a significant decline in 17β-estradiol (E2) and testosterone (T) production, and ectopic expression of male-biased markers (Dmrt1 and Gsdf) in gonads from the critical window of sex determination. Sex reversal was successfully rescued via T or E2 administration, and ovarian characteristics were maintained after termination of E2 supplementation in the absence of endogenous estrogen production in cyp17a1-/- XX fish. Likewise, deficiencies in T and 11-ketotestosterone (11-KT) production in both cyp17a1-/- XX sex-reversed males and cyp17a1-/- XY mutants resulted in meiotic initiation delays, vas deferens obstruction and sterility due to excessive apoptosis and abnormal mitochondrial morphology. However, 11-KT treatment successfully rescued the dysspermia to produce normal sperm in cyp17a1-/- male fish. Significant increases in gonadotropic hormone (gth) and gth receptors in cyp17a1-/- mutants may excessively upregulate steroidogenic gene expression in Leydig cells through a feedback loop. Taken together, our findings demonstrate that Cyp17a1 is indispensable for E2 production, which is fundamental for female sex determination and differentiation in XX tilapia. Additionally, Cyp17a1 is essential for T and 11-KT production, which further promotes spermatogenesis and fertility in XY males.

Lgr4 Regulates Oviductal Epithelial Secretion Through the WNT Signaling Pathway.

The WNT signaling pathway plays a crucial role in oviduct/fallopian development. However, the specific physiological processes regulated by the WNT pathway in the fallopian/oviduct function remain obscure. Benefiting from the Lgr4 knockout mouse model, we report the regulation of oviduct epithelial secretion by LGR4. Specifically, the loss of Lgr4 altered the mouse oviduct size and weight, severely reduced the number of oviductal epithelial cells, and ultimately impaired the epithelial secretion. These alterations were mediated by a failure of CTNNB1 protein accumulation in the oviductal epithelial cytoplasm, by the modulation of WNT pathways, and subsequently by a profound change of the gene expression profile of epithelial cells. In addition, selective activation of the WNT pathway triggered the expression of steroidogenic genes, like Cyp11a1 and 3β-Hsd1, through the activation of the transcriptional factor NR5A2 in an oviduct primary cell culture system. As demonstrated, the LGR4 protein modulates a WNT-NR5A2 signaling cascade facilitating epithelial secretory cell maturation and steroidogenesis to safeguard oviduct development and function in mice.

Re-Evaluation of Combinational Efficacy and Synergy of the Italian Protocol In Vitro: Are We Truly Optimizing Benefit or Permitting Unwanted Toxicity?

Introduction: Adrenocortical carcinoma (ACC) is a rare endocrine malignancy, with very poor prognosis as a majority of the patients have advanced disease at the time of diagnosis. Currently, adjuvant therapy for most patients consists of either mitotane (M) alone or in combination with multi-drug chemotherapeutics such as etoposide (E), doxorubicin (D), and cisplatin (P), known as the Italian protocol (IP; EDPM). This multi-drug treatment regimen, however, carries significant toxicity potential for patients. One way to improve toxicity profiles with these drugs in combination is to understand where their synergy occurs and over what dosing range so that lower dose regimens could be applied in combination with equal or improved efficacy. We hypothesize that a better understanding of the synergistic effects as well as the regulation of steroidogenic enzymes during combination therapy may provide more optimized combinational options with good potency and lower toxicity profiles. Methods: Two human ACC cell lines, NCI-H295R (hormonally active) and SW13 (hormonally inactive), were grown in 2D culture in appropriate growth medium. The viability of the cells after treatment with varying concentrations of the drugs (E, D, and P) either alone or in combinations with M was determined using the CellTiter Glow assay after 72 h, and the combination index for each was calculated using Compusyn by the Chou–Talalay method. The expression levels of enzymes associated with steroidogenesis were evaluated by RT-PCR in NCI-H295R. Results: When both cell lines were treated with M (ranging 25–50 μM), +E (ranging 18.75–75 μM), and +D (ranging 0.625–2.5 μM) we observed a synergistic effect (CI < 1) with potency equivalent to the full Italian protocol (IP), whereas combining M + P + D had an antagonistic effect (CI > 1) indicating the negative effect of adding cisplatin in the combination. Comparing the hormonally active and inactive cell lines, M + P + E was antagonistic in NCI-H295R and synergistic in SW13. Treatment of NCI-H295R cells with antagonistic combinations (M + P + D, M + P + E) resulted in a significant decrease in the levels of steroidogenic enzymes STAR, CYP11A1, and CYP21A2 compared to IP (p < 0.05) while M + E + D resulted in increased expression or no significant effect compared to IP across all genes tested. Conclusions: The synergistic effect for M + E + D was significant and equivalent in potency to the full IP in both cell lines and resulted in a steroidogenic gene expression profile similar to or better than that of full IP, warranting further evaluation. Future in vivo evaluation of the combination of M + E + D (with removal of P from the IP regimen) may lower toxicity while maintaining anticancer efficacy in ACC.