Steroidogenesis-related Genes Research Articles

Melatonin counteracts polyethylene microplastics induced adreno-cortical damage in male albino rats

There are various substances that can disrupt the homeostatic mechanisms of the body, defined as endocrine-disrupting chemicals (EDCs). The persistent nature of microplastics (MPs) is a cause for concern due to their ability to accumulate in food chains and widespread use, making their toxic effects particularly alarming. The potential of MPs for disrupting the endocrine system was observed in multiple tissues. Moreover, the adrenal gland is known to be extremely sensitive to EDCs, while with the effect of MPs on the adrenal gland has not previously been studied. This study aimed to highlight the potential polyethylene microplastics (PE-MPs) induced adreno-toxic effects rather than exploring the implicated mechanisms and concluding if melatonin (Mel) can afford protection against PE-MPs induced adreno-toxicity. To fulfill the goal, six groups of rats were used; control, Mel, PE-MPs (3.75 mg/kg), PE-MPs (15 mg/kg), PE-MPs (3.75 mg/kg) +Mel, and PE-MPs (15 mg/kg) +Mel. PE-MPs induced toxic changes in the adrenal cortex, which was evident by increased adrenal weight, histopathological examination, and ultrastructural changes detected by electron microscope. A reduction in serum cortisol and an increase in serum adrenocorticotropic hormone resulted from the adreno-toxic effects of PE-MPs. Mechanisms may include the reduction of steroidogenesis-related genes, as PE-MPs drastically reduce mRNA levels of StAR, Nr0b1, Cyp11A1, as well as Cyp11B1. Also, oxidative stress that results from PE-MPs is associated with higher rates of lipid peroxidation and decreased superoxide dismutase and glutathione. PE-MPs inflammatory effect was illustrated by elevated expression of IL-1β and NF-ķB, detected by immunohistochemical staining, in addition to increased expression of caspase-3 and mRNA of Bax, markers of proapoptotic activity. The impacts of PE-MPs were relatively dose-related, with the higher dose showing more significant toxicity than the lower one. Mel treatment was associated with a substantial amelioration of PE-MPs-induced toxic changes. Collectively, this study fills the knowledge gap about the MPs-induced adrenal cortex and elucidates various related toxic mechanisms. It also supports Mel’s potential protective activity through antioxidant, anti-inflammatory, anti-apoptotic, and gene transcription regulatory effects.

NTRK2 Promotes Sheep Granulosa Cells Proliferation and Reproductive Hormone Secretion and Activates the PI3K/AKT Pathway.

Neurotrophin receptor B (NTRK2), also named TRKB, belongs to the neurotrophic factor family. Previous studies have shown that NTRK2 is associated with high fertility in mammals. However, the molecular mechanism and regulatory pathway of this neurotrophic factor remain unclear. In this study, NTRK2 overexpression and NTRK2-siRNA were constructed to detect the effects of NTRK2 on the proliferation and hormone secretion of the ovarian granulosa cells (GCs) of sheep. We successfully isolated follicular phase granulosa cells in vitro from the ovaries of sheep in simultaneous estrus, and the immunofluorescence results confirmed that NTRK2 was expressed in the collected cells. Subsequently, the effect of NTRK2 on the proliferation of sheep granulosa cells was examined via cell transfection experiments. The results showed that the expression of CDK4 and CyclinD2 was significantly increased after NTRK2 overexpression, while the opposite trend was observed after the inhibition of NTRK2 expression (p < 0.05). The EdU and CCK-8 assays showed that the proliferation rate of sheep GCs was significantly increased after NTRK2 overexpression, while the opposite trend was observed after the inhibition of NTRK2 expression (p < 0.05). Moreover, NTRK2 significantly increased the expression of steroidogenesis-related genes, including steroidogenic acute regulatory protein (STAR) and hydroxy-δ-5-steroid dehydrogenase (HSD3B1), and cytochrome P450 family 19 subfamily A member 1 (CYP19A1). The ELISA results showed that the secretion levels of E2 and P4 significantly increased after NTRK2 overexpression, while the opposite trend was observed after the inhibition of NTRK2 expression (p < 0.05). Previous studies had confirmed that NTRK2 gene belongs to the PI3K-AKT signaling pathway and participates in the signaling of this pathway. This was demonstrated by protein-protein interaction analysis and NTRK2 belongs to the PI3K-AKT pathway. The modification of PI3K and AKT, markers of the PI3K-AKT pathway, via phosphorylation was increased after NTRK2 overexpression in the sheep GCs, while the opposite trend was observed after the inhibition of NTRK2 expression (p < 0.05). Overall, these results suggest that the NTRK2 gene regulates the proliferation of GCs and the secretion of steroid hormones in sheep, and that it influences the phosphorylation level of the PI3K/AKT signaling pathway. These findings provided a theoretical basis and new perspectives for exploring the regulation of NTRK2 gene in the development of ovine follicles.

Neonatal vitamin A supplementation improves sheep fertility potential.

This study aimed to explore the effects of neonatal vitamin A (VA) supplementation on testis development and spermatogenesis. A total of 32 newborn lambs were intramuscularly injected with corn oil (control group) or corn oil + 2500 IU/kg BW VA (VA group). They were slaughtered and sampled at 3 weeks and 8 months of age to analyze spermatogenesis, cell proliferation, hormone secretion, antioxidant status of the testis, and adult sheep sperm parameters. Compared with the control group, the expression of spermatogonial differentiation-related genes in VA group was up-regulated (P < 0.05). Testis weight, seminiferous tubule diameter, number of spermatogonium and spermatocyte, and sperm density increased significantly in VA group at 8 months of age (P < 0.05). Neonatal VA injection upregulated the expression of the cell proliferation marker PCNA and cell cycle-related genes in the testis (P < 0.05). VA increased the concentrations of testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) in the serum and upregulated steroidogenesis-related genes in the testis (P < 0.05). The antioxidant levels in the VA group were maintained at high levels. The total antioxidant capacity (T-AOC), antioxidant enzyme content and antioxidant-related genes were increased in the testis (P < 0.05). Furthermore, neonatal VA injection activated retinoic acid (RA) signaling to maintain the blood-testosterone barrier (BTB) in the testis of 3-week-old sheep. AMP-activated protein kinase (AMPK) and protein kinase B (AKT) signaling were also modulated in the sheep testis (P < 0.05). Taken together, VA supplementation in newborn rams promotes testis development and spermatogenesis to improve fertility.

The effects of low ambient temperature on steroidogenesis and mitochondrial functions in the testes of wild ground squirrels (Spermophilus dauricus)

Seasonal reproduction is a widely used breeding strategy in wildlife, especially vertebrates inhabiting temperate regions. Generally, ambient temperature is considered a significant factor influencing the reproductive status of animals. In the present study, wild ground squirrels (Spermophilus dauricus), typical seasonal breeders, were used as an animal model to investigate the mechanism behind the impact of low ambient temperature on testicular function. To simulate the winter environment of wild ground squirrels, we lowered the temperature gradient in the rearing environment to 4 °C. At sampling, the body surface temperature of the squirrels reared under normal ambient temperature (22 °C, NAT group) and the low ambient temperature (4 °C, LAT group) were 31.5 °C and 22.8 °C, respectively. Subsequently, we conducted immunohistochemical assays, qPCR, and enzyme-linked immunosorbent assays (ELISA) to examine the variations in testicular functions, as well as the dynamics and functions of mitochondria, in the squirrels of NAT and LAT groups. As a result, the levels of positive immunostaining for PCNA, P21, and P27 were significantly lower in the testes of LAT group, while the levels of immunostaining for Cleaved Caspase-3 and TUNEL were significantly higher. In addition, the low-temperature treatment reduced the expression level of steroidogenesis-related genes, including LHR, FSHR, GATA-4, P450scc, and P450arom, and decreased the testosterone concentration. Moreover, markers of mitochondrial fission and fusion, DRP1 and MFN2, respectively, were increased in the testes of LAT group. Additionally, the mRNA level of SOD1 was notably higher in the testes of LAT group. In conclusion, the low ambient temperature inhibited spermatogenesis, steroidogenesis, as well as mitochondrial dynamics and functions in the testes of wild ground squirrels.

REVERBA couples the circadian clock to Leydig cell steroidogenesis.

The involvement of the molecular clock in regulating cell physiological processes on a specific time scale is a recognized concept, yet its specific impact on optimizing androgen production in Leydig cells has been unclear. This study aimed to confirm the role of the REVERBA (NR1D1) gene in controlling the transcription of key genes related to Leydig cell steroid production. We investigated daily variations by collecting Leydig cells from rats at various times within a 24-h period. Chromatin immunoprecipitation study showed a time-dependent pattern for genes linked to steroid production (Nur77, Star, Cyp11a1, and Cyp17a1), which closely matched the 24-h REVERBA levels in Leydig cells, peaking between zeitgeber time (ZT) 7-11. To understand the physiological significance of REVERBA's interaction with promoters of steroidogenesis-related genes, Leydig cells from rats at two different times (ZT7 and ZT16; chosen based on REVERBA expression levels), were treated with either an agonist (GSK4112) or an antagonist (SR8278). The results revealed that the REVERBA agonist stimulated gene transcription, while the antagonist inhibited it, but only when REVERBA was sufficiently present, indicating a reliance on REVERBA's circadian fluctuation. Moreover, this REVERBA-dependent stimulation had a clear impact on testosterone production in the culture medium, underscoring REVERBA's involvement in the circadian regulation of testosterone. This study indicates that REVERBA, in addition to being a core component of the cellular clock, plays a key role in regulating androgen production in Leydig cells by influencing the transcription of critical steroidogenesis-related genes.

Protective Effects of Ginger Extract on Oxidative Stress and Steroidogenesis-related Genes in The Ovary of Streptozotocin-induced Diabetic Rats

Background and Aims: Ginger has anti-oxidant and anti-diabetic properties, but its beneficial effects have not been fully understood on ovarian disorders in diabetic conditions. In the current project, the impact of ginger extract was investigated on oxidative stress and steroidogenesis-related genes in streptozotocin (STZ)-induced female rats. &#x0D; Materials and Methods: The STZ-induced rats were utilized as a diabetic model and received 200 or 400 mg/kg/day ginger extract for eight weeks. The biochemical factors were measured by standard procedures in this study. Serum levels of insulin and sex hormones were assessed by the enzyme-linked immunosorbent assay (ELISA) technique, and the mRNA expression of target genes was assayed by real-time polymerase chain reaction.&#x0D; Results: An increase in the levels of glucose, testosterone, and malondialdehyde (MDA) as well as a decrease in the levels of insulin, glutathione peroxidase (GPx), 3β-hydroxysteroid dehydrogenase (3βHSD), steroidogenic acute regulatory protein (StAR), 17β-estradiol, and progesterone was observed in the diabetic rats. Ginger (200 mg/kg) exhibited a significant amelioration in the levels of glucose, testosterone, and MDA. Treatment with 200 mg/kg ginger enhanced the levels of GPx, StAR, and 17β-estradiol. Administration with 400 mg/kg ginger extract also ameliorated the level of glucose, testosterone, MDA, and elevated the level of insulin, GPx, 3βHSD, StAR, 17β-estradiol, and progesterone in the diabetic rats.&#x0D; Conclusion: The current study suggests ginger extract protects against ovarian damage in STZ-diabetic rats.

Reproductive toxicity following in utero and lactational exposure to a human-relevant phthalate mixture in rats.

This rodent (Wistar rats) study examined reproductive effects of in utero/lactational exposure to a mixture of 6 antiandrogenic phthalates (PMix): diisobutyl phthalate, di-n-butyl phthalate, diisopentyl phthalate, butylbenzyl phthalate, di-2-ethylhexyl phthalate, and diisononyl phthalate. The PMix was defined based on exposure data from pregnant women in Brazil. Experimental groups were established by extrapolating the estimated human dose to rats (0.1 mg/kg/day), followed by up to 3 additional doses corresponding to 5, 1000, and 5000 times the starting rat dose: 0 (control), 0.1, 0.5, 100, and 500 mg/kg/day. The fetal experiment assessed gestational exposure effects on fetal gonads, whereas the postnatal experiment evaluated reproductive parameters in males and females after in utero and lactational exposure. Prenatal exposure decreased fetal testicular testosterone production at 0.5 and 500 mg/kg/day. PMix 500 also reduced mRNA expression of steroidogenesis-related genes, upregulated transcript expression of the retinoic acid-degrading enzyme Cyp26b1, and increased multinucleated gonocytes incidence in fetal testes. Postnatal assessment revealed antiandrogenic effects at the highest dose, including reduced anogenital distance, nipple retention, and decreased weight of reproductive organs. Early puberty onset (preputial separation) was observed at the lowest dose in males. In contrast, females did not show significant changes in fetal and adult endpoints. Overall, the PMix recapitulated early and late male rat phthalate syndrome phenotypes at the highest dose, but also induced some subtle changes at lower doses, which warrant confirmation and mechanistic assessments. Our data support the use of epidemiologically defined mixtures for exposure risk assessments over traditional toxicological approaches.

Circadian clock gene BMAL1 regulates STAR expression in goose ovarian preovulatory granulosa cells

The ovarian circadian clock plays a regulatory role in the avian ovulation-oviposition cycle. However, little is known regarding the ovarian circadian clock of geese. In this study, we investigated rhythmic changes in clock genes over a 48-h period and identified potential clock-controlled genes involved in progesterone synthesis in goose ovarian preovulatory granulosa cells. The results showed that BMAL1, CRY1, and CRY2, as well as 4 genes (LHR, STAR, CYP11A1, and HSD3B) involved in progesterone synthesis exhibited rhythmic expression patterns in goose ovarian preovulatory granulosa cells over a 48-h period. Knockdown of BMAL1 decreased the progesterone concentration and downregulated STAR mRNA and protein levels in goose ovarian preovulatory granulosa cells. Overexpression of BMAL1 increased the progesterone concentration and upregulated the STAR mRNA level in goose ovarian preovulatory granulosa cells. Moreover, we demonstrated that the BMAL1/CLOCK complex activated the transcription of goose STAR gene by binding to an E-box motif. These results suggest that the circadian clock is involved in the regulation of progesterone synthesis in goose ovarian preovulatory granulosa cells by orchestrating the transcription of steroidogenesis-related genes.

Comparable bio-evaluation of curcumin and chitosan-encapsulated curcumin nanoparticles against the reprotoxic potential of fenpropathrin pyrethroid in rats: Genomic and morphometric prospectives.

This study delves into the intricate exploration of potential toxic effects resulting from subchronic exposure to fenpropathrin (FNP) on the reproductive system of male SD rats. Adding to the novelty, our study undertakes a pioneering comparison of the effects of curcumin (CUR) and curcumin-encapsulated chitosan nanoparticles (CS.CUR.NPs) on these toxic effects. The study involved a cohort of sixty male SD rats (six groups): vehicle control, CUR, Cs.CUR.NPs, FNP, and two combination groups (FNP with CUR or Cs.CUR.NPs). The synthesized Cs.CUR.NPs nanoparticles underwent meticulous characterization using Fourier Infrared spectroscopy (FT-IR) and transmission electron microscopy (TEM). The findings revealed that FNP caused oxidative stress, sperm abnormalities, reduced motility and sperm count FNP decreased serum LH, FSH, 17-β estradiol, and testosterone levels. FNP downregulated the mRNA expression of the spermatogenesis and steroidogenesis-related genes, While, downregulated hypothalamic KISS-1 and KISS-1r expression. Histopathological alterations were assessed and scored. Surprisingly, the treatment with CUR and Cs.CUR.NPs exhibited remarkable restorative effects on semen quality, sex hormone levels, antioxidant capacity, and mRNA expression of the targeted genes. Notably, Cs.CUR.NPs displayed superior properties when compared to CUR. Nevertheless, further research is imperative to evaluate their efficacy across various bodily tissues.

Steroidogenic activity of liposomal methylated resveratrol analog 3,4,5,4′-tetramethoxystilbene (DMU-212) in human luteinized granulosa cells in a primary three-dimensional in vitro model

PurposeSteroid hormone secretion is one of the key functions of granulosa cells (GCs). Resveratrol is a natural polyphenol, known for its beneficial health effects, such as improving reproductive health. However, its application is limited due to poor bioavailability. The methoxy derivative of resveratrol (DMU-212) was demonstrated to be more lipophilic, and therefore of greater bioavailability. However, since the addition of methoxy groups to the stilbene scaffold was found to make the molecule insoluble in water, DMU-212 was loaded into liposomes. This study aimed to evaluate how the liposomal formulation of DMU-212 (lipDMU-212) alters estradiol and progesterone secretion of human ovarian GCs in a primary three-dimensional cell culture model.MethodsDMU-212-loaded liposomes were prepared by thin film hydration followed by extrusion. Cell viability was measured after exposure of GCs spheroids to the liposomal formulation of DMU-212 using CellTiter-Glo® 3D Cell Viability Assay. The secretion of estradiol and progesterone was determined using commercial ELISA kits. RT-qPCR was conducted to analyze the expression of steroidogenesis-related genes. Finally, the western blot technique was used to analyze the effect of lipDMU-212 and FSH treatments on CYP11A1 and HSD3B1 protein levels.ResultslipDMU-212 was found to significantly increase estradiol and progesterone secretion in a dose-dependent manner by enhancing the expression of CYP11A1, HSD3B1, StAR, CYP17A1, CYP19A1, and HSD17B1 genes. We have also shown that lipDMU-212, used alone and in combination with FSH, significantly increased the expression of the HSD3B1 and CYP11A1 proteins in GCs. Furthermore, our study suggests that lipDMU-212 increases FSH activity.ConclusionsThis is the first study to describe the steroidogenic activity of liposomal formulation of DMU-212, possibly through increasing the StAR and CYP19A1 expression. These findings suggest that lipDMU-212 might have a beneficial effect in the treatment of disorders related to estrogen deficiency and hyperandrogenism, such as PCOS.

CircEpha5 regulates the synthesis and secretion of androgen in mouse preantral follicles by targeting miR-758-5p

Circular RNAs are involved in the pathogenesis of various diseases, although its expression pattern and role in polycystic ovary syndrome (PCOS), characterised by hyperandrogenism, are not very clear. This article assessed the circRNAs expression profile in the ovaries of PCOS mice by circRNAs high-throughput sequencing and explored the role of circEpha5 in hyperandrogenism. The results showed that the overexpression of circEpha5 in mouse preantral follicles could increase the expression of Cyp17a1, an androgen synthesis-related gene, which resulted in a higher serum level of testosterone. Dual-luciferase reporter gene studies identified miR-758-5p as a direct target of circEpha5. Consequently, miR-758-5p expression was downregulated upon circEpha5 overexpression. Ectopically expressed miR-758-5p reversed the stimulation effects of circEpha5 on steroidogenesis-related gene expression and testosterone release. Therefore, circEpha5 could sponge miR-758-5p to regulate the expression of Cyp17a1, thereby promoting the synthesis and secretion of androgen in the preantral follicles. This work is contributed to the understanding of the pathogenesis of hyperandrogenemia and lays the foundation for the development of therapeutic targets of PCOS hyperandrogenism.

Trichlorfon blocks androgen synthesis and metabolism in rat immature Leydig cells

Trichlorfon is a widely used organophosphorus insecticide. It has been reported that it has reproductive toxicity to animal models. However, whether trichlorfon affects testosterone biosynthesis and metabolism remains unclear. In this study, we explored the effects of trichlorfon on the steroidogenesis and the expression of genes in androgen biosynthetic and metabolic cascades in immature Leydig cells isolated from pubertal male rats. Immature Leydig cells were treated with trichlorfon (0.5–50 µM) for 3 h. Trichlorfon significantly inhibited total androgen output under basal condition at 5 and 50 μM, and under LH- and cAMP-stimulated conditions at 50 μM. Trichlorfon also downregulated the expression of Star, Sod2, and Gpx1 and their proteins at 5 and 50 μM and the expression of Cyp11a1, Hsd3b1, Cyp17a1, and Srd5a1 at 50 μM. Trichlorfon significantly inhibited total androgen output at 50 μM, which was partially reversed by 400 μg/ml vitamin E, which alone had no effects on androgen output. In conclusion, trichlorfon downregulates the expression of steroidogenesis-related genes and antioxidants, which leads to a decrease in androgen production in rat immature Leydig cells.

P-081 In utero exposure to diisopentyl phthalate (DiPeP) disrupts the expression of steroidogenesis-related genes and induces multinucleated gonocytes in the fetal rat testis

Abstract Study question Can the plasticizer diisopentyl phthalate (DiPeP) disrupt testosterone biosynthesis and alter fetal rat testis histology similarly to other common toxic phthalates? Summary answer DiPeP can dose-dependently suppress mRNA levels of steroidogenic genes and induce multinucleated gonocytes in the fetal rat testis, resembling the effects of other toxic phthalates. What is known already DiPeP is an uncommon phthalate, whose metabolites have been ubiquitously detected in urinary samples from Brazilian pregnant women and children. These results, which contrast with the undetected levels of DiPeP metabolites in other human biomonitoring studies worldwide, led us to investigate DiPeP reproductive and developmental toxicity in rats. Our preliminary data indicate that DiPeP is a potent antiandrogenic phthalate, but no studies have been conducted to assess the impact of DiPeP exposures on the incidence of multinucleated gonocytes in the rat fetal testis, a common feature of the rat phthalate syndrome. Study design, size, duration Pregnant Wistar rats (n = 7-9 dams/group) were treated orally (gavage) with DiPeP 0 (control), 11, 33, and 100 mg/kg/day between gestation days 14-20. Dose levels were based on prior rat phthalate toxicity studies and canola oil was used as a vehicle. On gestation day 20, dams were euthanized and up to three male fetuses from each litter were removed for the collection of fetal testes and the assessment of the study endpoints. Participants/materials, setting, methods Fetal rat testes were used for enzyme immunoassay quantification of testosterone production following ex vivo incubation of testes in culture media, analysis of mRNA levels of steroidogenic genes and insulin-like factor 3 (Insl3) by quantitative polymerase chain reaction (qPCR), and histological assessment of multinucleated gonocytes by optical microscopy. Main results and the role of chance No signs of systemic toxicity were observed as indicated by the lack of alterations in maternal body weights and the unchanged number, viability, and weight of fetuses. There were no significant changes in the ex vivo testosterone production at any DiPeP dose. On the other hand, DiPeP induced significant reductions in the mRNA expression of key steroidogenic genes, including suppressed mRNA levels of the steroidogenic acute regulatory protein (Star) and cytochrome P450 family 11 subfamily A member 1 (Cyp11a1) at 100 mg/kg/day and of cytochrome P450 family 17 subfamily A member 1 (Cyp17a1) at 33 and 100 mg/kg/day. Gene expression of Insl3, which is essential for the process of testis descent, was reduced at the highest dose by 58% in relation to control, but this change was not significant (p = 0.07). The number of multinucleated gonocytes, corrected for the number of testicular cord sections or the total area of analyzed testicular cords was significantly increased in the groups exposed to 33 and 100 mg/kg/day. Chance is an unlikely explanation for our results considering the observed dose-dependent responses and the consistency of our findings with prior phthalate reproductive toxicity data. Limitations, reasons for caution The tested doses are higher than the expected human exposure levels and animal-to-human extrapolation should be done with caution. Wider implications of the findings DiPeP suppresses the expression of steroidogenic genes and induces multinucleated gonocytes in the fetal rat testis. These endpoints seem more sensitive than inhibition of ex vivo testosterone production. Although human exposure levels are lower than the doses tested here, DiPeP can potentially act cumulatively with chemicals that share similar mechanisms. Trial registration number not applicable

Molecular cloning and characterization of STC1 gene and its functional analyses in yak (Bos grunniens) cumulus granulosa cells

Cumulus granulosa cells (CGCs), an important type of ovarian somatic cells, carries out various functions related to oogenesis, follicular development, and steroidogenesis. Studying the biological mechanisms involved in the development and function of CGCs makes a great contribution to understanding the reproductive regulation in female animals. Stanniocalcin-1 (STC1) is an important Ca2+-regulated glycoprotein hormone that exhibits high expression levels in ovaries. In this study, we cloned the coding sequence of the yak STC1, predicted the structure of STC1 protein, detected the expression and localization of STC1 in yak ovaries, and analyzed the functions of STC1 in yak CGCs. The CDS (coding sequence) region of yak STC1 gene was found to be 744 bp and encoded 247 amino acids. Homology comparison revealed that STC1 protein was highly conserved among mammals. The STC1 mRNA displayed dynamic expression profiles in different stages of yak ovaries, and the highest expression was found in the follicular phase. Regarding localization, STC1 protein was widely distributed in various kinds of yak ovarian cells, including oocytes, mural granulosa cells, CGCs, and thecal cells. Repressing the expression of STC1 resulted in defective proliferation and survival of yak CGCs. In addition, knockdown the expression of STC1 repressed the secretion of progesterone and promoted the secretion of estrogen. Overexpression of STC1 partially rescued the proliferation of CGCs and resulted in opposite effects on the secretion of progesterone and estrogen. Several apoptosis and steroidogenesis-related genes, including BAX, BCL2, HSD3B1, HSD17B1, CYP11A1 and CYP17A1 showed altered expressions after repressing or increasing the expression of STC1 in yak CGCs. To the best of our knowledge, this study is the first to focus on the role of STC1 in yak CGCs, and the outcomes offer fresh insights into the mechanism governing yak reproduction.

The role of platelet-derived growth factor BB signaling pathway in the regulation of stem and progenitor Leydig cell proliferation and steroidogenesis in male rats

Platelet-derived growth factor BB (BB) regulates cell proliferation and function. However, the roles of BB on proliferation and function of Leydig stem (LSCs) and progenitor cells (LPCs) and the underlying signaling pathways remain unclear. This study aimed to analyze the roles of PI3K and MAPK pathways in the regulation of proliferation-related and steroidogenesis-related gene expression in rat LSCs/LPCs. In this experiment, BB receptor antagonist, tyrosine kinase inhibitor IV (PKI), the PI3K inhibitor, LY294002, and the MEK inhibitor, U0126, were used to measure the effects of these pathways on the expression of cell cycle-related genes (Ccnd1 and Cdkn1b) and steroidogenesis-related genes (Star, Cyp11a1, Hsd3b1, Cyp17a1, and Srd5a1), as well as Leydig cell maturation gene Pdgfra [1]. These results showed that BB (10 ng/mL)-stimulated EdU-incorporation into LSCs and BB-mediated inhibition on its differentiation was mediated through the activation of its receptor, PDGFRB, as well as MAPK and PI3K pathways. The results of LPC experiment also showed that LY294002 and U0126 decreased BB (10 ng/mL)-upregulated Ccnd1 expression while only U0126 reversed BB (10 ng/mL)-downregulated Cdkn1b expression. U0126 significantly reversed BB (10 ng/mL)-mediated downregulation of Cyp11a1, Hsd3b1, and Cyp17a1 expression. On the other hand, LY294002 reversed the expression of Cyp17a1 and Abca1. In conclusion, BB-mediated induction of proliferation and suppression of steroidogenesis of LSCs/LPCs are dependent on the activation of both MAPK and PI3K pathways, which show distinct regulation of gene expression.

Function of secretoneurin in regulating the expression of reproduction-related genes in ovoviviparous black rockfish (Sebastes schlegelii).

Secretoneurin (SN), a conserved peptide derived from secretogranin-2 (scg2), also known as secretogranin II or chromogranin C, plays an important role in regulating gonadotropin in the pituitary, which affects the reproductive system. This study aimed to clarify the mode of action of scg2 in regulating gonad development and maturation and the expression of mating behavior-related genes. Two scg2 cDNAs were cloned from the ovoviviparity teleost black rockfish (Sebastes schlegelii). In situ hybridization detected positive scg2 mRNA signals in the telencephalon and hypothalamus, where sgnrh and kisspeptin neurons were reported to be located and potentially regulated by scg2. In vivo, intracerebral ventricular injections of synthetic black rockfish SNa affected brain cgnrh, sgnrh, kisspeptin1, pituitary lh and fsh and gonad steroidogenesis-related gene expression levels with sex dimorphism. In vitro, a similar effect was found in primary cultured brain and pituitary cells. Thus, SN could contribute to the regulation of gonadal development, as well as reproductive behaviors, including mating and parturition.

Exposure to nanoplastics induces mitochondrial impairment and cytomembrane destruction in Leydig cells

Plastic particle pollution poses an emerging threat to ecological and human health. Laboratory animal studies have illustrated that nano-sized plastics can accumulate in the testis and cause testosterone deficiency and spermatogenic impairment. In this study, TM3 mouse Leydig cells were in vitro exposed to polystyrene nanoparticles (PS-NPs, size 20 nm) at dosages of 50, 100 and 150 μg/mL to investigate their cytotoxicity. Our results demonstrated that PS-NPs can be internalized into TM3 Leydig cells and led to a concentration-dependent decline in cell viability. Furthermore, PS-NPs stimulation amplified ROS generation and initiated cellular oxidative stress and apoptosis. Moreover, PS-NPs treatment affected the mitochondrial DNA copy number and collapsed the mitochondrial membrane potential, accompanied by a disrupted energy metabolism. The cells exposed to PS-NPs also displayed a down-regulated expression of steroidogenesis-related genes StAR, P450scc and 17β-HSD, along with a decrease in testosterone secretion. In addition, treatment with PS-NPs destructed plasma membrane integrity, as presented by increase in lactate dehydrogenase release and depolarization of cell membrane potential. In summary, these data indicated that exposure to PS-NPs in vitro produced cytotoxic effect on Leydig cells by inducing oxidative injury, mitochondrial impairment, apoptosis, and cytomembrane destruction. Our results provide new insights into male reproductive toxicity caused by NPs.

Mitochondrial function and E2 synthesis are impaired following alteration of CLOCK gene expression in porcine ovarian granulosa cells

Circadian locomotor output cycles kaput (CLOCK) is a critical component of the mammalian circadian clock system and regulates ovarian physiology. However, the functions and mechanisms of CLOCK in porcine granulosa cells (GCs) are poorly understood. The present study focused on CLOCK's effects on estradiol synthesis. Similarity analysis showed that CLOCK is highly conserved between pigs and other species. The phylogenetic tree analysis indicated that porcine CLOCK was most closely related to that in Arabian camels. CLOCK significantly reduced E2 synthesis in GCs. CLOCK reduced the expression of steroidogenesis-related genes at the mRNA and protein levels, including CYP19A1, CYP11A1, and StAR. CYP17A1 levels were significantly downregulated. We demonstrated that CLOCK dramatically decreased ATP content, mitochondrial copy number, and mitochondrial membrane potential (MMP) and increased reactive oxygen species levels in GCs. We observed that mitochondria were severely damaged with fuzzy and fractured cristae and swollen matrix. These findings suggest that mitochondrial function and E2 synthesis are impaired following the alteration of CLOCK gene expression in porcine ovarian GCs.

Prepubertal exposure to copper oxide nanoparticles induces Leydig cell injury with steroidogenesis disorders in mouse testes

Copper oxide nanoparticles (CuONPs) are metallic multifunctional nanoparticles with good conductive, catalytic and antibacterial characteristics that have shown to cause reproductive dysfunction. However, the toxic effect and potential mechanisms of prepubertal exposure to CuONPs on male testicular development have not been clarified. In this study, healthy male C57BL/6 mice received 0, 10, and 25 mg/kg/d CuONPs by oral gavage for 2 weeks (postnatal day 22–35). The testicular weight was decreased, testicular histology was disturbed and the number of Leydig cells was reduced in all CuONPs-exposure groups. Transcriptome profiling suggested steroidogenesis was impaired after exposure to CuONPs. The steroidogenesis-related genes mRNA expression level, concentration of serum steroids hormones and the HSD17B3-, STAR- and CYP11A1-positive Leydig cell numbers were dramatically reduced. In vitro, we exposed TM3 Leydig cells to CuONPs. Bioinformatic analysis, flow cytometry analysis and western blotting analysis confirmed that CuONPs can dramatically reduce Leydig cells viability, enhance apoptosis, trigger cell cycle arrest and reduce cell testosterone levels. U0126 (ERK1/2 inhibitor) significantly reversed TM3 Leydig cells injury and testosterone level decrease induced by CuONPs. These outcomes indicate that CuONPs exposure activates the ERK1/2 signaling pathway, which further promotes apoptosis and cell cycle arrest in TM3 Leydig cells, and ultimately leads to Leydig cells injury and steroidogenesis disorders.

Green Synthesized Zinc Oxide Nanoparticles Using Moringa olifera Ethanolic Extract Lessens Acrylamide-Induced Testicular Damage, Apoptosis, and Steroidogenesis-Related Gene Dysregulation in Adult Rats

This study assessed the possible protective role of green synthesized zinc oxide nanoparticles using Moringa olifera leaf extract (MO-ZNPs) in acrylamide (ACR)-induced reproductive dysfunctions in male rats. ACR (20 mg/kg b.wt/day) and/or MO-ZNPs (10 mg/kg b.wt/day) were given orally by gastric gavage for 60 days. Then, sperm parameters; testicular enzymes; oxidative stress markers; reproductive hormones including testosterone, luteinizing hormone (LH)-estradiol, and follicle-stimulating hormone (FSH) concentration; testis histology; steroidogenesis-related gene expression; and apoptotic markers were examined. The findings revealed that MO-ZNPs significantly ameliorated the ACR-induced decline in the gonadosomatic index and altered the pituitary-gonadal axis, reflected by decreased serum testosterone and FSH with increased estradiol and LH, and sperm analysis disruption. Furthermore, a notable restoration of the tissue content of antioxidants (catalase and reduced glutathione) but depletion of malondialdehyde was evident in MO-ZNPs+ACR-treated rats compared to ACR-exposed ones. In addition, MO-ZNPs oral dosing markedly rescued the histopathological changes and apoptotic caspase-3 reactions in the testis resulting from ACR exposure. Furthermore, in MO-ZNPs+ACR-treated rats, ACR-induced downregulation of testicular steroidogenesis genes and proliferating cell nuclear antigen (PCNA) immune-expression were reversed. Conclusively, MO-ZNPs protected male rats from ACR-induced reproductive toxicity by suppressing oxidative injury and apoptosis while boosting steroidogenesis and sex hormones.