Apigenin improves testosterone synthesis by regulating endoplasmic reticulum stress.

Obesity is a growing epidemic among reproductive-age men, which can cause and exacerbate male infertility by means of associated comorbidities, endocrine abnormalities, and direct effects on the fidelity and throughput of spermatogenesis. A prominent consequence of male obesity is a reduction in testosterone levels. Natural products have shown tremendous potential anti-obesity effects in metabolic diseases. This study aimed to investigate the potential of apigenin (AP) to alleviate testicular dysfunction induced by a high-fat diet (HFD) and to investigate the underlying mechanisms, focusing on endoplasmic reticulum stress (ERS) and testosterone synthesis. A murine model of obesity was established using HFD-fed mice. The effects of AP on obesity, lipid metabolism, testicular dysfunction, and ERS were assessed through various physiological, histological, and molecular techniques. Administration of AP (10 mg/kg) ameliorated HFD-induced obesity and testicular dysfunction in a mouse model, as evidenced by decreased body weight, improved lipid profiles and testicular pathology, and restored protein levels related to testosterone. Furthermore, in vitro studies demonstrated that AP relieved ERS and recovered testosterone synthesis in murine Leydig cells (TM3) treated with free fatty acids (FFAs). It was also observed that AP rescued testosterone synthesis enzymes in TM3 cells, similar to that observed with the inhibitor of the PERK pathway (GSK2606414). In addition, ChIP, qPCR, and gene silencing showed that the C/EBP homologous protein (CHOP) bound directly to the promoter region of steroidogenic STAR and negatively modulated its expression. Collectively, AP has remarkable potential to alleviate HFD-induced obesity and testicular dysfunction. Its protective effects are attributable partly to mitigating ERS and restoring testosterone synthesis in Leydig cells.

METTL14 mediates nerve growth factor-stimulated testosterone synthesis in porcine theca cells.

Ovarian theca cells produce testosterone, which acts as a vital precursor substance for synthesizing estrogens during follicular development. Nerve growth factor (NGF) has been shown to participate in reproductive physiology, specifically to follicular development and ovulation. There is currently no available data on the impact of NGF on testosterone synthesis in porcine theca cells. Furthermore, m6A modification is the most common internal modification in eukaryotic mRNAs that are closely associated with female gametogenesis, follicle development, ovulation, and other related processes. It is also uncertain whether the three main enzymes associated with m6A, such as Writers, Erasers and Readers, play a role in this process. The present study, with an in vitro culture model, investigated the effect of NGF on testosterone synthesis in porcine theca cells and the role of Writers-METTL14 in this process. It was found that NGF activates the PI3K/AKT signaling pathway through METTL14, which regulates testosterone synthesis in porcine theca cells. This study will help to further elucidate the mechanisms by which NGF regulates follicular development and provide new therapeutic targets for ovary-related diseases in female animals.

Expression of the umami taste receptor T1R1/T1R3 in porcine testis of: Function in regulating testosterone synthesis and autophagy in Leydig cells.

Testosterone is a vital male hormone responsible for male sexual characteristics. The taste receptor family 1 subunit 3 (T1R3) regulates testosterone synthesis and autophagy in non-taste cells, and the links with the taste receptor family 1 subunit 1 (T1R1) for umami perception. However, little is known about these mechanisms. Thus, we aimed to determine the relationship between the umami taste receptor (T1R1/T1R3) and testosterone synthesis or autophagy in testicular Leydig cells of the Xiang pig. There was a certain proportion of spermatogenic tubular dysplasia in the Xiang pig at puberty, in which autophagy was enhanced, and the testosterone level was increased with a weak expression of T1R3. Silenced T1R3 decreased testosterone level and intracellular cyclic adenosine monophosphate (cAMP) content and inhibited the messenger RNA (mRNA) expression levels of testosterone synthesis enzyme genes [steroidogenic acute regulatory protein (StAR), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (3ß-HSD1), cytochrome P450 family 17 subfamily A member 1 (CYP17A1) and hydroxysteroid 17-beta dehydrogenase 3 (17ß-HSD3)]. In addition, T1R3 increased the number of acidic autophagy bubbles and upregulated the expression levels of autophagy markers [Microtubule-associated protein 1 A/1B-light chain 3 (LC3) and Beclin-1] in testicular Leydig cells of the Xiang pig. Using an umami tasting agonist (10 mM L-glutamate for 6 h), the activation of T1R1/T1R3 enhanced the testosterone synthesis ability by increasing the intracellular cAMP level and upregulated the expression levels of StAR, 3ß-HSD1, CYP17A1 and 17ß-HSD3 in Leydig cells. Furthermore, the number of acidic autophagy bubbles decreased in the T1R1/T1R3-activated group with the downregulation of the expression levels of the autophagy markers, including LC3 and Beclin-1. These data suggest that the function of T1R1/T1R3 expressed in testicular Leydig cells of the Xiang pig is related to testosterone synthesis and autophagy.

Protective effect of TNFAIP3 on testosterone production in Leydig cells under an aging inflammatory microenvironment.

BACKGROUND: The aging inflammatory microenvironment surrounding Leydig cells is linked to reduced testosterone levels in males. Tumor necrosis factor alpha-induced protein 3 (TNFAIP3) acts as a critical anti-inflammatory factor in various aging-related diseases. This study aims to investigate the protective effect of TNFAIP3 on testosterone production in Leydig cells under an aging inflammatory microenvironment. METHODS: Bioinformatics analysis examined TNFAIP3 expression differences in aging rat testes and validated the findings in aging mouse testes. In vitro models of inflammation were established using two Leydig cell lines, with tumor necrosis factor alpha (TNF-α) as the inflammatory factor. Lentiviral transduction was utilized to manipulate TNFAIP3 expression in these cell lines. Transcriptomic sequencing identified differentially expressed genes in TNFAIP3-overexpressing cells. RESULTS: Bioinformatics analysis and validation experiments revealed increased inflammatory signaling and elevated TNFAIP3 expression in aging rat and mouse testes. TNFAIP3 knockdown worsened testosterone synthesis inhibition and apoptosis in cells, while TNFAIP3 overexpression reversed these effects. Transcriptome analysis identified alterations in the P38MAPK pathway following TNFAIP3 overexpression. TNFAIP3 knockdown enhanced TNF-induced P38MAPK signaling, whereas its overexpression attenuated this effect. TNFAIP3 was found to regulate testosterone synthesis by upregulating CEBPB expression. CONCLUSIONS: TNFAIP3 exhibits inhibitory effects on apoptosis and promotes testosterone production in Leydig cells. The protective influence of TNFAIP3 on Leydig cells within an inflammatory microenvironment is likely mediated through by inhibiting the P38MAPK pathway and upregulating CEBPB expression.

Knockdown of ZnT4 Induced Apoptosis, Inhibited Proliferation and testosterone synthesis of TM3 cells.

Zinc deficiency has a huge impact on male reproduction. The zinc transporter (ZnT) family is involved in the maintenance of zinc homeostasis and testosterone synthesis. However, the underlying mechanisms remain to be investigated. Therefore, in this study, we aimed to determine the effect of zinc transporter 4 (ZnT4) on testosterone synthesis in male Kunming mice and mouse Leydig cells. The results of this study showed that compared with the zinc normal diet group (Con group), the zinc-deficient diet group (ZnD group) had decreased zinc content and increased ZnT4 expression in testicular tissues, and decreased serum testosterone levels, suggesting that ZnT4 may be involved in Leydig cell injury resulting from a zinc-deficient diet. Subsequently, mouse Leydig cell line TM3 cells were used to analyze the effect of ZnT4 downregulation on TM3 cell proliferation and apoptosis, on testosterone synthesis, and its underlying mechanisms. Here, we show that knockdown of ZnT4 can induce the accumulation of zinc, inhibit the viability, and induce apoptosis in TM3 cells. In addition, knockdown of ZnT4 downregulated testosterone concentration and expression of testosterone synthesis-related proteins steroidogenic acute regulatory protein (StAR) and 3ß-hydroxysteroid dehydrogenase/D5-D4 isomerase (3ß-HSD) in TM3 cells, while hCG could rescue their levels. We show that it is ZnT4 that plays a role in testosterone production through a mediated PI3K/Akt/mTOR autophagy pathway, whereas mTORC1 complex inhibitor (Rapa) blocks the decrease in testosterone levels caused by ZnT4 downregulation. In conclusion, the above results indicate that ZnT4 plays an important role in regulating testosterone synthesis.

VDR promotes testosterone synthesis in mouse Leydig cells via regulation of cholesterol side chain cleavage cytochrome P450 (Cyp11a1) expression.

BACKGROUND: The vitamin D receptor (VDR) mediates the pleiotropic biological actions that include osteoporosis, immune responses and androgen synthesis.VDR is widely expressed in testis cells such as Leydig cells, Sertoli cells, and sperm. The levels of steroids are critical for sexual development. In the early stage of steroidogenesis, cholesterol is converted to pregnenolone (precursor of most steroid hormones) by cholesterol side-chain lyase (CYP11A1), which eventually synthesizes the male hormone testosterone. OBJECTIVE: This study aims to reveal how VDR regulates CYP11A1 expression and affects testosterone synthesis in murine Leydig cells. METHODS: The levels of VDR, CYP11A1 were determined by quantitative real-time polymerase chain reaction (RT-qPCR) or western blot. Targeted relationship between VDR and Cyp11a1 was evaluated by dual-luciferase reporter assay. The levels of testosterone concentrations in cell culture media serum by enzyme-linked immunosorbent assay (ELISA). RESULTS: Phylogenetic and motif analysis showed that the Cyp11a1 family had sequence loss, which may have special biological functions during evolution. The results of promoter prediction showed that vitamin D response element (VDRE) existed in the upstream promoter region of murine Cyp11a1. Dual-luciferase assay confirmed that VDR could bind candidate VDREs in upstream region of Cyp11a1, and enhance gene expression. Tissue distribution and localizatio analysis showed that Cyp11a1 was mainly expressed in testis, and dominantly existed in murine Leydig cells. Furthermore, over-expression VDR and CYP11A1 significantly increased testosterone synthesis in mice Leydig cells. CONCLUSIONS: Active vitamin D3 (VD3) and Vdr interference treatment showed that VD3/VDR had a positive regulatory effect on Cyp11a1 expression and testosterone secretion. VDR promotes testosterone synthesis in male mice by up-regulating Cyp11a1 expression, which played an important role for male reproduction.

Effects of perfluorododecanoic acid on testicular function in mice.

Perfluorodecanoic acid (PFDoA) is a widely distributed environmental pollutant that can affect the functions of many organs. However, systematic evaluations of the effects of PFDoA on testicular functions are lacking. The aim of this study was to investigate the effects of PFDoA on mouse testicular functions, including spermatogenesis, testosterone synthesis, and stem Leydig cells (SLCs) in the interstitial tissue of the testis. PFDoA (0, 2, 5, 10 mg/kg/d) was administered via gavage to 2-month-old mice for 4 weeks. Serum hormone levels and sperm quality were assayed. Furthermore, to investigate the mechanisms by which PFDoA affects testosterone synthesis and spermatogenesis in vivo, the expression of StAR and P450scc in testicular tissue was measured by immunofluorescence staining and quantitative real-time PCR. In addition, the levels of SLC markers, including nestin and CD51, were studied. PFDoA decreased the luteinizing hormone concentration and sperm quality. Although the difference was not statistically significant, mean testosterone levels showed a downward trend. The expression of StAR, P450scc, CD51, and nestin was also suppressed in the PFDoA-treated groups compared with the control group. Our study suggested that PFDoA exposure can decrease testosterone biosynthesis, and even reduce the number of SLCs. These results indicated that PFDoA suppressed the main functions of testis, and further researches are required to identify strategies for preventing or reducing the effect of PFDoA on testicular function.

The alternate pathway of androgen metabolism and window of sensitivity.

Since the discovery in 1968 that dihydrotestosterone (DHT) is a major mediator of androgen action, a convincing body of evidence has accumulated to indicate that the major pathway of DHT formation is the 5α-reduction of circulating testosterone in androgen target tissues. However, we now know that DHT can also be formed in peripheral tissues by the oxidation of 5α-androstane-3α,17ß-diol (adiol). This pathway is responsible for the formation of the male phenotype. We discuss the serendipitous discovery in the tammar wallaby of an alternate pathway by which adiol is formed in the testes, secreted into plasma and converted in peripheral tissues to DHT. This alternate pathway is responsible for virilisation of the urogenital system in this species and is present in the testes at the onset of male puberty of all mammals studied so far. This is the first clear-cut function for steroid 5α-reductase 1 in males. Unexpectedly, the discovery of this pathway in this Australian marsupial has had a major impact in understanding the pathophysiology of aberrant virilisation in female newborns. Overactivity of the alternate pathway appears to explain virilisation in congenital adrenal hyperplasia CAH, in X-linked 46,XY disorders of sex development. It also appears to be important in polycystic ovarian syndrome (PCOS) since PCOS ovaries have enhanced the expression of genes and proteins of the alternate pathway. It is now clear that normal male development in marsupials, rodents and humans requires the action of both the classic and the alternate (backdoor) pathways.

Sweet Taste Receptor T1R3 Expressed in Leydig Cells Is Closely Related to Homeostasis of the Steroid Hormone Metabolism Profile.

Taste receptor type 1 subunit 3 (T1R3) is initially expressed in mammal tongue for recognition and response of sweet/umami tastants and is critical to nutrient absorption, even endocrine. In this study, down-regulation of related steroidogenic enzymes such as StAR, 3ß-HSD, CYP17A1, and 17ß-HSD with the decrease of T1R3 expression was found in Leydig cells treated by a T1R3 inhibitor (lactisole). The abundances of progesterone, 17a-hydroxyprogesterone, androstenedione, testosterone, and deoxycorticosterone were down-regulated by 2.3, 3.5, 1.4, 1.6, and 2.2 times, respectively, after T1R3 inhibition. In addition, opposite results were found in saccharin sodium treatment. T1R3 activation contributed to intracellular cyclic adenosine monophosphate (cAMP) accumulation (14.41 ± 0.58 vs 20.21 ± 0.65) and increased testosterone (20.31 ± 3.49 vs 50.01 ± 7.44) and steroidogenic metabolite levels. Coadministration of human chorionic gonadotropin and saccharin sodium resulted in elevating the testosterone and cAMP levels and enhancing the expression levels of steroidogenic-related factors. Similarly, intratesticular injection of lactisole and saccharin sodium further confirmed that T1R3 inhibition/activation affected the expression of related steroidogenic enzymes and the testosterone levels in mice. The above findings suggest that T1R3 plays a role in testicular steroidogenesis.

Selenium nanoparticles improve nickel-induced testosterone synthesis disturbance by down-regulating miR-708-5p/p38 MAPK pathway in Leydig cells.

The present study was designed to investigate the role of miR-708-5p/p38 mitogen-activated protein kinase (MAPK) pathway during the mechanism of selenium nanoparticles (Nano-Se) against nickel (Ni)-induced testosterone synthesis disorder in rat Leydig cells. We conducted all procedures based on in vitro culture of rat primary Leydig cells. After treating Leydig cells with Nano-Se and NiSO4 alone or in combination for 24 h, we determined the cell viability, reactive oxygen species (ROS) levels, testosterone production, and the protein expression of key enzymes involved in testosterone biosynthesis: steroidogenic acute regulatory (StAR) and cytochrome P450 cholesterol side chain cleavage enzyme (CYP11A1). The results indicated that Nano-Se antagonized cytotoxicity and eliminated ROS generation induced by NiSO4 , suppressed p38 MAPK protein phosphorylation and reduced miR-708-5p expression. Importantly, we found that Nano-Se upregulated the expression of testosterone synthase and increased testosterone production in Leydig cells. Furthermore, we investigated the effects of p38 MAPK and miR-708-5p using their specific inhibitor during Nano-Se against Ni-induced testosterone synthesis disorder. The results showed that Ni-inhibited testosterone secretion was alleviated by Nano-Se co-treatment with p38 MAPK specific inhibitor SB203580 and miR-708-5p inhibitor, respectively. In conclusion, these findings suggested Nano-Se could inhibit miR-708-5p/p38 MAPK pathway, and up-regulate the key enzymes protein expression for testosterone synthesis, thereby antagonizing Ni-induced disorder of testosterone synthesis in Leydig cells.

Role of ROS/JAK2/STAT3 signaling pathway in di-n-butyl phthalate-induced testosterone synthesis inhibition and antagonism of lycopene.

Di-n-butyl phthalate (DBP) causes adverse effects on male reproduction, especially testosterone synthesis inhibition. However, the specific mechanism of DBP-induced testosterone synthesis inhibition and its effective intervention measures of prevention and treatment are scarce presently. Lycopene (LYC) plays beneficial roles in male infertility because of its antioxidant activity. Nevertheless, it is unclear whether LYC could prevent DBP-induced male reproductive toxicity. By in vitro and in vivo investigations, this study demonstrated that DBP activated ROS/JAK2/STAT3 signaling pathway, promoted mitophagy and apoptosis, which in turn inhibited testosterone synthesis. Additionally, another major finding was that LYC supplement could reverse the above change, presenting as the restraint of ROS/JAK2/STAT3 signaling pathway, reduction of mitophagy and apoptosis, and improvement of testosterone synthesis. Our study facilitates deeper understandings of the mechanism in DBP-induced testosterone synthesis inhibition, and identifies LYC as the effective prevention and control strategies for DBP poisoning.

p38 mediates T-2 toxin-induced Leydig cell testosterone synthesis disorder.

T-2 toxin is an unavoidable food and feed contaminant that seriously threatens human and animal health. Exposure to T-2 toxin can cause testosterone synthesis disorder in male animals, but the molecular mechanism is still not completely clear. The MAPK pathway participates in the regulation of testosterone synthesis by Leydig cells, but it is unclear whether the MAPK pathway participates in T-2 toxin-induced testosterone synthesis disorders. In this research, testosterone synthesis capacity, testosterone synthase expression and MAPK pathway activation were examined in male mice and TM3 cells exposed to T-2 toxin. The results showed that T-2 toxin exposure decreased testicular volume and caused pathological changes in the microstructure and ultrastructure of testicular Leydig cells. T-2 toxin exposure also decreased testicular testosterone content and the protein expression of testosterone synthase. In vitro, T-2 toxin inhibited cell viability and decreased the expression of testosterone synthase in TM3 cells, and it decreased the testosterone contents in cell culture supernatants. Moreover, T-2 toxin activated the MAPK pathway by increasing the expression of p38, JNK and ERK as well as the expression of p-p38, p-JNK and p-ERK in testis and TM3 cells. The p38 molecular inhibitor (SB203580) significantly alleviated the T-2 toxin-induced decrease in testosterone synthase expression in TM3 cells and the T-2 toxin-induced reduction in testosterone content in TM3 cell culture supernatants. In summary, p38 mediates T-2 toxin-induced Leydig cell testosterone synthesis disorder.

DNA methylation-mediated inhibition of MGARP is involved in impaired progeny testosterone synthesis in mice exposed to DBP in utero.

The dibutyl phthalate (DBP) has been detected in fetuses and infants and can cause damage to the reproductive system in adulthood, but the exact mechanism remains unclear. Here, we aim to investigate the effects of intrauterine DBP exposure on offspring reproductive function and explore possible mechanisms. SPF C57BL/6 pregnant mice were given DBP (0.5, 5, 75 mg/kg/d) or corn oil from day 5 to day 19 by gavage. After weaning, the pups were fed a standard diet for 5 weeks. In addition, TM3 Leydig cell cultures were used to study the relevant mechanisms in vitro. The results showed that intrauterine DBP exposure could reduce sperm density and sperm motility, cause testicular tissue damage, down-regulate serum T and LH levels, and up-regulate serum FSH levels at 75 mg/kg/d. Western blot and methylation detection revealed intrauterine exposure to DBP down-regulated testosterone synthesis-related proteins StAR, P450scc, 3ß-HSD, PKA, and PKC expression, while up-regulated the levels of methyltransferase proteins expression and DNA 5-methylcytosine (5mC) in testicular tissue of mouse offspring at 75 mg/kg/d. Further detection found in utero 75 mg/kg/d DBP exposure down-regulated MGARP protein expression, and induced incomplete methylation of the MGARP gene. An in vitro analysis showed that MGARP inhibition is involved in an impaired testosterone synthesis in TM3 cells. Cell culture results suggest that MGARP down-regulation may be involved in impaired testosterone production in monobutyl phthalate-treated cells. The present study revealed that 75 mg/kg/d DBP exposure in utero resulted in testosterone synthesis disorders and reproductive function impairment in mouse offspring, and the mechanism may be related to DNA methylation-mediated down-regulation of MGARP in the testis.

MicroRNA profiling reveals the role of miR-133b-3p in promoting apoptosis and inhibiting cell proliferation and testosterone synthesis in mouse TM3 cells.

Late-onset hypogonadism (LOH) is an age-related clinical and biological syndrome in which serum testosterone deficiency is an important characteristic and diagnostic indicator. In this study, we firstly analyzed the difference in the expression level of three miR-133 s (including miR-133a-3p, miR-133a-5p, and miR-133b-3p) in rat testis samples, blood samples from mice before and 1 wk after testis removal, and mouse TM3 cells. Secondly, the mimics and inhibitors corresponding to the three miR-133 s of mouse were transfected into TM3 cells separately to determine the correlation between the three miRNAs. Finally, using mouse TM3 cells to analyze the effect of miR-133b overexpression or inhibition on the proliferation and apoptosis of mouse testicular Leydig cells, the effect on genes related to testosterone synthesis, and the effect on the level of testosterone in the culture medium. We found that, compared with the testis tissue of newborn rats, miR-133a-5p was increased in adult rats, and miR-133a-3p and miR-133b-3p were decreased. In addition, 1 wk after the testis was removed, the expression levels of these three miRNAs in the blood of adult mice decreased. The correlation of the three miRNAs was summarized, and it was found that miR-133b-3p played an important role in it. In TM3 cells, overexpression of miR-133b-3p suppressed the proliferation and promotes apoptosis of cells, suppressed the expression level of most genes related to cell proliferation and testosterone synthesis, and the concentration of testosterone in the culture medium decreased while these phenomena can be reversed by the inhibition of miR-133b-3p expression. It was found that miR-133b-3p can regulate testosterone production in TM3 cells at least by targeting FSCN1. The above results suggest that miR-133b-3p plays an important role in regulating testosterone synthesis. These findings also provide new candidate diagnostic indicators for late-onset hypogonadism in men and provide new clues for the further study of pathogenesis.

Evidences for Mutant Huntingtin Inducing Musculoskeletal and Brain Growth Impairments via Disturbing Testosterone Biosynthesis in Male Huntington Disease Animals.

Body weight (BW) loss and reduced body mass index (BMI) are the most common peripheral alterations in Huntington disease (HD) and have been found in HD mutation carriers and HD animal models before the manifestation of neurological symptoms. This suggests that, at least in the early disease stage, these changes could be due to abnormal tissue growth rather than tissue atrophy. Moreover, BW and BMI are reported to be more affected in males than females in HD animal models and patients. Here, we confirmed sex-dependent growth alterations in the BACHD rat model for HD and investigated the associated contributing factors. Our results showed growth abnormalities along with decreased plasma testosterone and insulin-like growth factor 1 (IGF-1) levels only in males. Moreover, we demonstrated correlations between growth parameters, IGF-1, and testosterone. Our analyses further revealed an aberrant transcription of testosterone biosynthesis-related genes in the testes of BACHD rats with undisturbed luteinizing hormone (LH)/cAMP/PKA signaling, which plays a key role in regulating the transcription process of some of these genes. In line with the findings in BACHD rats, analyses in the R6/2 mouse model of HD showed similar results. Our findings support the view that mutant huntingtin may induce abnormal growth in males via the dysregulation of gene transcription in the testis, which in turn can affect testosterone biosynthesis.

Alleviating effects of selenium on fluoride-induced testosterone synthesis disorder and reproduction toxicity in rats.

Fluoride (F) exists widely in food, water and other natural resources, and can cause damage to the reproductive system of human and animals. Studies have shown that selenium (Se) is a necessary trace element to maintain the normal male reproductive system. However, it is not clear whether it can alleviate the damage of reproductive system induced by F. Hence, sodium fluoride (NaF) was administered singly in drinking water at 100 mg L-1 alone and co-administered by drinking with sodium selenite (Na2SeO3) at 0.5, 1.0, 2.0 mg L-1 for 10 consecutive weeks. The results demonstrated that the sperm deformity rate were increased significantly by F, however, it was improved significantly after the addition of 2.0 mg L-1 Na2SeO3. The contents of glutathione peroxidase 4 (GPX-4), selenoprotein P (SePP), pregnenolone (PREG), androstenedione (ASD), and testosterone (T) were reduced obviously in the F group, however, it was increased significantly after adding 0.5, 1.0 and 2.0 mg L-1 Na2SeO3. F decreased noticeably the mRNA and protein expression levels of steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side chain lyase (P450scc), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), cytochrome P450 17α-hydroxylase (P450c17) and 17ß-hydroxysteroid dehydrogenase (17ß-HSD), which was increased obviously after the addition of 1.0 and 2.0 mg L-1 Na2SeO3. In summary, 2.0 mg L-1 Na2SeO3 can alleviate testosterone synthesis disorder induced by F via reducing oxidative stress, increasing the level of selenoprotein in testis and regulating the content of related hormones and enzyme activity during testosterone synthesis pathway.

The protective effect of L-carnitine on testosterone synthesis pathway, and spermatogenesis in monosodium glutamate-induced rats.

BACKGROUND: Monosodium glutamate (MSG) is a food ingredient that is increasingly used commercially. MSG leads to oxidative stress, consequently suppressing steroid hormone production that causes defects in male reproductive system. This study aimed to evaluate the effect of L-carnitine as an antioxidant on testicular damage in MSG-induced male rats. METHODS: Sixty adult male Spargue-Dawley rats were randomly divided into six groups of ten as follows: control (water), sham (normal saline), L-carnitine (200 mg/kg b.w), MSG (3 g/kg b.w), MSG + L-carnitine 100 (3 g/kg b.w of MSG and 100 mg/kg b.w of L-carnitine), and MSG + L-carnitine 200 (3 g/kg b.w of MSG and 200 mg/kg b.w of L-carnitine). The treatment was administered by oral gavage for six months. Serum levels of Malondialdehyde (MDA), Total Anti-oxidant Capacity (TAC), LH, FSH, testosterone, and mRNA expressions of Star, Cyp11a1, and Hsd17b3 genes, and histological and stereological changes were assessed. RESULTS: L-carnitine led to a significant decrease in the level of MDA and a significant rise in the serum levels of TAC, LH, FSH, and mRNA expression of Star and Cyp11a1 compared to the MSG group (p < 0.05). Furthermore, stereological results indicated a significant increment in the number of sexual lineage cells, the total volume of the testis, length, diameter, and volume of seminiferous tubules, the height of the germinal epithelium, sperm count, and sperm motility (p < 0.05) in MSG + L-carnitine 200 compare to MSG group. CONCLUSION: The study's findings demonstrated that L-carnitine due to its anti-oxidant properties, ameliorated the reproductive abnormalities in the male rats exposed to MSG.

Benzo[a]pyrene inhibits testosterone biosynthesis via NDUFA10-mediated mitochondrial compromise in mouse Leydig cells: Integrating experimental and in silico toxicological approaches.

Benzo[a]pyrene (B[a]P), a representative of polycyclic aromatic hydrocarbons (PAHs), is ubiquitously spread in the environment and showing deleterious impacts on male steroidogenesis, including testosterone synthesis disorder. However, the precise mechanisms involved in B[a]P-induced steroidogenesis perturbation remains obscure. In the present study, we integrated in vivo tests, transcriptome profiling, in vitro assays, and conjoint in silico toxicological approaches to delineate the detailed mechanisms. In mouse models, we observed that B[a]P administration remarkably inhibited testosterone synthesis accompanied by ultrastructural impairments of mitochondria and mitophagosome formation in mouse Leydig cells. Transcriptome profiling showed that B[a]P down-regulated the expression of Ndufa9, Ndufa6, Ndufa10, and Ndufa5 in mouse testes, which are identified as critical genes involved in the assembly and functionality of mitochondrial complex I. In the in vitro tests, the bioactive B[a]P metabolite BPDE induced perturbation of testosterone synthesis by NDUFA10-mediated mitochondrial impairment, which was further exacerbated by mitophagy in TM3 Leydig cells. The findings of in silico toxicological analyses were highly consistent with the experimental observations and further unveiled that B[a]P/BPDE-involved PPARα activation could serve as a molecular initiating event to trigger the decline in Ndufa10 expression and testosterone synthesis. Overall, we have shown the first evidence that mitochondrial compromise in Leydig cells is the extremely crucial target in B[a]P-induced steroidogenesis perturbation.

Natural Astaxanthin Improves Testosterone Synthesis and Sperm Mitochondrial Function in Aging Roosters.

Spermatogenesis, sperm motility, and apoptosis are dependent on the regulation of glandular hormones and mitochondria. Natural astaxanthin (ASTA) has antioxidant, anti-inflammatory, and anti-apoptotic properties. The present study evaluates the effects of ASTA on testosterone synthesis and mitochondrial function in aging roosters. Jinghong No. 1 layer breeder roosters (n = 96, 53-week old) were fed a corn−soybean meal basal diet containing 0, 25, 50, or 100 mg/kg ASTA for 6 weeks. The levels of plasma reproductive hormones and the mRNA and protein levels of molecules related to testosterone synthesis were significantly improved (p < 0.05) in the testes of the ASTA group roosters. In addition, antioxidant activities and free radical scavenging abilities in roosters of the ASTA groups were higher than those of the control group (p < 0.05). Mitochondrial electron transport chain complexes activities and mitochondrial membrane potential in sperm increased linearly with dietary ASTA supplementation (p < 0.05). The levels of reactive oxygen species and apoptosis factors decreased in roosters of the ASTA groups (p < 0.05). Collectively, these results suggest that dietary ASTA may improve testosterone levels and reduce sperm apoptosis, which may be related to the upregulation of the testosterone synthesis pathway and the enhancement of mitochondrial function in aging roosters.

Associations between isoflavone exposure and reproductive damage in adult males: evidence from human and model system studies†.

It is controversial whether exposure to isoflavones exerts male reproductive toxicity. The aim of this study was to investigate whether isoflavone exposure during adulthood could have deleterious impacts on male reproductive health by the cross-sectional study, animal experiments, and in vitro tests. In the cross-sectional study, we observed that urinary isoflavones were not significantly associated with semen quality including sperm concentrations, sperm count, progressive motility, and total motility, respectively. However, negative associations were found between plasma testosterone and urinary Σisoflavones, genistein, glycitein, and dihydrodaidzein. In the animal experiments, serum and intratesticular testosterone levels were decreased in mice exposed to several dosages of genistein. Genistein administration caused upregulation of estrogen receptor alpha and downregulation of cytochrome P45017A1 protein levels in testes of mice. In vitro tests showed that genistein caused a concentration-dependent inhibition of testosterone production by TM3 Leydig cells. Elevated protein expression of estrogen receptor alpha and decreased messenger RNA/protein level of cytochrome P45017A1 were also observed in genistein-treated cells. Protein level of cytochrome P45017A1 and testosterone concentration were significantly restored in the estrogen receptor alpha small interferring RNA-transfected cells, compared to cells that treated with genistein alone. The results demonstrate that exposure to isoflavones during adulthood may be associated with alterations of reproductive hormones. Particularly, genistein, which inhibits testosterone biosynthesis through upregulation of estrogen receptor alpha in Leydig cells of mice, might induce the disruption of testosterone production in human. The present study provides novel perspective into potential targets for male reproductive compromise induced by isoflavone exposure.