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Twenty-four-month-old male C57BU6 mice with low serum testosterone levels were used as a late-onset hypogonadism （LOH） animal model for examining the effects of velvet antler polypeptide （VAP） on sexual function and testosterone synthesis. These mice received VAP for 5 consecutive weeks by daily gavage at doses of 100, 200, or 300 mg kg-1 body weight per day （n = 10 mice per dose）. Control animals （n = 10） received the same weight-based volume of vehicle. Sexual behavior and testosterone levels in serum and interstitial tissue of testis were measured after the last administration of VAP. Furthermore, to investigate the mechanisms of how VAP affects sexual behavior and testosterone synthesis in vivo, the expression of steroidogenic acute regulatory protein （STAR）, cytochrome P450 cholesterol side-chain cleavage enzyme （P450scc）, and 3β-hydroxysteroid dehydrogenase （3βHSD） in Leydig cells was also measured by immunofluorescence staining and quantitative real-time PCR. As a result, VAP produced a significant improvement in the sexual function of these aging male mice. Serum testosterone level and intratesticular testosterone （ITT） concentration also increased in the VAP-treated groups. The expression of STAR, P450scc, and 3β-HSD was also found to be enhanced in the VAP-treated groups compared with the control group. Our results suggested that VAP was effective in improving sexual function in aging male mice. The effect of velvet antler on sexual function was due to the increased expression of several rate-limiting enzymes of testosterone synthesis （STAR, P450scc, and 3β-HSD） and the following promotion of testosterone syothesis in vivo.

Background: Aspermia caused by exogenous testosterone limit its usage in late-onset hypogonadism (LOH) patients desiring fertility. Saikokaryukotsuboreito (SKRBT) is reported to improve serum testosterone and relieve LOH-related symptoms. However, it is unclear whether SKRBT affects fertility. We aimed to examine the effects of SKRBT on spermatogenesis and fertility in aging male mice. Methods: Thirty aging male mice were randomly assigned to three groups. Mice were orally administered with phosphate-buffer solution or SKRBT (300 mg/kg, daily) or received testosterone by subcutaneous injections (10 mg/kg, every 3 days). Thirty days later, each male mouse was mated with two female mice. All animals were sacrificed at the end of 90 days. Intratesticular testosterone (ITT) levels, quality of sperm, expression of synaptonemal complex protein 3 (SYCP3), and fertility were assayed. Results: In the SKRBT-treated group, ITT, quality of sperm, and expression of SYCP3 were all improved compared with the control group (ITT: 85.50 ± 12.31 ng/g vs. 74.10 ± 11.45 ng/g, P = 0.027; sperm number: [14.94 ± 4.63] × 106 cells/ml vs. [8.79 ± 4.38] × 106 cells/ml, P = 0.002; sperm motility: 43.16 ± 9.93% vs. 33.51 ± 6.98%, P = 0.015; the number of SYCP3-positive cells/tubule: 77.50 ± 11.01 ng/ml vs. 49.30 ± 8.73 ng/ml, P < 0.001; the expression of SYCP3 protein: 1.23 ± 0.09 vs. 0.84 ± 0.10, P < 0.001), but fertility was not significantly changed (P > 0.05, respectively). In the testosterone-treated group, ITT, quality of sperm, and expression of SYCP3 were markedly lower than the control group (ITT: 59.00 ± 8.67, P = 0.005; sperm number: [4.34 ± 2.45] × 106 cells/ml, P = 0.018; sperm motility: 19.53 ± 7.69%, P = 0.001; the number of SYCP3-positive cells/tubule: 30.00 ± 11.28, P < 0.001; the percentage of SYCP3-positive tubules/section 71.98 ± 8.88%, P = 0.001; the expression of SYCP3 protein: 0.71 ± 0.09, P < 0.001), and fertility was also suppressed (P < 0.05, respectively). Conclusion: SKRBT had no adverse effect on fertility potential in aging male mice.

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. Summary Sentence The present study investigated the effect of NGF on testosterone synthesis in porcine theca cells. It was found that NGF activates the PI3K/AKT signaling pathway through METTL14, which regulates testosterone synthesis in porcine theca cells. Graphical Abstract

The insecticide acetamiprid is used to control noxious agricultural pests. However, it can cause mammalian toxicity. We evaluated the reproductive toxicity of acetamiprid in adult male Sprague Dawley rats. Rats were given oral acetamiprid alone or with vitamin E for 35 days. Rat plasma testosterone concentration and sperm quality decreased significantly as the levels of luteinizing hormone (LH) increased after exposure. At the same time, acetamiprid increased malondialdehyde and nitric oxide (NO) levels of Leydig cells. Further analysis showed that acetamiprid reduced the adenosine triphosphate (ATP) and cyclic adenosine monophosphate (cAMP) production of Leydig cells, but the expression of luteinizing hormone/choriogonadotropin receptor (LHCGR) and the activity of adenylyl cyclase were not changed. Acetamiprid exposure also significantly diminished protein levels of steroidogenic acute regulatory protein (STAR), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase cluster (HSD3B), and cytochrome P450, family 11, subfamily a, polypeptide 1 (CYP11A1), and testicular mRNA levels, which are cAMP-dependent proteins that are essential for steroidogenesis. Electron microscopy indicated mitochondrial membrane damage in the Leydig cells of the testes of exposed rats. Vitamin E ameliorated the impairment of acetamiprid on Leydig cells. Our results indicate that acetamiprid causes oxidative stress and mitochondrial damage in Leydig cells and inhibits the synthesis of testicular ATP and cAMP. Acetamiprid disrupts subsequent testosterone biosynthesis by decreasing the rate of conversion of cholesterol to testosterone and by preventing cholesterol from entering the mitochondria within the Leydig cells. These effects caused reproductive damage to the rats. Summary Sentence Mitochondrial inner membrane damage may be the mechanism by which acetamiprid inhibits the testosterone secretion of testicular interstitial Leydig cells.

Background/Objectives. Men experience Leydig cell and mitochondrial dysfunction due to the accumulation of reactive oxygen species during aging, leading to hormonal imbalances in the body. This results in symptoms of testosterone deficiency syndrome (TDS) as testosterone levels decline. Consequently, there is a growing need for alternative therapies, such as phytotherapy, to regulate testosterone secretion. Methods. In this study, we evaluated the potential of elderberry extract powder (KSB191) as a functional ingredient for improving TDS by analyzing its mechanism in regulating testosterone imbalance. The major compounds of KSB191 were rutin and fructose–leucine, and the efficacy of KSB191 was confirmed by observing increases in total testosterone, free testosterone, and sperm motility in an aged rat model with decreased testosterone levels. Additionally, we assessed safety by analyzing levels of prostate-specific antigen, alanine aminotransferase, aspartate aminotransferase, and creatinine. Results. To confirm the effectiveness of KSB191 in increasing testosterone synthesis and inhibiting its breakdown, we analyzed the expression levels of genes related to testosterone synthesis and degradation in the testis tissue. KSB191 not only increases the expression levels of enzymes (3β-HSD, CYP17A1, and 17β-HSD) that catalyze testosterone synthesis in Leydig cells, but also reduces the expression of enzymes (5α-reductase and CYP19A1) that degrade testosterone, thereby enhancing testosterone production in the body. Conclusions. KSB191 is predicted to be a novel functional ingredient that acts on Leydig cells and increases testosterone synthesis (particularly, the increase in free testosterone), ultimately alleviating the symptoms of TDS.

Stress is one of the leading causes of male infertility, but its exact function in testosterone synthesis has scarcely been reported. We found that adult male rats show a decrease in bodyweight, genital index and serum testosterone level after continual chronic stress for 21 days. Two‐dimensional gel electrophoresis (2‐DE) and MALDI‐TOF‐MS analysis identified 10 differentially expressed proteins in stressed rats compared with controls. A strong protein interaction network was found to be centred on Atp5a1 among these proteins. Atp5a1 expression significantly decreased in Leydig cells after chronic stress. Transfection of Atp5a1 siRNAs decreased StAR, CYP11A1, and 17β‐HSD expression by damaging the structure of mitochondria in TM3 cells. This study confirmed that chronic stress plays an important role in testosterone synthesis by regulating Atp5a1 expression in Leydig cells.

Circadian rhythm disorders caused by genetic or environmental factors lead to decreased male fertility but the mechanisms are poorly understood. The current study reports that the mechanism of Per1/Per2 Double knockout (DKO) reduced the reproductive capacity of elderly male mice. The sperm motility and spermatogenic capacity of male DKO mice were weak. Hormone-targeted metabolomics showed reduced plasma levels of free testosterone in DKO male mice compared with WT male mice. Transcriptomic analysis of testicular tissue showed the down-regulation of testosterone synthesis-related enzymes (Cyp11a1, Cyp17a1, Hsd17b3, Hsd3b1, and Star) in the steroid hormone synthesis pathway. Spermatogenesis genes, Tubd1 and Pafah1b were down-regulated, influencing tubulin dynamics and leading to impaired motility. Seleno-compound metabolic loci, Scly and Sephs2, were up-regulated and Slc7a11 and Selenop were down-regulated. Western-blotting showed that steroid acute regulatory protein (StAR) and p-CREB, PKA and AC1 were reduced in testicular tissue of DKO mice compared to WT. Therefore, Per1/Per2 disruption reduced testosterone synthesis and sperm motility by affecting the PKA-StAR pathway, leading to decreased fertility.

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. Summary Sentence Isoflavone exposure during adulthood is remarkably associated with reproductive hormone perturbation in males, which may be mediated through the estrogen receptor alpha/cytochrome P45017A1 pathway. Graphical Abstract

The prevalence of male infertility has been increasing globally, necessitating the search for safe and nontoxic active compounds to alleviate reproductive dysfunction. Although the precise mechanism remains unknown, Rupr. (CS) extract has protective effects on the reproductive system. The effect of C. songaricum Rupr. flavonoids (CSF) on reproductive injury and testicular mesenchymal stem cell viability in male mice and TM3 cells was investigated. We explored the possible association between these effects and the testosterone (T) synthesis pathway. Mice were administered cyclophosphamide to induce reproductive damage, followed by CSF administration. Body mass and organ index were recorded. Pathological changes in T and the epididymis were observed using hematoxylin-eosin staining. ELISA measured the serum levels of T, luteinizing hormone (LH), gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH), and estradiol (E ) in mice. Fructose and zinc ion levels in the seminal plasma were measured. TM3 cells were treated with Bisphenol A (BPA) and different concentrations of CSF, followed by proliferative evaluations using the CCK-8 assay and T and LH level assessments using ELISA. Furthermore, the expression of steroidogenic enzyme genes and proteins was investigated using western blotting and RT-PCR. CSF exhibited a notable reduction in reproductive damage and improved pathological changes in testicular and epididymal tissues. CSF group demonstrated substantially higher levels of seminal plasma fructose and zinc ions; markedly elevated serum levels of T, LH, GnRH, and FSH; and lower levels of E2 than those of the model group. Intracellular T content and secretion of T and LH increase with CSF while effectively mitigating BPA-induced damage to TM3 cells. CSF group exhibited substantially higher gene and protein expression of steroidogenic enzymes than those of the model group, both and . CSF ameliorates reproductive impairment by enhancing the expression of pivotal enzymes involved in synthesizing T. CSF ameliorates cyclophosphamide-induced reproductive impairment and bisphenol A-induced TM3 cell damage in mice by regulating sex hormone levels in the Hypothalamic-Pituitary-Gonadal Axis (HPG axis) and upregulating the expression of steroidogenic enzymes. Therefore, CS is a potential treatment for male reproductive impairment.

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 3β-hydroxysteroid dehydrogenase/D5-D4 isomerase (3β-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.