Leydig Research Articles

From Gene to Protein: Unraveling the Reproductive Blueprint of Male Grey Squirrels via Nerve Growth Factor (NGF) and Cognate Receptors

The grey squirrel, an invasive species, threatens the Eurasian red squirrel’s conservation, particularly in Umbria, Italy. Understanding its reproductive biology is essential to limiting its reproductive success. This study investigates the NGF system and its receptors (NTRK1 and p75NTR) in the testes of male grey squirrels, following prior research on female reproductive biology. NGF plays a role in testicular morphogenesis and spermiogenesis in animals and humans. As part of the LIFE Project U-SAVEREDS, eighteen squirrels were captured and classified into three morphotypes (immature, pubertal, and active spermatogenesis). NGF and its receptors were analyzed using real-time PCR, western blotting, immunohistochemistry, and plasma levels measured via ELISA. NGF qPCR expression levels were significantly higher during puberty compared to the immature and spermatogenesis stages (p < 0.01). Immunohistochemistry revealed NGF in Leydig cells, with stronger staining in pubertal and mature squirrels, while NTRK1 was found in Leydig cells in immature squirrels and germ cells in pubertal and mature ones. NGF receptors were observed in Sertoli cells in pubertal and mature squirrels. Plasma NGF levels showed a significant upregulation in pubertal squirrels (135.80 ± 12 pg/mL) compared to those in the immature (25.60 ± 9.32 pg/mL) and spermatogenesis stages (34.20 ± 6.06 pg/mL), with a p value < 0.01. The co-localization of NGF and its receptors suggests that NGF, produced by Leydig cells, regulates testis development and reproductive success through autocrine or paracrine mechanisms, potentially involving an unidentified pathway.

Reconstructing the Stem Leydig Cell Niche via the Testicular Extracellular Matrix for the Treatment of Testicular Leydig Cell Dysfunction

AbstractTherapies involving the use of stem Leydig cells (SLCs), as testicular mesenchymal stromal cells, have shown great promise in the treatment of Leydig cell (LC) dysfunction in aging males. However, the outcomes of these therapies are not satisfactory. In this study, it is demonstrated that the aging microenvironment of the testicular interstitium impairs the function of SLCs, leading to poor regeneration of LCs and, consequently, inefficient functional restoration. The study develops a decellularized testicular extracellular matrix (dTECM) hydrogel from young pigs and evaluates its safety and feasibility as a supportive niche for the expansion and differentiation of SLCs. dTECM hydrogel facilitates the steroidogenic differentiation of SLCs into LCs, the primary producers of testosterone. The combination of SLCs with a dTECM hydrogel leads to a significant and sustained increase in testosterone levels, which promotes the restoration of spermatogenesis and fertility in an LC‐deficient and aged mouse model. Mechanistically, collagen 1 within the dTECM is identified as a key factor contributing to these effects. Notably, symptoms associated with testosterone deficiency syndrome are significantly alleviated in aged mice. These findings may aid the design of therapeutic interventions for patients with testosterone deficiency in the clinic.

Age-related testosterone decline: mechanisms and intervention strategies.

Contemporary societies exhibit delayed reproductive age and increased life expectancy. While the male reproductive system demonstrates relatively delayed aging compared to that of females, increasing age substantially impacts its function. A characteristic manifestation is age-induced testosterone decline. Testosterone, a crucial male sex hormone, plays pivotal roles in spermatogenesis and sexual function, and contributes significantly to metabolism, psychology, and cardiovascular health. Aging exerts profound effects on the hypothalamic-pituitary-gonadal axis and Leydig cells, precipitating testosterone reduction, which adversely affects male health. Exogenous testosterone supplementation can partially ameliorate age-related testosterone deficiency; however, its long-term safety remains contentious. Preserving endogenous testosterone production capacity during the aging process warrants further investigation as a potential intervention strategy.

Germ Cell Dysfunction is Universal in Male Patients with β-Thalassemia Following Hematopoietic Stem Cell Transplantation During Childhood and Adolescence.

To assess gonadal function in adolescent male patients with β-thalassemia who underwent successful hematopoietic stem cell transplantation (HSCT) during childhood or adolescence. Fifty-two male patients with β-thalassemia, aged ≥10 years, who had undergone HSCT ≥2 years were included. Clinical data, such as age, genital Tanner (GT) stage at HSCT and enrollment, serum ferritin levels, and cumulative doses of alkylating agents, were collected. Gonadal function was evaluated through measurements of serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, inhibin B levels, and semen analysis. Age at enrollment and HSCT were 17 (10-31) and 9 (1-19) years, respectively. The duration from HSCT to enrollment was 7.5 (2-20) years. Of 52 patients, 46 (88%) exhibited Sertoli cell dysfunction. Thirty-one patients had relatively small testes for their GT stage, 34 of 44 with GT V had elevated FSH of ≥5 IU/L, and 20 of 49 with GT stages II-V had low serum inhibin B levels. None of the patients with GT stage V showed Leydig cell dysfunction or gonadotropin deficiency. Serum FSH ≥8 IU/L showed the best diagnostic accuracy for detecting oligo- and azoospermia. All 39 patients who underwent semen analysis had >1 abnormal parameters. Having relatively small testes for GT stage and serum FSH ≥8 IU/L were associated with oligo- and azoospermia (p <0.01). Male patients with β-thalassemia after HSCT experienced universal spermatogenesis impairment and frequent Sertoli cell dysfunction but their Leydig cell function appears to be preserved. The high likelihood of future subfertility should be informed before HSCT.

Transcriptome analysis reveals the molecular mechanisms of neonicotinoid acetamiprid in Leydig cells.

At present, the reproductive toxicology of neonicotinoids has received greater attention, however, its potential mechanisms are still not fully understood. Acetamiprid (ACE) is a new-generation neonicotinoid and has become a ubiquitous contaminant in the environment. This study aimed to investigate the toxic effects of ACE in TM3 Leydig cells based on transcriptome analysis. The viability and apoptosis of TM3 cells exposed to different concentrations of ACE were assessed by CCK8 and flow cytometry, respectively. After ACE exposure, transcriptome analysis was performed to screen differential expression genes (DEGs), followed by qPCR verification. Results showed that ACE exposure resulted in a time- and dose-dependent decrease in the viability of TM3 cells (p < .05). ACE also exerted a dose-dependent pro-apoptotic effect on TM3 cells. Results of RNA-seq showed that 1477 DEGs were obtained, of which 539 DEGs were up-regulated and 938 DEGs were down-regulated. GO and KEGG analyses of DEGs showed that DNA replication and cell cycle might be the key mechanisms for the cytotoxicity of ACE. qPCR results demonstrated that Mdm2, Cdkn1a (p21) and Gadd45 were significantly increased, and Pcna, Ccna2 (CycA), Ccnb1 (CycB), Ccne1 (CycE), and Cdk1 were significantly decreased, indicating that ACE exposure might promote G1/S and G2/M cell cycle arrest. Additionally, FoxO, p53, and HIF-1 signaling pathways and ferroptosis might play important roles in ACE-induced reproductive toxicity. Collectively, this study provides new perspectives into the mechanism of ACE-induced reproductive toxicity and lays a theoretical foundation for the in-depth study of non-target toxicity mechanisms of neonicotinoid insecticides.

Chemical castration in dogs using calcium chloride: effects on testicular hemodynamics and semen characteristic and serum levels of testosterone.

Dog overpopulation and stray dogs are global issues that are detrimental to public health and animal welfare. Thus, the goal of the current study was to provide alternatives for surgical castration. Therefore, calcium chloride was employed in this study, which might be an option for castration. Ten dogs were divided into two groups of five: a calcium chloride-treated group and a control group. The treated group received a single bilateral intratesticular injection of 1ml of sterile saline containing calcium chloride dihydrate (CaCl2•2 H2O) at a dose of 20mg/kg per testicle. While the control group was treated with 1ml of sterile saline solution, Semen and blood collection, as well as Doppler ultrasonography, were routinely carried out every week on days 0, 7, 14, 21, and 28 in order to evaluate the impact of the injection on semen parameters and testicular blood flow. The testicular volume and echogenicity in the CaCl2-treated group were significantly (P < 0.001) lower in weeks 2 through 4 than in the control group. Furthermore, in canine semen, CaCl2 dramatically decreased the amount, motility, and viability of sperm. When compared to vehicle-control animals, azoospermia was seen 2 weeks after the injection and persisted for the end of the study. The testes of all dogs were surgically removed at 30 days post-injection, and testes were put in 10% neutral buffered formalin for tissue processing. When compared to the control group, the average weight of testes in the chemical groups was dramatically reduced. Significant decreases in spermatogenic processes, necrosis, and degeneration of seminiferous tubules packed with necrotic debris, and fibrosed interstitial tissue, necrosed and calcified Sertoli, and Leydig cells were seen 30 days after CaCl2 injection. There was a significant decrease in testosterone levels compared to day 0 before CaCl2 injection and the control group. From weeks 1 through 4, there was a substantial decrease in both peak systolic velocity (PSV) and end-diastolic velocity (EDV) values (P < 0.001) following a single intratesticular injection of CaCl2. The resistance index (RI) and pulsatility index (PI) showed the opposite tendency. Based on the histopathological and semen evaluations in this investigation, the study concludes that a single intratesticular injection of CaCl2 appears to be a practical and generally applicable approach for chemical sterilization of dogs.

MORPHOFUNCTIONAL CHARACTERISTICS OF THE TESTES IN YOUNG SEXUALLY MATURE RATS FOLLOWING PRENATAL EXPOSURE TO LOW-LEVEL ELECTROMAGNETIC RADIATION

The aim of this study is to investigate the functional and morphological characteristics of the testes in young sexually mature rats exposed to low-intensity centimeter-range electromagnetic radiation during the intrauterine period (within the “mother-fetus” system). Materials and methods. Intrauterine exposure to electromagnetic radiation was simulated using a high-frequency generator G4-190-3/1, equipped with a P-6-23A radiating antenna. To evaluate the reproductive system, a morphological examination of the testes and measurement of blood testosterone levels were performed. Morphometric methods were employed to quantify histological findings. The area of Leydig cell nuclei was measured, and the optical density of stromal connective tissue was assessed using the PAS reaction. The proliferative activity of spermatogenic epithelium cells was determined through an immunohistochemical reaction to the Ki-67 antigen. Results. In this experiment, the consequences of intrauterine damage to the testicles of male offspring were observed. We can presume that there is not only intrauterine damage, which may undergo regeneration later in life, but also the formation of epigenetic changes of regulatory factors that affect the expression of genes determining the morphofunctional state of the spermatogenic epithelium. Epigenetic changes developed during intrauterine period can manifest throughout the offspring’s lifetime after birth. Conclusions. Functional and morphological changes were found in the testes of sexually mature young males, offspring of mothers exposed to low-intensity centimeter-level electromagnetic fields during pregnancy. Hypoplastic changes in the testicles, a decrease in the proliferative potential of the spermatogenic epithelium were revealed. Hormonal stimulation of spermatogenesis (hypertestosteronemia and an increase in the number of Leydig cells and the size of their nuclei) obviously have a compensatory nature. The obtained results provide the basis for large epidemiological studies with the involvement of a wide range of specialists in both medical and technical specialties.

Single-cell transcriptomic and cross-species comparison analyses reveal distinct molecular changes of porcine testes during puberty

The pig is an important model for studying human diseases and is also a significant livestock species, yet its testicular development remains underexplored. Here, we employ single-cell RNA sequencing to characterize the transcriptomic landscapes across multiple developmental stages of Bama pig testes from fetal stage through infancy, puberty to adulthood, and made comparisons with those of humans and mice. We reveal an exceptionally early onset of porcine meiosis shortly after birth, and identify a distinct subtype of porcine spermatogonia resembling transcriptome state 0 spermatogonial stem cells identified in humans, which were previously thought to be primate specific. We also discover the persistent presence of proliferating progenitors for myoid cells in postnatal testes. The regulatory roles of Leydig cell steroidogenesis and estrogen synthesis in supporting cell lineages are also explored, including the potential impact of estrogen on Sertoli cell maturation and spermatogenesis. Overall, this study offers valuable insights into porcine testicular development, paving the way for future research in reproductive biology, advancements in agricultural breeding, and potential applications in translational medicine.

The Effects of Bisphenol A and its Analogs on Steroidogenesis in MA-10 Leydig Cells and KGN Granulosa Cells†.

Bisphenols are a family of chemicals used in the manufacture of consumer products containing polycarbonate plastics and epoxy resins. Studies have shown that exposure to bisphenol A (BPA) may disrupt steroidogenesis and induce adverse effects on male and female reproduction, but little is known about BPA replacements. We determined the effects of six bisphenols on the steroidogenic function of MA-10 Leydig cells and KGN granulosa cells by measuring the levels of progesterone and estradiol produced by these cells as well as the expression of transcripts involved in steroid and cholesterol biosynthesis. MA-10 and KGN cells were exposed for 48hours to one of six bisphenols (0.01-50μM): BPA, bisphenol F, bisphenol S, bisphenol AF, bisphenol M, or bisphenol TMC under both basal and dibutyryl cAMP (Bu2cAMP)-stimulated conditions. In MA-10 cells, most bisphenols increased the Bu2cAMP-stimulated production of progesterone. In KGN cells, there was a general decrease in progesterone production, while estradiol levels were increased following exposure to many bisphenols. qRT-PCR analyses revealed that all six bisphenols (≥1μM) upregulated the expression of STAR, a cholesterol transporter, in both cell lines after stimulation. Key transcripts directly involved in steroid and cholesterol biosynthesis were significantly altered in a cell line, chemical, and concentration-dependent manner. The expression of upstream genes was also differentially affected. Thus, BPA and five of its analogs can disrupt steroid production in two steroidogenic cell lines and alter the levels of transcripts involved in this process. Importantly, BPA replacements do not appear to have fewer effects than BPA.

Glycyrrhizin alleviated cisplatin-induced testicular injury by inhibiting the oxidative, apoptotic, hormonal, and histological alterations.

To evaluate the potential contribution of glycyrrhizin (GLZ) to mitigate the testicular toxicity linked to cisplatin (CIS) intoxication. 40 mature male Wistar albino rats (Rattus norvegicus albinus) were randomly divided into 4 equal groups (n = 10) for 60 days: the control group, CIS-treated group (single dose of 7 mg/kg, IP), GLZ-treated group (25 mg/kg, PO), and GLZ plus CIS-treated group. Blood and testis samples were examined using biochemical, histological, and immunohistochemical techniques. Semen samples were also obtained, and any abnormalities were reported. Serum follicle-stimulating hormone, luteinizing hormone, and testosterone levels were all markedly reduced by CIS. Oxidative stress and a significant reduction in levels of the antioxidant enzymes glutathione peroxidase, superoxide dismutase, and catalase were linked to CIS. Immunohistochemically, CIS showed diffuse, significantly positive immunolocalizations against the anti-caspase 3 antibody, indicating widespread apoptosis within the testicular parenchyma. Histopathologically, CIS showed diffuse coagulative necrosis of spermatogenic cells, necrotic Sertoli cells, intertubular edema, and Leydig cell hyperplasia. Moreover, CIS revealed a noteworthy increase in sperm abnormalities. Pre-coadministration and posttreatment with GLZ mitigated the majority of these detrimental consequences, and serum levels of antioxidant enzymes, luteinizing hormone, follicle-stimulating hormone, and testosterone were significantly elevated. Glycyrrhizin has been proven to be a strong antioxidant as well as antiapoptotic and cytoprotective against CIS testicular damage. The described model is a tool to evaluate the testicular protective impact of GLZ.

Polystyrene microplastics cause reproductive toxicity in male mice

Microplastics are a common environmental pollutant that disrupts the reproductive system of living organisms. We investigated the reproductive toxicity of 2μm polystyrene microplastics (PS-MPs) in mice and treated them with PS-MPs for 6 weeks. We demonstrated that PS-MPs decreased the gonadosomatic index and the serum concentration of pituitary-testicular axis hormones (Follicle-stimulating hormone, Luteinizing hormone, and testosterone). The PS-MPs treatment also reduced viable epididymal sperm number and sperm motility. Our results also demonstrated a marked decrease in tubular differentiation index, spermatogenesis index, repopulation index, and steroidogenic foci. The PS-MPs treated mice demonstrated marketed tissue damage in the testis. We also found that reproductive abnormality in PS-MPs treated mice accompanied by reduced antioxidant capacity elevated oxidative stress, and, elevated apoptotic signaling. It was observed that Endoplasmic reticulum (ER) stress markers, including GRP78 and Chop, were upregulated. Based on these findings, oxidative stress and endoplasmic reticulum stress may contribute to the decline in the steroidogenic function of Leydig cells with PS-MPs treated.

Unlocking the therapeutic potential and personalized therapy of testosterone: a comprehensive review.

Testosterone, the primary male sex hormone, orchestrates various physiological processes including sex differentiation, development of male characteristics, sperm production, and fertility. Its synthesis primarily occurs in Leydig cells within the testes, with smaller contributions from the ovaries and adrenal glands, all derived from cholesterol. Current therapeutic use of testosterone is mainly confined totreating hypergonadotropic hypogonadism, with limited off-label usage for augmenting muscle growth. This review delves into numerous studies investigating testosterone's therapeutic potential across various medical conditions as depicted in the figure given below. Of all the studies in this review, which show a positive therapeutic result by using testosterone, the most promising areas of potential usage of testosterone are anxiety and diabetes mellitus, followed by obesity and depression. By the medium if this study, we want to not only enlist the various potential therapeutic uses of testosterone, butalso promote a optimal hormonal balance, which can lead toprevention and/or better treatment outcomes for the mentioned diseases.

Alpha-Linolenic Acid Supplementation Improves Testosterone Production in an Aged Breeder Rooster Model: Role of Mitochondrial Modulation and SIRT1 Activation.

Aging in males can lead to declines in testosterone production, essential for maintaining male reproductive health. To investigate the effects of dietary supplementation with alpha-linolenic acid (ALA) on testosterone production in aged breeder roosters and understand the underlying molecular mechanisms involved. An in vivo model is established to investigate the effects of dietary ALA supplementation on testosterone production in aged breeder roosters, and the Leydig cell culture is used to identify the potential molecular mechanism. Dietary supplementation with ALA increases in plasma testosterone. Congruently, ALA supplementation enhances the expression of testosterone biosynthesis-related enzymes. ALA supplementation exerts anti-apoptotic effects in testicular mitochondria, as evidenced by a lower expression of pro-apoptotic factors and a higher expression of the anti-apoptotic factor B-cell lymphoma 2 (Bcl-2). Moreover, In Leydig cells, ALA supplementation promotes mitochondrial biogenesis genes. The proposed mechanism is that ALA activates the sirtuin1 (SIRT1)pathway and is supported by higher SIRT1 transcript and protein in Leydig cells. Furthermore, blocking SIRT1 with siRNA reverses ALA's effects on testosterone biosynthesis and mitochondrial function-related genes. These findings indicate that dietary supplementation with ALA can improve testosterone production in aged breeder roosters, possibly by modulation of mitochondrial function via activating the SIRT1 pathway.

α-Linolenic acid promotes testosterone synthesis by improving mitochondrial function in primary rooster Leydig cells

The present study aimed to investigate the direct effects of α-Linolenic acid (ALA) on the in vitro production of testosterone and the expression of key enzymes and proteins related to steroidogenesis in Leydig cells of roosters. MethodsPurified primary Leydig cells isolated from 65-week-old roosters were purified and treated with different concentrations of ALA treatments: (0 μm/L [control], solvent control group (DMSO), 20 μM/L, 40 μM/L, and 80 μM/L) and cell counting-8 (CCK-8) for cell viability assay, Enzyme-linked immunosorbent assay (ELISA) kit for the determination of testosterone in cell supernatants, quantitative (real-time) PCR, and analysis of activities of antioxidants catalase (CAT), superoxide dismutase (SOD) and malondialdehyde (MDA), evaluation of mitochondrial membrane potential, pro- and anti-apoptotic proteins/genes Bcl-2, Bcl-2-associated X protein (Bax), apoptosis-inducing factor (AIF) were done respectively. ResultsOur results showed that ALA significantly increased testosterone secretion in primary rooster Leydig cells (P < 0.05), and 40 μM/L is the optimal dose. Leydig cells supplemented with ALA (20, 40, 80 μM) increased the expression of key enzymes and proteins 3β-hydroxysteroid dehydrogenase (3β-HSD), steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc) concerning steroidogenesis, enhanced antioxidant capability, improved mitochondrial biogenesis, and markedly improved the mitochondrial membrane potential (P < 0.05). Furthermore, the expression of the apoptosis-suppressive gene Bcl-2 was significantly increased, but Bax and AIF expression was decreased in the ALA group compared to that in the control group (P < 0.05). ConclusionALA promoted testosterone production, enhanced steroidogenic enzyme expression, improved mitochondrial function, and antioxidant capacity, and reduced apoptosis in primary rooster Leydig cells, with 40 μM/L identified as the optimal concentration.

New insights into the mechanisms underlying 5-hydroxymethylfurfural-induced suppression of testosterone biosynthesis in vivo and in vitro

5-hydroxymethylfurfural (HMF), produced by the Maillard reaction, can indicate thermal processes in food. Previous research has examined the cytotoxic, genotoxic, mutagenic, and carcinogenic characteristics of HMF and its derivatives in different organs. Nevertheless, there is currently no available evidence about the impact of HMF on male reproductive toxicity. In this study, the effects of HMF on testosterone biosynthesis in both mouse testis and TM3 Leydig cells were investigated. HMF was administered to mice at doses of 30 and 300 mg/kg/day for 21 days and to Leydig cells at concentrations of 0.1, 1, and 10 mM for 24 h. The mechanism of action of HMF on testosterone biosynthesis in both mouse testis and Leydig cells was revealed by measuring the amount of testosterone, 3′,5′-cyclic adenosine monophosphate (cAMP) levels, and the expression level of some important genes in the steroidogenic pathway. In addition, its effects on general testis were examined through histopathological evaluations. Upon examination of the results, it was observed that HMF had a significant impact on reducing testosterone and cAMP levels. Furthermore, HMF inhibited the expression of steroidogenic genes, including steroidogenic acute regulatory protein, cholesterol side-chain cleavage enzyme, 3β-hydroxy dehydrogenase, and 17β-hydroxy dehydrogenase, as well as transcription factors, such as steroidogenic factor-1, GATA binding protein-4, and nerve growth factor IB. HMF-administrated groups had germinal epithelium degradation, vacuolization, and disorders in the interstitial area. Consequently, it has been proven for the first time that HMF can damage the male reproductive system by detrimentally impacting the production of testosterone.

Investigating the Significance of the Transgenerational Impact of High and Repeated Doses of Ivermectin: Effects on Paternal Testis Histopathology, Pups' Development, and Sexual Behavior

Paternal exposure to environmental challenges is critical for the offspring's future health, and the transmission of acquired traits through generations increases the risk of offspring developing diseases. Ivermectin (IVM) is widely used in veterinary and human medicine to treat parasitosis. Our previous studies showed that IVM acute administration induced disorganization of the germinal epithelium and could cause damage to sperm production. Thus, this study investigated the effects of paternal exposure to repeated high ivermectin doses on paternal testis histology. After mating, their pups' development and sexual behavior in adult rats were examined. Method: Two groups of male rats were treated with IVM or its vehicle once a week for three weeks. We observed these males' body weight, organs and testis histology, and testosterone levels. These rats were mated with females without any treatment: the reproductive performance, the offspring development, and the male and female sexual behavior observed in adulthood. Relative to controls, the IVM paternal testis histology showed hypertrophy and hyperplasia of Leydig cells and increased diameter of the seminiferous tubules—no impairment in reproductive performance. In males and females, the physical and reflexes were modified. In adult age, female rats of the IVM group showed reduced sexual behavior and sexual preferences for the same sex, while male sexual behavior was not altered. Thus, it is possible that paternal exposure to IVM interfered with pups' hormonal and growth factors during development and in adult age. Further studies are needed to explore IVM transgenerational effects identifying possible mechanisms underpinning behavioral effects.

Mitochondrial malfunction-initiated Leydig cell premature senescence partially participates in 1-nitropyrene-evoked downregulation of steroidogenic synthases in testes

Serum testosterone (T) in males has been declining during the past decades. The previous reports found that 1-nitropyrene (1-NP) exposure suppressed testicular T synthesis. The purpose of the current study was to further explore whether premature senescence participates in 1-NP-triggered reduction of testicular T synthesis. Adult male mice were orally exposed to 1-NP (0, 100, and 500 μg/kg) daily for 14 days. Serum and testicular T contents were diminished in 1-NP-administered mice. Mitochondria-located steroidogenic synthases, including StAR, CYP11A1, and 3βHSD1, were downregulated in 1-NP-administered mouse testes and MLTC-1 cells. Mechanistically, 1-NP exposure increased acetylation modification of mitochondrial steroidogenic synthases by inhibiting the enzymatic activity of SIRT3, an NAD+-dependent deacetylase. Supplementing NAD+ precursor and Sirt3 overexpression relieved 1-NP-triggered reduction of steroidogenic synthase levels in mouse testes and MLTC-1 cells. By contrast, Sirt3 silencing aggravated 1-NP-evoked acetylation and reduction of steroidogenic synthase levels in MLTC-1 cells. Further experiments demonstrated that 1-NP exposure caused mitochondrial malfunction and premature senescence in mouse testes and MLTC-1 cells. Supplementation with mitochondria-directed antioxidant mitoquinone (MitoQ) prevented 1-NP-evoked Leydig cell premature senescence and downregulation of testicular steroidogenic synthases. These results suggest that mitochondrial malfunction-initiated Leydig cell premature senescence may partially participate in 1-NP-evoked reduction of steroidogenic synthase levels in testes.

Cordycepin Activates Autophagy to Suppress FGF9-induced TM3 Mouse Leydig Progenitor Cell Proliferation.

Fibroblast growth factor 9 (FGF9) is a member of the human FGF family known for its pivotal roles in various biological processes, such as cell proliferation, tissue repair, and male sex determination including testis formation. Cordycepin, a bioactive compound found in Cordyceps sinensis, exhibits potent antitumor effects by triggering apoptosis and/or autophagy pathways. Our research has unveiled that FGF9 promotes proliferation and tumorigenesis in MA-10 mouse Leydig tumor cells, as the phenomena are effectively countered by cordycepin through apoptosis induction. Moreover, we have observed FGF9-mediated stimulation of proliferation and tumorigenesis in TM3 mouse Leydig progenitor cells, prompting an investigation into the potential inhibitory effect of cordycepin on TM3 cell proliferation under FGF9 treatment. Hence, we hypothesized that cordycepin induces cell death via apoptosis and/or autophagy in FGF9-treated TM3 cells. TM3 cells were treated with cordycepin and/or FGF9, and the flow cytometry, immunofluorescent plus western blotting assays were used to determine how cordycepin regulated Leydig cell death under FGF9 treatment. Our findings reveal that cordycepin restricts cell viability and colony formation while inducing morphological alterations associated with cell death in FGF9-treated TM3 cells. Surprisingly, cordycepin fails to elicit the expression of key apoptotic markers, suggesting an alternate mechanism of action. Although the expression of certain autophagy-related proteins remains unaltered, a significant up-regulation of LC3-II, indicative of autophagy, is observed in cordycepin-treated TM3 cells under FGF9 influence. Moreover, the inhibition of autophagy by chloroquine reverses cordycepin-induced TM3 cell death, highlighting the crucial role of autophagy in this process. Our study demonstrates that cordycepin activates autophagy to induce cell death in TM3 cells under FGF9 treatment conditions.

Correction to: Melatonin attenuates detrimental effects of diabetes on the niche of mouse spermatogonial stem cells by maintaining Leydig cells

Correction to: Melatonin attenuates detrimental effects of diabetes on the niche of mouse spermatogonial stem cells by maintaining Leydig cells

Pathological and Hormonal Alterations of the Testis in Balb/C Mice Treated With Spirotetramat Insecticide

Background and Objectives: The side effects of insecticides are undeniable due to their excessive use by farmers. This study aims to investigate the impact of spirotetramat insecticide on pathological and hormonal alteration in BALB/c mice testes. Methods: In this experimental study, 24 adult BALB/c male mice, with a body weight range of 25-30 g, were obtained from the Animal House of Uremia University and examined. Mice were randomly divided into three groups of eight: control, experimental group 1, and experimental group 2. The mice of experimental group 1 received 2.5 mg/kg, and experimental group 2 received 10 mg/kg of spirotetramat. The control group received the same amount of distilled water for 21 days through the mouth by gavage. After this period, the mice were anesthetized and evaluated. Data were analyzed using a 1-way analysis of variance in SPSS version 19 software. Results: The results of the study showed that spirotetramat increased the thickness of the tunica albuginea, the diameter of the seminiferous tubules, the differentiation index of the testis tube, and the spermiogenesis index, but it decreased the thickness of the epithelium of the spermatogenic tubules, the replacement index of active spermatogonia, Leydig cells, and FSH, LH, and testosterone hormone levels. The thickness of interstitial tissue, Sertoli, and lymphocyte cells did not change significantly. Conclusion: By inhibiting the activity of acetyl coenzyme A, spirotetramat insecticide disrupts energy production and prevents the synthesis of lipids in the cells, finally decreasing spermatogenesis in mice.