Changes in the tight junctions of the testis during aging: Role of the p38 MAPK/MMP9 pathway and autophagy in Sertoli cells.

Impaired tight junction (TJ) function and autophagy and the activated p38 mitogen-activated protein kinase (MAPK)/matrix metalloproteinase 9 (MMP9) pathway in Sertoli cells cause spermatogenic disorders. However, it is unclear whether reduced TJ barrier function and autophagy and the activated p38 MAPK/MMP9 pathway in Sertoli cells are closely associated with age-related testicular dysfunction. Thus, we evaluated these changes in Sertoli cells using 6-, 12-, 18-, and 24-month-old Sprague-Dawley rats. The results showed that testicular morphology gradually degenerated, as evidenced by increased exfoliated germ cells, decreased seminiferous tubule diameter and seminiferous epithelium height, and reduced the numbers of spermatogonia, primary spermatocytes and spermatids during the process of aging. In addition, the TJs formed by adjacent Sertoli cells were progressively destroyed accompanied by an abnormal ultrastructure and decreased expression of the TJ proteins zonula occludens-1 (ZO-1), occludin, and claudin-11 with aging. Furthermore, the expression of phosphorylated p38MAPK and MMP-9 in Sertoli cells and testis gradually increased, and the expression of occludin co-localizated with MMP-9 progressively decreased. Meanwhile, autophagy levels also gradually decreased, including decreased autophagic vacuole formation and weak expression of light chain 3 (LC3) and autophagy-related 5 (Atg5) in Sertoli cells. Taken together, our results indicate that aging causes impaired TJ barrier function and degeneration of seminiferous tubules. The mechanism might be related to the activated p38MAPK/MMP9 pathway and inactivated autophagy in Sertoli cells.

Age-related histomorphometric and ultrastructural changes in the Sertoli cells of Japanese quail (Coturnix Coturnix japonica).

Prepared sections from pre-pubertal, pubertal, adult, and aged Japanese quail testes were examined by light microscopy and transmission electron microscopy (TEM) and measurements of seminiferous tubular diameter (STD), luminal diameter (SLD), epithelial height (SEH) and cross-sectional area of the seminiferous tubules (AST) were taken using an image analyzer. Apoptotic Sertoli cells with features such as cell shrinkage and chromatin condensation were observed in pre-pubertal and aged quail. There was a significant difference between the mean Sertoli cell number (SCN), SLD, SEH, STD and AST among the four age groups (P < 0.001). The highest SCN (mean ± standard error) was recorded in the adult (30.53 ± 0.42), with the aged group displaying the lowest mean (11.80 ± 0.27) SCN. Spearman's rho correlation coefficients demonstrated a strong relationship between the SCN and SEH in the pubertal (ρ=0.915; P < 0.001), adult (ρ=0.878; P < 0.001), and aged (ρ=0.858; P < 0.001) groups, while a significant moderate correlation was observed in the pre-pubertal (ρ=0.606; P < 0.001) group. There were significant moderate correlations between the SCN and STD in the pre-pubertal (ρ=0.445; P < 0.001), pubertal (ρ=0.653; P < 0.001), adult (ρ=0.440; P < 0.001), and aged (ρ=0.514; P < 0.001) groups. Furthermore, significant moderate correlations were estimated between the SCN and AST in the pre-pubertal (ρ=0.453; P < 0.001), pubertal (ρ=0.661; P < 0.001), adult (ρ=0.393; P = 0.001), and aged (ρ=0.498; P < 0.001) groups. This study provides baseline data on the morphology and development of the Sertoli cell, as well as testicular morphometry in avian species during the pre-pubertal, pubertal, adult, and aged stages using the Japanese quail as a model.

Deoxynivalenol and Zearalenone: Different Mycotoxins with Different Toxic Effects in the Sertoli Cells of Equus asinus.

(1) Background: Deoxynivalenol (DON) and zearalenone (ZEA) are type B trichothecene mycotoxins that exert serious toxic effects on the reproduction of domestic animals. However, there is little information about the toxicity of mycotoxins on testis development in Equus asinus. This study investigated the biological effects of DON and ZEA exposure on Sertoli cells (SCs) of Equus asinus; (2) Methods: We administered 10 µM and 30 µM DON and ZEA to cells cultured in vitro; (3) Results: The results showed that 10 µM DON exposure remarkably changed pyroptosis-associated genes and that 30 µM ZEA exposure changed inflammation-associated genes in SCs. The mRNA expression of cancer-promoting genes was remarkably upregulated in the cells exposed to DON or 30 µM ZEA; in particular, DON and ZEA remarkably disturbed the expression of androgen and oestrogen secretion-related genes. Furthermore, quantitative RT-PCR, Western blot, and immunofluorescence analyses verified the different expression patterns of related genes in DON- and ZEA-exposed SCs; (4) Conclusions: Collectively, these results illustrated the impact of exposure to different toxins and concrete toxicity on the mRNA expression of SCs from Equus asinus in vitro.

On the origin of germ cell neoplasia in situ: Dedifferentiation of human adult Sertoli cells in cross talk with seminoma cells in vitro.

Germ cell neoplasia in situ (GCNIS) is the noninvasive precursor of testicular germ cell tumors type II, the most common cancer in young men, which originates from embryonic germ cells blocked in their maturation. GCNIS is associated with impaired Sertoli cells (SCs) that express fetal keratin 18 (KRT18) and the pluripotency factor SRY-Box transcription factor 2 (SOX2). According to the current theory concerning the origin of GCNIS, these SCs are prepubertal cells arrested in their maturation due to (epi)genetic anomalies and/or environmental antiandrogens. Thus, they are unable to support the development of germ cells, which leads to their maturational block and further progresses into GCNIS. Alternatively, these SCs are hypothesized to be adult cells dedifferentiating secondarily under the influence of GCNIS. To examine whether tumor cells can dedifferentiate SCs, we established a coculture model of adult human SCs (FS1) and a seminoma cell line similar to GCNIS (TCam-2). After 2 wk of coculture, FS1 cells showed progressive expression of KRT18 and SOX2, mimicking the in vivo changes. TCam-2 cells showed SOX2 expression and upregulation of further pluripotency- and reprogramming-associated genes, suggesting a seminoma to embryonal carcinoma transition. Thus, our FS1/TCam-2 coculture model is a valuable tool for investigating interactions between SCs and seminoma cells. Our immunohistochemical and ultrastructural studies of human testicular biopsies with varying degrees of GCNIS compared to biopsies from fetuses, patients with androgen insensitivity syndrome, and patients showing normal spermatogenesis further suggest that GCNIS-associated SCs represent adult cells undergoing progressive dedifferentiation.

Genome-wide identification and functional analysis of long non-coding RNAs and mRNAs in male mice testes at the onset of puberty after low dose lead exposure.

Many researchers have studied the relationship between lead (Pb) and testis injury, but the underlying mechanisms are still unknown. The participation of long non-coding RNAs (lncRNAs) in biological processes has been proposed. To comprehensively gain insight into the molecular toxicity of Pb, expression patterns are analysed through RNA sequencing (RNA-seq) in male mice treated with 200 mg/L of Pb through the drinking water for 90 days at the onset of puberty. A total of 614 differentially expressed (DE) lncRNAs were included (p ≤ 0.05 and fold change ≥2), of which 288 were up-regulated, and 326 were down-regulated. A total of 2295 DE mRNAs (p ≤ 0.05 and fold change ≥2), including 1202 up-regulated and 1093 down-regulated ones, were found in the testes of Pb-exposed group. Functional analysis results showed that several lncRNAs might be implicated in the bio-pathway of mitogen-activated protein kinase (MAPK) signaling pathway. Finally, seven pairs of lncRNA-mRNA co-expression were established in mice testes and confirmed by RT-qPCR. Moreover, the DE genes were also altered in Sertoli cells. Therefore, our research might be helpful for future exploring the effects of Pb exposure on lncRNA in testis, as well as its function.

Mutations in non-muscle myosin 2A disrupt the actomyosin cytoskeleton in Sertoli cells and cause male infertility.

Mutations in non-muscle myosin 2A (NM2A) encompass a wide spectrum of anomalies collectively known as MYH9-Related Disease (MYH9-RD) in humans that can include macrothrombocytopenia, glomerulosclerosis, deafness, and cataracts. We previously created mouse models of the three mutations most frequently found in humans: R702C, D1424N, and E1841K. While homozygous R702C and D1424N mutations are embryonic lethal, we found homozygous mutant E1841K mice to be viable. However the homozygous male, but not female, mice were infertile. Here, we report that these mice have reduced testis size and defects in actin-associated junctions in Sertoli cells, resulting in inability to form the blood-testis barrier and premature germ cell loss. Moreover, compound double heterozygous (R702C/E1841K and D1424/E1841K) males show the same abnormalities in testes as E1841K homozygous males. Conditional ablation of either NM2A or NM2B alone in Sertoli cells has no effect on fertility and testis size, however deletion of both NM2A and NM2B in Sertoli cells results in infertility. Isolation of mutant E1841K Sertoli cells reveals decreased NM2A and F-actin colocalization and thicker NM2A filaments. Furthermore, AE1841K/AE1841K and double knockout Sertoli cells demonstrate microtubule disorganization and increased tubulin acetylation, suggesting defects in the microtubule cytoskeleton. Together, these results demonstrate that NM2A and 2B paralogs play redundant roles in Sertoli cells and are essential for testes development and normal fertility.

SOX9 is colocalized with paraspeckle protein NONO in cultured murine sertoli cells and features structural characteristics of intrinsically disordered proteins.

This study provides supporting evidence for the association between SOX9 and liquid-liquid phase separation. We show that SOX9 colocalized with a paraspeckle protein NONO in many, but not all, of the immortalized and primary murine Sertoli cells examined. In addition, we confirmed that SOX9 has structural characteristics of intrinsically disordered proteins.

Cytological features of spermatogenesis in Opsariichthys bidens (Teleostei, Cyprinidae).

Spermatogenesis is important for male fertility, but has not been well-studied in Opsariichthys bidens, an economically important freshwater fish in China. In this study, there was investigation of the cytological features of spermatogenesis in O. bidens using light microscopy, transmission electron microscopy, and immunofluorescence detection of microtubules. O. bidens has tubular testis. Spermatogenesis in O. bidens is of the cystic type, in which the spermatogenic cells develop into spermatozoa in cysts. There was asynchronous development of primary spermatocytes within a single cyst. Spermiogenesis was classified as Type I, which develops into a Type I aquasperm with an oval nucleus, a small and simple midpiece, a flagellum and no acrosome. There was a nuage in spermatogonia, spermatocytes, and spermatids in different developmental stages of spermatids which may have important functions in fish spermatogenesis. Furthermore, microtubule dynamics may be involved in spermatid reshaping, material transport, and polar distribution of organelles during spermiogenesis.

Low Dose of Genistein Alleviates Mono-(2-Ethylhexyl) Phthalate-Induced Fetal Testis Disorder Based on Organ Culture Model.

Mono-(2-ethylhexyl) phthalate (MEHP) and genistein have been classified as endocrine-disrupting chemicals (EDCs) which interfere with the differentiation and development of the male reproductive system. However, how these two EDCs would affect fetal rat testis development at a low dose was rarely studied. In this study, we established the organ culture system and applied it to evaluate testicular effects following multiple EDC exposure at a low dose. 15.5 days postcoitum fetal rat testes were dissected, cultured, and exposed to vehicle (control), GEN (1 µmol/L, G), MEHP (1 µmol/L, M), or GEN (1 µmol/L)+MEHP (1 µmol/L, G+M). Testicular cell markers, testosterone concentration, redox state, testicular histology, and testicular ultrastructure were evaluated. Our results showed that a low dose of MEHP suppressed the development of Sertoli cells, Leydig cells, and gonocytes by triggering oxidative injuries, which was consistent with the ultrastructural findings. However, coadministration of genistein at a low dose could partially attenuate MEHP-induced fetal testis damage through antioxidative action. Cotreatment of genistein at a low dose may have a promising future on its protecting role for attenuating other EDC-induced reproductive disorders during early life. Based on the results, it can be speculated that dietary intake of isoflavones may make the fetal testis less susceptible to phthalate-induced injury.

Pathological Findings in the Testes of COVID-19 Patients: Clinical Implications.

BACKGROUND: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), involves multiple organs. Testicular involvement is largely unknown. OBJECTIVE: To determine the pathological changes and whether SARS-CoV-2 can be detected in the testes of deceased COVID-19 patients. DESIGN, SETTING, AND PARTICIPANTS: Postmortem examination of the testes from 12 COVID-19 patients was performed using light and electron microscopy, and immunohistochemistry for lymphocytic and histiocytic markers. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the virus in testicular tissue. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Seminiferous tubular injury was assessed as none, mild, moderate, or severe according to the extent of tubular damage. Leydig cells in the interstitium were counted in ten 400× microscopy fields. RESULTS AND LIMITATIONS: Microscopically, Sertoli cells showed swelling, vacuolation and cytoplasmic rarefaction, detachment from tubular basement membranes, and loss and sloughing into lumens of the intratubular cell mass. Two, five, and four of 11 cases showed mild, moderate, and severe injury, respectively. The mean number of Leydig cells in COVID-19 testes was significantly lower than in the control group (2.2 vs 7.8, p < 0.001). In the interstitium there was edema and mild inflammatory infiltrates composed of T lymphocytes and histiocytes. Transmission EM did not identify viral particles in three cases. RT-PCR detected the virus in one of 12 cases. CONCLUSIONS: Testes from COVID-19 patients exhibited significant seminiferous tubular injury, reduced Leydig cells, and mild lymphocytic inflammation. We found no evidence of SARS-CoV-2 virus in the testes in the majority (90%) of the cases by RT-PCR, and in none by electron microscopy. These findings can provide evidence-based guidance for sperm donation and inform management strategies to mitigate the risk of testicular injury during the COVID-19 disease course. PATIENT SUMMARY: We examined the testes of deceased COVID-19 patients. We found significant damage to the testicular parenchyma. However, virus was not detected in testes in the majority of cases.

Sertoli cells in the freshwater pearl mussel Margaritifera laevis (Bivalvia: Margaritiferidae): A histological and ultrastructural study.

The developmental changes of Sertoli cells were examined and described in the freshwater pearl mussel Margaritifera laevis using light and transmission electron microscopy. Sertoli cells, which are located on the basal lamina of acini in the testis, include a large number of glycogen granules, electron-dense globules, lipid droplets, and sperm morulae. Electron-dense globules are the vacuoles into which the electron-dense material is condensed. In aging Sertoli cells, the content of the globules leaks out to the extracellular area. Large lipid droplets are formed by the deposition of smaller lipid droplets into a vacuole. After the disruption of the Sertoli cell, the lipid droplets are discharged to the extracellular area and fuse with to form a larger mass. The spermatogonia which were engulfed by the Sertoli cells begin to condense their chromatin and transform themselves into sperm morulae. The constituent cells of the sperm morulae proliferate and finally differentiate into the spermatozoa. After the disruption of the Sertoli cell, the spermatozoa produced from the sperm morulae are released into the acinus lumen. Numerous matured spermatozoa in the acini gather around the large lipid droplet, to form the sperm sphere. The completed sperm spheres are subsequently released through the exhalant siphon into the stream.

Melatonin protects the mouse testis against heat-induced damage.

Spermatogenesis, an intricate process occurring in the testis, is responsible for ongoing production of spermatozoa and thus the cornerstone of lifelong male fertility. In the testis, spermatogenesis occurs optimally at a temperature 2-4°C lower than that of the core body. Increased scrotal temperature generates testicular heat stress and later causes testicular atrophy and spermatogenic arrest, resulting in a lower sperm yield and therefore impaired male fertility. Melatonin (N-acetyl-5-methoxytryptamine), a small neuro-hormone synthesized and secreted by the pineal gland and the testis, is widely known as a potent free-radical scavenger; it has been reported that melatonin protects the testis against inflammation and reactive oxygen species generation thereby playing anti-inflammatory, -oxidative and -apoptotic roles in the testis. Nevertheless, the role of melatonin in the testicular response to heat stress has not been studied. Here, by employing a mouse model of testicular hyperthermia, we systematically investigated the testicular response to heat stress as well as the occurrence of autophagy, apoptosis and oxidative stress in the testis. Importantly, we found that pre-treatment with melatonin attenuated heat-induced apoptosis and oxidative stress in the testis. Also, post-treatment with melatonin promoted recovery of the testes from heat-induced damage, probably by maintaining the integrity of the Sertoli cell tight-junction. Thus, we for the first time provide the proof of concept that melatonin can protect the testis against heat-induced damage, supporting the potential future use of melatonin as a therapeutic drug in men for sub/infertility incurred by various testicular hyperthermia factors.

Clearing, immunofluorescence, and confocal microscopy for the three-dimensional imaging of murine testes and study of testis biology.

Optical clearing techniques provide unprecedented opportunities to study large tissue samples at histological resolution, eliminating the need for physical sectioning while preserving the three-dimensional structure of intact biological systems. There is significant potential for applying optical clearing to reproductive tissues. In testicular biology, for example, the study of spermatogenesis and the use of spermatogonial stem cells offer high-impact applications in fertility medicine and reproductive biotechnology. The objective of our study is to apply optical clearing, immunofluorescence, and confocal microscopy to testicular tissue in order to reconstruct its three-dimensional microstructure in intact samples. We used Triton-X/DMSO clearing in combination with refractive index matching to achieve optical transparency of fixed mouse testes. An antibody against smooth muscle actin was used to label peritubular myoid cells of seminiferous tubules while an antibody against ubiquitin C-terminal hydrolase was used to label Sertoli cells and spermatogonia in the seminiferous epithelium. Specimens were then imaged using confocal fluorescence microscopy. We were able to successfully clear testicular tissue and utilize immunofluorescent probes. Additionally, we successfully visualized the histological compartments of testicular tissue in three-dimensional reconstructions. Optical clearing combined with immunofluorescence and confocal imaging offers a powerful new method to analyze the cytoarchitecture of testicular tissue at histological resolution while maintaining the macro-scale perspective of the intact system. Considering the importance of the murine model, our developed method represents a significant contribution to the field of male reproductive biology, enabling the study of testicular function.

Ultrastructural feature of spermatogenic cells and spermatozoon in cultured burbot Lota lota.

Testis development and ultrastructure of spermatogenic cells and spermatozoa of burbot Lota lota, a commercially important cold freshwater fish, were studied by light and transmission electron microscopy. Spermatogonia, spermatocytes, spermatids, and spermatozoa are distributed along the seminiferous tubules. Electron-dense bodies appear in germ cells from primary spermatogonia to secondary spermatocytes. We identified three distinct stages of spermatid cell differentiation based on chromatin condensation, development of the flagellum, formation of a nuclear fossa, and elimination of excess cytoplasm. Spermatozoa were anacrosomal and characterized by location of the centrioles outside the nuclear fossa and incomplete perpendicular arrangement of the centrioles. The sperm flagellum displayed an axoneme with nine doublets of peripheral microtubules and two central microtubules. These results provide valuable information for burbot taxonomy and may clarify the process of spermatogenesis for this species.

Food-grade titanium dioxide (E171) by solid or liquid matrix administration induces inflammation, germ cells sloughing in seminiferous tubules and blood-testis barrier disruption in mice.

Food-grade titanium dioxide labeled as E171 has been approved for human consumption by the Food and Drug Administration (USA) and by the European Union for five decades. However, titanium dioxide has been classified as a possible carcinogen for humans by the International Agency of Research in Cancer raising concerns of its oral intake and the translocation to bloodstream, which could disturb barriers such as the blood-testis barrier. There is evidence that titanium dioxide by intragastric/intraperitoneal/intravenous administration induced alterations on testosterone levels, testicular function and architecture, but studies of the E171 effects on the testicle structure and blood-testis barrier are limited. E171 is contained not only in foods in liquid matrix but also in solid ones, which can exert different biological effects. We aimed to compare the effects of E171 consumption in a solid matrix (0.1%, 0.5% and 1% in pellets) and liquid suspension (5 mg/kg body weight) on testis structure, inflammation infiltrate and blood-testis barrier disruption of male BALB/c mice. Results showed that none of the administration routes had influence on body weight but an increase in germ cell sloughing and the infiltrate of inflammatory cells in seminiferous tubules, together with disruption of the blood-testis barrier were similar in testis of both groups even if the dose received in mice in liquid matrix was 136 or 260 times lower than the dose reached by oral intake in solid E171 pellets in 0.5% E171 and 1% E171, respectively. This study highlights the attention on matrix food containing E171 and possible adverse effects on testis when E171 is consumed in a liquid matrix.

ULK1 affects cell viability of goat Sertoli cell by modulating both autophagy and apoptosis.

Sertoli cells (SCs) are necessary for proper germ cell development and viability. Unc-51 like autophagy activating kinase (ULK1) protein kinase is an important regulator of autophagy activation. This study aims to investigate the role of autophagy promoter ULK1 on cell viability of goat SCs. Our results showed that ULK1 knockdown in goat SCs decreased autophagy activation, which was confirmed by decreased expression of autophagy-related markers including LC3, Beclin1, Atg5, and Atg7 (P < 0.05). Meanwhile, lower ULK1 levels resulted in decreased expressions of goat SC marker genes ABP, AMH, FASL, and GATA4. However, a reverse trend of these parameters occurred when the goat SCs were transfected with ULK1 overexpression construct; higher ULK1 levels in goat SCs also decreased the ratio of Bax/Bcl-2. Moreover, ULK1 overexpression in goat SCs activated the autophagy levels when cells were exposed to an environmental contaminant bisphenol A (BPA). The above results indicated that ULK1 gene might play important roles in goat SC function by regulating cell viability.

Exogenous oestradiol benzoate induces male mice azoospermia through modulation of oxidative stress and testicular metabolic cooperation.

In most cases, exogenous oestradiol benzoate (EB) inhibits spermatogenesis, however, the mechanism underlying this process has not been fully elucidated. The present study investigated the effect of EB on redox equilibrium and glycometabolism in mouse testes. Male Kunming mice were divided into 3 groups and injected with 0, 5 and 10 mg/kg EB, respectively. Histological analysis revealed no sperm and far fewer spermatogenic cells in the testes of EB­treated mice. Additionally, transmission electron microscopy revealed that mitochondria in Sertoli cells were transformed to vacuoles with irregular cristae in the EB­treated group. EB also significantly decreased the activities and mRNA expression of catalase, superoxide dismutase, and glutathione peroxidase and increased the activity of nitric oxide synthase and nitric oxide concentration in the testes compared with the control. These results indicated that oxidative damage was caused by EB treatment. With regard to glycometabolism, ATP content and activities of hexokinase and pyruvate kinase were significantly reduced in the EB­treated group. Although glucose and pyruvate concentrations were significantly increased by EB treatment, levels of lactate, the main energy source of spermatogenic cells, were unchanged. Monocarboxylate transporter 2 (MCT2) and MCT4, which are responsible for lactate transportation, were downregulated by EB. In conclusion, the results of the present study indicated that azoospermia induced by EB in male mice was associated with oxidative damage and the disorder of testicular metabolic cooperation.

Influence of Sialadenectomy on the Development of Spermatogenesis in Rats: Ultrastructural Characteristics of Spermatogenic Epithelium and Leydig Cells.

Sialadenectomy in young rats modifies the development of the spermatogenic and steroidogenic functions of the testes. Sialadenectomy causes ultrastructural changes in spermatogenic cells, sustentocytes, and Leydig cells that disappear by week 8 of the experiment due to realization of compensatory and adaptive mechanisms. The effects of endocrine factors of the greater salivary glands on the spermatogenic cells are realized directly and indirectly via interstitial endocrinocytes and sustentocytes.

Ethanol-Induced Mitochondrial Damage in Sertoli Cells is Associated with Parkin Overexpression and Activation of Mitophagy.

This study was conducted to elucidate the involvement of the PINK1-Parkin pathway in ethanol-induced mitophagy among Sertoli cells (SCs). In the research, adult rats were given intraperitoneal injections of ethanol (5 gm/kg) and sacrificed at various time periods within 24 h. Transmission electron microscopy was applied to reveal enhanced mitochondrial damage in SCs of the ethanol-treated rats (ETRs) in association with a significant increase in numbers of mitophagic vacuoles (mitophagosomes and autolysosomes) in contrast to very low levels in a control group treated with phosphate-buffered saline (PBS). This enhancement was ultra-structurally verified via observation of trapped mitochondria within LC3-labeled membranes, upregulation of LC3 protein levels, colocalization of LC3 and cytochrome c, and reduced expression of mitochondrial proteins. Importantly, Parkin expression was found to be upregulated in ETR SCs, specifically in mitochondria and mitophagosomes in addition to colocalization with PINK1 and pan-cathepsin, indicating augmented mitophagy. Transcription factor EB (TFEB, a transcription factor for autophagy and mitophagy proteins) was also found to be upregulated in nuclei of ETR SCs and associated with enhanced expression of iNOS. Enhanced Parkin-related mitophagy in ETR SCs may be a protective mechanism with therapeutic implications. To the authors' knowledge, this is the first report demonstrating the ultrastructural characteristics and molecular mechanisms of Parkin-related mitophagy in ETR SCs.

MitoQ ameliorates testis injury from oxidative attack by repairing mitochondria and promoting the Keap1-Nrf2 pathway.

Mitochondrial dysfunctions induced by oxidative stress could play a pivotal role in the development of testicular damage and degeneration, leading to impaired fertility in adulthood. MitoQ as mitochondria-targeted antioxidant has been used in many diseases for a long time, but its therapeutic effects on testicular injury 'have not been reported yet. Here, we examined the protective action mechanism of MitoQ on testicular injury from oxidative stress induced by triptolide (TP). Mice were orally administrated with MitoQ (1.3, 2.6 and 5 .2mg/kg, respectively) in a TP-induced model of testicular damage for 14 days. And then testis injuries were comprehensively evaluated in terms of morphological changes, spermatogenesis assessment, blood-testis barrier (BTB) integrity, and apoptosis. The results demonstrated MitoQ effectively increased testicular weight, maintained the integrity of BTB, protected microstructure of testicular tissue and sperm morphology by inhibition of oxidative stress. Further mechanism studies revealed that MitoQ markedly activates the Keap1-Nrf2 antioxidative defense system characterized by increasing the expression of Nrf2 and its target genes HO-1 and NQO1. Meanwhile, MitoQ upregulated the expression of mitochondrial dynamics proteins Mfn2 and Drp-1and exerted a protective effect on mitochondria. On this basis, the results from pharmacokinetic study indicated that the MitoQ could enter into testis tissues after oral administration in despite of the low absolute bioavailability, which provided the material basis for MitoQ in the treatment of testicular damage. More importantly, MitoQ reached mitochondria quickly and had an outstanding feature of mitochondria targeting in Sertoli cells. Therefore, these results provide information for the application of MitoQ against testicular injury diseases.