Search

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., (Copyright © 2022. Published by Elsevier Inc.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

(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.

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., (Copyright © 2021. Published by Elsevier Inc.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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, A E1841K /A E1841K 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., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Published by Elsevier Inc.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (Copyright © 2020 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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  μ mol/L, G), MEHP (1  μ mol/L, M), or GEN (1  μ mol/L)+MEHP (1  μ mol/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., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2020 Tong-Dian Zhang et al.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (© 2019 John Wiley & Sons, Ltd.)

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.

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 EB‑treated mice. Additionally, transmission electron microscopy revealed that mitochondria in Sertoli cells were transformed to vacuoles with irregular cristae in the EB‑treated 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 EB‑treated 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.

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.

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.

In a recent study, we reported that acute ethanol exposure enhanced autophagy in Sertoli cells (SCs) of adult rats. However, further research is needed to clarify the specific spermatogenic stage exhibiting the highest autophagic response, the mechanisms behind such specificity, and the related relevance to sperm. This brief report provides results indicating that stages VII⁻VIII (androgen-dependent or spermiation stages) of the spermatogenic cycle exhibited more marked autophagic response in acute-ethanol treated rats (ETRs) than other stages based on suppression of androgen receptor (AR), analysis of microtubule-associated protein 1 light chain 3 (LC3) (an autophagosomal marker) immunostaining in SCs, double labeling of LC3 and lysosomal proteins and electron microscopy. Ultrastructural observations and TUNEL method revealed a notable presence of phagocytosed apoptotic germ cells and retained sperm in SCs of ETRs at these specific stages-a finding rarely observed in control testes. In addition, PTEN-induced putative kinase 1 ( PINK1) (a sensor of mitochondrial damage and mitophagy) and giant lipid droplets were found to have accumulated in SCs of ETRs at same stages. Our data show novel findings indicating that stages VII⁻VIII of the spermatogenic cycle exhibit high levels of autophagy, specifically under stress conditions, as expressed by the term autophagic stages . This stage-specific upregulation of autophagy in SCs may be related to AR suppression, mitochondrial damage, lipid accumulation, and phagocytosis of apoptotic cells. The phenomenon may be an essential part of ensuring the viability of SCs and supporting germ cells in toxic environments.

Spermatogenesis in the freshwater pearl mussel Margaritifera laevis was investigated using light and electron microscopy. The testes of M. laevis are composed of numerous acini. We observed type A spermatogonia, large cells of irregular shape, solely near the acinus basal lamina. Type A spermatogonia proliferate and become type B spermatogonia, which are also irregular in shape and form clusters of germ cells of the same developmental stage. The numerous clusters differ with respect to developmental stage and are arranged randomly along the acinus periphery. The central region of the acinus was observed to contain only mature spermatozoa. This germ cell arrangement contrasts that of other bivalvians and may be characteristic of Margaritiferidae and Unionidae. We noted that each germ cell cluster is entirely covered throughout spermatogenesis by Sertoli cells that are loosely bound together. This report is the first to describe the involvement of Sertoli cells in Unionoidea spermatogenesis. Mature spermatozoa of M. laevis are of the primitive sperm type, having a cylindrical head with a discoidal acrosome and a midpiece with five spherical mitochondria., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We studied immunohistochemical and morphometric characteristics of the spermatogenic epithelium in rats against the background of peroral administration of nanoparticles containing titanium dioxide. Substantial degenerative changes in the spermatogenic epithelium were revealed: thinning, disorganization of layers, and detachment of sperm cells from the basement membrane. Immunohistochemical analysis revealed reduced proliferative activity and differentiation potential of epithelial cells, which was confirmed by changes in the expression of Ki-67 and c-kit markers. Our data attest to unfavorable effects of titanium dioxide nanoparticles on the structural and functional characteristics of the reproductive system in male rats leading to spermatogenesis disturbances.

Temperature is considered a crucial modulator of reproductive activity and testis homeostasis. It is well known that elevated temperatures cause several effects on testicular components, particularly on germ cells, which might lead to the impairment of spermatogenesis and loss of male fertility. The present study aimed to evaluate the effects of different environmental temperatures on several morphofunctional testis parameters, with emphasis on duration of spermatogenesis and spermatogenic efficiency. Thirty sexually mature Swiss mice (Mus musculus) were allocated in three different experimental groups, being kept in vivarium for three weeks at 16 °C, 23 °C (control group) and 32 °C. In order to estimate the duration of spermatogenesis, three animals per each group received intraperitoneal injections of tritiated thymidine and the testes were perfused-fixed and routinely processed for histological, morphometrical and immunoperoxidase analyses. Although the lower temperature (16 °C) did not change most of the evaluated testicular parameters, our findings showed that higher environmental temperature (32 °C) is able to alter important testis parameters, resulting for instance in acceleration of spermatogenesis, alterations in the stages frequencies, increased number of germ and Leydig cells apoptosis and reduced Sertoli cell and spermatogenic efficiencies. As in many conditions infertile men exhibit higher mean scrotal temperature, we believe that experimental studies with mice involving temperature might represent an interesting approach to better understand the mechanisms related to human testis function and sperm production., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Connexin43 (Cx43) is the predominant testicular gap junction protein and in cases of impaired spermatogenesis, Cx43 expression has been shown to be altered in several mammals. Amongst other functions, Cx43 is supposed to regulate junction formation of the blood-testis barrier (BTB). The aim of the present study was to investigate the expression pattern of different tight junction (TJ) proteins of the murine BTB using SC-specific Cx43 knockout mice (SCCx43KO). Adult homozygous male SCCx43KO mice (SCCx43KO-/-) predominantly show an arrest of spermatogenesis and SC-only tubules that might have been caused by an altered BTB assembly, composition or regulation. TJ molecules claudin-3, -5 and -11 were examined in adult wild type (WT) and SCCx43KO-/- mice using immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR). In this context, investigation of single tubules with residual spermatogenesis in SCCx43KO-/- mice was particularly interesting to identify a potential Cx43-independent influence of germ cells (GC) on BTB composition and dynamics. In tubules without residual spermatogenesis, a diffuse cytoplasmic distribution pattern for claudin-11 protein could be demonstrated in mutant mice. Nevertheless, claudin-11 seems to form functional TJ. Claudin-3 and -5 could not be detected immunohistochemically in the seminiferous epithelium of those tubules. Correspondingly, claudin-3 and -5 mRNA expression was decreased, providing evidence of generally impaired BTB dynamics in adult KO mice. Observations of tubules with residual spermatogenesis suggested a Cx43-independent regulation of TJ proteins by GC populations. To determine initial BTB formation in peripubertal SCCx43KO-/- mice, immunohistochemical staining and qRT-PCR of claudin-11 were carried out in adolescent SCCx43KO-/- and WT mice. Additionally, BTB integrity was functionally analysed using a hypertonic glucose fixative. These analyses revealed that SCCx43KO-/- mice formed an intact BTB during puberty in the same time period as WT mice, which however seemed to be accelerated., Competing Interests: The authors have declared that no competing interests exist.

Background: it has been demonstrated in various experimental studies that radiation exposure produces a negative impact on the processes of spermatogenesis associated with the disturbances of the microcirculation processes in the testes and the development of cellular and intracellular disintegration expressed as destructive changes in the cells leading to their death., Aim: The objective of the present study was to detect the ultrastructural abnormalities in the cells of Sertoli and spermatogonia under conditions of their exposure to radiation and to identify the peculiarities of their regeneration under the influence of the therapeutic and prophylactic application of low-intensity ultra-high frequency (UHF) electromagnetic radiation (EMR) and low-intensity low-frequency magnetic field (MF)., Material and Methods: The experiments were carried out on 28 non-pedigree mature male rats with the body weight 180-220 g that were divided into four groups. The first study group was comprised of the animals exposed to radiation followed by the application of low-intensity ultra-high frequency UHF electromagnetic radiation EMR. The rats in the second study group experienced effects of radiation and low-intensity low-frequency MF. The animals of the third (control) group were exposed to radiation alone, and those comprising the fourth group 1 (only radiation exposure) were considered to be intact., Results: The studies with the use of electron microscopy showed that the therapeutic and prophylactic application of low-intensity ultra-high frequency (UHF) electromagnetic radiation and low-intensity low-frequency magnetic field caused the decrease in the number and the severity of post-radiation defects in the treated cells together with the increase of the number and size of mitochondria as well as hyperplasia of ribosomes; moreover, it promoted cellular and intracellular regeneration. UHF electromagnetic radiation had a more pronounced stimulating effect on the regeneration processes as compared with low-frequency MF. Particularly active processes of intracellular regeneration evolved in Sertoli cells; they were manifested as the increase in the number and size of mitochondria, enhanced hyperplasia of ribosomes, and formation of polysomes and new membranes of the granular endoplasmic reticulum. In spermatogonia, intracellular regeneration was less pronounced than in the Sertoli cells but was accompanied by enhanced cell regeneration and a greater number of reserve stem/progenitor cells., Conclusions: The results of the present study provide a rationale for the possibility of the application of a low-frequency magnetic field and especially UHF electromagnetic radiation for the further development of the promising therapeutic and preventive technologies with a view to their introduction into routine clinical practice dealing with radiation-induced pathology.

The effects of Virgin coconut oil as an adjuvant to highly active antiretroviral therapy (HAART) were investigated on the testicular ultrastructure and biochemical markers in rats. Twenty male Sprague-Dawley rats, weighing 153-169 g were divided into four groups and treated as follows: control A (distilled water), B (HAART), C (HAART+Virgin coconut oil 10 ml/kg) and D (Virgin coconut oil [VCO] 10 ml/kg). Testicular segments were evaluated using transmission electron microscopy. Serum was assayed for testosterone, luteinising hormone, follicle stimulating hormone and testicular tissue for malondialdehyde and glutathione. Ultrastructure of basement membrane (Bm), mitochondria and spermatocytes was normal in the control group. HAART-treated group showed significant increase (p < .01) in Bm thickness with significant decrease in Leydig cell nuclear diameter (p < .05) and volume (p < .01) when compared with control group. Mitochondrial cristae appear collapsed, and Sertoli cells showed cytoplasmic vacuolations. HAART+VCO group showed improved ultrastructural details in Bm, and Sertoli cell and Leydig cells show abundant lipid droplets. Virgin coconut oil-treated group showed thinning of Bm with otherwise normal ultrastructural features of organelles. HAART-treated group showed significant increase (p < .01) in testosterone levels. There was no significant effect on malondialdehyde and glutathione levels. Virgin coconut oil improved testicular morphology and reversed HAART-induced ultrastructural alterations. Further studies on putative mechanism are required., (© 2017 Blackwell Verlag GmbH.)

Sertoli cells were discovered in the seminiferous tubules by Enrico Sertoli in 1865 (Morgagni 7:31-33, 1865). Intense phagocytosis is, in the context of spermatogenesis cycle, morphologically the most noticeable function of Sertoli cells. In this chapter the major principles of phagocytosis machinery and its specificities in the seminiferous tubules will be briefly reviewed, guidelines of analysis of main phagocytosis steps by confocal and transmission electron microscopy will be described, and a simplified method to assess phagocytosis rate in routine experiments will be given.

In this study, we aimed to investigate the testicular toxicity of two molecules derived from bee venom (BV): phospholipase A2 (PlA2) and melittin (Mlt). Ultrastructural effects of purified BV PlA2 and Mlt were assessed consecutive to repeated dose (30 days) and acute toxicity studies. For the subchronic treatment, PlA2 and Mlt were injected in daily doses equivalent to those released by a bee sting (105 μg PlA2/kg/day and 350 μg Mlt/kg/day), while in the acute treatment their doses corresponded to those released by 100 bee stings (9.3 mg PlA2/kg and 31 mg Mlt/kg). Both PlA2 and Mlt affected the Leydig cells and the cells in seminiferous tubules, the Sertoli cells first of all. PlA2 injection resulted in detachment of the Sertoli cells from the surrounding cells, and extracellular vacuolations, cytoplasmic vacuolations in their basal region and in branches as well, detachment of spermatids, residual bodies and sometimes even spermatocytes into the lumen, changes that had a higher magnitude after the acute treatment. Mlt injection induced similar ultrastructural alterations, but more severe, including degeneration of cellular organelles and cellular necrosis, resulting into rarefaction of the seminiferous epithelium; the ultrastructural changes had a higher magnitude after the 30 repeated dose treatment. We concluded that either of the two molecules tested here, PlA2 and Mlt, were Sertoli cells toxicants at the used doses, and they participated both in the BV testicular toxicity. We consider the observed changes as part of a preceding mechanism of the more severe alterations produced by the BV. It also remains possible that these early unspecific changes reported here could represent the response of the SCs not only to the components of bee venom, but to molecules of other venoms as well. The Sertoli cells were the primary target of PlA2 and Mlt in the spermatogenic epithelium, and their alteration led to further degenerative changes of the germ cells. Since the exposure to PlA2 and Mlt caused severe alteration, including cell death and detachment of immature germ cells into the lumen, we may also conclude that the bee venom molecules had a potential to interfere with normal progression of spermatogenesis. All the degenerative changes observed in the Sertoli cells were accompanied with changes of the Leydig cells., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

An emerging body of evidences based on in vitro studies indicate that mitophagy (selective autophagic clearance of damaged mitochondria) is a prosurvival mechanism, specifically under exposure to various stressors. Sertoli cells (SCs) play essential roles in maintenance of spermatogenesis via paracrine interactions with germ cells and other somatic cells in the testis; however, studies investigating mitophagy in SCs are still very few. In this chapter, we give a brief review of mechanisms and detection methods of mitophagy in SCs based on our recent publications on animal models of ethanol toxicity and current literature. In addition, we provide a method for induction and ultrastructural identification of mitophagy in SCs of adult Wistar rats using a single intraperitoneal injection (5 g/kg) of ethanol. Proper understanding of mitophagy features and mechanisms in SCs may have therapeutic implications for infertility associated with alcoholism and other diseases characterized by mitochondrial dysfunction.

We tested here the ability of bee venom (BV) to interfere with spermatogenesis in rats in two experimental conditions. The histopathological changes were assessed with brightfield microscopy using a novel staining technique, based on methylene blue, orange G and ponceau xylidine. Transmission electron microscopy was also used to identify fine subcellular changes. BV injection for 30days in daily doses of 700μg BV/kg resulted in reducing testicular weight, along with significant larger diameters of seminiferous tubules and reduced number of Sertoli cells (SCs). SCs were vacuolated, detached from the basement membrane, many necrosed, leading to the basement membrane denudation. Germ cells layers were separated by empty spaces conferring a rarefied aspect to the tissue, and spermatids were detached into lumen. Thus, the seminiferous epithelium was significantly thinned. Many Leydig cells (LCs) were in a necrotic state, with disrupted plasma membrane and without smooth endoplasmic reticulum. The acute treatment with a single LD50 of 62mgBV/kg, was followed by focal disruptions of the basement membrane and localized areas of necrosis, mainly affecting the SCs. Most of the observed SCs as well as some spermatogonia were highly vacuoled, empty spaces being observed within the epithelium. The SCs count was significantly decreased. Spermatids had also the tendency of separation from the SCs, and the significant larger diameter of the tubules found was associated with a thicker epithelium. Many LCs were necrosed, with disrupted plasma membrane, swollen mitochondria, no endoplasmic reticulum and implicitly showing rarefied cytoplasm. We concluded that BV was a testicular toxicant affecting both the LCs and the seminiferous tubules. The SCs cells represented the primary target site of BV whose effects were next extended upon the germ cells. In all cells, BV triggered unspecific degenerative changes that could impaire spermatogenesis. The present study also proposes an alternative staining technique very useful in assessing the histopathological aspects of spermatogenesis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Destruction of spermatogonial stem cells (SSCs) along the chemotherapy and radiotherapy is one of the side effects of cancer treatments that lead to infertility. In vitro propagation of hSSCs is necessary to obtain an adequate number of cells for successful transplantation. In this study, hSSCs were isolated from testis biopsies of the patients with maturation arrest and proliferated in DMEM in the presence of LIF and bFGF for 5 weeks. The various types of human spermatogonia were identified in culture system and compared with testis tissue using morphological criteria at the ultrastructural level. The results showed that although many various types of spermatogonia were identified, but no remarkable difference was observed between spermatogonial cells in culture system and testis tissue. Electron and light microscopic studies of hSSC colonies did not show differentiated SSCs in the culture system. The results also showed that probably the suitable time for transplanting of SSCs in recipient testis is 2-3 weeks after culture. Because apoptosis which may affect the development of germ cells has not started in colony cells at this time and the population of apoptotic cells are low., (© 2016 Blackwell Verlag GmbH.)

Introduction: Nanotechnology investigates materials at nanoscale level (0.1-100nm in diameter). There are many commercially nanoproducts such as silver, silicon, titanium, zinc, and gold. They are used in a variety of applications and released to the environment. Titanium dioxide (TiO 2 ) is one of the most commonly used nanoparticles (NP). In this study, the ultrastructural effects of TiO 2 -NP on zebrafish testis tissue were evaluated., Material and Method: Zebrafish were divided into four groups (N=60) as one control and 3 experimental groups (1mg/L, 2mg/L and 4mg/L TiO 2 ). Testis tissues were dissected after 5days of the exposure. Tissues were fixed with 2.5% glutaraldehyde at 4°C. After routine electron microscopy tissue processing, the testis were embedded in epon resin. Ultrathin sections were counterstained with 1% uranyl acetate and lead citrate and examined using a transmission electron microscope., Results: Mitochodrial degeneration with swelling and cristae loss were detected in Sertoli cells and spermatogonial cells of TiO2-NP treated groups in a dose-dependent manner. Moreover, autophagic vacuole accumulation were seen in Sertoli cell cytoplasms of the experimental groups. Necrosis was also detected in the 4mg TiO2-NP-treated group., Conclusion: TiO2-NP has been used in crop production, food additives, medicine, toothpastes, sunscreens, cosmetics, and in waste water treatment, which contaminated the environment. Our findings showed TiO 2 -NP-induced autophagy and necrosis at higher doses in Sertoli cells, which consequently negatively affected spermatogenic cells and testicular morphology of Zebrafish. It is important to give much more attention to the use of this NP to minimise the possible effects on nature and organisms., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background & Objectives: Bisphenol A (BPA) is an endocrine disruptor that is widely used in the manufacture of polycarbonate plastics, epoxy resins and dental sealants. It is known to have adverse effects on spermatogenesis in rodents. This study was aimed to evaluate the effects of BPA in adult common marmoset owing to its similarities with human spermatogenesis., Methods: Sixteen marmosets were divided into four groups (n=4 per group) and given oral doses of BPA (2.5, 12.5 and 25 μg/kg BW/day) for 70 days to cover two spermatogenic cycles, and the control group received only vehicle (honey). Testes were processed for histological and transmission electron microscopy studies., Results: Histology of the testis showed sloughing of germ cells into the lumen, increase in interstitial space and vacuolation of Sertoli cell cytoplasm. Ultrastructural analysis of the testis revealed several degenerative effects on the basement membrane, Sertoli cells, Leydig cells and other developing germ cells in the 12.5 and 25 μg/kg BW/day groups as compared to control., Interpretation & Conclusions: The observed ultrastructural changes caused by BPA in testicular morphology might be indicative of a perturbed sperm production. Considering the genetic and spermatogenic similarities of common marmoset (Callithrix jacchus) and humans, the study findings are of significance. Further studies are, however, needed to elucidate the mechanism of action.

To study how hydrogen-rich saline (HS) promotes the recovery of testicular biological function in a hemi-sectioned spinal cord injury (hSCI) rat model, a right hemisection was performed at the T11-T12 of the spinal cord in Wistar rats. Animals were divided into four groups: normal group; vehicle group: sham-operated rats administered saline; hSCI group: subjected to hSCI and administered saline; HRST group: subjected to hSCI and administered HS. Hind limb neurological function, testis index, testicular morphology, mean seminiferous tubular diameter (MSTD) and seminiferous epithelial thickness (MSET), the expression of heme oxygenase-1 (HO-1), mitofusin-2 (MFN-2), and high-mobility group box 1 (HMGB-1), cell ultrastructure, and apoptosis of spermatogenic cells were studied. The results indicated that hSCI significantly decreased the hind limb neurological function, testis index, MSTD, and MSET, and induced severe testicular morphological injury. The MFN-2 level was decreased, and HO-1 and HMGB-1 were overexpressed in testicular tissues. In addition, hSCI accelerated the apoptosis of spermatogenic cells and the ultrastructural damage of cells in the hypophysis and testis. After HS administration, all these parameters were considerably improved, and the characteristics of hSCI testes were similar to those of normal control testes. Taken together, HS administration can promote the recovery of testicular biological function by anti-oxidative, anti-inflammatory, and anti-apoptotic action. More importantly, HS can inhibit the hSCI-induced ultrastructural changes in gonadotrophs, ameliorate the abnormal regulation of the hypothalamic-pituitary-testis axis, and thereby promote the recovery of testicular injury. HS administration also inhibited the hSCI-induced ultrastructural changes in testicular spermatogenic cells, Sertoli cells and interstitial cells.

Alternative splicing has essential roles in development. Remarkably, spermatogenic cells express more alternatively spliced RNAs compared to most whole tissues; however, regulation of these RNAs remains unclear. Here, we characterize the alternative splicing landscape during spermatogenesis and reveal an essential function for the RNA-binding protein Ptbp2 in this highly regulated developmental program. We found that Ptbp2 controls a network of genes involved in cell adhesion, migration, and polarity, suggesting that splicing regulation by Ptbp2 is critical for germ cell communication with Sertoli cells (multifunctional somatic cells necessary for spermatogenesis). Indeed, Ptbp2 ablation in germ cells resulted in disorganization of the filamentous actin (F-actin) cytoskeleton in Sertoli cells, indicating that alternative splicing regulation is necessary for cellular crosstalk during germ cell development. Collectively, the data delineate an alternative splicing regulatory network essential for spermatogenesis, the splicing factor that controls it, and its biological importance in germ-Sertoli communication., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The endoplasmic reticulum (ER) in Sertoli cells is a component of unique adhesion junctions (ectoplasmic specializations-ESs) and is closely associated with structures termed tubulobulbar complexes (TBCs) that internalize intercellular junctions during sperm release and during the translocation of spermatocytes through the blood-testis barrier. A role for the ER in Ca2+ regulation at ESs and TBCs has been suspected, but evidence for this function has proved elusive. Using electron microscopy, we define two new ER-plasma membrane (PM) contact sites in apical Sertoli cell processes. One of these sites occurs at TBCs where flattened lamellar cisternae of ER envelope the swollen bulb regions of the complexes, and where the gap between adjacent membranes is 12 nm. The other is at the periphery of apical processes where the gap between membranes is 13-14 nm. Using immunolocalization at the light and electron microscopic levels, we demonstrate that Ca2+ regulatory machinery is present at the ESs attached to spermatid heads, and at ER-PM contacts. Sarco/endoplasmic reticulum Ca2+-ATPase 2 (ATP2A2, SERCA2) is present at ESs; transient receptor potential channel subfamily M member 6 (TRPM6), Homer1 (HOMER1), and inositol 1,4,5-trisphosphate receptor (ITPR, IP3R) are present at ER-PM contacts associated with TBC bulbs; and stromal interacting molecule 1 (STIM1), Orai1 (ORAI1), and ATP2A2 are present at the ER-PM contacts around the margins of Sertoli cell apical processes. In Sertoli cells, the molecular machinery associated with ER generated Ca2+ fluxes is present in regions and structures directly related to junction remodeling-a process necessary for sperm release., (© The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The aim was to study the ultrastructure of testicular parenchyma and define the morphological ultrastructure of spermatozoa of agoutis kept in captivity. Segments of testes from eight agouti males at prepubescence, prepuberty, pubescence and sexual maturity were fixed in glutaraldehyde. Laboratory procedures were performed for transmission electron microscopy. Spermatogonial cells of Type A - pale, Type A - dark, intermediate and Type B were found. Spermatocytes in the pachytene phase were abundant among primary spermatocytes. From the prepubertal phase, Sertoli cells exhibited invaginations in the nuclear membrane and lipid inclusions in the cytoplasm due to their phagocytic function. Leydig cells displayed higher metabolic activity during puberty as evidenced by the presence of lipid droplets. Spermatozoa were fully formed morphologically at prepuberty. The centriolar complex had partially degenerated and featured a centriolar space as in rodents. Sperm heads were tapered, without prominence of the acrosome or evidence of the perforatorium, differing from cavies, rats and mice. This is the first study to describe the ultrastructure of agouti spermatozoa. This research may assist as a basis for future work related to fertility and other biotechnologies applied to reproductive biology in agoutis.

This research explores the detrimental effect of nonylphenol (NP) to prepubertal Sprague-Dawley male rats in vivo and in vitro. Herein, forty-two 3-week-old rats were randomly divided into six groups, which were treated with NP (0, NAC, 25, 50, 100, 100+NACmg/kg/2d for 30 consecutive days) by intraperitoneal injection. NP induced a reduction in testosterone (15.58%, 17.23%, 13.38% in 25, 50, 100mg/kg group, respectively), triggered apoptosis related to oxidative stress, and disturbed mRNA and/or protein levels of PI3K, PTEN, PDK1, p-Akt, p-mTOR, p70S6K, caspase-3, LC3B. NP induced morphological abnormality in epididymal sperm (2.00-, 3.02-fold in 50, 100mg/kg group, respectively). Pretreatment with NAC, attenuated NP-induced ROS production; recovered testosterone in serum, and ameliorated toxic effect in epididymal sperm. Sertoli cells were isolated, purified, treated with NP (0, 10, 20, and 30μM) for 12h. NP disturbed mRNA and/or protein levels of caspase-3, cleave-caspase-3, LC3B involving the PI3K/Akt/mTOR pathway. It also decreased protein levels of ABP, FSHR, N-cadherin, transferrin, vimentin; disturbed the gene levels of all, but vimentin. Pretreatment with wortmannin, alleviated an NP-induced reduction in protein levels of PI3K and PTEN. In conclusion, excess NP exposure induces apoptosis and autophagy, causes reproductive lesions involving the PI3K/AKT/mTOR pathway both in vivo and in vitro. It also triggers oxidative stress and hormonal deficiency, reduces semen quality., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Head shaping in mammalian sperm is regulated by a number of factors including acrosome formation, nuclear condensation and the action of the microtubular manchette. A role has also been suggested for the attendant Sertoli cells and the perinuclear theca (PT). In comparison, relatively little information is available on this topic in birds and the presence of a PT per se has not been described in this vertebrate order. This study revealed that a similar combination of factors contributed to head shaping in the ostrich, emu and rhea, although the Sertoli cells seem to play a limited role in ratites. A fibro-granular structure analogous to the mammalian PT was identified, consisting of sub- and post-acrosomal components. The latter was characterized by stage-specific finger-like projections that appeared to emanate from the cytoplasmic face of the nuclear envelope. They were particularly obvious beneath the base of the acrosome, and closely aligned, but not connected to, the manchette microtubules. During the final stages of chromatin condensation and elongation of the sperm head the projections abruptly disappeared. They appear to play a role in stabilizing the shape of the sperm head during the caudal translocation of the spermatid cytoplasm., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Actin remodeling is a vital process for signaling, movement and survival in all cells. In the testes, extensive actin reorganization occurs at spermatid-Sertoli cell junctions during sperm release (spermiation) and at inter Sertoli cell junctions during restructuring of the blood testis barrier (BTB). During spermiation, tubulobulbar complexes (TBCs), rich in branched actin networks, ensure recycling of spermatid-Sertoli cell junctional molecules. Similar recycling occurs during BTB restructuring around the same time as spermiation occurs. Actin related protein 2/3 complex is an essential actin nucleation and branching protein. One of its subunits, Arpc1b, was earlier found to be down-regulated in an estrogen-induced rat model of spermiation failure. Also, Arpc1b was found to be estrogen responsive through estrogen receptor beta in seminiferous tubule culture. Here, knockdown of Arpc1b by siRNA in adult rat testis led to defects in spermiation caused by failure in TBC formation. Knockdown also compromised BTB integrity and caused polarity defects of mature spermatids. Apart from these effects pertaining to Sertoli cells, Arpc1b reduction perturbed ability of germ cells to enter G2/M phase thus hindering cell division. In summary, Arpc1b, an estrogen responsive gene, is a regulator of spermiation, mature spermatid polarity, BTB integrity and cell division during adult spermatogenesis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Gap junctions (GJ) are transmembrane channels that connect the cytoplasms of adjacent cells, thereby facilitating the rapid exchange of ions, second messengers, and metabolites smaller than 1kDa. Connexin 43, the best-studied GJ protein, is a component of the Sertoli cell barrier/blood-testis barrier (BTB). To gain insight into the biology of the BTB, we investigated the effects of interleukin 1alpha (IL1A), a pro-inflammatory cytokine that disrupts BTB function, on gap junctional communication (GJC) in Sertoli cells. Compared with controls, the levels of connexin 43 and connexin 43 (Ser 368) increased ~30- and 20-fold, respectively, at 24 h after IL1A treatment. To assess GJ function, we investigated fluorescence recovery in photobleached Sertoli cells after vehicle or IL1A treatment. Compared with the control, IL1A affected the ability of calcein to return to photobleached cells, indicating that GJC was compromised. To explain the effects of IL1A on GJ function, the involvement of the Sertoli cell cytoskeleton was investigated. Stress fibers aggregated at the periphery of Sertoli cells treated with IL1A. These results were substantiated by a biochemical assay that showed IL1A to disrupt the bundling of exogenous F-actin by Sertoli cells. In summary, IL1A regulates GJC in Sertoli cells, which is critical for BTB restructuring., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Swine testicular (ST) cell line is isolated from swine fetal testes and has been widely used in biomedical research fields related to pig virus infection. However, the potential benefit and utilization of ST cells in boar reproductive studies has not been fully explored. As swine fetal testes mainly contain multiple types of cells such as Leydig cells, Sertoli cells, gonocytes, and peritubular myoid cells, it is necessary to clarify the cell type of ST cell line. In this study, we identified ST cell line was a collection of Sertoli cells by analyzing the unique morphological characteristic with satellite karyosomes and determining the protein expression of two markers (androgen-binding protein, ABP; Fas ligand, FASL) of Sertoli cells. Then ST cells were further confirmed to be immature Sertoli cells by examining the expression of three markers (anti-Mullerian hormone, AMH; keratin 18, KRT18; follicle-stimulating hormone receptor, FSHR). In conclusion, ST cells are a collection of immature Sertoli cells which can be good experimental materials for the researches involved in Sertoli cell functions and maturation, or even in boar reproductions.

Meishan boars are known for their early sexual maturity. However, they exhibit a significantly smaller testicular size and a reduced proportion of Sertoli cells and daily sperm production compared with Duroc boars. The testes of Duroc and Meishan boars at 20, 75 and 270 days of age were used for histological and transcriptome analyses. Haematoxylin-eosin staining was conducted to observe histological structure of the testes in Duroc and Meishan boars at different ages. Although spermatogenesis occurred prior to 75 days in Meishan boars, the number of spermatogonia and Sertoli cells in Meishan boars were less than in Duroc boars at adulthood. The diameters of the seminiferous tubules of the testes differed significantly during the initiation of development of the seminiferous tubules between the two breeds. We obtained differentially expressed functional genes and analysed seven pathways involved in male sexual maturity and spermatogenesis using RNA-seq. We also detected four main alternative splicing events and many single nucleotide polymorphisms from testes. Eight functionally important genes were validated by qPCR, and Neurotrophin 3 was subjected to quantification and cellular localization analysis. Our study provides the first transcriptome evidence for the differences in sexual function development between Meishan and Duroc boars.

Titanium dioxide nanoparticles (TiO2 NPs), as largest production and use of nanomaterials, have been demonstrated to have a potential toxicity on reproductive system. However, the mechanism underlying male reproductive toxicity of TiO2 NPs remains limited. Thus, our study was designed to examine the cellular viability, apoptosis, oxidative stress, antioxidant capacity, and expression of apoptotic cytokines in primary cultured Sertoli cells isolated from mice under TiO2 NPs exposure. Results showed that TiO2 NPs exposure from 5 to 30 μg/mL resulted in reduction of cell viability, lactate dehydrogenase release, and induction of apoptosis or death on Sertoli cells. TiO2 NPs could migrate to Sertoli cells, which induced mitochondria-mediated or endoplasmic-reticulum-mediated apoptotic changes including elevation in reactive oxygen species (ROS) generation and reductions in superoxide dismutase, catalase, and glutathione peroxidase activities, decreases in mitochondrial membrane potential (ΔΨm), and releases of cytochrome c into the cytosol. In addition, upregulation of cytochrome c, Bax, caspase-3, glucose-regulated protein 78, and C/EBP homologous protein and caspase-12 protein expression, and downregulation of bcl-2 protein expression in primary cultured Sertoli cells induced by TiO2 NPs treatment. All of the results suggested that ROS generation may play a critical role in the initiation of TiO2 NPs-induced apoptosis by mediation of the disruption of ΔΨm, the cytochrome c release, and further the activation of caspase cascade and unfolded protein response signaling pathway., (© 2015 Wiley Periodicals, Inc.)

The aim of the research, carried out on three species of terrestrial isopods - Armadillidium granulatum, Halophiloscia hirsuta and Trichoniscus alexandrae - is to bring a first consistent contribution to the knowledge of the ultrastructural organization of the testis follicles. The testis follicles are seat of a remarkable dynamic activity of their cell components (somatic cells and germ cells) that results in a continuous variation, related to the trend of spermatogenesis, of their morphology, organization and of the relationships between the two cell populations. The somatic cells, known in literature as follicular cells, nurse cells or Sertoli cells, are arranged at the periphery of the follicle to form an epithelial layer of variable thickness resting on a thin basal lamina in turn surrounded by a discontinuous network of muscle cells. In A. granulatum and H. hirsuta, two types of Sertoli cells are present: a first type, the nurse cells, envelop the spermatids in cavities within their cytoplasm and through their secretion activity play a fundamental role in the formation of the spermatophores; moreover, they phagocytizes the residual cytoplasm of spermatids. A second type of Sertoli cells shows features that leave clearly identify its supporting role to the spermatophores in formation. In T. alexandrae, instead, only one type of Sertoli cells, the nurse cell, is present, whose features are widely superimposable to those observed in the other two species. Moreover, two septa of Sertoli cells depart from the periphery of the testis follicle to constitute an articulated compartmentalization of the follicle itself, probably targeted to realize at its inside a series of microenvironments functionally diversified in order to meets the needs of the different stages of the spermatogenic cycle., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objective: To investigate the effect of baicalein on the gap junction intercellular communication (GJIC) in the TM4 Sertoli cells of the mouse testis and its related mechanism., Methods: We measured the cytotoxicity of different concentrations of baicalein on the TM4 Sertoli cells in the mouse testis by MTT, detected the fluorescence transfer of the TM4 Sertoli cells by parachute assay, and determined the expression of the protein connexin 43 ( Cx43) in the baicalein-treated cells by Western blot and immunofluorescence assay., Results: Baicalein produced no obvious cytotoxicity on the TM4 Sertoli cells at the concentration below 60 µmol/L but significantly increased their GJIC at 0-20 µmol/L (P < 0.01). Western blot and immunofluorescence assay showed that 0-20 µmol/L baicalein remarkably elevated the expression of Cx43 in the TM4 cells (P < 0.01) and on the membrane of the TM4 cells., Conclusion: Baicalein at the concentration of 0-20 µmol/L can significantly enhance GJIC in mouse TM4 Sertoli cells by increasing the expression of the Cx43 protein.

The experiments on the outbred female rats using the electron microscopic technique have demonstrated that the application of ultrahigh frequency low-intensity electromagnetic radiation (LIEMR) with a flux density below 1 mCW/Cm2 and a frequency of approximately 1,000 MHz in the regime of primary prophylaxis and therapeutic-preventive action suppressed the development of the post-stress pathological ultrastructural changes and increased the activity of the regenerative processes in the Sertoli cells. It was shown that the developing adaptive and compensatory changes in the Sertoli cells most frequently involve the energy-producing structures (mitochondria) that undergo the enlargement of their average and total dimensions. Simultaneously, the amount of granular endoplasmic reticulum and the number of ribosomes increased while the intracellular links between the organelles strengthened and the reserve potential of the cells improved. It is concluded that the observed effects may be due to the action of both local and systemic regulation mechanisms.

N-ethyl-N-nitrosourea (ENU), a well known alkylating agent, is a powerful mutagen in mouse spermatogonia that is frequently used to generate mutant mice for the study of gene function. The present study was performed to investigate the toxic effects of a suggested ENU treatment protocol (100 mg/kg ENU once a week for three consecutive weeks) on the C57BL/6J mouse testis using light and transmission electron microscopy, with reference to testis weight and sperm count. Time-dependent changes in the weight of the testes, sperm counts and testicular morphology were observed, following an injury and recovery pattern; the most severe damage was observed in week four after the first injection of ENU, and then the testis gradually recovered. By the end of the experiment (week 12), the testis weights and sperm counts of the ENU-treated mice had restored to around 80% of the respective values in the control group. Histopathological alterations in the testis were identified by light and electron microscopy, which revealed that ENU led to a temporary depletion in the number of spermatogenic cells via direct and indirect toxic effects, including apoptosis and growth arrest in spermatogonia, Sertoli cell damage and peritubular cell injury. The results of this study complement the existing basic information on the toxicity of ENU in the testis, and provide scientific information for selecting the appropriate mating time for ENU-treated male mice.