Apoptotic Germ Cells Research Articles

Impaired Spermatogenesis in Infertile Patients with Orchitis and Experimental Autoimmune Orchitis in Rats.

Experimental autoimmune orchitis (EAO) is a well-established rodent model of organ-specific autoimmunity associated with infertility in which the testis immunohistopathology has been extensively studied. In contrast, analysis of testis biopsies from infertile patients associated with inflammation has been more limited. In this work, testicular biopsies from patients with idiopathic non-obstructive azoospermia diagnosed with hypospermatogenesis (HypoSp) [mild: n = 9, and severe: n = 11], with obstructive azoospermia and complete Sp (spermatogenesis) (control group, C, n = 9), and from Sertoli cell-only syndrome (SCOS, n = 9) were analyzed for the presence of immune cells, spermatogonia and Sertoli cell (SCs) alterations, and reproductive hormones levels. These parameters were compared with those obtained in rats with EAO. The presence of increased CD45+ cells in the seminiferous tubules (STs) wall and lumen in severe HypoSp is associated with increased numbers of apoptotic meiotic germ cells and decreased populations of undifferentiated and differentiated spermatogonia. The SCs showed an immature profile with the highest expression of AMH in patients with SCOS and severe HypoSp. In SCOS patients, the amount of SCs/ST and Ki67+ SCs/ST increased and correlated with high serum FSH levels and CD45+ cells. In the severe phase of EAO, immune cell infiltration and apoptosis of meiotic germ cells increased and the number of undifferentiated and differentiated spermatogonia was lowest, as previously reported. Here, we found that orchitis leads to reduced sperm number, viability, and motility. SCs were mature (AMH-) but increased in number, with Ki67+ observed in severely damaged STs and associated with the highest levels of FSH and inflammatory cells. Our findings demonstrate that in a scenario where a chronic inflammatory process is underway, FSH levels, immune cell infiltration, and immature phenotypes of SCs are associated with severe changes in spermatogenesis, leading to azoospermia. Furthermore, AMH and Ki67 expression in SCs is a distinctive marker of severe alterations of STs in human orchitis.

Synergistic enhancement of the mouse Pramex1 and Pramel1 in repressing retinoic acid (RA) signaling during gametogenesis

BackgroundPRAME constitutes one of the largest multi-copy gene families in Eutherians, encoding cancer-testis antigens (CTAs) with leucine-rich repeats (LRR) domains, highly expressed in cancer cells and gametogenic germ cells. This study aims to elucidate genetic interactions between two members, Pramex1 and Pramel1, in the mouse Prame family during gametogenesis using a gene knockout approach.ResultSingle-gene knockout (sKO) of either Pramex1 or Pramel1 resulted in approximately 7% of abnormal seminiferous tubules, characterized by a Sertoli-cell only (SCO) phenotype, impacting sperm count and fecundity significantly. Remarkably, sKO female mice displayed normal reproductive functions. In contrast, Pramex1/Pramel1 double knockout (dKO) mice exhibited reduced fecundity in both sexes. In dKO females, ovarian primary follicle count decreased by 50% compared to sKO and WT mice, correlating with a 50% fecundity decrease. This suggested compensatory roles during oogenesis in Pramex1 or Pramel1 sKO females. Conversely, dKO males showed an 18% frequency of SCO tubules, increased apoptotic germ cells, and decreased undifferentiated spermatogonia compared to sKO and WT testes. Western blot analysis with PRAMEX1- or PRAMEL1-specific antibodies on sKO testes revealed compensatory upregulation of each protein (30–50%) in response to the other gene’s deletion. Double KO males exhibited more severe defects in sperm count and litter size, surpassing Pramex1 and Pramel1 sKO accumulative effects, indicating a synergistic enhancement interaction during spermatogenesis. Additional experiments administering trans-retinoic acid (RA) and its inhibitor (WIN18,446) in sKO, dKO, and WT mice suggested that PRAMEX1 and PRAMEL1 synergistically repress the RA signaling pathway during spermatogenesis.ConclusionData from sKO and dKO mice unveil a synergistic interaction via the RA signaling pathway between Pramex1 and Pramel1 genes during gametogenesis. This discovery sets the stage for investigating interactions among other members within the Prame family, advancing our understanding of multi-copy gene families involved in germ cell formation and function.

Prenatal exposure to Di(2-ethylhexyl) phthalate and high-fat diet synergistically disrupts gonadal function in male mice†.

Prenatal exposure to Di (2-ethylhexyl) phthalate (DEHP) impairs the reproductive system and causes fertility defects in male offspring. Additionally, high-fat (HF) diet is a risk factor for reproductive disorders in males. In this study, we tested the hypothesis that prenatal exposure to a physiologically relevant dose of DEHP in conjunction with HF diet synergistically impacts reproductive function and fertility in male offspring. Female mice were fed a control or HF diet 7days prior to mating and until their litters were weaned on postnatal day 21. Pregnant dams were exposed to DEHP or vehicle from gestational day 10.5 until birth. The male offspring's gross phenotype, sperm quality, serum hormonal levels, testicular histopathology, and testicular gene expression pattern were analyzed. Male mice born to dams exposed to DEHP + HF had smaller testes, epididymides, and shorter anogenital distance compared with those exposed to HF or DEHP alone. DEHP + HF mice had lower sperm concentration and motility compared with DEHP mice. Moreover, DEHP + HF mice had more apoptotic germ cells, fewer Leydig cells, and lower serum testosterone levels than DEHP mice. Furthermore, testicular mRNA expression of Dnmt1 and Dnmt3a was two to eight-fold higher than in DEHP mice by qPCR, suggesting that maternal HF diet and prenatal DEHP exposure additively impact gonadal function by altering the degree of DNA methylation in the testis. These results suggest that the combined exposure to DEHP and high-fat synergistically impairs reproductive function in male offspring, greater than exposure to DEHP or HF dietalone.

Positive Effects of the Nutraceutical Association of Lycopene and Selenium in Experimental Varicocele.

Many natural substances commonly found in healthy diets have been studied for their potential to reduce male infertility associated with varicocele. A positive role of selenium (Se) or lycopene alone was demonstrated in experimental varicocele, while no data are available on their association. One group of male Sprague-Dawley rats was sham operated and daily treated with Se (3 mg/kg, i.p.), lycopene (1 mg/kg, i.p.), or their association. A second group underwent surgery to induce varicocele. Sham and half of the varicocele animals were sacrificed after twenty-eight days, while the residual animals were treated for one more month and then sacrificed. In varicocele animals, testosterone levels and testes weight were reduced, Hypoxia Inducible Factor-1α (HIF-1α) expression was absent in the tubules and increased in Leydig cells, caspare-3 was increased, seminiferous epithelium showed evident structural changes, and many apoptotic germ cells were demonstrated with TUNEL assay. The treatment with lycopene or Se alone significantly increased testis weight and testosterone levels, reduced apoptosis and caspase-3 expression, improved the tubular organization, decreased HIF-1α positivity of Leydig cells, and restored its tubular positivity. Lycopene or Se association showed a better influence on all biochemical and morphological parameters. Therefore, the nutraceutical association of lycopene plus Se might be considered a possible therapeutic tool, together with surgery, in the treatment of male infertility. However, long-term experimental and clinical studies are necessary to evaluate sperm quantity and quality.

GGA1 participates in spermatogenesis in mice under stress.

Infertility is recognized as a common and worrisome problem of human reproduction worldwide. Based on previous studies, male factors account for about half of all infertility cases. Exposure to environmental toxicants is an important contributor to male infertility. Bisphenol A (BPA) is the most prominent toxic environmental contaminant worldwide affecting the male reproductive system. BPA can impair the function of the Golgi apparatus which is important in spermatogenesis. GGA1 is known as Golgi-localized, gamma adaptin ear-containing, ARF-binding protein 1. Previously, it has been shown that GGA1 is associated with spermatogenesis in Drosophila, however, its function in mammalian spermatogenesis remains unclear. Gga1 knockout mice were generated using the CRISPR/Cas9 system. Gga1-/- male mice and wild-type littermates received intraperitoneal (i.p.) injections of BPA (40 µg/kg) once daily for 2 weeks. Histological and immunofluorescence staining were performed to analyze the phenotypes of these mice. Male mice lacking Gga1 had normal fertility without any obvious defects in spermatogenesis, sperm count and sperm morphology. Gga1 ablation led to infertility in male mice exposed to BPA, along with a significant reduction in sperm count, sperm motility and the percentage of normal sperm. Histological analysis of the seminiferous epithelium showed that spermatogenesis was severely disorganized, while apoptotic germ cells were significantly increased in the Gga1 null mice exposed to BPA. Our findings suggest that Gga1 protects spermatogenesis against damage induced by environmental pollutants.

P-001 ClpX is required in maintaining mitochondrial functions during meiosis and spermatogenesis

Abstract Study question What is the role of ClpX in spermatogonia differentiation and spermatogenesis? Summary answer ClpX is required to maintain normal mitochondrial functions in spermatocytes, deficiency of ClpX resulted in meiosisarrest and diruption of spermatogenesis in mice. What is known already ClpX is mitochondria-specific quality control protease, it maintains proteostasis via degrading misfolded or damagedproteins in mitochondrial matrix. ClpX is a AAA+ protease that uses the energy of ATP binding and hydrolysis to degradeunfolded or misfolded proteins. ClpX is reported working as a complex with ClpP, the complex consists of a hexamer ofClpX and a tetradecamer of ClpP. ClpX recognizes the protein substrates via binding with their unstructured peptidesequences, termed as degradation tags or recognition signals. The fragments of the cleaved polypeptides can then exitthe chamber and be further degraded. Study design, size, duration Study design: we generated a germ cell specific ClpX knockout mice line and evaluated size, weight, inner structure oftestis tissue. We also applied biochemical techniques and high-throughput sequencing techniques to investigate themechanism of ClpX in regulating spermatogenesis. Size: more than 50 mice were used in this study, including the control mice. Duration: 2 years Participants/materials, setting, methods Participants/materials: we generated a ClpX conditional knockout (cKO) mice line by CRISPR-gene editing and Cre-LoxP system, which specifically knockout ClpX in the germ cells of male mice. Setting: the siblings of ClpX WT mice and ClpX cKO mice were compared in various of experiments. Mehods: we performed morphology comparement to check the weight and size of tetes in the control and ClpX cKOmice, and applied immunofluorescence, western blot, histological study, high-throughput sequencing andpharmocological treatment. Main results and the role of chance We found ClpX deficiency reduced mitochondrial functions and quantity in spermatocytes, affected energy supply duringmeiosis and attenuated zygotene-pachytene transformation of the male germ cells. The affected spermatocytesexhibited disorder of chromosome synapsis and cross-over events. Their telomeres failed to attach to the nuclearenvelop, which was associated with failure of α-tubulin formation in the ClpX deficient spermatocytes. The dysregulatedspermatocytes finally underwent apoptosis resulting in decreased testicular size and vacuolar structures within theseminiferous tubules. Both transcriptome analysis and m6A-methylation sequencing analysis highlighted dysregulationof metabolic pathways including the mTOR signaling in the isolated spermatocytes. We confirmed over-activation of themTORC1 pathway with increased expression of phosphorylated S6 and 4EBP after deletion of ClpX in spermatocytes.Long-term inhibition of the mTORC1 signaling via rapamycin treatment in vivo partially rescue spermatogenesis in theseminiferous tubules with much less apoptotic germ cells and formation of late stage of meiotic germ cells, such asround spermatids and elongated spermatozoon. The data reveal the novel roles of ClpX in regulating meiosis andspermatogenesis. Limitations, reasons for caution This study is based on animal experiments, thus, these results only indicated the important roles of ClpX in micespermatogenesis rather than human beings. Clinical data has not yet found a Clpx gene mutation in reproductivedisease. Wider implications of the findings Clinical report has already found the mutation in ClpP can induce autosomal recessive Perrault syndrome, while themutation of ClpX has not been reported yet from clinical data. Our resutls demonstrated severe effect onspermatogenesis after knockingout of ClpX, this might give some insights to the clinic. Trial registration number not applicable

C9orf131 and C10orf120 are not essential for male fertility in humans or mice.

Male infertility affects approximately 7% of childbearing couples and is a major health issue. Although nearly 50% idiopathic infertile men are assumed to have a genetic basis, the underlying causes remain largely unknown in most infertility cases. Here, we report two rare homozygous variants in two previously uncharacterized genes, C9orf131 and C10orf120, identified in two unrelated men with asthenozoospermia. Both genes were predominantly expressed in the testes. Furthermore, C9orf131 and C10orf120 knockout mice were successfully generated using the CRISPR-Cas9 technology. However, both C9orf131-/- and C10orf120-/- adult male mice were fertile, with testis-to-body weight ratios comparable to those of wild-type mice. No overt differences were found between wild-type, C9orf131-/-, and C10orf120-/- mice regarding testicular/epididymal tissue morphology, sperm count, sperm motility, or sperm morphology. Moreover, TUNEL assays indicated that the number of apoptotic germ cells in testes was not significantly different between the three groups. In summary, these findings suggest that C9orf131 and C10orf120 are redundant genes in male infertility.

Cytoskeletal orchestration of glucose uptake in Sertoli cell to support efferocytosis of apoptotic germ cells

Efferocytosis of non-viable germ cells by Sertoli cells (SCs) constitutes a sentinel for testis homeostasis, yet how SCs signal for the metabolic and cytoskeletal adaption to this energetically costly process remains unexplored. Spectrin is membrane-associated periodic skeleton assembled into an actin-spectrin-based cytoskeletal structure with an interaction with glucose transporter Glut1. The contribution of spectrin to glucose uptake and efferocytosis is unknown. In this study, we identified a cross-regulation between glucose metabolism and efferocytosis in SCs. Pharmacological or genetic inhibition of glucose uptake or glycolysis compromises efferocytosis activity. We further found that βII-spectrin is a hitherto unappreciated regulator of glucose metabolism and cytoskeletal architecture. βII-spectrin deficiency impairs glucose uptake and lactate production in SCs. Moreover, a defective assembly of cytoskeleton and a loss of blood-testis barrier integrity are also featured by SCs deficient in βII-spectrin. The disruption in glucose metabolism and cytoskeletal organization synergistically lead to a defective efferocytosis. In vivo siRNA-mediated targeting of βII-spectrin in testis causes an obvious morphological aberration in seminiferous epithelium with the presence of exfoliated germ cells and multinucleated giant cells. Importantly, a decrease in expression of αII/βII-spectrin was observed in testes of Adjudin-induced infertility model. By exploring the functional relevance of βII-spectrin to the metabolic and cytoskeletal regulation of efferocytosis, our study proposes a potential link between βII-spectrin deregulation and male infertility.

Circ-CPSF1 Worsens Radiation-Induced Oxidative Stress Injury in Caenorhabditis elegans.

Radioactive substances have been used in various aspects in daily life. However, high-energy radiation could cause environmental problems, which would damage the human body. Circular RNA (CircRNA) has great potential in the minimization of ionizing radiation damage. To find a potential diagnostic and therapeutic target for reducing the damage of ionizing radiation, we selected circRNA cleavage and polyadenylation specificity factor subunit 1 (circ-CPSF1) based on its up-regulated expression after X-ray radiation and explored its effect on response to ionizing radiation using Caenorhabditis elegans (C. elegans). Circ-CPSF1 was screened out and its up-regulated expression was verified. The measurement of lifespan and germ cell apoptosis showed that circ-CPSF1 RNAi treatment extended lifespan and reduced apoptotic germ cells. ROS levels were significantly reduced after the interference of circ-CPSF1 in C. elegans with radiation. Mitochondrial membrane potential assay showed that the suppression of circ-CPSF1 could alleviate mitochondrial damage after radiation. Relative genes expression showed the involvement of circ-CPSF1 in radiation mediated DNA damage response pathways and apoptosis pathways. In conclusion, circ-CPSF1 exerts deleterious effects on lifespan, eggs production and germ cell apoptosis of C. elegans through oxidative stress, the DNA damage response (DDR) pathway, and the core apoptotic pathway after ionizing radiation, indicating the potential of circ-CPSF1 to be an important therapeutic target of radiation damage.

Histopathological examination of the protective effect of intense exercise in apoptotic germ cell damage due to diabetes

The aim of this study was to determine the protective and antioxidative effects of intensive exercise on streptozotocin (STZ)-induced testicular damage, apoptotic spermatognial cells death, and oxidative stress. 36 male Sprague Dawley rats were divided into three groups: control, diabetes, and diabetes+intensive exercise (IE) groups. Testicular tissues were examined histopathologically and antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and malondialdehyde (MDA) activity, as well as serum testosterone level, were measured. Seminiferous tubules and germ cells were found to be better in the testis tissue of the intense exercise group than in the diabetes group. Diabetes suppressed antioxidant enzymes CAT, SOD, GPx and testosterone levels were significantly decreased, and increased MDA level in the diabetic group compared to diabetes+IE group (p < 0.001). Following four weeks of treatment, intensive exercise improved the antioxidant defense, significantly decreased MDA activity, and increased testosterone levels in testicular tissue in the diabetic group compared to diabetes+IE group (p < 0.01). STZ-induced diabetes causes damage to the testis tissue. In order to prevent these damages, exercise practice has become very popular nowadays. In present study, our intensive exercise protocol, histological, and biochemical analysis of the effect of diabetes on the testicular tissues is shown.

Efeito protetor de Mucuna pruriens (L.) DC. var. extrato de semente de pruriens, em células germinativas apoptóticas em ratos machos etanólicos

Abstract Thai Mucuna pruriens (L.) DC. var pruriens (T-MP) seed containing levodopa (L-DOPA) and antioxidant capacity has been shown to improve sexual behavior and male reproductive parameters in rats treated with ethanol (Eth). However, its protective effect on testicular apoptotic germ cells has never been reported. This study aimed to investigate the potential effects of T-MP seed extract on expressions of caspase, proliferating cell nuclear antigen (PCNA), and dopamine D2 receptor (D2R) proteins in Eth rats. Thirty-six male Wistar rats were divided into four groups (9 animals/group), including control, Eth, T-MP150+Eth, and T-MP300+Eth, respectively. Control rats received distilled water, and Eth rats received Eth (3g/kg BW; 40%v/v). The T-MP groups were treated with T-MP seed extract at a dose of 150 or 300 mg/kg before Eth administration for 56 consecutive days. The results showed that the seminiferous tubule diameter and epithelial height were significantly increased in both T-MP treated groups compared to the Eth group. Additionally, the caspase-9 and -3, and PCNA expressions were decreased, but D2R expression was markedly increased in T-MP groups. It was concluded that T-MP seed extract could protect testicular apoptosis induced by Eth via changes in caspase, PCNA, and D2R protein expressions.

Absence of MerTK disrupts spermatogenesis in an age-dependent manner

Spermatogenesis is a highly specialized cell differentiation process regulated by the testicular microenvironment. During the process of spermatogenesis, phagocytosis performs an essential role in male germ cell development, and its dysfunction in the testis can cause reproduction defects. MerTK, as a critical protein of phagocytosis, facilitates the removal of apoptotic substrates from the retina and ovaries through cooperation with several phagocytosis receptors. However, its role in mammalian spermatogenesis remains undefined. Here, we found that 30-week-old MerTK−/− male mice developed oligoasthenospermia due to abnormal spermatogenesis. These mice showed damaged seminiferous tubule structure, as well as altered spermatogonia proliferation and differentiation. We also found that Sertoli cells from MerTK−/− mice had decreased phagocytic activity on apoptotic germ cells in vitro. Moreover, a transcriptomic analysis demonstrated that the pivotal genes involved in spermatid differentiation and development changed expression. These results indicate that MerTK is crucial for spermatogenesis, as it regulates the crosstalk between germ cells and Sertoli cells. This provides us insight into the molecular mechanism of MerTK on spermatogenesis and its implications for the diagnosis and treatment of human male infertility.

Smtnl2 regulates apoptotic germ cell clearance and lactate metabolism in mouse Sertoli cells

Smtnl2 is an epithelial Smoothelin that binds to actin filaments and is crucial for epithelial morphogenesis. We examined the role of Smtnl2 in Sertoli cells, which undergo dynamic cytoskeleton reorganization to phagocytose apoptotic germ cells, a process known as efferocytosis. We observed Smtnl2 expression in primary mouse Sertoli cells and the 15P1 Sertoli cell line. Smtnl2 expression increased in 15P1 cells committing efferocytosis. Smtnl2-deficient Sertoli cells exhibited defective ability to engulf apoptotic germ cells and importantly, the phenomenon occurred in the setting of an unaffected maturation of phagosome. We demonstrated that Smtnl2 regulates the engulfment process through the function of branched actin nucleation protein ARP3, an actin assembly dictator. Intriguingly, a shift in glucose metabolism that restricts lactate production in Sertoli cells was induced upon Smtnl2 depletion, leading to the activation of downstream AMPK and AKT signaling. Using an in vivo RNAi approach, we found that silencing of Smtnl2 in testis triggers an obvious disruption in cytoskeleton architecture and blood-testis barrier integrity across seminiferous epithelium, causing the detachment of massive germ cells from their nest, as evidenced by their exfoliation into the lumen. Overall, our study identifies Smtnl2 as a determinant for Sertoli cells’ functioning in supporting spermatogenesis.

Effect of Huaji Jianpi Decoction on the semen quality of high-fat diet-induced obese mice.

BackgroundAt present, the incidence of obesity is increasing. Several studies have shown that obesity can reduce male fertility by affecting spermatogenesis and semen quality. Traditional Chinese medicine (TCM) has fewer side effects and stable efficacy in the treatment of related diseases. This study aimed to investigate the effect of Huaji Jianpi Decoction on the semen quality of high-fat diet-induced obese mice.MethodsThe obese male mice model was constructed by using high-fat diet and the Huaji Jianpi Decoction was processed into an aqueous extract. Mice were allocated in the normal group (n=30) and five different treatment groups (n=50). Huaji Jianpi Decoction was applied in low-, medium- and high-dose [17.52 g/(kg·d), 35.04 g/(kg·d) and 70.07 g/(kg·d), respectively]. The body weight, body fat, testis wet weight, testis coefficient, and routine sperm parameters were detected and analyzed. Meanwhile, transmission electron microscope (TEM) was used to observe testis ultrastructure. reverse-transcription quantitative PCR (RT-qPCR) was used to measure the expression of tumour necrosis factor α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1).ResultsCompared with normal mice (ND), the testis wet weight and testis coefficient of mice in the blank group were significantly decreased, while the number of mitochondria was observed to be decreased on testis ultrastructure examination, and apoptotic cells and germ cells in the spermatogenic tubules were shed. After Huaji Jianpi Decoction administration, the body fat and blood lipid levels of obese mice were decreased, the testis wet weight and testis coefficient were increased, and semen parameters were increased. Different doses of Huaji Jianpi Decoction could improve testicular weight, sperm density, sperm motility, forward motility, and total sperm motility. Huaji Jianpi Decoction could also downregulate TNF-α and MCP-1 expression and inhibit germ cell apoptosis to improve semen quality.ConclusionsHuaji Jianpi Decoction can improve the semen quality of high-fat diet-induced obese mice by reducing weight and lipid levels.

Melatonin ameliorates testes against forced treadmill exercise training on spermatogenesis in rats.

It is well documented that some forced exercises can have bad effects on the genital system. Melatonin is a potent antioxidant that is effective in reducing the physical stress.

Reproductive Toxicity of Furfural Acetone in Meloidogyne incognita and Caenorhabditis elegans.

Furfural acetone (FAc) is a promising alternative to currently available nematicides, and it exhibits equivalent control efficiency on root-knot nematodes with avermectin in fields. However, its effect on the reproduction of root-knot nematode is poorly understood. In this study, the natural metabolite FAc was found to exhibit reproductive toxicity on Meloidogyne incognita and Caenorhabditis elegans. The number of germ cells of C. elegans was observed to decrease after exposure to FAc, with a reduction of 59.9% at a dose of 200 mg/L. FAc in various concentrations induced the germ-cell apoptosis of C. elegans, with an increase over six-fold in the number of apoptotic germ cells at 200 mg/L. These findings suggested that FAc decreased the brood size of nematode by inducing germ-cell apoptosis. Moreover, FAc-induced germ-cell apoptosis was suppressed by the mutation of gene hus-1, clk-2, cep-1, egl-1, ced-3, ced-4, or ced-9. The expression of genes spo-11, cep-1, and egl-1 in C. elegans was increased significantly after FAc treatment. Taken together, these results indicate that nematode exposure to FAc might inflict DNA damage through protein SPO-11, activate CEP-1 and EGL-1, and induce the core apoptosis pathway to cause germ-cell apoptosis, resulting in decreased brood size of C. elegans.

TNF-α/ENO1 signaling facilitates testicular phagocytosis by directly activating Elmo1 gene expression in mouse Sertoli cells.

Phagocytic clearance of apoptotic germ cells (GCs), as well as residual bodies released from developing spermatids, is critical for Sertoli cells (SCs) to maintain inner environment homeostasis within the testis. However, the molecular mechanisms controlling the phagocytosis are ill defined. Here, we identify a new role for alpha-enolase (ENO1), a key enzyme during glycolysis, as a molecule that facilitates testicular phagocytosis via transactivation of the engulfment and cell motility 1 (Elmo1) gene. Using immunohistochemistry and double-labeling immunofluorescence, ENO1 was observed to be expressed exclusively in the nuclei of SCs and its expression correlated with the completion of SC differentiation. By incubating TM4 cells with different pharmacological inhibitors and establishing TM4Tnfr1-/- cells, we demonstrated that SC-specific expression of ENO1 was under a delicate paracrine control from apoptotic GCs. In turn, persistent blockade of ENO1 expression by a validated small interfering RNA protocol resulted in the disturbance of spermatogenesis and impairment of male fertility. Furthermore, using ChIP, electrophoretic mobility shift and luciferase reporter assays, we showed that, in the presence of apoptotic GCs, ENO1 binds to the distal region of the Elmo1 promoter and facilitates transactivation of the Elmo1 gene. In agreement, overexpression of ELMO1 ameliorated ENO1 deficiency-induced impairment of phagocytosis in TM4 cells. These data reveal a novel role for SC-specific expression of ENO1 in regulating phagocytosis in testis, identify tumor necrosis factor-α and ELMO1 as critical upstream and downstream factors in mediating ENO1 action, and have important implications for our understanding of paracrine control of SC function by adjacent GCs.

Microinjection of antisense oligonucleotides into living mouse testis enables lncRNA function study

BackgroundLong non-coding RNAs (lncRNAs) have been the focus of ongoing research in a diversity of cellular processes. LncRNAs are abundant in mammalian testis, but their biological function remains poorly known.ResultsHere, we established an antisense oligonucleotides (ASOs)-based targeting approach that can efficiently knock down lncRNA in living mouse testis. We cloned the full-length transcript of lncRNA Tsx (testis-specific X-linked) and defined its testicular localization pattern. Microinjection of ASOs through seminiferous tubules in vivo significantly lowered the Tsx levels in both nucleus and cytoplasm. This effect lasted no less than 10 days, conducive to the generation and maintenance of phenotype. Importantly, ASOs performed better in depleting the nuclear Tsx and sustained longer effect than small interfering RNAs (siRNAs). In addition to the observation of an elevated number of apoptotic germ cells upon ASOs injection, which recapitulates the documented description of Tsx knockout, we also found a specific loss of meiotic spermatocytes despite overall no impact on meiosis and male fertility.ConclusionsOur study detailed the characterization of Tsx and illustrates ASOs as an advantageous tool to functionally interrogate lncRNAs in spermatogenesis.

Haemonchus contortus Transthyretin-Like Protein TTR-31 Plays Roles in Post-Embryonic Larval Development and Potentially Apoptosis of Germ Cells.

Transthyretin (TTR)-like proteins play multi-function roles in nematode and are important component of excretory/secretory product in Haemonchus contortus. In this study, we functionally characterised a secretory transthyretin-like protein in the barber’s pole worm H. contortus. A full-length of transthyretin-like protein-coding gene (Hc-ttr-31) was identified in this parasitic nematode, representing a counterpart of Ce-ttr-31 in Caenorhabditis elegans. High transcriptional levels of Hc-ttr-31 were detected in the egg and early larval stages of H. contortus, with the lowest level measured in the adult stage, indicating a decreased transcriptional pattern of this gene during nematode development. Localisation analysis indicated a secretion of TTR-31 from the intestine to the gonad, suggesting additional roles of Hc-ttr-31 in nematode reproduction. Expression of Hc-ttr-31 and Ce-ttr-31 in C. elegans did not show marked influence on the nematode development and reproduction, whereas Hc-ttr-31 RNA interference-mediated gene knockdown of Ce-ttr-31 shortened the lifespan, decreased the brood size, slowed the pumping rate and inhibited the growth of treated worms. Particularly, gene knockdown of Hc-ttr-31 in C. elegans was linked to activated apoptosis signalling pathway, increased general reactive oxygen species (ROS) level, apoptotic germ cells and facultative vivipary phenotype, as well as suppressed germ cell removal signalling pathways. Taken together, Hc-ttr-31 appears to play roles in regulating post-embryonic larval development, and potentially in protecting gonad from oxidative stress and mediating engulfment of apoptotic germ cells. A better knowledge of these aspects should contribute to a better understanding of the developmental biology of H. contortus and a discovery of potential targets against this and related parasitic worms.

Potential role of human chorionic gonadotropin supplementation in spermatogenesis in rats subjected to forced swimming exercise.

The aim of this study was to evaluate the supportive effect of human chorionic gonadotropin (hCG) on the quality of spermatogenesis, including count, motility, morphology, viability and apoptosis of sperm following forced swimming exercise. Twenty-four adult male Sprague-Dawley rats were used in this study. All rats were divided into four groups: control group; swimming exercise group (S); hCG administration group and swimming (SG) with hCG administration group (G). The experimental group was trained to force swimming stress for 10 min for 6 days. Then the sperm quality parameters were measured after dissection and epididymis removal. Spermatogenesis and germ cell apoptosis were evaluated by using Miller & Johnsen's score and TUNEL staining respectively. Results showed the count (control: 113±3.1, S: 74±1.9, G: 111±3, and SG: 103±2.4), motility (control: 93±2, S: 67±2.8,G: 90±2.7, and SG: 78±1), morphology (control: 89±3%, S: 47±2.4%, G: 90±3.1%, and SG: 67±1.1%), and viability of sperm (control: 91±2.9, S: 50±2, G: 91±1.9, and SG: 70±1.3) in swimming exercised-hCG administered group, significantly enhanced by hCG treatment compared to the swimming exercise group (p≤0.01). Also the number of apoptotic germ cells significantly decreased in swimming exercised-hCG administered group compared to the swimming exercised group. These results suggest that administration of hCG can protect the testes against the detrimental effect of forced swimming exercise in adult male rats.