Eef2k is not required for fertility in male mice

BackgroundEukaryotic elongation factor-2 kinase (Eef2k) is a protein kinase associated with the calmodulin-induced signaling pathway and an atypical alpha-kinase family member. Eef2k-mediated phosphorylation of eukaryotic translation elongation factor 2 (Eef2) can inhibit the functionality of this protein, altering protein translation. Prior work suggests Eef2k to be overexpressed in breast, pancreatic, brain, and lung cancers wherein it may control key processes associated with apoptosis, autophagy, and cell cycle progression. The functional importance of Eef2k in the testes of male mice, however, has yet to be clarified.MethodsA CRISPR/Cas9 approach was used to generate male Eef2k-knockout mice, which were evaluated for phenotypic changes in epididymal or testicular tissues through histological and immunofluorescent staining assays. In addition, TUNEL staining was conducted to assess the apoptotic death of cells in the testis. Fertility, sperm counts, and sperm motility were further assessed.ResultsMale Eef2k-knockout mice were successfully generated, and exhibited normal fertility and development. No apparent differences were observed with respect to spermatogenesis, sperm counts, or germ cell apoptosis when comparing male Eef2k^−/− and Eef2k^+/+ mice.ConclusionsMale Eef2k-knockout mice remained fertile and were free of any evident developmental or spermatogenic abnormalities, suggesting Eef2k to be dispensable in the context of male fertility.     

E3 ubiquitin ligase ASB17 is required for spermiation in mice

BackgroundA major goal of spermiation is to degrade the apical ectoplasmic specialization (ES) junction between Sertoli cells and elongating spermatids in preparation for the eventual disengagement of spermatids into the lumen. E3 ubiquitin ligases mediate the process of ubiquitination and the subsequent proteasomal degradation, but their specific role during spermiation remains largely unexplored.MethodsAnkyrin repeat and SOCS box protein 17 (Asb17)-knockout mice were generated via a CRISPR/Cas9 approach. Epididymal sperm parameters were assessed by a computer-assisted sperm analysis (CASA) system, and morphological analysis of testicular tissues were performed based on histological and immunostaining staining, and transmission electron microscopy (TEM). The interactions between ASB17 and Espin (ESPN) were predicted by HawkDock server and validated through protein pull-down and immunoprecipitation assays.ResultsWe report that ASB17, an E3 ligase, is required for the completion of spermiation and that mice lacking Asb17 are oligozoospermic owing to spermiation failure. ASB17-deficient mice are fertile; however, spermatids exhibit a disorganized ES junction, resulting in retention within the seminiferous epithelium. Mechanistically, ASB17 deficiency leads to excess accumulation of ESPN, an actin-binding essential structural component of the ES. We determined that ASB17 regulates the removal of the ES through ubiquitin mediated protein degradation of ESPN.ConclusionsIn summary, our study describes a role for ASB17 in the regulation of cell-cell junctions between germ cells and somatic cells in the testis. These findings establish a novel mechanism for the regulatory role of E3 ligases during spermatogenesis.     

Inhaled hydrogen gas therapy for prevention of testicular ischemia/reperfusion injury in rats

PurposeThis study evaluated whether 2% hydrogen (H₂) gas therapy protects against testicular ischemia/reperfusion injury which results in increased formation of reactive oxygen species and/or reactive nitrogen species, leading to testicular apoptosis and impaired spermatogenesis.MethodsPubertal six-week-old Spraque-Dawley rats were assigned to 5 groups (10 animals/group) as follows: group A was a sham operated group; groups B, C, D, and E underwent 5 hours of left testicular ischemia followed by 0, 30, 60, and 120 minutes of 2% H₂ gas therapy, respectively. Histological analysis was performed to verify structure and morphology of the testes and to investigate Johnsen scores, mean seminiferous tubule diameter, and the number of germ cell layers to classify spermatogenesis. Germ cell apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay and Bax/Bcl-2 ratio real-time polymerase chain reaction. We also investigated malondialdehyde levels as an indicator of lipid peroxidation.ResultsCompared to the sham group (A), germ cell apoptosis and lipid peroxidation in the ischemia group (B) were significantly increased with abnormal morphology and impaired spermatogenesis. In contrast, amelioration of testicular damages was evident in the H₂ therapy groups (C, D, and E).ConclusionsOur results showed that inhalation of 2% H₂ gas may be a promising therapy with anti-apoptotic and anti-oxidant properties in cases of testicular ischemia/reperfusion injury.     

Sirt1-deficient mice exhibit an altered cartilage phenotype

ObjectiveWe previously demonstrated that Sirt1 regulates apoptosis in cartilage in vitro. Here we attempt to examine in vivo cartilage homeostasis, using Sirt1 total body knockout (KO) mice.MethodArticular cartilage was harvested from hind paws of 1-week and 3-week-old mice carrying wild type (WT) or null Sirt1 gene. Knees of Sirt1 haploinsufficient mice also were examined, at 6 months. Joint cartilage was processed for histologic examination or biochemical analyses of chondrocyte cultures.ResultsWe found that articular cartilage tissue sections from Sirt1 KO mice up to 3 weeks of age exhibited low levels of type 2 collagen, aggrecan, and glycosaminoglycan content. In contrast, protein levels of MMP-13 were elevated in the Sirt1 KO mice, leading to a potential increase of cartilage breakdown, already shown in the heterozygous mice. Additional results showed elevated chondrocyte apoptosis in Sirt1 KO mice, as compared to WT controls. In addition to these observations, PTP1b (protein tyrosine phosphatase b) was elevated in the Sirt1 KO mice, in line with previous reports.ConclusionThe findings from this animal model demonstrated that Sirt1 KO mice presented an altered cartilage phenotype, with an elevated apoptotic process and a potential degradative cartilage process.     

Assessment of testicular volume: A comparison of fertile and sub-fertile West African men

BackgroundWhile the semen analysis appears to be the cornerstone in the evaluation of testicular function, the testicular volume has long been associated with testicular function. However, racial variations in testicular volume do exist. Neither the critical minimum testicular volume that guarantees adequate function, nor the optimal testicular volume that indicates peak testicular function are also known.ObjectiveTo evaluate the relationship between testicular volume and function using scrotal ultrasound scan in black West African men.Patients and methodsThe study examined 236 subjects over a period of one year. The subjects comprised of 136 patients with diagnosis of male infertility, as well as 100 healthy individuals as control. The relevant clinical history of each patient was extracted from their case notes. All the subjects had their testes examined using a high frequency (7.5 mHz) linear transducer of an ultrasound scanner. The results were expressed as percentages and tests of significance were done using the chi-square and Student's t-test. A P-value < 0.05 was considered statistically significant.ResultsThe mean testicular volume for the sub-fertile patients was 15.32 ml while it was 19.89 ml in the control group. There was a statistically significant difference between the testicular volumes in fertile and infertile men at different age groups, while there was an inverse relationship between testicular volume and severity of oligospermia. This was, however, not directly linear as a mean testicular volume of 18–20 ml was associated with highest semen density. Volumes higher than 20 ml and lower than 18 ml were associated with reduced sperm density. There was also a sharp decline in sperm density when the mean testicular volume reduced from 14 ml to 13 ml. Severe oligospermia (<5 million/ml) was associated with mean testicular volume less than 12 ml.ConclusionTesticular volume on scrotal ultrasound correlates well with severity of oligospermia in men with sub-fertility. While the critical mean testicular volume necessary for adequate spermatogenesis has not been determined, it appears there is an optimal testicular volume of 18–20 ml at which spermatogenesis is at its peak in sub-fertile Nigerian men.     

Quantification of survivin mRNA in testes of infertile patients and in testicular germ cell tumours: high levels of expression associated with normal spermatogenesis

Deregulated apoptosis of germ cells may contribute to male infertility as well as malignant transformation. Survivin, an inhibitor of apoptosis (IAP), is overexpressed in all the most common human malignancies, but barely detectable in normal tissues. We used real-time polymerase chain reaction (PCR) to quantify survivin mRNA expression in normal testes (n = 22), testes with defective spermatogenesis (n = 26) and testicular germ cell tumours (TGCTs; n = 16). Survivin was expressed at high levels in normal testes. Testicular survivin levels in infertile patients were related inversely to the severity of spermatogenic failure (p < 0.001), with a lack of expression in most specimens with pre-meiotic spermatogenic arrest and in all those with germ cell aplasia. Lower levels of expression were observed in TGCTs than in normal testes. While survivin expression was detected in most TGCTs with undifferentiated components (12 of 13), it was absent in all mature teratomas (n = 3). These data show that survivin is expressed in normal and transformed germ cells. Its downregulation in spermatogenic disorders indicates that survivin may contribute to the normal balance between germ cell proliferation and apoptosis. In TGCTs, survivin expression appears to be lost with somatic differentiation.     

Inhibition of mTOR pathway decreases the expression of pre‐meiotic and meiotic markers throughout postnatal development and in adult testes in mice

Rapamycin (mTOR inhibitor) has been reported to have negative effect on human male gonadal function. Previously, we showed that mTOR signalling molecules are expressed during early spermatogenesis in mice. The objective of this study was to investigate the role of mTOR signalling in meiosis both during the first wave of spermatogenesis and also during adult spermatogenesis. Day 5 post‐partum mice were administered rapamycin and retinoic acid (RA; a Stra8 activator), and expression of p‐p70S6K and Stra8 proteins was evaluated. p‐p70S6K and Stra8 protein expressions decreased in post‐natal testes after rapamycin treatment. Stra8 protein expression increased after RA and rapamycin+RA administrations in post‐natal testes. In adult mice, rapamycin was administrated for 1 or 4 weeks. Morphological analysis for testicular damage and TUNEL assay was performed. After rapamycin administration, germ cell loss increased in adult testes. Ultrastructural analysis revealed disorganised testicular morphology and vacuolisation. The number of apoptotic germ cells increased after 4 weeks rapamycin administration. Stra8 and Dmc1 expressions decreased in 4 weeks rapamycin group, whereas Sycp3 and VASA expression did not change. Our findings suggest that mTOR pathway has an important role in meiotic progress of male germ cells both during first wave of spermatogenesis and in adult mice.     

Volume alteration of undescended testes: Before and after orchiopexy

ObjectivesWe used ultrasound to investigate the volume of undescended testes before and after orchiopexy, and compared these data with normally descended testes.Materials and MethodsWe retrospectively reviewed boys in the age range of 0–18 years who had undergone unilateral or bilateral orchiopexy due to undescended testes (International Classification of Diseases-Ninth Revision, ICD-9 752.51) in National Taiwan University Hospital, Taipei, Taiwan between January 2010 and December 2013. A total of 116 boys received preoperative testicular ultrasound evaluation, and 75 of them received regular ultrasound during a mean follow-up period of 2.5 years. The volume of the testes was calculated by applying Hansen formula [testicular volume = length (L) × width (W)² × 0.52] and compared with a cohort of 92 boys constructed for normative values of testicular volume from The Netherlands.ResultsThe mean volume of the 145 undescended testes among 118 boys was 0.238 mL. The volume of the undescended testes was significantly smaller (p < 0.001) than the mean normative value of 0.418 mL. The volume of postorchiopexy undescended testes (0.356 mL) revealed a growing trend in the mean 2.5-year follow-up with a significance increase of size (p = 0.001), but has not yet reached the normal testicular size (0.604 mL).ConclusionThe preorchiopexy volumes of undescended testes are significantly smaller than normative values. The follow-up postorchiopexy volumes of undescended testes actually increased in size, although they were still smaller than normative values. These Taiwanese testicular growth curves should become reference values in pediatric clinical practice when evaluating testicular development.Keywords: cryptorchidism, orchiopexy, testicular volume, treatment outcome, undescended testis     

Hoxa-11 maintains cell proliferation in the mouse gubernaculum to facilitate testicular descent

Introduction

The gubernaculum is a structure vital for guiding testicular descent. The Homeobox gene, Hoxa-11, is involved in patterning embryonic structures and is necessary for gubernacular development, as Hoxa-11 knock-out mice exhibit abnormal gubernacula and undescended testes. We aimed to elucidate how testicular descent fails by examining cell proliferation and androgen receptor (AR) expression in Hoxa-11 KO mice gubernacula.

Methods

Postnatal day 2 wild type (n = 6) and Hoxa-11 KO mice (n = 6), were prepared for immunohistochemistry and confocal microscopy using antibodies against androgen receptor, slow skeletal myosin (My32), and Ki67, a marker of cell proliferation.

Results

The gubernacula of Hoxa-11 KO mice were hypocellular compared with WT. AR was present in the gubernaculum and abutting inguinal fat pad in both WT and Hoxa-11 KO with no difference in expression. Slow skeletal myosin was present in a clear ‘swirl’ in the growth centre of WT animals which was absent in the Hoxa-11 KO mice. Ki67, expressed in the growth centre and cremaster muscle in WT, was greatly decreased in Hoxa-11 KO.

Conclusion

Hoxa-11 may regulate fibroblast proliferation in the gubernaculum, as it does in human uterosacral ligaments, allowing formation of the 'growth centre' within the bulb and facilitating myogenesis and elongation to the scrotum. Polymorphisms in Hoxa-11 may contribute to the aetiology of human cryptorchidism.     

Regulation of cell adhesion in the testis： a new role for p73

The dramatic changes that male germ cells in the adult testis undergo in gene expression profile and morphology as they transition from spermatogonial stem cells through to mature spermatozoa is dependent upon their association with Sertoli cells. Sertoli cells are crucial for survival and maturation of male germ cells. Two recent papers, Holembowski et al？ and Inoue et a13 have described a surprising role for the p53 family member, p73, in regulation of germ cell-Sertoli cell adhesion.     

Influence of a leptin deficiency on testicular morphology, germ cell apoptosis, and expression levels of apoptosis-related genes in the mouse

Leptin-deficient (ob/ob) male mice are morbidly obese and exhibit impaired reproductive function. The objective of this study was to assess the effect of a leptin deficiency on testicular morphology, germ cell development, apoptotic activity within germ cells, and expression levels of apoptosis-related genes in the testis. Sixteen week-old ob/ob male mice (n = 8) and controls (n = 8) were killed, and their reproductive organs were weighed. Testes were processed for either histomorphological analysis (hematoxylin and eosin [H&E] staining), germ cell apoptosis assessment (deoxy-UTP-digoxigenin nick end labeling [TUNEL] method), or apoptosis-related gene expression analysis (microarray). Cross sections of the testes of leptin-deficient animals showed reduced seminiferous tubule area, fewer pachytene spermatocytes, and fewer tubules exhibiting elongated spermatids/mature spermatozoa. Condensation of germ cell nuclei and Sertoli cell vacuolization were evident in the testes of some ob/ob animals. Overall there was an elevation of apoptotic activity in the germ cells of ob/ob mice, particularly within the pachytene spermatocytes. With microarray technology, we identified 9 proapoptosis-related genes that were expressed at a significantly higher level in the testes of ob/ob mice than in the testes of the controls. Among these were members of the tumor necrosis factor receptor super family 1A and 5 (TNFR1 and 5) and peptidoglycan recognition proteins (associated with the extrinsic apoptotic pathway), and granzymes A and B, growth arrest and DNA damage inducible 45 gamma, sphingosine phosphate lyase 1, and caspase 9 (associated with the intrinsic apoptotic pathway). The results of the current study show that a leptin deficiency in mice is associated with impaired spermatogenesis, increased germ cell apoptosis, and up-regulated expression of proapoptotic genes within the testes.     

N-acetylcysteine protects against cadmium-induced germ cell apoptosis by inhibiting endoplasmic reticulum stress in testes

Cadmium (Cd) is a reproductive toxicant that induces germ cell apoptosis in the testes. Previous studies have demonstrated that endoplasmic reticulum (ER) stress is involved in Cd-induced germ cell apoptosis. The aim of the present study was to investigate the effects of N-acetylcysteine (NAC), an antioxidant, on Cd-induced ER stress and germ cell apoptosis in the testes. Male CD-1 mice were intraperitoneally injected with CdCl₂ (2.0 mg kg⁻¹). As expected, acute Cd exposure induced germ cell apoptosis in the testes, as determined by terminal dUTP nick-end labelling (TUNEL). However, the administration of NAC alleviated Cd-induced germ cell apoptosis in the testes. Further analysis showed that NAC attenuated the Cd-induced upregulation of testicular glucose-regulated protein 78 (GRP78), an important ER molecular chaperone. Moreover, NAC inhibited the Cd-induced phosphorylation of testicular eukaryotic translation initiation factor 2α (eIF2α), a downstream target of the double-stranded RNA-activated kinase-like ER kinase (PERK) pathway. In addition, NAC blocked the Cd-induced activation of testicular X binding protein (XBP)-1, indicating that NAC attenuates the Cd-induced ER stress and the unfolded protein response (UPR). Interestingly, NAC almost completely prevented the Cd-induced elevation of C/EBP homologous protein (CHOP) and phosphorylation of c-Jun N-terminal kinase (JNK), two components of the ER stress-mediated apoptotic pathway. In conclusion, NAC protects against Cd-induced germ cell apoptosis by inhibiting endoplasmic reticulum stress in the testes.     

The protective effects of grape seed extract on MDA, AOPP, apoptosis and eNOS expression in testicular torsion: an experimental study

Objectives

Grape seed proanthocyanidin extract (GSPE) is a potent antioxidant and a free radical scavenger. This study was designed to determine whether GSPE could protect against dysfunction and oxidative stress induced by torsion–detorsion injury in rat testis.

Methods

A total of 45 male Wistar albino rats were divided into five groups: control group, sham group, torsion–detorsion (T/D) group, T/D + GSPE group, GSPE group. GSPE was administrated 100 mg/kg/day with oral gavage over seven days before torsion. Testicular torsion was performed for 2 h, and afterward, detorsion was performed for 2 h. The rats were decapitated under ketamine anesthesia, and their testes tissues were removed. Tissue malondialdehyde, advanced oxidation protein products levels, eNOS expression, apoptosis and histopathological damage scores were then compared.

Results

Testicular torsion–detorsion caused significant increases in malondialdehyde level, apoptosis and eNOS expression level and caused a significant decrease in advanced oxidation protein product levels and testicular spermatogenesis in ipsilateral testes. GSPE prevented the rise in malondialdehyde, apoptosis and eNOS expression and improved testicular morphology and Johnsen’s score.

Conclusions

As a result, testicular torsion gives rise to serious damage in testes and GSPE is a potent antioxidant agent in preventing testicular injury.     

Tslc1 (nectin-like molecule-2) is essential for spermatozoa motility and male fertility

The nectin-like molecule-2 (TSLC1) is a cell-cell adhesion molecule expressed in testicular germ cells. To directly examine the role of Tslc1 in male fertility, we generated Tslc1+/- mice that have greater than 90% reduction in Tslc1 expression. Tslc1+/- males exhibited reduced fertility and rarely transmitted the Tslc1 mutant allele, whereas Tslc1+/- females were consistently able to transmit the mutant allele. Histologic and electron microscopic analyses of the testes in Tslc1+/- mice demonstrated disruption of the junctional scaffold between germ cells and Sertoli cells. Reduced Tslc1 expression had no effect on germ cell proliferation or apoptosis. While evidence of normal spermatozoal maturation was supported by Fluorescence Activated Cell Sorting (FACS) analysis, spermatozoa from Tslc1+/- mice demonstrated markedly reduced motility without compromised viability. Collectively, these results establish an essential role for Tslc1 in spermatozoal maturation and motility, distinct from other members of the nectin family.     

Ectopic porcine spermatogenesis in murine subcutis： tissue grafting versus cell-injection methods

Fragments of testis tissue from immature animals grow subcutaneous areas of immunodeficient mice. The same results testes of rodents are injected into the subcutis of nude mice. and develop spermatogenesis when grafted onto are obtained when dissociated cells from immature Those cells reconstitute seminiferous tubules and facilitate spermatogenesis. We compared these two methods, tissue grafting and cell-injection methods, in terms of the efficiency of spermatogenesis in the backs of three strains of immunodeficient mice, using neonatal porcine testicular tissues and cells as donor material. Nude, severe combined immunodeficient （SCID） and NOD/Shi- SCID, IL-2Rγc^null （NOG） mice were used as recipients. At 10 months after surgery, the transplants were examined histologically. Both grafting and cell-injection methods resulted in porcine spermatogenesis on the backs of recipient mice; the percentage of spermatids present in the transplants was 67% and 22%, respectively. Using the grafting method, all three strains of mice supported the same extent of spermatogenesis. As for the cell- injection method, although SCID mice were the best host for supporting reconstitution and spermatogenesis, any difference from the other strains was not signifcant. As NOG mice did not show any better results, the severity of immunodeficiency seemed to be irrelevant for supporting xeno-ectopic spermatogenesis. Our results confirmed that tubular reconstitution is applicable to porcine testicular cells. This method as well as the grafting method would be useful for studying spermatogenesis in different kinds of animals.     

Does a gonadotropin-releasing hormone analogue prevent cisplatin-induced spermatogenic impairment?

Summary The cytotoxic effect of cis-diamminedichloro-platinum II (cisplatin, CDDP) on spermatogenesis in BALB/C mice, and possible protection of the testes by leuprolide acetate (D-Leu-6 LHRH(1–9)-ethylamide, TAP-144, leuprolide), a synthetic gonadotropin-releasing hormone analogue, were examined. Temporary interruption of the pituitary-gonadal axis by the analogue and amelioration of the gonadal toxicity of cisplatin by reducing the cell division rate in spermatogenesis were expected. The results showed cisplatin to have a cytotoxic effect on spermatogenic cells in BALB/C mice. The administration of leuprolide had no effect on testicular weight or histological findings in the mouse testes. Pretreatment and simultaneous administration of leuprolide did not reduce the damaging effect of cisplatin on the testes in mice. This is at variance with a previously published report.     

Hodenhochstand

Undescended testis (UDT) is the most frequent congenital malformation affecting 1% of 1-year-old mature birth boys. If untreated UDT leads to progressive histological changes with impaired spermatogenesis and an increasing risk of testicular cancer. For clinical reasons palpable testes should be differentiated from impalpable testes. Spontaneous testicular descent can be expected only before 6 months of age. Subsequently, treatment should start without delay and be finished before the child has reached the age of 1 year. Surgery is the cornerstone of treatment. Inguinal approaches are standard practice for palpable testes, whereas laparoscopy is used in non-palpable testes. Hormonal treatment is ineffective for inducing testicular descent but facilitates germ cell maturation and an improvement in fertility potential. Only early reatment of UDT reduces germ cell loss, improves fertility and reduces the risk of testicular cancer.     

Resistance of CD28-deficient mice to autologous phase of anti-glomerular basement membrane glomerulonephritis

Background. The engagement of CD28 on T cells provides an essential costimulatory signal for T-cell activation and differentiation. Recent studies suggest that CTLA4Ig inhibits T-cell activation in vitro and in vivo, prevents acute and chronic allograft rejection, and induces tolerance in some experimental transplantation models.Methods. The present study examined the role of the CD28 costimulatory pathway in the induction of experimental anti-glomerular basement membrane (GBM) glomerulonephritis (GN). An accelerated type of anti-GBM GN was induced in wild-type mice and CD28-deficient (KO) mice. After 7 and 14 days, functional parameters, such as serum creatinine, amount of proteinuria, and serum mouse IgG levels were assessed. Histological studies were performed simultaneously to examine glomerular changes by light microscopy and immunoglobulin deposition by immunofluorescence staining. Flow cytometric analysis was undertaken to assess I-A^b antigen expression on spleen cells.Results. Anti-GBM GN induction was almost completely prevented in CD28-KO mice. CD28-KO mice had impaired ability to evoke B-cell activation, and developed lower mouse anti-rabbit IgG antibody titers in their serum than wild-type C57BL/6 mice. Furthermore, glomerular deposition of mouse anti-rabbit IgG was not detectable in CD28-KO mice after immunization with anti-GBM antibody.Conclusions. This is the first demonstration that the CD28 costimulatory pathway plays a critical role in autologous antibody production in anti-GBM GN, and suggests that effective inhibition of CD28-dependent autologous antibody production could be useful in the treatment of antibody-dependent GN in humans.