Hongliang Hu

Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study.

Recent studies have reported that induced pluripotent stem (iPS) cells from mice and humans can differentiate into primordial germ cells. However, whether iPS cells are capable of producing male germ cells is not known. The objective of this study was to investigate the differentiation potential of mouse iPS cells into spermatogonial stem cells and late-stage male germ cells. We used an approach that combines in vitro differentiation and in vivo transplantation. Embryoid bodies (EBs) were obtained from iPS cells using leukaemia inhibitor factor (LIF)-free medium. Quantitative PCR revealed a decrease in Oct4 expression and an increase in Stra8 and Vasa mRNA in the EBs derived from iPS cells. iPS cell-derived EBs were induced by retinoic acid to differentiate into spermatogonial stem cells (SSCs), as evidenced by their expression of VASA, as well as CDH1 and GFRα1, which are markers of SSCs. Furthermore, these germ cells derived from iPS cells were transplanted into recipient testes of mice that had been pre-treated with busulfan. Notably, iPS cell-derived SSCs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids, as shown by VASA and SCP3 expression. This study demonstrates that iPS cells have the potential to differentiate into late-stage male germ cells. The derivation of male germ cells from iPS cells has potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development.

Derivation of male germ cells from induced pluripotent stem cells in vitro and in reconstituted seminiferous tubules.

Previous studies have demonstrated that mouse‐ and human‐induced pluripotent stem (iPS) cells can differentiate into primordial germ cells in vitro. However, up to now it is not known whether iPS cells would be able to differentiate into male germ cells in vivo. The aim of this study was to explore differentiation potential of iPS cells to male germ cells in vitro and in vivo.

Differentiation of Induced Pluripotent Stem Cells into Male Germ Cells In Vitro through Embryoid Body Formation and Retinoic Acid or Testosterone Induction

Generation of germ cells from pluripotent stem cells in vitro could have great application for treating infertility and provides an excellent model for uncovering molecular mechanisms controlling gametogenesis. In this study, we explored the differentiation potential of mouse induced pluripotent stem (iPS) cells towards male germ cells. Embryoid body formation and retinoic acid/testosterone induction were applied to promote differentiation of mouse iPS cells into male germ cells in vitro. Quantitative RT-PCR and immunoflourescence were performed to characterize the iPS cell differentiation process, and notably there were different temporal expression profiles of male germ cell-associated genes. The expression of proteins, including MVH, CDH1, and SCP3, was remarkably increased. mRNA expression of Stra8, Odf2, Act, and Prm1 was upregulated in iPS cells by retinoic acid or testosterone induction, whereas Oct-4 transcription was reduced in these cells compared to the controls. Hormones were also measured in the EB medium. DNA content analysis by flow cytometry revealed that iPS cells could differentiate into haploid cells through retinoic acid or testosterone treatment. Collectively, our results suggest that mouse iPS cells possess the potency to differentiate into male germ cells in vitro through embryoid body formation and retinoic acid or testosterone induction.

Mesenchymal stem cells overexpressing Ihh promote bone repair

BackgroundIndian hedgehog (Ihh) signaling pathway is known to play key roles in various aspects of normal endochondral bone development. This study tested the potential roles of high Ihh signaling in the context of injury-induced bone regeneration.MethodsA rabbit tibia defect model was established to test the effects of the implant of Ihh/mesenchymal stem cells (MSCs)/scaffold complex. Computed tomography (CT), gross observation, and standard histological and immunohistological techniques were used to evaluate the effectiveness of the treatment. In vitro studies with MSCs and C3H10T1/2 cells were also employed to further understand the cellular and molecular mechanisms.ResultsWe found that the implanted Ihh/MSCs/scaffold complex promoted bone repair. Consistently, in vitro study found that Ihh induced the upregulation of chondrocytic, osteogenic, and vascular cell markers, both in C3H10T1/2 cells and MSCs.ConclusionsOur study has demonstrated that high Ihh signaling in a complex with MSCs enhanced bone regeneration effectively in a clinically relevant acute injury model. Even though the exact underlying mechanisms are still far from clear, our primary data suggested that enhanced chondrogenesis, osteogenesis, and angiogenesis of MSCs at least partially contribute to the process. This study not only has implications for basic research of MSCs and Ihh signaling pathway but also points to the possibility of direct application of this specific paradigm to clinical bone repair.

A new molecular diagnostic approach to assess Y chromosome microdeletions in infertile men.

OBJECTIVE: A key cause of spermatogenetic failure in infertile males is microdeletions in the azoospermia factor (AZF) regions of the Y chromosome. This study screened for microdeletions in the AZF regions using suspension array technology and compared the results with those from polymerase chain reaction (PCR). METHODS: Patients with spermatogenetic failure (n = 507) and healthy control sperm donors (n = 100) were recruited. DNA samples were analysed using both multiplex PCR with gel electrophoresis and suspension array technology. RESULTS: The suspension array method identified 45 infertile males with Y chromosome microdeletions, while none was found in the controls. Amongst the AZF subregions, two cases had deletions in AZFa, three in AZFb, 35 in AZFc, three in AZFbc and two in AZFabc. The results from 507 patients were identical when analysed with either suspension array or multiplex PCR, however suspension array technology offered improved sensitivity, may be more accurate and could give time and cost savings. CONCLUSIONS: Suspension array technology offers a rapid and high-throughput method for Y chromosome microdeletion screening in infertile men.

Ndrg2 regulates vertebral specification in differentiating somites.

It is generally thought that vertebral patterning and identity are globally determined prior to somite formation. Relatively little is known about the regulators of vertebral specification after somite segmentation. Here, we demonstrated that Ndrg2, a tumor suppressor gene, was dynamically expressed in the presomitic mesoderm (PSM) and at early stage of differentiating somites. Loss of Ndrg2 in mice resulted in vertebral homeotic transformations in thoracic/lumbar and lumbar/sacral transitional regions in a dose-dependent manner. Interestingly, the inactivation of Ndrg2 in osteoblasts or chondrocytes caused defects resembling those observed in Ndrg2(-/-) mice, with a lower penetrance. In addition, forced overexpression of Ndrg2 in osteoblasts or chondrocytes also conferred vertebral defects, which were distinct from those in Ndrg2(-/-) mice. These genetic analyses revealed that Ndrg2 modulates vertebral identity in segmented somites rather than in the PSM. At the molecular level, combinatory alterations of the amount of Hoxc8-11 gene transcripts were detected in the differentiating somites of Ndrg2(-/-) embryos, which may partially account for the vertebral defects in Ndrg2 mutants. Nevertheless, Bmp/Smad signaling activity was elevated in the differentiating somites of Ndrg2(-/-) embryos. Collectively, our findings unveiled Ndrg2 as a novel regulator of vertebral specification in differentiating somites.

Endothelial Nitric Oxide Synthase (eNOS) T-786C, 4a4b, and G894T Polymorphisms and Male Infertility: Study for Idiopathic Asthenozoospermia and Meta-Analysis

ABSTRACT Recent studies on the eNOS gene and male infertility show that expression of eNOS regulates normal spermatogenesis in the testis, and the eNOS gene variants (T-786C, 4a4b, and G894T) are potentially involved in impairment of spermatogenesis and sperm function. Thus, we conducted this association and meta-analysis study to further validate whether variants of those three loci affected the risk of idiopathic asthenozoospermia (AZS) and male infertility. Approximately 340 Chinese idiopathic AZS patients and 342 healthy men were included for this case-control study, genotyped by gel electrophoresis analysis or direct sequencing of PCR products. The eNOS mRNA isolated from the semen of patients was further examined by quantitative real-time PCR. Also, a meta-analysis of association between eNOS gene polymorphisms and male infertility was performed. A significant association was identified on allelic level between 4a4b variant and AZS in our study (chi-squared = 7.53, corrected P = 0.018, odds ratio (OR) = 1.808), while there were no significant difference of T-786C and G894T for asthenozoospermia in both genotype and allele distributions. In addition, expression of eNOS was up-regulated in patients compared with controls (about 2.4-fold, P < 0.001). Furthermore, the results of the meta-analysis support the conclusion that the T-786C and 4a4b loci were associated with male infertility in both Asian and Caucasian populations. Our study provides genetic evidence for the eNOS gene being a risk factor for idiopathic AZS and male infertility. Considering genetic differences among populations and complex pathogenesis of male infertility, more validating studies using independent samples are suggested in the future.

Association and meta-analysis of HLA and non-obstructive azoospermia in the Han Chinese population

The exact aetiology and pathogenesis of most non‐obstructive azoospermia (NOA) are still unknown. The previous two genomewide association studies (GWASs) have identified three different loci within the HLA region for NOA in the Han Chinese population, including rs3129878, rs498422 and rs7194. To further validate the risk of three GWAS‐linked loci for NOA, we conducted a case–control study of these three risk loci in an independent Han Chinese male population, with 603 NOA patients and 610 controls. Furthermore, we also performed a meta‐analysis of five studies on these three NOA‐risk loci. The case–control study strongly suggested a significant association between loci rs3129878, rs498422 and rs7194 and NOA (P = 6.75 × 10−21 (OR = 2.2586), P = 0.0060 (OR = 1.4013) and P = 0.0128 (OR = 1.2626) respectively). Our meta‐analyses also supported the susceptibility of these three risk loci to NOA (P < 0.01). The risk variants within the HLA region potentially have a strong effect on males at risk of NOA, and may serve as diagnostic markers for male infertility. However, considering genetic difference between different populations, future validating studies in larger independent samples and animal experiments are suggested.

AB145. Raman spectroscopy as an ex vivo non-invasive approach to distinguish complete and incomplete spermatogenesis within human spermatogenesis

Objective To evaluate the potential clinical application of Raman spectroscopy (RS) as a tool that may identify spermatogenesis within human seminiferous tubules. Design Raman spectroscopic scanning of human testicular tissue at different maturational stages; immunohistochemistry study and metabolomic analysis of non-obstructive azoospermic/obstructive azoospermic testes. Materials and methods A total of 52 patients with clinical indications of non-obstructive azoospermia (NOA) and obstructive azoospermia (OA) that underwent infertility evaluation and treatment were collected. This study was approved by the Reproductive Ethics Committee of Renji Hospital, Shanghai Jiao Tong University School of Medicine. Fresh testicular specimens were collected immediately after excision in the operating room and brought to the laboratory of Raman spectroscopy. For Raman scanning, a single seminiferous tubule was placed at the center of a glass bottomed dish and hydrated with PBS solution. Linear equidistant points (a total of 10 points with the interval of 5 µm) at the midline were scanned for each tubule with a 10 seconds period per point. Each testicular sample had ten randomly selected tubules scanned. After completion of Raman scanning, the testicular tissue was immediately fixed in Bouin’s solution and stored in 70% ethanol, embedded in paraffin, sectioned at 5 µm, and stained by hematoxylin and eosin (H&E). The 52 testicular tissue were categoried into three groups: spermatogenesis, maturational arrest (MA), and sertoli cell only syndrome (SCO). Meanwhile, the thickness of lamina propria (LP) was also measured. Then, testicular tissue were studied by immunohistochemistry of type-I, III, IV collagens and laminin. And the differences of metabolites between NOA and OA testes were examined by gas chromatography-mass spectrometer (GC-MS). For data analysis, the real-time Raman spectra were acquired automatically by OPUS 6.5 software and compared among different groups, then the spectral data were then loaded in the Matlab platform for k-means cluster analysis, which aimed to classify the spectra and predict the histological diagnosis for each spectrum. Results Tubules of OA patients had spectral intensities below 2,000 (au) while tubules of NOA patients had higher intensities, depending on the degree of spermatogenesis. Raman spectroscopy was able to separate samples of NOA and OA testicular tissue with a sensitivity of 90% and specificity of 85.71%. The LP of NOA tubules were thickened, and had increased deposition of type-I and type-III collagens. Twelve detected metabolites showed significant differences between NOA and OA testes. Conclusions Raman spectroscopy can non-invasively distinguish seminiferous tubules with complete and incomplete spermatogenesis, and may serve as a novel and potentially useful tool to guide surgeons performing micro-TESE to improve sperm retrieval. Support Science and Technology Commission of Shanghai Municipality (No: 10JCI409900), National Basic Research Program of China (No: 2011CB944504) and National Natural Science Foundation of China (Key Program: 31230048).