SRSF10 is essential for progenitor spermatogonia expansion by regulating alternative splicing.

Alternative splicing expands the transcriptome and proteome complexity and plays essential roles in tissue development and human diseases. However, how alternative splicing regulates spermatogenesis remains largely unknown. Here, using a germ cell-specific knockout mouse model, we demonstrated that the splicing factor Srsf10 is essential for spermatogenesis and male fertility. In the absence of SRSF10, spermatogonial stem cells can be formed, but the expansion of Promyelocytic Leukemia Zinc Finger (PLZF)-positive undifferentiated progenitors was impaired, followed by the failure of spermatogonia differentiation (marked by KIT expression) and meiosis initiation. This was further evidenced by the decreased expression of progenitor cell markers in bulk RNA-seq, and much less progenitor and differentiating spermatogonia in single-cell RNA-seq data. Notably, SRSF10 directly binds thousands of genes in isolated THY+ spermatogonia, and Srsf10 depletion disturbed the alternative splicing of genes that are preferentially associated with germ cell development, cell cycle, and chromosome segregation, including Nasp, Bclaf1, Rif1, Dazl, Kit, Ret, and Sycp1. These data suggest that SRSF10 is critical for the expansion of undifferentiated progenitors by regulating alternative splicing, expanding our understanding of the mechanism underlying spermatogenesis.

Human obstructive (postvasectomy) and nonobstructive azoospermia - Insights from scRNA-Seq and transcriptome analysis.

A substantial number of male infertility is caused by azoospermia. However, the underlying etiology and the molecular basis remain largely unknown. Through single-cell (sc)RNA sequencing, we had analyzed testis biopsy samples from two patients with obstructive azoospermia (OA) and nonobstructive azoospermia (NOA). We found only somatic cells in the NOA samples and explored the transcriptional changes in Sertoli cells in response to a loss of interactions with germ cells. Moreover, we observed a germ cell population discrepancy between an OA (postvasectomy) patient and a healthy individual. We confirmed this observation in a secondary study with two datasets at GSM3526588 and GSE124263 for detailed analysis wherein the regulatory mechanisms at the transcriptional level were identified. These findings thus provide valuable information on human spermatogenesis, and we also identified insightful information for further research on reproduction-related diseases.

A high-throughput, open-space and reusable microfluidic chip for combinational drug screening on tumor spheroids.

Screening drug combinations using tumor spheroids can play a vital role in the development of disease treatment and personalized medicine. However, current studies focus on drug gradients or combinations of two drugs in most cases, and it is difficult to find complex therapeutic combinations involving more drugs. The use of design-of-experiment (DOE) microfluidics is a potential strategy to study this area systematically. Here we develop a high-throughput, open-space multilayered PMMA microfluidic chip for combinational drug screening on tumor spheroids. This microchip is straightforward to fabricate, compatible with standard spheroid cultures, and friendly for end-users. The device consists of an inlet layer and multiple dispersing layers. In the inlet layer, different samples can be loaded into the chip simultaneously. The sample solutions flow into the dispersing layers to generate various combinations based on the specific DOE principle. We demonstrated that the chip performance is in quantitative agreement with the design, using water and doxycycline combinations as models. As a proof-of-concept study, we constructed a HeLa reporter cell line to quantify the autophagy of tumor spheroids and used the chip to identify critical factors relating to the growth of the spheroids. Specifically, we used L-glutamine, D-glucose, FBS, and cisplatin as the factors and studied the autophagy, growth curves, and spheroid sizes in response to different combinations of the four factors. We found that D-glucose can inhibit the effects of cisplatin on tumor spheroids, and cisplatin caused severe autophagy in 3D tumor spheroids compared to 2D monoculture cells. Our method has the potential to allow more drug combinations to be examined, and it can be extended to DOE approaches with seven or more inputs.

MicroRNA-126 engineered muscle-derived stem cells attenuates cavernosa injury-induced erectile dysfunction in rats.

BACKGROUND: Cavernosa injury is a common cause of organic erectile dysfunction (ED), which requires safe and effective treatments. In the present study, the therapeutic efficiency of muscle-derived stem cells (MDSCs) modified with microRNA-126 (miR-126) was determined in rats with cavernosa injury. METHODS: MDSCs were transfected with miR-126 and then were transplanted into rats with cavernosa injury. Erectile function, vascular function (western blot and immunofluorescence), extraction, and detection of exosomes were then undertaken. RESULTS: On the 28th day after transplantation, the highest value of intra-cavernous pressure (ICP)/mean arterial pressure (MAP) in rats of miRNA-126 group (0.84 ± 0.14) was observed (Control: 0.38 ± 0.07; MDSC: 0.54 ± 0.11, Vector: 0.60 ± 0.02; respectively). Treatment of miRNA-126-modified-MDSCs remarkably strengthened vascular structure, supported by hematoxylin-eosin staining. The expression of CD31, von Willebrand Factor and vascular endothelial factors were higher than those in other groups, indicating improved vascular function. In vitro mechanism studies showed that exosomes containing miR-126 isolated from MDSCs promoted angiogenesis and attenuated apoptosis of human umbilical venous endothelial cells. Finally, insulin receptor substrate 1 and Krüppel-like factor 10 were determined as the direct target genes of miR-126. CONCLUSIONS: MiR-126 engineered MDSCs notably repaired cavernosa injury in rats via vascular reconstruction by directly targeting IRS1 and KLF10, in which the exosomes secreted by MDSCs played a critical role.

Deciphering the autophagy regulatory network via single-cell transcriptome analysis reveals a requirement for autophagy homeostasis in spermatogenesis.

Background: Autophagy has been implicated as a crucial component in spermatogenesis, and autophagy dysfunction can lead to reproductive disorders in animal models, including yeast, C. elegans and mice. However, the sophisticated transcriptional networks of autophagic genes throughout human spermatogenesis and their biological significance remain largely uncharacterized. Methods: We profiled the transcriptional signatures of autophagy-related genes during human spermatogenesis by assessing specimens from nine fertile controls (including two normal persons and seven obstructive azoospermia (OA) patients) and one nonobstructive azoospermia (NOA) patient using single-cell RNA sequencing (scRNA-seq) analysis. Dysregulation of autophagy was confirmed in two additional NOA patients by immunofluorescence staining. Gene knockdown was used to identify the role of Cst3 in autophagy during spermatogenesis. Results: Our data uncovered a unique, global stage-specific enrichment of autophagy-related genes. Human-mouse comparison analysis revealed that the stage-specific expression pattern of autophagy-related genes was highly conserved in mammals. More importantly, dysregulation of some clusters of autophagy-related genes was observed in NOA patients, suggesting the association of autophagy with male infertility. Cst3, a human-mouse conserved and autophagy-related gene that is actively expressed in spermatogonia and early spermatocytes, was found to regulate spermatogonial stem cell (SSC) maintenance and subsequent male germ cell development. Knockdown of Cst3 increased autophagic activity in mouse SSCs and subsequently suppressed the transcription of SSC core factors such as Oct4, Id1, and Nanos3, which could be efficiently rescued by manipulating autophagic activity. Conclusions: Our study provides comprehensive insights into the global transcriptional signatures of autophagy-related genes and confirms the importance of autophagy homeostasis in SSC maintenance and normal spermatogenesis, opening new avenues for further dissecting the significance of the autophagy regulatory network in spermatogenesis as well as male infertility.

Adenylate kinase 1 deficiency disrupts mouse sperm motility under conditions of energy stress†.

Mammalian spermatozoa are highly polarized cells characterized by compartmentalized cellular structures and energy metabolism. Adenylate kinase (AK), which interconverts two ADP molecules into stoichiometric amounts of ATP and AMP, plays a critical role in buffering adenine nucleotides throughout the tail to support flagellar motility. Yet the role of the major AK isoform, AK1, is still not well characterized. Here, by using a proteomic analysis of testis biopsy samples, we found that AK1 levels were significantly decreased in nonobstructive azoospermia patients. This result was further verified by immunohistochemical staining of AK1 on a tissue microarray. AK1 was found to be expressed in post-meiotic round and elongated spermatids in mouse testis and subsequent mature sperm in the epididymis. We then generated Ak1 knockout mice, which showed that AK1 deficiency did not induce any defects in testis development, spermatogenesis, or sperm morphology and motility under physiological conditions. We further investigated detergent-modeled epididymal sperm and included individual or mixed adenine nucleotides to mimic energy stress. When only ADP was available, Ak1 disruption largely compromised sperm motility, manifested as a smaller beating amplitude and higher beating frequency, which resulted in less effective forward swimming. The energy restriction/recover experiments with intact sperm further addressed this finding. Besides, decreased AK activity was observed in sperm of a male fertility disorder mouse model induced by cadmium chloride. These results cumulatively demonstrate that AK1 was dispensable for testis development, spermatogenesis, or sperm motility under physiological conditions, but was required for sperm to maintain a constant adenylate energy charge to support sperm motility under conditions of energy stress.

Functional reconstruction of injured corpus cavernosa using 3D-printed hydrogel scaffolds seeded with HIF-1α-expressing stem cells.

Injury of corpus cavernosa results in erectile dysfunction, but its treatment has been very difficult. Here we construct heparin-coated 3D-printed hydrogel scaffolds seeded with hypoxia inducible factor-1α (HIF-1α)-mutated muscle-derived stem cells (MDSCs) to develop bioengineered vascularized corpora. HIF-1α-mutated MDSCs significantly secrete various angiogenic factors in MDSCs regardless of hypoxia or normoxia. The biodegradable scaffolds, along with MDSCs, are implanted into corpus cavernosa defects in a rabbit model to show good histocompatibility with no immunological rejection, support vascularized tissue ingrowth, and promote neovascularisation to repair the defects. Evaluation of morphology, intracavernosal pressure, elasticity and shrinkage of repaired cavernous tissue prove that the bioengineered corpora scaffolds repair the defects and recover penile erectile and ejaculation function successfully. The function recovery restores the reproductive capability of the injured male rabbits. Our work demonstrates that the 3D-printed hydrogels with angiogenic cells hold great promise for penile reconstruction to restore reproductive capability of males.

Pre-activation of mesenchymal stem cells with TNF-α, IL-1ß and nitric oxide enhances its paracrine effects on radiation-induced intestinal injury.

Conditioned medium from mesenchymal stem cells (MSC-CM) may represent a promising alternative to MSCs transplantation, however, the low concentrations of growth factors in non-activated MSC-CM hamper its clinical application. Recent data indicated that the paracrine potential of MSCs could be enhanced by inflammatory factors. Herein, we pre-activated bone-marrow-derived MSCs under radiation-induced inflammatory condition (MSC(IEC-6(IR))) and investigated the evidence and mechanism for the differential effects of MSC-CM(IEC-6(IR)) and non-activated MSC-CM on radiation-induced intestinal injury (RIII). Systemic infusion of MSC-CM(IEC-6(IR)), but not non-activated MSC-CM, dramatically improved intestinal damage and survival of irradiated rats. Such benefits may involve the modulation of epithelial regeneration and inflammation, as indicated by the regeneration of intestinal epithelial/stem cells, the regulation of the pro-/anti-inflammatory cytokine balance. The mechanism for the superior paracrine efficacy of MSC(IEC-6(IR)) is related to a higher secretion of regenerative, immunomodulatory and trafficking molecules, including the pivotal factor IGF-1, induced by TNF-α, IL-1ß and nitric oxide partially via a heme oxygenase-1 dependent mechanism. Together, our findings suggest that pre-activation of MSCs with TNF-α, IL-1ß and nitric oxide enhances its paracine effects on RIII via a heme oxygenase-1 dependent mechanism, which may help us to maximize the paracrine potential of MSCs.

Innovative magnetic rings for circumferential mucosectomy: preliminary research.

PURPOSE: To improve the procedures used to treat prolapse and hemorrhoids, novel magnetic rings were invented to use in circumferential mucosectomies to avoid the disadvantages of stapling techniques. METHODS: Thirty adult pigs were randomly divided into three groups: Group A (n = 10), which underwent circumferential mucosectomy with novel magnetic rings; Group B (n = 10), which underwent circumferential mucosectomy with conventional magnetic rings and Group C (n = 10), which underwent circumferential mucosectomy with a stapling technique. RESULTS: All pigs underwent the operation successfully, and the mean length of the procedure was similar among the three groups (p > 0.05). There was no bleeding in Group A or Group B, while there was a mean blood loss of 78.32 ± 26.03 ml in Group C (p < 0.01). Three cases of anastomotic stenosis were found in Group C (3/10); two cases were found in Group B (2/10) and no anastomotic stenosis was found in Group A (0/10). The difference between groups A and C was statistically significant (p < 0.05). The cost for the magnetic rings in groups A and B was noticeably lower than that of the stapling techniques in Group C (20.12 ± 3.35 vs. 15.76 ± 2.92 vs. 550.16 ± 29.71 US dollars, p < 0.001). The magnetic rings in groups A and B were spontaneously discharged from the body with the necrotic tissues within 1-2 weeks (8.20 ± 2.73 vs. 9.31 ± 3.62 days, p > 0.05), avoiding the permanent implantation of staples in Group C. The anastomosis site in Group A showed a smoother and more rapid healing process than that in Group B or C. CONCLUSIONS: The innovative magnetic rings we developed for circumferential mucosectomies provide a simple and novel surgical procedure for prolapse and hemorrhoids.

Engineering of corpus cavernosum using vascular endothelial growth factor-expressing muscle-derived stem cells seeded on acellular corporal collagen matrices.

OBJECTIVE: To explore the feasibility of developing vascularized tissue-engineered corpus cavernosum with vascular endothelial growth factor (VEGF)-expressing muscle-derived stem cells (MDSCs) as seed cells in a rabbit model. MATERIALS AND METHODS: MDSCs were isolated and expanded in vitro and transfected with human VEGF(165) lentiviral gene vector. The release of VEGF was confirmed using enzyme-linked immunosorbent assay. Acellular corporal collagen matrices (ACCMs) were obtained from donor rabbit penile tissue using an established decellularization process. Transfected and untransfected MDSCs were seeded on ACCMs. Histochemistry and scanning electron microscopy were performed to analyze the growth and distribution of MDSCs. After constructing animal models, we transplanted the seeded ACCMs to the excised corporal space. The neocorpora was harvested and assayed with Western blot and immunohistochemistry at the first and second month. RESULTS: Enzyme-linked immunosorbent assay indicated that the release of VEGF was significantly increased in the MDSC-VEGF group compared with the other groups. Histochemistry and scanning electron microscopy indicated that VEGF-expressing MDSCs showed better growth and adequate attachment than other groups on the ACCMs in vitro. Immunohistochemical staining showed that the expression of α-smooth muscle actin, von Willebrand factor, and CD31 in the MDSC-VEGF group was markedly increased at different points. The MDSC-VEGF group showed greater improvement on cavernosal contractile function than the other groups. The expression of endothelial nitric oxide synthase in the engineered corporal tissues was significantly greater in the MDSC-VEGF group than in the other groups at different measurement points. CONCLUSION: As seed cells, VEGF-overexpressing MDSCs could greatly increase the density of capillaries and the content of endothelial smooth muscle cells in constructing the engineered corporal tissues than can untransfected MDSCs.

The therapeutic role of VEGF-expressing muscle-derived stem cells in acute penile cavernosal injury.

INTRODUCTION: Traumatic penile injury is one of the urological emergencies. Surgery and conservative management are major treatment methods but are always accompanied by many complications. AIM: To investigate the feasibility of repairing cavernous tissues in acute rabbit penile cavernosal injury model with vascular endothelial growth factor (VEGF)-expressing muscle-derived stem cells (MDSCs). METHODS: MDSCs were isolated and transfected with hVEGF165 lentiviral gene vector in vitro. The expression of VEGF was confirmed using enzyme-linked immunosorbent assay (ELISA), Western blot, and real-time polymerase chain reaction (PCR) analyses. After animal models were constructed, animals were randomly divided into four groups, which were administrated with MDSCs/VEGF, MDSCs/vector, MDSCs, and normal saline, respectively. A month later, magnetic resonance imaging (MRI) and intracavernosal pressures (ICP) were performed on the animals. Then penile tissues were harvested and assayed with Western blot and immunohistochemistry. MAIN OUTCOME MEASURES: Real-time PCR, Western blot, ELISA, immunohistochemistry, MRI, and ICP were performed in our experiments. RESULTS: The expression of VEGF significantly increased in the VEGF-expressing MDSCs group compared with those in the MDSCs/vector and MDSCs groups. VEGF protein expression in the injury sites of cavernous tissues were significantly higher in the MDSCs/VEGF group compared with those in other three groups. Immunohistochemical staining showed that α-smooth muscle actin-positive cells, von Willebrand factor-positive cells and capillary density markedly increased in the MDSCs/VEGF group. Animals receiving MDSCs/VEGF showed a significant improvement in cavernosal contractile function and structural repair. CONCLUSIONS: The transplantation of VEGF-expressing MDSCs could repair the actuely injured cavernous tissue. We believed that it could be a novel therapeutic strategy for acute rabbit penile cavernosal injury.

Anti-fibrotic effects via regulation of transcription factor Sp1 on hepatic stellate cells.

BACKGROUND: Hepatic stellate cells (HSCs), the central cells in hepatic fibrosis, are characterized by sustaining activation, a process that consists in increased proliferation and over-expression of fibrotic genes. Transcription factor Sp1 mediates the expression of a variety of fibrotic genes expression and thereby play an important role in fibrosis. In addition, previous reports have indicated that Sp1 binding activity is greatly increased in activated HSCs. Thus, our aim was to investigate the anti-proliferative and anti-fibrotic effects of the oligonuceotide decoy of the transcription factor Sp1, ODN, a potent inhibitor of Sp1-activated transcription. METHODS: We optimized Lipofectamin 2000 (LF2000):ODN DNA ratio for the transfection of hepatic stellate cells HSC-T6. Then we measure the effect of transfected ODN on HSC-T6 cells' proliferation and fibrotic gene expression, and study the mechanism involved. RESULTS: At a DNA concentration of 1 µM and a ratio ODN DNA:LF2000 of 1:3, HSC-T6 cells have the maximal transfection efficiency with the lowest toxicity. Transfected ODN effectively blocks Sp1 binding to the promoter regions of cell cycle regulatory proteins cyclin D1, p27(KIP1) and fibrotic genes, including transforming growth factor (TGF)-ß1, Platelet-derived growth factor (PDGF)-BB, α-SMA, α1 (I) collagen and tissue inhibitor of metalloproteinases-1 (TIMP-1). ODN inhibits HSC-T6 proliferation and fibrotic genes expression in vitro. CONCLUSION: Sp1 is a key transcription factor that mediates proliferation and fibrotic gene synthesis of HSC-T6, inhibition of Sp1 with decoy ODN may be an effective approach to prevent the progression of hepatic fibrosis.

Inverted u-shaped purse and rotation flaps: correcting the inferoposterior deformity of reconstructed ears after canaloplasty of the external auditory meatus.

BACKGROUND: After patients with congenital microtia receive external ear canal plasty, the mastoid area usually has insufficient space for ear reconstruction. Hence, after ear reconstruction, an inferoposterior position deformity of the ear appears to some extent. Using inverted U-shaped purse and rotation flaps can correct this deformity effectively. METHODS: From May of 2009 to September of 2011, five patients received the described procedures in the authors' department. Inverted U-shaped purse and rotation flaps were used for all the patients. The inverted U-shaped purse flap was used to reduce the area of the canal orifice and to lower the position, and the rotation flap was applied to turn the ear in a more superoposterior position. Two patients also received full-thickness skin grafting to cover the secondary wound. In four patients, V-Y-plasty or Z-plasty was used to adjust the flap transition. RESULTS: For the five patients, the distances between the ear antihelix and canal orifice were shortened, and the areas of the canal orifice were diminished. The retroversion of the auricle was corrected in various degrees, and the angles of the long axis of the auricle and the horizontal line were increased an average of 14.4°. The vertical distance between the top of the helix and the center of the canal orifice was increased an average of 15.2 mm. A slight dog ear deformity in front of the crus of the helix was left after the operation, but it was alleviated in the follow-up period. CONCLUSION: By using inverted U-shaped purse and rotation flaps, the inferoposterior position deformity of the reconstructed ear after external ear canal plasty in congenital microtia can be resolved effectively. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors http://www.springer.com/00266.

[Percentage of uric acid calculus and its metabolic character in Dongjiang River valley].

OBJECTIVE: To study the percentage of uric acid calculus in uroliths and its metabolic character in Dongjiang River valley. METHODS: To analyze the chemical composition of 290 urinary stones by infrared (IR) spectroscopy and study the ratio changes of uric acid calculus. Uric acid calculus patients and healthy people were studied. Personal characteristics, dietary habits were collected. Conditional logistic regression was used for data analysis and studied the dietary risk factors of uric acid calculus. Patients with uric acid calculus, calcium oxalate and those without urinary calculus were undergone metabolic evaluation analysis. The results of uric acid calculus patients compared to another two groups to analysis the relations between the formation of uric acid calculus and metabolism factors. RESULTS: Uric acid calculi were found in 53 cases (18.3%). The multiple logistic regression analysis suggested that low daily water intake, eating more salted and animal food, less vegetable were very closely associated with uric acid calculus. Comparing to calcium oxalate patients, the urine volume, the value of pH, urine calcium, urine oxalic acid were lower, but uric acid was higher than it. The value of pH, urine oxalic acid and citric acid were lower than them, but uric acid and urine calcium were higher than none urinary calculus peoples. Blood potassium and magnesium were lower than them. CONCLUSIONS: The percentage of uric acid stones had obvious advanced. Less daily water intake, eating salted food, eating more animal food, less vegetables and daily orange juice intake, eating sea food are the mainly dietary risk factors to the formation of uric acid calculus. Urine volume, the value of pH, citric acid, urine calcium, urine uric acid and the blood natrium, potassium, magnesium, calcium, uric acid have significant influence to the information of uric acid stones.

[Protective effects of c-jun anti-sense gene recombinant transfection on rat cardiomyocytes inflicted by hypoxia and burn serum].

OBJECTIVE: To investigate the protective effects of c-jun antisense gene recombinant transfection on rat cardiomyocytes inflicted by hypoxia and burn serum. METHODS: Cardiomyocytes from Wistar rat were isolated and cultured before being divided into normal control (C), transfection (T) and non-transfection (NT) groups. C-jun antisense gene recombinant was constructed and transfected into cardiomyocytes, which were then treated by hypoxia and burn serum in T group, while those in NT group were simply treated by hypoxia and burn serum. The changes in the c-jun mRNA expression were determined by RT-PCR at 1, 3 and 7 hours after the cardiomyocytes being stimulated, and the changes in the expressions of c-jun protein, troponin-T (TnT) and beta-tubulin were assayed by Western blot. The morphological changes in the cardiomyocytes were observed by LM and EM. RESULTS: 1) The expressions of c-jun mRNA and protein in the NT group were increased evidently when compared with those in C and T groups. 2) The expressions of TnT and beta-tubulin in NT group were decreased evidently in contrast to those in C and T groups. In addition, there exhibited evident structural derangement, dissolution and fragmentation to granules of cardiomyocytes in NT group, while the myocardial cytoskeletal structure was well preserved with scarce fragmentation and dissolution in T group. CONCLUSION: Increased expression of c-jun in rat cardiomyocytes resulting in myocardial injury could be induced by combined treatment of hypoxia and burn serum, while c-jun antisense gene recombinant transfection might protect rat cardiomyocytes from injury.