Qing-Jun Chu

Background: Sertoli cell-only syndrome (SCOS) is the most serious pathological type of non-obstructive azoospermia. Recently, several genes related to SCOS have been identified, including FANCM, TEX14, NR5A1, NANOS2, PLK4, WNK3, and FANCA, but they cannot fully explain the pathogenesis of SCOS. This study attempted to explain spermatogenesis dysfunction in SCOS through testicular tissue RNA sequencing and to provide new targets for SCOS diagnosis and therapy. Methods: We analyzed differentially expressed genes (DEGs) based on RNA sequencing of nine patients with SCOS and three patients with obstructive azoospermia and normal spermatogenesis. We further explored the identified genes using ELISA and immunohistochemistry. Results: In total, 9406 DEGs were expressed (Log2|FC|≥ 1; adjusted P value < 0.05) in SCOS samples, and 21 hub genes were identified. Three upregulated core genes were found, including CASP4, CASP1, and PLA2G4A. Thus, we hypothesized that testis cell pyroptosis mediated by CASP1 and CASP4 might be involved in SCOS occurrence and development. ELISA verified that CASP1 and CASP4 activities in the testes of patients with SCOS were significantly higher than those in patients with normal spermatogenesis. Immunohistochemical results showed that CASP1 and CASP4 in the normal spermatogenesis group were mainly expressed in the nuclei of spermatogenic, Sertoli, and interstitial cells. CASP1 and CASP4 in the SCOS group were mainly expressed in the nuclei of Sertoli and interstitial cells because of the loss of spermatogonia and spermatocytes. CASP1 and CASP4 expression levels in the testes of patients with SCOS were significantly higher than those in patients with normal spermatogenisis. Furthermore, the pyroptosis-related proteins GSDMD and GSDME in the testes of patients with SCOS were also significantly higher than those in control patients. ELISA also showed that inflammatory factors (IL-1 β, IL-18, LDH, and ROS) were significantly increased in the SCOS group. Conclusions: For the first time, we found that cell pyroptosis-related genes and key markers were significantly increased in the testes of patients with SCOS. We also observed many inflammatory and oxidative stress reactions in SCOS. Thus, we propose that testis cell pyroptosis mediated by CASP1 and CASP4 could participate in SCOS occurrence and development.

Background and aims: Sertoli cell-only syndrome (SCOS) is the most serious pathological type of non-obstructive azoospermia. In recent years, some genes related to SCOS have been identified, including FANCM, TEX14, NR5A1, NANOS2, PLK4, WNK3, and FANCA, but they cannot explain the pathogenesis of SCOS completely. This study attempted to explain the spermatogenesis dysfunction in SCOS through testicular tissue RNA sequencing and provide new targets for SCOS diagnosis and therapy. Methods and results: We analyzed differentially expressed genes (DEGs) based on RNA sequencing of 9 SCOS patients and 3 obstructive azoospermia patients with normal spermatogenesis. A total of 9406 DEGs were expressed (Log2|FC| ≥ 1 and adjusted P value < 0.05) in SCOS patients, and 21 hub genes were identified among the DEGs. Three upregulated core genes were found in our study including CASP4, CASP1, and PLA2G4A. Thus, we hypothesized that testis cell pyroptosis mediated by CASP1 and CASP4 might be involved in SCOS occurrence and development. We verified that CASP1 and CASP4 activities in the testes of patients with SCOS were significantly higher than those in patients with normal spermatogenesis using ELISA. Immunohistochemical results showed that CASP1 and CASP4 in the normal spermatogenesis group were mainly expressed in the nuclei of spermatogenic cells, Sertoli cells, and interstitial cells. CASP1 and CASP4 in the SCOS group were mainly expressed in the nuclei of Sertoli cells and interstitial cells because of the loss of spermatogonia and spermatocytes. At the same time, CASP1 and CASP4 expression levels in the testes of patients with SCOS were significantly higher than those in normal spermatogenic patients. Furthermore, the pyroptosis-related proteins GSDMD and GSDME in the testes of patients with SCOS were also significantly higher than those in patients with normal spermatogenesis. ELISA also showed that inflammatory factors IL-1 β, IL-18, LDH, and ROS were significantly increased in the testes of SCOS patients. Conclusion: For the first time, we found that cell pyroptosis-related genes and key markers were significantly increased in the testes of patients with SCOS, and we observed a large number of inflammatory and oxidative stress reactions in SCOS. Thus, we considered that testis cell pyroptosis mediated by CASP1 and CASP4 could participate in SCOS occurrence and development.

Background. Nonobstructive azoospermia (NOA) is a complex disease characterized by the spermatogenic dysfunction of testicular tissues. The roles played by long noncoding RNAs (lncRNAs) in NOA pathogenesis have not been extensively studied. Methods. Microarray assays were performed on samples of testicular biopsy tissue obtained from patients with NOA for the purpose of identifying differentially expressed lncRNAs and messenger RNA (mRNA) transcripts, and the results were verified by quantitative real‐time polymerase chain reaction. Mouse‐derived GC‐1 spermatogonia (spg) cells undergoing treatment with Adriamycin (ADR) were used to investigate the biological functions of the selected lncRNAs in vitro. The target microRNAs (miRNAs) of lncRNAs and the target mRNAs of miRNAs were predicted by a bioinformatics analysis. Functional studies performed using the CCK‐8 assay, EdU incorporation assay, apoptosis detection, and senescence‐associated β‐galactosidase (SA‐β‐Gal) staining were conducted using GC‐1 spg cells. Results. Totals of 2,652 lncRNAs and 2,625 mRNAs were found to be differentially expressed in the testicular tissue of NOA patients when compared with patients in a control group. Dynamin 3 opposite strand (DNM3OS) was a provider of pe‐miR‐214‐5p that positively regulates miR‐214‐5p expression in GC‐1 spg cells. The E2 factor (E2F) family of transcription factor 2 (E2F2) was initially predicted and subsequently verified to be a downstream gene of miR‐214‐5p. E2F2 expression was upregulated after DNM3OS knockdown in ADR‐treated GC‐1 spg cells. Moreover, knockdown of either DNM3OS or miR‐214‐5p significantly alleviated ADR‐induced decreases in cellular activity and proliferation, as well as increases in apoptosis and senescence of mouse spermatogonial GC‐1 spg cells. Conclusions. DNM3OS was found to regulate the apoptosis and senescence of spermatogonia by providing miR‐214‐5p and decreasing E2F2 expression, suggesting it as a novel target for gene therapy of male infertility.

To explore a new method of in vitro culture and purification of rat corpus cavernosum endothelial cells (CCECs). Male Sprague-Dawley rats' penile tissue were digested with elastase or collagenase combined with mechanical extrusion to isolate and culture the CCECs. The fixed-point digestion method was used to purify the primary cells. High-purity CCECs were successfully isolated. Following the digestion of the primary CCECs by elastase or collagenase coupled with mechanical extrusion, the cells were paving stone- and cobblestone-shaped over 10 days. The cell purity yielded in the second generation (P2) CCECs after using the fixed-point digestion method was significantly high. Compared with primary CCECs extracted by elastase digestion combined with the mechanical extrusion method, CCECs cultured by collagenase digestion yielded higher purity and a more stable morphology after fixed-point digestion and purification. Immunofluorescence staining of the third generation CCECs and the expression results of endothelial cell-associated marker antibodies CD31 and VWF were positive, and flow cytometry showed the purity of CCECs was 96.9%. Enzymatic digestion combined with mechanical extrusion and fixed-point digestion is a simple, economical method for in vitro culture and purification of CCECs, which is conducive to studying the pathophysiological mechanisms of endothelial dysfunction and erectile dysfunction.

At present, infertile patients with maturation arrest (MA) are difficult to obtain mature sperm. Spermatogenesis and its molecular mechanism are still not clear. Patients with MA and normal spermatogenesis (NS) were collected. iTRAQ-based proteomic approach was performed to reveal the different proteins between them. To validate the confidence of proteome data, the individual samples were analyzed by Western blotting (WB), quantitative polymerase chain reaction (qPCR), and immunofluorescence. The miR-449a and CEP55 were determined by Luciferase assay. Mouse GC-1 cells were transfected with CEP55 siRNAs, miR-449a mimic, or inhibitor, and cell proliferation was determined. Compared with NS, 27 proteins were differentially expressed in MA, and CEP55 protein was the most significant difference. WB and qPCR showed that CEP55 levels were significantly elevated in NS than MA. In transfected cells, overexpression of miR-449a and knockdown of CEP55 both downregulated CEP55 expression and decreased cell proliferation. miR-449a suppresses mouse spermatogonia proliferation via inhibition of CEP55.

Scrotal hemorrhage after testicular sperm aspiration (TESA) is uncommon in clinical operation. Phosphodiesterase-5 inhibitors (PDE5i) are commonly given to men who have difficulty providing a sperm sample for assisted reproductive technique such as in vitro fertilization. In this study, we examine the incidence of scrotal hemorrhage after TESA in men who received a PDE5i. In this retrospective study, 504 men with TESA operation in Center for Reproductive Medicine, Nanfang Hospital, Southern Medical University were collected. Men in the drug group had taken orally PDE5i before TESA. Men in the control group only operated TESA. The testis volume, coagulation function were measured. Sonographic examination with Doppler imaging was performed when scrotal hemorrhage appeared. A total of 504 men with a mean age of 28.63 ± 4.22 years were included in the analysis. Of these, 428 did not receive a PDE5i prior to TESA and 76 received a PDE5i prior to TESA. Measures of coagulation function were not different between the groups. The incidence of hemorrhage was 0.0% in the control group and the drug group was 5.3%. The incidence of hemorrhage between two groups was different significantly (P = 0.000). In summary, the results of this study suggest that a PDE5i administration increases the risk of scrotal hemorrhage in men undergoing TESA, although the study design does not allow drawing a conclusion of cause and effect. Given the potential risk of scrotal hemorrhage after the ingestion of PDE5i, it may be wise not to administer it to men in whom a TESA may be performed.

In previous studies, it has been shown that the granulocyte macrophage‐colony stimulating factor (GM‐CSF) or interleukin‐2 (IL‐2) surface modified MB49 bladder cancer stem cells (MCSCs) vaccine could induce a specific antitumor immunity and against bladder cancer in mice model respectively. However, whether combined administration of GM‐CSF and IL‐2 could produce specific immune responses to cancer stem cells (CSCs) was uncertain. MCSCs were established and characterized. GM‐CSF and IL‐2 MCSCs vaccines were prepared and bioactivity was evaluated. The therapeutic, protective, specific, and memorial immune response animal experiments were designed. Tumor‐specific cytotoxic T lymphocytes assay, enzyme linked immunosorbent assay, flow cytometry assay were performed to indentify whether vaccine caused an antitumor immunity. Streptavidin (SA)‐GM‐CSF and SA‐IL‐2 MCSCs vaccines were prepared successfully. Such vaccines inhibited the volume of tumor and prolonged the survival of the mice in animal experiments. The express of IgG or IFN‐c, the portion of dendritic cells, CD8⁺ and CD4⁺ T cells were highest in the combined vaccines group than the SA‐GM‐CSF vaccine group, the SA‐IL‐2 vaccine group, the MCSCs group and the PBS group. The combined of GM‐CSF and IL‐2 vaccines could induce better antitumor immunity than a vaccine alone.