Loss-of-function CFTR p.G970D missense mutation might cause congenital bilateral absence of the vas deferens and be associated with impaired spermatogenesis / 亚洲男科学杂志(英文版)

Congenital bilateral absence of the vas deferens (CBAVD) is observed in 1%-2% of males presenting with infertility and is clearly associated with cystic fibrosis transmembrane conductance regulator (CFTR) mutations. CFTR is one of the most well-known genes related to male fertility. The frequency of CFTR mutations or impaired CFTR expression is increased in men with nonobstructive azoospermia (NOA). CFTR mutations are highly polymorphic and have established ethnic specificity. Compared with F508Del in Caucasians, the p.G970D mutation is reported to be the most frequent CFTR mutation in Chinese patients with cystic fibrosis. However, whether p.G970D participates in male infertility remains unknown. Herein, a loss-of-function CFTR p.G970D missense mutation was identified in a patient with CBAVD and NOA. Subsequent retrospective analysis of 122 Chinese patients with CBAVD showed that the mutation is a common pathogenic mutation (4.1%, 5/122), excluding polymorphic sites. Furthermore, we generated model cell lines derived from mouse testes harboring the homozygous Cftr p.G965D mutation equivalent to the CFTR variant in patients. The Cftr p.G965D mutation may be lethal in spermatogonial stem cells and spermatogonia and affect the proliferation of spermatocytes and Sertoli cells. In spermatocyte GC-2(spd)ts (GC2) Cftr p.G965D cells, RNA splicing variants were detected and CFTR expression decreased, which may contribute to the phenotypes associated with impaired spermatogenesis. Thus, this study indicated that the CFTR p.G970D missense mutation might be a pathogenic mutation for CBAVD in Chinese males and associated with impaired spermatogenesis by affecting the proliferation of germ cells.

Interaction between glycogen synthase kinase-3β and endoplasmic reticulum stress is involved in high glucose-induced injury in human umbilical vein endothelial cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the role of the interaction between glycogen synthase kinase-3β (GSK-3β) and endoplasmic reticulum stress (ERS) in the high glucose (HG)-induced injury in human umbilical vein endothelial cells (HUVECs).</p><p><b>METHODS</b>HUVECs treated with 40 mmol/L glucose for 24 h were examined for expression levels of GSK-3β, GRP78, CHOP and cleaved caspase-3 protein using Western blotting. The cell viability was examined using CCK-8 assay and cell apoptosis was detected with Hoechst 33258 nuclear staining and photofluorography. The intracellular level of reactive oxygen species (ROS) was measured with dichlorfluoresein staining and photofluorography. Mitochondrial membrane potential (MMP) was tested by rhodamine 123 (Rh123) staining and photofluorography.</p><p><b>RESULTS</b>Treatment of HUVECs with 40 µmol/L glucose for 3-24 h activated GSK-3β in a time-dependent manner, leading to significantly down-regulated expression of phosphorylated (p)-GSK-3β (P<0.05). HG exposure of the cells for 1-24 h induced ERS, evidenced by time-dependently up-regulated expression of GRP78 and CHOP (P<0.05). LiCl, an inhibitor of GSK-3β, attenuated HG-induced ERS and significantly lowered the expression levels of GRP78 and CHOP (P<0.01). 4-PBA, an inhibitor of ERS, obviously ameliorated the activation of GSK-3β by HG as shown by the increase in p-GSK-3β expression level (P<0.01). HG exposure for 24 h induced obvious injuries in HUVECs, which exhibited decreased cell viability, increased cell apoptosis, increased expression of cleaved caspase-3 and ROS generation, and loss of MMP. Pretreatment of the cells with LiCl or 4-PBA for 60 min before HG exposure significantly lessened the cell injuries (P<0.01).</p><p><b>CONCLUSION</b>Interactions between GSK-3β and ERS occur in HUVECs exposed to HG and participate in HG-induced cell injuries.</p>

Clinical study of western medicine combined with Chinese medicine based on syndrome differentiation in the patients with polarized hypertension / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effects and safety of Western medicine combined with Chinese medicine (CM) based on syndrome differentiation in the treatment of elderly polarized hypertension (PHPT), or isolated systolic hypertension with low diastolic blood pressure (DBP).</p><p><b>METHODS</b>A total of 125 elderly patients with PHPT were randomly assigned to two groups: 59 in the control group treated by Western medicine and 66 in the intervention group treated by Western medicine combined with CM treatment. Based on syndrome differentiation, the patients in the intervention group were further divided into subgroups of yang-qi deficiency and yin-qi deficiency. All subjects were treated with Western medicine of Amlodipine Besylate Tablets and Irbesartan Tablets (or Irbesartan and Hydrochlorothiazide Tablets), to decrease their systolic blood pressure (SBP) slowly to 125-135 mm Hg in 2-6 weeks. In the intervention group, Shiyiwei Shenqi Capsule was given additionally to the subgroup of yang-qi deficiency at the dosage of 3-5 capsules, thrice a day, while Dengzhan Shengmai Capsule was given additionally to the subgroup of yin-qi deficiency at the dosage of 2 capsules, 2-3 times per day. For all subjects, SBP, pulse pressure (PP), and DBP were measured before treatment and at the terminal of a 6-week treatment. For subjects in the intervention group, left ventricular ejection fraction (LVEF) was also recorded.</p><p><b>RESULTS</b>After a 6-week treatment, the SBP in the two groups and the PP in the intervention group decreased significantly compared to those before treatment (P<0.05), while the PP in the control group showed no significant difference between prior and post-treatment (P>0.05). After treatment, the DBP in the control group decreased (P>0.05), while the DBP and LVEF in the intervention group showed an increase tendency although it had no statistical significance (P>0.05). When subjects in the intervention group were classified further by the course of disease, the DBP and LVEF of subjects whose course of disease were less than 2 years, increased significantly after treatment (P<0.05).</p><p><b>CONCLUSION</b>Western medicine combined with CM treatment based on syndrome differentiation was safer and more effective than Western medicine alone in the treatment of elderly PHPT, because it not only reduced SBP but also improved DBP, which might lower the incidence of the cardiovascular and cerebrovascular events.</p>

NYD-SP27, a novel intrinsic decapacitation factor in sperm / 亚洲男科学杂志(英文版)

Prior to fertilization sperm has to undergo an activation process known as capaciation, leading to the acrosome reaction. Till now, little is known about the mechanism for preventing premature capacitation in sperm although decapacitation factors from various sources have been thought to be involved. In this study, we report that NYD-SP27, an isoform of phospholipase C Zeta 1 (PLCZ1), is localized to the sperm acrosome in mouse and human spermatozoa by immunofluorescence using a specific antibody. Western blot and double staining analyses show NYD-SP27 becomes detached from sperm, as they undergo capacitation and acrosome reaction. The absence of HCO3-, a key factor in activating capacitation, from the capacitation-inducing medium prevents the loss of NYD-SP27 from sperm. The anti-NYD-SP27 antibody also prevents the loss of NYD-SP27 from sperm, reduced the number of capacitated sperm, inhibited the acrosome reaction induced by ATP and progesterone, and inhibited agonist-induced PLC-coupled Ca2+ mobilization in sperm, which can be mimicked by the PLC inhibitor, U73122. These data strongly suggest that NYD-SP27 is a physiological inhibitor of PLC that acts as an intrinsic decapacitation factor in sperm to prevent premature capacitation and acrosome reaction.

Cloning, expression, and alternative splicing of the novel isoform of hTCP11 gene / 中国医学科学院学报

<p><b>OBJECTIVE</b>To identify a novel isoform of hTCP11 gene and investigate its expression and alternative splicing.</p><p><b>METHODS</b>According to the sequence of human ESTs which are highly homologous to hTCP11a, primers for PCR were synthesized. Then, the amplified fragments were cloned and sequenced; some methods including BLAST, ClustalW and RT-PCR were used for genomic analysis, study of alternative splicing and gene expression among multiple tissues and different testis tissues.</p><p><b>RESULTS</b>A novel isoform of hTCP11 gene was isolated. It encodes a 440 amino acid protein that is highly homologous to the mouse 566 amino acid protein which is important to sperm function because it encodes the receptor for fertilization promoting peptide (FPP). Among TCP11a, TCP11b and TCP11c, the complicated alternative splicing was found. RT-PCR analysis of RNA extracted from human tissues revealed that the gene is only expressed in fertile adult testes, but not in azoospermic patient testes, fetal testes or other human tissues.</p><p><b>CONCLUSION</b>Our results along with the mouse Tcp-11 function suggest that the isoforms of TCP11 gene play important roles in sperm function and fertility.</p>