Loss of PMFBP1 Disturbs Mouse Spermatogenesis by Downregulating HDAC3 Expression.

PURPOSE: Polyamine modulating factor 1 binding protein (PMFBP1) acts as a scaffold protein for the maintenance of sperm structure. The aim of this study was further to identify the new role and molecular mechanism of PMFBP1 during mouse spermatogenesis. METHODS AND RESULTS: We identified a profile of proteins interacting with PMFBP1 by immunoprecipitation combined with mass spectrometry and demonstrated that class I histone deacetylases, particularly HDAC3 and chaperonin-containing TCP1 subunit 3 (CCT3), were potential interaction partners of PMFBP1 based on network analysis of protein-protein interactions and co-immunoprecipitation. Immunoblotting and immunochemistry assays showed that loss of Pmfbp1 would result in a decline in HDACs and change the proteomic profile of mouse testis, in which differently expressed proteins are associated with spermatogenesis and assembly of flagella, which was proved by proteomic analysis of testis tissue obtained from Pmfbp1-/- mice. After integrating with transcriptome data for Hdac3-/- and Sox30-/- round sperm obtained from a public database, RT-qPCR confirmed ring finger protein 151 (Rnf151) and ring finger protein 133 (Rnf133) were key downstream response factors of the Pmfbp1-Hdac axis affecting mouse spermatogenesis. CONCLUSION: Taken together, this study indicates a previously unidentified molecular mechanism of PMFBP1 in spermatogenesis whereby PMFBP1 interacts with CCT3, affecting the expression of HDAC3, followed by the downregulation of RNF151 and RNF133, resulting in an abnormal phenotype of sperm beyond the headless sperm tails. These findings not only advance our understanding of the function of Pmfbp1 in mouse spermatogenesis but also provide a typical case for multi-omics analysis used in the functional annotation of specific genes.

A novel homozygous missense mutation in AK7 causes multiple morphological anomalies of the flagella and oligoasthenoteratozoospermia.

PURPOSE: To identify the genetic causes of multiple morphological anomalies of the flagella (MMAF) and oligoasthenoteratozoospermia (OAT). METHODS: Whole-exome sequencing (WES) was performed on the proband to identify pathogenic mutation for infertility. Western blotting and immunofluorescence analysis detected the expression level and localization of adenylate kinase 7 (AK7). RESULTS: We identified a novel homozygous missense mutation (NM_152327: c.1846G > A; p.E616K) in AK7 in two brothers with MMAF and OAT from a consanguineous family by WES. Western blotting and immunofluorescence experiments determined that the expression level of AK7 decreased in the sperm from the proband. The proband and his wife underwent two cycles of intracytoplasmic sperm injection (ICSI) treatment but got unfavorable outcomes. CONCLUSION: This study could provide precise genetic diagnosis for the patient and expand the spectrum of AK7 mutations.

A homozygous missense mutation in TBPL2 is associated with oocyte maturation arrest and degeneration.

The genetic causes in most of patients with oocyte maturation arrest remain largely unknown. In this study, we identified a homozygous missense mutation (c.895T>C; p.C299R) in TBPL2 (TATA box binding protein like 2) in two infertile sisters with oocyte maturation arrest and degeneration from a consanguineous family by whole-exome sequencing. The TBPL2 mutation is rare and pathogenic, and impaired the transcription initiation function of the protein. Our results showed that TBPL2 mutation might be associated with female infertility due to oocyte maturation arrest and degeneration.

A novel homozygous missense mutation of PMFBP1 causes acephalic spermatozoa syndrome.

PURPOSE: To identify the pathogenic mutation in PMFBP1 leading to acephalic spermatozoa syndrome. METHODS: Sanger sequencing was used to screen for mutations in the known pathogenic genes SUN5 and PMFBP1 in a patient with acephalic spermatozoa syndrome. Western blotting and immunofluorescence were used to detect the expression and localization of PMFBP1 in sperm. At the same time, a PMFBP1 mutant was constructed, and the expression level of PMFBP1 protein was further verified by in vitro experiments. RESULTS: We identified a novel homozygous PMFBP1 missense mutation, c.301A>C (p.T101P), in an infertile male from a consanguineous family. Our results showed that the expression of PMFBP1 mutant protein was decreased obviously in sperm of the patient. CONCLUSION: Our results showed that the novel homozygous missense mutation of PMFBP1 may be a cause of acephalic spermatozoa syndrome, which provided a basis for genetic counseling for the patient.

Pathogenesis of acephalic spermatozoa syndrome caused by splicing mutation and de novo deletion in TSGA10.

PURPOSE: To identify the genetic causes for acephalic spermatozoa syndrome. METHODS: Whole-exome sequencing was performed on the proband from a non-consanguineous to identify pathogenic mutations for acephalic spermatozoa syndrome. Quantitative real-time polymerase chain reaction and whole genome sequencing were subjected to detect deletion. The functional effect of the identified splicing mutation was investigated by minigene assay. Western blot and immunofluorescence were performed to detect the expression level and localization of mutant TSGA10 protein. RESULTS: Here, we identified a novel heterozygous splicing mutation in TSGA10 (NM_025244: c.1108-1G > T), while we confirmed that there was a de novo large deletion in the proband. The splicing mutation led to the skipping of the exon15 of TSGA10, which resulted in a truncated protein (p. A370Efs*293). Therefore, we speculated that the splicing mutation might affect transcription and translation without the dosage compensation of a normal allele, which possesses a large deletion including intact TSGA10. Western blot and immunofluorescence demonstrated that the very low expression level of truncated TSGA10 protein led the proband to present the acephalic spermatozoa phenotype. CONCLUSION: Our finding expands the spectrum of pathogenic TSGA10 mutations that are responsible for ASS and male infertility. It is also important to remind us of paying attention to the compound heterozygous deletion in patients from non-consanguineous families, so that we can provide more precise genetic counseling for patients.

Unraveling the Impact of the PROCA1 Mutation in Male Infertility: Incorporating Whole Exome Sequencing in Teratozoospermia Patients and Analyzing Proca1 Knockout Mice.

In the world, about 15% of couples are infertile, and nearly half of all infertility was caused by men. A large number of genetic mutations are thought to affect spermatogenesis by regulating acrosome formation. Here, we identified three patients harbouring the protein interacting with cyclin A1 (PROCA1) mutation by whole exome sequencing (WES) and Sanger sequencing among patients with predominantly acrosome-deficient teratozoospermia. However, the expression and roles of PROCA1 in infertile men remain unclear. We found that PROCA1 is predominantly expressed in the testis, where it is specifically localized to the acrosome of normal human sperm. Proca1 knockout (KO) mice were subsequently generated using CRISPR-Cas9 technology. However, Proca1 KO adult male mice were fertile, with testis-to-body weight ratios comparable to those of wild-type (WT) mice. Testicular tissue or sperm morphology were not significantly different in Proca1 KO mice compared to WT mice. Expression of the acrosome markers PNA and SP56 in the acrosome was comparable between Proca1 KO and WT mice. In summary, these findings suggested that the PROCA1 mutation identified in humans does not affect acrosome biogenesis in mice.

[Clinicopathological features and multivariate analysis of prognostic factors for patients with gastroenteropancreatic neuroendocrine tumors].

OBJECTIVE: To analyze the clinicopathologic characteristics of gastroenteropancreatic neuroendocrine neoplasm(GEP-NET) and explore the prognostic factors for patients with GEP-NET. METHODS: Retrospectively reviews were conducted for the charts of 68 patients diagnosed pathologically with GEP-NET and treated at Sichuan Cancer Hospital during January 2001 to June 2012. The information of prognostic factors was retrieved and analyzed. Kaplan-Meier method was used to estimate survival rates and plot patient survival curves of patients at different levels of predictive factors. The association between clinicopathologic characteristic and prognosis in GEP-NET patients was assessed with Log-rank test. Meanwhile Cox proportional hazard model was used to select independent risk factors of patient survival. RESULTS: Stomach (20/68,29.41%) and cardia (16/68,23.53%) were mostly involved. Frequent tumor sites for males were stomach and cardia (34/52,65.38%) while the most common site was intestinal canal for female (12/16) . Ages of disease onset were different significantly among patient groups of different sites (P = 0.023). The average age of intestinal NET was the highest while gastric NET had the lowest. During a median follow-up duration of 49 (3-120)months, there were 37 deaths (54.41%), including 30 from postoperative relapses. Postoperative survival time ranged from 4-115 months. The mean survival periods were (46 ± 7) months respectively. The 1, 3, and 5-year survival rates were 65.1%, 41.5% and 28.7% respectively. Univariate analyses showed that the risk factors of survival time were patient age over 60 years, tumor size > 2 cm, T2-4 stage of tumor, vascular invasion, lymph node metastasis, distant metastasis, positive surgical margin and pathological grades of neuroendocrine carcinoma (NEC) and mixed adenoneuroendocrine carcinoma (MANEC). Multivariate analysis indicated independent risk factors were tumor size > 2 cm and pathological grades of NEC and MANEC. CONCLUSIONS: GEP-NET may occur at multiple sites of digestive system and lack specific clinical manifestations. Tumor size, distant metastasis and pathological grades were independent prognostic factors for GEP-NET patients.

Genetic pathogenesis of acephalic spermatozoa syndrome: past, present, and future.

Acephalic spermatozoa syndrome (ASS) is one of the most severe spermatogenic failures of all infertility in men. The cognition of ASS has experienced a tortuous process. Over the past years, with the in-depth understanding of spermatogenesis and the emergence of new genetic research technologies, the unraveling of the genetic causes of spermatogenic failure has become highly active. From these advances, we established a genetic background and made significant progress in the discovery of the genetic causes of ASS. It is important to identify pathogenic genes and mutations in ASS to determine the biological reasons for the occurrence of the disease as well as provide genetic diagnosis and treatment strategies for patients with this syndrome. In this review, we enumerate various technological developments, which have made a positive contribution to the discovery of candidate genes for ASS from the past to the present. Simultaneously, we summarize the known genetic etiology of this phenotype and the clinical outcomes of treatments in the present. Furthermore, we propose perspectives for further study and application of genetic diagnosis and assisted reproductive treatment in the future.

Novel Mutation and Deletion in SUN5 Cause Male Infertility with Acephalic Spermatozoa Syndrome.

Acephalic spermatozoa syndrome (ASS) is a severe form of teratozoospermia, previous studies have shown that SUN5 mutations are the major cause of acephalic spermatozoa syndrome. This study is to identify the pathogenic mutations in SUN5 leading to ASS. PCR and Sanger sequence were performed to define the breakpoints and mutations in SUN5. Whole genome sequencing (WGS) was performed to detect heterozygous deletion. Western blotting and immunofluorescence analysis detected the expression level and localization of SUN5. Furthermore, the pathogenicity of the mutant SUN5 was predicted in silico and was verified by the experiments in vitro. We identified one novel homozygous missense mutation (c.775G>A; p.G259S) and one compound heterozygous including one reported missense mutation (c.1043A>T; p.N348I) and a large deletion that contains partial EFCAB8 ( NM_001143967 .1) and BPIFB2 ( NM_025227 ) and complete SUN5 ( NM_080675 ), and one recurrent homozygous splice-site mutation (c.340G>A; p.G114R) in SUN5 in three patients with ASS. Our results showed that SUN5 could not be detected in the patients' spermatozoa and the exogenous expression level of the mutant protein was decreased in transfected HEK-293T cells. This study expands the mutational spectrum of SUN5. We recommended a clinical diagnostic strategy for SUN5 genomic deletion to screen heterozygous deletions and indicated that the diagnostic value of screening for SUN5 mutations and deletions in infertile men with ASS.

Analysis of death causes of in-patients with malignant tumors in Sichuan Cancer Hospital of China from 2002 to 2012.

OBJECTIVES: To analyze underlying disease, fatality rate and the major causes of death of in-patients with malignant tumors in Sichuan Cancer Hospital. METHODS: Clinical data of in-patients from 2002 to 2012 were retrospectively analyzed. RESULTS: The top 10 tumors (82.0%of the total) of the malignant tumors of the in- patients were lung, cervical, esophagus, breast, colorectal, nasopharynx, liver and gastric cancers, lymphomas and ovarian cancers. The overall fatality rate was 2.7% during these eleven years, 3.4% and 2.0% for male and females, respectively with statistical significance for the difference (χ2=164.737, P<0.001). The top 10 death causes were lung cancer, liver cancer, colorectal cancer, esophagus cancer, gastric cancer, lymphoma, breast cancer, pancreatic cancer, ovarian cancer and nasopharynx cancer. In-patients with pancreatic cancer had the highest fatality rate (9.6%). There were different ranks of death causes in different sex groups and age groups. CONCLUSION: Prevention and control work of cancer should be enhanced not only for cancers with high incidence such as lung cancer, esophageal cancer but also for the cancers which have low incidence but high fatality rate, such as pancreatic cancer and gallbladder cancer, which would help to improve the survival rate and quality of life of cancer patients in the future.