Primary ciliary dyskinesia (PCD) is a rare hereditary orphan condition that results in variable phenotypes, including infertility. About 50 gene variants are reported in the scientific literature to cause PCD, and among them, dynein axonemal assembly factor 4 ( DNAAF4 ) has been recently reported. DNAAF4 has been implicated in the preassembly of a multiunit dynein protein essential for the normal function of locomotory cilia as well as flagella. In the current study, a single patient belonging to a Chinese family was recruited, having been diagnosed with PCD and asthenoteratozoospermia. The affected individual was a 32-year-old male from a nonconsanguineous family. He also had abnormal spine structure and spinal cord bends at angles diagnosed with scoliosis. Medical reports, laboratory results, and imaging data were investigated. Whole-exome sequencing, Sanger sequencing, immunofluorescence analysis, hematoxylin-eosin staining, and in silico functional analysis, including protein modeling and docking studies, were used. The results identified DNAAF4 disease-related variants and confirmed their pathogenicity. Genetic analysis through whole-exome sequencing identified two pathogenic biallelic variants in the affected individual. The identified variants were a hemizygous splice site c.784-1G>A and heterozygous 20.1 Kb deletion at the DNAAF4 locus, resulting in a truncated and functionless DNAAF4 protein. Immunofluorescence analysis indicated that the inner dynein arm was not present in the sperm flagellum, and sperm morphological analysis revealed small sperm with twisted and curved flagella or lacking flagella. The current study found novel biallelic variants causing PCD and asthenoteratozoospermia, extending the range of DNAAF4 pathogenic variants in PCD and associated with the etiology of asthenoteratozoospermia. These findings will improve our understanding of the etiology of PCD.
Adult ; Humans ; Male ; Asthenozoospermia/genetics* ; Dyneins/genetics* ; East Asian People ; Kartagener Syndrome/genetics* ; Mutation ; Proteins/genetics* ; Semen/metabolism*

Asthenoteratozoospermia is one of the most severe types of qualitative sperm defects. Most cases are due to mutations in genes encoding the components of sperm flagella, which have an ultrastructure similar to that of motile cilia. Coiled-coil domain containing 103 (CCDC103) is an outer dynein arm assembly factor, and pathogenic variants of CCDC103 cause primary ciliary dyskinesia (PCD). However, whether CCDC103 pathogenic variants cause severe asthenoteratozoospermia has yet to be determined. Whole-exome sequencing (WES) was performed for two individuals with nonsyndromic asthenoteratozoospermia in a consanguineous family. A homozygous CCDC103 variant segregating recessively with an infertility phenotype was identified (ENST00000035776.2, c.461A>C, p.His154Pro). CCDC103 p.His154Pro was previously reported as a high prevalence mutation causing PCD, though the reproductive phenotype of these PCD individuals is unknown. Transmission electron microscopy (TEM) of affected individuals' spermatozoa showed that the mid-piece was severely damaged with disorganized dynein arms, similar to the abnormal ultrastructure of respiratory ciliary of PCD individuals with the same mutation. Thus, our findings expand the phenotype spectrum of CCDC103 p.His154Pro as a novel pathogenic gene for nonsyndromic asthenospermia.
Asthenozoospermia/pathology* ; Dyneins/genetics* ; Homozygote ; Humans ; Male ; Microtubule-Associated Proteins ; Mutation ; Mutation, Missense ; Sperm Tail/metabolism*

Idiopathic asthenozoospermia, a common factor in male infertility, is characterized by altered sperm motility function in fresh ejaculate. Although the β-defensin 126 (DEFB126) protein is associated with asthenozoospermia, DEFB126 gene polymorphisms have not been extensively studied. Therefore, the association between DEFB126 gene polymorphisms and asthenozoospermia requires further investigation. Screening was performed by semen analysis, karyotype analysis, and Y microdeletion detection, and 102 fertile men and 106 men with asthenozoospermia in Chengdu, China, were selected for DEFB126 gene sequence analyses. Seven nucleotide mutations and two nucleotide deletions in the DEFB126 gene were detected. rs11467417 (317-318 del/del), rs11467497 (163-166 wt/del), c.152T>C, and c.227A>G were significantly different between the control and asthenozoospermia groups, likely representing high-risk genetic factors for asthenozoospermia among males. DEFB126 expression was not observed in sperm with rs11467497 homozygous deletion and was unstable in sperm with rs11467417 homozygous deletion. The rs11467497 four-nucleotide deletion leads to truncation of DEFB126 at the carboxy-terminus, and the rs11467417 binucleotide deletion produces a non-stop messenger RNA (mRNA). The above deletions may be responsible for male hypofertility and infertility by reducing DEFB126 affinity to sperm surfaces. Based on in silico analysis, the amino acids 51M and 76K are located in the highly conserved domain; c.152T>C (M51T) and c.227A>G (K76R) are predicted to be damaging and capable of changing alternative splice, structural and posttranslational modification sites of the RNA, as well as the secondary structure, structural stability, and hydrophobicity of the protein, suggesting that these mutations are associated with asthenozoospermia.
Male ; Humans ; Asthenozoospermia/metabolism* ; Sperm Motility/genetics* ; Homozygote ; Polymorphism, Single Nucleotide ; Semen ; Sequence Deletion/genetics* ; Spermatozoa/metabolism* ; Nucleotides/metabolism* ; beta-Defensins/metabolism*

ObjectiveTo investigate the effects of Qiangjing Tablets (QJT) on sperm quality and the MAPK signaling pathway in the SD rat model of asthenospermia (AS).

METHODSA total of 100 male SD rats were randomly divided into five groups of equal number, blank control, AS model control, high-dose QJT, medium-dose QJT, and low-dose QJT. All the rats were intragastrically administered ORN at 200 mg/kg/d for establishment of the AS model except those in the blank control group, which were given 1% CMC sodium solution at 1 ml/100 g by gavage. Meanwhile the animals of the high-, medium-, and low-dose QJT groups were gavaged with QJT at 6700, 3300 and 1700 mg/kg/d, respectively, qd 6 days a week for 20 days. Then the testis issue and the apoptosis of the testicular cells were observed under the electron microscope, the expression of vimentin in the testis was determined with the immunohistochemical SP method, that of ERK1/2 detected by Western blot, and the concentration of TGF-β1 in the semen measured by ELISA.

RESULTSThe AS model controls showed round nuclei of spermatocytes, homogeneously distributed chromatins, broken or lost mitochondria, and expanded rough endoplasmic reticulum in the testis tissue. In comparison, the rats of the high-, medium-, and low-dose QJT groups exhibited round nuclei of spermatocytes, homogeneously distributed chromatins, and well-structured mitochondria, rough endoplasmic reticulum and ribosome, which were all similar those of the blank controls. Compared with the blank controls, the AS model rats manifested significantly increased expressions of ERK1/2 (1.00 ± 0.00 vs 1.26 ± 0.10, P<0.01) and vimentin (0.16 ± 0.01 vs 0.17 ± 0.01, P<0.01) and apoptosis rate of cells in the testis tissue (［9.20 ± 3.07］ vs ［42.20 ± 9.17］ %, P<0.01), but decreased level of TGF-β1 in the semen (［627.67 ± 26.07］ vs ［566.73 ± 68.44］ ng/ml, P<0.05). In comparison with the model controls, the rats of the high- and medium- -dose QJT groups presented remarkably down-regulated expressions of ERK1/2 (1.26 ± 0.10 vs 1.14 ± 0.08, P<0.01; 1.26 ± 0.10 vs 1.18 ± 0.05, P<0.05) and vimentin (0.17 ± 0.01 vs 0.16 ± 0.01, P<0.01; 0.17 ± 0.01 vs 0.17 ± 0.09, P<0.05) and decreased rate of cell apoptosis (［42.20 ± 9.17］ vs ［21.60 ± 5.94］ %, P<0.01; ［42.20 ± 9.17］ vs ［33.95 ± 6.39］ %, P<0.05). The concentration of TGF-β1 in the semen was markedly lower in the high-dose QJT than in the AS model control group (［621.78 ± 30.80］ vs ［566.73 ± 68.44］ ng/ml, P < 0.05).

CONCLUSIONSQiangjing Tablets could improve semen quality in asthenospermia rats by acting against oxidative stress.

Animals ; Apoptosis ; Asthenozoospermia ; enzymology ; Drugs, Chinese Herbal ; pharmacology ; Male ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Mitogen-Activated Protein Kinases ; drug effects ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Semen ; Semen Analysis ; Signal Transduction ; Spermatozoa ; Testis ; metabolism ; ultrastructure ; Transforming Growth Factor beta1 ; metabolism ; Vimentin ; metabolism

ObjectiveTo investigate the expression and location of IQ motif-containing G (IQCG) in the human testis, compare its expression in normal-motility sperm with that in the sperm of asthenospermia patients, and explore its possible mechanisms and its correlation with fertility.

METHODSThe expression of the IQCG gene in the human testis was detected by RT-PCR and its location in the testis and sperm was determined by immunohistochemistry and immunofluorescence staining. Semen samples were collected from normal males, patients with asthenospermia, and fertile men that succeeded in artificial insemination with donor's sperm (AID), followed by analysis of the IQCG protein expression in different groups of samples by Western blot.

RESULTSImmunohistochemistry showed that IQCG was extensively expressed in the human testis, in the spermatocytes and spermatids, specifically in the sperm tail, weakly expressed or absent in the spermatogonial stem cells, and strongly expressed in the spermatogonial cells. The expression of IQCG was significantly lower in the asthenospermia patients than in the normal males (P= 0.041). Western blot manifested that IQCG was expressed in the semen of all the three groups of subjects, with statistically significant differences between the normal men and severe asthenospermia patients (P = 0.032) as well as between the fertile males and the severe asthenospermia group (P = 0.027) .

CONCLUSIONSIQCG may act on human sperm motility and its abnormal expression possibly reduces sperm motility and fertility. An insight into its action mechanisms may shed some new light on the etiology and treatment of asthenospermia.

Asthenozoospermia ; etiology ; metabolism ; therapy ; Fertility ; Humans ; Male ; Semen ; Sperm Motility ; Spermatozoa ; Testis ; metabolism

Spermatozoa are not mature until they transit the epididymis where they acquire motility and the ability to fertilize an egg through sequential modifications. The epididymis has three functional regions, caput, corpus, and cauda, and the luminal proteins of the epididymis play important roles in the above modifications. However, the proteins with differential enrichment between the caput and cauda are still largely unknown. To reveal the functions of the caput and cauda during sperm maturation, luminal proteins from caput and cauda of mice were analyzed by isobaric tag for relative and absolute quantitation (iTRAQ). Overall, 128 differentially enriched proteins were found, of which 46 were caput enriched and 82 were cauda enriched. Bioinformatic analysis showed that lipid metabolism was active in the caput; while anion- and cation-binding activity and phosphorus and organophosphate metabolism were active in the cauda. A new epididymal luminal protein, the caput-enriched PDZ domain containing 1 (Pdzk1), also named Na⁺/H⁺ exchange regulatory cofactor 3 (NHERF3), which plays a critical role in cholesterol metabolism and carnitine transport, was found in the lipid metabolism. Western blotting and immunofluorescence analyses showed that Pdzk1 was expressed in the epididymis but not in the testis, and localized at the middle piece of the sperm tail. Pdzk1 protein level was also reduced in the spermatozoa in case of asthenozoospermic patients compared with that in normozoospermic men, suggesting that Pdzk1 may participate in sperm maturation regulation and may be associated with male infertility. These results may provide new insights into the mechanisms of sperm maturation and male infertility.
Adult ; Animals ; Asthenozoospermia/metabolism* ; Carrier Proteins/metabolism* ; Case-Control Studies ; Epididymis/metabolism* ; Humans ; Intracellular Signaling Peptides and Proteins/metabolism* ; Male ; Membrane Proteins ; Mice ; Sperm Maturation ; Sperm Tail/metabolism* ; Spermatozoa/metabolism* ; Testis/metabolism*

Objective: To investigate the improving effect of astaxanthin (AST) on the sperm quality of rats with ornidazole (ORN)-induced oligoasthenozoospermiaand its action mechanism. METHODS: Forty adult male SD rats were equally randomized into groups A (solvent control), B (low-dose ORN ［400 mg/（kg·d）］), C (high-dose ORN ［800 mg/（kg·d）］), D (low-dose ORN ［400 mg/（kg·d）］ + AST ［20 mg/（kg·d）］), and E (high-dose ORN ［800 mg/（kg·d）］ + AST ［20 mg/（kg·d）］), all treated intragastrically for3 weeks.After treatment, the epididymal tails ononeside was taken for determination of sperm concentration and activity, and the epididymideson the other side harvested for measurement of the activities of GSH-Px, GR, CAT and SOD and the MDA contentin the homogenate. RESULTS: Compared with group A, sperm motilityin the epididymal tail andGSH-Px and SOD activities in theepididymiswere markedly decreased while the MDAcontent significantlyincreased in group B (P<0.05), spermmotility and concentrationin the epididymal tail, testisindex, and the activities of GSH-Px, GR, CAT and SOD in the epididymis were remarkably reduced while theMDA contentsignificantly increased in group C(P<0.05). In comparison with group B, group D showed markedly increased sperm motility (［45.3±8.7］% vs ［66.3±8.9］%, P<0.05) in the epididymal tail and SOD activity in the epididymis (［116.7±25.3］ U/mg prot vs ［146.1±23.8］ U/mg prot, P<0.05), decreased MDA content(［1.68±0.45］ nmol/mg prot vs ［1.19±0.42］ nmol/mg prot, P<0.05).Compared with group C, group Eexhibited significant increases in the weight gained (［89.0±9.5］ vs ［99.9±4.1］ %, P<0.05) and sperm motility (［17.9±3.5］% vs ［27.3±5.3］ %, P<0.05) but a decrease in the content of MDA (［2.03±0.30］ nmol/mg prot vs ［1.52±0.41］ nmol/mg prot, P<0.05). CONCLUSIONS AST can improve spermquality in rats with ORN-inducedoligoasthenozoospermia, which may be associated with its enhancing effect on the antioxidant capacity of the epididymis.
Animals ; Antioxidants ; pharmacology ; Asthenozoospermia ; prevention & control ; Epididymis ; drug effects ; metabolism ; Male ; Oligospermia ; prevention & control ; Ornidazole ; Oxidative Stress ; Protective Agents ; pharmacology ; Radiation-Sensitizing Agents ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sperm Count ; Sperm Motility ; Spermatozoa ; drug effects ; metabolism ; Xanthophylls ; pharmacology

Objective: To study the correlation of the gene expressions of Chk1 and Chk2 with sperm concentration and motility. METHODS: According to sperm concentration and motility (percentage of progressively motile sperm), we divided 80 semen samples into four groups of equal number: normal control, oligozoospermia (OS), asthenospermia (AS), and oligoasthenozoospermia (OAS). We detected the sperm DNA fragmentation index (DFI) and viability and determined the expressions of Chk1 and Chk2 in the sperm by RT-PCR and Western blot. RESULTS: Statistically significant differences were not found in sperm DFI among the control, OS, AS, and OAS groups (21.24±6.93, 19.67±7.64, 21.52±6.92, and 19.28±11.55, P>0.05), but observed in sperm concentration, progressive motility, and viability between the DFI >30% and DFI ≤30% groups (P<0.01). Compared with the normal control, sperm viability was remarkably decreased in the OS, AS, and OAS groups (［83.48±9.87］% vs ［63.86±9.16］%, ［50.45±16.99］%, and ［39.21±15.74］%, P<0.05). RT-PCR showed remarkable differences among the control, OS, AS, and OAS groups in the relative expression level of Chk1 mRNA (0.73±0.22, 0.62±0.14, 1.03±0.39, and 0.92±0.071, P<0.01), which was correlated positively with sperm concentration (b = 80.661, P<0.01) but negatively with sperm motility (b = －19.275, P < 0.01), as well as in that of Chk2 mRNA (0.66±0.30, 0.27±0.09, 0.59±0.19, and 0.42 ± 0.11, P<0.01), which was correlated negatively with sperm concentration (b = －90.809, P<0.01) but positively with sperm motility (b = 27.507, P <0.01). The relative expression levels of the Chk1 protein were significantly different among the four groups (0.63±0.05, 0.42±0.03, 1.13±0.08, and 0.87±0.07, P<0.01), which was correlated positively with sperm concentration (b = 55.74, P<0.01) but negatively with sperm motility (b =－22.649, P<0.01), and so were those of the Chk2 protein (1.23±0.36, 0.37±0.16, 0.87±0.08, and 0.68±0.12, P<0.01), which was correlated negatively with sperm concentration (b =－53.001, P<0.01) but positively with sperm motility (b = 16.676, P < 0.01). CONCLUSIONS Chk1 and Chk2 are significantly expressed in human sperm. In case of sperm DNA damage, up-regulated Chk1 expression may enhance sperm apoptosis and lead to asthenospermia, while increased Chk2 expression may inhibit spermatogenesis and result in oligospermia.
Apoptosis ; Asthenozoospermia ; genetics ; Checkpoint Kinase 1 ; genetics ; metabolism ; Checkpoint Kinase 2 ; genetics ; metabolism ; DNA Damage ; DNA Fragmentation ; Gene Expression ; Humans ; Male ; Oligospermia ; genetics ; Semen Analysis ; Sperm Count ; Sperm Motility ; genetics ; Spermatozoa ; physiology

Objective: To investigate the mRNA and protein expressions of outer dense fiber 2 (ODF2) in the sperm of the asthenospermia patient and their differences from those in normal healthy men. METHODS: According to the WHO criteria, we collected semen samples from 45 asthenozoospermia patients and 15 normal healthy volunteers. Using computer-assisted sperm analysis (CASA), we divided the semen samples from the asthenospermia patients into a mild, a moderate and a severe group, and determined the mRNA and protein expressions of ODF2 in different groups by RT-PCR and Western blot. RESULTS: Compared with the normal healthy men, the expression of the ODF2 gene showed no statistically significant difference in the mild asthenospermia group (1.112 0 ± 0.525 5 vs 0.688 0 ± 0.372 0, P >0.05) but remarkably decreased in the moderate (0.483 3 ± 0.186 3, P <0.05) and severe asthenospermia patients (0.448 3 ± 0.340 8, P <0.01). The OD value (ODF2/β-actin) of the ODF2 protein in the normal men exhibited no statistically significant difference from that in the mild asthenospermia group (0.458 7 ± 0.052 1 vs 0.326 1 ± 0.071 4, P >0.05), but markedly lower than in the moderate (0.145 4 ± 0.053 6, P <0.05) and severe asthenospermia patients (0.122 7 ± 0.045 7, P <0.01), which was consistent with the results of RT-PCR. CONCLUSIONS Decreased mRNA and protein expressions of ODF2 in the sperm are positively correlated with declined sperm motility of the asthenospermia patient, which is suggestive of the involvement of the ODF2 gene in the regulation of sperm motility.
Asthenozoospermia ; metabolism ; physiopathology ; Case-Control Studies ; Down-Regulation ; Heat-Shock Proteins ; genetics ; metabolism ; Humans ; Male ; RNA, Messenger ; metabolism ; Semen Analysis ; Sperm Motility ; Sperm Tail ; Spermatozoa ; metabolism

Objective: To investigate the expressions of solute carrier family 22 member 14 (SLC22A14) and sperm-associated antigen 6 (SPAG6) in the sperm of idiopathic asthenospermia men. METHODS: We collected semen samples from 50 idiopathic asthenozoospermia patients and another 50 normal sperm donors, purified the sperm by discontinuous density centrifugation on Percoll gradients, and then determined the mRNA and protein expressions of SLC22A14 and SPAG6 by RT-PCR and Western blot, respectively. RESULTS: Compared with the normal controls, the idiopathic asthenozoospermia patients showed significantly decreased mRNA expressions of SLC22A14 (0.77 ± 0.08 vs 0.53 ± 0.10, P<0.01) and SPAG6 (0.78 ± 0.09 vs0.52 ± 0.10 , P<0.01) and protein expressions of SLC22A14 (0.80 ± 0.09 vs 0.55 ± 0.10 , P<0.01) and SPAG6 (0.78 ± 0.09 vs 0.56 ± 0.09, P<0.01). CONCLUSIONS T The expressions of SLC22A14 and SPAG6 are reduced in the sperm of the patients with idiopathic asthenospermia, which may be one of the important causes of asthenospermia.
Asthenozoospermia ; metabolism ; Blotting, Western ; Ejaculation ; Humans ; Male ; Microtubule Proteins ; genetics ; metabolism ; Organic Cation Transport Proteins ; genetics ; metabolism ; Proteomics ; RNA, Messenger ; metabolism ; Sperm Motility ; Spermatozoa ; metabolism