Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenozoospermia categorized by immotile spermatozoa with abnormal flagella in ejaculate. Whole-exome sequencing (WES) is used to detect pathogenic variants in patients with MMAF. In this study, a novel homozygous frameshift variant (c.6158_6159insT) in dynein axonemal heavy chain 8 (DNAH8) from two infertile brothers with MMAF in a consanguineous Pakistani family was identified by WES. Reverse transcription-polymerase chain reaction (RT-PCR) confirmed DNAH8 mRNA decay in these patients with the DNAH8 mutation. Hematoxylin-eosin staining and transmission electron microscopy revealed highly divergent morphology and ultrastructure of sperm flagella in these patients. Furthermore, an immunofluorescence assay showed the absence of DNAH8 and a reduction in its associated protein DNAH17 in the patients' spermatozoa. Collectively, our study expands the phenotypic spectrum of patients with DNAH8-related MMAF worldwide.
Humans ; Male ; Consanguinity ; Pakistan ; Infertility, Male/metabolism* ; Semen/metabolism* ; Sperm Tail/metabolism* ; Spermatozoa/metabolism* ; Flagella/pathology* ; Mutation

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*

Objectives: To study the effects of Porphyromonas gingivalis (Pg) injected through tail vein on the molecular expression levels of biomarkers of neural stem cells (NSC) and neurons in the hippocampus of wild-type adult rats, and the effects on hippocampal neurogenesis. Methods: Eighteen male Sprague-Dawley (SD) rats were randomly divided into 3 groups based on the table of random numbers (n=6 in each group). In low-intensity group and high-intensity group, rats were injected intravenously through tail vein with 200 μl Pg ATCC33277 [1.0×10³ and 1.0×10⁸ colony forming unit (CFU), respectively] 3 times per week for 8 weeks. In the sham group, 200 μl of phosphate buffer saline (PBS) was given instead. Behavioral tests: the navigation and the exploration tests using Morris water maze (MWM) were applied to evaluate learning and memory ability of rats. Immunohistochemistry was performed to detect cells positively expressing nestin, doublecortin (DCX) and neuronal nuclei (NeuN) in the subgranular zone (SGZ) of rats in each group. Western blotting was used to evaluate the expression levels of nestin, DCX and NeuN in rat hippocampus. Results: Learning and memory abilities: on day 5 of navigation test, the lagency time was 22.83 (16.00, 38.34) s in the high-intensity group, significantly longer than the sham group [5.59 (5.41, 6.17) s] (t=-11.17, P<0.001). There were no significant differences between the low-intensity group [9.85 (8.75, 21.01) s] and the sham group (t=-6.83, P=0.080). Results in the exploration test showed that, in the high-intensity group, the number of fime crossing over the previous platform area within 60 s was 1.50 (1.00, 2.00), significantly less than the sham group [4.00 (2.75, 4.00)] (t=9.75, P=0.003); no significant differences between the low-intensity group [2.50 (2.00, 3.00)] and the sham one (t=4.50, P=0.382). Immunohistochemistry showed that the nestin⁺ cell density in the low-intensity group [(35.36±4.32) cell/mm²] and high-intensity group [(26.51±5.89) cell/mm²] were significantly lower than the sham group [(59.58±14.15) cell/mm²] (t=24.21, P=0.018; t=33.07, P=0.005); as for the mean absorbance of DCX⁺ cells, the low-intensity group (0.007±0.002) and the high-intensity group (0.006±0.002) were significantly lower than the sham group (0.011±0.001) (t=0.004, P=0.018; t=0.006, P=0.005); compared with the sham group [(1.13±0.14)×10³ cell/mm²], the density of NeuN⁺ neurons in the high-intensity group [(0.75±0.08)×10³ cell/mm²] was significantly reduced (t=0.38, P=0.017), and was not significantly changed in the low-intensity group [(0.88±0.19)×10³ cell/mm²] (t=0.25, P=0.075). Western blotting results showed that, compared with the sham group, the expression levels of nestin, DCX, and NeuN were significantly reduced in the high-intensity group (t=0.74, P<0.001; t=0.18, P=0.014; t=0.35, P=0.008), but were not statistically changed in the low-intensity group (t=0.18, P=0.108; t=0.08, P=0.172; t=0.19, P=0.077). Conclusions: Pg injected through tail vein may reduce learning and memory abilities of wild-type rats, and may reduce the number of nestin, DCX, and NeuN-positive cells, and the protein expression levels of the above molecules in the hippocampus.
Animals ; Biomarkers/metabolism* ; Hippocampus/metabolism* ; Male ; Nestin/metabolism* ; Neural Stem Cells/metabolism* ; Neurons/metabolism* ; Porphyromonas gingivalis/metabolism* ; Rats ; Rats, Sprague-Dawley ; Tail/metabolism*

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

OBJECTIVETo investigate the ameliorative effect of ginseng glycopeptide on cross-linking of rat tail tendon collagen.

METHODELISA was used to determine the inhibitory effect of ginseng glycopeptide on cross-linking of rat tail tendon collagen in vitro. After ginseng glycopeptide was intraperitoneally administrated to streptozotocin-induced diabetic rats for 12 weeks, the acid solubility, limited pepsin degradation properties and solubility in SDS-2-mercaptoethanol of the rat tail tendon collagen were determined, and the effect of ginseng glycopeptide on the tail tendon collagen cross-linking was evaluated.

RESULTGinseng glycopeptide inhibited significantly the cross-linking of rat tail tendon collagen in vitro. The solubility of the tail tendon collagen (in acid, pepsin and SDS-2-mercaptoethanol) was markedly decreased in diabetic rats and ginseng glycopeptide-treated diabetic rats had significantly an increase in the collagen solubility in the above-mentioned solutions, suggesting that ginseng glycopeptide decreased severity of the collagen cross-linking.

CONCLUSIONGinsengglycopeptide exhibits an significantly ameliorative effect on cross-linking of rat tail tendon collagen.

Animals ; Collagen ; metabolism ; Diabetes Mellitus, Experimental ; metabolism ; Female ; Glycopeptides ; isolation & purification ; pharmacology ; Male ; Panax ; chemistry ; Plants, Medicinal ; chemistry ; Rats ; Rats, Wistar ; Solubility ; Tail ; Tendons ; metabolism

OBJECTIVETo observe the effects of Yijingfang on CatSper1 in the mouse model of cyclophosphamide-induced oligoasthenospermia.

METHODSForty Kunming male mice were randomly divided into a control group (CG), a model group (MG), a small-dose Yijingfang group (SG), and a large-dose Yijingfang group (LG). The mice of CG were intraperitoneally injected with normal saline at 60 mg/kg once a day, while those of MG, SG and LG with cyclophosphamide, all for 5 days. During the next 34 days, the mice of SG and LG received intragastric administration of Yijingfang once a day, the former at a dose 2 times and the latter 5 times that of human routine usage, those of MG given the same volume of normal saline, and CG normally fed. At 35 days, we measured the sperm count, percentages of grades a + b and a + b + c sperm, and the expression of CatSper1 in the epididymal sperm of the mice.

RESULTSThe sperm counts of CG, MG, SG and LG were (5.20 +/- 1.34), (1.73 +/- 0.03), (2.08 +/- 0.01) and (3.31 +/- 0.56) x 10(6)/ml, respectively, significantly lower in MG than in CG (P < 0.05), but higher in LG than in MG (P < 0.05). The grade a + b sperm constituted (14.49 +/- 0.30), (6.64 +/- 1.88), (11.99 +/- 1.01) and (19.40 +/- 3.13)% in CG, MG, SG and LG, respectively, remarkably lower in MG than in CG (P < 0.05) but higher in LG than in MG (P < 0.05); the grade a + b + c sperm accounted for (68.39 +/- 15.13), (39.96 +/- 4.89), (62.28 +/- 4.43) and (73.61 +/- 5.05)%, respectively, obviously lower in MG than in CG (P < 0.05) but higher in LG than in MG (P < 0.05); the CatSper1 expressions were 0.76 +/- 0.05, 0.73 +/- 0.03, 0.75 +/- 0.12 and 0.85 +/- 0.04, respectively, markedly higher in LG than in MG (P < 0.05).

CONCLUSIONIntraperitoneal injection of cyclophosphamide decreases the sperm count, percentages of grades a + b and a + b + c sperm, and the expression of CatSper1 in mice, while large-dose Yijingfang can increase the above parameters, and hence contributes to the treatment of oligoasthenospermia.

Animals ; Calcium Channels ; drug effects ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Male ; Mice ; Mice, Inbred Strains ; Sperm Motility ; Sperm Tail ; drug effects ; metabolism

AIMTo characterize mouse capping protein alpha3 (CPalpha3) during spermatogenesis and sperm maturation.

METHODSWe produced rat anti-CPalpha3 antiserum and examined the expression of CPalpha3 in various mouse tissues using Western blot analysis and the localization of CPalpha3 in testicular and epididymal sperm using immunohistochemical analyses. We also examined how the localization of CPalpha3 and beta-actin (ACTB) in sperm changed after the acrosomal reaction by performing immunohistochemical analyses using anti-CPalpha3 antiserum and anti-actin antibody.

RESULTSWestern blot analysis using specific antiserum revealed that CPalpha3 was expressed specifically in testes. Interestingly, the molecular weight of CPalpha3 changed during sperm maturation in the epididymis. Furthermore, the subcellular localization of CPalpha3 in sperm changed dynamically from the flagellum to the post-acrosomal region of the head during epididymal maturation. The distribution of ACTB was in the post-acrosomal region of the head and the flagellum. After inducing the acrosomal reaction, the CPalpha3 and ACTB localization was virtually identical to the localization before the acrosomal reaction.

CONCLUSIONCPalpha3 might play an important role in sperm morphogenesis and/or sperm function.

Acrosome Reaction ; physiology ; Actins ; metabolism ; Animals ; Blotting, Western ; CapZ Actin Capping Protein ; metabolism ; Cells, Cultured ; Epididymis ; cytology ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Sperm Head ; metabolism ; Sperm Tail ; metabolism ; Spermatogenesis ; physiology ; Spermatozoa ; cytology ; metabolism ; Testis ; cytology ; 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*

PURPOSE: To clarify whether the bone resorption in femur measured by the expression of OPG & RANK-L was increased in tail suspended rat. MATERIALS AND METHODS: Four-week-old female Sprague Dawley rats were divided into two groups. The experimental group (n=79) was housed and fed with 2 weeks of tail suspension, and reloaded for 8 weeks without tail suspension. The control group (n=46) was housed and fed for 10 weeks without tail suspension. Bone mineral densities, serum levels of ALP and TRAP were measured in both groups. The expressions of the mRNAs of OPG and RANK-L were analyzed by RT-PCR. RESULTS: The ALP and TRAP were increased in the experimental group during both tail suspension and reloading, which reflected the increased bone metabolism in the experimental group. In femur of the experimental group, the expression of the mRNA of RANK-L was increased during tail suspension, and the expression the mRNA of OPG was decreased. With reloading, the expression of the mRNA of RANK-L in femur was decreased, while the expression of the mRNA of OPG was increased.
Animals ; Bone Density ; Bone Resorption ; Female ; Femur ; Hindlimb Suspension ; Humans ; Metabolism ; Osteoclasts ; Osteoporosis* ; Osteoprotegerin ; Rats* ; Rats, Sprague-Dawley ; RNA, Messenger ; Tail*

The CatSper channel is known as one of the most important Ca²⁺ channels on the cell membrane of mammalian sperm and plays a key role in the motility, hyperactivation and fertilization function of sperm. The CatSper protein, expressed exclusively in the principal piece of the sperm tail, is composed of CatSper1-4 and 5 auxiliary unitsβ,γ,δ and ε, and has an essential part in the functional and structural domains of Ca²⁺as well as in the spatiotemporal regulation of the P-Tyr protein, sperm hyperactivation, efficient sperm migration in the oviduct, egg penetration, and normal fertility. Recent studies show that functional deficiency of CatSper seriously affects sperm function,and the loss of any one of its 9 subunits may lead to male reproductive dysfunction. This paper outlines recent advances in the studies of the CatSperprotein, focusing on its expression, location, structure, and regulation,as well as itsinfluence on sperm hyperactivation and male reproduction.
Animals ; Calcium Channels ; chemistry ; physiology ; Humans ; Infertility, Male ; etiology ; Male ; Sperm Motility ; physiology ; Sperm Tail ; metabolism ; Sperm-Ovum Interactions ; physiology ; Spermatozoa ; physiology