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

Numerous genes have been associated with multiple morphological abnormalities of the sperm flagella (MMAF), which cause severe asthenozoospermia and lead to male infertility, while the causes of approximately 50% of MMAF cases remain unclear. To reveal the genetic causes of MMAF in an infertile patient, whole-exome sequencing was performed to screen for pathogenic genes, and electron microscope was used to reveal the sperm flagellar ultrastructure. A novel heterozygous missense mutation in the outer dense fiber protein 2 (ODF2) gene was detected, which was inherited from the patient's mother and predicted to be potentially damaging. Transmission electron microscopy revealed that the outer dense fibers were defective in the patient's sperm tail, which was similar to that of the reported heterozygous Odf2 mutation mouse. Immunostaining of ODF2 showed severe ODF2 expression defects in the patient's sperm. Therefore, it was concluded that the heterozygous mutation in ODF2 caused MMAF in this case. To evaluate the possibility of assisted reproductive technology (ART) treatment for this patient, intracytoplasmic sperm injection (ICSI) was performed, with the help of a hypo-osmotic swelling test and laser-assisted immotile sperm selection (LAISS) for available sperm screening, and artificial oocyte activation with ionomycin was applied to improve the fertilization rate. Four ICSI cycles were performed, and live birth was achieved in the LAISS-applied cycle, suggesting that LAISS would be valuable in ART treatment for MMAF.
Abnormalities, Multiple ; Animals ; Flagella ; Heat-Shock Proteins ; Humans ; Infertility, Male ; Male ; Mice ; Mutation ; Semen ; Sperm Tail ; Spermatozoa

Flagellin is a subunit protein of the flagellum, a whip-like appendage that enables bacterial motility. Traditionally, flagellin was viewed as a virulence factor that contributes to the adhesion and invasion of host cells, but now it has emerged as a potent immune activator, shaping both the innate and adaptive arms of immunity during microbial infections. In this review, we summarize our understanding of bacterial flagellin and host immune system interactions and the role flagellin as an adjuvant, anti-tumor and radioprotective agent, and we address important areas of future research interests.
Arm ; Flagella ; Flagellin ; Immune System ; Virulence

Cilia and flagella on eukaryotic cells are polarized organelles extending from the surfaces of cells, which participate not only in cell motility, but also in signal transduction and other processes. Structural or functional abnormalities of cilia can cause various human diseases, termed ciliopathies. Bardet-Biedl syndrome (BBS) is a ciliopathic human genetic disorder, and the pathogenesis is that mutated BBS genes result in abnormal cilia function. In order to study the pathogenic genes BBS8, we screened bbs8 mutant in Chlamydomonas reinhardtii and did a lot of physiology and biochemistry experiments. We affirmed that BBS8 protein was a cilia protein and had specific localization in the basal body by immunofluorescence (IF). The bbs8 mutant lost photokinesis, and it was defective in flagella shortening with drug induction. The results of silver staining and mass spectrometric analysis showed aberrant accumulation of flagellar proteins in the mutant flagella. We concluded that the BBS8 protein plays a significant role in flagellar membrane proteins transport, and the BBS8 protein might mediate retrograde transport to exert physiological function in the process.
Bardet-Biedl Syndrome ; Chlamydomonas reinhardtii ; Cilia ; Flagella ; Humans ; Protein Transport

Salmonellosis is one of the most common food born diseases in Korea. However, it takes more than 8 days and many expensive antiserums are used for the identification of Salmonella serovars since the microorganism easily undergoes phase variation. According to the data that 65.5% of Salmonella isolates in 2000~2004 year had monophasic flagella, we have developed a rapid serological identification method using a hin gene-specific polymerase chain reaction (PCR) for monophasic Salmonella isolates that does not require the time-consuming phase conversion experiments. Using our new method, 'hin specific PCR-based serological test', we could identify serovars of monophasic Salmonella in 4 days. For the purpose of rapid identification of salmonella serovars collected from outbreaks and sporadic cases, hin specific PCR-based serological tests will be a fast and efficient method.
Disease Outbreaks ; Flagella ; Korea ; Polymerase Chain Reaction* ; Salmonella Infections ; Salmonella* ; Serologic Tests

PURPOSE: We aimed to elucidate the expression and intracellular localization of sperm-specific cation channel CatSper in human spermatozoa. Moreover, the relationship between the expression of CatSper mRNA and the motility of ejaculated human spermatozoa were investigated. MATERIALS AND METHODS: Using cDNAs extracted from the ejaculated sperm of patients (n=39), the expression of CatSper mRNA was observed by RT-PCR. Semi-quantitative analysis of the CatSper mRNA expression was performed by comparing with the expression of GAPDH mRNA. To elucidate the expression and intracellular localization of CatSper protein, double fluorescent immunocytochemistry for CatSper and beta-tubulin was performed. RESULTS: The CatSper mRNA was expressed in all of the sperm samples. Using semi-quantitative analysis for the amount of CatSper mRNA expression, no significant difference was found between the normozoospermia and asthenozoospermia groups (1.5+/-0.6 vs. 1.4+/-0.6, p=0.623). Polyclonal antiserum, generated against a recombinant protein of the N-terminal 160 amino acids of human CatSper, was used. In double fluorescent immunocytochemistry, CatSper protein was found to be expressed in the flagellum of the ejaculated human spermatozoa, and localized in the connecting piece, mid-piece and principal piece, with the exception of the end piece of the flagellum. Moreover, the proportion of CatSper-positive sperm was similar in both the normozoospermia and asthenozoospermia groups. CONCLUSIONS: To the best of our knowledge, this is the first time ejaculated human spermatozoa have been shown to express the mRNA and protein of CatSper. The results of our RT-PCR and immunocytochemistry suggest that CatSper may play a role in the motility of ejaculated human spermatozoa.
Amino Acids ; Asthenozoospermia ; DNA, Complementary ; Flagella ; Humans* ; Immunohistochemistry ; RNA, Messenger ; Sperm Motility ; Spermatozoa* ; Tubulin

BACKGROUND: Trichomonas vaginalis is a pathogenic protozoa infecting human genitourinary tract. Metronidazole is currently the drug of choice to treat T. vaginalis infection. However, because of the side effects and the occurrence of resistant strains of metronidazole, it is needed to investigate alternatives. METHODS: The antiprotozoal effect of aquatic extract from Sophora flavescens on the growth and fine structure of T. vaginalis was examined by using trypan blue exclusion assay and electron microscopy. RESULTS: One hour after the addition of 4 mg/mL extract and half hour after the addition of 5 mg/mL showed antiprotozoal effect. One to two hours after the addition of 3 mg/mL extract, the movement of flagella and axostyle had disappeared, but death of the cells had not occurred until two hours after the addition. The fine structure of the cytoplasm was also changed half an hour to two hours after addition. The number of polyribosome decreased when that of single ribosomes in the cytoplasm increased. CONCLUSION: These results indicated that S. flavescens had the antiprotozoal effect on T. vaginalis by inhibition of cell multiplication as well as an impairment of protein synthesis.
Cell Proliferation ; Cytoplasm ; Flagella ; Humans ; Metronidazole ; Microscopy, Electron ; Polyribosomes ; Ribosomes ; Sophora* ; Trichomonas vaginalis* ; Trichomonas* ; Trypan Blue

Busulfan is an anticancer drug, which causes the apoptosis germ cells and azoospermia in humans and animals. Abnormal morphology of spermatozoa related to the male infertility. The sperm morphology is evaluation of sperm size, shape and appearance characteristics should be assessed by carefully observing a stained sperm sample under the microscope. Evaluation of sperm morphology has been considered as one of the most important factors for a successful fertilization and determining sperm quality. The mice were assigned to tow experimental groups: control and busulfan. Each group included six mice that were housed under standard conditions. The volume was estimated using the nucleator method. The sperm's flegellum and mid-piece length was estimated by counting the number of intersections between the tails and Merz grid test line in an unbiased counting frame, superimposed on live images of sperms. Our results demonstrated a significant different in the volume and surface of the sperm's head and the length of the sperm's flagellum in the control and busulfan groups. Busulfan can effect on the volume of the sperm's head and the length of the sperm's flagellum in rat.
Animals ; Apoptosis ; Azoospermia ; Busulfan* ; Fertilization ; Flagella ; Germ Cells ; Head ; Humans ; Infertility, Male ; Male ; Methods* ; Mice* ; Rats ; Spermatozoa* ; Tail

The bacterial flagellar structure can be divided into the basal body, the hook and the filament. Three minor components called hook associated proteins (HAP1, HAP2 and HAP3) form a junction between the hook and the filament (HAP1 and HAP3) and a capping structure at the distal end of flagellar filament (HAP2). Vibrio vulnificus is a halophilic pathogenic bacterium that is locomotive by means of a polar flagellum. From a V. vulnificus genome sequencing project, we obtained sequences of V. vulnificus flgK (Vv-flgK), flgL (Vv-flgL), and flaH (Vv-flaH) genes that encode HAP1, HAP3, and HAP2, respectively. To investigate roles of the HAP proteins, deletion mutants of the Vv-flgK, Vv-flgL and Vv-flaH were constructed. Electron microscopic analysis showed that the Vv-flgK or Vv-flgL mutant did not produce an intact polar flagellum while the Vv-flaH mutant produced a fragile flagellar structure. Western blot analysis against a major polar flagellin proposed that the null HAP1 and HAP3 mutations resulted in a failure of normal flagellar assembly since flagellins produced by the mutants were secreted out in the culture supernatants without long flagellar filaments. Motility was completely abolished by a single mutation in HAP1 or HAP3, and the HAP2 mutant showed a decreased motility. Also each of the mutants showed an impaired cytotoxicity and adherence to HeLa cell compared with the isogenic wild type strain. LD(50) increased by 10- and 11-fold in the V. vulnificus HAP3 and HAP2 mutant, respectively. These results suggest that the HAP proteins play important roles in polar flagellation and the virulence of V. vulnificus.
Bacterial Proteins ; Blotting, Western ; Electrons ; Flagella ; Flagellin ; Genome ; HeLa Cells ; Humans ; Proteins ; Sprains and Strains ; Vibrio ; Vibrio vulnificus

AIMTo investigate the human sperm oxygen/energy consumption and zinc content in relation to motility.

METHODSIn washed spermatozoa from 67 ejaculates, the oxygen consumption was determined. Following calculation of the total oxygen consumed by the Ideal Gas Law, the energy consumption of spermatozoa was calculated. In addition, the zinc content of the sperm was determined using an atomic absorption spectrometer. The resulting data were correlated to the vitality and motility.

RESULTSThe oxygen consumption averaged 0.24 micromol/10(6) sperm x 24h, 0.28 micromol/10(6) live sperm x 24h and 0.85 micromol/10(6) live motile sperm x 24h. Further calculations revealed that sperm motility was the most energy consuming process (164.31 mJ/10(6) motile spermatozoa x 24h), while the oxygen consumption of the total spermatozoa was 46.06 mJ/10(6) spermatozoa x 24h. The correlation of the oxygen/energy consumption and zinc content with motility showed significant negative correlations (r= -0.759; P<0.0001 and r=-0.441; P<0.0001, respectively). However, when correlating sperm energy consumption with the zinc content, a significant positive relation (r=0.323; P=0.01) was observed.

CONCLUSIONPoorly motile sperm are actually wasting the available energy. Moreover, our data clearly support the "Geometric Clutch Model" of the axoneme function and demonstrate the importance of the outer dense fibers for the generation of sperm motility, especially progressive motility.

Adult ; Energy Metabolism ; physiology ; Flagella ; physiology ; Humans ; Male ; Middle Aged ; Oxygen Consumption ; Sperm Motility ; physiology ; Spermatozoa ; metabolism ; Zinc ; metabolism