OBJECTIVE: To search for the possible pathogenic genes for multiple morphological anomalies of sperm flagella (MMAF).
METHODS: We performed whole exome sequencing (WES) of a typical case of MMAF and analyzed its possible pathogenic genes. We examined the semen sample from the patient and identified the ultrastructural characteristics of the sperm flagella under the scanning electron and transmission electron microscopes, and analyzed the expression pattern of cilia and flagela-associated protein 65 (CFAP65) in spermatogenesis by immunofluorescence assay.
RESULTS: The MMAF patient was found with a homozygous pathogenic mutation of the CFAP65 gene c.2675G>A(p.Trp892*). Scanning electron microscopy showed that the sperm of the patient had typical characteristics of MMAF, that is, without tails or with folded tails, curly tails, short tails or irregular tails. Transmission electron microscopy revealed the loss and disorder of the \"9+2\" structure in the sperm flagellum, with abnormal assembly of the fibrous sheath, accompanied by loss of central microtubules and dynamin arms. Cellular immunofluorescence assay suggested that the CFAP65 gene was expressed at all levels of mouse germ cells.
CONCLUSIONS: The CFAP65 gene is involved in the assembly of the sperm flagellum structure, and its mutation can cause the phenotype of MMAF, leading to male infertility.

Motile cilia, also called flagella, are found across a broad range of species; some cilia propel prokaryotes and eukaryotic cells like sperm, while cilia on epithelial surfaces create complex fluid patterns e.g., in the brain or lung. For sperm, the picture has emerged that the flagellum is not only a motor but also a sensor that detects stimuli from the environment, computing the beat pattern according to the sensory input. Thereby, the flagellum navigates sperm through the complex environment in the female genital tract. However, we know very little about how environmental signals change the flagellar beat and, thereby, the swimming behavior of sperm. It has been proposed that distinct signaling domains in the flagellum control the flagellar beat. However, a detailed analysis has been mainly hampered by the fact that current comprehensive analysis approaches rely on complex microscopy and analysis systems. Thus, knowledge on sperm signaling regulating the flagellar beat is based on custom quantification approaches that are limited to only a few aspects of the beat pattern, do not resolve the kinetics of the entire flagellum, rely on manual, qualitative descriptions, and are only a little comparable among each other. Here, we present SpermQ, a ready-to-use and comprehensive analysis software to quantify sperm motility. SpermQ provides a detailed quantification of the flagellar beat based on common time-lapse images acquired by dark-field or epi-fluorescence microscopy, making SpermQ widely applicable. We envision SpermQ becoming a standard tool in flagellar and motile cilia research that allows to readily link studies on individual signaling components in sperm and distinct flagellar beat patterns.

BACKGROUND: Sperm motility is an essential aspect of human fertility. Sperm contain an abundance of transcripts, thought to be remnants of mRNA, which comprise a genetic fingerprint and can be considered a historic record of gene expression during spermatogenesis. The aberrant expression of numerous genes has been found to contribute to impaired sperm motility; these include ROPN1 (rhophilin associated tail protein 1), which encodes a component of the fibrous sheath of the mammalian sperm flagella, and CABYR (calcium-binding tyrosine-(Y)-phosphorylation-regulated protein), which plays an important role in calcium activation and modulation. The aim of this study was to investigate ROPN1 and CABYR gene co-expression in asthenozoospermic semen samples in comparison with normozoospermic samples.
METHODS: We studied 120 semen samples (60 normozoospermic and 60 asthenozoospermic) from Caucasian patients attending our centre for an andrological check-up. Total RNA was extracted from purified spermatozoa with RNeasy mini kit. ROPN1 and CABYR mRNA expression was analysed using RT-qPCR. Continuous variables were described as means ± standard deviations.
RESULTS: ROPN1 and CABYR mRNA were simultaneously downregulated in asthenozoospermic in comparison with normozoospermic samples. There was also a positive correlation between total progressive motility and ROPN1 and CABYR gene expression and between total motile sperm number and ROPN1 and CABYR gene expression.
CONCLUSIONS: The results demonstrated downregulation of both ROPN1 and CABYR in asthenozoospermic samples and importantly, a positive correlation between the expression of the two genes, suggesting that ROPN1 and CABYR co-expression is a prerequisite for normal flagellar function and sperm motility.

Glyceraldehyde-3-phosphate dehydrogenase from human sperm (GAPDHS) provides energy to the sperm flagellum, and is therefore essential for sperm motility and male fertility. This isoform is distinct from somatic GAPDH, not only in being specific for the testis but also because it contains an additional amino-terminal region that encodes a proline-rich motif that is known to bind to the fibrous sheath of the sperm tail. By conducting a large-scale sequence comparison on low-complexity sequences available in databases, we identified a strong similarity between the proline-rich motif from GAPDHS and the proline-rich sequence from Ena/vasodilator-stimulated phosphoprotein-like (EVL), which is known to bind an SH3 domain of dynamin-binding protein (DNMBP). The putative binding partners of the proline-rich GAPDHS motif include SH3 domain-binding protein 4 (SH3BP4) and the IL2-inducible T-cell kinase/tyrosine-protein kinase ITK/TSK (ITK). This result implies that GAPDHS participates in specific signal-transduction pathways. Gene Ontology category-enrichment analysis showed several functional classes shared by both proteins, of which the most interesting ones are related to signal transduction and regulation of hydrolysis. Furthermore, a mutation of one EVL proline to leucine is known to cause colorectal cancer, suggesting that mutation of homologous amino acid residue in the GAPDHS motif may be functionally deleterious.

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The specific identification and systematic of triatomines have been based fundamentally on morphological observations. These organisms are classified into complexes and specific subcomplexes, principally for morphological parameters and geographical disposition. The use of cytogenetic analyzes has been represented as a tool in systematic and taxonomy of triatomines. Thus, the present work, through the analysis of spermiogenesis, aims to characterize this stage of spermatogenesis in triatomines little studied, and especially to compare it among the species Triatoma lenti and T. sherlocki, to assist in the diagnosis of differentiation of these insects. The presence of the heteropyknotic corpuscle is shown as a diagnostic tool to differentiate T. sherlocki and T. lenti, since it is absent in T. lenti. The analysis of the spermiogenesis in T. sherlocki also allowed us to address morphological differences between elongating cells, which were relatively smaller and more filamentous when compared to T lenti. Furthermore, the flagellum was observed in all stages of cell differentiation and elongation. This structure, which helps in the locomotion of the sperm, is hardly observed in cytogenetic analysis, especially throughout spermiogenesis. Thus, although other comparative approaches should be taken, this paper allowed emphasizing the analysis of spermiogenesis as an important cytotaxonomic tool that assists in the differentiation of morphologically related species, such as T. lenti and T. sherlocki.

OBJECTIVE: To compare subgroups of the human sperm hypoosmotic swelling test subgroups in both recurrent fertilization negative infertile cases with normal semen analysis and fertilization positive controls.
METHODS: This was a prospective case-controlled study performed with normospermic 33 previously fertile male (secondary infertility) and 41 infertile men who had undergone two or three unsuccessful in vitro fertilization attempts. HOS test was investigated in 4 subgroups including HOS 1, HOS 2, HOS 3, and HOS 4 according to the degree of sperm tail swelling and compared between the two groups.
RESULTS: Four subgroups were compared and statistical significance was demonstrated in HOS 1, HOS 3 and HOS 4 tests (p < 0.001) in fertile and infertile men. Highest HOS 1 and lowest HOS 4 grades were determined in Group A. However, no statistical significance was determined between two groups in HOS 2 test which was minimal swelling in sperm tails.
CONCLUSIONS: HOS 1, HOS 3 and HOS 4 subgroups of HOS test are reliable and useful methods providing important information regarding the sperm function. A high HOS test 1 grade plus a low HOS test 4 grade should suggest a fertility problem, despite a normal semen analysis. HOS test subgroups provide additional information in normospermic cases with unexplained infertility.

BACKGROUND: Although different techniques for sperm immobilization have been described, their value has not been assessed in an adequately powered randomized study. The aim of this study was to compare two types of sperm immobilization methods prior to ICSI and to test the hypothesis that triple touch immobilization (TTIm) would lead to a higher (5% -65% up to 70%) fertilization rate (FR) than single touch immobilization (STIm).
METHODS: A total of 3056 metaphase II (MII) oocytes, from 290 patients, were randomly assigned to the STIm group (n = 1528 oocytes; 145 cycles) or to the TTIm group (n = 1528 oocytes; 138 cycles). A total of 1478 oocytes (STIm group) and 1476 oocytes (TTIm group) were used in the statistical analysis. The primary outcome variable was FR. Secondary outcome variables included: number of good quality embryos (GQE) on day 2 and day 3, implantation rate (IR) and implantation with foetal heart beat rate (FHB). Statistical analysis was done using the Fisher Exact test with a significance level of 0.05.
RESULTS: The results showed no differences in FR between both groups. The proportion of good quality embryos on day 3, was significantly higher in the STIm group (37.5%) compared to the TTIm group (31.8%; p = 0.02).
CONCLUSIONS: In this RCT, the hypothesis that the post-ICSI FR would be higher after TTIm than after STIm was not confirmed and the number of good quality embryos on day 3 was significantly lower in the TTIm group than in the STIm group. These data suggest that more \'aggressive\' TTIm technique has no advantages compared to the STIm technique.

The aim of this study was to analyse the meiotic segregation and DNA fragmentation rates in ejaculated spermatozoa of Tunisian men who presented the macrocephalic sperm head syndrome and to compare the results with those from 20 fertile men with normal semen profiles. Sperm DNA fragmentation was evaluated by the terminal desoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labelling assay. Fluorescence in situ hybridisation for chromosomes X, Y and 18 was performed for the study of meiotic segregation. Despite a normal blood karyotype, patients with large-headed spermatozoa showed a significantly higher incidence of sperm chromosomal abnormalities compared with the control group. For all the patients, tetraploidy, triploidy and diploidy were the most observed abnormalities. A very high level of DNA fragmentation was shown for these patients. In conclusion, our results demonstrated that patients with large-headed, multiple-tailed spermatozoa had significantly higher incidence of sperm chromosomal abnormalities and very high level of DNA fragmentation. So intracytoplasmic sperm injection should not be recommended to these patients, not only because of its low chances of success rate but also because of its high genetic risk.

This study was designed to compare the performance of the kits Diff-Quick, Hemacolor and Spermac for staining the spermatozoa of rainbow trout. Automated sperm morphology analysis (ASMA) was performed using two image analysis programs to determine the sperm measurements: head size (length, width, area and perimeter), shape (ellipticity, rugosity, elongation and regularity) and tail length. Diff-Quick was found to be the best procedure for staining the trout spermatozoa. The use of this method rendered the highest number of cells correctly analyzed, and provided good colour intensity and contrast of the sperm head. No differences among the methods were detected in terms of tail length measurements. Mean values established using Diff-Quick for the main morphometric variables were: head length 2.93+/-0.13 microm; head width 2.33+/-0.15 microm and tail length 34.16+/-1.66 microm. Based on these findings, we recommend the Diff-Quick staining kit for its accurate and reproducible morphometric results. Notwithstanding, when analyzing the sperm tail of the rainbow trout, the Spermac method offers improved contrast.