Background Sperm morphology mainly refers to the shape of the head, the length of the flagellar segments, including the midpiece, principal piece and end piece, and the size of the accessory structures, including axonemes, outer dense fibers (ODFs), mitochondrial sheath (MS) and fibrous sheath (FS). Across species, there is considerable diversity in morphology. An established theory posits that the length of the sperm flagellum, especially the length of the midpiece, is a critical factor influencing sperm metabolism and velocity. However, our understanding of the relationships between sperm ultrastructures and the sperm flagellar length is incomplete. Methods The morphologies of sperm from 10 mammalian species, human, mouse, rat, dog, rabbit, goat, pig, bull, guinea pig and golden hamster, were examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). According to the SEM and TME images, the length of sperm heads and flagellar segments, the cross-sectional areas of the accessory structures and flagella and the width of sperm heads were measured using Image J software. The variation tendencies (referred to as slope) of the accessory structures along flagella were calculated by the linear regression method. Mitochondrial functions were measured using commercial kits. The velocities of sperm were measured using CASA software. Results The three-dimensional morphologies of sperm from 10 species and the slopes of internal accessory structures along flagella were obtained. The width of the axoneme tapered slightly from the base to the tip of the sperm flagellum, and slopes of the axonemes correlated negatively with the variability in flagellar length across species. Additionally, the cross-sectional areas of the ODFs and/or the MS were positively correlated with the lengths of the midpiece, principal piece, and total flagellum, as well as with sperm velocities. Mitochondrial volumes were positively correlated with ATP content and sperm swimming velocities. Conclusions Our results not only show the relationship between sperm internal structures, flagellar length and sperm physiology but also provide sizes of mitochondria and ODFs as new targets with which to study the regulation of sperm length and velocity. Electronic supplementary material The online version of this article (10.1186/s12958-019-0510-y) contains supplementary material, which is available to authorized users.
Outer dense fibers (ODFs), as unique accessory structures in mammalian sperm, are considered to play a role in the protection of the sperm tail against shear forces. However, the role and relevant mechanisms of ODFs in modulating sperm motility and its pathological involvement in asthenozoospermia were unknown. Here, we found that the percentage of ODF defects was higher in asthenozoospermic samples than that in control samples and was significantly correlated with the percentage of axoneme defects and non‐motile sperm. Furthermore, the expression levels of ODF major components (Odf1, 2, 3, 4) were frequently down‐regulated in asthenozoospermic samples. Intriguingly, the positive relationship between ODF size and sperm motility existed across species. The conditional disruption of Odf2 expression in mice led to reduced sperm motility and the characteristics of asthenozoospermia. Meanwhile, the expression of acetylated α‐tubulin was decreased in sperm from both Odf2 conditional knockout (cKO) mice and asthenozoospermic men. Immunofluorescence and biochemistry analyses showed that Odf2 could bind to acetylated α‐tubulin and protect the acetylation level of α‐tubulin in HEK293T cells in a cold environment. Finally, we found that lithium elevated the expression levels of Odf family proteins and acetylated α‐tubulin, elongated the midpiece length and increased the percentage of rapidly moving sperm in mice. Our results demonstrate that ODFs are beneficial for sperm motility via stabilization of the axoneme and that hypo‐expression of Odf family proteins is involved in the pathogenesis of asthenozoospermia. The lithium administration assay will provide valuable insights into the development of new treatments for asthenozoospermia.
It has been recently ascribed to several inflammatory cytokines (i.e. TGF-β3, TNF-α, and IL-1) a functional role in regulating Sertoli cell blood-testis barrier (BTB) dynamics. In the testis, IL-6 inhibits meiotic DNA synthesis during the seminiferous epithelium cycle, reduces sperm motility and influences the secretion of transferrin and inhibin B by Sertoli cells. Also, it has been shown that IL-6 affects tight junction permeability in Sertoli cells, but, little is known about its role in regulating the BTB. The aim of this study was to investigate the molecular mechanisms by which IL-6 affects BTB dynamics. We show that IL-6 perturbs the integrity of the BTB, and alters the normal localization and steady-state levels of BTB integral membrane proteins. We demonstrated that IL-6 regulates the BTB by inhibiting the degradation of BTB constitutive proteins and activating ERK-MAPK pathways. Our results provide mechanistic insight into the roles of IL-6 in regulating BTB dynamics.
Asthenozoospermia is a common cause of male infertility, but in most cases its etiology is unknown. The exocytic cell vesicles called seminal extracellular vesicles in the human seminal fluid have been reported to play a pivotal role in promoting the motility of spermatozoa, and functional disorder of seminal extracellular vesicles may cause male infertility. To determine whether abnormal seminal extracellular vesicles are involved in asthenozoospermia, the differential abundance proteins between normozoospermic (NSEV) and asthenozoospermic seminal extracellular vesicles (ASEV) samples were analyzed by iTRAQ coupled with two‐dimensional liquid chromatography–tandem mass spectrometry. A total of 3,699 proteins were identified in the seminal extracellular vesicles (false discovery rate <0.01). Overall, 11 proteins were significantly upregulated (>1.2) in ASEV and 80 were significantly downregulated (<0.833). Functional bioinformatic analysis showed that these proteins with differential abundance were mainly associated with transport, metabolism, and signal pathways. The changes of OPTN, SMYD2, EIF2B2, TRPV6, ACE, PRSS8, and PPAP2A in ASEV were verified by western blot analysis, and we found that the abundance of TRPV6 markedly reduced in the seminal extracellular vesicles and ejaculated spermatozoa of asthenozoospermic patients, which indicated trpv6 was important in sperm motility. This study provides deeper insight into the involvement of seminal extracellular vesicles in asthenozoospermia and should aid the search for novel biomarkers of male infertility.
Rare coding variants have been proven to be one of the significant factors contributing to spermatogenic failure in patients with non-obstructive azoospermia (NOA) and severe oligospermia (SO). To delineate the molecular characteristics of idiopathic NOA and SO, we performed whole-exome sequencing of 314 unrelated patients of Chinese Han origin and verified our findings by comparing to 400 fertile controls. We detected six pathogenic/likely pathogenic variants and four variants of unknown significance, in genes known to cause NOA/SO, and 9 of which had not been earlier reported. Additionally, we identified 20 novel NOA candidate genes affecting 25 patients. Among them, five (BRDT, CHD5, MCM9, MLH3 and ZFX) were considered as strong candidates based on the evidence obtained from murine functional studies and human single-cell (sc)RNA-sequencing data. These genetic findings provide insight into the aetiology of human NOA/SO and pave the way for further functional analysis and molecular diagnosis of male infertility.
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