Comparative analysis of mammalian sperm ultrastructure reveals relationships between sperm morphology, mitochondrial functions and motility

Abstract

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.

Keywords: Axoneme; Fibrous sheath; Flagellar lengths; Mitochondrial function; Mitochondrial sheath; Outer dense fibers; Sperm motility.

Fig. 1

Scanning electron micrographs of sperm from 10 species. a Human (Homo sapiens). b Rabbit (Oryctolagus cuniculus). c Dog (Canis familiaris). d Pig (Sus scrofa domesticus). e Guinea pig (Cavia porecellus). f Goat (Capra hircus). g Bull (Bos taurus). h Mouse (Mus musculus). i Golden hamster (Mesocricetus auratus). j Rat (Rattus norvegicus). In each panel, the left image shows sperm in whole-view, and the right images sequentially show the head, boundary of the midpiece-principal piece and the terminal of the principal piece in enlarged view. The scale bars are 5 μm in the left image and 2 μm in the right images. Please note the different lengths of the scale bars across the 10 species. The morphological features of the head and flagella are labelled by lines and arrows, respectively (n = 30 sperm/male, n = 3 males/species). The widths of positions 1–9 were measured as the diameters of flagella in these regions

Fig. 2

Transmission electron micrographs of flagellar segments from 10 species in cross-sectional view. a Diagram detailing key ultrastructures, including axoneme, ODFs, fibrous sheath, and mitochondria (red modes), in three major structural domains. The outer area is indicated as the area surrounded by the blue line; the ODF area is indicated as the area around by the purple line; the area of the axoneme is indicated as the area around by the yellow line; and the green mode indicates the FS area in the principal piece. The numbers show the numbers of odf fibers 1–9. b Human. c Rabbit. d Dog. e Pig. f Guinea pig. g Goat. h Bull. i Mouse. j Golden hamster. k Rat. The measured areas were labelled by coloured lines, as shown in panel J. The number of measured segments was more than 30 in each species. The scale bars are 400 nm

Fig. 3

Relationships between the slopes of mitochondria and t-odfs across species. Regression analysis shows a negative relationship between mitochondria and t-odfs (R² = 0.598, p < 0.05)

Fig. 4

Relationships between mitochondrial volume and ATP content, VSL. a Regression analysis shows a positive relationship between mitochondrial volume and ATP content (n = 3 replicates/sample, N = 3 male samples/species) (R² = 0.982, P < 0.001). b Regression analysis shows a positive relationship between mitochondrial volume and VSL (R² = 0.528, P = 0.017)