Background
Morphologic sperm traits, including the shape of the head and the length of the flagellum, are diverse in mammals. In general, the width and length of the sperm head vary due to variations in the size and organization of the acrosome and nuclei [
1]. Alternatively, the sperm flagellum can be divided into the following four distinct segments: the connecting piece, also termed the neck; the middle piece, or midpiece; the principal piece; and the end piece [
2]. The connecting piece is adjacent to the head and contains degenerated centrioles, which lack many structural characteristics and protein composition in comparison with typical centrioles in somatic cells [
3], and transitional structures linked with internal structures in the midpiece [
4]. The midpiece is defined by the cytoskeletal structures surrounded by packed mitochondria [
5]. The principal piece features the fibrous sheath (FS) that surrounds the cytoskeleton [
6]. The end piece is a prolongation of the principal piece without any accessory structures [
7]. The lengths of the connecting piece and the end piece are usually short across species, whereas the lengths of the midpiece and the principal piece contribute to the variation among sperm flagella in mammals [
8]. The mitochondria are the triphosphate adenosine (ATP) source in the sperm midpiece and produce and transport energy to the axoneme in the principal piece, which generates the driving force of the sperm [
9]. Thus, the lengths of the sperm midpiece and principal piece may be critical in determining sperm swimming velocity and the rate of fertilization [
10‐
13].
In addition to large variations in the shape of the head and the length of the flagellum, the morphology of the internal ultrastructures containing the mitochondrial sheath (MS), outer dense fibers (ODFs) and fibrous sheath (FS) differ widely between mammalian species [
14,
15]; there is little variation in axonemes between species. The axoneme exhibits a conserved 9 + 2 microtubule arrangement in cross-section, with nine doublet microtubules linking to a central pair complex that is composed of two singlet microtubules. The coordinated activities of multiple dynein are driven by hydrolysing ATP in the axoneme to drive flagellar motility [
16]. Traditionally, it is thought that the width of the axoneme is constant from the base to the tip of the sperm flagella, even across species [
17].
The MS defines the region of the midpiece and surrounds the ODFs and axoneme. The MS consists of different numbers of mitochondria, which are elongated and arranged in a helix pattern, called gyres, from species to species. For example, there are an estimated 10–12 gyres in humans, 15–17 in dogs, 97 in mice and 350 in rats [
14].
Ordinarily, the ODFs comprise a set of nine striated columns that start in the connecting piece and end at a specific position of the tail in certain species. In longitudinal view, each fiber is largest at the proximal end of the midpiece and tapers progressively along the flagellum, with each ending at different positions along the principal piece [
18]. Cross-sectionally, the shape and size of each fiber differ between and within species. Fibers 1, 5, 6, and sometimes 9, namely odfs 1, 5, 6, and 9, are generally larger than odfs 2, 3, 7 and 8 [
19]. It is thought that the ODFs not only protect the tail from damage caused by the shearing force during epididymal transport and ejaculation but also increase the stiffness of the flagellum and stabilize the axoneme [
20,
21].
The FS is a cytoskeletal structure of the principal piece. It consists of two longitudinal columns connected by semicircular ribs. The size of the longitudinal columns is largest at the anterior part of the principal piece and decreases gradually along the principal piece [
22]. Cross-sectionally, the shape and size of the longitudinal columns, but not the semicircular ribs, exhibit major variance across mammalian species. In the present study, the area of the FS is represented by the cross-sectional area of the longitudinal columns. It is believed that the FS serves as a scaffold for proteins participating in glycolysis, cAMP-dependent signalling transduction and mechanical support [
23,
24].
Although these structures of the flagellum have been characterized in many species, limited data about the three-dimensional nature of these internal structures between and within species have been reported. Furthermore, the relationships between the three-dimensional internal ultrastructures and sperm morphology, as well as sperm swimming velocity and mitochondrial functions, have been poorly explored.
To address these questions, sperm from ten common species, including guinea pig, mouse, rat, golden hamster, dog, bull, pig, rabbit, goat and human, were collected and analysed by transmission electron microscope (TEM) and scanning electron microscope (SEM) combined with biochemical assays and previous swimming velocity data. Our study not only provides quantitative data to describe differences in the three-dimensional ultrastructures of flagella and sperm morphologic characteristics but also resolves the relationships mentioned above.
Discussion
Morphological normality of sperm flagella is critical for sperm motility and fertilization processes. Sperm morphology assessments, including the morphologies of the sperm head, midpiece and other flagellar components, not only reflect the sperm quality and environmental factors during spermiogenesis but also become an important cue for diagnosis for male infertility in the clinic [
30,
31]. Additionally, we enhanced our understanding of the details and relationships between sperm micro- and macro-morphologies, which will advance understanding of the underlying mechanisms of sperm motility. For instance, the mitochondrial sheath not only produces optimal levels of ROS and ATP but also prevents the internal structures from buckling out from the flagellum during sperm swimming. ATP is transported to the axoneme and hydrolysed by dyneins, which are distributed in the axoneme asymmetrically and generate an imbalance force to drive flagellar motility [
32]. The functions of ODFs and FS are not fully understood. In general, the ODFs are considered as restraining structures that increase the stiffness of the flagellum and amplifiers that increase the bending torque generated by the axoneme’s bending movement [
33]. The FS also acts as a rigid structure that increases the stiffness of the flagellum; meanwhile, it is involved in glycolysis and Ca
2+ signalling transduction dependence with relevant enzymes and ion channels [
34‐
36]. Thus, descriptions of the three dimensions of sperm components, including head length, head width, midpiece length, principal length, flagellar length, areas of internal flagellar ultrastructures in each segment and the volume of mitochondria across 10 common species, may advance knowledge regarding sperm motility.
In this work, relationships between the internal flagellar ultrastructures, the lengths of the flagellar components and sperm motility were established. We also found that the areas of mitochondria and ODFs, as well as the major components of odf fibers, namely, odfs 1, 5, and 6, are positively correlated with the lengths of the flagellar components. In other words, the longer is the flagellar length, the thicker is the flagellar diameter. Speculatively, longer flagella have larger mitochondrial volumes. The flagellar lengths, especially the length of the midpiece, are positively associated with the volume of mitochondria across 10 species. The sperm midpiece length was largely considered a plausible predictor of sperm swimming velocity in house mice and primates [
10,
37], although some inconsistent results were reported for a passerine bird [
38]. Although the reasons for this discrepancy remain unknown, our findings further support the hypothesis that sperm with longer midpieces swim faster than do sperm with shorter midpieces, which might contribute to larger mitochondrial volume and function. Notably, ATP content is slightly positively associated with sperm swimming velocity, whereas the correlation coefficient between mitochondrial volume and swimming velocity was far smaller than the correlation coefficient between mitochondrial volume and ATP content. This suggests that other unidentified factors impact swimming velocity [
39]. The movement properties of the principal piece suggest that it may be a potential candidate. Unfortunately, we did not find any relationship between the area of the FS and swimming velocity or the length of any flagellar components.
Regarding sperm competition, beneficial changes in sperm morphology can increase sperm swimming velocity, which is a main determinant of fertilization success [
40,
41]. For instance, a positive relationship between sperm competition levels and sperm midpiece volume has been reported by Dixson and colleagues [
37]. However, the regulators of flagellar length and midpiece volume (which is equal to the sum of the mitochondrial and ODF volumes) are largely unexplored. Our study presents ODFs and/or mitochondria as potential determinants of flagellar length. The proteins involved in the development of ODFs, especially odfs 1, 5, and 6, and the size of mitochondria should be examined in further studies. Lower sperm motility, and even asthenozoospermia in humans, can occur due to deficiencies in the development of the ODFs and mitochondria [
27,
42]. In fact, we found that the lengths of sperm midpieces and principal pieces from asthenospermic samples were shorter, concomitant with higher defect rates of axonemes, ODFs and mitochondrial structures, in comparison with those from normospermic samples (Additional file
8: Figure S8).
Furthermore, we found that the area of the axoneme slightly tapered from the base to the tip of the flagellum in all 10 species, in contrast to existing beliefs that the width of the axoneme is constant along flagella, even across species. To confirm this result, we detected a similar change tendency by measuring the areas of the axoneme from 46 human normospermic and 25 asthenospermic sperm samples (data not shown). In addition, slopes of axonemes from 10 species were negatively correlated with the lengths of the components of sperm flagella. In other words, the longer is the flagellar length, the smaller is the slope of the axoneme along the flagellum. The physiological functions of the inconsistent width and slopes of the axonemes across species remain unclear.
Moreover, the area of the FS was not associated with any dimension of the sperm components. Additionally, it does not seem to be the major source of ATP, although glycolysis-related kinases are located in the FS. In our opinion, the functions of the FS may focus on the signal transductions for initiating sperm movements. For example, the Catsper family of proteins, which mediate Ca
2+ influx, are exclusively located in the FS [
43]. The size of the FS may be positively correlated with the expression levels of Catsper family proteins and Ca
2+ signal intensity during sperm capacitation [
36]. In addition, the principal piece is considered to be the source of propulsion forces [
44]. Speculatively, the size of the FS may be associated with the propulsion forces to mediate the swimming pattern after capacitation. This possibility should be validated in further studies.
Additionally, we did not find any relationships between the head dimension and sperm ultrastructures. In a previous study, it was reported that the head length and the head length/width ratio was associated with testes mass using data from 194 Entherian mammals [
28]. Indeed, we applied these data from Eduardo RS Roldan’s work to analyse the relationships between head dimension, sperm flagellar length, and VSL. We found that the head length was slightly associated with the midpiece length and VSL; the head width was also slightly associated with the midpiece length and VSL (data not shown). Therefore, these results regarding relationships between head dimension and sperm ultrastructures may apply to few species. More mammalian species should be examined to validate our results. Furthermore, our results cannot be applied directly to resolve the relationships between flagellar lengths and sperm ultrastructures at the within-species level. For example, Tim Birkhead and colleagues found that sperm with shorter midpieces contained the highest concentration of ATP in a passerine bird, though they also found that the lengths of sperm components were important for swimming velocity [
11,
38,
45], and other researchers found that faster velocities associated with higher ATP content and longer midpiece in many taxa may be due to increases in sperm competition levels [
46]. Additionally, these relationships found in the present study cannot be applied to non-mammalian species. For example, the ODFs are specific accessory structures in mammalian sperm that do not exist in invertebrate sperm such as the fruit fly, fish and sea squirt [
47,
48]. The longer sperm of the fruit fly (
Drosophila melanogaster) swim more slowly than do those with relatively shorter flagella [
49], though swimming velocity is unaffected by flagellar length in Atlantic salmon [
50].
Conclusion
Taken together, the common characteristics for the ten studied species are that flagella and internal ultrastructures, including MS, total Odfs, each Odf fiber, FS and axoneme, taper from the tip to end in ten species, whereas the slopes of these and shapes of sperm heads exhibit species-specific traits. Additionally, the structural description offers novel insights into the positive associations among internal structures, especially ODFs and mitochondria, and flagellar length, revealing relationships between structures and sperm physiology. Furthermore, our results not only provide original data to establish a three-dimensional view of sperm ultrastructures and morphology across 10 species at the level of electron microscopy but also highlight the potential targets. For instance, the proteins involved in the architectures of MS and ODF may also be determinants of flagellar length, which is considered to be a critical factor in sperm motility and sperm competition studies. The results of this study should enable the establishment of more precise mathematical models to better understand the development of sperm, the physics of sperm swimming and the asthenozoospermic pathogenesis.
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