2. Evolution of Mammalian Sperm
Although mammalian sperm share a common structural pattern across species, they differ considerably between taxa, both in shape and size. This begs the question as to how such diversity originates, both with regards to the mechanisms of sperm formation and to the selective pressures that drive the evolution of the male germ cell.
The general structure of the sperm cell consists of a head and a flagellum. The head carries the nucleus with the haploid chromosome set and a secretory granule, the acrosome, that contains enzymes which, upon release in the vicinity of the oocyte, help in penetration of its vestments. The flagellum, which attaches to the head via the neck or connecting piece, consists of three regions, known as the midpiece, the principal piece, and the terminal piece. The midpiece bears the mitochondria that supply energy via oxidative phosphorylation, and the principal piece propels the cell and also contributes to energy generation via glycolysis [2
Departures from this overall morphological pattern are seen in species that have sperm with no flagellum or that could have two or more. The acrosome may be lacking in some species of fish. In other species, the nucleus may be absent in a subpopulation of spermatozoa that could act as fillers that may carry the nucleus-containing spermatozoa to the site of fertilization. Major differences in sperm shapes make this the cell with the greatest morphological diversity of any cell type [3
]. In mammals, the majority of species have sperm heads that are round or oval, and symmetric but in some taxa (marsupials and rodents), there are major departures from this shape. The sperm head in these taxa could be asymmetric, falciform, with a displacement in the insertion of the flagellum, an acrosome that develops to a large size or to a very small one, and there may be an array of apical or basal appendices that further contribute to asymmetry. These modifications considerably influence the pattern of cell movement and its hydrodynamic efficiency. It is yet not clear how this array of shapes is generated during the period of sperm formation.
The sperm cell is the end result of a highly organized process, known as spermatogenesis, that takes place in the seminiferous tubules of the testis. The process involves three distinct phases. First, a phase of proliferation in which the diploid stem cells, known as spermatogonia, multiply by mitosis. Second, a meiotic phase, in which there is a reduction in the number of chromosomes and that ends with the formation of the haploid round spermatid. Third, the phase in which the cell differentiates from a spheroid to the shape and size typical of each species (spermiogenesis). During this latter period, which may take several days, there are several events including the formation of new organelles such as the acrosome, resulting from the fusion of vesicles originating in the Golgi apparatus, and the chromatoid body, which is composed of RNA. In addition, there is nuclear remodeling and shaping with chromatin condensation and elimination of residual cytoplasm. There is also assembly of the flagellum and relocalization of mitochondria. The process ends with spermiation, the release of the sperm cells into the lumen of the seminiferous tubules.
Spermatogenesis is highly conserved in general terms, although it seems that the first phases are more conserved than the latter ones [6
]. The phase of spermiogenesis seems to be more divergent among species, and this may relate to the time required to remodel the sperm nucleus and for the elongation of the flagellum, which appears to be more species specific. The underlying molecular mechanisms may also differ widely, particularly those occurring as part of the communication between the somatic and the germ cells.
The entire process of spermatogenesis occurs with the germ cells receiving support and nourishment from the Sertoli cells (specialized cells that orchestrate germ cell development and differentiation). This seems to be a limiting factor in the production of spermatozoa because the efficiency of cell–cell interactions may limit the number of spermatozoa being supported by Sertoli cells [7
]. In fact, the proportion of Sertoli cells in relation to the number of germ cells is directly correlated to the number of spermatozoa produced in the testes and present in epididymal reserves [8
Diversity in sperm shape and size may be linked to various selective processes. Individuals appear to have the plasticity to adjust the number of sperm produced and, to a certain extent, some morphological features such as sperm size, in response to the perceived risk of potential competition between males. This suggests that males may adjust the processes of sperm formation over a short period of time [9
], by modifying the relative size of the testis, its architecture (that is, the proportion of tissue devoted to sperm production) and kinetics [10
], and perhaps the molecular processes that underlie sperm differentiation.
At an evolutionary scale, selective forces could favor males that produce spermatozoa with certain shape or size if these traits are important determinants of their reproductive success [11
]. When females mate with two or more males, competition between spermatozoa occurs in the female tract, and males with more sperm or sperm cells with faster or more efficient swimming may have advantages in fertilizing oocytes. Females could also exercise some form of selection of one type of sperm over another or impose barriers that sperm need to overcome to reach the site of fertilization. These forces are collectively known as post-copulatory sexual selection [4
]. An additional powerful selective force is represented by the mode of fertilization. Species with external fertilization release their gametes and, in the rather uniform water environment, sperm need to quickly find the oocyte and engage in fertilization. In species with internal fertilization, the interaction between sperm and the oocyte has become more complex and, in particular, the oocytes are surrounded by thicker layers of cells or coats, and sperm have evolved a series of modified structures to engage in and succeed in fertilization. These modifications include, among others, an enlargement of the enzyme-containing acrosome and a tighter compaction of chromatin [12
]. One aspect of sperm biology that has probably been influenced by both sperm competition and the mode of fertilization is sperm swimming patterns. Sperm need to swim actively to negotiate barriers in the female tract. Furthermore, they develop a hyperactivated movement after a period of residence in the female tract that allows them to move progressively in the more viscous fluids of the tract. Hyperactivation is important for sperm to generate the propulsive force required to penetrate the zona pellucida. The demands for forceful movement are subserved by both the length of the flagellum and the shape of the sperm head. Sperm with greater size appear to swim faster, and so do spermatozoa with more elongated sperm heads [14
]. Thus, various selective forces appear to have influenced the design of spermatozoa both in relation to sperm head shape and the size of the different sperm components, particularly the flagellum. This, in turn, may have imposed a need to modify processes of sperm formation, in connection to spatial aspects of seminiferous tubule organization, the kinetics of sperm differentiation and, in addition, the molecular regulation of cellular remodeling.
At the molecular level, the diversity of sperm morphology and function has been influenced by the positive and negative selection of genes. Modifications in amino acid sequences have been selected during evolution [16
]. Genes implicated in male reproduction are evolving fast [17
], especially proteins essential for spermatogenesis, metabolism, flagellar motility, capacitation, acrosome reaction, and sperm–egg interaction [18
]. In particular, genes important for sperm differentiation and protein trafficking appear to be under positive selection. For example, SPAG17
was shown to be one of the genes that differentiates humans from Neanderthals [19
], and its encoded protein has been found to be positively selected [17
]. SPAG17 protein was originally characterized as a central pair protein present in the flagellar axoneme [20
]. However, this protein is also present in Golgi vesicles, the acrosome and manchette in developing spermatids and has been shown to be essential for sperm differentiation [21
]. The Spag6
gene, another gene originally described to play a role in the central pair of the axoneme [22
], has undergone positive selection [23
], although there is no change in the amino acid sequence of the protein, and this positive selection seems to be associated with transcriptional control [24
]. Recently, a duplication for the Spag6
gene was found in the mouse, where the Spag6
-BC061194 gene is the ancestral gene [25
]. In spermatids, SPAG6 protein localizes to Golgi vesicles, the acrosome and the manchette. Moreover, Spag6
knockout results in impaired spermiogenesis [26
]. The IFT88
gene was also selected during evolution [27
]. IFT88 protein is present in developing spermatids and localizes to Golgi vesicles, the acrosome–acroplaxome complex as well as the manchette and flagellum [28
mice display impaired spermiogenesis with spermatids carrying several anomalies [29
]. Other intraflagellar transport genes were similarly selected during evolution [27
] and are essential during spermiogenesis, including Ift20
] and Ift172
5. Conclusions and Future Directions
This review integrates recent findings regarding protein trafficking during sperm differentiation. The complete interactome of the proteins involved in Golgi transport and IMT mechanisms remains to be characterized. Here, we performed an in silico protein interaction analysis as a tool to generate models for the interactome based on the current literature. Future investigations should be performed to validate these two models.
As mentioned previously, super-resolution techniques are cutting edge technology that allow investigators to study protein trafficking. Several advances have been made using this technology, and it is expected that, in the near future, new algorithms will provide further improvement of the resolution of the images. Another technology that provides high resolution at the molecular level is electron cryotomography (Cryo-ET). The resolution of this technology reaches 1–4 nm, and the combination of series of 2D images generates a 3D view of the samples.
Several issues remain unresolved regarding protein trafficking in the manchette. For example, there is controversy about the nucleation of microtubules that originate the manchette assembly. Moreover, post-translational modifications of tubulin and actin filament tracks have not been fully characterized. Future studies should help elucidate the spatial and temporal coordination of the protein trafficking mechanisms, and how these processes regulate the differentiation of spermatids to spermatozoa.
Understanding the interactome and the protein trafficking mechanisms during sperm differentiation has clinical implications. Defects in sperm differentiation lead to male infertility, congenital disorders, and spontaneous abortion. Therefore, it is important to focalize our scientific efforts on developing new strategies to dissect the interactome of proteins and the mechanisms of protein transport in spermiogenesis.