When three-dimensional structures are bilaterally asymmetric, they become chiral, meaning that the two mirror-images cannot be superimposed. In animal body forms, chirality, either external, internal, or both, is a near-ubiquitous feature (reviewed in, for example, Ludwig, 1932; McManus, 2004). Well-known examples are coiled snails, claw asymmetry in crabs, eye torsion in flatfish, and vertebrate internal visceral organisation. However, the evolutionary developmental biology of symmetry breaking remains poorly understood (Palmer, 2004; Schilthuizen 2013; Davison et al., 2016).
Some symmetry-breaking developmental cascades have been partially resolved (Grande and Patel, 2009; Basu and Brueckner, 2008; Frasnelli et al., 2012). However, most of these occur at a relatively advanced stage of development, and evidence is mounting that the actual onset of symmetry breaking takes place much earlier in development and involves chiral molecules involved in the cytoskeleton (Brown and Wolpert, 1990; Vandenberg and Levin, 2010). For example, Davison et al. (2016) showed that, in the pond snail, Lymnaea stagnalis (Linnaeus, 1758), and the frog, Xenopus laevis Daudin, 1802, the upstream action of formins, proteins that regulate the polymerization of cytoskeletal actin filaments (Evangelista et al., 2003), and, hence, cell polarity (Nelson, 2003), translated downstream into reversed or randomized whole-body chirality. These observations support the “F-molecule” hypothesis of Brown and Wolpert (1990), which suggests that a chiral molecule in the zygote sets in motion directionally asymmetric development during ontogeny, and that such a mechanism may be acting in animals generally (Oliverio et al., 2010). In gastropods, this would agree with the single-gene, delayed inheritance, in which the offspring’s coiling is determined by the mother’s, not by the offspring’s genotype (Sturtevant, 1923; Schilthuizen and Davison, 2005).
Evaluating if and how molecular, intracellular chirality may be connected to organismal chirality is laborious and time-consuming, especially if this is to be done across multiple species. Methods for rapid assessment of cell polarity would facilitate this work. Recently, techniques to visualise cellular chirality by allowing cell populations to grow on ring-shaped micropatterns (Wan et al., 2011) have become available. In some taxa, the striking morphology of mature sperm cells may also help. In many Mollusca, Gastrotricha, Kinorhyncha, Tardigrada, and in several groups of arthropods and vertebrates, spermatozoa have a helical structure (Ludwig, 1932; Baccetti and Afzelius, 1976; Jamieson, 2007), and cell polarity may be readily determined by inspection of their chirality. In helical asymmetry, the two mirror-image forms are called dextral, right-handed, or clockwise coiled (D) and sinistral, left-handed, or anti-clockwise coiled (L), where helix coiling direction is determined by the rotation (clockwise or anti-clockwise) as the helix is traced starting at the end nearest the observer when viewed end-on. Systematic analyses of spermatozoon dextrality and/or sinistrality may provide further insights into connections between cytoskeleton chirality and asymmetric development.
During spermiogenesis of helical sperm cells, a sheath of microtubules forms, which subsequently develops into a microtubular helix that envelops the spiral acrosome and nucleus, and, in some cases, midpiece, and is then shed (Healy, 2001; Aire, 2014). It has been a matter of contention which molecular cell component drives the formation of this helical structure. Some authors have implicated the microtubular helix (e.g., Kondo et al., 1988). However, because the contact between the microtubuli and the spiral nucleus is minimal and transient, this is no longer considered tenable, and, instead, the crystalline conformation of DNA-histone molecules in the nucleus and/or the contents of the acrosome are currently thought to organise in a helical arrangement, with the microtubular helix secondary to that (Fawcett et al., 1971; Threadgold, 2017).
Although numerous studies have documented helical shape of sperm cells, few pay explicit attention to the coiling direction of these helices. In fact, as Ludwig (1932) lamented, a considerable number of authors seem to have been unconcerned about the true coiling direction of the sperm cells in their study organism, and have depicted either a left- or right-handed spiral based on personal preference rather than biological reality. Photographic images also do not often give a reliable indication of coiling direction. By their nature, transmission light or electron microscope images do not reveal whether a gyre is clockwise or anticlockwise, whereas scanning electron microscope images are sometimes mirror-imaged during the publication process (see Discussion). Consequently, very little reliable information is available on the degree of intra-individual, interspecific, and intraspecific variability in sperm cell coiling direction.
We therefore used scanning electron microscopy on relatively large numbers of mature sperm cells from multiple species of two taxa with helical sperm cells that have figured prominently in the sperm morphology literature, namely land snails and slugs (Stylommatophora) and songbirds (Passeriformes). With some provisos, we confirm Ludwig’s (1932) suspicion that in land snails, spermatozoa appear to be consistently dextral, whereas in songbirds, they are sinistral.