Flagellar Mechanics and Sperm Guidance

Motility is a characteristic function of the male gamete, which allows spermatozoa to actively reach and penetrate the female gamete in organisms with internal and external fertilization. Sperm motility is acquired under the control of many extrinsic and intrinsic factors and is based on the specialized structure of the sperm flagellum. An overview of how the sperm flagellum is organized and works to support cell motility is presented, with special focus on the molecular mechanisms and factors involved in the development and maintenance of sperm motility. Data obtained both in organisms with external fertilization, such as sea urchins, ascidians or fishes as well as those relying on internal fertilization, such as mammals, are critically analyzed.

In most animal species, sperm motility is dependent on a long appendage called flagellum. Flagella are essential organelles found in most eukaryotic cells: their basic structure is the axoneme, built of a scaffold of microtubules and responsible for movement generation in an autonomous manner provided energy in the form of ATP is present. Beating of flagella allows movement, using thrust on the milieu surrounding sperm cells and is responsible of the translational drive of spermatozoa either in the fluid or by contact with structures cells or tissues. The present paper aims to describe:

1. The biochemical and structural elements of the “9+2” flagellar structure, so called axoneme, a complicated arrangement of at least 250 different protein subunits which sustains motility.

2. The mechanisms of wave generation and propagation along the axoneme of flagella, stating that in paradigms of wave propagation, a clear distinction is made between the dynein dependent microtubule sliding (oscillatory motor) and the bending mechanism (including regulator of wave propagation). The waves propagation is supported by a bending/relaxing cyclic mechanism which propagates in register, but frame-shifted with the powering action of the dynein-ATPase motors all along the axoneme. While knowledge has been largely accumulated on the motor components, little is known about the elements regulating the bending processes.

3. Guidance of spermatozoa is closely dependent on flagellar behavior. An overview of the various ways by which a spermatozoon can orient itself or be oriented to the corresponding egg in order to improve fertilization success is presented. As specific guidance mechanisms occur in response to chemicals such as Ca2+ ions controlling asymmetry of flagella beating special emphasis is devoted to such regulatory aspects. A discussion is also devoted to the way a cell elaborates its own flagellum and details possible hypothesis able to explain the origin of the axoneme, recognized as an ancestral structure with a high degree of conservation, as well as phylological aspects of this unique mechanical device used in an extremely large variety of situations to insure efficient cell movements of the male gametes, but also many unicells, such as the green algae Chlamydomonas, used as models to better understand the properties of this ubiquitous organelle due to its high degree of structure and function conservation all along evolutionary tree.

A variety of broadcast-spawning marine organisms require sophisticated sperm guidance mechanisms, named chemotaxis, to locate the egg and fertilize it. In sea urchins, oocytes release small peptides from the egg outer envelope that bind to their sperm flagellar receptors and trigger a signaling pathway that results in intracellular Ca2+ concentration fluctuations. Each transient Ca2+ increase leads to a momentary elevation of flagellar bending asymmetry which results in a pronounced turn essential for chemotaxis. In addition, this process needs a precise spatiotemporal coordination between the Ca2+-dependent turns, the form of chemoattractant gradient and periods of straighter swimming. Chemotaxis results when spermatozoa are able to undergo Ca2+-dependent turns when swimming down the chemoattractant gradient, while they suppress turning events when swimming up the gradient. This chapter summarizes the sequence of events and known components of the signaling pathway leading to chemotaxis in sea urchin spermatozoa, and the strategies that are being employed to unravel this fascinating and fundamental process.

Urochordates are marine animals, and spermatozoa of many urochordates show chemotactic behavior toward conspecific eggs during fertilization. Sperm chemotaxis of the ascidian Ciona intestinalis has been particularly well investigated. The sperm-activating and sperm-attracting factor (SAAF) of the phlebobranchian ascidians are secreted from the egg cells, and identified as polyhydroxylsterolsulfates. The molecular structure of SAAFs differs in different species, and the differences may cause species-specific chemotactic responses. Ascidian spermatozoa appear to sense a SAAF concentration decrease, resulting in a transient increases in intracellular Ca2+ in the flagellum and quick changes in the swimming direction of the sperm. In this chapter, we will introduce the features and molecular mechanisms of sperm chemotaxis in urochordates, and particularly those in ascidians.

Pacific herring (Clupea pallasi) are estuarine fish that spawn in reduced salinity water. The spermatozoa are virtually motionless upon spawning into water of varying salinities and only initiate motility upon contact with a chorion-bound glycoprotein. Sperm can remain in the water column for up to 24 hrs, yet are still capable of fertilizing eggs. Immotility in the environment is maintained as a result of herring sperm utilizing reverse sodium (Na)-calcium (Ca) exchange (Ca2+ in, Na+ out) as a mechanism for increasing intracellular calcium at motility initiation. The primary initiator of motility, Sperm Motility Initiation Factor (SMIF) requires protein kinase C activation that in turn appears to increase the reverse Na-Ca exchanger. A nondiffusible chemoattractant, Micropylar Sperm Attractant (MSA) is also present on the chorion immediately surrounding the micropyle opening in herring (as well as other fish and insects) that induces a rapid increase in intracellular Ca2+ when sperm come in contact with it and this causes sperm to make sudden turns toward the canal opening. As such, herring sperm appear to undergo at least two increases in intracellular Ca2+: one at motility initiation by SMIF, and a further increase as they contact MSA at the micropylar opening.

The specific guidance mechanisms occurring in response to chemical substances (often via Ca2+ controlled asymmetry of beating) or using guides developed in various species because of special structures or tissues as well as by eggs in the vicinity of a unique entrance point on egg surface, the micropyle present in some species, are explored in this chapter. In order to avoid an excess of overlap with other chapters, several situations are described either, in the case of "simple spermatozoa" in species with external fertilisation. Part of this review chapter presents a tentative synthesis of the distribution of chemotaxis during the course of evolution.

In the recent two decades mammalian spermatozoa were demonstrated to perform chemotaxis, thermotaxis and rheotaxis. It is believed that in the Fallopian tube spermatozoa are first guided to the fertilization site by long-range mechanisms, thermotaxis and rheotaxis, and there they are guided to the egg by two processes of chemotaxis, considered a short-range mechanism. The occurrence of chemotaxis and thermotaxis in additional locations along the female genital tract cannot be excluded.

Sperm cells evolved to acquire a sophisticated motility structure called a flagellum which gives spermatozoa a self-propelling force. Nonetheless, in internal fertilizing species such as mammals, the length and complexity of the female genital tract mean that sperm motility is insufficient to accomplish the sperm’s mission of transferring genetic material to the oocyte. Thus, a long-standing question in the reproductive biology field has been how spermatozoa are delivered to the oocyte surface. Several mechanisms have been proposed to help sperm transport, such as oviduct peristalsis, chemotaxis, thermotaxis and rheotaxis. In this chapter we will discuss the state of the art of sperm chemotaxis.

Sperm thermotaxis, the active orientation of sperm swimming according to a temperature gradient has been suggested to act as a long-range guidance mechanism in the oviduct during fertilization, between the cooler sperm storage site and the warmer fertilization site. In this process capacitated spermatozoa can sense even very shallow temperature gradients. They respond to the changing temperature by modulating their flagellar beating. The outcome is a higher frequency of turns and hyperactivation events when the temperature drops, and a rather linear swimming when they sense a temperature increase. In this way they are guided towards the warmer temperature.

Spermatozoa face the Herculean task of finding an egg and are beset with numerous challenges, not least reaching their target without getting lost. Furthermore, the sperm flagellum not only allows rapid swimming but also an ability to steer and thus navigate, invariably coupling sperm motility and guidance cue response. Thus in the following we briefly review the mechanics of how sperm swim, together with modelling strategies for developing computational simulations of swimming sperm. We proceed to consider how these simulations can inform our understanding of sperm guidance together with the limitations of modelling approaches, as well as perspectives of future studies that can be tackled with present modelling frameworks and where fundamental advances in our biological understanding are required for further progress.

Sperm chemotactic behavior is based on the control of swimming direction. Transient conversion of the asymmetry in the flagellar waveform is the most used regulatory mechanism to change the swimming direction. Direct regulation of outer arm dynein by a neuronal calcium sensor type of Ca2+-binding protein, calaxin, is a prerequisite for the regulation of chemotaxis. Bikont species, such as green algae, brown algae, dinoflagellates, ciliates and excavates, also show similar directional movements, including phototaxis, chemotaxis, and responses to mechanical stimuli. These behaviors depend on changes in flagellar motility in response to the gradient or direction of chemical or physical stimuli. However calaxin is not present in bikont species; instead they appear to use another Ca2+-sensor similar to the outer arm dynein light chain LC4 in Chlamydomonas. In this chapter, we briefly describe the mechanism of sperm chemotaxis, compare it with flagellar regulation seen in several taxis of bikont species, many of them model species for understanding flagellar mechanics, and discuss the common and divergent strategies tuning the control of flagellar response during eukaryotic taxis.

This chapter comes in continuity with chapter 10 in a sense that it tentatively aims to provide a synthetic overview of problematic posed by sperm guidance in a large variety of species and summarize globally the diversity of mechanisms individually adopted in each group where sperm attraction was characterized, while discerning common strategies.

This book is aiming to present a compilation of most data presently explaining how a flagellum operates and how it governs the direction of the sperm cell that it propels. Sum of knowledge in these fields and general laws that can be formulated are briefly developed in this conclusion.