A Comparative Study of Snake and Lizard Tongue-flicking,with an Evolutionary Hypothesis

Many lizards and all snakes flick their tongues. It is known that this unique behavioral pattern serves to collect airborne and substrate chemicals which give the animal information via Jacobson's Organ about the location of food, conspecifics, and possibly other environmental factors. However, a comparative topographic analysis of tongue movements in squamate reptiles is lacking, and it might shed light on the evolution of this behavior. In this study, a survey was made of the lizards and snakes which tongue-flick. Observations and films were made of 25 lizard species representing 10 families and 30 snake species representing 5 families. The information from observations and film analyses of representative species was used to hypothesize the steps of the evolution of tongue-flicking from the simple downward extensions of primitive lizards to the complex multiple oscillations of snakes.     

Ultrastructure of the tongue and anterior process of the sublingual plica in four species of venomous snakes

The general histology and ultrastructure of the tongue and anterior process of the sublingual plica of four Taiwanese venomous snakes, the Chinese cobra (Naja naja atra), banded krait (Bungarus multicinctus), Taiwan habu (Trimeresurus mucrosquamatus), and bamboo snake (Trimeresurus stejnegeri stejnegeri) are described. The tongue fork exhibits a mid-dorsal invagination that broadens gradually toward its base. No mid-ventral invagination is observed. The epithelial cells on both dorsal and ventral aspects of the tongue fork have large and small microfacets, micropores and microvilli. The cell size, distribution pattern of the large microfacets, and the number of small microfacets present on both sides of the fork are essentially the same within a species, but vary among species. The function of these ultrastructures on the cell surface might be for the capture of chemical substances. The large microfacets are raised areas of the cell membrane, each with a pale granule contained within. The chemical nature of the pale granule is not yet known. The small pores surrounding the large microfacets are shallow hollows left after the release of the pale granules from the microfacets. The basic histological pattern of the tongue fork of these species is similar, being composed of a mucosal layer outside and dense musculature inside. No taste buds are discernible. The anterior processes are concave-like expansions of the anteriormost portions of the sublingual plicae. The oblique folds and micropapillae of this organ might be helpful for receiving the chemicals collected on the tongue, when the tongue makes contact with the elevated processes. The elevated processes may penetrate the ducts of Jacobson's organs to effect the final transfer.     

Suggestions for the functional relationship between differently located taste buds and vomeronasal olfaction in several mammals

In almost all mammals a well developed, paired and blind ending vomeronasal Organ (VNO) situated within the basement of the nasal septum, communicates with the oral cavity. This contact is established by two nasopalatine ducts, which penetrate the rostral palate close to the incisors. These ducts open orally into the sulcus which moulds the palatine papilla. In several mammals taste buds were found in the epithelium of the patatine papilla located within the nasopalatine ducts or close to their oral openings. Presumably these taste buds interact with the vomeronasal olfaction. It is likely that they are leading to a chemosensory sensation comparable to the combination of normal taste and smell. As not all mammals with a functionable VNO possess taste buds in this position, an inspection of the rostral part of the tongue which touches the palatine papilla presented an interesting situation concerning the distribution of taste buds. This region of the tongue is almost completely free of taste buds in species like Tupaia glis and Didelphis marsupialis virginiana, which have taste buds in the epithelium of their palatine papilla. In Lemur catta however, where the palatine papilla is lacking taste buds, the respective tongue part is densely covered with them. In this case it appears likely that they in a way of substitution functionally are connected with vomeronasal olfaction.     

Tongue manipulation of the palate assists estrous detection in the bovine

To understand the mechanisms for introducing urine or vaginal secretions into the vomeronasal organ, we used 16 mm cinematography and a freeze frame/slow motion technique to analyze the mouth and tongue movements of Brahman bulls while they examined the vulvas of restrained, estrogen-primed cows. Prior to flehmen, the mouth slowly opened, the curled tip of the tongue compressed the hard palate and the body of the tongue protruded from the mouth. The tongue maintained this form and moved forward. Once the tip of the tongue reached the incisive papilla, the body of the tongue retracted and the tip of the tongue relaxed. This tongue compression stroke (TCS) of the hard palate occurred 2 to 6 times, lasting 1 4 to 1 2 sec/stroke. Pressure changes in the vomeronasal organ are assumed to occur during and following TCSs, resulting in aspiration of any liquid in the incisive pit into the incisive and vomeronasal ducts. Such aspiration probably does not occur during flehmen because the tongue is relaxed and on the floor of the mouth.     

Prey Transport Mechanisms in Blindsnakes and the Evolution of Unilateral Feeding Systems in Snakes

Most snakes ingest and transport their prey via a jaw ratchetingmechanism in which the left and right upper jaw arches are advancedover the prey in an alternating, unilateral fashion. This unilateraljaw ratcheting mechanism differs greatly from the hyolingualand inertial transport mechanisms used by lizards, both of whichare characterized by bilaterally synchronous jaw movements.Given the well-corroborated phylogenetic hypothesis that snakesare derived from lizards, this suggests that major changes occurredin both the morphology and motor control of the feeding apparatusduring the early evolution of snakes. However, most previousstudies of the evolution of unilateral feeding mechanisms insnakes have focused almost exclusively on the morphology ofthe jaw apparatus because there have been very few direct observationsof feeding behavior in basal snakes. In this paper I describethe prey transport mechanisms used by representatives of twofamilies of basal snakes, Leptotyphlopidae and Typhlopidae.In Leptotyphlopidae, a mandibular raking mechanism is used,in which bilaterally synchronous flexions of the lower jaw serveto ratchet prey into and through the mouth. In Typhlopidae,a maxillary raking mechanism is used, in which asynchronousratcheting movements of the highly mobile upper jaws are usedto drag prey through the oral cavity. These findings suggestthat the unilateral feeding mechanisms that characterize themajority of living snakes were not present primitively in Serpentes,but arose subsequently to the basal divergence between Scolecophidiaand Alethinophidia.     

Experimental induction of palate shelf elevation in glutamate decarboxylase 67-deficient mice with cleft palate due to vertically oriented palatal shelf

BACKGROUND: Gamma-aminobutyric acid is an inhibitory neurotransmitter, synthesized by two isoforms of glutamate decarboxylase (GAD), GAD65 and -67. Unexpectedly, inactivation of GAD67 induces cleft palate in mice. Reduction of spontaneous tongue movement resulting from decreased motor nerve activity has been related to the development of cleft palate in GAD67(-/-) fetuses. In the present study, development of cleft palate was examined histologically and manipulated with culture of the maxilla and partial resection of fetal tongue. METHODS: GAD67(-/-) mice and their littermates were used. Histological examination and immunohistochemistry were performed conventionally. Organ culture of the maxilla was carried out as reported previously. Fetuses were maintained alive under anesthesia and tips of their tongues were resected. RESULTS: Elevation of palatal shelves, the second step of palate formation, was not observed in GAD67(-/-) mice. In wild-type mice, GAD67 and gamma-aminobutyric acid were not expressed in the palatal shelves, except in the medial edge epithelium. During 2 days of culture of maxillae dissected from E13.5-E14.0 GAD67(-/-) fetuses, elevation and fusion of the palatal shelves were induced. When E13.5-15.5 mutant fetuses underwent partial tongue resection, the palatal shelves became elevated within 30 min. CONCLUSIONS: These results suggest that the potential for palate formation is maintained in the palatal shelves of GAD67(-/-) fetuses, but it is obstructed by other, probably neural, factors, resulting in cleft palate.     

Feeding behaviour in Cylindrophis and its bearing on the evolution of alethinophidian snakes

Cylindrophis ruffus ingests prey using two distinct mechanisms. During initial phases of prey transport, lateral movements of the rear of the braincase combine with small unilateral movements of the toothed bones of each side; prey is usually constricted during this phase to permit the snake to push its head over the prey. Once transport has carried the leading part of the prey into the anterior oesophagus, Cylindrophis begins to use bilaterally synchronized movements of the jaw apparatus combined with low-amplitude, short wave-length flexions of the anterior vertebral column. Transport of prey is many times faster during the bilateral phase than during the unilateral phase.
Radiographic and cinematographic evidence indicates that the mandibular tips of Cylindrophis do not separate more than 1.5–2.0 times the resting distance between the dentary tips. Although this limits potential gape size, the intramandibular joint is highly mobile, allowing the mandibles to conform to a variety of prey shapes. Manipulations of anaesthetized and fresh, dead specimens revealed that the palatomaxillary arches are tightly attached to the ventral bones of the snout, movements of each arch being reflected in equivalent movements of the ipsilateral elements of the snout.
Cylindrophis represents a functional stage intermediate between most lizards with limited palatomaxillary kinesis and advanced snakes with considerable palatomaxillary mobility. Contrary to previous hypotheses, however, upper jaw liberation in Cylindrophis is due to liberation of the ventral snout, not to reduction of attachments to the braincase and snout. This suggests that the nose played a crucial role in the evolution of the feeding apparatus in alethinophidian snakes.     

The internal cranial anatomy of the Plesiosauria (Reptilia, Sauropterygia): evidence for a functional secondary palate

In the late 19th Century, the choanae (or internal nares) of the Plesiosauria were identified as a pair of palatal openings located rostral to the external nares, implying a rostrally directed respiratory duct and air path inside the rostrum. Despite obvious functional shortcomings, this idea was firmly established in the scientific literature by the first decade of the 20th Century. The functional consequences of this morphology were only re-examined by the end of the 20th Century, leading to the conclusion that the choanae were not involved in respiration but instead in underwater olfaction, the animals supposedly breathing with the mouth agape. Re-evaluation of the palatal and internal cranial anatomy of the Plesiosauria reveals that the traditional identification of the choanae as a pair of fenestrae situated rostral to the external nares appears erroneous. These openings more likely represent the bony apertures of ducts that lead to internal salt glands situated inside the maxillary rostrum. The 'real' functional choanae (or caudal interpterygoid vacuities), are situated at the caudal end of the bony palate between the sub-temporal fossae, as was suggested in the mid-19th Century. The existence of a functional secondary palate in the Plesiosauria is therefore strongly supported, and the anatomical, physiological, and evolutionary implications of such a structure are discussed.     

Hummingbird tongues are elastic micropumps

Pumping is a vital natural process, imitated by humans for thousands of years. We demonstrate that a hitherto undocumented mechanism of fluid transport pumps nectar onto the hummingbird tongue. Using high-speed cameras, we filmed the tongue–fluid interaction in 18 hummingbird species, from seven of the nine main hummingbird clades. During the offloading of the nectar inside the bill, hummingbirds compress their tongues upon extrusion; the compressed tongue remains flattened until it contacts the nectar. After contact with the nectar surface, the tongue reshapes filling entirely with nectar; we did not observe the formation of menisci required for the operation of capillarity during this process. We show that the tongue works as an elastic micropump; fluid at the tip is driven into the tongue''s grooves by forces resulting from re-expansion of a collapsed section. This work falsifies the long-standing idea that capillarity is an important force filling hummingbird tongue grooves during nectar feeding. The expansive filling mechanism we report in this paper recruits elastic recovery properties of the groove walls to load nectar into the tongue an order of magnitude faster than capillarity could. Such fast filling allows hummingbirds to extract nectar at higher rates than predicted by capillarity-based foraging models, in agreement with their fast licking rates.     

Sublingual structures of primates. II. Hominoidea, review, summary and literature

1. In Homo and the great apes (Pongidae) there occurs, besides the plica sublingualis a plica fimbriata at the ventral surface of the tongue. This duplicature of the mucosa does not occur in the Hylobytidae and in the other primates. 2. Some taste buds could be found in the epithelium of the plica sublingualis of the Pongidae. 3. There are many taste buds in the epithelium of the plica fimbriata of the Pongidae. On this sublingual structure there were counted 1776 taste buds in Pongo, 592 in Gorilla and 280 in Pan. A few taste buds could also be found on the plica fimbriata of a human newborn. 4. A glandula apicis linguae occurs in Homo, Pan, Gorilla and Pongo. 5. The fresh saliva of the glandula apicis linguae and the saliva on the floor of the mouth can be tested by the taste buds in the epithelium of the plica fimbriata, of papillae lenticulares and of areae gustatoriae at the ventral surface of the tongue. 6. It might be the function of the sublingual taste buds to taste the fresh saliva as a gradient for the central nervous comparison with the taste of the saliva on the dorsal surface of the tongue. 7. Because of the complete absence of a sublingua in the Platyrrhini and in the Cercopithecinae it is unlikely that the plica fimbriata of Homo and the great apes can be interpreted as a homalogon of the sublingua in the prosimians. 8. Because of the absence of a sublingua in other ordines of the Mammalia (Insectivora, Carnivora, Rodentia, Chiroptera, Ungulata) it is unlikely as well that the sublingua in the prosimians can be interpreted as a homologon of the tongues of the lower vertebrates. The sublingual structures occuring in the Marsupialia have to be investigated. 9. Because of these reasons the new development of the sublingua in the prosimians and the plica fimbriata in the Hominoidea, in complete independence from one another, seems to be a better explanation of the 2 structures and less contradictionary to anatomical and phylogenetic arguments. The different function of both structures in the recent primates gives a hint for the possible reason for their development during the process of evolution.     

The fluid mechanics of bolus ejection from the oral cavity

The squeezing action of the tongue against the palate provides driving forces to propel swallowed material out of the mouth and through the pharynx. Transport in respose to these driving forces, however, is dependent on the material properties of the swallowed bolus. Given the complex geometry of the oral cavity and the unsteady nature of this process, the mechanics governing the oral phase of swallowing are not well understood. In the current work, the squeezing flow between two approaching parallel plates is used as a simplified mathematical model to study the fluid mechanics of bolus ejection from the oral cavity. Driving forces generated by the contraction of intrinsic and extrinsic lingual muscles are modeled as a spatially uniform pressure applied to the tongue. Approximating the tongue as a rigid body, the motion of tongue and fluid are then computed simultaneously as a function of time. Bolus ejection is parameterized by the time taken to clear half the bolus from the oral cavity, t_1/2. We find that t_1/2 increases with increased viscosity and density and decreases with increased applied pressure. In addition, for low viscosity boluses (μapproximately 1000 cP), viscosity dominates. A transition region between these two regimes is found in which both properties affect the solution characteristics. The relationship of these results to the assessment and treatment of swallowing disorders is discussed.     

Mechanics of drinking in the mallard (Anas platyrhynchos,anatidae)

Water drinking in the mallard is accomplished by a fine-tuned set of movements of upper and lower jaw and of the tongue. During immersion of the tips of the bill, the oral cavity is formed into smaller volumes containing water and into connecting tubes. Two mechanisms serve the water transport: (1) lingual and jaw movements press water from the water-containing spaces into the tubes; (2) a quantitative simulation of the shape of the oral cavity during immersion shows that the two tubes are so narrow that capillary action also contributes to water transport. Thereafter, the tips of the bill are raised until they point upward. In this “tip-up” position, water flows into the esophagus because of gravity. We conclude that, in addition to normal tip-up drinking observed in almost all Passeriformes and Galliformes, a second type of tip-up drinking may be distinguished in Anseriformes. The integration of the drinking mechanism, keeping the water inside the mouth, and the straining mechanism, expelling the water along the beak rims, is effected by specific actions of the elaborate lingual apparatus.     

A Model of Tracer Transport in Airway Surface Liquid

This study is concerned with reconciling theoretical modelling of the fluid flow in the airway surface liquid with experimental visualisation of tracer transport in human airway epithelial cultures. The airways are covered by a dense mat of cilia of length ∼ 6 μm beating in a watery periciliary liquid (PCL). Above this there is a layer of viscoelastic mucus which traps inhaled pathogens. Cilia propel mucus along the airway towards the trachea and mouth. Theoretical analyses of the beat cycle smithd, fulb predict small transport of PCL compared with mucus, based on the assumption that the epithelium is impermeable to fluid. However, an experimental study coord indicates nearly equal transport of PCL and mucus. Building on existing understanding of steady advection-diffusion in the ASL (Blake and Gaffney, 2001; Mitran,2004) numerical simulation of an advection-diffusion model of tracer transport is used to test several proposed flow profiles and to test the importance of oscillatory shearing caused by the beating cilia. A mechanically derived oscillatory flow with very low mean transport of PCL results in relatively little ‘smearing’ of the tracer pulses. Other effects such as mixing between the PCL and mucus, and significant transport in the upper part of the PCL above the cilia tips are tested and result in still closer transport, with separation between the tracer pulses in the two layers being less than 9%. Furthermore, experimental results may be replicated to a very high degree of accuracy if mean transport of PCL is only 50% of mucus transport, significantly less than the mean PCL transport first inferred on the basis of experimental results.     

Invasion of black-tailed deer by nose bot fly larvae (Diptera: Oestridae: Oestrinae)

Invasion of black-tailed deer (Odocoileus hemionus columbianus) by larvae of the nose bot flies (Cephenemyia apicata and C. jellisoni) was investigated by obssrving their expulsion by larviparous females and their subsequent activity on the host. The drying uterine fluid encasing larvae ( = larval packet) delays desiccation, ensures adhesion to hairs, and immediately dissolves upon contact with saliva. Contrary to the widely accepted nasal mode of invasion, larvae placed on muzzles of deer crawl ventrally toward the upper lip, enter the mouth, and then crawl caudally along the hard palate or tongue toward the throat. Hair located between the muzzle and nostril prevents larvae from entering the nostrils. A natural per os mode of invasion, heretofore unrecognized, is proposed. This is initiated by: (1) females depositing larval packets on the muzzle of deer, or (2) deer licking larval packets from contaminated areas around the muzzle. The positive thermotropism of larvae is compatible with such a per os mode of entry into the host.     

Structure de la glande nasale externe deTiliqua rugosa (Reptilia,Scincidae) et rapports avec sa fonction

Summary The scincid lizardTiliqua rugosa possesses a large external nasal gland which is located intraconchally. Highly ramified tubules, imbedded primarily in the periphery of the gland, unite to form collecting ducts which empty into a short excretory canal. The diameter of the tubules increases progressively from 30. at the distal extremity of the gland to over 200 at the level of the collecting ducts. The intraglandular portion of the excretory canal is often dilated to form an ampulla. The thickness of the epithelium increases from 12 at the level of the tubules to 25–30 in the excretory canal.The excretory canal is lined with an epidermal epithelium close to the point where it enters the vestibule. In all the rest of the gland the tubules are lined with two cell types: large, typical muco-serous cells and striated cells. At the distal end of the tubules the striated cells are narrow and poorly differentiated and alternate more-or-less regularly with the muco-serous cells. The relative proportion of these striated cells increases progressively, as does their size, as one moves proximally down the tubule. In the gland as a whole the striated cells are approximately twice as numerous as the muco-serous cells but, due to their smaller size, they occupy less than one third of the tubular volume.Electron microscopy of the striated cells ofTiliqua rugosa revealed the presence of extensive lateral interdigitations and expansions of the basal cytoplasmic membrane, anatomical specialisations which are normally indicative of active salt transport. These modifications are less marked however than in the external nasal glands of the lizardsLacerta muralis andVaranus griseus, which do not appear to function as salt glands. In addition there are few mitochondria present, although they are of large size. The combination of these ultrastructural features, plus the fact that the striated cells are intermixed with muco-serous cells in the tubules, makes it most unlikely that the external nasal gland ofTiliqua rugosa is capable of elaborating an hyperosmotic fluid. What is more, this has never been conclusively demonstrated in this species in physiological studies.The progressive specialisation of the striated cells from the distal to the proximal section of the tubules poses the problem of the origin and differentiation of this cell type.A review of results obtained from the study ofTiliqua rugosa and other species of lizards shows that the nature of the relationship between structure and function of the external nasal gland is far from clear. The existence of salt glands, capable of excreting hyperosmotic solutions, is invariably linked with the presence in the gland of well-developed striated segments composed almost entirely of cells possessing extensive interdigitations of the lateral membranes. Amongst terrestrial lizards, nasal salt glands are usually found in herbivorous species and they are primarily adapted to the extrarenal excretion of potassium ions. The problem for carnivorous species is more often that of an excess of sodium rather than potassium ions and with the possible exceptionAcanthodactylus species, functional nasal salt glands have not been demonstrated in terrestrial carnivores, despite the presence in some cases of well-developed striated segments in the gland having a similar structure to those found in herbivores. In humid regions, carnivorous lizards probably never require extrarenal excretory mechanism and in arid regions their survival is assured by their capacity to tolerate hypernatraemia when confronted with excessive salt loads. Salt glands capable of eliminating sodium ions to any extent have only been described in two littoral species, an herbivorous iguanid and a carnivorous varanid. Unfortunately the structure of their respective nasal glands has not yet been described and their further study would be desirable.     

Consensus nomenclature for dyneins and associated assembly factors

Dyneins are highly complex, multicomponent, microtubule-based molecular motors. These enzymes are responsible for numerous motile behaviors in cytoplasm, mediate retrograde intraflagellar transport (IFT), and power ciliary and flagellar motility. Variants in multiple genes encoding dyneins, outer dynein arm (ODA) docking complex subunits, and cytoplasmic factors involved in axonemal dynein preassembly (DNAAFs) are associated with human ciliopathies and are of clinical interest. Therefore, clear communication within this field is particularly important. Standardizing gene nomenclature, and basing it on orthology where possible, facilitates discussion and genetic comparison across species. Here, we discuss how the human gene nomenclature for dyneins, ODA docking complex subunits, and DNAAFs has been updated to be more functionally informative and consistent with that of the unicellular green alga Chlamydomonas reinhardtii, a key model organism for studying dyneins and ciliary function. We also detail additional nomenclature updates for vertebrate-specific genes that encode dynein chains and other proteins involved in dynein complex assembly.

IntroductionDynein family motor proteins form multiple different dynein complexes in mammals, with important roles in a wide range of cellular functions (King, 2017; Osinka et al., 2019; Roberts, 2018). Dyneins can be broadly classified into two groups: cytoplasmic and axonemal. Dynein complexes “walk” toward the minus ends of microtubules; while doing so, they can transport a variety of cargoes within cells (Trokter et al., 2012). The motor activity of these complexes allows them to play key roles in enabling motility of whole cells, generating fluid flow across cell surfaces, and transporting organelles and other components within the cytoplasm.Dynein subunits are classified by mass into four categories: heavy (∼520 kD), intermediate (∼70 –140 kD), light intermediate (∼53–59 kD), and light (∼10–30 kD) chains (Pfister et al., 2006). The heavy and intermediate chains are specific to certain dynein complexes, while the light chains may be components of both cytoplasmic and axonemal dynein machinery, and in some cases, nondynein complexes. The light intermediate chains are present only in the cytoplasmic dynein class.Dynein-based movement is powered by the ATP-driven dynein heavy chain subunits (Schmidt and Carter, 2018). 15 genes in the human genome encode dynein heavy chains: 1 for each of the 2 cytoplasmic dynein complexes and 13 that encode heavy chain components of the various axonemal dynein complexes. A dynein-related gene, DNHD1 (dynein heavy chain domain 1) has been referred to as a “ghost gene”: it may be a remnant of an earlier duplication that has not decayed at a normal rate, as a truncated version might poison cytoplasmic dynein heavy chain dimerization and thus be lethal (Gibbons, 2018; Schmidt and Carter, 2018). DNHD1 is currently classified as an “orphan” dynein heavy chain-encoding gene (Kollmar, 2016) but may be in the process of becoming a pseudogene (Wickstead, 2018).Cytoplasmic dyneinsDynein 1 complexThe cytoplasmic dynein 1 complex (Table S1) is present throughout eukaryotes, with some notable exceptions such as green plants and red algae (Wickstead and Gull, 2007). It is involved in a wide variety of intracellular transport activities, transporting cargoes including chromosomes, mRNA, and protein complexes (Reck-Peterson et al., 2018). The dynein 1 complex also acts in cell division, helping to form and orient the mitotic spindle (Torisawa and Kimura, 2020), establish cell polarity (Lu and Gelfand, 2017), and position organelles (Allan, 2014; Oyarzún et al., 2019; Palmer et al., 2009).A dimer of DYNC1H1-encoded heavy chains forms the core of the cytoplasmic dynein 1 complex (Fig. 1 a) and acts as its ATPase motor (Palmer et al., 2009; Pfister et al., 2005). Each heavy chain contains six AAA+ domains, an antiparallel coiled-coil region with a microtubule-binding domain at its tip, and a C-terminal domain (Bhabha et al., 2016; Carter, 2013; Reck-Peterson et al., 2018; Roberts et al., 2013). Immediately N-terminal of the AAA1 domain is a linker that traverses the plane of the AAA ring and changes conformation during the ATPase cycle to drive motor activity. AAA1 exhibits ATP hydrolytic activity, acting as an ATPase and powering the dynein motor complex (Silvanovich et al., 2003), while nucleotide binding at several other AAA domains appears to modify how conformational change propagates through the AAA ring and affects microtubule-binding activity. The coordinated activity of both heavy chains within the dynein complex is required for processivity (Reck-Peterson et al., 2006).Open in a separate windowFigure 1.Cytoplasmic dynein complexes. (a) The cytoplasmic dynein 1 complex. The DYNC1H1 protein heavy chains have large globular heads at the C-termini that are composed of a ring of six AAA+ domains. The microtubule-binding domains are located at the tips of antiparallel coiled coils that derive from AAA4. The linker/N-terminal domains connect the AAA rings and the intermediate and light chains. (b) The cytoplasmic dynein 2 complex. The DYNC2H1 protein heavy chains power retrograde IFT and have the same general domain organization as DYNC1H1. However, the tails of the two heavy chains fold differently due to an asymmetry imposed by the two different intermediate chains: one is straight while the other forms a zigzag shape and interacts with the IFT-B train (Toropova et al., 2019). The linker/N-terminal domain connects the AAA ring and the intermediate and light chains. *It remains unknown whether the DYNLT2B protein forms a homodimer or a heterodimer with another Tctex-type light chain. (c) Schematic showing the interaction between the dynein 1 and dynactin complexes. The adapter molecule affects the type of cargo bound; in this figure, the hook microtubule tethering protein 3 (HOOK3)–encoded protein is acting as a cargo adapter.The intermediate chains of metazoan dynein 1 connect it to another multi-subunit complex known as dynactin (Loening et al., 2020). Dynactin is built around a filament of the protein encoded by ACTR1A (actin-related protein 1A). It activates dynein and regulates its binding to vesicles and organelles to be transported (Ketcham and Schroer, 2018). A coiled coil–containing cargo adaptor protein is required for dynein 1 activation (Fig. 1 c). A single adaptor protein sandwiches between dynactin and dynein, where it interacts with the dynein heavy chain tails and the light intermediate chain and along the length of the dynactin complex (Gonçalves et al., 2019; Reck-Peterson et al., 2018). There are currently ≥12 known cargo adaptor proteins, which are encoded by HOOK1, HOOK2, HOOK3, BICD2, BICDL1, BICDL2, RAB11FIP3, RASEF, CRACR2A, NIN, NINL, and SPDL1 (Barisic et al., 2010; Casenghi et al., 2005; Dona et al., 2015; Gonçalves et al., 2019; Horgan et al., 2010; Lee et al., 2018; Loening et al., 2020; Olenick et al., 2016; Vallee et al., 2021; Wang et al., 2019). Other protein cofactors may also be required for dynein recruitment to their cargoes. For example, the protein encoded by PAFAH1B1 (platelet activating factor acetylhydrolase 1b regulatory subunit 1; HUGO Gene Nomenclature Committee [HGNC] ID: 8574), also published using the alias LIS1 (lissencephaly 1) is required along with dynactin and BICD2 for dynein 1 to traffic many cargoes, such as nuclei, along microtubules (Faulkner et al., 2000; Splinter et al., 2012). LIS1 has most recently been suggested to stabilize the “open” conformation of cytoplasmic dynein 1 such that the heavy chains are able to undergo a mechanochemical cycle and cannot adopt the autoinhibited or “closed” state where movement of key mechanical elements is abrogated by interactions between heavy chains (Markus et al., 2020).The dynein light chains can be divided into three subfamilies: the t-complex associated (Tctex)–type family (encoded by DYNLT1, DYNLT2, DYNLT2B, DYNLT3, DYNLT4, and DYNLT5), the LC8-type family (encoded by DYNLL1, DYNLL2, and DNAL4), and the roadblock-type family (encoded by DYNLRB1 and DYNLRB2; Bowman et al., 1999; King et al., 1998; King et al., 1996; King and Patel-King, 1995). Most of these protein chains can be found in both cytoplasmic dynein complexes: the exceptions are DYNLT2, which is an axonemal dynein subunit; DYNLT2B, which is found in the dynein 2 complex and the I1/f inner dynein arm (IDA); DYNLT4 and DYNLT5, which are not well characterized; and DNAL4, which is present only in outer dynein arms (ODAs).Several proteins originally identified as dynein light chains are also found in numerous multimeric complexes unrelated to dyneins and appear to act as general dimerization engines or hubs (Williams et al., 2018). The LC8-type light chains (DYNLL1 and DYNLL2) are present in many enzymes including myosin V (Benashski et al., 1997; Espindola et al., 2000) and neuronal nitric oxide synthase (Jaffrey and Snyder, 1996). They also play a role in regulating apoptosis via an interaction with the BCL2 family protein encoded by BCL2L11 (Puthalakath et al., 1999). The DYNLT1 protein has reported roles in actin remodeling and neurite outgrowth (Chuang et al., 2005) and hypocretin signaling (Duguay et al., 2011). DYNLRB1 interacts with Rab6 family member proteins in the Golgi apparatus (Wanschers et al., 2008), and both roadblock-type dynein light chains are reportedly involved in a TGFβ signaling pathway (Jin et al., 2009).Dynein 2 complexCilia are highly complex microtubule-based organelles that extend from the cell surface and can be classified as either primary (or nonmotile) or motile (Satir and Christensen, 2008). Most eukaryotic cells, excluding blood cells and those actively dividing, have an associated primary or nonmotile cilium. These act as sensory organelles, detecting a broad range of signaling molecules (Kopinke et al., 2021; Mykytyn and Askwith, 2017; Saternos et al., 2020).The dynein 2 complex (also known as the intraflagellar transport [IFT] dynein or cytoplasmic dynein 1b in Chlamydomonas reinhardtii; Table S2) is found only in cells with associated cilia or flagella, where it locates within and around the base of these structures (Höök and Vallee, 2006). IFT trains are multiprotein complexes required for the assembly and function of cilia and flagella in eukaryotes (Dutcher, 2019; Wingfield et al., 2017). The anterograde IFT motor complex kinesin 2 moves IFT trains and associated cargoes plus the dynein 2 complex along microtubules, from the base to the tip of a cilium or flagellum (Toropova et al., 2019; Vuolo et al., 2020). The retrograde IFT motor complex dynein 2 transports IFT trains and associated factors from the tip back to the base (Hou and Witman, 2015; Pazour et al., 1998). The dynein 2 complex is required for the assembly of cilia and flagella (Pazour et al., 1999; Pfister et al., 2006) and also has key roles in ciliary signaling functions (Vuolo et al., 2020).The core of dynein 2 is composed of a dimer of two DYNC2H1-encoded heavy chains (Fig. 1 b). The tails of these identical heavy chains are directed into two different conformations by the other subunits in the complex (Toropova et al., 2019). Each heavy chain is stabilized by its interaction with a DYNC2LI1-encoded protein subunit. The C-terminal helix of one of these light intermediate subunits associates with a DYNC2I1 (previously WDR60)-encoded protein with a DYNLRB-encoded subunit, to enforce a distinct conformation on one heavy chain (Toropova et al., 2019; Vuolo et al., 2020).The DYNC2I1- and DYNC2I2-encoded intermediate chains bind the heavy chains via their C-terminal β-propeller domains. The N-terminal regions of these intermediate chains are dimerized by three DYNLL1/2 dimers and one of each of the other light chain dimers: DYNLT1/3, DYNLRB1/2, and DYNLT2B (Toropova et al., 2019). The DYNLT2B-encoded light chain is a unique accessory component of the dynein 2 complex. Whether it forms a homodimer or heterodimer with another light chain remains to be confirmed, although there is evidence to suggest that, unlike the other light chains, the DYNLT2B subunit may be monomeric (DiBella et al., 2001). Recent structural studies of Tetrahymena ODAs have revealed a Tctex-family heterodimer (Rao et al., 2021).Axonemal dyneinsMotile cilia (sometimes termed flagella when they occur singly or in small numbers on a cell) are more restricted to certain cell types. Their movement enables sperm to swim (Linck et al., 2016), respiratory cilia on epithelial cells to sweep away mucus containing trapped pathogens (Hansson, 2019), and oviduct epithelial cells to waft an ovum along a fallopian tube toward the uterus (Spassky and Meunier, 2017). Multiciliated cells in the brain help move the cerebrospinal fluid and also influence neuronal migration (Brooks and Wallingford, 2014). In the male reproductive tract, the epithelial cells of the efferent ducts are densely covered with multiple motile cilia necessary for the transport of sperm cells (Aprea et al., 2021a). Motility of nodal cilia in the embryonic left–right organizer is necessary for the determination of correct left–right body asymmetry (Nonaka et al., 1998).An axoneme is the microtubule superstructure core of the cilium and contains many tightly associated components. A motile cilium has a highly conserved “9 + 2” structure: 9 microtubule doublets that surround a central pair of 2 microtubule singlets (the “central apparatus”; Fig. 2). Axonemal dyneins are the motor complexes that drive a sliding motion between ciliary doublet microtubules, enabling movement. Motile cilia have IDAs and ODAs and radial spokes that are thought to be involved in signal transduction between the central pair and the outer microtubule ring (Ishikawa, 2017). Nonmotile cilia have only the outer doublet ring and have a 9 + 0 microtubule arrangement, although the number of outer doublets decreases and their arrangement changes beyond the proximal part of the cilium (Kiesel et al., 2020).Open in a separate windowFigure 2.Axonemal dynein complexes. (a) Axonemal ODA. The blue text denotes subunits found in ODA complexes in respiratory cilia, and red text denotes subunits found in ODA complexes in sperm flagella. (b) Axonemal inner arm I1/f complex subunits (IDA). (c) Monomeric IDAs. Each inner arm species is constructed around a distinct monomeric heavy chain associated with an actin monomer and either DNALI1 or centrin; species d contains two additional components. In most cases, the precise equivalence between the human and C. reinhardtii monomeric heavy chain species is uncertain.The ODAs (Table S3 and Fig. 2 a) and IDAs (Table S4 and Table S5, and Fig. 2, b and c) in motile cilia are arranged in two rows with a complex 96-nm repeat organization. They are permanently attached to the A-tubule of one outer doublet microtubule (see Fig. 3, a and b) and transiently interact in an ATP-dependent manner with the B-tubule of the adjacent doublet to generate a sliding force (King, 2017). IDAs with a single heavy chain are termed monomeric (Table S4), while the I1/f IDA (Table S5) is dimeric, with two nonidentical heavy chains. These different types of dyneins vary in terms of their enzymatic and motor properties, likely reflecting their precise roles in the generation of ciliary motility (King, 2017).Open in a separate windowFigure 3.Organization of a mammalian motile cilium. (a) The diagram illustrates the general 9 + 2 microtubule arrangement within the ciliary axoneme. The inner and outer rows of dynein arms generate the force required for ciliary beating. The N-DRC complex is a key regulatory structure that interconnects the doublet microtubules. The radial spokes regulate the beat of cilia by transducing signals between the doublets and the central microtubule pair. (b) Tomographic image of an averaged 96-nm repeat for a single human ciliary doublet microtubule, revealing the microtubule-associated dynein arms, N-DRC, and radial spoke. The scale bar represents 25 nm. This image was generated by Jason Schrad (Nicastro laboratory) using data from Lin et al. (2014). (c and d) Cross-section (c) and longitudinal (d) views of the 48-nm repeat organization of a bovine doublet microtubule. The components of the ODA-DC are individually colored and indicated. This ribbon diagram was generated with the PyMol molecular graphics system (Schrödinger) using Protein Data Bank accession no. 7RRO (Gui et al., 2021).ODA docking complex (ODA-DC)The correct functioning of cilia and flagella in most eukaryotes is dependent on the ODA chains attaching to the outer doublet microtubules at 24-nm intervals (Dean and Mitchell, 2015; King, 2017). The ODA-DC facilitates binding and may also play a role in regulating the activity of the ODAs (Takada et al., 2002). The ODA-DC in C. reinhardtii consists of three protein subunits, encoded by DCC1 (DC1), DCC2 (DC2), and DLE3 (DC3). In mammals, it consists of five protein subunits (Gui et al., 2021) encoded by five genes, now named ODAD1, ODAD2, ODAD3, ODAD4, and CLXN (calaxin; Fig. 2 a and Fig. 3, b and c). CLXN (previously EFCAB1) has been assigned the alias symbol ODAD5, and authors may refer to it as such in publications if they wish, referencing the approved gene symbol at least once to aid data retrieval. Only ODAD1 and ODAD3 have orthologues in C. reinhardtii (DCC2 and DCC1, respectively).Dynein axonemal assembly factors (DNAAFs)Genes encoding proteins that act as axonemal dynein assembly factors are named using the root symbol DNAAF. These proteins play an important role in the preassembly of IDAs and ODAs in the cytoplasm before their transport to cilia (Fabczak and Osinka, 2019; King, 2021).Historically, the DNAAF root has been used only for proteins directly involved in the preassembly of axonemal dynein arms in the cytoplasm. We wrote to authors who have published on the genes that we are reporting in this publication as newly updated DNAAFs (see their symbols in bold in Table S6) and discussed this issue with our specialist advisors for this gene group (https://www.genenames.org/data/genegroup/#!/group/1627). This effort resulted in an agreement to use the term “DNAAF” more broadly. Therefore, a DNAAF symbol can now also be assigned to genes encoding proteins that play a role in trafficking dynein arms from the cytoplasm to cilia.Association with human phenotypesHumans have four described cilia types, and defects in all types are associated with various diseases: motile 9 + 2 cilia (e.g., respiratory cilia, ependymal cilia, sperm flagella); motile 9 + 0 cilia (e.g., nodal cilia); nonmotile 9 + 2 cilia (e.g., the kinocilium of hair cells and the proximal region of olfactory cilia); and nonmotile 9 + 0 cilia (e.g., renal monocilia and the connecting cilia of photoreceptor cells). Cilia are located on almost all polarized cell types of the human body; therefore, cilia-related disorders (ciliopathies) affect many organ systems (Fliegauf et al., 2007). Genetic mutations that impair cilia and/or flagella beating cause a heterogeneous group of rare disorders referred to as motile ciliopathies (Wallmeier et al., 2020). The pathogenic mechanisms, clinical symptoms, and severity of the diseases depend on the specific affected genes and the tissues in which they are expressed. Defects in ependymal cilia can result in hydrocephalus. Reduced fertility can be due to defective cilia in the fallopian tubes or the efferent ducts as well as sperm flagella. The malfunction of motile monocilia on the left–right organizer during early embryonic development can lead to laterality defects such as situs inversus and heterotaxy. Severe impairment of mucociliary clearance in the respiratory tract leads to chronic bronchial problems. Primary ciliary dyskinesia (PCD), which can present with a variety of these features, is the most common motile ciliopathy.The genetic disorder PCD is heterogeneous and has been linked to variants in genes encoding dyneins, axonemal dynein assembly factors, and ODA-DC subunits (Wallmeier et al., 2020). PCD-associated phenotypes include chronic respiratory problems, recurrent middle ear infections, male infertility, and subfertility in females (Leigh et al., 2019). Roughly 50% of PCD patients are diagnosed with Kartagener syndrome, a subtype defined by a triad of symptoms: chronic sinusitis, bronchiectasis, and situs inversus, where the positions of major body organs are reversed (Zariwala et al., 2011). Situs inversus totalis is observed when all thoracic and abdominal viscera are reversed; individuals with situs inversus or situs ambiguus show more variable organ positioning (seen in ≥6% of PCD cases; Kennedy et al., 2007; Sempou and Khokha, 2019).Table 1.Human phenotypes associated with variants of genes encoding dyneins and dynein-associated proteins

The cell biology of fertilization: Gamete attachment and fusion

Fertilization is defined as the union of two gametes. During fertilization, sperm and egg fuse to form a diploid zygote to initiate prenatal development. In mammals, fertilization involves multiple ordered steps, including the acrosome reaction, zona pellucida penetration, sperm–egg attachment, and membrane fusion. Given the success of in vitro fertilization, one would think that the mechanisms of fertilization are understood; however, the precise details for many of the steps in fertilization remain a mystery. Recent studies using genetic knockout mouse models and structural biology are providing valuable insight into the molecular basis of sperm–egg attachment and fusion. Here, we review the cell biology of fertilization, specifically summarizing data from recent structural and functional studies that provide insights into the interactions involved in human gamete attachment and fusion.

IntroductionDuring sexual reproduction, the oocyte and sperm fuse to generate a new and unique embryo. The journey of a sperm to an egg ends in the ampulla of the female oviduct. From there, the sperm must overcome a number of physical and biochemical barriers. After undergoing the acrosome reaction and binding the ova, the sperm penetrates through the cumulus oophorus cells and the zona pellucida (ZP) to reach the perivitelline space (PVS) and oocyte membrane. Upon fusion of the sperm and egg membranes, the sperm nucleus and organelles are incorporated into the egg cytoplasm.An understanding of the mechanisms of mammalian fertilization is crucial to treat infertility and develop new methods of birth control. Infertility affects 15% of couples globally, and in one third of these cases, the underlying cause is unknown (Gelbaya et al., 2014). Developments in assisted reproductive technologies have provided couples with new options to conceive but may have epigenetic side effects (Mani et al., 2020). Furthermore, only 40% of couples manage to have a child despite 2 yr of treatment. Safety, efficacy, and acceptability of contraceptives are also critically important, but many current female contraceptive methods have side effects that limit long-term use (Aitken et al., 2008), while male contraceptives are limited to condoms or vasectomy (Kanakis and Goulis, 2015). A better understanding of the molecular players involved in fertilization is necessary to drive innovation in both assisted reproductive technologies and contraception.In this review, we will first briefly review the events that prepare the gametes for fertilization. We will then discuss how recent studies of genetically altered mice and structural biology efforts have shed light on the molecular mechanisms of sperm–egg attachment and fusion. We will also discuss the gaps in current knowledge and suggest new perspectives and future directions in the search for other protein factors involved at the gamete fusion synapse.Cell biology of gametesFertilization requires proper gametogenesis (oogenesis in the female and spermatogenesis in the male), which produces haploid cells and introduces diversity. Primordial germ cells (PGC) are the embryonic precursors to spermatocytes and ova. The cells produced by the first few divisions of the fertilized egg are totipotent and capable of differentiating into any cell type, including germ cells. PGCs originate within the primary ectoderm of the embryo and then migrate into the yolk sac. Between weeks 4 and 6, the PGCs migrate back into the posterior body wall of the embryo, where they stimulate cells of the adjacent coelomic epithelium and mesonephros to form primitive sex cords and induce the formation of the genital ridges and gonads. The sex (gonadal) cords surround the PGCs and give rise to the tissue that will nourish and regulate the development of the maturing sex cells (ovarian follicles in the female and Sertoli cells in the male).EggOogenesis is a complex differentiation process by which mature functional ova develop from germ cells (Fig. 1 A; Edson et al., 2009). In humans, oogenesis begins in the ovary at 6–8 wk of fetus development, when PGCs differentiate into oogonia. By the 12th week, several million oogonia enter prophase, the first meiotic division and become dormant until shortly before ovulation (Hayashi et al., 2020). Due to their large and watery nuclei, these cells are referred to as germinal vesicles (Pan and Li, 2019). These primary oocytes become enclosed by follicle cells to form primordial follicles. The number of primordial follicles peaks at ∼7 million by the fifth month of fetal life, with ∼700,000 left at birth and 400,000 by puberty (Marcozzi et al., 2018). All of the egg cells that the ovaries will release are already present at birth.Open in a separate windowFigure 1.Gametogenesis and fertilization. (A–C) Illustration of oogenesis and follicle development (A), spermatogenesis (B), and the major steps in fertilization (C): (1) initial contact, (2) acrosome reaction, (3) ZP penetration, (4) sperm–egg fusion, (5) entry of sperm nucleus, (6) cortical reaction, and (7) fusion of the sperm and egg nuclei. The oocyte with its ZP measures 130 μm in diameter. Created with BioRender.During each menstrual cycle, hormones from the hypothalamic–pituitary–gonadal axis restart the division of the primary oocytes in meiosis I and follicular development (Atwood and Vadakkadath Meethal, 2016). Primary follicles develop into secondary follicles, containing each growing oocyte surrounded by two or more layers of proliferating follicle cells. ZP glycoproteins are secreted by the oocyte of the primary follicle and possibly the follicular cells (Törmälä et al., 2008). Although these glycoproteins form a physical barrier between the follicle cells and the oocyte, follicle cells and the oocyte remain connected through transzonal cytoplasmic projections from the follicle cells until fertilization (Makabe et al., 2006). A reciprocal dialog between the oocyte and its surrounding follicular cells coordinates the different phases of follicular development and the maintenance of meiotic arrest (Dalbies-Tran et al., 2020). Oocyte-derived microvilli control female fertility by optimizing ovarian follicle selection in mice (Zhang et al., 2021). The epithelium of 5–12 primary follicles proliferates to form a multilayered capsule around the oocyte. A few of these growing follicles continue to enlarge in response to follicle-stimulating hormone (FSH; Visser and Themmen, 2014). A single follicle becomes dominant, and the others degenerate by atresia (Atwood and Vadakkadath Meethal, 2016). Meiosis of the oocyte in the mature preovulatory follicle is blocked until a surge in levels of FSH and luteinizing hormone that occurs midway through the menstrual cycle. The membrane of the germinal vesicle nucleus breaks down, the chromosomes align in metaphase, and the oocyte expels its first polar body. The secondary oocyte then begins the second meiotic division, which is arrested at the meiotic metaphase II stage until ovulation (Gougeon, 1996). Ovulation depends on the breakdown of the follicle wall and occurs ∼38 h after the increase in levels of FSH and luteinizing hormone (Holesh et al., 2021). The disruption of the follicle wall expulses the oocyte, which is captured by the fimbriated mouth of the oviduct and moved into the ampulla. The oocyte retains its ability to be fertilized for ∼24 h and completes meiosis only if it is fertilized.SpermIn contrast to oogenesis, which is complete before birth, spermatogenesis is a continuous process that begins at puberty (Fig. 1 B). In humans, spermatogenesis takes 74 d to complete; thus, multiple spermatogenesis events occur simultaneously to allow for continual sperm production. Spermatogenesis occurs in the testis in a stepwise manner, beginning with diploid spermatogonia at the basal surface of seminiferous tubules and ending with mature elongated spermatozoa that are released in tubule lumens in a process called spermiation (Clermont, 1972; Yang and Oatley, 2014). During spermatogenesis, mitosis results in gene amplification, meiosis results in genome reduction, and finally maturation occurs (Hess and Renato de Franca, 2008). At this stage, sperm are not motile and are fertilization incompetent. Two additional sperm maturational processes are required outside the testis. First, sperm undergo a maturation process during epididymal transit (Bedford et al., 1973) involving posttranslational modifications of previously synthesized proteins and acquisition of proteins from the epididymal epithelium (James et al., 2020; see text box). After ejaculation into the female reproductive tract, dilution triggers additional changes in sperm, collectively termed capacitation (see text box), that prepare the sperm for the acrosome reaction.Epididymal maturationSperm exchange with the epididymal epithelium occurs by direct interaction with epithelial cells, by interaction with soluble proteins in the epididymal fluid or via extracellular exosome-like vesicles released by epithelial cells called epididymosomes (James et al., 2020). The purposes of this exchange are to redistribute sperm proteins and change the composition and lipid balance of the sperm membrane. These changes take place during the transit from the epididymis initial segment, through the caput and the corpus, to the cauda where sperm are stored (Cornwall, 2009). Epididymal transit lasts 10–12 d in mammals, but storage is dependent on sexual activity. Since fertilization is not immediate, fertilizing capacities of the spermatozoa are preserved by decapacitation factors that are active in the epididymis. An example of a decapacitation factor is SPINK3, which is secreted by seminal vesicles; it impairs sperm membrane hyperpolarization and calcium influx through CatSper (Zalazar et al., 2020). Epididymal plasma and sperm represent only a small fraction (5%) of semen in men (Batruch et al., 2011). Two thirds of the volume of semen comes from the seminal vesicles and the other third from the prostate. These secretions protect the sperm and prevent early maturation.Sperm capacitationMore than 70 yr ago, Austin and Chang described capacitation as the changes required for sperm to fertilize oocytes in vivo (Austin, 1952; Chang, 1951). Once sperm enter the female reproductive tract, they undergo capacitation. Capacitation results in hyperactivation of sperm movement and initiation of the acrosome reaction (Saling et al., 1979; Florman and First, 1988). During capacitation, stabilizing or decapacitation factors that are adsorbed on the sperm plasma membrane are removed (Bedford and Chang, 1962). These agents that initiate removal of decapacitation factors are electrolytes, energy substrates, and proteins such as seminal plasma protein or albumin. Removal of decapacitation factors increases sperm plasma membrane fluidity, allowing an increase in the permeability to calcium, chloride, and bicarbonate ions (Gangwar and Atreja, 2015). Sperm motility depends on the membrane potential, intracellular pH, and balance of intracellular ions (reviewed in Nowicka-Bauer and Szymczak-Cendlak, 2021). The most important ion for this function is Ca²⁺ (Hwang et al., 2019). This secondary messenger is an important signaling pathways activator that regulates sperm motility (Finkelstein et al., 2020). The activation of soluble adenyl cyclases generates cyclic adenosine monophosphate that in turn activates serine/threonine protein kinase A, which induces a cascade of protein phosphorylation initiating the induction of sperm motility (Chen et al., 2000). Protein phosphorylation, sperm hyperactivation, and the acrosome reaction are used in vitro to evaluate capacitation. Capacitation can be induced in vitro by incubation in medium containing calcium, bicarbonate ions, and serum albumin (Touré, 2019).Mammalian sperm capacitation occurs during sperm migration in the female tract. Mammalian males ejaculate millions of sperm cells into the female reproductive tract, but only a few hundred sperm at most reach the oocytes. This massive elimination process likely prevents polyspermy (reviewed in Kölle, 2015). Selection of human sperm during the journey begins in the acidic environment of the vagina. In the cervix, only morphologically normal sperm can migrate. Some sperm immediately pass into the cervical mucus, whereas the remaining sperm becomes a part of the coagulum. The next selection occurs at the uterus–tubal junction, the connection between the uterus and the oviduct that represents a major obstacle for sperm migration (Kölle, 2015). Experiments in mice indicate that sperm motility alone is insufficient for sperm migration through the uterus–tubal junction (Fujihara et al., 2018). Uterine contractions facilitate sperm transport as do molecular interactions. Several proteins, such as ADAM3 and other ADAM family members, are known to be involved in this step in mice (Yamaguchi et al., 2009; Xiong et al., 2019); most ADAM proteins have human orthologues.Spermatogenesis takes place in a species-specific cycle called the seminiferous epithelial cycle and is regulated in particular through the hypothalamic–pituitary–testicular axis. Indeed, at puberty, the testes (interstitial steroidogenic Leydig cells) secrete an increased amount of testosterone, which triggers growth of the testes, maturation of the seminiferous tubules, and the commencement of spermatogenesis. The Sertoli cells are the major somatic cells present in the seminiferous tubules and are considered to be the main regulators of spermatogenesis. They orchestrate spermatogenesis by supporting spermatogonial stem cells, determining the testis size, organizing meiotic and postmeiotic development and sperm output, supporting androgen production by maintaining the development and function of Leydig cells, and regulating other aspects of testis function like peritubular myoid cells, immune cells, and the vasculature, which participate in the maintenance of the spermatogonial stem cell niche.Acrosome reactionThe acrosome is a secretory vesicle located on the anterior region of sperm that originates from the spermatid Golgi apparatus. An acrosomal granule is formed by the fusion of proacrosomal vesicles in the vicinity of the nucleus. The region increases in size and spreads over the anterior part of the nucleus. The acrosome reaction is driven by SNARE complexes and results in the exocytosis of the contents of the acrosome upon fusion of the plasma membrane with the outer acrosomal membrane (Fig. 1 C; reviewed in Okabe, 2016; De Blas et al., 2005). The timing of the acrosome reaction is critical. Only sperm that have undergone this reaction are fertilization competent, but when a high proportion of sperm undergo the acrosome reaction prematurely, success of in vitro fertilization is low (Wiser et al., 2014). Several studies indicate that only a fraction of sperm is capable of undergoing spontaneous acrosome reaction. In human and mice sperm samples, 15–20% of cells undergo spontaneous acrosome reaction (Nakanishi et al., 2001), whereas only 20–30% undergo progesterone-induced acrosome reaction (Stival et al., 2016), suggesting physiological heterogeneity of sperm population. In addition, Inoue et al. demonstrated that acrosome-reacted mouse spermatozoa recovered from the PVS can fertilize other eggs (Inoue et al., 2011).Based on in vitro data, it was thought that the acrosome reaction occurs when the sperm contacts the ZP, particularly the ZP3 protein (Litscher and Wassarman, 1996). Using transgenic mice that express fluorescent markers in the acrosome (Nakanishi et al., 1999) and the midpiece mitochondria (Hasuwa et al., 2010), real-time observation of acrosomal exocytosis was possible. These experiments showed that most mouse spermatozoa capable of fertilization had undergone the acrosome reaction before contact with the oocyte ZP (Jin et al., 2011). Most spermatozoa begin to react in the isthmus of the oviduct before reaching the ampulla (Hino et al., 2016; La Spina et al., 2016). Contact with the ZP in vitro probably makes it possible to complete a partial acrosome reaction. The most important function of the acrosome reaction is to induce changes in the sperm membrane (Okabe, 2016). The relocations of IZUMO1 and SPACA6, proteins essential for sperm–egg fusion, that occur after the acrosome reaction are illustrative examples of these changes (Sosnik et al., 2009; Barbaux et al., 2020; Satouh et al., 2012). The presence of these proteins on the sperm membrane, in addition to the classic markers Pisum sativum agglutinin, Peanut agglutinin lectins, or CD46, can be used as markers for the acrosome reaction (Ito and Toshimori, 2016). The acrosome and its disruption are both crucial for effective fertilization, as low fertilization rates are observed upon intracytoplasmic sperm injection of acrosome-intact sperm (Morozumi and Yanagimachi, 2005) or round spermatozoa lacking acrosomes (Dávila Garza and Patrizio, 2013).ZP penetrationThe ZP is a physical barrier between the oocyte and the follicular cells that forms from glycoproteins secreted from the primary follicles (Fig. 1 C). The human ZP consists of four glycoproteins (hZP1–hZP4; Harris et al., 1994). Mice, which have been used for most of the ZP studies in mammals, express only three ZP glycoproteins (mZP1–mZP3; Litscher and Wassarman, 2007). Analysis of mouse lines expressing human ZP proteins demonstrated that only hZP2 is important in human sperm–egg binding (Gupta, 2021). Experiments using purified native or recombinant human ZP proteins have shown that hZP1, hZP3, and hZP4 bind to the capacitated human spermatozoa and induce the acrosome reaction (Gupta, 2021). ZP1 is required for the structural integrity of the ZP (Chakravarty et al., 2008). To better understand the roles of ZP glycoproteins, further studies, particularly on ZP protein glycosylation, are needed. The species-specific binding of the ZP to sperm is presumably related to these carbohydrate moieties (Clark, 2014). The sialyl-Lewis(x) sequence is the major carbohydrate ligand for human sperm–egg binding (Pang et al., 2011). The current hypothesis that hZP1, hZP3, and hZP4 bind to capacitated sperm and hZP2 binds to sperm with intact acrosomes will need to be revisited due to the recent demonstration that the acrosome reaction takes place before ZP contact. Regardless, the role of the ZP in preventing polyspermy is clear. Indeed, ZP hardening is due to ZP2 cleavage by ovastacin, a protease released into the PVS by cortical granules after the first sperm–egg fusion (Burkart et al., 2012).Sperm–egg attachment and membrane fusionAfter penetration of the ZP, the sperm enters the PVS and can attach and fuse with the egg plasma membrane. The development of genetic knockout animal models has proven critical in determining the importance of various sperm and egg proteins in sperm–egg attachment and fusion. Surprisingly, genetic knockout studies revealed that many factors originally thought to be important for fertilization were in fact not necessary (reviewed in Okabe, 2018, 2015). The proteins from sperm and egg that are essential for sperm–egg membrane interaction and fusion are listed in