The sperm of Matsucoccus feytaudi (Insecta,Coccoidea): Can the microtubular bundle be considered as a true flagellum?

In the present work the spermiogenesis and sperm structure of Matsucoccus feytaudi, a primary pest of the maritime pine in southern eastern Europe, is studied. In addition to the already known characteristics of coccid sperm, such as the absence of the acrosome and mitochondria, and the presence of a bundle of microtubules responsible for sperm motility, a peculiar structure from which the microtubule bundle takes origin is described. Such a structure – a short cylinder provided with a central hub surrounded by several microtubules with a dense wall – is regarded as a Microtubule Organizing Centre (MTOC). During spermiogenesis, quartets of fused spermatids are formed; from each spermatid, a bundle of microtubules, generated by the MTOC, projects from the cell surface. Each cell has two centrioles, suggesting the lack of a meiotic process and the occurrence of parthenogenesis. At the end of the spermiogenesis, when the cysts containing bundles of sperm are formed, part of the nuclear material together with the MTOC structure is eliminated. Based on the origin of the microtubular bundle from the MTOC, the nature of the bundle as a flagellum is discussed.     

An ultrastructural analysis of mature sperm from the crayfish Pacifastacus leniusculus,Dana

Sperm from the crayfish, Pacifastacus leniusculus, resemble other reptantian sperm in that they are composed of an acrosome, subacrosomal region, nucleus, membrane lamellar complex, and spikes which radiate from the nuclear compartment. The acrosome (PAS positive vesicle) can be subdivided into three regions: the apical cap, crystalline inner acrosomal material, and outer acrosomal material which is homogeneous except for a peripheral electron dense band. The nucleus contains uncondensed chromatin and bundles of microtubules which project into the spikes. The orientation of the microtubule bundles relative to the nuclear envelope near the base of the subacrosomal region suggests that the nuclear envelope may function in the organization of the spike microtubules.     

Ultrastructure of spermiogenesis and spermatozoa in Marchalina hellenica (Gennadius) (Hemiptera: Sternorrhyncha,Marchalinidae)

The spermiogenesis, the sperm structure and the sperm motility of Marchalina hellenica (Gennadius) were examined. In the early spermiogenesis a centriolar apparatus was identified, but this structure is not involved in the production of the sperm flagellum. As in other Coccoidea, the flagellar axoneme originates by the activity of the thickened tip of the numerous microtubules surrounding the nuclear anterior region close to the periphery of the cell. This region pushes against a narrow cytoplasmic layer, giving rise to a papilla. In this region a novel structure, consisting of a regular network of thin filaments, arranged orthogonally to the bundle of microtubules, is visible. The sperm flagellum consists of a series of about 260 microtubules, regularly arranged in rings around the axial nucleus. This latter extends in the middle part of the sperm length. As usual in scale insects, sperm form a bundle, which in M. hellenica is composed of 64 sperm cells, surrounded by somatic cyst cells. The sperm bundle has an helicoidal array, with a cap of dense material at its apex, lending the anterior and the posterior region of the sperm bundle with a different structural organization. This difference is responsible of the different speed gradient observed in the helical wave propagating along the sperm bundle.     

Association of protein kinase A type I with detergent-resistant structures of mammalian sperm cells

The finding that flagellar movement in detergent-permeabilized sperm cells is restored when Mg ATP and cAMP are added implicated detergent-resistant protein kinase A (PKA) in the regulation of sperm motility. It is widely believed that only the PKA regulatory subunit RII can associate with the cytoskeleton and/or organelles. In this paper we used monoclonal antibodies against the PKA catalytic subunit and RI subunit and demonstrated that PKA type I is also associated with the sperm cytoskeleton. To our knowledge, this is the first report showing anchored PKA type I. This association was found in sperm of nonrodent mammalian species and, to a lesser extent, also in mouse sperm. The PKA catalytic subunit is bound to the cytoskeleton secondarily via its complex with the regulatory subunit. The detergent-resistant complexes of RI and catalytic subunits localize predominantly to the flagellum. Ultrastructural immunogold labeling revealed the association of detergent-resistant PKA type I with outer dense fibers (ODF) and the fibrous sheath (FS) but not with microtubules. This location is consistent with a proposed role of PKA in regulation of FS sliding on underlying ODF. Mol. Reprod. Dev. 50:79–85, 1998. © 1998 Wiley-Liss, Inc.     

MICROTUBULES IN RELATION TO THE MOTILITY OF A SPERM SYNCYTIUM IN AN ARMORED SCALE INSECT

Male scale insects of the species Parlatoria oleae Colvée (Homoptera: Coccoidea) produce motile sperm bundles. The bundle is a syncytium consisting of 10 to 20 closely packed, filamentous spermatozoa, which share a common cytoplasm and are enclosed in a common membrane. The individual spermatozoon is not surrounded by a plasma membrane, but is delimited by a scroll-like sheath composed of 45 to 50 microtubules. The microtubules run parallel to the long axis of the spermatozoon and are arranged in a spiral pattern as seen in transection. The outside diameter measures approximately 140 to 220 A and the inside diameter, 70 to 100 A. The spermatozoon is about 300 µ long and tapers gradually from a diameter of approximately 0.3 µ anteriorly to 0.1 µ posteriorly. The anterior half (150 µ) has a threadlike core of chromatin about 0.07 µ in diameter. A homogeneous cytoplasm surrounds the nuclear core and fills the posterior half of the spermatozoon. Neither osmium tetroxide nor glutaraldehyde fixation revealed the presence of a nuclear envelope, acrosomal membranes, mitochondria, flagellum, or centrioles. In spite of the apparent lack of orthodox cell organelles, the spermatozoon is actively motile upon release from the bundle. It exhibits capactiy for motility throughout its entire length. Since the sheath of microtubules is the only structure which extends the full length of the spermatozoon, it probably plays a significant role in spermatozoan motility.     

A microtubule organizing centre (MTOC) is responsible for the production of the sperm flagellum in Matsucoccus feytaudi (Hemiptera: Coccoidea)

A microtubule organizing centre (MTOC) has been described in the spermatid of the hemipteran Matsucoccus feytaudi (Coccoidea). This structure, revealed as a fluorescent ring by treatment with γ-tubulin antibody, gives rise to a bundle of microtubules which surrounds the elongated cylindrical nucleus. This microtubule bundle has been considered an atypical sperm flagellum provided with sperm motility. A comparison of the M. feytaudi MTOC with the material associated with the centriole of Drosophila melanogaster spermatids confirms the great similarity between the two structures, both involved in the nucleation of microtubules. Like the D. melanogaster material associated with the centriole, the M. feytaudi MTOC is a transient structure which disappears or degenerates at the end of spermiogenesis and is no longer visible in the mature sperm.     

Characterization of lake minnow Eupallasella percnurus semen in relation to sperm morphology,regulation of sperm motility and interpopulation diversity

Sperm morphology and regulation of sperm motility of lake minnow Eupallasella percnurus, an endangered cyprinid, were investigated. Milt characteristics from two isolated populations of E. percnurus were compared to characterize the interpopulation diversity. Electron microscopic studies revealed that E. percnurus spermatozoa comprise simple, uniflagellate, anacrosomal aquasperm with species‐specific features as an eccentrically located implantation of nuclear fossa and eccentric insertion of flagellum. Sperm motility was significantly inhibited by relatively low ion concentrations (150, 150 and 8 mM for NaCl, KCl and CaCl₂, respectively). Sperm maintained a high motility rate over a wide pH range (5·5–10·5), which may reflect adaptation to a highly variable environment. The two E. percnurus populations were markedly different in milt volume, sperm concentration, seminal plasma pH, sperm motility and beat cross frequency, which may result from genetic differences and environmental conditions.     

The sperm structure of Cryptocercus punctulatus Scudder (Blattodea) and sperm evolution in Dictyoptera

Sperm of the dictyopteran key taxon Cryptocercus punctulatus was examined. It has largely maintained a blattodean groundplan condition, with a three‐layered acrosome, an elongate nucleus, a single centriole, a conspicuous centriole adjunct material, two connecting bands (=accessory bodies), and a long functional flagellum with a 9+9+2 axoneme provided with accessory tubules with 16 protofilaments and intertubular material. These sperm characters are shared with several other polyneopterans. The sperm of C. punctulatus is very similar to what is found in Periplaneta americana and species of other groups of roaches, including the sperm of Loboptera decipiens described here for the first time. The general sperm organization here described can be assumed for the groundplan of Insecta and Pterygota. The following evolutionary path can be suggested: after the split between Cryptocercidae and the common ancestor of Isoptera, the typical pattern of sperm formation was altered very distinctly, resulting in a duplication or multiplication (Mastotermitidae) of the centrioles. Mastotermes has maintained a certain sperm motility, but with a very unusual apparatus of multiple flagella with a 9+0 axoneme pattern. After the split into Mastotermitidae and the remaining Isoptera, sperm motility was completely abandoned, and different modifications of sperm components occurred, and even the loss of the sperm flagellum. J. Morphol. 276:361–369, 2015. © 2014 Wiley Periodicals, Inc.     

Ultrastructure of the sperm of Dolania americana Edmunds and Traver (Ephemeroptera : Behningiidae)

The ultrastructure of the mature sperm of the mayfly, Dolania americana Edmunds and Traver (Ephemeroptera : Behningiidae), is described from scanning and transmission electron microscopy. The head is 0.7–1 μm wide and 4.6–6.9 μm long, rodlike, and topped by a short, rounded acrosome 0.4 μm long and 0.6 μm wide. The flagellum is 5–6 times the head length and is flattened, except for a thin, tubelike terminal portion. The axoneme pattern is 9-9-1 (9 outer singlet microtubules, 9 doublet microtubules, and a central dark element) and is new for Ephemeroptera. The inner dynein arms are conspicuous and outer arms are lacking, and radial spokes and a central sheath are prominent. A densely-staining and bi-lobed accessory body lies adjacent to the axoneme. A mitochondrial derivative with regularly arranged transverse-to-oblique cristae lies adjacent to the accessory body.     

Ultrastructure of spermiogenesis and synthesis of enigmatic cytoplasmic inclusions in the spirally shaped spermatozoon of the polychaete Spio setosa (Annelida: Spionidae)

The sperm of Spio setosa (Polychaeta, Spionidae) are known to be very unusual in form; here, spermiogenesis and the structure of the spermatozoon in this species are described by transmission electron microscopy. While spermiogenesis is similar to that described for many other polychaetes, two notable exceptions to this process include the synthesis of abundant ring‐shaped and tubular, membrane‐bounded cytoplasmic inclusions in the midpiece, and the differentiation of a spirally shaped sperm head. Spermatids develop as free‐floating tetrads in the male's coelom. A microtubular manchette does not develop during chromatin condensation and nuclear elongation, and the spiral acrosome forms as a single Golgi‐derived vesicle that migrates anteriorly to become housed in a deep anterior nuclear fossa. Early in spermiogenesis, numerous Golgi‐derived, membrane‐bounded cytoplasmic inclusions appear in the cytoplasm; these ultimately occupy the sperm midpiece only. The mature spermatozoon in the male has a 15‐μm‐long head consisting of a nucleus coiled like a spring and a spiral acrosome with differentiated substructure, the posterior two thirds of which sits in an anterior nuclear fossa. The midpiece is wider than the rest of the spermatozoon and contains 9–10 spherical mitochondria surrounding the two centrioles, as well as numerous membrane‐bounded conoid and tubular cytoplasmic inclusions. The axoneme has a 9 + 2 arrangement of microtubules. By contrast, stored sperm in the female's seminal receptacles have lost the midpiece inclusions but contain an abundance of glycogen. The function of the midpiece inclusions remains unresolved, and the significance of their absence in stored sperm within the seminal receptacle and the appearance of midpiece glycogen stores remains unclear and requires additional investigation.     

Ultrastructure of spermiogenesis, sperm, and the spermatheca in Terebrasabella heterouncinata (Polychaeta: Sabellidae: Sabellinae)

Abstract. The sabellid polychaete, Terebrasabella heterouncinata, forms burrows in gastropod shells. It is a small, intratubular brooder that breeds semi‐continuously. It has been shown to self‐fertilize, but its reproductive biology suggests that some form of sperm transfer must occur between individuals. To gain an understanding of its fertilization biology, the ultrastructure of spermiogenesis and the sperm in T. heterouncinata was described, and the animal examined for sperm storage structures. Spermiogenesis occurs in clusters of eight spermatids. The mature sperm has an elongate nucleus and a bilaterally symmetrical acrosome with twisted subacrosomal spaces. The midpiece is short, with three crescent‐shaped mitochondria, and forms a tight sheath around the axoneme. A single spermatheca, which opens on the inner ventral part of the crown near the buccal region, is present. It is a simple blind‐ending duct that runs below the ventral nerve cord and is longer than 100 μm. This is the first record of a single spermatheca in Sabellidae. The shape of the sperm and the presence of a spermatheca confirm that individuals of T. heterouncinata produce ent‐aquasperm and would normally cross‐fertilize.     

Collection and evaluation of epididymal sperm in captive agoutis (Dasyprocta aguti)

The objective was to establish a protocol for the collection and evaluation of epididymal sperm in agoutis. Eight males (1-2 y old) underwent left orchidectomy and epididymal sperma were collected by retrograde flush. Average values were flush volume 32 μL, pH 6.9, sperm concentration 748 x 10⁶ sperm/mL, with motility 86.5% and vigor 4.6. Viable sperm were present in all flush samples; 66% of sperm were alive, and 41.9% of sperm responded positively to the hypoosmotic test (using distilled water). There were 21.1% morphologically abnormal sperm, of which 2.0 and 19.1% were primary and secondary defects, respectively. The acrosome was intact in 99.5% of sperm. The sperm head was 4.89 ± 0.41 μm long and 3.13 ± 0.35 μm wide, with an area of 13.01 ± 2.01 μm². Midpieces were 5.33 ±0.44 μm long and 0.98 ± 0.13 wide, sperm tails were 29.91 ± 2.29 μm, and overall sperm length was 40.12 ± 2.44 μm. In conclusion, epididymal sperm collection from agoutis was satisfactory; the collected sperm has the potential to be stored, facilitating development of other reproductive biotechnologies for this species.     

The fine structure of the spermatozoa of Siphonaria algesirae (Gastropoda,Pulmonata)

The sperm of Siphonaria algesirae (Gastropoda, Pulmonata), a species with internal fertilization, was studied by light and electron microscopy. The spermatozoon is a very long, uniflagellate cell composed of a conical head with an apical acrosome, a midpiece with a helically coiled external sheath containing a complex mitochondrial derivative with a wavelength of ～ 5.5 μm, and an endpiece. There are no axonemal microtubules. Instead, nine homogeneous coarse fibers with transverse striations in the apical zone project toward the anterior section of the midpiece. In the posterior zone of the midpiece the coarse fibers are differentiated in a common microtubular axoneme. The complex mitochondrial derivative of the midpiece shows an organized group of 100 Å diameter spherical particles. Externally the midpiece is surrounded along its length by a cylinder formed by two membranes. A complex structure separates the transitional zone between the midpiece and the endpiece.     

Fine structure of the giant aflagellate spermatozoon in pseudostomum quadrioculatum (leuckart) (platyhelminthes,prolecithophora)

The giant aflagellate spermatozoa of P. quadrioculatum are composed of two different parts: a thicker head piece and a more slender tail piece. In the head there exist a large elongated nucleus and an elongated mitochondrial derivative situated in a groove-like cavity of the nucleus. In mature spermatozoa the nuclear material is arranged in many small membrane bounded areas. Both structures, nucleus and mitochondrial derivative, are spirally coiled. The outer part of the membrane in the mitochondrial derivative forms many loop-like foldings. Both organelles continue to the tail in form of two small, helically coiled ribbons; the nucleus is anchored within the mitochondrial derivative by an electron-opaque process. A sheath of spirally-orientated cortical microtubules starting from the tip of the head runs to the tip of the tail under the cell membrane. In addition, a second sheath of tubules occurs in the tail region, these tubules also run parallel to each other, but in the opposite direction to the microtubules of the outer sheath.The possible relations between the structures observed and the motility of the spermatozoa are discussed; in addition, some phylogenetic comments are attempted.Abbreviations c — cerebrum - com — cortical microtubules - cop — copulatory organ - fm — foldings of the mitochondrial membrane - l — lattice - mid — mitochondrial derivative - mt — microtubules - n — nucleus - ne — nuclear envelope - ph — pharynx - pn — protonephidium - rp — ribbon-like nuclear process - te — testis - tt — testis - tt — tip of the tail - vi — vitellarium - vs — vesicula seminalis     

Comparison of flagellated and nonflagellated sperm in plants

Differences among flagellated and nonflagellated sperm in land plants are striking, but close examination reveals similarities in pattern of cytoskeleton and in nuclear structure. The microtubular cytoskeleton of flowering plant sperm consists of microtubule bundles arranged obliquely around the nucleus, terminating in cellular extensions. Microtubules are linked into bundles that branch and rejoin along the axis of the sperm cell, forming a cytoskeleton that determines cell shape but does not actively participate in cell movement. Generative cells and sperm share a pattern of microtubules not found in somatic cells. This pattern is initiated in the generative cell, one division before sperm formation, a situation parallel to spermatogenous cell development in vascular plants with flagellated sperm. Chromatin in flagellated and nonflagellated sperm is condensed by specialized histones.     

Prohibitin involvement in the generation of mitochondrial superoxide at complex I in human sperm

Prohibitin (PHB), a major mitochondrial membrane protein, has been shown earlier in our laboratoryto regulate sperm motility via an alteration in mitochondrial membrane potential (MMP) in infertile men with poor sperm quality. To test if PHB expression is associated with sperm mitochondrial superoxide (mROS) levels, here we examined sperm mROS levels, high MMP and lipid peroxidation in infertile men with poor sperm motility (asthenospermia, A) and/or low sperm concentrations (oligoasthenospermia, OA). The diaphorase‐type activity of sperm mitochondrial complex I (MCI) and PHB expression were also determined. We demonstrate that mROS and lipid peroxidation levels are significantly higher in sperm from A and OA subjects than in normospermic subjects, whereas high MMP and PHB expression are significantly lower. A positive correlation between mROS and lipid peroxidation and a negative correlation of mROS with PHB expression, high MMP, and sperm motility were found in these subjects. The finding of similar diaphorase‐type activity levels of sperm MCI in the three groups studied suggests that the catalytic subunits of MCI in the matrix arm may produce mROS on its own. There may be a dysfunction of electron transport at MCI associated with decreased expression of PHB in sperm with poor quality. We conclude that mROS level is increased and associated with decreased PHB expression, and it may regulate sperm motility via increases in low MMP and lipid peroxidation. This is the first report on the involvement of PHB in human sperm motility loss associated with increased generation of mROS at MCI.     

The 3-dimensional fine structure of the mitotic spindle in Trypanosoma cruzi

The fine structure of nuclear division in the hemoflagellate Trypanosoma cruzi has been studied with serial sections and three-dimensional reconstructions of each divisional stage. After a preliminary stage in which the chromatin becomes dispersed, there is an equatorial stage defined by the appearance of an arranged set of ten dense plaques located about the equatorial region of the nucleus. At this stage a regular microtubular spindle is formed in the nucleus. Each plaque has a symmetrical structure formed by transverse bands and the bands are formed by tightly packed fibrillar material. The wide faces of the plaques are associated with tangential microtubules coming from the poles while the front and rear edges are free to associate with chromatin. Although structural continuity between chromatin fibers and the material of the plaques is possible, this continuity has not been proved. The equatorial spindle is formed by about 120 microtubules arranged in two sets of about 60 microtubules running from each pole to the dense plaques and divided into discrete bundles which reach a single plaque. The microtubules of each bundle may pass tangential to the wide faces of the plaque and end about 0.2 m beyond it, or they may end at the pole-facing edges of the plaque. No continuous, interpolar microtubules were observed at this stage. At the beginning of the elongational stage the dense plaques split into halves and each set of half-plaques migrates to one pole. During mid-elongational stage the pole-converging microtubules and the polar bulges disappear and microtubules become rearranged between the two sets of half-plaques. During late elongational stages, continuous microtubules run between the two sets of half-plaques and maximum nuclear elongation is attained. Chromatin remains dispersed throughout nuclear division. Two main movements have been observed in these mitotic nuclei: the migration of half-plaques to the poles and the elongation of the nucleus. Both these movements are accompanied by large changes in the architecture of the microtubular spindle and are probably dependent on microtubular function. It is concluded that the dense plaques play a kinetochore-like role and thus T. cruzi would have ten chromosomal units.     

Structural and immunocytochemical characterization of the Ginkgo biloba L. sperm motility apparatus

Summary. Ginkgo biloba and the cycads are the only extant seed plants with motile sperm cells. However, there has been no immunocytochemical characterization of these gametes to determine if they share characteristics with the flagellated sperm found in bryophytes and pteridophytes or might give clues as to the relationships to nonflagellated sperm in all other seed plants. To determine characteristics of proteins associated with the motility apparatus in these motile sperm, we probed thin sections of developing spermatogenous cells of Ginkgo biloba with antibodies to acetylated and tyrosinated tubulin and monoclonal antibodies that recognize mammalian centrosomes and centrin. The blepharoplast that occurs as a precursor to the motility apparatus consists of an amorphous core, pitted with cavities containing microtubules and a surface studded with probasal bodies. The probasal bodies and microtubules within the blepharoplast cavities are labeled with antibodies specific to acetylated tubulin. Positive but weak reactions of the blepharoplast core occur with the centrosomereactive antibodies MPM-2 and C-9. Reactions to centrin antibodies are negative at this developmental stage. From this pre-motility apparatus structure, an assemblage of about 1000 flagella and associated structures arises as the precursor to the motility apparatus for the sperm. The flagellar apparatus consists of a three-layered multilayered structure that subtends a layer of spline microtubules, a zone of amorphous material similar to that in the blepharoplast, and the flagellar band. Centrin antibodies react strongly with the multilayered structure, the transition zone of the flagella, and fibrillar material near the flagellar base at the surface of the amorphous material. Both the spline microtubules and all of the tubules in the flagella react strongly with the antibodies to acetylated tubulin. These localizations are consistent with the localizations of these components in pteridophyte and bryophyte spermatogenous cells, although the blepharoplast material surrounding and connecting flagellar bases does not occur in the seedless (nonseed) land plants. These data indicate that despite the large size of ginkgo gametes and the taxonomic separation between pteridophytes and Ginkgo biloba, similar proteins in gametes of both groups perform similar functions and are therefore homologous among these plants. Moreover, the presence of acetylated tubulin in bands of microtubules may be a characteristic shared with more derived non-flagellated sperm of other conifers and angiosperms. Correspondence and reprints: Southern Weed Science Research Unit, USDA Agricultural Research Service, P.O. Box 350, Stoneville, MS 38776, U.S.A.     

Microfluidic mixing for sperm activation and motility analysis of pearl Danio zebrafish

Sperm viability in aquatic species is increasingly being evaluated by motility analysis via computer-assisted sperm analysis (CASA) following activation of sperm with manual dilution and mixing by hand. User variation can limit the speed and control over the activation process, preventing consistent motility analysis. This is further complicated by the short interval (i.e., less than 15 s) of burst motility in these species. The objectives of this study were to develop a staggered herringbone microfluidic mixer to: 1) activate small volumes of Danio pearl zebrafish (Danio albolineatus) sperm by rapid mixing with diluent, and 2) position sperm in a viewing chamber for motility evaluation using a standard CASA system. A herringbone micromixer was fabricated in polydimethylsiloxane (PDMS) to yield high quality smooth surfaces. Based on fluorescence microscopy, mixing efficiency exceeding 90% was achieved within 5 s for a range of flow rates (from 50 to 250 μL/h), with a correlation of mixing distances and mixing efficiency. For example, at the nominal flow rate of 100 μL/h, there was a significant difference in mixing efficiency between 3.5 mm (75 ± 4%; mean ± SD) and 7 mm (92 ± 2%; P = 0.002). The PDMS micromixer, integrated with standard volumetric slides, demonstrated activation of fresh zebrafish sperm with reduced user variation, greater control, and without morphologic damage to sperm. Analysis of zebrafish sperm viability by CASA revealed a statistically higher motility rate for activation by micromixing (56 ± 4%) than manual activation (45 ± 7%; n = 5, P = 0.011). This micromixer represented a first step in streamlining methods for consistent, rapid assessment of sperm quality for zebrafish and other aquatic species. The capability to rapidly activate sperm and consistently measure motility with CASA using the PDMS micromixer described herein will improve studies of germplasm physiology and cryopreservation.     

Environmental osmolality influences sperm motility activation in an anuran amphibian

Evolutionary theory predicts that selection will favour sperm traits that maximize fertilization success in local fertilization environments. In externally fertilizing species, osmolality of the fertilization medium is known to play a critical role in activating sperm motility, but there remains limited evidence for adaptive responses to local osmotic environments. In this study, we used a split‐sample experimental design and computer‐assisted sperm analysis to (i) determine the optimal medium osmolality for sperm activation (% sperm motility and sperm velocity) in male common eastern froglets (Crinia signifera), (ii) test for among‐population variation in percentage sperm motility and sperm velocity at various activation‐medium osmolalities and (iii) test for among‐population covariation between sperm performance and environmental osmolality. Frogs were obtained from nine populations that differed in environmental osmolality, and sperm samples of males from different populations were subjected to a range of activation‐medium osmolalities. Percentage sperm motility was optimal between 10 and 50 mOsm kg^?1, and sperm velocity was optimal between 10 and 100 mOsm kg^?1, indicating that C. signifera has evolved sperm that can function across a broad range of osmolalities. As predicted, there was significant among‐population variation in sperm performance. Furthermore, there was a significant interaction between activation‐medium osmolality and environmental osmolality, indicating that frogs from populations with higher environmental osmolality produced sperm that performed better at higher osmolalities in vitro. This finding may reflect phenotypic plasticity in sperm functioning, or genetic divergence resulting from spatial variation in the strength of directional selection. Both of these explanations are consistent with evolutionary theory, providing some of the first empirical evidence that local osmotic environments can favour adaptive sperm motility responses in species that use an external mode of fertilization.