Motor apparatus in human spermatozoa that lack central pair microtubules

Electron microscopic examination of the spermatozoa from a man suffering from asthenozoospermia (poor or low sperm motility) showed that approximately 92% of the sperm flagella lacked central pair microtubules but possessed dynein arms and radial spokes while a small percentage of the spermatozoa had complete flagella. The characteristics of the motor apparatus of the spermatozoa and the effects of caffeine on the sperm motility were examined, as were the reactivation of demembranated spermatozoa and the sliding of doublet microtubules. Almost all spermatozoa were immotile in a Tyrode solution while only a small percentage of spermatozoa showed slow forward movement or feeble flagellar vibration, whereas addition of caffeine to the sperm suspension induced forward swimming of approximately half of the spermatozoa. The reactivation of demembranated spermatozoa with MgATP(2-) could not succeed because of disintegration of the demembranated flagella. However, when the demembranated spermatozoa were exposed to MgATP(2-) and then treated with elastase, the microtubular doublets of approximately half the number of the flagella slid from the end or middle of the flagella. These results suggest that the motor apparatus in the sperm flagella that lack the central pair microtubules is functionally assembled and intrinsically capable of undergoing flagellar movement but not strong enough to beat normally.     

Fine structure of scorpion spermatozoa (Buthus occitanus; buthidae,scorpiones)

The mature spermatozoa of Buthus occitanus are threadlike in shape and divided into sperm head, middle piece, and end piece. The sperm head is corkscrew shaped anteriorly and in this region bears an unusual acrosomal complex consisting of a ring-shaped acrosomal vacuole associated with a subacrosomal filament and a perinuclear amorphous component. The subacrosomal filament extends posteriorly into a tube-like invagination of the elongated nucleus. The middle piece is characterized by elongated mitochondria which spiral around the anterior part of the flagellum in an extended collar separated from the flagellum by an extracellular cleft, termed the central flagellar tunnel. In addition to the usual 9 × 2 + 2 axonemal pattern in flagella, 9 × 2 + 1 and 9 × 2 + 3 patterns also were observed. The end piece is represented by the free flagellum. Similarities and diversities of scorpionid spermatozoa are discussed with respect to systematic relationships.     

Characterization and subcellular localization of Tektin 3 in rat spermatozoa

Mammalian sperm flagella have filament‐forming Tektin proteins (Tektin 1–5) reported to be involved in the stability and structural complexity of flagella. Male mice null for Tektin3 produce spermatozoa with reduced forward progression and increased flagellar structural bending defects. The subcellular localization of Tektin3 (TEKT3) in spermatozoa, however, has not been clarified at the ultrastructural level. To elucidate the molecular localization of TEKT3 in flagella of rat spermatozoa, we performed extraction studies followed by immunoblot analysis, immunofluorescence microscopy, and immunogold electron microscopy. Extraction of sperm flagella from the cauda epididymis resulted in complete removal of axonemal tubulins, while TEKT3 was resistant to extraction with the same S‐EDTA (1% SDS, 75 mM NaCl, 24 mM EDTA, pH 7.6) solution, suggesting that TEKT3 might be present in the peri‐axonemal component and not directly associated with axonemal tubulins. Resistance to S‐EDTA extraction might be due to disulfide bond formation during epididymal maturation since concentrations of DTT greater than 5 mM drastically promoted release of TEKT3 from flagella. Immunofluorescence microscopy and pre‐embedding immunoelectron microscopy revealed that TEKT3 was predominantly associated with the surface of mitochondria and outer dense fibers in the middle piece. In addition, TEKT3 was found to be present at the equatorial segment region of the acrosome membrane in sperm heads. TEKT3 might not only work as a flagellar constituent required for flagellar stability and sperm motility but also may be involved in acrosome‐related events, such as the acrosome reaction or sperm–egg fusion. Mol. Reprod. Dev. 78:611–620, 2011. © 2011 Wiley‐Liss, Inc.     

Ultrastructural studies of late-stage spermatids and mature spermatozoa of the puffer fish, Takifugu niphobles (Tetraodontiformes) and the effects of osmolality on spermatozoan structure

Ultrastructures of late-stage spermatids and spermatozoa, and of spermatozoa after exposure to various osmolalities, were studied in the puffer, Takifugu niphobles. The mature spermatozoa consisted of a head, a midpiece of many mitochondria and a flagellum with sharp sidefins, had many ring-structures just inside of the plasma membrane of cytoplasmic sleeve and triangular-structures projecting into cytoplasmic canal at the base of flagellum. In late spermatids, the rings and projections were present, but the side-fins had round ends and the cytoplasm of flagellum was amorphous. When spermatozoa were exposed to seawater, the plasma membrane became swollen in the head-midpiece region but shrank in the tail region. In 1/2 seawater, swelling in the tail occurred in some spermatozoa. In 1/3 seawater approximately isotonic to the seminal plasma, there was little change. In 1/10 seawater, the plasma membrane swelled slightly in the head region, but swelled much more in the tail region. In buffer solution, the membrane swelled in all regions, surrounding the nucleus and many sections of axoneme. Thus, function of the plasma membrane in the head-region may be different from that in the tail-region. Spermatozoa of marine fish may fertilize the eggs when the osmolality surrounding the sperm, which changes due to the mixing of seminal plasma and seawater, reaches the correct level for the spermatozoa to obtain correct structure.     

Ultrastructure of the biflagellate spermatozoon of tomopteris helgolandica greef, 1879 (annelida,polychaeta)

The spermatozoon of the polychaete Tomopteris helgolandica is of an aberrant type with two flagella, each measuring about 40μm. The nucleus is roughly conical and weakly bent. At the anterior end it is rounded and covered only by the nuclear and plasma membranes. Membraneous, electron-dense structures are applied laterally to the nucleus. These structures may have a helical arrangement. The middle piece contains about ten mitochondria, two centrioles, and two centriolar satellite complexes. The centriolar regions are connected with the posterior part of the nucleus. The axonemes of the two tail flagella lack the usual central complex with central tubules, radial spokes, or related structures. No arms seem to be present on the A tubules of the doublets. In the middle piece the tail flagella are surrounded by invaginations of the plasma membrane forming flagellar canals. The sperm has a bilateral symmetry whereas the primitive sperm has a radial symmetry. The occurrence of two tail flagella in this spermatozoon has no phylogenetical connection with biflagellate spermatozoa in other animal groups. A series of mutations has resulted in the development of two flagella emerging from the two centrioles, the lack of a central complex in the axoneme, and the lack of a typical acrosome. In the Polychaeta, sperm structure is generally more related to function that to phylogenetics. During swimming the spermatozoon of Tomopteris rotates around its longitudinal axis.     

Effects of osmolality on sperm morphology, motility and flagellar wave parameters in Northern pike (Esox lucius L.)

Northern pike (Esox lucius L.) spermatozoa are uniflagellated cells differentiated into a head without acrosome, a midpiece and a flagellar tail region flanked by a fin structure. Total, flagellar, head and midpiece lengths of spermatozoa were measured and show mean values of 34.5, 32.0, 1.32, 1.17 μm, respectively, with anterior and posterior widths of the midpiece measuring 0.8 and 0.6 μm, respectively. The osmolality of seminal plasma ranged from 228 to 350 mOsmol kg⁻¹ (average: 283.88 ± 33.05). After triggering of sperm motility in very low osmolality medium (distilled water), blebs appeared along the flagellum. At later periods in the motility phase, the tip of the flagellum became curled into a loop shape which resulted in a shortening of the flagellum and a restriction of wave development to the proximal part (close to head). Spermatozoa velocity and percentage of motile spermatozoa decreased rapidly as a function of time postactivation and depended on the osmolality of activation media (P < 0.05). In general, the greatest percentage of motile spermatozoa and highest spermatozoa velocity were observed between 125 and 235 mOsmol kg⁻¹. Osmolality above 375 mOsmol kg⁻¹ inhibited the motility of spermatozoa. After triggering of sperm motility in activation media, beating waves propagated along the full length of flagella, while waves appeared dampened during later periods in the motility phase, and were absent at the end of the motility phase. By increasing osmolality, the velocity of spermatozoa reached the highest value while wave length, amplitude, number of waves and curvatures also were at their highest values. This study showed that sperm morphology can be used for fish classification. Sperm morphology, in particular, the flagellar part showed several changes during activation in distilled water. Sperm motility of pike is inhibited due to high osmolality in the seminal plasma. Osmolality of activation medium affects the percentage of motile sperm and spermatozoa velocity due to changes in flagellar wave parameters.     

Molecular Cloning and Subcellular Localization of Tektin2-Binding Protein 1 (Ccdc 172) in Rat Spermatozoa

Tektins (TEKTs) are composed of a family of filament-forming proteins localized in cilia and flagella. Five types of mammalian TEKTs have been reported, all of which have been verified to be present in sperm flagella. TEKT2, which is indispensable for sperm structure, mobility, and fertilization, was present at the periphery of the outer dense fiber (ODF) in the sperm flagella. By yeast two-hybrid screening, we intended to isolate flagellar proteins that could interact with TEKT2, which resulted in the isolation of novel two genes from the mouse testis library, referred as a TEKT2-binding protein 1 (TEKT2BP1) and -protein 2 (TEKT2BP2). In this study, we characterized TEKT2BP1, which is registered as a coiled-coil domain-containing protein 172 (Ccdc172) in the latest database. RT-PCR analysis indicated that TEKT2BP1 was predominantly expressed in rat testis and that its expression was increased after 3 weeks of postnatal development. Immunocytochemical studies discovered that TEKT2BP1 localized in the middle piece of rat spermatozoa, predominantly concentrated at the mitochondria sheath of the flagella. We hypothesize that the TEKT2-TEKT2BP1 complex might be involved in the structural linkage between the ODF and mitochondria in the middle piece of the sperm flagella.     

The effect of sulfhydryl oxidation on the morphology of immature hamster epididymal spermatozoa induced to acquire motility in vitro

Immotile spermatozoa from the caput epididymidis become progressively motile when incubated in medium containing theophylline, seminal plasma, and albumin. We previously reported that under these incubation conditions the spermatozoa induced to acquire motility exhibited a marked flagellar angularity, with the sperm head or midpiece bent 90-180 degrees towards the tail. In addition, we demonstrated that sperm flagellar bending did not occur when the sulfhydryl oxidant diamide was added to the motility induction medium. In the present study, we examined further the effect of sulfhydryl oxidation on the morphology and sulfhydryl content of immature caput spermatozoa induced to acquire motility in vitro. We found that flagellar bending was prevented and sperm flagellar straightness was maintained in a dose-dependent manner by diamide. Moreover, flow cytometric analysis of caput sperm sulfhydryls using the sulfhydryl reagent monobromobimane (mBBr) revealed that 1) diamide oxidizes caput sperm sulfhydryls, and 2) less than 15% of the total reactive sperm sulfhydryls were oxidized at diamide concentrations capable of preventing sperm angulation. Sodium tetrathionate (NaTT), another sulfhydryl oxidant, and hamster cauda epididymal fluid (CEF) containing sulfhydryl oxidase enzyme activity also maintained flagellar straightness in induced caput spermatozoa and oxidized sperm sulfhydryls. The flagellar straightness in caput spermatozoa treated with sulfhydryl oxidants, however, was temporary; with extended incubation, diamide- or CEF-treated spermatozoa exhibited flagellar bending. Additional studies showed that the flagellar straightness observed in sulfhydryl-oxidized spermatozoa was sustained when nitrofurantoin, an inhibitor of glutathione reductase, was included in the induction medium. Flow cytometric analysis of nitrofurantoin-treated spermatozoa showed that nitrofurantoin maintained the sperm disulfides formed by diamide and prevented the reduction of sperm disulfides back to sulfhydryls. Taken together, these studies demonstrate the significance of sulfhydryl oxidation in maintaining the morphology of immature caput epididymal spermatozoa induced to acquire motility in vitro and suggest that sulfhydryl oxidation may be important in the development of motility during sperm epididymal maturation in vivo.     

Changes in sperm plasma membrane lipid diffusibility after hyperactivation during in vitro capacitation in the mouse

We have used the technique of fluorescence recovery after photobleaching to measure the diffusibility of the fluorescent lipid analogue, 1,1'-dihexadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate on the morphologically distinct regions of the plasma membranes of mouse spermatozoa, and the changes in lipid diffusibility that result from in vitro hyperactivation and capacitation with bovine serum albumin. We found that, as previously observed on ram spermatozoa, lipid analogue diffusibility is regionalized on mouse spermatozoa, being fastest on the flagellum. The bovine serum albumin induced changes in diffusibility that occur with hyperactivation are also regionalized. Specifically, if we compare serum incubated in control medium, which maintains normal motility, with those hyperactivated in capacitating medium, we observe with hyperactivation an increase in lipid analogue diffusion rate in the anterior region of the head, the midpiece, and tail, and a decrease in diffusing fraction in the anterior region of the head.     

Calcium regulation of flagellar curvature and swimming pattern in triton X-100--extracted rat sperm

Free Ca2+ changes the curvature of epididymal rat sperm flagella in demembranated sperm models. The radius of curvature of the flagellar midpiece region was measured and found to be a continuous function of the free Ca2+ concentration. Below 10(-7) M free Ca2+, the sperm flagella assumed a pronounced curvature in the same direction as the sperm head. The curvature reversed direction at 2.5 x 10(-6) M Ca2+ to assume a tight, hook-like bend at concentrations of 10(-5) to 10(-4) M free Ca2+. Sodium vanadate at 2 x 10(-6) M blocked flagellar motility, but did not inhibit the Ca2+-mediated change in curvature. Nickel ion at 0.2 mM and cadmium ion at 1 microM interfered with the transition and induced the low Ca2+ configuration of the flagellum. The forces that maintain the Ca2+-dependent curvature are locally produced, as dissection of the flagella into segments did not significantly alter the curvature of the excised portions. Irrespective of the induced pattern of curvature, the sperm exhibited coordinated, repetitive flagellar beating in the presence of ATP and cAMP. At 0.3 mM ATP the flagellar waves propagated along the principal piece while the level of free Ca2+ controlled the overall curvature. When Ca2+-treated sperm models with hooked midpieces were subjected to higher concentrations of ATP (1-5 mM), some cells exhibited a pattern of movement similar to hyperactivated motility in capacitated live sperm. This type of motility involved repetitive reversals of the Ca2+-induced bend in the midpiece, as well as waves propagated along the principal piece. The free Ca2+ available to the flagellum therefore appeared to modify both the pattern of motility and the flagellar curvature.     

Motility activation and metabolism characteristics of spermatozoa of the black-lip-pearl oyster Pinctada margaritifera var: cumingii (Jameson, 1901)

Motility of Pinctada margaritifera (Linnaeus, 1758); var: cumingii (Jameson, 1901) (P. margaritifera) spermatozoa collected from gonads are not immediately activated at spawning in seawater (SW) but motility occurs when spermatozoa are transferred into alkaline seawater (pH ranging from 9.0 to 11.4). This motility-activating effect of alkaline pH is reversed when pH is shifted back to more acidic values. In both cases, activity of sperm (% motile cells) increases gradually after alkaline pH activation then lasts for several minutes. The characteristics of these fully motile spermatozoa are described in details at the level of flagella: the wave amplitude and wave-length range 5 to 6 μm and 15 μm respectively, while the flagellar beat frequency is approximately 49 Hz. The velocity of sperm displacement is from 220 to 230 μm/sec. The general swimming pattern is almost circular: the head trajectories describe portions of circles intercalated with small linear segments. Spermatozoa saved in natural seawater at 4°C retain potent motility for several days and can be subsequently activated by alkaline seawater. Respiration and ATP concentration were measured in 3 conditions: regular seawater (pH 7.8), artificial diluent (pH 8.2), and alkaline Tris-buffered seawater (pH 10.5). Results show that sperm respiration rates are higher whereas ATP levels are lower in the latter two media.     

Diffusion and regionalization in membranes of maturing ram spermatozoa

An essential feature of the "fluid mosaic model" (Singer, S. J., and G. L. Nicolson , 1972, Science (Wash. DC)., 175:720-731) of the cell plasma membrane is the ability of membrane lipids and proteins to diffuse laterally in the plane of the membrane. Mammalian sperm are capable of overcoming free random diffusion and restricting specific membrane components, both lipid and protein, to defined regions of the sperm's surface. The patterns of these regionalizations evolve with the processes of sperm differentiation: spermatogenesis, epididymal maturation, and capacitation. We have used the technique of fluorescence recovery after photobleaching to measure the diffusion of the lipid analogue 1,1'- dihexadecyl 3,3,3',3'- tetramethylindocarbocyanine perchlorate ( C16dil ) on the different morphological regions of testicular and ejaculated ram spermatozoa. We have found: (a) that the major morphologically distinct regions (head, midpiece, and tail) of the plasma membrane of both testicular and ejaculated spermatozoa are also physically distinct as measured by C16dil diffusibility; (b) that despite regional differences in diffusibility there is exchange of this lipid analogue by lateral diffusion between the major morphological regions of the plasma membrane; and (c) that epididymal maturation results in changes in C16dil diffusibility in the different regions of the sperm plasma membrane. In particular, the plasma membranes of the anterior and posterior heads become physically distinct.     

Hyperactivation of monkey spermatozoa is triggered by Ca2+ and completed by cAMP

Digital image analysis of the flagellar movements of cynomolgus macaque spermatozoa hyperactivated by caffeine and cAMP was carried out to understand the change in flagellar movements during hyperactivation. The degree of flagellar bending increased remarkably after hyperactivation, especially at the base of the midpiece. Mainly two beating patterns were seen in the hyperactivated monkey sperm flagella: remarkably asymmetrical flagellar bends of large amplitude and relatively symmetrical flagellar bends of large amplitude. The asymmetrical bends were often seen in the early stage of hyperactivation, whereas the symmetrical bends executed nonprogressive, figure-of-eight movement. Beat frequency of the hyperactivated spermatozoa significantly decreased while wavelength of flagellar waves roughly doubled. To determine the conditions under which the axonemes of hyperactivated sperm flagella have asymmetrical or symmetrical bends, the plasma membranes of monkey spermatozoa were extracted with Triton X-100 and motility was reactivated with MgATP(2-) under various conditions. The asymmetrical flagellar bends were brought about by Ca(2+), whereas the symmetrical flagellar bends resulted from low levels of Ca(2+) and high levels of cAMP. Under these conditions, beat frequency and wavelength of flagellar waves of demembranated, reactivated spermatozoa were similar to those of the hyperactivated spermatozoa. These results suggest that during hyperactivation of monkey spermatozoa intracellular Ca(2+) concentrations first rise, and then decrease while cAMP concentrations increase simultaneously.     

Real-time analysis of the role of Ca(2+) in flagellar movement and motility in single sea urchin sperm

Eggs of many marine and mammalian species attract sperm by releasing chemoattractants that modify the bending properties of flagella to redirect sperm paths toward the egg. This process, called chemotaxis, is dependent on extracellular Ca(2+). We used stroboscopic fluorescence imaging to measure intracellular Ca(2+) concentration ([Ca(2+)]i) in the flagella of swimming sea urchin sperm. Uncaging of cyclic GMP induced Ca(2+) entry via at least two distinct pathways, and we identified a nimodipine-sensitive pathway, compartmentalized in the flagella, as a key regulator of flagellar bending and directed motility changes. We found that, contrary to current models, the degree of flagellar bending does not vary in proportion to the overall [Ca(2+)]i. Instead we propose a new model whereby flagella bending is increased by Ca(2+) flux through the nimodipine-sensitive pathway, and is unaffected by [Ca(2+)]i increases through alternative pathways.     

Nm23/NDP kinases in human male germ cells: role in spermiogenesis and sperm motility?

Nucleoside diphosphate (NDP) kinases, responsible for the synthesis of nucleoside triphosphates and produced by the nm23 genes, are involved in numerous regulatory processes associated with proliferation, development, and differentiation. Their possible role in providing the GTP/ATP required for sperm function is unknown. Testis biopsies and ejaculated sperm were examined by immunohistochemical and immunofluorescence microscopy using specific antibodies raised against Nm23-H5, specifically expressed in testis germinal cells and the ubiquitous NDP kinases A to D. Nm23-H5 was present in sperm extract, together with the ubiquitous A and B NDP kinases (but not the C and D isoforms) as shown by Western blotting. Nm23-H5 was located in the flagella of spermatids and spermatozoa, adjacent to the central pair and outer doublets of axonemal microtubules. High levels of NDP kinases A and B were observed at specific locations in postmeiotic germinal cells. NDP kinase A was transiently located in round spermatid nuclei and became asymmetrically distributed in the cytoplasm at the nuclear basal pole of elongating spermatids. The distribution of NDP kinase B was reminiscent of the microtubular structure of the manchette. In ejaculated spermatozoa, the proteins presented specific locations in the head and flagella. Nm23/NDP kinase isoforms may have specific functions in the phosphotransfer network involved in spermiogenesis and flagellar movement.     

The development of regionalized lipid diffusibility in the germ cell plasma membrane during spermatogenesis in the mouse

The lipids and proteins of sperm cells are highly regionalized in their lateral distribution. Fluorescence recovery after photobleaching studies of sperm membrane component lateral diffusibility have shown that the sperm plasma membrane is also highly regionalized in the extents and rates of diffusion of its surface components. These studies have also shown that regionalized changes in lateral diffusibility occur during the differentiative processes of epididymal maturation and capacitation. Unlike mammalian somatic cells, sperm cells exhibit large nondiffusing lipid fractions. In this paper, we will show that both regionalized lipid diffusibility and nondiffusing lipid fractions develop with the morphogenesis of cell shape during spermatogenesis in the mouse. Pachytene spermatocytes and round spermatids show diffusion rates and the nearly complete recoveries (80-90%) typical of mammalian somatic cells. In contrast, stage 10-11 condensing spermatids, testicular spermatozoa, cauda epididymal spermatozoa, as well as the anucleate structures associated with these later stages of spermatogenesis (residual bodies and the cytoplasmic droplets of condensing spermatids and testicular spermatozoa), exhibit large nondiffusing fractions. Both the diffusion rates and diffusing fractions observed on the anterior and posterior regions of the head of stage 10-11 condensing spermatids are the same as the values obtained for these regions on testicular spermatozoa. Possible mechanisms of lipid immobilization and possible physiological implications of this nondiffusing lipid are discussed.     

Ultrastructure of Thunnus thynnus and Euthynnus allettevatus spermatozoa

The spermatozoa of Thunnus thynnus and Euthynnus alletteratus consist of an acrosome-less head (comprising the ovoid nucleus and the short midpiece) and a long flagellar tail that contains the conventional 9 + 2 axoneme and lacks lateral fins. The centrioles are arranged at approximately right angles and lie outside of a shallow nuclear groove. The flagellum inserts laterally on the nucleus, therefore the spermatozoon is asymmetrical. The midpiece contains a few mitochondria which are separated from the axoneme by the cytoplasmic canal; they are spherical in T. thynnus and elongate, somewhat irregular in E. alletteratus . Although the main ultrastructural characteristics of the spermatozoa appear to indicate a great homogeneity in the sperm morphology within the family Scombridae, small species-specific divergences may be of use in systematics.     

The ultrastructure of amberjack (Seriola dumerilii) sperm

Scanning and transmission electron microscopy were used to investigate the fine structure of sperm of the Mediterranean amberjack Seriola dumerilii. Each spermatozoon has an ovoid head which lacks an acrosome, a short, irregularly-shaped midpiece and a long flagellar tail. The midpiece houses eight spherical mitochondria, which are separated from the axoneme by the cytoplasmic canal. The centrioles are arranged approximately at right angles to each other. The proximal centriole lies inside, and the distal centriole outside, the nuclear fossa. The flagellum is inserted eccentrically into the head and is tangential to the nucleus, so that the spermatozoon is asymmetrical. It contains the conventional 9 + 2 axoneme, shows intratubular differentiations in the A microtubules of doublets 1, 2, 5 and 6, and possesses one pair of lateral fins. On the basis of its ultrastructural organization, the amberjack sperm resembles type II sperm as defined previously, except for the presence of the proximal centriole inside the nuclear fossa. This could result from a partial rotation of the nucleus during spermiogenesis.     

Fine structure of the spermatozoon of the viviparous teleost, Cymatogaster aggregata

The approximately 50 μm long sperm of Cymatoguster aggregata is composed of an elongate head (4 μm), an elongate mitochondria1 midpiece (3.5 μm) and a tail flagellum (roughly 40 μm). The sperm lacks an acrosome. Contained within depressions on one surface of the compressed head are a proximal centriole and a distal centriole separated by an electron dense, intercentriolar body. The anterior portion of the tail flagellum originates at the basal body (distal centriole) and is contained within an extracellular, flagellar tunnel within the mitochondria1 midpiece. The morphological similarity of C. uggregutu sperm to sperm of other internally fertilizing fishes supports the hypothesis that spermatozoan morphology is related to the mode of fertilization and that an elongate head and midpiece are specializations for internal fertilization.     

Partially irreversible cold-induced lipid phase transitions in mammalian sperm plasma membrane domains: freeze-fracture study

In an effort to investigate the nature of the cellular injury caused when mammalian spermatozoa are cooled prior to cryopreservation, the occurrence of thermal phase transitions amongs the lipid components of the sperm plasma membrane was investigated by the use of freeze-fracture electron microscopy. The mechanisms by which glycerol and egg yolk exert protective effects during semen cooling and freezing were also examined. Ram and blackbuck spermatozoa, maintained at 30 degrees C prior to fixation at this temperature, exhibited randomly distributed intramembranous particles over the acrosomal, postacrosomal, and flagellar regions of the plasma membrane. In contrast, spermatozoa fixed at 5 degrees C after slow cooling to this temperature exhibited particle clustering over the postacrosomal region of the head as well as over the tail. These effects were not influenced by the presence of egg yolk or glycerol during the cooling procedure, although these substances protected the spermatozoa against loss of motility. Particle clustering over the sperm tail, induced by the slow cooling process, was found to be only partially reversible. The extensive areas of particle-free lipid, noted to result from the cooling procedure, were absent if the spermatozoa were rewarmed to 30 degrees C; however, the original distribution of particles was not restored and numerous small particle-free domains persisted. It is proposed that this type of irreversible change within the sperm plasma membrane may contribute to the loss of motility and fertility suffered by spermatozoa after cooling and freezing. Furthermore, it is suggested that protective substances such as egg yolk may exert their effects by countering these deleterious changes, rather than by preventing their occurrence.