The crater defect in boar spermatozoa: A correlative study with transmission electron microscopy,scanning electron microscopy,and light microscopy

Nuclear vacuoles resembling the “crater defect” described in bull spermatozoa were observed in 14 boars. Both the incidence of the defect and semen quality were monitored with phase contrast microscopy over a three-month period. The percentages of cratered spermatozoa varied widely both among boars and in ejaculates from the same boar taken on different days. The presence of cratered spermatozoa at a level of 5% or more appeared to be associated with low semen quality. The defect was studied with scanning and transmission electron microscopy and was found to consist of nuclear invaginations, about 0.5 μm in diameter, containing some scanty amorphous electron-dense material. In boars showing a high incidence of spermatozoa with crater defects, abnormalities of the acrosome and perforatorium were common.     

Sperm volumetric dynamics during in vitro capacitation process in bovine spermatozoa

Previous studies have demonstrated that sperm head morphometry can be used as a potential diagnostic tool for detecting biophysical changes associated with sperm viability in bovine spermatozoa. In this study, sperm head morphometry was used to investigate its value as a biophysical marker for detecting volumetric changes in bovine spermatozoa under in vitro capacitating and non-capacitating incubation conditions. To further test this hypotesis, aliquots of pooled, washed bovine sperm were incubated in either Tyrode’s complete medium with heparin (TCMH; a capacitating medium containing Ca²⁺, NaHCO₃ and heparin), Tyrode’s complete medium heparin-free (TCM; a medium containing just Ca²⁺ and NaHCO₃) or Tyrode’s basal medium (TBM; a non-capacitating medium free of Ca²⁺, NaHCO₃ and heparin, used as control). Aliquots of sperm were processed for morphometric analysis at different incubation-time intervals (0, 3 and 6 h at 38°C), and the chlortetracycline assay was used simultaneously to confirm the ability of the sperm to undergo capacitation (B pattern) and the acrosome reaction (AR pattern) status in each medium. After 3 h of incubation under TCMH conditions, a significant increase was observed in the percentage of B and AR patterns and a significant decrease was found in all sperm morphometric parameters (P<0.01). Interestingly, after 6 h of incubation in TCMH, the percentage of B and AR patterns increased drastically over time and marked differences were found in the dimensional and shape parameters, which were significantly smaller compared with TBM or TCM media (P<0.001). Significant correlations were observed between sperm size and AR pattern (r=−0.875, P<0.01). In conclusion, sperm head morphometry can be used as a potential biophysical marker for detecting volumetric changes during capacitation process in bovine spermatozoa.     

Quantitative analysis of flagellar movement in hyperactivated and acrosome-reacted golden hamster spermatozoa

Caudal epididymal spermatozoa of golden hamsters were incubated in capacitation medium. Their movement patterns changed as they became hyperactivated and underwent the acrosome reaction. To understand the basic mechanism by which changes in movement pattern are brought about, digital image analysis was carried out on the flagellar movements recorded with a video system. The degree of flagellar bending increased with incubation time, especially in the proximal midpiece. The hyperactivated spermatozoa had remarkably asymmetrical flagellar waves of large amplitude because either the bends in the same direction as the hook of the head (referred as the "pro-hook bend") or the bends in the opposite direction to the hook of the head (referred as the "anti-hook bend") extremely increased their curvature; whereas, the acrosome-reacted spermatozoa had relatively symmetrical flagellar waves of large amplitude because both the pro- and anti-hook bends remarkably increased their curvature. Beat frequency significantly decreased while wavelength of flagellar waves increased after hyperactivation and further after the acrosome reaction. These results suggest that both extreme pro- and anti-hook bends are essential in the acrosome-reacted spermatozoa even though beat frequency decreased markedly.     

Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid

Human ejaculated spermatozoa were washed through a Percoll gradient, preincubated for 10 hr in a defined medium containing serum albumin, and then induced to undergo rapid acrosome reactions by addition of human follicular fluid or a Sephadex G-75 column fraction of the fluid. Induction by follicular fluid did not occur when the spermatozoa were preincubated for only 0 or 5 hr. The reactions were detected by indirect immunofluorescence using a monoclonal antibody directed against the human sperm acrosomal region. The percentage of acrosomal loss counted by transmission electron microscopy agreed with that counted by immunofluorescence. The apparent molecular weight of the Sephadex G-75 fraction containing the peak of acrosome reaction-inducing activity was 45,000 ± 4,200 (SD). The occurrence of physiological acrosome reactions was supported by: assessing motility (no significant loss of motility occurred during the treatment period when sperm were preincubated with bovine serum albumin), transmission electron microscopy (the ultrastructural criteria for the acrosome reaction were met), and zona-free hamster oocyte binding and penetration (spermatozoa pretreated with the active fraction of follicular fluid, then washed and incubated with oocytes, showed significantly greater binding to and penetration of oocytes). The stimulation of the acrosome reaction by follicular fluid is apparently not due to blood serum contamination; treatment of preincubated spermatozoa with sera from the follicular fluid donors had no effect on the spermatozoa. The nature of the active component(s) in that fraction is currently being investigated.     

Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoa

Actin cytoskeleton remodeling is a critical process for the acquisition of fertilizing capacity by spermatozoa during capacitation. However, the molecular mechanism that regulates this process has not been fully elucidated. In somatic cells, Ras-related C3 botulinum toxin substrate 1 protein (Rac1) promotes the polymerization of actin by participating in the modeling of two structures: lamellipodia and adhesion complexes linked with the plasma membrane. Rac1 is expressed in mammalian spermatozoa; however, the role of Rac1 in sperm physiology is unknown. This study aimed to elucidate the participation of Rac1 in capacitation and acrosome reaction (AR). Rac1 was found to be dispersed throughout the acrosome and without changes in the middle piece. After 60 minutes of capacitation, Rac1 was found in the apical region of the acrosome only, which concurred with an increase in Rac1-GTP. Rac1 inhibition prevented such changes. In the middle piece, Rac1 localization remained unchanged. Besides, Rac1 inhibition blocked capacitation and AR. The present study demonstrates that Rac1 participates only in the actin cytoskeleton remodeling that occurs in the acrosomal apical region during capacitation, a region where a large amount of actin is polymerized and shaped in a diadem-like structure. Our data also show that this actin cytoskeleton organized by Rac1 interacts with filamin-1, and such interaction was blocked by the inhibition of Rac1, which led to a different organization of the actin cytoskeleton. All these outcomes imply that the formation of an F-actin cytoskeleton in the acrosomal apical region is a necessary event for capacitation and AR, and which is Rac1 driven.     

Selection of a high quality subpopulation of frozen thawed bovine spermatozoa by filtration in a Sephadex column. An assessment of functional effects of progesterone

It is accepted that cryopreservation exerts deleterious effects on functional characteristics of mammalian spermatozoa. Conventional procedures for processing frozen-thawed gametes, such as centrifugation, produce additional damage. In the present work, we investigated the efficacy of processing bovine cryopreserved semen by filtration in a Sephadex column (SF group) or by washing by centrifugation (100 g, 10 min, twice) (W group); the results obtained from both procedures were compared to untreated samples (C group). The effects of in vitro addition of progesterone (10 μM, 20 min) upon sperm functional activity were studied also. The evaluated sperm parameters were concentration, motility (progressive or non progressive cells), viability and acrosome reaction. They were measured at time 0 (immediately after processing) or after 4 h incubation in capacitating conditions. Sperm concentration was (× 10⁻⁶): 37.5 ± 5.4 in C, 8.3 ± 2.1 in W and 12.5 ± 2.9 in SF. The percentages of motile, progressive, viable or acrosome intact gametes were significantly higher in SF than in W or in C. in SF group, after 4 h incubation in capacitating conditions, progesterone increased significantly the population of acrosome reacted cells whereas this parameter was not modified when the cells were incubated in absence of heparin. Motility and viability were not modified by the hormone. We conclude that Sephadex filtration method is an adequate tool to obtain a subpopulation of spermatozoa with superior quality, as assessed by motility, viability and acrosomal integrity; besides, our results strongly support that, as in other species, progesterone would be a physiological inductor of acrosome reaction in bovine.     

Circular DNA in human and boar spermatozoa

Circular DNA molecules were isolated from human and boar whole spermatozoa or spermatozoal nuclei and measured for size by electron microscopy. The DNA molecules derived from both mammals were heterogeneous in size ranging from 0.07 to 17 μm; nearly 75% of the molecules were ?0.5 μm in length. The mean lengths were 1.0 μm and 1.5 μm for circular DNAs isolated from human and boar spermatozoa, respectively. The origin and function of these molecules remains unknown.     

Comparative distribution of short dystrophin superfamily products in various guinea pig spermatozoa domains

In this study, the presence and cellular distribution of dystrophin family products (i.e. Dp71d, Dp71f-like protein and dystrobrevin) was examined by indirect immunofluorescence and Western blotting in guinea pig spermatozoa. Two dystrophin-associated proteins, beta-dystroglycan and alpha-syntrophin, and nNOS a protein frequently associated with alpha-syntrophin, were determined. In spermatozoa lacking plasma membrane and acrosome, Dp71f-like protein was found in the postacrosomal perinuclear theca and also in the middle piece of the flagellum. In the flagellum, Dp71f-like protein is localized together with alpha-syntrophin and nNOS. Dp71d was present in the plasma membrane of the middle piece with beta-dystroglycan, alpha-syntrophin and nNOS. Dp71d was also present in plasma membrane of the post acrosomal region, but only with nNOS. Finally, dystrobrevin was located all along skeletal flagellum structures and in the subacrosomal hemisphere of the perinuclear theca. This distinct and complementary distribution in various domains of spermatozoa may reveal a specific function for each short dystrophin family product, in the stabilization of the domains where they are located.     

Observations on the acrosome reaction of human sperm in vitro

Human sperm were incubated in vitro in serum or the defined medium TMPA and were periodically assessed for acrosome reactions using two new methods of assay. The first method, FITC-RCA labeling, was previously shown to be valid for estimating the percentage of normal acrosome reactions of human sperm. The second method, a triple staining technique, is shown in this study to give results comparable to those obtained with FITC-RCA labeling. The percentage of acrosome-reacted sperm was determined at 0, 2.5, 5, and 7 hr of incubation. In both media, some sperm had reacted by 2.5 hr; a maximum percentage of reactions occurred between 5 and 7 hr. The maximum percentage never exceeded 20–25%, which represents only one-third of the live sperm, ie, those potentially able to undergo normal acrosome reactions. It will be important in future studies to determine if this low-peak percentage is due to the fact that: (1) Commonly used culture media are suboptimal or (2) only about 25% of the sperm in a human ejaculate are capable of undergoing normal acrosome reactions.     

Bacteria induce expression of apoptosis in human spermatozoa

An increased number of sperm undergoing apoptosis has been observed during inflammatory processes in the male genital tract, which might be associated with elevated reactive oxygen species (ROS) levels. However, another factor to stimulate apoptosis could be the direct contact with bacteria or its products, even in the absence of ROS. The aim of this study was to investigate whether bacteria can directly initiate apoptosis in human spermatozoa. Human spermatozoa selected by density gradient centrifugation were incubated with polymorphonuclear granulocytes (PMN) isolated from blood and/or E. faecalis, E. coli or S. aureus. As ROS inductor in PMN, phorbol-12-myristate-13-acetate was used. After incubating the cells for 60 min at 37C, ROS were determined by chemiluminescence and phosphatidyl serine (PS) externalization was analyzed by flow cytometry with Annexin V-FITC and propidium iodide (PI). The increase in the percentage of spermatozoa Annexin V-FITC-positive/ PI-negative (early event of late apoptosis) was significant after the incubation with PMN plus PMA, PMN plus E. coli and E. coli alone. The percentage of spermatozoa Annexin V-FITC-positive/ PI-positive (apoptosis/necrosis) increased significantly in sperm incubated with E. coli and S. aureus(20.3% ± 3 and 13.6% ± 3.2 compared to sperm alone, 6% ± 0.5). Sperm incubated with PMN-PMA activated showed only a relative increase in apoptosis/necrosis (8.4% ± 1). Our results show that bacteria directly increase the PS externalisation in ejaculated human sperm. This way of inducing apoptosis does not require external ROS and may result from anyone of the molecular mechanisms that account for changes in motility, vitality and DNA integrity, that are characteristics of spermatozoa in male genital tract infection.     

Zinc does not inhibit the capacitation of human spermatozoa in vitro

The effects of zinc on human sperm motility, fertilizing capacity (as assessed by penetration of human spermatozoa into the zona pellucida-free hamster oocyte), and nuclear chromatin decondensation were investigated using spermatozoa from four fertile donors. Both sperm motility and the penetration of sperm into zona-free hamster ova were consistently impaired in media containing 1,000 μM zinc. Spermatozoa from one man were similarly affected at a concentration of 500 μM zinc, but no adverse effects were noted at this zinc concentration in experiments with other donors. Since decreased fertilizing capacity in response to zinc was always accompanied by a significant decline in both the percentage of motile cells and mean swimming speeds, it appears that all of these results reflect a general toxic effect on the cells. At lower concentrations (125–250 μM), zinc had no effect on human sperm motility nor their ability to undergo capacitation and penetrate zona-free hamster ova in vitro. For some donors, zinc (125–500 μM) stimulated both the attachment of spermatozoa to the hamster vitellus and the incorporation of spermatozoa into the hamster ooplasm. The decondensation of human sperm nuclear chromatin in sodium dodecyl sulfate was largely inhibited when zinc was added to the medium, but no significant changes in nuclear stability were apparent after capacitation in zinc-free medium. We conclude that zinc, when present in subtoxic concentrations, does not adversely affect the ability of human spermatozoa to undergo capacitation and penetrate zona-free hamster ova in vitro.     

Phosphorylation state of protamines 1 and 2 in human spermatids and spermatozoa

The basic nuclear proteins of a fraction of elongating spermatids from human tests and of a fraction of motile spermatozoa from the ejaculate, separated by ion-exchange chromatography, were compared. Analysis by acetic acid-urea polyacrylamide gel electrophoresis (PAGE) showed that, in both fractions, four proteins of lower mobility were coeluted with protamine 1 by 23% guanidinium chloride (GuCI) while protamine 2 alone was eluted by 50% GuCI. Treatment with alkaline phosphatase identified those four proteins as phosphorylated protamines, and cyanogen bromide (CNBr) treatment of the dephosphorylated protamines distinguished them as variants of protamine 2 and not of protamine 1. Thus far, phosphorylated forms of protamine 1 have not been detected in either spermatids or spermatozoa. Those observations indicate that protamine 2 functions in the cycle of phosphorylation-dephosphorylation, which is essential to the process of sperm chromatin condensation, while the role of protamine 1 in human spermiogenesis is not yet defined. The presence of phosphorylated protamine in motile, presumably mature spermatozoa appears to be characteristic of human sperm but not of the sperm of other mammals and is probably the basis for the heterogeneity of chromatin condensation frequently observed in human spermatozoa.     

GABA和孕酮对人及豚鼠精子的体外获能作用

为了探讨γ－氨基丁酸（ＧＡＢＡ）是否参与人及豚鼠精子体外获能的调节,将生育男子和豚鼠清子分别悬浮于ＢＷＷ和低Ｃａ＾２＋最小获能培养基（ＬＣａ＾２＋－ＭＣＭ）中,加入ＧＡＢＡ、孕酮（Ｐ４）、ＧＡＢＡＡ受体激动剂及其拮抗剂,在５％ＣＯ２孵箱３８．５℃培养２ｈ。然后用ｉｏｎｏｐｈｏｒｅ Ａ２３１８７激发精子顶体反应（ＡＲ）和超激活运动（ＨＡＭ）。以精子与金霉素（ＣＴＣ）荧光结合类型、ＡＲ和ＨＡＭ为指标来     

Expression of the human antigen SPAG2 in the testis and localization to the outer dense fibers in spermatozoa

Antisperm antibodies (ASAs) have been implicated in some instances of infertility. To characterize sperm antigens relevant to immunologic and immunocontraceptive development, SPAG2 (sperm-associated antigen 2) was identified by screening a human testis cDNA library with human sera positive for ASAs. Subsequently, two isoforms, SPAG2–1 and SPAG2–2, were identified in testis and placenta libraries, respectively. In the current study, Southern analysis of human genomic DNA with a probe common to the two SPAG2 isoforms indicated a single SPAG2 gene; therefore, alternative splicing is a likely mechanism for production of variant mRNAs. In situ hybridization of human testis sections demonstrated the expression of SPAG2 in primary spermatocytes, with decreased or arrested expression in postmeiotic cells. Immunofluorescence of Triton X-100–extracted spermatozoa with an anti-SPAG2 peptide antiserum indicate that SPAG2 is an intracellular component of the sperm flagellum. Electron microscopy refined this localization to the outer dense fibers (ODFs), structural filaments associated with the mammalian sperm axoneme. The ODFs have been reported to be composed of keratinlike intermediate filament proteins. However, SPAG2 does not exhibit the molecular characteristics of such proteins, nor does SPAG2 demonstrate sequence homology with previously characterized ODF proteins. Therefore, SPAG2 represents a novel protein of human sperm ODFs. Characterization of SPAG2 will further our understanding of ODF function in normal sperm motility and of flagellar abnormalities that lead to male infertility. Mol. Reprod. Dev. 50:284–293, 1998. © 1998 Wiley-Liss, Inc.     

Partial characterization of a fraction from human follicular fluid that initiates the human sperm acrosome reaction in vitro

A human follicular fluid (HFF) fraction prepared by Sephadex G-75 column chromatography has been previously shown by this laboratory to initiate the human sperm acrosome reaction (AR) in vitro. In the present report, the apparent molecular weight (MW) of this AR activity determined by a longer G-75 column than was used in the previous work was 50,000 ± 5,106. The G-75 Sephadex void volume fractions of some but not all HFF samples were also found to contain some AR-initiating activity. The occasional void volume activity was less potent than that of the 50,000 MW fraction and was not studied further. Further characterization of the 50,000 MW fraction was carried out. A time-course study demonstrated that maximum AR were obtained within 5 min following the addition of the 50,000 MW fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining revealed that the 50,000 MW fraction was still a relatively crude preparation. Treatment of the 50,000 MW fraction with chloroform:methanol did not extract the AR-initiating activity into the lipid phase. The AR-initiating activity of the untreated 50,000 MW fraction was precipitated when it was boiled, but the activity was partially resistant to boiling after overnight incubation. Treatment of the 50,000 MW fraction with pronase E or with several glycosaminoglycan hydrolases did not destroy the activity. Pronase treatment resulted in a higher amount of boiling-resistant AR-initiating activity. The AR-initiating activity of the untreated 50,000 MW fraction was partially dialyzable, but the activity of an undialyzed fraction did not pass through an ultrafiltration membrane with a 10,000 MW cut-off. However, treatment of the 50,000 MW fraction with protease, peptide:N-glycosidase F, and to a lesser extent chondroitinase ABC yielded an active lower MW activity which could pass through such an ultrafiltration membrane. The lower MW activity released by peptide:N-glycosidase F eluted in the included volume (5,000–1,000) of a Sephadex G-25 column. Neutral hexose but not protein or peptide was detected in the G-25 peak of AR-initiating activity. These results suggest that the AR-initiating activity present in the 50,000 MW fraction of HFF: (1) is present either as two different AR factors (a high-MW factor and a low-MW, noncovalently bound factor) or as a single factor responsible for both the nondialyzable and dialyzable AR-initiating activities (the latter being enzymatically released from the former), and (2) may be at least partially associated with N-linked oligosaccharides of a glycoprotein or proteoglycan.     

Lipid peroxidation in human spermatozoa as relatd to midpiece abnormalities and motility

The formation of malondialdehyde (MDA), a product of lipid peroxidation (LPO), was measured in human spermatozoa from 27 subjects with normal sperm characteristics. Peroxidation of lipids in washed spermatozoa was induced by catalytic amounts of ferrous ions and ascorbate and malondiaidehyde dctermint-d by thiobarbituric method. MDA formation varied considerably from one sample to another. The studied population showed a strong correlation between lipid peroxidation potential (amount of MDA formed by 10⁸ spermatozoa after 1 hour of incubation) and 1) initial motility r = ?0.623, P = 0.001; and 2) morphologic abnormalities of the midpiece r = 0.405, P = 0.05. These results suggest that poor motility is linked with membrane fragility and that spermatozoa with midpiece abnormalities probably have membrane and/or cytoplasmic antiperoxidant system defects. Because LPO potential is related to the two most important characteristics of fertility, it seems possible that it has the potential to become a good biochemical index of semen quality.     

Study on the role of calmodulin in sperm function through the enrichment and identification of calmodulin-binding proteins in bovine ejaculated spermatozoa

Calmodulin is a small, highly conserved acidic protein present at high levels in spermatozoa that mediates numerous intracellular Ca²⁺-dependent events. Sperm motility and fertilizing ability results from an array of biochemical pathways under Ca²⁺ control, in which the importance of calmodulin is not fully understood. The role of calmodulin in sperm function has been mostly assessed using antagonists. Nevertheless, few known calmodulin-regulated enzymes have been described in spermatozoa regarding their involvement in sperm function. To further understand the role of this important Ca²⁺ mediator in spermatozoa, different studies were also undertaken to investigate and to identify sperm calmodulin-binding proteins and determine their localization and subcellular distribution as an attempt to elucidate the role of this important Ca²⁺ mediator. In the present study, sperm calmodulin-binding proteins were identified by mass spectrometry after Ca²⁺-dependent biotinylated-calmodulin binding on sperm head proteins subjected to 2D electrophoresis and transferred on a polyvinylidene difluoride membrane. Calmodulin binding protein identification was also done on detergent extracted whole sperm proteins pulled down in a Ca²⁺-dependent manner by calmodulin-conjugated sepharose beads. In this latter group, 300 proteins were identified in at least two experiments out of three, and those identified in the three independent experiments were analyzed for overrepresented biological processes using the Bos taurus Gene Ontology database. Proteins with known function in reproductive processes, fertilization, sperm-egg recognition, sperm binding to the zona pellucida, regulation of sperm capacitation, and sperm motility were identified and further emphasize the importance of calmodulin in sperm function.     

Sperm-oocyte membrane fusion in the human during monospermic fertilization

Sperm-oocyte membrane fusion has been observed during monospermic fertilization of a human oocyte in vitro. Women were stimulated with both clomiphene citrate and human menopausal gonadotropin and were given human chorionic gonadotropin before a LH-surge. Twelve oocytes, collected at laparoscopy from six women who became pregnant by IVF, were allowed to mature for 7–14 hours in vitro and inseminated with preincubated sperm, fixed between 1–3 hours after insemination, and examined by transmission electron microscopy. Membrane fusion had occurred in one ovum 2 hours after insemination, and the oocyte had resumed maturation and was at anaphase II of meiosis. Cortical granules had been exocytosed, and some of their contents were visible at the surface close to the oolemma all around the oocyte. The sperm that fused with this oocyte was acrosome-reacted and had been partly incorporated into the ooplasm, while the anterior two-thirds of its head was phagocytosed by a tongue of cortical ooplasm. Membrane fusion had occurred between the oolemma and the plasma membrane overlying the postacrosomal segment of the sperm head, posterior to the equatorial vestige. Sperm chromatin had not decondensed, and serial sections revealed a midpiece attached to the basal plate and a tail located deeper in the ooplasm, all devoid of plasma membrane. Supplementary sperm penetrating the inner zona, approaching the perivitelline space, had undergone the acrosome reaction but had a persistent vestige of the equatorial segment of the acrosome with intact plasma membrane. Evidence of sperm chromatin decondensation was seen in other oocytes, 3 hours after insemination, which were at telophase II of meiosis. Eight oocytes penetrated by sperm were monospermic, while four were unfertilized. The general pattern of sperm fusion and incorporation appears to conform to that seen in most other mammals. The study also reveals that sperm have to complete the acrosome reaction before fusing with the egg.     

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.