Early degradation of paternal mitochondria in domestic pig (Sus scrofa) is prevented by selective proteasomal inhibitors lactacystin and MG132

Ubiquitin-dependent proteolysis has been implicated in the recognition and selective elimination of paternal mitochondria and mitochondrial DNA (mtDNA) after fertilization in mammals. Initial evidence suggests that this process is contributed to by lysosomal degradation of the ubiquitinated sperm mitochondrial membrane proteins. The present study examined the role of the proteasome-dependent protein degradation pathway of the ubiquitin system, as opposed to lysosomal proteolysis of the ubiquitinated proteins, in the regulation of sperm mitochondrion elimination after fertilization. Boar spermatozoa prelabeled with vital fluorescent mitochondrial probes MitoTracker were used to trace the degradation of paternal mitochondria after in vitro fertilization (IVF) of porcine oocytes. The degradation of sperm mitochondria in the cytoplasm of fertilized oocytes started very rapidly, i.e., within 12-20 h after insemination. Four stages of paternal mitochondrial degradation were distinguished, ranging from an intact mitochondrial sheath (type 1) to complete degradation (type 4). At 27-30 h postinsemination, 96% of zygotes contained the partially (type 3) or completely (type 4) degraded sperm mitochondria. Highly specific peptide inhibitors of the ubiquitin-proteasome pathway, lactacystin (10 and 100 microM) and MG132 (10 microM), efficiently blocked the degradation of the sperm mitochondria inside the fertilized egg when applied 6 h after insemination. Using 10 microM MG132, only 13.6% of fertilized oocytes screened 27-30 h after IVF displayed type 3 sperm mitochondria, and there was no incidence of type 4, completely degraded mitochondria. Although lactacystin is not a reversible agent, the effect of MG132 was fully reversible: zygotes transferred to regular culture medium after 24 h of culture with 10 microM MG132 resumed development and degraded sperm mitochondria within the next cell cycle. Surprisingly, penetration of the zona pellucida (ZP) was also inhibited by MG-132 and lactacystin when the inhibitors were added at insemination. Altogether, these data provide the first evidence of the participation of proteasomes in the control of mammalian mitochondrial inheritance and suggest a new role of the ubiquitin-proteasome pathway in mammalian fertilization.     

An improved, efficient method for analyzing human sperm chromosomes using zona-free hamster ova

We have developed an improved method for analyzing human sperm chromosome, using zona-free hamster ova. Our main improvements of methodology are as follows: (1) Fertilization rate of hamster oocytes by human spermatozoa was markedly raised by successive treatments of the spermatozoa with 5-15 microM ionophore A23187 solutions and a capacitation medium (BWW medium) containing 3.5% HSA. The HSA most effective in inducing capacitation was selected from several kinds of HSA products commercially available. (2) Monospermic fertilization was ensured by inseminating oocytes with highly capacitated spermatozoa at a low concentration for a short time. (3) TC medium 199 was used for postinsemination culture of the eggs. (4) A medium containing podophyllotoxin and vinblastine (0.04 micrograms/ml each) was used to block karyogamy and first-cleavage spindle formation. (5) Chromosome slides were prepared with our gradual fixation-air-dry method instead of Tarkowski's method. Ninety-two to 177 spermatozoa corresponding in number to 43%-79% (mean: 62%) of the inseminated oocytes were successfully karyotyped in each experiment. In spite of above-mentioned quantitative improvements, quality of Q-banding was not necessarily satisfactory in our slides. Improvement of banding technique is an important problem to be solved in our method. Spontaneous incidence of chromosome aberrations was studied in a total of 1,091 spermatozoa obtained from nine semen samples from four donors. Incidences of aneuploidy and structural anomaly were 0.9% (hyperhaploidy, 0.45%; hypohaploidy, 0.45%) and 13.0%, respectively. Structural aberrations included breaks (45.1%), fragments (32.4%), exchanges (21.8%), and deletions (0.7%). Ratio of X-sperm to Y-sperm was 53% to 47%. These results were discussed in comparison with those reported previously.     

Evaluation of chromosomal risk following intracytoplasmic sperm injection in the mouse

To investigate whether cytogenetic risks occur using the mouse intracytoplasmic sperm injection (ICSI) technique, the incidence of chromosome aberrations was compared in one-cell embryos produced by ICSI technique and those by conventional in vitro fertilization (IVF) technique. Spermatozoa were incubated in TYH medium for 1.5-2 h before IVF insemination. For the ICSI technique, spermatozoa were incubated in five different media: TYH, Hepes-buffered TYH (H-TYH), modified CZB (mCZB), Hepes-buffered mCZB (H-mCZB), and PB1 for 0.5 h, 2-2.5 h, and 6 h before injection into metaphase II oocytes. The incidence of IVF embryos with structural chromosome aberrations was 2%, whereas the occurrence of structural chromosome aberrations in ICSI embryos was dependent on the kind of medium and sperm incubation time. When spermatozoa were incubated in TYH medium for 2 h or more, the aberration rates in the resultant ICSI embryos (4%) were not significantly different from that of IVF embryos. However, there was a significant increase in aberration rates in ICSI embryos derived from spermatozoa that were incubated in other culture conditions (6%-28%). In addition, a time-dependent increase in aberration rates was found in ICSI embryos when H-TYH, H-mCZB, and PB1 were used for sperm incubation. There was no significant difference in incidence of aneuploidy between IVF and ICSI embryos. The chromosome analysis results of one-cell embryos were reflected by the performance of postimplantation embryo development. The causal mechanism of chromosome damage in ICSI embryos was discussed in relation to the plasma membrane cholesterol, the acrosome, and in vitro aging of spermatozoa.     

Human sperm chromosomes in zona-free hamster eggs: Pretreatment of spermatozoa with calcium ionophore facilitates later examination of chromosomes

Freshly ejaculated human spermatozoa exposed to 10 μM calcium ionophore A23187 for 30 minutes quickly bound to and penetrated into zona-free hamster eggs. When these eggs were cultured for 13 hours in the presence of low concentrations of vinblastin and podophyllotoxin, then fixed and spread with the use of a cold fixation technique, haploid sets of sperm chromosomes were demonstrated. The number of spermatozoa that were attached to the egg surface at insemination influenced the success rate of chromosome demonstration. The best results (an average of about 0.7 chromosome set per egg) were obtained when insemination was controlled so that an average of seven spermatozoa attached to each egg at the end of a 15–30-minute insemination period. This improved method for demonstrating human sperm chromosomes would be useful in the analysis of paternal genetic contributions in reproduction as well as in assessing the effects of environmental genotoxic agents or conditions on spermatozoa.     

An ultrastructural study of epididymal mouse spermatozoa binding to zonae pellucidae in vitro: sequential relationship to the acrosome reaction

Mouse sperm bind to the zona pellucida of the egg prior to penetration of the zona and entry into the perivitelline space. The question then arises: when does the acrosome reaction occur relative to these processes? An ultrastructural study of mouse epididymal sperm bound to the surface of the zona and in the privitelline space was undertaken to clarify this point. Cumulus-free mouse eggs were inseminated in either a complete defined culture medium capable of supporting in vitro fertilization or in Tris/NaCl buffer containing Ca+2. Both media support sperm binding to the zona to the same extent; binding is complete in 15 minutes. Unbound sperm were removed by a step gradient density centrifugation to yield a preparation of eggs with sperm firmly bound. All sperm in the perivitelline space had undergone the acrosome reaction. Sperm bound at the surface of the zonae pellucidae of eggs recovered at ten minutes after insemination all had intact acrosomes. At 40 minutes after insemination, half of the sperm were intact; the other half were in the initial stages of the acrosome reaction. At 90 minutes after insemination, 12% of the sperm had undergone the full acrosome reaction and were starting to penetrate the zona; of the balance, half were in various stages of the acrosome reaction, while half were still intact. These findings support the hypothesis that the sequence of the early reactions leading to fertilization in the mouse is: intact sperm binding to zona; acrosome reaction at the zona surface; penetration of the zona.     

Improved method for artificial insemination in the great apes

Artificial insemination in the great apes has not achieved its potential as a tool in maintenance of the endangered captive population. Three factors can influence the success rate of artificial insemination: sperm preparation, site of insemination, and timing of insemination. We have tried to optimize methods regarding these three steps. A modified method for insemination is described which has resulted in a 21% success rate (six term pregnancies from 29 inseminations) in the chimpanzee and which has successfully initiated a pregnancy in a gorilla.     

Fertilization cones of inseminated sea urchin (Arbacia punctulata) oocytes: Development of an asymmetry in plasma membrane topography

Electron microscopic and cytochemical investigations were carried out on inseminated Arbacia oocytes comparing structural and chemical properties of their microvillous surface and fertilization cones. Early fertilization cones (up to 4 min postinsemination) were relatively small, smooth surface projections of cytoplasm that engulfed the incorporated sperm nucleus. However, in contrast to surrounding microvillous areas of the oocyte surface, enlarged fertilization cones (8 to 15 min postinsemination) had a distinctive crenated appearance that persisted until their regression. When examined by various cytochemical techniques, membrane delimiting fertilization cones had a much lower affinity for agents that stain negatively charged and carbohydrate moieties (cationized ferritin, concanavalin A, ruthenium red, and alcian blue) than did other regions of the oocyte surface. This difference in surface properties of membrane delimiting the site of sperm-egg fusion was not due solely to incorporated sperm plasma membrane and did not occur when inseminated oocytes were incubated with cytochalasin B. Little or no difference in the membrane of the fertilization cone versus microvillous areas was observed when inseminated eggs were freeze-fractured or prepared with agents (filipin and polymixin B) to demonstrate β-hydroxysterols and anionic phospholipids. These observations indicate that membrane delimiting the fertilization cone differs from the remainder of the oocyte surface and suggests that following insemination significant rearrangements of surface molecules take place within the egg plasmalemma that give rise to asymmetries in membrane topography.     

Polyspermic mouse eggs can dispose of supernumerary sperm

Zona-free mouse eggs inseminated with capacitated epididymal sperm in a modified Krebs-Ringer bicarbonate medium exhibited unusual kinetics of sperm incorporation. At a sperm concentration of 10⁵ cells/ml or higher, the mean number of sperm per egg reached a maximum and then decreased with time. This decrease was correlated with the abstriction of sperm in cytoplasmic blebs which formed during or slightly after second polar body abstriction, 1.5–2.5 hr postinsemination. A correlation was apparent between the degree of polyspermy and the total number of sperm lost by this mechanism. Of 82 dispermic eggs studied, 36 underwent sperm loss by blebbing, a process that restored the monospermic condition. The sequential steps in the abstriction process are depicted in micrographs of whole mounts of fixed eggs. A sperm head or male pronucleus could be seen in isolated blebs. The prevention of bleb formation by exposure of penetrated eggs to cytochalasin B largely eliminated any difference in sperm number when the mean number of sperm per egg was compared at 2, 4, and 6 hr postinsemination. Sperm abstriction may be a novel sperm exclusion mechanism employed by mammalian eggs. Evidence is also presented that an unknown mechanism of sperm exclusion is operative in mouse eggs, since sperm loss by abstriction did not account for all sperm loss.     

Penetration in vitro of bovine oocytes during maturation by frozen-thawed spermatozoa

Bovine immature oocytes cultured for various times in TC-199 medium were inseminated with frozen-thawed spermatozoa in Medium BO with caffeine (5 mM) and heparin (10 micrograms/ml). Very high penetration rates (95-100%) were obtained in all oocytes which had been cultured for 0-20 h. When oocytes cultured for 0 and 4 h were inseminated, 100% of them were penetrated and had a decondensing sperm head and most of the oocytes remained at the stage of condensed germinal vesicle (GV) to telophase-I 20-22 h after insemination. The formation of male and female pronuclei was first observed in oocytes inseminated 8 h after culture. The proportions of polyspermy and average number of spermatozoa in penetrated oocytes gradually decreased as oocyte maturation proceeded. Penetration of at least one spermatozoon with a decondensing head into oocytes at the GV stage (without culture) was almost completed up to 8 h after insemination and at that time most of the penetrated oocytes were still at the stage of GV or condensed GV. These results indicate that maturation of bovine oocytes is not required for sperm penetration into the vitellus or for sperm nuclear decondensation under the in-vitro conditions used.     

Incubation of mouse sperm with lactate delays capacitation and hyperactivation and lowers fertilization levels in vitro

Mouse sperm were incubated in medium with or without 24 mM lactate and assessed for (1) motility characteristics including hyperactivation—a computer-assisted motion analysis system was used; (2) capacitation—a chlortetracycline fluorescent dye binding assay was used; and (3) ability to penetrate oocytes. Lactate affected all aspects of motility and delayed the rates of both hyperactivation and capacitation. When a concentration of 8 × 10³ sperm/ml was used for insemination in vitro, sperm preincubated 60–90 minutes in medium with lactate prior to insemination in lactate-free medium fertilized fewer oocytes than did sperm preincubated in lactate-free medium. Use of a calcium-sensitive electrode demonstrated that lactate chelated appreciable amounts of calcium in the medium. Capacitation was assayed in sperm incubated 60 minutes in medium with various concentrations of lactate or CaCl₂. When medium containing lactate was compared to medium without lactate but having a similar level of free calcium, the level of capacitation of sperm incubated with lactate was less than half that of sperm incubated without lactate. These results demonstrate that including 24 mM lactate in the medium can have detrimental effects on mouse sperm hyperactivation and capacitation. The detrimental effects on capacitation are partly but not completely due to the chelation of calcium by lactate.     

Cortical granule exocytosis in sea urchin eggs is inhibited by drugs that alter intracellular calcium stores

In sea urchin eggs fertilization is accompanied by cortical granule exocytosis, a secretory event thought to be initiated by release of intracellularly sequestered calcium. We have examined the effect of two drugs on this process: chlortetracycline (CTC), a known chelator of intracellular calcium, and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), an antagonist of intracellular calcium release in both skeletal and smooth muscle. Preincubation of eggs for 10 min with either CTC or TMB-8 blocked sperm entry, inhibited the burst of 45Ca2+ efflux normally seen postinsemination, and prevented fertilization envelope elevation. Half-maximal inhibition occurred with 200 microM CTC and 60 microM TMB-8. Electron microscopy confirmed that cortical granule exocytosis had been blocked, although inhibition was not due to a direct effect on exocytosis. CTC and TMB-8 had no effect on Ca2+-stimulated granule fusion in isolated egg cortices. Rather, these drugs block the early events in egg activation: sperm incorporation and triggering of exocytosis. These two effects appear to be independent since addition of either drug just before insemination permits sperm entry but inhibits calcium release and cortical granule exocytosis.     

金鱼精子入卵过程的扫描电镜观察

本文采用扫描电镜观察了金鱼(Carassius auratus)卵壳膜(chorion)表面结构和精子入卵过程。在壳膜的卵膜孔(micropyle)区有5—10条沟和嵴。位于精孔管下面,卵的质膜为一束较长的微绒毛组成的精子穿入部(sperm entry site)。授精5s,精子头的顶部已附着于精子穿入部,随即两者的质膜发生融合,而围于精子头部四周的微绒毛迅速伸长形成一受精锥,它不断将精子头部包裹。授精110s,精子的头部和颈部已完全进入卵内,受精锥本身也渐趋消失,但精子尾部仍平躺于卵的表面。皮层小泡是在授精30s后才开始破裂并释放其内含物,导致卵子表面呈蜂窝状,并在无膜内表面附着了大量球状物。     

Maturation and sperm penetration of canine ovarian oocytes in vitro.

Canine ovarian oocytes were cultured in a medium consisting of TC medium 199, fetal calf serum and antibiotics. Ninety-nine percent of the apparently healthy oocytes were in the germinal vesicle (dictyate) stage when recovered from the ovaries; 25% of them reached metaphase I or II by 72 hours of culture. Washed ejaculated spermatozoa were added to BWW medium containing oocytes which had either been removed directly from the follicles or which had been cultured for 24--72 hours. The earliest acrosome reaction and zona penetration by spermatozoa were seen at seven hours after insemination. Seventy-four percent of the oocytes examined between 11 and 24 hours after insemination showed evidence of zona penetration by spermatozoa. Neither the condition of the oocyte vitellus nor the stage of nuclear maturation influenced the incidence of zona penetration. Decondensing sperm nuclei were found in the vitellus of 27% of the oocytes which had not been cultured and in the vitellus of 20% of those which had been cultured for 24--72 hours and were in various stages of maturation. These results indicate that (1) canine ovarian oocytes can be matured in vitro, (2) the spermatozoa require capacitation which takes approximately seven hours in vitro and (3) maturation of the oocytes is not required for sperm passage through the zona pellucida or entry into the vitellus nor for sperm nuclear decondensation.     

Integration of sperm and egg plasma membrane components at fertilization

Studies examining the integration of the sperm and egg plasma membranes, subsequent to gamete fusion in the surf clam, Spisula solidissima, were carried out employing the concanavalin A-horseradish peroxidase-diaminobenzidine procedure (Con A-HRP-DAB). When unfertilized Spisula eggs were incubated in Con A, either prior to or after aldehyde fixation and reacted with HRP-DAB, enzymatic precipitate was found associated with the vitelline layer and plasmalemma. The plasma membranes of sperm treated in a similar manner failed to stain. The plasma membranes of fertilized eggs reacted with Con A-HRP-DAB and examined by 1 min postinsemination were associated uniformly with enzymatic precipitate except at sites of sperm incorporation. These portions of unstained plasma membrane were derived from the spermatozoon and delimited the contents of the fertilization cone. From 2 to 4 min postinsemination, HRP-DAB reaction product became associated with the plasma membrane delimiting the fertilization cone. By 4 min postinsemination no difference in staining of the plasma membranes derived from the egg or the sperm (plasmalemma delimiting the fertilization cone) was detected. Evidence is presented suggesting that the acquisition of HRP-DAB reaction product by the former sperm plasmalemma is due to the movement of Con A binding sites from the egg plasma membrane.     

Male chromosomes of sea urchin hybrid andromerogones created with cryopreserved sperm

We developed a method for preparing male chromosomes from sea urchin hybrid andromerogones created with cryopreserved sperm. We obtained hybrid andromerogones by heterospermic insemination of Hemicentrotus pulcherrimus non-nucleate egg fragments produced by centrifuging unfertilized eggs in a stepwise saccharose density gradient. The hybrid andromerogones showed cleavage rates of 1%-93%, cleaved successively into two- and four- blastomeres and developed to early blastulae. The morulae or early blastulae were treated with colchicine (0.1-1.0 mg/ml), dissociated into single blastomeres by pippeting, swollen with 7%-10% sodium citrate for 10 min and fixed with methanol:acetic acid (3:1). The fixed cells were dropped on slides and air-dried. The andromerogones for 5 sperm species showed a half of their respective diploid chromosome numbers without chromosome elimination. This method is applicable for analysis of the haploid male chromosome complement in sea urchin species for which only sperm can be obtained.     

Sperm-egg interaction in the painted frog (Discoglossus pictus): An ultrastructural study

The ultrastructure of sperm changes and penetration in the egg was studied in the anuran Discoglossus pictus, whose sperm have an acrosome cap with a typical tip, the apical rod. The first stage of the sperm apical rod and acrosome reaction (AR) consists in vesiculation between the plasma membrane and the outer acrosome membrane. The two components of the acrosome cap are released in sequence. The innermost component (component B) is dispersed first. The next acrosome change is the dispersal of the outermost acrosome content (component A). At 30 sec postinsemination, when the loss of component B is first observed, holes are seen in the innermost jelly coat (J1), surrounding the penetrating sperm. Therefore, this acrosome constituent might be related to penetration through the innermost egg investments. At 1 min postinsemination, during sperm penetration into the egg, a halo of finely granular material is observed around the inner acrosome membrane of the spermatozoon, suggesting a role for component A at this stage of penetration. Gamete-binding and fusion take place between D1 (the egg-specific site for sperm interaction) and the perpendicularly oriented sperm. Spermatozoa visualized at their initial interaction (15 sec postinsemination) with the oolemma are undergoing vesiculation. The first interaction is likely to occur between the D1 glycocalyx and the plasma membrane of the hybrid vesicles surrounding the apical rod. As fusion is observed between the internal acrosome membrane and the oolemma, it can be postulated that gametic interaction might be followed by fusion of the latter with the apical rod internal membrane that extends posteriorly into the inner acrosome membrane. Insemination of the outermost jelly layer (J3) dissected out of the egg, and observations of the ultrastructural changes of spermatozoa in this coat, indicate that J3 rather than the vitelline coat (VC) induces the AR. Interestingly, at the late postinsemination stage, VC fibrils are seen crosslinking the inner acrosome membrane. The role of this binding is here discussed. Mol. Reprod. Dev. 47:323–333, 1997. © 1997 Wiley-Liss, Inc.     

鲫鲤杂种一代(F1)自交二代(F2)的受精细胞学研究

荷包红鲫(♀)×湘江野鲤(♂)杂交产生的杂种一代(F     

Effects of fetal calf serum in culture medium on development of bovine oocytes matured and fertilized in vitro

Bovine oocytes matured and fertilized in vitro were cultured in a chemically defined bovine embryo culture medium (BECM) or polyvinylalcohol (PVA)-free BECM supplemented with 10% heat-treated or unheated fetal calf serum (FCS). When PVA in BECM was replaced with FCS 8 h postinsemination, the proportion of >/=-cell-stage embryos 48 h postinsemination was significantly lower in heat-treated FCS (74%) than in PVA (91%) medium, but the value (84%) in unheated FCS was not different from the values in PVA and heat-treated FCS. However, the addition of either heat-treated FCS or unheated FCS to PVA-free BECM did not depress or increase the proportions (63 to 70%) of >/=-cell-stage embryos 96 h postinsemination and stimulated blastocyst formation 144 (21 to 28%) and 192 (47 to 54%) h postinsemination. Blastocyst formation was also stimulated when heattreated FCS or unheated FCS was added to PVA-free BECM 120 h postinsemination. The mean cell number of blastocysts developed in PVA-free BECM with heat-treated FCS (150 cells) or unheated FCS (152 cells) significantly increased compared with that (119 cells) developed without FCS. These results indicate that, although BECM can support bovine embryo development, a high proportion (about 50%) of morphologically normal blastocysts can be produced from oocytes matured and fertilized in vitro when they are cultured in PVA-free BECM containing FCS.     

Effects of radiographic contrast media on domestic cat epididymidal sperm

Laparoscopic artificial insemination has an important role in felid conservation but it is costly and includes surgical risk. Therefore, radiographic contrast medium combined with non-surgical transcervical AI to verify intrauterine gamete placement could be a viable alternative. Gamete-rescued fresh and frozen-thawed sperm were extended with one of two commercial contrast media (nonionic and ionic), with osmolarity adjusted to 320-330 mOsm, or feline optimized culture medium (control). Percent motility, forward progression status, and acrosomal integrity were recorded every 30 min for 4 h. Sperm penetration abilities were assessed by coincubating treated sperm with conspecific in vitro matured oocytes for 18 to 20 h, and presumptive zygotes and embryos were fixed and stained to determine sperm penetration and fertilization rate. There was reduced motility and acrosomal integrity in frozen-thawed versus fresh sperm (P < 0.05). Neither radiographic contrast medium induced adverse effects on fresh sperm motility relative to control medium (P > 0.05), but motility of frozen-thawed sperm decreased when treated with nonionic radiographic contrast medium compared to control medium (P < 0.05). There were no differences in acrosomal integrity between radiographic contrast and control media in fresh (P > 0.05) or frozen sperm (P > 0.05). Neither radiographic contrast media decreased the numbers of morphologically normal sperm (P > 0.05) or reduced the ability of domestic cat sperm to penetrate (P > 0.05) or fertilize (P > 0.05) conspecific oocytes. Ionic radiographic contrast medium can be added to fresh or frozen-thawed domestic cat sperm with no adverse effect on motility, morphology, acrosomal integrity or oocyte penetration rates, and thus may be used to facilitate further development of transcervical AI procedures.     

Evidence for the function of hyperactivated motility in sperm

After insemination, mammalian sperm undergo a striking change in flagellar beat pattern, termed hyperactivation. In low-viscosity culture medium, nonhyperactivated sperm flagella generate relatively symmetrical, low-amplitude waves, while hyperactivated sperm flagella generate an asymetrical beating pattern that results in nonprogressive movement. Since sperm encounter highly viscous and viscoelastic fluids in the female reproductive tract, the progress of hyperactivated sperm was compared with that of nonhyperactivated and transitional sperm in media of increasing viscosity. Hamster sperm obtained from the caudal epididymis were incubated in a medium that promotes capacitation. After 0, 3, and 4 h of incubation, the majority of the sperm exhibited, respectively, activated, transitional, and hyperactivated motility. At each of these time points, aliquots of sperm were removed from incubation and added to solutions of 0, 5%, 10%, 20%, and 30% Ficoll in medium. Samples containing mostly hyperactivated sperm (4 h) maintained higher swimming and flagellar velocities and were able to generate greater forces in response to increased viscous loading than activated sperm (0 h). Transitional sperm (3 h) showed an intermediate response. The paths of hyperactivated sperm through solutions of 20% and 30% Ficoll were considerably straighter than those made through medium alone. This is the first demonstration that hyperactivation can confer a mechanical advantage upon sperm in the oviduct where they may encounter viscous oviductal fluid and a viscoelastic cumulus matrix.