Study of bovine sperm motility in shear-thinning viscoelastic fluids

To elucidate the process whereby sperm arrive at an egg in the female reproductive organs, it is essential to investigate how rheological properties of the fluid around mammalian spermatozoa affect their motility. We examined the motility and flagellar waveform of bovine sperm swimming in a fluid with similar rheological properties as mammalian cervical mucus. The results indicated that the surrounding rheological properties largely affected the flagellar waveform of mammalian spermatozoa; in particular, shear-thinning viscoelastic fluid increased the progressive motility of the sperm. To investigate the influence of flagellar waveform on sperm motility in more detail, the waveform was expressed as a function and the progressive thrust of the sperm was calculated based on the empirical resistive force theory. The results of this study showed that the progressive thrust increased with the curvature of the flagellar tip. Moreover, we calculated the thrust efficiency of motile sperm. Results showed that the thrust efficiency in shear-thinning viscoelastic fluids was larger than that in Newtonian fluids, regardless of viscosity. This suggests that motile sperm in cervical mucus move efficiently by means of a motion mechanism that is suited to their surrounding environment.     

Factors regulating mammalian sperm migration through the female reproductive tract and oocyte vestments

Mechanisms of mammalian sperm migration through the female reproductive tract and ovum vestments are described. The perspective is biophysical as well as biochemical and morphological, and the focus is upon the role of sperm motility in these processes. Sperm forward progression is characterized as an interactive process between the the cell and its environment, and the mediation of flagellar bend propagation by the physical properties of its surroundings is described. These properties, together with flagellar beat kinematics, sperm morphology, and surface properties, determine the magnitude of the forces generated by sperm and their consequent rate of progression. Sperm interactions with the cervical mucus, the cumulus oophorus, and the zona pellucida are described. The poorly understood affinity of the sperm surface for the macromolecules of the mucus, cumulus, and zona is stressed, as is the viscoelastic structural mechanical resistance of these biopolymers to sperm motion. The kinematics and consequences of hyperactivated sperm motion are presented, with emphasis on objective characterization of such motion (as a biomarker), along with analysis of the mechanical advantage that such motion may confer on spermatozoa during egg–vestment interaction.     

Assessment of human sperm function after recovery from the female reproductive tract

The physiology and fertile life of human spermatozoa in the female reproductive tract have received little previous attention. A technique was developed for recovering spermatozoa from human cervical mucus at various intervals after artificial insemination. The functions of these cells as measured by penetration of the human zona pellucida and fusion with the zona-free hamster oocytes were examined. Penetration into the zona pellucida was consistently observed when sperm were recovered from 1 to 80 h after insemination. Penetration through the zona into the perivitelline space (PVS) was seen from 1 to 72 h after insemination. Fusion of human sperm with zona-free hamster oocytes was observed from 1 to 48 h after insemination. Motile sperm were recovered 112 and 120 h after insemination with swimming speeds comparable to freshly capacitated spermatozoa. Concentrations of recovered sperm at these longer intervals from insemination were insufficient for sperm-oocyte assays. These studies demonstrate that human spermatozoa aged in vivo may be recovered from cervical mucus for physiologic study, and suggest that the fertile life of human sperm may be 80 h or more.     

Mechanical hypothesis of sperm penetration.

Sperm have generally been supposed to penetrate the zona pellucida surrounding the mammalian egg with the aid of a protease, acrosin. Difficulties associated with this view are discussed and an alternative, mechanical hypothesis introduced. The calculated force exerted by individual sperm is too small to permit the rupture of any but the weakest of secondary chemical bonds. Mechanical progress through the zona must rely on stress relaxation in a viscoelastic medium. The known properties of the zona appear to be consistent with such a mechanism of penetration.     

Ewe breed differences in fertility after cervical AI with frozen-thawed semen and associated differences in sperm penetration and physicochemical properties of cervical mucus

The objective of this study was to examine sperm penetration through cervical mucus and associated physicochemical properties of cervical mucus from Belclare and Suffolk ewes - two breeds with divergent pregnancy rate following cervical AI using frozen-thawed semen. In Experiment 1, sperm penetration through cervical mucus was assessed in 15 Belclare and 15 Suffolk ewes at 30, 48 and 57h post sponge removal. In Experiment 2, rheological properties of mucus from 17 Belclare and 19 Suffolk ewes at 48 and 57h post sponge removal were determined. In Experiment 3, 20 Belclare and 20 Suffolk ewes were used to assess mucus ferning and pH collected at 42, 48, 57 and 65h post sponge removal. In Experiment 1, a higher number of sperm penetrated cervical mucus from Belclare ewes at 48h, reflected by a breed by time interaction (P=0.05). In Experiment 2, mucus from Suffolk ewes tended to have higher elastic and complex moduli than that from Belclare ewes (P=0.06) regardless of time of collection. There was no effect of ewe breed on the viscous modulus. In Experiment 3, there was a significant effect of time post sponge removal on ferning (P<0.01), but there was no effect of breed. There was no effect of time or breed on mucus pH. It is concluded that breed differences in the rheological properties of cervical mucus affect the ability of sperm to swim through cervical mucus and this may explain breed differences in fertility observed after cervical AI using frozen-thawed semen.     

Correlation between the spermatozoal characteristics and sperm penetration distance in polyacrylamide gel and bovine cervical mucus

Correlation between the spermatozoal characteristics and the sperm penetration distance in polyacrylamide gel was assessed, utilizing frozen thawed semen samples obtained from 6 bulls, and it was compared with the correlation between sperm penetration in bovine cervical mucus and spermatozoal characteristics. In vitro sperm penetration tests were performed with mucus and gel. The sperm penetration in gel and mucus was significantly and positively correlated with post-thaw motility (r=0.81; r=0.89:P<0.01) and acrosome integrity (r=0.88; r=0.94:P<0.01). A significant negative correlation with abnormal spermatozoa (r=-0.84;r=0.83:P<0.01) was observed. Both sperm concentration and post-thaw live spermatozoa were not significantly correlated. A significant multiple regression between sperm penetration and the spermatozoal characteristics both in gel (R2=0.87; F=40.27; P<0.01) and mucus (R2=0.91; F=60.48; P<0.01) was observed. The major spermatozoal characteristics determining the capacity of spermatozoa to penetrate gel were post-thaw motility, percentage of abnormal spermatozoa and acrosome integrity. The acrosome integrity has a more significant contribution. The correlation established with sperm penetration in gel was very similar to that of sperm penetration in mucus. The utility of gel as a mucus substitute in in vitro sperm penetration tests was discussed.     

Studies on bonnet monkey cervical mucus the effect of proteases on mucus glycoproteins of macaca radiata

The influence of proteinases on monkey cervical glycoproteins was investigated to assess their effect on cervical mucus and, thereby, on sperm penetration. The major component of periovulatory cervical mucus, a high molecular weight glycoprotein, was treated with Pronase, trypsin, chymotrypsin, papain, and bovine seminal peptidase, and the enzyme-resistant glycoprotein was purified by gel filtration on Sepharose 2B. A macromolecular component in high yield was recovered containing carbohydrate and protein moieties. Asialoglycoprotein, on treatment with Pronase, trypsin, and bovine seminal peptidase released more than one glycoprotein fragment. The carbohydrate and amino acid components of the native and degraded glycoproteins were similar in composition with variations in proportions. The structure of the carbohydrate-rich, pronase-resistant glycoprotein, further purified on Sepharose 2B, was examined. Sequential Smith degradation and methylation of the degraded glycoprotein fragment established a structure that shows some differences to that of the native glycoprotein. The influence of proteinases on cervical-mucus glycoproteins and a possible mechanism of sperm penetration through Pronase-treated glycoproteins is discussed.     

Comparison of ram sperm interaction with bovine cervical mucus

Ram sperm penetration in estrous bovine cervical mucus was evaluated from ejaculates collected during long (16L:8D) and short (8L:16D) photoperiods with varying ambient temperatures. The distance traveled by vanguard sperm was affected by an interaction of the photoperiod and temperature (P<0.001). Sperm migration distance in a capillary tube filled with mucus (22.5 to 23.2 mm) was greater when sperm were collected from rams on short days and when the ambient temperature was between 10 and 31 degrees C than when sperm were collected under either long or short days (15.5 to 17.8 mm), when ambient temperatures were between 1 to 9 degrees C. Incidence of head-to-head agglutination of sperm differed by temperature (P<0.05) and photoperiodic (P<0.09) conditions. The percentage of ejaculates with evidence of sperm agglutination in the mucus was higher in long (62.5%) vs short (45.2) days, and it was greater in sperm collected in warm (61%) vs cold (44%) days. Physical interaction of cervical mucus with spermatozoa was examined. The binding of an iodinated protein from lyophilized mucus to a detergent soluble extract of washed or unwashed sperm was observed. These data show that both photoperiod and temperature affect the interaction of ram sperm with bovine cervical mucus.     

Sperm penetration through cumulus mass and zona pellucida

Mammalian fertilization requires sperm to penetrate the cumulus mass and egg zona pellucida prior to fusion with the egg. Although sperm penetration through these physical barriers is essential, the molecular mechanism has not yet been completely elucidated. In addition to sperm motility, hyaluronan-hydrolyzing and proteolytic enzymes of sperm have been suggested to participate in the penetration events. Here we focus on the functional roles of hyaluronidase and protease in sperm passage through the cumulus mass and zona pellucida.     

Interaction of human spermatozoa with the human zona pellucida and zona-free hamster oocyte following capacitation by exposure to human cervical mucus

The functional capacity for sperm interaction with the human zona pellucida and zona-free hamster oocyte was tested after human spermatozoa were capacitated by passage through a column of human cervical mucus. The results were compared with those obtained when spermatozoa from an aliquot of the same semen sample were capacitated by the standard laboratory methods involving sequential washing by dilution and centrifugation of the semen. Washed-capacitated sperm suspensions were more successful than mucus-capacitated sperm in attaching to the zona-free hamster oocyte and in fusing with its oolemma. However, the ability of mucus-capacitated sperm to penetrate the human zona pellucida was equal to washed capacitated sperm. These experiments demonstrate an approach that may be useful in comparative studies of human sperm capacitation in vivo and in vitro.     

The evolution of hamster sperm motility during capacitation and interaction with the ovum vestments in vitro

Movement characteristics of golden hamster spermatozoa were studied upon collection from the cauda epididymis, during an incubation which capacitates the spermatozoa in vitro, during penetration of the cumulus, and during attachment to and penetration of the zona pellucida. High-speed videomicrography was employed to quantitate flagellar beat frequency and shape. The status of the acrosome was also assessed. During capacitation, hamster spermatozoa become increasingly invigorated before the onset of hyperactivated motility. Within the cumulus, beat frequency and curvature are reduced, apparently in response to the physical resistive properties of the matrix material. These properties appear to vary within the cumulus. Initial attachment to the zona precedes completion of the acrosome reaction, is non-rigid, and is accompanied by increased beat frequency and curvature. Subsequently, the onset of rigid binding to the zona, completion of the acrosome reaction, and increased flagellar beat frequency are very closely associated in time. The latter produces an increase in thrust against the zona. Preliminary results indicate that ensuing zona penetration requires not more than five minutes, is at oblique angles, and is associated with a continuation of vigorous flagellar beating.     

Effects of surrounding fluid on motility of hyperactivated bovine sperm

Mammalian spermatozoa in organisms with internal fertilization are required to swim in the cervical and oviductal mucus, whose rheological properties differ substantially from those of water. Moreover, on the way to the oviduct, a change in sperm motility called hyperactivation may occur. In the present study, we focused on the motion characteristics of hyperactivated bovine sperm and investigated the effect of the surrounding fluid on motility. We prepared two kinds of polyacrylamide with high-viscosity non-Newtonian fluid properties, similar to the actual cervical and oviductal mucus. Using semen from Japanese cattle, we evaluated curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP). Additionally, we estimated linearity (LIN), straightness (STR), and wobble (WOB) as sperm motility parameters for several surrounding fluids. We successfully induced hyperactivation of bovine sperm in high-viscosity non-Newtonian fluid. Hyperactivation resulted in an increase in VCL and a decrease in VSL. In the high-viscosity non-Newtonian fluid, the hyperactivated sperm moved in a zig-zag pattern with regularity, different from the movement observed in a diluted solution. The increase in WOB in the non-Newtonian fluid suggests that hyperactivated sperm efficiently progress along the groove that exists on the oviductal mucus wall. These results improve our understanding of the motility of bovine sperm when they undergo hyperactivation in the actual cervical and oviductal mucus.     

In vitro penetration of hamster oocyte-cumulus complexes using physiological numbers of sperm

We have compared the ability of uncapacitated, capacitated acrosome intact, and acrosome-reacted hamster sperm to penetrate the cumulus and corona radiata of fresh hamster oocyte-cumulus complexes (OCC) in vitro. This was done using physiological numbers (1-20) of sperm so that cumulus and corona radiata cells did not disperse during challenge. Uncapacitated sperm did not penetrate to the zona pellucida surface; most (74%) uncapacitated sperm bound to cumulus cells at the periphery of the OCC. Capacitated acrosome-intact sperm penetrated to the zona pellucida surface; a significant percentage of these sperm arrived at the zona pellucida without showing evidence of initiating an acrosome reaction. Most capacitated acrosome-reacted sperm did not enter the extracellular matrix between cumulus and corona radiata cells; those which did penetrated to the zona surface with difficulty, if at all. These results suggest that the changes which occur in the sperm surface during capacitation are more important than the acrosome reaction in enabling hamster sperm to penetrate the cumulus and corona radiata. The effects of gold sodium thiomalate (GST) and polyphloretin phosphate (PPP) (inhibitors of hyaluronidase) on penetration of the OCC by capacitated sperm were also examined. Both synthetic inhibitors blocked sperm penetration to the zona pellucida, but the effective concentrations of inhibitors were far in excess of what was needed to block hyaluronidase activity. Reasons for concluding that the action of these inhibitors is nonspecific are discussed. These data show that hamster sperm with intact acrosomes can penetrate the cumulus and corona radiata cell layers of fresh OCC in vitro and support the hypothesis that the acrosome reaction occurs on the zona pellucida surface.     

Intra- and interspecies comparison of sperm migration through polyacrylamide gel as an index of spermatozoal viability

Sperm migration distance through a polyacrylamide gel mimicking cervical mucus was compared among seven species: dog, cat, goat, ram, clouded leopard, dorcas gazelle, and Eld's deer. Freshly collected spermatozoa were evaluated microscopically and then assayed for migration distance in gel-filled capillary tubes in raw or diluted concentrations or after longevity declines in sperm motility and progressive status. Sperm count per milliliter of ejaculate and sperm percent motility ratings varied (P < 0.05) among selected species. Undiluted ejaculated spermatozoa from all species, except the clouded leopard, penetrated the polyacrylamide gel. Migration distance (mm) was different (P < 0.05) among species and not clearly correlated to concentration or motility factors. Although sperm concentrations in the dog and Eld's deer were similar, average migration distance of deer sperm was more than fourfold greater (P < 0.01) than that of dog sperm. The sperm motility rating for the gazelle was greater (P < 0.05) than that for the cat; however, penetration distance in the cat was more than twice as great (P < 0.05) as that in the gazelle. Species also varied in migration response after altering sperm concentration: Halving the sperm count of the dog, gazelle, and Eld's deer had no effect, but the same procedure decreased (P < 0.05) penetration distance in the goat and ram. No migration was observed in any species at a sperm concentration of 25 × 10⁶ cells or less. Within species a decline in sperm percent motility-progressive status ratings was correlated (P < 0.05) to a subsequent decrease in penetration distance. These results provide a comparative assessment of sperm cell migration through a synthetic cervical mucus and suggest that this test may be a useful adjunct in evaluating reproductive potential. However, the assay is specific in interpretive merit, as sperm penetration distance within a single batch of gel is markedly variant among species and in some species dependent on sperm concentration. This interspecies specificity in sperm migration through a homogeneous gel suggests intrinsic species variance in sperm cell-cervical mucus interaction, suggesting that this assay could be valuable in future studies of the mechanism of sperm transport.     

Sperm binding to the zona pellucida is not sufficient to induce acrosome exocytosis

At fertilization, spermatozoa bind to the zona pellucida (ZP1, ZP2, ZP3) surrounding ovulated mouse eggs, undergo acrosome exocytosis and penetrate the zona matrix before gamete fusion. Following fertilization, ZP2 is proteolytically cleaved and sperm no longer bind to embryos. We assessed Acr3-EGFP sperm binding to wild-type and huZP2 rescue eggs in which human ZP2 replaces mouse ZP2 but remains uncleaved after fertilization. The observed de novo binding of Acr3-EGFP sperm to embryos derived from huZP2 rescue mice supports a ;zona scaffold' model of sperm-egg recognition in which intact ZP2 dictates a three-dimensional structure supportive of sperm binding, independent of fertilization and cortical granule exocytosis. Surprisingly, the acrosomes of the bound sperm remain intact for at least 24 hours in the presence of uncleaved human ZP2 regardless of whether sperm are added before or after fertilization. The persistence of intact acrosomes indicates that sperm binding to the zona pellucida is not sufficient to induce acrosome exocytosis. A filter penetration assay suggests an alternative mechanism in which penetration into the zona matrix initiates a mechanosensory signal transduction necessary to trigger the acrosome reaction.     

Movement of cynomolgus and rhesus monkey spermatozoa collected from the lower female reproductive tract

Postcoital (pc) cervical mucus was collected in 73 menstrual cycles of cynomolgus monkeys and in 43 cycles of rhesus monkeys at 2,6,10,30 hr pc. Videomicrography was used to analyze sperm numbers and movement in the mucus. Both cynomolgus and rhesus monkeys had comparable populations of motile sperm in the mucus at 2 hr pc. However, by 6 hr pc, cervical mucus from cynomolgus monkeys contained twice as many total sperm and motile sperm as mucus from rhesus monkeys (P <.05). Mean swimming speeds of the free-swimming cervical sperm were similar for the two species at this time. No motile sperm were recovered in mucus from rhesus monkeys at 30 hr pc. In cynomolgus monkeys, however, 14 of the 26 animals examined at 30 hr pc had motile sperm in their mucus. These sperm exhibited lower percent molility, percent free-swimming sperm, and swimming speed than those sperm observed at 6 hr pc. Uterine sperm were collected by transcervical or transuterine aspiration from cynomolgus monkeys. In the transcervical technique, sperm were successfully obtained in four of nine animals examined at 6 hr and in four of five animals at 30 hr pc. The percentage of motile sperm in the uterine fluid was high, 82% ± 4%, and the swimming speeds (86 ± 2μm/sec) were higher than those observed in cervical mucus. Approximately 5–10% of the uterine sperm exhibited swimming motions similar to the hyperactivated motility seen in most mammals. These findings indicate that the sperm cervical mucus interaction in vivo in cynomolgus monkeys has more similarities to the human situation than does the interaction in rhesus monkeys.     

Mechanics of sperm-egg interaction at the zona pellucida.

Mammalian sperm traverse several layers of egg vestments before fertilization can occur. The innermost vestment, the zona pellucida, is a glycoprotein shell, which captures and tethers the sperm before they penetrate it. We report here direct measurements of the force required to tether a motile human sperm as well as independent calculations of this force using flagellar beat parameters observed for sperm of several species on their homologous zonae. We have compared these sperm-generated forces with the calculated tensile strength of sperm-zona bonds, and found that a motile sperm can be tethered, at least temporarily, by a single bond. Therefore, sperm can be captured by the first bond formed and tethered permanently by a few. The sperm cannot subsequently penetrate the zona unless the bonds are first eliminated. However, premature elimination would simply allow the sperm to escape. Therefore, not only must the bonds be eliminated, but the timing of this must be regulated so that the sperm is already oriented toward the egg and beginning to penetrate as the bonds are broken.     

Effect of medium milieu on sperm penetration and pronuclear formation of bovine oocytes matured in vitro

The purpose of the present study was to investigate the optimal concentration of osmolarity, calcium and bicarbonate for sperm penetration and formation of pronuclei (PN), and to investigate the time required for capacitation, penetration across the zona pellucida and formation of PN in bovine cumulus-free oocytes matured in vitro. Bovine follicular oocytes collected at slaughter were matured and fertilized in vitro. Bovine sperm penetrated the zona pellucida in medium containing 240 to 440 mOsm, whereas PN formation was observed in a narrow range of osmolarities, from 280 to 360 mOsm. Maximal penetration by spermatozoa and PN formation was obtained in the medium with 2.5 mM calcium. High rates of spermatozoa penetration were observed in the medium with 37 to 49 mM NaHCO3. However, PN were formed regardless of the concentration of NaHCO3. The times required for sperm capacitation and penetration through the zona pellucida were 260 and 50 min, respectively. The first development of PN was recorded at 120 min after sperm penetration. Therefore, our study suggests that fertilization ability of spermatozoa in vitro appears to be more stable in high concentrations of NaCI. Oocytes are more sensitive to osmotic stress than spermatozoa. Calcium is required for both sperm penetration and PN formation in cumulus-free oocytes, but bicarbonate may be needed mainly for the penetration of spermatozoa.     

The physiology of sperm recovered from the human cervix: acrosomal status and response to inducers of the acrosome reaction

Cervical mucus was collected from 35 women after artificial insemination. Mucus collections were performed at 1 h, 1 day, 2 days, or 3 days following insemination. Sperm viability was greater than 80% at all recovery times as assessed by exclusion of the supravital dye Hoechst 33258. Virtually 100% of the viable sperm were acrosome-intact at all times as assessed with a fluorescein isothiocyanate-conjugated pea lectin. Sperm were recovered from the mucus after migration into the Biggers, Whittin, and Whittingham medium in vitro. Sperm did not undergo the acrosome reaction in response to human follicular fluid immediately after migration from the mucus but did respond to this agonist after 6 h of incubation in vitro. Sperm recovered at all times after insemination had the same pattern of response to follicular fluid. Sperm that penetrated a column of cervical mucus in vitro also responded to follicular fluid with an increase in acrosome reactions after migration from the mucus and incubation for 6 h in vitro. Unlike the sperm that migrated from cervical mucus, sperm that were separated from semen by Percoll density centrifugation did not undergo the acrosome reaction when challenged with follicular fluid after 6 h but did respond after 24 h incubation. Sperm that migrated from cervical mucus had a similar increase in acrosome reactions after 6 h incubation, regardless of whether the acrosome reaction agonist was follicular fluid or disaggregated human zona pellucida.(ABSTRACT TRUNCATED AT 250 WORDS)