精子的运动特性

精子的运动特性与生育力有密切关系。本文从超微结构水平和分子生物学基础出发,阐述精子运动机制的微管滑动学说;介绍了精子在液体介质中的运动形式、检测精子活动力的主要技术、影响精子活动力的因素、从异常精液中选择正常精子的技术,以及精子活动力在雄性和雌性生殖道中的演变过程。     

Immunodetection of acrosin during the acrosome reaction of hamster, guinea-pig and human spermatozoa.

Mammalian sperm acrosomes contain a trypsin-like protease called acrosin which causes limited and specific hydrolysis of the extracellular matrix of the mammalian egg, the zona pellucida. Acrosin was localized on hamster, guinea-pig and human sperm using monoclonal and polyclonal antibodies to human acrosin labelled with colloidal gold. This was visualized directly with transmission electron microscopy, and with light and scanning microscopy after silver enhancement of the colloidal gold probe. Four distinct labelling patterns were found during capacitation and the acrosome reaction in hamster and guinea-pig spermatozoa, and three patterns were found in human spermatozoa. In the hamster, acrosin was not detected on the inner acrosomal surface after the completion of the acrosome reaction, thus correlating with the observation that hamster spermatozoa lose the ability to penetrate the zona after the acrosome reaction. With guinea-pig and human spermatozoa, acrosin was still detected after the completion of the acrosome reaction, thus correlating with the observation that acrosome reacted guinea-pig spermatozoa bind to and penetrate the zona pellucida.     

Relationship between heparin binding characteristics and ability of human spermatozoa to penetrate hamster ova

Heparin binding site affinity and density on human spermatozoa were compared between fertile and infertile men with normal or abnormal results in the zona-free hamster ova-sperm penetration assay (SPA). A portion of fresh semen from fertile donors and potentially infertile men was processed through the SPA while the remainder of the ejaculate was used to quantitate heparin binding on spermatozoa. Saturation binding assays with [3H]heparin (15-375 nM) were analysed for 3 groups of men: (1) infertile patients with abnormal SPA results, (2) infertile patients with normal SPA results and (3) fertile donors. The heparin binding site density was significantly higher in men who possessed normal SPA results (infertile men and fertile donors) than in infertile men with abnormal scores in the SPA. There was no difference in heparin binding affinity between the three groups. These findings suggest that the heparin binding-site density may be related to the ability of human spermatozoa to undergo successfully the acrosome reaction.     

The movement characteristics of rabbit spermatozoa before and after activation

Spermatozoa were flushed with mineral oil from the lower isthmus of the rabbit oviduct at four hours postcoitus (pc) and 11 hours pc. Videotapes were made of sperm behavior in the native isthmic fluid and after dilution of the fluid with culture medium. The tapes showed that, initially, spermatozoa in the native isthmic fluid were virtually immotile, but immediately resumed movement on contact with the culture medium. Isthmic sperm motility then evolved over a five- to 10-minute interval into the characteristic biphasic pattern of activated movement. Cine films of isthmic spermatozoa taken with a high-speed camera were analyzed to determine flagellar beat frequency, maximum flagellar curvature, and swimming velocity. Progressiveness ratios and hydrodynamic power outputs were then calculated for individual spermatozoa. Two phases of activated sperm movement, a whiplash phase and a progressive phase, were identified and characterized. The power output of activated spermatozoa increased twentyfold in comparison with the preactivated state. The power output of activated spermatozoa did not differ between the two phases of activated movement.     

Influence of epididymal maturation on the capacity of hamster and rabbit spermatozoa for complement activation

Hamster and rabbit spermatozoa released from the epididymis were tested for the ability to activate complement via the alternative pathway. While hamster spermatozoa were more active, the spermatozoa of both species reduced complement activity in homologous and also human serum previously adsorbed to remove sperm antibodies, and they bound C3 in the presence of EGTA + Mg2+. Hamster spermatozoa from the caput epididymidis were more anticomplementary and bound more C3 than did cauda spermatozoa and, though less marked, a similar difference was evident between caput and cauda spermatozoa from the rabbit epididymis.     

Ability of hamster spermatozoa to digest their own DNA

Mammalian sperm chromatin is bound by protamines into highly condensed toroids with approximately 50 kilobases (kb) of DNA. It is also organized into loop domains of about the same size that are attached at their bases to the proteinaceous nuclear matrix. In this work, we test our model that each sperm DNA-loop domain is condensed into a single protamine toroid. Our model predicts that the protamine toroids are linked by chromatin that is more sensitive to nucleases than the DNA within the toroids. To test this model, we treated hamster sperm nuclei with DNase I and found that the sperm chromatin was digested into fragments with an average size of about 50 kb, by pulse-field gel electrophoresis (PFGE). Surprisingly, we also found that spermatozoa treated with 0.25% Triton X-100 (TX) and 20 mM MgCl2 overnight resulted in the same type of degradation, suggesting that sperm nuclei have a mechanism for digesting their own DNA at the bases of the loop domains. We extracted the nuclei with 2 M NaCl and 10 mM dithiothreitol (DTT) to make nuclear halos. Nuclear matrices prepared from DNase I-treated spermatozoa had no DNA attached, suggesting that DNase I digested the DNA at the bases of the loop domains. TX-treated spermatozoa still had their entire DNA associated with the nuclear matrix, even though the DNA was digested into 50-kb fragments as revealed by PFGE. The data support our donut-loop model for sperm chromatin structure and suggest a functional role for this type of organization in that sperm can digest its own DNA at the sites of attachment to the nuclear matrix.     

The binding of sterol sulfates to hamster spermatozoa.

Spermatozoa obtained from the cauda epididymidis possess twice the ability to take up sterol sulfates in vitro when compared to sermatozoa obtained from the caput. This would suggest that a modification of the membrane composition of the spermatozoa occurs during passage through the epididymis. Free sterols are taken up in a similar pattern. Radioautographic studies reveal that, for sterol sulfates, this uptake occurs selectively in the regions of the head and mid-piece of the spermatozoa whereas the free sterols are distributed evenly throughout the length of the spermatozoa. The binding of sterol sulfates to spermatozoa appears to involve sites that are unsaturable. The possibility exists that sterol sulfates, previously implicated in membrane stabilization, may play a similar role in spermatozoa.     

Desmosteryl sulfate and desmosterol in hamster epididymal spermatozoa

Desmosteryl sulfate composes 95% of the sterol sulfate fraction of hamster epididymal spermatozoa at a level of 10 mg/g dry weight. Desmosterol represents as much as 40% of the free sterol fraction of these cells. Only cholesterol was detected in the free sterol fraction from plasma and liver of the same animals. The present report details the evidence for proof of structure of the two compounds isolated.     

Relationship between the movement characteristics of human spermatozoa and their ability to penetrate cervical mucus and zona-free hamster oocytes

In a group of normospermic donors exhibiting hamster oocyte penetration scores of 0-100%, multiple regression analysis indicated that only 20% of the variation in fertilizing potential could be explained by differences in the movement characteristics of the spermatozoa following incubation in vitro. When the movement characteristics of the spermatozoa in semen were considered this figure was reduced to 6.8% as a result of significant differences in the motility patterns exhibited by the seminal and post-incubation sperm populations. A much closer relationship was observed between the movement characteristics of human spermatozoa in semen and their ability to penetrate cervical mucus. When differences in motile sperm densities were taken into account, 76% of the variation in cervical mucus penetration could be accounted for by the existence of linear correlations with certain aspects of sperm movement (multiple R = 0.874). Of the various attributes of sperm motility measured (linear velocity of progression, frequency of rotation, amplitude of sperm head displacement, % rolling and % yawing), a failure to exhibit an adequate amplitude of lateral sperm head displacement was consistently found to be the most significant factor determining the success of sperm-cervical mucus interaction (R2 = 0.53).     

Thapsigargin stimulates acrosomal exocytosis in hamster spermatozoa

Thapsigargin (TG), a plant-derived sesquiterpene lactone, inhibits several isoforms of both the sarcoplasmic and endoplasmic reticulum Ca²⁺-ATPases. Thus, intracellular Ca²⁺ stores found in the endoplasmic reticulum can be released by this compound. The mammalian sperm acrosome reaction (AR) depends on influx of extracellular Ca²⁺. However, few reports have presented evidence for the involvement of putative Ca²⁺ stores and intracellular Ca²⁺ mobilization in the AR. Thus, we designed experiments to evaluate the effect of TG on the hamster sperm AR. Thapsigargin stimulated—in a dose-dependent manner—the AR of spermatozoa previously capacitated for at least 3 hr, not affecting sperm motility. A maximal stimulatory effect was apparent 3 min after addition of TG to spermatozoa previously capacitated for 4 hr and was dependent on external Ca²⁺ since ethyleneglycol-bis-(b-amino-ethyl ether) N,N′-tetra-acetic acid added 1 min before TG completely inhibited AR stimulation. The Ca²⁺ channel blockers diltiazem and nifedipine also abolished the TG-stimulatory effect when added to capacitated spermatozoa 10 min before the inhibitor. In addition, the trypsin inhibitors p-nitrophenyl-p′-guanidine-benzoate hydrochloride and benzamidine added to the sperm suspensions 10 min before TG inhibited by 70–80% the TG-induced AR. These results indicate that putative Ca²⁺ stores release may be involved in stimulation of extracellular Ca²⁺ influx required for the occurrence of the AR. In addition, a sperm trypsin-like protease may be part of the mechanism by which TG induces the hamster sperm AR. Mol. Reprod. Dev. 51:84–91, 1998. © 1998 Wiley-Liss, Inc.     

Zona-induced acrosome reaction of hamster spermatozoa

It is well established that the zonae pellucidae of mature unfertilized eggs have the ability to induce the acrosome reaction of capacitated spermatozoa. To determine if this capacity of the zona is species-specific, hamster spermatozoa were allowed to attach to the zonae of homologous and heterologous eggs and examined for the acrosome reaction. The zonae of eggs from six different species were tested and the zona of hamster egg was found to have the strongest capacity to induce the acrosome reaction of hamster spermatozoa, followed by human and rat zonae. The zonae and mouse, guinea pig, and domestic fowl eggs were incapable of inducing the acrosome reaction of hamster spermatozoa. The acrosome reaction-inducing ability of the hamster zona was found to increase during maturation in the ovary. The zona of mature unfertilized hamster eggs maintained their acrosome reaction-inducing ability even after aldehyde fixation or storage in a highly concentrated solution of ammonium sulfate.     

Influence of ejaculation frequency of stallions on characteristics of semen and output of spermatozoa.

Approximately 1 week was required to stabilize the extragonadal sperm reserves in stallions ejaculated daily for 10 weeks. The true daily sperm output of a stallion was equal to the mean daily sperm output of seven ejaculates +/- 1-35 X 10(9) spermatozoa. Mean concentrations of spermatozoa/ml and number of spermatozoa/ejaculate were higher (P less than 0-01) for X1 and X3/week ejaculation frequencies than for a X6/week frequency. Sperm output/week was nearly identical for a X6/week frequency. Sperm output/week was nearly identical for the X3 and X6 frequencies and higher (P less than 0-01) than the X1 frequency. Increase of ejaculation frequency from one to two ejaculates/day twice weekly significantly (P less than 0-01) raised the output of spermatozoa/week. Gel-free semen volume, spermatozoa/ml, and number of spermatozoa/ejaculate were higher (P less than 0-01) in the first, than in the second, ejaculate. Collection of semen on alternate days would be a practical ejaculation frequency for inseminating mares. Two ejaculates collected twice a week would be a practical ejaculation frequency for long-term storage of stallion semen.     

Development of ability to penetrate the cumulus oophorus by hamster spermatozoa capacitated in vitro,in relation to the timing of the acrosome reaction

Hamster spermatozoa were tested for their ability to penetrate the intact cumulus matrix at low sperm:egg ratios (approximately 3:1). Uncapacitated spermatozoa attached to the surface of the cumulus and could not penetrate. Spermatozoa capacitated in vitro began to be able to penetrate after about 2 hr of preincubation, coincidentally with the first appearance of hyperactivation and spontaneous acrosome reactions. In all, 628 in vitro incubated spermatozoa were evaluated on and in cumuli: 270 could penetrate, but only ten of these were judged to have intact, “unmodified” acrosomes. Almost all spermatozoa capable of penetrating showed optically “modified” and swelling acrosomal caps, and this confirms previous observations on cumulus penetration in vivo. Penetration appeared limited to a phase in capacitation prior to completion of the acrosome reaction, as spermatozoa that had lost the acrosomal cap penetrated poorly and showed reduced viability. Penetration of the cumulus was inhibited by the hyaluronidase inhibitor sodium aurothiomalate. Cumulus penetrating ability could result either from a change in surface properties of the sperm at capacitation, which renders them less “sticky” to the matrix, or from release or activation of a “cumulus lysin.” We conclude that the ability to enter the cumulus matrix coincides with physiological changes in spermatozoa that occur during a terminal phase of capacitation preceding complete loss of the acrosomal cap, and that initiation of this process in vivo must precede sperm-zona contact.     

Movement characteristics of bovine epididymal spermatozoa: effects of forward motility protein and epididymal maturation

Movement characteristics of untreated bovine caudal epididymal spermatozoa were compared by high-speed cinemicrography with those of theophylline-activated caput epididymal spermatozoa with and without added forward motility protein (FMP). Comparison of individual movement characteristics clearly established the importance of FMP in converting the nonprogressive motility of theophylline-activated caput sperm into the progressive swimming of mature caudal sperm. Although the total or curvilinear distance traveled in 1 sec by theophylline-activated caput sperm was not changed by the addition of FMP, the linear progression was doubled and the percentage of progressively motile sperm was tripled by this protein. Untreated caudal sperm were 80% motile and theophylline-activated caput sperm were nearly 50% motile; the percentage of motile sperm that were progressive was the same for theophylline-activated caput sperm with FMP and for untreated caudal sperm. Caput sperm without FMP roll infrequently, if at all, but caput sperm with FMP and caudal sperm roll at 4.7 Hz. The beat frequency increases significantly with the addition of FMP and is even higher for caudal sperm. The hydrodynamic power output rises concomitantly with the beat frequency. Perhaps the most striking difference between caput sperm without FMP and those with it is in the swimming paths they follow. Caput sperm without FMP exhibit frequent reversals in direction, or yawing of the sperm heads as they loop back and cross over their tails in an apparently very flexible bending. Their average swimming paths are circles. Caput sperm with FMP and caudal sperm do not show this behavior, but swim in average paths which are linear. The minimum radius of curvature of the tail of caput sperm without FMP is much smaller than that for the other two cell types. These studies clarify the role of FMP in epididymal development of sperm motility.     

Movement characteristics and acrosomal status of rabbit spermatozoa recovered at the site and time of fertilization

Rabbit spermatozoa were recovered from the oviductal ampullae 11 h postcoitus by an oil microflush technique. Their movement was evaluated in the ampullar fluid, or in ampullar fluid diluted with in vitro fertilization medium, in slide preparations which were approximately 25 micron or 100 micron deep. The movement of these sperm was compared with the movement of ejaculated sperm in diluted semen. Movement parameters measured from videotapes recorded by a high-speed camera were coded according to treatment and entered into a microcomputer for statistical analysis. A total of 157 spermatozoa were recovered from the oviducts of 16 does: 152 were motile and 126 were free-swimming. Nearly all of the free-swimming sperm swam in trajectories whose average paths were circular. The flagellar beat pattern of the circular swimmers was asymmetric and nearly planar, and the sperm did not roll. Spermatozoa observed in 25-micron slide preparations produced smaller flagellar bends than sperm swimming in 100-micron preparations and tended to swim in larger circles which were oriented in the plane of the slide. Spermatozoa observed within the cumulus matrix moved in a slow, erratic, sinuous manner, but resumed rapid circling upon leaving the matrix. It was concluded that the ampullar sperm were hyperactivated, retaining this physiological condition as they entered the cumulus. The movement qualitatively resembled that of hyperactivated guinea pig and hamster spermatozoa because these species effectively swim in circles. In contrast, 80% of the ejaculated spermatozoa swam in linear trajectories, resulting from relatively symmetrical, flagellar beat patterns. The percentage of rolling spermatozoa and the rolling frequencies were less in the 25-micron than the 100-micron slide preparations. Thus, the movement parameters of both ampullar and ejaculated spermatozoa were affected by the geometry of their observation chambers. This influence should be taken into account when observing sperm motility in vitro. It could also be important in vivo, where changes in sperm movement in response to epithelial surfaces might provide an advantage for reaching the cumulus mass. Ninety-eight percent of the motile ampullar sperm were observed to have acrosomes, including all spermatozoa found within the cumulus matrix.     

Affinity binding of hamster oviductin to spermatozoa and its influence on in vitro fertilization

The aim of this study was to determine the influence of hamster oviductal glycoprotein (Oviductin) on in vitro gamete interaction. Oviductin was purified from the oviducts using lithium 3,5-diiodosalicylate, followed by phenol extraction. Immunocytochemistry using indirect fluorescence staining revealed that oviductin binds to the sperm anterior acrosomal region. The specific binding of oviductin resulted in inhibition of in vitro fertilization in studies using cumulus-free oocytes. The inhibitory effect was dependent on the concentration of oviductin and occurred in both ovarian and oviductal oocytes but not zona-free oocytes, indicating that sperm-zona interaction was interferred by oviduction. However, the inhibitory effect of oviductin sperm-zona interaction was reduced when cumulus-enclosed oocytes from ovaries and oviducts were used, indicating that the egg investment including cumulus oophorus has some effect on oviductin-sperm complex and maintaining the fertilizing ability. © Wiley-Liss, Inc.