宫颈粘液对人精子甘露糖受体表达的影响

目的：研究人宫颈粘液是否影响人精子甘露糖受体的表达。方法：正常人精子穿透宫颈粘液2h后．用金霉素（CTC）荧光染色法鉴定其获能及顶体状态,并用异硫氰酸荧光素标记的甘露糖化牛血清白蛋白（FITC-DMA）检测精子甘露糖受体的表达。对照组分别用获能培养基BWW培养0、2、6h后用相同方法检测。结果：人精子穿透宫颈粘液后“获能”型精子百分率较穿透前增高,而“顶体反应”型精子百分率及甘露糖受体的标记阳性率与穿透前无差别。结论：宫颈粘液促进精子获能,但不诱导顶体反应,且不影响其甘露糖受体的表达。     

Effect of human cervical mucus on human sperm motion and hyperactivation in vitro.

The aim of our experiment was to examine the effect of exposure to human cervical mucus on quantitative sperm motility with specific reference to hyperactivated sperm motility. Human spermatozoa were allowed to penetrate cervical mucus for 20 min before swimming into Earle's balanced salt solution tissue culture medium for 25 min. The sperm motion characteristics were compared to those which had been obtained from a direct swim-up for 45 min. Spermatozoa treated with mucus were more 'active' than the control group. Multivariate statistical analysis indicated that cervical mucus promotes hyperactivated motility and that sperm sub-populations exposed to cervical mucus are very heterogeneous, as indicated by the numbers and motility characteristics of spermatozoa.     

Andrology: Effects of pentoxifylline and progesterone on human sperm capacitation and acrosome reaction

This study was designed to test the effects of pentoxifyllineand progesterone upon capacitation of fresh human spermatozoa.Capacitation and acrosomal integrity were assessed using thefluorescent probe chlortetracycline on spermatozoa co-stainedwith a supravital fluorescent dye, Hoechst 33258. Hyperactivatedmotility was measured using computer-assisted movement analysis.After exposure to pentoxifylline (1 mg/ml; 30 min), the fluorescent‘B’ pattern, characteristic of capacitated, acrosome-intactcells, increased significantly (P < 0.01), though no increasein ‘AR’ pattern, characteristic of acrosome-reactedcells, was detected. There was a significant increase in hyperactivemotility (P < 0.001). Exposure to progesterone (1µg/ml;60 min) resulted in a significant increase in ‘B’pattern (P < 0.05) and ‘AR’ pattern (P < 0.005),though no effect on the expression of hyperactivation was detected.No effect upon hyperactivation was detected on exposure of freshor cryopreserved spermatozoa to a physiological range of progesteroneconcentrations (0.1–1000 ng/ml). Sequential exposure topentoxifylline then progesterone resulted in a significant increasein ‘B’ pattern, acrosome loss and hyperactivation.Sperm viability was not affected in any treatment group. Theseobservations suggest that pentoxifylline and progesterone affectcapacitation through independent mechanisms. Stimulation ofboth capacitation and acrosome reaction resulted from sequentialexposure to pentoxifylline and progesterone. This may have implicationsfor sperm handling for assisted reproductive techniques.     

A comparison of three methods for detecting the acrosome reaction in human spermatozoa

This study was designed to compare three different fluorescentprobes to assay the acrosome reaction in human spermatozoa:chlortetracycline (CTC), mannosylated bovine serum albumin (BSA)labelled with fluorescein (MAF), and quinacrine (QN)- Normalhuman sperm ejaculates were washed and allowed to swim up for30–60 min. Samples were examined under epifluorescencefor the percentage of the acrosome reacted spermatozoa, as detectedby the three probes. There was no significant difference betweensamples of fresh, uncapadtated spermatozoa evaluated with CTC,MAF or QN; all gave <10% reacted. Following capacitationfor 3 h, the percentage of spontaneously reacted spermatozoawas higher than in fresh spermatozoa; CTC and MAF gave the samepercentage (12%), while QN indicated a higher percentage (18%)of reacted spermatozoa (P < 0.001). Following exposure toionophore A23187 at 1 h, the percentage of acrosome reactionsincreased to a mean of 31% as detected with CTC or MAF; themean percentage (45%) was significantly higher with QN (P <0.0001). Further incubation up to 2 h with A23187 did not changethese percentages. These results suggest that the QN probe detectsthe onset stage of the acrosome reaction, whereas the CTC andMAF probes detect the later stages in which the acrosomal capis lost. Use of the two types of probe provides a means forfiner resolution of the time course of the acrosome reactionin the human spermatozoa.     

Chemical composition and protein source in the capacitation medium significantly affect the ability of human spermatozoa to undergo follicular fluid induced acrosome reaction

We studied the effect of media composition on sperm capacitation,using Biggers—Whitten—Whittingham (BWW) medium,Ham's-F10 and a modified Tyrode's medium (HSM) supplementedwith bovine serum albumin (BSA) or fetal cord serum (FCS). Weevaluated the effect of chemical environment and protein supplementationon the sperm motion parameters of curvilinear velocity and linearity,and on the ability of incubated spermatozoa to undergo follicularfluid induced acrosome reaction. Neither chemical compositionnor protein supplementation of capacitation media greatly affectedmotion parameters after 2 h incubation. Furthermore, chemicalcomposition had only a small effect on the ability of spermatozoato undergo the acrosome reaction upon exposure to follicularfluid. A higher proportion of spermatozoa underwent acrosomereaction after incubation in HSM (8% control (C); 28% follicularfluid) than in BWW (8% C, 17% follicular fluid) or Ham‘sF-10 (6% C, 19% follicular fluid). By contrast, protein sourceproved critical in determining acrosome reaction inducibility.Spermatozoa incubated in BSA-supplemented media showed a 4-foldincrease in acrosomal discharge when exposed to follicular fluid(6% C, 22% follicular fluid) compared to controls while spermatozoaincubated in FCS were unable to undergo acrosome reaction (6%C, 6% follicular fluid). Simultaneous addition of FCS to BSAcapacitation medium blocked acrosome reaction inducibility andthe late addition of BSA, after sperm incubation in FCS, didnot facilitate acrosome reaction. We propose that an inhibitorof sperm capacitation is present in FCS and therefore, the selectionof optimum incubation conditions for spermatozoa may be of criticalimportance when evaluating or treating infertile patients.     

An evaluation of the role of relaxin in the penetration of cervical mucus by spermatozoa

Relaxin-like immunoreactivity was measured in seminal plasmafrom men who were separated into two groups, on the basis ofa previous positive or negative result in a postcoital cervicalmucus penetration test. There was no difference in the relaxinconcentration between the groups. The effect of exogenous porcinerelaxin (0, 10 or 100 ng/ml) on human cervical mucus penetrationin vitro by washed human spermatozoa was studied using a capillarytube preparation. In the positive postcoital test group thehighest relaxin concentration (100 ng/ml) tended to inhibitcervical mucus penetration, although this effect was only significantfor one of the parameters measured (number of spermatozoa penetratingto the 10-mm mark). The same trend was apparent for the negativepostcoital test group, but no differences were significant.The results are in direct contrast to previous reports thatrelaxin can stimulate human spermatozoa motility and cervicalmucus penetration.     

The effect of three anti-oestrogen drugs on cervical mucus quality and in-vitro sperm--cervical mucus interaction in ovulatory women

The anti-oestrogens, clomiphene citrate, tamoxifen and cyclofenilare commonly used in the treatment of female infertility. Theirrole in the management of anovulation is well established butthere is continuing controversy about their relevance to otherareas of management. We have studied the effects of each ofthese drugs on cervical mucus and sperm—cervical mucusinteraction among 23 patients with unexplained infertility.Each patient received all three drugs in an alternative monthtreatment regime and in addition acted as her own control. Thestarting point in each patient was randomized. Luteinizing hormone(LH) and oestradiol were measured daily from day 10, and folliclescanning was also undertaken. Cervical mucus quality and sperm—cervical mucus interaction were studied on the day ofonset of the LH surge. The use of clomiphene and tamoxifen resultedin a significant reduction in cervical mucus score and sperm— cervical mucus interaction as judged by the distancetravelled by the vanguard spermatozoa. Cyclofenil had no effecton these parameters.     

Fertilization promoting peptide, a tripeptide similar to thyrotrophin-releasing hormone, stimulates the capacitation and fertilizing ability of human spermatozoa in vitro

Recent studies have demonstrated that a prostatic tri-peptidesimilar in structure to thyrotrophin-releasing hormone (TRH)can stimulated the in-vitro capacitation and fertilizing abilityof epididymal mouse spermatozoa. Therefore we have proposedthat this tripeptide be referred to as fertilization promotingpeptide (FPP). Using chlortetracycline fluorescence analysisand the hamster oocyte penetration test (HOPT), we have obtainedevidence that FPP can also promote the capacitation and fertilizingability of ejaculated human spermatozoa in vitro. FPP (25–200nM) caused a significant increase in the proportion of B-patterncapacitated cells and a decrease in the proportion of F-patternuncapacitated cells, with no significant stimulation of acrosomalexocytosis. Comparison of FPP with two structurally similartripeptides, TRH and pyro-glutamyl phenylalanylprolineamide,at 50 nM revealed that only FPP could significantly promotecapacitation. Finally, after a brief exposure to progesteroneto induce acrosomal exocytosis in capacitated cells, FPP-treatedsuspensions penetrated a significantly higher proportion ofoocytes than the untreated controls when assessed in the HOPT.The presence of FPP in human seminal plasma at concentrationssimilar to those used here suggests that, in vivo, FPP may playa positive role in promoting human sperm function.     

The spontaneous acrosome reaction of human spermatozoa incubated in vitro

Motile human sperm populations were prepared from liquefiedsemen (10 donors x 3 replicates) using Percoll density gradientsat 30–60 min post-ejaculation. Sperm suspensions wereincubated in a complex ’synthetic tubal fluid‘ culturemedium (STF) at 37°C under 5% CO₂ in air for up to 36 h.Parallel aliquots were incubated with 50 µM A23187 toinduce maximum acrosome loss (AR_max). Acrosome reactions wereassessed using both the triple-stain (TS) technique and fluorescentpeanut agglutinin (PNA) lectin-labelling. During incubation,the proportion of TS acrosome reacted spermatozoa increasedfrom 9.1 to 54.3% with AR_max being 68.3%. Spermatozoa showingintact acrosomes by PNA labelling decreased from 68.4 to 26.1%over 36 h of incubation (AR_max = 13.8%). Simultaneously, spermatozoashowing complete acrosomal loss (no PNA labelling) increasedfrom 8.1 to 27.0% (AR_max= 46.3%). Therefore, while only 23.5%of cells were actually undergoing acrosomal changes at the startof incubation, this had increased to 46.9% after 36 h (AR_max=40.7%). These experiments clearly show that even in selectedpopulations, not all human spermatozoa are capable of undergoingan acrosome reaction. However, the incidence of acrosomal changesafter 36 h of incubation did approach the AR_max. These levelsof spontaneous occurrence of the human sperm acrosome reactionwere higher than those reported in many other in-vitro incubationstudies: an improvement that may be attributable to the morephysiological nature of the STF culture medium     

Comparison of human cervical mucus and artificial sperm penetration media.

The cervical mucus penetration tests aid research and determine the clinical importance of positive sperm antibody tests. Limited availability and variability of human cervical mucus have instigated the search for mucus substitutes for these tests. This study compares sperm migration in cervical mucus with that in artificial media including hyaluronate solution, egg white and albumin Tyrode solution. Results were quantified by measuring the migration distance (the maximum distance of capillary migration from a semen reservoir by spermatozoa after 1 h) and the sperm concentration at half the migration distance. The mean of both measures for cervical mucus and hyaluronate solution were equivalent [4.4 +/- 1.1 (SD) versus 4.3 +/- 1.0 cm and 118 +/- 51 versus 111 +/- 44x10(3)/ml], and higher than in egg white and albumin Tyrode solution. Antisperm antibodies impaired sperm penetration in cervical mucus and hyaluronate solution in a similar manner (r = 0.92). These results suggest that hyaluronate solution sufficiently resembles human cervical mucus in terms of penetrability that it may be used as a substitute for mucus in capillary tube tests of sperm function. The higher penetrability of cervical mucus and hyaluronate solution is probably related to a channelling effect due to their polymeric structure.     

Changes in the sialylglycoconjugate distribution on the human sperm surface during in-vitro capacitation: partial purification of a 20 kDa sialylglycoprotein of capacitated spermatozoa

Changes in the distribution of sialylglycoconjugates on thesurface of uncapacitated and in-vitro capacitated human spermatozoawere studied by means of two sialic acid specific lectins [Maackiaamurensis agglutinin and Sambucus nigra agglutinin). On theuncapacitated sperm surface, sialylglycoconjugates were foundbe localized from the post-acrosomal region of the sperm headto the tail middle piece, whereas after invitro capacitationthese molecules were only found in a small area of the post-acrosomalregion. The surface of capacitated human spermatozoa was alsoinvestigated by specifically radiolabelling its terminal sialicacid residues. A 20 kDa glycoprotein, which was partially purifiedby anion-exchange chromatography, was the main component ofthe sialylglycoconjugate pattern after in-vitro capacitation.     

Human and bovine cervical mucus penetration as a test of sperm function for in-vitro fertilization

Human and bovine cervical mucus penetration tests (n = 57) wereperformed preceding IVF to test their prognostic value as spermfunction tests for IVF. This evaluation also induded resultsfrom conventional semen analysis and from a computerized spermanalysis system. The bovine cervical mucus penetration testwas shown to be at least as valuable as the human cervical mucuspenetration test in evaluating sperm function. The migrationdistance of the vanguard sperm (P < 0.001) and the spermdensity at a fixed migration distance in the mucus column (P< 0.05) correlated most closely with the IVF results. A clearparallelism with the out come of the ‘swim up’ techniquewas also found. Of the sperm parameters examined, only spermmotility In the ejaculate (P < 0.05) correlated significantlywith the results of IVF. It is concluded that the outcome ofa bovine cervical mucus penetration test depends on the samesperm functions as re quired for IVF. Therefore, this test maybe of predictive value in an IVF programme.     

Variation in the cholesterol/phospholipid ratio in human spermatozoa and its relationship with capacitation

The cholesterol/phospholipid (C/PL) ratio was determined for spermatozoa from eight men with normal semen parameters. There was a close correlation between the C/PL ratio and the rate of sperm capacitation as assessed by the hamster egg penetration test. A lower C/PL ratio correlated with a faster capacitation time. This supports the notion that the loss or reduction of membrane cholesterol constitutes an important step of capacitation in human spermatozoa.     

The role of follicular fluid in inducing the acrosome reaction of human spermatozoa incubated in vitro

Motile human sperm populations were prepared from liquefiedsemen (5 donors x 3 replicates) using Percoll gradients at 30–60 min post-ejaculation and preincubated in a complex ‘syntheitictubal fluid’ culture medium (STF) at 37° C under 5%CO₂ in air for 6h. Aliquots of these suspensions were then incubatedfor a further 2 h in STF containing 0, 5, 25, 50, 75 and 100%(v/v) pooled human follicular fluid (FF). Another aliquot wastreated with 10 µm A23187 in STF for 20min and then incubatedin fresh STF medium for a futher 2h to induce maximal acrosomeloss. Acrosome reactions were assessed using both the triple-staintechnique and fluorescent peanut agglutinin lectin-labelling.Sperm motility and movement characteristics were assessed fromvideorecordings using digital image analysis (CellSoft). Exposureto FF caused only relatively small proportions of the preincubatedspermatozoa to undergo acrosome reactions. The size of theseresponsive sub-populations was smaller than that capable ofresponding to a Ca²⁺ influx generated by A23187. Increased FFconcentrations induced a progressive loss of motility and trendsfor changes in movement characteristics that may have been relatedto reduced intracellular Ca²⁺. This interpretation of theseobervations is that while FF may act to stimulate or promotethe human sperm acrosome reaction it does not appear to be aspecific inducer of it. Consequently, a precise role for FFat the relatively low concentrations that would be expectedto be present in the tubal ampulla in the physiological regulationof human fertilization remains unproven     

Induction of acrosome reaction in human spermatozoa used for subzonal insemination.

Human spermatozoa were injected into the perivitelline space of oocytes from 43 couples (44 cycles) in whom fertilization had failed in conventional in-vitro fertilization (IVF). The spermatozoa were treated to enhance the percentage of acrosome-free spermatozoa either by incubation for 24 h in T6 medium with 50% follicular fluid (v/v) or by incubation for 24 h in T6 medium followed by electroporation and incubation for a few hours in T6 medium with 3.5 mM pentoxifylline. After these two procedures, the mean percentage of acrosome-free spermatozoa increased to 35.5 and 53.9% respectively. Up to three spermatozoa were injected into the perivitelline space of metaphase II oocytes; few oocytes were damaged during the injection procedure. The overall fertilization rate was 30.9% of the 433 oocytes that were intact after subzonal insemination. Only 3% of the injected oocytes had more than two pronuclei. The cleavage rate of the fertilized oocytes was 80%. There was no difference in the fertilization and cleavage rates between the two sperm treatment procedures. One, two or three embryos were replaced in 34 cycles and seven patients became pregnant. In three of the four ongoing pregnancies, prenatal diagnosis by amniocentesis indicated a normal karyotype.     

Maturation of human spermatozoa (from selected epididymides of prostatic carcinoma patients) with respect to their morphology and ability to undergo the acrosome reaction

Spermatozoa were recovered form three regions of the epididymisof six prostatic carcinoma patients. After washing and incubatingfor 3 h in Ham's F-10 medium, with or without 5 µM A23187for the last 30 min, spermatozoa were tested for vitality byhypotonic swelling and permeated with methanol to detect theacrosome with peanut agglutinin. Whereas the extent of spontaneousacrosome reactions was similar for spermatozoa from all regionsof the duct, 17 and 28% of spermatozoa from all regions of theduct, 17 and 28% of spermatozoa from the corpus and cauda epididymidisrespectively, responded to stimulation by A23187 with acrosomereactions but there was no stimulation by A23187 of spermatozoafrom the efferent ducts. The percentage of morphologically normalspermatozoa increased stepwise towards the distal regions, withabnormalities being mostly enlarged heads in more proximal regions:they were largely absent form the cauda epididymidis. Spermhead swelling was similarly observed in cynomolgus monkey spermatozoafrom the caput epididymidis but not the more distal regions.These forms were not observed when spermatozoa were fixed beforesmearing, indicating that they were artefacts of sperm preparation.The changes in the susceptibility of non-fixed epididymal spermatozoato produce morphological artefacts and the gain in their acrosomalresponse to ionophore demonstrate maturational changes of spermatozoain the human epididymis.     

Improvement in motion characteristics and acrosome status in cryopreserved human spermatozoa by swim-up processing before freezing

The purpose of this study was to examine if selecting a sperm population with improved motion characteristics before freezing reduces the deleterious effects of cryopreservation. Semen specimens from 15 normal donors were divided into two equal aliquots. The first aliquot received no treatment (control), and the second was processed by swim-up from a washed sperm preparation to select a sperm population with better motility and motion characteristics (swim-up). Both aliquots were cryopreserved by the liquid nitrogen vapour method. Percentage motility and motion characteristics were evaluated by computer-assisted semen analysis. Acrosome integrity as well as spontaneous and calcium ionophore-induced acrosome reactions before freezing and after thawing were assessed by fluorescein isothiocyanate conjugated peanut agglutinin combined with a supra vital dye (Hoechst-33258). Swim-up processing enabled selection of a sperm population with better motion characteristics, percentage motility and viability before freezing (P < 0.001), but with no difference in percentage of acrosome-intact spermatozoa (P = 0.63). After thawing, the swim-up specimens exhibited faster velocity and progression than untreated specimens (P < 0.001). They also had higher percentages of spermatozoa with intact acrosomes and spermatozoa able to undergo acrosome reaction in response to calcium ionophore (P < 0.05). Selecting a highly motile sperm population before freezing enhances overall post-thaw spermatozoa quality.     

Artificial induction of the acrosome reaction in human spermatozoa

This study investigated the use of human follicular fluid andpentoxifylline as inducers of the human sperm acrosome reactionin vitro. Motile sperm suspensions were prepared using a discontinuousPercoll gradient, preincubated for 3 h, divided into aliquotsand exposed to various concentrations of non-heart-inactivatedfollicular fluid for 1 and 24 h and pentoxifylline for 30 min.Detection of the acrosome reaction involved the combined useof a fluorescent vital stain, H33258 [GenBank] , and fluorescein isothiocyanate-conjugatedpeanut agglutinin (FITC-PNA). A short (1 h) exposure to follicularfluid at concentrations of 50% or more, did not compromise spermmotility and significantly increased the proportion of spermatozoahaving completed the acrosome reaction. Similarly, a 30 minexposure to pentoxifylline also significantly increased theproportion of spermatozoa having completed the acrosome reaction.     

Induction of capacitation in human spermatozoa in vitro by an endometrial sialic acid-binding protein

A Ca²⁺-dependent sialic acid-binding protein (SABP) of humanendometrium, which specifically bound to human sperm head plasmamembrane in vitro, was found to increase the percentage motilityand acrosome-reacted pattern of uncapacitated spermatozoa. Theprotein was synthesized in the endometrium and secreted intothe uterine fluid. This intra-uterine factor, which is apparentlyadvantageous in vitro in inducing human sperm capacitation,may play a significant role in promoting the postrelease maturationof ejaculated spermatozoa by enhancing ⁴⁵Ca uptake into spermatozoaby a pathway which is insensitive to calcium-channel blockers.However, the ⁴⁵Ca uptake could be enhanced on exposure to thedivalent cation ionophore A23187 and inhibited in the presenceof the calmodulin inhibitor trifluoperazine. The SABP also inducesan increase in intracellular Ca²⁺ in spermatozoa, as seen byFURA-2 AM studies. Furthermore, overlay studies show human SABPto be a Ca²⁺-binding protein. The data presented here suggestthat SABP induces invitro sperm capacitation and the subsequentacrosome reaction by increasing intracellular Ca²⁺ concentration.     

Sperm-cervical mucus interaction, in particular in the presence of antispermatozoal antibodies

Disturbances of the interaction between spermatozoa and cervicalmucus can cause subfertility or infertility. The diagnosis ofsuch a disturbed interaction is possible with simple laboratorytests. Oligomucorrhoea and dysmucorrhoea are the most frequentcauses of a disturbed sperm-cervical mucus interaction. AntispermatozoalIgA plays a quantitatively limited, but qualitatively importantrole. Sophisticated time-consuming laboratory investigationshave mostly only additional value for the diagnosis.