生育与不育男性精子顶体反应释放的顶体酶活性比较

目的比较生育与不育男性精子顶体反应释放的顶体酶活性,以此来评价精子顶体反应。方法30例不育男性和10例健康生育男性精液,用Percoll密度梯度分离法优选精子,FITC-PSA荧光染色法分析AR;用分光光度比色法测定顶体酶活性。结果少精子症组、弱精子症组和畸形精子症组精子顶体反应率和顶体反应释放的顶体酶活性均明显低于生育组(P<0.05),前3组中顶体反应率和顶体反应释放的顶体酶活性以畸形精子症组最低,但不育各组间比较均没有明显差异。将顶体反应释放的顶体酶活性与其相应的顶体反应率进行相关分析,两者存在明显正相关关系(P<0.001)。结论用顶体反应释放的顶体酶活性可以评价精子顶体反应功能。     

The acrosome reaction in human sperm from men of proven fertility

Suspensions of motile sperm were prepared from semen samplesdonated by 40 men of recent, proven fertility and incubatedunder capacitating conditions for 24 h. At selected time-pointsaliquots were removed, assessed for motility, fixed and examinedwith the electron microscope to determine the rate of acrosomeloss. Data indicate that acrosome loss increases significantlywith time, but absolute values are relatively low. After 24h a mean of 15.4% sperm had initiated the acrosome reaction;this figure included 9.7% which had completed it. The proportionof cells at intermediate stages was similar throughout incubation({small tilde}5%), indicating that initiation of the acrosomereaction occurs at a fairly constant rate. In four samples motilitydeclined over 24 h and in six, contaminating cells were observed.In the majority of these 10, acrosome loss was higher than thatobserved in the remaining 30 samples. Additionally, the assessmentof <25 000 cells during this study made it possible to evaluatespecific ultrastructural features of the normal acrosome reactionin human sperm. Six stages were identified, with the intermediateones involving loss of acrosomal matrix material while outermembranes appear to retain their integrity; this contrasts sharplywith the current view of the generalized mammalian sperm acrosomereaction.     

Effect of fibronectin on proteasome activity, acrosome reaction, tyrosine phosphorylation and intracellular calcium concentrations of human sperm

BACKGROUND: Previously we showed that the human sperm proteasome plays significant roles during mammalian fertilization. Here we studied the effect of fibronectin (Fn), an extracellular matrix protein present in the cumulus oophorus of the oocyte, on proteasome activity, acrosome reaction, intracellular calcium concentration ([Ca(2+)](i)) and protein tyrosine phosphorylation of human sperm. METHODS: Aliquots of motile sperm were incubated for 15 min (T0), 5 h (T5) and 18 h (T18), at 37 degrees C, 5% CO(2) and 95% air with Fn (0-100 microg/ml). The chymotrypsin- and trypsin-like activity of the proteasome was measured using the fluorogenic substrates, Suc-Leu-Leu-Val-Tyr-AMC and Boc-Gln-Ala-Arg-AMC, respectively. At T18, sperm aliquots were incubated for 15 min with Fn and/or progesterone in the presence or absence of epoxomicin (a proteasome inhibitor). The percentage of viable acrosome reacted sperm was evaluated using the Fluorescein isothiocyanate (FITC)-labeled Pisum sativum agglutinin. Tyrosine phosphorylation was evaluated by western blot and [Ca(2+)](i) using fura 2. RESULTS: Fn stimulated both enzymatic activities of the proteasome and the acrosome reaction of human sperm. Progesterone enhanced and epoxomicin drastically inhibited the effect of Fn. Fn treatment also increased the [Ca(2+)](i). Western blot analysis revealed that Fn increased tyrosine protein phosphorylation and that some proteasome subunits became tyrosine phosphorylated upon Fn treatment. CONCLUSIONS: These results suggest that Fn activates the proteasome and induces the acrosome reaction in human sperm. This effect may involve binding with specific receptors (integrins) on the sperm surface and the activation of tyrosine kinases.     

Andrology: Osmo-sensitivity of the human sperm acrosome reaction

The osmo-sensitivity of the human sperm acrosome reaction wasinvestigated by determining the effect of hyper- and hypo-osmolalconditions on the ionophore A23187- and dbcAMP-induced reactionof both capacitated and non-capacitated spermatozoa. Hyper-osmolalconditions inhibited the agonist-induced reactions of both typesof spermatozoa. Hypo-osmolal conditions caused a spontaneousloss of acrosomes from capacitated but not from non-capacitatedspermatozoa. The loss of acrosomes under hypo-osmolal conditionswas further enhanced by dbcAMP but not by ionophore A23187.Although significant decreases in sperm viability were occasionallyobserved at the high and low osmolalities, these decreases werenot consistent and could not account for the observed loss ofacrosomes. It is concluded that the human sperm acrosome reactionis osmo-sensitive. The acrosome reaction stimulated by ionophoreA23187 (raises intracellular Ca²⁺) and dbcAMP (activates proteinkinase A which causes protein phosphorylation) appears to involvewater entry downstream from the action of these agonists. Preincubationin albumin (capacitation) causes human spermatozoa to lose theiracrosomes under hypo-osmolal conditions. Finally, capacitationis not an essential prerequisite to the acrosome reaction aslong as agonists are used that by-pass certain membrane-relatedevents.     

Human sperm capacitation and the acrosome reaction.

A model is presented that describes the mechanism of human sperm capacitation and the acrosome reaction. The processes of capacitation and the acrosome reaction are proposed to function in control of the activation/release of acrosomal enzyme(s) involved in sperm penetration through the zona pellucida. During capacitation, the sperm head membranes are biochemically modified, allowing the acrosome reaction to take place when the spermatozoon approaches or reaches the zona pellucida, resulting in the localized activation and release of the appropriate enzyme(s). Further, capacitation is presented as a continuing process that occurs during sperm transport through the female genital tract and is physiologically not completed until the spermatozoon reaches the oocyte (unless the spermatozoa are kept at a particular genital tract site for prolonged periods). The biochemical alterations that occur during capacitation are discussed. It is suggested that extensive modifications in the lipid bilayer structure, e.g. in the cholesterol or phospholipid content, are not part of capacitation because such changes would prematurely destabilize the membranes. Rather, such changes occur during the acrosome reaction. It is also proposed that the human sperm acrosome reaction has many similarities to the somatic cell exocytotic events which occur during the regulated pathway of secretion. One or more oocyte stimuli result in the activation of protein kinases, likely (but not necessarily) via activation of G-protein coupled receptors on the sperm plasma membrane and the formation of second messengers. The kinases phosphorylate and activate proteins, continuing the biochemical cascade that ultimately results in the acrosome reaction. The role of other enzyme systems such as those involved in ion transport, proteolysis, phospholipid metabolism (including that of arachidonic acid) and other metabolic events, is discussed. Calcium ion influx as initiator of the acrosome reaction is reconsidered. The proposed model also takes into consideration the structural events of membrane fusion.     

Appropriate timing of the acrosome reaction is a major requirement for the fertilizing spermatozoon

This study was undertaken to determine the site of the acrosomereaction of spermatozoa penetrating into freshly inseminatedhuman oocytes. The inseminated oocytes were treated with ananti-acrosin monoclonal antibody and the bound antibody wasvisualized at the ultrastructural level with the use of a secondperoxidase-conjugated antibody. Quantitative analysis of serialthin sections cut throughout the specimens showed that the numberof spermatozoa within the zona pellucida (potentially fertilizingones) corresponded to the number of acrosin deposits associatedwith acrosomal ghosts on the zona pellucida surface. As it isknown that a large acrosin bundle is liberated from a spermatozoonat a well-defined point of the acrosome reaction, these findingsindicate that the acrosome reaction of the fertilizing spermatozoonmust be exactly synchronized with its penetration through theegg vestments, apparently by the action of specific acrosomereaction-promoting substances in the oocyte/cumulus complex.These results represent a theoretical basis for evaluation ofdirect and indirect laboratory tests for human sperm acrosomereaction. ‘Good’ sperm samples should display elevatedlevels of acrosome-reacted spermatozoa after the administrationof an appropriate stimulus and low levels of spontaneous acrosomereactions.     

Andrology: The influence of sperm morphology and the acrosome reaction on fertilization outcome after sub-zonal injection (SZI) of human spermatozoa

The usefulness of sub-zonal injection (SZI) for the treatmentof severe male factor infertility has been restricted by lowand unpredictable fertilization rates and the high risk of polyspermyafter the injection of multiple spermatozoa. In this prospectivestudy, we have evaluated whether sperm morphology and the percentageof acrosome-reacted spermatozoa at the time of injection canbe used to predict SZI fertilization outcomes. Populations ofmotile spermatozoa equivalent to those injected were collectedfrom the medium/oil interface immediately after SZI of eachcohort of oocytes. Morphology was assessed using the World HealthOrganization 1987 criteria and the acrosomal status of spermatozoawas determined after staining with rhodamine-conjugated Pisumsativum agglutinin. A fertilization index (FI) was calculatedto express the actual fertilizing potential of the spermatozoainjected. In all, 67 patients underwent 72 SZI cycles. The overallfertilization and polyspermy rates were 36 and 47% respectively,and a clinical pregnancy rate per transfer of 22% was achieved.Linear regression analysis demonstrated a statistically significantrelationship between morphology and the FI (r = 0.506, P <0.0001). Patients with <10% normal morphology always hada FI < 10%, and this was reflected by low fertilization andpolyspermy rates and the high number (32%) of cycles with completefailure of fertilization in this group. In patients with >10% normal morphology, there were two patterns: low (10% FI)or high (>10% FI) fertility. This was evident in the fertilization(23 and 85%, respectively) and polyspermy (25 and 68%, respectively)rates of these two patient sub-groups. While the percentageof acrosome-reacted spermatozoa at the time of injection wasweakly correlated with the FI (r = 0.292, P < 0.05), it couldnot be used to predict differences in fertilization potentialbetween patient sub-groups. We conclude that sperm morphologyand acrosomal status at the time of injection are of limiteduse in predicting SZI fertilization outcomes, although patientswith poor morphology ( 10% normal) have lower fertilizationand polyspermy rates.     

In vivo assessment of the human sperm acrosome reaction and the expression of glycodelin-A in human endometrium after levonorgestrel-emergency contraceptive pill administration

BACKGROUND: The objectives were firstly to assess acrosome reaction (AR) status of spermatozoa following uterine flushing, secondly to measure levonorgestrel (LNG) levels in serum and in uterine flushing fluid and finally to measure endometrial glycodelin-A expression after administration of LNG as a form of emergency contraception (EC). METHODS: Forty-eight experiments were conducted on 15 regularly menstruating women. Four groups were formed based on different intercourse to treatment interval and treatment to recovery of spermatozoa and the biopsies. RESULTS: Twenty-four and forty-eight hours after treatment, there were 14.5 +/- 3.9 x 10(6) and 17.3 +/- 6.8 x 10(6) sperm recovered from the uterus, respectively. There were no differences between the AR rate and the endometrial glycodelin-A staining intensity in an LNG or placebo treated cycles. The LNG in uterine flushing medium represented 1.38% of the values observed in serum 24 h after the LNG intake. CONCLUSIONS: Twenty-four and forty-eight hours after administration of EC, neither the proportion of AR sperm, nor the glycodelin-A level was influenced by 1.5 mg of LNG. LNG did not impair the cervical mucus either because viable spermatozoa were found in the genital tract 36-60 h after coitus and 24-48 h after LNG intake. The mechanism of action of LNG as EC remains unknown.     

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     

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     

Hyperactivation of capacitated human sperm correlates with the zona pellucida-induced acrosome reaction of zona pellucida-bound sperm

BACKGROUND: The aim of this study was to determine the relationship between human sperm hyperactivation (HA), sperm-zona pellucida (ZP) binding and the ZP-induced acrosome reaction (AR) of ZP-bound sperm in vitro. METHODS: Sperm samples from 129 infertile men were studied. Motile sperm (2 x 10(6)) selected by Pure sperm were incubated with four oocytes in 1 ml human tubal fluid supplemented with 10% human serum. After 2-h incubation, the number of sperm bound to the ZP and the AR of ZP-bound sperm were examined. Velocities and HA of sperm in insemination medium were assessed by Hamilton-Thorn Sperm Analyzer. RESULTS: The HA was highly correlated with the ZP-induced AR in all the subjects (rho = 0.626, P < 0.001). In the 69 men with < or = 100 sperm bound/ZP, allowing accurate counts, HA was not significantly correlated with sperm-ZP binding. Men with <7% HA sperm were more likely to have very low ZP-induced AR. Only normal sperm morphology was significantly correlated with sperm-ZP binding (rho = 0.346 and 0.446 in semen and insemination medium, respectively, both P < 0.001). Sperm motility and velocities were significantly correlated with sperm morphology but not with either sperm-ZP binding or the ZP-induced AR. CONCLUSIONS: The correlation of HA with the ZP-induced AR of ZP-bound sperm suggests a mechanistic link between HA and the physiological AR in humans. Assessment of HA of capacitated sperm in vitro may be a useful clinical test for male infertility associated with defective ZP-induced AR that does not require the use of human oocytes.     

精子顶体酶活性与体外受精关系的探讨

目的探讨精子顶体酶活性与体外受精的关系。方法将236个体外受精-胚胎移植（IVF-ET）治疗周期根据顶体酶活性分成三组：A组≥48.2μIU/106精子,共95周期;B组：30～48.1μIU/106精子,共92周期;C组〈30μIU/106精子,共49周期;比较三组间的受精率、卵裂率、优质胚胎率。结果 A、B、C组的受精率分别为77.02%、74.11%和48.69%,卵裂率分别为97.80%、96.65%和90.29%,优质胚胎率分别为41.32%、40.79%和33.42%。三组间卵裂率、优质胚胎率比较,差异无统计学意义（P〉0.05）。C组的受精率明显低于A组（P〈0.05）,且完全不受精的发生率高于A、B组（P〈0.05）。B组与C组相比受精率下降,但差异无统计学意义（P〉0.05）。结论当顶体酶活性低于30μIU/10^6精子时受精率显著下降,可考虑行部分卵胞浆内单精子注射治疗。     

Andrology: A new predictive test for in-vitro fertilization based on the induction of sperm acrosome reaction by N-acetylglucosamine-neoglycoprotein

Neoglycoproteins with N-acetylglucosamine residues (BSA-GIcNAc)induced specifically the acrosome reaction (AR) in human spermatozoa.Our objective was to investigate the relationship between thisphenomenon and the in-vitro fertilization (IVF) rate. Spermsuspensions from IVF protocols were incubated with BSA-GlcNAc(t), using calcium ionophore (i) or medium alone (c) as positiveor negative controls. When the normalized AR percentage ratio(STIM) (%ARt-%ARc): (%ARi-%ARc) was compared with fertilizationrate in 31 couples from our IVF programme, a positive correlationwas found (r = 0.46, P < 0.01). The fertilization rate inpatients with STIM 0.2 was higher than in non-responders (STIM< 0.2); 72 ± 7% compared with 5 ± 3%. The overallpredictive value of this test for adequate fertilization rate(>30%) was 87%, sensitivity 91% and specificity 78%. Falsepositives were 9% and false negatives 22%. For successful fertilizationrates (>60%), the results were: overall predictive value,84%; sensitivity 100%; specificity 64%. False positives were23% and no false negatives were found. The results indicatedthat the induction of AR in human spermatozoa by GlcNAc-neoglycoproteinscould be used to predict their fertilizing ability in vitro.     

Fertilization and early embryology: Progesterone-induced acrosome reaction: potential role for sperm acrosome antigen-1 in fertilization

We have established a monoclonal antibody (mAb) AG7 defininga sperm acrosome antigen-1 (SAA-1) on spermatozoa from the humanand several mammalian species. MAb AG7 inhibits fertilizationof mouse eggs in vitro and in vivo. An important characteristicof mAb AG7 is its inhibition of the rise in intracellular calciuminduced by progesterone in human spermatozoa. Here we show that,following the acrosome reaction, SAA-1 is lost from the capof human spermatozoa but remains detectable in the equatorialregion. Acrosome reaction assays demonstrated a clear differencebetween progesterone- and A23187-induced acrosome reactions.For induction of the acrosome reaction with progesterone, aminimum capacitation time of 6 h was required. A23187 inducedthe acrosome reaction regardless of capacitation time. MAb AG7completely inhibited the progesterone-induced acrosome reaction,but not the A23187-induced acrosome reaction in human spermatozoa.Differences in the pattern of calcium flux induced by the twoagents might account for this phenomenon. The inhibition ofthe progesterone-induced acrosome reaction by mAb AG7 impliesa regulatory function of SAA-1 during the human sperm acrosomereaction.     

A simple method for assessment of the human acrosome reaction of spermatozoa bound to the zona spellucida: lack of relationship with ionophore A23187-induced acrosome reaction

Acrosome reactions induced by the calcium ionophore A23187 [GenBank] andzona pellucid a (ZP) were studied. Sperm samples were obtainedfrom fertile men or men with normal semen analysis and normalsperm-ZP binding. Oocytes were obtained, with the consent ofthe patients, after the failure of fertilization in vitro. Motilespermatozoa selected by a swim-up technique were incubated with10 µM A23187 [GenBank] for 1 h, four oocytes for 2 h or solubilizedZP (4 ZP/µl) for 2 h. Spermatozoa bound to the ZP weredislodged and collected in a small volume of phosphate-bufferedsaline by aspirating the oocytes with a glass pipette with aninner diameter (120 µm) slightly smaller than the diameterof the oocyte. The acrosome status of the spermatozoa was determinedusing fluorescein-labelled Pisum sativum agglutinin. The proportionof spermatozoa undergoing the acrosome reaction on the ZP at2 h varied over a wide range (5–99%), but the agreementbetween results for the same semen sample exposed to differentgroups of oocytes was good: the standard deviations of the differencesbeing 9%. Pre-incubation of spermatozoa for 2 h did not increasethe ZP-induced acrosome reaction. Re-incubation of ZP with thesame sperm suspension for 2 h after removing ZP-bound spermatozoafrom the first 2 h incubation produced a significantly lowerZP-induced acrosome reaction in the second incubation (22 ±16%) than in the first incubation (30 ± 14%; P < 0.001,n = 20). There was no significant difference in the ZP-inducedacrosome reaction with oocytes with ZP which had or had notbeen penetrated by spermatozoa during the in-vitro fertilizationinsemination. Pre-incubation of spermatozoa with solubilizedZP blocked sperm-ZP binding. However, the acrosome reactioninduced by solubilized ZP (4 ZP/µl) was significantlylower than the acrosome reaction induced by intact ZP (10 ±5 and 30 ± 13% respectively, n = 11, P < 0.001), butthere was a high correlation (Spearman r = 0.822, P < 0.01)between the results. On the other hand, although the averageof the acrosome reaction was similar for A23187 [GenBank] (42%) and forZP (43%), there was no significant correlation between the resultsfor the two stimuli (n = 60). In conclusion, a useful methodfor assessing the ZP-induced acrosome reaction has been developedusing oocytes which failed to fertilize in vitro. The lack ofa relationship between the results of the chemical (A23187 [GenBank] )and physiological (ZP) stimali for the acrosome reaction inthe same subjects questions the biological basis of using A23187 [GenBank] for tests of sperm function. Solubilized human ZP in a concentrationthat blocks sperm-ZP binding is a less efficient inducer ofthe acrosome reaction than is intact ZP. It is possible thatthe three-dimensional structure of the ZP is important for inductionof the acrosome reaction or that spermatozoa which bind to theZP are more likely to acrosome react Assessment of the physiologicalacrosome reaction for diagnosis of sperm defects which interferewith the fertilization process should be concentrated on thespermatozoa which are capable of binding to the ZP.     

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.     

Effects of cryopreservation on human sperm acrosomes.

Total acrosin activity and acrosomal status were determined before and after cryopreserving human spermatozoa. Three different cryopreservation protocols were used. Both acrosin activity and the incidence of intact acrosomes decreased during cryopreservation. The magnitudes of the decreases were weakly but significantly correlated (r = 0.29, P less than 0.05), suggesting that acrosomal loss contributed to the decrease in acrosin activity. The effects of the three cryopreservation protocols were not significantly different. Motility decreased more (average 43%) than did the percentage of spermatozoa with intact acrosomes (27%) and the total acrosin activity (24%). These measurements suggested that acrosomal damage may have been secondary to cell death. This hypothesis was tested by determining the acrosomal status of spermatozoa that survived cryopreservation. Spermatozoa that were motile after thawing averaged 96% acrosome-intact; their acrosin activity, however, was significantly less than that of motile, unfrozen spermatozoa. These observations support the idea that the acrosomal loss due to cryopreservation is associated with cell death but also demonstrate decreased total acrosin activity of the acrosome-intact spermatozoa that survive cryopreservation.     

The incidence of spontaneous acrosome reaction in homogeneous populations of hyperactivated human spermatozoa.

The incidence of spontaneous acrosome reaction occurring in 1314 individually selected hyperactivated (HA) human spermatozoa was compared to that occurring in 8226 individually selected non-hyperactivated spermatozoa (non-HA) sampled over an incubation time course to allow for capacitation. Two-way analysis of variance showed a significant difference between HA and non-HA spermatozoa for the mean percent acrosome reacted (R), partially acrosome reacted (PR) and combined total (R+PR) (P < 0.001). One-way analysis showed that among the HA spermatozoa there were marked differences among the proportion showing R+PR at the various time points (P = 0.005). Using the same end point, there was no significant evidence of change with time for the non-HA spermatozoa. The overall data indicated that HA human spermatozoa have a greater propensity for spontaneous acrosomal loss than non-HA spermatozoa during incubation in synthetic culture media.     

甲氧滴滴涕对大鼠精子顶体反应的影响

目的:探讨甲氧滴滴涕(methoxychlor,MXC)对雄性大鼠精子顶体反应的影响。方法:在体外培养的精子悬液中加入不同浓度的MXC和孕酮处理,通过精子考马斯亮蓝染色,镜检计数顶体反应型精子的百分率。结果:与空白组比较,单纯经MXC处理组的顶体反应型精子百分率明显增加,另加孕酮的各处理组顶体反应型精子的百分率增加尤为明显,MXC终浓度≤6.25μg/ml的处理组顶体反应型精子百分率不增加。结论:MXC可促进大鼠精子发生顶体反应且与其剂量有关,当MXC终浓度≤6.25μg/ml不诱发顶体反应。MXC能加强孕酮诱发精子顶体反应的作用。     

The use of anticlusterin monoclonal antibodies for the combined assesment of human sperm morphology and acrosome integrity

Clusterin is an abundant protein in the human male reproductivetract which appears to be produced by the testis, epididymisand the seminal vesicles. Using monoclonal antibodies and anamplified immunoperoxidase technique, we have identified twoapparently biochemically distinct forms of clusterin on humanspermatozoa. Morphologically abnormal spermatozoa have an extensivesurface coating of conventional 80 kDa native clusterin, butthis form of clusterin is not detectable on normal spermatozoa.Normal spermatozoa, however, contain within the acrosomal capa different form of clusterin, reactive with an anticlusterina-chain antibody. Agglutinated spermatozoa, most of which aregrossly abnormal, were intensely labelled with the antibodyagainst conventional 80 kDa clusterin, suggesting that the ‘clustering’properties of this protein may play a role in the aggregationof abnormal spermatozoa. Anticlusterin monoclonal antibodiesmay be useful for semen analysis. Staining spermatozoa withanticlusterin monoclonal antibodies is a technically simplemethod which provides a visually obvious means of assessingspermatozoa morphology and acrosome status simultaneously. Thecurrent data also suggest that different functions of clusterinin the reproductive tract may be attributed to different molecularforms of the protein.