Mechanism of sperm capacitation and the acrosome reaction： role of protein kinases

Mammalian sperm must undergo a series of biochemical and physiological modifications, collectively called capacitation, in the female reproductive tract prior to the acrosome reaction （AR）. The mechanisms of these modifications are not well characterized though protein kinases were shown to be involved in the regulation of intracellular Ca2＋ during both capacitation and the AR. In the present review, we summarize some of the signaling events that are involved in capacitation. During the capacitation process, phosphatidyl-inositol-3-kinase （PI3K） is phosphorylated/activated via a protein kinase A （PKA）-dependent cascade, and downregulated by protein kinase C a （PKCa）. PKCa is active at the beginning of capacitation, resulting in PI3K inactivation. During capacitation, PKCa as well as PP172 is degraded by a PKA-dependent mechanism, allowing the activation of PI3K. The activation of PKA during capacitation depends mainly on cyclic adenosine monophosphate （cAMP） produced by the bicarbonate-dependent soluble adenylyl cyclase. This activation of PKA leads to an increase in actin polymerization, an essential process for the development of hyperactivated motility, which is necessary for successful fertilization. Actin polymerization is mediated by PIP2 in two ways： first, PIP2 acts as a cofactor for phospholipase D （PLD） activation, and second, as a molecule that binds and inhibits actin-severing proteins such as gelsolin. Tyrosine phosphorylation of gelsolin during capacitation by Src family kinase （SFK） is also important for its inactivation. Prior to the AR, gelsolin is released from PIP2 and undergoes dephosphorylation/activation, resulting in fast F-actin depolymerization, leading to the AR.

Insulin and leptin enhance human sperm motility, acrosome reaction and nitric oxide production

Aim： To investigate the in vitro effects of insulin and leptin on human sperm motility, viability, acrosome reaction and nitric oxide （NO） production. Methods： Washed human spermatozoa from normozoospermic donors were treated with insulin （10 μIU） and leptin （10 nmol）. Insulin and leptin effects were blocked by inhibition of their intracellular effector, phosphotidylinositol 3-kinase （PI3K）, by wortmannin （10 μmol） 30 min prior to insulin and leptin being given. Computer-assisted semen analysis was used to assess motility after 1, 2 and 3 h of incubation. Viability was assessed by fluorescence-activated cell sorting using propidium iodide as a fluorescent probe. Acrosome-reacted cells were observed under a fluorescent microscope using fluorescein-isothiocyanate-Pisum sativum agglutinin as a probe. NO was measured after treating the sperm with 4,5-diaminofluorescein-2/diacetate （DAF-2/DA） and analyzed by fluorescence-activated cell sorting. Results： Insulin and leptin significantly increased total motility, progressive motility and acrosome reaction, as well as NO production. Conclusion： This study showed the in vitro beneficial effects of insulin and leptin on human sperm function. These hormones could play a role in enhancing the fertilization capacity of human spermatozoa.

Acrosome reaction： relevance of zona pellucida glycoproteins

During mammalian fertilisation, the zona pellucida （ZP） matrix surrounding the oocyte is responsible for the binding of the spermatozoa to the oocyte and induction of the acrosome reaction （AR） in the ZP-bound spermatozoon. The AR is crucial for the penetration of the ZP matrix by spermatozoa. The ZP matrix in mice is composed of three glycoproteins designated ZP1, ZP2 and ZP3, whereas in humans, it is composed of four （ZP1, ZP2, ZP3 and ZP4）. ZP3 acts as the putative primary sperm receptor and is responsible for AR induction in mice, whereas in humans （in addition to ZP3）, ZP1 and ZP4 also induce the AR. The ability of ZP3 to induce the AR resides in its C-terminal fragment. O-linked glycans are critical for the murine ZP3-mediated AR. However, N-linked glycans of human ZP1, ZP3 and ZP4 have important roles in the induction of the AR. Studies with pharmacological inhibitors showed that the ZP3-induced AR involves the activation of the Gi-coupled receptor pathway, whereas ZP1- and ZP4-mediated ARs are independent of this pathway. The ZP3-induced AR involves the activation of T-type voltage-operated calcium channels （VOCCs）, whereas ZP1- and ZP4-induced ARs involve both T- and L-type VOCCs. To conclude, in mice, ZP3 is primarily responsible for the binding of capacitated spermatozoa to the ZP matrix and induction of the AR, whereas in humans （in addition to ZP3）, ZP1 and ZP4 also participate in these stages of fertilisation.

The human acrosome reaction

We developed tests of sperm-oocyte interaction: sperm-zona binding, zona-induced acrosome reaction, sperm-zona penetration and sperm-oolemma binding, using oocytes which failed to fertilise in clinical in vitro fertilization(IVF). Although oocyte defects contribute to failure of sperm oocyte interaction, rarely are all oocytes from one wom-an affected. Low or zero fertilization in standard IVFwas usually caused by sperm abnormalities. Poor sperm-zona pel-lucida binding was frequently associated with failure of standard IVF and obvious defects of sperm motility or morpholo-gy. The size and shape of the acrosome is particularly important for sperm binding to the oocyte. The proportion ofacrosome intact sperm in the insemination medium was related to the IVF rote. Inducing the acrosome reaction with acalcium ionophore reduced sperm-zona binding. Blocking acrosome dispersal with an acrosin inhibitor prevented sperm-zona penetration. Sperm-zona penetration was even more highly related to IVF rates than was sperm-zona binding.Some patients had low or zero fertilization rates with standard IVF but normal sperm by conventional tests and normalsperm-zona binding. Few of their sperm underwent the acrosome reaction on the surface of the zona and none penetrat-ed the zona. In contrast, fertilization and pregnancy rates were high with intmcytoplasmic sperm injection. We call thiscondition defective zona pellucida induced acrosome reaction. Discovery of the nature of the abnormalities in the signaltmnsduction and effector pathways of the human zona pellucida induced acrosome reaction should result in simpler testsand treatments for the patients and also provide new leads for contraceptive development.

F^ole and regulation of EGFR in actin remodeling in sperm ：apacitation and the acrosome reaction

To bind and fertilize the egg, the spermatozoon should undergo few biochemical and motility changes in the female reproductive tract collectively called capacitation. The capacitated spermatozoon binds to the egg zona pellucida, and then undergoes the acmsome reaction （AR）, which allows its penetration into the egg. The mechanisms regulating sperm capacitation and the AR are not completely understood. In the present review, we summarize some data regarding the role and regulation of the epidermal growth factor receptor （EGFR） in these processes. In the capacitation process, the EGFR is partially activated by protein kinase A （PKA）, resulting in phospholipase D （PLD） activation and actin polymerization. Protein kinase C alpha （PKCα）, which is already activated at the beginning of the capacitation, also participates in PLD activation. Further activation of the EGFR at the end of the capacitation enhances intracellular Ca2＋ concentration leading to F-actin breakdown and allows the AR to take place. Under in vivoconditions, the EGFR can be directly activated by its known ligand epidermal growth factor （EGF）, and indirectly by activating PKA or by transactivation mediated by G protein-coupled receptors （GPCRs） activation or by ouabain. Under physiological conditions, sperm PKA is activated mainly by bicarbonate, which activates the soluble adenylyl cyclase to produce cyclic adenosine monophosphate （cAMP）, the activator of PKA. The GPCR activators angiotensin II or Ivsoohosphatidic acid, as well as ouabain and EGF are phvsioloeical comoonents oresent in the female reoroductive tract.

Clinical pregnancies and livebirths achieved by intracytoplasmic injection of round headed acrosomeless spermatozoa with and without oocyte activation in familial globozoospermia： case report

We report the successful outcome of intracytoplasmic sperm injection （ICSI） treatment in two siblings with familial globozoospermia. After controlled ovarian hyperstimulation and oocyte pick-up, retrieved oocytes were mechanically activated before ICSI and a fertilization rate of 33.3% was achieved in the first case. The second couple underwent ICSI without oocyte activation and a 9.1% fertilization rate was obtained. The transfer of two grade I embryos in the first couple and one grade I embryo in the second couple resulted in clinical pregnancies with healthy livebirths. It was concluded that the main problem of cases with globozoospermia is a low fertilization rate, and even though ICSI and oocyte activation can increase this rate it is not necessarily needed to achieve a pregnancy. （Asian J Androl 2008 Mar; 10： 332-336）

Carbohydrates mediate sperm-ovum adhesion and triggering of the acrosome reaction

The fertilization process is the net result of a complex sequence of events that collectively result in the fusion of theopposite gametes. The male gamete undergoes continuous morphological and biochemical modifications during spermdevelopment in the testis (spermatogenesis), maturation in the epididymis, and capacitation in the female reproductivetract. Only the capacitated spermatozoa are able to recognize and bind to the bioactive glycan residue(s) on the ovum'sextracellular coat, the zona pellucida (ZP). Sperm-zona binding in the mouse and several other species is believed totake place in two stages. First, capacitated (acrosome-intact) spermatozoa loosely and reversibly adhere to the zona-in-tact ovum. In the second stage tight irreversible binding occurs. Both types of bindings are attributed to the presence ofglycan- binding proteins (receptors) on the sperm plasma membrane and their complementary bioactive glycan units(ligands) on the surface of the ZP. The carbohydrate-mediated adhesion event initiates a signal transduction cascade re-sulting in the exocytosis of acrosomal contents. This step is believed to be prerequisite which allows the hyperactivatedacrosome-reacted spermatozoa to penetrate the ZP and fertilize the ovum. This review focuses on the role of carbohy-drate residues in sperm-ovum interaction, and triggering of the acrosome reaction. I have attempted to discuss extensiveprogress that has been made to enhance our understanding of the well programmed multiple molecular events necessaryfor successful fertilization. This review will identify these events, and discuss the functional significance of carbohy-drates in these events.

Inhibition of mouse acrosome reaction and sperm-zona pellucida binding by anti-human sperm membrane protein 1 antibody

Aim： To investigate the possible functions of human sperm membrane protein （hSMP-1） in the process of fertilization. Methods： A 576-bp cDNA fragment of HSD-1 gene coding for the extracellular domain of hSMP-1 was cloned and expressed. The localization of this protein on human and mouse sperm was determined by indirect immunofluorescent staining by using anti-recombinant hSMP-1 （anti-rhSMP-1） antibodies. Sperm acrosome reaction and spermzona pellucida （ZP） binding assay were carried out in 10-week-old BALB/c mice. Results： Recombinant hSMP-1 was successfully cloned and expressed. The expression of the native protein was limited on the acrosome of human and mouse sperm. Treatment of anti-rhSMP-1 antibodies significantly decreased the average number of sperms bound to each egg. Meanwhile, the percentage of acrosome reaction was decreased in comparison to pre-immune control after treatment with anti-rhSMP-1 （P 〈 0.05）. Conclusion： The results suggest that anti-rhSMP-1 antibody inhibited mouse acrosome reaction and sperm-ZP binding.

Evaluation on Sperm Acrosome Integrity of Infertile Men with Varicocele

Objective To determine the sperm acrosome integrity of samples from infertile men with varicocele. Methods Forty-nine infertile men with varicocele were divided into three groups according to the grade of varicocele. Group A （grade Ⅰ）, B （grade Ⅱ）, and C （grade Ⅲ） consisted of 15, 18, and 16 cases, respectively. Besides, 15 semen samples from normospermic donors were used as the control. The acrosome integrity of sperm was examined with fluorescein-labeled Pisum sativum agglutinin. Acrosomal ultrastructure was observed with transmission electron microscopy. Results In three varicocele groups, most samples had high sperm abnormal morphology rates. There were significant differences in acrosome integrity rates between each varicocele group and the control （P〈0.01）. Group C had the lowest acrosome integrity rate among the three groups. Ultrastructural observation showed that acrosome malformations revealed acrosomal membranes defects, swelling, hypoplasia, and dissolution of the matrix. Conclusions Infertile men with varicocele had low level of acrosome integrity. Severe varicocele for infertile men might be associated with severe acrosomal defects. Evaluating sperm acrosome should aid the understanding of the sperm structural state and benefit the treatment for infertile men.

Destabilization of acrosome and elastase influence mediate the release of secretory phospholipase A2 from human spermatozoa

Aim： To determine the cellular distribution of secretory phospholipase A2 （sPLA2） in dependence on the acrosomal state and under the action of elastase released under inflammatory processes from leukocytes. Methods： Acrosome reaction of spermatozoa was triggered by calcimycin. Human leukocyte elastase was used to simulate inflammatory conditions. To visualize the distribution of sPLA2 and to determine the acrosomal state, immunofluorescence techniques and lectin binding combined with confocal laser scanning fluorescence microscopy and flow cytometry were used. Results： Although sPLA2 was detected at the acrosome and tail regions in intact spermatozoa, it disappeared from the head region after triggering the acrosome reaction. This release of sPLA2 was associated with enhanced binding of annexin V-fluoroscein isothiocyanate （FITC） to spermatozoa surfaces, intercalation of ethidium-homodimer I, and binding of FITC-labelled concanavalin A at the acrosomal region. Spermatozoa from healthy subjects treated with elastase were characterized by release of sPLA2, disturbance of acrosome structure, and loss of vitality. Conclusion： The ability of spermatozoa to release secretory phospholipase A2 is related to the acrosomal state. Premature destabi- lization of the acrosome and loss of sPLA2 can occur during silent inflammations in the male genital tract. The distribution pattern of sPLA2 in intact spermatozoa might be an additional parameter for evaluating sperm quality.

Flotillin-2 is an acrosome-related protein involved in mouse spermiogenesis

Spermatogenesis is a complex process of terminal differentiation by which mature sperms are generated,and it can be divided into three phases：mitosis,meiosis and spermiogenesis.In a previous study,we established a series of proteomic profiles for spermatogenesis to understand the regulation of male fertility and infertility.Here,we further investigated the localization and the role of flotillin-2 in spermiogenesis.Flotillin-2 expression was investigated in the testis of male CD1 mice at various developmental stages of spermatogenesis by using Western blotting,immunohistochemistry and immunofluorescence.Flotillin-2 was knocked down in vivo in three-week-old male mice using intratesticular injection of small inhibitory RNA（siRNA）,and sperm abnormalities were assessed three weeks later.Flotillin-2 was expressed at high levels in male germ cells during spermatogenesis.Flotillin-2 immunoreactivity was observed in pachytene spermatocytes as a strong dot-shaped signal and in round spermatids as a sickle-shaped distribution ahead of the acrosome.Immunofluorescence confirmed flotillin-2 was localized in front of the acrosome in round spermatids,indicating that flotillin-2 was localized to the Golgi apparatus.Knockdown of flotillin-2 in vivo led to a significant increase in head sperm abnormalities isolated from the cauda epididymis,compared with control siRNA-injected testes.This study indicates that flotillin-2 is a novel Golgi-related protein involved in sperm acrosome biogenesis.

Desmosterol, the main sterol in rabbit semen： distribution among semen subfractions and its role in the in vitro spermatozoa acrosome reaction and motility

Sterols are essential components of the cell membrane lipid bilayer that include molecules such as cholesterol and desmosterol, which are significantly found in the spermatozoa of several animal species. However, the presence of desmosterol in rabbit semen has never been investigated. The aims of this study were to characterize the sterol composition of subfractions of ejaculated rabbit semen and evaluate the in vitro effects of sterol on the spermatozoa acrosome reaction and motility. Two sterols, occurring prevalently in the free form （94.3%）, were identified in whole semen collected from 10 fertile New Zealand White rabbits, specifically desmosterol （58.5% of total sterols） and cholesterol （35.9% of total sterols）. Desmosterol was the predominant sterol found in all subfractions of rabbit semen, varying from 56.7% （in the prostatic secretory granules, PSGs） to 63.8% （in the seminal plasma）. Spermatozoa contained an intermediate proportion of desmosterol （59.8%）, which was asymmetrically distributed between the heads （52.0% of the total content of sterols） and the tails （81.8%）. Results showed that both desmosterol and cholesterol can be transferred from the PSGs to the spermatozoa and are equally effective in inhibiting in vitro spermatozoa capacitation at a concentration higher than 1 mg L^-1. In contrast, neither desmosterol nor cholesterol had a significant effect on spermatozoa motility. Thus, it was concluded that, the various fractions of rabbit seminal fluid differ from each other in sterol composition and quantity, probably due to their different functional properties, and these fractions may undergo significant sterol changes depending on the stage of spermatozoa capacitation.

Effect of genistein on acrosome reaction and zona pellucida binding independent of protein tyrosine kinase inhibition in bull

Aim： To investigate if the phytoestrogen, genistein, affects essential functions of cryopreserved bovine spermatozoa. Methods： The effect of genistein upon motility was assessed by computer-assisted motion analysis. Hemizona assay was performed to detect the ability of spermatozoa binding to the zona pellucida. The inducibility of the acrosome reaction using progesterone and ZP3-6 peptide was analysed by fluorescein-conjugated Pisum sativum agglutinin （FITC-PSA）/Hoechst 33258 double staining. Capacitation after incubation with genistein was assessed by the chlortetracycline （CTC） assay. Immunoblots showed the pattern of protein tyrosine phosphorylation of cryopreserved bovine spermatozoa. Results： Immunodetection of tyrosine-phosphorylated proteins showed that genistein did not affect tyrosine phosphorylation in cryopreserved bovine spermatozoa. However, genistein significantly reduced the progesterone- and ZP3-6 peptide-mediated induction of the acrosome reaction and led to a dose-dependent inhibition of sperm-zona pellucida binding; while sperm motility and capacitation were not affected by this phytoestrogen, as indicated by computer-assisted sperm motion analysis and the CTC assay, respectively. Conclusion： Our results suggest that in cryopreserved bovine spermatozoa, genistein affects a protein tyrosine phosphorylation-independent signal transduction pathway that is involved in sperm capacitation, the acrosome reaction and sperm-zona pellucida binding.

Role of ions and ion channels in capacitation and acrosome reaction of spermatozoa

Capacitation and acrosome reaction are important prerequisites of the fertilization process. Capacitation is a highlycomplex phenomenon occurring in the female genital tract, rendering the spermatozoa capable of binding and fusionwith the oocyte. During capacitation various biochemical and biophysical changes occur in the spermatozoa and thespermatozoal membranes. Ions and ion channels also play important roles in governing the process of capacitation bychanging the fluxes of different ions which in turn controls various characteristics of capacitated spermatozoa. Alongwith the mobilization of ions the generation of free radicals and efflux of cholesterol also plays an important role in thecapacitation state of the spermatozoa. The generation of free radical and efflux of cholesterol change the mechano-dynamic properties of the membrane by oxidation of the polyunsaturated lipids and by generating the cholesterol freepatches. The process of capacitation renders the spermatozoa responsive to the inducers of the acrosome reaction. Theglycoprotein zona pellucida 3 (ZP3) of the egg coat zona pellucida is the potent physiological stimulator of the acro-some reaction; progesterone, a major compoent of the follicular fluid, is also an induce of the acrosome reaction.The inducers of the acrosome reaction cause the activation of the various ion-channels leading to high influxes of calci-um, sodium and bicarbonate. The efflux of cholesterol during the process of capacitation alters the permeablity of themembrane to the ions and generate areas which are prone to fusion and vesculation process during the acrosome reac-tion. Ths review focuses mainly on effects of the ion and ion-channels, free radicals, and membrane fluidity changesduring the process of capacitation and acrosome reaction. (Asian J Androl 1999 Sep; 1: 95-107)

Effect of NO on spontaneous acrosome reaction in AsAb positive rat spermatozoa

Objective: To explore the effect of NO on the spontaneous acrosome reaction in antisperm antibody (AsAb) positive rat spermatozoa. Methods: The rat model of AsAb was set up by artificial immunization. The level of AsAb in blood serum was determined by TAT and ELISA. Rat spermatozoa was visualized by staining the acrosome with Coomassie brilliant blue. The NO concentration in rat spermatozoa was assayed by HPLC. Results: The percentage of acrosome reaction, NO concentration, superoxide dismutase (SOD) and Na^+-K^+ATPase activity in AsAb positive rat spermatozoa were significantly decreased compared with the control group. Low dose of NO (SNP 10^(-9)～10^(-8) mol/L) increased the percentage of acrosome reaction and SOD activity, but had no effect on Na^+-K^+ATPase activity. High dose of NO (SNP 10^(-6)～10^(-4) mol/L) decreased the three items. Conclusion: The decrease in acrosome reaction in positive AsAb rat spermatozoa might be related to a decrease in NO and increase in O_2. (the SOD activity was decreased) in sperm. A low dose of NO might increase the percentage of acrosome reaction in AsAb positive rat by inhibiting the superoxide, while a high dose of NO was harmful to sperm function. [Reprod Contracep (in Chinese) 2002; 22: 203]

Roles of tumor necrosis factor alpha on sperm acrosin activity and acrosome reaction

Aim: To study the roles of tumor necrosis factor alpha (TNF-α) on the sperm acrosin activity and acrosome reaction. Methods: The sperm acrosin activity was tested by the method of BAEE/ ADH Unity and the acrosome reaction by the Triple-stain technique. Results: TNF-α decreased the sperm acrosin activity and acrosome reaction (P<0.01; P<0.01, respectively); it also inhibited the Ca2+-ATPase activity and SOD activity in sperm (P< 0.05; P<0.001, respectively), but increased the NOS activity and the amount of NO in sperm (P<0.001; P<0.001, respectively). While it had a less significant effect on the activity of Na+-K+-ATPase (P>0.05). Conclusion: TNF-α inhibits the sperm acrosin activity and acrosome reaction.

Effects of penicillamine, hypotaurine, and epinephrine on motility, hyperactivity, acrosome reaction of fresh ram sperm

Objective:To determine the effects of different concentrations of penicillamine, hypotaurine, and epinephrine (PHE) as well as incubation time on motility, hyperactivity and acrosome reaction (AR) of ram spermin vitro.Methods: Freshly ejaculated spermatozoa from three ram were collected, pooled and subjected to swim up technique in modified sperm Tyrode's albumin lactate pyruvate medium supplemented with different concentrations of PHE (10, 20, 30, 40, 50, 75 and 100 mM/mL). Then best concentrations were compared and examined for motility, hyperactivity and AR.Results: A high concentrations of PHE (30, 40, 50, 75 and 100mM/mL) showed a significant increase in motility when compared to control immediately after dilution and exist for the first and second hour of incubation period. However, when longer incubation time were used, a significant (P<0.05) decrease in motility was achieved. Similar finding was observed in hyperactivity. Stained semen samples showed a maximum percentage of incomplete AR after 1 h incubation corresponding to 50 and 75 mM/mL of PHE;however, spermatozoa treated with 75 mM/mL had a higher tendency to undergo complete AR after further incubation up to 4 h. A dose dependent relationship was detected where the maximum value of total AR was shown in spermatozoa treated with 75 mM/mL PHE for 4 h. Conclusions: To obtain better motility, hyperactivity and AR, treatment of ram spermatozoa with 75 mM/mL PHE for 4 h before being used in insemination was considered the best concentration of PHE to be used in the process ofin-vitro fertilization.

Effect of GABA on sperm acrosome reation in antisperm antibody positive patients

To investigate the effect of gamma-aminobutyric acid (GABA) on the rate of sperm acrosome reaction both in normal and antisperm antibody (AsAb) positive men. Methods: The sperm acrosome reaction was tested with triplestain technique in two groups of 18 men each. Results: (1) GABA increased the rate of sperm acrosome reaction both in normal and AsAb positive subjects (P<0.01); (2) GABA increased the Na^+-K^+-ATPase activity of sperm (P<0.01); (3)GABA increased the Ca^(2+)-ATPase activity of sperm (P<0.05); (4) GABA decreased the production of MDA and oxygen free radicals of sperm. Conclusion: GABA could regulate the rate of sperm acrosome reaction. (Chin J Andro12002; 16: 355)

The Relationship between Some lons and Acrosome Reaction of Human Sperm

The acrosome reaction of sperm was induced by calcium ionophore A 23187.The letionship between some ions and acrosome reaction by removing Na+ from the medium.or by adding angtagonist of K+.TEA chloride,or antagonist of Ca++,verapamil,or anlagonist of Na+-K+-ATPase,acetyl strophanthithidin is studied.The results show that Na+,H,Ca++ and Na+ pump are necessary.for acrosome reaclion of human sperm.The Ca++ might not enter the sperms through the channel of Ca++.

Effects of ubiquitin-protea-some pathway on mouse sperm capacitation, acrosome reaction and in vitro fertilization

Chlortetracycline (CTC) fluorescence patterns were used to study changes in the patterns B and AR of mouse sperm after incubation with reagents that would block the UPP. They were the monoclonal antibody againstubiquitinated proteins——UCPi; the polyclonal antibodyagainst ubiquitin-anti-Ub, and a special inhibitor againstproteasome——ALLN. Furthermore, we treated the capaci-tated sperm or the eggs with these reagents separately and tested whether the normal in vitro fertilization was blocked or not. Results illustrate that UCP1, anti-Ub, and ALLN have little effects on sperm capacitation and acrosome reaction, but they do inhibit fusion of mouse sperm with eggs, which suggests that UPP play an important role in mouse in vitro fertilization.