Zona Pellucida-induced Acrosome Reaction Research Articles

Progesterone Accelerates the Completion of Sperm Capacitation and Activates CatSper Channel in Spermatozoa from the Rhesus Macaque.

During transit through the female reproductive tract, mammalian spermatozoa are exposed to increasing concentrations of progesterone (P4) released by the cumulus oophorus. P4 triggers massive calcium influx into human sperm through activation of the sperm-specific calcium channel CatSper. These properties of human spermatozoa are thought to be unique since CatSper is not progesterone sensitive in rodent sperm. Here, by performing patch clamp recording from spermatozoa from rhesus macaque for the first time, we report that they express P4-sensitive CatSper channel identically to human sperm and react to P4 by inducing responsiveness to zona pellucida, unlike human sperm, which respond directly to P4. We have also determined the physiologic levels of P4 capable of inducing capacitation-associated changes in macaque sperm. Progesterone (1 μM) induced up to a 3-fold increase in the percentage of sperm undergoing the zona pellucida-induced acrosome reaction with the lowest threshold as low as 10 nM of P4. Submicromolar levels of P4 induced a dose-dependent increase in curvilinear velocity and lateral head displacement, while sperm protein tyrosine phosphorylation was not altered. Macaque spermatozoa exposed to 10 μM of P4 developed fully hyperactivated motility. Similar to human sperm, on approaching cumulus mass and binding to zona pellucida, macaque spermatozoa display hyperactivation and undergo an acrosome reaction that coincides with the rise in the sperm intracellular calcium. Taken together, these data indicate that P4 accelerates the completion of capacitation and provides evidence of spermatozoa "priming" as they move into a gradient of progesterone in search for the oocyte.

Heparin and Heparin Binding Proteins: Potential Relevance to Reproductive Physiology

Glycosaminoglycans (GAGs) have crucial roles in cell-cell interaction and communication. The communication between sperm and egg during fertilization is the finest example of intercellular communication involving a protein-carbohydrate recognition system. GAGs, especially heparin, are implicated in various processes, such as capacitation, acrosome reaction (AR), and sperm nuclei decondensation by interacting with a wide range of proteins, leading to fertilization. Seminal plasma (SP) comprises of multiple proteins that bind to heparin and related GAGs. Heparin binding proteins (HBPs) originating from secretions of the male accessory sex glands are known to play a vital role during fertilization events. They interact with GAGs present in the female genital tract and enhance the subsequent zona pellucida-induced AR, and thus have been correlated with fertility in some species. Several carbohydrate and zona pellucida-binding proteins, many of which belong to the spermadhesin family, are identified as HBPs. Many studies have been documented about the potential physiological role of some HBPs in various steps of fertilization. However, there is insufficient knowledge about functions executed by various HBPs and their exact mechanism and pathways. An in-depth knowledge of HBPs and their role in fertilization is of fundamental importance to resolve biological pathways and protein interactions at the molecular level. This review surveys some of the relevant findings supporting the potential role of heparin and HBPs in reproduction. It also describes consensus heparin binding sites emerging from known literature on HBPs that interact with heparin.

Quinn’s advantage fertilization medium enhances zona pellucida-induced acrosome reaction compared with human tubal fluid medium

The aim of this study was to compare sperm velocity, hyperactivation, zona pellucida (ZP) binding and ZP-induced acrosome reaction (AR) between Quinn’s advantage fertilization (QAF), human tubal fluid (HTF) and Ham’s F10 media. Semen samples were obtained from normozoospermic men and motile spermatozoa were prepared by gradient centrifugation (PureSperm). Unfertilized oocytes from clinical IVF were used for spermatozoa–oocyte interaction tests. Sperm velocity and hyperactivation were assessed using a Hamilton-Thorn motility analyser. When media were supplemented with human albumin, sperm motility and velocity and sperm binding were not significantly different between QAF and HTF. However, ZP-induced AR was significantly higher with QAF than HTF (42 ± 22 versus 21 ± 18, P < 0.001). Sperm velocity, hyperactivation and sperm binding were also significantly higher in QAF than Ham’s F10 media. Supplementation of media with either human serum or human albumin showed no difference in effect on all sperm test results. In conclusion, QAF medium significantly enhances ZP-induced AR which is essential for sperm penetration. Thus QAF appears to be a better medium than HTF for sperm fertilizing ability in conventional IVF. The aim of this study was to compare sperm velocity, hyperactivated motility, sperm binding and zona pellucida (ZP)-induced acrosome reaction (AR) between Quinn’s advantage fertilization (QAF) and human tubal fluid (HTF) and Ham’s F10 media. Semen samples were obtained from men with normal semen analysis results and all test results were compared in paired control samples between different media. When media were supplemented with human albumin, sperm motility, velocity and sperm binding were not significantly different between QAF and HTF. However, ZP-induced AR was significantly higher with QAF than HTF. Sperm velocity, hyperactivated motility and sperm binding were all significantly higher in QAF than Ham’s F10 media. Supplementation with either human serum or human albumin in media had no different effects on all sperm test results. In conclusion, Quinn’s advantage fertilization medium significantly enhances ZP-induced AR, which is essential for sperm penetration. Thus QAF appears to be a better medium than HTF for sperm fertilizing ability in conventional IVF.

TRPM8 in mouse sperm detects temperature changes and may influence the acrosome reaction

Changes in the concentration of intracellular Ca(2+) ([Ca(2+) ]i) trigger and/or regulate principal sperm functions during fertilization, such as motility, capacitation, and the acrosome reaction (AR). Members of the large TRP channel family participate in a variety of Ca(2+) -dependent cell signaling processes. The eight TRPM channel members constitute one of the seven groups belonging to this family. Here we document using RT-PCR experiments the presence of Trpm2, 4, 7, and 8 in mouse spermatogenic cells. Trpm8 transcription is up-regulated after day 30. The localization of TRPM8 protein in mouse sperm was confirmed by immunocytochemistry and Western blots. Patch clamp recordings in testicular mouse sperm revealed TRPM8 agonist (menthol and icilin) activated currents sensitive to TRPM8 inhibitors N-(4-t-Butylphenyl)-4-(3-Chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide (BCTC) and capsazepine. These findings are consistent with the presence of functional TRPM8 in mouse sperm. Furthermore, menthol induced a [Ca(2+) ]i increase and the AR in these cells, that were inhibited by capsazepine (20 µM) and BCTC (1.6 µM). Notably, the progesterone and zona pellucida-induced AR was significantly (>40%) inhibited by BCTC and capsazepine, suggesting the possible participation of TRPM8 channels in this reaction. TRPM family members present in sperm could be involved in other important signaling events, such as thermotaxis, chemotaxis, and mechanosensory transduction.

Acrosome Reaction Induced by Zona Pellucida Is Mediated by Alpha-7 Nicotinic Acetylcholine and Epidermal Growth Factor Receptors.

In order to penetrate into the egg, the capacitated spermatozoon should bind to the zona pellucida (ZP) and undergo the acrosomal reaction. The acrosome reaction is mediated by receptor activation that leads to calcium influx into the sperm. In the present study we showed that isolated ZP activates sperm alpha7-nicotinic-achetylcholine receptor (á7nAChR) leading to the trans-activation of epidermal-growth-factor-receptor (EGFR), calcium influx and the acrosome reaction. The data revealed that alpha7nAChR agonist initiates the acrosome reaction which was inhibited by EGFR-antagonist, suggesting that EGFR mediates this reaction. Moreover, the ZP induced acrosome reaction was significantly inhibited by alpha-bungarotoxin (alpha7 antagonist) and by tyrphostin (EGFR antagonist), suggesting that EGFR and the alpha7 subunit are activated by ZP. Furthermore, ZP or EGF did not induce the acrosome reaction in sperm from alpha7 null mice, suggesting that ZP-induced acrosome raction depends on the existance of sperm alpha7 and mediated by EGFR. Inhibition of alpha7 in wild type sperm revealed only partial inhibition of the acrosome reaction induced by EGF, supporting the idea that the alpha7nAChR is localized up-stream to the EGFR. Recent studies suggest that Src kinase can activate EGFR in sperm. It was also shown that Src co-localized with the alpha7 in human sperm. We showed here that Src inhibitor blocked the alpha7-dependent acrosome reaction and EGFR-phosphorylation suggesting that SRC-family mediate these processes. Moreover, EGFR phosphorylation on tyrosine-845 induced by ZP is also inhibited by SRC-family inhibitor suggesting that this kinase mediate the ZP-induced EGFR phosphorylation. It was recently shown in our lab that EGFR activation leads to calcium influx. Here we showed that agonist of the alpha7, EGFR or ZP induced calcium influx that leads to the acrosome reaction. However, in alpha7 null mice, these agonists did not induce calcium influx or acrosome reaction, and this blockade could be bypassed using calcium ionophore supporting the role of alpha7 in calcium influx induced by ZP. In conclusion we suggest that activation of alpha7 by ZP leads to SRC-family dependent-EGFR activation, Ca2+ influx and the occurrence of the acrosome reaction. (poster)

Zona pellucida-induced acrosome reaction in human spermatozoa is potentiated by glycodelin-A via down-regulation of extracellular signal-regulated kinases and up-regulation of zona pellucida-induced calcium influx

Glycodelin-A interacts with spermatozoa before fertilization, but its role in modulating sperm functions is not known. Zona pellucida-induced acrosome reaction is crucial to fertilization and its dysfunction is a cause of male infertility. We hypothesized that glycodelin-A, a glycoprotein found in the female reproductive tract, potentiates human spermatozoa for zona pellucida-induced acrosome reaction. Glycodelin isoforms were immunoaffinity purified. The sperm intracellular cAMP concentration, protein kinase-A (PKA) and extracellular signal-regulated kinase (ERK) activities, and intracellular calcium were measured by ELISA, kinase activity assay kits and Fluo-4AM technique, respectively. The phosphorylation of inositol 1,4,5-trisphosphate type-1 receptor (IP3R1) mediated by ERK was determined by western blotting. Zona pellucida-induced acrosome reaction was detected by Pisum sativum staining. Pretreatment of spermatozoa with glycodelin-A significantly up-regulated adenylyl cyclase/PKA activity and down-regulated the activity of ERK and its phosphorylation of IP3R1, thereby enhancing zona pellucida-induced calcium influx and zona pellucida-induced acrosome reaction. Glycodelin-F or deglycosylated glycodelin-A did not have these actions. Treatment of spermatozoa with a protein kinase inhibitor abolished the priming activity of glycodelin-A, whilst ERK pathway inhibitors mimic the stimulatory effect of glycodelin-A on zona pellucida-induced acrosome reaction. Glycodelin-A in the female reproductive tract sensitizes spermatozoa for zona pellucida-induced acrosome reaction in a glycosylation-specific manner through activation of the adenylyl cyclase/PKA pathway, suppression of extracellular signal-regulated kinase activation and up-regulation of zona pellucida-induced calcium influx. The action of glycodelin-A may be important in vivo to ensure full responsiveness of human spermatozoa to the zona pellucida.

Effects of glycodelins on functional competence of spermatozoa

Glycodelin is a glycoprotein with 4 known glycoforms, namely glycodelin-S, glycodelin-A, glycodelin-F and glycodelin-C. The glycoforms are present in the female reproductive tract which the spermatozoa must pass through before fertilizing the oocyte. Thus the spermatozoa interact with each of these glycoforms in succession. The glycoforms have different effects on sperm function. Glycodelin-S in the seminal plasma suppresses albumin-induced cholesterol efflux from the spermatozoa and thereby regulates the initiation of capacitation. Glycodelin-A in the oviductal fluid suppresses extracellular signal-regulated kinase making the spermatozoa more sensitive to zona pellucida-induced acrosome reaction. Follicular fluid glycodelin-F suppresses progesterone-induced acrosome reaction and prevents premature acrosome reaction. Glycodelin-A and glycodelin-F also inhibit spermatozoa-zona pellucida binding by interacting with sperm surface fucosyltransferase-5, which also binds to zona pellucida glycoproteins. The physiological implication of this phenomenon remains to be determined. The inhibitory activities of glycodelin-A and glycodelin-F on spermatozoa-zona pellucida binding is removed by glycodelin-C in the cumulus matrix. Glycodelin-C not only displaces sperm-bound glycodelin-A and glycodelin-F, it also enhances spermatozoa-zona pellucida binding. These different biological activities of the glycodelin isoforms are determined by glycosylation of the proteins. Deglycosylation abolishes the binding and therefore the action of the glycodelins on spermatozoa. Knowledge of the mechanism of actions of glycodelins may enable development of novel strategies for fertility regulation.

Release of DEFB126 from macaque sperm and completion of capacitation are triggered by conditions that simulate periovulatory oviductal fluid

Capacitation of macaque sperm in vitro has been achieved efficiently only with the addition of both cyclic nucleotides and methylxanthines. The use of these exogenous sperm activators clouds an understanding of the normal mechanisms underlying capacitation and may slow early embryo development following in vitro fertilization (IVF). We demonstrate that culture medium which simulates periovulatory oviductal fluid with respect to bicarbonate (HCO(3)(-)) and glucose concentration induces capacitation in a high percentage of macaque sperm as determined by the ability of sperm to undergo both the release of coating protein DEFB126 and the zona pellucida-induced acrosome reaction (AR). Few sperm were able to undergo the AR following 6 hr incubation in medium containing either 35 mM HCO(3)(-) (approximately 7.2 pH) or 90 mM HCO(3)(-) (approximately pH 7.8) with 5 mM glucose. When glucose concentration was lowered to 0.5 mM to match levels reported for women at midcycle, the AR rate increased significantly in sperm incubated in both levels of HCO(3)(-), indicating that glucose interferes with sperm responsiveness to increasing HCO(3)(-) concentration observed in the primate oviduct during ovulation. Even greater synchronization of capacitation could be achieved with nonphysiologic extremes of alkalinity or energy substrate deprivation. In the latter case, sperm achieved high rates of IVF. A shift in pH from 7.2 to 7.8 in a HEPES-buffered medium was sufficient to remove DEFB126 from the surface of most sperm after only 3 hr. The loss of DEFB126 from sperm under periovulaory fluid conditions has implications for the timing of release of sperm from the oviductal reservoir.

Mouse Sperm Lacking Serine Protease Prss21/Tesp5 Barely Undergo the Zona Pellucida-Induced Acrosome Reaction, but Do Fertilize the Egg.

Mouse Sperm Lacking Serine Protease Prss21/Tesp5 Barely Undergo the Zona Pellucida-Induced Acrosome Reaction, but Do Fertilize the Egg.

Involvement of Cystic Fibrosis Transmembrane Conductance Regulator in Mouse Sperm Capacitation

Mammalian sperm acquire fertilizing ability in the female tract during a process known as capacitation. In mouse sperm, this process is associated with increases in protein tyrosine phosphorylation, membrane potential hyperpolarization, increase in intracellular pH and Ca2+, and hyperactivated motility. The molecular mechanisms involved in these changes are not fully known. Present evidence suggests that in mouse sperm the capacitation-associated membrane hyperpolarization is regulated by a cAMP/protein kinase A-dependent pathway involving activation of inwardly rectifying K+ channels and inhibition of epithelial sodium channels (ENaCs). The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that controls the activity of several transport proteins, including ENaCs. Here we explored whether CFTR is involved in the regulation of ENaC inhibition in sperm and therefore is essential for the capacitation-associated hyperpolarization. Using reverse transcription-PCR, Western blot, and immunocytochemistry, we document the presence of CFTR in mouse and human sperm. Interestingly, the addition of a CFTR inhibitor (diphenylamine-2-carboxylic acid; 250 microM) inhibited the capacitation-associated hyperpolarization, prevented ENaC closure, and decreased the zona pellucida-induced acrosome reaction without affecting the increase in tyrosine phosphorylation. Incubation of sperm in Cl- -free medium also eliminated the capacitation-associated hyperpolarization. On the other hand, a CFTR activator (genistein; 5-10 microM) promoted hyperpolarization in mouse sperm incubated under conditions that do not support capacitation. The addition of dibutyryl cyclic AMP to noncapacitated mouse sperm elevated intracellular Cl-. These results suggest that cAMP-dependent Cl- fluxes through CFTR are involved in the regulation of ENaC during capacitation and thus contribute to the observed hyperpolarization associated with this process.

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

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. 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. 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. 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.

Recovery of human sperm motility and ability to interact with the human zona pellucida after more than 28 years of storage in liquid nitrogen

Six donor semen samples were evaluated after 28 years cryopreservation in liquid nitrogen. The results showed that the samples retained good postthaw motility recovery and normal levels of binding to the human zona pellucida and that four of the five samples tested also gave normal levels of zona-induced acrosome reaction. In conclusion, human sperm can survive very long-term storage, which is pertinent information for clinicians referring boys and young men for sperm banking before chemotherapy.

Phospholipase Cδ4 Associates with Glutamate Receptor Interacting Protein 1 in Testis

We reported previously that phospholipase C (PLC) delta4 is required for calcium mobilization in the zona pellucida-induced acrosome reaction in sperm. Here we focused on the function of the C2 domain of PLCdelta4 and report that glutamate receptor-interacting protein1 (GRIP1) was identified as a binding protein of the PLCdelta4-C2 domain on yeast two-hybrid screening. Physiological interaction of GRIP1 with PLCdelta4 in mouse testis was confirmed by immunoprecipitation with anti-PLCdelta4 antibodies and the association seemed to correlate with the maturation stage of sperm. We also determined that a PDZ-binding motif at the C-terminus of the PLCdelta4-C2 domain is responsible for GRIP1 binding, whereas the sixth or seventh PDZ domain of GRIP1 is essential and sufficient for association with the PLCdelta4-C2 domain. These results indicate that PLCdelta4 binds via its C2 domain to the PDZ6 or PDZ7 domain of GRIP1, and that this association may play a role in spermatogenesis.

Normal range and variation of the zona pellucida–induced acrosome reaction in fertile men

Objective To investigate the normal range of the zona pellucida (ZP)–induced acrosome reaction (AR) in fertile men. Design A study of normative data. Setting Academic research and teaching tertiary hospital. Intervention(s) None. Patient(s) Fertile men (n = 111) who had children or whose female partner was currently pregnant. Main outcome measure(s) Semen analysis, sperm–ZP binding, and the ZP-induced AR. Result(s) There was wide range for the ZP-induced AR: 20%–98% with a mean of 48%. The ZP-induced AR was significantly correlated with sperm concentration and normal morphology. The ZP-induced AR was significantly lower in men with sperm concentration ≤60 × 10 6/mL than those with sperm concentration >60 × 10 6/mL. Similarly, men with normal sperm morphology ≥15% had significantly higher ZP-induced AR than men with normal morphology <15%. Conclusion(s) There is a wide range of the ZP-induced AR in fertile men that is correlated with sperm concentration and normal morphology.

Failed fertilization: is it predictable?

The purpose of this review is to critically examine the ability of screening tests to predict fertilization failure. Failed fertilization occurs in 5-10% of in-vitro fertilization cycles and 2-3% of intracytoplasmic sperm injection cycles. Failed fertilization may result from impaired spermatozoa, oocyte deficiencies or defects in the in-vitro sperm/oocyte medium. In the in-vitro fertilization setting most cases are caused by male factor deficiencies, whereas failure of oocyte activation is the most common cause of failed fertilization after intracytoplasmic sperm injection. Although the standard semen analysis has limited ability to predict fertilization failure, strict sperm morphology criteria, sperm-zona binding ratios and zona pellucida induced acrosome reaction tests provide increased capacity to avoid this outcome. The quality of the semen sample on the day of oocyte retrieval and fertilization performance in previous in-vitro fertilization cycles may also guide the appropriate use of intracytoplasmic sperm injection. However, the routine use of the latter technique in cases of non-male factor infertility is contraindicated. The ever improving techniques highlighted in this analysis offer improved ability to predict failed fertilization.

Involvement of a Na+/HCO [formula omitted]Cotransporter in Mouse Sperm Capacitation

Mammalian sperm are incapable of fertilizing eggs immediately after ejaculation; they acquire fertilization capacity after residing in the female tract for a finite period of time. The physiological changes sperm undergo in the female reproductive tract that render sperm able to fertilize constitute the phenomenon of "sperm capacitation." We have demonstrated that capacitation is associated with an increase in the tyrosine phosphorylation of a subset of proteins and that these events are regulated by an HCO(3)(-)/cAMP-dependent pathway involving protein kinase A. Capacitation is also accompanied by hyperpolarization of the sperm plasma membrane. Here we present evidence that, in addition to its role in the regulation of adenylyl cyclase, HCO(3)(-) has a role in the regulation of plasma membrane potential in mouse sperm. Addition of HCO(3)(-) but not Cl(-) induces a hyperpolarizing current in mouse sperm plasma membranes. This HCO(3)(-)-dependent hyperpolarization was not observed when Na(+) was replaced by the non-permeant cation choline(+). Replacement of Na(+) by choline(+) also inhibited the capacitation-associated increase in protein tyrosine phosphorylation as well as the zona pellucida-induced acrosome reaction. The lack of an increase in protein tyrosine phosphorylation was overcome by the presence of cAMP agonists in the incubation medium. The lack of a hyperpolarizing HCO(3)(-) current and the inhibition of the capacitation-dependent increase in protein tyrosine phosphorylation in the absence of Na(+) suggest that a Na(+)/HCO(3)(-) cotransporter is present in mouse sperm and is coupled to events regulating capacitation.

Relationship between zona pellucida–induced acrosome reaction, sperm morphology, sperm–zona pellucida binding, and in vitro fertilization

Objective To evaluate the possible relationships between sperm morphology, acrosome responsiveness to solubilized human zona pellucida, and sperm–zona binding potential among [1] consecutive andrology referrals and [2] randomly selected in vitro fertilization (IVF) cases. Design Prospective analytical study. Setting Academic training hospital. Patient(s) Randomly selected couples consulting for infertility. Intervention(s) Acrosome reaction response to solubilized human zona pellucida was recorded. Main outcome measure(s) We determined the difference in the percentage of sperm that acrosome reacted after exposure to solubilized zona pellucida and spontaneous acrosome reaction. The results were expressed as percentage zona induced acrosome reaction (ZIAR). Result(s) Data were analyzed using correlation coefficients (r) and receiver operator characteristics (ROC curve analyses). The ROC curve analyses indicated ZIAR to be a sensitive indicator for fertilization failure during IVF therapy, with sensitivity and specificity of 81% and 75%, respectively. For andrology referrals, a positive and statistically significant correlation existed between ZIAR data and sperm morphology (r = 0.65) and sperm–zona binding (r = 0.57). Conclusion(s) ZIAR results provide further information regarding dysfunctional sperm and can be used as an additional diagnostic test. Our results predicted fertilization failure during IVF treatment.

Disordered zona pellucida–induced acrosome reaction and failure of in vitro fertilization in patients with unexplained infertility

Objective To determine the relationship between the zona pellucida (ZP)–induced acrosome reaction (AR) and fertilization rate and pregnancy rate in standard IVF and the frequency of disordered ZP-induced AR (DZPIAR) in patients with unexplained infertility. Design Prospective study. Setting Academic research and teaching tertiary hospital. Patient(s) Patients with unexplained infertility with normal semen analysis. Intervention(s) None. Main outcome measure(s) Semen analysis, the ZP-induced AR, and measurements of fertilization rate and pregnancy rate with in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Result(s) A statistically significant correlation was found for the ZP-induced AR and fertilization rate with standard IVF (n = 65). Patients with DZPIAR (ZP-induced AR of ≤16%) had an average fertilization rate of 23%, in contrast to patients with ZP-induced AR of >16%, who had an average fertilization rate of 61%. The sensitivity and specificity of DZPIAR for prediction of IVF rates <30% and ≥30% were 80% and 86%, respectively. Of 260 patients screened, the frequency of DZPIAR was 29%. Ten patients with DZPIAR had an average fertilization rate of 15% and no pregnancy with initial IVF cycles, and a fertilization rate of 61% (with three live-birth pregnancies) with subsequent ICSI cycles. Another 33 patients with DZPIAR were treated with ICSI alone, with an average fertilization rate of 71% and a live-birth pregnancy rate of 17% per embryo transfer. Sixteen of the patients had live-birth pregnancies (including one set of twins) after undergoing an average of 3.2 embryo transfers. Conclusion(s) Patients with DZPIAR have a low or zero fertilization rate with standard IVF but high fertilization and pregnancy rates with ICSI. Up to 29% of patients with unexplained infertility with normal semen analysis may have this condition, which should be diagnosed and treated with ICSI rather then standard IVF.

Zona pellucida-induced acrosome reaction in human sperm: dependency on activation of pertussis toxin-sensitive G(i) protein and extracellular calcium, and priming effect of progesterone and follicular fluid.

In these studies, we aimed to characterize the effects of the physiological, homologous agonists of the acrosome reaction, i.e. the zona pellucida (ZP) and progesterone/follicular fluid, on human sperm. The specific aims of our studies were: (i) to examine the dependency of the solubilized ZP-induced acrosome reaction on G(i) protein activation and presence of extracellular calcium; and (ii) to determine whether progesterone/follicular fluid exert a priming or synergist effect on the solubilized ZP-induced acrosome reaction. Highly motile sperm from fertile donors were exposed to the agonists in a microassay and the acrosomal status of live sperm was determined by indirect immunofluorescence using PSA-FITC/Hoechst double-staining. Pretreatment with pertussis-toxin (100 ng/ml) and EGTA (2.5 mmol/l) significantly inhibited the ZP-induced acrosome reaction without affecting the spontaneous rate of exocytosis. Progesterone (1.25 microg/ml) and human follicular fluid (10%) exerted a priming, time-dependent effect on the ZP-induced acrosome reaction. These studies demonstrated that: (i) acrosomal exocytosis of capacitated human sperm triggered by the homologous ZP is dependent on the activation of G(i) proteins (pertussis toxin-sensitive) and the presence of extracellular calcium; and (ii) progesterone and follicular fluid exert a priming effect on the ZP-induced acrosome reaction.

Phorbol myristate acetate induces ruffling of the acrosome of human sperm

Objective: To determine the effect of phorbol myristate acetate (PMA) on human acrosome morphology and the acrosome reaction. Design: Controlled experiments on sperm and unfertilized oocytes from volunteers. Setting: Academic research and teaching tertiary hospital. Patient(s): Sperm samples were from normospermic men and unfertilized oocytes from IVF patients. Main Outcome Measure(s): Acrosome morphology was assessed by using transmission and scanning electron microscopy. The acrosome reaction was assessed by using fluorescein-labeled Pisum sativum agglutinin. Result(s): PMA induced acrosome ruffling, indicated by a marked wavy appearance. A significant correlation was found between PMA-induced ruffling and PMA enhancement of the zona pellucida-induced acrosome reaction. Protein kinase C inhibitors bisindolylmalemide I and sangivamycin had no effect on PMA-induced acrosomal ruffling, but actin polymerization inhibitors cytochalasin B and cytochalasin D significantly decreased PMA-induced acrosomal ruffling. In contrast, bisindolylmalemide I, sangivamycin, cytochalasin B, and cytochalasin D significantly decreased both the zona pellucida-induced acrosome reaction and the PMA enhancement of the zona pellucida-induced acrosome reaction. Conclusion(s): PMA-induced acrosomal ruffling involves actin polymerization, possibly independent of conventional protein kinase C. Acrosomal ruffling is involved in the PMA augmentation of the zona pellucida-induced acrosome reaction.