Spontaneous Acrosome Reaction Rate Research Articles

116 Effect of different energy sources on motility and calcium ionophore-induced acrosome reaction in equine sperm

Equine sperm appears to differ from sperm of other species, preferring oxidative phosphorylation over glycolysis for energy production. However, there is little information about the effects of different energy sources on capacitation-related events in equine sperm. We evaluated stallion sperm incubated in the presence of different energy sources at a pH (~8.3) known to support some aspects of capacitation. Fresh stallion semen (n=9 ejaculates) was washed, then diluted to 30×106 spermmL−1 with modified Whitten's medium containing 25mM bicarbonate, 7mgmL−1 bovine serum albumin (BSA), and only one energy source (5mM glucose, 10mM lactate, or 10mM pyruvate). The diluted sperm were incubated in air for 0.5, 2, or 4h, then exposed to 5 µM calcium ionophore A23187 (CaI) or vehicle (V) for 10min. Sperm were washed and incubated in the assigned medium for 30min, then evaluated by computer-assisted sperm motility analysis and by live/dead staining (Fixable Live/Dead Red, Molecular Probes) followed by fixation (2% v/v paraformaldehyde) and staining for acrosome reaction, using Pisum sativum agglutinin (FITC-PSA, Sigma Aldrich). Stained sperm were evaluated via flow cytometry to detect live acrosome-reacted sperm. Data were analysed using ANOVA with Tukey post hoc comparison (SAS 9.4, SAS Institute). Medium pH at 4h was 8.37 to 8.48. There were no significant differences between CaI and V treatments for any experimental endpoint (P>0.05). At 4h, pyruvate values for total motile sperm (46±5%) and curvilinear velocity (113±8µm s−1) were significantly higher than those for lactate (32±5% and 86±5µm s−1) or glucose (27±4% and 90±5µm s−1). The percentage of live sperm that were acrosome-reacted (%L-AR) did not increase significantly until 4 h; at this time,%L-AR was higher for lactate (46±3%) than for pyruvate (21±3%) or glucose (18±3%; P<0.05). To the best of our knowledge, this is the first critical evaluation of the effect of different energy sources on motility and acrosome reactivity of equine sperm. The higher motility in pyruvate supports the concept that stallion sperm utilises oxidative phosphorylation more effectively than glycolysis. The apparent lack of effect of CaI on%L-AR may be related to the 1- to 2-h delay typically seen after its use in equine sperm; the present data show that incubation even for 4h in potentially capacitating conditions does not prepare equine sperm to respond quickly to CaI. Surprisingly, a high rate of spontaneous acrosome reaction was achieved in the lactate treatment. The notably higher%L-AR with lactate than with pyruvate suggests that this effect is not due to utilisation of substrate for oxidative phosphorylation but may be associated with conversion of lactate to pyruvate via lactate dehydrogenase, or possibly with effects of lactate through other pathways. Although pyruvate was associated with greater motility, this could be related to higher oxidative stress, which could have influenced the%L-AR. Further work is needed to determine whether the acrosomal changes observed are related to functional capacitation.

Correlationship between hunman spermatozoa spontaneous acrosome reaction rate and sperm survival rate and total motility and abnormal rate

Objective To study the exploring of correlationship between hunman spermatozoa spontaneous acrosome reaction rate and sperm survival rate and total motility and abnormal rate and to evaluate the effect of hunman spermatozoa spontaneous acrosome reaction rate on male fertility. Methods A total of 223 semen samples in our hospital from December 2017 to May 2018 were selected.The patient was abstinent for 2-7 days, and all semen was collected in a disposable sterile and non-toxic container.All the standards were from WHO laboratory manual for the examination and processing of human sperm(fifth edition). Routine analysis of semen was performed using the CASA system.Sperm survival rate and total motility were calculated.The sperm abnormality rate after papanicolaou staining was observed.The spermatozoa spontaneous acrosome reaction rate was tested by PSA-FITC staining.According to the positive rate of acrosome reaction (AR) than 10 was abnormal, two groups were divided into AR>10 and AR no more than 10. Correlationship between hunman spermatozoa spontaneous acrosome reaction rate and sperm survival rate and total motility and abnormal rate were explored by SPSS 22.0 statistical software. Results There were significant differences in sperm survival rate and total motility between normal and abnormal groups(P<0.05). The hunman spermatozoa spontaneous acrosome reaction rate was negatively correlated with sperm survival rate and total motility(r=-0.249, P<0.05; r=-0.256, P<0.05). There was no significant correlation between hunman spermatozoa spontaneous acrosome reaction rate and sperm abnormal rate. Conclusions The hunman spermatozoa spontaneous acrosome reaction rate is related the sperm survival rate and total motility, and it can be used as one of the indicator of male reproductive function. Key words: Semen; Acrosome Reaction; Survival Rate; Teratozoospermia

New Insight into Sperm Capacitation: A Novel Mechanism of 17β-Estradiol Signalling.

17β-estradiol (estradiol) is a natural estrogen regulating reproduction including sperm and egg development, sperm maturation—called capacitation—and sperm–egg communication. High doses can increase germ cell apoptosis and decrease sperm count. Our aim was to answer the biological relevance of estradiol in sperm capacitation and its effect on motility and acrosome reaction to quantify its interaction with estrogen receptors and propose a model of estradiol action during capacitation using kinetic analysis. Estradiol increased protein tyrosine phosphorylation, elevated rate of spontaneous acrosome reaction, and altered motility parameters measured Hamilton-Thorne Computer Assisted Semen Analyzer (CASA) in capacitating sperm. To monitor time and concentration dependent binding dynamics of extracellular estradiol, high-performance liquid chromatography with tandem mass spectrometry was used to measure sperm response and data was subjected to kinetic analysis. The kinetic model of estradiol action during sperm maturation shows that estradiol adsorption onto a plasma membrane surface is controlled by Langmuir isotherm. After, when estradiol passes into the cytoplasm, it forms an unstable adduct with cytoplasmic receptors, which display a signalling autocatalytic pattern. This autocatalytic reaction suggests crosstalk between receptor and non-receptor pathways utilized by sperm prior to fertilization.

Human sperm acrosomal status, acrosomal responsiveness, and acrosin are predictive of the outcomes of in vitro fertilization: A prospective cohort study

The sperm acrosome reaction (AR) is a physiological secretory course of membrane fusion and hydrolytic enzymes, as well as matrix protein release, enabling spermatozoa to penetrate the egg surroundings. An instable acrosomal status before a specific stimulus, insufficient acrosomal responsiveness, or inadequate enzymatic activity of acrosomal content can be detrimental to male fertility. This prospective cohort study was designed to determine whether three human sperm acrosome evaluation parameters—including spontaneous AR rate, AR after calcium ionophore A23187 challenge (ARIC) rate, and modified Kennedy acrosin activity—can predict fertilization outcomes in vitro and are correlated with male characteristics. A total of 485 eligible couples undergoing in vitro fertilization (IVF) therapy were included in two phases of this study. In a ‘construction phase’, three acrosome evaluation parameters were determined simultaneously in 132 cases, whereas in a ‘validation phase’, the spontaneous AR rate was determined in 353 cases. The results of the ‘construction phase’ revealed that the spontaneous AR rate was the only significant predictor of fertilization outcome (unadjusted odds ratio [OR] = 0.68, 95% confidence interval [CI]: 0.53–0.88, P = 0.003; adjusted OR = 0.64, 95% CI: 0.43–0.95, P = 0.03), and the cut-off value for total fertilization failure (TFF) prediction, determined by ROC curve analysis, was 9.91%; higher acrosin activity was shown to predict a higher fertilization rate only when patients were divided into groups (≥25 μIU/106 spermatozoa, 14–25 μIU/106 spermatozoa, <14 μIU/106 spermatozoa). The spontaneous AR rate was negatively correlated with sperm motility, forward progression motility, and normal morphology; modified Kennedy acrosin activity was positively correlated with normal morphology; and the ARIC rate was not correlated with any of the male characteristics. A similar result was obtained for the spontaneous AR rate in the ‘validation phase’, and the cut-off value in predicting TFF was calibrated for 9.52%. Clinically, patients can voluntarily choose spontaneous AR rate alone or in combination with modified Kennedy acrosin activity to predict TFF, and early rescue intracytoplasmic sperm injection (ICSI), half ICSI, or full ICSI should be considered in advance for men with spontaneous AR rates ≥9.52% or spontaneous AR rates ≥9.52% and AE activities <25 μIU/106 spermatozoa.

ITRAQ-based analysis of sperm proteome from normozoospermic men achieving the rescue-ICSI pregnancy after the IVF failure

BackgroundIn the assisted reproduction, the infertile molecules of spermatozoa from normozoospermic men who underwent the unexplained failure of in vitro fertilization (IVF) due to the lack of sperm binding to the normal zona pellucida, and then achieved pregnancy with the rescue intracytoplasmic sperm injection (R-ICSI) remain unclear. More works are still necessary to explore this male infertile mechanism.MethodsNormozoospermicmen with the IVF pregnancy and normozoospermic men with the R-ICSI pregnancy after the conventional IVF failure were collected. iTRAQ-based proteomic approach were performed to reveal the new infertile causes between the IVF pregnancy men and the R-ICSI pregnancy men. To validate the confidence of proteome data, the individual samples were analyzed by western blot and immunofluorescence. Further, the spontaneous acrosome reactions were measured to evaluate the sperm quality.ResultsCompared with IVF pregnancy group, 56 sperm proteins were differentially expressed in the R-ICSI pregnancy group. Bioinformatic analyses (PANTHER, DAVID, PubMed and STRING) indicated these altered sperm proteins were involved in various molecular functions: reproduction, chromosome organization, and sperm-oocyte interaction. Moreover, the confidence of proteome data was confirmed by western blot and immunofluorescence using the individual samples, which were consistent with our proteomic data. Additionally, an increased rate of the spontaneous acrosome reaction rate was found in the R-ICSI pregnancy group.ConclusionsThe sealtered sperm proteins and the increased spontaneous acrosome reaction rate might account for this unexplained male infertility in the R-ICSI pregnancy patients. The present proteomic results will throw light on the better understanding of the unexplained infertile mechanisms underlying these normozoospermic man who achieved R-ICSI pregnancy after IVF failure.

Value Analysis of Sperm Spontaneous Acrosome Reaction in Male Fertility Evaluation

Objective: Using correlation analysis between the sperm spontaneous acrosome reaction rate (SARR) and routine semen parameters in male infertility patients, we explored the value of the sperm spontaneous acrosome reaction in the evaluation of sperm functions. Methods: The participants comprised of 219 male infertility patients who had visited the Center for Reproductive Medicine, Shandong University from October 2016 to March 2017. According to the sperm SARR, we classified the patients with infertility into the control group and the case group. According to the WHO human semen examination and processing laboratory manual, fifth edition, recommendations and standards. We obtained routine semen parameters including semen volume, sperm concentration, total motility, and progressive sperm rate, and we measured sperm motility rate and the proportion of sperm with normal morphology. Results: With regard to the total motility, progressive sperm rate, sperm survival rate and the proportion of sperm with normal morphology, there were statistically significant differences between the two groups (p 0.05). Conclusion: Sperm SARR in patients with infertility is closely related to routine semen parameters and plays a supplementary reference in male fertility evaluation.

Application of spontaneous acrosome reaction of sperm in prediction of outcome of in-vitro fertilization and embryo transfer

To investigate the clinical application of spontaneous acrosome reaction (AR) rate of sperm in predicting the outcome of in-vitro fertilization and embryo transfer (IVF-ET). The spontaneous AR rate of the sperm of patients who underwent IVF-ET treatment in our center during the period from November to December 2014 were studied. The cut-off value from 6% to 12% were set and analyzed its association between the IVF-ET outcomes (including fertility rates, normal fertilization rates and high-quality embryo rates). For those who underwent fresh embryo transplantation, the rates of chemical pregnancy and clinical pregnancy were calculated, and compared the spontaneous AR rates and quantity of acrosomal enzyme according to the pregnancy outcome. There were 202 patients in this study and the mean spontaneous AR rate was 5.99%±5.18%. For patients with the spontaneous AR rate ≥9% versus <9%, the fertility rate, normal fertilization rate and high-quality embryo rate were 81.33% vs 83.85%, 60.53% vs 60.99%, and 51.10% vs 59.67%, respectively, with statistically significant difference in the high-quality embryo rate (P=0.02). For patients who underwent fresh embryo transplantation, when comparison was made between those with spontaneous AR rate ≥8% and those <8%, the rate of chemical pregnancy and clinical pregnancy were 48.57% (17/35) vs 69.64% (78/112) and 37.14% (13/35) vs 63.39% (71/112), respectively, both with statistically significant difference (P=0.02 and P<0.01). The patients with clinical pregnancy had lower spontaneous AR rate than those without clinical pregnancy (5.41%±3.87% vs 7.40%±6.79%, P=0.04), while the quantity of acrosomal enzyme showed no significant difference [(131.79±68.50) vs (153.62±59.59) μU/10(6,) P=0.06]. Logistic regression analysis demonstrated association between spontaneous AR rates and clinical pregnancy (OR=0.93, 95%CI: 0.87-0.99, P=0.03). The spontaneous AR rate of sperm may have clinical significance in predicting the outcome of IVF-ET, as it is reversely correlated with IVF high-quality embryo rate and pregnancy rate. The quantity of acrosomal enzyme may not have significant predictive value for the outcome of IVF.

45 ACROSOME REACTION AND HETEROLOGOUS ZONA BINDING ASSAY OF FROZEN STALLION SPERM AFTER HYPERACTIVATION

During cryopreservation and due to the large portion of seminal plasma removal, there is a decrease in equine sperm antioxidant protection. Lactoferrin and catalase in seminal plasma play an antioxidant role. The fertilizing ability of equine sperm has been analysed in vitro using sperm-zona binding assays with heterologous oocytes. The results have been correlated with in vivo fertility by means of acrosome reaction (AR) and the number of attached sperm to the zona pellucida (ZP). The aim of the present work was to estimate the potential fertilizing ability of stallion sperm frozen with INRA82 extender (Battelier et al. 1997) with lactoferrin and catalase, and after hyperactivation with procaine and calcium ionophore A-23187 (Ca-I) by determining the AR rate and number of attached sperm to the bovine ZP. Semen from 6 stallions was frozen with 3 extenders: (T1) control, INRA 82; (T2) T1 + 500 μg mL–1 lactoferrin; and (T3) T1 + 200 IU mL–1 catalase. After semen thawing, the sperm were selected by swim-up and distributed in 3 aliquots according to the hyperactivation treatments: (H1) control, after thawing; (H2) capacitating Whitten’s medium + 5 mM procaine chloride; and (H3) capacitating Whitten’s medium + 5 μM Ca-I. To the zona binding assays, bovine oocytes derived from abattoir ovaries were incubated at 38.5°C with 5% CO2 (1 h), and 5 oocytes were poured into each treatment droplet under mineral oil. Sperm were stained with Hoechst 33342 dye (35 μg mL–1), and after 2 h co-culture, the number of sperm attached to the ZP was determined with epi-fluorescent microscopy. The rate of sperm AR was determined after freezing-thawing (control) and hyperactivation treatments with propidium iodide and fluorescein isothiocyanate/peanut agglutinin dies with a flow cytometer. The green fluorescent (peanut agglutinin+) and not red stained (propidium iodide) sperm were considered acrosome reacted. Means of ZP attached sperm and percentage of AR sperm were analysed by ANOVA and Tukey test. A probability of P &lt; 0.05 was considered significant. The mean of ZP attached sperm (4.2 ± 3.5) and AR sperm rate (4.4 ± 3.7%) did not differ among the extenders (P &gt; 0.05). The rate of sperm AR after hyperactivation with procaine (5.2 ± 2.4%) did not differ to the Ca-I (6.1 ± 3.7%); however, they were higher than the spontaneous AR rate (1.1 ± 0.5%, P &lt; 0.05). Lower number of ZP attached sperm was observed by the Ca-I induced hyperactivation protocol (1.9 ± 2.1) compared with the procaine (5.9 ± 3.7; P &lt; 0.05), although they did not differ to the control (3.3 ± 2.7). In conclusion stallion frozen sperm were better hyperactivated with procaine than with Ca-I, and therefore, it is a more suitable sperm hyperactivation inductor to study equine IVF protocols with frozen semen. Acknowledgments are extended to CAPES, Brazil, for the financial support.

Progress of sperm IZUMO1 relocation during spontaneous acrosome reaction

It has been recently shown in mice that sperm undergo acrosome reaction (AR) by passing through cumulus cells; furthermore, the acrosome-reacted sperm can bind to zona pellucida and consequently fertilise the egg. During AR, the relocation of the primary fusion protein IZUMO1 into the equatorial segment is crucial for sperm-egg fusion. There is a high rate of spontaneous AR in rodents, with up to 60% in promiscuous species. The aim of this study was to clarify whether the IZUMO1 relocation in sperm after spontaneous and induced AR is the same, and whether there is a correlation between the speed of IZUMO1 relocation and species-specific mating behaviour in field mice. Immunofluorescent detection of IZUMO1 dynamics during the in vitro capacitation, spontaneous, calcium ionophore and progesterone-induced AR was monitored. Our results show that during spontaneous AR, there is a clear IZUMO1 relocation from the acrosomal cap to the equatorial segment, and further over the whole sperm head. In addition, there is positive tail tyrosine phosphorylation (TyrP) associated with hyperactive motility. Moreover, the beginning and the progress of IZUMO1 relocation and tail TyrP positively correlate with the level of promiscuity and the acrosome instability in promiscuous species. The findings that crucial molecular changes essential for sperm-egg fusion represented by dynamic movements of IZUMO1 also happen during spontaneous AR are vital for understanding fertilisation in mice.

Assessment of sperm hyperactivated motility and acrosome reaction can discriminate the use of spermatozoa for conventionalin vitrofertilisation or intracytoplasmic sperm injection: Preliminary results

Basic semen analysis is insufficient for determining the fertility potential. The aim of this study was to determine if hyperactivated motility (HAM) and acrosome reaction (AR) can be useful tests for evaluating semen quality during male infertility evaluations and to help the clinician decide whether regular insemination or intracytoplasmic sperm injection (ICSI) is preferable during in vitro fertilisation. A prospective study was conducted. Patients with normal sperm according to World Health Organization guidelines who underwent IVF treatment and planned regular insemination were asked to participate. A portion of sperm sample was evaluated for HAM and AR on day of ovum pick up. In HAM assessment, 93.3% of patients with increased HAM had a high fertilisation rate compared with 64% in the group without increased HAM (P = 0.059). For the AR evaluation, 91.7% of samples with a low rate of spontaneous AR had a high fertilisation rate compared with 39.3% in the group with a high rate of spontaneous AR (P = 0.004).

Sperm acrosome reaction detection in intrauterine insemination

To investigate the relationship between sperm acrosome reaction (AR) and the clinical pregnancy rate of intrauterine insemination (IUI). We detected the sperm spontaneous AR rate and Ca2+ ionophore A23187-induced AR rate in 128 patients who accepted IUI treatment, collected their clinical data and analysed the relationship between sperm AR rate and clinical pregnancy rate of IUI. There was no statistical difference between the spontaneous AR rates in the pregnant group and the non-pregnant group (7.7% vs. 7.0%, P>0.05), but there was statistical difference between the induced AR rates(51.9 % vs. 43.5%, P<0.05). There was statistical difference in the clinical pregnancy rate among the 3 IUI groups divided by induced AR rate (≤20.0%, 20.1%-49.9%, and ≥50.0%; 4.8% vs. 12.5% vs. 18.6%, P<0.05). The spontaneous AR rate has nothing to do with the clinical pregnancy rate of IUI, but the induced AR rate is associated with the clinical pregnancy rate of IUI.

Characterization of the Endocannabinoid System in Human Spermatozoa and Involvement of Transient Receptor Potential Vanilloid 1 Receptor in Their Fertilizing Ability

Human spermatozoa express type-1 cannabinoid receptor (CB1), whose activation by anandamide (AEA) affects motility and acrosome reaction (AR). In this study, we extended the characterization of the AEA-related endocannabinoid system in human spermatozoa, and we focused on the involvement of the AEA-binding vanilloid receptor (TRPV1) in their fertilizing ability. Protein expression was revealed for CB1 ( approximately 56 kDa), TRPV1 ( approximately 95 kDa), AEA-synthesizing phospholipase D (NAPE-PLD) ( approximately 46 kDa), and AEA-hydrolyzing enzyme [fatty acid amide hydrolase (FAAH), approximately 66 kDa]. Both AEA-binding receptors (CB1 and TRPV1) exhibited a functional binding activity; enzymatic activity was demonstrated for NAPE-PLD, FAAH, and the purported endocannabinoid membrane transporter (EMT). Immunoreactivity for CB1, NAPE-PLD, and FAAH was localized in the postacrosomal region and in the midpiece, whereas for TRPV1, it was restricted to the postacrosomal region. Capsazepine (CPZ), a selective antagonist of TRPV1, inhibited progesterone (P)-enhanced sperm/oocyte fusion, as evaluated by the hamster egg penetration test. This inhibition was due to a reduction of the P-induced AR rate above the spontaneous AR rate, which was instead increased. The sperm exposure to OMDM-1, a specific inhibitor of EMT, prevented the promoting effect of CPZ on spontaneous AR rate and restored the sperm responsiveness to P. No significant effects could be observed on sperm motility. In conclusion, this study provides unprecedented evidence that human spermatozoa exhibit a completely functional endocannabinoid system related to AEA and that the AEA-binding TRPV1 receptor could be involved in the sperm fertilizing ability.

Absence of spermatozoal CD46 protein expression and associated rapid acrosome reaction rate in striped field mice (Apodemus agrarius)

BackgroundIn rodents, the cell surface complement regulatory protein CD46 is expressed solely on the spermatozoal acrosome membrane. Ablation of the CD46 gene is associated with a faster acrosome reaction. Sperm from Apodemus flavicollis (yellow-necked field mice), A. microps (pygmy field mice) and A. sylvaticus (European wood mice) fail to express CD46 protein and exhibit a more rapid acrosome reaction rate than Mus (house mice) or BALB/c mice. A. agrarius (striped field mice) belong to a different Apodemus subgenus and have pronounced promiscuity and large relative testis size. The aim of this study was to determine whether A. agrarius sperm fail to express CD46 protein and, if so, whether A. agrarius have a faster acrosome reaction than Mus.MethodsReverse transcription polymerase chain reaction (RT-PCR) was used to assess whether A. agrarius transcribe testicular CD46 mRNA. RT-PCR was supplemented with 3'- and 5'-rapid amplification of cDNA ends to determine the complete nucleotide sequence of A. agrarius CD46. Fluorescence microscopy was used to assess whether CD46 protein is expressed by A. agrarius sperm. The acrosome status of A. agrarius sperm was calculated over time by immunocytochemistry using peanut agglutinin lectin.ResultsWe demonstrate that A. agrarius mice transcribe two unique alternatively spliced testicular CD46 mRNA transcripts, both lacking exon 7, which differ from those described previously in other Apodemus species. The larger A. agrarius CD46 transcript has an insert between exons 10 and 11 which, if translated, would result in a novel cytoplasmic tail. In addition, A. agrarius CD46 transcripts have an extended AU-rich 3'-untranslated region (UTR) and a truncated 5'-UTR, resulting in failure to express spermatozoal CD46 protein. We show that A. agrarius has a significantly faster spontaneous acrosome reaction rate than A. sylvaticus and Mus.ConclusionAbsence of CD46 protein expression is associated with acrosomal instability in rodents. A. agrarius mice express novel CD46 transcripts, resulting in the trade of spermatozoal CD46 protein expression for a rapid acrosome reaction rate, in common with other species of field mice. This provides a strategy to increase competitive sperm advantage for individuals, leading to faster fertilisation in this highly promiscuous genus.

Severe Subfertility in Mice with Androgen Receptor Inactivation in Sex Accessory Organs But Not in Testis

Androgen action on sex accessory organs influences rodent fertility, but the mechanisms remain unclear and investigation is difficult without the ability to restrict androgen action in specific tissues. We used Cre-LoxP technology to generate male mice with prostate epithelial-specific androgen receptor deficiency (denoted PEARKO). In addition to prostate, these males have reduced androgen action due to tissue-selective androgen receptor inactivation in seminal vesicle, epididymis, and vas deferens, whereas the testis is unaffected. We find that fertility of PEARKO males was severely reduced, compared with littermates with prominent defects in copulatory plug formation, which were smaller, softer, and more friable than controls. Despite normal testis sperm production, sperm numbers were reduced in caput but increased in cauda epididymis, suggesting alterations in sperm epididymal transit kinetics associated with increased rate of spontaneous acrosome reaction and abnormal flagellar morphology in PEARKO cauda epididymal sperm. Whereas the quantitative in vitro fertilizing ability of PEARKO epididymal sperm was normal, fewer fertilized oocytes were flushed from the oviducts of females after natural mating with PEARKO males. These data show that sperm formed in mice with impaired androgen action restricted to accessory glands and epididymis are quantitatively normal in number and in vitro fertilizing function but that severe in vivo subfertility reflects other functions related to sperm transport and survival in female reproductive tract that determine fertility in vivo.

Mice Deficient in CHRNA7, a Subunit of the Nicotinic Acetylcholine Receptor, Produce Sperm with Impaired Motility1

In this study we investigate the role of the CHRNA7 subunit (also known as the alpha7 subunit) of the nicotinic acetylcholine receptor in mouse sperm function. We confirm by reverse-transcription-polymerase chain reaction the expression in adult mouse testis of Chrna7 mRNA and demonstrate the subunit's presence in mouse sperm by immunoblot. Alpha-bungarotoxin binds a range of nicotinic acetylcholine receptor subunits, including the CHRNA7 subunit. Localization studies using a fluorescent alpha-bungarotoxin-tetramethyl-rhodamine conjugate revealed specific binding sites on the midpiece of mouse sperm with fainter alpha-bungarotoxin binding on the remainder of the flagellum. Mice engineered with a double-null disruption of the Chrna7 gene displayed only faint fluorescence on the midpiece, suggesting that the CHRNA7 contributed the majority of the observed alpha-bungarotoxin binding sites. The location of alpha-bungarotoxin binding suggested that nicotinic acetylcholine receptors may play an ionotropic role in sperm motility. Sperm from Chrna7(-/-) mice display no difference in number, morphology, viability or spontaneous acrosome reaction rate compared with Chrna7(+/+) sperm. Studies using computer-assisted sperm analysis indicate the motility of Chrna7(-/-) sperm is significantly impaired. This impairment is characterized by significantly reduced swimming velocities, failure to maintain vigorous swimming, and lower levels of hyperactivated swimming patterns in Chrna7(-/-) sperm compared with Chrna7(+/+) sperm. This is the first genetic evidence that sperm nicotinic acetylcholine receptors are important for maintenance of normal sperm motility.

Effects of diadenosine polyphosphates and seminal fluid vesicles on rabbit sperm cells.

Membrane vesicles were isolated from rabbit seminal plasma. Electron microscopy analyses showed the presence of numerous small, round vesicles with a diameter of about 70 nm. Determination of enzyme activities was carried out by high performance liquid chromatography and showed that the vesicles can degrade the diadenosine polyphosphates (ApnA), Ap3A and Ap4A and ATP and ADP, but not AMP. Studies of the degradation of diadenosine compounds by the vesicles present in seminal fluid showed an increasing production of AMP as the by-product and a time-dependent generation of dephosphorylated products consistent with the presence of ecto-ATP diphosphophosphatase (ecto-apyrase). In the presence of rabbit spermatozoa, AMP did not accumulate because 5'nucleotidase and adenosine deaminase, present at the surface of sperm cells, transformed AMP into adenosine and inosine. The effects of seminal fluid vesicles and diadenosine compounds on the acquisition of fertilizing capacity by rabbit spermatozoa were evaluated by Pisum sativum agglutinin fluorescein isothiocyanate conjugated staining. The results obtained with uncapacitated spermatozoa showed that the capacitating effector BSA could be substituted efficiently by the addition of diadenosine compounds and vesicles previously incubated for 2 h to the capacitative medium. Under these experimental conditions, the spontaneous acrosome reaction rate was not increased. Capacitated rabbit spermatozoa did not undergo acrosome reaction when l-alpha-lysophosphatidylcholine was substituted by diadenosine compounds previously incubated with vesicles. In conclusion, this study has shown that rabbit seminal fluid vesicles can degrade diadenosine compounds to AMP and that the addition of the vesicles and diadenosine compounds to uncapacitated rabbit spermatozoa favours the acquisition of the fertilizing capacity.

The Effect of Cryopreservation on the Spontaneous Acrosome Reaction of Human Spermatozoa

Objectives: Cryopreserved spermatozoa are widely used in assisted reproduction programs. While processing and incubation increase the rate of spontaneous acrosome reaction in human spermatozoa, little is known about the effect of cryopreservation. This study was conducted to assess the effect of cryopreservation on the spontaneous acrosome reaction of human spermatozoa. To the best of our knowledge, this is the first study to include a period of incubation before assessing the rate of acrosome reaction.

Effects of products of activated immune cells and recombinant cytokines on spontaneous and ionophore-induced acrosome reaction.

The purpose of this study was to examine whether the products of activated immune cells influence spontaneous and ionophore-induced sperm acrosome reaction. The spontaneous and ionophore-induced acrosome reaction were evaluated by staining with fluorescein isothiocyanate (FITC) Pisum sativam agglutinin after incubation in capacitating media supplemented with either supernatants from Con-A activated leukocyte cultures or human recombinant (r) IL-1 beta, TNF-alpha, and INF-gamma. The supernatants from Con A-activated peripheral blood leukocyte cultures at 1:1 and 1:10 dilution significantly increased the rate of spontaneous acrosome reaction (P < 0.001 and P < 0.01). Along with displayed abnormally elevated levels of spontaneous acrosome loss, sperm cells showed an insufficient ability to undergo acrosome reaction in response to the ionophore treatment. Recombinant IL-1 beta at increasing concentrations from 30 to 3 x 10(4) U/ml did not have an effect on spontaneous and ionophore-induced acrosome reaction. In contrast, spermatozoa that underwent capacitation in media with 7 x 10(3), 7 x 10(4), and 7 x 10(5) U/ml of rINF-gamma showed a significant increase in spontaneous and induced acrosome reaction compared to the control (P < 0.001). Recombinant TNF-alpha at concentrations of 3.5 x 10(3) U/ml and 3.5 x 10(4) U/ml significantly inhibited ionophore-induced acrosome reaction (P < 0.001). Both rINF-gamma and rTNF-alpha together revealed an effect on the acrosome reaction similar to Con-A generated supernatants (1:1 and 1:10 dilution) only at the highest concentrations. Some cases of infertility may result from a defective acrosome reaction (premature acrosome loss or insufficient acrosome response to the stimulants) caused by products of activated lymphocytes and macrophages that are released into the male and female reproductive tracts.

Relationship between acrosome reactions and hamster egg penetration after ionophore challenge in absence of teratozoospermia

To determine the relationship between acrosome reactions and hamster egg penetration after ionophore challenge in nonteratozoospermic semen samples. A tertiary care center, the Andrologic Clinic, Department of Internal Medicine, University of L'Aquila. Twenty-five nonteratozoospermic patients with infertile marriages and nine fertile men. The hamster egg penetration assay and the acrosome reaction assessment were performed on capacitated sperm suspensions in basal conditions and after ionophore challenge with ionomycin. The relationship between the acrosome reactions and the hamster egg penetration was analyzed. Although in basal conditions the spontaneous acrosome reaction rate was not correlated with the hamster egg penetration, after ionophore challenge both the induced acrosome reaction rate and the acrosome reaction increase (induced minus spontaneous acrosome reaction rate) correlated with the hamster egg penetration. The highest correlation was observed between the induced acrosome reaction rate and the penetration index. Considering a penetration index = 2 as the threshold of a good response of the hamster egg penetration assay to the ionophore challenge, significantly lower acrosome reaction rates and increases were associated with poor hamster egg penetration. However, different penetration indexes occurred at the same level of induced acrosome reaction rate. The capability of sperm to react after ionophore challenge reflects to some extent the capability of the acrosome-reacted sperm to undergo the changes of the plasma membrane that are necessary for sperm-oocyte fusion. The simple evaluation of the acrosome reactions may represent a useful complement rather than a substitute for the hamster egg penetration assay in monitoring the responses of human sperm to the ionophore challenge. An impaired inducibility of the acrosome reactions may account for poor hamster egg penetrations exhibited by nonteratozoospermic semen samples.