Physiological action of oestradiol on the acrosome reaction in human spermatozoa

The acrosome is a secretory vesicle located in the sperm head. The acrosome reaction consists in the fusion of the sperm plasma membrane with the external acrosomal membrane. It has been observed that this reaction does not take place in spermatozoa incubated in cervical mucus, hydrogel that contains high concentrations of oestradiol in the peri-ovulatory period. The objective of the present study was to analyse the influence of oestradiol on the acrosome reaction in human spermatozoa to evaluate the possible inhibitory effect of this hormone. Spermatozoa were incubated in progesterone (10.1 nmol l⁻¹); oestradiol plus progesterone (oestradiol at 840 pmol l⁻¹ and progesterone at 10.1 nmol l⁻¹), oestradiol (840 pmol l⁻¹) and control (without steroidal hormones) for 30 min, 60 min, 240 min and 24 h. The acrosome reaction was evaluated by stain with Hoechst 33258 and fluorescein isothiocyanate-conjugated Pisum sativum agglutinin lectin. Progesterone-incubated spermatozoa showed the highest percentage of acrosome reaction ( P  <   0.05). Spermatozoa incubated with oestradiol and oestradiol plus progesterone showed the lowest percentage of acrosome reaction. The present study demonstrates the inhibitory role of oestradiol on the acrosome reaction, stimulated by progesterone in human spermatozoa under physiological conditions.     

Evaluation of the acrosome reaction in human spermatozoa: comparison of cytochemical and fluorescence techniques

The acrosome reaction, which is essential for fertilization, includes fusion and vesiculation of the plasma membrane with the outer acrosomal membrane of spermatozoa, thereby releasing the acrosomal content. Determination of the ability of spermatozoa to undergo the acrosome reaction has proved to be a useful parameter in evaluation of infertile patients. The objective of this study was to compare cytochemical techniques, such as double stain (Giemsa/trypan blue) and triple stain (Bismarck brown/rose bengal/trypan blue), with a fluorescence method using Pisum sativum agglutinin fluorescein conjugate and Hoechst dye N degrees 33258 (double fluorescence). Whereas the cytochemical methods are easy to perform in general laboratories, the fluorescence technique requires special and costly instrumentation. In semen obtained from fertile donors, spermatozoa were selected by the swim-up technique and the acrosome reaction was induced by incubation at low temperature. The percentages of vital and acrosome-reacted spermatozoa were determined after incubation at 4 degrees C and at room temperature. No statistically significant difference was found between double fluorescence (viability 86.3%, acrosome reaction 14.7%) and triple stain (viability 85.3%, acrosome reaction 17%) (P > 0.05). On the other hand, the double stain technique showed different values for viability (70.3%) and acrosome reaction (42.5%) (P < 0.05). In conclusion, triple stain yielded results similar to those obtained by the fluorescence technique in evaluating the acrosome reaction and can therefore easily be used in general or research laboratories.     

Sperm acrosome antigen-1, a molecule intimately involved in the regulation of the acrosome reaction: analysis of expression on spermatozoa from infertile couples

Summary.  Sperm acrosome antigen-1 (SAA-1) is a molecule on the acrosomal cap of sperm from the human and a number of mammalian and lower species. SAA-1 was initially characterized by a monoclonal antibody (mab) AG7 directed against SAA-1. Previous studies indicate that SAA-1 may play an important role in the regulation of the acrosome reaction in the human and other species. Unselected couples seeking infertility treatment were subjected to an analysis of the amount of SAA-1 present on washed husband sperm. Using indirect immunonuorescence as well as radioimmunobinding assay, the expression of SAA-1 on patient spermatozoa was found to be significantly decreased compared to a group of healthy sperm donors. The decrease in SAA-1 did not correlate well with sperm morphology. Couples entered into the study were followed for an average of 12 months, while they received infertility treatment. Most couples conceived after a variable number of treatment cycles. It is concluded that a decrease of SAA-1 expression may contribute to subfertility, which can be overcome by the aid of assisted reproduction.     

Association between freezing agent and acrosome damage of human spermatozoa from subnormal and normal semen

This experimental study compares the effects of human sperm preservation medium (HSPM) with TEST-yolk buffer (TYB) as cryoprotectants of human spermatozoa with respect to the integrity of the acrosome after the freeze-thawing procedure. Fifty-six semen samples were included in this study; 18 were subnormal (G1) and 38 were normal (G2) based on World Health Organization criteria, except for morphology, which was evaluated according to strict criteria. Each semen sample was divided into two parts: the first part was prepared for cryopreservation by the addition of HSPM (1:1) and the second by addition of TYB (1:1). Freezing was performed in liquid nitrogen vapour. Smears were made before freezing and after the thawing process for evaluation of acrosome integrity using fluorescent-lectin labelling. The mean percentage of spermatozoa with intact acrosomes in the subnormal group was 77.0 +/- 7.2% before freezing and decreased significantly (P < 0.001) after thawing: to 63.7 +/- 8.2% with the use of HSPM and 66.8 +/- 8.7% with the use of TYB. The corresponding values in the normal semen samples were 83.4 +/- 9.2%, 76.0 +/- 8.8% and 77.9 +/- 9.2%, respectively. It is obvious that the decrease in the mean percentage of spermatozoa with intact acrosome was significantly higher when using HSPM in comparison with TYB, not only for G1 (-14.9 +/- 1.9% versus -11.8 +/- 1.4%) but also for G2 samples (-13.8 +/- 1.5% versus -11.9 +/- 1.3%). In conclusion, TYB should be recommended for freeze-thawing of human spermatozoa as the first-choice cryoprotectant, for normal as well as subnormal semen samples, in order to protect the sperm acrosome from the deleterious effects of the freeze-thawing procedure.     

The zona pellucida-induced acrosome reaction of human spermatozoa involves extracellular signal-regulated kinase activation

Extracellular signal-regulated kinases (ERKs), belonging to the family of mitogen-activated protein kinases (MAPKs), are cytoplasmic and nuclear serine/threonine kinases involved in the signal transduction of several extracellular effectors. Recent evidence indicates the presence of p21 Ras and the phosphorylation of ERK1 and ERK2, suggesting the occurrence of the Ras/ERK cascade in mammalian spermatozoa. The present article describes the biological role of ERK during the acrosome reaction of human spermatozoa on stimulation with zona pellucida (ZP). The mitogen-activated protein-kinase inhibitor PD098059 was used as a pharmacological tool to study the involvement of extracellular signal-regulated kinases in the induction of the acrosome reaction in human spermatozoa. This compound significantly inhibited the acrosome reaction induced by both ZP and the calcium ionophore A23187. These results suggest that ERKs are involved in the signal transduction pathway through which ZP stimulation works during the process of fertilization.     

Effect of norethisterone and its A-ring reduced metabolites on the acrosome reaction in porcine spermatozoa

The synthetic progestin, norethisterone (NET), has been reported as a contragestational postcoital agent in humans, rodents and rabbits. The effect and molecular mechanisms of NET and its A-ring reduced metabolites, 5alpha-NET and 3beta5alpha-NET, on the acrosome reaction (AR) are unknown. The aim of this study was to assess the effect of these compounds on an in vitro progesterone-induced AR in porcine spermatozoa. The spermatozoa were obtained from semen ejaculated by proven fertile adult pigs. Seminal plasma removed and incubated under capacitating conditions was performed in TALP-Hepes medium for 4 h. Progesterone (P4) and three different progestins: norethisterone (NET), 5alpha-norethisterone (5alpha-NET) and 3beta5alpha-NET were then added at equimolar doses, and the spermatozoa were incubated for 15 min. Double-staining with PSA-FITC and Hoechst-33258 assessed the AR and sperm viability. Both P4 and NET induced the AR, while 5alpha-NET not only did not induce this process, but was able to block the effect of P4 on the spermatozoa. 3beta5alpha-NET was not able to inhibit P4 action. These results suggest that NET and its A-ring reduced metabolites act in different ways on the progesterone-induced AR in porcine spermatozoa.     

Effects on the quality of frozen-thawed alpaca （Lama pacos） semen using two different cryoprotectants and extenders

Aim： To evaluate two extenders and two cryoprotectant agents （CPA） for alpaca semen cryopreservation. Methods： Semen samples were obtained from four adult alpacas （Lama pacos） and frozen using extender Ⅰ （TRIS, citrate, egg yolk and glucose） or extender Ⅱ （skim milk, egg yolk and fructose）, each containing either glycerol （G） or ethylene glycol （EG） as CPA. Consequently, four groups were formed： 1） extender Ⅰ-G; 2） extender Ⅰ-EG; 3） extender Ⅱ-G; and 4） extender Ⅱ-EG. Semen was diluted in a two-step process： for cooling to 5 ℃ （extenders without CPA）, and for freezing （extenders with CPA）. Viability and acrosome integrity were assessed using trypan blue and Giemsa stains. Results： When compared, the motility after thawing was higher （P 〈 0.05） in groups Ⅱ-EG （20.0 %±6.7 %） and Ⅱ-G （15.3 %±4.1%） than that in groups Ⅰ-G （4.0 %±1.1%） and Ⅰ-EG （1.0 %±1.4 %）. Viable spermatozoa with intact acrosomes in groups Ⅱ-EG （18.7 %±2.9 %） and Ⅱ-G （12.7 %±5.9 %） were higher than that in groups Ⅰ-G （5.7 %±1.5 %） and Ⅰ-EG （4.0 %±1.0 %）. Conclusion： The skim milk- and egg yolk-based extenders containing ethylene glycol or glycerol to freeze alpaca semen seems to promote the survival of more sperm cells with intact acrosomes than the other extenders. （Asian J Androl 2005 Sep; 7： 303-309）     

Assessment of the effect of testosterone on the acrosome reaction of human spermatozoa

In the acrosome reaction, the spermatozoon plasma membrane fuses with the outer acrosomal membrane, resulting in the release of the acrosomal content. Several compounds, such as sex steroids, are known to modulate the acrosomal exocytosis. Testosterone regulates various functions in male reproductive physiology; however, little is known about the relationship between testosterone and the acrosome reaction. Thus, our objective was to study the effect of testosterone on the acrosome reaction of human spermatozoa. To evaluate the acrosomal exocytosis, spermatozoa were incubated with testosterone (0.2, 2.0 and 20 nmol l(-1)), progesterone and control medium for 60, 120, 240 and 1440 min. The acrosome reaction was assessed by staining with Hoechst 33258 and fluorescein isothiocyanate-conjugated P. sativum agglutinin lectin. In general, spermatozoa incubated with progesterone had the highest percentage of acrosomal exocytosis. The percentage of acrosome reaction obtained in the three treatments with testosterone differed from that observed for progesterone at 120, 240 and 1440 min (24 h). Additionally, significant differences were found between testosterone (2.0 and 20 nmol l(-1)) and progesterone after 60 min. Differences between control and the three testosterone treatments studied were obtained only at 1440 min. In general terms, these results show that testosterone exerts no inductor effects on the acrosome reaction of human spermatozoa.     

Platelet-activating factor induces acrosome reaction via the activation of extracellular signal-regulated kinase in human spermatozoa

Platelet-activating factor (PAF) affects capacitation, acrosome reaction and fertilisation potential of spermatozoa. This study investigated the underlying mechanism(s) through which PAF regulated sperm function. Our data demonstrated that PAF dose-dependently induced, whilst lyso-PAF (PAF precursor) showed no effect on acrosome reaction of capacitated human spermatozoa. Treatment with PAF for 90 min enhanced tyrosine phosphorylation and expression of extracellular signal-regulated protein kinases (ERK) 1 and 2 in human spermatozoa. Moreover, pre-treatment with the ERK inhibitor U0126 significantly and dose-dependently suppressed PAF-induced acrosome reaction. Therefore, PAF may be actively involved in the modulation of sperm acrosome reaction by interacting with ERK. The role of PAF in fertilisation warrants further investigation.     

A new method for evaluation of the acrosome reaction in viable human spermatozoa

The acrosome reaction of human spermatozoa was induced by changes of temperature. Spermatozoa were collected from fertile donors and a patient group, and selected by the "swim-up" method. The spermatozoa were treated in two different ways: Protocol I: 24 hours at room temperature followed by additional incubation at 37 degrees C for 3 hours (control), and protocol II: 24 hours at 4 degrees C followed by additional incubation at 37 degrees C for 3 hours. The acrosome reaction of the viable spermatozoa was evaluated by a new method utilizing indirect immunofluorescence with anti-outer acrosomal membrane antibodies and exposure to a hypo-osmotic medium. In fertile donors as well as in the patient group, significant induction of the acrosome reaction (20%) was evident after exposure to low temperature (4 degrees C). The spontaneous rate of acrosome reaction in the control group was below 7%.     

Reactive oxygen species influence the acrosome reaction but not acrosin activity in human spermatozoa

It is now widely accepted that the higher levels of reactive oxygen species (ROS) produced by damaged or deficient spermatozoa are associated with a loss of motility and a decreased capacity for sperm-oocyte fusion. Furthermore, earlier studies show, under physiological conditions, that some ROS may be involved in capacitation and hyperactivation of human spermatozoa. We measured ROS levels, acrosome reaction (AR) and acrosin activity (AA) in semen samples from suspected subfertile men to reveal the influence of ROS on AR and AA of human spermatozoa. Semen samples were obtained from 60 patients. Samples with > or = 1 x 10(6) leukocytes/mL were excluded from the study. ROS production was determined using a chemiluminescence technique. AR was determined using a triple stain technique. The percentage of acrosome-reacted spermatozoa after low temperature induction of the AR (test value), and the inducibility of AR (= the difference between the test value and the control), were calculated. The AA was analysed by determining the proteolytic potential of spermatozoa on gelatin plates. The mean halo diameter and percentage of halo formation in each sample were measured as AA parameters. Scatter plots of ROS levels and AR parameters showed that the percentage of acrosome reacted spermatozoa and AR inducibility were better in samples with low rather than high ROS levels. On the other hand, there were no apparent similarities between ROS and the AA parameters. Therefore, the percentage of acrosome-reacted spermatozoa and AR inducibility were significantly higher in the low than in the high ROS group (p = 0.028, p = 0.0001, respectively). In addition, there was no significant difference in AA parameters between groups. These findings suggest that lower ROS in semen may have a role in AR but excessive ROS may exert a negative influence on AR, while ROS in semen has no relationship to AA.     

Correlation between tyrosine phosphorylation intensity of a 107 kDa protein band and A23187-induced acrosome reaction in human spermatozoa

This study, performed using semen samples from 10 men, investigated the relationship between sperm protein tyrosine phosphorylation and acrosomal status in conditions supporting in vitro capacitation. Percoll-selected spermatozoa (cells from the 95% fraction) were incubated for 3 h at 37 degrees C under an atmosphere of 5% CO2 in air, in a polyvinyl alcohol (1 mg ml(-1)) containing Biggers-Whitten-Whittingham's medium, nonsupplemented or supplemented with either bovine serum albumin (BSA; fatty acid free, 3 mg ml(-1)) or 2-hydroxy-propyl-beta-cyclodextrin (2-OH-p-beta-CD; 0.5, 1, 2 mmol l(-1)). Sperm suspension in each medium was split into two aliquots. The first was used to evaluate the acrosomal status by staining with the fluorescein isothiocyanate Pisum sativum agglutinin after induction of the acrosome reaction (AR) for 45 min with 10 micromol l(-1) of A23187 calcium ionophore. The second aliquot was used for sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting, followed by a densitometric analysis. Compared with the nonsupplemented medium, BSA- or 2-OH-p-beta-CD-supplementation induced an increase in both the percentage of live acrosome-reacted sperm and the tyrosine phosphorylation intensity of the main phosphorylated 107 kDa protein. A correlation between the percentage of live acrosome-reacted sperm and the 107-kDa protein phosphotyrosine intensity was observed. Therefore, the 107 kDa protein-phosphotyrosine level measurement would bring additional information to conventional semen parameters in the assessment of the human sperm functionality.     

Inhibition of the acrosome reaction (AR) and fertilization capacity of mouse spermatozoa by norethisterone A-ring reduced metabolite (5alpha-NET)

The contra gestational effects of norethisterone and its main metabolites, 5alpha-NET and 3beta5alpha-NET, has been demonstrated in several species. However, the focus has been mainly on their effects in the uterus. We previously reported that 5alpha-NET inhibits the progesterone-induced AR in pig spermatozoa and induces severe morphological damage to fertilized mouse oocytes. In the present study, we analysed the effects of these compounds on the fertilization process in vitro. Oocytes and spermatozoa were obtained from Balb/c female and C57BL/6J male mice, respectively. Both, the AR assays and the fertilization experiments were performed under different steroid treatment schemes using progesterone as a control. Results showed that norethisterone induced the AR, while 5alpha-NET reduced the percentage of spermatozoa that had undergone progesterone-induced AR. Both 17beta-estradiol and 3beta5alpha-NET induced the AR in a considerably lower percentage of spermatozoa than progesterone. In addition, when 5alpha-NET was added to the medium simultaneously with progesterone at the moment of fertilization, the percentage of fertilized oocytes (two-cell stage) decreased by as much as 77% as compared with the control progesterone-treated group. All results suggest that these compounds can have important effects on the fertilization process.     

Clinical importance of a micro-assay for the evaluation of sperm acrosome reaction using homologous zona pellucida

This study aimed to develop an acrosome reaction assay using microvolumes of solubilized human zonae pellucidae among 35 couples attending an in vitro fertilization programme. The sperm morphology of the men was classified as g-pattern (5-14% normal forms) and/or normal pattern (> 14% normal forms). All the couples had a history of repeated poor or failed in vitro fertilization rates from previous attempts. A zona-induced acrosome reaction test was performed using homologous 0.25 zona pellucida microl-1 incubated with spermatozoa to induce the acrosome reaction. Acrosome reactions were measured with FITC-PSA staining, and expressed as the difference between zona-induced and spontaneous acrosome reaction spermatozoa. The results indicated that microvolumes of solubilized human zona pellucida could successfully be used to determine the acrosome reaction status of spermatozoa. The results were compared with in vitro fertilization rates of metaphase II oocytes, and analysed with the receiver operating characteristics curve. Receiver operating characteristics analyses divided the patients into two groups: i.e. zona-induced acrosome reaction < 15% and > 15%. The sensitivity and specificity for zona-induced acrosome reaction results versus fertilization were 93% and 100%, respectively. The correlation coefficient between zona-induced acrosome reaction and in vitro fertilization was r = 0.94 (P < 0.0001). Zona-induced acrosome reaction data can be used as an indicator for fertilization failure, thus helping clinicians to refine the therapeutic approach for infertile couples prior to the onset of the treatment.     

Preincubation in peritoneal fluid decreases the follicular fluid-induced acrosomal reactivity of human spermatozoa

Summary. The aim of this study was to determine the effects of preincubation in peritoneal fluid on the follicular fluid-induced acrosomal reactivity of human spermatozoa in vitro. Thirty women participating in our IVF-EL program were given a GnRH-analogue, highly purified FSH and hCG in order to induce superovulation. Peritoneal and follicular fluids were aspirated during pick-up laparoscopy, centrifuged, filtered and frozen until use. An aliquot of swim-up suspension from nor-mospermic semen specimens ( n =30) was incubated with peritoneal fluid or HAM-F10 for 30–180 min, and follicular fluid (in volumetric proportion approximately 50/50 with peritoneal fluid) was subsequently added. The percentage of acrosomally-reacted spermatozoa was assessed using the FITC-conjugated Pisum sativum lectin before and after incubation in peritoneal fluid or control medium, as well as after follicular fluid addition. Peritoneal fluid was not able to stimulate acrosomal reactivity; further, preincubation in peritoneal fluid decreased, but not abolished, the follicular fluid-induced acrosomal reactivity. A longer pre-incubation in peritoneal fluid was associated with a lower percentage of reacted spermatozoa in response to the addition of follicular fluid. In conclusion, our data suggest that peritoneal fluid acts maintaining spermatozoa in an unreacted status in the upper female genital tract. After mixing with follicular fluid, a phenomenon that is likely to occur at ovulation, peritoneal fluid reduces, but does not abolish, the stimulating effect of follicular fluid on acrosomal reactivity.