Acrosome Reaction Rate Research Articles

Group X phospholipase A2 is released during sperm acrosome reaction and controls fertility outcome in mice

Ejaculated mammalian sperm must undergo a maturation process called capacitation before they are able to fertilize an egg. Several studies have suggested a role for members of the secreted phospholipase A2 (sPLA2) family in capacitation, acrosome reaction (AR), and fertilization, but the molecular nature of these enzymes and their specific roles have remained elusive. Here, we have demonstrated that mouse group X sPLA2 (mGX) is the major enzyme present in the acrosome of spermatozoa and that it is released in an active form during capacitation through spontaneous AR. mGX-deficient male mice produced smaller litters than wild-type male siblings when crossed with mGX-deficient females. Further analysis revealed that spermatozoa from mGX-deficient mice exhibited lower rates of spontaneous AR and that this was associated with decreased in vitro fertilization (IVF) efficiency due to a drop in the fertilization potential of the sperm and an increased rate of aborted embryos. Treatment of sperm with sPLA2 inhibitors and antibodies specific for mGX blocked spontaneous AR of wild-type sperm and reduced IVF success. Addition of lysophosphatidylcholine, a catalytic product of mGX, overcame these deficiencies. Finally, recombinant mGX triggered AR and improved IVF outcome. Taken together, our results highlight a paracrine role for mGX during capacitation in which the enzyme primes sperm for efficient fertilization and boosts premature AR of a likely phospholipid-damaged sperm subpopulation to eliminate suboptimal sperm from the pool available for fertilization.

Effect of selenium and vitamin E on acrosome reaction in porcine spermatozoa.

Selenium (Se) and vitamin E (Vit-E), as an integral part of antioxidant systems, play an important role in the motility and acrosome reaction (AR) of mammalian spermatozoa. The purpose of this study was to investigate the effect of Se and Vit-E on motility, viability, AR and accumulation of ammonia in the culture medium during different incubation periods in porcine sperm. Sperm samples were washed, swum-up and incubated at 37°C for 1 and 3 h in Sp-TALP medium supplemented with sodium selenite (SS), seleon l-methionine (SeMet) and Vit-E in the presence or absence of ammonia. Sperm motility was determined on the basis of movement quality examined by phase microscopy. Viability and AR of spermatozoa were assessed by Hoechst 33258 and chlortetracycline (CTC) staining technique, and accumulation of ammonia was measured by the indophenol method. The incorporation of 14C(U)-glucose was assessed with a liquid scintillation counter. In experiment 1, the sperm motility, viability, AR and incorporation of 14C(U)-glucose increased significantly (P < 0.05) in SS, SeMet and Vit-E (5, 5 μg/l and 1.0 mM, respectively) compared with the control. In experiment 2, treatment of the sperm with SeMet and SeMet + Vit-E in the presence of 300 μM ammonia also resulted in a significant increase (P < 0.05) in the rate of motility, viability, AR and incorporation of 14C(U)-glucose. In contrast, the accumulation of ammonia was reduced by SeMet and SeMet + Vit-E compared with the other treatments. These findings indicate that SeMet and SeMet + Vit-E may play an important role in reducing the accumulation of ammonia and subsequently in increasing the rate of AR and the utilization of glucose in porcine spermatozoa.

Cholesterol addition protects membrane intactness during cryopreservation of stallion sperm

Addition of cholesterol to sperm membranes improved equine sperm stability during semen cryopreservation; however, it also reduced in vivo fertility. The objective of the present study was to determine the effects of adding cholesterol to stallion sperm prior to freezing, and subsequently removing it from frozen–thawed sperm. Semen from 12 stallions was subjected to four treatments: (T1) control, semen was diluted with Kenney extender, centrifuged, and resuspended to 100 × 10 6 spermatozoa/mL in INRA 82 freezing extender, packaged into 0.5-mL straws, cooled to 5 °C, and cryopreserved in liquid nitrogen; (T2) T1 with the addition of cholesterol before cooling (the cholesterol was incorporated to the sperm membranes with the methyl-β-cyclodextrin-cholesterol complex); (T3) T2 with post-thaw removal of the cholesterol with 0.052 mg methyl-β-cyclodextrin/50 × 10 6 sperm; and (T4) T3 with 0.156 mg methyl-β-cyclodextrin/50 × 10 6 sperm. Sperm progressive motility and functional integrity of sperm plasma membranes were evaluated microscopically and by the hyposmotic swelling test, respectively. Using flow cytometry, physical integrity of sperm plasma membranes was assessed with propidium iodide, acrosomal integrity with fluoresceinated lectin peanut agglutinin, and rate of sperm acrosome reaction induced with of the calcium ionophore A23187. Cholesterol inclusion (T2) increased the proportion of frozen–thawed sperm with intact plasma membrane. Nevertheless, sperm from T2 (9.3 ± 5.9%) had a lower rate of acrosome reaction after induction, compared to the control group (16.5 ± 11.0%). After cholesterol removal, there was no increase in the induced acrosome reaction rate (T3: 11.3 ± 7.1% and T4: 11.8 ± 9.9%). Perhaps the cyclodextrin concentrations used were too low to remove sufficient cholesterol from sperm membranes to restore the ability of cryopreserved sperm to undergo an acrosome reaction. Regardless, the addition of cholesterol to improve post-thaw sperm integrity, and its subsequent removal, still has potential for cryopreservation of stallion sperm.

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.

Effects of an Environmentally Relevant Organochlorine Mixture and a Metabolized Extract of This Mixture on Porcine Sperm Parameters In Vitro

Organochlorine chemicals are present in the environment worldwide; however, populations living in the Far North are particularly at risk because their traditional diets are mainly composed of contaminated animals (fish, seals, whales, and polar bears). It has been suggested that male fertility is globally declining, possibly because of chronic, low-level exposure to environmental contaminants. This study was designed to assess the effects on fresh sperm fertility parameters using the porcine model of 1) an environmentally relevant mixture of 15 organochlorines and 2) the metabolized extract of this mixture. In the first experiment, the organochlorine mixture (at relative concentrations of 10.5, 14.7, and 21 microg/mL polychlorinated biphenyls [PCBs]) reduced sperm total motility, progressive motility, and viability, and increased capacitation, spontaneous acrosome reaction rates, and cytosolic calcium levels, suggesting that the mixture alters the sperm membrane and is detrimental to sperm function. In the second experiment, the metabolized extract of this organochlorine mixture (at relative concentrations of 0.9, 1.8, 2.7, 3.6, and 4.5 microg/L OH-PCBs) tended to decrease only sperm total motility. In an in vitro porcine model, the mixture of organochlorines, as found in the Arctic food chain, was rapidly detrimental to sperm function at concentrations above environmental levels. In contrast, short and physiologically relevant exposure to the metabolized extract of this mixture induced only limited adverse effects on sperm motility.

Acrosome Reaction of Sperm in the Mud Crab Scylla serrata as a Sensitive Toxicity Test for Metal Exposures

In order to test the sensitivity of the sperm cell of the mud crab Scylla serrata to heavy metals, the toxic effects of Ag+, Cd2+, Cu2+, and Zn2+ on the acrosome reaction (AR) were studied by artificially inducing the AR of sperm exposed to heavy metals, counting the AR rates by light microscopy, and observing structural changes in sperm by transmission electron microscopy. The AR in S. serrata occurs at two stages. The first stage (ARI) is the eversion of the subacrosomal material. The second stage (ARII) is the ejection of the acrosomal filament. The results showed the EC50 values of the AR based on (ARI + ARII)% for Ag+, Cd2+, Cu2+, and Zn2+ were 10.02, 2.14, 13.69, and 2.21 microg/L, and the EC50 values based on ARII % of Ag+, Cd2+, Cu2+, and Zn2+ were 1.96, 0.20, 1.46, and 0.34 microg/L. The order of toxicity is Cd2+ > Zn2+ > Cu2+ > Ag+ based on the percentage of reacted sperm at the second stage. Sperm cells exposed to heavy metals showed an increased rate of swelling, shape irregularities, and the acrosomal filament of some sperm cells was, crooked, ruptured, and even dissolved. The AR of the sperm cell from S. serrata is more sensitive to the tested heavy metals compared to sea urchin sperm cell toxicity tests.

Selective isolation of acrosome-reacted human spermatozoa with progressive motility by using cell affinity chromatography on concanavalin A Sepharose.

In order to ensure fertility, mammalian spermatozoa have to undergo acrosome reaction, the most obvious morphological change during this being the exposure of the inner acrosomal membrane. In the present study, the acrosome-reacted human spermatozoa were successfully separated without loss of viability by using cell affinity chromatography on Concanavalin A (Con A) Sepharose. Con A demonstrated affinity for both the intact and the acrosome-reacted spermatozoa regardless of their viability; the latter, however, gave higher affinity than the former against Con A. Prior to the column chromatography, the immotile spermatozoa and the seminal plasma were excluded by means of a modified swim-down procedure and the resulting spermatozoa were subsequently immobilized by slow rate cooling in ice-cold water. Cell affinity chromatography was performed at 4 degrees C. To prevent mechanical trapping of the spermatozoa among the packed gel beads, the column was interconnected with a reservoir, the vertical drive of which was allowed to lose the gel bed and thereby release the trapped spermatozoa. Stepwise competitive elution with 5.0 microM mannose and 25% heat-inactivated human serum was capable of separating the intact spermatozoa and the acrosome-reacted spermatozoa from each other. The acrosome reaction rate of sperm fraction which was adsorbed to Con A Sepharose and eluted with 25% serum was found to be 83 +/- 2.3%, and motility and viability of these fractions were measured to be 80 +/- 6.3% and 83 +/- 7.6%, respectively (n = 8, mean +/- SD). The status of the acrosome in a final preparation (motility 92%, acrosome reaction rate 88%) was observed by scanning electron microscopy, and 81% spermatozoa lost their acrosome cap.

Monoclonal antibodies to canine intra-acrosomal sperm proteins recognizing acrosomal status during capacitation and acrosome reaction.

Monoclonal antibodies Ds-1 and Ds-2 specifically labelling dog sperm acrosome were prepared by immunization of mice with acetic acid extracts of dog spermatozoa. Electron microscopy and indirect immunofluorescence localized the site of Ds-1 and Ds-2 proteins inside the acrosomal vesicle. Ds-1 antibody detected 55, 76, 115, 120 and 190 kDa proteins under non-reducing conditions, and 73 kDa and 54 kDa proteins after reduction (p73/Ds-1 and p54/Ds-1). 92 kDa and 40 kDa proteins recognized by Ds-2 (p92/Ds-2 and p40/Ds-2) migrated at > 200 kDa in the absence of reducing agent. In vivo, p73/Ds-1 and p54/Ds-1 are therefore likely to be present both in free and complexed form, while all of p92/Ds-2 and p40/Ds-2 form disulfide-bonded complexes. Decrease in the rate of acrosomes stained with Ds-1 and Ds-2 was correlated with the progress of capacitation resulting in the increased rate of spontaneous acrosome reactions, as suggested by a dramatic effect of A23187. Monoclonal antibody to boar acrosin (ACR-2) recognized dog sperm acrosin homologue. A higher rate of ACR-2-negative spermatozoa was observed after capacitation and A23187 treatment compared to Ds-1 and Ds-2, indicating that proteins recognized by Ds-1 and Ds-2 are localized in a specific compartment of acrosome, distinct from acrosin and possibly representing fraction of acrosomal matrix.

Acrosomal status of human spermatozoa after follicular fluid or calcium ionophore challenge in relation to semen parameters and fertilizing capacity in vitro.

The proportion of spermatozoa that undergo spontaneous acrosome reaction in vitro is relatively low. The proportion can be enhanced by incubation with either biological inducers such as follicular fluid or chemicals like calcium ionophore. It has been suggested that improper acrosomal reaction may be a cause of fertilization failure in vitro. The objectives of the present study were to assess the acrosomal status of human sperm following follicular fluid or calcium ionophore treatment and to analyse the relationship between spontaneous and induced acrosome reaction and fertilization rates in vitro by standard in vitro fertilization (IVF) technology. In all, 53 semen samples (22 normal and 31 subnormal) were studied. The effect of calcium ionophore A 23187 and follicular fluid was assessed using the fluorescence activated cell sorter. IVF results were evaluated in relation to the acrosome status of the sperm samples. Our results demonstrate that the effect of follicular fluid on the acrosomal status correlated positively with the effect obtained by the calcium ionophore (Pearson's correlation r = 0.45). A significantly higher percentage of maximal acrosome change (P < 0.02) was found in cases where fertilization occurred (19/27), than in sperm samples that did not achieve fertilization in vitro (8/27). The present finding that follicular fluid induced acrosome reaction can serve as a predictive tool which is as good as the ionophore treatment for assessing IVF outcome, supports the use of this method for clinical purposes.

Assessment of hyperactivation, acrosome reaction and motility characteristics of spermatozoa from semen of men of proven fertility and unexplained infertility.

Semen from men of proven fertility was compared with that of men with unexplained infertility to determine differences in spermatozoal functions such as hyperactivation and acrosome reaction and spermatozoal motility characteristics. The hyperactivated spermatozoa in both groups could be visualised on the monitor of the Computer Assisted Semen Analyser and they exhibited 'circling', 'thrashing', 'starspin' and 'helical' motility patterns and the mean hyperactivation rates were not significantly different. However, 20% of the men with unexplained infertility did not exhibit hyperactivation compared to only 4% in the fertile group. Furthermore, the semen from infertile men when evaluated for hyperactivation could be categorised into two groups with those having lower hyperactivation (< 10% or < 6% after 4 and 6 h of incubation respectively), forming the first group, and those having a higher hyperactivation rate constituting the second group. In the fertile men such distinct groups were not visible and the percentage hyperactivation ranged from 1 to 16%. No significant differences were observed in the rate of acrosome reaction of fertile and unexplained infertile men. The non-hyperactivated spermatozoa from unexplained infertile men showed a significant increase in path velocity (VAP), curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) and a decrease in linearity (LIN) and straightness (STR) compared to spermatozoa from fertile men. Furthermore, the hyperactivated spermatozoa from infertile men also showed an increase in progressive velocity (VSL) (only after 2 h of incubation) and LIN and decrease in ALH and beat cross frequency (BCF) compared to spermatozoa from fertile men. The results are discussed in the light of the importance of the above spermatozoal functions and spermatozoal parameters in fertilization.

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%.

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.

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.

Effects of taurine and hypotaurine supplementation and ionophore concentrations on post-thaw acrosome reaction of dog spermatozoa

The aim of this work was to study of the effect of the amino acids (AA) taurine (T) and hypotaurine (H) and of different calcium ionophore concentrations on the ability of capacitated frozen-thawed dog sperm to undergo the acrosome reaction (AR). Fifteen ejaculates grouped into five pools were used. Sperm was frozen at a concentration of 80 × 10 6 sperm cells/mL in the Uppsala Equex extender (UE) supplemented with 25, 50 and 75 mM of either AA. The UE extender without T or H was used as control. After thawing, sperm was capacitated with Canine Capacitation Medium for 20 min. Sperm was then challenged with calcium ionophore A23178 at 0, 2.5 and 10 μM concentration and evaluated for integrity of plasma and acrosome membranes after 5, 15 and 30 min of incubation, utilizing PI/Fitc-PNA fluorescent staining and flow cytometry. Sperm cryopreserved in UE supplemented with 50 mM T (UE 50T) had higher AR rates than sperm cryopreserved with UE 75T, UE 25H and UE 50H, but AR rates were similar to semen frozen with the control extender. Challenges with 2.5 and 10 μM/L of calcium ionophore increased AR in frozen-thawed sperm incubated for 5, 15 and 30 min. The combination of calcium ionophore concentration and incubation time resulting in the highest AR rate was 10 μM and 15 min.

Effect of amino acids and dipeptides on the acrosome reaction and accumulation of ammonia in porcine spermatozoa.

Aim: The present study was designed to investigate the effect of amino acids and their dipeptides in the medium related to the urea cycle on the motility, viability, acrosome reaction (AR) and accumulation of ammonia in the medium over different incubation periods in porcine spermatozoa and to assess the utilization of glucose. Methods: Porcine spermatozoa were washed, swim-up and incubated at 37°C for 0-4h in mTALP medium supplemented with 75-600µmol/L ammonia. Amino acids (1.0mmol) or their dipeptides (2.0mmol) were added individually to the mTALP medium containing either no ammonia or 300µmol/L of ammonia. The viability and AR of porcine spermatozoa were assessed using the triple-staining technique and the accumulation of ammonia in the medium was measured using the indophenol method. Results: The motility, viability and AR were adversely affected (P<0.05) by concentrations of ammonia ≥300µmol/L compared with the control. Supplementation of l-alanyl-l-glutamine (AlaGln), l-glycyl-l-glutamine (GlyGln) and AlaGln+GlyGln in the presence of 300µmol/L ammonia significantly increase (P<0.05) the rate of motility, viability, AR, incorporation, accumulation of ammonia and oxidation of 14C(U)-glucose compared with the ammonia supplement control. Conclusion: AlaGln and GlyGln in mTALP medium were more stable and effective than the individual amino acids in reducing the accumulation of ammonia, and subsequently increasing the rate of AR and the utilization of glucose in porcine spermatozoa. (Reprod Med Biol 2008; 7: 123-131).

Characterization of Human Sperm Antigens Reacting With Anti‐Sperm Antibodies From an Infertile Female Patient's Serum

Identification of sperm antigens that elicit immunoglobulin (Ig) production and knowledge of their roles in sperm transport and fertilization may enhance diagnosis and treatment of immunologic infertility. Sperm antigens recognized by a female patient's serum anti-sperm antibodies were characterized using an indirect immunobead-binding test, immunoblot analysis, and immunochemical labeling. The anti-sperm antibodies' effect on sperm function was evaluated by acrosome induction by calcium ionophore. Immunobeads specific for IgG were bound to the head of 79% of motile donor sperm. Immunochemical labeling of antibody-binding sites was restricted to the plasma membrane over the acrosomal crescent. No labeling was observed on the inner acrosomal membrane of acrosome-reacted sperm. The antibodies reacted with 35-, 40-, 47-, and 65-kd proteins extracted from acrosome-intact donor sperm. Sperm incubated in 1:4, 1:8, 1:16, and 1:32 dilutions of anti-sperm antibody-positive serum had similar rates of spontaneous acrosome reaction and significantly lower rates of ionophore-induced acrosome reaction compared with sperm incubated in control serum. These results suggest that sperm antigens recognized by the patient's serum anti-sperm antibodies are restricted to the acrosomal region of the plasma membrane. The antibodies may impair fertility by compromising the sperm's ability to undergo capacitation and/or acrosome reaction.

Enzymatic Activity of Proprotein Convertase 4 is Important for Mammalian Fertilization.

Proprotein convertase (PC) is a member of the subtilisin/kexin family of serine proteases. PC4 is expressed selectively in male germ cells and has a preferred cleavage motif of H/R/K-(X/)n-X/K/R-R↓ (X = any amino acid except Cys, n = 1 or 3). Although Pc4 knockout (KO) male mice produce sperm, they have minimal fertilizing ability, and this may be the cause of subfertility in these KO mice. However, it is not clear from these results whether sperm PC4 is an active enzyme, whose activity is required for fertilization. Therefore, we first determined the PC activity on mouse acrosome intact (AI) and acrosome reacted (AR) live sperm and acrosomal vesicles (AV) and in the soluble acrosomal content (AC), using a PC specific fluorogenic substrate (pERTKR-MCA; p: pyro, MCA: 4-methylcoumarin-7-amide). PC cleavage of this substrate generates AMC (7-amino-4-methylcoumarin), which can be measured spectrofluorometrically. Since sperm also contain trypsin, which, at a high level, would cleave pERTKR-MCA to yield AMC, we first determined the level of sperm trypsin activity using a trypsin substrate, Boc-QAR-MCA. Our results indicated trypsin activity equivalent to 2-4 ng purified (Sigma) trypsin in 105 sperm used in the PC activity assay. These amounts of purified trypsin, however, did not cleave the PC substrate to yield AMC. Therefore, this PC substrate could be used for assaying mouse sperm PC activity. Our assays indicated that live AI and AR sperm and AV, but not the AC, contained PC activity. The differential presence of PC4 in AI/AR sperm and AV was confirmed by immunoblotting using anti-PC4 antibody. To further prove that sperm PC activity was essential for fertilization, a peptide inhibitor specific for PC4 was used for gamete treatment. Accumulated evidence indicates that PC prodomain contains a PC cleavage site that allows auto-processing of proPC to the mature active PC form. These proPC peptides, however, can inhibit PC activity. Therefore, proPC475-90 was chemically synthesized; its inhibitory effect on recombinant PC4 activity was then demonstrated (Ki= 5 µM). We further revealed that proPC475-90inhibited sperm PC activity and mouse in vitro fertilization dose-dependently, with the highest inhibitions at 500 µM. The two inhibitory effects had a high correlation coefficient (0.82), suggesting that PC activity was important for fertilization. Significantly, the sperm capacitation and acrosome reaction rates were inhibited by proPC475-90, implicating that proPC475-90acted on these two steps, pertinent to fertilization. To further understand the molecular mechanisms of proPC475-90 inhibitory action on sperm fertilizing ability, we searched for a PC natural substrate(s). We focused on sperm surface proteins known for their roles in fertilization, which contain PC cleavage motifs. Based on these criteria, ADAM2 is a promising PC candidate. Further, processing of ADAM2 from 90 kDa in testicular germ cells to 45 kDa in caudal epididymal sperm and then to 27 kDa in acrosome reacted sperm has been reported. Here we showed that the processing of the 45 kDa to the 27 kDa form actually occurred during capacitation and this processing was inhibited proPC475-90,present in the capacitating sperm suspension. Therefore, PC4 may exert its action on ADAM2 processing and the ADAM2 processed form (27 kDa) may be important for sperm fertilizing ability. Funded by CIHR, Thailand Research Fund and Ministry of Education, Thailand.

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.

Protective effect of antioxidant supplementation in sperm-preparation medium against oxidative stress in human spermatozoa

Oxidative stress induced by reactive oxygen species (ROS) is associated with an impaired fertilization ability of spermatozoa. We investigated the effects of adding antioxidants to a sperm preparation medium on the functional parameters of the spermatozoa. Spermatozoa were washed with Ham's F-10 media containing the antioxidants, ethylenediaminetetraacetic acid (EDTA) and catalase, at various concentrations, and then the ROS levels in sperm suspensions, and the forward motility, acrosome reaction, DNA integrity and lipid peroxidation of the spermatozoa were assessed. The ROS levels were significantly lower in sperm suspensions washed with the antioxidants (196 approximately 312 rlu; relative light units) than in control sperm (604 rlu, P < 0.05). The addition of 10 microM EDTA to the sperm preparation medium significantly improved the motility of the spermatozoa compared with the control group, the groups containing EDTA at other concentrations and the groups containing catalase. Catalase significantly increased the acrosome reaction rate of the spermatozoa. Both EDTA and catalase significantly decreased the DNA fragmentation rate of the spermatozoa. However, the antioxidants did not reduce lipid peroxidation. Supplementing sperm preparation medium with EDTA or catalase significantly improved the overall functional parameters of the spermatozoa by reducing the ROS levels.

The impact of spermatozoa preincubation time and spontaneous acrosome reaction in intracytoplasmic sperm injection: a controlled randomized study

To determine the optimum time interval between semen processing and incubation before intracytoplasmic sperm injection (ICSI) and correlate it with the acrosomal reaction rate. Controlled randomized study. The Egyptian IVF-ET Center. Couples with male factor infertility undergoing ICSI using ejaculated semen. The patients were prospectively randomized according to differences in sperm preincubation time before ICSI into 1-hour, 3-hour, and 5-hour groups. The status of the acrosome was studied using electron microscopy. The primary outcome measures were fertilization rate and acrosome reaction rate. Secondary outcome measures were the implantation and pregnancy rates. The rate of acrosomally reacted spermatozoa was the highest (68.2%) after 5 hours of incubation and lowest (25.6%) after 1 hour of incubation. The difference was statistically significant. The fertilization rate was the highest (74%) using spermatozoa incubated for 3 hours as compared with 1 hour (70%) and 5 hours (67%), but the difference was not statistically significant. Acrosome reaction is time dependent; the optimum incubation time of spermatozoa before ICSI was 3 hours, which resulted in the highest fertilization rate.