Percentage Of Acrosome Reaction Research Articles

Effect of bicarbonate and polyvinyl alcohol on in vitro capacitation and fertilization ability of cryopreserved equine spermatozoa.

Factors contributing to the limited success of in vitro fertilization in horses remain to be studied. In this work, we elucidated the effect of different essential capacitation media components, bicarbonate, and bovine serum albumin or polyvinyl-alcohol, and the incubation microenvironment on sperm parameters associated with capacitation, acrosome reaction, and their ability to activate oocytes via heterologous intracytoplasmic spermatozoa injection in equine cryopreserved spermatozoa. Frozen-thawed spermatozoa underwent incubation at different time intervals in either Tyrode's albumin lactate pyruvate medium (non-capacitating; NC) or Tyrode's albumin lactate pyruvate supplemented with bicarbonate, bicarbonate and polyvinyl-alcohol, bicarbonate and bovine serum albumin, polyvinyl-alcohol and bovine serum albumin alone. Protein kinase A-phosphorylated substrates and tyrosine phosphorylation levels, sperm motility, and acrosome reaction percentages were evaluated. After determining the best condition media (capacitating; CAP), heterologous intracytoplasmic spermatozoa injection on pig oocytes was performed and the phospholipase C zeta sperm localization pattern was evaluated. Incubation of frozen-thawed equine spermatozoa with bicarbonate and polyvinyl-alcohol in atmospheric air for 45min induced an increase in protein kinase A-phosphorylated substrates and tyrosine phosphorylation levels compared to NC condition. Sperm incubation in bicarbonate and polyvinyl-alcohol medium showed an increase in total motility and progressive motility with respect to NC (p ≤ 0.05). Interestingly, three parameters associated with sperm hyperactivation were modulated under bicarbonate and polyvinyl-alcohol conditions. The kinematic parameters curvilinear velocity and amplitude of lateral head displacement significantly increased, while straightness significantly diminished (curvilinear velocity: bicarbonate and polyvinyl-alcohol=120.9±2.9 vs. NC=76.91±6.9µm/s) (amplitude of lateral head displacement: bicarbonate and polyvinyl-alcohol=1.15±0.02 vs. NC=0.77±0.03µm) (straightness: bicarbonate and polyvinyl-alcohol=0.76±0.01 vs. NC=0.87±0.02) (p ≤ 0.05). Moreover, the spontaneous acrosome reaction significantly increased in spermatozoa incubated in this condition. Finally, bicarbonate and polyvinyl-alcohol medium was established as CAP medium. Although no differences were found in phospholipase C zeta localization pattern in spermatozoa incubated under CAP, equine spermatozoa pre-incubated in CAP condition for 45min showed higher fertilization rates when injected into matured pig oocytes (NC: 47.6% vs. CAP 76.5%; p ≤ 0.05). These findings underscore the importance of bicarbonate and polyvinyl-alcohol in supporting critical events associated with in vitro sperm capacitation in the horse, resulting in higher oocyte activation percentages following heterologous intracytoplasmic spermatozoa injection. This protocol could have an impact on reproductive efficiency in the equine breeding industry.

Black Tea Aqueous Extracts Improve Human Sperm Functions: An In Vitro Study

Infertility affects about 25% of couples worldwide, and oxidative stress (OS) is linked to its idiopathic etiology. Green, black, white, and oolong teas are produced from Camellia sinensis, depending on their oxidation level, and contain antioxidant properties that may enhance male reproductive functions. The study aimed to investigate the effects of black tea aqueous extract on human sperm functions in vitro. Semen samples were collected from donors, liquefied, analyzed, and divided into normal (n = 40) and abnormal (n = 19) groups using the World Health Organization 2010 criteria. Samples were washed and incubated with black tea aqueous extracts (0, 0.4, 4, 40, 405 μg/ml) for 1 hr and analyzed. Along with a considerable decrease in intracellular reactive oxygen species (ROS) production, DNA-fragmented spermatozoa, and acrosome reaction, the percentage of sperm vitality and intact mitochondrial membrane potential (MMP) increased (p<0.05). Furthermore, compared to the normal group, a substantial increase in the percentage of acrosome reaction, ROS production, and percentage of spermatozoa with fragmented DNA, while a reduction in the percentage of intact MMP and sperm vitality, was noted in the abnormal group (p<0.05). Compared to the controls, there was no significant change in motility between the normal and abnormal groups (p>0.05). Black tea’s antioxidant activity, caffeine concentration, or both may have contributed to its improvement in human sperm function in vitro.

Capacitation and Acrosomal Reaction Parameters of Fresh and Cryopreserved Rooster Semen

Background: Semen cryopreservation induces alterations in spermatozoa and changes in the plasma membrane, limiting the acrosome reaction and fertilizing ability. The research aim was to study the sperm capacitation indicators and acrosome reaction capacity in rooster semen, comparing fresh semen and post-thaw semen. Methods: Fifty ejaculates were obtained from five roosters through dorsal-ventral massage. Semen was diluted and preserved fresh or cryopreserved in Beltsville Poultry Semen Extender. In both groups of preserved semen, basic seminal parameters, capacitation and acrosome reaction were assessed. The latter were evaluated both with and without a perivitelline layer as the acrosome reaction inducer, using chlortetracycline staining. Result: The results revealed a higher percentage of sperm progressive motility in fresh semen (85.2±1.5%) compared with thawed semen (44.8±1.7%; P less than 0.05). The percentages were similar when comparing sperm with an acrosome reaction in fresh semen (10.6±1.1%) to sperm incubated without a perivitelline layer (13.2±1.7%; P greater than 0.05). However, when incubated with a perivitelline layer for 40 minutes, fresh semen spermatozoa exhibited a higher percentage of acrosome reaction (59.2±2.1%) compared with thawed semen incubated with a perivitelline layer (40.6±2.3%; P less than 0.05).

Cortisol modulates Ca2+ signaling and acrosome reaction in human sperm.

Stress and elevated cortisol levels have negative effects on fertility, although there is controversy about the effect of cortisol on human sperm. One study reported that hydrocortisone (HC), the synthetic form of cortisol, does not activate CatSper channel but is able to inhibit its activation by progesterone (Pg). However, subsequent reports showed that HC has an agonist effect on CatSper, producing intracellular Ca2+ ([Ca2+ ]i ) increases. These studies were performed using only electrophysiological techniques and fluorometric measurements of Ca2+ . Therefore, it is important to evaluate the effects of different HC concentrations on human sperm physiology using other approaches and techniques. To analyze the effects of different HC concentrations on human sperm physiology and to evaluate the association of perceived stress, anxiety and depression with acrosome reaction (AR), and semen quality. In capacitated human sperm, we tested the effects of HC on [Ca2+ ]i mobilization and AR. Furthermore, the perceived stress and symptoms of anxiety and depression were evaluated with standardized psychometric tests. HC induces a low increase in [Ca2+ ]i compared to Pg response while, at higher concentrations, HC inhibits the Pg-induced Ca2+ response. Also, low concentrations of HC induce AR and when co-incubating it with Pg, the percentages of AR are decreased. Finally, the anxiety symptoms are negatively correlated with Pg induced AR and semen quality. HC has an agonist effect on [Ca2+ ]i mobilization and AR. However, our data also suggest that HC may compete for the binding site of Pg. Additionally, the association between anxiety and decreased AR could be explained by high cortisol levels. Our data showed that HC interferes with the Pg action. Therefore, the negative correlation between anxiety symptoms and Pg-induced AR is due to the accompanying high cortisol levels of this condition and its competitive action with Pg.

CONTROL OF Neospora caninum IN SEMEN OF BULL

O objetivo do presente estudo foi desenvolver um diluente de sêmen de touros eficiente no controle de Neospora caninum. O teste dos antimicrobianos (experimento 1) - trimetoprim isolado (TRI) e associado a sulfadiazina (SDT) e ao sulfametoxazol (SMT), claritromicina (CLA), eritromicina (ERI), azitromicina (AZI) e clindamicina (CLI) - foi realizado utilizando cultivo celular e avaliado quanto à presença de taquizoítas livres e ao efeito citopático na monocamada. Os antimicrobianos que se mostraram eficazes no controle dos taquizoítos in vitro foram utilizados na preparação do diluente para o sêmen. Cada antimicrobiano foi avaliado quanto à toxicidade dos espermatozóides (experimento 2) sendo 8 grupos (7 grupos receberam antibiótico no diluente, exceto 1 grupo- grupo controle). Em relação à análise morfológica, os grupos SMT e AZI apresentaram mais defeitos de acrossoma e de cauda.Os grupos SDT, SMT e AZI apresentaram valores mais baixos da Velocidade de Trajeto. A Velocidade Progressiva foi menor nos grupos SMT e AZI e a Frequência de Batimentos de cauda também foi menor nos grupos CLI e AZI. Os valores de Velocidade Curvilinear dos grupos SDT, SMT e CLA foram menores. Os grupos SMT, ERI e AZI apresentaram valores mais baixos de Motilidade Total e Motilidade Progressiva. Quanto à citometria de fluxo, os grupos SDT, SMT e AZI apresentaram percentagens maiores de reação acrossomal e membrana plasmática lesada. Os antibióticos que podem ser adicionados ao diluente de sêmen bovino foram clindamicina, claritromicina e trimetoprim.

112 The effect of biological extenders on invitro induction of the acrosome reaction in bovine spermatozoa

Biological extenders in semen are used to protect the integrity of the sperm cells during cryopreservation. The two most common extenders are egg yolk and milk, and initial experiments describing heparin capacitation for IVF were conducted with semen frozen in egg yolk extender. However, semen of many bulls used for IVF is frozen in milk-based extender, and it is not clear whether this affects the response to heparin capacitation. This study aims to assess whether the use of milk- or egg-based extenders in frozen-thawed bovine semen affects sperm's ability to undergo the acrosome reaction, utilising either the endogenous progesterone pathway or calcium ionophore A23187 to induce the acrosome reaction following heparin capacitation. Frozen semen from 12 bulls (6 using egg yolk citrate-based extender containing 20% egg yolk, and 6 using milk-based extender) was used for no less than 3 replicates each. All semen was commercially processed and cryopreserved using the same methodology. Semen was thawed and separated using a Percoll gradient and centrifugation at 400×g for 10min, then incubated for 4h in a capacitating medium containing heparin (10μgmL−1) at a concentration of 1×106 spermmL−1. Capacitated sperm were then introduced to progesterone (10μM) or A23187 (10 μM) for acrosome induction and stained with fluorescein isothiocyanate-conjugated peanut agglutinin (5μgmL−1). After treatment and staining, the sperm were analysed using flow cytometry to determine the percentage of populations with fluorescent acrosomes, indicating an exposed acrosome and an ongoing acrosome reaction. Fluorescent microscopy was also used to confirm fluorescence in acrosome-reacting samples. Results were analysed as a 2×3 factorial design by ANOVA with extender and acrosome induction treatment as factors. For egg yolk-extended semen, the mean percent of acrosome-reacted sperm was 83.7 (±6.8), 81.8 (±7), and 15.0 (±7.5) for sperm treated with progesterone, A23187, and heparin only, respectively. Compared to milk-extended semen, for which the mean percent acrosome-reacted was 15.0 (±8.2), 14.8 (±7.4), and 12.0 (±6.8) for progesterone, A23187, and heparin only, respectively. Significant differences were observed between extenders, with egg yolk-extended semen having a significantly greater response to acrosome reaction-inducing agents than semen extended with milk-based extenders (P<0.05). Also, it was observed that progesterone induced a similar percentage of acrosome reactions as A23187. Variation between bulls in the response to heparin capacitation has been observed and cannot be eliminated in this study. The difference observed between extenders warrants further study with a split ejaculate design. The possibility that the semen extender used during cryopreservation affects the response to heparin capacitation suggests that this should be accounted for in IVF protocols to increase the efficiency of this technology.

Integrated Analyses of Phenotype and Quantitative Proteome of CMTM4 Deficient Mice Reveal Its Association with Male Fertility

The chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing family (CMTM) is a gene family that has been implicated in male reproduction. CMTM4 is an evolutionarily conserved member that is highly expressed in the testis. However, its function in male fertility remains unknown. Here, we demonstrate that CMTM4 is associated with spermatogenesis and sperm quality. Using Western blotting and immunohistochemical analyses, we found CMTM4 expression to be decreased in poor-quality human spermatozoa, old human testes, and testicular biopsies with nonobstructive azoospermia. Using CRISPR-Cas9 technology, we knocked out the Cmtm4 gene in mice. These Cmtm4 knockout (KO) mice showed reduced testicular daily sperm production, lower epididymal sperm motility and increased proportion of abnormally backward-curved sperm heads and bent sperm midpieces. These mice also had an evident sub-fertile phenotype, characterized by low pregnancy rates on prolonged breeding with wild type female mice, reduced in vitro fertilization efficiency and a reduced percentage of acrosome reactions. We then performed quantitative proteomic analysis of the testes, where we identified 139 proteins to be downregulated in Cmtm4-KO mice, 100 (71.9%) of which were related to sperm motility and acrosome reaction. The same proteomic analysis was performed on sperm, where we identified 3588 proteins with 409 being differentially regulated in Cmtm4-KO mice. Our enrichment analysis showed that upregulated proteins were enriched with nucleosomal DNA binding functions and the downregulated proteins were enriched with actin binding functions. These findings elucidate the roles of CMTM4 in male fertility and demonstrates its potential as a promising molecular candidate for sperm quality assessment and the diagnosis or treatment of male infertility.

Testicular histopathology and phosphorylated protein changes in mice with diabetes induced by multiple-low doses of streptozotocin: An experimental study

The streptozotocin (STZ)-induced diabetic model is widely used to evaluate the adverse effects of diabetes mellitus (DM) on spermatogenesis and testicular steroidogenesis. However, the actual mechanism of sub/infertility in DM males needs to be elucidated. To conduct a detailed examination of the testicular histopathology, sperm acrosome reaction (AR) status, and tyrosine-phosphorylated protein expression in the testis of male mice induced with STZ. Ten ICR mice were divided into two groups (n=5/each): control and diabetes induced by multiple low doses of streptozotocin (MLD-STZ). The control mice were intraperitoneally injected with citrate buffer, whereas MLD-STZ mice were injected with STZ at 40 mg/kg body weight for five consecutive days. At the end of the experiment (day 40), reproductive parameters, AR status, and the histopathology of the testis and epididymis were evaluated. The expression of testicular tyrosine phosphorylated proteins was examined. Blood glucose levels, AR percentages, and sperm abnormality of STZ group were significantly higher (p=0.003, 0.001, 0.000), while sperm concentration was significantly lower (p=0.001) compared to control. Histopathology of the seminiferous tubule was classified into 7 types. Additionally, abundant round cells were found in the epididymal lumen of the MLD-STZ mice. Moreover, the intensities of testicular phosphorylated proteins (170, 70, 36, 30, and 25 kDas) were markedly higher and a 120 kDa protein band was noticeably lower in the MLD-STZ mice. MLD-STZ-induced DM causes many testicular histopathologies, precocious sperm AR, and increased expression of testicular phosphorylated proteins. These findings may clarify some mechanisms of sub/infertility in DM males.

Semen evaluation and in vivo fertility in a Northern Italian pig farm: Can advanced statistical approaches compensate for low sample size? An observational study

The evaluation of sperm functionality and morphology allows discerning between high and low quality ejaculates, but does not give detailed predictive information regarding in vivo fertility. The current developments in statistical modeling have helped in carrying out reproductive studies, but their biggest limitation is in the size of the dataset to be used. The aim of the present observational study was to evaluate whether advanced statistical approaches, such as mixed effects regression models and bootstrap resampling, can help in assessing the predictive ability of semen parameters in terms of in vivo fertility (farrowing rate and litter size), on a small/medium farm with a limited number of animals.Data regarding 33 ejaculates, including viability, subjective motility and acrosome reaction, were collected. Two hundred and thirty-five sows were inseminated with an outcome of 167 deliveries and 1734 newborn piglets. In order to evaluate the relationships among the parameters measured and fertility, mixed effects regression statistical models were used. Once the covariates to be included in the final models were identified, non-parametric bootstrapping was used. The results showed that the farrowing rate was highly associated with the total number of spermatozoa and subjective motility, while litter size was associated with percentage of acrosome reaction. In conclusion, the proposed statistical approach seemed to be suitable for studies regarding reproduction and fertility, even for relatively small sample sizes. Nonetheless, larger data sets are still preferable and required in order to achieve higher reliability.

The Effect of Mahkota Dewa (Phaleria macrocarpa) Pulp Extract by Peroral Administration Toward The Percentage of Capacitation and Acrosome Reaction in Rat (Rattus norvegicus)

This research was conducted to find outthe influence of mahkota dewa (Phaleria macrocarpa) pulp extract against the percentage of capacitation and acrosome reaction of male rat (Rattus norvegicus). The experimental animals used are 20 male rats with the average body weight of 200g and were divided into four different groups. P0 group was the control group and given only CMCNa 0.5%. P1, P2,and P3 was given mahkota dewa pulp extract with a dosage of 7.5 mg/200gr BW/ day, 15mg/200gr BW/day,and 30 mg/200gr BW/day respectively through peroral administration for 14 days. The research used Completely Randoized Design (CRD) and the data was then analyzed by one way ANOVA (Analysis of Variance) followed by Duncan test with the significance of 0.5. The results show that the mahkota dewa pulp extract shows significant impact on capacitation and acrosome reaction of rat. P2 group shows the highest percentage capacitation significance the a mean of 30.00 ± 4.24 and 5.60 ± 0.89 for acrosome reaction percentage. Keywords: Phaleria macrocarpa pulp extrtact; capasitation; acrosome reaction; sperm potention

Effect of Centrifugation on Motility, Sperm Capacitation and Acrosome Reaction in Soy Bean and Avocado Seed Milk Extenders of Cryopreserved Goat Spermatozoa

Abstract Removal of seminal plasma by centrifugation (0 centrifugation, 1 centrifugation, 2 centrifugations, 3 centrifugations) and preservation in two different tris-extenders viz., avocado seed milk (ASM) and soy bean milk (SBM) based extenders were studied for their ability to support motility, in vitro capacitation and acrosome reaction of spermatozoa obtained from West African Dwarf (WAD) goat bucks during cryopreservation. Semen samples collected with the aid of artificial vagina were centrifuged for one, two and three times. The centrifuged samples were diluted with the two tris-extenders each containing 20 mL of avocado seed milk and soybean milk and cryopreserved for 30 days. The results showed higher (P < 0.05) sperm motility (P < 0.05) with increased centrifugation times. Spermatozoa that were centrifuged had higher (P < 0.05) percentage of acrosome reaction and capacitation with increased centrifugation times compared to the control. Optimal improvement in these parameters was obtained with increased centrifugation times. The findings revealed that removal of seminal plasma by centrifugation improved sperm quality of WAD goat bucks during cryopreservation and optimum improvement was achieved consistently with 3 centrifugations.

Peroxiredoxins prevent oxidative stress during human sperm capacitation.

Do peroxiredoxins (PRDXs) control reactive oxygen species (ROS) levels during human sperm capacitation? PRDXs are necessary to control the levels of ROS generated during capacitation allowing spermatozoa to achieve fertilizing ability. Sperm capacitation is an oxidative event that requires low and controlled amounts of ROS to trigger phosphorylation events. PRDXs are antioxidant enzymes that not only act as scavengers but also control ROS action in somatic cells. Spermatozoa from infertile men have lower levels of PRDXs (particularly of PRDX6), which are thiol-oxidized and therefore inactive. Semen samples were obtained from a cohort of 20 healthy nonsmoker volunteers aged 22-30 years old over a period of 1 year. Sperm from healthy donors was capacitated with fetal cord serum ultrafiltrate (FCSu) in the absence or presence of thiostrepton (TSP), inhibitor of 2-Cys PRDXs or 1-Hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol lithium (MJ33), inhibitor of calcium independent-phospholipase A2 (Ca2+-iPLA2) activity of PRDX6, added at different times of incubation. Capacitation was also induced by the dibutyryl cAMP+3-isobuty1-1-methylxanthine system. Sperm viability and motility were determined by the hypo-osmotic swelling test and computer-assisted semen analysis system, respectively. Capacitation was determined by the ability of spermatozoa to undergo the acrosome reaction triggered by lysophosphatidylcholine. Percentages of acrosome reaction were obtained using the FITC-conjugated Pisum sativum agglutinin assay. Phosphorylation of tyrosine residues and of protein kinase A (PKA) substrates were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblotting with specific antibodies. Actin polymerization was determined by phalloidin labeling. TSP and MJ33 prevented sperm capacitation and its associated actin polymerization in spermatozoa incubated with 10% FCSu (capacitation inducer) compared to non-capacitated controls (P < 0.05) without altering sperm viability. PKA substrates and tyrosine phosphorylations were prevented in FCSu-treated spermatozoa in a differential fashion depending on the type and the time of addition of the inhibitor used compared to non-capacitated controls (P < 0.05). TSP and MJ33 promoted an increase of lipid peroxidation in spermatozoa (P < 0.01) and these levels were higher in those spermatozoa incubated with the inhibitors and FCSu compared to those capacitated spermatozoa incubated without the inhibitors (P < 0.0001). Inhibition of 2-Cys PRDXs by TSP generated an oxidative stress in spermatozoa, affecting their viability compared to controls (P < 0.05). This oxidative stress was prevented by nuclephile D-penicillamine (PEN). MJ33 also promoted an increase of lipid peroxidation and impaired sperm viability compared to non-treated controls (P < 0.05) but its effect was not circumvented by PEN, suggesting that not only peroxidase but also Ca2+-iPLA2 activity of PRDX6 are necessary to guarantee viability in human spermatozoa. Not applicable. We focused on the global effect of PRDXs inhibitors on human sperm capacitation and in two of its associated phosphorylation events. Thus, other phosphorylation events and mechanisms necessary for capacitation may also be affected. PRDXs are the major antioxidant system in ejaculated spermatozoa and are necessary to allow spermatozoon to achieve fertilizing ability (capacitation and acrosome reaction). This research was supported by Canadian Institutes of Health Research (MOP 133661) and the Fonds de Recherché en Santé Quebec (FRSQS #22151) to C.O. The authors have nothing to disclose.

Heparin binding proteins and their relationship with vital sperm function tests vis-à-vis fertility of buffalo bull semen

ObjectiveTo characterize HBP of seminal plasma, fresh- and frozen-thawed sperm extracts and determine their relationship with post-thaw sperm function tests and bull fertility. MethodsBoth fresh (1–2 ml) and frozen semen (50 straws per bull) were collected from thirty breeding Murrah buffalo bulls and subjected to immunoblotting. Further, frozen-thawed semen was evaluated for first service conception rate (FSCR), percent acrosome reaction, hypoosmotic swelling test (HOST), viability, DNA integrity and total motility and linked to HBP. ResultsFourteen immunoreactive bands in seminal plasma (135, 75, 70, 65, 60, 55, 45, 37, 33, 31, 28, 24, 18 and 16 kDa), twelve in fresh sperm extracts (75, 70, 65, 55, 48, 37, 31, 28, 24, 20, 16 and 11 kDa) and thirteen in frozen-thawed spermatozoa (135, 100, 75, 70, 65, 55, 48, 45, 37, 31, 28, 24 and 20 kDa) were detected in western blots. In seminal plasma, fresh- and frozen-sperm extracts, bulls positive for 70 and 18 kDa; 55 kDa and 135, 75, 55, 45, 28 and 24 kDa, respectively, had higher (P < 0.05) FSCR as compared to their negative counterparts and had also higher (P < 0.05) percentages of most seminal parameters in positive ones. The antibody binding was most prevalent in acrosomal and postacrosomal regions of head in majority of spermatozoa. ConclusionWe have identified buffalo bull seminal HBP that influence semen quality and subsequent fertility of bulls.

Identification of Heparin Binding Proteins in Frozen-thawed Capacitated/Acrosome-reacted Spermatozoa and Their Relationship with Fertility of Buffalo Bull Semen

The present was conducted to separate heparin binding proteins (HBP) of capacitated sperm extracts and determine their relationship with acrosome reaction and semen fertility of bulls (n=30). Fourteen immunoreactive bands in capacitated spermatozoa of bulls (135, 100, 75, 70, 65, 55, 48, 45, 33, 31, 26, 24, 20 and 18 kDa) were detected in western blots following incubation with anti-HBP antibody. The frozen-thawed semen was evaluated for first service conception rate (FSCR) and percent acrosome reaction and linked to HBP. In capacitated spermatozoa, bulls positive for 70, 31, 20 and 18 kDa had significantly higher (P 0.05) in bulls positive for 135 and 100 kDa HBP than in their negative contemporary mates and exhibited an increase of 7.4% for the two proteins. A significantly higher (P < 0.05) rate of acrosome reaction was recorded in bulls with HBP of 135, 100, 70 and 18 kDa in comparison to their contemporary mates with a difference of 7.4, 7.4, 8.7 and 9.4%, respectively. Alternatively, FSCR and acrosome reaction of bulls with 75, 55 and 48 kDa were lower (P < 0.05) than their corresponding counterparts. In conclusion, HBP of 135, 100, 70 and 18 kDa did seem to activate in vitro sperm acrosome reaction and subsequent fertility of bulls.

Involvement of Protein cAMP-dependent Kinase, Phospholipase A2 and Phospholipase C in Sperm Acrosome Reaction of Chinchilla lanigera.

The mechanisms involved in fertilization are the centre of attention in order to determine the conditions required to reproduce in vitro the events that take place in vivo, with special interest in endangered species. Previous data from mouse sperm, where acrosome reaction (AR) occurs more often in the interstitium of the cumulus oophorus, contribute to strengthen the use of progesterone as a physiological inducer of this process. We studied the participation of protein kinase A (PKA), phospholipases A2 and C (PLA2 , PLC) in the AR induced by progesterone from Chinchilla epididymal spermatozoa. The addition of db-cAMP to the incubation medium caused an increase of 58% in the AR, while the use of H89 (30 μm), a PKA inhibitor, reflected a decrease of 40% in the percentage of reacted gametes. The assays conducted with arachidonic acid showed a maximum increase of 23% in the AR. When gametes were pre-incubated with PLA2 inhibitors, a dose-dependent inhibitory effect was observed. The addition of phorbol12-myristate13-acetate (10 μm) revealed higher percentages of AR induction (60%). When PLC was inhibited with neomycin and U73122, a dose-dependent decrease in AR percentages was observed. Combined inhibition of PKA, PLA2 and PLC, AR values similar to control were obtained. This work shows evidence, for the first time in Chinchilla, that progesterone activates the AC/cAMP/PKA system as well as sperm phospholipases and that these signalling pathways participate jointly and cooperatively in AR. These results contribute to the understanding of the complex regulation that is triggered in sperm after the effect of progesterone.

Oxidative stress impairs function and increases redox protein modifications in human spermatozoa.

Oxidative stress, generated by excessive reactive oxygen species (ROS) or decreased antioxidant defenses (and possibly both), is associated with male infertility. Oxidative stress results in redox-dependent protein modifications, such as tyrosine nitration and S-glutathionylation. Normozoospermic sperm samples from healthy individuals were included in this study. Samples were incubated with increasing concentrations (0-5 mM) of exogenous hydrogen peroxide, tert-butyl hydroperoxide, or diethylamine NONOate (DA-NONOate, a nitric oxide (NO∙) donor) added to the medium. Spermatozoa treated with or without ROS were incubated under capacitating conditions and then levels of tyrosine phosphorylation and percentage of acrosome reaction (AR) induced by lysophosphatidylcholine were determined. Modified sperm proteins from cytosolic, triton-soluble, and triton-insoluble fractions were analyzed by SDS-PAGE immunoblotting and immunocytochemistry with anti-glutathione and anti-nitrotyrosine antibodies. Levels of S-glutathionylation increased dose dependently after exposure to hydroperoxides (P<0.05) and were localized mainly to the cytosolic and triton-soluble fractions of the spermatozoa. Levels of tyrosine-nitrated proteins increased dose dependently after exposure to DA-NONOate (P<0.05) and were mainly localized to the triton-insoluble fraction. ROS-treated spermatozoa showed impaired motility without affecting viability (hypo-osmotic swelling test). These treated spermatozoa had tyrosine phosphorylation and AR levels similar to that of non-capacitated spermatozoa following incubation under capacitating conditions, suggesting an impairment of sperm capacitation by oxidative stress. In conclusion, oxidative stress promotes a dose-dependent increase in tyrosine nitration and S-glutathionylation and alters motility and the ability of spermatozoa to undergo capacitation.Free Spanish abstractA Spanish translation of this abstract is freely available at http://www.reproduction-online.org/content/149/1/113/suppl/DC1.

Effect of Spermine-NONOate on acrosome reaction and associated protein tyrosine phosphorylation in Murrah buffalo (Bubalus bubalis) spermatozoa

The present study was conducted to know the role of Nitric Oxide (NO) on the acrosome reaction (AR) in Murrah buffalo (Bubalus bubalis) spermatozoa. Ejaculated buffalo spermatozoa were washed, suspended in sp-TALP media containing 6mg BSA/mL and cell concentration was adjusted to 50×106cells/mL. The cells were incubated for 6h in the absence or presence of heparin (10μg/mL) to induce capacitation. Fully capacitated spermatozoa were incubated in presence of 100μg/mL Lysophosphatidyl choline (LPC, T1) or 100μM Spermine-NONOate (T2) or 100mM l-NAME (T3) or 100μM Spermine-NONOate+100mM l-NAME (T4) or 1mM db-cAMP+0.1mM IBMX (T5) or 100μM H-89 (T6) or 100μM Spermine-NONOate+100μM H-89 (T7) in combination to induce acrosome reaction. The extent of AR was assessed by dual-staining of spermatozoa with trypan blue/Giemsa stain. AR-associated tyrosine-phosphorylated proteins were detected by SDS-PAGE followed by immunoblotting using monoclonal anti-phosphotyrosine antibody. Significant (P<0.05) number of spermatozoa were acrosome reacted in Spermine-NONOate (T2) treated cells but it was significantly (P<0.05) lower than LPC (T1) induced AR. Addition of Spermine-NONOate+l-NAME (T4) resulted in non significant (P>0.05) decrease in acrosome reaction. On addition of H-89+Spermine-NONOate (T7) to sperm culture medium, resulted in significant (P<0.05) decrease in the percent acrosome reaction. Conversely, addition of db-cAMP+IBMX (T5, cAMP analogue) resulted in the significantly (P<0.05) higher number of acrosome reacted spermatozoa. Pattern of sperm protein tyrosine phosphorylation was also different in NO induced acrosome reaction compared to that of LPC. The present study concluded that nitric oxide is involved in acrosome reaction of buffalo spermatozoa by causing the tyrosine phosphorylation of proteins mainly p17 and p20 and through activation of cAMP/PKA pathway.

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.

Effect of oviductal secretion components on the fertilizing capacity of amphibian sperm: Biological and ultrastructural studies

The present study was carried out to analyze the fertilization-supporting activity of Rhinella arenarum egg-jelly components. Spermatozoa were treated with diffusible factor (DF) constituted by the components released from the jelly coat into deionized water or with full jelly (FJ) containing all the components secreted by the oviductal pars convoluta (PC) during the transit of the oocytes through the duct, or with washed jelly (WJ) constituted only by structural components. Both jellies were solubilized by ultraviolet irradiation. These gametes were used for ultrastructural and biological studies in order to determine the acrosome state and the fertilizing capacity. Additional experiments were performed by using Ca 2+, a diffusible cation present in R. arenarum jelly envelopes. Results demonstrated a marked increase in the acrosome reaction (AR) of sperm treated with FJ or DF compared to the controls (Ringer's solution), no significant differences being observed between both treatments, while WJ showed low AR percentages similar to the ones obtained with the controls. The addition of Ca 2+ induced an increase in this parameter in a dose-dependent manner, although the values reached with FJ or DF were not attained. The results of the “ in vitro” fertilization show a strong inverse association to the acrosome reaction (AR) rate. Treatment with Ca 2+ at the concentration present in the jelly (6.3 ± 0.9 mM) inhibited the fertilizing capacity as a function of incubation time, showing that at 2 min there was a decrease in the fertilization percentages compared to 10% Ringer's. Data indicated that Ca 2+ present in jelly is involved in the AR induction but suggests the possible participation of other diffusible and/or structural components of the oviductal secretion in this process.

Cholesterol and desmosterol in two sperm populations separated on Sil‐Select gradient

Sperm lipids are important for sperm viability, maturity and function. This study aimed to identify cholesterol and desmosterol composition of human spermatozoa of two sperm populations separated on Sil-Select gradient. Forty-eight males were divided into four groups namely healthy men (n = 13), asthenozoospermia (n = 11), asthenoteratozoospermia (n = 10) and oligoasthenoteratozoospermia (n = 14). Sperm cholesterol and desmosterol were estimated in two human sperm population separated by centrifugation in a discontinuous Sil-Select gradient. The results showed that cholesterol and desmosterol were the major sterols in human spermatozoa. Spermatozoa recovered from upper/lower layer interface (fraction I) had low fertilization potential, while those from the base (fraction II) had high fertilization potential. Median values of cholesterol and desmosterol in fraction I were 2.55 micromol and 0.77 micromol/10(9) spermatozoa and in fraction II were 1.16 micromol and 0.27 micromol/10(9) spermatozoa. Cholesterol/desmosterol ratio was significantly higher in fraction II than I (4.8 vs. 3.2, p < 0.01). Cholesterol, desmosterol, total phospholipids and sterols/phospholipids were negatively correlated with sperm concentrations, sperm motility, linear velocity, normal sperm morphology and acrosome reaction percentage whereas cholesterol/desmosterol ratio was positively correlated with these parameters. It is concluded that the difference in sterol composition of sperm subpopulations separated on Sil-Select gradient suggests that composition of sterols is related to sperm functions.