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

2016 ◽  
Vol 28 (2) ◽  
pp. 152
Author(s):  
M. A. Lagares ◽  
H. S. Martins ◽  
M. R. Souza ◽  
C. F. A. M. Penna ◽  
F. O. P. Leme ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Propidium Iodide ◽  
Acrosome Reaction ◽  
Fluorescent Microscopy ◽  
Calcium Ionophore ◽  
Peanut Agglutinin ◽  
Binding Assays ◽  
Frozen Semen ◽  
Fertilizing Ability

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

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

2020 ◽  
Vol 32 (2) ◽  
pp. 183
Author(s):  
L. Gatenby ◽  
K. R. Bondioli
Keyword(s):  
Acrosome Reaction ◽  
Fluorescent Microscopy ◽  
Calcium Ionophore ◽  
Fluorescein Isothiocyanate ◽  
Egg Yolk ◽  
Calcium Ionophore A23187 ◽  
Induction Treatment ◽  
Ionophore A23187 ◽  
Frozen Semen ◽  
The Mean

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&lt;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.

Sperm from beta 1,4-galactosyltransferase-null mice are refractory to ZP3-induced acrosome reactions and penetrate the zona pellucida poorly

Development ◽  
1997 ◽  
Vol 124 (20) ◽  
pp. 4121-4131 ◽  
Author(s):  
Q. Lu ◽  
B.D. Shur
Keyword(s):  
Acrosome Reaction ◽  
Direct Evidence ◽  
Calcium Ionophore ◽  
Wild Type ◽  
Sperm Surface ◽  
Gamete Recognition ◽  
Dependent Signal ◽  
Relative Inability ◽  
Egg Coat

A variety of sperm surface components have been suggested to mediate gamete recognition by binding to glycoside ligands on the egg coat glycoprotein ZP3. The function of each of these candidate receptors is based upon varying degrees of circumstantial and direct evidence; however, the effects on fertilization of targeted mutations in any of these candidate receptors have not yet been reported. In this paper, we describe the effects of targeted mutations in beta1,4-galactosyltransferase, the best studied of the candidate receptors for ZP3. Surprisingly, galactosyltransferase-null (gt[−/−]) males are fertile; however, sperm from gt(−/−) males bind less radiolabeled ZP3 than wild-type sperm, and are unable to undergo the acrosome reaction in response to either ZP3 or anti-galactosyltransferase antibodies, as do wild-type sperm. In contrast, gt(−/−) sperm undergo the acrosome reaction normally in response to calcium ionophore, which bypasses the requirement for ZP3 binding. The inability of gt(−/−) sperm to undergo a ZP3-induced acrosome reaction renders them physiologically inferior to wild-type sperm, as assayed by their relative inability to penetrate the egg coat and fertilize the oocyte in vitro. Thus, although ZP3 binding and subsequent induction of the acrosome reaction are dispensable for fertilization, they impart a physiological advantage to the fertilizing sperm. A second strain of mice was created that is characterized by a loss of of the long galactosyltransferase isoform responsible for ZP3-dependent signal transduction, but which maintains normal levels of Golgi galactosylation. Sperm from these mice show that the defective sperm-egg interactions in gt(−/−) mice are due directly to a loss of the long galactosyltransferase isoform from the sperm surface and are independent of the state of intracellular galactosylation during spermatogenesis.

92 EVALUATION OF BOAR SPERM FUNCTIONALITY AFTER A CUSHIONED CENTRIFUGATION TECHNIQUE

2006 ◽  
Vol 18 (2) ◽  
pp. 154
Author(s):  
J. Gadea ◽  
S. Martínez-Miró ◽  
G. Decuadro-Hansen ◽  
C. Matás
Keyword(s):  
Seminal Plasma ◽  
Acrosome Reaction ◽  
Sperm Viability ◽  
Lipid Packing ◽  
Lipid Disorder ◽  
Merocyanine 540 ◽  
Centrifuge Tube ◽  
Before And After ◽  
Centrifugation Technique

Separation of sperm from seminal plasma is required in most semen freezing procedures. Semen is typically subjected to centrifugation to concentrate sperm into a pellet and allow removal of the seminal plasma prior to dilution in freezing extender. Centrifugation is a relatively effective method to recover sperm, however, the process also causes considerable sperm damage. The use of a dense, inert, and isotonic solution as a cushion in the bottom of the centrifuge tube allows a greater centrifugation speed to be applied and results in greater sperm recovery. The aim of the present work was to evaluate the effects of this cushioned centrifugation technique on in vitro sperm viability and functionality. Sperm-rich fractions from 16 fertile boars were diluted and cooled to 15�C; then subsamples were centrifuged by one of two different techniques. A standard method (SM), 800 g for 10 min in 50-mL tubes (Westendorf et al. 1975 Dtsch. Tier�rztl. Wschr. 82, 261-267) and a cushioned method (CM), 1000 g for 20 min using 45 mL of diluted semen on 5 mL of an isotonic iodixanol solution (60% w/v gradient) were performed. Sperm samples were stained with merocyanine 540 (M540) and Yo-Pro 1 (Harrison et al. 1996 Mol. Rep. Dev. 45, 378-391) to detect changes in lipid packing disorder of the plasma membrane. Another set of sperm samples was incubated in the presence of (0.7 �M) 22,72-dichlorodihydrofluorescein diacetate (Gadea et al. 2005 J. Androl. 26, 396-404) to estimate production of reactive oxygen species (ROS). A final set of sperm samples was stained with peanut aggultinin-fluorscein isothiocyanate (PNA-FITC) and propidium iodide to evaluate the acrosome reaction. All of these parameters were evaluated by flow cytometry before and after centrifugation. ANOVA analysis revealed that centrifugation altered lipid packing disorder and viability. Raw semen (RS) had a larger number of viable low lipid disorder sperm than centrifuged semen (RS = 86.9a vs. SM = 81.64b vs. CM = 80.6b, P < 0.01) and a decreased number of dead sperm cells (RS = 9.5a vs. SM = 15.0b vs. CM = 16.3b, P < 0.01). However, the cushioned and standard centrifugation methods yielded similar results for all the parameters measured. No significant differences were found for generation of ROS or in the number of sperm exhibiting the acrosome reaction. In conclusion, compared to the standard centrifugation method, this simple cushioned modification is a more efficient means of processing boar semen for freezing because significantly less sample losses are detected; also, it provides similar levels of sperm viability and functionality, and consequently a higher number of doses per ejaculation can be produced.

303 FERTILIZATION-RELATED PARAMETERS OF FROZEN - THAWED SPERMATOZOA FROM SUBFERTILE JAPANESE BLACK CATTLE

2007 ◽  
Vol 19 (1) ◽  
pp. 266
Author(s):  
K. Kuroda ◽  
M. Fukushima ◽  
M. Miyake ◽  
H. Harayama
Keyword(s):  
Acrosome Reaction ◽  
Economic Losses ◽  
Japanese Black Cattle ◽  
Cell Stage ◽  
Causal Factors ◽  
Mean Values ◽  
Progressive Movement ◽  
Male Subfertility ◽  
Fertilizing Ability

The subfertility derived from male factors is a problem of concern in domestic animals, because it could cause a disintegration of the breeding system and large economic losses, particularly when the subfertility affects genetically superior male animals. Therefore, it is urgent that causal factors of male subfertility be determined. Recently, an increasing number of subfertile bulls have been found among Japanese Black cattle, which is a representative breed of Japanese beef cattle. The purpose of the present study was to elucidate causal factors of male subfertility in Japanese Black cattle. Frozen–thawed spermatozoa from 8 subfertile (S1-S8) and 7 fertile (F1–F7, control) bulls were used for the assessment of fertilization-related parameters. The data obtained from each subfertile bull in the following experiments were individually compared with the mean values of the fertile bull group. In Experiment 1, sperm motility was observed in samples that were frozen-thawed and subsequently washed in PBS. Many spermatozoa (higher than 65%) exhibited flagellar movement in all samples from fertile and subfertile bulls. However, the percentages of progressively motile spermatozoa from 2 subfertile bulls were significantly lower (S2: 6%; S7: 7%; P &lt; 0.05, ANOVA and Tukey&apos;s multiple range tests) than those from fertile bulls (average: 37&percnt;). Moreover, rapidly progressive movement was not observed in the spermatozoa from 4 subfertile bulls (S1, S2, S6, and S7). These data suggest abnormality in the motility system of sperm flagella in these 4 subfertile bulls. In Experiment 2, the capacitation&ndash;acrosome reaction state of frozen&ndash;thawed spermatozoa was examined by the CTC-staining assay. More than 50&percnt; of the frozen&ndash;thawed spermatozoa from 4 subfertile bulls (S5&ndash;S8) were prematurely progressing in the capacitation state immediately after washing and resuspension in the medium lacking CaCl2. Moreover, the addition of CaCl2 to the medium induced acrosomal loss in these sperm samples (percentages of spermatozoa without the acrosome: 36&ndash;49&percnt;). These findings indicate the occurrence of premature capacitation and a spontaneous acrosome reaction in spermatozoa from these 4 subfertile bulls. In Experiment 3, the in vitro fertilizing ability of frozen&ndash;thawed spermatozoa was evaluated by the IVF test. The percentages of fertilized eggs with both male and female pronuclei or developmental rates of fertilized eggs to the 2-cell or 4-cell stage were significantly lower in the spermatozoa from S6 to S8 bulls than in those from fertile bulls (P &lt; 0.05, chi-squared tests). This may suggest that spermatozoa from these 3 subfertile bulls hardly accomplish the normal fertilization process. In summary, low progressive motility and low in vitro fertilizing ability because of premature capacitation were found in the spermatozoa from subfertile bulls. It is therefore possible that these are causal factors for the subfertility of male Japanese Black cattle.

322 EFFECT OF SODIUM NITROPRUSSIDE ON BUFFALO SPERM CAPACITATION IN VITRO

2007 ◽  
Vol 19 (1) ◽  
pp. 276 ◽  
Author(s):  
L. Boccia ◽  
L. Attanasio ◽  
A. De Rosa ◽  
G. Pellerano ◽  
R. Di Palo ◽  
...  
Keyword(s):  
Sodium Nitroprusside ◽  
Acrosome Reaction ◽  
Production Efficiency ◽  
Control Group ◽  
Sperm Capacitation ◽  
Promoting Effect ◽  
Vitro Fertilization ◽  
Domestic Species ◽  
Frozen Semen

The overall in vitro embryo production efficiency in buffalo is hampered by the poor fertilization rate. It is known that the quality of the frozen semen may affect fertilization efficiency. However, it is not possible to rule out that the process of capacitation, required by spermatozoa to acquire the fertilizing ability, is impaired in the in vitro fertilization (IVF) system. Although several agents have been proven to induce sperm capacitation in vitro, heparin treatment is still the most efficient method in most of the domestic species. There is evidence that capacitation is part of an oxidative process and that nitric oxide (NO) acts as a capacitation inducer in human (Herrero et al. 1999 Biol. Reprod. 61, 575–581) and bovine (Rodriguez et al. 2005 Anim. Reprod. Sci. 85, 231–242) spermatozoa. The aim of the present study was to evaluate whether sodium nitroprusside (SNP), a well-known generator of NO in vitro, improves buffalo sperm capacitation in vitro. Frozen–thawed sperm from a bull previously tested for IVF were treated by swim-up in order to select only the motile population. Spermatozoa were incubated in the presence of 0.01 mM heparin (control group) for 1 h (n = 266), 2 h (n = 270), and 3 h (n = 306), and in the presence of 10 �M SNP for 1 h (n = 302), 2 h (n = 286), and 3 h (n = 260). The concentration of SNP was chosen on the basis of a preliminary dose-response trial (0.1 �M, 1 �M, and 10 �M). Following incubation with these agents, sperm were exposed for 15 min to 60 �g mL-1 of lysophosphatidylcholine, an agent known to induce acrosome reaction only on capacitated spermatozoa. Trypan blue was used first to differentiate live from dead spermatozoa and the dried smears were then fixed in 37% formaldehyde and stained with Giemsa for acrosome evaluation by microscopic examination. The proportion of acrosome-reacted spermatozoa in each group was used to assess the efficiency of capacitation under different incubation conditions. Differences between groups were analyzed by chi-squared test. No dead spermatozoa were found in all groups. Following 1-h sperm treatment with either heparin or SNP, the proportion of acrosome-reacted spermatozoa was similar (35.3% vs. 28.5%, respectively). However, extending the incubation time to 2 h, SNP significantly (P &lt; 0.01) increased the incidence of acrosome reaction compared to heparin (60.1% vs. 44.1%, respectively). Analogously, when the sperm treatment was prolonged to 3 h, SNP gave a significantly (P &lt; 0.01) higher percentage of acrosome reaction compared to the control (68.8% vs. 36.6%, respectively). In conclusion, sperm treatment with SNP for either 2 or 3 h significant improved the efficiency of buffalo sperm capacitation in vitro compared with heparin, that is, the capacitating agent currently used in the IVF system. The promoting effect of SNP indirectly indicates that NO acts as a capacitation inducer in buffalo spermatozoa. Finally, these results suggest the need to evaluate the effect of SNP on the fertilizing capability of buffalo spermatozoa in vitro.

In vitro induction of the acrosome reaction in ovine spermatozoa by calcium ionophore A23187

2003 ◽  
Vol 51 (1) ◽  
pp. 103-109 ◽  
Author(s):  
Ö. Uçar ◽  
T. J. Parkinson
Keyword(s):  
Phase Contrast ◽  
Acrosome Reaction ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Contrast Microscopy ◽  
The Relationship ◽  
In Vitro Induction ◽  
Artificial Vagina

The relationship between concentration of calcium ionophore A23187 and incubation time upon the proportion of spermatozoa undergoing acrosome reaction (AR) in vitro was investigated in rams from a commercial artificial insemination (AI) program. Two ejaculates were collected by artificial vagina from each of nine rams of three breeds (Finn Dorset, Charolais and Suffolk) aged 8-36months. Each ejaculate was diluted in a skimmed milk extender. Spermatozoa were thereafter incubated for 45 or 60min in modified Tyrode's medium (TALP) which contained either zero, 0.1 or 1.0µM/l A23187. After fixing in 10% formaldehyde, the number of spermatozoa that had undergone AR was determined by phase contrast microscopy. In pre-incubation samples, 21.3± 3.3% of spermatozoa had undergone AR. Percentages of acrosome reacted spermatozoa were significantly (P<0.001) increased after incubation with A23187. After incubation with 0.1µM/l A23187 for 45 and 60min there were 22.4±3.0% and 31.7±4.3% acrosome reacted spermatozoa, respectively. After incubation with 1.0µM/l A23187 for 45 and 60min there were 46.2±6.5% and 53.8±5.9% acrosome reacted spermatozoa, whilst corresponding numbers in control samples were 17.0±2.7% and 22.3±4.2%. There was also a significant (P<0.001) effect of individual animals upon the responses to different concentrations of A23187. These findings indicate that (i) A23187 can be used to assess the AR of ovine spermatozoa in vitro and (ii) there are effects of individual animals upon the proportion of spermatozoa undergoing AR.

scholarly journals Progress of sperm IZUMO1 relocation during spontaneous acrosome reaction

Reproduction ◽  
2014 ◽  
Vol 147 (2) ◽  
pp. 231-240 ◽  
Author(s):  
Natasa Sebkova ◽  
Lukas Ded ◽  
Katerina Vesela ◽  
Katerina Dvorakova-Hortova
Keyword(s):  
Fusion Protein ◽  
Acrosome Reaction ◽  
Mating Behaviour ◽  
Calcium Ionophore ◽  
Cumulus Cells ◽  
Sperm Head ◽  
High Rate ◽  
Field Mice ◽  
Species Specific

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

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