scholarly journals Changes in content and localization of proteins phosphorylated at tyrosine, serine and threonine residues during ram sperm capacitation and acrosome reaction

Reproduction ◽  
2009 ◽  
Vol 137 (4) ◽  
pp. 655-667 ◽  
Author(s):  
Patricia Grasa ◽  
Carmen Colas ◽  
Margarita Gallego ◽  
Luís Monteagudo ◽  
Teresa Muiño-Blanco ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Acrosome Reaction ◽  
Equatorial Region ◽  
Sperm Capacitation ◽  
Subsequent Increase ◽  
Phosphorylated Proteins ◽  
Preferential Localization ◽  
Ram Sperm ◽  
Control Samples

Previously, we reported the involvement of tyrosine phosphorylation in events that lead to ram sperm capacitation. In this study, we carried out a comparative analysis of the localization of tyrosine, serine and threonine phosphoproteins in different functional stages of ram spermatozoa (after the swim-up procedure,in vitrocapacitation, and ionophore-induced acrosome reaction) by immunofluorescence, immunocytochemistry and confocal microscopy. Capacitation increased protein tyrosine, serine and threonine phosphorylation whereas the induction of the acrosome reaction resulted in significantly decreased phosphorylation, mainly in those proteins that increased following capacitation. Control samples showed tyrosine-phosphorylated proteins restricted to the head, mainly distributed at the equatorial region with some cells also displaying an acrosomal and/or post-acrosomal localization.In vitrocapacitation promoted both tail and acrosome phosphorylation, and the acrosome reaction induced the loss of labeling on the acrosome and the subsequent increase in the post-acrosomal region and flagellum. The preferential localization of serine- and threonine-phosphorylated proteins in the equatorial and acrosomal regions found in control samples changed during capacitation, which induced tail phosphorylation in a sequential manner. After the acrosome reaction, the labeling of both phosphoamino acids decreased in the acrosome and increased in the post-acrosome. The obtained results were proved by two immunodetection techniques and strengthened by confocal microscopy, and indicate that changes in phosphorylated proteins during capacitation and acrosome reaction of ram spermatozoa may have physiological significance in consolidating certain phosphorylated proteins to specific sperm regions involved in acrosomal exocytosis and zona pellucida recognition, binding and penetration.

scholarly journals Penicillamine prevents ram sperm agglutination in media that support capacitation

Reproduction ◽  
2016 ◽  
Vol 151 (2) ◽  
pp. 167-177 ◽  
Author(s):  
T Leahy ◽  
J P Rickard ◽  
R J Aitken ◽  
S P de Graaf
Keyword(s):  
Sperm Capacitation ◽  
Dibutyryl Camp ◽  
Phosphorylated Proteins ◽  
Bicarbonate Ions ◽  
Merocyanine 540 ◽  
Highly Effective ◽  
Lactate And Pyruvate ◽  
Ram Sperm ◽  
Sperm Population

Ram spermatozoa are difficult to capacitate in vitro. Here we describe a further complication, the unreported phenomenon of head-to-head agglutination of ram spermatozoa following dilution in the capacitation medium Tyrodes plus albumin, lactate and pyruvate (TALP). Sperm agglutination is immediate, specific and persistent and is not associated with a loss of motility. Agglutination impedes in vitro sperm handling and analysis. So the objectives of this study were to investigate the cause of sperm agglutination and potential agents which may reduce agglutination. The percentage of non-agglutinated, motile spermatozoa increased when bicarbonate was omitted from complete TALP suggesting that bicarbonate ions stimulate the agglutination process. d-penicillamine (PEN), a nucleophilic thiol, was highly effective at reducing agglutination. The inclusion of 250 μM PEN in TALP reduced the incidence of motile, agglutinated spermatozoa from 76.7±2.7% to 2.8±1.4%. It was then assessed if PEN (1 mM) could be included in existing ram sperm capacitation protocols (TALP +1 mM dibutyryl cAMP, caffeine and theophylline) to produce spermatozoa that were simultaneously capacitated and non-agglutinated. This protocol resulted in a sperm population which displayed high levels of tyrosine phosphorylated proteins and lipid disordered membranes (merocyanine-540) while remaining motile, viable, acrosome-intact and non-agglutinated. In summary, PEN (1 mM) can be included in ram sperm capacitation protocols to reduce sperm agglutination and allow for the in vitro assessment of ram sperm capacitation.

scholarly journals Steroid hormone receptors and direct effects of steroid hormones on ram spermatozoa

Reproduction ◽  
2017 ◽  
Vol 154 (4) ◽  
pp. 469-481 ◽  
Author(s):  
S Gimeno-Martos ◽  
M González-Arto ◽  
A Casao ◽  
M Gallego ◽  
J A Cebrián-Pérez ◽  
...  
Keyword(s):  
Estrogen Receptors ◽  
Steroid Hormones ◽  
Acrosome Reaction ◽  
Hormone Receptors ◽  
Female Genital Tract ◽  
Equatorial Region ◽  
Steroid Hormone Receptors ◽  
Apical Region ◽  
Ram Sperm

This study was based on the assumption that steroid hormones present in the female genital tract may have a rapid effect on ram spermatozoa by interaction with specific surface receptors. We demonstrate the presence of progesterone (PR) and estrogen (ER) receptors in ram spermatozoa, their localization changes duringin vitrocapacitation and the actions of progesterone (P4) and 17β-estradiol (E2) on ram sperm functionality. Immunolocalization assays revealed the presence of PR mainly at the equatorial region of ram spermatozoa. Western blot analyses showed three bands in ram sperm protein extracts of 40–45 kDa, compatible with those reported for PR in the human sperm membrane, and both classical estrogen receptors (66 kDa, ERα and 55 kDa, ERβ). ERα was located in the postacrosomal region of all the spermatozoa and ERβ on the apical region of 63.7% of the cells. The presence of ERβ was correlated with the percentage of non-capacitated spermatozoa evaluated by chlortetracycline staining (R = 0.848,P < 0.001). This significantly decreased afterin vitrocapacitation and nearly disappeared when acrosome reaction was induced. The addition of P4 and E2 beforein vitrocapacitation resulted in a higher (P < 0.001) acrosome-reacted sperm rate compared with the control (13.0%), noticeably greater after 3 h and when added to a high-cAMP medium (37.3% and 47.0% with E2 and P4, respectively). In conclusion, the results of this study demonstrate for the first time that ovine spermatozoa have progesterone and estrogen receptors and that both steroid hormones are related with the induction of the acrosome reaction.

scholarly journals Does Melatonin Exert Its Effect on Ram Sperm Capacitation Through Nitric Oxide Synthase Regulation?

2020 ◽  
Vol 21 (6) ◽  
pp. 2093
Author(s):  
Sara Miguel-Jiménez ◽  
Melissa Carvajal-Serna ◽  
Silvia Calvo ◽  
Adriana Casao ◽  
José Álvaro Cebrián-Pérez ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Acrosome Reaction ◽  
No Production ◽  
Sperm Capacitation ◽  
Synthase Regulation ◽  
Oxide Synthase ◽  
Nos Isoforms ◽  
Ram Sperm

Nitric oxide (NO·), synthesized from L-arginine by nitric oxide synthase (NOS), is involved in sperm functionality. NOS isoforms have been detected in spermatozoa from different species, and an increment in NOS activity during capacitation has been reported. This work aims to determine the presence and localization of NOS isoforms in ram spermatozoa and analyse their possible changes during in vitro capacitation. Likewise, we investigated the effect of melatonin on the expression and localization of NOS and NO· levels in capacitated ram spermatozoa. Western blot analysis revealed protein bands associated with neuronal NOS (nNOS) and epithelial NOS (eNOS) but not with inducible NOS (iNOS). However, the three isoforms were detected by indirect immunofluorescence (IFI), and their immunotypes varied over in vitro capacitation with cAMP-elevating agents. NO· levels (evaluated by DAF-2-DA/PI staining) increased after in vitro capacitation, and the presence of L-arginine in the capacitating medium raised NO· production and enhanced the acrosome reaction. Incubation in capacitating conditions with a high-cAMP medium with melatonin modified the NOS distribution evaluated by IFI, but no differences in Western blotting were observed. Melatonin did not alter NO· levels in capacitating conditions, so we could infer that its role in ram sperm capacitation would not be mediated through NO· metabolism.

scholarly journals Signal transduction mechanisms involved in in vitro ram sperm capacitation

Reproduction ◽  
2006 ◽  
Vol 132 (5) ◽  
pp. 721-732 ◽  
Author(s):  
Patricia Grasa ◽  
José Álvaro Cebrián-Pérez ◽  
Teresa Muiño-Blanco
Keyword(s):  
Tyrosine Phosphorylation ◽  
Calcium Influx ◽  
Calcium Entry ◽  
Calcium Entry Blocker ◽  
Protein Tyrosine Phosphorylation ◽  
Sperm Capacitation ◽  
Protein Tyrosine ◽  
Entry Blocker ◽  
Ram Sperm

We validate the chlortetracycline (CTC) technique for the evaluation of capacitation and acrosome reaction-like changes in ram sperm, carrying out a double estimation of the acrosome status after treatment with lysophosphatidylcholine, using fluorescein isocyanate (FITC)-RCA/ethidium homodimer 1 (EthD-1) and CTC/EthD-1. Highly consistent results and a positive correlation between the results of acrosome-reacted sperm evaluated with both techniques were obtained. In this study, we evaluate the effects of ram sperm capacitation of BSA, Ca2+, NaHCO3and cAMP agonists and their influence on the associated protein tyrosine phosphorylation. We found a time-dependent increase in capacitation related to protein tyrosine phosphorylation, either in the absence or the presence of BSA. The addition of an increasing concentration of cholesterol to samples containing BSA did not influence results. The effect of bicarbonate was concentration-dependent, with a significantly lowered value of non-capacitated sperm in the presence 18 and 25 mM. The addition of extracellular calcium did not significantly increase either the proportion of capacitated sperm or the protein tyrosine phosphorylation signalling, although a significantly higher value of acrosome-reacted sperm was found in samples containing 4 mM Ca2+. cAMP agonists increased capacitated sperm and protein tyrosine phosphorylation signalling. The inhibition of protein kinase A by H-89 caused a decrease in sperm capacitation. Addition of a calcium-entry blocker (Verapamil; Sigma) did not influence results, which suggests that the calcium entry blocker was unable to inhibit the calcium influx associated with capacitation in ram sperm. Our findings might benefit our understanding of the biochemical mechanisms involved in mammalian sperm capacitation and ultimately, fertility.

Testase 1 (ADAM 24) a plasma membrane-anchored sperm protease implicated in sperm function during epididymal maturation or fertilization

2001 ◽  
Vol 114 (9) ◽  
pp. 1787-1794 ◽  
Author(s):  
G.Z. Zhu ◽  
D.G. Myles ◽  
P. Primakoff
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Acrosome Reaction ◽  
Phosphorylation Site ◽  
Equatorial Region ◽  
Proteolytic Processing ◽  
Mature Sperm ◽  
Kinase C

Plasma membrane-anchored proteases have key roles in cell signaling, migration and refashioning the cell surface and its surroundings. We report the first example of a plasma membrane-anchored protease on mature sperm, testase 1 (ADAM 24). Unlike other studied sperm ADAMs (fertilin (α) and (β), cyritestin) whose metalloprotease domains are removed during sperm development, we found testase 1 retains an active metalloprotease domain, suggesting it acts as a protease on mature sperm. Testase 1 is a glycoprotein (molecular mass 88 kDa), localized to the equatorial region of the plasma membrane of cauda epididymal sperm. Typically, proteolytic removal of the pro-domain is an initial activation step for ADAM proteases. The pro-domain of the testase 1 precursor (108 kDa) is proteolytically removed as sperm transit the caput epididymis to produce processed (mature) testase 1 (88 kDa). Testase 1 is unique among all studied ADAMs in that its proteolytic processing occurs on the sperm plasma membrane instead of at an intracellular site (the Golgi). Using GST-fusion proteins and a synthetic testase 1 C-terminal peptide, we found that the cytoplasmic tail of testase 1 could be phosphorylated in vitro by protein kinase C (PKC). Thus testase 1 apparently has a cytoplasmic PKC phosphorylation site(s). Protein kinase C is known to stimulate other ADAMs' protease activity. Because events of the acrosome reaction include PKC activation, we speculate that testase 1 protease function could be important in sperm penetration of the zona pellucida after sperm PKC is activated during the acrosome reaction.

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.

Effect of estrogens on sperm capacitation and acrosome reaction in vitro

2009 ◽  
Vol 81 (2) ◽  
pp. 155
Author(s):  
L. Ded ◽  
A. Dorosh ◽  
P. Dostalova ◽  
J. Peknicova
Keyword(s):  
Acrosome Reaction ◽  
Sperm Capacitation

scholarly journals Acrosome reaction inhibitor released during in vitro sperm capacitation

2003 ◽  
Vol 26 (5) ◽  
pp. 296-304 ◽  
Author(s):  
Simone G. Martins ◽  
Patricia V. Miranda ◽  
Adriano Brandelli
Keyword(s):  
Acrosome Reaction ◽  
Sperm Capacitation

Influence of progesterone on boar sperm capacitation

1995 ◽  
Vol 144 (1) ◽  
pp. 13-18 ◽  
Author(s):  
B Barboni ◽  
M Mattioli ◽  
E Seren
Keyword(s):  
Pisum Sativum ◽  
Direct Effect ◽  
Acrosome Reaction ◽  
Sperm Capacitation ◽  
Fertilizing Ability ◽  
Boar Sperm ◽  
The Status ◽  
Boar Semen ◽  
Fertilization System

Abstract This research investigates the effect of progesterone (P4) on boar sperm capacitation. Ejaculated spermatozoa were washed and incubated under capacitating conditions with or without P4. At different times of incubation samples of sperm were exposed to solubilized zonae pellucidae (ZP) and the degree of capacitation was evaluated by the incidence of zona-induced acrosome reaction (AR). The status of the acrosome was studied by using an FITCconjugated lectin (Pisum sativum agglutinin; FITC-PSA). The effect of P4 on the fertilizing ability of semen was then evaluated in an in vitro fertilization system by exposing in vitro matured oocytes to sperm preincubated for 2 or 4 h with or without P4, under capacitating conditions. PSA staining showed that P4 does not affect the incidence of spontaneous AR. By contrast, spermatozoa incubated with P4 showed a higher percentage of AR than controls after the exposure to solubilized ZP. This enhanced reactivity to ZP suggests a direct effect of P4 on sperm capacitation. The in vitro fertilization assay was consistent with these results demonstrating a higher fertilizing ability in sperm preincubated with P4 than in controls while the steroid was without effect when added only during the fertilization step. These results demonstrate that P4 improves the fertilizing ability of boar semen essentially by facilitating the process of capacitation. Journal of Endocrinology (1995) 144, 13–18

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