Bailey JL Factors regulating sperm capacitation. Structural events and molecular mechanisms that promote mammalian sperm acrosomal exocytosis and motility. Chang MC Fertilizing capacity of spermatozoa deposited into the fallopian tubes. Chang MC Fertilization of rabbit ova in vitro. Harayama H Roles of intracellular cyclic AMP signal transduction in the capacitation and subsequent hyperactivation of mouse and boar spermatozoa. Kavanagh JP Sodium, potassium, calcium, magnesium, zinc, citrate and chloride content of human prostatic and seminal fluid.
Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. Development — PubMed Google Scholar. This is highly relevant since spermatozoa are inactive at both the transcriptional and the translational levels, excluding the possibility of increased gene expression accounting for rises in the relative abundance of some proteins in sperm lysates and placing more emphasis on dynamic molecular changes due, perhaps, to protein modification, degradation or translocation Signorelli et al.
We found that incubation with capacitation medium induced quantitative changes in proteins involved mainly in sperm motility and signaling, which are processes regulated by PTMs. While several proteomic reports can be found focused on sperm capacitation, this is the first study applying advanced proteomic strategies to analyze protein changes following the release of the acrosomal content. It is important to take into consideration that, although calcium influx is required to initiate the process, the use of a calcium ionophore does not mimic the natural trigger of the acrosome reaction, which requires binding to the zona pellucida Liu and Baker, a , b.
However, tests simulating a zona-mediated physiological acrosome reaction are restricted by the limited availability of enough biological material for proteomic studies. Calcium ionophore A is therefore an acceptable alternative when aiming to describe protein changes, since the results in terms of the quantitative loss of acrosomal contents should be equivalent. We found 9 proteins with a lower abundance after the acrosome reaction compared with lysates from CAP sperm, while 5 proteins seemed to increase their relative protein levels.
Of note, none of the peptides from proteins with a lower abundance after the acrosome reaction contained residues with potential PTMs in their sequence. In contrast, all proteins with relatively higher abundance in AR lysates could be targets of protein modifiers.
These results suggest that all proteins with apparently lower abundance in AR lysates may be released or relocated during the acrosome reaction, while those with relative increases in abundance may represent modified proteins involved in signaling processes that have lost the corresponding PTM.
In support of this hypothesis, many of the proteins with diminished protein levels after induction of the acrosome reaction are known to be components of the acrosomal vesicle, including ACR, ACRBP, ARV1, and SPACA7 and, therefore, those might be released together with the acrosomal content. However, potential changes in the distribution of sperm proteins should also be considered, as it has been observed in previous studies using a number of different methods Torabi et al.
The lysis buffer used in this study to solubilize sperm proteins most likely gained access to compartments of the sperm during and following capacitation that were hidden beforehand. A similar effect would lead to the release of proteins following the acrosome reaction where the acrosomal vesicle is lost. This does not imply that our data is a technical artifact notwithstanding the status of albumin and semenogelin, both disregarded ; instead, we propose that it is mainly the dynamic changes in cellular and structural aspects of the sperm occurring during capacitation and the acrosome reaction that is the main driver of these results, although active enzymatic destruction of sperm proteins during these processes may also contribute.
It should also be recognised that successive transitional stages take place before the complete release of the acrosome contents Buffone et al. Therefore, further studies considering different time points during the acrosome reaction would shed light into the functional involvement of these proteins. The finding of alterations in the abundance of sperm proteins already known to be involved in sperm capacitation and the acrosome reaction supports the reliability of the strategy followed by this study.
Moreover, it also increases the interest in those altered proteins never associated previously with sperm functionality. For example, our results suggest that, in our in vitro conditions, sperm maturation induced changes in the ER lipid raft-associated 2 ERLIN2 protein, which is known to mediate degradation of inositol 1,4,5-triphosphate receptors Wang Y.
Only an involvement of GGH in the metabolism of folic acid is reported in the literature DeVos et al. However, since both GGH and CCT2 showed significant alterations in protein abundance after the process of acrosome reaction, GGH is suggested as a potential candidate for further study. Another novel protein thought to be involved in the process of the acrosome reaction is the Transmembrane em24 domain-containing protein 10 TMED10 , which, although not previously associated with sperm function, may be involved in vesicular trafficking in other cellular systems Nakano et al.
Sperm capacitation and the acrosome reaction are complex processes involving many proteins and signaling pathways. Indeed, the sheer complexity precludes our obtaining a complete proteomic picture of these processes in just one MS-based study. A combination of data from high-throughput studies applying different approaches, such as enrichment in peptides with specific PTMs, the conduction of cell fractionation or the use of isobaric tags for protein quantification, among others, is required Amaral et al.
In this study, we have applied TMT labeling to quantify changes in sperm proteins in response to stimuli leading to an in vitro simulation of post-gonadal sperm maturation. We believe that our results increase the current knowledge about maturation of ejaculated sperm and suggest new players in the process. However, further experiments at peptide level are now required. By dissected the process of capacitation and the acrosome reaction at the peptide level, there is the possibility of understanding the roles of these proteins during the acquisition of competence to fertilize the oocyte and to confirm both the presence of modifications and alterations thereof in infertile patients.
In addition, our results highlight the limitations of the currently available in vitro methods to mimic the in vivo situation. Capacitation and the acrosome reaction occur within the female reproductive system and thus, secretions from and components of the female tract may have an impact on sperm acquisition of fertilizing capacity.
Therefore, the development of 3-D culture systems that mimic the oviduct and provide an in vivo- like environment to the sperm cell are desirable Ferraz et al. A better understanding of the molecular mechanism mediating fertilization is essential in order to improve current infertility diagnosis and treatment.
The donors provided their written informed consent to participate in this study. JB was involved in donor samples collection. JE performed the proteomic technology. All authors critically reviewed and approved the final version of the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We thank Raquel Ferreti and Alicia Diez for their assistance in the collection and routine analysis of semen samples.
The mean of the ratio between TMT intensities of the three donors and the internal control are shown for each protein at each sperm condition. FIGURE S4 Protein—protein interaction networks between proteins found with altered abundance in this study and all those proteins associated with Gene Ontology terms related to capacitation and acrosome reaction.
Proteins known to be functionally associated to the processes of capacitation and acrosome reaction were retrieved from the Gene Ontology Consortium database and submitted to STRING database together with the list of proteins found in this study with statistical significant differences in abundance.
The number of identified peptides, PSMs at both TMT runs, quantified peptides in all samples and quantified peptides that fit the strict selection criteria for statistics are indicated. P -values after one way ANOVA for repeated measures and t -test with Holm—Sidak adjustment are provided for both proteins and peptides.
Amaral, A. The combined human sperm proteome: cellular pathways and implications for basic and clinical science. Update 20, 40— Identification of proteins involved in human sperm motility using high-throughput differential proteomics. Proteome Res. Azpiazu, R. High-throughput sperm differential proteomics suggests that epigenetic alterations contribute to failed assisted reproduction. Bailey, J. Use of phosphoproteomics to study tyrosine kinase activity in capacitating boar sperm.
Theriogenology 63, — Baker, M. Proteomics 10, — Baldi, E. Human sperm activation during capacitation and acrosome reaction: role of calcium, protein phosphorylation and lipid remodelling pathways. Barrachina, F. Novel and conventional approaches for the analysis of quantitative proteomic data are complementary towards the identification of seminal plasma alterations in infertile patients.
Proteomics mcRA Barros, C. Membrane vesiculation as a feature of the mammalian acrosome reaction. Cell Biol. Battistone, M. Evidence for the involvement of proline-rich tyrosine kinase 2 in tyrosine phosphorylation downstream of protein kinase a activation during human sperm capacitation. Benoff, S. Fertilization potential in vitro is correlated with head-specific mannose-ligand receptor expression, acrosome status and membrane cholesterol content.
Bogle, O. Identification of protein changes in human spermatozoa throughout the cryopreservation process. Andrology 5, 10— Breitbart, H. Regulation of sperm capacitation and the acrosome reaction by PIP 2 and actin modulation. Asian J. Brucker, C. The human sperm acrosome reaction: physiology and regulatory mechanisms. An update. Update 1, 51— Buffone, M. The role of the acrosomal matrix in fertilization. Heads or tails? Structural events and molecular mechanisms that promote mammalian sperm acrosomal exocytosis and motility.
Carr, D. Identification of sperm-specific proteins that interact with A-kinase anchoring proteins in a manner similar to the Type II regulatory subunit of PKA. Castillo, J. The contribution of human sperm proteins to the development and epigenome of the preimplantation embryo. Update 24, — Codina, M. Advances in sperm proteomics: best-practise methodology and clinical potential.
Expert Rev. Proteomics 12, — Acrosome reaction as a preparation for gamete fusion. Dacheux, J. Estrogen concentrations 0. Every sample contained a protein equivalent of 10 6 cells. Representative results shown. The SDS—PAGE results from the whole sperm lysate obtained during set time points of capacitation in vitro show an increasing effect, dependent on the time of capacitation and estrogen concentration. A higher concentration of estrogens in the capacitating medium and a greater number of proteins phosphorylated on tyrosine residues were detected.
Very similar results were obtained for the 0. The results show a very similar response to selected concentrations of all four estrogens in terms of the detection of protein TyrP from the whole sperm lysate at experimental capacitation times. The proportion of spontaneous and CaI-induced acrosome reaction in control sperm samples at all experimental times during sperm capacitation in vitro in shown in Fig.
The comparison of spontaneous and CaI-induced acrosome reaction in control samples. Each data point represents five separate experimental observations. Estrogens were considered only as female hormones, but they play an important role in the male reproductive system too.
Testosterone and androstenedione are precursors for the synthesis of E 2 and estrone by cytochrome P aromatase. Consequent changes take place in the liver, where estrone is changed to E 3. This implies that estrogens are also produced in males and not only in females; therefore, they influence male reproductive parameters Broeder et al.
An estrogen action and specific cellular response are triggered through binding of these hormones to complementary ERs. ERs play an important role in activating signaling pathways leading to sperm capacitation, essential for further successful fertilization. ERs have been detected in the sperm of many species, for example, human Aquila et al.
Moreover, localization of ERB was defined by immunofluorescent labeling as a thin sickle localized over the apical region of sperm head, covering also the apical hook region.
While the presence of ERB on mature mouse epididymal sperm was confirmed, it could be considered to take part in activating signaling pathways leading to TyrP, during capacitation. GPR30 receptor could be another receptor responsible for interaction with estrogen hormones in sperm Aquila et al. Protein TyrP that occurs during mammalian sperm capacitation is crucial for sperm to obtain the ability to undergo acrosome reaction.
These events are, therefore, indicators of the sperm reproductive fitness leading to successful fertilization of the ova. TyrP is regulated by different intracellular pathways Visconti et al. This paper addresses the question, whether a rising concentration of estrogens such as E 2 , estrone, E 3 , and 17A-ethynylestradiol influences mouse sperm capacitation and acrosome reaction in vitro. Based on the presented results it can be concluded that these studied estrogens significantly stimulate the capacitation progress in a concentration-dependent manner.
The number of sperm capable of undergoing head TyrP, as well as the overall TyrP, was generally higher compared with the control. Except E 3 , all other estrogens increased the TyrP mainly at higher experimental concentrations. Synthetic estrogen 17A-ethynylestradiol affected TyrP in the sperm head in all experimental times and its effect is different from other estrogens. However, one could expect that in the beginning of the sperm capacitation the amount of sperm positive for sperm head TyrP would be lower than that in a fully capacitated population.
This was not, however, so obvious in our study. This could be explained by the fact that even at the starting time a sperm suspension was exposed to a complete capacitating medium for almost a minute; therefore, sperm were still subjected to all relevant ions and proteins.
For this reason, we cannot rule out the possibility that signaling pathways could have been activated. Also, in our study, the sperm from a very distal region of cauda epididymis were used in contrast to study of Asquith et al. The presented results show that estrogens in general increase sperm TyrP, however, each estrogen can trigger a response of different strength with respect to its concentration and also capacitation time.
A possible explanation of a non-identical estrogenic response may be due to the fact that estrogens activate diverse types or parts of signaling pathways, or bind to and activate different receptors triggering pathways leading to sperm TyrP during capacitation.
Simultaneously the effect of estrogens on the number of acrosome-reacted sperm after CaI-induced acrosome reaction was studied. CaI is usually used for the activation of releasing lytic proteins from the acrosome and simulates the in vivo zona pellucida triggered acrosome reaction Yamagata et al.
All studied estrogens affected the onset of acrosome reaction; however, in this case, estrogens significantly reduced the percentage of sperm that completed the acrosome reaction. These results correlate with recent studies Baldi et al. Similarly, our results summarizing the state of the acrosome after CaI-induced acrosome reaction correlate with an elevated amount of sperm head TyrP during sperm capacitation in presence of estrogens. On the other hand, it was presented Adeoya-Osiguwa et al.
On the other hand, those sperm capacitated with natural estrogens for a longer period did not show differences to the control, suggesting either the slowing down of capacitation or an effect that is lost or compensated in fully capacitated sperm. This proposes its prolonged adverse effect on the sperm-fertilizing ability. In mice, there is a high spontaneous acrosome reaction Johnson et al. The proportion of spontaneous and CaI-induced acrosome reaction gives, therefore, important information to assess the magnitude of the actual effect of estrogens on CaI-induced acrosome reaction.
The decrease of sperm ability to undergo the induced acrosome reaction in presence of selected estrogens falls in early capacitating times below the spontaneous acrosome rate, which may emphasize the actual effect of estrogens on ability of sperm to undergo the acrosome reaction. The results of TyrP from immunofluorescent analysis were confirmed by immunoprotein detection of the whole sperm samples. The characteristics of specific changes in the sperm head are crucial for judging the sperm ability to fertilize Stewart-Savage For this reason, we have predominantly focused on the effect of different concentrations of selected estrogens on the TyrP in the sperm head.
On the other hand, the status of the whole sperm considering TyrP in the flagellum could not be ignored. In correlation with previously published results Visconti et al.
The results of protein phosphorylation from sperm capacitated with selected estrogens showed a significant increase in the number of proteins phosphorylated on tyrosine residues in higher concentrations for E 2 , estrone, and 17A-ethynylestradiol, and in a lower concentration for E 3. As protein TyrP is ongoing in the sperm head and the flagellum, results from immunoprotein detection of the whole sperm lysate samples and the immunofluorescence detection of TyrP in the sperm head cannot be fully compared.
Nevertheless, some correlation could be made and a parallelism can be seen between these two groups of results. Before sperm—egg fusion, sperm undergoes molecular changes, which are indicated by the status of TyrP Visconti et al. These processes strictly follow one another in a precisely set order. Among many, TyrP triggers actin polymerization, which prevents the premature fusion of the plasma and outer acrosomal membranes leading to acrosome reaction Brener et al.
If the ERs are hyperstimulated by an excessive amount of estrogens, the activity of the protein tyrosine kinase is elevated and the phosphorylation on tyrosine residues remains triggered. Therefore, the phospholipase D is constantly activated leading to a consequent polymerization of actin Breitbart et al.
However, if in the beginning of the acrosome reaction the phospholipase D remains constantly upregulated by protein tyrosine kinase, the activity of protein kinase C can be possibly delayed and depolymerization of actin slowed down.
The timing of all the processes involved in the molecular changes leading to capacitation and acrosome reaction is very important and inaccurate time setting of consecutive events can result in a reduction in the sperm-fertilizing ability. One or more oocyte stimuli result in the activation of protein kinases, likely but not necessarily via activation of G-protein coupled receptors on the sperm plasma membrane and the formation of second messengers.
The kinases phosphorylate and activate proteins, continuing the biochemical cascade that ultimately results in the acrosome reaction. The role of other enzyme systems such as those involved in ion transport, proteolysis, phospholipid metabolism including that of arachidonic acid and other metabolic events, is discussed.
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