Introduction
Capacitation is a physiological process that spermatozoa must experience in the female reproductive tract or in vitro to obtain the ability to bind, penetrate and fertilize the egg [
1‐
3]. The capacitation is based on many molecular processes including changes in the intracellular calcium concentration [
4], rearrangement of the acrosomal matrix [
5], rearrangement of the sperm cytoskeleton [
6‐
8], phosphorylation of sperm proteins [
9,
10] and changes in the sperm plasma membrane [
11].
Since the discovery of capacitation, several methods have been developed to characterize this complex biological process. There are four major fluorescent methods, to be mentioned, and they all target different sperm characteristics: 1. CTC method detects the redistribution of the intracellular calcium in the sperm head during capacitation [
12,
13]; 2. ACR.2 recognizes the rearrangement of the acrosomal matrix by detecting changes in the accessibility of acrosin epitopes. The higher accessibility of the acrosin epitopes is a significant marker of capacitation progress [
14]; 3. FITC-phall) binds to F-actin, as actin polymerization significantly increases during capacitation progress [
15]; 4. Fluorescein isothiocyanate-conjugated antibodies, such as anti-phosphotyrosine (pY) antibody (anti-pY), detecting a capacitation dependent phosphorylation of various proteins [
16,
17].
All accounted methods can be used in various experimental protocols, e.g. CTC in fluorimetry, ACR.2 in ELISA, anti-Y in western blot etc. Fluorescent analysis is a general method suitable for all detection procedures and generally, there are two ways to perform fluorescent analysis on a cellular level: 1. by fluorescent microscopy and 2. flow cytometry. The physiological acrosome reaction (AR) is triggered by glycolytic extracellular matrix of the egg called
zona pellucida (
ZP) [
18].
A standardize and reliable evaluation of capacitation and the selection of a reliable detection methods is a methodological prerequisite for the quality assessment of fertilizing potential of individual sperm and sperm population exposed to physiological or environmental factors. In our study, we focused in detailed on analyzing the capacitation process of boar sperm through fluorescent detection using both fluorescent microscopy and flow cytometry. The aim of this work was to assess the ability of individual methods to detect relevant molecular changes during sperm capacitation; to compare their advantages and disadvantages in order to select a suitable method for evaluation of sperm capacitation and estimate the potential of individual methods to predict sperm ability to undergo ZP triggered AR and subsequently fertilize the oocyte.
Discussion
Spermatozoa have to undergo series of controlled molecular changes in female reproductive tract or in vitro before being able to bind, penetrate and fertilize the egg [
1‐
3]. Nevertheless, many molecular and physiological aspects of capacitation are still waiting to be discovered or characterized. In our study, we targeted depiction of capacitation process dynamics by multiple fluorescent techniques and compare their detection outcome. Moreover, we were able to address the ability of individual methods to detect the measurable physiological status of capacitated sperm.
CTC is considered as gold standard in fluorescent microscopy analysis of sperm capacitation state [
12,
13,
21‐
23]. Notable disadvantage of this method is the difficult assessment of individual cell fluorescent patterns under the fluorescent microscope [
12] and a relative low fluorescent intensity in combination with fast photobleaching, which make analysis difficult for human eye. On the other hand, ACR.2 antibody analysis of the specimen is much easier for the human evaluator, due to a strong positive signal and prominent acrosomal patterns. Although anti-pY and FITC-phall are able to detect changes during capacitation in fluorescent intensity in the sperm head and tail, the major disadvantage of these methods is the absence of a specific fluorescent pattern corresponding to the capacitation progress and subsequent necessity to set an intensity threshold, which is subjective. However, this disadvantage may be overcome by using a computer picture analyzer [
24]. The described challenges using anti-pY and FITC-phall methods for detection of the capacitation state resulted in lowest correlation of the data compares to those obtained by CTC and ACR.2.
The other obvious way how to overcome subjective analysis of the fluorescent intensities is to use flow cytometry. On very positive terms, in general, the flow cytometry data corresponded with those from fluorescent microscopy, with few important remarks. CTC assay may not be suitable for fluorescence detection by flow cytometry. During capacitation, the prominent change in the CTC fluorescent analysis is the appearance of the dark postacrosomal segment, a fluorescent pattern, which is not well distinguishable by cytometer detector. On the other hand, data from anti-pY and FITC-phall express much better statistical differences among individual capacitation times using flow cytometry, which might be likely due to the fact, that strong point of the flow cytometry analysis is the ability to precisely measure small differences in the fluorescent intensity. Finally, the strength of analysis using ACR.2 antibody is in the regular presence of three easily distinguishable fluorescent intensity peaks, which enables to gate them to acquire another set of useful data for statistical analysis. In general, flow cytometry generates different type of statistical parameters (e.g. arithmetical, geometrical mean of fluorescent intensity, number of accidents in set gates etc.), which are accessible for subsequent statistical analysis (e.g., comparing multiple groups by ANOVA) [
17,
23,
25‐
27]. In our study, we analyzed the percentages of sperm in the appropriate gate for ACR.2, pY and Phall and arithmetical means of fluorescent intensity for CTC. In general, the strong point of flow cytometry analysis is ability to analyze thousands of cells per sample, objective analysis and capability to precisely measure the fluorescent intensity, which changes correlate with the physiological process. The relative weaknesses of the method are the cost of the instrument and analysis and inability to exactly asses the specific morphological fluorescent patterns, a drawback, which can be now almost overcome by sophisticated cytometers, which combine the advantages of both flow cytometers and fluorescent microscopes [
28,
29].
The combination of the data from fluorescent microscopy and flow cytometry enables us to describe the temporal changes and succession of the molecular processes detected by individual analytical methods. According to our results, the first observable change is the redistribution of calcium ions (CTC FM [
30];), accompanied by highest accessibility of acrosin epitopes (ACR.2 FM, FC), which resulted from enzymatic and proteomic changes in acrosomal matrix. At later capacitation stages (180 min), the phosphorylation of sperm proteins [
31] and actin polymerization [
6,
7] are also well detectable by presented methods. At this point it is important to mention, that collecting samples at only five different times during capacitation is not sufficient for detailed characterization of the molecular changes, on which physiological process of capacitation is based and sperm life imaging is more appropriate method to study this in detail. For example, fast changes in calcium concentration should be measured by methods other than CTC [
32,
33]. Similarly, changes in actin polymerization should be measured by multiple analytical methods, since staining with FITC-phall can reflect rather changes in accessibility of actin epitopes than actin polymerization and depolymerization itself. On the other hand, CTC, contrary to methods measuring fast changes in calcium concentration, is able to reflect global changes of sperm cellular calcium homeostasis thus, likewise other methods used in this work, play important role in studying capacitation as cellular physiological process.
Due to the fact that capacitation is the physiological process, which results in the ability of sperm to undergo AR in the presence of
zona pellucida, we tested the ability of individual methods to predict number of physiologically capacitated sperm. According to the results presented in Fig.
4 and Table
2, all used methods with well-thought of experimental design (fluorescent microscopy and flow cytometry) show a good correlation with the number of cells after
zona pellucida induced AR, but there are major differences in their ability to predict the percentage of cells undergoing acrosome reaction in the presence of
zona pellucida in boars. FM CTC and FM ACR.2 are best in prediction of status physiologically capacitated sperm showing the lowest bias in Bland-Altman analysis and thus can be used as a useful tool for optimization of capacitating media [
34] or for studying effect of various compounds with the pro-, or anti-capacitation effect [
14]. On the other hand, pY method showed the lowest agreement (the highest bias) between the number of cells detected as capacitated at 240 min and the number of cells detected as AR after
ZP-induced acrosomal reaction and therefore in our arrangement highly underestimate the % of cells which will undergo the
ZP-induced AR.
Despite the fact, that our experimental approach enabled to compare four methods used for characterization of capacitation process in boar sperm, and broaden up the knowledge on interpretation of obtained data, there are still several limitations, which need to be addressed in future studies. The first is related to the evaluation of individual cells in a sample by multiple analytical methods. The co-staining of the individual samples by for example ACR.2 and anti-pY antibody would enable to conclude if individual cells are detected by both methods as non-capacitated, capacitated or AR and rigorously calculate methods agreement on the level of individual cells. This approach would not be technically possible for the CTC method since sample processing and evaluation by FM differs from antibody or FITC-phall staining. The second limitation is similar but related to AR prediction. The experimental approach used in the current study also does not allow to determine if individual cells detected as capacitated by individual methods would be exactly those undergoing AR when exposed to solubilized
ZP. The presented ratios and agreements of cells detected as capacitated by CTC and ACR.2 and cells detected as AR by PSA after
ZP-induced AR suggest that cells detected as capacitated by these two methods will undergo AR after exposure to solubilized
ZP. However, such conclusion can not be drawn for anti-pY and FITC-Phall methods. A possible approach to probe this in further detail would be to induced AR by
ZP during several times of incubation where the ratios of cells detected as non-capacitated and capacitated are different and using FC observed what population of cells (non-capacitated/capacitated) will undergo AR. However, there are again several technical limitations, since ACR.2 antibody displays intermediate fluorescent intensity peaks in earlier stages of the incubation and there are gates overlaps for anti-pY and FITC-phall, as shown in Fig.
3. Similarly, the presented approach would not suit the CTC method.
To summarize, the multiple fluorescent methods used in our study to monitor boar sperm capacitation proved to be able to detect the temporal changes of the capacitation process. However, for some methods, flow cytometry is more appropriate than fluorescent microscopy and vice versa, and this should be considered in an experimental design. Data from individual analytical methods significantly correlates, albeit there are notable differences in the correlation coefficient between them. Furthermore, a change in the temporal dynamics in individual molecular processes detected by appropriate methods were observed. These individual observations and assessments are crucial, as the differences in temporal changes allow us to make rough model of chronological succession of processes underlying capacitation. Finally, using a correlation analysis with data from ZP-induced acrosome reaction was shown, that described methods are able to predict the number of spermatozoa undergoing AR after exposure to ZP but there were major differences among individual methods. The detailed knowledge of limits of these methods commonly used for evaluation of capacitation status and prediction of sperm ability to undergo the AR should help to standardize individual results and lead to production of good comparable data among scientific laboratories.
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