Assessment of In Vitro Capacitation in Stallion Spermatozoa
Barend M Gadella,^1,3 Rahul Rathi,² Mart M Bevers,¹ and Ben Colenbrander^1,2
Department of Farm Animal Health¹ and of Equine Sciences² of the Graduate School of Animal Health, Department of Biochemistry and Cell Biology³ of the Institute of Biomembranes 
Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

Introduction

Capacitation is an important, but rather incompletely understood phenomenon that a spermatozoon undergoes before it can fertilize the oocyte. It is a complex of a number of sub-phenomena such as: hyperactivation, plasma membrane alterations, ion fluxes, etc. leading to more complicated internal changes by way of signal transduction. A reliable and easy procedure to assess the capacitation state of sperm cells is not yet available. The only accepted capacitation assays are the chlortetracycline (CTC) staining and the detection of hypermotility. This paper discusses the validity of capacitation assays in stallion spermatozoa. Moreover, new sophisticated flow cytometric assays are compared to the classic assays to detect capacitation and acrosome reactions in stallion sperm cells.

Hypermotility Assessment is Inaccurate

Hypermotility occurs spontaneously in sperm cells that are exposed to in vitro capacitation media that contain bicarbonate. Bicarbonate activates adenylate cyclase directly and thereby elevates intracellular cAMP concentration, which has been shown to induce hyperactivation in sperm of several mammalian species. Hence, it was hypothesized that incubation in medium with bicarbonate would lead to stallion sperm hyperactivation. Hyperactivation has been tried to be defined objectively in human spermatozoa on the basis of different motility parameters obtained from computer assisted sperm analysis (CASA) systems like average lateral head displacement, curvelinear velocity, straight line velocity, average path velocity and  beat cross frequency as well as other derived motility parameters (like wobble, linearity and straightness of sperm movements). Such definitions do not exist for stallion spermatozoa. High curvelinear velocity and average path velocity were found to be most contributing factors for hypermotile stallion sperm although, other motility parameters like wobble, linearity, and straightness of sperm movement were extra tools to identify hyperactive spermatozoa. The capacitative treatments did not lead to a clear induction of stallion sperm hypermotility; the percentage of hyperactive spermatozoa increased and/or decreased with time inconsistently and it varied between ejaculates. The calcium dependence of the induction of hypermotility of stallion sperm cells was also tested. Addition of Ca²⁺ ionophore allowed entry of extracellular Ca²⁺ in capacitated sperm cells. However, it also diminished stallion sperm motility almost entirely (this phenomenon was also observed in semen of other mammalian species by other research groups), whereas, more than 35% of the cells remained vital even after a 5 hr incubation experiment. The collapse of cell motility and the increased cell deterioration induced by Ca²⁺ ionophore are probably due to the unphysiological elevations of intracellular Ca²⁺ (>0.5mM). This high Ca²⁺ can overwhelm both mitochondrial and plasma membrane pumps allowing free entry of calcium ions with a coincident rise in respiratory activity and probably an effect on sperm motility. The detection of stallion sperm hyperactivation in vitro is also surrounded with shortcomings of the observation technology: (i) Hyperactive cells showed a typical biphasic motility characteristic in which the hyperactive spermatozoon first makes its characteristic vigorous non-progressive movement (i.e. with high curvelinear velocity and average linear path) but then suddenly becomes static, remains static for some time before showing hypermotility again. This sperm behavior might contribute to underestimating the percentage of hyperactive spermatozoa obtained by CASA analysis of samples assessed for less than 1 second. (ii) Some spermatozoa moved at a speed too high to be captured in CASA system to keep track of the movement. Therefore it is possible that observed data of two or more tracks may in fact be derived from one cell with extreme movements, which may lead to errors in the estimation of sperm hypermotility characteristics. In order to overcome these pitfalls the option was to visually analyze all individual tracks, and to analyze around 200 tracks per assay. However, in a practical environment this option is not feasible. The hyperactivation assessments by CASA are therefore considered to be unsuitable to be used as capacitation assays for stallion sperm samples.

Better Alternatives for the CTC Assessment

Chlortetracycline (CTC) is a fluorescent antibiotic, which on a Ca²⁺ dependent manner binds to the surface of sperm cells. The CTC-Ca²⁺complex preferentially binds to hydrophobic regions such as the cell membrane, and capacitation-induced changes in labeling patterns are now widely considered to reflect capacitated state of sperm cells. However, the CTC method is laborious because it cannot be applied on a flow-cytometer. This is due to the fact that, unlike the clear shift in surface distribution of CTC, the amount of CTC staining is not unambiguously changing upon sperm capacitation and the acrosome reaction. Moreover, CTC staining should be performed on fixed cells, which implies that the cells have first to be labeled with a membrane impermeable DNA stain for discrimination of live (not fluorescent) and deteriorated (fluorescent) cells. Subsequently, after removing unbound DNA stain from the sperm cells, the cells can be fixed and stained with CTC. Obviously this procedure may introduce artifacts such as cellular and/or acrosomal deterioration (also because activated sperm cells are more fragile than freshly ejaculated sperm cells). On top of this, the molecular basis of CTC staining to sperm cells is not understood. However, the major drawback of CTC staining is its Ca²⁺ dependent binding to the sperm surface. This Ca²⁺ dependency implies that CTC cannot be used to detect Ca²⁺ independent capacitational changes in sperm cells. Another parameter that can be evaluated with CTC is the intactness of the acrosome. For this evaluation reliable methods employing acrosome specific fluorescent lectins have also been described (e.g. with PNA-FITC).

Therefore, the CTC method of detecting simultaneously sperm capacitation and acrosome reaction was compared with two separate flow cytometric assays developed to monitor capacitation dependent changes in membrane fluidity and acrosomal status in live stallion sperm cells, respectively. Flow cytometric detection of capacitation changes on membrane architecture using merocyanine 540 as a reporter probe and acrosomal status using FITC-PNA staining have some clear advantages above the CTC staining method: (i) Given the obvious differences in fluorescent intensities of control and capacitated/acrosome reacted cells flow cytometric method allows very rapid objective discrimination of the status of high amounts of sperm cells (in this paper we analyzed 10,000 sperm cells per time-point in a few seconds). (ii) Prior to analysis the sperm suspension only requires simultaneous addition of appropriate amounts of PI and FITC-PNA or Yo-Pro-1 and merocyanine 540 and 10 minutes of labeling (instead of multiple staining, washing and fixation steps which are required for CTC). It should be noted that these membrane impermeable DNA stains gave nearly identical sperm viability results. The cells can be analyzed in the flow cytometer in unfixed state at relatively physiological conditions (i.e. at 37⁰ C, in absence or presence of 15 mM bicarbonate, 5%CO₂). These conditions are required for minimizing the risk of cell deterioration especially in capacitated sperm cells. For the purpose of analysis a decrease in percentage of non-capacitated/acrosome-intact spermatozoa was considered rather than an increase in capacitated/acrosome-reacted cells. This enabled a more realistic approach, as there would be a higher chance of capacitated/ acrosome-reacted cells of dying and thus being missed out from analysis.

A: Detection of membrane fluidity with merocyanine 540

CTC staining gave significantly different results from merocyanine 540 in terms of capacitation-state of the spermatozoa when incubated in Tyrode’s medium containing 15 mM bicarbonate (Tyr+bic). Merocyanine 540 detected a more rapid increase in membrane fluidity at ½ hr time-point, which plateaued at the same level as CTC detectable capacitation changes, which were seen only after 3 hrs. Most likely the differences are due to the fact that the membrane related changes detected by merocyanine 540 precede Ca²⁺ influx (on which CTC binding depends). This is suggested by our data on CTC and merocyanine 540 staining patterns observed in presence of Ca²⁺ ionophore: (i) A much more pronounced and rapid increase in capacitated sperm cells was detected with CTC when compared to incubation in absence of Ca ²⁺ ionophore. (ii) The presence of Ca²⁺ ionophore did not influence merocyanine 540 staining patterns. (iii) In presence of Ca²⁺ ionophore the capacitation response mirrored the merocyanine 540 response, whereas, in absence of Ca²⁺ ionophore CTC response was only moderate when compared to merocyanine 540.

In absence of bicarbonate merocyanine 540 response was minimal. The data indicate that bicarbonate induced a change in the lipid packing of the plasma-membrane that can be monitored by merocyanine 540 (in boar sperm cells this phenomenon has been linked with  phospholipid scrambling). Only a sub-population of the responsive stallion sperm cells also showed the CTC response. This is due to the fact that the rise of intracellular Ca²⁺ required for CTC binding is a later event in sperm capacitation than the bicarbonate mediated lipid scrambling. The two methods gave similar results in presence of Ca²⁺ ionophore due to the fact that all merocyanine 540 responding cells now have appropriate Ca²⁺ for CTC binding.

B: Detection of acrosome reaction with PNA-FITC

Another event that can be evaluated with CTC is the acrosome reaction. The latter is a calcium dependent process, and high intracellular Ca²⁺ is required for the fusion between the sperm plasma membrane with the outer acrosome membrane. After increased intracellular Ca²⁺level the acrosome reaction of capacitated sperm cells may proceed. Only after this secretory event FITC-PNA is able to label its binding epitope on the outer acrosomal membrane. Therefore like CTC, FITC-PNA staining is Ca²⁺ dependent (albeit in an indirect manner) which may explain that acrosome reaction assessments gave similar results according to both staining protocols: A moderate induction of acrosome reaction was observed in Tyr + Bic whereas the response was nihil in absence of bicarbonate (Tyr). Addition of Ca²⁺ ionophore further induced the acrosome reaction in Tyr+bic incubated sperm suspensions by facilitating the rise of intracellular Ca²⁺ required for the secretory event. This suggests that bicarbonate and ionophore show a synergistic effect towards the acrosome reaction, and that bicarbonate, alone, is sufficient to cause acrosome reactions, but calcium ionophore alone is not. It also gives some insight into the train of events leading to the acrosome reaction. It is inferred that bicarbonate causes increase in plasma membrane fluidity, which probably mediates higher permeability to Ca²⁺. A clear advantage of the FITC-PNA/ PI assay is the rapid, objective and simultaneous analysis of the acrosomal status and the viability of a large number of sperm cells (n=10,000 in few seconds), whereas the CTC detection method requires several labeling, washing and fixation steps and is not suitable for flow cytometry.

Conclusion

Conclusively, in this paper we have compared several methods to detect sperm capacitation, acrosome status, sperm viability and motility properties under conditions where either the sperm membrane fluidity was affected or the acrosome reaction was induced. The major aim was to relate CTC staining patterns on fixed sperm cells (used for simultaneous assessment of capacitation and acrosome reaction) to new flow cytometric assays for detection of membrane changes in a Ca²⁺ independent manner. FITC-PNA is superior to CTC because it is well established as a marker lectin for the outer acrosomal membrane and the membrane specificity of CTC is not known. Furthermore, merocyanine 540 and FITC-PNA are to be preferred over CTC staining because flow cytometric assays (merocyanine 540 and FITC-PNA) are easier to perform than fluorescence microscopic assays (CTC), and also because washing and fixation steps are not required thus eliminating number of deterioration factors. Simultaneous detection of sperm viability can be done with a membrane impermeable DNA stain (all the three stains used in this study gave identical results). The two flowcytometric assays are also superior to hyperactivation because of technical limitations of CASA on capacitated cell suspensions.