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a The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia 23507
b Department of Biological Sciences, Hampton University, Hampton, Virginia 23688
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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150 µm/sec; amplitude of lateral head displacement (ALH) 8.0 µm, and linearity (LIN) 
60%. Using these criteria, caffeine and dbcAMP significantly stimulated HA (61 ± 8%) compared to control conditions (12 ± 2%), p < 0.01, with a concomitant increase in PY-20 labeling (88 ± 12%) vs. control (13 ± 2%), p < 0.01. PY-20 labeling significantly correlated with HA (r = 0.75, p < 0.01) and with some motion characteristics used for HA sorting including ALH (r = 0.86, p = 0.0013) and LIN (r = -0.88, p < 0.001) but not VCL (r = 0.21). Treatment with genistein (10 µM) had no effect on HA or PY-20 immunocytochemistry in the absence of caffeine and dbcAMP, but the tyrosine kinase inhibitor significantly decreased caffeine- and dbcAMP-stimulated HA and PY-20 labeling in a dose-dependent manner (p < 0.01). These results demonstrate that tyrosine phosphorylation of sperm tail proteins is an integral signaling pathway modulating some but not all of the motion characteristics associated with cAMP- and caffeine-stimulated HA in cynomolgus monkey spermatozoa.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Spermatozoa from the cynomolgus monkey (Macaca fascicularis) and other species of the genus Macaca exhibit unique requirements for successful fertilization in vitro. Neither capacitation nor capacitation-related HA occurs spontaneously, and completion of both processes is dependent upon exogenous stimulation with the sperm activators caffeine and dibutyryl (db)cAMP [5]. With this absolute requirement for sperm activation, macaque sperm provide a controlled system for studying intracellular mechanisms involved in capacitation. The uniqueness of this model is further evidenced by studies indicating that in macaque sperm, hyperactivation occurs under different stimulating conditions than induction of the acrosome reaction [6, 7]. With the separation of these two capacitation-dependent processes, their specific signaling pathways can begin to be delineated.
In the present study, we first defined the sorting criteria for automated sorting of macaque sperm exhibiting HA. Once these sorting criteria were defined, we used them to provide a rapid, physiological endpoint in the study of capacitation-related cellular events that occur in macaque spermatozoa during hyperactivation. Protein phosphorylation of tyrosine residues on sperm proteins is one important intracellular mechanism regulating sperm function that has also been suggested as a meaningful indicator of capacitation [8, 9]. We provide evidence demonstrating that tyrosine phosphorylation is an integral part of caffeine- and dbcAMP-stimulated macaque sperm capacitation and some but not all of the motion characteristics associated with HA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult proven breeder male cynomolgus monkeys (Macaca fascicularis) (n = 10) weighing 5–8 kg were used in this study. Monkeys were housed in individual cages with a room temperature of 22°C and 12L:12D, and fed a diet of commercially available monkey chow and water ad libitum. Serum testosterone levels were determined for each monkey before inclusion in this study. Before these studies were initiated, protocols were approved by the Animal Care and Use Committee of Eastern Virginia Medical School and were in accordance with the Guiding Principles for the Care and Use of Research Animals issued by the Society for the Study of Reproduction.
Sperm Processing
Semen specimens were collected by electroejaculation directly into Tyrode's albumin lactate pyruvate (TALP)-HEPES [10] that had been pre-equilibrated to room temperature (approximately 25°C). Monkey spermatozoa were prepared for motion assessment according to previously reported techniques [11]. Sperm were diluted into TALP-HEPES medium supplemented with 0.3% BSA and centrifuged at 400 x g for 7 min. After the supernatant was removed, TALP-HEPES medium was overlaid onto the pellet, and the pellet was loosely dislodged. After a 1-h incubation at room temperature, the motile sperm fraction was collected and assessed by computer-assisted motion analysis.
Sperm Activation with Caffeine and dbcAMP
After collection of the motile fraction of macaque sperm, the sperm concentration of each control and experimental group was adjusted to 20 million/ml and incubated for an additional hour at room temperature. The motile sperm fraction was next pelleted, and the supernatant was replaced with TALP medium supplemented with 0.3% BSA that had been pre-equilibrated by an 18-h incubation at 37°C and 5% CO2 in water-saturated air. Sperm were activated with a combination of caffeine (1 mM) and dbcAMP (1 mM) as described by Boatman and Bavister [5]. Baseline or untreated sperm, a subset of sperm from the same monkey specimen, were processed in a similar manner in the absence of activators. Both untreated and treated spermatozoa were incubated at 37°C and 5% CO2 in water-saturated air for 0.5 h before sperm motion and protein tyrosine phosphorylation assessments.
Sperm Motion Assessment
Sperm motion was evaluated with the HTM-IVOS Motion Analyzer (Hamilton-Thorn Research, Danvers, MA) as described previously for human sperm [12] and modified for assessment of cynomolgus monkey sperm [11, 13]. The changes in the settings take into account the increased velocity of monkey sperm compared to human sperm. All sperm samples were equilibrated to and assessed at 37°C to allow for standardization between treatments. The pertinent settings used during the HTM assessment were as follows: frames acquired = 30, frame rate = 60 Hz, minimum contrast = 80, minimum cell size = 5 pixels; "slow cells" were accepted as motile. At the outset of each experiment, we verified that the settings permitted the accurate differentiation of motile sperm vs. nonmotile sperm or debris by using the "playback" option. During "playback," the motions of sperm in the previous field were replayed: a green dot was located over the heads of all motile sperm for each frame, and a red dot was positioned over the heads of nonmotile spermatozoa. When errors were detected, the settings were adjusted until the problem was corrected.
The following motion characteristics were compared between the control and treated sperm groups: curvilinear velocity or track speed (VCL, the velocity derived from all 30 head positions); progressive velocity (VSL, the velocity based on the first and last head positions only); maximal amplitude of lateral head displacement (ALH, a measure of the side-to-side movement of the head); and linearity (LIN, VSL/VCL, a measure of the straightness of trajectory).
To optimize the motion assessments, the sperm concentration of each sperm group was adjusted to 20 million/ml and analyzed using a MicroCell counting chamber (Conception Technologies, San Diego, CA) with a chamber depth of 20 µm. Using these experimental conditions prevented the crossing of sperm tracks that would result in errors in data collection. Approximately 100 sperm were evaluated per baseline (untreated) and caffeine- and dbcAMP-treated group.
Tyrosine-phosphorylated Protein Immunoreactivity
For the assessment of phosphorylated tyrosine residues in macaque spermatozoa, control and treated groups of sperm were washed by centrifugation with PBS containing sodium azide (0.5%) and PMSF (0.1 µM). Washed sperm were air-dried onto polytetrafluoroethylene-coated spot slides, methanol-fixed, and stored at -70°C until evaluated. After thawing to room temperature, nonspecific binding sites were blocked with PBS with 1% BSA. Control and treated sperm were next incubated for 1 h with the antibody PY-20, a monoclonal antibody raised against phosphorylated tyrosine residues on proteins and fluorescein isothiocyanate (FITC)-conjugated (Calbiochem, LaJolla, CA). Determination of tyrosine phosphorylation in sperm was completed in a blinded fashion by two evaluators at x600 magnification with epifluorescence microscopy. At least 100 sperm per group were evaluated.
Treatment of Sperm with the Tyrosine Kinase Inhibitor Genistein
To further examine the relationship between macaque sperm HA and tyrosine phosphorylation, in two additional experiments sperm were preincubated with the tyrosine kinase inhibitor genistein (Calbiochem). In the first experiment, the motile fraction of sperm from three monkeys was incubated for 2 h at room temperature with genistein (10 µM) in TALP-HEPES medium. Control (untreated) and genistein-treated sperm were next transferred to TALP medium and incubated with and without the sperm activators caffeine and dbcAMP, as described above with one exception. The experimental sperm groups (with genistein and without activators, and with genistein and with activators) were exposed to genistein throughout the experiment by transferring to TALP medium containing genistein (10 µM) with and without caffeine and dbcAMP, respectively. In the second experiment, sperm from three monkeys were incubated in medium either without genistein or in medium containing increasing concentrations of genistein (0.1, 1.0, 10, 20 µM). Control and treated groups were treated with the sperm activators as described above. Sperm were evaluated for HA and phosphotyrosine immunoreactivity.
Statistical Evaluations
Data were analyzed by Student's paired t-test and ANOVA followed by Bonferroni's post-test or Pearson's rank order correlation; p 0.05 was considered significant. All results are expressed as the mean ± standard error of the mean. For each experiment, the number of replicates is presented, and this number represents ejaculates obtained from different monkeys.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The motion characteristics of sperm from nine monkeys were examined without (baseline) and with the addition of the activators caffeine and dbcAMP. For each characteristic, ALH, LIN, VSL, and VCL, a mixed-model ANOVA was used to compare the baseline and treated means and to determine whether the predicted means for each characteristic differed significantly. The predicted means are presented in Table 1. A significant difference between the mean values of the baseline and treated groups was observed for all of the motion characteristics examined (ALH and LIN, p = 0.0001; VSL and VCL, p < 0.05).
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To discriminate hyperactivated sperm from nonhyperactivated sperm, confidence levels were determined for each motion characteristic. For ALH and LIN, there was no overlap in the confidence intervals. Therefore, using the 99.9% confidence intervals for ALH, a cutoff of 8 µm was used to differentiate hyperactivated from nonhyperactivated sperm. For LIN, the cutoff was 60. Because of the overlap in the upper and lower 95% confidence intervals for VSL and VCL, it was not possible to use these values to set the minimal threshold levels for these two motion characteristics. The lower 95% cutoff of 150 µm/sec for VCL was used as the minimal threshold level for automated sorting of hyperactivated sperm. Since VSL decreased with caffeine and dbcAMP treatment, no minimal threshold level was included for this motion characteristic. Using these sorting criteria to differentiate hyperactivated sperm, a significant increase in hyperactivated sperm from 11.0 ± 1.0% to 58.0 ± 1.6% was observed in the treated group (p < 0.002).
Tyrosine Phosphorylation of Macaque Sperm Proteins
The presence of tyrosine-phosphorylated proteins was localized with PY-20 immunoreactivity to the tail region of methanol-fixed cynomolgus monkey sperm (Fig. 1). Specifically, labeling was primarily observed in the principal piece region of the flagellum and a small region of the neck of macaque spermatozoa. Labeling of these tyrosine-phosphorylated sperm tail proteins was very intense; however, in approximately 10% of labeled sperm, the labeling intensity was light compared to that of the majority of labeled sperm.
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Sperm from five cynomolgus monkeys were treated with caffeine and dbcAMP to examine the association between tyrosine phosphorylation of sperm tail proteins and the proportion of sperm exhibiting HA. Treatment with activators resulted in a significant increase in the proportion of sperm exhibiting tyrosine-phosphorylated proteins as evidenced by PY-20 immunoreactivity (p < 0.01) (Table 2). Concomitantly, the proportion of macaque exhibiting HA using the established sorting criteria significantly increased with caffeine and dbcAMP stimulation (p < 0.01).
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A significant correlation existed between the proportion of sperm exhibiting tyrosine-phosphorylation of sperm tail proteins and HA (r = 0.75, p < 0.01). This PY-20 immunoreactivity also significantly correlated with the motion characteristics of ALH (r = 0.86, p < 0.0013), LIN (r = -0.88, p < 0.007), and VSL (r = -0.71, p < 0.02) (Table 2). However, no correlation existed between PY-20 immunoreactivity and either percent motility (r = -0.17) or VCL (r = 0.21).
Sperm Treatment with Genistein
Sperm activated with caffeine and dbcAMP in the absence of genistein exhibited the expected significant increase in the motion characteristics VCL (p < 0.05) and ALH (p < 0.01), and the expected decrease in LIN (p < 0.05). Both HA and PY-20 immunoreactivity also significantly increased with activation (p < 0.01). Macaque sperm treated with increasing concentrations of genistein reacted in a concentration-dependent manner, displaying an inhibition of HA and PY-20 immunoreactivity (Fig. 2, A and B). With the highest concentrations of genistein tested (10 and 20 µM), there was a significant inhibition in HA (p < 0.05) and phosphotyrosine (PY-20) immunoreactivity (p < 0.01). This resulting reduction for both HA and PY-20 labeling was to near baseline levels. In the absence of caffeine and dbcAMP, genistein had no effect on the proportion of sperm exhibiting HA or phosphotyrosine immunoreactivity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These alterations in macaque sperm motion characteristics were observed only upon stimulation with the sperm activators caffeine and dbcAMP; they were not spontaneous as observed for other mammalian species including humans [3]. Another difference between primate species is the proportion of the sperm populations exhibiting hyperactivation. In vitro capacitation in human sperm is asynchronous [14, 15], as evidenced by the low proportion of a sperm population exhibiting HA at any given time [16]. In many species, in vitro capacitation that in many instances approximates that of oviductal fluid can be achieved in balanced salt solutions containing appropriate concentrations of electrolytes, metabolic energy sources, and serum albumin [17]. Since macaque sperm do not capacitate in vitro either in the TALP medium or in the Biggers, Whitten, and Whittingham (BWW) medium in use in other laboratories [5, 7], it is safe to suggest that some component(s) do not mimic the in vivo environment. Macaque sperm treated with caffeine and dbcAMP display more synchrony as evidenced by the large proportion exhibiting HA; this synchrony may be due in part to these exogenous stimulators bypassing the initial receptor-mediated events that would elicit the appropriate signaling cascade.
Studies of several mammalian species have since indicated unique relationships between protein tyrosine kinase and cAMP signaling pathways during capacitation [18–22]. Protein tyrosine phosphorylation has been proposed as a consistent indicator of intracellular changes preceding and/or associated with capacitation. Our results indicate that treatment with the exogenous cyclic nucleotide analogue dbcAMP and with caffeine, conditions known to induce capacitation in macaque sperm [5–7], significantly increased tyrosine phosphorylation of sperm tail proteins. This immunoreactivity was also positively associated with macaque HA. In particular, phosphotyrosine was positively correlated with increased amplitude of ALH and negatively associated with LIN. This result was not unexpected since it is presumed that hyperactivation is tightly correlated with capacitation.
The identity of the sperm tail proteins that are tyrosine phosphorylated is necessary to further delineate the signaling pathways involved in macaque sperm hyperactivation. Tyrosine-phosphorylated proteins were localized to the principal region of the tail and to a small region near the neck of the sperm. The labeling patterns observed in macaque sperm were similar to those reported for mouse and human sperm [23, 24]. In those species, these phosphotyrosine proteins were identified as the major fibrous sheath protein, A kinase anchoring protein (AKAP82), and the precursor, proAKAP82. Future studies are planned to identify the macaque sperm tail proteins that are phosphorylated during caffeine and dbcAMP stimulation of hyperactivation. AKAP82 and proAKAP82 are potential candidates. It should be remembered that in the present study detection of phosphotyrosine immunoreactivity was completed on methanol-fixed macaque sperm. Future studies are planned that examine tyrosine phosphorylation in live unfixed sperm.
Treatment with genistein, a tyrosine kinase inhibitor, significantly blocked tyrosine phosphorylation of sperm tail proteins in a dose-dependent manner, therefore presumably blocking capacitation and subsequently HA. It is interesting to note that treatment with genistein did not completely block either tyrosine phosphorylation or HA. This incomplete inhibition may be due to insufficient concentrations of the inhibitor entering the cell. However, other signaling pathways such as calcium influx are involved in capacitation and its related events [17, 25]. Genistein has been reported to affect calcium influx in pancreatic cells [26] as well as glucose uptake [27]. If this tyrosine kinase inhibitor acts in a similar manner in mammalian sperm, then its usefulness in delineating intracellular mechanisms involved in capacitation is limited, and other more selective inhibitors will be required.
In conclusion, macaque sperm have highly defined criteria for achievement of capacitation and the resultant HA. These criteria provide a unique model for delineating the cellular events occurring during capacitation and its related events. Such a system has potential application as a means to study methods of fertility enhancement and intervention in humans, as well as conservation of endangered nonhuman primates.
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FOOTNOTES |
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2 Correspondence: Mary C. Mahony, The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, 601 Colley Avenue, Norfolk, VA 23507. FAX: 757 446 8998; marym{at}jones1.evms.edu
Accepted: December 15, 1998.
Received: May 29, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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