| Summary: | 碩士 === 國立臺灣大學 === 臨床醫學研究所 === 90 === Summary
Introduction
Upon release from the male, mammalian sperm are nonfertilizing, but become functionally competent cells with the capacity to fertilize physiologically both in the female reproductive tract and / or under appropriate conditions in vitro, a process so-called “capacitation”. Capacitation involves both sperm surface and intracellular changes. Certain requirements for the modulation of capacitation have been identified :
Fertilization promoting peptide ( FPP; pGlu-Glu-ProNH2 ), a tripeptide produced by the prostate gland and secreted into seminal plasma in several mammals, is a potential modulator of capacitation in vivo. At ejaculation, nonfertilizing mammalian sperm come into contact with FPP, which elicits capacitation — dependant responses, i.e. stimulating capacitation in uncapacitated sperm and then inhibiting spontaneous acrosome reaction in capacitated cells. FPP thus initiates and maintains fertilizing potential of sperm until they contact an oocyte in the female reproductive tract.
Adenosine, another compound found in mammalian seminal plasma, elicits similar capacitation — dependant responses in sperm cells to FPP. The combination of FPP plus adenosine, whether mixed at low, non-stimulatory concentrations or high, maximally-stimulatory concentrations, was more effective in promoting capacitation than either compound used individually. This suggests that the two molecules act via separate external receptors and modulate a common signal transduction pathway.
Adequate sperm motility is known to be an essential prerequisite for successful fertilization. Pentoxifylline, clinically used to enhance motility of sperm from infertile human subjects, is a phosphodiesterase inhibitor leading to an increase in intracellular cAMP level. In hamsters, pentoxifylline has been shown to induce capacitation, hyperactivation and acrosome reaction, and also to improve fertilization rates in vitro. The application of pentoxifylline could contributes to enhancement of post — thawed sperm motility and selection of viable sperm in intracytoplasmic sperm injection.
Both FPP and adenosine may act as a first messenger, via adenylate cyclase ( AC ) / cAMP signaling pathway, to modulate the fertilizing ability of mammalian sperm. Cyclic AMP, therefore, plays an essential role in the mechanisms of capacitation and acrosome reaction, which are modulated by either external signal molecules such as FPP and adenosine, or by artificial sperm stimulants such as pentoxifylline. Herein, we try to evaluate the effects of FPP, adenosine and pentoxifylline on the fertilizing ability and motility characteristics of frozen — thawed human sperm.
Materials and Methods
Semen Sample
Semen obtained by masturbation was provided by volunteer donors. After liquefaction and basic seminalysis, the donated semen sample was stored by cryopreservation. On the day of examination, the frozen semen sample was thawed, and the post — thawed sperm suspension was then divided into 2 to 4 aliquotes, one of which was used as the control. Just after thawing, the other aliquots were treated to make 100nM FPP, 10μM adenosine, or 1 mg/mL pentoxyfilline sperm suspensions. Thereafter, we assessed the effects of these three reagents on frozen — thawed human sperm at 1 hour and 4 hours of incubation.
Part I. Chlortetracycline ( CTC ) Fluorescence Assessment
We collected 16 semen samples, all which were divided into 4 aliquotes of sperm suspension after thawing. One of the 4 aliquotes was used as the control, and the other aliquots were added with reagents to make 100nM FPP - treated, 10μM adenosine - treated, and 1 mg/mL pentoxyfilline - treated sperm suspensions respectively.
Most cytological techniques uased to determine the effects of treatment on sperm capacitation only identify acrosome — intact cells and acrosome — reacted cells。With CTC, the acrosome — intact category can be subdivided into two on the basis of apparent differences in their functional state, i.e. uncapacitated and capacitated:
F, with uniform fluorescence over the entire head, characteristic of uncapicitated, acrosome-intact cells
B, with a fluorescence - free band in the post-acrosomal region, characteristic of capicitated, acrosome - intact cells
AR, with dull or absent fluorescence over the sperm head, characteristic of capicitated, acrosome - reacted cells
In addition to CTC , we use Hoechst 33258 to assess the live / dead status of the sperm.
In each sample, 200 Hoechst 33258 negative cells were assessed for CTC staining patterns under Nikon’s eclipse E600 microscope equipped with phase — contrast and epifluorescent optics.
Part II. Computer Aided Sperm Analyzer ( CASA )
Depending on the post — thawed semen qualities, we divided the sperm suspensions into 2 to 4 aliquotes. One aliquote was used as the control, and the other 1 to 3 aliquots were treated to make 100nM FPP, 10μM adenosine, or 1 mg/mL pentoxyfilline sperm suspensions. There were 27 cases with FPP — treated aliquotes, 16 cases with adenosine — treated aliquotes, and 23 cases with pentoxyfilline — treated aliquots in this experiment.
Using CASA, we assessed the following motion parameters in each sample:
Straight — line velocity ( VSL )：the straight — line distance between the beginning and the end of the track divided by the time elapsed
Curvilinear velocity ( VCL )：the total distance between each position of the cell center of brightness ( CB ) for a given cell during the acquisition, divided by the time elapsed
Average path velocity ( VAP )：the smoothed average position of the CB and gives an average cell path velocity
Amplitude of lateral head displacement ( ALH )：the mean width of the head oscillation as the cell swims
Beat cross frequency ( BCF )：the frequency with which the cell track crosses the cell path in either direction
Straightness ( STR )：the departure of the cell path from a straight line
Linearity ( LIN )：the departure of the cell track from a straight line
Statistic Analyses
All statistic analyses were carried out by paired t — test. Percentage data of the CTC results were subjected to arc — sine transformation before statistic analyses. Data were expressed as mean ± SEM and P≦0.05 was considered to be statistically significant.
Results
Part I. Chlortetracycline ( CTC ) Fluorescence Assessment
CTC fluorescence assessment showed no significant differences between control group and FPP — treated, adenosine — treated, pentoxifylline — treated groups. All FPP, adenosine, and pentoxifylline failed to improve the fertilizing ability of frozen — thawed human sperm.
Part II. Computer Aided Sperm Analyzer ( CASA )
At 1 hour of incubation, the VCL of adenosine — treated and pentoxifylline — treated groups were significantly greater than that of control group. In addition, pentoxifylline significantly lessened LIN up to 4 hours of incubation.
Although FPP did not speed the frozen — thawed human sperm, it did modify the motility characteristics. At 1 hour of incubation, the BCF of FPP — treated group was significantly smaller than that of control group. At 4 hours of incubation, FPP made the STR and LIN much less than that of the control group.
Pentoxifylline did not affect the motility rate of the frozen — thawed human sperm. However, the motility rates of both the FPP — treated and adenosine — treated groups were unexpectedly lower than that of the control group at 1 hour of incubation.
Discussion
CTC fluorescence assessment showed that none of FPP, adenosine and pentoxifylline have significant effects on the capacitation and acrosome status of frozen — thawed human sperm. While comparing the motility characteristics of reagent - treated cells and non - treated cells with CASA, however, we found that all these three reagents did modify the swimming behaviors of frozen — thawed human sperm. The results of our studies gave us some hints of mechanisms about the modulation of sperm fertilizing ability:
After 1 hour of incubation, the VCL of adenosine — treated and pentoxifylline — treated cells are significantly greater than that of non — treated cells. This indicated that the intracellular cAMP level determines swimming velocity of sperm. Within the physiologically effective range, the higher the intracellular cAMP level, the larger the VCL. Because the swimming velocities of frozen — thawed cells did not be affected by the addition of FPP, we concluded that FPP, as a first messenger, may act on AC / cAMP signaling pathway indirectly. Despite of no effects on the velocities of the post — thawed sperm cells, FPP made the departure of cell tracks and paths significantly farther away from a straight line after 4 hours of incubation. Such a finding suggested that FPP is more closely than adenosine related to the alterations of swimming tracks associated with hyperactivation, which most of post- thawed sperm failed to express because of the damages resulting from freezing and thawing procedure. In addition, it is reasonable that the motility rates reflected the overall energy reserve of sperm population in our study. By activating adenylate cyclase, adenosine converted ATP to cAMP and thus contributed to the significant fall in the motility rate of post — thawed cells after 1 hour of incubation.. In contrast to pentoxifylline, which accelerated the movement of frozen — thawed cells without affecting motility rate, FPP caused a significant decrease in motility rate without speeding the sperm cells. There must be additional energy expenses other than those required for swimming in the FPP — treated cells. These additional energy expenses, which resulted in intracellular “ energy shift “, also accounted for the less active sports ( less BCF without greater velocities ) in the FPP —treated cells after 1 hour of incubation. After 4 hours of incubation, either of the adenosine — treated and FPP — treated groups had a smaller, but not significantly different motility rate from that of control group. This indicated that a substantial proportions of cryopreservation — damaged cells, because of energy exhaustion resulting from the addition of FPP or adenosine, did not survive as long as if not exposed to any reagent post — thawed.
Sperm cell membrane plays an essential role in capacitation and acrosome reaction. Cryopreservation causes damage to the integrity and function of the sperm cell membrane and thus alters the permeabilities of ion and water, leading to sperm intracellular ion concentrations out of the physiologic ranges optimal for fertilization. It is well known that the intracellular Ca＋＋ concentration is closely correlated with the fertilizing ability of spermatozoa, and rearrangements of cytoskeletal elements modulated by intracellular Ca＋＋ / calmodulin — dependent protein kinases lead to modifications of cell movement. Because the previous literature revealed that FPP is a potential modulator of capacitation and hyperactivation, and our studies showed that FPP could modify swimming behaviors with additional energy expenses in frozen — thawed sperm cells with non - optimal intracellular ion concentrations, we propose that FPP potentiates fertilizing ability and prevents spontaneous acrosome loss, via regulating membrane — bound Na＋ - K＋ATPase and / or Ca＋＋ATPase, by keeping the sperm intracellular Ca＋＋ concentration within the “ capacitation “ range. In addition to cAMP, the intracellular Ca＋＋ is another second messenger in modulation of capacitation and acrosome reaction of mammalian spermatozoa.
FPP failed to speed frozen — thawed cells because the intracellular “ energy shift “ resulted in no significant increase in the cAMP level. However, pentoxifylline also made the departure of cell tracks significantly farther away from a straight line as FPP did. Therefore, there may be “ cross — talk “ between Ca＋＋ / calmodulin and AC / cAMP signaling pathway.
While acting on the specific receptor on the sperm cell membrane, FPP may make conformational changes of the cytoplasmic domain of its receptor which then activate G proteins. Some of these G proteins help modulate the intracellular Ca＋＋ level, activate the Ca＋＋ / calmodulin — dependent protein kinases, and thus lead to rearrangements of cytoskeletal elements associated with hyperactivation. Other G proteins modulate AC / cAMP and the consequent protein phosphorylation. Both Ca＋＋ / calmodulin and AC / cAMP signaling pathway interact with each other and thus modulate the expression of capacitation and hyperactivation.
Although Green et al. reported that FPP induces capacitation in fresh human sperm within one hour of incubation, our study showed that the physiologic effects on the motion characteristics of frozen — thawed cells caused by FPP appeared beyond one hour of incubation. Such a delay resulted from a longer time it took for FPP to modulate the intracellular ion concentrations, which might be far away from the physiologic ranges, in the viable frozen — thawed sperm cells to the optimal levels for fertilization. Because cryopreservation disrupted sperm cell membrane and cytoskeleton, only a few “ healthier “ post — thawed sperm cells could express capacitation and hyperactivation. This is why none of the three reagents used in our studies made any difference in the fertilizing ability between reagent —treated and control groups.
We suggest that FPP resets the intracellular ion concentrations of sperm cells at levels optimal for fertilization at ejaculation. Both the intracellular Ca＋＋ and cAMP act as second messengers in the modulation of sperm fertilizing ability. There may be branching and cross — talk between pathways involved in capacitation to regulate and coordinate a sperm cell’s response to specific signal molecules such as FPP and adenosine. It consumes energy to regulate the intracellular ion concentrations and to amplify the cell’s specific response to the incoming information, so is the physiologic significance of the phenomenon of “ capacitation “.
Perspectives
To verify the mechanism of FPP in modulating the fertilizing ability of mammalian sperm, we could try to use digitalis and vanadate to evaluate the possible effects of FPP on Na＋ - K＋ATPase and Ca＋＋ATPase on the sperm cell membrane. Digitalis is an inhibitor of Na＋ - K＋ATPase, which leads to an elevation of intracellular Ca＋＋ level. By stablizing the structure of Ca＋＋ATPase, vanadate slows down the passage of Ca＋＋. Comparisons of the effects of FPP, digitalis / vanadate, and FPP ＋ digitalis / vanadate on spermatozoa, we will clarify the possible mechanisms of FPP we proposed on the sperm intracellular ion concentrations.
In addition, it was recently demonstrated that ceramide stimulates the Ca＋＋ATPase in a dose — dependent manner and sphingosine has conversely an inhibitory effect on Ca＋＋ATPase activity. Such sphingolipids may also be considered in assessing whether or not FPP has any effects on membrane — bound Ca＋＋ATPase of sperm.
Calcium channel blockers such as nifedipine, verapamil, and diltiazem may also help us understand the role of calcium channel in capacitation and acrosome reaction.
By using microarray, we may try to specify the proteins and their functions in the signal pathways responded to FPP and adenosine.
Once proved to contribute to regulation of membrane — bound Na＋ - K＋ATPase and Ca＋＋ATPase, FPP could be widely applied in biomedical science.
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