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Effect of follicular fluid on sperm movement characteristics
FERTILITY AND STERILITY Copyright c 1990 The American Fertility Society
Vol. 54, No.6, December 1990 Printed on acid-free paper in U.S.A.
Effect of follicular fluid on sperm movement characteristics
Carmen Mendoza, Ph.D_* Jan Tesarik, M.D., D-Sc-t Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Granada, Granada, Spain, and lnstitut National de Ia Sante et de Ia Recherche Medicale (INSERM), Unite 187, Department of Obstetrics and Gynecology, Hopital Antoine-Bee/ere, Clamart, France
Human spermatozoa previously washed from seminal plasma and incubated in capacitating medium were exposed to human follicular fluid (FF) for different time periods and their movement characteristics were examined by computer-assisted digital image analysis. No differences from control samples were observed after short exposures (15 minutes and~ hours). When the exposure was prolonged to 6 hours, spermatozoa incubated in the presence of FF displayed significant differences in some movement characteristics as compared with samples incubated without FF supplement. When only rapidly moving sperm subpopulations were considered, these effects ofFF were similar to those produced by a short incubation with the solubilized cumulus oophorus matrix as described previously. After 24 hours of incubation, there was a remarkable beneficial effect of FF on motility preservation. Possible physiological and clinical implications are discussed. Fertil Steril54:1135, 1990
The potential roles of ovarian follicular components that are released at ovulation together with the oocyte as signals modulating different aspects of sperm function are recently under scrutiny mainly for two reasons. First, examination of sperm capability to respond to these factors may offer additional diagnostic means for the andrologic laboratory and second, inclusion of these factors to culture media may improve sperm fertilizing ability in some conditions. 1 Both the cumulus oophorus 2- 4 and follicular fluid (FF), 2•5- 7 which accompany the ovulated oocyte after follicle rupture, have been reported to stimulate the acrosome reaction of human spermatozoa, and both of them are also potent accelerators of the activation of the main sperm proteolytic enzyme acrosin. 3 •8 Moreover, we have shown recently that components of
the human cumulus oophorus intercellular matrix exert a specific effect on the pattern of human sperm movement, acting selectively on capacitated spermatozoa.9 The experiments described here were undertaken to examine the effects of the other major by-product of ovulation, FF, on sperm movement characteristics. We demonstrate that FF can induce changes in human sperm movement characteristics similar to those resulting from the action of the cumulus oophorus but only after relatively long incubations. In addition, the inclusion of FF to culture medium significantly improves motility scores of sperm samples maintained in vitro for extended time periods.
Received January 11, 1990; revised and accepted August 1, 1990. *Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Granada, University Campus, Fuentenueva_ t Reprint requests: Jan Tesarik, M.D., D_Sc_, INSERM U. 187, Department of Obstetrics and Gynecology, Hopital Antoine-Beclere, 92141 Clamart, France.
Source and Preparation of Spermatozoa
VoL 54, No_ 6, December 1990
MATERIALS AND METHODS
Semen samples were obtained from eight donors with normal sperm parameters. 10 The procedures for selection of motile spermatozoa and in vitro capacitation were essentially the same as described. 9 Briefly, sperm were washed from seminal plasma Mendoza and Tesarik FF and sperm motion
1135
by repeated centrifugation and resuspension in Tyrode's medium (Difco, Detroit, Ml), 300 ,uL of B2 medium (API System, Montalieu-Vercieu, France) were carefully layered onto the final sperm pellet, and actively moving cells were recovered with the upper 200 ,uL of this medium after 30 minutes of incubation. Spermatozoa were then incubated for an additional 2 to 3 hours in B2 medium under 5% C0 2 in air for in vitro capacitation. Source and Preparation of FF
Samples of FF were collected by ultrasonically guided follicle aspiration from 10 women undergoing in vitro fertilization (IVF) treatment. Ovarian stimulation was carried out with gonadotropins after administration of a gonadotropin-releasing hormone agonist (GnRH-a)Y Only fluid obtained from follicles bearing a healthy-appearing oocyte rated as Grade 3 of oocyte maturation 12 and surrounded by a fully mucified cumulus oophorus rated as preovulatory 13 was used in this study. Samples showing any pink coloration were discarded, and only the first aspirates from follicles were used. All samples were centrifuged at 1500 X g for 10 minutes immediately after oocyte recovery, supernatants were pooled, filtered through a Millipore (0.45 ,urn pore size; Millipore S.A., Molsheim, France), and stored frozen at -20oC until used. Sperm Incubation with FF
Aliquants of motility-selected spermatozoa previously incubated for in vitro capacitation (see above) were pelleted again, resuspended in 1 mL fresh B2 medium supplemented with 20% FF (final sperm concentration of 1 to 5 X 106/mL), and left at 37oC under a gas phase of 5% C0 2 in air for periods up to 24 hours. Nonsupplemented B2 medium alone was used in control incubations.
10
120
* 8
100 ~
u
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80
~
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en .....
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:::1.
60
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4
~
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Ill
>
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:::1. ...J
<
40 2 20 0
0 VSL
ALH
Movement Characteristics
Figure 1 Progressive velocity ( VSL) and lateral head displacement (ALH) values (mean± SD for each donor sample) measured in whole sperm populations (D and 1!21 bars) and in their rapid (curvilinear velocity> 90 Jlm/s) subpopulations (D and ill bars) after 6 hours of control incubation in B2 alone (D and D bars) and in B2 supplemented with 20% FF (1!21 and • bars). *Values significantly different from corresponding controls (P < 0.01).
search, Inc., Danvers, MA). The parameter set-up and measurement conditions were those described previously. 9 The following characteristics of sperm motion were determined (for explanation of terms see Mortimer et al. 14 ): curvilinear velocity, progressive velocity, path velocity, linearity, straightness, lateral head displacement, and beat cross frequency. All measurements were carried out at a constant temperature of 3TC. The time of each measurement was limited to the maximum of 2 minutes to minimize changes because of medium evaporation. Under these conditions, 100 to 150 sperm tracks were recorded in each individual analysis. Two replicates of each analysis were made so that tracks of at least 200 motile spermatozoa were analyzed for each specimen and each time point. Statistical Analysis
Sperm Movement Analysis
Samples (200 ,uL) of sperm suspensions were taken at different times of incubation and concentrated by mild centrifugation (500 X g for 5 minutes) followed by decanting the supernatant and resuspension of the remaining cell pellet in 20 ,uL of the corresponding supernatant. Ten-microliter samples of these concentrated sperm suspensions were then put into a Makler chamber (Sefi-Medical Instruments, Haifa, Israel) and immediately introduced into a fully automated computer-assisted semen analyzer (HTM 2000; Hamilton Thorn Re1136
Mendoza and Tesarik
FF and sperm motion
All analyses were performed using the StatView II statistical package (Abacus Concepts, Inc., Berkeley, CA). Intragroup and intergroup variance was first assessed using ANOVA. Data for groups in which the ANOV A indicated significant differences were analyzed by Student's t-test and x2 test for normally distributed populations. Pearson and likelihood ratio x2 analysis were used to determine significance between non-normally distributed groups. Paired statistical comparisons were made for paired aliquots of each specimen incubated under different experimental conditions. Fertility and Sterility
Table 1 Motile and Rapid Spermatozoa After Different Times of Incubation in B2 Medium Alone and in B2 Supplemented With 20% FF• Motile sperm b
Rapid sperm c
Group
2h
6h
24h
B2 B2 + FFd
65 ± 9 72 ± 10
48 ± 7 62 ± 8
12 ± 4 27 ± 10
2h
6h
24h
25 ± 8 30 ± 8
29 ± 8 24 ± 7
0±0 9± 3
%
• Values are means± SD for each donor sample (n = 8). b Curvilip.ear velocity> 10 p.m/s. c Curvilinear velocity> 90 p.m/s. d Percentages of motile sperm in the B2 + FF group are significantly different from B2 for the 6-hour and 24-hour time points (P < 0.05). Percentages of rapid sperm are different for the 24-hour time point only (P < 0.05).
RESULTS The inclusion of 20% FF did not affect the values of curvilinear velocity as compared with B2 medium alone at any time examined. Similarly, the value of progressive velocity did not change in response to FF during the first 2 hours of incubation. However, after 6 hours of incubation, FF produced a significant alteration of this movement characteristic (Fig. 1). It was noteworthy that values were shifted in an opposite sense if measured in whole sperm populations and in their rapidly moving (curvilinear velocity> 90 f.Lm/s) subpopulations. In the whole populations, a decrease in progressive velocity was observed in the presence ofFF as compared with B2 alone, whereas the values of this motility parameter for samples exposed to FF were increased when only the rapid sperm subpopulations were considered (Fig. 1). The rapid subpopulations represented about 25% of the entire sperm populations at the 6-hour time point (Table 1). The same character of changes as for progressive velocity was observed for path velocity and for the two composite parameters of sperm movementlinearity and straightness. Differences between
samples incubated with FF and those in B2 alone were only significant after 6 hours of incubation, whereas the values did not differ after 15 minutes and 2 hours of incubation. The first significant modification of lateral head displacement in the presence of FF was also detected after 6 hours of incubation; in this case the values were augmented in whole sperm populations but reduced in the rapid sperm subpopulations (Fig. 1). Beat cross frequency measured in whole sperm populations did not change in response to FF at any time examined. However, in the rapid sperm subpopulations there was a significant increase in beat cross frequency after 6 hours of exposure to FF as compared with B2 alone (17.2 ± 1.9 versus 13.9·± 1.5). When spermatozoa were incubated in vitro for 24 hours, spectacular differences in all sperm movement characteristics examined were found between sampl~s incubated in the presence of FF and those incubated in B2 alone (Table 2). Although there was a remarkable decline in all parameters of sperm motility after 24 hours in B2 alone as compared with swim-up samples, the inclusion ofFF in medium had a significant moderating effect on these alterations and, in fact, the values measured after 24 hours in the presence of FF approached those of the original swim-up sperm populations (Table 2). When only rapid spermatozoa (curvilinear velocity > 90 f.Lm/s) were taken into account, there was even a significant increase in progressive velocity (84.5 ± 24.6 versus 70.2 ± 18. 7) and beat cross frequency (17.2 ± 2.9 versus 13.8 ± 2.4) in samples incubated for 24 hours with FF as compared with swim-up spermatozoa. The presence of FF increased the percentage of motile sperm at the 6-hour and 24-hour time points (Table 1). The effects of FF on the percentage of rapidly moving spermatozoa (curvilinear velocity > 90 f.Lm/s) after 2 hours and 6 hours of incubation
Table 2 Comparison of Changes in Movement Characteristics of Human Spermatozoa Incubated for 24 Hours in B2 Medium Alone and in B2 Substituted With 20% FF as Compared With Swim-up Sperm Populations Values of movement characteristics•
Group
Curvilinear velocity
Progressive velocity
Path velocity
Linearity
Swim-up B2 B2 +FFb
84.8± 11.5 30.0 ± 14.1 65.9 ± 35.2
68.1 ± 10.5 18.9 ± 14.5 55.3 ± 34.6
79.7 ± 18.2 21.1 ± 22.0 55.5 ± 34.8
73.1 ± 26.2 63.5 ± 22.0 77.6 ± 22.8
• Values are means± SD for each donor sample (n
Vol. 54, No.6, December 1990
=
8).
Straightness 87.0 ± 14.1 82.7 ± 10.9 87.8 ± 11.9
Lateral head displacement
Beat cross frequency
4.1 ± 1.1 2.0 ± 1.0 3.2 ±2.0
12.1 ± 2.8 7.6 ± 4.1 12.6 ± 4.1
b Except for straightness, all values are significantly different from B2 (P < 0.05).
Mendoza and Tesarik FF and sperm motion
1137
were negligible, but this rapid sperm subpopulation totally disappeared after 24 hours in B2 alone, whereas the proportion of rapid sperm cells after 24 hours of incubation still approached 10% of the whole sperm populations (Table 1). DISCUSSION
The present observations did not show any rapid response of human spermatozoa previously incu. bated for in vitro capacitation to subsequent exposure to FF. This is in contrast to the immediate effect of the cumulus oophorus intercellular matrix described recently. 9 However, when only rapidly moving spermatozoa were analyzed, the changes in sperm movement characteristics observed in this study after 6 hours of incubation with FF resembled those previously reported for capacitated spermatozoa incubated with the solubilized cumulus intercellular matrix for only 5 minutes. 9 This resemblance suggests that small quantities of factors responsible for the cumulus-specific modifications of sperm motion may be released to FF, but their concentration in FF may not be sufficient to elicit a rapid response. A similar situation seems to exist for cumulus-derived factors stimulating the acrosome reaction because the effect of FF on human sperm acrosome reaction is most considerable after relatively long incubations.6•7 It remains to be seen, however, whether the slow effects reported in this study are really caused by a low concentration of the same factors that have a rapid effect in the cumulus. It cannot be excluded that FF may contain other factors that affect sperm function in important ways. In fact, the observed effects ofFF may have resulted from a combined action of cumulus-derived molecules and other yet undetermined factors present in this biological material. The opposite effects of FF on some sperm movement characteristics observed in whole sperm populations and in their rapid subpopulations may be caused by different factors. First of all, these results may have been partly influenced by the effects of FF on sperm motility. If, as documented in Table 1, motility decreases more considerably in the absence of FF than in its presence, measured mean values of sperm velocity may have been affected by a faster depletion of slower swimming (presumably moribund) cells from the motile sperm populations in control incubations as compared with samples incubated with FF in which survival of such cells may have been prolonged. However, the absence of 1138
Mendoza and Tesarik FF and sperm motion
corresponding changes in values of curvilinear velocity speaks against this possibility. The known stimulating effect of FF on the acrosome reaction may have also contributed to these differences because acrosome-reacting spermatozoa are thought to rapidly lose their motility. 15 It appears therefore that acrosome-reacting spermatozoa were relatively under-represented in the rapid sperm subpopulations. Finally, the presence of FF may influence the time course of some other changes during sperm capacitation, a process that is still incompletely understood. From the practical viewpoint, the most interesting finding was the maintenance of a highly motile sperm subpopulation over incubation periods as long as 24 hours when FF was added to culture medium. This observation is in contradiction with some recent studies in which an impairment of sperm motility in the presence of unheated human FF has been reported, 6•16 an effect that could not, however, be confirmed by other investigators. 7•17 The source of these inconsistencies is not known. It may be that the fluid from some follicles is indeed toxic for spermatozoa and this condition may be responsible for some cases of infertility. If this correlation is confirmed, the examination of sperm response to FF can be considered as an additional diagnostic means in infertility management. It is noteworthy that almost 10% of spermatozoa incubated for 24 hours in the presence of FF in this study had curvilinear velocity over 90 ~J-m/s and progressive velocity of this rapid sperm subpopulation was even higher than that of the corresponding subpopulation of swim-up spermatozoa. The values of linearity, lateral head displacement, and beat cross frequency characterizing this sperm subpopulation approached the values typical of the cumulus-related motility pattern,9 the type of movement supposed to be related to the actual sperm ability to penetrate the ovum. Samples of FF previously tested for eventual sperm toxicity may thus be used as a medium supplement in all cases when a prolonged maintenance of sperm fertilizing ability is needed, such as for repeated artificial inseminations using the same semen sample, combination of gamete intrafallopian transfer and intrauterine insemination, or delayed in vitro insemination of oocytes showing signs of immaturity at the time of recovery. REFERENCES 1. Tesarik J, Testart J: Human sperm-egg interactions and
their disorders: implications in the management of infertility. Hum Reprod 4:729, 1989
Fertility and Sterility
2. Tesarik J: Comparison of acrosome reaction-inducing activities of human cumulus oophorus, follicular fluid and ionophore A23187 in human sperm populations of proven fertilizing ability in vitro .. J Reprod Fertil 74:383, 1985 3. Tesarik J, Pilka L, Drahorad J, Cechova D, Veselsky L: The role of cumulus cell-secreted proteins in the development of human sperm fertilizing ability: implication in IVF. Hum Reprod 3:129, 1988 4. Siiteri JE, Dandekar P, Meizel S: Human sperm acrosome reaction-initiating activity associated with the human cumulus oophorus and mural granulosa cells. J Exp Zool 246: 71,1988 5. Suarez SS, Wolf DP, Meizel S: Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid. Gamete Res 14:107, 1986 6. Mortimer D, Camenzind AR: The role of follicular fluid in inducing the acrosome reaction of human spermatozoa incubated in vitro. Hum Reprod 4:169, 1989 7. Stock CE, Bates R, Lindsay KS, Edmonds DK, Fraser LR: Extended exposure to follicular fluid is required for significant stimulation of the acrosome reaction in human spermatozoa. J Reprod Fertil86:401, 1989 8. Drahorad J, Cechova D, Tesarik J: Activation of acrosin by a locally produced component of human follicular fluid. J Reprod Fertil83:599, 1988 9. Tesarik J, Mendoza C, Testart J: Effect of the human cumulus oophorus on human sperm movement characteristics. J Reprod Fertil 88:665, 1990 10. World Health Organization: Laboratory Manual for the
Vol. 54, No.6, December 1990
11.
12.
13.
14.
15.
16.
17.
Examination of Human Semen and Semen -Cervical Mucus Interaction. 2nd edition. Cambridge, The Press Syndicate of the University of Cambridge, 1987, p 6 Testart J, Belaisch-Allart JC, Forman R, Gazengel A, Strubb N, Hazout A, Frydman R: Influence of different stimulation treatments on oocyte characteristics and in vitro fertilizing ability. Hum Reprod 4:192, 1989 Marrs RP, Saito H, Yee B, Sato F, Brown J: Effect of variation of in vitro culture techniques upon oocyte fertilization and embryo development in human in vitro fertilization procedures. Fertil Steril41:519, 1984 Laufer N, Tarlatzis BC, DeCherney AH, Masters JT, Haseltine FP, MacLusky N, Naftolin F: Asynchrony between human cumulus-corona cell complex and oocyte maturation after human menopausal gonadotropin treatment for in vitro fertilization. Fertil Steril 42:366, 1984 Mortimer S, Serres C, Mortimer ST, Jouannet P: Influence of image sampling frequency on the perceived movement characteristics of progressively motile human spermatozoa. Gamete Res 20:313, 1988 Bedford JM: Significance of the need for sperm capacitation before fertilization in eutherian mammals. Biol Reprod 28:108, 1983 Langlais J, Kan FWK, Granger L, Raymond L, Bleau G, Roberts KD: Identification of sterol acceptors that stimulate cholesterol efilux from human spermatozoa during in vitro capacitation. Gamete Res 20:185, 1988 Yee B, Cummings LM: Modification of the sperm penetration assay using human follicular fluid to minimize false negative results. Fertil Steril50:123, 1988
Mendoza and Tesarik FF and sperm motion
1139
Vol. 54, No.6, December 1990 Printed on acid-free paper in U.S.A.
Effect of follicular fluid on sperm movement characteristics
Carmen Mendoza, Ph.D_* Jan Tesarik, M.D., D-Sc-t Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Granada, Granada, Spain, and lnstitut National de Ia Sante et de Ia Recherche Medicale (INSERM), Unite 187, Department of Obstetrics and Gynecology, Hopital Antoine-Bee/ere, Clamart, France
Human spermatozoa previously washed from seminal plasma and incubated in capacitating medium were exposed to human follicular fluid (FF) for different time periods and their movement characteristics were examined by computer-assisted digital image analysis. No differences from control samples were observed after short exposures (15 minutes and~ hours). When the exposure was prolonged to 6 hours, spermatozoa incubated in the presence of FF displayed significant differences in some movement characteristics as compared with samples incubated without FF supplement. When only rapidly moving sperm subpopulations were considered, these effects ofFF were similar to those produced by a short incubation with the solubilized cumulus oophorus matrix as described previously. After 24 hours of incubation, there was a remarkable beneficial effect of FF on motility preservation. Possible physiological and clinical implications are discussed. Fertil Steril54:1135, 1990
The potential roles of ovarian follicular components that are released at ovulation together with the oocyte as signals modulating different aspects of sperm function are recently under scrutiny mainly for two reasons. First, examination of sperm capability to respond to these factors may offer additional diagnostic means for the andrologic laboratory and second, inclusion of these factors to culture media may improve sperm fertilizing ability in some conditions. 1 Both the cumulus oophorus 2- 4 and follicular fluid (FF), 2•5- 7 which accompany the ovulated oocyte after follicle rupture, have been reported to stimulate the acrosome reaction of human spermatozoa, and both of them are also potent accelerators of the activation of the main sperm proteolytic enzyme acrosin. 3 •8 Moreover, we have shown recently that components of
the human cumulus oophorus intercellular matrix exert a specific effect on the pattern of human sperm movement, acting selectively on capacitated spermatozoa.9 The experiments described here were undertaken to examine the effects of the other major by-product of ovulation, FF, on sperm movement characteristics. We demonstrate that FF can induce changes in human sperm movement characteristics similar to those resulting from the action of the cumulus oophorus but only after relatively long incubations. In addition, the inclusion of FF to culture medium significantly improves motility scores of sperm samples maintained in vitro for extended time periods.
Received January 11, 1990; revised and accepted August 1, 1990. *Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Granada, University Campus, Fuentenueva_ t Reprint requests: Jan Tesarik, M.D., D_Sc_, INSERM U. 187, Department of Obstetrics and Gynecology, Hopital Antoine-Beclere, 92141 Clamart, France.
Source and Preparation of Spermatozoa
VoL 54, No_ 6, December 1990
MATERIALS AND METHODS
Semen samples were obtained from eight donors with normal sperm parameters. 10 The procedures for selection of motile spermatozoa and in vitro capacitation were essentially the same as described. 9 Briefly, sperm were washed from seminal plasma Mendoza and Tesarik FF and sperm motion
1135
by repeated centrifugation and resuspension in Tyrode's medium (Difco, Detroit, Ml), 300 ,uL of B2 medium (API System, Montalieu-Vercieu, France) were carefully layered onto the final sperm pellet, and actively moving cells were recovered with the upper 200 ,uL of this medium after 30 minutes of incubation. Spermatozoa were then incubated for an additional 2 to 3 hours in B2 medium under 5% C0 2 in air for in vitro capacitation. Source and Preparation of FF
Samples of FF were collected by ultrasonically guided follicle aspiration from 10 women undergoing in vitro fertilization (IVF) treatment. Ovarian stimulation was carried out with gonadotropins after administration of a gonadotropin-releasing hormone agonist (GnRH-a)Y Only fluid obtained from follicles bearing a healthy-appearing oocyte rated as Grade 3 of oocyte maturation 12 and surrounded by a fully mucified cumulus oophorus rated as preovulatory 13 was used in this study. Samples showing any pink coloration were discarded, and only the first aspirates from follicles were used. All samples were centrifuged at 1500 X g for 10 minutes immediately after oocyte recovery, supernatants were pooled, filtered through a Millipore (0.45 ,urn pore size; Millipore S.A., Molsheim, France), and stored frozen at -20oC until used. Sperm Incubation with FF
Aliquants of motility-selected spermatozoa previously incubated for in vitro capacitation (see above) were pelleted again, resuspended in 1 mL fresh B2 medium supplemented with 20% FF (final sperm concentration of 1 to 5 X 106/mL), and left at 37oC under a gas phase of 5% C0 2 in air for periods up to 24 hours. Nonsupplemented B2 medium alone was used in control incubations.
10
120
* 8
100 ~
u
Cl>
80
~
6
en .....
E
:::1.
60
:I:
4
~
...J
Ill
>
E
:::1. ...J
<
40 2 20 0
0 VSL
ALH
Movement Characteristics
Figure 1 Progressive velocity ( VSL) and lateral head displacement (ALH) values (mean± SD for each donor sample) measured in whole sperm populations (D and 1!21 bars) and in their rapid (curvilinear velocity> 90 Jlm/s) subpopulations (D and ill bars) after 6 hours of control incubation in B2 alone (D and D bars) and in B2 supplemented with 20% FF (1!21 and • bars). *Values significantly different from corresponding controls (P < 0.01).
search, Inc., Danvers, MA). The parameter set-up and measurement conditions were those described previously. 9 The following characteristics of sperm motion were determined (for explanation of terms see Mortimer et al. 14 ): curvilinear velocity, progressive velocity, path velocity, linearity, straightness, lateral head displacement, and beat cross frequency. All measurements were carried out at a constant temperature of 3TC. The time of each measurement was limited to the maximum of 2 minutes to minimize changes because of medium evaporation. Under these conditions, 100 to 150 sperm tracks were recorded in each individual analysis. Two replicates of each analysis were made so that tracks of at least 200 motile spermatozoa were analyzed for each specimen and each time point. Statistical Analysis
Sperm Movement Analysis
Samples (200 ,uL) of sperm suspensions were taken at different times of incubation and concentrated by mild centrifugation (500 X g for 5 minutes) followed by decanting the supernatant and resuspension of the remaining cell pellet in 20 ,uL of the corresponding supernatant. Ten-microliter samples of these concentrated sperm suspensions were then put into a Makler chamber (Sefi-Medical Instruments, Haifa, Israel) and immediately introduced into a fully automated computer-assisted semen analyzer (HTM 2000; Hamilton Thorn Re1136
Mendoza and Tesarik
FF and sperm motion
All analyses were performed using the StatView II statistical package (Abacus Concepts, Inc., Berkeley, CA). Intragroup and intergroup variance was first assessed using ANOVA. Data for groups in which the ANOV A indicated significant differences were analyzed by Student's t-test and x2 test for normally distributed populations. Pearson and likelihood ratio x2 analysis were used to determine significance between non-normally distributed groups. Paired statistical comparisons were made for paired aliquots of each specimen incubated under different experimental conditions. Fertility and Sterility
Table 1 Motile and Rapid Spermatozoa After Different Times of Incubation in B2 Medium Alone and in B2 Supplemented With 20% FF• Motile sperm b
Rapid sperm c
Group
2h
6h
24h
B2 B2 + FFd
65 ± 9 72 ± 10
48 ± 7 62 ± 8
12 ± 4 27 ± 10
2h
6h
24h
25 ± 8 30 ± 8
29 ± 8 24 ± 7
0±0 9± 3
%
• Values are means± SD for each donor sample (n = 8). b Curvilip.ear velocity> 10 p.m/s. c Curvilinear velocity> 90 p.m/s. d Percentages of motile sperm in the B2 + FF group are significantly different from B2 for the 6-hour and 24-hour time points (P < 0.05). Percentages of rapid sperm are different for the 24-hour time point only (P < 0.05).
RESULTS The inclusion of 20% FF did not affect the values of curvilinear velocity as compared with B2 medium alone at any time examined. Similarly, the value of progressive velocity did not change in response to FF during the first 2 hours of incubation. However, after 6 hours of incubation, FF produced a significant alteration of this movement characteristic (Fig. 1). It was noteworthy that values were shifted in an opposite sense if measured in whole sperm populations and in their rapidly moving (curvilinear velocity> 90 f.Lm/s) subpopulations. In the whole populations, a decrease in progressive velocity was observed in the presence ofFF as compared with B2 alone, whereas the values of this motility parameter for samples exposed to FF were increased when only the rapid sperm subpopulations were considered (Fig. 1). The rapid subpopulations represented about 25% of the entire sperm populations at the 6-hour time point (Table 1). The same character of changes as for progressive velocity was observed for path velocity and for the two composite parameters of sperm movementlinearity and straightness. Differences between
samples incubated with FF and those in B2 alone were only significant after 6 hours of incubation, whereas the values did not differ after 15 minutes and 2 hours of incubation. The first significant modification of lateral head displacement in the presence of FF was also detected after 6 hours of incubation; in this case the values were augmented in whole sperm populations but reduced in the rapid sperm subpopulations (Fig. 1). Beat cross frequency measured in whole sperm populations did not change in response to FF at any time examined. However, in the rapid sperm subpopulations there was a significant increase in beat cross frequency after 6 hours of exposure to FF as compared with B2 alone (17.2 ± 1.9 versus 13.9·± 1.5). When spermatozoa were incubated in vitro for 24 hours, spectacular differences in all sperm movement characteristics examined were found between sampl~s incubated in the presence of FF and those incubated in B2 alone (Table 2). Although there was a remarkable decline in all parameters of sperm motility after 24 hours in B2 alone as compared with swim-up samples, the inclusion ofFF in medium had a significant moderating effect on these alterations and, in fact, the values measured after 24 hours in the presence of FF approached those of the original swim-up sperm populations (Table 2). When only rapid spermatozoa (curvilinear velocity > 90 f.Lm/s) were taken into account, there was even a significant increase in progressive velocity (84.5 ± 24.6 versus 70.2 ± 18. 7) and beat cross frequency (17.2 ± 2.9 versus 13.8 ± 2.4) in samples incubated for 24 hours with FF as compared with swim-up spermatozoa. The presence of FF increased the percentage of motile sperm at the 6-hour and 24-hour time points (Table 1). The effects of FF on the percentage of rapidly moving spermatozoa (curvilinear velocity > 90 f.Lm/s) after 2 hours and 6 hours of incubation
Table 2 Comparison of Changes in Movement Characteristics of Human Spermatozoa Incubated for 24 Hours in B2 Medium Alone and in B2 Substituted With 20% FF as Compared With Swim-up Sperm Populations Values of movement characteristics•
Group
Curvilinear velocity
Progressive velocity
Path velocity
Linearity
Swim-up B2 B2 +FFb
84.8± 11.5 30.0 ± 14.1 65.9 ± 35.2
68.1 ± 10.5 18.9 ± 14.5 55.3 ± 34.6
79.7 ± 18.2 21.1 ± 22.0 55.5 ± 34.8
73.1 ± 26.2 63.5 ± 22.0 77.6 ± 22.8
• Values are means± SD for each donor sample (n
Vol. 54, No.6, December 1990
=
8).
Straightness 87.0 ± 14.1 82.7 ± 10.9 87.8 ± 11.9
Lateral head displacement
Beat cross frequency
4.1 ± 1.1 2.0 ± 1.0 3.2 ±2.0
12.1 ± 2.8 7.6 ± 4.1 12.6 ± 4.1
b Except for straightness, all values are significantly different from B2 (P < 0.05).
Mendoza and Tesarik FF and sperm motion
1137
were negligible, but this rapid sperm subpopulation totally disappeared after 24 hours in B2 alone, whereas the proportion of rapid sperm cells after 24 hours of incubation still approached 10% of the whole sperm populations (Table 1). DISCUSSION
The present observations did not show any rapid response of human spermatozoa previously incu. bated for in vitro capacitation to subsequent exposure to FF. This is in contrast to the immediate effect of the cumulus oophorus intercellular matrix described recently. 9 However, when only rapidly moving spermatozoa were analyzed, the changes in sperm movement characteristics observed in this study after 6 hours of incubation with FF resembled those previously reported for capacitated spermatozoa incubated with the solubilized cumulus intercellular matrix for only 5 minutes. 9 This resemblance suggests that small quantities of factors responsible for the cumulus-specific modifications of sperm motion may be released to FF, but their concentration in FF may not be sufficient to elicit a rapid response. A similar situation seems to exist for cumulus-derived factors stimulating the acrosome reaction because the effect of FF on human sperm acrosome reaction is most considerable after relatively long incubations.6•7 It remains to be seen, however, whether the slow effects reported in this study are really caused by a low concentration of the same factors that have a rapid effect in the cumulus. It cannot be excluded that FF may contain other factors that affect sperm function in important ways. In fact, the observed effects ofFF may have resulted from a combined action of cumulus-derived molecules and other yet undetermined factors present in this biological material. The opposite effects of FF on some sperm movement characteristics observed in whole sperm populations and in their rapid subpopulations may be caused by different factors. First of all, these results may have been partly influenced by the effects of FF on sperm motility. If, as documented in Table 1, motility decreases more considerably in the absence of FF than in its presence, measured mean values of sperm velocity may have been affected by a faster depletion of slower swimming (presumably moribund) cells from the motile sperm populations in control incubations as compared with samples incubated with FF in which survival of such cells may have been prolonged. However, the absence of 1138
Mendoza and Tesarik FF and sperm motion
corresponding changes in values of curvilinear velocity speaks against this possibility. The known stimulating effect of FF on the acrosome reaction may have also contributed to these differences because acrosome-reacting spermatozoa are thought to rapidly lose their motility. 15 It appears therefore that acrosome-reacting spermatozoa were relatively under-represented in the rapid sperm subpopulations. Finally, the presence of FF may influence the time course of some other changes during sperm capacitation, a process that is still incompletely understood. From the practical viewpoint, the most interesting finding was the maintenance of a highly motile sperm subpopulation over incubation periods as long as 24 hours when FF was added to culture medium. This observation is in contradiction with some recent studies in which an impairment of sperm motility in the presence of unheated human FF has been reported, 6•16 an effect that could not, however, be confirmed by other investigators. 7•17 The source of these inconsistencies is not known. It may be that the fluid from some follicles is indeed toxic for spermatozoa and this condition may be responsible for some cases of infertility. If this correlation is confirmed, the examination of sperm response to FF can be considered as an additional diagnostic means in infertility management. It is noteworthy that almost 10% of spermatozoa incubated for 24 hours in the presence of FF in this study had curvilinear velocity over 90 ~J-m/s and progressive velocity of this rapid sperm subpopulation was even higher than that of the corresponding subpopulation of swim-up spermatozoa. The values of linearity, lateral head displacement, and beat cross frequency characterizing this sperm subpopulation approached the values typical of the cumulus-related motility pattern,9 the type of movement supposed to be related to the actual sperm ability to penetrate the ovum. Samples of FF previously tested for eventual sperm toxicity may thus be used as a medium supplement in all cases when a prolonged maintenance of sperm fertilizing ability is needed, such as for repeated artificial inseminations using the same semen sample, combination of gamete intrafallopian transfer and intrauterine insemination, or delayed in vitro insemination of oocytes showing signs of immaturity at the time of recovery. REFERENCES 1. Tesarik J, Testart J: Human sperm-egg interactions and
their disorders: implications in the management of infertility. Hum Reprod 4:729, 1989
Fertility and Sterility
2. Tesarik J: Comparison of acrosome reaction-inducing activities of human cumulus oophorus, follicular fluid and ionophore A23187 in human sperm populations of proven fertilizing ability in vitro .. J Reprod Fertil 74:383, 1985 3. Tesarik J, Pilka L, Drahorad J, Cechova D, Veselsky L: The role of cumulus cell-secreted proteins in the development of human sperm fertilizing ability: implication in IVF. Hum Reprod 3:129, 1988 4. Siiteri JE, Dandekar P, Meizel S: Human sperm acrosome reaction-initiating activity associated with the human cumulus oophorus and mural granulosa cells. J Exp Zool 246: 71,1988 5. Suarez SS, Wolf DP, Meizel S: Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid. Gamete Res 14:107, 1986 6. Mortimer D, Camenzind AR: The role of follicular fluid in inducing the acrosome reaction of human spermatozoa incubated in vitro. Hum Reprod 4:169, 1989 7. Stock CE, Bates R, Lindsay KS, Edmonds DK, Fraser LR: Extended exposure to follicular fluid is required for significant stimulation of the acrosome reaction in human spermatozoa. J Reprod Fertil86:401, 1989 8. Drahorad J, Cechova D, Tesarik J: Activation of acrosin by a locally produced component of human follicular fluid. J Reprod Fertil83:599, 1988 9. Tesarik J, Mendoza C, Testart J: Effect of the human cumulus oophorus on human sperm movement characteristics. J Reprod Fertil 88:665, 1990 10. World Health Organization: Laboratory Manual for the
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Examination of Human Semen and Semen -Cervical Mucus Interaction. 2nd edition. Cambridge, The Press Syndicate of the University of Cambridge, 1987, p 6 Testart J, Belaisch-Allart JC, Forman R, Gazengel A, Strubb N, Hazout A, Frydman R: Influence of different stimulation treatments on oocyte characteristics and in vitro fertilizing ability. Hum Reprod 4:192, 1989 Marrs RP, Saito H, Yee B, Sato F, Brown J: Effect of variation of in vitro culture techniques upon oocyte fertilization and embryo development in human in vitro fertilization procedures. Fertil Steril41:519, 1984 Laufer N, Tarlatzis BC, DeCherney AH, Masters JT, Haseltine FP, MacLusky N, Naftolin F: Asynchrony between human cumulus-corona cell complex and oocyte maturation after human menopausal gonadotropin treatment for in vitro fertilization. Fertil Steril 42:366, 1984 Mortimer S, Serres C, Mortimer ST, Jouannet P: Influence of image sampling frequency on the perceived movement characteristics of progressively motile human spermatozoa. Gamete Res 20:313, 1988 Bedford JM: Significance of the need for sperm capacitation before fertilization in eutherian mammals. Biol Reprod 28:108, 1983 Langlais J, Kan FWK, Granger L, Raymond L, Bleau G, Roberts KD: Identification of sterol acceptors that stimulate cholesterol efilux from human spermatozoa during in vitro capacitation. Gamete Res 20:185, 1988 Yee B, Cummings LM: Modification of the sperm penetration assay using human follicular fluid to minimize false negative results. Fertil Steril50:123, 1988
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