Accelerated mouse sperm penetration in vitro in the

Transcription

1 Accelerated mouse sperm penetration in vitro in the presence of caffeine Lynn R. Fraser Department of Human Biology, Basic Medical Sciences Group, Chelsea College, Manresa Road, London SW3 6LX, U.K. Summary. Evidence for accelerated mouse sperm penetration into eggs in vitro, and hence for accelerated capacitation, was obtained when the fertilizing ability of sperm suspensions was tested after preincubation, for a period of time insufficient to permit full capacitation under standard conditions, in the presence or absence of caffeine. When eggs were fixed after 1 h, the majority (76\m=.\7%)in the caffeine-containing medium were fertilized compared with only 32\m=.\1%in the caffeine-free medium. When fixation was later, after 1 h 15 min, fertilization levels were high (approximately 90%) in both groups, but significant differences in the stages of egg activation and sperm head decondensation reached were observed. In the absence of caffeine, early stages of nuclear development were predominant, while in the presence of caffeine, a significant proportion of eggs had reached terminal stages of activation. There was also some evidence for precocious sperm, but not egg, nuclear development when fertilization occurred in caffeine-containing medium. Introduction Mammalian sperm motility has been reported as being enhanced and/or prolonged in the presence of caffeine (Garbers, First, Sullivan & Lardy, 1971a; Garbers, Lust, First & Lardy, 1971b; Hicks, Martinez-Manautou, Pedron & Rosado, 1972; Schoenfeld, Amelar & Dubin, 1975). In some instances, caffeine has also been able to initiate motility in non-motile spermatozoa, e.g. from the testis (Brackett, Hall & Oh, 1978). Since caffeine can act as a cyclic nucleotide phosphodiesterase inhibitor and hence affect the intracellular level of cyclic AMP by inhibiting its enzymic breakdown (Casillas & Hoskins, 1970), cyclic AMP has been implicated in the observed motility increases (Garbers et al, 1971b; Hicks et al, 1972). While it is generally true that non-motile spermatozoa are non-fertilizing, the converse that motile spermatozoa are able to fertilize is not necessarily true (Harrison, 1977). Whether the stimulation of motility by caffeine has any effect on sperm capacitation and hence fertilizing ability has yet to be determined. However, the participation of cyclic AMP in the capacitation of spermatozoa has also been suggested (Morton & Albagli, 1973; Toyoda & Chang, 1974; Reyes, Goicoechea & Rosado, 1978), thus raising the possibility that caffeine, in fact, might have an effect on capacitation; if so, this should be detectable. Using an in-vitro fertilization system for which the temporal requirements for sperm capacitation and fertilization have been carefully studied (Fraser & Drury, 1976; Fraser, 1979), the effect of caffeine was examined to determine whether there is any influence on both motility and capacitation, the latter being assessed by determination of sperm penetration rates in vitro and subsequent early nuclear development.

2 Materials and Methods Fertilization in vitro Egg donors were adult, 2-4-month-old (C57BL/10 CBA) F! females induced to superovulate with an injection of 7-5 i.u. PMSG (Gestyl: Organon), followed approximately 48 h later by an injection of 5-0 i.u. hcg (Pregnyl: Organon). AU females were killed 13 h after the hcg injection. Sperm donors were adult, 2-4-month-old TO males, with 2 males being used in each experiment. Sperm suspensions were obtained by extruding spermatozoa from the cauda epididymidis into various media, detailed below, under paraffin oil (Boots); all dishes received spermatozoa^from both donors. Sperm suspensions were preincubated at 37 C in an atmosphere of 5% C02, 5% 02, 90% N2 for 30 min or 2 h, diluted 10-fold to give a final sperm concentration of approximately 2 106/ml and unfertilized eggs were added (Fraser & Drury, 1975). The medium used throughout was that described by Fraser & Drury (1975), and contained 30 mg BSA (Armour)/ml. Where noted, the medium also contained 2 or 6 mm-caffeine. Three series of experiments were carried out. In Series I, initial sperm suspensions were prepared in caffeine-free medium, as well as media containing 2 or 6 mm-caffeine. After preincubation for 30 min, a time insufficient for complete capacitation (Fraser & Drury, 1976), suspensions were diluted into medium of the same composition as used for the preincubation and eggs were added. In Series II, sperm suspensions were prepared as above, but only one concentration of caffeine, 2 mm, was used and the preincubation period was 2 h, a time sufficient for complete capacitation (Fraser & Drury, 1976). Sperm suspensions were then diluted into the same medium used for preincubation and eggs were added. A third medium, containing caffeine and lacking glucose, was also tested. In Series III, sperm suspensions were prepared in caffeine-free ( caffeine) and 2 mincaffeine-containing medium (+ caffeine) and preincubated for 30 min. Suspensions were diluted into the same medium used for preincubation, and, additionally, spermatozoa in the caffeine-free medium were diluted into caffeine-containing medium ( to + caffeine). Eggs were then added. Assessment offertilization and nuclear development In the first experiments in Series I, eggs were incubated in the sperm suspensions for approximately 50 min, washed once and placed in a small droplet of medium under paraffin oil. At 1 h after mixing all eggs were fixed by adding an excess of buffered formalin to each droplet; in this way, fixation of all groups was carried out within a few seconds. In all other experiments, eggs were incubated for just over 1 h, washed as above and fixed at 1 h 15 min. The eggs were then stained with 0-5% aceto-orcein and examined. If an egg proved to be fertilized, i.e. had resumed meiosis II and possessed a fertilizing sperm head, the stages of egg activation and of sperm head decondensation were assessed (Fraser, 1979). The former stages include: very early anaphase, early anaphase, mid-late anaphase, telophase and second polar body. Sperm head stages are: (1) just beginning to decondense; (2) approximately half-decondensed; (3) fully decondensed. Within a single egg therefore the egg and sperm nuclear development could be assessed independently. Assessment ofmotility In addition to the above, assessment was made of the sperm motility patterns at the time of sperm dilution, i.e. at the beginning of potential gamete interaction. The various suspensions

3 - were rated according to the proportion of single, motile spermatozoa which exhibited the typical whiplash tail activity which is associated with fertilizing ability and hence capacitation (Fraser, 1977). Series I Results When spermatozoa were preincubated in fertilization medium with and without caffeine and eggs were added, then fixed after 1 h, there was a significant difference ( 2, < 0-001) in the proportion of eggs fertilized, 9/28 (32-1%) in caffeine-free and 23/30 (76-7%) in caffeinecontaining medium. However, when the eggs were fixed after 1 h 15 min, the majority of eggs in the caffeine-free medium were fertilized (Table 1) and the stages of nuclear development could therefore be compared with those occurring in caffeine-containing medium. In caffeine-free medium the majority of eggs were at very early or early anaphase (62-7%) and the majority of sperm heads were at stage 1, i.e. just beginning to decondense. When spermatozoa from the same males were preincubated in fertilization medium containing 2 or 6 mm-caffeine, the proportion of eggs fertilized after 1 h 15 min was similar to that in caffeine-free medium. Striking differences were noted in the egg activation and sperm head decondensation stages, however (Table 1). In both groups, the majority of eggs had progressed beyond early anaphase and the proportion of eggs at telophase in each group was significantly higher ( 2, < 0-001) than in the caffeine-free medium. Similarly, approximately 50% of the sperm heads in Groups and C had undergone extensive decondensation and were at stages 2 or 3. The proportion at stage 3 was again significantly higher (P < 0-001) in the presence of caffeine (Groups and C), compared with that in Group A. Because there were some differences between the 2 caffeine-containing media, with a slightly higher proportion of eggs at telophase and sperm heads at stage 3 (P < 0-05) in Group than in Group C, only this concentration was used for the other 2 series. Table 1. Accelerated sperm penetration of mouse eggs in vitro in the presence of caffeine Egg stage (%) Sperm head stage, % No. of eggs- No. of fertilized/ Very early Early Mid-late Group exps. no. tested (%) anaphase anaphase anaphase Telophase A C Preincubation and fertilization in caffeine-free medium 102/ (87-9) (42-1) (20-6) (23-5) (13-7) Preincubation and fertilization in 2 mm-caffeine-containing medium 101/ (88-6) (6-9) (2-0) (30-7) (60-4) Preincubation and fertilization in 6 mm-caffeine-containing medium 86/ (89-6) (11-6) (5-8) (39-5) (43-0) Sperm suspensions were preincubated for 30 min, then diluted; eggs were fixed 1 sperm suspensions. h 15 min after mixing with Series II When spermatozoa were preincubated for 2 h in caffeine-free medium, fertilization levels 1 h 15 min after gamete mixing were again high (Table 2). The majority of eggs had advanced to telophase-second polar body and the majority of sperm heads had undergone extensive

4 - decondensation. After a 2-h preincubation in the presence of 2 mm-caffeine, fertilization levels were high and both egg activation and sperm head decondensation had progressed to late stages. There were no significant differences detectable between the caffeine-free and caffeine-containing groups. When glucose was omitted from the caffeine-containing medium, no fertilization (0/30) was obtained. Table 2. Sperm penetration of mouse eggs in vitro after 2 h preincubation in medium with and without 2 mm-caffeine Egg stage (%) Sperm head stage, % No. of eggs No. of fertilized/ Early Mid-late Telophase- Group exps. no. tested (%) anaphase anaphase polar body A Preincubation and fertilization in caffeine-free medium 68/ * «ai 4 (90-7) (2-9) (20-6) 23'6 15'3 6' 1 (76-5) Preincubation and fertilization in 2 mm-caffeine-containing medium 61/ < 38 -ion -mi 5 (86) (13-1) (24-6) (62-3) Eggs were fixed 1 h 15 min after mixing with sperm suspensions. 29' 24'2 46'8 Table 3. Accelerated sperm penetration of mouse eggs in vitro in the presence of 2 mm-caffeine Egg stage (%) No. of eggs- Sperm head stage, S No. of fertilized/ Very early Early Mid-late Group exps. no. tested (%) anaphase anaphase anaphase Telophase Preincubation and fertilization in caffeine-free medium 59/ (93-7) (45-7) (22-0) (25-4) (6-8) Preincubation and fertilization in 2 mm-caffeine-containing medium 72/ (94-7) (8-3) (2-8) (34-7) (54-2) C Preincubation in caffeine-free medium, fertilization in 2 mm-caffeine-containing medium 68/ (91-9) (23-5) (16-2) (26-5) (33-8) 54'2 25' 20'8 Sperm suspensions were preincubated for 30 min, then diluted; eggs were fixed 1 h 15 min after mixing with sperm suspensions. Series III As in Series I, preincubation in caffeine-free medium for 30 min yielded high fertilization levels (Table 3), with the majority of eggs at early anaphase and the majority of sperm heads at stage 1. Preincubation for 30 min in the presence of caffeine again yielded significantly higher proportions of eggs at telophase (P < 0-001) and sperm heads at stage 3 (P < 0-001) when compared with the first group. When spermatozoa were preincubated in caffeine-free medium and diluted into caffeine-containing medium ( to + caffeine), the fertilization levels were also high. Assessment of egg activation and sperm head decondensation revealed proportions at the various stages to be intermediate between those in Groups A and B. The proportion of eggs at telophase in Group C was significantly higher (P < 0-001) than in Group A and significantly lower (P < 0-05) than in Group B. Similarly, comparisons for sperm heads at stage 3 in Group

5 C indicated a higher proportion ( < 0-01) than in Group A and a lower proportion (P < 0-001) than in Group. Relationship between egg activation and sperm head stages In addition to the significantly higher proportion of advanced egg activation and sperm head stages noted when fertilization occurred in the presence of caffeine after a 30-min preincubation of the sperm suspensions (Tables 1 and 3), a careful analysis of the correlation between stages within individual eggs indicated that in the presence of caffeine the sperm head stages were more advanced than in eggs which were at the same activation stage but had been fertilized in the absence of caffeine. These analyses are shown in Table 4. Since all eggs in all groups which were at early anaphase possessed sperm heads at stage 1, only stages of mid-anaphase and beyond are considered. In Series I and III, all eggs fertilized in the absence of caffeine and at midanaphase when fixed had sperm heads at stage 1, while some eggs at the same stage but fertilized in the presence of caffeine had sperm heads at stages 2 or 3. In eggs at late anaphase, stage 3 sperm heads were noted only in eggs fertilized in the presence of caffeine. Only sperm suspensions tested after 30-min preincubation have been considered because the differences between groups disappeared when a 2-h preincubation was allowed to permit full capacitation (Table 2). Table 4. Accelerated sperm head decondensation (stages 1-3) in mouse eggs at various stages of nuclear development in the presence of caffeine Caffeine (mm) Mid-anaphase Late anaphase Telophase Series I 0 (16) 100 (8) (14) (11) (20) (61) (16) (18) (37) Series HI 0 (4) 100 (11) (4) ^2 (5) (13) (23) (3) (22) (39) The total number of eggs at each egg stage is given in parentheses; other values are %. Motility patterns Epididymal mouse spermatozoa are very motile when released into a medium capable of supporting capacitation. For this reason, detecting any significant effect of caffeine on the speed of sperm movements is difficult; only if the spermatozoa have become sluggish will the addition of caffeine have a noticeable effect on this aspect of sperm motility. On the other hand, assessment of sperm motility patterns after a 30-min preincubation revealed striking differences. In the absence of caffeine, very few of the spermatozoa (<5%) exhibited the whiplash tail movements described in mouse sperm suspensions capable of immediate fertilization (Fraser, 1977). When caffeine was present during the preincubation period, the majority of spermatozoa (>50%) showed the motility patterns consistent with capacitation. When spermatozoa were preincubated in caffeine-free medium and diluted into caffeine-containing medium, a significant proportion of the spermatozoa (25-50%) exhibited the whiplash motility pattern within a short time.

6 Discussion When mouse spermatozoa were preincubated for 30 min in caffeine-free medium and eggs were then added, within 1 h 15 min the proportion of eggs which had been fertilized was very high, approximately 90%. The finding that the majority of these eggs, after fixation and staining, proved to be at early stages of egg activation and possessed sperm heads just beginning to decondense indicated that although the spermatozoa were not fully capacitated at the time of gamete mixing, they became capable of fertilizing during the later part of the incubation with eggs. This is confirmed by the lower fertilization levels (32-1%) observed when eggs in this group were fixed earlier, i.e. after 1 h. The spread in stages indicates that the acquisition of fertilizing ability by individual spermatozoa in the total population was not completely synchronous. The longer preincubation time of 2 h, however, should allow capacitation to be completed before the incubation with eggs (Fraser & Drury, 1976) and a more synchronous pattern of fertilization would be expected. Indeed, such a synchrony was noted, with the majority of eggs at terminal stages of activation and sperm heads fully decondensed (Table 2; Fraser, 1979). In contrast to the results in the caffeine-free medium, the presence of caffeine during the 30 min preincubation time resulted in significantly accelerated sperm penetration. In 2 mm-caffeine, the majority of eggs were at telophase and possessed sperm heads that had undergone considerable decondensation at the time of fixation, indications that fertilization had occurred within a short time of gamete mixing. Furthermore, there appeared to be greater synchrony of fertilization and development, which would be consistent with the suggestion that the sperm populations were nearly fully capacitated at the time of egg-sperm mixing. Indeed, there were relatively few differences between stages obtained when spermatozoa were preincubated for as short a time as 30 min or as long as 2 h in the presence of caffeine, there being merely a slightly greater shift toward the terminal stages of assessment (Tables 1, 2 and 3). In contrast, striking differences were found between the 2 preincubation times in the rate of penetration when caffeine was not present in the medium. When using a system such as fertilization in vitro to evaluate the performance of one gamete, here the spermatozoon, one must always bear in mind that the final assessable results depend on the interaction of two gametes and hence a possible effect on the egg must also be considered. That caffeine was affecting spermatozoa rather than eggs is indicated by the experiments where eggs were fixed after 1 h. At this time, the majority of eggs in the caffeine-containing medium were fertilized while those in the caffeine-free medium were unfertilized. Furthermore, in Series III, 2 of the groups of spermatozoa were diluted into caffeine-containing medium so that the eggs were released into media with similar concentrations of caffeine, yet there were significant differences in the rates of sperm penetration. These rates were dependent on the presence or absence of caffeine during the initial preincubation period and thus the detectable effects were due to changes in the spermatozoa and not in the eggs. Finally, in Series II when both groups of sperm suspensions had been preincubated for 2 h to permit full capacitation, the presence of caffeine had no detectable accelerating effect on the stages of nuclear development; similar terminal stages were observed in both groups. This conclusion is consistent with the sperm motility pattern assessments, where the observed ranking of sperm groups from that showing the highest proportion of whiplash motility to least was identical to the ranking based on rates of fertilization and subsequent nuclear development, i.e. + caffeine > to + caffeine > caffeine. The almost immediate effect of caffeine on motility patterns indicates a specific effect on spermatozoa, rather than eggs, which is associated with fertilizing ability (Fraser, 1977) and not simply an increase in general motility. In addition to the accelerated rate of penetration, there was some evidence of accelerated sperm head decondensation when fertilization occurred in the presence of caffeine. Under standard conditions, the finding of mid-anaphase stage eggs with stage 2 sperm heads is relatively unusual and stage 3 sperm heads have not been seen previously in such eggs (L. R.

7 Fraser, unpublished observation). Similarly, although stage 1 and 2 sperm heads have been found frequently in eggs at late anaphase, stage 3 heads are very rare. Such unusual egg stagesperm head stage combinations were noted in Series I and III, but the reason for such precocious decondensation is not immediately obvious. While it seems clear that, in the presence of caffeine, spermatozoa become capacitated more rapidly, and hence penetrate into eggs more rapidly, once inside the egg the sperm head comes under the influence of the egg cytoplasm. In the absence of caffeine, the rates at which mouse spermatozoa both penetrate eggs and undergo subsequent nuclear development are dependent, to a certain extent, on the age of the egg, with the processes being accelerated in older eggs (Fraser, 1979). In the present experiments, however, the eggs were all recovered at the same time after hcg injection and fertilization in the different groups would appear to have occurred within about 45 min of each other (based on timing values in Fraser, 1979). The egg age should not therefore have been a major factor and the effect of caffeine must have been on the sperm head, but whether directly or via the egg cytoplasm is at present unclear. Since the predominant range of egg activation and sperm head stages mirrored the length of time the spermatozoa, rather than the egg, had been in caffeinecontaining medium, there was no obvious acceleration of egg stage by caffeine. Otherwise, similar stages of egg activation would be expected in both the caffeine-containing groups in Series III (Table 3). These experimental results demonstrate that caffeine accelerates the rate at which mouse spermatozoa become capacitated in vitro and capable of fertilizing eggs and therefore suggest that cyclic AMP may be a key factor in sperm capacitation. Such an hypothesis has been proposed by a number of workers (Harrison, 1977). In order for a cell to produce cyclic AMP from ATP, the enzyme adenylate cyclase is required and adenylate cyclases have been demonstrated in the spermatozoa for a number of mammals, including hamster (Morton & Albagli, 1973), man (Hicks et al, 1972) and monkey (Casillas & Hoskins, 1971). Furthermore, the activity of adenylate cyclase in capacitated hamster spermatozoa is greater than in uncapacitated spermatozoa, based on production of cyclic AMP per unit time (Morton & Albagli, 1973), suggesting that during capacitation there may be an increase in the concentration of intracellular cyclic AMP. By precociously increasing this concentration, caffeine may accelerate the rate at which capacitation occurs, as observed in the present experiments. Additionally, the present results are consistent with in-vitro fertilization studies with the rat (Toyoda & Chang, 1974) and rabbit (Reyes, Goicoechea & Rosado, 1978) which have suggested that addition of cyclic AMP to the medium can shorten the time required for capacitation. It should be stressed that caffeine has not been shown to induce capacitation per se, but rather to accelerate the rate at which it occurred when spermatozoa were incubated in an environment permitting capacitation, albeit more slowly. Hoppe (1976) has shown that glucose is required for fertilization in vitro in standard medium, i.e. caffeine-free. The present experiments have demonstrated that caffeine-containing medium, in the absence of glucose, was unable to support fertilization and thus was unable to change non-permissive conditions into permissive ones. A similar conclusion was drawn by Morton & Chang (1973) when caffeine was added to non-capacitating medium containing hamster spermatozoa. The rate of motility was significantly increased, but the spermatozoa proved unable to fertilize eggs and hence would appear to have been uncapacitated. Resolution of the specific function(s) of cyclic AMP, assuming this to be the active agent in caffeine-affected systems, still remains. Since cyclic AMP is apparently involved in a myriad of detectable processes, further experiments are obviously required to identify, if possible, the critical pathway in this particular system. In addition to stimulation of motility, caffeine also has been shown to increase the rates of respiration (Garbers et al, 1971a, b) and glycolysis (Hoskins, 1973) of spermatozoa. More extensive studies may indicate that one of these is the route whereby caffeine accelerates capacitation.

8 References Brackett, B.G., Hall, J.L. & Oh, Y. (1978) In vitro fertilizing ability of testicular, epididymal and ejaculated rabbit spermatozoa. Fert. Steril. 29, Casillas, E.R. & Hoskins, D.D. (1970) Activation of monkey spermatozoa adenyl cyclase by thyroxine and triiodothyronine. Biochem. Biophys. Res. Com mun. 40, Casillas, E.R. & Hoskins, D.D. (1971) Adenyl cyclase activity and cyclic 3',5'-AMP content of ejaculated monkey spermatozoa. Archs Biochem. Biophys. 147, Fraser, L.R (1977) Motility patterns in mouse sper matozoa before and after capacitation. J. exp. Zool. 202, Fraser, L.R. (1979) Rate of fertilization in vitro and subsequent nuclear development as a function of the post-ovulatory age of the mouse egg. J. Reprod. Fert. 55, Fraser, L.R. & Drury, L. (1975) The relationship between sperm concentration and fertilization in vitro of mouse eggs. Biol. Reprod. 13, Fraser, L.R. & Drury, L. (1976) Mouse sperm genotype and the rate of egg penetration in vitro. J. exp. Zool. 197, Garbers, D.L., First, N.L., Sullivan, JJ. & Lardy, H.A. (1971a) Stimulation and maintenance of ejaculated bovine spermatozoan respiration and motility by caffeine. Biol. Reprod. 5, Garbers, D.L., Lust, W.D., First, N.L. & Lardy, H.A. (1971b) Effects of phosphodiesterase inhibitors and cyclic nucleotides on sperm respiration and motility. Biochemistry, N.Y. 10, Harrison, R.A.P. (1977) The metabolism of mammalian spermatozoa. In Frontiers in Reproduction and Fertility Control, Part 2, pp Eds R. O. Greep & M. A. Koblinsky. MIT Press, Cambridge, Massachusetts. Hicks, J.J., Martinez-Manautou, J., Pedron, N. & Rosado, A. (1972) Metabolic changes in human spermatozoa related to capacitation. Fert. Steril. 23, Hoppe, P.C. (1976) Glucose requirement for mouse sperm capacitation in vitro. Biol. Reprod. 15, Hoskins, D.D. (1973) Adenine nucleotide mediation of fructolysis and motility in bovine epididymal sper matozoa. /. biol. Chem. 248, Morton, B. & Albagli, L. (1973) Modification of hamster sperm adenyl cyclase by capacitation in vitro. Biochem. Biophys. Res. Commun. 50, Morton, B. & Chang, T.S.K. (1973) The effect of fluid from the cauda epididymidis, serum components and caffeine upon the survival of diluted epididymal hamster spermatozoa. J. Reprod. Fert. 35, Reyes,., Goicoechea, B. & Rosado, A. (1978) Calcium requirement for rabbit spermatozoal capacitation and enhancement of fertilizing ability by ionophore A and cyclic adenosine 3':5'-monophosphate. Fert. Steril. 29, Schoenfeld, C, Amelar, R.D. & Dubin. L. (1975) Stimulation of ejaculated human spermatozoa by caffeine. Fert. Steril. 26, Toyoda, Y. & Chang, M.C. (1974) Capacitation of epididymal spermatozoa in a medium with high K/Na ratio and cyclic AMP for the fertilization of rat eggs in vitro. J. Reprod. Fert. 36, Received 8 February 1979