- Email: [email protected]
Pentoxifylline improves sperm capacitation and in vitro fertilization of oocytes in the golden hamster
PENTOXIFYLLINE IMPROVES SPERM CAPACITATION FERTILIZATION OF OOCYTES IN THE GOLDEN
A. Rupasri’,
AND IN VITRO HAMSTER
D. Jayaprakash’, A.T.Peter2, M.S. Sreenivasa’, M. Kumart and P.B. Seshagirilya
‘Center for Reproductive Biology and Molecular Endocrinology Indian Institute of Science, Bangalore 560 012, India 2Department of Veterinary Clinical Sciences Purdue University, West Lafayette, IN, USA
Received
January 27, 1995 Mar& IO, 1995
for publication: Accepted:
ABSTRACT Pentoxifylline (PF) is used to improve motility of spermatozoa from subfertile or nonfertile males to accomplish in vitro fertilization in humans. The possible adverse effect of PF on pre- and peri- implantation stage embryo development in a suitable rodent model, such as the golden hamster, is yet to be determined. In this study, hamster cauda epididymal spermatozoa were exposed to different concentrations (0.23 to 3.6 mM) of PF, and their quantitative [percentage of motility] and qualitative [Score 0 to 51 motility were assessed and values expressed as the sperm motility index. Upon addition of spermatozoa to dishes containing PF, an immediate increase in sperm motility and sperm motility index was evident, which increased up to 4 to 6 h and then declined. The sperm motility index increase by PF was dose-dependant, and al.8 mM PF was detrimental after 4 h. The optimum dose of PF was found to be 0.45 mM. To assess the fertilizing ability of PF-treated spermatozoa, in vitro fertilization was carried out. Fertilization rates for spermatozoa treated with 3.6 mM PF were lower (53.8 2 7.8) than for the controls (69.5 ? 10.2), whereas treatment with 0.45 mM PF increased the rates (91.6 + 4.3) compared with that of the controls (80.2 f 5.9). In conclusion, low concentrations (0.23 to 0.45 mM) of PF improve sperm capacitation and fertilization of oocytes in vitro in the golden hamster. Key words:
hamster,
in vitro fertilization,
pentoxifylline,
sperm motility
Acknowledgments We thank the Indian Institute of Science for financial support and the MUCIA, Purdue University International Programs for travel support (to ATP). aCorrespondence
and reprint requests.
Theriogenology 44:553-562, 1995 0 1995 by Elsevier Science inc. 655 Avenue of the Americas, New York, NY 10010
0093-691 x/95/$1 0.00 SSDI 0093-691X(95)00226-X
554
Theriogenology INTRODUCTION
In humans, the causes of infertility in the subfertile male may oligospermia, asthenozoospermia and teratozoospermia. To accomplish assisted using such spermatozoa, various sperm motility factors need to be assessed. (PF), a methyl xanthine derivative, is a sperm motility enhancing compound is being tested in clinical trials with encouraging results (1,6,12,15,17,21,22). adverse effects of PF on pre- and post- implantation viability of embryos defined.
be those of reproduction Pentoxifylline and its effect The possible are not well
Most studies with human spermatozoa used 3.6 mM or higher doses of PF (7,22). Information on the optimum dose, its possible adverse effect on fertilization and embryo development in a suitable rodent model is needed since experiments with humans are not feasible for ethical and practical reasons. Hence, we initiated investigations into the effect of PF on gamete maturation, fertilization and pre- and peri- implantation embryo development in the golden hamster. Hamsters are an ideal rodent model since they provide several major advantages for the studies on early mammalian development (4). In this study, we present data on the dose-dependent enhancement of sperm capacitation and fertilization by PF in the golden hamster. MATERIALS
AND METHODS
Adult (3 to 5 mo) male and female golden hamsters (Mesocricetus auratus) used for the present study were either from our colony or from the Laboratory Animals Information Service Centre, Hyderabad, India. They were housed under normal temperature (24-26OC) and lighting schedule (12L: 12D). The culture medium for the sperm assay and for in vitro fertilization was a modified Tyrode’s medium containing bovine serum albumin (BSA, 3 mg/ml); sodium lactate (10 mM); pyruvate, (0.25 mM); and 1 mg/ml polyvinyl alcohol (TALP-PVA) supplemented with penicillamine, hypotaurine and epinephrine (PHE), as described elsewhere (4,5). All components and pentoxifylline were purchased from Sigma Chemical Company (St. Louis, MO, USA) or HiMedia Labs (Bombay, India). A stock solution of 360 mM PF was prepared in T’LPVA (TALP-PVA devoid of albumin and pyruvate) and the required dilutions were made on the day of experiment. The medium used for the experiments had an osmolality of 285 to 300 mOsmols/kg water and a pH of 7.4 after equilibration in 5% CO, in air at 37°C. The sperm motility assay was carried out according to the published procedures (5). Briefly, male hamsters with large, turgid caudae epididymides were sacrificed by cervical dislocation. Epididymides were dissected out, collected in TL-HEPES buffer (pH 7.4). cleared from adherent tissues, and the distal tubules were punctured by a sterile 26.5-g needle. These 2 ~1 of the released epididymal spermatozoa (approximately 3 to 4 x 106/ml)
Theriogenology
555
were transferred to each 35-mm Falcon dish (#3001) containing 2 ml of preequilibrated TALP-PVA medium with or without desired concentrations (0.23 to 3.6 mM) of PF, and incubated in a humidified atmosphere of 5% CO, in air at 37’C. Observations on the percentage of sperm motility and quality of motility (Score 0 to 5) were made every 1 or 2 h for a duration of 6 to 8 h. Values were expressed as sperm motility index, SMI: % motility x [quality score 0-512. The dishes were scored independently by at least 2 persons, and the mean values were calculated at the end of each experiment. The experiments were repeated with at least 2 to 3 batches of media. Insemination drops (100 ~1) of TALP-PVA supplemented with PHE were placed in 35-mm Falcon dishes and overlaid with silicone oil (Aldrich Chemical Co., Milwaukee, WI). Adult females were superstimulated with 25 IU, ip of PMSG on the day of the post-estrus discharge, followed 56 h later by 25 IU hCG. The animals were sacrificed at 16 to 18 h posthCG treatment, and the oviducts were removed and collected in preequilibrated mineral oil. The swollen ampullae were punctured with a fine forceps, and the contents were allowed to extrude out. The cumulus cells were removed by transferring into a 100 ~1 drop of hyaluronidase (1 mg/ml) containing 0.01 mg/ml of soybean trypsin inhibitor in TL-HEPES. Oocytes were washed twice with TALP-PVA + PHE and transferred to insemination drops, 5 or 10 ~1 of untreated or PF-treated sperm suspensions were introduced into each drop. Only unwashed spermatozao were used, and the PF concentrations during coincubation were 23 or 45 PM and 180 or 360 PM when 0.45 mM and 3.6 mM PF- treated sperm samples were used, respectively. After 3 to 4 h of coincubation, the oocytes were washed and examined for the presence of 2 pronuclei (4), using an Olympus IMT-2 inverted microscope equipped with Nomarski DIC objectives (x10 and x20). Values expressed in figures and tables were means ? SEM from a minimum experiments. Statistical analysis was carried out by Student’s t-test.
of 5
RESULTS An immediate increase in the motility of cauda epididymal spermatozoa was observed in medium containing PF, with maximal sperm motility index reaching by 2 to 4 h (P < 0.01) then declining by 6 h. In contrast, the sperm motility index of spermatozoa in PF-free medium showed a gradual increase until 6 h (Figure 1). Exposure of spermatozoa to lower concentrations (2.7 and 1.8 mM) of PF resulted in higher sperm motility index compared with that observed with 3.6 mM PF. However, with 0.9 mM concentration there was no increase in sperm motility index at 2 h, but it increased significantly by 4 h (P < 0.001) and by 6 h (P < 0.01). Observations indicate that spermatozoa exhibited hyperactivation following 2 h of incubation with PF (1.8 to 3.6 mM) and it persisted up to 4 h.
556
w
TALP
ALONE
O---J
TALP + 3.6 mM PF
-
TALP + 2.7 mM PF
O-.-J
TALP+1.8mMPF
O---O
TALP + 0.9 ml4 PF,
I
0
1
1
I
2
4
6
TIME Figure 1.
(hours)
Effect of varying concentrations of pentoxifylline on hamster sperm motility. Each value represents mean +. SEM (n=5). Level of significance: l = P < 0.05, * = P CO.01, n = P < 0.001.
When lower concentrations, i.e., 0.9, 0.45 and 0.23 mM of PF were tested in a separate set of experiments (Figure 2), a further increase in sperm motility index was evident. The maximum sperm motility indices were obtained with 0.45 mM followed by 0.23 and 0.9 mM concentrations of PF which were significantly (P
557
Theriogenology
16001
1400-
-
TALP
e.,.d
TALP +o.g
ALONE
+--*
TALP+0.45
C.-O
TALP+O.
mM PF
mM PF 23
mM Plj,
X $
1200-
g -I
lOOO-
S t
800-
I 0
1
1
I
2
4
6
TIME
Figure 2.
(hours)
Effect of varying concentrations of pentoxifylline on hamster sperm motility. Each value represents mean + SEM (n = 12). Level of significance: * = P < 0.01, n = P < 0.001.
Insemination of oocytes with 3.6 mM PF-treated spermatozoa slightly decreased the fertilization rates (53.8 f 7.8) compared with that (69.5 + 10.2) obtained with untreated spermatozoa. There was no significant difference in fertilization rates when 2 different concentrations (5 x lo4 or 10 x 104) of spermatozoa were tested (Table 1).
558 Table 1.
Treatment
Theriogenology Assessment of in vitro fertilizability pentoxifylline-treated spermatozoaa
Sperm concentration
of golden hamster
No. of oocytes inseminated
No. of oo tes fertilizedYJ
Percentage of fertilization Mean k SEM
5 x lo4
64
51
69.9 f 10.6’
10 x lo4
29
21
75.02 08.3c
Total
93
72
69.5 2 10.2d
5x lo4
59
34
52.6+ 7.8e
10 x lo4
29
18
66.7? 4.7e
Total
88
52
53.8a
(per ml)
No Pentoxixifylline
Pentoxifyllinef (3.6 mM)
oocytes by 3.6 mM
7.Sd
aData are from 9 IVF procedures using 9 superovulated females. bEvidence of fertilization: presence of 2 pronuclei in the ooplasm at 3 to 4 hours post insemination (4). Observations were made using an Olympus IM’I-2 microscope with Nomarski DIC objective lenses (x10 and x20). ‘Mean values were not statistically significant (P > 0.2). dMean values were not statistically significant (P < 0.2). eMean values were not statistically significant (P > 0.2). fThe concentrations of pentoxifylline in insemination drops were 180 and 360 PM when 50 and 100 thousand spermatoza were used, respectively.
In a separate set of experiments, IVF with 0.45 mM PF-treated spermatozoa was carried out. An improvement in the fertilization rate was observed with 0.45 mM PF samples (91.6 + 4.3) when compared with that of the controls (SO.2 + 5.9). When 2 different concentrations (5 x lo4 or 10 x 104) of spermatozoa were used for insemination, Fertilized oocytes were normal by the fertilization rates were similar (Table 2). morphological criteria and contained 2 pronuclei and 2 polar bodies (Figure 3).
559
Theriogenology Table 2.
Treatment
No
Assessment of in vitro fertilizability petoxifylline-treated spermatozoaa
of golden hamster
oocytes by 0.45 mM
Percentage of fertilization Mean t SEM
No. of oocytes inseminated
No. of oo tes fertilizedx
5 x lo4
56
49
81.7 ? 6.5”
10 x lo4
35
30
84.7 + 4.3c
Total
91
79
80.2 * 5.9d
5 x lo4
59
53
90.8 + 4.32
10 x lo4
36
30
83.7 * 7.4”
Total
95
83
91.6 ? 4.3d
Sperm concentration (per ml)
pentoxifylline
Pentoxifyllinef (0.45 mM)
aData from 7 IVF procedures using 7 superovulated females. bEvidence of fertilization: presence of 2 pronuclei in the ooplasm at 3 to 4 hours post insemination (4). Observations were made using an Olympus IMT-2 microscope with Nomarski DIC objective lenses (x10 and x20). ‘Mean values were not statistically significant (P > 0.20). dMean values were not statistically significant (P < 0.20). eMean values were not statistically significant (P > 0.20). fThe concentration of pentoxifylline in insemination drops were 180 and 360 PM when 50 and 100 thousand spermatoza were used, respectively.
Figure 3.
Photomicrographic appearance of oocytes fertilized in vitro with untreated (Panel a) and 0.45 mM pentoxifylline-treated (Panel b) spermatozoa in the golden hamster. Arrows indicate locations of pronuclei. Photographed with Nomarski DIC objectives (x20).
560
Theriogenology DISCUSSION
Our study shows, for the first time, that pentoxifylline (PF, so.45 mM) improves capacitation of spermatozoa and in vitro fertilization of oocytes in the golden hamster. The sperm motility enhancing effect of PF is dose-dependent; low concentrations enhance longevity of motility, and its prolonged exposure at high concentrations (~1.8 mM) is detrimental to sperm motility (Figures 1 and 2), as observed with human (12) and mouse (21) spermatozoa. An immediate effect, within 10 min of exposure, of PF on the enhancement of motility of hamster spermatozoa was observed. Similar to observations with normospermic human samples (13), exposure of hamster spermatozoa to PF did not result in appreciable improvement in quantitative motility. The quality of motilty was, howerver, different. The hyperactivated motility of spermatozoa is achieved after 2 h and persists up to 4 h. These features are similar to those observed with normo-, astheno- and terato-spermic human samples (7,10,16,22). Microscopic observations indicate that hamster spermatozoa exhibit curvilinear (hyperactivated) motility by 2 to 4 h and are able to fertilize oocytes in vitro (Tables 1 and 2). Computer-assisted semen analysis of PF-treated human spermatozoa reveal increased curvilinear velocity but not straight line velocity (2,9). The curvilinear velocity profile of PF-treated hamster spermatozoa by Computer-assisted semen analysis remains to be investigated. The drastic decline in hamster sperm motility observed after 4 h of treatment with ~2.7 mM PF (Figure 1) may be attributed to the toxic effect on sperm plasma membrane at the ultrastructural level, as observed with human spermatozoa (9,10,13,16). The other possibility for the decline is that spermatozoa could have undergone acrosome reaction resulting in the release of hydrolytic enzymes which may affect their integrity and viability. Although BSA is well recognized as an acrosome reaction-inducing factor (3,11), it is likely that PF could also have been induced acrosome reaction independently of BSA. This remains to be investigated in hamsters. The data in Tables 1 and 2 show that fertilization could be achieved with unwashed spermatozoa in the presence of 23,45, 180 and 360 PM PF during insemination. Although a statistically significant difference in fertilization rates was not discernible, there was a slight reduction in rates with 3.6 mM PF-treated spermatozoa, as reported in the mouse (20,21), and an improvement with 0.45 mM PF-treated spermatozoa. The slight difference, although not statistically significant, in fertilization rates between control samples in 2 different sets of experiments (Tables 1 and 2) could be due to the use of different groups of animals from the commercial supplier. These data show that removal of PF from the sperm suspension medium prior to insemination may not be required, and continued presence of ~360 PM PF during insemination may not inhibit fertilization in the hamster. Since the aim of the present study was to evaluate the fertilizing ability of the PFtreated spermatozoa and to confirm the fertilization status of the inseminated oocytes, culture of fertilized oocytes and an assessment of their preimplantation development were
561
Theriogenology
not carried out. Our ongoing studies, however, indicate that the first cleavage division of IVF oocytes inseminated with PF-treated spermatozoa are not affected. We also found that continuous presence of 50.9 mM PF in hamster embryo culture medium (14) does not affect cultured 8-cell embryo development, and the presence of 3.6 mM PF retards embryo development (Rupasri and Seshagiri, unpublished observation). Human and mouse embryo development in vitro is arrested by PF in a dose-dependent manner and at lower concentrations the effect of PF is less profound (8,18,19,20). Although there is considerable controversy on the use of PF as a sperm motility stimulant in assisted reproduction in humans (6,17,22), extensive investigation in suitable rodent models is essential not only to evaluate the long-term safety of PF during pregnancy but also to understand its molecular mechanism of action in improving sperm motility and its clearcut influence on pre- and peri- implantation embryo development. Pentoxifyiline could prove to be an acceptable, useful sperm motility stimulant (alternative to caffeine/heparin) for use in humans and other species, particularly to improve the motility of inherently poor quality spermatozoa of a few domestic species such as the buffalo.
REFERENCES
Aparicio NJ, Schwarzstein L, de Turner EA. Pentoxifylline (BL 191) by oral administration in the treatment of asthenospermia. Andrologia 1980;12:228-231. Barlow P, Delvigne A, Van Dromme J, Van Hoeck J, Vandenbosch K, Leroy F. Predictive value of classical and automated sperm analysis for in vitro fertilization. Hum Reprod 1991;6:1119-1124. Bavister BD. Bovine serum albumin is requred for efficient capacitation of hamster spermatozoa in vitro. Biol Reprod 1981;24(Suppl 1):37abstr. Bavister BD. A consistently successful procedure for in vitro fertilization of golden hamster eggs. Gamete Res 1989; 23:139-158. Bavister BD, Andrews JC. A rapid sperm motility bioassay procedure for qualitycontrol testing of water and culture media. J In Vitro Fertil Embryo Transfer 1988;5:67-75. Hammitt DG, Bedia E, Rodgers PR, Syrop CH, Donovan JF, Williamson RA. Comparison of motility stimulants for cryopreserved human semen. Fertil Steril 1989;52:495-502. Kaskar K, Franken DR, Van der Horst G, Kruger TF. The effect of pentoxifylline on sperm movement characteristics and zona pellucida binding potential of teratozoospermic men. Hum Reprod 1994;9:477-481. Lacham-kaplan 0, Trounson AO. Embryo development capacity of oocytes fertilized by immature sperm and sperm treated with motility stimulants. Reprod Fertil Dev 1994;6:113-116. Lewis SEM, Moohan JM, Thompson W. Effects of pentoxifylline on human sperm motility in normospermic individuals using computer-assisted analysis. Fertil Steril 1993;59:418-423.
562 10 11 12 13 14 15 16 17’ 18 19 20 21 22
Theriogenology Mbizvo MT, Johntson RC, Baker GHW. The effect of the motility stimulants, caffeine, pentoxifylline and 2-deoxyadenosine on hyperactivation of cryopreserved human sperm. Fertil Steril 1993;59:1112-1117. Miyamoto H, Chang MC. The importance of serum albumin and metabolic intermediates for capacitation of spermatozoa and fertilization of mouse eggs in vitro. J Reprod Fertil 1973; 32:193-205. Moohan JM, Winston RML, Lindsay KS. Variability of human sperm response to immediate and prolonged exposure to pentoxifylline. Hum Reprod 1993;8:1696-1700. Rees JM, Ford WCL, Hull MGR. Effect of caffeine and of pentoxifylline on the motility and metabolism of human spermatozoa. J Reprod Fertil 1990;90:147-156. Seshagiri PB, Bavister, BD. Phosphate is required for inhibition by glucose of development of hamster 8-cell embryos in vitro. Biol Reprod 1989;40:607-614. Shen MR, Chiang PH, Yang RC, Hong CY, Chen SS. Pentoxifylline stimulates human sperm motility both in vitro and after oral therapy. Br J Clin Pharmacol 1991;31:711-714. Tesarik J, Thebault A, Testart J. Effect of pentoxifylline on sperm movement characteristics in normozoospermic and asthenozoospermic specimens. Hum Reprod 1992;7:1257-1263. Toumaye H, Janssens R, Camus M, Staessen C, Devroey P, Van Steirteghem AC. Pentoxifylline is not useful in enhancing sperm function in cases with previous in vitro fertilization failure. Fertil Steril 1993;59:210-215. Tournaye H, Van der Linden M, Van den Abbeel E, Devroey P, Steirteghem AV. Effects of pentoxifylline on in-vitro development of preimplantation mouse embryos. Hum Reprod 1993;8: 1475- 1480. Toumaye H, Van der Linden M, Van den Abbeel E, Devroey P, w AV. Effect of pentoxifylline on implantation and post-implantation development of mouse embryos in vitro. Hum Reprod 1993;8:1948-1954. Tournaye H, Van der Linden M, Van den Abbeel E, Devroey P, Steirteghem AV. The effect of pentoxifylline o mouse in vitro fertilization and early embryonic development. Hum Reprod 1994;9:1903-1908. Yovich JL. Pentoxifylline: actions and applications in assisted reproduction. Hum Reprod 1993;8:1786-1791. Yovich JM, Edirisinghe WR, Ctmnnins JM, Yovich JL. Influence of pentoxifylline in severe male factor infertility. Fertil Steril 1990;53:715-722.
A. Rupasri’,
AND IN VITRO HAMSTER
D. Jayaprakash’, A.T.Peter2, M.S. Sreenivasa’, M. Kumart and P.B. Seshagirilya
‘Center for Reproductive Biology and Molecular Endocrinology Indian Institute of Science, Bangalore 560 012, India 2Department of Veterinary Clinical Sciences Purdue University, West Lafayette, IN, USA
Received
January 27, 1995 Mar& IO, 1995
for publication: Accepted:
ABSTRACT Pentoxifylline (PF) is used to improve motility of spermatozoa from subfertile or nonfertile males to accomplish in vitro fertilization in humans. The possible adverse effect of PF on pre- and peri- implantation stage embryo development in a suitable rodent model, such as the golden hamster, is yet to be determined. In this study, hamster cauda epididymal spermatozoa were exposed to different concentrations (0.23 to 3.6 mM) of PF, and their quantitative [percentage of motility] and qualitative [Score 0 to 51 motility were assessed and values expressed as the sperm motility index. Upon addition of spermatozoa to dishes containing PF, an immediate increase in sperm motility and sperm motility index was evident, which increased up to 4 to 6 h and then declined. The sperm motility index increase by PF was dose-dependant, and al.8 mM PF was detrimental after 4 h. The optimum dose of PF was found to be 0.45 mM. To assess the fertilizing ability of PF-treated spermatozoa, in vitro fertilization was carried out. Fertilization rates for spermatozoa treated with 3.6 mM PF were lower (53.8 2 7.8) than for the controls (69.5 ? 10.2), whereas treatment with 0.45 mM PF increased the rates (91.6 + 4.3) compared with that of the controls (80.2 f 5.9). In conclusion, low concentrations (0.23 to 0.45 mM) of PF improve sperm capacitation and fertilization of oocytes in vitro in the golden hamster. Key words:
hamster,
in vitro fertilization,
pentoxifylline,
sperm motility
Acknowledgments We thank the Indian Institute of Science for financial support and the MUCIA, Purdue University International Programs for travel support (to ATP). aCorrespondence
and reprint requests.
Theriogenology 44:553-562, 1995 0 1995 by Elsevier Science inc. 655 Avenue of the Americas, New York, NY 10010
0093-691 x/95/$1 0.00 SSDI 0093-691X(95)00226-X
554
Theriogenology INTRODUCTION
In humans, the causes of infertility in the subfertile male may oligospermia, asthenozoospermia and teratozoospermia. To accomplish assisted using such spermatozoa, various sperm motility factors need to be assessed. (PF), a methyl xanthine derivative, is a sperm motility enhancing compound is being tested in clinical trials with encouraging results (1,6,12,15,17,21,22). adverse effects of PF on pre- and post- implantation viability of embryos defined.
be those of reproduction Pentoxifylline and its effect The possible are not well
Most studies with human spermatozoa used 3.6 mM or higher doses of PF (7,22). Information on the optimum dose, its possible adverse effect on fertilization and embryo development in a suitable rodent model is needed since experiments with humans are not feasible for ethical and practical reasons. Hence, we initiated investigations into the effect of PF on gamete maturation, fertilization and pre- and peri- implantation embryo development in the golden hamster. Hamsters are an ideal rodent model since they provide several major advantages for the studies on early mammalian development (4). In this study, we present data on the dose-dependent enhancement of sperm capacitation and fertilization by PF in the golden hamster. MATERIALS
AND METHODS
Adult (3 to 5 mo) male and female golden hamsters (Mesocricetus auratus) used for the present study were either from our colony or from the Laboratory Animals Information Service Centre, Hyderabad, India. They were housed under normal temperature (24-26OC) and lighting schedule (12L: 12D). The culture medium for the sperm assay and for in vitro fertilization was a modified Tyrode’s medium containing bovine serum albumin (BSA, 3 mg/ml); sodium lactate (10 mM); pyruvate, (0.25 mM); and 1 mg/ml polyvinyl alcohol (TALP-PVA) supplemented with penicillamine, hypotaurine and epinephrine (PHE), as described elsewhere (4,5). All components and pentoxifylline were purchased from Sigma Chemical Company (St. Louis, MO, USA) or HiMedia Labs (Bombay, India). A stock solution of 360 mM PF was prepared in T’LPVA (TALP-PVA devoid of albumin and pyruvate) and the required dilutions were made on the day of experiment. The medium used for the experiments had an osmolality of 285 to 300 mOsmols/kg water and a pH of 7.4 after equilibration in 5% CO, in air at 37°C. The sperm motility assay was carried out according to the published procedures (5). Briefly, male hamsters with large, turgid caudae epididymides were sacrificed by cervical dislocation. Epididymides were dissected out, collected in TL-HEPES buffer (pH 7.4). cleared from adherent tissues, and the distal tubules were punctured by a sterile 26.5-g needle. These 2 ~1 of the released epididymal spermatozoa (approximately 3 to 4 x 106/ml)
Theriogenology
555
were transferred to each 35-mm Falcon dish (#3001) containing 2 ml of preequilibrated TALP-PVA medium with or without desired concentrations (0.23 to 3.6 mM) of PF, and incubated in a humidified atmosphere of 5% CO, in air at 37’C. Observations on the percentage of sperm motility and quality of motility (Score 0 to 5) were made every 1 or 2 h for a duration of 6 to 8 h. Values were expressed as sperm motility index, SMI: % motility x [quality score 0-512. The dishes were scored independently by at least 2 persons, and the mean values were calculated at the end of each experiment. The experiments were repeated with at least 2 to 3 batches of media. Insemination drops (100 ~1) of TALP-PVA supplemented with PHE were placed in 35-mm Falcon dishes and overlaid with silicone oil (Aldrich Chemical Co., Milwaukee, WI). Adult females were superstimulated with 25 IU, ip of PMSG on the day of the post-estrus discharge, followed 56 h later by 25 IU hCG. The animals were sacrificed at 16 to 18 h posthCG treatment, and the oviducts were removed and collected in preequilibrated mineral oil. The swollen ampullae were punctured with a fine forceps, and the contents were allowed to extrude out. The cumulus cells were removed by transferring into a 100 ~1 drop of hyaluronidase (1 mg/ml) containing 0.01 mg/ml of soybean trypsin inhibitor in TL-HEPES. Oocytes were washed twice with TALP-PVA + PHE and transferred to insemination drops, 5 or 10 ~1 of untreated or PF-treated sperm suspensions were introduced into each drop. Only unwashed spermatozao were used, and the PF concentrations during coincubation were 23 or 45 PM and 180 or 360 PM when 0.45 mM and 3.6 mM PF- treated sperm samples were used, respectively. After 3 to 4 h of coincubation, the oocytes were washed and examined for the presence of 2 pronuclei (4), using an Olympus IMT-2 inverted microscope equipped with Nomarski DIC objectives (x10 and x20). Values expressed in figures and tables were means ? SEM from a minimum experiments. Statistical analysis was carried out by Student’s t-test.
of 5
RESULTS An immediate increase in the motility of cauda epididymal spermatozoa was observed in medium containing PF, with maximal sperm motility index reaching by 2 to 4 h (P < 0.01) then declining by 6 h. In contrast, the sperm motility index of spermatozoa in PF-free medium showed a gradual increase until 6 h (Figure 1). Exposure of spermatozoa to lower concentrations (2.7 and 1.8 mM) of PF resulted in higher sperm motility index compared with that observed with 3.6 mM PF. However, with 0.9 mM concentration there was no increase in sperm motility index at 2 h, but it increased significantly by 4 h (P < 0.001) and by 6 h (P < 0.01). Observations indicate that spermatozoa exhibited hyperactivation following 2 h of incubation with PF (1.8 to 3.6 mM) and it persisted up to 4 h.
556
w
TALP
ALONE
O---J
TALP + 3.6 mM PF
-
TALP + 2.7 mM PF
O-.-J
TALP+1.8mMPF
O---O
TALP + 0.9 ml4 PF,
I
0
1
1
I
2
4
6
TIME Figure 1.
(hours)
Effect of varying concentrations of pentoxifylline on hamster sperm motility. Each value represents mean +. SEM (n=5). Level of significance: l = P < 0.05, * = P CO.01, n = P < 0.001.
When lower concentrations, i.e., 0.9, 0.45 and 0.23 mM of PF were tested in a separate set of experiments (Figure 2), a further increase in sperm motility index was evident. The maximum sperm motility indices were obtained with 0.45 mM followed by 0.23 and 0.9 mM concentrations of PF which were significantly (P
557
Theriogenology
16001
1400-
-
TALP
e.,.d
TALP +o.g
ALONE
+--*
TALP+0.45
C.-O
TALP+O.
mM PF
mM PF 23
mM Plj,
X $
1200-
g -I
lOOO-
S t
800-
I 0
1
1
I
2
4
6
TIME
Figure 2.
(hours)
Effect of varying concentrations of pentoxifylline on hamster sperm motility. Each value represents mean + SEM (n = 12). Level of significance: * = P < 0.01, n = P < 0.001.
Insemination of oocytes with 3.6 mM PF-treated spermatozoa slightly decreased the fertilization rates (53.8 f 7.8) compared with that (69.5 + 10.2) obtained with untreated spermatozoa. There was no significant difference in fertilization rates when 2 different concentrations (5 x lo4 or 10 x 104) of spermatozoa were tested (Table 1).
558 Table 1.
Treatment
Theriogenology Assessment of in vitro fertilizability pentoxifylline-treated spermatozoaa
Sperm concentration
of golden hamster
No. of oocytes inseminated
No. of oo tes fertilizedYJ
Percentage of fertilization Mean k SEM
5 x lo4
64
51
69.9 f 10.6’
10 x lo4
29
21
75.02 08.3c
Total
93
72
69.5 2 10.2d
5x lo4
59
34
52.6+ 7.8e
10 x lo4
29
18
66.7? 4.7e
Total
88
52
53.8a
(per ml)
No Pentoxixifylline
Pentoxifyllinef (3.6 mM)
oocytes by 3.6 mM
7.Sd
aData are from 9 IVF procedures using 9 superovulated females. bEvidence of fertilization: presence of 2 pronuclei in the ooplasm at 3 to 4 hours post insemination (4). Observations were made using an Olympus IM’I-2 microscope with Nomarski DIC objective lenses (x10 and x20). ‘Mean values were not statistically significant (P > 0.2). dMean values were not statistically significant (P < 0.2). eMean values were not statistically significant (P > 0.2). fThe concentrations of pentoxifylline in insemination drops were 180 and 360 PM when 50 and 100 thousand spermatoza were used, respectively.
In a separate set of experiments, IVF with 0.45 mM PF-treated spermatozoa was carried out. An improvement in the fertilization rate was observed with 0.45 mM PF samples (91.6 + 4.3) when compared with that of the controls (SO.2 + 5.9). When 2 different concentrations (5 x lo4 or 10 x 104) of spermatozoa were used for insemination, Fertilized oocytes were normal by the fertilization rates were similar (Table 2). morphological criteria and contained 2 pronuclei and 2 polar bodies (Figure 3).
559
Theriogenology Table 2.
Treatment
No
Assessment of in vitro fertilizability petoxifylline-treated spermatozoaa
of golden hamster
oocytes by 0.45 mM
Percentage of fertilization Mean t SEM
No. of oocytes inseminated
No. of oo tes fertilizedx
5 x lo4
56
49
81.7 ? 6.5”
10 x lo4
35
30
84.7 + 4.3c
Total
91
79
80.2 * 5.9d
5 x lo4
59
53
90.8 + 4.32
10 x lo4
36
30
83.7 * 7.4”
Total
95
83
91.6 ? 4.3d
Sperm concentration (per ml)
pentoxifylline
Pentoxifyllinef (0.45 mM)
aData from 7 IVF procedures using 7 superovulated females. bEvidence of fertilization: presence of 2 pronuclei in the ooplasm at 3 to 4 hours post insemination (4). Observations were made using an Olympus IMT-2 microscope with Nomarski DIC objective lenses (x10 and x20). ‘Mean values were not statistically significant (P > 0.20). dMean values were not statistically significant (P < 0.20). eMean values were not statistically significant (P > 0.20). fThe concentration of pentoxifylline in insemination drops were 180 and 360 PM when 50 and 100 thousand spermatoza were used, respectively.
Figure 3.
Photomicrographic appearance of oocytes fertilized in vitro with untreated (Panel a) and 0.45 mM pentoxifylline-treated (Panel b) spermatozoa in the golden hamster. Arrows indicate locations of pronuclei. Photographed with Nomarski DIC objectives (x20).
560
Theriogenology DISCUSSION
Our study shows, for the first time, that pentoxifylline (PF, so.45 mM) improves capacitation of spermatozoa and in vitro fertilization of oocytes in the golden hamster. The sperm motility enhancing effect of PF is dose-dependent; low concentrations enhance longevity of motility, and its prolonged exposure at high concentrations (~1.8 mM) is detrimental to sperm motility (Figures 1 and 2), as observed with human (12) and mouse (21) spermatozoa. An immediate effect, within 10 min of exposure, of PF on the enhancement of motility of hamster spermatozoa was observed. Similar to observations with normospermic human samples (13), exposure of hamster spermatozoa to PF did not result in appreciable improvement in quantitative motility. The quality of motilty was, howerver, different. The hyperactivated motility of spermatozoa is achieved after 2 h and persists up to 4 h. These features are similar to those observed with normo-, astheno- and terato-spermic human samples (7,10,16,22). Microscopic observations indicate that hamster spermatozoa exhibit curvilinear (hyperactivated) motility by 2 to 4 h and are able to fertilize oocytes in vitro (Tables 1 and 2). Computer-assisted semen analysis of PF-treated human spermatozoa reveal increased curvilinear velocity but not straight line velocity (2,9). The curvilinear velocity profile of PF-treated hamster spermatozoa by Computer-assisted semen analysis remains to be investigated. The drastic decline in hamster sperm motility observed after 4 h of treatment with ~2.7 mM PF (Figure 1) may be attributed to the toxic effect on sperm plasma membrane at the ultrastructural level, as observed with human spermatozoa (9,10,13,16). The other possibility for the decline is that spermatozoa could have undergone acrosome reaction resulting in the release of hydrolytic enzymes which may affect their integrity and viability. Although BSA is well recognized as an acrosome reaction-inducing factor (3,11), it is likely that PF could also have been induced acrosome reaction independently of BSA. This remains to be investigated in hamsters. The data in Tables 1 and 2 show that fertilization could be achieved with unwashed spermatozoa in the presence of 23,45, 180 and 360 PM PF during insemination. Although a statistically significant difference in fertilization rates was not discernible, there was a slight reduction in rates with 3.6 mM PF-treated spermatozoa, as reported in the mouse (20,21), and an improvement with 0.45 mM PF-treated spermatozoa. The slight difference, although not statistically significant, in fertilization rates between control samples in 2 different sets of experiments (Tables 1 and 2) could be due to the use of different groups of animals from the commercial supplier. These data show that removal of PF from the sperm suspension medium prior to insemination may not be required, and continued presence of ~360 PM PF during insemination may not inhibit fertilization in the hamster. Since the aim of the present study was to evaluate the fertilizing ability of the PFtreated spermatozoa and to confirm the fertilization status of the inseminated oocytes, culture of fertilized oocytes and an assessment of their preimplantation development were
561
Theriogenology
not carried out. Our ongoing studies, however, indicate that the first cleavage division of IVF oocytes inseminated with PF-treated spermatozoa are not affected. We also found that continuous presence of 50.9 mM PF in hamster embryo culture medium (14) does not affect cultured 8-cell embryo development, and the presence of 3.6 mM PF retards embryo development (Rupasri and Seshagiri, unpublished observation). Human and mouse embryo development in vitro is arrested by PF in a dose-dependent manner and at lower concentrations the effect of PF is less profound (8,18,19,20). Although there is considerable controversy on the use of PF as a sperm motility stimulant in assisted reproduction in humans (6,17,22), extensive investigation in suitable rodent models is essential not only to evaluate the long-term safety of PF during pregnancy but also to understand its molecular mechanism of action in improving sperm motility and its clearcut influence on pre- and peri- implantation embryo development. Pentoxifyiline could prove to be an acceptable, useful sperm motility stimulant (alternative to caffeine/heparin) for use in humans and other species, particularly to improve the motility of inherently poor quality spermatozoa of a few domestic species such as the buffalo.
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