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Dose-dependent effect of heparin on fertilizing ability of goat spermatozoa
DOSE-DEPENDENT
EFFECT OF HEPARIN ON FERTILIZING OF GOAT SPERMATOZOA
ABILITY
J.F. Cox, F. Saravia, M. Briones and A. Santa Maria Department of Animal Production, Faculty of Veterinary Medicine University of Conception, Casilla 537, Chillan, Chile Received for publication: Accepted:
January 27, 1994 20, 1995
March
ABSTRACT Intact bovine oocytes were used to study the effect of heparin on goat IVF. Oocytes were matured in Medium 199 plus estrous sheep serum. Fresh semen was incubated for 4 h at room temperature, and spermatozoa were then resuspended in medium Talp plus serum and incubated further for 1 h at 39 “C in 5% CO2 in air. Later, spermatozoa were resuspended in Talp plus serum and heparin and were then incubated in microdrops until the oocytes were matured. In Experiment 1, the effect of heparin on spermatozoa from individual males was studied by a dose-response curve. In Experiment 2, the timing of sperm penetration in matured oocytes was studied to assess the stage at which the action of heparin could be expressed in the fertilization process. In Experiment 3, heparin from the same source but at different grades of bioactivity was adjusted for bioactivity and its effect on spermatozoa was compared in terms of penetration rates in order to identify heparin-dependent variations on goat IVF. In Experiment 4, the influence of calcium on the effect of heparin at different levels of bioactivity on the fertilizing ability spermatozoa was assessed as in Experiment 3. In Experiment 5, different batches of heparin from the same source and grade of bioactivity were compared as above. The results suggest that 1) heparin stimulates fertilization rates following a comparable pattern between males; 2) the most probable site of action is at the stage of sperm capacitation; and 3) provided that the source and grade of bioactivity is preserved, heparin maintains the efficiency of sperm penetration into matured oocytes. Key words: heparin, in vitro fertilization,
heterologous
fertilization,
goats
____ Acknowledgments The authors thank Dr. M. Cid for reviewing the written English language of the manuscript and Mr. J. Avila for his technical support. The study was funded by Fondecyt 91-318 and DIUC 94.153.02-l.
Theriogenology 44:451460, 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0093-691X/95/$1 0.00 SSDI 0093-691X(95)00199-1
452
Theriogenology INTRODUCTION
Heparin has been shown to improve sperm fertilizing ability in cattle (20), sheep (3) and goats (5). In cattle, where its activity has been most studied, heparin, promotes sperm capacitation by mechanisms still not clearly understood, but that seem to depend on sperm adsorption of seminal proteins at ejaculation (6,16). This event increases the ability of spermatozoa to bind heparin through its sulfate residue, a basic requirement for triggering the heparin capacitation-promoting effect (14,15). Parrish (29) suggests that heparin could remove decapacitating factors adhered to the sperm plama membrane, such as calmodulin-binding proteins (11,13), which in turn could facilitate the influx of calcium into the cell. In addition, calcium is needed to allow for the heparin effect on capacitation (7). The effect of heparin on goat fertilization is less well known. It has been shown that heparin stimulates the penetration of goat spermatozoa of intact goat, cattle and sheep matured oocytes under in vitro conditions, resulting in similar trends of penetration (5). But it is not known what stage of sperm preparation for fertilization is primarily affected, or whether there are any differences between males in the doseresponse curve, as has been observed in cattle (10,12). The heparin currently used in IVF is obtained as a sodium salt, most usually from porcine intestinal mucosa and from other suitable tissues of domestic animals (18). The activity of heparin is assessed by a standard bioassay that measures the antifactor Xa activity of the molecule, and is expressed in USP Heparin Units, which are defined by the USP Heparin Sodium Reference Standard. It is not known whether the extraction and purification procedure which potentiates the anticoagulant moiety of the heparin affects the fertilization-stimulating property seen in ruminant spermatozoa. We used a heterologous fertilization system validated for the study of sperm function at fertilization in goats (5). We sought 1) to characterize the effect of heparin on sperm penetration in matured oocytes, and 2) to identify heparin-dependent variations on sperm ability to fertilize matured oocytes in vitro. MATERIALS
AND METHODS
Oocyte Collection and Culture Ovaries were collected from cows at a local slaughterhouse and brought to the laboratory in PBS at about 35 “C within 2 h. Cumulus-oocyte complexes (COC) were obtained by aspirating follicles 1 to 5 mm in diameter with a 21-g needle connected to a 5-ml syringe. After washing the COC twice with Hepes-199 (Medium 199 with Earle’s salts and 25 mM Hepes supplemented with 10% v/v inactivated estrus sheep serum (ESS) and 75 ug/ml of kanamycin pH: 7.3), they were cultured for 24 h in 50 fl microdrops of maturation medium (5 to 10 COG/drop) under light paraffin oil at 39
Theriogenology
453
“C and 5% CO2 in air. The maturation medium consisted of Medium 199 plus 20% v/v ESS, 20 pg/ml insulin (Sigma Chemical Co, St. Louis, MO), 10 mM Hepes and 75 ug/ml kanamycin (pH: 7.5). Sperm Preparation
and Capacitation
Fresh semen was collected by artificial vagina from 4 Anglo Nubian bucks. After 4 h of incubation at laboratory temperature (about 20 “C), the semen was washed twice in calcium-free Talp (20), and spermatozoa were recovered by centrifugation at 300 g for 5 min. Next, the sperm concentration was adjusted to 1.5 x lo8 sperm/ml and incubated in Talp IVF medium (Talp plus 10% ESS, 2.5 ug/ml heparin and 2.7 mM CaC12) for 60 min at 39 “C and 5% CO2 in air. The sperm concentration was then adjusted to about 1x106 sperm/ml, and 50~1 microdrops under paraffin oil (at least 2 per replicate) were prepared using the same medium, according to the experimental protocols. The sperm suspension was then incubated at 39 “C and 5% of CO2 in air, until the introduction of matured oocytes. Heparin was prepared weekly and was dissolved in Talp calcium-free medium, sterilized by filtration using a 0.22 u disposable filter (Sigma) and stored in freezing vials (Sigma) at -20 “C until utilization. Comparisons between the different heparin bioactivity levels were done either by dryed-mass weight basis adjusted for bioactivity grade (Experiments 3 and 4) or by dryed-mass weight basis alone. All chemicalsused Louis, MO, USA).
in culture media were obtained from Sigma Chemical Co (St
In Vitro Fertilization After incubation, the COCs were washed twice in calcium-free Talp and were then completely denuded by passing them through a fine pipette. Thereafter, matured oocytes were assigned randomly to the already incubated sperm suspensions. Only oocytes with an expanded cumulus and evenly granulated cytoplasm were used in the experiments. Exueriment 1. To establish a dose-response curve of heparin for each male used in the study, sperm preparations were treated with 0.0, 2.5, 5.0, 10,O and 25.0 lrg of heparin/ml of IVF medium. Matured oocytes were then incubated in the sperm microdrops for 18 h at 39 “C and 5% of CO2 in air. Experiment. To establish the timing of sperm penetration in matured oocytes, spermatozoa were treated for capacitation and incubated in separated plates under paraffin oil. Matured oocytes were then incubated in sperm microdrops ‘either for 1,3,6 or 18 h. Exueriment 3. Heparins from porcine and ovine intestinal mucosa with different grades of bioactivity (3 from porcine and 1 from ovine) were assessed in their ability
Theriogenology
454 to stimulate penetration in matured oocytes by goat spermatozoa. incubated for 18 h at 39 “C and 5% of CO2 in air.
Gametes were
Exneriment 4. Heparins from the same source but of different bioactivity were assessed for their ability to facilitate oocyte penetration, as in Experiment 3, but at 3 levels of calcium in the IVF medium (1.0, 2.0 and 3.0 mM CaClz). Sperm concentrations were adjusted to allow for approximately 50% fertilization rate in the medium with high calcium. Exoeriment 5. Three different batches of heparin from the same source and of the same bioactivity were assessed for their capability to stimulate sperm penetration in matured oocytes. Gamete interaction was maintained for 18 h at 39 “C and 5% of CO2 in air. Microscopic
Evaluation
After sperm-oocyte interaction, oocytes were released mechanically from attached spermatozoa by passing them through a glass pipette and fixing themin a 1:3 v/v aceto-alcohol solution. Then, 24 to 48 h later, the oocytes were stained with a 1% acetolacmoid to assess sperm penetration into the cytoplasm. Oocytes were considered to be penetrated when pronuclear development and the sperm tail could be observed in the cytoplasm. Statistical Analysis Penetration rates were calculated and percentages were arc-sin transformed and analyzed by an ANOVA and Newman Keuls tests, using the Statview@ computer program. A value of P< 0.05 was considered to be significant. RESULTS Experiment 1. Dose-Effect of Heparin on Fertilizing Ability of Goat Spermatozoa Penetration rates were calculated discarding germinal vesicles or broken oocytes (less than 5%). The heparin dose-response curve of the males in the study showed the ability of spermatozoa to fertilize bovine oocytes was stimulated by heparin according to a similar pattern for the various bioactivity grades (Figure 1). The fertilization rate was rapidly improved by adding heparin to the IVF medium at values that varied between 2.5 to 10 ug/ml depending on the male. Although the fertilization rates were similar between males (P> 0.05), the following experiments were performed with pooled semen from the most fertile males and at a heparin concentration of 10 ug/ml in the IVF medium.
455
Theriogenology
Male 1 Male 2 Male 3 -+P-+. Male 4 n= 1180 oocytes
”
I
lb
2b 3; Heparin @g/ml) Figure 1. Effect of heparin on penetration rate of bovine spermatozoa (5 replicates/male). 0
Experiment 2. Effect Bovine Oocytes
of Heparin
on the Timing
of Sperm
oocytes
by goat
Penetration
in
To establish the timing of sperm penetration in matured oocytes, separated plates (10 to 15 oocytes/group) were removed at set time from incubation, and the oocytes were fixed and stained (Figure 2). The results in Figure 2 show that at 3 h of sperm-oocyte incubation there is a significant increase in the sperm penetration rate induced by heparin that continues up to a plateau at 6 h of gamete incubation. Considering that penetration rates in untreated controls remain at basal values, the pattern of sperm penetration seen in the experiment suggests that heparin stimulates fertilization rates in goats by affecting the sperm capacitation process. Experiment 3. Effect of Heparin Spermatozoa
Bioactivity
Grade on Fertilizing
Ability of Goat
Heparins graded differentially according to their anticoagulant potency (different USP Units/mg dry mass) but made comparable by adjusting their dry mass weights were assessed in their capability to stimulate fertilization by goat spermatozoa. The results in Table 1 show that there were no differences (I+ 0.05) in the sperm penetration rate of bovine oocytes or in the polispermy rate (results not shown). This could mean that providedthe bioactivity grades are comparable, heparins prepared by similar extraction and purification procedures maintain their ability to stimulate the fertilization potential of goat sperm populations.
Theriogenology
456
100
1
With heparin Without heparin
II
n= 420 oocytes
a
0
10
0
20
Incubation period (h) Figure 2. Effect of heparin on the timing of sperm penetration in matured oocytes (4 replicates per experimental group). atbJ Different superscripts indicate significant differences between treated sperm (PcO.05).
Table 1. Effect of heparins with different biological activities on sperm penetration of bovine oocvtes Heparin
Sperm penetration
product
Number of
(grade) 18F-05001 ( 1-A )
replicate& 5
Penetration
efficiency ratec
(%) 51/62 (82.3)
Range (%) 60.0 - 92.9
9OH-0261
( 1)
5
56/65 (86.2)
73.5 - 100
&IF-00161
(2)
5
46/63 (73.0)
45.5 - 90.9
51H-0310 ( l-ov )a 5 59/66 (89.4) Heparin from ovine intestinal mucosa. b At least 10 oocytes per group in each replicate. c Non-significant difference (P>O.O5).
76.9 - 100
Experiment 4. Effect of Calcium on Fertilization-Promoting Different Bioactivity in Goat Spermatozoa
Ability of Heparins of
a
The variations observed in fertilization rates obtained with Grade-l and Grade-2 heparins were further studied by modifying the calcium levels in the IVF medium
Theriogenology
457
and simultaneously adjusting the sperm concentration the experiment. These results are shown in Figure 3.
to increase the sensitivity
of
Calcium increased the fertilization rate in a dose-dependent manner when using Heparin I, but a such pattern was not observed when Heparin II was used. The marginal results obtained with low concentrations of calcium was only reversed when 3 mM calcium were added to the IVF medium. Although there is a certain tendency for higher fertilization rates when using Heparin I, the only significant difference was obtained when 2 mM of calcium were added to the IVF medium. The results of this experiment suggest that Heparin I tends to improve the efficiency of fertilization by facilitating the utilization of calcium for sperm capacitation.
60
Heparin grade I Heparin grade II n: 450 oocytes
1
2
3
Calcium (CaCl, mM) Figure 3. Effect of heparins at different bioactivity levels on the fertilization rate of matured bovine oocytes by goat spermatozoa treated with 3 concentrations of calcium in the IVF medium (5 replicates per experimental group). a/b Different superscripts in columns indicate significant differences (P < 0.02).
Experiment 5. Effect of Different Batches of Heparin on the Penetration Goat Spermatozoa
Efficiency of
According with the results showed in Table 1, different batches of heparins with the same bioactivity grade should yield comparable sperm penetration rates of matured oocytes. The results in Table 2 show that there were no differences penetration rates when batches of heparins with similar anticoagulant potency (I% 0.05) were compared, norwere any differences seen between batches in the polispermy rate (results not shown).
458
Theriogenology DISCUSSION
The heterologous system we have standardized uses denuded bovine oocytes because we have shown that the absence of granulosa cells attached to the zona pellucida facilitates penetration by goat spermatozoa (4). The pattern of sperm penetration seen in the first experiment is similar to that observed previously using cattle (10) and goat spermatozoa (5) but differs from that of ram spermatozoa in which the fertilization rates initially improve but at the higher concentrations, in the range of concentrations used in the present study, they are inhibited (3). Table 2. Effect of different batches of heparin of the same grade of bioactivity in the penetration rates of bovine oocytes by goat spermatozoa Heparin
Sperm penetration Penetration
efficiency
batch
Number of
(grade)
replicatesa
rateb
Range
(%)
(%)
A(1)
5
56/60 (93.3)
83.3 - 100
B(1)
5
55/66 (82.1)
63.3 - 100
5 53/62 (85.5) C(1) a At least 10 oocytes per group in each replicate. b Non-significant difference (P>O.O5).
72.7 - 100
Heparin seems to stimulate the fertilization rate by affecting the capacitation of goat spermatozoa. Looking at the timing of sperm penetration (Experiment 2), it can be seen that the sperm penetration efficiency is affected only after 1 h of spermoocyte interaction. To our knowledge, there is no information on minimal in vivo capacitation time for goats as exists for cattle, sheep and various laboratory species (10,21). But considering that heparin stimulates sperm capacitation in cattle (20), that the minimal capacitation time for sheep is 1 to 1.5 h (lo), and that acrosome reaction and the sperm penetration through the oocyte vestments usually take less than 1 h (21), the most probable site of action of heparin is during sperm capacitation. Heparin is the most potent glycosaminoglycan in inducing capacitation in bovine spermatozoa (8) and such ability seems to be related to the sulfate content of the molecule (1). In fact, the desulfation of heparin negates its ability to bind and capacitate bovine spermatozoa, whereas the resulfation allows the molecule to regain its ability to bind spermatozoa (14). Sulfate residue may also mediate the anticoagulant effect of heparin (17), and therefore may affect the bioassay used to grade the molecule. According to Ax and Lenz (1), heparin may have 1.6 to 3.0 sulfates per disaccharide, so the source of heparin and the extraction and purification procedures used by commercial companies may become a source of variation in the efficiency of the molecule to stimulate sperm ability to fertilize matured oocytes. In Experiments 3 and 5, we looked at this fact by comparing heparins of different grades but similar bioactivity, and by comparing heparins of similar grades but from different batches, and we did not find significant variations in terms of sperm
459
Theriogenology
penetration rates. However, an important variation in the sperm penetration rate was seen when Grade II heparin was used. When we compared Heparins I and II under more sensitive conditions (Experiment 4), we again did not find statistical differences at calcium concentrations equivalent to the standard IVF system, but Heparin I was superior when calcium concentrations in the IVF medium were lower. Previous experiments in cattle showed that calcium is essential during the 4-h period of heparin-induced sperm capacitation (7). This cation may have multiple effects on sperm capacitation, including the interaction between heparin and proteins (2), the activation of calmodulin-dependent events (13), or the regulation of several intracellular events required for fetilization (21). Under restricted calcium availability, any of the mechanisms underlying sperm capacitation can be impaired. Therefore, we suggest that the improvement obtained with the use of Grade I heparin in Experiment 4 may be due to a better utilization of calcium induced by the molecule. The cumulative results suggest that heparin stimulates caprine IVF in a pattern similar between all the males in our study and by improving the efficiency of the capacitation process. REFERENCES 1. Ax RL, Lenz RW. Glycosaminoglycans as probes to monitor differences in fertility of bulls. J Dairy Sci 1987; 70:1477-1486. 2. Ayotte L, Perlin AS. N.M.R. Spectroscopic observations related to the function of sulfate groups in heparin. Calcium binding vs biological activity. Carbohydr Res 1986; 145:267-277. 3. Cox JF, Saravia F. Use of a multiple sperm penetration assay in ovines. Proc. XVII Annual Meeting of the Chilean Society of Animal Production 1992 Ref 61. 4. Cox JF, Hormazabal J, Santa Maria A. Effect of the cumulus on in vitro fertilization of bovine matured oocytes. Theriogenology 1993; 40:1259-1267. 5. Cox JF, Avila J, Saravia F, Santa Maria A. Assessment of fertilizing ability of goat spermatozoa by in vitro fertilization of cattle and sheep intact oocytes. Theriogenology 1994; 41:1621-1630. 6. Florman HM, First NL. The regulation of acrosomal exocytosis, I. Sperm capacitation is required for the induction of acrosome reactions by the bovine zona pellucida. Dev Biol 1988; 128:453-463. 7. Handrow RR, First NL, Parrish JJ. Calcium requirement and increased association with bovine sperm during capacitation by heparin. J Exp Zoo1 1989; 252:174-182. 8. Handrow RR, Lenz RW, Ax RL. Structural comparisons among glycosaminoglycans to promote an acrosome reaction in bovine spermatozoa. Biochem Biophys Res Comm 1982; 107:1326-1332. 9. Hunter RHF. The Fallopian tubes. Their role in fertility and infertility. Berlin: Springer Verlag, 1988; 81-108. 10. Lancaster R, Catt J, Rhodes S, Polge C. Bull and Breed specific effects of heparincapacitated bovine sperm on in vitro fertilization of in vitro matured follicular
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Theriogenology
oocytes. 6th Scientific Meeting of the European Embryo Transfer Association 1990; 166 abstr. 11. Leclerc I’, Sirard MA, Chafouleas JG, Lambert RD. Decreased binding of calmodulin to bull sperm proteins during heparin-induced capacitation. Biol Reprod 1990; 42:483-489. 12. Leibfried-Rutledge ML, Critser ES, Parrish JJ, First NL. In vitro maturation and fertilization of bovine oocytes. Theriogenology 1989; 31:61-74. 13. Manjunath L, Chandonnet L, Baillargeon L, Roberts KD. Calmodulin-binding proteins in bovine semen. J Reprod Fertil 1993; 97:75-81. 14. Miller DJ, Ax RL. Chemical N-desulfation of heparin negates its ability to capacitate bovine spermatozoa. Gamete Res 1989; 23:451-465. 15. Miller DJ, Ax RL. Carbohydrates and fertilization in animals. Molec Reprod Develop 1990; 26:184-198. 16. Miller DJ, Winer MA, Ax RL. Heparin-binding proteins from seminal plasma bind to bovine spermatozoa and modulate capacitation by heparin. Biol Reprod 1990; 42:899-915. 17. Ofosu FA, Buchanan MR, Anvari N, Smith LM, Blajchman MA. Plasma anticoagulant mechanisms of heparin, heparan sulfate, and dermatan sulfate. AM NY Acad Sci 1989; 556:123-131. 18. O’Reilly RA. Anticoagulant, antitrombolic and trombolytic drugs. In: Gilman AG, Goodman LS, Rall TW, Murad F. The Pharmacological Basis of Therapeutics. New York: Macmillan Publishing Co, 1985; 1271-1290. 19. Parrish JJ. Bovine in vitro fertilization. In: Dunbar BS, O’Rand MG. A Comparative Overview of Mammalian Fertilization. New York: Plenum Press, 1991; 351-361. 20. Parrish JJ, Susko-Parrish J, Winer MA, First NL. Capacitation of bovine sperm by heparin. Biol Reprod 1988; 38:1171-1180. 21. Yanagimachi R. Mammalian fertilization. In: Knobil E, Neil J (eds), The Physiology of Reproduction. New York: Raven Press, 1988; 135-185.
EFFECT OF HEPARIN ON FERTILIZING OF GOAT SPERMATOZOA
ABILITY
J.F. Cox, F. Saravia, M. Briones and A. Santa Maria Department of Animal Production, Faculty of Veterinary Medicine University of Conception, Casilla 537, Chillan, Chile Received for publication: Accepted:
January 27, 1994 20, 1995
March
ABSTRACT Intact bovine oocytes were used to study the effect of heparin on goat IVF. Oocytes were matured in Medium 199 plus estrous sheep serum. Fresh semen was incubated for 4 h at room temperature, and spermatozoa were then resuspended in medium Talp plus serum and incubated further for 1 h at 39 “C in 5% CO2 in air. Later, spermatozoa were resuspended in Talp plus serum and heparin and were then incubated in microdrops until the oocytes were matured. In Experiment 1, the effect of heparin on spermatozoa from individual males was studied by a dose-response curve. In Experiment 2, the timing of sperm penetration in matured oocytes was studied to assess the stage at which the action of heparin could be expressed in the fertilization process. In Experiment 3, heparin from the same source but at different grades of bioactivity was adjusted for bioactivity and its effect on spermatozoa was compared in terms of penetration rates in order to identify heparin-dependent variations on goat IVF. In Experiment 4, the influence of calcium on the effect of heparin at different levels of bioactivity on the fertilizing ability spermatozoa was assessed as in Experiment 3. In Experiment 5, different batches of heparin from the same source and grade of bioactivity were compared as above. The results suggest that 1) heparin stimulates fertilization rates following a comparable pattern between males; 2) the most probable site of action is at the stage of sperm capacitation; and 3) provided that the source and grade of bioactivity is preserved, heparin maintains the efficiency of sperm penetration into matured oocytes. Key words: heparin, in vitro fertilization,
heterologous
fertilization,
goats
____ Acknowledgments The authors thank Dr. M. Cid for reviewing the written English language of the manuscript and Mr. J. Avila for his technical support. The study was funded by Fondecyt 91-318 and DIUC 94.153.02-l.
Theriogenology 44:451460, 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0093-691X/95/$1 0.00 SSDI 0093-691X(95)00199-1
452
Theriogenology INTRODUCTION
Heparin has been shown to improve sperm fertilizing ability in cattle (20), sheep (3) and goats (5). In cattle, where its activity has been most studied, heparin, promotes sperm capacitation by mechanisms still not clearly understood, but that seem to depend on sperm adsorption of seminal proteins at ejaculation (6,16). This event increases the ability of spermatozoa to bind heparin through its sulfate residue, a basic requirement for triggering the heparin capacitation-promoting effect (14,15). Parrish (29) suggests that heparin could remove decapacitating factors adhered to the sperm plama membrane, such as calmodulin-binding proteins (11,13), which in turn could facilitate the influx of calcium into the cell. In addition, calcium is needed to allow for the heparin effect on capacitation (7). The effect of heparin on goat fertilization is less well known. It has been shown that heparin stimulates the penetration of goat spermatozoa of intact goat, cattle and sheep matured oocytes under in vitro conditions, resulting in similar trends of penetration (5). But it is not known what stage of sperm preparation for fertilization is primarily affected, or whether there are any differences between males in the doseresponse curve, as has been observed in cattle (10,12). The heparin currently used in IVF is obtained as a sodium salt, most usually from porcine intestinal mucosa and from other suitable tissues of domestic animals (18). The activity of heparin is assessed by a standard bioassay that measures the antifactor Xa activity of the molecule, and is expressed in USP Heparin Units, which are defined by the USP Heparin Sodium Reference Standard. It is not known whether the extraction and purification procedure which potentiates the anticoagulant moiety of the heparin affects the fertilization-stimulating property seen in ruminant spermatozoa. We used a heterologous fertilization system validated for the study of sperm function at fertilization in goats (5). We sought 1) to characterize the effect of heparin on sperm penetration in matured oocytes, and 2) to identify heparin-dependent variations on sperm ability to fertilize matured oocytes in vitro. MATERIALS
AND METHODS
Oocyte Collection and Culture Ovaries were collected from cows at a local slaughterhouse and brought to the laboratory in PBS at about 35 “C within 2 h. Cumulus-oocyte complexes (COC) were obtained by aspirating follicles 1 to 5 mm in diameter with a 21-g needle connected to a 5-ml syringe. After washing the COC twice with Hepes-199 (Medium 199 with Earle’s salts and 25 mM Hepes supplemented with 10% v/v inactivated estrus sheep serum (ESS) and 75 ug/ml of kanamycin pH: 7.3), they were cultured for 24 h in 50 fl microdrops of maturation medium (5 to 10 COG/drop) under light paraffin oil at 39
Theriogenology
453
“C and 5% CO2 in air. The maturation medium consisted of Medium 199 plus 20% v/v ESS, 20 pg/ml insulin (Sigma Chemical Co, St. Louis, MO), 10 mM Hepes and 75 ug/ml kanamycin (pH: 7.5). Sperm Preparation
and Capacitation
Fresh semen was collected by artificial vagina from 4 Anglo Nubian bucks. After 4 h of incubation at laboratory temperature (about 20 “C), the semen was washed twice in calcium-free Talp (20), and spermatozoa were recovered by centrifugation at 300 g for 5 min. Next, the sperm concentration was adjusted to 1.5 x lo8 sperm/ml and incubated in Talp IVF medium (Talp plus 10% ESS, 2.5 ug/ml heparin and 2.7 mM CaC12) for 60 min at 39 “C and 5% CO2 in air. The sperm concentration was then adjusted to about 1x106 sperm/ml, and 50~1 microdrops under paraffin oil (at least 2 per replicate) were prepared using the same medium, according to the experimental protocols. The sperm suspension was then incubated at 39 “C and 5% of CO2 in air, until the introduction of matured oocytes. Heparin was prepared weekly and was dissolved in Talp calcium-free medium, sterilized by filtration using a 0.22 u disposable filter (Sigma) and stored in freezing vials (Sigma) at -20 “C until utilization. Comparisons between the different heparin bioactivity levels were done either by dryed-mass weight basis adjusted for bioactivity grade (Experiments 3 and 4) or by dryed-mass weight basis alone. All chemicalsused Louis, MO, USA).
in culture media were obtained from Sigma Chemical Co (St
In Vitro Fertilization After incubation, the COCs were washed twice in calcium-free Talp and were then completely denuded by passing them through a fine pipette. Thereafter, matured oocytes were assigned randomly to the already incubated sperm suspensions. Only oocytes with an expanded cumulus and evenly granulated cytoplasm were used in the experiments. Exueriment 1. To establish a dose-response curve of heparin for each male used in the study, sperm preparations were treated with 0.0, 2.5, 5.0, 10,O and 25.0 lrg of heparin/ml of IVF medium. Matured oocytes were then incubated in the sperm microdrops for 18 h at 39 “C and 5% of CO2 in air. Experiment. To establish the timing of sperm penetration in matured oocytes, spermatozoa were treated for capacitation and incubated in separated plates under paraffin oil. Matured oocytes were then incubated in sperm microdrops ‘either for 1,3,6 or 18 h. Exueriment 3. Heparins from porcine and ovine intestinal mucosa with different grades of bioactivity (3 from porcine and 1 from ovine) were assessed in their ability
Theriogenology
454 to stimulate penetration in matured oocytes by goat spermatozoa. incubated for 18 h at 39 “C and 5% of CO2 in air.
Gametes were
Exneriment 4. Heparins from the same source but of different bioactivity were assessed for their ability to facilitate oocyte penetration, as in Experiment 3, but at 3 levels of calcium in the IVF medium (1.0, 2.0 and 3.0 mM CaClz). Sperm concentrations were adjusted to allow for approximately 50% fertilization rate in the medium with high calcium. Exoeriment 5. Three different batches of heparin from the same source and of the same bioactivity were assessed for their capability to stimulate sperm penetration in matured oocytes. Gamete interaction was maintained for 18 h at 39 “C and 5% of CO2 in air. Microscopic
Evaluation
After sperm-oocyte interaction, oocytes were released mechanically from attached spermatozoa by passing them through a glass pipette and fixing themin a 1:3 v/v aceto-alcohol solution. Then, 24 to 48 h later, the oocytes were stained with a 1% acetolacmoid to assess sperm penetration into the cytoplasm. Oocytes were considered to be penetrated when pronuclear development and the sperm tail could be observed in the cytoplasm. Statistical Analysis Penetration rates were calculated and percentages were arc-sin transformed and analyzed by an ANOVA and Newman Keuls tests, using the Statview@ computer program. A value of P< 0.05 was considered to be significant. RESULTS Experiment 1. Dose-Effect of Heparin on Fertilizing Ability of Goat Spermatozoa Penetration rates were calculated discarding germinal vesicles or broken oocytes (less than 5%). The heparin dose-response curve of the males in the study showed the ability of spermatozoa to fertilize bovine oocytes was stimulated by heparin according to a similar pattern for the various bioactivity grades (Figure 1). The fertilization rate was rapidly improved by adding heparin to the IVF medium at values that varied between 2.5 to 10 ug/ml depending on the male. Although the fertilization rates were similar between males (P> 0.05), the following experiments were performed with pooled semen from the most fertile males and at a heparin concentration of 10 ug/ml in the IVF medium.
455
Theriogenology
Male 1 Male 2 Male 3 -+P-+. Male 4 n= 1180 oocytes
”
I
lb
2b 3; Heparin @g/ml) Figure 1. Effect of heparin on penetration rate of bovine spermatozoa (5 replicates/male). 0
Experiment 2. Effect Bovine Oocytes
of Heparin
on the Timing
of Sperm
oocytes
by goat
Penetration
in
To establish the timing of sperm penetration in matured oocytes, separated plates (10 to 15 oocytes/group) were removed at set time from incubation, and the oocytes were fixed and stained (Figure 2). The results in Figure 2 show that at 3 h of sperm-oocyte incubation there is a significant increase in the sperm penetration rate induced by heparin that continues up to a plateau at 6 h of gamete incubation. Considering that penetration rates in untreated controls remain at basal values, the pattern of sperm penetration seen in the experiment suggests that heparin stimulates fertilization rates in goats by affecting the sperm capacitation process. Experiment 3. Effect of Heparin Spermatozoa
Bioactivity
Grade on Fertilizing
Ability of Goat
Heparins graded differentially according to their anticoagulant potency (different USP Units/mg dry mass) but made comparable by adjusting their dry mass weights were assessed in their capability to stimulate fertilization by goat spermatozoa. The results in Table 1 show that there were no differences (I+ 0.05) in the sperm penetration rate of bovine oocytes or in the polispermy rate (results not shown). This could mean that providedthe bioactivity grades are comparable, heparins prepared by similar extraction and purification procedures maintain their ability to stimulate the fertilization potential of goat sperm populations.
Theriogenology
456
100
1
With heparin Without heparin
II
n= 420 oocytes
a
0
10
0
20
Incubation period (h) Figure 2. Effect of heparin on the timing of sperm penetration in matured oocytes (4 replicates per experimental group). atbJ Different superscripts indicate significant differences between treated sperm (PcO.05).
Table 1. Effect of heparins with different biological activities on sperm penetration of bovine oocvtes Heparin
Sperm penetration
product
Number of
(grade) 18F-05001 ( 1-A )
replicate& 5
Penetration
efficiency ratec
(%) 51/62 (82.3)
Range (%) 60.0 - 92.9
9OH-0261
( 1)
5
56/65 (86.2)
73.5 - 100
&IF-00161
(2)
5
46/63 (73.0)
45.5 - 90.9
51H-0310 ( l-ov )a 5 59/66 (89.4) Heparin from ovine intestinal mucosa. b At least 10 oocytes per group in each replicate. c Non-significant difference (P>O.O5).
76.9 - 100
Experiment 4. Effect of Calcium on Fertilization-Promoting Different Bioactivity in Goat Spermatozoa
Ability of Heparins of
a
The variations observed in fertilization rates obtained with Grade-l and Grade-2 heparins were further studied by modifying the calcium levels in the IVF medium
Theriogenology
457
and simultaneously adjusting the sperm concentration the experiment. These results are shown in Figure 3.
to increase the sensitivity
of
Calcium increased the fertilization rate in a dose-dependent manner when using Heparin I, but a such pattern was not observed when Heparin II was used. The marginal results obtained with low concentrations of calcium was only reversed when 3 mM calcium were added to the IVF medium. Although there is a certain tendency for higher fertilization rates when using Heparin I, the only significant difference was obtained when 2 mM of calcium were added to the IVF medium. The results of this experiment suggest that Heparin I tends to improve the efficiency of fertilization by facilitating the utilization of calcium for sperm capacitation.
60
Heparin grade I Heparin grade II n: 450 oocytes
1
2
3
Calcium (CaCl, mM) Figure 3. Effect of heparins at different bioactivity levels on the fertilization rate of matured bovine oocytes by goat spermatozoa treated with 3 concentrations of calcium in the IVF medium (5 replicates per experimental group). a/b Different superscripts in columns indicate significant differences (P < 0.02).
Experiment 5. Effect of Different Batches of Heparin on the Penetration Goat Spermatozoa
Efficiency of
According with the results showed in Table 1, different batches of heparins with the same bioactivity grade should yield comparable sperm penetration rates of matured oocytes. The results in Table 2 show that there were no differences penetration rates when batches of heparins with similar anticoagulant potency (I% 0.05) were compared, norwere any differences seen between batches in the polispermy rate (results not shown).
458
Theriogenology DISCUSSION
The heterologous system we have standardized uses denuded bovine oocytes because we have shown that the absence of granulosa cells attached to the zona pellucida facilitates penetration by goat spermatozoa (4). The pattern of sperm penetration seen in the first experiment is similar to that observed previously using cattle (10) and goat spermatozoa (5) but differs from that of ram spermatozoa in which the fertilization rates initially improve but at the higher concentrations, in the range of concentrations used in the present study, they are inhibited (3). Table 2. Effect of different batches of heparin of the same grade of bioactivity in the penetration rates of bovine oocytes by goat spermatozoa Heparin
Sperm penetration Penetration
efficiency
batch
Number of
(grade)
replicatesa
rateb
Range
(%)
(%)
A(1)
5
56/60 (93.3)
83.3 - 100
B(1)
5
55/66 (82.1)
63.3 - 100
5 53/62 (85.5) C(1) a At least 10 oocytes per group in each replicate. b Non-significant difference (P>O.O5).
72.7 - 100
Heparin seems to stimulate the fertilization rate by affecting the capacitation of goat spermatozoa. Looking at the timing of sperm penetration (Experiment 2), it can be seen that the sperm penetration efficiency is affected only after 1 h of spermoocyte interaction. To our knowledge, there is no information on minimal in vivo capacitation time for goats as exists for cattle, sheep and various laboratory species (10,21). But considering that heparin stimulates sperm capacitation in cattle (20), that the minimal capacitation time for sheep is 1 to 1.5 h (lo), and that acrosome reaction and the sperm penetration through the oocyte vestments usually take less than 1 h (21), the most probable site of action of heparin is during sperm capacitation. Heparin is the most potent glycosaminoglycan in inducing capacitation in bovine spermatozoa (8) and such ability seems to be related to the sulfate content of the molecule (1). In fact, the desulfation of heparin negates its ability to bind and capacitate bovine spermatozoa, whereas the resulfation allows the molecule to regain its ability to bind spermatozoa (14). Sulfate residue may also mediate the anticoagulant effect of heparin (17), and therefore may affect the bioassay used to grade the molecule. According to Ax and Lenz (1), heparin may have 1.6 to 3.0 sulfates per disaccharide, so the source of heparin and the extraction and purification procedures used by commercial companies may become a source of variation in the efficiency of the molecule to stimulate sperm ability to fertilize matured oocytes. In Experiments 3 and 5, we looked at this fact by comparing heparins of different grades but similar bioactivity, and by comparing heparins of similar grades but from different batches, and we did not find significant variations in terms of sperm
459
Theriogenology
penetration rates. However, an important variation in the sperm penetration rate was seen when Grade II heparin was used. When we compared Heparins I and II under more sensitive conditions (Experiment 4), we again did not find statistical differences at calcium concentrations equivalent to the standard IVF system, but Heparin I was superior when calcium concentrations in the IVF medium were lower. Previous experiments in cattle showed that calcium is essential during the 4-h period of heparin-induced sperm capacitation (7). This cation may have multiple effects on sperm capacitation, including the interaction between heparin and proteins (2), the activation of calmodulin-dependent events (13), or the regulation of several intracellular events required for fetilization (21). Under restricted calcium availability, any of the mechanisms underlying sperm capacitation can be impaired. Therefore, we suggest that the improvement obtained with the use of Grade I heparin in Experiment 4 may be due to a better utilization of calcium induced by the molecule. The cumulative results suggest that heparin stimulates caprine IVF in a pattern similar between all the males in our study and by improving the efficiency of the capacitation process. REFERENCES 1. Ax RL, Lenz RW. Glycosaminoglycans as probes to monitor differences in fertility of bulls. J Dairy Sci 1987; 70:1477-1486. 2. Ayotte L, Perlin AS. N.M.R. Spectroscopic observations related to the function of sulfate groups in heparin. Calcium binding vs biological activity. Carbohydr Res 1986; 145:267-277. 3. Cox JF, Saravia F. Use of a multiple sperm penetration assay in ovines. Proc. XVII Annual Meeting of the Chilean Society of Animal Production 1992 Ref 61. 4. Cox JF, Hormazabal J, Santa Maria A. Effect of the cumulus on in vitro fertilization of bovine matured oocytes. Theriogenology 1993; 40:1259-1267. 5. Cox JF, Avila J, Saravia F, Santa Maria A. Assessment of fertilizing ability of goat spermatozoa by in vitro fertilization of cattle and sheep intact oocytes. Theriogenology 1994; 41:1621-1630. 6. Florman HM, First NL. The regulation of acrosomal exocytosis, I. Sperm capacitation is required for the induction of acrosome reactions by the bovine zona pellucida. Dev Biol 1988; 128:453-463. 7. Handrow RR, First NL, Parrish JJ. Calcium requirement and increased association with bovine sperm during capacitation by heparin. J Exp Zoo1 1989; 252:174-182. 8. Handrow RR, Lenz RW, Ax RL. Structural comparisons among glycosaminoglycans to promote an acrosome reaction in bovine spermatozoa. Biochem Biophys Res Comm 1982; 107:1326-1332. 9. Hunter RHF. The Fallopian tubes. Their role in fertility and infertility. Berlin: Springer Verlag, 1988; 81-108. 10. Lancaster R, Catt J, Rhodes S, Polge C. Bull and Breed specific effects of heparincapacitated bovine sperm on in vitro fertilization of in vitro matured follicular
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oocytes. 6th Scientific Meeting of the European Embryo Transfer Association 1990; 166 abstr. 11. Leclerc I’, Sirard MA, Chafouleas JG, Lambert RD. Decreased binding of calmodulin to bull sperm proteins during heparin-induced capacitation. Biol Reprod 1990; 42:483-489. 12. Leibfried-Rutledge ML, Critser ES, Parrish JJ, First NL. In vitro maturation and fertilization of bovine oocytes. Theriogenology 1989; 31:61-74. 13. Manjunath L, Chandonnet L, Baillargeon L, Roberts KD. Calmodulin-binding proteins in bovine semen. J Reprod Fertil 1993; 97:75-81. 14. Miller DJ, Ax RL. Chemical N-desulfation of heparin negates its ability to capacitate bovine spermatozoa. Gamete Res 1989; 23:451-465. 15. Miller DJ, Ax RL. Carbohydrates and fertilization in animals. Molec Reprod Develop 1990; 26:184-198. 16. Miller DJ, Winer MA, Ax RL. Heparin-binding proteins from seminal plasma bind to bovine spermatozoa and modulate capacitation by heparin. Biol Reprod 1990; 42:899-915. 17. Ofosu FA, Buchanan MR, Anvari N, Smith LM, Blajchman MA. Plasma anticoagulant mechanisms of heparin, heparan sulfate, and dermatan sulfate. AM NY Acad Sci 1989; 556:123-131. 18. O’Reilly RA. Anticoagulant, antitrombolic and trombolytic drugs. In: Gilman AG, Goodman LS, Rall TW, Murad F. The Pharmacological Basis of Therapeutics. New York: Macmillan Publishing Co, 1985; 1271-1290. 19. Parrish JJ. Bovine in vitro fertilization. In: Dunbar BS, O’Rand MG. A Comparative Overview of Mammalian Fertilization. New York: Plenum Press, 1991; 351-361. 20. Parrish JJ, Susko-Parrish J, Winer MA, First NL. Capacitation of bovine sperm by heparin. Biol Reprod 1988; 38:1171-1180. 21. Yanagimachi R. Mammalian fertilization. In: Knobil E, Neil J (eds), The Physiology of Reproduction. New York: Raven Press, 1988; 135-185.