Materials and Methods. Spermatozoa. Experiment 2. Experiment 3. Experiment 1

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2 8 MOTOISHI et al. of a thiol, can induce nuclear decondensation of human but not of bull ejaculated spermatozoa [15, 16]. Since heparin is a naturally occurring substance and since the in vitro decondensation occurs under physiological temperature and ph, heparin might act in the same manner in vivo as well [17]. Recently, Jager et al. [18] reported that relatively low concentrations of heparin (in the presence of a thiol) induced a rapid decondensation of human, mouse and bovine sperm nuclei, but detailed studies were conducted only for human spermatozoa. The present investigation examined the nature of nuclear decondensation of bull and human sperm nuclei by treating them in vitro with (1) dithiothreitol (DTT, a thiol-reducing agent), (2) heparin (a polyanion known to dissociate DNAprotamine complex of sperm nuclei), (3) DTT plus heparin, (4) DTT followed by heparin or (5) heparin followed by DTT. Materials and Methods Spermatozoa Ejaculated spermatozoa were collected from one bull by standard techniques and stored in French straws (0.5 ml, sperm/ml) at 196 C. After thawing at 35 C, the spermatozoa were expelled from the straw into BO medium [19] devoid of bovine serum albumin (mbo, 8 ml) and were centrifuged two times (600 g, 5 min) in this medium. The pellet was mixed with a small volume (1 ml) of mbo medium, and the resultant sperm suspensions were divided into 50 µl aliquots. The aliquots were subjected to two cycles of freezing and thawing (without cryoprotectant) to disrupt the membranes and were stored at 20 C. Human ejaculated spermatozoa were obtained from a normal, fertile donor via masturbation. The spermatozoa were washed two times with mbo medium by centrifugation (600 g, 5 min). The pelleted spermatozoa were then diluted to an approximate concentration of 10 7 sperm/ml by adding mbo medium. Diluted spermatozoa were divided into 50 µl aliquots, and aliquots were stored at 20 C after two cycles of freezing and thawing. Experiment 1 In Experiment 1, bull and human spermatozoa were treated with high concentrations of DTT and/ or heparin to clearly examine the differences between species in nuclear decondensation. Frozen bull spermatozoa (50 µl/tube) were thawed at room temperature (26 C), and 50 µl of mbo medium containing either heparin (20 mg/ml), DTT (1000 mm) or heparin (20 mg/ml) plus DTT (1000 mm) were added, resulting in final concentrations of heparin, DTT and heparin plus DTT of 10 mg/ml, 500 mm and 10 mg/ml plus 500 mm, respectively. For control spermatozoa (50 µl/tube), 50 µl of mbo medium was added. Decondensation of bull sperm nuclei was examined at 1, 3, 5, and 30 min after treatment of spermatozoa at room temperature (26 C). Ten µl of sperm suspension was removed from each tube and mixed with 20 µl of eosin B solution (2% in PBS, ph 6.7). Addition of the eosin B solution immediately stopped the nuclear decondensation of spermatozoa. The mixture was applied onto a slide and was covered with a coverglass. The length and width of 20 randomly selected sperm heads were examined in wet preparations under a differential interference contrast microscope at 600 magnification. The surface area of each sperm nucleus was calculated from the formula: area=length width π/4. Nuclear decondensation of human sperm nuclei was examined in an identical manner except that the eosin B solution was added at 3, 10, 60 or 180 sec after treatment because nuclear decondensation of human spermatozoa started immediately and proceeded rapidly, making the sperm heads become invisible within a very short time. Experiment 2 Decondensation of bull sperm nuclei was examined by adding simultaneously with various concentrations of heparin (0, 1, 10, 100 µg/ml; final concentration) to a fixed concentration of DTT (10 mm). The concentration of DTT (10 mm) was selected based on our preliminary experiment (data is not shown). The procedure used for adding DTT and/or heparin to the sperm suspension was identical to that used in Experiment 1. After 5 and 30 min of treatment at 26 C, nuclear decondensation of 10 randomly selected spermatozoa was evaluated by the method described in Experiment 1. Experiment 3 Decondensation of human sperm nuclei was examined by adding various concentrations of DTT (0, 0.1, 1.0, 10 mm) or heparin (0, 10, 100, 1000 µg/ ml) in separate dose-response trials. After 30 min

3 DECONDENSATION OF BULL AND HUMAN SPERM NUCLEI IN VITRO 9 of treatment at 26 C, nuclear decondensation of 10 randomly selected spermatozoa was evaluated as described in Experiment 1. Experiment 4 In this experiment DTT and heparin were added sequentially in two different orders to examine the mechanism of DTT and heparin on nuclear decondensation. Based on the result of Experiment 1 and our preliminary experiment (data is not shown), two doses of DTT or heparin incapable of inducing nuclear decondensation of bull sperm by itself were selected. Frozen-thawed bull spermatozoa were pretreated with heparin (100 µg/ml) or DTT (100 mm) for 30 min at 26 C. To remove heparin or DTT, the spermatozoa were washed two times in mbo medium by centrifugation at 600 g for 5 min. The alternate compound of two different concentrations (10 or 100 mm DTT ; 10 or 100 µg/ml heparin) was then added to the pellet (spermatozoa), and the nuclear decondensation of 10 randomly selected spermatozoa was examined after 30 min using the method described in Experiment 1. Experiment 5 DTT and heparin were added sequentially in two different orders in this experiment. Based on the result of Experiment 3, lower doses of DTT (0.1 mm) or heparin (10 µg/ml) incapable of inducing nuclear decondensation of human sperm by itself were used. Frozen-thawed human spermatozoa were pretreated with DTT (0.1 mm) or heparin (10 µg/ml) for 30 min at 26 C. To remove DTT or heparin, the spermatozoa were washed twice as described for Experiment 4. The alternate compound at the same concentrations used for pretreatment was then added to the pellet (spermatozoa), and the nuclear decondensation of 10 randomly selected spermatozoa was examined after 5 and 30 min. Statistical analysis When we examined more than 200 spermatozoa before and after freezing, both bovine and human spermatozoa in our sample (each from one normal, fertile donor) were fairly homogeneous in their size (Type I errors can be neglected), so one-factor analysis of variance was used to determine whether statistically significant difference between groups existed. Means within a treatment were compared with the control value using Duncan s multiple range test. In Experiment 1, time 0 value (value before treatment) was compared with later time values (treatment value) within a treatment. In Experiment 2, 0-dose (0 mm DTT and 0 µg/ml heparin) value was compared with treatment values within a treatment. In Experiment 3, non-treatment value (0-dose value) was compared with treatment values. In Experiment 4, non-treatment values (value before pretreatment) was compared with treatment value within a treatment. In Experiment 5, non-treatment value (value before treatment) was compared with treatment values. Results Neither DTT alone (500 mm) nor heparin alone (10 mg/ml) was able to induce bull sperm nuclei decondensation irrespective of the duration of treatment (Table 1). However, simultaneous treatment with DTT and heparin caused decondensation after only 3 min of treatment. In contrast, decondensation of human sperm nuclei started immediately in response to either DTT alone or heparin alone, and decondensation proceeded so rapidly that within 3 min the sperm heads had become invisible (Table 2). The minimum concentrations of DTT and heparin added together to form a mixture capable of inducing nuclear decondensation of bull sperm appear to be 10 µg/ml heparin and 10 mm DTT (Fig. 1), but a 30-min treatment duration is necessary to achieve decondensation. Nuclear decondensation of human sperm was caused by 10 mm DTT alone or 1000 µg/ml heparin alone (Fig. 2), but lower doses were ineffective at inducing decondensation. Pretreatment of bull sperm with 100 mm DTT for 30 min followed by 100 µg/ml heparin for 30 min caused nuclear decondensation, but decondensation was not induced by any other pretreatment/treatment combinations (Fig. 3). Decondensation of human sperm nuclei was caused by a 30-min pretreatment with very low concentrations of DTT (0.1 mm) or heparin (10 µg/ml) followed by treatment for 5 or 30 min with the alternate compound at the same low concentration used for pretreatment (Fig. 4).

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5 DECONDENSATION OF BULL AND HUMAN SPERM NUCLEI IN VITRO 11 Fig. 3. Bull sperm head area (µm 2 ) following a 30-min pretreatment with heparin or DTT and subsequent treatment with alternate compound for 30 min. Control, neither heparin nor DTT was added. Data are presented as mean ± SEM (n=10). *P<0.01 compared with control value. Discussion Very different results were obtained for decondensation of bull and human sperm nuclei following treatment with DTT or heparin. When DTT or heparin alone was added at high concentration, bull sperm nuclei did not decondense. Contrarily, the nuclear decondensation of human sperm started rapidly after the addition of DTT or heparin, and the sperm heads became invisible rather quickly. When both DTT and heparin were added together at high concentrations, however, both bull and human sperm nuclei decondensed in vitro. Pretreatment of bull sperm with 100 mm DTT followed by treatment with heparin (100 µg/ml) resulted in the decondensation of sperm nuclei, but the decondensation was not induced when heparin was added first followed by DTT. These results suggest that disulfide bond reduction (caused by the thiol-reducing agent, DTT) is required for the bull sperm nuclei to decondense in response to heparin. In contrast, decondensation of human sperm nuclei was induced by a low concentration of DTT or heparin alone. Furthermore, pretreatment of human sperm with 0.1 mm DTT decreased the concentration of heparin required for inducing nuclear decondensation of human sperm to as low as 10 µg/ml, a concentration incapable of inducing decondensation by itself. This Fig. 4. Human sperm head area (µm 2 ) following a 30-min pretreatment with DTT (0.1 mm) or heparin (10 µg/ ml) and subsequent treatment with alternate compound for 5 or 30 min. Control, neither DTT nor heparin was added. Data are presented as mean ± SEM (n=10). *P<0.01 compared with control value. indicates that the disulfide bond reduction is also preferable for human as well as bovine sperm decondensation. Interspecies differences in sperm nuclear stability have been reported, and these differences may be related to the number and/or arrangement of disulfide bonds [11, 20, 21]. This in turn is determined by the type of protamines present in the sperm nucleus. Bull sperm nuclei contain only protamine I [6], which is maximally crosslinked by disulfide bridges [7]. Bull sperm nuclei are, therefore, probably very stable. Human sperm nuclei, on the other hand, contain protamine II and protamine I [6, 8, 9, 10]. Since protamine II contains less cysteine, human sperm nuclei can be expected to have a lower concentration of disulfide bridges and thus be less stable than bull sperm nuclei. In the present study, heparin could induce nuclear decondensation of human sperm independent of DTT, but the same was not true of bull sperm. This suggests that heparin can act directly on the chromatin mass of human but not of bovine sperm. This is probably due to the differences in the structure of DNA-protamine complex between the two species. Jager et al. [18] suggested that heparininduced decondensation of human sperm is probably due to a direct reaction of heparin with the chromatin because the decondensation started immediately upon addition of the heparin (provided that the sperm nucleus was exposed, either by freezing and thawing or by treatment with a detergent). Heparin is a polyanion known to have a strong affinity for protamine molecules. Chargaff and

6 12 MOTOISHI et al. Olson [22] demonstrated that heparin and protamine molecules combine to form an insoluble complex. Since the strong condensation of the sperm nucleus is the result of binding of protamines to DNA, dissociation of the protamine-dna complex (caused by heparin) will lead to decondensation of the chromatin and thus to an increase in the nuclear volume [18]. However, bull sperm did not decondense by heparin treatment alone. A significant decondensation of bull sperm nuclei occurred when they were pretreated with DTT, followed by treatment with heparin. These results suggest that disulfide bond reduction is required for heparin molecules to combine with protamine molecules in the bovine. Therefore, the decondensation mechanisms of bull and human sperm nuclei appear to be different. Bull sperm showed DTTdependent nuclear decondensation whereas human sperm showed DTT-independent nuclear decondensation. However, human sperm nuclei decondensed at very low concentration of heparin (one which could not induce decondensation by itself) when they were pretreated with DTT, indicating the disulfide bond reduction is also preferable for human as well as bull sperm. In summary, our results support the idea that the decondensation process involves disulfide bond reduction and that the species differences in degree of nuclear decondensation is probably due to the differences in the number of disulfide bonds. Bull sperm nuclei have more disulfide bonds than those of human sperm; therefore, higher concentrations of DTT and heparin are required for nuclear decondensation to occur. However, the biological process of nuclear decondensation appears to be basically the same for bull and human sperm, namely disulfide bond reduction followed by DNA-protamine dissociation. Acknowledgments The authors wish to thank Dr. C.R. Youngs, Iowa State University, for his critical reading of this paper. This study was supported in part by a grant-in-aid ( ) for Scientific Research (B) from the Ministry of Education, Science and Culture of Japan. References 1. Goto K, Kinoshita A, Takuma Y, Ogawa K. Fertilization of bovine oocytes by the injection of immobilized, killed spermatozoa. Vet Rec 1990; 127: Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocytes. Lancet 1992; 340: Van Steirteghem AC, Nagy Z, Joris H, Liu J, Staessen C, Smitz J, Wisanto A, Devroey P. High fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod 1993; 8: Keefer CL, Younis AI, Brackett BG. Cleavage development of bovine oocytes fertilized by sperm injection. Mol Reprod Dev 1990; 25: Li X, Iwasaki S, Nakahara T. Investigation of various conditions in microfertilization of bovine oocytes and subsequent changes in nuclei and development to embryos. J Reprod Dev 1993; 39: Calvin HI. Comparative analysis of the nuclear basic proteins in rat, human, guinea pig, mouse and rabbit spermatozoa. Bioch Biophys Acta 1976; 434: Balhorn R. A model for the structure of chromatin in mammalian sperm. J Cell Biol 1982; 93: Balhorn R, Weston S, Thomas C, Wyrobek A. DNA packaging in mouse spermatids; synthesis of protamine variants and four transition proteins. Exp Cell Res 1984; 150: McKay DJ, Renaux BS, Dixon GJ. The amino acid sequence of human sperm protamine P1. Bio Rep 1985; 5: McKay DJ, Renaux BS, Dixon GJ. Human sperm protamines, amino-acid sequences of two forms of protamine P2. Euro J Bioch 1986; 156: Perreault SD, Barbee RR, Elstein KH, Zucker RM, Keefer CL. Interspecies differences in the stability of mammalian sperm nuclei assessed in vivo by sperm microinjection and in vitro by flow cytometry. Biol Reprod 1988; 39: Wiesel S, Schultz GA. Factors which may affect removal of protamine from sperm DNA during fertilization in the rabbit. Gamete Res 1981; 4: Calvin HI, Grosshans K, Blake EJ. Estimation and manipulation of glutathione levels in prepubertal mouse ovaries and ova: Relevance to sperm nucleus transformations in the fertilized egg. Ga-

7 DECONDENSATION OF BULL AND HUMAN SPERM NUCLEI IN VITRO 13 mete Res 1986; 14: Perreault SD, Barbee RR, Slott VL. Importance of glutathion in the acquisition and maintenance of sperm nuclear decondensing activity in maturing hamster oocytes. Dev Biol 1988; 125: Delgado NM, Huacuja L, Merchant H, Reyes R, Rosado A. Species specific decondensation of human sperm nuclei by heparin. Arch Androl 1980; 4: Caranco A, Reyes R, Magdaleno VM, Huacuja L, Hernandez O, Rosado A, Mechant H, Delgado NM. Heparin-induced nuclei decondensation of mammalian epididymal spermatozoa. Arch Androl 1983; 10: Reyes R, Rosado A, Hernandez O, Delgado NM. Heparin and glutathione: Physiological decondensing agents of human sperm nuclei. Gamete Res 1989; 23: Jager S, Wijchman J, Kremer J. Studies on the decondensation of human, mouse, and bull sperm nuclei by heparin and other polyanions. J Exp Zool 1990; 256: Brackett BG, Oliphant G. Capacitation of rabbit spermatozoa in vitro. Biol Reprod 1975; 12: Bedford JM, Bent MJ, Calvin HI. Variations in the structural character and stability of the nuclear chromatin in morphologically normal human spermatozoa. J Reprod Fertil 1973; 33: Mahi CA, Yanagimachi R. Induction of nuclear decondensation of mammalian spermatozoa in vitro. J Reprod Fertil 1975; 44: Chargaff E, Olson KB. Studies on the chemistry of blood coagulation. VI. Studies on the action of heparin and other coagulants. The influence of protamine on the anticoagulant effect in vivo. J Biol Chem 1938; 122: