Effect of amino acids on cryopreservation of cynomolgus monkey (macaca fascicularis) sperm.код для вставкиСкачать
American Journal of Primatology 59:159–165 (2003) BRIEF REPORT Effect of Amino Acids on Cryopreservation of Cynomolgus Monkey (Macaca fascicularis) Sperm YAHUI LI1–3, WEI SI1,2, XIUZHEN ZHANG1,2, ANDRAS DINNYES4, and WEIZHI JI1n 1 Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, People’s Republic of China 2 Graduate School, Chinese Academy of Sciences, Beijing, People’s Republic of China 3 College of Food Science and Technology, Yunnan Agricultural University, Kunming Yunnan, People’s Republic of China 4 Research Group for Applied Animal Genetics and Biotechnology, Hungarian Academy of Sciences and Szent Istvan University, Godollo, Hungary The effects of three amino acids (proline, glutamine, and glycine) added to the freezing medium Tes-Tris-egg yolk (TTE) for cryopreservation of cynomolgus monkey (Macaca fascicularis) spermatozoa were studied. This is the first report on the effects of amino acids on nonhuman primate sperm cryopreservation. The addition of 5 mM proline, 10 mM glutamine, and 10 or 20 mM glycine each significantly improved post-thaw sperm motility and membrane and acrosome integrity compared with the control (TTE alone). However, a significant decrease in motility and membrane/acrosome integrity was observed when amino acid concentrations increased to 60 mM for proline and glutamine, and 80 mM for glycine. The results suggest that adding a limited amount of amino acids to the freezing media is beneficial for freezing cynomolgus monkey sperm. Am. J. Primatol. 59:159–165, 2003. r 2003 Wiley-Liss, Inc. Key words: amino acid; sperm; cryopreservation; Macaca fascicularis INTRODUCTION Sperm cryopreservation is an important element in preserving rare or endangered animals, treating human infertility, and further developing reproductive techniques, such as artificial insemination (AI) and in vitro fertilization (IVF). Quite a few spermatozoa are damaged during the freeze-thaw process. Many factors, such as the cryoprotectant used, influence this cryodamage. Glycerol has been widely used as a cryoprotectant during sperm cryopreservation. Various other compounds have also been demonstrated to have the ability to Contract grant sponsor: China National Science Foundation; Contract grant number: 39970117; Contract grant sponsor: Chinese Academy of Sciences; Contract grant number: KZ952-J1-109. n Correspondence to: Weizhi Ji, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 Estern Jiaochang Road, Kunming, Yunnan 650223, People’s Republic of China. E-mail: firstname.lastname@example.org(W.Ji) Received 3 December 2002; revision accepted 14 February 2003 DOI 10.1002/ajp.10073 Published online in Wiley InterScience (www.interscience.wiley.com). r 2003 Wiley-Liss, Inc. 160 / Li et al. protect sperm during freezing [Chen et al., 1993; De Leeuw et al., 1993; Sztein et al., 2001; Woelders et al., 1997], and an intensive search for new cryoprotectants continues. Certain plants are known to accumulate the amino acid proline to resist cold temperatures [Stewart & Lee, 1974], and it has also been reported that some amino acids protect animal cells against hypothermia [Kruuv & Glofcheski, 1992]. Protective effects of the amino acids glutamine, proline, glycine, alanine, taurine, and hypotaurine during cryopreservation of spermatozoa in rams [Kundu et al., 2001], stallions [Lindeberg et al., 1999; Trimeche et al., 1999], bulls [Chen et al., 1993], and humans [Renard et al., 1996] have been described. However, no reports have been published on nonhuman primate sperm preservation using amino acids as cryoprotectants. In the present study, we investigated the cryoprotective potential of three amino acids for spermatozoa of the cynomolgus monkey, one of the most widely used nonhuman primate models. METHODS Media Preparation All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated. The base for the cryoprotectant solutions was Tes-Tris-egg yolk (TTE), as described by Sankai et al. . Before the experiments were conducted, Lproline was added to the TTE at concentrations of 1, 3, 5, 10, 20, 40, or 60 mM. Both L-glutamine and L-glycine were added separately to the medium at 3, 5, 10, 20, 40, 60, and 80 mM. All freezing media were adjusted to pH 7.0–7.2. The osmotic pressures of the media were measured with an automatic osmometer (Osmette A, 5002; Precision Systems, Inc., Natick, MA). Finally, 10% glycerol was added to prepare the freezing medium (the final concentration for sperm freezing was 5% glycerol). Semen Collection and Cryopreservation of Spermatozoa Fifteen sperm samples were collected by penile electroejaculation [Si et al., 2000] from four anesthetized cynomolgus monkeys (5–11 years old) provided by the Laboratory Animal Center of the Kunming Institute of Zoology, Chinese Academy of Sciences. Semen was collected into a disposable plastic test tube containing 2 ml of TALP-Hepes [Bavister et al., 1983], which was kept at 371C in a water bath for 30 min to allow the clot to liquefy. After the clot liquified, a small sample was examined for sperm motility and membrane and acrosome integrity; the rest was washed twice with 9 volumes of TALP-Hepes and centrifuged at 200 g for 10 min. The supernatant was then aspirated, and the sperm pellet was dispersed and mixed with a Pasteur pipette before it was frozen. The sperm freezing procedure was identical to that described by Sankai et al. . After they were stored in liquid nitrogen for more than 7 days, 0.25-ml plastic straws (IMV, L’Aigle, France) containing frozen spermatozoa were thawed in a 371C water bath for 2 min. Thawed sperm suspension was diluted with 5 volumes of TALP-Hepes, 5 times at 30-sec intervals, and then washed twice with TALP-Hepes by centrifugation at 200 g for 10 min. The sperm pellet was dispersed immediately. Amino Acids and Sperm Cryopreservation / 161 Examination of Sperm Motility and Membrane/Acrosome Integrity Sperm motility. With the use of a hemocytometer counting chamber, fresh and thawed sperm samples were assessed for percentage of progressive motility by counting 200 spermatozoa, in duplicate. Membrane integrity. Sperm membranes were stained with Hoechst 33258 according to the method of Mortimer et al. . At least 200 spermatozoa were scored for each sample. Acrosome integrity. Acrosome staining was performed as described by Valcarcel et al. , with the exception that in the present study only FITC-PNA was used. A minimum of 200 spermatozoa per sample were counted. Statistical Analysis All data are expressed as mean 7 STD. The sperm motility and membrane/ acrosome integrity data were subjected to arcsine (square root) transformation and analyzed by analysis of variance (ANOVA) and Tukey’s test. Po0.05 was considered statistically significant. RESULTS Effect of L-Proline The effect of L-proline on cynomolgus monkey sperm cryopreservation is shown in Table I. When compared with the control, sperm motility and membrane/acrosome integrity were significantly increased by the addition of 5 mM proline. However, the addition of 60 mM proline significantly decreased sperm motility and membrane/acrosome integrity. Effect of L-Glutamine The effect of L-glutamine on cynomolgus monkey sperm cryopreservation is presented in Table II. The addition of 10 mM glutamine improved sperm motility, and 5–40 mM glutamine increased membrane integrity significantly. Furthermore, 3–10 mM glutamine significantly improved acrosome integrity. Overall, 10 mM glutamine provided the best cryoprotection for sperm. It was found that 80 mM glutamine had negative effects for all three parameters examined. Effect of L-Glycine The results of L-glycine addition are shown in Table III. The addition of 10–40 mM glycine significantly improved post-thaw motility. Membrane and 162 / Li et al. acrosome protection was provided by both 10 and 20 mM glycine. At a concentration of 80 mM, the three values decreased significantly. Overall, 10 mM glycine produced the highest values of sperm motility and membrane/ acrosome integrity. TABLE I. Effect of Proline Addition on Cynomolgus Monkey Sperm Cryopreservation Proline addition Osmotic pressure (mOsm/kg) Fresh sperm 0 (TTE control) 1 mM 400 402 3 mM 403 5 mM 406 10 mM 408 20 mM 418 40 mM 442 60 mM 465 F(7, 32) a,b,c,d,e values Motility (%) 80.672.2 55.071.0ae 48.373.4b (P=0.022) 51.571.3ab (P=0.576) 64.073.2c (P=0.001) 60.671.9ce (P=0.071) 60.770.8ce (P=0.065) 55.173.7ae (P=1.000) 38.074.8d (P=0.000) 41.159 Membrane intact (%) 76.572.0 45.673.2ae 43.971.8e (P=0.915) 47.271.6ce (P=0.918) 54.573.1b (P=0.000) 49.971.6ac (P=0.55) 49.371.1ac (P=0.126) 44.771.9e (P=0.998) 36.471.7d (P=0.000) 31.606 Acrosomal integrity (%) 91.770.5 70.671.3ac 68.473.6c (P=0.955) 69.172.1c (P=0.993) 77.971.4be (P=0.007) 75.274.2ae (P=0.211) 75.172.8ae (P=0.246) 67.074.8c (P=0.631) 58.871.7d (P=0.000) 19.607 Groups with different superscripts in the same column are significantly different (Po 0.05). TABLE II. Effect of Glutamine Addition on Cynomolgus Monkey Sperm Cryopreservation Glutamine addition Osmotic pressure (mOsm/kg) Fresh sperm 0 (TTE control) 3 mM 400 402 5 mM 403 10 mM 408 20 mM 420 40 mM 440 60 mM 460 80 mM 482 F(7, 32) a,b,c,d,e values Motility (%) 81.471.4 55.174.7a 59.471.4ac (P=0.298) 60.171.0abc (P=0.145) 65.372.7b (P=0.000) 57.573.3ac (P=0.893) 54.371.0ac (P=1.000) 45.873.2d (P=0.000) 46.273.5d (P=0.001) 27.087 Membrane intact (%) 78.272.4 45.470.9ac 49.172.4ce (P=0.092) 50.372.4e (P=0.008) 58.872.4b (P=0.000) 49.871.7e (P=0.027) 49.672.2e (P=0.036) 41.671.4ad (P=0.076) 38.871.7d (P=0.000) 48.299 Acrosomal integrity (%) 92.970.8 69.573.7ae 74.772.7c (P=0.042) 75.471.2c (P=0.014) 80.571.6b (P=0.000) 74.572.5ce (P=0.055) 73.573.7ce (P=0.216) 64.871.4ad (P=0.101) 60.071.2d (P=0.000) 34.202 Groups with different superscripts in the same column are significantly different (Po 0.05). Amino Acids and Sperm Cryopreservation / 163 TABLE III. Effect of Glycine Addition on Cynomolgus Monkey Sperm Cryopreservation Glycine addition Osmotic pressure (mOsm/kg) Fresh sperm 0 (TTE control) 3 mM 400 402 5 mM 403 10 mM 410 20 mM 421 40 mM 440 60 mM 462 80 mM 482 F(7, 32) a,b,c,d,e,f values Motility (%) 81.672.7 54.571.0ac 53.471.5c (P=0.992) 53.973.0c (P=1.000) 64.672.4b (P=0.000) 64.271.7be (P=0.000) 59.871.2de (P=0.022) 56.773.2acd (P=0.829) 40.273.1f (P=0.000) 55.885 Membrane intact (%) 78.771.2 50.172.9acde 47.271.4c (P=0.598) 49.571.4cd (P=1.000) 59.972.6b (P=0.000) 59.473.0b (P=0.000) 53.771.2de (P=0.316) 51.374.0cd (P=0.994) 41.971.6f (P=0.000) 30.001 Acrosomal integrity (%) 93.170.7 70.570.9a 63.675.7bd (P=0.018) 70.572.9ae (P=1.000) 78.771.5c (P=0.001) 77.473.2cf (P=0.007) 73.170.5aef (P=0.808) 71.572.9ae (P=0.999) 60.971.5d (P=0.000) 23.259 Groups with different superscripts in the same column are significantly different (Po 0.05). DISCUSSION In this study we have shown that the addition of amino acids to spermfreezing media can be beneficial in the cryopreservation of cynomolgus monkey sperm. A few previous reports demonstrated that amino acids had certain cryoprotective effects on sperm [Chen et al., 1993; Kundu et al., 2001; Lindeberg et al., 1999; Renard et al., 1996; Trimeche et al., 1999]; however, none of these studies involved nonhuman primates. Our study showed that proline, glutamine, and glycine have a positive effect on sperm cryopreservation. Different amino acids had different optimal concentrations (B5–20 mM), which were lower than those reported in previous studies for other species (40–80 mM). Aside from species differences, these contrasting findings can be explained by differences in the extenders and freezing procedures used. In most previous studies, the sperm was cooled by a programmed freezing machine at a rate of 10–201C/min, whereas in our study it was cooled much faster (at a rate of approximately 601C/min). In the present study, the concentration of egg yolk in the TTE was 20%; in Trimeche et al.’s  study (in which semen was cooled at a similarly fast rate) it was 2%. The reasons for the cryoprotection provided by the three amino acids tested in this study remain unclear. Kundu et al.  suggested that the protective effects of amino acids may stem from their ability to form a layer on the sperm surface, as these positively charged molecules can combine with the phosphate groups of the sperm plasma membrane phospholipids. In the present study, however, at pH 7.0–7.2 of freezing media, proline, glutamine, and glycine are negatively charged. According to Alvarez and Storey , some amino acids can prevent lipid peroxidation of sperm membrane during 164 / Li et al. cryopreservation; however, whether this occurred in the present study remains to be proved. It is interesting that the addition of 1 mM proline significantly decreased sperm motility while 3 mM glycine decreased acrosome integrity significantly. There is currently no explanation for this, and it merits further study. Trimeche et al.  observed that high concentrations of glutamine are detrimental to the freezing of sperm, and suggested that this may be the result of high osmotic pressure when the concentration of glutamine rises. In our study, sperm damage also occurred at high amino acid concentrations, in accordance with the findings of Trimeche et al. . In conclusion, we have shown that the three amino acids studied had positive effects on cynomolgus monkey sperm cryopreservation. 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