N-乙基-N-亚硝基脲对巴马香猪精液品质及精子发生影响的研究
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摘要
N-乙基-N-亚硝基脲(N-Ethyl-N-nitrosourea, ENU)是一种较强的基因诱变剂,能在相对较短的时间内诱导产生大量随机突变表型,进一步通过表型筛选及遗传试验可能获得具有特殊表型的突变系动物,可用于建立人类遗传性疾病研究的新模型,在一定程度上还可以作为验证基因功能的一种参考方法。
ENU具有较大的细胞毒性,给药后往往会导致雄性实验动物生殖能力的下降甚至不育,目前ENU诱变的实验动物一般为小鼠,而小鼠的解剖与生理功能与人相差较大,并且小鼠的精液不易采集,很难对其生殖功能进行长期监控。猪的解剖与生理功能与人较为相似,特别是小型猪已成为医学实验上重要的实验材料。利用小型猪巴马香猪做为实验动物,通过ENU诱导建立大型哺乳动物基因突变模型和人类疾病动物模型,在理论和医学实践上均具有重要价值。小鼠诱变实验的结果表明,ENU剂量的选择十分重要,一个适宜的剂量不仅能诱导DNA突变,而且还要保障实验动物的生殖功能在停药后可以逐渐恢复,从而使获得的突变能够遗传下去。本研究针对ENU诱变过程适宜剂量筛选这一关键问题,在研究巴马香猪精液品质特征的基础上,长期监控ENU处理对公猪生殖功能的影响,筛选到了既能诱导可疑表型,又能保证在ENU停药后,公猪生殖功能能基本恢复正常的适宜剂量。
试验首先检测了90头健康成年巴马香猪新鲜精液质量,并探索液冷冻保存的方法。成年巴马香猪射精量、精液活力、密度、畸形率、抗力指数分别为95.31m1,75.33%,0.78x108,5.13%及581,其中,巴马香猪的射精量、精子活力和密度均低于杜洛克和约克夏等商品猪。在巴马香猪精液冷冻研究中,采用正交实验设计,在探讨低密度脂蛋白(LDL)浓度、甘油浓度、海藻糖浓度作用的基础上,进一步结合15℃平衡时间和解冻方法对冷冻解冻后精子活率的影响,进行巴马香猪精液最佳冻存方案的优化。结果表明方案1(A3B4C2D3E1)为最佳方案,其解冻后精子活率为52.26%。为分析稀释液与冷冻液中LDL、甘油和海藻糖不同联用组合的保护效果,在方案1的基础上,共设计了4种组合,以精子运动能力、顶体完整性、质膜完整性以及DNA损伤率等为判定指标,结果表明,9%的LDL、200mM海藻糖与2%的甘油联用时(即组合4)显著优于其他组合的保护效果(P<0.05),并进一步证明方案1是巴马香猪精液冷冻保存的最佳方案。
巴马香猪ENU给药当天各剂量组猪只采食量与对照组相比极显著地降低(P<0.01),然后逐渐(给药后第5-6天)恢复直至正常(与对照组无显著差异,P>0.05)；待第2周(次)、第3周(次)重复给药时,会出现同样的变化趋势。ENU给药期间巴马香猪采食量会受到较大影响,采食量的减少具有浓度依赖性,采食量恢复时间随剂量的增加而延长。给药后各剂量组公猪的生长速度都有一定程度降低。随ENU给药剂量的增加,巴马香猪的死亡率逐渐升高,可育率逐渐降低。结果表明,巴马香猪对85mg/kg及以下剂量可耐受。
巴马香猪ENU给药后12月内射精量呈现先下降后上升的趋势,给药后第12月,65mg/kg及以下剂量组巴马香猪射精量基本恢复正常；75-105mg/kg剂量组射精量均不能恢复到给药前水平,但75mg/kg剂量组公猪射精量恢复情况好于85-105mg/kg剂量组公猪(P<0.05),85-105mg/kg剂量组公猪之间射精量无显著性差异(P<0.05)。巴马香猪精子活力先下降后上升,给药后第12月,45mg/kg和65mg/kg剂量组公猪精子活力能够恢复到给药前水平,75-105mg/kg剂量组公猪均不能恢复到给药前水平,75mg/kg和85mg/kg剂量组公猪精子活力恢复情况好于90-105mg/kg剂量组公猪(P<0.05),90-105mg/kg剂量组公猪之间精子活力无显著性差异(P<0.05)。巴马香猪精液密度也表现为先下降后上升,45mg/kg和65mg/kg剂量组公猪精液密度能够恢复到给药前水平,75-105mg/kg剂量组公猪均不能恢复到给药前水平,其中75mg/kg剂量组公猪精液密度恢复情况好于85mg/kg剂量组(P<0.05),后者精液密度恢复好于90-105mg/kg剂量组公猪(P<0.05)。巴马香猪精子畸形率先上升后下降,45mg/kg和65mg/kg剂量组公猪精子畸形率能够恢复到给药前水平,75-105mg/kg剂量组公猪均不能恢复到给药前水平,其中75mg/kg和85mg/kg剂量组精子畸形率恢复好于90-105mg/kg剂量组公猪(P<0.05)。给药后18月公猪射精量显著高于给药后12月(P<0.05),表明公猪副性腺的精浆分泌功能有所恢复,其他精液指标无显著差异,表明ENU给药后12月公猪精液生成和精子发生的能力恢复情况已经基本稳定。
ENU给药后公猪配种记录表明,45mg/kg和65mg/kg剂量组公猪的与配母猪妊娠率与对照组无显著差异(P>0.05),其他各剂量组均与对照组均有显著差异(P<0.05),并且有随着剂量增大与配母猪妊娠率降低趋势更为明显的现象；断奶仔猪成活率的差异未表现出与给药剂量之间的相关性：各剂量组公猪(F0代)所生产的F1代仔猪可疑表型发生率均与对照组有显著差异(P<0.05),其中45mg/kg和65mg/kg剂量组仔猪可疑表型发生率显著低于105mg/kg剂量组(P<0.05),75-95mg/kg剂量组与105mg/kg剂量组仔猪可疑表型发生率无显著差异(p>0.05)。结合公猪对ENU的耐受性与仔猪可疑异常表型发生率考虑,75-85mg/kg属于巴马香猪ENU诱变的合适剂量。
ENU给药后18个月对巴马香猪生殖器官和副性腺进行组织学检测,结果表明,巴马香猪的睾丸系数、附睾系数和精囊腺系数与对照组相比,除了45mg/kg和65mg/kg剂量组外,85-105mg/kg剂量组巴马香猪附睾系数和精囊腺系数均显著低于对照组,表明生殖器官和副性腺已受到了损伤；随着ENU给药剂量的增加,公猪睾丸内正常曲细精管减少,部分变性萎缩和完全变性萎缩的曲细精管比例上升；曲细精管内各级生精细胞与支持细胞的比例也随着ENU剂量的增加而降低。ENU给药对巴马香猪前列腺无明显影响。透射电镜下给药组可见部分精原细胞核出现空泡、精子核解聚,严重者可见部分精原细胞核解聚,部分精子细胞核膜消失。免疫组化实验结果表明,曲细精管中GDNF蛋白的表达量随着ENU剂量的增大而降低,表明ENU剂量越大,曲细精管损伤程度越大,公猪支持细胞分泌GDNF蛋白的能力越低,精原干细胞自我更新的能力也因此而下降,随之各级生精细胞数量减少,公猪表现为生精障碍和精液品质的降低。随着ENU给药剂量的增加,曲细精管内生精细胞凋亡数量逐渐升高,也是公猪生精能力降低的主要原因之一。
激素水平跟踪检测结果表明,ENU注射前各剂量组公猪血清T和E2的水平无显著差异(P>0.05)；给药后4月,除45mg/kg剂量组公猪血清T和E2水平与对照组无显著差异(P>0.05)外,其他剂量组与对照组均有显著差异(P<0.05),T水平显著低于对照组(P<0.05),而E2水平显著高于于对照组(P<0.05)。表明ENU给药后,随着巴马香猪睾丸细胞的变性,T和E2的分泌均受到了较大影响,而这2种生殖内分泌激素水平的异常既是公猪精液品质变化的标志,也是引起精液品质变化的原因之一。
试验最后对ENU影响巴马香猪精子发生的分子机制进行了研究。睾丸分泌雄性激素和产生精子这两种功能的实现需要在相关基因的调控下完成,其中,StAR基因和P450scc基因主要与睾酮合成相关；GDNF基因、SCF基因和CREM基因主要与精子发生相关；在精子发生过程中,同时也伴随着生精细胞的凋亡,在外源性因素作用下,细胞凋亡的速度会有所变化,在细胞凋亡的线粒体介导途径中,Bcl-2家族中抑制细胞凋亡的Bcl-2基因和促进细胞凋亡的Bax基因的表达水平对细胞的生存与死亡起到决定性的作用。实验结果表明,除105mg/kg剂量组公猪睾丸组织中StAR基因表达量显著低于对照组(P<0.05)外,其他剂量组所受影响不大；45mg/kg和65mg/kg剂量组P450scc基因的表达量与与对照组均无显著差异(P>0.05),75和85mg/kg剂量组的表达量显著低于对照组(P<0.05),90-105mg/kg剂量组公猪睾丸内P450scc基因的表达量显著低于其他各剂量组(P<0.05),提示ENU给药后公猪睾酮水平的下降可能主要与P450scc基因表达的降低有关。GDNF基因和CREM基因的表达情况大致相同,即45mg/kg和65mg/kg剂量组公猪睾丸内GDNF基因的表达量基本未受影响(P>0.05),75-105mg/kg剂量组公猪睾丸内GDNF基因的表达量均显著降低(P<0.05),而且随剂量的增加其表达量降低的幅度越大,表明随着ENU给药剂量的增加,公猪睾丸曲细精管内精原干细胞的自我更新能力逐渐减弱,成熟的精子细胞变少,剂量达到90mg/kg以上时,公猪精原干细胞自我更新及精子成熟会严重受阻。45mg/kg和65mg/kg剂量组公猪睾丸内BAX基因的表达量与对照组无显著差异(P>0.05),75-105mg/kg剂量组BAX基因的表达量显著高于对照组(P<0.05),并且差异随ENU剂量的加大而升高(P<0.05),而各剂量组Bcl-2基因的表达量与对照组均无显著差异(P>0.05),提示除45和65mg/kg剂量组外,公猪睾丸内生精细胞的凋亡程度随剂量的增加而加大,并且当ENU给药剂量增加至90mg/kg以上时,生精细胞凋亡水平较高,生精细胞数量减少,其精液质量有较大水平的降低。
综上所述,本研究可以得到如下结论：
(1)成年巴马香猪正常的射精量、精液活力、密度、畸形率、抗力指数分别为95.31ml,75.33%,0.78x108,5.13%及581；(2)巴马香猪最佳精液冷冻-解冻方法是精液稀释液中添加9%的LDL和200mM海藻糖,冷冻液中添加2%的甘油,降温过程中于15℃平衡3小时,并于37℃下解冻45s；(3)ENU给药后巴马香猪的采食量下降,生长速度变慢,死亡率和不育率随给药剂量的增加而升高,对90mg/kg及以上剂量不耐受；(4)随着ENU给药剂量的增加,公猪血清T水平下降,E2水平上升,公猪睾丸、附睾和精囊腺受损程度加重,巴马香猪的精液质量降低；(5)ENU给药后,公猪睾丸组织P450scc基因、GDNF基因和CREM基因表达下降,BAX基因表达水平上升,公猪睾丸生精能力受损；(6)根据巴马香猪对ENU可耐受性和生精功能变化的综合分析,最佳ENU诱变剂量可初步确定为75-85mg/kg。
N-Ethyl-N-nitrosourea (ENU) is generally accepted as a very strong mutagenic agent. ENU can induce large amount of random mutation phenotypes within a relatively short period, mutant strain animals with special phenotypes may be obtained after further phenotypic screening and genetic tests, and genetic basic tests can be thus carried out on special phenotypes of mutated animals. It has shown excellent prospects in the studies on mice and can be used as a reference method to confirm the functions of genes. ENU mutagenesis can also be used to establish new models for the studies on genetic diseases in human. The management database for ENU mutagenic mice of G1generation has been established now, and genetic resources of mutagenic mice are effectively preserved by using embryo cryopreservation technique, sperm freezing, in vitro fertilization and other techniques.
The experimental animal carriers for ENU mutagenesis are normally mice. The results from the mutagenesis tests on mice indicate that ENU not only can induce single base mutation in DNA, but also is a kind of cytotoxic agent, which can lead to death of large amount of spermatogenous cells in male mice. After ENU is withdrawn, the genital functions can be gradually recovered. The administration dosage in the ENU mutagenesis tests is very important. High dosages may lead to increase in the mutation rate, but the treatments may lead to intolerance, infertility or death in male mice; in contrast, low dosages may decrease the mutation rate, and the results of the tests may be not satisfactory. Since the strains of mice and the administration methods utilized by the researchers are different, the research results are not comparable, and the "proper dosage" of ENU is still not determined. Normally, the dosage of ENU can be evaluated from the three aspects as below:(1) the percentage of infertile male mice;(2) the length of average infertility period;(3) the number of dead male mice within a relatively short period after the genital capacity is recovered. The experimental data for ENU mutagenesis for big experimental animals are still insufficient now. The present study was carried out to examine the effects of ENU on spermatism and spermatogenesis in Bama miniature pigs in order to investigate a proper dosage for ENU mutagenesis of Bama miniature pig.
The present study firstly examined the quality of fresh sperm from90adult Bama miniature pigs and explored the method for sperm cryopreservation. Ejaculation amount, sperm vitality, density, abnormal rate and resistance of adult Bama miniature pigs were95.31ml,75.33%,0.78×108,5.13%and581, among which the ejaculation amount, sperm vitality and density were all lower than Duroc, Yorkshire and other commercial available pigs, indicating that the cryopreservation technique for its sperm still had certain difficulty. Orthogonal experimental design was used for the sperm cryopreservation studies on Bama miniature pigs, and the program for sperm cryopreservation of Bama miniature pigs was optimized on the basis of investigating the functions of LDL concentration, glycerol concentration and trehalose concentration by combining with the effects of equilibrium time at15℃and thawing method on the motility rate after freezing and thawing. The results showed that the program1(A3B4C2D3E1) was the optimal program, and the motility rate after thawing was52.26%. In order to analyze the protective effects of different combinations of LDL, glycerol and trehalose in the dilution and the refrigerating solution, four kinds of combinations were designed, and sperm motility, acrosomal integnity, plasma membrane integrity, DNA injury rate and other parameters were used as the judging parameters. The results indicated that the protective effects for the combination with9%LDL,200mM trehalose and2%glycerol (the combination4) were significantly better than those of other combinations (P<0.05), which further indicated that the program1was the optimal program for sperm cryopreservation of Bama miniature pigs.
During the administration stage after ENU mutagenesis for Bama miniature pigs, the food intake for the pigs in different dosage groups on the day of administration extremely significantly decreased in comparison to that in the control group (P<0.01), afterwards, it gradually recovered (5-6days after administration)(the difference in comparison to that in the control group was not statistically significant, P>0.05); the same tendency may appear after repeated administration on the second and third administration (at the second and third week). The food intake of Bama miniature pigs may be significantly affected during the administration with ENU, the decrease in food intake was dose-dependent, and the recovery time for food intake increased with the increase in the dosage. The growth rates of male pigs in different dosage groups after drug administration showed decreases to different extents. The mortality of Bama miniature pigs gradually increased with the increase in the administration dosage of ENU, and the fertility gradually decreased. The results indicated that Bama miniature pigs can tolerate a dosage no higher than85mg/kg.
The ejaculation amount of Bama miniature pigs within12months after ENU mutagenesis showed a decreasing and subsequently increasing tendency, and the ejaculation amounts of Bama miniature pigs almost recovered in the dosage groups of no higher than65mg/kg at12months after drug administration; the ejaculation amounts in the75-105mg/kg groups could not recover the level before drug administration, but the recovery of ejaculation amount in the75mg/kg group was better than those in the85-105mg/kg groups (P<0.05), and the differences in the ejaculation amounts among the85-105mg/kg groups were not statistically significant (P<0.05). The sperm motility of Bama miniature pigs firstly decreased and then increased, and the sperm motility in the45mg/kg and65mg/kg group at12weeks after administration can recover the level before administration, while the sperm motility in the75-105mg/kg groups can not recover the level before administration, the recovery in sperm motility in the75mg/kg and85mg/kg groups was better than that in the90-105mg/kg groups (P<0.05), and the differences in the sperm motility among the90-105mg/kg groups were not statistically significant (P<0.05). The sperm density of Bama miniature pigs also firstly decreased and then increased, the sperm density in the45mg/kg and65mg/kg group can recover the level before administration, the sperm density in the75-105mg/kg groups can not recover the level before administration, among which the recovery in sperm density in the75mg/kg group was better than that in the85mg/kg group (P<0.05), the recovery in sperm density in the latter group was better than that in the90-105mg/kg groups (P<0.05). The sperm abnormality rate of Bama miniature pigs firstly increased and then decreased, the sperm abnormality rates in the45mg/kg and65mg/kg groups can recover the level before administration, while the sperm abnormality rates in the75-105mg/kg groups can not recover the level before administration, among which the sperm abnormality rates in the75mg/kg and85mg/kg groups were better than those in the90-105mg/kg groups (P<0.05). The ejaculation amount18months after administration was significantly higher than that of12months (P<0.05), indicating that the functions of accessory gonads of the male pigs recovered and no statistically significant difference was detected in other sperm parameters. The recovery in the capabilities of spermatism and spermatogenesis12months after ENU administration had become stable.
The breeding records for the male pigs after ENU mutagenesis indicated that no statistically significant difference was detected in the pregnancy rate of female pigs for breeding in the45mg/kg and65mg/kg groups (P>0.05), and the differences of other dosage groups in comparison to that in the control group were all statistically significant (P<0.05), and it became more evident that the pregnancy rates of the female pigs for breeding decreased with the increase in the dosage; no statistically significant difference was detected in the litter sizes of the male pigs in different dosage groups (P>0.05); the difference in the survival rates of weaned baby pigs did not show any correlation with the administration dosage; the differences in the incidence rates of suspectable phenotypes in the pigs of F1generation reproduced by the male pigs in different dosage groups (FO generation) were all statistically significant in comparison to that in the control group (P<0.05), among which the incidence rates of suspectable phenotypes in the baby pigs in the45mg/kg and 65mg/kg groups were significantly lower than that in the105mg/kg group (P<0.05), and the differences in the incidence rates of suspectable phenotypes in the baby pigs between the75-95mg/kg groups and the105mg/kg group were not statistically significant (P>0.05). Combining the tolerance of the male pigs to ENU and the incidence rate of suspectable phenotypes in the baby pigs,75-85mg/kg were proper dosages for ENU mutagenesis of Bama miniature pigs.
Histological examinations were carried out on the genital organs and the acccessory gonads in the Bama miniature pigs18months after ENU administration. The results showed that the epididymis index and the seminal vesicle index in the85-105mg/kg groups were significantly lower than those of the control group except the45and65mg/kg groups, indicating that the genital organs and the acccessory gonads had been damaged; with the increase in the administration dosage of ENU, the normal contorted seminiferous tubules in the testis of the male pigs decreased, the percentage of contorted seminiferous tubule showing partial and complete degenerative atrophy decreased; the percentages of different grades of spermatogenic cells and supporting cells in the contorted seminiferous tubules also decreased with the increase in ENU dosage. ENU administration had no significant effects on the prostate of Bama miniature pigs. It can be observed under the transmission electron microscope that vacuoles can be detected in some spermatogenous cells and sperm nucleus depolymerization can be detected, and the nuclear membrane of some spermatids disappeared. The results from the follow-up detection on hormonal level showed that the differences in serum T and E2levels in the male pigs in different dosage groups before ENU injection were not statistically significant (P>0.05); statistically significant differences can be detected in serum T and E2levels in the male pigs between the dosage groups and the control group except the45mg/kg group four months after administration (P>0.05), and the T level was significantly lower than that in the control group (P<0.05), while the E2level was significantly higher than that in the control group (P<0.05), indicating that T and E2secretion was significantly affected with the degeneration of testicular cells of Bama miniature pigs. The decrease in T level and the increase in E2level are not only the symbol for the changes in sperm quality of male animals, but also the reason inducing the changes in sperm quality.
Testicles secrete male hormones and produce sperms, and these two functions should be accomplished under the regulations of related genes. Among which, StAR and P450scc are mainly related to testosterone synthesis, while GDNF, SCF and CREM are mainly related to spermatogenesis. Apoptosis of spermatogenic cells is also concurrent with spermatogenesis, and the rate of cell apoptosis may change under the effects of exogenous factors. Bax and Bcl-2are representatives for the genes promoting and inhibiting apoptosis respectively. The experimental results indicated that except that the expression level of StAR in the testicular tissues of the male pigs in the105mg/kg group was significantly lower than that in the control group (P<0.05), other dosage groups were not significantly affected; no statistically significant difference was detected in the expression level of P450scc in the45and65mg/kg groups and the control group (P>0.05), the expression levels in the75and85mg/kg groups were significantly lower than that in the control group (P<0.05), and the expression levels of P450scc in the testicles of the male pigs in the90-105mg/kg groups were significantly lower than those in other dosage groups (P<0.05, Figure6-2). The expression levels of GDNF in the testicles of the45and65mg/kg groups were not affected (P>0.05), the expression levels of GDNF and CREM were almost the same, in other words, the expression levels of GDNF in the testicles of the male pigs in the75-105mg/kg groups significantly decreased (P<0.05), while the decreasing amplitude in the expression level increased with the increase in the dosage, indicating that the self-renewal capability of stem spermatogonium in the contorted seminiferous tubules in the testicles of the male pigs gradually decreased and the mature sperm cells decreased. When the dosage reached90mg/kg or even higher, self-renewal of stem spermatogonium and sperm maturation of the male pigs were seriously inhibited. The differences in the expression level of BAX in the testicles of the male pigs in the45and65mg/kg groups in comparison to the control group were not statistically significant (P>0.05). the expression levels of BAX in the75-105mg/kg groups were significantly higher than that in the control group (P<0.05). and the differences increased with the increase in ENU dosage (P<0.05), while the differences in the expression levels of Bcl-2in different dosage groups in comparison to the control group were not statistically significant, indicating that the apoptosis of spermatogenic cells in the testicles of the male pigs increased with the increase in the dosage except the45and65mg/kg groups, and the apoptosis of spermatogenic cells was maintained at a relatively high level when the administration dosage of ENU increased to a dosage higher than90mg/kg, spermatogenic cells decreased and the sperm quality significantly decreased.
In general, the conclusions as followed can be drawn from the present study:
(1) Normal ejaculation amount, sperm vitality, density, abnormality rate and resistance of adult Bama miniature pigs were95.31ml.75.33%,0.78×108,5.13%and581respectively;
(2) The optimal method for semen freezing of Bama miniature pigs was the program1, in other words, supplement with9%LDL,200mM trehalose and2%glycerol. equilibrium at15℃for3h and thawing at37℃for45s;
(3) The food intake of Bama miniature pig decreased after administration with ENU, the growth rate decreased, the death rate and the infertility increased with the increase in the administration dosage, and they can not tolerate a dosage no lower than90mg/kg;
(4) With the increase in the administration dosage of ENU, the sperm quality of Bama miniature pigs gradually decreased, the damages in testicles, epididymis and seminal vesicle of male pigs were gradually aggravated, the amplitudes in the decrease in serum T level and the increase in serum E2level of the male pigs both increased, the expression level of GDNF in the contorted seminiferous tubules gradually decreased, the apoptotic spermatogenic cells gradually increased;
(5) the decrease in serum testosterone level in the male pigs after administration with ENU was mainly related to the decrease in the expression level of P450scc, the decreases in the expression levels of GDNF and CREM led to the decrease in the self-renewal capability of stem spermatogonium and the blockage of sperm maturation in the male pigs, the increase in the expression level of BAX led to the increase in the apoptotic spermatogenic cells, the decrease in the number of spermatogenic cells and the damages in spermatogenic capability;
(6) According to the comprehensive analysis on the tolerance of Bama miniature pigs to ENU and the changes in spermatogenic functions, and the optimal dosage for ENU mutagenesis can be preliminarily determined as75-85mg/kg.
ENU具有较大的细胞毒性,给药后往往会导致雄性实验动物生殖能力的下降甚至不育,目前ENU诱变的实验动物一般为小鼠,而小鼠的解剖与生理功能与人相差较大,并且小鼠的精液不易采集,很难对其生殖功能进行长期监控。猪的解剖与生理功能与人较为相似,特别是小型猪已成为医学实验上重要的实验材料。利用小型猪巴马香猪做为实验动物,通过ENU诱导建立大型哺乳动物基因突变模型和人类疾病动物模型,在理论和医学实践上均具有重要价值。小鼠诱变实验的结果表明,ENU剂量的选择十分重要,一个适宜的剂量不仅能诱导DNA突变,而且还要保障实验动物的生殖功能在停药后可以逐渐恢复,从而使获得的突变能够遗传下去。本研究针对ENU诱变过程适宜剂量筛选这一关键问题,在研究巴马香猪精液品质特征的基础上,长期监控ENU处理对公猪生殖功能的影响,筛选到了既能诱导可疑表型,又能保证在ENU停药后,公猪生殖功能能基本恢复正常的适宜剂量。
试验首先检测了90头健康成年巴马香猪新鲜精液质量,并探索液冷冻保存的方法。成年巴马香猪射精量、精液活力、密度、畸形率、抗力指数分别为95.31m1,75.33%,0.78x108,5.13%及581,其中,巴马香猪的射精量、精子活力和密度均低于杜洛克和约克夏等商品猪。在巴马香猪精液冷冻研究中,采用正交实验设计,在探讨低密度脂蛋白(LDL)浓度、甘油浓度、海藻糖浓度作用的基础上,进一步结合15℃平衡时间和解冻方法对冷冻解冻后精子活率的影响,进行巴马香猪精液最佳冻存方案的优化。结果表明方案1(A3B4C2D3E1)为最佳方案,其解冻后精子活率为52.26%。为分析稀释液与冷冻液中LDL、甘油和海藻糖不同联用组合的保护效果,在方案1的基础上,共设计了4种组合,以精子运动能力、顶体完整性、质膜完整性以及DNA损伤率等为判定指标,结果表明,9%的LDL、200mM海藻糖与2%的甘油联用时(即组合4)显著优于其他组合的保护效果(P<0.05),并进一步证明方案1是巴马香猪精液冷冻保存的最佳方案。
巴马香猪ENU给药当天各剂量组猪只采食量与对照组相比极显著地降低(P<0.01),然后逐渐(给药后第5-6天)恢复直至正常(与对照组无显著差异,P>0.05)；待第2周(次)、第3周(次)重复给药时,会出现同样的变化趋势。ENU给药期间巴马香猪采食量会受到较大影响,采食量的减少具有浓度依赖性,采食量恢复时间随剂量的增加而延长。给药后各剂量组公猪的生长速度都有一定程度降低。随ENU给药剂量的增加,巴马香猪的死亡率逐渐升高,可育率逐渐降低。结果表明,巴马香猪对85mg/kg及以下剂量可耐受。
巴马香猪ENU给药后12月内射精量呈现先下降后上升的趋势,给药后第12月,65mg/kg及以下剂量组巴马香猪射精量基本恢复正常；75-105mg/kg剂量组射精量均不能恢复到给药前水平,但75mg/kg剂量组公猪射精量恢复情况好于85-105mg/kg剂量组公猪(P<0.05),85-105mg/kg剂量组公猪之间射精量无显著性差异(P<0.05)。巴马香猪精子活力先下降后上升,给药后第12月,45mg/kg和65mg/kg剂量组公猪精子活力能够恢复到给药前水平,75-105mg/kg剂量组公猪均不能恢复到给药前水平,75mg/kg和85mg/kg剂量组公猪精子活力恢复情况好于90-105mg/kg剂量组公猪(P<0.05),90-105mg/kg剂量组公猪之间精子活力无显著性差异(P<0.05)。巴马香猪精液密度也表现为先下降后上升,45mg/kg和65mg/kg剂量组公猪精液密度能够恢复到给药前水平,75-105mg/kg剂量组公猪均不能恢复到给药前水平,其中75mg/kg剂量组公猪精液密度恢复情况好于85mg/kg剂量组(P<0.05),后者精液密度恢复好于90-105mg/kg剂量组公猪(P<0.05)。巴马香猪精子畸形率先上升后下降,45mg/kg和65mg/kg剂量组公猪精子畸形率能够恢复到给药前水平,75-105mg/kg剂量组公猪均不能恢复到给药前水平,其中75mg/kg和85mg/kg剂量组精子畸形率恢复好于90-105mg/kg剂量组公猪(P<0.05)。给药后18月公猪射精量显著高于给药后12月(P<0.05),表明公猪副性腺的精浆分泌功能有所恢复,其他精液指标无显著差异,表明ENU给药后12月公猪精液生成和精子发生的能力恢复情况已经基本稳定。
ENU给药后公猪配种记录表明,45mg/kg和65mg/kg剂量组公猪的与配母猪妊娠率与对照组无显著差异(P>0.05),其他各剂量组均与对照组均有显著差异(P<0.05),并且有随着剂量增大与配母猪妊娠率降低趋势更为明显的现象；断奶仔猪成活率的差异未表现出与给药剂量之间的相关性：各剂量组公猪(F0代)所生产的F1代仔猪可疑表型发生率均与对照组有显著差异(P<0.05),其中45mg/kg和65mg/kg剂量组仔猪可疑表型发生率显著低于105mg/kg剂量组(P<0.05),75-95mg/kg剂量组与105mg/kg剂量组仔猪可疑表型发生率无显著差异(p>0.05)。结合公猪对ENU的耐受性与仔猪可疑异常表型发生率考虑,75-85mg/kg属于巴马香猪ENU诱变的合适剂量。
ENU给药后18个月对巴马香猪生殖器官和副性腺进行组织学检测,结果表明,巴马香猪的睾丸系数、附睾系数和精囊腺系数与对照组相比,除了45mg/kg和65mg/kg剂量组外,85-105mg/kg剂量组巴马香猪附睾系数和精囊腺系数均显著低于对照组,表明生殖器官和副性腺已受到了损伤；随着ENU给药剂量的增加,公猪睾丸内正常曲细精管减少,部分变性萎缩和完全变性萎缩的曲细精管比例上升；曲细精管内各级生精细胞与支持细胞的比例也随着ENU剂量的增加而降低。ENU给药对巴马香猪前列腺无明显影响。透射电镜下给药组可见部分精原细胞核出现空泡、精子核解聚,严重者可见部分精原细胞核解聚,部分精子细胞核膜消失。免疫组化实验结果表明,曲细精管中GDNF蛋白的表达量随着ENU剂量的增大而降低,表明ENU剂量越大,曲细精管损伤程度越大,公猪支持细胞分泌GDNF蛋白的能力越低,精原干细胞自我更新的能力也因此而下降,随之各级生精细胞数量减少,公猪表现为生精障碍和精液品质的降低。随着ENU给药剂量的增加,曲细精管内生精细胞凋亡数量逐渐升高,也是公猪生精能力降低的主要原因之一。
激素水平跟踪检测结果表明,ENU注射前各剂量组公猪血清T和E2的水平无显著差异(P>0.05)；给药后4月,除45mg/kg剂量组公猪血清T和E2水平与对照组无显著差异(P>0.05)外,其他剂量组与对照组均有显著差异(P<0.05),T水平显著低于对照组(P<0.05),而E2水平显著高于于对照组(P<0.05)。表明ENU给药后,随着巴马香猪睾丸细胞的变性,T和E2的分泌均受到了较大影响,而这2种生殖内分泌激素水平的异常既是公猪精液品质变化的标志,也是引起精液品质变化的原因之一。
试验最后对ENU影响巴马香猪精子发生的分子机制进行了研究。睾丸分泌雄性激素和产生精子这两种功能的实现需要在相关基因的调控下完成,其中,StAR基因和P450scc基因主要与睾酮合成相关；GDNF基因、SCF基因和CREM基因主要与精子发生相关；在精子发生过程中,同时也伴随着生精细胞的凋亡,在外源性因素作用下,细胞凋亡的速度会有所变化,在细胞凋亡的线粒体介导途径中,Bcl-2家族中抑制细胞凋亡的Bcl-2基因和促进细胞凋亡的Bax基因的表达水平对细胞的生存与死亡起到决定性的作用。实验结果表明,除105mg/kg剂量组公猪睾丸组织中StAR基因表达量显著低于对照组(P<0.05)外,其他剂量组所受影响不大；45mg/kg和65mg/kg剂量组P450scc基因的表达量与与对照组均无显著差异(P>0.05),75和85mg/kg剂量组的表达量显著低于对照组(P<0.05),90-105mg/kg剂量组公猪睾丸内P450scc基因的表达量显著低于其他各剂量组(P<0.05),提示ENU给药后公猪睾酮水平的下降可能主要与P450scc基因表达的降低有关。GDNF基因和CREM基因的表达情况大致相同,即45mg/kg和65mg/kg剂量组公猪睾丸内GDNF基因的表达量基本未受影响(P>0.05),75-105mg/kg剂量组公猪睾丸内GDNF基因的表达量均显著降低(P<0.05),而且随剂量的增加其表达量降低的幅度越大,表明随着ENU给药剂量的增加,公猪睾丸曲细精管内精原干细胞的自我更新能力逐渐减弱,成熟的精子细胞变少,剂量达到90mg/kg以上时,公猪精原干细胞自我更新及精子成熟会严重受阻。45mg/kg和65mg/kg剂量组公猪睾丸内BAX基因的表达量与对照组无显著差异(P>0.05),75-105mg/kg剂量组BAX基因的表达量显著高于对照组(P<0.05),并且差异随ENU剂量的加大而升高(P<0.05),而各剂量组Bcl-2基因的表达量与对照组均无显著差异(P>0.05),提示除45和65mg/kg剂量组外,公猪睾丸内生精细胞的凋亡程度随剂量的增加而加大,并且当ENU给药剂量增加至90mg/kg以上时,生精细胞凋亡水平较高,生精细胞数量减少,其精液质量有较大水平的降低。
综上所述,本研究可以得到如下结论：
(1)成年巴马香猪正常的射精量、精液活力、密度、畸形率、抗力指数分别为95.31ml,75.33%,0.78x108,5.13%及581；(2)巴马香猪最佳精液冷冻-解冻方法是精液稀释液中添加9%的LDL和200mM海藻糖,冷冻液中添加2%的甘油,降温过程中于15℃平衡3小时,并于37℃下解冻45s；(3)ENU给药后巴马香猪的采食量下降,生长速度变慢,死亡率和不育率随给药剂量的增加而升高,对90mg/kg及以上剂量不耐受；(4)随着ENU给药剂量的增加,公猪血清T水平下降,E2水平上升,公猪睾丸、附睾和精囊腺受损程度加重,巴马香猪的精液质量降低；(5)ENU给药后,公猪睾丸组织P450scc基因、GDNF基因和CREM基因表达下降,BAX基因表达水平上升,公猪睾丸生精能力受损；(6)根据巴马香猪对ENU可耐受性和生精功能变化的综合分析,最佳ENU诱变剂量可初步确定为75-85mg/kg。
N-Ethyl-N-nitrosourea (ENU) is generally accepted as a very strong mutagenic agent. ENU can induce large amount of random mutation phenotypes within a relatively short period, mutant strain animals with special phenotypes may be obtained after further phenotypic screening and genetic tests, and genetic basic tests can be thus carried out on special phenotypes of mutated animals. It has shown excellent prospects in the studies on mice and can be used as a reference method to confirm the functions of genes. ENU mutagenesis can also be used to establish new models for the studies on genetic diseases in human. The management database for ENU mutagenic mice of G1generation has been established now, and genetic resources of mutagenic mice are effectively preserved by using embryo cryopreservation technique, sperm freezing, in vitro fertilization and other techniques.
The experimental animal carriers for ENU mutagenesis are normally mice. The results from the mutagenesis tests on mice indicate that ENU not only can induce single base mutation in DNA, but also is a kind of cytotoxic agent, which can lead to death of large amount of spermatogenous cells in male mice. After ENU is withdrawn, the genital functions can be gradually recovered. The administration dosage in the ENU mutagenesis tests is very important. High dosages may lead to increase in the mutation rate, but the treatments may lead to intolerance, infertility or death in male mice; in contrast, low dosages may decrease the mutation rate, and the results of the tests may be not satisfactory. Since the strains of mice and the administration methods utilized by the researchers are different, the research results are not comparable, and the "proper dosage" of ENU is still not determined. Normally, the dosage of ENU can be evaluated from the three aspects as below:(1) the percentage of infertile male mice;(2) the length of average infertility period;(3) the number of dead male mice within a relatively short period after the genital capacity is recovered. The experimental data for ENU mutagenesis for big experimental animals are still insufficient now. The present study was carried out to examine the effects of ENU on spermatism and spermatogenesis in Bama miniature pigs in order to investigate a proper dosage for ENU mutagenesis of Bama miniature pig.
The present study firstly examined the quality of fresh sperm from90adult Bama miniature pigs and explored the method for sperm cryopreservation. Ejaculation amount, sperm vitality, density, abnormal rate and resistance of adult Bama miniature pigs were95.31ml,75.33%,0.78×108,5.13%and581, among which the ejaculation amount, sperm vitality and density were all lower than Duroc, Yorkshire and other commercial available pigs, indicating that the cryopreservation technique for its sperm still had certain difficulty. Orthogonal experimental design was used for the sperm cryopreservation studies on Bama miniature pigs, and the program for sperm cryopreservation of Bama miniature pigs was optimized on the basis of investigating the functions of LDL concentration, glycerol concentration and trehalose concentration by combining with the effects of equilibrium time at15℃and thawing method on the motility rate after freezing and thawing. The results showed that the program1(A3B4C2D3E1) was the optimal program, and the motility rate after thawing was52.26%. In order to analyze the protective effects of different combinations of LDL, glycerol and trehalose in the dilution and the refrigerating solution, four kinds of combinations were designed, and sperm motility, acrosomal integnity, plasma membrane integrity, DNA injury rate and other parameters were used as the judging parameters. The results indicated that the protective effects for the combination with9%LDL,200mM trehalose and2%glycerol (the combination4) were significantly better than those of other combinations (P<0.05), which further indicated that the program1was the optimal program for sperm cryopreservation of Bama miniature pigs.
During the administration stage after ENU mutagenesis for Bama miniature pigs, the food intake for the pigs in different dosage groups on the day of administration extremely significantly decreased in comparison to that in the control group (P<0.01), afterwards, it gradually recovered (5-6days after administration)(the difference in comparison to that in the control group was not statistically significant, P>0.05); the same tendency may appear after repeated administration on the second and third administration (at the second and third week). The food intake of Bama miniature pigs may be significantly affected during the administration with ENU, the decrease in food intake was dose-dependent, and the recovery time for food intake increased with the increase in the dosage. The growth rates of male pigs in different dosage groups after drug administration showed decreases to different extents. The mortality of Bama miniature pigs gradually increased with the increase in the administration dosage of ENU, and the fertility gradually decreased. The results indicated that Bama miniature pigs can tolerate a dosage no higher than85mg/kg.
The ejaculation amount of Bama miniature pigs within12months after ENU mutagenesis showed a decreasing and subsequently increasing tendency, and the ejaculation amounts of Bama miniature pigs almost recovered in the dosage groups of no higher than65mg/kg at12months after drug administration; the ejaculation amounts in the75-105mg/kg groups could not recover the level before drug administration, but the recovery of ejaculation amount in the75mg/kg group was better than those in the85-105mg/kg groups (P<0.05), and the differences in the ejaculation amounts among the85-105mg/kg groups were not statistically significant (P<0.05). The sperm motility of Bama miniature pigs firstly decreased and then increased, and the sperm motility in the45mg/kg and65mg/kg group at12weeks after administration can recover the level before administration, while the sperm motility in the75-105mg/kg groups can not recover the level before administration, the recovery in sperm motility in the75mg/kg and85mg/kg groups was better than that in the90-105mg/kg groups (P<0.05), and the differences in the sperm motility among the90-105mg/kg groups were not statistically significant (P<0.05). The sperm density of Bama miniature pigs also firstly decreased and then increased, the sperm density in the45mg/kg and65mg/kg group can recover the level before administration, the sperm density in the75-105mg/kg groups can not recover the level before administration, among which the recovery in sperm density in the75mg/kg group was better than that in the85mg/kg group (P<0.05), the recovery in sperm density in the latter group was better than that in the90-105mg/kg groups (P<0.05). The sperm abnormality rate of Bama miniature pigs firstly increased and then decreased, the sperm abnormality rates in the45mg/kg and65mg/kg groups can recover the level before administration, while the sperm abnormality rates in the75-105mg/kg groups can not recover the level before administration, among which the sperm abnormality rates in the75mg/kg and85mg/kg groups were better than those in the90-105mg/kg groups (P<0.05). The ejaculation amount18months after administration was significantly higher than that of12months (P<0.05), indicating that the functions of accessory gonads of the male pigs recovered and no statistically significant difference was detected in other sperm parameters. The recovery in the capabilities of spermatism and spermatogenesis12months after ENU administration had become stable.
The breeding records for the male pigs after ENU mutagenesis indicated that no statistically significant difference was detected in the pregnancy rate of female pigs for breeding in the45mg/kg and65mg/kg groups (P>0.05), and the differences of other dosage groups in comparison to that in the control group were all statistically significant (P<0.05), and it became more evident that the pregnancy rates of the female pigs for breeding decreased with the increase in the dosage; no statistically significant difference was detected in the litter sizes of the male pigs in different dosage groups (P>0.05); the difference in the survival rates of weaned baby pigs did not show any correlation with the administration dosage; the differences in the incidence rates of suspectable phenotypes in the pigs of F1generation reproduced by the male pigs in different dosage groups (FO generation) were all statistically significant in comparison to that in the control group (P<0.05), among which the incidence rates of suspectable phenotypes in the baby pigs in the45mg/kg and 65mg/kg groups were significantly lower than that in the105mg/kg group (P<0.05), and the differences in the incidence rates of suspectable phenotypes in the baby pigs between the75-95mg/kg groups and the105mg/kg group were not statistically significant (P>0.05). Combining the tolerance of the male pigs to ENU and the incidence rate of suspectable phenotypes in the baby pigs,75-85mg/kg were proper dosages for ENU mutagenesis of Bama miniature pigs.
Histological examinations were carried out on the genital organs and the acccessory gonads in the Bama miniature pigs18months after ENU administration. The results showed that the epididymis index and the seminal vesicle index in the85-105mg/kg groups were significantly lower than those of the control group except the45and65mg/kg groups, indicating that the genital organs and the acccessory gonads had been damaged; with the increase in the administration dosage of ENU, the normal contorted seminiferous tubules in the testis of the male pigs decreased, the percentage of contorted seminiferous tubule showing partial and complete degenerative atrophy decreased; the percentages of different grades of spermatogenic cells and supporting cells in the contorted seminiferous tubules also decreased with the increase in ENU dosage. ENU administration had no significant effects on the prostate of Bama miniature pigs. It can be observed under the transmission electron microscope that vacuoles can be detected in some spermatogenous cells and sperm nucleus depolymerization can be detected, and the nuclear membrane of some spermatids disappeared. The results from the follow-up detection on hormonal level showed that the differences in serum T and E2levels in the male pigs in different dosage groups before ENU injection were not statistically significant (P>0.05); statistically significant differences can be detected in serum T and E2levels in the male pigs between the dosage groups and the control group except the45mg/kg group four months after administration (P>0.05), and the T level was significantly lower than that in the control group (P<0.05), while the E2level was significantly higher than that in the control group (P<0.05), indicating that T and E2secretion was significantly affected with the degeneration of testicular cells of Bama miniature pigs. The decrease in T level and the increase in E2level are not only the symbol for the changes in sperm quality of male animals, but also the reason inducing the changes in sperm quality.
Testicles secrete male hormones and produce sperms, and these two functions should be accomplished under the regulations of related genes. Among which, StAR and P450scc are mainly related to testosterone synthesis, while GDNF, SCF and CREM are mainly related to spermatogenesis. Apoptosis of spermatogenic cells is also concurrent with spermatogenesis, and the rate of cell apoptosis may change under the effects of exogenous factors. Bax and Bcl-2are representatives for the genes promoting and inhibiting apoptosis respectively. The experimental results indicated that except that the expression level of StAR in the testicular tissues of the male pigs in the105mg/kg group was significantly lower than that in the control group (P<0.05), other dosage groups were not significantly affected; no statistically significant difference was detected in the expression level of P450scc in the45and65mg/kg groups and the control group (P>0.05), the expression levels in the75and85mg/kg groups were significantly lower than that in the control group (P<0.05), and the expression levels of P450scc in the testicles of the male pigs in the90-105mg/kg groups were significantly lower than those in other dosage groups (P<0.05, Figure6-2). The expression levels of GDNF in the testicles of the45and65mg/kg groups were not affected (P>0.05), the expression levels of GDNF and CREM were almost the same, in other words, the expression levels of GDNF in the testicles of the male pigs in the75-105mg/kg groups significantly decreased (P<0.05), while the decreasing amplitude in the expression level increased with the increase in the dosage, indicating that the self-renewal capability of stem spermatogonium in the contorted seminiferous tubules in the testicles of the male pigs gradually decreased and the mature sperm cells decreased. When the dosage reached90mg/kg or even higher, self-renewal of stem spermatogonium and sperm maturation of the male pigs were seriously inhibited. The differences in the expression level of BAX in the testicles of the male pigs in the45and65mg/kg groups in comparison to the control group were not statistically significant (P>0.05). the expression levels of BAX in the75-105mg/kg groups were significantly higher than that in the control group (P<0.05). and the differences increased with the increase in ENU dosage (P<0.05), while the differences in the expression levels of Bcl-2in different dosage groups in comparison to the control group were not statistically significant, indicating that the apoptosis of spermatogenic cells in the testicles of the male pigs increased with the increase in the dosage except the45and65mg/kg groups, and the apoptosis of spermatogenic cells was maintained at a relatively high level when the administration dosage of ENU increased to a dosage higher than90mg/kg, spermatogenic cells decreased and the sperm quality significantly decreased.
In general, the conclusions as followed can be drawn from the present study:
(1) Normal ejaculation amount, sperm vitality, density, abnormality rate and resistance of adult Bama miniature pigs were95.31ml.75.33%,0.78×108,5.13%and581respectively;
(2) The optimal method for semen freezing of Bama miniature pigs was the program1, in other words, supplement with9%LDL,200mM trehalose and2%glycerol. equilibrium at15℃for3h and thawing at37℃for45s;
(3) The food intake of Bama miniature pig decreased after administration with ENU, the growth rate decreased, the death rate and the infertility increased with the increase in the administration dosage, and they can not tolerate a dosage no lower than90mg/kg;
(4) With the increase in the administration dosage of ENU, the sperm quality of Bama miniature pigs gradually decreased, the damages in testicles, epididymis and seminal vesicle of male pigs were gradually aggravated, the amplitudes in the decrease in serum T level and the increase in serum E2level of the male pigs both increased, the expression level of GDNF in the contorted seminiferous tubules gradually decreased, the apoptotic spermatogenic cells gradually increased;
(5) the decrease in serum testosterone level in the male pigs after administration with ENU was mainly related to the decrease in the expression level of P450scc, the decreases in the expression levels of GDNF and CREM led to the decrease in the self-renewal capability of stem spermatogonium and the blockage of sperm maturation in the male pigs, the increase in the expression level of BAX led to the increase in the apoptotic spermatogenic cells, the decrease in the number of spermatogenic cells and the damages in spermatogenic capability;
(6) According to the comprehensive analysis on the tolerance of Bama miniature pigs to ENU and the changes in spermatogenic functions, and the optimal dosage for ENU mutagenesis can be preliminarily determined as75-85mg/kg.
引文
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