摘要
流式细胞高速分选术是被生产的后代证实有效的性别控制技术,可以获得高纯度的X、Y精子。该方法基于X和Y精子染色体DNA含量差别,采用活细胞荧光染料与精子DNA结合,分选获得活精子,随后用于子宫内人工授精或性控胚胎移植。分选冷冻精液低剂量人工授精在牛已被证明是一种有效的手段,能够生产表观正常的后代。而对其他物种、甚至人类,研究改进其精液分选和授精将促进该技术的推广应用,流式细胞分选的X和Y精子同时也是寻找其他性别控制方法的良好研究材料。本研究旨在将精子专用高速的流式细胞分选技术用于梅花鹿、马鹿和山羊,初步建立了梅花鹿、马鹿和山羊分选精液的低剂量授精技术体系和进一步对高纯度分选奶牛精液采用扣除杂交筛选,初步获得牛XY精子间的差异表达基因。获得如下结果:
1.对16头种公马鹿电刺激采精,筛选比对天鹿3号稀释液的冷冻方法和甘油浓度,经冷冻处理,液氮保存,对冷冻精液品质和受胎效果进行评定。结果表明:马鹿对电刺激采精有耐受性,麻醉保定的采精频率每周2次最佳。甘油浓度6%的天鹿3号稀释液冷冻效果最优,细管冻精活率48.14±13.15%,顶体完整率为56.34±14.84%,126头诱导同期发情和107头自然发情马鹿人工授精的情期产羔率分别达到50.80%和78.50%。
2.对电刺激或假阴道采集的马鹿、梅花鹿和山羊精液运输后流式分选、冷冻和低剂量授精。(1)马鹿X、Y精子纯度分别为93. 1%和95. 2%,解冻活率为46±5%和43±5%与常规对照精子活率43±6%没有明显差异,采用分选的106个有效精子对同期化发情的母鹿通过直肠把握授精生产15头性控后代,两性后代性别符合率均为100%,初生重和85d重及妊娠期与对照组比较均没有显著差异。(2)梅花鹿XY精子纯度分别为91.2%和94.1%,活率为45±4%和43±3%与常规对照精子活率43±4%没有明显差异,采用分选的106个有效精子对同期化发情的母花鹿通过腹腔镜授精生产20头性控后代,雌性后代性别符合率100%(5/5),雄性为92.9%(14/15)。初生重和85d重及妊娠期与对照组比较均没有显著差异。(3)萨能奶山羊XY精子纯度分别为94.4%和96. 2%,解冻活率为43±4%和42±4%,通过腹腔镜用分选的106个有效Y精子对同期化发情的母羊优势卵泡侧子宫角输精,生产出4头雄性后代,性别符合率为100%。初生重和70d重及妊娠期与对照组比较均没有显著差异。采用分选的低剂量106个有效精子通过腹腔镜或直肠把握子宫角授精可以生产健康的马鹿、梅花鹿和山羊的预知性别后代。
3.流式细胞分选的冷冻马鹿精子对超数排卵供体采用直肠把握方法单次子宫体输精(对照组108有效精子,n=6;未分选组107有效精子,n=6;Y精子组107有效精子,n=6),成功生产出可供移植的性控雄性胚胎25枚;建立了一种有效的基于牙釉质基因DNA序列片段巢式扩增分析的胚胎性别鉴定的方法,对15枚常规马鹿胚胎样本(未分选精液授精)性别鉴定为8M:7F,4枚性控胚胎(分选Y精液授精)全部为雄性;移植后常规胚胎组受胎率(47.6%,20/42)和雄性胚胎组(42.9%,8/21)的差异不显著,雄性胚胎组的8头后代全部为雄性,明显偏离常规胚胎组11M:9F的后代性别比。
4.采用Trizol结合E.Z.N.A.TM Total RNA kitⅡ离心层析柱方法对上游纯化的奶牛细管冷冻精子总RNA在2h内提取得到总RNA,OD260:OD280比值1.8,产量0.92μg/107精子,纯化精子总RNA的23SrRNA、Lep和Sry基因片段的RT-PCR产物电泳均获得目的条带且没有污染体细胞18S rRNA,可以用于后续的精子基因表达方面的研究。
5.用流式细胞术分离的高纯度牛X精子和Y精子作为材料,在固相支持物磁珠上进行Y精子对X精子扣除杂交以富集性别差异表达基因,对扣除得到的Y-X差异mRNA采用Super SMART技术扩增,构建Y-X扣除杂交cDNA质粒库;克隆菌落PCR鉴定后,对27个有插入片段的克隆测序,BLAST分析均可以搜索到匹配的牛基因组序列,在线分析测得序列的同源性,结果比对上高度同源的4个EST序列,其中1个为牛已知Sry基因,3个为牛未知基因。
The Beltsville sperm sexing technology is currently the only effective means of altering the sex ratio of offspring in livestock. The method is based on the flow-cytometric separation of X- and Y-chromosome-bearing sperm based on X/Y DNA content difference. The method involves treating sperm with a DNA-binding fluorochrome, Hoechst 33342, and flow-cytometrically sorting them into separate X and Y populations that can subsequently be used for intrauterine insemination, deep-uterine insemination or producing sexed embryos for transfer. Insemination of lower sperm numbers in cattle has proven to be an effective means of utilizing the sexing technology. Offspring produced at bovine using the technology have been morphologically normal and reproductively capable in succeeding generations. Improvements in the technology will no doubt lead to much greater usage of sexed sperm for applications such as endangered species, laboratory animals, hobby or pet species, even human beings. Flow-sorted X- and Y-sperm are also the good research material to study other methods for sex control. Thus semen from wapiti, sika, goat and bovine were collected and separated into X- and Y- chromosome-bearing sperm after analysis and reanalysis using a Cytomation MoFlo high-speed cell sorter modified for sperm, and then inseminated or produced sexed-embryos for transfer. Further research in screening low abundances of differentially expressed genes in X or Y sperm by subtractive hybridization with high-purity bovine X and Y sperm sorted by flow cytometry were studied. The main results were showed as following:
1. Sixteen wapiti stags were electroejaculated and their semen were cryopreservated. Semen evaluation and their fertility were assessed and recorded. TIANLU 3 diluent for stag was screened of the freezing method and final concentration of glycerol. The results of the experiment show that the best frequency of electroejaculation was 2 times each week for wapiti stag. The voltage had risen year by year; the electro ejaculation patience of wapiti stags had been proved. It was excellent for TIANLU 3 diluent contained 6% glycerol to crypreserve wapiti sperm. The post-thaw motility reached up to 48.14±13.15%. Acrosome integrity reached up to 56.34±14.84%. 126 hinds (estrus synchronization) and 107 hinds (normal estrus) had been applied insemination with this frozen thawed semen, and the calving rate reached up to 50.80% and 78.50%, respectively.
2. Semen was collected by electro-ejaculation or artificial vagina from wapiti stags, sika stags and goat, and transported to the laboratory and separated into X- and Y- chromosome-bearing sperm after analysis and reanalysis using a modified high-speed cell sorter for sperm. Wapiti hinds, sika hinds and nanny goats induced estrus synchronization with intra-vaginal progesterone-impregnated CIDR devices and administrations of PMSG were intra-uterine inseminated by rectum manipulation or laparoscopy with a low numbers (106 motile sperm) of sex-sorted cryopreserved sperm. (1) Wapiti: no significant differences in the post-thaw motility of control (43±6%), X-sorted (46±5%) and Y-sorted (43±5%) samples were recorded, sex ratio of sperm were 93.1% and 95.2%, respectively. Ultimately 7 out of the 7 calves produced by wapiti hinds inseminated with Y-sorted sperm were male (100%) and 8/8 calves or 100% of the calves from hinds inseminated with X-sorted sperm were female. The birth body weight, 85d body weight and gestation period for male calves were not significantly lower than those of the control, respectively. (2) Sika: no significant differences in the post-thaw motility of control (43±4%), X-sorted (45±4%) and Y-sorted (43±3%) samples were recorded, sex ratio of sperm were 91.2% and 94.1%, respectively. Ultimately 14 out of the 15 calves produced by Sika hinds inseminated with Y-sorted sperm were male (92.9%) and 5/5 calves or 100% of the calves from Sika hinds inseminated with X-sorted sperm were female. The birth body weight, 85d body weight and gestation period for male calves were not significantly lower than those of the control, respectively. (3) Goat: post thaw motility of sorted frozen X- and Y-chromosome-bearing sperm were 43±4% and 42±4%, sex ratio of sperm were 94.4% and 96.2%, respectively. The birthrate after insemination with sorted frozen thawed Y-sperm was significantly lower (25%, 4/16) than that of the control (90%, 9/10), and 4 kids were all male. The birth body weight, 70d body weight and gestation period for male kits were not significantly lower than those of the control, respectively. Normal calves or healthy kids of the predicted sex can be produced after intra-uterine insemination conducted by laparoscopy or rectum manipulation with low numbers of sex-sorted cryopreserved sperm in wapiti, sika and goat.
3. Embryo was produced in superovulated wapiti hinds induced by eight declining doses of FSH starting on days 9-12 of the estrus cycle and inseminated with 1×10~7 unsorted (unsorted, n=6) or 1×10~7 Y-sorted frozen-thawed (Y, n=6) and 1×10~8non-sorted frozen-thawed (a commercial dose control, n=6) semen via a single time rectum manipulation. Twenty-five embryos of predicted sex were produced. Sex identification of samples from wapiti based PCR amplification using the bovine amelogenin gene primers was developed. The sex ratio of the embryos from hinds inseminated with Y sperm (4M:0F) was significantly (P<0.05) deviated for the 53.3% (male, 8/15) and 46.7% (female, 7/15) in the non-sexed group. No significant differences in the birthrate of control (non-sexed embryo) and sexed embryo groups were recorded.The sex ratio of the offspring from sexed embryo (8M:0F) was significantly (P<0.05) deviated for the 55% (male, 11/20) and 45% (female, 9/20) in the non-sexed embryo group.
4. The laminar analysis column method and Trizol single-step method were combined for RNA extraction from swimming-up bovine frozen-thaw sperm. The bovine sperm swimming-up from frozen straws were not contaminated by somatic cells, and high-quality total RNA was obtained in about 2 hours. The value of OD260/OD280 was 1.8, and the≥200bp total RNA extracted from bovine sperm yielded 0.92μg/107sperm.Following RT-PCR, clear bands of SRY, LEP, and 23S rRNA of sperm cDNA were shown in a garose gel electrophoresis respectively, not a band of 18S rRNA.The purity and integrality of total RNA isolated from the bovine sperm of frozen straw with the method of combined the laminar analysis column with Trizol single-step were significantly satisfactory for the demands of molecular biological experiments.
5. The low abundances of differentially expressed genes in X or Y sperm were screened by subtractive hybridization with high-purity bovine X and Y sperm sorted by flow cytometry. Twenty-seven clones isolated from Y-X subtracted cDNA library which had different insert fragments were sequenced. All sequences from 27 clones can be found matching the cow genome sequence following searching the Bos taruus genomic BLAST databases. There exist 4 bovine ESTs in Y-X subtracted cDNA library. One of them is known in bovine, which is the sequence of sry gene, and three are unknown in bovine.
引文
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