创建转基因犬的相关基础研究
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摘要
2005年底完成的犬全基因组图谱,揭示了其基因与人类的相似程度高于人与小鼠等多数动物,同时发现犬有360多种遗传疾病与人类相似。因此,在生物医药研究中,犬(Canis familiaris)是最重要的实验动物之一,转基因犬或基因敲除犬是研究人类基因功能、遗传性疾病的理想模型动物。虽然人类已成功获得转基因小鼠、大鼠、豚鼠、地鼠、牛、羊、猪、兔等十多种哺乳动物动物或家畜,越来越多的转基因动物已运用到生物医药领域,但由于犬生殖器官结构和生殖生理的特殊性,其基础生物学、生殖的人工调控研究进展非常缓慢。但到目前为止,人类尚未获得转基因犬。本文以Beagle(毕格)犬为研究载体,分别以雌性动物和雄性动物为对象,开展创建转基因犬的相关基础研究。研究分为以下三个部分。
1 Beagle母犬的生殖生物学研究
传统的转基因动物研究是以雌性动物为对象,拟创建转基因犬的首选技术路线也是如此。为了给该途径的实验提供可供参考的基础资料,本部分对Beagle母犬的基础生殖生物学进行研究。对扬州和苏州两个Beagle犬养殖基地、共463只·胎次、2-5胎母犬连续3年的观察和资料分析,发现母犬一年四季均有发情和产仔,但呈一定的季节分布,春秋两季为发情旺季,两地分别占全年发情总数的71.0%和72.8%,发情的平均间隔时间为34周,平均妊娠期为62.9天,平均胎产仔数5.35只。扬州和苏州两地母犬的上述繁殖性能未见明显差异。
母犬生殖器官大体解剖显示,卵巢被卵巢囊包裹,弯曲而短小的输卵管行走于卵巢囊壁内。卵泡随着发育而凸至卵巢表面,发情期卵巢为瓣状结构。组织学研究显示:初生时,卵巢的皮质部含有大量的原始卵泡,3月龄出现初级卵泡,6月龄出现次级卵泡,9月龄出现成熟卵泡。不同月龄的卵巢均可见多卵卵泡,其数量随年龄增长呈下降趋势。在初生时,子宫腺体的体积小、数量少,以后不断发育增生,腺体周围的基质也逐渐密集。初生至9月龄的母犬输卵管粘膜仅形成初级皱襞,12月龄后开始出现较多次级皱襞,几乎充满整个管腔。
卵巢的类固醇激素—雌二醇(E_2)和孕酮(P)是雌性动物最重要的生殖激素,其不仅调控着动物性器官发育、成熟、性行为产生、妊娠、分娩等生理活动,也是检测、观察动物生殖行为的重要指标。放射免疫分析法测定初生至性成熟前及其发情前期至分娩,Beagle母犬的卵巢类固醇激素分泌的动态变化,结果显示:血清中E_2的浓度在初生时很低,3月龄开始逐渐升高,9月龄后一直维持于20 pg/ml左右;初生时检不出血清中P,以后虽然有所升高,但一直维持于较低水平(＜0.6ng/ml)。性成熟后的经产母犬,在乏情期,E_2和P均维持在较低的水平。进入发情前期后,E_2浓度逐渐升高,至接受交配前的第4天出现第一个分泌高峰,浓度达75pg/ml,并维持数天,以后有所下降;直至交配受孕后的第18天,由于黄体的分泌作用,出现第二个高峰;在妊娠的最后阶段降至发情前期开始之前的水平。发情前期开始后,P浓度缓慢升高;发情前期的晚期迅速升高,从排卵配种前的第4天至排卵配种日,P从2.7ng/ml升高到10.9ng/ml;以后P不断升高,至妊娠14天达到39.4 ng/ml的峰值,并以较高浓度维持一段时间;在妊娠后期至分娩,其逐渐下降,分娩时孕酮的浓度已降至1ng/ml以下。
雌性动物性器官的发育与生殖活动,除与外周血中的性激素水平有关,还与生殖器官、腺垂体中雌激素受体(ER)和孕酮受体(PR)的数量有关。非同位素原位杂交(ISH)法检测犬生殖器官、垂体中雌激素受体α(ERα)mRNA和孕酮受体(PR)mRNA的相对表达丰度,结果显示:ERαmRNA阳性斑点主要出现在各级卵泡的颗粒细胞和内膜细胞、子宫腺体细胞和垂体的腺体细胞,卵巢中大卵泡的阳性率明显高于小卵泡,即卵泡的发育程度与ERmRNA表达丰度存在密切关系。卵巢在3月龄开始表达ERαmRNA,以后逐渐增加,至9月龄后维持在较高的水平;发情前期和发情期卵巢的ERαmRNA表达水平高于其它时期。子宫ERαmRNA的阳性率,从初生至15月龄,随内膜腺体细胞的数量和体积同步增加,阳性率随日龄增长而增加;由发情前期过渡到发情期后,ERαmRNA表达呈下降趋势。垂体腺体细胞的ERαmRNA在3月龄后一直维持在较高的水平;在发情周期,发情前期的晚期开始,ERαmRNA表达呈下降趋势。PRmRNA主要原位杂交阳性信号集中于各级卵泡的颗粒细胞、有腔卵泡与排卵前卵泡的内膜细胞和黄体细胞中、子宫腺体细胞和脑垂体的腺体细胞。初生未见卵巢PRmRNA的表达,3～18月龄之间的阳性率呈增长趋势;PRmRNA表达水平在发情前期较高,第9天达到较高水平。12月龄前的母犬子宫,几乎不表达PRmRNA;在发情前期开始后,PRmRNA表达呈上升趋势。初生时,母犬脑垂体PRmRNA几乎不表达,随着月龄的增加,PRmRNA在3月龄和6月龄表达水平较高,后表达水平下降。在发情周期PRmRNA一直维持在较低的水平。
以上犬基础生殖生物学研究结果,不仅丰富了犬生殖生物学资料,更重要的是为犬诱导发情、同步发情、超数排卵研究和动物实验安排提供了理论依据。
2以雌性动物为对象创建转基因犬相关辅助生殖技术研究
以雌性动物为对象创建转基因犬研究,首先要有大量的原核期受精卵用于胚胎操作。但犬为单发情动物,有较长的乏情期,人工诱导发情困难;在解剖上,犬的卵巢被厚厚的卵巢囊包裹,很难用腹腔镜技术从卵巢取卵。利用淘汰犬卵巢取卵,体外培养成熟、体外受精是一项重要的辅助生殖技术(ART)。我们以切割法采集卵巢上未成熟卵母细胞,建立了犬卵母细胞体外成熟培养的方法,获得了成熟的卵母细胞。结果显示:发情前期和发情期母犬卵巢的一级卵母细胞回收率较高,3岁犬平均回收的数量最多,而1岁以下幼犬和7岁以上老犬的回收率最低。在以M199作为基础培养液中,联合添加内皮生长因子(EGF)和雌二醇(E_2),可促进犬卵母细胞成熟,以发育至MII期的卵母细胞比例为指标,其成熟率高于单独添加组和对照组(22.58%vs 18.00%、5.56%,4.76%)。添加20%的FBS,其卵成熟率与添加20%的EDS无显著差异(17.14%vs 17.95%),但均显著高于对照组(0%)。EGF虽可促进卵丘细胞的扩展,但对卵母细胞的成熟无剂量依赖性。卵母细胞体外成熟培养的时间以72h为优。
人工诱导发情是又一项重要的辅助生殖技术。本研究以肌肉注射促性腺激素的方法对乏情期犬、青年不发情犬和老龄不育犬进行人工诱导发情实验。结果显示:外源激素1200IU PMSG+500IU HCG能有效地诱导乏情期犬和青年不发情犬的发情,而对老龄不育犬的诱导效果不明显。同时发现在PMSG总剂量(1200IU)不变的情况下,一次给药的总诱导发情、成功配种率为40%(6/15),其中乏情期犬的成功配种率为50%(4/8);少量多次给药诱导发情的总成功率为50%(15/30),其中乏情期犬的成功配种率为69%(11/16),并有31%(5/16)的母犬妊娠产仔,产仔母犬表现出与自然繁殖母犬同样的激素变化规律。提示PMSG+HCG组合、少量多次给药的方法可有效诱导乏情期经产母犬的发情,缩短发情间隔时间,为转基因犬制作提供更多的发情母犬。
胚胎移植是以雌性动物路线创建转基因犬的关键辅助生殖技术。我们以自然发情同步母犬配对供体和受体的策略,采取外科手术的方法开展犬受精卵输卵管移植的研究。并首次获得成活仔犬。在移入49枚1-8细胞受精卵的12只受体中,有3只母犬于卵母细胞受精(供体初次配种)后的第61天～65天成功分娩,产仔6只,分娩率为25%(3/12)。
3以雄性动物为对象创建转基因犬的部分基础研究
在以雌性动物为对象创建转基因犬的努力屡屡遭受挫折后,本课题又对以雄性动物为对象创建转基因犬的技术进行了初步探索。该转基因策略可分为精原干细胞的体内基因转移与体外基因转移,后者又包括精原干细胞的分离培养、受体内源性精原干细胞的消除、转基因精原干细胞的移植重建、人工采精及其阳性精子筛选、体外显微受精等主要环节。本文对体外途径的若干程序作了探索性研究。在精原干细胞的体外培养技术中,以外科手术摘取4-5月龄Beagle公犬睾丸,体外酶消化睾丸组织制作细胞悬液,以贴壁差异法纯化分离精原干细胞(SSCs),DMEM为基础培养基体外培养纯化的精原干细胞,并在体外对培养的SSCs进行EGFP基因转染。结果显示:4-5月龄的Beagle公犬可获得较多的SSCs;0.2mg/ml胶原酶Ⅳ+0.25%胰蛋白酶两步酶消化法的分离效果最好,每100mg睾丸组织分离的SCCs总数和成活细胞比例分别为6.67×10~6和95.5%;经AKP染色法鉴定,以贴壁差异法纯化SSCs,其纯度达50.7%;在DMEM基础培养液中添加10%胎牛血清(FBS)和20ng/ml EGF显著提高了SCCs集落数。SCCs体外转基因实验显示,EGFP基因可以在SCCs上表达,其中阳离子聚合物转染法的表达效率明显优于磷酸钙转染法(22.1%vs 8.1%)。除精原干细胞的体外培养与基因转移技术外,其他以雄性动物为对象创建转基因犬的相关辅助生殖技术正在研究中。
Canine is one of the most important laboratory animals in the biomedicine research. Canine entire genome atlas was completed at the end of 2005 year. Compared with other animals, such as mouse, canine gene was much more similar to human gene. About 360 kinds of canine genetic diseases are similar to human's. Therefore, the gene knock-in or gene knock-out dog was one of the ideal animal models to study human gene functions and the genetic diseases. People had succeeded in creating more than 10 of kinds transgenic animals such as mouse, rat, cavy, hamster, cattle, sheep, pig, rabbit and so on. More and more transgenic animals had been applied to the biological science research. Progress is slow on canine reproductive biology because of the specificity of its reproductive organs and reproductive physiology. The transgenic dog still hasn't been reported by now. This dissertation demonstrated the basic research on creating the transgenic dog. This paper is divided into three parts.
1. Study on the reproductive biology of bitches
We investigated 463 Beagle bitches/parturitions for 3 years, which were bred in Yangzhou and Suzhou respectively. We discovered that the bitches could be in estrous at all the year round, but especially in spring and autumn. The bitches were monoestrous. The average interval time of oestrus was 34 weeks. But the percent of the estrous bitches had certain differences in each season. The oestrus rate of the spring and autumn was 71% and 72.8% respectively on the two farms, which indicated that the bitch wasn't the classic seasonal oestrus animal. The gestation period was 62.9 days on average, mostly between 61～64 days. Each bitche gave birth to 5.35 puppies on average each time, mostly between 3～7 puppies and sometimes up to 10 puppies. There was no significant difference in the reproductive performance between the two farms.
By dissecting the bitches' generative organs, we could see that the ovary was packaged by the bursa ovarica. The bent and short oviduct was in the bursa ovarica's parietes. Along with the follicle's growth and development, the ovary's surface showed a petal structure. The histological research on the ovary of postnatal bitches demonstrated as following: Ovarian cortex sections of newborn bitches had large numbers of primordial follicles. With the age increasing, the primary follicles appeared in 3 mounth-old, the secondary follicles appeared in 6 month-old and the mature follicle appeared in 9 month-old. The endometrial gland developed and proliferated continuously after newborn, then gradually developed. At the same time, the stroma became denser. The oviduct had formed primary mucosal folds from newborn to 9 month-old, lots of secondary mucosal folds emerged after 12 months.
The dynamic change of estradiol (E_2) and progesterone (P) in bitches' serum was tested by radioimmunoassay (RIA) to bitches in puberty and estrous cycle. The results as following: The E_2 concentration was extremely low at birth, then ascended gradually in 3 month-old and maintained constantly about 20pg/ml after 9 month-old. The P concentration was zero in the newborn, but later increased and stayed at a lower level ( < 0.6 ng/ml). The concentration of E_2 and P both maintained at a lower level during the anestrus. At the onset of proestrus, E_2 increased gradually, then reached the first peak (75pg/ml) in the 4th day before mating, but later decreased to some extent. Until the 18th days after pregnancy, the second peak appeared due to the secretion of corepus luteum. P rised slowly in the early stage of proestrus, but rapidly in the later stage. It increased from 2.7ng/ml of the 4th day before the mating to 10.9ng/ml in the mating, later increased continually. The 14th day after the pregnancy, it got to the peak 39.4 ng/ml and later maintained for a period of time. P declined gradually from the end stage of pregnancy to the parturition, and it reach 1ng/ml below in the parturition.
The method of in situ hybridization (ISH) was first applied to detect relative expression abundance of estrogen receptor a (ERα) mRNA and progesterone receptor (PR) mRNA both the reproductive organ and the pituitary gland of the bitches. This study explained their relativities between sex hormonal concentration in serum, morphopoiesis of reproductive organ and expression abundance of ERa and PR mRNA. It laid the foundation to further explore the relationship between the reproductive capacity and the difference in the level of ER and PR gene transcription and translation. Results of image manipulation analysis system indicated that ER mRNA almost are not detected in the ovary at birth. ER mRNA expression increased from 3rd to 9th month-old, and then declined slightly. Postive staining was mainly detected in the granule cells of the various follicles, thicker in big follicles than in small follicles. Ovary expression of ERamRNA remained high during the proestrous and estrous . In uterus, ERamRNA was mainly detected in endometrium gland and lesser in the stroma. In estrus cycle, ERamRNA expression showed a downward trend. ERamRNA was detected mainly in the pituitary gland cells. ERamRNA were almost not detected at birth. With the increase of age, ERamRNA had been maintained at a higher level. In the estrous cycle, ERamRNA expression showed a downward trend in the pituitary.
Above results of canine reproductive biology is help for improving canine fertility and arranging the experimentates reasonably. What's more, they have provided the theory evidence of inducing estrus, superovulation of dogs and so on.
2. Related study on the assisted reproductive technology for creating a transgenic dog with the female
The research was based on the feminity to creating a transgenic dog. First, lots of zygotes were needed. Actually the artificial inducing estrus of bitch was extremely difficult now, due to ovaries were packaged by bursa ovarica ,and oocytes' recovery was unable by laparoscope. In vitro maturation and artificial inducing estrus were crucial assisted reproductive technologies for canine, which was the important way to create transgenic animals. But at present, the canine ART cann't satisfy the need of the transgenic dog production. This study was concern with in vitro oocytes maturation, inducing estrus and embryo transfer in oviduct of bitch. The embryo transfer in oviduct technology established a platform for generating transgenic animal.
Massive procaryon zygotes were required in embryo operatation. We sliced ovaries to release immature oocytes. We had established the oocyte culture system. We examined the number of good oocytes collected from bitches ovaries at diffenent ages and various stages of the estrous cycle by slicing the ovaries, then cultured them to do comparative experiment in vitro with the different culture medium additive, such as serum, EGF and E_2, which could promote the oocytes maturation. The standard of oocytes maturation was achieving MII by using hoechest 33342 staining technique. Results demonstrated that, the recover-ratio of COCs in proestrus and oestrus was higher than other periods. The average returning quantity from 3-year-old bitches was the highest, but <1 and >7-year-old bitches was the lowest. The nuclear maturation rates of oocytes in the culture medium supplmented with EGF and E_2 were higher than that supplemented with EGF or E_2 and the control (22.58% vs 18.00%, 5.56%; 22.58% vs 4.76%). There's no difference in the maturation rates of oocytes between 20% FBS and 20% EDS trial (17.14% vs 17.95%), which were higher than the control (0%). EGF had effect on cumulus expansion, but the rate of oocytes was not depend on the EGF dose. It showed that 72h was suitable for IVM of canine oocytes. The cumulus cells had important effect on oocytes maturation, as the oocytes with cumulus had higher maturation rates than oocytes without cumulus (15.56% vs 3.13%).
We carried out experiments of inducing estrus of anoestrum, young of nonestrus and sterile agedness bitches by i.m. PMSG + hCG. The results as following: extraneous hormone (1200IU PMSG + 500IU hCG) could induce effectively the anoestrus and young of nonestrus bitches to estrous, but useless for the sterile agedness dogs. In an invariable dose PMSG 1200IU, the inducing estrus rate was 40% (6/15) by given medicine once, but that was 50% (4/8) in the anoestrus group. That was 50% (15/30) by given medicine several times but a little ounce each time, the inducing estrous rate was 69% (11/16). Furthermore, The deliver puppies rate of bitches was 31% (5/16) . These bitches' hormone variation rule was the same as generally breeding bitches'. The results showed that this way was feasible to supply with more estrous bitches.
Embryo transfer was also one of the assisted reproductive technologies. We transfer zygotes of 1-8 cells stage into 12 recipient's oviducts. There were 3 receivers giving birth to 6 puppies at the 61th-65th day after the donor's oocytes fertilization, the successful rate was 25%(3/12). it was the first to obtain living puppies in this way.
3. Some fundamental research to create a transgenic dog based on male
Creating the transgenic animals is mediated by the Spermatogonial stem cell (SSCs), including transgenosis in vivo and in vitro. The latter included SSCs culture in vitro, eliminating endogenous SSCs, SSCs transfer and so on. Testes were collected by surgery operation from 4-5 month-old Beagle dogs. The SSCs were isolated by enzymatic combinations. According to the differentiation of velocity of cells sticking to the wall in vitro, SSCs were purified and then cultivated with DMEM containing different concentration of serum or EGF respectively. By counting the clusters every day, the growth efficiency of SSCs can be readily detected within 1 week in a semi-quantitative manner. The SSCs were transfected with pEGFP-N1 plasmids by the cationic polymer transfection reagen and calcium phosphate transfection reagen. The results as following: collagenase IV+ trypin combination was an effective method to dissociate canine SSCs, the total number of dissociated cells was 6.67×10~6/100mg testis parenchyma and rate of living SSCs was 96.5%. It Is necessary to add FBS to the culture medium for SSCs culture in vitro . Compared with EGF group, the clusters of cell in the 20ng/ml group increased markedly. The gene transfection efficiency of the cationic polymer transfection reagen method was much higher than that of calcium phosphate method (22.1% vs 8.1%) .In conclusion, we had developed an efficient and economical method to isolate the SSCs, through which we could obtain enough viable germ cells. The technique of SSCs culture demonstrated that dog SSCs could be maintained in culture medium in vitro for approximately 1 week. The clusteres came from a single cell indicated that SSCs are able to achieve growth and proliferation in the culture system in vitro. We have succeeded in the EGFP gene transfection in vitro, which was prepared for the further SSCs transplantation technique to get the transgenic puppies.
1 Beagle母犬的生殖生物学研究
传统的转基因动物研究是以雌性动物为对象,拟创建转基因犬的首选技术路线也是如此。为了给该途径的实验提供可供参考的基础资料,本部分对Beagle母犬的基础生殖生物学进行研究。对扬州和苏州两个Beagle犬养殖基地、共463只·胎次、2-5胎母犬连续3年的观察和资料分析,发现母犬一年四季均有发情和产仔,但呈一定的季节分布,春秋两季为发情旺季,两地分别占全年发情总数的71.0%和72.8%,发情的平均间隔时间为34周,平均妊娠期为62.9天,平均胎产仔数5.35只。扬州和苏州两地母犬的上述繁殖性能未见明显差异。
母犬生殖器官大体解剖显示,卵巢被卵巢囊包裹,弯曲而短小的输卵管行走于卵巢囊壁内。卵泡随着发育而凸至卵巢表面,发情期卵巢为瓣状结构。组织学研究显示:初生时,卵巢的皮质部含有大量的原始卵泡,3月龄出现初级卵泡,6月龄出现次级卵泡,9月龄出现成熟卵泡。不同月龄的卵巢均可见多卵卵泡,其数量随年龄增长呈下降趋势。在初生时,子宫腺体的体积小、数量少,以后不断发育增生,腺体周围的基质也逐渐密集。初生至9月龄的母犬输卵管粘膜仅形成初级皱襞,12月龄后开始出现较多次级皱襞,几乎充满整个管腔。
卵巢的类固醇激素—雌二醇(E_2)和孕酮(P)是雌性动物最重要的生殖激素,其不仅调控着动物性器官发育、成熟、性行为产生、妊娠、分娩等生理活动,也是检测、观察动物生殖行为的重要指标。放射免疫分析法测定初生至性成熟前及其发情前期至分娩,Beagle母犬的卵巢类固醇激素分泌的动态变化,结果显示:血清中E_2的浓度在初生时很低,3月龄开始逐渐升高,9月龄后一直维持于20 pg/ml左右;初生时检不出血清中P,以后虽然有所升高,但一直维持于较低水平(＜0.6ng/ml)。性成熟后的经产母犬,在乏情期,E_2和P均维持在较低的水平。进入发情前期后,E_2浓度逐渐升高,至接受交配前的第4天出现第一个分泌高峰,浓度达75pg/ml,并维持数天,以后有所下降;直至交配受孕后的第18天,由于黄体的分泌作用,出现第二个高峰;在妊娠的最后阶段降至发情前期开始之前的水平。发情前期开始后,P浓度缓慢升高;发情前期的晚期迅速升高,从排卵配种前的第4天至排卵配种日,P从2.7ng/ml升高到10.9ng/ml;以后P不断升高,至妊娠14天达到39.4 ng/ml的峰值,并以较高浓度维持一段时间;在妊娠后期至分娩,其逐渐下降,分娩时孕酮的浓度已降至1ng/ml以下。
雌性动物性器官的发育与生殖活动,除与外周血中的性激素水平有关,还与生殖器官、腺垂体中雌激素受体(ER)和孕酮受体(PR)的数量有关。非同位素原位杂交(ISH)法检测犬生殖器官、垂体中雌激素受体α(ERα)mRNA和孕酮受体(PR)mRNA的相对表达丰度,结果显示:ERαmRNA阳性斑点主要出现在各级卵泡的颗粒细胞和内膜细胞、子宫腺体细胞和垂体的腺体细胞,卵巢中大卵泡的阳性率明显高于小卵泡,即卵泡的发育程度与ERmRNA表达丰度存在密切关系。卵巢在3月龄开始表达ERαmRNA,以后逐渐增加,至9月龄后维持在较高的水平;发情前期和发情期卵巢的ERαmRNA表达水平高于其它时期。子宫ERαmRNA的阳性率,从初生至15月龄,随内膜腺体细胞的数量和体积同步增加,阳性率随日龄增长而增加;由发情前期过渡到发情期后,ERαmRNA表达呈下降趋势。垂体腺体细胞的ERαmRNA在3月龄后一直维持在较高的水平;在发情周期,发情前期的晚期开始,ERαmRNA表达呈下降趋势。PRmRNA主要原位杂交阳性信号集中于各级卵泡的颗粒细胞、有腔卵泡与排卵前卵泡的内膜细胞和黄体细胞中、子宫腺体细胞和脑垂体的腺体细胞。初生未见卵巢PRmRNA的表达,3～18月龄之间的阳性率呈增长趋势;PRmRNA表达水平在发情前期较高,第9天达到较高水平。12月龄前的母犬子宫,几乎不表达PRmRNA;在发情前期开始后,PRmRNA表达呈上升趋势。初生时,母犬脑垂体PRmRNA几乎不表达,随着月龄的增加,PRmRNA在3月龄和6月龄表达水平较高,后表达水平下降。在发情周期PRmRNA一直维持在较低的水平。
以上犬基础生殖生物学研究结果,不仅丰富了犬生殖生物学资料,更重要的是为犬诱导发情、同步发情、超数排卵研究和动物实验安排提供了理论依据。
2以雌性动物为对象创建转基因犬相关辅助生殖技术研究
以雌性动物为对象创建转基因犬研究,首先要有大量的原核期受精卵用于胚胎操作。但犬为单发情动物,有较长的乏情期,人工诱导发情困难;在解剖上,犬的卵巢被厚厚的卵巢囊包裹,很难用腹腔镜技术从卵巢取卵。利用淘汰犬卵巢取卵,体外培养成熟、体外受精是一项重要的辅助生殖技术(ART)。我们以切割法采集卵巢上未成熟卵母细胞,建立了犬卵母细胞体外成熟培养的方法,获得了成熟的卵母细胞。结果显示:发情前期和发情期母犬卵巢的一级卵母细胞回收率较高,3岁犬平均回收的数量最多,而1岁以下幼犬和7岁以上老犬的回收率最低。在以M199作为基础培养液中,联合添加内皮生长因子(EGF)和雌二醇(E_2),可促进犬卵母细胞成熟,以发育至MII期的卵母细胞比例为指标,其成熟率高于单独添加组和对照组(22.58%vs 18.00%、5.56%,4.76%)。添加20%的FBS,其卵成熟率与添加20%的EDS无显著差异(17.14%vs 17.95%),但均显著高于对照组(0%)。EGF虽可促进卵丘细胞的扩展,但对卵母细胞的成熟无剂量依赖性。卵母细胞体外成熟培养的时间以72h为优。
人工诱导发情是又一项重要的辅助生殖技术。本研究以肌肉注射促性腺激素的方法对乏情期犬、青年不发情犬和老龄不育犬进行人工诱导发情实验。结果显示:外源激素1200IU PMSG+500IU HCG能有效地诱导乏情期犬和青年不发情犬的发情,而对老龄不育犬的诱导效果不明显。同时发现在PMSG总剂量(1200IU)不变的情况下,一次给药的总诱导发情、成功配种率为40%(6/15),其中乏情期犬的成功配种率为50%(4/8);少量多次给药诱导发情的总成功率为50%(15/30),其中乏情期犬的成功配种率为69%(11/16),并有31%(5/16)的母犬妊娠产仔,产仔母犬表现出与自然繁殖母犬同样的激素变化规律。提示PMSG+HCG组合、少量多次给药的方法可有效诱导乏情期经产母犬的发情,缩短发情间隔时间,为转基因犬制作提供更多的发情母犬。
胚胎移植是以雌性动物路线创建转基因犬的关键辅助生殖技术。我们以自然发情同步母犬配对供体和受体的策略,采取外科手术的方法开展犬受精卵输卵管移植的研究。并首次获得成活仔犬。在移入49枚1-8细胞受精卵的12只受体中,有3只母犬于卵母细胞受精(供体初次配种)后的第61天～65天成功分娩,产仔6只,分娩率为25%(3/12)。
3以雄性动物为对象创建转基因犬的部分基础研究
在以雌性动物为对象创建转基因犬的努力屡屡遭受挫折后,本课题又对以雄性动物为对象创建转基因犬的技术进行了初步探索。该转基因策略可分为精原干细胞的体内基因转移与体外基因转移,后者又包括精原干细胞的分离培养、受体内源性精原干细胞的消除、转基因精原干细胞的移植重建、人工采精及其阳性精子筛选、体外显微受精等主要环节。本文对体外途径的若干程序作了探索性研究。在精原干细胞的体外培养技术中,以外科手术摘取4-5月龄Beagle公犬睾丸,体外酶消化睾丸组织制作细胞悬液,以贴壁差异法纯化分离精原干细胞(SSCs),DMEM为基础培养基体外培养纯化的精原干细胞,并在体外对培养的SSCs进行EGFP基因转染。结果显示:4-5月龄的Beagle公犬可获得较多的SSCs;0.2mg/ml胶原酶Ⅳ+0.25%胰蛋白酶两步酶消化法的分离效果最好,每100mg睾丸组织分离的SCCs总数和成活细胞比例分别为6.67×10~6和95.5%;经AKP染色法鉴定,以贴壁差异法纯化SSCs,其纯度达50.7%;在DMEM基础培养液中添加10%胎牛血清(FBS)和20ng/ml EGF显著提高了SCCs集落数。SCCs体外转基因实验显示,EGFP基因可以在SCCs上表达,其中阳离子聚合物转染法的表达效率明显优于磷酸钙转染法(22.1%vs 8.1%)。除精原干细胞的体外培养与基因转移技术外,其他以雄性动物为对象创建转基因犬的相关辅助生殖技术正在研究中。
Canine is one of the most important laboratory animals in the biomedicine research. Canine entire genome atlas was completed at the end of 2005 year. Compared with other animals, such as mouse, canine gene was much more similar to human gene. About 360 kinds of canine genetic diseases are similar to human's. Therefore, the gene knock-in or gene knock-out dog was one of the ideal animal models to study human gene functions and the genetic diseases. People had succeeded in creating more than 10 of kinds transgenic animals such as mouse, rat, cavy, hamster, cattle, sheep, pig, rabbit and so on. More and more transgenic animals had been applied to the biological science research. Progress is slow on canine reproductive biology because of the specificity of its reproductive organs and reproductive physiology. The transgenic dog still hasn't been reported by now. This dissertation demonstrated the basic research on creating the transgenic dog. This paper is divided into three parts.
1. Study on the reproductive biology of bitches
We investigated 463 Beagle bitches/parturitions for 3 years, which were bred in Yangzhou and Suzhou respectively. We discovered that the bitches could be in estrous at all the year round, but especially in spring and autumn. The bitches were monoestrous. The average interval time of oestrus was 34 weeks. But the percent of the estrous bitches had certain differences in each season. The oestrus rate of the spring and autumn was 71% and 72.8% respectively on the two farms, which indicated that the bitch wasn't the classic seasonal oestrus animal. The gestation period was 62.9 days on average, mostly between 61～64 days. Each bitche gave birth to 5.35 puppies on average each time, mostly between 3～7 puppies and sometimes up to 10 puppies. There was no significant difference in the reproductive performance between the two farms.
By dissecting the bitches' generative organs, we could see that the ovary was packaged by the bursa ovarica. The bent and short oviduct was in the bursa ovarica's parietes. Along with the follicle's growth and development, the ovary's surface showed a petal structure. The histological research on the ovary of postnatal bitches demonstrated as following: Ovarian cortex sections of newborn bitches had large numbers of primordial follicles. With the age increasing, the primary follicles appeared in 3 mounth-old, the secondary follicles appeared in 6 month-old and the mature follicle appeared in 9 month-old. The endometrial gland developed and proliferated continuously after newborn, then gradually developed. At the same time, the stroma became denser. The oviduct had formed primary mucosal folds from newborn to 9 month-old, lots of secondary mucosal folds emerged after 12 months.
The dynamic change of estradiol (E_2) and progesterone (P) in bitches' serum was tested by radioimmunoassay (RIA) to bitches in puberty and estrous cycle. The results as following: The E_2 concentration was extremely low at birth, then ascended gradually in 3 month-old and maintained constantly about 20pg/ml after 9 month-old. The P concentration was zero in the newborn, but later increased and stayed at a lower level ( < 0.6 ng/ml). The concentration of E_2 and P both maintained at a lower level during the anestrus. At the onset of proestrus, E_2 increased gradually, then reached the first peak (75pg/ml) in the 4th day before mating, but later decreased to some extent. Until the 18th days after pregnancy, the second peak appeared due to the secretion of corepus luteum. P rised slowly in the early stage of proestrus, but rapidly in the later stage. It increased from 2.7ng/ml of the 4th day before the mating to 10.9ng/ml in the mating, later increased continually. The 14th day after the pregnancy, it got to the peak 39.4 ng/ml and later maintained for a period of time. P declined gradually from the end stage of pregnancy to the parturition, and it reach 1ng/ml below in the parturition.
The method of in situ hybridization (ISH) was first applied to detect relative expression abundance of estrogen receptor a (ERα) mRNA and progesterone receptor (PR) mRNA both the reproductive organ and the pituitary gland of the bitches. This study explained their relativities between sex hormonal concentration in serum, morphopoiesis of reproductive organ and expression abundance of ERa and PR mRNA. It laid the foundation to further explore the relationship between the reproductive capacity and the difference in the level of ER and PR gene transcription and translation. Results of image manipulation analysis system indicated that ER mRNA almost are not detected in the ovary at birth. ER mRNA expression increased from 3rd to 9th month-old, and then declined slightly. Postive staining was mainly detected in the granule cells of the various follicles, thicker in big follicles than in small follicles. Ovary expression of ERamRNA remained high during the proestrous and estrous . In uterus, ERamRNA was mainly detected in endometrium gland and lesser in the stroma. In estrus cycle, ERamRNA expression showed a downward trend. ERamRNA was detected mainly in the pituitary gland cells. ERamRNA were almost not detected at birth. With the increase of age, ERamRNA had been maintained at a higher level. In the estrous cycle, ERamRNA expression showed a downward trend in the pituitary.
Above results of canine reproductive biology is help for improving canine fertility and arranging the experimentates reasonably. What's more, they have provided the theory evidence of inducing estrus, superovulation of dogs and so on.
2. Related study on the assisted reproductive technology for creating a transgenic dog with the female
The research was based on the feminity to creating a transgenic dog. First, lots of zygotes were needed. Actually the artificial inducing estrus of bitch was extremely difficult now, due to ovaries were packaged by bursa ovarica ,and oocytes' recovery was unable by laparoscope. In vitro maturation and artificial inducing estrus were crucial assisted reproductive technologies for canine, which was the important way to create transgenic animals. But at present, the canine ART cann't satisfy the need of the transgenic dog production. This study was concern with in vitro oocytes maturation, inducing estrus and embryo transfer in oviduct of bitch. The embryo transfer in oviduct technology established a platform for generating transgenic animal.
Massive procaryon zygotes were required in embryo operatation. We sliced ovaries to release immature oocytes. We had established the oocyte culture system. We examined the number of good oocytes collected from bitches ovaries at diffenent ages and various stages of the estrous cycle by slicing the ovaries, then cultured them to do comparative experiment in vitro with the different culture medium additive, such as serum, EGF and E_2, which could promote the oocytes maturation. The standard of oocytes maturation was achieving MII by using hoechest 33342 staining technique. Results demonstrated that, the recover-ratio of COCs in proestrus and oestrus was higher than other periods. The average returning quantity from 3-year-old bitches was the highest, but <1 and >7-year-old bitches was the lowest. The nuclear maturation rates of oocytes in the culture medium supplmented with EGF and E_2 were higher than that supplemented with EGF or E_2 and the control (22.58% vs 18.00%, 5.56%; 22.58% vs 4.76%). There's no difference in the maturation rates of oocytes between 20% FBS and 20% EDS trial (17.14% vs 17.95%), which were higher than the control (0%). EGF had effect on cumulus expansion, but the rate of oocytes was not depend on the EGF dose. It showed that 72h was suitable for IVM of canine oocytes. The cumulus cells had important effect on oocytes maturation, as the oocytes with cumulus had higher maturation rates than oocytes without cumulus (15.56% vs 3.13%).
We carried out experiments of inducing estrus of anoestrum, young of nonestrus and sterile agedness bitches by i.m. PMSG + hCG. The results as following: extraneous hormone (1200IU PMSG + 500IU hCG) could induce effectively the anoestrus and young of nonestrus bitches to estrous, but useless for the sterile agedness dogs. In an invariable dose PMSG 1200IU, the inducing estrus rate was 40% (6/15) by given medicine once, but that was 50% (4/8) in the anoestrus group. That was 50% (15/30) by given medicine several times but a little ounce each time, the inducing estrous rate was 69% (11/16). Furthermore, The deliver puppies rate of bitches was 31% (5/16) . These bitches' hormone variation rule was the same as generally breeding bitches'. The results showed that this way was feasible to supply with more estrous bitches.
Embryo transfer was also one of the assisted reproductive technologies. We transfer zygotes of 1-8 cells stage into 12 recipient's oviducts. There were 3 receivers giving birth to 6 puppies at the 61th-65th day after the donor's oocytes fertilization, the successful rate was 25%(3/12). it was the first to obtain living puppies in this way.
3. Some fundamental research to create a transgenic dog based on male
Creating the transgenic animals is mediated by the Spermatogonial stem cell (SSCs), including transgenosis in vivo and in vitro. The latter included SSCs culture in vitro, eliminating endogenous SSCs, SSCs transfer and so on. Testes were collected by surgery operation from 4-5 month-old Beagle dogs. The SSCs were isolated by enzymatic combinations. According to the differentiation of velocity of cells sticking to the wall in vitro, SSCs were purified and then cultivated with DMEM containing different concentration of serum or EGF respectively. By counting the clusters every day, the growth efficiency of SSCs can be readily detected within 1 week in a semi-quantitative manner. The SSCs were transfected with pEGFP-N1 plasmids by the cationic polymer transfection reagen and calcium phosphate transfection reagen. The results as following: collagenase IV+ trypin combination was an effective method to dissociate canine SSCs, the total number of dissociated cells was 6.67×10~6/100mg testis parenchyma and rate of living SSCs was 96.5%. It Is necessary to add FBS to the culture medium for SSCs culture in vitro . Compared with EGF group, the clusters of cell in the 20ng/ml group increased markedly. The gene transfection efficiency of the cationic polymer transfection reagen method was much higher than that of calcium phosphate method (22.1% vs 8.1%) .In conclusion, we had developed an efficient and economical method to isolate the SSCs, through which we could obtain enough viable germ cells. The technique of SSCs culture demonstrated that dog SSCs could be maintained in culture medium in vitro for approximately 1 week. The clusteres came from a single cell indicated that SSCs are able to achieve growth and proliferation in the culture system in vitro. We have succeeded in the EGFP gene transfection in vitro, which was prepared for the further SSCs transplantation technique to get the transgenic puppies.
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