H-Y抗体对小鼠胚胎H-Y抗原表达的研究

BALB/C雄性小鼠脾细胞悬液腹腔注射免疫同系母鼠 ,精子细胞毒性试验筛选抗血清 ,效价高的抗血清分别用于胚胎培养的毒性试验和制备单克隆抗体再辅以间接免疫荧光和PCR对胚胎进行性别鉴别。结果表明 ,直接用H Y抗血清培养的胚胎 ,胚胎退化率达 43 3 % ,与自然性比差异不显著 ;PCR验证间接免疫荧光法鉴定的胚胎性别 ,雄性胚胎准确率为 83 % ,雌性胚胎准确率为 94%。     

Migration and differentiation of gonadal germ cells under cross-sex germline chimeras condition in domestic chickens

A series of experiments was conducted to investigate migration, proliferation and differentiation of gonadal germ cells (GGCs) collected from the gonads of 7-day-old chick embryos under cross-sex germline chimera conditions. The migratory and proliferative abilities of exogenous GGCs were examined by transferring 50 fluorescently labeled GGCs collected from White Leghorn (WL) embryos into the blood of 2-day-old Rhode Island Red (RIR) embryos. No significant difference was observed in the number of fluorescently labeled GGCs in the gonads of recipient embryos among any of the four possible donor and recipient sex combinations. Cross-sex germline chimeras were produced to examine the differentiation of GGCs by transferring 100 GGCs from WL embryos into 2-day-old RIR embryos. Exogenous-GGC-derived progeny were obtained from both male and female recipients, except when female GGCs were transferred into male recipients. The migratory ability of GGCs recovered from the 7-day-old embryonic gonad was not influenced by cross-sex germ cell transfer conditions, whereas the differentiation of the GGCs was affected by the sex combinations of GGCs donors and recipients.     

过表达FOXL2基因对鸡胚性腺分化的影响

试验旨在研究FOXL2基因对鸡胚性腺分化的影响。本试验分为试验1组、试验2组和空白对照组（鸡胚数量分别为260、100、20枚），试验组通过胚盘下腔注射的方法分别将pLV-FOXL2慢病毒重组质粒、pLV空质粒注入胚胎期第2天的鸡胚，空白对照组不做处理并与试验组一起孵化至出雏，利用CHD1基因遗传性别鉴定的方法对出雏的雏鸡进行性别检测，分析其性腺解剖学、组织学结构变化，并利用免疫组化的方法检测性腺FOXL2和CYP19A1蛋白表达量。结果显示，试验1组遗传性别为公的23只，遗传性别为母的18只，表型性别为公的21只，表型性别为母的18只，其中有2只表型性别不典型，左侧性腺发生变化，朝卵巢结构转变；试验2组遗传性别为公的9只，遗传性别为母的12只，表型性别与遗传性别一致。阳性PCR检测结果显示，试验1组获得阳性个体10个，阳性率为24.4%（10/41）；试验2组获得阳性个体8个，阳性率为38.1%（8/21）。性腺解剖学结果显示，阳性pLV-FOXL2雄性鸡胚左侧性腺体积明显大于右侧性腺，表现膨松状态；组织切片结果显示，雄性鸡胚性腺具有典型的卵巢皮质层和髓质层结构；阳性pLV-FOXL2雌性鸡胚性腺的发育无明显变化。免疫组化结果显示，FOXL2和CYP19A1蛋白在试验1组左右侧睾丸中的表达量与空白对照组母鸡卵巢中的表达量相似，显著高于试验2组（P<0.05）。以上结果表明，FOXL2基因可能促进鸡雄性性腺的性反转，在鸡性腺分化和发育过程中发挥着重要的作用。     

Effect of Overexpression of FOXL2 Gene on Gonadal Differentiation in Chicken Embryo

The aim of this study was to investigate the effect of FOXL2 gene on gonadal differentiation in chicken embryos.This test was divided into the experiment groups 1,2 and blank control group(chicken embryos were 260,100 and 20,respectively).In the experimental group,pLV-FOXL2 lentivirus recombinant plasmids and pLV empty plasmids were injected into the chicken embryo on the second day of the embryonic stage by subpaneoidal injection.The blank control group was not treated and incubated with the experimental groups until the chick was hatched.CHD1 gene genetic sex identification method was used to detect the sex of chicks,the changes of gonadal anatomy and histological structure were analyzed,and the expression levels of FOXL2 and CYP19A1 proteins were detected by immunohistochemistry.The results showed that in the experiment group 1,there were 23 male and 18 female of genetic sex,21 male and 18 female of phenotypic sex,and two of them had atypical phenotypic gender,with changes in the left gonadal gland toward ovarian structure.The phenotypic sex was consistent with the genetic sex in experiment group 2,with 9 male and 12 female.Positive PCR results showed that 10 positive individuals were obtained in the experiment group 1,with a positive rate of 24.4% (10/41),and 8 positive individuals were obtained in the experiment group 2,with a positive rate of 38.1% (8/21).The anatomical structure of the gonad showed that the volume of the left gonad was significantly larger than that of the right gonad in the positive plV-FOXL2 male embryos.The results of tissue sections showed that the gonad of male chicken embryo had typical structure of ovarian cortex and medulla.There was no significant change in the development of gonad in female embryos with positive pLV-FOXL2.Immunohistochemical results showed that the expression levels of FOXL2 and CYP19A1 proteins in the left and right testicle of experiment group 1 were similar to that in the hen ovary of the blank control group,and were significantly higher than that in experiment group 2 (P<0.05).These results suggested that FOXL2 gene might promote the sexual inversion of chicken gonads and played an important role in the differentiation and development of chicken gonads.     

兔早期胚胎PCR性别鉴定体系的建立

根据兔SRY基因序列设计两对引物作为兔雄性特异性引物,根据兔APP基因序列设计1对引物作为内标引物,分别建立了兔早期胚胎性别鉴定的双重PCR和巢式PCR反应体系,在不同浓度的基因组DNA和兔早期胚胎上进行性别鉴定应用,同时,对兔SRY巢式PCR引物特异性进行了分析。结果表明,多重PCR扩增兔基因组DNA可以准确判定其性别,扩增灵敏度为100pg基因组DNA;多重PCR鉴定24枚兔32细胞桑椹胚性别,只能对整胚成功鉴定。巢式PCR,公兔基因组DNA扩增出282bp的SRY基因片段,母兔没有扩增产物,扩增灵敏度为10pg;对24枚兔32细胞桑椹胚性别鉴定结果表明,巢式PCR可以对少至4个胚胎细胞进行准确鉴定,同一胚胎结果符合率为100%(24/24)。SRY引物只对兔雄性基因组DNA特异,而其他动物(人、牛、绵羊、小鼠)雄性DNA及兔的冲卵液,均无PCR产物。     

Nondestructive sex-specific monitoring of early embryonic development rate in white layer chicken eggs using visible light transmission

ABSTRACT

1. Sex-specific variations in early embryonic development rates may pre-empt later variations in embryonic development through to pipping and hatching. Given that erythropoiesis (blood production) can be equated with early embryonic growth rate, it was hypothesised that blood pigment haemoglobin can act as a specific spectral fingerprint for changes in growth rate. Moreover, by measuring longitudinal, rather than lateral, spectral transmission through the egg, a more consistent spectrum with a higher signal-to-noise ratio could be captured.

2. Longitudinal visible transmission (T575/T598 ratio), which is sensitive to haemoglobin, was used to monitor sex-specific early embryonic development rate in white layer chicken eggs from d 0 to 8 of incubation. The sex of these eggs was subsequently confirmed two days after hatching.

3. Embryonic development was detectable from d 3 (72 h) of incubation, 36 h earlier than previously reported lateral spectral measurements, supporting the greater sensitivity of longitudinal measurements.

4. At d 3, the mean T575/T598 ratio for male embryos was significantly lower (P < 0.001) (i.e. higher absorbance of haemoglobin) than for female embryos, which was thought to be due to sex-differences in early embryogenesis. On the other hand, female embryos had a significantly lower (P < 0.05) mean T575/T598 ratio than male embryos at d 7 of incubation, presumably due to the combined effects of oestrogen synthesis receptors and enzymes on erythropoiesis in female embryos at this time.

5. In conclusion, the proposed methodology has the sensitivity to differentiate sex-specific embryonic development rates during early incubation and the potentiality to advance precision incubation management and poultry research.     

鉴定牛体外培养胚胎性别的一种新PCR方法及其应用

根据已知序列设计了2对引物,分别在体外扩增SRY基因及1个常染色体基因,能同时扩增出2个基因的胚胎为雄性,只能扩增出常染色体基因的胚胎为雌性。通过该方法对50个卵母细胞进行PCR验证,有49个扩增出1条常染色体基因,准确率为98％;鉴定了48枚用普通精液受精所得胚胎及57枚用经SRY抗体处理的精液受精所得胚胎的性别,雌性比例分别为56％和82％。     

The tryptophan requirement of broiler chicks.

The "available" tryptophan requirements of male and female broiler chicks were determined at 7-d intervals from 0 to 56 d, using a diet-dilution technique. Availability of tryptophan in the diets was estimated by growth assay with chicks. 2. The tryptophan requirements were 2.4 (males) and 2.2 g/kg of diet (female) from 0-7 d, and 1.7 g/kg (males and females) from 7-35 and 35-56 d. The absolute requirement of the chick for tryptophan increased with age and was significantly different for male and female birds. 3. A highly significantly predictive equation relating tryptophan requirement to mean body weight and gain was established.     

昆明小鼠不同性别早期胚胎体外培养发育潜力观察

自主设计2对引物,提取已知性别小鼠肝细胞DNA,扩增雄性小鼠特有的Sry基因及雌、雄小鼠共有的ZFX基因,建立双重PCR性别鉴定方法.提取小鼠早期8细胞胚胎单卵裂球、双卵裂球DNA,分别进行双重PCR性别鉴定,选择出早期8细胞胚胎的适宜模板量;将已测性别的8细胞胚胎按照雌、雄性别分开培养,观察统计雌、雄8细胞胚胎发育至囊胚的比率.试验结果显示,与单个卵裂球DNA模板量相比,双卵裂球的模板量检出率高,两者之间差异显著(75.00%Vs 93.33%,P<0.05);经过性别鉴定的雄性8细胞胚胎比雌性8细胞胚胎发育至囊胚的比率高,两者之间差异显著(53.33%Vs 33.33%,P<0.05).结果表明,采用双重PCR性别鉴定方法,以双卵裂球的DNA量为模板能够有效的对小鼠早期8细胞胚胎进行性别鉴定;雄性胚胎在体外培养条件下比雌性胚胎具有更好的发育潜力.     

H-Y单抗检测方法初探

优化间接ELISA条件 ,提高灵敏度 ,建立检测H Y抗体的检测方法。利用BALB/c公鼠免疫同系母鼠 ,制备H Y单抗。PCR验证间接免疫荧光法鉴定胚胎性别准确率。结果表明 :鉴定雄性胚胎的准确率为 83% ( 1 5 / 1 8)。鉴定雌性胚胎的准确率为 94 % ( 1 5 / 1 6)。     

Cryopreservation and sexing of in vivo- and in vitro-produced bovine embryos for their practical use

My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GL-Tip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of 0.1-1.0% deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-PCR and purification of DNA product before standard sexing PCR of biopsy samples from Day 3-4 in vitro-derived embryos allowed accurate sex determination, and Day-7 blastocysts developed from Day 3-4 embryos were cryopreserved by GL-Tip vitrification without a loss of their viability. Thus the field application of bovine embryo transfer is in part supported by improvements of technologies in embryo cryopreservation and sex pre-determination.     

家禽胚胎性别鉴定方法研究进展

在家禽生产中,孵化时蛋用禽希望雌性雏较多,而肉用禽却希望雄性雏较多,且在蛋用禽中,雄性雏的处理也需要花费很大的资金。因此在孵化时,如果可以尽早的鉴别出胚蛋的性别,不仅可以节约孵化时的花费,更可以节约废雏的处理费用。作者主要介绍了几种家禽早期胚胎性别鉴定的方法,以期为国内此方面的研究提供借鉴。     

牛胚胎性别鉴定荧光定量PCR方法的优化与应用

旨在建立一种可检测牛早期胚胎性别的复合探针体系,减少常规PCR方法电泳所带来的污染,提高鉴定准确率,降低鉴定成本。本研究以SRY为牛胚胎性别鉴定的目标基因,以进口YCD-PCR性别鉴定试剂盒为对照组（n=9 543）,荧光定量PCR的单引物分别扩增体系（荧光单扩,FSPSA,n=6 570）和双引物混合扩增体系（荧光双扩,FDPMA,n=22 238）分别做试验组,从性别鉴定效率、无反应率、雌雄胚胎百分率和比值、移植妊娠率、雌性胚胎母犊率、不同细胞取样数下的性别鉴定结果和鉴定成本等方面进行各组间比较。结果表明,荧光单扩组的雌性胚胎百分率和性别鉴定效率极显著高于其他两组（P<0.01）,无反应率极显著低于其他两组（P<0.01）,雌雄胚胎比值与其他两组差异较大（1.03∶1 vs 1∶1.03和1∶1.02）,雌性胚胎母犊率极显著低于荧光双扩组和对照组（P<0.01）;荧光双扩组的雌性胚胎百分率、雄性胚胎百分率和雌性胚胎母犊率均与对照组无显著差异（P>0.05）,雌雄胚胎比值与对照组相似（1∶1.03和1∶1.02）,无反应率极显著低于对照组（P<0.01）,性别鉴定效率极显著高于对照组（P<0.01）。取样细胞4~6组和7~10组的性别鉴定效率极显著高于1~3组和SD（separated & died cells）组（P<0.01）,而1~3组和SD组之间及4~6组和7~10组之间差异不显著（P>0.05）。移植妊娠率4~6细胞组最高（49.68%）,但各取样组间无显著差异（P>0.05）。荧光双扩法与对照组相比,鉴定成本下降了46.76%。结果显示,荧光双扩方法产母犊准确率最高,鉴定成本较低,取样4~6细胞时的移植妊娠率最高,是一种更可行的牛胚胎性别鉴定技术,更有利于产业化推广和应用。     

Y are you not pregnant: identification of Y chromosome segments in female cattle with decreased reproductive efficiency

Reproductive efficiency is of economic importance in commercial beef cattle production, since failure to achieve pregnancy reduces the number of calves marketed. Identification of genetic markers with predictive merit for reproductive success would facilitate early selection of females and avoid inefficiencies associated with sub-fertile cows. To identify regions of the genome harboring variation affecting reproductive success, we applied a genome-wide association approach based on the >700,000 SNP marker assay. To include the largest number of individuals possible under the available budget, cows from several populations were assigned to extremes for reproductive efficiency, and DNA was pooled within population and phenotype before genotyping. Surprisingly, pools prepared from DNA of low reproductive cattle returned fluorescence intensity data intermediate between fertile females and males for SNP mapped to the Y chromosome (i.e., male sex chromosome). The presence of Y-associated material in low reproductive heifers or cows was confirmed by Y-directed PCR, which revealed that 21 to 29% of females in the low reproductive category were positive by a Y chromosome PCR test normally used to sex embryos. The presence of the Y chromosome anomaly was further confirmed with application of additional Y-specific PCR amplicons, indicating the likelihood of the presence of some portion of male sex chromosome in female cattle in various beef cattle herds across the U.S. Discovery of this Y anomaly in low reproductive females may make an important contribution to management of reproductive failures in beef cattle operations.     

鹌鹑早期胚胎bcl-2基因表达的差异及发育性变化

采用RT-PCR方法,用Wpkci和β-actin引物进行多重PCR鉴定66～120 h鹌鹑胚胎样本性别后,选取不同时间点雌、雄胚胎各4枚,以β-actin为内标,测定bcl-2 mRNA的相对丰度;探讨bcl-2对鹌鹑早期胚胎发育的影响。结果表明：①雄性胚胎在66～90 h bcl-2 mRNA表达量一直维持在较低水平,96～102 h bcl-2 mRNA有所增加,但96与102 h间差异不显著（P＞0.05）,在108 h显著降低（P＜0.05）;与108 h相比,114 h显著升高,120 h维持在较高水平。雄性胚胎在66～108 h（72 h除外）bcl-2 mRNA表达均比雌性胚胎的表达量低,在114～120 h bcl-2 mRNA表达水平均比雌性胚胎的高。②雌性胚胎在66～90 h bcl-2 mRNA表达量一直维持在较低水平;与90 h相比,96 h显著升高,96～120 h逐渐降低,其中96～108 h下降,但三者间差异不显著（P＞0.05）;与108 h相比,114～120 h显著下降（P＜0.05）。③相同时间点雌、雄胚胎间的比较结果表明,84 h雌性胚胎bcl-2 mRNA表达显著高于雄性胚胎（P＜0.05）,90、108 h雌性胚胎bcl-2 mRNA表达极显著高于雄性胚胎（P＜0.01）;而在120 h雄性胚胎bcl-2 mRNA表达极显著高于雌性胚胎（P＜0.01）。鹌鹑胚胎发育早期,雌、雄样本bcl-2 mRNA表达存在一定的时序性,说明bcl-2对雌、雄鹌鹑早期分化及早期胚胎发育有重要影响。     

应用H—Y单克隆抗体鉴定小鼠和家兔胚胎性别

以Ｃ５７ＢＬ／６雄性小鼠的脾细胞免疫同雌性小鼠，选择免疫应答反应较好的雌性小鼠４只，取其脾细胞与ＳＰ２／０骨髓瘤细胞进行融合的杂交瘤细胞经过３次克隆，筛选出了２株能够分泌Ｈ－Ｙ抗体的杂交瘤细胞系ＨＹＡ１和ＨＹＡ２，其分泌的相应抗体分别名为ＨＹａ１和ＨＹａ２。琼脂双扩散试验表明，ＨＹａ１和ＨＹａ２分别属于ＩｇＭ和ＩｇＧ亚类。胚间接免疫荧炮分析及胚胎细胞毒试验表明，ＨＹａ１和ＨＹａ２分别属于ＩｇＭ和Ｉ     

Experiences of Using PCR for Sexing Bovine Embryos*

Bovine embryos were used to evaluate the accuracy and the efficiency of sexing embryos by amplifying male specific DNA using the polymerase chain reaction. Blind tests with biopsies and biopsied embryos suggested that the method is accurate. However, often the amplification was unsuccessful and the assay therefore uninformative. We were able to amplify male specific DNA from degenerated embryos as well as from embryos stained with Hoechst 33342.     

Fadrozole对公鸡的生长发育和精清激素浓度的影响

芳香化酶抑制剂Fadrozole能诱导雌性鸡胚雄性化,而不影响出生时雄性鸡胚的性别,但Fadrozole是否影响公鸡出雏后的生长发育和繁殖能力尚不清楚。在性别分化前(E3.0),向农大3号矮小商品代鸡胚注入PBS和不同剂量的Fadrozole(0.、10.3、0.5、1.0mg和1.3mg分别为1F、F2、F3、F4和F5),以出雏后的公鸡作为试验材料,探索Fadrozole对出雏后公鸡的生长发育、精清中的性激素和生长轴激素的影响。结果表明:高剂量组F4和F5分别为33.33%和13.33%,出雏率显著低于对照组P(<0.05),而低剂量组F1、F2和F3与对照组相比,出雏率无显著变化(P>0.05);各剂量Fadrozole处理组和对照组的体重和胫长在8周龄和20周龄时无显著差异P(>0.05);性成熟后(30周龄),F5精清中的睾酮(T)和雌二醇(E2)显著高于对照组和其他各处理组(P<0.05),但ln(T/E2)在各处理组和对照组差异均不显著(P>0.05);本试验说明Fadrozole不影响雄性胚胎的性腺分化,但高浓度的Fadrozole不利于鸡胚的发育;Fadrozole不影响出生后公鸡的生长发育、精清中的生长轴激素激分泌和精清中睾酮与雌二醇的比值。     

Effects of donor cells’ sex on nuclear transfer efficiency and telomere lengths of cloned goats

The aim of this study was to investigate the effects of donor cells’ sex on nuclear transfer efficiency and telomere length of cloned goats from adult skin fibroblast cells. The telomere length of somatic cell cloned goats and their offspring was determined by measuring their mean terminal restriction fragment (TRF) length. The result showed that (i) reconstructed embryos with fibroblast cells from males Boer goats obtained significantly higher kids rate and rate of live kids than those of female embryos and (ii) the telomere lengths of four female cloned goats were shorter compared to their donor cells, but five male cloned goats had the same telomere length with their donor cells, mainly due to great variation existed among them. The offspring from female cloned goats had the same telomere length with their age‐matched counterparts. In conclusion, the donor cells’ sex had significant effects on nuclear transfer efficiency and telomere lengths of cloned goats.     

Introduction of exogenous DNA into gonads of chick embryos by lipofection and electroporation of stage X blastoderms in vivo

1. In order to introduce exogenous DNA into gonads of chick embryos, stage X blastoderms of freshly laid and unincubated eggs were transfected by lipofection and electroporation in vivo. 2. The introduced DNA, green fluorescence protein (GFP) gene, was efficiently expressed in the blastoderms incubated for 24 h (78.8%, 78/99). 3. The GFP gene was present in most of the embryonic bodies and extra-embryonic membranes died by d 10 of incubation, when analysed by polymerase chain reaction. On d 16 to 20 of incubation, the GFP gene was detected in 7.0 to 20.9% of embryos in the heart, liver, stomach and brain, but not in the sartorius muscle. For the gonads, the GFP gene was detected in 22.2% (6/27) of the testes and 6.3% (1/18) of the ovaries examined. 4. These results suggest that it is possible to introduce exogenous DNA into gonads of chick embryos by lipofection and electroporation of stage X blastoderms in vivo.