奶牛妊娠早期血液中PAG及相关基因表达的研究
摘要
奶牛配种后,尽早地判断其妊娠与否,以便对已妊娠者加强饲养管理,对未妊娠者找出原因,及时补配或进行必要的处理。这对缩短奶牛产犊间隔,提高其繁殖力具有重要意义。本试验分别运用了半定量PCR技术和mRNA差异显示技术,研究了以奶牛血液中PAG基因的表达量为依据的早期妊娠诊断方法以及空怀牛和妊娠牛血液相关基因表达的差异,从分子水平上了解早期妊娠诊断过程中基因表达的变化规律,以期为哺乳动物早期妊娠诊断的新方法研究提供参考。结果如下:
空怀牛和已妊娠21天的妊娠牛血液中PAG基因均有表达,妊娠牛PAG基因表达量略高于空怀牛,但是两者差异不显著,t=2.36950.05。这也就说明配种后21天奶牛血液中PAG基因的表达量不能被作为妊娠早期诊断的依据。
在妊娠母牛血液中共筛选到了6条分别特异表达的片段,经过测序及对比分析,表明有3条带与已知的功能基因或调控基因相似,其余条带为未知功能基因或无相似基因。其中:S1序列与转化因子TGF-β2具有较高的同源性(98%),TGF-β2对牛滋养层和子宫内膜上皮有促进增生的作用,而对上皮细胞生长和倾入滋养层有抑制作用;TGF-β2还对转型变异的人子宫内膜和家畜上皮有促增长作用。S4序列与集刺落因子1有较高的同源性(97%),CSF能刺激早期胎盘生长,还能抑制NK细胞的功能,减少流产的发生。S5序列与胰岛素生长因子具有较高的同源性(98%),IGF主要通过膜受体起作用,可刺激胎盘促乳素的释放,调节其它类固醇激素的合成与分泌。IGFs与胚泡植入、胎盘形成和功能、胎儿生长发育有关,具有促进滋养细胞浸润、利于胚胎着床等作用;参与了早期妊娠多个环节的调节,是胚胎发育的重要影响因子。
After breeding, judging the cow whether is pregnant as soon as possible, may promote pregnant cows’raise and management, breed again in time or take some necessary measures.It is important to shorten procreative interval and advance the reproduced ability. This experiment has utilized the semi-quantitative PCR technology and the mRNA difference display technology separately, to exploringly study the early pregnancy diagnosis methods based on the PAG gene expression quantity in the cows’blood as well as the non-pregnant and the pregnant cows’gene expression difference in the blood. From the molecular level,to understand gene expression's change rule during the early pregnant diagnosis process, which provides the reference to the research of mammal early pregnancy diagnosis's new method. The results are as follows:
The PAG gene had the expressed both in non-pregnant and pregnant cows’blood, the PAG gene expression quantity in pregnant cows’blood is slightly higher than non-pregnant cows, but the difference is not remarkable, t=2.36950.05. This also shows that judging whether the cow is pregnant by examining the PAG gene expression quantity in blood on the 21st day after breeding is unpractical.
Altogether screened six special expression fragments separately in the pregnant cows’blood, passed through the sequence and the contrastive analysis, indicated that had 3 bandings and the known function gene or the regulative gene is similar, other bandings for unknown function gene or not similar gene. And:
The S1 sequence has the high homology (98%) with the transforming growth factor2 (TGF-β2), TGF-β2 has the promotion proliferation function to cows’trophoblast and the endometrial epidermis, but has the inhibitory function to the growth of the epithelial cell and plunge the trophoblast; it also has the promotion proliferation function to reforming variated endometrium of human and the domestic animal epidermis .
The S4 sequence has the high homology (97%) with the clony- stimulating factor 1 (CSF 1), CSF 1can stimulate the early placenta to grow, but can also suppress the NK cell's function, reduce miscarries.
The S5 sequence has the high homology (97%) with the Insulin like growth factor 1(IGF1), IGF1 mainly has an effect through the membrane acceptor, may stimulate the placenta to release the prolactin, adjust the synthesis and the secretion of other steroid hormone.IGF1 has relationship with the implantation of blastocyst, the form and function of placenta, and the growing development of embryo, it also can promote the soakage of trophocyte, and is propitious to the preimplantation; it participated in the many link's adjustments during early pregnancy, and is the important effect factor of embryonic development.
空怀牛和已妊娠21天的妊娠牛血液中PAG基因均有表达,妊娠牛PAG基因表达量略高于空怀牛,但是两者差异不显著,t=2.3695
在妊娠母牛血液中共筛选到了6条分别特异表达的片段,经过测序及对比分析,表明有3条带与已知的功能基因或调控基因相似,其余条带为未知功能基因或无相似基因。其中:S1序列与转化因子TGF-β2具有较高的同源性(98%),TGF-β2对牛滋养层和子宫内膜上皮有促进增生的作用,而对上皮细胞生长和倾入滋养层有抑制作用;TGF-β2还对转型变异的人子宫内膜和家畜上皮有促增长作用。S4序列与集刺落因子1有较高的同源性(97%),CSF能刺激早期胎盘生长,还能抑制NK细胞的功能,减少流产的发生。S5序列与胰岛素生长因子具有较高的同源性(98%),IGF主要通过膜受体起作用,可刺激胎盘促乳素的释放,调节其它类固醇激素的合成与分泌。IGFs与胚泡植入、胎盘形成和功能、胎儿生长发育有关,具有促进滋养细胞浸润、利于胚胎着床等作用;参与了早期妊娠多个环节的调节,是胚胎发育的重要影响因子。
After breeding, judging the cow whether is pregnant as soon as possible, may promote pregnant cows’raise and management, breed again in time or take some necessary measures.It is important to shorten procreative interval and advance the reproduced ability. This experiment has utilized the semi-quantitative PCR technology and the mRNA difference display technology separately, to exploringly study the early pregnancy diagnosis methods based on the PAG gene expression quantity in the cows’blood as well as the non-pregnant and the pregnant cows’gene expression difference in the blood. From the molecular level,to understand gene expression's change rule during the early pregnant diagnosis process, which provides the reference to the research of mammal early pregnancy diagnosis's new method. The results are as follows:
The PAG gene had the expressed both in non-pregnant and pregnant cows’blood, the PAG gene expression quantity in pregnant cows’blood is slightly higher than non-pregnant cows, but the difference is not remarkable, t=2.3695
Altogether screened six special expression fragments separately in the pregnant cows’blood, passed through the sequence and the contrastive analysis, indicated that had 3 bandings and the known function gene or the regulative gene is similar, other bandings for unknown function gene or not similar gene. And:
The S1 sequence has the high homology (98%) with the transforming growth factor2 (TGF-β2), TGF-β2 has the promotion proliferation function to cows’trophoblast and the endometrial epidermis, but has the inhibitory function to the growth of the epithelial cell and plunge the trophoblast; it also has the promotion proliferation function to reforming variated endometrium of human and the domestic animal epidermis .
The S4 sequence has the high homology (97%) with the clony- stimulating factor 1 (CSF 1), CSF 1can stimulate the early placenta to grow, but can also suppress the NK cell's function, reduce miscarries.
The S5 sequence has the high homology (97%) with the Insulin like growth factor 1(IGF1), IGF1 mainly has an effect through the membrane acceptor, may stimulate the placenta to release the prolactin, adjust the synthesis and the secretion of other steroid hormone.IGF1 has relationship with the implantation of blastocyst, the form and function of placenta, and the growing development of embryo, it also can promote the soakage of trophocyte, and is propitious to the preimplantation; it participated in the many link's adjustments during early pregnancy, and is the important effect factor of embryonic development.
引文
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[2] Zoli A P, Beckers J F,WoutersBallman P, et al. Purification and characterization of a bovine pregnacy-associated glycoprotein. Biol Reprod[J]. 1991, 45 (1) :1～10.
[3]刘泽隆,张涌.家畜胎盘生物活性物质的研究进展.西北农业大学学报[J].1999,27(3):82～86.
[4] Gonzalez F, Batista M, Quesada E, et al. Pregnancy-associated glycoproteins (PAG) and ultrasonography for diagnosis of pregnancy, hydrometra and embryonic mortality in dairy goat. Proceedings of the 161h Scientific Meeting of the European Embryo Transfer Association (Santander, Spain) [M] 2000:156.
[5]李裕强.家畜妊娠相关糖蛋白的研究进展.动物医学进展[J].1999,20(3):21～23.
[6]孙庆申,田文儒,侯振中.胎盘的功能最新研究进展.黑龙江畜牧兽医[J].2001(9):44～45.
[7] Roberts R M, Xie S, Mathialagan N. Materanl recognition of pregnancy. Bio l Reprod[J].1996,(54): 294～302.
[8] G.Haugejorden, S.Waage, E.Dahl, et al. Pregnancy associated glycoproteins (PAG) in postpartum cows, ewes, goats and their offspring. Theriogenology[J].66 (2006) :1976～1984.
[9] Xie S, Low B G, Kramer K , et al. Identification of the major pregnancy-specific antigens of cattle and sheep as inactive members of the aspartic proteinase family. Proc Natl A cad Sci USA[J] .1991, 88: 10247～10251.
[10]S P. Breukelman , Otto Szenci , JF Beckers , et al. Ultrasonographic appearance of the conceptus, fetal heart rate and profiles of pregnancy-associated glycoproteins (PAG) and prostaglandin F2a-metabolite (PGF2a-metabolite) after induction of fetal death with aglepristone during early gestation in cattle. Theriogenology [J].64 (2005): 917～933.
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