| As a major country in sheep breeding,China has a large number of local breeds.Sheep are singleton animals that reproduce seasonally,which cause the limitation of a large number of breeds with excellent economic traits in the lack of production capacity due to lambing rate.In so doing,improving lambing rate has been a constant pursuit in the farming industry.Apoptosis plays a crucial role in mammalian embryonic development and fetal growth.Survivin,as the most powerful member of the IAP family(NAIP,c-IAP1,c-IAP2,XIAP,Survivin,BRUCE,Livin,h ILP)to inhibit apoptosis,is highly expressed during embryonic and fetal development.Especially,the Survivin gene can promote neovascularization,which is relevant to the study of endometrium in embryonic implantation and embryonic development.This is of guiding significance for studies related to endometrium in embryonic implantation and embryonic development.In the present study,we select the Small Tail Han Sheep with multiple fecundity and Qianhua Merino sheep(Qianhua sheep)with both meat and wool as the experimental subjects.The expression of the Survivin gene at the endometrium,oviduct and ovary at 2-month-old,6-month-old and 12-month-old is investigated by Realtime PCR and Western Blot to demonstrate whether the Survivin gene has a regulatory role in the development of the female reproductive organs.Immunofluorescence(IF)is used to quantify the microvessel density(MVD)in the endometrium of two sheep at three periods and compare it with Survivin mRNA expression.The objective is to verify the possibility of Survivin gene as a candidate gene for multiple birth traits in sheep and the association of this gene with multiple birth traits in sheep.The following findings are obtained from these studies:1.The results of immunohistochemistry show that Survivin protein is positively expressed on the endometrium,oviduct and ovary of 2-month-old,6-month-old and 12-month-old Small Tail Han Sheep and Qianhua sheep.2.The results of Real-time PCR and Western Blot assay show that Survivin mRNA expression and Survivin protein expression are found in the endometrium,oviduct and ovary of 2-month-old,6-month-old and 12-month-old Small Tail Han Sheep and Qianhua sheep,and there are inter-month and inter-organ differences.(1)Survivin mRNA expression in the uterus of Qianhua sheep is highly significant(P < 0.01)between 2-month-old and 6-month-old,significant(0.01 < P < 0.05)between2-month-old and 12-month-old,and not significant(P > 0.05)between 6-month-old and12-month-old;Survivin gene mRNA expression in the oviduct and ovary of Qianhua sheep is not significantly different between the three periods(P > 0.05).The differences in Survivin protein expression in the uterus of Qianhua sheep are not significant between 2-month-old,6-month-old and 12-month-old;In the ovaries of Qianhua sheep,the differences are highly significant between 2-month-old and 6-month-old(P < 0.01),significant between 2-month-old and 12-month-old(0.01 < P < 0.05),and not significant between 6-month-old and 12-month-old(P > 0.05);In Qianhua sheep oviducts,the difference between 2-month-old and 6-month-old,2-month-old and 12-month-old is highly significant(P < 0.01).The difference between 6-month-old and 12-month-old is not significant(P > 0.05).(2)As for Survivin mRNA expression in 2-month-old Qianhua sheep,the difference between uterus and oviduct is highly significant(P < 0.01),the difference between uterus and ovary is not significant(P > 0.05),and the difference between oviduct and ovary is significant(0.01 < P < 0.05);The expression of Survivin mRNA in 6-monthold Ganhua sheep is not significantly different from the uterus and fallopian tubes(P>0.05),the difference between the uterus and ovaries is highly significant(P<0.01),and the difference between the uterus and ovaries is significant.(0.01<P<0.05);The expression of Survivin mRNA in 12-month-old Ganhua sheep is not significantly different from the uterus and fallopian tubes(P>0.05),the difference between uterus and ovary is significant(0.01<P<0.05),and the difference between fallopian tube and ovary is highly significant(P<0.01).The expression of Survivin protein in 12-monthold Ganhua sheep has significant differences between uterus and ovaries,as well as uterus and fallopian tubes(0.01<P<0.05),but there is no significant difference between ovaries and fallopian tubes(P>0.05);The expression of Survivin protein in6-month-old Ganhua sheep has no significant differences between endometrium,fallopian tube,and ovary(P>0.05).The expression of Survivin protein in 2-monthold Ganhua sheep has significant differences between ovary and fallopian tube as well as ovary and uterus.(0.01<P<0.05)The difference between uterus and fallopian tube is not significant(P>0.05).(3)Survivin mRNA expression in the uterus of Small Tail Han Sheep is not significantly different between 2-month-old,6-month-old and 12-month-old(P > 0.05);Survivin mRNA expression in the oviduct of Small Tail Han Sheep is not significantly different between 2-month-old and 6-month-old(P > 0.05),and is highly significant between 2-month-old and 12-month-old as well as between 6-month-old and 12-monthold(P < 0.01)Survivin mRNA expression in the ovaries of Small Tail Han Sheep differed significantly(0.01 < P < 0.05)between 2-month-old and 6-month-old,not significantly(P > 0.05)between 2-month-old and 12-month-old,6-month-old and 12-month-old.The expression of Survivin protein is not significantly different in the three stages of uterus,ovary and fallopian tube in Small Tail Han Sheep(P>0.05).(4)The expression of Survivin mRNA in 2-month-old small tail Han sheep is not significantly different from the uterus and fallopian tubes(P>0.05),and there are significant differences between the uterus and ovaries as well as fallopian tubes and ovaries(P<0.01);Survivin mRNA is not significantly different in 6-month-old small tail Han sheep.The expression of sheep is not significantly different from the uterus and ovary(P>0.05),while the difference between the uterus and fallopian tube,fallopian tube and ovary is significant(0.01<P<0.05);The expression of Survivin mRNA in 12-month-old small tail Han sheep,the uterus The difference between the fallopian tube and the fallopian tube is not significant(P>0.05),the uterus and the ovary(P<0.01)as well as the fallopian tube and the ovary(0.01<P<0.05)are significantly different.The expression of Survivin protein is not significantly different in the three parts of 2-month-old,6-month-old and 12-month-old Small Tail Han Sheep(P>0.05).(5)The expression of Survivin mRNA in the same parts of the same period of time is not significantly different between the 2-month-old uterus and the 12-month-old endometrium(P > 0.05).the 6-month-old endometrium is significantly different(0.01< P < 0.05);The differences between the 2-month-old oviduct,the 6-month-old oviduct,the 12-month-old oviduct,the 2-month-old ovary,the 6-month-old ovary,and the 12-month-old ovary are not significant(P > 0.05).Survivin protein expression in 3-monthold ovary,3-month-old oviduct,6-month-old endometrium,12-month-old oviduct,12-month-old ovary,and 12-month-old ovary are not significant(P > 0.05);Survivin protein expression in 3-month-old uterus and 12-month-old ovary are significant(P <0.05)in both small tail Han sheep and Qianhua sheep.(0.01 < P < 0.05).3.Microvessel density(MVD)is obtained in three periods in Qianhua and small Tail Han sheep by detecting positive expression of CD34 in the endometrium using immunofluorescence method.The study shows that Survivin gene mRNA is positively correlated with MVD in three periods in Qianhua Meat Merino Sheep and small Tail Han sheep. |
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