The Study Of The Reason And Mechanism Of Delayed Procreation In UT-B Null Female Mouse

Urea transporter (UTs) proteins are central to modulating urea movementacross biological membrane. To date, all of the known facilitative UT proteinsare the product of two genes, UT-A (Solute Carrier Family 14α2, slc14α2) andUT-B (slc14α1). The two UT genes occur in tandem on chromosome 18q12-21.UT-B is strongly expressed in red blood cells and kidney, as well as manynon-renal tissues, such as heart, brain and testis. Although major steps havebeen made in defining the molecular identity and basic functionalcharacteristics of UT-B, little information was known about their physiologicalfunction. Therefore, researchers have generated UT-B null mice to study thephysiological function and they have found that the urine concentrating abilityof the UT-B null mice decreased because the urea countercurrentmultiplication process was blocked. Moreover, they found Jknull that is JknullRBCs are easily screened out by increased resistance to lysis in 2 M ureasolution. However, little information was known about their physiologicalfunction in other organs of the UT-B null mice and this paper aim directly atfemale procreate system.Objective :1 We used UT-B null male mice to mate with female mice and producedUT-B null female mice. The physiological features of the UT-B null femalemice were studied.2 To explain the mechanism of the delayed procreation for UT-B nullfemale mice.Methods:1 UT-B null mice's origin procreation and identification(1)The UT-B null male mice were accorded by Professor Alan. SVerkman University of California, San Francisco USA in Sep.10th 2004 andauthorized us to use(.the authorizing paper is attached )(2)According to thecleaning breeding standard, the mice were raised and reproduced in the AnimalDepartment of Jilin University .(3)Genotype identification: when the micewere 20d old, we cut their tails and extract the genome DNA, PCRamplification, and identified by agarose gel electrophoresis. ( 4 ) Thedifference for the physiological feature between the UT-B null female mice(UT-B -/-)and the wild type(UT-B +/+) was studied: ① 2mol/L ureahemolysis test was used to observe the erythrocytic reactiveness of the UT-B-/-and UT-B +/+ female mice. ② The UT-B gene and protein expression inthe female reproductive organ was detected by RT-PCR, hybridization in situ,western blot and immunohistochemistry techniques. ③ Urea content in theamniotic fluid of the mice which were preganant for 18d was determined bybiochemistry methods.2.The cause and mechanism of the delayed procreation in the UT-Bnull female mice(1)Using the reproductive competition experiment, We select the samegenetic background UT-B-/-female mouse and UT-B+/+ female mouse andfeed them with a mature male mouse in the same cage. And we observe theconception rate, the time that they give birth to the young mice and thequantity and the condition of the young mice. (2)The body weight, uterineweight and the organ coefficient of the female mice in the different days weredetermined and calculated. ( 3 ) The morphology observation andsemi-quantitative analysis:selecting ovaries, uterus, and oviduct of the 4w,6w and 9w old female mice for both UT-B-/-female mouse and UT-B+/+female mouse, and then fixed them with neutral formalin then paraffinimbedding, HE dyeing ,at last we observed them with light microscope to lookfor the difference between the two groups.(4)The plasma estrogen (E2)content in the 4w,6w and 9w old female mice for both UT-B-/-female mouseand UT-B+/+ female mouse were determined with radio-immunity technique.(5)We also tested the hypothalamus GnRH mRNA, pituitary ovaries GnRHRmRNA and FSH, ovaries FSHR mRNA and ER-βexpression by RT-PCR andwestern blot techniques(.6)In addition, the AQP8 mRNA and AQP1 protein inthe ovaries, uterus, oviduct and placenta were also studied by RT-PCR andwestern blot techniques.Results and discussion:1.Genotype identification and biology characteristics of the UT-B nullfemale mouse1.1 The result for the genotype identification of the UT-B null femalemouse:Under the condition of the UT-B gene null mice from University ofCalifornia, San Francisco USA, by procreating we got the heterozygote mice,and then we mated the heterozygote mice with the heterozygote mice thenwe've got the UT-B -/-and UT-B +/+ mice.When the mice were 20d old, we identified their genotype. Theelectrophoresis for the gnome DNA suggest that they were UT-B -/-mice onlywhen there was amplification product at 250bp, and they were UT-B+/+ miceonly when there was amplification product at 400bp. If there were two bandsat 250 and 400bp, they must be heterozygote mice. And the first filialgeneration of the heterozygote mice were three genotypes:UT-B+/+,heterozygote type and UT-B -/-. And the ratio is in line with Mendel's law ofinheritance.1.2 UT-B-/-mice showed the hemolysis resistance to 2mol/L ureasolution: the erythrocyte from the UT-B-/-female mice add to the 2mol/L ureasolution, the cell shrinked immediately, and then swelled gradually until mosterythrocyte hemolyzed after 10min. While the erythrocyte from the UT-B+/+female mice add to the 2mol/L urea solution, the cell hemolyzed immediately.There is significant difference between the two groups. This result is accordedwith the genotype result. That is to say we have successfully built the genotypeidentification system for the UT-B -/-mice.1.3 The contrast study on the UT-B gene and protein expression in thefemale reproductive organ: we determined the UT-B gene and proteinexpression in the ovaries, uterus, oviduct and placenta by hybridization in situ,western blot and immunohistochemistry techniques. And we found the UT-Bgene and protein expressed with the different level in the UT-B+/+ mice,especially expressed significantly in uterus, oviduct and placenta. This willexplain UT-B plays an important role in the urea transporting in the femalereproductive system.. The UT-B gene and protein expression in the UT-B-/-mice were negative. This result will also prove that we have built the UT-Bgene null animal model, this animal model will set the foundation of the UT-Bfunction in the genital system.1.4 The urea content in the amniotic fluid: we have found that the BUNcontent of the UT-B-/-female mice which are pregnant for 18d is evidenthigher than that in the UT-B +/+ mice. The accumulating urea will illustratethat the UT-B express in the placenta will help the foetus to clean themetabolic product .2. The cause and mechanism of the delayed procreation2.1 UT-B-/-female mice reproduce delayed, the time of the UT-B-/-female mice give birth to the young mice for the first time is 4.5±0.3d laterthan the UT-B+/+ female mice. There is no difference between the UT-B-/-female mice and UT-B+/+ female mice in conception rate, the number of theyoung mice and the mice's common condition.2.2 The contrast observation between the two groups about femalegonadial morphology: ① The uterine wet weight of the UT-B -/-is creasedsignificantly in the 4w,6w and 9w old. The uterine wet weight of the UT-B -/-with 4w and 6w old is lower than that in the UT-B+/+ female mice, but there isno difference in the organ coefficient of the two groups. The body weight ofthe UT-B -/-female mice after 4w old is lower than that of the UT-B+/+female mice. ②The ovaries and uterus development condition of the 4w oldUT-B +/+ mice's is prior to that of the UT-B -/-female mice, but there is nodifference between the two groups after 9w old by the HE dyeing .Observation with the light microscope observation, we found that: (1)ovaries:The ovaries of the UT-B +/+ female mice is coated with simplesquamous epithelium, and the cortical part of the ovaries is made of follicle indifferent stage and follicle connective tissue. The medulla of the ovaries ismade of loose connective tissue, and there are many fusiform shape matrix celland blood vessel in the medulla. The ovaries' formation of the UT-B +/+ andUT-B-/-female mice is same. But the ovaries' volume of the UT-B-/-mice issmaller than that of the UT-B+/+ mice, and the ovotid development conditionof the UT-B+/+ is more active than the UT-B+/+ female mice. When they are9w old, there is no difference in the ovotid development condition. (2)Uterus:the construction of the UT-B+/+ female mice is the same as that of the UT-B-/-female mice, but the uterine development condition of the 4w UT-B-/-is worsethan the UT-B+/+ female mice. After they are mature there is no different. Theresults above provide the morphological evidence for the reproducing delay.2.3 Serum estrogen content: The serum estrogen content of the UT-B-/-mice for 4w and 6w is lower than that of the UT-B+/+ mice, at the time of 9wthere is no difference . The female mice's ovarian structure of the UT-B-/-mice for 4w is the same as that of the UT-B+/+ mice, but the ovarian volumeis smaller than the latter, and the ovotid development is later than the latter. Soit is not the reason that the ovaries per se pathocrinia caused the estrogenichormone difference. Estrogen(E2)can facilitate the sexual organ formationand secondary sex characteristics development of the impuberal animal.Therefore, estrogenic hormone level decrement perhaps is the direct cause tothe reproducing delay for the UT-B-/-female mice.2.4 We also detect the hypothalamus GnRH mRNA, pituitary GnRHRmRNA, FSH mRNA and ovaries FSHR expression by semi-quantitateRT-PCR and detect the ER-β protein expression in the ovaries by Western blot:The level of the hypothalamus GnRH mRNA , pituitary GnRHR mRNA, FSHmRNA and ovaries FSHR in the 4w UT-B -/-female mice is greatly lowerthan that of the UT-B+/+ female mice by PCR production electrophoresis grayscale scanned. Western blot showed that ER-β protein level in 4w UT-B -/-female mice is lower than UT-B+/+ female mice. There is no difference in thehormone and receptor for 9w mice between the two groups. The femaleprocreate hormone and receptor level in the hypothalamic-pituitary-gonadalaxis (HPGA) is important to the female reproductive development. That willexplain that estrogen decreased in the reproductive delay of the UT-B -/-female mice is caused by the function of the hypothalamic-pituitary-gonadalaxis (HPGA) is abnormal. The mechanism of the UT-B gene null affected thehypothalamic-pituitary-gonadal axis(HPGA) need study later.2.5 Semi-quantitative RT-PCR detected AQP8 mRNA expression in theovaries and oviducts: AQP8 mRNA expression in the ovaries and oviducts ofthe UT-B -/-female mice is remarkably higher than that of the UT-B +/+female mice.We detected the AQP1 protein expression in the ovaries, uterus, placentaand oviducts by Semi-quantitative Western blot method: AQP1 proteinexpression in the uterus UT-B -/-female mice is higher than that in the UT-B+/+ female mice, and there is no difference in the other organs. It is knownthat UT-B have the ability to transport water, AQPs expression is creased bycompensating mechanism in the UT-B-/-female mice. Therefore, AQP8expression in the oviduct and AQP1 expression in the uterus is increased,which is probably a compensating mechanism that UT-B may play animportant role in the transporting water in these parts.ConclusionOn the basis of building the technological platform of genotypeidentification of the UT-B null female mouse successfully, this paper studiedthe UT-B function in female reproductive system. ①The UT-B null femalemouse have significant hemolysis resistant phenotype. ② There are theUT-B gene and protein expression in the ovaries,uterus, oviduct and placentaetc. female reproductive organ while in the UT-B+/+ they are negativeexpression. ③ UT-B-/-female mice giving birth to the young mice for thefirst time is 4.5±0.3d later than the UT-B+/+ mice, so they are typicalreproduction delay animal model. ④The ovaries and uterine volume ofUT-B-/-female mice which are 4w old are smaller than the UT-B+/+ femalemice, and also the development condition of the ovum is worse than the latter.But when they are mature, (after 9w) there is no difference between the twogroups. ⑤The reason which caused the delayed procreation and reproductiveorgan growth retardation is related with the serum estrogen contentdecreased in the 4w UT-B-/-mice, while the decrease of the serum estrogencontent is related with the regulation of the hypothalamic-pituitary-gonadalaxis(HPGA). ⑥The content of the AQP8 mRNA in the UT-B -/-oviduct,AQP1 protein level in the UT-B -/-uterus are obviously higher than the UT-B+/+ female mice, perhaps they are a kind of compensating change.The report is the first one in the world that the UT-B gene and proteinexpress in the female reproductive organ and in the placenta and the discoveryof the delayed procreation and the mechanism in the UT-B-/-mice. The resultsof this study also supplied the experimental evidence for the female delayedprocreation mechanism as well as modulation and set the foundation of theUT-B function in the genital system and neuroendocrine system.