| IntroductionEstrogen and its receptor signaling system play very important roles in themorphogenesis and functional differentiation of mammary glands as well as theirpathogenesis process. The benign and malignant breast diseases such as breast cystichyperplasia and breast cancer are associated with disorders of the ER signal system.Studies on ER have not been interrupted since Elwood V Jensen theoretically confirmedthe existence of the estrogen receptor for the first time in the1960s. The estrogen receptorhas five structural domains with three main activation pathways including classical ligandactivation pathway, non-classical activation pathway and phosphorylation activationpathway. ER can form the ligand-receptor complex by combining with estrogen to causeconformational changes of the estrogen receptor and form dimers within the cell nucleus,so as to combine with the functional element ERE on the target gene to activatetranscription; in addition, ER also can activate transcription by combining with AP1andSP1. There are multiple phosphorylation sites on estrogen receptor, which are mainly inthe AF1area, for example, the phosphorylation of serine305can activate ER withoutcombining with ligand. However, at present, the relationship between phosphorylation sies of estrogen receptor and the mammogenesis and carcinogenesis of breast has still not beenclearly illuminated.Knockin mouse is a very effective way and tool to study the functions of specificgenes/protein site. It has played an important role in creating human disease-relatedanimal models, investigating the pathogenesis of diseases, analyzing related genes,preventing disease, also as an important evaluation system in innovative drug research anddevelopment. Thus, gene-modified mouse is not only conducive to the development offorefront in the life sciences, but also plays a leading role in medical research.Early laboratory study discovered the305Ser site of ER (ER S305) is aphosphorylation site. Pak1kinase and PKA kinase could phosphorylate this site. Thephosphorylation of this site can activate ER without binding to the ligand, thus it might beinvolved in mammary gland carcinogenesis, progression and development of resistanceto anti-estrogen therapy. The study intends to produce the mutant mice integrated with thepoint mutation ER-S305A, to observe how this will affect mammary gland developmentand carcinogenesis if this phosphorylation site is lost，as well as to elucidate the exactregulatory mechanisms of ER, which will serve as further evidences to breast cancerchemoprevention and play an active role in the entry of potential therapeutic target.ObjectivesThis research mutates serine into alanine by producing ER S309A mutant mice, toobserve mammary gland development of these mice. By observing homozygous mice,including mammary gland development, estrous cycle before/after treatment with estrogen,this study aims at proving whether the mutated ER is functional.Methods1. Establish ER-S309A gene replacement mice, extract the total RNA, and make genesequencing after reverse transcription to confirm whether the establishment of replacementmice is successful.2. Genotyping mice with PCR combined with restriction assay, and observe whether homozygous-type female mice and male mice have breeding ability, if yes, observefeeding capacity of female mice.3. Homozygous type and wild type female mice were separately measured bloodconcentrations of estradiol.4. Take the third and fourth pairs of mammary glands of homozygous-type andwild-type female mice of6,9and12weeks old, conduct azocarmine whole mountstaining to observe the development of mammary ductal system, and conducthematoxylin-eosin (HE) staining to observe the changes of estrus cycle.5. After bilateral ovary castration surgery on wild-type mice of3weeks old, divide theminto estrogen-giving group and control group; Give estrogen to homozygous-type micefrom3-week old on, then observe the fourth pair of mammary glands of mice of6,9,12weeks old in hormone-supplementing group and control group respectively: Conductazocarmine staining to observe the development situation, and conduct hematoxylin-eosin(HE) staining to observe the change of estrus cycle.6. Homozygous-type ovaries and fallopian tubes have obvious morphological change.Results1. The establishment of ER S309A gene replacement mice is successful. Sequencinganalysis showed that the309-serine was replaced by alanine.2. Homozygous male mice are infertile. However, the histology of the testes of theseanimals looks normal. The homozygous female mice are fertile but the fecundity islow..3. The blood estradiol concentration of female mutant mice was not significantlydifferent from that of wild type mice..4. The ducts of female mutant mouse mammary glands grew much slower than that ofwild type mice. There were less branches, terminal end buds and glandular acini in mutantmice. The estrous cycle of mutant mice was in disorder and the number of keratinocyteswere markedly decreased.5. The mammary glands of wild type castrated female mice can grow normally if they are given estrogen, and keratinocytes appear obviously during estrous cycle; there is notobvious ducts generated in mammary gland with carmine dye of control group withoutbeing given estrogen, and they have no estrous cycle. The mammary gland ducts ofmutantfemale mice grow faster after they are given estrogen from the third week; and thebranches increase; the number and area of terminal buds increase; a large number ofkeratinocytes appear during estrous cycle.6. Homozygous mice ovaries and fallopian tubes did not see abnormalities.Conclusions1. The generation of the ER-S309A gene replacement mouse was successful;2. The function of estrogen receptors is compromised and the male mice lost theirreproductive function when the ER-S309phosphorylation site was lost; while thereproductive function of female mice was decreased.3.The estrous cycle of mutant female mice was in disorder, the keratinized vianialepithelial cells were reduced in mutant female mice. Ectopic supplement of estrogenrestored keratinization of vaginal epithelial cells.4. In the process of mammogenesis, the growth rate of the mammary duct of the S309Agene replacement mice was decreased due to the reduced function of estrogen receptor.5. The blood estradiol level of female mutant mice is normal. |
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