Demonstration of 24p3 Protein in Mouse Reproductive Tracts and Regulation of its Expression in Uterus by Ovarian Steroids

• Main Authors: Hsien-Lu Huang, 黃憲祿
• Other Authors: Yee-Hsiung Chen
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/23805408366494656606

博士 === 國立臺灣大學 === 生化科學研究所 === 87 === Abstract A glycoprotein in mouse uterine luminal fluid was isolated by Sephadex G-100 chromatography and purified to homogeneity by HPLC on a reverse-phase C18 column. Automated Edman degradation was unable to determine the N-terminal residue of this glycoprotein and the partial sequences determined from its trypsin digests were found to be identical with the protein sequences deduced from 24p3 cDNA. The core protein and the total amount of carbohydrate together gave a molecular mass of 26 kDa. Results from the characterization of the glycopeptide bond indicated the presence of N-linked carbohydrate but no O-linked carbohydrate in the protein. A sandwich ELISA method was employed to examine the distribution of 24p3 protein in the mouse reproductive tract. The protein was not detectable in blood. It was present in a considerable amount in epididymis, vagina, cervix or uterus, although it was not found in ovary, testis, seminal vesicle, prostate, vas deferens or coagulating gland. We examined 24p3 expression in mouse uterus at various stages of the natural estrous cycle and during the preimplantation period. The level of 24p3 mRNA appeared intensively in proestrus and estrus, declined sharply from metestrus to diestrus. Consistent with this observation, 24p3 protein was abundant in proestrus, decreased from estrus to metestrus and declined to a very low level in diestrus. The uterine 24p3 expression closely overlapped with the estradiol (E2) surge in proestrus and estrus but it was suppressed when progesterone (P4) rose to a high level during the reproductive cycle. Neither the protein nor its transcript of 24p3 gene was detected in the uterus of immature mice or ovariectomized adult animals. While an injection of P4 to these animals was unable to initiate the uterine 24p3 expression, administration of estrogenic steroids to these animals markedly stimulated the gene expression. Treatment of these animals with E2 together with P4, on the other hand, did not stimulate the gene expression. In the day 1(D1) pregnant animals, 24p3 mRNA remained at a high level on D1 and D2 but dropped to an almost undetectable level on D3 and D4. This accompanied with decrease in 24p3 protein from D1 to D2 and decline in the protein to undetectable level from D3 to D4. The staining patterns of both the immunohistochemical localization of 24p3 protein and in situ hybridization for the detection of 24p3 mRNA in the uterine sections showed that 24p3 expression took place mainly in the luminal and glandular epithelial cells of endometrium. This together with our previous observation in which 24p3 protein was shown in uterine luminal fluid indicates that the protein is secreted primarily from these cells to their respective luminal surfaces during proestrus and estrus. Among the caput, corpus and cauda of epididymis in adult mice, 24p3 protein was immunodetected by Western blot procedure. Immunohistochemical staining patterns of the epididymal section showed that the 24p3 protein appeared only in the epithelial cells of ductuli efferentes of the proximal caput. 24p3 protein in the caput of epididymis disappeared when the animals were castrated and this could not be restored by administration of DES or testosterone.