Expression Of Aquaporins In Human Endometrium And Study On The Ovarian Steroid Hormone Regulation

Water is the major component of cells and and it composes 70% of the total weight. Most physiological and biochemical reactions take place in waterycondition.Water movement across cell membrane is a fundamental property of life. How water transport through a transmembrance route is a basically long-standing problem in the life science. Water may move across an epithelium by: (1) diffusion;(2) as a by-product of co-transporter activity;or (3) facilitated by aquaporins (AQPs) or water channels. Diffusion is an effective way to move water across epithelia, but it is dependent upon the steepness of the osmotic gradient across that epithelium. AQPs move water under the physiological state of near iso-osmolar gradients.Aquaporins are a family of hydrophobic intrinsic membrane proteins that efficiently and selectively transport water. Since the discovery of first water channel(AQPl) from the human red blood membrane by Agre et al. in 1992, rapid and serial progresses have been made in characterization of AQP structure and function. At least 12 homologous members(AQP0~AQP12) have been molecularly identified in mammals.AQPs are expressed in various epithelium and endothelium involving fluid secretion and absorption, and in many cell types such as erythrocytes, white blood cells, adipocytes, and muscle fibers that have no obvious relationship with fluid transport. The extensive expression pattern of AQPs may indicate functional importance in multiorgan physiology and pathophysiology.The movement of fluids across cell membranes in the uterus is an importantaspect in the menstrual cycle and implantation. Transcellular water movement through proteinaceous water channels, termed aquaporins (AQPs), is a crucial mechanism for reproductive physiology. These AQPs are able to support a large volume of water flow. In the female reproductive tract, tissue fluid shifts result in uterine edema at different stages of the menstrual cycle. The cycling endometrium undergoes recurrent uterine stromal edema in response to hormonal stimuli . The uterus also manifests an inflammatory-like response at the time of insemination that is marked by generalized edema and hyperemia. Fluid produced and secreted by endometrial cells of uterus at the secretory phase provides a physiological medium for embryonic implantation and development.The molecular mechanism that controls formation and volume of endometrial fluid remains to be determined. The purpose of the present study was 1) to investigate the profile expression of AQP1-AQP9 in human uterine endometrium;2) to determine whether the expression of uterine AQP2 presented a periodicity going with the menstrual cycle;and whether uterine AQP2 expression had correlation with serum 17 3 -estradiol (E2) and/or progesterone (P) levels.3) to probe the regulative mechanisms by ovarian steroid hormones.Part One: Expression of Aquaporin 1~ 9 in Human EndometriumObjective: To investigate the expression profile of aquaporin 1-9 (AQP1-AQP9) in human normal endometrium.Method: The transcription of 10 isoforms of aquaporins (AQPs) that have been reported in the female mammalian reproductive system in normal human endometrium were first detected by reverse transcriptase-polymerase chain reaction (RT-PCR), including 10 cases of proliferative and 10 cases of secrectory phases, respectively. And immunohistochemistry was used to examine the translation and location of AQP 1,-2 ,-8.Results: Among thelO members of AQP family, AQP 1,-2 ,-8 expressed at the mRNA level in the normal human endometrium 1 including both the proliferative andthe secretory phases. Immunohistochemistry was used to examine the translation of AQPl,-2 ,-8. The immunohistochemical method detected that AQP1 was located in microvascular and small vessel epithelial cells. AQP2 and AQP8 was prominent in the glandular epithelial cells, and AQP2 was also evident in the luminal epithelial cells. Both of AQP2 and AQP8 had weak immunoreactivity in the stromal cells.Conclusion: AQP1 maybe associated with the water permeability of the microvascular and small vessels in human endometrium. And AQP2 and AQP8 maybe take a part in uterine gland secretion and absorption, while AQP2 could play a role in the formation and clearance of uterine cavity fluid.Part Two: Cyclical Expression of Aquaporin 2 in Human Endometrium and its Correlation with Serum Ovarian Steroid HormonesObjective: To examine the expression of aquaporin 2 (AQP2) in human uterineendometrium at the different menstrual phases and its relationship with the serum levels of ovarian steroid hormones.Method:Western Blotting, immunohistochemistry, and real time RT-PCR were employed in the study. RT-PCR detected its messenger RNA and the product was sequenced. Serum concentration of estradiol(E2) and progesterone(P) were measured by electrochemiluminescence method at the same time.Results: AQP2 was localized to the luminal and glandular epithelial cells, and there was weak immunoreaction in the stromal cells in the endometrium. In the proliferative phases, the expression of AQP2 peaked up in the late proliferative phase(1.95±0.13) compared with that of the early-mid phase(1.08±0.09)(P<0.01), and in the secretory phases its average level increased at the middle phase(1.634± 0.153) compared with that of the early(1.325±0.139) and late phases(1.09± 0.06).Message RNA was positive in all cases by RT- PCR and the PCR product was confirmed in nearly exact consistency with the Genbank by sequencing. However, The mRNA levels of AQP2 were not significantly altered at different menstrual cycles. Serum E2 level, but not P and E2/P level, was positively correlated with the normalized intensity of AQP2 in all phases^ = 0.6183, P<0.01;r2 =0.0310, P >0.05;r2 =0.058, P>0.05).Conclusion: The expression of AQP2 in human endometrium varied cyclically, with the highest in the late proliferative phase and then the mid-secretory phase.The highest level of endometrial AQP2 in the late proliferative phase suggests AQP2 maybe involve in the great reduction in viscosity of the luminal fluid, and the higher level in mid-secretory phase implies that AQP2 might play a physiological role in the receptivity of human uterus.Part Three: Study on the Regulation of Ovarian Steroid Hormones on Aquaporin 2 Expression in Cultured Human Endometrial Epithelial CellsObjective: To investigate the effect s of 17 3 -estradiol ( E2) and progesterone ( P4) on the expression of aquaporin 2 (AQP2) in cultured human endometrial epithelial cells.Method: The level s of AQP2 mRNA and protein in cultured human endometrial epithelial cells were analyzed by using RT-real time PCR and Western Blotting respectively. Endometrial cells was treated with 17 P -estradiol(E2,10"8mol/L), progesterone(P4,10-6mol/L), E2(10-8mol/L)+P4(10-6mol/L), E2(10-8mol/L)+ ICIi82780 (10"7mol/L) , respectively for 72h.The control group was received no special treatment.Results: A specific AQP2 band of 282 bp was visualized by RT-PCR analysis and 29 kDa was found by Western Blotting in all groups. Significant expression of AQP2 mRNA and protein was only seen in the cultured endometrial glandular cells treated with E2;P4 alone or plus E2 and E2 plus ICI182780 didn't induce the up-regualtion of AQP2 mRNA and protein. Both ICI182780 and P4 can inhibit the up-regulation induced by E2 based on the priming of E2, respectively. One cell swelling assay was used to investigate water permeabilityof cultured endometrial glandular cells in response to E2 .For the flow cytometry-based cell swelling assay, a population of cells enriched for cultured endometrial glandular cells were harvested and resuspended in PBS. The cell size distribution was then analyzed by flow cytometry. Water was then added to adjust the osmolarity to 210 mOsM. Following a 2min incubation, the cell size distribution was again analyzed by flow cytometry, and histograms of initial and final cell volumes were overlaid for comparison. These results show that the cultured endometrial glandular cells of the control group display similar initial and final volumes, whereas the cultured endometrial glandular cells from E2-treated uteri swell substantially after 2min, and such E2-treated swell can be inhibited by priming use mercury chloride.Conclusion: AQP2 expression in cultured endometrial epithelial cells was upregulated by estrogen.The stimulative effect of estrogen on AQP2 expression is inferred to be through the estrogen receptor. Estrogen maybe enhance water permeability of the cultured human endometrial epithelial cells through AQP2.