Structure And Function Of Aquaporin Water Channels

Aquaporins (AQPs) are water channel proteins that are expressed in procaryotic and eukaryotic cell membrane and transport water specifically and rapidly. Thirteen aquaporins have been identified in mammals and named AQP1-AQP12. Aquaporins are expressed in various tissues and organs and play important physiological roles. Extensive studies on the structure and function of aquaporins have been performed, and the approaches and methods of the studies are diverse. One of the approaches is study on gene targeting mouse model, which result is direct, reliable, and reflects the gene function in integral body. In the present dissertation, studies on the structure and function of aquaporins were performed using or establishing aquaporin gene targeting mouse models.There are two sections in the present dissertation. In the first section, we discovered a new function of AQP8 in female reproductive system using AQP8 knock out mice, and demonstrated the mechanism. First, we found signi?cantly increased number of offspring delivered by AQP8-/- mothers compared with wild-type mothers in cross-mating experiments. Comparison of ovulation in the two genotypes demonstrated that AQP8-/- ovaries released more oocytes. Histological analysis showed increased number of corpus luteums in mature AQP8-/- ovaries, suggesting increased maturation and ovulation of follicles. By RT-PCR, western blot and immunohistochemistry analyses, we determined the expression of AQP8 in mouse ovarian granulosa cells. Granulosa cells isolated from AQP8-/- mice showed 45% of decreased membrane water permeability than wild-type mice. As the atresia of ovarian follicles is primarily due to apoptosis of granulosa cells, we analyzed the apoptosis of isolated granulosa cells from wild-type and AQP8-/- mice. The results indicated signi?cantly lower apoptosis rate in AQP8-/- granulosa cells (21.3±3.6% vs. 32.6±4.3% in AQP8+/+ granulosa cells). Taken together, we conclude that AQP8 de?ciency increases the number of mature follicles by reducing the apoptosis of granulosa cells, thus increasing the fertility of female mice. Another important phenotype of AQP8-/- mice is the increased number of multi-oocyte follicles in ovaries, suggesting that AQP8 involves in the formation of follicles. This discovery may offer new insight of the control of female fertility by regulating aquaporins. The second section of the present dissertation is the establishment of AQP4-A25Q knock-in mouse model. AQP4 is an important water channel expressed in central nervous system, and play significant roles in cerebrospinal fluid. AQP4 knock-out mice showed obstructive hydrocephalus and altered brain edema status. A special character of AQP4 protein in membrane is the formation of orthogonal arrays of particles (OAPs), which function is unknown. The amino acid mutation AQP4-A25Q was reported to disturb the formation of OAP. In the present study, we plan to establish the AQP4-A25Q knock-in mouse model, and then research the functions of OAP using this model. First, an insertion targeting vecter was constructed, then was transfected in ES cells. After selection of the transfected ES cells, we identified the correctly recombinated ES cell clones and micro-injected them in blastocysts. Chimera mice then were mating and were performed the Cre-LoxP excision. Finally the homozygous AQP4-A25Q knock-in mouse model was established. The analysis as genotyping, RT-PCR, DNA sequencing and immunohistochemistry were performed on the mice and identified the success of AQP4-A25Q mutation on genomic, mRNA and protein levels. Further studies are detection of the OAP disorder and the researches of OAP functions. Our work of establishment of AQP4-A25Q knock-in mouse model offers effective model for the studies of AQP4 OAP function in nervous system.