The Role Of AQP2/5for Fluid Homeostasis In Mice Reproductive System

The aquaporins (AQPs) are a family of membrane water channels that are conservedthroughout the animal and plant kingdoms, as well as lower organisms. So far, thirteenmembers have been identified in mammalian, which are widely distributed in specific celltypes in many organs and tissues. The primary function of most aquaporins is to facilitatewater transmembrane transport, playing an important role in organism water homeostasis.These aquaporins showed tissue-specific distribution in body, and a lot of reports haverevealed their expression and function in the kidney, brain, lung, intestine and other organs.However, much less is known about of AQPs for fluid homeostasis in the female reproductivesystem. Ovary and uterus are important components in female reproductive system. In rodentspecies, the ovary and the end of oviduct are encapsulated by a thin membrane so-calledovarian bursa. The biological function of ovarian bursa remains unknown. Its structureappears to facilitate oocytes transport into oviduct. The normal intrauterine fluid environmentis essential for embryo implantation. For instance, in hydrosalpinx patients, the implantationand pregnancy rates are markedly decreased after in vitro fertilization-embryo transfer(IVF-ET), while salpingectomy could significantly improve the pregnancy rates. The leakageof hydrosalpinx fluid into the endometrial cavity was supposed to be the major cause forimpaired fertility. However, the underlying mechanisms of hydrosalpinx fluids onimplantation and ongoing pregnancy were not fully understood and remain controversialregarding its toxicity.In this study, using a variety of molecular biology and physiology methods, we explore:1) Hormonal regulation of ovarian bursa fluid in mice and the aquaporins expression pattern;2) The expression of aquaporins in uterus, aberrant implantation and pregnancy outcome dueto excessive mice intrauterine fluid.1. In the present study, we observed a rapid fluid accumulation and reabsorption withinthe ovarian bursa after ovarian stimulation (PMSG-primed hCG injection).There is a rapidfluid accumulation at1h after hCG, followed by gradual reabsorption at2~5h after hCGinjection. The rapid fluid turn over within ovarian-bursa compartment had led us tohypothesize that the aquaporin family might play an active role around the timing of ovulation.By means of RT-PCR, Real-time PCR, immunofluorescence and surgical isolation ovary and bursa, we detected the expression and location of aquaporins in the ovarian tissue and inisolated ovarian bursa. Aqp2/5showed dynamic changes, which was closely related with theintra-bursa fluid dynamics. Further immunofluorescence examination of AQP2and AQP5inthe ovarian bursa revealed that AQP2is specifically localized in the outer layer (peritonealside), whereas AQP5localized in the inner layer (ovarian side) of the bursa. These resultssuggested that they might be involved in efficient water transport through ovarian bursanearby ovulation.2. Though further analysis of the proteins interacted with AQP5, by usingco-immunoprecipitation, Western Blot, SDS-PAGE, sliver and MS, we found that AQP5interaction with ACTIN, TMP1, TMP3, MYL12B, MYL6, suggesting that these proteins wererelated to the cellular trafficking of AQP5.3. By infusing different volume of non-toxic fluid (0.9%saline) into uterine lumenbefore embryo implantation in mice, we established a model of hydrometra, by which wefound that despite the embryos were not flushed out from the uteri, the timing of implantationdelayed and normal intrauterine distribution (embryo spacing) was disrupted. The abnormalimplantation at early pregnancy further lead to embryo growth retardation, miscarriage andincreased pregnancy loss. In this model, via Real-time PCR, frozen sections and othermethods, we further examine the expression of aquaporin and receptivity related gene inuterine. The results showed that the expression of aquaporin were not changed, but theexpression of integrin α(v) and β(3) were increased in the infused mouse uteri, implicating acompensatory effect to cope with the excessive fluid environment in order to facilitate theattachment. Besides, this animal model is similar to the adverse effects observed inhydrosalpinx patients, whose implantation and pregnancy rates are markedly decreased afterIVF-ET. Our data showed that the volume of fluid in uterine lumen is critical before embryoimplantation in mice, suggesting the excessive fluid might be the major cause of decreasedimplantation rate in the patients with hydrosalpinx.Taken together, our findings suggest that expression of AQP2and AQP5under ovulationhormones is actively involved in bursa fluid homeostasis in female reproductive system,which might be related to5cytoskeletal proteins that interact with AQP5. In addition, theunconspicuous change of aquapoins in the excessive intrauterine fluid model implicated thatan existence of compensatory effect to the excessive fluid environment, which might bereveal the major cause of decreased implantation rate in IVF-ET patients with hydrosalpinx.