Effect Of Controlled Ovarian Hyperstimulation On Expression Of Aquaporin-3 In Mouse Oocytes Using Semi-quantitative Real-time PCR Analysis

BackgroundWater movement across cells occurs by passive transport through either the lipid bilayer or protein water channels termed aquaporins (AQPs). These water channels were found in tissues that exhibit rapid and regulated movement of water, such as the kidney and lung. Aquaporins are members of the major intrinsic protein (MIP) family of transmembrane channel proteins. Up to now, 13 aquaporins have been identified in mammalian tissuses such as kidney, lung, brain, and pancreas (AQPs0-12). All mammalian aquaporins contain six trans-membrane-spanning domains and form an hourglass structure. In all of the mammalian aquaporins, excluding AQPs-4 and -7, there are extracellular mercurial-sensitive sites in which Hg²⁺ binds an extracellular cysteine residue and inhibits water movement through the pore. Previous studies indicated specific aquaporins were expressed in both male and female reproductive tissues of the rat, mouse, and human. For example, aquaporin-3 and -7 have beenshown expressions in the mouse oocytes. In the cryopreservation of cells, the movement of water and cryoprotectants across plasma membranes play a crucial role in the survival of the cells. Controlled Ovarian Hyperstimulation (COH) has been extensively utilized in ART for the stimulation of growth of large numbers of follicles and the retrieval of many oocytes used in IVF-ET. But previous studies have shown that COH is unfavorable for uterine receptivity and maybe damage it. These studies show that COH is disadvantageous for mammalian reproduction, but at the same time it can stimulate the growth of large numbers of follicles. Previous studies have shown that the fertilization rate in vitro was decreased after superovulation with high-dose stimulation. On the other hand, embryo freezing has been a successful practice, but previous attempts of oocyte cryopreservation achieved poorer results due to the low rates of survival, fertilization, and cleavage.Based on these observations, we designed this experiment to test our hypothesis stating COH is disadvantageous for mammalian oocytes quality. For this purpose, we investigated the expression of mRNAs of aquaporin-3 in mouse Mil oocytes after ovulation induction with PMSG by semi-quantitative real-time PCR and observed the rate of fertilization in vitro.ObjectiveThe aims of the present study are to evaluate the expression of mRNA of aquaporin-3 in mouse Mil oocytes after Controlled Ovarian Hyperstimulation with PMSG by semi-quantitative real-time PCR and observe the rate of fertilization in vitro to investigate the influences of Controlled Ovarian Hyperstimulation on mouse oocytes and the rate of fertilization in vitro.Material & MethodsThe animals used in these experiments were virgin 6⁷ weeks old female ICR mice. The mice were housed in 12/12 hours light/dark cycle (light on from 0:00 to 12:00) at 25 ± 0.5 °C and 50⁶0% of humidity. The mice were fed with a standard pellet diet and water. Sixty mice were randomly allocated into 2 groups labelled the COH group and the control group. Mice in the COH group were superovulated at 6 pm by intraperitoneal injection of 7.5 IU pregnant mare serum gonadotropin ( PMSG) and followed by 5 IU human chorionic gonadotropin (HCG) after 46⁴8 h. In the control group, the mice estrous cycle was identified by vaginal discharge and smear observed at 9am everyday. After 12—14h following HCG or next day 8:30am after estrum exhibited, mice were killed by cervical dislocation, and oviducts were excised. Cumulus masses were recovered from the dilated ampullae under a dissecting microscope and digested granulosa cells using hyaluronidase. The naked oocytes were washed repeatedly with Phosphate-buffered saline (PBS). The oocytes suspended in a small amount of PBS (about 0.5ul) were transffered to a 0.5 ml microcentrifuge tube, frozen with liquid nitrogen, and stored at -70 "C until RNA extraction. Oocytes swelling assay and Real-time reverse transcription polymerase chain reaction (RT-PCR) were used to determine the expression of aquaporin-3 mRNA in mouse Mil oocytes. In vitro fertilization was performed to calculate the rate of fertilization.Results1. Inhibition of swelling of oocytes in hypotomic medium after incubation in PBS with 50uM HgCb: HgCb prevented Mil oocytes swelling in hypotonicmedium after incubation in 50uM HgCk (P < 0.05).2. Comparison of aquaporin-3 mRNA expression in COH group and thecontrol group with real-time, semi-quantitative PCR: the expression of aquaporin-3 mRNA in mouse MII oocytes was confirmed in both groups. We demonstrated that the expression of AQP3 mRNA was significantly decreased (P < 0.05) in COH group (ACT=0.65±0.23) compared to the control group (ACT=2.49±0.05).3. The fertilization rate in vitro: The fertilization rate in COH group (66.27%) was significantly lower (P < 0.05) than in the control group (83.67%) in IVF.Conclusions1. Aquaporin-3 mRNA are expressed in mouse MII oocytes.2. Controlled Ovarian Hyperstimulation decreased the expression of AQP3 mRNA in mouse oocytes.3. The decrease of the rate of fertilization in vitro in COH group may be mediated by the down-regulation of AQP3.