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1 IUBMB Life, 62(11): , November 2010 Research Communication Increased Female Fertility in Aquaporin 8-Deficient Mice Weiheng Su 1, Ying Qiao 1, Fei Yi 1, Xingang Guan 1, Di Zhang 1, Shuzhi Zhang 1, Feng Hao 1, Yinghong Xiao 1, Hongguo Zhang 1, Lei Guo 1, Longfei Yang 2, Xuechao Feng 1 and Tonghui Ma 1,2 1 Membrane Channel Research Laboratory, Northeast Normal University, Changchun, China 2 Central Research Laboratory, Jilin University Bethune Second Hospital, Changchun, China Summary Aquaporin-8 (AQP8) is a water channel expressed extensively in male and female reproductive systems. But its physiological functions are largely unknown. In the present study, we first found significantly increased number of offspring delivered by AQP8 2/2 mothers compared with wild-type mothers in crossmating experiments. Comparison of ovulation in the two genotypes demonstrated that AQP8 2/2 ovaries released more oocytes ( vs in normal ovulation and vs in superovulation). Histological analysis showed increased number of corpus luteums in mature AQP8 2/2 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 2/2 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 2/2 mice. The results indicated significantly lower apoptosis rate in AQP8 2/2 granulosa cells ( % vs % in AQP8 1/1 granulosa cells). Taken together, we conclude that AQP8 deficiency increases the number of mature follicles by reducing the apoptosis of granulosa cells, thus increasing the fertility of female mice. This discovery may offer new insight of improving female fertility by reducing granulosa cell apoptosis through AQP8 inhibition. Ó 2010 IUBMB IUBMB Life, 62(11): , 2010 Keywords AQP8; fertility; granulosa cell; water permeability; apoptosis. INTRODUCTION Mammalian aquaporins (AQPs) are a family of water-transporting membrane proteins selectively expressed in cell types of various organs where they may play important physiological functions (1, 2). Received 8 October 2010; accepted 29 October 2010 Address correspondence to: Tonghui Ma, Membrane Channel Research Laboratory, Northeast Normal University, Changchun , China. Tel: Fax: math108@gmail.com Among the 13 members of mammalian AQP family, AQP8 has been shown to express extensively in the reproductive system (in testis and sperm in the male reproductive system and in ovary, oviduct, uterus, placenta, amnion, chorion, and cervix in the female reproductive system) (3 10). Yang et al. (11) studied the role of AQP8 in male reproductive function in a knockout mouse model. They reported that the weight and size of the testes in AQP8 2/2 mice were remarkably increased. However, no abnormalities in fertility and sperm count or morphology were found. The function of AQP8 in female reproductive physiology remains unknown. Folliculogenesis is a process from the primordial follicles, through primary, secondary, and antral stages to the largest Graafian or preovulatory follicles (POFs). In response to the luteinizing hormone surge, the POFs ovulate and release oocytes. After ovulation, the granulosa cells and theca cells remaining within the POFs differentiate into the corpus luteum (CL) (12). Granulosa cells play a nurturing role in supporting oocyte development and follicle maturation by providing essential nutrients and estrogen (13, 14). In each cycle of folliculogenesis, most primordial follicles that initially grown undergo atresia caused by apoptosis of granulosa cells, only small number of them is destined to become POFs (15). A previous study reported expression of AQP7, AQP8, and AQP9 in rat follicular granulosa cells and indicated that water permeability of antral follicles from rat ovary was predominantly transcellular and mediated by AQPs (6). However, the physiological importance of the AQPs in female fertility is unknown. In the present study, we identified a novel phenotype of increased female fertility in an AQP8 knockout mouse model. AQP8 deletion significantly decreased granulosa cell water permeability and protected granulosa cells from apoptosis, which may be responsible for the increased fertility in AQP8 knockout female mice. MATERIALS AND METHODS Mice AQP8 2/2 mice used in this study were described previously (11). The mice were maintained in a specific pathogen-free grade animal facility on a 12-h light/dark cycle. All experiments ISSN print/issn online DOI: /iub.398

2 INCREASED FEMALE FERTILITY IN AQP8-DEFICIENT MICE 853 Table 1 Fertility of female AQP8 2/2 mice Groups Genotypes Litters Pups Pups per litter 1 Wild-type female 3 wild-type male AQP8 2/2 female 3 wild-type male a 3 Wild-type female 3 AQP8 2/2 male AQP8 2/2 female 3 AQP8 2/2 male b a Significant difference compared with Group 1 (P \ 0.01). b Significant difference compared with Group 3 (P \ 0.01). were performed on age-matched wild-type and AQP8 2/2 mice in a C57BL/6 genetic background. Protocols for mouse experiments were approved by the Committee on Animal Research of Northeast Normal University. Immunohistochemistry Ovaries of AQP8 1/1 and AQP8 2/2 mice were fixed in 4% paraformaldehyde, and then embedded in paraffin. Continuous sections of 3 lm were prepared and processed by standard procedures. For immunohistochemistry, the sections were blocked by 5% normal goat serum for 30 min, and then incubated with anti- AQP8 antibody (Millipore, 1:200) diluted in 0.1 M PBS with 1% bovine serum albumin and 0.5% Triton X-100 at 25 8C for1h. Fertility of Female AQP8 2/2 Mice A 10-week-old female AQP8 1/1 and AQP8 2/2 mice were subjected to mating study as following: 1, wild-type female 3 wildtype male; 2, AQP8 2/2 female 3 wild-type male; 3, wild-type female 3 AQP8 2/2 male; 4, AQP8 2/2 female 3 AQP8 2/2 male. One female mouse was housed with one 12-week-old known fertile male mouse, and male mice were rotated weekly. Cages were monitored daily, and the number of pups and litters were recorded. Ovulation, Superovulation, and Oocyte Count Ovulation and superovulation were carried out on 10-week-old female AQP8 1/1 and AQP8 2/2 mice. To observe whether ovulation happened, one female mouse was mated with one fertile male mouse. The female mouse with vaginal plug next morning was euthanized, and the oviducts were excised. The cumulus cell oocyte complex (COC) or oocytes were harvested from the oviducts. After treatment with 0.3% hyaluronidase (Sigma) for 10 min, oocytes were counted. Photomicroscopy was performed by using an OLYMPUS DP72 system. The superovulation was performed by successive injections of pregnant mare serum gonadotropin (Sigma, 10 units/mouse) and human chorionic gonadotropin (Sigma, 10 units/mouse) with a 48 h interval. The oocytes were harvested 18 h later and counted by the same procedure. Histology Ovaries of AQP8 1/1 and AQP8 2/2 mice were processed for hematoxylin and eosin staining by standard procedures. The number of follicles for each ovary was counted from 10 of the largest sections and was normalized by the total ovarian area in the section as described previously (16, 17). Corpus luteum had a diameter greater than 175 lm in ovarian cross section. The ovary area was measured with PHOTOSHOP cs2 Version 9.0 (Adobe systems). Figure 1. Oocytes released from female AQP8 1/1 and AQP8 2/2 mice after ovulation. A: Representative images of oocytes or COCs released after ovulation. Scale bar, 500 lm. B: Statistical analysis of oocyte counts after ovulation (n 5 12). *, P \ C: Rates of COCs released after ovulation. 1/1, wild-type; 2/2, AQP8 2/2.

3 854 SU ET AL. Figure 2. The number of corpus luteums in ovaries from AQP8 1/1 and AQP8 2/2 mice. A C: HE-stained ovary sections from female mice at different ages (left) and statistical analysis of the number of corpus luteums (right) (n 5 8). *, P \ /1, wildtype; 2/2, AQP8 2/2. CL, corpus luteum; W, week. Scale bar, 400 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] The sections were washed with PBS and incubated with HRPlabeled sheep anti-rabbit secondary antibody for 1 h at 25 8C. Peroxidase activity was visualized by reaction with diaminobenzidine. Granulosa Cell Harvest and culture Granulosa cells were harvested from the largest follicles of ovaries by needle puncture, treated with 0.5% hyaluronidase for 10 min, centrifuged, and resuspended in Dulbecco s modified Eagle s medium (DMEM, sigma) supplemented with 10% fetal bovine serum and 100 lg/ml penicillin and 100 lg/ml streptomycin. The cells were cultured at 37 8C, 5% CO 2 when needed. Reverse Transcription Polymerase Chain Reaction (RT-PCR) Total RNA from freshly isolated granulosa cells was extracted using an RNeasy micro kit (Qiagen), and cdna was reverse transcribed from 2 lg total RNA using a SuperScript First-strand Synthesis System (Invitrogen). The cdna was used as a template for PCR amplification using primers flanking a 352 bp region of mouse AQP8 coding sequence: sense primer, 5 0 -TTGGGGCTCATCATTGCTACC-3 0 ; antisense primer, AGAAGCCAATGGAGAATGGG-3 0. The PCR products were analyzed by agarose gel electrophoresis. Immunoblotting Freshly isolated granulosa cells were dissolved in lysis buffer (150 mm NaCl, 20 mm Tris, 5 mm EDTA ph7.5, 1% Triton X-100, and supplemented with 1 mm PMSF). Twenty micrograms of proteins were resolved on 10% SDS-PAGE, and transferred to PVDF membranes. After blocking with 5% (w/v) nonfat milk and washing in Tris-buffered saline Tween solution, membranes were incubated with primary rabbit anti-aqp8 polyclonal antibody (Millipore, 1:200) for 1 h, washed and then incubated with secondary goat anti-rabbit IgG antibody conjugated to horseradish peroxidase (Sigma, 1:5000) and detected using an enhanced chemoluminescence kit (Amersham). Water Permeability Measurement The osmotic water permeability of granulosa cell plasma membranes was measured using a calcein fluorescence quenching method described previously (18), with some modification. Briefly, granulosa cells from wildtype and AQP8 2/2 mice were cultured on round glass coverslips precoated with polylysine for 24 h. Cells were incubated with 5 lmol/l calcein-am (Sigma) for 15 min and then mounted in a perfusion chamber designed for rapid solution exchange. The time course of cytoplasmic calcein fluorescence in response to an osmotic gradient was monitored by exchanging perfusate osmolality between 300 mosmol (PBS) and 500 mosmol (with added sucrose). Shrinking rate of cells is presented as a reciprocal exponential time constant (1/s), which is proportional to osmotic water permeability, where s is the time needed from the beginning of the osmotic switch to the point when the cytoplasmic calcein fluorescence reaches its maximum.

4 INCREASED FEMALE FERTILITY IN AQP8-DEFICIENT MICE 855 Figure 3. Analyses of AQP8 expression and granulosa cell water permeability. A: Granulosa cells isolated from AQP81/1 and AQP82/2 mice. Scale bar, 50 lm. B: RT-PCR analysis using AQP8 primers. b-actin was used as reference gene. C: Immunoblot using AQP8 antibody. D: Images of immunohistochemistry showing AQP8 staining of granulosa cells in ovarian follicles. Scale bar, 100 lm. E (left): Representative curves of granulosa cells water permeability analysis showing the time-course of calcein fluorescence in response to changes in perfusate osmolality from 300 to 500 mosmol. (right): Cell shrinking rates of granulosa cells presented by reciprocal exponential time constants (1/s) (n 5 6). 1/1, wild-type; 2/2, AQP82/2. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Apoptosis Assay by Flow Cytometry Granulosa cells ( cells) freshly isolated from AQP81/1 and AQP82/2 mice were cultured for 24 h in mm polystyrene tubes in 0.5 ml DMEM medium without or with 40 mm paclitaxel (Sigma). Annexin V-FITC/ propidium iodide (PI) assay for estimating cells undergoing apoptosis was performed as described previously (19). Briefly, granulosa cells after incubation were washed in PBS and labeled with 5ul FITC-conjugated annexin V according to the manufacturer s instructions (Beyotime, Shanghai, China). After incubation in dark for 10 min and then labeled with PI, the samples were immediately analyzed on a Flow Cytometer (Beckman Coulter, Epics XL). Statistical Analysis Statistical analysis was performed by SPSS statistics (version 17.0) using two-tailed Student t test. Data are expressed as the mean 6 SD.

5 856 SU ET AL. RESULTS Increased Fertility in Female AQP8 2/2 Mice In mating studies, female AQP8 2/2 mice exhibited increased fertility by showing a significant increase of litter size regardless of the genotypes of male mates (Table 1). AQP8 deletion in male mice did not affect their fertility, which is consistent with previous finding of normal sperm count and morphology in male AQP8 2/2 mice (11). Increased Numbers of Oocytes in Ovulation of Female AQP8 2/2 Mice To test the hypothesis that the enhanced fertility in female AQP8 2/2 mice is due to increased ovulation, we counted the number of oocytes released in normal ovulation and gonadotropin-induced superovulation in 10-week-old female AQP8 1/1 and AQP8 2/2 mice. There was a significant increase in the number of released oocytes both in normal ovulation ( vs in AQP8 1/1 female, P \ 0.05) and in superovulation ( vs in AQP8 1/1 female, P \ 0.05) (Figs. 1A and 1B). Another interesting finding was that oocytes from all female AQP8 2/2 mice during normal ovulation appeared as COC. Oocytes harvested from only 43% female AQP8 1/1 mice appeared as COCs (Figs. 1A and 1C). In superovulation, all oocytes released from either AQP8 1/1 or AQP8 2/2 mice are in COC form. Increased Number of Corpora Luteas in the AQP8 2/2 Ovaries Ovaries from AQP8 1/1 and AQP8 2/2 mice at 5, 10, and 20 weeks of age were compared. As shown in Fig. 2, there were significantly more corpora luteas (CLs) in AQP8 2/2 ovaries at age of 10 weeks ( per mm 2 vs in AQP8 1/1 mice, P \ 0.05) and 20 weeks ( per mm 2 vs in AQP8 1/1 mice, P \0.05), supporting increased ovulation. Expression and Water-Transporting Function of AQP8 in Granulosa Cells Granulosa cells were freshly isolated from the ovaries of AQP8 1/1 and AQP8 2/2 mice. There was no obvious difference of cell size (diameter of lm inaqp8 2/2 vs lm inaqp8 1/1 cells, n 5 100) and morphology between the two genotypes (Fig. 3A). RT-PCR and immunoblotting analysis of freshly isolated granulosa cells identified AQP8 mrna and protein expression in AQP8 1/1 but not AQP8 2/2 cells (Figs. 3B and 3C). Immunohistochemistry using AQP8 antibody exclusively stained granulosa cells in AQP8 1/1 but not AQP8 2/2 ovaries (Fig. 3D). Figure 3E shows the significantly decreased plasma membrane water permeability of AQP8 2/2 granulosa cells. Relative plasma membrane water permeability of AQP8 2/2 granulosa cells was approximately 45% lower than that of AQP8 1/1 granulosa cells. Figure 4. Flow cytometry analysis of granulosa cell apoptosis. A: upper left, uninduced AQP8 1/1 granulosa cells; upper right, uninduced AQP8 2/2 granulosa cells; lower left, AQP8 1/1 granulosa cells incubated with paclitaxel; lower right, AQP8 2/2 granulosa cells incubated with paclitaxel. B: Statistical analysis of the apoptosis rates of the four groups in A (n 5 6). *, P \ **, P \ /1, wildtype; 2/2, AQP8 2/2. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Decreased Apoptosis Rate of AQP8 2/2 Granulosa Cells Previous studies suggested that higher plasma membrane water permeability promotes apoptosis (20, 21). We analyzed the apoptosis of freshly isolated granulosa cells by flow cytometry. As shown in Fig. 4, paclitaxel-induced apoptosis rate of AQP8 2/2 granulosa cells was significantly lower than that of AQP8 1/1 ( % vs %, P \ 0.01). The baseline apoptosis rate was also lower in AQP8 2/2 granulosa cells ( % vs %, P \ 0.05). DISSCUSION In the present study on AQP8 knockout mice, we have identified a novel role of AQP8 in female reproductive physiology.

6 INCREASED FEMALE FERTILITY IN AQP8-DEFICIENT MICE 857 Evidence from mating experiments, ovulation studies and ovary histological analysis consistently indicated enhanced female fertility in AQP8 2/2 mice. AQP8 is expressed exclusively in granulosa cells in mouse ovary, suggesting that altered granulosa cell function by AQP8 deletion is responsible for the increased ovulation. Although previous studies reported expression of other AQPs including AQP7 and AQP9 in granulosa cells, our study indicated that AQP8 is the major water channel in granulosa cell, because AQP8 deletion decreased its water permeability by 45%. Granulosa cells play an important role in oocyte development and follicle maturation (13, 14). During follicular growth and development, more than 99% of follicles disappeared, primarily due to apoptosis of granulosa cells (22 25). A previous study by Jablonski et al. (20) indicated that AQP expression in plasma membrane promoted apoptosis of various cell types. Overexpression of AQP1 in Chinese hamster ovary cells enhanced their rate of apoptosis. On the other hand, inhibition of AQP-mediated water permeability blocks the apoptotic volume decrease (AVD) of granulosa cells. They concluded that AQP-mediated water loss is important for the AVD and downstream apoptotic events and that the water permeability of the plasma membrane can control the rate of apoptosis. Our apoptosis analysis of AQP8 2/2 versus AQP8 1/1 granulosa cells clearly confirmed such a mechanism. Therefore, we believed that the decreased apoptosis rate of AQP8 2/2 granulosa cells probably led to decreased atresia of growing follicles, followed by increased maturation of POFs and increased number of released oocytes during ovulation. Furthermore, the increased incidence of COCs in AQP8 2/2 mice may be also associated with the decreased apoptosis rate of granulosa cells. Further studies are needed to elucidate the molecular mechanism and cellular events of altered granulosa cell function in the absence of AQP8 during folliculogenesis. In conclusion, we have identified an important function of AQP8 in female reproductive physiology. The enhanced female fertility in AQP8-deficient mice is due to increased ovulation associated with altered granulosa cell function and follicular development. The present study may provide new insights into the molecular mechanism of folliculogenesis and female birth control. ACKNOWLEDGEMENTS The authors thank Lianhui Dou for animal care, Shuqin Pan for tissue sectioning and histological work. REFERENCES 1. Carbrey, J. M. and Agre, P. (2009) Discovery of the aquaporins and development of the field. Handb. Exp. Pharmacol. 190, Verkman, A. S. (2008) Mammalian aquaporins: diverse physiological roles and potential clinical significance. Expert Rev. Mol. Med. 10, Calamita, G., Mazzone, A., Cho, Y. S., Valenti, G. and Svelto, M. 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