Prostacyclin enhances mouse embryo development and hatching but not increased embryonic cell number and volume

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1 Prostacyclin enhances mouse embryo development and hatching but not increased embryonic cell number and volume Chung-Hsien Liu, M.D., Ph.D., a Maw-Sheng Lee, M.D., Ph.D., a,b,c Ching-Hung Hsieh, M.D., M.S., d Chun-Chia Huang, M.S., c,e,f Hui-Mei Tsao, M.S., c and Yih-Shou Hsieh, Ph.D. e a Department of Obstetrics and Gynecology and e Institute of Biochemistry, College of Medicine, Chung-Shan Medical University Hospital, Taichung; b Department of Medicine, China Medical University, Taichung; c Infertility Clinic, Lee Women s Hospital, Taichung; d Department of Obstetrics and Gynecology, Taipei City Hospital, Taipei; and f Department of Biotechnology, Central Taiwan University of Science and Technology, Taichung, Taiwan Objective: To evaluate in vitro effects of prostacyclin (PGI 2 ), we cultured mouse embryos with a PGI 2 analogue, because human fallopian tube cells synthesize abundant amounts of PGI 2. Design: Animal model. Setting: Animal study in a private infertility clinic. Animal(s): Mouse embryos. Intervention(s): In vitro effects of PGI 2 analogue on mouse embryos. Main Outcome Measure(s): Development rate, blastocyst volume, rate of complete hatching, and cell number of hatched blastocysts. Result(s): Exposure to PGI 2 analogue during the four-cell to morula stages was critical to enhanced embryo development and hatching but did not increase blastocyst volume or cell number of hatched blastocysts. The effects of PGI 2 analogue were statistically significant at 1.0 mol/l and 2.0 mol/l in human tubal fluid medium, with or without 1% bovine serum albumin. Conclusion(s): Prostacyclin analogue enhanced embryo development and hatching, but PGI 2 did not increase number of cells in hatched blastocysts or blastocyst volume. (Fertil Steril 2006;86(Suppl 3): by American Society for Reproductive Medicine.) Key Words: Prostacyclin, embryo development, PGI 2 Received November 28, 2005; revised and accepted May 3, Supported by the Chinese Infertility Foundation, Taiwan. Authors C.-H.L. and M.-S.L. contributed equally to the work and both should be considered to be the first author. Reprint requests: Yih-Shou Hsieh, Ph.D., Institute of Biochemistry, Medical College, Chung-Shan Medical University, No. 263 Pei-Tung Road, Taichung 406, Taiwan (FAX: ; msleephd@giga. net.tw). Prostacyclin (PGI 2 ) traditionally has been thought to be involved in maintaining blood and vascular homeostasis (1). However, a recent study discovered that decidualization of the endometrium was completely abolished in cyclooxygenase (COX)-2 knockout mice (2). To some extent, the disordered decidualization was rescued by exogenous PGI 2 analogue (3). Huang et al. (4, 5) reported in 2002 and 2004, respectively, that both human and mouse oviduct epithelial cells express enzymes that are essential for the synthesis of PGI 2, namely, COX-1 or COX-2 and PGI 2 synthase. Abundant PGI 2 was produced when [ 14 C]arachidonic acid was incubated with microsomes that were prepared from human (4) or mouse oviduct (5). These data suggested that PGI 2 has multiple functions in several aspects of female reproduction. The environment within the mammalian oviduct enhances the fertilization potential of sperm and promotes development of cleaving embryos. Embryos cocultured with oviduct epithelial cells have been shown to have improved development, hatching, and implantation (6, 7). A recent report indicated that the response of embryos to iloprost (PGI 2 analogue) was specific to developmental stage and coincided with expression of IP (PGI 2 receptor) (8). These critical periods for responsiveness coincided with the sojourn of mouse embryos through the oviduct, during which time the fertilized egg develops into a morula. The role of PGI 2 in embryonic development has not been fully elucidated. The relevant developmental stages also coincide with activation of the genome in human and mouse embryos, an event that takes place between the four- and eight-cell stages and after the two-cell stage, respectively (9 11). In our previous study with a mouse model, we suggested that the implantation rate for the hatching group in a different grade of blastocysts was the highest. Embryo volume was larger, and the number of inner cell mass blastomeres was greater (12). Because the first 72 hours of embryo development take place in the oviduct, in the current study we cultured embryos with PGI 2 analogue to investigate PGI 2 effects on early embryo development rate and other parameters regarding potential implantation such as size and blastomere numbers for blastocysts /06/$32.00 Fertility and Sterility Vol. 86, Suppl 3, October 2006 doi: /j.fertnstert Copyright 2006 American Society for Reproductive Medicine, Published by Elsevier Inc. 1047

2 MATERIALS AND METHODS Animals All mice were obtained from the National Laboratory Animal Center (Taipei, Taiwan) and housed in humidity- (40% 60%) and temperature- (22 C 2 C) controlled rooms with a 12:12-hour light dark photoperiod. Mice were given food and water ad libitum. All procedures were approved by the Lee Women s Hospital Institutional Animal Care and Use Committee and were performed in accordance with the Guiding Principles of Lee Women s Hospital for the Care and Use of Laboratory Animals. Preparation of Embryos Superovulation was induced in 6- to 8-week-old virgin mice (B6CBF1 strain; C57BL/6 CBA) by intraperitoneal injection of equine chorionic gonadotropin (5 IU; Sigma, St. Louis, MO) and hcg (5 IU; Serono, Rome, Italy) 48 hours later. Each superovulated mouse was placed overnight in a cage with a sexually mature male of the same strain. Successful mating was determined by the presence of a copulation plug in the vagina. The mated female mice were checked for a vaginal plug 18 hours after overnight breeding and were killed 20 hours after hcg injection; zygotes were collected from the oviducts. Zygotes cumulus cells were removed by exposure to hyaluronidase (80 IU/mL; Sigma). The zygotes then were placed into wells with fresh basal human tubal fluid (HTF) medium (13). The formula of HTF medium was NaCl, 97.6 mm; KCl, 4.7 mm; KH 2 PO 4, 0.37 mm; MgSO 4 7H 2 O, 0.2 mm; sodium lactate 60% syrup, 21.4 mm; sodium pyruvate, 0.33 mm; glucose, 2.8 mm; NaHCO 3, 25 mm; CaCl 2 2H 2 O, 2.04 mm; penicillin-g, 100 U/mL; streptomycin sulfate, 50 g/ ml; and phenol red, 0.1%. Human tubal fluid medium was prepared with the following process in our laboratory: a small amount of pure water was used to dissolve the calcium chloride in a 100-mL beaker. Then, roughly 80% of the final required volume of pure water was measured, and the other components of the medium were added. The mixture was stirred until all powder was dissolved and mixed evenly with the calcium chloride solution. Additional water was added to bring the solution to final volume. The medium then was sterilized immediately by filtration with a membrane with a porosity of 0.22 m (Millipore Co., Bedford, MA). Medium for culturing embryos was equilibrated overnight in a 5% CO 2 incubator. Two-pronucleus (2PN) embryos that could be observed clearly under 40 phase-contrast microscopy (defined as day 1 embryos) were transferred into dishes with preequilibrated HTF medium with or without 1% bovine serum albumin (BSA). The experimental embryos were exposed to the various concentrations of the PGI 2 analogue iloprost (Schering AG, Germany), diluted by HTF medium with or without 1% BSA. The embryos of control groups were placed in dishes with an equivalent amount of medium without PGI 2 analogue. Embryos were examined, and parameters of development stage were recorded every 24 hours. Blastocysts were examined for volume by measuring the diameter with a ruler with 200 phase-contrast microscopy after 96 hours of culture (day 5). After 120 hours of culture (day 6), each embryo was examined for the presence of the zona pellucida. Embryos that were completely free of the zona pellucida were counted as completely hatched. The rate of complete hatching was determined by dividing the number of completely hatched embryos by the total number of 2PN embryos. Complete hatching of embryos was chosen as an endpoint instead of blastocyst formation or embryo hatching because the latter two markers have not been correlated experimentally with establishment of a viable pregnancy (14). Mouse Embryos Exposed to 2.0 M Iloprost on the Basis of Stages of Embryo Development Two-pronuclear stage embryos were flushed from oviducts and cultured in HTF medium. All embryos were cultured in HTF medium before being exposed to 2.0 M iloprost. All 2PN-stage mouse embryos were divided into four groups. Embryos in the first group were changed to HTF medium with 2.0 M iloprost at the 2PN stage and were cultured through hatching. Embryos in the second group were changed at the two-cell stage, 24 hours after embryos were flushed from oviducts. Embryos in the third group were changed at the four-cell stage, 48 hours after embryos were flushed from oviducts. Embryos in the forth group were changed at the morular stage, 72 hours after embryos were flushed from oviducts. Embryos were examined, and parameters of development stage were recorded every 24 hours. Blastocysts were examined for volume by measuring the diameter with a ruler with 200 phase-contrast microscopy after 96 hours of culture (day 5). Differential Staining of Trophectoderm and Inner Cell Mass Cells in the trophectoderm and inner cell mass of completely hatched embryos were counted after differential staining of nuclei using a modification of Piekos method (12, 15). Briefly, zona-free blastocysts were incubated for 10 minutes at 5 C in M16 medium (Sigma) containing 10 mm trinitrobenzenesulfonic acid, 4.0 mg/ml of polyvinylpyrrolidone, and 0.015% Triton X-100. Subsequent to a wash in M2 medium (Sigma), the blastocysts were incubated in 0.1 mg/ml antidinitrophenol-bsa (INC, CA) at 37 C for 15 minutes and washed again three times with M2 medium. The blastocysts then were incubated at 37 C for 15 minutes in M2 medium containing a 1:10 dilution of guinea pig complement serum (Sigma) and 10.0 g/ml of propidium iodide (Sigma) and then were washed three times in Dulbecco s phosphate-buffered saline (GIBCO). After fixing in absolute ethanol containing 22.0 g/ml of bisbenzimide (Sigma) at 5 C overnight, individual blastocysts were mounted in glycerol on microscope slides and were compressed manually before visualization by epifluorescence using Nikon filter blocks UV-2A and G-2A (Nikon, Japan) Liu et al. PGI 2 enhances mouse embryo development Vol. 86, Suppl 3, October 2006

3 Statistical Analysis Rates were expressed as percentages. Differences between embryo development rates were compared using the 2 test. The data for cell numbers and diameter of embryos were expressed as mean SD and were compared using Student s t tests. P.05 was considered to be statistically significant. RESULTS Prostacyclin Enhancement of Embryo Development Rate Both the day 5 blastocyst formation rate and the day 6 hatching rate of our mouse embryos were enhanced by iloprost in the HTF medium (Fig. 1). The day 5 blastocyst formation rate was statistically significantly increased at concentrations of 2.0, 2.5, and 3.0 M iloprost in HTF medium. The day 6 hatching rate was statistically significantly increased at concentrations of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 M iloprost in HTF medium. In contrast, the day 5 blastocyst formation rate and day 6 hatching rate were not enhanced by iloprost in HTF medium with 1% BSA (data not shown). Prostacyclin Enhancement of Complete Hatching but Not of Embryo Volume The complete hatching rate of mouse embryos was enhanced by iloprost in a dose-dependent manner. The effects were statistically significant when a concentration of iloprost higher than 2.0 M was present in HTF medium (Fig. 1). The day 6 complete hatching rate was statistically significantly increased to 50.0%, 57.1%, 51.5%, and 51.4% at 2.0, 2.5, 3.0, and 3.5 M iloprost in HTF medium, respectively. In contrast, only 20.5% of embryos in the control group of only HTF medium hatched completely. Furthermore, the day 6 complete hatching rate was statistically significantly increased to 63.6%, 67.5%, 52.4%, 68.0%, 57.5%, and 55.6% at 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 M iloprost in HTF medium with 1% BSA, FIGURE 1 Development rate of mouse embryos exposed to different concentrations of iloprost. *Statistically significantly different from control group. D2/2C embryos developed to two-cell stage or to greater than the two-cell stage on day 2; 4C four-cell stage; MOR morula; BL blastocyst; HA hatching; CHA complete hatching. Liu. PGI 2 enhances mouse embryo development. Fertil Steril Fertility and Sterility 1049

4 TABLE 1 Mouse embryos exposed to 2 M iloprost on the basis of stages of embryo development observed in HTF medium. Start of 2 M iloprost exposure (stage of embryo development) Parameter Control 2PN Two-cell Four-cell Morula 2PN (n) Two-cell 100 (36/36) 100 (30/30) 93.3 (28/30) 97.4 (38/39) 100 (36/36) Four-cell 97.2 (35/36) 100 (30/30) 90.0 (27/30) 97.4 (38/39) 94.4 (34/36) Morula 97.2 (35/36) 100 (30/30) 90.0 (27/30) 97.4 (38/39) 94.4 (34/36) Blastocyst 77.8 (28/36) 100 a (30/30) 96.7 a (29/30) 97.4 a (38/39) 91.7 (33/36) Complete hatching 22.2 (8/36) 46.7 a (14/30) 46.7 a (14/30) 46.2 a (18/39) 33.3 (12/36) Note: Other than in first row, data are percentage of developed embryos (number that developed/2pn number). a Statistically significantly different from control group (P.05; 2 test). Liu. PGI 2 enhances mouse embryo development. Fertil Steril respectively. In contrast, only 30.0% of embryos hatched completely in medium plus BSA. The diameter of day 5 blastocysts was m in HTF medium. The diameters of day 5 blastocysts were , , 129 9, , , 122 9, and m with 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 M iloprost in HTF medium, respectively. Blastocyst volume was measured on the basis of diameter; the volume of day 5 blastocysts did not significantly increase in the presence of iloprost. Critical Periods for Exposure to 2.0 M Iloprost That Enhanced Complete Hatching Although Not Blastomere Number Because embryos undergo several developmental stages before entry into the uterus, this study was designed to investigate developmental stage specific responses to iloprost. The results indicate that the four-cell to morula stages are more critical than others. Exposure to iloprost from the two-cell to hatching stage or four-cell to hatching stage yielded the same rates of complete hatching as did exposure to iloprost from 2PN to hatching stage in HTF medium (Table 1). Although exposure to iloprost enhanced complete hatching, the total cell number was not significantly increased in embryos placed in HTF medium (Table 2). We also observed that exposure to iloprost from the twocell to hatching stage or four-cell to hatching stage yielded the same rates of complete hatching as did exposure to iloprost from 2PN to hatching stage, when exposure was in HTF medium with 1% BSA. Although exposure to iloprost enhanced complete hatching, total cell number was not significantly increased in embryos in HTF medium with 1% BSA (data not shown). TABLE 2 Results for differentially stained embryos exposed to 2 M iloprost on the basis of stages of embryo development observed in HTF medium. Start of 2 M iloprost exposure (stage of embryo development) Parameter Control 2PN Two-cell Four-cell Morula Complete hatching (n) No. of blastomeres No. of cells in ICM No. of cells in TE ICM TE cell ratio (%) Note: Data are mean SD unless otherwise indicated. ICM inner cell mass; TE trophectoderm. Liu. PGI 2 enhances mouse embryo development. Fertil Steril Liu et al. PGI 2 enhances mouse embryo development Vol. 86, Suppl 3, October 2006

5 DISCUSSION Blastocyst implantation is a required event for pregnancy, and its failure is a leading cause of infertility in women. Prostaglandins have been implicated as critical regulators of important reproductive events, including implantationassociated changes during early pregnancy. Prostacyclin is the most abundant prostaglandin in the early pregnant mouse uterus, and its concentration is higher at implantation sites than in interimplantation sites (16, 17). In this study, we showed that supplementing culture medium with PGI 2 analogue positively affected the complete hatching rate of embryos. This is in agreement with a previous report that showed that PGI 2 analogue enhanced complete hatching of mouse embryos in culture medium (7). However, culture medium with 1% BSA has been shown to increase rates of cleavage, blastocyst formation, and total cell number in blastocysts of porcine embryos (18). In other species, BSA has been shown to significantly increase the rate of partial hatching and may even have a small effect on the rate of blastocyst formation for mouse (19) and rabbit embryos (20). The effects, as seen in our study, were statistically significant when an iloprost concentration of 2.0 M was present in HTF medium (Fig. 1) or when a concentration of 1.0 M was present in HTF medium with 1% BSA (data not shown). In our study, supplementing medium with both 1% BSA and PGI 2 analogue did not produce a significant increase in embryo development rate, but the day 6 complete hatching rate was improved. When culture medium was free from any source of protein, the embryo development rate was improved by the presence of PGI 2 analogue. We believe that embryos exposed to oviduct-derived PGI 2 during early development retain enhanced hatching potential when they reach the uterus. The observed response of embryos to iloprost is developmental-stage specific (7). Mouse embryos express COX-1, COX-2, and PGI 2 in the four-cell stage; beyond that, enzyme activity is present in the inner cell mass and trophectoderm of the blastocyst (21). Prostacyclin analogue supplementation from the 2PN to four-cell stages increased the blastocyst rate in our study. It has been shown that PGI 2 may be essential from the 2PN stage in mouse preimplantation embryos. Mouse blastocyst grade can be used to predict implantation rate by volume, size, and number of blastomeres, as shown in our previous study (12). In our differentially stained, hatched embryos, embryonic cell number and volume were not significantly increased. Different mechanisms are involved in the hatching of mouse embryos in vitro and in vivo. Hatching in vitro involves mouse blastocyst expansion, causing a global zonal thinning before zonal rupture, whereas hatching in vivo involves global zonal lysis by uterine or trophectodermal lysins (22). Our results are not consistent with the findings of Xu et al. (6) and Yeung et al. (23) that embryos cocultured with epithelial oviduct cells had a higher cell number. Because embryonic cell number and volume were not significantly increased in our study, we conclude that the hatching-rate increase with PGI 2 analogue in vitro may occur through the mechanism suggested by Perona and Wassarman (24) and by Sawada et al. (25), which is that increased production of trypsin-like proteases by the trophectoderm may lyse the zona. Although HTF media (13) were not designed specifically for mouse IVF, we routinely have used this media to grow mouse embryos as a quality control in our laboratory, and the blastocyst development rate for strain of B6CBF1 mice were about 80%. Culture media have been modified to improve embryo development in vitro. Culture media supplemented with different nutrients or growth factors have been used to study possible effects on in vitro development. 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