Human Reproduction, Vol. 18, No. 12, 2582-2589,
December 2003
© 2003 European Society of Human Reproduction and Embryology
Prostacyclin enhances embryo hatching but not sperm motility
1 Obstetrics and Gynecology–Division of Reproductive Endocrinology and 2 Vascular Biology Research Center and Internal Medicine–Division of Hematology, University of Texas–Houston Health Science Center, Houston, TX 77030 and 3 Obstetrical and Gynecological Associates, Houston, TX 77030, USA
4 Present address: Johns Hopkins University, Baltimore, MD 21218, USA
5 Present address: College of St Scholastica, Duluth, MN 55811, USA
6 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology–Division of Reproductive Endocrinology, University of Texas–Houston Health Science Center, 6431 Fannin, MSB 3.604, Houston, TX 77030, USA. e-mail: jaou-chen.huang{at}uth.tmc.edu
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Abstract |
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BACKGROUND: Recently we discovered that the human oviduct synthesizes abundant prostacyclin (PGI2). Gene knock-out studies suggest that PGI2 is essential to endometrial decidualization, but the effects of PGI2 on sperm and embryos have not been reported. METHODS: The effects of PGI2 on human sperm were analysed by a computer-assisted semen analysis system. The effects of PGI2 on mouse embryos were examined based on the rates of complete hatching. The expression of PGI2 receptor (IP) was evaluated by Western blot analysis and immunohistochemistry. The binding of PGI2 to embryos was confirmed by radioligand binding assay. Finally, cAMP levels were assessed in PGI2-challenged embryos. RESULTS: Iloprost (a stable PGI2 analogue) did not affect the motility or the overnight survivability of human sperm. Western blot analysis did not detect IP in the sperm plasma membrane. In contrast, the hatching of mouse embryos was enhanced by iloprost (ED50 6.7 nmol/l). Exposure to iloprost during 8-cell to morulae or morulae to early blastocyst stages was critical to enhanced hatching. This coincided with the developmental stage-specific expression of IP. Although iloprost bound to blastocysts, it did not significantly increase cAMP. CONCLUSION: PGI2 enhanced the hatching of mouse embryos but not the motility of human sperm.
Key words: cAMP/co-culture/prostaglandin
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Introduction |
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The environment within the oviduct enhances the fertilization potential of sperm and promotes the development of cleaving embryos. Soluble factor(s) secreted by oviductal epithelial cells have been reported to prolong sperm survival, increase sperm motility, and induce the acrosome reaction (Anderson et al., 1994
; Kervancioglu et al., 1994; Ménézo et al., 1997; Yao et al., 1999). Embryos co-cultured with oviductal epithelial cells have been shown to have improved development, hatching and implantation (Xu et al., 2000, 2001).
Prostacyclin (PGI2) is traditionally thought to be involved in maintaining blood and vascular homeostasis. However, recent observations on gene knock-out mice suggest that it may have other physiological functions. The decidualization of the endometrium was completely abolished in cyclooxygenase (COX)-2 knock-out mice (Lim et al., 1997) but could be, to some extent, rescued by exogenous PGI2 analogue (Lim et al., 1999).
The role of PGI2 in the development of embryos is not clear. Although PGI2 receptor (IP) knockout mice were fertile, their litter size has not been compared with that of the wild type (Murata et al., 1997). The genotypic distribution of the pups arising from mating heterozygous IP knock-out mice did not conform to Mendel’s Law: the prevalences of male and female pups with homozygous IP knock-out genotype were 37 and 20% respectively less than expected (Murata et al., 1997).
The effects of prostaglandin (PG) E and F on sperm motility have been studied previously: PGE2 and PGF2
enhanced the motility of sperm in vitro (Grunberger et al., 1981; Aitken et al., 1985; Colon et al., 1986; Delamere et al., 1990). A receptor for PGE2 that mediates calcium influx was recently identified in the plasma membrane of human sperm (Schaefer et al., 1998). IP has not been reported in the human sperm membrane, although a report associated low PGI2 in the seminal fluid with decreased sperm motility (Schlegel et al., 1986).
Our recent results indicate that human (Huang et al., 2002) and mouse (unpublished data) oviductal epithelial cells express enzymes essential to the synthesis of PGI2, i.e. COX-1 or COX-2 and PGI2 synthase. Abundant PGI2 was produced when [14C]arachidonic acid was incubated with microsomes prepared from human (Huang et al., 2002) or mouse oviduct (unpublished data). Since the sperm travel to distal oviduct to fertilize the egg and the first 72 h of embryo development takes place in the oviduct, these findings prompted us to investigate the effects of PGI2 on sperm and the embryo.
In the present study, the effects of PGI2 on human sperm motility and overnight survival were analysed by a computer-assisted semen analysis (CASA) system and the impact of PGI2 on the embryos was evaluated by observing the complete hatching of mouse embryos cultured with PGI2 analogue. To further elucidate the mechanism, the expression of IP and the effector coupled to IP were also studied.
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Source of reagents and institutional approval
Unless stated otherwise, reagents were purchased from Sigma Co. (USA). Use of human semen samples was approved by the Committee for the Protection of Human Subjects (equivalent to an Institutional Review Board). The care of the laboratory animals and the research protocols were approved by the Animal Welfare Committee.
Sperm preparation and incubation
Five normal and three subnormal (
30% motile sperm after washing) semen samples were collected from different individuals by masturbation. After complete liquefaction, motile sperm were separated by centrifugation over a two-layer density gradient (40 and 80% PureSperm®; Nidacon International AB, Sweden). The pellet was washed with -minimum essential medium (MEM)–HEPES media (Irvine Scientific, USA) supplemented with 10% serum protein substitute (SPS; Sage Biopharma, USA) and then was resuspended in the same media at a concentration of 20x106/ml. Aliquots were incubated with or without 1 µmol/l iloprost, a stable PGI2 analogue, at 37°C under 5% CO2. Because cAMP has been reported to increase sperm motility (Wade et al., 2003), we used 1 mmol/l 8-bromo cAMP as a positive control. Vehicle (water) was used as a negative control. The sperm motility was analysed after 30 and 60 min. These two time-points were previously determined to be optimal for analysing sperm motility. Because the overnight survivability of human sperm has been reported to correlate with fertilization potential (Franco et al., 1993), we also compared the motility of sperm after overnight culture in the presence or absence of iloprost.
Computer-assisted semen analysis
Sperm motility was evaluated by a CASA system (Hamilton Thorn IVOS system, USA). The microscope stage and the housing were maintained at 37°C. To examine various elements of sperm movement (see below), aliquots (5 µl at 20x106/ml) were transferred to a 20 µm deep chamber (Micro-Cell®; Fertility Technologies, Inc., USA) prewarmed to 37°C. Because hyperactivated motility of sperm was reported to correlate with fertilization potential (Kay et al., 1998), we also determined the percentage of hyperactivated sperm. To examine sperm displaying characteristics of hyperactivation, a 40 µm deep chamber was used. Approximately 1500 sperm, sampled from 15 representative fields, were analysed for each sample.
The following elements of sperm movement were analysed: curvilinear velocity (VCL, µm/s), straight line velocity (VSL, µm/s), average path velocity (VAP, µm/s), linearity (LIN, VSL divided by VCL), amplitude of lateral head displacement (ALH, µm), beat cross-frequency (BCF, Hz), and percentage of sperm exhibiting moving pattern of hyperactivation (%). Hyperactivated sperm were defined as those displaying a LIN value <65 µm/s, a VCL value >100 µm/s, and an ALH value >7 µm.
Preparation of plasma membrane from human sperm
The plasma membrane of human sperm was prepared as described previously (Althouse et al., 1995). Briefly, washed sperm from four normal samples (each containing >95% motile sperm) were pooled, placed in 4 ml of phosphate-buffered saline (PBS) with 20 mmol/l HEPES (pH 7.4) and a cocktail of protease inhibitors: 1 mmol/l 4–2-aminoethyl benzene sulphonyl fluoride hydrochloride, 0.8 µmol/l aprotinin, 50 µmol/l betastatin, 15 µmol/l E-64, 20 µmol/l leupeptin hemisulphate, 10 µmol/l pepstatin A (Calbiochem-Novabiochem Corp., USA). The suspension was pressurized with nitrogen gas to 650 lb/inch2 in a cell disruptor (Parr Instrument Company, USA) for 10 min at room temperature and then rapidly depressurized. The disrupted membrane was subjected to a series of centrifugations at 5°C: 6000 g for 10 min, followed by 35 000 g for 15 min, and finally 300 000 g for 60 min. Supernatant was collected for each successive centrifugation. The pellet of the final centrifugation was washed in PBS and resuspended in 20 mmol/l Tris–HCl, pH 7.4 using a hand homogenizer. The protein concentration was determined using bovine serum albumin (BSA) as standard (Micro BCA; Pierce Chemical Co., USA).
Harvest and culture of mouse embryos
Mice were kept under controlled temperature, humidity and light cycle (12 h light/12 h dark cycle) conditions with free access to water and food. Three week old C57Bl/6 female mice were purchased from Harlan (USA). Eight week old C3H male mice were purchased initially from Harlan and later from The Jackson Laboratory (USA). Superovulation in the female mice was achieved by intraperitoneal injection of pregnant mare serum gonadotropin (5 IU), followed by hCG (5 IU) 46 h later. After receiving hCG, each female mouse was paired with one fertile male mouse. Forty-eight hours later, 2-cell embryos were harvested from the oviduct into -MEM media supplemented with 25 mmol/l HEPES and 1% BSA (Irvine Scientific).
Embryos (17–20 per group) were cultured at 37°C under 5% CO2 in a four-well dish (Nalge Nunc International, USA) containing 600 µl of medium in each well. The HTF and the -MEM media were used sequentially during the 96 h period to meet the changing nutritional requirements of the cleaving embryos (Gardner, 1998). The HTF medium (SAGE Biopharma) was used during the first 48 h, and the -MEM medium (Irvine Scientific), with Earle’s salts and 2 mmol/l glutamine, was used during the second 48 h. Both media were supplemented with 10% SPS. The experimental embryos received iloprost in water and the control embryos received an equivalent amount of water. Preliminary experiments showed that >95% of the embryos became blastocysts by 96 h, similar to those cultured in KSOM media (Specialty Media; Cell and Molecular Technologies, Inc., USA). After 96 h culture, each embryo was examined for the presence of the zona pellucida. Embryos completely free of the zona pellucida were counted as having completely hatched. The rate of complete hatching was determined by dividing the number of completely hatched embryos by the total number of embryos. Complete hatching of embryos was chosen as an endpoint instead of blastocyst formation or embryo hatching because the latter two markers are not correlated with establishment of a viable pregnancy (Lane et al., 1997).
Western blot analysis
The deduced amino acid sequences of mouse IP (417 a.a., Genebank_BAA05144) and human IP (386 a.a., Genebank_ BAA06110) are highly homologous (Katsuyama et al., 1994). Preliminary studies confirmed that an affinity-purified polyclonal peptide antibody (a gift from Dr Ke-He Ruan, University of Texas Health Science Center) cross-reacted with mouse IP. Western blot analysis was performed as described previously (Huang et al., 2002). Briefly, plasma membrane protein from human sperm (40 µg) or total cell lysate from 60 mouse blastocysts was separated by electrophoresis on a 10% acrylamide gel (PAGE) and transferred to a nitrocellulose membrane (Schleicher & Schuell, Inc., USA). Immunoreactive protein was detected by incubation with the antibody and visualization with enhanced chemi-fluorescence (Amersham Biosciences, USA), detected using a STORM 860 laser scanner (Amersham Biosciences). Human platelet microsomes were used as positive controls. The antibody specificity was confirmed in parallel experiments using pre-absorbed antibody.
Immunohistochemistry, fluorescence microscopy and confocal microscopy
Mouse embryos were fixed in 4% paraformaldehyde (pH 7.4) at 4°C for 30 min. After three washes in PBS, the embryos were blocked for 20 min at room temperature in Tris-buffered saline (pH 7.4) containing 0.05% Tween-20, 5% powder milk and 0.1% Triton X-100. The embryos were incubated with IP antibody (5 ng/ml) in blocking buffer for 2 h, then with goat anti-rabbit IgG coupled with Alexa 488 (2.5 µg/ml; Molecular Probes, USA) for 30 min at 37°C. Cell nuclei were counterstained with 10 µg/ml propidium iodide at room temperature for 20 min. The embryos were mounted in Fluoromount-G® (Southern Biotechnology Associates Inc., USA). For fluorescence microscopy, blastocysts were placed in the mounting media and overlaid with a coverslip. For confocal microscopy, a spacer of 50 µm was placed between the slide and the coverslip to maintain the three-dimensional morphology of the embryo. For negative controls, embryos were incubated with 10 ng/ml non-immune rabbit IgG (i.e. twice the concentration of primary antibody).
Fluorescence microscopy was performed using a Zeiss AxioPlan 2 microscope (Carl Zeiss, Germany) equipped with appropriate filters. Images were captured using a CCD camera and processed by the AxioVision program (Version 3.0.6). Confocal microscopy was performed using a BioRod Radiance 2000 confocal system (Bio-Rad Laboratories, USA) attached to an Olympus BX-50 microscope. The images were processed using the Image-Pro+ program (Media-Cybernetics, USA).
Whole embryo radioligand binding assay
Analysis of the binding of [3H]iloprost to blastocysts was performed as previously described (Arbab et al., 2002) with slight modification. A total of 232 hatched and hatching blastocysts were washed three times in binding buffer (10 mmol/l MnCl2 in 10 mmol/l HEPES, pH 7.4) and then transferred to 100 µl of binding buffer with or without unlabelled iloprost (5 µmol/l). The reaction was started by adding an equal amount of binding buffer containing [3H]iloprost (200 nmol/l, specific activity 11.0 Ci/mmol; Amersham Biosciences) and incubating for 60 min at room temperature. The reaction was terminated by transferring the blastocysts to 2 ml of ice-cold wash buffer (0.01% BSA in 10 mmol/l HEPES, pH 7.4). The buffer and the blastocysts were filtered through glass fibre filters (Whatman GF/C 2.4 cm) and the filters were washed three times with 2 ml of wash buffer. The filters were dried in an oven before the radioactivity was determined by scintillation counting with 5 ml of scintillation fluid.
Determination of cAMP in embryos
Levels of cAMP in the embryos were determined based on a method described previously (Manejwala et al., 1986) with some modification. Embryos (10 in each group) were pre-incubated in -MEM media containing 20 mmol/l HEPES, 0.2 mmol/l 3-isobutyl-1-methylxanthine (IBMX) and 100 µmol/l EDTA for 10 min before they were transferred to the same media containing 10 mg/ml forskolin and 2 mg/ml cholera toxin (positive control) or vehicle (negative control) or 1 µmol/l iloprost. After a 2 h incubation at 37°C, embryos were transferred to 10 µl of 0.1 N HCl and stored at –70°C until assay. Prior to assay, the pH was neutralized with 10 µl of 0.1 N NaOH and the samples were acetylated and assayed according to the manufacturer’s protocol (Biotrak, Amersham Biosciences).
Statistical analysis
Student’s t-test or one-way ANOVA followed by Dunnett’s test were used where appropriate. P < 0.05 was considered statistically significant. Construction of dose–response curves and calculation of ED50 values (with the Hill slope set at 1.0) were completed with GraphPad Prism® (USA) software.
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Results |
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Motility and overnight survival of human sperm were not affected by PGI2
The effects of iloprost on the motility and the overnight survival of sperm were evaluated by CASA in five normal and three subnormal semen samples. The percentages of motile sperm and hyperactivated sperm were enhanced by 8-bromo cAMP but not affected by iloprost (Figure 1A and 1B). All aspects of movement, except LIN, such as VCL, VSL, VAP, ALH, and BCF were increased by 8-bromo cAMP but not affected by iloprost (data not shown). The percentage of motile sperm after overnight culture was also not affected by iloprost (data not shown). Thus, neither the motility nor the overnight survivability of normal or subnormal sperm was affected by PGI2.
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IP was not detected in sperm plasma membrane
To examine the expression of IP by human sperm, purified sperm plasma membrane (40 µg) was separated by PAGE and probed with an affinity-purified polyclonal antibody against human IP. The Western blot analysis did not detect IP in the human sperm plasma membrane (Figure 2).
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PGI2 enhanced the complete hatching of mouse embryos
The complete hatching of mouse embryos was enhanced by iloprost in a concentration-dependent manner. The effects were statistically significant at
0.1 µmol/l (Figure 3). Maximum augmentation of complete embryo hatching occurred at 1 µmol/l, where 81 ± 7% (mean ± SD, n = 3) of the experimental embryos hatched completely. In contrast, only 49 ± 14 % (mean ± SD, n = 5) of the control embryos hatched completely. The ED50 value derived from the dose–response curve was 6.7 nmol/l (Figure 3). The saturable, concentration-dependent responses and the ED50 value of 6.7 nmol/l suggest that the effects of iloprost were mediated by a receptor.
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Duration of PGI2 exposure and the developmental stages of embryos critical to the enhanced hatching
Because embryos undergo several developmental stages before entering the uterus, we investigated developmental stage-specific responses to iloprost. Our results indicate that some periods during the 96 h culture were more critical than others. Exposure to iloprost during the first 24 h of culture (during this period most of the 2-cell embryos developed into 8-cell embryos) did not enhance hatching (Figure 4). On the other hand, exposure to iloprost 0–72 or 24–72 h after harvest yielded the same rates of complete hatching as did 0–96 h exposure (Figure 4).
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The two critical periods of exposure to iloprost that ensured the full effects of iloprost were 24–42 and 42–72 h after harvest, corresponding to the transformation from 8-cell embryos to morulae and from morulae to early blastocysts respectively (table at bottom of Figure 4). Our data suggest that brief exposure of embryos to oviduct-derived PGI2 confers an enhanced potential for complete hatching even after the embryos leave the oviduct.
Mouse embryos express IP
To investigate the developmental stage-specific expression of IP in mouse embryos, we performed Western blot analysis on blastocysts and immunohistochemistry on embryos at different developmental stages. Western blot analysis showed that a protein of the expected molecular weight was detectable by the affinity-purified antibody against IP (Figure 5). Fluorescence microscopy showed IP staining was present in morulae and blastocysts (Figure 6A–E) but not in unfertilized oocytes, nor in 1-, 2-, 4- and 8-cell embryos (not shown). The IP staining in morulae and blastocysts shared the same fine, reticular pattern. Confocal microscopy images suggest that IP was preferentially expressed in the trophectoderm (Figure 6G). Thus, the stage-specific expression of IP by the mouse embryo coincided with the responsiveness to iloprost.
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20 µm.Radiolabelled iloprost binds to mouse embryos
The concentration-dependent enhancement of complete embryo hatching by iloprost suggested that the effect was receptor-mediated. We performed a radioligand binding assay to further confirm the binding of mouse embryos by iloprost. We elected to quantify iloprost binding at a 0.1 µmol/l level of the ligand, where augmented hatching was first observed and found that
3.04 fmol [3H]iloprost bound specifically to 116 blastocysts (Figure 7).
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IP in the mouse embryos is not coupled to Gs
To examine the possible coupling of Gs to IP in mouse blastocysts, we first confirmed the presence of activatable adenylate cyclase in the mouse blastocysts (positive control). Separately, blastocysts were challenged with iloprost (experiment) or vehicle (water, negative control). There was a nearly 70-fold increase in cAMP (from 0.016 fmol to 1.1 fmol per embryo) when embryos were challenged by forskolin and cholera toxin (Figure 8). There was no difference in cAMP levels between control and experimental embryos (Figure 8), indicating that IP was not coupled to Gs.
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Discussion |
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Hyperactivation (Kay et al., 1998
) and overnight survival (Franco et al., 1993) have been reported to correlate with the fertilization potential of human sperm. Our data suggest that PGI2 did not enhance either of these two sperm properties. Our findings are corroborated by the inability to detect IP in the plasma membrane of human sperm and by the unchanged cAMP levels after iloprost challenge (data not shown).
Contrary to findings in sperm, PGI2 did affect the embryos and enhanced their complete hatching. This concurs with previous reports that co-culture of mouse embryos with epithelial cells from human oviducts increased embryo cell number, reduced apoptosis, and improved embryo hatching (Piekos et al., 1995; Xu et al., 2000, 2001). Thus, oviduct-derived PGI2 and endothelium-derived PGI2 exert their effects in a similar, paracrine fashion. The former enhances embryo hatching; the latter prevents platelet aggregation. The concentration-dependent response was consistent with a receptor-mediated event and the ED50 value of 6.7 nmol/l was similar to the reported Kd value of solublized IP (8 nmol/l) from human platelets (Tsai et al., 1989).
The response of embryos to iloprost was developmental stage-specific and coincided with the expression of IP (Figure 4). These critical periods for responsiveness coincided with the sojourn of mouse embryos in the oviduct, during which time the fertilized eggs develop into morulae. The relevant developmental stages also coincided with the activation of genome in human and mouse embryos, which takes place between the 4- and 8-cell stages and after the 2-cell stage respectively (Tesarik et al., 1986, 1988; Braude et al., 1988).
Thus, embryos exposed to oviduct-derived PGI2 during early development retain the enhanced hatching potential when they reach the uterus. The embryos, therefore, actively prepare themselves for implantation while they are in the oviduct. From the perspective of an embryo, oviduct-derived PGI2 is complementary to endometrial PGI2, which mediates endometrial decidualization to ensure receptivity (Lim et al., 1999).
Despite the presence of activatable adenylate cyclase in the embryos, our data do not support the coupling of IP to Gs, such as can be seen in the oviductal smooth muscle cells (Arbab et al., 2002). Coupling of IP to effectors other than Gs or to multiple effectors has been reported. In the rat kidney, the IP in cells from the thick ascending limb is reportedly coupled to Gi (Hebert et al., 1998). In transfected cell lines overexpressing IP, both Gs and Gq couple to IP (Smyth et al., 2000; Lawler et al., 2001). Transient increase of intracellular calcium has been reported to accelerate the outgrowth of trophoblast in vitro and facilitate embryo implantation in utero (Stachecki et al., 1994). We thus speculate that the IP in the embryonic cells may be coupled to Gq, which increases the calcium levels but does not change cAMP levels.
Although the expression of IP coincided with the response of embryos to iloprost, the role of IP in the iloprost-mediated enhanced hatching is not conclusive. Peroxisome proliferator-activated receptor (PPAR) 
, a nuclear receptor, has been postulated to mediate the effects of PGI2 in endometrial decidualization (Lim et al., 2000). As a specific IP antagonist is not available, other approaches, such as constructing an IP knock-out mouse or RNA interference techniques (Wianny et al., 2000), will be required to provide conclusive evidence.
The enhanced hatching mediated by PGI2 may be due to an increased number of embryonic cells. Different mechanisms are involved in the hatching of mouse embryo in vitro and in vivo. Hatching in vitro involves blastocyst expansion, causing a global zonal thinning prior to zonal rupture, whereas hatching in vivo involves global zonal lysis by uterine or trophectodermal lysins (Montag et al., 2000). Therefore, a sufficiently high number of embryonic cells is required to accomplish hatching in vitro. In this respect, our results are consistent with findings that embryos co-cultured with epithelial cells of the oviduct had more cells, less cell death, and improved hatching (Yeung et al., 1992; Xu et al., 2000). In addition, PGI2 may increase production of trypsin-like proteases by the trophectoderm to lyse the zona (Perona et al., 1986; Sawada et al., 1990).
The low implantation potential of IVF embryos (10–20% per embryo) (Center for Disease Control 2001) was attributed in part to suboptimal culture conditions (De Vos et al., 2000). Culture media have been modified to improve embryo development in vitro (Gardner 1998). Based on our data, supplementing media with PGI2 analogue such as iloprost may provide an improved environment for the developing embryos and increase their implantation potential.
In conclusion, PGI2 enhanced the complete hatching of cultured mouse embryos but not the motility of human sperm in vitro. The stage-specific expression of IP by the mouse embryos coincided with their responsiveness to PGI2.
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Acknowledgements |
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The authors wish to thank Ms. Dana Whittaker for her secretarial assistance. J.-C. Huang is a Women’s Reproductive Health Research Scholar (NICHD HD01277).
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Submitted on June 17, 2003; accepted on August 27, 2003.
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