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EARLY PREGNANCY: Biology and Medicine Editor-in-Chief: Eytan R. Barnea MD, FACOG

January 2000
Volume IV, Number 1
ISSN: 1537-6583
Pages: 030-038

Platelet-Activating Factor Increases Intracellular Calcium Levels In Preimplantation Stage Embryos

Joseph E. Logan and William E. Roudebush*

Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Medical University of South Carolina, Charleston, South Carolina, 29425 USA

Short Title: Embryonic calcium and PAF

Key Words: Platelet-activating factor; Intracellular calcium; Preimplantation embryos; Mouse

Correspondence:* Tel. (843)792-8348, Fax. (843)792-0533, Email. wer@primate.musc.edu

Acknowledgements: The authors would like to thank Drs. Alix Darden (Research Coordinator) and Robert Baldwin (Chairman) of the Biology Department, The Citadel, Charleston, SC for coordinating and permitting JEL to conduct his research requirement at MUSC. This research was supported in part by NICHD-1RO3HD3523301 and Medical University of South Carolina Institutional Funds of 1996-1997.

Abstract

Platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine;PAF] has an active role in preimplantation embryo development. PAF has been shown to act via the receptor mediated inositol triphosphate-diacylglycerol (IP₃/DAG) pathways in non-reproductive cells to increase intracellular calcium ([Ca++]i) levels. Molecular evidence on the presence of the PAF-receptor in mouse preimplantation embryos has recently been reported, however the effect of PAF on embryonic [Ca++]i is unclear. Therefore, the study objective was to determine the effect of PAF on [Ca++]i in the mouse preimplantation embryo. Two-cell embryos were collected from PMSG/hCG primed mature female CFW mice, washed in modified M16 (phenol red free) and loaded with FURA-2AM (0.2 µM). Background [Ca++]i levels were measured for a minimum of 120 seconds prior to treatment. PAF or lyso-PAF (the biological inactive form) were added [10^-7 M final concentration] and [Ca++]i levels measured. Background, nonstimulated, [Ca++]i levels had a mean of 131.4 nM. [Ca++]i levels began to increase by 4.6 seconds with maximum levels reached by 179.9 seconds after PAF exposure, baseline levels returned by 460 seconds. Maximum [Ca++]i levels (405.9 nM mean) were 3X that of non-PAF or lyso-PAF exposure. The results further demonstrate the magnitude of PAFs' action in the preimplantation embryo period. PAF's mechanism of action, in preimplantation mouse embryos, appears to involve a PAF-receptor mediated increase in intracellular calcium.

Introduction

Platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine;PAF] is a potent signaling phospholipid that is produced by, and influences, an assortment of cell types (Hanahan, 1986; Braquet et al., 1987). PAF is involved in a number of reproductive and developmental processes (Harper, 1989; Pike et al., 1992; Minhas et al., 1996), including ovulation (Kikukawa et al., 1991), fertilization (Roudebush et al. 1990), preimplantation development (Roberts et al., 1993), implantation (Ryan et al., 1987), and parturition (Silver et al., 1992). PAF is found in uterine fluid, being produced by uterine epithelium and preimplantation stage embryos (Angle et al., 1988). It appears that PAF's action in the embryo may be receptor-mediated since PAF-antagonists completely inhibit its action (Nishi et al., 1995). PAF is believed to bind with cell surface receptors causing the formation of inositol triphosphate (IP₃) and diacylglycerol followed by an increase in intracellular calcium (Roudebush et al., 1997).

Diffusion of calcium [Ca++] in the cytosol is much slower than any other second messenger mainly due to the presence of large number of almost immobile [Ca++] binding sites (Pozzan et al., 1994). Thus, upon cell stimulation, when [Ca++] enters the cell from the extracellular medium, it would reach very slowly or not at all, the deeper regions of the cytosol (Pozzan et al., 1994). On the contrary, the presence of distributed, rapidly exchanging intracellular calcium ([Ca++]i) deposits are able to release [Ca++] in response to either a rapidly diffusing second messenger, such as IP₃, and ensures a more diffuse and timely coordinated increases in [Ca++]i. Furthermore, if there is a large increase in [Ca++]i, then this increase is expected to return to normal [Ca++]i levels. Increased calcium levels (intracellular and intranuclear) are quantitatively correlated with transcription factor expression (Ghosh et al., 1994) and cell cycle progression (Takuwa et al., 1995). A large rise of [Ca++]i , if prolonged, results in an irreversible damage of a number of cell functions (Pozzan et al., 1994). This is prevented by a very fast return of [Ca++]i to resting level sustained by [Ca++] accumulation in organelles, such as mitochondria and the endoplasmic reticulum (Pozzan et al., 1994).

Calcium also has been proven to be an important second messenger that controls or influences a variety of cellular functions, including oocyte maturation, spermatazoa capacitation, acrosome reaction, hormone secretion and muscle growth (Alberts et al., 1983). In addition, studies have indicated that [Ca++]i release in mouse morulae occurs predominantly through the IP₃ receptor, and that alterations of [Ca++]i levels can accelerate or delay embryonic growth and regulation of oocyte and embryonic development (Stachecki and Armant, 1996).

The objective of this present study was to determine the effect of PAF on preimplantation stage embryo intracellular calcium levels in the mouse.

Materials And Methods

Two cell stage embryos were collected from pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) (10 IU ea) primed female (1 month of age) Swiss-Webster (CFW; Charles River) mice mated with fertile CFW male (6 months of age) mice. Interval between PMSG and hCG was 46-48 hours.

The effect of PAF on intracellular calcium levels in exposed two-cell CFW mouse embryos was determined as previously described (Roudebush et al., 1997). Briefly, embryos were extracted and collected in modified M16 (phenol red free) medium at the one cell stage. After the cells were incubated for 24 hours, to the 2-cell stage, the embryos were loaded with 0.1 µM of Fura-2 AM (0.2 µM) for 30 minutes at 37°C. Fura-2 AM (0.2 µM) (an ultraviolet-excitable ratiometric intracellular calcium indicator) was selected for use since upon binding to intracellular calcium, Fura-2 AM (0.2 µM) will exhibit an absorption shift that is observed by excitation spectrum scanning (between 300 and 400 nm) while emission is monitored (at 510 nm). A slow titration of the dye is required to reduce the level of entrapped Fura-2 AM (0.2 µM) in organelles. Loading the embryos at 37°C reduces dye endocytosis. These steps minimize the level of unhydrolyzed dye in the mouse embryo, thus avoiding erroneous readings. After loading, the embryos were collected into individual imaging dishes with M16 and placed into an Attofluor Digital Fluorescence system (ADF; Carl Zeiss Co., Thornwood, NY). The ADF was set at wavelengths of 340/380 nm (slit, 5nm) and emission at 510 nm (slit, 10 nm). Internal controls for minimum and maximum fluorescence values were run on each sample. The background intracellular calcium levels were obtained for a minimum of 90 seconds prior to treatment. PAF or lyso-PAF (the biological inactive form) [0.1 µM; final concentration] were added directly to the ADF chamber and intracellular calcium levels were recorded at five second intervals until values normalized. Data were analyzed by Students' t-test.

Results

Baseline calcium levels, response times, and time to reach maximum response and the maximum intracellular calcium levels following PAF exposure are presented in Table 1. The non-stimulated intracellular calcium levels were recorded to have a mean concentration of 149.4 nM. Non-stimulated background [Ca++]i levels began to increase by an average of 4.3 seconds after PAF exposure with maximum calcium levels were reached by an average of 176.9 seconds. The mean maximum [Ca++]i levels after PAF exposure was 456.1 nM, which was approximately three times (3X) that of non PAF or lyso-PAF exposure (P<0.001).

The effect of PAF and lyso-PAF on [Ca++]i in mouse preimplantation two-cell stage embryos is presented in Table 2. The mean base [Ca++]i level in mouse preimplantation two-cell stage embryos was 116.0 nM. After exposure to lyso-PAF, the mean [Ca++]i was recorded at 116.2 nM, indicating no change in [Ca++]i levels. Following PAF exposure, these same cells obtained a mean [Ca++]i level of 285.0 nM, increasing two times (2X) over their base intracellular calcium levels. The mean time for these 2-cell stage embryos to reach a maximum [Ca++]i level was 229.8 seconds, which was quicker than the non lyso-PAF exposed 2-cell stage embryos. The mean maximum [Ca++]i level was obtained at a P<0.05 level of significance.

A typical 2-cell stage mouse embryo response to PAF exposure is presented in Figure 1. In this single example, a base level of [Ca++]i was obtained for 90 seconds and was measured to be 134nM. Following baseline [Ca++]i determination, exogenous PAF (10^-7 M final concentratiion) was added (as indicated by the arrow) to the ADF chamber and [Ca++]i levels were continually obtained. This particular two-cell stage mouse embryo responded almost instaneously to the exogenous PAF with increasing [Ca++]i levels overtime. The embryo obtained a maximum [Ca++]i level of 499nM, 104 seconds after exgenous PAF exposure (as indicated by the arrow). After the maximum [Ca++]i level was attained, the embryo began to re-accumulate [Ca++] back into cellular organelles, therefore showing a steady decline in [Ca++]i as was expected (Pozzan et al., 1994), until it reached near baseline levels.

Discussion

Human, mouse, sheep and rabbit preimplantation embryos produce and release PAF (O'Neill, 1985; Collier et al., 1988; Battye et al., 1991; Pike et al., 1992). In addition, PAF production by the human embryo is related to its' subsequent pregnancy potential (O'Neill et al., 1987). Enhanced embryo development has also been reported in rabbit oocytes fertilized in vitro with PAF treated spermatozoa (Roudebush et al., 1993). PAF antibodies also inhibit embryo development (Roudebush et al., 1995) and antagonists inhibit implantation (Spinks et al., 1988; Andu et al., 1990), providing evidence on the presence and requirement of embryo-derived PAF during the preimplantation period.

Cellular response to different ligands is typically via protein receptors located on the cell membrane. The ligand-receptor binding initiates the release of a second messenger which then influences the cell activity. PAF's mechanism of action, in preimplantation embryos, appears to be a receptor mediated increase of intracellular calcium (Roudebush et al., 1997). PAF will bind with cell surface receptors causing the formation of inositol triphosphate (IP₃) and diacylglycerol (DAG), as secondary messengers (Lapetina, 1982), which induces the increase of intracellular calcium (Vargraftig and Barquet, 1987). The receptor-activated calcium mobilization by the inositol-calcium signaling involves two main phases: (a) calcium release from an intracellular store; and (b) extracellular calcium entry by channels (Putney, 1987).

Calcium has been also proven to be an important secondary messenger that controls or influences a variety of cellular functions, including, but not limited to: oocyte maturation, spermatazoa capacitation, acrosome reaction, hormone secretion, and muscle growth (Alberts et al., 1983). In addition, intracellular calcium regulates NAD kinase activity, and protein function (Hanson et al., 1994) and protein synthesis (Gilchrist et al., 1994). In this study, the embryonic cells responded to PAF with an immediate rapid diffusion of [Ca++]i. This type of response supports the fact that calcium responds to IP₃ as a secondary messanger (Pozzan et al., 1994). Furthermore IP₃ may effect embryonic development by regulation of intracellular calcium levels (Roudebush et al., 1997). Intracellular calcium release in mouse morulae occurs predominantly through the IP₃ receptor, and intracellular calcium levels can either accelerate or delay, embryonic growth and differentiation, providing a link between the regulation of oocyte and embryonic development (Stachecki and Armant, 1996).

The results of this study demonstrate the magnitude of PAF's action in the preimplantation stage embryo. PAF exposure of two-cell stage mouse embryos resulted in a three fold increase of intracellular calcium over background levels (131.4nM). After [Ca++]i maximized, the calcium level began to decline and re-accumulate in orgenelles as expected. A prolong increase of intracellular calcium can cause irreversible damage to cell function Pozzan et al., 1994). This is consistent with our previous study (Roudebush et al., 1997), where we found that PAF exposure will increase (four times) intracellular calcium over background levels (30nM). However, a major difference between the two data sets, is in the baseline [Ca++]i levels. A plausible explanation for this is in the previous study, the lower basal intracellular calcium concentration may be due to that calcium free medium was used, thus depleting calcium stores (Thorton et al., 1992).

The significance of this study further indicates the mechanism of action of PAF and its' affects on preimplantation mouse embryos. PAF is released and subsequently influences embryonic cells by increasing intracellular calcium levels. An increase in intracellular calcium in preimplantation embryos will facilitate development (Alberts et al., 1983).

Additional studies are required to elucidate the influence of PAF on intracellular calcium of preimplantation stage mouse embryos at all developmental stages (e.g. four cell stage, morula stage and blastocyst stage). Studies may also be needed to further prove that PAF's effect on preimplantation development is receptor mediated and involves the inositol triphosphate system. Finally, calmodulin has also been shown to be a primary downstream receptor for calcium (Stachecki and Armant, 1996). Further studies may be needed to clarify calmodulin activity needed for developmental acceleration that follows calcium signaling.

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Table 1

Intracellular Calcium Levels (nM) in Two-Cell Stage Preimplantation Mouse Embryos: Baseline and Following PAF Exposure

Table 2

Intracellular Calcium Levels (nM) in Two-Cell Stage Preimplantation Mouse Embryos: Baseline and Following Lyso-PAF and PAF Exposure

*: P=0.010

Figure 1

Effect of PAF on Intracellular Calcium in a Single Embryo