Embryotrophic factor-3 from human oviductal cells enhances proliferation, suppresses apoptosis and stimulates the expression of the {beta}1 subunit of sodium–potassium ATPase in mouse embryos

J.S. Xu1, Y.L. Lee1, K.F. Lee1, K.L. Kwok1, W.M. Lee2, J.M. Luk3 and W.S.B. Yeung1,4

1 Department of Obstetrics and Gynaecology, 2 Department of Zoology, Queen Mary Hospital, The University of Hong Kong, Hong Kong and 3 Department of Surgery, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China

4 To whom correspondence should be addressed. Email: wsbyeung{at}hkucc.hku.hk


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Embrytrophic factor-3 (ETF-3) from human oviductal cells enhanced the development of mouse preimplantation embryos. This report studied the embryotrophic mechanisms of the molecule. METHODS AND RESULTS: Mouse embryos were incubated with ETF-3 for 24 h at different stages of development. ETF-3 treatment between 96 and 120 h post-HCG increased the cell count of blastocysts, whilst treatment between 72 and 96 h post-HCG enhanced the expansion and hatching of the blastocysts. ETF-3 increased the cell number of the embryos by suppressing apoptosis and increasing proliferation as determined by TUNEL and bromodeoxyuridine uptake assays, respectively. Real-time quantitative PCR showed that the in vivo developed and ETF-3-treated blastocysts had a significantly higher mRNA copy number of Na/K-ATPase-{beta}1, but not of hepsin, than that of blastocysts cultured in medium alone. The former gene was associated with cavitation of blastocysts while the latter was related to hatching of blastocyst. The beneficial effect of ETF-3 on blastocyst hatching was also seen when ETF-3-supplemented commercially available sequential culture medium for human embryo culture was used to culture mouse embryos. CONCLUSIONS: ETF-3 improves embryo development by enhancing proliferation, suppressing apoptosis and stimulating expression of genes related to blastocyst cavitation. Supplementating human embryo culture medium with ETF-3 may improve the success rate in clinical assisted reproduction.

Key words: apoptosis/embryotrophic factor/oviduct/proliferation/sodium–potassium ATPase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Oviductal cells produce a number of factors, including factors of known and unknown identity, to enhance the development of embryos (Yeung et al., 2002Go). Three large molecular size embryotrophic factors, termed ETF-1, -2 and -3, have been isolated from the spent medium of human oviductal cell culture (Liu et al., 1998Go; Xu et al., 2001Go). Although ETF-3 is the most abundant factor among the three ETFs isolated, the amount collected from primary oviductal cell culture is minute. Therefore, an immortalized oviductal cell line has been established (Y.L.Lee et al., 2001Go) to produce sufficient ETF-3 for characterization.

ETF-3 enhances the development of mouse blastocysts resulting in blastocysts, with more trophectoderm (TE) cells, larger diameter, increased ability to escape from the zona pellucida (hatching), and hatched blastocysts with higher attachment potential and larger trophoblast outgrowth (Xu et al., 2001Go; Lee et al., 2003Go). How ETF-3 affects embryo development is unclear. The increase in the blastocyst cell count could be a result of a decrease in cell death and/or an increase in cell proliferation of the embryo. Apoptosis is a common phenomenon in the development of embryos of various mammalian species (Hardy, 1997Go). Its incidence is significantly higher in cultured mouse blastocysts than in those developed in vivo (Devreker and Hardy, 1997Go). Compared with studies on apoptosis in embryos, there are fewer studies on cell proliferation in embryos (Herrler et al., 1998Go; Makarevich and Markkula, 2002Go). The effect of ETF-3 on apoptosis and proliferation in embryos is unknown.

ETF-3 alters the gene expression of the resulting blastocyst in an mRNA differential display study (Lee et al., 2003Go). The formation of TE involves the expression of specific gene products (Watson and Barcroft, 2001Go). Therefore, it is possible that the increase in size and hatching rate of ETF-3-treated blastocysts is due to changes in the expression of genes related to the development of TE.

Cavitation of blastocyst depends on the development of the ion transport systems in the TE (Baltz et al., 1997Go). Sodium–potassium ATPase (Na/K-ATPase) is one of the well-characterized enzymes involved in this process. It is found in the basolateral membranes of the TE cells. The main increase in Na/K-ATPase activity occurs after the morula stage in the mouse (Van Winkle and Campione, 1991Go), supporting the involvement of the enzyme in cavitation (MacPhee et al., 2000Go). The enzyme contains three subunits, {alpha}, {beta} and {gamma}. The catalytic {alpha}-subunit is responsible for ion transport and has been studied intensively in mouse embryos (Watson et al., 1990Go; MacPhee et al., 1994Go). The {beta}-subunit is important in the functioning of the enzyme by facilitating the correct folding of the {alpha}-subunit in the membrane (Geering, 1991Go).

Before implantation, the expanded blastocyst escapes from the zona pellucida, possibly by using membrane-bound serine protease (Perona and Wassarman, 1986Go). Hepsin is a serine protease. It is present in the mouse preimplantation embryo as early as the 2-cell stage and peaks at the blastocyst stage prior to hatching (Vu et al., 1997Go). Therefore, the enzyme has been suggested to be involved in the hatching process of preimplantation embryos.

In this study, we further characterize the biological activities of ETF-3 in embryo development, aiming to delineate the mechanisms of action of the molecule. There are four objectives in this report. They are (i) to define the window of action of ETF-3 by treating the embryo for 24 h only, at different stages of embryonic development; (ii) to study the action of ETF-3 on cell proliferation and apoptosis in the embryo; (iii) to determine the expression of Na/K-ATPase-{beta}1 and hepsin mRNA in blastocysts with or without ETF-3 treatment; and (iv) to compare the activity of ETF-3 in different culture systems.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Preparation of oviductal cells
The Ethics Committee of the University of Hong Kong approved the protocol for this study. Human Fallopian tube tissue was obtained from patients who consented to the removal of their Fallopian tubes during a total abdominal hysterectomy for uterine fibroids. The oviductal cells were obtained by trypsin/EDTA (Gibco, NY) dispersion of the oviductal epithelium and cultured in Dulbecco's modified Eagle's medium/Ham's F12 medium (1:1 v/v) (Sigma, St Louis, MO) supplemented with penicillin, streptomycin, glutamine, sodium bicarbonate and 15% human pre-ovulatory serum (sDMEM/F12) at 37°C under an atmosphere of 5% CO2 in air (Yeung et al., 1992Go). After confluent growth, the cells were trypsinized and stored in liquid nitrogen.

Purification of ETF-3
ETF-3 was purified from the spent DMEM/F12 medium supplemented with 3 mg/ml (w/v) bovine serum albumin (BSA) after culturing human oviductal cells for 24 h (conditioned medium, CM) as described previously (Liu et al., 1995Go, 1998Go). Briefly, CM was passed through a concanavalin A (ConA) affinity column. The >100 kDa components that bound to the lectin column containing the embryotrophic activity (Liu et al., 1998Go) were collected by centrifugation of the ConA-eluted fraction through a Centricon-100 concentrator (Amicon, Austin, TX) which retained molecules with size >100 kDa, and were fractionated further with stepwise elution from a Mono-Q ion-exchange column. ETF-3 was eluted with 0.3 mol/l NaCl. It was concentrated and reconstituted in the embryo culture media at a concentration of 10 µg/ml, which was the minimum concentration found to stimulate blastocyst formation of mouse embryos in vitro (data not shown).

Embryo culture and assessment of embryo development
Zygotes were collected from 6- to 8-week-old MF-1 females after superovulation by an i.p. injection of 5 IU of pregnant mare's serum gonadotrophin (Sigma) followed 48 h later by an injection of 5 IU of HCG (Sigma). The females were placed with the fertile BALB/c males after the HCG injection. The resulting embryos were used in our bioassay system for screening of oviductal ETFs because the development of the embryos in vitro was not too robust or too fragile. The former would make the bioassay insensitive to the action of the ETF(s) while the latter would cause instability in the embryo culture and decrease the precision of the bioassay. Cumulus-enclosed zygotes were collected at 22–23 h post-HCG and denuded with 0.3 mg/ml hyaluronidase in 20 mmol/l HEPES-buffered CZB medium (CZB + H) containing 81.62 mmol/l NaCl, 4.83 mmol/l KCl, 1.18 mmol/l KH2PO4, 1.18 mmol/l MgSO4 7H2O, 25.12 mmol/l NaHCO3, 1.70 mmol/l CaCl2 2H2O, 31.30 mmol/l sodium lactate, 0.27 mmol/l sodium pyruvate, 0.11 mmol/l EDTA, 1 mmol/l glutamine, 5 mg/ml BSA, 100 IU/ml sodium penicillin and 0.7 mmol/l streptomycin (Chatot et al., 1989Go). The zygotes were washed three times in CZB + H and twice in pre-gassed CZB medium before pooling and randomly allocating to different treatment groups. They were cultured up to 72 h post-HCG in the CZB medium at 5% CO2 in humidified air. These embryos were transferred and cultured in glucose (5 mmol/l) containing CZB (CZB + G) up to 144 h post-HCG. In order to study the influence of culture conditions on the embryotrophic activity of ETF-3, embryos were also cultured in a commercially available sequential embryo culture system, G1.2/G2.2 (Vitrolife, Göteborg, Sweden), with or without ETF-3 supplementation.

To define the window of action of ETF-3, embryos were treated with ETF-3 (10 µg/ml) for 24 h at different developmental stages (Figure 1). Embryos treated between 24 and 48, 48 and 72, 72 and 96, and 96 and 120 h post-HCG were labelled as E24/48, E48/72, E72/96 and E96/120, respectively. The age of the embryos was timed with reference to the time after HCG administration. The rate of embryo development, and allocation of inner cell mass (ICM) and TE in blastocysts and outgrowth in vitro were determined.



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Figure 1. Experimental design. Mouse preimplantation embryos at different stages of development were treated with ETF-3 for 24 h.

 
Differential staining of the TE and the ICM was performed by the method of Handyside and Hunter (1984)Go with modifications. Briefly, acid Tyrode's solution was used to remove the zona pellucida of blastocysts. The zona-free blastocysts were washed in phosphate-buffered saline (PBS), incubated in rabbit anti-mouse whole serum for 15 min at room temperature, washed in PBS six times, incubated in guinea pig complement with 10 µg/ml propidium iodide for 5–6 min at 37°C, rinsed quickly in PBS, fixed in absolute ethanol, and stained with Hoechst 33258 (25 µg/ml) for 2 h. The embryos were then observed under fluorescence microscopy. The total cell number (TCN) of the blastocysts was counted using the Hoechst filter set (340/460, Ex/Em), whilst the TE cells were counted using the propidium iodide filter set (540/610, Ex/Em). The number of cells in the ICM was calculated by subtracting the number of TE cells from the TCN.

The MetaMorph Imaging System (Universal Imaging, PA) was used to determine the size of blastocysts at 120 h post-HCG after capturing of the image of the blastocysts with a digital camera (Photometrics, AZ). The blastocysts were then cultured in 20 µl droplets of DMEM/F12 containing 15% human serum under paraffin oil for 2 days. Human serum was used because of its ready availability from our assisted reproduction clinics. The number of embryos attached to the culture dish was counted. The MetaMorph Imaging System was used to determine the spreading area of the blastocyst outgrowth.

Blastomere proliferation and apoptosis
The In Situ Cell Proliferation Kit (Roche, Indianapolis, IN, Germany) was used to study blastomere proliferation. Morula and blastocysts at 96 and 120 h post-HCG, respectively, were incubated in the embryo culture medium containing 10 µmol/l bromodeoxyuridine (BrdU) in an atmosphere of 5% CO2 in air at 37°C for 1 h. The embryos were then washed in PBS, fixed in 4% paraformaldehyde for 30 min, digested with 0.05% trypsin in PBS containing 0.05% CaCl2 for 15 min, denatured in 4 mol/l HCl for 20 min, and labelled with fluorescein isothiocyanate (FITC)-conjugated anti-BrdU monoclonal antibody. The total number of blastomeres per embryo was determined by staining with propidium iodide (PI, 1 µg/ml in PBS).

TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) staining was used to detect apoptosis as described (Xu et al., 2000Go). Briefly, embryos were incubated at 37°C for 15 min in CZB containing PI (1 µg/ml). They were then washed three times successively in PBS, fixed in 4% formaldehyde in PBS (pH 7.4) for 30 min, washed six times in PBS, permeated in 0.1% Triton X-100 at 4°C for 1 min, washed three times in PBS and incubated in TUNEL reaction cocktail (in situ cell death detection system; Boehringer Mannheim, Germany) at 37°C for 1 h. The total number of blastomeres per embryo was determined by staining with Hoechst 33258 (20 µg/ml in PBS).

Analysis of Na/K-ATPase-{beta}1 and hepsin mRNA
mRNAs from mouse embryos were extracted by the Dynabeads mRNA Direct Kit (Dynal AS, Oslo, Norway) as described previously (K.F.Lee et al., 2001Go). Reverse transcription was performed according to the First Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, Uppsala, Sweden). The quality of the mRNA obtained with the method was ascertained by PCR analysis of the {beta}-actin gene. The intensity of the {beta}-actin signal was similar among different groups of blastocysts (data not shown). The reversed-transcribed mRNA was used for quantitative PCR (qPCR) of mouse Na/K-ATPase-{beta}1 (GenBank accession number: NM_009721) and hepsin (GenBank accession number: AF030065). The primers and probes for their specific amplification (Table I) were designed using the Primer Express software program (PE Applied Biosystems, Foster City, CA). The probes were labelled with the fluorescent reporter dye 5-carboxyfluorescein at the 5' end and with the quencher N,N,N,N'-tetramethyl-6-carboxyrhodamine (TAMRA) at the 3' end. Standards of hepsin and Na/K-ATPase-{beta}1 of size 78 and 70 bp, respectively, were prepared by cloning of the PCR-amplified products for mouse Na/K-ATPase-{beta}1 and hepsin into pGEM-T easy vector (Promega Corp., Madison, WI). Plasmid DNAs were purified and sequenced. The standards of Na/K-ATPase-{beta}1 and hepsin for real-time PCR were 20–2 x 106 molecules per reaction.


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Table I. Primers and probes used for real-time PCR

 
The quantification of Na/K-ATPase-{beta}1 and hepsin mRNA in in vivo developed blastocysts obtained by flushing from the mouse uterus at 102 h post-HCG, and in expanded blastocysts cultured in CZB medium alone or in ETF-3-supplemented CZB was performed by real-time PCR detection using an ABI PRISM 7700 Sequence Detector (P E Applied Biosystems). In each experiment, eight expanded blastocysts per group were used. The experiment was repeated four times. By comparing with the standard scale of Na/K-ATPase-{beta}1 and hepsin, the ‘threshold cycle’ (CT) at which each fluorescent signal was first detected above the background was used as a measure of the absolute copy number of the two mRNA templates present in each mouse blastocyst sample. PCRs were carried out using a 96-well plate setup. A 2.5 µl aliquot of reverse-transcribed cDNA derived from the same number of blastocysts from the three groups was amplified in a 25 µl volume containing 12.5 µl of 2x Universal PCR Master Mix (PE Applied Biosystems), 900 nmol/l of each primer and 250 nmo/l of TaqMan probes. After an initial denaturation step at 95°C for 10 min, 40 cycles of PCR were carried out at 95 °C for 15 s and at 60°C for 1 min. A standard curve was constructed for each gene using the plasmid DNA containing the same inserted fragment amplified by the TaqMan system. Each sample was run in duplicate and two no template control wells were included in each PCR experiment. The PCR products of the standard and mouse blastocyst samples were confirmed as single bands using gel electrophoresis stained with ethidium bromide.

Data analysis
The data from three replicate experiments were combined and analysed by the SigmaStat (Jandel Scientific, San Rafael, CA) software package. {chi}2 test and ANOVA where appropriate were used to compare the effect of different treatments among various groups. Newmen Keuls' test was applied as a post-test of ANOVA to compare the cell number of TE and ICM, TCN, blastocyst size, in vitro spreading area, blastomere proliferation, apoptosis and mRNA transcript number of different groups of embryos.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
ETF-3 treatment for 24 h at different stages of embryo development
The incidence of expanded and hatching blastocyst formation at 120 and 144 h post-HCG, respectively, is shown in Figure 2. In CZB, 15% (14 out of 95) of the 2-cell embryos reached the hatching blastocyst stage. Twenty-four hours of ETF-3 treatment between 72 and 96 h post-HCG (E72/96) significantly increased the hatching rate to 31% (29 out of 94). Although the rate of blastocyst formation in E72/96 was also increased when compared with the control, the difference was not significant. The results for other treatment groups were comparable with the control.



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Figure 2. Embryo development at 120 and 144 h post-HCG after ETF-3 treatment for 24 h at different developmental stages. More embryos underwent hatching after being treated with ETF-3 from 72 to 96 h post-HCG. Numbers in parentheses represent the number of 2-cell embryos used in each group. (*P<0.05).

 
Two unpublished observations showed that the embryotrophic effect of ETF-3 was not due to contaminants from BSA in the CM. First, a control experiment had been performed in which the same amount of fresh culture medium as that of the oviductal cell CM used in this study was passed through the ETF-3 purification process. The fraction from the fresh medium corresponding to the ETF-3 fraction from the CM had no embryotrophic activity. Secondly, ETF-3 could be purified from oviductal cells cultured in protein-free medium.

The allocations of ICM and TE, as well as the TCN in blastocysts at 120 h post-HCG are shown in Figure 3. Blastocysts treated from 96 to 120 h post-HCG (E96/120) had significantly more TCN (44.7±2.1) than those cultured in CZB alone (39.7±2.5). This increase was due to a significant increase (P<0.05) in the number of TE cells (ETF-3, 35.6±1.8; control, 30.9 = 1.4). The number of ICM cells in this group (9.1±1.5) was comparable with that of the control (9.0±1.7). These parameters in other groups were similar to those in the control. Only blastocysts in E72/96 had significantly larger size and spreading area than the control embryos (Table II).



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Figure 3. Total cell numbers per blastocyst and cell allocation to the ICM and TE in mouse embryos after ETF-3 treatment for 24 h at different developmental stages. Treatment from 96 to 120 h increased the cell count in the TE and the total cell number. Numbers in parentheses represent the number of blastocysts used in each group. (*P<0.0001).

 

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Table II. Blastocyst size, attachment and outgrowth of embryos after treatment with ETF-3 for 24 h at different stages of development (mean±SEM)

 
Blastomere proliferation and apoptosis
In this experiment, embryos were treated with ETF-3 continuously from 24 to 120 h post-HCG. ETF-3 significantly increased (P<0.05) the percentage of proliferating blastomeres at the morula (ETF-3, 67.7±1.1%; control, 53.6±1.2%) and the blastocyst (ETF-3, 23.1±1.6%; control, 16.2±1.5%) stages (Figure 4). On the other hand, it significantly decreased (P<0.05) the incidence of apoptosis at these stages (morula: ETF-3, 7.3±1.5%, control, 8.9±1.7%; blastocyst: ETF-3, 18.3±1.9%; control, 23.1±2.7%) (Figure 4). The high apoptotic rate of the blastocysts cultured in the CZB medium reflected the poor in vitro developmental potential of the mouse strain used, as a similar apoptotic rate was found when the embryos were grown in a better culture system, G1.2/G2.2 (Xu et al., 2004Go)



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Figure 4. Proliferation and apoptosis of blastomeres in morula and blastocysts after continuous ETF-3 treatment from 24 to 120 h post-HCG. Each bar represents the mean±SEM. Numbers in parentheses represent the number of embryos used in each group. (*P<0.05).

 
Expression of Na/K-ATPase-{beta}1 and hepsin
In this experiment, analyses were confined to the expanded blastocyst only, which was defined as an embryo with a blastocoel >50% of the size of the blastocyst and showing zona thinning at 120 h post-HCG. Figure 5 shows the mean mRNA copy number of Na/K-ATPase-{beta}1 and hepsin in one blastocyst equivalent found in the control, ETF-3-treated and in vivo developed blastocysts in four independent experiments. Significantly higher (P<0.05) Na/K-ATPase-{beta}1 copy number per blastocyst was found in the ETF-3-treated blastocysts (5748±815) and in the in vivo developed blastocysts (6081±755) when compared with the control (2634±400). A low copy number of hepsin mRNA was found in all the blastocysts. There was no significant difference among all the groups (control, 82±27; ETF-3, 108±16; in vivo, 54±25, P>0.05). The inter-assay errors for qPCR of hepsin and Na/K-ATPase-{beta}1 ranged from 0.13 to 1.74% and from 0.51 to 2.2%, respectively. The corresponding intra-assay errors ranged from 0 to 1.1% and from 0 to 3.74%, respectively. Agarose gel electrophoresis of the qPCR products showed a single band of the expected size for both genes (Na/K-ATPase-{beta}1, 78 bp; hepsin, 70 bp) (data not shown).



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Figure 5. Absolute quantification of the hespin and Na/K-ATPase-{beta}1 mRNA copy number per blastocyst in control medium, ETF-3 treatment and in vivo developed mouse blastocysts. The asterisks represent values that are significantly different from the control (P<0.03).

 
Action of ETF-3 in different culture systems
Embryos cultured in CZB supplemented with ETF-3 had a significantly higher blastulation rate than those cultured without ETF-3 treatment, but this was similar to those cultured in G1.2/G2.2 with or without ETF-3 supplementation (Table III). The hatching rate was significantly higher in ETF-3-treated embryos than the rate of those in the corresponding culture media without ETF-3. Blastocysts cultured in the G1.2/G2.2 system had significantly more cells than those cultured in the CZB system (Table IV). Supplementation with ETF-3 significantly increased the development of TE cells of blastocysts cultured in the CZB medium to a level equivalent to those cultured in the G1.2/G2.2 system. The number of TE cells in blastocysts derived from the G1.2/G2.2 system with or without ETF-3 treatment was similar.


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Table III. Development of mouse embryos in the G1.2/G2.2 system with or without ETF-3 supplementation

 

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Table IV. Cell allocation in blastocysts cultured in the G1.2/2.2 system with or without ETF-3 supplementation

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A previous report showed that ETF-3 enhanced the blastulation and hatching of embryos when the treatments covered 72–120 h post-HCG (Xu et al., 2001Go). The present study confirmed these beneficial effects of ETF-3, and further demonstrated that there were differential effects of ETF-3 on embryonic development within this window of action. Whilst treatment between 96 and 120 h post-HCG (E96/120) increased the TCN of the resulting blastocysts, treatment covering 72–96 h post-HCG (E72/96) enhanced the expansion and hatching process of the blastocysts. It is unclear why the influence of ETF-3 is confined only to these two treatment periods. The fact that ETF-3 did not have a significant effect on day 1 and day 2 embryos was consistent with the predominant effect of the factor being on the TE, which is not formed until day 3.

Although removal of blastomeres from 4- and 8-cell mouse and human embryos has no effect on blastulation (Wilton and Trounson, 1989Go; Hardy et al., 1990Go; Tarin et al., 1992Go), a sufficient number of blastomeres is required for blastocyst expansion and successful hatching of mouse embryos in vitro (Montag et al., 2000Go). Fragmentation in human embryos, which could be equivalent to loss of blastomeres, is common in vitro. A high degree, but not low degree, of fragmentation in human embryos is associated with a decrease in the TCN and the blastulation rate (Hardy et al., 2003Go), suggesting that blastulation and TCN in a blastocyst may not necessarily be related when a minimum number of cells is present. The differential temporal effect of ETF-3 on hatching and TCN is in line with these observations that the number of blastomeres and blastulation are under different controls, and ETF-3 affects both controlling mechanisms in temporal succession.

The cell count in an embryo depends on apoptosis and proliferation of blastomeres. ETF-3 increases the TCN by increasing proliferation and reducing apoptosis. Embryos possess apoptotic genes (Spanos et al., 2002Go). Oviductal cell co-culture stimulates cell proliferation (Xu et al., 2004Go), suppresses apoptosis and caspase activity, and maintains the mitochondrial potential of embryos (Xu et al., 2003Go). The molecular action of ETF-3 on proliferation and apoptosis is unclear. It stimulates the expression of HSP70 and Cul-1 (Lee et al., 2003Go). The former may protect the embryo from undergoing apoptosis (Neuer et al., 1999Go; Matwee et al., 2001Go). Cul-1 is related to cell cycling by regulating the abundance of cell cycling proteins at the G1–S phase (Skowyra et al., 1999Go).

ETF-3 preferentially enhances the development of TE. The cell numbers in the TE and ICM of human blastocysts are under different control (Hardy et al., 2003Go). Increased fragmentation was associated with lower blastocyst cell numbers. At low levels of fragmentation, the decrease in cell number is at the expense of the TE cells with a steady number of ICM cells (Hardy et al., 2003Go). Differential susceptibility of ICM and TE cells to apoptosis in rodent embryos has also been suggested (Pampfer, 2000Go). The present results show that ETF-3 selectively increases the development of the TE, leading to the formation of larger blastocysts with higher hatching rate. To study the mechanism of action of ETF-3, we used qPCR assay to quantify the expression of two genes, Na/K-ATPase-{beta}1 and hepsin, in single mouse blastocyst equivalents; the former is involved in blastocyst cavitation whilst the latter is related to the hatching process.

Na/K-ATPase isoforms play critical roles in the formation of the blastocoel (Watson et al., 1999Go; MacPhee et al., 2000Go). The {alpha}1{beta}1 isozyme is primarily responsible for blastocoel formation (MacPhee et al., 2000Go). The amount of Na/K-ATPase-{alpha}1 mRNA per cell is relatively constant from the 2-cell to the blastocyst stage (Watson et al., 1990Go). However, the {beta}-subunit is detected at the late morula stage and becomes more abundant in the early blastocyst (Gardiner et al., 1990Go; Kidder, 1993Go; Betts et al., 1997Go). Therefore, it has been suggested that the {beta}-subunit is more significant in the functional expression of Na/K-ATPase (Geering, 1991Go).

The present results show that the Na/K-ATPase-{beta}1 mRNA level in mouse blastocysts after ETF-3 treatment is doubled and is comparable with the level found in blastocysts developed in vivo. This is consistent with the known ETF-3 effect on the TE (Xu et al., 2001Go) and with the differential expression of Na/K-ATPase-{alpha}1 and {beta}1 isoforms in mouse blastocyst; abundant in TE but low in ICM (MacPhee et al., 2000Go). The increase in the expression of Na/K-ATPase-{beta}1 mRNA in blastocysts may stimulate cavitation in the ETF-3-treated embryos. This has to be confirmed by determination of the Na/K-ATPase activity in embryos with or without ETF-3 treatment.

It is possible that the increase in Na/K-ATPase-{beta}1 mRNA copy number of ETF-3-treated blastocysts results from the increase in the TE cell number after ETF-3 treatment (Xu et al., 2001Go). The present study demonstrates that the Na/K-ATPase-{beta}1 mRNA copy number in blastocysts is doubled after ETF-3 treatment, whereas the TE cell number is only increased by 40% under similar culture conditions (Xu et al., 2001Go). Thus, the increase in TE cell number cannot fully account for the increase in mRNA copy number, suggesting that ETF-3 increases the mRNA expression of Na/K-ATPase-{beta}1 of the TE cells.

Studies demonstrated that an embryo-derived trypsin-like protease participated in the hatching process of mouse blastocysts (Dabich and Andary, 1976Go; Denker, 1977Go; Sawada et al., 1990Go; O'Sullivan et al., 2001aGo,bGo). Serine protease is present in the preimplantation embryo (Perona and Wassarman, 1986Go). Hepsin is an endogenous mouse serine protease with increased expression in the early mouse blastocyst and has been suggested to assist hatching (Vu et al., 1997Go). In this study, the mRNA expression of hepsin is low and is unaffected by ETF-3 treatment. The effect of ETF-3 on other hatching-related proteases such as implantation serine protease (O'Sullivan et al., 2001aGo) remains to be investigated.

Impairment of the hatching process is a possible cause of the low implantation rate in human-assisted reproduction programmes (Cohen et al., 1990Go). Thus the beneficial effect of ETF-3 on hatching could be useful in assisted reproduction. To address this possibility, it is important to demonstrate that the embryotrophic activity of ETF-3 still exists in a good culture system. It has been reported that one of the commonly used human embryo sequential culture systems, G1.2/G2.2, is better than the standard mouse embryo culture system, KSOMaa, in producing mouse embryos with higher blastulation rate, more cells per blastocyst and more cells in the ICM (Gardner and Lane, 2002Go). Bovine embryos cultured in the G1.2/G2.2 system have a similar blastulation rate and pregnancy rate after embryo transfer, but a higher TCN and ICM cell count than those co-cultured with the BRL cells (Lane et al., 2003Go). Therefore, we compared the development of mouse embryos cultured in the G1.2/G2.2 system with and without ETF-3 supplementation. The present study showed that although the blastulation rates of mouse embryo in the G1.2/G/2.2 system with or without ETF-3 treatment were similar, the hatching rate after ETF-3 treatment was significantly higher, supporting the use of ETF-3 in clinically assisted reproduction. The G1.2 and G2.2 culture media contain amino acids, which preferentially stimulate proliferation of cells in the ICM (Biggers et al., 2000Go). The selective action of ETF-3 on the TE may well be a good complement to the G1.2/G2.2 system in supporting embryo development in vitro.

A number of observations suggest that the effect of ETF-3 on the hatching process is unlikely to be due to improvement in the overall health of the embryo and the general quality of the culture conditions. First, ETF-3-treated blastocysts have a higher hatching rate irrespective of the culture system used, CZB or G1.2/G2.2. The latter has been reported to be better than the standard mouse embryo culture system (Gardner and Lane 2002Go). Secondly, the action of ETF-3 is likely to be cell type specific as ETF-3 affects mainly the development of the TE cells, which is manifested as an increase in the number of TE cells, but of the ICM cells after treatment (Xu et al., 2001Go). Thirdly, the localization of ETF-3 immunoreactivity mainly to the TE (Lee et al., 2004Go) and the enhancement of Na/K-ATPase-{beta}1 expression (this study) of ETF-3-treated blastocysts are consistent with the cell type-specific action of ETF-3. It is likely that ETF-3 improves the development of TE via a variety of mechanisms because ETF-3 affects the expression of a number of genes in the treated embryos (Lee et al., 2003Go) including Na/K-ATPase-{beta}1 (this study) and ezrin (Lee et al., 2003Go); both have been implicated in blastocyst cavitation (MacPhee et al., 2000Go; Dard et al., 2001Go).

The search for a better embryo culture system is ongoing. One approach is to mimic the oviductal conditions. Oviductal cells produce a number of embryotrophic factors that enhance embryo development via different mechanisms. We recently have identified ETF-3 as a complement protein C3 derivative (Lee et al., 2003Go). Mouse embryos are likely to encounter this factor during preimplantation development in vivo, as mouse oviductal epithelium expresses C3 immunoreactivity cyclically with peak expression at the estrus and metestrus stages (Lee et al., 2004Go). Evidence is accumulating that granulocyte–macrophage colony-stimulating factor (GM-CSF) from the oviduct also acts physiologically to promote the growth and development of preimplantation embryos. While GM-CSF is similar to ETF-3 in suppressing apoptosis of blastocysts, it has more effect on ICM cells (Sjoblom et al., 2002Go). Further understanding of the physiological action of these embryotrophic factors is not only of academic importance but is extremely helpful in designing better culture systems.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank the clinical staff in the Department of Obstetrics and Gynaecology, The University of Hong Kong for supplying the human oviductal samples. This project is supported by grants from Research Grant Council, Hong Kong (HKU7319/01M) and CRGC, University of Hong Kong to W.S.B.Y.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on January 5, 2004; resubmitted on April 13, 2004; accepted on August 4, 2004.





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