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Regular Article |
a Departments of Obstetrics and Gynaecology and
b Zoology, University of Hong Kong, Hong Kong, China
ABSTRACT
Mouse embryos at different stages of development were cocultured with human oviduct cells or cultured in the presence of oviduct-derived embryotrophic factor-1, -2, and -3 (ETF-1, -2, and -3) for various amounts of time within the preimplantation period. Cocultures that included the period from 48 to 72 h post-hCG stimulated cell division and increased the cell numbers in the inner cell mass (ICM) of the exposed blastocyst. Exposure of embryos to oviductal cells from 96 to 120 h post-hCG increased the cell number in the trophectoderm (TE), blastocyst size, hatching rate, attachment, and in vitro spreading of the blastocyst. ETF-1 and ETF-2 affected embryos between 48 and 72 h post-hCG by increasing the number of cells in the ICM. In contrast, ETF-3 had a more profound effect on embryos that were exposed from 96 to 120 h post-hCG, where it mostly affected the development of TE cells, leading to higher hatching rate. Human oviductal cells improved mouse embryo development partly by the production of high molecular weight embryotrophic factors. These factors had differential effects on mouse embryo development.
early development, embryo, female reproductive tract, fallopian tubes, oviduct
INTRODUCTION
The development of embryos cultured in a single chemically defined medium is inferior to that of their counterparts in vivo in terms of developmental rate, blastomere number, and implantation potential [1, 2]. The reproductive tract creates a dynamic environment for the development of mammalian embryos in vivo. The cyclic morphologic and physiologic changes in the oviduct and the uterus suggest that these organs offer different microenvironments for the preimplantation embryo at different stages of development. The nutrient requirement of the embryo also changes as it grows, as reflected by the alteration in its energy metabolism [3] and its differential requirement for amino acids [4].
Numerous peptide growth factors, receptors, and binding proteins are spatially and temporally expressed in the oviduct of human, mouse, and other domestic animals. During preimplantation development, many of these factors and their receptors are also expressed in the embryos. Addition of growth factors to the culture medium facilitates embryo development [5–7], suggesting that paracrine pathways exist between the oviduct and the preimplantation embryos.
Apart from growth factors, oviduct-specific glycoproteins are present and have been characterized in several species, including rhesus monkey [8], baboon [9], and human [10]. Oviduct-specific glycoprotein binds to the zona pellucida and blastomere of hamster embryo [11], which strongly suggests that the oviduct secretes specific factors affecting embryonic development. Coculture experiments on embryos with oviduct tissue/cells also support this hypothesis [12, 13]. Coculture techniques with oviductal cells have been developed to imitate the in vivo environment and to improve embryo development in vitro. We and others have demonstrated that coculture with human oviductal cells improves the hatching rate of human embryos [14] and the development of mouse embryos in vitro [15, 16].
One of the goals in reproduction research is to develop an understanding of the mechanism of embryotrophic activity of human oviductal cells and its effects on human embryo development. Because human embryos are not available for obvious ethical reasons, mouse embryos are used for research. Characterization of oviductal embryotrophic factors using coculture has been difficult for two reasons. First, oviductal cells only produce minute amounts of embryotrophic factors, making characterization of these factors difficult. Second, the actions of these oviductal factors are facilitatory and not obligatory. The development of many mouse strains is so robust that they need very little or no support under standard culture conditions to develop to a morphologically normal blastocyst, the usual endpoint for embryologic studies. Hence, the sensitivity for detecting any possible embryotrophic activity will be lower when these mouse strains are used because enhanced developmental changes after coculture in these mice would not be easily noticeable. To overcome this difficulty, we used a stain of mouse with low developmental potential in commonly used mouse culture medium, Chatot Ziomek Bavister medium (CZB) [17], high potassium simplex optimized medium (KSOM) [18], and mouse tubal fluid medium (mTF) [19]. The use of this mouse strain allowed us to determine whether cocultures improved early embryo development. Using this model, we detected the presence of three embryotrophic factors from human oviductal cells in conditioned medium [20].
Two objectives were defined for the present study. The first objective was to determine the effect of oviductal cells at different stages of preimplantation embryo development. Minami et al. [21] showed that two- to four-cell-stage mouse embryos were sensitive to the embryotrophic coculture effect with mouse oviductal tissue. However, they did not investigate the action of oviductal tissue on embryo development later than 63 h post-hCG. The second objective was to determine whether the three recently isolated embryotrophic fractions from human oviductal cells have different biologic effects on embryonic development. To address these issues, the temporal effects of human oviductal cells and their partially purified embryotrophic fractions on mouse embryo development were investigated.
MATERIALS AND METHODS
Preparation of Oviductal Cells
The Ethics Committee of the University of Hong Kong approved the protocol for this study. Human oviductal cells were prepared as described previously [14]. Human fallopian tube tissue was obtained from patients who gave informed consent for removal of their fallopian tubes during a total abdominal hysterectomy for uterine fibroids. The oviduct epithelium was teased off, washed, minced, and dispersed in trypsin/EDTA (Gibco, Grand Island, NY). The oviduct cells obtained were cultured in Dulbecco minimal essential medium/Ham F-12 medium (DMEM/F12; 1:1 v/v; Sigma, St. Louis, MO) supplemented with penicillin, streptomycin, glutamine, sodium bicarbonate, and 15% human preovulatory serum (sDMEM/F12) at 37°C under an atmosphere of 5% CO2 in air. When the growth of the cells reached confluence, the cells were trypsinized and stored in liquid nitrogen.
Conditioned Medium
Conditioned medium (CM) was the spent sDMEM/F12 medium after culturing the oviductal cells for 24 h. Frozen oviductal cells were thawed and seeded in sDMEM/F12. After 3–4 days of culture, the cells reached 
80% confluence. The culture medium was replaced with fresh DMEM/F12 supplemented with 3 mg/ml (w/v) BSA. CM was collected 24 h later. After centrifugation at 1500 x g for 30 min, the supernatant were filtered through a 0.4-µm filter to remove cell debris and was frozen at -70°C until purification of embryotrophic factors.
Purification of Embryotrophic Factors
Three embryotrophic glycoproteins were purified as described previously [20]. CM was passed through a concanavalin A (ConA) affinity column, and the high-molecular-mass components (>100 kDa) of the fraction binding to ConA were obtained with the use of a Centricon100 (Amicon, Bedford, MA). The >100-kDa fraction was then further separated into several fractions after stepwise elution from an ion-exchange column. Fractions that were eluted with 0.1 M, 0.2 M, or 0.3 M NaCl were concentrated and reconstituted in embryo culture medium. These fractions were labeled embryotrophic fraction (ETF)-1, ETF-2, and ETF-3. The protein concentration in ETFs was determined by a protein assay kit (Bio-Rad, Hercules, CA). The ETFs were then diluted to the required concentration with CZB or CZB supplemented with 5 mM glucose (CZB+G) according to the experimental design.
Embryo Culture Medium
Mouse zygotes were cultured up to 72 h post-hCG in CZB medium [17], which contained 81.62 mM NaCl, 4.83 mM KCl, 1.18 mM KH2PO4, 1.18 mM MgSO4·7H2O, 25.12 mM NaHCO3, 1.70 mM CaCl2·2H2O, 31.30 mM sodium lactate, 0.27 mM sodium pyruvate, 0.11 mM EDTA, 1 mM glutamine, 5 mg/ml BSA, 100 IU/ml sodium penicillin, and 0.7 mM streptomycin. The development of embryos from this mouse strain in CZB was similar to that in KSOM and was much better than that of embryos cultured in mTF (unpublished data). These embryos were then transferred to CZB+G for the rest of the culture period up to 144 h post-hCG. Mouse zygotes were handled in CZB containing 20 mM Hepes (CZB+H). All the chemicals used were purchased from Sigma. Salts, EDTA, and phenol red were of cell culture grade, and the other reagents were of embryo culture grade.
Mice and Embryos
Zygotes were collected from 6- to 8-wk-old MF-1 females after induction of superovulation by an i.p. injection of 5 IU eCG (Sigma) followed 48 h later by 5 IU hCG (Sigma). Immediately following the second injection, the females were placed overnight with BALB/c males of proven fertility. Mating was confirmed by the presence of a vaginal plug the following morning. Cumulus-enclosed zygotes were collected 22–23 h post-hCG in CZB+H and denuded with 0.3 mg/ml hyaluronidase in CZB+H. The zygotes were then washed three times in CZB+H and twice in pregassed CZB before being pooled and then randomly allocated to different treatment groups.
Coculture Conditions
Two days prior to coculture, cryopreserved oviductal cells were thawed and washed in PBS. Thawed oviductal cells were seeded at a concentration of 1 x 104 cells in a 20-µl droplet of DMEM/F12 supplemented with 15% human preovulatory serum and were then covered with paraffin oil. Two hours prior to experimentation, the oviductal cells were rinsed three times with the appropriate embryo culture medium. Embryos were cocultured in 5% CO2 at 37°C in humidified air. They were transferred to fresh culture droplets every 24 h.
Morphology Assessment
The age of the embryos was timed with reference to the time after hCG administration. Embryo morphology was assessed at 48, 72, 96, 120, and 144 h post-hCG. Embryos were classified according to the following: two-cell stage, three- to four-cell stage, morula, blastocyst, and hatching blastocyst that had a clear herniation of the zona pellucida by the trophectoderm (TE).
Differential Labeling of TE and Inner Cell Mass
Differential staining of the TE and the inner cell mass (ICM) was performed by the method of Hardy et al. [22] with modifications. Acid Tyrode solution was used to remove the zona pellucida of blastocysts. The zona-free blastocysts were then washed in 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 Hochest (25 µg/ml) for 2 h. The embryos were then observed under a fluorescence microscope (Nikon, Tokyo, Japan) after mounting in glycerol.
Blastocyst Size and Trophoblast Outgrowth
Images of blastocysts at 120 h post-hCG were captured by a digital camera (Photometrics, Tucson, AZ) and analyzed by the MetaMorph Imaging System (Universal Imaging, West Chester, PA). The blastocysts were then transferred into 20-µl droplets of DMEM/F12 containing 15% human serum under paraffin oil. The number of embryos attached to the culture dish was counted 2 days later. The MetaMorph Imaging System was used to determine the spreading area of blastocyst outgrowth.
Experiment 1: Temporal Effect of Human Oviductal Cells
Embryos at different developmental stages were cocultured with human oviductal cells for various time periods. Two sets of experiments were performed (Fig. 1). In experiment A, coculture treatment started at 24 h post-hCG and ended at 48 h (A24/48), 72 h (A24/72), 96 h (A24/96), or 120 h (A24/120) post-hCG. In experiment B, coculture started at 24 h (B24/120), 48 h (B48/120), 72 h (B72/120), and 96 h (B96/120) post-hCG and ended at 120 h post-hCG.
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Experiment 2: Temporal Effect of ETFs
ETF-1, -2, and -3 containing proteins at a concentration of 10 µg/ml were prepared and used to culture mouse embryos at different stages of development for various time periods as described for experiment 1.
Data Analysis
Data from three replicate experiments were combined and analyzed using an ANOVA. Our unpublished data showed that only about 50% of the ovulated oocytes would be fertilized in vivo and that over 90% of the fertilized zygotes would cleave in the present culture system. Therefore, the incidence of a particular stage of development was calculated with reference to the number of two-cell embryos obtained on the second day of culture. A chi-square test for a contingency table and an ANOVA where appropriate were used to compare the effect of different treatments among various groups. The chi-square test was then used to compare the percentages of embryos reaching various developmental stages. The Student t-test was applied to compare the mean TE, ICM, total cell number per blastocyst (TCN), ICM/TE ratio, blastocyst size, and in vitro spreading area of different groups of embryos.
RESULTS
Morphologic Development
There was no difference in the percentage of three- to four-cell embryos and morulae at 72 and 96 h post-hCG, respectively, between the control and various treatment groups (data not shown). When coculture was initiated at 24 h post-hCG, the percentage of hatching blastocysts increased as the duration of coculture increased (Fig. 2a). Both the blastulation rate and hatching rate of the A24/72, A24/96, and A24/120 cultures were significantly higher than those of the control and A24/48 cultures. The same trend was observed when coculture was started at different stages of development (Fig. 2b). The blastulation rates and hatching rates of B24/120, B48/120, and B72/120 cultures were higher than those of the control.
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Cell Number and Allocation of ICM and TE
The allocation of ICM and TE of blastocysts at 120 h after various treatments is shown in Figure 3. In experiment A (Fig. 3a), the TCN and the cell number in the ICM and TE of each blastocyst increased along with the duration of coculture. Embryos that had been cocultured for 48 h or more had a significantly higher TCN than did those embryos cultured in medium alone. The blastocysts in the A24/72 group had significantly more cells in the ICM but not in the TE than was observed in controls. Further increases in the duration of coculture (A24/96, A24/120) significantly increased cell numbers in both the ICM and the TE. In experiment B (Fig. 3b), coculture significantly increased the number of cells in the TE independent of coculture duration (Fig. 3b). However, only cocultures that included the period of 48–72 h post-hCG (i.e., B24/120 and B48/120) had a significant increase in the number of cells in the ICM when compared with the control embryos.
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Expansion and Outgrowth of Blastocysts In Vitro
The size of the blastocyst at 120 h post-hCG, the incidence of blastocyst attachment and the spreading area of the trophoblast in vitro are shown in Table 1. Regardless of the treatment duration, embryos that had been cocultured between 96 and 120 h post-hCG had significantly larger blastocysts (P < 0.0001) when compared with the control embryos. In addition, A24/96 treatment also increased the size of the resulting blastocysts (P < 0.0001, Table 1).
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Because the actual number of blastocysts used in this experiment was low, there were no significant differences in the percentage of attached blastocysts among all the groups in the two experiments. However, an increasing percentage of attached blastocysts was seen when the duration of coculture was longer and the coculture included the 96- to 120-h post-hCG period.
The spreading area of embryos treated with coculture between 96 and 120 h post-hCG and of those cocultured for more than 48 h (A24/96) was significantly larger than that of the control (Table 1).
Temporal Effect of ETFs
Three ETFs (ETF-1, ETF-2, and ETF-3) were isolated [15–20]. They corresponded to the fractions eluted with 0.1 M, 0.2 M, and 0.3 M NaCl, respectively, from the ion-exchanged column.
ETF-1 ETF-1 had no effect on the morphologic development of two-cell embryos, three- to four-cell embryos, morulae, blastocysts, and hatching blastocysts at 48, 72, 96, 120, or 144 h post-hCG, respectively (data not shown). However, ETF-1 did affect the allocation of the blastomere in the resulting blastocyst (Fig. 4).
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When treatment was started at 24 h post-hCG and continued for various time periods (Fig. 4a), all embryos except those in A24/48 cultures had a significantly higher (P < 0.05) TCN, ICM:TE ratio, and number of cells in the ICM compared with the control. These parameters were similar in blastocysts derived from the A24/72, A24/96, and A24/120 groups. The embryos treated with ETF-1 for 4 days (A24/120) also had significantly more cells in the TE (P < 0.0001).
When embryos were treated starting at different developmental stages, up to 120 h post-hCG (Fig. 4b), only blastocysts from the B24/120 and B48/120 groups had significantly higher (P < 0.00001) TCNs, ICM:TE ratios, and numbers of cells in the ICM than did the controls. The B24/120 group also had more TE cells (P < 0.0001) than did the control embryos.
ETF-2 ETF-2 had no significant effect on the morphologic development of two-cell embryos, three- to four-cell embryos, morulae, blastocysts, and hatching blastocysts (data not shown). Its temporal effects on the allocation of the ICM and the TE of blastocysts at 120 h post-hCG are shown in Figure 5.
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When embryos were treated from 24 h post-hCG for different time periods (Fig. 5a), the resulting blastocysts except those in the A24/48 group had higher TCNs, ICM:TE ratios, and numbers of cells in the ICM than did the control embryos (P < 0.0001). Blastocysts from the A24/120 group also had significantly more TE cells than did the control embryos (P < 0.0005).
When the treatment started at various developmental stages up to 120 h post-hCG (Fig. 5b), blastocysts in the B24/120 and B48/120 groups had significantly higher (P < 0.0001) TCNs, ICM:TE ratios, and numbers of cells in the ICM than did the control group. Treatment that lasted from 72 to 120 h post-hCG also induced more cells in the ICM and thus resulted in a higher ICM:TE ratio than that of the controls.
ETF-3 ETF-3 enhanced the formation and hatching of blastocysts (Fig. 6). When treatment started at 24 h post-hCG, the blastulation and hatching rate were significantly higher in the A24/120 and A24/96 groups than in the control (P < 0.05; Fig. 6a). The A24/120 group also had a significantly higher blastulation and hatching rate than either the A24/28 or A24/72 groups. When treatment started at different developmental stages and ended at 120 h post-hCG, the groups that included Day 3 of culture (i.e., B24/120, B48/120, and B72/120) all had significantly higher blastulation and hatching rates than did the control group (Fig. 6b).
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The effects of ETF-3 on the allocation of the ICM and the TE in the blastocyst are shown in Figure 7. When treatment started at 24 h post-hCG, the A24/96 and A24/120 groups had higher TCNs and more TE cells than did the control group (P < 0.0001). Blastocysts from the A24/120 group also had significantly more ICM cells than did the control. However, the ICM:TE ratio was not affected by ETF-3 treatment.
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When treatment was initiated at different developmental stages and ended at 120 h post-hCG, all the experimental groups had higher TCNs and more TE cells (Fig. 7b). The number of cells in the ICM was also significantly increased in the B24/120 and B48/120 groups when compared with the control group.
DISCUSSION
These results confirmed those of previous studies from this and other laboratories that oviductal cells improve embryo development with respect to blastocyst formation and hatching [14–23]. The beneficial effect of human oviductal cells on mouse blastocysts was also manifested in this study by increases in the number of cells in the TE and ICM, blastocyst size, and trophoblast outgrowth after attachment in vitro.
The present results show that the embryotrophic effect of coculture is enhanced when exposure to oviductal cells is longer (Fig. 2). The effects are most pronounced when the coculture lasts for 4 days. Based on the present temporal experiment, there were two periods when mouse embryos were most responsive to coculture exposure. These two periods were 48–72 h and 72–120 h post-hCG. However, the effects of the coculture exposure during these two periods were different; the former led to enhanced ICM development, and the latter led to enhanced TE development.
When the coculture covered 48–72 h post-hCG (i.e., two- to four-cell stage), the TCN and number of cells in the ICM of the treated blastocyst increased (Fig. 3). Cocultures that did not include this period did not show a significant effect on ICM development. This finding is consistent with that of another study [21], in which mouse oviductal embryotrophic activity was evident between the late two-cell stage and just before the four-cell stage of embryo development, which coincides with the timing of the switch from maternal to embryonic genome control in the mouse [24–27].
The relationship between the embryotrophic effect of oviductal cells and the onset of embryonic genome activation remains to be investigated. Because the second cell cycle is longer than the subsequent cycles during embryo development [28], the embryo at this stage may be more susceptible to influence by culture conditions than are embryos at other developmental stages. Coculture has been shown to decrease the length of the second cell cycle [29]. The mechanisms for increasing the blastomere count of embryos after coculture remain unclear. One possibility is that human oviductal cells reduce apoptotic cell death in mouse embryos [16], which may result in an increase in blastomere number.
Cell differentiation of embryos into the ICM and the TE occurs at the morula stage. In this study, coculture and ETF-1 and ETF-2 treatment before the three- to four-cell stage affected the allocation of ICM in the blastocyst. Edwards and Beard [30] hypothesized that cell determination in embryos occurs very early in development before the postulated time for embryonic genome activation. This cell determination is manifested morphologically as polarity of the oocyte and early embryo. In support of their hypothesis are the findings that the transcription levels at the one-cell stage [31, 32] are critical for later development. Recent studies also demonstrated the occurrence of polarity in early human embryos [33, 34] and that early pronucleus morphology is related to subsequent blastocyst development and implantation [35]. The effects of ETF-1 and ETF-2 on the early transcription and polarity of treated embryos should be explored.
When coculture covers 72–120 h post-hCG, it specifically increases the number of TE cells in the resulting blastocyst. The effect seems to be more pronounced between 72 and 96 h post-hCG. Thus, the blastulation rate is significantly higher than that of the control when coculture covers 72–120 h post-hCG (B72/120; Fig. 2) but not when it covers only 96–120 h post-hCG (B96/120; Fig. 2). Our preliminary data with short coculture (24 h) throughout the preimplantation period also support the conclusion that the embryos at 72–96 h post-hCG are most responsive to the coculture effect on blastocyst formation. This sensitive period coincides with the in vivo period when the embryo undergoes compaction in the oviduct and cell differentiation to form the ICM and TE.
Blastulation depends on the activity of Na+-K+ ATPase located on the TE cells [36]. The increase in TE cell number after coculture is likely to lead to an increase in Na+-K+ ATPase activity in the blastocyst. Therefore, it is not surprising that when embryos are cocultured during this period, the size of the blastocyst increases, mainly because of the presence of an enlarged blastocoel. The enhanced development of the TE after coculture was also suggested in another study in which cocultured human blastocysts secreted more hCG than did those in routine culture [37].
With enhanced development of the TE, more of these embryos undergo hatching. Several factors, including a trypsinlike hatching enzyme [38], extracellular factor [39], and actin filaments [40], may be involved in embryo hatching. Schiewe et al. [39] demonstrated that TE vesicles with no ICM could hatch. This finding is in agreement with the present observation that the percentage of hatching embryos is higher when the number of TE cells increases after coculture. The improved development of the TE also leads to a higher percentage of attached blastocysta and enhanced trophoblast outgrowth in vitro.
Apart from the current study, there is only one other published report on the temporal effect of oviductal tissue on embryo development. Minami et al. [21] reported that the influence of mouse ampullae on blastulation of mouse embryos was restricted to a period at approximately 55–56 h post-hCG. This finding differs from those of the present study, where it was shown that human oviductal cells can affect mouse embryo development at two stages. The discrepancy between these two studies may be due to the use of different endpoints for assessing embryotrophic activity. In the previous study, only blastulation rate was used. However, more parameters probably should be used to examine the efficiency of embryo culture [41]. In the current study, several endpoints were used, including ICM and TE development, which are intimately related to embryo viability [42]. The other differences between these two studies are the use of different somatic cells and culture media for coculture. Embryos may respond differently when cocultured with different cell types [43].
The coculture effects obtained in this study are at least partly due to the production of ETF-1, -2, and -3. This study is the first to demonstrate the temporal effect of partial purified oviductal ETFs on embryo development, indicating that the oviduct plays an active role in supporting early embryonic development.
The activities of ETF-1 and ETF-2 were mainly on the development of the ICM. These two ETFs have similar molecular sizes [17]. Therefore, the molar concentrations of these factors used in the present study were similar. Comparing their embryotrophic activities, ETF-1 seems to be more potent than ETF-2 in affecting ICM development. This difference was indicated by a doubling of the ICM cell number after ETF-1 treatment but only a 60% increase in the ICM cell number after ETF-2 treatment (Figs. 6 and 7). The lower potency of ETF-2 is also partly reflected by the lower ICM:TE ratio in this group of embryos (range: 0.34–0.36) when compared with that after ETF-1 treatment (range: 0.5–0.53).
When ETF-1 and ETF-2 treatments covered 48–72 h post-hCG, the number of cells in the ICM was significantly higher than that in the corresponding control culture. Inclusion of the culture periods of 24–48 h and 96–120 h post-hCG does not have an additional effect on the number of ICM cells. These data suggest that the window of action for ETF-1 and ETF-2 on ICM development is likely to be between 48 and 72 h post-hCG. Prolonged treatment with ETF-1 (48–120 h post-hCG) and ETF-2 (24–120 h post-hCG) also affected the allocation of the TE. Treatments that covered only part of this window did not have an effect.
In contrast to ETF-1 and ETF-2, ETF-3 affects embryo development from 72 h post-hCG onwards. When embryos were treated with ETF-3 for the first 2 days of culture (24–72 h post-hCG), the blastulation rate of the treated embryos was similar to that of the control. ETF-3 enhances blastulation and hatching of the treated embryos when the treatment covers 72–120 h post-hCG and enhances the development of TE cells more than that of ICM cells. Only those embryos treated with ETF-3 from 48 to 120 h post-hCG had significantly more ICM cells than did the control.
The identities of ETFs are not known. Growth factors are known to be produced by the oviduct [44] and to enhance embryo development in vitro. However, ETFs are unlikely to be common growth factors because ETFs have much larger molecular masses (ETF-1, 154 kDa; ETF-2, 164 kDa; ETF-3, 207 kDa) [20]. Our unpublished data show that antibodies against transforming growth factor (TGF) 
, TGFß, insulin-like growth factor (IGF) II, and IGF binding protein III cannot abolish the embryotrophic activity of the ETFs. Desai et al. [45] also reported that vero cell coculture was better than leukemia inhibitory factor, interleukin 6, platelet-derived growth factor, IGF-I, IGF-II, and TGFß in supporting the postthaw development of cryopreserved mouse morulae. The molecular masses of ETFs are also larger than that of human oviduct-specific glycoprotein [10]. These ETFs may be de novo maternal signals for embryo differentiation and metabolism.
Human oviductal cells have differential effects on mouse embryos at different stages of preimplantation development. These effects can partly be accounted for by the secretion of three ETFs from the oviductal cells. Our preliminary unpublished chromatographic data suggest that oviductal cells produce more ETF-3 upon progesterone stimulation. The identity and the regulation of the secretion of these factors would be important for furthering our understanding of the role of the oviduct in early preimplantation development.
ACKNOWLEDGMENTS
The authors are grateful to the clinicians in the Department of Obstetrics and Gynaecology, University of Hong Kong, for supplying the fallopian tube samples.
FOOTNOTES
1 This work was supported fully by a grant (HKU241/95M) to W.S.B.Y. from the Research Grant Council, Hong Kong. 
2 Correspondence: W.S.B. Yeung, Department of Obstetrics and Gynaecology, University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, China. FAX: 8522 855 0947;wsbyeung{at}hkucc.hku.hk
Accepted: June 28, 2001.
Received: March 19, 2001.
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 Y.L. Lee, K.F. Lee, J.S. Xu, K.L. Kwok, J.M. Luk, W.M. Lee, and W.S.B. Yeung Embryotrophic Factor-3 from Human Oviductal Cells Affects the Messenger RNA Expression of Mouse Blastocyst Biol Reprod, February 1, 2003; 68(2): 375 - 382. [Abstract] [Full Text] [PDF]
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