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Endocrinology, doi:10.1210/en.2007-1277
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Endocrinology Vol. 149, No. 3 1268-1276
Copyright © 2008 by The Endocrine Society

Preimplantation Embryos Cooperate with Oviductal Cells to Produce Embryotrophic Inactivated Complement-3b

Pui-Keung Tse1, Yin-Lau Lee1, Wang-Ngai Chow, John M. C. Luk, Kai-Fai Lee and William S. B. Yeung

Departments of Obstetrics and Gynaecology (P.-K.T., Y.-L.L., W.-N.C., K.-F.L., W.S.B.Y.) and Surgery (J.M.C.L.), The University of Hong Kong, Hong Kong Special Administrative Region, China

Address all correspondence and requests for reprints to: W. S. B. Yeung, Department of Obstetrics and Gynaecology, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, China. E-mail: wsbyeung{at}hkucc.hku.hk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human oviductal epithelial (OE) cells produce complement protein 3 (C3) and its derivatives, C3b and inactivated complement-3b (iC3b). Among them, iC3b is the most potent embryotrophic molecule. We studied the production of iC3b in the oviductal cell/embryo culture system. In the immune system, C3 convertase converts C3 into C3b, and the conversion of C3b to iC3b requires factor I (fI) and its cofactors, such as factor H or membrane cofactor protein. Human oviductal epithelium and OE cells expressed mRNA and protein of the components of C3 convertase, including C2, C4, factor B, and factor D. The OE cell-conditioned medium contained active C3 convertase activity that was suppressed by C3 convertase inhibitor, H17 in a dose and time-dependent manner. Although the oviductal epithelium and OE cells produced fI, the production of its cofactor, factor H required for the conversion of C3b to iC3b, was weak. Thus, OE cell-conditioned medium was inefficient in producing iC3b from exogenous C3b. On the contrary, mouse embryos facilitated such conversion to iC3b, which was taken up by the embryos, resulting in the formation of more blastocysts of larger size. The facilitatory activity was mediated by complement receptor 1-related gene/protein Y (Crry) with known membrane cofactor protein activity on the trophectoderm of the embryos as anti-Crry antibody inhibited the conversion and embryotrophic activity of C3b in the presence of fI. In conclusion, human oviduct possesses C3 convertase activity converting C3 to C3b, and Crry of the preimplantation embryos may be involved in the production of embryotrophic iC3b on the surface of the embryos.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CULTURED EMBRYOS are sensitive to environmental conditions. Suboptimal culture condition could lead to irreversibly long-term consequences of postnatal development (1). The development of optimal human preimplantation embryo culture condition is critical for not only the assisted reproduction program (2) but is also beneficial to the derivation of human embryonic stem cells (3).

Preimplantation embryos grow in the oviduct for most of the preimplantation period in vivo. In vitro experiments suggest that the oviductal cell secretes a variety of components that enhance embryo development (4). We have previously demonstrated that one of the human oviduct-derived embryotrophic factors (ETFs), ETF-3 (5, 6), is a mixture containing complement protein 3 (C3), and its derivatives, C3b and iC3b (7). Among the various components in ETF-3, inactivated complement-3b (iC3b) is most potent in stimulating embryo development.

C3 is a central molecule in the complement system (8). It is traditionally thought to be involved in the antipathogenic activity and removal of dead or dysfunctional spermatozoa in the female reproductive tract. However, evidence shows that C3 also has novel roles in reproduction. Derivatives of C3 stimulate preimplantation embryo development (7). It is proposed that C3 facilitates fertilization by enhancing spermatozoa-egg membrane apposition (9). Indeed, nature uses a number of molecules involved in the immune system for reproduction. Many of them are embryotrophic, including macrophage colony stimulating factor (CSF), CSF-1 (10), granulocyte-macrophage CSF (11), leukemia inhibitory factor (12), and platelet activating factor (13). The correlation between the developmental potential of preimplantation embryo with the expression of soluble human leukocyte antigen-G (14, 15) and preimplantation embryo development gene, ped (16, 17), further indicates the unconventional roles of immune molecules in reproduction.

Studies on the conversion of C3 to embryotrophic iC3b are important for the understanding of the mechanism of action of ETF-3 on regulating embryo development in the oviduct. In the immune pathway, upon activation, C3 molecules are cleaved into C3b by C3 convertases that are synthesized through classical, alternative, or mannan-binding lectin pathways (18). C3b is subsequently cleaved to iC3b by factor I (fI) in the presence of factor H (fH) or other cofactors, such as complement receptor type 1 (CR1) or membrane cofactor protein (MCP) (19) (Fig. 1Go). In mouse, the rodent-specific transmembrane protein, complement receptor 1-related gene/protein Y (Crry), possesses human MCP activity and, thus, can act as a cofactor for fI-mediated cleavage of C3b (20).


Figure 1
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  		FIG. 1. The conversion and protein structure of C3, C3b, and iC3b. C3 is first converted into C3b by C3 convertases derived from classical/lectin pathway (C4b2a) or alternative pathway (C3bBb). C3b is further cleaved into iC3b by fI in the presence of cofactors (CR1, fH, or MCP). The unique fragments of C3, C3b, and iC3b are 113 kDa ({alpha}-113), 106 kDa ({alpha}-106), and 40 kDa ({alpha}-40), respectively.

 
There is cross talk between the early embryos and the maternal reproductive tract (21). In mouse, the production of oviductal ETF could be stimulated by the presence of embryos (22). We hypothesize that the human oviductal cells cooperate with the embryo in the production of embryotrophic iC3b. To test this hypothesis, the pathway for the conversion of C3 to iC3b in the oviduct/embryo culture system was determined. We demonstrated that the preimplantation embryos play an important role in such conversion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental subjects
Human oviductal tissues were obtained from patients admitted for hysterectomy due to uterine fibromyoma. The Ethics Committee of the University of Hong Kong approved the study protocol. The animal research protocol was also approved by the Committee on the Use of Live Animals in Teaching and Research of the University. Human oviductal epithelial (OE) cells were isolated as previously described (23). Briefly, fresh human oviductal epithelium was teased off, washed and minced in PBS (Sigma-Aldrich, St. Louis, MO), followed by trypsinization in 0.05% trypsin/EDTA (GIBCO BRL, Rockville, MD) at 37 C for 45 min. The cell suspension was spun at 300 g for 2 min, and the cell pellet obtained was further washed three times with 3 ml PBS at 37 C. The oviductal cells were cultured in DMEM/F12 (1:1 vol/vol; Sigma-Aldrich) supplemented with streptomycin (75 IU/ml), penicillin (100 IU/ml), glutamine (2.5 mM), sodium bicarbonate (26 mM), and 10% heat-inactivated fetal bovine serum (GIBCO/BRL). Immediately after confluent growth, the cells were subcultured for immediate use or trypsinized and cryopreserved in liquid nitrogen until use.

Expression of human C2, C4, factor B (fB), factor D (fD), fI, and fH RT-PCR
Total RNA from human oviductal (OE) cell was isolated using TRIZOL reagent (Invitrogen, Carlsbad, CA) and reverse transcribed into cDNA using the First-strand cDNA Synthesis Kit (Amersham Pharmacia, Buckinghamshire, UK) according to the manufacturers’ protocols. The resulting cDNA together with cDNA of human placenta, prostate, testis, ovary, leukocyte, skeletal muscle, liver, and positive control from the Human Multiple Tissue cDNA panels I and II (Clontech, Palo Alto, CA) were subjected to PCR analysis with primers listed in Table 1Go (24, 25, 26, 27, 28). Water instead of cDNA was used as negative control.


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  		TABLE 1. Sequences of the PCR primers used in determination for the presence of mRNA for complement C2, C4, fB, and fD

 
Immunohistochemistry and Western blot analysis
Formalin-fixed and paraffin-embedded human oviductal tissue sections and OE cells grown in chamber slide (Nunc, Inc., Naperville, IL) were investigated by immunohistochemistry as described previously (7) using antibodies against C2 (1:500; Calbiochem, San Diego, CA), C4 (1:500; Calbiochem), fB (1:500; Calbiochem), fD (1:50; Abcam, Cambridge, UK), fI (1:1000; Calbiochem), and fH (1:1000; Calbiochem) after antigen retrieval by immersing the slides in Target Retrieval Solution (Dako Corp., Carpinteria, CA) and heated to 95 C for 20 min. The stained sections were observed under a fluorescent microscope (Nikon Corp., Tokyo, Japan) after labeling the primary antibodies with fluorescent-labeled second antibody and the nuclei counterstained with 10 µg/ml propidium iodide (Sigma-Aldrich) and Hoechst stain (Sigma-Aldrich). To confirm the protein expression in the oviductal cells, 20 µg proteins extracted from OE cells with Cytobuster (Novagen, Madison, WI) was subjected to SDS-PAGE, and Western blotting was performed using antibodies against C2, C4, fB, fD, fI, and fH.

Biological activity of C3 convertases in OE cells
To determine the presence of biologically active C3 convertases, OE and fibroblast cells were grown in 96-well plates (IWAKI, Tokyo, Japan) until 80–90% confluence in DMEM/F12 medium supplemented with 10% fetal bovine serum. The cells were then washed with the coculture medium, CZB (29), and exogenous C3 (10 µg/ml; Calbiochem) in CZB medium was added. Conditioned media were collected after 0, 6, 12, and 24 h, and were analyzed by Western blotting using polyclonal antihuman C3 (1:20,000; Calbiochem) antibody. C3 alone in CZB medium incubated in 37 C for 6, 12, and 24 h was included as control.

The biological activity of C3 convertases in OE cells was confirmed by studying the effect of inhibitor of classical/lectin and alternative pathway, H17 fragment (HEVKIKHFSPY) of complement C2 receptor inhibitor trispanning (30) on the conversion of C3 to C3b. H17 binds specifically to C2 in the classical pathway (30) and fB in the alternative pathway (31). The inhibitor was added to final concentrations of 5, 10, and 25 nM together with the exogenous C3.

Conversion of C3b to iC3b by fI and/or fH in OE cell-conditioned media
Conditioned medium after treating OE cells with C3 (10 µg/ml) for 24 h as mentioned previously was collected. The conversion of C3b to iC3b by fI and/or fH was determined by mixing the conditioned medium with either fI (0.5 µg) and/or fH (4 µg) in 35 µl conditioned media. The mixtures were incubated at 37 C for 5, 15, and 60 min before being subjected to Western blotting using monoclonal ETF3-C14 antibody (1:100) (7), as described previously.

Conversion of C3b to iC3b by fI and/or fH in preimplantation embryo culture
The role of preimplantation embryos in the conversion of C3b to iC3b was studied by examining the development of preimplantation embryos in C3b with or without fI and/or fH. Embryo culture was performed as described (25). Mature Institute of Cancer Research (ICR), female mice (6–8 wk old) were superovulated with 5 IU pregnant mare’s serum gonadotropin (Sigma-Aldrich), followed by an injection of 5 IU human chorionic gonadotropin (hCG) (Sigma-Aldrich) 46 h later. ICR female mice were then mated with proven fertile BALB/c males. The day with the presence of a vaginal plug was regarded as d 1. One-cell embryos were collected from ICR female mice and cultured in the presence of C3b (10 µg/ml) with or without fI (5 µg/ml) and/or fH (5 µg/ml) in 20 µl potassium simplex optimization medium (KSOM) supplemented with amino acids (KSOMaas) (CHEMICON International, Temecula, CA) under mineral oil in an atmosphere of 5% CO2 in air at 37 C for 4 d. The number of embryos reaching expanded blastocyst stage was recorded at 120 h after hCG. A phase-contrast inverted microscope (Nikon) was used to capture the image of the expanded blastocysts, and the diameter of the expanded blastocysts was determined by the Image Pro Plus imaging system (Media Cybernetics, Bethesda, MD).

The conversion of C3b to iC3b in the aforementioned embryo culture system was determined. To increase the sensitivity of detection of C3 fragments in the embryo culture system, C3b was biotinylated in vitro using the FluoReporter Mini Biotin-XX Protein Labeling Kit according to the manufacturer’s protocol (Invitrogen). The biotinylated C3b was used immediately for culture or frozen at –80 C for future use. There were 20–30 one-cell embryos cultured in 20-µl droplets as described previously, except that biotinylated C3b was used instead of C3b. The embryo culture-conditioned media were collected, and 10 µl for each sample was subjected to Western blotting. The resulting expanded blastocysts in groups of 14 were also subjected to SDS-PAGE. Western blotting was performed using alkaline phosphatase conjugated streptavidin (Sigma Chemical Co., St. Louis, MO).

Crry protein expression on mouse blastocyst
Mouse blastocysts were flushed from the uterus of ICR females 3.5 d after coitum. The zona pellucida was removed with acid Tyrode solution, and the blastocysts were fixed in 4% paraformaldehyde in PBS (pH 7.35) for 30 min. They were then permeated with 0.1% Triton X-100 on ice for 1 min and blocked with 10% rabbit serum. Primary antibody incubation was performed at 4 C overnight using anti-Crry antibody (1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) with or without blocking peptide (Santa Cruz Biotechnology). After being washed five times in PBS containing 0.5% Tween 20 for 15 min each, the blastocysts were incubated with fluorescein isothiocyanate-labeled rabbit antigoat IgG antibody (1:100; Zymed Laboratories, Inc., San Francisco, CA) at room temperature for 1 h, and examined under a confocal microscope (MRC-600; Bio-Rad Laboratories, Hercules, CA).

Conversion of C3b to iC3b in the presence of anti-Crry antibody in embryo culture
To study the involvement of Crry on embryo in the conversion of C3b into iC3b, one-cell mouse embryos were first incubated with anti-Crry antibody (5 µg/ml) in KSOMaa for 30 min before transferring to droplets containing biotinylated C3b and fI with anti-Crry antibody and cultured for 4 d. Embryos were also cultured in droplets containing biotinylated C3b and fI without anti-Crry antibody. The embryo culture media from the two groups were collected for SDS-PAGE, and Western Blotting was performed as described previously.

F(ab')2 generation of anti-Crry antibody for embryo culture
The biological activity of C3b and fI in the presence of anti-Crry on preimplantation embryo development was studied. Preliminary data in this laboratory demonstrated that some IgG molecules affected embryo development, and the effect was reduced by removal of the Fc region of the antibodies. To remove the Fc region of anti-Crry antibody, the ImmunoPure F(ab')2 Preparation Kit (Pierce, Rockford, IL) was used. The resulting anti-Crry-F(ab')2 at a concentration of 0.1 µg/ml was used to pretreat the one-cell mouse embryos for 30 min before transferring to droplets containing either anti-Crry-F(ab')2 alone or C3b, fI and anti-Crry-F(ab')2 together. The embryos were cultured for 4 d. The blastulation rate and the size of the expanded blastocyst were determined as described previously.

Statistical analysis
The data obtained from three replicates of embryo culture were combined and analyzed by the {chi}2 test for the development of embryos and one-way ANOVA for the comparison of the areas of the blastocysts. P < 0.05 was considered a significant difference.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Human oviduct contains components of C3 convertases
mRNA expression of C2 (201 bp), C4 (202 bp), fB (356 bp), fD (446 bp), fI (722 bp), and fH (347 bp) was present in OE, placenta, prostate, testis, ovary, skeletal muscle, liver, and the positive control. Leukocytes expressed C2 and fD only. Bands of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (452 bp) were obtained in all the tissues examined, and no band was detected in the negative control (Fig. 2AGo).


Figure 2
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  		FIG. 2. Expression of C2, C4, fB, fD, fI, and fH in human tissues. A, Expected PCR products with sizes of C2 (201 bp), C4 (202 bp), fB (356 bp), fD (446 bp), fI (722 bp), and fH (347 bp) were obtained in OE cells (lane 1), placenta (lane 2), prostate (lane 3), testis (lane 4), ovary (lane 5), skeletal muscle (lane 7), liver (lane 8), and positive control (lane 9). Leukocyte expressed C2 and fD only (lane 6). Bands of GAPDH (452 bp) were obtained in lanes 1–10 for normalization of the signals. No band was obtained in negative control (H2O, lane 10). B, Immunohistochemical staining of human oviduct showing the presence of C2, C4, fB, and fI in the stromal and epithelial region. fD was expressed mainly in the epithelium. The expression of fH was very low in the epithelium, and some patients did not express fH. C, Immunocytochemical staining detected positive cytoplasmic immunoreactivity of C2, C4, fB, fD, fI, and fH in the OE cells. No signal was found in the negative control when the primary antibodies were omitted, and antigoat (G) and antisheep (S) secondary antibodies were used. D, Western blotting showed that the C2 fragments (63 kDa), whole C2 molecule (93 kDa), {gamma}-chain of complement C4 (33 kDa), fB (93 kDa), and fD (24 kDa) and components of fI (38, 50, and 88 kDa) were observed in OE cell lysates. The fH band was undetectable in the OE cell lysate.

 
The spatial expression of the proteins in human oviductal tissue sections (Fig. 2BGo) and OE cells (Fig. 2CGo) was determined. Although the immunoreactivities of C2, C4, and fB were detected in both the stromal and epithelial region of human oviduct, fD and fI protein was expressed mainly in the epithelium. The expression level of fH in the oviductal epithelium was very weak, and some patients did not express fH immunoreactivity. All six proteins were detected in the cytoplasm, and not in the nucleus of the cultured OE cells (Fig. 2CGo). Again fH was the protein with the weakest signal. No signal was detected with the omission of the primary antibodies (data not shown).

The protein expression was confirmed by Western blotting analysis of the cultured OE cells (Fig. 2DGo). Bands of C2 (102 kDa), C4 (33, 75, and 93 kDa), fB (90 kDa), fD (24 kDa), and fI (38, 50, and 88 kDa) were obtained in OE cell lysate. In contrast to the immunocytochemistry results, no fH signal was obtained in the OE cell lysate.

Human oviductal cells produce active C3 convertase
Active C3 convertases from OE cells converted exogenous C3 to C3b. The production of C3b was determined by the presence of its specific fragment of the {alpha}-chain, {alpha}-106 (106 kDa) in the spent medium after OE cell culture. Exogenous C3 contains two chains, the {alpha}-113 (113 kDa) and β-75 (75 kDa) chains. Western blot analysis using anti-C3 antibody (Fig. 3AGo) demonstrated that these chains remained intact for 24 h in embryo culture medium, CZB alone (Fig. 3A.aGo). In the absence of exogenous C3, OE cells produced C3 into the conditioned medium in a time-dependent manner (Fig. 3A.bGo). When OE cells were cultured in the presence of exogenous C3 (Fig. 3A.dGo), an extra band of 106 kDa ({alpha}-106) appeared with an amount that increased from 6- to 24-h culture. No {alpha}-106 band was detected in fibroblast cell culture at 6 and 12-h culture, though a very faint band was seen at 24-h culture (Fig. 3A.cGo). The C3 convertase activity of the OE cell culture was confirmed using the inhibitor for C3 classical and alternative pathways, H17 (30, 31). When different concentrations of H17 peptides (5, 10, and 25 nM) were used, the intensity of the {alpha}-106 band decreased in a dose-dependent manner (Fig. 3BGo).


Figure 3
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  		FIG. 3. Western blot detection of C3 convertase activity in the conditioned media of OE cell culture using anti-C3 antibody. A, After incubating exogenous C3 in media alone without cells for 6–24 h, C3 fragments ({alpha}-113 and β-75) remained intact (a). The {alpha}-113 and β-75 fragment of C3 was secreted from the OE cells in a time-dependent manner when no exogenous C3 was added (b). When exogenous C3 protein was incubated with OE cells, a band of 106 kDa ({alpha}-106) was formed in a time-dependent manner (d). C3 fragments remained intact when it was incubated with fibroblasts for 6 and 12 h. A very faint band of {alpha}-106 was detected at 24-h culture (c). B, Western blotting of conditioned media after treating OE cells with exogenous C3 and different concentrations of synthetic peptide (H17). Compared with the control without H17 addition, the intensity of the {alpha}-106 band decreased at 24-h culture when H17 was added in increasing concentrations of 5, 10, and 25 nM.

 
Oviductal cell-conditioned medium with low fH activity is inefficient in converting C3b to iC3b
The production of iC3b was determined by the detection of its specific fragment, {alpha}-40 (40 kDa) using monoclonal ETF-C14 antibody that recognized only the {alpha}-40 fragments of the molecule (Fig. 4Go). In the spent media after culturing OE cells with exogenous C3 for 24 h, a weak {alpha}-40 band was detected (Fig. 4Go, lane CM). When fI alone was added to the conditioned medium, the {alpha}-40 band increased slightly after 5-min incubation and remained constant thereafter for 60 min. However, when fH alone was added, the intensity of the {alpha}-40 band increased significantly in a time-dependent manner. As expected, the intensity of this band was strongest with simultaneous addition of fI and fH.


Figure 4
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  		FIG. 4. Conversion of C3b into iC3b in the presence of fI and/or fH. Using monoclonal antibody, ETF-C14 in Western blot analysis, a weak band of {alpha}-40 was observed in the conditioned medium after culturing OE cells with C3 for 24 h (CM). When fI was added, the intensity of the {alpha}-40 band increased slightly after 5, 15, and 60-min incubation (fI). When fH was added, the {alpha}-40 band increased significantly (fH) in a time-dependent manner. The intensity of this band was strongest with simultaneous addition of fI and fH (fI + fH).

 
Preimplantation embryos convert C3b to iC3b in the presence of fI
The data on embryo development in different culture conditions are shown in Table 2Go. The blastulation rate was significantly higher when the embryos were cultured in the presence of C3b + fI (60%) than those cultured in the presence of C3b alone (34%) and in KSOMaa (44%). The embryo treated with C3b + fI + fH produced significantly more blastocysts (51%) than those treated with C3b alone. The size of the blastocysts from the C3b + fI group (9759 ± 356 µm2) was significantly larger than those from all other groups (P < 0.05). Thus, C3b + fI exhibited the most potent embryotrophic effects.


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  		TABLE 2. The development of mouse embryo cultured in KSOMaa with C3b in the presence of fI and/or fH

 
The conversion of C3b into iC3b in the embryo culture system was studied by culturing one-cell mouse preimplantation embryos in medium containing biotinylated C3b, fI, and/or fH (Fig. 5AGo). Addition of fI and fH to biotinylated C3b in PBS produced iC3b as indicated by the appearance of the {alpha}-40 band (lane 2) when compared with C3b alone (lane 1). A strong {alpha}-40 band was detected when C3b was incubated with fI and fH in embryo culture medium, KSOMaa with (lane 8) or without (lane 7) preimplantation embryos. No {alpha}-40 band was detected when KSOMaa contained C3b alone, regardless of the absence (lane 3) or presence of (lane 4) embryos. A strong {alpha}-40 band appeared after culturing preimplantation embryos in the presence of C3b and fI (lane 6). However, the band was very weak in the absence of preimplantation embryo (lane 5). The detection of the other iC3b specific band of 63 kDa, {alpha}-63 was not possible because BSA with a similar molecular size in the culture medium masked such detection. The intensities of C3b specific band, {alpha}-106 band were decreased in lanes 6–8 when compared with lanes 3–5, where no {alpha}-40 band was detected.


Figure 5
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  		FIG. 5. Western blot analysis using alkaline phosphatase-conjugated streptavidin that recognized the biotinylated C3b and its derivatives in embryo culture. A, A {alpha}-40 band was detected in the embryo culture media containing C3b, fI, and fH, regardless of the presence (lane 8) or absence (lane 7) of embryo. No such band was detected in PBS containing C3b alone (lane 1) and in KSOMaa containing C3b with (lane 4) or without (lane 3) preimplantation embryo. A strong {alpha}-40 band was detected in medium after culturing preimplantation embryos with C3b and fI alone (lane 6), and the band was undetectable in the absence of the embryos (lane 5). The appearance of the {alpha}-40 band was associated with a decrease in the intensity of the {alpha}-106 band (lanes 6–8). B, Bands of {alpha}-40 and {alpha}-63 were found in the expanded blastocysts treated with C3b, fI, and fH in KSOMaa (lane 3), but not with KSOMaa alone (lane 2). A control lane in which C3b was incubated with fI and fH in KSOMaa was included for reference (lane 1).

 
The expanded blastocysts from three replicates of embryo cultures were washed in PBS supplemented with Tween 20 to remove BSA, and were pooled and subjected to Western blotting analysis (Fig. 5BGo). Two iC3b fragments, {alpha}-40 and {alpha}-63, were detected in blastocysts treated with C3b, fI, and fH (lane 3, n = 14), but not in blastocysts cultured in KSOMaa alone (lane 2, n = 14). A control lane in which C3b was incubated with fI and fH in KSOMaa was included for reference (lane 1).

Crry on mouse embryo is involved in cleaving C3b
Crry immunoreactivity was found in the trophectoderm but not in the inner cell mass (arrow) of mouse blastocysts. No signal was found when the anti-Crry antibody was preabsorbed with the blocking peptide (PAB-anti-Crry) (Fig. 6AGo). To determine whether Crry on embryo was involved in the conversion of C3b into iC3b, anti-Crry antibody was added into the embryo culture medium supplemented with C3b and fI. The results of Western blotting analysis of the spent medium are shown in Fig. 6BGo. As expected, in the absence of anti-Crry antibody, a strong {alpha}-40 band was observed in the spent medium after culturing the embryo with C3b and fI (lane 4), which is similar to the pattern when C3b was incubated with fI and fH in PBS (lane 2). After incubating the mouse embryos with anti-Crry antibody, C3b and fI for 4 d, only the {alpha}-106 and β-75 bands were detected in the spent medium (lane 3). The pattern was similar to the lane containing C3b alone in PBS (lane 1).


Figure 6
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  		FIG. 6. The involvement of mouse embryonic Crry on the conversion of C3b to iC3b. A, Crry immunoreactivity was found in the trophectoderm, but not in the inner cell mass (arrow) of mouse blastocysts using anti-Crry antibody under a confocal microscope. No staining was found in the blastocyst using blocking peptide preabsorbed anti-Crry antibody (PAB anti-Crry). Phase contrast images were also included. Bar, 20 µm. B, Western blotting of embryo culture spent media supplemented with anti-Crry antibody. A {alpha}-40 band was obtained in the spent embryo culture medium containing C3b and fI (lane 4). The band disappeared when 5 µg/ml anti-Crry antibody was added (lane 3). C3b alone (lane 1) and C3b with fI and fH (lane 2) in PBS were included as control.

 
The development of the embryo treated with C3b + fI in the presence of anti-Crry-F(ab')2 is shown in Table 3Go. The blastulation rate was significantly higher when the embryos were cultured in the presence of C3b + fI (56%) than those cultured in the presence of C3b alone (45%) and in KSOMaa (39%). The addition of anti-Crry-F(ab')2 significantly inhibited C3b + fI-induced blastulation (43%). The size of the blastocysts from the C3b + fI group (10668 ± 236 µm2) was significantly larger than those from all of the other groups (P < 0.05). Thus, anti-Crry-F(ab')2 treatment nullified the beneficial effect of C3b + fI on blastulation rate and blastocyst size.


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  		TABLE 3. The development of mouse embryo cultured in KSOMaa with C3b in the presence of fI and/or antibody against Crry, anti-Crry-F(ab')2

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Human oviductal cells produce ETF-3 that contains mainly C3 and its derivative, C3b. The amount of iC3b in ETF-3 is minimal, as indicated by the low proportion of the {alpha}-40 fragment in ETF-3 (7). However, iC3b is the most potent embryotrophic C3 derivative tested, whereas C3 itself is not embryotrophic. Based on these observations and the presence of C3 immunoreactivity in the oviductal epithelium (7), it was hypothesized that C3 was converted to embryotrophic iC3b in the oviduct and that the molecules in the immune system might be involved in the conversion.

Upon activation of the immune system, C3 is successively cleaved to C3b by C3 convertases (32), and to iC3b by fI and its cofactors (19). Thus, the expressions of components of C3 convertases in the classical/lectin (C2 and C4) and alternative (fB and fD) pathways were studied in the human oviduct. Data showed that both the mRNA and protein of these molecules were present in the human oviduct and OE cells. Although there are reports on the presence of complement components in many reproductive organs in different species (7, 33, 34, 35, 36), there is no study on the biological activity of these proteins in the oviduct.

The proteins of C2, C4, and fB were found in both the stromal and epithelial region of human oviduct, whereas fD was mainly localized to the epithelium. The main site of synthesis of C2, C4, and fB in the body is the liver, whereas that of fD is the adipose tissue (37). Because proteins in the oviductal luminal fluid can be derived either as transudate of serum proteins or from the secretory epithelium of the oviduct (38, 39), it is highly possible that the signal of C2, C4, and fB in the stromal region is derived from plasma. Further experiments using in situ hybridization or combined use of laser capture microdissection and RT-PCR to study their mRNA expression would confirm the origin of these stromal immunoreactivities in the oviduct.

The production of C2, C4, fB, and fD in the oviductal epithelium is confirmed by the presence of their mRNA and proteins in OE cells in vitro. Two observations demonstrated that these components of C3 convertases were biologically active. First, OE cells convert exogenous C3 to C3b, resulting in a time-dependent increase in the formation of the unique fragment of C3b, {alpha}-106. This activity was specific to the OE cells because fibroblasts derived from human oviduct could not produce {alpha}-106 to a similar extent under the same condition. Second, H17 peptide decreased the {alpha}-106 signal in a dose and time-dependent manner. The conversion of C3 to C3b is a natural process in the porcine oviduct because C3b but not C3 is mainly found in the oviductal fluid (40).

H17 peptide inhibits the formation of C3 convertase through the classical/lectin pathway-mediated complement activation by competing with C4b for C2 (30), and that through the alternative pathway by binding to fB leading to blockage of the fD-mediated fB cleavage (31). Although the present results cannot determine the contribution of the three pathways in the production of C3b, it is unlikely that the classical and lectin pathways are operating because the activation of these pathways involve antibodies and mannan-binding lectins, respectively (41), which are absent in the OE cell culture system. On the other hand, C3 can be hydrolyzed spontaneously to form a metastable C3(H2O) molecule that has C3b characteristics, and can activate the C3 convertase of the alternative pathway in the presence of fB and fD to generate C3b from C3 (41, 42, 43). Three observations suggest that the alternative pathway was operating in the OE culture system. First, the OE cells produce the molecules, C3, fB, and fD essential for the pathway. Second, H17 peptide blocked the conversion of C3 to C3b by OE cells. Third, fibroblasts lacking fB and fD (unpublished observation) did not produce C3b from C3 efficiently. It has been demonstrated that the maternal alternative pathway of complement activation was the primary contributor to the embryonic lethality in Crry-deficient mice (44). Thus, it is tempting to speculate that the alternative pathway also contributes significantly to the production of C3b in the oviduct in vivo.

fI in the presence of its cofactors such as fH cleaves C3b. Although the oviductal epithelium and cultured OE cells contained the mRNA and protein of fI, their expression of fH protein was weak, suggesting that the OE cells might not be efficient in producing iC3b from C3b. Weak expression of fH was detected by immunocytochemical staining but not by Western blot analysis in this study. The discrepancy could be due to the fact that the fH signal was concentrated within each cultured cell of small size, requiring observation under microscope, whereas that the signal in the Western blot analysis was spreading over a much bigger area that could be seen with the naked eye. The weak expression of fH is in line with the data that the OE cell-conditioned medium could produce a small amount of iC3b after 24-h incubation with C3b and in the presence of exogenous fI. In contrast, supplementation of fH alone significantly increased the production of {alpha}-40 in a time-dependent manner. As expected, the production of {alpha}-40 was highest in the presence of both exogenous fI and fH. These observations indicated that the availability of fH was rate limiting in the production of embryotrophic iC3b by the OE cells.

OE cell coculture stimulates mouse embryo development in vitro (45, 46). Based on the aforementioned findings, the possibility that the embryo was involved in converting C3b to iC3b was tested. This proposed pathway has the advantage that the embryotrophic iC3b is formed close to the embryo, allowing rapid action of the molecule on embryo development. It was believed that the amount of iC3b produced would be minute because of the limited availability of suitable machinery in the embryos. Therefore, exogenous C3b was biotinylated to increase the sensitivity of detection. Such modification did not affect the ability of fI and fH to cleave the biotinylated C3b, and a strong {alpha}-40 band was detected in the embryo culture media when both components were present. Interestingly, a strong {alpha}-40 band was also obtained when the preimplantation embryos were cultured with biotinylated C3b and fI but without fH. The intensity of the band was further increased when both fI and fH were added. In the absence of fI and fH, the embryo could not produce iC3b from exogenous C3b. These results demonstrated that C3b was converted to iC3b by the cofactor present on the mouse preimplantation embryos in the presence of exogenous fI.

The production of iC3b by the embryos treated with C3b and fI was associated with the formation of more and larger blastocysts, consistent with a previous report that iC3b increases the developmental rates and the size of the treated blastocysts (7). Although the highest intensity of the {alpha}-40 fragment was detected in the spent medium containing C3b + fI + fH, its embryotrophic activities were less potent than that containing C3b + fI in terms of hatching rate and blastocyst size. This difference could be due to a rapid conversion of a significant proportion of the C3b into iC3b in the early part of the culture period in the C3b + fI + fH group. The biological activity of this iC3b would have decreased in the later part of culture period because iC3b is unstable in vitro (47). Our unpublished data also showed that embryotrophic activity of purified iC3b decreased with time of storage. The observed lowered embryotrophic activity was further aggravated by the fact that ETF-3 exerted its greatest effect in the later part of the culture period (6). On the other hand, the limited availability of embryonic cofactor of fI in the C3b + fI group might provide a gradual and continuous conversion of C3b into fresh bioactive iC3b. Moreover, the conversion occurred next to the embryo, making the newly formed iC3b more efficient in supporting embryo development.

Data showed that the blastocysts took up iC3b from the medium as embryos possessed two biotinylated iC3b fragments, {alpha}-40 and {alpha}-63, after C3b + fI + fH treatment. This observation is consistent with the presence of C3 immunoreactivity in the trophectoderm of iC3b-treated blastocyst (7). The fact that the monoclonal antibody against the {alpha}-40 fragment of iC3b nullified the embryotrophic effect of ETF-3 suggests that the fragment contained the domain critical to the embryotrophic activity of iC3b. It is interesting to note that C3 deposition without inflammatory response was detected in the extraembryonic tissue of the normal pregnant mouse (44), consistent with the possible nonimmune function of the molecule.

Apart from fH, fI can cleave C3b into iC3b in the presence of other cofactors, such as MCP and CR1. In mouse, Crry has MCP-like activity and regulates the deposition of activated C3 on the surface of autologous cells in vitro (48, 49). Mouse embryos constitutively expressed mRNA of Crry throughout preimplantation development (7). This is confirmed in the present study demonstrating the presence of Crry immunoreactivity in the mouse blastocysts. The restricted expression of Crry protein in the trophectoderm cells, but not the inner cell mass, is consistent with the detection of ETF-3 immunoreactivity only in the trophectoderm of mouse embryos treated with ETF-3 in vitro (7). Addition of anti-Crry antibody into embryo culture medium containing C3b and fI suppressed the production of {alpha}-40 by the preimplantation embryos, and abolished the beneficial effect of C3b and fI treatment, supporting that Crry is the molecule on mouse preimplantation embryo converting C3b to iC3b in the presence of fI.

In summary, we propose the following conversion pathway of C3 to iC3b in the context of preimplantation embryo and oviduct cooperation (Fig. 7Go). The oviductal cells secrete C3, which is hydrolyzed spontaneously into C3b-like molecule, C3(H2O). In the presence of oviductal fB and fD, more C3 is converted into C3b as a result of activation of the alternative pathway. On the surface of the preimplantation embryos and in the presence of oviductal fI, Crry converts C3b into iC3b and stimulates the development of the embryo. In this scheme, the preimplantation embryo cooperates with the oviductal cells in the production of active iC3b on the surface of the embryo, ensuring that the unstable embryotrophic iC3b can act on the embryo efficiently. Although species-specific differences cannot be excluded, circumstantial evidence supports that the system probably works in human in vivo as well. This evidence includes the detection of protein expression of C3 (7) and components of C3 convertase in human oviduct tissue. Human embryo expresses MCP (50), which can convert C3b into iC3b in the presence of fI. The potential use of iC3b in a human-assisted reproduction program needs further investigation.


Figure 7
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  		FIG. 7. A schematic representation of the proposed C3 embryotrophic pathways involved in the oviduct. The presence of C3 immunoreactivity in the cytoplasm of human oviductal epithelium is shown. The oviductal-derived C3 is hydrolyzed to the metastable C3(H2O), which is converted to C3b by C3 convertases in the alternative pathway. fI together with embryonic Crry convert C3b into iC3b, which exert its embryotrophic effects on embryo development.

 


   Acknowledgments
 
We thank the clinical staff of the Department of Obstetrics and Gynaecology, University of Hong Kong, for supplying the human oviductal samples.


   Footnotes
 
This work was supported by Committee on Research and Conference and Competitive Earmarked Research (HKU7411/04M) grants (to K.-F.L. and Y.-L. L.).

Disclosure Statement: The authors have nothing to declare.

First Published Online November 26, 2007

1 P.-K.T. and Y.-L.L. contributed equally to the study. Back

Abbreviations: CE, Complement protein 3; CR1, complement receptor type 1; Crry, complement receptor 1-related gene/protein Y; CSF, colony stimulating factor; ETF, embryotrophic factor; fB, factor B; fD, factor D; fH, factor H; fI, factor I; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hCG, human chorionic gonadotropin; KSOM, potassium simplex optimization medium; KSOMaa, potassium simplex optimization medium supplemented with amino acid; MCP, membrane cofactor protein; OE, oviductal epithelial.

Received September 18, 2007.

Accepted for publication November 12, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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