Demilune Cell and Parotid Protein from Murine Oviductal Epithelium Stimulates Preimplantation Embryo Development
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《内分泌学杂志》
Department of Obstetrics and Gynaecology (K.-F.L., J.-S.X., Y-L.L., W.S.B.Y.) and Center of Reproduction, Development and Growth (K.-F.L., W.S.B.Y.), Hong Kong Jockey Club Clinical Research Centre, Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, People’s Republic of China
Abstract
In mammals, fertilization and early preimplantation embryo development occur in the oviduct. We hypothesized that interaction exists between the developing embryos and the maternal genital tract, such that the embryos modulate the physiology and gene expression of the oviduct so that it is conducive to their development. By comparing the gene expression patterns in mouse oviducts containing transferred preimplantation embryos with those of oviducts containing oocytes, we report here the characterization of demilune cell and parotid protein (Dcpp), which was up-regulated in the embryo-containing oviduct. Dcpp mRNA was highly expressed in the oviductal epithelium at the estrus stage. The Dcpp gene codes for a protein of 150 amino acids and contains a signal peptide suggestive of secretory function. The Dcpp mRNA level was maintained in the oviductal epithelium of pregnant females but decreased continuously in those of pseudopregnant mice. Exogenous estrogen stimulated the expression of Dcpp mRNA and protein in ovariectomized mice. The effect was abolished by an estrogen antagonist, ICI 182,780. Dcpp protein was present in mouse oviductal fluid but not in uterine fluid. More importantly, Dcpp immunoreactivity was found in embryos recovered from the oviduct but not in mature oocytes from the ovary. Supplementation of Dcpp to culture medium stimulated the development of mouse embryos to the blastocyst stage. Anti-Dcpp antibody decreased the beneficial effect of Dcpp on implantation of two-cell mouse embryos transferred to the oviducts of the foster mothers. In summary, our data demonstrated that Dcpp is highly expressed in the oviductal lumen in the presence of preimplantation embryos. It stimulates the growth of preimplantation embryos and may play an important role in embryo-maternal dialogue.
Introduction
IN MAMMALS, FERTILIZATION and early embryonic development occur in the oviduct. The metabolic needs of preimplantation embryos change during development (1). To cope with the changing needs of the developing embryos, the microenvironment in the female reproductive tract undergoes biochemical and physiological changes (2, 3) to provide the best support for the development of the embryos. Evidence is accumulating that interaction exists between the gametes or embryos and the oviduct during the preimplantation period (4, 5, 6, 7). This interaction can be achieved via the paracrine factors (reviewed in Ref.8), including IGF-binding proteins (9, 10), IL-1 (11), vascular endothelial growth factor and receptors (12), TGF (13), platelet-activating factor (7), leptin (14), and other factors or peptides of unknown identity (3, 15, 16, 17, 18).
The microenvironment of the oviduct is regulated by steroid hormones. The ratio of the ciliated cells to the secretory cells in the mammalian oviduct changes in a cyclical manner (19), and the ciliated cells may be transformed to secretory cells after loss of their cilia (20). These changes are associated with oviductal biosynthetic activity, which is modulated by estrogen and progesterone (reviewed in Ref.2). Porcine oviductal epithelial cells secrete at least 14 major proteins into the culture medium (21), including oviduct-specific glycoprotein, tissue inhibitor of metalloproteinase, and plasminogen activator inhibitor-1 (2). Interestingly, these proteins are expressed in a unique temporal and spatial pattern consistent with the existence of physiological interactions of the oviduct with the embryos during early embryonic development (22).
It has been reported that the hamster oviduct can distinguish between developing embryos and oocytes (23) and between embryos at different ages (24), as evidenced by the ability of the oviduct to transport the oocytes and embryos to the uterus at different rates. However, Freeman et al. (25) demonstrated that it was the embryos themselves that controlled the oviductal transport in mares. However, the molecular mechanisms regulating oviductal transport remain unknown.
Studies of embryonic-maternal interaction have been advanced by the use of both transcriptomic and proteomic approaches to delineate the role of the oviduct in preimplantation embryo development (5) and gamete interactions (4). Recently Bauersachs et al. (17) compared the gene expression patterns between the ipsilateral and contralateral bovine oviductal epithelial cells by suppression subtraction hybridization (SSH) at d 3.5 after standing heat. They identified a number of differentially expressed genes related to immunity or proteins involved in cell-cell interactions. These genes and their protein products can potentially shed detailed light on embryonic-maternal interaction.
We previously demonstrated that oviductal cells affect the gene expression of developing embryos in vitro (26), partly via embryotrophic factor-3 (27). On the other hand, developing embryos have also altered gene expression in the oviduct (6, 28). By comparing gene expression in the oocyte-containing oviduct with that in the embryo-containing oviduct, we successfully isolated more than a dozen genes differentially expressed in the embryo-containing oviduct (6, 29). In the present study, we characterized one of these oviductal genes, OD135, which is highly expressed in mouse oviducts and is homologous to the demilune cell and parotid protein (Dcpp) found in the mouse salivary gland. Dcpp is highly expressed in the sublingual gland and to a lesser extent the parotid and submandibular gland (30). However, its roles in salivary gland development and oviduct are still unknown. In this study, the expression of the Dcpp gene during pregnancy, regulation by estrogen in the ovariectomized mice, and the effect of protein on in vitro embryo culture were investigated. It is suggested that the maternal genital tract recognizes developing embryos and secretes embryotrophic factor such as Dcpp to promote the development of the embryo in vivo.
Materials and Methods
Tissue collection and RNA isolation
The model we used for studying the effect of embryos on oviductal gene expression has been reported previously (6). MF1 female mice were superovulated by successive ip injection of pregnant mare’s serum gonadotropin (Sigma, St. Louis, MO) and human chorionic gonadotropin (hCG; Sigma). The fertilized zygotes and oocytes were collected from these animals with and without mating at 24 h after hCG, respectively, and were transferred to a different oviduct of the same pseudopregnant female mice. After 48 h, oviducts containing three to four cell embryos or oocytes were collected if they were previously transferred with one-cell embryos (embryo containing oviduct) or oocytes (oocyte containing oviduct), respectively. The mRNA of the oocyte-containing oviducts and embryo-containing oviducts were subjected to SSH with the PCR-Select cDNA subtraction kit (CLONTECH Laboratories Inc., Palo Alto, CA). The research protocol was approved by the University of Hong Kong’s Committee on the Use of Live Animals in Teaching and Research. The oviducts from the estrus cycle (proestrus, estrus, metestrus, and diestrus) of pregnant and pseudopregnant mice (n = 5) from d 1–4 were collected. Ovariectomized mice were injected with 100 ng/mouse estradiol (E2) or 1 mg/mouse progesterone, or E2 plus 3 mg/kg ICI 182,780 (Tocris, Bristol, UK), or progesterone plus 1 mg/mouse RU486 (Tocris) at 24 h before tissue collection. Half of the collected oviducts were fixed in 4% paraformaldehyde in PBS for in situ hybridization, and the remaining halves were used for RNA extraction. Poly(A)+ RNA and total RNA from mouse tissues were isolated by the QuickPrep micro-mRNA purification kit (Amersham Biosciences, Piscataway, NJ) and the Absolutely RNA RT-PCR miniprep kit (Stratagene, La Jolla, CA), respectively, in accordance with the manufacturer’s instructions. The RNA was quantified by spectrophotometry and stored at –70 C until use.
Northern blot and in situ hybridization
Northern blot hybridization was performed in accordance with a protocol previously described (13). For in situ hybridization, paraffin-embedded mouse oviducts were sectioned at 5 μm thick and subjected to in situ hybridization using the mRNA locator in situ hybridization kit (Ambion Inc., Austin, TX) as described (29). The dewaxed tissues were washed in PBS and treated with proteinase K for 10 min at room temperature. The tissues were washed sequentially with PBS for 5 min, 1.32% triethanolamine for 3 min, triethanolamine with 0.25% acetic anhydride for 10 min, and PBS for another 5 min at room temperature. The sense and antisense probes for Dcpp (Table 1) were generated using the SP6/T7 riboprobe combination system (Promega, Madison, WI). The tissue sections were allowed to hybridize with the probe in hybridization buffer (1 x 106 cpm/slide) overnight in a humidified chamber at 55 C. After hybridization, the slides were washed extensively to remove nonspecifically bound riboprobe. Tissues were washed with 4x standard saline citrate (SSC) buffer [1x SSC containing 0.15 M NaCl and 15 mM sodium citrate; and 1 mM dithiothreitol (DTT)] for 5 min at 55 C, followed by 2x SSC/1 mM DTT for 30 min at 55 C, and then treated with RNase A (20 μg/ml) at 37 C for 15 min. Slides were then washed in 2x SSC/1 mM DTT for 30 min at 55 C and then 0.1x SSC for another 30 min at 55 C before being dehydrated in ethanol and air dried. RNase A-resistant hybrids were detected after 1–2 wk of autoradiography using Kodak NTB-2 (Amersham BioSciences) liquid emulsion. The slides were poststained with hematoxylin. Tissues were examined with a Zeiss Axioskop microscope (Photometrics Sensys, Roper Scientific, Tucson, AZ) under bright- and dark-field optics.
Tissue distributions
Normalized mouse cDNA panels (CLONTECH) were used to examine the expression of Dcpp in other mouse tissues. Five microliters of cDNA were mixed with 45 μl 1x PCR buffer [10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, and 0.1% Triton X-100], 200 nM primers (Table 1), 200 μM deoxynucleotide triphosphates, and 5 U Taq DNA polymerase (Roche Molecular Biochemicals, Indianapolis, IN). The PCR conditions were 26–38 cycles at 95 C for 15 sec and 68 C for 5 min. The amplified PCR products were separated on a 2% agarose gel, stained with ethidium bromide, and visualized by UV illumination.
Real-time PCR
Total RNA template was reverse transcribed with first-strand cDNA synthesis kit (Amersham Biosciences) and used for real-time PCR in a 20-μl reaction volume containing 1x SYBR Green PCR mix (Applied Biosystems Inc., Foster City, CA) in an iCyclerIQ real-time PCR system (Bio-Rad Laboratories, Hercules, CA). The primer sequences used is shown in Table 1. The PCR amplification conditions were as follows: 95 C for 5 min to activate the polymerase, followed by 45 cycles at 95 C for 30 sec for denaturation, 60 C for 30 sec for annealing, and 72 C for 45 sec for polymerization. The relative amount of gene expression was calculated using the expression of -actin as an internal standard. The PCR cycle number that generated the first fluorescence signals above a threshold value [threshold cycle (CT)] was determined. Threshold was calculated as a value 10 times SD above the mean fluorescence generated during the baseline cycles. A comparative CT method (2–CT method) was used to detect the relative gene expression (31, 32). The following formula was used to calculate the relative amount of the transcript of interest in the treated sample (X) and the control sample (Y), both normalized to an endogenous reference (-actin): 2–CT, where CT is the difference in CT between the gene of interest and -actin, and CT is the difference for sample X = CT, X – CT, Y. Pregnancy d 1 or proestrus stage was used as calibrators for different PCR experiments.
Dcpp antibody production, immunohistochemistry, and Western blot analysis
Antibodies against Dcpp were raised from two rabbits immunized with keyhole limpet hemocyanin-conjugated Dcpp peptide (NM_019910: CQFNYNNEDGQVYGS, 61–74 amino acids) by Zymed Laboratory Inc. (South San Francisco, CA). This peptide sequence is unique in the protein database. The antibodies were affinity purified with anti-Dcpp peptide column. The antibody titers were measured by ELISA method. A 1:100 and 1:2000 (0.5 μg/ml) dilutions were made for immunohistochemistry and Western blot analysis respectively. For immunohistochemical staining, either fluorescein isothiocyanate-/Cy-3 conjugated antirabbit antibody for fluorescent microscopy or biotin-conjugated antirabbit IgG secondary antibody for light microscopy was used. For Western blot analysis, horseradish peroxidase-conjugated antirabbit IgG secondary antibody was used, and the specific signal was visualized by the enhanced chemiluminescence method.
Expression and purification of Dcpp recombinant protein
The full-length cDNA of the Dcpp (accession no. NM_019910) was PCR amplified and cloned into pET100-D/TOPO vector (Invitrogen, Carlsbad, CA). Expression of the Dcpp fusion protein was performed in Escherichia coli BL21 (DE3) Star by 0.5 mM isopropyl-1-thio--D-galactopyranoside induction for 3 h. Dcpp protein was expressed as inclusion bodies in the bacteria and purified with the protein refolding kit (Novagen, EMD Biosciences, Madison, WI), in accordance with the manufacturer’s protocol. In brief, bacterial cell pellet was resuspended in 0.1 culture volume of 1x IB wash buffer [20 mM Tris-HCl (pH 7.5), 10 mM EDTA, 1% Triton X-100], and lysozyme at a final concentration of 100 μg/ml was added. The mixture was incubated at 30 C for 15 min and sonicated for 5 x 20 sec on ice. Inclusion bodies were collected by centrifugation at 10,000 x g for 10 min, washed twice with 1x IB wash buffer, and dissolved in 1x IB solubilization buffer [1 mM DTT, 50 mM 3-(cyclohexylamino)-1-propanesulfonic acid (pH 11.0)] supplemented with 0.3% N-lauroylsarcosine at 10–20 mg/ml protein concentration. The soluble Dcpp protein fraction in the supernatant was collected after centrifugation and dialyzed with 500 ml of 1x dialysis buffer [0.1 mM DTT, 20 mM Tris-HCl (pH 8.5) and then 500 ml of 20 mM Tris-HCl (pH 8.5)]. The soluble Dcpp protein was added into the potassium simplex optimized medium (KSOM) for embryo culture experiment.
Collection of mouse oviductal and uterine flushing
Five mature ICR female mice aged 6–8 wk were used. Ten oviducts and uteri were collected separately from the mice. For tubal flushing, a curved blunt-end metal capillary connected to a 1-ml syringe was used. The oviducts were flushed with 60–80 μl of flushing medium (PBS supplemented with 0.3% polyvinylpyrrolidone) from infundibulum to the uterotubal junction. The flushing medium was used repeatedly to concentrate the oviductal fluid. The flushing of uterine fluid was performed in a similar manner by injecting 100–120 μl of medium into the tip of the uterine horn.
Embryo collection and culture
The in vivo-developed oocytes, zygotes, and embryos were recovered at different time points, as described previously (26). The procedure for obtaining superovulated embryos was essentially the same as before (27). Mature ICR female mice (age, 6–8 wk) were superovulated with 5 IU pregnant mare’s serum gonadotropin (Sigma), followed by an injection of 5 IU of hCG (Sigma) 46 h later. The ICR female mice were then mated with proven-fertile BALB/c males. The day with the presence of vaginal plug was regarded as d 1. The zygotes were recovered 24 h after hCG from the oviductal ampullae into HEPES-buffered KSOM (KSOM/HEPES) containing 0.8 mg/ml hyaluronidase (Sigma) to remove the cumulus mass. They were washed three times in 250 μl KSOM/HEPES and once in KSOM and were then pooled and allocated randomly in groups of 20–30 for culturing in KSOM with or without Dcpp supplementation. The Dcpp protein (10 μg/ml) was supplemented to KSOM containing 5 mM glucose for embryo culture in the following 120 h. The percentages of embryos reaching the fully expanded blastocyst stage were recorded at 120 h after hCG. The data obtained from at least three replicate experiments were combined and analyzed by 2 test or Student t test when appropriate.
Implantation study
Twelve-week-old pseudopregnant ICR females were used as recipients for embryo transfer. Pseudopregnancy was induced by mating with proven, vasectomized BALB/c males. Ten in vivo-developed two-cell embryos (five embryos per horn) in Chatot, Ziomek, Bavister (CZB) medium (33) containing 10 μg/ml Dcpp protein, with or without anti-Dcpp antibody (100 μg/ml), or antibody alone were transferred to the oviduct of the pseudopregnant females on d 2 of pregnancy, as previously described (34). After embryo transfer, each mouse was placed in a separate cage supplied with ad libitum foodstuff and water. The number of implantation sites was counted at d 8 of pregnancy.
Statistical analysis
All results are expressed as means ± SEM. Statistical comparisons were performed by one-way ANOVA followed by Tukey test. A probability of P < 0.05 was used to indicate a statistically significant difference.
Results
Identification of Dcpp from the subtracted oviductal library
Two mouse oviduct cDNA libraries were constructed for the enrichment of transcripts preferentially expressed in the embryo-containing oviduct relative to the oocyte-containing oviduct. Positive signals with higher expression in the embryo-containing oviductal samples were detected in 97 of 250 reamplified clones, as reported previously (6, 29). One of the clones, OD135, was found to be the mouse Dcpp. It had more than 90% DNA sequence homology with the published sequence (Dcpp, accession no. NM_019910). The expression and the function of Dcpp in the mouse oviduct were characterized in this study.
Expression and localization of Dcpp in mouse tissues
Northern blot and multiple tissue cDNA panel PCR analysis were used to determine the transcript sizes and the tissue distribution of the Dcpp clone. A transcript of about 0.6 kb in size was observed in the oviduct only by Northern blot analysis (Fig. 1A). Using a more sensitive PCR method, Dcpp was also highly expressed in the skeletal muscle and lymph node. The expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were similar for all the cDNA samples studied in this experiment (Fig. 1B). SignalP 3.0 program determined that the potential cleavage site of the signal peptide of Dcpp was at amino acid position 24. The location of Dcpp mRNA expression in the oviduct was investigated by in situ hybridization (ISH). Mouse oviduct sections from animals at different stages of the estrus cycle were hybridized with a 450-bp 35S-labeled antisense or sense RNA probe containing sequences from Dcpp cDNA. A hybridization signal was observed at the luminal epithelium of the oviduct from all stages, with the strongest expression at the estrus stage (Fig. 2A). A very weak or no hybridization signal was detected when the sense probe was used. To quantify the expression of Dcpp at different stages of the estrus cycle, real-time PCR was used. The expression levels of Dcpp peaked in the estrus stage (Fig. 2B). The signal declined in the metestrus and diestrus stages. -Actin gene was used as the RNA control in these samples.
Dcpp expression in the mouse oviduct during pregnancy
We further investigated the expression levels of Dcpp in pregnant and pseudopregnant mouse oviducts at d 1–4 of pregnancy by real-time PCR. The level of Dcpp mRNA remained constant in the pregnant mouse oviducts but decreased continuously in the pseudopregnant mouse oviducts (Fig. 2C). The decrease from d 1 reached statistical significance (P < 0.05) on d 4 in the pseudopregnant oviducts, confirming that the embryos could modulate the expression of the Dcpp gene in mouse oviducts.
Localization of Dcpp protein in the mouse tissues
Antibody against Dcpp peptide was raised from rabbits. Western blot analysis showed a specific 17-kDa Dcpp protein from the mouse oviduct (Fig. 3A). This band disappeared when the Dcpp antibody was preabsorbed with the corresponding peptide. Positive staining of Dcpp was found in the epithelial lining of the mouse oviduct (Fig. 3B). The Dcpp immunoreactivity was stronger at estrus (data not shown). This change in immunoreactivity was positively correlated with the mRNA expression levels (Fig. 2B), being highest at estrus and lowest at diestrus. No signal was found when the antibody was preabsorbed with Dcpp peptide (Fig. 2B) and confirmed the specificity of the antibody.
Dcpp gene is hormonally regulated in ovariectomized mice
The hormonal regulation of Dcpp mRNA expression was investigated by using an antiestrogen, ICI 182,780, and an antiprogesterone/glucocorticoid antagonist, RU486. Animals were ovariectomized and injected with vehicle (sesame oil), estrogen, progesterone, or a combination of estrogen and ICI 182,780 or progesterone and RU486. By using ISH, Dcpp mRNA expression was not observed in vehicle-treated ovariectomized animals (Fig. 4). Administration of estrogen-induced Dcpp expression in the mouse oviduct. ICI 182,780 drastically declined estrogen-induced Dcpp mRNA expression in these animals. Progesterone administration slightly increased the expression of Dcpp mRNA, which was suppressed by RU486. Combined treatment of the animals with estrogen and progesterone induced the expression of Dcpp mRNA to a level similar to estrogen alone treatment (Fig. 4). Estrogen or combined estrogen and progesterone treatment strongly induced the expression of Dcpp protein in ovariectomized mice. The effect was abolished when antiestrogen ICI 182,780 was used (Fig. 4). A moderate induction of Dcpp protein was observed when progesterone was used. RU486 antagonizes this effect of progesterone on ovariectomized mice.
Embryotrophic activity of Dcpp
The development of mouse embryos with or without Dcpp treatment is shown in Table 2. The rate of embryo development was based on the number of two-cell embryos after 24 h of culture. The embryos incubated with Dcpp for 4 d had significantly more expanded blastocysts (P < 0.05) than those cultured in medium alone (34 vs. 23%). The size of the expanded blastocyst after Dcpp treatment, as determined by the area of the expanded blastocyst, was also significantly larger than in the untreated group (9472 ± 279 vs. 8409 ± 251, P < 0.05).
Dcpp is present in the developing embryos
Mouse oviductal and uterine fluid were collected and subjected to Western blot analysis using the Dcpp antibody. We found a band of 17 kDa in size, corresponding to the predicted native Dcpp protein in oviductal but not in uterine fluid (Fig. 5A). We also studied the expression and localization of Dcpp proteins in mouse oocytes and embryos (Fig. 5). Mature oocytes were collected from hormonally stimulated mouse ovaries at 2300 h on the day of hCG injection. In vivo developed mouse embryos from three to four cells to blastocyst stages were collected from the oviduct and uterus at various time points. These embryos were subjected to Dcpp immunostaining. Interestingly, immunoreactivity was found in the embryos recovered from the mouse oviducts at the three to four cells, morula, and blastocyst stages but not in the germinal vesicle oocytes or mature oocytes taken from mouse ovaries. The immunoreactivity was abolished when preabsorbed antibody was used. Higher Dcpp immunostaining was found at the morula stage. This suggests that Dcpp was expressed and secreted from mouse oviducts and was taken up by the developing embryo.
Effect of Dcpp antibody on the implantation rate of transferred embryos
Table 3 presents the implantation rate of two-cell embryos after their transfer to the oviduct of the foster mother in medium containing Dcpp, anti-Dcpp antibody, or Dcpp and anti-Dcpp antibody. Treatment with anti-Dcpp antibody resulted in a 46% implantation rate. This was significantly lower (P < 0.05) than the implantation rate of embryos transferred with Dcpp protein (70%). Addition of anti-Dcpp antibody neutralized the beneficial effect of Dcpp on embryo implantation (48 vs. 70%, P < 0.05). This neutralizing effect was found to be specific because the addition of an irrelevant antibody did not have the same effect (data not shown).
Discussion
The oviduct provides an optimal microenvironment for the development of preimplantation embryos. Whereas it is generally accepted that maternal-embryo dialogue is important to provide the best environment for such development, direct evidence demonstrating this concept is lacking. Various oviductal derived factors are known to enhance embryo development in vitro (16, 18, 35), but it remains unclear how their production is regulated or whether the embryo is involved in such regulation. Similarly, we cannot fully explain the mechanisms of the differential transport of oocytes and embryos in rat and hamster oviducts (23, 24).
To address this question, we previously used SSH to compare the gene expression profiles between oviducts containing preimplantation embryos and those containing oocytes in a mouse model and successfully isolated 97 clones that were differentially up-regulated in the embryo-containing oviducts (6). Seventeen were putative secretory products with signal peptides (29). Dcpp is one of these clones. In this article, we provided evidence for its regulation and demonstrated that it might be one of the molecules mediating embryo-maternal dialogue in mice.
The gene coding for Dcpp was first identified from the mouse salivary gland (30) and is highly homologous to the common salivary protein found in mice (89% identity in 128 amino acids) and rats (42% identity in 160 amino acids) (36). The mouse Dcpp gene is located on chromosome 17A3.3. It contains three exons encoding a protein with 150 amino acids. It has a jacalin-like lectin domain. Proteins containing this domain are lectins that may be involved in cell-cell communications, host-pathogen interactions, cancer metastasis, embryogenesis, and tissue development (37). This jacalin-like lectin domain has been found in the animal prostatic spermine-binding protein (38), which is under Hoxb13 regulation for normal ventral prostate morphogenesis and epithelial differentiation (39).
In the present study, Northern blot and cDNA panel analysis showed that Dcpp mRNA was highly expressed in the oviduct, although it is also present in skeletal muscle, lymph nodes, and 17-d-old embryos. ISH confirmed the expression of Dcpp in the oviduct and further located it to the oviductal epithelium throughout the estrus cycle. The maximal expression of Dcpp at the estrus stage suggests that the molecule is under steroid regulation. Indeed, our experiment on ovariectomized mice with or without treatment with estrogen, progesterone, and their antagonists demonstrated that estrogen significantly up-regulated the expression of Dcpp mRNA and protein. This conclusion was confirmed by the abolition of this effect with the use of estrogen antagonist. Estrogen has been suggested to be the most important steroid in modifying the oviductal environment in mammals during late follicular development and estrus (2). How estrogen regulates Dcpp expression is not clear. Our unpublished data show the presence of half estrogen-responsive elements in the promoter region of the Dcpp gene. Whether these elements and/or other transcriptional factors are involved in the regulation of Dcpp expression remains to be investigated.
Progesterone also slightly increased the expression of Dcpp. This effect was nullified by the progesterone/glucocorticoid antagonist, RU486. However, its contribution was low in comparison with that of estrogen. The simultaneous administration of estrogen and progesterone to ovariectomized mice produced the same effect as estrogen alone.
We also demonstrated in this study that the embryos in the reproductive tract modulated the expression of Dcpp. Compared with the pregnant mouse oviducts in the first 4 d of pregnancy, the Dcpp mRNA copy number as determined by real-time PCR continuously decreased in pseudopregnant mouse oviducts containing no embryo, and the difference reached statistical significance (P < 0.05) at d 4 of pregnancy or pseudopregnancy. Because pregnant and pseudopregnant mice have a similar hormonal profile, the ability of pregnant mice to maintain the expression of Dcpp mRNA in the oviduct is highly likely to be due to the presence of developing preimplantation embryos. This is consistent with our previous report on differential gene expression between embryo- and oocyte-containing oviducts (29). The presence of an oocyte-cumulus complex has also been shown to influence the gene expression profile in bovine oviducts in the postovulation period (17).
The embryos and oviducts possess the necessary biochemical machinery for communication, although the detailed mechanism of action is not known. The oviduct produces a number of factors, and many of their corresponding receptors are present in embryos (3). Several reports have demonstrated the beneficial effect of oviductal factors on embryo development. Granulocyte-macrophage colony-stimulating factor (40), leptin (14), and complement protein component 3 (18) are examples of the more recently identified embryotrophic oviductal factors. Their potential receptors have been detected in embryos (14, 18, 41). Recently the estrogen-regulated oviduct-specific glycoprotein has also been reported to enhance the formation of bovine blastocysts (35). However, its receptor in the embryo remains unknown.
There are fewer reports on embryonic factors affecting oviductal physiology. The best-known example is embryo-derived platelet activating factor (42). Its receptor is present in the oviducts of humans (43) and cows (44). Platelet-activating factor has been shown to affect the electrophysiology of human oviducts (45) and increase the intracellular calcium concentration (46) and proliferation (47) of bovine oviductal cells. In mares, embryonic prostaglandin E2 has been proposed as the agent responsible for the differential transport of embryos and oocytes in the oviduct (48). How oviductal embryos modulate the expression of Dcpp remains to be investigated.
This is the first report demonstrating the embryotrophic activity of Dcpp. The beneficial effect of Dcpp on embryo development manifested itself as an increase both in the rate of blastocyst formation and in the size of the resulting blastocysts in culture. These in vitro data were supported by in vivo data showing that simultaneous oviductal transfer of two-cell embryos with Dcpp had a higher implantation rate and lower abortion rate than the transfer of the embryos with anti-Dcpp antibody. Indeed, anti-Dcpp antibody could abolish the effect of Dcpp on implantation and abortion in vivo. The positive influence of Dcpp on implantation may be due to a direct effect of Dcpp on implantation or an indirect effect on improving embryo quality before implantation. Our experiment was unable to distinguish between these possibilities.
Consistent with the embryotrophic activity of Dcpp, immunohistochemistry revealed that Dcpp immunoreactivity was found in embryos retrieved from mouse oviducts but not in oocytes from the ovary. The intensity of the signal peaked at the morula stage and decreased at the blastocyst stage. These data suggest that Dcpp secreted from the oviduct was taken up by the embryos during the transfer in the oviduct. The identity of the Dcpp receptor in the embryos has yet to be determined.
Similar to other oviductal embryotrophic factors (e.g. oviduct-specific glycoprotein and complement protein component 3), Dcpp is unlikely to be crucial to preimplantation embryo development in vivo. This is supported by the inability of anti-Dcpp antibody to inhibit implantation completely and the recent demonstration that mice deficient in oviduct-specific glycoprotein and complement protein component 3 are fertile (49, 50). However, the presence of these factors greatly enhances the development of the preimplantation embryo.
The long-term effect of culturing embryos in medium containing simple salt solution is being vigorously discussed. In animal models, culture conditions affect the frequency of large offspring syndrome in farm animals (51), the expression of the imprinting gene H19 in mice (52), and the behavior of adults derived from cultured embryos (53). Improving culture conditions by supplementing oviductal embryotrophic factors may eliminate or reduce the detrimental effects of suboptimal culture conditions. Several recent studies suggested that there is an increase in imprinting defects in children born after assisted reproduction (54, 55, 56). Whether culture conditions play a role in the generation of these abnormalities is not known.
In conclusion, we have demonstrated that Dcpp is an estrogen-regulated oviductal embryotrophic factor. Mouse oviducts maintain the production of Dcpp in the presence of preimplantation embryos, which subsequently improves embryo development. The study strongly suggests that Dcpp might be an important mediator for preimplantation embryo-maternal dialogue. The potential application of Dcpp in the culture of human embryos deserves further exploration.
Acknowledgments
We are grateful to Mr. K. L. Kwok and Ms. J. F. C. Chow (Department of Obstetrics and Gynaecology, The University of Hong Kong) for their technical assistance. We are also grateful to Dr. D. J. Wilmshurst (the university’s technical writer) for commenting on our draft.
Footnotes
This work was supported in part by the University of Hong Kong’s Committee on Research and Conference Grants (10202566 and 10204873) and the Hong Kong Research Grant Council Grants HKU7327/00M, HKU7436/03M, and HKU7411/04M.
First Published Online October 20, 2005
Abbreviations: CT, Threshold cycle; CZB, Chatot, Ziomek, Bavister; Dcpp, demilune cell and parotid protein; DTT, dithiothreitol; E2, estradiol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hCG, human chorionic gonadotropin; ISH, in situ hybridization; KSOM, potassium simplex optimized medium; SSC, standard saline citrate; SSH, suppression subtraction hybridization.
Accepted for publication October 7, 2005.
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Abstract
In mammals, fertilization and early preimplantation embryo development occur in the oviduct. We hypothesized that interaction exists between the developing embryos and the maternal genital tract, such that the embryos modulate the physiology and gene expression of the oviduct so that it is conducive to their development. By comparing the gene expression patterns in mouse oviducts containing transferred preimplantation embryos with those of oviducts containing oocytes, we report here the characterization of demilune cell and parotid protein (Dcpp), which was up-regulated in the embryo-containing oviduct. Dcpp mRNA was highly expressed in the oviductal epithelium at the estrus stage. The Dcpp gene codes for a protein of 150 amino acids and contains a signal peptide suggestive of secretory function. The Dcpp mRNA level was maintained in the oviductal epithelium of pregnant females but decreased continuously in those of pseudopregnant mice. Exogenous estrogen stimulated the expression of Dcpp mRNA and protein in ovariectomized mice. The effect was abolished by an estrogen antagonist, ICI 182,780. Dcpp protein was present in mouse oviductal fluid but not in uterine fluid. More importantly, Dcpp immunoreactivity was found in embryos recovered from the oviduct but not in mature oocytes from the ovary. Supplementation of Dcpp to culture medium stimulated the development of mouse embryos to the blastocyst stage. Anti-Dcpp antibody decreased the beneficial effect of Dcpp on implantation of two-cell mouse embryos transferred to the oviducts of the foster mothers. In summary, our data demonstrated that Dcpp is highly expressed in the oviductal lumen in the presence of preimplantation embryos. It stimulates the growth of preimplantation embryos and may play an important role in embryo-maternal dialogue.
Introduction
IN MAMMALS, FERTILIZATION and early embryonic development occur in the oviduct. The metabolic needs of preimplantation embryos change during development (1). To cope with the changing needs of the developing embryos, the microenvironment in the female reproductive tract undergoes biochemical and physiological changes (2, 3) to provide the best support for the development of the embryos. Evidence is accumulating that interaction exists between the gametes or embryos and the oviduct during the preimplantation period (4, 5, 6, 7). This interaction can be achieved via the paracrine factors (reviewed in Ref.8), including IGF-binding proteins (9, 10), IL-1 (11), vascular endothelial growth factor and receptors (12), TGF (13), platelet-activating factor (7), leptin (14), and other factors or peptides of unknown identity (3, 15, 16, 17, 18).
The microenvironment of the oviduct is regulated by steroid hormones. The ratio of the ciliated cells to the secretory cells in the mammalian oviduct changes in a cyclical manner (19), and the ciliated cells may be transformed to secretory cells after loss of their cilia (20). These changes are associated with oviductal biosynthetic activity, which is modulated by estrogen and progesterone (reviewed in Ref.2). Porcine oviductal epithelial cells secrete at least 14 major proteins into the culture medium (21), including oviduct-specific glycoprotein, tissue inhibitor of metalloproteinase, and plasminogen activator inhibitor-1 (2). Interestingly, these proteins are expressed in a unique temporal and spatial pattern consistent with the existence of physiological interactions of the oviduct with the embryos during early embryonic development (22).
It has been reported that the hamster oviduct can distinguish between developing embryos and oocytes (23) and between embryos at different ages (24), as evidenced by the ability of the oviduct to transport the oocytes and embryos to the uterus at different rates. However, Freeman et al. (25) demonstrated that it was the embryos themselves that controlled the oviductal transport in mares. However, the molecular mechanisms regulating oviductal transport remain unknown.
Studies of embryonic-maternal interaction have been advanced by the use of both transcriptomic and proteomic approaches to delineate the role of the oviduct in preimplantation embryo development (5) and gamete interactions (4). Recently Bauersachs et al. (17) compared the gene expression patterns between the ipsilateral and contralateral bovine oviductal epithelial cells by suppression subtraction hybridization (SSH) at d 3.5 after standing heat. They identified a number of differentially expressed genes related to immunity or proteins involved in cell-cell interactions. These genes and their protein products can potentially shed detailed light on embryonic-maternal interaction.
We previously demonstrated that oviductal cells affect the gene expression of developing embryos in vitro (26), partly via embryotrophic factor-3 (27). On the other hand, developing embryos have also altered gene expression in the oviduct (6, 28). By comparing gene expression in the oocyte-containing oviduct with that in the embryo-containing oviduct, we successfully isolated more than a dozen genes differentially expressed in the embryo-containing oviduct (6, 29). In the present study, we characterized one of these oviductal genes, OD135, which is highly expressed in mouse oviducts and is homologous to the demilune cell and parotid protein (Dcpp) found in the mouse salivary gland. Dcpp is highly expressed in the sublingual gland and to a lesser extent the parotid and submandibular gland (30). However, its roles in salivary gland development and oviduct are still unknown. In this study, the expression of the Dcpp gene during pregnancy, regulation by estrogen in the ovariectomized mice, and the effect of protein on in vitro embryo culture were investigated. It is suggested that the maternal genital tract recognizes developing embryos and secretes embryotrophic factor such as Dcpp to promote the development of the embryo in vivo.
Materials and Methods
Tissue collection and RNA isolation
The model we used for studying the effect of embryos on oviductal gene expression has been reported previously (6). MF1 female mice were superovulated by successive ip injection of pregnant mare’s serum gonadotropin (Sigma, St. Louis, MO) and human chorionic gonadotropin (hCG; Sigma). The fertilized zygotes and oocytes were collected from these animals with and without mating at 24 h after hCG, respectively, and were transferred to a different oviduct of the same pseudopregnant female mice. After 48 h, oviducts containing three to four cell embryos or oocytes were collected if they were previously transferred with one-cell embryos (embryo containing oviduct) or oocytes (oocyte containing oviduct), respectively. The mRNA of the oocyte-containing oviducts and embryo-containing oviducts were subjected to SSH with the PCR-Select cDNA subtraction kit (CLONTECH Laboratories Inc., Palo Alto, CA). The research protocol was approved by the University of Hong Kong’s Committee on the Use of Live Animals in Teaching and Research. The oviducts from the estrus cycle (proestrus, estrus, metestrus, and diestrus) of pregnant and pseudopregnant mice (n = 5) from d 1–4 were collected. Ovariectomized mice were injected with 100 ng/mouse estradiol (E2) or 1 mg/mouse progesterone, or E2 plus 3 mg/kg ICI 182,780 (Tocris, Bristol, UK), or progesterone plus 1 mg/mouse RU486 (Tocris) at 24 h before tissue collection. Half of the collected oviducts were fixed in 4% paraformaldehyde in PBS for in situ hybridization, and the remaining halves were used for RNA extraction. Poly(A)+ RNA and total RNA from mouse tissues were isolated by the QuickPrep micro-mRNA purification kit (Amersham Biosciences, Piscataway, NJ) and the Absolutely RNA RT-PCR miniprep kit (Stratagene, La Jolla, CA), respectively, in accordance with the manufacturer’s instructions. The RNA was quantified by spectrophotometry and stored at –70 C until use.
Northern blot and in situ hybridization
Northern blot hybridization was performed in accordance with a protocol previously described (13). For in situ hybridization, paraffin-embedded mouse oviducts were sectioned at 5 μm thick and subjected to in situ hybridization using the mRNA locator in situ hybridization kit (Ambion Inc., Austin, TX) as described (29). The dewaxed tissues were washed in PBS and treated with proteinase K for 10 min at room temperature. The tissues were washed sequentially with PBS for 5 min, 1.32% triethanolamine for 3 min, triethanolamine with 0.25% acetic anhydride for 10 min, and PBS for another 5 min at room temperature. The sense and antisense probes for Dcpp (Table 1) were generated using the SP6/T7 riboprobe combination system (Promega, Madison, WI). The tissue sections were allowed to hybridize with the probe in hybridization buffer (1 x 106 cpm/slide) overnight in a humidified chamber at 55 C. After hybridization, the slides were washed extensively to remove nonspecifically bound riboprobe. Tissues were washed with 4x standard saline citrate (SSC) buffer [1x SSC containing 0.15 M NaCl and 15 mM sodium citrate; and 1 mM dithiothreitol (DTT)] for 5 min at 55 C, followed by 2x SSC/1 mM DTT for 30 min at 55 C, and then treated with RNase A (20 μg/ml) at 37 C for 15 min. Slides were then washed in 2x SSC/1 mM DTT for 30 min at 55 C and then 0.1x SSC for another 30 min at 55 C before being dehydrated in ethanol and air dried. RNase A-resistant hybrids were detected after 1–2 wk of autoradiography using Kodak NTB-2 (Amersham BioSciences) liquid emulsion. The slides were poststained with hematoxylin. Tissues were examined with a Zeiss Axioskop microscope (Photometrics Sensys, Roper Scientific, Tucson, AZ) under bright- and dark-field optics.
Tissue distributions
Normalized mouse cDNA panels (CLONTECH) were used to examine the expression of Dcpp in other mouse tissues. Five microliters of cDNA were mixed with 45 μl 1x PCR buffer [10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, and 0.1% Triton X-100], 200 nM primers (Table 1), 200 μM deoxynucleotide triphosphates, and 5 U Taq DNA polymerase (Roche Molecular Biochemicals, Indianapolis, IN). The PCR conditions were 26–38 cycles at 95 C for 15 sec and 68 C for 5 min. The amplified PCR products were separated on a 2% agarose gel, stained with ethidium bromide, and visualized by UV illumination.
Real-time PCR
Total RNA template was reverse transcribed with first-strand cDNA synthesis kit (Amersham Biosciences) and used for real-time PCR in a 20-μl reaction volume containing 1x SYBR Green PCR mix (Applied Biosystems Inc., Foster City, CA) in an iCyclerIQ real-time PCR system (Bio-Rad Laboratories, Hercules, CA). The primer sequences used is shown in Table 1. The PCR amplification conditions were as follows: 95 C for 5 min to activate the polymerase, followed by 45 cycles at 95 C for 30 sec for denaturation, 60 C for 30 sec for annealing, and 72 C for 45 sec for polymerization. The relative amount of gene expression was calculated using the expression of -actin as an internal standard. The PCR cycle number that generated the first fluorescence signals above a threshold value [threshold cycle (CT)] was determined. Threshold was calculated as a value 10 times SD above the mean fluorescence generated during the baseline cycles. A comparative CT method (2–CT method) was used to detect the relative gene expression (31, 32). The following formula was used to calculate the relative amount of the transcript of interest in the treated sample (X) and the control sample (Y), both normalized to an endogenous reference (-actin): 2–CT, where CT is the difference in CT between the gene of interest and -actin, and CT is the difference for sample X = CT, X – CT, Y. Pregnancy d 1 or proestrus stage was used as calibrators for different PCR experiments.
Dcpp antibody production, immunohistochemistry, and Western blot analysis
Antibodies against Dcpp were raised from two rabbits immunized with keyhole limpet hemocyanin-conjugated Dcpp peptide (NM_019910: CQFNYNNEDGQVYGS, 61–74 amino acids) by Zymed Laboratory Inc. (South San Francisco, CA). This peptide sequence is unique in the protein database. The antibodies were affinity purified with anti-Dcpp peptide column. The antibody titers were measured by ELISA method. A 1:100 and 1:2000 (0.5 μg/ml) dilutions were made for immunohistochemistry and Western blot analysis respectively. For immunohistochemical staining, either fluorescein isothiocyanate-/Cy-3 conjugated antirabbit antibody for fluorescent microscopy or biotin-conjugated antirabbit IgG secondary antibody for light microscopy was used. For Western blot analysis, horseradish peroxidase-conjugated antirabbit IgG secondary antibody was used, and the specific signal was visualized by the enhanced chemiluminescence method.
Expression and purification of Dcpp recombinant protein
The full-length cDNA of the Dcpp (accession no. NM_019910) was PCR amplified and cloned into pET100-D/TOPO vector (Invitrogen, Carlsbad, CA). Expression of the Dcpp fusion protein was performed in Escherichia coli BL21 (DE3) Star by 0.5 mM isopropyl-1-thio--D-galactopyranoside induction for 3 h. Dcpp protein was expressed as inclusion bodies in the bacteria and purified with the protein refolding kit (Novagen, EMD Biosciences, Madison, WI), in accordance with the manufacturer’s protocol. In brief, bacterial cell pellet was resuspended in 0.1 culture volume of 1x IB wash buffer [20 mM Tris-HCl (pH 7.5), 10 mM EDTA, 1% Triton X-100], and lysozyme at a final concentration of 100 μg/ml was added. The mixture was incubated at 30 C for 15 min and sonicated for 5 x 20 sec on ice. Inclusion bodies were collected by centrifugation at 10,000 x g for 10 min, washed twice with 1x IB wash buffer, and dissolved in 1x IB solubilization buffer [1 mM DTT, 50 mM 3-(cyclohexylamino)-1-propanesulfonic acid (pH 11.0)] supplemented with 0.3% N-lauroylsarcosine at 10–20 mg/ml protein concentration. The soluble Dcpp protein fraction in the supernatant was collected after centrifugation and dialyzed with 500 ml of 1x dialysis buffer [0.1 mM DTT, 20 mM Tris-HCl (pH 8.5) and then 500 ml of 20 mM Tris-HCl (pH 8.5)]. The soluble Dcpp protein was added into the potassium simplex optimized medium (KSOM) for embryo culture experiment.
Collection of mouse oviductal and uterine flushing
Five mature ICR female mice aged 6–8 wk were used. Ten oviducts and uteri were collected separately from the mice. For tubal flushing, a curved blunt-end metal capillary connected to a 1-ml syringe was used. The oviducts were flushed with 60–80 μl of flushing medium (PBS supplemented with 0.3% polyvinylpyrrolidone) from infundibulum to the uterotubal junction. The flushing medium was used repeatedly to concentrate the oviductal fluid. The flushing of uterine fluid was performed in a similar manner by injecting 100–120 μl of medium into the tip of the uterine horn.
Embryo collection and culture
The in vivo-developed oocytes, zygotes, and embryos were recovered at different time points, as described previously (26). The procedure for obtaining superovulated embryos was essentially the same as before (27). Mature ICR female mice (age, 6–8 wk) were superovulated with 5 IU pregnant mare’s serum gonadotropin (Sigma), followed by an injection of 5 IU of hCG (Sigma) 46 h later. The ICR female mice were then mated with proven-fertile BALB/c males. The day with the presence of vaginal plug was regarded as d 1. The zygotes were recovered 24 h after hCG from the oviductal ampullae into HEPES-buffered KSOM (KSOM/HEPES) containing 0.8 mg/ml hyaluronidase (Sigma) to remove the cumulus mass. They were washed three times in 250 μl KSOM/HEPES and once in KSOM and were then pooled and allocated randomly in groups of 20–30 for culturing in KSOM with or without Dcpp supplementation. The Dcpp protein (10 μg/ml) was supplemented to KSOM containing 5 mM glucose for embryo culture in the following 120 h. The percentages of embryos reaching the fully expanded blastocyst stage were recorded at 120 h after hCG. The data obtained from at least three replicate experiments were combined and analyzed by 2 test or Student t test when appropriate.
Implantation study
Twelve-week-old pseudopregnant ICR females were used as recipients for embryo transfer. Pseudopregnancy was induced by mating with proven, vasectomized BALB/c males. Ten in vivo-developed two-cell embryos (five embryos per horn) in Chatot, Ziomek, Bavister (CZB) medium (33) containing 10 μg/ml Dcpp protein, with or without anti-Dcpp antibody (100 μg/ml), or antibody alone were transferred to the oviduct of the pseudopregnant females on d 2 of pregnancy, as previously described (34). After embryo transfer, each mouse was placed in a separate cage supplied with ad libitum foodstuff and water. The number of implantation sites was counted at d 8 of pregnancy.
Statistical analysis
All results are expressed as means ± SEM. Statistical comparisons were performed by one-way ANOVA followed by Tukey test. A probability of P < 0.05 was used to indicate a statistically significant difference.
Results
Identification of Dcpp from the subtracted oviductal library
Two mouse oviduct cDNA libraries were constructed for the enrichment of transcripts preferentially expressed in the embryo-containing oviduct relative to the oocyte-containing oviduct. Positive signals with higher expression in the embryo-containing oviductal samples were detected in 97 of 250 reamplified clones, as reported previously (6, 29). One of the clones, OD135, was found to be the mouse Dcpp. It had more than 90% DNA sequence homology with the published sequence (Dcpp, accession no. NM_019910). The expression and the function of Dcpp in the mouse oviduct were characterized in this study.
Expression and localization of Dcpp in mouse tissues
Northern blot and multiple tissue cDNA panel PCR analysis were used to determine the transcript sizes and the tissue distribution of the Dcpp clone. A transcript of about 0.6 kb in size was observed in the oviduct only by Northern blot analysis (Fig. 1A). Using a more sensitive PCR method, Dcpp was also highly expressed in the skeletal muscle and lymph node. The expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were similar for all the cDNA samples studied in this experiment (Fig. 1B). SignalP 3.0 program determined that the potential cleavage site of the signal peptide of Dcpp was at amino acid position 24. The location of Dcpp mRNA expression in the oviduct was investigated by in situ hybridization (ISH). Mouse oviduct sections from animals at different stages of the estrus cycle were hybridized with a 450-bp 35S-labeled antisense or sense RNA probe containing sequences from Dcpp cDNA. A hybridization signal was observed at the luminal epithelium of the oviduct from all stages, with the strongest expression at the estrus stage (Fig. 2A). A very weak or no hybridization signal was detected when the sense probe was used. To quantify the expression of Dcpp at different stages of the estrus cycle, real-time PCR was used. The expression levels of Dcpp peaked in the estrus stage (Fig. 2B). The signal declined in the metestrus and diestrus stages. -Actin gene was used as the RNA control in these samples.
Dcpp expression in the mouse oviduct during pregnancy
We further investigated the expression levels of Dcpp in pregnant and pseudopregnant mouse oviducts at d 1–4 of pregnancy by real-time PCR. The level of Dcpp mRNA remained constant in the pregnant mouse oviducts but decreased continuously in the pseudopregnant mouse oviducts (Fig. 2C). The decrease from d 1 reached statistical significance (P < 0.05) on d 4 in the pseudopregnant oviducts, confirming that the embryos could modulate the expression of the Dcpp gene in mouse oviducts.
Localization of Dcpp protein in the mouse tissues
Antibody against Dcpp peptide was raised from rabbits. Western blot analysis showed a specific 17-kDa Dcpp protein from the mouse oviduct (Fig. 3A). This band disappeared when the Dcpp antibody was preabsorbed with the corresponding peptide. Positive staining of Dcpp was found in the epithelial lining of the mouse oviduct (Fig. 3B). The Dcpp immunoreactivity was stronger at estrus (data not shown). This change in immunoreactivity was positively correlated with the mRNA expression levels (Fig. 2B), being highest at estrus and lowest at diestrus. No signal was found when the antibody was preabsorbed with Dcpp peptide (Fig. 2B) and confirmed the specificity of the antibody.
Dcpp gene is hormonally regulated in ovariectomized mice
The hormonal regulation of Dcpp mRNA expression was investigated by using an antiestrogen, ICI 182,780, and an antiprogesterone/glucocorticoid antagonist, RU486. Animals were ovariectomized and injected with vehicle (sesame oil), estrogen, progesterone, or a combination of estrogen and ICI 182,780 or progesterone and RU486. By using ISH, Dcpp mRNA expression was not observed in vehicle-treated ovariectomized animals (Fig. 4). Administration of estrogen-induced Dcpp expression in the mouse oviduct. ICI 182,780 drastically declined estrogen-induced Dcpp mRNA expression in these animals. Progesterone administration slightly increased the expression of Dcpp mRNA, which was suppressed by RU486. Combined treatment of the animals with estrogen and progesterone induced the expression of Dcpp mRNA to a level similar to estrogen alone treatment (Fig. 4). Estrogen or combined estrogen and progesterone treatment strongly induced the expression of Dcpp protein in ovariectomized mice. The effect was abolished when antiestrogen ICI 182,780 was used (Fig. 4). A moderate induction of Dcpp protein was observed when progesterone was used. RU486 antagonizes this effect of progesterone on ovariectomized mice.
Embryotrophic activity of Dcpp
The development of mouse embryos with or without Dcpp treatment is shown in Table 2. The rate of embryo development was based on the number of two-cell embryos after 24 h of culture. The embryos incubated with Dcpp for 4 d had significantly more expanded blastocysts (P < 0.05) than those cultured in medium alone (34 vs. 23%). The size of the expanded blastocyst after Dcpp treatment, as determined by the area of the expanded blastocyst, was also significantly larger than in the untreated group (9472 ± 279 vs. 8409 ± 251, P < 0.05).
Dcpp is present in the developing embryos
Mouse oviductal and uterine fluid were collected and subjected to Western blot analysis using the Dcpp antibody. We found a band of 17 kDa in size, corresponding to the predicted native Dcpp protein in oviductal but not in uterine fluid (Fig. 5A). We also studied the expression and localization of Dcpp proteins in mouse oocytes and embryos (Fig. 5). Mature oocytes were collected from hormonally stimulated mouse ovaries at 2300 h on the day of hCG injection. In vivo developed mouse embryos from three to four cells to blastocyst stages were collected from the oviduct and uterus at various time points. These embryos were subjected to Dcpp immunostaining. Interestingly, immunoreactivity was found in the embryos recovered from the mouse oviducts at the three to four cells, morula, and blastocyst stages but not in the germinal vesicle oocytes or mature oocytes taken from mouse ovaries. The immunoreactivity was abolished when preabsorbed antibody was used. Higher Dcpp immunostaining was found at the morula stage. This suggests that Dcpp was expressed and secreted from mouse oviducts and was taken up by the developing embryo.
Effect of Dcpp antibody on the implantation rate of transferred embryos
Table 3 presents the implantation rate of two-cell embryos after their transfer to the oviduct of the foster mother in medium containing Dcpp, anti-Dcpp antibody, or Dcpp and anti-Dcpp antibody. Treatment with anti-Dcpp antibody resulted in a 46% implantation rate. This was significantly lower (P < 0.05) than the implantation rate of embryos transferred with Dcpp protein (70%). Addition of anti-Dcpp antibody neutralized the beneficial effect of Dcpp on embryo implantation (48 vs. 70%, P < 0.05). This neutralizing effect was found to be specific because the addition of an irrelevant antibody did not have the same effect (data not shown).
Discussion
The oviduct provides an optimal microenvironment for the development of preimplantation embryos. Whereas it is generally accepted that maternal-embryo dialogue is important to provide the best environment for such development, direct evidence demonstrating this concept is lacking. Various oviductal derived factors are known to enhance embryo development in vitro (16, 18, 35), but it remains unclear how their production is regulated or whether the embryo is involved in such regulation. Similarly, we cannot fully explain the mechanisms of the differential transport of oocytes and embryos in rat and hamster oviducts (23, 24).
To address this question, we previously used SSH to compare the gene expression profiles between oviducts containing preimplantation embryos and those containing oocytes in a mouse model and successfully isolated 97 clones that were differentially up-regulated in the embryo-containing oviducts (6). Seventeen were putative secretory products with signal peptides (29). Dcpp is one of these clones. In this article, we provided evidence for its regulation and demonstrated that it might be one of the molecules mediating embryo-maternal dialogue in mice.
The gene coding for Dcpp was first identified from the mouse salivary gland (30) and is highly homologous to the common salivary protein found in mice (89% identity in 128 amino acids) and rats (42% identity in 160 amino acids) (36). The mouse Dcpp gene is located on chromosome 17A3.3. It contains three exons encoding a protein with 150 amino acids. It has a jacalin-like lectin domain. Proteins containing this domain are lectins that may be involved in cell-cell communications, host-pathogen interactions, cancer metastasis, embryogenesis, and tissue development (37). This jacalin-like lectin domain has been found in the animal prostatic spermine-binding protein (38), which is under Hoxb13 regulation for normal ventral prostate morphogenesis and epithelial differentiation (39).
In the present study, Northern blot and cDNA panel analysis showed that Dcpp mRNA was highly expressed in the oviduct, although it is also present in skeletal muscle, lymph nodes, and 17-d-old embryos. ISH confirmed the expression of Dcpp in the oviduct and further located it to the oviductal epithelium throughout the estrus cycle. The maximal expression of Dcpp at the estrus stage suggests that the molecule is under steroid regulation. Indeed, our experiment on ovariectomized mice with or without treatment with estrogen, progesterone, and their antagonists demonstrated that estrogen significantly up-regulated the expression of Dcpp mRNA and protein. This conclusion was confirmed by the abolition of this effect with the use of estrogen antagonist. Estrogen has been suggested to be the most important steroid in modifying the oviductal environment in mammals during late follicular development and estrus (2). How estrogen regulates Dcpp expression is not clear. Our unpublished data show the presence of half estrogen-responsive elements in the promoter region of the Dcpp gene. Whether these elements and/or other transcriptional factors are involved in the regulation of Dcpp expression remains to be investigated.
Progesterone also slightly increased the expression of Dcpp. This effect was nullified by the progesterone/glucocorticoid antagonist, RU486. However, its contribution was low in comparison with that of estrogen. The simultaneous administration of estrogen and progesterone to ovariectomized mice produced the same effect as estrogen alone.
We also demonstrated in this study that the embryos in the reproductive tract modulated the expression of Dcpp. Compared with the pregnant mouse oviducts in the first 4 d of pregnancy, the Dcpp mRNA copy number as determined by real-time PCR continuously decreased in pseudopregnant mouse oviducts containing no embryo, and the difference reached statistical significance (P < 0.05) at d 4 of pregnancy or pseudopregnancy. Because pregnant and pseudopregnant mice have a similar hormonal profile, the ability of pregnant mice to maintain the expression of Dcpp mRNA in the oviduct is highly likely to be due to the presence of developing preimplantation embryos. This is consistent with our previous report on differential gene expression between embryo- and oocyte-containing oviducts (29). The presence of an oocyte-cumulus complex has also been shown to influence the gene expression profile in bovine oviducts in the postovulation period (17).
The embryos and oviducts possess the necessary biochemical machinery for communication, although the detailed mechanism of action is not known. The oviduct produces a number of factors, and many of their corresponding receptors are present in embryos (3). Several reports have demonstrated the beneficial effect of oviductal factors on embryo development. Granulocyte-macrophage colony-stimulating factor (40), leptin (14), and complement protein component 3 (18) are examples of the more recently identified embryotrophic oviductal factors. Their potential receptors have been detected in embryos (14, 18, 41). Recently the estrogen-regulated oviduct-specific glycoprotein has also been reported to enhance the formation of bovine blastocysts (35). However, its receptor in the embryo remains unknown.
There are fewer reports on embryonic factors affecting oviductal physiology. The best-known example is embryo-derived platelet activating factor (42). Its receptor is present in the oviducts of humans (43) and cows (44). Platelet-activating factor has been shown to affect the electrophysiology of human oviducts (45) and increase the intracellular calcium concentration (46) and proliferation (47) of bovine oviductal cells. In mares, embryonic prostaglandin E2 has been proposed as the agent responsible for the differential transport of embryos and oocytes in the oviduct (48). How oviductal embryos modulate the expression of Dcpp remains to be investigated.
This is the first report demonstrating the embryotrophic activity of Dcpp. The beneficial effect of Dcpp on embryo development manifested itself as an increase both in the rate of blastocyst formation and in the size of the resulting blastocysts in culture. These in vitro data were supported by in vivo data showing that simultaneous oviductal transfer of two-cell embryos with Dcpp had a higher implantation rate and lower abortion rate than the transfer of the embryos with anti-Dcpp antibody. Indeed, anti-Dcpp antibody could abolish the effect of Dcpp on implantation and abortion in vivo. The positive influence of Dcpp on implantation may be due to a direct effect of Dcpp on implantation or an indirect effect on improving embryo quality before implantation. Our experiment was unable to distinguish between these possibilities.
Consistent with the embryotrophic activity of Dcpp, immunohistochemistry revealed that Dcpp immunoreactivity was found in embryos retrieved from mouse oviducts but not in oocytes from the ovary. The intensity of the signal peaked at the morula stage and decreased at the blastocyst stage. These data suggest that Dcpp secreted from the oviduct was taken up by the embryos during the transfer in the oviduct. The identity of the Dcpp receptor in the embryos has yet to be determined.
Similar to other oviductal embryotrophic factors (e.g. oviduct-specific glycoprotein and complement protein component 3), Dcpp is unlikely to be crucial to preimplantation embryo development in vivo. This is supported by the inability of anti-Dcpp antibody to inhibit implantation completely and the recent demonstration that mice deficient in oviduct-specific glycoprotein and complement protein component 3 are fertile (49, 50). However, the presence of these factors greatly enhances the development of the preimplantation embryo.
The long-term effect of culturing embryos in medium containing simple salt solution is being vigorously discussed. In animal models, culture conditions affect the frequency of large offspring syndrome in farm animals (51), the expression of the imprinting gene H19 in mice (52), and the behavior of adults derived from cultured embryos (53). Improving culture conditions by supplementing oviductal embryotrophic factors may eliminate or reduce the detrimental effects of suboptimal culture conditions. Several recent studies suggested that there is an increase in imprinting defects in children born after assisted reproduction (54, 55, 56). Whether culture conditions play a role in the generation of these abnormalities is not known.
In conclusion, we have demonstrated that Dcpp is an estrogen-regulated oviductal embryotrophic factor. Mouse oviducts maintain the production of Dcpp in the presence of preimplantation embryos, which subsequently improves embryo development. The study strongly suggests that Dcpp might be an important mediator for preimplantation embryo-maternal dialogue. The potential application of Dcpp in the culture of human embryos deserves further exploration.
Acknowledgments
We are grateful to Mr. K. L. Kwok and Ms. J. F. C. Chow (Department of Obstetrics and Gynaecology, The University of Hong Kong) for their technical assistance. We are also grateful to Dr. D. J. Wilmshurst (the university’s technical writer) for commenting on our draft.
Footnotes
This work was supported in part by the University of Hong Kong’s Committee on Research and Conference Grants (10202566 and 10204873) and the Hong Kong Research Grant Council Grants HKU7327/00M, HKU7436/03M, and HKU7411/04M.
First Published Online October 20, 2005
Abbreviations: CT, Threshold cycle; CZB, Chatot, Ziomek, Bavister; Dcpp, demilune cell and parotid protein; DTT, dithiothreitol; E2, estradiol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hCG, human chorionic gonadotropin; ISH, in situ hybridization; KSOM, potassium simplex optimized medium; SSC, standard saline citrate; SSH, suppression subtraction hybridization.
Accepted for publication October 7, 2005.
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