Molecular Human Reproduction, Vol. 6, No. 12, 1099-1105,
December 2000
© 2000 European Society of Human Reproduction and Embryology
Uterine physiology |
In-vivo transfection of the female reproductive tract epithelium
Centro de Investigaciones Biológicas, CSIC, Velázquez 144, 28006 Madrid, Spain
Abstract
Mouse female genital tract was transfected in vivo using the ß-galactosidase reporter gene. To transfect the female tract, DNA/liposome complexes were injected through the infundibulum of the oviducts of adult, immature, and pseudopregnant females. Females which were in different stages of the ovarian cycle were also employed. Transfection was analysed using histochemical, immunological and molecular (Southern blotting, polymerase chain reaction and gene sequencing) procedures. The lower region of the uterine glands and the oviduct epithelium in the isthmus and juncture regions were the most conspicuous transfected areas. The greatest numbers of transfected cells were 6% in the oviduct and 9% in the uterus, meanwhile the duration of expression reached a maximum of 7 days in the oviduct and 14 days in the uterus. The hormonal stage of the genital tract epithelium directly affected transfection, as the highest number of successful transfections occurred during the meta-oestrus and pseudopregnancy stages.
female genital tract/gene transfer/oviduct transfection/uterus
Introduction
The first transfection experiments were undertaken in the early 1960s, when different authors demonstrated that cultured cells could gain radioactively labelled DNA (Wolff and Lederberg, 1994
). From then on, the transfection of different cells and tissues has become widespread. Transfection experiments have been done in cultured cells, but in recent years in-vivo transfection has also been developed (Wolff and Lederberg, 1994; Anderson, 1998) as an important procedure to analyse both gene expression (Scherule and Cheng, 1996) and gene regulation (Kitsis et al., 1991; Danko et al., 1997), as well as for gene therapy purposes (Nabel et al., 1993; Anderson, 1998).
Different approaches have been used to deliver foreign genes into animal cells in vivo (Anderson, 1998). Although most of the research on gene transfer has used recombinant viruses, non-viral vectors have also been used for in-vivo transfection (Scherule and Cheng, 1996). Non-viral methods employ DNA carriers consisting of lipids, proteins, peptides or polymeric matrixes as well as ligands capable of directing the DNA complexes to some cell-surface receptors on the target cell (Scherule and Cheng, 1996). Non-viral systems have been successfully used in vivo to deliver genes to lung (Fortunati et al., 1996), muscle (Wolff et al., 1990; Morishita et al., 1995), liver (Wilson et al., 1992), endothelial (Cuevas et al., 1996) or tumour cells (Nabel et al., 1993).
Transfection of the female genital tract is a major goal in reproductive biology and medicine and would allow the expression of proteins which control different aspects of the reproductive process. On the other hand, transfection of the uterine epithelium could be employed to deliver anti-proliferative signals and provide a sound basis for future clinical trials of gene therapy applied to uterine cancer and other pathological disorders. In-vitro transfection of primary cultures of human endometrial cells has been widely reported, using non-viral methods, e.g. calcium phosphate (Brosens et al., 1999; Gao et al., 1999) or liposomes (Charnock-Jones et al., 1997). In-vivo transfections of the mammalian reproductive tract have been reported in the mouse uterus (Charnock-Jones et al., 1997) and in the oviduct (Relloso and Esponda, 1998). In both cases, the ß-galactosidase reporter gene complexed to cationic liposomes was used, and the expression was observed in uterine gland cells and in the oviduct isthmus and juncture epithelium. In these studies on oviduct and uterus transfection, the capacity of oviductal and uterine cells to be transfected was reported (Charnock-Jones et al., 1997; Relloso and Esponda, 1998), while in the present report, the effectiveness of transfection is analysed and the influence of the stages of the oestrus cycle, sexual maturity and pseudopregnant stage on transfection efficiency is evaluated.
Materials and methods
Animals
Mice (CD1 strain) were maintained at constant temperature in a 12 h light:12 h dark cycle and with food and water ad libitum. Immature (22 days old) and mature (45 days old) females were used, and the stages of the ovarian cycle were determined by vaginal smears (Allen, 1922; Rugh, 1990). Pseudopregnant females were produced after mating normal females with vasectomized males.
The animal protocol used was in accordance with the law 223/88 on Animal Protection of Spain, and the European Union Agreement about Vertebrate Animal Protection (3/18/1986). The work was in accordance with the NIH guidelines for the care and use of laboratory animals and the CSIC ethical committee.
Reporter plasmid and liposome preparation
The reporter plasmid construction used was pCMV-ß (Clontech, Palo Alto, CA, USA), which expresses the Escherichia coli ß-galactosidase enzyme under the control of the cytomegalovirus early promoter. In order to produce the DNA/liposome complexes, 100 ng of DNA and 2 µg of Lipofectamine (Gibco BRL, Gaithersburg, MD, USA) were mixed in 20 µl of phosphate-buffered saline (PBS) and left for 15 min at room temperature before use.
In-vivo gene transfer
Adult, immature and pseudopregnant females were injected with DNA/liposome complexes in the left oviduct using a thin microcapillar (Rafferty, 1970; Dickmann, 1971). 20 µl of the DNA/liposomes solution was injected into the infundibular region of the left oviduct. In order to study the efficiency of transfection, animals were killed at different time periods (from 2 days to several weeks) and the female reproductive tract was isolated and used for ß-gal localization. To analyse the influence of the ovarian cycle in the DNA/liposome complexes uptake, adult female mice and pseudopregnant females were injected at the different stages of the ovarian cycle. The oviducts and uteri of these animals were isolated and assayed for ß-gal expression, 2 days after the injection.
Histochemical and immunocytochemical localization of ß-gal
Uteri and oviducts were isolated and fixed for 15 min in a solution of 1% glutaraldehyde diluted in PBS. After fixation, tissue blocks were rinsed and treated for 15–20 h at room temperature with the X-gal staining solution diluted in Tris-buffered saline (TBS) as previously reported (Charnock-Jones et al., 1997). After several washings, the X-gal stained samples were sectioned at 10 µm using a cryostat. In several cases, serial sections (10 µm) were used and then the complete female tract organs were viewed under the microscope. Sections were stained with neutral red solution, mounted and observed under bright field microscopy. Controls consisting of uteri and oviducts from animals injected with the liposome solution, with PBS only, or from non-injected animals, were also treated with the X-gal staining assay.
To estimate the transfection efficiency, 10 sections were analysed from each sample. In the oviducts, the ß-gal positive and negative cells were counted in several fields randomly selected from each section. A total of 2000–5000 cells were checked. In the uteri, 250–500 positive and negative uterine glands were analysed in several fields randomly selected from each sample.
For immunocytochemistry, some uteri and oviducts from treated and control animals were fixed for 1 h in PBS containing 1% paraformaldehyde, and then washed in PBS. Samples were sectioned at 10 µm using a cryostat and sections were treated with a blocking solution of 0.5% skimmed milk in TBS, and then incubated with a 1:100 (v/v) dilution of the anti-ß-gal rabbit immunoglobulin G (IgG; Clontech) in blocking solution overnight at 4°C. After several washes in 0.2% Tween 20 in TBS, a fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (Sigma, St Louis, MO, USA) diluted 1:500 in the blocking solution was used for 1 h at room tempereature. Slides were mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA) to avoid quenching the fluorescence, and analysed using epifluorescence microscopy.
Immunoprecipitation and Western blotting
Oviducts and uteri were homogenized under liquid N2. Proteins were pooled and immunoprecipitated with 2 µl of anti-ß-gal rabbit IgG (Clontech) and 50 µl of Sepharose-conjugated G protein (Pharmacia Biotech, Europe). Samples were loaded onto 6% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) gels and transferred to nitrocellulose filters. Filters were blocked in a solution of TBS containing 3% bovine serum albumin (BSA), 5% skimmed milk and 0.5% Tween 20, and then incubated with a monoclonal anti-ß-gal antibody (Promega, Madison, WI, USA) diluted 1:1000 in TBS. After several washings, filters were treated with an anti-IgG mouse peroxidase conjugated (Sigma), and developed by the SuperSignal Chemiluminiscent Substrate (Pierce, Rockford, IL, USA). Control samples were protein extracts from oviducts and uteri of non-treated animals.
Southern blotting, PCR and DNA sequencing
Episomal and genomic DNA was extracted from tissues of treated and control animals, 3 days after the injection of the DNA/liposomes solution into the oviduct using a previously described procedure (Wilson et al., 1992). DNA samples were digested with EcoRI and transferred to nitrocellulose filters. A [32P]-labelled DNA fragment from the ß-gal gene was used as a probe for the Southern blots.
Episomal DNA extracted from uteri and oviducts of treated and control animals was used for PCR amplification. Two specific primers from the ß-gal gene were used: the forward primer was 5'-GCGAAGAGGCCCGCACCGTC-3' and the reverse primer was 5'-CAGTACAGCGCGGCTGAAAT-3'. The thermocycler programme was 5 min/94°C followed by 30 cycles of 1 min at 94°C, 1 min at 52°C, and 1 min 30 s at 72°C, with the reaction ending with a 7 min incubation at 72°C. The amplified fragments of the reaction of pCMV-ß plasmid and episomal DNA of uteri from treated animals were run in a 1% agarose gel. Later, the products of the reaction were excised out of the gel and purified using the QIA-quick Gel Extraction Kit (Qiagen, Hilden, Germany). The DNA sequence was determined using the dideoxy chain termination method (Sanger et al., 1997). The sequencing reactions were analysed using a 377 automated DNA sequencer (Applied Biosystems Inc, Foster City, CA, USA) and sequences were aligned on-line employing the Genetics Computer Group software package.
Results
Localization of ß-gal gene expression
Histochemical detection using the ß-gal enzymatic reaction showed that transfection and reporter gene expression had occurred. In the oviduct, some epithelial cells from treated animals presented the characteristic blue staining of the X-gal reaction (Figures 1A,B). Some glandular cells in the uterus also showed the same characteristics (Figure 1D,E). The transfected cells in the oviduct epithelium usually appeared in randomly distributed groups (Figure 1A) and the muscular layer did not show ß-gal expression (Figure 1A,B). The oviductal isthmus and juncture and the bottom of the uterine glands were the regions in which the highest number of transfected cells appeared. Transfected cells reached a maximum of ~6% in the oviduct epithelium and ~9% in the uterine glands. In both oviductal and uterine epithelial cells, the reaction was uniformly distributed in the cytoplasm, and the nuclei were negative. On the other hand, uterine cervix and vaginal epithelium did not show any ß-gal expression. Control preparations did not show ß-gal staining either in the oviduct epithelium (Figure 1C) or in the uterine glandular cells (Figure 1F).
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Immunohistochemistry of samples from treated animals produced a positive reaction for the ß-gal enzyme. The yellow–green fluorescence indicates the presence of the reporter gene expression in the oviductal epithelium (Figure 1 G,H
) and in the glandular cells of uterus (Figure 1 K,L) with a similar location to that observed with the X-gal enzymatic reaction. The antibody reaction revealed that ß-gal expression was homogeneously distributed in the cytoplasm while nuclei were in all cases negatively stained (Figure 1 H,L). Control preparations did not show fluorescence (Figure 1 I, J, M and N).
Immunohistochemistry and histochemistry results were confirmed by Western blot analyses which showed that a positive 115 kDa band corresponding to ß-gal appeared in protein extracts from the oviducts and uteri of treated animals, while no positive reaction was observed when protein extracts from non-treated animals were employed (Figure 2 A,B).
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DNA studies
Southern blot analysis indicated that the DNA transfected was detected in the episomal fraction from treated animals (Figure 2 C,D
). This analysis did not rule out the existence of randomly integrated transgenes undetectable by this assay (data not shown). Non-injected females did not show hybridization signal either in the oviductal or in the uterine DNA extracts (Figure 2 C,D). Furthermore the PCR technique used to detect the foreign DNA showed that the ß-gal gene appeared in the episomal fraction of the oviducts and uteri from treated animals (Figure 1E). PCR and Southern blotting from the genomic DNA fraction did not show a positive reaction. Comparison of the sequences of the 580 bp PCR fragment from treated uteri with that of the pCMV-ß plasmid showed a 99% identity.
Effect of age and oestrous cycle on reporter gene expression
To determine the duration of the reporter gene expression, the ß-gal expression at different times after DNA/liposome complexes injection in the oviduct was analysed. ß-gal expression was detected for up to 7 days in the oviduct and for up to 14 days in the uterus of adult female mice. Moreover, in all cases more animals expressed the transfected gene in the uterus than in the oviduct (Table I). The immature females showed similar percentages of ß-gal positive animals for both uterine and oviductal transgene expression, and the duration of the expression was similar to that of adult groups (Table II). The pseudopregnant group showed the highest number of animals positive for ß-gal expression in the oviduct (Tables III and IV), but in this case the duration of expression was only for up to 4 days (Table III). With regard to the effect of ovarian cycle on ß-gal gene expression, the highest number of positive animals for both the uterus and oviduct appeared during meta-oestrus and pseudopregnant stages (Table IV).
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Discussion
Methods of transfecting cells and tissues have been developed over the last few years (Wolff and Lederberg, 1994). Gene transfer into somatic cells represents a useful tool for the study of gene function, as well as a way to test promoter activity and gene fusion expression. In-vivo transfection may be used to validate gene constructs before the production of transgenic animals or their use in human gene therapy (Nabel et al., 1993; Anderson, 1998). In-vivo transfection of the female reproductive tract has been reported in the uterine epithelium, using an injection of DNA/liposome complexes into the base of the uterine horn (Charnock-Jones et al., 1997) and in the oviductal epithelium, with an injection through the infundibulum (Relloso and Esponda, 1998). In both reports, only an analysis of the ß-gal expression using the X-gal staining reaction was done. Our results show a preferential expression of the transfected gene in the isthmus and/or juncture regions of the oviduct and in the lower part of the uterine glands. The reason for this preferential expression of transfected gene is still not clear. Several authors have suggested different hypotheses that would influence the expression of the exogenous gene. Important factors could include the stability of the reporter protein (Charnock-Jones et al., 1997) in different cells, the promoter activity depending of the cell type (Scherule and Cheng, 1996; Charnock-Jones et al., 1997) and the accessibility of the complexes to the transfected areas (Danko et al., 1997). Nevertheless, the transfected areas in the oviduct and the uterus are scarce, with low percentages of transfected cells (~6% in the oviduct epithelium and ~9% in the uterine glands). In the case of uterine transfection, when an injection of DNA/liposome complexes into the base of the uterine horn was employed, ~70% of cells appeared transfected (Charnock-Jones et al., 1997). Differences between those results and the present report are probably because the amount of DNA/liposome complexes which comes in contact with the cells would be higher when the injection is done in the uterine horn than when the gene/liposome complexes are introduced through the oviduct. In this regard, it is important to note that the utero–tubal junction possesses a very narrow lumen (Hunter, 1988) and would be an obstacle for the diffusion of the complexes to the uterus. Thus, in our case the quantity of DNA/liposome complexes that reach the uterus would be smaller than when they are deposited directly into the uterine lumen.
Different experiments have demonstrated that the percentage of in-vivo transfected cells is very variable. Low percentages of transfected cells have been reported in different tissues such as respiratory epithelial cells (Grubb et al., 1994; Katkin et al., 1995), or muscle (Acsadi et al., 1991) where a variable percentage of cells (1–30%) expressed the foreign DNA. In any case, the overall efficiency of gene transfer and expression in mammals is within a low range. Some authors have claimed that it could be the result of an evolutionary process of the organisms which have developed systems to protect themselves from viral aggression (Anderson, 1998). This protection would act against the incorporation and expression of foreign DNA into their cells. For this reason, the transfection by either viral or non-viral vectors could lead to a low transfection efficiency.
Southern blotting and PCR studies have determined that transfected DNA can be detected in the episomal fraction in both the uterus and oviduct, although these analyses do not rule out the existence of small amounts of integrated DNA which would be undetectable by these assays. In other tissues, e.g. muscle (Wolff et al., 1992) or liver (Wilson et al., 1992), the transfected DNA was also detected in the episomal fraction and the integrated transgene was never detected in the host cell chromosome. Since the transfected gene only appears as an episomal form, the transfected DNA would be eliminated from the cell in the successive mitotic processes, concomitantly with the decrease of gene product. This may explain the transient expression of the transfected genes in all cases studied (Wolff et al., 1992; Ledley and Ledley, 1994; Scherule and Cheng, 1996).
In the present report, we have analysed the kinetics of expression of the transfected gene in the uterus and oviduct. We have used immature, mature and pseudopregnant females. The longest exogenous DNA expression was observed in immature and adult animals (7 days in oviduct and 14 in uterus). The pseudopregnant group showed the shortest length of expression (4 days). In general, the duration of transgene expression in mammals after transfection procedures is disappointingly low. For example in the thyroid, the exogenous gene expression drops within 40 h (Sikes et al., 1994). A similar pattern of elimination of the DNA and short expression of the gene product has been observed in synovial tissue after intra-articular administration of plasmid DNA (Yovandich et al., 1995). After i.v. administration of cationic lipids, gene expression was observed in the lung for 7–21 days (Ledley, 1995). Several factors that would affect the duration of transgene expression have been suggested, such as various intrinsic kinetic processes, e.g. the distribution and extracellular half-life of DNA, the efficiency of DNA uptake into cells, the rate of degradation of DNA within the cell, the rate of transcription, the stability of the mRNA and the rate of translation (Ledley and Ledley, 1994). Other factors include the attenuation of gene expression by methylation of foreign sequences or by the influence of lymphokines and cytokines on the exogenous promoter (Paillard, 1997; Anderson, 1998). However, the main mechanism seems to be cell death, either because the reporter protein is toxic or because the immune system recognizes and eliminates the foreign gene products together with the cells expressing them (Danko et al., 1997; Anderson, 1998).
Regarding the influence of the oestrous cycle in the transfection of the female tract, our results clearly show that the hormonal stage of epithelium affects transfection. The highest number of ß-gal positive animals were found in meta-oestrus and pseudopregnant groups, in which the hormonal stage (high concentrations of progesterone and lower concentrations of oestradiol) and epithelial conditions are similar. The lowest percentage of ß-gal-positive animals occurred in immature, di-oestrus, pro-oestrus or oestrus groups. These differences seem to be undoubtedly related to the stage of the female tract epithelium produced by the effect of progesterone. During pseudopregnancy and meta-oestrus, oviduct epithelium is well-developed, with deciliation processes and a low level of secretion occurring (Hunter, 1988; Croxatto and Villalón, 1995). On the other hand, the transfection was quite unsuccessful in young animals and during di-oestrus in which the oviduct epithelium was inactive or growing (Brenner and Maslar, 1988; Kim-Björklund et al., 1991), as well as in pro-oestrus or oestrus in which the lumen is full of secretions (Hunter, 1988; Croxatto and Villalón, 1995). The same situation occurs for the uterine epithelium, because when the uterine epithelium is mature and the diameter of the gland is greatest (as occurs during meta-oestrus and in pseudopregnant females; Brenner and Maslar, 1988; Kim-Börklund et al., 1991), transfection was more efficient. Nevertheless, in young animals or during di-oestrus when the epithelium is inactive, and during pro-oestrus and oestrus in which a growing process occurs, transfection is quite unsuccessful.
In summary, these results show that the ovarian cycle is involved in the level of expression through the changes occurring in the female tract epithelium. Thus, transfection is more efficient when the epithelium is developed and the accessibility conditions favour the contact between the DNA/liposome complexes and the cells (i.e. low secretion in the oviduct and wide gland diameter in the endometrium).
Acknowledgments
The authors thank C.Bernabeu, M.L.Botella, R.Carballada, A.Corbí and A.González Díaz, (Centro de Investigaciones Biológicas, CSIC) for their help and advice. This work was partially supported by Grant PB96-0808 from DGICYT (Spain).
Notes
1 To whom correspondence should be addressed: E-mail: esponda{at}cib.csic.es 
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Submitted on May 17, 2000; accepted on September 18, 2000.
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