Journal of Reproduction and Development, Vol. 53, No. 2, 2007

—Full Paper—

Increased Th1/Th2 (IFN-γ/IL-4) Cytokine mRNA Ratio of Rat in the Pregnant Mouse Uterus

Chang-Long NAN1,2), Zi-Li LEI1,2), Zhen-Jun ZHAO1,2), Li-Hong SHI1,2), Ying-Chun OUYANG1), Xiang-Fen SONG1), Qing-Yuan SUN1) and Da-Yuan CHEN1)

1)State Key Laboratory of Reproductive Biology, Institute of Zoology and 2)Graduate School, Chinese Academy of Sciences, #25 Bei-Si-Huan-Xi Lu, Hai Dian, Beijing 100080, P. R. China

Abstract. Somatic cell nuclei can be dedifferentiated in ooplasm from another species, and interspecies cloned embryos can be implanted into the uteri of surrogates. However, no full have been achieved through interspecific mammalian cloning. Rat transferred into mouse uteri provide a unique model for studying the causes of interspecific failure. In this study, intraspecific pregnancy (mouse-mouse) and interspecific pregnancy (rat-mouse) models were established. On Day 9 of pregnancy, the fetoplacental units were separated from the uterine implantation sites and the expression of messenger (m)RNA was quantitated by real- time PCR. We compared the mRNA expression levels of type-1 T helper (Th1) and type-2 T helper (Th2) cytokines, interferon-gamma (IFN-γ), and interleukin-4 (IL-4) in fetoplacental units between intraspecific and interspecific pregnancy groups. The mRNA expression of IFN-γ in the fetoplacental units of the interspecific pregnancy group was significantly higher than that of the intraspecific pregnancy group (P<0.05). The mRNA expression of IL-4 in the interspecific pregnancy group was significantly lower than that in the intraspecific pregnancy group (P<0.05). We also analyzed the ratio of IFN-γ/IL-4 mRNA, and an increased IFN-γ/IL-4 mRNA ratio was observed in the interspecific pregnancy compared with that in the intraspecific pregnancy group. The IFN-γ and IL-4 mRNA expressions indicate that there is a Th1/Th2 imbalance in the feto-maternal interface of interspecific pregnancies. Bias of Th1 cytokine dominance may be a barrier to reproductive success between species. Key words: Interferon (IFN)-γ, Interleukin (IL)-4, Interspecific pregnancy, Mouse, Rat (J. Reprod. Dev. 53: 219–228, 2007)

o far, somatic cell nuclear transfer (SCNT) has explored to produce cloned offspring, and usually succeeded in generating live animals in many includes interspecific somatic cell nuclear transfer species, including sheep, , mice, goats, pigs, and interspecific pregnancy. In previous research, rabbits, , rats, mules, , dogs, and ferrets the oocytes of bovine, sheep, and rabbits have been [1–4]. However, it is impractical to clone used for iSCNT, and interspecific reconstructed endangered species by intraspecific SCNT because embryos have been obtained [5–13]. Interspecific of insufficient numbers of oocytes and surrogate reconstructed embryos can implant into females. So, interspecific SCNT (iSCNT) has been interspecific uteri, but only three speices have been successfully cloned by iSCNT [14–16]. Bovine Accepted for publication: October 14, 2006 Published online: November 29, 2006 ooplasm was capable of reprogramming the Correspondence: D-Y Chen (e-mail: [email protected]) banteng [Bos javanicus, listed as a threatened 220 NAN et al. species by the IUCN Red List (2002)] nucleus to recurrent spontaneous abortion [30, 31], achieve stage embryos, and two preeclampsia [32–34], intrauterine growth pregnancies were established with heartbeats retardation [35], preterm delivery, and premature detectable at day 30, but both pregnancies were rupture of membranes [36, 37]. Shift of the Th1/ subsequently lost [17]. In another study, two Th2 balance to Th1 dominance contributes to the pregnancies were established after iSCNT using failure of pregnancy. domestic sheep (Ovis aries) for recipient ooplasts In order to investigate the importance of the and an exotic argali (Ovis ammon) as a karyoplast Th1/Th2 balance in the failure of interspecific donor, but these two pregnancies were lost by day pregnancy, we transferred rat or mouse blastocysts 59 of gestation [8]. In our laboratory, we have into mouse uteri in this study to produce models of reconstructed approximately 2300 panda intraspecific and interspecific pregnancy. The (Ailuropoda melanoleuca, a highly endangered mRNA expressions of IFN-γ and IL-4 at the feto- species) -rabbit interspecific cloned embryos and maternal interface of intraspecific and interspecific transferred them into the oviducts of 100 recipient pregnancies were quantitated by real-time PCR. cats. We found that panda-rabbit cloned embryos Thus, for the purposes of this study, the ratio of could be implanted into the uteri of a third species, IFN-γ mRNA to IL-4 mRNA was accepted as the but pregnancies were lost after 45 days [10]. The Th1/Th2 ratio [38] in order to determine whether high failure rate of interspecific pregnancies has incorrect modulation of the Th1/Th2 balance become a major obstacle to successful interspecific occurs in interspecific pregnancy. cloning. Therefore, it is necessary to understand the reason for interspecific pregnancy failure. Studies of transfer between rats and mice Materials and Methods have shown that rat embryos can adhere closely to the epithelium of mouse uteri [18], and rat embryos Collection of blastocysts can survive up to the ninth day of pregnancy [19]. Adult females of outbred Kunming mice (5–6 This provides us with a good model for weeks old) and outbred SD rats (10–14 weeks old) investigating the mechanisms of interspecific were superovulated with 10 IU and 40 IU pregnant pregnancy failure. mare serum gonadotropin (PMSG, Tianjin T lymphocytes can be classified as type-1 T Experimental Animal Center, Tianjin, P.R. China), helper (Th1) cells and type-2 T helper (Th2) cells. followed by 10 IU and 40 IU human chorionic Th1 cells produce mainly interleukin (IL)-2, gonadotropin (hCG; Institute of Zoology, CAS, interferon (IFN)-γ, and tumor necrosis factor (TNF)- Beijing, P.R. China) 48 h later, respectively. Then, β, i.e. Th1-type cytokines, and induce cellular the superovulated females were mated with male immunity. Th2 cells synthesize IL-4, IL-5, IL-6, IL- animals of the same species. The females were 10, and IL-13, i.e. Th2-type cytokine, and induce checked the next morning for the presence of a antibody production [20]. During pregnancy, the vaginal plug to confirm successful mating. The first cytokines produced by Th2 cells predominate and morning that a vaginal plug was observed was suppress Th1-type immune reaction. Reversal of designated Day 1 of pregnancy. Day 4 female mice the Th2 cytokines bias, which was referred to as “a and Day 5 female rats were euthanized post-coitus Th2 phenomenon” by Wegmann et al. [21], plays an by cervical dislocation, and embryos were flushed important role in normal pregnancy. Many clinical from the uteri with M2 medium [39]. Mouse and laboratory findings support Wegmann’s blastocysts were transferred into droplets of CZB hypothesis [22–28]. Excessive amounts of Th1 medium [40] and rat blastocysts were transferred cytokines could hamper the immunological into mR1ECM medium [41]. Both were then tolerance of the female to the foreign antigens of the incubated at 37 C in 5% CO2 in a humidified fetus. Increased expression of Th1 cytokines or an chamber before . increased ratio of Th1/Th2 cytokines has been observed at the feto-maternal interface in abortion- Embryo transfer and sample collection prone mice [29], and this occurs not only at the feto- The foster mothers for embryo transfer were maternal interface but also in the peripheral blood Kunming females mated with vasectomized males. in many human pregnancy failures, such as Five or six rat blastocysts in 1–3 µl mR1ECM were TH1/TH2 RATIO IN RAT-MOUSE PREGNANCY 221 surgically transferred into a single uterine horn of ATT-TTC-ATG-3’ (3’ primer); 5’-ACA-GGA-GAA- Day 3 pseudopregnant mice. The same number of GGG-ACG-CCA-T-3’ (5’ primer) and 5’-GAA- mouse blastocysts was transferred into a single GCC-CTA-CAG-ACG-AGC-TCA-3’ (3’ primer); 5’- uterine horn of Day 3 pseudopregnant mice to be CCC-TAA-GGC-CAA-CCG-TGA-A-3’ (5’ primer) used as a control group. The pregnant animals and 5’-CAG-CCT-GGA-TGG-CTA-CGT-ACA-3’ (3’ were enthanized by cervical dislocation on Day 9, primer), respectively. The cycling conditions were and the uterine horns with conceptuses were 94 C for 30 sec, 55 C for 30 sec, and 72 C for 30 sec dissected and isolated. They were opened for 40 cycles with Pyrobest DNA polymerase longitudinally, and the fetoplacental units were (TaKaRa, Ohtsu, Japan). Each of these amplicons separated from the uterine implantation sites, was purified on silica columns (Qiagen, Valencia, immediately frozen in liquid nitrogen, and stored CA, USA). Plasmid standards for each target gene at –80 C for later use. of interest were constructed by cloning a partial cytokine cDNA fragment into a pGEM-T easy RNA extraction and cDNA synthesis plasmid vector (TaKaRa). The exact sequences of Frozen tissues from fetoplacental units were the cloned amplicons were analysed by cycle pulverized in liquid nitrogen. Total RNA sequencing. Concentrations of standard plasmids extractions were performed using TRIzol reagent were measured by UV-spectrophotometry, and (Invitrogen, Carlsbad, CA, USA) according to the copy numbers were calculated as described by Manufacturer’s instructions, and were diluted in Overbergh [42]. RNase-free water. Residual genomic DNA was removed by treatment with 2 units of DNase I Real-time PCR (Promega, Madison, WI, USA) at 37 C for 30 min Total tissue RNA of fetoplacental units was followed by inactivation at 65 C for 10 min. The extracted from 22 mouse-mouse and 22 rat-mouse samples were re-extracted with an equal volume of pregnancies. Then, cDNA was synthesized and TRIzol reagent. The aqueous phase containing the subjected to real-time PCR analysis. IFN-γ and IL-4 RNA was precipitated with isopropyl alcohol, and specific primer sequences were obtained from the RNA was dissolved in RNase-free water. Total another study [42], and the primer sequence for β- RNA was qualified by electrophoresis. Two µg of actin was designed using the Primer Express extracted total RNA was reverse transcribed with computer program (Perkin Elmer Applied M-MuLV reverse transcriptase (New England Biosystems, Foster City, CA, USA). All primer Biolabs, Ipswich, MA, USA) at 42 C for 60 min and sequences were located in two different exons. The incubated at 94 C for 5 min. The cDNA was kept at primer sequences are presented in Table 1. Real- –20 C prior to PCR amplification. time PCR reactions were performed in 96-well optical PCR plates using an ABI Prism 7000 cDNA standards Sequence Detection System (version 1.2) according Lymphocytes were separated from mouse to the manufacturer’s instructions. Reactions were spleens, washed three times with medium RPMI conducted in a 50 µl volume of reaction mix with 1640, and then placed in RPMI 1640 containing 10% 0.5 µM forward and 0.5 µM reverse primers, 3 mM 6 FCS at a concentration of 10 cells/ml in a 6-well MgCl2, 0.2 mM dNTP mixture (2 mM each), 1.5U plate. The splenocytes were stimulated for 24–48 h TaKaRa Ex Taq HS, 1 × PCR buffer and 1 × SYBR for cytokine production with 10 µg/ml Con A Green I (Amresco, Solon, OH, USA). The typical (Sigma Chemical, St. Louis, MO, USA) at 37 C in a protocol for real-time PCR was as follows: initial humidified atmosphere of 5% CO2 . After incubation at 95 C to active modified Taq incubation, the cells were resuspended in TRIzol polymerase followed by 40 cycles of denaturation reagent. Total RNA was extracted according to the at 95 C for 15 sec, annealing at the specific manufacturer’s protocol. Partial cDNA fragments annealing temperature for 15 sec, extension at 72 C of IFN-γ, IL-4, and β-actin were generated by RT- for 30 sec, and plate reading at the specific plate PCR using the primers below. The primer sets used read temperature for 15 sec. Detection of the to amplify IFN-γ, IL-4, and β-actin cDNA standards fluorescent products was carried out at the period were 5’-TCA-AGT-GGC-ATA-GAT-GTG-GAA- of reading plate, and the dissociation curve was GAA-3’ (5’ primer) and 5’-TGG-CTC-TGC-AGG- typically generated post-run using a Sequence 222 NAN et al.

Table 1. Oligonucleotide primer sequences, length of products and amplification conditions

Accession No.a Primer sequences (5’–3’) Product (bp)b Anneal Reading plate forward and reverse temp. (C) temp. (C) β-actin FW:CCCTAAGGCCAACCGTGAA 83 56 76 NM_007393 RV:CAGCCTGGATGGCTACGTACA IFN-γ FW:TCAAGTGGCATAGATGTGGAAGAA 92 55 76 NM_008337 RV:TGGCTCTGCAGGATTTTCATG IL-4 FW:ACAGGAGAAGGGACGCCAT 95 56 76 NM_021283 RV:GAAGCCCTACAGACGAGCTCA

aGenBank accession number of cDNA and the corresponding gene, available at http://www.ncbi.nlm.nih.gov/. bPCR product length in base pairs. FW: forward primer. RV: reverse primer. IL: interleukin. IFN-γ: interferon-γ.

Table 2. Comparison of implantation of transferred rat and mouse embryos into mouse uteri on Day 9 Model of pregnancy No. of No. of recipients * No. (%) of No. (%) of transferred embryos pregnant recipients implantation sites

Mouse-mouse pregnancy 274 47 35 (74.5)a 186 (67.9)a Rat-mouse pregnancy 433 76 32 (42.1)b 141 (32.6)b Values with different superscripts within each column are significantly different (P<0.05). *Five or six blastocysts were transferred into a single uterine horn of each mouse recipient.

Detection System. The threshold cycle (Ct) value was conducted using Chi-square analysis. As the for each sample was recorded by the sequence mRNA expression of IFN-γ and IL-4 did not have a detector. normal distribution (even after logarithmic Standard curves were generated using 10-fold transformation), differences in transcript levels serial dilutions (covering 103 to 108 copies) of cDNA between the intraspecies and interspecies standard plasmids for each gene. The Ct values are pregnancy groups were analyzed by Mann- inversely proportional to the log of the copy Whitney U test. Group data is expressed as the number on the standard curves. The Ct values for mean ± SD. A probability of less than 5% was the IFN-γ, IL-4 and β-actin transcripts from considered to be statistically significant. fetoplacental specimens were plotted on standard curves, and the amounts (copies) of each transcript were calculated. The amounts of the IFN-γ and IL-4 Results transcripts (copies) expressed relative to that of β- actin (copies) were used to compare the IFN-γ and Implantation of mouse or rat embryos in mouse uteri IL-4 mRNA level in the different groups. Each A total of 433 rat blastocysts were transferred sample obtained from the same mouse was tested into 76 Kunming mouse recipients, and 32 in duplicate, and the average was used in this pregnancies (42.1%) were detected after 9 days of study. The ratios of IFN-γ/IL-4 mRNA were gestation (Table 2). There was a significantly lower analyzed to investigate the differences in Th1/Th2 pregnancy rate in the rat-mouse pregnancy group cytokine mRNA between the mouse-mouse than in the mouse-mouse pregnancy group (74.5%). intraspecific and rat-mouse interspecific Moreover, 32.6% of the rat embryos implanted into pregnancies. mouse uteri, and this was lower than that of mouse- mouse intraspecific implantation (67.9%). By Day Statistical analysis 9, the transferred rat embryos in the mouse uteri Comparision of the pregnancy and implantation had a normal appearances as compared with the percentage data of the mouse-mouse intraspecific mouse embryos in the mouse uteri, but the volume and rat-mouse interspecific pregnancies on Day 9 of the fetoplacental units in the rat-mouse TH1/TH2 RATIO IN RAT-MOUSE PREGNANCY 223

Fig. 1. The development of mouse-mouse intraspecific and rat-mouse interspecific pregnancies on Day 9. a) Mouse embryos in a mouse uterus on Day 9 of pregnancy. b) Rat embryos in a mouse uterus on Day 9 of pregnancy. Intraspecific and interspecific pregnancies were established by transfer of six blastocysts into one uterine horn of each mouse, and five conceptuses were found in both implanted horns.

Fig. 2. Fetoplacental units isolated from uterine implantation sites on Day 9. a) Mouse- mouse pregnancy. b) Rat-mouse pregnancy. Bar=1 mm. pregnancy group was smaller than that of the levels of IFN-γ mRNA expression were significantly mouse-mouse pregnancy group (Figs. 1 and 2). elevated in the fetoplacental units of the interspecific pregnancy group in comparision to Expression of IFN-γ and IL-4 mRNA in fetoplacental those of the intraspecific pregnancy group (P<0.05) units (Fig. 3). However, the levels of IL-4 mRNA IFN-γ and IL-4 mRNA were detected in all tissue expression were significantly lower in the samples, and the amounts of IFN-γ and IL-4 mRNA fetoplacental units of the interspecific pregnancy transcripts were determined by real-time group than in the intraspecific pregnancy group quantitative PCR. The results revealed that the after being normalized to β-actin (P<0.05) (Fig. 4). 224 NAN et al.

Fig. 3. IFN-γ mRNA expression in the fetoplacental units of Fig. 4. IL-4 mRNA expression in the fetoplacental units of mouse-mouse intraspecific and rat-mouse mouse-mouse intraspecific and rat-mouse interspecific pregnancies on Day 9 of pregnancy. M- interspecific pregnancy on Day 9 of pregnancies. M- M represents mouse-mouse intraspecific pregnancy M represents mouse-mouse intraspecific pregnancy (n=22). R-M represents rat-mouse interspecific (n=22). R-M represents rat-mouse interspecific pregnancy (n=22). RNA was isolated from pregnancy (n=22). RNA was isolated from fetoplacental units. IFN-γ and β-actin mRNA fetoplacental units. IL-4 and β-actin mRNA expression levels were determined by quantitative expression levels were determined by quantitative RT-PCR. IL-4 expression levels were adjusted to those RT-PCR. IFN-γ expression levels were adjusted to of β-actin. those of β-actin.

Table 3. Ratio of Th1/Th2 mRNA in the fetoplacental units of the intraspecific and interspecific pregnancy groups on Day 9

No. IFN-γ/β-actin IL-4/β-actin IFN-γ/IL-4* (× 10–3)(× 10–3)

Mouse-mouse intraspecific pregnancy 22 1.81 ± 0.59a 0.89 ± 0.57a 2.76 ± 1.76a Rat-mouse interspecific pregnancy 22 2.43 ± 0.72b 0.50 ± 0.32b 8.87 ± 8.22b Values with different superscripts within each column are significantly different (P<0.05). *Values for the IFN-γ/IL-4 ratio were the expressions of the IFN-γ and IL-4 transcripts after normalization to β-actin.

Th1/Th2 mRNA ratio in the intraspecific and another species has been performed in basic studies interspecific pregnancy groups on the development of interspecific gestation [43] The mean level of IFN-γ/IL-4 mRNA ratios in the and in animal conservation programs [44] where mouse-mouse pregnancy group (2.76 ± 1.76) was there is a lack of suitable numbers of recipients. significantly lower than that in the rat-mouse The vast majority of interspecific embryo transfers pregnancy group (8.87 ± 8.22) (Table 3). have been unsuccessful, and although a few pregnancies have been achieved in previous cases, most do not develop to term. In this study, rat- Discussion mouse interspecific pregnancies were established successfully, but the mouse-mouse intraspecific Embryo transfer of one species to the uteri of pregnancy group had significantly higher rates of TH1/TH2 RATIO IN RAT-MOUSE PREGNANCY 225 pregnancy and implantation than the rat-mouse Previously, a model of interspecific pregnancy interspecific pregnancy group. was established using embryo transfer between rats The mechanisms that enable or prevent a and mice. Tarkowski [18] found that the upper successful outcome in interspecific pregnancies are time limit for the survival of rat blastocysts in far from clear. Inappropriate maternal immune mouse uteri was the seventh day of pregnancy. rejection of the fetus has been considered the cause Later, Dai et al. [19] found the upper time limit for of failure of many interspecific pregnancies [45–47]. the survival of rat blastocysts in mouse uteri was Using a rodent interspecific pregnancy model the ninth day of pregnancy when using different established by embryo transfer between M. culture media and lineages of rats and mice. The musculus and M. caroli, Clark et al. [48] explained histological structures of the transferred rat that the failure of immunocompetent M. musculus embryos were intact on Day 9, but a large amount females to support the development of M. caroli of blood appeared in deciduas, which were porotic, embryos was due to the presence of activated on Day 10. In mice, the changes in IFN-γ released natural killer (NK) cells that selectively by the cells at the feto-maternal interface were discriminate against M. caroli embryos early in normal during gestation, with the maximal level on pregnancy. In mice, NK cell-like granulated Day 6 and declining levels on Days 12 and 18. The metrial gland cells in the uterus are a subset of ratio of IL-4/IFN-γ dramatically increased after lymphocytes belonging to the NK cell lineage [49], Day 6 [22], and a Th2-like bias of the maternal and IFN-γ activates NK cells and enhances immune response occurred in normal mouse cytotoxic activity [50, 51]. The Th1-type cytokine pregnancies after Day 6. In this study, we used the IFN-γ also induces apoptosis of [52], same rats and mice lineages described in the abortion [53], and fetal resorption [24]. In this previous research [19] to establish intraspecific and study, increased IFN-γ mRNA expression was interspecific pregnancy models, keeping in mind observed in the interspecific pregnancy group that Th2 bias occurs in normal pregnancies and that compared with the intraspecific pregnancy group, the structure of embryos remains intact in rat- and this may be harmful to implantation and mouse pregnancies on Day 9, the midpoint development of transferred embryos. between Days 6 and 12, which is in agreement with IL-4 is involved in many events that occurs in the the observation of Th1/Th2 balance alternation in feto-maternal interface. IL-4 secreted by intraspecific and interspecific pregnancies. -infiltrating lymphocytes stimulates Therefore, Day 9 was selected for cytokine production of leukemia inhibitory factor in determination. endometrial tissue [54], which plays a crucial role Wegmann et al. [21] proposed an immunotrophic in implantation and in several species hypothesis suggesting that during physiological [55]. Soluble IL-4 enhances -induced matrix pregnancy, the Th1/Th2 activity balance is strongly metalloproteinase (MMP)-1 and inhibits MMP-9 shifted toward Th2 activity (the so called “Th2 production at both the protein and mRNA levels phenomenon”). The normal shift toward Th2 [56]. MMPs are key molecules involved in the dominance in pregnancy has been postulated to invasion that degrades the extracellular matrix [57]. play a crucial role in protecting the fetus from IL-4 promotes hCG release in trophoblasts [58], and rejection and in maintenance of normal pregnancy, the released hCG induces production of indicating that the maternal immune system progesterone from the corpus luteum, which redirects maternal immunity away from cell- promotes prodution of Th2 cytokines [59], inhibits mediated immunity towards humoral NK activity, and displays an anti-abortive effect responsiveness. It has been found that Th2-type [60]. In this study, IL-4 mRNA expression in the cytokines produced by maternal cells are fetoplacental units of the interspecific pregnancy predominant at the feto-maternal interface during group was found to be significantly lower than that normal pregnancy in mice [22] and humans [33]. of the intraspecific pregnancy group. This result Excessive Th1 cytokine production, on the other suggests that insufficient production of IL-4 may hand, has been shown to evoke rejection responses have a deleterious effect on interspecific pregnancy directed against fetoplacenal semiallografts [61]. and may weaken the developmental competence of Th1/Th2 imbalance has been found in association rat embryos in mouse uteri. with multiple failures of recurrent spontaneous 226 NAN et al. abortions [30, 31], preeclampsia [32–34], pregnancies in this study, and the IFN-γ/IL-4 intrauterine growth retardation [35], preterm mRNA ratio increased compared with that of delivery, and premature rupture of membranes [36, intraspecific pregnancies. The IFN-γ/IL-4 mRNA 37]. In this study, a significant increase in the Th1/ ratio indicates that there is a Th1/Th2 imbalance in Th2 ratio was observed in the interspecific the feto-maternal interface of rat-mouse pregnancy group when compared to the pregnancies. Th1/Th2 bias to Th1 dominance may intraspecific pregnancy group, suggesting a bias be a barrier to reproductive success between state for Th1/Th2 in interspecific pregnancies. This species. result is in agreement with a previous study on interspecific gestation of horses and donkeys [62]. Failure to down-regulate cell-mediated cytotoxicity Acknowledgements to interspecific paternal antigens was observed in pregnancies obtained using horses and donkeys, We are grateful to Drs. Jun-Cheng Huang, Yan suggesting a incompetent shift of immune Jiang, Yi-Liang Miao and Li-Ying Yan for their reactivity away from cellular and toward humoral technical assistance. This study were supported by immunity during interspecific pregnancies, the Climbing Project of the Ministry of Science and although a Th1/Th2 ratio was not provided. Technology of China and the Knowledge In conclusion, increased IFN-γ mRNA expression Innovation Project of the Chinese Academy of and decreased IL-4 mRNA expression were Sciences. observed in the fetoplacental units of rat-mouse

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