Veterinary Research Communications, 31(Suppl. 1) (2007) 205–207 DOI: 10.1007/s11259-007-0095-3
# Springer 2007
Fetal Microchimerism in Normal and Embryo Transfer Bovine Pregnancies L. Turin1,*, G. Tribbioli1, P. Invernizzi2, F.R. Grati2,3, S. Crema1, G. Laible4 and F. Riva1 1 Dipartimento di Patologia Animale, Igiene e Sanita` Pubblica Veterinaria, Universita` di Milano, Via Celoria 10, 20133, Milano, Italy; 2Dipartimento di Medicina, Chirurgia e Odontoiatria, Universita` di Milano, Milano, Italy; 3TOMA, Advanced Biomedical Assays S.p.A., Busto Arsizio, (VA), Italy; 4AgResearch Ruakura Research Centre, Hamilton, New Zealand * Correspondence: E-mail:
[email protected]
Turin, L., Tribbioli, G., Invernizzi, P., Grati, F.R., Crema, S., Laible, G. and Riva, F., 2007. Fetal microchimerism in normal and embryo transfer bovine pregnancies. Veterinary Research Communications, 31(Suppl. 1), 205–207 Keywords: bovine, microchimerism, Real-Time PCR, transgenic
INTRODUCTION Microchimerism indicates the presence, within one individual, of a small population of cells or DNA from another genetically distinct individual. The phenomenon can occur in humans as a result of the bi-directional exchange of cells between fetus and mother during pregnancy. In the human species, fetal DNA has been detected in the maternal circulation from early pregnancy to years after delivery and has been correlated to some auto-immune disorders such as systemic sclerosis and Hashimoto_s thyroiditis (Bianchi et al., 1996; Invernizzi et al., 2002). In contrast to the highly invasive hemocorial placenta of primates, which allows direct contact of fetal and maternal blood, the epitheliochorial placenta of ruminants includes multiple layers, which separate the two vascular systems. Nevertheless, mother-to-fetus transplacental DNA leakage in cattle has been reported in nuclear transfer pregnancies (Hiendleder et al., 2004). Indeed, placental development failures have been reported for nuclear transfer pregnancies, which may explain the passage of cells form the mother to the fetus (Hill et al., 2000). The potential impact of microchimerism in cattle has to be considered in the context of the key role that they play in the human food chain. Cattle represent an attractive target for biotechnology applications such as transgenesis, with the aim of using and improving their production characteristics, such as disease resistance or biopharming production (Niemann and Kues, 2003; Brophy et al., 2003). The generation of transgenic cattle lines involves often the in vitro production of embryos, which are subsequently transferred into synchronized recipient females for development to term. Transplacental passage of cells from a transgenic fetus to the cow would have implications on the use of recipients as food animals for human consumption. Although this is not a major issue for small scale research projects, where transgenic animals are physically contained and cannot enter
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the human food chain, it might become a significant financial hurdle once such biotechnology will be applied on a larger scale. Our group was the first to initiate an investigation on the feto-maternal passage of DNA during bovine pregnancy. We detected fetally derived DNA sequences in the maternal blood of cows at different time-points during and after pregnancy by means of Real-Time PCR assay. Preliminary analysis of maternal tissues indicates that persistent fetally derived DNA might originate from cell populations engrafted into maternal tissues.
MATERIALS AND METHODS The study was based on two groups of animals, naturally mated heifers at their first male pregnancy and conventional recipient cows pregnant with in vitro produced transgenic embryos for the chimeric "/.-casein (CSN2/3) (Brophy et al., 2003). All the cows in the study were analyzed (blood) at mid-gestation, (d150), pre-calving (d260), at calving (within 2 days) and 4 months post-calving. Mothers of transgenics were also tested (blood) 26 months after delivery and two of them were sacrificed at about 30 months post-calving for collection of tissues (muscle, heart, thyroid, uterus, udder, kidney, lung, adrenal gland, spleen, liver, lymph nodes and skin). The blood (500 2l) and the tissues (200 2g) underwent DNA extraction (three independent extractions) using a commercial kit (Qiagen). The electrophoretically quantified DNA was tested by Real-Time quantitative PCR (Taqman) assay (ABI Prism 7000, Applied Biosystem), with specific sets of primers and probes for amplification and quantification of male-specific Y chromosome S4 sequences (Y-S4), transgene sequences (TG) and the "-globin gene (normalizator). Results were statistically analyzed using ANOVA and Two-Sample t test.
RESULTS AND DISCUSSION Feto-maternal leakage was determined in bovine by means of Real-Time PCR both for male-specific Y-S4 and transgene sequences. As we have recently published, we detected Y-S4 DNA in some of the conventional heifers at all time-points tested, 25% at mid-gestation, 40% at pre-calving, 73% al calving and 40% 4 months post-calving. The transgenic CSN2/3 sequence, undetectable at mid-gestation was identified in the blood samples at all later stages until over two years after delivery. CSN2/3 sequence were present in 20% of blood samples obtained at pre-calving, 11% at calving, 50% 4 months post-calving and 10% over 2 years later. Although a significantly higher proportion of cows with microchimerism was detected for the Y-S4 sequence rather than for TG, the absolute amount of fetal DNA measured in maternal blood was markedly higher for the transgenic CSN2/3 sequence compared to the Y-S4 one. The magnitude of microchimerism that we detected in cow blood (average of 0.04 ng/ml for Y-S4 and 0.24 ng/ml for CSN2/3) was in the same order of magnitude reported for Y-specific sequences in humans (Invernizzi et al., 2002). After the initial demonstration that feto-maternal cross-talk in cattle exists from the mother to the fetus by Hiendleder and coworkers there is now proof that the communi-
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cation functions both ways, with evidence from our group for the opposite direction. The positive results obtained for naturally mated heifers show that microchimerism can occur independently of placental anomalies correlated with in vitro reproductive technologies. The implications of microchimerism in livestock animals needs to be further evaluated. In order to commence elucidation of the possible engraft of the fetally derived sequences into specific tissues, we further assayed for the presence of TG sequences in different tissues and organs from two cows with transgenic calves, which were sacrificed 30 months post-calving. In the absence of any false positive PCR results in the negative controls, we detected TG sequences in the heart, lung and lymph nodes. Although these data are still preliminary and need to be verified using different techniques and a larger number of animals, they are consistent with human studies, where engrafted fetal cells were detected in the heart, lymphoid organs and lung.
ACKNOWLEDGEMENTS We thank the members of reproductive technologies group and farm staff at AgResearch. This study was supported by the New Zealand Foundation of Research and Technology and AgResearch.
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