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. 2006 Sep 26;103(39):14390-5.
doi: 10.1073/pnas.0603836103. Epub 2006 Sep 15.

Endogenous retroviruses regulate periimplantation placental growth and differentiation

Kathrin A Dunlap  1 , Massimo Palmarini, Mariana Varela, Robert C Burghardt, Kanako Hayashi, Jennifer L Farmer, Thomas E Spencer
Affiliations

Endogenous retroviruses regulate periimplantation placental growth and differentiation

Kathrin A Dunlap et al. Proc Natl Acad Sci U S A. .

Abstract

Endogenous retroviruses (ERVs) are fixed and abundant in the genomes of vertebrates. Circumstantial evidence suggests that ERVs play a role in mammalian reproduction, particularly placental morphogenesis, because intact ERV envelope genes were found to be expressed in the syncytiotrophoblasts of human and mouse placenta and to elicit fusion of cells in vitro. We report here in vivo and in vitro experiments finding that the envelope of a particular class of ERVs of sheep, endogenous Jaagsiekte sheep retroviruses (enJSRVs), regulates trophectoderm growth and differentiation in the periimplantation conceptus (embryo/fetus and associated extraembryonic membranes). The enJSRV envelope gene is expressed in the trophectoderm of the elongating ovine conceptus after day 12 of pregnancy. Loss-of-function experiments were conducted in utero by injecting morpholino antisense oligonucleotides on day 8 of pregnancy that blocked enJSRV envelope protein production in the conceptus trophectoderm. This approach retarded trophectoderm outgrowth during conceptus elongation and inhibited trophoblast giant binucleate cell differentiation as observed on day 16. Pregnancy loss was observed by day 20 in sheep receiving morpholino antisense oligonucleotides. In vitro inhibition of the enJSRV envelope reduced the proliferation of mononuclear trophectoderm cells isolated from day 15 conceptuses. Consequently, these results demonstrate that the enJSRV envelope regulates trophectoderm growth and differentiation in the periimplantation ovine conceptus. This work supports the hypothesis that ERVs play fundamental roles in placental morphogenesis and mammalian reproduction.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Design and effects of morpholinos on enJSRV Env expression in vitro. (A) MAO-env was designed to inhibit splicing and translation of enJSRV env mRNA but not expression of full-length genomic RNA (which expresses the viral Gag). (B) 293T cells were mock-transfected (lane 1) or transfected with pSV-En2EnvFlag, a simian virus 40-driven expression plasmid for enJS5F16 env cDNA tagged with a Flag epitope at the C terminus (lanes 2–4). Cells were then treated with MAO-env (lane 2) or MAO-5mis (lane 3) and MAO-std as controls (lane 4). All morpholinos were complexed with the Endo-Porter delivery reagent and used at a final concentration of 80 μM. After 48 h, enJS5F156 Env expression was determined by immunoprecipitation (IP) and Western blot analysis (WB). Note that the full-length retroviral Env is processed into a surface domain and a transmembrane domain (TM). (C) 293T cells were mock-transfected (lane 1) or transfected with pSV-En2EnvFlag as above. Cells were then treated with Endo-Porter alone (lane 2), MAO-std as a control (lane 3), MAO-5mis as a control (lanes 4–6; 20, 40, and 80 μM, respectively), or MAO-env (lanes 7–9; 20, 40, and 80 μM, respectively). All morpholinos were complexed with Endo-Porter delivery reagent. After 24 h, enJS5F16 Env expression was determined by immunoprecipitation and Western blot analysis as in B. (D) 293T cells were mock-transfected (lane 1) or cotransfected with pSV-En2EnvFlag and pCMV2 en56A1 expressing the full-length en56A1 clone (lanes 2–4). Cells were then treated Endo-Porter alone (lanes 1 and 2), MAO-5mis as a control (lane 3), or MAO-env (lane 4). All morpholinos were complexed with Endo-Porter delivery reagent and used at a final concentration of 80 μM. After 48 h, enJSRV Env expression (Upper) was determined by immunoprecipitation and Western blot analysis as in B and C and Gag expression by Western blot analysis (Lower).
Fig. 2.
Fig. 2.
Effects of morpholinos on periimplantation conceptus trophoblast growth and differentiation. MAO-std, MAO-5mis, or MAO-env was injected into the uterine lumen on day 8 after mating, and conceptuses were recovered on day 16 (see Materials and Methods for experimental details). (A) Morphology of the conceptuses was examined by using an inverted microscope. Micrographs are shown at the same magnification. Note the retarded growth in the conceptus recovered from a MAO-env-treated ewe. (B) Portions of the conceptuses were fixed in paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. (Width of each field of view is 420 μm with the Inset at 85 μm.) (C and D) Trophoblast giant binucleate cells (BNCs) in conceptuses were detected by pregnancy-associated glycoproteins (PAGs) (C) and CSH1 (alias placental lactogen) (D) in the conceptus. Immunoreactive PAG and CSH1 proteins were detected in paraformaldehyde-fixed, paraffin-embedded sections of conceptuses by using a rabbit anti-ovine PAG or anti-ovine CSH1 antibody. (Width of each field of view is 420 μm with the Inset at 210 μm.) Data are representative of conceptuses from all ewes. MTC, mononuclear trophectoderm cell.
Fig. 3.
Fig. 3.
Delivery and effectiveness of morpholinos in vivo. MAO-std, MAO-5mis, or MAO-env was injected into the uterine lumen of sheep on day 8 after mating, and the conceptuses were removed on day 16 (see Materials and Methods for experimental details). (A) Portions of the conceptuses were frozen in optimal cutting temperature (OCT) compound and sectioned. Sections were rinsed in PBS, and a coverslip was affixed by using DAPI-containing mounting medium. Fluorescence microscopy was used to detect the rhodamine-labeled morpholino (orange/red) and DAPI nuclei (blue). (B and C) Conceptuses and uteri were sectioned and analyzed for enJSRV Env protein by immunofluorescence analysis using a rabbit antiserum toward the JSRV Env (B) or Gag (C) with a FITC-labeled secondary antibody. (Width of each field of view is 140 μm.) Data are representative of conceptuses from all ewes. LE, luminal epithelium; sGE, superficial glandular epithelium; S, stroma; Tr, trophectoderm.
Fig. 4.
Fig. 4.
Effects of morpholinos on in vitro ovine trophectoderm growth. Mononuclear trophectoderm cells were isolated from day 15 conceptuses. (A) Morpholino delivery. Rhodamine-labeled MAO-std was complexed with Endo-Porter aqueous delivery reagent and added to cells in culture. Fluorescence microscopy was used to visualize the labeled MAO in cells. [Width of each field of view is 870 μm (Left) and 90 μm (Right).] (B) Effect of morpholinos on enJSRV Env and Gag protein in cultured trophectoderm cells. Cells were grown on glass slides and mock-treated or treated with MAO-std, MAO-5mis, or MAO-env for 48 h. Immunofluorescence analysis determined that synthesis of enJSRV Env, but not Gag, protein was inhibited in cells treated with MAO-env but not the other morpholinos. Results are representative of three experiments. (Width of each field of view is 140 μm.) (C) Effect of morpholinos on trophectoderm cell growth. Cells were grown in culture dishes until 30% confluency and mock-treated (no morpholino) or treated with MAO-std, MAO-5mis, or MAO-env for 48 h. Cell number was reduced (P < 0.05) by 33% in cultures treated with the MAO-env relative to control morpholinos. Results are from three independent experiments, and data are expressed as the percentage of cell number in mock-treated cultures.
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References

    1. DeMartini JC, Carlson JO, Leroux C, Spencer T, Palmarini M. Curr Top Microbiol Immunol. 2003;275:117–137. - PubMed
    1. Palmarini M, Sharp JM, De las Heras M, Fan H. J Virol. 1999;73:6964–6972. - PMC - PubMed
    1. Palmarini M, Mura M, Spencer TE. J Gen Virol. 2004;85:1–13. - PubMed
    1. Palmarini M, Gray CA, Carpenter K, Fan H, Bazer FW, Spencer TE. J Virol. 2001;75:11319–11327. - PMC - PubMed
    1. Dunlap KA, Palmarini M, Adelson DL, Spencer TE. Biol Reprod. 2005;73:271–279. - PubMed

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