Fig. 9. Treatment of neonatal ovaries with LV-sh436 knocks down Fxna mRNA expression without generating an interferon-like response. Newborn rat ovaries were placed in organ culture and exposed for 4 days to LV-EGFP, LV-sh436 or LV-sh436 carrying several mismatches. The ovaries were then processed for either immunohistochemistry or RNA extraction. (A) LV-sh436 markedly reduced Fxna mRNA abundance. The bars are mean±s.e.m., and the numbers in parenthesis indicate the number of ovary pools per group (three ovaries per pool). ^**, P<0.01. The inset shows a representative gel. (B) Absence of an interferon response 4 days after infection of 1-day-old ovaries with LV-sh436 in organ culture, as determined by the normal content of Oas1 mRNA measured in the treated ovaries. LV-EGFP, lentiviral vector alone; LV-sh436 Mism, LV carrying a sh436 sequence with nucleotide mismatches; LV-sh436, lentiviral vector carrying Fxna shRNA 436. (C,D) Lentiviral infection of neonatal rat ovaries in organ culture. The ovaries were explanted on the day of birth and exposed for 4 days to LV-EGFP. The glands were then fixed and subjected to immunohistofluorescence using polyclonal antibodies to EGFP. Cell nuclei were stained with Hoescht 33258 (blue). Low (C) and higher (D) magnification images depicting viral infection of both somatic cells and oocytes. (E-H) LV-sh436 infection of neonatal ovaries disrupts the structural organization of the ovary. The ovaries were treated as indicated above. Hoescht-stained cell nuclei are shown in light blue in E and G, and in magenta in F and H. Note the presence of primary follicles (arrows) in ovaries treated with LV-EGFP (E,F), and the aggregates of LV-sh436-expressing somatic cells (arrowheads) near isolated oocytes (arrows) in ovaries exposed to LV-sh436 (G,H). Scale bars: C, 200 µm; D, 10 µm; E-H, 25 µm.