Mice deficient in oocyte-specific oligoadenylate synthetase-like protein OAS1D display reduced fertility

Abstract

The double-stranded RNA (dsRNA)-induced interferon response is a defense mechanism against viral infection. Upon interferon activation by dsRNA, 2',5'-oligoadenylate synthetase 1 (OAS1A) is induced; it binds dsRNA and converts ATP into 2',5'-linked oligomers of adenosine (called 2-5A), which activate RNase L that in turn degrades viral and cellular RNAs. In a screen to identify oocyte-specific genes, we identified a novel murine cDNA encoding an ovary-specific 2',5'-oligoadenylate synthetase-like protein, OAS1D, which displays 59% identity with OAS1A. OAS1D is predominantly cytoplasmic and is exclusively expressed in growing oocytes and early embryos. Like OAS1A, OAS1D binds the dsRNA mimetic poly(I-C), but unlike OAS1A, it lacks 2'-5' adenosine linking activity. OAS1D interacts with OAS1A and inhibits the enzymatic activity of OAS1A. Mutant mice lacking OAS1D (Oas1d(-/-)) display reduced fertility due to defects in ovarian follicle development, decreased efficiency of ovulation, and eggs that are fertilized arrest at the one-cell stage. These effects are exacerbated after activation of the interferon/OAS1A/RNase L pathway by poly(I-C). We propose that OAS1D suppresses the interferon/OAS/RNase L-mediated cellular destruction by interacting with OAS1A during oogenesis and early embryonic development.

FIG. 1.

Oas1d mRNA is exclusively expressed in growing oocytes in the ovary. (A) Northern blot analysis of Oas1d mRNA expression in multiple mouse tissues, including brain (Br), heart (He), kidney (Ki), liver (Li), lung (Lu), intestine (In), spleen (Sp), stomach (St), testis (Te), Ovary (Ov), Gdf9^−/− ovary (GDF9^−/− Ov), and uterus (Ut). The membrane was stripped and reprobed with an 18S rRNA cDNA probe as a loading control. (B) Real-time quantitative RT-PCR analysis of Oas1d and Oas1a mRNA expression in multiple mouse tissues. (C) In situ hybridization analysis of mouse ovarian sections. Bright (left panel) and dark (right panel) field images are shown (magnification, ×60). AF, antral follicle; PF, primary follicle.

FIG. 2.

OAS1D is a cytoplasmic protein exclusively expressed in oocytes and early embryos. (A) OAS1D is detected exclusively in the ovary, whereas OAS1A is ubiquitously expressed. Preimmune serum does not react with recombinant OAS1D (lane A in the upper panel), and anti-OAS1D serum does (lane B in the upper panel). Anti-OAS1A serum detects the 46 kD recombinant OAS1A band (lane A in the lower panel) and preimmune serum does not (lane B in the lower panel). (B) Immunohistochemical localization of OAS1D and OAS1A in the mouse ovary. OAS1D immunoreactivity is detected exclusively in the cytoplasm of oocytes in the wild-type or Gdf9^−/− ovary, whereas OAS1A immunoreactivity is detected not only in oocytes but also in granulosa cells. Preimmune sera did not detect immunoreactive material. (C) Immunofluorescence analysis of OAS1D and OAS1A in oocytes and early embryos. OAS1D and OAS1A are stained red; DNA is stained blue with DAPI. Expression in oocytes (GV stage and metaphase II), two-cell embryos, and eight-cell embryos was analyzed on the same slide. OAS1A displays constant expression in the cytoplasm of oocytes through eight-cell embryos, whereas OAS1D levels drastically decrease after mitotic division.

FIG. 3.

Biochemical characteristics of OAS1D. (A) Poly(I-C) induces Oas1a but not Oas1d mRNA expression. Adult female and male mice were injected intraperitoneally with (+) or without (−) 250 μg of poly(I-C). In some mice, poly(I-C) was injected intratesticularly (Te, +*) or intracranially (Brain, +*). Total RNA was isolated from tissues 24 h after treatment and analyzed by Northern blot analysis with Oas1a, Oas1d, or 18S rRNA probes. Oas1a is induced ubiquitously, whereas Oas1d is not induced and continues to be expressed only in the ovary (Ov). Tissue abbreviations are the same as in the Fig. 1 legend. (B) dsRNA binding assay. Aliquots of native (N1 and N2) or fully denatured (D) His-tagged recombinant OAS1A or OAS1D were incubated with poly(I-C)-agarose, followed by washing and Western blot analysis with anti-His antibody. The same aliquot from each reaction was subjected to SDS-PAGE before incubation with poly(I-C)-agarose and was used as a loading control. (C) 2′,5′-oligoadenylate synthetase activity assay. Poly(I-C)-agarose-bound recombinant proteins were incubated with [γ-³²P]ATP (20 μCi) in a reaction buffer containing 20 mM HEPES-KOH, 50 mM KCl, 25 mM magnesium acetate, 7 mM 2-mercaptoethanol, and 5 mM ATP for the indicated times in hours at 33°C. The reaction mixture was centrifuged, and the supernatants were fractionated on a 20% polyacrylamide gel containing 7 M urea. The gel was dried, followed by autoradiography.

FIG. 4.

Generation of Oas1d knockout mice. (A) Oas1d genomic locus and targeting vector for generation of an Oas1d-null allele. A deletion of exon 1 (containing the start codon) and partial exon 2 of the Oas1d gene was achieved by homologous recombination in AB2.2 ES cells. 5′ and 3′ probes were used to distinguish wild-type and mutant alleles. (H3, Hind III; B2, Bgl II; SM, SmaI; NA, Nar I; BH, BamH I; CL, ClaI). (B) Southern blot analysis of Bgl II-digested tail DNA from a litter of pups using the 3′ probe. The probe detects a 7.1-kb wild-type allele and a 6.2-kb mutant allele. (C) Western blot analysis of ovarian tissues from wild-type (+/+), Oas1d heterozygous (+/−), and Oas1d homozygous (−/−) mutant mice. ACTIN was detected as the loading control.

FIG. 5.

Histological analyses of wild-type and Oas1d^−/− ovaries. (A to D) Histology of the wild-type (A) and Oas1d^−/− (B to D) ovaries. The wild-type ovary contains multiple corpus lutea (CL) and follicles at different growing stages. The Oas1d^−/− ovary contains fewer CL and many degenerating follicles (arrows). High-power images (magnification, ×200) of the Oas1d^−/− ovary show two areas containing several degenerating (arrows) follicles (C and D). (E to H) TUNEL assay of the wild-type (E and G) and Oas1d^−/− (F and H) ovaries with (G and H) or without (E and F) poly(I-C) treatment. More TUNEL-positive follicles are present in the Oas1d^−/− ovary than in the wild-type ovary with or without poly(I-C) treatment. Magnification, ×50. Insets in panels F and H show high-power images (magnification, ×400) of TUNEL-positive follicles.

FIG. 6.

Induction of IFN-γ by poly(I-C), interaction of OAS1D with OAS1A, and inhibition of OAS1A enzymatic activity by OAS1D. (A) Induction of Ifng (IFN-γ) and Oas1a by poly(I-C) treatment in eight-cell embryos. The PCR conditions are described in Materials and Methods. (B) OAS1D binds OAS1A in vitro. Recombinant OAS1A and OAS1D were incubated in a binding buffer, followed by immunoprecipitation with OAS1A antibody. The precipitates were washed and subjected to Western blot analysis with OAS1D antibody. (C) Total protein lysates from ovary (50 μg) or oocytes (232 to 245 oocytes) were immunoprecipitated with OAS1A antibody, followed by Western blot analysis with OAS1D antibody. Lanes: 1, wild-type ovary without poly(I-C) treatment; 2, wild-type oocytes without poly(I-C) treatment; 3, Oas1d^−/− ovary without poly(I-C) treatment; 4, wild-type oocytes after poly(I-C) treatment; 5, Oas1d^−/− ovary after poly(I-C) treatment; 6, wild-type ovary immunoprecipitated with OAS1A preimmune serum; 7, wild-type ovary after poly(I-C) treatment. (D) Inhibition of OAS1A activity by OAS1D. Six reactions containing a fixed amount of OAS1A and increasing amounts of OAS1D (0, 0.5×, 1×, 2×, 4×, or 8×) were analyzed, and OAS1D was found to completely abolish OAS1A enzymatic activity when presented in excessive amount (2× to 4×). As a specificity control, when OAS1D was replaced with excessive amounts (2× and 4×) of bovine serum albumin (BSA), no effect on the OAS1A activity was observed.