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First published online 31 January 2007
doi: 10.1242/dev.02795

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Fxna, a novel gene differentially expressed in the rat ovary at the time of folliculogenesis, is required for normal ovarian histogenesis

Cecilia Garcia-Rudaz^1,*, Felix Luna^1,*,, Veronica Tapia^1,, Bredford Kerr¹, Lois Colgin², Francesco Galimi^3,4, Gregory A. Dissen¹, Neil D. Rawlings⁵ and Sergio R. Ojeda^1,

¹ Division of Neuroscience, Oregon National Primate Research Center/Oregon Health and Science University, 505 N.W. 185th Avenue, Beaverton, OR, USA.
² Division of Animal Resources, Oregon National Primate Research Center/Oregon Health and Science University, 505 N.W. 185th Avenue, Beaverton, OR, USA.
³ University of Sassari Medical School/INBB, Italy.
⁴ The Salk Institute, San Diego, CA, USA.
⁵ The Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

View larger version (40K): [in this window] [in a new window]   Fig. 1. A novel mRNA differentially expressed in the developing ovary of the rat is also expressed in several other tissues. (A) Autoradiograph of a sequencing gel showing that the signal intensity of a PCR product (C5-530a2, arrow) derived from the gene differential display amplification of total perinatal rat ovarian RNA is greater in samples from PN-48-hour than F21 ovaries. Each PCR reaction was electrophoresed in duplicate. (B) Northern blot analysis of polyA+ RNA extracted from different tissues of 2-day-old female rats identifies a ubiquitous 5.4 kb mRNA species (arrow) and a longer, much less abundant transcript in ovary, kidney and adrenal gland. The cRNA probe used was transcribed from a C5-530a2 cDNA template. Each lane contains 5 µg of polyA+ RNA. Cyclophilin mRNA (cyclo) detected subsequently on the same blot was used as a control for procedural variability. Migration of the 4.7 and 1.8 kb ribosomal RNA species detected by ethidium bromide staining is indicated on the left side of the blot. Note that cyclophilin mRNA expression is not constant across tissues. Ov, 2-day-old rat ovary; Ut, uterus; Kd, kidney; Ad, adrenal gland; Lv, liver; Pit, pituitary gland; Hy, hypothalamus; Hi, hippocampus; Cc, cerebral cortex.

View larger version (22K): [in this window] [in a new window]   Fig. 2. Genomic structure of the C5-530a2 (Fxna) gene. Boxes represent exons, and the horizontal line connecting them represents introns. Gray boxes denote 5'- and 3'-untranslated regions. Exon number is given above each box; numbers beneath indicate the size (bp) of each exon.

View larger version (29K): [in this window] [in a new window]   Fig. 3. Changes in C5-530a2 mRNA expression during early postnatal development of the rat ovary, as assessed by an RNase-protection assay. (A) Above: the hypothetical gene (BC031519) predicted to encode a 317 amino acid protein; beneath: C5-530a2 (NM_184050) that was demonstrated experimentally to have a longer open reading frame. The predicted coding regions are depicted in gray and the untranslated regions in black. The approximate locations of the two probes used for RNase-protection assay are shown as white rectangles. The two putative ATG sites are indicated. (B) Left panel shows a representative autoradiogram depicting the increase in C5-530a2 mRNA abundance (detected by RNase-protection assay using probe A) between F21 and PN 48 hours, and the decrease towards adult values seen thereafter. Probe A consists of 289 nt transcribed from a C5-530a2 cDNA template plus 88 nt derived from transcribed vector sequences. The cyclophilin probe (Cyclo) is 211 nt in length, of which 158 nt correspond to transcribed vector sequences. The mRNA species protected by probe A, and the cyclophilin cRNA probe, are arrowed. MM, molecular weight markers (32P-labeled RNA ladder); UP, undigested probe; DP, digested probe; A, adult ovaries. Right panel is a densitometric analysis of the changes in C5-530a2 mRNA levels detected by RNase-protection assay. RNA abundance is expressed as arbitrary units (AU) calculated using the individual C5-320a2/cyclophilin mRNA ratios from each sample. **, P<0.02, 48 hour group versus all other groups. Bars are mean values for each group and vertical lines represent s.e.m. (C) The same RNase-protection assay analysis as in B, but using probe B and samples pooled from selected developmental ages. Probe B consists of 492 nt, of which 406 correspond to transcribed vector sequences.

View larger version (155K): [in this window] [in a new window]   Fig. 4. Alignment of selected sequences from peptidase family M28. Sequences have been selected from an alignment of all known peptidase members of family M28. Sequences are numbered according to the type example for the family, aminopeptidase S (Streptomyces griseus). Active site residues are indicated by asterisks, and metal ligands by hash (#) symbols. The cysteines involved in the known disulfide bridge in aminopeptidase S are indicated (+). Cysteines taking part in the same disulfide bridge are denoted as/1 and 1\. Key to sequences: (a) aminopeptidase S (Streptomyces griseus); (b) aminopeptidase Ap1 (Vibrio proteolyticus); (c) glutamate carboxypeptidase II (Homo sapiens); (d) agCP10156 protein (Anopheles gambiae); (e) agCG47216 protein (Anopheles gambiae); (f) Fxna peptidase (Rattus norvegicus); (g) Fxna peptidase (Mus musculus); (h) Fxna peptidase (Pan troglodytes); (i) Fxna peptidase (Homo sapiens); (j) Fxna peptidase (Canis familiaris); (k) Fxna peptidase (Gallus gallus); (l) IAP aminopeptidase (Escherichia coli).

View larger version (166K): [in this window] [in a new window]   Fig. 5. Fxna functional domains. The deduced amino acid sequence is shown in capital letters and the nucleotide sequence in lower case letters. The peptidase unit is underlined; the catalytic residues (Asp 201, Glu 245) are circled, and the metal-binding amino acids (His 199, Asp 211, Glu 246, Glu 272 and His 348) within squares. Heavy underlining identifies the polyadenylation signal.

View larger version (61K): [in this window] [in a new window]   Fig. 6. The cellular localization of Fxna. (A) Predicted cellular localization domains of Fxna. The graph depicts the probability of different regions of the Fxna protein being located on the outer or inner side of a cellular membrane, or embedded within the membrane. Red, membrane-associated domains; green line, outer side of membrane; blue line, inner side of membrane. The catalytic domain is located on the outer side of a cellular membrane between amino acid residues 163-393. (B-D) Double immunohistofluorescence images showing the colocalization of Fxna-Flag (green) with PDI, an ER-specific marker (red). COS-7 cells were transfected with Fxna-Flag 24 hours prior to staining with a monoclonal antibody against Flag and a polyclonal antibody against PDI. Note that non-transfected cells are only positive for PDI. Cell nuclei are stained with Hoescht 33258 (blue). Scale bar: 20 µm.

View larger version (148K): [in this window] [in a new window]   Fig. 7. Fxna mRNA is expressed in granulosa cells of the developing rat ovary. The mRNA was detected by in situ hybridization using 35S-UTP-labeled Fxna cRNA probe B. (A) Low magnification dark-field image of a PN-48-hour ovary. Cell nuclei were stained with thionin (blue-purple); the hybridization signal in newly formed follicles is seen as white grains. (B) Higher magnification, bright-field image showing the presence of Fxna mRNA (black grains) in granulosa cells, but not oocytes (arrows) of a PN-48-hour ovary. (C) Low magnification, dark-field image of a 21-day-old ovary showing the presence of Fxna mRNA in granulosa cells of both preantral and antral follicles. Arrows point to atretic follicles devoid of hybridization signal. The inset is a higher magnification, bright-field image of the boxed area, showing that the apparent localization of Fxna mRNA to the oocyte is most likely due to an abundance of hybridization signal in granulosa cells of the cumulus oophorus. (D) Bright-field image of C. (E) High magnification, dark-field image of a 21-day-old ovary reiterating the presence of Fxna mRNA in granulosa cells of antral and preantral follicles, and the absence of hybridization (arrows) in oocytes. (F) Bright-field image of E. Scale bars: A, 100 µm; B,E,F, 20 µm; C,D, 400 µm; inset, 50 µm.

View larger version (50K): [in this window] [in a new window]   Fig. 8. Knock-down of Fxna expression using siRNAs and shRNAs. (A) Left panel shows that siRNAs decrease Fxna mRNA levels in RK3E cells 48 hours after transfection, as measured by semi-quantitative PCR. Each well was transfected with a different siRNA and the cellular RNA extracted 48 hours later. An 886 bp cDNA fragment was then PCR-amplified using primers annealing to sequences in the coding region of Fxna mRNA. Right panel is a quantitation of the changes detected in the gel shown in the left panel. (B) Left panel is a gel showing that mutating the third base of two codons in the sequence of Fxna mRNA targeted by LV-sh436 rescues Fxna mRNA from siRNA-induced silencing. The mutation creates a silent mutation as it does not change the encoded amino acids. 293T cells were transfected with a plasmid encoding wild-type Fxna mRNA or an Fxna mRNA carrying a silent mutation of the mRNA region targeted by LV-sh436. Six hours later the cells were infected with a lentiviral vector producing LV-sh436 or were left uninfected. The cells were then cultured for 4 days before extracting the RNA for semi-quantitative PCR. Right panel is a quantitation of the changes illustrated in the left panel. Fxna WT, wild-type Fxna mRNA; Fxna-Mut, Mutated Fxna mRNA. Bars are mean±s.e.m. and numbers in parentheses indicate the number of wells per group. **, P<0.01 versus both control groups.

View larger version (93K): [in this window] [in a new window]   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.

View larger version (121K): [in this window] [in a new window]   Fig. 10. Loss of Fxna disrupts ovarian histogenesis and reduces follicular formation. Neonatal rat ovaries were exposed for 4 days in organ culture to LV-EGFP or LV-sh436. One ovary from each animal was treated with LV-EGFP and the contralateral ovary was treated with LV-sh436. The glands were then fixed, serially sectioned and stained as described in Materials and methods. (A,B) Low magnification view of a control ovary (A) and of a Fxna knock-down gland (B) showing immature regions. Primary follicles with normal appearance (arrows) are fewer in number in LV-sh436-treated ovaries. Scale bars, 100 µm. (C,D) Higher magnification images illustrating the presence of multiple primary follicles (one layer of granulosa cells; arrows) in control (LV-EGFP-treated) ovaries (C), and the disorganization of somatic and germ cells in Fxna knock-down ovaries treated with LV-sh436 (D). Note the aggregates of somatic cells not associated with oocytes (pair of arrows) and the clusters of oocytes encapsulated by a rim of somatic cells (arrows). Scale bars, 20 µm. (E) The number of naked oocytes is similar in Fxna-deficient (black bars) and control (white bars) ovaries (left), but the total number of follicles per ovary is decreased in Fxna knock-down ovaries (right). (F) The number of follicles at all stages of development (primordial, primary and secondary) is reduced in Fxna-deficient ovaries (black bars). Bars are mean±s.e.m. Numbers in parentheses indicate the number of ovaries per group. *, P<0.01 versus LV-EGFP-treated group.