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. 2012 Apr 27;86(4):127.
doi: 10.1095/biolreprod.111.094938. Print 2012 Apr.

Rhox13 is translated in premeiotic germ cells in male and female mice and is regulated by NANOS2 in the male

Christopher B Geyer  1 , Rie Saba, Yuzuru Kato, Amy J Anderson, Vesna K Chappell, Yumiko Saga, Edward M Eddy
Affiliations

Rhox13 is translated in premeiotic germ cells in male and female mice and is regulated by NANOS2 in the male

Christopher B Geyer et al. Biol Reprod. .

Abstract

Male and female germ cells enter meiosis in response to an extrinsic cue by retinoic acid (RA), but the pathways downstream of RA signaling that regulate early gametogenesis remain uncertain. We identified a novel reproductive homeobox gene, Rhox13, transcribed in the prenatal ovary and testis beginning on Embryonic Day (E) 13.5. Translation of RHOX13 also begins in female germ cells on E13.5 but is suppressed in male germ cells until Postnatal Day 3. Translation of RHOX13 coincides with initiation of RA signaling in both male and female gonads in vivo but occurs precociously in neonatal testes exposed to RA in vitro or in fetal male germ cells when NANOS2 is absent in vivo. Conversely, RHOX13 translation in female germ cells is suppressed in the presence of ectopically induced NANOS2. These results strongly suggest that RHOX13 expression is regulated at a posttranscriptional step by direct interaction of NANOS2 with Rhox13 mRNA to suppress translation.

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Figures

FIG. 1
FIG. 1
Expression of RHOX13 in the fetal, neonatal, and adult ovary. A–F) Immunohistochemistry was performed on gonadal sections of the ovaries at the ages indicated using anti-RHOX13. Light immunostaining of RHOX13 was first detectable in nuclei of a subset of oogonia at E13.5 (B, indicated by black arrows), which increased in E14.5 preleptotene oocytes (C). RHOX13 was faintly detectable at E15.5 (D) in leptotene/zygotene oocytes (Lep/Zyg, indicated by arrows), but no signal was present in pachytene oocytes (examples are circled) at E15.5. No RHOX13-positive cells were seen after initiation of folliculogenesis by P3 (E) or in the adult ovary (F). Bars = 50 μm.
FIG. 2
FIG. 2
Expression of RHOX13 in the pre-, neo-, and postnatal testis. A–H) Ages are indicated on each section. Immunohistochemistry reveals that RHOX13 protein is first found in a subset of spermatogonia at P3 (D, indicated by black arrows). By P5–P6, approximately 50% of spermatogonia express RHOX13, are smaller in diameter, and have the nuclear morphology of differentiating type B spermatogonia (F and G). The larger type A spermatogonia are RHOX13-negative and consistently located at the periphery of the testis cords (G, white arrows). I) Developmental immunoblot showing that RHOX13 protein is first detectable at P3–P4, confirming the results from IHC. ACTB was utilized as a loading control. Bar = 25 μm.
FIG. 3
FIG. 3
RA induction of RHOX13 expression in P1 testis explants. A) RT-PCR was performed using primers to amplify Stra8, Rhox13, and Actb from testes at E15.5, P3, P4, P5, and P>60. NT = no template control. B) For explant culture, testes were dissected from neonatal mice, cut into small pieces, and cultured for 24 h without RA (−RA) or with RA (+RA). Retinoic acid was dissolved in ethanol, and an equivalent volume of ethanol was added to the −RA sample. C) Quantitative PCR was performed to quantitate Stra8, Rhox13, and Nanos2 mRNA levels from explants grown with (RA+) or without (RA−) added RA. D–F) Positive staining was present in gonocytes in P1 testis explants cultured in the presence of RA (F, cells in green). Primary antibody was excluded from the negative control (D). Bar = 25 μm.
FIG. 4
FIG. 4
RHOX13 is precociously translated in Nanos2−/− fetal testes. A) Immunohistochemistry was performed on E15.5 testis sections using anti-RHOX13, and no signal was evident in Nanos2+/− gonocytes. B–C) A strong signal was detected in gonocytes in spermatic cords (black arrows) as well as in the adjacent mesonephros (white arrow) in the Nanos2−/− testis. D) Preincubation of anti-RHOX13 with the immunizing peptide resulted in loss of signal in the Nanos2−/− testis. Bars = 25 μm. E) Rhox13 mRNA levels were assessed by qRT-PCR in Nanos2+/− and Nanos2−/− testes at E13.5, E14.5, and E15.5. P values (Student t-test) for each stage at E13.5, E14.5, and E15.5 are 0.12, 0.00, and 0.11, respectively. Statistically significant differences are indicated by an asterisk (E14.5).
FIG. 5
FIG. 5
Transgenic NANOS2 inhibits RHOX13 expression in oocytes. Tamoxifen was injected at E10.5 to pregnant female double transgenic mice harboring both CAG-lox-CAT-lox-FLAG-Nanos2 and Pou5f1-CreERT2, and ovaries were prepared at E12.5 (A–C) and E14.5 (D–I). Ovaries from E12.5 mice were sectioned and coimmunostained with anti-NANOS2 and anti-TRA98 (A–C). Ovaries from E14.5 mice were sectioned and coimmunostained with anti-DMC1 and either anti-NANOS2 (D–F) or anti-RHOX13 (G–I). D–F) White dashed lines encircle germ cells that express NANOS2 (E), and all have reduced levels of DMC1 (D). F) A merged image of D and E is presented, indicating that there is minimal coexpression. G–I) Germ cells that have NANOS2-induced reduction of DMC1 (G) also do not express RHOX13 (H), indicating that NANOS2 is capable of repressing meiosis and RHOX13 expression. I) A merged image of G and H indicates that cells that do not express DMC1 also do not express RHOX13; cells that express both DMC1 and RHOX13 are yellow. Nuclei were stained with DAPI and can be seen in blue (C, F, I). Bar = 100 μm for A–C and 50 μm for D–I.
FIG. 6
FIG. 6
Rhox13 mRNA directly binds to NANOS2 in the fetal testis. Male gonadal extracts from wild-type (WT) and transgenic (Tg) mice expressing FLAG-NANOS2 at E14.5 were subjected to IP with anti-FLAG antibody. RNA precipitants were analyzed by RT-PCR for each of the genes indicated. The numbers of amplification cycles used for each gene are listed in parentheses to the right.
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