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. 2013 Nov;100(5):1468-75.
doi: 10.1016/j.fertnstert.2013.07.1978. Epub 2013 Aug 28.

Bone morphogenetic protein 4 promotes mammalian oogonial stem cell differentiation via Smad1/5/8 signaling

Eun-Sil Park  1 , Dori C Woods, Jonathan L Tilly
Affiliations

Bone morphogenetic protein 4 promotes mammalian oogonial stem cell differentiation via Smad1/5/8 signaling

Eun-Sil Park et al. Fertil Steril. 2013 Nov.

Abstract

Objective: To test whether bone morphogenetic protein 4 (BMP4) directly regulates differentiation of adult mouse ovary-derived oogonial stem cells (OSCs) in vitro.

Design: Animal study.

Setting: Research laboratory.

Animal(s): Adult C57BL/6 female mice.

Intervention(s): After purification from adult ovaries by fluorescence-activated cell sorting, OSCs were cultured without or with BMP4 in the absence or presence of the BMP4 antagonist, Noggin.

Main outcome measure(s): Rates of in vitro-derived (IVD) oocyte formation and changes in gene expression were assessed.

Result(s): Cultured OSCs expressed BMP receptor (BMPR) 1A (BMPR1A), BMPR1B, and BMPR2, suggesting that BMP signaling can directly affect OSC function. In agreement with this, BMP4 significantly increased the number of IVD oocytes formed by cultured OSCs in a dose-dependent manner, and this response was inhibited in a dose-dependent fashion by cotreatment with Noggin. Exposure of OSCs to BMP4 was associated with rapid phosphorylation of BMPR-regulated Smad1/5/8 proteins, and this response was followed by increased expression of the meiosis initiation factors, stimulated by retinoic acid gene 8 (Stra8), muscle-segment homeobox 1 (Msx1), and Msx2. In keeping with the IVD oocyte formation data, the ability of BMP4 to activate Smad1/5/8 signaling and meiotic gene expression in OSCs was abolished by cotreatment with Noggin.

Conclusion(s): Engagement of BMP4-mediated signaling in adult mouse ovary-derived OSCs cultured in vitro drives differentiation of these cells into IVD oocytes through Smad1/5/8 activation and transcriptional up-regulation of key meiosis-initiating genes.

Keywords: BMP4; germ cells; oocyte; oogenesis; stem cells.

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Figures

Figure 1
Figure 1
(A) Detection of Bmpr1a, Bmpr1b and Bmpr2 mRNAs in cultured OSCs (no RT, PCR analysis of RNA sample without reverse transcription used as a negative control to exclude genomic DNA contamination; β-actin, internal sample loading control). (B–D) Representative immunofluorescence-based detection of BMPR1A, BMPR1B and BMPR2 proteins (green) in cultured OSCs counterstained with DAPI (blue) to visualize nuclear DNA (scale bars, 10 μm).
Figure 2
Figure 2
(A–C) Effect of BMP4 and Noggin, alone (A, B) or in combination (C), on numbers of IVD-oocytes formed per well of 2.5 × 104 OSCs after 16 hours of treatment (mean ± SEM, n = 3–4 independent cultures; different letters, P < 0.05). (D) Lack of effect of BMP4 (100 ng/mL) and Noggin (150 ng/mL), alone or in combination, on proliferation of OSCs (2.5 × 104 cells initially seeded per well) over a 40-hour culture period (mean ± SEM, n = 4 independent cultures; no significant differences detected).
Figure 3
Figure 3
Representative immunoblot depicting the effect of BMP4 (100 ng/mL) and Noggin (150 ng/mL), alone or in combination, on Smad1/5/8 phosphorylation (p-Smad1/5/8) in wells of 5 × 104 OSCs after the indicated durations of treatment. Note that total Smad1 protein and β-actin protein levels remain comparable across treatment groups.
Figure 4
Figure 4
(A) Conventional RT-PCR analysis of Stra8, Msx1 and Msx2 mRNA levels in OSCs (2.5 × 104 cells per well) treated for 24 hours without or with 100 ng/mL BMP4, alone or in combination with 150 ng/mL Noggin (no RT, PCR analysis of RNA sample without reverse transcription used as a negative control to exclude genomic DNA contamination; β-actin, internal sample loading control). (B) Representative examples of immunofluorescence-based detection of Stra8 protein (green, arrows) in cultured OSCs, counterstained with rhodamine phalloidin (red) and DAPI (blue) to visualize cytoplasmic F-actin and nuclear DNA, respectively (scale bars, 10 μm). (C–E) Real-time (quantitative) RT-PCR analysis of changes in Stra8 (C), Msx1 (D) and Msx2 (E) mRNA levels normalized to β-actin mRNA levels in OSCs (2.5 × 104 cells per well) treated for 24 hours without or with 100 ng/mL BMP4, alone or in combination with 150 ng/mL Noggin (mean ± SEM, n = 4 independent cultures; different letters, P < 0.05).
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References

    1. Zou K, Yuan Z, Yang Z, Luo H, Sun K, Zhou L, et al. Production of offspring from a germline stem cell line derived from neonatal ovaries. Nat Cell Biol. 2009;11:631–6. - PubMed
    1. Pacchiarotti J, Maki C, Ramos T, Marh J, Howerton K, Wong J, et al. Differentiation potential of germ line stem cells derived from the postnatal mouse ovary. Differentiation. 2010;79:159–70. - PubMed
    1. Zou K, Hou L, Sun K, Xie W, Wu J. Improved efficiency of female germline stem cell purification using Fragilis-based magnetic bead sorting. Stem Cells Dev. 2011;20:2197–204. - PubMed
    1. White YA, Woods DC, Takai Y, Ishihara O, Seki H, Tilly JL. Oocyte formation by mitotically active germ cells purified from ovaries of reproductive age women. Nat Med. 2012;18:413–21. - PMC - PubMed
    1. Zhang Y, Yang Z, Yang Y, Wang S, Shi L, Xie W, et al. Production of transgenic mice by random recombination of targeted genes in female germline stem cells. J Mol Cell Biol. 2011;3:132–41. - PubMed

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