FMRP associates with cytoplasmic granules at the onset of meiosis in the human oocyte

Germ cell development and primordial follicle formation during fetal life is critical in establishing the pool of oocytes that subsequently determines the reproductive lifespan of women. Fragile X-associated primary ovarian insufficiency (FXPOI) is caused by inheritance of the FMR1 premutation allele and approximately 20% of women with the premutation allele develop ovarian dysfunction and premature ovarian insufficiency. However, the underlying disease mechanism remains obscure, and a potential role of FMRP in human ovarian development has not been explored. We have characterised the expression of FMR1 and FMRP in the human fetal ovary at the time of germ cell entry into meiosis through to primordial follicle formation. FMRP expression is exclusively in germ cells in the human fetal ovary. Increased FMRP expression in germ cells coincides with the loss of pluripotency-associated protein expression, and entry into meiosis is associated with FMRP granulation. In addition, we have uncovered FMRP association with components of P-bodies and stress granules, suggesting it may have a role in mRNA metabolism at the time of onset of meiosis. Therefore, this data support the hypothesis that FMRP plays a role regulating mRNAs during pivotal maturational processes in fetal germ cells, and ovarian dysfunction resulting from FMR1 premutation may have its origins during these stages of oocyte development

Immunohistochemical co-localisation of FMRP and primordial germ cell or meiosis markers in fetal ovaries at 14-16wga.

<p>A. FMRP and the pluripotency marker OCT4 are expressed in distinct germ cell subpopulations with only few germ cells co-expressing OCT4 and FMRP. OCT4-positive cells are located closer to the cortex and FMRP-positive cells are closer to the medulla. Note the granular distribution of FMRP (white arrowheads). B. As with OCT4, there is limited overlap between FMRP and the primordial germ cell-specific marker LIN28. Note that in germ cells co-expressing FMRP and LIN28, FMRP distribution is not granular (white arrow). C. Expression of the meiosis marker SYCP3 correlates with FMRP granulation (white arrowheads) in most but all of germ cells, suggesting that FMRP granulation precedes SYCP3 expression. D. Diagram outlining FMRP expression and granulation in germ cell development from primordial germ cells (PGCs) to meiotic oocytes. Abbreviations: ct, cortex; md, medulla. Scale bars: 50μm. Pictures are representatives from at least five different ovaries.</p

Immunohistochemical co-localisation of FMRP and P-body or stress granule markers in 2^nd trimester human fetal ovaries.

<p>Yellow signal indicates co-localisation. A. FMRP and the P-body marker DCP1a are expressed in distinct compartments within germ cells and no expression overlap is evident. B. The distribution of the P-body marker DDX6 is generally diffuse; some degree of co-localisation between FMRP granules and DDX6 is evident, possibly the result of random association of DDX6 with FMRP. C. The distribution of the P-body marker GW182 is distinctly granular in the human fetal ovary and there is a degree of association of GW182 and FMRP granules (white arrowheads). D. The distribution of the stress granule marker PABP1 is more diffuse towards the cortex and more granular towards the medulla; as with GW182, some PABP1 granules co-localise with FMRP granules. E. The distribution of the stress granule marker G3BP is distinctly granular. A portion of FMRP granules co-localise with G3BP Abbreviations: ct, cortex; md, medulla. Scale bars: 50μm. Pictures are representatives from at least five different ovaries.</p

Primary antibodies, secondary antibodies and fluorescent dyes used.

<p>Primary antibodies, secondary antibodies and fluorescent dyes used.</p

Immunohistochemical co-localisation of FMRP, GW182 and G3BP in human fetal ovaries at 15 wga.

<p>A. Yellow signal indicates co-expression of G3BP and GW182. Cyan signal indicates co-expression of GW182 and FMRP. Magenta signal indicates co-expression of G3BP and FMRP. White signal indicates co-expression of G3BP, GW182 and FMRP. B. Venn diagram depicting the relationship between granule components. The majority of granules positive for both G3BP and GW182 contained FMRP (75.5%).</p