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, 10 (3), e0119819
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GDF9 Is Transiently Expressed in Oocytes Before Follicle Formation in the Human Fetal Ovary and Is Regulated by a Novel NOBOX Transcript

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GDF9 Is Transiently Expressed in Oocytes Before Follicle Formation in the Human Fetal Ovary and Is Regulated by a Novel NOBOX Transcript

Rosemary A L Bayne et al. PLoS One.

Abstract

During human fetal ovary development, the process of primordial follicle formation is immediately preceded by a highly dynamic period of germ cell and somatic cell reorganisation. This is regulated by germ-cell specific transcription regulators, by the conserved RNA binding proteins DAZL and BOLL and by secreted growth factors of the TGFβ family, including activin βA: these all show changing patterns of expression preceding follicle formation. In mice, the transcription factor Nobox is essential for follicle formation and oocyte survival, and NOBOX regulates the expression of GDF9 in humans. We have therefore characterised the expression of GDF9 in relation to these known key factors during follicle formation in the human fetal ovary. mRNA levels of GDF9, BMP15 and NOBOX were quantified by qRT-PCR and showed dramatic increases across gestation. GDF9 protein expression was localised by immunohistochemistry to the same population of germ cells as those expressing activin βA prior to follicle formation but did not co-localise with either BOLL or DAZL. A novel NOBOX isoform was identified in fetal ovary that was shown to be capable of up-regulating the GDF9 promoter in reporter assays. Thus, during oogenesis in humans, oocytes go through a dynamic and very sharply demarcated sequence of changes in expression of these various proteins, even within individual germ cell nests, likely to be of major functional significance in determining selective germ cell survival at this key stage in ovarian development. Transcriptional variation may contribute to the range of age of onset of POI in women with NOBOX mutations.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. GDF9 is expressed in the human fetal ovary.
qRT-PCR analysis of GDF9 (A), BMP15 (B) and NOBOX (C) mRNA expression in human fetal ovary across the gestational range of 8 to 20 weeks. Ovaries (n = 5–7 per group) were grouped according to developmental stage and transcript levels measured relative to those of RPL32. Bars indicate mean±sem. Statistically different levels are indicated by asterisks above the columns, thus expression of GDF9 at 18–20 weeks was significantly higher than at 8–11 weeks (p<0.005) as was expression of BMP15 and of NOBOX (both p<0.01). DAB immunohistochemical detection of GDF9: 19 week (D, E) and 20 week (F) human fetal ovary stained with anti-GDF9 antibody or normal goat IgG negative control (F inset)—positive staining is brown. Thick arrows indicate primordial follicles and thin arrows germ cells that are not stained for GDF9 while the arrowheads indicate primordial follicles that are positive for GDF9. Scale bars are 50μm (D and F) and 20μm (E).
Fig 2
Fig 2. Co-localisation of GDF9 with activin βA but not DAZL or BOLL prior to follicle formation.
(A) Double immunohistochemistry of 18 week fetal ovary stained for GDF9 (green) and activin βA (red), thus in the merged image co-expression is yellow. Unstained germ cells are indicated with arrows. Counterstain is TOPRO. (B) Triple fluorescent immunohistochemistry for GDF9 (green), DAZL (blue) and BOLL (red) in 20 week human fetal ovary with DAPI as counterstain (grey). Split channel and merged images in (A) and (B) are shown as are merged images of non-immune serum negative control (NEG). Scale bars are 20μm. (C) Nuclear diameters of DAZL, BOLL and GDF9 stained germ cells indicates that GDF9 positive cells are significantly larger (p<0.001) than DAZL but not BOLL expressing cells (bars indicate mean ± sem). (D) Higher magnification merged image of GDF9/DAZL/BOLL immunohistochemistry showing one large primordial follicle is positive for both GDF9 and DAZL but other follicles are positive only for DAZL.
Fig 3
Fig 3. Structure of the human NOBOX gene and expression of exons in the human fetal ovary.
(A) Database analysis of human NOBOX transcripts identified 3 possible transcripts for human NOBOX. Only those exons marked in black have been confirmed previously at the experimental level [41]. Exon and intron sizes are indicated and primers used for RT-PCR are shown above each exon. (B) Agarose gel analysis of RT-PCR products using primer pairs as indicated above the lanes. (+) and (-) indicate RT+ and RT- fetal ovary cDNA template. Size marker is the 100bp ladder (Promega) where the 500bp band is more intense. The arrow indicates the position of the weak band identified with the 2Fa + 3Rb primer pair. (C) Structure of the mouse Nobox locus for comparison with the human sequences.
Fig 4
Fig 4. Protein sequence of the new human NOBOX isoform aligned with mouse Nobox.
Human NOBOX protein (NP_001073882.2) and mouse Nobox protein (NP_570939.1) sequences are aligned. Alternating exons are coloured red and black, with the exon that is split in mouse coloured blue. The homeodomain is shaded grey.
Fig 5
Fig 5. Expression of human NOBOX enhances expression from promoters containing NBEs in luciferase assays.
Luciferase assays were performed on HEK293 cells transfected with a selection of promoter-luciferase plasmids containing (GDF9 and ZP3) or lacking (ZP2) a putative NOBOX Binding Element (NBE) in combination with either NOBOX expression plasmid or empty vector. Data represent the mean (±sem) activity from at least 5 separate experiments. Statistically significant differences are denoted by asterisks.

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