<i>Figla</i>-<i>Cre</i> Transgenic Mice Expressing Myristoylated EGFP in Germ Cells Provide a Model for Investigating Perinatal Oocyte Dynamics

<div><p>FIGLA (Factor in the germline, alpha) is a bHLH transcription factor expressed abundantly in female and less so in male germ cells. Mice lacking FIGLA do not form primordial follicles in the ovary and females are sterile, but there is no obvious phenotype in males. Using the <i>Figla</i> promoter to express Cre recombinase, we have established <i>mEGFP/mTomato</i> reporter mice with green germ cells and red somatic tissue. These mice were crossed into the <i>Figla</i> null background to accelerate perinatal oocyte loss. Live imaging of cultured newborn ovaries provides evidence that few oocytes egress and the vast majority disappear within the confines of the ovary. Although a cohort of mobile, phagocytic cells was observed, macrophage depletion in <i>Csf1^op/op</i> mice did not affect oocyte loss. Investigations with TUNEL assays and caspase inhibitors suggest that apoptosis plays a role in the perinatal loss of oocyte in female mice. These results establish the utility of <i>Figla-EGFP/Cre; mTomato/mEGFP</i> in investigating germ cell dynamics in prepubertal mice.</p></div

Oocyte apoptosis in <i>Figla</i> null ovary is caspase dependent.

<p>Newborn ovary pairs from <i>Figla</i> null (A) and normal mice (B) were cultured <i>in vitro</i> with or without 100 µM caspase inhibitors (pan caspase: Z-VAD-fmk; caspase 2: Z-VDVAD-fmk; caspase 3: Z-DEVD-fmk; caspase 8: Z-IETD-fmk; caspase 12: Z-ATAD-fmk), 2 µM Bafilomycin A1, or 100 µM bpV(pic) plus 500 µg/mL 740Y-P for 2 (except for pancaspase which was for 7) days at 37 °C. (A) Oocyte loss in <i>Figla</i> null ovaries was significantly inhibited in the presence of the pancaspase inhibitor and to a lesser extent with inhibitor for caspase 8 and 3. (B) The presence of inhibitors did not morphologically or physiologically affect normal oocytes. In these assays, only ovary pairs with comparable numbers of healthy oocytes were selected for <i>in vitro</i> culture. The hilum of each ovary was juxtaposed on the filter (Fig. 1D) and images were obtained by laser scanning confocal microscopy from the opposite side of the ovary. Z projections of EGFP fluorescence intensity were collapsed into a single plane to visualize the number of germ cells before and after treatment with inhibitors. Scale bar, 100 µm.</p

EGFP expression in <i>Figla-EGFP/Cre</i> transgenic mice.

<p>(A) Female (1) and male (2) embryonic gonads at E14.5 and E16.5, respectively, were dissected from hemizygous <i>Figla-EGFP/Cre</i> transgenic mice and whole-mount images were obtained by laser scanning confocal microscopy. (B) After crossing into <i>mTomato/mEGFP</i> reporter mice, EGFP was detected in either ovary (1,2) or testis (3) of newborn mice where it was more intense at the membrane than in the cytoplasm. It was not detected in heart (4), kidney (5) or liver (6). Strong auto-fluorescence was present in hepatic tissues.</p

FIGLA, a Basic Helix-Loop-Helix Transcription Factor, Balances Sexually Dimorphic Gene Expression in Postnatal Oocytes▿

Maintenance of sex-specific germ cells requires balanced activation and repression of genetic hierarchies to ensure gender-appropriate development in mammals. Figla (factor in the germ line, alpha) encodes a germ cell-specific basic helix-loop-helix transcription factor first identified as an activator of oocyte genes. In comparing the ovarian proteome of normal and Figla null newborn mice, 18 testis-specific or -enhanced proteins were identified that were more abundant in Figla null ovaries than in normal ovaries. Transgenic mice, ectopically expressing Figla in male germ cells, downregulated a subset of these genes and demonstrated age-related sterility associated with impaired meiosis and germ cell apoptosis. Testis-associated genes, including Tdrd1, Tdrd6, and Tdrd7, were suppressed in the transgenic males with a corresponding disruption of the sperm chromatoid body and mislocalization of MVH and MILI proteins, previously implicated in posttranscriptional processing of RNA. These data demonstrate that physiological expression of Figla plays a critical dual role in activation of oocyte-associated genes and repression of sperm-associated genes during normal postnatal oogenesis

Gonad-specific expression of Cre in the <i>Figla-EGFP/Cre</i>; <i>mTomato/mEGFP</i> mice.

<p>(A) Transgene designed for bicistronic expression of both EGFP and Cre under the control of <i>Figla</i> promoter (3.8 kb) was used to generate the <i>Figla-EGFP/Cre</i> transgenic mice. EGFP is present in the cytoplasm (Cyto) of germ cells. (B) Reporter mice with floxed <i>mTomato</i> (myristoylated Tomato) and <i>mEGFP</i> driven by the universal β-actin/CMV promoter (1.7 kb). <i>mTomato</i> is expressed in somatic and germ cells where it is anchored to the membrane (Memb). (C) Mice with both (A) and (B) transgenes express mTomato in all somatic cells including those in the gonads. Both cytoplasmic (from A) and membrane bound (from B) EGFP are present in germ cells in double transgenic mice (dTg). (D) Isolated mouse ovaries were imaged by confocal microscopy using a 20× objective and a petri dish with a glass coverslip. The ovary was placed under a tilted Millicell Cell Culture Insert with a filter bottom that was filled with 1.2% agarose (DMEM/FBS) and surrounded a ring of agarose gel to maintain moisture. The cover and petri dish were tightly opposed with a tight-fitting parafilm pad and wrapped in parafilm with pores allowing gas exchange with an outside environmental chamber (37 °C, 5% CO₂).</p

Macrophages involved in clearance of dead oocytes in normal and <i>Figla</i> null ovaries.

<p>(A) Time-lapse recording of a normal <i>Figla-EGFP/Cre</i>; <i>mTomato/mEGFP</i> newborn ovary captured a destruction process of an oocyte (circle). A somatic cell (arrowhead) closely associates with the oocyte and appears to phagocytize the remains of the degrading oocyte. (B) Normal or <i>Figla</i> null newborn ovary sections were immuno-stained with F4/80, a macrophage specific marker. Ovarian tissue is outlined by dotted line. (C) Oocytes and macrophages were stained by Mvh (red) or F4/80 (green), respectively. Macrophage-engulfed degrading oocytes were observed in <i>Figla</i> null (dotted circles), but not normal ovaries.</p