Characterization of identified LT-LRCs

<p>(<b>12 week chase</b>) <b>in mouse distal oviduct.</b> Immunofluorescent double-labeling was performed for GFP and ERα (A), GFP and PR (B), GFP and Ki67 (C), GFP and CD44 (D) and CD44 and ERα (E). DAPI staining was performed to display all cell nuclei. White arrowheads indicate Ki67 positive cells, the yellow arrowhead indicates a cell which is positive for CD44 as well as GFP.</p

Differentiation of spheroids towards specific cell lineages of the female reproductive tract.

<p>Early spheroids (2 days of culture, A), late spheroids (2 weeks of culture, B) and spheroids which were stimulated to differentiate (10% FCS added to the culture medium for 2 days, C) were stained for ERα, CD44, PR and PAEP expression and compared to stained sections from mice distal oviduct (D), proximal oviduct (E) and endometrium (F). The red arrows indicate ERα positive cells, the black arrows indicate ERα negative cells. In panel A, 4 different spheroids were used, in panel B two (ER and PR, CD44 and PEAP stainings were performed on the same spheroid, respectively) and stainings in panel C are on consecutive sections from one spheroid. Images are representative for three different experiments containing more than 10 early and late spheroids and 2 – 3 differentiated spheroids.</p

Isolated LT-LRC can form self-renewing spheroids in culture.

<p>Oviducts from 12 week chased mice were dissected and a single cell suspension was analyzed by FACS. From three different 12 week chased mice, GFP⁺ and GFP-negative (GFP−) cells were isolated by FACSorting and cultured in Matrigel under serum-free conditions to assay their capacity to form spheroids (A). Self-renewal was assayed by dissociating spheroids and plating the unsorted cell suspension to form new spheroids as indicated in the Materials and Methods section. In short, primary spheroids (i.e. obtained directly from the chased animals) were reduced to single cell suspensions and plated again (unsorted) in matrigel/serum-free culture conditions. The corresponding secondary organoids were then dissociated and plated to obtain tertiary ones (B). Using limiting dilutions, individual GFP⁺ cells were cultured for 0 to 20 days in Matrigel under serum-free conditions, thus forming a polarized epithelial organoid. The bars represent 50 μM (C).</p

Spheroids differentiation assay.

<p>20 day old spheroids derived from oviducts of 12 week chased mice were pulsed for one day with doxycycline and were subsequently allowed to differentiated for 0 (A), 1 (B and C), 2 (D and E), 3 (F) or 6 days (G) in the presence of 10% FCS. The two columns of images on the left hand side of the figure represent confocal images taken at two different planes (bottom and middle). The phase-contrast images in the third colum from the left represents a detail from the confocal image. C represents a spheroid derived from a different animal which was induced to differentiate for 1 day, displaying formation of multiple cell layers as indicated by nuclear DAPI staining and immunofluorescence for CK8. In E attachment of the spheroid to the culture dish is shown. H and I represent spheroids isolated from different animals that were allowed to differentiate for 9 days (H) and for 20 days (I) showing complex structures (nuclei were stained with Hoechst 34580).</p

Pulse-chase experiment using the doxycycline-inducible H2B-GFP system.

<p>A schematic representation of the experiment is given in A, where a 7-day treatment with doxycycline (pulse) results in expression of H2B-GFP throughout the entire organism. At different chase time points (12 and 47 weeks after pulse), the GFP signal is progressively diluted from dividing cells and retained in quiescent cells in the distal oviduct (B). In the proximal oviduct (C) and the endometrium (D), label-retaining cells (LRCs) are lost at both 12 and 47 weeks chase time points. FACSorting was performed on single cell digestions of oviducts from different mice. In (E), the GFP signal is plotted against the number of cells. Animals used were: untreated mice as negative controls (black line, Negative); mice pulsed for 7 days (no chase) as positive controls (red line, Pulse); mice pulsed for 7 days and chased for 12 weeks (green line, 12 weeks); and mice pulsed for 7 days and chased for 47 weeks (light-green line, 47 weeks). The scale bar represents 50 μM.</p

Number of different subtypes of meiotic nuclei and frequency of pachytene nuclei with a pseudo-XY body in E16.5 and E17.5 oocytes from <i>Spo11^+/+</i> and <i>Spo11^+/YF</i>embryos.

*<p><b>percentage of the total number of counted nuclei.</b></p

Relocalisation of persistent radiation-induced DSBs into a pseudo XY body.

<p>(A) Irradiated <i>Spo11^YF/YF</i> spermatocytes were collected 1 h, 48 h and 120 h upon irradiation and immunostained for RAD51 (green), SYCP3 (red), and γH2AX (blue). Spermatocytes that were irradiated at the leptotene stage, should have reached zygotene and pachytene with respect to the pattern of γH2AX, at 48 and 120 h following irradiation, respectively. (B) Fraction of cells showing a pseudo XY body upon irradiation at the analysed time-points (n = 50). (C) Immunostaining of <i>Spo11^YF/YF</i> spermatocyte 120 hours after irradiation with anti-RNA pol II (green) and anti-γH2AX (red). The intense γH2AX domain (pseudo XY body) corresponds to a nuclear area depleted for RNA pol II.</p

SPO11-dependent and -independent RAD51 foci in mouse meiocytes.

<p>(A–C) The number of RAD51 foci decreases from leptotene to zygotene in <i>Spo11^+/+</i> and <i>Spo11^+/YF</i> spermatocytes, whereas a few foci are detected in <i>Spo11^YF/YF</i> spermatocytes and oocytes at both stages. (A–B) Double immunostaining with anti-SYCP3 (red), anti-RAD51 (green) of spermatocyte (A) and oocyte (B) nuclei from <i>Spo11^+/+</i> (A–B, left panel) and <i>Spo11^YF/YF</i> (A–B, right panel) mice. Arrowheads point to RAD51 foci in <i>Spo11^YF/YF</i> spermatocytes and oocytes, both leptotene and zygotene. Extensive accumulation of RAD51 along axial elements of one or few chromosomes (arrows) can be observed in both <i>Spo11^+/+</i> and <i>Spo11^YF/YF</i> oocyte nuclei (B, lower panel). Size bars represent 10 µm. (C) The number of RAD51 foci was counted in <i>Spo11^+/+</i>, <i>Spo11^+/YF</i>, and <i>Spo11^YF/YF</i> leptotene and zygotene spermatocytes and oocytes. Each dot represents the focus count of one nucleus. Black bars indicate mean number of foci. P values for the indicated comparisons (Mann-Whitney, two-tailed), and genotypes are indicated in the plot. (D) The number of MLH1 foci in pachytene spermatocyte nuclei was counted in <i>Spo11^+/+</i>and <i>Spo11^+/YF</i> mice. Black bars indicate the mean values. (E) Number of RAD51 foci at E17.5 in <i>Spo11^+/+</i> and <i>Spo11^YF/YF</i> oocytes.</p

Enrichment of DNA repair markers in the pseudo XY body of <i>Spo11^YF/YF</i> spermatocytes.

<p>(A) Nuclei of <i>Spo11^YF/YF</i>zygotene spermatocytes were divided in four subgroups depending on their positivity for the pseudo XY body and for foci of one of the three DNA repair proteins RAD51 (n = 120), DMC1 (n = 227) or RPA (n = 108) as follows: 1) with pseudo XY body and with foci, 2) with pseudo XY body and without foci, 3) without pseudo XY body and with foci, 4) without pseudo XY body and without foci. Spermatocyte nuclei were immunostained with anti-SYCP3 (red), anti-γH2AX (blue), and one of the following antibodies: anti-RAD51 (green, upper panel), anti-DMC1 (green, middle panel) or RPA (green, lower panel). Every panel shows a representative nucleus for each of the four subgroups mentioned above. Numbers in the bottom left corner of every picture represent the percentage of nuclei of this type in the analyzed cell population. (B) The average number of RAD51, DMC1 and RPA foci per nucleus was counted in spermatocytes of the first subgroup (outlined in red). The table also shows the percentage of foci located within a pseudo XY body and the percentage of pseudo XY bodies which contained at least one focus. (C) Scatter plot representing the colocalization percentage in relation to the fraction of the nuclear area occupied by the pseudo XY body. Every dot represents a nucleus. Pearson linear correlation coefficient [Pcorr] = 0.0741.</p

Pseudo XY bodies containing synapsed axes in wild type embryonic oocytes.

<p>(A–B) Triple immunostaining with anti-HORMAD1 (red), anti-TEX12 (green) and anti-γH2AX (blue) of oocyte nuclei from E17.5 wild type embryos. In the lower right corner percentages are reported, representing the frequency of each type of cell in the pachytene oocyte population (n = 244). (A) Representative pictures of early zygotene (EZ), late zygotene (LZ), early pachytene (EP) and pachytene (P) oocytes, from left to right. HORMAD1 levels are decreasing while TEX12 accumulates as synapsis progresses. Parallel to the increase of synapsis and HORMAD1 removal, γH2AX accumulation decreases. (B) Representative pictures of pachytene oocytes with a pseudo XY body. HORMAD1 positive axes totally (left picture) or partially (middle picture) colocalize with the pseudo XY body, or are not present (right picture) in the pseudo XY body. (C) Scatter plot of the total length of synapsed axes in E17.5 wild type pachytene oocytes, belonging to the following categories: HORMAD1 and pseudo XY body absent (blue); HORMAD1 absent and pseudo XY body present (light blue); presence of both HORMAD1 and a pseudo XY body. Every dot represents a nucleus. Black bars indicate the mean values. P values for the indicated comparison (Mann-Whitney, two-tailed) are shown in the plot. (D–E) Triple immunostaining with anti-TEX12 (white), anti-RNA polymerase II (green), and anti-γH2AX (red) of pachytene oocytes from E17.5 wild type embryos, imaged with the Zeiss LSM700 confocal microscope. Depletion of RNA pol II can be observed in the area of the the pseudo XY body marked by γH2AX, both when synapsis is complete (D) and when unsynapsed axes (E) are present in this region (E). Size bars represent 10 µm.</p