Loss of Kdm5/Lid results in high H3K4me3 and abnormal karyosomes independently from the meiotic recombination checkpoint.

<p>(A) Schematic representation of <i>Drosophila</i> oogenesis. A germline stem cell produces a cystoblast which undergoes four rounds of pre-meiotic mitosis to form a cyst consisting of 16 cells. In region 2a, up to four cells in a cyst initiate meiosis and form the SC. In region 2b, two cells (pro-oocytes) maintain the meiotic state. By region 3, one of these cells is finally selected as the oocyte, and all other cells have become nurse cells. Karyosome forms at stage 2–3 of oogenesis. At stage 3 and later, the SC gradually disassembles from chromosome arms, except in centromeric regions. At stage 14, the oocyte completes maturation and arrests in meiotic metaphase I. (B) The spatial distribution and quantified level of H3K4me3 in control and <i>Kdm5/lid</i> RNAi oocytes. The total H3K4me3 signal intensity was significantly higher in <i>Kdm5/lid</i> RNAi oocytes at all stages (p<0.001). n≥7. Error bars represent the standard errors of the mean. reg; region, st; stage. (C) Karyosome defects caused by two different shRNAs (<i>lid</i> RNA1 and <i>lid</i> RNAi2) and a <i>Kdm5/lid</i> mutant (<i>lid</i>^{<i>10424/k06801</i>}). The karyosome morphologies at stages 3–9 were classified into three categories: "spherical" when the karyosome shows a spherical shape, "mildly distorted" when spherical shape was distorted but largely maintained, and "distorted" when spherical shape was largely disrupted. *** indicates a significant difference from the control (p<0.001). n≥18. (D) γH2Av foci which mark DSBs in meiotic nuclei in region 2a, 2b and region 3 of control and <i>Kdm5/lid</i> RNAi ovaries. DSBs are repaired by region 3 in both. Meiotic nuclei were identified by C(3)G staining. n≥12. (E) Karyosome morphology at stage 4/5 in control RNAi, <i>Rad51/spnA</i>, <i>Kdm5/lid RNAi</i>, <i>Chk2/mnk</i>^<i>p</i>6/+ <i>spnA</i> double mutant, <i>Chk2/mnk</i>^p6/+ mutant with <i>Kdm5/lid</i> RNAi, and <i>Kdm5/lid p53</i> double RNAi. The frequency of abnormal karyosomes at stage 3–9 was quantified for each genotype. n≥16. Suppression of meiotic checkpoint does not rescue the karyosome defect of <i>Kdm5/lid</i> RNAi (p = 1.00). Scale bars = 5 μm.</p

Kdm5/Lid demethylase activity is dispensable for meiotic chromatin reorganisation.

<p>(A) Distribution and intensity of H3K4me3 on meiotic chromosomes in a <i>Kdm5/lid</i> mutant (<i>lid</i>^{<i>10424/k06801</i>}) without a transgene (–), the <i>Kdm5/lid</i> mutant carrying a wild-type transgene (<i>lid[WT])</i> and <i>the Kdm5/lid</i> mutant carrying a catalytically inactive transgene (<i>lid[JmjC*]</i>). Images of the karyosome in stage-5 oocytes were taken and the contrast has been enhanced using identical conditions. The total signal intensity of H3K4me3 on the karyosome was measured as described in Materials and Methods. Error bars indicate standard errors. n≥6. The signal intensity in the <i>Kdm5/lid</i> mutant carrying no transgenes (–) or the <i>lid[JmjC*]</i> transgene is significantly different from the one carrying the <i>lid[WT]</i> transgene at all stages (p<0.001). (B) Rescue of karyosome defects of the <i>Kdm5/lid</i> mutant by both the wild-type transgene (<i>lid[WT]</i>) and demethylase-inactive transgene (<i>lid[JmjC*]</i>). Karyosome morphology was observed in the <i>Kdm5/lid</i> mutant oocytes carrying no transgenes (–), the <i>lid[WT]</i> transgene or the <i>lid[JmjC*]</i> transgene, and was classified as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006241#pgen.1006241.g001" target="_blank">Fig 1C</a>. *** indicates a significant difference in the pattern of distribution from the control (p<0.001). n≥18. (C) Centromere pairing and clustering do not require the demethylase activity of Kdm5/Lid. Centromeres highlighted by the Cid antibody are indicated by arrows, and foci of pericentromeric dodeca satellite specific to chromosome 3 (periCen3) are indicated by arrowheads. The numbers of centromere (Cid) clusters were counted for each group of stages (n≥12). Oocyte nuclei with tightly paired (<1 μm) or separate (≥1 μm) periCen3 foci were counted for each group of stages (n≥11). ***, ** and * indicate significant differences (p<0.001, p<0.01 and p<0.05, respectively). (D) SC morphology is not affected by loss of the Kdm5/Lid demethylase activity. Filamentous structures of C(3)G observed in region 3 oocytes from the <i>Kdm5/lid</i> mutant carrying the <i>lid[WT]</i> or <i>lid[JmjC*]</i> transgene, and a spotty appearance observed in the <i>Kdm5/lid</i> mutant alone. The SC component C(3)G and H3K4me3 were co-stained, imaged and contrast-enhanced using identical conditions. The morphology of the C(3)G-containing structure was categorised and counted for each group of stages (n≥10). ***, ** and * indicate significant differences (p<0.001, p<0.01 and p<0.05, respectively). Scale bars = 5 μm in all images.</p

Homologous centromere pairing is disrupted upon loss of Kdm5/Lid.

<p>(A) Centromere clustering in pre-meiotic nuclei of 16-cell cysts (16 cc) in wild-type and <i>Kdm5/lid</i> mutant ovaries. Parts of the germarium containing 16-cell cyst (circled) are shown in the left panels (Scale bar = 5 μm), and magnified images of one cell, in each case, are shown in the right panels (Scale bar = 3 μm). The anterior end of the germarium is oriented towards the top. The stage of each cyst was determined using the morphology of fusome visualised by Hts. (B) Increased number of centromere clusters in <i>Kdm5</i>/<i>lid</i> mutant oocytes in comparison to wild type in region 3. The SC component C(3)G was used to identify oocytes. Scale bars = 3 μm. (C) The number of centromere clusters in pre-meiotic and meiotic nuclei in various oogenesis stages of wild type and the <i>Kdm5/lid</i> mutant. cc; cell cyst, reg; region, st; stage. ** and *** (p<0.01 and 0.001) indicate significant differences from wild type in terms of the frequency of nuclei with one or two centromere clusters. ≥32 nuclei and meiotic nuclei were quantified for each pre-meiotic stage and region 2a, while ≥16 germaria were quantified for each later meiotic stage. (D) A schematic diagram showing three levels of centromere association in oocytes, cohesion of sister-centromeres, pairing of homologous centromeres and clustering of non-homologous centromeres. (E) Closely paired signals of the pericentromeric dodeca satellite specific to chromosome 3 (periCen3) that co-localise with CenpA/Cid foci in the wild-type oocyte at stage 6, and which are clearly separated into two foci in the <i>Kdm5/lid</i> RNAi oocyte at stage 6. Arrows indicate Cid and periCen3 foci. Scale bars = 5 μm. (F) Schematic representation of the behaviour of the pericentromere 3 signals (periCen3) in control and <i>Kdm5/lid</i> RNAi oocytes. (G) The proportion of paired or separate pericentromeric signals (periCen3) in control and <i>Kdm5/lid</i> RNAi oocytes. Two signals separated by ≥1 μm were defined as "separate" in this quantification. ** indicates significant difference from the control (p<0.01). n≥17.</p

Loss of Kdm5/Lid leads to instability of chromosome cores along arms but does not affect persistence at centromeres.

<p>(A) SMC1 signals in meiotic nuclei in region 2a and 2b of control and <i>Kdm5/lid</i> RNAi ovaries, showing filamentous patterns in control, and fragmented filaments in region 2a and mainly diffuse pattern in region 2b of <i>Kdm5/lid</i> RNAi ovaries. (B) Quantification of SMC1 staining pattern in control and <i>Kdm5/lid</i> RNAi meiotic nuclei. *** indicates a significant difference in the pattern distribution from the control (p<0.001). n≥7. Chromosome cores visualised by filamentous SMC staining fails to be maintained in <i>Kdm5/lid</i> RNAi. (C) Centromeric SMC1/3 localisation in control and <i>Kdm5/lid</i> oocytes at stage 4. Colocalisation between Cid and SMC1/3 foci was observed in all oocytes examined (n = 13 for stage 4–6). Arrows indicate Cid and SMC1/3 foci. Scale bars = 5 μm.</p

Loss of Kdm5/Lid leads to partial formation and instability of the SC along chromosome arms but does not affect persistence of a transverse protein at centromeres.

<p>(A) Schematic representation of early meiotic events. In region 2a, the SC starts to assemble to promote synapsis which results in crossover and chiasmata formation. The filamentous structure of the SC mostly disassembles by stage 6 of oogenesis except at centromeres. (B) A control region-3 oocyte with filamentous C(3)G staining, and a <i>Kdm5/lid</i> RNAi oocyte with spots of C(3)G staining. (C) Quantification of C(3)G staining pattern in control and <i>Kdm5/lid</i> RNAi oocytes. For region 2a, we classified each germarium based on the majority of the SC morphology in multiple nuclei which accumulate C(3)G. For region 2b where the two nuclei accumulate C(3)G, each germarium was scored for the morphology of the better formed SC. ** and *** indicate significant differences in the pattern distribution from control (p<0.01 and p<0.001, respectively). n≥14. (D) Centromeric C(3)G localisation in control and <i>Kdm5/lid</i> RNAi oocytes at stage 5. Cid, the <i>Drosophila</i> CenpA, highlights all centromeres. The arrows indicate colocalisation of the Cid signals with centromeric C(3)G. Colocalisation was observed in all oocytes examined (n = 27 for stage 4–6). Scale bars = 5 μm.</p

Abnormal chromosome positioning and orientation in prometa/metaphase I and a potential reduction in crossovers in oocytes lacking Kdm5/Lid.

<p>(A) Mis-positioned chromosomes with normal spindle morphology in <i>Kdm5/lid</i> RNAi oocytes in prometa/metaphase I in comparison to control RNAi. Chromosome orientation was assessed by <i>in situ</i> hybridisation using dodeca satellite as a pericentromere 3 probe (arrows). Scale bar = 5 μm. (B) Quantification of chromosome configuration in control and <i>Kdm5/lid</i> RNAi oocytes. The “other” category includes a meiotic figure with more than two foci of the pericentromere 3 signal. The frequency of mis-oriented pericentromere 3 in <i>Kdm5/lid</i> RNAi is significantly different from the control RNAi (p<0.05). n≥45. (C) Localisation and signal intensity of Vilya-3xHA foci in meiotic nuclei in region 2a and 2b of control and <i>Kdm5/lid</i> ovaries expressing HA-tagged Vilya. Scale bar = 5 μm. (D) The signal intensity of Vilya^3xHA foci in region 2a and 2b meiotic nuclei of control and <i>Kdm5/lid</i> RNAi ovaries expressing HA-tagged Vilya. The graphs show the numbers of foci per meiotic nucleus with the maximum signal intensity in indicated ranges. Foci with a signal intensity lower than 30 are significantly more frequent in <i>Kdm5/lid</i> RNAi than in control (p<0.001). Intensities of ≥66 Vilya^3XHA foci have been measured for each region of each genotype.</p

Characterization of Self-reported Improvements in Knowledge and Health Among Users of Flo Period Tracking App: Cross-sectional Survey

BackgroundResearch shows that poor knowledge and awareness of menstrual and pregnancy health among women are associated with adverse reproductive health and pregnancy outcomes. Menstrual cycle– and pregnancy-tracking mobile apps are promising tools for improving women’s awareness of and attitudes toward their reproductive health; however, there is little information about subscribers’ perceptions of app functionality and its impact on their knowledge and health. ObjectiveThis study aimed to explore knowledge and health improvements related to menstrual cycle and pregnancy, as well as improvements in general health among Flo app users. We also investigated what components of the Flo app were associated with the abovementioned improvements and evaluated whether those improvements differed based on education level, country of residence (low- and middle-income vs high-income countries), free or premium subscription to the app, short- or long-term use of the app, and frequency of use. MethodsFlo subscribers who had been using the app for no less than 30 days, completed a web-based survey. A total of 2212 complete survey responses were collected. The survey included demographic questions and questions about motivations guiding the use of the Flo app and which components of the app improved their knowledge and health, as well as to what extent. ResultsMost study participants reported improvements in menstrual cycle (1292/1452, 88.98%) and pregnancy (698/824, 84.7%) knowledge from Flo app use. Participants with higher levels of education and those from high-income countries reported using the app predominantly for getting pregnant (χ21=4.2, P=.04; χ21=52.3, P<.001, respectively) and pregnancy tracking (χ21=19.3, P<.001; χ21=20.9, P=.001, respectively). Participants with less education reported using the app to avoid pregnancy (χ21=4.2; P=.04) and to learn more about their body (χ21=10.8; P=.001) and sexual health (χ21=6.3; P=.01), while participants from low- and middle-income countries intended to mainly learn more about their sexual health (χ21=18.2; P<.001). Importantly, the intended use of the app across education levels and country income levels matched areas in which they had gained knowledge and achieved their health goals upon use of the Flo app. Period, fertile days, and ovulation predictions as well as symptom tracking were consistently the top 3 components in the app that helped users with their cycle knowledge and general health. Reading articles or watching videos helped with users' education regarding their pregnancy. Finally, the strongest improvements in knowledge and health were observed in premium, frequent, and long-term users. ConclusionsThis study suggests that menstrual health apps, such as Flo, could present revolutionary tools to promote consumer health education and empowerment on a global scale