Journal: Nature Communications

Article Title: Oocyte-derived microvilli control female fertility by optimizing ovarian follicle selection in mice

doi: 10.1038/s41467-021-22829-2

Figure Lengend Snippet: a Illustration of the strategy to label the derivation of the cellular communicating structures in the ZP by cellular specifically expressed CreER T2 or Cre recombinase. Membrane-localized red-fluorescent protein (mT) switches to green-fluorescent protein (mG) in GCs of Foxl2-CreER T2 ;mTmG follicles and oocytes of Gdf9-Cre;mTmG follicles. b Images of Foxl2-CreER T2 ;mTmG ovarian sections showing the high density of GC-TZPs (arrows), and labeling of a single GC showing the messy tree root-like structure of GC-TZPs in detail (right). c Images of Gdf9-Cre;mTmG ovarian sections, the mushroom-like Oo-Mvi (arrowheads) distributed in a low density (left) and high magnification showing the vesicle tips (arrowheads) of Oo-Mvi (right). d The developmental dynamics of Oo-Mvi (arrowheads), showing the establishment of Oo-Mvi accompanied by follicle growth. e Time-lapse imaging of Oo-Mvi in living oocytes showing the breakdown of vesicles (arrowheads) of Oo-Mvi (Supplementary Movie ). All experiments were repeated at least three times, and representative images are shown. Oo oocyte. GCs granulosa cells. ZP zona pellucida. b – e The colors were inverted to black/white (b/w) to highlight GC-TZPs ( b ) and Oo-Mvi ( c – e ). Scale bars: 5 μm ( b – d ) and 2 μm ( e ).

Article Snippet: To detect ERM family or GDF9 protein expression in the ovary, we incubated sections with the following primary antibodies: the anti-RDX antibody (rabbit, 1:100, ab52495, Abcam), anti-p-ERM antibody (rabbit, 1:100, mAb#3726, Cell Signaling Technologies), anti-EZRIN antibody (rabbit, 1:100, ab4069, Abcam), anti-MOESIN antibody (rabbit, 1:100, ab52490, Abcam), and anti-GDF9 antibody (goat, 1:50, AF739, R&D).

Techniques: Labeling, Imaging

Journal: Nature Communications

Article Title: Oocyte-derived microvilli control female fertility by optimizing ovarian follicle selection in mice

doi: 10.1038/s41467-021-22829-2

Figure Lengend Snippet: a Isolated ZP from Gdf9-Cre;mTmG oocytes with Oo-Mvi (arrowheads) kept in the samples for MS analysis. b MS analysis revealing the expression of RDX in the isolated ZP. c Relative mRNA levels of Rdx in different follicle components and tissues showing high Rdx expression in oocytes. n = 3 and P -value (Ovary vs. Oo) = 0.0014. d Western blot showing that RDX and p-ERM are highly expressed in oocytes. e Immunostaining showing RDX protein localized in the oocytes of follicles from the dormant primordial (arrowheads) to growing (arrow) stages. g p-ERM highly expresses on the membrane of oocytes in growing follicles (arrows) but not on that of dormant oocytes in primordial follicles (arrowheads). f , h High-magnification images showing the localization of RDX ( f ) or p-ERM ( h ) in the Oo-Mvi (arrowheads). All experiments were repeated at least three times, and representative images are shown. Data are presented as the mean ± SD with experiments performed in triplicate. Data were analyzed by two-tailed unpaired Student’s t -test and *** P < 0.001. Oo oocyte. GCs granulosa cells. ZP zona pellucida. Scale bars: 25 μm ( a ), 100 μm ( e , g ), 10 μm ( f , h ).

Article Snippet: To detect ERM family or GDF9 protein expression in the ovary, we incubated sections with the following primary antibodies: the anti-RDX antibody (rabbit, 1:100, ab52495, Abcam), anti-p-ERM antibody (rabbit, 1:100, mAb#3726, Cell Signaling Technologies), anti-EZRIN antibody (rabbit, 1:100, ab4069, Abcam), anti-MOESIN antibody (rabbit, 1:100, ab52490, Abcam), and anti-GDF9 antibody (goat, 1:50, AF739, R&D).

Techniques: Isolation, Expressing, Western Blot, Immunostaining, Two Tailed Test

Journal: Nature Communications

Article Title: Oocyte-derived microvilli control female fertility by optimizing ovarian follicle selection in mice

doi: 10.1038/s41467-021-22829-2

Figure Lengend Snippet: a Schematic representation of the deletion of Rdx exons 4 and 5 by Gdf9-Cre -mediated recombination in oocytes of Oo -Rdx −/− . b Immunofluorescence detection of RDX and p-ERM showing successful deletion of RDX in oocytes (arrowheads) of Oo -Rdx −/− . n = 6 ovaries per group. c , d Imaging the living oocytes ( c ) and ovarian tissue sections ( d ) showing a failure of the construction of Oo-Mvi on the oocyte surface in Oo- Rdx −/− ;mTmG females. n = 40 Oo- Rdx +/+;mTmG and 16 Oo- Rdx −/− ;mTmG oocytes ( c ), 12 Oo- Rdx +/+;mTmG and 9 Oo- Rdx −/−;mTmG ovaries ( d ). e Fertility check showing a subfertility phenotype in the Oo- Rdx −/− females with significantly decreased litter size and number and a shortened reproductive lifespan ( n = 8) compared to that in Oo- Rdx +/+ mice ( n = 9) during 40 weeks of mating. f A significantly lower ovulated oocyte number in Oo- Rdx −/− females ( n = 5) compared to that in Oo- Rdx +/+ mice ( n = 4), indicating that the abnormal development of oocytes caused subfertility in Oo- Rdx −/− females. P -value = 0.0036. Representative images are shown. Data are presented as the mean ± SD. Data were analyzed by two-tailed unpaired Student’s t -test and ** P < 0.01. Scale bars: 100 μm ( b , f ), 25 μm ( c , d ).

Article Snippet: To detect ERM family or GDF9 protein expression in the ovary, we incubated sections with the following primary antibodies: the anti-RDX antibody (rabbit, 1:100, ab52495, Abcam), anti-p-ERM antibody (rabbit, 1:100, mAb#3726, Cell Signaling Technologies), anti-EZRIN antibody (rabbit, 1:100, ab4069, Abcam), anti-MOESIN antibody (rabbit, 1:100, ab52490, Abcam), and anti-GDF9 antibody (goat, 1:50, AF739, R&D).

Techniques: Immunofluorescence, Imaging, Two Tailed Test

Journal: Nature Communications

Article Title: Oocyte-derived microvilli control female fertility by optimizing ovarian follicle selection in mice

doi: 10.1038/s41467-021-22829-2

Figure Lengend Snippet: a , b PCNA staining (arrows) of GC proliferation ( a ), showing a significant decrease in GC proliferation in the early growing follicles of Oo- Rdx −/− ovaries ( b ). Each point represents the GC-PCNA rate in a single follicle (PF: control n = 105, Oo- Rdx −/− n = 77, P -value = 0.0000011; SF: control n = 46, Oo- Rdx −/− n = 49, P -value = 0.0072). c , d TUNEL detection (arrows) of apoptosis ( c ), showing a significantly increased ratio of follicles with TUNEL-positive GCs in Oo- Rdx −/− ovaries ( n = 6). P -value = 0.0007 (PF), 0.02 (SF) ( d ). e F-actin staining GC-TZPs (arrows) showing a failure of GC-TZP construction in Oo- Rdx −/− oocytes. f Quantification of GC-TZPs confirmed a dramatically decreased density of GC-TZPs in Oo- Rdx −/− oocytes ( n = 18) compared to that in Oo- Rdx +/+ controls ( n = 7). P -value = 0.0000008. g Supplying GDF9 rescued the defect of GC-TZP formation in Oo- Rdx −/− ovaries. h Quantification of GC-TZPs in Oo- Rdx +/+ ( n = 20), Oo- Rdx −/− ( n = 19), and Oo- Rdx −/− +GDF9 ( n = 21) oocytes, showing the successful rescue of GC-TZP defect by GDF9 supplementation. P -value: 0.000000027 (Oo- Rdx +/+ vs. Oo- Rdx −/− ); 0.21 (Oo- Rdx +/+ vs. Oo- Rdx −/− +GDF9). Data are presented as the mean ± SD. PF primary follicle, SF secondary follicle. Representative images are shown. Data were analyzed by two-tailed unpaired Student’s t -test and *** P < 0.001, ** P < 0.01, * P < 0.05, n.s. P ≥ 0.05. Scale bars: 25 μm.

Article Snippet: To detect ERM family or GDF9 protein expression in the ovary, we incubated sections with the following primary antibodies: the anti-RDX antibody (rabbit, 1:100, ab52495, Abcam), anti-p-ERM antibody (rabbit, 1:100, mAb#3726, Cell Signaling Technologies), anti-EZRIN antibody (rabbit, 1:100, ab4069, Abcam), anti-MOESIN antibody (rabbit, 1:100, ab52490, Abcam), and anti-GDF9 antibody (goat, 1:50, AF739, R&D).

Techniques: Staining, TUNEL Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Oocyte-derived microvilli control female fertility by optimizing ovarian follicle selection in mice

doi: 10.1038/s41467-021-22829-2

Figure Lengend Snippet: a GDF9 was localized in the vesicles of Oo-Mvi. Immunostaining showing GDF9 protein (Red, arrowheads) localized in the oocytes and the vesicles of Oo-Mvi (Green, RDX). n = 29 ovarian follicles. b Rhodamine-labeled GDF9 (R-GDF9) or BSA (R-BSA) was microinjected into living oocytes at GV stage ( n = 15 per group), and the R-GDF9 intensity and distribution were detected at 15 or 30 min after injections (Supplementary Movies and ). The R-GDF9 spots surrounding the oocytes were pointed by arrowheads. c , d A time-dependent decrease of R-GDF9 intensity in oocytes ( c ) and increase of the number of R-GDF9 spots ( d ) were observed after injections. P -value: 0.0000014 ( c ), 0.00064 ( d ). e ER-tracker staining showed a cloudy ER distribution (top) in the oocyte cytoplasm, and the many ER bubbles (top, arrowheads) surrounding oocytes (Supplementary Movie ), which were similar to the R-GDF9 distribution ( b , arrowheads) and Oo-Mvi vesicles (bottom, arrowheads). f Quantification of the number of ER bubbles ( n = 17) and Oo-Mvi vesicles ( n = 14). P -value = 0.81. g Quantification of the fluorescence intensity of ER in oocyte cytoplasm and bubbles ( n = 20). P -value = 0.00000009. h Relative mRNA levels of Gdf9 , Bmp15, and Fgf8 in Oo- R dx +/+ and Oo- Rdx −/− oocytes showing a decreased consumption of OSFs in the oocytes of Oo- Rdx −/− females. P -value: 0.000006 ( Gdf9 ), 0.0001 ( Bmp15 ), 0.0019 ( Fgf8 ). i The proposed model of the function of Oo-Mvi in follicle development and female reproduction, showing that Oo-Mvi in the ZP determine the fate of individual follicles by regulating oocyte-GC communication. Data are presented as the mean ± SD with experiments performed in triplicate, and representative images are shown. Data were analyzed by two-tailed unpaired Student’s t -test and *** P < 0.001, ** P < 0.01, and n.s. P ≥ 0.05. Scale bars: 5 μm ( a ); 20 μm ( b , e ).

Article Snippet: To detect ERM family or GDF9 protein expression in the ovary, we incubated sections with the following primary antibodies: the anti-RDX antibody (rabbit, 1:100, ab52495, Abcam), anti-p-ERM antibody (rabbit, 1:100, mAb#3726, Cell Signaling Technologies), anti-EZRIN antibody (rabbit, 1:100, ab4069, Abcam), anti-MOESIN antibody (rabbit, 1:100, ab52490, Abcam), and anti-GDF9 antibody (goat, 1:50, AF739, R&D).

Techniques: Immunostaining, Labeling, Staining, Fluorescence, Two Tailed Test