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First published online 11 November 2008
doi: 10.1242/jcs.036400

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Effect of cell shape and packing density on granulosa cell proliferation and formation of multiple layers during early follicle development in the ovary

¹ Institute of Reproductive and Developmental Biology, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK
² Department of Histopathology, Charing Cross Hospital, Fulham Palace Road, London W6 8RF, UK
³ Department of Mathematics and Centre for Integrative Systems Biology at Imperial College (CISBIC), Imperial College London, London SW7 2AZ, UK

View larger version (77K): [in this window] [in a new window]   Fig. 1. (A) Summary of cellular changes that take place during initiation of follicle growth. (B) Stages of follicle development analysed in this study. (C) Ovary section stained with H&E, showing examples of follicles at primordial (p), transitional (t) and primary-plus stage. In the primary-plus follicle the majority of GCs are columnar and form a single layer (GCs), although there are some areas that start to show signs of multilayering (e.g. boxed area). Note basement membrane (white arrowheads), theca cells (black arrowheads) and GCs undergoing mitosis (black arrow). Scale bar: 20 µm.

View larger version (153K): [in this window] [in a new window]   Fig. 2. (A-J) Transmission electron micrographs (A,B,D,G,H), light micrographs (C,F,J) and confocal images (E,I) showing change in GC shape from the primordial to the secondary stages of follicle development. (A) Primordial follicle in day 12 ovary. Three flat GCs are visible in this section, with flattened nuclei (arrows). The GCs are bounded by a basal lamina (double arrow). Note thin GCs between adjacent oocytes (arrowhead); n, oocyte nucleus, o, oocyte cytoplasm, gc, granulosa cell. Scale bar: 10 µm. (B) Desmosome-like junction (arrowhead) between GC and oocyte in day 10 primordial follicle. Note formation of zona pellucida (zp). Scale bar: 500 nm. (C) Primary follicle stained using H&E with a single layer of cuboidal GCs (black arrows). Position of basement membrane marked (white arrowheads). Scale bar: 30 µm. (D) Columnar, polarised GC spanning from basal lamina (bl, arrows) to oocyte (o), with transzonal processes (TZPs) (black arrowhead) traversing zp. GC nucleus (n) abuts basal lamina and GC membrane tracked by white arrowheads. Scale bar: 2 µm. (E) Confocal image of primary-plus follicle immunolabelled for β-catenin (green) with second GC layer developing (black arrowhead) and columnar GCs (arrowed). Note polarised nuclei labelled with DAPI (false-coloured red; white arrowheads) close to basal lamina. Scale bar: 30 µm. (F) Primary plus follicle with a new second layer of GCs (black arrows) developing close to the oocyte. Note the continuity and tight packing of the first layer on the basal lamina (white arrowheads), and developing theca layer (black arrowheads). Scale bar: 30 µm. (G) GC (gc) on basal lamina, with lamina densa (LD) clearly visible, as are hemidesmosomes (white arrowheads) and collagen fibrils (black arrows). Scale bar: 500 nm. (H) TZPs (black arrowheads) from GC (gc) traversing zona pellucida (zp). Note desmosome-like junction (black arrow) at contact with oocyte (o). Scale bar: 500 nm. (I) High-magnification view of columnar GC (asterisk) and TZPs (arrowed) contacting oocyte (o). Note β-catenin staining of TZPs (green), with bright punctate spots where TZPs contact the oocyte (arrowheads). Scale bar: 10 µm. (J) Secondary follicle with two complete layers of GCs (arrowed). Note the developing theca layer (black arrowheads). Scale bar: 30 µm.

View larger version (11K): [in this window] [in a new window]   Fig. 3. (A-C) Growth trajectory of follicles during initiation of follicle growth. Oocyte diameter (A), number of GCs (B) and GC height (C) in the largest cross section on days 12 (open circles and white bars) and 21 (black circles, hatched bars); p, primordial; t, transitional; 1°, primary; 1°+, primary with a second layer forming; 2°, secondary; 2°+, secondary with third layer forming; new 1°+, primary with < five second-layer cells. Values are presented as the mean ± 95% confidence interval. *** P<0.0001, ** P<0.01 and * P<0.05: significant differences between days 12 and 21. For all three variables there was a significant difference between values at successive stages (supplementary material Table S1), except oocyte diameter between 2° to 2°+ on day 12.

View larger version (43K): [in this window] [in a new window]   Fig. 4. Ki-67 immunolabelling of follicles. (A) Low-magnification view of day 12 ovary immunolabelled for Ki67 (brown) showing widespread labelling of GCs that is increased in larger follicles. Scale bar, 100 µm. (B,C) Proportion of follicles with one or more Ki67-positive GCs (B) and proportion of Ki67-positive GCs on days 12 (open bars) and 21 (hatched bars) (C). p, primordial; t, transitional; 1°, primary; 1°+, primary with a second layer forming; 2°, secondary; 2°+, secondary with third layer forming. Values are means and 95% confidence interval. Asterisks on the right shoulder of the day 12 bars denote significant differences between days 12 and 21, asterisks on horizontal lines mark significant differences between stages; *** P<0.0001, ** P<0.01, * P<0.05. In B, differences between transitional and primary, and between subsequent stages, are impossible to compare by regression when the proportion =1. (D) Primordial follicle with unlabelled GCs (arrowheads). Scale bar, 10 µm. (E) Primary follicle immunolabelled for Ki67 (green) and MCM2 (red), with nuclei labelled with DAPI (blue). Nuclei indicated by asterisks are MCM2-positive, nucleus indicated by white arrow is Ki67- and MCM2-positive; white arrowheads indicate Ki67- and MCM2-negative nuclei (DAPI only). Scale bar:10 µm.

View larger version (35K): [in this window] [in a new window]   Fig. 5. Relationship between GC shape and Ki67 immunolabelling. (A) Proportion of flat and cuboidal GCs that are Ki67-positive on days 12 (open bars) and 21 (hatched bars). (B) Proportion of flat and cuboidal GCs that are Ki67-positive at the primordial, transitional and primary stages, on days 12 and 21. Values are presented as the mean ± 95% confidence interval. *** P<0.0001, ** P<0.01, * P<0.05: significant differences between days 12 and 21. (C) High-magnification view of primordial follicle with no Ki67-positive GCs. (D) Ki67-positive primordial follicle with labelled flat GC (arrowed). (E,F) Ki67-positive transitional (E) and primary (F) follicles with labelled cuboidal cells (examples indicated by arrows). Scale bars: 10 µm.

View larger version (30K): [in this window] [in a new window]   Fig. 6. (A) Oocyte diameter and proportion of cuboidal cells in transitional follicles on days 12 (open circles, dotted line) and 21 (filled circles, continuous line). Lines are linear regression fits with no significant slope and no difference between days 12 and 21. (B,C) Oocyte diameter (B) and number of GCs (C) are similar in Ki67-negative (open bars) and Ki67-positive (hatched bars) primordial follicles on days 12 and 21. Values are presented as the mean ± 95% confidence interval.

View larger version (44K): [in this window] [in a new window]   Fig. 7. (A) Increasing proportion of Ki67-positive GCs with increasing oocyte diameter on days 12 (blue) and 21 (red). Dotted lines are for all GCs, continuous lines are for just cuboidal GCs. Values are presented as the mean ± 95% confidence interval. (B) Relationship between oocyte diameter and the number of GCs on day 21. Lines are linear regression fits of unilaminar (blue line) and multilayered (green line) follicles. For day 12 see supplementary material Fig. 1B. (C) Percentage of Ki67-positive GCs on the basal lamina (BL) and inner layers (inner) on days 12 and 21. (D) Day 21 follicle at the secondary-plus stage with sparse Ki67 labelling on the basal lamina (white arrowheads), and more on the inner layers (black arrowheads).

View larger version (83K): [in this window] [in a new window]   Fig. 8. (A) Scattergram of widths of GCs on basal lamina on both day 12 and 21. Note reduction of widths from primordial to primary stage. Significant differences between means (horizontal lines) are marked. (B) Scattergram of GC width against GC height in follicles from primordial to the `new second layer' stage on both day 12 and 21. (C-G) Electron micrographs of basal lamina; gc, granulosa cell; o, oocyte; t, theca cell; white arrowheads indicate lamina densa; black arrowheads indicate hemidesmosomes; black arrows indicate collagen fibrils. (C) Day 21 primordial follicle. Note clear lamina densa (arrowheads). Scale bar: 2 µm. (D) Day 10 primordial follicle. Note occasional and sparse collagen fibrils (arrows). Scale bar: 500 nm. (E) Day 10 follicle at the primary plus stage. Basal lamina closely following GC indentations. Occasional multiple layers of lamina densa are visible (arrowheads), as are anchoring filaments (af) between hemidesmosome (black arrowhead) and lamina densa (see insert). Note more numerous collagen fibrils (arrows). Scale bar: 500 nm. (F) Day 21 multilayered follicle. Note thick layer of collagen fibrils (square bracket). Bar: 2 µm. (G) High power of collagen fibrils surrounding a day 21 primary follicle. Scale bar: 500 nm. (H) F-actin expression (red) in a day 21 primary-plus follicle, note strong cortical staining in theca cells. Nuclei are blue (DAPI). Scale bar: 30 µm.

View larger version (70K): [in this window] [in a new window]   Fig. 9. (A) Diagram illustrating axes of mitoses at different stages of follicle development and in different layers of the GCs. Thicker lines mark predominant angle. (B-G) H&E stained sections of day 12 (B, C, E–F) and 21 (D) ovary showing angle of mitoses (arrows). (B) Mitosis parallel to basal lamina and oocyte surface in transitional follicle. (C) Mitosis perpendicular to basal lamina and oocyte surface in primary follicle. (D and E) Perpendicular mitosis in GC spanning between GC and oocyte. (F) Perpendicular mitosis in a basal GC. (G) Mitosis in an inner GC parallel to oocyte surface. Scale bars: 20 µm.

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