Stage-specific patterns of basal microfilaments
We analysed in detail, during vitellogenic stages S8–12, the bMF-organisation in the cuboidal and columnar FCE (Fig. 3) and detected, despite of some variation, characteristic stage-specific patterns (Fig. 4). The bMF-bundles in the cuboidal FCE of S8 are highly polarised perpendicular to the antero-posterior (a-p) axis of the follicle (circumferential organisation). This parallel alignment, both within individual FC and in relation to neighbouring FC, disappears in part during S9. In the flattening FC (the prospective cFC) near the border between NC and Oo, condensations of bMF become obvious. The bMF-bundles in the remaining columnar FCE surrounding the Oo retain their parallel alignment within individual FC, but they become more disordered relative to neighbouring FC. During S10a, the bMF-bundles in cFC are again aligned in parallel and oriented circumferentially. Subsequent morphological changes during S10b, like thickening of the dorsal FCE and elongation of inwardly migrating cFC, are accompanied by bMF-condensations that first appear in dorsal cFC and spread out over mbFC to pFC during S11. In S11, a peculiar bMF-organisation showing crescent- or fan-shaped condensations becomes obvious, wheras during S12, a new pattern of dense parallel bMF oriented circumferentially appears (Fig. 4).
Stage-specific patterns of microtubules
A detailed analysis of the MT-organisation also revealed characteristic stage-specific patterns during S8–12 (Fig. 5). In S8, similar to bMF, the preferred orientation of MT in the cuboidal FC is perpendicular to the a-p axis of the follicle. From S9 onward, diffuse MT surround the FC nuclei in a basket-like arrangement. In the flattening cFC, a longitudinal orientation of MT along the a-p axis first becomes obvious. During S10a-12, this longitudinal pattern continuously spreads out to mbFC and pFC. During S9–12, the MT of squamous FC covering the NC are organised in typical web-like structures enclosing the nuclei (Fig. 5).
Bioelectrical patterns correlate with cytoskeletal patterns
We have shown previously [15, 16] that, during the course of development, ovarian follicles undergo significant changes in their pHi- and Vmem-patterns caused by varying activities of asymmetrically distributed or activated ion-transport mechanisms. In the present study, we analysed in detail how the cytoskeletal organisation in the FCE alters during vitellogenesis (Figs. 4 and 5). It is obvious that stage-specific changes of pHi and Vmem correlate spatially and temporally with structural modifications of bMF and MT (summarised in Fig. 6). These alterations are accompanied by cell migrations, cell rearrangements, or cell-shape changes like, e.g., cell flattening or cell stretching. In S8, the uniformly cuboidal FCE exhibits relatively homogeneous pHi- and Vmem-patterns as well as homogeneous bMF- and MT-patterns [16]. During S9, gradients of pHi and Vmem develop with relatively acidic and relatively depolarised cFC [16]. At this stage, the bMF in the flattening cFC lose their circumferential orientation and condense, while the MT change their orientation from d-v to a-p. In S10a, the bMF-bundles of the columnar FCE are aligned in parallel circumferentially again. During further development, d-v gradients of pHi and Vmem develop [16]. In S10b, the dorsal FCE is relatively hyperpolarised and relatively acidic compared to the ventral FCE and, as a result, dorsal cFC and neighbouring FC are the most acidic FC. In these, in part, inwardly migrating cells, the bMF condense again. During this process, in late S10b/11, a strong depolarisation of dorsal cFC and neighbouring FC becomes apparent. Unlike the bMF-pattern, the MT-organisation alters gradually along the a-p axis, but not along the d-v axis. In pFC, which are relatively alkaline and depolarised [16], no longitudinal alignment of MT was found.
Modifying pHi and Vmem with inhibitors of ion-transport mechanisms
We used six inhibitors of ion-transport mechanisms, which we have recently shown to affect either pHi, Vmem or both parameters in the FCE during S10b [16]. We found that each tested inhibitor also exerted influence on the cytoskeletal organisation (Figs. 7, 8, 9 and 10). Certain groups of inhibitors giving rise to similar effects on pHi and/or Vmem caused similar changes in the bMF- and/or MT-patterns. Therefore, we conclude that the observed cytoskeletal changes depended on the induced pHi- and/or Vmem-changes, and not on effects of the involved ions.
As described in detail previously ([16], summarised in Fig. 2), alkalisation was caused by furosemide, glibenclamide, 9-anthroic acid or verapamil. Furosemide and glibenclamide resulted in the strongest overall increase of pHi and also in enhanced angles of the a-p and the d-v gradient. 9-Anthroic acid led to an enhanced angle of the a-p gradient, but to a reduced angle of the d-v gradient. Amiloride or bafilomycin resulted in acidification and in reduction of the angles of the a-p and the d-v gradient. Vmem was influenced to the greatest extent by verapamil (strong hyperpolarisation), followed by glibenclamide (hyperpolarisation), and both inhibitors reduced the angles of the a-p and the d-v gradient. Furosemide, 9-anthroic acid, amiloride and bafilomycin, respectively, had no consistent effects on Vmem and on both gradients.
Changes in pHi and Vmem affect the organisation of basal microfilaments
Inhibition experiments were performed using S10b-follicles of the wild-type as well as of the transgenic strain Lifeact-GFP. The bMF-patterns in the FCE of both strains were very similar (Figs. 7 and 8), only a slight difference in the thickness of bMF-bundles was obvious: The bMF-bundles of fixed phalloidin-stained wild-type follicles were thinner than those of living Lifeact-GFP follicles. Furthermore, Lifeact-GFP follicles often showed a weakly fluorescent area in the FCE that seemed to result from squeezing during microscopic observation.
Despite of some variation, the effects of inhibitors on the bMF-patterns were also similar in both strains (Figs. 7 and 8). Strong alkalisation, either without a distinct effect on Vmem (furosemide) or combined with hyperpolarisation (glibenclamide), retained a highly polarised bMF-pattern consisting of parallel aligned, but thinner bMF-bundles, whereas condensations of bMF, as in the controls, were rarely observed (Fig. 8a,b). The bMF-bundles of furosemide-treated follicles appeared to be even thinner and partially disintegrated compared to those of glibenclamide-treated follicles. Presumably, this difference depends on the fact that furosemide showed no clear influence on Vmem. Furosemide and glibenclamide both led to alkalisation in all FC, but especially in pFC and ventral FC, thus enhancing the angles of the a-p and the d-v pHi-gradient (cf. Figure 2). Both Vmem-gradients were either maintained or reduced resulting in a larger area of relatively hyperpolarised FC.
Slight alkalisation together with no clear effect on Vmem (9-anthroic acid) reduced the frequency of bMF-condensations (Fig. 8a,b). This treatment led to an enhanced angle of the a-p pHi-gradient but to a reduced angle of the d-v pHi-gradient, since the ventral cFC became less alkalised (cf. Figure 2). Slight alkalisation combined with strong hyperpolarisation (verapamil) resulted either in depolymerisation or in condensation of bMF throughout the entire columnar FCE (Fig. 8b). This seems to be due to the fact that the angles of both the a-p and the d-v Vmem-gradient were reduced, which led to more homogeneous electrochemical properties throughout the FCE (cf. Figure 2).
Acidification combined with an unchanged Vmem (amiloride, bafilomycin) led to an increase in bMF condensation and disintegration. The angles of the a-p and the d-v pHi-gradient of both amiloride- and bafilomycin-treated follicles were reduced (cf. Figure 2), and the relatively acidic area of the FCE showing condensed bMF was enlarged (Fig. 8a,b).
Taken together, we found that alkalisation prevented condensation of bMF and stabilised their parallel alignment, while the bMF-bundles became thinner. In contrast, acidification led to increasing condensations of bMF in both the a-p and the d-v direction, while the bMF-bundles became thicker and more disordered. When strong alkalisation was combined with hyperpolarisation, disintegration of bMF was absent. Thus, hyperpolarisation had a stabilising effect on bMF (Figs. 7 and 8).
Changes in pHi and Vmem affect the organisation of microtubules
Inhibition experiments were performed using S10b-follciles of the wild-type as well as of the transgenic strain αTub84B-GFP. While, in living αTub84B-GFP follicles, the α-subunits of all MT in the FCE were labelled, only a subset of MT was stained in fixed wild-type follicles treated with an antibody against acetylated α-tubulin. Thus, in αTub84B-GFP, a denser network of MT-bundles was revealed and the overall longitudinal alignment of MT became more evident (Figs. 9 and 10).
Alkalisation, caused by furosemide, glibenclamide or 9-anthroic acid, resulted either in reduction (glibenclamide, 9-anthoric acid) or in loss (furosemide) of the longitudinal orientation of MT as well as in their partial disintegration. In furosemide-treated follicles (strong alkalisation, no clear effect on Vmem), disintegration of MT was most prominent compared to follicles treated with glibenclamide (strong alkalisation, slight hyperpolarisation) or with 9-anthoric acid (slight alkalisation, no clear effect on Vmem). Strong alkalisation combined with no clear effect on Vmem (furosemide) resulted in spherical FC, presumably due to weakend cell-cell contacts, which was particularly visible in αTub84B-GFP. This phenomenon was less pronounced with glibenclamide, presumably due to a stabilising effect of hyperpolarisation. Slight alkalisation combined with strong hyperpolarisation (verapamil) preserved the longitudinal orientation, while the MT-bundles appeared to be thicker (Figs. 9 and 10).
In addition, furosemide, glibenclamide or 9-anthroic acid led to an enhanced angle of the a-p pHi-gradient and to a reduced angle of the a-p Vmem-gradient (cf. Figure 2). This means that the cFC became more alkaline compared to the mbFC, while the area of relatively hyperpolarised FC became enlarged. The altered pHi-gradient resulted in loss of the longitudinal MT-alignment in the mbFC and cFC. Verapamil also led to an enhanced angle of the a-p pHi-gradient and to a reduced angle of the a-p Vmem-gradient (cf. Figure 2). But the effect of verapamil on pHi in general as well as on its gradients was small, so that it had no impact on MT-organisation. In addition, the strong hyperpolarising effect of verapamil and the reduced Vmem-gradients both preserved the longitudinal alignment of MT (Figs. 9 and 10).
Acidification in the whole FCE as well as reduced angles of both pHi-gradients combined with no clear effects on Vmem caused by amiloride and bafilomycin (cf. Figure 2) did not alter the MT-organisation (not shown).
Therefore, we conclude that hyperpolarisation as well as acidification exerted stabilising effects on the longitudinal orientation of MT-bundles. Strong alkalisation resulted in loss of this MT-arrangement and in partial disintegration of MT. These effects were reduced when alkalisation was combined with hyperpolarisation, which preserved the longitudinal orientation of MT-bundles (Figs. 9 and 10).