Three-dimensional organization of transzonal projections and other cytoplasmic extensions in mouse ovarian follicles

Each mammalian oocyte is nurtured by its own multi-cellular structure, the ovarian follicle. We used new methods for serial section electron microscopy to examine entire cells and their projections in mouse antral ovarian follicles. It is already known that cumulus cells send towards the oocyte thin cytoplasmic projections called transzonal projections (TZPs), which are crucial for normal oocyte development. We found that most TZPs do not reach the oocyte, and that they often branch and make gap junctions with each other. Furthermore, the connected TZPs are usually contacted on their shaft by oocyte microvilli. Mural granulosa cells were found to possess randomly oriented cytoplasmic projections that are strikingly similar to free-ended TZPs. We propose that granulosa cells use cytoplasmic projections to search for the oocyte, and cumulus cell differentiation results from a contact-mediated paracrine interaction with the oocyte.


Introduction 51
The mammalian ovarian follicle is a complex tissue structure that nurtures the 114 To visualize TZPs in three dimensions, we collected ~600 serial sections of 40-115 45 nm thickness (total depth 27 μm) and imaged volumes of ~43 x 43 μm (x, y) at a 3.5-116 5 nm per pixel resolution. The imaged volumes were centered to contain the zona 117 pellucida, oocyte surface, and cumulus cell bodies ( Figure 1B). We were able to trace 118 every TZP sent by individual cells (Video 1 and Figure 1C), and record their interactions 119 with other TZPs and with the oocyte surface (described below). As can be seen in video 120 2, TZPs arise from cumulus cells located at varying distances from the oocyte.

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We found that the majority of TZPs do not reach the surface of the oocyte ( Figure   122 2A, C, D). On average, each cumulus cell had 31 ± 5 (n = 8) TZPs that do not reach the 123 surface of the oocyte, and 9 ± 2 (n = 8) TZPs that reach the oocyte and make a junction 124 with it ( Figure 2B, C, D) (data shown as mean ± standard error of the mean, unless 125 specified otherwise). We will refer to these as free-ended and connected TZPs, 126 respectively.

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We measured the lengths of connected and free-ended TZPs in serial electron 128 micrographs ( Figure 2E). The lengths of connected TZPs had a bell curve-type 129 distribution with an average length of 7.9 ± 1.9 μm (n = 70) (mean ± standard deviation).

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Free-ended TZPs had a different kind of distribution, in which the number of projections 131 decreased with length. The first, second, and third quartiles were 1.1, 2.2, and 4.1 μm, 132 respectively. In other words, 25% of the projections were shorter than 1.1 μm, the 133 median (50%) was 2.2 μm, and 25% of the projections were longer than 4.1 μm. The 134 shortest and longest lengths were 0.2 μm and 11.4 μm (see Figure 8C).

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TZPs often contact each other and make gap junctions.

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Our data revealed novel characteristics of TZPs throughout the zona pellucida.

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For instance, we found that ~12% of the TZPs analyzed branched into one or more TZPs form contacts indiscriminately, in other words, two TZPs about to make a contact 168 are not restricted whether they are derived from the same cell or from two different cells.

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Gap junctions within the zona pellucida have previously been observed by 170 immunofluorescence (Simon et al., 2006). However, due to the limited resolution of SEM 171 (~3 nm for these studies), it was not possible to identify small gap junctions in our data.

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To investigate if the TZP-TZP contacts consisted of gap junctions, we collected sections 173 from the same follicles that had been previously analyzed, and used transmission 174 electron microscopy (TEM) to image the zona pellucidae. We found that most, but not 175 all, contacts in the upper half of the zona pellucida were gap junctions ( Figure 3C).

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Supporting the idea that TZPs can form gap junctions at their endings, we found that 177 some TZPs (18 of 325) ended in an invaginated annular junction within a cumulus cell

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Connected TZPs and oocyte microvilli make contacts with each other.

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As described previously (Li and Albertini, 2013;Motta et al., 1994), TZPs end in 183 junctions at the oocyte surface that contain adherens junctions, as identified by an 184 electron-dense deposit ( Figure 4A, B) (Niessen and Gottardi, 2008). We reconstructed 185 12 of these junctions and found that they range from 0.39 μm to 3.59 μm in length, and    et al., 2007). In our analysis, oocyte microvilli were generally uniformly distributed along 190 the oocyte surface, and were 1.06 ± 0.09 μm (n = 45) long. Interestingly, we often 191 noticed areas in the zona pellucida where microvilli appeared "clumped" ( Figure 4D).

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Serial section analysis revealed that these clumped areas consisted of one or two TZPs 193 connected to the oocyte, which were closely associated with 3-6 oocyte microvilli ( Figure  5 4E and Video 5). Video 5 shows an example of a connected TZP that is almost 195 continuously coupled with a long microvillus and then becomes surrounded by 5-6 short 196 microvilli as it gets close to the oocyte surface. In some cases, microvilli were seen To test whether TZP-microvilli contacts were gap junctions, we inspected thin 201 sections by TEM as described in the previous section. The oocyte cytoplasm and 202 microvilli can usually be distinguished from TZPs by a difference in electron density (see 203 example in Video 3) or by the presence of precipitate that often forms in the oocyte 204 cytoplasm and microvilli, but not on TZPs (see example in Video 5). This allowed us to 205 identify possible TZP-microvilli contacts in the TEM images. In contrast to contacts seen 206 in the upper half of zona pellucida, these did not appear to be gap junctions (data not 207 shown).

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Cytoplasmic projections are also found outside of the zona pellucida (non-TZPs).

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During our analysis of TZPs, we found that many cumulus cells had some

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Cumulus cells were identified as any cell that was connected to the oocyte by 216 means of TZPs. As described before, these cells differed based on whether the cell body 217 was located adjacent to the zona pellucida or displaced away from it ( Figure 5A, B).

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Cumulus cells that were directly adjacent to the zona pellucida extended most 219 projections toward the oocyte as TZPs, and only a few away from it ( Figure 5C). These 220 cells had an average of 51 ± 4 TZPs and 10 ± 3 non-TZP cytoplasmic projections (n = 5)

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(connected and free-ended TZPs were pooled together for these studies). Displaced

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To analyze the projections of mural granulosa cells, we imaged volumes of ~71 x 232 71 x 27 μm (x, y, z) at a resolution of 5-6 nm per pixel, centered on mural granulosa cells 233 of the follicles that were previously used to study TZPs. Cells chosen for analysis were 234 selected if they were centrally located within the field-of-view and the cell body was

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Other common ending types were inside an invagination in a neighboring cell (24%) 263 (Video 10), or as a free end in the extracellular space (26%). Less common endings 264 seen were as an end-to-end contact with a projection from a different cell (7%), as an 265 invaginated annular junction (5%), or as a small linear gap junction (<1%). Examples of 266 all these endings can be seen in videos 1, 6, and 8.

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Common features of projections throughout the follicle.

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Free-ended TZPs and the cytoplasmic projections of granulosa cells showed a 270 similar overall appearance; both were usually devoid of organelles, and their lengths 271 were strikingly similar. As with free-ended TZPs, we found that the number of non-TZP 272 cytoplasmic projections from the cumulus, inner mural, and outer mural cells decreased 273 with length ( Figure 8B). The first, second, and third quartiles were 1.1, 2.5, and 4.4 μm, 274 respectively (described above). The shortest and longest lengths were 0.2 μm and 13.7 275 μm. Figure 8C shows a detailed summary of these findings. The length distribution and 276 thickness of these projections, which was 76 ± 2 nm (n = 67), did not change based on 277 their location within the follicle.

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The striking similarity between the lengths of free-ended TZPs and non-TZP 279 cytoplasmic projections from every cell group suggested to us that all granulosa cells are 280 programmed to seek out the oocyte by extending cytoplasmic projections. If so, some of 281 these projections should be longer than the thickness of the zona pellucida. Consistent 282 with this idea, we found that at least a quarter of the projections from every somatic cell 283 within the follicle were longer than the thickness of the zona pellucida, which was 4.1 ± 284 0.3 μm (n = 3) (as mentioned above) ( Figure 8B, C). Our findings suggest that every 285 somatic cell in the follicle is able to probe a distance that is longer than the thickness of

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We found that most TZPs that reach the oocyte surface are contacted by several 310 oocyte microvilli on their shaft ( Figure 4D-F and Video 5). This previously undetected 311 association is significant because there are several critical interactions between the 312 oocyte and cumulus cells and one or more of these interactions could occur at these 313 contact sites. We initially considered whether these were the sites of gap junctions 314 between cumulus cells and the oocyte. We tentatively conclude that the microvilli / TZP 315 contact sites are not gap junctions because we did not find them in this region of the 316 zona pellucida by TEM. An interaction that may be occurring at the microvilli / TZP 317 contact sites is discussed below.

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Free-ended TZPs and dynamics 320 Although TZPs have been known for many years, their dynamics have not yet 321 been characterized. A recent study has focused on this issue using a reconstituted 322 system (El-Hayek et al., 2018). When cumulus cells that have been stripped from their 323 innate oocyte are reaggregated with a donor oocyte, they make new TZPs, which form 324 junctions with the oocyte. This clearly demonstrates that TZPs are dynamic structures.

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Our serial section data is consistent with dynamic TZPs in-situ. We show for the    -Raymond, 1986;Fantin et al., 2015). Upon contact with axons, dendritic 367 filopodia were observed to become dendritic spine synapses (Ziv and Smith, 1996).

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In the ovarian follicle, filopodia could be involved in transducing paracrine or

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In summary, new methods for serial section electron microscopy enabled us to 408 examine whole cells and their relationship to other cells with ultrastructural resolution.

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Our study shows that this kind of structural information may be useful in understanding               920 Figure 3B shows a reconstruction of TZPs in which two branching points can be seen.