The development of a healthy oocyte within the ovarian follicle requires continuing contact between the germ cell and the somatic granulosa cells that surround it (El-Hayek and Clarke, 2016; Clarke, 2018; Richani et al., 2021; Robert, 2021; Li and Albertini, 2013; Doherty et al., 2022; Xie et al., 2023; Marchais et al., 2022). Signals from the granulosa cells initiate the increase in the volume of the oocyte when primordial (non-growing) follicles are activated to enter the growth phase (Zhang et al., 2014; Saatcioglu et al., 2016; Nagamatsu et al., 2019). Subsequently, the granulosa cells provide nucleotides, amino acids, and pyruvate to the growing oocyte via gap junctions that connect the two cell types (Xie et al., 2023; Clarke, 2018; Marchais et al., 2022). When gap junctional communication between the two cell types is genetically disrupted, oocytes do not develop normally and cannot be fertilized (Gittens and Kidder, 2005; Simon et al., 1997; Winterhager and Kidder, 2015; Carabatsos et al., 2000). Once the oocyte reaches full size, it acquires the ability to undergo the final stage of development, termed meiotic maturation, but is prevented from precociously doing so by cyclic GMP that is produced and transferred to it by the granulosa cells, also via gap junctions (Jaffe and Egbert, 2017; Wigglesworth et al., 2013; Norris et al., 2009; Zhang et al., 2010). In vitro, oocytes can develop normally when enclosed by granulosa cells but not when the granulosa cells are removed and provided in co-culture, further emphasizing the crucial importance of physical contact between the two cell types (O’Brien et al., 2003; Eppig, 1979; Herta et al., 2021).

In primordial follicles, a single layer of squamous granulosa cells surrounds the oocyte and contact it via adherens junctions (Mora et al., 2012; Jorgensen, 2013). Following activation of growth, a glycoprotein-rich coat termed the zona pellucida becomes established around the oocyte (Litscher and Wassarman, 2020; Wassarman and Litscher, 2018). The zona pellucida steadily thickens as growth continues, reaching a final thickness of about 7 μm in mice and 15 μm in humans. Despite the barrier imposed by the zona pellucida, the granulosa cells and oocyte maintain direct contact by a dense network of thin filopodia-like projections, termed transzonal projections (TZPs), that grow out from the granulosa cells through the zona pellucida (Clarke, 2022; Macaulay et al., 2014; Baena and Terasaki, 2019; Motta et al., 1994; Zhang et al., 2021; Albertini and Rider, 1994). The tips of the TZPs establish contact with the oocyte plasma membrane through heterotypic adherens junctions comprising N-cadherin (granulosa cell) and E-cadherin (oocyte) and harbour the gap junctions that link the two cell types (Mora et al., 2012) and likely also through focal adhesions (McGinnis and Kinsey, 2015). New TZPs are generated throughout follicular growth, thereby increasing intercellular molecular trafficking (El-Hayek et al., 2018a). A genetically imposed decrease in the number of TZPs leads to defects in oocyte gene expression and to abnormal meiotic maturation and embryonic development (Crozet et al., 2023). Despite the essential role of TZPs during oocyte development in mammals, as well as the presence of analogous structures in non-mammalian species (Kessel et al., 1985; Schroeder, 1981), the molecular mechanisms that govern their generation and stability are not yet understood.

The TGFβ signalling pathway regulates the functions and interactions of a wide range of cell types under various physiological and pathological conditions (Derynck and Budi, 2019; Peng et al., 2022). Binding of the ligand to a complex of type I and type II receptors located at the cell surface activates signalling within the cell, most commonly through the members of the SMAD family of transcription factors, although SMAD-independent signalling can also occur (Aashaq et al., 2022; Derynck and Budi, 2019). Upon phosphorylation of the tetrameric type II and type I receptors and subsequent phosphorylation of receptor-activated SMAD proteins (R-SMADs; SMAD1, 2, 3, 5, and 8), a heterotrimeric complex with SMAD4 is formed which enters the nucleus and regulates gene expression by binding to specific SMAD-binding DNA elements. Although R-SMADs can signal in the absence of SMAD4, evidence indicates that the presence of SMAD4 in the heterotrimer amplifies TGFβ cell signalling by stabilizing the complex and by favouring the binding of other transcriptional co-repressors or co-activators of target genes (Aragón et al., 2019; Guglielmi et al., 2021).

Genetic, pharmacological, and in vitro studies have revealed multiple roles for TGFβ/SMAD signaling during follicular growth and development (Patton et al., 2021; Pangas and Rajkovic, 2015). TGFβ signalling via SMAD3 in granulosa cells acts initially in primordial follicles to maintain their quiescence (Granados-Aparici et al., 2019; Kang et al., 2014) and later, following their activation, becomes excluded from the nucleus in association with rapid granulosa cell growth and proliferation (Hardy et al., 2018). The TGFβ superfamily members, growth-differentiation factor (GDF) 9, which signals through SMAD2/3, and bone morphogenetic protein (BMP) 15, which signals through SMAD1/5/8, are produced by the oocyte (Dong et al., 1996; Yan et al., 2001). Female mice lacking GDF9 rarely generate more than a single layer of granulosa cells (Elvin et al., 1999b; Elvin et al., 1999a; Dong et al., 1996). This defect can be rescued by preventing the production of inhibin, and the doubly mutant follicles can develop at least to the pre-antral stage (Wu et al., 2004). Genetic inactivation of SMAD4 in the granulosa cells from growing follicles leads to a reduction in the number of antral follicles and ovulated eggs, as well as the appearance of luteinized follicles containing entrapped oocytes (Pangas et al., 2006). Similar phenotypes are observed when both SMAD2 and SMAD3 are inactivated in the granulosa cells (Li et al., 2008). These effects are likely due at least in part to a failure of the cumulus layer to expand in response to the ovulatory surge of LH (Pangas et al., 2006; Li et al., 2008), as expansion is known to require SMAD signalling and to be required for ovulation (Dragovic et al., 2007; Hao et al., 2022; Mottershead et al., 2015; Peng et al., 2013; Yu et al., 2013).

Two lines of evidence point to a potential role for SMAD signalling in the generation or maintenance of TZPs. First, deletion of GDF9 leads to both a decrease in the number of TZPs and a failure of a proportion of the TZPs to correctly orient towards the oocyte (Carabatsos et al., 1998). Second, TZP number is reduced in follicles grown in vitro following RNAi-mediated depletion of GDF9 in the oocyte (El-Hayek et al., 2018a). To test the role of SMAD4 specifically, we used a Cre-lox strategy to inactivate Smad4 in granulosa cells, as previously described (Jorgez et al., 2004; Pangas et al., 2006). Using image analysis tools and three-dimensional reconstructions, we show that depletion of SMAD4 in the granulosa cells decreases the number of TZPs that project from individual granulosa cells in both intact granulosa cell-oocyte complexes (GOCs) and in reaggregated complexes. Morphometric parameters including length and orientation are also altered. The decrease in TZP number in intact GOCs is associated with a reduction in the amount of N-cadherin and Notch2 in the granulosa cells, suggesting that the composition of the granulosa cell surface is perturbed in the absence of SMAD4. We propose that SMAD4, through its activity to increase expression of target genes, stabilizes TZPs by promoting their adhesion to the oocyte surface, thereby helping to increase the number of TZPs and communication between the growing oocyte and its follicular microenvironment.

We began by verifying the expression of SMAD4 during folliculogenesis. As shown in Figure 1A and consistent with prior reports, SMAD4 was detectable in primordial and growing follicles as well as in the granulosa cells collected from pre-antral follicles. SMAD2 and SMAD3 were also present, with SMAD2 being the major species detected in oocytes. Two strategies were tested to deplete SMAD4 from the granulosa cells of growing follicles. First, mice were generated carrying two floxed Smad4 alleles and a transgene encoding Cre recombinase under the control of the Amhr2 promoter (Jorgez et al., 2004; Pangas et al., 2006; Figure 1B). These mice also carried a transgene (mTmG, see Methods) constructed such that Tomato fluorescent protein is expressed in the absence of Cre activity, whereas Cre-mediated recombination switches expression to green fluorescent protein (GFP), thus allowing us to identify cells where Cre had been active. Upon immunostaining of ovarian sections or whole-mount intact follicles, only a small number of cells expressed GFP (Figure 1C, left). Consistent with this observation, less than 10% of FACS-sorted granulosa cells were GFP-positive (Figure 1C, right). Thus, in our system, Amhr2-Cre did not mediate efficient recombination of floxed DNA in granulosa cells. This is consistent with results described in previous reports (Pangas et al., 2006; Li et al., 2008).

Figure 1

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We, therefore, turned to mice that expressed Cre under the control of a tamoxifen-inducible form of the estrogen receptor (ER) gene promoter (Rosa26^CreERT2, hereafter termed ER-Cre). Because ER is expressed in pituitary gonadotropes and SMAD4 regulates the transcription of the Fshb gene encoding the β-subunit of follicle-stimulating hormone (Li et al., 2018), this model could not be used in vivo. GOCs from mid-growth stage follicles (diameter 100–120 μm) were, therefore, isolated, exposed to tamoxifen for one day and then incubated in tamoxifen-free medium for another five days. Under these conditions, a substantial fraction of the granulosa cells underwent Cre-mediated recombination, as indicated by the expression of GFP in immunostained GOCs and by FACS analysis (Figure 1E). This in vitro system was used for the experiments described here.

To examine the efficiency of Cre-mediated Smad4 recombination, the mRNA level was assessed in intact GOCs using the RNAscope technique and in purified granulosa cells using qRT-PCR. Following tamoxifen treatment and culture, there was an approximately 60% reduction in the number of foci in the granulosa cells from 5.78+/-0.36 (mean, SEM, n=21) in ER-Cre^-; Smad4^fl/fl mice to 2.16+/-0.26 (n=26) in ER-Cre⁺; Smad4^fl/fl mice (p<0.0001), and a 70% reduction in the amount of PCR product from granulosa cells (n=5, p=0.0001) (Figure 1F). Immunoblotting of granulosa cells revealed a corresponding 70% reduction in SMAD4 protein in the granulosa cells of ER-Cre⁺; Smad4^fl/fl mice compared to ER-Cre^-; Smad4^fl/fl controls (n=11, p<0.0001) (Figure 1G). Thus, the in vitro ER-Cre strategy substantially reduced although did not eliminate SMAD4 from the granulosa cells.

To examine the effect of SMAD4 depletion on the TZPs, GOCs were isolated and cultured as above, fixed and stained using anti-GFP to label cells where Cre had been active and phalloidin to label the TZPs, and imaged them using confocal microscopy. First, the TZPs were analyzed as a population. Because TZPs arise from both the innermost and more distal granulosa cell layers (Baena and Terasaki, 2019), it was not possible to reliably determine how many granulosa cells contributed to the GFP-positive TZPs in a GOC. The total number of phalloidin-stained TZPs – irrespective of whether they were GFP-positive – that projected to the oocyte in equatorial optical sections of GOCs from control (Smad4^wt/wt) and experimental (Smad4^fl/fl) mice were, therefore, counted (Figure 2A and B). TZP number was normalized to the diameter of the corresponding oocyte, although the mean oocyte diameter did not differ between the groups (Figure 2B), We observed a 20.4+/-0.05% reduction in the number of TZPs in the GOCs from the Smad4^fl/fl mice (n=63 [control], 59 [experimental], p=0.002).

Figure 2

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A limitation of quantifying TZPs in intact GOCs is that the granulosa cells, depending on their number and spatial distribution, can variably impair their resolution and discrimination even using confocal microscopy. Therefore, the number of TZPs was also counted after mechanically removing the granulosa cells from the GOCs. This analysis revealed a striking 51.1+/-0.04% reduction in the normalized number of TZPs in the GOCs from the Smad4^fl/fl mice (n=44 [control], 47 [experimental], p<0.0001) (Figure 2C and D). As observed using intact GOCs, the mean oocyte diameter did not differ (Figure 2D). To verify the difference in TZP-number, we also compared ER-Cre⁺; Smad4^fl/fl to a different control genotype, ER-Cre^-; Smad4^fl/fl mice. A quantitatively similar 43.6+/-0.04% decrease in TZPs within the ER-Cre⁺ population ( +=164 [control], 172 [experimental], p<0.0001) (Figure 2D) was observed following tamoxifen treatment and culture. Together, these results indicate that depletion of SMAD4 in the granulosa cells is associated with a decrease in the number of TZPs.

TZPs provide the physical platform that enables essential gap junctional communication between the granulosa cells and the oocyte. To test whether the reduction in TZP number in SMAD4-depleted granulosa cells impaired this communication, we used a fluorescence recovery after photobleaching (FRAP) assay. Control and experimental GOCs were treated with tamoxifen and incubated as above. After five days, they were exposed to calcein-AM, a fluorescent dye that can pass through gap junctions. The fluorescence in the oocyte of each GOC was bleached using the laser of a confocal microscope and its recovery was then recorded. As shown in Figure 2E, which plots the fluorescence in the oocyte as a function of time after photobleaching and at the end of the recording period normalized to the initial fluorescence, we observed no difference between groups in the rate of transfer of fluorescence from the granulosa cells to the oocyte. Thus, despite the decreased number of TZPs, depletion of SMAD4 did not detectably impair gap junctional communication between the granulosa cells and the oocyte.

As TZPs project not only from the cells immediately adjacent to the zona pellucida but also from those in more distal layers, it was possible that the reduced number of TZPs in ER-Cre⁺; Smad4^fl/fl complexes reflected an impaired ability of the granulosa cells to proliferate. ER-Cre⁺; Smad4^wt/wt and ER-Cre⁺; Smad4^fl/fl GOCs were incubated for 5 hr in the presence of the thymidine analogue, EdU, added five days after the tamoxifen exposure. No difference between the groups in the fraction of EdU-positive cells was observed (Figure 2F), however, implying that the reduced number of TZPs was not due to impaired proliferation of the granulosa cells.

We then focused our analysis on individual granulosa cells. GOCs where individual GFP-positive cells could be readily identified and the TZPs were easily attributable to a particular cell (Figure 3A) were selected, serial optical sections were obtained using confocal microscopy, and three-dimensional images were reconstructed using Imaris software. Figure 3B shows one such reconstruction, where GFP-positive granulosa cells are pseudo-coloured in green. We observed a 25% decrease in the mean volume of Smad4^fl/fl granulosa cells compared to wild-type cells (323.2+/-11.2 vs 242.6+/-10.0; n=293, 322; p<0.0001), but no difference between genotypes in the sphericity of the cells (Figure 3C). The TZPs in a subset of the GFP-positive granulosa cells were then analyzed (Figure 3D). In this subset, there was a 15% difference in mean cell volume (392.2+/-20.8 vs 334.0+/-21.2; n=55, 63) which did not reach statistical significance (p=0.054). However, whereas wild-type cells harbored a mean of 15.8+/-0.87 (n=55) TZPs per cell, this number was reduced by 35% in Smad4^fl/fl cells to 10.5+/-0.71 (n=65, p<0.0001). As compared to those of wild-type cells, the TZPs of the Smad4^fl/fl cells were about 10% longer (4.64+/-0.08 μm vs 5.19+/-0.09 μm; n=893, 720; p<0.0001) and very slightly narrower (0.40+/-0.003 μm vs 0.38+/-0.003 μm; n=870, 719; p<0.0001)(Figure 3E). We did not detect other differences between the genotypes in TZP morphology. These results obtained using reconstructed 3-dimensional images of GFP-positive cells establish that the number of TZPs is reduced in individual SMAD4-depleted granulosa cells.

Figure 3

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A limitation of analyzing TZPs in GFP-positive cells within GOCs is that this strategy cannot distinguish TZPs that were generated in a low-SMAD4 environment (i.e. after the Cre recombinase was expressed) from those generated earlier in a normal-SMAD4 environment. To ensure that we examined only TZPs that were generated after Cre had been expressed, we used our previously published reaggregation assay (El-Hayek et al., 2018a; Figure 4A). GOCs obtained from mid-growth stage follicles of ER-Cre; mTmG mice carrying the appropriate Smad4 alleles were exposed to tamoxifen for one day and then incubated for an additional four days. The granulosa cells were then separated from the oocyte and combined with an oocyte that did not carry the mTmG gene. These reaggregated GOCs were incubated for an additional six days, then fixed and stained using anti-GFP (Figure 4B).

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When the entire population of granulosa cells in the reaggregated GOCs was analyzed, there was a ~30% reduction in mean volume in the Smad4^fl/fl cells as compared to Smad4^wt/wt cells (394.5+/-18.8 vs 279.8+/-16.6; n=185, 159; p<0.0001) and no difference in cell sphericity (Figure 4C), as observed using intact GOCs. There was no difference between genotypes, however, in mean volume among the cells whose TZPs were characterized (Figure 4D). Within this group, the mean number of TZPs per cell was reduced by ~50%, from 14.2+/-2.0 (n=16) in the Smad4^wt/wt cells to 6.84+/-1.1 (n=25) in the Smad4^fl/fl cells (Figure 4D, p=0.001). A ~20% increase in the mean length of the TZPs of Smad4^fl/fl cells (3.71+/-0.17 vs 4.40+/-0.28; n=174, 154; p=0.03) was also detected, as in intact GOCs, but no difference in the mean width (Figure 4E).

The increased mean length of the TZPs of Smad4^fl/fl cells compared to Smad4^wt/wt cells prompted us to analyze the trajectory of individual TZPs. We traced their path from their point of origin to their tip, then measured the distance between the point of origin and the oocyte and subtracted from this the distance between the tip and the oocyte. A large positive difference indicates that the tip of the TZP is relatively close to the oocyte; conversely, a small positive or a negative difference indicates that that tip is relatively far from the oocyte (Figure 4F). This analysis revealed that the mean difference was 50% greater in the Smad4^fl/fl cells compared to Smad4^wt/wt cells (1.61+/-0.16 vs 2.40+/-0.21; n=174, 157; p=0.003) (Figure 4G). This indicates that the tips of the TZPs of the Smad4^fl/fl cells were, on average, closer to the oocyte than those of the Smad4^wt/wt cells. We also found that the proportion of TZPs that were oriented away from the oocyte trended towards being greater in the Smad4^wt/wt cells, although the difference did not reach statistical significance (Figure 4G). Taken together, the trajectory analyses provide additional evidence that the TZPs of Smad4^fl/fl cells differ from those of Smad4^wt/wt cells.

The observation that TZP-number was modestly reduced when intact GOCs were analyzed, yet markedly reduced when oocytes from which the granulosa cells had been stripped were analyzed, suggested that the attachment between the TZPs and oocyte might be weakened when SMAD4 was depleted. To evaluate this, the amount of N-cadherin, which is the principal cell adhesion molecule that has been identified in granulosa cells, was compared between Smad4^wt/wt and Smad4^fl/fl granulosa cells. Consistent with the previous results (see Figure 1E), SMAD4 was reduced by 73.5+/-0.05% in the Smad4^fl/fl granulosa cells (Figure 5A, n=6, p<0.0001). Strikingly, N-cadherin was reduced by 52.7+/-0.04% (n=6, p=0.0004) in the Smad4^fl/fl granulosa cells (Figure 5A). Moreover, Notch2, a transmembrane receptor expressed in granulosa cells that associates with membrane-bound Jagged1 expressed on the oocyte, was reduced by 52.6+/-0.05% (n=5; p=0.0003) in these cells (Figure 5B). In contrast to the protein, however, there was no detectable reduction in the amount of encoding mRNA in either case as assayed using qRT-PCR, although Smad4 was reduced in the same samples (Figure 5C). Thus, a reduction in SMAD4 in granulosa cells is associated with a reduction in certain transmembrane proteins, including those responsible for cell adhesion.

Figure 5

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As we previously reported that depletion of GDF9, which is secreted by the oocyte and signals in the granulosa cells through SMAD4, was associated with a reduction in mRNAs encoding structural components of TZPs – Myo10, Fscn1, and Daam1 (El-Hayek et al., 2018a), we measured the quantity of these mRNAs using qRT-PCR. We found that they were not depleted in the Smad4^fl/fl granulosa cells compared to the Smad4^wt/wt cells (Figure 5D). This implies either that the SMAD4 which remained was sufficient to induce expression of these genes or the GDF9 regulates their expression through a SMAD4-independent pathway.

We have examined the role of SMAD4 in establishing the TZPs that connect the granulosa cells to the oocyte. Using a tamoxifen-inducible Cre recombinase strategy, we were able to reduce SMAD4 in the granulosa cells to about 30% of its wild-type level. This reduction was associated with a decrease ranging from 20–50% in the number of TZPs in the granulosa cells assessed as a population (i) in intact GOCs and (ii) after removal of the granulosa cell bodies to increase optical resolution, and as individual cells (iii) in intact GOCs and (iv) in reconstructed complexes. Previous studies where SMAD4, or both SMAD2 and SMAD3, was depleted from granulosa cells described a loose or loss association of the cumulus cells with the oocyte in some follicles (Pangas et al., 2006; Li et al., 2008). Moreover, as described in the Introduction, blocking the production of GDF9 by the oocyte leads to a decrease in the number of TZPs (Carabatsos et al., 1998; El-Hayek et al., 2018a). Together these observations suggest that SMAD4 regulates the size of the population of TZPs in growing follicles.

The population of TZPs presumably reflects the net result of the generation of new TZPs and the retraction of existing TZPs, as is the case for filopodia (Gallop, 2020; Heckman and Plummer, 2013; Mattila and Lappalainen, 2008). SMAD4 could thus promote either or both processes. The observation that GFP-expressing granulosa cells in reaggregated complexes generated new TZPs implies that SMAD4 is not required for this process, although it does not rule out a role in increasing the rate of generation. A clue to the function of SMAD4 may lie in the observation that the decrease in the number of TZPs in SMAD4-depleted cells was quantitatively greater when the granulosa cell bodies were removed prior to analysis. This suggests that the process of removing the cell bodies also removed the TZPs more easily in the SMAD4-depleted cells than in wild-type cells.

In this context, it is intriguing that N-cadherin, which is thought to stabilize the attachment of the TZP tips to the oocyte surface, was depleted in the mutant cells. We propose that, by increasing the quantity of N-cadherin in the granulosa cells, SMAD4 increases the strength of adhesion of the TZPs to the oocyte surface (Figure 6). This in turn would decrease the probability of retraction, thereby increasing the steady-state number of TZPs. This model is consistent with the observation that the non-attached (free-ended) TZPs of SMAD4-depleted cells were on average longer and closer to the oocyte than those of wild-type cells. Specifically, if the ability of the TZPs to establish a stable contact with the oocyte wer impaired in SMAD4-depleted cells, a relatively large proportion might continue to ‘search’ for an attachment and to lengthen during the searching process.

Figure 6

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Mechanistically, some reports have linked SMAD4 to transcription of the Cdh2, which encodes N-cadherin (Kang et al., 2014; Yang et al., 2015), but we did not detect a difference in mRNA quantity between normal and SMAD4-depleted granulosa cells. In view of the reduced quantity in the latter of Notch2, also a membrane-associated protein, it may be speculated that SMAD4 might act more broadly to regulate the expression of genes whose products maintain the normal configuration of the granulosa cell plasma membrane. An alternative explanation is raised by observations that granulosa-cell depletion of SMAD4 is associated with the presence of follicles containing luteinized granulosa cells enclosing an entrapped oocyte and increased expression of markers of luteinization, including Cyp11a1, Sfrp4, Star, and Ptgfr (Pangas et al., 2006). This suggests that the SMAD4-depleted granulosa cells in the GOCs studied here might have become luteinized, and that luteinization in turn caused a reduction in the number of TZPs. We did not detect increased expression of the luteinization markers (unpublished data), suggesting that luteinization did not occur under the conditions of our experiments. This difference may arise because we examined GOCs that had been maintained in vitro, whereas the previous study examined follicles in intact ovaries of prepuberal mice following the injection of an FSH analogue.

Regardless of the mechanism linking SMAD4 to TZP number, it is noteworthy that the number of TZPs that project from an individual granulosa cells – about 15 in this study and about 40 in a previous study where more advanced (antral) follicles were examined (Baena and Terasaki, 2019) – considerably exceeds the number of filopodia that are present on individual blastomeres of mouse embryos (5-6) (Fierro-González et al., 2013) or on chick embryonic cells (~4) (Pröls et al., 2015). SMAD4 may, therefore, act in granulosa cells to amplify an innate ability to generate filopodia, which become TZPs and increase the efficiency of communication between the growing germ cell and its somatic microenvironment.

Original data describing TZP characteristics have been deposited at Dryad https://doi.org/10.5061/dryad.tht76hf6s.

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Author details

1. Sofia Granados-Aparici

   1. Research Institute, McGill University Health Centre, Montreal, Canada
   2. Present address: Cancer CIBER (CIBERONC), Madrid, Spain
   3. Present address: Pathology Department, Medical School, University of Valencia-INCLIVA, Valencia, Spain

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Methodology, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   sograap@gmail.com

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4448-3254

2. Qin Yang

   Research Institute, McGill University Health Centre, Montreal, Canada

   Contribution

   Data curation, Validation, Investigation, Methodology

   Competing interests

   No competing interests declared

3. Hugh J Clarke

   1. Research Institute, McGill University Health Centre, Montreal, Canada
   2. Departments of Obstetrics and Gynecology and Biology, Division of Experimental Medicine, McGill University, Montréal, Canada

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   hugh.clarke@mcgill.ca

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2626-244X

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