Ectopic hAMH-driven SOX17 expression induces hyperplastic Sertoli valve formation in mouse testes

Reviewer #1 (Public review):

Summary:

This manuscript is an excellent follow-up to your 2022 study, in which Sox17 expression was localized to the rete testis and shown to be required for proper formation of the Sertoli cell valve (transition region). By using Nr5a1-Cre to drive conditional deletion of Sox17 specifically in rete testis cells, you demonstrate that testis weights remain normal at 2 weeks of age but become significantly reduced by 8 weeks in Sox17-cKO males. At the later time point, the seminiferous epithelium is severely disrupted, with apparent arrest of spermiogenesis: the epididymal lumen is essentially devoid of sperm, and most tubules lack elongated spermatids.

Strengths:

Clearly shows the role of Sox17 in Sertoli cells being important to the SV function. The SV (transition region) between the rete testis and seminiferous tubules remains an understudied domain of testicular biology. The present work, together with your prior study, highlights intriguing mechanisms operating in this specialized niche.

Weaknesses:

The available data do not fully explain either the developmental assembly of the Sertoli valve or the precise consequences of its functional disruption. These studies are nonetheless valuable precisely because they raise more questions than they answer; the conceptual implications are thought-provoking.

Reviewer #2 (Public review):

This manuscript investigates the role of SOX17 in the formation and function of the Sertoli valve (SV) at the interface between seminiferous tubules and the rete testis (RT). Building on previous work showing that rete testis-specific deletion of Sox17 disrupts SV formation, leading to defective spermiogenesis and male infertility, the authors explore how SOX17 overexpression in Sertoli cells regulate SV of rodent testes.

Using transgenic mouse models with ectopic Sox17 expression in Sertoli cells, the study demonstrates that SOX17 is not only required but can also modulate SV formation. Ectopic expression in Sertoli cells induces expansion of the SV structure and partially rescues SV defects and spermatogenesis in RT-specific Sox17 conditional knockout animals. The data support a model in which SOX17 acts through paracrine signaling to regulate SV formation, although the precise mechanisms remain to be clarified.

Overall, this is a well-executed study with novel and significant findings. The ability to experimentally manipulate SV size is particularly compelling and provides a valuable framework to study fluid dynamics and epithelial interactions in the testis. This work will be of broad interest to the reproductive biology and developmental biology communities.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public review):

Summary:

This manuscript is an excellent follow-up to your 2022 study, in which Sox17 expression was localized to the rete testis and shown to be required for proper formation of the Sertoli cell valve (transition region). By using Nr5a1-Cre to drive conditional deletion of Sox17 specifically in rete testis cells, you demonstrate that testis weights remain normal at 2 weeks of age but become significantly reduced by 8 weeks in Sox17-cKO males. At the later time point, the seminiferous epithelium is severely disrupted, with apparent arrest of spermiogenesis: the epididymal lumen is essentially devoid of sperm, and most tubules lack elongated spermatids.

Strengths:

The study clearly shows the role of Sox17 in Sertoli cells as being important to SV function. The SV (transition region) between the rete testis and seminiferous tubules remains an understudied domain of testicular biology. The present work, together with the authors' prior study, highlights intriguing mechanisms operating in this specialized niche.

Weaknesses:

At the same time, the available data do not yet fully explain either the developmental assembly of the Sertoli valve or the precise consequences of its functional disruption. These studies are nonetheless valuable precisely because they raise more questions than they answer; the conceptual implications are thought-provoking.

Reviewer #2 (Public review):

This manuscript investigates the role of SOX17 in the formation and function of the Sertoli valve (SV) at the interface between seminiferous tubules and the rete testis (RT). Building on previous work showing that rete testis-specific deletion of Sox17 disrupts SV formation, leading to defective spermiogenesis and male infertility, the authors explore how SOX17 overexpression in Sertoli cells regulates the SV of rodent testes.

Using transgenic mouse models with ectopic Sox17 expression in Sertoli cells, the study demonstrates that SOX17 is not only required but can also modulate SV formation. Ectopic expression in Sertoli cells induces expansion of the SV structure and partially rescues SV defects and spermatogenesis in RT-specific Sox17 conditional knockout animals. The data support a model in which SOX17 acts through paracrine signaling to regulate SV formation, although the precise mechanisms remain to be clarified.

Overall, this is a well-executed study with novel and significant findings. The ability to experimentally manipulate SV size is particularly compelling and provides a valuable framework to study fluid dynamics and epithelial interactions in the testis. This work will be of broad interest to the reproductive biology and developmental biology communities.

Reviewer #3 (Public review):

Summary:

These studies are based on previously published work that showed that deletion of expression of the Sox17 gene in the testis essentially deleted the formation of the Sertoli valve in the Rete testis. The authors extended this work by constructing a vector that resulted in increased Sox17 expression by Sertoli cells and enhanced formation of the Sertoli valve in both wild type and Sox17 knockout mice. The work provides strong evidence supporting the requirement for Sox17 expression to allow formation of the Sertoli valve.

Strengths:

The general approach was to express Sox17 from a Tg mouse that expressed Sox17 from Sertoli cells. This Tg mouse was bred into both the WT and the Sox17 KO mouse. The Sertoli valve was enhanced in both the WT/Tg mouse and KO/Tg mouse, showing that ectopic Sox17 could compensate in the Sox17 Ko and act in a concentration-dependent manner in the WT mouse. The results are strong and support the conclusions from the authors. The results were as expected from the original paper describing the KO of Sox 17. These results strengthen these conclusions and provide ideas for additional conclusions. These studies were technically challenging, and the authors provided a very solid manuscript.

Weaknesses:

The authors refer several times to high or low expression, but it all appears to be based on immunohistochemistry, and there is no real quantification using PCR, for example. The process used for cell quantification lacks a rationale for why certain numbers were assigned.

We sincerely thank the reviewers for their careful evaluation of our manuscript and for their constructive and encouraging comments. We are grateful for the recognition of the significance of the Sertoli valve as an understudied transition region between the rete testis and seminiferous tubules, as well as for the positive assessment of our genetic approach and the evidence that ectopic SOX17 expression can modulate SV formation. We have carefully considered all points raised in the assessment and have revised the manuscript accordingly. The major revisions include:

(1) Clarification of the scope and limitations of the study (Reviewers #1 and #2):

In response to the comments that the developmental assembly of the Sertoli valve and the precise consequences of its functional disruption remain incompletely understood, we clarified the scope and limitations of the present study at the end of 7th paragraph in the Discussion. Although our findings support a model in which SOX17 regulates SV formation through paracrine signaling, the downstream effectors and precise molecular mechanisms remain to be identified. We therefore revised the Discussion to avoid overinterpretation of the molecular mechanisms and to emphasize that comprehensive mechanistic analyses, including transcriptomic analyses using the Tg mouse model, represent an important direction for future research. We also added histological analyses of the earliest detectable lesions at 4 weeks of age and low-magnification images of adult Sox17 cKO testes (new Figure S1), revealing selective sloughing of round spermatids despite preserved Sertoli cell architecture and subsequent mosaic spermatogenic defects among individual seminiferous tubules. These observations provide additional insights into the altered luminal microenvironment and suggest that spermatogenic defects may progress in a tubule-by-tubule manner.

(2) Clarification of quantitative analysis and methodology (Reviewer #3):

In response to concerns regarding the basis and methodology of cell quantification, we revised the Methods to provide detailed information on tissue preparation, fixation, orientation of the rete testis–Sertoli valve region, and the criteria used for quantitative analysis of SV-associated Sertoli cells (new Figure S4). We clarified that Sertoli cells were counted within the SV region extending approximately 100 μm from the RT boundary, including Sertoli cells protruding into the RT lumen, based on previously established criteria (Aiyama et al., 2015).

(3) Clarification of the limitations of expression-level assessment (Reviewer #3):

In response to concerns regarding the quantitative assessment of SOX17 and other SV-associated molecules, we clarified the technical limitations of selectively isolating the very small SV region and obtaining sufficient material for quantitative molecular analyses such as qPCR at the end of 7th paragraph in the Discussion. We therefore clarified that expression of SV-associated molecules in the present study was primarily evaluated using histological and immunohistochemical approaches and added the relevant text to acknowledge these limitations.

We also made additional revisions to clarify each mouse Tg line, phenotypic descriptions, standardize gene nomenclature, improve methodological descriptions, and refine the relevant Discussion where appropriate.

We sincerely appreciate the reviewers’ thoughtful and constructive comments. Their feedback has helped us clarify the scope of our conclusions, strengthen the methodological descriptions, and improve the overall presentation of the study. All changes have been incorporated into the revised manuscript.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(i) Although the current paper is not responsible for interpreting the 2022 findings, both datasets show reduced spermatid production accompanied by multinucleated giant germ-cell syncytia. This phenotype has been attributed to backflow of tubular fluid and consequent microenvironmental perturbation. While this is a reasonable hypothesis, it is not entirely consistent with earlier experimental observations. Complete ligation of the efferent ductules reliably produces giant cells, whereas estrogen-receptor knockout, which also causes massive luminal fluid accumulation, does not. In addition, ligation of the testicular artery itself can induce giant-cell formation. Although this may have already been answered in the papers, can you be sure that a direct or indirect effect on the vasculature can be excluded in the Sox17-cKO model?

We thank the reviewer for this important comment. In our models, SOX17 expression was manipulated specifically in the Sertoli cell lineage, either by SF1-Cre-mediated Sox17 deletion or by ectopic SOX17 expression under the hAMH-promoter. SOX17-expressing vascular endothelial cells were not targeted in either model, making a direct effect of Sox17 manipulation on the testicular vasculature unlikely. Moreover, the partial rescue of the Sox17 cKO phenotype by hAMH-Sox17 supports the interpretation that the phenotype primarily results from altered SOX17 function in Sertoli cells and RT epithelia.

However, indirect effects on the vascular or interstitial environment by aberrant luminal flow cannot be completely excluded, particularly with the substantial accumulation of sloughed round spermatids (giant cells) within the rete testis. Addressing the potential for an initial luminal flow defect, we newly added histological images of 4-week-old testes (Figure S1), where selective post-meiotic germ cell sloughing occurs despite preserved Sertoli cell process architecture, suggesting an altered adluminal microenvironment that impairs Sertoli–spermatid adhesion. Furthermore, low-magnification images of adult mature Sox17 cKO testes (Figure S1B) display a mosaic pattern of spermatogenic defects across individual tubules. While 3D reconstruction was not conducted, this structural pattern supports the view that spermatogenic failure progresses on a tubule-by-tubule basis, potentially linked to the structural integrity of individual Sertoli valves.

(ii) A related and important unresolved issue is the total number of Sertoli cells per testis in cKO males. The number of Sertoli cells per tubule cross-section is reported to be equivalent to controls; however, the substantial reduction in testis weight implies a corresponding reduction in tubule length. Under these conditions, maintenance of a normal per-cross-section count would still be compatible with an overall decrease in total Sertoli-cell number. Although it is generally accepted that murine Sertoli cells exit the cell cycle around postnatal day 15, continued growth of the testis may still occur in the Sertoli valve region, where Sertoli cells retain proliferative capacity. Your discussion of possible heterogeneity in the embryonic origin of Sertoli cells near the rete testis is therefore particularly intriguing and commendable. Should this hypothesis be substantiated, it would raise the possibility that Sertoli cells derived from the valve region, especially those that migrate into the seminiferous tubules, are intrinsically less competent to support full spermatogenesis than those of classic gonadal-ridge origin.

To help readers appreciate the overall severity and topographic distribution of the spermatogenic defect (particularly in tubule segments distant from the rete), inclusion of a low-magnification photomicrograph of a well-fixed (Bouin's) testicular cross-section would be very useful.

We thank the reviewer for this important comment. We agree that the maintenance of Sertoli cell numbers per seminiferous tubule cross-section does not necessarily indicate preservation of the total Sertoli cell number per testis, particularly given the substantial reduction in testis size and potential reduction in overall seminiferous tubule length. Although total Sertoli cell numbers can theoretically be estimated using stereological approaches, such analyses are technically demanding and beyond the scope of the present study.

We also appreciate the reviewer’s insightful suggestion regarding potential heterogeneity among Sertoli cell populations. Sertoli cells associated with the Sertoli valve region may have distinct developmental origins or functional properties compared with classical gonadal ridge-derived Sertoli cells, which could potentially influence their capacity to support complete spermatogenesis. Although this hypothesis was not directly tested in this study, we have expanded the Discussion to highlight the developmental and functional heterogeneity of Sertoli cell populations associated with the Sertoli valve as an important topic for future investigation.

In addition, as requested, we have added a low-magnification image of well-preserved testicular cross-sections in Supplementary Figure S1B to better illustrate the overall severity and topographic distribution of spermatogenic defects throughout the testis.

Specific Comments:

(1) Figure 3A and associated fertility/histology data. The results state that epididymal spermatozoa were detected in only 2 of 7 cKO;Tg males at 8 weeks of age, yet Materials and Methods indicate that spermatogenesis was evaluated in only 5 males. a) Were the remaining two males also examined histologically? b) It would be interesting to determine if the severity of pathological changes was the same in regions more distant from the rete testis, or possibly different tubules. See: Nakata H, Wakayama T, Sonomura T, Honma S, Hatta T and Iseki S (2015). "Three-dimensional structure of seminiferous tubules in the adult mouse." J Anat 227(5): 686-694. c) In addition, mating trials were performed with four independent cKO;Tg males, two of which sired offspring. It is unclear whether the testes of these four mating males were included among the five (or seven) animals evaluated for histology, and whether the two fertile males correspond exactly to the two individuals that retained epididymal sperm. Please clarify these relationships explicitly so that readers can correctly interpret the link between histological findings and fertility.

We thank the reviewer for this important comment. We apologize that the relationship among the groups of animals used for histological analysis, epididymal sperm detection, and fertility assessment was not sufficiently clear in the original manuscript. Because this study focused specifically on the anatomically minute RT–SV region, our sampling strategy had to prioritize the maximal utilization of this limited tissue. In this study, the RT–SV region, the remaining testicular tissue, and the epididymis were processed separately as three tissue blocks for each animal (Figure S4) and were independently evaluated for distinct analysis sets. Briefly, the proximal quarter containing the rete testis and Sertoli valve region was used for SV analysis, whereas the remaining three-quarters of the testis were used for evaluation of spermatogenesis, and the epididymis was analyzed separately for the presence of spermatozoa. Therefore, due to these technical requirements, tissue allocation, and independent analytical evaluation, the numbers of animals used for RT–SV analysis, testicular histology, epididymal sperm detection, and fertility testing were not identical.

For quantitative histological analyses, we also used virgin males to minimize potential variation associated with mating experience and to allow comparison with age-matched littermate controls. Therefore, these animals were not used for fertility testing. Fertility assessment was performed using an independent cohort of cKO; Tg males that were subjected to long-term mating trials with wild-type females. Thus, fertility outcomes and histological findings were not designed to be directly matched at the individual level.

In response to the reviewer’s suggestion, we have clarified the selection of experimental animals and the relationship among fertility assessment and histological analyses in the Materials and Methods and added a schematic illustration of the sampling strategy in Figure S4. We also corrected the citation for Nakata H et al., 2015 in the revised manuscript.

(2) Page 8, line 301 (Sertoli-cell quantification). The description of the counting method-"counted in each ... (~100 μm from the edge of the RT; Fig. 4C)"-is ambiguous.

(a) Does this mean that cells were counted beginning at the rete boundary and extending radially outward for approximately 100 μm, or is a circumferential sampling area intended? (b) Figure 4C shows a large standard deviation, indicating substantial variability with the current approach. An alternative strategy (for example, counting Sertoli cells within standardized areas or per tubule specifically within the valve region) might reduce variability and improve reproducibility. Regardless of the method ultimately chosen, a more precise, step-by-step description of the quantification protocol is required so that it can be reliably replicated by other laboratories.

We thank the reviewer for pointing out that the description of the Sertoli cell quantification method was not sufficiently clear. The Sertoli cell quantification was performed using the same criteria as previously described (Aiyama et al., 2015; Uchida et al., 2022), in which SOX9-positive Sertoli cell nuclei within the SV-associated region were counted.

In the revised manuscript, we have clarified that the SV region was operationally defined as comprising (i) the terminal 100 μm segment of the seminiferous tubule immediately adjacent to the rete testis (RT) and (ii) the protruded SV extending into the RT lumen. Based on the distribution of spermatogonial stem cells, the ~100 μm region extending from the RT boundary along the seminiferous tubule toward the ST side was defined as the SV region (Aiyama et al., 2015). Only sagittal sections showing a continuous RT–SV–ST axis and sectioning the SV approximately through its mid-sagittal plane were included for quantitative analysis.

Furthermore, to improve reproducibility, we have added a more detailed description of the tissue preparation and quantification procedures in the Materials and Methods and provided a schematic illustration of the quantification strategy in the new Figure S4.

Reviewer #2 (Recommendations for the authors):

(1) Phenotypic differences between transgenic lines: the phenotypic differences between the tg26 and tg27 lines are intriguing and warrant further clarification. While tg27 mice exhibit infertility and defective spermatogenesis, tg26 animals remain fertile with SV expansion. Could the authors elaborate on the underlying causes of these differences? In particular, is infertility in tg27 mice due to excessive SOX17 expression impairing Sertoli cell function? A comparison of Sox17 expression levels between tg26 and tg27 lines would be informative. In addition, it would be useful to assess whether acetylated tubulin (Ac-Tub) expression is present in the Sertoli cells of the tg27 mouse testis.

We thank the reviewer for this highly constructive and insightful comment. We clarified in the revised manuscript that the analysis of the Tg27 mouse was performed using the F0 founder male and added an explanation that only the Tg26 line could be established as its heterogenous SOX17 expression in Sertoli cells did not impair overall fertility. We agree that the phenotypic differences between the Tg26 line and the Tg27 mouse provide important clues regarding the dosage-dependent effects of SOX17 in Sertoli cells. Unfortunately, we were unable to establish a stable, multi-generational transgenic line from this Tg27 founder (F0) male. Consequently, we could not perform detailed molecular or immunohistochemical analyses on this line beyond the initial histological evaluation of the F0 generation presented in Figure 1. For this reason, we cannot provide a quantitative comparison of Sox17 expression levels or evaluate acetylated tubulin (Ac-Tub) expression in Tg27 Sertoli cells.

To address the reviewer's concern without overstepping the available data, we removed direct quantitative comparisons of Sox17 expression levels between the two lines from the text. Instead, we added a clear description of their contrasting cellular expression patterns - specifically, the mosaic, heterogeneous SOX17 expression in Tg26 Sertoli cells versus the ectopic, uniform SOX17 expression in the infertile #27 F0 male - in the 'Animals' section of Materials and Methods. This mosaic pattern in Tg26 testes suggests the presence of Sertoli cells with low or undetectable SOX17 levels, which may be associated with sustaining overall fertility.

(2) Mechanism of SOX17 action: although SOX17 is a transcription factor, the author's studies indicate it regulates SV formation via paracrine and/or autocrine signaling. The underlying mechanisms remain unclear. Which downstream factors mediate this effect? The observed upregulation of RSPO1 and WNT4 is suggestive, but more direct evidence would strengthen this conclusion. For example, does SV expansion in tg26 mice depend on the activation of RSPO1/WNT signaling? Additional molecular analyses, such as bulk RNA-seq comparing control and transgenic testes, could help identify pathways regulated by SOX17 and clarify its mode of action.

We thank the reviewer for this important and insightful suggestion. At present, comprehensive analyses, including scRNA-seq of Sox17 cKO and littermate control testes, have not identified definitive downstream targets of SOX17 (Uchida et al., 2022). As the reviewer rightly points out, the Tg26 mouse model generated in this study represents a valuable tool for investigating SOX17-dependent molecular pathways. To this end, we are currently conducting transcriptomic analyses of Tg26 seminiferous tubules to identify genes altered in SOX17+ Sertoli cells. However, determining whether these candidate genes represent direct transcriptional targets of SOX17 and whether they function specifically in the rete testis-associated region during Sertoli valve formation will require extensive functional and expression studies. Therefore, we feel it would be premature to draw definitive conclusions regarding the underlying molecular mechanisms, including the precise involvement of the RSPO1/WNT signaling pathway, in the present manuscript. Accordingly, rather than overinterpreting the available data, we have revised the Discussion to clarify this limitation (at the end of 7th paragraph in the Discussion). Furthermore, incorporating initial insights from our ongoing Tg26 transcriptomic analyses, we have added a brief discussion, supported by relevant literature, on the possibility that SOX17 may regulate Sertoli valve formation by modulating cell adhesion and extracellular matrix (ECM) organization and altering the responsiveness of SOX17-positive Sertoli cells to morphogenetic signals originating from the rete testis (new 5th paragraph in Discussion).

(3) Minor comment: Gene nomenclature should be standardized: e.g. line 245, Sox17 and hAMH should be italicized.

We thank the reviewer for pointing this out. All gene names have been italicized throughout the manuscript.

Reviewer #3 (Recommendations for the authors):

No suggestions except to quantify some of the changes in concentration of agents by PCR rather than eyeball levels with immunocytochemistry. Verify the cell quantification procedure used.

We thank the reviewer for this comment. The Sertoli valve (SV) is an extremely small transitional structure, with only approximately 20 sites per mouse testis. As a result, selective isolation of the SV region to collect sufficient material for molecular analyses, such as quantitative PCR, remains technically challenging. We have therefore added this limitation to the Discussion.

Regarding the cell quantification procedure, we have clarified the methodology in the revised Materials and Methods and added a schematic illustration in Figure S4. Specifically, the Sertoli cell number in the SV region was quantified by counting SOX9-positive Sertoli cell nuclei within a standardized SV-associated region in RT–SV–ST sagittal sections.