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:

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.

Author response:

(1) Clarification of the scope of the present study and future mechanistic analyses

We agree that the downstream molecular mechanisms by which SOX17 regulates Sertoli valve formation remain to be elucidated. Our findings are consistent with a model in which SOX17 regulates Sertoli valve formation through paracrine signaling; however, the downstream effectors have not yet been identified. Despite extensive analyses of Sox17 conditional knockout and wild-type mice, including single-cell RNA sequencing, identifying the downstream molecular targets of SOX17 has remained challenging (Uchida et al., 2022). The transgenic mouse model generated in the present study now provides a valuable experimental platform for investigating SOX17-dependent molecular pathways. We are currently performing transcriptomic analyses using this model to identify candidate downstream pathways and genes regulated by SOX17. However, further investigation will be required to determine whether these candidates represent direct transcriptional targets of SOX17 and whether they function specifically within the rete testis during Sertoli valve formation.

Accordingly, we will avoid overinterpreting the molecular mechanisms in the present study and will revise the Discussion to more clearly acknowledge these limitations while emphasizing that elucidation of these mechanisms represents an important direction for future research. We therefore believe that a comprehensive mechanistic analysis is beyond the scope of the present study.

(2) Clarification of the quantitative methodology

We will provide a more detailed description of the methodology used for Sertoli cell quantification. Specifically, Sertoli cells were counted within the SV region extending 100 μm from the rete testis (RT) boundary, and Sertoli cells protruding into the RT lumen were also included in the analysis. The sampling procedure for sagittal RT-SV-seminiferous tubule (ST) sections will be described more explicitly in the revised Methods to improve reproducibility.

(3) Clarification regarding expression levels

We appreciate the reviewer's comment regarding the quantitative assessment of SOX17 and other SV-associated molecules.

The Sertoli valve (SV) is an extremely small transitional structure, with only approximately 20 SVs present in each mouse testis. In addition, Sertoli cells within the SV are tightly interconnected. Consequently, selectively isolating the SV without contamination from adjacent tissues while obtaining sufficient material for quantitative molecular analyses, such as quantitative PCR, remains technically challenging.These technical limitations partly explain why the Sertoli valve has remained an understudied structure in testicular biology. Therefore, in the present study, the expression of SV-associated molecules was primarily evaluated by histological and immunohistochemical analyses. We will clarify these technical limitations in the revised manuscript and revise the relevant text accordingly.

(4) Additional revisions

We will address the remaining comments, including clarification of the phenotypic differences between Tg26 (established line) and Tg27 (F0), standardization of gene nomenclature, correction of methodological descriptions, and improvements to the Discussion and figure presentation where appropriate.