The Slingshot phosphatase 2 is required for acrosome biogenesis during spermatogenesis in mice

Essential revisions:

1) Provide Phalloidin staining on tubule squashes to examine actin cytoskeleton.

We appreciate the insightful comment by the editor and apologize for the lack of flourescent stain marks. We indicated the actin cytoskeleton which is the rhodamine phalloidin in the panels of Figure 5B in the originally submitted manuscript, namely “the representative images of Phalloidin staining performed on tubule squashes”. To clarify, we have now highlighted the mark of Phalloidin and added some indicators regarding the structures of disorganized F-actin in the images of a new Figure 5B in the revised manuscript. Furthermore, we have replaced the images of Phalloidin staining on tubule squashes with more representative ones in Figure 5B in the revised manuscript which demonstrated clearly that actin filament remodeling is disrupted in the round spermatids of Ssh2 KO mice and solidified the conclusion that the actin remodeling was disordered in Ssh2 KO spermatids.

2) Provide EM analyses of the round spermatids to detect the cytoplasmic distribution of actin filaments in the wild type and disrupted actin filament remodeling in KO mice, if possible.

Thank you for your comment. Previously we already have conducted a transmission electron microscopy (TEM) analysis on the testes samples to discover the distribution and ultrastructural organization of F-actin in WT and Ssh2 KO round spermatids. Unfortunately, even at high magnification (30,000x, right panel of Author response image 1) by TEM of the testicular section no diminutive actin filament was observed in the cytoplasm of round spermatids except for the acroplaxome-an actin-rich specialized structure anchors the acrosome-in WT spermatids as well as some thick bundle-like structures located at the acrosomal region of Ssh2 KO spermatids (Author response image 1). According to their unique characteristic of appearance, we interpreted these electron-dense bundles as the aberrantly aggregated actin filaments whose lengths are in accordance with the lengths of COFILIN-saturated F-actin fragments (Bamburg, Minamide, Wiggan, Tahtamouni, and Kuhn, 2021), suggesting the disrupted actin filament remodeling during acrosome biogenesis resulted from Ssh2 KO. However, due to the technological limitations of TEM and the complexity of the intracellular environment of round spermatids, we only recognized a few aggregated actin bundles with the loss of filamentous appearance in Ssh2 KO spermatids and no typical diminutive actin filament was detected which had been imaged under high-resolution cryo-TEM (Haviv, Gov, Ideses, and Bernheim-Groswasser, 2008) or live-cell total internal reflection fluorescence microscopy (Johnson et al., 2015) on the purified actin bundles and cultured cells. Given the lack of effective approaches to culture murine round spermatids in vitro, confocal microscopy of flourescence-labelled F-actin (e.g., IF staining by FITC-phalloidin) is a more accessible method for visualizing the disruption of actin remodeling than EM in murine spermatids as the actin-related findings that several other studies demonstrated (Djuzenova et al., 2015).

Author response image 1

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Reviewer #2 (Recommendations for the authors):

Nice work and it could enhance the impact of this work by examining carefully the defect of actin cytoskeleton remodeling, proacrosomal vesicle trafficking, or any other subcellular organelles in round spermatids with EM. It is important to distinguish the general effect of the actin cytoskeleton and specific effects on acrosomal biogenesis such as defects in Golgi and proacrosomal vesicle fusion.

We appreciate and thank the referee for the positive feedback which helps us to keep these suggestions in our mind to perform the experiments in our future projects.

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