Differential RA responsiveness among subsets of mouse late progenitor spermatogonia

in Reproduction
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  • 1 Department of Biology, University of Texas at San Antonio, San Antonio, Texas, USA

Correspondence should be addressed to B P Hermann; Email: brian.hermann@utsa.edu
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Initiation of spermatogonial differentiation in the mouse testis begins with the response to retinoic acid (RA) characterized by activation of KIT and STRA8 expression. In the adult, spermatogonial differentiation is spatiotemporally coordinated by a pulse of RA every 8.6 days that is localized to stages VII–VIII of the seminiferous epithelial cycle. Dogmatically, progenitor spermatogonia that express retinoic acid receptor gamma (RARG) at these stages will differentiate in response to RA, but this has yet to be tested functionally. Previous single-cell RNA-seq data identified phenotypically and functionally distinct subsets of spermatogonial stem cells (SSCs) and progenitor spermatogonia, where late progenitor spermatogonia were defined by expression of RARG and Dppa3. Here, we found late progenitor spermatogonia (RARGhigh KIT−) were further divisible into two subpopulations based on Dppa3 reporter expression (Dppa3-ECFP or Dppa3-EGFP) and were observed across all stages of the seminiferous epithelial cycle. However, nearly all Dppa3+ spermatogonia were differentiating (KIT+) late in the seminiferous epithelial cycle (stages X–XII), while Dppa3− late progenitors remained abundant, suggesting that Dppa3+ and Dppa3− late progenitors differentially responded to RA. Following acute RA treatment (2–4 h), significantly more Dppa3+ late progenitors induced KIT, including at the midpoint of the cycle (stages VI–IX), than Dppa3− late progenitors. Subsequently, single-cell analyses indicated a subset of Dppa3+ late progenitors expressed higher levels of Rxra, which we confirmed by RXRA whole-mount immunostaining. Together, these results indicate RARG alone is insufficient to initiate a spermatogonial response to RA in the adult mouse testis and suggest differential RXRA expression may discriminate responding cells.

Supplementary Materials

    • Supplementary Figure 1. (A-B) Representative immunofluorescence of DPPA3-EGFP (Green), PLZF (Red) and DAPI (Blue) in cryosections from adult Dppa3-Egfp mice. Asterisk: PLZF+/EGFP-, arrowhead: PLZF+/EGFP+, circle: PLZF-/EGFP+. (C-D) Negative control staining (for both panels A-B and Fig. 1B) were performed both with normal (non-immune) IgGs and omission of primary antibodies. Bar = 40µm.
    • Supplementary Figure 2. (A) Flow cytometry analysis of Dppa3-ECFP (left panel) or Dppa3-EGFP (right panel) and RARG in seminiferous tubule cells in undifferentiated spermatogonia (①; PLZFlow/KIT-). (B) Flow cytometry analysis of Dppa3-ECFP (left panel) or Dppa3-EGFP (right panel) in seminiferous tubule cells in undifferentiated spermatogonia (②; PLZFhigh/KIT-). (C) Flow cytometry analysis of Dppa3-ECFP (left panel) or Dppa3-EGFP (right panel) and RARG in seminiferous tubule cells in differentiating spermatogonia (③; PLZFhigh/KIT+). (D) Flow cytometry analysis of Dppa3-ECFP (left panel) or Dppa3-EGFP (right panel) and RARG in seminiferous tubule cells in differentiating spermatogonia (④; PLZFlow/KIT+). (E) Flow cytometry analysis of Dppa3-ECFP (left panel) or Dppa3-EGFP (right panel) and RARG in seminiferous tubule cells in differentiating spermatogonia (⑤; PLZF-/KIT+). (F-J) Flow cytometry analysis of GFP and RARG in seminiferous tubule cells from wild-type (non-transgenic) adult C57BL/6 mice in undifferentiated spermatogonia (F: ①; PLZFlow/KIT-, G: ②; PLZFhigh/KIT-) and differentiating spermatogonia (H: ③; PLZFhigh/KIT+, I: ④; PLZFlow/KIT+, J: ⑤; PLZF-/KIT+). (K-L) Flow cytometry analysis of wild-type (non-transgenic) testis cells from C57BL/6 mice stained with the noted isotype negative control antibodies to establish positive staining gates for KIT and PLZF or SOX3 and RARG.
    • Supplementary Figure 3. (A) Whole-mount immunofluorescence (WM-IIF) of DPPA3-ECFP (Green), RARG (Red) and GFRA1 (White) in Stages I-V of seminiferous tubules from adult Dppa3-Ecfp mice. Bar = 20µm. Arrowhead: ECFP- late progenitors (RARGhigh/ECFP-), asterisk: ECFP+ late progenitors (RARGhigh/ECFP+). (B) WM-IIF of DPPA3-ECFP (Green), RARG (Red) and GFRA1 (White) in Stages VI-IX of seminiferous tubules from adult Dppa3-Ecfp mice. Bar = 20µm. Arrowhead: ECFP- late progenitors (RARGhigh/ECFP-), asterisk: ECFP+ late progenitors (RARGhigh/ECFP+). (C) WM-IIF of DPPA3-ECFP (Green), RARG (Red) and GFRA1 (White) in Stages X-XII of seminiferous tubules from adult Dppa3-Ecfp mice. Bar = 20µm. Arrowhead: ECFP- late progenitors (RARGhigh/ECFP-), asterisk: ECFP+ late progenitors or early differentiating spermatogonia (RARGhigh/ECFP+). (D) Quantification of the proportion of RARGhigh/ECFP- (blue bars) or RARGhigh/ECFP+ (red bars) that are GFRA1+ in Stages I-V from A, Stages VI-IX from B, and Stages X-XII from C.
    • Supplementary Figure 4. (A) Whole-mount immunofluorescence (WM-IIF) of Dppa3-EGFP (Green), RARG (Red) and KIT (White) in Stages I-V of seminiferous tubules from adult Dppa3-Egfp mice. Bar = 20µm. Arrowhead: EGFP- late progenitors (RARGhigh/EGFP-), asterisk: EGFP+ late progenitors (RARGhigh/EGFP+). (B) WM-IIF of Dppa3-EGFP (Green), RARG (Red) and KIT (White) in Stages VI-IX of seminiferous tubules from adult Dppa3-Egfp mice. Bar = 20µm. Arrowhead: EGFP- late progenitors (RARGhigh/EGFP-), asterisk: EGFP+ late progenitors (RARGhigh/EGFP+). (C) WM-IIF of Dppa3-EGFP (Green), RARG (Red) and KIT (White) in Stages X-XII of seminiferous tubules from adult Dppa3-Egfp mice. Bar = 20µm. Arrowhead: EGFP- late progenitors (RARGhigh/EGFP-), arrow: early differentiating spermatogonia (RARGhigh/EGFP+/KIT+). (D) Negative control WM-IIF with either 1) normal non-immune IgGs (top row) from the same host species as the antibodies used in panels A-C and Fig. 3A-C or 2) omission of primary antibodies (bottom row).
    • Supplementary Figure 5 (A) RNA velocity clustering of individual adult late progenitors spermatogonia (Suzuki et al., 2021) projected onto a tSNE plot. Dots indicate cell state (steady-state mRNA levels) and vectors indicate direction and magnitude of predicted future cell state based on un-spliced (nascent) transcriptomes. (B) Heatmap of marker gene expression collapsed from each cluster with mRNA levels according to the Z-score scale. (C) Whole-mount immunofluorescence (WM-IIF) of Dppa3-ECFP (Green), RXRA (Red) and KIT (White) in Stages VI-IX of seminiferous tubules from adult Dppa3-Ecfp mice. Bar = 20µm. Arrowhead: RXRA+/ECFP-/KIT- (likely early progenitors), asterisk: RXRA+/ECFP+/KIT- late progenitors.

 

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