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

Contributor Notes

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.

Abstract

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.

Introduction

Spermatogenesis in mammalian testes is a highly productive process leading to production of millions of gametes per gram of parenchyma per day (Johnson et al. 1980). Productivity of the spermatogenic lineage is enabled by a large number (12 (mouse) or 5 (human)) of coordinated, clonal amplifying divisions between spermatogonial stem cells (SSCs) and sperm (reviewed by Fayomi & Orwig 2018). During steady-state adult spermatogenesis, germ cell development also occurs in a highly ordered and repeating fashion, termed as the cycle of the seminiferous epithelium, which is characterized by a defined set of cellular associations between different spermatogenic cell types (Clermont et al. 1972, de Rooij & Russell 2000). Each set of associations between different types of spermatogonia, spermatocytes and spermatids constitutes a 'stage' of the cycle of the seminiferous epithelium – there are 12 stages in mice (Oakberg 1956) and monkeys (Clermont & Leblond 1959) but only six stages in humans (Clermont 1963, Amann et al. 2008, Nihi et al. 2017). Consequently, the seminiferous cycle is coordinated in time such that a cycle is completed every 8.6 days in mouse (Oakberg 1956, Clermont & Trott 1969) and 16 days in the human (Heller & Clermont 1963) and, ultimately, it takes 34.5 days (mouse) and 64 days to produce spermatozoa from SSCs. During this time, four key developmental transitions take place, including spermatogonial differentiation, meiotic initiation, spermatid elongation during spermiogenesis, and spermiation (release of spermatozoa into the seminiferous tubule lumen), in a spatiotemporally coordinated fashion such that they occur during stages VII–VIII of the mouse seminiferous epithelial cycle (Oakberg 1956, Meng et al. 2001).

A pulse of retinoic acid (RA) production at the mid-point of the mouse seminiferous epithelial cycle (stages VII–VIII) drives spermatogonial differentiation and coincides with meiotic initiation and spermatid release (spermiation) (Endo et al. 2015, Hogarth et al. 2015). RA is an essential metabolite of vitamin A (retinol) and a long history of literature indicates that RA is required for these major developmental transitions in the spermatogenic lineage (de Rooij & Russell 2000, Busada et al. 2015, Endo et al. 2015) and for male fertility (Chung et al. 2004, Bowles et al. 2006, Anderson et al. 2008). Indeed, spermatogenesis in mice or rats subjected to restricted dietary intake of RA (the vitamin A-deficient (VAD) model) is blocked at the undifferentiated spermatogonia stage, causing infertility (Morales & Griswold 1987). Inhibition of spermatogenesis in VAD is also reversible because supplying exogenous RA drives arrested Aaligned spermatogonia to differentiate into A1 spermatogonia that reinitiate spermatogenesis and male fertility (Morales & Griswold, 1987). Since the half-life of RA in the mouse is less than 1 h (Nau 1986) and the peak pulse of RA occurs at stages VIII–IX of the seminiferous epithelium (Hogarth et al. 2015), RA levels must be tightly controlled by enzymatic biosynthesis and catabolism. Indeed, a pharmacological block to spermatogenesis and male infertility can also be achieved by inhibiting the rate-limiting enzymes responsible for testicular RA biosynthesis, ALDH1A1/A2, with the Bis-(dichloroacetyl)-diamine (BDAD) WIN18,446 (Heller et al. 1961). Reciprocally, potentiation of RA action through treatment with RA metabolism-blocking agents (RAMBAs) that have broad CYP inhibitory activity (ketoconazole) or which are specific to CYP26A1/B1 (talarozole) (Nelson et al. 2013) leads to enhanced spermatogonial differentiation in vitro and in vivo during the first wave of spermatogenesis (Velte et al. 2019). Thus, bioavailability of RA plays an important role as a rheostat on spermatogenic progression.

RA action is mediated by binding one of the three type II nuclear receptors, retinoic acid receptor alpha, beta or gamma (RARA, RARB, and RARG) that form requisite heterodimers with Retinoid X receptors alpha, beta and gamma (RXRA, RXRB, and RXRG) and bind to DNA at direct repeats of retinoic acid response elements (RAREs) (Chambon 2005, Germain et al. 2006, Huang et al. 2014, Stevison et al. 2017). In the absence of ligand (e.g. RA), RARE-bound RAR-RXR heterodimers complex with co-repressors (e.g. N-CoR) that prevent transcriptional activation of target genes. The presence of ligand favors RARE-bound RAR-RXR heterodimer interaction with co-activator proteins to facilitate RNA polymerase II-dependent transcription. In the testis, RARA expression is predominantly localized to Sertoli cells (but also detectable in some spermatogonia, spermatocytes and spermatids), RARB is not detectable, and RARG is restricted to a subset of spermatogonia (Gely-Pernot et al. 2012, Ikami et al. 2015). Among spermatogonia, RARG appears to be restricted to progenitor spermatogonia (Ikami et al. 2015, Hermann et al. 2018, Lord & Oatley 2018, Suzuki et al. 2021) and RARG+ progenitors in stages VII to VIII respond to RA and produce differentiating spermatogonia (Gely-Pernot et al. 2012, Ikami et al. 2015). However, the homogeneity of the progenitor response to RA during steady-state spermatogenesis has not been carefully studied.

Previous single-cell RNA-seq analyses of adult mouse spermatogonia (Hermann et al. 2018) facilitated identification of phenotypically and functionally distinct subsets of progenitor spermatogonia (Suzuki et al. 2021). Specifically, progenitor spermatogonia were phenotypically segregated into early progenitors with lower levels of prototypical progenitor markers than late progenitors (Suzuki et al. 2021). Functionally, while early progenitors lacked RARG expression, most RARG high late progenitors transitioned to KIT+ differentiating spermatogonia within 24 h after exogenous RA treatment, yet surprisingly, a significant proportion of spermatogonia exhibiting high levels of RARG protein escaped the differentiation-favoring influence of RA (Endo et al. 2015, Suzuki et al. 2021).

To further investigate this interesting observation, we obtained two independent lines of transgenic mice expressing Dppa3 fluorescent reporters (Payer et al. 2006, Seki et al. 2007, Ohinata et al. 2008), since Dppa3 mRNA is specifically expressed in late progenitors (Suzuki et al. 2021). These tools allowed us to classify the RARGhigh/KIT− late progenitors into two subpopulations based on presence or absence of Dppa3 reporter expression and linked these to differential response to endogenous and exogenous RA across the cycle of the seminiferous epithelium. Our results demonstrate that Dppa3-expressing late progenitors (RARGhigh/KIT−) preferentially differentiate in response to RA, indicating that RARG alone is insufficient to specify which late progenitors will differentiate when RA is not limiting. Further, we discovered differential expression of RXRA in Dppa3+/RARGhigh late progenitors, which may indicate this receptor mediates response to RA among this subset of undifferentiated spermatogonia.

Materials and methods

Mice

All experiments utilizing animals were approved by the Institutional Animal Care and Use Committee of the University of Texas at San Antonio (Assurance D16-00357) and were performed in accordance with the National Research Council's 'Guide for the Care and Use of Laboratory Animals'. All animals were maintained under conditions of ad libitum water and food with constant light–darkness cycles. Cryopreserved mouse embryos bearing either a Dppa3-Egfp transgene (Accession No. CDB0407T, (Payer et al. 2006, Seki et al. 2007)) or a Dppa3-Ecfp transgene (Accession No. CDB0465T, (Ohinata et al. 2008)) were obtained from the RIKEN Center for Biosystems Dynamics Research (http://www.clst.riken.jp/arg/mutant%20mice%20list.html) and re-derived at the Washington State University Animal Production Core. Male C57BL/6 (The Jackson Laboratories) or transgenic reporter mice were used as adults (2–6 months old). Some mice in this study were treated with a single 100 μL bolus of 7.5 mg/mL all-transRA (Sigma-Aldrich) as described (Endo et al. 2015) and euthanized 2 or 4 h later.

Flow cytometry analysis

Single-cell suspensions from adult mouse testes were generated as previously described (Hermann et al. 2018), with minor modifications. Briefly, testicular parenchyma from each adult mouse was digested with 1 mg/mL Collagenase Type IV (Sigma) for 15 min at 37°C, washed with DPBS to remove interstitial cells, digested with 0.25% trypsin/EDTA (ThermoFisher Scientific) for 3 min at 37°C, digestion stopped by addition of Hank’s Buffered Salt Solution (HBSS) containing 1% FBS, and pipetted until the cells were dissociated. Dissociated cells were fixed with 4% PFA for 10 min at RT. After washing in DPBS, fixed cells were permeabilized by incubation in DPBS containing 0.3% Triton X-100 for 10 min RT and then washed in DPBS. Permeabilized cells were subsequently incubated for 30 min at RT in DPBS supplemented with 1% FBS (DPBS/FBS) containing primary antibodies (or isotype controls). Primary antibodies were as follows: mouse anti-PLZF (4 μg/mL, clone 2A9, Active Motif), rat anti-GFP (1 μg/mL, clone GF090R, Nakarai Tesque), rabbit anti-RARG (4.5 μg/mL, clone D3A4; #8965, Cell Signaling Technology), and goat anti-KIT (0.8 μg/mL, AF1356, R&D Systems). Isotype control antibodies were: mouse IgG (4 µg/mL, BD), rat IgG (1 μg/mL, Invitrogen), rabbit IgG (4.5 μg/mL, BD), and goat IgG (0.8 μg/mL, Invitrogen). After washing twice in DPBS/FBS, cells were incubated in DPBS/BSA containing secondary antibodies and conjugated antibodies for 30 min at RT. Fluorophore-conjugated secondary antibodies were as follows: Alexa Fluor 405 Donkey anti-mouse IgG (8 μg/mL, ab175658, Abcam), Alexa Fluor 488 Donkey anti-rat IgG (4 μg/mL, Invitrogen), Alexa Fluor 568 Donkey anti-rabbit IgG (4 μg/mL, Invitrogen), and Alexa Fluor 647 Donkey anti-goat IgG (8 μg/mL, Invitrogen). After washing in DPBS/FBS, fluorescent signals were detected with an LSRII cytometer (BD Biosciences) in the UTSA Cell Analysis Core and data were analyzed using FlowJo v10 software (BD Biosciences).

Immunostaining of cryosections

Testes were fixed with 4% paraformaldehyde (PFA) for 3 h at 4°C, washed 3× in DPBS, and then bathed sequentially in 10% sucrose, 20% sucrose, and 30% sucrose before freezing in OCT compound and used to generate 8 µm coronal cryosections. The sections were mounted on charged slides, washed with DPBS, blocked in DPBS containing 0.3% Triton X-100, 10% FBS and 2% BSA for 2 h at 4°C and then incubated overnight at 4°C with primary antibodies (or isotype controls) diluted in DPBS supplemented with 1% BSA (DPBS/BSA). Primary antibodies were as follows: goat anti-PLZF (0.8 μg/mL, AF2944, R&D Systems), rat anti-GFP (1 μg/mL, clone GF090R, Nakarai Tesque) and rabbit anti-DDX4 (1 μg/mL, ab13840, Abcam). Isotype control antibodies were: goat IgG (0.8 μg/mL, Invitrogen), rat IgG (1 μg/mL, Invitrogen) and rabbit IgG (1 μg/mL, BD). After washing three times in DPBS for 15 min each, samples were incubated in DPBS/BSA containing secondary antibodies for 1 h at RT. Secondary antibodies were as follows: Alexa Fluor 488 Donkey anti-rat IgG (2 μg/mL, Invitrogen), Alexa Fluor 568 Donkey anti-goat IgG (2 μg/mL, Invitrogen), and Alexa Fluor 647 Donkey anti-rabbit IgG (8 μg/mL, Invitrogen). After washing three times in DPBS/BSA for 15 min each, nuclei were stained in DPBS/BSA containing 1 µg/mL DAPI for 10 min and samples were mounted in 50% Glycerol. Positive immunoreactivity was validated by both omitting the primary antibodies or replacing the primary antibodies with normal, non-immune, isotype control antibodies as noted above. Fluorescent signals on stained sections were detected at 20× magnification using an AxioImager M1 (Zeiss) and an AxioCam MRm (Zeiss).

Whole mount immunofluorescence

Seminiferous tubules were mechanically teased apart and rinsed in DPBS, fixed with 4% PFA for 3 h at 4°C, washed 3X in DPBS, blocked in DPBS containing 0.3% Triton X-100, 10% FBS and 2% BSA for 2h at 4°C and then incubated overnight at 4°C with primary antibodies or isotype negative control antibodies diluted in DPBS supplemented with DPBS/BSA. Primary antibodies were as follows: either goat anti-c-KIT (0.8 μg/mL, AF1356, R&D Systems) or goat anti-GFRA1 (0.8 μg/mL, AF560, R&D Systems), plus rat anti-GFP (1 μg/mL, clone GF090R, Nakarai Tesque) and either rabbit anti-RARG (4.536 μg/mL, clone D3A4; #8965, Cell Signaling Technology) or rabbit anti-RXRA (0.4 μg/mL, clone D6H10; # 3085, Cell Signaling Technology). Isotype control antibodies were as follows: goat IgG (0.8 μg/mL, Invitrogen), rat IgG (1 μg/mL, Invitrogen) and rabbit IgG (1 μg/mL, BD Biosciences). After washing three times in DPBS for 15 min each, samples were incubated in DPBS/BSA containing secondary antibodies and Lectin PNA from Arachishypogaea (peanut) conjugated to Alexa Fluor 647 (PNA, ThermoFisher) for 1 h at RT. Secondary antibodies were as follows: Alexa Fluor 405 Donkey anti-goat IgG (8 μg/mL, Ab175664, Abcam), Alexa Fluor 488 Donkey anti-rat IgG (2 μg/mL, Invitrogen) and Alexa Fluor 568 Donkey anti-rabbit IgG (4 μg/mL, Invitrogen). After washing three times in DPBS/BSA for 15 min each, samples were mounted in 50% Glycerol. Positive immunoreactivity was validated by both omitting the primary antibodies or replacing the primary antibodies with normal, non-immune, isotype control antibodies as noted above. Fluorescent signals in stained tubules were detected at 63× magnification using an AxioImager M1 (Zeiss) and an AxioCam MRm (Zeiss).

Statistical analyses

Each experiment was repeated at least three times with testes from independent adult animals. All data are expressed as the mean ± s.e.m. Statistical analysis of the data was performed by analysis using the Student’s t-test. P values < 0.05 were considered to be statistically significant.

Results

Dppa3 subdivides late progenitors into two subpopulations

In order to study the molecular mechanisms underlying preparation for differentiation among progenitor spermatogonia, we sought a specific marker that labels spermatogonia at the late progenitor stage. At the mRNA level, a number of genes are recognized as markers that discriminate progenitor spermatogonia from SSCs, including Nanos3, Neurog3, Pou5f1 and Rarg (Suzuki et al. 2009, Nakagawa et al. 2010, Ikami et al. 2015, La et al. 2018b), several of which are also specifically expressed much earlier in germline development at the primordial germ cell (PGC) stage (Kehler et al. 2004, Suzuki et al. 2008). Consequently, we examined expression of other PGC markers, such as Dppa3 (Saitou et al. 2002, Sato et al. 2002), Tfap2c (Weber et al. 2010), and Utf1 (Kasowitz et al. 2017), in single-cell transcriptomes from adult Id4-EGFPBright spermatogonia, which contain a mixture of SSCs, progenitor spermatogonia and differentiating spermatogonia (Hermann et al. 2018). Our previous analyses defined subsets of SSCs (quiescent and activated) and progenitors (early and late) in this dataset and established a sequential developmental relationship between these cells and differentiating spermatogonia (Suzuki et al. 2021) (Fig. 1A). We found that both Dppa3 and Nanos3 were uniquely activated in late progenitors, while expression of other candidates, including Pou5f1, Tfap2c, and Utf1 encompassed both early and late progenitors (Fig. 1A). Immunolabeling studies have previously shown that NANOS3 protein is broadly detected in most undifferentiated spermatogonia (Suzuki et al. 2009) and, thus, would not be useful for specifically investigating late progenitor spermatogonia. To visualize Dppa3 (aka: Stella), we utilized available transcriptional reporters (Payer et al. 2006, Ohinata et al. 2008), since we have not been able to find a suitable DPPA3 antibody. We found that epifluorescence from both transgenes was too low for reliable detection by microscopy or cytometry and, thus, to investigate the expression patterns of Dppa3 reporters in adult mouse testis, we performed immunostaining for ECFP or EGFP with an anti-GFP antibody and co-labeled with DDX4 to define germ cells and PLZF to define spermatogonia. ECFP or EGFP positive cells were always localized to the seminiferous tubule basement membrane, co-labeled with DDX4 and exhibited partial overlap PLZF, indicating preferential expression by undifferentiated spermatogonia (Fig. 1B and Supplementary Fig. 1, see section on supplementary materials given at the end of this article).

Figure 1
Figure 1

Dppa3 marks germ cells on the basement membrane of seminiferous tubules. (A) Heatmap of primordial germ cells marker gene (Dppa3, Nanos3, Pou5f1, Rarg, Tfap2c, and Utf1) expression collapsed from the annotation (Suzuki et al. 2021). Levels of mRNAs are according to the indicated Z-score scale. (B) Representative immunofluorescence of Dppa3-ECFP (Green), , DDX4 (white), PLZF (Red) and DAPI (Blue) in cryosections from adult Dppa3-Ecfp mice. Asterisk: PLZF+/ECFP−, arrowhead: PLZF+/ECFP+, circle: PLZF−/ECFP+. Bar = 40 µm.

Citation: Reproduction 161, 6; 10.1530/REP-21-0031

To further classify Dppa3-reporter expressing cells, we examined reporter overlap with populations of spermatogonia in the adult mouse testis highly enriched for SSCs (①: PLZFlow/KIT−), progenitor spermatogonia (②: PLZFhigh/KIT−), early differentiating spermatogonia (③ and ④: PLZF+/KIT+) and late differentiating spermatogonia (⑤: PLZF−/KIT+) and subdivided these populations based on RARG labeling (Suzuki et al. 2021) (Fig. 2A and B). In all populations, we found that the intensity of Dppa3-ECFP labeling was higher than that of Dppa3-EGFP labeling (compare left and right panels in Supplementary Fig. 2A, B, C, D and E). A small proportion (13.1% ECFP and 9.27% EGFP) of cells in the SSC-enriched population ① exhibited ECFP or EGFP labeling and 95.6% (ECFP) or 89.9% (EGFP) of those were RARGhigh and in a transition state (Fig. 2C and D and Supplementary Fig. 2A). A larger proportion (49.4% ECFP and 42.1% EGFP) of the progenitor-enriched population ② were ECFP+ or EGFP+, of which 89.9% (ECFP) or 84.1% (EGFP) were RARGhigh late progenitors (Fig. 2C and D and Supplementary Fig. 2B). Early differentiating spermatogonia in populations ③, ④ and ⑤ were strongly ECFP+ or EGFP+ (Fig. 2C and D and Supplementary Fig. 2C, D and E). These results indicate that the Dppa3-reporters are expressed by both progenitors and differentiating spermatogonia.

Figure 2
Figure 2

Dppa3-ECFP positive cells are highly enriched in late progenitor spermatogonia. (A) Flow cytometry analysis of isolated seminiferous tubule cells from adult Dppa3-Ecfp mouse testes sequentially gated for undifferentiated spermatogonia (①; PLZFlow/KIT−, C: ②; PLZFhigh/KIT−) and differentiating spermatogonia (③; PLZFhigh/KIT+, ④; PLZFlow/KIT+, ⑤; PLZF−/KIT+). Results are also representative of labeling cells from Dppa3-Egfp mouse testes. (B) Diagram indicating the relationship between populations of spermatogonia in the adult testis (beginning with quiescent SSC interconversion with activated SSCs, a potentially reversible transition from activated SSCs to early progenitor spermatogonia, and RA-dependent late progenitor production of KIT positive differentiating spermatogonia. (C) Quantification of flow cytometry from the left-hand panels of Supplementary Fig. 2A, B, C, D and E. Data are mean ± s.e.m. (n = 3 adult Dppa3-Ecfp mice). (D) Quantification of flow cytometry from the right-hand panels of Supplementary Fig. 2A, B, C, D and E. Data are mean ± s.e.m. (n = 3 adult Dppa3-Egfp mice). Negative control flow cytometry gating using WT testes (for both C and D) are in Supplementary Fig. 2F, G, H, I and J, and labeling with isotype negative control antibodies is found in Supplementary Fig. 2K and L.

Citation: Reproduction 161, 6; 10.1530/REP-21-0031

Since the transition between the undifferentiated and differentiating states occurs precisely at stages VII–VIII of the cycle of the seminiferous epithelium, we characterized expression driven by the Dppa3 promoter in intact seminiferous tubules in which stage can be recognized (Suzuki et al. 2021). First, we confirmed that Dppa3-ECFP reporter expression was absent from GFRA1+ SSCs and early progenitors throughout the cycle of the seminiferous epithelium (Supplementary Fig. 3). Secondly, we co-labeled for ECFP/EGFP, RARG and KIT to distinguish early progenitors (RARGlow/−/KIT−), late progenitors (RARGhigh/KIT−) and differentiating spermatogonia (KIT+) and observed similar results with both the ECFP and EGFP transgene (Fig. 3 and Supplementary Fig. 4). Among RARG+ late progenitors in early stages of the cycle of the seminiferous epithelium, a portion were Dppa3-ECFP+, and nearly all were KIT−, with no significant difference between the Dppa3-ECFP+ and Dppa3-ECFP- subsets (Fig. 3A and D). Similarly, in stages VI–IX where both ECFP+/RARG+ and ECFP-/RARG+ late progenitors were evident, 76.1% of ECFP-/RARG+ were KIT−, but significantly fewer (69.7%) of the ECFP+/RARG+ were KIT− (Fig. 3B and D), indicating more of the ECFP+ late progenitors had begun differentiating. Strikingly, only 5.6% of ECFP+/RARG+ late progenitors remained KIT− in stages X–XII (nearly all had become KIT+ differentiating spermatogonia), while 69.1% of ECFP-/RARG+ late progenitors remained KIT−, in similar proportion to preceding stages VI–IX (Fig. 3C and D). These results indicate that the Dppa3 reporters distinguish two subsets of RARGhigh late progenitors and suggest the intriguing possibility that Dppa3+/RARGhigh late progenitors are poised to differentiate, while Dppa3-/RARGhigh late progenitors have reduced competence to differentiate in response to RA (Fig. 3E).

Figure 3
Figure 3

Dppa3 distinguishes novel subsets of RARG+ late progenitors in the adult mouse testis. (A) Whole-mount immunofluorescence (WM-IIF) of Dppa3-ECFP (Green), RARG (Red) and KIT (White) in Stages I–V of seminiferous tubules from adult Dppa3-Ecfp mice. Bar = 20 µm. Arrowhead: ECFP− late progenitors (RARGhigh/ECFP−), asterisk: ECFP+ late progenitors (RARGhigh/ECFP+). (B) WM-IIF of Dppa3-ECFP (green), RARG (red) and KIT (white) in stages VI–IX of seminiferous tubules from adult Dppa3-Ecfp mice. Bar = 20 µm. Arrowhead: ECFP− late progenitors (RARGhigh/ECFP−), asterisk: ECFP+ late progenitors (RARGhigh/ECFP+). (C) WM-IIF of Dppa3-ECFP (green), RARG (red) and KIT (white) in stages X–XII of seminiferous tubules from adult Dppa3-Ecfp mice. Bar = 20 µm. Arrowhead: ECFP− late progenitors (RARGhigh/ECFP-), arrow: early differentiating spermatogonia (RARGhigh/ECFP+/KIT+). (D) Quantification of the proportion of and in stages I−V from A, stages VI−IX from B, and stages X–XII from C. (E) Diagram showing the expression of profiles of RARG (red nuclei), Dppa3-ECFP (green cytoplasm) and KIT (dark green plasma membrane) by stage of the cycle of the seminiferous epithelium in mice. The boxes illustrate our hypothesis: solid red box = late progenitors competent for spermatogonial differentiation (black arrows), dashed red box = late progenitors incompetent to differentiate in response to RA.

Citation: Reproduction 161, 6; 10.1530/REP-21-0031

Dppa3-negative late progenitors exhibit a delayed differentiation response to RA

To test the hypothesis that the Dppa3-ECFP+ late progenitors and Dppa3-ECFP− late progenitors differ in their capacity to respond to RA, we administered exogenous RA and measured the in vivo acute response at 2 and 4 h after treatment, which is temporally sufficient to induce KIT and Stra8 (Evans et al. 2014, Koli et al. 2017). Compared with untreated controls, flow cytometry measurement of spermatogonial differentiation showed no significant change in the percentage of KIT positivity among PLZF+/RARGhigh/ECFP− spermatogonia either 2 or 4 h after RA treatment, but a significantly greater proportion of PLZF+/RARGhigh/ECFP+ spermatogonia were KIT+ at both time-points (Fig. 4A and B). Wholemount immunostaining demonstrated that neither PLZF+/RARGhigh/ECFP− nor PLZF+/RARGhigh/ECFP+ late progenitor subsets in stages I–V became KIT+ 2 h after RA treatment (Fig. 4B and F), indicating that RARG alone was insufficient to permit rapid differentiation in response to RA. Further, similar to controls, nearly no ECFP+/RARG+ spermatogonia remained KIT− in stages X–XII 2 h after RA, while only 70% of ECFP–/RARG+ late progenitors remained KIT− (Fig. 4E and F). However, the proportion of ECFP+/RARG+ late progenitors at stage VI−IX that remained KIT− 2 h after RA was significantly lower than controls, while the proportion of mid-cycle ECFP−/RARG+ spermatogonia that remained KIT− was not significantly changed (Fig. 4D and F). Overall, these results support a novel concept that RARG is insufficient to permit rapid late progenitor differentiation in response to RA in the adult testis.

Figure 4
Figure 4

Differential response of RARG+ late progenitors to exogenous RA stimulation correlates with Dppa3 expression. Quantification of KIT levels by flow cytometry analysis in isolated seminiferous tubule cells (A) (PLZF+/RARGhigh/GFP−) and (B) (PLZF+/RARGhigh/GFP+) from adult Dppa3-Ecfp mouse testes gated for untreated and RA for 2 or 4 h (n = 3 adult Dppa3-Ecfp mice). (C) Whole-mount immunofluorescence (WM-IIF) of Dppa3-ECFP (green), RARG (red) and KIT (white) in stages I–V of seminiferous tubules from RA-treated (2 h) adult Dppa3-Ecfp mice. Bar = 20 µm. Arrowhead: ECFP− late progenitors (RARGhigh/ECFP−), asterisk: ECFP+ late progenitors (RARGhigh/ECFP+). (D) WM-IIF of Dppa3-ECFP (green), RARG (red) and KIT (white) in stages VI–IX of seminiferous tubules from RA-treated (2 h) adult Dppa3-Ecfp mice. Bar = 20 µm. Arrowhead: ECFP− late progenitors (RARGhigh/ECFP−), arrow: early differentiating spermatogonia (RARGhigh/ECFP+/KIT+). (E) WM-IIF of Dppa3-ECFP (green), RARG (red) and KIT (white) in stages X–XII of seminiferous tubules from RA-treated (2 h) adult Dppa3-Ecfp mice. Bar = 20 µm. Arrowhead: ECFP− late progenitors (RARGhigh/ECFP−), arrow: early differentiating spermatogonia (RARGhigh/ECFP+/KIT+). (F) Quantification of the proportion of ECFP− late progenitors and ECFP+ late progenitors that are KIT− in control mice and 2 h after exogenous RA in stages I–V (from C), stages VI–IX (from D), and stages X–XII (from E). (G) Diagram depicting the relative differentiation competence of late progenitor subsets (distinguished by presence of absence of Dppa3 reporter expression) in at the midpoint of the cycle of the seminiferous epithelium.

Citation: Reproduction 161, 6; 10.1530/REP-21-0031

Finally, to uncover the molecular mechanisms responsible for the differential response to RA among RARG-expressing late progenitors, we turned to our existing single-cell transcriptomes and subdivided late progenitors into three clusters (Supplementary Fig. 5A) (Hermann et al. 2018, Suzuki et al. 2021). Late progenitors in cluster 3 were localized to the end-point of vectors from RNA velocity analysis (Supplementary Fig. 5A), suggesting they were most advanced. Levels of Dppa3 were highest in clusters 2 and 3 (Supplementary Fig. 5B), suggesting these two clusters represent the functionally distinct late progenitor subpopulations identified above. We then compared levels of RA-related genes (Rara, Rarg, Rxra, Rxrb, Rxrg, Cyp26a1, Cyp26b1, Crabp1, and Crbp2) among these clusters. As expected, levels of RA-responsive Cyp26a1, Cyp26b1, Crabp1, and Crbp2 were all significantly elevated in early differentiating spermatogonia, with limited expression in progenitors (Supplementary Fig. 5B). Late progenitors in clusters 2 and 3 expressed Rara and Rarg mRNA, and Rxrb was higher in cluster 2, while Rxra was significantly higher in the more advanced cluster 3 (Supplementary Fig. 5B). In confirmation, we observed nuclear immunostaining with an RXRA+ antibody among Dppa3-ECFP+/KIT− spermatogonia in stages VI–IX of the seminiferous epithelium (Supplementary Fig. 5C). These results support a novel theory where RARG/RXRA heterodimers regulate spermatogonial differentiation competence in the mouse testis.

Discussion

RA is essential for spermatogenic progression and is known to be required for spermatogonial differentiation (Tegelenbosch & de Rooij 1993, Zhou et al. 2008, Busada et al. 2015, Endo et al. 2015, Ikami et al. 2015). Indeed, a pulse of endogenous RA in stages VII–VIII of the seminiferous epithelial cycle is the inducing factor that drives waves of spermatogenic maturation and continual spermatogenesis (Sada et al. 2012, Endo et al. 2015, Hogarth et al. 2015). Although the cellular source of this RA pulse in the adult mouse testis remains an open question, possible contributors include Leydig and Sertoli cells, which express Aldh1a1, and/or late pachytene spermatocytes at epithelial stages VII–X and in diplotene spermatocytes at stage XI, which express high levels of Aldh1a2 (Vernet et al. 2006). Regardless of the source of the ligand, spermatogonial response to RA is considered to largely be dictated by expression of RARG. Forced expression of RARG driven from the Gfra1 promoter was sufficient to drive differentiation of the majority of the Aundiff pool in response to endogenous RA (Ikami et al. 2015). Yet, subpopulations of RARG+ spermatogonia are able to escape the differentiation influence of RA (Endo et al. 2015, Ikami et al. 2015, Suzuki et al. 2021). Results of the current study challenge the long-accepted notion that nearly all progenitor spermatogonia (classically described in the literature as Aaligned spermatogonia) differentiate at stages VII–VIII in response to a pulse of RA to become type A1 differentiating spermatogonia. Specifically, we found that Dppa3-negative late progenitors, which are themselves defined by RARG immunolabeling, at stages VI–IX exhibit a significantly reduced response to exogenous RA compared with controls (while significantly more Dppa3-expressing late progenitors responded). In addition, late progenitors exhibiting RARG expression at stages I–V and X–XII of the cycle are likewise incapable of rapid differentiation in response to exogenous RA. This raises the exciting possibility that another, unknown, germ cell-intrinsic feature linked to Dppa3 expression serves as a rheostat to RA response in late progenitors at the time when these cells are ordinarily exposed to RA, in vivo.

Since our results indicate that RARG expression is insufficient to distinguish which spermatogonia will respond to RA, it seems likely that some other aspect of receptor complement may differ between responding and non-responding late progenitors. Knockout mouse studies, both constitutional and cell-type specific, have previously determined that testes produce a normal first-wave of spermatogenesis and have histologically normal spermatogenesis in many seminiferous tubules for roughly 1 year in the absence of RARG (Gely-Pernot et al. 2012). Another report indicated that RARA may also play a role in spermatogonial proliferation (Peer et al. 2018), so RARA may compensate for loss of RARG in spermatogonia. Surprisingly, our results appear to be at odds with data that suggest RARG is sufficient for spermatogonial differentiation in response to RA (Gely-Pernot et al. 2012, Ikami et al. 2015). Since we identified differential Rxra expression among subsets of Rarg+ late progenitors that are more or less advanced, our results suggest that RARG/RXRA heterodimer formation may determine which late progenitors can rapidly become KIT+ in response to RA. Immunostaining results confirmed presence of RXRA in both ECFP+ and ECFP− germ cells at the mid-point of the cycle, although we have been unable to test colocalization of RARG and RXRA due to lack of compatible antibodies. Still, RARG/RXRA heterodimers are already known to directly regulate expression of Sall4 (Gely-Pernot et al. 2015), which is involved in spermatogonial differentiation (Chan et al. 2017). Thus, hierarchical differentiation competence among progenitors may include acquisition of both RXR and RAR complements to enable rapid late progenitor response to RA.

Several fluorescent reporter lines have been reported for various types of male germ cells, including Neurog3-Egfp (Yoshida et al. 2004, Nakagawa et al. 2010), Oct4-GFP (La et al. 2018b), and Id4-Egfp (Chan et al. 2014) in order to allow for monitoring of cell state in vivo. Yet, none had previously been reported which was suitable for distinguishing early and late progenitor spermatogonia. Here, we found that Dppa3-EGFP and Dppa3-ECFP are suitable tools for selecting late progenitors and early differentiating spermatogonia using antibodies that recognize the fluorophores. Although prior studies reported that Dppa3-ECFP expression in PGCs occurred slightly later than Dppa3-EGFP expression (Payer et al. 2006, Ohinata et al. 2008), our characterization of transgene expression in the adult mouse was largely congruent. Our results do demonstrate, however, that the labeling intensity of the ECFP transgene, derived from a smaller plasmid than the EGFP transgene, was generally stronger. It is important to consider that the half-life of EGFP is ∼26  h (Corish & Tyler-Smith 1999) and that of ECFP is ∼29 h (Corre et al. 2014) and, thus, the temporal expression pattern may diverge from native DPPA3 protein. We have been unable to identify a suitable DPPA3 antibody to confirm transgene fidelity in the adult mouse testis. Despite our best efforts, we were also unable to reliably detect native epifluorescence with any microscopy or flow cytometry method, which unfortunately limits the ability to perform downstream studies, such as molecular and biochemical characterization or live cell experiments, in vitro.

In conclusion, until recently, phenotypic heterogeneity among undifferentiated spermatogonia and the complexity of the spatiotemporal arrangement across the cycle of the seminiferous epithelium has made characterizing the molecular mechanisms of spermatogonial differentiation a very difficult task (Nakagawa et al 2010, Shirakawa et al. 2013, Ikami et al. 2015, La et al. 2018a, Cheng et al. 2020). One challenge was a lack of reporter transgenes that distinguish SSCs from progenitors and allow direct examination of the transition from progenitors to differentiating spermatogonia. In this study, we report that Dppa3 reporter transgenes (colabeled with other markers) are able to distinguish between KIT-negative early and late progenitor spermatogonia, and using these tools, we discovered that RARG expression among progenitors is insufficient to predict which progenitors will differentiate in response to RA.

Supplementary materials

This is linked to the online version of the paper at https://doi.org/10.1530/REP-21-0031.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) through grants R01 HD090007 to BPH and P50 HD98593 to JRM. Data were generated in the Cell Analysis Core which is supported by a grant from the NIH (G12MD007591).

Author contribution statement

Experiments were designed by SS and BH, data were collected and analyzed by SS, results were interpreted and conclusion made by SS, JM and BH, and manuscript was written by SS and BH.

Acknowledgements

The authors thank the Washington State University Animal Production Core for help re-deriving thee transgenic mice used in this study from cryopreserved embryos.

References

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Supplementary Materials

 

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    Dppa3 marks germ cells on the basement membrane of seminiferous tubules. (A) Heatmap of primordial germ cells marker gene (Dppa3, Nanos3, Pou5f1, Rarg, Tfap2c, and Utf1) expression collapsed from the annotation (Suzuki et al. 2021). Levels of mRNAs are according to the indicated Z-score scale. (B) Representative immunofluorescence of Dppa3-ECFP (Green), , DDX4 (white), PLZF (Red) and DAPI (Blue) in cryosections from adult Dppa3-Ecfp mice. Asterisk: PLZF+/ECFP−, arrowhead: PLZF+/ECFP+, circle: PLZF−/ECFP+. Bar = 40 µm.

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    Dppa3-ECFP positive cells are highly enriched in late progenitor spermatogonia. (A) Flow cytometry analysis of isolated seminiferous tubule cells from adult Dppa3-Ecfp mouse testes sequentially gated for undifferentiated spermatogonia (①; PLZFlow/KIT−, C: ②; PLZFhigh/KIT−) and differentiating spermatogonia (③; PLZFhigh/KIT+, ④; PLZFlow/KIT+, ⑤; PLZF−/KIT+). Results are also representative of labeling cells from Dppa3-Egfp mouse testes. (B) Diagram indicating the relationship between populations of spermatogonia in the adult testis (beginning with quiescent SSC interconversion with activated SSCs, a potentially reversible transition from activated SSCs to early progenitor spermatogonia, and RA-dependent late progenitor production of KIT positive differentiating spermatogonia. (C) Quantification of flow cytometry from the left-hand panels of Supplementary Fig. 2A, B, C, D and E. Data are mean ± s.e.m. (n = 3 adult Dppa3-Ecfp mice). (D) Quantification of flow cytometry from the right-hand panels of Supplementary Fig. 2A, B, C, D and E. Data are mean ± s.e.m. (n = 3 adult Dppa3-Egfp mice). Negative control flow cytometry gating using WT testes (for both C and D) are in Supplementary Fig. 2F, G, H, I and J, and labeling with isotype negative control antibodies is found in Supplementary Fig. 2K and L.

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    Dppa3 distinguishes novel subsets of RARG+ late progenitors in the adult mouse testis. (A) Whole-mount immunofluorescence (WM-IIF) of Dppa3-ECFP (Green), RARG (Red) and KIT (White) in Stages I–V of seminiferous tubules from adult Dppa3-Ecfp mice. Bar = 20 µm. Arrowhead: ECFP− late progenitors (RARGhigh/ECFP−), asterisk: ECFP+ late progenitors (RARGhigh/ECFP+). (B) WM-IIF of Dppa3-ECFP (green), RARG (red) and KIT (white) in stages VI–IX of seminiferous tubules from adult Dppa3-Ecfp mice. Bar = 20 µm. Arrowhead: ECFP− late progenitors (RARGhigh/ECFP−), asterisk: ECFP+ late progenitors (RARGhigh/ECFP+). (C) WM-IIF of Dppa3-ECFP (green), RARG (red) and KIT (white) in stages X–XII of seminiferous tubules from adult Dppa3-Ecfp mice. Bar = 20 µm. Arrowhead: ECFP− late progenitors (RARGhigh/ECFP-), arrow: early differentiating spermatogonia (RARGhigh/ECFP+/KIT+). (D) Quantification of the proportion of and in stages I−V from A, stages VI−IX from B, and stages X–XII from C. (E) Diagram showing the expression of profiles of RARG (red nuclei), Dppa3-ECFP (green cytoplasm) and KIT (dark green plasma membrane) by stage of the cycle of the seminiferous epithelium in mice. The boxes illustrate our hypothesis: solid red box = late progenitors competent for spermatogonial differentiation (black arrows), dashed red box = late progenitors incompetent to differentiate in response to RA.

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    Differential response of RARG+ late progenitors to exogenous RA stimulation correlates with Dppa3 expression. Quantification of KIT levels by flow cytometry analysis in isolated seminiferous tubule cells (A) (PLZF+/RARGhigh/GFP−) and (B) (PLZF+/RARGhigh/GFP+) from adult Dppa3-Ecfp mouse testes gated for untreated and RA for 2 or 4 h (n = 3 adult Dppa3-Ecfp mice). (C) Whole-mount immunofluorescence (WM-IIF) of Dppa3-ECFP (green), RARG (red) and KIT (white) in stages I–V of seminiferous tubules from RA-treated (2 h) adult Dppa3-Ecfp mice. Bar = 20 µm. Arrowhead: ECFP− late progenitors (RARGhigh/ECFP−), asterisk: ECFP+ late progenitors (RARGhigh/ECFP+). (D) WM-IIF of Dppa3-ECFP (green), RARG (red) and KIT (white) in stages VI–IX of seminiferous tubules from RA-treated (2 h) adult Dppa3-Ecfp mice. Bar = 20 µm. Arrowhead: ECFP− late progenitors (RARGhigh/ECFP−), arrow: early differentiating spermatogonia (RARGhigh/ECFP+/KIT+). (E) WM-IIF of Dppa3-ECFP (green), RARG (red) and KIT (white) in stages X–XII of seminiferous tubules from RA-treated (2 h) adult Dppa3-Ecfp mice. Bar = 20 µm. Arrowhead: ECFP− late progenitors (RARGhigh/ECFP−), arrow: early differentiating spermatogonia (RARGhigh/ECFP+/KIT+). (F) Quantification of the proportion of ECFP− late progenitors and ECFP+ late progenitors that are KIT− in control mice and 2 h after exogenous RA in stages I–V (from C), stages VI–IX (from D), and stages X–XII (from E). (G) Diagram depicting the relative differentiation competence of late progenitor subsets (distinguished by presence of absence of Dppa3 reporter expression) in at the midpoint of the cycle of the seminiferous epithelium.

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