Abstract
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Marker expression reveals heterogeneity of spermatogonia in the neonatal mouse testis
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Abstract
Prospermatogonia transition into type A spermatogonia, which provide the source for the spermatogonial stem cell (SSC) pool. A percentage of these type A spermatogonia then differentiate to enter meiosis as spermatocytes by ~P10. It is currently unclear when these distinct populations are initially formed in the neonatal testis, and when the expression of markers both characteristic of and required for the adult undifferentiated and differentiating states are established. In this study, we compared expression of known spermatogonial cell fate markers during normal development and in response to the differentiation signal provided by retinoic acid (RA). We found that some markers for the undifferentiated state (ZBTB16/PLZF and CDH1) were expressed in nearly all spermatogonia from P1 through P7. In contrast, differentiation markers (STRA8 and KIT) appeared in a subset of spermatogonia at P4, coincident with the onset of RA signaling. GFRA1, which was present in nearly all prospermatogonia at P1, was only retained in STRA8/KIT− spermatogonia. From P4 through P10, there was a great deal of heterogeneity in the male germ cell population in terms of marker expression, as markers characteristic of the undifferentiated (except GFRA1) and differentiating states were co-expressed through this interval. After P10, these fate markers diverged to mark distinct populations of undifferentiated and differentiating spermatogonia, and this pattern was maintained in the juvenile (P18) and adult (P>60) testis. Taken together, these results reveal that the spermatogonia population is heterogeneous during the first wave of spermatogenesis, and indicate that neonatal spermatogonia may not serve as an ideal substitute for studying the function of adult spermatogonia.
Introduction
In the mouse, prospermatogonia (also called gonocytes and, less commonly, prespermatogonia) proliferate briefly after sex determination in the fetal testis, and then enter a prolonged quiescent period from approximately embryonic day (E)14.5 until postnatal day (P)1–2 (Vergouwen et al. 1991, Western et al. 2008). At that point, neonatal prospermatogonia begin to move to the periphery of the testis cords and resume mitosis as spermatogonia, marking the initiation of spermatogenesis (Nagano et al. 2000, Drumond et al. 2011). Spermatogonia become flanked by Sertoli cells within the cord and myoid cells outside the cord, and respond to juxtacrine and paracrine signals from these somatic cells in this “niche” to either remain undifferentiated (Aundiff) or differentiate (Adiff) to ultimately enter meiosis by ~P10 (de Rooij 2001). This is termed the first wave of spermatogenesis, and it does not rely upon stem cell function, indicating that the first complement of sperm develop directly from this first group of spermatogonia (Yoshida et al. 2006). In subsequent waves, the SSC population provides a consistent source of progenitor spermatogonia that differentiate to ensure fertility for the remainder of the male reproductive lifespan.
Aundiff and Adiff spermatogonia are further characterized based on their topology; the most well-accepted current model predicts that stem cell potential predominates in individual As spermatogonia, and this potential progressively diminishes as they divide into clones with retained intercellular bridges (Apr−Aal) (Yang & Oatley 2014). Although spermatogonial development begins shortly after birth in the mouse, it is unclear how similar development during the first wave of spermatogenesis is to that during steady-state spermatogenesis in the adult. This is an important point, as numerous studies utilize isolated spermatogonia from neonatal mice (especially at P7, where they are a relatively high percentage of the testicular cell population) as a substitute for adult spermatogonia. Recent studies suggest that functional differences exist between neonatal and adult spermatogonia. Replication-dependent viruses more readily transduced spermatogonia from neonatal mice than their adult counterparts, suggesting that these spermatogonia divide more rapidly (Nagano et al. 2001, Nagano et al. 2002). In support of this concept, de Rooij and colleagues showed that spermatogonial development is accelerated in juvenile rats and hamsters (van Haaster & de Rooij 1993). A comparison of germ cell transplantation results from neonatal (P6) and adult mice revealed that the recolonization index for pup SSCs was half that of the adult, indicating delayed proliferation/expansion after transplantation and suggesting a difference in the ability of these spermatogonia to self-renew and differentiate (Ebata et al. 2007).
It is unclear whether prospermatogonia transition to SSCs, which then differentiate, or whether both populations arise directly from prospermatogonia. In addition, little is currently known regarding the cell fate decisions that take place in the neonatal testis that result in creation of a functional stem cell population. In the adult testis, Type A spermatogonia are classified functionally as stem cell and progenitor (both undifferentiated) and differentiating. These adult populations are distinguishable at the histological level by their morphology and the stage of the seminiferous epithelium in which they reside. In addition, a number of protein markers have been identified that are differentially expressed in spermatogonia that are undifferentiated (e.g. ZBTB16/PLZF, ID4, GFRA1, RET, NGN3) and differentiating (e.g. STRA8, KIT, SOHLH1, SOHLH2) (reviewed in (Yang & Oatley 2014)). Immunostaining results from several studies using adult tissues revealed that there is heterogeneity among the spermatogonial population (Grisanti et al., 2009; Nakagawa et al., 2007; Suzuki et al., 2009). However, it is currently unclear whether this has phenotypic consequence or whether heterogeneity is observed as the spermatogonial population is created in the neonatal testis.
We undertook this study to answer several important questions that have not been addressed in the neonatal testis. First, using established adult spermatogonial cell fate markers, are there homogenous or heterogeneous populations during the first wave of spermatogenesis? Second, when do markers of cell fate diverge during the first wave of spermatogenesis? Third, how do neonatal spermatogonia respond to a differentiation signal (RA) by modifying expression of cell fate markers? Here, we report that spermatogonia constitutively express certain markers of undifferentiated fate (CDH1 and ZBTB16) through ~P10, but express the receptors GFRA1 or KIT (or neither) during this interval. This suggests that there are 3 subpopulations of spermatogonia in the neonatal testis: GFRA1+/KIT− (undifferentiated, can respond to GDNF), GFRA1−/KIT+ (differentiating, can respond to KITL), or GFRA1−/KIT− (potentially uncommitted to a specific cell fate). This third subpopulation progressively decreases as the KIT+ population increases, suggesting that these spermatogonia are differentiating at later points to generate the asynchrony seen in the adult testis.
Materials and Methods
Animal Care
CD-1 mice were used for all analyses except where indicated otherwise. In particular, the Id4-GFP mice were backcrossed onto a C57Bl/6 background (Chan et al. 2014). Animal procedures were performed in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of East Carolina University (AUP #A178a). RA injections were done as previously reported, and mice were humanely euthanized by decapitation (P0–P10) or by CO2 asphyxiation followed by thoracotomy (P18 and P>60) (Busada et al. 2014b).
Tissue processing for histological analysis
Testes for histological analysis were fixed for 1 h to overnight in Bouin’s solution (Ricca Chemical) at 4°C, washed in multiple changes of 1× PBS, dehydrated through an ethanol series, and processed for paraffin embedding. Five µm sections were cut and stained with hematoxylin and eosin (H&E) using standard methods. Images were taken on an Axio Observer A1 microscope (Carl Zeiss Microscopy, LLC) equipped with a XL16 digital camera and Exponent version 1.3 software (Dage-MTI).
Immunostaining frozen sections
Testes were fixed for 2 h to overnight in 4% PFA, washed multiple times in 1× PBS, transferred to 30% sucrose, embedded in O.C.T, and stored at −80°C. Five µm thick sections were cut and placed on positively-charged slides and stored at −20°C prior to use. Immunolabeling was performed using standard methods. Briefly, blocking and antibody incubations were done at room temperature in 1× PBS containing 3% BSA + 0.1% Triton X-100, and stringency washes were done with 1× PBS +0.1% Triton X-100. Sections were blocked for 1 h prior to 1 h incubation in primary antibody (see Table 1). Secondary antibodies (1:500, Alexa Fluor-488 or -555, Invitrogen) plus phalloidin-635 (1:500, Invitrogen) were incubated for 1 h at room temperature. Primary antibody was omitted as a negative control. Coverslips were mounted with Vectastain containing DAPI (Vector Laboratories), and images obtained using a Fluoview FV1000 confocal laser-scanning microscope (Olympus America). Immunolabeling was performed in triplicate on testes from at least 2 different animals. ImageJ (U. S. National Institutes of Health) was used to assess images used for quantitation. Between 21–30 cords from at least 2 different animals were counted. Each marker was co-stained with DDX4 to label all germ cells. Cells where counted as positive for a marker when selected by the threshold tool using the default algorithm. Thresholds for each marker counted were as follows: STRA8 = 70–255, KIT =70–255, DDX4 = 100–255, ZBTB16 = 50–255, CDH1 = 100–255, GFRA1 = 50–255.
Table 1
Protein | Spermatogonia Expression | Reference | Antibody |
---|---|---|---|
STRA8 | Adiff (A1-) | (Zhou et al. 2008b) | Abcam rabbit polyclonal (49602), 1:3,000 |
KIT | Adiff (A1-) | (Mithraprabhu & Loveland 2009) | Santa Cruz goat polyclonal (sc-1494), 1:1,000 Cell Signaling rabbit polyclonal (3074S), 1:1,000 |
ZBTB16/PLZF | Aundiff (As, Apr, Aal) | (Buaas et al. 2004, Costoya et al. 2004) | Santa Cruz rabbit polyclonal (sc-22839), 1:500 |
DDX4/VASA/MVH | Aundiff and Adiff | Abcam rabbit polyclonal (ab-13840), 1:250 R&D Systems goat polyclonal (AF2030), 1:800 | |
CDH1/ECAD | Aundiff (As, Apr, Aal) | (Tokuda et al. 2007) | Cell Signaling rabbit polyclonal (3195P), 1:200 |
GFRA1 | Aundiff (As, Apr) | (Naughton et al. 2006, Grasso et al. 2012) | R&D Systems goat polyclonal (AF560), 1:200 |
Whole mount immunostaining of testis cords
Testes were first decapsulated, and then cords were mechanically separated. Cords were fixed for 1 h in 4% paraformaldehyde and washed multiple times in 1× PBS. Individual cords were transferred to slides and IIF performed using standard methods. Briefly, blocking and antibody incubations were done at room temperature in 1× PBS containing 3% BSA + 0.1% Triton X-100, and stringency washes were done with 1× PBS + 0.1% Triton X-100. Secondary antibodies (1:500, Alexa Fluor-488 or -555, Life Technologies) were incubated for 1 h at room temperature. Primary antibody was omitted as a negative control, and fluorescent images obtained as described above.
Results
Heterogeneity in neonatal germ cell morphology
Male germ cell development in neonatal mice (~P0–P7) has been described largely based on changes in cellular morphology. Some studies have suggested homogenous populations exist during this time (e.g. (Bellve et al. 1977)), while others reported type A spermatogonia with distinct morphology (e.g. (Kluin & de Rooij 1981)). We carefully examined the morphology and nuclear diameter of germ cells in CD-1 mice, which we have used for our recent studies. Germ cells are readily identified based on established morphological characteristics (Kluin & de Rooij 1981), and identification can be verified by immunostaining for ZBTB16/PLZF (Fig. S1A–C), which we previously reported was detectable in all germ cells in the neonatal testis (Busada et al. 2014b). The prospermatogonia population at P1 appeared homogenous, with germ cells sharing similar nuclear diameters and chromatin morphology. In contrast, at P4 the spermatogonia population appeared heterogeneous, with differing nuclear diameters and chromatin morphology (Fig. 1), which supports the potential for multiple spermatogonial types as early as P4.
Cell fate marker expression in neonatal prospermatogonia (P1)
There have been no published comprehensive analyses of neonatal spermatogonia development using established adult fate markers. Therefore, it is not clear when specific spermatogonial cell types appear in the neonatal testis, as it is difficult to distinguish undifferentiated and differentiating type A spermatogonia based solely on morphological criteria. Shortly after birth (P1), the testis cords contain peripherally-located dividing Sertoli cells and centrally-located quiescent prospermatogonia (de Rooij & Griswold 2012). To determine what percentage of prospermatogonia express certain spermatogonial fate markers, we co-immunolabeled with antibodies against CDH1, ZBTB16, and GFRA1 (undifferentiation markers) and KIT and STRA8 (differentiation markers) along with an antibody against the pan germ cell marker DDX4, which enabled us to determine the percentage of germ cells expressing various cell fate markers. Nearly 100% of P1 prospermatogonia had detectable levels of CDH1, ZBTB16, and GFRA1 but almost none had detectable expression of the differentiation markers KIT or STRA8 (Fig. 2, Fig. S2).
Cell fate marker expression in P4 – P10 spermatogonia
We next examined marker expression at P4, a time when the testis contains morphologically distinct populations of spermatogonia ((Kluin & de Rooij 1981), Fig. 1). We hypothesized that the presence of morphological differences indicates various degrees of spermatogonial differentiation. To examine this, we co-localized ZBTB16, CDH1, and STRA8 with GFRA1 and KIT, canonical markers of undifferentiated and differentiating spermatogonia, respectively (Oulad-Abdelghani et al. 1996, Buaas et al. 2004, Costoya et al. 2004, Buageaw et al. 2005, Prabhu et al. 2006, Tokuda et al. 2007, Zhou et al. 2008b, Grisanti et al. 2009, Mithraprabhu & Loveland 2009, Grasso et al. 2012). At P4, there were largely distinct subpopulations of spermatogonia that were either GFRA1+ (60%) or KIT+ (35%), and <10% of the population was GFRA1+/KIT+ (Fig. 3, Figs. S2–4). All spermatogonia stained positive for both ZBTB16 and CDH1, accepted markers of the undifferentiated fate. In addition, nearly all KIT+ spermatogonia were STRA8+ (Busada et al. 2014a), indicating that these cells had been exposed to RA. Considering only these markers, it is clear that at least two unique populations of spermatogonia exist in the testis at P4 (GFRA1+/KIT−/CDH1+/ZBTB16+/STRA8− and GFRA1−/KIT+/CDH1+/ZBTB16+/STRA8+). In addition, based on germ cell identification by DAPI staining, it is apparent that small populations of spermatogonia are KIT−/GFRA1−.
By P7, all differentiating (KIT+) spermatogonia remained CDH1+ and 80% were still ZBTB16+, and there was almost no overlap in GFRA1 and KIT expression (17% and 42% of DDX4+ cells, respectively). We observed this same relationship in spermatogonia in C57Bl/6 testes at P6 (Fig. S3). However, a correlation between staining intensity became apparent in a subset of spermatogonia (e.g. strong KIT+ with weak ZBTB16+). In addition, there was less observed STRA8 and KIT co-expression seen in P4 spermatogonia, which resulted in populations that were KIT+/STRA8+, KIT+/STRA8−, and KIT−/STRA8+ (Fig. 4). Analysis of these markers alone indicated multiple distinct populations of germ cells exist at P7 (GFRA1+/KIT−/CDH1+/ZBTB16+/STRA8−, GFRA1−/KIT+/CDH1+/ZBTB16+/STRA8+, GFRA1−/KIT+/CDH1+/ZBTB16+/STRA8−, GFRA1−/KIT−/CDH1+/ZBTB16+/STRA8+, and GFRA1−/KIT−/CDH1+/ZBTB16−/STRA8+ and GFRA1−/KIT+/CDH1+/ZBTB16−/STRA8−).
By P10, GFRA1 and KIT were still detectable in distinct populations (8% and 75% of DDX4+ cells, respectively), but there was a stark divergence in the expression of undifferentiated markers ZBTB16 and CDH1. All spermatogonia that were GFRA1+ were CDH1+/ZBTB16+, but most KIT+ cells were now CDH1− (Fig. 5). In addition, the staining intensity began to correlate with presumed cell fate; for example, KIT+ cells had weak staining for ZBTB16, and the opposite was also true (strong ZBTB16 correlated with weak or no staining for KIT). As at P7, there were three distinct populations of differentiating cells that were KIT+/STRA8+, KIT+/STRA8−, and KIT−/STRA8+. The latter population was centrally located in the testis cords, and likely are preleptotene spermatocytes, which have the highest levels of STRA8 (Zhou et al. 2008b). We co-immunostained testis cords with ZBTB16 and GFRA1 or KIT to determine whether GFRA1 or KIT staining correlated with spermatogonial chain length. As expected, GFRA1 was primarily detected in As and Apr spermatogonia, while KIT was detected in longer chains (Fig. S4).
It was recently shown that ID4 was enriched in As spermatogonial stem cells (SSCs), and that transgenic mice faithfully expressed GFP downstream of an Id4 promoter in SSCs (Oatley et al. 2011, Chan et al. 2014). We obtained testes from Id4-GFP mice at P6 and colocalized GFP with GFRA1 and KIT, since these largely marked unique populations of spermatogonia. We found that, in general, GFRA1 signal was strongest in spermatogonia with bright GFP, and that KIT signal was strongest in spermatogonia with dim or absent GFP signal (Fig. S5).
Cell fate marker expression in the P18 and adult (P>60) testis
By P18 in the mouse, the blood-testis barrier has formed, Sertoli cells no longer divide, lumina have appeared, and the seminiferous epithelia contains spermatogonia and spermatocytes at various stages of progression through meiosis, but haploid round spermatids are not yet present (Griswold 1998, Hess & Renato de Franca 2008). The examination of fate markers revealed that KIT staining was weak or absent in nearly all ZBTB16+, CDH1+, and GFRA1+ spermatogonia (Fig. 6). There were STRA8+/KIT+, STRA8+/KIT-, and STRA8−/KIT+ germ cells, but these populations were present in separate tubules (Fig. 6). Therefore, there was significant separation of the classic spermatogonial cell fate markers by this time, and this continued into adulthood (Fig. S6).
How does marker expression change in response to a differentiation signal?
Spermatogonia differentiate in response to RA, beginning in the neonatal testis at P3–4 and then throughout the remainder of the male reproductive lifespan (van Pelt & de Rooij 1991, Gaemers et al. 1998, Zhou et al. 2008b, Snyder et al. 2011, Busada et al. 2014b). We utilized an in vivo model of spermatogonial differentiation, in which we administered exogenous RA at P1, 2–3 days prior to their normal exposure (Zhou et al. 2008b, Snyder et al. 2011, Busada et al. 2014b). As shown previously (Busada et al. 2014b), exogenous RA induced the precocious expression of differentiation markers STRA8 and KIT by 24 h (Fig. 7A). However, mRNA levels also transiently increased for Cyp26a1 and Cyp26b1 (Busada et al. 2014b), which encode two RA-degrading enzymes, and by 48 h after treatment, STRA8 and KIT were absent (Fig. 7B–C), presumably due to loss of all testicular RA signaling. The Cyp26a1 and Cyp26b1 levels began to normalize by 72 h after RA exposure, suggesting that the testicular RA environment returned to normal, and germ cells eventually entered meiosis in similar numbers in RA-exposed animals, albeit 2 days later than vehicle-treated controls (Busada et al. 2014b). Results from that experiment suggested that expression of STRA8 and KIT did not result in an irreversible commitment to differentiation, but rather that spermatogonia must receive multiple consistent signals to differentiate. To assess this, we followed changes in expression of spermatogonial cell fate markers over the 144 h following RA exposure at P1.
Exogenous RA induced the precocious appearance of STRA8+/KIT+ spermatogonia by 24 h (Fig. 7A, (Busada et al. 2014b)) but, by 48 and 72 h post-RA injection, STRA8 and KIT both became undetectable in spermatogonia (Fig. 7B–C). There were no apparent changes in the expression of the undifferentiated markers ZBTB16 and CDH1 over this interval (Fig. 7E–L), suggesting that the spermatogonia population had returned to an undifferentiated state following the single exposure to RA. At 144 h after RA injection, STRA8 and KIT again became detectable in spermatogonia, indicating a resumption of RA signaling (Fig. 7D). Although specific testis cords were either KIT+ or KIT− in vehicle-treated controls, as in WT (Fig. S7), this was largely lost in testes of RA-treated mice (Fig. 7D, H, L). This resulting similarity in the expression of fate markers suggests homogeneity in differentiation status, provides an explanation for the widespread synchronization of the seminiferous epithelial cycle seen in adult mice treated with RA as neonates (Snyder et al. 2011, Hogarth et al. 2013).
Discussion
Here, we examined the expression of established markers of spermatogonial cell fate in spermatogonia during the first wave of spermatogenesis in the mouse. Importantly, we found that the receptors GFRA1 and KIT, which mark undifferentiated and differentiating spermatogonia, were predominantly localized to distinct cells from P4 onward. This is in contrast to ZBTB16 and CDH1, markers for undifferentiated spermatogonia in the adult, which were present in nearly all germ cells from P1 through P7. These were also co-localized with the differentiation markers STRA8 and KIT in a subset of spermatogonia at P4, concomitant with the onset of RA signaling. This co-localization of undifferentiated and differentiated markers persisted through P10, after which fate markers divergence was seen at P18 and in the adult testis. Taken together, our findings reveal that neonatal spermatogonia are a heterogeneous population as early as P4, and exhibit significant differences in marker expression in comparison with juvenile and adult spermatogonia. This data exposes molecular differences between neonatal and adult spermatogonia that may in part explain functional differences in terms of colonization ability and cell cycle length. In addition, data from this study reveal that caution should be exercised when making conclusions about spermatogonial behavior when utilizing neonatal spermatogonia as a substitute for adult spermatogonia.
The neonatal transition from prospermatogonia to spermatogonia is critical for establishment of spermatogenesis and lifelong male fertility. However, it was unclear when male germ cells begin to show signs of heterogeneity in terms of cell fate markers. We did not detect significant heterogeneity in cell morphology or the expression of cell fate markers in prospermatogonia at P1. Almost none of the prospermatogonia exhibited detectable levels of the differentiation markers STRA8 and KIT, while nearly all prospermatogonia had detectable GFRA1, ZBTB16, and CDH1. This is in agreement with some reports (e.g. ZBTB16 and CDH1 (Costoya et al. 2004, Tokuda et al. 2007)) but in contrast with others (e.g. ~50% RET+ or GFRA1+ at P0, although data not shown for GFRA1 (Naughton et al. 2006)). It is possible that, in the latter study, overnight fixation or the harsh tissue processing steps preceding paraffin embedding compromised some of the epitopes for GFRA1 and prevented their detection.
Our data supports a model (see Fig. 8A) by which spermatogonia constitutively express undifferentiated markers, and the differentiation program is initiated in response to RA in a subset of cells. Except for GFRA1, these undifferentiated markers persist until at least P7, after which there is significant separation. A similar pattern was seen for SALL4, which is detectable in all spermatogonia through P7, but then diverges to be expressed in undifferentiated spermatogonia in the adult (Gassei & Orwig 2013). After exposure to RA in this study, nearly all spermatogonia became STRA8+/KIT+. However, by 48 h these differentiation markers were lost, and only undifferentiated markers were constitutively expressed during this period. This suggests that the program of differentiation is initiated in undifferentiated spermatogonia, and that perhaps the most important aspect of commitment to a stem cell fate is avoiding exposure to a differentiation signal such as RA. Indeed, nearly all prospermatogonia exposed to RA (either by culture in excess RA or deletion of the RA-degrading enzyme Cyp26b1) prematurely expressed differentiation markers such as Stra8, Dmc1, Sycp3, Sohlh1, and Kit and attempt to enter meiosis (MacLean et al. 2007, Trautmann et al. 2008, Zhou et al. 2008a, Zhou et al. 2008b, Li et al. 2009, Busada et al. 2014b). However, we recently found that the signal provided by a one-time bolus of RA was not sufficient to maintain the differentiated state; by 48 h after RA injection, all of the neonatal spermatogonia that were previously STRA8+/KIT+ had lost expression of both markers and only had detectable undifferentiated markers for at least 3 more days (Busada et al. 2014b). Since spermatogonia express both undifferentiated and differentiating markers simultaneously, it is reasonable to predict that these cells have not yet committed to the differentiated fate (Fig. 8B). Our results support the concept that multiple, or continuous exposure(s) to RA is necessary to maintain STRA8 and KIT expression.
Beginning at P4, there are at least 2 distinct populations of spermatogonia that express either the KIT or GFRA1 receptor. The separate expression of these 2 receptors provides these distinct populations of spermatogonia with the ability to respond differentially to the ligands GDNF and KITL, which are provided by neighboring Sertoli and peritubular myoid cells (Hofmann 2008, Chen et al. 2014). GDNF binds to the GFRA1 and RET co-receptors to regulate spermatogonial stem cell maintenance both in vitro and in vivo (Meng et al. 2000, Viglietto et al. 2000, Meng et al. 2001, Kubota et al. 2004, He et al. 2007, Kubota et al. 2011). In contrast, KITL binding to KIT is required for spermatogonial differentiation and their subsequent entry into meiosis (Besmer et al. 1993, Kissel et al. 2000, Mithraprabhu & Loveland 2009). It is potentially the gain of these cell surface receptors that informs a spermatogonium to remain undifferentiated or to differentiate to eventually enter meiosis. While binding of either ligand to their cognate receptor(s) activates the PI3K/AKT signaling pathway, the distinct outcomes indicate the involvement of different components of this complex pathway.
Taken together, our results indicate that prospermatogonia transition to an initial heterogeneous population of undifferentiated and differentiating spermatogonia by ~P4. This supports the model whereby prospermatogonia directly give rise to the foundational pool of SSCs (which function to ensure lifelong fertility) as well as the undifferentiated progenitors and differentiating spermatogonia that become spermatozoa during the first wave of spermatogenesis.
Supplementary Material
1
Figure S1. ZBTB16 identifies germ cells in the neonatal testis:IHC was performed on Bouin’s-fixed testis sections from mice at P4 (A–B) and P7 (C). Primary antibody was omitted in A, and the brown signal in B–C represents ZBTB16. Sections were counterstained with hematoxylin (blue). Scale bar = 60 µm.
2
Figure S2. Calculating the percentage of KIT+ and GFRA1+ cells and GFRA1/ZBTB16 in the developing testis:Co-immunolabeling was performed on testis cryosections from mice aged P1–P10 to detect KIT (green) and the pan germ cell marker DDX4 (red) or GFRA1 (green) and DDX4 (red) or ZBTB16 (red). F-actin was labeled with phalloidin to outline cord structures (blue). Ages are indicated at the bottom of each column, and the fluorescence channels are separated in the top two rows but combined in the bottom. Scale bar = 60 µm.
3
Figure S3. Detection of KIT and STRA8 in the P6 C57Bl/6 testis:Co-immunolabeling was done to localize KIT (green) and STRA8 (red) in P6 spermatogonia, and nuclei were labeled with DAPI (in blue). Scale bar = 60 µm.
4
Figure S4. Whole mount co-immunostaining ZBTB16 with either KIT or GFRA1:Testis cords from P7 mice were co-immunostained to detect the following: (A–C) KIT (green) and ZBTB16 or (D–F) GFRA1 (green) and ZBTB16 (red). Scale bar = 100 µm.
5
Figure S5. Detection of GFRA1 and KIT in P6 Id4-eGFP testes:GFP epifluorescence was detected (green), and immunostaining was performed to visualize GFRA1 (red, A–B) or KIT (red, C–D). The green channel was omitted in A and C. F-actin was labeled with phalloidin (in blue). White arrows indicate KIT+ spermatogonia (C–D). Scale bar = 60 µm.
6
Figure S6. Fate marker detection in the adult testis:Immunostaining was performed to detect the following proteins in the P>60 testis: (A) DDX4 (green) and KIT (red), (B) GFRA1 (green) and KIT (red), and (C) KIT (green) and STRA8 (red). White arrows (in B) indicate GFRA1+ spermatogonia (C–D). Scale bar = 60 µm.
7
Figure S7. Fate marker detection in the vehicle-treated neonatal testis:Immunostaining was performed on testes at indicated intervals after vehicle administration to detect KIT (green) and CDH1 (red) in A–D and KIT (green) and ZBTB16 (red) in E–H. Actual age of mouse at time of sacrifice is listed on each image in parentheses. Scale bar = 60 µm.
Acknowledgements
We thank Joani Zari-Oswald (Brody School of Medicine at East Carolina University) for technical assistance and Dr. Brian Hermann (University of Texas at San Antonio) for testes from Id4-GFP mice (originally from Dr. Jon Oatley, Washington State University).
Funding
Supported by a grant from the NIH/NICHD (HD072552) to CBG.
Footnotes
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.
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Funding
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NICHD NIH HHS (2)
Grant ID: R15 HD072552
Grant ID: HD072552