Although the importance of macrophages for the initial stages of testis cord formation in the fetal testis is widely accepted, there is a gap in understanding which other immune cells contribute to testis growth after the cords first form up to shortly after birth, when spermatogenesis is initiated. In addition, macrophage origins and roles in postnatal testis growth and ongoing adult spermatogenesis in homeostatic conditions remain key discussion points in the field (DeFalco et al. 2014; Mossadegh-Keller et al. 2017; Mossadegh-Keller and Sieweke, 2018; Lokka et al. 2020; Wang et al. 2021). The present study extends outcomes from previous investigations of fetal testis macrophages by examining their phenotype and distribution in the interval following sex determination through birth in the mouse testis. The documentation of CD3+ and Ly6G+ cell emergence is an important finding which highlights the dynamic nature of immune cell subpopulations in the fetal testis, revealing their close association with germ cells. The potential presence of B cells in the fetal mouse testis at E13.5 was examined previously using the B220 marker in sections, and no B cells were detected (DeFalco et al. 2014). We also checked for B cells using CD19 as a marker by IF, and no positive cells were identified at any age (data not shown). An important caveat to this study is the well-documented heterogeneity of each immune cell population, including macrophages, granulocytes and T cells, so that the detection of a single marker must be interpreted with caution. For example, T cell lineage markers have been reported on macrophages and other myeloid cells (Chávez-Galán et al. 2015), and Ly6G is found on some macrophages (Wang et al. 2021) in experiments using Flow cytometry. However, histological evaluation of the cells expressing each of these markers remains an effective approach to providing cell type identification.
The changing landscape of immune cell subpopulations in the fetal mouse testis
Quantitative analysis of sections examined using indirect immunofluorescence documented the increasing density of immune cells (CD45+), highlighting changes in the relative contributions of macrophages (F4/80+), T cells (CD3+) and neutrophils (Ly6G+) (Figure 1). While each subpopulation increases significantly in density between E13.5 and PND0, indicating their numerical elevation, the proportion of F4/80+:CD45+ cells decreased significantly, while Ly6G+ cells were rare to absent at E13.5. Thus, while our E13.5 data agree with the earlier report that 90% of CD45+ cells in the E12.5 testis are macrophages (DeFalco et al. 2014), it was surprising that, by PND0, about half of CD45+ cells in the testis were Ly6G+. The detection of both Ly6G+ and CD3+ cells in the newborn testis indicates that the immune milieu is altered dramatically by the time when spermatogenesis commences, shortly after birth.
Two phenotypes of fetal testicular macrophages
Based on a previous ontological investigation, F4/80Hi macrophages detectable before E14.5 are known to be yolk-sac-derived, while F4/80Int are fetal liver-derived and emerge by E16.5 (Lokka et al. 2020). In agreement with this, we detected only elongated F4/80Hi macrophages at E13.5, while at E15.5 and PND0, both elongated F4/80Hi and small rounded F4/80Dim macrophages were noted. The dim expression of CD45 on testicular macrophages at E13.5 is consistent with previous reports (DeFalco et al. 2014; Lokka et al. 2020), while higher levels were observed at E15.5 and PND0. Adult kidney and microglia also contain two CD45+ macrophage subpopulations, with “intermediate” and “high” signals, which are proposed to have different origins (Lee et al. 2018; Rangaraju et al. 2018). The low level of CD45 on testicular macrophages during fetal life indicates this is not an appropriate marker for first gating in flow cytometry or cell sorting techniques to study testicular macrophages at these developmental stages. In contrast CD45 on Ly6G+ cells which appeared to be neutrophils was present at high and readily detectable levels.
The previous report of fetal testicular macrophages concentrated near the gonad–mesonephros boundary at E10.5 and E13.5 demonstrated that a substantial proportion of macrophages originate and enter the testis from the mesonephros (DeFalco et al. 2014). We also observed macrophages at E13.5 and E15.5 on the mesonephros side of the testis, and note that they frequently appeared to be attached to and crossing through the capsule. Intriguingly, these later profiles appeared highly similar to leukocyte extravasation (Muller, 2013). The subsequent redistribution of macrophages from perimeter to the interior region of the testis contrasts with the localisation of neutrophils. Ly6G+ cells are rare at E13.5 but increased from E15.5 onwards; they appeared asymmetrically distributed across the fetal testis sections at E15.5. Also, the lack of macrophages detected inside the lumen of blood vessels at E13.5, E15.5 and PND0 contrasts with the frequent observation of Ly6G+ cells inside vessels, particularly at PND0, a distinction that merits further careful analysis.
While confirming the previous report of F4/80+ polymorphonuclear cells in the fetal and newborn testis (Lokka et al. 2020), we also identified some Ly6G+ granulocytic cells, most likely neutrophils, that were F4/80+. In agreement with previous investigations, no MHC class II+ macrophages were detected in the fetal testis, with only rare CD206+/MHCII+ macrophages (0-1 per section) at PND0 (Mossadegh-Keller et al., 2017; Lokka et al. 2020; Wang et al. 2021). The main population of E13.5 macrophages, consisting of large, elongated, CD45Dim/F4/80Hi/CD206+/MHCII− cells, became less prominent with the emergence of smaller, rounded CD45Int/F4/80Dim/CD206-/MHCII- cells at E15.5. This may reflect the imperative for a shift from a predominantly immunosuppressive macrophage population towards one that includes more immune-reactive macrophages at birth. These findings consolidate the understanding that there are at least two distinct populations of macrophages in newborn mouse testis, as well as several other immune cell types.
Cell-cell interactions of immune cells in the fetal testis
The full functional significance of the close localisation and apparent contact between different immune cell subsets is unknown, but may include roles in fetal testis morphogenesis, immune regulation, and germ cell removal and development, all of which are integral to the foundations of male fertility. The phagocytosis of germ and Sertoli cells by macrophages in E11.5 and E12.5 mouse testes was previously demonstrated (DeFalco et al. 2014), and we also observed germ cells outside the cords apparently engulfed by macrophages at the mesonephro-testis border at E13.5. Also consistent with the report of macrophages inside testis cords at E11.5 and E12.5 (DeFalco et al. 2014), and in accord with the detection of macrophages inside cords up to two weeks after birth (Lokka et al. 2020), we found macrophages inside cords, but at a declining incidence from E13.5 to birth. In contrast, Ly6G+ cells were observed inside cords, but only at PND0. We also observed immune cells in close contact with other immune cell types from E13.5 to PND0, with between 4 and 10 immune cell clusters in each section, appearing more frequently with advanced age development. While the functional significance of these contacts is unknown, we speculate macrophages may process germ cell antigens that are subsequently exposed to regulatory T cells for induction and maintenance of immune tolerance during adult life (Tung et al. 2017; Fijak et al., 2018; O'Donnell et al. 2021) or they may produce factors, such as activins or CSF1, that influence germ and/or Sertoli cell development, as identified for the adult mouse testis (DeFalco et al. 2015). Based on others’ observations from early fetal testis development (DeFalco et al. 2014; Mossadegh-Keller et al, 2017), and longstanding evidence of close structural and functional relationships between macrophages and Leydig cells in postnatal testes (Hutson, 1992; Gaytan et al. 1994; Fadel and Sarzotti, 2000) we knowledge that the important interactions between these immune cells and the developing Leydig cells and vasculature may be highly relevant to identifying points of vulnerability that affect postnatal testis growth and function.
Testicular CD3+ cells
We observed few CD3+ cells at E15.5, aligned with the report of DeFalco and colleagues (DeFalco et al. 2014). This was unsurprising because T cell development occurs late in fetal life, while the first thymic T cells are detectable in murine lymph nodes at E18–E20 (Fadel and Sarzotti, 2000). Several T cells in the PND0 testis appeared to contact macrophages. However, the interaction of macrophages and T cells is not likely to be through MHC class II (H2-Eb1) because almost all macrophages at PND0 appeared to lack this marker. Most of the small number of MHCII+ cells in PND0 testes were F4/80-, indicating either that another antigen presenting molecule is present on testicular macrophages, or that antigen presentation through MHCII is not the function of T cell-macrophage interactions at PND0. The finding of T cells in the fetal testis both adjacent to cords and in interstitial regions at PND0 suggests that similar to adult macrophages (DeFalco et al. 2015; Mossadegh-Keller et al. 2017), there are at least two functionally distinct subpopulations.
Ly6G+ cells in the fetal testis
Neutrophils constitute a key Ly6G+ cell population that plays critical roles in antibacterial immune responses, inducing inflammation, regulation of the hematopoietic niche, and angiogenesis, but their role in morphogenesis during development is much less well-defined (McGrath et al. 2015; Christoffersson and Phillipson et al. 2018; Casanova-Acebes et al. 2018; Carnevale et al., 2020) An early report indicated that neutrophils are not detected in the adult rodent testis under normal condition but can enter from the blood supply in pathophysiological conditions (O'Bryan et al. 2000). Neutrophils have reported in mouse and rat testes with infection or testicular torsion, including in ischaemia–reperfusion models (Lysiak et al. 2001, 2004; Celebi and Paul, 2008; Michel et al. 2016; Fijak et al. 2018) and in human testicular tumors (Akhtar et al. 1996), all relating to the adult testis.
In this report we have made the remarkable identification of Ly6G+ cells as the second-most frequent immune cell population in the newborn mouse testis. The only previous evaluation of neutrophils in the fetal mouse testis, conducted using flow cytometry, identified 9% of CD45+ cells as neutrophils (Gr-1+ cells) at E12.5 samples (DeFalco et al. 2014), while our analysis of tissue sections identified neutrophils as rare to absent at E13.5 (0-3 cells per section). The difference between these two observations may reflect the sensitivity of these distinct approaches. Murine fetal neutrophils are present in both the liver and bloodstream by E11.5 and expand rapidly in both compartments; neutrophil phenotypes in the E12.5 liver are consistent with early through late neutrophil maturation stage phenotypes. The initiation of haematopoiesis in the bone marrow at E18.5 (Sugiyama et al. 2011) fits with the present finding that the Ly6G+ population increases in the mouse testis to compromise more than half of the CD45+ population by birth. The remarkable drop in Ly6G+ cell prevalence in the testis shortly after birth, and their near absence from the adult mouse testis, provide a strong indication that these cells serve a highly specific role in a particular stage of testis development. The abundance of Ly6G+ cells in this period of late fetal testis development and at the onset of spermatogenesis coincides with the movement of germ cells from the cord centre to the perimeter, as they transform from gonocytes into spermatogonia. Their function may be related to a transient modification of the peri-cord environment or perhaps even the Sertoli cells, aligned with their known role in tissue remodelling (Peiseler and Kubes, 2019).
In contrast to the finding that macrophages are frequent near the mesonephros at E13.5 and E15.5, Ly6G+ cells are mostly absent from this location. Their predominant location in the section interior, close to and inside vessels from E15.5 onwards, may reflect a role in vascularisation of the fetal testis, essential for normal testis development, including formation of fetal Leydig cells, smooth muscle cells and pericytes (Lysiak et al., 2001; Coveney et al. 2008; Kumar and DeFalco, 2018). It will be important to consider their mechanism of recruitment to the developing testis, particularly as signalling molecules such as CXCL12, which recruits and maintains CXCR4+ neutrophils in lung (Carnevale et al., 2020), are also central to early germline development (Heckmann et al. 2018). A signalling link, such as the common potential for germ cells and neutrophils to respond to the Sertoli cell-derived products including CXCL12 and activin A, may explain why we observed neutrophils and germ cells co-located within the fetal testis.
Neutrophils have been increasingly identified as being heterogeneous in both structure and function, with a capacity for functional plasticity, and are predominantly reported in relationship to adult pathologies (Christoffersson and Phillipson, 2018; Carnevale et al. 2020). Changes in neutrophil maturation status are distinguished by their cell and nuclear shape (Hong, 2017). Here we make the intriguing observation of Ly6G+ cells with two distinct phenotypes: those with round, kidney-shaped and segmented nuclei were detected at E15.5, while at PND0 the dominant population was that with segmented nuclei. Based on their phenotypes, the Ly6G+ cells appeared to represent early- and mid-developmental neutrophil stages at E15.5 and the late differentiated stage at PND0. The application of additional markers will be required to track the developmental and functional trajectory of these cells within the nascent testis.
We observed further evidence of Ly6G+ cell heterogeneity in the early testis, indicating there are at least two populations: one clearly F4/80+ positive and one negative/dim for F4/80. While the transcript encoding F4/80, Adgre1, is detectable in murine neutrophils, the protein is not normally expressed on the cell surface (Sasmono et a. 2007). However, purified Ly6G+ cells express CSF-1R after overnight culture and can subsequently differentiate to form F4/80+ macrophages in response to CSF-1 (Sasmono et al., 2007). It will be exciting to learn how the complex milieu of the developing testis may attract and instruct granulocyte maturation and function.
In conclusion, this study documents for the first time the progressive emergence of neutrophils and T cells, the frequency and localisation of macrophages, T cells, neutrophils and their association with germ cells in the developing fetal testis. In particular, it establishes an understanding of the changing proportions of germline and immune cells that occur during fetal testis development in the mouse. By revealing the frequent, close cellular contacts between macrophages, T cells and neutrophils during testis development, we provide information that will be key to understanding how immune cells function during development of the fetal testis and influence maturation of the earliest male germ cells. Information gained from this study should be considered in the development of strategies to support in vitro germline growth, for example by adding immune cells into scaffolds or organoids.