Macrophages are present in all tissues, and have heterogenous phenotypes with distinct niche-specific functions arising from their different ontogenies and microenvironmental stimuli (Bleriot et al., 2020). As in other tissues, macrophages are essential for the development and homeostatic regulation of the testis, and for directing and controlling inflammatory responses to infection and immunity. Heterogeneity of the testicular macrophage population was first established in studies on the adult rat testis (Wang et al., 1994, Winnall et al., 2011). Subsequently, heterogeneity in the mouse testis was described by identification of two F4/80+ subpopulations, namely peritubular macrophages, located adjacent to the seminiferous tubules on the interstitial side of the myoid peritubular cell layer, and the more easily visualised interstitial tissue macrophages, which are closely associated with the Leydig cells and blood vessels (DeFalco et al., 2015). They also differ in morphology, ontogeny and function and express heterogenous phenotypic markers. The interstitial macrophages are present from birth, predominantly express the canonical anti-inflammatory marker, CD206, and lack expression of major histocompatibility class II antigens (MHCII), while peritubular macrophages appear at two weeks postnatally, and generally lack CD206, but constitutively express high levels of MHCII (DeFalco et al., 2015; Mossadegh-Keller et al., 2016; Lokka et al., 2020; Wang et al., 2021). The interstitial macrophages are implicated in regulating structural remodeling, Leydig cell function and steroidogenesis, blood and lymph flow, and modulating potentially damaging inflammatory responses. The peritubular macrophages, due to their location surrounding the seminiferous epithelium and constitutive expression of MHCII, are implicated in control of the spermatogonial niche and interaction with circulating T lymphocytes to support immune tolerance to the spermatogenic cell antigens,

Studies on testicular macrophages have typically concentrated on the parenchyma encompassing the seminiferous tubules, whilst minor testicular compartments, specifically the rete testis, transition region, efferent ducts and the capsule/tunica albuginea, have received much less attention. The rete connects the seminiferous tubules to the efferent ducts via anastomosing channels lined by flattened or squamous to cuboidal-shaped epithelial cells, which are structurally different from the Sertoli cells (Dym, 1974, Malolina and Kulibin, 2019). The transition region, or tubuli recti, lies between the rete and seminiferous tubules with an epithelium consisting of cells, referred to as ‘modified’ or ‘transition region’ Sertoli cells (Figueiredo et al., 2021, Major et al., 2021).

Tung and colleagues were the first to report that F4/80+ macrophages were more abundant in the rete and subcapsule region adjacent to the rete of the adult mouse, and that many of these macrophages were MHCII+ (Tung et al., 1987). The efferent ducts were also surrounded by numerous F4/80+MHCII+ macrophages. Crucially, the rete and subcapsule are the primary sites of disease initiation in models of murine experimental auto-immune orchitis (EAO). In mice actively immunised with testicular homogenate, orchitis was initiated in seminiferous tubules under the testicular capsule, distal to the rete, while adoptive transfer of the activated T cells induced orchitis in the straight tubules, rete and efferent ducts (Tung et al., 1987). In studies using immunisation with syngeneic testicular germ cells, EAO was shown to start in the transition region with subsequent spread to surrounding seminiferous tubules (Itoh et al.,1991).

Activins are members of the transforming growth factor-β family of cytokines, which regulate development, tissue remodeling and immunity in multiple tissues, including the testis (Hedger and Winnall, 2012). In a previous study, it was demonstrated that activin A supports total F4/80+ macrophage numbers in the intertubular and peritubular regions surrounding the seminiferous tubules and in the subcapsule region of the adult mouse (Biniwale et al., 2022). Conversely, chronically elevated activin A reduced MHCII expression by CD206+ macrophages in the testis, whereas reduced activin A increased MHCII gene expression (Indumathy et al., 2020, Biniwale et al., 2022).

The aim of the present study was to examine the distribution and phenotypes of macrophages in the compartments particularly susceptible to orchitis, the rete, transition region, efferent ducts, subcapsule and tunica albuginea, and compare their phenotypes, and their responses to activin and testis infection, with the more characterised parenchymal macrophage subsets.