Testicular macrophage regulation of Leydig cell development and function

Abstract

The unique microenvironment structure of the testis affects the function of Leydig Cells (LCs) both physically and physiologically. The testicular macrophages are located adjacent to the LCs in the interstitial space and the two cell types share a close physiological and functional relationship. Macrophages first appear in the testis in prenatal life, and increase in number during both prenatal and postnatal development when they support the development and function of the testis. The dynamics of macrophage population expansion correlates with generation of the adult Leydig cell population in postnatal life. From these observations I hypothesise that testicular macrophage numbers have a consistent ratio to the number of LCs, and therefore manipulating testicular macrophage numbers may modulate LC number and testosterone (T) production by LCs. As such, manipulation of testicular macrophages represents a viable and novel mechanism by which LC function can be improved. To test this, markers for distinct macrophage populations in the testes were identified, namely c-fms-GFP, Mac2 and CD163. The number of either Mac2+ or CD163+ cell populations was determined at key stages throughout postnatal life, and the ratios of these cells to LC number were calculated at each age. This showed a consistent ratio between macrophages and LCs in the testis throughout postnatal life. The stimulatory effect of macrophages during LC development was then determined, by increasing the number of macrophages through cytokine treatment with recombinant CSF1-Fc. This model was then analysed for changes in testicular macrophage number, LC function and LC number. CSF1-Fc increased macrophage numbers in the developing testis. Macrophage number was increased following CSF1-Fc treatment at stem LC, progenitor LC and immature LC stages of LC development, and in adulthood. Importantly, increasing macrophage number during development led to early maturation of the LC population, suggesting macrophages may function as a driver of LC maturation. In adulthood, testicular macrophage numbers were reduced via treatment with an anti-CSF1 antibody to further determine the role of testicular macrophages in LC number and function. Whilst CD163+ macrophage number was reduced, no change in LH or T was observed. In contrast CSF1-Fc treatment induced an increase in macrophage number and LC number, with an elevated T level. Results suggest that macrophage support of steroidogenesis in adulthood is dispensable or can be compensated through LH/T feedback, but CSF1-Fc can contribute to LC function, LC number and T production through action at the level of the brain and the testis. Finally, to determine the potential clinical significance of increasing testicular macrophage support, experiments were completed on animals with pathological conditions: LC androgen receptor knockout mice (LCARKO) (LCs fail to fully mature) and ageing mice (cumulative free radical damage). Delivery of CSF1-Fc was observed to improve LC maturation in LCARKO mice, but failed to modulate LCs in ageing animals, suggesting CSF1-Fc may have clinical application in specific pathologies related to LC dysfunction. In summary, these studies further define the testicular macrophage population as important supporting cell types for LC development, function and maturation, and identifies possible mechanisms by which enhancing macrophage action can support or improve poor LC development and function.