Three-dimensional imaging of vascular development in the mouse epididymis

Abstract

Long considered an accessory tubule of the male reproductive system, the epididymis is proving to be a key determinant of male fertility. In addition to its secretory role in ensuring functional maturation and survival of spermatozoa, the epididymis has a complex immune function. Indeed, it must manage both peripheral tolerance to sperm antigens foreign to the immune system and the protection of spermatozoa as well as the organ itself against pathogens ascending the epididymal tubule. Although our knowledge of the immunobiology of this organ is beginning to accumulate at the molecular and cellular levels, the organization of blood and lymphatic networks of this tissue, important players in the immune response, remains largely unknown. In the present report, we have taken advantage of a VEGFR3:YFP transgenic mouse model. Using high-resolution three-dimensional (3D) imaging and organ clearing coupled with multiplex immunodetections of lymphatic (LYVE1, PDPN, PROX1) and/or blood (PLVAP/Meca32) markers, we provide a simultaneous deep 3D view of the lymphatic and blood epididymal vasculature in the mature adult mouse as well as during postnatal development.

Spatiotemporal Expression Patterns of Vascular Endothelial Growth Factor and Receptors in Rat Testis and Epididymis throughout Postnatal Development

“…We observed VEGF expression only in some spermatogonia and Sertoli cells in the seminiferous tubules. On the other hand, the expression of VEGFR-1 and 2 was detected in elongated spermatids (speci cally stages [10][11][12][13][14][15][16][17][18][19] as well as spermatogonia. Leydig cells, vessel walls, and peritubular myoid cells in the intertubular area had positive immunostaining for VEGF, as well as VEGFR-1 and 2.…”

Tissue clearing and imaging approaches for in toto analysis of the reproductive system

“…Solvent-based tissue clearing methods that are compatible with immunofluorescent staining include BABB (benzyl alcohol/benzyl benzoate) [ 51 , 69–73 ], 3DISCO (3D imaging of solvent-cleared organs) [ 15 , 52 , 74 , 75 ], iDISCO+ (immunolabeling-enabled 3D imaging of solvent-cleared organs) [ 76–80 ], and ECi (ethyl cinnamate) [ 80 , 81 ]. These protocols can generally be broken down into five steps ( Figure 1 ).…”

“…There are three main imaging modalities that allow for in toto imaging of the reproductive system: laser scanning confocal microscopy and SCDM [ 52 , 63 , 73 , 75 ]; multiphoton microscopy [ 86 , 87 ]; and light sheet microscopy [ 78 ]. All of these methods perform optical scanning through the thickness (Z-axis) of the sample, and the resulting Z-stack of optical slices can be projected in the 3D space to render 3D images of the sample.…”

“…These methods allow for precise analysis of tissue shape and context during development. The power of these approaches is illustrated in many recent studies, including an in-depth time-course of mouse ovary morphogenesis [ 79 ] and the analysis of vascular development in the mouse epididymis [ 75 ].…”

“…For example, at the cellular level, object segmentation has been applied to study germ cells, by segmenting individual oocytes from 3D images of perinatal mouse and adult medaka ovaries, as well as germ cells in the fetal mouse testis [ 80 , 86 , 93 , 118 , 119 ]. Larger scale object segmentation has been applied to (1) groups of cells to investigate uterine gland architecture, testis cord morphogenesis, and epididymis vasculature [ 51 , 71 , 75 , 90 , 120 , 121 ]; (2) systemic networks to investigate vasculature, innervation, and lymphatics [ 69 , 75 , 77 , 103 ]; and (3) whole organs to investigate mouse ovary and testis morphogenesis and developing gonads in the context of the whole embryo [ 15 , 51 , 78 , 79 ].…”