Three-dimensional reconstruction of efferent ducts in wild-type and Lgr4 knock-out mice

Abstract

We have recently shown that Lgr4 knock-out (LGR4KO) male mice are infertile due to a developmental defect of the reproductive tract. Spermatozoa do not reach the epididymis and accumulate at the rete testis and efferent ducts (ED). We have proposed that in LGR4KO, ED might fail to connect resulting in blind-ended tubes that preclude the normal transit of sperm cells. To explore this possibility, we reconstructed the three-dimensional (3D) structure of the organ from serial microphotographs. The resulting model allowed to individualize and follow each ED from the testis up to the epididymis, and to display the spatial distribution of their content. The transit of spermatozoa is indeed blocked in LGR4KO mice but, contrary to the expectation, the ducts connect normally to each other, forming a single tube that flows into the epididymis, as in the wild-type animals. In the KO however, transit of the sperm is abruptly blocked at the same level syncytial-like aggregates appear in the luminal space. The model also allowed calculating, for the first time, morphometric parameters of the mouse ED, such as total volume, surface, radius, and length. These data unambiguously showed that ED in the mutant mouse are dramatically shortened and less convoluted than in the wild-type animal, providing an explanation to the phenotype observed in LGR4KO. Combined with in situ immunodetection or RNA in situ hybridization, 3D reconstruction of serial histological sections will provide an efficient mean to study expression profiles in organs which do not lend themselves to whole-mount studies.