Computer assisted image analysis to assess colonization of recipient seminiferous tubules by spermatogonial stem cells from transgenic donor mice

Abstract

Transplantation of spermatogonial stem cells from fertile, transgenic donor mice to the testes of infertile recipients provides a unique system to study the biology of spermatogonial stem cells. To facilitate the investigation of treatment effects on colonization efficiency an analysis system was needed to quantify colonization of recipient mouse seminiferous tubules by donor stem cell-derived spermatogenesis. In this study, a computer-assisted morphometry system was developed and validated to analyze large numbers of samples. Donor spermatogenesis in recipient testes is identified by blue staining of donor-derived spermatogenic cells expressing the E. coli lacZ structural gene. Images of seminiferous tubules from recipient testes collected three months after spermatogonial transplantation are captured, and stained seminiferous tubules containing donor-derived spermatogenesis are selected for measurement based on their color by color thresholding. Colonization is measured as number, area, and length of stained tubules. Interactive, operator-controlled color selection and sample preparation accounted for less than 10% variability for all collected parameters. Using this system, the relationship between number of transplanted cells and colonization efficiency was investigated. Transplantation of 10(4) cells per testis only rarely resulted in colonization, whereas after transplantation of 10(5) and 10(6) cells per testis the extent of donor-derived spermatogenesis was directly related to the number of transplanted donor cells. It appears that about 10% of transplanted spermatogonial stem cells result in colony formation in the recipient testis. The present study establishes a rapid, repeatable, semi-interactive morphometry system to investigate treatment effects on colonization efficiency after spermatogonial transplantation in the mouse.