Exploring Metabolic Configurations Across the Seminiferous Epithelium

Abstract

The seminiferous epithelium is a spatially organized tissue in which developing germ cells occupy distinct metabolic microenvironments throughout spermatogenesis. While transcriptomic and proteomic studies have characterized changes in molecular composition during germ cell development, they do not directly assess enzyme functional capacity while preserving tissue architecture. Here, an in situ lactate dehydrogenase (LDH) activity assay was developed to characterize the spatial distribution of LDH activity in unfixed mouse testis sections. Fluorescent counterstaining with Hoechst, phalloidin, and SCP3 enabled segmentation of seminiferous tubule cross-sections into regions containing mitotic and meiotic cells and spermiogenic cells while maintaining native tissue morphology. Quantitative analysis of area-normalized formazan deposition demonstrated epithelial stage-dependent differences in LDH activity within these cellular compartments. Linear mixed-effects modeling revealed significantly increased LDH activity in the mitotic and meiotic compartment during epithelial stage groups VI–VIII and IX–XII relative to stages I–V, whereas the spermiogenic compartment exhibited reduced activity during stages VI–VIII compared with stages I–V and IX–XII. Examination of Ldhc wild-type, heterozygous, and knockout mice further demonstrated a marked reduction in overall LDH activity following loss of LDHC expression. These findings establish a workflow for spatially resolved dehydrogenase activity measurements in unfixed tissue and demonstrate that LDH functional capacity varies across epithelial stages, spermatogenic cell subpopulations, and Ldhc genotype. This approach complements transcriptomic and proteomic analyses by providing functional measurements of metabolic enzyme capacity while preserving the native spatial organization of complex tissues.