In order to detect precious clinical FFPE samples at single-cell resolution and fully retain spatial localization information, we optimized a method suitable for low-input spatial proteomics based on laser capture microdissection (LCM) and successfully identified nearly 2000 proteins in about 100 spermatocytes cut from formalin-fixed paraffin-embedded (FFPE) tissue sections. We used the optimized spatial proteomics technology to research on prophase (pachytene, diplotene)/metaphase transition of spermatocytes in meiosis I in both mouse and human testicular tissues. It was found that the protein characteristics of mouse pachytene and diplotene spermatocytes were more similar and new key proteins MPI and UBE2N were discovered during this transition. Spatial proteomics of pachytene spermatocytes with normal spermatogenesis in patients with obstructive azoospermia (OA) and pachytene spermatocytes with prophase/metaphase transition disorder in patients with meiotic-arrest (MA) azoospermia revealed that the upregulated proteins in pachytene spermatocytes of MA all belong to the glutathione metabolic pathway. Inhibition of IDE and IDH2, two human-mouse homologous proteins upregulated in pachytene spermatocytes of OA, affected metaphase I formation.