Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses

Abstract

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. Differentiation of germ cells takes place in the cytoplasmic pockets of somatic Sertoli cells that host 4 to 5 generations of germ cells simultaneously and coordinate and synchronize their development. Therefore, the composition of germ cell types within a cross-section is constant, and these cell associations are also known as stages (I-XII) of the seminiferous epithelial cycle. Importantly, stages can also be identified from intact seminiferous tubules based on their differential light absorption/scatter characteristics revealed by transillumination, and the fact that the stages follow each other along the tubule in a numerical order. This article describes a transillumination-assisted microdissection method for the isolation of seminiferous tubule segments representing specific stages of mouse seminiferous epithelial cycle. The light absorption pattern of seminiferous tubules is first inspected under a dissection microscope, and then tubule segments representing specific stages are cut and used for downstream applications. Here we describe immunostaining protocols for stage-specific squash preparations and for intact tubule segments. This method allows a researcher to focus on biological events taking place at specific phases of spermatogenesis, thus providing a unique tool for developmental, toxicological, and cytological studies of spermatogenesis and underlying molecular mechanisms.

C11orf94/Frey is a key regulator for male fertility by controlling Izumo1 complex assembly

“…1F ). Because maturation of round spermatids involves several morphologically distinct stages that are associated with extensive rearrangement of acrosomal compartments, we aimed to delineate the exact stage of expression of C11orf94 by costaining with the acrosomal marker peanut agglutinin (PNA)–fluorescein isothiocyanate (FITC) as described in detail in ( 25 ). Following this approach, applying confocal imaging C11orf94 could only be detected in round spermatids belonging to stages V to VII/VIII with no visible expression in earlier or later stages ( Fig.…”

Identification of Rat Testicular Leydig Precursor Cells by Single-Cell-RNA-Sequence Analysis

“…To eliminate possible contamination from blood cells, the testicular artery was cannulated and perfused with DMEM/F12 culture medium containing 2.2 g/L Hepes, 0.1% BSA, 0.7 g/L sodium bicarbonate, pH 7.4). Interstitial tissue and seminiferous tubules of the testes were then separated with fine forceps under a transillumination dissection microscope ( Mäkelä et al, 2020 ). Interstitial tissue from the testes of each of three animals was combined, and then digested with 1 mg/ml collagenase-IV in DMEM/F12 medium at 34C for 30 min with slow shaking (90 cycles/min).…”

SMG6 localizes to the chromatoid body and shapes the male germ cell transcriptome to drive spermatogenesis

“…Mouse testes were decapsulated in Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham (D8437, Sigma). Segments of the seminiferous epithelial cycle representing stages VII–VIII of spermatogenesis were cut as previously described ( 39 , 40 ). The isolated pieces of tubules were incubated on glass slides in 30 μl of medium supplemented with 1 mM ethynyl uridine (EU) at 34°C for 10 h in a highly humidified atmosphere containing 5% CO 2 .…”

“…The isolated pieces of tubules were incubated on glass slides in 30 μl of medium supplemented with 1 mM ethynyl uridine (EU) at 34°C for 10 h in a highly humidified atmosphere containing 5% CO 2 . Spermatogenic cells were spread out of the cultured seminiferous tubules to monolayers using the squash technique ( 39 , 40 ), snap-frozen in liquid nitrogen, and fixed in 96% EtOH for 3 min. The nascent RNA was visualized using the Click-iT RNA Alexa Fluor 488 Imaging Kit (Molecular Probes, Invitrogen, C10329).…”