Advanced immunostaining approaches to study early male germ cell development

Abstract

Mammalian male germ cell development takes place in the testis under the influence of a variety of somatic cells and an incompletely defined paracrine and endocrine influences. Since it is not recapitulated well in vitro, researchers studying spermatogenesis often manipulate the germline by creating transgenic or knockout mice or by administering pharmaceutical agonists/antagonists or inhibitors. The effects of these types of manipulations on germline development can often be determined following microscopic imaging, both of stained and immunostained testis sections. Here, we describe approaches for microscopic analysis of the developing male germline, provide detailed protocols for a variety of immunostaining approaches, and discuss transgenic fluorescent reporter lines for studying the early stages of spermatogenesis.

Immunofluorescence Staining of Paraffin Sections Step by Step

“…The technique can be applied for other tissue types, both paraffin-embedded sections and cryosections. However, we believe, as also others ( Niedenberger and Geyer, 2018 ), that the tissue quality and cellular morphology are much well-preserved in paraffin-embedded tissues. In addition, it has been reported that non-specific background staining is less common for paraffin-embedded tissue sections than for frozen sections ( Larsson, 1988 ; Elias, 2003 ).…”

“…Immunofluorescence (IF) staining is a method of choice in studying the subcellular localization of proteins in fixed biological samples (Zaglia et al, 2016;Niedenberger and Geyer, 2018;Smith and Gabriel, 2018). It provides researchers with an easy tool to detect and compare the distribution of proteins in the cells and tissues of various model organisms.…”

“…In the case of direct immunostaining, the primary antibody against the antigen of interest is directly conjugated to a fluorophore that enables direct fluorescent detection using a fluorescent microscope. In the indirect immunostaining approach, a fluorophore-conjugated secondary antibody against the unconjugated primary antibody is applied ( Becheva et al, 2018 ; Niedenberger and Geyer, 2018 ). This method can be performed on cultured cells (immunocytochemistry, ICC) and tissues (immunohistochemistry, IHC) ( Maity et al, 2013 ).…”

Sertoli cell and spermatogonial development in pigs

“…To pinpoint the specific expression of UCHL1 in pro- and undifferentiated spermatogonia and the timepoint of spermatogonial differentiation in pigs, we next performed double immunostaining for UCHL1 and VASA or KIT on testis sections. VASA, also known as DDX4 or MVH, is a conserved pan-germ cell marker in testes [ 28 , 29 ]. Immunofluorescence staining results showed that UCHL1 shared an identical localization pattern with VASA in testes from d 7 to 90, i.e., UCHL1 + cells were also positive for VASA, and vice versa.…”

Acyl-CoA synthetase 6 enriches seminiferous tubules with the ω-3 fatty acid docosahexaenoic acid and is required for male fertility in the mouse

“…Indirect immunofluorescence was done as described previously (53). Briefly, testes were fixed overnight in 4% paraformaldehyde, washed in 1ϫ PBS, and then incubated overnight in 30% sucrose, all at 4°C.…”