Germline stem cell lineage tracing by free-floating immunofluorescent assay of mouse seminiferous tubule.
Whole-mount immunohistochemistry (whole-mount IH) of the seminiferous tubule is widely used to investigate the self-renewal and differentiation of spermatogonial stem cells (SSCs). Examination of the length of spermatogonial cysts is critical for tracing SSCs lineage by using Whole-mount IH. However, it is difficult for antibody molecules to penetrate into the depth of seminiferous epithelium because its thickness and the tight peritubular myoid and basement membrane outside. Here, we developed a free-floating immunofluorescent procedure of mouse seminiferous tubules using regular incubation time and normal antibody concentration. Microscopic results showed that undifferentiated spermatogonia were positively labeled by promyelocytic leukemia zinc finger protein, E-cadherin, and glial cell line-derived neurotrophic factor family receptor alpha 1, respectively. Spermatogonial cysts in varied length were revealed clearly and spermatogonia subpopulations including A(single) (A(s)), A(paired) (A(pr)), and A(aligned) (A(al)) were distinguished in lower background images. This method provides us an alternate simple way to trace the lineage of individual SSCs and show their three-dimensional locations and distributions within their niches anatomically in next step.
Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Sciences and Veterinary Medicine, Jilin University, Changchun 130062, China.
Bo
B
Jin
Bin
B
Guo
Guanyu
G
Che
Yixue
Y
Sun
Yang
Y
Liu
Xueming
X
Zhang
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D015820:Cadherins / Q000378:metabolism; D002454:Cell Differentiation; D019070:Cell Lineage; D005455:Fluorescent Antibody Technique; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors / Q000378:metabolism; D008297:Male; D051379:Mice; D012670:Seminiferous Epithelium / Q000378:metabolism; D012671:Seminiferous Tubules / Q000166:cytology* / Q000378:metabolism; D013093:Spermatogonia / Q000378:metabolism; D013194:Staining and Labeling; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism
D000906:Antibodies; D015820:Cadherins; C494988:Gfra1 protein, mouse; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors