Reconstruction of a seminiferous tubule-like structure in a 3 dimensional culture system of re-aggregated mouse neonatal testicular cells within a collagen matrix.
Male gonad development is initiated by the aggregation of pre-Sertoli cells (SCs), which surround germ cells to form cords. Several attempts to reconstruct testes from dissociated testicular cells have been made; however, only very limited morphogenesis beyond seminiferous cord formation has been achieved. Therefore, we aimed to reconstruct seminiferous tubules using a 3-dimensional (D) re-aggregate culture of testicular cells, which were dissociated from 6-dpp neonatal mice, inside a collagen matrix. We performed a short-term culture (for 3 days) and a long-term culture (up to 3 wks). The addition of KnockOut Serum Replacement (KSR) promoted (1) the enlargement of SC re-aggregates; (2) the attachment of peritubular myoid (PTM) cells around the SC re-aggregates; (3) the sorting of germ cells inside, and Leydig cells outside, seminiferous cord-like structures; (4) the alignment of SC polarity inside a seminiferous cord-like structure relative to the basement membrane; (5) the differentiation of SCs (the expression of the androgen receptor); (6) the formation of a blood-testis-barrier between the SCs; (7) SC elongation and lumen formation; and (8) the proliferation of SCs and spermatogonia, as well as the differentiation of spermatogonia into primary spermatocytes. Eventually, KSR promoted the formation of seminiferous tubule-like structures, which accompanied germ cell differentiation. However, these morphogenetic events did not occur in the absence of KSR. This in vitro system presents an excellent model with which to identify the possible factors that induce these events and to analyze the mechanisms that underlie cellular interactions during testicular morphogenesis and germ cell differentiation.
Department of Biological Science, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto 860-8555, Japan. Electronic address: jidong@kumamoto-u.ac.jp.
Jidong
J
Zhang
Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan. Electronic address: jhatakey@kumamoto-u.ac.jp.
Jun
J
Hatakeyama
Department of Biological Science, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto 860-8555, Japan. Electronic address: etoko@gpo.kumamoto-u.ac.jp.
Ko
K
Eto
Department of Biological Science, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto 860-8555, Japan. Electronic address: abeshin@gpo.kumamoto-u.ac.jp.
Shin-Ichi
S
Abe
D000818:Animals; D000831:Animals, Newborn; D002449:Cell Aggregation / Q000187:drug effects; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation; D002478:Cells, Cultured; D003094:Collagen / Q000494:pharmacology*; D006801:Humans; D007985:Leydig Cells / Q000166:cytology / Q000187:drug effects; D008297:Male; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D011944:Receptors, Androgen / Q000378:metabolism; D012671:Seminiferous Tubules / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D013090:Spermatocytes / Q000166:cytology / Q000187:drug effects; D019108:Tight Junctions / Q000378:metabolism
D011944:Receptors, Androgen; D003094:Collagen
3 Dimensional culture; Collagen; KnockOut Serum Replacement (KSR); Mouse; Re-aggregated testicular cells; Seminiferous tubule