Morphogenetic restructuring and formation of basement membranes by Sertoli cells and testis peritubular cells in co-culture: Inhibition of the morphogenetic cascade by cyclic AMP derivatives and by blocking direct cell contact

doi:10.1016/0012-1606(87)90112-6

Developmental Biology

Volume 120, Issue 1, March 1987, Pages 139-153

https://doi.org/10.1016/0012-1606(87)90112-6 Get rights and content

Abstract

Addition of dibutyryl cyclic AMP (dbcAMP), methylisobutylxanthine (MIX), or cytochalasin D to co-cultures of Sertoli cells and testicular peritubular myoid cells blocks a series of morphogenetic changes which otherwise occur during culture. When Sertoli cells are plated directly onto preexisting layers of peritubular cells maintained under basal conditions, structures form which display many of the characteristics of germ cell-depleted seminiferous tubules. The presence of dbcAMP, MIX, or cytochalasin D, added at varying times after plating Sertoli cells, results in the inhibition of each successive stage of in vitro remodeling: the inhibition of migration of Sertoli cells, the inhibition of initial ridge formation, the blockage of subsequent formation of mounds and nodules of compacted Sertoli cell aggregates, the prevention of the formation of basal lamina and associated layers of extracellular matrix between Sertoli cell aggregates and surrounding peritubular cells, and the inhibition of tubule formation. The presence of dbcAMP also inhibits the migration of peritubular cells, contractions by these cells, and compaction of Sertoli cell aggregates. When intimate cell apposition is prevented by plating the two cell types on either side of a membrane filter, the morphogenetic cascade is blocked, and no formation of a germ cell-depleted seminiferous tubule-like structure occurs. Other effects of dbcAMP on cell shape, cell movement, and cell association patterns during co-culture are described. Possible mechanisms by which dbcAMP, MIX, or cytochalasin D blocks restructuring are discussed. Since each elicits perturbations of the cytoskeleton, we offer the interpretation that cytoskeletal changes may be correlated with the prevention of closely apposing cell compact and the inhibition of basement membrane formation. Interactions observed between Sertoli cells and peritubular cells during co-culture are postulated to be analogous to those occurring in other types of mesenchymal cell-epithelial cell interactions during organogenesis and during tubulogenesis in the fetal testis. Speculatively, the blockage by dbcAMP of the morphogenetic cascade in the co-cultured system may be related to the inhibition by dbcAMP of testis cord formation in organ cultures of fetal gonads reported by others.

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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.
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^☆: This work was supported by a grant (MT 3292) from the Medical Research Council of Canada, to whom we express our deep gratitude.

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Full paperMorphogenetic restructuring and formation of basement membranes by Sertoli cells and testis peritubular cells in co-culture: Inhibition of the morphogenetic cascade by cyclic AMP derivatives and by blocking direct cell contact☆

Abstract

J. Ultrastruct. Res

Dev. Biol

Cell. Diff

Cell

Exp. Cell Res

Dev. Biol

Dev. Biol

Molecular Biology of the Cell

Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro

Contraction and organization of collagen gels by cells cultured from periodontal ligament, gingiva and bone suggest functional differences between cell types

J. Cell Sci

Basal lamina in epithelial mesenchymal morphogenetic interactions

Differentiation of peritubular myoid cells of the testis: Effects of intratesticular implantation of newborn mouse testes into normal and hypophysectomized adult

Biol. Reprod

Connectin: Cell surface protein that binds both laminin and actin

Epithelial-mesenchymal interactions in hormone-induced development

Regulation of microfilaments and microtubules by calcium and cyclic AMP

The blood-testis barrier in the rat and the physiological compartmentation of the seminiferous epithelium

Biol. Reprod

Role of interactions between peritubular cells and Sertoli cells in mammalian testicular functions

Fluorescence microscopy reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate

Science

The absence of specific interactions of Sertoli cell secreted proteins with antibodies directed against H-Y antigen

Cell

Extracellular matrix regulates Sertoli cell differentiation, testicular cord formation and germ cell development in vitro

J. Cell Biol

Role of basement membrane in development and differentiation

Full paper
Morphogenetic restructuring and formation of basement membranes by Sertoli cells and testis peritubular cells in co-culture: Inhibition of the morphogenetic cascade by cyclic AMP derivatives and by blocking direct cell contact☆