Fact or fiction

Figures & data

Table 1. Overview of studies addressing in vitro spermatogenesis.

Culture methods	Treatment/ Culture conditions	References	Outcome
Culture of testicular tissue fragments: Maintain the spatial structure of the seminiferous tubule but not the oxygen supply	Hanging drop of hemolymph	Goldschmidt, 1915	Maturation of spermatocytes into spermatozoa stage in moths (Samia cecropia L.).
	Agar arranged on a steel grid (gas-liquid interphase method), Serum containing Vitamin A, E and C	Steinberger et al., 1964	Development of gonocytes into the stage of primary type A spermatogonia in rats (rattus).
	Gas-liquid interphase method, doubling of the concentration of glutamine in the culture medium	Steinberger,1965	Spermatogenesis passes to the pachytene stage in rats.
	Gas-liquid interphase method	Steinberger E. 1967	Maturation of human preleptotene spermatocytes into pachytene stage.
	Without testosterone/ FSH substitution	Parvinen et al., 1983	Completion of meiosis of late pachytene spermatocytes and induction of early spermiogenic events.
	Substitution with FSH	Boitani et al., 1993	Differentiation of type A spermatogonia into pachytene spermatocytes in rats.
	Mechanical dissociated from encapsulating somatic cysts cells	Kawamoto et al., 2008	Differentiation of 16-cell spermatogonial clones of larval testes into motile, spermatids in Drosophila melanogaster.
	Agar gel half-soaked in the medium (gas-liquid interphase method), KSR medium	Sato et al., 2011a b	Complete process of spermatogenesis in mice (Mus musculus) and achievement of functional gametes.
Conventional testicular cell culture: Do not maintain the spatial structure	Co-culture with Sertoli cells and the alternating presence of high/low FSH	Tres and Kierszenbaum, 1983	Differentiation of rat cells in the meiotic prophase.
	Drops of culture medium, Substitution with rFSH	Tesarik et al., 1998	Increase in the percentge of secondary spermatocytes and round spermatids in obstructive azoospermic patients.
	Co-culture with Vero cells	Cremades et al., 1999	Maturation of round spermatids into elongating spermatids in azoospermic patients .
	Co-culture with Sertoli cells, substitution with rFSH and testosterone	Sousa et al., 2002	Meiosis and spermiogenesis in non-obstructive azoospermic patients.
	Co-culture with Vero cells	Tanaka et al., 2003	Meiotic differentiation primary spermatocytes into round spermatids in azoospermic men, arrested at the level of primary spermatocytes.
	Co-culture with Sertoli cells	Virgier et al., 2004	Positive effect on meiotic divisions of pachytene spermatocytes into round spermatids of rats.
	Co-culture with Sertoli cells	Iwanami et al., 2006	Differentation of type A spermatogonia of rats into round spermatid-like cells with abnormal gene expression pattern.
	Co-culture with Sertoli cells	Sakai, 2006	Complete process of spermatogenesis in the zebra fish (Danio rerio).
	Co-culture with Sertoli cells, medium supplemented with FBS, substitution with testosterone	Xie et al., 2010	Spermatogonia differentiate into morphological normal spermatids in buffaloes (Bubalus bubalis).
Testicular 3-D cell culture: Do not maintain the natural structure but try to resemble the spatial environment	Collagen gel matrix, shaking during seeding increased the viability of cells	Lee JH et al., 2006b	Meiosis and differentiation of rat germ cells into post-meiotic stages.
	Calcium alginate capsules	Lee DR et al., 2006a	Expression of genes specific for postmeiotic haploid state in non-obstructive azoospermic patients.
	Collagen gel matrix	Lee JH et al., 2007	Differentiation of spermatocytes into spermatids in non-obstructive azoospermic patients.
	Soft-Agar-Culture-System/ Methylcellulose matrix system, substitution with hCG and FSH	Stukenborg et al., 2008/ 2009	Differentation and meiosis of spermatogonia into morphologically mature spermatozoa in mice.
	Soft-Agar-Culture-System, medium supplemented with FCS	Abu Elhija et al., 2011	Differentation and meiosis of spermatogonia into morphologically mature spermatozoa in mice.

Figure 1. (A) Cytology of the mouse testis showing the relationship and the function of the interstitial, basal, intraepithelial, and adluminal compartment. (B) Schematic diagram of different approaches for male germ cell differentiation in vitro and their outcomes so far.

Figure 2. Artificial collagen sponges seeded with isolated testicular cells of mice (7 dpp). (A) Scanning electron microscope image of a collagen sponge prior to culture. (B) Scanning electron microscope image of a colonized collagen sponge. Isolated testicular cells (2 million) established aggregates in the structure of the sponge after one day of culture. (C) Testicular cells of eGFP mice on a collagen sponge after three days of culture. The cells have colonized the collagen scaffold along the given structure. Scale bar represents 100 µm. (D) Micrographs of a section of a paraffin embedded collagen sponge (hematoxylin staining) Scale bar represents 100 µm. (E, F) Histological micrographs for morphological identification of mouse testicular cell types after seven days of culture. Samples were embedded in resin (Technovit, Heraeaus Kulzer, GmbH, Wehrheim, Germany); stained with perjodic acid Schiff reagent; and cut to 3µm sections (protocol according to ref. 49). Scale bar represents 100 µm. (E) Cell clusters consist of a mixture of testicular cells. i.e peritubular cells (o), Sertoli cells (*) and some undifferentiated germ cells (#). (F) During colonization of the scaffolds, first signs of tubulogenic reassembly occur. The cells utilize the scaffold for structural reorganization when attaching to the collagen fibers (arrowheads).

Wistuba J, Brinkworth MH, Schlatt S, Chahoud I, Nieschlag E. Intrauterine bisphenol A exposure leads to stimulatory effects on Sertoli cell number in rats. Environ Res 2003; 91:95 - 103; http://dx.doi.org/10.1016/S0013-9351(02)00019-1; PMID: 12584010

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