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. 2012 Mar;14(2):285-93.
doi: 10.1038/aja.2011.112. Epub 2011 Nov 7.

Differentiation of Murine Male Germ Cells to Spermatozoa in a Soft Agar Culture System

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Free PMC article

Differentiation of Murine Male Germ Cells to Spermatozoa in a Soft Agar Culture System

Mahmoud Abu Elhija et al. Asian J Androl. .
Free PMC article

Abstract

Establishment of an in vitro system that allows the development of testicular germ cells to sperm will be valuable for studies of spermatogenesis and future treatments for male infertility. In the present study, we developed in vitro culture conditions using three-dimensional agar culture system (SACS), which has the capacity to induce testicular germ cells to reach the final stages of spermatogenesis, including spermatozoa generation. Seminiferous tubules from testes of 7-day-old mice were enzymatically dissociated, and intratubular cells were cultured in the upper layer of the SACS in RPMI medium supplemented with fetal calf serum (FCS). The lower layer of the SACS contained only RPMI medium supplemented with FCS. Colonies in the upper layer were isolated after 14 and 28 days of culture and were classified according to their size. Immunofluorescence and real-time PCR were used to analyse specific markers expressed in undifferentiated and differentiated spermatogonia (Vasa, Dazl, OCT-4, C-Kit, GFR-α-1, CD9 and α-6-integrin), meiotic cells (LDH, Crem-1 and Boule) and post-meiotic cells (Protamine-1, Acrosin and SP-10). Our results reveal that it is possible to induce mouse testicular pre-meiotic germ cell expansion and induce their differentiation to spermatozoa in SACS. The spermatozoa showed normal morphology and contained acrosomes. Thus, our results demonstrate that SACS could be used as a novel in vitro system for the maturation of pre-meiotic mouse germ cells to post-meiotic stages and morphologically-normal spermatozoa.

Figures

Figure 1
Figure 1
Scheme of the SACS. The SACS was composed of two layers: the solid lower layer (0.5% (w/v) agar) and the soft upper layer (0.37% (w/v) agar), which were cultured in 24-well plates. Testicular tissue from immature mice (a) was mechanically separated to obtain interstitial tissue and tubules (b). The tubules were enzymatically digested (c), and the isolated tubular cells (d) were used for culture in the upper phase of the SACS (e). Tubular cells (106 cells per well per 200 µl) were cultured in the upper layer of the soft agar medium. Cultures were incubated in 5% CO2 incubator at 37 °C. FCS, fetal calf serum; SACS, Soft Agar Culture System.
Figure 2
Figure 2
Characterisation of isolated tubular cells before culture in SACS. Isolated tubular cells were examined by RT-PCR analysis using specific markers for pre-meiotic (Nanog, Vasa, OCT-4, C-Kit, GFR-α-1, CD9 and α-6-integrin), meiotic (Crem-1 and LDH) and post-meiotic stages (Protamine) and also for Sertoli cells (ABP) and peritubular cells (P) (α-Sm) (a). Immunofluorescence analysis for testicular tubular cells from 7-day-old mice was used to identify cells positive for pre-meiotic (C-Kit, GFR-α-1, α-6-integrin and CD9), meiotic (Boule, Crem-1 and LDH) and post-meiotic markers (Protamine) (b). Scale bars=10 µm. LDH, lactate dehydrogenase; SACS, Soft Agar Culture System; SC, Sertoli cell.
Figure 3
Figure 3
Tubular cell colonies development in the SACS. Tubular cells (106 cells per well) were cultured in the upper layer of the SACS. The lower layer of the SACS consisted of RPMI containing 20% FCS. The size of the colonies in the upper layer was evaluated after 14 and 28 days of culture. (a) Colonies were designated as small (S) when they contained around 50 cells; (b) medium (M) when they contained around 150 cells; and (c) large (L) when they contained more than 300 cells. (d) The capacity of tubular cells to form S, M or L colonies in SACS was examined after 14 and 28 days of culture. *P<0.05, ***P<0.001, compared with 14 days. (e) The expression of markers for Sertoli, peritubular, macrophage and Leydig cells (ABP, α-Sm, CD11-b and LHR respectively) was examined by PCR analysis using specific primers for each marker. Scale bars=10 µm. BC, before culture; AC, after culture (colonies); PC, positive control (RNA from the testis of an 8-week-old mouse). FCS, fetal calf serum; SACS, Soft Agar Culture System.
Figure 4
Figure 4
Immunofluorescence staining of cells from colonies developed in the SACS and testicular tissue from immature and mature mice. Colonies that developed within 28 days in culture in SACS were isolated and stained by specific antibodies for different markers of germ cell development by immunofluorescence, including Vasa, Dazl, C-Kit, GFR-α-1, CD9, α-6-integrin (a), Boule, Crem-1, LDH, Protamine, Acrosin, and the negative control (NC) (b). The presence of Vasa, Dazl, CD9, GFR-α-1, C-Kit, α-6-integrin, Crem-1, LDH and Protamine, was examined in parallel in testicular tissue from 7-day-old (c) and 8-week-old mice (d). Scale bars=10 µm. LDH, lactate dehydrogenase; SACS, Soft Agar Culture System.
Figure 5
Figure 5
Differentiation of tubular cells to spermatozoa in SACS. Tubular cells were cultured in the SACS as described in Figures 1 and 2 and evaluated as described in the section on ‘Materials and methods'. The presence of differentiated germ cells (a), including spermatozoa (b), in the SACS was examined under the microscope after H&E staining. More than 10 spermatozoa were determined in each slide (each well). The presence (c) of acrosomes was examined by PNA-FITC staining (green-colour acrosomes). DAPI (blue) staining indicated the heads of the sperm (c). Scale bars=3–5 µm. Arrowheads in the upper panel indicate the developing flagellum. H&E, haematoxylin and eosin; SACS, Soft Agar Culture System.

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