Effects of mouse embryonic stem cells (OG2 cells expressing the GFP-transgene) on cord formation of rat testicular cells.

<p>3A) Micrographs showing immunofluorescence of GFP-signal (upper panels) and merged images (phase contrast plus immunofluorescence) of primary rat (7-day-old) Sertoli cells and peritubular cells seeded at an initial density of 10⁶ cells/cm² on flat PDMS substrates after culture for one day (upper two panels), three days (middle two panels) and six days (lower two panels) in DMEM. A variable number of OG2 cells (10, 10², 10³, 10⁴, 10⁵ cells/cm²) was added to the primary cells at the initiation of cell cultures. No OG2 cells were added to controls. No obvious change of cellular arrangements was observed at day 1. On days 3 and 6 of culture cord-like structure formation is observed which intensified with increasing numbers of OG2 cells. OG2 cells formed expanding colonies in contact with the cord-like structures. 3B) Analysis of the size of cord-like structures at day 6 of culture. Cord-like structures were significantly larger compared to all other experimental groups depending on the initial density of OG2 cells on flat PDMS substrates at day 6. In the control group and after addition of only 10 OG2 cells no cord like structures were encountered.</p

Cell specific and density dependent response of testicular cells to nanogratings.

<p>Micrographs showing immunostaining of primary rat (7-day-old) Sertoli cells and peritubular cells. The cells were seeded at an initial density of 10⁶ cells/cm² in DMEM and cultured for one week. Cells were fixed at day 7. Peritubular cells are marked for α-smooth muscle actin (brown precipitate). Nuclei were stained blue with hematoxylin. 1A–D) Cells cultured on flat PDMS substrate (1A), and substrates carrying nanogratings of 200 nm (1B), 350 nm (1C) and 5 um (1D) dimensions. Red arrows indicate the direction of nanogratings. 1E) Quantitatitive analysis of directional changes in peritubular cells on different substrates. All nanogratings evoked changes in the orientation of peritubular cells. No visible influence on Sertoli cells was noted. 1F–H) Cells cultured on 350 nm PDMS substrate after inhomogeneous seeding creating diversity in plating density. Areas of high cell density (1F) revealed a random distribution of Sertoli cells in contrast to an aligned orientation towards nanogratings in low cell density zones (1G) on the same substrate in the same well. Peritubular cells were always oriented in accordance with the direction of the nanogratings. Quantification of cellular orientation in Sertoli cells (1H). Sertoli cell density was determined in randomly selected microscopic frames (as seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060054#pone-0060054-g001" target="_blank">Figs. 1F–G</a>) and the predominant cellular characteristic (either randomly shaped or spindle shaped (aligned)) was recorded for each frame. Seven recordings were performed per experiment and seven independent experiments were analyzed. We established that the threshold to respond to the nanogratings occurred at a density of approximately 1000 cells/mm². Scale bar = 50 um.</p

Cord like-structure formation in testicular cell cultures (see also Video S1).

<p>2A) Two series of three micrographs presenting the stages of cord-like structure formation in testicular cells cultured on 350 nm and flat PDMS substrates in DMEM at days 14, 14.5 and 15 of culture. Primary rat (7-day-old) Sertoli and peritubular cells were seeded at an initial density of 10⁶ cells/cm². At two weeks, cultures consisted of confluent mixed monolayers with several Sertoli cell clusters. Imaging was started at day 14 when first indications of cellular migration were observed. During the observation period active movements triggered a change of the confluent monolayers into cord-like structures on both 350 nm (upper panel) and flat PDMS substrates (lower panel). The orientation of cord-like structures did not follow the direction of nanogratings (red arrows). 2B) Testicular cell cultures performed under identical conditions as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060054#pone-0060054-g002" target="_blank">Fig. 2A</a> except that cells were seeded at a lower initial density of 10⁵ cells/cm² either on nanograting (left panel) or flat (right panel) PDMS substrates. Micrographs were taken at day 15 of culture. The orientation of cord-like structures was following the direction of nanogratings (red arrow). Scale bar = 200 um. 2C) Analysis of changes in the direction of cord-like structures seeded at high (10⁶ cells/cm²) or low (10⁵ cells/cm²) initial density on 350 nm PDMS substrates at day 15. 2D) Analysis of the size of individual cord-like structures seeded at high (10⁶ cells/cm²) or low (10⁵ cells/cm²) initial density on 350 nm and flat PDMS substrates at day 15.</p

Effects of Nanostructures and Mouse Embryonic Stem Cells on <i>In Vitro</i> Morphogenesis of Rat Testicular Cords

<div><p>Morphogenesis of tubular structures is a common event during embryonic development. The signals providing cells with topographical cues to define a cord axis and to form new compartments surrounded by a basement membrane are poorly understood. Male gonadal differentiation is a late event during organogenesis and continues into postnatal life. The cellular changes resemble the mechanisms during embryonic life leading to tubular structures in other organs. Testicular cord formation is dependent on and first recognized by SRY-dependent aggregation of Sertoli cells leading to the appearance of testis-specific cord-like structures. Here we explored whether testicular cells use topographical cues in the form of nanostructures to direct or stimulate cord formation and whether embryonic stem cells (ES) or soluble factors released from those cells have an impact on this process. Using primary cell cultures of immature rats we first revealed that variable nanogratings exerted effects on peritubular cells and on Sertoli cells (at less than <1000 cells/mm²) by aligning the cell bodies towards the direction of the nanogratings. After two weeks of culture testicular cells assembled into a network of cord-like structures. We revealed that Sertoli cells actively migrate towards existing clusters. Contractions of peritubular cells lead to the transformation of isolated clusters into cord-like structures. The addition of mouse ES cells or conditioned medium from ES cells accelerated this process. Our studies show that epithelial (Sertoli cell) and mesenchymal (peritubular cells) cells crosstalk and orchestrate the formation of cords in response to physical features of the underlying matrix as well as secretory factors from ES cells. We consider these data on testicular morphogenesis relevant for the better understanding of mechanisms in cord formation also in other organs which may help to create optimized in vitro tools for artificial organogenesis.</p> </div

Combined effects of nanostructures and OG2 cell conditioned medium on cord formation of testicular cells.

<p>5A) Phase contrast micrographs of primary rat (7-day-old) testicular cells seeded at an initial density of 10⁶ cells/cm² on 350 nm (right panel) and flat (left panel) PDMS substrates in conditioned medium (Three day culture of OG2 cells at a density of 10⁵ cells/cm²) at day 1 (upper panel) and day 3 (lower panel) of culture. At day 1 orientation of cells in the direction of nanogrids was visible when compared to flat PDMS substrates. At day 3 of culture cellular aggregation and cord-like structure formation occurred on both flat and 350 nm PDMS substrates. The direction of cord-like structure was aligned with nanogratings on 350 nm PDMS substrates but not on flat PDMS substrates. Red arrows indicate the direction of nanogratings. Scale bar = 200 um. 5B) Analysis of the direction of cord-like structures in conditioned medium and after exposure to 350 nm or flat PDMS substrates at day 3 of culture. The quantitative results confirm the microscopic observation that nanogratings have an impact on the orientation of cord-like structures. Cord-like structures on flat PDMS show a random orientation. 5C) Analysis of the size of cord-like structures at day 3 of culture. Cord-like structures were of similar size irrespective of the exposure to nanogratings.</p

Gene expression patterns of <i>Lin28a</i>, <i>Ddx4</i> and of SSC niche-associated factors in adult mouse testes after cytotoxic treatments.

<p>Changes in transcript levels of <i>Lin28a</i> (A), <i>Ddx4</i> (B), <i>Cxcr7</i> (C), <i>Erm</i> (D), <i>Itgb1</i> (E), <i>Cxcr4</i> (F), <i>Gdnf</i> (G), <i>Amh</i> (H) and <i>Cxcl12</i> (I) on days 1, 3, 7, 21 and 28 after DMSO (•) and busulfan (▪, 38 mg/kg) treatment (n = 10 per time point and treatment). Results were calculated using luciferase as external standard and values from the sham-treated control group (n = 8) as calibrator and are presented as fold change in gene expression per testis (RQ per testis). Significant differences between the DMSO and the busulfan groups are marked with asterisks (*, P = 0.01 to 0.05; **, P = 0.001 to 0.01 or ***, P<0.001).</p

Profiling of Cxcl12 Receptors, Cxcr4 and Cxcr7 in Murine Testis Development and a Spermatogenic Depletion Model Indicates a Role for Cxcr7 in Controlling Cxcl12 Activity

<div><p>In mice the chemokine Cxcl12 and its receptor Cxcr4 participate in maintenance of the spermatogonial population during postnatal development. More complexity arises since Cxcl12 also binds to the non-classical/atypical chemokine receptor Cxcr7. We explored the expression pattern of Cxcl12, Cxcr4 and Cxcr7 during postnatal development in mouse testes and investigated the response of Cxcl12, Cxcr4, Cxcr7 and SSC-niche associated factors to busulfan-induced germ cell depletion and subsequent recovery by RNA expression analysis and localization of the proteins. In neonatal testes transcript levels of <i>Cxcl12</i>, <i>Cxcr4</i> and <i>Cxcr7</i> were relatively low and protein expression of Cxcr7 was restricted to gonocytes and spermatogonia. During development, RNA expression of <i>Cxcl12</i> remained stable but that of <i>Cxcr4</i> and <i>Cxcr7</i> increased. Cxcr7 was expressed in germ cells located at the basement membrane of the seminiferous tubules. In adult testes, transcript levels of <i>Cxcl12</i> were highest while the localization of Cxcr7 did not change. Following germ cell depletion, a significantly increased expression of <i>Cxcl12</i> and a decreased expression of <i>Cxcr7</i> were observed. Germ cells repopulating the seminiferous tubules were immunopositive for Cxcr7. We conclude that Cxcr7 expression to be restricted to premeiotic germ cells throughout postnatal testicular development and during testicular recovery. Hence, the spermatogonial population may not only be simply controlled by interaction of Cxcl12 with Cxcr4 but may also involve Cxcr7 as an important player.</p></div

Expression pattern of Cxcr7 during testicular germ cell development.

<p>Changes in testicular weight (A) as well as changes in transcript levels of <i>Cxcl12</i> (B), <i>Cxcr4</i> (C) and <i>Cxcr7</i> (D) during postnatal development in mouse testes ((1d<i>pp</i> and <37d<i>pp</i> (n = 3); 7d<i>pp</i>, 14 d<i>pp</i>, 21 d<i>pp</i> (n = 4)), 2 ^(−ΔCt) per testis, relative to <i>luciferase</i>). Results are shown as mean ± SD. Representative images showing immunofluorescence stainings for Cxcr7 (red) and Hoechst (blue) on days 1, 7, 14, 21,>37 of postnatal testicular development (first column, E–I). Within the inserts (second column) expression of Cxcr7 was observed in gonocytes (green arrow), spermatogonia (white arrows) and interstitial cells (asterisks). Sertoli cells are indicated with white arrowheads. Results of the respective negative controls using nonspecific IgG antibodies are shown in the right column (IgG/Hoechst). Scale bars represent 10 µm and 40 µm, respectively.</p

Cxcr4 and Cxcr7 expression in adult mouse testes.

<p>Representative images show immunofluorescence staining for Cxcr4 (green, A) in adult mouse testes. Cxcr4 is expressed by testicular cells at the basement membrane (arrow) of seminiferous tubules (indicated by dotted lines). Co-stainings revealed no Cxcr4 expression in Lin28a-positive (C) and Cxcr7-positive (D) spermatogonia. Incubation with corresponding IgG antibodies was used as negative control (B, C and D right column). All sections were counterstained with Hoechst (blue). Scale bars represent 10 and 15 µm, respectively.</p

Expression of Lin28a and Ddx4 in adult mouse testes following busulfan treatment.

<p>Representative images showing immunohistochemical stainings for Lin28a (left column) and Ddx4 (right column) on testis sections of DMSO (A, B) and busulfan treated (C–L) adult mice. For Lin28a and Ddx4 stainings on days 1 (C), 3 (E), 7 (G), 21 (I) and 28 (K) after busulfan treatment higher magnifications are shown for each time point (D, F, H, J, L). Germ cells are indicated by arrows and Sertoli cells by arrow heads. As negative control, stainings with nonspecific IgGs were performed (A; Insert). Scale bars represent 25 µm and 50 µm.</p