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

Cxcr7 and Cxcr4 expression in germ cell-depleted mouse testes.

<p>Representative immunofluorescence images of testicular tissue section 28 days after busulfan treatment of adult mice. Cxcr7 expression (B, red, arrow) is restricted to Sall4-positive spermatogonia (A, green, arrow) but Cxcr4 expression (F, green, arrowheads) is not solely observed in Lin28-positive spermatogonia (E, red, arrows). The respective merged images are shown in (C) and (G). Hoechst (blue) was used as nuclear counterstain. Incubation with corresponding IgG antibodies was used as negative control and a representative image is shown in (D, H). Scale bars represent 15, 20 and 30 µm.</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

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

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