SALL4 Expression in Gonocytes and Spermatogonial Clones of Postnatal Mouse Testes

The spermatogenic lineage is established after birth when gonocytes migrate to the basement membrane of seminiferous tubules and give rise to spermatogonial stem cells (SSC). In adults, SSCs reside within the population of undifferentiated spermatogonia (Aundiff) that expands clonally from single cells (Asingle) to form pairs (Apaired) and chains of 4, 8 and 16 Aaligned spermatogonia. Although stem cell activity is thought to reside in the population of Asingle spermatogonia, new research suggests that clone size alone does not define the stem cell pool. The mechanisms that regulate self-renewal and differentiation fate decisions are poorly understood due to limited availability of experimental tools that distinguish the products of those fate decisions. The pluripotency factor SALL4 (sal-like protein 4) is implicated in stem cell maintenance and patterning in many organs during embryonic development, but expression becomes restricted to the gonads after birth. We analyzed the expression of SALL4 in the mouse testis during the first weeks after birth and in adult seminiferous tubules. In newborn mice, the isoform SALL4B is expressed in quiescent gonocytes at postnatal day 0 (PND0) and SALL4A is upregulated at PND7 when gonocytes have colonized the basement membrane and given rise to spermatogonia. During steady-state spermatogenesis in adult testes, SALL4 expression overlapped substantially with PLZF and LIN28 in Asingle, Apaired and Aaligned spermatogonia and therefore appears to be a marker of undifferentiated spermatogonia in mice. In contrast, co-expression of SALL4 with GFRα1 and cKIT identified distinct subpopulations of Aundiff in all clone sizes that might provide clues about SSC regulation. Collectively, these results indicate that 1) SALL4 isoforms are differentially expressed at the initiation of spermatogenesis, 2) SALL4 is expressed in undifferentiated spermatogonia in adult testes and 3) SALL4 co-staining with GFRα1 and cKIT reveals distinct subpopulations of Aundiff spermatogonia that merit further investigation. © 2013 Gassei, Orwig

Differential expression of SALL4 isoforms during postnatal testis development.

<p>(A–D) Co-staining for DAZL (all germ cells, red) and SALL4A/B (green). (A, B) Some SALL4-negative gonocytes (white arrows) were observed at PND 0. Autofluorescence was observed in the interstitium (asterisk). All germ cells strongly express SALL4 at PND 7 (C) and PND 14 (D) (E–H) Co-staining for VASA/DDX4 (all germ cells, red) and SALL4A (green). (E,F) SALL4A was absent from VASA-positive germ cells at PND 0 and 3. SALL4A was strongly expressed in all germ cells at PND 7 (G) and PND 14 (H). Scale bars = 10 µm. (I) Western blot analysis of total testis protein isolated from different postnatal ages showed differential expression of SALL4 isoforms. Protein from R1 mouse embryonic stem cells is shown as positive control. Blots were reprobed with anti-beta actin IgG to ensure equal loading of proteins.</p

SALL4 is expressed in undifferentiated spermatogonia in adult mouse seminiferous tubules.

<p>Double immunohistochemistry of tissue sections was used to examine the localization of SALL4 in comparison to PLZF (A–D) and LIN28 (E–H), two known markers for undifferentiated spermatogonia. (D, H) Data are expressed as the average number of single or double stained cells per tubular cross section ± SEM. (I–K) SALL4 was not expressed in GATA4-positive Sertoli cells. (L) SALL4, PLZF and LIN28-positive cells were normalized to 1000 Sertoli cells to express the relative size of the respective spermatogonial populations. The basement membrane of each tubule is marked by a dashed line. Scale bars = 10 µm. For quantitative analysis, 100 circular tubule cross sections in testes from at least 3 adult male mice were scored.</p

SALL4 is expressed by undifferentiated spermatogonia in postnatal day 7 and postnatal day 14 testes.

<p>(A–F) SALL4 expression is restricted to LIN28 positive undifferentiated spermatogonia at PND 7 and PND 14. (G–L) SALL4 expression is restricted to PLZF positive undifferentiated spermatogonia at PND 7 and PND 14. Scale bars = 10 µm.</p

SALL4 is co-expressed with PLZF and LIN28 in clones of undifferentiated spermatogonia.

<p>(A–D) SALL4 and PLZF were co-expressed in most undifferentiated spermatogonia. Small fractions of A_s and A_pr spermatogonia showed molecular heterogeneity (*). (E–H) SALL4 also largely overlaps with LIN28 expression, with heterogeneity of expression observed in few A_s and A_pr spermatogonial clones (*). Scale bars = 50 µm. Data obtained from n = 3 animals, presented as average ± SEM. Asym: Number of clones with molecular asymmetry.</p

Differential expression of GFRα1 and SALL4 in A_s and A_pr spermatogonia.

<p>Expression of GFRα1 was limited to short chains of A_undiff and more restricted than expression of SALL4. (A–D) Examples of GFRα1 and SALL4 expressing spermatogonial clones in whole mount seminiferous tubules. Scale bars = 10 µm. Panel D was modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053976#pone.0053976-Phillips1" target="_blank">[1]</a> with permission. (E) Quantitative evaluation of GFRα1 and SALL4 expression in clones of undifferentiated spermatogonia. Among A_s spermatogonia, 50% of cells co-expressed both markers, but substantial populations of SALL4-only and GFRα1-only cells were also observed. The number of SALL4-only cells increased in longer chains, when GFRα1 expression ceased. Asym: Number of clones with molecular asymmetry.</p

SALL4A and SALL4B co-expression in juvenile germ cells.

<p>SALL4A and SALL4B are expressed in the same population of germ cells at PND 7 (A–C) and PND 14 (D–F). Scale bars = 10 µm.</p

Donor-Host Involvement in Immature Rat Testis Xenografting into Nude Mouse Hosts1

Immature testicular tissue of a wide variety of mammalian species continues growth and maturation when ectopically grafted under the dorsal skin of adult nude mouse recipients. Tissues from most donor species fully mature, exhibiting complete spermatogenesis within months. The connection to the recipient's vascular system is mandatory for graft development, and failure of vascularization leads to necrosis in the grafted tissue. In the present study, we analyze to what extent 1) the xenografted immature donor tissue and 2) the recipient's cells and tissues contribute to the functional recovery of a “testicular xenograft.” We address whether recipient cells migrate into the testicular parenchyma and whether the circulatory connection between the donor testicular tissue and the recipient is established by ingrowing host or outgrowing donor blood vessels. Although this issue has been repeatedly discussed in previous xenografting studies, so far it has not been possible to unequivocally distinguish between donor and recipient tissues and thus to identify the mechanisms by which the circulatory connection is established. To facilitate the distinction of donor and recipient tissues, herein we used immature green fluorescent protein-positive rat testes as donor tissues and adult nude mice as graft recipients. At the time of graft recovery, donor tissues could be easily identified by the GFP expression in these tissues, allowing us to distinguish donor- and recipient-derived blood vessels. We conclude that the circulatory connection between graft and host is established by a combination of outgrowing small capillaries from the donor tissue and formation of larger vessels by the host, which connect the graft to subcutaneous blood vessels