BAX-mediated cell death affects early germ cell loss and incidence of testicular teratomas in Dnd1(Ter/Ter) mice

Abstract

A homozygous nonsense mutation (Ter) in murine Dnd1 (Dnd1(Ter/Ter)) results in a significant early loss of primordial germ cells (PGCs) prior to colonization of the gonad in both sexes and all genetic backgrounds tested. The same mutation also leads to testicular teratomas only on the 129Sv/J background. Male mutants on other genetic backgrounds ultimately lose all PGCs with no incidence of teratoma formation. It is not clear how these PGCs are lost or what factors directly control the strain-specific phenotype variation. To determine the mechanism underlying early PGC loss we crossed Dnd1(Ter/Ter) embryos to a Bax-null background and found that germ cells were partially rescued. Surprisingly, on a mixed genetic background, rescued male germ cells also generated fully developed teratomas at a high rate. Double-mutant females on a mixed background did not develop teratomas, but were fertile and produced viable off-spring. However, when Dnd1(Ter/Ter) XX germ cells developed in a testicular environment they gave rise to the same neoplastic clusters as mutant XY germ cells in a testis. We conclude that BAX-mediated apoptosis plays a role in early germ cell loss and protects from testicular teratoma formation on a mixed genetic background.

Figure 1. Expression of Dnd1 is restricted to germ cells of the developing testis

Dnd1 in situ expression in a whole mount (A,B) and sectioned (C,D) E14.5 WT testis is restricted to testis cords, and disappears in a Busulfan treated testis (B,D). Busulfan is a chemical that eliminates germ cells. (E,F) RT-PCR on Sox9-ECFP positive and negative cells, sorted from E12.5–E15.5 gonads. Dnd1 expression is only detected in the ECFP-negative fraction (containing the Oct4 positive germ cell population) and is excluded from the pure Sertoli cell fractions (exclusively Sox9 positive).

Figure 2. Teratomas form in mutants on the 129Sv/J background but germ cells are lost completely on other genetic backgrounds

(A)Large bilateral testicular teratomas from a P17 129Sv/J Dnd1^Ter/Ter mutant compared to (B) a mutant from the same litter that did not develop teratomas. On the 129Sv/J background testes develop teratomas at a rate of ~95% while the remainder simply lose their germ cells. Scale bars represent 1cm. (C) P11 wildtype testis section in which germ cells within testis cords are immunostained with antibodies against GCNA (red) and MVH (green). (D) P11 Dnd1^Ter/Ter testis section on a mixed genetic background contains no germ cells that stain positive for either GCNA or MVH within testis cords by this stage. Scale bars represent 50 μm.

Figure 3. On a Bax mutant background, germ cells are partially rescued in Dnd1^Ter/Ter embryos, and these double-mutants develop testicular teratomas on a mixed genetic background

(A) Germ cells (immunostained with MVH in green) are grouped inside testis cords in a wildtype testis at E13.5. Germ cell numbers are greatly reduced in a Dnd1^Ter/Ter testis (C), and somewhat increased in a Dnd1^Ter/+ mouse carrying two loss of function alleles of Bax (E). (G) A Dnd1^Ter/Ter;Bax^−/− double-mutant testis shows a significant increase in germ cell number compared to C. Scale bars represent 50μm. (B) P8 wildtype testes. On a mixed genetic background, Dnd1^Ter/Ter mutant testes are reduced in size as a result of germ cell loss (D as compared to B). Loss of Bax on a Dnd1^+/+ background leads to an increase in testis size (F), whereas loss of Bax on a Dnd1^Ter/Ter background results in teratoma formation (H). Scale bars represent 0.5cm.

Figure 4. Dnd1^Ter/Ter;Bax^−/− ovaries have more oocytes than Dnd1^Ter/Ter ovaries

Representative sections from the thickest part of (A) a P16 Dnd1^Ter/Ter ovary and (B) a P18 Dnd1^Ter/Ter;Bax^−/− ovary stained with hematoxylin and eosin. Scale bars represent 100 μm.

Figure 5. XX germ cells in an XX Sry^MYC testis form neoplastic clusters like XY germ cells in a testis

XY germ cells are MVH positive (green) in an E18.5 control XY testis (A), and in an XX Sry^MYC (sex-reversed) testis (B) although their numbers are somewhat reduced. (C) At E18.5, the XY Dnd1^Ter/Ter testis lacks MVH-positive cells, but an E-cadherin-positive (red) neoplastic cluster is detected. These clusters typically give rise to nascent teratomas (Cook et al., in preparation). (D) At E18.5 the XX Sry^MYC Dnd1^Ter/Ter testis also lacks MVH-positive germ cells but contains a similar E-cadherin positive neoplastic cluster. All testis samples are on a 129Sv/J background. Scale bars represent 50 μm.