Inhibition of primordial germ cell proliferation by the medaka male determining gene Dmrt I bY

Abstract

Background: Dmrt I is a highly conserved gene involved in the determination and early differentiation phase of the primordial gonad in vertebrates. In the fish medaka dmrt I bY, a functional duplicate of the autosomal dmrt I a gene on the Y-chromosome, has been shown to be the master regulator of male gonadal development, comparable to Sry in mammals. In males mRNA and protein expression was observed before morphological sex differentiation in the somatic cells surrounding primordial germ cells (PGCs) of the gonadal anlage and later on exclusively in Sertoli cells. This suggested a role for dmrt I bY during male gonad and germ cell development.

Results: We provide functional evidence that expression of dmrt I bY leads to negative regulation of PGC proliferation. Flow cytometric measurements revealed a G2 arrest of dmrt I bY expressing cells. Interestingly, also non-transfected cells displayed a significantly lower fraction of proliferating cells, pointing to a possible non-cell autonomous action of dmrt I bY. Injection of antisense morpholinos led to an increase of PGCs in genetically male embryos due to loss of proliferation inhibition.

Conclusion: In medaka, dmrt I bY mediates a mitotic arrest of PGCs in males prior to testes differentiation at the sex determination stage. This occurs possibly via a cross-talk of Sertoli cells and PGCs.

Figure 1

Dmrt1bY overexpression modifies cell cycle pattern in cell culture as well as in live embryos. A and B: GFP fluorescence is cytoplasmic in mouse TM4 cells transiently transfected with p CMV:EGFP-SV40UTR control plasmid (A) while nuclear localized when transfected with pCMV:Dmrt1bY:GFP-SV40UTR construct (B). C and D: Radar histograms representing the DNA content distribution of Dmrt1bY:GFP relative to GFP (control) transfected cells expressed in percentage. "Ctr" and "GFP Ctr, GFP+" represent GFP negative and positive control cells in the plate tranfected with control GFP plasmid; similarly "Dmrt1bY, GFP+" and "Dmrt1b, GFP-" represent GFP negative and positive cells In the plate tranfected with Dmrt1bY:GFP plasmid. E and F: Cell cycle distribution reflected by DNA content in control versus stage 10 (MBT) and post-MBT (stage 13) Dmrt1bY-injected embryos.

Figure 2

Nuclear localization of Dmrt1bY:GFP and ΔDmrt1bY fusion proteins and modulation of cell proliferation by Dmrt1bY overexpression. A and B: When capped RNAs encoding fusion protein of GFP with either Dmrt1bY (A) or ΔDmrt1bY (B) were injected into one cell stage embryos (75 ng/μL) strong nuclear localization is observed (A); mainly cytoplasmic (B) at stage 8. D, E and F: Green fluorescence produced by Dmrt1bY:GFP fusion protein colocalizes with DAPI staining of nuclei. GFP fluorescence (D), DAPI fluorescence (E), overlay (F). C and C': Embryos injected with high doses of Dmrt1bY:GFP, once arrested shortly after MBT, remain quiescent and become necrotic (C'). C: GFP-injected control embryo. G and H: Dmrt1bY injected embryos were stained for the mitotic marker phospho-Histone H3. Wild type embryo (G), Dmrt1bY injected embryo (H). Scale bars: 25 μM in A, B, D, E, F and 120 μM in C, C', G, H.

Figure 3

Delay in early development in transgenic fish expressing Dmrt1bY under control of the cytoskeletal actin promoter. A, D, G, J and M: normal wild type embryo at 16 cell stage, late blastula, 90% epiboly, late neurula and 22 somite stage corresponding to stages 6, 11, 16, 18 and 26 respectively. B, E, H, K and N: Dmrt1bY transgenic line embryo. C, F, I and L: GFP expression during early development of the Dmrt1bY transgenic line. Wild type and Dmrt1bY transgenic embryos at the same time of embryonic development were obtained in the same clutch of fertilized eggs by crossing a Dmrt1bY:GFP heterozygous parental fish to a non-transgenic fish. Scale bars: 100 μM in A to L and 50 μM in M and N.

Figure 4

Loss of replicating cells in transgenic fish expressing Dmrt1bY. A, B and C: Cell replication status was quantified at 80% epiboly (stage 17) by pH3 immunostaining. Wild type (A), CSKA:Dmrt1bY:GFP transgenic line. pH3 positive cells were counted for a constant given area (A, B) and revealed a highly significant (p < 0.001) decrease of 25.5% replicating cells in the transgenic line when compared to wild type (C). Scale bars: 120 μM.

Figure 5

Differences in primordial germ cell number and shape diagnostic for the male and female gonad primordium anlage. A and B: Green fluorescence of the Olvas medaka primordial gonad at hatching stage (stage 39). Male (A), female (B). Both sexes have a significantly higher number of PGCs in the right PGC lateral clump. C: Comparison of the Olvas PGC phenotype versus genotype (Dmrt1bY) at hatching stage. D: Offspring obtained by mating a Y^Awr/Y^Awr female with an Olvas X/Y^HdrR male. E: Comparison of the Olvas primordial gonad phenotype at hatching stage versus genotype (Y^Awr or Y^HdrR). Identification of genetic sex in each individual was performed by PCR analysis for dmrt1bY presence or absence after genomic DNA extraction. The dmrt1bY fragments from either both Awr or Hd-rR-specific alleles were amplified with diagnostic primer sets. Scale bars: 7.5 μM.

Figure 6

Shift of male primordial gonad replication pattern towards female by down regulation of Dmrt1bY (BrdU incorporation). Replication pattern of the primordial gonad was followed by BrdU incorporation. A: female primordial gonad phenotype at hatching stage. B: male primordial gonad phenotype at hatching stage. C, D and E: fluorescence produced by BrdU incorporation colocalizes with GFP fluorescence of primordial germ cells in the Olvas transgenic line. GFP fluorescence (C), BrdU fluorescence (D), overlay (E). F: primordial gonad phenotype at hatching stage after Dmrt1bY morpholino injection in a genetic male.

Figure 7

Shift of male primordial gonad phenotype towards female by down regulation of Dmrt1bY. A: Male primordial gonad phenotype at hatching stage (stage 39). B1 and B2: Partially shifted male to female primordial gonad phenotype at hatching stage after MO-Dmrt1bY morpholino injection. C: Female and totally reversed male primordial gonad phenotype at hatching stage after MO-Dmrt1bY morpholino injection. D: Comparison of the primordial gonad phenotype versus genotype after Dmrt1bY morpholino injection. Scale bars: 7.5 μM.