Figure 6.

MFN2 Regulates Both Mitochondrial and ER Functions in Germ Cells

(A and B) Fluo4-AM fluorescent intensity was measured by flow cytometry on non-adherent germ cells (A), as well as sorted CD9+/KIT− undifferentiated and KIT+ differentiating spermatogonia (B) from testes of P11 mice. Data are presented as mean ± 1 SEM from three biological replicates. ^∗∗∗p < 0.001; N.S., no statistical significance.

(C) IF on non-adherent germ cells from P11 testes with green Fluo3-AM probe (to indicate Ca²⁺ level) and a red mitochondrial tracker.

(D) Mitochondrial and ER architectures were examined by TEM on testicular sections from Mfn2^f/+ and Mfn2^f/f; Ddx4-Cre testes of P5 mice. Red arrows indicate ER. N, nucleus.

(E) Expression levels of chaperon proteins were examined by western blots on testes from P12 mice.

(F) Histological studies of testicle sections from Mfn2^f/f; Ddx4-Cre mice at day 30 after viral introduction of MFN2 tagged with mitochondrion (MFN2^ActA) or ER (MFN2^Cb5) localization signals. Red stars indicate the seminiferous tubules containing haploid spermatids. Scale bar, 50 μm.

(G) IHF analyses with an antibody against acrosin (ACR), counterstained with peanut agglutinin (PNA) and DAPI on Mfn2^f/f; Ddx4-Cre testis sections at day 30 after viral introduction with ActA- or Cb5-tagged MFN2 through seminiferous tubules. Ctrl, control viruses produced with the empty vector. Scale bar, 20 μm.

(F and G) Three to four mice per group were used for viral injection. See also Figures S5 and S6.