PMC

A. Expression of unique sequence piRNAs in MitoPLD-/- testes relative to MitoPLD+/+ testes at E16.5. The data for the piRNAs from the top 19 piRNA clusters producing the largest numbers (amounts) of piRNA in wildtype testes are shown. The positions of these clusters are shown in . The piRNA counts were normalized by the miRNA counts.
B. Expression of piRNAs from retrotransposons in MitoPLD-/- testes relative to MitoPLD+/+ testes at E16.5. The data for the piRNAs from the retrotransposons of top 10 piRNA sources are shown. The retrotransposons are ordered from left to right according to the number of piRNAs they produce in wildtype. The total miRNA counts were used for normalization.
C. Northern blotting analysis of piRNAs in MitoPLD+/- and -/- testes at E16.5. pi-7-1 is a piRNA generated from the largest embryonic piRNA cluster on chr7. LTR+ piRNA has a sequence of IAP1 retrotransposon.
D. Expression of unique sequence piRNAs in MitoPLD-/- testes relative to MitoPLD+/+ testes at P10. See (A).
E. Expression of piRNAs from retrotransposons in MitoPLD-/- testes relative to MitoPLD+/+ testes at P10. The data for the top 8 sources of piRNAs in wildtype are shown. Retrotransposons are ordered from left to right according to the number of piRNAs that they produce. See (B).
F. Northern blotting analysis of piRNAs in MitoPLD+/+, +/- and -/- testes at P10. pi-19-1 is a unique piRNA from a piRNA cluster. pi-SINE1, 3 and 4 have sequences of SINE B1.
G. The piRNAs from SINE B1 are bound to MILI. The MILI complex was immunoprecipitated from P10 testes and the bound RNAs were probed with oligonulceotides for pi-SINE1 and pi-SINE4 in (F). RNA from five pairs of testes was loaded in each lane.
H. piRNAs from MitoPLD-/- testes tend to lose the characteristics of the primary piRNA (1st U) but gain that of the secondary piRNA (complementarity between 1st base and 10th base). The nucleotide compositions of the 1st and 10th nucleotide are shown for piRNAs from clusters and repeat from P10 testes.
I. The distance between the 5’ ends of complementary piRNAs. piRNAs from P10 testes that matched the consensus sequence of IAP1 retrotransposon with up to 3 mismatches were used. A peak at 10 nt in MitoPLD-/- testes suggests that the ping-pong cycle (the secondary pathway) is still active.

Apoptosis and increased serine protease activity in Spink2 mutant testes. A, germ cell apoptosis in Spink2^+/+ and Spink2^gt/gt testes. Sections of testes were TUNEL-stained; positive cells are indicated by arrows. Scale bar, 50 μm. B, comparison of TUNEL-positive germ cells in Spink2^+/+ and Spink2^gt/gt testes. The percentage of apoptotic germ cells per seminiferous tubule in Spink2^+/+ testes is shown in the y axis. Values are means ± S.E. (error bar). Statistical significance (*, p < 0.05; n = 4) was assessed using Student's t test. The average percentage of apoptotic germ cells in Spink2^gt/gt testes was 529.88% ± 77.28% of that in WT testes. C, increased serine protease activity in Spink2^gt/gt testes. Testis extracts from Spink2^+/+ and Spink2^gt/gt males were incubated with the rhodamine-conjugated substrate, and serine protease activity was measured. Values are means ± S.D. (n = 3). Statistical significance (*, p < 0.01) was assessed using Student's t test. Each experiment was repeated at least three times. Increased activity in Spink2^gt/gt testes was 133.36 ± 2.09% of that in WT testes. D, caspase-3 activation in the testes. Immunoblot of whole cell lysates of Spink2^+/+ and Spink2^gt/gt testes was performed with anti-caspase-3 antibody. An anti-α-tubulin antibody was used as a control for sample loading.

(A) Sperm parameters of testes with a Dmxl2 cell-specific KO. Epididymal sperm concentration in germ cell cKO (Dmxl2^loxP/-; Vasa-Cre) testes was 71% lower than that in control testes (Dmxl2^loxP/loxP) (pValue = 0.021), and that in Sertoli and germ cell dcKO (Dmxl2^loxP/-; Vasa-Cre; Amh-Cre) testes was 61% lower than that in control testes (pValue = 0.035). No significant difference was detected between germ cell cKO and dcKO testes (pValue = 0.7429). The motility of the spermatozoa was unaffected. (B) Histological appearance and (C) stereological analyses of control, Sertoli cell cKO, germ cell cKO and Sertoli and germ cell dcKO (dcKO) testes. The fraction of the volume occupied by the Sertoli cell cytoplasm (c) in the lumen is 41.7% greater in Dmxl2 dcKO testes than in control testes (pValue = 0.02), whereas the lumen area fraction (l) presents a mirror-image 36.4% decrease (pValue = 0.001). A similar effect was observed in comparisons of germ cell cKO and control testes (pValues of 0.02 and 0.03, respectively). Slight differences in seminiferous epithelium thickness (se) were observed in Dmxl2 dcKO testes relative to control (pValue = 0.01). Significant differences are indicated by different letters.

(A) Diagram of JNK signaling pathway. (B) Testis from 3-day-old puc-lacZ^A251 male immunostained for β-galatosidase (green), co-stained for Zfh-1 (red) and Vasa (blue). Arrowheads indicate the LacZ-positive early cyst cells. (C-E) Testes from 4w-old puc-lacZ^A251 males immunostained for β-galactosidase, co-stained for FasIII (asterisks). Testes exhibiting class 1 and class 2 of extra puc-lacZ-positive cells are shown in D and E. (F) The percentages of testes with extra puc-lacZ-positive cells were much higher in mated males than unmated males. N: number of testes scored. (G) Percentages of 4w-old testes with extra puc-lacZ-positive cells. Overproduction of puc-lacZ-positive cells was observed in mated testes, but not in unmated testes at both 25°C and 29°C. N: number of testes scored. (H) Box-and-whisker plots showing the relative anti-β-galactosidase intensity in single early cyst cells in 4w-old puc-lacZ testes. N: number of the puc-lacZ-positive cells scored. (I-K) Testes of TRE-EGFP immunostained for GFP (green), co-stained for Arm (red). (I and J) Reduced TRE-EGFP expression in cyst cells of the unmated 4w-old males (I) compared to the mated 4w-old males (J). (K) The relative TRE-EGFP intensity in early cyst cells of unmated and mated 4w-old males. N: number of the cyst cells scored. P-values were calculated by Mann-Whitney test in H and K, and by Chi-squared test in G, and P. ns: p>0.05, *p<0.05, **p<0.01, and ***p<0.001. Asterisks indicate the hubs. Mass-mating was conducted for experiments shown in D-H, J, and K. Unmated of 30 males per vial was in C, F, G, H, I, and K.