Fig 1.
CTR1 is expressed in pachytene spermatocytes and Sertoli cells in a stage specific manner.
Immunohistochemical localization of CTR1 in adult (PND 60) Ctr1fl/fl mouse testis cross sections (A, B). Arrow indicates expression on primary pachytene spermatocytes. Arrowheads indicate SC apical and basal cytoplasm. CTR1 expression on the apical surface of the epithelial cell in the intestine of adult mice indicated by arrows (D). Negative secondary only control of testis (C) and intestine (E) shown. Scale bar = 50 μm. Seminiferous epithelium at stages II-VI displayed the highest CTR1 expression on pachytene spermatocytes (F). Stages VII-XII (G, H and I) display the least CTR1 staining on primary spermatocytes (arrows) but high CTR1 localization on SCs (indicated by arrowheads) within the seminiferous tubule cross sections. Three different tubule cross sections for each specified stage are shown below each grouped of stages (i-iii). Scale bar = 100 μm.
Fig 2.
Ctr1ΔGC mice exhibit severe loss of germ cell with increasing age.
Total testis weight (A), and testis to body weight (TW/BW) ratios (B) of WT and Ctr1ΔGC mice over time with a representative image of PND 41 testis (C) of both WT and Ctr1ΔGC mice. (N = 3 for each age; *p <0.05 and *** p < 0.001). Histological cross sections of WT (D, E and F) and Ctr1ΔGC (G, H and I) seminiferous tubules on PND 14 (left), PND 28 (middle), and PND 41 (right) with PAS-H staining. Scale bar = 50 μm. Ctr1ΔGC testis at PND 14 (D and G) arrowhead indicating zygotene spermatocytes and arrow indicating pachytene spermatocytes. Ctr1ΔGC testis at PND 28 (E and H) black arrow indicates round spermatids; white arrow indicates pachytene spermatocytes; black arrowhead indicates apoptotic GCs; white arrowhead indicates elongated spermatids. Ctr1ΔGC testis at PND 41 (F and I) white arrowhead indicates residual pre-meiotic (preleptotene and spermatogonia) GCs; arrow indicates SCs; black arrowhead indicates apoptotic cells; star sign indicates SC vacuole. (J) Apoptotic index in WT or Ctr1ΔGC on each PNDs (N = 3 for each age; *p < 0.05). (K) TUNEL staining indicating apoptotic cells (arrow) in the seminiferous tubules of PND 28 WT or Ctr1ΔGC mice at peak apoptosis. Scale = 100 μm.
Fig 3.
Ctr1ΔGC mice at PND 41 display presence of undifferentiated spermatogonial cells and normal number of Sertoli cells in adult testes of Ctr1ΔGC mice.
Immunohistochemical nuclear staining for PCNA in GCs of both (A) WT and (B) Ctr1ΔGC testes. Presence of FOXO-1 expressing undifferentiated spermatogonial cells at PND 41 in (C) WT and (D) Ctr1ΔGC testes. Average number of SOX9-positive cells per seminiferous tubules (E). On right, representative immunohistochemical staining from SOX9 analysis of WT and Ctr1ΔGC; arrows indicating SOX9-positive SCs (Average ±SD, N = 3 for each genotype). Scale bar = 100 μm. Histological analysis on Ctr1ΔGC testis at PND 70 indicating multiple clusters of SCs in the lumen indicated by circles (F) with PAS-H staining. SOX9-positive SCs clarifying the SCs clusters in the lumen (G). (H) Presence of FOXO-1-positive spermatogonial cells, indicated by arrows, within the tubules of Ctr1ΔGC testis at PND 70. Scale = 50 μm.
Fig 4.
Ctr1ΔSC mice at PND 60 are indistinguishable from WT littermates.
Immunohistochemical analysis of CTR1 protein expression within the Ctr1ΔSC and WT testis. (A) CTR1 localization on within the seminiferous tubules in WT mice expressed on the SCs (arrowheads) and spermatocytes (arrow), and CTR1 localization on the spermatocytes only (arrows) in the Ctr1ΔSC testis (C). Negative secondary only control of WT and Ctr1ΔSC testis shown on the right (B and D, respectively). (E) Q-PCR on Ctr1 mRNA expression normalized to Hprt1 gene indicates up to 70% reduced expression in Ctr1ΔSC SCs compared to WT (average ± SEM). (F) Immunoblot analysis on crude membrane extract on WT and Ctr1ΔSC SC isolates against CTR1 antibody shows undetectable CTR1 expression in Ctr1ΔSC compared to WT mice. (G) Testicular weight (TW), testes to body weight ratio (TW/BW) and testicular spermatid head counts (TSC) all displayed similar to their WT littermates. The graph represents relative measurements of Ctr1ΔSC mice compared to WT mice, dashed line represents 100% of each measurement in WT mice (average ± SD). (H) SOX9-positive cells within the seminiferous tubules of both WT and Ctr1ΔSC testes displayed similar number of SCs. (I) Representative images of SOX9 staining indicated by arrows in both WT and Ctr1ΔSC testis. Each data point is representative of 4–5 mice of each genotype. Scale bar = 100 μm.
Fig 5.
Ctr1ΔSC mice exhibit testicular copper deficiency.
(A) Relative Cu, Fe and Zn levels in Ctr1ΔSC testis and in (B) Ctr1ΔSC seminiferous tubules compared to WT testis. Data represent percent concentration (μg/g dry tissue weight) of Ctr1ΔSC tissues compared to WT tissues (average ±SD). Dashed line represents 100% of each metal in WT tissues. For each genotype N = 4–5, *P<0.05, **p<0.005. (C) Immunoblot analysis on CCS and GAPDH is shown as loading control. Three animals were randomly selected and analyzed from each WT and Ctr1ΔSC mice. Each number above represents an animal from each genotype. (D) Quantified protein expression of CCS normalized to GAPDH expression (average ± SEM, N = 3, p = 0.0656). (E) Cytochrome c oxidase (CCO) activity of WT and Ctr1ΔSC testis. Tissue lysates from randomly selected three WT and Ctr1ΔSC mice were analyzed to measure CCO activity. Graph represents the rate of cytochrome c (cyt c) oxidation (μM/min/mg of protein lysate) of Ctr1ΔSC and WT testis and normalized to citrate synthase (CS) activity for each sample (average ± SD, N = 3, ***p<0.001). (F) Immunoblot analysis on the COX5A, a CCO complex subunit, and GAPDH is shown as loading control.
Fig 6.
Loss of CTR1 in Sertoli cells display normal copper levels but copper deficient phenotype.
(A) Relative Cu, Fe and Zn levels in primary SCs isolated from WT and Ctr1ΔSC testes. Data represents percent concentration (μg of metal per 10,000 cells) of Ctr1ΔSC SCs compared to WT SCs. Dashed line represents 100% of each metal in WT SCs. (average ± SD, N = 5–7). (B) Immunoblot analysis (C) quantified protein expression of CCS normalized to GAPDH expression. Primary SCs isolated from three animals analyzed from each WT and Ctr1ΔSC mice (average ± SEM, N = 3, *p<0.05). (D) CCO activity of WT and Ctr1ΔSC primary SCs. Cell lysates from three different WT and Ctr1ΔSC mice were analyzed to measure CCO activity. Graph represents the rate of cyt c oxidation (μM/min/mg of protein lysate) of WT and Ctr1ΔSC SCs and normalized CS activity for each sample (average ± SD, N = 3, *p<0.05). (E) Immunoblot analysis on the COX5A expression of primary SCs derived from three individual mice from each genotype. GAPDH is shown as loading control.