Objective: ATP6V0d2 is a subunit of the vacuolar-type H+-ATPase (V-ATPase) that pumps H+ ions into lysosomes. TRPML1 (MCOLN1/Mcoln1) transports cations out of lysosomes.Mcoln1^(-/-) mice recapitulate the lysosomal sto...Objective: ATP6V0d2 is a subunit of the vacuolar-type H+-ATPase (V-ATPase) that pumps H+ ions into lysosomes. TRPML1 (MCOLN1/Mcoln1) transports cations out of lysosomes.Mcoln1^(-/-) mice recapitulate the lysosomal storage disorder mucolipidosis type IV (MLIV) phenotype. We previously demonstrated thatMcoln1^(-/-) female mice quickly became infertile at 5 months old (5M) with degenerating corpora lutea (CL) and progesterone (P4) deficiency. We tested our hypothesis thatAtp6v0d2 deficiency could partially compensate forMcoln1 deficiency to restore CL functions inAtp6v0d2^(-/-)Mcoln1^(-/-) mice.Methods: Control andAtp6v0d2^(-/-)Mcoln1^(-/-) female mice underwent fertility test from 2M to 7M. A subset of them was dissected at 5M on day 3.5 post-coitum (D3.5). The D3.5 ovaries from 5M control,Mcoln1^(-/-), andAtp6v0d2^(-/-)Mcoln1^(-/-) mice were evaluated for CL morphology, lipid droplet staining, and markers of mitochondria and P4 steroidogenesis in the luteal cells.Results: The fertility test ofAtp6v0d2^(-/-)Mcoln1^(-/-) female mice (2M–7M) revealed normal mating activity but reduced fertility compared with the control;yet ~25% of them remained fertile at 5M to 7M but with dystocia. We analyzed a subset of 11Atp6v0d2^(-/-)Mcoln1^(-/-) mice (5M) in the fertility test on D3.5: three (27.3%) had normal P4 levels and all examined CL parameters, indicating full restoration of CL function compared withMcoln1^(-/-), whereas eight had P4 deficiency, with two (18.2%) infertile and six (54.5%) once fertile. In contrast toMcoln1^(-/-) CLs, which had extensive amorphous cellular debris, indicating cell degeneration,Atp6v0d2^(-/-)Mcoln1^(-/-) CLs had reduced amorphous cellular debris regardless of P4 levels. However, similar toMcoln1^(-/-) CLs, P4-deficientAtp6v0d2^(-/-)Mcoln1^(-/-) CLs showed impaired differentiation, enlarged lipid droplets, disorganized expression of endothelial basal lamina marker collagen IV, and reduced expression of mitochondrial marker heat shock protein 60 (HSP60) and steroidogenesis rate-limiting protein StAR, indicating that additionalAtp6v0d2 deficiency compensates forMcoln1 deficiency-induced cell degeneration, but is insufficient to restore luteal cell differentiation and P4 steroidogenesis in P4-deficientAtp6v0d2^(-/-)Mcoln1^(-/-) CLs.Conclusion: This study shows thatAtp6v0d2^(-/-)Mcoln1^(-/-) CLs had varied improvements compared withMcoln1^(-/-) CLs, and it providesin vivo genetic evidence of the coordination between different lysosomal channels in CL function.展开更多
基金funded by NIH R01HD065939(co-funded by ORWH and NICHD)NIH R03HD097384NIH R03 HD100652 to X.Y.
文摘Objective: ATP6V0d2 is a subunit of the vacuolar-type H+-ATPase (V-ATPase) that pumps H+ ions into lysosomes. TRPML1 (MCOLN1/Mcoln1) transports cations out of lysosomes.Mcoln1^(-/-) mice recapitulate the lysosomal storage disorder mucolipidosis type IV (MLIV) phenotype. We previously demonstrated thatMcoln1^(-/-) female mice quickly became infertile at 5 months old (5M) with degenerating corpora lutea (CL) and progesterone (P4) deficiency. We tested our hypothesis thatAtp6v0d2 deficiency could partially compensate forMcoln1 deficiency to restore CL functions inAtp6v0d2^(-/-)Mcoln1^(-/-) mice.Methods: Control andAtp6v0d2^(-/-)Mcoln1^(-/-) female mice underwent fertility test from 2M to 7M. A subset of them was dissected at 5M on day 3.5 post-coitum (D3.5). The D3.5 ovaries from 5M control,Mcoln1^(-/-), andAtp6v0d2^(-/-)Mcoln1^(-/-) mice were evaluated for CL morphology, lipid droplet staining, and markers of mitochondria and P4 steroidogenesis in the luteal cells.Results: The fertility test ofAtp6v0d2^(-/-)Mcoln1^(-/-) female mice (2M–7M) revealed normal mating activity but reduced fertility compared with the control;yet ~25% of them remained fertile at 5M to 7M but with dystocia. We analyzed a subset of 11Atp6v0d2^(-/-)Mcoln1^(-/-) mice (5M) in the fertility test on D3.5: three (27.3%) had normal P4 levels and all examined CL parameters, indicating full restoration of CL function compared withMcoln1^(-/-), whereas eight had P4 deficiency, with two (18.2%) infertile and six (54.5%) once fertile. In contrast toMcoln1^(-/-) CLs, which had extensive amorphous cellular debris, indicating cell degeneration,Atp6v0d2^(-/-)Mcoln1^(-/-) CLs had reduced amorphous cellular debris regardless of P4 levels. However, similar toMcoln1^(-/-) CLs, P4-deficientAtp6v0d2^(-/-)Mcoln1^(-/-) CLs showed impaired differentiation, enlarged lipid droplets, disorganized expression of endothelial basal lamina marker collagen IV, and reduced expression of mitochondrial marker heat shock protein 60 (HSP60) and steroidogenesis rate-limiting protein StAR, indicating that additionalAtp6v0d2 deficiency compensates forMcoln1 deficiency-induced cell degeneration, but is insufficient to restore luteal cell differentiation and P4 steroidogenesis in P4-deficientAtp6v0d2^(-/-)Mcoln1^(-/-) CLs.Conclusion: This study shows thatAtp6v0d2^(-/-)Mcoln1^(-/-) CLs had varied improvements compared withMcoln1^(-/-) CLs, and it providesin vivo genetic evidence of the coordination between different lysosomal channels in CL function.
