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
The lysosome is the most acidic membrane-bound intracellular organelle.Lysosomal acidity is primarily maintained by vacuolar H+-ATPase(V-ATPase)and counter ion channels.There are>60 hydrolytic enzymes in the lysoso...The lysosome is the most acidic membrane-bound intracellular organelle.Lysosomal acidity is primarily maintained by vacuolar H+-ATPase(V-ATPase)and counter ion channels.There are>60 hydrolytic enzymes in the lysosome for its fundamental digestive role.Lysosomes also play important roles in endocytosis,exocytosis,autophagy,and cell death.Studies that have implicated roles of lysosomes in the female reproductive system are reviewed here.In the ovary,lysosomes are implicated in the preparation of free cholesterol for steroidogenesis and degradation of regulators of steroidogenesis,regulation of follicular atresia,follicle rupture during ovulation,luteal cell survival,and luteal regression.In the oviduct,lysosomes are involved in endocytosis of both serum and oviductal luminal components.In the uterus during the menstrual/estrous cycle,lysosomes are associated with endometrial secretion,apoptosis,and menstruation.In the uterus during early pregnancy,lysosomes are involved in the temporal and directional changes of endocytosis,uterine epithelial acidification upon embryo implantation initiation,and embryo-maternal mutual communications via extracellular vesicles.In the placenta,lysosomes are implicated in nutrient transport and placental separation from the uterus for parturition.In the mammary gland,lysosomes are important for mammary gland development and involution.These findings suggest/demonstrate functions of lysosomes in multiple processes of female reproduction,from ovulation to ovarian steroidogenesis for pregnancy maintenance,and from essential in utero nurturing of developing embryos/fetuses via embryo/fetal-maternal communications,to optional postpartum nurturing of newborns via lactation.Future studies using genetically or modified animal models and pharmacological approaches will provide novel insights into the functions and mechanisms of lysosomes in the mammalian female reproductive system.展开更多
Objective:FemaleMcoln1^(-/-)mice exhibit progressive progesterone(P4)deficiency,luteal cell degeneration,and premature embryo implantation failure at 5 months old.We attempted to rescue embryo implantation in non-virg...Objective:FemaleMcoln1^(-/-)mice exhibit progressive progesterone(P4)deficiency,luteal cell degeneration,and premature embryo implantation failure at 5 months old.We attempted to rescue embryo implantation in non-virginMcoln1^(-/-)mice(5-6 months old)with exogenous P4 treatment on days 1.5 post-coitum(D1.5),D2.5,and D3.5,and observed partially restored luteal cell morphology on D4.5,but unexpectedly found 17β-estradiol(E2)contamination in the P4 working solution.In this study,we aim to investigate exogenous P4 and/or E2 for the partial recovery of luteal cell morphology in infertileMcoln1^(-/-)mice.Methods:Control and non-virginMcoln1^(-/-)mice(5-6 months old)were treated with newly ordered vehicle,P4,E2,or P4+E2 on D1.5 and D2.5 and dissected on D3.5 for P4 and E2 measurements,ovary histology,immunofluorescence,lipid droplet staining,and transmission electron microscopy.Results:E2 treatment significantly increased serum P4 levels in D3.5Mcoln1^(-/-)mice.E2 and P4+E2 treatments,but not P4 treatment alone,largely improved the morphology of D3.5Mcoln1^(-/-)corpora lutea,indicated by a more contiguous web-like collagen IV expression pattern,increased heat shock protein 60 expression,and reduced accumulation of large lipid droplets.Transmission electron microscopy revealed extremely enlarged autophagosomes and lipid droplets,lysosomes with lamellar structures,and mitochondria with reduced cristae in vehicle-treated D3.5Mcoln1^(-/-)luteal cells,while in E2-treated D3.5Mcoln1^(-/-)luteal cells,extremely enlarged autophagosomes and lipid droplets were reduced,indicating improved luteal cell ultrastructure.Conclusion:These findings reveal protective effects of high levels of exogenous E2 on P4 production and lysosomal function inMcoln1^(-/-)luteal cells.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.
基金support from Interdisciplinary Toxicology Program,Department of Physiology and Pharmacology,College of Veterinary Medicine,and Office of the Vice President for Research at University of Georgia,as well as the National Institutes of Health(R01HD065939(co-funded by ORWH and NICHD),R03HD097384,and R03HD100652).
文摘The lysosome is the most acidic membrane-bound intracellular organelle.Lysosomal acidity is primarily maintained by vacuolar H+-ATPase(V-ATPase)and counter ion channels.There are>60 hydrolytic enzymes in the lysosome for its fundamental digestive role.Lysosomes also play important roles in endocytosis,exocytosis,autophagy,and cell death.Studies that have implicated roles of lysosomes in the female reproductive system are reviewed here.In the ovary,lysosomes are implicated in the preparation of free cholesterol for steroidogenesis and degradation of regulators of steroidogenesis,regulation of follicular atresia,follicle rupture during ovulation,luteal cell survival,and luteal regression.In the oviduct,lysosomes are involved in endocytosis of both serum and oviductal luminal components.In the uterus during the menstrual/estrous cycle,lysosomes are associated with endometrial secretion,apoptosis,and menstruation.In the uterus during early pregnancy,lysosomes are involved in the temporal and directional changes of endocytosis,uterine epithelial acidification upon embryo implantation initiation,and embryo-maternal mutual communications via extracellular vesicles.In the placenta,lysosomes are implicated in nutrient transport and placental separation from the uterus for parturition.In the mammary gland,lysosomes are important for mammary gland development and involution.These findings suggest/demonstrate functions of lysosomes in multiple processes of female reproduction,from ovulation to ovarian steroidogenesis for pregnancy maintenance,and from essential in utero nurturing of developing embryos/fetuses via embryo/fetal-maternal communications,to optional postpartum nurturing of newborns via lactation.Future studies using genetically or modified animal models and pharmacological approaches will provide novel insights into the functions and mechanisms of lysosomes in the mammalian female reproductive system.
基金the Offfce of the Vice President for Research,Interdisciplinary Toxicology Program,and Department of Physiology and Pharmacology at the University of Georgia,and the National Institutes of Health(grants NIH R01HD065939[co-funded by ORWH and NICHD],R03HD097384,and R03 HD100652 to X.Y.)for ffnancial supportSerum P4 and E2 levels were determined at The University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core,which is supported by the Eunice Kennedy Shriver NICHD/NIH(NCTRI)Grant P50-HD28934.
文摘Objective:FemaleMcoln1^(-/-)mice exhibit progressive progesterone(P4)deficiency,luteal cell degeneration,and premature embryo implantation failure at 5 months old.We attempted to rescue embryo implantation in non-virginMcoln1^(-/-)mice(5-6 months old)with exogenous P4 treatment on days 1.5 post-coitum(D1.5),D2.5,and D3.5,and observed partially restored luteal cell morphology on D4.5,but unexpectedly found 17β-estradiol(E2)contamination in the P4 working solution.In this study,we aim to investigate exogenous P4 and/or E2 for the partial recovery of luteal cell morphology in infertileMcoln1^(-/-)mice.Methods:Control and non-virginMcoln1^(-/-)mice(5-6 months old)were treated with newly ordered vehicle,P4,E2,or P4+E2 on D1.5 and D2.5 and dissected on D3.5 for P4 and E2 measurements,ovary histology,immunofluorescence,lipid droplet staining,and transmission electron microscopy.Results:E2 treatment significantly increased serum P4 levels in D3.5Mcoln1^(-/-)mice.E2 and P4+E2 treatments,but not P4 treatment alone,largely improved the morphology of D3.5Mcoln1^(-/-)corpora lutea,indicated by a more contiguous web-like collagen IV expression pattern,increased heat shock protein 60 expression,and reduced accumulation of large lipid droplets.Transmission electron microscopy revealed extremely enlarged autophagosomes and lipid droplets,lysosomes with lamellar structures,and mitochondria with reduced cristae in vehicle-treated D3.5Mcoln1^(-/-)luteal cells,while in E2-treated D3.5Mcoln1^(-/-)luteal cells,extremely enlarged autophagosomes and lipid droplets were reduced,indicating improved luteal cell ultrastructure.Conclusion:These findings reveal protective effects of high levels of exogenous E2 on P4 production and lysosomal function inMcoln1^(-/-)luteal cells.
