Atp6v0d2 deficiency partially restores defects in Mcoln1-deficient mouse corpus luteum

Abstract

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 that Mcoln1 ^-/- female mice quickly became infertile at 5 months old (5M) with degenerating corpora lutea (CL) and progesterone (P4) deficiency. We tested our hypothesis that Atp6v0d2 deficiency could partially compensate for Mcoln1 deficiency to restore CL functions in Atp6v0d2 ^-/- Mcoln1 ^-/- mice.

Methods: Control and Atp6v0d2 ^-/- 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 ^-/-, and Atp6v0d2 ^-/- 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 of Atp6v0d2 ^-/- 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 11 Atp6v0d2 ^-/- 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 with Mcoln1 ^-/-, whereas eight had P4 deficiency, with two (18.2%) infertile and six (54.5%) once fertile. In contrast to Mcoln1 ^-/- CLs, which had extensive amorphous cellular debris, indicating cell degeneration, Atp6v0d2 ^-/- Mcoln1 ^-/- CLs had reduced amorphous cellular debris regardless of P4 levels. However, similar to Mcoln1 ^-/- CLs, P4-deficient Atp6v0d2 ^-/- 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 additional Atp6v0d2 deficiency compensates for Mcoln1 deficiency-induced cell degeneration, but is insufficient to restore luteal cell differentiation and P4 steroidogenesis in P4-deficient Atp6v0d2 ^-/- Mcoln1 ^-/- CLs.

Conclusion: This study shows that Atp6v0d2 ^-/- Mcoln1 ^-/- CLs had varied improvements compared with Mcoln1 ^-/- CLs, and it provides in vivo genetic evidence of the coordination between different lysosomal channels in CL function.

Keywords: ATP6V0D2/Atp6v0d2; Atp6v0d2−/−Mcoln1−/− mice; Corpus luteum; Progesterone; TRPML1/Mcoln1.

Fig. 1.

Fertility test in Atp6v0d2^−/−Mcoln1^−/− female mice. (A) Plugging latency from cohabitation to detection of the first vaginal plug. Control: gray dots, n = 29; Atp6v0d2^−/−Mcoln1^−/−: red triangle, n = 40. (B) Pregnancy rates at 2M, 5M, and 6M–7M. Pregnancy age was based on the time when a vaginal plug was detected. *P <0.05, compared with the respective control group; #P <0.05, compared with the 2M Atp6v0d2^−/−Mcoln1^−/− group. Control: n = 29/29 (2M), 16/17 (5M), and 9/9 (6M–7M); Atp6v0d2^−/−Mcoln1^−/−: n = 29/40 (2M), 5/18 (5M), and 3/13 (6M–7M). The reduction of 12 mice in the control cohort from 2M to 5M was attributable to previous pregnancy and nursing (three mice) therefore not in mating; sickness therefore euthanized (one mouse); dissection for D3.5 serum collection at 5M (four mice); and being sacrificed following COVID-19 lockdown policy (four mice). The reduction of eight mice in the control cohort from 5M to 6–7M was because of previous pregnancy and nursing (one mouse) therefore not in mating; and being sacrificed following COVID-19 lockdown policy (seven mice). The reduction of 22 mice in the Atp6v0d2^−/−Mcoln1^−/− cohort from 2M to 5M was because of previous pregnancy and nursing (one mouse) therefore not in mating; death (one mouse); dissection for D3.5 serum collection at 5M (11 mice); and being sacrificed following COVID-19 lockdown policy (nine mice). The reduction of five mice in the Atp6v0d2^−/−Mcoln1^−/− cohort from 5M to 6M–7M was because of death before 6M (two mice); and being sacrificed following COVID-19 lockdown policy (three mice). (C) Dystocia rates at 2M, 5M, and 6M–7M. *P <0.05, compared with the respective control group; #P <0.05, compared with the 2M Atp6v0d2^−/−Mcoln1^−/− group. (D–F) Body weight change during pregnancy at pregnancy ages of 2M (D), 5M (E), and 6M–7M (F). The numbers of pregnant mice with body weight data were 22, 11, and 5 for the control groups and 26, 4, and 3 for the Atp6v0d2^−/−Mcoln1^−/− groups, respectively. One missing data point in (E) was because of incomplete collection of the body weight data. Error bar, standard deviation; *P <0.05, compared with the respective control group at the same post-coitum day, two-tailed unequal variance student t test; or P = 0.001 (2M, D), P <0.001 (5M, E), and P <0.001 (6M–7M, F), two-way analysis of variance.

Fig. 2.

Serum P4 level (A) and estrogen (E₂) level (B). A subset of mice at 5M was randomly selected from the cohort in the fertility test. Serum was collected at post-coitum day 3.5 (D3.5). n = 4 for control mice, black dots; and n = 11 for Atp6v0d2^−/−Mcoln1^−/− mice, red triangles. Atp6v0d2^−/−Mcoln1^−/− mice were divided into three subgroups as follows: low P4 and infertile (n = 2), low P4 and once fertile (n = 6), and normal P4 (n = 3). P4: progesterone.

Fig. 3.

Histology of CL and Col IV immunofluorescence in the ovaries. Ovaries were from D3.5 5M mice that were in the fertility cohort (control and Atp6v0d2^−/−Mcoln1^−/−) or not in the fertility cohort but were non-virgin (Mcoln1^−/−). Five groups of mice were included: control mice, Mcoln1^−/− mice, and three subgroups of Atp6v0d2^−/−Mcoln1^−/− mice (low P4 and infertile, low P4 and once fertile, and normal P4). (A–H1) Hematoxylin and eosin staining in fixed ovaries. Black arrow in (B1) and (C1), amorphous cellular debris; (F1) and (G1), regressing CL from a previous cycle; red arrow in (F1) and (G1), empty cytoplasm; green arrow in (F1) and (G1), dense nucleus. (I) Percentage of mice with extensive amorphous cell debris in the CL of the control, Mcoln1^−/−, and Atp6v0d2^−/−Mcoln1^−/− groups. n = 4–6/group; * P <0.05, compared with both the control and Atp6v0d2^−/−Mcoln1^−/− groups. (J–N1) Col IV staining in frozen ovaries. (A, F, J) Control; (B, G, K) Mcoln1^−/−; (C, H, L) Atp6v0d2^−/−Mcoln1^−/− low P4 and infertile; (D, M) Atp6v0d2^−/−Mcoln1^−/− low P4 and once fertile; (E, N) Atp6v0d2^−/−Mcoln1^−/− normal P4. (A1–H1) and (J1–N1) enlarged from the boxed area in (A–H) and (J–N), respectively. Scale bar: 400 µm in (F) and (G); 200 µm in (A–E), (H), (J–N); and 25 µm in (A1–H1) and (J1–N1); *CL; green * in M, most likely a regressing CL from a previous cycle, with lower Col IV staining than other CLs in the section but higher than that in follicles (red * in M). No specific staining in the follicle or CL but autofluorescence in the interstitial areas of the negative control ovaries (data not shown). CL: corpus luteum; Col IV: collagen IV; DAPI: 4′,6′-diamino-2-phenylindole; P4: progesterone.

Fig. 4.

Nile red staining of lipid droplets and immunofluorescence detection of HSP60 and StAR in frozen ovaries from 5M mice at D3.5. Five groups of mice were included: control mice, Mcoln1^−/− mice, and three groups of Atp6v0d2^−/−Mcoln1^−/− mice (low P4 and infertile, low P4 and once fertile, and normal P4). (A–E1) Nile red staining. (F–J1) HSP60. (K–O1) StAR. (A, F, K) Control; (B, G, L) Mcoln1^−/−; (C, H, M) Atp6v0d2^−/−Mcoln1^−/− low P4 and infertile; (D, I, N) Atp6v0d2^−/−Mcoln1^−/− low P4 and once fertile; (E, J, O) Atp6v0d2^−/−Mcoln1^−/− normal P4 level. (A1–O1) Enlarged from the CL labeled with * in (A–O), respectively. Scale bar, 400 µm in (A–O), and 25 µm in (A1–O1). No specific staining in the follicle or CL but autofluorescence in the interstitial areas of the negative control ovaries (data not shown). CL: corpus luteum; HSP60: heat shock protein 60; P4: progesterone; StAR: steroidogenic acute regulatory protein.

Fig. 5.

Proposed model for varied rescuing effects in Atp6v0d2^−/−Mcoln1^−/− CL compared with Mcoln1^−/− CL. Atp6v0d2 encodes one of the two ATP6V0d subunits for V-ATPase, which pumps H⁺ from the cytosol to the lysosomal lumen. Mcoln1 encodes TRPML1, a cation counter ion channel that transports cations, including H⁺, from the lysosomal lumen to the cytosol. TRPML1 deficiency leads to MLIV, a progressive and severe lysosomal storage disorder with a slow onset. The proposed model is for D3.5 CLs from 5M mice that have been in mating since 2M. (A) Control. Balanced lysosomal lumen ionic homeostasis, indicated by the green lumen. (B) Mcoln1^−/−. Imbalanced lysosomal ionic components, expected to be caused by accumulation of cations in the lysosomal lumen resulted from TRPML1 deficiency, indicated by the white lumen. (C) Atp6v0d2^−/−Mcoln1^−/−. Gradient rescue of lysosomal lumen ionic homeostasis by additional ATP6V0d2 deficiency. In the CLs where ATP6V0d2 deficiency leads to more impairment of V-ATPase activity in pumping H⁺ in the lysosomal lumen, the imbalance caused by TRPML1 deficiency could be canceled out to reach a new balance for the lysosome to be fully functional, indicated by gradient reddish V-ATPase (more impairment), grayish arrow (less H⁺ pumping), and greenish lysosomal lumen (functional lysosome). In the CLs where ATP6V0d2 deficiency has no or minor impairment of V-ATPase activity, the imbalance caused by TRPML1 deficiency could not or could only be partially corrected, indicated by gradient whitish V-ATPase, reddish arrow, and white lysosomal lumen. CL: corpus luteum; MLIV: mucolipidosis type IV; TRPML1: transient receptor potential cation channel, mucolipin subfamily, member 1.