Association of luteal cell degeneration and progesterone deficiency with lysosomal storage disorder mucolipidosis type IV in Mcoln1-/- mouse model†

Abstract

Transient receptor potential cation channel, mucolipin subfamily, member 1 (TRPML1) (MCOLN1/Mcoln1) is a lysosomal counter ion channel. Mutations in MCOLN1 cause mucolipidosis type IV (MLIV), a progressive and severe lysosomal storage disorder with a slow onset. Mcoln1-/- mice recapitulate typical MLIV phenotypes but roles of TRPML1 in female reproduction are unknown. Despite normal mating activities, Mcoln1-/- female mice had reduced fertility at 2 months old and quickly became infertile at 5 months old. Progesterone deficiency was detected on 4.5 days post coitum/gestation day 4.5 (D4.5). Immunohistochemistry revealed TRPML1 expression in luteal cells of wild type corpus luteum (CL). Corpus luteum formation was not impaired in 5-6 months old Mcoln1-/- females indicated by comparable CL numbers in control and Mcoln1-/- ovaries on both D1.5 and D4.5. In the 5-6 months old Mcoln1-/- ovaries, histology revealed less defined corpus luteal cord formation, extensive luteal cell vacuolization and degeneration; immunofluorescence revealed disorganized staining of collagen IV, a basal lamina marker for endothelial cells; Nile Red staining detected lipid droplet accumulation, a typical phenotype of MLIV; immunofluorescence of heat shock protein 60 (HSP60, a mitochondrial marker) and in situ hybridization of steroidogenic acute regulatory protein (StAR, for the rate-limiting step of steroidogenesis) showed reduced expression of HSP60 and StAR, indicating impaired mitochondrial functions. Luteal cell degeneration and impaired mitochondrial functions can both contribute to progesterone deficiency in the Mcoln1-/- mice. This study demonstrates a novel function of TRPML1 in maintaining CL luteal cell integrity and function.

Keywords: StAR; TRPML1/Mcoln1; corpus luteum; heat shock protein 60; lipid accumulation; progesterone.

Figure 1

Normal mating activity but impaired fertility in Mcoln1^−/− female mice. (A) Plugging rate. N = 30 (2M), 10 (4M), or 7–8 (5M). (B) Plugging latency from cohabitation to detection of a vaginal plug. Each black dot (C, control, Mcoln1^+/+ and Mcoln1^+/−) or red triangle (−/−, Mcoln1^−/−): an individual mouse; 2M, 4M, 5M: 2, 4, and 5 months old. (C) Term pregnancy rate (2M, 4M) or implantation rate on gestation day 4.5 (D4.5) (5M) in the mated mice. ^* P < 0.05. (B and C) N = 28–30 (2M), 9–10 (4M), or 7–8 (5M). (D) Number of pups at birth (2M, 4M) or number of implantation sites on 4.5 days post coitum/gestation day 4.5 (D4.5, 5M) in the mated mice. The number on top of each bar: the number of mice delivered pups (2M and 4M) or with implantation sites (5M)/the total number of mated mice in each group; black for control and red for Mcoln1^−/−; ^* P < 0.05; error bar, standard deviation.

Figure 2

Serum progesterone (P4) and estrogen (E2) levels in D4.5 control and Mcoln1^−/− female mice at 5 months (5M) old. Each dot or triangle: an individual mouse from fertility test at 5 months old in Figure 1C; black dot or triangle for P4 levels and blue dot or triangle for E2 levels; red rectangle: median for each group; C: control, Mcoln1^+/+ and Mcoln1^+/−, N = 8; −/−: Mcoln1^−/−, N = 7; ^* P < 0.05. Note: seven out of eight control serum samples had P4 levels exceeding the detection limit of 40 ng/ml.

Figure 3

Numbers and histology of CLs at 5–6 months old. (A) Numbers of CLs in control (Con) and Mcoln1^−/− ovaries on D1.5 and D4.5. N = 4 (D1.5) and 10–20 (D4.5); error bar, standard deviation. (B, B1, B2) Control. (C, C1, C2) Mcoln1^−/−. (B1 and C1) Corpus luteum enlarged from (B) and (C), respectively; (B2 and C2) Luteal cells enlarged from (B1) and (C1), respectively; scale bar: 400 μm (B–C), 100 μm (B1–C1), or 25 μm (B2–C2); yellow arrow in (C2): cell debris; black arrow in (C2): vacuolated cell. H & E staining.

Figure 4

Detection of TRPML1 in D3.5 wild type mouse ovary using immunohistochemistry. (A) Negative control without primary antibody. (B–F) Transient receptor potential cation channel, mucolipin subfamily, member 1 expression in the ovary, with enlarged view of a primary follicle (C), a pre-antral follicle (D), an antral follicle (E), and a corpus luteum (F). CL: corpus luteum; Lu: luteal cells; Gr: granulosa cells; Te: theca cells; Ge: germinal epithelium; scale bar: 100 μm (A and B) or 20 μm (C and F); TRPML1 signal: brown staining; hematoxylin counterstaining: blue.

Figure 5

Immunofluorescence detection of collagen IV (Col IV) in corpra lutea of D3.5 control and Mcoln1^−/− mice. (A) Control ovary. (B) Mcoln1^−/− ovary. (A1 and B1) representative images of a corpus luteum enlarged from A and B, respectively; green: Col IV staining; blue: DAPI staining of nuclei; red #: corpus luteum; white ^*: follicle; scale bar: 200 μm (A–B) or 50 μm (A1–B1). (C) Relative intensity of Col IV staining in the CL/interstitial tissue in the ovary. N = 3–5; ^* P < 0.05; error bar, standard deviation.

Figure 6

Nile red staining of lipid droplets in D3.5 control and Mcoln1^−/− ovaries. (A) Control ovary. (B) A Mcoln1^−/− ovary with mild lipid accumulation. (C) A Mcoln1^−/− ovary with severe lipid accumulation. (A1–C1) Representative images of luteal cells enlarged from (A) to (C), respectively; green: Nile red staining of lipid droplets; blue, DAPI staining of nuclei; red #: corpus luteum; white ^*: follicle; scale bar: 400 μm (A–C) or 12.5 μm (A1–C1). (D) Number of lipid droplets in a standard reference frame (1041×780 pixel image). (E) Size of lipid droplets (square pixels). (D and E) N = 5; ^* P < 0.05; error bar, standard deviation.

Figure 7

Immunofluorescence detection of a mitochondrial marker heat shock protein 60 (HSP60) in D4.5 control and Mcoln1^−/− ovaries. A. Control ovary. B. Mcoln1^−/− ovary. A1 and B1: representative images of CLs enlarged from A and B, respectively; green: HSP60 staining; red #: corpus luteum; white ^*: follicle; scale bar: 400 μm (A–B) or 50 μm (A1–B1). C. Relative intensity of HSP60 staining in the CL/interstitial compartment in the ovary. N=3–4; ^*P < 0.05, compared to age-matched control; error bar, standard deviation.

Figure 8

Detection of StAR mRNA expression in D4.5 ovaries by in situ hybridization using a StAR antisense probe. A. StAR expression in an ovary of a 3 months old pseudo-pregnant control mouse. B. StAR expression in an ovary of a 3 months old pregnant Mcoln1^−/− mouse. C. StAR expression in an ovary of a 5 months old non-pregnant Mcoln1^−/− mouse. A1-C1: Enlarged from the boxed area in A–C, respectively; red #: corpus luteum; black ^*: follicle; scale bar: 200 μm (A–C) or 50 μm (A1–C1); no specific staining in a control section using a StAR sense probe (data not shown).