Scavenger receptor-B1 and luteal function in mice

Abstract

During luteinization, circulating high-density lipoproteins supply cholesterol to ovarian cells via the scavenger receptor-B1 (SCARB1). In the mouse, SCARB1 is expressed in cytoplasm and periphery of theca, granulosa, and cumulus cells of developing follicles and increases dramatically during formation of corpora lutea. Blockade of ovulation in mice with meloxicam, a prostaglandin synthase-2 inhibitor, resulted in follicles with oocytes entrapped in unexpanded cumulus complexes and with granulosa cells with luteinized morphology and expressing SCARB1 characteristic of luteinization. Mice bearing null mutation of the Scarb1 gene (SCARB1(-/-)) had ovaries with small corpora lutea, large follicles with hypertrophied theca cells, and follicular cysts with blood-filled cavities. Plasma progesterone concentrations were decreased 50% in mice with Scarb1 gene disruption. When SCARB1(-/-) mice were treated with a combination of mevinolin [an inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR)] and chloroquine (an inhibitor of lysosomal processing of low-density lipoproteins), serum progesterone was further reduced. HMGR protein expression increased in SCARB1(-/-) mice, independent of treatment. It was concluded that theca, granulosa, and cumulus cells express SCARB1 during follicle development, but maximum expression depends on luteinization. Knockout of SCARB1(-/-) leads to ovarian pathology and suboptimal luteal steroidogenesis. Therefore, SCARB1 expression is essential for maintaining normal ovarian cholesterol homeostasis and luteal steroid synthesis.

Fig. 1.

Protein expression of SCARB1 in the ovarian theca (A), granulosa (B), cumulus oophorus (C), and luteal cells (D) of mature mice undergoing normal estrous cycles. A–D: Confocal microscopy images of fluorescent immunohistochemistry staining of SCARB1. E–H: Confocal microscopy images of DAPI staining of nuclei. I–L: Merged image of SCARB1 and DAPI. Bars: 10 µm. Images are representative of the ovaries of three or more animals. DAPI, 4′,6-diamidino-2-phenylindole; SCARB1, scavenger receptor-BI.

Fig. 2.

Relative abundance of SCARB1 mRNA (A) (means ± SEM) and protein (B, C) in ovarian cells collected by laser microdissection from ovaries collected at different stages of the estrous cycle from WT mice, as determined by qPCR and immunoblotting. SCARB1, scavenger receptor-BI; WT, wild type.

Fig. 3.

Bright field microscopy images of hematoxylin-eosin stained sections of ovaries from immature mice stimulated with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) and collected 18 h (A, E), 24 h (B, F) or 36 h after hCG (C, G). Mice received 0 (A–D) or 6 mg/g body weight meloxicam, a prostaglandin synthase-2 activity inhibitor (E–H). D: An enlargement of a CL at 36 h after hCG. H: Intact follicle with luteinized granulosa cells and entrapped oocyte at 36 h after hCG in a meloxicam-treated mouse. F insert: An oocyte with expanded cumulus oophorus. Bars: 150 µm. C, cumulus oophorus; CL, corpus luteum; G, granulosa cells, O, oocyte T, theca.

Fig. 4.

Relative abundance of SCARB1 mRNA (means ± SEM) as determined by qPCR in whole ovaries collected 18, 24, or 36 h after hCG of immature mice stimulated with eCG and hCG. Mice received 0 (Control; open bars) or 6 µg/g BW meloxicam, a cyclooxygenase-2 activity inhibitor (Meloxicam; solid bars). eCG, equine chorionic gonadotropin; hCG, human chorionic gonadotropin; SCARB1, scavenger receptor-B1.

Fig. 5.

Protein expression of SCARB1 in representative corpora lutea (A–C, G, H) and in follicles (D–F) of immature mice stimulated with eCG and hCG and collected 18 h (A, D), 24 h(B, E, G) or 36 h after hCG (C, F, H). Mice received 0 (A–C) or 6 µg/g BW meloxicam, a prostaglandin synthase-2 activity inhibitor (D–H). Merged confocal microscopy images of fluorescent immunohistochemistry staining of SCARB1 and DAPI staining of nuclei. Panel C insert: An example of negative control (first antibody replaced by BSA). Bars: 50 µm. eCG, equine chorionic gonadotropin; hCG, human chorionic gonadotropin; SCARB1, scavenger receptor-B1.

Fig. 6.

Plasma progesterone concentrations (LS means ± SEM) from WT or SCARB1^−/− mature mice stimulated with eCG and hCG and collected 18 h after hCG. Mice received placebo injections (Control) or a combination of 20 mg/g BW mevinolin (Mev; an HMGR inhibitor) and 100 mg/g BW chloroquine (Chloro; an inhibitor of lysosomal processing of LDL). hCG, human chorionic gonadotropin; HMGR, 3-hydroxy-3-methylglutaryl CoA reductase; eCG, equine chorionic gonadotropin; hCG, human chorionic gonadotropin; SCARB1, scavenger receptor-B1; WT, wild type.

Fig. 7.

Bright field microscopy images of atypical structures in hematoxylin-eosin-stained (A–C) or periodic acid-Schiff (PAS)-stained (D) sections of ovaries from SCARB1^−/− mature mice stimulated with eCG and hCG and collected 18 h after hCG. A: Follicular cyst-like structure; bar: 200 µm. B: Small luteinized structure; bar: 100 mm. C: Partially luteinized follicle; bar: 50 µm. D: Partially luteinized follicle showing intact basal lamina (arrow); bar: 50 µm. CL, corpus luteum; eCG, equine chorionic gonadotropin; G, granulosa cells; hCG, human chorionic gonadotropin; SCARB1, scavenger receptor-B1; T, theca.

Fig. 8.

Bright field microscopy images of hematoxylin-eosin-stained sections of ovaries from WT (A, C) or SCARB1^−/− mature mice stimulated with eCG and hCG and collected 18 h after hCG. Mice received placebo injections (A, B) or a combination of 20 µg/g BW mevinolin (Mev; an HMGR inhibitor) and 100 µg/g BW chloroquine (Chloro; an inhibitor of lysosomal processing of LDL) (C, D). Bars: 200 µm. CL, corpus luteum; eCG, equine chorionic gonadotropin; F, follicle; hCG, human chorionic gonadotropin; HMGR, 3-hydroxy-3-methylglutaryl CoA reductase; SCARB1, scavenger receptor-B1; WT, wild type.

Fig. 9.

Immunohistochemistry of HMGR, the rate-limiting enzyme in cholesterol synthesis, in corpora lutea from WT (A, C) or SCARB1^−/− mature mice (B, D) stimulated with eCG and hCG and collected 18 h after hCG. Mice received placebo injections (A, B) or a combination of 20 µg/g BW mevinolin (Mev; an HMGR inhibitor) and 100 µg/g BW chloroquine (Chloro; an inhibitor of lysosomal processing of LDL) (C, D). A insert: Negative control (NC) for the SCARB1 immunofluorescence staining reaction where the first antibody was omitted. Bars: 50 µm. eCG, equine chorionic gonadotropin; hCG, human chorionic gonadotropin; HMGR, 3-hydroxy-3-methylglutaryl CoA reductase; SCARB1, scavenger receptor-B1; WT, wild type.