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. 2023 Feb;64(2):100327.
doi: 10.1016/j.jlr.2022.100327. Epub 2022 Dec 31.

Serum opacity factor rescues fertility among female Scarb1-/- mice by reducing HDL-free cholesterol bioavailability

Corina Rosales  1 , Dedipya Yelamanchili  2 , Baiba K Gillard  3 , Jing Liu  2 , Antonio M Gotto Jr  4 , Henry J Pownall  3
Affiliations

Serum opacity factor rescues fertility among female Scarb1-/- mice by reducing HDL-free cholesterol bioavailability

Corina Rosales et al. J Lipid Res. 2023 Feb.

Abstract

Human female infertility, 20% of which is idiopathic, is a public health problem for which better diagnostics and therapeutics are needed. A novel cause of infertility emerged from studies of female mice deficient in the HDL receptor gene (Scarb1). These mice are infertile and have high plasma HDL cholesterol (C) concentrations, due to elevated HDL-free cholesterol (FC), which transfers from HDL to all tissues. Previous studies have indicated that oral delivery of probucol, an HDL-lowering drug, to female Scarb1-/- mice reduces plasma HDL-C concentrations and rescues fertility. Additionally, serum opacity factor (SOF), a bacterial virulence factor, disrupts HDL structure, and bolus SOF injection into mice reduces plasma HDL-C concentrations. Here, we discovered that delivering SOF to female Scarb1-/- mice with an adeno-associated virus (AAVSOF) induces constitutive SOF expression, reduces HDL-FC concentrations, and rescues fertility while normalizing ovary morphology. Although AAVSOF did not alter ovary-FC content, the ovary-mol% FC correlated with plasma HDL-mol% FC in a fertility-dependent way. Therefore, reversing the abnormal plasma microenvironment of high plasma HDL-mol% FC in female Scarb1-/- mice rescues fertility. These data provide the rationale to search for similar mechanistic links between HDL-mol% FC and infertility and the rescue of fertility in women by reducing plasma HDL-mol% FC.

Keywords: HDL receptor gene; Infertility; bacterial virulence factor; cholesterol transport; gene therapy; high-density lipoproteins; ovary morphology; ovary-free cholesterol content; plasma HDL cholesterol; serum opacity factor delivery.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

None
Graphical abstract
Fig. 1
Fig. 1
Serum opacity factor reaction. A: Kinetics of reaction versus HDL from WT and Scarb1−/− mice as labeled. Dashed and solid lines are the experimental data and the data fitted lines, respectively, to a rising exponential fit. HDL (1 mg/ml) and SOF (1 μg/ml) were mixed at 37°C, and opacification was monitored by right-angle light scattering. B–E: SEC analysis of aliquots of the SOF reaction products versus HDL that were collected after completion of the kinetic studies. SOF incubated with HDL from WT (B, D) and Scarb1−/− (C, E) mice. Profiles are expressed as protein absorbance (280 nm, B, C) and cholesterol concentration (D, E). The profiles with red fill are the protein absorbance (280 nm) profiles of HDL before treatment with SOF. SOF, serum opacity factor; SEC, size-exclusion chromatography.
Fig. 2
Fig. 2
SEC of (A) mouse plasma at low (L—), medium (M—), and high (H—) AAVSOF doses. B: SEC of 250 μl HDL (1 mg/ml) after incubation with plasma (5 μl) from mice receiving L-, M-, and H-dose AAVSOF. Gray-fill, HDL. C: Plasma total cholesterol 14 days after M-AAVSOF dosing. ∗P < 0.05. D: PCR analysis of multiple tissues using primers for SOF, LDLR, and GAPDH as labeled for WT mice, Scarb1-/- mice not treated with AAVSOF (NT SKO), and Scarb1-/- mice treated with AAVSOF (SOF SKO). SEC, size-exclusion chromatography; SOF, serum opacity factor.
Fig. 3
Fig. 3
Fertility among female Scarb1-/- mice receiving probucol or AAVSOF. Four markers of fertility were measured in three separate studies in which >10 mice per treatment were followed according to (A) the number of days until the first litter following treatment, (B) number of pups per litter, (C) percent of fertile females, and (D) percent surviving pups. Data are mean ± SD. Other details are in the Materials and methods section.
Fig. 4
Fig. 4
AAVSOF normalizes ovary morphology. Panels show the representative histology of ovaries from (A) WT mice, (B) Scarb1−/− mice, (C) Scarb1−/− receiving AAVSOF. Primordial follicle, PF (black); primary follicle, P1F (blue); secondary follicle, S2F (green); corpus luteum, CL (red). Notably, the corpus luteum, which is not formed in ovaries from Scarb1−/− mice, is restored by AAVSOF. The ages of the mice studied ranged from 12 to 20 weeks and there was no meaningful within-group difference among the ovaries.
Fig. 5
Fig. 5
Lipid compositions of plasma (A–C) and HDL (D–F) from WT and Scarb1-/- mice. As labeled, the mice were grouped as WT, Scarb1−/− untreated, Scarb1−/− treated with AAVSOF and fertile, and Scarb1-/- treated with AAVSOF and infertile. (A) Plasma lipid compositions, (B) Plasma-mol% FC, (C) Plasma-FC/TC weight ratios, (D) HDL lipid compositions, (E) HDL-mol% FC, (F) HDL-FC/TC weight ratios. Mol% plasma FC = 100 × molesFC/(molesFC + molesPL). The respective mean age ± SD and age ranges (weeks) were as follows: WT—20.4 ± 2.5 and 16–23; Scarb1−/−—19.5 ± 3.7 and 14–23; AAVSOF Scarb1−/− Fertile—39.5 ± 4.5 and 27–43; AAVSOF Scarb1−/− Infertile—42.8 ± 1.4 and 41–45. The respective number of mice per group were 12, 11, 12, and 13. FC, free cholesterol; TC, total cholesterol.
Fig. 6
Fig. 6
Lipid compositions of ovaries from WT and Scarb1−/− mice. As labeled, the mice were grouped as WT, Scarb1−/−, Scarb1−/− receiving AAVSOF and fertile, and Scarb1−/− receiving AAVSOF and infertile. (A) Total protein per ovary, (B) ovary lipid compositions, (C) ovary mol% FC, (D) ovary FC/TC weight ratios. (E) Ovary CE content versus HDL-CE (m = −19 + 11; r2 = >0.05; P = 0.09), (F) Ovary FC content versus HDL-FC (m = 0.22 + 2.4; r2 = 0.33; P = 0.37), (G) Ovary mol% FC versus HDL mol% FC (m = 0.51 + 0.11; r2 = 0.33; P < 0.0001). Ovary mol%-FC = 100 × molesFC/(molesFC + molesPL). Ages and number of mice per group are the same as in Figure 5 legend. FC, free cholesterol; TC, total cholesterol.
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