Estrogen promotes Leydig cell engulfment by macrophages in male infertility
Wanpeng Yu, … , Ituro Inoue, Xiangdong Li
Wanpeng Yu, … , Ituro Inoue, Xiangdong Li
Published April 24, 2014
Citation Information: J Clin Invest. 2014;124(6):2709-2721. https://doi.org/10.1172/JCI59901.
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Research Article Endocrinology

Estrogen promotes Leydig cell engulfment by macrophages in male infertility

Abstract

Male infertility accounts for almost half of infertility cases worldwide. A subset of infertile men exhibit reduced testosterone and enhanced levels of estradiol (E2), though it is unclear how increased E2 promotes deterioration of male fertility. Here, we utilized a transgenic mouse strain that overexpresses human CYP19, which encodes aromatase (AROM+ mice), and mice with knockout of Esr1, encoding estrogen receptor α (ERαKO mice), to analyze interactions between viable Leydig cells (LCs) and testicular macrophages that may lead to male infertility. In AROM+ males, enhanced E2 promoted LC hyperplasia and macrophage activation via ERα signaling. E2 stimulated LCs to produce growth arrest–specific 6 (GAS6), which mediates phagocytosis of apoptotic cells by bridging cells with surface exposed phosphatidylserine (PS) to macrophage receptors, including the tyrosine kinases TYRO3, AXL, and MER. Overproduction of E2 increased apoptosis-independent extrusion of PS on LCs, which in turn promoted engulfment by E2/ERα-activated macrophages that was mediated by AXL-GAS6-PS interaction. We further confirmed E2-dependant engulfment of LCs by real-time 3D imaging. Furthermore, evaluation of molecular markers in the testes of patients with nonobstructive azoospermia (NOA) revealed enhanced expression of CYP19, GAS6, and AXL, which suggests that the AROM+ mouse model reflects human infertility. Together, these results suggest that GAS6 has a potential as a clinical biomarker and therapeutic target for male infertility.

Authors

Wanpeng Yu, Han Zheng, Wei Lin, Astushi Tajima, Yong Zhang, Xiaoyan Zhang, Hongwen Zhang, Jihua Wu, Daishu Han, Nafis A. Rahman, Kenneth S. Korach, George Fu Gao, Ituro Inoue, Xiangdong Li

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Figure 5

Estrogen effects on PS asymmetry, apoptosis, MMP, cell proliferation, and scramblase expression in AROM+ LCs and in mLTC-1 cells.

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Estrogen effects on PS asymmetry, apoptosis, MMP, cell proliferation, an...
(A and B) PS exposure on the surface of mLTC-1 LCs (passage 16) was measured by annexin V–FITC staining (x axis) without (A) or with (B) 10 nM E2 treatment, and apoptosis was measured by PI staining (y axis). (C) Dividing annexin V+ mLTC-1 LCs after E2 treatment were observed by phase-contrast microscopy after 36 hours of incubation at 37°C. Scale bar: 100 μm. (D) Pcna mRNA levels, normalized to Rpl19, in annexin V+ mLTC-1 LCs without (Cont) or with 10 nM E2, 10 nM E2 plus 1 μM ICI, 10 nM PPT, or 10 nM DPN treatment. (E) MMP of control (with or without DMSO) or E2-treated mLTC-1 LCs was measured using the JC-1 dye. Phosphatidylcholine-treated cells (PC) were used as positive controls for mitochondrial dissipation. (F) Intracellular [Ca2+] in mLTC-1 cells, treated as in D, was measured using the Ca2+ indicator Fura2/AM. (G) Plscr1–Plscr4 mRNA levels of 5-month-old WT and AROM+ mouse testes, normalized to Rpl19. (H) Plscr1–Plscr4 mRNA levels, normalized to Rpl19, in mLTC-1 cells treated as in D. Each sample was assessed in triplicate, and each experiment was repeated 3 individual times. *P < 0.05, **P < 0.01, ***P < 0.001 vs. respective control (no DMSO), or as indicated by brackets.