NELL2-mediated lumicrine signaling through OVCH2 is required for male fertility

Abstract

The lumicrine system is a postulated signaling system in which testis-derived (upstream) secreted factors enter the male reproductive tract to regulate epididymal (downstream) pathways required for sperm maturation. Until now, no lumicrine factors have been identified. We demonstrate that a testicular germ-cell-secreted epidermal growth factor-like protein, neural epidermal growth factor-like-like 2 (NELL2), specifically binds to an orphan receptor tyrosine kinase, c-ros oncogene 1 (ROS1), and mediates the differentiation of the initial segment (IS) of the caput epididymis. Male mice in which Nell2 had been knocked out were infertile. The IS-specific secreted proteases, ovochymase 2 (OVCH2) and A disintegrin and metallopeptidase 28 (ADAM28), were expressed upon IS maturation, and OVCH2 was required for processing of the sperm surface protein ADAM3, which is required for sperm fertilizing ability. This work identifies a lumicrine system essential for testis-epididymis-spermatozoa (NELL2-ROS1-OVCH2-ADAM3) signaling and male fertility.

Fig. 1.. Testicular NELL2 is indispensable for ROS1 mediated differentiation of epididymal initial segment.

(A) In vitro binding of ROS1 with NELL2 and several other proteins. (B) RT-PCR analyses of Nell2 expression in adult organs and in postnatal testis. Hprt is also shown as internal controls. (C) Litter sizes of Nell2 KO mice. Average and S.D. are shown. (D-E) HE staining of caput epididymis of 14w old WT (D) and Nell2 KO (E) mice. Bars, 50 μm. (F-H) Phospho-ERK immunoblot (F) and immunofluorescence analyses of WT (G) and Nell2 KO (H) 14w caput epididymis. Bars, 50 μm. (I) Expression of transcription factors downstream of ERK signaling in 14w WT and Nell2 KO caput epididymis. RPKM, Reads Per Kilobase per Million.

Fig. 2.. NELL2 is required for ADAM3 maturation and subsequent sperm fertilizing ability.

(A-F) Migration of GFP-tagged WT (A-C) and Nell2 KO (D-F) sperm ejaculated into WT female reproductive tract. (G, H) Sperm-ZP binding assay with WT (G) and Nell2 KO (H) spermatozoa. Bars, 100 μm. (I) Immunoblot detection of cauda epididymal sperm lysates. (J) Immunoblot detection of ADAM3 in TGC, TS (testicular spermatozoa), caput, corpus, and cauda epididymal spermatozoa. (K) Expression of genes encoding proteases in WT and Nell2 KO caput epididymis. Only genes whose average read counts in WT are >100 are shown. (L) Immunoblot analysis of caput epididymal protein lysates from WT, Nell2 KO, and Ros1 KO males.

Fig. 3.. Epididymal secreted protease, OVCH2, is required for ADAM3 processing and male fertility.

(A, B) HE staining of initial segment sections of epididymis from WT (A) and Ovch2 KO (B) mice. Bar, 100 μm. (C) Immunoblot detection of cauda epididymal sperm lysates. (D) Immunoblot detection of ADAM3 in TGC, TS, caput, corpus, and cauda epididymal spermatozoa. (E) Litter size analysis of Ovch2 KO mice. Average and S.D. are shown. (F-K) Migration of dsRed2-tagged WT (F-H) and Ovch2 KO (I-K) spermatozoa ejaculated into WT female reproductive tract. (L, M) Sperm-ZP binding assay with WT (L) and Nell2 (M) KO spermatozoa. Bars, 100 μm.

Fig. 4.. Localization of OVCH2 in the caput epididymis and schematic lumicrine model representing testicular NELL2 regulation of epididymis-dependent sperm maturation.

(A) Precise localization of OVCH2 to the caput epididymis, the region most proximal to the testis. Staining of OVCH2 (magenta), F-actin (green), and nuclei (cyan) staining are shown. Bar, 1 mm. (B) In juvenile males, epididymal IS is undifferentiated. (C) During sexual maturation and in adult males, NELL2 is transported from testis to epididymis through the luminal space. IS epithelial cells are fully differentiated by the NELL2-ROS1-ERK signaling pathway and induces the secretion of proteases including OVCH2. OVCH2 subsequently promotes sperm surface ADAM3 processing, which is indispensable for sperm maturation. (D) In Nell2 KO males, IS differentiation and OVCH2 protease secretion are impaired. As a consequence, ADAM3 processing is aberrant, and males are infertile.