Mammalian sperm translate nuclear-encoded proteins by mitochondrial-type ribosomes

Abstract

It is widely accepted that spermatozoa are translationally silent. The present study demonstrates, for the first time, incorporation of labeled amino acids into polypeptides during sperm capacitation, which was completely inhibited by mitochondrial translation inhibitors but not by the cytoplasmic translation inhibitor. Unlike 80S cytoplasmic ribosomes, 55S mitochondrial ribosomes were present in polysomal fractions, indicating that these ribosomes are actively involved in protein translation in spermatozoa. Inhibition of protein translation significantly reduced sperm motility, capacitation and in vitro fertilization rate. Thus, contrary to the accepted dogma, nuclear genes are expressed as proteins in sperm during their residence in the female reproductive tract until fertilization.

Figure 1.

Protein synthesis in spermatozoa. (A) Autoradiogram of an electrophoretic gel with [³⁵S]Met–[³⁵S]Cys-labeled polypeptides from human, bovine, mouse, and rat sperm incubated for 6, 4, 3, and 3 h, respectively. Sperm were incubated under capacitation conditions with no additional treatment (C) or after treatment with CP (0.1 mg/mL) or CH(1 mg/mL). (B) Fluorescence photograph of bovine sperm with BODIPY-lysine-tRNA^Lys-labeled polypeptides photographed at 0 h (panel 1), or after 1 h capacitation without (panels 2,4) or with (panel 3) CP treatment. Bar: panels 1–3, 40 μm; panel 4, 10 μm. (C) Time course of [³⁵S]Met–[³⁵S]Cys incorporation into polypeptides in bovine sperm under capacitation conditions. (D) Immunoprecipitation of [³⁵S]Met–[³⁵S]Cys-labeled: angiotensin II type I receptor (AT₁-R) (lanes 1), progesterone receptor (PR) (lanes 2), and PKCα after 4 h under capacitation conditions with no additional treatment (C) or after treatment with CP (0.1 mg/mL) or CH (1 mg/mL) (lanes 3). (E) Immunocytochemistry of AT₁-R (row 1) and PR (row 2) in bovine sperm at 0 h and at 4 h of capacitation ± CP (0.1 mg/mL). Bar, 20 μm.

Figure 2.

Identification and localization of mRNAs and proteins in sperm. (A) RT–PCR of sperm-related mRNA. Sperm RNA was purified and amplified by RT–PCR using specific primers (Supplementary Table S2). PCR products are mouse PKA-Cs (lane 1), bovine PKA-Cs (lane 2), human PKCα (lane 3), bovine AKAP 110 (lane 4), human PKCβI (lane 5), mouse CatSper (lane 6), human CatSper (lane 7), mouse CatSper II (lane 8), human ATPase α4 (lane 9), rat ATPase α4 (lane 10), human AT₁-R (lane 11), bovine AT₁-R (lane 12), and control—PCR with mRNA but without RT (lane 13). (B) Immunostaining of CatSper mRNA in mouse sperm by in situ hybridization. The head (H), midpiece (M), and principal piece (PP) regions of the sperm are indicated. (C) Immunogold localization of bovine AT1-R (top row), mouse Catsper (middle row), and mouse PKA-Cs (bottom row) mRNAs and proteins by electron microscopy. Cross-section is through the midpiece of bovine and mouse sperm, and the arrows indicate gold particles localized inside the mitochondria. Bar, 0.2 μm.

Figure 3.

Identification of active sperm ribosomes. Sperm ribosomal fraction was allowed to sediment using sucrose gradient, then the optical density tracing at 260 nm was determined, 20 fractions were collected, and the absorbance measured at 260 nm in each fraction was measured again. RNA was isolated from each fraction and treated with DNase. All fractions were detected for the presence of rRNAs. 12S rRNA of the 55S mitochondrial ribosome (a) and 18S rRNA of the 80S cytoplasmic ribosomes (b) were identified by RT–PCR at the linear phase of the PCR. (c) mRNA of AKAP 110 was identified by RT–PCR. The S values were determined using standards for 55S mitoribosomes and 80S cytoplasmic ribosomes from HeLa cells.

Figure 4.

Inhibition of sperm functions by CP. Bovine sperm were incubated in capacitation medium for the specified times with (gray columns) or without (white column) 0.1 mg/mL CP. At the end of the incubation, sperm motility, actin polymerization, acrosome reaction, and in vitro fertilization rates were determined. Data are expressed as percentage of control ± SD from four independent experiments.