Cysteine dioxygenase is essential for mouse sperm osmoadaptation and male fertility

Abstract

Sperm entering the epididymis are immotile and cannot respond to stimuli that will enable them to fertilize. The epididymis is a highly complex organ, with multiple histological zones and cell types that together change the composition and functional abilities of sperm through poorly understood mechanisms. Sperm take up taurine during epididymal transit, which may play antioxidant or osmoregulatory roles. Cysteine dioxygenase (CDO) is a critical enzyme for taurine synthesis. A previous study reported that male CDO^-/- mice exhibit idiopathic infertility, prompting us to investigate the functions of CDO in male fertility. Immunoblotting and quantitative reverse transcription-polymerase chain reaction analysis of epididymal segments showed that androgen-dependent CDO expression was highest in the caput epididymidis. CDO^-/- mouse sperm demonstrated a severe lack of in vitro fertilization ability. Acrosome exocytosis and tyrosine phosphorylation profiles in response to stimuli were normal, suggesting normal functioning of pathways associated with capacitation. CDO^-/- sperm had a slight increase in head abnormalities. Taurine and hypotaurine concentrations in CDO^-/- sperm decreased in the epididymal intraluminal fluid and sperm cytosol. We found no evidence of antioxidant protection against lipid peroxidation. However, CDO^-/- sperm exhibited severe defects in volume regulation, swelling in response to the relatively hypo-osmotic conditions found in the female reproductive tract. Our findings suggest that epididymal CDO plays a key role in post-testicular sperm maturation, enabling sperm to osmoregulate as they transition from the male to the female reproductive tract, and provide new understanding of the compartmentalized functions of the epididymis.

Keywords: epididymal maturation; fertilization; sperm; taurine.

Figure 1

Characterization of epididymal cysteine deoxygenase (CDO) expression. (A) Histology of mouse epididymides collected from sexually mature mice showing segments 1–8. (B) Comparison of CDO protein expression in the epididymis of mature and immature (4-week-old) male mice. CDO in the liver (Lv) is shown for comparison [22]. Each lane was loaded with 100 μg epididymal proteins. Immunoblotting for CDO revealed that CDO expression (arrow) was abundant in segments 1–3 of the caput region, but was dramatically lower in subsequent segments towards the cauda region. Higher CDO expression was found in mature versus immature mice. CDO expression in mature mice was accompanied by the appearance of the non-cofactor (arrow) and cysteine-tyrosine cross-linked cofactor (arrow head) forms (B, top). In contrast, only the non-cofactor form of CDO was detected in immature mice (B, bottom). (C) CDO mRNA quantification in sections of epididymides from sexually mature mice was consistent with the high CDO expression level in the caput compared with that in the corpus and cauda regions. Data are means ± standard error (SE) of five independent replicates.

Figure 2

CDO localization in the epididymis. Immunohistochemistry was performed in wild-type (WT) (A) and CDO^−/− mice (G, H). The entire epididymis, consisting of segments 1–8 (see Fig. 1), was captured using sets of tiled images. CDO was expressed specifically in the narrow (arrow) and basal cells (arrow head) adjacent to smooth muscle cells in segment 1 (b, inset). CDO was most abundant in segment 2, consistent with Figure 1(B and C). Notably, CDO was localized in both the cytoplasm and nucleus. Segment 3 exhibited less CDO expression compared with that in segment 2 (D). CDO localization was observed in basal cells in segments 3 and 4 (arrowhead; D and E, inset). However, CDO expression in basal cells disappeared in segment 8 (F, inset). As a control for staining specificity, CDO was not detected in either the caput (G) or corpus and cauda regions (H) of CDO^−/− epididymis. Bars = 1 mm (A, G, H), 100 μm(B, D, E, F), 50 μm (C).

Figure 3

In vitro fertilization (IVF) with cumulus intact (CC+) and zona pellucida-free (ZP−) oocytes. (A) CDO^−/− sperm had lower fertilizing ability compared with that of WT sperm using either CC+ or ZP− oocytes (*P < 0.05). Data are means ± SE of five independent replicates for each experiment. (B) Acrosome exocytosis (AE) of WT and CDO^−/− sperm co-incubated with CC+ for 6 h were not significantly different. Data are means ± SE from four independent replicates. (C) Tyrosine phosphorylation of WT and CDO^−/− sperm proteins after incubation under non-capacitating (NC) or capacitating conditions (CAP). No difference was observed in the capacitation-induced phosphorylation profile of WT and CDO^−/− sperm. A representative immunoblot is shown from three replicate experiments. (D) Hyperactivation of WT and CDO^−/− sperm incubated under CAP condition. No difference was observed between genotypes. Data are means ± SE from four independent replicates.

Figure 4

Morphological analysis. The morphologies of WT and CDO^−/− sperm were classified as normal, deformed sperm head (head), midpiece deformed or bent at the distal midpiece or annulus ring (Mid), principal piece deformed or kinked (PP), or containing cytoplasmic droplets (CD). (A) Gross abnormalities were higher in CDO^−/− versus WT sperm (*P < 0.05). Data are means ± SE from five independent replicates. (B) Morphological classification of abnormal sperm. CDO^−/− sperm had a higher rate of head segment abnormalities compared with WT sperm (*P < 0.05). Data are means ± SE.

Figure 5

Quantification of epididymal taurine and hypotaurine contents. (A) Epididymal intraluminal fluid. CDO^−/− mice had significantly less taurine and hypotaurine in the intraluminal fluid compared with WT mice (*P < 0.05). Data are means ± SE from four independent replicates. (B) Sperm cytosolic fraction. Cytosolic taurine and hypotaurine contents were greater in WT versus CDO^−/− sperm. Data are means ± SE of four independent replicates.

Figure 6

Tail angulation assay as a proxy for defects in volume regulation. (A) Tail angulation in WT and CDO^−/− sperm incubated for 2 h under NC conditions (260 mOsm). Following the incubation, more CDO^−/− sperm manifested bent tails compared with WT sperm (*P < 0.05). Data are means ± SE of three independent replicates. (B) Tail angulation in WT and CDO^−/− sperm after a 15-min incubation in TYH of different osmotic pressures (420, 320, or 160 mOsm). To assess whether exogenous taurine could rescue/prevent sperm tail angulation at the lower tonicities, CDO^−/− sperm were also incubated in the presence of 5 mM taurine. The number of sperm with bent tails increased dramatically in CDO^−/− compared with WT sperm when they were incubated at 320 or 160 mOsm (^*P < 0.05, WT versus CDO^−/−). Supplementing with 5 mM taurine completely abolished the increased tail angulation in CDO^−/− sperm at the lower osmotic pressures. Data are means ± SE from five independent replicates for the sperm tail angulation observations under different osmotic pressures, and four independent replicates for the taurine-rescue experiment. (C) IVF of WT and CDO^−/− sperm. To examine the roles of taurine in sperm, CDO^−/− sperm were pre-incubated and inseminated under the presence or absence of 5 mM taurine. CDO^−/− sperm showed lower fertility than WT sperm. However, supplementing taurine to pre-incubation and insemination rescued the impaired fertility (^abP < 0.05). Data are means ± SE from five independent replicates for IVF experiment.