Flotillin-2 is an acrosome-related protein involved in mouse spermiogenesis

Abstract

SPERMATOGENESIS IS A COMPLEX PROCESS OF TERMINAL DIFFERENTIATION BY WHICH MATURE SPERMS ARE GENERATED, AND IT CAN BE DIVIDED INTO THREE PHASES: mitosis, meiosis and spermiogenesis. In a previous study, we established a series of proteomic profiles for spermatogenesis to understand the regulation of male fertility and infertility. Here, we further investigated the localization and the role of flotillin-2 in spermiogenesis. Flotillin-2 expression was investigated in the testis of male CD1 mice at various developmental stages of spermatogenesis by using Western blotting, immunohistochemistry and immunofluorescence. Flotillin-2 was knocked down in vivo in three-week-old male mice using intratesticular injection of small inhibitory RNA (siRNA), and sperm abnormalities were assessed three weeks later. Flotillin-2 was expressed at high levels in male germ cells during spermatogenesis. Flotillin-2 immunoreactivity was observed in pachytene spermatocytes as a strong dot-shaped signal and in round spermatids as a sickle-shaped distribution ahead of the acrosome. Immunofluorescence confirmed flotillin-2 was localized in front of the acrosome in round spermatids, indicating that flotillin-2 was localized to the Golgi apparatus. Knockdown of flotillin-2 in vivo led to a significant increase in head sperm abnormalities isolated from the cauda epididymis, compared with control siRNA-injected testes. This study indicates that flotillin-2 is a novel Golgi-related protein involved in sperm acrosome biogenesis.

Keywords: Golgi apparatus; RNA interference; acrosome biogenesis; flotillin-2.

Fig. 1. Western blotting analysis of flotillin-2 protein expression at different developmental stages in the mouse testis.

Flotillin-2 is expressed in the testis during the first wave of spermatogenesis. Similar results were observed in triplicate experiments.

Fig. 2. Immunohistochemical localization of flotillin-2 in the mouse testis at different developmental stages.

Spematogonia in the testis of 4 d (A) and primary spermatocytes in the testis of 10 d (B) old mice displayed weak flotillin-2 immunoreactivity. A flotillin-2 dot-shaped immunoreactive signal was obvious in the pachytene spermatocytes of 3 weeks old mice (C), and as the round spermatids appeared, the signal elongated ahead of the acrosome. Flotillin-2 immunoreactivity was similar in the testis of 4 weeks old mice (D) and in adult mice (E), it was observed at high levels in pachytene spermatocytes as a strong dot-shaped signal and also in round spermatids as a sickle-shaped distribution in the acrosome. Control (F) showed negative signal. All scale bars are 10 µm.

Fig. 3. Flotillin-2 is located ahead of the developing acrosome.

Immunofluorescent analysis of flotillin-2 in the testis of adult mouse. A: Flotillin-2 (green); B: PNA (red); C: merge; D: Higher magnification image. All scale bars are 10 μm.

Fig. 4. Efficiency of flotillin-2 siRNAs in GC2-spd cells.

Western blotting analysis(A and B) of the effect of siRNA (MSS204354; Invitrogen) on flotillin-2 protein expression at 72 h. The data represent an average of three independent experiments, and are expressed as fold changes relative to the negative control. C: Immunofluorescent analysis of the effect of siRNA (MSS204354) on flotillin-2 protein expression at 72 h. All scale bars are 10 µm. Similar results were observed in three independent experiments.

Fig. 5. Efficiency of flotillin-2 siRNAs in vivo.

A: Trypan blue staining was used as a marker of siRNA injection; 30–40% of the seminiferous tubules were injected with siRNA. B: Trypan blue staining (Red) showed siRNA injection did not lead to seminiferous tubule damage. Scale bar is 10 µm. C:Western blotting analysis of flotillin-2 protein expression in 3 week old mouse testis 48 h after injection of flotillin-2 siRNA. D: Quantification of Western blotting analysis. Data are the mean fold change relative to the control siRNA-injected mice from three independent experiments.

Fig. 6. Knockdown of flotillin-2 in vivo affects sperm morphology in the cauda epididymis.

The testes of three weeks old mice were injected with flotillin-2 siRNA and sperm were isolated from the cauda epididymis three weeks later. A: Sperm abnormalities. The ratios were subjected to arcsine square root transformation prior to Student's t test; *P < 0.05. Representative images of the shape of sperm from the cauda epididymis of mice injected with flotillin-2 siRNA (B) or control siRNA (C). The arrows indicate sperm with an abnormal shape. All scale bars are 10 µm. Similar results were observed in three independent experiments.

Fig. 7. Knockdown of flotillin-2 in vivo induced acrosome abnormalities.

Fluorescent staining of the acrosome with peanut agglutinin (red) and nuclear staining with Hoechst (blue) of normal sperm (A) and sperm with abnormal heads (B-F) from the cauda epididymis of mice injected with flotillin-2 siRNA. The sperm with abnormal heads contained an abnormal acrosome. Similar results were observed in triplicate experiments.