Dynamics of Sun5 localization during spermatogenesis in wild type and Dpy19l2 knock-out mice indicates that Sun5 is not involved in acrosome attachment to the nuclear envelope

Abstract

The acrosome is an organelle that is central to sperm physiology and a defective acrosome biogenesis leads to globozoospermia, a severe male infertility. The identification of the actors involved in acrosome biogenesis is therefore particularly important to decipher the molecular pathogeny of globozoospermia. We recently showed that a defect in the DPY19L2 gene is present in more than 70% of globozoospermic men and demonstrated that Dpy19l2, located in the inner nuclear membrane, is the first protein involved in the attachment of the acrosome to the nuclear envelope (NE). SUN proteins serve to link the nuclear envelope to the cytoskeleton and are therefore good candidates to participate in acrosome-nucleus attachment, potentially by interacting with DPY19L2. In order to characterize new actors of acrosomal attachment, we focused on Sun5 (also called Spag4l), which is highly expressed in male germ cells, and investigated its localization during spermatogenesis. Using immunohistochemistry and Western blot experiments in mice, we showed that Sun5 transits through different cellular compartments during meiosis. In pachytene spermatocytes, it is located in a membranous compartment different to the reticulum. In round spermatids, it progresses to the Golgi and the NE before to be located to the tail/head junction in epididymal sperm. Interestingly, we demonstrate that Sun5 is not, as initially reported, facing the acrosome but is in fact excluded from this zone. Moreover, we show that in Dpy19l2 KO spermatids, upon the detachment of the acrosome, Sun5 relocalizes to the totality of the NE suggesting that the acrosome attachment excludes Sun5 from the NE facing the acrosome. Finally, Western-blot experiments demonstrate that Sun5 is glycosylated. Overall, our work, associated with other publications, strongly suggests that the attachment of the acrosome to the nucleus does not likely depend on the formation of SUN complexes.

Fig 1. Specificity of antibodies targeting Sun5.

(A) Western blot of protein extracts from HEK cells heterologously expressed with or without plasmid containing mouse short isoform of Sun5 and revealed with anti-Sun5 antibodies 1 (Ab1). Below, gel of protein showing that both lanes were similarly loaded. (B) Similar experiment but revealed with anti-Sun5 antibodies 2 (Ab2). (C) Western blot of protein extracts from HEK cells heterologously expressed with a plasmid containing human short isoform of SUN5-DDK tagged and revealed with anti-DDK antibodies. (D) HEK cells were transfected with mouse short isoform of Sun5 and stained with Hoechst (blue, D1) and anti-Sun5 Ab1 (D2, green). D3 corresponds to overlay + phase contrast. (E) Similar experiment performed with NIH/3T3 cells.

Fig 2. Sun5 is located in the cytoplasm in pachytene spermatocyte.

(A) Pachytene spermatocyte co-stained with Hoechst (blue) and anti-Sun5 Ab1 (green) (A1). A2 corresponds to overlay + phase contrast. (B) Pachytene spermatocytes were purified by unit gravity sedimentation from spermatogenic cell suspension obtained from sexually mature males. The purity of the fraction (B1, observed with optical microscope), was assessed by counting the cells exhibiting characteristic Hoechst nuclear staining under a fluorescent microscope (B2). (C) Western blot of cytoplasmic proteins extracted from the pachytene spermatocytes showing that Anti-Sun5 Ab immunodecorates a single band around 40 KDa.

Fig 3. Sun5 is located in the nuclear envelope (NE) in round spermatid and is excluded from the NE facing the acrosome.

(A) Round spermatid stained with anti-Sun5 Ab1 (A2, green) and counterstained with Hoechst to evidence the nucleus (A1, blue). A3 corresponds to overlay of A1 and A2; Arrow head indicates the Sun5 staining which was not bound to the NE. A4 corresponds to overlay with phase contrast. (B) Round spermatid co-stained with anti-Sp56 (B1, red) and anti-Sun5 Ab1 (B2, green) and counterstained with Hoechst to evidence the nucleus (blue). B3 corresponds to overlay of B1 and B2; arrow head indicates the Sun5 staining which was not bound to the NE. Note that Sun5 staining is located in a more external location than Sp56 staining. B4 corresponds to overlay with phase contrast.

Fig 4. Sun 5 progresses through the Golgi apparatus and presents post-translational modifications during spermiogenesis.

(A) Round spermatid co-stained with anti-Sun5 Ab1 (A1, green) and anti-GM130 antibodies a marker of the Cis-Golgi (A2, red) and counterstained with Hoechst to evidence the nucleus (blue). A3 corresponds to overlay. A4 corresponds to overlay with phase contrast (B) Western blots of nuclear protein extracts from testis revealed with anti-Sun5 Ab1 (B1, left) and anti-Sun5 Ab2 (B2, right), showing a similar pattern evidenced by both antibodies. (C) Protein extracts (load, lane 1) were incubated 15 hours without (−, lane 2) or with a deglycosylation enzyme mix (+ Ez, lane 3) and the impact on Sun5 was studied in Western blot with Ab2 antibodies. (D) Protein extract was split in two identical fractions, and one fraction was boiled. The band at 95 KDa observed with Sun5 antibodies in Fig. 4B was no longer present when denaturation conditions were strengthened by boiling the sample in Laemmli buffer. Protein loads controlled with TGX stain free precast gels are showed in panel C of S5 Fig.

Fig 5. Sun5 is removed during spermiogenesis along an antero-caudal axis.

(A) In early condensing spermatids, Sun5 is located at the base of the nucleus. Elongating spermatids co-stained with anti-Sp56 antibodies (A1, red) and anti-Sun5 Ab1 (A2, green) and counterstained with Hoechst to evidence the nucleus (blue). Arrow heads show specific Sun5 staining at the implantation fossa in late condensing spermatids. (B) In early condensing spermatids, Sun5 is no longer present in Golgi apparatus. Elongating spermatids co-stained with anti-GM130 antibodies (B1, purple) and anti-Sun5 Ab1 (B2, green) and counterstained with Hoechst to evidence the nucleus (blue). (C) In round spermatids (white arrows), Lamin B1 (purple) is not located in front of the acrosomal vesicle (evidenced by anti-Sp56 antibodies green), showing that lamin B1 and Sun5 were both excluded from the acrosomal area. In early condensing spermatids (yellow arrow heads), Lamin B1 (purple) is absent. Cells were counterstained with Hoechst to evidence the nucleus (blue).

Fig 6. Epididymal sperm presents a punctiform staining at the implantation fossa.

(A, B) Sun5 staining is located at the implantation fossa in epididymal sperm (arrow heads). (C) Sperm were fractionated by mild sonication allowing separating head and flagella fractions and resolved proteins by SDS PAGE of both fractions were subjected to Western blotting analysis with Ab2 Sun5 antibody. As control, testis nuclear protein extracts were subjected to Western blotting analysis in the same trial.

Fig 7. Detachment of the acrosome in spermatids from Dpy19l2 KO males leads to a relocalization of Sun5.

(A, B) Round spermatids co-stained with anti-Sp56 antibodies (A1, B1, red) and anti-Sun5 Ab1 (A2, B2, green) and counterstained with Hoechst to evidence the nucleus (blue). A3 corresponds to overlay and A4 and B3 correspond to overlay + phase contrast. (C) Sun5 removal during spermiogenesis along the antero-caudal axis is not modified in Dpy19l2 KO condensed spermatids. Elongating Dpy19l2 KO spermatids co-stained with anti-Sp56 antibody (C1, red) and anti-Sun5 Ab1 (C2, green) and counterstained with Hoechst to evidence the nucleus (blue); C3 overlay. (D) Similar stainings in fully condensed spermatids showing the almost complete vanishing of Sun5. (E) Absence of Sun5 staining in Dpy19l2 KO sperm head. (F) Western blot of nuclear protein extracts from WT and Dpy19l2 KO testis revealed with anti-Sun5 Ab1. The absence of Dpy19l2 did not modify Sun5 glycosylation pattern.