t-SNARE Syntaxin2 (STX2) is implicated in intracellular transport of sulfoglycolipids during meiotic prophase in mouse spermatogenesis

Abstract

Syntaxin2 (STX2), also known as epimorphin, is a member of the SNARE family of proteins, with expression in various types of cells. We previously identified an ENU-induced mutation, repro34, in the mouse Stx2 gene. The Stx2(repro34) mutation causes male-restricted infertility due to syncytial multinucleation of spermatogenic cells during meiotic prophase. A similar phenotype is also observed in mice with targeted inactivation of Stx2, as well as in mice lacking enzymes involved in sulfoglycolipid synthesis. Herein we analyzed expression and subcellular localization of STX2 and sulfoglycolipids in spermatogenesis. The STX2 protein localizes to the cytoplasm of germ cells at the late pachytene stage. It is found in a distinct subcellular pattern, presumably in the Golgi apparatus of pachytene/diplotene spermatocytes. Sulfoglycolipids are produced in the Golgi apparatus and transported to the plasma membrane. In Stx2(repro34) mutants, sulfoglycolipids are aberrantly localized in both pachytene/diplotene spermatocytes and in multinucleated germ cells. These results suggest that STX2 plays roles in transport and/or subcellular distribution of sulfoglycolipids. STX2 function in the Golgi apparatus and sulfoglycolipids may be essential for maintenance of the constriction between neighboring developing spermatocytes, which ensures ultimate individualization of germ cells in later stages of spermatogenesis.

Keywords: Stx2; glycolipids; meiosis; mouse; spermatogenesis.

Fig. 1

First wave of spermatogenesis of the Stx2^repro34 mutant and STX2 expression. A, B) At P14, no significant differences were observed between wild-type (A) and Stx2^repro34 mutants (B). C, D) At P16, pachytene spermatocytes were observed in normal testes (C), and the mutant testes included syncytial multinucleated spermatocytes (D, arrows). E, F) At P32, the seminiferous epithelium in wild type was filled with round and elongating spermatids (E), whereas no spermatids were observed in the seminiferous epithelium of the mutants (F). Arrows in F indicate multinucleated cells. G) Western blotting of STX2 in the testis revealed a weak expression at P6–P14. At P16 and later days, higher expressions were seen. No expression was detected in the adult mutant testis. SG, spermatogonia; L/Z, leptotene or zygotene spermatocytes; P, pachytene spermatocyte; RS, round spermatid; ES, elongated spermatid. Arrows indicate degenerated germ cells. Bar = 100 μm.

Fig. 2

Immunohistochemical analysis of Stx2^repro34 mutant testis. A–D) The ring-shaped expression of TEX14, a component of the intercellular bridge of germ cells, was observed in the connecting regions of the cells in both wild-type (A, C) and mutant testis (B). Note positive reactions around multinucleated germ cells (D, arrowheads). Arrows in B indicate multinucleated cells. Bars in A and B = 50 μm.

Fig. 3

Multinucleation and chromosomes. Germ cells collected by squash technique from the wild-type (A, C) and Stx2^repro34 mutant testes (B, D) were immunostained with antibodies against SCP3 (A, B) and α-TUBULIN (C, D). A, B) Spots of SCP3 in metaphase spermatocyte showed chiasma formation (red spots in A and B). Note more spots were observed in the mutant than in the wild type (B). C, D) Spindle formation in metaphase spermatocyte was shown by α-TUBULIN. Wild-type metaphase spermatocyte showed a single pair of spindle bodies (C), while multipolar formation of spindle bodies was observed in the mutant syncytial cell (D). The nuclei were counterstained with DAPI. Original magnification ×1000.

Fig. 4

Subcellular localization of STX2 in the developing germ cells. A) In the testicular section, STX2 (red) and TEX14 (green) were colocalized to the intercellular bridges of the spermatids (A, inset). B–G) Germ cells obtained from the seminiferous tubular segment at stages I–IV by squash preparation revealed localization of STX2 to the intercellular bridges of spermatogonia and pachytene spermatocytes (arrows in D and G, respectively), which was colocalized with TEX14 (B, E). H–J) Subcellular localization of STX2 was observed in the perinuclear region of pachytene spermatocytes obtained from the segment at stages IX–X (H). The proacrosomal region of round spermatids from the segment at stages I–VII (I) and the acrosomes of elongated spermatids from post-stage VIII (J) also exhibited STX2 localization. Arrows in H indicate perinuclear localization of STX2, arrows in I indicate localization of STX2 to the proacrosomal region, and arrows in J indicate acrosomal localization of STX2. Nuclei were counterstained with DAPI. Roman numerals show stages of the spermatogenic cycle. SG, spermatogonia; In, intermediate spermatogonia; P, pachytene spermatocytes; RS, round spermatid; ES, elongated spermatid. Original magnification ×400 (A) and ×1000 (B–J).

Fig. 5

Localization of sulfoglycolipids in the seminiferous epithelium. A, B) Granular immunoreaction of sulfoglycolipids (green) was seen in the plasma membrane of early pachytene spermatocytes (A) and leptotene/zygotene spermatocytes (arrowheads in C). In the Stx2^repro34 mutant, dotty reactions were distributed to the seminiferous epithelium (arrows in B). A weak reaction was seen entirely in the cytoplasm of syncytial spermatocytes at the diplotene stage (arrows in D). E–H) Ring-shaped sulfoglycolipid reaction in normal pachytene spermatocytes (arrows in E, G) and round and elongating spermatids (arrowhead in E) obtained by squash preparation showed colocalization with TEX14 (E, G). Normal pachytene spermatocytes were surrounded by dot sulfoglycolipid reactions (G). Sulfoglycolipids were barely observed around Stx2^repro34 mutant pachytene spermatocytes (F), but localized to the intercellular bridges (arrows in F, H). Many Stx2^repro34 mutant pachytene spermatocytes showed abnormal accumulation of sulfoglycolipids around the nucleus (arrowheads in H). The nuclei were counterstained with DAPI. Roman numerals show stages of the mouse spermatogenic cycle. L/Z, leptotene or zygotene spermatocytes; P, pachytene spermatocyte; D, diplotene spermatocytes; RS, round spermatid; ES, elongated spermatid. Bar = 50 μm.