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.

Nuclear localization of PRDM9 and its role in meiotic chromatin modifications and homologous synapsis

“…In some experiments, after isolation of specifically staged seminiferous tubules, a squash method was used for the cytological preparation. Squash preparations of germ cells were prepared based on previously described methods (Fujiwara, et al 2013; Kotaja, et al 2004), with some modifications. Briefly, mouse testes were placed in PBS and detunicated.…”

An ENU-induced mutation in the mouse Rnf212 gene is associated with male meiotic failure and infertility

“…Sections (5 m) were stained with hematoxylin and eosin (HE) for evaluation of spermatogenesis. Immunohistochemistry was performed as previously described (Fujiwara et al 2013). Mouse monoclonal anti-human PCNA primary antibody and anti-rat IgG HRP labeled secondary antibody (Table 1) were used to label the histological sections.…”

STX2is a causative gene for nonobstructive azoospermia

“…In 2013, Fujiwara et al. () demonstrated that nullizygosity of Stx2 results in universal maturation arrest in mice. Stx2 mutant mice characteristically exhibit spermatogenic failure with multinucleated spermatocytes.…”

“…Stx2 mutant mice characteristically exhibit spermatogenic failure with multinucleated spermatocytes. STX2 (MIM# 132350, NM_001980.4) encodes a transporter for sulfoglycolipids, which are indispensable in the maintaining of intercellular bridges of developing spermatocytes (Fujiwara et al., ). To date, however, STX2 mutations have not been identified in humans.…”

“…Although the mutation‐positive patient carried additional substitutions in SPATA17 and MEI1 , which may be associated with the risk of nonobstructive azoospermia (Nakamura et al., ), multinucleation of the germ cells is more consistent with STX2 mutations than SPATA17 and MEI1 mutations. Actually, universal maturation arrest and multinucleated spermatocyte have been observed in mice lacking Stx2 (Fujiwara et al., ). Although multinucleation is a nonspecific feature reflecting spermatocyte degradation (Miething, ; Yin, Hawkins, DeWolf, & Morgentaler, ), the coexistence of maturation arrest and multinucleation has been documented almost exclusively in mice with defective sulfoglycolipid metabolism (Fujimoto et al., ; Fujiwara et al., ; Honke et al., ).…”