The male germ cells must adopt the correct morphology at each differentiation stage for proper spermatogenesis. The spermatogonia regulates its differentiation state by its own migration. The male germ cells differentiate and mature with the formation of syncytia, failure of forming the appropriate syncytia results in the arrest at the spermatocyte stage. However, the detailed molecular mechanisms of male germ cell morphological regulation are unknown. Here, we found that EXOC1, a member of the Exocyst complex, is important for the pseudopod formation of spermatogonia and spermatocyte syncytia in mice. EXOC1 contributes to the pseudopod formation of spermatogonia by inactivating the Rho family small GTPase Rac1 and also functions in the spermatocyte syncytia with the SNARE proteins STX2 and SNAP23. Since EXOC1 is known to bind to several cell morphogenesis factors, this study is expected to be the starting point for the discovery of many morphological regulators of male germ cells.

Germ cell development is essential for maintaining reproduction in animals. In postpubertal females, oogenesis is a highly complicated event for producing fertilizable oocytes. It starts when dormant primordial oocytes undergo activation to become growing oocytes. In postpubertal males, spermatogenesis is a differentiation process for producing sperm from spermatogonial stem cells. To obtain full understanding of the molecular mechanisms underlying germ cell development, the Cre/loxP system has been widely applied for conditional knock‐out mouse studies. In this study, we established a novel knock‐in mouse line, B6‐Ddx4 em1(CreERT2)Utr, which expresses CreERT2 recombinase under the control of the endogenous DEAD‐box helicase 4 (Ddx4) gene promoter. Ddx4 was specifically expressed in both female and male germ cell lineages. We mated the CreERT2 mice with R26GRR mice, expressing enhanced green fluorescent protein (EGFP) and tDsRed before and after Cre recombination. We found tDsRed signals in the testes and ovaries of tamoxifen‐treated B6‐Ddx4 em1(CreERT2)Utr::R26GRR mice, but not in untreated mice. Immunostaining of their ovaries clearly showed that Cre recombination occurred in all oocytes at every follicle stage. We also found 100% Cre recombination efficiency in male germ cells via the progeny test. In summary, our results indicate that B6‐Ddx4 em1(CreERT2)Utr is beneficial for studying female and male germ cell development.

31Spermatogenesis requires high regulation of germ cell morphology. The 32 spermatogonia regulates its differentiation state by its own migration. The male 33 germ cells differentiate and mature with the formation of syncytia, failure of 34 forming the appropriate syncytia results in the arrest of spermatogenesis at the 35 spermatocyte stage. However, the detailed molecular mechanisms of male germ 36 cell morphological regulation are unknown. Here, we found that EXOC1 is 37 important for the pseudopod formation of spermatogonia and spermatocyte 38 syncytia in mice. We found that while EXOC1 contributes to the inactivation of 39Rac1 in the pseudopod formation of spermatogonia, in spermatocyte syncytium 40 formation, EXOC1 and SNAP23 cooperate with STX2. Our results showed that 41 EXOC1 functions in concert with various cell morphology regulators in 42 spermatogenesis. Since EXOC1 is known to bind to several cell morphogenesis 43 factors, this study is expected to be the starting point for the discovery of many 44 morphological regulators of male germ cells.

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