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(18) AKT LOCALIZES IN THE MEIOTIC NUCLEUS AND PHOSPHORYLATES H2AX DURING SPERMATOGENESIS.

Feng, Li-Xin ¹, Geng, Yixun¹, Dym, Martin¹, ¹ Georgetown University, Washington, DC

ABSTRACT- Spermatogenesis is a complicated and continuous process of cellular differentiation that produces the haploid sperm. This occurs in the seminiferous tubules where spermatogonia reside on the basement membrane and are flanked by the supportive Sertoli cells. Sertoli cells produce a number of growth factors that regulate spermatogonial differentiation. Among them, stem cell factor (SCF) is required for spermatogonial differentiation as well as meiotic initiation. We and other groups have revealed that the PI3K/AKT pathway is triggered by SCF to regulate spermatogonial differentiation. Since c-kit, the receptor for SCF, is also expressed in early spermatocytes, the SCF/c-kit/PI3K/AKT pathway may be involved in the regulation of the early stages of meiosis. We examined the localization of AKT by immunofluorescent staining in adult mouse testis. We found that AKT localizes in the meiotic nucleus rather than in cytoplasm, where AKT exists in most other types of cells. Thus, the SCF/c-kit/PI3K/AKT pathway may target a nuclear-located molecule in the regulation of meiosis. Interestingly, double immunofluorescent staining demonstrated that AKT co-localizes with phosphorylated H2AX (gamma-H2AX) in spermatocytes. Furthermore, gamma-H2AX was pulled down by AKT antibody, and the phosphorylation of H2AX was abolished by the PI3K inhibitor, Wortmannin, but restored by overexpression of the constitutively active v-AKT. In the testis, gamma-H2AX foci are present in preleptotene to zygotene spermatocytes and remain in the sex body in pachytene spermatocytes. Functional studies demonstrated that gamma-H2AX binds to the meiotic double DNA breaks and then is involved in the initiation and completion of meiosis in spermatocytes. Thus, our study indicates that the PI3K/AKT pathway mediates the SCF/c-kit signaling in the regulation of meiosis via phosphorylating H2AX.

KEY WORDS: AKT, Meiosis, H2AX, Spermatogenesis