Abstract
Spermatogenesis is a complex process involving cell division and differentiation of Spermatogonial Stem Cells (SSCs), mediated by coordinated expression of a number of genes. The study of SSC biology was challenging due to the unavailability of proper SSC markers and their culture methods. In recent years, these hurdles have been somewhat overcome. A number of markers for undifferentiated spermatogonia are known to be involved in the process of SSC self-renewal. Oct-4 is one such marker expressed from Primordial Germ Cells (PGCs) of prenatal stages till undifferentiated spermatogonia of postnatal adult testes. Though, expressed by undifferentiated spermatogonia, its role in the process of their self-renewal, proliferation and differentiation is not clearly understood. In the current study, using shRNA mediated gene silencing approach, we show that Oct-4 silencing in undifferentiated type A spermatogonia leads to phenotypic differentiation of these cells to type A1-A4 spermatogonia, marked by the increased c-KIT expression, and decreased proliferation, marked by decreased PCNA expression. The study also demonstrates that Oct-4 silencing causes down-regulation of Plzf, Gfra-1, c-Ret, Bcl6b and Etv5 mRNA and protein. Therefore the current study suggests that Oct-4 does play an important role in deciding the fate of undifferentiated spermatogonia by controlling their proliferation and differentiation. In conclusion, the present study may provide useful insights into understanding the involvement of Oct-4 in proliferation and differentiation of undifferentiated spermatogonia in mice.
Highlights
Spermatogenesis is a complex process that involves continuous self-renewal of subset of type A spermatogonia called Spermatogonial Stem Cells (SSCs), which further undergo differentiation to give rise to spermatozoa thereby maintaining continual spermatogenesis from puberty till old age in males
To analyze whether Oct-4 plays an essential role in the undifferentiated spermatogonial cell proliferation and differentiation, undifferentiated type A spermatogonia were subjected to Oct4 silencing
In order to knock-down the expression of Oct-4 in undifferentiated type A spermatogonial cells, three short hairpin RNA (shRNA) sequences were used for targeting different regions of Oct-4 mRNA
Summary
Spermatogenesis is a complex process that involves continuous self-renewal of subset of type A spermatogonia called Spermatogonial Stem Cells (SSCs), which further undergo differentiation to give rise to spermatozoa thereby maintaining continual spermatogenesis from puberty till old age in males. Study by Kanatsu-Shinohara et al [7] has shown that cultures of “Germline Stem” (GS) cells, derived from neonatal testes can be successfully expanded in culture for years while maintaining SSC colonization activity. These cells express markers of undifferentiated spermatogonia such as OCT4 and PLZF, as well as several cell surface markers, including RET, GFRa1, α6 and β1 integrins [6,8,9,10]. This is essential for their possible use as a therapy for patients undergoing chemotherapy so as to preserve their fertility by maintaining the germline [14,15]
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