The RNA-binding protein NANOS2 is required to maintain murine spermatogonial stem cells RNA結合タンパク質NANOS2は、マウス精原幹細胞の未分化性を維持する




    • 佐田, 亜衣子 サダ, アイコ



The RNA-binding protein NANOS2 is required to maintain murine spermatogonial stem cells




佐田, 亜衣子


サダ, アイコ




博士 (理学)







&nbsp;&nbsp;Spermatogonial stem cells represent a stem cell population in adult testes, and their biological activities provide a base of continuous production of spermatozoa. The stem cell function resides in undifferentiated spermatogonia, consisting of types Asingle, Apaired and Aaligned. These cells transform into type A1 differentiating spermatogonia, which subsequently undergo six mitotic and two meiotic divisions to form haploid spermatozoa within 35 days. The entire developmental process from spermatogonia to spermatozoa occurs within seminiferous tubules of testes. In the tubules, all types of spermatogonia are located on the basement membrane, and the subsequent differentiating cell types are arranged in a sequential order towards the lumen. The spermatogenesis is also supported by close interaction of the germ cells with somatic Sertoli cells.<br/>&nbsp;&nbsp;Currently, several factors have been implicated in the regulation of spermatogonial stem cells. For example, a growth factor GDNF (glial cell-line derived neurotrophic factor) is secreted by Sertoli cells and acts as one of the major niche signals for spermatogonial stem cells. A transcription factor PLZF (promyelocytic leukemia zinc finger) is needed in a cell autonomous fashion for the maintenance of the spermatogonial stem cells, since male mice lacking Plzf expression exhibit progressive germ cell loss and testis atrophy with age, causing infertility. However, the previous loss of function studies had some limitations in terms of understanding the mechanisms by which stem cells are lost upon the gene deletion, as it could be caused by cell death, a defective proliferation, a premature differentiation, or other mechanisms. <br/>&nbsp;&nbsp;A precise identification of stem cells is another important issue in stem cell biology. According to“Asingle model”, Asingle spermatogonia represent spermatogonial stem cells: this type is recognized as single cells without any intercellular connection with others, whereas Apaired and Aaligned spermatogonia are connected by intercellular cytoplasmic bridges and committed to differentiation. However, the stem cell activity of the Asingle spermatogonia has not been tested due to a lack of their specific molecular markers. <br/>&nbsp;&nbsp;NANOS, a zinc-finger RNA-binding protein, has been proposed as an evolutionarily conserved factor for germline cell function. Among three NANOS homologs in mice, NANOS2 is essential for the male-type differentiation of embryonic germ cells. In adult testes, NANOS2 is predominantly expressed in the Asingle and Apaired spermatogonia, suggesting that NANOS2 may also be involved in the spermatogonial stem cell function. However, the postnatal function of NANOS2 has not been studied, because the majority of Nanos2-null male germ cells undergo apoptosis before birth. <br/>&nbsp;&nbsp;In the first part of my doctoral thesis, I report my findings indicating that the RNA-binding protein NANOS2 is a key regulator for the maintenance of spermatogonial stem cells. First, by lineage-tracing analyses, I revealed that undifferentiated spermatogonia expressing Nanos2 retained abilities to self-renew and generate the entire spermatogenic cell lineage. Hence, such spermatogonia can be referred to as spermatogonial stem cells. Next, I addressed if NANOS2 plays a role in these stem cells by using a conditional gene knockout system. I showed that conditional disruption of postnatal Nanos2 led to depletion of spermatogonial stem cell reserves and resulted in the complete depletion of germ cells within a few cycles of spermatogenesis. These results indicate that NANOS2 is expressed in spermatogonial stem cells and is required for the maintenance of these cells. <br/>&nbsp;&nbsp;The current view of the regulation of stem cell state or “stemness” depicts following cellular processes: (1) cell proliferation to expand stem cell population; (2) maintenance of the undifferentiated state; and (3) cell death/survival. NANOS2 should control these events for maintaining the proper state of spermatogonial stem cells. To further determine the role of NANOS2 in the stem cell maintenance, I generated transgenic mice, which allowed continuous expression of NANOS2 in germ cell lineages. I found that mouse testes in which Nanos2 had been overexpressed accumulated spermatogonia with undifferentiated, stem cell-like properties. Furthermore, these Nanos2-overexpressing cells showed lower proliferation rates and similar levels of apoptosis compared to the control spermatogonia. These results indicate that NANOS2 maintains spermatogonial stem cells by suppressing proliferation and differentiation of these cells. <br/>&nbsp;&nbsp;Studies in diverse stem cell systems indicate that the stem cell regulations are dependent on not only stem cell-intrinsic factors but also extrinsic signals from the microenvironment known as niche signals. However, it remains unclear how extrinsic signals and intrinsic stem cell factors intersect in stem cells to control their cellular state. During murine spermatogenesis, GDNF signals from Sertoli cells and germ cell-intrinsic factor NANOS2 represent key regulators for the maintenance of spermatogonial stem cells. In the second part of my thesis, I examined the possible genetic interaction between NANOS2 and GDNF signal transduction pathway. <br/>&nbsp;&nbsp;First, I conducted conditional knockout of Gfra1, a receptor for GDNF, to decipher roles of the GDNF signaling during adult spermatogenesis. The absence of Gfra1 resulted in rapid loss of spermatogonial stem cells, which might result from deficits in proliferation and/or in the maintenance of undifferentiated state. Next, I asked whether the overexpression of Nanos2 could negate the stem cell loss phenotype caused by the Gfra1-deletion. I found that overexpressed Nanos2 could not support Gfra1-mutant stem cells permanently, because proliferation of spermatogonial stem cells was severely impaired by the lack of GDNF signaling. This result indicates that stem cell proliferation is highly dependent on the GDNF-stimulated pathway. <br/>&nbsp;&nbsp;On the other hand, NANOS2 did prevent precocious differentiation of spermatogonial stem cells caused by the Gfra1-deficiency, indicating that the stem cell differentiation could be suppressed solely by NANOS2 independent of GDNF signaling. The GDNF-independent NANOS2 function was further assessed by the ectopic expression of Nanos2 in GFRA1-negative spermatogonia. The results support my idea that NANOS2 is capable of maintaining undifferentiated state of spermatogonial stem cells in the absence of GDNF-signaling pathway. Taken together, I suggest that the stem cell-intrinsic factor NANOS2 and extrinsic GDNF signals coordinately maintain spermatogonial stem cells by acting through different cellular and/or molecular mechanisms. My doctoral studies thus offer a novel insight into understanding how stem cells are maintained during murine spermatogenesis. <br/>



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