This study investigated the functional importance of VRK1 for the maintenance of mammalian spermatogonial stem cells. In view of current genetics analyses, VRK1 is important to the process of gametogenesis. Previous reports have analyzed the function of VRK in Caenorhabditis elegans
and Drosophila melanogaster
in which single VRK orthologs exist. siRNA-mediated VRK depletion in C. elegans
results in early embryonic lethality caused by cell cycle arrest during the earliest cell division 
. In vrk-1 mutants of C. elegans
that express truncated VRK-1 proteins, sterility was observed from defects in the cell cycle 
. NHK1, the VRK1 homolog in D. melanogaster
, is important for karyosome formation and metaphase arrest during meiosis. Mutations in NHK1 lead to sterility in both male and female flies due to abnormal meiotic spindle formation 
. NHK1 mutations in oocytes also cause the formation of multiple spindles and increased chromosome condensation during prophase 
. These reports indicate that VRK1 plays an important role in germ cell development. In contrast to C. elegans
and D. melanogaster
, mammalian genomes encode three forms of VRK whose functions are more diverse and complex. Among the VRK families, a hypomorphic mouse model for VRK1 has been recently reported 
. VRK1 mutant mice generated by the gene trap method showed progressive germ cell loss and resulting infertility. Germ cell nuclear antigen (GCNA), proliferating cell nuclear antigen (PCNA), and c-kit-positive germ cells were all greatly reduced in VRK1-deficient seminiferous tubules. Whereas adult mice showed obvious germ cell defects, testes from prepubertal mice seemed normal. The authors concluded that VRK1 deficiency did not affect the maintenance of spermatogonial stem cells. This conclusion was suggested by counting the number of Plzf-positive cells in the seminiferous tubules during the first wave of spermatogenesis. Previous studies also reported that mice in which essential genes related to the maintenance of spermatogonial stem cells have been knocked out do not frequently show abnormalities in germ cell production during the first wave of spermatogenesis. Few factors other than Plzf 
and TATA box-binding protein-associated factor 4b (Taf4b) 
are known to be essential for the maintenance of spermatogonial stem cells. Disruption of the transcriptional repressor Plzf in mice results in a progressive depletion of germ cells, resulting in a phenotype that consists only of Sertoli cells. Similarly, inactivation of Taf4b results in severe germ cell depletion characterized by tubules of only Sertoli cells and male infertility 
. Although these mice can complete spermatogenesis, leading to the production of normal sperm during the first wave of spermatogenesis, the number of germ cells is reduced, and spermatogonial proliferation is defective at an early age. All of these effects cause infertility in these animals.
In the present research, we analyzed the first wave of spermatogenesis in VRK1-deficient mice and found that the functional defect in spermatogenesis occurred at an early age. Within 2 weeks, the number of actively proliferating spermatogonia was significantly reduced as indicated by cyclin D1 immunostaining (). Moreover, the expression levels of marker genes, including Plzf, POU5f1(Oct-4), POU3f1(Oct-6), Ret, Ngn3, and Lin28, in undifferentiated spermatogonia were markedly downregulated as indicated by microarray analysis and quantitative RT-PCR by day 8 (). Therefore, these results suggest that the impairment of important genes in undifferentiated spermatogonia impede the normal meiotic development of spermatocytes as shown by the GO data ( and Table S1
), indicating that VRK1 plays a crucial role in normal spermatogenesis in neonatal mice.
First, we suppose that VRK1 may be involved in the maintenance of spermatogonial stem cells because VRK1-deficient testes have tubules containing cells at degenerated stages of spermatogenesis or a Sertoli cell-only phenotype as previously reported 
. These phenotypes typically occur in testes undergoing stem cell loss 
. Wiebe et al. 
also expected that degeneration of germ cells is induced by the dysfunctional division of differentiated spermatogonial cells, not by spermatogonial stem cells. However, we revealed that VRK1 deficiency caused a loss of spermatogonial stem cells. We believe that this discrepancy comes from the different methodologies for the quantification of spermatogonial stem cells. Previous reports counted the number of Plzf-positive cells in cross-sections of seminiferous tubules. Although this examination can determine the status of stem cells, it is ambiguous because analyses using cross-sections cannot count all spermatogonial stem cells. Spermatogonial stem cells are a very rare population of all the germ cells, and only a fraction of stem cells can be detected in cross-sectioned tubules. Therefore, most studies quantify spermatogonial stem cells by flow cytometry analysis with antibodies against specific surface marker proteins, which enable the concentration of spermatogonial stem cells for further functional analyses 
. Our quantification results obtained by flow cytometry provide evidence for the impairment of spermatogonial stem cells in VRK1-deficient mice (). Moreover, we found that in 5-week-old VRK1 deficient testes, loss of chained undifferentiated spermatogonia is apparent compared to wild type (). It seems that the proliferation of undifferentiated spermatogonia requires VRK1. Although it is unclear whether VRK1 directly participates in the self-renewal of As
spermatogonial stem cells, a great reduction in the number of spermatogonial stem cells appeared in VRK1-deficient mice. After 8 weeks, VRK1-deficient testes showed a great reduction in spermatogonial stem cells by flow cytometry analysis. In addition, Plzf-positive cells were rarely detected after 9 weeks by whole mount immunohistochemisty of seminiferous tubules (Figure S3
). These results indicate that VRK1 is required for the maintenance of spermatogonial stem cells that are regulated by self-renewal.
Spermatogonial stem cells can be regulated by both intrinsic and extrinsic factors; therefore, we asked whether VRK1 directly causes a loss of undifferentiated spermatogonia. Since it is unknown which spermatogonial subpopulation expresses VRK1, we confirmed the expression of VRK1 in undifferentiated spermatogonia by immunocytochemistry after MACS and flow cytometry analysis. We determined that undifferentiated spermatogonia expressing Oct-4 and Plzf but not c-kit also expressed VRK1 (). Among the undifferentiated spermatogonia specified as Ep-CAM+/α6-integrin+/c-kit−, approximately 66% of cells expressed VRK1 (). These data suggest the existence of a specific subpopulation in undifferentiated spermatogonia that expresses VRK1. Another possibility is that the level of VRK1 changes during the phases of the cell cycle. As VRK1 is highly expressed during the G2-M phases of the cell cycle in somatic cells, the expression levels of VRK1 might also oscillate during the cell cycle in proliferating spermatogonia. This idea might contribute to the detection of undifferentiated spermatogonia that are in the G2-M phase. Together, we suggest that VRK1 is a crucial intrinsic factor for the maintenance of spermatogonial stem cells.
Here, we suggest VRK1 mainly exists in spermatogonia, but not in Sertoli cells. VRK1 protein was detected in spermatogonia with anti-mouse VRK1 antibody but not in Sertoli cells (). In the previous study, GT12/GT12 (a gene-trap vector insertion within intron 12 of Vrk1
) tubule was stained with x-gal 
. Although this data confirmed the promoter activity of VRK1in spermatogonia and Sertoli cells, it does not determine whether VRK1 protein exists or not. Proteolysis is a crucial mechanism for the regulation of VRK1 
. But mutant VRK1 deleted in exon 13–15 may not be normally regulated because C-terminal region of VRK1 has been suggested as a regulatory domain interacting with other proteins 
. Therefore, strong staining for beta-galactosidase in Sertoli cells may be due to the accumulation of mutant VRK1 protein.
Although VRK1 is not detected in Sertoli cells by immunohistochemistry, undetectable amount of VRK1 protein might exist. In this case, it cannot be overlooked that an impairment in the maintenance of undifferentiated spermatogonia can be caused by a malfunction of the stem cell niche supported by Sertoli cells. Wiebe et al. showed that expression levels of the kit ligand were unaltered in VRK1-deficient testes, suggesting that VRK1-deficient Sertoli cells retain their ability to secrete important paracrine factors 
. To prove that VRK1 is an intrinsic factor for the maintenance of undifferentiated spermatogonia, repopulation assays using transplantation of VRK1-deficient germ cell are needed.
Overall, our present study showed that the proliferative capability of undifferentiated spermatogonia, including spermatogonial stem cells, is compromised at an early postnatal age by VRK1-deficiency. Furthermore, in view of the specific biochemical role of VRK1 in the progression of the cell cycle, these data implicate VRK1 in the mitotic regulation of spermatogonial stem cell maintenance. To elucidate the molecular mechanisms of VRK1 in male germ cells, further investigation is expected to clarify this important issue.