The mammalian vaccinia-related kinases (VRK1, VRK2, and VRK3) form a distinct branch of the casein kinase family. A single VRK homolog is encoded in the genomes of D. melanogaster
and C. elegans,
and the early developmental arrest that accompanies ablation of its expression indicates that this kinase is essential [15
]. With regard to dVRK, also known as NHK-1, the most severe alleles cause an embryonic lethal phenotype, whereas milder, hypomorphic alleles spare viability but cause male and female sterility [15
]. Diminished NHK-1 function causes meiotic defects associated with aberrant chromatin modification, spindle formation, chromosome condensation, and karyomere formation. RNAi-mediated depletion of NHK-1 in cultured S2 cells has adverse effects on mitosis, leading to irregular chromosome condensation and to the presence of unaligned and lagging chromosomes [15
]. Similar phenotypes have been observed for C. elegans
, in which siRNA-mediated depletion of cVRK was shown to cause aberrant meiotic and mitotic spindles as well as altered chromatin organization [30
In mammals, VRK1 is the ortholog most closely related to the D. melanogaster
and C. elegans
VRK enzymes, in that it is catalytically active and exhibits a nuclear localization. The present study represents to our knowledge the first genetic analysis of the function of VRK1 in mice. The GT3 allele contains a βgeo gene-trap insertion within intron 3 that should lead to the expression of a highly truncated fusion protein encoding only 72 amino acids of VRK1 fused to βgeo. As is often seen [21
], splicing to the gene trap is incomplete, and approximately 15–20% of the Vrk1
transcripts splice around the trap and express full-length VRK1. Thus, the GT3/GT3 mice are, in fact, hypomorphic for VRK1.
Examination of hypomorphic models has been extremely useful for highlighting cell types and pathways that are most sensitive to the loss of a protein while leaving many other tissues unaffected. For example, complete disruption of the LKB1 and PDK1 kinases causes embryonic arrest, but hypomorphic (10–20% of wild-type levels) expression of these kinases is sufficient to support development and allow further study of the kinases [33
]. Likewise, our work shows that diminished VRK1 expression has no apparent impact on development, is compatible with viability, and is well tolerated by many tissues and organs. Given the demonstration that VRK is essential in other model organisms, we believe that the hypomorphic nature of the GT3 allele enabled us to avoid an embryonic lethal phenotype and uncover the vital role of VRK1 in spermatogenesis. The fact that the mice are viable and healthy may suggest VRK1 is less important in other tissues, perhaps because of redundancy within the VRK family or with other Ser/Thr protein kinases. The protein most similar to VRK1 is VRK2, which may be able to fulfill some of the roles played by VRK1. Interestingly, a possible role for VRK2 itself in spermatogenesis was suggested by the fact that Pog
−/− mice, which lack the Pog
(proliferation of germ cells) gene, have a milder infertility defect than gsd
(germ-cell deficient) mice, which lack both Pog
and the adjacent Vrk2
Our observation that VRK1 was expressed in Sertoli cells and spermatogonia extends an initial report of human VRK1 expression within seminiferous tubules [36
]. Our expression data, combined with the infertility of GT3/GT3 mice, led us to examine the impact of VRK1 deficiency on spermatogenesis. Gross examination of GT3/GT3 testes indicated that in postpubertal mice, these testes were significantly smaller than those of their +/+ counterparts. Subsequent histological analysis of these testes was extremely informative, because the cell types responsible for spermatogenesis can be readily observed in each seminiferous tubule. Briefly, undifferentiated (type A) spermatogonial stem cells are found at the periphery of the tubule along with the Sertoli cells. Spermatogonial stem cells are capable of both self-renewal and differentiation; expression of the transcription factor ZBTB16 is a defining characteristic of these cells [28
]. Differentiation produces type B spermatogonia; these cells express markers such as KIT and PCNA and form the population that will undergo meiosis. As the early meiotic cells known as primary spermatocytes become secondary spermatocytes and then spermatids, they move continually closer to the lumen of the tubule. By determining which cell types were absent in the GT3/GT3 testes, we sought to pinpoint the steps at which VRK1 expression was critical. In young males 2–6 wk of age, little difference could be observed between the +/+ and GT3/GT3 testes. Both +/+ and GT3/GT3 mice possessed all cell types expected to be present at a particular time in development, leading us to conclude that VRK1 deficiency does not impede the first postnatal wave of spermatogenesis that occurs during this time. However, during the next few weeks, the GT3/GT3 mice exhibit a progressive loss of GCNA+, PCNA+, and KIT+ premeiotic cells. As a result, all later stages of meiotic cells were also progressively depleted. However, the pool of undifferentiated ZBTB16+ cells remained constant over this time period. Thus, VRK1 is not necessary to maintain the earliest spermatogonial lineage, unlike the transcriptional repressor ZBTB16 [28
]. Therefore, we postulate that VRK1 is needed either for the differentiation of type A spermatogonial stem cells into type B spermatogonia, the ongoing proliferation of type B cells required for the homeostasis of the precursor population, or both. In this regard, the phenotype of VRK1-deficient mice is similar to that of mice lacking either the Sohlh-1 or -2 transcriptional regulators; the testes of mice lacking these proteins retain ZBTB16+ cells but lack other differentiated cell types [37
]. However, whereas the expression of Sohlh-1 or -2 is limited to spermatogonia, VRK1 appears to be expressed both in Sertoli cells and in the basal layer of spermatogonia. This expression profile indicates that the loss of the differentiating pool of spermatogonia in VRK1-deficient testes might be the result of a cell-autonomous role for VRK1 in spermatogonial differentiation and/or proliferation. Alternatively, wild-type levels of VRK1 expression in the supporting Sertoli cells might be needed for them to provide the requisite signals to the spermatogonia. Such a requirement has been found for the Sertoli-specific factors ERM and Kit ligand (KITL), the absence of which can lead to a “Sertoli Cell Only” phenotype in the seminiferous tubules of adult mice [39
]. Distinguishing between these two possibilities is an important goal for future studies. However, semiquantitative RT-PCR analysis revealed no significant difference in the expression of KL between +/+ and GT3/GT3 testis (Supplemental Fig. S6), suggesting that the VRK1-deficient Sertoli cells retain the ability to express at least this critical paracrine factor.
On a molecular level, the impact of VRK1 depletion likely is multifactorial. The VRK1/BAF pathway has been examined in mammalian tissue culture and has been documented via genetic analysis of model organisms [16
]. VRK1-mediated phosphorylation of BAF inhibits its ability to bind DNA and interact with protein partners. Thus, dynamic phosphorylation of BAF likely is a positive regulator of nuclear envelope breakdown and a negative regulator of nuclear envelope reformation and of BAF-mediated recruitment of chromatin to the nuclear periphery. Indeed, in C. elegans
, VRK1 depletion has been shown to reduce BAF phosphorylation and to cause defects in nuclear envelope reformation at the end of mitosis. Aberrant nuclear envelope structures were also observed upon expression of a BAF mutant that exhibited reduced phosphorylation in comparison to the wild-type protein, providing correlative evidence that the VRK1-BAF pathway is causing the observed phenotype. In D. melanogaster
, comparable sterility phenotypes are seen upon depletion of VRK1 or expression of a nonphosphorylatable variant of BAF [23
]. In both cases, formation of the meiotic karyosome is impeded by aberrant recruitment of the chromosomes to the nuclear periphery. It seems highly likely that the VRK/BAF axis will be evolutionarily conserved, and that future studies will confirm VRK1-mediated phosphorylation of BAF plays important roles in mitosis and meiosis by regulating nuclear envelope dynamics.
Murine VRK1 may exert a pro-proliferative role by phosphorylating other substrates in mitosis or, indeed, during distinct phases of the cell cycle. Phosphorylation of histone H3 has been reported; this phosphorylation would be likely to affect chromosome condensation during mitosis. VRK1 has recently been shown to interact with RAN and with polo-like kinase 3 [42
]. RAN not only is involved in nucleocytoplasmic transport but also plays an important role in mitotic spindle assembly and chromosome segregation [44
]; PLK3 is implicated in a signaling cascade that affects microtubule dynamics [45
] as well as Golgi fragmentation [46
]. VRK1 has also been reported to phosphorylate and activate the transcription factor CREB, which stimulates the expression of cyclin D, an important mediator of the G1
/S transition. This pathway may also be important in the pro-proliferative role of VRK1.
Although the present study is focused solely on the male infertility defect in the hypomorphic Vrk1 mice, it is also intriguing that VRK1 is highly expressed in the spleen and that the proliferative markers PCNA and phosphohistone H3(Ser10) are greatly reduced in the spleens of VRK1-deficient mice. These data suggest that other proliferative pathways, such as erythropoiesis, which occurs in the murine spleen, might be impaired in the VRK1-deficient mice. Moreover, our studies indicate that female mice homozygous for the hypomorphic Vrk1 allele are also infertile. It will be of great interest to determine the basis for this infertility. The studies described herein indicate that VRK1 plays essential and evolutionarily conserved roles in gametogenesis and fertility in worms, flies, and mammals. The VRK1-deficient line of mice that we have established will provide a fruitful system with which to examine the important contributions of this kinase to cell proliferation and gametogenesis.