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The kinase VRK1 is required for normal meiotic progression in mammalian oogenesis

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The kinase VRK1 is required for normal meiotic progression in mammalian oogenesis
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  The kinase VRK1 is required for normal meiotic progressionin mammalian oogenesis Carolyn S. Schober  a , Fulya Aydiner  b,1 , Carmen J. Booth  c , Emre Seli  b , Valerie Reinke  a, * a Department of Genetics, Yale University School of Medicine, New Haven, CT, USA b Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, CT, USA c Section of Comparative Medicine, Yale University School of Medicine, New Haven, CT, USA A R T I C L E I N F O Article history: Received 2 December 2010Received in revised form19 January 2011Accepted 20 January 2011Available online 26 January 2011 Keywords: VRK1OogenesisMeiosisp53MouseA B S T R A C TThe kinase VRK1 has been implicated in mitotic and meiotic progression in invertebratespecies, but whether it mediates these events during mammalian gametogenesis is notcompletely understood. Previous work has demonstrated a role for mammalian VRK1 inproliferation of male spermatogonia, yet whether VRK1 plays a role in meiotic progres-sion, as seen in  Drosophila , has not been determined. Here, we have established a mousestrain bearing a gene trap insertion in the VRK1 locus that disrupts  Vrk1  expression. Inaddition to the male proliferation defects, we find that reduction of VRK1 activity causesa delay in meiotic progression during oogenesis, results in the presence of lagging chro-mosomes during formation of the metaphase plate, and ultimately leads to the failure of oocytes to be fertilized. The activity of at least one phosphorylation substrate of VRK1,p53, is not required for these defects. These results are consistent with previously definedfunctions of VRK1 in meiotic progression in  Drosophila  oogenesis, and indicate a con-served role for VRK1 in coordinating proper chromosomal configuration in femalemeiosis.   2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Gametogenesis is a fundamental biological process and anecessary precursor to sexual reproduction. In mammals,the production of oocytes in females or sperm in males in-volves a sexually dimorphic progression of germ cells throughmeiosis. However, errors that arise during this intricate pro-cesscanresultininfertility.Errorsduringfemalemeiosismostoften lead to aneuploidy, which is estimated to cause approx-imately one-third of human spontaneous abortions, or resultin physical and/or mental disabilities if the conception resultsinafulltermpregnancy(HassoldandHunt,2001).Defectsthatoccurduringtheprocessofspermatogenesisinmalescanalsolead to infertility, although most diagnoses are often idio-pathicinnature(MatzukandLamb,2002,2008).Giventhesen-sitive nature of infertility, along with the ethical implicationsof working with human material, the study of mammaliangametogenesis has relied heavily upon rodent models. How-ever, many of the genes that govern this process, particularlythose controlling meiotic events, remain elusive (Handel andSchimenti, 2010). Consequently, gametogenesis requires con-tinued investigation in order to further understand the prop-erly coordinated production of oocytes and sperm andultimately, infertility.Vaccinia related kinase-1 (VRK1) is a serine/threoninekinase that has recently been implicated in gametogenesis 0925-4773/$ - see front matter    2011 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.mod.2011.01.004* Corresponding author.E-mail address: valerie.reinke@yale.edu (V. Reinke). 1 Present address: Department of Obstetrics, Gynecology and Reproductive Sciences, Dartmouth Hitchcock Medical Center, Lebanon,NH, USA. M E C H A N I S M S O F D E V E L O P M E N T  128 (2011) 178  –  190 available at www.sciencedirect.comjournal homepage: www.elsevier.com/locate/modo  in multiple species. Evidence from the study of the  Drosophila homolog of VRK1, nucleosomal histone kinase-1 (NHK-1), hasdemonstrated that mutations in NHK-1/VRK1 lead to abnor-mal chromosome structure and improper spindle formationduring female meiosis (Cullen et al., 2005; Ivanovska et al.,2005). Furthermore, a recent report has proposed that NHK-1/VRK1 acts as a downstream effector of the meiotic check-point in  Drosophila  oocytes (Lancaster et al., 2010). Additionalevidence implicating a role for VRK1 in germ cell develop-ment has also been demonstrated in the germline stem cellsof   Caenorhabditis elegans  where a reduction in proliferation,but not an increase in apoptosis, causes the sterility defectobserved in  vrk-1  mutants (Gorjanacz et al., 2007; Waterset al., 2010). In mammals, VRK1 has been shown to affectgerm cell proliferation in males, leading to sterility (Choiet al., 2010; Wiebe et al., 2010). Female mice lacking VRK1function are reported to be sterile as well, but the basis forthis phenotype is unknown.Multiple candidate phosphorylation substrates have beenidentified for VRK1 (reviewed in (Klerkx et al., 2009)). Mitoticchromatin modifications attributed to VRK1 include phos-phorylation of histones H3 at Ser10 (Kang et al., 2007) andH2A at Thr119 (Aihara et al., 2004; Brittle et al., 2007). Phos-phorylation of H2A at Thr119 is dependent upon NHK-1/VRK1 during female meiosis in  Drosophila  (Ivanovska et al.,2005). The nuclear pore protein barrier-to-autointegrationfactor (BAF), which also functions during mitosis to regulatethe association of chromatin with the nuclear envelope, isanother phosphorylation substrate of VRK1 (Gorjanaczet al., 2007; Lancaster et al., 2007; Nichols et al., 2006). Final-ly, VRK1 has been shown to affect transcription factors in-volved in cell cycle progression by targeting CREB (Kang et al., 2008), ATF2 (Sevilla et al., 2004b), c-JUN (Sevilla et al., 2004a), and the tumor suppressor p53 (Lopez-Borgesand Lazo, 2000). Most recently, the functional relationshipbetween p53 and VRK1 was investigated in vivo within thegerm line of   C. elegans , where VRK1 acts to inhibit the cellcycle arrest activity of p53 (Waters et al., 2010). However,whether this inhibitory relationship between VRK1 andp53 is conserved within the mammalian germ line remainsunknown.In this study, we investigated the role of VRK1 during mammalian gametogenesis by establishing mice carrying agene trap insertion within the  Vrk1  locus that disrupts  Vrk1 expression. Our results show that a reduction in VRK1causes sterility in both male and female mice. Male micehomozygous for the  Vrk1  gene trap allele, while fertile atfirst, display a progressive loss of spermatogenesis that re-sults in early onset sterility, similar to previous reports (Choiet al., 2010; Wiebe et al., 2010). Female homozygous mice arecompletely sterile and display multiple defects during oocytedevelopment, including delays in meiotic progression andlagging chromosomes during metaphase II. We also evalu-ated the relationship between VRK1 and p53, and found thatin contrast to the inhibitory relationship seen in  C. elegans germ cells, VRK1 and p53 have additive effects in mamma-lian germ cells. In sum, our results demonstrate that VRK1has both conserved and species-specific functions ingametogenesis. 2. Results 2.1. Generation of Vrk1 mutant mice In an effort to expand our studies of   Vrk1  into a mamma-lian system, we acquired the ES cell line RRR178 from theInternational Gene Trap Consortium. This cell line is hetero-zygous for an insertion between exons three and four of the Vrk1  locus of the pGT0Lxf vector, which includes a strong splice acceptor at the 5 0 end, a  b geo fusion transcript, and apoly A site at the 3 0 end (Fig. 1A). Splicing of the trap into Vrk1  resultsin an mRNA transcript that contains only the firstthree exons of Vrk1 followed by the  b geo sequence, therebyexcluding the kinase active domain, which is found in exonseven of the  Vrk1  locus. Based on the work described below,this truncated transcript is sufficient to cause a reduction of  Vrk1  wild-type expression. RRR178 ES cells were injected intomouseblastocysts andchimeric male offspringwith germcelltransmission were crossed to B6 females in order to establisha colony of mice containing the  Vrk1 Gt(RRR178)Byg allele (hereaf-ter referred to as  Vrk1 Gt ).We determined the exact site of the gene trap within  Vrk1 by conducting PCR with primer pairs spanning the length of the intron using DNA from a sample of the ES cells and thensequencing the product. The site of the gene trap insertionwas at +6176 of intron 3. We then established a PCR genotyp-ing strategy (Fig. 1B). Offspring from crosses between hetero-zygous  Vrk1 Gt micewere born at the expected Mendelian ratioof 1:2:1 of wild type ( Vrk1 +/+ ), heterozygous ( Vrk1 Gt/+ ) andhomozygous mutant ( Vrk1 Gt/Gt ) genotypes. All genotypeswereviable and healthy and both sexes were equally represented. 2.2. VRK1 is broadly expressed with high expression inthe testes and ovaries We first determined the expression profile of VRK1. Semi-quantitative RT-PCR conducted on  Vrk1 +/+ somatic tissues and Fig. 1 – Generation of   Vrk1  gene trapped mice. (A) The  Vrk1 locus is disrupted by insertion of the gene trap within thethird intron. Exons are depicted as numbered boxes.Primers used for genotyping are shown as lettered arrows.(B) An example of the products produced from a genotypingPCR using the primer set shown in A. Primers ‘a’ and ‘b’span the gene trap insertion site and create a product in theabsence of the gene trap from either one or both of the  Vrk1 alleles present in tail biopsies. Primers ‘a’ and ‘c’ create aproduct in the presence of the gene trap. M E C H A N I S M S O F D E V E L O P M E N T  128 (2011) 178  –  190  179  LacZ staining of whole mount  Vrk1 Gt/Gt embryonic day 13.5embryos revealed broad expression of VRK1 throughout themouse (data not shown, (Choi et al., 2010; Wiebe et al.,2010)). Northern blot analysis utilizing a probe directed tothe 3 0 end of   Vrk1  demonstrated that the transcript is ex-pressed in all tissues tested, with highest expression in boththe testes and ovaries aswell as in the thymus (Fig. 2A). Whencompared to  Vrk1 +/+ testes,  Vrk1 Gt/Gt testes have approxi-mately 70% reduction of Vrk1 expression, and  Vrk1 Gt/Gt ova-ries display almost 80% reduction. This expression patternand reduction of Vrk1 is consistent with a previous report of an independently generated mouse line carrying the samegene trap (Wiebe et al., 2010).Previous analysis of   Vrk1  expression within the testes indi-cated that Vrk1 is present in both the somatic Sertoli cells aswell as in the spermatogonia (Choi et al., 2010; Wiebe et al.,2010). We analyzed Vrk1 expression in the ovary of   Vrk1 Gt/Gt females by staining for LacZ, and found that LacZ staining was restricted to the developing oocyte, with no staining within the surrounding somatic granulosa cells (Fig. 2B).Notably, this staining only reflects the cells in which the Vrk1 Gt transcript is expressed, and does not reflect endoge-nous protein localization, as the truncated  Vrk1 Gt transcriptlacks the nuclear localization signal found in exon 11 of  Vrk1 . Nevertheless, this LacZ staining indicates that  Vrk1  isexpressed specifically within the oocyte. 2.3. Vrk1 Gt/Gt males display early onset sterility No initial phenotypes were observed in  Vrk1 Gt/Gt mice atbirth.However, afterreaching sexual maturity,  Vrk1 Gt/Gt maleswere only briefly fertile, and became sterile by ten weeks of age. In order to determine the severity of this phenotype,eight  Vrk1 Gt/Gt males and eight  Vrk1 Gt/+ males were each ma-ted with either  Vrk1 +/+ or  Vrk1 Gt/+ females over an eightmonth period beginning at 35 d.p.p. While the  Vrk1 Gt/+ maleswere able to sire litters with 100% success through 200 d.p.p.,and 75% success through 275 d.p.p., the  Vrk1 Gt/Gt males neversired a litter after 69 d.p.p. (Fig. 3A), despite the continuedpresence of copulatory plugs. Notably, while the male Vrk1 Gt/Gt mice used in this study were all fertile at the onsetof sexual maturity, an independently derived mouse line thatutilized the same gene trap resulted in a slightly more severephenotype, in which only a single litter was obtained fromone homozygous mutant male (Wiebe et al., 2010). The slightdiscrepancy between studies is most likely owing to the nat-ure of the gene trap and the amount of wild-type VRK1 thatis expressed due to leaky splicing around the gene trap inser-tion in the two separate mouse lines.Vrk1 Gt/Gt testes weigh significantly less than  Vrk1 +/+ testes(Fig. 3B), although there is no significant difference in bodymass between  Vrk1 +/+ and  Vrk1 Gt/Gt males at any age (  p  > 0.1,data not shown). The  Drosophila  homolog of VRK1, NHK-1, isrequired during meiosis, and has been implicated in the dis-assembly of the synaptonemal complex (SC) (Ivanovskaet al., 2005; Lancaster et al., 2010). The SC mediates properpairing, synapsis and recombination of homologous chromo-somes prior to exit from meiotic prophase and entry intometaphase I (G2/MI transition), and failure to make this tran-sition during mammalian spermatogenesis results in a mei-otic arrest at prophase I and a decrease in testes weight(Sun et al., 2010). To address whether  Vrk1 Gt/Gt males mighthave meiotic defects, we assessed the formation of the SCboth prior to sexual maturity at 17 d.p.p. as well as at Fig. 2 – Expression analysis of   Vrk1  gene trapped mice. (A) Northern blot depicting knockdown of the  Vrk1  transcript (top)using  b actin as a loading control. Normalized expression is shown below. Tissue abbreviations are as follows: Te, testis; Ov,ovary; Sm In, small intestine; Li, liver; Ki, kidney; He, heart; Th, thymus; Lu, lung; and Br, brain. (B) LacZ expression of the Vrk1  gene trap is observed within the oocytes of frozen ovarian sections from a  Vrk1 Gt/Gt  mouse (right), but not in controlsections from a  Vrk1 +/+ mouse (left). Original magnification is 25 · . 180  M E C H A N I S M S O F D E V E L O P M E N T  128 (2011) 178  –  190  77 d.p.p., after the onset of sterility. Using an antibody againstthe lateral unit of the SC, we found that the SC appears toform normally on chromosomes in  Vrk1 Gt/Gt spermatocytes(Fig. 3C).Meiotic defects that prevent a proper G2/MI transition canlead to an arrest of spermatogenesis at prophase I, whichwould result in the presence of only spermatogonia and sper-matocytes within the seminiferous tubules. However, we ini-tially observe all stages of spermatogenesis, including maturespermatids, within the seminiferous tubules of   Vrk1 Gt/Gt males during the first several weeks after sexual maturity(Fig. 3D, 6 and 10 weeks). Instead, histologic evaluation of tes-tes sections revealed a progressive reduction of all stages of spermatogenesis within  Vrk1 Gt/Gt testes beginning at10 weeks, that resulted in a significant loss of germ cells atall stages of meiosis and differentiation, with marked loss/degeneration of the seminiferous tubular epithelium, by14 weeks (Fig. 3D, 10 and 14 weeks). In sum, we observe nodetectable meiotic arrest, but rather a progressive loss of germ cells at all stages of differentiation.Two previous reports demonstrated that the spermatogen-esis defect in mice with decreased VRK1 function in the germline can be attributed to a loss of both differentiated andundifferentiated spermatogonia, and a decrease in overallproliferation within the testes (Choi et al., 2010; Wiebe et al.,2010). This data is consistent with our findings, althoughthe onset of sterility is slightly delayed in our  Vrk1 Gt/Gt mice.Overall the phenotypes closely mirror each other, and provideindependent evidence that a deficiency in VRK1 activity im-pedes the process of spermatogenesis over time. Fig. 3 –  Vrk1 Gt/Gt  males become sterile due to spermatogenesis defects. (A) Sterilityof   Vrk1 Gt/Gt  maleswas assessed during theage ranges shown and successful mating attempts were recorded.  n  = 8 per genotype. (B) Testes from mice were obtained at the ages shown and weighed. Error bars indicate standard deviation.  n P 4 per each genotype per each age. Student’s  t   test   p  values  *  p  < 0.0035,  **  p  < 0.00034. (C) Spermatocytes from  Vrk1 +/+ and  Vrk1 Gt/Gt  micewere stainedwith an anti-SCP1(green) andDAPI.Scalebarsare 10  l m. (D)Representativesectionsoftestes from  Vrk1 +/+ and  Vrk1 Gt/Gt  micereveal a progressivedeclineinspermatogenesis and loss/degeneration of the seminiferous tubular epithelium (arrowheads). Representative spermatids arecircled. Scale bars are 50  l m. M E C H A N I S M S O F D E V E L O P M E N T  128 (2011) 178  –  190  181  2.4. Vrk1 Gt/Gt  females are infertile Unlike the male  Vrk1 Gt/Gt mice, the female  Vrk1 Gt/Gt micenever produced any litters. To monitor this phenotype moreprecisely, we set up a mating study in which  Vrk1 Gt/Gt femaleswere mated with  Vrk1 +/+ males of proven fertility over thecourse of six months. To assess female fertility at the earliestpoint upon sexual maturity, six  Vrk1 Gt/Gt females were firstmated at five weeks of age. Additionally, five  Vrk1 Gt/Gt femaleswere first mated at eight weeks of age, for a total of eleven Vrk1 Gt/Gt females tested. Deposition of seminal fluid was ob-served each time a female was placed with a male, indicating that a copulation event took place, yet there were no signs of pregnancy and no litters ever resulted.To further explore this phenotype, we examined ovarianhistology.  Vrk1 Gt/Gt ovaries contain all stages of folliculogene-sis including corpora lutea, which indicate successful ovula-tion events. Histologic comparison of   Vrk1 +/+ and  Vrk1 Gt/Gt ovaries did not uncover any obvious differences (Fig. 4). Thisobservation led us to hypothesize that the sterility of   Vrk1 Gt/ Gt females might occur because of post-ovulation defects. 2.5. Oocytes from Vrk1 Gt/Gt  females are not fertilized In order to assess embryonic development, we matedsuperovulated females with  Vrk1 +/+ males and then obtainedpotentially fertilized oocytes from the ampullae of plugged fe-males the following morning. These oocytes were culturedover a four day period to monitor embryonic development.Embryos from both the  Vrk1 +/+ and  Vrk1 Gt/+ females devel-oped past the two-cell and morula stages and formed blasto-cysts (Fig. 5A and B). However, no embryonic developmentwas detected in most oocytes from  Vrk1 Gt/Gt mice. Some oo-cytes obtained from  Vrk1 Gt/Gt mice did progressto the two-cellstage, but not beyond. Moreover, unmated  Vrk1 +/+ control fe-males produced similarnumbersof two-cellembryos (  p  > 0.2),indicating that such embryos were probably the result of parthenogenesis.To investigate whether oocytes from  Vrk1 Gt/Gt femaleswere fertilized, we examined formation of pronuclei. Whilezygotes with visible maternal and paternal pronuclei were re-trieved from both  Vrk1 +/+ and  Vrk1 Gt/+ mice, we never ob-served both maternal and paternal pronuclei in oocytesobtained from  Vrk1 Gt/Gt mice (Fig. 5C). Moreover, we notedthat many oocytes from  Vrk1 Gt/Gt mice failed to reach MII ar-rest, as there were also earlier stages of meiosis represented(Fig 5B and note that the oocyte in Fig. 5C is actually a GVoo- cyte). Therefore, oocytes from  Vrk1 Gt/Gt mice do not appear tobe fertilized despite the fact that sperm are observed withinthe oviduct of   Vrk1 Gt/Gt females, and in contact with oocytes(black arrowheadsin Fig. 5A), when oocytes are obtained fromthe ampulla. Together, these findings led us to conclude thatdefects in oogenesis impair fertilization but not ovulation.This conclusion differs from  Drosophila , where disruption of NHK-1/VRK1 does not impede fertilization (Ivanovska et al.,2005). 2.6. Oocytes from Vrk1 Gt/Gt mice have developmentaldeficiencies Superovulation of   Vrk1 +/+ and  Vrk1 Gt/Gt mice producescomparable numbers of oocytes (22.1 ± 9.5 versus 21.5 ± 13.6,respectively,  n  = 13 stimulations). However, the prevalence of oocytes displaying proper polar body extrusion was signifi-cantly less in oocytes from  Vrk1 Gt/Gt mice compared to Vrk1 +/+ oocytes (25% versus 87.7%, respectively,  p  < 0.0005).To assess oocyte development more precisely, we hormonallystimulated  Vrk1 +/+ and  Vrk1 Gt/Gt mice, obtained GV stage oo-cytes from antral follicles, and followed oocyte developmentin culture.After incubation for 20 h invitro, we found a signif-icant difference in developmental progression between oo-cytes from  Vrk1 +/+ and  Vrk1 Gt/Gt mice. While 59.9% of theoocytes from  Vrk1 +/+ females displayed polar body extrusion,only 27.9% of oocytes from  Vrk1 Gt/Gt females progressed tothis stage (  p  < 0.0005). Although germinal vesicle breakdown(GVBD) appeared to take place normally in oocytes from Vrk1 Gt/Gt mice (  p  > 0.02), 67.7% progressed through GVBD butfailed to extrude a polar body versus only 39.2% from  Vrk1 +/ + females (  p  < 0.0005), indicating meiotic delays.To distinguish more precisely the stage at which these de-lays arise duringoogenesisin  Vrk1 Gt/Gt mice, we followedmei-otic oocyte development in vitro from the GV stage throughmetaphase II (Fig. 6A). We initially detected similar numbersof rimmed and condensed oocytes, as described in (Canet al., 2003) from both  Vrk1 +/+ and  Vrk1 Gt/Gt mice. Subse-quently, significantly fewer oocytes from  Vrk1 Gt/Gt femalesreached the ‘‘parachute’’ stage, a point at the completion of GVDB during which bivalents clump together prior to en-trance into prometaphase I as described in (Debey et al.,1993), within 2.5 h, compared to  Vrk1 +/+ females (15.6% versus49.1% respectively,  p  < 0.0005). Yet, we again found compara-ble numbers of oocytes from  Vrk1 +/+ and  Vrk1 Gt/Gt females atlater time points corresponding to prometaphase and meta-phase I, indicating that the mutant oocytes had recovered Fig. 4 – Ovarian histopathology from  Vrk1 +/+ and  Vrk1 Gt/Gt  mice. Shown here are representative sections of ovariesstained with hematoxylin and eosin from 4 week-old  Vrk1 +/ + (left) and  Vrk1 Gt/Gt  mice (right). No significant morphologicdifferences in preantral follicles (arrowheads), maturefollicles ( * ), or corpora lutea (CL) were detected in serialsections of ovaries from multiple mice from each genotype.Scale bars are 500  l m. 182  M E C H A N I S M S O F D E V E L O P M E N T  128 (2011) 178  –  190
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