Premature ovarian failure (POF), a major cause of female infertility, is a complex disorder, but the molecular mechanisms underlying the disorder are only poorly understood. Here we report that protein kinase CK2 contributes to maintaining follicular survival through PI3K/AKT pathway and DNA damage response pathway. Targeted deletion of CK2β in mouse oocytes from the primordial follicle stage resulted in female infertility, which was attributed to POF incurring by massive follicle atresia. Downregulated PI3K/AKT signaling was found after CK2β deletion, indicated by reduced level of phosphorylated AKT (S473, T308, and S129) and altered AKT targets related to cell survival. Further studies discovered that CK2β-deﬁcient oocytes showed enhanced γH2AX signals, indicative of accumulative unrepaired DSBs, which activated CHK2-dependant p53 and p63 signaling. The suppressed PI3K/AKT signaling and failed DNA damage response signaling probably contribute to large-scale oocyte loss and eventually POF. Our ﬁndings provide important new clues for elucidating the mechanisms underlying follicle atresia and POF. chemotherapy Introduction , with genetic factors being the main cau- Premature ovarian failure (POF) refers to amenorrhea in ses. Accumulating genes in the X chromosome such as 4,10 11 12–14 women of less than 40 years of age accompanied by ele- FMR1 , FMR2 , BMP15 and genes in the auto- 15,16 17 18 vated menopausal levels of serum gonadotropins (follicle- some such as FOXL2 , FSHR , LH receptor , and 19,20 stimulating hormone, FSH > 40 IU/l) and decreased inhibin A are known to be involved in POF; however, estrogen . POF is an ovarian dysfunction characterized by for many years, the underlying mechanisms of POF have premature depletion of ovarian follicles in ~1% of women largely remained unknown. under the age of 40 years and 0.1% under the age of 30 Protein kinase CK2 is a ubiquitous serine/threonine years , which usually leads to female infertility. The causes protein kinase that is a heterotetramer consisting of two of POF vary and are complex, and it includes genetic regulatory subunits, CK2β, and two catalytic subunits, 3–5 6,7 21 aberrations , autoimmune ovarian damage , ther- CK2α and CK2α’ . Depending on speciﬁc functions, the apeutic interventions such as radiotherapy and catalytic and regulatory subunits may exist in the forms of 22,23 α2β2, α’2β2, αα’β2, or unassembled molecules . CK2 displays functions in numerous cellular processes by Correspondence: Q-Y. Sun (email@example.com) or W-P. Qian (qianweipingsz@126. participating in multiple signaling pathways, including com) 24–27 28–31 PI3K/AKT , Wnt/β-catenin , and the DNA Department of Reproductive Medicine, Peking University Shenzhen Hospital, 32–36 damage response pathway . Extensive studies have 518036 Shenzhen, Guangdong, China State Key Laboratory of Stem Cell and Reproductive Biology, Institute of shown that CK2 is involved in the promotion of cell Zoology, Chinese Academy of Sciences, 100101 Beijing, China survival and anti-apoptotic functions in normal and Full list of author information is available at the end of the article These authors contributed equally: Qiu-Xia Liang, Zhen-Bo Wang, Fei Lin. Edited by P. Agostini © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Ofﬁcial journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; Liang et al. Cell Death and Disease (2018) 9:508 Page 2 of 11 checkpoint by targeting or interacting with proteins such 22 48 49 as CHK1 , CHK2 and BRCA1 . Generally, CK2 func- tions as a monitoring hub of the cell cycle and apoptosis, integrating diverse signals into the appropriate cellular responses. −/− CK2α embryos die in mid-gestation, with defects in 50 −/− heart and neural tube . CK2α’ females show normal fertility, but males are infertile. Male mice lacking CK2α’ show extensive germ cell apoptosis characterized by nuclear abnormalities ranging from spermatogonia to 51,52 −/− early spermatids . CK2β mice die shortly after implantation with no signs of apoptosis but reduced cell −/− proliferation. CK2β blastocysts cannot develop an inner cell mass in vitro . Zygote-speciﬁc knockout of CK2β is destructive for embryonic stem cells and primary embryonic ﬁbroblasts . The above studies demonstrate that CK2β is indispensable for cell survival. However, the roles of CK2β in folliculogenesis/oogenesis are largely unknown. Here we targeted CK2β for deletion in oocytes ﬂ/ﬂ from the primordial follicle stage by crossing Ck2β mice with Gdf9-Cre mice. We found that CK2β was essential for female fertility and loss of CK2β caused ovarian follicle atresia and POF. Results Fig. 1 The expression and oocyte-speciﬁc deletion of CK2β.a Oocyte-speciﬁc deletion of Ck2β gene causes mice Immunoblotting showing the expression pattern of CK2β in mouse infertility oocytes at different developmental stages. A total 150 oocytes were collected after being cultured for 0, 4, 8, and 12 h, corresponding to Toward determining the potential roles of CK2β in the GV, GVBD, MI, and MII stages, respectively. β-actin was detected as an female reproductive system, expression patterns of internal control. b Immunohistochemistry analysis of the expression CK2β in ovaries and oocytes at different developmental pattern of CK2β in primordial, primary, secondary, and tertiary follicles stages were assessed through immunoblotting and at PD21. c Immunohistochemistry detection of CK2β loss in oocytes of ﬂ/ﬂ + immunohistochemistry. Immunoblotting revealed that Ck2β ;GCre mice. Scale bar: 50 μm CK2β was stably and highly expressed in GV, GVBD, MI, and MII oocytes (Fig. 1a). Within the ovary, immunohistochemistry revealed that CK2β located in cancer cells. In HEK-293T cells, CK2 phosphorylates the nuclei of oocytes and granulosa cells from pri- AKT/PKB at Ser129, preventing AKT Thr308 depho- mordial follicles to antral follicles (Fig. 1b). These data sphorylation and enhancing the catalytic activity of AKT/ suggest that CK2β potentially functions in folliculo- 24,30,37 PKB , which in turn phosphorylates β-catenin at genesis/oogenesis. Ser552 and promotes its nuclear localization, eventually To conﬁrm our hypothesis, we generated oocyte- strengthening resistance to apoptosis by upregulating speciﬁc CK2β mutant mice by crossing Ck2β mice in anti-apoptotic survivin gene transcription . CK2 reg- which exon I–II were targeted with transgenic mice ulates PI3K signaling by phosphorylating PTEN C ter- expressing Gdf9 promotor-driven Cre recombinase 38,39 minus , which enhances the stability and inhibits the (Supplementary Figure1). In Gdf9-Cre mice, Cre was 38–40 activity of PTEN . In leukemia cells, CK2 inhibits expressed from primordial to later follicular stages. His- ﬂ/ﬂ + Ikaros, a tumor suppressor, which can repress the tran- tological analysis of Ck2β ;GCre mouse ovaries showed scription of genes promoting the PI3K pathway . Since loss of CK2β localization in nuclei of oocytes, indicating CK2 was ﬁrst reported to facilitate DNA single-strand functional deletion of CK2β (Fig. 1c). breaks repair , accumulating evidence has shown that To observe the effect of oocyte-speciﬁc deletion of CK2 is involved in DNA damage repair pathway. CK2 CK2β on fertility, a breeding assay was conducted by ﬂ/ﬂ ﬂ/ﬂ + regulates the G1/S DNA damage checkpoint by targeting mating Ck2β or Ck2β ;GCre female mice with wild- 41,42 43,44 p53 , p53 regulatory proteins (e.g., MDM-2) and type males of tested fertility for 6 months. Continuous WAF1/CIP1 45,46 ﬂ/ﬂ + Cdk-inhibitory proteins such as p21 and breeding assessment indicated that Ck2β ;GCre KIP1 47 p27 . CK2 also contributes to the G2/M damage females were completely infertile (Fig. 2a). Ofﬁcial journal of the Cell Death Differentiation Association Liang et al. Cell Death and Disease (2018) 9:508 Page 3 of 11 ﬂ/ﬂ + ﬂ/ﬂ ﬂ/ﬂ Fig. 2 Infertility and follicle atresia in Ck2β ;GCre mice. a Comparison of the accumulative number of pups per Ck2β female and Ck2β ; + ﬂ/ﬂ ﬂ/ﬂ GCre female for 6 months. At least ﬁve mice of each genotype were used in this assay. b Ovary weight to body weight ratio of Ck2β and Ck2β ; GCre mice at 3, 4, 6, and 8 weeks of age after birth. For each time point, at least three mice of each genotype were used for analysis. Data are ﬂ/ﬂ ﬂ/ﬂ + presented as the mean ± SEM. P < 0.05(*), 0.01(**) or 0.001(***). c–j Representative ovarian histology of Ck2β and Ck2β ;GCre mice of 3, 4, 6, and 8 weeks of age, respectively. Images c’–j’ correspond to the partial magniﬁcation of images c–j. Yellow arrowheads in f’, h’, and j’ indicate atretic follicles. For each time point, at least three mice of each genotype were used for analysis. Scale bar: 100 μm Ablation of Ck2β gene expression in oocytes at the 0.0124%, and 0.0071%. These data revealed that CK2β primordial follicle stage results in follicle atresia and POF deletion resulted in atrophy of ovaries in mice. To determine whether the infertility was due to ovarian To clarify the cause of ovarian atrophy, we next exam- ﬂ/ﬂ dysfunction and consequential functional oocyte loss, we ined the morphology of ovaries from both Ck2β and ﬂ/ﬂ ﬂ/ ﬂ/ﬂ + ﬁrst measured the size of ovaries from Ck2β and Ck2β Ck2β ;GCre mice. At 3 weeks of age, histological ﬂ + ﬂ/ﬂ ;GCre mice. As shown in Fig. 2b, the size of the ovaries assessment revealed that Ck2β mice showed normal ﬂ/ﬂ of Ck2β mice continued to increase from week 3 to ovarian morphology characterized by the presence of week 8, with a mean ovarian weight ratio of 0.0316%, primordial and activated follicles including primary, sec- 0.0326%, 0.0424%, and 0.0598% corresponding to week 3, ondary, and antral follicles (Fig. 2c, c’). All of these ﬂ/ﬂ + 4, 6, and 8, respectively. Compared to the control, the structures were also found in the Ck2β ;GCre mouse ﬂ/ﬂ + ovary size of Ck2β ;GCre mice decreased slightly in ovaries. Although a few follicles underwent atresia, the week 3 and decreased sharply from week 4 to week 8, with ovaries looked healthy on the whole (Fig. 2d, d’). At mean ovarian weight ratio of only 0.0221%, 0.0196%, 4 weeks of age, all types of follicles could be found in both Ofﬁcial journal of the Cell Death Differentiation Association Liang et al. Cell Death and Disease (2018) 9:508 Page 4 of 11 ﬂ/ﬂ ﬂ/ﬂ + Ck2β mice (Fig. 2e, e’) and Ck2β ;GCre mice (Fig. 2f, Ck2β deletion causes PI3K/AKT signaling hypoactivation ﬂ/ﬂ ﬂ/ﬂ f’). However, most follicles in the ovaries of Ck2β ; According to the above-mentioned research, Ck2β ; + + GCre mice showed signs of atresia (Fig. 2f’, yellow GCre mice displayed defects in primordial follicle sur- arrows) in contrast to control ovaries that contained vival. Accumulating literature indicates that PI3K/AKT substantial healthy-looking follicles (Fig. 2e’). At 6 weeks, signaling plays a vital role in regulating the survival of 54–56 the time of sexual maturity, massive atretic follicles primordial follicles during their long dormancy .In ﬂ/ (Fig. 2h’, yellow arrows) appeared in the ovaries of Ck2β view of the involvement of CK2 in the PI3K/AKT pathway ﬂ + ﬂ/ﬂ ;GCre mice (Fig. 2h, h’) compared to Ck2β mice at a cellular level, we ﬁrst focus our attention on the PI3K/ (Fig. 2g, g’). Although most atretic follicles maintained AKT pathway. Accordingly, we performed immunoblot- ﬂ/ﬂ ﬂ/ﬂ follicular structures, the oocytes in atretic follicles were ting analysis using ovaries from Ck2β and Ck2β ; eliminated and the space was ﬁlled with granulosa cells GCre mice at 2 weeks of age. It showed that the levels of ﬂ/ﬂ (Fig. 2h, h’). By 8 weeks of age, in contrast to Ck2β phosphorylated AKT (S473 and S129) decreased slightly, ovaries (Fig. 2I, i’), almost all types of follicles had been whereas phosphorylated AKT (T308) was markedly ﬂ/ﬂ + depleted in Ck2β ;GCre ovaries, and it was difﬁcult to reduced (Fig. 5a); when considering the elevated total ﬂ/ﬂ + ﬁnd follicular structures in the ovaries of Ck2β ;GCre level of AKT1/2/3 protein (Fig. 5a), the relative level of mice (Fig. 2j, j’). phosphorylated AKT (S473, T308, and S129) was Quantitative analysis revealed that the numbers of primary, decreased more obviously in CK2β mutant ovaries. PI3K/ ﬂ/ﬂ secondary, and antral follicles in the ovaries of Ck2β ; AKT regulator PTEN was also detected and it showed + ﬂ/ﬂ GCre mice were similar to those of Ck2β mice at 3 and decreased expression (Fig. 5a). Subsequently, the down- 4 weeks, but the number of primordial follicles within the stream of PI3K/AKT pathway was detected. We ﬁrst ﬂ/ﬂ + ovaries of Ck2β ;GCre mice was markedly reduced as examined the TSC2/mTOR signaling which was critical to ﬂ/ﬂ compared to Ck2β mice (Fig. 3a, b). At 6 weeks, in addi- oocyte survival. The results showed that the activity of tion to primordial follicles, signiﬁcant differences were also mTOR/rpS6 signaling pathway was enhanced in mutant observed in the secondary and antral follicles (Fig. 3c). By ovaries, as indicated by elevated levels of phosphorylated ﬂ/ﬂ 8 weeks of age, all types of follicles in ovaries of Ck2β ; mTOR (S2448), phosphorylated S6K (T389), and phos- GCre mice were signiﬁcantly decreased (Fig. 3d). In general, phorylated rpS6 (Ser240/244) (Fig. 5b). However, the the primordial follicles reduction occurred at 3 weeks of age levels of phosphorylated TSC2 (S1387) displayed no dif- and continued decreasing until depletion of the primordial ference between mutant and control ovaries (Fig. 5b). ﬂ/ﬂ + follicle pool at young adulthood (8 weeks) in Ck2β These results were inconsistent when considering that ;GCre ﬂ/ﬂ mice compared to Ck2β mice (Fig. 3e). The activated mTOR was negatively regulated by TSC2. The upregu- follicle reduction appeared at the time of onset of sexual lated mTOR/S6K/rpSK signaling may result from feed- maturity (4 weeks) and displayed a similar decreased trend back effects on defective follicle survival. Afterward, other from week 4 to week 6 for primordial follicles in the ovaries AKT substrates contributing to cell survival were further ﬂ/ﬂ + of Ck2β ;GCre mice (Fig. 3f). This phenotype resembles detected. As the ﬁrst reported AKT substrate which is premature ovarian failure (POF) in humans. important to cell survival, two isoforms of glycogen syn- The histological analysis indicated that absence of CK2β in thase kinase 3 (GSK3), GSK3α and GSK3β, were detected. oocytes caused follicular atresia and POF. To conﬁrm these The results showed that the level of GSK3α expressed in observations, we performed immunohistochemistry of the ovaries was higher than GSK3β (Fig. 5c), and the germ cell marker MVH on 3- and 8-week-old ovarian sec- expression levels of both phosphorylated GSK3α/β (S21/ ﬂ/ﬂ + tions. As shown in Fig. 4a, in ovaries of Ck2β ;GCre mice 9) and total GSK3α/β protein displayed no variation in at 3 weeks of age, most follicles including primordial, pri- control and mutant mice (Fig. 5c). We next detected mary, secondary, and antral follicles showed MVH-positive FOXO proteins which are also important cell survival- staining, suggesting that these follicles were healthy. How- related AKT substrates. The examination of the levels of ever, at 8 weeks of age, there were few MVH-positive pri- phosphorylated FOXO1 (T24)/FOXO3a (T32) found that mordial or primary follicles scattering in the cortical region in they were signiﬁcantly decreased (Fig. 5c), while the levels ﬂ/ﬂ + ovaries of Ck2β ;GCre mice, which was symptomatic of of total FOXO1 protein did not show obvious changes POF. TUNEL assay on ovarian sections showed that (Fig. 5c). increased granulosa cell apoptosis occurred in ovaries of ﬂ/ﬂ + Ck2β ;GCre mice at 4 weeks of age compared to ovaries Ck2β depletion results in accumulated DNA damage in ﬂ/ﬂ in Ck2β mice, which was caused by growing follicle atresia oocytes (Fig. 4b). The above data demonstrate that the accelerated Double-strand breaks derived from unrepaired meiotic demise of primordial follicles and defective survival of or environmental stress could result in oocyte elimination ﬂ/ﬂ growing follicles may be responsible for infertility of Ck2β ; and female infertility through CHK2-dependent activation + 57 GCre female mice. of p53 or p63 . Considering that CK2 is involved in the Ofﬁcial journal of the Cell Death Differentiation Association Liang et al. Cell Death and Disease (2018) 9:508 Page 5 of 11 ﬂ/ﬂ + Fig. 3 Decreased number of primordial and activated follicles in Ck2β ;GCre mice. a–d Quantiﬁcation of numbers of different types of follicles in ovaries at 3 weeks, 4 weeks, 6 weeks, and 8 weeks, respectively. Primordial, primary, secondary, and antral follicles were counted. For each time point, at least three mice of each genotype were used for analysis. Data are presented as the mean ± SEM. P < 0.05(*), 0.01(**) or 0.001(***). e–f Quantiﬁcation of numbers of primordial follicles (e) and activated follicles (f) in ovaries at 3 weeks, 4 weeks, 6 weeks, and 8 weeks, respectively. For each time point, at least three mice of each genotype were used for analysis. Data are presented as the mean ± SEM. P < 0.05(*), 0.01(**) or 0.001(***) 32,34–36 ﬂ/ﬂ DNA damage response pathway , we wonder whe- (S15) showed no difference in ovaries of Ck2β and −/− ﬂ/ﬂ + ther Ck2β oocyte depletion relates to this pathway. Ck2β ;GCre mice. However, by 4 weeks of age, the Thus, we ﬁrst performed immunoblotting using ovary level of p63 was downregulated while phosphorylated p53 ﬂ/ﬂ ﬂ/ﬂ + ﬂ/ﬂ + lysates from Ck2β and Ck2β ;GCre mice at 2 weeks (S15) was upregulated in ovaries of Ck2β ;GCre mice of age. As shown in Fig. 6a, the levels of phosphorylated (Fig. 6b), consistent with the antagonizing relationship CHK2 (T68) were slightly increased and γH2AX was between p53 and p63 . Moreover, we conﬁrmed our ﬂ/ﬂ + signiﬁcantly upregulated in ovaries of Ck2β ;GCre ﬁndings using immunoﬂuorescence analysis. As indicated mice, whereas the levels of p63 and phosphorylated p53 in Fig. 6c, the γH2AX signals in small oocytes from Ofﬁcial journal of the Cell Death Differentiation Association Liang et al. Cell Death and Disease (2018) 9:508 Page 6 of 11 ﬂ/ﬂ + Ck2β ;GCre mice at 2 weeks of age were remarkably enhanced. Taken together, these data suggest that CK2β depletion causes DSBs accumulation and failed activation of the DNA damage response pathway. Oocyte-speciﬁc deletion of CK2β causes a striking reduction in CK2α but not CK2α’ expression To explore the forms of CK2 functions in the mouse ovary, immunoblotting was carried out to detect protein ﬂ/ﬂ levels of CK2α, CK2α', and CK2β in ovaries of Ck2β ﬂ/ﬂ + and Ck2β ;GCre mice (Fig. 7). As expected, the level of CK2β protein was dramatically reduced in ovary extracts ﬂ/ﬂ + prepared from Ck2β ;GCre mice compared with ﬂ/ﬂ Ck2β mice. The low level of CK2β protein that was ﬂ/ﬂ detected in the ovary extracts collected from Ck2β ; GCre mice likely came from granulosa cells in which CK2β was not deleted. Meanwhile, the level of CK2α protein was signiﬁcantly downregulated in ovaries of ﬂ/ﬂ + Ck2β ;GCre mice, while the levels of CK2α' protein in ﬂ/ﬂ + ovaries of Ck2β ;GCre mice showed no variation ﬂ/ﬂ compared with Ck2β mice. Immunoblotting analysis of phospho-CK2 substrate using ovaries from control and mutant mice found that CK2 activity was largely reduced (Fig. 7b). These data suggest that CK2 is presumably functioning in oocytes in the forms of α β and the 2 2 ﬂ/ﬂ reduced expression of CK2α protein in ovaries of Ck2β ; ﬂ/ﬂ Fig. 4 Premature ovarian failure in Ck2β ;GCre+ mice. a Germ GCre mice is probably due to degradation resulting from ﬂ/ﬂ cells marker MVH immunohistochemistry of ovaries of Ck2β and decreased stability of CK2α protein without CK2β. ﬂ/ﬂ + Ck2β ;GCre at 3 and 8 weeks after birth. At least three mice of each genotype were used in this assay. Scale bar: 100 μm. b TUNEL ﬂ/ﬂ ﬂ/ﬂ Discussion immunoﬂuorescence staining of the ovaries of Ck2β and Ck2β ; In humans, the primordial germ cells (PGCs) migrate to GCre at 4 weeks after birth. Green: TUNEL-positive signal; Blue: DAPI. At least three mice of each genotype were used for analysis. Scale bar: gonadal ridges and are enclosed by pregranulosa cells to 100 μm form primordial follicles. Most ovarian primordial follicles ﬂ/ﬂ + Fig. 5 Downregulation of PI3K/AKT signaling in ovaries from Ck2β ;GCre mice. Immunoblotting detection of PI3K/AKT signaling in ovaries of ﬂ/ﬂ ﬂ/ﬂ + Ck2β and Ck2β ;GCre at 2 weeks after birth. The ovary lysates were collected at least from three mice of each genotype and immunoblotted for p-AKT (S473), p-AKT (T308), p-AKT (S129), AKT1/2/3, PTEN, p-TSC2 (S1387), TSC2, p-mTOR (S2448), p-S6K (T389), p-rpS6 (S240/244), p-GSKα/β (S21/9), GSKα/β, p-FOXO1 (T24)/FOXO3a (T32), FOXO1, and β-actin. Levels of β-action were used as internal control. Each experiment was repeated at least 2–3 times. Molecular mass is given in kilo Daltons Ofﬁcial journal of the Cell Death Differentiation Association Liang et al. Cell Death and Disease (2018) 9:508 Page 7 of 11 ﬂ/ﬂ + Fig. 6 Impaired follicle survival in Ck2β ;GCre mice involved in DNA damage response. a Immunoblotting analysis of DNA damage ﬂ/ﬂ ﬂ/ﬂ + response signaling in ovaries of Ck2β and Ck2β ;GCre mice at 2 weeks after birth. The ovary lysates were collected at least from three mice of each genotype and immunoblotted for γH2AX, H2AX, p-CHK2 (T68), CHK2, p63, p-p53 (S15), p53 and β-actin. Level of β-actin was detected as internal control. Each experiment was repeated at least 2–3 times. Molecular mass is given in kilo Daltons. b Immunoblotting analysis of the expression of p53 ﬂ/ﬂ ﬂ/ﬂ + and p63 in ovaries of Ck2β and Ck2β ;GCre at 4 weeks after birth. The ovary lysates were obtained from at least three mice of each genotype and immunoblotted for p63, p-p53 (S15), p53 and β-actin. Level of β-actin was used as internal control. Each experiment was repeated at least three ﬂ/ﬂ ﬂ/ times. Molecular mass is given in kilo Daltons. c MVH and γH2AX immunoﬂuorescent staining of 2-week-old ovarian sections from Ck2β and Ck2β ﬂ + ;GCre mice. Green: γH2AX; Red: MVH; Blue: DAPI. At least three mice of each genotype were used in this assay. Scale bar: 50 μm are maintained in a quiescent state, providing as a reserve with histological observations and follicle counts. At for a woman’s reproductive life . Premature depletion of 3 weeks, most follicles looked healthy but the primordial the ovarian reserve incurs cessation of ovarian function, follicles in ovaries of CK2β mutant mice were markedly resulting in POF . Elucidating the mechanisms that reduced compared to control mice. Starting at 4 weeks, control the dormancy and survival of primordial follicles the ovaries of CK2β mutant mice showed increasing is critical for understanding of ovarian biology. In this numbers of atretic follicles, which were eliminated study, using Gdf9 promotor-driven Cre recombinase, we quickly, resulting in the decrease of the number of successfully deleted CK2β in oocytes from the primordial activated follicles. follicle stage, which facilitated investigation on the roles of Previous researches reveal that PI3K signaling is critical CK2β in folliculogenesis. We found that CK2β mutant to control the survival of primordial follicles, since sup- females showed defective follicular survival and sterility. pressed or elevated PI3K/AKT signaling leads to pre- 54–56 Morphological observation revealed that the ovary size mature depletion of follicles, causing POF . In this of CK2β mutant mice started to decrease from 3 weeks study, we systematically analyze the PI3K/AKT signaling. after birth and eventually became reduced to about 1/4 Immunoblotting results showed that Ck2β depletion of that in control mice at 8 weeks, which was consistent caused downregulation of phosphorylated AKT (S473, Ofﬁcial journal of the Cell Death Differentiation Association Liang et al. Cell Death and Disease (2018) 9:508 Page 8 of 11 ﬂ/ﬂ + Fig. 7 Detection of expression of CK2 subunits and CK2 activity in Ck2β ;GCre mouse ovaries. a Immunoblotting detection of the ﬂ/ﬂ ﬂ/ﬂ + expression of CK2α and CK2α’ in ovaries of Ck2β and Ck2β ;GCre at 2 weeks after birth. b Immunoblotting detection of CK2 activity in ovaries of ﬂ/ﬂ ﬂ/ﬂ + Ck2β and Ck2β ;GCre at 2 weeks after birth. The obviously altered bands were marked with red arrows. The ovary lysates were obtained from at least three mice of each genotype and immunoblotted for CK2α, CK2α’, CK2β, and phospho-CK2 substrate, β-actin. Level of β-actin was used as internal control. Each experiment was repeated at least three times. Molecular mass is given in kilo Daltons T308, and S129). Previous studies ﬁnd that CK2 can follicular survival at least partly via PI3K/AKT/FOXO phosphorylate AKT/PKB at Ser129 and such phosphor- pathway, although details remain to be elucidated. ylation of AKT prevents AKT Thr308 dephosphorylation The oocytes derived from primordial follicle are arres- 24,30,37 and enhances the catalytic activity of AKT/PKB ted at the diplotene stage of prophase of ﬁrst meiotic Previous studies also show that CK2 can phosphorylate division for months, even years, after birth depending on PTEN at several sites and such phosphorylation prevents species. The oocytes at the diplotene stage have ﬁnished 38–40 PTEN degradation and inhibits its activity . Conse- synapsis that relies on homologous recombination, a high- quently, deletion of CK2β causes low stability and high ﬁdelity DNA double-strand breaks (DSBs) repair process. activity of PTEN, leading to reduced level of PTEN pro- Any homolog synapsis or DSB repair errors would tein and downregulated AKT phosphorylation. prompt DNA damage checkpoints to eliminate defective Studies of targets of AKT found that the activity of TSC2 meiotic oocytes, which is mediated by the CHK2-p53/p63 57,63 was not changed but its downstream pathway mTOR/S6K/ pathway . Considering the involvement of CK2 in the 32,34–36 rpSK signaling was signiﬁcantly enhanced in CK2β mutant DNA damage response pathway in other species , ovaries. These ﬁndings are in conﬂict with previous reports we then studied this pathway in CK2β mutant ovaries. In which suggest that elevated mTOR/S6K/rpSK signaling is our study, oocyte-speciﬁc deletion of CK2β from the responsible for oocyte growth and follicular activation and primordial follicle stage caused elevated γH2AX and suppressed mTOR/S6K/rpSK signaling leads to loss of phosphorylated CHK2 (T68) signals in ovaries and ele- 54,55 primordial follicles . The upregulated mTOR/S6K/ vated γH2AX signals in small oocytes from mice at rpSK signaling may result from feedback effects to defec- 2 weeks of age, indicating accumulated DSBs. By 4 weeks tive follicle survival. Further studies of other targets of of age, the DNA damage response pathway was sig- AKT found that the levels of phosphorylated GSK3α/β niﬁcantly elevated, indicated by the upregulated level of ﬂ/ﬂ + (S21/9) were not changed. However, phosphorylated phosphorylated p53 (S15) in ovaries of Ck2β ;GCre FOXO1 (T24)/FOXO3a (T32) were downregulated in mice. Accordingly, the follicular atresia at least early fol- CK2β mutant mice. CK2β deletion downregulates acti- licle atresia may be partly dependent on DNA damage vated AKT, which in turn inhibits FOXO phosphorylation response pathway. 60,61 and keeps FOXO1 staying in nucleus . Unpho- In this study, as expected, the level of CK2β protein was sphorylated FOXO proteins trigger expression of genes signiﬁcantly reduced in CK2β mutant ovaries compared that are crucial for the induction of apoptosis, such as to controls. However, the level of CK2α also showed an FASL and BIM . From above results, CK2β regulates obvious decrease despite of no difference in the levels of Ofﬁcial journal of the Cell Death Differentiation Association Liang et al. Cell Death and Disease (2018) 9:508 Page 9 of 11 CK2α’ in CK2β mutant ovaries and control ovaries. This is (9464; Cell Signaling Technology, Inc.); rabbit monoclonal −/− consistent with a previous report in which CK2α’ anti-FOXO1 antibody (2880; Cell Signaling Technology, females show normal fertility . The above data reveal that Inc.); rabbit monoclonal anti-γH2AX antibody (9718; Cell CK2 presumably functions in oocytes in the forms of α β , Signaling Technology, Inc.); rabbit monoclonal anti- 2 2 −/− 50 but since CK2α embryos die at the embryonic stage , H2AX antibody (7631; Cell Signaling Technology, Inc.); oocyte-speciﬁc knockdown of CK2α is necessary to vali- rabbit polyclonal anti-p-CHK2 (T68) antibody (BS4043; Bioworld Technology, Inc.); rabbit polyclonal anti-CHK2 date the hypothesis. In summary, we identiﬁed CK2β as a key protein safe- antibody (BS6791; Bioworld Technology, Inc.); rabbit guarding mouse follicle survival and fertility. Our data monoclonal anti-p-p53 (S15) (12571; Cell Signaling provide new insights into occurrence, diagnosis, and Technology, Inc.); rabbit polyclonal anti-p53 antibody treatment of POF. (BS3156; Bioworld Technology, Inc.); rabbit monoclonal anti-p63 (ab124762; abcam); mouse monoclonal anti- Materials and methods CK2α antibody (ab70774; abcam); rabbit polyclonal anti- Mice CK2α’ antibody (BS6571; Bioworld Technology, Inc.); ﬂ/ﬂ Mice lacking Ck2β in oocytes (referred to as Ck2β ; rabbit monoclonal anti-phospho-CK2 substrate [(pS/pT) GCre ) were obtained by crossing previously reported DXE] (8738; Cell Signaling Technology, Inc.); secondary ﬂ/ﬂ 53 Ck2β mice with Gdf9-Cre (C57BL6 background) antibodies were purchased from Zhongshan Golden 64 ﬂ/ﬂ mice .The Ck2β female mice were used as control Bridge Biotechnology Co, Ltd (Beijing). group. DNA extraction from mouse tail was used to gen- Δ ﬂ otype Ck2β , Ck2β , and Gdf9-Cre alleles, respectively. The Immunoblotting primer pair for Ck2β allele was forward: 5′-GAGGGCA- Ovary extracts were prepared using a homogenizer in TAGTAGATATGAATCTG-3′ and reverse: 5′-GGA- RIPA buffer supplemented with protease and phosphatase TAGCAAACTCTCTGAG-3′. The primer pair for Ck2β inhibitor cocktail (Roche Diagnostics). After transient allele was forward: 5′-ATGAGTAGCTCTGAGGAGGTG- ultrasound, the ovary lysates were incubated on ice for 30 3′ and reverse: 5′-GGATAGCAAACTCTCTGAG-3′.The min and then centrifuged at 4 °C, 12,000 rpm for 20 min. primer pair for Gdf9-Cre allele was forward: AGG- The supernatant was transferred to a new tube and equal CATGCTTGAGGTCTGAT, and reverse: CACAGT- volume loading buffer was added. After being boiled at 95 CAGCAGGTTGGAGA. All animal operations were °C for 10 min, the protein lysates were used for immu- approved by the Animal Research Committee principles of noblotting analysis. Immunoblotting was performed as the Institute of Zoology, Chinese Academy of Sciences. described previously . Brieﬂy, the separated proteins in SDS-PAGE were electrically transferred to a poly- Antibodies vinylidene ﬂuoride membrane. After incubation with Rabbit monoclonal anti-CK2β antibody (AJ1128b; primary and secondary antibodies, the membranes were ABGENT); mouse monoclonal anti-β-actin antibody (sc- scanned with Bio-Rad ChemiDoc XRS+. 47778; Santa Cruz); mouse monoclonal anti-MVH anti- body (ab27591; abcam); rabbit monoclonal anti-p-Akt Hematoxylin and eosin staining and quantiﬁcation of (S473) antibody (4046; Cell Signaling Technology, Inc.); ovarian follicles ﬂ/ﬂ ﬂ/ﬂ rabbit monoclonal anti-p-Akt (T308) (13038; Cell Sig- Ovaries were dissected from Ck2β and Ck2β ; naling Technology, Inc.); rabbit monoclonal anti-p-Akt GCre mice immediately after killing. The ovaries were (S129) (ab133458; abcam); rabbit monoclonal anti-AKT1/ ﬁxed in 4% formaldehyde overnight at 4 °C, dehydrated in 2/3 antibody (ab32505; abcam); rabbit monoclonal anti- an ethanol series and embedded in parafﬁn. Parafﬁn- PTEN antibody (9559; Cell Signaling Technology, Inc.); embedded ovaries were cut into sections of 8-μm thick- rabbit polyclonal anti-p-TSC2 (S1387) antibody (5584; ness and mounted on glass slides. After 48 °C overnight Cell Signaling Technology, Inc.); rabbit monoclonal anti- drying, the sections were deparafﬁnized in xylene, TSC2 antibody (4308; Cell Signaling Technology, Inc.); hydrated by a graded alcohol series and stained with rabbit monoclonal anti-p-mTOR (S2448) antibody (5536; hematoxylin and eosin for histological analyses. Ovarian Cell Signaling Technology, Inc.); rabbit monoclonal anti- primordial, primary, secondary, and antral follicles were p-S6K (T389) antibody (9234; Cell Signaling Technology, counted in every ﬁfth section of an ovary. Quantiﬁcation Inc.); rabbit monoclonal anti-p-rpS6 (S240/244) antibody of ovarian follicles was performed as previously repor- 66,67 (5364; Cell Signaling Technology, Inc.); rabbit polyclonal ted . In each section, follicles that contained oocytes anti-GSK3α/β (S21/9) antibody (9331; Cell Signaling with clearly visible nuclei were scored and the cumulative Technology, Inc.); rabbit monoclonal anti-GSK3α/β number of follicles were multiplied by a correction factor antibody (5676; Cell Signaling Technology, Inc.); rabbit of 5 to represent the estimated number of total follicles in polyclonal anti-p-FOXO1 (T24)/FOXO3a (T32) antibody an ovary. Ofﬁcial journal of the Cell Death Differentiation Association Liang et al. Cell Death and Disease (2018) 9:508 Page 10 of 11 Immunohistochemistry and immunoﬂuorescence Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 100101 Beijing, China. University of Chinese Academy of Sciences, 100101 Ovaries used for immunostaining were ﬁxed in 4% par- Beijing, China. Center for Reproductive Medicine, Nanjing Drum Tower aformaldehyde (pH 7.4) overnight at 4℃, dehydrated, and Hospital, The Afﬁliated Hospital of Nanjing University Medical School, 210008 embedded in parafﬁn. Parafﬁn-embedded ovaries were cut Nanjing, China. Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211, USA. INSERM U1036, Institute de Recherches en into sections of 5-μm thickness. Then, the sections were Technologies et Sciences pour le Vivant/Biologie du Cancer et de l’Infection, deparafﬁnized, immersed in sodium citrate buffer (pH 6.0), Commissariat à l’Énergie Atomique et aux Énerigies Alternatives Grenoble, and heated for 15 min in a microwave for antigen retrieval. Grenoble, France. KinaseDetect, Krusaa, Denmark After blocking with 5% donkey serum albumin, sections Conﬂict of interest were incubated with primary antibodies at 4 °C overnight. The authors declare that they have no conﬂict of interest. For immunohistochemistry, the sections were treated with 3% H O to eliminate internal peroxidase activity and 2 2 Publisher's note incubated with an appropriate horseradish peroxidase Springer Nature remains neutral with regard to jurisdictional claims in (HRP)-conjugated secondary antibody. Finally, the signal published maps and institutional afﬁliations. of primary antibody was detected by the Vectastain ABC Supplementary Information accompanies this paper at (https://doi.org/ kit (Vector Laboratories, CA, USA) and the sections were 10.1038/s41419-018-0505-1). counterstained with hematoxylin. Images were captured using a Nikon DS-Ri1 CCD camera. 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