Chk1/2 inhibitor AZD7762 blocks the growth of preantral follicles by inducing apoptosis, suppressing proliferation, and interfering with the cell cycle in granulosa cells

Xiao-ming Liu (  lxm19860405@163.com ) Yuying Children's Hospital of Wenzhou Medical College: Wenzhou Medical University Second A liated Hospital https://orcid.org/0000-0002-7062-2468 Fang Chen Wenzhou Medical College Chashan Campus: Wenzhou Medical University Fan Zhang The Second A liated Hospital and Yuying Children's Hospital of Wenzhou Medical University Jun-Zhao Zhao Wenzhou Medical University Second A liated Hospital


Introduction
In mammals, oocytes arrested in the diplotene stage of the rst meiotic prophase are surrounded by a single, squamous layer of somatic cells to form a nite population of non-growing primordial follicles [1].
Primary follicles are recruited from the primordial pool as oocytes grow. These cells continue to proliferate to form many layers surrounding the oocyte and eventually become granulosa cells [2]. This transition is associated with participation in the subsequent phases of follicular growth, as the measured recruitment of primordial follicles from the resting pool of follicles is crucial for the development of folliculogenesis throughout the reproductive lifespan of mammals [3]. However, apoptosis reduces this endowment by two-thirds before birth. In addition, granulosa cells apoptosis is the main cause of follicular atresia at different stages of their growth [4,5].
When atresia occurs, pyknotic nuclei are rst observed in granulosa cells. Then a detachment of granulosa cell layer and fragmentation of basal membrane occurs, ultimately resulting in hypertrophied thecal cells and disruption of thecal integration and thecal vessels [6]. Granulosa cell apoptosis may occur much earlier than the morphological changes in follicular atresia, which can be observed only when granulosa cell apoptosis reaches a certain degree [7]. Generally, proliferation and differentiation of granulosa cells lead to follicular maturation and ovulation, whereas apoptosis and degeneration of granulosa cells result in follicular atresia [8]. Many apoptosis-related factors have been implicated in follicular atresia, including death ligands and receptors, intracellular pro-and anti-apoptotic molecules, cytokines, growth factors, and several apoptosis-related genes [9]. Although new regulatory factors are continuously being identi ed, comprehensive knowledge of the signaling networks that function during granulosa cell apoptosis remains limited.
Checkpoint kinases are threonine/serine that can be divided into two subtypes, Chk1 and Chk2, which have a critical role in DNA damage responses, cell cycle control, and cell survival [10]. In response to DNA damage, Chk1 and Chk2 are activated by PI3 kinase-related kinases ATM and ATR, respectively, aiming at many downstream substrates that coordinate cell cycle checkpoint activation, DNA restitution, and apoptosis [11]. Moreover, Chk1/2 has also been implicated in anaphase entry, chromosome condensation, and maintenance of genome integrity in somatic cells in the absence of DNA damage [12].
Chk1 knockout mice are embryonically lethal, suggesting that Chk1 is an important molecule during early embryonic development [13]. Moreover, embryonic stem cells speci c Chk1 knockout mice display premature activation of Cdc2/cyclin B and mitotic catastrophe [14]. At the same time, Chk2-de cient cells show signi cant defects in UV-induced apoptosis and G1/S arrest [15]. Preliminary unpublished observations from our laboratory showed that the expression of Chk1/2 uctuates during follicular development, suggesting the importance of Chk1/2 in folliculogenesis; yet, the exact role in follicular development is not fully understood.
In the present study, Chk1/2 inhibitor (AZD7762) was used to further investigate the role of Chk1/2 during preantral follicular development and cellular proliferation and apoptosis.

Animals
Female Kunming white mice of 12-14 (9 g-10 g) or 21-23 (12 g-14 g) days old were obtained from the Centre of Laboratory Animals of Hubei Province (Wuhan, PR China). Mice were housed in an environment with a temperature of 24 ± 1 ºC, relative humidity of 50 ± 1%, and a light/dark cycle of 12/12 hr, and given food and water ad libitum. All animal studies (including the mice euthanasia procedure) were done in compliance with the regulations and guidelines of the Hubei Research Center of Experimental Animals and conducted according to the AAALAC and the IACUC guidelines (Approval ID: SCXK (Hubei) 2008-0005).
Isolation and culture of preantral follicles and granulosa cells Preantral follicles (100 μm-120 μm) obtained from the ovaries of 12-14 days old female mice were gently separated using the 1 ml syringe needle under a stereomicroscope (CKX41SF; Olympus Optical Technology Philippines Inc., Lapu-Lapu City, Philippines), and observed under an inverted microscope (TE2000-U; Nikon). Follicles with two or three layers of granulosa cells and a diameter between 100 and 120 µm were collected and cultured in α-Minimum Essential Media (α-MEM, Gibco) containing 10 µg/mL of ITS (0.55 mg/mL human transferrin, 1.0 mg/mL recombinant human insulin, and 0.5 µg/mL sodium selenite) and 100 mIU/mL of follicle-stimulating hormone (FSH, Sigma Chemical Company, St. Louis, MO) with one follicle per well in 96-well culture plates in a humidi ed atmosphere containing 5%CO 2 /95% air at 37ºC for 48 h. After that, follicles were cultured in α-MEM medium containing ITS with or without 1 μM of AZD7762 (Axon Medchem BV, Cat. No. Axon 1399) for an additional 96 h and observed under microscopy (TE2000-U; Nikon) for assessment of morphology and follicular growth, as indicated by follicular diameter (F.D). The concentration of the Chk1/2 inhibitor used in our study was selected based on previous study results [16].

Cell proliferation assay
Cell proliferation assay was measured using a WST-1 Cell Proliferation Assay kit (Beyotime, Wuhan, China). Brie y, granulosa cells were cultured in a 96-well culture plate (4x10 3 cells/well) for 24 h. Cells were then exposed to a gradually increased concentration of AZD7762 (1, 5, 10, 20, and 50 μM) for 24 h, 48 h, and 72 h. After each time point, 10 µl of freshly prepared WST-1 solution was added to each well, along with the culture medium. The absorbance of the samples was measured after 1 h at 37°C using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 450 nm.
Cell cycle assay For cell cycle analysis, pre-GCs were cultured in a 6-well culture plate with 0 μM or 1 μM AZD7762 for 48 h. After being washed with PBS, the cells were digested and harvested using the Cell Cycle Detection kit (KeyGen Biotech Co., Ltd., Nanjing, China). Cells were then xed in 70% ethanol at 4°C overnight, washed with PBS, and incubated with 100 μL RNase A at 37°C for 30 min. Then, the cells were stained with 400 μL PI in the dark for 30 min at 4°C and analyzed through ow cytometry using a BD FACS Calibur RT-PCR analysis RT-PCR analysis was performed to con rm that inhibition of Chk1/2 by AZD7762 could regulate genes expression, PCNA, and Bax. Pre-GCs were cultured with 0 μM or 1 μM AZD7762 for 48 h, after which a total RNA was extracted using the RNAprep pure Cell/Bacteria Kit (TIANGEN, Beijing), and in vitro transcription was performed through RevertAid TM First-strand cDNA Synthesis kit (Thermo, Wuhan). RT-PCR was quanti ed using special primer pairs (Table 1)

Western blot
After AZD7762 treatment for 48 h, cells were harvested in RIPA buffer (Santa Cruz), which contained 10 mg/mL protease inhibitors cocktail (Santa Cruz) and 10 mM phenylmethylsulfonyl uoride (PMSF; Ding-Guo, Beijing). The concentration of total protein was determined by bicinchoninic acid (BCA) assay (Pierce, Rockford, USA), and 20 mg of total protein was subjected to gel electrophoresis as previously described [17].

Statistical analysis
Experiments were independently performed at least three times, and data are presented as mean±SD.
Differences between each group were analyzed by one-way ANOVA followed by Tukey's Honesty Signi cant Difference (HSD) test using SPSS (Version 17.0; SPSS, Chicago, IL, USA); P< 0.05 was regarded as a statistically signi cant difference.

Results
Chk1/2 are essential for preantral follicular development In order to assess the role of Chk1/2 during follicular development, we cultured preantral follicles with Chk1/2 broad-spectrum inhibitor (AZD7762) in vitro. In the control group, the gradual growth of follicles was observed, while follicles cultured with AZD7762 showed no growth or cell number reduction ( Figure  1A and 1B, P<0.05). Meanwhile, as shown in Figure 1C, the granulosa cells of follicles treated with AZD7762 around the outer layer showed cell shrinkage and weak connection between cells compared with the control follicles. Thus, we predicted that the arrested development of preantral follicles and abnormalities of the morphology of follicles might be related to the granulosa cells status, including proliferation and apoptosis.

Inhibition of Chk1/2 induces granulosa cells apoptosis
As shown in Figure 2A, granulosa cells cultured with AZD7762 showed abnormal cell morphology. As seen in Figure 2B, the apoptosis rate in the AZD7762 group was signi cantly higher than in the control group (P<0.05). In addition, the expression of Bax, a marker of apoptosis, was signi cantly up-regulated in both mRNA and protein levels ( Figure 2C and 2D, P<0.001 and P<0.05).

Inhibition of Chk1/2 reduces granulosa cell proliferation
In order to study whether Chk1/2 affect granulosa cells' proliferation, cells were cultured with different concentration of AZD7762 for 24, 48, or 72 h, respectively. When cells were cultured for 24 h, the percentage of proliferation was similar, and no signi cant difference between the control group and 1 µM AZD7762 was seen (P>0.05); yet, the percentage of proliferation was signi cantly reduced when cells were treated with a higher concentration of AZD7762 (5, 10, 20, and 50 µM, P< 0.01, Figure 3A). However, after 48 or 72 h, the percentage of proliferation was also signi cantly reduced in 1 µM AZD7762 ( Figure  3A, P<0.01), while no signi cant difference was found among a higher concentration of AZD7762 (5, 10, 20, and 50 µM, Figure 3A) In addition, RT-PCR was applied to detect the expression level of PCNA, which is a marker of proliferation. After culturing cells with AZD7762 for 48 h, the expression of PCNA mRNA was signi cantly decreased compared with control cells (Figure 3B, P<0.01). Furthermore, Western blot results also showed decreased expression of PCNA protein in AZD7762 cultured cells ( Figure 3C, P<0.01).
Inhibition of Chk1/2 arrests cell cycle at S and G2/M stages Next, we explored the effect of Chk1/2 arrests cell cycle at S and G2/M stages. As shown in Figure 4, the percent of the G1 stage in the AZD7762 group was signi cantly lower than the control cells (P<0.001), while the percent of S and G2 stages were signi cantly increased in the AZD7762 group than control (P<0.001). Therefore, our results indicated that inhibition of Chk1/2 by AZD7762 could suppress the proliferation of cells and disturb the normal cell cycle. Taken together, inhibition of Chk1/2 resulted in inhibition of proliferation and promotion of apoptosis of granulosa cells.

Discussion
In this study, AZD7762 was used to inhibit the function of both Chk1 and Chk2. Preantral follicles treated with AZD7762 showed a developmental abnormality. Moreover, granulosa cells treated with AZD7762 showed decreased cell growth, increased apoptosis, and abnormal cell cycle distributions. These results suggest that Chk1/2 has an important role in preantral follicular development and the growth of granulosa cells.
The development of preantral follicles includes oocyte growth, granulosa cell proliferation, differentiation, and apoptosis. However, more than 99% of follicles disappear, primarily due to the apoptosis of granulosa cells, and the majority of follicles become atretic during the early antral stage of development [18]. Thus, we selected the preantral follicles in this experiment, which were then treated with a Chk1/2 inhibitor to monitor the follicular development. Activated Chk1 and Chk2 have a full spectrum of substrates that are key cell cycle regulators. In the control group, the gradual growth of follicles was observed, while follicles cultured with AZD7762 showed no growth or even negative growth (Fig. 1). These results suggested that Chk1/2 is essential for follicular development.
Gonadotropin can promote the differentiation of the granulosa cells, making them vulnerable to apoptosis. Thus, pre-GCs were selected to study the role of Chk1/2 in regulating the development of granulosa cells. The cells treated with AZD7762 showed decreased cell growth and increased cell apoptosis (Fig. 2 and Fig. 3). Similarly, a previous study has suggested that Chk1 is required for mitotic progression and proliferation of Hela cells through negative regulation of polo-like kinase 1 Plk1 [19]. Meanwhile, Chk1-depleted lobuloalveolar mammary epithelial cells do not proliferate and undergo apoptosis, suggesting that cell proliferation is important for apoptosis [20]. Likewise, in our study, a proliferation of granulosa cells was signi cantly inhibited and showed an uncoordinated cell cycle (Fig. 4). The link between apoptosis and proliferation suggests that death resulting from Chk1 depletion may involve mitotic alteration [21]. However, in some circumstances, Chk2 appeared to be at least in part able to make up for the loss of Chk1 in some cells [22]. Our preliminary studies of Chk1/2 inhibition in mouse oocytes supported this hypothesis [23]. Moreover, the cell cycle of pre-GCs was disturbed by inhibition of Chk1/2 and showed increased G2 and S stages (Fig. 4). As Chk1/2 are the key cell cycle checkpoint kinase, and the major function of Chk1 is to coordinate the cell cycle checkpoint response, including G1, S, G2/M, and M phase [24], Chk2 is needed for the optimal G2/M delay of G2 phase cells; Chk2-de cient cells show G1/S arrest [25]. Our study showed that Chk1/2 might affect ovarian function by regulating the state (proliferation or apoptosis) of GCs and the fate (growth or atresia) of follicular development.
Future studies should investigate the exact function of Chk1 and Chk2 in follicular development and the regulatory mechanism of the Chk1/2 network responsible for follicular development. Studies have shown that Chk1 is a potential target for treating cancer [24], so another important issue is evaluating the possibility of Chk1/2 in reducing follicular atresia. Therefore, we propose that Chk1/2 could represent an option for suppressing follicular atresia.

Conclusions
Our present results provide insight into the roles of Chk1/2 in mouse ovaries, including follicular development, granulosa cells proliferation, and apoptosis. These results suggest that Chk1/2 may have an important role in follicular development and ovarian functions. Furthermore, future research on the security application of AZD7762 as drugs in clinical therapy of cancer (especially female patients) is warranted.

Funding
This study was supported by the Natural Science Foundation of Zhejiang (Program NO. LQ18H040008). Abnormal morphology and structure of preantral follicles cultured with Chk1/2 inhibitor AZD7762. Scale bars, 100 µm.  The same batch of protein samples was used in Figure 2D, so the lane of β-actin was the same. The value expressed by each bar represents the mean± SD. a vs. b, P<0.01.