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HDAC6 regulates primordial follicle activation through mTOR signaling pathway

HDAC6 regulates primordial follicle activation through mTOR signaling pathway Primordial follicle pool established perinatally is a non-renewable resource which determines the female fecundity in mammals. While the majority of primordial follicles in the primordial follicle pool maintain dormant state, only a few of them are activated into growing follicles in adults in each cycle. Excessive activation of the primordial follicles accelerates follicle pool consumption and leads to premature ovarian failure. Although previous studies including ours have emphasized the importance of keeping the balance between primordial follicle activation and dormancy via molecules within the primordial follicles, such as TGF-β, E-Cadherin, mTOR, and AKT through different mechanisms, the homeostasis regulatory mechanisms of primordial follicle activation remain unclear. Here, we reported that HDAC6 acts as a key negative regulator of mTOR in dormant primordial follicles. In the cytoplasm of both oocytes and granulosa cells of primordial follicles, HDAC6 expressed strong, however in those activated primordial follicles, its expression level is relatively weaker. Inhibition or knockdown of HDAC6 significantly promoted the activation of limited primordial follicles while the size of follicle pool was not affected profoundly in vitro. Importantly, the expression level of mTOR in the follicle and the activity of PI3K in the oocyte of the follicle were simultaneously up- regulated after inhibiting of HDAC6. The up-regulated mTOR leads to not only the growth and differentiation of primordial follicles granulosa cells (pfGCs) into granulosa cells (GCs), but the increased secretion of KITL in these somatic cells. As a result, inhibition of HDAC6 awaked the dormant primordial follicles of mice in vitro. In conclusion, HDAC6 may play an indispensable role in balancing the maintenance and activation of primordial follicles through mTOR signaling in mice. These findings shed new lights on uncovering the epigenetic factors involved physiology of sustaining female reproduction. Introduction state for as long as 50 years in humans or for more than 1 Ovarian follicles are the basic and non-renewable year in mice. Only a small portion of dormant primordial 1,2 reproductive unit of female mammals . In mammalian follicles are progressively recruited into the growing fol- ovaries, the majority of follicles reserved are primordial licle pool each time in adults, which is called primordial follicles, which are maintained at dormant or quiescent follicle activation . Obviously, the balance between quiescent and activated state of the primordial follicles is crucial for female fecundity. Excessive activation of pri- Correspondence: Hua Zhang (huazhang@cau.edu.cn)or mordial follicles will lead to exhaustion of the resting Chao Wang (wangcam@126.com) Key Laboratory of Ministry of Education for Conservation and Utilization of follicle reserve and result in premature ovarian failure Special Biological Resources in the Western China, College of Life Science, (POF), which seriously affect female physical and mental Ningxia University, 750021 Yinchuan, China health . Unfortunately, there is hardly any effective mea- Guizhou Provincial Key Laboratory of Pathogenesis & Drug Research on Common Chronic Diseases, Department of Physiology, School of Basic Medical surements to help these patients so far because the Sciences, Guizhou Medical University, 550009 Guiyang, Guizhou, China acknowledgement of the mechanisms governing the Full list of author information is available at the end of the article These authors contributed equally: Tuo Zhang, Meina He, Lihua Zhao Edited by G.M. 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Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Zhang et al. Cell Death and Disease (2021) 12:559 Page 2 of 12 activation and dormant of primordial follicles is still overexpression of Hdac6 retarded the activation of pri- obscure. mordial follicle. HDAC6 regulates primordial follicles The primordial follicle consists of a quiescent oocyte quiescent through regulating the expression of mTOR and surrounding flattened primordial follicle granulosa signaling. Collectively, we hypothesize that HDAC6 plays cells. Previous studies including ours have implied that an indispensable role in sustaining primordial follicles oocyte derived molecules, such as phosphatidylinositol 3- through affecting the expression of mTOR signaling pathway. kinase (PI3K), cell division cycle 42 (CDC42), E-cadherin/ N-adherin (E-cad/N-cad), and newborn ovary homeobox 5–11 (NOBOX) , as well as oocyte and pfGCs expressed Materials and methods Sirtuin 1 (SIRT1) and transforming growth factor β (TGF- Animal 12,13 β) , are pivotal for the sustainment of primordial fol- Adult CD-1 mice were purchased from Beijing Vital licles after birth. On the other hand, the activation of River Laboratory Animal Technology Co., Ltd. and bred primordial follicle is a complex and coordinated process to male mice of the same strain. Mice were housed under among individual oocyte, pfGCs, and even the sur- controlled lighting (12 h light, 12 h dark) and temperature rounding micro-environment . Actually, many molecules (24–26 °C) conditions with free access to food and water. mentioned above are involved in the activation process of Vaginal plug detection was considered as 0.5 day post primordial follicle originated from the pfGCs, of which coitus (dpc). The first 12 h after birth was considered as mTORC1 signaling in pfGCs and PI3K signaling in 0 day postpartum (dpp). All procedures were conducted oocytes is the dominant pathway . in accordance with the guidelines of and approved by the Although the technique of in vitro activation (IVA) of Animal Research Committee of the China Agricultural primordial follicles based on these findings have achieved University. to help POF patients producing fertilizable oocytes for 16,17 successful reproduction, the efficiency is quite low . Whole ovary and cortex fragments isolation And it is hard to unveil the pathological mechanism of The newborn mice were sacrificed at the designated POF so far because there is very limited knowledge about times. The ovaries of the newborn mice were micro- how primordial follicle dormancy is finely tuned dissected in cold phosphate buffered saline (PBS) under after birth. microscope. Make sure that the structure of whole ovaries Most recently, we have proved that SIRT1, one of the was not damaged during the separation procedure. When members of histone deacetylase family, contributes to isolating the cortex fragments under a steroid microscope, sustain primordial follicles in mice, implying the impor- a syringe of 1 ml with needle was applied to carefully tance of epigenetic modification effect in the process .In separate the ovarian cortex from the medulla tissue. line with this, an overexpression of histone deacetylase 6 (Hdac6) extends reproductive lifespan of mice since an Ovary organ culture increased folliculogenesis and particularly secondary and The dissected ovary was cultured in six-well culture antral follicles are found in 19-month-old mice compared plates (Nest, Jiangsu, China) in 1200 μl of Dulbecco with 16-month wild-type mice . HDAC6 belongs to class Modified Eagle Medium/Ham F12 nutrient mixture IIb of HDAC family, and is a microtubule-associated (DMEM/F12) (Gibco, Life Technologies, CA) with deacetylase . Physiologically, it predominantly functions insulin-transferrin-sodium selenite (Sigma, 1:100, USA) in the cytoplasm by deacetylating various substrates so as and penicillin–streptomycin solution at 37 °C, 5% CO to regulate cell migration, cell cytoskeleton, cell motility, and saturated humidity. The female mice ovaries were and cell immune synapse formation, and even misfolded randomly assigned. Same number of ovaries in each 20–26 proteins degradation . HDAC6 contains two intact group. The culture medium was exchanged every other catalytic domains that located respectively at the N- day. The final concentrations of Tubastatin A (TubA in terminal and the central regions of the protein, which is short) (S8049; Selleck, Shanghai, China), Rapamycin (Rap different from those of SIRT1. Additionally, it is not only in short) (S1039; Selleck, Shanghai, China), and ISCK03 important for regulating chromatin compaction and (I6410; Sigma, USA) were 4 μM, 2.5 μM, and 5 μM, meiotic apparatus assembly in mouse oocytes, but plays respectively. The control group was supplemented with 27–30 essential part in ARID1A-mutated ovarian cancer . the same volume of dimethyl sulfoxide (DMSO). Therefore, we wonder if HDAC6 was also involved in sustaining primordial follicles. Immunofluorescence and immunohistochemistry Briefly, we found that HDAC6 participates in sustaining Freshly isolated ovaries were fixed in cold 4% paraf- primordial follicle quiescent in newborn mice. We proved ormaldehyde overnight, dehydrated in gradient alcohol, that either inhibiting or knocking down HDAC6 pro- embedded in paraffin, and sectioned at 5 μm. Ovarian moted the activation of primordial follicles, while sections were deparaffinized, rehydrated and subjected to Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death and Disease (2021) 12:559 Page 3 of 12 antigen retrieval with 0.01% sodium citrate buffer (pH 6.0) Real-time PCR analysis at high temperature (95–98 °C) for 16 min. The sections Total RNA obtained from at least six ovaries was were then rinsed thoroughly with PBS and blocked with extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, normal donkey serum in PBS for 1 h at room temperature USA) according to the manufacturer’s protocol. First- and incubated with primary antibodies overnight at 4 °C. strand cDNA was synthesized by reverse transcription The primary antibodies used are listed in Table S1. Next, using 1 µg of total RNA (Promega Reverse Transcription ovarian sections was rinsed thoroughly with PBS and System, Promega, USA). Quantitative-RT-PCR was per- incubated with Alexa Fluor 488- or 555- conjugated sec- formed using SYBR Select Master Mix (Applied Biosys- ondary antibody for 1 h at 37 °C. The ovarian sections were tems, Life Technologies, USA) and operated by Applied rinsed thoroughly with PBS, stained with DAPI for 5 min Biosystems 7500 Real Time PCR System (Applied Bio- and sealed in anti-fade fluorescence mounting medium systems, Life Technologies, USA). The data were nor- (C1210, Applygen, China) with coverslips. Immunohis- malized by β-actin. Primers used are listed in Table S2. tochemistry was performed using Histostain™-SP Kits (PV-9001, ZSGB-BIO, China) and DAB peroxidase sub- Gene knockdown and overexpression strate kits (ZLI-9017, ZSGB-BIO, China) according to the The vectors of either for knockdown or overexpression manufacturer’s protocols. Sections were examined and of Hdac6 was respectively mixed with trypan blue solu- photographed using Nikon 80i or Nikon A1 laser. tion (vector/trypan blue solution: 10/1) and then injected into isolated 1 dpp mouse ovaries using glass pipettes Total follicle, primordial follicle, and primary follicle under a stereomicroscope. After the ovaries were injected counting with the blue liquid, electrotransfection was performed by The serial sections of each ovary (8 μm thick) were the application of three 5-ms-long quasi-square pulses at stained with hematoxylin. Only when follicles containing a pulse-field strength of up to 30 V/cm. The Hdac6 clearly visible nucleus of oocytes in each individual sec- knockdown vector was constructed by cloning Hdac6- tion of every ovary were counted. The serial sections of shRNA with sequence: GGTACTTCCCATCGCC- each ovary were counted before statistical analysis. Max- TATGA. The Hdac6 overexpression vector was con- imum and minimum were excluded. A double-blind structed by cloning the open reading frame of Hdac6 into experiment was performed during follicle number the pCMV-C-His (D2650, Beyotime Biotechnology, counting. The follicles were distinguished from each other China). Golden star t6 super PCR mix were purchased as follows: primordial follicle (a single oocyte surrounded from Beijing Tsingke Co., Ltd. All of the constructs were by several flattened pre-granulosa cells) and primary fol- verified by sequencing. Primers are listed in Table S3. licle (an enlarged oocyte surround by a mixture of squa- mous and cuboidal granulosa cells). Statistical analysis All experiments were repeated at least three times. The Western blotting analysis values are presented as the means ± SEM. The data were Each protein sample obtained from at least six ovaries analyzed by either t test or ANOVA, and considered were extracted in WIP Tissue and cell lysis solution statistically significant at P < 0.05. containing 1 mM phenylmethylsulfonyl fluoride (8553 S, Cell Signaling Technologies, USA) according to the Results manufacturer’s instructions. The protein concentration HDAC6 is indispensable for preventing primordial follicle was measured by a BCA assay (P0012, Beyotime, China). activation in vitro The samples were separated on 10% SDS-PAGE and then To explore the potential role of HDAC6 in either sus- transferred to polyvinylidene fluoride membranes taining the dormancy or activation of primordial follicle, (IPVH00010, Millipore, USA). The membranes were immunofluorescence staining was firstly performed to blocked with 5% skim milk powder for 1 h at room tem- detect the cellular localization and expression dynamics of perature and then incubated with appropriate primary HDAC6 in newborn and adult mouse ovaries. It showed antibodies overnight at 4 °C. The primary antibodies used that HDAC6 was predominantly localized to the cyto- are listed in Table S1. The membranes were rinsed plasm of oocytes and granulosa cells in newborn and adult thoroughly with TBST. The membranes were incubated mice ovaries (Figs. 1A and S1). with the secondary antibody (1:5000, ZSGB-BIO, China) We found that the signal of HDAC6 was homogenous for 1 h at room temperature and rinsed thoroughly with stronger in the majority of primordial follicles, but was TBST. The membranes were visualized using a Super- relatively weaker in some primordial follicles, which may Signal West Pico Chemiluminescent Detection System be on the way to be activated. This phenomenon is (Prod 34080, Thermo, USA). β-actin (CW0096M, observed in both ovaries of adult and newborn mice Cwbiotech, China) was used as the intrinsic control. unanimously (Figs. S1 and S2). The statistics results show Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death and Disease (2021) 12:559 Page 4 of 12 Fig. 1 The expression pattern of HDAC6 in the neonatal mouse ovaries. A Cellular localization of HDAC6 in newborn ovaries. Newborn mice ovaries were stained for HDAC6 (green) and the oocyte specific marker DDX4 (red) at the indicated time points. The nuclei were counter-stained by DAPI (blue). HDAC6 was mainly localized to the cytoplasm of both oocytes and granulosa cells in either primordial follicles or growing follicles. B qRT- PCR assay showed that the expression level of Hdac6 mRNA was increased from 1 dpp to 5 dpp and decreased on 7 dpp. C Western blotting assay showed that the level of HDAC6 protein was increased from 1 dpp to 5 dpp and decreased on 7 dpp as well. The experiments were repeated at least three times. And representative images are shown. The data are presented as the means ± SEM. Scale bars: 50 μm. that 3.5 ± 0.2% of primordial follicles with weaker HDAC6 the mRNA and protein of HDAC6 increase significantly expression are presented in adult ovaries, while 4 ± 0.4% along with the establishment of the primordial follicle of primordial follicles with weaker HDAC6 expression are pool at 3 dpp, and retain at a relative higher level from 5 presented in 5 dpp ovaries (Fig. S3). dpp to 7 dpp, during which time primordial follicle acti- In order to clarify whether the primordial follicles with vation is generally initiated (Fig. 1B, C). weaker HDAC6 signal are being activated, the 5 dpp To further determine the expression pattern of HDAC6 ovaries were stained for HDAC6 and Foxo3a in adjacent in primordial follicles from 1 dpp to 7 dpp, a model of serial sections. The results showed that over 60% of pri- ovarian cortex fragments isolated from the intact ovary mordial follicles with weaker expression of HDAC6 were was established. We noticed that only primordial follicles simultaneously the ones whose FOXO3a was located in were found in the isolated cortical fragments in 1 dpp–7 the cytoplasm (Fig. R6). The results suggest that 65.1 ± dpp ovaries (Figs. S5–S9). The western blotting results 5.3% of primordial follicles with weaker HDAC6 signal are showed that the HDAC6 expression decreased from 1 dpp being activated. and then maintained at a relatively weaker level from 3 Then the qRT-PCR and western blotting analyses of dpp to 7 dpp in ovarian cortex fragments. Interestingly, whole ovary samples revealed that the expressions of both the general protein level of HDAC6 in 5 dpp whole ovary Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death and Disease (2021) 12:559 Page 5 of 12 was higher than in 3 dpp–7 dpp ovarian cortex fragments levels of both total and phosphorylated mTOR which are (Fig. S10). Collectively, these results suggest that the the downstream molecules of TSCs, were up-regulated in expression of HDAC6 in primordial follicles is homo- TubA treatment (Fig. 3A). To further confirm the reg- genous, whereas the expression of HDAC6 becomes ulation effect of decreased HDAC6 on mTOR, the 1 dpp weaker in some primordial follicles that were to be acti- ovaries were either injected with Hdac6-KD or Hdac6-OE vated. These results indicated that HDAC6 plays a vector respectively and cultured for additional 3 days. Western blotting results showed that the levels of both potential role in primordial follicle activation versus quiescent. total and phosphorylated mTOR were either up-regulated To study the function of HDAC6 in primordial follicle in Hdac6-KD treatment or down-regulated in Hdac6-OE in newborn mouse ovaries, an in vitro culture model was treatment, respectively (Fig. 3B, C). These results sug- established. The histological and statistical analysis results gested that HDAC6 may be pivotal for balancing pri- showed that the ovaries cultured in vitro develop normally mordial follicle between dormancy and activation through as the morphology as well as the numbers of different influencing the mTOR signaling pathway. stage of follicles and granulosa cells were consistent with To further confirm the above conclusion, Rapamycin the simultaneously developed ovaries in vivo (Fig. S11). (Rap), the specific inhibitor of mTOR, was used to verify if Based on this model, 2 dpp ovaries were cultured with it could attenuate the expression of mTOR induced by TubA, one of the inhibitors of HDAC6, for 2 days. The TubA treatment. For this purpose, 2 dpp ovaries were results showed that acetylated alpha-tubulin was cultured with either TubA or Rap for 3 days before increased and the level of HDAC6 was decreased com- examination. The histological analysis and whole ovary pared to the control (Fig. 2A). Based on these findings, the counting results showed that fewer growing follicles were treated ovaries were furtherly cultured for 3 days before observed in TubA plus Rap treatment than those in the histological analysis. The results showed that more TubA treatment (Fig. 3D–F). Inhibition of mTOR by Rap growing follicles were observed in TubA treatment reversed the follicular development caused by TubA. compared to the control, while the number of total folli- These results suggested that HDAC6 in the primordial cles within each ovary was comparable to the control (Fig. follicles of new born ovaries prevent follicles from pre- 2B, C). These results implied that HDAC6 participates in mature activation. preventing primordial follicles from premature activation in vivo. Downregulation of HDAC6 is necessary for activating PI3K- To further confirm the function of HDAC6 in regulat- AKT pathway in oocytes in vitro ing primordial follicle development, both Hdac6-shRNA To study the potential relationship between down- (Hdac6-KD) vector and Hdac6-overexpression (Hdac6- regulated HDAC6 and the PI3K pathway within oocytes OE) vector were respectively constructed, and injected of primordial follicles, 2 dpp ovaries were cultured with or into 1 dpp ovaries respectively. The injected ovaries were without TubA for 2 days. Western blotting results showed cultured for 3 days, the results showed that the endo- that the total levels of either AKT or Foxo3a were genous HDAC6 protein levels were either efficiently unchanged, while the levels of both phosphorylated AKT down-regulated by Hdac6-KD, or up-regulated in Hdac6- and Foxo3a were up-regulated in TubA treatment (Fig. OE group, respectively (Fig. 2D, G). Then, the injected 3A). At the same time, the conclusion was further con- ovaries were cultured for 4 days, the histological analysis firmed by results applying Hdac6-KD and Hdac6-OE, showed that more growing follicles were observed in respectively (Fig. 4B, C). In addition, immunofluorescence Hdac6-KD group and fewer growing follicles were staining and quantitative results showed that Foxo3a, observed in Hdac6-OE group, respectively (Fig. 2E, H). which shuttled from the nucleus to the cytoplasm, was The quantitative results further confirmed the phenotype increased in TubA treated ovaries (Fig. 4D). These results observed in Figs. 2F, I. These results indicated that indicated that inhibition of HDAC6 expression in mice downregulating the level of HDAC6 was necessary for ovaries was indispensable for the activation of PI3K mice primordial follicles activation in vitro. pathway. HDAC6 prevents primordial follicle activation through Down-regulated HDAC6 in new born ovaries governed inhibiting mTOR signaling pathway primordial follicle activation through KITL-KIT in vitro To investigate how decreased HDAC6 participates in According to the existed hypothesis, it is somatic cell primordial follicle activation, the changes of the mTOR that initiates primordial follicle activation. That is, the up- signaling pathway were detected. Before examination, 2 regulated mTORC1 signaling in pre-granulosa cells trig- dpp ovaries were cultured with or without TubA for gers the awakening of dormant oocytes through secreting 2 days. The western blotting results showed that both KIT ligand (KITL). The coupling between mTORC1- TSC1 and TSC2 were unchanged (Fig. S12) whereas the KITL in pfGCs and KIT-PI3K signaling in oocytes Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death and Disease (2021) 12:559 Page 6 of 12 Fig. 2 HDAC6 is involved in the primordial follicle activation. A Inhibition efficiency analysis of TubA. The 2 dpp ovaries were cultured with or without TubA for 2 days. The ac-Tubulin was increased while HDAC6 was decreased in TubA treatment. ac-Tubulin: acetylated alpha-tubulin. B, C The histological analysis and follicle counting results showed that inhibition of HDAC6 expression by TubA accelerated primordial follicles activation without affecting the number of total follicles. The 2 dpp ovaries were cultured with or without TubA for 3 days. D Knockdown efficiency analysis of Hdac6-KD. The 1 dpp ovaries were transfected with Hdac6-KD vector and cultured for additional 3 days. The level of HDAC6 was significantly decreased in Hdac6-KD treatment. E, F The histological analysis and follicle counting results showed that knock down of Hdac6 expression accelerated primordial follicles activation without affecting the number of total follicles. The 1 dpp ovaries were transfected with Hdac6-KD vector and cultured for 4 days to assess the influence of RNAi on follicle development. G Overexpression efficiency analysis of Hdac6-OE. The 1 dpp ovaries were transfected with Hdac6-OE vector and cultured for 3 days. The level of HDAC6 was significantly increased in Hdac6-OE treatment. H, I The histological analysis and follicle counting results showed that overexpression of Hdac6 retarded the activation of primordial follicles without affecting the number of total follicles. The 1 dpp ovaries were transfected with Hdac6-OE vector and cultured for 4 days to assess the influence of overexpression of Hdac6 on follicle development. The experiments were repeated at least three times. And representative images are shown. The letters indicate there is a significant difference between the control and the specific treatment group. The data are presented as the means ± SEM. Scale bars: 50 μm. awakens primordial follicle finally . In line with this, we follicles by activating mTOR and improving pfGCs dif- therefore further examined the signal cascade within the ferentiation into granulosa cells followed by activating oocytes in our TubA supplementing assays. The results oocytes. To prove this hypothesis, the following assays showed that in addition to the activation of mTOR, the were performed. PI3K-AKT pathway activity was increased in TubA On the one hand, 2 dpp ovaries were cultured for 1 day treatment accordingly. with TubA and collected to examine the growth of Therefore, we hypothesized that down-regulated ovaries. The western blotting results showed that the HDAC6 may be involved in the activation of primordial expression of PCNA protein within ovaries was up- Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death and Disease (2021) 12:559 Page 7 of 12 Fig. 3 HDAC6 is responsible for primordial follicle activation by regulating mTOR signaling pathway. A–C The mTOR signaling pathway in the ovaries was affected by either inhibiting or elevating the level of HDAC6. Western blotting results showed that both total-mTOR and p-mTOR were increased in the TubA group or Hdac6-KD group compared with those in the respective controls. On contrary, Hdac6-OE treatment decreased the levels of both total-mTOR and p-mTOR compared with the control. D, E The histological analysis and counting results showed that the primordial follicle activation was reversed in TubA plus Rap treatment compared with TubA treatment alone. The experiments were repeated at least three times. And representative images are shown. The data are presented as the means ± SEM. Scale bars: 50 μm. regulated in TubA treatment (Fig. 5A), which implied that proteins, is pivotal for preserving fertility of mice through the growth of the whole ovaries was accelerated by TubA. inhibiting mTOR signaling in pfGCs of mice primordial Immunofluorescence staining and counting of PCNA follicles. Therefore, the detailed findings from this study positive cell results also showed that the PCNA positive supplied additional proofs to deeper understand the signal was significantly increased in pfGCs (Fig. 5B, C). underlined mechanisms of primordial follicle quiescent. These results suggested that the growth of ovaries, espe- Our study approved that the expression level of HDAC6 cially the growth of pfGCs, was accelerated by TubA. in primordial follicles is heterogeneous within the ovary. On the other hand, we blocked the communication Because those primordial follicles with weaker HDAC6 network between pfGCs and oocyte by ISCK03, one of the expression are being activated ones while those with KIT inhibitors, in TubA assay. The results showed that stronger HDAC6 are quiescent ones, it is possible that the both p-AKT and p-Foxo3a were decreased (Fig. 6A, B) decreased level of HDAC6 in primordial follicles indicates despite that mTOR and KITL were increased in TubA the fate of the follicles is activation. During the process, plus ISCK03. In which case, the primordial follicle acti- the decline of HDAC6 may be transient because the levels vation was retarded according to the histomorphology of HDAC6 elevates in primary follicles soon. According to and statistical results (Fig. 6C, D). The results suggested previous studies, short-term activation treatment with that HDAC6 controls primordial follicle activation one of the activators of either AKT, CDC42, or SIRT1 through KITL-KIT signaling. promotes the activation of mouse primordial follicles 9,11,12,17,31,32 in vitro . Therefore, the transition of pri- Discussion mordial follicles from the dormant state to the activated Primordial follicle pool established perinatally determines state may be a very short process. the length of the female reproductive lifespan. In this study, The activation of primordial follicles is a relatively bal- we found that HDAC6, one of the epigenetic modification ancing process. Properly controlling the speed of Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death and Disease (2021) 12:559 Page 8 of 12 Fig. 4 HDAC6 participates in the maintenance and activation of PI3K signaling. A–C Western blotting results showed that both p-Foxo3a and p-AKT were increased in either TubA or Hdac6-KD group compared with those in the controls, respectively. On contrary, the levels of both p-Foxo3a and p-AKT were down-regulated by Hdac6-OE treatment compared with the control. D The immunostaining stain showed that the cytoplasmic translocation of Foxo3 was increased in primordial follicles after treated with Tub A. Red arrow indicates cytoplasm Foxo3a. Black arrow indicates nucleus Foxo3a. E The statistical results showed that the cytoplasmic translocation of Foxo3 was increased in primordial follicles after treated with TubA. At least 500 primordial follicles were counted in each group. Scale bars: 50 μm. Fig. 5 HDAC6 regulates the proliferation of primordial follicle granulosa cell. A The western blotting results showed PCNA was increased after 2 dpp ovaries was cultured with TubA for 1 day. B Immunostaining stain of PCNA results showed that the growth of pfGCs in primordial follicles was increased compared with the control after 2 dpp ovaries were cultured with or without TubA for 1 day. C PCNA counting results showed that PCNA positive signaling was increased in pfGCs after with TubA compared with the control. The experiments were repeated at least three times. And representative images are shown. Scale bars: 50 μm. Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death and Disease (2021) 12:559 Page 9 of 12 Fig. 6 HDAC6 regulated primordial follicle activation through mTOR-KITL signaling. A The western blotting results showed the protein expression of mTOR, p-Foxo3a, Foxo3a, p-AKT, and AKT in TubA group, ISCK03 group and TubA plus ISCK03 group, respectively. B The Kitl mRNA relative level in TubA group, ISCK03 group and TubA plus ISCK03 group, respectively. C, D The histological analysis and counting results showed that the primordial follicle activation was reversed in TubA plus ISCK03 treatment compared with TubA treatment. The experiments were repeated at least three times. And representative images are shown. The data are presented as the means ± SEM. Scale bars: 50 μm. primordial follicle activation prolongs the reproductive HDAC6. According to Wang et al. , down-regulated 33–35 life of the ovaries . According to previous report, the TGF-β results in primordial follicle activation. However, ovarian reproductive lifespan was extended in Hdac6 whether HDAC6 is the upstream or downstream mole- overexpression mice ovary . It is assumed that over- cular of TGF-β within cells is controversial based on 36,37 expression of Hdac6 may retard the activation of pri- specific cell types been researched . Besides, although mordial follicles and slow down the rate of follicle we have proved that high level of E-cadherin in oocytes, consumption, which prolongs the lifespan of mice ovaries. and lower level of Sirt1in both oocytes and pfGCs, are also Although our study has uncovered the independent role positive molecules to inactivate primordial follicles, we of HDAC6 in primordial follicle activation process in did not find any relationship between HDAC6, SIRT1, mice ovaries in vitro, whether HDAC6 regulates the and E-cadherin so far (data not shown), implying that activation speed of primordial follicles to extend repro- there may have multiple signal pathways existing in pri- ductive life requires further study. mordial follicle. Additional studies are needed to clarify Interestingly, according to this study, the action of the complex relationship between these molecules. HDAC6 on sustaining primordial follicle dormancy in This study re-emphasizes the importance of epigenetic mice seems to overlap the findings of TGF-β according to modification in sustaining the primordial follicle pool our previous study . We noticed that not only the in vivo. Growing evidences from ours and others have expression pattern of TGF-β and HDAC6 in new born proved that the development of either germ cells or early mice ovaries are similar to each other, but the level of embryos are under controlled by epigenetic modification TGF-β, as well as its downstream key protein, namely the proteins, like histone deacetylases including HDAC1, activated SMAD3, decreased in response to inhibitor of HDAC2, HDAC3, SIRT1, HDAC6, as well as histone Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death and Disease (2021) 12:559 Page 10 of 12 38–45 demethylase like LSD1 . We have proved that the findings provided further proofs demonstrating that epi- common target protein of both SIRT1 and HDAC6 in genetic modification participates in homeostatic of pri- pfGCs is unexceptionally mTOR . On the other hand, mordial follicle reserve. since the present hypothesis referring to primordial fol- Acknowledgements licle activation underlines the importance of the com- The authors wish to thank each member of Xia Lab for their valuable munication between the somatic cells and oocytes within discussion. follicle , our study has supplied additional proofs to Funding support the theory soundly. Our study also indicates that This study was supported by grants from the National Key Research & there are multiple signal pathways governing the process Developmental Program of China grant number 2018YFC1003700 and of activating primordial follicles, which worth further 2017YFC1001100 to G.X.; 2018YFC1003801 to C.W.; the National Basic Research Program of China grant numbers 2013CB945501 to G.X.; Institution of Higher study based on more complicated in vitro and in vivo Education Projects of Building First-class Discipline Construction in Ningxia models. Region (Biology) grant number NXYLXK2017B05 to G.X.; the National Natural The levels of activated mTOR as well as PI3K proteins Science Foundation of China grant numbers 31872792 to C.W.; 31371448 and 31571540 to G.X.; the Beijing Natural Science Foundation grant numbers determine the fate of primordial follicles, as has been 5182015 to C.W., 7182090 to G.X.; Project of State Key Laboratory of proved by this study and others. First, upregulation of Agrobiotechnology grant numbers 2015SKLAB4-1 to C.W., 2016SKLAB-1 to G.X. mTOR in either pfGCs or oocyte accelerates primordial 46,47 follicle activation . Conditional knockout of mTOR in Author details oocytes of primordial follicle causes defective follicular Key Laboratory of Ministry of Education for Conservation and Utilization of development leading to progressive degeneration of Special Biological Resources in the Western China, College of Life Science, 48 2 Ningxia University, 750021 Yinchuan, China. Guizhou Provincial Key oocytes , whereas conditional knockout of Tsc1, the Laboratory of Pathogenesis & Drug Research on Common Chronic Diseases, negatively regulator of mTOR, in either oocytes or pfGCs Department of Physiology, School of Basic Medical Sciences, Guizhou Medical of primordial follicles resulted in systematic premature University, 550009 Guiyang, Guizhou, China. State Key Laboratory of 14,31 Agrobiotechnology, College of Biological Sciences, China Agricultural activation of primordial follicle pool . Second, University, 100193 Beijing, China. Department of Biology, School of Basic increasing evidences from others and ours have indicated Medical Science, Guizhou Medical University, 550025 Guiyang, Guizhou, China. the importance of the activity of PI3K signaling pathway, Department of Pathology and Hepatology, the 5th Medical Centre, Chinese People’s Liberation Army General Hospital, 100039 Beijing, China. RDFZ Xishan in determining if oocytes within primordial follicles could School, 100193 Beijing, China be activated, no matter it is activated by signals from oocyte itself or from granulosa cells of the primordial Author contributions 8,9,11,12,15 follicle . The activity of AKT in response to T.Z., M.H., and L. Z. designed the research; T.Z., M.H., S.Q., Z.Z., X.D., B.Z., Y.Y., and X.L. performed the experiment. T.Z., M.H. G.X. T.C., and Y.W. analyzed the data. inhibition of HDAC6 seems to be cell type dependent . T.Z., H.Z. and C.W. wrote the manuscript. G.X. revised the manuscript. All In this study, HDAC6 inhibition results in increased AKT authors read and approved the final manuscript. kinase activity indirectly in newborn mice ovary through activated mTOR-KITL pathway in pfGCs. Further studies Conflict of interest The authors declare no competing interests. are needed to clarify the exact factors within the niche of primordial follicles which are the key signals for sustain- Ethics statement ing the quiescent of primordial follicles before a clearer All animal procedures were conducted in accordance with the guidelines of and approved by the Animal Research Committee of the China Agricultural model of primordial follicle sustaining be figured out. University. In vitro activation (IVA) of primordial follicle technique based on above mentioned theory has been established to Publisher’s note help solve the problems of POF patients recently . The Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. main target for the tech is to inhibit the activity of PTEN or activate PI3K in oocytes . From this study, we have Supplementary information The online version contains supplementary provided an alternative target, the HDAC protein in material available at https://doi.org/10.1038/s41419-021-03842-1. pfGCs, for activating mTOR-KIT-PI3K signaling cascades Received: 9 June 2020 Revised: 2 March 2021 Accepted: 8 March 2021 between somatic cells and oocytes within individual pri- mordial follicle. 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HDAC6 modulates cell motility by altering the acetylation level and activation of primordial follicles. Hum. Mol. Genet. 19,397–410 (2009). of cortactin. Mol. Cells 27,197–213 (2007). 48. Guo, J. et al. Oocyte stage-specific effects of MTOR determine granulosa cell 25. Li, Y., Shin, D. & Kwon, S. H. Histone deacetylase 6 plays a role as a distinct fate and oocyte quality in mice. Proc. Natl Acad. Sci. USA 115,5326–5333 regulator of diverse cellular processes. FEBS J. 280,775–793 (2013). (2018). Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death and Disease (2021) 12:559 Page 12 of 12 Official journal of the Cell Death Differentiation Association http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Death & Disease Springer Journals

HDAC6 regulates primordial follicle activation through mTOR signaling pathway

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2041-4889
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10.1038/s41419-021-03842-1
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Abstract

Primordial follicle pool established perinatally is a non-renewable resource which determines the female fecundity in mammals. While the majority of primordial follicles in the primordial follicle pool maintain dormant state, only a few of them are activated into growing follicles in adults in each cycle. Excessive activation of the primordial follicles accelerates follicle pool consumption and leads to premature ovarian failure. Although previous studies including ours have emphasized the importance of keeping the balance between primordial follicle activation and dormancy via molecules within the primordial follicles, such as TGF-β, E-Cadherin, mTOR, and AKT through different mechanisms, the homeostasis regulatory mechanisms of primordial follicle activation remain unclear. Here, we reported that HDAC6 acts as a key negative regulator of mTOR in dormant primordial follicles. In the cytoplasm of both oocytes and granulosa cells of primordial follicles, HDAC6 expressed strong, however in those activated primordial follicles, its expression level is relatively weaker. Inhibition or knockdown of HDAC6 significantly promoted the activation of limited primordial follicles while the size of follicle pool was not affected profoundly in vitro. Importantly, the expression level of mTOR in the follicle and the activity of PI3K in the oocyte of the follicle were simultaneously up- regulated after inhibiting of HDAC6. The up-regulated mTOR leads to not only the growth and differentiation of primordial follicles granulosa cells (pfGCs) into granulosa cells (GCs), but the increased secretion of KITL in these somatic cells. As a result, inhibition of HDAC6 awaked the dormant primordial follicles of mice in vitro. In conclusion, HDAC6 may play an indispensable role in balancing the maintenance and activation of primordial follicles through mTOR signaling in mice. These findings shed new lights on uncovering the epigenetic factors involved physiology of sustaining female reproduction. Introduction state for as long as 50 years in humans or for more than 1 Ovarian follicles are the basic and non-renewable year in mice. Only a small portion of dormant primordial 1,2 reproductive unit of female mammals . In mammalian follicles are progressively recruited into the growing fol- ovaries, the majority of follicles reserved are primordial licle pool each time in adults, which is called primordial follicles, which are maintained at dormant or quiescent follicle activation . Obviously, the balance between quiescent and activated state of the primordial follicles is crucial for female fecundity. Excessive activation of pri- Correspondence: Hua Zhang (huazhang@cau.edu.cn)or mordial follicles will lead to exhaustion of the resting Chao Wang (wangcam@126.com) Key Laboratory of Ministry of Education for Conservation and Utilization of follicle reserve and result in premature ovarian failure Special Biological Resources in the Western China, College of Life Science, (POF), which seriously affect female physical and mental Ningxia University, 750021 Yinchuan, China health . Unfortunately, there is hardly any effective mea- Guizhou Provincial Key Laboratory of Pathogenesis & Drug Research on Common Chronic Diseases, Department of Physiology, School of Basic Medical surements to help these patients so far because the Sciences, Guizhou Medical University, 550009 Guiyang, Guizhou, China acknowledgement of the mechanisms governing the Full list of author information is available at the end of the article These authors contributed equally: Tuo Zhang, Meina He, Lihua Zhao Edited by G.M. Fimia © The Author(s) 2021 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():,; 1234567890():,; 1234567890():,; Zhang et al. Cell Death and Disease (2021) 12:559 Page 2 of 12 activation and dormant of primordial follicles is still overexpression of Hdac6 retarded the activation of pri- obscure. mordial follicle. HDAC6 regulates primordial follicles The primordial follicle consists of a quiescent oocyte quiescent through regulating the expression of mTOR and surrounding flattened primordial follicle granulosa signaling. Collectively, we hypothesize that HDAC6 plays cells. Previous studies including ours have implied that an indispensable role in sustaining primordial follicles oocyte derived molecules, such as phosphatidylinositol 3- through affecting the expression of mTOR signaling pathway. kinase (PI3K), cell division cycle 42 (CDC42), E-cadherin/ N-adherin (E-cad/N-cad), and newborn ovary homeobox 5–11 (NOBOX) , as well as oocyte and pfGCs expressed Materials and methods Sirtuin 1 (SIRT1) and transforming growth factor β (TGF- Animal 12,13 β) , are pivotal for the sustainment of primordial fol- Adult CD-1 mice were purchased from Beijing Vital licles after birth. On the other hand, the activation of River Laboratory Animal Technology Co., Ltd. and bred primordial follicle is a complex and coordinated process to male mice of the same strain. Mice were housed under among individual oocyte, pfGCs, and even the sur- controlled lighting (12 h light, 12 h dark) and temperature rounding micro-environment . Actually, many molecules (24–26 °C) conditions with free access to food and water. mentioned above are involved in the activation process of Vaginal plug detection was considered as 0.5 day post primordial follicle originated from the pfGCs, of which coitus (dpc). The first 12 h after birth was considered as mTORC1 signaling in pfGCs and PI3K signaling in 0 day postpartum (dpp). All procedures were conducted oocytes is the dominant pathway . in accordance with the guidelines of and approved by the Although the technique of in vitro activation (IVA) of Animal Research Committee of the China Agricultural primordial follicles based on these findings have achieved University. to help POF patients producing fertilizable oocytes for 16,17 successful reproduction, the efficiency is quite low . Whole ovary and cortex fragments isolation And it is hard to unveil the pathological mechanism of The newborn mice were sacrificed at the designated POF so far because there is very limited knowledge about times. The ovaries of the newborn mice were micro- how primordial follicle dormancy is finely tuned dissected in cold phosphate buffered saline (PBS) under after birth. microscope. Make sure that the structure of whole ovaries Most recently, we have proved that SIRT1, one of the was not damaged during the separation procedure. When members of histone deacetylase family, contributes to isolating the cortex fragments under a steroid microscope, sustain primordial follicles in mice, implying the impor- a syringe of 1 ml with needle was applied to carefully tance of epigenetic modification effect in the process .In separate the ovarian cortex from the medulla tissue. line with this, an overexpression of histone deacetylase 6 (Hdac6) extends reproductive lifespan of mice since an Ovary organ culture increased folliculogenesis and particularly secondary and The dissected ovary was cultured in six-well culture antral follicles are found in 19-month-old mice compared plates (Nest, Jiangsu, China) in 1200 μl of Dulbecco with 16-month wild-type mice . HDAC6 belongs to class Modified Eagle Medium/Ham F12 nutrient mixture IIb of HDAC family, and is a microtubule-associated (DMEM/F12) (Gibco, Life Technologies, CA) with deacetylase . Physiologically, it predominantly functions insulin-transferrin-sodium selenite (Sigma, 1:100, USA) in the cytoplasm by deacetylating various substrates so as and penicillin–streptomycin solution at 37 °C, 5% CO to regulate cell migration, cell cytoskeleton, cell motility, and saturated humidity. The female mice ovaries were and cell immune synapse formation, and even misfolded randomly assigned. Same number of ovaries in each 20–26 proteins degradation . HDAC6 contains two intact group. The culture medium was exchanged every other catalytic domains that located respectively at the N- day. The final concentrations of Tubastatin A (TubA in terminal and the central regions of the protein, which is short) (S8049; Selleck, Shanghai, China), Rapamycin (Rap different from those of SIRT1. Additionally, it is not only in short) (S1039; Selleck, Shanghai, China), and ISCK03 important for regulating chromatin compaction and (I6410; Sigma, USA) were 4 μM, 2.5 μM, and 5 μM, meiotic apparatus assembly in mouse oocytes, but plays respectively. The control group was supplemented with 27–30 essential part in ARID1A-mutated ovarian cancer . the same volume of dimethyl sulfoxide (DMSO). Therefore, we wonder if HDAC6 was also involved in sustaining primordial follicles. Immunofluorescence and immunohistochemistry Briefly, we found that HDAC6 participates in sustaining Freshly isolated ovaries were fixed in cold 4% paraf- primordial follicle quiescent in newborn mice. We proved ormaldehyde overnight, dehydrated in gradient alcohol, that either inhibiting or knocking down HDAC6 pro- embedded in paraffin, and sectioned at 5 μm. Ovarian moted the activation of primordial follicles, while sections were deparaffinized, rehydrated and subjected to Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death and Disease (2021) 12:559 Page 3 of 12 antigen retrieval with 0.01% sodium citrate buffer (pH 6.0) Real-time PCR analysis at high temperature (95–98 °C) for 16 min. The sections Total RNA obtained from at least six ovaries was were then rinsed thoroughly with PBS and blocked with extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, normal donkey serum in PBS for 1 h at room temperature USA) according to the manufacturer’s protocol. First- and incubated with primary antibodies overnight at 4 °C. strand cDNA was synthesized by reverse transcription The primary antibodies used are listed in Table S1. Next, using 1 µg of total RNA (Promega Reverse Transcription ovarian sections was rinsed thoroughly with PBS and System, Promega, USA). Quantitative-RT-PCR was per- incubated with Alexa Fluor 488- or 555- conjugated sec- formed using SYBR Select Master Mix (Applied Biosys- ondary antibody for 1 h at 37 °C. The ovarian sections were tems, Life Technologies, USA) and operated by Applied rinsed thoroughly with PBS, stained with DAPI for 5 min Biosystems 7500 Real Time PCR System (Applied Bio- and sealed in anti-fade fluorescence mounting medium systems, Life Technologies, USA). The data were nor- (C1210, Applygen, China) with coverslips. Immunohis- malized by β-actin. Primers used are listed in Table S2. tochemistry was performed using Histostain™-SP Kits (PV-9001, ZSGB-BIO, China) and DAB peroxidase sub- Gene knockdown and overexpression strate kits (ZLI-9017, ZSGB-BIO, China) according to the The vectors of either for knockdown or overexpression manufacturer’s protocols. Sections were examined and of Hdac6 was respectively mixed with trypan blue solu- photographed using Nikon 80i or Nikon A1 laser. tion (vector/trypan blue solution: 10/1) and then injected into isolated 1 dpp mouse ovaries using glass pipettes Total follicle, primordial follicle, and primary follicle under a stereomicroscope. After the ovaries were injected counting with the blue liquid, electrotransfection was performed by The serial sections of each ovary (8 μm thick) were the application of three 5-ms-long quasi-square pulses at stained with hematoxylin. Only when follicles containing a pulse-field strength of up to 30 V/cm. The Hdac6 clearly visible nucleus of oocytes in each individual sec- knockdown vector was constructed by cloning Hdac6- tion of every ovary were counted. The serial sections of shRNA with sequence: GGTACTTCCCATCGCC- each ovary were counted before statistical analysis. Max- TATGA. The Hdac6 overexpression vector was con- imum and minimum were excluded. A double-blind structed by cloning the open reading frame of Hdac6 into experiment was performed during follicle number the pCMV-C-His (D2650, Beyotime Biotechnology, counting. The follicles were distinguished from each other China). Golden star t6 super PCR mix were purchased as follows: primordial follicle (a single oocyte surrounded from Beijing Tsingke Co., Ltd. All of the constructs were by several flattened pre-granulosa cells) and primary fol- verified by sequencing. Primers are listed in Table S3. licle (an enlarged oocyte surround by a mixture of squa- mous and cuboidal granulosa cells). Statistical analysis All experiments were repeated at least three times. The Western blotting analysis values are presented as the means ± SEM. The data were Each protein sample obtained from at least six ovaries analyzed by either t test or ANOVA, and considered were extracted in WIP Tissue and cell lysis solution statistically significant at P < 0.05. containing 1 mM phenylmethylsulfonyl fluoride (8553 S, Cell Signaling Technologies, USA) according to the Results manufacturer’s instructions. The protein concentration HDAC6 is indispensable for preventing primordial follicle was measured by a BCA assay (P0012, Beyotime, China). activation in vitro The samples were separated on 10% SDS-PAGE and then To explore the potential role of HDAC6 in either sus- transferred to polyvinylidene fluoride membranes taining the dormancy or activation of primordial follicle, (IPVH00010, Millipore, USA). The membranes were immunofluorescence staining was firstly performed to blocked with 5% skim milk powder for 1 h at room tem- detect the cellular localization and expression dynamics of perature and then incubated with appropriate primary HDAC6 in newborn and adult mouse ovaries. It showed antibodies overnight at 4 °C. The primary antibodies used that HDAC6 was predominantly localized to the cyto- are listed in Table S1. The membranes were rinsed plasm of oocytes and granulosa cells in newborn and adult thoroughly with TBST. The membranes were incubated mice ovaries (Figs. 1A and S1). with the secondary antibody (1:5000, ZSGB-BIO, China) We found that the signal of HDAC6 was homogenous for 1 h at room temperature and rinsed thoroughly with stronger in the majority of primordial follicles, but was TBST. The membranes were visualized using a Super- relatively weaker in some primordial follicles, which may Signal West Pico Chemiluminescent Detection System be on the way to be activated. This phenomenon is (Prod 34080, Thermo, USA). β-actin (CW0096M, observed in both ovaries of adult and newborn mice Cwbiotech, China) was used as the intrinsic control. unanimously (Figs. S1 and S2). The statistics results show Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death and Disease (2021) 12:559 Page 4 of 12 Fig. 1 The expression pattern of HDAC6 in the neonatal mouse ovaries. A Cellular localization of HDAC6 in newborn ovaries. Newborn mice ovaries were stained for HDAC6 (green) and the oocyte specific marker DDX4 (red) at the indicated time points. The nuclei were counter-stained by DAPI (blue). HDAC6 was mainly localized to the cytoplasm of both oocytes and granulosa cells in either primordial follicles or growing follicles. B qRT- PCR assay showed that the expression level of Hdac6 mRNA was increased from 1 dpp to 5 dpp and decreased on 7 dpp. C Western blotting assay showed that the level of HDAC6 protein was increased from 1 dpp to 5 dpp and decreased on 7 dpp as well. The experiments were repeated at least three times. And representative images are shown. The data are presented as the means ± SEM. Scale bars: 50 μm. that 3.5 ± 0.2% of primordial follicles with weaker HDAC6 the mRNA and protein of HDAC6 increase significantly expression are presented in adult ovaries, while 4 ± 0.4% along with the establishment of the primordial follicle of primordial follicles with weaker HDAC6 expression are pool at 3 dpp, and retain at a relative higher level from 5 presented in 5 dpp ovaries (Fig. S3). dpp to 7 dpp, during which time primordial follicle acti- In order to clarify whether the primordial follicles with vation is generally initiated (Fig. 1B, C). weaker HDAC6 signal are being activated, the 5 dpp To further determine the expression pattern of HDAC6 ovaries were stained for HDAC6 and Foxo3a in adjacent in primordial follicles from 1 dpp to 7 dpp, a model of serial sections. The results showed that over 60% of pri- ovarian cortex fragments isolated from the intact ovary mordial follicles with weaker expression of HDAC6 were was established. We noticed that only primordial follicles simultaneously the ones whose FOXO3a was located in were found in the isolated cortical fragments in 1 dpp–7 the cytoplasm (Fig. R6). The results suggest that 65.1 ± dpp ovaries (Figs. S5–S9). The western blotting results 5.3% of primordial follicles with weaker HDAC6 signal are showed that the HDAC6 expression decreased from 1 dpp being activated. and then maintained at a relatively weaker level from 3 Then the qRT-PCR and western blotting analyses of dpp to 7 dpp in ovarian cortex fragments. Interestingly, whole ovary samples revealed that the expressions of both the general protein level of HDAC6 in 5 dpp whole ovary Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death and Disease (2021) 12:559 Page 5 of 12 was higher than in 3 dpp–7 dpp ovarian cortex fragments levels of both total and phosphorylated mTOR which are (Fig. S10). Collectively, these results suggest that the the downstream molecules of TSCs, were up-regulated in expression of HDAC6 in primordial follicles is homo- TubA treatment (Fig. 3A). To further confirm the reg- genous, whereas the expression of HDAC6 becomes ulation effect of decreased HDAC6 on mTOR, the 1 dpp weaker in some primordial follicles that were to be acti- ovaries were either injected with Hdac6-KD or Hdac6-OE vated. These results indicated that HDAC6 plays a vector respectively and cultured for additional 3 days. Western blotting results showed that the levels of both potential role in primordial follicle activation versus quiescent. total and phosphorylated mTOR were either up-regulated To study the function of HDAC6 in primordial follicle in Hdac6-KD treatment or down-regulated in Hdac6-OE in newborn mouse ovaries, an in vitro culture model was treatment, respectively (Fig. 3B, C). These results sug- established. The histological and statistical analysis results gested that HDAC6 may be pivotal for balancing pri- showed that the ovaries cultured in vitro develop normally mordial follicle between dormancy and activation through as the morphology as well as the numbers of different influencing the mTOR signaling pathway. stage of follicles and granulosa cells were consistent with To further confirm the above conclusion, Rapamycin the simultaneously developed ovaries in vivo (Fig. S11). (Rap), the specific inhibitor of mTOR, was used to verify if Based on this model, 2 dpp ovaries were cultured with it could attenuate the expression of mTOR induced by TubA, one of the inhibitors of HDAC6, for 2 days. The TubA treatment. For this purpose, 2 dpp ovaries were results showed that acetylated alpha-tubulin was cultured with either TubA or Rap for 3 days before increased and the level of HDAC6 was decreased com- examination. The histological analysis and whole ovary pared to the control (Fig. 2A). Based on these findings, the counting results showed that fewer growing follicles were treated ovaries were furtherly cultured for 3 days before observed in TubA plus Rap treatment than those in the histological analysis. The results showed that more TubA treatment (Fig. 3D–F). Inhibition of mTOR by Rap growing follicles were observed in TubA treatment reversed the follicular development caused by TubA. compared to the control, while the number of total folli- These results suggested that HDAC6 in the primordial cles within each ovary was comparable to the control (Fig. follicles of new born ovaries prevent follicles from pre- 2B, C). These results implied that HDAC6 participates in mature activation. preventing primordial follicles from premature activation in vivo. Downregulation of HDAC6 is necessary for activating PI3K- To further confirm the function of HDAC6 in regulat- AKT pathway in oocytes in vitro ing primordial follicle development, both Hdac6-shRNA To study the potential relationship between down- (Hdac6-KD) vector and Hdac6-overexpression (Hdac6- regulated HDAC6 and the PI3K pathway within oocytes OE) vector were respectively constructed, and injected of primordial follicles, 2 dpp ovaries were cultured with or into 1 dpp ovaries respectively. The injected ovaries were without TubA for 2 days. Western blotting results showed cultured for 3 days, the results showed that the endo- that the total levels of either AKT or Foxo3a were genous HDAC6 protein levels were either efficiently unchanged, while the levels of both phosphorylated AKT down-regulated by Hdac6-KD, or up-regulated in Hdac6- and Foxo3a were up-regulated in TubA treatment (Fig. OE group, respectively (Fig. 2D, G). Then, the injected 3A). At the same time, the conclusion was further con- ovaries were cultured for 4 days, the histological analysis firmed by results applying Hdac6-KD and Hdac6-OE, showed that more growing follicles were observed in respectively (Fig. 4B, C). In addition, immunofluorescence Hdac6-KD group and fewer growing follicles were staining and quantitative results showed that Foxo3a, observed in Hdac6-OE group, respectively (Fig. 2E, H). which shuttled from the nucleus to the cytoplasm, was The quantitative results further confirmed the phenotype increased in TubA treated ovaries (Fig. 4D). These results observed in Figs. 2F, I. These results indicated that indicated that inhibition of HDAC6 expression in mice downregulating the level of HDAC6 was necessary for ovaries was indispensable for the activation of PI3K mice primordial follicles activation in vitro. pathway. HDAC6 prevents primordial follicle activation through Down-regulated HDAC6 in new born ovaries governed inhibiting mTOR signaling pathway primordial follicle activation through KITL-KIT in vitro To investigate how decreased HDAC6 participates in According to the existed hypothesis, it is somatic cell primordial follicle activation, the changes of the mTOR that initiates primordial follicle activation. That is, the up- signaling pathway were detected. Before examination, 2 regulated mTORC1 signaling in pre-granulosa cells trig- dpp ovaries were cultured with or without TubA for gers the awakening of dormant oocytes through secreting 2 days. The western blotting results showed that both KIT ligand (KITL). The coupling between mTORC1- TSC1 and TSC2 were unchanged (Fig. S12) whereas the KITL in pfGCs and KIT-PI3K signaling in oocytes Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death and Disease (2021) 12:559 Page 6 of 12 Fig. 2 HDAC6 is involved in the primordial follicle activation. A Inhibition efficiency analysis of TubA. The 2 dpp ovaries were cultured with or without TubA for 2 days. The ac-Tubulin was increased while HDAC6 was decreased in TubA treatment. ac-Tubulin: acetylated alpha-tubulin. B, C The histological analysis and follicle counting results showed that inhibition of HDAC6 expression by TubA accelerated primordial follicles activation without affecting the number of total follicles. The 2 dpp ovaries were cultured with or without TubA for 3 days. D Knockdown efficiency analysis of Hdac6-KD. The 1 dpp ovaries were transfected with Hdac6-KD vector and cultured for additional 3 days. The level of HDAC6 was significantly decreased in Hdac6-KD treatment. E, F The histological analysis and follicle counting results showed that knock down of Hdac6 expression accelerated primordial follicles activation without affecting the number of total follicles. The 1 dpp ovaries were transfected with Hdac6-KD vector and cultured for 4 days to assess the influence of RNAi on follicle development. G Overexpression efficiency analysis of Hdac6-OE. The 1 dpp ovaries were transfected with Hdac6-OE vector and cultured for 3 days. The level of HDAC6 was significantly increased in Hdac6-OE treatment. H, I The histological analysis and follicle counting results showed that overexpression of Hdac6 retarded the activation of primordial follicles without affecting the number of total follicles. The 1 dpp ovaries were transfected with Hdac6-OE vector and cultured for 4 days to assess the influence of overexpression of Hdac6 on follicle development. The experiments were repeated at least three times. And representative images are shown. The letters indicate there is a significant difference between the control and the specific treatment group. The data are presented as the means ± SEM. Scale bars: 50 μm. awakens primordial follicle finally . In line with this, we follicles by activating mTOR and improving pfGCs dif- therefore further examined the signal cascade within the ferentiation into granulosa cells followed by activating oocytes in our TubA supplementing assays. The results oocytes. To prove this hypothesis, the following assays showed that in addition to the activation of mTOR, the were performed. PI3K-AKT pathway activity was increased in TubA On the one hand, 2 dpp ovaries were cultured for 1 day treatment accordingly. with TubA and collected to examine the growth of Therefore, we hypothesized that down-regulated ovaries. The western blotting results showed that the HDAC6 may be involved in the activation of primordial expression of PCNA protein within ovaries was up- Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death and Disease (2021) 12:559 Page 7 of 12 Fig. 3 HDAC6 is responsible for primordial follicle activation by regulating mTOR signaling pathway. A–C The mTOR signaling pathway in the ovaries was affected by either inhibiting or elevating the level of HDAC6. Western blotting results showed that both total-mTOR and p-mTOR were increased in the TubA group or Hdac6-KD group compared with those in the respective controls. On contrary, Hdac6-OE treatment decreased the levels of both total-mTOR and p-mTOR compared with the control. D, E The histological analysis and counting results showed that the primordial follicle activation was reversed in TubA plus Rap treatment compared with TubA treatment alone. The experiments were repeated at least three times. And representative images are shown. The data are presented as the means ± SEM. Scale bars: 50 μm. regulated in TubA treatment (Fig. 5A), which implied that proteins, is pivotal for preserving fertility of mice through the growth of the whole ovaries was accelerated by TubA. inhibiting mTOR signaling in pfGCs of mice primordial Immunofluorescence staining and counting of PCNA follicles. Therefore, the detailed findings from this study positive cell results also showed that the PCNA positive supplied additional proofs to deeper understand the signal was significantly increased in pfGCs (Fig. 5B, C). underlined mechanisms of primordial follicle quiescent. These results suggested that the growth of ovaries, espe- Our study approved that the expression level of HDAC6 cially the growth of pfGCs, was accelerated by TubA. in primordial follicles is heterogeneous within the ovary. On the other hand, we blocked the communication Because those primordial follicles with weaker HDAC6 network between pfGCs and oocyte by ISCK03, one of the expression are being activated ones while those with KIT inhibitors, in TubA assay. The results showed that stronger HDAC6 are quiescent ones, it is possible that the both p-AKT and p-Foxo3a were decreased (Fig. 6A, B) decreased level of HDAC6 in primordial follicles indicates despite that mTOR and KITL were increased in TubA the fate of the follicles is activation. During the process, plus ISCK03. In which case, the primordial follicle acti- the decline of HDAC6 may be transient because the levels vation was retarded according to the histomorphology of HDAC6 elevates in primary follicles soon. According to and statistical results (Fig. 6C, D). The results suggested previous studies, short-term activation treatment with that HDAC6 controls primordial follicle activation one of the activators of either AKT, CDC42, or SIRT1 through KITL-KIT signaling. promotes the activation of mouse primordial follicles 9,11,12,17,31,32 in vitro . Therefore, the transition of pri- Discussion mordial follicles from the dormant state to the activated Primordial follicle pool established perinatally determines state may be a very short process. the length of the female reproductive lifespan. In this study, The activation of primordial follicles is a relatively bal- we found that HDAC6, one of the epigenetic modification ancing process. Properly controlling the speed of Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death and Disease (2021) 12:559 Page 8 of 12 Fig. 4 HDAC6 participates in the maintenance and activation of PI3K signaling. A–C Western blotting results showed that both p-Foxo3a and p-AKT were increased in either TubA or Hdac6-KD group compared with those in the controls, respectively. On contrary, the levels of both p-Foxo3a and p-AKT were down-regulated by Hdac6-OE treatment compared with the control. D The immunostaining stain showed that the cytoplasmic translocation of Foxo3 was increased in primordial follicles after treated with Tub A. Red arrow indicates cytoplasm Foxo3a. Black arrow indicates nucleus Foxo3a. E The statistical results showed that the cytoplasmic translocation of Foxo3 was increased in primordial follicles after treated with TubA. At least 500 primordial follicles were counted in each group. Scale bars: 50 μm. Fig. 5 HDAC6 regulates the proliferation of primordial follicle granulosa cell. A The western blotting results showed PCNA was increased after 2 dpp ovaries was cultured with TubA for 1 day. B Immunostaining stain of PCNA results showed that the growth of pfGCs in primordial follicles was increased compared with the control after 2 dpp ovaries were cultured with or without TubA for 1 day. C PCNA counting results showed that PCNA positive signaling was increased in pfGCs after with TubA compared with the control. The experiments were repeated at least three times. And representative images are shown. Scale bars: 50 μm. Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death and Disease (2021) 12:559 Page 9 of 12 Fig. 6 HDAC6 regulated primordial follicle activation through mTOR-KITL signaling. A The western blotting results showed the protein expression of mTOR, p-Foxo3a, Foxo3a, p-AKT, and AKT in TubA group, ISCK03 group and TubA plus ISCK03 group, respectively. B The Kitl mRNA relative level in TubA group, ISCK03 group and TubA plus ISCK03 group, respectively. C, D The histological analysis and counting results showed that the primordial follicle activation was reversed in TubA plus ISCK03 treatment compared with TubA treatment. The experiments were repeated at least three times. And representative images are shown. The data are presented as the means ± SEM. Scale bars: 50 μm. primordial follicle activation prolongs the reproductive HDAC6. According to Wang et al. , down-regulated 33–35 life of the ovaries . According to previous report, the TGF-β results in primordial follicle activation. However, ovarian reproductive lifespan was extended in Hdac6 whether HDAC6 is the upstream or downstream mole- overexpression mice ovary . It is assumed that over- cular of TGF-β within cells is controversial based on 36,37 expression of Hdac6 may retard the activation of pri- specific cell types been researched . Besides, although mordial follicles and slow down the rate of follicle we have proved that high level of E-cadherin in oocytes, consumption, which prolongs the lifespan of mice ovaries. and lower level of Sirt1in both oocytes and pfGCs, are also Although our study has uncovered the independent role positive molecules to inactivate primordial follicles, we of HDAC6 in primordial follicle activation process in did not find any relationship between HDAC6, SIRT1, mice ovaries in vitro, whether HDAC6 regulates the and E-cadherin so far (data not shown), implying that activation speed of primordial follicles to extend repro- there may have multiple signal pathways existing in pri- ductive life requires further study. mordial follicle. Additional studies are needed to clarify Interestingly, according to this study, the action of the complex relationship between these molecules. HDAC6 on sustaining primordial follicle dormancy in This study re-emphasizes the importance of epigenetic mice seems to overlap the findings of TGF-β according to modification in sustaining the primordial follicle pool our previous study . We noticed that not only the in vivo. Growing evidences from ours and others have expression pattern of TGF-β and HDAC6 in new born proved that the development of either germ cells or early mice ovaries are similar to each other, but the level of embryos are under controlled by epigenetic modification TGF-β, as well as its downstream key protein, namely the proteins, like histone deacetylases including HDAC1, activated SMAD3, decreased in response to inhibitor of HDAC2, HDAC3, SIRT1, HDAC6, as well as histone Official journal of the Cell Death Differentiation Association Zhang et al. Cell Death and Disease (2021) 12:559 Page 10 of 12 38–45 demethylase like LSD1 . We have proved that the findings provided further proofs demonstrating that epi- common target protein of both SIRT1 and HDAC6 in genetic modification participates in homeostatic of pri- pfGCs is unexceptionally mTOR . On the other hand, mordial follicle reserve. since the present hypothesis referring to primordial fol- Acknowledgements licle activation underlines the importance of the com- The authors wish to thank each member of Xia Lab for their valuable munication between the somatic cells and oocytes within discussion. follicle , our study has supplied additional proofs to Funding support the theory soundly. Our study also indicates that This study was supported by grants from the National Key Research & there are multiple signal pathways governing the process Developmental Program of China grant number 2018YFC1003700 and of activating primordial follicles, which worth further 2017YFC1001100 to G.X.; 2018YFC1003801 to C.W.; the National Basic Research Program of China grant numbers 2013CB945501 to G.X.; Institution of Higher study based on more complicated in vitro and in vivo Education Projects of Building First-class Discipline Construction in Ningxia models. Region (Biology) grant number NXYLXK2017B05 to G.X.; the National Natural The levels of activated mTOR as well as PI3K proteins Science Foundation of China grant numbers 31872792 to C.W.; 31371448 and 31571540 to G.X.; the Beijing Natural Science Foundation grant numbers determine the fate of primordial follicles, as has been 5182015 to C.W., 7182090 to G.X.; Project of State Key Laboratory of proved by this study and others. First, upregulation of Agrobiotechnology grant numbers 2015SKLAB4-1 to C.W., 2016SKLAB-1 to G.X. mTOR in either pfGCs or oocyte accelerates primordial 46,47 follicle activation . Conditional knockout of mTOR in Author details oocytes of primordial follicle causes defective follicular Key Laboratory of Ministry of Education for Conservation and Utilization of development leading to progressive degeneration of Special Biological Resources in the Western China, College of Life Science, 48 2 Ningxia University, 750021 Yinchuan, China. Guizhou Provincial Key oocytes , whereas conditional knockout of Tsc1, the Laboratory of Pathogenesis & Drug Research on Common Chronic Diseases, negatively regulator of mTOR, in either oocytes or pfGCs Department of Physiology, School of Basic Medical Sciences, Guizhou Medical of primordial follicles resulted in systematic premature University, 550009 Guiyang, Guizhou, China. State Key Laboratory of 14,31 Agrobiotechnology, College of Biological Sciences, China Agricultural activation of primordial follicle pool . Second, University, 100193 Beijing, China. Department of Biology, School of Basic increasing evidences from others and ours have indicated Medical Science, Guizhou Medical University, 550025 Guiyang, Guizhou, China. the importance of the activity of PI3K signaling pathway, Department of Pathology and Hepatology, the 5th Medical Centre, Chinese People’s Liberation Army General Hospital, 100039 Beijing, China. RDFZ Xishan in determining if oocytes within primordial follicles could School, 100193 Beijing, China be activated, no matter it is activated by signals from oocyte itself or from granulosa cells of the primordial Author contributions 8,9,11,12,15 follicle . The activity of AKT in response to T.Z., M.H., and L. Z. designed the research; T.Z., M.H., S.Q., Z.Z., X.D., B.Z., Y.Y., and X.L. performed the experiment. T.Z., M.H. G.X. T.C., and Y.W. analyzed the data. inhibition of HDAC6 seems to be cell type dependent . T.Z., H.Z. and C.W. wrote the manuscript. G.X. revised the manuscript. All In this study, HDAC6 inhibition results in increased AKT authors read and approved the final manuscript. kinase activity indirectly in newborn mice ovary through activated mTOR-KITL pathway in pfGCs. Further studies Conflict of interest The authors declare no competing interests. are needed to clarify the exact factors within the niche of primordial follicles which are the key signals for sustain- Ethics statement ing the quiescent of primordial follicles before a clearer All animal procedures were conducted in accordance with the guidelines of and approved by the Animal Research Committee of the China Agricultural model of primordial follicle sustaining be figured out. University. In vitro activation (IVA) of primordial follicle technique based on above mentioned theory has been established to Publisher’s note help solve the problems of POF patients recently . The Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. main target for the tech is to inhibit the activity of PTEN or activate PI3K in oocytes . From this study, we have Supplementary information The online version contains supplementary provided an alternative target, the HDAC protein in material available at https://doi.org/10.1038/s41419-021-03842-1. pfGCs, for activating mTOR-KIT-PI3K signaling cascades Received: 9 June 2020 Revised: 2 March 2021 Accepted: 8 March 2021 between somatic cells and oocytes within individual pri- mordial follicle. 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