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. 2021 May 29;12(6):559.
doi: 10.1038/s41419-021-03842-1.

HDAC6 regulates primordial follicle activation through mTOR signaling pathway

Tuo Zhang #  1   2   3 , Meina He #  4 , Lihua Zhao #  5 , Shaogang Qin  3 , Zijian Zhu  3 , Xinhua Du  3 , Bo Zhou  3 , Yi Yang  1 , Xinfeng Liu  1 , Guoliang Xia  1   3 , Tengxiang Chen  2 , Yuanxi Wang  6 , Hua Zhang  7 , Chao Wang  8
Affiliations

HDAC6 regulates primordial follicle activation through mTOR signaling pathway

Tuo Zhang et al. Cell Death Dis. .

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.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
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.
Fig. 2
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.
Fig. 3
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.
Fig. 4
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
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.
Fig. 6
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.
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