RAB37-mediated autophagy guards ovarian homeostasis and function

Abstract

Loss of ovarian homeostasis is associated with ovary dysfunction and female diseases; however, the underlying mechanisms responsible for the establishment of homeostasis and its function in the ovary have not been fully elucidated. Here, we showed that conditional knockout of Rab37 in oocytes impaired macroautophagy/autophagy proficiency in the ovary and interfered with follicular homeostasis and ovary development in mice. Flunarizine treatment upregulated autophagy, thus rescuing the impairment of follicular homeostasis and ovarian dysfunction in rab37 knockout mice by reprogramming of homeostasis. Notably, both the E2F1 and EGR2 transcription factors synergistically activated Rab37 transcription and promoted autophagy. Thus, RAB37-mediated autophagy ensures ovary function by maintaining ovarian homeostasis.Abbreviations: AMH: anti-Mullerian hormone; ATG: autophagy related; BECN1: beclin 1; cKO: conditional knockout; Cre: cyclization recombination enzyme; dpp: days postpartum; E2: estradiol; E2F1: E2F transcription factor 1; EBF1: EBF transcription factor 1; EGR2: early growth response 2; FSH: follicle stimulating hormone; LH: luteinizing hormone; mpp: months postpartum; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; RAB37: RAB37, member RAS oncogene family; SQSTM1: sequestosome 1; TFEB: transcription factor EB; Zp3: zona pellucida glycoprotein 3.

Keywords: Gene knockout; RAB37; homeostasis; mice; ovary; transcription regulation.

Figure 1.

Conditional knockout of Rab37 in oocytes impairs follicular homeostasis and ovarian development. (A) conditional knockout strategy for Rab37 via the Cre-LoxP system. Two LoxP sites are located in introns 2 and 8 of the Rab37 gene. Zp3-Cre mediates deletion of exons between two LoxP sites. (B) breeding programme for rab37 homozygous oocyte-specific knockout mice. (C) genotyping of rab37 knockout mice via PCR. (D) Western blot analysis of the RAB37 protein in Rab37^Flox/Flox and rab37^-/- ovaries. GAPDH was used as an internal control. (E) immunofluorescence analysis of RAB37 localization in Rab37^Flox/Flox and rab37^-/- oocytes. The nuclei were stained with DAPI, and the FITC signals (green) indicate the RAB37 protein. The enlarged images (i and ii) originate from the white squares in the merged panels. Scale bar: 25 μm (original) or 10 μm (enlarged). (F) H&E staining of paraffin sections of ovaries from Rab37^Flox/Flox and rab37^-/- mice at 14 days and 1, 2, 3, and 4 months after birth. dpp, days postpartum; mpp, months postpartum. Scale bar: 500 μm. (G) the ovarian indices (ovary weight : body weight × 10⁻⁴) of Rab37^Flox/Flox and rab37^-/- mice at 14 days and 1, 2, 3, and 4 months after birth. n > 3, *, p < 0.05; **, p < 0.01. (H) schematic diagram of follicle development. Primordial follicles are first activated and subsequently develop into primary, secondary, and mature follicles. (I-M) comparisons of primordial (I), activated (J), primary (K), secondary (L), and mature follicles (M) numbers per ovary between Rab37^Flox/Flox and rab37^-/- mice at 14 days and at 1, 2, 3, and 4 months after birth. n = 5; *, p < 0.05; **, p < 0.01. (N-Q) serum concentrations of estradiol (E2) (N), follicle stimulating hormone (FSH) (O), luteinizing hormone (LH) (P), and anti-Mullerian hormone (AMH) (Q) in Rab37^Flox/Flox and rab37^-/- mice at 14 days, 1, 2, 3, and 4 months after birth; n = 6; *, p < 0.05; **, p < 0.01.

Figure 2.

Autophagy is impaired during ovarian development in Rab37 knockout mice. (A) Western blot analysis of RAB37 and autophagy-associated protein levels in Rab37^Flox/Flox and rab37^-/- ovaries at 14 days and 1, 2, 3, and 4 months after birth. GAPDH was used as an internal control. (B-D) quantification of SQSTM1 (B), LC3B-II (C), and RAB37 (D) protein levels from (A). The data are presented as the means ± S.D., *, p < 0.05; **, p < 0.01 (n = 3). (E) immunofluorescence analysis of LC3B puncta in Rab37^Flox/Flox and rab37^-/- oocytes at 14 days and 1, 2, 3, and 4 months. The nuclei were stained with DAPI (blue). FITC signals (green) indicate the LC3B protein. Scale bar: 25 μm (original) or 10 μm and 2 μm (enlarged). (F) statistics of autophagic puncta per oocyte from (E). The puncta were counted from at least 14 oocytes. **, p < 0.01.

Figure 3.

Double knockouts of the E2F1 and EGR2 binding sites inhibit RAB37-mediated autophagy. (A) sequence comparisons of the binding sites of transcription factors between the wild-type and double knockouts of E2F1+EGR2 sites in the Rab37 promoter in NIH-3T3 cells. The ovals represent the E2F1 and EGR2 binding sites, the base sequences in red indicate the core binding sites, and the dotted lines indicate the knockout region and binding sites in the Rab37 promoter region. (B) RT-PCR analysis of the transcription levels of Rab37 in WT and double knockouts of E2F1+EGR2 sites when E2F1 and EGR2 are overexpressed upon starvation. The cells were cultured in normal or EBSS medium for 1 h (starved for 1 h). actb was used as an internal control. (C) RT-PCR quantification of Rab37 levels relative to that of Actb. The data are presented as the means ± S.D. **, p < 0.01 (n = 3 independent experiments). (D) the transcription factors E2F1 and EGR2 promote RAB37-mediated autophagy. Western blot analysis was used to detect the expression levels of RAB37 and autophagy-associated proteins in WT and double knockouts of E2F1+EGR2 sites. The cells were cultured in normal or EBSS medium for 1 h (starved for 1 h). GAPDH was used as an internal control. (E-G) quantification of RAB37 (E), LC3B-II (F), and SQSTM1 (G) relative levels. The data are presented as the means ± S.D. *, p < 0.05; **, p < 0.01 (n = 3). (H) immunofluorescence analysis of autophagic puncta in WT and double knockouts of E2F1+EGR2 sites. The cells were cultured in normal or EBSS medium for 1 h (starved 1 h). The nuclei were stained with DAPI (blue). Endogenous RAB37 (green) and LC3B (red) were detected with anti-RAB37/FITC-conjugated ImmunoPure goat anti-rabbit IgG and anti-LC3B-conjugated Alexa Fluor® 647, respectively. The enlarged images originate from the white squares in the merged panels. Scale bar: 10 μm (original) and 1 μm (enlarged). (I) statistics of autophagic puncta per cell from (H). The number of autophagic puncta was counted for 20 cells. *, p < 0.05; **, p < 0.01.

Figure 4.

Flunarizine promotes RAB37-mediated autophagy in mouse embryonic fibroblast cell line. (A) construction of rab37 KO lines in NIH/3T3 cells (NIH Swiss mouse embryonic fibroblast cells) by CRISPR-Cas9 technology. The sgRNA site (blue letters with the PAM site in red) and sequence alignments between the edited region in the WT and 3 KO cell lines (RAB37 KO-25, −29, and − 61) are shown. The dotted lines represent the knockout region. (B) schematic diagram of the protein coding regions of RAB37 in the WT and 3 KO cell lines. Truncated sequences are shown in boxes with frameshift sequences in dashed lines. The numbers refer to the amino acid positions. (C) effect of flunarizine on the expression levels of RAB37 and autophagy-associated proteins in WT and rab37 KO cells. The cells were treated with flunarizine (20 μM) or DMSO (control) for 2 h, respectively. GAPDH was used as an internal control. (D-F) quantification of SQSTM1 (D), LC3B-II (E), and RAB37 (F) relative levels. The data are presented as the means ± S.D. *, p < 0.05; **, p < 0.01 (n = 3). (G) immunofluorescence analysis of the effect of flunarizine on autophagy. The cells were treated with flunarizine (20 μM) or DMSO (control) for 2 h, respectively. Endogenous LC3B proteins (red) were detected by anti-LC3B/TRITC-conjugated ImmunoPure goat anti-rabbit IgG. The nuclei were stained with DAPI (blue). The enlarged images originate from the white squares in the merged panels. Scale bar: 10 μm (original) and 2 μm (enlarged). (H) statistical analysis of autophagic puncta per cell in (G). The puncta were counted from 31 cells. *, p < 0.05; **, p < 0.01.

Figure 5.

Flunarizine promotes RAB37-mediated autophagy in ovary cell line. (A) rab37 KO was performed in Chinese hamster ovary cells (CHO) by CRISPR-Cas9 technology. The sgRNA site (blue letters with the PAM site in red) and sequence alignments between editing regions in the WT and 3 edited cell lines (RAB37 KO-2, −7, and − 22) are shown. The dotted lines represent deletion region in the cells RAB37 KO-2 and RAB37 KO-7. RAB37 KO-22 has a insertion of 2 bases. (B) schematic diagram of the protein coding regions of RAB37 in the WT and 3 KO cell lines. Truncated sequences are shown in boxes with frameshift sequences in dashed lines. The numbers refer to the amino acid positions. (C) effect of flunarizine on the expression levels of autophagy-associated proteins and RAB37 in WT and RAB37 KO-2 cells. The cells were treated with flunarizine (300 μM) or DMSO (control) for 2 h, respectively. GAPDH was used as an internal control. (D-F) quantification of SQSTM1 (D), LC3B-II (E), and RAB37 (F) relative levels. The data are presented as the means ± S.D. *, p < 0.05; **, p < 0.01 (n = 3). (G) immunofluorescence analysis of the effect of flunarizine on autophagy. The cells were treated with flunarizine (300 μM) or DMSO (control) for 2 h, respectively. Endogenous LC3B proteins (red) were detected by anti-LC3B/TRITC-conjugated ImmunoPure goat anti-rabbit IgG. The nuclei were stained with DAPI (blue). The enlarged images originate from the white squares in the merged panels. Scale bar: 5 μm (original) and 1 μm (enlarged). (H) statistical analysis of autophagic puncta per cell in (G). The puncta were counted from 50 cells. **, p < 0.01.

Figure 6.

Flunarizine-induced autophagy rescues the impairment of follicular homeostasis and dysfunction of ovaries lacking Rab37. (A) Western blot assays showing the effect of flunarizine on RAB37 and autophagy-associated protein levels in the ovaries of Rab37^Flox/Flox and rab37^-/-. The mice were injected intraperitoneally daily with flunarizine (10 mg/kg) or DMSO (control) for one month. GAPDH was used as an internal control. (B-D) quantification analysis of SQSTM1 (B), LC3B-II (C), and RAB37 (D) relative levels. The data are presented as the means ± S.D. **, p < 0.01 (n = 3). (E) immunofluorescence analysis of LC3B puncta in oocytes from Rab37^Flox/Flox and rab37^-/- mice induced by flunarizine. The nuclei were stained with DAPI (blue). FITC signals (green) indicate LC3B expression. Scale bar: 25 μm (original) or 10 μm and 2 μm (enlarged). (F) statistics of autophagic puncta per oocyte from (E). The puncta were counted from at least 14 oocytes. **, p < 0.01. (G) H&E staining of paraffin sections of ovaries from Rab37^Flox/Flox and rab37^-/- mice induced by flunarizine or DMSO (control). Scale bar: 500 μm. (H) the ovarian indices (the ratio of ovarian weight to body weight × 10⁻⁴) of Rab37^Flox/Flox and rab37^-/- mice induced by flunarizine and DMSO (control). n = 8, **, p < 0.01. (I-M) comparisons of primordial (I), activated (J), primary (K), secondary (L), and mature follicles (M) number between Rab37^Flox/Flox and rab37^-/- mice induced by flunarizine or DMSO (control). n = 5, *, p < 0.05; **, p < 0.01. (N-Q) serum concentrations of estradiol (E2) (N), follicle stimulating hormone (FSH) (O), luteinizing hormone (LH) (P), and anti-Müllerian hormone (AMH) (Q) in Rab37^Flox/Flox and rab37^-/- mice induced by flunarizine or DMSO (control). n = 8; *, p < 0.05; **, p < 0.01.

Figure 7.

RAB37-mediated autophagy regulates follicular homeostasis and maintains ovarian function. The transcription factors E2F1 and EGR2 cooperatively activate the expression of RAB37 in mice. RAB37 promotes the formation of the ATG12–ATG5-ATG16L1 complex in a GTP-dependent manner, thus facilitating the lipidation of LC3B to enhance autophagy. RAB37-regulated autophagy promotes primordial follicle activation and maintains follicular homeostasis and ovarian function. Conditional knockout of Rab37 in mouse oocytes inhibits LC3B lipidation and autophagy, impairs follicle activation and homeostasis, and disrupts ovary function. Flunarizine treatment rescues the impairment of follicular homeostasis and abnormal function of ovaries lacking Rab37 by upregulating autophagy and thus maintaining follicular homeostasis and ovarian function.