Germline quality control: eEF2K stands guard to eliminate defective oocytes

Abstract

The control of germline quality is critical to reproductive success and survival of a species; however, the mechanisms underlying this process remain unknown. Here, we demonstrate that elongation factor 2 kinase (eEF2K), an evolutionarily conserved regulator of protein synthesis, functions to maintain germline quality and eliminate defective oocytes. We show that disruption of eEF2K in mice reduces ovarian apoptosis and results in the accumulation of aberrant follicles and defective oocytes at advanced reproductive age. Furthermore, the loss of eEF2K in Caenorhabditis elegans results in a reduction of germ cell death and significant decline in oocyte quality and embryonic viability. Examination of the mechanisms by which eEF2K regulates apoptosis shows that eEF2K senses oxidative stress and quickly downregulates short-lived antiapoptotic proteins, XIAP and c-FLIPL by inhibiting global protein synthesis. These results suggest that eEF2K-mediated inhibition of protein synthesis renders cells susceptible to apoptosis and functions to eliminate suboptimal germ cells.

Figure 1. Intense phosphorylation of eEF2 in mouse ovaries

(A) Immunofluorescent staining of p-eEF2 in eEF2K+/+ mouse ovary. Scale bars represent 25 μm. F, developing follicle; AF, atretic follicle; PF, primordial follicle; GC, granulosa cells. Red, p-eEF2 staining; Blue, DAPI staining. (B) Immunohistochemical staining of p-eEF2 in eEF2K+/+ and eEF2K−/− mouse ovaries. Scale bars represent 100 μm. AnF, antral follicle. Brown, p-eEF2 staining; Blue, Hematoxylin staining. (C) The levels of eEF2K protein in various mouse tissues lysates by Western blot analysis. eEF2K is strongly expressed in the mouse ovaries. Refer also to Figure S1.

Figure 2. Knockout of eEF2K preserves follicles and reduces oocyte quality in aged female mice

(A) Hematoxylin and eosin staining of 20-month-old mouse ovary sections. Scale bars represent 500 μm. F, follicle; CL, corpus luteum. (B) Hematoxylin and eosin staining of antral follicle (left panel) with an unhealthy, sickle-shaped oocyte (arrowhead), and preantral follicle (right panel) with an unhealthy oocyte with blebs (arrowhead) from 20-month-old eEF2K−/− mice. Scale bars represent 100 μm. (C) Iron hematoxylin/aniline blue staining of ovary from 17-month-old eEF2K−/− mouse. (Left panel) Preovulatory-like follicles with a diameter greater than 500 μm in the eEF2K−/− ovary. Scale bars represent 500 μm. (Right panel) Preovulatory-like follicles contain granulosa cells undergoing mitosis. Red arrows indicate granulosa cells in metaphase of the cell cycle. Scale bars represent 25 μm. (D) Dissected uterus and hematoxylin and eosin staining of uterine tissue from 20-month-old eEF2K+/+ and eEF2K−/− mice. Black scale bars represent 500 μm, green scale bars represent 50 μm. Refer also to Figure S2-3, Supplemental Experimental Procedures.

Figure 3. Knockout of eEF2K reduces apoptosis levels in mouse ovaries

(A) Quantification of total pyknotic nuclei per ovary in eEF2K+/+ and eEF2K−/− mice. (B) Quantification of pyknotic nuclei per large atretic follicle with a diameter of over 250 μm in eEF2K+/+ and eEF2K−/− mice. (C) Quantification of cleaved caspase-3 (CC3) positive granulosa cells per ovary. (D) Immunohistochemisty of CC3 in follicles of eEF2K+/+ and eEF2K−/− mice. Scale bars represent 50 μm. (E) Quantification and analysis of follicles at different developmental stages in eEF2K+/+ and eEF2K−/− ovaries. (F) Quantification of superovulated oocytes from 15-month-old eEF2K+/+ and eEF2K−/− mice. Data are represented as mean +/− S.E.M. and “ * ” represents P< 0.05 (Mann-Whitney U-test). (G) Granulosa cells isolated from eEF2K+/+ and eEF2K−/− mice were treated with TNF-α and cycloheximide. Pyknotic nuclei counts were determined by nuclear condensation or chromatin fragmentation with Hochest 33342 staining. Over 200 nuclei were counted for each experimental group. (H) Percentage of apoptosis induced by doxorubicin (DOX) in eEF2K+/+ and eEF2K−/− oocytes. Metaphase II oocytes were collected and treated without (DMSO) or with 200 nM or 1 μM doxorubicin. Oocytes with apoptotic morphology (cytoplasmic fragmentation) were quantified. The total number of oocytes analyzed per group was 16-24. (I) Morphological changes in doxorubicin (DOX)-treated eEF2K+/+ and eEF2K−/− oocytes. Oocytes were treated with doxorubicin for 24 hours, and then fixed and stained with DAPI. Yellow boxes enlarge the image of an oocyte with cellular fragmentation. Scale bars represent 100 μ m. Refer also to Figure S3.

Figure 4. Deficiency of EFK-1, the homolog of eEF2K in C. elegans, reduces germ cell apoptosis and oocyte quality

(A) Whole-mount immunostaining of phosphorylated EEF-2 by EFK-1 in the gonads of N2 and efk-1(ok3609) adult C. elegans. Scale bars represent 100 μm (top and bottom panels) and 20 μm (middle panel). DG, distal gonad; PG, proximal gonad; E, embryo; S, spermatheca. (B) Quantification of germ cell corpses per gonad in the N2, efk-1(ok3609), and ced-3(n717) by SYTO-12 staining. (C) Quantification of germ cell corpses per gonad by SYTO-12 staining after RNAi (control or efk-1) in the ced-1(e1754) mutant which retains corpses due to a defect in cell engulfment. (D) Percentage of small-sized eggs produced during reproductive lifespan in N2, efk-1(ok3609), and ced-3(n717). (E) Percentage of unhatched eggs produced during reproductive lifespan in N2 and efk-1(ok3609). Data are represented as mean +/− S.E.M. and “ *** “ represents P<0.001 (Mann-Whitney U-test). Refer also to Figure S4, Figure S6.

Figure 5. eEF2K is activated during apoptosis and regulates it through downregulation of short-lived anti-apoptotic proteins

(A) Western blot analysis of p-eEF2 levels in H₂O₂-treated NIH-3T3 cells or doxorubicin (DOX)-treated MEFs. (B) Immunostaining of p-eEF2 in NIH3T3 cells exposed to 400 μM H₂O₂ for 3h compared to untreated cells. Scale bars represent 20 μm. (C) Immunofluorescent staining in HeLa cells treated with 1μM doxorubicin for 18h; p-eEF2, DAPI and TUNEL staining are shown. Scale bars represent 10 μm. (D) Analyzed of cleaved caspase-3 levels by Western blot in H₂O₂ and doxorubicin-treated knockout MEFs expressing vector alone (−eEF2K) or vector containing eEF2K cDNA (+eEF2K). (E) Apoptosis analyzed by TUNEL assay in H₂O₂ and doxorubicin-treated wild-type (eEF2K+/+), knockout (eEF2K−/−), and knockout MEFs expressing vector alone (−eEF2K) or vector containing eEF2K cDNA (+eEF2K). Data are represented as mean +/− S.E.M. and “ *** “ represents P< 0.002 (two-tailed T-test). (F) Measurement of protein synthesis in knockout MEFs expressing vector alone (−eEF2K) or vector containing eEF2K cDNA (+eEF2K) treated with doxorubicin for 12h and labeled with ³⁵S-methionine. (G) Western blot analysis of p-eEF2, c-FLIP_L, XIAP, Mcl-1, tBID and α-tubulin in doxorubicin-treated MEFs expressing vector alone (−eEF2K) or vector containing eEF2K cDNA (+eEF2K). (H) Western blot analysis of eEF2, XIAP, c-FLIP_L, and GAPDH in eEF2 knockdown MEFs. Refer also to Figure S5.

Figure 6. Germline maintenance by eEF2K

(A) Model of ovarian cell death in mouse ovaries. In this model, inhibition of protein synthesis by eEF2K sensitizes granulosa cells and oocytes to apoptotic stimuli and promotes follicle atresia. Insufficient follicle atresia in eEF2K−/− ovaries results in the accumulation of aberrant follicles with unhealthy oocytes. (B) Model of germ cell death in C. elegans. In this model, eEF2K regulates protein synthesis thereby adjusting the threshold for apoptosis during germline development and selection. The high activity of eEF2K results in a more restrictive cellular environment, which increases selective pressure and lowers the threshold for triggering apoptosis in order to produce high quality germ cells.