Oocyte stage-specific effects of MTOR determine granulosa cell fate and oocyte quality in mice

Abstract

MTOR (mechanistic target of rapamycin) is a widely recognized integrator of signals and pathways key for cellular metabolism, proliferation, and differentiation. Here we show that conditional knockout (cKO) of Mtor in either primordial or growing oocytes caused infertility but differentially affected oocyte quality, granulosa cell fate, and follicular development. cKO of Mtor in nongrowing primordial oocytes caused defective follicular development leading to progressive degeneration of oocytes and loss of granulosa cell identity coincident with the acquisition of immature Sertoli cell-like characteristics. Although Mtor was deleted at the primordial oocyte stage, DNA damage accumulated in oocytes during their later growth, and there was a marked alteration of the transcriptome in the few oocytes that achieved the fully grown stage. Although oocyte quality and fertility were also compromised when Mtor was deleted after oocytes had begun to grow, these occurred without overtly affecting folliculogenesis or the oocyte transcriptome. Nevertheless, there was a significant change in a cohort of proteins in mature oocytes. In particular, down-regulation of PRC1 (protein regulator of cytokinesis 1) impaired completion of the first meiotic division. Therefore, MTOR-dependent pathways in primordial or growing oocytes differentially affected downstream processes including follicular development, sex-specific identity of early granulosa cells, maintenance of oocyte genome integrity, oocyte gene expression, meiosis, and preimplantation developmental competence.

Keywords: Sertoli-like cells; female infertility; granulosa cell; meiosis; oocyte-expressed MTOR.

Fig. 1.

Infertility and compromised oocyte quality in cKO mice. (A) Schematic illustration of the stages at which GDF9-Cre and ZP3-Cre are expressed. Expression of GDF9-Cre and ZP3-Cre starts in primordial and growing oocytes, respectively. (B) IF staining of MTOR in 21-d-old WT, Mtor-GcKO, and Mtor-ZcKO ovaries. MTOR and DNA are stained in magenta and blue, respectively. Arrows point to oocytes in primordial follicles; asterisks indicate growing oocytes. (Scale bars, 50 μm.) (C) Western blot analysis of the expression of MTOR and the activated form of its major downstream effectors—pRPS6KB1, pRPS6, pEIF4EBP1, and p-AKT^Ser473—and the internal control ACTB in WT, Mtor-GcKO, and Mtor-ZcKO FGOs. (D) Number of pups born and number of oocytes ovulated by WT and Mtor-cKO females and the rate of two-cell and blastocyst formation by ovulated WT and Mtor-cKO oocytes after IVF. *P < 0.05, compared with the WT or control by student’s t test. Data represent the mean ± SEM.

Fig. 2.

Defective follicle and granulosa cell development in Mtor-GcKO ovaries. (A) Micrographs of periodic acid-Schiff (PAS)-stained 21-d-old WT and Mtor-GcKO ovarian sections. (B, Upper) Photographs of whole bodies (Left) and ovaries (Right) of 6-mo-old WT and Mtor-GcKO mice. (Lower) Micrographs of PAS-stained ovarian sections of 6-mo-old WT (Left) and Mtor-GcKO 9 (Right) mice. (C) qRT-PCR analyzing the expression of a cohort of genes characteristic of ovarian granulosa cells (Top) and testicular Sertoli and/or Leydig cells (Bottom) in 6-mo-old WT and Mtor-GcKO ovaries. (D, Upper) Transmission electron microscopic imaging of a 6-mo-old Mtor-GcKO mouse ovarian follicle with abnormal somatic cells that resemble immature Sertoli-like cells. (Lower) Magnified view of the boxed area in the Upper image indicated as ectoplasmic specialization (ES). BM, basal membrane; N, nucleus; Nu, nucleolus; TJ, tight junction. (E) IF staining of CLDN5 in 6-mo-old Mtor-GcKO ovaries. CLDN5, ZP2, and DNA are stained magenta, green, and blue, respectively. (F, Upper) IF staining of γH2AX in 5-wk-old WT and Mtor-GcKO ovaries. γH2AX and DNA are stained green and blue, respectively. (Lower) The bar graph shows the quantification of the γH2AX staining. *P < 0.05, compared with the WT or control by student’s t test. Data represent the mean ± SEM. (Scale bars, A, B, E, and F, 100 μm.)

Fig. 3.

Defective meiotic progression to MII in ovulated oocytes of both cKO mice. (A) First polar body (PB1) rate in ovulated oocytes. (B) Quantification of the meiotic stages of ovulated oocytes. TI, telophase I. (C) Micrographs showing typical meiotic stages prevalent in WT (a), Mtor-GcKO (b–d), and Mtor-ZcKO (e–h) ovulated oocytes. (a) Normal MII stage. (b and e) Abnormal MII with a deformed spindle and misaligned chromosomes. (c, d, g, and h) Stages with defective cytokinesis. (f) Telophase I. Microtubules, chromosomes, and F-actin are stained green, blue, and magenta, respectively. (Scale bars, 20 μm.) *P < 0.05, compared with the WT or control by student’s t test. Data represent the mean ± SEM.

Fig. 4.

Differential effect of Mtor cKO on the integrity of transcriptome of FGOs. (A) Venn diagram illustrating the relationship of the changed transcripts identified by RNA-seq in Mtor-GcKO and Mtor-ZcKO FGOs. WT–GcKO: WT vs. Mtor-GcKO; WT–ZcKO: WT vs. Mtor-ZcKO; GcKO–ZcKO: Mtor-GcKO vs. Mtor-ZcKO. The total number of changed transcripts is indicated in parentheses. (B) Venn diagram illustrating the relationship of up- and down-regulated transcripts identified by RNA-seq in Mtor-GcKO and Mtor-ZcKO FGOs. WT–GcKO_Up: up-regulated in Mtor-GcKOs compared with WT; WT–ZcKO_Up: up-regulated in Mtor-ZcKOs compared with WT; WT–GcKO_Down: down-regulated in Mtor-GcKOs compared with WT; WT–ZcKO_Down: down-regulated in Mtor-ZcKOs compared with WT. The number of changed transcripts in each group is shown in parentheses. (C) Real-time qRT-PCR validating changes in representative transcripts selected from RNA-seq data. (D) Heatmaps illustrating the enriched terms (GO/KEGG terms or canonical pathways) associated significantly with changed transcripts identified by RNA-seq in Mtor-GcKO and Mtor-ZcKO FGOs. GcKO–ZcKO: Mtor-GcKO vs. Mtor-ZcKO; WT–GcKO: WT vs. Mtor-GcKO; WT–ZcKO: WT vs. Mtor-ZcKO. (E) Heatmaps illustrating differences between WT and Mtor-GcKO FGOs in the expression of a cohort of transcripts involved in various processes.

Fig. 5.

Proteomic analysis of Mtor-ZcKO ovulated oocytes. (A) Distribution of significantly changed proteins at various magnitudes of difference in expression levels between Mtor-ZcKO and WT ovulated oocytes detected by LC-MS. The number of changed proteins in each category of fold change is indicated above each bar. (B) Western blot analysis of RICTOR, TRIM36, PRC1, and ACTB expression in WT and Mtor-ZcKO (cKO) ovulated oocytes. (C) Venn diagrams illustrating the relationship of the proteomic dataset with polysome RNA array datasets on mRNAs translated in WT FGOs at all stages, i.e., GV, MI, and MII stages, indicated as “Polysome” (Left) or translated in oocytes only during the transition from GV to MII, indicated as “Polysome_Up” (Right) (44). (D) Heatmaps illustrating differences between WT and Mtor-ZcKO ovulated oocytes in the expression of proteins that are in common between the Proteomics dataset and the Polysome_up dataset.

Fig. 6.

Localization and function of PRC1 in oocytes during meiotic maturation. (A) PRC1 localization in WT oocytes at different meiotic stages. (B) Effect of PRC1 knockdown on oocyte meiotic progression in WT cells. (Upper Left) Knockdown of PRC1 by Prc1-morpholino (MO). (Lower Left) The graph shows the percentage of oocytes in which meiosis progressed normally to MII. (Right) Micrographs demonstrate typical cytokinesis defects in PRC1–knocked-down oocytes. PRC1/F-actin, microtubules, and chromosomes are stained magenta, green, and blue, respectively. (C and D) Rescuing cytogenesis defects of Mtor-ZcKO oocytes by PRC1 overexpression. Mtor-ZcKO GV oocytes were microinjected with Prc1 mRNA and matured in vitro for 18 h. Meiotic status was then analyzed by IF staining of chromosomes and spindles. (C, Upper) The Western blot gel image detecting PRC1 expression. (Lower) The graph shows the quantification of Western blot results. (D) The quantification of oocytes at normal MII stage. “+mRNA” indicates microinjection with Prc1 mRNA. *P < 0.05, compared with the cKO-group by student’s t test. (Scale bars, 20 μm.)