MTOR-mediated interaction between the oocyte and granulosa cells regulates the development and function of both compartments in mice†.

Coordinated development of the germline and the somatic compartments within a follicle is an essential prerequisite for creating a functionally normal oocyte. Bi-directional communication between the oocyte and the granulosa cells enables the frequent interchange of metabolites and signals that support the development and functions of both compartments. Mechanistic target of rapamycin (MTOR), a conserved serine/threonine kinase and a widely recognized integrator of signals and pathways key for cellular metabolism, proliferation, and differentiation, is emerging as a major player that regulates many facets of oocyte and follicle development. Here, we summarized our recent observations on the role of oocyte- and granulosa cell-expressed MTOR in the control of the oocyte's and granulosa cell's own development, as well as the development of one another, and provided new data that further strengthen the role of cumulus cell-expressed MTOR in synchronizing oocyte and follicle development. Inhibition of MTOR induced oocyte meiotic resumption in cultured large antral follicles, as well as cumulus expansion and the expression of cumulus expansion-related transcripts in cumulus-oocyte complexes in vitro. In vivo, the activity of MTOR in cumulus cells was diminished remarkably by 4 h after hCG administration. These results thus suggest that activation of MTOR in cumulus cells contributes to the maintenance of oocyte meiotic arrest before the LH surge. Based on the observations made by us here and previously, we propose that MTOR is an essential mediator of the bi-directional communication between the oocyte and granulosa cells that regulates the development and function of both compartments.

Echinoderm Microtubule Associated Protein Like 1 Is Indispensable for Oocyte Spindle Assembly and Meiotic Progression in Mice.

Completion of the first meiosis is an essential prerequisite for producing a functionally normal egg for fertilization and embryogenesis, but the precise mechanisms governing oocyte meiotic progression remains largely unclear. Here, we report that echinoderm microtubule associated protein (EMAP) like 1 (EML1), a member of the conserved EMAP family proteins, plays a crucial role in the control of oocyte meiotic progression in the mouse. Female mice carrying an ENU-induced nonsense mutation (c.1956T > A; p.Tyr652∗) of Eml1 are infertile, and the majority of their ovulated oocytes contain abnormal spindles and misaligned chromosomes. In accordance with the mutant oocyte phenotype, we find that EML1 is colocalized with spindle microtubules during the process of normal oocyte meiotic maturation, and knockdown (KD) of EML1 by specific morpholinos in the fully grown oocytes (FGOs) disrupts the integrity of spindles, and delays meiotic progression. Moreover, EML1-KD oocytes fail to progress to metaphase II (MII) stage after extrusion of the first polar body, but enter into interphase and form a pronucleus containing decondensed chromatins. Further analysis shows that EML1-KD impairs the recruitment of γ-tubulin and pericentrin to the spindle poles, as well as the attachment of kinetochores to microtubules and the proper inactivation of spindle assembly checkpoint at metaphase I (MI). The loss of EML1 also compromises the activation of maturation promoting factor around the time of oocyte resumption and completion of the first meiosis, which, when corrected by WEE1/2 inhibitor PD166285, efficiently rescues the phenotype of oocyte delay of meiotic resumption and inability of reaching MII. Through IP- mass spectrometry analysis, we identified that EML1 interacts with nuclear distribution gene C (NUDC), a critical mitotic regulator in somatic cells, and EML1-KD disrupts the specific localization of NUDC at oocyte spindles. Taken together, these data suggest that EML1 regulates acentrosomal spindle formation and the progression of meiosis to MII in mammalian oocytes, which is likely mediated by distinct mechanisms.

A novel homozygous nonsense mutation in zona pellucida 1 (ZP1) causes human female empty follicle syndrome.

PURPOSE: To identify a pathogenic gene mutation in a female infertility proband characterized by empty follicle syndrome (EFS) and explore the genetic cause of EFS. METHODS: Whole exome sequencing (WES) was performed to identify the candidate pathogenic mutation. Sanger sequencing was used to validate the mutation in family members. The pathogenicity of the identified variant and its possible effects on the protein were evaluated with in silico tools. Immunofluorescence staining was used to study the possible mechanism of the mutation on affected oocyte. RESULTS: We identified a family with a novel homozygous nonsense mutation in zona pellucida 1 (ZP1) (c.199G > T [p.Glu67Ter]). Based on bioinformatics analysis, the mutation was predicted to be pathogenic. This variant generates a premature stop codon in exon 2 at the 199th nucleotide, and was inferred to result in a truncated ZP1 protein of 67 amino acids at the ZP-N1 domain. An in vitro study showed that the oocyte of the EFS proband was degenerated and the zona pellucida was absent. Additionally, the mutant ZP1 proteins were localized in the cytoplasm of the degenerated oocyte but not at the surface. CONCLUSIONS: The novel mutation in ZP1 is a genetic cause of female infertility characterized by EFS. Our finding expands the genetic spectrum for EFS and will help justify the EFS diagnosis in patients.

Insufficient Cumulus Expansion and Poor Oocyte Retrieval in Endometriosis-Related Infertile Women.

Endometriosis (EMs) is a common cause for female infertility, leading to the need for in vitro fertilization (IVF). In clinics, we found the operative oocyte retrieval to be more or less difficult in women with EMs. We hypothesized that EMs may be involved in the insufficient cumulus expansion that partially explained the lower oocyte retrieval in EMs-related infertile women undergoing assisted reproductive technology (ART). To explore whether the insufficient cumulus expansion exists in EMs-related infertile women and whether there is a possible relationship between the insufficient cumulus expansion and the clinical phenomenon of difficulty in oocyte retrieval. Those infertile women undergoing IVF recorded in our database between January 2013 and October 2017 were included. The expression levels of cumulus expansion-related genes (HAS2/PTGS2/PTX3/TNFAIP6) in the cumulus cells (CCs) from 19 infertile women with EMs and 24 controls were analyzed by real-time PCR. After that, 635 women with EMs-associated infertility (the EMs group) and 4634 women with male factor-associated infertility (the control group) were included in the retrospective analysis. The clinical outcomes were compared between the two groups. The relative mRNA levels of cumulus expansion-related genes were significantly decreased in the CCs from those infertile women with EMs when compared to the control group (all p < 0.05), especially the expression of PTGS2. The mean oocyte retrieval rates (proportion of obtained oocytes in punctured follicles) were (76.33 ± 2.58)% and (71.80 ± 0.58)% (p < 0.01). The mean numbers of flushing times per follicle were 1.11 ± 0.65 and 3.86 ± 1.53 (p < 0.001). The lower expression of cumulus expansion-related genes in CCs suggests the insufficient cumulus expansion in EMs-related infertile women, which partially explains a possible mechanism related to poor oocyte retrieval.

Hfm1 participates in Golgi-associated spindle assembly and division in mouse oocyte meiosis.

HFM1 (helicase for meiosis 1) is widely recognized as an ATP-dependent DNA helicase and is expressed mainly in germ-line cells. HFM1 is a candidate gene of premature ovarian failure (POF), hence it is also known as POF9. However, the roles of HFM1 in mammalian oocytes remain uncertain. To investigate the functions of HFM1, we established a conditional knockout (cKO) mouse model. Specific knockout of Hfm1 in mouse oocytes from the primordial follicle stage resulted in depletion of ovarian follicular reserve and subfertility of mice. In particular, abnormal spindle, misaligned chromosomes, loss of cortical actin cap, and failing polar body extrusion were readily observed in Hfm1-cKO oocytes. Further studies indicated that in addition to its cytoplasmic distribution, Hfm1 accumulated at the spindle poles, colocalized with the Golgi marker protein, GM130. Generally, GM130 signals overlapped with p-Mapk at the two spindle poles to regulate meiotic spindle assembly and asymmetric division. In this research, centrosome associated proteins, such as GM130 and p-Mapk, detached from the spindle poles in Hfm1-cKO oocytes. In conclusion, our data suggest that Hfm1 participates in Golgi-associated spindle assembly and division in mouse oocyte meiosis. These findings provide clues for pathogenesis of POF.

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

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.

Oocyte-dependent activation of MTOR in cumulus cells controls the development and survival of cumulus-oocyte complexes.

Communication between oocytes and their companion somatic cells promotes the healthy development of ovarian follicles, which is crucial for producing oocytes that can be fertilized and are competent to support embryogenesis. However, how oocyte-derived signaling regulates these essential processes remains largely undefined. Here, we demonstrate that oocyte-derived paracrine factors, particularly GDF9 and GDF9-BMP15 heterodimer, promote the development and survival of cumulus-cell-oocyte complexes (COCs), partly by suppressing the expression of Ddit4l, a negative regulator of MTOR, and enabling the activation of MTOR signaling in cumulus cells. Cumulus cells expressed less Ddit4l mRNA and protein than mural granulosa cells, which is in striking contrast to the expression of phosphorylated RPS6 (a major downstream effector of MTOR). Knockdown of Ddit4l activated MTOR signaling in cumulus cells, whereas inhibition of MTOR in COCs compromised oocyte developmental competence and cumulus cell survival, with the latter likely to be attributable to specific changes in a subset of transcripts in the transcriptome of COCs. Therefore, oocyte suppression of Ddit4l expression allows for MTOR activation in cumulus cells, and this oocyte-dependent activation of MTOR signaling in cumulus cells controls the development and survival of COCs.