Granulosa cells, the ovarian support cells that nurture developing oocytes, may retain a molecular memory of reproductive ageing even after being chemically reprogrammed into induced pluripotent stem cells, according to a new study published in Science China Life Sciences. The research shows that cells taken from reproductively aged mice can be converted into chemically induced pluripotent stem cells, or CiPSCs, with apparently normal pluripotency characteristics. Yet when those cells are directed toward the primordial germ cell-like state that precedes egg formation, their performance falls sharply. The findings identify abnormal activation of the ERK/MAPK signalling pathway as a major contributor to this age-associated differentiation failure and suggest that manipulating this pathway could improve future strategies for generating germ cells from older donors.

Granulosa cells are essential components of the ovarian follicle. They surround the oocyte, provide metabolic and developmental support, regulate follicular growth, and produce hormones including estrogen and progesterone. Because they are somatic rather than germ cells, their conversion into pluripotent stem cells offers a potential route toward generating new germ cells from a patient’s own tissues. Previous work by the research team showed that granulosa cells collected from young mice could be chemically reprogrammed into GC-CiPSCs. These cells were subsequently differentiated into primordial germ cell-like cells, or PGCLCs, and eventually into functional oocytes capable of producing healthy offspring. The new study asked whether the same process would remain effective when the starting granulosa cells had already undergone reproductive ageing.

To investigate this question, the researchers established GC-CiPSC lines from granulosa cells isolated from female mice 11 to 12 months old. At this age, mice exhibit substantial reproductive decline, making them a useful model for studying cellular ageing in the ovary. The investigators first confirmed that the aged granulosa cells displayed multiple hallmarks of senescence and mitochondrial deterioration. Compared with granulosa cells from young animals, the aged cells showed altered expression of PCNA and the senescence-associated protein p16, reduced levels of DRP1, lower mitochondrial DNA copy numbers, diminished ATP production, increased reactive oxygen species, and a loss of mitochondrial membrane potential. Together, these features indicated that the donor cells carried extensive metabolic and structural damage before reprogramming began.

Despite this ageing profile, the cells could still be converted into pluripotent stem cells. The resulting aged GC-CiPSCs expressed core pluripotency genes at levels comparable to embryonic stem cells and GC-CiPSCs generated from young granulosa cells. This result is important because it demonstrates that successful acquisition of pluripotency does not necessarily mean that every aspect of cellular age has been erased. Chemical reprogramming can reset broad transcriptional and epigenetic features associated with cell identity, allowing a mature somatic cell to return to a stem-like state. However, the process may not completely eliminate persistent mitochondrial damage, altered regulatory networks, or age-related changes embedded in the nuclear environment. Such residual defects may remain invisible during the generation and maintenance of iPSCs but become apparent when the cells are challenged to undertake a highly specialised developmental programme.

The difference emerged during PGCLC induction. When young GC-CiPSCs, aged GC-CiPSCs, and embryonic stem cells were exposed to conditions that promote primordial germ cell specification, the aged donor-derived cells generated substantially fewer PGCLCs. PGCLCs are laboratory-produced counterparts of primordial germ cells, the embryonic precursors that ultimately give rise to sperm or eggs. Their formation requires coordinated changes in gene expression, suppression of somatic programmes, and activation of a germline-specific developmental network. The reduced output from aged GC-CiPSCs therefore suggested that reprogramming had restored pluripotency without fully restoring developmental competence. In practical terms, the cells could “look” pluripotent while remaining less capable of responding correctly to signals that establish germline identity.

The researchers next tested whether mitochondrial dysfunction was the principal cause of the defect. They supplemented the differentiation system with nicotinamide mononucleotide, or NMN, and alpha-ketoglutarate, compounds associated with metabolic support and mitochondrial function. They also used sodium palmitate to activate DRP1, a protein involved in mitochondrial fission and the remodelling of mitochondrial networks. None of these interventions substantially improved PGCLC induction from aged GC-CiPSCs. The negative results do not imply that mitochondria are unimportant for germ-cell development. Germline specification places considerable demands on energy metabolism and redox control. Instead, they suggest that mitochondrial impairment is only one part of a wider ageing-associated programme, and that correcting mitochondrial activity alone cannot overcome changes in nuclear signalling or developmental gene regulation.

Transcriptomic analysis provided a more specific explanation. Genes expressed at higher levels in aged GC-CiPSCs were enriched for components of the ERK/MAPK signalling pathway. This pathway transmits extracellular growth-factor signals through a cascade involving RAS, RAF, MEK, and ERK, ultimately influencing transcription, proliferation, differentiation, and cell fate. During early development, the timing and intensity of ERK/MAPK activity can help determine whether pluripotent cells remain in a stem-like state, adopt somatic identities, or enter the germline pathway. Excessive or inappropriate pathway activity may therefore divert cells away from germ-cell specification or make them less responsive to the molecular cues used in culture. The age-associated elevation of MAPK-related transcription suggested that this signalling imbalance could be a functional barrier rather than merely a consequence of ageing.

To test that possibility, the team added PD0325901, a selective inhibitor of MEK, the kinase immediately upstream of ERK. Inhibition of MEK nearly doubled the efficiency of PGCLC induction from aged GC-CiPSCs. The improvement was also visible at the protein level. Cultures treated with PD0325901 contained more cells expressing STELLA and VASA, markers associated with germ-cell identity and development, while fewer cells expressed GATA4, a marker linked to somatic differentiation. These changes indicate that ERK/MAPK inhibition did not simply increase the total number of cells; it shifted the balance of cell fate decisions toward the germline programme. The result also demonstrates that part of the developmental defect remained reversible, even though the donor cells had originated from aged ovaries and retained other age-related abnormalities.

Additional transcriptomic comparisons showed that PD0325901 reversed expression changes associated with MAPK cascade regulation and cellular senescence in PGCLCs derived from aged GC-CiPSCs. At the same time, the treatment increased the activity of gene networks related to mitochondrial metabolism. This finding may help explain why direct mitochondrial supplementation was ineffective: the relevant metabolic changes may depend on the correct developmental state and its associated transcriptional programme, rather than on simply adding metabolic cofactors or stimulating mitochondrial remodelling. By adjusting signalling at the stage when germ-cell identity is being established, MEK inhibition may allow cells to activate metabolic pathways in a developmentally coordinated manner.

The study highlights both the promise and the limitations of using aged somatic cells as a source for germ-cell regeneration. Chemical reprogramming was sufficient to produce apparently competent pluripotent stem cells from senescent granulosa cells, but it did not fully restore their ability to follow the germline pathway. The results point to a layered model of cellular age memory in which mitochondrial dysfunction, senescence-associated nuclear regulation, and altered signal responsiveness interact. ERK/MAPK inhibition can partially relieve the barrier, but it is unlikely to represent a complete solution. Future approaches may need to combine pathway-specific control with interventions that repair mitochondrial quality, improve genome and epigenome stability, and reset age-associated transcriptional programmes. Such work could eventually inform autologous reproductive technologies, particularly for older women, although the findings remain preclinical and were obtained in a mouse model. The study was co-authored by Dai Heng and Kairang Jin and supported by China’s National Key Research and Development Program and the National Natural Science Foundation of China.

Subject of Research: Age-related defects in the differentiation of granulosa-cell-derived chemically induced pluripotent stem cells into primordial germ cell-like cells, and the role of ERK/MAPK signalling in overcoming those defects.

Article Title: Not provided in the supplied content.

Web References: https://doi.org/10.1007/s11427-025-3284-8

References: Science China Life Sciences, DOI: 10.1007/s11427-025-3284-8

Keywords: granulosa cells, reproductive ageing, chemically induced pluripotent stem cells, GC-CiPSCs, primordial germ cell-like cells, PGCLCs, oocyte regeneration, ERK/MAPK signalling, MEK inhibition, PD0325901, mitochondrial dysfunction, cellular senescence, germ-cell differentiation, reproductive biology

Tags: age-related decline in germ cell formationERK/MAPK pathway inhibitiongerm-cell differentiation impairmentgranulosa cell-derived induced pluripotent stem cellsgranulosa cells in ovarian follicle supportimproving germ cell generation from older donorsovarian support cell reprogrammingpluripotency in aged stem cellsprimordial germ cell-like differentiation failurereproductive ageing molecular memoryreprogramming of aged somatic cellssignaling pathway role in germ cell development