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A non-canonical role of somatic Cyclin D/CYD-1 in oogenesis and in maintenance of reproductive fidelity, dependent on the FOXO/DAF-16 activation state [1]

['Umanshi Rautela', 'Molecular Aging Laboratory', 'National Institute Of Immunology', 'Aruna Asaf Ali Marg', 'New Delhi', 'Gautam Chandra Sarkar', 'Ayushi Chaudhary', 'Debalina Chatterjee', 'Mohtashim Rosh', 'Molecular Genetics Laboratory']

Date: 2024-12

For the optimal survival of a species, an organism coordinates its reproductive decisions with the nutrient availability of its niche. Thus, nutrient-sensing pathways like insulin-IGF-1 signaling (IIS) play an important role in modulating cell division, oogenesis, and reproductive aging. Lowering of the IIS leads to the activation of the downstream FOXO transcription factor (TF) DAF-16 in Caenorhabditis elegans which promotes oocyte quality and delays reproductive aging. However, less is known about how the IIS axis responds to changes in cell cycle proteins, particularly in the somatic tissues. Here, we show a new aspect of the regulation of the germline by this nutrient-sensing axis. First, we show that the canonical G1-S cyclin, Cyclin D/CYD-1, regulates reproductive fidelity from the uterine tissue of wild-type worms. Then, we show that knocking down cyd-1 in the uterine tissue of an IIS receptor mutant arrests oogenesis at the pachytene stage of meiosis-1 in a DAF-16-dependent manner. We observe activated DAF-16-dependent deterioration of the somatic gonadal tissues like the sheath cells, and transcriptional de-regulation of the sperm-to-oocyte switch genes which may be the underlying reason for the absence of oogenesis. Deleting DAF-16 releases the arrest and leads to restoration of the somatic gonad but poor-quality oocytes are produced. Together, our study reveals the unrecognized cell non-autonomous interaction of Cyclin D/CYD-1 and FOXO/DAF-16 in the regulation of oogenesis and reproductive fidelity.

The conserved insulin-IGF-1 signaling (IIS) pathway in Caenorhabditis elegans is a neuroendocrine cascade that surveils the availability of nutrients as well as exposure to environmental and cellular stressors to regulate somatic development, reproduction and aging, both cell autonomously and non-autonomously. Lowering IIS flux activates the downstream FOXO transcription factor DAF-16 that mediates most of these responses. How this pathway responds to perturbations in cell cycle proteins to influence reproductive decision is less known. Here we show that the G1-S phase cyclin, Cyclin D/CYD-1 and the IIS nutrient-sensing axis crosstalk to regulate oogenesis and germ cell quality. In the wild-type worms, we found that CYD-1 in the uterine tissue regulates oocyte health and reproductive fidelity. Knocking down cyd-1, only in the somatic uterine tissue of IIS mutants, leads to a DAF-16-dependent arrest of oogenesis. Interestingly, we observed DAF-16-dependent destruction of the somatic gonadal tissues and de-regulation of the sperm-to-oocyte switch genes when CYD-1 is depleted in the uterus of animals with lower IIS. We speculate that defects in cell cycle proteins in the uterus may be interpreted by activated DAF-16 as a potential threat to progeny health that is unlikely to be rectified. So, our study unveils a new function of activated FOXO/DAF-16 and Cyclin D/CYD-1 in maintaining oocyte health and reproductive fidelity.

Funding: This project was partly funded by the National Bioscience Award for Career Development (BT/HRD/NBA/38/04/2016) and extramural grant (BT/PR27603/GET/119/267/2018) from the Department of Biotechnology, Government of India ( https://dbtindia.gov.in/ ), Science and Engineering Research Board-Science and Technology Award for Research (SERB-STAR) award (STR/2019/000064), Jagadish Chandra Bose National Fellowship (JCB/2022/000021) and extramural grant (CRG/2022/000525) from the Ministry of Science and Technology, Government of India ( https://serb.gov.in/page ), as well as core funding from the National Institute of Immunology (to AM). GCS is supported by an ICMR SRF fellowship (RMBH/FW/2020/19), and UR by DBT-JRF fellowship DBT/2018/NII/1035. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Together, our study highlights the non-canonical, cell non-autonomous function of Cyclin D/CYD-1 in reproductive fitness and the unconventional role of activated FOXO/DAF-16 in causing tissue damage to stall oogenesis. Considering the conserved nature of these players, it is tempting to speculate that such mechanisms may be conserved during evolution.

In this study, we report two novel and unanticipated observations: 1) cyd-1 knock-down solely in the somatic uterine tissues of wild-type (WT) worms, compromises reproductive fidelity, leading to poor quality oocytes and endomitotic oocytes in the germline, and 2) cyd-1 knock-down in the uterine tissues causes DAF-16-dependent arrest of the germline in the pachytene stage of meiosis-I specifically in the daf-2 mutant (where DAF-16 is in an activated state). We also show an unexpected link between activated DAF-16 and dramatic somatic gonad defects with concomitant downregulation of genes important for the sperm-to-oocyte switch that may cause the germline pachytene arrest and halted oogenesis.

To identify unexplored interactors of the IIS pathway, we had earlier performed a reverse genetic screen and identified a cyclin-dependent kinase CDK12/CDK-12 that regulates oogenesis cell non-autonomously [ 40 ]. To broaden our understanding of other cell cycle regulators that may have non-canonical roles in reproductive aging and if they crosstalk with the IIS pathway, we screened for cyclins that regulate germline development. Interestingly, this led to the identification of Cyclin D/CYD-1 [a core cell-cycle protein that regulates the G1 to S phase transition] [ 41 , 42 ] as a novel interactor of the IIS pathway. Beyond their involvement in cell cycle progression, cyclins and CDKs exhibit diverse biological functions [ 43 , 44 ]. Cyclin D is particularly noteworthy for its involvement in a broad spectrum of biological functions, encompassing metabolic regulation [ 45 , 46 ], organ development [ 47 , 48 ], DNA damage repair [ 49 ], and transcriptional control [ 50 – 52 ].

Germline is the most precious tissue of an organism that ensures perpetuation of a species. However, with aberrant metabolism brought about by aging and metabolic disorders, the reproductive tissues deteriorate leading to poor quality of oocytes, adversely affecting reproductive outcomes and progeny fitness [ 1 – 5 ]. To ensure optimal growth of the gametes and the fertilized oocytes, the process of oogenesis takes inputs from other reproductive and somatic niches in the body. For example, oogenesis is firmly regulated by the nutrient-sensing [ 6 – 8 ] and stress-responsive pathways [ 9 , 10 ]. In C. elegans, the insulin/IGF-1 signaling (IIS) pathway, a conserved neuro-endocrine signaling axis that is central to nutrient sensing and stress resilience [ 11 – 14 ], regulates both somatic [ 15 , 16 ] as well as reproductive aging [ 17 , 18 ] in a cell-autonomous as well as non-autonomous manner [ 18 – 22 ]. In mammals, IIS is crucial for the activation, and growth of the primordial oocyte follicles [ 23 – 25 ]. The imbalance in IIS underpins pathophysiologies of multiple ovarian dysfunctions including PCOS and infertility [ 26 – 29 ]. The IIS-PI3K-AKT axis, when activated, maintains the FOXO transcription factor in the cytoplasm through inhibitory phosphorylation [ 30 – 32 ]. The overactivation of the PI3K-AKT pathway that is downstream of the IIS receptor has been shown to cause global activation of the oocyte follicles and depletes the ovarian reserve, leading to premature reproductive aging and infertility in mice [ 33 ]. On the other hand, when the signaling through the IIS pathway is low, FOXO is released from its cytoplasmic anchor and enters the nucleus to activate gene expression [ 14 , 30 – 32 ]. It has been noticed that FOXO activation during the early stages of oocyte development preserves the ovarian reserve and extends reproductive capacity in mice [ 34 , 35 ]. In C. elegans, where the mechanisms of aging are relatively well worked out [ 36 – 38 ] mutations in the daf-2 (the IIS receptor ortholog) lower signaling flux through this pathway, leading to a long life and a slower reproductive aging [ 17 , 18 , 39 ]. The daf-2 worms have a delayed decline in oocyte quality [ 18 , 39 ] and germline stem cell pool with age [ 22 ]. Interestingly, activated DAF-16 is required in the intestine and muscle for oocyte quality maintenance [ 18 ] while DAF-16 in the somatic gonad delays age-related germline stem cell loss [ 22 ]. It is important to note that in most cases, activated FOXO is pro-longevity and it preserves reproductive fidelity.

Results

In a recent study, we described the crosstalk of the cyclin-dependent protein kinase, CDK-12 with the IIS pathway to regulate germ cell development [40]. Interestingly, in that study, the function of the CDK-12 was found to be independent of its canonical cyclin, cyclin K/CCNK-1 [53]. Following up on this study, we asked if other cyclins may crosstalk with the IIS pathway to regulate germline development. For this, we knocked down the annotated cyclins in C. elegans in daf-2(e1370) (referred to as daf-2) and found that the RNAi-mediated depletion/knock-down (KD) of cyclin D/cyd-1 and cyclin E/cye-1, genes that encode core cell-cycle proteins governing the transition from the G1 to S phase, caused a DAF-16 dependent sterility specifically in a daf-2 mutant (S1A Fig). To determine the effect of cyd-1 and cye-1 KD on germline development, the gonads of day-1 adults were stained with DAPI to mark the nucleus. The KD of cye-1 drastically reduced the germ cell number in daf-2 worms, as is expected from its known function in cell division (S1B Fig), whereas cyd-1 KD demonstrated a non-canonical role in germ cell development without dramatic changes in mitosis, as described below. The loss-of-function cyd-1 alleles are arrested during early larval development and are sterile [42]. Consequently, they could not be used for our experiments to study interaction with the IIS pathway and so, we focused our efforts on investigating the role of CYD-1 in germ cell development and quality assurance using RNAi.

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1011453

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