Exceptional longevity of mammalian ovarian and oocyte macromolecules throughout the reproductive lifespan

Reviewer #1 (Public Review):

Summary:

This manuscript by Bomba-Warczak describes a comprehensive evaluation of long-lived proteins in the ovary using transgenerational radioactive labelled 15N pulse-chase in mice. The transgenerational labeling of proteins (and nucleic acids) with 15N allowed the authors to identify regions enriched in long-lived macromolecules at the 6 and 10-month chase time points. The authors also identify the retained proteins in the ovary and oocyte using MS. Key findings include the relative enrichment in long-lived macromolecules in oocytes, pregranulosa cells, CL, stroma, and surprisingly OSE. Gene ontology analysis of these proteins revealed enrichment for nucleosome, myosin complex, mitochondria, and other matrix-type protein functions. Interestingly, compared to other post-mitotic tissues where such analyses have been previously performed such as the brain and heart, they find a higher fractional abundance of labeled proteins related to the mitochondria and myosin respectively.

Strengths:

A major strength of the study is the combined spatial analyses of LLPs using histological sections with MS analysis to identify retained proteins.

Another major strength is the use of two chase time points allowing assessment of temporal changes in LLPs associated with aging.

The major claims such as an enrichment of LLPs in pregranulosa cells, GCs of primary follicles, CL, stroma, and OSE are soundly supported by the analyses, and the caveat that nucleic acids might differentially contribute to this signal is well presented.

The claims that nucleosomes, myosin complex, and mitochondrial proteins are enriched for LLPs are well supported by GO enrichment analysis and well described within the known body of evidence that these proteins are generally long-lived in other tissues.

Weaknesses:

One small potential weakness is the lack of a mechanistic explanation of if/why turnover may be accelerating at the 6-10 month interval compared to 1-6.

A mild weakness is the open-ended explanation of OSE label retention. This is a very interesting finding, and the claims in the paper are nuanced and perfectly reflect the current understanding of OSE repair. However, if the sections are available and one could look at the spatial distribution of OSE signal across the ovarian surface it would interesting to note if label retention varied by regions such as the CLs or hilum where more/less OSE division may be expected.

Reviewer #2 (Public Review):

Summary:

The manuscript by Bomba-Warczak et al. applied multi-isotope imaging mass spectrometry (MIMS) analysis to identify the long-lived proteins in mouse ovaries during reproductive aging, and found some proteins related to cytoskeletal and mitochondrial dynamics persisting for 10 months.

Strengths:

The manuscript provides a useful dataset about protein turnover during ovarian aging in mice.

Weaknesses:

The study is pretty descriptive and short of further new findings based on the dataset. In addition, some results such as the numbers of follicles and ovulated oocytes in aged mice are not consistent with the published literature, and the method for follicle counting is not accurate. The conclusions are not fully supported by the presented evidence.

Reviewer #3 (Public Review):

Summary:

In this study, Bomba-Warczak et al focused on reproductive aging, and they presented a map for long-lived proteins that were stable during reproductive lifespan. The authors used MIMS to examine and show distinct molecules in different cell types in the ovary and tissue regions in a 6 month mice group, and they also used proteomic analysis to present different LLPs in ovaries between these two timepoints in 6-month and 10-month mice. The authors also examined the LLPs in oocytes in the 6-months mice group and indicated that these were nuclear, cytoskeleton, and mitochondria proteins.

Strengths:

Overall, this study provided basic information or a 'map' of the pattern of long-lived proteins during aging, which will contribute to the understanding of the defects caused by reproductive aging.

Weaknesses:

The 6-month mice were used as an aged model; no validation experiments were performed with proteomics analysis only.