P-219 Single-follicle spatial proteomics of the whole ovary by MALDI mass spectrometry imaging: towards the identification of protein-markers of the follicle's quality
Authors: Zuccotti M , Fiorentino G , Smith A , Nicora G , Bellazzi R , Cimadomo D , Vaiarelli A , Ubaldi F M , Rienzi L , Magni F , Garagna S 
Published: June 2023

Abstract

Study question
Can we improve our knowledge of the single-follicle proteome signature by MALDI mass spectrometry imaging?

Summary answer
We identified follicle-type proteins that function during follicle growth (NUMA1, TPM2), GV-to-MII transition (SFPQ, ACTBL, MARCS, NUCL), ovulation (GELS, CO1A2) and preimplantation development (TIF1B, KHDC3).

What is known already
The acquisition of oocytes developmental competence occurs through a bidirectional exchange of molecular information between the oocyte, the companion follicle cells, the stroma, and the vascular network. This reciprocal relationship is still poorly understood and particularly scarce is our knowledge of the proteins that are playing a regulative role during folliculogenesis.
Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) is a powerful tool that allows the study of the proteomic landscape of small morphological structures inside a tissue, but in the field of reproduction has been only used to analyse rodent spermatogenesis.

Study design, size, duration
Nano-scale liquid chromatography-electrospray ionisation-tandem mass spectrometry (nLC-ESI-MS/MS) was combined with MALDI-MSI to identify the proteomic landscape and to map the changes occurring throughout folliculogenesis. We performed single-follicle spatial proteomics on all the follicles, from the secondary to the fully-grown preovulatory, present in histological serial sections of an entire prepubertal 25-day-old mouse ovary. Cross-referencing our results with those obtained by previous proteomics and transcriptomics analyses allowed to strengthen their significance.

Participants/materials, setting, methods
A single 25-day-old mouse ovary was fixed in 10% formalin, dehydrated, embedded in paraffin, and sectioned to obtain 133 6-μm serial sections. After paraffin removal, trypsin digestion and matrix deposition, mass spectra were acquired using a rapifleX MALDI Tissuetyper™ (Bruker) with a raster sampling of 20 μm in both x and y axes. Then, tryptic peptides were extracted from sections and analysed with nLC-ESI-MS/MS using a Dionex-UltiMate-3000 LC-nano-system coupled with an Impact HD™ UHR-QqToF (Bruker).

Main results and the role of chance
This study proposes a spatial proteomics workflow to investigate the proteome of a whole prepubertal 25-day-old mouse ovary, preserving the spatial relationship of the peptides in this complex histological context. A total of 401 proteins were identified by nLC-ESI-MS/MS, 69 with a known function in ovary biology. Enrichment analysis highlighted significant KEGG and Reactome pathways, with apoptosis, developmental biology, PI3K-Akt, epigenetic regulation of gene expression, and extracellular matrix organisation being well represented. Then, correlating these data with the spatial information provided by MALDI-MSI on 276 follicles highlighted 94 proteins that were detected throughout the secondary to the pre-ovulatory transition. Of these, 37 proteins showed a gradual quantitative change during follicle differentiation, comprising 10 with a known role in follicle growth (NUMA1, TPM2), oocyte GV-to-MII transition (SFPQ, ACTBL, MARCS, NUCL), ovulation (GELS, CO1A2) and preimplantation development (TIF1B, KHDC3).
The proteome landscape identified includes molecules of known function in the ovary, but also those whose specific role is emerging. Altogether, this work demonstrates the utility of performing spatial proteomics in the context of the ovary and offers sound bases for more in-depth investigations that aim to further unravel its spatial proteome.

Limitations, reasons for caution
A limitation of MALDI-MSI is its 20 µm/pixel resolution which 1) leads to the acquisition of spectra comprising follicular and extra-follicular tissue, particularly with small follicles (15-30µm in diameter); and 2) does not distinguish among the different follicular cell types, thus precluding its application for single-cell in situ proteomics.

Wider implications of the findings
MALDI-MSI is a potent technology to study the proteome of ovary specimens, but it could also be used for the analysis of their lipidic or metabolic profiles. Our pipelline aim to identify protein-markers of the follicle's quality and, in turn, of the oocyte's developmental competence.

Trial registration number
not appplicable