Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome

Sarah R. Oca&#241;as; Jos&#233;  V. V. Isola; Tatiana D. Saccon; Kevin D. Pham; Ana J. Chucair-Elliott; Augusto Schneider; Willard M. Freeman; Michael B. Stout

doi:10.3791/64765

Zellspezifische paarweise Abfrage des Ovarialepigenoms und des Transkriptoms der Maus

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Biology

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Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome

Zellspezifische paarweise Abfrage des Ovarialepigenoms und des Transkriptoms der Maus

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12:25 min

February 24, 2023

DOI: 10.3791/64765-v

Sarah R. Ocañas*^1,2,3, José V. V. Isola*^1,3, Tatiana D. Saccon¹, Kevin D. Pham², Ana J. Chucair-Elliott², Augusto Schneider⁴, Willard M. Freeman^2,3, Michael B. Stout^1,3

¹Aging and Metabolism Research Program,Oklahoma Medical Research Foundation, ²Genes & Human Disease Research Program,Oklahoma Medical Research Foundation, ³Oklahoma City Veterans Affairs Medical Center, ⁴Nutrition College,Federal University of Pelotas

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Overview

This study optimizes the translating ribosome affinity purification (TRAP) and isolation of nuclei tagged in specific cell types (INTACT) methods for paired interrogation of the ovarian transcriptome and epigenome in a mouse model. The approach enables analysis of specific ovarian cell populations without cell sorting, facilitating insights into cell type-specific changes associated with aging.

Key Study Components

Research Area

Cell type-specific analysis
Ovarian aging
Transcriptomic and epigenomic investigation

Background

Understanding ovarian aging is crucial for addressing female infertility.
Current methods often require cell sorting, which can limit throughput.
This technique offers a streamlined way to analyze ovaries from the same mouse.

Methods Used

Optimized TRAP and INTACT methods
NuTRAP mouse model crossed with Cyp17a1-Cre line
Single ovary collection and specific protocols for nuclear isolation

Main Results

Enabled paired analysis of transcriptomic and epigenomic data.
Uncovered cell type-specific changes in the ovaries related to aging.
Validated the potential of this method for broader application across various cell types.

Conclusions

The method demonstrates effectiveness in isolating nucleic acids from specific ovarian cell populations.
This approach has significant implications for studies focused on female reproductive health.

Frequently Asked Questions

What are the advantages of this method?

It increases throughput, reduces costs, and eliminates the need for specialized cell sorting equipment.

Can this method be applied to other cell types?

Yes, as long as a specific Cre driver is available for the cell type of interest.

What biological effects were observed in the study?

The study identified specific changes in ovarian cells associated with aging, relevant for understanding reproductive senescence.

Why is cell specificity important?

It ensures that the analysis accurately reflects changes within the targeted cell populations, which is crucial for understanding their biological roles.

What preliminary steps are involved in this method?

The method begins with precise collection and homogenization of the ovary, followed by centrifugation to isolate nuclei.

How significant are the findings regarding female infertility?

The findings enhance the understanding of ovarian aging, which is essential for developing treatments for female infertility.

In diesem Protokoll wurden die translatierende Ribosomenaffinitätsreinigungsmethode (TRAP) und die Isolierung von Zellkernen, die in bestimmten Zelltypen markiert wurden (INTACT), für die paarweise Abfrage des zellspezifischen ovariellen Transkriptoms und Epigenoms unter Verwendung des NuTRAP-Mausmodells, das mit einer Cyp17a1-Cre-Mauslinie gekreuzt wurde, optimiert.

Diese Methode bietet eine Technik, um Nukleinsäuren aus bestimmten Eierstockzellpopulationen derselben Maus zu isolieren, ohne dass eine Zellsortierung erforderlich ist. Diese Technik kann dabei helfen, herauszufinden, wie sich bestimmte Zellpopulationen unter verschiedenen Bedingungen verändern. Zum Beispiel mit dem Altern.

Der Hauptvorteil dieser Technik besteht darin, dass sie eine paarweise epigenomische und transkriptomische Analyse eines bestimmten ovariellen Zelltyps ermöglicht, ohne dass eine Zellsortierung erforderlich ist. Dies erhöht den Durchsatz erheblich, senkt die Kosten und macht spezielle Geräte bei der Durchführung von zelltypspezifischen Analysen aus Eierstockgewebe überflüssig. Unser Ziel ist es, die zelltypspezifischen Veränderungen zu identifizieren, die mit der Alterung der Eierstöcke vor der reproduktiven Seneszenz auftreten, was Auswirkungen auf die Behandlung der weiblichen Unfruchtbarkeit hat.

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