Evidence of Fluctuating Integrated Stress Response Activity in Murine Primordial Ovarian Follicles

doi:10.21203/rs.3.rs-1682172/v1

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Evidence of Fluctuating Integrated Stress Response Activity in Murine Primordial Ovarian Follicles

https://doi.org/10.21203/rs.3.rs-1682172/v1

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We are evaluating the action of the Integrated Stress Response (ISR) pathway and how its enforcement of cell cycle arrest and enhancement of a cytoprotective state contribute to the rate of primordial follicle (PF) loss. Physiological stress and damage are variable locally/regionally within the ovary, thus we predict that ISR activity within PFs will be locations-dependent. We therefore tested whether levels of the marker of the active ISR, serine 51-phosphorylation of eukaryotic Initiation Factor 2 alpha subunit (P-eIF2α), differed between oocytes and pregranulosa cells within PFs, between PFs in the same ovary, and between PFs in ovaries collected from different animals. Quantitative fluorescence analysis revealed that P-eIF2α levels correspond tightly between oocytes and pregranulosa cells within the same PFs, but that different follicles in the same ovary have varied levels of the ISR activity marker; this was consistent between animal replicates. The variation of ISR activity in PFs may therefore directly reflect local/regional differences in physiological factors that induce cell damage and stress. This feature of the dynamically changing intra-ovarian environment may influence the likelihood that PFs stay dormant or begin to grow.

Cell damage

Cell stress

eukaryotic Initiation Factor 2 alpha (eIF2α)

Ovary

Ovarian Aging

Integrated Stress Response Pathway (ISR)

Primordial Follicles

Mechanisms that control ovarian primordial follicle growth activation (PFGA) and thus the rate of loss of primordial follicles (PFs) are beginning to be understood [1, 2], The impetus for understanding these mechanisms arises from the clinically significant problem of accelerated ovarian aging (in some instances, primary ovarian insufficiency; POI) [3], or early loss of ovarian function via either naturally occurring or iatrogenic (e.g. cancer chemotherapy) processes [4, 5]. In recent studies evaluating the ovarian response to cell-damaging agents, we observed that markers of cell damage were often present under normal, physiological conditions [6]. We therefore probed whether physiological regulation of the Integrated Stress Response (ISR) pathway [7] contributes to ovarian function, starting with the control of follicle growth. In the initiation of the ISR, different classes of cellular stress activate four ISR eukaryotic initiation Factor 2 (eIF2) subunit alpha kinases (Fig. 1), which in turn phosphorylate and inactivate eIF2α (phosphoserine 51-eIF2α, P-eIF2α).

ISR activation both upregulates the translation of cytoprotective/DNA damage response (DDR) and cell-cycle control proteins, and functionally overlaps with DDR pathways (Fig. 1, [8, 9]). Resolution of cell stress and DNA damage then allows cell cycle resumption and growth, referred to as ISR checkpoint resolution. While our prior studies used an immortalized granulosa cell (GC) line as a model to characterize ISR action, here we test the hypothesis that ISR activity fluctuates in within PFs in an intact mouse. If correct, ISR activity would differ between PFs in the same ovary at the same time. Such differences between ISR activity in PFs could then be explained by differences in regional signaling, stress, and damage. We performed immunofluorescence staining for P-eIF2α in histological preparations of mouse ovaries, generated electronic images of PFs, and quantified the signal in pregranulosa cells and, separately, oocytes. Signal in each cell type was compared within PFs, between PFs in the same ovary, and between PFs in ovaries collected from different animals.

Mice were handled in compliance with an approved protocol (University of Utah Institutional Animal Care and Use Committee Protocol #00001431). Ovaries from were collected from 5 separate 30 day-old wildtype (WT) C57Bl/6/Ola 129 mice and fixed in 10% non-buffered formalin for 12 hours at room temperature. After storage in 70% alcohol, specimens were paraffin-embedded and sectioned by the Research Histology Core Facility of the ARUP Laboratories (Salt Lake City, UT, USA). Two adjacent five micron-thick sections were probed per slide as follows, per [6]. One section was used to evaluate P-eIF2α levels (primary antibody rabbit anti-P-eIF2α, Cell Signaling Technologies, #3398, RRID:AB_2096481, diluted 1:100) and the other was treated identically but primary antibody was omitted (secondary antibody-only controls). Alexa Fluor 647-conjugated anti-rabbit secondary antibody (Abcam, #ab150075; 1:500) was applied to all sections..

Electronic images were collected using Nikon Widefield microscope (Nikon Corporation, Americas) at 20X using identical camera and device settings. Background autofluorescence from secondary antibody-only controls were subtracted from primary antibody-stained samples using Nikon NIS-Elements Analysis software. PFs and their cell types were selected in the DAPI channel by their morphology, and pregranulosa cells and oocytes were analyzed separately for each follicle. Only sections where visible nuclei were present for both cell types were used. Staining intensities were quantified by measuring average signal intensity in the region of interest. In each case, signal was divided by the measured area for each region of interest.

Data are shown in Fig. 2 with a representative PF shown in panel a: P-eIF2α signal in purple channel, DNA blue channel. White lines shown denote boundaries drawn for signal quantification in images, where the solid line is the boundary of the pregranulosa cell layer, and the dotted line is the boundary of the primordial oocyte. P-eIF2α signal shown in a' for comparison to second-antibody only control in a''). ISR activity was observed to vary between PFs, both in pregranulosa cells (b, plotted on x-axis) and in oocytes (b, y-axis), and this was observed consistently across unique ovary specimen replicates. It was also notable that there was a positive relationship between the P-eIF2α signal in the pregranulosa cells and oocytes of the same PFs (b).

Variable ISR activity in PFs in the same ovary at the same time is consistent with our working model, wherein ISR activity corresponds to local levels of cellular stress and damage. Unlike other organs with relatively stable architecture, the ovary is unique due to its constantly changing content. Over time, the growth and atresia of follicles, and the adult processes of ovulation, repair of ovulatory “wounds” and development and regression of corpora lutea mean that no two snapshots of the same ovary will ever be the same. In addition, all of these processes correspond to changes in the ovarian stromal compartment during reproductive aging, that themselves reflect tissue damage over time [10].

Data presented here showing that dormant PFs in the same ovary exhibit a range of P-eIF2α levels indicate at least that ISR activation is not identical between PFs, and imply that spatial differences in stress-inducing conditions are occurring. It is plausible that oxygen tension, glucose availability, endoplasmic reticulum (ER) stress, and the concentrations of factors like Tnfα (Fig. 1 table, [6]) are variable in different regions within the ovary. Because these conditions are dynamic over time, the ISR likely responds dynamically. These data further support the model ([6]; Fig. 1, left) where a default state of ISR activation contributes to PF arrest and slow follicle growth after “recruitment”, and where rare ISR checkpoint resolution favors cell cycle entry and the rare growth activation of PFs.

Author Contributions:

SHL, JR, KL, JJ conception and design of research; SHL performed experiments; ELC, JR, IL, JJ analyzed data, SHL, JR, ELC, IL, AP, JJ interpreted results of experiments; JJ drafted the manuscript; SHL, JJ prepared figures; JJ, AJP, KL edited and revised manuscript; SHL, JR, ELC, IL, AJP, KL, JJ approved final version of manuscript.

Funding

J.J. is supported by CU-Anschutz Department of Obstetrics and Gynecology Research Funds and McPherson Family Funds. K.L. and S.H.L. are supported by R01HD089933 to PI: Lai.

Acknowledgements

Drs. Nanette Santoro and Benjamin Bitler are gratefully acknowledged for providing feedback on the manuscript prior to submission.

Conflicts of Interest

No conflicts of interest were present for any author during the execution of these studies, nor during the preparation of the manuscript.

Ethics approval

University of Utah IACUC approval (Lai Laboratory) provided, Protocol #00001431. Approved on 04/14/2022 with the expiration date 04/14/2025

Consent to participate

Not applicable.

Consent for publication

All study authors have provided their consent for publication.

Availablity of data and material

Raw study data (e.g., image files and raw data used for analysis) are available upon request.

Code availability

Not applicable.

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Reviewers agreed at journal
15 Jul, 2022
Reviewers invited by journal
08 Jun, 2022
Editor assigned by journal
26 May, 2022
First submitted to journal
22 May, 2022

You are reading this latest preprint version

Evidence of Fluctuating Integrated Stress Response Activity in Murine Primordial Ovarian Follicles

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