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# 3D imaging-based analysis of the germline in teleost

Marlène Davilma, Stéphanie Gay, Manon Thomas, Laurence Dubreil,
Frédérique Clément, Violette Thermes

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► To cite this version:

Marlène Davilma, Stéphanie Gay, Manon Thomas, Laurence Dubreil, Frédérique Clément, et al. 3D imaging-based analysis of the germline in teleost. From cell to organism: When cell biology meets development, Oct 2024, Paris, France. hal-04745244

HAL Id: hal-04745244

https://hal.inrae.fr/hal-04745244v1

Submitted on 20 Oct 2024

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Distributed under a Creative Commons Attribution 4.0 International License
# 3D imaging-based analysis of the germline in teleost

Marlène Davilma¹, Stéphanie Gay¹, Manon Thomas¹, Laurence Dubreil², Frédérique Clément³, Violette Thermes¹

1- Team SOCS, LPGP INRAE, Campus de Beaulieu, 35042 Rennes

2- Oniris, INRAE, APEX, PAnTher, 44300 Nantes, France

3- Joint CNRS-INRAE-INRIA project-team MUSCA, Centre INRIA de Saclay

## Context and objectives

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* In mammals, the stock of oocytes is predetermined at birth, whereas in teleosts, the oocyte reserve is thought to have the potential to be renewed throughout life

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* The reserve is used in adults to produce mature oocytes and replenished from germline stem cells

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* Germline stem cells are organized in germline cradles surrounded by somatic cells expressing sox9 [1]

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## Is the renewal of the oocyte reserve by germline stem cells continuous throughout the female lifespan, and how is it controlled?

Exploration of the oogenesis renewal process and its control using the medaka as a model organism and the development of a 3D imaging strategy for the whole ovary, to provide **quantitative data to study the cell dynamics of germinal cradles**.

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* Study oocyte reserve formation and renewal (size, distribution and composition)

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* Identify the molecular regulators impacting the oocyte reserve

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Changes in follicle distribution under the influence of hormonal regulators (FSH, LH, AMH): what regulatory mechanisms affect germline cradles (feedback loops? miRNAs?) and hence maintenance of oocyte reserve?

## A potential regulator of this reserve

### KO of miR-187 :

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* Fewer eggs laid, lower female fecundity

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* Increase in the number of stage I follicles

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90 days
juvenile

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WT

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MUT

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**Hypothesis :** involvement of miR187 in the transition from stage I to stage II, that is, in the exit from the germinal cradle and consequently in the formation of the oocyte reserve

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* Study of germ cell proliferation (PH3 labeling)

## Imaging and image analysis

Development of a whole ovary immunolabeling protocol combined with the C-ECi clearing protocol to image the germinal cradles and follicles. Completion of the protocol followed by the transfer of samples to agarose for **3D imaging using Light Sheet Blaze imaging** (APEX platform, ONIRIS, Nantes).

Denoising and segmentation of images using the **N2V** module and **Cellpose** for the study of follicle distribution, followed by error correction and diameter measurement with **AMIRA** software. Adaptation of these analysis steps for the quantitative study of germinal cradles.

### 1- Study the distribution and number of cradles in medaka ovaries

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* Denoising with N2V

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* Segmentation of vasa-positive germline stem cell clusters surrounded by somatic cells expressing sox9 with Cellpose_Napari

### 2- Study the composition of germinal cradles

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* Difficulty : vasa-labeling is cytoplasmic so that these cells are difficult to segment individually

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* Use of MG core labeling to individually separate vasa-positive cells with Cellpose_Napari

### 3- Study the proliferation of germ cells

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Juvenile

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Adult

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* Combination of PH3-labeling with vasa-labeling to verify that the mitotic cells in germinal cradles are indeed germ cells

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* Segmentation of PH3-positive germ cells to count the number of dividing germ cells per germinal cradle

<figure><img src="image_30.png" /><figcaption>Nuclei in magenta / somatic cells expressing sox9 in cyan / germ cells expressing vasa in yellow</figcaption></figure>

Cell in mitosis within a
cradle

Cell in mitosis
(out of the germinal cradles)

## Prospects

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* Develop workflow for germline cradles image analysis (numbering, distribution and composition)

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* Develop workflow for study germ cells proliferation by using PH3 labeling

## References

1- Nakamura, Shuhei, et al. "Identification of germline stem cells in the ovary of the teleost medaka." *Science* 328.5985 (2010): 1561-1563.

2- Gay, Stéphanie, et al. "MiR-202 controls female fecundity by regulating medaka oogenesis." *PLoS genetics* 14.9 (2018): e1007593.

3- Lesage, Manon, et al. "C-ECi: a CUBIC-ECi combined clearing method for three-dimensional follicular content analysis in the fish ovary." *Biology of Reproduction* 103.5 (2020): 1099-1109.

4- Lesage, Manon, et al. "An end-to-end pipeline based on open source deep learning tools for reliable analysis of complex 3D images of ovaries." *Development* 150.7 (2023): dev201185.