Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse.
Journal
Journal of visualized experiments : JoVE
ISSN: 1940-087X
Titre abrégé: J Vis Exp
Pays: United States
ID NLM: 101313252
Informations de publication
Date de publication:
03 09 2021
03 09 2021
Historique:
entrez:
20
9
2021
pubmed:
21
9
2021
medline:
16
10
2021
Statut:
epublish
Résumé
Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells entering meiotic prophase I are eliminated during Fetal Oocyte Attrition (FOA) and the first week of postnatal life. Three major mechanisms regulate the number of oocytes that survive during development and establish the ovarian reserve in females entering puberty. In the first wave of oocyte loss, 30-50% of the oocytes are eliminated during early FOA, a phenomenon that is attributed to high Long interspersed nuclear element-1 (LINE-1) expression. The second wave of oocyte loss is the elimination of oocytes with meiotic defects by a meiotic quality checkpoint. The third wave of oocyte loss occurs perinatally during primordial follicle formation when some oocytes fail to form follicles. It remains unclear what regulates each of these three waves of oocyte loss and how they shape the ovarian reserve in either mice or humans. Immunofluorescence and 3D visualization have opened a new avenue to image and analyze oocyte development in the context of the whole ovary rather than in less informative 2D sections. This article provides a comprehensive protocol for whole ovary immunostaining and optical clearing, yielding preparations for imaging using multiphoton microscopy and 3D modeling using commercially available software. It shows how this method can be used to show the dynamics of oocyte attrition during ovarian development in C57BL/6J mice and quantify oocyte loss during the three waves of oocyte elimination. This protocol can be applied to prenatal and early postnatal ovaries for oocyte visualization and quantification, as well as other quantitative approaches. Importantly, the protocol was strategically developed to accommodate high-throughput, reliable, and repeatable processing that can meet the needs in toxicology, clinical diagnostics, and genomic assays of ovarian function.
Identifiants
pubmed: 34542534
doi: 10.3791/62972
pmc: PMC8911993
mid: NIHMS1780479
doi:
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Video-Audio Media
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : NICHD NIH HHS
ID : R01 HD093778
Pays : United States
Organisme : NICHD NIH HHS
ID : T32 HD007065
Pays : United States
Références
Front Genet. 2013 Jun 24;4:117
pubmed: 23805152
Reproduction. 2004 May;127(5):569-80
pubmed: 15129012
Reproduction. 2021 Feb;161(2):R13-R35
pubmed: 33170803
Dev Biol. 2001 Jun 15;234(2):339-51
pubmed: 11397004
F1000Res. 2020 Sep 7;9:
pubmed: 32934798
Reproduction. 2012 Feb;143(2):139-49
pubmed: 22065859
Cell. 2014 Apr 24;157(3):726-39
pubmed: 24746791
Biol Reprod. 2015 Nov;93(5):111
pubmed: 26423124
Genesis. 2006 Dec;44(12):622-32
pubmed: 17146778
Hum Reprod Update. 2014 May-Jun;20(3):353-69
pubmed: 24287894
J Embryol Exp Morphol. 1981 Aug;64:133-47
pubmed: 7310300
Semin Reprod Med. 2013 Nov;31(6):399-415
pubmed: 24101221
Nat Neurosci. 2015 Oct;18(10):1518-29
pubmed: 26368944
J Anat. 1966 Oct;100(Pt 4):761-76
pubmed: 4961727
Development. 2014 Nov;141(22):4406-14
pubmed: 25344073
Biol Reprod. 2015 Nov;93(5):113
pubmed: 26423126
Mol Cell. 2017 Sep 21;67(6):1026-1036.e2
pubmed: 28844861
Hum Reprod Update. 2019 Nov 5;25(6):673-693
pubmed: 31600388
PLoS One. 2014 Oct 13;9(10):e108343
pubmed: 25310678
PLoS One. 2015 Mar 26;10(3):e0120242
pubmed: 25812007
Hum Genet. 1981;57(1):86-92
pubmed: 7262874
Mol Hum Reprod. 2021 Feb 27;27(3):
pubmed: 33544861
Hum Reprod Update. 2012 Sep-Oct;18(5):525-35
pubmed: 22647504
Mol Hum Reprod. 2009 Dec;15(12):795-803
pubmed: 19710243
Nat Commun. 2020 Jan 16;11(1):330
pubmed: 31949138
J Vis Exp. 2012 Jul 30;(65):
pubmed: 22871843
Dev Biol. 1997 Jul 1;187(1):107-13
pubmed: 9224678
J Ovarian Res. 2020 Oct 14;13(1):121
pubmed: 33054849
Science. 2014 Jan 31;343(6170):533-6
pubmed: 24482479
Dev Biol. 2001 Mar 15;231(2):291-320
pubmed: 11237461
PLoS One. 2010 Jan 27;5(1):e8772
pubmed: 20111701
Proc Natl Acad Sci U S A. 2020 Aug 18;117(33):20015-20026
pubmed: 32759216
Reprod Biol Endocrinol. 2003 Feb 06;1:11
pubmed: 12646064
Development. 2002 Mar;129(5):1155-64
pubmed: 11874911
Proc R Soc Lond B Biol Sci. 1963 Oct 22;158:417-33
pubmed: 14070052
Sci Rep. 2018 Sep 10;8(1):13499
pubmed: 30202115
Climacteric. 2021 Oct;24(5):438-443
pubmed: 33522318
Reproduction. 2010 Feb;139(2):349-57
pubmed: 19846484
Front Neuroanat. 2019 Mar 27;13:35
pubmed: 30971902
Biol Reprod. 2020 Apr 24;102(5):1080-1089
pubmed: 31965156
Genetics. 2017 Aug;206(4):1823-1828
pubmed: 28576861
Development. 1998 Sep;125(17):3323-8
pubmed: 9693136
Reproduction. 2008 Jan;135(1):3-12
pubmed: 18159078
Front Genet. 2015 Oct 15;6:308
pubmed: 26528328
Nat Rev Mol Cell Biol. 2001 Nov;2(11):838-48
pubmed: 11715050
J Vis Exp. 2020 Oct 16;(164):
pubmed: 33135690
Reprod Biol Endocrinol. 2018 Aug 2;16(1):72
pubmed: 30071873
Dev Cell. 2014 Jun 9;29(5):521-533
pubmed: 24882376