Spatial Characterization of Bioenergetics and Metabolism of Primordial to Preovulatory Follicles in Whole Ex Vivo Murine Ovary.
Previous work characterizing ovarian bioenergetics has defined follicular metabolism by measuring metabolic by-products in culture media. However, culture conditions perturb the native state of the follicle, and these methods do not distinguish between metabolism occurring within oocytes or granulosa cells. We applied the phasor approach to fluorescence lifetime imaging microscopy (phasor FLIM) at 740-nm two-photon excitation to examine the spatial distribution of free and protein-bound nicotinamide adenine dinucleotide hydride (NADH) during primordial through preovulatory stages of follicular development in fresh ex vivo murine neonatal and gonadotropin stimulated prepubertal ovaries. We obtained subcellular resolution phasor FLIM images of primordial through primary follicles and quantified the free/bound NADH ratio (relative NADH/NAD+) separately for oocyte nucleus and oocyte cytoplasm. We found that dynamic changes in oocyte nucleus free/bound NADH paralleled the developmental maturation of primordial to primary follicles. Immunohistochemistry of NAD+-dependent deacetylase SIRTUIN 1 (SIRT1) in neonatal ovary revealed that increasing SIRT1 expression in oocyte nuclei was inversely related to decreasing free/bound NADH during the primordial to primary follicle transition. We characterized oocyte metabolism at these early stages to be NADH producing (glycolysis/Krebs). We extended the results of prior studies to show that cumulus and mural granulosa cell metabolism in secondary through preovulatory follicles is mainly NADH producing (glycolysis/Krebs cycle), while oocyte metabolism is mainly NADH consuming (oxidative phosphorylation). Taken together, our data characterize dynamic changes in free/bound NADH and SIRT1 expression during early follicular development and confirm results from previous studies defining antral and preovulatory follicle metabolism in culture.
Department of Developmental and Cell Biology, University of California Irvine, Irvine, California.
Rachel
R
Cinco
Laboratory for Fluorescence Dynamics, University of California Irvine, Irvine, California.|Department of Biomedical Engineering, University of California Irvine, Irvine, California.
Michelle A
MA
Digman
Laboratory for Fluorescence Dynamics, University of California Irvine, Irvine, California.|Department of Biomedical Engineering, University of California Irvine, Irvine, California.
Enrico
E
Gratton
Department of Developmental and Cell Biology, University of California Irvine, Irvine, California uluderer@uci.edu.|Department of Medicine, University of California Irvine, Irvine, California.|Program in Public Health, University of California Irvine, Irvine, California.
Ulrike
U
Luderer
D000818:Animals; D004734:Energy Metabolism; D005260:Female; D006107:Granulosa Cells / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D009243:NAD / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism*; D006080:Ovarian Follicle / Q000378:metabolism*; D010053:Ovary / Q000378:metabolism*; D010085:Oxidative Phosphorylation; D056564:Sirtuin 1 / Q000378:metabolism
D009243:NAD; D056564:Sirtuin 1
FLIM; Krebs cycle; NADH; cumulus cells; folliculogenesis; glycolysis; granulosa cells; oocyte; oxidative phosphorylation; phasor approach to fluorescence lifetime imaging microscopy; sirtuin 1
Mol Reprod Dev. 2009 Mar;76(3):231-8
18618608
Science. 2003 Jul 11;301(5630):215-8
12855809
Mitochondrion. 2007 Sep;7(5):330-9
17576101
Reprod Domest Anim. 2009 Sep;44 Suppl 3:50-8
19660080
Am J Physiol Endocrinol Metab. 2014 Apr 15;306(8):E893-903
24569591
Sci Rep. 2012;2:568
22891156
Oncogene. 2009 Aug 13;28(32):2882-93
19503100
Proc Natl Acad Sci U S A. 2004 Apr 20;101(16):5880-5
15079084
Nature. 2005 Mar 3;434(7029):113-8
15744310
Biochim Biophys Acta. 1997 Apr 11;1319(2-3):293-300
9131050
J Biomed Opt. 2012 Nov;17(11):116023
23214184
Annu Rev Pharmacol Toxicol. 1984;24:451-81
6428300
Cell. 2004 Feb 20;116(4):551-63
14980222
Int J Dev Biol. 2012;56(10-12):799-808
23417402
Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1271-5
1741380
Hum Reprod. 1994 Apr;9(4):617-23
8046012
Hum Reprod. 2007 Nov;22(11):2842-50
17872911
Carcinogenesis. 2006 Mar;27(3):409-18
16221732
Pharmacol Rev. 2012 Jan;64(1):166-87
22106091
Biophys J. 2008 Jan 15;94(2):L14-6
17981902
Biol Reprod. 1993 Apr;48(4):798-806
8485244
Lab Invest. 2013 May;93(5):528-42
23459371
Free Radic Biol Med. 2004 Jun 1;36(11):1366-77
15135172
Oxid Med Cell Longev. 2015;2015:659687
26075037
J Reprod Fertil. 1968 Dec;17(3):555-7
5715685
Biol Reprod. 2010 Dec;83(6):909-18
20686180
Cancer Res. 2005 Oct 1;65(19):8766-73
16204046
J Neurosci. 2010 Jul 21;30(29):9695-707
20660252
Dev Biol. 2008 Sep 1;321(1):197-204
18601916
Oncol Lett. 2012 Dec;4(6):1151-1157
23226794
Sci Rep. 2015 May 20;5:9848
25993434
FEBS Lett. 2003 Apr 10;540(1-3):3-6
12681474
FEBS Lett. 2011 Jun 6;585(11):1651-6
21443875
Biotechniques. 2000 Jul;29(1):52, 54
10907076
Proc Natl Acad Sci U S A. 1967 Aug;58(2):560-7
5233459
Proc Natl Acad Sci U S A. 2011 Aug 16;108(33):13582-7
21808026
Cell Mol Life Sci. 2015 Jan;72(2):251-71
25280482
J Biol Chem. 2004 Dec 3;279(49):50754-63
15381699
Hum Reprod. 1994 Apr;9(4):604-9
8046010
J Biophotonics. 2011 Sep;4(9):592-9
21425242
PLoS One. 2011 Apr 26;6(4):e19194
21541336
Mech Dev. 2002 Jul;115(1-2):157-60
12049782
PLoS One. 2008 Feb 13;3(2):e1571
18270565
Gene. 2013 Jul 1;523(1):82-7
23566837
Biophys J. 2012 Jul 3;103(1):L7-9
22828352
Cell Metab. 2013 Jun 4;17(6):838-50
23747243
Aging Clin Exp Res. 2012 Apr;24(2):125-33
21502801
Biol Reprod. 2013 May 02;88(5):111
23536372
J Biol Chem. 2005 Apr 22;280(16):16456-60
15716268
Am J Physiol Cell Physiol. 2007 Feb;292(2):C615-40
16943239
Genes Dev. 2004 Jan 1;18(1):12-6
14724176
J Exp Zool. 1976 Dec;198(3):375-82
1003146
Biochemistry. 2011 Oct 4;50(39):8264-9
21888353
Science. 2004 Jul 2;305(5680):99-103
15232110
EMBO J. 2014 Jul 1;33(13):1454-73
24825347
Hum Reprod. 1990 Nov;5(8):915-9
2081802
Dev Biol. 2005 Mar 1;279(1):20-30
15708555
Biochim Biophys Acta. 2014 Dec;1846(2):617-29
25157892
Hum Reprod Update. 2014 Jan-Feb;20(1):29-39
24097804
Curr Opin Cell Biol. 2003 Apr;15(2):241-6
12648681
J Biol Chem. 2014 Mar 21;289(12):8299-311
24515103
Reproduction. 2007 Sep;134(3):415-24
17709560
Biomark Med. 2010 Apr;4(2):241-63
20406068
Hum Reprod. 2014 Sep;29(9):2006-17
24963165