Maternal low-protein diet programmes low ovarian reserve in offspring

in Reproduction
Restricted access

The ovarian reserve of primordial follicle oocytes is formed during in utero development and represents the entire supply of oocytes available to sustain female fertility. Maternal undernutrition during pregnancy and lactation diminishes offspring ovarian reserve in rats. In mice, maternal oocyte maturation is also susceptible to undernutrition, causing impaired offspring cardiovascular function. We aimed to determine whether programming of the ovarian reserve is impacted in offspring when maternal undernutrition extends from preconception oocyte development through to weaning. C57BL6/J female mice were fed normal protein (20%) or low-protein (8%) diet during preconception, pregnancy and lactation periods. Maternal ovaries were harvested at weaning and offspring ovaries were collected at postnatal day (PN)21 and 24 weeks of age. Total follicle estimates were obtained by histologically sampling one ovary per animal (n = 5/group). There was no impact of diet on maternal follicle numbers. However, in offspring, maternal protein restriction significantly depleted primordial follicles by 37% at PN21 and 51% at 24 weeks (P < 0.05). There were no effects of diet on other follicle classes. Histological analysis showed no differences in the proportion of proliferative follicles (pH3 positive), but increased atresia (cleaved caspase-3-positive, or TUNEL-positive) was detected in ovaries of protein-restricted offspring at both ages (P < 0.05). Our data show that maternal diet during the preconception period, in utero development and early life has significant impacts on follicle endowment and markers of follicle health later in life. This highlights the need for further investigation into the importance of maternal preconception diet for offspring reproductive development and health.

 

An official journal of

Society for Reproduction and Fertility

Sections

Figures

  • View in gallery

    Analysis of maternal pregnancy and ovaries. (A) The time to litter since pairing with a male stud was recorded in days (n = 11–12/group). (B) The number of live pups per litter was recorded (n = 11–12/group). Maternal ovaries (~18 weeks; n = 5/group) from mice fed (C) control diet or (D) low-protein diet were randomly selected and stained with PAS. Bars are 200 μm. Representative images of (E) primordial follicle oocyte; bar = 10 μm, (F) transitional follicle; bar = 10 μm, (G) primary follicle bar = 20 μm, (H) secondary follicle; bar = 50 μm, and (I) antral follicle; bar = 100 μm. (J) Total numbers of primordial, transitional, primary, secondary and antral follicles per ovary, (K) ovarian volume (μm3), (L) numbers of corpora lutea per ovary and (M) maternal weight was recorded following weaning. Data are mean ± s.e.m., t-test, *P < 0.05, ***P < 0.001.

  • View in gallery

    Analysis of offspring ovaries at PN21. PAS stained PN21 ovaries from offspring of mothers fed (A) control diet or (B) low-protein diet. Bars are 200 μm. (C) Total numbers of primordial, transitional, primary, secondary and antral follicles, (D) ovarian volume (μm3) and (E) offspring weight at PN21. Data are mean ± s.e.m., t-test, n = 5 mice/group; *P < 0.05.

  • View in gallery

    Analysis of adult offspring ovaries at 24 weeks. PAS-stained ovaries from adult offspring of mothers fed (A) control diet or (B) low-protein diet. Bars are 200 μm. (C) Total numbers of primordial, transitional, primary, secondary and antral follicles (D), ovarian volume (μm3) and (F) corpora lutea were quantified and (E) offspring weight recorded at 24 weeks. Data are mean ± s.e.m., t-test, n = 5 mice/group; *P < 0.05, **P < 0.01.

  • View in gallery

    Analysis of follicle atresia. Representative photomicrographs of TUNEL stained ovaries from PN21 offspring (A, B, C and D) or adult offspring (E, F, G and H) of mothers fed control or low-protein diet. Representative photomicrographs of cleaved caspase-3 immunostained ovaries from PN21 offspring (I, J, K and L) or adult offspring (M, N, O and P) of mothers fed control or low-protein diet. Insets are negative controls. Bars are 500 μm (A, B, E, F, I, J, M, N), or 200 μm (C, D, G, H, K, L, O, P). The number of (Q) TUNEL and (R) cleaved caspase-3 positive stained follicles were quantified in 3–5 sections per ovary and expressed as a percentage of total follicles. Positive follicles were defined by positive oocyte staining and/or ≥4 positive granulosa cells per follicle, where the ooplasm was visualised. Data are mean ± s.e.m., t-test, n = 5 mice/group; *P < 0.05, **P < 0.01.

  • View in gallery

    Analysis of follicle proliferation. Representative photomicrographs of phosphorylated histone H3 (pH3) immunostained ovaries from PN21 offspring (A, B, C and D) or adult offspring (E, F, G and H) of mothers fed control diet or low-protein diet. Insets are negative controls. Bars are 500 μm (A, B, E, F), or 200 μm (C, D, G, H). (I) The number of pH3 positive stained follicles was quantified in 3–5 sections per ovary and expressed as a proportion of total follicles (%). Positive follicles were defined by positive oocyte staining and/or ≥4 positive granulosa cells per follicle, where the ooplasm was visualised. Data are mean ± s.e.m., t-test, n = 5 mice/group.

  • View in gallery

    Analysis of follicle mitochondrial and lysosome distribution. Representative photomicrographs of MTCO1 immunostained ovaries from PN21 (A and B) and adult offspring (C and D) of mothers fed control diet or low-protein diet. Follicles with positive staining and ooplasm present were categorised based on homogenous (arrow head), or abnormal (arrow) mitochondrial staining. (E) The number of follicles with homogenous or abnormal staining was expressed as the proportion of total follicles (%), analysed in 3–5 sections per ovary. 465 oocytes were analysed in 20 mice (n = 5/group). Data are mean ± s.e.m., t-test, ***P < 0.001. Representative photomicrographs of LAMP-1 immunofluorescence from PN21 (F and G) or adult offspring (H and I) of mothers fed control diet, or low-protein diet. Lysosomes (positive staining, white arrow heads) did not correspond with apparent vacuoles (yellow outline) in low-protein diet offspring oocytes. Insets are negative controls. Bars are 10 μm.

  • View in gallery

    Analysis of follicle DNA damage. Representative photomicrographs of γH2AX immunostained ovaries from PN21 (A and B) or adult offspring (C and D) of mothers fed control diet or low-protein diet. Insets are negative controls. Bars are 200 μm. (E) The number of γH2AX-positive stained follicles was quantified in 3–5 sections per ovary and expressed as a proportion of total follicles (%). Positive follicles were defined by positive oocyte staining and/or ≥4 positive granulosa cells per follicle, where the nucleus was visualised. 455 oocytes were analyses in 20 mice (n = 5/group). Data are mean ± s.e.m., Mann–Whitney test (PN21), t-test (24 week), *P < 0.05.

References

AikenCETarry-AdkinsJLOzanneSE 2016 Transgenerational effects of maternal diet on metabolic and reproductive ageing. Mammalian Genome 27 430439. (https://doi.org/10.1007/s00335-016-9631-1)

BernalABVickersMHHamptonMBPoyntonRASlobodaDM 2010 Maternal undernutrition significantly impacts ovarian follicle number and increases ovarian oxidative stress in adult rat offspring. PLoS ONE 5 e15558. (https://doi.org/10.1371/journal.pone.0015558)

BlackmoreHLPiekarzAVFernandez-TwinnDSMercerJRFiggNBennettMOzanneSE 2012 Poor maternal nutrition programmes a pro-atherosclerotic phenotype in ApoE-/- mice. Clinical Science 123 251257. (https://doi.org/10.1042/CS20110487)

BorenJBrindleKM 2012 Apoptosis-induced mitochondrial dysfunction causes cytoplasmic lipid droplet formation. Cell Death and Differentiation 19 15611570. (https://doi.org/10.1038/cdd.2012.34)

BronsonFHMarstellerFA 1985 Effect of short-term food deprivation on reproduction in female mice. Biology of Reproduction 33 660667. (https://doi.org/10.1095/biolreprod33.3.660)

da Silva FariaTde Bittencourt BrasilFSampaioFJda Fonte RamosC 2010a Effects of maternal undernutrition during lactation on estrogen and androgen receptor expressions in rat ovary at puberty. Nutrition 26 993999. (https://doi.org/10.1016/j.nut.2009.09.027)

da Silva FariaTde Bittencourt BrasilFSampaioFJda Fonte RamosC 2010b Maternal malnutrition during lactation affects folliculogenesis, gonadotropins, and leptin receptors in adult rats. Nutrition 26 10001007. (https://doi.org/10.1016/j.nut.2009.09.026)

FindlayJKHuttKJHickeyMAndersonRA 2015 How is the number of primordial follicles in the ovarian reserve established? Biology of Reproduction 93 111. (https://doi.org/10.1095/biolreprod.115.133652)

FlawsJAAbbudRMannRJNilsonJHHirshfieldAN 1997 Chronically elevated luteinizing hormone depletes primordial follicles in the mouse ovary. Biology of Reproduction 57 12331237. (https://doi.org/10.1095/biolreprod57.5.1233)

GluckmanPDHansonMACooperCThornburgKL 2008 Effect of in utero and early-life conditions on adult health and disease. New England Journal of Medicine 359 6173. (https://doi.org/10.1056/NEJMra0708473)

GonzalezPNGasperowiczMBarbeito-AndresJKleninNCrossJCHallgrimssonB 2016 Chronic protein restriction in mice impacts placental function and maternal body weight before fetal growth. PLoS ONE 11 e0152227. (https://doi.org/10.1371/journal.pone.0152227)

GuzmanCCabreraRCardenasMLarreaFNathanielszPWZambranoE 2006 Protein restriction during fetal and neonatal development in the rat alters reproductive function and accelerates reproductive ageing in female progeny. Journal of Physiology 572 97108. (https://doi.org/10.1113/jphysiol.2005.103903)

GuzmanCGarcia-BecerraRAguilar-MedinaMAMendezIMerchant-LariosHZambranoE 2014 Maternal protein restriction during pregnancy and/or lactation negatively affects follicular ovarian development and steroidogenesis in the prepubertal rat offspring. Archives of Medical Research 45 294300. (https://doi.org/10.1016/j.arcmed.2014.05.005)

HuttKJMcLaughlinEAHollandMK 2006 KIT/KIT ligand in mammalian oogenesis and folliculogenesis: roles in rabbit and murine ovarian follicle activation and oocyte growth. Biology of Reproduction 75 421433. (https://doi.org/10.1095/biolreprod.106.051516)

JinZLKimNH 2017 RAD51 maintains chromosome integrity and mitochondrial distribution during porcine oocyte maturation in vitro. Journal of Reproduction and Development 63 489496. (https://doi.org/10.1262/jrd.2017-078)

Karbalay-DoustSNoorafshanA 2012 Stereological estimation of ovarian oocyte volume, surface area and number: application on mice treated with nandrolone decanoate. Folia Histochemica et Cytobiologica 50 275279. (https://doi.org/10.5603/FHC.2012.0037)

KereliukSMBrawermanGMDolinskyVW 2017 Maternal macronutrient consumption and the developmental origins of metabolic disease in the offspring. International Journal of Molecular Sciences 18 E1451. (https://doi.org/10.3390/ijms18071451)

KerrJBHuttKJCookMSpeedTPStrasserAFindlayJKScottCL 2012 Cisplatin-induced primordial follicle oocyte killing and loss of fertility are not prevented by imatinib. Nature Medicine 18 11701172. (https://doi.org/10.1038/nm.2889)

KirkegaardKTaylorMPJacksonWT 2004 Cellular autophagy: surrender, avoidance and subversion by microorganisms. Nature Reviews Microbiology 2 301314. (https://doi.org/10.1038/nrmicro865)

LiewSHVaithiyanathanKCookMBouilletPScottCLKerrJBStrasserAFindlayJKHuttKJ 2014 Loss of the proapoptotic BH3-only protein BCL-2 modifying factor prolongs the fertile life span in female mice. Biology of Reproduction 90 77. (https://doi.org/10.1095/biolreprod.113.116947)

LiewSHNguyenQNStrasserAFindlayJKHuttKJ 2017 The ovarian reserve is depleted during puberty in a hormonally driven process dependent on the pro-apoptotic protein BMF. Cell Death and Differentiation 8 e2971. (https://doi.org/10.1038/cddis.2017.361)

MachtingerRGaskinsAJMansurAAdirMRacowskyCBaccarelliAAHauserRChavarroJE 2017 Association between preconception maternal beverage intake and in vitro fertilization outcomes. Fertility and Sterility 108 10261033. (https://doi.org/10.1016/j.fertnstert.2017.09.007)

McGeeEAHsuehAJ 2000 Initial and cyclic recruitment of ovarian follicles. Endocrine Reviews 21 200214. (https://doi.org/10.1210/edrv.21.2.0394)

MitchellMSchulzSLArmstrongDTLaneM 2009 Metabolic and mitochondrial dysfunction in early mouse embryos following maternal dietary protein intervention. Biology of Reproduction 80 622630. (https://doi.org/10.1095/biolreprod.108.072595)

NguyenQNZerafaNLiewSHMorganFHStrasserAScottCLFindlayJKHickeyMHuttKJ 2018 Loss of PUMA protects the ovarian reserve during DNA-damaging chemotherapy and preserves fertility. Cell Death and Differentiation 9 618. (https://doi.org/10.1038/s41419-018-0633-7)

National Health and Medical Research Council 2013 Australian dietary guideliness. Canberra: National Health and Medical Research Council

OrtizREcheverriaOMSalgadoREscobarMLVazquez-NinGH 2006 Fine structural and cytochemical analysis of the processes of cell death of oocytes in atretic follicles in new born and prepubertal rats. Apoptosis 11 2537. (https://doi.org/10.1007/s10495-005-3347-0)

SchubertKAVaanholtLMStavasiusFDemasGEDaanSVisserGH 2008 Female mice respond differently to costly foraging versus food restriction. Journal of Experimental Biology 211 22142223. (https://doi.org/10.1242/jeb.017525)

TillyJL 2003 Ovarian follicle counts – not as simple as 1, 2, 3. Reproductive Biology and Endocrinology 1 11. (https://doi.org/10.1186/1477-7827-1-11)

TingenCMBristol-GouldSKKiesewetterSEWellingtonJTSheaLWoodruffTK 2009 Prepubertal primordial follicle loss in mice is not due to classical apoptotic pathways. Biology of Reproduction 81 1625. (https://doi.org/10.1095/biolreprod.108.074898)

WallaceWHKelseyTW 2010 Human ovarian reserve from conception to the menopause. PLoS ONE 5 e8772. (https://doi.org/10.1371/journal.pone.0008772)

WatkinsAJWilkinsACunninghamCPerryVHSeetMJOsmondCEckertJJTorrensCCagampangFRClealJ et al. 2008 Low protein diet fed exclusively during mouse oocyte maturation leads to behavioural and cardiovascular abnormalities in offspring. Journal of Physiology 586 22312244. (https://doi.org/10.1113/jphysiol.2007.149229)

WatkinsAJLucasESWilkinsACagampangFRFlemingTP 2011 Maternal periconceptional and gestational low protein diet affects mouse offspring growth, cardiovascular and adipose phenotype at 1 year of age. PLoS ONE 6 e28745. (https://doi.org/10.1371/journal.pone.0028745)

WinshipALBakaiMSarmaULiewSHHuttKJ 2018 Dacarbazine depletes the ovarian reserve in mice and depletion is enhanced with age. Scientific Reports 8 6516. (https://doi.org/10.1038/s41598-018-24960-5)

YardeFBroekmansFJvan der Pal-de BruinKMSchonbeckYte VeldeERSteinADLumeyLH 2013 Prenatal famine, birthweight, reproductive performance and age at menopause: the Dutch hunger winter families study. Human Reproduction 28 33283336. (https://doi.org/10.1093/humrep/det331)

Index Card

PubMed

Google Scholar

Related Articles

Altmetrics

Metrics

All Time Past Year Past 30 Days
Abstract Views 66 66 66
Full Text Views 298 298 66
PDF Downloads 83 83 17