Although it is becoming increasingly evident that maternal starvation during pregnancy can have permanent effects on a range of physiological processes in the offspring, scant information is available about the consequence of such condition for oogenesis and hence for lifetime reproductive success of progeny in mammals. In the present study, we address this topic by starving pregnant mice at the time of ovarian differentiation (12.5 days post coitum (dpc)) for three consecutive days and analyzed the consequence ﬁrst on the survival of the fetal oocytes and their capability to progress throughout the stages of meiotic prophase I (MPI) and then on the postnatal folliculogenesis of the offspring. The results showed that maternal starvation increased apoptosis in the fetal ovaries, resulting in reduction of the oocyte number. Moreover, MPI progression was slowed down in the surviving oocytes and the expression of DNA repair players in the starved ovaries increased. Transcriptome analysis identiﬁed 61 differentially expressed genes between control and starved ovaries, the most part of these being involved in metabolic processes. A signiﬁcant decrease in the percentage of oocytes enclosed in primordial follicles and the expression of oocyte genes critically involved in folliculogenesis such as Nobox, Lhx8 and Sohlh2 in the 3 days post partum (dpp) starved ovaries were found. Finally, at the time of juvenile period (21 dpp), the number of oocytes and antral follicles resulted signiﬁcantly lower in the ovaries of the offspring from starved mothers in comparison to controls. Our ﬁndings support the notion that maternal starvation can affect ovary development in the offspring that could adversely affect their reproductive success in the adult life. Introduction is available about the effect of starvation during pregnancy Adequate and correct diet during pregnancy are critical on the offspring reproductive functions in mammals. 1–3 for the health of mother and newborns . As a matter of In humans, three studies examined whether exposure to fact, it had been clearly established that some offspring acute, severe famine in utero during the Dutch famine pathologies (for instance, obesity, diabetes and cardio- 1944–1945 affected a women’s subsequent reproduction. vascular disease) might have their origins in inadequate Lumey and Stein found that exposure to fetal famine was 4,5 nutrition during pregnancy . However, little information sufﬁcient to result in a 300 g decrease in mean birth- weight; however, these individuals did not suffer from adverse effects on their subsequent fecundity in adult- Correspondence: Wei Shen (firstname.lastname@example.org)(email@example.com) hood, but were more likely to give birth to offspring of Institute of Reproductive Sciences, College of Life Sciences, Qingdao reduced birthweight. This reduced birthweight in the Agricultural University, Qingdao 266109, China Center for Reproductive Medicine, Qingdao Women’s and Children’s Hospital, second generation was associated with a high frequency of Qingdao University, Qingdao 266034, China early infant mortality. Painter et al. , who interviewed the Full list of author information is available at the end of the article These authors contributed equally: Jun-Jie Wang, Xiao-Wei Yu. Edited by E. Candi © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Ofﬁcial journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; Wang et al. Cell Death and Disease (2018) 9:452 Page 2 of 13 same sample of women at a mean age of 50 years, but experienced a starvation period of 3 days, and the con- used a different sample of controls, found a small but sequences on the ovary development during the fetal signiﬁcant decrease in the prevalence of nulliparity. More period and early folliculogenesis were investigated. recently, Yarde et al. , in an independent sample of women born after the same famine, reported earlier Results and discussion menopause. Elias et al. found a slight decrease in age of Many animals alter their reproductive strategies in menopause following famine exposure during early response to environmental stress. For example, in female childhood. In a follow-up study of women born in Eng- Drosophila and Caenorhabditis elegans, starvation acti- land in the ﬁrst half of the twentieth century, Cresswell vates apoptotic checkpoints and autophagy in oogenesis 10 11 26,27 et al. and Hardy and Kuh also found that menstruation and reduces the production of mature oocytes . In this ceased at an earlier age in those who had low weight gain regard, we recently found that nutrient deﬁciency at birth during their ﬁrst or the second year of life (but see Treloar could generate a number of adaptive metabolic and oxi- 12 13 et al. , for the opposite result). Steiner et al. reported a dative responses in the ovaries causing increased apop- weak association between birthweight and age at meno- tosis and autophagy in both the somatic cells and oocytes, pause. Some authors observed that low birthweight leading to a delay of germ cell cyst breakdown and follicle 21,28 infants with prematurity or growth retardation tend to assembly . Here we investigated the consequences of 14,15 have fewer offspring , and that retarded fetus growth starvation of pregnant mice for 3 days at the time of the can impair ovarian development, which may have impli- fetal gonad sex differentiation and beginning of meiosis cations for the timing of menopause . Despite such on the ovary development during the fetal period and apparent contradictory results in humans, animal models early folliculogenesis. support the notion that starvation during pregnancy can have adverse effect on the offspring reproductive cap- Mother’s starvation impairs fetal growth ability. A reduction of lifetime reproductive capacity after Starvation for a relative short pregnancy time, from 12.5 prenatal undernutrition has been reported in female to 15.5 dpc, caused a signiﬁcant decrease of the mother’s 17 18 mice and sheep . Food restriction during the second body weight at the end of treatment (Fig. 1a), paralleled to half of pregnancy in rats resulted in premature repro- reduced concentrations of glucose, total cholesterol, ductive senescence in female offspring . In single- progesterone (PROG) and E2 in the blood (Fig. 1b–e). ovulating species, a study found evidence that maternal The greatly reduced level of PROG was likely responsible dietary restriction inﬂuences ovarian reserve in bovine for the slightly higher probability of pregnant female Because female reproductive capacity in rodents is largely abortion at 15.5 dpc (data not shown). Moreover, the body deﬁned by the number and quality of primordial follicles weight of the surviving fetuses was signiﬁcantly lower in developed in the ovary during the neonatal period, termed the starved (0.43 ± 0.008 g) compared to control (0.47 ± the ovarian reserve, it can be hypothesized that reduced 0.007 g) groups (Fig. 1f, g, P < 0.01). Finally, the weight of amount of nutrients during this crucial process can impair placenta also resulted signiﬁcantly lower in the starved the formation of the ovarian reserve with adverse con- (0.12 ± 0.004 g) than in the control (0.14 ± 0.006 g) groups sequence for reproduction. Actually, several studies (Fig. 1h). For non-abortion mice after starvation, however, showed that apoptosis and autophagy are part of the there was no signiﬁcant difference in the number of the starvation cell response, possibly triggered by oxidative delivered pups compared with that of control group stress in damaged cells and that insulin-like growth (11.25 ± 0.63 vs 10.00 ± 0.91) (Fig. 1i). factor-1 signaling plays an important role in such pro- cesses . In this regard, we recently found that starvation Mother’s starvation increases apoptosis in the fetal ovaries at birth impairs germ cell cyst breakdown and increases and causes reduction of the oocyte number 21 21 autophagy and apoptosis in mouse oocytes . Interest- In line with our previous results reported above and ingly, diet can also cause epigenetic changes in gene general observation that starvation, as a result of meta- expression with possible consequence for the correct bolic stress, increases autophagy and/ or apoptosis in 23 29 development of cells and tissues . Epigenetics involves several tissues , we found evidence of increased level of heritable changes in gene expression via post-translational apoptosis in the ovaries of the starved 15.5 dpc fetuses. In and post-transcriptional modiﬁcations without altering fact, the number of TUNEL (terminal deoxynucleotidyl DNA base sequence. For instance, in the mouse, gestational transferase dUTP nick end labeling)-positive cells scored nutritional restriction has been reported to alter the level of per unit area in tissue sections of ovaries of the starved DNA methylation in the sperm of male offspring, resulting fetuses was almost double (15.30 ± 1.39) in comparison to 23–25 in metabolic diseases in the next generations . control (7.73 ± 0.86) (Fig. 2a, b). At the same time, the In the present study, pregnant mice at the time of the Bax/Bcl-2 ratio, evaluated at both the messenger RNA gonad sex differentiation (12.5 days post coitum (dpc)) (mRNA) and protein levels, resulted signiﬁcantly higher Ofﬁcial journal of the Cell Death Differentiation Association Wang et al. Cell Death and Disease (2018) 9:452 Page 3 of 13 B D E Total PROG E2 (g) Control Cholesterol 8 1.5 1.0 1.0 Starvation (n=3) 0.8 0.8 6 * 1.0 0.6 0.6 ** 0.4 0.4 0.5 2 (n=3) (n=6) (n=4) (n=4) 0.2 0.2 ** ** 0 0.0 0.0 0.0 12.5 13.5 14.5 15.5 12.5 13.5 14.5 15.5 Time of pregnancy (days) Time of pregnancy (days) G H I (g) (g) 0.6 0.20 p=0.30 ** 0.15 0.4 10 ** 0.10 0.2 0.05 (n=22) (n=11) (n=4) 0.0 0.00 Fig. 1 Effects of starvation on mothers and fetuses. a Body weight of pregnant females from 12.5 to 15.5 dpc in control and starved groups. b–e Amount of glucose, total cholesterol, PROG and E2 in the blood of 15.5 dpc pregnant females in control and starved groups. f The 15.5 dpc fetuses and placentas. g, h Body and placenta weights of 15.5 dpc fetuses. i Number of fetus of 15.5 dpc pregnant females; n = number of mothers or fetuses (g, h) used for the analyses. Results are presented as mean ± SEM. *P < 0.05; **P < 0.01 apoptosis in the ovaries of the starved fetuses in com- to progress through the meiosis prophase I (MPI) parison to control (Fig. 2c–e). stages . To evaluate if starvation affects these processes, The increased levels of apoptosis markers in the starved we analyzed MPI stages by oocyte cytospreads. The ovaries were associated to a slight but statistically sig- results showed that MPI progression was altered in niﬁcant decreased number of oocytes assessed in tissue starved (oocytes at zygotene 75.92 ± 3.25%; oocytes at sections after staining with the germ cell-speciﬁc anti- pachytene: 5.61 ± 3.41%) compared with control ovaries mouse VASA homolog (MVH) antibody (489.30 ± 10.16 (oocytes at zygotene: 62.21 ± 2.73%; oocytes at pachytene: vs 532.40 ± 9.48, P < 0.05) (Fig. 2f, g). It remained to 26.56 ± 0.37%) (Fig. 3a, b). Moreover, quantitative real- determine if such reduced oocyte number was due to time PCR (qRT-PCR) analyses showed that the transcripts increased apoptotic rate in oocytes or an indirect con- for the germ cell-speciﬁc genes, Mvh and deleted in sequence of increased apoptosis in the surrounding azoospermia-like (Dazl), and of the meiosis-related genes, somatic cells. stimulated by retinoic acid 8 (Stra8), synaptonemal complex protein 1 and 3 (Scp1 and Scp3), were all sig- Mother’s starvation delays meiotic progression in fetal niﬁcantly decreased in the starved ovaries (Fig. 3c). These oocytes and increases the expression of DNA repair results were also conﬁrmed at the protein level for MVH, players in the starved ovaries STRA8 and SCP3 (Fig. 3d, e). Reduction of the oocyte In the fetal ovaries of the CD-1 mice, between 12.5 and number and delay in MPI progression in the starved 14.5 dpc, most part of germ cells enter meiosis and begin ovaries could both explain such decreased gene Ofﬁcial journal of the Cell Death Differentiation Association Body weight of pregnant mice Starvaon Control Body weights of pregnant mice 40 45 50 55 60 65 Blood glucose (mM) 0 2 4 6 8 Body weight of fetuses 0.0 0.2 0.4 0.6 Relave amount 0.0 0.5 1.0 1.5 Placenta weight 0.00 0.10 0.20 0.25 Relave amount 0.0 0.2 0.4 0.6 0.8 1.0 Number of fetuses Relave amount 0 5 10 15 0.0 0.2 0.4 0.6 0.8 1.0 Wang et al. Cell Death and Disease (2018) 9:452 Page 4 of 13 Merge Merge+Hoechst TUNEL MVH 20 μm 20 μm 20 μm 20 μm 20 μm 20 μm 20 μm 20 μm BD C E BAX/BCL2 Bax/Bcl2 20 2.5 ** ** 2.0 ** 1.5 BAX 21 kDa 1.0 BCL2 28 kDa 0.5 (n=3) (n=3) (n=5) ACTIN 42 kDa 0 0.0 0 F G Control Starvaon (n=6) 20 μm 20 μm Fig. 2 Apoptotic markers increase and oocyte numbers decrease in 15.5 dpc ovaries from fetuses of starved mothers. a TUNEL immunohistochemistry (green) in MVH-positive oocytes (red); cell nuclei was stained with Hoechst (blue). b Number of TUNEL-positive cells. c–e WB and qRT-PCR for BAX and BCL2. f, g MVH immunoﬂuorescence (green) and number of oocytes in control and starved ovaries; n = number of ovaries (g) or independent repeats used for the analyses. *P < 0.05; **P < 0.01 expressions. The absence of signiﬁcant difference in the genes involved in DNA repair and homologous recom- percentages of oocytes at different MPI stages in the ovary bination such as Atm (ataxia telangiectasia mutated), of control and treated groups at 18.5 dpc (see below), Mlh1 (mutL homolog 1) and Brca1 (breast cancer type suggested, however, a recovery in meiotic progression 1 susceptibility protein) (Fig. 3c). after the mother resuming the correct diet in the last Double staining of oocyte cytospreads for SCP3 and period of pregnancy. γH2AX (Fig. 4a) showed that while there was no sig- Interestingly, western blot (WB) analyses showed sig- niﬁcant difference in the total percentage of γH2AX- niﬁcant higher levels of γH2AX (a marker of DNA positive oocytes between the control (97.34 ± 0.80%) and double-strand breaks) and RAD51 (involved in DNA the starved (97.11 ± 1.60%) groups (Fig. 4b), in the latter, double-strand breaks repair), in the starved 15.5 dpc at all MPI stages analyzed, the percentages of oocytes ovaries (Fig. 3d, e). Moreover, besides Rad51, we found showing strong γH2AX was much higher at varying that starvation increased the transcript levels of other degrees except diplotene stage (Fig. 4c). Finally, double Ofﬁcial journal of the Cell Death Differentiation Association Control Starvaon TUNEL posive cells Relave protein level 0 0.5 1.0 1.5 2.0 2.5 Number of MVH-posive oocytes per four secons 0 200 400 600 Relave mRNA level 0 1 2 3 Wang et al. Cell Death and Disease (2018) 9:452 Page 5 of 13 Leptotene Zygotene Pachytene Diplotene A B (%) Control (n=3) Starvaon 20 μm 20 μm 20 μm 20 μm 20 μm 20 μm 20 μm 20 μm Dazl Rad51 Mvh Stra8 Scp1 Scp3 Brca1 Mlh1 Atm 100 100 100 100 100 250 150 250 200 ** ** 80 80 80 80 80 200 ** 200 60 60 60 150 60 60 150 ** ** ** 40 40 40 40 40 100 100 ** 50 20 20 20 20 20 50 50 ** 0 0 0 0 0 0 0 0 0 D E MVH 76 kDa MVH STRA8 SCP3 γH2AX RAD51 1.0 1.0 1.0 4 2.0 ** STRA8 0.8 0.8 0.8 45 kDa 3 1.5 0.6 0.6 0.6 ** 2 1.0 SCP3 28-39 kDa ** 0.4 0.4 0.4 1 0.5 0.2 γH2AX 0.2 (n=3) 0.2 17 kDa (n=3) (n=3) (n=4) (n=3) 0.0 0.0 0.0 0 0.0 RAD51 37 kDa ACTIN 42 kDa Fig. 3 Slowed MPI progression and increased DNA repair players in 15.5 dpc ovaries from fetuses of starved mothers. a SCP3 immunoﬂuorescence (red) of oocyte cytospread at various MPI stages; nuclei was counterstained with Hoechst (blue). b Percentages of oocyte at different MPI of 15.5 dpc ovaries. c–e qRT-PCR and WB analyses of the expressions of germ cell-speciﬁc, meiotic- and DNA damage-related genes in control and starved ovaries; n = number of independent repeats used for the analyses. *P < 0.05; **P < 0.01 staining for SCP3 and RAD51 revealed that RAD51- histone structure, DNA methylation, miRNA modulation 31,32 positive rate signiﬁcantly increased (P < 0.01; Fig. 5a, b), and DNA repair . but there were not signiﬁcant differences in the number of positive oocytes between the control and starved groups Transcriptome analysis and comparison identify at each MPI stage (Fig. 5c). differentially expressed genes between control and These last results evidence that defects in MPI likely starved ovaries occur because of alteration of homologous recombination In transcriptome analyses, we found that control and and DNA repair in the oocytes of the starved ovaries. This starved ovaries showed 61 signiﬁcant differentially supports the important notion that nutrients might expressed genes (DEGs) (Fig. S1A-S1C) and that most modulate cellular DNA damage response. The relevance part of these genes were involved in metabolic process of diet and nutrition in human health and disease is well (Fig. S1D). established. Actually, basic laboratory researches, clinical For DEGs that resulted more constantly affected by trials and epidemiological studies demonstrated that maternal starvation, a heatmap was generated (Fig. 6a). nutrient-rich bioactive foods can induce epigenetic qRT-PCR was then performed for 10 of these genes changes and alter gene expression by the alteration of the potentially relevant for reproductive processes (Fig. 6b). Ofﬁcial journal of the Cell Death Differentiation Association (n=3) Merge SYCP3 Relave of mRNA level (%) 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 Relave of protein level 0 20 40 60 80 100 0 0.2 0.4 0.6 0.8 1.0 0 20 40 60 80 100 0 0.2 0.4 0.6 0.8 1.0 0 50 100 150 200 250 0 0.2 0.4 0.6 0.8 1.0 Percentages of meioc stages 0 20 40 60 80 100 0 50 100 150 0 1 2 3 4 0 50 100 150 200 250 0 0.5 1.0 1.5 2.0 0 50 100 150 200 Wang et al. Cell Death and Disease (2018) 9:452 Page 6 of 13 Fig. 4 γH2AX staining in oocytes at various MPI stages. a Representative SCP3 (red) and γH2AX (green) staining of oocyte cytospread at various MPI stages. b, c Quantiﬁcation of γH2AX-positive oocytes in the ovaries of fetuses from control and starved mothers; n = number of independent repeats or counting oocytes (c) used for the analyses. *P < 0.05; **P < 0.01 The transcripts of most of these genes, such those for Mother’s starvation causes reduction in the number of the enolase 1 (Eno1) , macrophage migration inhibitory fac- primordial follicles and impairs follicle development in the 34 35 tor (Mif) , pyruvate dehydrogenase kinase 1 (Pdk1) , offspring ovaries lactate dehydrogenase A (Ldha) , melatonin receptors To investigate whether the defects in the oogenic 37 38 Mt1 and Mt2 , reproductive homeobox 8 (Rhox8) and process found in the starved 15.5 dpc fetal ovaries phosphoglycerate kinase 1 (Pgk1) , were signiﬁcantly impaired the ovary development at later stages, increased. Conversely, mRNA amount of doublecortin- we examined MPI in oocytes obtained from 18.5 like kinase 1 (Dclk1) was decreased while those of vas- dpc starved ovaries, and folliculogenesis in 3 and 21 days cular endothelial growth factor A (Vegfa) was not post partum (dpp) ovaries of offspring of starved affected. mothers. Ofﬁcial journal of the Cell Death Differentiation Association Wang et al. Cell Death and Disease (2018) 9:452 Page 7 of 13 AB Ne Ne ga gve a ve Merge SCP3 RAD51 (%) Posi ve Posi ve 20 μm ** (n=3) 20 μm Normal Positive None Posi ve (%) n=153 n=316 20 μm (%) n=525 n=596 20 μm 20 μm (%) n=803 n=297 20 μm n=428 n=493 (%) 20 μm 20 μm Fig. 5 RAD51 staining in oocytes at various MPI stages. a Representative SCP3 (red) and RAD51 (green) staining of oocyte cytospread at various MPI stages. b, c Quantiﬁcation of RAD51-positive oocytes in the ovaries of fetuses from control and starved mothers; n = number of independent repeats or counting oocytes (c) used for the analyses. **P < 0.01 While, as reported above, at 18.5 dpc, no signiﬁcant dpp, we scored a slight decrease in the oocyte number difference in the oocyte MPI stages between control and (Fig. 7c, d), but above all a marked lower percentage of starved ovaries was found (Fig. 7a, b), in the latter, at 3 oocytes enclosed in primordial follicles in comparison to Ofﬁcial journal of the Cell Death Differentiation Association Pachytene Zygotene Leptotene Diplotene None None None Posi ve Posi ve Posi ve None Posi ve Zygotene Leptotene Diplotene Pachytene Leptotene Diplotene zygotene Pachytene Percentage of oocytes percentage of oocytes Percentage of oocytes percentage of oocytes percentage of oocytes Percentage of oocytes percentage of oocytes Percentage of oocytes Percentage of oocytes 0 2040 6080 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 Wang et al. Cell Death and Disease (2018) 9:452 Page 8 of 13 A B Eno1 Mif Pdk1 Vegfa Ldha 5 3 8 2.0 3 ** 6 1.5 2 2 ** 4 1.0 2 0.5 (n=4) (n=4) (n=3) (n=3) (n=5) 0 0 0 0.0 0 Mt1 Mt2 Rhox8 Pgk1 Dclk1 4 4 4 1.2 3 3 * 4 0.8 2 2 2 2 0.4 1 1 1 (n=4) (n=5) (n=3) (n=3) (n=3) 0 0 0 0 0.0 Fig. 6 Transcriptome analysis of 15.5 dpc ovaries from fetuses of control and starved mothers. a Heatmap of differentially expressed genes (DEGs) constantly affected by maternal starvation. b qRT-PCR analyses of 10 genes showed in the heatmap in 15.5 dpc control and starved ovaries; n = number of independent repeats used for the analyses. *P < 0.05; **P < 0.01 the control (24.31 ± 1.15% vs 44.65 ± 1.01%, P < 0.01; fetuses can disturb oogenic processes with adverse con- Fig. 7e). The parallel decreased amount of mRNA of sequence for folliculogenesis. oocyte genes involved in the germ cell cyst breakdown and primordial follicle formation such as Nobox (newborn Materials and methods ovary homeobox), Sohlh2 (spermatogenesis and oogenesis Animal breeding and treatment speciﬁc basic helix-loop-helix2) (Fig. 7f) and Lhx8 (LIM All animals used in this study were CD-1 mice, which homeobox 8, protein level in Fig. 7g) in the 3 dpp starved were kept in self-help feeding way with light and dark ovaries offered a likely explanation of this defect (P < 0.05 cycle of half day and raised according to the national or P < 0.01). guideline and Ethical Committee of Qingdao Agricultural Finally, at 21 dpp, the number of follicles in tissue University. Mating was arranged regularly at 4:30–5:30 p. sections resulted signiﬁcantly lower in starved ovaries in m., vaginal plug was checked at 8:30–9:30 a.m. on next comparison to control (440.5 ± 25.94 vs 544.6 ± 17.67) (P morning; the presence of vaginal plug was considered as < 0.05; Fig. 8a, b), while the number of antral follicles 0.5 dpc. showed a signiﬁcant decrease of almost one half in the Due to the sensibility to food deprivation between 12.5 ovaries of the offspring from starved mothers (22.67 ± and 15.5 dpc, which is crucial for the ovarian sex differ- 1.67 vs 42.00 ± 1.53) (P < 0.01; Fig. 8c). Although these last entiation and meiosis initiation, starvation began at observations are predictive of reduced fertility perfor- 11:00–12:00 a.m. at 12.5 dpc and ceased at the same time mance, at the moment no detailed information is available at 15.5 dpc. During this process of starvation, the water about such possible defect. was provided as usual and food supply was cut down, and On the other hand, the litter size and birthweight of F2 after that the diet of starved females came back to normal. offspring produced from F1 females scarcely declined The total number of starved mothers was 120. These (Fig. 9a–c), while there was an apparent imbalance in the mothers, unless otherwise indicated, were killed to sex ratio at birth (P < 0.05; Fig. 9d). Moreover, the per- remove the fetuses. centages of oocyte enclosed in cysts and follicles sug- gested a small depletion in the formation of primordial Detection of blood sugar, total cholesterol and sex follicles at 3 dpp (P < 0.05; Fig. 9e, f), whereas the total hormone levels number of follicles and progression of follicular growth Biochemical indicators designed to validate the effect of between starved and control groups had no signiﬁcant starvation on the pregnant mothers were: blood sugar, defects at 21 dpp (Fig. 9g–i). total cholesterol, PROG and 17β-estradiol (E2) levels. The In conclusion, our study represents a ﬁrst indication blood sugar concentration was determined by collecting that even a relatively short period of mother starvation blood samples with the ACCU-CHEK Active of Roche during critical stages of the ovary development in the (Roche, Germany) and corresponding test strips. The Ofﬁcial journal of the Cell Death Differentiation Association Relave mRNA level Relave mRNA level 0 2 4 6 0 1 2 3 4 5 0 1 2 3 0 4 8 12 15 20 0 0.5 1.0 1.5 2.0 0 2 4 6 8 0 0.5 1.0 1.5 2.0 0 1 2 3 4 0 1 2 3 0 0.4 0.8 1.2 Wang et al. Cell Death and Disease (2018) 9:452 Page 9 of 13 A Leptotene Zygotene Pachytene Diplotene (%) (n=3) Legend Legend 20 μm 20 μm 20 μm 20 μm D F (%) (%) Nobox Lhx8 (%) Sohlh2 Control 2000 100 100 100 p=0.26 80 80 80 ** 60 60 60 Cyst 1000 40 40 40 ** 20 20 Follicle (n=3) (n=3) (n=3) (n=3) 0 0 0 0 100 μm Starva on (%) Cyst LHX8 (%) ** 41 kDa Follicle LHX8 ACTIN 42 kDa ** 100 μm (n=3) (n=4) Fig. 7 MPI oocyte stages and folliculogenesis in the ovaries of 18.5 dpc fetuses and 3 dpp pups from control and starved mothers. a Representative oocyte cytospreads stained for SCP3 (green); nuclei counterstained with Hoechst (blue). b Percentages of oocyte in MPI stages. c Sections of 3 dpp ovaries showing MVH-positive oocytes (green) in cysts (white arrows) or in primordial follicles (white arrowheads). d, e Number of oocytes and percentages of oocyte in cysts or in primordial follicles in 3 dpp ovaries. f, g qRT-PCR and WB of oocyte-speciﬁc genes involved in folliculogenesis; n = number of ovaries (d, e) or independent repeats used for the analyses. *P < 0.05; **P < 0.01 levels of total cholesterol (Comin Biotechnology, Suzhou, (TAKARA, RR820A, Japan) and Light Cycler real-time China), PROG (LanpaiBIO, shanghai, China) and E2 PCR instrument of Roche 480 (Roche, Germany). Each (Jinma Biotechnology, Shanghai, China) were tested fol- sample extracted from 3 to 6 ovaries was ampliﬁed in lowing the manufacturer’s instructions. triplicate to normalize the system and pipetting error, and the relative mRNA expression levels of all genes were −ΔΔCt Quantitative real-time PCR calculated by the formula of 2 and normalized with The complementary DNA (cDNA) prepared for PCR β-actin. was synthesized by TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, AT311-03, Analysis of meiotic stages Beijing, China) from total extracted RNA using RNAprep The meiotic stages of fetal oocytes were analyzed using 42,43 pure Micro Kit (Aidlab, RN07, Beijing, China). All primers cytospreads as previously described . Fixed samples used for analysis were listed in Table S1. PCR ampliﬁca- were blocked with antibody dilution buffer (ADB) at 37 °C tion was conducted with SYBR Premix Ex Taq™ II for 30 min and incubated with primary antibodies Ofﬁcial journal of the Cell Death Differentiation Association Merge SYCP3 Oocyte percent Number of oocyte per ﬁve sec ons 0 20 40 60 80 100 0 500 1000 1500 2000 Percentage of MPI stages Rela ve mRNA level 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 Rela ve protein level 0 20 40 60 80 100 0 20 40 60 80 100 Wang et al. Cell Death and Disease (2018) 9:452 Page 10 of 13 Control Primordial Follicle Primary Follicle 200 μm 50 μm 50 μm Starvaon Secondary Follicle Antral Follicle 200 μm 50 μm 100 μm B C (n=3) (n=3) Control Control Starvation Starvaon * 300 ** 0 0 Fig. 8 Analysis of folliculogenesis in 21 dpp ovaries of offspring of starved pregnant mothers. a Representative immunohistochemistry of MVH-positive oocytes (dark brown) in 21 dpp ovaries. b, c Quantiﬁcation of the number of oocytes and different classes of follicles in 21 dpp ovaries of offspring of control and starved mothers; n = number of ovaries used for the analyses. *P < 0.05; **P < 0.01 (Table S2) at 37 °C for 8 h in a 1:200 dilution. After three on deparafﬁnated sections after antigen retrieval at 96 ° washes with Tris-buffered saline (TBS), they were blocked C. Sections were blocked and incubated with anti-MVH at 4 °C overnight in ADB. Finally, the cells were incubated protein antibody as reported above for primary anti- in dark with secondary antibodies of CY3 (anti-rabbit, bodies, then FITC (CY3 in TUNEL assay)-labeled goat Beyotime, A0562, Nantong, China; anti-mouse, Beyotime, anti-rabbit IgG and nucleic acid was stained with pro- A0568) or ﬂuorescein isothiocyanate (FITC)-labeled goat pidium iodide (Solaibio, P8080-10, Beijing, China) or IgG (anti-rabbit, Beyotime, A0516; anti-mouse, Beyotime, Hoechst 33342 (in TUNEL assay). For immunohis- A0521) at 37 °C for 1.5 h and stained with Hoechst 33342 tochemistry with enzyme substrates, the samples must (Beyotime, C1022) for 5 min. Slides were sealed and be treated with 3% H O before primary antibody 2 2 prepared for ﬂuorescence microscope observation incubation, and then the secondary antibody was horse (Olympus, BX51, Japan). The number of ovaries of each radish peroxidase (HRP)-conjugated goat anti-rabbit sample was 3 to 4 and every independent experiment for IgG (Beyotime, A0258); the DAB kit was purchased oocyte counting was at least 300. from ZSGB-BIO company (Beijing, China). Considering that the oocyte number ﬂuctuated largely in ovaries of Immunohistochemistry and TUNEL assay different size, the oocyte of each experimental ovary was Ovaries were ﬁxed in 4% paraformaldehyde (Solaibio, counted in every ﬁve sections and at total three sections P1110, Beijing, China) and processed according to from different fetuses or six for pups. Then, Image J standard methods for parafﬁn samples. Sections were software was used to calculate areas of these ovary taken every 5 µm and immunohistochemistry performed sections, and accordingly the oocyte counting was Ofﬁcial journal of the Cell Death Differentiation Association Number of follicle per ﬁve secons 0 200 400 600 800 Number of follicle per ﬁve secons 0 100 200 300 400 500 Wang et al. Cell Death and Disease (2018) 9:452 Page 11 of 13 (g) 15 p=0.33 n=62 n=23 2.2 Control Starvaon 2.0 1.8 1.6 1.4 (n=3) 1.2 1.0 D E F MVH MVH+PI Male Cyst Control Control (%) (%) Female Follicle 100 100 Follicle 80 80 60 100 μm 60 Cyst * 40 40 Starvaon Starvaon 20 Follicle (n=3) (n=3) Cyst 100 μm Control Starvaon (n=3) (n=3) Control 800 Control Starvation Starvaon 200μm 200μm Fig. 9 Analysis of folliculogenesis F2 offspring of starved pregnant mothers. a Photograph of F2 offspring of control and starved mothers. b Litter size of F1 mothers. c Body weight of F2 fetus at 0 dpp. d Sex ratio of F2 offspring. e, f MVH-positive oocytes (green) in cysts (white arrows) or in primordial follicles (white arrows) in 3 dpp ovaries. g–i Quantiﬁcation of the number of follicles and percentages of different classes of follicles in 21 dpp ovaries from control and F2 offspring of starved ovaries; n = number of ovaries or fetuses (c) or independent repeats (b, d) used for the analyses. *P < 0.05 adjusted to the same level on the basis of ovarian section follicle was a kind of follicle with a ﬂuid-ﬁlled cavity adja- areas. cent to the oocyte : the total number of each follicle class For the estimate of the number of the different classes of was counted in every ﬁve sections for a total of 8 or follicles in postnatal ovaries, we deﬁned the primordial and 10 sections for each of three ovaries from different mice. primary follicle of a follicle containing an intact MVH- The TUNEL assay was performed on parafﬁn ovary positive oocyte surrounded by a single layer of ﬂat or sections as before , using the BrightGreen Apoptosis cuboidal granulosa cells, the secondary and mature follicles Detection Kit (Vazyme, A112-02, Nanjing, China) were two or multiple layers of granulosa cells, and the antral according to the manufactures’ instructions. Ofﬁcial journal of the Cell Death Differentiation Association Sex ratio of newborn mice in F2 0 20 40 60 80 100 Number of follicle per ﬁve secons 0 200 400 600 800 Number of fetuses 0 5 10 15 Number of follicle per ﬁve secons 0 100 200 300 400 500 Body weights of F2 fetus 1.0 1.2 1.4 1.6 1.8 2.0 2.2 Follicle percentage of 3 dpp-mice 0 20 40 60 80 100 Wang et al. Cell Death and Disease (2018) 9:452 Page 12 of 13 Western blot Authors' contributions J.-J.W., X.-W.Y, R.-Y.W., X.-F.S., S.-F.C., W.G., J.-C.L., Y.-P.L., J.L. and S.-H..Z. For each sample, proteins were extracted from 3 to 4 conducted the experiments; J.-J.W and X.-W.Y. analyzed the data; J.-J.W., W.S. ovaries using the RIPA lysis solution (Beyotime, P0013C) and M.D.F. wrote the manuscript; W.S. and M.D.F. designed the experiments. for sodium dodecyl sulfate–polyacrylamide gel electro- All authors reviewed the manuscript. phoresis. They were then transferred onto a poly- SQ Conﬂict of interest vinylidene ﬂuoride membrane (immobilon-P transfer The authors declare that they have no conﬂict of interest. membranes, Millipore, ISEQ00010, USA). After blocking in TBST buffer (TBS with Tween-20) containing 5% Publisher's note bovine serum albumin, the membrane was incubated in Springer Nature remains neutral with regard to jurisdictional claims in primary antibody (Table S2) overnight at 4 °C, washed and published maps and institutional afﬁliations. incubated with HRP-conjugated goat anti-rabbit (Beyo- Supplementary Information accompanies this paper at https://doi.org/ time, A0208) or anti-mouse IgG (Beyotime, A0216) 10.1038/s41419-018-0492-2. diluted in TBST at room temperature for 1.5 h. Ulti- mately, the BeyoECL Plus Kit (Beyotime, A0018) was used Received: 22 November 2017 Revised: 10 March 2018 Accepted: 15 March for chemiluminescence; β-ACTIN was used as house- keeping protein as previous reported . 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