The effect of maternal body condition on in vivo production of zygotes and behavior of delivered offspring in mice

Theriogenology 83 (2015) 577–589

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The effect of maternal body condition on in vivo production of zygotes and behavior of delivered offspring in mice


s a, Ján Burku
s a, Du
san Fabian a, *, Janka Kubandová a, Stefan Ciko b b
a Czikková a, Juraj Koppel a ceková , Son Kamila Fabianová , Enikö Ra
a b

Institute of Animal Physiology, Slovak Academy of Sciences, Ko
sice, Slovakia Institute of Neurobiology, Slovak Academy of Sciences, Ko
sice, Slovakia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 August 2014 Received in revised form 21 October 2014 Accepted 25 October 2014

This study investigated the effects of maternal body condition on oocyte quality and zygote production. Additionally, we examined the possible consequences on somatic parameters and behavior of naturally delivered offspring. We used an experimental model based on overfeeding of outbred mice during intrauterine and early postnatal development to produce the following four types of females: physiological (7%–8%), slightly increased (8%–11%), highly increased (>11%), and low (<7%) body fat content (Echo Magnetic Resonance Imaging). The fertilized females with slightly increased body fat showed increased numbers of spontaneously ovulated oocytes and an increased fertilization index compared with control animals. On the contrary, mice with slightly and highly increased body fat showed increased numbers of isolated immature oocytes and degenerates. Furthermore, animals with increased body fat had significantly decreased deposits of neutral lipids in the cytoplasm of mature oocytes (Nile red staining) and showed lower reduction in DNA cytosine methylation signal in parental pronuclei (5-methylcytosine immunohistochemistry). The highly increased amount of body fat in mothers was accompanied with lower weights in newborn pups and 5-week-old offspring. We also observed several deviations from normal behavior (open-field test and forced swimming test). The females with low body fat displayed a lower fertilization index, a lower percentage of zygotes at pronuclear stage 4 with demethylated DNA cytosine in parental pronuclei, and lower newborn weights. Although delivered offspring were able to gain normal weight by the fifth week of life, there were several deviations from normal behavior observed. Our results show that periconceptional status of maternal body condition adversely affects the quality of oocytes and might be correlated with significant changes during postnatal offspring development. The data documenting later onset of DNA demethylation in zygotes and decreased amounts of neutral lipids in oocytes suggest that the observed alterations in offspring might originate in modifications established at the earliest stages of conceptus development. Ó 2015 Elsevier Inc. All rights reserved.

Keywords: Oocyte Zygote Fat deposit DNA methylation Offspring behavior

1. Introduction It is known that an adequate body fat mass is important for the onset of reproductive function, particularly in

* Corresponding author. Tel.: þ421 55 727 6274; fax: þ421 55 728 7842. E-mail address: [email protected] (D. Fabian). 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.10.025

females. Furthermore, physical status is one of the major factors determining reproductive success in productive animals [1]. Numerous human clinical studies have reported the following findings in obese women: frequent cycle cancellations, increased gonadotropin requirement during ovarian stimulation, fewer collected oocytes, reduced oocyte quality, lower pregnancy rate after IVF, reduced function of the corpus

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luteum, and increased risk of miscarriage. However, other studies did not find any disorders in ovarian stimulation or any differences in either the oocyte fertilization rate or the numbers of successful pregnancies when comparing obese and nonobese patients (reviewed in [2,3]). Experimental studies on rodents have proven defective ovarian function, increased follicular apoptosis, poor oocyte quality, decreased oocyte size, oocyte meiotic aneuploidy, impaired oocyte mitochondrial function, and significant delays after in vitro cleavage in animals with diet-induced obesity [4–8]. Furthermore, during later development, decreased expression of embryonic Insulin-like growth factor I receptor, increased glucose consumption, and growth retardation of fetuses and pups have been documented in mouse dams fed a high-fat diet [5–7]. The effect of maternal leanness or undernutrition on the in vivo production of oocytes has not been studied so profoundly. However, there is a negative effect on the postpartum fertilization rate in cows with excess body fat. Additionally, impaired oocyte competence and oocyte quality have also been documented [1]. However, in most epidemiologic studies and animal models, it is difficult to determine which outcomes are because of factors associated with maternal metabolic profile and which are because of the consumption of a specific diet. A two-generation model based on overnutrition of experimental animals during intrauterine and early postnatal development to produce adult female mice with four different body types has been developed in our laboratory. The body types include normal controls with physiological body weight and amount of body fat, mice with slightly increased body fat, mice with highly increased body fat and weight (obesity-like phenotype), and lean mice with decreased body fat and weight [9,10]. Using this model, we demonstrated the effect of altered maternal body condition on the development of in vivo–derived preimplantation embryos. The embryos isolated from dams with highly increased and highly decreased maternal body fat had slowed development and an increased incidence of apoptosis. Consistent with previous reports, our model possesses several important advantages. Specifically, our model simulates population heterogeneity in a manner analogous to naturally reproducing mammalian subjects (it uses outbred mice). Our model also allows for the study of maternal body condition and at the same time minimizing the impact of the composition of actual nutrition (during reproductive process, all animals are fed standard diet only), the impact of maternal aging (all females are at the early adulthood at the age standard used for reproductive studies), and the impact of hormonal treatment (only spontaneously ovulating donors of oocytes and pups are used) [10]. The aim of present study was to evaluate the relationship between maternal periconceptional body condition and in vivo production of oocytes in mice using a standardized experimental model. The model simulates natural variability in monogastric mammals including the human population and minimizes confounding adverse effects that may mask changes induced by maternal condition. In addition to assessing basic reproductive parameters (ovulation rate and fertilization index), we evaluated specific and novel features of oocyte quality, including the accumulation of lipid deposits in oocytes and the process of active DNA

demethylation of the zygotic genome. Furthermore, we investigated the possible consequences of the observed changes on the somatic development and behavior of offspring naturally delivered from dams grouped according to body fat percentage. 2. Materials and methods 2.1. Animals and experimental design All experiments were performed on mice of the outbred ICR (CD-1 IGS) strain (Velaz, Prague, Czech Republic) and the entire experimental design was repeated four times. A two-generation dietary model based on overfeeding of experimental mice during prenatal and early postnatal development was used to produce females with various body conditions (Fig.1) [10]. Adult female mice (30–35 day’s old) of the parental generation (P) underwent hormonal synchronization (pregnant female’s serum gonadotropin [eCG 5 IU intraperitoneally, Folligon; Intervet International, Boxmeer, Holland], followed 47 hours later by the administration of hCG [4 IU intraperitoneally, Pregnyl; Organon, Oss, Holland]). These females were mated with males of the same strain overnight. The fertilized mice were randomly divided into the control and experimental groups and were individually housed in plexiglass cages under standard conditions (temperature 22  2  C, humidity 55  5%, 12:12-hour light-dark cycle with lights on at 6:00 AM with free access to food and water). During the gestation period (21 days) and the lactation period (from birth to weaning: 21 days) dams in the control group (C) were fed standard pellet diet (M1, 3.2 cal/g; Ricmanice, Czech Republic). The dams in the experimental group were fed standard diet M1 with the addition of highenergy liquid product Ensure Plus (1.5 cal/mL; Abbott Laboratories, Hoofddorp, The Netherlands [11]) ad libitum. To

Fig. 1. Experimental design. Diagram shows dietary regime of three generations of mice used in the study (P, parental [\]; F1, the first filial [\]; F2, the second filial generation [\ þ _)]). M1, standard diet; ENSURE, diet supplement with Ensure PLUS; C, control mice; EX, experimental mice; CN, dams with the physiological amount of body fat; CL, lean control females with decreased body fat; EXN, experimental mice with slightly increased body fat; EXF, experimental mice with highly increased body fat.

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assign normal nutrition in control pups and further overnutrition in experimental pups, the litter size was adjusted on the eighth day after birth up to 10 pups per nest (usually to eight). After weaning, mice of the F1 generation in both groups were fed standard diet only. Then, on Day 34, the delivered females were individually measured for their weight and scanned by Echo Magnetic Resonance Imaging (Whole Body Composition Analyzer; Echo Medical System, Houston, TX, USA) to evaluate body fat deposits (in grams). The mice were then allocated into the following four groups according to the percentage of their body fat: CN (n ¼ 57), normal controls with physiological amounts of body fat (7%– 8%) and physiological body weight; EXN (n ¼ 55), normal experimental mice with slightly increased body fat (8%–11%,) and physiological body weight; EXF (n ¼ 54), fat experimental mice with highly increased body fat (>11%) and elevated body weight; and CL (n ¼ 60), lean controls with decreased body fat (<7%) and body weight. The percentage of body fat was calculated as body fat (g)/body weight (g)  100. Adult F1 females displaying the four different types of body condition were used in the reproductive study. Starting on Day 35, the spontaneously ovulating female mice were mated with males of the same strain during one or more nights. Three days before the first overnight mating, estrus was synchronized in each group by exposing the females to bedding contaminated with male urine and preputial gland secretions [12]. A successful mating was confirmed by the presence of a vaginal plug, on examination every morning at 7:30 AM, and this was designated Day 1 of pregnancy. On Day 1 of pregnancy, approximately 3 of 4 fertilized dams from all groups were sacrificed by cervical dislocation or decapitation and subjected to oocyte and zygote isolation (CL [n ¼ 35], CN [n ¼ 34], EXN [n ¼ 35], and EXF [n ¼ 37]). Blood samples were collected from randomly selected decapitated dams. The blood was centrifuged and used to measure leptin and adiponectin content. The collected serum was transferred to clean vials and stored at 80  C until examination. The hormone concentrations were quantified using commercial ELISA kits (mouse leptin 96-well plate assay, mouse adiponectin 96-well plate assay; Millipore, Billerica, MA, USA), according to the manufacturer’s instructions. The remaining mated mice were allowed to deliver and suckle their pups when on standard diets (CL [n ¼ 8], CN [n ¼ 7], EXN [n ¼ 7], and EXF [n ¼ 7]). The offspring (F2 generation) were weaned, placed on standard diet, and at fifth week of life evaluated for basic somatic and behavioral parameters. All animal experiments were reviewed and approved by Ethical Committee for animal experimentation of the Institute of Animal Physiology and were approved by State Veterinary and Food Administration of the Slovak Republic (Ro 1261/10–221c). All studies were performed in accordance with Slovak legislation based on European Union Directive 2010/63/European Union on the protection of animals used for experimental and other scientific purposes. 2.2. Evaluation of in vivo–produced eggs Oocytes and zygotes were recovered by flushing the oviduct of F1 dams using a flushing-holding medium

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containing 1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) at two time points: 1.8:00 hours (0.5 hours after plug identification, which was approximately 6 hours after supposed spontaneous ovulation) and 2.13:00 hours (5.5 hours after plug identification, approximately 11 hours after supposed spontaneous ovulation). The two time points were chosen to minimize the adverse effects of physiological asynchrony in fertilization times in naturally mated females. Immediately after isolation, the collected oocytes and zygotes were subjected to preliminary stereomicroscopic classification (Nikon SMZ 745T; Nikon, Tokyo, Japan), and the number of isolates per dam was determined. Zygotes were selected for the pronuclear (PN) genome demethylation assay (5-methylcytosine immunodetection) and oocytes were subjected to lipid accumulation assays (Nile red fluorescence staining). The exact classification of all isolates was performed after DNA staining in both assays. The isolates with one polar body and an acentric mitotic spindle (metaphase chromosomes) were classified as unfertilized MII oocytes. The isolates with one or two polar bodies displaying two pronuclei or equatorially localized mitotic spindles were classified as zygotes. The developmental advance of zygotes (PN stage 1–5 [PN1– PN5 stage]) was evaluated according to the distance and the size of maternal and paternal pronuclei [13]. All onecell isolates showing nontypical morphology (absent polar body, highly translucent cytoplasm with nonhomogenous structure, cytoplasmic autolysis, cytoplasmic fragmentation, etc.) were classified as immature or degenerated oocytes. To ensure that the compared isolates were at the same developmental stage, only unfertilized MII oocytes were evaluated in the lipid accumulation assay and only zygotes at PN1 to PN5 stage were evaluated during the immunodetection of 5-methylcytosine in paternal pronuclei. 2.2.1. Lipid accumulation quantification We used Nile red staining and then assessed the digital images obtained by fluorescence microscopy to quantify the accumulation of neutral lipids (cholesterol, lipoproteins, and triglycerides) in intracellular fat vacuoles [14–16]. The oocytes were fixed in 4% paraformaldehyde, washed (PBS with BSA), and stained with Hoechst 33342 dye (10 mg/mL in PBS; Sigma-Aldrich). The oocytes were then placed overnight into Nile red dye (10 mg/mL; Life Technologies, Carlsbad, CA, USA). The oocytes were then rinsed and mounted on a slide with Vectashield (Vector laboratories, Burlingame, CA, USA). An epifluorescence microscope (Nikon Eclipse 80i; Nikon, Tokyo, Japan) was used to excite fluorescent stains (Nile red: excitation 485 nm/emission 525 nm; Hoechst 33342: excitation 350 nm/emission 461 nm) and to capture digital photographs of the equatorial part of the oocyte using a 20 objective. An identical exposure time (200 ms) was used for all Nile red–stained objects. During the evaluation of microphotographs (Fig. 2), the fluorescence intensity from 0 to 255 shades for each pixel (0, no fluorescence; 255, maximum fluorescence) was

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Fig. 2. Lipid accumulation in oocytes. Digital images were obtained using fluorescence microscopy and subjected to assessment of integrated density of Nile red fluorescence signal. Original magnification: 200. Oocytes were isolated from (A) lean control females with decreased body fat, (B) control dams with the physiological amount of body fat, (C) experimental mice with slightly increased body fat, and (D) experimental mice with highly increased body fat.

measured with Ellipse software (ViDiTo, Ko
sice, Slovakia). The fluorescence units were regularly adjusted by considering background in which no embryo was present as zero to compensate for possible variation of the fluorescence lamp intensity. Finally, the integrated density of the Nile red fluorescence signal for each unfertilized MII oocyte was derived from the software analysis product of mean gray level (the mean value of fluorescence units of all the pixels in the selection) in the selected area (the number of pixels in inside of closed contour object). 2.2.2. 5-Methylcytosine Immunodetection Nuclear DNA methylation was visualized using indirect immunofluorescence with an antibody to 5-methylcytosine. The zygotes were fixed in 4% paraformaldehyde and permeabilized in 0.3% Triton-X (Sigma-Aldrich). After extensive washing with 0.05% Tween 20, the zygotes were subjected to 2 M of hydrochloric acid for 30 minutes at 37  C to denature the DNA. The acid was then neutralized with 100 mM Tris/HCl buffer (pH 8.5) (Sigma-Aldrich). The zygotes were blocked overnight at 4  C in 2% BSA (Roche) and the methylated DNA was incubated with a mouse monoclonal antibody against 5-methylcytosine (Calbiochem, La

Jolla, CA, USA; 2.5 mg/mL in the blocking solution for 1 hour at 37  C). The zygotes were then incubated in goat antimouse immunoglobulin G conjugated to Texas Red (Jackson ImmunoResearch; 3.75 mg/mL for 1 hour at room temperature). The zygotes were stained with 100 nM YOYO-1 iodide (Invitrogen), mounted on slides, and observed using a fluorescence microscope (Nikon Eclipse 80i; Nikon). The same exposure parameters were used for each image recording. The fluorescence intensities of 5-methylcytosine signal in both maternal and paternal pronuclei were measured using Ellipse software (Fig. 3). In each zygote, the value of the mean gray in the methylated maternal pronucleus was determined as 100% signal. A paternal pronucleus displaying staining throughout the whole nuclear area with a mean gray level of 95% to 100% of the signal measured in the maternal pronucleus was classified as methylated. A paternal pronucleus showing a reduction in the staining signal (<95%) was classified as demethylated (i.e., undergoing active demethylation process). The exact level (%) of the DNA demethylation in the male pronucleus was derived from the paternal PN to maternal PN signal ratio.

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Fig. 3. Demethylation of DNA in zygotes. Illustrative images were obtained using fluorescence microscopy (original magnification: 200). The presence of DNA methylation was visualized by 5-methylcytosine immunostaining (A1, B–F) and PN morphology was visualized by YOYO-1 iodide chromatin staining (A2). (A1, A2) Zygote (merge of two optical sections). The smaller maternal pronucleus with methylated DNA is closer to the polar body (pb). (B, C, D1) Zygotes at PN1, PN2, and PN3 stages with methylated DNA in both pronuclei. (D2) Zygote at PN3 stage with decreased signal of 5-methylcytosine in paternal pronucleus. (E1, E2, F) Zygotes at PN4 and PN5 stages with various degrees of DNA demethylation in paternal pronucleus. PN, pronuclear.

2.3. Evaluation of in vivo–derived offspring The following procedures were performed on offspring (F2 generation) delivered from dams with the four different types of body condition (CL, CN, EXN, and EXF). The number of pups and their natal body weight were assessed on the day of birth. At Day 13 after birth, we assessed the sex of pups. The sex ratio was calculated as follows: the number of male or female pups/total number of pups in one group  100. At Day 32 after birth, the offspring were again individually weighed and scanned using Echo Magnetic Resonance Imaging to determine the

exact amount of body fat deposits. The percentage of body fat was calculated as body fat (g)/body weight (g)  100. To investigate the possible changes in animal behavior between Days 32 and 36, we selected 60 random offspring (30 males þ 30 females) from each group (CL, CN, EXN, and EXF). The animals were then subjected to the forced swimming test (FST) and the open-field test. 2.3.1. Forced swimming test On the first day of testing, mice from the F2 generation were forced to swim in a glass cylinder (height 15 cm and

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diameter 12 cm) containing water (23  C) 10 cm deep for 15 minutes. On the second day of testing, mice were forced to swim in the same glass cylinder for 5 minutes and the time of their immobility was measured using a stopwatch. In the FST the animals display “behavioral despair,” as indicated by increased immobility and less escape-oriented behaviors. When forced to swim in the cylinder filled with water, the mice eventually cease escape attempts and become immobile. The increasing immobility time reflects a state of helplessness and despair [17]. 2.3.2. Open-field test Mice from the F2 generation first received two familiarization sessions that involved placing the mice in the test apparatus for 20 minutes. The habituation sessions allowed any anxiety because of novelty to dissipate before testing and also gave an indication of baseline behavioral measures. In the afternoon of the third day, the open-field tests were conducted in a normally lit room. Before the test, all mice were habituated to the behavior room for 2 hours. The open field apparatus consists of a wooden base covered with washable waterproof black foil and transparent acrylic walls (60  45  36 cm). A color charge-coupled device camera (Panasonic VW CP484; Panasonic) was installed in the center above the apparatus. The back of each mouse was painted red and the color charge-coupled device camera followed the red point. The mice were placed in the center of the open field and the behavior of the mice was recorded for 5 minutes. After each record, the mouse was returned to its home cage and the open-field arena was cleaned with disinfectant solution and paper towels. Data were analyzed with the Ethovision XT 7.0 (Noldus Information and Technology BV, Wageningen, The Netherlands) software for automatic behavioral scoring. The following parameters were assessed: the total distance moved by the animal, its average velocity, the time spent in the central or the peripheral zone, and the frequency of entries into the central zone. An observer scored the number of rears, amount of scratching on the base of the apparatus, cleaning frequency, cleaning duration, and rest duration. 2.4. Statistical analysis All statistical analyses were performed using Statistica (StatSoft, Tulsa, USA). The results are expressed as the mean values  SEM. An ANOVA followed by Tukey’s post hoc test was used to detect differences in the following parameters:

average body fat and weight of dams and offspring, blood concentration of leptin and adiponectin, the integrated density of Nile red staining in oocytes, the average decrease in 5-methylcytosine signal in male pronuclei in zygotes, the average number of isolates per dam, in average litter size per dam, and in all parameters obtained during FST and open-field behavioral tests. To detect differences in the distribution of fertilized, unfertilized and degenerated eggs, a chi-square test with two degrees of freedom was used. A chi-square test with one degree of freedom was used to detect differences in the fertilization index, the differences in the demethylation processes in zygotes, and differences in the sex ratio of pups. For all results, P < 0.05 was considered statistically significant. 3. Results 3.1. Somatic parameters of mothers On Day 34, the control female mice (F1) in the group with physiological body fat (CN) showed 7.48  0.06% of body fat and an average body weight of 22.45  0.31 g. The blood samples collected randomly from several fertilized control mice (on Days 36–40) had 0.78  0.20 ng/mL leptin and 12,347  1266 ng/mL adiponectin in the serum (Table 1). The mice in the EXN group showed a significantly increased percentage of body fat (P < 0.001), but no differences in body weight, leptin, or adiponectin blood concentration compared with controls (P > 0.05). The mice in the EXF group showed significantly increased amounts of body fat (P < 0.001) and body weight (P < 0.05) compared with the CN. During the dissection of sacrificed EXF females, we observed a massive accumulation of fat in the abdominal and perirenal areas. Most EXF mice also showed slight hyperleptinemia and hyperadiponectinemia. However, there were no significant differences in the average blood concentration of leptin or adiponectin compared with normal controls (P > 0.05). The group of control female mice spontaneously exhibiting lean body condition (CL) showed a significantly decreased percentage of body fat (P < 0.001) and body weight (P < 0.001). In this group, the average blood concentration of leptin was similar to the normal control group. The concentration of adiponectin showed a decreasing trend. However, there was no statistical significance (P > 0.05). The concentration of adiponectin in the CL group was significantly lower than the EXF group (P < 0.01).

Table 1 Average value of body fat and body weight and concentrations of serum leptin and adiponectin in experimental female mice of F1 generation (Day 34). Group (% of body fat)

CL (<7%)

CN (7%–8%)

EXN (8%–11%)

EXF (>11%)

Female mice (F1) (n) Body fat (%) Body weight (g) Evaluated female mice (n) Leptin (ng/mL) Evaluated female mice (n) Adiponectin (ng/mL)

82 5.91  0.07Y 17.39  0.57Y 12 0.77  0.11 10 9774  723a

80 7.48  0.06a 22.45  0.31a 12 0.78  0.20 11 12,347  1266a,b

78 9.54  0.13b[ 23.01  0.54a,b 12 0.71  0.16 11 12,008  1231a,b

77 13.06  0.24c[ 24.55  0.37b[ 12 1.28  0.30 11 16,834  2020b

Data are expressed as arithmetical means  SEM, different letters in superscript indicate statistical difference, arrows indicate significant increase or decrease when compared with CN group (Tukey’s post hoc test following ANOVA, values P < 0.05 are considered as statistically significant). Abbreviations: CL, controls with decreased body fat and weight; CN, controls with the physiological amount of body fat and weight; EXN, experimental mice with slightly increased body fat and physiological body weight; EXF, experimental mice with highly increased body fat and weight.

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Table 2 Average number of isolated oocytes and zygotes per fertilized F1 mouse and an average amount of fat deposits in matured oocytes (integrated density of Nile Red fluorescence staining). Group (% of body fat)

CL (<7%)

CN (7%–8%)

EXN (8%–11%)

EXF (>11%)

Donor female mice (F1) (n) Average number of isolates (n) Evaluated isolates (n) Integrated density (105)

35 11.76  0.55a,b 144 28.35  0.45

34 11.37  0.54a 137 28.27  0.48

35 13.36  0.45b[ 172 25.71  0.40aY

37 11.41  0.60a,b 107 25.06  0.55aY

Data are expressed as arithmetical means  SEM, different letters in superscript indicate statistical difference, arrows indicate significant increase or decrease when compared with CN group (Tukey’s post hoc test following ANOVA, values P < 0.05 are considered as statistically significant). Abbreviations: CL, controls with decreased body fat and weight; CN, controls with the physiological amount of body fat and weight; EXN, experimental mice with slightly increased body fat and physiological body weight; EXF, experimental mice with highly increased body fat and weight.

3.2. Effect of maternal body condition on oocyte fertilization and fat deposits A stereomicroscopic evaluation of the oviductal contents showed that the average number of isolates collected from all fertilized CN was 11.37  0.54 per dam. There was a significant increase in the number of isolated oocytes and zygotes in females with slightly increased body fat (EXN) compared with controls (P < 0.05) (Table 2.). The fluorescence DNA staining of isolates from dams with physiological amounts of body fat (CN) showed that at early isolation (approximately 6 hours after supposed ovulation) 41.79% of oocytes were fertilized (at zygote stage), 51.49% were at oocyte MII stage, and 6.72% displayed immature or degenerated morphology (Fig. 4A). We found that half of early isolated zygotes were at PN1 or PN2 stage (50.00%; Fig. 3B, C). At the late isolation time point (approximately 11 hours after supposed ovulation) the proportion of fertilized oocytes in control dams increased to 79.84%. The proportion of oocytes at MII stage decreased to 18.55% and there were only 1.61% immature or degenerated oocytes (Fig. 4B). Most late isolated zygotes reached PN3 or PN4 stage (64.10%; Fig. 3D, E). In mothers with slightly and highly increased amount of body fat (EXN and EXF), there were significant differences in

the distribution of all obtained isolates (P < 0.001 for EXN, P < 0.01 for EXF; Fig. 4C). These females produced higher numbers of immature and degenerated oocytes compared with controls. Conversely, a significantly higher fertilization index (showing the percentage of successful fertilization in the group of mature oocytes only) was recorded in the EXN and EXF animals (P < 0.001 for EXN, P < 0.05 for EXF). Furthermore, in the EXN group, the highest number of late isolated zygotes reached PN5 stage (37.29% vs. 23.08% in CN and 23.91% in EXF; Fig. 3F). However, the differences in zygotic-stage distribution between groups did not show statistical significance (P > 0.05). In mothers with decreased amount of body fat (CL), the overall distribution of isolates was similar to controls (P > 0.05; Fig. 4C). However, Figure 4 shows these females produced significantly lower numbers of fertilized oocytes (fertilization index, P < 0.05). The results of the lipid accumulation assay showed that unfertilized MII oocytes isolated from dams with slightly and highly increased amounts of body fat (EXN and EXF) displayed lower integrated density of Nile red fluorescence signal than control mice (CN) (P < 0.001 for both cases, Table 2). The average number of lipid deposits in isolates produced by mothers with decreased body fat (CL) was similar to controls.

Fig. 4. Proportion of fertilized, unfertilized, and immature (or degenerated) oocytes. Oocytes were obtained from lean control females with decreased body fat and weight [CL, n ¼ 139 (A), n ¼ 132 (B), together n ¼ 271 (C)], controls with the physiological amount of body fat and weight [CN, n ¼ 134 (A), n ¼ 124 (B), together n ¼ 258 (C)], experimental mice with slightly increased body fat and physiological body weight [EXN, n ¼ 112 (A), n ¼ 199 (B), together n ¼ 311 (C)], and fat experimental mice with highly increased body fat and weight [EXF, n ¼ 137 (A), n ¼ 118 (B), together n ¼ 255 (C)] (A) at early isolation (6 hours after supposed ovulation), (B) at late isolation (11 hours after supposed ovulation). Significant differences between groups were determined using chi-square tests: *P < 0.05; **P < 0.01; ***P < 0.001; and NSP > 0.05. Asterisks at the top of columns indicate differences in distribution of mature fertilized, mature unfertilized, and immature (rarely degenerated) oocytes [chi-square tests with two degrees of freedom] and asterisks in italics next to columns indicate the differences in fertilization index (i.e., successful fertilization in mature oocytes only [chi-square tests with one degree of freedom]) between control (CN) and experimental groups (EXN, EXF, CL).

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Table 3 Demethylation (DM) of DNA in male pronuclei of zygotes isolated from F1 mice. Group (% of body fat)

CL (<7%)

CN (7%–8%)

EXN (8%–11%)

EXF (>11%)

PN3 zygotes (n) % of DM PN3 zygotes Signal decrease in _ PN (%) PN4 zygotes (n) % of DM PN4 zygotes Signal decrease in _ PN (%) PN5 zygotes (n) % of DM PN5 zygotes

32 59.38b 20.26  1.67 28 71.43aY 19.95  1.94aY 7 100.00

28 75.00ab 29.24  4.56 35 94.74b 38.67  3.14 9 100.00

37 86.49a 28.13  2.14 36 91.67b 25.25  2.77aY 22 100.00

33 63.64b 27.02  3.71 32 78.95ab 25.51  2.11aY 11 100.00

Data are expressed as arithmetical means  SEM, different letters in superscript indicate statistical difference, arrows indicate significant decrease when compared with CN group (values P < 0.05 are considered as statistically significant). PN3, PN4, PN5: zygotes at particular pronuclear stages; % of DM zygotes: percentage of zygotes containing demethylated male pronucleus in group (chi-square test); signal decrease in _ PN: average % decrease in 5-methylcytosine signal in male pronuclei derived from male PN to female PN signal ratio (5-methylcytosine signal in female pronucleus was determined as 100%) (Tukey’s post hoc test following ANOVA). Abbreviations: CL, controls with decreased body fat and weight; CN, controls with the physiological amount of body fat and weight; EXN, experimental mice with slightly increased body fat and physiological body weight; EXF, experimental mice with highly increased body fat and weight.

3.3. Effect of maternal body condition on demethylation of DNA in zygotes

3.4. Effect of maternal body condition on somatic parameters of offspring

In all evaluated zygotes the maternal pronuclei displayed homogeneous methylation staining in the nucleus. In mothers with physiological amounts of body fat (CN), the demethylation of DNA cytosine in paternal pronuclei was observed in 0.00% of zygotes at PN1 or PN2 stage, 75.00% of zygotes at PN3 stage, 94.74% of zygotes at PN4 stage, and 100.00% of zygotes at PN5 stage (Table 3). In the PN3 stage zygotes, the 5-methylcytosine staining in paternal pronuclei was approximately 30% lower than the signal in maternal pronuclei. In the PN4 stage zygotes the signal was reduced to approximately 40%. In mothers with slightly and highly increased amounts of body fat (EXN and EXF), we found a similar distribution of zygotes with demethylated cytosine in paternal pronuclei compared with controls (P > 0.05). However, in the PN4 stage zygotes, the average signal reduction in paternal pronuclei was significantly lower (approximately 25%, P < 0.01 for both cases; Table 3). Mothers with decreased body fat (CL) showed a significantly lower percentage of zygotes at PN4 stage with demethylated DNA cytosine in paternal pronuclei (P < 0.01) and a significantly lower reduction in 5-methylcytosine signal (P < 0.001). This result suggests a significant delay and lower extent of demethylation in zygotes isolated from CL females (Table 3).

Pregnant mice from both the control and experimental groups delivered similar numbers of pups on average (Table 4). All pups were born alive. There were no significant differences in the sex ratio of offspring (P > 0.05). Compared with the CN, pups delivered from dams with highly increased body fat (EXF) showed a significantly lower weight on the day of birth (P < 0.05) and at early adulthood (Day 32, P < 0.001). We found decreased birth weight in pups delivered from lean dams (CL) with decreased body fat (P < 0.001). However, during early postnatal development these offspring were able to reach a weight comparable with the weight of offspring delivered from control dams with a physiological amount of body fat (CN). The offspring in all groups showed approximately the same amount of body fat deposits on average at Day 32 (approximately 7%, Table 5). There was a slight decrease in the average percentage of body fat in males delivered from fat dams (EXF group, 6.76  0.22 vs. 8.50  0.20; P < 0.01) and in females delivered from lean dams (CL group, 5.24  0.21 vs. 6.33  0.20; P < 0.01) compared with CN. 3.5. Effect of maternal body condition on the behavior of offspring In offspring delivered from CN, the average immobility time recorded during the FST was approximately 63 seconds

Table 4 Average number and sex ratio of F2 pups delivered from F1 mothers with different body condition. Group (% of body fat)

CL (<7%)

CN (7%–8%)

EXN (8%–11%)

EXF (>11%)

Donor dams (F1) (n) Delivered pups (F2) (n) Average litter size per dam Birth weight of pups (g) Sex ratio of pups (%)

8 87 10.88  0.52 1.61  0.01aY 56.32%_:43.68%\

7 86 12.29  0.75 1.72  0.02b 54.65%_:45.35%\

7 80 11.43  1.60 1.69  0.02b,c 43.75%_:56.25%\

7 83 11.86  0.55 1.64  0.02a,cY 51.81%_:48.19%\

Data are expressed as arithmetical means  SEM, different letters in superscript indicate statistical difference, arrows indicate significant increase or decrease when compared with CN group (Tukey’s post hoc test following ANOVA average litter size and birth weight, chi-square tests for sex ratio, values P < 0.05 are considered as statistically significant). Abbreviations: CL, controls with decreased body fat and weight; CN, controls with the physiological amount of body fat and weight; EXN, experimental mice with slightly increased body fat and physiological body weight; EXF, experimental mice with highly increased body fat and weight.

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Table 5 Average value of body fat and body weight in F2 offspring chosen for behavioral tests (Day 32). Group

CL

CN

EXN

EXF

Tested offspring (n) Body fat (%) Body weight (g)

60 (30_:30\) 6.46  0.23a,b 24.14  0.45a

60 (30_:30\) 6.93  0.16a,b 22.59  0.52a,b

60 (30_:30\) 7.19  0.17a 21.84  0.49b

60 (30_:30\) 6.70  0.14b 18.52  0.31Y

Data are expressed as arithmetical means  SEM, different letters in superscript indicate statistical difference, arrows indicate significant increase or decrease when compared with CN group (Tukey’s post hoc test following ANOVA, values P < 0.05 are considered as statistically significant). Abbreviations: CL, controls with decreased body fat and weight; CN, controls with the physiological amount of body fat and weight; EXN, experimental mice with slightly increased body fat and physiological body weight; EXF, experimental mice with highly increased body fat and weight.

with a lean body (CL group with decreased amount of body fat and weight) despite the standardized nutritional and breeding conditions and the absence of any manifesting illnesses. In addition to the phenotype differences, in EXF group the tendencies to increase in leptin and adiponectin blood concentrations were recorded, and in CL group a tendency to decrease in adiponectin blood concentration was observed. As shown in our previous study, fat dams showed significantly higher blood glucose (both EXN and EXF groups) and insulin (EXF group) concentrations than did controls (CN) [10]. The females with these four types of conditions were used as donor dams for the in vivo production of oocytes, zygotes (present study), preimplantation embryos (previous study [10]), and for the natural delivery of offspring (present study). The selection of females resistant to weight gain in response to prenatal and early postnatal overnutrition (EXN group) allowed us to interpret the results in a fat-level–dependent manner and to distinguish between the effect of nutrition and maternal metabolic profile (once they were weaned, all F1 females received standard pellet diet only). Our results show a relatively high similarity between outcomes obtained in CN and EXN in several evaluated parameters. Nevertheless, a slight periconceptional increase in body fat deposits of donor mice females (9.54% vs. 7.48% at average) positively affected the average number of ovulated oocytes per animal. There was also a positive effect on the fertilization index and average number of isolated preimplantation embryos per dam [10]. These findings did not influence the average number of delivered pups. However, the number of evaluated pregnant females per group was relatively low. Thus, the statistical power of the results could

(Table 6). This result was significantly higher in mice delivered from lean dams (CL) (approximately 94 seconds; P < 0.01). A similar tendency was observed in descendants of fat dams (EXF group, 79 seconds), but the result was not statistically significant (P > 0.05). There were several differences noted between groups during the open-field test (Table 6). The offspring delivered from experimental dams with highly increased body fat (EXF) showed higher total distance movements in the apparatus (P < 0.001), a higher number of entries to the central zone (P < 0.05), and higher average resting time (P < 0.05) than CN. In offspring delivered from lean control dams with decreased body fat (CL), we found a lower frequency and shorter time of cleaning (P < 0.05 for both parameters). There were no significant differences between groups in any other parameters (average time spent in central zone, average velocity of movement, and total number of rears). 4. Discussion Heterogenous populations include animals that are naturally susceptible or resistant to the development of obesity or other metabolic diseases [18,19]. In our study, the use of an outbred strain of ICR (CD-1 IGS) mice in a standardized dietary model allowed us to produce female mice (F1 generation) with the following four types of body conditions: (1) a group of normal controls with physiological amounts of body fat and weight (CN), (2) a group of experimental mice showing slightly increased amounts of body fat but normal body weight (EXN), (3) a group of experimental mice showing highly increased amounts of body fat and weight (EXF), and (4) a group of control mice

Table 6 Behavioral analysis in F2 offspring (n ¼ 60 in each group) delivered from F1 mothers with different body condition using forced swimming test (FST) and open-field test. Group

CL

Immobility time (FST) (s) Total distance moved (m) Time in central zone (s) Center entries’ frequency (n) Velocity (cm/s) Rears (n) Scratching (n) Cleaning frequency (n) Cleaning duration (s) Rest duration (s)

94.33 20.01 15.91 10.95 12.61 32.62 2.50 3.13 11.19 18.83

CN          

7.00a[ 1.48 3.25 1.09a,b 0.59 1.80 0.30 0.40aY 1.25aY 4.34a,b

63.78 19.35 15.58 10.65 11.46 34.15 2.72 4.75 20.95 12.92

EXN          

7.29b 1.66 1.62 1.11a 0.44 2.00 0.37 0.50b 2.88b 2.70a

57.15 19.15 9.98 7.22 12.39 33.70 2.95 4.25 24.7 21.67

EXF          

6.57b 1.84 0.86 0.74a 1.66 2.29 0.38 0.31a,b 2.56b 4.29a,b

79.22 33.02 17.14 15.27 13.44 32.80 1.84 4.77 17.58 34.37

         

6.58a,b 2.29a[ 1.69 1.65b[ 0.75 2.91 0.42 0.55b 3.18a,b 7.03b[

Data are expressed as arithmetical means  SEM, different letters in superscript indicate statistical difference, arrows indicate significant increase or decrease when compared with CN group (Tukey’s post hoc test following ANOVA, values P < 0.05 are considered as statistically significant). Abbreviations: CL, controls with decreased body fat and weight; CN, controls with the physiological amount of body fat and weight; EXN, experimental mice with slightly increased body fat and physiological body weight; EXF, experimental mice with highly increased body fat and weight.

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be improved. We found that mice with slightly increased body fat produced a higher number of immature oocytes or degenerates, and their matured oocytes displayed significantly lower deposits of neutral lipids in the cytoplasm. Additionally, their zygotes at PN4 stage showed less DNA demethylation in parental pronuclei. However, the somatic and behavioral parameters of delivered offspring were comparable with the normal control group. On the contrary, a high increase in the amount of body fat in females (13.06% at average) had a negative effect on most evaluated parameters. The mice in the EXF group showed an increased number of isolated immature oocytes or degenerates and a significantly lower number of neutral lipid deposits in the cytoplasm of mature oocytes. The EXF group also had the following characteristics: less DNA demethylation in parental pronuclei of zygotes at PN4 stage, slower preimplantation development of embryos [10], an increased apoptotic index in blastocysts [10], lowered weight of newborns, lower offspring weight at the age of 5 weeks, and decreased body fat deposits in 5-week-old females. Furthermore, in offspring delivered from dams with highly increased body fat there were several deviations from normal behavior including higher locomotion activity, lowered anxiety (represented by higher frequency of entries to central zone), and performance of slightly more comfort behaviors (represented by higher resting time). Most evaluated parameters were impaired in the animals spontaneously displaying lean body condition (CL). The female mice with decreased body fat (5.91% at average) had a lower fertilization index, later onset, and lower extent of active demethylation in male pronuclei of PN4 stage zygotes, slower embryo transition from oviduct to uterus [10], slower embryo development [10], increased incidence of apoptosis in blastocysts [10], and lower weight of newborns compared with normal controls. Although the offspring delivered from lean dams were able to gain normal weight by the fifth week, there were deviations from normal behavior recorded including depressive-like behavior (represented by higher immobility time during FST) and performance of less comfort behavior (represented by lower cleaning duration and frequency). There have been several studies examining the physiology of maternal fat metabolism and its endocrine functions. However, the mechanisms mediating the effect of obesity and leanness on developing conceptuses remain largely unknown. Oocytes, zygotes, and embryos are apparently able to process external signals. The presence of receptors for leptin, adiponectin, insulin, insulin-like factors, and many other metabolic hormones has been documented at both the mRNA and protein levels. Furthermore, the effect of various mediators and substrates of lipid metabolism on oocyte maturation, fertilization, and cleavage has been shown in numerous in vitro studies [2,3]. The potential effect of four mediators (leptin, adiponectin, insulin, and glucose) on oocyte and embryo development has been discussed in our previous papers [9,10,20,21]. These mediators showed altered serum levels in EXN and EXF dams produced in our two-generation mouse model. Nevertheless, because several mediators might be involved in the process, further investigations at the genetic and metabolic levels are necessary.

4.1. The effect of maternal body condition on fertility and lipid accumulation in oocytes The effect of maternal periconceptional overnutrition (obesity) or undernutrition (leanness) on the ovulation rate and on the IVF or in vivo fertilization has been previously described in various experimental and epidemiologic studies. In the case of overnutrition (obesity), conflicting effects on both parameters were documented. Several studies have identified a reduced rate of collected and in vitro fertilized oocytes from obese women [2,3,22]. Similarly, the presence of lower follicular activity and smaller and fewer mature oocytes was reported in mice with diet-induced obesity after hormonally induced ovulation [6,23]. Conversely, our results indicate no effect or a positive effect of overnutrition (obesity) on the ovulation and fertilization rates. In accordance with our outcomes, several studies have reported that there are no differences in the number of collected and IVF produced oocytes or the number of isolated zygotes between obese and nonobese donors in both women and experimental animals [3,7,23,24]. There was no effect of overnutrition found on ovulation activity and fertilization rate in vivo and in vitro either in cows or ewes [25–27]. Finally, a positive effect on the ovulation and fertilization rates has been observed in only a few experimental studies. Minge et al. [4] showed that a diet containing high fatty acid content increases the total number of oocytes collected from female mice unaffected with hormonal synchronization. Additionally, Ferguson et al. [28] have reported an increased number of MII oocytes in overfed and synchronized gilts. Wakefield et al. [5] have documented a trend of more zygotes isolated from superovulated mice fed an omega-3 polyunsaturated fatty acid– enriched diet. These data suggest that moderate maternal obesity might have a positive impact on the ovulation rate and fertilization in vivo. However, the significance of the effect is dependent on various factors, such as maternal age, individual or strain genetic predisposition, actual nutrition, or hormonal treatment. Regardless of recorded positive effect of maternal periconceptional body fat increase in ICR mice on ovulation rate (EXN group) and fertilization rate (EXN and EXF group), the average litter size was not increased in our study. Similarly, no effect on pregnancy rates in high-fat diet fed inbred C57BL/6J mice was documented by Tortoriello et al. [23]. However, in the same study, fat DBA/2J strain females showed decreased pregnancy rates. This result suggests a strain-dependent effect. A decreased live birth rate was reported also in most human clinical studies [22,29]. As mentioned previously, if more donor dams were used in this study then some alterations in this parameter could have occurred. The data regarding maternal undernutrition (leanness) are more consistent. In our study (CL group) and other reports there is a negative effect of poor maternal condition on the fertilization rate in vivo. It has been shown that cows that suffer excess body condition score loss early postpartum are less likely to ovulate, have a reduced artificial insemination success rate, and a lower conception rate at

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first service. Additionally, these cows have an increased likelihood for pregnancy loss and an increased calving-toconception interval [1]. These cows also have impaired oocyte competence and quality and a decreased oocyte cleavage rate [30,31]. A lean body condition was negatively correlated with pregnancy rates in cows [1]. However, experimental mice with spontaneous leanness (present study) or subjected to a low-protein diet during oocyte maturation [32] showed no effects on litter size. Previous studies on mammalian oocytes and embryos have reported that the amount of intracellular lipids may reflect the embryo quality and developmental potential. Intracellular lipids are stored mainly in the form of lipid droplets [33]. The different types of lipids play important roles in numerous cellular processes. For example, oocytes may use intracellular triglycerides as an energy source and different types of phospholipids regulate oocyte maturation. The ratio of phospholipids to cholesterol also influences the physical properties of their biological membranes [16,34,35]. Previous in vitro experiments have shown that maturing oocytes can accumulate fatty acids from their microenvironments and the presence of serum in culture media significantly increased the amount of cytoplasmic lipids [33,36,37]. Recently, a new and reliable technique was developed to evaluate the lipid content of single murine, bovine, and pig oocytes in a semiquantitative way (relative quantification of the lipid content) using Nile red staining [14]. Using this approach, we found a direct relationship between maternal condition (metabolic profile) and lipid metabolism of in vivo developing murine oocytes. To our knowledge, only Barceló-Fimbres and Seidel [15] have compared lipid content in oocytes isolated from culled mature cows and fattened feedlot heifers. However, in that case, there was no significant difference between groups. Because experimentally induced fat gain in our model was not accompanied with increased levels of serum triglycerides or cholesterol (a significant increase was found only in serum insulin and glucose [9]), we did not expect increases in oocyte lipid deposits. However, we found that oocytes and zygotes isolated from fertilized female mice with both slightly and highly increased body fat (EXN and EXF groups) showed significantly less neutral lipids on average compared with CN. This result suggests the existence of an unknown mechanism (maternal or gametogenic) that adjusts fat deposits in developing oocytes (perhaps even the primordial germ cells). This mechanism is negatively correlated with maternal body condition (or nutrition) and might be the consequence of a “predictive adaptive response,” which would affect the character of energetic metabolism of a conceptus during its later embryonic, fetal, or even postnatal development. A decrease in body weight and fat in mouse offspring might also be correlated with this result. 4.2. The effect of maternal body condition on offspring Our results showed that the changes in maternal metabolism in both experimentally induced fat gain and spontaneously developed lean dams (F1 generation) affected several somatic parameters of delivered offspring and caused various deviations from their normal behavior (F2

587

generation). The concept that maternal health and nutrition during pregnancy can have long-term effects on offspring is commonly known as the Barker hypothesis, or the hypothesis of developmental origins of adult diseases [38]. In the case of maternal diet-induced obesity, most previous studies documented fetal growth retardation (gilts overfed before pregnancy [34]; mice dams fed high-fat diet (HFD) before and during pregnancy [6]; and mice dams fed HFD before pregnancy [8]) or decreases in birth weight and fat deposits in pups (rabbit does fed hyperlipidic diet before and during pregnancy [39]). In our study, a decrease in birth weight was recorded in F2 pups delivered from mice showing highly increased body fat during the periconceptional period (EXF group). Such offspring showed significantly decreased body weight even in early adulthood. Furthermore, a decrease in average body fat was found in males. In a similar study, Jungheim et al. [6] did not find any alterations in the average body weight of 25-day-old mice delivered from obese dams (fed HFD before and during pregnancy). However, these offspring demonstrated glucose intolerance and increased cholesterol. These results suggest the early development of metabolic-type syndrome. In the case of maternal undernutrition (leanness), the effects on offspring are not well studied. In addition to our study (CL group), a negative effect on the birth weight of pups has only been documented by Kwong et al. [40] in rat dams fed a low-protein diet during the preimplantation period of pregnancy. Conversely, Watkins et al. [32] and Rae et al. [41] did not observe any alterations in birth weight of newborn or older mice delivered from dams fed a lowprotein diet during oocyte maturation or lambs delivered from ewes fed a reduced diet during the first 95 days of gestation, respectively. In our study, 5-week-old F2 offspring delivered from dams with decreased body fat (CL) showed body weights comparable with controls (offspring of CN dams). However, decreased deposits of body fat were recorded in females. This finding suggests that there are long-term effects of altered periconceptional or intrauterine conditions in lean dams on fat metabolism of adult female offspring as well. The evaluation of potential relationships between changes in maternal homeostasis during pregnancy and altered offspring behavior is the major theme of numerous interdisciplinary studies. It has been shown that offspring exposed to maternal obesity and high-fat diet consumption during development are more susceptible to developing mental health and behavioral disorders such as anxiety, depression, attention deficit hyperactivity disorder, and autism spectrum disorders [42]. However, most of these studies on primates or animals were focused on maternal nutrition during the perinatal period and the mothers of the tested offspring were fed a high-fat diet during the gestation and lactation periods [43,44]. To our knowledge, this is the first study to show deviations in the behavior of offspring delivered from dams displaying an obesity-like phenotype with altered metabolic status during early pregnancy. The neural system is formed during later periods of development and there is most likely no direct causality for this relationship. However, brain developmental abnormalities in fetuses originating from transferred embryos produced by fat mice

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dams fed a high-fat diet before pregnancy has been described previously [8]. These changes might be related to our results. Furthermore, the effect of maternal leanness during the periconceptional period on offspring behavior has been recorded previously (in mouse dams fed low-protein diet during preimplantation period [32], in rat dams undernourished during whole gestation period [45], and in sheep undernourished during periconception period [46]). The potential mechanisms of long-term effects because of changes in maternal homeostasis during the periconceptional period or early pregnancy are intensively studied. Alterations in epigenetic processes are well studied in this context. During mammalian development, there are two rounds of physiological DNA demethylation and remethylation. Before gametogenesis, the genome is demethylated in the primordial germ cells and subsequently remethylated during gametogenesis to establish gender specific imprints [47]. After fertilization, another round of DNA demethylation and remethylation occurs, but without affecting the imprinted regions. In the mouse zygote, the paternal genome is rapidly and actively demethylated [48]. The maternal genome shows a gradual loss of methylation S-phases and is passively demethylated as a consequence of the lack of DNA (cytosine-5)-methyltransferase 1 activity [49]. De novo DNA methylation occurs in a species-specific manner, and in mouse this methylation starts at the blastocyst stage [50]. A genome-wide loss of 5-methylcytosine signal by antibody staining in the paternal pronucleus is known to occur beginning at the PN3 stage, which is the time when zygotic DNA replication begins [13,51]. In our study, no demethylation processes were recorded before the PN3 stage. Gradual reduction in the 5-methylcytosine signal was observed in 75% of zygotes at PN3 stage, 90% of zygotes at PN4 stage, and 100% of zygotes at PN5 stage isolated from control F1 females (CN). When compared with controls, parental pronuclei in the PN4 stage zygotes isolated from dams displaying slightly increased (EXN), highly increased (EXF), or decreased (CL) body fat showed a significantly increased 5-methylcytosine signal. In the CL group, the proportion of zygotes at PN4 stage with demethylated DNA was significantly lower. A similar trend was observed in the EXF group and in zygotes at PN3 stage isolated from CL and EXF mothers. However, the findings were not statistically significant in these cases. The observed delayed onset and decreased demethylation activity suggest that epigenetic mechanisms could regulate of effect of maternal body condition on conceptuses at the earliest stages of development. The effect of maternal obesity (induced by 12-week preconceptional high-fat diet) on DNA methylation of particular metabolism-related genes in oocytes was also shown in a recent study by Ge et al. [52]. Previous studies have reported that not only obesity, but also periconceptional undernutrition causes significant changes in DNA methylation that are maintained in adult offspring [53]. 4.3. Conclusions We conclude that alterations in maternal body condition affect reproductive processes at several steps including the

period of ovulation, fertilization, and early embryo development in vivo. Furthermore, the mother’s body condition might affect the somatic phenotype and behavior of delivered offspring. The effect of an obesity-like phenotype is dependent on its level (i.e., amount of maternal body fat deposits) and might impact particular reproductive parameters in opposite manners. Nevertheless, a negative effect dominates. The data on later onset and lower extent of DNA demethylation in zygotes isolated from dams with highly increased or decreased body fat and the altered lipid deposits in oocytes isolated from dams with increased body fat suggest that changes in offspring might originate in epigenetic or metabolic modifications established at the earliest stages of conceptus development. Acknowledgments This work was supported by the Slovak Research and Development Agency (http://www.apvv.sk) under contract APVV-0815-11 and the Slovak Academy of Sciences (http:// www.vega.sav.sk) under contracts VEGA 2/0049/11 and VEGA 2/0001/14. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors, except K.F., E.R. and S.C., are members of the COST Action FA1201 Epiconcept: Epigenetics and Periconception Environment (http://www.

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