Parents ethanol use impairs ethanol-naive offspring development and reproduction

in Reproduction
Authors:
Vanessa Caroline FioravanteDepartment of Structural and Functional Biology, Institute of Biosciences of Botucatu (IBB), UNESP – Univ Estadual Paulista, São Paulo, Brazil
General and Applied Biology Program, Institute of Biosciences of Botucatu (IBB), UNESP – Univ Estadual Paulista, São Paulo, Brazil

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Alana Rezende GodoiDepartment of Structural and Functional Biology, Institute of Biosciences of Botucatu (IBB), UNESP – Univ Estadual Paulista, São Paulo, Brazil
General and Applied Biology Program, Institute of Biosciences of Botucatu (IBB), UNESP – Univ Estadual Paulista, São Paulo, Brazil

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Victória Mokarzel de Barros CamargoDepartment of Structural and Functional Biology, Institute of Biosciences of Botucatu (IBB), UNESP – Univ Estadual Paulista, São Paulo, Brazil
General and Applied Biology Program, Institute of Biosciences of Botucatu (IBB), UNESP – Univ Estadual Paulista, São Paulo, Brazil

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Renata Steffany do NascimentoDepartment of Structural and Functional Biology, Institute of Biosciences of Botucatu (IBB), UNESP – Univ Estadual Paulista, São Paulo, Brazil

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Patricia Fernanda Felipe PinheiroDepartment of Structural and Functional Biology, Institute of Biosciences of Botucatu (IBB), UNESP – Univ Estadual Paulista, São Paulo, Brazil

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Francisco Eduardo MartinezDepartment of Structural and Functional Biology, Institute of Biosciences of Botucatu (IBB), UNESP – Univ Estadual Paulista, São Paulo, Brazil

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Correspondence should be addressed to F E Martinez; Email: fe.martinez@unesp.br
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Parental ethanol consumption can influence the offspring phenotype. In this way, we analyzed the impairments of maternal and paternal high ethanol consumption during postpuberty on the physical development, feeding pattern, puberty onset and reproductive function of ethanol-naive offspring to birth to adulthood. Female and male UChB rats (voluntary 10%, v/v ethanol consumer) were divided into a control group (C) and an ethanol exposed group (E) from 65 to 80 days of age. The C and E were mated at 100 days. The maternal parameters and offspring development and reproduction parameters were monitored. We observed reduced feeding intake and body weight in the dams of E group throughout gestation and lactation period. Delay in physical development, lower body weight and altered feeding pattern were observed in female and male offspring of E group. In addition, the puberty onset was delayed in both sexes, with lower testosterone levels in the juvenile and pubertal males. There was a prolongation on the estrous and proestrus phases in females from E but the estrous cycle duration did not change between groups. Ovary and uterus weight were reduced in pubertal and adult females from E group. Reduced epididymis and seminal vesicle weight, increased sperm abnormalities, decrease in the daily sperm production and accelerated epididymal transit time were observed in E males. The high maternal and paternal ethanol use on postpuberty impairs the parameters of ethanol-naive offspring inducing alteration on development and reproduction.

Abstract

Parental ethanol consumption can influence the offspring phenotype. In this way, we analyzed the impairments of maternal and paternal high ethanol consumption during postpuberty on the physical development, feeding pattern, puberty onset and reproductive function of ethanol-naive offspring to birth to adulthood. Female and male UChB rats (voluntary 10%, v/v ethanol consumer) were divided into a control group (C) and an ethanol exposed group (E) from 65 to 80 days of age. The C and E were mated at 100 days. The maternal parameters and offspring development and reproduction parameters were monitored. We observed reduced feeding intake and body weight in the dams of E group throughout gestation and lactation period. Delay in physical development, lower body weight and altered feeding pattern were observed in female and male offspring of E group. In addition, the puberty onset was delayed in both sexes, with lower testosterone levels in the juvenile and pubertal males. There was a prolongation on the estrous and proestrus phases in females from E but the estrous cycle duration did not change between groups. Ovary and uterus weight were reduced in pubertal and adult females from E group. Reduced epididymis and seminal vesicle weight, increased sperm abnormalities, decrease in the daily sperm production and accelerated epididymal transit time were observed in E males. The high maternal and paternal ethanol use on postpuberty impairs the parameters of ethanol-naive offspring inducing alteration on development and reproduction.

Introduction

Ethanol is one of the main abuse drugs ingested worldwide (Rehm et al. 2009, Balddin et al. 2018) and responsible for approximately 5.2% of global deaths (GBD 2018). Chronic alcohol intake can result in female and male reproductive pathologies confirmed in experimental models (Oremosu & Akang 2015, Srivastava et al. 2018) and humans (Eggert et al. 2004, Sansone et al. 2018). Its toxic molecules can harm the gonads and lead to oxidative stress which damages the integrity of the germinative DNA and, consequently, embryo development (Jana et al. 2010, Fan et al. 2017).

Maternal alcohol consumption is capable of restricting fetal growth and impairing the offspring development (Weinberg et al. 2008, Brix et al. 2020). Thus, an adverse intrauterine environment can program the growth and development of the main physiological or behavioral systems to improve the adaptation and survival of adulthood offspring (Zhang et al. 2005). Different phenotypes can be produced by development programming, highlighting the plasticity of the offspring adaptation (Zhang et al. 2005, Rando & Simmons 2015). However, these early adaptations may increase the propensity of disorders in adulthood (Hochberg et al. 2011). Additionally, besides fetal programming conducted by the uterine environment, it has been established that paternal exposure also plays an important role in offspring development (Lane et al. 2014, Bedi et al. 2019).

In this perspective, preconception experiences, maternal or paternal, as the nutrition, metabolic status and toxicants exposure have been associated with embryonic, fetal and postnatal development (Lane et al. 2014, Öst et al. 2014). Thus, lifestyle can define the offspring development patterns. However, the molecular mechanisms which hereditarily influence the phenotypes are poorly understood (Bedi et al. 2019). Ethanol-induced epigenetic mechanisms, capable of modifying the expression pattern of different tissues (Asimes et al. 2017), is one of the mechanisms related to changes in descendants’ phenotypes.

Studies in rodents have shown that prepuberty, puberty, or conception ethanol exposure can be harmful to pups, however, there is no knowledge about postpuberty. We hypothesized that postpubertal parents’ ethanol intake, with ethanol withdrawn after this period, could harm ethanol-naive offspring development and reproduction. Therefore, we evaluated the impacts of postpubertal maternal and paternal high ethanol use on physical development, body growth, feeding pattern, puberty onset, reproductive function and hormones levels of female and male offspring. Our investigations report on the damage caused to descendants due to parents’ ethanol abuse in postadolescence, raising awareness about the impacts of our lifestyle on future generations.

Materials and methods

Animals and experimental design

The experiments were in accordance with the Ethical Principles in Animal Research and approved by the Bioscience Institute/UNESP Ethical Committee for Animal Research (protocol no 1185). Twelve males (231 ± 10.7 g/55 days old) and 12 females (171 ± 6.3 g/55 days old) UChB rats obtained from the Anatomy Department of Botucatu Bioscience Institute/UNESP were used. The animals were housed in polypropylene cages (32 cm × 40 cm × 18 cm) and maintained under controlled conditions (25 ± 1°C, humidity 55 ± 5%, and light from 6 to 18 h) with access to commercial feed and water ad libitum. In this study, we employed a voluntary model of ethanol exposure, UChB variety. This model is adequate because it avoids the stress associated with forced feeding and provides knowledge about the effects of voluntary ethanol consumption as observed in the society (Gapp et al. 2014).

At 65 days old, the rats were randomly distributed in two groups: a control group (C) and an ethanol exposed group (E). The females and males of E were exposed to ethanol for 15 days consecutively, thus, free access of 10% (v/v) ethanol solution was provided for them from 65 to 80 days old, corresponding to postpuberty (Picut et al. 2015). Ethanol was withdrawal after this period. The consumption was measured by (ethanol consumed (mL)/15 × 100)/body weight (g). The rats UChB consumes a high amount of ethanol and only animals with ingestion > 2 g/kg/day were selected to continue in the experiment (Mardones & Segovia-Riquelmi 1983). The average ethanol consumption was 2.89 ± 1.05 g/kg/day for females and 2.63 ± 0.54 g/kg/day for males.

Females of C (n = 6) and E (n = 6) were mating to males of C (n = 6) and E (n = 6), respectively, at 100 days old in the overnight (one female and one male/cage). Vaginal smear was carried out daily in the morning and the first day of pregnancy was considered when spermatozoa were found. After the pregnancy detection, the dams were individualized and monitored. At birth, the offspring were cut to eight pups (four females and four males) per dam. The offspring were weaned at postnatal day (PND) 21 and two rats of the same sex were housed per cage for monitoring. The female (n = 24/group) and male (n = 24/group) offspring from C and E were monitored at birth to adulthood and randomly distributed in three phases: juvenile, pubertal, and adult. All phases were composed of eight females and eight males per group. The experimental design is diagrammed in Fig. 1.

Figure 1
Figure 1

Diagram of the experimental design. GD, gestational day; LD, lactational day; PND, postnatal day. Rats were divided into a control group (C) and an ethanol (10%, v/v) exposed group (E) from PND 65-80 (postpuberty), with withdrawal after this period. The rats were mated at PND 100. The dams were monitored. At the birth of pups (n = 24/sex/group), the physical development, body weight, and feed and water intake were monitored. The organs and reproductive hormones were analyzed in three phases: juvenile (PND 30), pubertal (PND 44/female, PND 55/male) and adult (PND 100). The female and male reproductive function was observed by the estrous cycle and sperm parameters.

Citation: Reproduction 161, 2; 10.1530/REP-20-0316

Maternal parameters

The gestation duration, litter size and ratio of female and male offspring were evaluated. Body weight and feed and water consumption on gestation and lactation were weekly measured. Feeding efficiency was validated by the food efficiency coefficient (FEC: (Final weight – Initial weight)/Feed consumed amount) and weight gain for caloric consumption (WGCC: (Final weight – Initial weight)/Kilocalories consumed amount) (Campbell 1963).

Landmarks of physical development, anogenital and nasoanal distance of offspring

The pinna unfolding, hair growth, incisors teeth eruption, and eye-opening were observed daily from birth to PND 20. Anogenital distance (AGD) was measured at PND 1 (neonate), and nasoanal length (NAL) at PND 1 (neonate), PND 55 (pubertal) and PND 91 (adult) by digital pachymeter. AGD corresponds to the distance between the anus to the genital tubercle and NAL the anus to the nostril. Relative AGD was calculated by DAG/∛peso (Gallavan et al. 1999).

Body weight, feed and water consumption and feeding efficiency of offspring

The body weight was measured from birth to PND 100 and weight gain was also calculated. After the weaning, the feed and water consumption and feeding efficiency were calculated.

External signs of puberty onset of offspring

The females were daily evaluated from PND 30 to verify the vaginal opening. On the vaginal opening, 10 µL of 0.9% saline was inserted into the vagina then aspirated (Borges et al. 2017). Vaginal fluids were placed on the slide and analyzed under a light microscope (200× magnification) to detect the day of first estrus. In the males, the testicular descent and preputial separation were observed daily from PND 15 and PND 38, respectively (Mylchreest et al. 2000).

Reproductive organs weight and plasm reproductive hormone levels of offspring

The females and males were weighted and sacrificed by CO2 inhalation followed by decapitation for the collection of blood samples from cervical vessels. Rats were killed in three phases: juvenile (PND 30), pubertal (PND 55 to males and PND 44 to females, after the vaginal opening and first estrus) and adult (PND 100). Pubertal and adult females were killed during the estrus phase. The testis, epididymis, ventral prostate, and seminal vesicle (with fluid) in the males, and ovaries and uterus in the females were removed, dissected, and weighted on analytical balance. The sexual glands were not separated in the juvenile males; thus, the ventral prostate and seminal vesicle were weighted together. The relative organ weight was calculated by organ weight (mg)/body weight (g).

The blood samples were allocated into EDTA tubes and centrifuged (2486 g for 15 min at 4°C). Plasma concentration of testosterone (mg/mL) in the males and 17-beta estradiol (pg/mL) in the females were determined by the competitive ELISA method. The ELISA kit: testosterone (catalog no E-EL-0155, 96T, Elabscience Biotechnology Co., EUA, sensitivity of 0.17 ng/mL, detection range between 0.31 and 20 ng/mL, coefficient of variation <10%) and estradiol (catalog no E-EL-0065, 96T; Elabscience Biotechnology Co., EUA, sensitivity of 25 pg/mL, detection range between 40 and 1500 pg/mL, coefficient of variation ≤15%) were read on a spectrophotometer at 450 nm.

Estrous cycle of female offspring

The estrous cycle was assessed based on vaginal smears collected every morning for 15 days from PND 65 to PND 80. The samples were analyzed under a light microscope and estrous cycle phases were classified as metestrus (leukocytes, and cornified and nucleated epithelial cells), diestrus (leukocytes), proestrus (nucleated epithelial cells) and estrus (anucleate cornified cells) (Marcondes et al. 2002). The estrous cycle duration was calculated by the number of days between one estrus phase to the next and the number of estrous cycles during the assay (Borges et al. 2017).

Sperm count, daily sperm production and epididymal transit time in pubertal and adult male offspring

Homogenization-resistant testicular spermatids and sperm in the caput/corpus and cauda epididymal were obtained from vas deferens (left side) and were counted as described for Robb et al. (1978). The sperm count was determined using the Neubauer chambers. Two Neubauer chambers, divided into 2 antimeres, were prepared per animal, accounting 20 fields/animal. Spermatids numbers were obtained by sperm count mean multiplied by the dilution factor. Sperm concentration (spermatids/g testis) was obtained by the spermatids count mean divided by the weight of testicular parenchyma. Daily sperm production was obtained by dividing the total number of homogenization-resistant spermatids per testis by 6.1, the number of days in which these spermatids are present on germinative epithelium (Robb et al. 1978). Transit time through the caput/corpus and cauda epididymis was calculated by dividing the number of sperm within each of these regions by the daily sperm production (Robb et al. 1978).

Sperm morphology in adult male offspring

The volume of ten microliters of semen was obtained from vas deferens. Seminal fluid was placed on the slide, dried at room temperature for 10 min and evaluated under phase-contrast microscopy (400×, total magnification). Two hundred sperm per animal were evaluated for head or flagellar defects (Seed et al. 1996). Anomalies were classified into head anomalies (neither typical nor isolated hook) or tail anomalies (broken or tail headless) and the data were expressed in percentage (Filler 1993).

Statistical analysis

The data were analyzed by the software GraphPad Prism® (version 7, GraphPad Software). A two-way ANOVA was used in the dam’s parameters and offspring’s parameters (nasoanal length, feed and water consumption, body and reproductive organ weight, hormone levels and male reproductive function). Post hoc analysis was performed when there was a significant interaction among factors by two-way ANOVA (Sidak’s multiple comparison test). Time, treatment, and interaction values were expressed in the figure and table legends. Unpaired t-test for parametric data or Mann–Whitney test for non-parametric data were employed in the gestation duration, litter size, sex ratio, landmarks of physical development, anogenital distance, external signs of puberty onset, estrous cycle and sperm morphology. Results were expressed as mean ± s.d. The differences were considered significant when P < 0.05.

The statistical analyses were performed based on the number of neonates (n = 24/sex/group). However, in addition, analysis based on the pregnant dams as the experimental unit (n = 6/group) was also realized. There was no relevant difference between these analyses.

Results

Postpubertal ethanol use decreased body weight and feed and water consumption of dams

The gestation duration (C: 23.3 ± 0.47, E: 21.83 ± 0.69), littler size (C: 10.10 ± 2.64, E: 8.30 ± 2.11) and sex ratio of offspring (females C: 51.35 ± 2.41%, E: 51.10 ± 3.62%; males C: 48.65 ± 2.41%, E: 48.90 ± 3.62%) did not differ between the groups. The body weight and gestational weight gain of E dams were lower (Fig. 2A and B). We also observed maternal body weight after pups’ delivery. The body weight loss of GD 21 compared to the delivery day was lower in E dams (C: 59.89 ± 9.76; E: 49.23 ± 9.72).

Figure 2
Figure 2

Parameters of gestation and lactation of control (C) and postpubertal ethanol exposed (E) dams. A) Body weight, B) body weight gain, C) feed consumption and D) water consumption of C (n = 6) and E (n = 6). Values expressed as mean ± SD. P values were calculated using a two-way ANOVA. *Significant differences between groups (P < 0.05) from post-hoc Sidak's multiple comparison test. Panel A: PInter = 0.0129, PTime < 0.0001, PTreat < 0.0026; Panel B: PInter = 0.1683, PTime < 0.0001, PTreat < 0.0001; Panel C: PInter = 0.4915, PTime < 0.0001, PTreat < 0.0001 and Panel D: PInter = 0.1071, PTime < 0.0001, PTreat < 0.0001.

Citation: Reproduction 161, 2; 10.1530/REP-20-0316

Reduced feed and water consumption were also observed (Fig. 2C and D). However, there were no differences in feeding efficiency on gestation (FEC C: 0.13 ± 0.02, E: 0.14 ± 0.02; WGCC C: 0.04 ± 0, E: 0.05 ± 0) and lactation (FEC C: 0.02 ± 0, E: 0.02 ± 0; WGCC C: 0.01 ± 0, E: 0.01 ± 0).

Postpubertal ethanol use impaired the physical development, body weight and feeding pattern of ethanol-naive offspring

The female and male offspring from E delayed all the landmarks of physical development evaluated (Table 1).

Table 1

Comparison of the mean day of landmarks of physical development in the female (n = 24/group) and male (n = 24/group) offspring from control (C) and ethanol exposed (E) groups. Values expressed as mean ± S.D. Data analyzed by Mann–Whitney test.

Parameters Females Males
C E C E
Pinna unfolding 2.3 ± 0.5 3.3 ± 0.8* 2.3 ± 0.4 3.4 ± 0.6*
Hair growth 5.0 ± 0.5 5.7 ± 0.5* 5.0 ± 0.6 5.8 ± 0.4*
Incisor eruption 9.2 ± 0.8 10.3 ± 1.2* 9.3 ± 0.9 10.4 ± 1.1*
Eye-opening 13.6 ± 0.7 14.4 ± 0.7* 13.5 ± 0.6 14.5 ± 0.6*

*Significant differences between groups (P < 0.05).

There was a decrease in the relative AGD in the males at PND 1 (E: 2.41 ± 0.18, C: 2.29 ± 0.27), but not in females (C: 1.26 ± 0.14, E: 1.18 ± 0.22) from E group. The NAL was lower at PND 1 and PND 55 in both sexes (Table 2).

Table 2

Absolute nasoanal length (cm) at postnatal day (PND) 1, PND 55 and PND 91 in the female (n = 24 – 8/group) and male (n = 24 – 8/group) offspring from control (C) and ethanol exposed (E) groups. Values expressed as mean ± S.D. P values were calculated using a two-way ANOVA.

PND Females Males
C E C E
1  5.43 ± 0.18 5.17 ± 0.26* 5.55 ± 0.14 5.25 ± 0.28*
55 19.77 ± 0.37 18.16 ± 0.56* 20.90 ± 0.58 19.12 ± 0.78*
91 21.04 ± 0.73 21.51 ± 0.98 23.7 ± 0.35 23.16 ± 0.73

*Significant differences between groups (P < 0.05) from post hoc Sidak’s multiple comparison test. Females: PInter < 0.0001, PTime < 0.0001, PTreat < 0.0001. Males: PInter < 0.0001, PTime < 0.0001, PTreat < 0.0001.

The body weight in the offspring from E was lower at birth (females C: 6.26 ± 0.54, E: 5.87 ± 0.27; males C: 7.02 ± 0.89; E: 6.04 ± 0.06) and throughout preweaning in both females (Fig. 3A), 1st and 3rd weeks, and males (Fig. 4A), 1st, 2nd and 3rd. There was an increase in body weight gain on the 4th week in both sexes, correspondent to postweaning (Figs 3B and 4B). Feed and water consumption were increased in the females (Fig. 3C and D) and decreased in the males (Fig. 4C and D) from E. There were no differences in feeding efficiency in females (FEC C: 0.15 ± 0.01, E: 0.15 ± 0.01; WCGG C: 0.05 ± 0, E: 0.05 ± 0). However, there was a significant decrease in males (FEC C: 0.19 ± 0.01, E: 0.17 ± 0.02; WCGG C: 0.06 ± 0, E: 0.05 ± 0.01).

Figure 3

Parameters of female offspring from control (C) and ethanol exposed (E) groups. A) Body weight (n = 24 - 8 / group), B) body weight gain (n = 24 - 8 / group), C) feed consumption (n = 8 / group) and D) water consumption (n = 8 / group) of females from C and E from birth to adulthood. Values expressed as mean ± S.D. P values were calculated using a two-way ANOVA. *Significant differences between groups (P < 0.05) from post-hoc Sidak's multiple comparison test. Panel A: PInter < 0.0001, PTime < 0.0001, PTreat = 0.8409; Panel B: PInter < 0.0001, PTime < 0.0001, PTreat = 0.0089; Panel C: PInter < 0.0001, PTime < 0.0001, PTreat = 0.0153 and Panel D: PInter < 0.0001, PTime < 0.0001, PTreat < 0.0001.

Citation: Reproduction 161, 2; 10.1530/REP-20-0316

    Figure 4
    Figure 4

    Parameters of male offspring from control (C) and ethanol exposed (E) groups. A) Body weight (n = 24 - 8 / group), B) body weight gain (n = 24 - 8 / group), C) feed consumption (n = 8 / group) and D) water consumption (n = 8/group) of males from C and E from birth to adulthood. Values expressed as mean ± S.D. P values were calculated using a two-way ANOVA. *Significant differences between groups (P < 0.05) from post-hoc Sidak's multiple comparison test. Panel A: PInter < 0.0001, PTime < 0.0001, PTreat < 0.0001; Panel B: PInter < 0.0001, PTime < 0.0001, PTreat < 0.0001; Panel C: PInter < 0.0001, PTime < 0.0001, PTreat = 0.0394 and Panel D: PInter < 0.0001, PTime < 0.0001, PTreat = 0.0002.

    Citation: Reproduction 161, 2; 10.1530/REP-20-0316

    Postpubertal ethanol use delayed the puberty onset and decreased reproductive organs weight and testosterone levels of ethanol-naive offspring

    Reproductive parameters of offspring from E group were affected. There was a delay in the vaginal opening (C: 41.77 ± 1.57; E: 43.75 ± 2.55) and first estrus (C: 42.40 ± 1.96, E: 45.00 ± 3.46) in the females, testicular descent (C: 25.27 ± 0.87, E: 26.69 ± 0.96) and preputial separation (C: 44.33 ± 1.40, E: 46.44 ± 1.72) in the males. In the same way, reproductive organs were also harmed as observed by the reduced organ weights in both females (Table 3) and males (Table 4) in different ages. The weight of sexual glands was lower in the males from E in the juvenile phase (C: 2.36 ± 0.40, E: 1.60 ± 0.24). There were no differences in plasma estradiol levels in females, nevertheless, testosterone levels were reduced in the males from E in the juvenile and pubertal phases.

    Table 3

    Body weight, relative reproductive organs weight, and plasma estradiol levels at postnatal day – PND 30 (juvenile), PND 44 (pubertal) and PND 100 (adult) in the female offspring from control (C, n = 8/phase) and ethanol exposed (E, n = 8/phase) groups. Values expressed as mean ± S.D. P values were calculated using a two-way ANOVA.

    Body weight (g) Ovaries (mg/g) Uterus (mg/g) Estradiol levels (pg/mL)
    Juvenile (PND 30)
     C 84.00 ± 5.21 0.40 ± 0.05 0.99 ± 0.31 129.30 ± 9.78
     E 69.75 ± 5.39* 0.37 ± 0.04 0.78 ± 0.16 128.50 ± 4.87
    Pubertal (PND 44)
     C 150.80 ± 4.98 0.52 ± 0.04 1.80 ± 0.20 154.80 ± 15.99
     E 150.60 ± 2.26 0.41 ± 0.03* 1.57 ± 0.17* 160.00 ± 16.24
    Adult (PND 100)
     C 257.60 ± 5.81 0.60 ± 0.08 2.19 ± 0.14 155.40 ± 14.37
     E 245.40 ± 1.92* 0.39 ± 0.04* 1.86 ± 0.28* 158.7 0 ± 19.83

    *Significant differences between groups (P < 0.05) from post hoc Sidak’s multiple comparison test. Body weight: PInter = 0.0006, PTime < 0.0001, PTreat < 0.0001; Ovaries: PInter < 0.0001, PTime < 0.0001, PTreat < 0.0001; Uterus: PInter = 0.6938, PTime < 0.0001, PTreat = 0.0002 and Estradiol levels: PInter = 0.4843, PTime < 0.0001, p=PTreat = 0.2577.

    Table 4

    Body weight, relative reproductive organs weight, and plasma testosterone levels at postnatal day – PND 30 (juvenile), PND 55 (pubertal) and PND 100 (adult) in the male offspring from control (C: n = 8/phase) and ethanol exposed (E: n = 8/phase) groups. Values are expressed as mean ± S.D. P values were calculated using a two-way ANOVA.

    Juvenile (PND 30) Pubertal (PND 55) Adult (PND 100) Comparison tests
    C E C E C E PInter PTime PTreat
    Body weight, g 80.13 ± 7.14 70.13 ± 9.85 228.80 ± 15.60 207.30 ± 5.70* 407.90 ± 14.77 368.90 ± 27.95 0.0757 <0.0001 <0.0001
    Testis, mg/g 3.10 ± 0.31 2.89 ± 0.21 4.88 ± 0.16 4.79 ± 0.24 3.73 ± 0.37 3.99 ± 0.30 0.0140 <0.0001 0.9481
    Epididymis, mg/g 0.60 ± 0.07 0.49 ± 0.06* 1.11 ± 0.18 0.76 ± 0.07* 1.80 ± 0.05 1.49 ± 0.14* 0.0064 <0.0001 <0.0001
    Ventral prostate, mg/g 1.81 ± 0.36 1.84 ± 0.65 2.70 ± 0.50 3.22 ± 0.53 0.1633 <0.0001 0.1910
    Seminal vesicle, mg/g 2.50 ± 0.64 1.95 ± 0.30 6.33 ± 0.96 4.64 ± 1.70* 0.1464 <0.0001 0.0081
    Testosterone, ng/mL 1.18 ± 0.23 0.48 ± 0.08* 8.80 ± 2.53 2.64 ± 0.46* 7.01 ± 1.59 6.85 ± 1.11 < 0.0001 <0.0001 <0.0001

    *Significant differences between groups (P < 0.05) from post hoc Sidak’s multiple comparison test.

    Postpubertal ethanol use compromised the reproductive function of ethanol-naive offspring

    The estrous cycle length evaluated from PND 65 to PND 80 did not differentiate in the female offspring from C (4.9 ± 0.2) and E (5.1 ± 0.3). Regarding estrous cycle regularity, 25% of control females had prolonged estrus phase, while 50% of females whose parents drank ethanol had prolonged estrus and proestrus phases. On the other hand, there was no change in the sequence of estrous cycle. The reproduction function of male from the E was affected. Pubertal males showed a significant decrease in the count sperm and daily sperm production per testis, with the acceleration of total sperm transit time. The shorter total sperm transit time was also observed on adult males associated with the acceleration of transit time in the epididymal cauda (Table 5). Furthermore, there was an increase in the percentage of sperm with morphologic abnormalities (C: 19.94 ± 8.55%, E: 32.31 ± 11.49%), including a higher incidence of tail anomalies (C: 5.19 ± 1.65%, E: 8.44 ± 3.29%).

    Table 5

    Sperm parameters at postnatal day – PND 55 (pubertal) and PND 100 (adult) in the male offspring from control (C: n = 8/phase) and ethanol exposed (E: n = 8/phase) groups. Values expressed as mean ± S.D. P values were calculated using a two-way ANOVA.

    Parameters Pubertal (PND 55) Adult (PND 100) Comparison tests
    C E C E PInter PTime PTreat
    Testis
     Spermatid number (×106/g/day) 85.63 ± 4.97 71.29 ± 11.9* 92.47 ± 17.79 97.27 ± 21.77 0.0384 <0.0012 0.2028
     Daily sperm production (×106/g/testis/day) 14.04 ± 0.82 11.69 ± 1.95* 15.16 ± 2.92 15.95 ± 3.57 0.0492 0.0021 0.2290
    Caput /corpus epididymal
     Sperm number (×106/g/organ) 89.29 ± 22.4 61.25 ± 30.47 192.3 ± 28.71 197.30 ± 40.37 0.1283 <0.0001 0.3216
     Sperm transit time (days) 0.99 ± 0.35 0.54 ± 0.39 4.22 ± 1.49 3.41 ± 1.25 0.5208 <0.0001 0.0762
    Cauda epididymal
     Sperm number (×106/g/organ) 30.71 ± 13.36 27.14 ± 10.94 569.7 ± 142.80 539.4 ± 116.30 0.6863 <0.0001 0.6152
     Sperm transit time (days) 0.14 ± 0.08 0.11 ± 0.08 9.74 ± 3.35 5.32 ± 2.06* 0.0120 <0.0001 0.0111
    Total sperm transit time (days) 1.14 ± 0.42 0.65 ± 0.40* 13.96 ± 4.62 8.74 ± 3.22* 0.0411 <0.0001 0. 0238

    *Significant differences between groups (P < 0.05) from post hoc Sidak’s multiple comparison test.

    Discussion

    This study highlights how lifestyle after adolescence influences future generations outcomes. Although studies highlight the risks of using ethanol during pregnancy, the impacts of postpuberty use are poorly known. This is the first study to conduct an extensive investigation on the development and reproduction parameters of ethanol-naive offspring. The maternal and paternal high alcohol use during postpuberty, besides affecting dams previously exposed, also impaired offspring parameters throughout life.

    Although ethanol intake did not alter the gestation duration and litter size, we observed a lower body weight and feed intake after 2nd gestational week and lower gestational weight gain in dams exposed to ethanol on postpuberty (E). Body weight index, gestational weight gain and food consumption influence pregnant and fetal outcomes (Kind et al. 2006, Brett et al. 2014). Preconception nutrition is also a factor that affects maternal metabolic response to pregnancy (Kind et al. 2006). It is known that high ethanol intake could interfere energy balance, thermogenic metabolism and lead to a deficient nutritional status (Addolorato et al. 1998, Sebastiani et al. 2018). Plasma leptin levels increased by alcohol withdrawal have also been observed (Kiefer et al. 2005). We found lower body weight loss after delivery in E dams. Thus, we correlated some of these differences in maternal weight loss with gravid uterine weight (Kind et al. 2006). As litter size was similar between C and E dams, we suggest lower fetal and placental weight since offspring E had low birth weight and fetal and placental weight are positively correlated near birth (Brett et al. 2014). Taken together, we associate the lower body weight and feed intake of dams to the late effects of alcohol exposure that were accentuated during pregnancy, a period of metabolic and physiological changes.

    We observed a lower body weight at birth and throughout preweaning in the female and male offspring from E group. The weight gain and nasoanal length were also lower. Lower body weight at birth can indicate an adverse fetal environment (Rando & Simmons 2015) and it may relate to fetal programming (Hanson & Gluckman 2008). Adolescence alcohol use can lead to long-term effects in adulthood, as impairment of the maternal endocrine system and maternal–fetal interface (Zhang et al. 2005). Besides, alcohol can alter the fetal supply of nutrients through multiple mechanisms including the inadequate absorption and distribution of nutrients and increased urine excretion of key micronutrients (Sebastiani et al. 2018). Lower body weight of offspring (Jabbar et al. 2016) and inadequate placental function, observed by changes in placental morphology, gene expression and fetal growth restriction programming (Gardebjer et al. 2014) were consequences to preconception maternal ethanol intake. Placental dysfunctions can suppress essential fetal nutrients, even with adequate maternal nutrient availability (Brett et al. 2014). Taken together, offspring outcomes have been highly associated with maternal preconception nutrition which is recognized for influencing the oocyte or embryo and thereby the competency of the fetus and its postnatal life (Kind et al. 2006). Nevertheless, paternal experiences also play an additional role (Robertson 2005, Rando & Simmons 2015). Chang et al. (2017), using chronic paternal ethanol exposure, observed a reduction of the gestational sac weight and a decrease in placental efficiency. The seminal fluid stimulates the female reproductive tract to produce growth factors and cytokines which protect the embryo (Robertson 2005) and changes in the seminal signalization due to environmental exposure are capable of influencing descendants’ phenotype (Bromfield et al. 2014).

    The insulin-like growth factor (IGF) is important for fetal and postnatal development and IGF deficiency implicates signaling pathways and normal body growth (Kanaka-Gantenbein et al. 2003). Similarly, the endothelial growth factor (EGF) plays a role in regulating the activity of epidermal and epithelial tissues as eye-opening, hair growth, and incisor eruption (Smart et al. 1989, Calamandrei & Alleva 1989). Although Emanuele et al. (2001) did not report changes in the IGF offspring from paternal alcoholic intake, we observed delay in all landmarks of physical development and lower body growth in offspring from E. In this way, we hypothesized possible endocrine and metabolic programming highlighted by both maternal and paternal ethanol intake.

    Interestingly, the programming ethanol-induce hypothesis could be supported by the feeding pattern of offspring from the E, since reduced feed and water intake in males were similar to the dams. However, the female feeding pattern was independent of maternal results. The offspring feeding pattern may be correlated, partially, to the modified learning because of parents’ toxic agent exposure (Knee et al. 2004). We also noticed an acute increase in body weight, weight gain and feed consumption in both females and males from the E following postweaning. This attempt to re-establishment can be associated with catch-up growth, characterized by an accelerated growth of weight or length following a period of slower development (Ozanne & Hales 2004). The lower neonatal body weight can lead to activating catch-up growth (Ozanne & Hales 2004), as observed in our results.

    The lower AGD at PND 1 of males from E indicates either decreased androgens production or action during fetal development, and our results agree with a clinical study in children sired by paternal ethanol use (Xia et al. 2018). The AGD is androgen-dependent for perineal muscle growth, thus, reduced androgen levels lead to smaller AGD (Xia et al. 2018, Welsh et al. 2010). Besides, the decreased sexual glands and epididymis weight from juvenile to adult, delayed puberty onset, and impairment on sperm parameters were observed in the males from E. We correlated these findings with the decreased testosterone levels, observed in the juvenile and pubertal periods. Lower testosterone on sexual maturation leads to delay maturation on Leydig cells and the hypothalamic–pituitary–gonadal (HPG) axis (Rando & Simmons 2015, Asimes et al. 2018). Modifications in male sexual biomarkers, as observed in this study, are risk factors for disorders that could emerge in adulthood, such as cancer and infertility (Skakkebaek et al. 2001).

    The decreased spermatid count and daily sperm production in the male offspring from the E suggests damage to the spermatogenic process which can be a consequence of reduced testosterone levels, reported in our findings. In addition, the acceleration in total sperm transit time through epididymis observed in the pubertal males was also noticed in the adult. Reduced testicular, and epididymal germ cells concentration can contribute to faster sperm transit time as well as the reduction in the epididymis weight (Oliva et al. 2006). The increased percentage of sperm with morphological abnormalities of offspring from E can be either due to failures in the spermatogenic process or in sperm maturation. Inadequate signalization of epididymal factors which plays a role in maturation can drive to these abnormalities (Koch et al. 2015, Zi et al. 2015), while low testosterone levels limit the spermatogenic process (Zi et al. 2015). Taken together, the impairments in the male whose parents consumed ethanol could lead to inadequate reproduction function. However, we do not know how harmful this damage is for reproductive capacity. Although some parameters were re-established in adulthood, highlighting its modulation according to testosterone levels, the early hormonal imbalance apparently harms the long-term reproduction.

    Despite evaluating a smaller number of reproductive parameters in the females compared to males, the postpubertal parental ethanol consumption also induces reproductive adaptations in female offspring. There were prolonged estrus and proestrus phases in the E females, delayed puberty onset and reduced ovarian and uterus weight in the pubertal and adult period. Thus, these observations suggest a hormone level imbalance or inadequate HPG axis function (Picut et al. 2015). Nevertheless, the decreased reproductive organs can not be directly correlated to estradiol because we did not report changes in this hormone. As reported by Sliwowska et al. (2016), female offspring whose dams consumed ethanol had a Kiss-1 expression decreased, impairing reproduction control and excitatory pathways signaling. Thus, deficiency Kiss-1 can affect the HPG axis (Navarro et al. 2004). One of the limitations of this study was due to the fact that we did not have the ovarian tissue to evaluate the ovarian morphology and function. Since estradiol levels were similar in the groups, we associated the delay puberty onset with reduced ovarian mass and we hypothesized an inadequate response between ovarian receptors and gonadotrophic hormones. The decrease of the IGF could be an additional factor for reproductive impairment in females (Cohick et al. 2015).

    Alterations on the development and reproduction parameters of offspring by parents ethanol exposure can be also associated with maternal care (Walker et al. 2004, Amorim et al. 2011). Using the voluntary ethanol consumption model, our laboratory reported lower maternal care on dams that were early exposed to ethanol, representing long-term impairs for the pups (Amorim et al. 2011). In addition, epigenetic mechanisms have been one of the main factors which lead to changes in offspring. Desynchronization of puberty factors and the lower GnRH expression have been reported in offspring due to paternal alcohol-induced epigenetic mechanism (Asimes et al. 2018). However, studies have not been strongly capable of correlating sexual DNA methylation with progeny outcomes, suggesting a DNA methylation independent mechanism in the transmission of epigenetic errors induced by preconception alcohol exposure (Chang et al. 2017). Thus, maternal, paternal, or both parents exposure should be considered for the descendant outcomes, once the methylation pattern could change according to the individual or both contributions (Asimes et al. 2017).

    In summary, the results presented here suggest the potentially alarming possibility in which exposure to alcohol on postpuberty produces changes that result in profound alterations to the offspring’ parameters, since the high ethanol use on postpuberty adversely interferes with parameters of dams exposed previously and harms the ethanol-naive female and male offspring. Delay in physical development, lower body weight and reduced reproductive organs were the main results reported. Interestingly, our findings were similar to fetal ethanol exposure studies (Weinberg et al. 2008), highlighting that the postpuberty is also a vulnerable period to ethanol use. Given the limitation of study design, we cannot distinguish which parent contributed the most to observed changes. However, our previous laboratory studies along with published data about preconception maternal ethanol exposure strongly suggest a maternal influence as the main, as observed by low maternal care, interruptions on maternal-fetal communication, irregular placental efficiency and fetal growth programming. Despite the limitations, our approach conducted to the interesting initial results, demonstrating that high postpubertal ethanol intake influences descendant’s parameters.

    Future studies are needed to identify the mechanisms responsible for ethanol-naive offspring outcomes as well as to explore epigenetic modifications in parents’ germline. We also support studies conducted with different amounts of ethanol and separation of maternal and paternal contribution. In this regard, preventive measures can be established aiming awareness about the importance of an adequate lifestyle and the consequences of postpuberty high ethanol use on consumer’s health and on the development and reproduction parameters of future generations.

    Declaration of interest

    The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

    Funding

    This study was financed by the Grant 2018/12354-5, São Paulo Research Foundation (FAPESP), and Coordination for the Improvement of Higher Education Personnel – Brazil (CAPES) – Finance Code 001.

    Author contribution statement

    V C F conceived the study, performed experiments, analyzed and finished data, and wrote the paper. A R G, V M B C, and R S N performed experiments. P F F P and F E M provided training to perform the experiments and intellectual input for the experimental design and data analysis. All authors contributed to editing the paper.

    Acknowledgements

    The authors would like to acknowledge Jorge W F Barros (Institute of Biosciences of Botucatu/UNESP) for assistance with experimental procedures and Sérgio A A Santos (Institute of Biosciences of Botucatu/UNESP) for analysis of plasma hormones.

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      Figure 1

      Diagram of the experimental design. GD, gestational day; LD, lactational day; PND, postnatal day. Rats were divided into a control group (C) and an ethanol (10%, v/v) exposed group (E) from PND 65-80 (postpuberty), with withdrawal after this period. The rats were mated at PND 100. The dams were monitored. At the birth of pups (n = 24/sex/group), the physical development, body weight, and feed and water intake were monitored. The organs and reproductive hormones were analyzed in three phases: juvenile (PND 30), pubertal (PND 44/female, PND 55/male) and adult (PND 100). The female and male reproductive function was observed by the estrous cycle and sperm parameters.

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      Figure 2

      Parameters of gestation and lactation of control (C) and postpubertal ethanol exposed (E) dams. A) Body weight, B) body weight gain, C) feed consumption and D) water consumption of C (n = 6) and E (n = 6). Values expressed as mean ± SD. P values were calculated using a two-way ANOVA. *Significant differences between groups (P < 0.05) from post-hoc Sidak's multiple comparison test. Panel A: PInter = 0.0129, PTime < 0.0001, PTreat < 0.0026; Panel B: PInter = 0.1683, PTime < 0.0001, PTreat < 0.0001; Panel C: PInter = 0.4915, PTime < 0.0001, PTreat < 0.0001 and Panel D: PInter = 0.1071, PTime < 0.0001, PTreat < 0.0001.

    • Figure 3

      Parameters of female offspring from control (C) and ethanol exposed (E) groups. A) Body weight (n = 24 - 8 / group), B) body weight gain (n = 24 - 8 / group), C) feed consumption (n = 8 / group) and D) water consumption (n = 8 / group) of females from C and E from birth to adulthood. Values expressed as mean ± S.D. P values were calculated using a two-way ANOVA. *Significant differences between groups (P < 0.05) from post-hoc Sidak's multiple comparison test. Panel A: PInter < 0.0001, PTime < 0.0001, PTreat = 0.8409; Panel B: PInter < 0.0001, PTime < 0.0001, PTreat = 0.0089; Panel C: PInter < 0.0001, PTime < 0.0001, PTreat = 0.0153 and Panel D: PInter < 0.0001, PTime < 0.0001, PTreat < 0.0001.

    • View in gallery
      Figure 4

      Parameters of male offspring from control (C) and ethanol exposed (E) groups. A) Body weight (n = 24 - 8 / group), B) body weight gain (n = 24 - 8 / group), C) feed consumption (n = 8 / group) and D) water consumption (n = 8/group) of males from C and E from birth to adulthood. Values expressed as mean ± S.D. P values were calculated using a two-way ANOVA. *Significant differences between groups (P < 0.05) from post-hoc Sidak's multiple comparison test. Panel A: PInter < 0.0001, PTime < 0.0001, PTreat < 0.0001; Panel B: PInter < 0.0001, PTime < 0.0001, PTreat < 0.0001; Panel C: PInter < 0.0001, PTime < 0.0001, PTreat = 0.0394 and Panel D: PInter < 0.0001, PTime < 0.0001, PTreat = 0.0002.

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