Ethanol-treated Females Research Articles

Maternal Chronic Ethanol Exposure Decreases Stress Responses in Zebrafish Offspring

In humans, prenatal alcohol exposure can cause serious health issues in children, known collectively as Foetal Alcohol Spectrum Disorders (FASD). Despite the high prevalence of FASD and a lack of effective treatments, the underlying mechanisms causing the teratogenic action of ethanol are still obscure. The limitations of human studies necessitate the use of animal models for identifying the underlying processes, but few studies have investigated the effects of alcohol in the female germline. Here, we used the zebrafish Danio rerio to investigate the effects of chronic (repeated for seven days) exposure to alcohol. Specifically, we tested whether the offspring of females chronically exposed to ethanol during oogenesis exhibited hormonal abnormalities when subjected to a stressor (alarm cue) as larvae, and if they exhibited anxiety-like behaviours as adults. Exposure to alarm cue increased whole-body cortisol in control larvae but not in those of ethanol-treated females. Furthermore, adult offspring of ethanol-treated females showed some reduced anxiety-like behaviours. These findings suggest that the offspring of ethanol-treated females had reduced stress responses. This study is the first to investigate how maternal chronic ethanol exposure prior to fertilisation influences hormonal and behavioural effects in a non-rodent model.

Conditioned social preference and reward value of activating oxytocin-receptor-expressing ventral tegmental area neurons following repeated daily binge ethanol intake.

Individuals with alcohol use disorder (AUD) exhibit a disruption of social behavior and dysregulation of oxytocin signaling in the brain, possibly reflecting decreased activation of oxytocin receptors (OxTRs) in reward pathways in response to social stimuli. We hypothesize that daily binge ethanol intake causes a deficit in social reward and oxytocin signaling in the ventral tegmental area (VTA). After 9weeks of daily binge ethanol intake (blood ethanol concentration >80mg%), OxTR-cre mice underwent conditioned place preference for social reward. Separate groups of mice were tested for the effects of binge ethanol on voluntary social interactions, food reward, locomotion, and anxiety-like behaviors. A subset of mice underwent transfection of OxTR-expressing VTA neurons (VTAOxtr ) with a light-sensitive opsin, followed by operant training to respond to light delivered to VTA. Ethanol-naïve male mice increased the time spent on the side previously paired with novel mice while ethanol-treated mice did not. Binge ethanol did not affect conditioned place preference for food reward in males, but this response was weakened in ethanol-treated females. Ethanol treatment also caused a sex-specific impairment of voluntary social interactions with novel mice. There were minimal differences between groups in measures of anxiety and locomotion. Ethanol-naïve mice had significantly greater operant responding for activation of VTAOxtr than sham-transfected mice but ethanol-treated mice did not. There was no difference in the number of VTAOxtr after binge ethanol. Daily binge ethanol causes social reward deficits that cannot be explained by nonspecific effects on other behaviors, at least in males. Only ethanol-naïve mice exhibited positive reinforcement caused by activation of VTAOxtr while daily binge ethanol did not alter the number of VTAOxtr in either males or females. Thus, subtle dysregulation of VTAOxtr function may be related to the social reward deficits caused by daily binge ethanol.

Reactive, Adult Neurogenesis From Increased Neural Progenitor Cell Proliferation Following Alcohol Dependence in Female Rats.

Hippocampal neurodegeneration is a consequence of excessive alcohol drinking in alcohol use disorders (AUDs), however, recent studies suggest that females may be more susceptible to alcohol-induced brain damage. Adult hippocampal neurogenesis is now well accepted to contribute to hippocampal integrity and is known to be affected by alcohol in humans as well as in animal models of AUDs. In male rats, a reactive increase in adult hippocampal neurogenesis has been observed during abstinence from alcohol dependence, a phenomenon that may underlie recovery of hippocampal structure and function. It is unknown whether reactive neurogenesis occurs in females. Therefore, adult female rats were exposed to a 4-day binge model of alcohol dependence followed by 7 or 14 days of abstinence. Immunohistochemistry (IHC) was used to assess neural progenitor cell (NPC) proliferation (BrdU and Ki67), the percentage of increased NPC activation (Sox2+/Ki67+), the number of immature neurons (NeuroD1), and ectopic dentate gyrus granule cells (Prox1). On day seven of abstinence, ethanol-treated females showed a significant increase in BrdU+ and Ki67+ cells in the subgranular zone of the dentate gyrus (SGZ), as well as greater activation of NPCs (Sox2+/Ki67+) into active cycling. At day 14 of abstinence, there was a significant increase in the number of immature neurons (NeuroD1+) though no evidence of ectopic neurogenesis according to either NeuroD1 or Prox1 immunoreactivity. Altogether, these data suggest that alcohol dependence produces similar reactive increases in NPC proliferation and adult neurogenesis. Thus, reactive, adult neurogenesis may be a means of recovery for the hippocampus after alcohol dependence in females.

Oxidative stress and cellular and tissue damage in organogenic outbred mouse embryos after moderate perigestational alcohol intake.

Perigestational alcohol consumption by CF-1 mouse, from before mating up to the period of embryo organogenesis, leads to retarded early embryo development and neural tube defects. Here, we addressed if perigestational alcohol ingestion up to Day 10 of pregnancy induces oxidative stress and changes in macromolecules and organ tissues of early organogenic embryos. Adult CF-1 female mice were administered 10% ethanol in their drinking water for 17 days prior to mating and until Day 10 of gestation, whereas control females were administered ethanol-free water. Our results demonstrated significantly reduced Catalase abundance and activity and increased glutathione content in the embryos of ethanol-treated females. The nitrite level was significantly reduced, but TBARS (thiobarbituric acid reactive substances) content, an index of lipid peroxidation, did not change. Embryos derived from ethanol-treated females also showed higher abundance of 3-nitrotyrosine (3-NT)-containing proteins in all tissues, compared to the control group. Apoptosis was significantly increased in the ectoderm and mesoderm, but not in the heart-although this organ did contain more cleaved Caspase-3-positive cardiomyocytes per area of ventricular myocardium than controls. In sum, moderate perigestational alcohol ingestion up to Day 10 of gestation in mice induces oxidative stress by altering radical nitrogen species and antioxidant enzymatic and non-enzymatic mechanisms in embryos. Further, generalized protein nitration, due to unbalanced nitric oxide levels associated with tissue-specific apoptosis, was detected in embryos, suggesting that oxidative mechanisms may play an important role in the perigestational alcohol-induced malformation of organogenic embryos exposed to ethanol.

Effects of chronic alcohol consumption and withdrawal on the response of the male and female hypothalamic–pituitary–adrenal axis to acute immune stress

The hypothalamic–pituitary–adrenal (HPA) axis plays a central role in the response to stress, and its activity is sexually dimorphic and modulated by sex steroids. Recent work indicates that HPA axis functioning is disturbed by chronic alcohol consumption and subsequent withdrawal in rats of both sexes, but particularly in females. To examine the influence of sex steroid hormones in HPA axis response to acute stress after ingestion of a 20% ethanol solution over 6months and subsequent withdrawal (2months), intact males, and estradiol- and oil-injected ovariectomized females received a single intraperitoneal injection of lipopolysaccharide (LPS). Six hours after LPS administration, corticosterone concentrations were increased in all male groups; however, in ethanol-treated rats they remained below those of control and withdrawn rats. mRNA levels of corticotrophin-releasing hormone (CRH) increased, and were identical in all groups after LPS stimulation, whereas those of vasopressin, although increased, remained below control levels. LPS stimulation elevated corticosterone concentrations in all oil-injected female groups, but did not alter those of estradiol-injected females. In oil- and estradiol-injected ethanol-treated females, CRH mRNA levels did not change in response to LPS stimulation, whereas those of vasopressin increased, but stayed below control levels. In withdrawn oil- and estradiol-injected females, CRH and vasopressin gene expression increased, but did not reach control levels. These data show that prolonged alcohol consumption produces long-lasting, possibly irreversible, changes in the neuroendocrine system that regulates the production of corticosteroids, and that these consequences are more profound in females, particularly when estrogen levels are low.

Periconceptional alcohol consumption-induced changes in embryonic prostaglandin E levels in mouse organogenesis: Modulation by nitric oxide

The mechanisms of the teratogenic effects of maternal alcohol consumption remain unclear. The aim of the present work was to study the organogenic PGE 2 levels and the modulation of PGE 2 levels by NO after periconceptional alcohol ingestion. Female mice were intoxicated with a 10% ethanol in drinking water before pregnancy and up to day 10 of gestation. The PGE 2 released from organogenic embryos was measured by radio immunoassay following incubation with or without the addition of either a NO donor or a NO synthase (NOS) inhibitor. In the ethanol-treated females, we found increased percentages of retarded embryos, associated with a significantly elevated resorption rate ( p < 0.05 ), very high quantities of morphologically abnormal E.10 embryos ( p < 0.001 ) and significantly increased PGE 2 release, as compared to the embryo parameters of control females. While in the control-derived E.10 embryos the NO donor produced significantly increased PGE 2 release, in the ethanol-derived embryos decreased quantities of PGE 2 were observed. L-NMMA inhibited PGE 2 release in both control and ethanol-derived embryos at different concentrations, whereas it decreased PGE 2 content in controls but not in ethanol-derived embryos. The periconceptional alcohol ingestion produced excessive PGE 2 release, decreased PGE 2 content and disruption of the regulatory NO–PGE 2 pathways. These PGs alterations may be related to delayed organogenesis and abnormal neural tube development after chronic periconceptional consumption of alcohol.

Alterations in preimplantation in vivo development after preconceptional chronic moderate alcohol consumption in female mice.

Although many studies have explored the effects of acute or chronic ethanol exposure during the postimplantation period on embryo/fetal development, few reports have described the ethanol effects on preimplantation embryo development. Little is known about the effects of ethanol consumption prior to gestation on embryo growth. Recently, we have shown that chronic moderate ethanol intake by prepubertal female mice reduces the ovulatory response and impairs in vitro fertilization and in vitro embryo preimplantation development. The purpose of the present work was to evaluate the effects of preconceptional chronic moderate ethanol ingestion on preimplantation embryo morphology and differentiation, the timing of cleavage and embryo growth in vivo, and to determine the time pattern in which alterations appear. Prepubertal female mice were treated with 10% (w/v) ethanol for 30 days prior to conception. After inducing ovulation on day 27 and 29 of the ethanol treatment, females were mated with control males and the day of presence of vaginal plug was day 1. On day 1, a decreased percentage of normal fertilized oocytes, elevated parthenogenetic oocyte activation and unfertilized eggs with abnormal metaphase II were found in ethanol-treated, compared to control females. On day 2, while any differences in the total percentage of 2-cell embryos were observed, the treated females had a significantly higher percentage of morphologically abnormal embryos, compared to control females. On day 3, the preconceptional consumption of ethanol produced significantly reduced percentages of compacted morulae and an increased percentage of uncompacted morulae. The total percentage of morulae in the treated females was lower than in controls. On day 4, ethanol-treated females showed significantly decreased percentages of hatched attached blastocysts and increased early blastocyst and morula percentages, compared to controls. Thus, preconceptional chronic moderate ethanol ingestion by prepubertal female mice produced retarded development, impaired blastocyst hatching, abnormal embryo morphology and embryo loss by fragmentation due to alterations induced in the female gamete.

Low chronic ethanol consumption affects ovulation and PGE synthesis by the cumulus cell masses in mice

Central and gonadal function can be affected by chronic consumption of high and moderate doses of ethanol. Few studies have been conducted to determine the effect of ethanol intake at ovarian and gamete level. Previously, we showed that fertilization rates of low chronic ethanol treated female mice were diminished. Also, our recent results indicated that moderate chronic intake of ethanol by immature females could alter the ovulatory quantity and produce morphological alterations in the superovulated oocytes. Furthermore, PGE production by oocyte cumulus complexes (OCCs) was reduced in the females treated with 10% (w/v) ethanol. In the present investigation, we studied the effects of 5% ethanol treatment given to immature mice for 30 days on the quality and quantity of oocytes superovulated at 16 h posthuman chronic gonadotrophin. Treated females had impaired ovulation rates (P ⪢ 0.05) as compared to the controls. The percentage of activated and morphologically abnormal oocytes was elevated in the ethanol-treated females (P⪢ 0.05). PGE synthesis by the OCCs was higher than in the controls (P⪢ 0.01). In summary, the administration of long-term ethanol at a relatively low dose to immature females produces decreased ovulation rates, abnormal oocyte morphology with high spontaneous activation and altered levels of PGE production by the oocytes' cumulus complexes. The relationship between the oocyte quality and abnormal synthesis of PGE is discussed.

Mouse oocyte quality and prostaglandin synthesis by cumulus oocyte complex after moderate chronic ethanol intake

Chronic ingestion of ethanol produces a variety of effects on female reproductive function, depending on the dose and the exposure time but the mechanism of alcohol-induced ovarian failure has been little studied. Also the effects of chronic ethanol consumption on the oocyte quality in relation to morphological alterations and PGE synthesis by the oocyte cumulus complexes (OCCs) have not been described. In this study, immature female mice were treated with 10% ethanol in drinking water for 30 days. Then they were induced to superovulate, and at 14, 16 and 20 h post-hCG the quality of the ovarian and oviductal oocytes and PGE production by OCC was determined. At 14 h post-hCG, the percentage of oviductal immature oocytes was increased in the ethanol-treated females ( P < 0.05). At 16 h post-hCG, the percent of oviductal activated oocytes was higher in the treated females ( P < 0.05), and the ovarian immature oocytes were decreased as compared to the control females ( P < 0.05). At 20 h post-hCG, the ethanol-treated females had higher percents of activated oocytes in the oviducts and in the ovaries ( P < 0.05) with respect to the controls. PGE synthesis by OCCs, assessed by RIA, was decreased in the treated female mice ( P < 0.001). In summary, moderate chronic ethanol treatment in immature female mice can produce morphologic abnormalities in the oocytes (high parthenogenetic activated rates) and altered PGE production in the OCCs.

Effects of low chronic ethanol exposure on prostaglandin E synthesis by preimplantation mouse embryos

Embryo prostaglandin (PG) synthesis plays a role in the modulation of embryo metabolism and viability, and in the beginning of the implantation. The effects of ethanol consumption seem to be mediated at least in part by PGs. Increased PG production of postimplantation embryos is associated with retardation and abnormalities in the gestational period. The aim of this study was to find out the effects of low chronic ethanol ingestion by mice, previous to pregnancy, on the PGE released by in vitro and in vivo derived embryos. Immature females or adult males were treated with 5% ethanol for 30 days. After fertilization and mating, two-cell embryos, morulae and blastocysts were collected. The PGE synthesis and release were measured by radioimmunoassay. PGE production by in vitro derived two-cell embryos from ethanol-treated females was lower than in the control group ( P < 0.01). Also, PGE production was reduced when two-cell embryos came from ethanol-treated males ( P < 0.01). There were no differences in PGE synthesis by in vitro derived morulae and blastocysts in these groups. Two-cell embryos derived from mating produced lower quantities of PGE when they came from ethanol-treated females mated with control males, as compared to the control group. PGE release by in vivo derived blastocysts from ethanol-treated females was reduced significantly, as compared to the control group ( P < 0.01). We conclude that a low concentration of ethanol administered chronically to immature females reduces PGE synthesis and release by two-cell embryos from culture in vitro, and by embryos of days 2 and 4 from in vivo development.

Impaired mouse fertilization by low chronic alcohol treatment.

Little is known about the effects of low chronic alcohol intake on fertility, particularly in females. Recently, we have shown that chronic 10% (w/v) ethanol treatment affects in-vitro fertilization of mouse female gamete. The aim of this study was to solve questions concerning the lowest dose and duration of ethanol treatment required to alter the fertility of immature and adult female and adult male mouse. Mice were treated with 5% and 2.5% (w/v) ethanol in drinking water for 4 weeks. The in-vitro fertilization rates were significantly decreased with the 5% ethanol when oocytes from prepubertal and pubertal ethanol-treated females were inseminated with spermatozoa from adult control males. The in-vitro fertilization rates were not diminished when oocytes from control females were inseminated with spermatozoa from adult ethanol-treated males. Haploid oocytes were increased when oocytes came from immature females treated with ethanol. The in-vitro fertilization rates were not decreased in adult treated females. The in-vivo fertilization rates were not modified when prepubertal ethanol-treated females were mated with adult control males. Fragmented oocytes, in the in-vitro fertilization experiments, were significantly increased when they came from prepubertal and adult treated females inseminated with ethanol-treated males. These results show that there is a threshold of the ethanol dose to produce an effect. Chronic low ethanol ingestion by immature female mice has a deleterious effect on their in-vitro fertilization. Furthermore, acute ethanol ingestion by adult females during the induction of ovulation resulted in high parthenogenetic activation and fragmentation of mouse oocytes.

Prenatal ethanol and the prepubertal sexually dimorphic nucleus of the preoptic area

The volume of the sexually dimorphic nucleus of the preoptic area of the hypothalamus (SDN-POA) was determined in 14–31-day-old male and female rats whose mothers received a liquid diet containing 5% w/v ethanol from day 8 of gestation to parturition. Pair-fed dams received as a nutritional control an equal volume of an isocaloric liquid diet with maltose-dextrin in place of ethanol. Normal controls had laboratory rat chow and water available ad lib. The SDN-POA volume of ethanol-exposed males was significantly reduced compared to the pair-fed and normal males, and became indistinguishable from the SDN-POA volumes of the pair-fed and normal females. Ethanol-treated females also had a markedly reduced SDN-POA volume compared to the pair-fed and normal females. Our findings indicate that the SDN-POA of prepubertal rats of both sexes is sensitive to the effects of in utero ethanol exposure. While plasma testosterone, progesterone and estradiol titers, which we measured in fetuses on gestation day 22, were differentially affected by maternal ethanol consumption, the alterations by themselves cannot adequately explain the effects of prenatal ethanol exposure on the developing SDN-POA.

Central nervous system myelination in rats exposed to ethanol in utero.

Pregnant rats were pair-fed using isocaloric control or 6.6% (v/v) ethanol liquid diets. At 18, 25, and 53 days, the in vivo incorporation of [3H] leucine and [14C] glucose into proteins and lipids of three central nervous system (CNS) myelin subfractions was examined in the offspring (control and ethanol pups) of control and ethanol-treated females. With few exceptions, the pattern of CNS myelination appeared near-normal in the ethanol pups. The ethanol pups had brain weights and total myelin protein content within the normal range. In addition, the amount of protein in light, medium, and heavy myelin was normal at 18 and 25 days. However, the ethanol pups had an excess of the chemically and morphologically immature heavy fraction at 53 days. The incorporation of [3H] leucine and [14C] glucose into myelin subfractions and separated proteins and lipids was similar in control and ethanol pups. The findings of near-normal CNS myelination in the offspring of female rats fed an ethanol liquid diet during gestation differ from our previous findings of a premature onset and slowdown of active CNS myelination in the offspring of female rats that consumed an ethanol liquid diet for one month prior to conception as well as during gestation.

The effect of chronic maternal alcohol consumption on the development of central nervous system myelin subfractions in rat offspring

Female Sprague-Dawley rats were pair-fed using isocaloric control or 6.6% (v/v) ethanol liquid diets for one month prior to conception and throughout gestation. The development of three central nervous system (CNS) myelin subfractions was examined in the 18- to 54-day-old offspring (control and ethanol pups) of control and ethanol-treated females. During the same age period, the in vivo incorporation of [3H] leucine into myelin subfraction proteins and of [14C] glucose into myelin subfraction lipids was also studied.Despite the fact that the ethanol pups consistently had smaller brain and body weights than normal rats, 18- and 25-day-old ethanol pups had more CNS myelin than control pups. However, the increased myelin content was due solely to an excess of the chemically and morphologically immature heavy myelin fraction. Fifty-four-day-old ethanol pups had a small deficit of total CNS myelin.Developing ethanol pups also demonstrated abnormal rates of incorporation of [3H] leucine and [14C] glucose into the myelin subfractions. At 18 and 25 days, the ethanol pups incorporated more of both precursors into all fractions of myelin, while at 54 days they demonstrated decreased incorporation.Although the quantity and rate of synthesis of individual myelin subfractions was abnormal in ethanol pups, the protein composition of the separated subfractions was normal. In addition, the ethanol pups had a normal distribution of 3H and 14C radioactivity, in separated myelin subfraction proteins and lipids, respectively.The results of the present study suggest that the consumption of a 6.6% (v/v) ethanol liquid diet prior to conception and throughout gestation resulted in a premature onset and slow-down of active myelination.