DELETERIOUS EFFECTS OF CHRONIC MODERATE ALCOHOL INTAKE BY FEMALE MICE ON PREIMPLANTATION EMBRYO GROWTH IN VITRO

Elisa Cebral*, Andrea Lasserre, Valeria Rettori and Martha A. de Gimeno

Centro de Estudios Farmacológicos y Botánicos (CEFYBO-CONICET), Serrano 669 (1414), Capital Federal, Argentina

Received 7 August 1998; in revised form 6 January 1999; accepted 24 February 1999


    ABSTRACT
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The susceptibility of preimplantation stages of embryo development to preconceptional alcohol ingestion by females has had little investigation. We have recently shown that chronic 10% (w/v) ethanol intake by young female mice reduces the ovulatory response and impairs the quality of the oocytes. The aim of this study was to investigate the effects of 10% ethanol administration for 30 days on immature female mice on the day of in-vitro fertilization (day 1) and on preimplantation embryo development. Female mice were ovulated on days 27 and 29 of ethanol treatment and in-vitro fertilization was performed 16 h post-human chorionic gonadotrophin administration (day 30). The oocytes from the ethanol-treated females inseminated with spermatozoa from control males, showed a significantly higher percentage of parthenogenetic activation compared to the control females. An increased percentage of fragmented oocytes was found after insemination, compared to control females. When the embryos were cultured, the percentage of 2-cell (day 2), 4-cell (day 3) embryos, and compacted morulae (day 4) was significantly reduced in treated females, compared to control females. On day 5, we found a highly significant decreased percentage of early and expanded blastocysts in the ethanol-treated females. The percentage of hatching and hatched (extruded) blastocysts was also reduced significantly in treated females at days 6 and 7 (blastocyst stages). An increased percentage of morphologically abnormal embryos was found on days 5 and 6 in ethanol-treated females compared with controls. We conclude that chronic moderate ethanol ingestion by young female mice results in decreased fertilization, embryo growth retardation, cleavage arrest, and abnormal embryo development in vitro.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Chronic alcohol consumption during pregnancy produces a variety of deleterious effects in developing embryos/fetuses, from growth impairment to birth abnormalities (Kotch and Sulik, 1992Go). The most severe manifestation of ethanol teratogenesis is the fetal alcohol syndrome (FAS) (Weston et al., 1994Go; Abel, 1995Go), which can occur in a substantial proportion of infants born to mothers who are chronic heavy daily drinkers (Kaufman, 1997Go). The deleterious effects of ethanol have been demonstrated in rodents when a liquid ethanol diet was administered to pregnant mice during the period of organogenesis (Randall and Taylor, 1979Go). There has been little emphasis on the effects of maternal alcohol intake on early embryo development. Checiu and Sandor (1982, 1986) reported reduced implantation with impaired oviductal embryo transport and retarded and abnormal development of mouse embryos associated with acute maternal intoxication of ethanol during the preimplantation period. Chernoff (1980) reported that female mice exposed to a continuous diet of ethanol both prior to, and throughout, gestation produced several fetal anomalies.

It is known that the effects of alcohol on embryo development and fetal growth are dependent on the dose, time, and duration of exposure. Most studies in animal models have been conducted with doses of ethanol that produce high ethanol-blood/tissue levels. The effects of moderate doses of alcohol are particularly important, since this is the common pattern of usage by the general population.

Some reports have examined the ethanol-induced fertility alterations in males, both in man and in laboratory animals (Anderson et al., 1983Go, 1987Go; Cicero et al., 1994Go). In females, chronic ethanol consumption impairs the reproductive cycle and alters ovarian function (Van Thiel et al., 1978Go; Eskay et al., 1981Go; Cebral et al., 1998aGo). In women, there is an elevated risk of infertility with high alcohol consumption (Mello, 1988Go), and even moderate alcohol use can affect fertility (Grodstein et al., 1994Go). Few studies have reported on the effects of alcohol administered to female mice prior to conception. It has been shown that alcohol given i.p. at specific times after ovulation can alter the quality of oocytes and increase parthenogenesis (Dyban and Khozhai, 1980Go). Exposure to alcohol at the appropriate stages of gametogenesis might be one of the causes of spontaneous abortion in which chromosome malsegregation may occur (Kaufman and O'Neill, 1988Go). Recently, we have shown that chronic 10% (w/v) ethanol in drinking water ingested by female mice during the onset of sexual maturity can alter the quality of oocytes (Cebral et al., 1998 aGo,bGo). The aim of this work was to study the influence of moderate chronic ethanol ingestion by female mice on the preimplantation development in vitro. We also examined the pronucleus formation of in vitro fertilized oocytes to examine the possible alterations in embryo development with fertilization and the gamete quality post-insemination.


    MATERIALS AND METHODS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Animals
Groups of three to four hybrid F1 mice (C57Bl x CBA) from our colony were kept in plastic cages and maintained under controlled room temperature (25°C) and light cycles (14 h light/10 h dark; lights on at 06:00). They were fed ad libitum with a commercial mouse chow diet (Diet No. 1, from Nutrimentos S.A., Buenos Aires, Argentina). Daily caloric intake was estimated by the calorific value of the diet used (2900 kcal/kg). The amount of food consumed by female mice was determined by the difference between the chow offered and the remaining food plus the amount spilled.

Adult male 75-day-old mice (average body weight ± SEM: 30.4 ± 0.78 g) were used. Females were immature (prepubertal, 30-day-old, average body weight: 16.8 ± 0.45 g) at the start of the ethanol administration period. Control female mice were the same age and initial weights as the ethanol-treated mice.

Ethanol treatment
Immature female mice were treated with 10% (w/v) ethanol in drinking water for 30 days. Controls received water, and ethanol was substituted by maltose–dextrin (3.8 kcal/g) in a proportion of 56 g to 300 ml water, to be isocaloric with 10% ethanol; where water was the only drinking resource. The body weights were recorded daily throughout the treatment. The amount of daily liquid intake was determined by volume differences between the offered and the remaining volume. Calories derived from ethanol were estimated as 7.1 kcal/g. From these data, daily patterns of caloric intake and the percentage of ethanol derived calories (%EDC) were determined.

The effects of chronic moderate ethanol ingestion were examined on in-vitro fertilization (pronucleus formation and fragmentation rates) and on in-vitro embryo development.

Blood-ethanol measurement
A group of five immature female mice chronically treated with ethanol as described above were decapitated and trunk blood was collected into heparinized Eppendorf tubes at 06:00 on day 30 of treatment. Samples were maintained at 4°C, for blood-ethanol determinations within 4 h of collection. Ethanol was measured by gas chromatography as described previously (Cebral et al., 1997Go, 1998aGo).

In vitro fertilization
Source and collection of spermatozoa. Male mice were killed by cervical dislocation on the morning of day 30. One epididymis of a male was dissected and the cauda placed in a 200-µl drop of modified fertilization medium (FM) (Fraser and Drury, 1975Go) supplemented with 30 mg/ml bovine serum albumin (BSA 3%, Sigma Chemical Co., St Louis, MO, USA) and overlaid with mineral oil (Sigma). Spermatozoa were obtained by making small cuts in the cauda. The dense mass of spermatozoa was allowed to disperse for 5 min. The tissue was removed and the sperm concentration was determined using a Neubauer chamber. The sperm suspension was then incubated for 90 min in the same medium in a humidified tissue culture incubator (37°C and 5% CO2 in air) to allow capacitation.

Source and collection of oocytes. Female mice were induced to superovulate with 10 IU of pregnant mare's serum gonadotrophin (PMSG, Sigma) given at 18:00 on day 27 of the ethanol treatment and 10 IU of human chorionic gonadotrophin (HCG, Sigma) 48 h later (day 29). Females were killed by cervical dislocation 16–17 h after HCG injection when the ethanol treatment was stopped (day 30). Both oviducts were removed and placed in phosphate-buffered saline (PBS). The cumulus masses containing the oocytes were released from an ampulla into a 150-µl drop of FM [one oocyte –cumulus complex (OCC)/drop] and overlaid with mineral oil.

In vitro insemination. One OCC from one female was inseminated with 1–2 x 106 spermatozoa/ml from one male. The following groups were studied: (1) Group I: oocytes from control females inseminated with spermatozoa from control males; (2) Group II: oocytes from ethanol-treated females inseminated with spermatozoa from control males.

Evaluation of in vitro fertilization. After 5 h insemination, the oocytes from each female were recovered and washed to remove the cumulus cells and the adherent spermatozoa. They were placed into 100 µl fertilization medium (FM), with BSA 3% and overlaid with mineral oil. Oocytes were observed under an inverted phase-contrast microscope and classified as ‘activated' when the second polar body (II PB) was present, intact (morphologically normal or abnormal oocyte) and fragmented and/or necrotic (lysed) (recorded as degenerated).

At 8 h post-insemination, the activated oocytes were transferred to 100 µl of M16 medium (Whittingham, 1971Go) supplemented with BSA 3%, containing the vital fluorocrome Hoechst 33342 (0.5 µg/ml, Sigma) and overlaid with mineral oil. They were incubated for 1 h and pronucleus formation examined under a fluorescence microscope. Oocytes were normally fertilized when the II PB was present with two pronuclei (2PN). Haploid [activated oocytes with one pronucleus (1PN), triploid (polyspermic, with II PB and 3PN] or anucleated (with 0PN) oocytes were assessed. In each group a total of eight female mice were used.

In vitro development. In-vitro development was performed separately from in vitro fertilization experiments.

Immature female mice were treated with 10% ethanol for 30 days as described above. On day 27, they were superovulated with PMSG (10 IU) and HCG (10 IU) 48 h later. At 16–17 h post-HCG, in vitro fertilization was performed. At 6 h post-insemination, morphologically intact and activated (II PB) oocytes derived from one female were washed and placed in a 100-µl drop of M16 medium supplemented with BSA 3% and overlaid with mineral oil. The eggs were cultured in a humidified tissue culture incubator. The fragmented or dead eggs were discarded. The following groups were studied: (1) Group I: embryos derived from oocytes of control females inseminated with spermatozoa from control males; (2) Group II: embryos derived from oocytes of ethanol-treated females inseminated with spermatozoa from control males.

The in vitro development experiments were replicated five times. Fifteen females were used from each group.

  1. Evaluation of general development. The embryos were examined under an inverted phase-contrast microscope. Day 1 of in vitro development was considered to be 24–32 h post-HCG (pronuclei and zygotes formation). The stages of embryo development were assessed. We obtained on day 2, 2-cell embryos; day 3, 2- and 4-cell embryos; day 4, uncompacted morulae or compacted morulae; day 5, early blastocysts (with initial blastocele), expanded blastocysts, and some compacted morulae; day 6, hatching blastocysts appeared (zona-escaping blastocysts with rupture and penetration of the zona pellucida by trophoectodermal projections); day 7, blastocysts were hatching or hatched (extruded or zona free embryo). Morphologically normal and abnormal embryo quantities in the culture were considered as general development.
  2. Evaluation of embryo quality. The morpho-logically abnormal embryos were assessed from day 2 to day 6 of in vitro development. The quality was studied by a combination of characteristics and general appearance. The following abnormalities were taken into account: extracellular fragmentation (presence of small clumps of cytoplasm surrounded by membrane in the perivitelline space); no intact blastomeres (with necrotic signs or lysis); blastomeres of unequal size; decompacted blastomeres in compacted morulae; abnormal cavitation (two or more blastoceles); vesicles in ectoderm; small inner cell mass.

Statistics
Group differences were examined by the {chi}2 test for in vitro fertilization and embryo development data, and analysis of variance (ANOVA, Student's t-test); for other measures, the Instat Program (GraphPAD software, San Diego, CA, USA) was used. P < 0.05 was considered as significant.


    RESULTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Average daily intakes of water, food, and ethanol in females (expressed in ml, g, and kcal/mouse respectively) are shown in Table 1Go. The %EDC was 27%. The body weight reached at the end of the treatment was 26.1 ± 0.4 g for control females and 27.2 ± 0.7 g for ethanol-treated females (mean ± SEM). The total caloric intake was similar in both groups, although the amount of solid and liquid intake was significantly different between groups.


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Table 1. Daily intake of food, fluid, and percentage of ethanol-derived calories in female mice chronically treated with 10% ethanol
 
Blood-ethanol levels on the morning of the last day of treatment were 50 ± 2.5 mg/dl of blood (mean ± SEM for 5 mice).

In vitro fertilization
Evaluation at 5 h post-insemination. Table 2Go shows that chronic ethanol (10% w/v) treatment produced significantly lower activation percentage (oocytes with II PB) in the treated females compared to control females (P < 0.001). The percentages of intact and degenerated oocytes were significantly increased in the treated females with respect to oocytes from control females (P < 0.001). The total number of oocytes obtained from eight treated females (215) was less than that from eight control females (316).


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Table 2. Effect of chronic 10% ethanol intake on in vitro fertilization (5 h post-insemination)
 
Evaluation at 8 h post-insemination. The percentage of fertilized oocytes expressed as oocytes with II PB and 2PN was significantly (P < 0.01) decreased in the ethanol-treated females compared to control females (Table 3Go). The percentage of haploid oocytes (II PB and 1PN) was higher in the treated females than in the control females (P < 0.01). The percentages of anucleated (0PN) and polyspermic (3PN) oocytes were similar between the control and treated females (Table 3Go).


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Table 3. In vitro fertilization at 8 h post-insemination: evaluation of pronucleus formation in activated oocytes (with II PB)
 
In vitro development and embryo quality
Evaluation of general development. Table 4Go(A) shows the embryo stages on days 2, 3, and 4. On day 2, there was a significantly decreased percentage of 2-cell embryos in the ethanol-treated females compared to control females (P < 0.01). On day 3, the 4-cell embryo percentage was reduced in the ethanol-treated group (P < 0.001) with respect to the control group, while there was a highly significant increase in the percentage of 2-cell embryos compared to the control females. On day 4, a reduced percentage of embryos reached the compacted morula stage in the ethanol-treated group compared to the control group (P < 0.001).


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Table 4. Influence of 10% ethanol consumption on general in vitro development
 
Later in vitro development is shown in Table 4Go(B). On day 5, the embryos of the control group were in early and expanded blastocyst stages, a small percentage was in the compacted morula stage. The ethanol-treated group had very few blastocysts compared to the control females (P < 0.001). However, there was a higher percentage of compacted morulae in the treated females compared to the control females (P < 0.001). On day 6, most embryos from the control females were in the expanded blastocyst stage and hatching blastocysts were also found. There was a significantly decreased percentage of expanded and hatching blastocysts in the ethanol-treated group (P < 0.001), whereas the percentage of early blastocysts was increased in the ethanol-treated females compared to the control females (P < 0.01). On day 7, there were very few embryos in the hatching and hatched blastocyst stages in the ethanol-treated group compared to the control group (P < 0.001).

Evaluation of embryo quality. Figure 1Go shows abnormal embryo percentages, recorded during in vitro development. On day 2, ethanol-treated females showed a higher percentage of abnormal 2-cell embryos compared to the control group (P < 0.05), whereas on days 3 (4-cell embryos) and 4 (morulae), there were no significant differences between the groups. The ethanol-treated females had significantly increased percentages of abnormal blastocysts on days 5 and 6 compared to control females (P < 0.001).



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Fig. 1. Evaluation of embryo quality.

Morphologically abnormal embryos were assessed every day in the culture as described in Materials and methods. Columns represent the percentage of abnormal embryos. Solid columns, control females; hatched columns, ethanol-treated female and control male embryos. *P < 0.05, ***P < 0.001, {chi}2-test.

 

    DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Our hypothesis was that chronic ethanol consumption by female mice prior to conception could affect not only the in vitro fertilization rate but also the embryo development in vitro. After 5 h of in vitro insemination, oocytes from ethanol-treated female mice showed lower activation rates than oocytes from control females. Furthermore, when we evaluated the presence of pronuclei in the activated oocytes at 8 h post-insemination, the percentage of fertilized oocytes was reduced in the ethanol-treated females because there was an elevated percentage of haploid oocytes (1PN). In a previous paper, we have shown that consumption of 10% ethanol long term produces a high percentage of oviductal activated oocytes at 16 and 20 h post-HCG (Cebral et al., 1998bGo). The increased percentage of 1 PN oocytes found after insemination may be caused by a high proportion of activated oocytes contained in the OCC before in vitro fertilization. The 1 PN-activated and intact (metaphase II) oocytes cannot be penetrated by spermatozoa. The possible mechanism(s) of these ethanol effects is not clear. It may involve impaired chromosome segregation, interference with cell division (Kaufman, 1997Go), and impairment of spindle apparatus functioning (O'Neill and Kaufman, 1989Go). A sensitive period for a relatively high incidence of aneuploidy around the time of ovulation, due to ethanol consumption, has also been described (Kaufman, 1985Go). The exposure of female mice to alcohol during the meiotic divisions of the cycle before ovulation induced disrupted chromosome segregation (Kaufman and Bain, 1984 aGo,bGo).

We found high rates of oocyte fragmentation in the treated females after in vitro fertilization. Fragmentation can occur in cleaved unfertilized ova that undergo parthenogenetic activation (Takase et al., 1995Go). We have previously demonstrated that the OCC, recovered at 16 h post-HCG from treated females, contained an increased percentage of fragmented oocytes after it was maintained for 5 h in culture medium without spermatozoa (Cebral et al., 1998bGo). However, the ethanol-treated females showed reduced numbers of oocytes in in vitro fertilization compared to control females. This result is in accord with our previous findings which showed that treated females ovulated fewer oocytes per female at 16 h post-HCG than controls but a similar number of oocytes at 20 h post-HCG. The follicular maturation and ovulation time was therefore retarded in the 10% ethanol-treated females (Cebral et al., 1998bGo). Eskay et al. (1981) suggested that ethanol in the range of 50–100 mg/dl can act as a direct gonadal toxin. We hypothesized that the present ethanol level of 50 mg/dl was toxic to oocyte development in vivo.

Many studies have described the effects of ethanol administration during the preimplantation period (Checiu and Sandor, 1986Go), before mating and through the preimplantation development (Sandor et al., 1980Go) or exposure during culture in vitro of mouse embryos (Wiebold and Becker, 1987Go; Kalmus and Buckenmaier, 1989Go; Kowalczyk et al., 1996Go). However, we studied the in-vitro growth of embryos derived from oocytes of female mice treated chronically with moderate ethanol doses before fertilization.

Our work shows for the first time that chronic ingestion of alcohol by females prior to gestation can produce early retarded and arrested development. When embryo development was examined, impaired growth was found in the ethanol-treated females at all the embryo stages studied. There was an embryo loss from day 2 and retardation from day 3 (2- and 4-cell embryos). Oocytes can be activated by non-specific exogenous stimuli and begin parthenogenetic development (Pickering et al., 1988Go; Winston et al., 1991Go). Retarded embryo development in the ethanol-treated females may be a consequence of augmented parthenogenetic development. Several authors (Kaufman, 1990Go; Henery and Kaufman, 1992Go) have found that parthenogenetically activated single pronuclear haploid mouse embryos have a slower cleavage rate during the preimplantation period than heterozygous diploid parthenogenetic embryos and fertilized control diploid embryos. Since we have also found high parthenogenetic activation after fertilization, we believe that the retarded embryo growth of ethanol-treated females was a parthenogenetic development. We also found reduced percentages of blastocysts and impaired hatching in vitro. The exact reason(s) why a proportion of haploid parthenogenones fail to blastulate is unknown. It was reported that these embryos contain a higher proportion of delayed blastomeres (Kaufman, 1990Go). However, since we found an increased proportion of morphologically abnormal blastocysts in the ethanol-treated females, we think that there is a relation with the nuclear status of embryos, produced by ethanol ingestion. It has been shown that monosomic and trisomic embryos induced by maternal exposure to ethanol are morphologically abnormal and are only capable of surviving to the morula stage (Kaufman and Bain, 1984bGo).

The developing embryos that did not reach a normal stage either arrested or became fragmented. We found very early embryo loss (day 2) and increased fragmentation later. Little is known about the mechanisms underlying the degeneration of oocytes and embryos, but it has been suggested that parthenogenetic embryos can undergo an apoptotic process and/or fragmentation (Takase et al., 1995Go).

This paper shows that chronic moderate ethanol ingestion by young female mice has deleterious effects on pronucleus formation and on preimplantation development of in-vitro fertilized embryos resulting in arrested and retarded development, morphological abnormalities, and embryo losses.


    ACKNOWLEDGEMENTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The authors thank Dr Samuel M. McCann and Dr Marcelo Viggiano for revising this paper and suggestions regarding the statistical analysis. This work was supported by Grant PIP 4076 from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and Bago S.A. (Argentina).


    FOOTNOTES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
* Author to whom correspondence should be addressed. Back


    REFERENCES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
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